Journal of Parasitology

Volume 93, Issue 4 (August 2007), pp.727- 976

BIOCHEMISTRY-PHYSIOLOGY

COMPARISON OF THE PEPTIDASE ACTIVITY IN THE ONCOSPHERE EXCRETORY/SECRETORY PRODUCTS OF TAENIA SOLIUM AND TAENIA SAGINATA

Mirko J. Zimic, Jesús Infantes, César López, Jeanette Velásquez, Marilú Farfán, Mónica Pajuelo, Patricia Sheen, Manuela Verastegui, Armando Gonzalez, Hector H. García, and Robert H. Gilman

727

ECOLOGY-EPIDEMIOLOGY

EFFECTS OF ACANTHOCEPHALUS LUCII (ACANTHOCEPHALA) ON INTERMEDIATE HOST SURVIVAL AND GROWTH: IMPLICATIONS FOR EXPLOITATION STRATEGIES

Daniel P. Benesh and E. Tellervo Valtonen

735

PROXIMATE FACTORS AFFECTING THE LARVAL LIFE HISTORY OF ACANTHOCEPHALUS LUCII (ACANTHOCEPHALA)

Daniel P. Benesh and E. Tellervo Valtonen

742

FIELD EVIDENCE OF HOST SIZE-DEPENDENT PARASITISM IN TWO MANIPULATIVE PARASITES

Yannick Outreman, Frank Cézilly, and Loïc Bollache

750 HELMINTH COMMUNITIES IN FIVE SPECIES OF SYMPATRIC AMPHIBIANS FROM THREE ADJACENT EPHEMERAL PONDS IN SOUTHEASTERN WISCONSIN

H. Randall Yoder and James R. Coggins

755

ECTOPARASITOLOGY

NEW AND PREVIOUSLY DESCRIBED SPECIES OF DACTYLOGYRIDAE (MONOGENOIDEA) FROM THE GILLS OF PANAMANIANFRESHWATER FISHES (TELEOSTEI)

Edgar F. Mendoza-Franco, M. Leopoldina Aguirre-Macedo, and Víctor M. Vidal- Martínez

761

THREE NEW SPECIES OF LIGOPHORUS (MONOGENEA: DACTYLOGYRIDAE) ON THE GILLS OF MUGIL CEPHALUS (TELEOSTEI: MUGILIDAE) FROM THE JAPAN SEA

Nataliya Yu. Rubtsova, Juan A. Balbuena, and Volodimir L. Sarabeev

772

TWO NEW SPECIES OF HALIOTREMA (MONOGENOIDEA: DACTYLOGYRIDAE) FROM ACANTHURUS NIGROFUSCUS AND ACANTHURUS OLIVACEUS (TELEOSTEI: ACANTHURIDAE) IN THE SOUTH CHINA SEA

Yuan Sun, Delane C. Kritsky, and Tingbao Yang

781

IN MEMORIAM

ROBERT L. CALENTINE 1929–2007

Bruce M. Christensen

787

ECTOPARASITOLOGY

THE FIRST CHONDRACANTHID (COPEPODA: CYCLOPOIDA) REPORTED FROM CULTURED FINFISH, WITH A REVISED KEY TO THE SPECIES OF CHONDRACANTHUS

Danny Tang, Melanie Andrews, and Jennifer M. Cobcroft

788

GENETICS-EVOLUTION

IMPACT OF POPULATION STRUCTURE ON GENETIC DIVERSITY OF A POTENTIAL VACCINE TARGET IN THE CANINE HOOKWORM (ANCYLOSTOMA CANINUM)

Jennifer M. Moser, Ignazio Carbone, Prema Arasu, and Greg Gibson

796

IMMUNOLOGY

IDENTIFICATION OF FASCIOLA HEPATICA RECOMBINANT 15-KDA FATTY ACID–BINDING PROTEIN T-CELL EPITOPES THAT PROTECT AGAINST EXPERIMENTAL FASCIOLIASIS IN RABBITS AND MICE

Antonio Muro, Patricia Casanueva, Julio López-Abán, Vicente Ramajo, Antonio R. Martínez-Fernández, and George V. Hillyer

817

RENEWED HOPE FOR A VACCINE AGAINST THE INTESTINAL ADULT TAENIA SOLIUM

Edda Sciutto, Gabriela Rosas, Carmen Cruz-Revilla, Andrea Toledo, Jacquelynne Cervantes, Marisela Hernández, Beatríz Hernández, Fernando A. Goldbaum, Aline S. de Aluja, Gladis Fragoso, and Carlos Larralde

824

INVERTEBRATE-PARASITE RELATIONSHIPS

SURFACE MEMBRANE PROTEINS OF BIOMPHALARIA GLABRATA EMBRYONIC CELLS BIND FUCOSYL DETERMINANTS ON THE TEGUMENTAL SURFACE OF SCHISTOSOMA MANSONI PRIMARY SPOROCYSTS

Maria G. Castillo, Xiao-Jun Wu, Nathalie Dinguirard, A. Kwame Nyame, Richard D. Cummings, and Timothy P. Yoshino

832

REDESCRIPTION OF TWO DICYEMENNEA (PHYLUM: DICYEMIDA) FROM ROSSIA PACIFICA (MOLLUSCA: CEPHALOPODA: DECAPODA)

Hidetaka Furuya

841

LIFE CYCLES-SURVEY

CHIMPANZEE PINWORM, ENTEROBIUS ANTHROPOPITHECI (NEMATODA: OXYURIDAE), MAINTAINED FOR MORE THAN TWENTY YEARS IN CAPTIVE CHIMPANZEES IN JAPAN

Hideo Hasegawa and Toshifumi Udono

850

PATHOLOGY

DEVELOPMENT AND PATHOLOGY OF ECHINOSTOMA CAPRONI IN EXPERIMENTALLY INFECTED MICE

Carla Muñoz-Antoli, Javier Sotillo, Carlos Monteagudo, Bernard Fried, Antonio Marcilla, and Rafael Toledo

854

SYSTEMATICS-PHYLOGENETICS

A NEW GENUS AND SPECIES OF MACRODEROIDIDAE, AND OTHER DIGENEANS FROM FISHES OF LAKE MALAWI, AFRICA

Rodney A. Bray and Sherman S. Hendrix

860 NEW SPECIES OF SKRJABINODON (NEMATODA: PHARYNGODONIDAE) IN URACENTRON FLAVICEPS (SQUAMATA: IGUANIDAE) FROM ECUADOR AND PERU

Charles R. Bursey and Stephen R. Goldberg

866

REDESCRIPTION, SYNONYMY, AND NEW RECORDS OF VEXILLATA NOVIBERIAE (DIKMANS, 1935) (NEMATODA: TRICHOSTRONGYLINA), A PARASITE OF RABBITS SYLVILAGUS SPP. (LEPORIDAE) IN THE UNITED STATES

María Celina Digiani, John M. Kinsella, Thomas B. Kass, and Marie-Claude Durette- Desset

870

A COLLECTION OF FISH LEECHES (HIRUDINIDA: PISCICOLIDAE) FROM JAPAN AND SURROUNDING WATERS, INCLUDING REDESCRIPTIONS OF THREE SPECIES

Sharon Furiness, Julianne I. Williams, Kazuya Nagasawa, and Eugene M. Burreson

875

PARACOSMOCERCELLA ROSETTAE N. GEN. ET N. SP. (NEMATODA: COSMOCERCOIDEA: COSMOCERCIDAE) COLLECTED FROM THE JAPANESE TREE FROG, HYLA JAPONICA (ANURA: HYLIDAE), IN JAPAN

Hideo Hasegawa and Yatsukaho Ikeda

884

DIVERSITY AND PHYLOGENY OF MITOCHONDRIAL CYTOCHROME B LINEAGES FROM SIX MORPHOSPECIES OF AVIAN HAEMOPROTEUS (HAEMOSPORIDA: HAEMOPROTEIDAE)

Olof Hellgren, Asta Križanauskiene, Gediminas Valki nas, and Staffan Bensch

889

A NEW SPECIES OF MACROBOTHRIIDAE (CESTODA: DIPHYLLIDEA) FROM THORNBACK RAY PLATYRHINA SINENSIS IN CHINA

Hongtao Li and Yanhai Wang

897

A NEW SPECIES OF COMEPHORONEMA (NEMATODA: CYSTIDICOLIDAE) FROM THE STOMACH OF THE ABYSSAL HALOSAUR HALOSAUROPSIS MACROCHIR (TELEOSTEI) FROM THE MID-ATLANTIC RIDGE

František Moravec and Sven Klimpel

901

A NEW SPECIES OF PARACAPILLARIA (NEMATODA: CAPILLARIIDAE) FROM THE INTESTINE OF THE TOAD DUTTAPHRYNUS MELANOSTICTUS (ANURA) FROM THE MALAYAN PENINSULA

František Moravec, David Modrý, and Miloslav Jirk

907

TWO NEW SPECIES OF COSMOCERCIDS (ASCARIDIDA) IN THE TOAD CHAUNUS ARENARUM (ANURA: BUFONIDAE) FROM ARGENTINA

Geraldine Ramallo, Charles R. Bursey, and Stephen R. Goldberg

910

NEW SPECIES OF HAPLOMETROIDES (DIGENEA: PLAGIORCHIIDAE) FROM PHALOTRIS NASUTUS (GOMES, 1915) (SERPENTES, COLUBRIDAE)

Reinaldo José da Silva, Vanda Lúcia Ferreira, and Christine Strüssmann

917

A NEW SPECIES OF PARACAPILLARIA (NEMATODA: CAPILLARIIDAE) PARASITIZING THE BRAZILIAN SANDPERCH, PINGUIPES BRASILIANUS (PISCES: PINGUIPEDIDAE), FROM ARGENTINA

Juan T. Timi, María A. Rossin, Ana L. Lanfranchi, and Jorge A. Etchegoin

922 THERAPEUTICS-DIAGNOSTICS

CHEMICAL INACTIVATION OF TOXOPLASMA GONDII OOCYSTS IN WATER

Katlyn E. Wainwright, Melissa A. Miller, Bradd C. Barr, Ian A. Gardner, Ann C. Melli, Tim Essert, Andrea E. Packham, Tin Truong, Manuel Lagunas-Solar, and Patricia A. Conrad

925

RESEARCH NOTES

Multiplication of Trypanosoma pacifica (Euglenozoa: Kinetoplastea) in English Sole, Parophrys vetulus, From Oregon Coastal Waters

Eugene M. Burreson and Egil Karlsbakk

932

Contribution of NADH Dehydrogenase Subunit I and Cytochrome C Oxidase Subunit I Sequences Toward Identifying a Case of Human Coenuriasis in France

Jocelyne Collomb, Marie Machouart, Marie-France Biava, Mélanie Brizion, Karine Montagne, François Plénat, and Bernard Fortier

934

Checklist of Helminth Parasites of the Cane Toad Bufo marinus (Anura: Bufonidae) From Mexico

Arlett Espinoza-Jiménez, Luis García-Prieto, David Osorio-Sarabia, and Virginia León- Règagnon

937

Parasitic Helminths of Free-Ranging Mink (Neovison vison mink) From Southern Florida

Garry W. Foster, Mark W. Cunningham, John M. Kinsella, and Mike Owen

945

Viability of the Encysted Metacercariae of Echinostoma caproni Judged by Light Microscopy Versus Chemical Excystation

Bernard Fried and Robert C. Peoples

947

Coprology of Panthera tigris altaica and Felis bengalensis euptilurus From the Russian Far East

P. González, E. Carbonell, V. Urios, and V. V. Rozhnov

948

Seroprevalence of Toxoplasma gondii in Pigs From Vietnam

Lam Thi Thu Huong and J. P. Dubey

951

Seroprevalence of Toxoplasma gondii in Cats From St. Kitts, West Indies

L. Moura, P. Kelly, R. C. Krecek, and J. P. Dubey

952

Bartonella and Rickettsia From Fleas (Siphonaptera: Ceratophyllidae) of Prairie Dogs (Cynomys spp.) From the Western United States

Will K. Reeves, Thomas E. Rogers, and Gregory A. Dasch

953

Prevalence of Antibodies to Leishmania infantum and Trypanosoma cruzi in Wild Canids From South Carolina

Alexa C. Rosypal, Richard R. Tidwell, and David S. Lindsay

955

Distribution Pattern of Phthirapterans Infesting Certain Common Indian Birds

A. K. Saxena, Sandeep Kumar, Nidhi Gupta, J. D. Mitra, S. A. Ali, and Roshni Srivastava

957 Feline Cuterebrosis Caused by a Lagomorph-Infesting Cuterebra spp. Larva

Frank Slansky

959

Molecular Identification of Two Strains of Third-Stage Larvae of Contracaecum rudolphii Sensu Lato (Nematoda: Anisakidae) From Fish in Poland

Beata Szostakowska and Hans-Peter Fagerholm

961

Genetic and Immunological Characterization of the 14-3-3 Molecule From Schistosoma bovis

N. Uribe, A. Muro, C. Vieira, J. Lopez-Aban, E. del Olmo, L. Suárez, A. R. Martínez- Fernández, and M. Siles-Lucas

964

CRITICAL COMMENT

From the Roots of Parasitology: Hippocrates' First Scientific Observations in Helminthology

Constantinos Trompoukis, Vasilios German, and Matthew E. Falagas

970

IN MEMORIAM

Wilbur L. Bullock 1922–2007

Patrick M. Muzzall and David G. Huffman

973

BOOK REVIEW

Cisticercosis: Guía para Profesionales de la SaludEditor's Note …

975

J. Parasitol., 93(4), 2007, pp. 727–734 ᭧ American Society of Parasitologists 2007

COMPARISON OF THE PEPTIDASE ACTIVITY IN THE ONCOSPHERE EXCRETORY/SECRETORY PRODUCTS OF TAENIA SOLIUM AND TAENIA SAGINATA

Mirko J. Zimic, Jesu´ s Infantes, Ce´sar Lo´ pez, Jeanette Vela´squez, Marilu´ Farfań, Mo´ nica Pajuelo, Patricia Sheen, Manuela Verastegui, Armando Gonzalez*, Hector H. Garcıá†, and Robert H. Gilman‡ Laboratorios de Investigacioń y Desarrollo, Facultad de Ciencias, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, SMP, Lima, Peru´. e-mail: [email protected]

ABSTRACT: We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluoro- metrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.

Taeniasis/cysticercosis is caused by infection with the ces- cause human cysticercosis, with those of T. saginata, which do tode Taenia solium. It is an important public health problem in not, to determine whether the enzymes present in the onco- developing countries (Garcia et al., 2003), and it is recognized sphere may be the reason for the difference in pathogenicity in as an increasing cause of human disease (Schantz, 1989). Neu- humans. rocysticercosis is a major cause of morbidity and hospitalization Peptidase activity is widely used by many parasites to pen- in these areas, and it accounts for more cases of epilepsy than etrate host tissue, i.e., helminths that secrete serine, cysteine, any other parasitic disease (Garcia et al., 1993). and metallo- and aminopeptidases for this purpose (Halawa and Taenia solium has a life cycle involving 2 hosts. Humans are Jakacka, 1977; Fukase et al., 1984, 1985; McKerrow, 1989; the only known definitive host for the adult worm, and pigs are Marco and Nieto, 1991; White et al., 1992; Song and Chappell, the usual intermediate hosts for the larval form. Pigs can ingest 1993; Kong et al., 1994). complete proglottids or T. solium eggs when consuming human Similarly, in T. saginata, serine, cysteine, and aminopepti- feces or food contaminated with fecal matter, resulting in in- dase activities have been identified in the E/S products of on- vasion of oncospheres and development of muscular and brain cospheres (White et al., 1996). In the present study, we com- cysts. Similarly, ingestion of T. solium eggs by humans through pared the peptidase activity of E/S antigens to determine wheth- fecal oral contamination causes human cysticercosis. er a difference in the profile of these proteins may account for Upon ingestion in both pigs and humans, oncospheres are the differential invasion. stimulated by the intestinal tract environment, hatch from the eggs, cross the intestinal wall, and migrate to different parts of MATERIALS AND METHODS the body through the bloodstream. The mechanism used by T. Hatching and activation of oncospheres solium oncospheres to penetrate the human intestinal wall is unknown, but it has important implications for potential vac- Gravid T. solium and T. saginata proglottids were collected from newly diagnosed patients who had received a standard oral taeniacide cines. treatment of 2 g of niclosamide (Jeri et al., 2004). The feces were Taenia saginata is a cestode closely related to T. solium in collected for 48 hr after treatment. The proglottids were collected by which cattle and related animals are the main intermediate sieving and washing thoroughly with distilled water, and they were then hosts. Humans are the definitive hosts that harbor the tapeworm stored in 25% glycerol, supplemented with penicillin (1,000 IU/ml), gentamicin (100 ␮g/ml), and amphotericin B (25 ␮g/ml) at 4 C until and release eggs in the feces. Only cattle can develop cysticer- used. Differentiation of species was made by histology and polymerase cosis after ingestion of eggs; humans are not susceptible to this chain reaction-restriction enzyme analysis (Mayta et al., 2000). infection. Due to the inability of T. saginata to cause human To release eggs, gravid proglottids were mechanically macerated with cysticercosis, it is important to contrast the peptidase activity a glass homogenizer. To reduce contamination with fecal material, eggs of the excretory/secretory (E/S) antigens of T. solium, which were maintained in 2.5% potassium dichromate solution (Sigma, St. Louis, Missouri) at 4 C for 24 hr. After 15 min of incubation at room temperature, eggs in the supernatant were recovered with a Pasteur pi- Received 1 June 2006; revised 31 December 2006; accepted 2 January pette. Eggs were washed 3 times in distilled water before hatching. In 2007. vitro hatching of oncospheres was performed by adding 0.75% sodium * Facultad de Medicina Veterinaria, Universidad Nacional Mayor de hypochlorite and incubating for 10 min at4Casdescribed previously San Marcos, Cuadra 29 Av. Circunvalacion s/n, San Borja, Lima 42, (Laws, 1968; Stevenson, 1983; Ito et al., 1997; Verastegui et al., 2000). Peru. Oncospheres were washed 3 times in RPMI 1640 medium (Sigma), † Cysticercosis Unit, Instituto de Ciencias Neurolo´gicas. Jr. Ancash resuspended in this medium, and counted using a Neubauer chamber 1271, Hospital Santo Toribio de Mogrovejo, Barrios Altos, Lima 1, (Fisher Scientific, Atlanta, Georgia). Peru. To stimulate an optimal secretion of peptidases, oncospheres were ‡ Present address: Department of International Health, Bloomberg activated with artificial intestinal fluid containing pancreatin (1% pan-

School of Public Health, The Johns Hopkins University, Baltimore, creatin, 1% fresh pig bile, and 0.2% Na2CO3 in RPMI 1640 medium, Maryland 21205. To whom correspondence should be addressed. pH 8) for 1 hr at 37 C while shaking (Read and Simmons, 1963).

727 728 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

To contrast the effect of pancreatin on activation, a fraction of T. tidase interference, thus detecting mainly trypsin-like activity. MeoSuc-

solium oncospheres was activated with a pancreatin-free medium (0.8% Ala-Ala-Pro-Met-AMC was tested without CaCl2 to reduce chymotryp- cholesterol, 2% fresh pig bile, and 1% Na2CO3 in RPMI 1640 medium, sin-like interference, thus detecting mainly elastase-like activity. pH 8), for 1 hr at 37 C while shaking (White et al., 1996). Each reaction consisted of 480 ␮l of the specific buffer, 10 ␮lof The oncospheres activated with pancreatin medium (OAPM) and the E/S fraction, and 10 ␮l of substrate. The background fluorescence of oncospheres activated with pancreatin-free medium (OAPFM) were AMC was controlled with a blank, prepared by replacing the E/S an- washed 3 times in 12 ml of RPMI 1640 medium. In each washing, the tigens with a similar volume of buffer. In addition, confirmation that solution was homogenized by inversion, centrifuged at 537 g for 5 min, the oncosphere culture medium lacked any significant peptidase activity and the supernatant was frozen at Ϫ70 C for measuring peptidase ac- before use was completed for all the substrates tested. Reactions were tivity. conducted at 37 C and followed for 24 hr, with measurements of the To determine the effect of centrifugation in the viability of onco- accumulated fluorescence taken approximately every 10 min during the spheres, the numbers of total and viable oncospheres were counted at first 2 hr of reaction and every hour during the next 4 hr of reaction, the beginning and at the end of the third wash for both OAPM and completing the last measurement at the 24 hr of reaction. OAPFM. The E/S batches from 3 different taenias were prepared for both T. solium and T. saginata. Due to E/S availability limitations, the amino- Oncosphere E/S antigen preparation peptidase B-like activity was tested only in 1 batch for both parasites. The average hydrolysis rates over the 24-hr reaction period for each of ϫ 6 After washing, 1–5 10 activated oncospheres were transferred into the E/S fractions (24 hr, 48 hr, 72 hr, and 96 hr) were estimated from 24-well Falcon plates with minimal essential medium (Sigma), contain- a linear regression between the accumulated concentration of hydro- ␮ ing penicillin (1,000 IU/ml) and gentamicin (100 g/ml), and then they lyzed substrates and the time since the start of the reaction, using the incubated at 37 C in an atmosphere of 5% CO2 with frequent shaking. pooled data of the 3 batches of oncospheres. The data included in the The oncospheres were monitored daily by microscopy to determine regression included every reading since the start of the reaction until viability and appearance. The concentration of viable oncospheres in the first reading before the first out-of-range fluorescence reading. the culture at time 0 was estimated using 0.4% trypan blue (Sigma) to A normalized activity was calculated by dividing the average hydro- stain 100–150 oncospheres. Oncospheres that excluded the dye were lysis rate (slope of the linear regression between the hydrolyzed AMC- considered viable. Every 24 hr, the medium plus oncospheres was re- substrate and the reaction time) by the average concentration of viable covered, placed in a 1.5-ml conical screw top tube, and centrifuged at oncospheres present over the time period. The normalized activity in- 425 g for 5 min. The supernatant was removed and used in subsequent dicates the mean number of substrates hydrolyzed per viable oncosphere protease assays; the pellet containing oncospheres was resuspended in per minute under the defined conditions. new media. To prevent crystallization and protein damage, 1% dimethyl To confirm the specificity of the peptide substrates, peptidase reac- sulfoxide (DMSO) was added to the harvested media, which was then tions were repeated in the presence of class-specific and nonspecific Ϫ stored at 70 C (Beynon and Bond, 1989). This process was repeated inhibitors, acquired from Sigma. For cysteine peptidase substrates we 4 times to obtain the E/S antigens corresponding to the 24-, 48-, 72-, used E64, leupeptin, and chymostatin. For serine protease substrates, and 96-hr spent media fractions. we used phenylmethylsulfonyl fluoride (PMSF), chymostatin, tosyl- To determine the effect of the culture period and the centrifugation phenylalanylchloromethane (TPCK), 4-[2-aminoethyl]benzenesulfonyl on the viability, the number of viable oncospheres was counted at the fluoride (Pefabloc), and leupeptin. EDTA was used with the metallo- start and at the end of the 24-hr period of culture as well as after the protease substrate. A pool of similar volumes of 24- and 48-hr E/S centrifugation. fractions from T. solium OAPM was tested. Concentrations of inhibitors were used as suggested by the manufacturer, i.e., 10 ␮M chymostatin, Protease assays 100 ␮MPMSF,10␮M TPCK, 100 ␮M Pefabloc, 10 ␮M leupeptin, 10 ␮ Serine-like and cysteine-like peptidase as well as aminopeptidase B M E64, and 10 mM EDTA. The adequate concentrations of chymo- activities were measured in each of the E/S fractions of T. solium and statin, TPCK, Pefabloc, E64, and leupeptin were verified as the mini- T. saginata. For these measurements, peptide substrates coupled to mum concentration able to achieve a significant inhibition in a dose– 7-amino-4-methylcoumarin (AMC) were used. The free AMC produced response curve. The percentage of inhibition was determined by com- after the cleavage of the peptide was fluoresced, and the intensity of paring the normalized hydrolysis rates after inhibition with that of the the fluorescence as detected in a TD-700 fluorometer (Turner Designs, uninhibited reaction. The reduction of the hydrolysis rate after adding Sunnyvale, California); 360-nm excitation and 460-nm emission wave- the inhibitor was compared with the uninhibited rate to estimate the lengths were used to determine the peptidase activity. The accumulated percentage of inhibition. concentration of hydrolyzed peptide substrates at a particular time was To account for contamination of E/S enzymes by cell lysis and the estimated by interpolating the detected fluorescence in a calibration release of cytosolic enzymes during sample manipulation, oncospheres curve of free AMC. were sonicated after the recovery of the 96-hr E/S fraction. Activity of Substrates used were Z-Arg-Arg-AMC (a specific substrate for ca- representative peptidases chymotrypsin- and cathepsin L-like after son- thepsin B-like peptidase), Z-Phe-Arg-AMC (a substrate for cathepsin ication, representing total intercellular activity, was measured using the L-, cathepsin B-like cysteine peptidases, and trypsin-like serine pepti- same fluorescent assays. dases), Mu-Leu-Tyr-AMC (a specific substrate for calpaine-like peptidases), Suc-Leu-Leu-Val-Tyr-AMC (a substrate for chymotrypsin-like Gelatin zymography serine peptidases and calpaine-like peptidases), Z-Arg-AMC (a substrate To further characterize the peptidases present in the oncosphere E/S for trypsin-like serine peptidases and cysteine peptidases), MeoSuc-Ala- products, gelatinolytic activity of a pool of 24-, 48-, 72-, and 96-hr Ala-Pro-Met-AMC (a substrate for elastases and chymotrypsin-like ser- E/S fractions of T. solium was determined using gelatin-based gel elec- ine peptidases), and Arg-AMC (a substrate for aminopeptidase B and trophoresis. The E/S fraction was resolved on an 11% polyacrylamide metalloproteases). All substrates were obtained from MP Biomedicals gel copolymerized with 0.12 mg/ml gelatin for approximately 3.5 hr (Aurora, Ohio), and they were diluted in a 5 mM solution in DMSO. (buffer included 25 mM Tris, 190 mM glycine, and 0.1% sodium do- We followed individual protocols for each substrate. These assays were decyl sulfate). After electrophoresis, the gel was washed in 2.5% Triton done following the conditions detailed in the technical sheet of the X for 1 hr, and then they were rinsed twice in low salt collagenase manufacturer that are specific for each peptidase (www.mpbio.com). buffer (55 mM Tris base, 200 mM sodium chloride, and 5 mM calcium Z-Phe-Arg-AMC was tested with 8 mM dithiothreitol (DTT) at pH 5 chloride, pH 7.2) and incubated at 37 C for approximately 16 hr in to reduce trypsin-like interference and with 4 M urea to reduce cathep- collagenase buffer. sin B-like interference, thus detecting mainly cathepsin L-like activity, Gelatinolytic activity was revealed as clear bands against a blue back- as confirmed by inhibition with trans-epoxysuccinyl-L-leucylamido-(4- ground by a single step stain–destain method using 0.02% Coomassie guanidino)butane (E64). Suc-Leu-Leu-Val-Tyr-AMC was tested at pH Blue (Pharmacia, Stockholm, Sweden) in 1:3:6 ratio of acetic acid: 7.8 to reduce calpaine-like interference, thus detecting mainly chymo- methanol:water. Inhibitions in gelatin zymography were obtained by trypsin-like activity, as confirmed by inhibition with chymostatin. preincubating the E/S pool with the previously described inhibitors in Z-Arg-AMC was tested at pH 7.3 without DTT to reduce cysteine pep- similar concentrations as used in the fluorescence assay. ZIMIC ET AL.—PEPTIDASE ACTIVITY IN T. SOLIUM AND T. SAGINATA 729

FIGURE 1. Comparison of the peptidase activity of the washing supernatants and E/S fractions between the OAPM and the OAPFM of Taenia solium ([AMC] minϪ1 10Ϫ8).

Statistical analysis RESULTS To verify the validity of using linear regression to estimate the hy- Comparison of the peptidase activity after activation with drolysis rate within the 24-hr follow up period, the Pearson–Wilkins correlation parameter was calculated. The comparison of the hydrolysis and without pancreatin rates was performed by constructing 95% conﬁdence intervals (CI) The serine peptidase activity of the activation medium con- around the slope parameter of the linear regression (Neter and Wasser- man, 1974). If these intervals overlap, we conclude that there is no taining pancreatin was much higher than that of the activation evidence to suggest a difference in the hydrolysis rates. All the analysis medium free of pancreatin. As seen in Figure 1, the recovered was performed with Stata 9.0 (Stata Corporation, 2005). supernatant after the third wash, for both the OAPM and the 730 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Normalized activity of the 24-, 48-, 72-, and 96-hr E/S fractions of Taenia solium and Taenia saginata. The substrates used to test each activity were Suc-Leu-Leu-Val-Tyr-AMC (chymotrypsin), Z-Arg-AMC (trypsin), MeoSuc-Ala-Ala-Pro-Met-AMC (elastase), Z-Phe-Arg-AMC (cathepsin L), Z-Arg-Arg-AMC (cathepsin B), Mu-Leu-Tyr-AMC (calpaine), and Arg-AMC (aminopeptidase B).

24-hr E/S 48-hr E/S 72-hr E/S 96-hr E/S Substrate (activity) T. solium T. saginata T. solium T. saginata T. solium T. saginata T. solium T. saginata

Chymotrypsin 1,020* 101.0 195 58.3 120 63.1 21.8 84.9 [585, 1460] [20.2, 181] [173, 216] [15.8, 101] [80.4, 160] [2.1, 124] [9.6, 34.1] [72.4, 97.3] Trypsin 24.8 1.9 18.3 NS† 14.8 NS 9.4 NS [17.7, 31.9] [1.1, 2.8] [10.4, 26.1] [10.9, 18.7] [6.2, 12.7] Elastase 5.9 178 3.1 4.3 6.0 1.9 36.8 4.5 [4.1, 7.6] [166, 190] [1.6, 4.5] [1.8, 6.8] [4.3, 7.7] [0.8, 2.9] [33.8, 39.1] [3.3, 5.8] Cathepsin L 13.0 85.2 13.6 29.3 8.7 1.9 11.4 NS [10.2, 15.8] [65.0, 105.0] [6.6, 20.6] [24.9, 33.7] [2.1, 15.2] [1.3, 2.4] [2.4, 20.3] Cathepsin B 0.5 NS NS NS NS NS NS NS [0.4, 0.6] Calpaine 9.5 8.8 74.2 10.1 NS 15.3 1.0 NS [5.7, 13.3] [0.6, 17.0] [26.9, 122] [4.2, 16.1] [1.2, 30.5] [0.5, 1.4] Amino 5.1 6.5 NS 2.4 NS 0.4 ‡ NS Peptidase B [4.8, 5.4] [6.1, 6.9] [2.2, 2.5] [0.3, 0.5]

* Mean [95% CI] of the peptidase activity (no. hydrolyzed AMC substrates ϫ minϪ1 ϫ viable oncosphereϪ1 ϫ 10Ϫ12). † NS, activity was not signiﬁcant. ‡ Activity was not tested.

OAPFM, had similar low peptidase activity for every substrate Using these activities, means and 95% confidence intervals tested. All the peptidase activities tested showed an increase in of the normalized peptidase activities for each of the 4 E/S the 24-hr E/S compared with the third wash, and they were fractions were calculated for each substrate for T. solium and similar in both the OAPM and the OAPFM fractions. These T. saginata, and results are shown in Table I and Figure 2. In results show that any peptidase residual activity, due to the all the cases, the correlation parameter between the concentra- pancreatin, was significantly removed after the third wash. After tion of hydrolyzed substrates and time ranged between 0.70 and the activation and during the third wash of the oncospheres, the 0.95, supporting the use of a linear regression to estimate the centrifugation indicated that 13.2% of the viable oncospheres mean hydrolysis rate within the 24-hr follow-up period. died or were lost. In general, a significant decreasing trend of the normalized During the 24-hr E/S culture, the OAPM showed a higher peptidase activity was observed with consecutive E/S fractions percentage of viability (number of viable oncospheres ending (P Ͻ 0.01) for both T. solium and T. saginata. The only ex- the 24-hr E/S culture/number of total oncospheres beginning) ceptions to this trend were for calpaine, which peaked in activ- than the OAPFM (0.73 vs. 0.60, P Ͻ 0.00001). Similarly, the ity in the 48-hr E/S fraction of T. solium, and elastase, which mobility of the OAPM was qualitatively observed to be more showed a slight increase in activity in the 96-hr E/S fraction of vigorous than that of the OAPFM. T. solium. During the production of the OAPM-E/S antigens at 24 hr, All peptidases’ activities tested were present in both T. so- 48 hr, 72 hr, and 96 hr, the loss of viability after the culture lium and T. saginata. Of all types, except calpaine-like in T. period was 20.2%, 9.1%, 9.1%, and 14.7%, respectively, and solium, the highest activities corresponded to the 24-hr E/S the loss of viability after the centrifugations was 8.1%, 2.8%, fraction. The greatest activity for both T. solium and T. saginata 3.1%, and 3.8%, respectively. corresponded to the chymotrypsin-like peptidase. For the 24-hr E/S in T. solium, the chymotrypsin-like activity was more than Comparison of oncosphere E/S peptidase activity 40 times higher than all other classes of peptidases, and more between Taenia solium and Taenia saginata than 10 times larger than the chymotrypsin-like activity in T. To achieve the best stimulation and induction of E/S antigens, saginata. Other peptidase types that were more active in T. we activated T. solium and T. saginata with media containing solium E/S fractions included trypsin-like, calpaine-like, and pancreatin. The 2 parasite oncosphere E/S fractions were then cathepsin B-like, the last of which had only slight activity in compared for the level of their peptidase activity at various T. solium and was inactive in T. saginata. Of these peptidases, periods. As expected, the concentration of viable T. solium and all were statistically significantly more active in the first 24 hr, T. saginata oncospheres systematically decreased across the except calpaine-like peptidases. Peptidase classes that were E/S fractions from hour 24 to 96 due to death and loss during more active in T. saginata included elastase-like, cathepsin manipulations. The mean concentrations of T. solium onco- L-like, and aminopeptidase B, all of which showed statistically spheres were 2,593, 801, 668, and 534 oncospheres/␮l during significant increased activity in the 24-hr E/S fraction compared the 24-hr, 48-hr, 72-hr, and 96-hr E/S fractions, respectively. with their activity in T. solium. Similarly, the mean concentrations of T. saginata oncospheres The highest ratio of the 24-hr E/S activity between T. solium/ were 2,319, 629, 429, and 462 oncospheres/␮l during the 24- T. saginata was found in trypsin-like (13.1, P Ͻ 0.01) and hr, 48-hr, 72-hr, and 96-hr E/S fractions, respectively. chymotrypsin-like (10.1, P Ͻ 0.01) peptidases, whereas elas- ZIMIC ET AL.—PEPTIDASE ACTIVITY IN T. SOLIUM AND T. SAGINATA 731

FIGURE 2. Variation of the normalized activities for the different E/S fractions collected (24 hr, 48 hr, 72 hr, and 96 hr), for Taenia solium and Taenia saginata ([AMC] minϪ1 [variable oncosphere (onc.)]Ϫ1 10Ϫ12). tase-like peptidases showed a large ratio (29.6, P Ͻ 0.01) fa- To verify the speciﬁcity of the peptide substrates for their voring T. saginata. The largest absolute difference in the 24-hr designated peptidases, the ﬂuorescence assays also were con- E/S activity (T. solium Ϫ T. saginata) corresponded to chy- ducted in the presence of inhibitors. The percentages of inhi- motrypsin-like activity (1,020–101, P Ͻ 0.01). bition of the chymotrypsin-like activity were 93.9% for chy- 732 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. (a) Zymography assay of the several E/S fractions of Taenia solium and Taenia saginata. The proteolytic activity was determined in collagenase buffer, pH 7.6. (b) Zymography of T. solium E/S pool, in the presence of several inhibitors. The proteolytic activity was determined in collagenase buffer, pH 7.6. Asterisk (*), molecular weight markers. mostatin, 90.3% for TPCK, 90.8% for Pefabloc, and 59.8% for tential contamination of the E/S fractions with cytosolic pro- leupeptin, whereas the trypsin-like activity was reduced by teins, sonicated T. solium oncospheres recovered after the 96- 100% by using each of the above-mentioned inhibitors. The hr E/S fraction culture were tested for peptidase activity. Nor- elastase-like activity was partially inhibited by chymostatin malized activity of chymotrypsin-like was 2,360 (95% CI ϭ (41.7%) and leupeptin (63.2%). Similarly, the percentage of in- 1,600–3,130) and cathepsin L-like was 2,670 (95% CI ϭ hibition of the cathepsin L-like activity was 80.8% for E64, 1,040–4,300) (hydrolyzed AMC-peptides per minute per viable 80.2% for leupeptin, and 83.7% for chymostatin, whereas cal- oncosphere ϫ 10Ϫ12). These figures represent approximately paine-like was only 29.3% inhibited by leupeptin. Aminopep- 2.3-fold the chymotrypsin-like activity and 130-fold the ca- tidase B was 100% inhibited by EDTA. Given the prolonged thepsin L-like activity, respectively, of the 24-hr E/S fraction duration of the reactions and the instability of PMSF in aqueous of T. solium. solution, this inhibitor was unable to affect the serine peptidases Gelatin zymography was then used to obtain general infor- in our system. mation about the molecular weight of major peptidases present The 24-hr E/S fractions of both T. solium and T. saginata, in the E/S fractions of both T. solium and T. saginata. The i.e., cysteine peptidases such as cathepsin L-like and calpaine- results are shown in Figure 3a. Zymography assays showed a like enzymes when tested at pH 5 (for highest assay sensitivity), 26-kDa intense band of activity when a collagenase buffer with generally had lower activities than those of serine peptidases pH 7.6 was used. This particular enzyme was present in every such as chymotrypsin-like and trypsin-like when tested at pH E/S fraction of T. solium and T. saginata. In addition, an 80- 7.6. When tested at pH 7.6 to better model small intestinal kDa band was noted in T. solium E/S fractions, showing the environment, the activities of these cysteine peptidases were maximum activity in the 48-hr E/S antigens followed by the even lower. 72-hr and 96-hr E/S fractions. To examine the overall intracellular expression and the po- Finally, to learn about the specific enzymatic activity of the ZIMIC ET AL.—PEPTIDASE ACTIVITY IN T. SOLIUM AND T. SAGINATA 733 proteins in the T. solium bands, zymography gels were run with is not easily explained by these data. Specifically, no single protease inhibitors used in the fluorescent inhibition assays. A peptidase showed high levels of activity in one species and an pool of 24-, 48-, and 72-hr E/S antigens of T. solium was tested absence of activity in the other. Instead, part of this specificity and run in the gel with the collagenase buffer, pH 7.6, after a may be conferred by a complex and changing series of excreted preincubation for 5 min at room temperature with selected in- peptidases, or it may be due to other mechanisms, such as dif- hibitors. PMSF was not considered because of the instability ferences in adherence to the intestinal epithelium. problems. As shown in Figure 3b, chymostatin, TPCK, and Pe- Previous investigators have tested all substrates in a general fabloc, notably inhibited the 26-kDa band, suggesting this may PBS buffer, pH 7.5, to mimic the intestinal environment. In be a serine peptidase. The 80-kDa band was inhibited by leu- contrast, in our study, each fluorescence assay was conducted peptin, partially inhibited by Pefabloc, and less inhibited by under conditions to maximize the specificity of the peptide sub- chymostatin. strate to a particular peptidase. The cathepsin L-like and cal- Although there is a partial inhibition of the 80-kDa protein paine-like activities of all the E/S products for both T. solium by Pefabloc, it is not possible to characterize with confidence and T. saginata were much lower when tested in basic pH con- this protein based on inhibition zymography. EDTA did not ditions than at their normally acid pH, confirming that, in the inhibit either band; thus, none of these enzymes are metallo- intestinal environment, chymotrypsin-like activity may play an proteases or calpaine, which is inhibited by EDTA by chelating even more dominate role among the secreted proteases. calcium. Hydrolysis rates among the 3 batches of each Taenia species were variable. Variability can be due to measurement error of DISCUSSION viability, differences between batches of oncospheres, and differences in the time from harvest to the time of ES preparation, This study demonstrates that there is a variety of peptidase because of the need to pool oncospheres from multiple patients. activity in the T. solium and T. saginata oncosphere E/S anti- It is important to highlight that during the collection of the gens, with a chymotrypsin-like enzyme exhibiting the highest E/S fractions, there is a certain rate of oncosphere loss. This activity. Moreover, there was no difference in the enzyme types loss is due both to oncospheres adherent to the surfaces of tubes that were present in the 2 species, although there were differ- used in the transfer and to the death of old oncospheres during ences in their activity. Our investigation demonstrates it is un- manipulation. In death, oncospheres may have released cyto- likely that a unique enzyme found in T. solium, but not in T. solic material, contaminating the E/S with cytosolic proteins. saginata, is responsible for the invasion of this oncosphere into To address this potential contamination, the total intracellular the human intestinal wall. activity of a peptidase from each general class was measured Media containing pancreatin was used to activate the onco- in sonicated oncospheres after harvesting the 96-hr E/S fraction. spheres. Pancreatin contains many of the enzymes we were The resulting level of activity represents the total intracellular measuring in the E/S media. By comparing the level of enzyme peptidase activity for each class. In cathepsin L, the total intra- in our OAPM versus OAPFM in the last of 3 washes, we were cellular activity was 2,670, whereas the highest activity of this able to demonstrate that any effect due to pancreatin contami- enzyme in the E/S fractions was just 13.6, suggesting that no nation was no longer present. greater than 0.5% of oncospheres were lysed. Therefore, the Among taeniid cestodes, only 1 other study has examined 24-hr chymotrypsin E/S fraction could only be contaminated by oncosphere-secreted proteases in detail. Thus, Baig and White 0.5% of viable oncospheres, meaning that only 1% of the ac- demonstrated in T. saginata the presence of serine, cysteine, tivity could have resulted from intracellular contamination. and aminopeptidase activity in a 24-hr E/S fraction (White et The present study identifies the major peptidases excreted/ al., 1996). Our studies have extended their findings to T. solium secreted by T. solium and T. saginata. Although both parasites and included controlling for the viability of oncospheres as well had the same complement of enzymes, the activity of chymo- as demonstrating the temporal sequence of these enzyme re- trypsin was 10-fold higher in T. solium and was detected in the actions. Other studies have not compared across species or 24-hr E/S. Our data suggest that the observed chymotrypsin- across culture fractions collected serially in time. To make this like peptidase is likely to be a 26-kDa protein. The different comparison, we normalized the hydrolysis rates for the concen- levels of T. solium and T. saginata oncosphere E/S proteases tration of viable oncospheres at the start of each E/S fraction suggest a potential role in the host specificity for larval invasion culture medium, thereby permitting direct comparison of activ- through penetration of the intestinal mucosa; however, the spec- ities between species at each time point and across different ificity of each parasite may be explained by several other rea- E/S fractions. With few exceptions, normalized peptidase activ- sons, e.g., slight differences in the biological substrates. Wheth- ity in both species of Taenia fell throughout the 4-day culture er this difference in activity of chymotrypsin is responsible for period. Oncospheres of both species showed highest levels of the invasion of the human intestine by T. solium requires further activity in serine proteases, which are most active at high pHs, study such as those found in the small intestine. Oncospheres invade the intestine 15 to 48 hr after ingestion; thus, our finding that ACKNOWLEDGMENTS protease activity declines after 24 hr is consistent with this rapid invasion (Yoshino, 1933). This work was partially funded by research grants P01 AI51976 from Although statistical differences do exist between levels of the National Institutes of Health and 23981 from The Bill and Melinda Gates Foundation; grants U01 AI35894 and TW05562 from the Na- specific peptidases in the 2 species, the differential specificity tional Institutes of Health; grant 01107 from the United States Food of invasion of the oncospheres of T. solium and T. saginata for and Drug Administration, and grant 063109 from The Wellcome Trust. the human/porcine and the bovine small intestine, respectively, The sponsors had no role in the design or writing of this article. 734 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

LITERATURE CITED Echinococcus granulosus. International Journal for Parasitology 21: 743–746. BEYNON, R. J., AND J. S. BOND. 1989. Proteolytic enzymes: A practical MAYTA, H., A. TALLEY,R.H.GILMAN,J.JIMENEZ,M.VERASTEGUI,M. approach. IRL Press, Oxford, U.K., 244 p. RUIZ,H.H.GARCIA, AND A. GONZALEZ. 2000. Differentiating Tae- FUKASE, T., Y. MATSUDA,S.AKIHAMA, AND H. ITAGAKI. 1984. Some nia solium and Taenia saginata infections by simple hematoxylin- hydrolyzing enzymes, especially arginine amidase, in plerocercoids eosin staining and PCR-restriction enzyme analysis. Journal of of Spirometra erinacei (Cestoda; Diphyllobothriidase). Japanese Clinical Microbiology 38: 133–137. Journal of Parasitology 33: 283–290. MCKERROW, J. H. 1989. Parasite proteases. Experimental Parasitology 68: 111–115. ———, ———, ———, AND ———. 1985. Purification and some NETER, J., AND W. W ASSERMAN. 1974. Applied linear statistical models. properties of cysteine protease of Spirometra erinacei plerocercoid Richard D. Irwin, Inc., Homewood, Illinois, 842 p. (Cestoda; Diphyllobothriidase). Japanese Journal of Parasitology READ,C.P.,AND J. E. SIMMONS,JR. 1963. Biochemistry and physiology 34: 351–360. of tapeworms. Physiological Reviews 43: 263–305. GARCIA, H. H. 2004. Species identification after treatment for human SCHANTZ, P. M. 1989. Surveillance and control programs for cestode taeniasis. Lancet 363: 949–950. diseases. In Parasitic diseases: Treatment and control, M. J. Miller ———, R. GILMAN,M.MARTINEZ,V.C.TSANG,J.B.PILCHER,G. and E. J. Love (eds.). CRC Press, Boca Raton, Florida, p. 275– HERRERA,F.DIAZ,M.ALVARADO, AND E. MIRANDA. 1993. Cysti- 290. cercosis as a major cause of epilepsy in Peru. Lancet 341: 197– SONG,C.Y.,AND C. L. CHAPPELL. 1993. Purification and partial char- 200. acterization of cysteine proteinase from Spirometra mansoni ple- ———, A. E. GONZALEZ,C.A.EVANS, AND R. H. GILMAN. 2003. Taenia rocercoids. Journal of Parasitology 79: 517–524. solium cysticercosis. Lancet 362: 547–556. STATA CORPORATION. 2005. Stata Statistical Software: Release 9. HALAWA, B., AND B. JAKACKA. 1977. Proteolytic activity of Taenia sa- StataCorp, College Station, Texas. ginata oncospheres. Wiadmosci Parazytologiczne 23: 363–367. STEVENSON, P. 1983. Observations on the hatching and activation of ITO, A., M. ITO,K.S.EOM,W.C.CHUNG,C.CHEN,L.MA,S.ENDO, fresh Taenia saginata eggs. Annals of Tropical Medicine and Par- AND P. C. FAN. 1997. In vitro hatched oncospheres of Asian Taenia asitology 77: 399–404. from Korea and Taiwan develop into cysticerci in the peritoneal VERASTEGUI, M., A. GONZALEZ,R.H.GILMAN,C.GAVIDIA,N.FALCON, cavity of female scid (severe combined immunodeficiency) mice. T. B ERNAL, AND H. H. GARCIA. 2000. Experimental infection model International Journal for Parasitology 27: 631–633. for Taenia solium cysticercosis in swine. Veterinary Parasitology 94: 33–44. JERI, C., R. H. GILMAN,A.G.LESCANO,H.MAYTA,M.E.RAMIREZ,A. WHITE, A. C., S. BAIG, AND P. R OBINSON. 1996. Taenia saginata onco- E. GONZALEZ,R.NAZERALI, AND H. H. GARCIA. 2004. Species iden- sphere excretory/secretory peptidases. Journal of Parasitology 82: tification after treatment for human taeniasis. Lancet, March 20 7–10. 363: 949–950. WHITE,JR., A. C., J. L. MOLINARI,A.V.PILLAI, AND A. A. REGE. 1992. KONG, Y., Y. B. CHUNG, AND S. Y. CHO. 1994. Cleavage of immuno- Detection and preliminary characterization of Taenia solium meta- globulin G by excretory-secretory cathepsin S-like proteases of Spi- cestode proteases. Journal of Parasitology 78: 281–287. rometra mansoni plerocercoid. Parasitology 109: 611–621. YOSHINO, K. 1933. Studies on the post-embryonal development of Tae- LAWS, G. F. 1968. The hatching of taeniid eggs. Experimental Parasi- nia solium: III. On the development of Cysticercus cellulosae with- tology 23: 1–10. in the definitive intermediate host. Journal of the Formosa Medical MARCO,M.,AND A. NIETO. 1991. Metalloproteinases in the larvae of Association 32: 166–169. J. Parasitol., 93(4), 2007, pp. 735–741 ᭧ American Society of Parasitologists 2007

EFFECTS OF ACANTHOCEPHALUS LUCII (ACANTHOCEPHALA) ON INTERMEDIATE HOST SURVIVAL AND GROWTH: IMPLICATIONS FOR EXPLOITATION STRATEGIES

Daniel P. Benesh and E. Tellervo Valtonen Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of Jyva¨skyla¨, Finland. e-mail: [email protected].ﬁ

ABSTRACT: Intermediate host exploitation by parasites is presumably constrained by the need to maintain host viability until transmission occurs. The relationship between parasitism and host survival, though, likely varies as the energetic requirements of parasites change during ontogeny. An experimental infection of an acanthocephalan (Acanthocephalus lucii) in its isopod intermediate host (Asellus aquaticus) was conducted to investigate host survival and growth throughout the course of parasite development. Individual isopods were infected by exposure to ﬁsh feces containing parasite eggs. Isopods exposed to A. lucii had reduced survival, but only early in the infection. Mean infection intensity was high relative to natural levels, but host mortality was not intensity dependent. Similarly, a group of naturally infected isopods harboring multiple cystacanths did not have lower survival than singly infected isopods. Isopods that were not exposed to the parasite exhibited sexual differences in survival and molting, but these patterns were reversed or absent in exposed isopods, possibly as a consequence of castration. Further, exposed isopods seemed to have accelerated molting relative to unexposed controls. Infection had no apparent effect on isopod growth. The effects of A. lucii on isopod survival and growth undermine common assumptions concerning parasite- induced host mortality and the resource constraints experienced by developing parasites.

For parasites with complex life cycles, intermediate hosts Acanthocephalus lucii and its isopod intermediate host, Asellus serve a variety of functions, 2 of the most important being that aquaticus. Acanthocephalus lucii exhibits a typical acantho- (1) they are an energy resource for parasites, allowing growth cephalan life cycle (Schmidt, 1985); the definitive host is a and in some cases asexual reproduction, and (2) they are vessels vertebrate, commonly European perch (Perca fluviatalis), and for transmitting parasites to the next host in the life cycle, com- an arthropod, in this case an isopod (A. aquaticus), serves as monly through predation (Parker, Chubb, Ball, and Roberts, intermediate host. Adult worms mate in the intestine of fish, 2003). These 2 roles of intermediate hosts presumably impose and females release eggs into the environment with the feces. conflicting selective pressures on parasites. Parasite growth and Isopods become infected by ingesting eggs. The parasite de- maturation requires consumption of host resources, and heavy velops in the isopod to the infective cystacanth stage, and the exploitation of intermediate hosts may benefit parasites by in- life cycle is completed when an infected isopod is eaten by an creasing growth rates, production of infective stages, and/or appropriate definitive host. Brattey (1986) found that isopods adult success. However, damage done is likely constrained by exposed to A. lucii eggs experienced higher mortality than un- the need to keep the intermediate host alive long enough to exposed isopods. Conversely, Hasu et al. (2006) observed that mature and be successfully transmitted to the next host. There- infected isopods actually survive better than controls. Given fore, the optimal level of host exploitation (virulence by some this contradictory evidence, additional investigation into the definitions) is expected to reflect a tradeoff between the benefits consequences of A. lucii infection on intermediate host survival of resource consumption and the costs of reduced host viability seems warranted. (Ebert and Herre, 1996; Poulin, 1998). The specific goals of this study were (1) to assess how A. There are examples of parasite-induced reductions in inter- lucii infection affects isopod survival throughout parasite de- mediate host viability within all major helminth groups, e.g., velopment and (2) to evaluate whether A. lucii affects host nematodes (Ashworth et al., 1996), trematodes (Sorensen and growth, or molting behavior, or both. Minchella, 2001), cestodes (Rosen and Dick, 1983), and acanthocephalans (Hynes and Nicholas, 1958). On the other hand, MATERIALS AND METHODS there are also systems in which neither parasitism nor infection Animal collection and maintenance intensity affects host survival (e.g., Uznanski and Nickol, 1980; Isopods were collected at the end of August 2005 with a dip net from Wedekind, 1997; Hurd et al., 2001). Thus, the host exploitation Niemija¨rvi, a small pond in central Finland (62Њ12ЈN, 25Њ45ЈE) in which strategies employed by larval helminths seem to vary widely. the only fish species present is Carassius carassius, the crucian carp. Recognizing which factors mediate the relationship between Thus, all isopods were uninfected because the definitive host of the parasitism and intermediate host survival in different systems parasite is not present in the pond. In the laboratory, isopods were fed on a diet of leaves, primarily of alder (Alnus glutinosa). Leaves were is a necessary step toward explaining this variability. For in- conditioned in aerated lake water for at least 2 wk to allow microbial stance, host sex (Shostak et al., 1985), host condition (Krist et colonization prior to being offered to isopods. Animals were maintained al., 2004), and infection intensity (Fredensborg et al., 2004) at approximately 18 C under constant illumination. may all exacerbate or buffer parasite-induced mortality. More- Experimental infection over, the probability of host death can vary with parasite development (Shostak et al., 1985; Schjetlein and Skorping, 1995; To collect A. lucii eggs, perch were isolated in a large tank during August 2005. Water flow through the tank was set at a very low level Sorensen and Minchella, 1998; Duclos et al., 2006). to allow feces accumulation. Perch feces were collected and stored in The aim of this study was to examine intermediate host ex- refrigerated lake water. Owing to mortality and the subsequent addition ploitation, as well as some potential factors influencing it, by of new individuals, the number of fish in the tank fluctuated with time, an acanthocephalan. The host-parasite system examined was but there were usually more than 50 fish. Some of these perch were dissected (n ϭ 93, mean length ϭ 12.99 cm, standard deviation [SD] Ϯ 2.4), and A. lucii prevalence was 75% with a mean infection intensity Received 6 October 2006; revised 14 January 2007; accepted 15 Jan- of 4.8 (SD Ϯ 4.8). Some of the collected feces were examined with a uary 2007. light microscope to confirm the presence of mature eggs.

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Individual isopods larger than 5 mm were isolated in plastic contain- categorical covariate. Additionally, isopod sex and relative antenna ers, 10 ϫ 15 ϫ 5 cm, containing 400 ml of lake water. Individuals were length were included in the model. The terms included in each of the either exposed to fish feces (n ϭ 339) or sham exposed with distilled 3 Cox regression models were evaluated using both forward and back- water (n ϭ 185). All the collected feces were combined in 5-L lake ward selection procedures. water to make a single suspension. For the exposed isopods, the fecal Isopod growth and molting: Isopod growth was evaluated with an suspension was vigorously mixed before 10 ml was added to each con- analysis of covariance (ANCOVA) using isopod size as the dependent tainer. Ten milliliters of distilled water was added to each container with variable, exposure as an independent factor, and time, i.e., days of sur- unexposed, control isopods. Fish feces were left in the containers for vival, as a covariate. An identical analysis was conducted to test wheth- 10 days before the exposure was terminated by removing all remaining er naturally infected and uninfected isopods differed in growth. Natu- feces. Control isopods were given conditioned leaves 5 days after being rally infected isopods harboring either a single or multiple cystacanths sham exposed. After 10 days and until the end of the experiment, all were pooled for this analysis. isopods were maintained on conditioned leaves. Every 5 days, the water Molting was only recorded for the isopods from the experimental in each container was replaced with fresh, aerated lake water. infection. It was considered to have 2 components, frequency and timing. That is, an isopod molted a particular number of times during the Data collection experiment, and those molts transpired during a given number of days Molting was monitored throughout the experiment for all isopods. after exposure. Both the quantity and timing of molts was dependent When shed exoskeletons were observed, they were removed from the on the length of time individual isopods were observed, i.e., days of containers and the date was recorded. Isopods were checked daily to survival. Thus, molting frequency was made relative by dividing the determine survival. Dead isopods were sexed by examining their ple- number of times an isopod molted by the number of days it survived. opods, and their length was measured to the nearest 0.5 mm. In A. This value will be referred to as the molting rate. Timing of molts was aquaticus, male antenna length is a secondary sexual character (Bertin summarized by calculating the average days postexposure (PE) on and Cezilly, 2003), and it was also recorded from dead individuals. which an isopod molted. Specifically, for individual isopods, the days Dead isopods were dissected to determine infection status, and parasites PE on which each observed molt occurred were summed and divided from infected isopods were counted. The experiment was terminated by the total number of molts. This value was made relative by dividing 101 days after the initial exposure. it by the number of days an isopod survived, and will henceforth be referred to as molt timing. Two variables were thus used to summarize Naturally infected isopods the molting behavior of individual isopods, number of molts per day survival, i.e., molting rate, and the average day PE on which molting The survival of naturally infected isopods was investigated to deter- occurred divided by days of survival, i.e., molt timing. mine whether conclusions based on the experimental infection also To examine whether exposed and unexposed isopods differed in molt- seem applicable to natural infections. Naturally infected and uninfected ing behavior, molting rate and molt timing were used as dependent isopods were collected in October 2005 from Lake Jyva¨sja¨rvi, Central variables in multivariate analysis of variance (MANOVA). Molting rate Finland (62Њ14ЈN, 25Њ44ЈE). Isopods infected with A. lucii cystacanths was ln transformed to homogenize variance. Exposure and isopod sex have darkened respiratory opercula (Brattey, 1983), so they can be iden- were used as independent factors in the analysis. When all the isopods tified relatively easily. However, because only isopods infected with that had molted at least once were included in the analysis, the as- cystacanths can be recognized and collected, the effects of earlier acan- sumption of equal covariance was violated (Box’s test, P Ͻ 0.001). This thocephalan ontogeny on host survival cannot be determined. Instead, was considered problematic because more male isopods were sampled monitoring the survival of isopods harboring cystacanths allows the than female isopods, and, when covariance matrices are heterogeneous, long-term effects of parasitism on host survival to be evaluated. Unin- biased sample sizes can distort P values (Zar, 1999). However, when fected and infected isopods were isolated individually in plastic con- only isopods that molted at least twice were used in the analysis, the tainers and maintained in a manner identical to the experimentally ex- null hypothesis of equal covariance was not rejected (P ϭ 0.26). Thus, posed isopods. Isopod size, sex, antenna length, and infection status, the MANOVA only included isopods that molted at least twice. i.e., presence/absence of A. lucii and cystacanth intensity, were recorded upon isopod death. This group of isopods was observed for 75 days All analyses were conducted with SPSS 12.0.1 (SPSS Inc., Chicago, before surviving isopods were killed and dissected. Illinois) statistical software, and alpha values less than 0.05 were considered statistically significant. Untransformed values are shown in fig- Data analyses ures. Survival: The factors affecting isopod survival in the experimental infection were assessed with Cox regression, a method commonly used RESULTS for survival analyses (Andersen, 1991). An assumption of Cox regression models is that the ratio of the hazard function for any 2 individuals Survival of all isopods is dependent on their covariate values and the baseline hazard function, but not time (proportional hazards assumption). This assumption ap- After 101 days, 427 of 524 isopods (82%) had died. Antenna peared to be violated, however, because some factor effects seemed to length, corrected for body size, had a weak, positive effect on vary with time. Thus, a time-dependent covariate was incorporated into the model. Exposure (unexposed vs. exposed to A. lucii) and isopod sex survival, i.e., the hazard function decreased 6% for each unit were included in the model as categorical covariates. For each individ- increase in residual antenna length (Table I). There was a sig- ual, the lengths of the first antennae were averaged and used as the nificant interaction between exposure and the time-dependent dependent variable in a linear regression with isopod size as the inde- covariate, suggesting the baseline hazard function differed be- pendent variable. The residuals of this regression, which are a measure of antenna length corrected for body size, were used as a covariate in tween unexposed and exposed isopods (Table I). Specifically, the model. exposed isopods seem to have lower survival during the initial A separate Cox regression was conducted with only exposed isopods 40 days or so of the experiment (Fig. 1). After approximately to examine the factors affecting the survival of infected isopods. Infec- 40 days, the survival functions of unexposed and exposed iso- tion intensity and antenna length, corrected for isopod size, were included as covariates. Isopod sex was incorporated into the model as a pods appear quite similar (Fig. 1). Male isopods survived slight- categorical covariate. As before, a time-dependent covariate was added ly longer than female isopods, on average (47 vs. 44 days). to the model. However, the effects of isopod sex on survival were both time A third Cox regression was used to evaluate the factors affecting and exposure dependent (Table I). For unexposed isopods, fe- survival of naturally infected isopods. Three categories of isopods were males exhibited higher mortality than males during the middle defined: (1) uninfected (n ϭ 64); (2) isopods with a single cystacanth (n ϭ 41); and (3) isopods with more than 1 cystacanth (n ϭ 17; mean of the experiment (between days 20 and 50 approximately; Fig. intensity ϭ 2.4). Infection status was incorporated into the model as a 2A). In contrast, exposed male isopods had slightly greater mor- BENESH AND VALTONEN—HOST EXPLOITATION BY A. LUCII 737

TABLE I. Terms included and excluded from the ﬁnal model produced by Cox regression survival analysis of all isopods (n ϭ 524). Forward and backward selection procedures gave the same best model. Odds ratio indicates the ratio of the estimated survival probabilities of contrasted classes, e.g., the survival probability of male isopods was estimated to be 1.4 times higher than female isopods.

Terms Wald score df P Odds ratio

Terms in the ﬁnal model Antenna length corrected for body size 5.29 1 0.021 0.94 Isopod sex 4.13 1 0.042 1.37 Exposure ϫ time-dependent covariate 50.33 1 Ͻ0.001 Isopod sex ϫ exposure ϫ time-dependent covariate 7.95 1 0.005 Terms excluded from the ﬁnal model Exposure 0.00 1 1.00 Isopod sex ϫ time-dependent covariate 0.19 1 0.89 Isopod sex ϫ exposure 0.45 1 0.83 tality than females during the early part of the experiment (Fig. males tended to have lower survival early in the experiment 2B). (Fig. 2B).

Survival of exposed isopods Survival of naturally infected isopods Around 16 days PE, parasites were large enough for infection Only 39 of 122 isopods (32%) died during the 75-day ob- intensity to be reliably quantified. Thus, the analysis of exposed servation period. The survival of these isopods was not affected isopod survival only included individuals that lived at least 16 by infection status, isopod sex, relative antenna length, or any days (n ϭ 252). Infection prevalence was 98% with only 4 of of their interactions (all P Ͼ 0.18). 252 isopods uninfected, and the mean infection intensity was 13.5 (SD Ϯ 8.8) parasites. Contrary to expectations, infection Isopod growth and molting intensity had a significant positive effect on survival (Table II). For the experimental isopods, there was a significant positive Furthermore, there was a significant interaction between infec- relationship between time (days survival) and isopod size (F1,520 tion intensity and the time-dependent covariate, suggesting the ϭ 537.7, P Ͻ 0.001). The intercept (exposure in ANCOVA, effects of intensity on survival varied with time (Table II). This ϭ ϭ ϫ F1,520 0.47, P 0.49) and slope (exposure time interaction, interaction is perhaps best exemplified by the fact that the re- ϭ ϭ F1,520 0.001, P 0.97) of this correlation were similar for lationship between intensity and survival was better described unexposed and exposed isopods, suggesting their pattern of ϭ Ͻ by a curve than a line (F1,250 22.07, P 0.01). Intensity had growth did not differ. Similarly, the relationship between time a small, positive effect on survival initially, but, as the exper- and isopod size was positive for uninfected and naturally in- iment progressed, intensity had negligible impact on survival ϭ Ͻ fected isopods (F1,118 135.3, P 0.001), but intercept and (Fig. 3). There was also a significant interaction between isopod slope parameters were unaffected by infection status (infection sex and the time-dependent covariate (Table II), since exposed ϭ ϭ ϫ ϭ status, F1,118 1.49, P 0.23; infection status time, F1,118 0.59, P ϭ 0.44). Of the 524 isopods observed, 394 (75%) molted at least once during the experiment. The number of times an isopod molted ranged from 1 to 7, with an average of 1.8 molts per isopod. For those isopods molting multiple times, the average interval between molts was 17.9 (SD Ϯ 7.6) and 23.6 (SD Ϯ 11.3) days for exposed (n ϭ 122) and unexposed (n ϭ 85) isopods, respectively. The intermolt period did not change over the course of the experiment for either exposed or unexposed isopods, e.g., the interval between the first and second molt did not significantly differ from the interval between the second and third molt, etc. (paired t-tests, all P Ͼ 0.1). ϭ ϭ Exposure had no effect on molting rate (F1,203 0.17, P ϭ ϭ 0.68), but isopod sex did (F1,203 6.35, P 0.01). On average, females tended to have a higher molting rate than males. How- ever, there was a significant interaction between exposure and ϭ ϭ isopod sex (F1,203 7.79, P 0.01). Unexposed females had a higher molting rate than unexposed males, whereas exposed male and female isopods had very similar molting rates (Fig. 4A). Furthermore, unexposed females molted more frequently FIGURE 1. Cumulative survival of 185 unexposed and 339 exposed than exposed isopods, while unexposed males molted less fre- isopods over the course of 101 days. quently than exposed isopods (Fig. 4A). 738 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. The relationship between intensity and isopod survival. A curvilinear (quadratic) function (Y ϭϪ2.59 ϩ 0.57x ϩϪ0.004x2; R2 ϭ 0.21) provided a better ﬁt to the data than a line (Y ϭ 7.4 ϩ 0.12x; R2 ϭ 0.14).

ϭ 3.56, P ϭ 0.06), unexposed females tended to molt later than all other isopods (Fig. 4B). Molt timing did not differ between exposed males and females (Fig. 4B).

DISCUSSION Parasites are generally assumed to decrease the survival of their intermediate hosts. In accordance with this premise, increasing infection intensity should negatively affect host survival. Isopods exposed to A. lucii experienced a higher mortality rate than control isopods but only during the early part of the infection. Furthermore, survival did not have the expected negative relationship with intensity; if anything, it was positive. Thus, some of the common assumptions concerning host-par- FIGURE 2. Cumulative survival of male and female isopods that were either (A) unexposed or (B) exposed to Acanthocephalus lucii. asite interactions may not apply to this system. Brattey (1986) found isopods exposed to A. lucii to have consistently reduced survival during 60 days of observation. Exposed isopods molted sooner than unexposed isopods The exposed isopods observed here also had lower survival, ϭ Ͻ (F1,203 9.66, P 0.01), but isopod sex did not affect molt but it was not consistently lower; late in the infection exposed ϭ ϭ timing (F1,203 2.85, P 0.09). Although the interaction be- and unexposed isopods exhibited similar survival. In contrast, tween isopod sex and exposure was not quite signiﬁcant (F1,203 Hasu et al. (2006) found infected isopods to survive better than

TABLE II. Terms included and excluded from the ﬁnal Cox regression survival analysis of exposed isopods which lived at least 16 days (n ϭ 252). Forward and backward selection procedures gave the same best model.

Terms Wald score df P Odds ratio

Terms in the ﬁnal model Infection intensity 44.99 1 Ͻ0.001 0.87 Infection intensity ϫ time-dependent covariate 29.58 1 Ͻ0.001 Isopod sex ϫ time-dependent covariate 4.92 1 0.027 Terms excluded from the ﬁnal model Antenna length corrected for body size 0.97 1 0.33 Isopod sex 1.24 1 0.27 Isopod sex ϫ infection intensity 0.37 1 0.54 Isopod sex ϫ infection intensity ϫ time-dependent covariate 0.13 1 0.72 BENESH AND VALTONEN—HOST EXPLOITATION BY A. LUCII 739

The relative growth rate of A. lucii is nonlinear; parasite volume increases rapidly initially, but the growth rate slows over time (Benesh and Valtonen, 2007). Therefore, later infections may be less energetically burdensome for the host, resulting in lower mortality. The similar survival rates of cystacanth-infected and uninfected isopods collected from nature support this notion. Periods of rapid parasite growth have been observed to be det- rimental to intermediate host viability in other systems as well (Shostak et al., 1985; Duclos et al., 2006). Alternatively, given that intermediate hosts respond immunologically to acanthocephalan infections (Robinson and Strickland, 1969; Nickol and Dappen, 1982), host defenses and their associated costs could also play a role in reducing host survival during early infections (Hasu et al., 2006). Surprisingly, antenna length relative to body size had a weak positive effect on survival. The mechanism underlying the relationship between mortality and shorter antennae is not clear, but it might reﬂect the number of antagonistic conﬂicts experienced by an isopod, or it may be a consequence of immunological costs associated with wounding (Plaistow et al., 2003). Contrary to expectations, intensity positively affected survival, particularly early in the experimental infection. A common explanation for parasite-induced increases in host survival is that parasites divert host resources away from reproduction (Hurd et al., 2001; Sorensen and Minchella, 2001). Like several other Acanthocephalus species (Oetinger, 1987; Dezfuli et al., 1994; Kakizaki et al., 2003), A. lucii is a castrating parasite, at least in female hosts (Brattey, 1983). Whether castration is responsible for the apparent positive effect of intensity in early infections is unknown, and better knowledge of the mechanism and time course involved in resource reallocation would be needed to assess this phenomenon. It should be noted that considerable variation was observed in the relationship between intensity and survival; the quadratic function in Figure 3 explained only 21% of the variation in this association. Conse- quently, any statements about the effects of intensity on survival in this system are tenuous. Nonetheless, the survival of both experimentally and naturally infected isopods did not decrease with infection intensity, as expected. Other acanthocephalan species similarly fail to conform to the anticipated negative relationship between intensity and host viability (Lackie, 1972; Uznanski and Nickol, 1980). The parasite burdens in this study were far higher than those found in nature; the majority of infected isopods collected from nature harbor a single parasite (Brattey, 1986). Consequently, the results concerning host survival and the effects of intensity

FIGURE 4. (A) The number of molts per day survival (molting rate) may not reflect the situation in nature. On the other hand, some of exposed and unexposed isopods of each sex. Molt timing (B), defined of the trends observed in the experimentally infected isopods as the average day of molting divided by the number of days survival, were also observed in the naturally infected isopods, e.g., lack of exposed and unexposed isopods of each sex. Only isopods that molt- of intensity-dependent mortality and similar growth patterns be- ed at least twice were included (n ϭ 207). Bars represent standard error. tween uninfected and infected isopods. Thus, the observed effects of A. lucii infection on isopod viability seem likely to controls. However, their study focused exclusively on gravid, operate at lower intensities as well. Additionally, some acan- female isopods, which may differ in their response to infection. thocephalan populations exhibit very high infection levels in Nickol (1985) hypothesized that early mortality in laboratory their intermediate hosts (Seidenberg, 1973; Gleason, 1987; acanthocephalan infections results from massive penetration of Brown and Pascoe, 1989; Sparkes et al., 2004), so the conse- acanthors through the intermediate host gut. This could explain quences of heavy infections may be relevant to some natural some of the initial mortality of exposed isopods, but additional situations. mechanisms seem necessary to explain the depressed survivor- Sexual differences in isopod mortality depended on exposure ship of exposed isopods during the first few weeks of infection. to A. lucii. For unexposed isopods, females suffered higher 740 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 mortality than males, but this trend was reversed for exposed if host growth is not obstructed, the resources available to de- isopods; males had slightly higher mortality than females. Un- veloping parasites are not temporally constrained; there may exposed females molted more frequently and at a later point in always be an influx of resources permitting additional parasite the experiment than unexposed males, which could reflect at- growth. The size of several acanthocephalan species, including tainment of sexual maturity because female A. aquaticus, like A. lucii (Benesh and Valtonen, 2007), is positively correlated all freshwater isopods, must undergo a nuptial molt to become with host size (Awachie, 1966; Dezfuli et al., 2001; Steinauer sexually mature (Jormalainen, 1998). Indeed, late in the exper- and Nickol, 2003), so it would seem advantageous not to ham- iment, some control females were observed bearing eggs in per host growth. their marsupium. Thus, the lower survival of unexposed fe- The impact of A. lucii on its intermediate host’s viability, males may stem from resource allocation toward reproduction. growth, and molting undermines common assumptions con- Exposed females, on the other hand, were likely castrated as a cerning host-parasite interactions. First, the effects of A. lucii consequence of A. lucii infection (Brattey, 1983), so energy on host mortality were not consistent over time, so the pre- potentially dedicated to host reproduction was perhaps available sumed tradeoff between host exploitation and host viability may for host maintenance instead. This might explain the slightly primarily operate at particular times of parasite ontogeny. Sec- greater survival of exposed females in the first few weeks of ond, host mortality was not intensity dependent. Third, the the- the experiment. The difference in survival of exposed male and oretical, resource-based, maximum size attainable by parasites female isopods was not a result of dissimilar infection intensi- in their intermediate hosts (Parker, Chubb, Roberts et al., 2003) ties, since the sexes appear rather similar in susceptibility to A. may not be temporally fixed. Host growth was not impeded by lucii (Brattey, 1986; Hasu et al., 2007). Host castration may A. lucii, and host molting was accelerated, so the asymptotic, also account for the similar molting pattern exhibited by ex- maximum parasite size, if it is defined by host size, may in- posed males and females; inability to achieve sexual maturity crease over time. The observations on this system, as well as could disrupt normal, sex-specific molting behavior. others like it (Uznanski and Nickol, 1980), demonstrate that Exposed isopods tended to molt slightly more frequently than assumptions concerning the interaction of acanthocephalans unexposed, male isopods. Additionally, they tended to molt ear- with their intermediate hosts cannot be made lightly. lier during the experiment than both male and female unexposed isopods. These results suggest that infection with A. lucii ACKNOWLEDGMENTS increases host molting. Previous studies have found molting to We would like to thank T. Hasu for sharing her experiences with be inhibited (Thomas et al., 1996; Kokkotis and McLaughlin, infecting and maintaining isopods in the laboratory and for providing 2006) or unaffected by parasites (Calado et al., 2005). The ac- useful comments on earlier drafts of this manuscript. Eero Vestola celerated molting of exposed isopods did not seem to translate helped in the maintenance of isopods. D.P.B. was supported by Ful- bright and CIMO grants. into a larger size at the end of the experiment, suggesting that molting may not reflect isopod growth (Marcus, 1990). Inter- LITERATURE CITED estingly, the size of A. lucii was positively related to molting rate, indicating that increased host molting is favorable for par- ANDERSEN, P. K. 1991. Survival analysis 1982–1991: The second decade asite growth and may, therefore, represent adaptive manipula- of the proportional hazards regression model. Statistics in Medicine 10: 1931–1942. tion (Benesh and Valtonen, 2007). Alternatively and/or addi- ASHWORTH, S. T., C. R. KENNEDY, AND G. BLANC. 1996. Density-depen- tionally, earlier molting may serve the purpose of repairing dent effects of Anguillicola crassus (Nematoda) within and on its damage done to the intestine by invading acanthors. Early in copepod intermediate hosts. Parasitology 113: 303–309. the experiment, many exposed isopods shed exoskeletons that AWACHIE, J. B. E. 1966. The development and life-history of Echino- rhynchus truttae Schrank 1788 (Acanthocephala). Journal of Hel- included intestines still packed with food, suggesting that molt- minthology 40: 11–32. ing was premature and it was either induced, or necessary, or BENESH,D.P.,AND E. T. VALTONEN. 2007. Proximate factors affecting both. This was never observed in control isopods. Beyond sim- the larval life history of Acanthocephalus lucii (Acanthocephala). ply mending damage done to the intestine, accelerated molting Journal of Parasitology 93: 742–749. could reduce parasite burden by removing young acanthors at- BERTIN,A.,AND F. C EZILLY. 2003. Sexual selection, antennae length and the mating advantage of large males in Asellus aquaticus. Journal tached to the intestine. However, since neither molting rate nor of Evolutionary Biology 16: 698–712. timing seemed to affect intensity (data not shown), this hypoth- BRATTEY, J. 1983. The effects of larval Acanthocephalus lucii on the esis was not supported. pigmentation, reproduction and susceptibility to predation of the The relationship between isopod size and time did not differ isopod Asellus aquaticus. Journal of Parasitology 69: 1172–1173. ———. 1986. Life history and population biology of larval Acantho- between uninfected and infected isopods, so the initial size and cephalus lucii (Acanthocephala: Echinorhynchidae) in the isopod subsequent growth of these groups were very similar. In con- Asellus aquaticus. Journal of Parasitology 72: 633–645. trast, juvenile isopods, smaller than those used here, seem to BROWN,A.F.,AND D. PASCOE. 1989. Parasitism and host sensitivity to exhibit accelerated growth after exposure to A. lucii (Hasu et cadmium: An acanthocephalan infection of the freshwater amphi- al., 2007). Thus, the effects of A. lucii on host growth may be pod Gammarus pulex. Journal of Applied Ecology 26: 473–487. CALADO, R., C. BARTILOTTI, AND L. NARCISCO. 2005. Short report on dependent on the size of the host at the time of infection. Some the effect of a parasitic isopod on the reproductive performance of acanthocephalans reduce the growth of their intermediate hosts a shrimp. Journal of Experimental Marine Biology and Ecology (Hynes and Nicholas, 1958; Awachie, 1966), but others do not 321: 13–18. (Uznanski and Nickol, 1980). Thus, in a situation analogous to DEZFULI, B. S., L. GIARI, AND R. POULIN. 2001. Costs of intraspecific and interspecific host sharing in acanthocephalan cystacanths. Par- survival, the effects of acanthocephalans on host growth appear asitology 122: 483–489. to vary between species. This has important consequences for ———, E. ROSSETTI,E.A.FANO, AND R. ROSSI. 1994. Occurrence of parasite development and exploitation strategies. For example, larval Acanthocephalus anguillae (Acanthocephala) in Asellus BENESH AND VALTONEN—HOST EXPLOITATION BY A. LUCII 741

aquaticus (Crustacea, Isopoda) from the River Brenta. Italian Jour- ala) on morphometrics and reproduction of Caecidotea intermedius nal of Zoology 61: 77–81. (Crustacea: Isopoda). Transactions of the American Microscopical DUCLOS, L. M., B. J. DANNER, AND B. B. NICKOL. 2006. Virulence of Society 106: 240–248. Corynosoma constrictum (Acanthocephala: Polymorphidae) in Hy- PARKER, G. A., J. C. CHUBB,M.A.BALL, AND G. N. ROBERTS. 2003. alella azteca (Amphipoda) throughout parasite ontogeny. Journal Evolution of complex life cycles in helminth parasites. Nature 425: of Parasitology 92: 749–755. 480–484. EBERT,D.,AND E. A. HERRE. 1996. The evolution of parasitic diseases. ———, ———, G. N. ROBERTS,M.MICHAUD, AND M. MILINSKI. 2003. Parasitology Today 12: 96–101. Optimal growth strategies of larval helminths in their intermediate FREDENSBORG, B. L., K. M. MOURITSEN, AND R. POULIN. 2004. Intensity- hosts. Journal of Evolutionary Biology 16: 47–54. dependent mortality of Paracalliope novizealandiae (Amphipoda: PLAISTOW, S. J., Y. OUTREMAN,Y.MORET, AND T. R IGAUD. 2003. Vari- Crustacea) infected by a trematode: Experimental infections and ation in the risk of being wounded: An overlooked factor in studies field observations. Journal of Experimental Marine Biology and of invertebrate immune function? Ecology Letters 6: 489–494. Ecology 311: 253–265. POULIN, R. 1998. Evolutionary ecology of parasites. Chapman and Hall, GLEASON, L. N. 1987. Population dynamics of Pomphorhynchus bul- London, U.K., 212 p. bocolli in Gammarus pseudolimnaeus. Journal of Parasitology 73: ROBINSON,E.S.,AND B. C. STRICKLAND. 1969. Cellular responses of 1099–1101. Periplaneta americana to acanthocephalan larvae. Experimental HASU, T., J. C. HOLMES, AND E. T. VALTONEN. 2007. Isopod (Asellus Parasitology 50: 694–697. aquaticus) size and acanthocephalan (Acanthocephalus lucii) infec- ROSEN, R., AND T. A . D ICK. 1983. Development and infectivity of the tions. Journal of Parasitology 93: 450–457. procercoid of Triaenophorus crassus Forel and mortality of the first ———, E. T. VALTONEN, AND J. JOKELA. 2006. Costs of parasite resis- intermediate host. Canadian Journal of Zoology 61: 2120–2128. tance for female survival and parental care in a freshwater isopod. SCHJETLEIN, J., AND A. SKORPING. 1995. The temperature threshold for Oikos 114: 322–328. development of Elaphostrongylus rangiferi in the intermediate HURD, H., E. WARR, AND A. POLWART. 2001. A parasite that increases host: An adaptation to winter survival? Parasitology 111: 103–110. host lifespan. Proceedings of Royal Society of London, Series B SCHMIDT, G. D. 1985. Development and life cycles. In Biology of the 268: 1749–1753. Acanthocephala, B. B. Nickol and D. W. T. Crompton (eds.). Cam- HYNES,H.B.N.,AND W. L. NICHOLAS. 1958. The resistance of Gam- bridge University Press, Cambridge, U.K., p. 273–306. marus spp. to infection by Polymorphus minutus (Goeze, 1782) SEIDENBERG, A. J. 1973. Ecology of the acanthocephalan, Acanthoce- (Acanthocephala). Annals of Tropical Medicine and Parasitology phalus dirus (Van Cleave, 1931) in its intermediate host, Asellus 52: 376–383. intermedius Forbes (Crustacea: Isopoda). Journal of Parasitology JORMALAINEN, V. 1998. Precopulatory mate guarding in crustaceans: 59: 957–962. Male competitive strategy and intersexual conflict. Quarterly Re- SHOSTAK, A. W., R. B. ROSEN, AND T. A . D ICK. 1985. The use of growth view of Biology 73: 275–304. curves to assess the crowding effect on procercoids of the tape- KAKIZAKI, T., T. SAITO,A.OHTAKA, AND K. NAGASAWA. 2003. Effects worm Triaenophorus crassus in the copepod host Cyclops bicus- of Acanthocephalus sp. (Acanthocephala: Echinorhynchidae) on pidatus thomasi. Canadian Journal of Zoology 63: 2343–2351. the body size and reproduction of isopods (Asellus hilgendorfi). SORENSEN,R.E.,AND D. J. MINCHELLA. 1998. Parasite influences on Limnology 4: 43–46. host life history: Echinostoma revolutum parasitism of Lymnaea KOKKOTIS,T.,AND J. D. MCLAUGHLIN. 2006. Pathogenicity of the hy- elodes snails. Oecologia 115: 188–195. menolepidid cestode Microsomacanthus hopkinsi in its intermedi- ———, AND ———. 2001. Snail-trematode life history interactions: ate host Hyalella azteca: Implications for transmission, host fitness, Past trends and future directions. Parasitology 123: S3–S18. and host populations. Canadian Journal of Zoology 84: 32–41. SPARKES, T. C., V. M. WRIGHT,D.T.RENWICK,K.A.WEIL,J.A.TALK- KRIST, A. C., J. JOKELA,J.WIEHN, AND C. M. LIVELY. 2004. Effects of INGTON, AND M. MILHALYOV. 2004. Intraspecific host sharing in the host condition on susceptibility to infection, parasite developmental manipulative parasite Acanthocephalus dirus: Does conflict occur rate, and parasite transmission in a snail-trematode interaction. over host modification? Parasitology 129: 335–340. Journal of Evolutionary Biology 17: 33–40. STEINAUER,M.L.,AND B. B. NICKOL. 2003. Effect of cystacanth body LACKIE, J. M. 1972. The course of infection and growth of Moniliformis size on adult success. Journal of Parasitology 89: 251–254. dubius (Acanthocephala) in the intermediate host Periplaneta THOMAS, F., O. VERNEAU,F.SANTALLA,F.CEZILLY, AND F. R ENAUD. 1996. Americana. Parasitology 64: 95–106. The influence of intensity of infection by a trematode parasite on MARCUS, J. H. 1990. Moulting as an indicator of growth in Asellus the reproductive biology of Gammarus insensibilis (Amphipoda). aquaticus (L., 1758) and A. meridianus Racovitza, 1919 (Isopoda). International Journal for Parasitology 26: 1205–1209. Crustaceana 58: 136–143 UZNANSKI, R. L., AND B. B. NICKOL. 1980. Parasite population regula- NICKOL, B. B. 1985. Epizootiology. In Biology of the Acanthocephala, tion: Lethal and sublethal effects of Leptorhynchoides thecatus B. B. Nickol and D. W. T Crompton (eds.). Cambridge University (Acanthocephala: Rhadinorhynchidae) on Hyalella azteca (Amphi- Press, Cambridge, U.K., p. 307–346. poda). Journal of Parasitology 66: 121–126. ———, AND G. E. DAPPEN. 1982. Armadillidium vulgare (Isopoda) as WEDEKIND, C. 1997. The infectivity, growth and virulence of the cestode an intermediate host of Plagiorhynchus cylindraceus (Acantho- Schistocephalus solidus in its first intermediate host, the copepod cephala) and isopod response to infection. Journal of Parasitology Macrocyclops albidus. Parasitology 115: 317–324. 68: 570–575. ZAR, J. H. 1999. Biostatistical analysis. Prentice-Hall, Upper Saddle OETINGER, D. F. 1987. Effects of Acanthocephalus dirus (Acanthoceph- River, New Jersey, 929 p. J. Parasitol., 93(4), 2007, pp. 742–749 ᭧ American Society of Parasitologists 2007

PROXIMATE FACTORS AFFECTING THE LARVAL LIFE HISTORY OF ACANTHOCEPHALUS LUCII (ACANTHOCEPHALA)

Daniel P. Benesh and E. Tellervo Valtonen Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of Jyva¨skyla¨, Finland. e-mail: [email protected].ﬁ

ABSTRACT: The growth and eventual size of larval helminths in their intermediate hosts presumably has a variety of fitness consequences. Therefore, elucidating the proximate factors affecting parasite development within intermediate hosts should provide insight into the evolution of parasite life histories. An experimental infection that resulted in heavy intensities of an acanthocephalan (Acanthocephalus lucii) in its isopod intermediate host (Asellus aquaticus) permitted the examination of parasite developmental responses to variable levels of resource availability and intraspecific competition. Isopods were infected by exposure to egg-containing fish feces, and larval infrapopulations were monitored throughout the course of A. lucii development. The relative rate of parasite growth slowed over time, and indications of resource constraints on developing parasites, e.g., crowding effects, were only observed in late infections. Consequently, the factors likely representative of resource availability to larval parasites (host size and molting rate) primarily affected parasite size in late infections. Moreover, at this stage of infection, competitive interactions, gauged by variation in worm size, seemed to be alleviated by greater resources, i.e., larger hosts that molted more frequently. The relatively rapid, unconstrained growth of young parasites may be worse for host viability than the slower, resource-limited growth of larger parasites.

Many parasite life history traits, e.g., growth rates, are pre- To elucidate the evolutionary forces shaping parasite growth sumably determined by the level of host resource consumption. in their intermediate hosts, the proximate factors affecting par- Therefore, the exploitation strategies employed by larval hel- asite development must be understood. Resource availability, minths in their intermediate hosts should be reflected in parasite possibly represented by host size, condition, and/or growth, is growth rates and body sizes. The potential fitness benefits one such factor presumably affecting the rate of parasite ontog- linked to larger larval size may include better establishment eny. Another factor is the number of conspecifics present. The success in the definitive host (Rosen and Dick, 1983; Steinauer response of developing parasites to variable resource pools and and Nickol, 2003), higher adult fecundity (Fredensborg and infection intensities can thus provide insight into the effects of Poulin, 2005), and less developmental time to sexual maturity competition on parasite life history strategies. An experimental (Poulin, 1998). Consequently, selection should promote faster infection with an acanthocephalan (Acanthocephalus lucii)in larval growth and greater ultimate size. However, the direction- its isopod intermediate host produced high infection intensities al selection on parasite size is probably stabilized by the general (Benesh and Valtonen, 2007b). This provided an opportunity to need to maintain host viability until transmission occurs (Laf- examine parasite growth under conditions of high yet variable ferty and Kuris, 2002). levels of intraspecific competition. This evolutionary tradeoff has been modeled to predict growth strategies of larval helminths in their intermediate hosts (Parker et MATERIALS AND METHODS al., 2003). Specifically, it was suggested that growth patterns may Animal collection, maintenance, and experimental infection not be entirely a function of resources, but instead they may reflect The collection site, maintenance of isopods, and experimental infec- flexible, adaptive life history strategies. That is, individual para- tion protocol were described previously (Benesh and Valtonen, 2007b). sites in the presence of competing conspecifics decrease their size, Briefly, adult isopods (Ͼ5 mm) of the species Asellus aquaticus were and thus the parasite burden on the host, not as a response to individually exposed to European perch (Perca fluviatilis) feces con- limited resources, but to maintain host viability. Parker et al. taining acanthocephalan (A. lucii) eggs. The exposure was terminated after 10 days, and the course of infection was monitored over a period (2003) attempted to address the predictions of their model by ex- of 101 days. amining data taken from the literature on experimental infections of copepods with pseudophyllidean cestodes, but too little infor- Data collection mation was available to make comparisons robust. Recent exper- Isopod molting was followed throughout the experiment, and the date imental work, though, suggests that the larval growth of the ces- of observed molts was recorded. Isopods were checked daily to deter- tode Schistocephalus solidus may vary in an adaptive manner (Mi- mine survival. The sex and length of isopods were recorded upon death. Dead isopods were dissected, and parasites from infected isopods were chaud et al., 2006). Like cestodes, there is a paucity of information counted and measured. Parasites were examined with a compound mi- on the growth strategies of acanthocephalans in their intermediate croscope, and measurements were taken using an ocular micrometer. hosts. Intermediate host size and the presence of competitors are During the early stages of the experiment, i.e., before 50 days or so, known to affect acanthocephalan development in some systems the length and width of individual worms was measured to the nearest (Awachie, 1966; Uznanski and Nickol, 1980; Pilecka-Rapacz, 0.004 mm. Later in the experiment, because worms were much larger, the length and width of individual worms were measured to the nearest 1986; Dezfuli et al., 2001; Poulin et al., 2003; Steinauer and Nick- 0.01 mm. When possible, worms were sexed. After 101 days postex- ol, 2003), but the pervasiveness and magnitude of these phenom- posure (PE), all surviving isopods were killed and dissected. ena are poorly known. Therefore, further empirical work is necessary to assess theoretical expectations and reach general conclu- Data analyses sions concerning life history strategies of parasites in intermediate The volume of individual worms was used as a measure of worm size. hosts. If worms were greater than or equal to 1 mm in length, they were considered cylindrical in shape and their volume was calculated using the formula (␲lw2)/4, where l is worm length and w is worm width. Worms less than Received 6 October 2006; revised 14 January 2007; accepted 15 Jan- 1 mm were approximately ovoid in shape, and their volume was computed uary 2007. using the formula (␲lw2)/6. For each infected isopod, 4 variables were

742 BENESH AND VALTONEN—LARVAL A. LUCII GROWTH PATTERNS 743

calculated to characterize the infrapopulation, i.e., total worm volume (TWV), average worm volume (AWV), maximum worm volume (MWV), and the coefficient of variation in worm volume (CV, standard deviation of worm volume divided by the AWV). TWV, AWV, and MWV were dependent on the age of infection because worms grew throughout the experiment. On the other hand, CV is relative to the AWV of the infrapopulation, so it is not necessarily dependent on infection age. To make all infrapopulations comparable, it was necessary to ‘‘correct’’ values of TWV, AWV, and MWV for the age of infection. Each of these variables was ln transformed and plotted against time, i.e., days of survival. Linear, logarithmic, and quadratic functions were fitted to the data (Figs. 1A–C). A logarithmic function, of the form Y ϭ a ϩ m ϫ ln(X), provided the best fit to the data for all 3 variables (lowest residual sum of squares and consequent P value). Therefore, the residuals from each of the 3 logarithmic functions were taken as values of TWV, AWV, and MWV independent of infection age. In all cases, residuals were normally distributed with homogenous variance. These 3 sets of residuals were used as dependent variables in a multivariate analysis of covariance (MANCOVA). Also, ln-transformed CV was included as a fourth dependent variable. To assess whether the factors affecting infrapopulation characteristics changed over the course of the experiment, a categorical variable was generated representing the time isopods died and were dissected. Specifically, infrapopulations were designated as being from isopods that died less than 40 days PE, called early infections, or from those that died more than 40 days PE, dubbed late infections. Day 40 was chosen as the splitting point because it effectively halved the data, creating nearly equal sample sizes in each group. Along with infection age, host size was used as an independent factor. Isopods were assigned to 3 size classes, small (Ͻ7.5 mm), intermediate (7.5–8.5 mm), and large (Ͼ8.5mm), designated to have equal sample sizes within each category. Infection intensity and molting rate (see Benesh and Valtonen [2007b] for description and calculation of molting rate) were used as covariates. Interactions between infection age and host size, infection age and intensity, and infection age and molting rate were also included in the model to evaluate whether the effects of these factors varied with time. Host sex was initially included in the MANCOVA model, but it was removed from the final analysis because the equality of covariances assumption was not met (Box’s test, P Ͻ 0.001). When there are dissimilar sample sizes in factor levels, as was the case for host sex, i.e., the data were male biased (males: n ϭ 174, females: n ϭ 56), violation of this assumption can distort P values (Zar, 1999). Samples in the levels of the other factors, by contrast, were similar. Thus, the model excluding host sex was considered more robust. In any case, when included in the model, host sex did not have any main effects on the dependent variables, and all the other results were qualitatively un- changed, so removal of this factor did not seem to affect conclusions. Parasite growth patterns may differ between isopods that died (n ϭ 196) during the experiment and those that did not (n ϭ 34). Thus, the MANCOVA analysis was also conducted on a data set excluding hosts that survived 101 days. Also, in some isopods, shriveled, melanized, and obviously dead worms were observed alongside live worms. Be- cause dead worms cannot interact with live, cooccurring conspecifics, they may bias some infrapopulation characteristics. Therefore, the MANCOVA was rerun after excluding obviously dead worms from the data, recalculating estimates of worm volume, and adjusting intensity to only include live worms. The MANCOVA was thus rerun twice, once excluding surviving isopods and once excluding dead worms. In both cases, the dependent variables, independent factors, and covariates were the same as described above. An additional confounder is that A. lucii is sexually dimorphic, i.e., females are larger than males as both adults and larvae. Thus, female-

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FIGURE 1. Ln total worm volume (TWV; A), ln average worm volume (AWV; B), and ln maximum worm volume (MWV; C) in cubic millimeters plotted against days survival (n ϭ 230). For all 3 infrapopulation characteristics, the logarithmic function (Y ϭ b ϩ ln[X]) provided a better ﬁt to the data than linear or quadratic functions. The residuals from each of the logarithmic functions were taken as relative values of TWV, AWV, and MWV adjusted for time. 744 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

biased infrapopulations may have misleadingly high TWV, AWV, TABLE I. Summary of the MANCOVA analysis of 230 infrapopulations MWV, and CV. To check for this, the extent that sex ratio correlated in which all measured worms were used. The 4 dependent variables with any of the 4 infrapopulation characteristics was determined. An used to characterize each infrapopulation were total worm volume alternative scenario in which worm sex ratio could bias results is if it (TMV), average worm volume (AWV), maximum worm volume changes with time. For example, the sex ratio may shift toward a male (MWV), and the coefficient of variation in worm volume (CV). Infec- excess during the experiment if isopods harboring more female worms, tion age, whether infrapopulations were observed before or after 40 which may be more energetically demanding, die earlier. Therefore, the days PE, and isopod size, i.e., small, intermediate, or large, were cate- degree that sex ratio changed over time was evaluated with regression gorical factors. Molting rate and infection intensity were covariates. An analysis. Only infrapopulations in which more than half of the worms identical analysis in which obviously dead worms were excluded from were large enough to be sexed were used to investigate the potential the data set produced the same results. Significant terms are indicated biasing effects of sex ratio. In all tests, the log transformation of sex in bold. ratio was used. Alpha values less than 0.05 were considered statistically significant. F P SPSS 12.0.1 statistical software was used to conduct all analyses (SPSS df Inc., Chicago, Illinois). Infection age TWV 0.082 1 0.775 RESULTS AWV 1.080 1 0.300 MWV 2.962 1 0.087 Data from 3,202 worms in 230 infrapopulations were used CV 9.973 1 0.002 in the MANCOVA. This sample is less than the number of infected isopods (n ϭ 248) for 2 reasons. First, in 9 infrapop- Isopod size ulations it was possible to count the worms, but their condition TWV 1.308 2 0.272 was too poor for accurate measurements of worm size to be AWV 3.377 2 0.036 made. Second, there were 9 isopods that harbored only 1 par- MWV 1.127 2 0.326 CV 1.866 2 0.157 asite, so it was not possible to calculate the CV. The analysis with the whole data set produced the same qualitative results Infection intensity as the analyses that either excluded surviving hosts or dead TWV 26.759 1 Ͻ0.001 worms. Thus, only the results from analysis of the whole data AWV 13.652 1 Ͻ0.001 set are presented. MWV 0.196 1 0.658 Ͻ Dead worms were observed in 40 isopods. The average size CV 24.374 1 0.001 of dead worms was 0.036 mm3, which is roughly the size of Molting rate early acanthellae. The variance to mean ratio of intensity was TWV 10.832 1 0.001 greater for dead worms than for live worms, 7.14 vs. 4.29, AWV 12.074 1 Ͻ0.001 suggesting dead worms were generally aggregated into fewer MWV 11.155 1 Ͻ0.001 hosts than live worms. The sex ratio of dead worms was female CV 0.083 1 0.773 biased, 1.96 &:1 ( (112 females, 57 males), and a chi-square Infection age ϫ Molting rate test indicated that this ratio was significantly different from TWV 3.417 1 0.066 ␹2 ϭ Ͻ 1:1 ( 1 17.90, P 0.001). AWV 2.788 1 0.096 The sex ratio of all the sexed worms was slightly female MWV 0.914 1 0.340 biased at 1.1 &:1 ( (785 females, 710 males). A chi-square CV 5.338 1 0.022 test suggested this was a marginally significant departure from Infection age ϫ Isopod size ␹2 ϭ ϭ the expected 1:1 ratio ( 1 3.76, P 0.05). There was no TWV 3.206 2 0.042 relationship between worm sex ratio and any of the 4 infrapop- AWV 5.414 2 0.005 ulation characteristics (Pearson correlations, n ϭ 97 in each MWV 2.342 2 0.099 case, all P Ͼ 0.094). Furthermore, infrapopulation sex ratio did CV 4.766 2 0.009 not change with time, i.e., days of survival (r ϭ 0.03, P ϭ Infection age ϫ Intensity 0.77). Thus, sex ratio is considered to have little confounding TWV 23.008 1 Ͻ0.001 effect on the analysis of larval infrapopulations. AWV 12.912 1 Ͻ0.001 The age of infection, late or early, had a significant effect on MWV 15.945 1 Ͻ0.001 the CV (Table I). Specifically, variation in worm size was greater CV 0.732 1 0.393 in late infections, i.e., after 40 days. There was no main effect of infection age on TWV, AWV, or MWV (Table I). Isopod size had a significant positive effect on AWV, but not on TWV, MWV, or affect AWV in early infections, but in late infections a negative CV (Table I). Infection intensity had different main effects on the correlation between AWV and intensity was observed (Fig. 2B). 4 dependent variables. Intensity was positively related to TWV In early infections, there was a slight positive relationship be- and CV (Table I; Fig. 2A, D, respectively). In contrast, it had a tween MWV and intensity, but in late infections this relation- negative effect on AWV (Table I; Fig. 2B). There was no main ship was negative (Fig. 2C). For CV, there was not a significant effect of intensity on MWV (Table I; Fig. 2C). interaction between infection age and intensity, suggesting that There were significant interactions between intensity and in- the relationship between CV and intensity did not change with fection age for 3 of the infrapopulation characteristics (TWV, time, i.e., it was consistently positive (Fig. 2D). AWV, and MWV), however, indicating the effects of intensity Molting rate was positively correlated with TWV, AWV, and were time dependent. TWV increased with intensity in early MWV (Table I; Fig. 3A–C). Infection age and molting rate infections, but not in late infections (Fig. 2A). Intensity did not significantly interacted to determine CV (Table I). In late in- BENESH AND VALTONEN—LARVAL A. LUCII GROWTH PATTERNS 745

FIGURE 2. The relationships between 4 infrapopulation characteristics and intensity. In the case of total worm volume (A), average worm volume (B), and maximum worm volume (C), the data are relative to time, i.e., they are the residuals from the logarithmic regressions in Figure 1A–C. The coefficient of variation in worm volume (D) is relative to average worm volume, so it is independent of time. Open circles represent early infections (infrapopulations observed before 40 days PE), whereas closed triangles depict late infections (infrapopulations observed after40 days PE). Dashed lines are fitted to the data from early infections, and solid lines correspond to late infections. fections, the relationship between CV and intensity was slightly For TWV, AWV, and CV, there was a significant interaction negative, but in early infections it was weakly positive (Fig. between infection age and isopod size, suggesting the relation- 3D). The interaction between infection age and molting rate ship between these infrapopulation characteristics and size de- was not significant for TWV, AWV, or MWV (Table I). How- pends on the time of the infection (Table I). In particular, TWV ever, for these 3 characteristics, the positive relationship with and AWV exhibit strong positive relationships with isopod size molting rate seemed to be greater in late infections than in early late in infections, but they do not seem to be affected by size infections (Fig. 3A–C). in early infections (Fig. 4A, B). MWV showed a similar trend 746 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. The relationships between 4 infrapopulation characteristics and molting rate, the number of molts per day survival. Total worm volume (A), average worm volume (B), and maximum worm volume (C) data are relative to time, i.e., they are the residuals from the logarithmic regressions in Figure 1A–C. The coefﬁcient of variation in worm volume (D) is relative to average worm volume, so it is independent of time. Open circles represent early infections (infrapopulations observed before 40 days PE), whereas closed triangles depict late infections (infrapopulations observed after 40 days PE). Dashed lines are ﬁtted to the data from early infections, and solid lines correspond to late infections.

(Fig. 4C), but the interaction between infection age and isopod DISCUSSION size was not signiﬁcant (Table I). In early infections, isopod size had no effect on CV (Fig. 4D). For small and intermediate The relationship between time and the 3 measures of worm size isopods, CV was much higher in late infections, but this size (TWV, AWV, and MWV) was not linear. Instead, it was was not the case for large isopods (Fig. 4D); CV in large iso- slightly curved, indicating that the relative rate of parasite pods was similar before and after 40 days. growth decreased as the experiment progressed. Consequently, BENESH AND VALTONEN—LARVAL A. LUCII GROWTH PATTERNS 747

FIGURE 4. The relationships between 4 infrapopulation characteristics and isopod size. In the case of total worm volume (A), average worm volume (B), and maximum worm volume (C), the data are relative to time, i.e., they are the residuals from the logarithmic regressions in Figure 1A–C. The coefficient of variation in worm volume (D) is relative to average worm volume, so it is independent of time. Open circles represent mean values from early infections (infrapopulations observed before 40 days PE), whereas closed triangles depict the mean values from late infections (infrapopulations observed after 40 days PE). Bars represent standard error. the impact of the factors affecting larval infrapopulations was ol, 2003; Poulin et al., 2003). However, when the impact of time dependent. For example, intensity had a positive main ef- intensity is considered in the context of time, more complex fect on TWV, but a negative main effect on AWV. This con- patterns emerge. Early on, higher intensities did yield higher forms to expectations; more worms should produce more worm TWV, but this relationship disappeared in late infections. This biomass, and the negative effect of intensity on AWV represents suggests that there is a threshold parasite biomass that can be the anticipated crowding effect observed in other acanthoceph- supported by an individual isopod, regardless of the number of alans (Awachie, 1966; Dezfuli et al., 2001; Steinauer and Nick- parasites. For AWV, the expected crowding effect was ob- 748 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 served, but only in late infections. The average size of young normally large or small individuals increases. Some variation worms, however, did not seem to be affected by the presence probably also stemmed from age differences between parasites, of conspecifics. The observation of crowding suggests that re- which may range up to 10 days given the length of exposure. sources, or perhaps even space, are limited in late infections Much parasite size variation, however, likely derived from in- when worms are larger. Temporal depletion of resources could traspecific competition, i.e., individuals were not equal in their put worms under pressure to exploit resources aggressively ear- ability to acquire nutrients and grow. Competitive interactions ly during development, since they may become more sparse between developing parasites can take 2 forms, i.e., exploita- later. tion/indirect competition, or interference/direct competition. In The consequences of this pressure may be manifested in the the former case, competitive interactions are predicted to vary relationship between maximum worm volume (MWV), inten- with resource abundance, whereas in the latter case competition sity, and time. In early infections, MWV was positively related is expected to be largely independent of resources. Variation in to intensity. The presence of multiple competing conspecifics worm size tended to increase as resources decreased, i.e., CV may promote rapid initial exploitation of host resources while was greater in late infections. Furthermore, CV was negatively they are still abundant (Parker et al., 2003). This scramble com- related to the 2 factors (molting rate and isopod size) likely petition may result in some parasites growing larger than might representing resource availability for developing parasites. be expected. On the other hand, the positive association be- These factors only affected CV in late infections when resourc- tween intensity and MWV could be entirely probabilistic; if es were presumably constrained, suggesting that competition more worms are measured, the chance of recording an abnor- between larval parasites may be diffused in larger or faster mally large one increases. The negative correlation between growing hosts. Variation in worm size, thus, seems to vary with MWV and intensity in late infections is probably analogous to resource availability, which suggests that exploitation compe- the situation with AWV in that it represents a crowding effect tition, as opposed to interference, is primarily operating among caused by limited resources. growing A. lucii. The impact of isopod size on parasite growth was also time Dead parasites, almost exclusively young acanthellae, oc- dependent. Isopod size seemed to have little effect on A. lucii curred in some infrapopulations. Dead acanthocephalans in size in early infections, but TWV and AWV increased with host their intermediate hosts, particularly early developmental stag- size in late infections. In early infections, the impact of host es, have been reported previously (e.g., Hynes and Nicholas, size on worm size may be negligible because resources are 1958; Crompton, 1967; Robinson and Strickland, 1969; Nickol relatively abundant, but as worms grow and resources dwindle, and Dappen, 1982; Gleason, 1989). Because the larval mortality the energetic constraints imposed on parasites increase. Larger of A. lucii, as well as other species, seems to be greater during hosts may ease these constraints, thereby allowing more para- early development, the time spent in these ‘‘risky’’ stages site growth. In several other systems, intermediate host size has should be minimized. Thus, the high initial relative growth rate also been recognized as an important determinant of acantho- may be advantageous, even though it might negatively affect cephalan size, at least for late developmental stages (Awachie, host survival (Benesh and Valtonen, 2007b). There are 2 non- 1966; Dezfuli et al., 2001; Steinauer and Nickol, 2003). MWV exclusive possible explanations for the size-specific mortality. increased with isopod size in late, but not early, infections. First, the host immune response is most effective at killing However, this trend was not significant, suggesting that host young acanthellae, or, second, the parasites are heavily con- size, and by association resource availability, does not affect strained by resources at this stage. The overrepresentation of MWV to the same degree as TWV and AWV. The reason for females among the dead worms suggests that if females require this is unclear, but it could represent the competitive superiority more energy to develop, limiting resources are the cause of of some individual parasites in that, regardless of the available worm death. However, the aggregation of dead worms into rel- resource base, they can achieve a relatively large size. atively few hosts also could imply that some isopods have an Isopods that molted more frequently tended to harbor larger especially efficient immune response against A. lucii. worms, suggesting the growth or physiological processes as- Another unexpected result was that the A. lucii sex ratio was sociated with molting enhance parasite development. The pos- slightly female biased. Given that the departure from 1:1 was itive impact of host molting on parasite growth has not been slight and the sex ratio of acanthocephalans from natural and previously documented in other systems, though host growth laboratory infections of intermediate hosts is generally 1:1 seems to increase parasite growth in some systems (Barber, (Crompton, 1985), this result must be interpreted with caution. 2005). Hosts that molt more presumably have higher food in- Despite the apparent female bias in the component population, take, assuming molting is energetically costly, and may, there- sex ratio did not seem to affect the characteristics of infrapop- fore, provide more resources to developing parasites. Though ulations. The rather high parasite burdens may have dampened the interactions were not significant, the impact of molting on any biasing effects of sex ratio on worm sizes. parasite size seemed to be greater in late infections. This could All the proximate factors (host size, molting rate, and inten- result from the observation of more molts as time progressed, sity) affecting A. lucii growth in some way revolved around thereby making the correlation easier to detect in late infections. resource availability, though the relevance of resources seemed Another possibility is that, like the situation with isopod size, to change over time. Interestingly, the temporal changes in re- molting rate primarily affects parasite size in late infections source availability and parasite growth rates may affect host because resources have become less abundant at this stage. survival. Infected isopods had reduced survival, relative to un- The variability in worm size increased with infection inten- infected isopods, during the early stages of A. lucii develop- sity. Some of this may be attributable to infrapopulation size; ment, but not at later stages of infection (Benesh and Valtonen, if more worms are observed, the likelihood of measuring ab- 2007b). Though the mechanism underlying this mortality is un- BENESH AND VALTONEN—LARVAL A. LUCII GROWTH PATTERNS 749 known, it is possible that relatively rapid and apparently re- Nickol and D. W. T. Crompton (eds.). Cambridge University Press, source-independent growth by young parasites is worse for host Cambridge, U.K., p. 213–272. DEZFULI, B. S., L. GIARI, AND R. POULIN. 2001. Costs of intraspecific viability than the slower, resource-limited growth of larger par- and interspecific host sharing in acanthocephalan cystacanths. Par- asites. This goes against some hypothetical expectations in that asitology 122: 483–489. heavier parasite burdens (total parasite mass) are assumed to FREDENSBORG, B. L., AND R. POULIN. 2005. Larval helminthes in inter- result in greater host mortality (Parker et al., 2003). The null mediate hosts: Does competition early in life determine the fitness of adult parasites? International Journal for Parasitology 35: 1061– relationship between total worm volume and intensity in late 1070. infections suggests that developing A. lucii approached and/or GLEASON, L. N. 1989. Movement of Pomphorhynchus bulbocolli larvae reached an upper size limit defined by each individual host, yet from the hemocoel to the peripheral circulation of Gammarus pseu- this did not result in lower host survival. The growth patterns dolimnaeus. Journal of Parasitology 75: 982–985. of several pseudophyllidean cestode species, on the other hand, HYNES,H.B.N.,AND W. L. NICHOLAS. 1958. The resistance of Gam- marus spp. to infection by Polymorphus minutus (Goeze, 1782) suggest that parasites are not exploiting host resources at max- (Acanthocephala). Annals of Tropical Medicine and Parasitology imum levels, possibly reflecting adaptive life history strategies 52: 376–383. (Parker et al., 2003; Michaud et al., 2006). Such submaximal LAFFERTY,K.D.,AND A. M. KURIS. 2002. Trophic strategies, animal growth may also occur in single A. lucii infections, particularly diversity, and body size. Trends in Ecology and Evolution 17: 507– 513. for male parasites (Benesh and Valtonen, 2007a), leaving open MICHAUD, M., M. MILINSKI,G.A.PARKER, AND J. C. CHUBB. 2006. the possibility that larval growth patterns are not exclusively Competitive growth strategies in intermediate hosts: Experimental determined by host resources (Parker et al., 2003). In any case, tests of a parasite life-history model using the cestode, Schistoce- identifying the proximate factors impacting the growth of dif- phalus solidus. Evolutionary Ecology 20: 39–57. NICKOL,B.B.,AND G. E. DAPPEN. 1982. Armadillidium vulgare (Iso- ferent parasites, such as resource availability, host defenses, and poda) as an intermediate host of Plagiorhynchus cylindraceus host mortality, and evaluating them within a comparative (Acanthocephala) and isopod response to infection. Journal of Par- framework may reveal the selective forces shaping the evolu- asitology 68: 570–575. tion of parasite life history strategies. PARKER, G. A., J. C. CHUBB,G.N.ROBERTS,M.MICHAUD, AND M. MILINSKI. 2003. Optimal growth strategies of larval helminths in their intermediate hosts. Journal of Evolutionary Biology 16: 47– ACKNOWLEDGMENTS 54. PILECKA-RAPACZ, M. 1986. On the development of acanthocephalans of T. Hasu graciously shared her experiences with isopods in the labo- the genus Acanthocephalus Koelreuther, 1771, with special atten- ratory and gave insightful criticisms on a draft of this manuscript. Eero tion to their influence on intermediate host, Asellus aquaticus L. Vestola helped in the maintenance of isopods. D.P.B. was provided fi- Acta Parastologica Polonica 30: 233–350. nancial support by Fulbright and CIMO grants. POULIN, R. 1998. Evolutionary ecology of parasites. Chapman and Hall, London, U.K, 212 p. LITERATURE CITED ———, K. NICHOL, AND A. D. M. LATHAM. 2003. Host sharing and host manipulation by larval helminthes in shore crabs: Cooperation AWACHIE, J. B. E. 1966. The development and life-history of Echino- or conflict? International Journal for Parasitology 33: 425–433. rhynchus truttae Schrank 1788 (Acanthocephala). Journal of Hel- ROBINSON,E.S.,AND B. C. STRICKLAND. 1969. Cellular responses of minthology 40: 11–32. Periplaneta americana to acanthocephalan larvae. Experimental BARBER, I. 2005. Parasites grow faster in faster growing fish hosts. Parasitology 50: 694–697. International Journal for Parasitology 35: 137–143. ROSEN, R., AND T. A . D ICK. 1983. Development and infectivity of the BENESH,D.P.,AND E. T. VALTONEN. 2007a. Sexual differences in larval procercoid of Triaenophorus crassus Forel and mortality of the first life history traits of acanthocephalan cystacanths. International intermediate host. Canadian Journal of Zoology 61: 2120–2128. Journal for Parasitology 37: 191–198. STEINAUER,M.L.,AND B. B. NICKOL. 2003. Effect of cystacanth body ———, AND ———. 2007b. Effects of Acanthocephalus lucii (Acan- size on adult success. Journal of Parasitology 89: 251–254. thocephala) on intermediate host survival and growth: Implications UZNANSKI, R. L., AND B. B. NICKOL. 1980. A sequential ranking system for exploitation strategies. Journal of Parasitology 93: 735–741. for developmental stages of an acanthocephalan, Leptorhynchoides CROMPTON, D. W. T. 1967. Studies on the haemocytic reaction of Gam- thecatus, in its intermediate host, Hyalella azteca. Journal of Par- marus spp., and its relationship to Polymorphus minutus (Acantho- asitology 66: 506–512. cephala). Parasitology 57: 389–401. ZAR, J. H. 1999. Biostatistical analysis. Prentice-Hall, Upper Saddle ———. 1985. Reproduction. In Biology of the Acanthocephala, B. B. River, New Jersey, 929 p. J. Parasitol., 93(4), 2007, pp. 750–754 ᭧ American Society of Parasitologists 2007

FIELD EVIDENCE OF HOST SIZE-DEPENDENT PARASITISM IN TWO MANIPULATIVE PARASITES

Yannick Outreman, Frank Ce´zilly, and Loı¨c Bollache*† UMR CNRS 5561 Biogeosciences, Universite´ de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France. e-mail: [email protected]

ABSTRACT: The distribution of parasites within host natural populations has often been found to be host age-dependent. Host mortality induced by parasites is the commonest hypothesis proposed for explaining this pattern. Despite its potential importance in ecology, the parasitism intensity in relation with the host age has rarely been studied in the ﬁeld. The 2 manipulative acanthocephalans, Polymorphus minutus and Pomphorhynchus laevis, use the amphipod Gammarus pulex as an intermediate host, and their infection intensity and incidence among G. pulex populations were examined by analyzing 2 large samples of hosts collected in eastern France. Both parasites had low prevalence in the host populations, but their mean abundances were highly related with gammarid age. For the 2 acanthocephalans, results reported a disappearance or an absence of heavily infected hosts in the older host age classes. These results suggested that parasites that alter intermediate host behavior for enhancing their transmission success to the deﬁnitive host reduce the survival of their intermediate host. In conclusion, manipulative parasites might act as a mechanism regulating the density of gammarid populations.

Recently, several theoretical, experimental, and field studies through the ability of the parasite to alter the behavior of its have recognized parasites as an important determinant of host intermediate host’s behavior (Lafferty, 1999). Parasite-induced population dynamics and animal community structure (Min- manipulation of host behavior is particularly common in acan- chella and Scott, 1991; Poulin, 1999; Tompkins et al., 2001). thocephalans (Bethel and Holmes, 1973; Ce´zilly et al., 2000; The distribution of parasites within host natural populations is Perrot-Minnot, 2004). Manipulations by these parasites may in- the result of various host-related or parasite-related processes, volve both physiological (Helluy and Holmes, 1990; Thompson such as host exposure to the parasites, and host resistance and and Kavaliers, 1994) and behavioral alterations (Hindsbo, 1972; susceptibility to the infestation (Anderson and May, 1979), and Moore, 1984). In Burgundy (eastern France), 2 acanthocepha- it is often found to be age-dependent (Kennedy, 1984; Thomas lans, Polymorphus minutus and Pomphorhynchus laevis, use the et al., 1995; Krasnov et al., 2006). Among the numerous hy- amphipod Gammarus pulex as an intermediate host. These par- potheses proposed to explain the age-dependent patterns in the asites differ markedly in their final host, a fish for P. laevis and parasite distribution within host populations, parasite-induced a bird for P. minutes, and in their effect on the intermediate host mortality, suggested by Anderson and Gordon (1982), is host phenotype. Compared with uninfected gammarids that are one of the most popular (Rousset et al., 1996; Latham and Pou- photophobic and tend to remain at the bottom of the water lin, 2002). column, P. laevis-infected gammarids are attracted by light, However, evidence for parasite-induced host mortality from whereas P. minutus-infected individuals swim close to the water field data remains difficult to obtain for 2 main reasons: (1) surface (Ce´zilly et al., 2000). Given these behavioral alter- dead hosts are rarely found, and, even if they are, the death ations, the influence of these parasites on the survival of G. cause can rarely be attributed unequivocally to the parasite pulex could be high. The aim of the present study was to in- (Price, 1980; Gordon and Rau, 1982; Scott and Dobson, 1989; vestigate the effects of these manipulative parasites on the sur- McCallum and Dobson, 1995); and (2) to relate alterations of vival of their intermediate host, G. pulex, by examining the fecundity or survival with effects on the host population re- relationship between parasitism abundance and age of hosts quires quantitative field measures (Scott and Dobson, 1989). sampled in the field. These difficulties have led to the development of techniques whereby existence of parasite-induced mortality can be deter- MATERIALS AND METHODS mined indirectly (Anderson and Gordon, 1982; Rousset et al., Two large samples of G. pulex were collected on different dates 1996) by examining changes in parasitism as a function of host (March 1999 and August 1999) at the same site (Tille River, Arc/Tille, age, i.e., when parasitism induces host mortality, a maximum Burgundy, eastern France). Individuals were randomly collected in the level of infection occurs in hosts of intermediate age and falls aquatic vegetation using the kick-sampling method described by Hynes in the older age classes. Nevertheless, some other causes may (1954), and they were immediately preserved in 70% alcohol. In the laboratory, G. pulex individuals were sexed and measured using linear also drive to age-specific distribution such as negative effect of dimensions (distance from the fourth coxal plate basis to individual parasite on the host growth, age-dependent exposure or resis- dorsal limit) by stereoscopic microscopy (Nikon SMZ-10A; Nikon, To- tance to the parasites (Kennedy, 1984; Goater and Ward, 1992; kyo, Japan) and a video analysis system (VTO 232; Linkam Scientific Duerr et al., 2003). Instruments, Surrey, U.K.) (Bollache et al., 2000). Gammarids were then Many parasites have complex life cycles that include a tran- dissected to count the number of P. minutus and P. laevis cystacanths. Growth in gammarids roughly conforms to a logistic curve, but the sient phase in an intermediate host, followed by passive transfer relationship between size and age depends largely on temperature, food, to the definitive host through a predator–prey system. In some and sex (Sutcliffe et al., 1981). Male and female gammarids were then species, transmission to the definitive host can be enhanced analyzed separately and allocated to 1 of 12 length classes, assuming a good relationship between size and age. In classes 2–11, size steps are equal, i.e., male ϭ 0.15 mm, female ϭ 0.13 mm. Class 1 includes all Received 30 October 2006; revised 4 January 2007; accepted 4 Jan- those individuals that are too small to be in class 2, i.e., male Ͻ1.50 uary 2007. mm, female Ͻ1.40 mm, and class 12 includes all those individuals that * Equipe Ecologie Evolutive, UMR CNRS 5561 Biogeósciences, Uni- are too large to be in class 11, i.e., male Ͼ3.0 mm, female Ͼ2.7 mm. versite´ de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France. For each parasite species and each host size class, the prevalence, † To whom correspondence should be addressed. i.e., proportion of infected individuals, the mean parasite abundance,

750 OUTREMAN ET AL.—SIZE-INTENSITY INFECTION IN G. PULEX 751

FIGURE 1. Prevalences of Polymorphus minutus and Polymorphus laevis in relation to host length class in Gammarus pulex. Males individuals denoted by filled circles and females by open circles. i.e., number of parasites divided by number of hosts examined, and the plained by regression models was not significant in either male variance to mean abundance ratio, i.e., measure of parasite aggregation or female gammarids. (Bush et al., 1997), were estimated. The first 2 indices of infection were tested against 3 fixed effects: sampling date, host size, and parasite species. Analysis was carried out by means of generalized linear models DISCUSSION using the S-plus statistical software (MathSoft, Cambridge, Massachu- setts). To determine the relationship between both the mean parasite Several models in ecology and evolution assume that para- abundance and the variance-to-mean abundance ratio and host age, we sites adversely affect fitness of their hosts (Anderson and May, ϭ ␤ ϩ ␤ ϩ ␤ 2 fitted linear or polynomial, i.e., quadratic model: y 0 1x 2x , 1979; Hamilton, 1980; Anderson and Gordon, 1982; Holt and regressions to the set of points and retained the polynomial model if Pickering, 1985; Rousset et al., 1996), although some parasi- the quadratic term of the regression that represents the amount of curvature in the data, i.e., ␤ , was significantly different from 0. tologists contend that parasites are relatively benign (Noble et 2 al., 1989). Our study suggests that P. minutus and P. laevis conform to the former view. Indeed, the most parsimonious RESULTS explanation for the decline in the mean abundance of parasites For each host size class, data on parasite prevalence are given in the older age classes of hosts, concomitant with a decline in in Figure 1, and the mean parasite abundance and variance to aggregation, is the presence of mortality induced by these par- mean abundance ratio for P. minutus and P. laevis are presented asites in host populations, such as infected individuals are re- in Figures 2 and 3, respectively. Prevalence and mean abun- moved from the population at higher rates than uninfected in- dance of P. minutus and P. laevis in gammarids differed sig- dividuals (Anderson and Gordon, 1982; Rousset et al., 1996). nificantly among the 2 samples (Table I). In both male and Thus, when host mortality is positively correlated with parasite female gammarids, prevalence and abundance of P. laevis were accumulation (Anderson and Gordon, 1982; Rousset et al., significantly higher than for P. minutus (Table I; Figs. 1, 2). 1996) or simply with parasite presence (Rousset et al., 1996), For both sexes, prevalence and mean abundance of the 2 ma- a maximum level of infection occurs in hosts of intermediary nipulative parasites differed significantly with host size; these age. changes were similar for the 2 acanthocephalan species (Table However, convex size–abundance curves could be generated I). Both prevalence and mean abundance increased steadily with by other factors and may complicate interpretation of such pat- host size, attaining their highest value in hosts of intermediate tern. First, parasite mortality as a result of host responses may size, and then they fell off in higher length classes. For mean occur (Crompton and Nickol, 1985), but, in general, dead and parasite abundance, best regression models were always the encapsulated parasites are still observable at host dissection polynomial models because the quadratic terms of the regres- (Anderson and Gordon, 1982), and, as such, they are recorded sion were significantly different from 0 (Figs. 2, 3). Mean abun- in measurement of parasite abundance. No encapsulated para- dance tended to peak in the medium-sized individuals. For var- sites were found in our samples. Second, when size is used as iance-to-mean abundance ratios, percentage of variation ex- a measure of age, reduced growth in infected individuals may 752 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 2. Mean parasite abundance and variance to mean abundance of Polymorphus minutus in relation to host length class in Gammarus pulex. The number of hosts in each length class is indicated above each point. r2 ϭ percentage of variance explained; P ϭ signiﬁcance of the polynomial model. explain the decline of both abundance value and aggregation the larger host size classes. However, Rigaud and Moret (2003) indices in larger length classes. Hynes and Nicholas (1963) sug- have shown that P. laevis and P. minutus can evade their ingest that acanthocephalans may reduce the growth rate of gam- termediate host immune response through immunosuppression, marids. Although direct evidence is still lacking, such an ex- whatever the host size. In addition, these authors found that planation can not be excluded. Third, increased resistance with immune responses of female gammarids to P. laevis infection age may explain the decline in the mean infection levels among were stronger than those in the males, whereas level of para-

FIGURE 3. Mean parasite abundance and variance to mean abundance of Polymorphus laevis in relation to host length class in Gammarus pulex. The number of host in each length class is indicated above each point. r2 ϭ percentage of variance explained; P ϭ signiﬁcance of the polynomial model. OUTREMAN ET AL.—SIZE-INTENSITY INFECTION IN G. PULEX 753

TABLE I. Generalized linear models showing the effects of sampling tality of 2 closely related amphipod species, i.e., Gammarus date, host size, and parasite species (Polymorphus minutus and Poly- aequicauda and Gammarus insensibilis, Thomas et al. (1995) morphus laevis) on the prevalence and mean parasite abundance of these 2 acanthocephalans in males (a) and females (b) of their inter- reported that mean parasite abundance and infection aggrega- mediate host Gammarus pulex. tion increased with host size in G. aequicauda compared with G. insensibilis. It has been shown that this parasite does not Variables (distribution family) induce behavioral alteration in the former species compared with the latter species (Helluy, 1983). Using a similar approach, Prevalence Abundance Medoc et al. (2006) reported that P. minutus, known to actively (binomial) (Poisson) manipulate the behavior of Gammarus roeseli (Bauer et al., Source df FPvalue FPvalue 2005), had a significant effect on the mortality of its host. Thus, (a) Males the pattern of mortality between gammarids infected by P. minutus or P. laevis, and those infected with nonmanipulative par- Sampling date 1 21.80 Ͻ0.001 33.13 Ͻ0.001 Parasite species 1 99.56 Ͻ0.001 78.04 Ͻ0.001 asites, supports the idea that manipulation has a real impact on Host size 11 5.46 Ͻ0.001 4.78 Ͻ0.001 the survival of infected hosts; thus, gammarids with altered be- Error 7,145 havior are more susceptible to predation by the definitive host Total 7,169 (Moore and Gotelli, 1990; Poulin, 1995). In summary, by af- (b) Females fecting host survival, male reproduction success (Bollache et al., 2001), and female fecundity (Bollache et al., 2002), P. lae- Sampling date 1 8.11 Ͻ0.01 13.57 Ͻ0.001 Parasite species 1 75.74 Ͻ0.001 70.73 Ͻ0.001 vis and P. minutus can be recognized as potential regulatory Host size 11 4.32 Ͻ0.001 3.85 Ͻ0.001 factors of the density of G. pulex populations, even when these Error 6,347 2 acanthocephalans are present in low prevalences. Total 6,371 ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Contrat de Plan sitism as a function of host size found here was similar between Etat-Re´gion Bourgogne and the Programme National Biodiversite´ volet females and males. ‘Interactions durables.’ Recently, Duclos et al. (2006) have shown that infection by the acanthocephalan Corynosoma constrictum reduced survival LITERATURE CITED of their host, the amphipod Hyallela azteca. Their results, from ANDERSON,R.M.,AND D. M. GORDON. 1982. Processes influencing the experimental infection, suggest that developing parasites satisfy distribution of parasite numbers within host populations with special emphasis on parasite-induced host mortalities. Parasitology 85: energy requirements by depriving amphipod hosts of resources, 373–398. and they force premature host death, particularly when infection ———, AND R. M. MAY. 1979. Population biology of infectious dis- intensities are high. However, previous results do not support eases: Part I. Nature 280: 361–367. this hypothesis, and they show that glycogen and lipid reserves BAUER, A., E. R. HAINE,M.J.PERROT-MINNOT, AND T. R IGAUD. 2005. in G. pulex, indicators of good physiological condition in in- The acanthocephalan parasite Polymorphus minutus alters the geo- tactic and clinging behaviours of two sympatric amphipods hosts: vertebrates, are not affected by the infection of P. laevis (Plais- The native Gammarus pulex and the invasive Gammarus roeseli. tow et al., 2001). Journal of Zoology London 267: 39–43. The primary evidence suggesting that increased mortality in BETHEL,W.M.,AND J. C. HOLMES. 1973. Altered evasive behaviour and the infected G. pulex is due to more than pathology is that responses to light in amphipods harbouring acanthocephalan cystacanths. Journal of Parasitology 65: 667–669. acanthocephalans are manipulative, making gammarids more BOLLACHE, L., G. GAMBADE, AND F. C E´ ZILLY. 2000. The influence of susceptible to predation (Moore, 1984, 2002; Lafferty, 1992). micro-habitat segregation on size assortative pairing in Gammarus Ce´zilly et al. (2000) found that P. minutus and P. laevis cys- pulex (L.) (Crustacea, Amphipoda). Archiv fu¨r Hydrobiologie 157: tacanths are able to manipulate the behavior of gammarids. Ac- 547–558. cording to the parasite manipulation hypothesis, such changes ———, ———, AND ———. 2001. The effects of two acanthocephalan parasites, Pomphorhynchus laevis and Polymorphus minutus,on in intermediate host phenotype can be interpreted as an adaptive pairing success in male Gammarus pulex (L.) (Crustacea: Amphi- strategy of the parasite to promote its transmission to the defin- poda). Behavioral Ecology and Sociobiology 49: 296–303. itive host (Holmes and Bethel, 1972; Moore and Gotelli, 1990; ———, T. RIGAUD, AND F. C E´ ZILLY. 2002. Effects of two acanthoceph- Poulin, 1995), especially for parasites relying on trophic trans- alan parasites on the fecundity and pairing status of female Gam- marus pulex (Crustacea: Amphipoda). Journal of Invertebrate Pa- mission to reach their definitive host (Poulin, 1998, Lafferty, thology 79: 102–110. 1999). For the 2 acanthocephalans in the present study, the ob- BUSH,A.O.,K.D.LAFFERTY,J.M.LOTZ, AND A. W. SHOSTAK. 1997. served curves of infection parameters as a function of host size Parasitology meets ecology on its own terms: Margolis et al. re- were convex, suggesting that the 2 manipulative acanthoceph- visited. Journal of Parasitology 83: 575–583. ´ ´ alans reduce survival of infected gammarids in nature by in- CEZILLY, F., A. GREGOIRE, AND A. BERTIN. 2000. Conflict between cooccurring manipulative parasites? An experimental study of joint creasing their risk of predation by predators. Previous studies influence of two acanthocephalan parasites on the behaviour of support our hypothesis of host mortality induced by manipu- Gammarus pulex. Parasitology 120: 625–630. lative parasites. For example, Perrot-Minnot et al. (2007) CROMPTON,D.W.T.,AND B. B. NICKOL. 1985. Biology of the Acan- showed a 10-fold effect of Pomphorhynchus tereticollis, a very thocephala. Cambridge University Press, Cambridge, U.K., 519 p. DUCLOS, L. M., B. J. DANNER, AND B. B. NICKOL. 2006. Virulence of close species to P. laevis, on the predation rate of G. pulex by Corynosoma constrictum (Acanthocephala: Polymorphidae) in Hy- the definitive host Cottus gobio. In the study on the effect of allela azteca (Amphipoda) throughout parasite ontogeny. Journal Microphallus papillorobustus, a trematode parasite, on the mor- of Parasitology 92: 749–755. 754 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

DUERR,H.P.,K.DIETZ, AND M. EICHNER. 2003. On the interpretation an acanthocephalan parasite strategy. American Naturalist 123: of age-intensity profiles and dispersion patterns in parasitological 572–577. surveys. Parasitology 126: 87–101. ———. 2002. Parasites and the behavior of animals. Oxford University GOATER,C.P.,AND P. I. WARD. 1992. Negative effect of Rhabdias bu- Press, New York, New York, 315 p. fonis (Nematoda) on the growth and survival of toads (Bufo bufo). ———, AND N. J. GOTELLI. 1990. A phylogenetic perspective on the Oecologia 89: 161–165. evolution of altered host behaviours: A critical look at the manip- GORDON,D.M.,AND M. E. RAU. 1982. Possible evidence for mortality ulation hypothesis. In Parasitism and host behaviour, C. J. Barnard, induced by the parasite Apatemon gracilis in a population of brook and J. M. Behnke (eds.). Taylor and Francis, London, U.K., p. 193– sticklebacks. Parasitology 84: 41–47. 233. HAMILTON, W. D. 1980. Sex versus non-sex versus parasite. Oikos 35: NOBLE,E.R,G.A.NOBLE,G.A.SCHAD, AND A. J. MCINNES. 1989. 282–290. Parasitology: The biology of animal parasites, 6th ed. Lea and Fei- HELLUY, S. 1983. Relations hoˆtes-parasites du Tre´matode Microphallus biger, Philadelphia, Pennsylvania, 574 p. papillorobustus (Rankin, 1940). II. Modifications du comportement PERROT-MINNOT, M. J. 2004. Larval morphology, genetic divergence, des Gammarus hoˆtes interme´diaires et localisation des me´tacercai- and contrasting levels of host manipulation between forms of Pom- res. Annales de Parasitologie Humaine et Compareé 58: 1–17. phorhynchus laevis (Acanthocephala). International Journal for Par- ———, AND J. C. HOLMES. 1990. Serotonin, octopamine and the cling- asitology 34: 45–54. ing behaviour induced by the parasite Polymorphus paradoxus ———, N. KALDONSKI, AND F. C E´ ZILLY. 2007. Increased susceptibility (Acanthocephala) in Gammarus lacustris (Crustacea). Canadian to predation and altered anti-predator behaviour in an acanthoceph- Journal of Zoology 68: 1214–1220. alan-infected amphipod. International Journal for Parasitology 37: HINDSBO, O. 1972. Effects of Polymorphus (Acanthocephala) on colour 645–651. and behaviour of Gammarus lacustris. Nature 238: 333. PLAISTOW, S. J., J. P. TROUSSARD, AND F. C E´ ZILLY. 2001. The effect of HOLT,R.D.,AND J. PICKERING. 1985. Infectious disease and species the acanthocephalan parasite Pomphorhynchus laevis on the lipid coexistence: A model of Lokta-Volterra form. American Naturalist and glycogen content of its intermediate host Gammarus pulex. 126: 196–211. International Journal for Parasitology 31: 346–351. HUDSON, P. J., AND J. GREENMAN. 1998. Competition mediated by par- POULIN, R. 1995. ‘‘Adaptative’’ change in the behaviour of parasitized asites: Biological and theoretical progress. Trends in Ecology and animals: A critical review. International Journal for Parasitology Evolution 13: 387–390. 25: 1371–1383. ———. 1999. The functional importance of parasites in animal com- HYNES, H. B. N. 1954. The ecology of Gammarus duebeni Lilljeborg and its occurrence in freshwater in western Britain. Journal of An- munities: Many roles at many levels? International Journal for Par- imal Ecology 23: 38–84. asitology 29: 903–914. PRICE, P. W. 1980. Evolutionary biology of parasites. Princeton Univer- ———, AND W. L. NICHOLAS. 1963. Importance of the acanthocephalan sity Press, London, U.K., 237 p. Polymorphus minutus as a parasite of domestic ducks in the United RIGAUD,T.,AND Y. M ORET. 2003. Differential phenoloxidase activity Kingdom. Journal of Helminthology 3: 185–198. between native and invasive gammarids infected by local acantho- KENNEDY, C. R. 1984. The use of frequency-distributions in an attempt cephalans: Differential immunosuppression? Parasitology 127: to detect host mortality induced by infections of diplostomatid 571–577. metacercariae. Parasitology 89: 209–220. ROUSSET, F., F. THOMAS,T.DE MEEU¨ S, AND F. R ENAUD. 1996. Inference KRASNOV, B. R., M. STANKO, AND S. MORAND. 2006. Age-dependent of parasite-induced host mortality from distributions of parasite flea (Siphonaptera) parasitism in rodents: A host’s life history mat- loads. Ecology 77: 2203–2211. ters. Journal of Parasitology 92: 242–248. SCOTT, M. E., AND A. DOBSON. 1989. The role of parasites in regulating LAFFERTY, K. D. 1999. The evolution of trophic transmission. Parasi- host abundance. Parasitology Today 5: 176–183. tology Today 15: 111–115. SUTCLIFFE,D.W.,T.R.CARRICK, AND L. G. WILLOUGHBY. 1981. Effects LATHAM,A.D.M.,AND R. POULIN. 2002. Field evidence of the impact of diet, body size, age and temperature on growth rates in the of two acanthocephalan parasites on the mortality of three species amphipod Gammarus pulex. Freshwater Biology 11: 183–214. of New Zealand shore crabs (Brachyura). Marine Biology 141: THOMAS, F., F. RENAUD,F.ROUSSET,F.CE´ ZILLY, AND T. DE MEEU¨ S. 1995. 1131–1139. Differential mortality of two closely related host species induced MCCALLUM,H.,AND A. DOBSON. 1995. Detecting disease and parasite by one parasite. Proceedings of the Royal Society London 260: threats to endangered species and ecosystems. Trends in Ecology 349–352. and Evolution 10: 190–194. THOMPSON,S.N.,AND M. KAVALIERS. 1994. Physiological bases for MEDOC, V., L. BOLLACHE, AND J. N. BEISEL. 2006. Host manipulation parasite-induced alterations of host behaviour. Parasitology 109: of a freshwater crustacean (Gammarus roeseli) by an acanthoceph- 119–138. alan parasite (Polymorphus minutus) in a biological invasion con- TOMPKINS,D.M.,A.P.DOBSON,P.ARNEBERG,M.E.BEGON,I.M. text. International Journal for Parasitology 36: 1351–1358. CATTADORI,J.V.GREENMAN,A.P.HEESTERBEEK,P.J.HUDSON,D. MINCHELLA,D.J.,AND M. E. SCOTT. 1991. Parasitism: A cryptic deter- NEWBORN,A.PUGLIESE,A.P.RIZZOLI,R.ROSA,F.ROSSO, AND K. minant of animal community structure. Trends in Ecology and Evo- WILSON. 2001. Parasites and host population dynamics. In The lution 6: 250–254. ecology of wildlife diseases, P. J. Hudson (ed.). Oxford University MOORE, J. 1984. Altered behavioural responses in intermediate hosts– Press, Oxford, U.K., p. 45–62. J. Parasitol., 93(4), 2007, pp. 755–760 ᭧ American Society of Parasitologists 2007

HELMINTH COMMUNITIES IN FIVE SPECIES OF SYMPATRIC AMPHIBIANS FROM THREE ADJACENT EPHEMERAL PONDS IN SOUTHEASTERN WISCONSIN

H. Randall Yoder* and James R. Coggins Department of Biological Sciences, University of Wisconsin–Milwaukee, Milwaukee, Wisconsin 53201. e-mail: [email protected]

ABSTRACT: Representatives of 5 amphibian species (313 individuals), including eastern American toads (Bufo americanus), wood frogs (Rana sylvatica), spring peepers (Pseudacris crucifer), blue-spotted salamanders (Ambystoma laterale), and central newts (Notophthalmus viridescens louisianensis), were collected from 3 ephemeral ponds during spring 1994, and they were inspected for helminth parasites. The component communities of anurans were more diverse than those of caudates. Infracommunities of all host species were isolationist and depauperate, due mostly to host ectothermy and low vagility. Toad infracommunities were dominated by skin-penetrating nematodes, and they had the highest values of mean total parasite abundance, mean species richness, and overall prevalence. This was likely due to their greater vagility compared with other host species. Infracommunities of wood frogs and blue-spotted salamanders had intermediate values for these measures of parasitism, whereas spring peeper and newt infracommunities had the lowest values. In addition to relative vagility, feeding habits and habitat preference were likely important in helminth community structure. Body size also seemed to play a role because mean wet weight of host species followed the same general trend as values of parasitism. However, effects of size were variable within host species and difﬁcult to separate from other aspects of host ecology.

Brandt (1936) and Rankin (1937) were the first to explore ties in amphibian hosts and suggested various aspects of host effects of amphibian habitat use on their parasite assemblages. biology as important factors in parasite community structure. Subsequently, few studies were carried out on the ecology of The current report describes helminth communities of 5 am- amphibian parasites until Kennedy et al. (1986) compiled and phibian species from southeastern Wisconsin. Collections were analyzed data on parasitic helminth communities of a taxonom- made during a single breeding season from 3 ephemeral ponds ically wide range of fish and birds. They found that fish tend in proximity. This allowed collections of fairly large numbers to harbor fewer helminth parasites and less diverse helminth of hosts that are highly cryptic during most of the year, and it communities than birds and mammals. From this, they identi- limited the effects of seasonal variation. Using ponds in prox- fied several important factors in helminth community structure, imity to each other also limited sampling to hosts migrating including complexity and/or physiology of host alimentary ca- from the surrounding areas for breeding and thus limited the nal, host vagility, host diet, and exposure to direct life cycle effects of geographical, and, to some extent, spatial variation. helminths that enter by penetration. They predicted that hel- The goal of this study was to describe and compare helminth minth communities in reptiles and amphibians should be iso- parasite communities in a wide range of amphibian hosts, with- lationist rather than interactive. in a narrow set of temporal and spatial parameters, to elucidate Several studies have been published on amphibian parasite factors important in the structure of those communities. communities in response to the work of Kennedy et al. (1986), including Goater et al. (1987), Aho (1990), Muzzall (1991a, MATERIALS AND METHODS 1991b), Barton and Richards (1996), McAlpine (1997), and Eastern American toads (Bufo americanus;nϭ 39), wood frogs McAlpine and Burt (1998). The most comprehensive of these (Rana sylvatica;nϭ 78), spring peepers (Pseudacris crucifer;nϭ 79), blue-spotted salamanders (Ambystoma laterale;nϭ 54), and central was the study of Aho (1990) who compiled and analyzed orig- newts (Notophthalmus viridescens louisianensis;nϭ 63) were collected inal and previously published data on a wide range of amphib- from 3 temporary ponds on, or adjacent to, the University of Wiscon- ian and reptile hosts. Supportive of the predictions of Kennedy sin–Milwaukee Field Station, Ozaukee County, Wisconsin (43Њ23ЈN, et al. (1986), Aho attributed depauperate, isolationist helminth 88Њ2ЈW). The ponds were 8,000 m2 (pond 1), 1,200 m2 (pond 2), and 2 communities largely to host ectothermy. Aho further concluded 1,000 m (pond 3) in area. The shortest distance between ponds 1 and 2 was 79 m. Pond 3 was 310 m from pond 1 and 372 m from pond 2. that relatively low vagility limits exposure to a variety of par- All collections were made during the 1994 spring breeding migration asites whose transmission depends on food web interactions but (6 April–26 April). Most species were collected by 30.5-m drift fences suggested that relatively sedentary host behavior could allow with pitfall traps, while migrating toward breeding ponds. Spring peep- for successful colonization by skin-penetrating nematodes. Sim- ers and some toads were collected by dip-net. Amphibians were transported to the laboratory and killed in ethyl m-aminobenzoate methane ilar to Brandt (1936) and Rankin (1937), Aho (1990) found that sulfonic acid. Snout-to-vent length (svl; centimeters) and wet weight helminth species richness and abundance varied with host hab- (grams) were recorded. Each individual was identified and frozen until itat. necropsy was performed. Amphibians were dissected and sexed by go- Other studies have concentrated on smaller groups of host nadal observation. Buccal cavity, heart, lungs, liver, gall bladder, stomach, small intestine, large intestine, kidney, urinary bladder, body cavity, species within narrow geographic ranges (Goater et al., 1987; brain, eyes, and leg muscles were inspected for helminth parasites using Muzzall, 1991a, 1991b; Barton and Richards, 1996; McAlpine, stereomicroscopy. 1997; McAlpine and Burt, 1998). Like Aho (1990), all sup- Nematodes were preserved in 70% ethanol and mounted temporarily ported the idea of depauperate, isolationist parasite communi- in glycerin for identification. Adult trematodes and larval platyhelminths were fixed in alcohol-formalin-acetic acid, stained with acetic carmine, and mounted in Canada balsam. Parasites were identified via observation and measurement of morphological characteristics based on Received 29 September 2006; revised 18 January 2007; accepted 22 descriptions from the literature. Voucher specimens have been deposited January 2007. at the H. Manter Helminth Collection, University of Nebraska–Lincoln * Present address: Department of Biology, Lamar University, P.O. Box (HWML 15249–15260). 10037, Beaumont, Texas 77710. Use of ecological terms follows Bush et al. (1997). Jaccard’s coeffi-

755 756 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 cient was used to assess similarity of helminth species composition of DISCUSSION component communities between each possible pair of host species (Krebs, 1989). Pearson’s product-moment correlation coefficients were In comparing helminth species composition at the component calculated to elucidate relationships of parasite abundance and helminth species richness with host svl. Mean abundance (for all countable par- community level, greatest similarities were seen among the asite species) and mean species richness were compared among host most closely related hosts. All 3 anurans were parasitized by species using Kruskal–Wallis 1-way analysis of variance (ANOVA). nematodes belonging to the same 3 genera. and they were gen- The same tests were used to compare wet weight among host species. erally intermediate hosts to the same group of larval worms. These tests were used in lieu of their parametric analogs because vari- Component communities of the 3 caudate hosts were very spe- ances were not homogeneous. Values for prevalence of infection (by any and all parasite species) were compared among host species using cies poor, consisting of only 2 or 3 species. Muzzall (1991a) R ϫ C contingency tables and G-tests for goodness of fit. All statistical and Bolek and Coggins (2003) found similar results, and they tests were performed according to Sokal and Rohlf (1981). attributed them at least partially to smaller size, lower vagility, and less diverse diet in caudates compared with anurans. Ad- RESULTS ditionally, this may reveal some importance of phylogenetic In total, 313 amphibians were collected between 6 April and relationships in terms of which of the parasites available in the 26 April 1994 and found to be infected with at least 14 helminth compound community are capable of infecting the various host taxa. Five nematode and 4 trematode species occurred as adults species. in these infracommunities. Unidentified encysted nematode lar- The role of host specificity was variable in these communi- vae, 3 larval trematode forms, and 1 type of metacestode cyst ties. Many of the helminth species were generalists, infecting also were found (Table I). more than 1 host. However, specialists such as R. americanus, In comparing measures of parasitism among host species, C. variabilis, and P. americanum were important within their toads had the greatest values for overall prevalence of infection, host taxa. In comparing measures of parasitism at the compo- total mean helminth abundance, and mean species richness. nent and infracommunity levels, host physiology, anatomy, and Wood frogs and blue-spotted salamanders had values that were ecology seem to be important in determining community struc- much lower than those of toads, but similar to each other. ture. Spring peepers followed the aforementioned host species and As predicted by Kennedy et al. (1986), the infracommunities newts had the lowest values (Fig. 1). of all 5 host species were isolationist and depauperate, which The R ϫ C table and G-test for goodness of fit rejected in- is also consistent with other reports of helminth communities ϭ Ͻ in amphibians. Physiology of host (ectothermy), host vagility, dependence of prevalence and host species (Gadj 59.7, P 0.001). Kruskal–Wallis 1-way ANOVA showed significant dif- host diet, and exposure to monoxenous helminths that enter by ferences in total mean helminth abundance (H ϭ 96.2, P Ͻ penetration all seem to be important in the structure of these 0.001) and species richness (H ϭ 77.2, P Ͻ 0.001) among host helminth communities. Other studies also have suggested the species. Kruskal–Wallis 1-way ANOVA also revealed signifi- importance of host size (Muzzall, 1991b; McAlpine, 1997) and cant variation in wet weight (grams) due to host species (Fig. habitat use (Brandt, 1936; Rankin, 1937; Aho, 1990; Muzzall, 1). 1991b; McAlpine, 1997), which seem to be important in these The component communities of B. americanus, R. sylvatica, communities as well. Many, if not all, of these factors tend to P. crucifer, A. laterale, and N. viridescens consisted of 7, 7, 9, interact and overlap in their influence on parasite communities 2, and 3 helminth species, respectively (Table I). Values for in ways that are difficult to distinguish. The depauperate nature Jaccard’s similarity coefficient (Table II) ranged from 11 to of these communities was, as suggested by Aho (1990), prob- 75%, with highest degree of similarity occurring between the ably due to ectothermy and low vagility, but the latter varies ϭ among host species. It is important to note that these hosts were component communities of R. sylvatica and P. crucifer (Sj 75%) and lowest degree of similarity between P. crucifer and collected during early spring breeding on their way to, or in, ϭ the ponds from surrounding areas. Therefore, the helminths pre- A. laterale (Sj 11%) as well as between P. crucifer and N. ϭ sent are limited for the most part to those that overwintered viridescens (Sj 11%). Multiple species infections were most common in B. ameri- successfully in the hosts. This probably also contributed to the canus infracommunities, with 0, 1, 2, 3, 4, and 5 species co- depauperate nature of the communities. Additionally, the occurring in 3, 11, 13, 4, 7, and 1 host, respectively. This was ephemeral nature of these ponds might affect aspects of host followed by 0, 1, 2, 3, and 4 helminth species occurring to- migration. The ponds are also free of fish, which could limit gether in 33, 21, 20, 3, and 1 host, respectively, in R. sylvatica, the number of generalist helminths available in the compound and 0, 1, and 2 species occurring in a single P. crucifer in 48, community. However, it should be noted that these ponds also 22, and 9 cases, respectively. The 2 helminth species found in are used for breeding by the tiger salamander (Ambystoma ti- A. laterale occurred together in the same host in only 4 of 31 grinum), spotted salamander (A. maculatum), chorus frog (P. infected individuals. Uninfected hosts were common and mul- triseriata), and gray tree frog (Hyla versicolor; Reinartz, 1986). tiple species infections were rare in N. viridescens, with 0, 1, The majority of helminth taxa making up helminth commu- and 2 species co-occurring in 49, 12, and 2 cases, respectively. nities from these locations exhibited heteroxenous life cycles. Pearson’s product moment correlation revealed significant, Among these were the adult trematodes whose recruitment, in positive relationships between svl and helminth species richness most species, depends upon food web interactions, which, in for B. americanus (r ϭ 0.6, P Ͻ 0.01) and R. sylvatica (r ϭ turn, are affected by vagility and size in gape-limited predators 0.4, P Ͻ 0.01) and between svl and total mean helminth abun- such as the Amphibia. This group also included larval platy- dance for B. americanus (r ϭ 0.8, P Ͻ 0.01), R. sylvatica (r ϭ helminths and larval nematodes. Although heteroxenous para- 0.3, P Ͻ 0.05), and P. crucifer (r ϭ 0.3, P Ͻ 0.05). sites were most speciose, the most abundant species were often YODER AND COGGINS—HELMINTHS OF WISCONSIN AMPHIBIA 757

TABLE I. Prevalence (%), abundance Ϯ SD (range), and location of infection for helminths parasitizing amphibian hosts (n ϭ host sample size).

Host species Pseudacris Ambystoma Notophthalmus Bufo americanus Rana sylvatica crucifer laterale viridescens Parasite taxon n ϭ 39 n ϭ 78 n ϭ 79 n ϭ 54 n ϭ 63 Location

Rhabdias americanus 85————L 4.7 Ϯ 8.7 (0–52) — — — — Rhabdias ranae —403——L — 0.8 Ϯ 1.3 (0–6) 0.03 Ϯ 0.2 (0–1) — — Oswaldocruzia pipiens 30 28 10 — — SI 2.1 Ϯ 4.6 (0–17) 0.4 Ϯ 0.8 (0–4) 0.2 Ϯ 0.9 (0–6) — — Cosmocercoides variabilis Larvae 44 — — — — L, SI 3.9 Ϯ 10.3 (0–60) — — — — Adults 36 — — — — LI 3.2 Ϯ 6.9 (0–30) — — — — Total 54 — — — — 7.1 Ϯ 14.9 (0–78) — — — — Cosmocercoides sp. Larvae — 1 — 22 11 L, SI — 0.04 Ϯ 0.3 (0–3) — 1.2 Ϯ 3.3 (0–17) 0.1 Ϯ 0.4 (0–2) Adults — 4 4 40 8 LI — 0.1 Ϯ 0.3 (0–2) 0.04 Ϯ 0.2 (0–1) 0.9 Ϯ 1.6 (0–9) 0.1 Ϯ 0.3 (0–2) Total — 4 4 50 19 — 0.1 Ϯ 0.5 (0–4) 0.04 Ϯ 0.2 (0–1) 2.0 Ϯ 3.7 (0–17) 0.2 Ϯ 0.5 (0–2) Gorgoderina bilobata 13————UB 1.7 Ϯ 7.0 (0–42) — — — — Glypthelmins pennsylvanien- sis — — 11 — — SI — — 0.1 Ϯ 0.4 (0–3) — — Haematoloechus varioplexus —41——L — 0.4 Ϯ 3.1 (0–27) 0.01 Ϯ 0.1 (0–1) — — Fibricola texensis† 13 3 13 — — LM 1.5 Ϯ 4.1 (0–15) 0.04 Ϯ 0.3 (0–2) 0.7 Ϯ 2.3 (0–14) — — Alaria mustelae†——1——LM — — 0.04 Ϯ 0.3 (0–3) — — Unidentiﬁed metacercariae† 8 5 5 — 2 K, OM, LM ‡‡‡—‡ Mesocestoides sp.† 8 12࿣ 3§ — — LM, OM ‡‡‡—— Phyllodistomum americanum — — — 15§ — UB — — — 0.2 Ϯ 0.6 (0–3) — Nematode cysts† — — — — 5 LM, OM — ———‡

* L, lungs; SI, small intestine; LI, large intestine; UB, urinary bladder; LM, leg musculature; K, kidneys; OM, organ mesentery. † Larval stages. ‡ Counts were not made. § Host record. ࿣ Host locality record (Wisconsin). monoxenous nematodes that infect by way of skin penetration tal moisture and temperature (Vogt, 1981). Unlike most am- and for which recruitment was likely affected by vagility and phibian species, categorized as ‘‘ambush predators,’’ toads the terrestrial nature of hosts. move about and actively search for food (Seale, 1987). This At the infracommunity level, toads had highest values for behavior is supported by a dry, sculptured skin possessing epi- species richness and abundance of parasitism. Thus, their par- dermal channels that draw water from the substrate by capillary asite infracommunities were the least depauperate and isola- action, as well as water-absorbing surfaces in their pelvic re- tionist. They are also highly terrestrial and the most vagile of gions. Toads also possess more highly developed lungs and the host species in this study. higher aerobic respiratory ability than most anurans (Seale, After spring breeding, adult toads leave water and forage in 1987). These adaptations limit desiccation and aid in thermo- a variety of habitats, with activity dependent upon environmen- regulation and sustained movement. This ability to move rela- 758 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

atode, found in non-bufonid anurans and caudates, is assumed in most cases to have been C. dukae. The biology of C. variabilis infecting toads from this location has been shown to be distinct from that of C. dukae. The former species is a naturally occurring parasite of bufonids and hylids, whereas the latter species has been considered primarily a parasite of terrestrial gastropods (Ogren, 1953, 1959; Anderson, 1960; McGraw, 1968), which persists in the rectum of amphibians that feed upon infected molluscs (Vanderburgh and An- derson, 1987b; Anderson, 1992). Adult males of the 2 species differ somewhat morphologically. However, values for the 2 species overlap, and sample sizes were too small in the present study to use these criteria for identification. Finally, larval Cos- mocercoides were found in the lungs of several non-bufonid hosts in the present study. This suggests that, in some cases, either C. variabilis is successfully infecting non-bufonids, at FIGURE 1. Total mean helminth abundance, mean helminth species richness, overall prevalence (%) of helminths, and mean host wet weight least up to the point of entering the lungs, or C. dukae is making for Bufo americanus, Rana sylvatica, Ambystoma laterale, Pseudacris a lung migration, and presumably infecting by skin penetration. crucifer, and Notophthalmus viridescens. Bars ϭ standard error. The parasite component community of red spotted newts was also extremely depauperate at the present location. Although adult newts in general are highly aquatic, their life histories are tively freely through the terrestrial environment, and possibly affected by environmental variation (Pfingsten and Downs, sit for long periods without being covered by moist material, 1989). If ponds dry in autumn or freeze solid during winter, brings them into contact with skin-penetrating nematodes, both of which occur at the present location, adults leave to which dominated their infracommunities. Bolek and Coggins resume a terrestrial existence until ponds refill (Morin, 1983; (2000, 2003) reached similar conclusions regarding parasite communities of Wisconsin toads. Pfingsten and Downs, 1989), or thaw. Gill (1978) reported pop- The component communities of wood frogs and spring peep- ulations that underwent an autumn migration to the terrestrial ers were the most similar to each other in terms of species environment and spring migration back to ponds. This seems composition. However, levels of parasitism at the infracom- to be the scenario at the present location. The absence of larval munity level tended to be much lower in spring peepers. Neither and adult trematodes reported from other studies (Muzzall, of these species possesses the antidesiccation or respiratory ad- 1991a) and the presence of Cosmocercoides sp. point to a more aptations of toads. Adult wood frogs are terrestrial ambush terrestrial existence of this host in the current study. Addition- predators (Dickerson, 1906; Vogt, 1981), and they are less vag- ally, in unstable environments, the terrestrial eft stage is omitted ile than toads, presumably limiting exposure to skin-penetrating from the life history (Pfingsten and Downs, 1989), which is nematodes. Spring peepers were the most arboreal host species thought to be the case in Wisconsin (Vogt, 1981). Therefore, in this study (Vogt, 1981), contributing to the low prevalence parasites that might be recruited during that life history stage and abundance of skin-penetrating nematodes relative to other in other locations would not have been encountered. Muzzall anurans. Reports on parasites of wood frogs are summarized in et al. (2003) reported a similar situation in Michigan. McAllister et al. (1995) and Yoder and Coggins (1996). Aho (1990) found composition of helminth infracommunities The species-poor component community observed in blue- to be more similar among newts from the same location than spotted salamanders from this location is in line with existing between populations, and more similar among permanent or reports (Coggins and Sajdak, 1982; Muzzall and Schinderle, ephemeral ponds (lower species richness in ephemeral ponds) 1992; Bolek, 1997). Blue-spotted salamanders are the most fos- than among all ponds combined. From this, Aho concluded that sorial, and probably the least vagile, hosts represented in this ‘‘ecological factors influencing variation in life history traits in study. They are residents of the forest floor, and the occurrence local populations potentially play a major role in regulating of Cosmocercoides sp. is probably a function of terrestrially helminth community structure.’’ Muzzall (1991a, 1991b) and either through contact with soil, or a diet consisting largely of Muzzall et al. (2001, 2003) described helminth communities terrestrial slugs (Gilhen, 1974; Bolek, 1997), or both. This nem- from newts and anurans from 2 locations in Michigan and con-

TABLE II. Jaccard’s similarity coefﬁcient (%) comparing helminth component communities between pairs of host species.

Notophthalmus Bufo americanus Rana sylvatica Pseudacris crucifer Ambystoma laterale viridescens

B. americanus — R. sylvatica 33 — P. crucifer 27 75 — A. laterale 01411— N. viridescens 0141133— YODER AND COGGINS—HELMINTHS OF WISCONSIN AMPHIBIA 759 cluded (Muzzall et al., 2003) that ‘‘. . . newts are not suitable ACKNOWLEDGMENTS hosts for many helminth species as frogs are and newts are Collections were made under permits issued by the Wisconsin De- infected less commonly than frogs ....’’ They attributed this partment of Natural Resources and with permission of a private land- to ecological factors, including greater vagility, gape size, and owner. We thank Thomas Slawski for assistance in design and assembly surface area in anurans as well as ecological and physiological of drift fences and Matt Bolek for many discussions of the topic. Fi- host specificity. They also suggested, like Aho (1990), that local nally, we thank the committee and staff of the University of Wisconsin– Milwaukee Field Station for logistical support of the project. ecological factors are important in determining helminth community structure in newts at a particular site. In the current study, the ephemeral nature of the ponds, and, possibly, the LITERATURE CITED absence of fish seem to be important factors. Migrations of AHO, J. M. 1990. Helminth communities of amphibians and reptiles: newts are due to changes in the physical habitat as well as Comparative approaches to understanding patterns and processes. breeding ecology, which limits interaction of newts with infec- In Parasite communities: Patterns and processes, G. Esch, A. Bush, tive stages of both aquatic and terrestrial parasites and contrib- and J. Aho (eds.). Chapman and Hall, New York, New York, p. 157–196. utes to extremely depauperate helminth communities. ANDERSON, R. C. 1960. On the development and transmission of Cos- Host body size also seems to have been important in struc- mocercoides dukae of terrestrial mollusks in Ontario. Canadian turing helminth communities in these hosts. In making com- Journal of Zoology 38: 801–825. parisons among host species, there was a general trend in which ———. 1992. Nematode parasites of vertebrates. CAB International, Oxon, U.K., 578 p. species with larger body size had higher values of parasitism. BAKER, M. R. 1978. Development and transmission of Oswaldocruzia Within host species, relationships between svl and abundance, pipiens Walton, 1929 (Nematoda: Trichostrongylidae) in amphibi- or species richness, varied with positive correlations detected ans. Canadian Journal of Zoology 56: 1026–1031. only in the anurans, and they were strongest in toad infracom- ———. 1979. Seasonal population changes in Rhabdias ranae Walton, 1929 (Nematoda: Rhabdiasidae) in Rana sylvatica of Ontario. Ca- munities. Increasing parasite species richness and helminth nadian Journal of Zoology 57: 179–183. abundance with increasing size of anuran hosts has been ob- BARTON,D.P.,AND S. J. RICHARDS. 1996. Helminth infracommunities served by other researchers (Muzzall, 1991b; McAlpine, 1997). in Litoria genimaculata (Amphibia: Anura) from Birthday Creek, Muzzall (1991b) suggested that this had to do with a larger an upland rainforest stream in northern Queensland, Australia. In- number and wider range of intermediate hosts eaten by older ternational Journal for Parasitology 26: 1381–1385. BOLEK, M. G. 1997. Seasonal occurrence of Cosmocercoides dukae and (larger) individuals, a longer period to become infected, and prey analysis in the blue-spotted salamander, Ambystoma laterale, greater surface area for colonization of parasites. The first ex- in southeastern Wisconsin. Journal of the Helminthological Society planation is relevant to adult trematodes whose recruitment of- of Washington 64: 292–295. ten depends upon food web interactions. For example, in the ———, AND J. R. COGGINS. 2000. Seasonal occurrence and community structure of helminth parasites from eastern American toad, Bufo current study, small gape size limits odonates in the diet of americanus americanus, from southeastern Wisconsin. Compara- spring peepers, resulting in lower prevalence and abundance of tive Parasitology 67: 202–209. H. varioplexus compared with wood frogs. Muzzall’s (1991b) ———, AND———. 2003. Helminth community structure of sympatric latter explanations are most applicable to infection by skin-pen- eastern American toad, Bufo americanus americanus, northern leopard frog, Rana pipiens, and blue-spotted salamander, Ambys- etrating nematodes. Importance of exposure time would depend toma laterale, from southeastern Wisconsin. Journal of Parasitol- on longevity of worms in their hosts, which is unknown at the ogy 89: 673–680. present location. However, these nematodes are known to be BRANDT, B. B. 1936. Parasites of certain North Carolina Salientia. Eco- capable of overwintering in anuran hosts (Baker, 1978, 1979; logical Monographs 6: 491–532. Vanderburgh and Anderson, 1987a). McAlpine (1997) wrote BUSH,A.O.,K.D.LAFFERTY,J.M.LOTZ, AND A. W. SHOSTAK. 1997. Parasitology meets ecology on its own terms: Margolis et al. re- ‘‘. . . the ecology of the host (diet and habitat in particular) and visited. Journal of Parasitology 83: 575–583. parasite (transmission dynamics) confounds any simple rela- COGGINS,J.R.,AND R. A. SAJDAK. 1982. A survey of helminth parasites tionship between the diversity of helminth communities and in the salamanders and certain anurans from Wisconsin. Proceed- size ....’’This statement can be readily applied to the present ings of the Helminthological Society of Washington 49: 99–102. DICKERSON, M. C. 1906. The frog book. Dover Publications, Inc., New study as well. York, New York, 253 p. In conclusion, anurans in this study hosted richer helminth GILHEN, J. 1974. Distribution, natural history, and morphology of the communities than caudates. Parasite communities in all hosts blue-spotted salamanders, Ambystoma laterale and A. tremblayi in were depauperate and isolationist in nature. This was likely due Nova Scotia. Nova Scotia Museum Curatorial Report 22: 1–38. GILL, D. E. 1978. Effective population size and interdemic migration largely to ectothermy and is supportive of the hypotheses of rates in a metapopulation of the red-spotted newt, Notophthalmus Kennedy et al. (1986) and the conclusions of several other re- viridescens (Rafinesque). Evolution 32: 839–849. searchers. Although infracommunities of toads were most often GOATER, T. M., G. W. ESCH, AND A. O. BUSH. 1987. Helminth parasites dominated by skin-penetrating nematodes, parasite abundance of sympatric salamanders: Ecological concepts at the infracom- in all other hosts was so low as to call into question any use munity, component, and compound community levels. American Midland Naturalist 118: 289–299. of the term dominance. Feeding habits, in terms of gape size, KENNEDY,C.R.,A.O.BUSH, AND J. M. AHO. 1986. Patterns in helminth range of prey species and foraging behavior, relative vagility, communities: Why are birds and fish different? Parasitology 93: and habitat preference were all aspects of host biology identi- 205–215. fied as important in the structure of parasitic helminth com- KREBS, C. J. 1989. Ecological methodology. Harper & Rowe Publishers, New York, New York, 654 p. munities at this location. Host size was also important in the MCALLISTER, C. T., S. J. UPTON,S.E.TRAUTH, AND C. R. BURSEY. 1995. structure of helminth communities, but its effects were difficult Parasites of wood frogs, Rana sylvatica (Ranidae), from Arkansas, to distinguish from other aspects of host and parasite biology. with a description of a new species of Eimeria (Apicomplexa: Ei- 760 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

meriidae). Journal of the Helminthological Society of Washington OGREN, R. E. 1953. A contribution to the life cycle of Cosmocercoides 62: 143–149. in snails (Nematoda: Cosmorcecidae). Transactions of the Ameri- MCALPINE, D. F. 1997. Helminth communities in bullfrogs (Rana ca- can Microscopical Society 72: 87–91. tesbeiana), green frogs (Rana clamitans), and leopard frogs (Rana ———. 1959. The nematode Cosmocercoides dukae as a parasite of pipiens) from New Brunswick, Canada. Canadian Journal of Zo- the slug. Proceedings of the Pennsylvania Academy of Science 33: ology 75: 1883–1890. 236–241. ———, AND M. D. B. BURT. 1998. Helminths of bullfrogs, Rana ca- PHINGSTEN,R.A.,AND F. L. DOWNS. 1989. Salamanders of Ohio. Col- tesbeiana, green frogs, R. clamitans, and leopard frogs, R. pipiens lege of Biological Sciences, The Ohio State University, Columbus, in New Brunswick. Canadian Field-Naturalist 112: 50–68. Ohio, 315 p. MCGRAW, J. C. 1968. A study of Cosmocercoides dukae (Holl, 1928) RANKIN, J. S. 1937. An ecological study of parasites of some North Wilkie, 1930 (Nematoda: Cosmoceridae) from amphibians in Ohio. Carolina salamanders. Ecological Monographs 7: 169–269. Ph.D. Dissertation. Ohio State University, Columbus, Ohio, 98 p. REINARTZ, J. A. 1986. A guide to the natural history of the Cedarburg MORIN, P. J. 1983. Comparative and predatory interactions in natural Bog part II. The University of Wisconsin–Milwaukee Field Station and experimental populations of Notophthalmus viridescens dor- Bulletin 19: 1–53. salis and Ambystoma tigrinum. Copeia 1983: 628–639. SEALE, D. B. 1987. Amphibia. In Animal energetics, vol. 2, T. J. Pandian MUZZALL, P. M. 1991a. Helminth communities of the newt, Notoph- and F. J. Vanberg (eds.). Academic Press, San Diego, California, p. thalmus viridescens, from Turkey Marsh, Michigan. Journal of Par- 467–552. asitology 77: 87–91. SOKAL, R. R., AND F. J. ROHLF. 1981. Biometry, 2nd ed. W. H. Freeman ———. 1991b. Helminth infracommunities of the frogs Rana cates- and Co., New York, New York, 859 p. beiana and Rana clamitans from Turkey Marsh, Michigan. Journal VANDERBURGH, D. J., AND R. C. ANDERSON. 1987a. Preliminary observations on seasonal changes in prevalence and intensity of Cos- of Parasitology 77: 366–371. mocercoides variabilis (Nematoda: Cosmocercoidea) in Bufo amer- ———, AND D. B. SCHINDERLE. 1992. Helminths of the salamanders icanus (Amphibia). Canadian Journal of Zoology 65: 1666–1667. Ambystoma t. tigrinum and Ambystoma laterale (Caudata: Ambys- ———, AND———. 1987b. The relationship between nematodes of the tomatidae) from southern Michigan. Journal of the Helmintholog- genus Cosmocercoides Wilkie, 1930 (Nematoda: Cosmocercoidea) ical Society of Washington 59: 201–205. in toads (Bufo americanus) and slugs (Deroceras laeve). Canadian ———, M. G. GILLILLAND III, C. S. SUMMER, AND C. J. MEHNE. 2001. Journal of Zoology 65: 1650–1661. Helminth communities of green frogs Rana clamitans Latreille, VOGT, R. C. 1981. Natural history of amphibians and reptiles of Wis- from Turkey Marsh, Michigan. Journal of Parasitology 87: 962– consin. The Milwaukee Public Museum and Friends of the Muse- 968. um, Inc., Milwaukee, Wisconsin, 205 p. ———, J. D. PETERSON, AND M. G. GILLILLAND III. 2003. Helminths YODER,H.R.,AND J. R. COGGINS. 1996. Helminth communities in the of Notophthalmus viridescens (Caudata: Salamandridae) from northern spring peeper, Pseudacris c. crucifer Wied, and the wood 118th Pond, Michigan, U.S.A. Comparative Parasitology 70: 214– frog, Rana sylvatica Le Conte, from southeastern Wisconsin. Jour- 217. nal of the Helminthological Society of Washington 63: 211–214. J. Parasitol., 93(4), 2007, pp. 761–771 ᭧ American Society of Parasitologists 2007

NEW AND PREVIOUSLY DESCRIBED SPECIES OF DACTYLOGYRIDAE (MONOGENOIDEA) FROM THE GILLS OF PANAMANIAN FRESHWATER FISHES (TELEOSTEI)

Edgar F. Mendoza-Franco*, M. Leopoldina Aguirre-Macedo, and Vıćtor M. Vidal-Martıńez Laboratory of Parasitology, Centro de Investigacioń y de Estudios Avanzados (CINVESTAV, Unidad Me´rida), Carretera Antigua a Progreso Km. 6, Apartado Postal 73 ‘‘Cordemex,’’ C.P. 97310 Me´rida, Yucatań, Mexico. e-mail: [email protected]

ABSTRACT: During an investigation of the diversity of metazoan parasites of 7 freshwater fish species from 3 localities in central Panama, the following gill dactylogyrid (Monogenoidea) species were found: Aphanoblastella chagresii n. sp. from Pimelodella chagresi (Heptapteridae); Aphanoblastella travassosi (Price, 1938) Kritsky, Mendoza-Franco, and Scholz, 2000 from Rhamdia quelen (Heptapteridae); Diaphorocleidus petrosusi n. sp. from Brycon petrosus (Characidae); Gussevia asota Kritsky, Thatcher, and Boeger, 1989, from Astronotus ocellatus (Cichlidae); Sciadicleithrum panamensis n. sp. from Aequidens coeruleopunctatus (Cichlidae); Urocleidoides flegomai n. sp. from Piabucina panamensis (Lebiasinidae); and Urocleidoides similuncus n. sp. from Poecilia gillii (Poeciliidae). Consideration of the comparative morphology and distribution of these parasites along with the evolutionary history of the host fishes suggests that diversification may be associated with geotectonic events that provided isolation of the Central American fauna with the uplift of the Panamanian Isthmus during early Pliocene (3 mya).

Central America comprises a tectonically dynamic part of the surements were obtained from unflattened specimens fixed in hot (ഠ90 ഠ world that has interested biogeographers for many years (Ro- C) or ambient temperature ( 30 C) formalin (4%), stained in Gomori trichrome and mounted in Canada balsam. Drawings were made with sen, 1976; Gayet et al., 1992; Iturralde and MacPhee, 1999). the aid of a drawing tube using an Olympus microscope with Nomarski This diverse region has served as a corridor for organismic interference contrast. Measurements, all in micrometers, represent dispersal from South America upwards (and vice versa), and straight-line distances between extreme points and are expressed as the much attention has been focused on the Isthmus of Panama as mean followed by the range and number (n) of structures measured in parentheses; body length includes that of the haptor. Numbering of hook a barrier to dispersal of marine organisms (Bermingham and pairs follows the scheme illustrated in Mendoza-Franco, Violante-Gon- Martin, 1998; Perdices et al., 2002). Within this scenario, za´lez, and Vidal-Martıńez (2006). Type and voucher specimens are de- knowledge of the diversity of the parasite fauna of fishes is still posited in the United States National Parasite Collection, Beltsville, limited, as is the case for the monogenoidean parasites (Scholz Maryland (USNPC); National Helminthological Collection of Mexico et al., 1999; Aguirre-Macedo et al., 2001; Vidal-Martinez et al., (CNHE), Institute of Biology, National Autonomous University of Mex- ico, Mexico; and the helminthological collection of the Institute of Par- 2001; Choudhury et al., 2002; Mendoza-Franco et al., 2003; asitology, Cˇ eske´ Budeˇjovice, Czech Republic (IPCAS), as indicated in Aguirre-Macedo and Scholz, 2005). Although species in this the respective descriptions. Host names follow those in the Food and group of flatworms have already been extensively studied in Agriculture Organization Fish Base (http://www.fishbase.org). fish from South America (Kritsky et al., 1986; Boeger and Krit- sky, 1988; Kritsky et al., 1989, 1992; Boeger et al., 1994; Krit- DESCRIPTION sky et al., 1996, 1997; Agarwal and Kritsky, 1998; Kohn and Aphanoblastella chagresii n. sp. Cohen, 1998; Kritsky and Gutie´rrez, 1998; Boeger and Kritsky, (Figs. 1–8) 2003), these same species are largely unstudied in Central Diagnosis: Body fusiform, greatest width near posterior trunk. Ce- America. Monogenoidean species were found during a study on phalic margin narrow; cephalic lobes poorly to moderately developed; metazoan parasites carried out in April 2006 in freshwater en- 3 bilateral pairs of head organs; cephalic glands indistinct. Eyes 4, pos- vironments from several localities in the surroundings of Sob- terior pair larger, closer together than anterior pair; accessory granules eranıá National Park in central Panama. In the present study, 7 usually uncommon in cephalic region. Pharynx subspherical; esophagus dactylogyrid species (5 new) are described and/or reported from moderately long. Peduncle broad; haptor subhexagonal. Ventral anchor with well-developed roots, straight shaft, elongated curved point. Dorsal the gills of 7 fish species, and a possible explanation of the anchor with short deep root, protruding superficial root, straight shaft origin and speciation of these parasites in Central America moderately elongated, elongate curved point. Ventral bar, delicate, based on their geographical distribution is provided. broadly V-shaped, with a poorly developed posteromedial protuberance; dorsal bar broadly V-shaped, bulbous ends directed laterally. Hooks MATERIALS AND METHODS similar, each with, protruding thumb, delicate shaft and point, fine shank; hook pair 1, reduced in size; FH loop about 40% shank length Fish were collected using beach seines, hook and line, and trammel (pairs 2–7), 50% shank length (pair 1). Male copulatory organ (MCO) nets from Rio Frijolito (09Њ09Ј53ЉN, 79Њ45Ј16ЉW), Lago Gatun a coil of ϳ2.5 counterclockwise rings, base of male copulatory organ (09Њ06Ј871ЉN, 79Њ41Ј721ЉW), and Lago Alajuela (09Њ15Ј00ЉN, with small sclerotized plate. Accessory piece, comprising variable 79Њ34Ј59.88ЉW) in the Chagres River Basin in central Panama. Live sheath along distal shaft of copulatory organ. Testis subspherical; sem- fish were brought to the laboratory, killed by pithing the brain, and inal vesicle as dilation of vas deferens, indistinct, fusiform; one prostatic examined for monogenoids. Worms were removed from gills, examined, reservoir. Germarium subspherical to ovate, comprising comparatively and measured as temporary or permanent mounts fixed with ammonium few cells, slightly overlapping gonads; oviduct, ootype not observed; picrate (Ergens, 1969). Additional specimens were mounted unstained uterus delicate. Vaginal aperture sinistroventral, simple; vaginal canal in glycerin jelly for study of the sclerotized structures. All other mea- tubular, straight to slightly slanted posteriorly into pyriform seminal receptacle. Measurements of 21 specimens studied from this host provided in Table I. Received 25 September 2006; revised 22 February 2007; accepted 22 February 2007. Taxonomic summary * Current address: Smithsonian Tropical Research Institute, Naos Island Laboratories, Apartado Postal 0843-03092 Balboa, Ancon, Panama Type host: Catfish Pimelodella chagresi (Steindachner, 1877) (Sil- City, Republic of Panama. uriformes: Heptapteridae).

761 762 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 1–8. Aphanoblastella chagresii n. sp. (1) Whole mount (composite, ventral view). (2) Ventral bar. (3) Hook (pair 1). (4) Dorsal bar. (5) Hook (pairs 2–7). (6) Copulatory complex (dorsal view). (7) Ventral anchor. (8) Dorsal anchor. All ﬁgures are drawn to the 20-␮m scale, except 1 (100-␮m). MENDOZA-FRANCO ET AL.—DACTYLOGYRIDS FROM PANAMA 763

TABLE I. Measurements of species of Aphanoblastella from species of Pimelodella and Rhamdia (Siluriformes) in the tropics.*

A. chagresii† A. travassosi‡ A. travassosi§ A. travassosi࿣ A. travassosi† P. chagresi P. laticeps R. guatemalensis R. quelen R. quelen

Body length 236 (175–350; n ϭ 16) 295 (180–375) 282 (204–364; n ϭ 34) 390 (321–472) 364 (295–432; n ϭ 11) Greatest width 63 (42–82; n ϭ 12) 85 (65–100) 104 (77–127; n ϭ 32) 85 (75–91) 95 (80–112; n ϭ 11) Pharynx width 19 (14–22; n ϭ 15) 20 (15–25) 28 (21–33; n ϭ 23) 26 (24–28) 24 (21–29; n ϭ10) Haptor width 50 (40–60; n ϭ 8) — 55 (45–63; n ϭ 31) 55 (42–61) 56 (51–74; n ϭ 9) Ventral anchor length 20 (19–20; n ϭ 28) 18 (15–20) 22 (21–24; n ϭ 13) 20 (19–21) 26 (24–29; n ϭ 27) Ventral anchor width 10 (9–15; n ϭ 11) — 16 (14–17; n ϭ 11) 14 (13–14) 15 (14–17; n ϭ 8) Dorsal anchor length 16 (16–17; n ϭ 21) 15 (14–20) 24 (21–27; n ϭ 11) 17 (16–17) 27 (25–30; n ϭ 20) Dorsal anchor width 8 (8–10; n ϭ 13) — 16 (14–18; n ϭ 12) 13 (12–14) 16 (14–18; n ϭ 7) Ventral bar length 23 (20–28; n ϭ 18) 29 (23–40) 32 (29–37; n ϭ 10) 30 (29–31) 30 (26–33; n ϭ 17) Dorsal bar length 24 (20–28; n ϭ 11) 30 (25–42) 37 (31–44; n ϭ 9) 23 (22–25) 35 (31–39; n ϭ 9) Hooks pairs 1, 3–7 14 (13–14; n ϭ 21) 15 (10–17) 13 (12–14; n ϭ 23) 9 (8–10) 12 (11–13; n ϭ 20) Hooks pair 2 10 (10–11; n ϭ 7) — — — — MCO# length 16 (15–17; n ϭ 5) 85 (70–110) 41 (38–45; n ϭ 5) — Accessory piece 18 (15–22; n ϭ 14) 30 (25–35) 31 (28–36; n ϭ 4) 29 (24–32) 32 (32–33; n ϭ 3) Germarium length 24 (19–28; n ϭ 8) — 28 (20–44; n ϭ 23) 390 (321–472) 30 (22–37; n ϭ 8) Germarium width 25 (20–44; n ϭ 9) — 22 (18–25; n ϭ 23) — 21 (11–30; n ϭ 8) Testis length 29 (28–31; n ϭ 7) — 51 (40–59; n ϭ 19) — 58 (50–66; n ϭ 6) Testis width 27 (20–40; n ϭ 7) — 35 (25–46; n ϭ 18) — 28 (24–37; n ϭ 5)

* Measurements (in ␮m) are mean, with range in parentheses; n ϭ number of measurements. † Present study. ‡ Measurements of A. travassosi from Argentina (Suriano, 1986). § Measurements of A. travassosi from southeast Mexico (Kritsky et al., 2000). ࿣ Measurements of A. travassosi from Trinidad (Molnar et al., 1974). # Male copulatory organ.

Site of infection: Gills. same geographic range of their hosts, Pimelodella spp., and both are Type locality/collection date: Rio Frijolito (09Њ09Ј53ЉN, relatively similar in the size of their ventral (length 19–20 vs. 15–20 in 79Њ45Ј16ЉW), Republic of Panama, April 2006. A. travassosi from P. laticeps) and dorsal (length 16–17 vs. 14–20) Specimens deposited: Holotype (CNHE 5784); 10 paratypes (CNHE anchors (see Table I) and in the morphology of the accessory piece 5786), 5 paratypes (IPCAS M-431), and 5 paratypes (USNPC 99625). (compare figures 6 [present study] and 15 from the original redescrip- Etymology: The specific name is derived from its host. tion of A. travassosi in Suriano, 1986). Further, the hook measurements (length 10–17) provided by Suriano for A. travassosi fit into the ranges Remarks (length 10–14) of A. chagresii n. sp. All above mentioned suggests that A. travassosi from P. laticeps may be a synonym of A. chagresii n. sp., Aphanoblastella was erected by Kritsky et al. (2000) to accommodate in other words, the same parasite species (A. chagresii n. sp.) on Pi- dactylogyrid species possessing tandem gonads, a coiled MCO with melodella spp. If so, then a split between Pimelodella and Rhamdia counterclockwise rings, unmodified anchors, a ventral bar with postero- resulted in speciation of A. chagresii n. sp. and A. travassosi, respec- medial projection, a nonarticulated MCO, and accessory piece simple tively (see phylogeny of Rhamdia in Perdices et al., 2002). Confirmation and similar hooks with undilated shanks. Since then, 3 species of of the synonymy of A. travassosi from P. laticeps in Argentina with Aphanoblastella have been recognized from the gills of neotropical cat- respect to A. chagresii n. sp. from P. chagresi in Panama will require fish species of Rhamdia (Siluriformes). These include: Aphanoblastella further study of new specimens collected from P. laticeps and/or mo- travassosi (Price, 1938) Kritsky, Mendoza-Franco, and Scholz, 2000 lecular research. (type species) from Rhamdia rogersi (Regan, 1907), R. sebae (Valen- ciennes, 1840), and R. quelen (Quoy and Gaimard, 1824) (ϭ R. gua- Aphanoblastella travassosi (Price, 1938) temalensis in Perdices et al., 2002) in Costa Rica and Trinidad, respec- Kritsky, Mendoza-Franco and Scholz, 2000 tively, and from R. guatemalensis (Gu¨nther, 1864) in Mexico; from Pimelodella laticeps Eigenmann, 1917 in Argentina; A. robustus (Miz- Diagnosis: Comparative measurements by host presented in Table I. elle and Kritsky, 1969) Kritsky, Mendoza-Franco and Scholz, 2000 from Rhamdia sp. in the Amazon River of Brazil, and A. mastigatus Taxonomic summary (Suriano, 1986) Kritsky, Mendoza-Franco and Scholz, 2000 from R. Host: Silver catfish, Rhamdia quelen (Quoy and Gaimard, 1824) (Sil- sapo (Valenciennes, 1840) in Argentina (see Molna´r et al., 1974; Sur- uriformes: Heptapteridae). iano, 1986; Kritsky et al., 2000). Based on comparative haptor mor- Site of infection: Gills. phology, A. chagresii n. sp. most closely resembles the type species, A. Locality/collection date: Lago Alajuela (09Њ15Ј00ЉN, 79Њ34Ј59.88ЉW), travassosi (as redescribed on the basis of specimens found in R. gua- Republic of Panama, April 2006. temalensis [Kritsky et al., 2000]). Aphanoblastella chagresii n. sp. dif- Specimens deposited: 3 reference specimens (CNHE 5786), 5 in fers from A. travassosi by having hooks of different size and an ex- IPCAS (M-353), and 5 in USNPC (99626). panded accessory piece (rod-shaped in A. travassosi) and by lacking a posteromedial process on the ventral bar. Additionally, it differs in its Remarks shorter ventral (length 19–20 vs. 21–24 in A. travassosi) and dorsal (length 16–17 vs. 21–27) anchors and accessory piece (length 15–22 The specimens fit the diagnosis of A. travassosi, the type species of vs. 28–36) (see Table I). It is noteworthy that A. chagresii n. sp. from the genus, which was redescribed by Kritsky et al. (2000) on the basis P. chagresi appears to be morphometrically similar to A. travassosi of specimens found in R. guatemalensis from cenotes (ϭ sinkholes) in reported from P. laticeps in Argentina (Suriano, 1986; Kritsky et al., southeastern Mexico. Comparison of the present material with a vouch- 2000). Aphanoblastella chagresii n. sp. and A. travassosi occur on the er of A. travassosi from R. guatemalensis from the Parasitology Labo- 764 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 ratory, CINVESTAV, Merida, Mexico (CHCM 314), did not reveal any 2003), but none of these parasite species show similarity with Dia- differences. The present finding of A. travassosi on R. quelen in Panama phorocleidus petrosusi n. sp. is a new geographical record. Gussevia asota Kritsky, Thatcher, and Boeger, 1989 Diaphorocleidus petrosusi n. sp. ϭ (Figs. 9–15) Diagnosis: Ventral anchor 25–26 long. Dorsal anchor 26 (n 2) long; base 10–11 wide. Ventral bar 30 long; dorsal bar 28 long. Hook Diagnosis: Body 298 (242–337; n ϭ 8) long, fusiform; greatest width 10–11 long. Proximal ring diameter of the MCO 16 long. Accessory 68 (57–75; n ϭ 8) near midlength. Cephalic margin broad; cephalic piece 31 long. lobes moderately developed; 3 bilateral pairs of head organs; cephalic glands indistinct. Eyes 4; members of posterior pair with conspicuous Taxonomic summary lens, larger, closer together than members of anterior pair; eye granule variable in size, usually elongate ovate; accessory granules in cephalic, Host: Red oscar, Astronotus ocellatus (Agassiz, 1831) (Perciformes: anterior trunk regions. Pharynx spherical 17 (15–18; n ϭ 7) diameter; Cichlidae). esophagus short. Peduncle broad; haptor subhexagonal 66 (58–73; n ϭ Site of infection: Gills. 7) wide. Anchors similar; each with poorly defined deep root, elongate Locality/collection date: Lago Gatun (09Њ06Ј871ЉN, 79Њ41Ј721ЉW), superficial root, straight shaft, point short; ventral anchor 32 (30–34; n Republic of Panama, April 2006. ϭ 26) long, base 18 (16–20; n ϭ 17) wide; dorsal anchor 22 (21–23; Specimens deposited: 1 reference specimen (CNHE 5789). n ϭ 15) long, base 12 (11–13; n ϭ 11) wide. Ventral bar 24 (22–26; n ϭ 15) long, straight to broadly U-shaped, ends enlarged, usually with Remarks anteromedial indentation; dorsal bar 20 (22–26; n ϭ 15) long, broadly U-shaped. Hook 17 (15–18; n ϭ 32) long, with 2 subunits; FH loop Measurements and the morphology of the sclerotized structures of about 30% shank length. Copulatory organ a coil with about one and the present specimen do not differ significantly from that figured in the half counterclockwise rings, coil diameter of the first ring 16 (13–20; n original description. Gussevia asota is 1 of 3 originally described spe- ϭ 12). Accessory piece 18 (16–20; n ϭ 3) long, comprising a pincer cies (G. astronoti Kritsky, Thatcher, and Boeger, 1989 and G. rogersi shape distally that appears as 2 supporting processes. Gonads overlap- Kritsky, Thatcher, and Boeger, 1989) on native A. ocellatus in South ping; testis elongate, fusiform, dorsoposterior to germarium, 12 (10–15; America (Brazil) and also reported from the same host species held in n ϭ 3) long, 6 (5–8; n ϭ 3) wide, ovate; seminal receptacle as expansion an aquarium in the United States (Kritsky et al., 1989). The finding of of vas deferens, sigmoid, pyriform; one prostatic reservoir. Germarium a single specimen of G. asota suggests that A. ocellatus has lost their slightly oval, elongated, 44 (32–67; n ϭ 6) long, 16 (13–20; n ϭ 7) original monogenoids since its colonization to Panama from South wide; oviduct, ootype not observed; submarginal sinistral vaginal ap- America. Astronotus ocellatus from Lago Gatun in Panama had only 2 erture, a nondilated sclerotized tube into large medial seminal receptacle specimens (one measured) on 6 fish. Thus, probably seasonality, i.e., lying anterior to germarium; vitellaria limited in trunk, absent in regions low infection prevalences, could help to explain the absence of these of reproductive organs. South American monogenoids on A. ocellatus in Panama.

Taxonomic summary Sciadicleithrum panamensis n. sp. (Figs. 16–22) Type host: Sa´balo pipon Brycon petrosus Meek and Hildebrand, 1913 (Characiformes, Characidae). Diagnosis: Body fusiform; greatest width near midlength. Cephalic Site of infection: Gills. margin broad; cephalic lobes moderately developed; 3 bilateral pairs of Type locality/collection date: Rio Frijolito (09Њ09Ј53ЉN, head organs; cephalic glands indistinct. Eyes 4; members of posterior 79Њ45Ј16ЉW), Republic of Panama, April 2006. pair closer together than members of anterior pair; eye granule usually Specimens deposited: Holotype (CNHE 5787); 3 paratypes (CNHE elongate ovate. Pharynx spherical; esophagus short to moderately long. 5788), 4 paratypes (IPCAS M-432), and 3 paratypes (USNPC 99627). Peduncle broad; haptor subhexagonal. Ventral anchor 25 (23–27; n ϭ Etymology: This species is derived from the specific name of its host. 15) long, with slightly appressed roots, evenly curved shaft, short point; base 11 (11–13; n ϭ 11) wide. Dorsal anchor 23 (22–27; n ϭ 13) long, Remarks with slightly depressed superficial root, poorly differentiated deep root, evenly curved shaft, short point; base 9 (8–9; n ϭ 8) wide. Ventral bar Placement of this new species in Diaphorocleidus is based on the 26 (23–28; n ϭ 9) long, broadly V-shaped, with small enlarged ends; generic diagnosis provided by Jogunoori et al. (2004), i.e., species with dorsal bar 23 (22–27; n ϭ 8) long, yoke-shaped, with slightly enlarged overlapping gonads, a coiled copulatory organ with counterclockwise ends, prominent anteromedial expansion. Hooks similar, each 12 (11– rings, submarginal sinistral vaginal aperture, and hook shank with 2 13; n ϭ 34) long, with upright thumb, delicate point, shank; FH loop subunits. The morphology of the features of the haptor and the copu- about 80% shank length. Gonads overlapping; testis ovate, dorsopos- latory complex distinguish Diaphorocleidus petrosusi n. sp. from the terior to germarium; seminal vesicle as expansion of vas deferens, fu- other 4 species of the genus that occur on fish of the Characidae (Dia- siform; one prostatic reservoir, fusiform. MCO a coiled tube comprising phorocleidus affinis (Mizelle, Kritsky, and Crane, 1968) Jogunoori, 1.5 rings, base with slight flange. Accessory piece with broad basal Kritsky, and Venkatanarasaiah, 2004, from Bryconops affinis (Gunther, portion terminating in diagonal opening, distal portion slightly pointed, 1864); Diaphorocleidus armillatus Jogunoori, Kritsky, and Venkatan- lying within second shaft of the copulatory organ. Germarium with arasaiah, 2004, (type species) from Gymnocorymbus ternetzi (Boulen- irregular margin; oviduct, ootype, uterus not observed. Vagina dextro- ger, 1895); Diaphorocleidus kabatai (Molna´r, Hanek, and Fernando, ventral, a slight sclerotized tube opening into small medial seminal re- 1974), Jogunoori, Kritsky and Venkatanarasaiah, 2004, from Astyanax ceptacle; vitellaria dense throughout trunk, except absent in regions of bimaculatus (Linnaeus, 1758), and Diaphorocleidus microstomus (Miz- reproductive organs. elle, Kritsky and Crane, 1968), Jogunoori, Kritsky and Venkatanara- saiah, 2004, from Hemigrammus microstomus Durbin, 1918) (Jogunoori Taxonomic summary et al., 2004). Diaphorocleidus petrosusi n. sp. most closely resembles D. armillatus from which it differs by having a distally pincer-shaped Type host: Chogorro Aequidens coeruleopunctatus (Kner, 1863) (Per- accessory piece (tortuous in D. armillatus), and by the comparative ciformes: Cichlidae). morphology of the dorsal anchors. Four dactylogyrid species have been Site of infection: Gills. described in South America: 3 from Brycon melanopterus (Cope, 1872) Type locality/collection date: Rio Frijolito (09Њ09Ј53ЉN, (Jainus amazonensis Kritsky, Thatcher, and Kayton, 1980; Tereancis- 79Њ45Ј16ЉW), Republic of Panama, April 2006. trium kerri Kritsky, Thatcher, and Kayton, 1980, and Trinibaculum bra- Specimens deposited: Holotype (CNHE 5790); 2 paratypes (CNHE ziliensis Kritsky, Thatcher and Kayton, 1980) and 1 from Brycon ce- 5791) and 3 paratypes (USNPC 99628). phalus (Gu¨nther, 1869) (Annulotrematoides bryconi Cuglianna, Silva Etymology: This species is named for the country from which it was Cordeiro, and Luque, 2003) (Kritsky et al., 1980; Cuglianna et al., collected. MENDOZA-FRANCO ET AL.—DACTYLOGYRIDS FROM PANAMA 765

FIGURES 9–15. Diaphorocleidus petrosusi n. sp. (9) Whole mount (composite, ventral view). (10) Hook. (11) Copulatory complex (ventral view). (12) Ventral anchor. (13) Dorsal anchor. (14) Ventral bar. (15) Dorsal bar. All ﬁgures are drawn to the 25-␮m scale, except 9 (100-␮m). 766 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 16–22. Sciadicleithrum panamensis n. sp. (16) Whole mount (composite, ventral view). (17) Ventral anchor. (18) Ventral bar. (19) Hook. (20) Dorsal bar. (21) Dorsal anchor. (22) Copulatory complex (ventral view). All ﬁgures are drawn to the 25-␮m scale, except 16 (100-␮m). MENDOZA-FRANCO ET AL.—DACTYLOGYRIDS FROM PANAMA 767

Remarks Remarks Features of the haptor (ventral bar with cavities, hook with slender Based on the presence of sinistral vaginal sclerite, overlapping go- shank and upright thumb) and copulatory sclerites (MCO with clock- nads, MCO with counterclockwise rings, and hooks with enlarged wise rings) clearly indicate that present specimens comprise an unde- shanks (pairs 1, 5 usually reduced in size), this species is considered a scribed species of Sciadicleithrum. This species resembles its 9 con- new member of Urocleidoides (Mizelle and Price, 1964) Kritsky, geners from South America by sharing the following features: a sinis- Thatcher, and Boeger, 1986. Urocleidoides includes a group of 7 trop- troventral vaginal aperture, overlapping gonads, absence of longitudinal ical species, which parasitize fishes of the Characidae (Characidium lateral grooves on shaft and point of anchors, no disjunction between caucanum Eigenmann, 1912), Anostomidae (Rhytiodus microlepis Kner, roots of ventral and dorsal anchors, and a copulatory organ comprising 1858), Curimatidae (Curimata argentea [ϭ Steindachnerina argentea] less than 2 rings (see Mendoza-Franco and Vidal-Martıńez, 2005). [Gill 1858]), and Erythrinidae (Hoplias malabaricus (Bloch, 1794)) While 2 other species of Sciadicleithrum (S. aequidens (Price and within Characiformes and Poeciliidae (Poecilia reticulata (Peters, 1859) Schlueter, 1967) Kritsky, Thatcher and Boeger, 1989 and S. cavanaughi and Xiphophorus helleri (Heckel, 1848)) of the Cyprinodontiformes (Price, 1966) Kritsky, Thatcher, and Boeger, 1989) have apparently spe- (Kritsky et al., 1986; Suriano, 1997; Jogunoori et al., 2004). Uroclei- ciated from the South American species of Aequidens maroni (ϭ Cleith- doides flegomai n. sp. found in P. panamensis represents the first record racara maroni) (Steindachner, 1881) (Kritsky et al., 1989), speciation of Urocleidoides in another family (Lebiasinidae) within Characiformes. of S. panamensis n. sp. seems to have occurred through its host geo- Based on the comparative morphology of the copulatory complexes and graphic range, A. coeruleopunctatus in Central America (the Atlantic bars, the closest relatives of U. flegomai n. sp. are U. vaginoclaustrum slope of Panama and Pacific slope of Costa Rica [Food and Agriculture Jogunoori, Kritsky, and Venkatanarasaiah, 2004 from X. helleri; U. er- Organization Fish Base, http://www.fishbase.org]). Additionally, it has emitus Kritsky, Thatcher, and Boeger, 1986 from H. malabaricus; and been demonstrated that Aequidens is a secondary fish (i.e., ability of the host to survive high salinities), with populations not genetically U. anops Kritsky and Thatcher, 1974 from C. caucanum from the Neo- isolated from each other in Panama. Similarly, that ability in the com- tropics (compare Figs. 24–28 [present study] and figs. 18, 22–25 for U. mon ancestor of A. coeruleopunctatus to cross short distances (e.g., to vaginoclaustrum in Jogunoori et al. [2004]; figs. 3, 6, and 7 for U. Panama from South America) through salt water could have allowed eremitus in Kritsky et al. [1986]; and 14, 16, and 17 for U. anops in isolated populations of this host species with the consequent speciation Kritsky and Thatcher [1974]). Considering that the Characiformes (200 of its monogenoid, S. panamensis n. sp. species in Africa and more than 1,200 species in the Neotropics in about 14–16 families) and Cyprinodontiformes (850 species in about 110 gen- Urocleidoides flegomai n. sp. era) comprise the most speciose assemblages of fishes in the tropics (Figs. 23–31) and North America (Costa, 1998), high diversification of Urocleidoides on these potential host species is to be expected. Diagnosis: Body fusiform 256 (200–357; n ϭ 15) long, with parallel lateral margins; greatest width 55 (45–72; n ϭ 13) usually at level of testis. Cephalic margin broad; cephalic lobes well developed; 3 bilateral Urocleidoides similuncus n. sp. pairs of head organs; cephalic glands distinct. Eyes 4; subequal; eye (Figs. 32–40) granules frequently dissociated, small, usually ovate; accessory granules Diagnosis: ϭ (granules not associated with the eyes) present in cephalic region and Body 177 (168–192; n 4) long, robust; greatest width anterior trunk. Pharynx spherical 16 (11–20; n ϭ 16) in diameter; 85–110 usually at level of testis. Cephalic margin broad; cephalic lobes esophagus moderately long. Peduncle broad; haptor hexagonal, 59 (51– well developed; 4 bilateral pairs of head organs; cephalic glands indis- 74; n ϭ 13). Ventral anchor 31 (28–36; n ϭ 20) long, with elongate tinct. Four eyes poorly developed, subequal; members of posterior pair slightly depressed superficial root, short deep root, curved shaft, elon- usually farther apart than those of anterior pair; eye granules usually ϭ gate point; base 16 (15–18; n ϭ 13) wide. Dorsal anchor 25 (22–29; n ovate. Pharynx spherical 19 (17–22; n 5); esophagus short (contracted ϭ 18) long, with well-differentiated roots, curved shaft, elongate point; specimens). Peduncle broad; haptor subhexagonal. Anchors similar; base 13 (12–15; n ϭ 14) wide. Ventral bar 29 (24–36; n ϭ 15) long, each with robust base, truncate superficial root, well-developed deep broadly V-shaped with enlarged terminations and a slight anteromedial root, angular bends at junctions of base and shaft and shaft and point; indentation; dorsal bar 25 (21–32; n ϭ 15) long, broadly U- or short point; ventral anchor 21 (20–22; n ϭ 5) long; base 14 (14–15; n V-shaped, with terminations directed laterally. Hooks similar, each with ϭ 4) wide; dorsal anchor 19 (18–20; n ϭ 6) long; base 15 (14–15; n protruding thumb, delicate shaft and point, dilated shank; hook pairs 1, ϭ 5) wide. Ventral bar 25 (23–26; n ϭ 3) long, with bulbous termina- 5 reduced in size; FH loop about 30% shank length (pairs 2, 3, 4, 6, tions; dorsal bar 22 (20–25; n ϭ 3) long, rod-shaped to slightly arced, 7), 50% shank length (pair 1); hook pairs 2, 3, 4,7—23(21–25; n ϭ with enlarged ends. Hooks similar, each 10 (9–10; n ϭ 7) long, with 14) long; hook pairs 1, 5 — 15 (14–15; n ϭ 10) long; hook pair 6 — delicate shaft and point, protruding thumb, slightly dilated shank; FH 19 (17–20; n ϭ 19) long. Male copulatory organ a coil of ϳ4.5 rings, loop about 80% shank length. Male copulatory organ a coil of ϳ3.25 base with lateral flange, tube delicate, 13 (11–14; n ϭ 9) diameter of rings, 19 (18–20; n ϭ 3) diameter of the first ring, base with lateral the first ring. Accessory piece flabellate, 17 (15–20; n ϭ 3) long. Vagina flange, tube delicate. Accessory piece 20–22 long, flabellate. Gonads sinistral, a tortuous tube with distal coil connecting to a bulb prior to overlapping, germarium ovate; testis dorsal, ovate (lateral margins in- discharching into small medial seminal receptacle anterior to germar- distinct); seminal vesicle a distal enlargement (expansion) of vas defer- ium. Gonads overlapping, germarium 13 (10–20; n ϭ 10) long; testis ens; 1 prostatic reservoir; oviduct, ootype, uterus not observed. Vagina dorsal, slightly visible at end of germarium; seminal vesicle a distal sinistral, an elongate narrow sclerotized tube coiled prior to opening enlargement (expansion) of vas deferens; 1 prostatic reservoir; oviduct, into irregular medial seminal receptacle; vaginal sclerite 23 (22–26; n ootype, uterus not observed. Vaginal sclerite 21 (20–25; n ϭ 14) long, ϭ 4) long, a robust rod with distal hook, subterminal short projection, composed of grooved rod with distal hook, subterminal short projection. proximal portion with longitudinal groove. Vitellaria dense throughout Vitellaria scattered throughout trunk, except absent in regions of repro- trunk, except absent in regions of reproductive organs. ductive organs. Taxonomic summary Taxonomic summary Type-host: Candela Piabucina panamensis Gill, 1877 (Characifor- Type host: Molly Poecilia gillii (Kner, 1863) (Cyprinodontita, Poe- mes: Lebiasinidae). ciliidae). Site of infection: Gills. Site of infection: Gills. Type locality/collection date: Rio Frijolito (09Њ09Ј53ЉN; Type locality/collection date: Rio Frijolito (09Њ09Ј53ЉN, 79Њ45Ј16ЉW), Republic of Panama, April 2006. 79Њ45Ј16ЉW), Republic of Panama, April 2006. Specimens deposited: Holotype (CNHE 5792); 5 paratypes (CNHE Specimens deposited: Holotype (CNHE 5794); 4 paratypes (CNHE 5793), 5 paratypes (IPCAS M-433), and 5 paratypes (USNPC 99629). 5795) and 2 paratypes (USNPC 99631). Etymology: The specific name is from Greek (flegomai ϭ blaze, Etymology: The specific name is from Latin (simil/is ϭ similar ϩ flame) and refers to the vernacular Spanish name of its host, candela. uncus ϭ hook) and refers to the similar size of hooks. 768 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 23–31. Urocleidoides ﬂegomai n. sp. (23) Whole mount (composite, ventral view). (24) Ventral anchor. (25) Copulatory complex (dorsal view). (26) Ventral bar. (27) Dorsal anchor. (28) Dorsal bar. (29) Hook. (30) Vaginal sclerite. (31) Vagina. All ﬁgures are drawn to the 25-␮m scale as Figure 29, except 23 (100-␮m) and 25 (25-␮m). MENDOZA-FRANCO ET AL.—DACTYLOGYRIDS FROM PANAMA 769

FIGURES 32–40. Urocleidoides similuncus n. sp. (32) Whole mount (composite, ventral view). (33) Ventral anchor. (34) Vaginal sclerite. (35) Copulatory complex (ventral view). (36) Dorsal anchor. (37) Ventral bar. (38) Hook. (39) Dorsal bar. (40) Vagina. All ﬁgures are drawn to the 25-␮m scale, except 32 (50-␮m) and 40 (30-␮m).

Remarks (Poeciliidae), Perciformes (Cichlidae), and Siluriformes (Pi- Urocleidoides similuncus n. sp. differs from congeneric species by melodidae ϭ Heptapteridae) were found. Molecular and mor- possessing similar anchors, each with a robust base, superficial root with phological comparisons suggest that South American fish taxa U. fle- truncate tip, and by having hooks of similar size. It resembles are the most basal groups in characid, cichlid, and pimelodid gomai n. sp., U. eremitus, and U. anops in the general morphology of the copulatory complex. Specimens of U. similuncus n. sp. from P. gillii phylogeny, followed by its Neotropical forms, creating putative were strongly contracted, apparently a result of premature fixation while monophyletic sister groups (Perdices et al., 2002). Based on the worms were still alive (see Fig. 32). That contraction is reflected in this conclusion and considering the morphology and host range the comparatively shorter body lengths, measuring only 168–192 in specimens from P. gillii in the Frijolito River compared with those of of the monogenoidean species found in this study (species of U. flegomai n. sp. (length 200–357) from P. panamensis in the same Urocleidoides on Characiformes, species of Sciadicleithrum locality. This is the only described species of Urocleidoides with hooks and Aphanoblastella on Cichlidae and Pimelodidae) the hy- of similar size. pothesis of a common evolutionary history with the South American clade is supported. The current zoogeographical dis- DISCUSSION tribution of monogenoidean species (except for G. asota) from In the present study, 7 species (5 new) of monogenoids in- different genera found in Panama may be initially explained by festing the gills of fish species of 5 families within the Chara- dispersal of a common ancestor of each genus to this region, ciformes (Characidae and Lebiasinidae), Cyprinodontiformes i.e., by primary and/or secondary host species from parent 770 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 drainages in South America. This ability of hosts to invade new tory to infer the evolutionary landscape of lower Central America. geographical areas could have allowed sufficient evolutionary Molecular Ecology 7: 499–517. BOEGER,W.A.,AND D. C. KRITSKY. 1988. Neotropical Monogenoidea. time for them to overlap into different biogeographic regions 12. Dactylogyridae from Serrasalmus nattereri (Cypriniformes, to produce their own endemic lineages along with their para- Serrasalmidae) and aspects of their morphologic variation and dis- sites in Central America. For instance, based on molecular data, tribution in the Brazilian Amazon. Proceedings of the Helminthol- it has been suggested that Rhamdia sp. colonized Central Amer- ogical Society of Washington 55: 188–213. ———, AND ———. 2003. Parasites, fossils and geologic history: His- ica (late Pliocene, 5 mya), much later than Rivulus spp. (Cy- torical biogeography of the South American freshwater croakers, prinodontiformes) (15.9–18.4 mya) and the heroine cichlids Plagioscion species (Teleostei, Sciaenidae). Zoologica Scripta 32: (11.3–13 mya) (Perdices et al., 2002; Chakrabarty, 2006). The 3–11. 2 latter groups are secondary freshwater fishes, which may have ———, ———, AND E. BELMONT-JE´ GU. 1994. Neotropical Monoge- dispersed through brackish or marine water before the uplift of noidea. 20. Two new species of oviparous Gyrodactylidea (Polyon- choinea) from Loricariid catfishes (Siluriformes) in Brazil and the the Isthmus of Panama (Perdices et al., 2002). This could result phylogenetic status of Ooegyrodactylidae Harris, 1983. Journal of in loss of the monogenoids due to a lack of tolerance to high Helminthological Society of Washington 61: 34–44. salinities experienced during colonization. Consistent with this CHAKRABARTY, P. 2006. Systematics and historical biogeography of hypothesis, only 1 of the 3 monogenoidean species (G. asota, Greater Antillean Cichlidae. Molecular Phylogenetic and Evolution 39: 619–627. G. astronoti, and G. rogersi) from the gills of A. ocellatus from CHOUDHURY, A., R. HARTVIGSEN-DAVERDIN, AND D. R. BROOKS. 2002. native habitats in South America (Brazil) was found in Panama. Wallinia chavarriae sp n. (Trematoda: Macroderoididae) in Asty- Furthermore, Cichla ocellaris harbors 3 species of Gussevia in anax aeneus (Gunther, 1860) and Bryconamericus scleroparius South America, but none of them was found on the 5 C. ocel- (Regan, 1908) (Osteichthyes: Characidae) from the area de conser- laris individuals we examined. Although this also could be ex- vacion, Guanacaste, Costa Rica. Journal of Parasitology 88: 107– 112. plained by sampling error, parasitological studies on Mexican COSTA, W. J. E. M. 1998. Phylogeny and classification of the Cypri- cichlids conducted since 1987 have showed only 4 species of nodontiformes (Euteleostei: Atherinomorpha): A reappraisal. In Sciadicleithrum compared to its 9 congeners from South Amer- Phylogeny and classification of Neotropical fishes, L. R. Malabar- ica (Vidal-Martinez et al., 2001; Mendoza-Franco and Vidal- ba, R. E. Reis, R. P. Vari, Z. M. Lucena, and C. A. S. Lucena (eds.). Edipucrs, Porto Alegre, Brazil, p. 537–560. Martinez, 2005). Therefore, the possibility exists that the low CUGLIANNA, A. M., N. DA SILVA CORDEIRO, AND J. L. LUQUE. 2003. number and/or absence of monogenoidean species in native Annulotrematoides bryconi sp. n. (Monogenea: Dactylogyridae) cichlids from Central America and the Greater Antilles (e.g., parasitic on Brycon cephalus (Osteichthyes: Characidae) from Bra- Cuba) (Mendoza-Franco, Vidal-Martıńez et al., 2006) by in- zil. Folia Parasitologica 50: 272–274. vasion of its secondary host species or its derivatives to these ERGENS, R. 1969. The suitability of ammonium picrate-glycerin in preparing slides of lower Monogenoidea. Folia Parasitologica 16: 320. areas from South America (Chakrabarty, 2006) could be a result GAYET, M., J. C. RAGE,T.SEMPERE, AND P. Y. G AGNIER. 1992. Mode of of loss of parasites (Mendoza-Franco and Vidal-Martinez, interchanges of continental vertebrates between North and South 2005). Historical factors such as geotectonic events within Cen- America during the late Cretaceous and Paleocene. Bulletin de la tral America would help to explain how these fish groups have Socie´te´ Geologique de France 6: 781–791. ITURRALDE-VINENT,M.A.,AND R. D. E. MACPHEE. 1999. Paleogeog- radiated from basal lineages within Central America exhibiting raphy of the Caribbean region: Implications for Cenozoic bioge- their own distinct fauna and probably endemic species of mon- ography. Bulletin of the American Museum of Natural History 238: ogenoids, e.g., Brycon petrosus as the probable original host 1–95. for those monogenoidean species (e.g., D. kabatai) infesting JOGUNOORI, W., D. C. KRITSKY, AND J. VENKATANARASAIAH. 2004. Neo- Astyanax spp. in the Neotropics. tropical Monogenoidea. 46. Three new species from the gills of introduced aquarium fishes in India, the proposal of Heterotylus n. g. and Diaphorocleidus n. g., and the reassignment of some pre- ACKNOWLEDGMENTS viously described species of Urocleidoides Mizelle & Price, 1964 (Polyonchoinea: Dactylogyridae). Systematic Parasitology 58: We acknowledge the Smithsonian Tropical Research Institute (STRI) 115–124. for financial and logistical support. We thank R. G. Reina (STRI) for KOHN,A.,AND S. C. COHEN. 1998. South American Monogenea — List assistance with the fish collections and identifications. The final prep- of species, hosts and geographical distribution. International Jour- aration of this contribution was conducted during the postdoctoral stay nal for Parasitology 28: 1517–1554. of E.F.M.F. at STRI, Republic of Panama. KRITSKY,D.C.,W.A.BOEGER, AND M. JE´ GU. 1996. Neotropical Mon- ogenoidea. 28. Ancyrocephalinae (Dactylogyridae) of piranha and LITERATURE CITED their relatives (Teleostei, Serrasalmidae) from Brazil and French Guiana: Species of Notozothecium Boeger and Kritsky, 1988, and AGARWAL,N.,AND D. C. KRITSKY. 1998. Neotropical Monogenoidea. Mymarothecium gen. n. Journal of the Helminthological Society of 33. Three new species of Ancistrohaptor n. g. (Dactylogyridae, Washington 63: 153–175. Ancyrocephalinae) on Triportheus spp. (Teleostei, Characidae) ———, ———, AND ———. 1997. Neotropical Monogenoidea. 29. from Brazil, with checklist of ancyrocephalines recorded from Neo- Ancyrocephalinae (Dactylogyridae) of piranha and their relatives tropical characiform fishes. Systematic Parasitology 39: 59–69. (Teleostei, Serrasalmidae) from Brazil: Species of Amphitecium AGUIRRE-MACEDO,M.L.,AND T. S CHOLZ. 2005. Culuwiya cichlidorum Boeger and Kritsky, 1988, Heterothecium gen. n. and Pithanothe- n. sp. (Digenea: Haploporidae) from the Black belt cichlid Vieja cium gen. n. Journal of the Helminthological Society of Washington maculicauda (Pisces: Cichlidae) from Nicaragua. Journal of Para- 64: 25–54. sitology 91: 1379–1384. ———, ———, ———, AND L. R. VAN EVERY. 1992. Neotropical ———, ———, D. GONZA´ LEZ-SOLI´S,V.M.VIDAL-MARTIŃEZ,P.POSEL, Monogenoidea. 17. Anacanthorus Mizelle and Price, 1965 (Dac- G. ARJONA-TORRES,E.SIU ESTRADA, AND S. DUMAILO. 2001. Larval tylogyridae, Anacanthorinae) from characoid fishes. Journal of the helminths parasitizing freshwater fishes from the Atlantic Coast of Helminthological Society of Washington 59: 25–51. Nicaragua. Comparative Parasitology 68: 42–51. ———, AND P. A . G UTIE´ RREZ. 1998. Neotropical Monogenoidea. 34. BERMINGHAM, E., AND A. P. MARTIN. 1998. Comparative mtDNA phy- Species of Demidospermus (Dactylogyridae, Ancyrocephalinae) logeography of Neotropical freshwater fishes: Testing shared his- from the gills of pimelodids (Teleostei, Siluriformes) in Argentina. MENDOZA-FRANCO ET AL.—DACTYLOGYRIDS FROM PANAMA 771

Journal of the Helminthological Society of Washington 65: 147– ———, AND E. W. PRICE. 1964. Studies on monogenetic trematodes. 159. XXVII. Dactylogyrid species with the proposal of Urocleidoides ———, E. F. MENDOZA-FRANCO, AND T. S CHOLZ. 2000. Neotropical gen. n. Journal of Parasitology 50: 579–584. Monogenoidea. 36. Dactylogyrids from the gills of Rhamdia gua- MOLNA´ R, K., G. HANEK, AND C. H. FERNANDO. 1974. Ancyrocephalids temalensis (Siluriformes: Pimelodidae) from cenotes of the Yucatan (Monogenea) from freshwater fishes of Trinidad. Journal of Para- Peninsula, Mexico, with proposal of Ameloblastella gen. n. and sitology 60: 914–920. Aphanoblastella gen. n. (Dactylogyridae: Ancyrocephalinae). Com- PERDICES, A., E. BERMINGHAM,A.MONTILLA, AND I. DOADRIOB. 2002. parative Parasitology 67: 76–84. Evolutionary history of the genus Rhamdia (Teleostei: Pimelodi- ———, AND V. E. THATCHER. 1974. Monogenetic Trematodes (Mono- dae) in Central America. Molecular Phylogenetics and Evolution pisthocotylea: Dactylogyridae) from freshwater fishes of Colombia, 25: 172–189. South America. Journal of Helminthology 48: 59–66. PRICE, E. W. 1938. The monogenetic trematodes of Latin America. Liv- ———, ———, AND W. A. B OEGER. 1986. Neotropical Monogenea. 8. ro Jubilar Professor Travassos, Rio de Janeiro, Brazil, 3: 407–413. Revision of Urocleidoides (Dactylogyridae, Ancyrocephalinae). ———. 1966. Urocleidus cavanaughi, a new monogenetic trematode Proceedings of the Helminthological Society of Washington 53: 1– from the gills of the keyhole cichlid, Aequidens maroni (Stein- 37. dachner). Bulletin of the Georgia Academy of Science 24: 117– ———, ———, AND ———. 1989. Neotropical Monogenoidea. 15. 120. Dactylogyrids from gills of Brazilian Cichlidae with proposal of ———, AND E. A. SCHLUETER. 1967. Two new monogenetic Trematodes Sciadicleithrum gen. n. (Dactylogyridae). Proceedings of the Hel- from South America. Journal of the Tennessee Academy of Science minthological Society of Washington 56: 128–140. 42: 23–25. ROSEN, D. E. 1976. A vicariance model of Caribbean biogeography. ———, ———, AND R. J. KAYTON. 1980. Neotropical Monogenoidea. 3. Five new species from South America with the proposal of Ter- Systematic Zoology 24: 431–464. eancistrium gen. n. and Trinibaculum gen. n. (Dactylogyridae: An- SCHOLZ, T., M. L. AGUIRRE-MACEDO,G.SALGADO-MALDONADO,J.VAR- GAS-VA´ ZQUEZ,V.M.VIDAL-MARTIŃEZ,R.WOLTER,R.KUCHTA, cyrocephalinae). Acta Amazonica 10: 411–417. AND W. K O¨ RTING. 1999. Redescription of Pseudoacanthostomum MENDOZA-FRANCO,E.F.,P.POSEL, AND S. DUMAILO. 2003. Monogeneans panamense Caballero, Bravo-Hollis and Grocott, 1953 (Digenea: (Dactylogyridae, Ancyrocephalinae) of freshwater fishes from the Acanthostomidae), a parasite of Siluriform fishes of the family Ari- Caribbean coast of Nicaragua. Comparative Parasitology 70: 1–11. idae, with notes on its biology. Journal of the Helminthological ´ ———, AND V. M . V IDAL-MARTINEZ. 2005. Phylogeny of species of Society of Washington 66: 146–154. Sciadicleithrum (Monogenoidea: Ancyrocephalinae), and their his- SURIANO, D. M. 1986. El genero Urocleidoides Mizelle y Price, 1964 torical biogeography in the Neotropics. Journal of Parasitology 91: (Monogenea: Ancyrocephalidae). Anatomıá y posicioń sistema´tica. 253–259. Urocleidoides mastigatus sp. nov. y U. travassosi (Price, 1934) ´ ———, ———, Y. CRUZ-QUINTANA, AND F. L. PRATS-LEON. 2006. Molnar, Hanek y Fernando, 1974 parasitas de Rhamdia sapo (Va- Monogeneans from freshwater fishes from Cuba with description lenciennes, 1840) Eigenmann y Eigenmann, 1888 y Pimelodella of Salsuginus cubensis sp. n. from the Limia vittata. Systematic laticeps Eigenmann, 1917 (Pisces: Siluriformes) de la Laguna de Parasitology 64: 181–190. Chascomus, Republica Argentina. Physis (Buenos Aires), Seccioń ———, J. VIOLANTE-GONZA´ LEZ, AND V. M . V IDAL-MARTIŃEZ. 2006. B 44: 73–80. Cornutohaptor nigrescensi n. gen. and n. sp. (Monogenoidea: Di- ———. 1997. The genus Urocleidoides Mizelle and Price, 1964 plectanidae) from the gills of the black snook Centropomus ni- (Monogenea: Ancyrocephalidae) parasitizing characoidei fishes in grescens (Perciformes, Centropomidae) of the Pacific coast of Mex- Argentina. Physis (Buenos Aires), Section B 53: 1–6. ico. Journal of Parasitology 92: 481–485. VIDAL-MARTIŃEZ, V. M., M. L. AGUIRRE-MACEDO,T.SCHOLZ,D.GON- MIZELLE,J.D.,AND D. C. KRITSKY. 1969. Studies on monogenetic ZALEZ-SOLIS, AND E. F. MENDOZA-FRANCO. 2001. Atlas of the hel- Trematodes. XXXIX. Exotic species of Monopisthocotylea with the minth parasites of cichlid fishes of Mexico. Academy of Sciences, proposal of Archidiplectanum gen. n. and Longihaptor gen. n. Prague, Czech Republic, 185 p. American Midland Naturalist 81: 370–386. ———, T. SCHOLZ, AND M. L. AGUIRRE-MACEDO. 2001. Dactylogyridae ———, ———, AND J. W. CRANE. 1968. Studies on monogenetic Trem- of cichlid fishes from Nicaragua, Central America with descriptions atodes. XXXVIII. Ancyrocephalinae from South America with the of Gussevia herotilapiae and three new species of Sciadicleithrum proposal of Jainus gen. n. American Midland Naturalist 80: 186– (Monogenea: Ancyrocephalinae). Comparative Parasitology 68: 198. 76–86. J. Parasitol., 93(4), 2007, pp. 772–780 ᭧ American Society of Parasitologists 2007

THREE NEW SPECIES OF LIGOPHORUS (MONOGENEA: DACTYLOGYRIDAE) ON THE GILLS OF MUGIL CEPHALUS (TELEOSTEI: MUGILIDAE) FROM THE JAPAN SEA*

Nataliya Yu. Rubtsova, Juan A. Balbuena†, and Volodimir L. Sarabeev Department of Biology, Zaporizhzhya National University, 66 Zhukovskogo Street, 69600 Zhaporizhzhya, Ukraine. e-mail: [email protected]

ABSTRACT: Comparative morphology and multivariate morphometric analysis of monogeneans collected on flathead mullets Mugil cephalus from 2 Russian localities of the Japan Sea revealed the presence of 3 new species of Ligophorus, namely, L. domnichi n. sp., L. pacificus n. sp., and L. cheleus n. sp., which are described herein. So far, only 1 species of dactylogyrid monogenean identified as Ligophorus chabaudi was known on flathead mullets in this sea, but after comparison with the present material, we propose that this form actually represents L. domnichi n. sp. Results support previous zoogeographical evidence, suggesting that flathead mullets from different seas harbor different species complexes of Ligophorus. One interesting finding is that the 3 new species have a U-shaped ovary, whereas ovate ovaries have been reported in previous descriptions of species of the genus, e.g., L. vanbenedenii, L. parvicirrus, L. imitans, and L. chongmingensis. The U-shaped ovary was revealed only when the worms were observed in lateral view. The additional examination of L. vanbenedenii, L. parvicirrus, L. imitans, and L. pilengas specimens from our collections also revealed a U-shaped ovary in these forms as well. Further studies should establish whether or not this character is shared by all members of the genus.

Data on the monogenean parasites of mullets (Mugilidae) I). Fish were examined microscopically for monogeneans within the day from the Japan Sea are very scarce. To our knowledge, only 4 of capture, or after thawing if frozen. This study is based on 89 specimens belonging to 3 new species of Ligophorus (Table I). Specimens species have been recorded or described: Gyrodactylus mugili were mounted in glycerin jelly, following Gussev (1983). Specimens Zhukov, 1970; Ligophorus kaohsianghsieni (Gussev, 1962); L. were stained in iron acetocarmine, passed through a series of increasing pilengas Sarabeev and Balbuena, 2004, and L. chabaudi Euzet ethanol concentrations (from 70 to 100%), cleared in dimethyl phthal- and Suriano, 1977 (Gussev, 1955; Zhukov, 1970; Gussev, 1985; ate, and mounted in Canada balsam to study details of their internal Sarabeev and Balbuena, 2004, and our unpublished data). The anatomy. We measured 35 characters, 25 of them belonging to sclerotized first 3 species occur on the so-iuy mullet Mugil soiuy Basi- structures. Nineteen sclerotized characters were measured as defined in lewsky, 1855 and have also been reported in the Azov and the Sarabeev and Balbuena (2004) and Rubtsova et al. (2006), whereas the Black Seas after the introduction of this host in this area (Dmi- remainder (ventral and dorsal anchors shaft lengths; distal part length trieva, 1996; Miroshnichenko and Maltsev, 1998; Sarabeev and and width, shaft length of upper lobe, and lower lobe length of acces- Balbuena, 2004). The fourth species has been reported on the sory piece of male copulatory complex) were determined as shown on Figure 1. These 6 additional characters were chosen because they flathead mullet Mugil cephalus Linnaeus, 1758. It was first re- seemed useful for species discrimination after a preliminary study of corded as Ancyrocephalus vanbenedenii (Parona and Perugia, the present specimens. Dimensions of soft internal organs and body 1890) but, after the erection of Ligophorus by Euzet and Sur- represent their largest measurement. The following abbreviations for the iano (1977), these authors transferred it to L. chabaudi Euzet characters are used throughout the text: BL, body length; BW, body and Suriano, 1977. However, in the light of recent zoogeograph- width; VAA, ventral anchor total length; VAB, ventral anchor main part length; VAC, ventral anchor outer root length; VAD, ventral anchor ic evidence suggesting that flathead mullets from different geo- inner root length; VAE, ventral anchor point length; VAF, ventral anchor graphic areas harbor different species of Ligophorus (Sarabeev shaft length; DAA, dorsal anchor total length; DAB, dorsal anchor main et al., 2005), and because L. chabaudi is commonly found on part length; DAC, dorsal anchor outer root length; DAD, dorsal anchor flathead mullets from the Mediterranean (Rubtsova et al., 2006), inner root length; DAE, dorsal anchor point length; DAF, dorsal anchor the occurrence of this species in the Japan Sea was in need of shaft length; UTL, uncinulus total length; USHL, uncinulus shaft length; USL, uncinulus sickle length; VBL, ventral bar length; VBAP, distance scrutiny. During a parasitological survey of flathead mullets in between membranous anterior processes (protuberances) of ventral bar; 2 Russian localities of the Japan Sea, several dactylogyrid DBL, dorsal bar length; PAPL, penis accessory piece total length; PA- monogeneans were collected, and a detailed morphological and PUL, penis accessory piece distal portion of upper lobe length; PAPUW, morphometric study revealed that they could be ascribed to 3 penis accessory piece distal portion of upper lobe width; PAPSHL, pe- new species of Ligophorus, which are described herein. In ad- nis accessory piece upper lobe shaft length; PAPLL, penis accessory dition, after comparison with the description of specimens orig- piece lower lobe length; PL, total length of penis; VL, vagina length. Measurements are given in micrometers as mean Ϯ standard devia- inally identified as A. vanbenedenii from the Japan Sea, we tion with range in brackets, and number of specimens measured for propose that this form actually conforms to 1 of these new each character in parentheses. Measurements and drawings were made species. with the use of a Leica DMLB2 microscope and a Nikon Optiphot-2 microscope with interference contrast (magnification ϫ10ϫ10 for the MATERIALS AND METHODS body and ϫ10ϫ100 [under immersion oil] for sclerotized structures and internal organs). Photographs of sclerotized structures of the haptor and Adult flathead mullets were collected in 2 Russian localities of the Japan Sea, i.e., Kiyevka Bay and Delta of the Razdol’naya River (Table male copulative apparatus were made with a Leica DMR microscope with interference contrast and a Leica DC300 camera. Because variation ranges of all metric characters of the 3 new species Received 3 March 2006; revised 5 June 2006; accepted 5 June 2006. overlapped, a principal-components analysis (PCA) based on the overall * The term ‘‘Japan Sea’’ is used in conformity with the current usage covariation matrix was used to visualize and evaluate the morphometric set by the International Hydrographic Organization, and does not nec- differentiation between the species. Because correlation between most essarily reflect the authors’ stance over the international dispute about of the morphometric characters of Ligophorus spp. have been observed the naming of this sea. (Mariniello et al., 2004), PCA seems a suitable tool to circumvent the † Marine Zoology Unit, Cavanilles Institute of Biodiversity and Evo- problem of multicollinearity of metric traits (Ter Braak, 1995) and their lutionary Biology, University of Valencia, P.O. Box 22085, 46071 discrimination. The PCA was performed on all but 1 (VL), log-trans- Valencia, Spain. formed metric characters of 79 specimens for which data for all vari-

772 RUBTSOVA ET AL.—THREE NEW SPECIES OF LIGOPHORUS 773

TABLE I. Zoogeographical and host information on the specimens of Ligophorus collected from M. cephalus in the Japan Sea used in the present study.

Species Locality* Date N Host length range (mm)

Ligophorus domnichi n. sp. KB 42Њ51ЈN 133Њ40ЈE June 2004 31 445–480 Ligophorus paciﬁcus n. sp. RD 43Њ20ЈN 131Њ47ЈE June 2003 1 388 L. paciﬁcus n. sp. KB 42Њ51ЈN 133Њ40ЈE June 2004 30 435–480 Ligophorus cheleus n. sp. RD 43Њ20ЈN 131Њ47ЈE June 2003 5 466–470 L. cheleus n. sp. KB 42Њ51ЈN 133Њ40ЈE June 2004 22 398–480

* Locality abbreviations: KB—Kiyevka Bay; RD—Razdol’naya Delta.

ables measured were available. VL was excluded from the analyses Type locality: Kiyevka Bay (42Њ51ЈN, 133Њ40ЈE) Japan Sea, Russia. because 14% of individuals contained missing values for this trait. To Specimens deposited: Four syntypes: The Natural History Museum, test the consistency of eigenvalues and PCA coefficients, we used a London, U.K., Reg. No. 2006.6.1.2. jackknife cross-validation procedure in which the classification function Etymology: The specific name is proposed to honor Dr. Inna Dom- is computed from all possible subsamples generated by leaving 1 in- nich, a parasitologist from Zaporizhzhya National University (Ukraine). dividual out in each replicated computation, allowing confidence intervals of approximately 95% to be set (Gibson et al., 1984; Crowley, Remarks 1992). The PCA computations were performed with PAST 1.33 (Ham- Ligophorus domnichi n. sp. belongs to the group of Ligophorus spe- mer and Harper, 2005). cies identified by Rubtsova et al. (2006) with a penis entering at the distal end of a claw-shaped accessory piece of the male copulatory DESCRIPTIONS complex, ventral and dorsal anchors similar in size and shape, a short point of both ventral and dorsal anchors, and a prominent median pro- Ligophorus domnichi n. sp. cess of the ventral bar. These characters are met by L. chabaudi, L. (Figs. 1, 2A–C) pilengas Sarabeev and Balbuena, 2004; and L. cephali Rubtsova, Bal- Description (based on 32 whole mounts; morphometric measure- buena, Sarabeev, Blasco and Euzet, 2006. Ligophorous domnichi n. sp. ments of sclerotized structures are presented on Table II): Body 686 Ϯ is distinguishable from these 3 species by its well-developed postero- 157 [410–960] (30) long, fusiform; maximum width 140 Ϯ 18 [99– median process in the dorsal bar and the heavily sclerotized bulb at the 190] (30) usually at level of gonads. Cephalic region moderate; 1 ter- distal portion of the upper lobe of the accessory piece. Additionally, minal and 2 bilateral cephalic lobes; 3 bilateral pairs of head organs; the new species differs from L. chabaudi by the straight ventral bar and cephalic glands indistinct, posterolateral to pharynx. Each eye with con- the bowed upper lobe of the male copulatory accessory piece, as well spicuous lens. Pharynx subspherical 35.7 Ϯ 4.4 [28–42] (21) ϫ 31.8 Ϯ as by the shorter ventral and dorsal bars, the ventral anchor outer root, 2.7 [27–38] (21). Testis 55.4 Ϯ 6.2 [50–67] (7) ϫ 29.3 Ϯ 4.3 [21–35] and from L. cephali and L. pilengas by the hammer-shaped upper lobe (7), ovate to pyriform; seminal vesicle tapered, posteromedial to penis; of accessory piece of male copulatory complex. In addition, the position prostatic reservoir pyriform. Male copulatory complex consists of tu- of the vaginal aperture of L. cephali is dextroventral and L. pilengas bular, C-shaped penis (about 1 ␮m in diameter at middle part) with has longer ventral and dorsal bars (Euzet and Suriano, 1977; Sarabeev well-developed heel and claw-shaped accessory piece; the latter bilobed and Balbuena, 2004; Balbuena et al., 2005; Rubtsova et al., 2006). at the distal half and directs distal portion of penis. Lower lobe elongate, The forms redrawn and identified as A. vanbenedenii from M. ce- bowed, concave, along upper lobe, shorter than latter, distal tip of lower phalus from the Tumen-Ula River (Japan Sea, Russia) by Gussev lobe extending or slightly not to level of that upper lobe. Upper lobe (1955), and later transferred to Ligophorus as L. chabaudi (Euzet and hammer shaped, arched, tubular, thin walled, with membranous funnel- Suriano, 1977), seem to correspond to L. domnichi n. sp., because they shaped opening on top of distal part; the latter bowed with heavily show a bowed, heavily sclerotized bulb at the distal portion of the upper sclerotized bulb at distal portion; shaft of upper lobe arched, elongate lobe of the accessory piece, and a well-developed posteromedian pro- tube with or without flanges. Penis enters membranous funnel-shaped cess in the dorsal bar (see Figs. 2–3 in Gussev, 1955). opening at distal end of accessory piece. Ovary 76.7 Ϯ 9.2 [65–101] (13) ϫ 34.8 Ϯ 5.3 [24–42] (13) elongate, U-shaped (Fig. 1B). Vaginal Ligophorus pacificus n. sp. aperture midventral, funnel shaped; vaginal canal long, thin, coiled or (Figs. 2D–F, 3A–E) winding, heavily sclerotized, leading to ovate seminal receptacle. Vi- Description (based on 31 whole mounts; morphometric measure- telline follicles dense. Peduncle broad, short, subequal in width and ments of sclerotized structures are shown on Table II): Worms with length, tapering posteriorly; haptor subhexagonal, 59.6 Ϯ 9.4 [44–78] ϫ Ϯ characters of genus as defined by Euzet and Sanfilippo (1983) and sup- (22) 92.6 15.5 [62–128] (22) armed, with 14 uncinuli, 2 pairs of plemented by Sarabeev and Balbuena (2004). Body 681 Ϯ 128 [450– anchors, 2 transverse bars. Ventral anchor with inner root longer and Ϯ ϭ Ϯ 920] (29) long, fusiform; maximum width 145 33 [100–250] (30) heavier than outer root (VAD/VAC 2.6 0.5 [1.8–4.2] [30]), arched usually at level of gonads. One or 2 terminal and 2 bilateral cephalic blade bent at middle. Base markedly thicker than blade, separated by lobes. Each eye with lens. Pharynx subspherical 35.5 Ϯ 3 [28–39] (11) notch; filament present. Ventral anchors connected by transverse ventral ϫ 39.4 Ϯ 4.2 [32–47] (11). Testis 71.2 Ϯ 21.5 [48–95] (5) ϫ 36.6 Ϯ bar. Ventral bar straight or slightly bowed with 2 membranous protu- 16 [22–60] (5); male copulatory complex consists of tubular, C-shaped berances, contiguous to heavily sclerotized median process between penis (about 1 ␮m in diameter at middle part) with well-developed heel them, the latter not reaching level of tip of protuberances. Dorsal anchor and claw-shaped accessory piece, which directs distal portion of penis. similar to ventral, but slightly longer with smaller ratio between inner ϭ Ϯ Accessory piece bilobed, bifurcate at the distal half. Lower lobe mod- and outer roots (DAD/DAC 1.9 0.3 [1. 5–2.8] [30]) and different erately elongate, slightly bowed or straight, concave, along upper lobe, in shape of blade, which bent at the distal third. Dorsal transversal bar shorter than latter, distal tip of lower lobe not reaching level of that V shaped with posteromedian process, connects dorsal anchors. All 14 upper lobe. Upper lobe hammer-shaped, bowed, tubular, thin-walled uncinuli similar in shape and size with straight shaft and sickle. Fila- with membranous funnel-shaped opening on top of distal half; the latter ment loop of the sickle coils close to distal quarter of shaft. straight with membranous subtriangular expansion; shaft of upper lobe moderately elongated tube with or without flanges. Penis enters mem- Taxonomic summary branous funnel-shaped opening at distal end of accessory piece. Ovary Type host: Mugil cephalus Linnaeus, 1758, flathead mullet. 86.4 Ϯ 15.3 [70–115] (9) ϫ 37 Ϯ 8.3 [28–50] (9) elongate, U-shaped. Site of infection: Gill secondary lamellae. Vagina long, thin, coiled or winding, heavily sclerotized tube; vaginal 774 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 1. Ligophorus domnichi n. sp.: overall view, ovary, haptor, and genital sclerotized structures. (A) Whole worm, ventral view. (B) Ovary, lateral view. (C) Male copulatory complex: penis and accessory piece. (D) Vaginal armament. (E) Marginal hook. (F) Ventral bar and anchors. (G) Dorsal bar and anchors. For anchors and accessory piece sclerotized structures, the new metric variables added in the present study are shown (see Materials and Methods section for abbreviations of measurements). RUBTSOVA ET AL.—THREE NEW SPECIES OF LIGOPHORUS 775

FIGURE 2. Photomicrographs of sclerotized elements of haptor and male copulatory complex of Ligophorus domnichi n. sp. (A–C) and Ligophorus pacificus n. sp. (D–F). A, D. Male copulatory complex. B, E. Ventral bar and anchors. C, F. Dorsal bar and anchors. Scale bar ϭ 10 ␮m. aperture submidventral, funnel-shaped. Peduncle broad, short, subequal which is bent at the distal third. Dorsal transversal bar bowed with in width and length, tapering posteriorly. Haptor subhexagonal 63 Ϯ inconspicuous posteromedian process, connects dorsal anchors. All 14 12.5 [48–85] (12) ϫ 104.9 Ϯ 21.5 [68–144] (12) armed, with 14 un- uncinuli similar in shape and size with straight shaft and sickle. Fila- cinuli, 2 pairs of anchors, 2 transverse bars. Ventral anchor with inner ment loop of the sickle coils close to distal quarter of shaft. root longer and heavier than outer root (VAD/VAC ϭ 1.9 Ϯ 0.3 [1.3– 2.3] [31]); point and outer root subequal in length; arched blade bent Taxonomic summary at middle. Base markedly thicker than blade, separated by notch; fila- Type host: Mugil cephalus Linnaeus, 1758, flathead mullet. ment present. Ventral anchors connected by transverse ventral bar. Ven- Site of infection: Gill secondary lamellae. tral bar straight or slightly bowed with 2 membranous protuberances, Type locality: Kiyevka Bay (42Њ51ЈN, 133Њ40ЈE) Japan Sea, Russia, contiguous to heavily sclerotized elongate median process between and Delta of Razdol’naya River (43Њ20ЈN, 131Њ47ЈE) Japan Sea, Russia. them, the latter extending to level of tip of protuberances. Dorsal anchor Specimens deposited: Four syntypes: The Natural History Museum, similar to ventral, but slightly longer and different in shape of blade, London, U.K., Reg. No. 2006.6.1.3-4. 776 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE II. Comparison of sclerotized metrical characters of 3 new species of Ligophorus from the Japan Sea. See Materials and Methods section for abbreviations of metric variables.

Species Ligophorus domnichi n. sp. Ligophorus paciﬁcus n. sp. Ligophorus cheleus n. sp. Characters Mean SD Range N Mean SD Range N Mean SD Range N

VAA 38.3 2 34–42.5 30 39.1 2 34–43 31 39.3 1.9 35.3–43 25 VAB 24.8 1.6 22–28 30 25.4 2.1 20–30 31 28.3 1.7 22.5–31 25 VAC 7.8 1.3 5–10.5 30 11.2 1.6 9–16 31 11.3 1 9.5–13 25 VAD 19.7 1.3 17–22.5 30 21.3 1.5 18–24 31 18.6 1.3 16–21 25 VAE 11.1 0.6 10–12.3 30 10.9 0.8 9–12 31 10 0.7 9–11 25 VAF 16.1 1.1 14–19 30 16.4 1.5 14–19 31 19.9 1 17.4–21.5 25 DAA 39.7 1.8 36–43 30 41.8 2.6 36–47 30 39.2 2.4 32–45 26 DAB 28 1.4 24–30 30 29.1 1.5 27–33 30 28.9 1.1 26–31 26 DAC 10 1.2 7.5–12 30 11.6 1.1 9–13.5 30 9.9 0.8 8–12 26 DAD 18.4 1.7 16–23 30 19.8 2.1 15–24 30 18.6 1.3 16–21 26 DAE 8.6 0.8 7–10 30 9.3 0.8 7–10 29 8.3 0.7 7–9 26 DAF 19.2 0.9 17–22 30 19.5 1.5 17–24 30 20.7 1.3 18–24 26 UTL 13.6 0.4 12.8–14.5 29 13 0.7 11–14 28 13.2 0.6 12.5–15 18 USHL 7.3 0.5 6.5–8 29 7 0.5 6–8 28 7.2 0.4 6.5–8 18 USL 6.3 0.3 6–7 29 5.9 0.4 5–7 28 6.0 0.4 5–7 18 VBL 38.7 2.3 32–42 30 40.6 4.2 32–55 29 38.9 2.9 34–46 22 VBAP 8.4 1.3 6–10 30 10.5 1.7 8–15 25 9.2 1 7–11 22 DBL 36.1 2.2 32–39 31 42.3 4.2 32–51 30 39.7 3.3 34–46 24 PAPL 48.9 5.1 39–57 32 40.5 4.7 30–49 31 40.6 3.6 34–47 28 PAPSHL 29.3 3.2 22–36 32 25.2 4.8 16–35 31 26.2 3.1 21.7–33 27 PAPUL 19.7 2.1 15.5–23 31 15.6 1.6 12–18.5 31 15 1.8 11–18 27 PAPUW 6.2 0.9 4.5–8 31 8.6 1.3 7–11 29 3 0.3 2.5–4 18 PAPLL 17.4 1.5 15.5–21 31 12.7 1.5 9–15.5 31 10.8 0.9 9–13 27 PL 100.8 8.9 87–120 32 115.7 9.6 93–130 31 90.6 10 70–114 28 VL 49.7 5.7 40–64 25 66.6 11.7 47–102 29 41 5.8 32–50 15

Etymology: The specific designation pacificus refers to the geograph- tubular, C-shaped penis (about 1 ␮m in diameter at middle part) with ical situation of the type locality in the Pacific Ocean. well-developed heel and claw-shaped accessory piece, which directs distal portion of penis. Accessory piece bilobed bifurcate at the distal Remarks half. Lower lobe short, straight, concave, turns away from upper lobe, shorter than latter, distal tip of lower lobe not reaching level of that Ligophorus pacificus n. sp. belongs to the same morphological group upper lobe. Upper lobe, bowed, tubular, thick walled with membranous of Ligophorus as L. domnichi n. sp. In this group, L. pacificus n. sp. is funnel-shaped opening on top of distal half. Penis enters membranous easily distinguishable from L. domnichi n. sp. by the shape of the distal funnel-shaped opening at distal end of accessory piece. Ovary 71.4 Ϯ half of the upper lobe of the accessory piece of male copulatory com- 17.9 [45–97] (9) ϫ 38.7 Ϯ 13.7 [25–59] (9) elongate, U-shaped. Vagina plex, which is straight with a membranous medial bulb, a shorter lower long, thin, convoluted to straight, heavily sclerotized tube; vaginal ap- lobe of the accessory piece and an inconspicuous posteromedian process erture submidventral, funnel shaped. Peduncle broad, short, subequal in on dorsal bar. It also differs from L. pilengas and L. cephali by the width and length, tapering posteriorly. Haptor subhexagonal 58.8 Ϯ hammer-shaped upper lobe of the accessory piece, and additionally 10.4 [37–80] (12) ϫ 91.1 Ϯ 14.4 [65–120] (12) armed, with 14 uncinuli, from L. pilengas by the shorter lower lobe than the upper lobe of the 2 pairs of anchors, 2 transverse bars. Ventral anchor with inner root accessory piece, and from L. cephali by the straight lower lobe of the longer and heavier than outer root (VAD/VAC ϭ 1.6 Ϯ 0.2 [1.4–2.1] accessory piece and submidventral position of vaginal aperture. Ligo- [25]); point and outer root subequal in length; arched blade bent at the phorous pacificus n. sp. is distinguished from L. chabaudi by the sub- distal half. Base markedly thicker than blade, separated by notch; fila- triangular membranous expansion of upper lobe of copulatory complex ment present. Ventral anchors connected by transverse ventral bar. Ven- (bulbous in L. chabaudi ), the arched upper lobe, the longer lower lobe tral bar slightly bowed or V-shaped with 2 membranous protuberances, of the accessory piece and the elongate anteromedian process of ventral contiguous to heavily sclerotized short median process between them, bar, which extends to level of tip of protuberances. (Euzet and Suriano, the latter not reaching level of tip of protuberances. Dorsal anchor sim- 1977; Sarabeev and Balbuena, 2004; Rubtsova et al., 2006). ilar to ventral, but with slightly higher ratio between inner and outer roots (DAD/DAC ϭ 1.9 Ϯ 0.2 [1. 5–2.3] [26]). Dorsal transversal bar Ligophorus cheleus n. sp. bowed, connects dorsal anchors. All 14 uncinuli similar in shape and (Figs. 3F–J, 4) size with straight shaft and sickle. Filament loop of the sickle coils at Description (based on 28 whole mounts; morphometric measure- about shaft middle. ments of sclerotized structures are presented on Table II): Worms with characters of genus as defined by Euzet and Sanfilippo (1983) and sup- Taxonomic summary plemented by Sarabeev and Balbuena (2004). Body 586 Ϯ 98.8 [420– Type host: Mugil cephalus Linnaeus, 1758, flathead mullet. 860] (26) long, fusiform; greatest width 109 Ϯ 28.9 [58.5–170] (26) Site of infection: Gill secondary lamellae. usually at level of gonads. One or 2 terminal and 2 bilateral cephalic Type locality: Kiyevka Bay (42Њ51ЈN, 133Њ40ЈE) Japan Sea, Russia lobes. Posterior pair of eyes with lens. Pharynx subspherical 31.2 Ϯ 3.2 and Delta of Razdol’naya River (43Њ20ЈN, 131Њ47ЈE) Japan Sea, Russia. [25–35] (10) ϫ 29.4 Ϯ 4.9 [24–40] (10). Testis 60.1 Ϯ 12.1 [49–78] Specimens deposited: Four syntypes: The Natural History Museum, (8) ϫ 23.3 Ϯ 3.5 [19–30] (8). Male copulatory complex consists of London, U.K., Reg. No. 2006.6.1.1. RUBTSOVA ET AL.—THREE NEW SPECIES OF LIGOPHORUS 777

FIGURE 3. Haptor and genital sclerotized structures of Ligophorus paciﬁcus n. sp. (A–E) and Ligophorus cheleus n. sp. (F–J). A, F. Male copulatory complex: penis and accessory piece. B, G. Vaginal armament. C, H. uncinulus. D, I. Ventral bar and anchors. E, J. Dorsal bar and anchors. 778 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 5. PCA the 2 first components, performed on 26 morphometric variables of 79 specimens of L. domnichi n. sp. (Ⅺ), L. pacificus n. sp. (⅜) and L. cheleus n. sp. (᭝). Ellipses encompass 95% confidence limits for each species.

bulb on the upper lobe of the accessory piece, and additionally from L. domnichi n. sp. and L. paciﬁcus n. sp. by the shorter lower lobe of the accessory piece, the absence of posteromedian process on the dorsal bar and the uncoiled vagina, and from L. chabaudi by the shorter ventral bar. The new species is also distinguished from L. pilengas and L. cephali by the uncoiled vagina and the shorter lower lobe of the accessory piece. In contrast, L. pilengas has a lower lobe longer than the upper lobe, and the lower lobe in L. cephali is arched and reaching the upper lobe of the accessory piece. The latter species differs additionally from L. cheleus n. sp. by the submidventral position of the vaginal aperture.

Multivariate analyses The plot of the 79 specimens against the 2 first principal components of the PCA (PC1 and PC2) shows 3 distinct groups, which correspond to the 3 new species described here (Fig. 5). This pattern was consistently observed in each of the 79 jackknife runs of the PCA (results not shown). The eigenvalues of the first 2 principal components from the actual sample and the jackknife runs were similar and accounted for more than 60% of the total variance (Table III). The mean jackknifed pseudovalues indicated moderate quantitative differences in the association between the original coefficients for both PC1 and PC2 (Table III), thereby suggesting stability of the PCA analysis. However, as revealed by the 95% jackknifed confidence intervals, most pseudocoef- ficients on PC1 (BL, VAA, VAC, DAA, DAB, DAD, DAF, UTL, FIGURE 4. Photomicrographs of sclerotized elements of haptor and USHL, USL, VBL, VBAP, DBL, PAPL, and PAPSHL) and on PC2 male copulatory complex of Ligophorus cheleus n. sp. (A) Male cop- (BL, BW, VAA, VAB, VAD, VAE, VAF, DAA, DAB, DAD, DAE, ulatory complex. (B) Ventral bar and anchors. (C) Dorsal bar and an- DAF, UTL, USHL, USL, and VBL) did not differ significantly from 0 chors. Scale bar ϭ 10 ␮m. (Table III), and thus the contribution of these variables to their respective principal component is unclear. The first component was mostly influenced by the measurements of PAPUW and reflects the pronounced difference between L. cheleus n. sp. and the 2 other species (L. pacificus Etymology: The specific name is derived from Latin, cheleus (having n. sp. and L. domnichi n. sp.). The second component was largely de- a claw-shaped mechanism), referring to the claw-shaped accessory termined by VAC and PAPLL, and primarily distinguishes L. pacificus piece of male copulatory complex characteristic of this new species. n. sp. from L. domnichi n. sp. This means that the species described here are mainly distinguished by the morphology of the male copulatory Remarks complex (2 of 3 most important features along the 2 first principal Like the previous species, this 1 belongs to the same morphologic components are characters of the copulatory complex). The metric dif- group. In this group, L. cheleus n. sp. is easily distinguished from L. ferences between L. pacificus n. sp. and L. domnichi n. sp. are less than chabaudi, L. domnichi n. sp., and L. pacificus n. sp. by the absence of between these 2 species and L. cheleus n. sp. RUBTSOVA ET AL.—THREE NEW SPECIES OF LIGOPHORUS 779

TABLE III. Principal component analysis of 26 morphometric variables from 79 specimens of 3 species L. domnichi n. sp., L. pacificus n. sp., and L. cheleus n. sp. Mean pseudovalues and their 95% confidence intervals (CI) for the first (PC1) and second (PC2) principal components were obtained through a jackknife procedure. See Materials and Methods for abbreviations of metric variables.

PC1 PC2 Pseudovalues Pseudovalues Variable Coefﬁcient Mean 95% CI Coefﬁcient Mean 95% CI

BL 0.125 0.132 0.035 0.215 Ϫ0.007 Ϫ0.005* Ϫ0.214 0.200 BW 0.277 0.275 0.148 0.405 0.009 0.003* Ϫ0.183 0.201 VAA Ϫ0.009 Ϫ0.009* Ϫ0.035 0.016 Ϫ0.043 Ϫ0.044 Ϫ0.083 Ϫ0.003 VAB Ϫ0.108 Ϫ0.113 Ϫ0.137 Ϫ0.078 Ϫ0.059 Ϫ0.062* Ϫ0.126 0.007 VAC Ϫ0.113 Ϫ0.112* Ϫ0.228 0.003 Ϫ0.540 Ϫ0.559 Ϫ0.658 Ϫ0.423 VAD 0.101 0.105 0.066 0.135 Ϫ0.073 Ϫ0.074 Ϫ0.125 Ϫ0.020 VAE 0.071 0.074 0.040 0.102 0.050 0.052* Ϫ0.003 0.102 VAF Ϫ0.181 Ϫ0.177 Ϫ0.214 Ϫ0.148 Ϫ0.057 Ϫ0.059* Ϫ0.125 0.011 DAA 0.030 0.033 0.002 0.058 Ϫ0.066 Ϫ0.067 Ϫ0.098 Ϫ0.033 DAB Ϫ0.011 Ϫ0.009* Ϫ0.030 0.008 Ϫ0.044 Ϫ0.044 Ϫ0.080 Ϫ0.008 DAC 0.098 0.104 0.046 0.150 Ϫ0.161 Ϫ0.159 Ϫ0.252 Ϫ0.070 DAD 0.029 0.029* Ϫ0.012 0.069 Ϫ0.089 Ϫ0.091 Ϫ0.155 Ϫ0.022 DAE 0.076 0.080 0.033 0.118 Ϫ0.071 Ϫ0.071 Ϫ0.134 Ϫ0.008 DAF Ϫ0.067 Ϫ0.065 Ϫ0.093 Ϫ0.042 Ϫ0.034 Ϫ0.033* Ϫ0.077 0.009 UTL Ϫ0.00004 0.000* Ϫ0.021 0.021 0.061 0.063 0.032 0.090 USHL Ϫ0.010 Ϫ0.010* Ϫ0.039 0.018 0.053 0.055 0.007 0.100 USL 0.008 0.008* Ϫ0.020 0.036 0.075 0.077 0.035 0.114 VBL 0.034 0.036* Ϫ0.002 0.071 Ϫ0.068 Ϫ0.069 Ϫ0.119 Ϫ0.017 VBAP 0.050 0.056* Ϫ0.030 0.130 Ϫ0.294 Ϫ0.299 Ϫ0.409 Ϫ0.179 DBL 0.021 0.025* Ϫ0.030 0.073 Ϫ0.192 Ϫ0.196 Ϫ0.245 Ϫ0.139 PAPL 0.065 0.066* Ϫ0.005 0.135 0.262 0.270 0.168 0.356 PAPSHL 0.015 0.013* Ϫ0.061 0.092 0.212 0.217 0.076 0.349 PAPUL 0.120 0.128 0.037 0.204 0.333 0.346 0.255 0.410 PAPUW 0.834 0.844 0.774 0.894 Ϫ0.218 Ϫ0.220 Ϫ0.400 Ϫ0.035 PAPLL 0.243 0.242 0.143 0.343 0.467 0.484 0.385 0.548 PL 0.190 0.187 0.134 0.245 Ϫ0.127 Ϫ0.130 Ϫ0.199 Ϫ0.055 Eigenvalue 0.051 0.050 0.040 0.062 0.023 0.023 0.018 0.028 % Variance explained 43.1 42.4 36.4 49.8 19.5 19 14.9 24.1

* Not signiﬁcantly different from 0.

DISCUSSION species were never observed on this host (data not shown), whereas L. kaohsianghsieni and L. pilengas were not found on Our results support the presence of 3 new sympatric species flathead mullets. This observation supports the Euzet and Sur- of Ligophorus on flathead mullets from the Japan Sea; they are iano (1977) view of species of Ligophorus as being predomi- well distinguished by features of the haptor and genital scler- nantly oioxenic. otized structures, which are meaningful for discrimination of The interspecific variability of most metric characters of the Ligophorus species (Euzet and Suriano, 1977; Mariniello et al., 2004; Sarabeev and Balbuena, 2004). In addition, we consider 3 new species is high and the variation ranges of all metric that the Ligophorus form drawn by Gussev (1955) from M. features overlapped among species. Only 3 characters, 2 of cephalus from the Tumen-Ula River (see Figs. 2, 3) and trans- them used for the first time in the present study for metric ferred by Euzet and Suriano (1977) to L. chabaudi actually analysis, seemed useful for species discrimination, namely, PA- corresponds to L. domnichi n. sp. (see above). Thus, the re- PUW for separation of L. cheleus n. sp. from L. pacificus n. sp. sulting distribution pattern would be congruent with previous and L. domnichi n. sp., and VAC and PAPLL for distinguishing evidence suggesting that flathead mullets from different seas the latter 2 species (Tables II and III). The problem of species harbor different species complexes of Ligophorus (Sarabeev et identification in Ligophorus stems from the high interspecific al., 2005), resulting from open oceans acting as geographical variability and wide overlapping variation ranges of the metric barriers for the coastal flathead mullet populations (Esch and characters, as revealed by previous studies resorting to multi- Fernańdez, 1993). variate analyses for specimen discrimination (Mariniello et al., The description of L. cheleus n. sp., L. pacificus n. sp., and 2004; Sarabeev and Balbuena, 2004; Rubtsova et al., 2006). In L. domnichi n. sp. from the flathead mullet raises the number the present study, the PCA analysis and jackknife procedure of Ligophorus species from mullets in the Japan Sea to 5. The fully support the ascription based on morphological features of other 2 species, L. kaohsianghsieni and L. pilengas, occur on the specimens on the flathead mullet from the Sea of Japan to M. soiuy. In an ongoing survey of so-iuy mullets from the same 3 species. localities of the Japan Sea as in the present study, the 3 new One interesting morphological finding of our study is the 780 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 presence of a U-shaped ovary in all 3 new species, because raneé. Bulletin du Museúm National d’Histoire Naturelle, 3e se´rie, previous descriptions of other species of Ligophorus report oval Zoologie 472: 799–821. FERNA´ NDEZ, J. 1987. Los para´sitos de la lisa Mugil cephalus L., en ovaries (see, for instance, Fig. 2 in Euzet and Suriano, 1977 Chile: sistema´tica y aspectos poblacionales (Perciformes: Mugili- concerning L. vanbenedenii [Parona and Perugia, 1890]; Fig. 1 dae). Gayana Zoolo´gica 51: 3–58. in Euzet and Sanfilippo, 1983 concerning L. parvicirrus Euzet GIBSON, A. R., A. J. BAKER, AND A. MOEED. 1984. Morphometric var- and Sanfilippo, 1983; Fig. 1 in Hassan et al., 1990 concerning iation in induced populations of the common myna (Acridotheres tristis): An application of the jackknife to principal component L. imitans Euzet and Suriano, 1977; Fig. 4 in Fernańdez, 1987 analysis. Systematic Zoology 33: 408–421. concerning L. huitrempe Fernańdez, 1987; Fig. 1 in Hu and Li, GUSSEV, A. V. 1955. Ancyrocephalus (S. L.) vanbenedenii (Par. et Per.) 1992 concerning L. chongmingensis Hu and Li, 1992; and Fig. (Monogenoidea) and its geographic distribution. Zoologicheskiy 1A in Sarabeev and Balbuena, 2004 concerning L. pilengas). Zhurnal 34: 291–294. ———. 1983. Methods for collection and preparation of monogeneans However, the U-shaped ovary of our specimens was revealed parasitizing fish. Nauka, Leningrad, USSR, 46 p. only when the worms were observed in lateral view, whereas ———. 1985. Order Dactylogyridea. In Keys to the parasites of fresh- the ovary looked oval in both ventral and dorsal views. This water fish of the USSR fauna. Metazoan parasites, vol. 2, A. V. led us to examine additional specimens of L. vanbenedenii, L. Gussev, M. N. Dubinina, O. N. Pugachev, E. V. Raykova, I. A. Hotenovskiy, and R. Ergens (eds.). Nauka, Leningrad, USSR, p. parvicirrus, L. imitans, and L. pilengas from our collections. 15–251. The ovary was also U-shaped in these species. Thus, the HAMMER,O.,AND D. A. T. HARPER. 2005 PAST. Paleontological data U-shaped ovary does not seem unusual for the species of Lig- analysis. http://folk.uio.no/ohammer/past. ophorus, and we suggest that further studies should establish HASSAN,S.H.,A.A.KHIDR, AND O. A. ABU SAMAK. 1990. Some helminth parasites of mugilid and sciaenid fishes from Mediterranean whether or not this character is shared by all members of the Sea in Egypt. Journal of the Egyptian–German Society of Zoology genus. 1: 155–167. HU, Z., AND D. LI. 1992. Two new species of monogenetic trematodes of marine fishes Mugil cephalus from the Chongming Island, ACKNOWLEDGMENTS Shanghai, China. Journal of Shanghai Teachers University (Natural Sciences) 21: 67–70. We thank Dr. Ludmila Shvetsova, Sector of Hydrobiont Diseases, MARINIELLO, L., M. ORTIS,S.D’AMELIO, AND V. P ETRARCA. 2004. Mor- Pacific Research Fisheries Centre, Vladivostok, Russia, for her help in phometric variability between and within species of Ligophorus collecting the material. This study was funded by the International As- Euzet & Suriano, 1977 (Monogenea: Ancyrocephalidae) in the sociation for the Promotion of Cooperation with Scientists from the Mediterranean Sea. Systematic Parasitology 57: 183–190. New Independent States of the Former Soviet Union (Project No. MIROSHNICHENKO, A. I., AND V. N . M ALTSEV. 1998. New for the Azov 03-51-5998). VLS benefited a postdoctoral fellowship (No. SB2003- and Black Sea basin species of gyrodactylus (Monogenea, Gyro- 0334) from the State Secretary of Universities and Research of the dactylidae) from haarder. Proceedings of the Southern Scientific Ministry of Education and Science of Spain. Research Institute of Marine Fisheries and Oceanography 44: 116– 125. RUBTSOVA, N. Y., J. A. BALBUENA,V.L.SARABEEV,I.BLASCO, AND L. LITERATURE CITED EUZET. 2006. Description and morphometrical variability of a new species of Ligophorus and of Ligophorus chabaudi (Monogenea: BALBUENA, J.A., N. YU.RUBTSOVA, AND V. L. SARABEEV. 2006. Ligo- phorus pilengas Sarabeev & Balbuena, 2004 (Monogenea: Ancy- Dactylogyridae) on Mugil cephalus (Teleostei) from the Mediter- rocephalidae) proposed as senior synonym of Ligophorus gussevi ranean Basin. Journal of Parasitology 92: 486–495. SARABEEV,V.L.,AND J. A. BALBUENA. 2004. Ligophorus pilengas n. Miroshnichenko & Maltsev, 2004. Systematic Parasitology 63: 93– sp. (Monogenea, Ancyrocephalidae) from the introduced so-iuy 96. mullet Mugil soiuy (Teleostei, Mugilidae) in the Sea of Azov and ROWLEY, P. H. 1992. Resampling methods for computation-intensive C the Black Sea. Journal of Parasitology 90: 222–228. data analysis in ecology and evolution. Annual Review of Ecology ———, ———, AND L. EUZET. 2005. Taxonomic status of Ligophorus and Systematics 23: 405–447. mugilinus (Hargis, 1955) (Monogenea, Ancyrocephalidae), with a DMITRIEVA, E. V. 1996. Fauna of Monogenea of the Far-East Mugil description of Ligophorus mediterraneus n. sp. from Mugil ce- soiuy in the Black Sea. Vestnik Zoologii 4-5: 95–97. phalus (Teleostei, Mugilidae) from the Mediterranean Basin. Jour- ESCH,G.W.,AND J. C. FERNA´ NDEZ. 1993. A functional biology of par- nal of Parasitology 91: 1444–1451. asitism. Chapman & Hall, London, U.K., 337 p. TER BRAAK, C. J. F. 1995. Ordination. In Data analysis in community EUZET, L., AND D. SANFILIPPO. 1983. Ligophorus parvicirrus n. sp. and landscape ecology, R. G. H. Jongman, C. J. F. Ter Braak, and (Monogenea, Ancyrocephalidae) parasite de Liza ramada (Risso, O. F. R. Van Tongeren (eds.). Cambridge University Press, Cam- 1826) (Teleostei, Mugilidae). Annales de Parasitologie Humaine et bridge, U.K., p. 91–173. Compareé 58: 325–335. ZHUKOV, E. V. 1970. New trematoda species from marine and fresh- ———, AND D. M. SURIANO. 1977. Ligophorus n. g. (Monogenea, An- water fish from basins of the Japan and the Yellow Seas. Parasi- cyrocephalidae) parasite des Mugilidae (Te´leósteéns) en Me´diter- tologiya 2: 155–161. J. Parasitol., 93(4), 2007, pp. 781–786 ᭧ American Society of Parasitologists 2007

TWO NEW SPECIES OF HALIOTREMA (MONOGENOIDEA: DACTYLOGYRIDAE) FROM ACANTHURUS NIGROFUSCUS AND ACANTHURUS OLIVACEUS (TELEOSTEI: ACANTHURIDAE) IN THE SOUTH CHINA SEA

Yuan Sun, Delane C. Kritsky*, and Tingbao Yang† Research Center for Parasitic Organisms and State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshan University, Guangzhou, 510275, China. e-mail: [email protected]

ABSTRACT: Haliotrema leporinus n. sp. and Haliotrema pratasensis n. sp. are described from the gills of surgeonﬁshes (Acan- thuridae) off the Pratas Islands in the South China Sea. Haliotrema leporinus n. sp. differs from all other members of the genus by possessing a rabbit-head–shaped sclerotized piece in the haptor. Haliotrema pratasensis n. sp. differs from its congeners by having a copulatory organ with a clockwise coil and a funnel-shaped base and by lacking an accessory piece.

Many studies on the dactylogyrid parasites of the 81 valid cyrocephalus longispicularis Yamaguti, 1968 and Pseudancy- species of fishes comprising the Acanthuridae (Froese and Pau- rocephalus nasonis Yamaguti, 1968 from N. brevirostris. ly, 2005) have been carried out. MacCallum (1915) described Young (1968) reported 4 dactylogyrids from Australian acan- Diplectanum teuthis MaCallum, 1915 from Teuthis hepatus, thurids: Haliotrema dempsteri (Mizelle and Price, 1964) Young, Linnaeus (ϭParacanthurus hepatus (Linnaeus, 1766)), from the 1968 from A. mata, A. dussumieri, and Acanthurus xanthopte- New York Aquarium. Yamaguti (1940) reported Haliotrema xe- rus Valenciennes; Haliotrema ctenochaeti Young, 1968 from C. suri Yamaguti, 1940 from Xesurus scalprum Cuvier et Valen- strigosus; Haliotrema lineati Young, 1968 from Acanthurus li- ciennes (ϭPrionurus scalprum Valenciennes) from Mie Prefec- neatus (Linnaeus); Haliotrema parahaliotrema (Mizelle and ture, Japan. Mizelle (1963) described Cleithrarticus cleithrar- Price, 1964) Young, 1968, from Z. veliferum and Acanthurus ticus Mizelle, 1963 from Acanthurus olivaceus Bloch and grammoptilus Richardson. Young (1968) also transferred D. Schneider from the Southwest Pacific Ocean; this species was teuthis to Haliotrema, raised Ancyrocephalus teuthis eilaticus found by Young (1980) from the same host in Australia. An- to specific rank and transferred it to Haliotrema as Haliotrema cyrocephalus teuthis eilaticus on Acanthurus sp. was described eilatica, and proposed Parahaliotrema Mizelle and Price, 1964, by Paperna (1965) from the Gulf of Eilat, Israel. a junior synonym of Haliotrema Johnston and Tiegs, 1922. Yamaguti (1968) recorded 16 new dactylogyrid species and Mizelle and Kritsky (1969), unaware of the nearly concurrent redescribed 1 species from 14 acanthurid species in Hawaii: paper by Young, erected Parahaliotrema zebrasoma Mizelle Haliotrema acanthuri Yamaguti, 1968 from Acanthurus sand- and Kritsky, 1969, from Z. veliferum from Hawaii. Vala et al. vicensis Streets (ϭAcanthurus triostegus (Linnaeus)); Haliotre- (1982) transferred this species to Haliotrema based on Young’s ma angulare Yamaguti, 1968 from Acanthurus nigroris Valen- (1968) suggestion. ciennes; Haliotrema brotulae Yamaguti, 1968 from Zebrasoma Paperna (1972) found H. eilatica and H. ctenochaeti from flavescens (Bennett); Haliotrema ctenochaeti Yamaguti, 1968 unidentified acanthurids in the northwest Eilat Gulf, Red Sea. from Ctenochaetus strigosus (Bennett) and Acanthurus nigro- Machida (1979) recorded P. duplicatus and Nasoancyroce- phalus diorchis N. unicornis fuscus (Forsska˚l); Haliotrema flexicirrus Yamaguti, 1968 from Machida, 1979 from from Oki- nawa, Japan. Mailard and Dossou (1979) described Cleithrar- A. nigroris, A. nigrofuscus, and Acanthurus leucopareius (Jen- ticus beninensis Mailard and Dossou, 1979 (the genus name kins); Haliotrema macracantha Yamaguti, 1968 from A. ni- was misspelled Cleitharticus in this paper) from Acanthurus groris, Acanthurus thompsoni (Fowler), Zebrasoma veliferum monroviae Steindachner off the Gulf of Benin (Africa). Zhukov (Bloch), and Z. flavescens; Haliotrema palmatum Yamaguti, (1980) reported Haliotrema ampliomacrohamus Zhukov, 1980 1968 from Acanthurus dussumieri Valenciennes and Acanthu- and Haliotrema ampliomicrohamus Zhukov, 1980 from Acan- rus mata Cuvier; Haliotrema serpenticirrus Yamaguti, 1968 thurus chirurgus (Bloch) and H. teuthis from A. chirurgus and from A. dussumieri; Haliotrema sigmoidocirrus Yamaguti, 1968 Acanthurus coeruleus Bloch and Schneider from the Gulf of from A. mata and A. dussumieri; Haliotrema zancli Yamaguti, Mexico. Dyer et al. (1989) recorded Haliotrema alatum Ya- 1968 from A. dussumieri, A. mata, A. olivaceus, and C. stri- maguti, 1942 from Acanthurus bariene Lesson; Haliotrema gosus; Cleithrarticus bulbovagina Yamaguti, 1968 from A. dus- upenei Yamaguti, 1953 from A. nigrofuscus; and Pseudohali- sumieri C. cleithrarticus A. olivaceus A. dussumieri ; from , , and otrema sphincteroporus Yamaguti, 1953 from A. lineatus and A. mata; Pseudancyrocephalus duplicatus Yamaguti, 1968 from A. olivaceus from Okinawa, Japan. Naso unicornis (Forsska˚l); Pseudancyrocephalus kala Yama- During a study of the monogenoids of surgeonfishes (Acan- guti, 1968 from Naso brevirostris (Cuvier) and Naso hexacan- thuridae) collected from the water surrounding the Pratas Is- thus (Bleeker); Pseudancyrocephalus longicirrus Yamaguti, lands in the South China Sea, 2 undescribed species of Hali- 1968 from N. brevirostris and N. hexacanthus; and Pseudan- otrema were found. In this paper, the 2 new species are described. Received 18 October 2006; revised 3 January 2007; accepted 8 Feb- ruary 2007. MATERIALS AND METHODS * Department of Health and Nutrition Sciences, College of Health Pro- fessions, Campus Box 8090, Idaho State University, Pocatello, Idaho Fish hosts were collected near the Pratas Islands in the South China 83209. Sea (20Њ33Ј–21Њ35ЈN, 115Њ43Ј–117Њ07ЈE) by fishermen and transported † To whom correspondence should be addressed. to Nan’ao, Shenzhen, in May 2004. Gills baskets of each fish were

781 782 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 separately removed and placed in seawater, and dactylogyrids were col- oo¨type; oo¨type short, opening into thick-walled uterus. Vaginal aperture lected from the gills with a fine needle under a dissecting microscope. dextromarginal, vaginal vestibule pyriform to globose, with longitudinal Specimens were lightly flattened under a coverslip, mounted in am- muscles; vaginal duct narrow, running posteriorly to join subpyrimidal monium picrate glycerine for examination of sclerotized structures, and seminal receptacle. Vitellarium dense throughout trunk, absent in re- subsequently removed from the slides, then stained with Gomori’s tri- gions of other reproductive organs. chrome and mounted in Canada balsam for soft-anatomy studies and permanent preservation (Yang et al., 2004). Illustrations were prepared Taxonomic summary using an Olympus BX41 microscope with the aid of a drawing apparatus. Measurements, all in micrometers, are expressed as the mean Type host: Acanthurus nigrofuscus (Forsska˚l). Њ Ј Њ Ј followed by the range and number (n) of structures measured in paren- Type locality: Pratas Islands, South China Sea (20 33 –21 35 N, Њ Ј Њ Ј theses. Body length includes that of haptor. The measurement of the 115 43 –117 07 E). copulatory organ represents an approximation of total length obtained Site of infestation: Gills. by means of a map meter CV- 9 Jr (Koizumi Sokki Mfg. Co., Ltd., Type specimens: Holotype (ZSU 20040519-1), 12 paratypes (ZSU Japan). Dimensions of other internal structures represent straight-line 20040519-2-13), 1 paratypes (USNPC 99553), 1 paratype (BMNH distances between extreme ends; the body width is that of the greatest 2007.2.21.4). ϭ width of the trunk. Numbering of hook pairs follows that recommended Etymology: The specific name is from Latin (leporis a hare) and by Mizelle (1936) and Mizelle and Price (1963). Type and voucher refers to the shape of the sclerotized piece associated with the ventral specimens are deposited in the School of Life Sciences, Zhongshan extrinsic adductor muscles in the haptor. University (ZSU), Guangzhou, People’s Republic of China. Some paratypes are deposited in the U.S. National Parasite Collection, Beltsville, Remarks Maryland (USNPC) and the Natural History Museum, London, U.K. The tubular copulatory organ without an accessory piece of Hali- (BMNH). For comparative purposes, the following specimens were ex- otrema leporinus n. sp. resembles those of Haliotrema bilobatus; Hal- amined: holotype, Haliotrema flexicirrus Yamaguti, 1968 (USNPC iotrema parvihamus; Haliotrema neobilobatus Bychowsky and Nagi- 63625); holotype, Pseudohaliotrema zanclus Mizelle and Price, 1964; bina, 1970; Haliotrema caesionis Yamaguti, 1953; Haliotrema kurodai and holotype, Pseudohaliotrema canescens Mizelle and Price, 1964 Ogawa and Egusa, 1978; and Haliotrema spiculare Yamaguti, 1968. (USNPC 60015). However, the haptoral sclerites differentiate H. leporinus n. sp. from these species. The dorsal bar with rounded ends in H. leporinus n. sp. DESCRIPTIONS serves to distinguish it from H. bilobatus, H. parvihamus, H. neobilobatus, and H. spiculare (ends bifid in latter species). The broadly U- Haliotrema leporinus n. sp. shaped ventral bar and straight dorsal bar of H. leporinus n. sp. differ (Figs. 1–9) from those of H. kurodai and H. caesionis (in H. kurodai, the bars are Description (based on the holotype and 14 paratypes collected from nearly straight; in H. caesionis, the dorsal bar has a distinct median A. nigrofuscus): Body fusiform, 703 (583–875; n ϭ 9) long; greatest notch). The most remarkable feature of the new species is the rabbit- width 165 (115–212; n ϭ 9) usually at level of testis. Cephalic region head–shaped sclerotized piece in the haptor, which is a unique morpho- broad; cephalic lobes moderately developed; 3 bilateral pairs of head logical feature in Haliotrema and finally helps to differentiate H. le- organs; bilateral pair of cephalic glands at pharyngeal level. Eyespots porinus n. sp from those above-mentioned species and other congeners. absent; accessory chromatic granules minute, scattered in cephalic, an- ϭ terior trunk regions. Pharynx subspherical, 48 (37–68; n 13) in great- Haliotrema pratasensis n. sp. est width; esophagus short; intestinal bifurcation postpharyngeal, ceca (Figs. 10–16) confluent posterior to gonads, lacking diverticula. Peduncle broad, short, tapered posteriorly. Description (based on the holotype and 14 paratypes collected from Haptor 131 (85–151; n ϭ 8) long, 174 (132–236; n ϭ 9) wide, dif- A. olivaceus): Body fusiform, 574 (458–842; n ϭ 12) long; greatest ferentiated from body proper. A rabbit-head–shaped sclerotized piece width 176 (129–246; n ϭ 15), usually at level of testis. Cephalic region located medially in haptor, 46 (40–49; n ϭ 12) long, 34 (26–47; n ϭ broad, short; cephalic lobes moderately developed; 3 bilateral pairs of 12) wide, with 4 tapered bilateral projections, members of anterior pair head organs; bilateral pair of cephalic glands at pharyngeal level. Eye- longer and more slender than those of posterior pair. Ventral extrinsic spots absent; accessory chromatic granules minute, scattered in cephal- adductor muscle (Kearn, 1971) well developed, passing through this ic, anterior trunk regions. Pharynx subspherical, 46 (37–55; n ϭ 15) in sclerotized piece and connecting to 2 lateral small half-moon–shaped greatest width; esophagus broad, elongate; intestinal bifurcation anterior structures near tips of superficial roots of ventral anchors. to male copulatory organ; ceca confluent posterior to gonads, lacking Ventral anchor 92 (88–95; n ϭ 13) long, with moderately elongate diverticula. Peduncle broad, short. superficial root, short deep root, curved shaft; base 38 (33–41; n ϭ 13) Haptor 94 (85–107; n ϭ 12) long, 129 (102–171; n ϭ 12) wide, wide. Dorsal anchor 82 (72–93; n ϭ 6) long, with small fenestration, slightly marked from trunk, with 10 short bilaterally paired lobes each hinged base, shaft slender, occasionally bent 90Њ from the base along containing a hook; ventral extrinsic adductor muscle well developed, diagonal hinge, elongate superficial root, poorly defined deep root; base connecting to two half-moon–shaped pieces near tips of superficial roots 40 (27–50; n ϭ 6) wide. Anchor wings fine, conspicuous. Ventral bar of ventral anchors. Ventral anchor 55 (51–58; n ϭ 11) long, fenestrated, 102 (100–105; n ϭ 13) long, inverted, broadly U-shaped, ends rounded with short superficial and deep roots, curved shaft; base 26 (23–28; n with terminal scales. Dorsal bar straight, 97 (94–101; n ϭ 12) long, ϭ 11) wide. Dorsal anchor 61 (58–64; n ϭ 10) long, fenestrated, with with bilateral broad subterminal posterior protrusions, 2 bilateral ante- elongate superficial root, poorly developed deep root, hinged base; base rior protuberances originating more medially from the bar extremities. 27 (19–32; n ϭ 10) wide. Anchor wings fine, conspicuous. Ventral bar Hook similar, 11–12 (n ϭ 47) long, with terminally depressed thumb, 49 (46–53; n ϭ 12) long, broadly U-shaped, with 2 large anterior round- slender shank comprising 1 subunit, delicate point; filamentous hooklet ed expansions. Dorsal bar 45 (42–47; n ϭ 12) long, straight, with pos- loop about shank length. teriorly knobbed ends. Hooks similar, 12 (11–12; n ϭ 24) long, with Testis 106 (80–122; n ϭ 8) long, 90 (61–144; n ϭ 8) wide, sub- depressed acute thumb, delicate point, slender shank comprising 1 subspherical, intercecal, posterior to body equator. Vas deferens thick, loop- unit; filamentous hooklet loop about shank length. ing left intestinal cecum, turning back on itself anteriorly forming lon- Testis 62 (49–89; n ϭ 10) long, 39 (30–50; n ϭ 9) wide, bacilliform, gitudinal seminal vesicle lying alongside prostatic reservoir. Copulatory intercecal, lying in posterior third of body; proximal vas deferens not organ 90 (85–98; n ϭ 12) long, tubular, bent near base and then ante- observed, distal vas deferens passing ventrally to left intestinal cecum, riorly directed, slightly bowed along its distal portion; base funnel- turning back on itself to form seminal vesicle. Prostatic reservoir sig- shaped; accessory piece absent. Prostatic reservoir large, reniform, ta- moid. Copulatory organ 169 (149–181; n ϭ 15) long, a coil of 1¼ pered distally, prostatic glands conspicuous, radiating around proximal clockwise rings, with funnel-shaped base; accessory piece absent. Ova- end of prostatic reservoir. ry 85 (46–129; n ϭ 14) long, 46 (23–73; n ϭ 15) wide, subovate, Ovary 57 (33–79; n ϭ 9) long, 38 (30–60; n ϭ 9) wide, subspherical, pretesticular, slightly overlapping testis; oviduct short; oo¨type large, sur- pretesticular, slightly overlapping testis; oviduct passing immediately to rounded by Mehlis’ gland; uterus arising from oo¨type. Vaginal aperture SUN ET AL.—TWO NEW DACTYLOGYRID SPECIES 783

FIGURES 1–9. Haliotrema leporinus n. sp. (1) Whole worm (dorsal, composite). (2) Copulatory complex (dorsal view). (3) Ventral bar. (4) Dorsal bar. (5) Rabbit-head–shaped sclerotized piece. (6) Ventral anchor. (7, 8) Doral anchor. (9) Hook. 784 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 10–16. Haliotrema pratasensis n. sp. (10) Whole worm (ventral, composite). (11) Copulatory complex. (12) Ventral bar. (13) Dorsal bar. (14) Ventral anchor. (15) Doral anchor. (16) Hook. SUN ET AL.—TWO NEW DACTYLOGYRID SPECIES 785 dextromarginal; vaginal canal narrow, weakly sclerotized, running Haliotrema remained polyphyletic. Plaisance et al. (2005) in- obliquely posteriorly to join small inconspicuous seminal receptacle. vestigated the phylogenetic relationship among species of Eu- Vitellarium dense throughout trunk, absent in regions of other reproductive organs. ryhaliotrematoides, Aliatrema, and Haliotrema from the Chae- todontidae based on sequences of 18S rDNA, 28S rDNA (D1– Taxonomic summary D3), and 16S rDNA. They concluded that these dactylogyrids Type host: Acanthurus olivaceus, Bloch and Schneider, 1801. are divided into 2 independent lineages: one clade contains spe- Type locality: Pratas Islands, South China Sea (20Њ33Ј–21Њ35ЈN, cies of Aliatrema and Euryhaliotrematoides and the other in- 115Њ43Ј–117Њ07ЈE). cludes species of Haliotrema. These studies supported Plais- Site of infestation: Gills. ance and Kritsky’s classification. Type specimens: Holotype (ZSU 20040520-1), 12 paratypes (ZSU 20040520-2-13), 1 paratype (USNPC 99552), 1 paratype (BMNH Haliotrema pratasensis n. sp. resembles species of Aliatrema 2007.2.21.3). Plaisance and Kritsky, 2004, which is characterized mainly by Etymology: The specific name refers to Pratas Island, near which the species having a counterclockwise coiled copulatory organ un- host was collected. supported by an accessory piece and with a funnel-shaped base, dextral vagina, and hooks with upright acute thumb. Aliatrema Remarks includes only 1 species, Aliatrema cribbi, collected from mem- Haliotrema pratasensis n. sp. differs from all other species of Hali- bers of the Chaetodontidae. However, H. pratasensis n. sp. otrema by the male copulatory organ (coiled, funnel-shaped base and lacking accessory piece) and the morphology of anchors and bars. Hal- lacks eyes and has a clockwise copulatory organ, which distin- iotrema pratasensis n. sp. resembles Haliotrema longitubocirrus By- guishes it from species of Aliatrema that possess 2–4 eyes and chowsky and Nagibina, 1971 and Haliotrema tiegsi Bychowsky and a counterclockwise copulatory organ. Moreover, the anatomical Nagibina, 1970, by the shape of male copulatory organ. The copulatory structures of Aliatrema species, such as the intestinal ceca and organ of Haliotrema pratasensis n. sp. differs from that of H. longitu- vas deferens, have not been described. bocirrus by its smooth distal portion, whereas in H. longitubocirrus the distal portion is with an accessory piece. In H. tiegsi, Bychowsky and Kearn (1971) reported 2 prominent extrinsic muscles passing Nagibina (1970) described an obvious accessory piece at the distal por- through a muscular or fibrous ring and diverging sharply from tion of the male copulatory organ, but Zhang et al. (2001) did not one another in the haptor of Haliotrema balisticus (Hargis, describe this structure in their redescription of H. tiegsi with the spec- 1955) Yamaguti, 1963. In the haptor of Haliotrema leporinus imens collected from the same host from the South China Sea. Our examination to Zhang et al.’s (2001) and our newly collected specimens n. sp., we found a large rabbit-head–shaped sclerotized piece from the same host verified the existence of an accessory piece, which with 4 projections in the middle of the haptor, which serves as is consistent with Bychowsky and Nagibina’s description. Nonetheless, the ring. Two bilateral extrinsic muscles pass through this scler- the new species can also be distinguished from H. tiegsi by the broadly otized piece between its anterior pair of projections, then sep- U-shaped ventral bar and the straight dorsal bar, whereas in H. tiegsi arate from each other along posterior pair members and finally the ventral bar is straight and dorsal bar is V-shaped. connect to 2 small half-moon–shaped pieces near the tips of the superficial roots of the ventral anchors. This large sclero- DISCUSSION tized piece is only found in H. leporinus n. sp. at present, but Haliotrema was erected by Johnston and Tiegs (1922) for the small pieces near the tips of the superficial roots of the Haliotrema australe Johnston and Tiegs, 1922, from the black- ventral anchors were also found in H. pratasensis n. sp.; Hal- spot goatfish Upeneus signatus (Gu¨nther, 1867) (Mullidae) iotrema ampliocuspidis; Haliotrema geminatohamula; H. lon- (ϭParupeneus signatus (Gu¨nther, 1867)), from Australia. It was gitubocirrus; Haliotrema subtilihamula; Haliotrema surculocir- diagnosed by having a haptor armed with dorsal and ventral rus; Haliotrema tenuicirrus Bychowsky and Nagibina, 1971; pairs of anchors with each pair joined by a transverse bar, con- Haliotrema magnigastrohamus Zhukov, 1976; Haliotrema au- fluent intestinal ceca, vas deferens looping the left intestinal ribaculum; Haliotrema myripristisi; and Haliotrema tenuiha- cecum, and the vagina opening dextrally. The genus Haliotrema mus Zhukov, 1980; and Haliotrema dicollinum Zhang et al. now contains 157 species, including our 2 new species (Kritsky 2001. However, the small pieces in H. dicollinum, H. auriba- and Stephens, 2001; Zhang et al., 2001; Wang et al., 2003; culum, H. myripristisi, and H. tenuihamus are near the super- Plaisance et al., 2004; Li and Chen, 2005; Li, Zhang et al., ficial roots of dorsal anchors. 2005; Li, Yan et al., 2005). Euzet and Suriano (1977) transferred 2 Haliotrema species ACKNOWLEDGMENTS from mugilids to Ligophorus Euzet and Suriano, 1977, which We would like to thank Zhang Jianying of South China Normal Uni- is characterized by a long tubular copulatory organ with an versity, China, for providing literature. Yang Hongzhi of the Fishery accessory piece and a sclerotized vagina. Kritsky and Boeger Institute of Longgang District, Shenzhen, Guangdong Province allowed us to use the laboratory and other relevant facilities during field collec- (2002) established Euryhaliotrema for dactylogyrids from the tions. Financial support was provided by grants from the National Nat- Sciaenidae, Sparidae, Lutjanidae, and Haemulidae. The genus ural Science Fund (30370242), the National 863 Science and Technol- was diagnosed by a coiled counterclockwise copulatory organ ogy Project (2001AA621010), and the Natural Science Fund of Guang- with a bulbous base and supported by an accessory piece. In dong Province (2002C20312). this study, the authors considered the morphological boundaries of Haliotrema not well established and transferred 9 Haliotrema LITERATURE CITED species to Euryhaliotrema. Plaisance and Kritsky (2004) pro- BYCHOWSKY, B. E., AND L. F. NAGIBINA. 1970. On the new and little posed Euryhaliotrematoides and Aliatrema for parasites of spe- known species of the genus Haliotrema Johnston et Tiegs, 1922 (Monogenoidea). 1. Zoologicheskii Zhurnal 49: 1789–1801. cies of the Chaetodontidae, transferred 4 Haliotrema species to ———, AND ———. 1971. New and little known species of the genus Euryhaliotrematoides, and suggested that Ligophorus, Euryhal- Haliotrema Johnston et Tiegs, 1922 (Monogenoidea). 2. Zoologi- iotrema, and Euryhaliotrematoides were monophyletic, whereas cheskii Zhurnal 50: 25–40. 786 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

DYER, W. G., E. H. WILLIAMS,JR., AND L. B. WILLIAMS. 1989. Mono- obtained from the Japanese black sea bream, Acanthopagrus schle- geneans from marine fishes of Okinawa, Japan. Proceedings of the geli (Bleeker). Bulletin of the Japanese Society of Scientific Fish- Helminthological Society of Washington 56: 64–68. eries 44: 1329–1332. EUZET, L., AND D. M. SURIANO. 1977. Ligophorus n. g. (Monogenea, PAPERNA, I. 1965. Contribution to the knowledge of the Red Sea No. Ancyrocephalidae) parasite des Mugilidae (Te´leósteéns) en Me´di- 32. Monogenetic trematodes from the gills of Red Sea fishes. Bul- terraneé. Bulletin du Museúm National d’Histoire Naturelle (Zool- letin Sea Fishes Researches Station Israel 39: 1–10. ogie) 329: 799–822. ———. 1972. Monogenea of Red Sea Fishes. III. Dactylogyridae from FROESE,R.,AND D. PAULY. 2005. FishBase. Available at: www. littoral and reef fishes. Journal of Helminthology 46: 47–62. fishbase.org. Accessed 25 October 2005. PLAISANCE, L., S. BOUAMER, AND S. MORAND. 2004. Description and HARGIS, W. J. 1955. Monogenetic trematodes of Gulf of Mexico fishes. redescription of Haliotrema species (Monogenoidea: Poloyoncho- II. The superfamily Gyrodactyloidea (continued). Journal of Para- inea: Dactylogyridae) parasitizing butterfly fishes (Teleostei: Chae- sitology 41: 185–193. todontidae) in the Indo-West Pacific Ocean. Parasitology Research JOHNSTON,T.H.,AND O. W. TIEGS. 1922. New gyrodactyloid trematodes 93: 72–78. from Australian fishes, together with a reclassification of the su- ———, AND D. C. KRITSKY. 2004. Dactylogyrids (Platyhelminthes: perfamily Gyrodactyloidea. Proceedings of the Linnaean Society of Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodon- New South Wales 47: 83–131. tidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia KEARN, G. C. 1971. The attachment of the ancyrocephalid monogenean and Australia: Species of Euryhaliotrematoides n. gen. and Alia- Haliotrema balisticus to the gills of the trigger fish, Balistes ca- trema n. gen. Journal of Parasitology 90: 328–341. priscus (ϭcarolinensis). Parasitology 63: 157–162. ———, D. T. LITTLEWOOD,P.D.OLSON, AND S. MORAND. 2005. Mo- KRITSKY, D. C., AND W. A. B OEGER. 2002. Neotropical Monogenoidea. lecular phylogeny of gill monogeneans (Platyhelminthes, Mono- 41: New and previously described species of Dactylogyridae genea, Dactylogyridae) and colonization of Indo-West Pacific but- (Platyhelminthes) from the gills of marine and freshwater perciform terflyfish hosts (Perciformes, Chaetodontidae). Zoologica Scripta fishes (Teleostei) with proposal of a new genus and a hypothesis 34: 425–436. on phylogeny. Zoosystema 24: 7–40. VALA, J. C., C. MAILLARD, AND R. M. OVERSTREET. 1982. Haliotrema ———, AND F. S TEPHENS. 2001. Haliotrema abaddon n. sp. (Monoge- (Monogenea: Ancyrocephalinae) from ostraciid fishes in Guade- noidea: Dactylogyridae) from the gills of wild and maricultured loupe, West Indies. Journal of Parasitology 68: 1130–1137. West Australian dhufish Glaucosoma hebraicum (Teleostei: Glau- WANG, W. B., L. LIU, AND Y. X . Z HOU. 2003. Description of four new cosomatidae), in Australia. Journal of Parasitology 87: 749–754. species of Haliotrema Johnston and Tiegs (Monogenea, Ancyro- LI,H.Y.,AND Y. J. CHEN. 2005. Description of two new species of the cephalidae). Acta Zootaxonomica Sinica 28: 24–29. genus Haliotrema from marine fishes in Guangdong. Marine Sci- YAMAGUTI, S. 1940. Studies on the helminth fauna of Japan. Part 31. ences 29: 50–53. Trematodes of fishes, VII. Japanese Journal of Zoology 9: 35–108. ———, X. Z. YAN,X.W.YU,Q.L.LAN, AND X. Y. HUANG. 2005. ———. 1942. Studies on the helminth fauna of Japan. Part 37. Trem- Description of two new species of Ancyrocephalidae (Monogeno- atodes of fishes, VIII. Proceeding from Japanese Journal of Medical idea). Journal of Guangxi Normal University 23: 86–89. Sciences, VI. Bacteriology and Parasitology 2: 105–130. ———. 1953. Parasitic worms mainly from Celebes. Part 2. Monoge- ———, J. Y. ZHANG,L.E.CHEN, AND Y. J. CHEN. 2005. Description of two new species of Ancyrocephalidae (Monogenoidea). Acta netic trematodes of fishes. Acta Medicinae Okayama 8: 203–256. Scientiarum Naturalium Universitatis SunYat-Seni 44: 91–93. ———. 1963. Systema helminthum. Volume IV: Monogenea and As- pidocotylea. Interscience Publishers, New York, 669 p. MACCALLUM, G. A. 1915. Some new species of ectoparasitic trema- ———. 1968. Monogenetic trematodes of Hawaiian fishes. University todes. Zoologica 1: 395–410. of Hawaii Press, Honolulu, Hawaii, 287 p. MACHIDA, M. 1979. Dactylogyrid monogeneans from surgeonfish of YANG, T. B., D. C. KRITSKY AND Y. S UN. 2004. Revision of Allobene- southern Japan. Bulletin of the National Science Museum Series A denia Yamaguti, 1963 (Monogenoidea: Capsalidae) with the de- (Zoology) 5: 83–87. scription of A. zhangi n. sp. from Epinephelus fasciatus (Teleostei: MAILARD, C., AND C. DOSSOU. 1979. Cleitharticus beninensis n.sp. Serranidae) in the South China Sea. Systematic Parasitology 59: (Monogenea), a gill parasite of Acanthurus monroviae (Teleostei) 223–233. from the Gulf of Benin. Bulletin de l’Institut Fondamental YOUNG, P. C. 1968. Ten new species of Haliotrema (Monogenoidea: d’Afrique Noire 41: 317–323. Dactylogyridae) from Australian fish and a revision of the genus. MIZELLE, J. D. 1936. New species of trematodes from the gills of Illinois Journal of Zoology (London) 154: 41–75. fishes. American Midland Naturalist 17: 785–806. ———. 1980. The taxonomy of some Dactylogyrid Monogenoidea ———. 1963. Studies on monogenetic trematodes, XXIV. A new dac- from Australian fishes. Zoologischer Anzeigen 180: 269–279. tylogyrid genus from Acanthurus olivaceus Bloch. Journal of Par- ZHANG, J. Y., T. B. YANG, AND L. LIU. 2001. Monogeneans of Chinese asitology 49: 752–753. marine fishes. Agriculture Press, Beijing, People’s Republic of Chi- ———, AND D. C. KRITSKY. 1969. Studies on monogenetic trematodes. na, 400 p. XL. New species from marine and freshwater fishes. American ZHUKOV, E. V. 1976. New monogenean species of the genus Haliotrema Midland Naturalist 82: 417–428. Johnston and Tiegs, 1922, from the Gulf of Mexico fishes of the ———, AND C. E. PRICE. 1963. Additional haptoral hooks in the genus Fam. Lutianidae, in fauna systematics and phylogeny of Monoge- Dactylogyrus. Journal of Parasitology 49: 1028–1029. noidea. Proceedings, Institute of Biology and Pedology, Far-East ———, AND ———. 1964. Studies on monogenetic trematodes. XXV. Science Centre, Academy of Sciences of the U.S.S.R., New Series Six new species of Ancyrocephalinae from the gills of Zanclus 35: 33–47. canescens (Linnaeus) with a key to the genera of Ancyrocephali- ———. 1980. Monogenea of the genus Haliotrema Johnston et Tiegs, nae. Journal of Parasitology 50: 81–89. 1922 from the gills of fishes fam. Holocentridae and fam. Acan- OGAWA,K.AND S. EGUSA. 1978. Haliotrema kurodai n. sp. (Monoge- thuridae of the Gulf of Mexico. Parazitologiya (Leningrad) 29: 41– nea: Dactylogyridae, Ancyrocephalinae), a monogenean parasitic 52. J. Parasitol., 93(4), 2007, p. 787 ᭧ American Society of Parasitologists 2007

IN MEMORIAM

ROBERT L. CALENTINE 1929–2007

Dr. Robert Leland ‘‘Bob’’ Calentine passed away on 10 May 2007 their fish and aquatic oligochaete hosts. Numerous publications dealing at his home in River Falls, Wisconsin. Bob was born to Royce L. and with the caryophyllid genera Archigetes, Biacetabulum, Monobothrium, Anna M. Calentine on 17 October 1929 in Cylinder, Iowa, and grew Khawia, Glaridacris, and Penarchigetes resulted from these studies, and up working on his parents’ farm in Ruthven, Iowa. Upon graduating as several of these papers were coauthored with undergraduate students. his high school’s valedictorian in 1948, he began his undergraduate In addition to being an accomplished parasitologist, Bob was truly a education at Iowa State University. His father’s death during his first biologist in the broadest sense. His knowledge of vertebrate and inver- year required him to interrupt his schooling to manage the family farm. tebrate taxonomy and natural history was exceptional, making his ‘‘old Bob enrolled at Iowa State Teacher’s College–Cedar Falls in 1950 and school’’ field biology class difficult yet extremely popular with students. obtained his B.A. in Math and Chemistry in 1953. Following service Spending an afternoon with Bob in the field provided an education in the U.S. Army from 1953–1955, he returned to Iowa State Teacher’s experience that could not be matched in any classroom. College and received an M.A. in Science Education in 1956. That same Although the University of Wisconsin–River Falls did not offer a year he was hired to teach all biological science courses at Ellsworth doctoral degree, Bob’s dedication to, and enthusiasm for, parasitology Community College in Iowa Falls. The following year, 1957, he married research inspired in numerous undergraduates a passion for parasitolog- Marilyn Campbell. After three years, his interest in fisheries prompted ical research—several of us in the American Society of Parasitologists him to begin studies for his doctorate at Iowa State University, but owe our careers to Bob’s enthusiasm for science and his tutelage and during this process he became enthralled with fish parasitology; con- encouragement during the time we were students in biology at the Uni- sequently, he conducted his doctoral studies under the direction of Dr. versity of Wisconsin–River Falls. Martin J. Ulmer. These studies culminated in the awarding of a Ph.D. Bob retired in May 1992 to pursue his other loves. He was an avid in 1963 based on dissertation research titled ‘‘The Life Cycle of Ar- and accomplished gardener and an award-winning photographer. His chigetes iowensis (Cestoda: Caryophyllaeidae).’’ photographs can be found in numerous biology textbooks and in the Following graduation, Bob took a faculty position at the University archives of the Nikon Small World photo pictorials. Bob also raised of Wisconsin–River Falls, in large part due to the location of River German Wirehaired Pointers and spent many hours with these dogs, and Falls near the Kinnickinnic, Willow, and St. Croix rivers. These waters fortunate friends, in pursuit of ring-necked pheasants and ruffed grouse. proved to be rich sources of catostomid and cyprinid fishes, aquatic Bob was preceded in death by both parents and one sister, Ruby oligochaetes, and, of course, caryphyllaeid cestodes. Despite heavy Bartlett. He is survived by his three children, Lori, Bobbi, and Randy teaching loads that included courses in general biology, histology, par- Calentine, and his sister Ruth Johnson. He will be sadly missed by all asitology, field biology, and microscopy, as well as a tour of duty as who knew him. Chair of the Department of Biology, Bob obtained Public Health Ser- vice grant support and continued a major research program on cary- Bruce M. Christensen, Department of Pathobiological Sciences, Uni- ophyllaeid tapeworms and the ecological relationships they have with versity of Wisconsin–Madison, Madison, Wisconsin 53706

787 J. Parasitol., 93(4), 2007, pp. 788–795 ᭧ American Society of Parasitologists 2007

THE FIRST CHONDRACANTHID (COPEPODA: CYCLOPOIDA) REPORTED FROM CULTURED FINFISH, WITH A REVISED KEY TO THE SPECIES OF CHONDRACANTHUS

Danny Tang, Melanie Andrews*, and Jennifer M. Cobcroft* School of Animal Biology (M092), The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. e-mail: [email protected]

ABSTRACT: A new species of the Chondracanthidae (Copepoda: Cyclopoida), Chondracanthus goldsmidi, is described based on material collected from the nasobranchial region of striped trumpeter (Latris lineata [Forster]) cultured at the Tasmanian Aqua- culture and Fisheries Institute, Marine Research Laboratories, Australia. This represents the first report of a chondracanthid copepod infecting cultured finfish and the first metazoan parasite from cultured striped trumpeter. Chondracanthus goldsmidi n. sp. can be distinguished from its female congeners by the absence of lateral processes on the head and the presence of 3 pairs of lateral trunk outgrowths, 3 middorsal body outgrowths (of which the first 2 are rounded), a small and subcylindrical antennule, and unornamented legs 1 and 2. A revised key to the 39 valid species of Chondracanthus is provided.

Latris lineata (Forster), known commonly in the southern MATERIALS AND METHODS hemisphere as striped trumpeter, is a demersal teleost species Parasites were collected from striped trumpeter L. lineata juveniles inhabiting the coasts of southeastern Australia, New Zealand, and adults that were either cultured or wild-caught animals held in cap- and South America (Kailola et al., 1993). This species, which tivity at the TAFI, Marine Research Laboratories in Tasmania. Fish were held in 25,000-L tanks supplied with unfiltered seawater at ambient grows to at least 1.2 m total length and 25 kg, has high-quality temperature (annual range 9–19 C). Fish were routinely removed from flesh and is amenable to culture conditions; it is currently being the tanks, anaesthetized (0.02% 2-phenoxyethanol, Sigma-Aldrich, St. investigated as a candidate for commercial aquaculture at the Louis, Missouri) and examined for chondracanthid copepods on 9 sep- Tasmanian Aquaculture and Fisheries Institute (TAFI), Marine arate occasions (Table I). Twenty-four transformed adult female copepods (13 each with 1 attached male; 1 with 2 attached males; 2 without Research Laboratories, Australia (Kailola et al., 1993; Trotter attached males) were removed from striped trumpeter, preserved in ei- et al., 2001; Morehead and Hart, 2003; Bransden et al., 2005; ther 10% formalin or 70% ethanol, and later soaked in lactic acid for Brown et al., 2005; Battaglene and Cobcroft, in press). at least 24 hr prior to examination with an Olympus BX50 compound To date, only 1 parasite, namely the myxozoan Kudoa neu- microscope. Three female and 4 male specimens were measured with the use of an ocular micrometer. Three specimens from each sex were rophila (Grossel, Dykova`, Handlinger and Munday, 2003), is dissected and examined according to the wooden slide procedure of known to cause disease in cultured striped trumpeter. This my- Humes and Gooding (1964). All drawings were made with the aid of a xozoan species specifically targets the tissues of the central ner- camera lucida. Anatomical terminology follows Boxshall and Halsey vous system of postlarval striped trumpeter, resulting in behav- (2004). ioral abnormalities such as loss of spatial control (Grossel et DESCRIPTION al., 2003). Recent routine examination of healthy and moribund striped trumpeter reared at the Tasmanian Aquaculture and Chondracanthus goldsmidi n. sp. Fisheries Institute resulted in the discovery of a new parasite, (Figs. 1–4) a species of cyclopoid copepod, Chondracanthus Delaroche, Adult female: Body (Figs. 1A, B) divided into head, short neck, and stout trunk. Total length (from anterior margin of head to distal end of 1811. The sessile parasites were commonly attached to tissue posterior processes on trunk) 4.92 Ϯ 0.35 mm; trunk width 2.57 Ϯ 0.43 within the gill cavity, occasionally on the gills themselves, or mm; head length 1.23 Ϯ 0.03 mm and head width 1.53 Ϯ 0.20 mm. in the nares. Host tissue appeared swollen at the attachment site Head composed of cephalosome only, broader posteriorly, lacking pro- and, in a severe infection of a population of 84 juveniles (250 cesses. Neck region composed of first pediger only, with a rounded outgrowth on middorsal surface. Pedigerous somites 2, 3, and 4 fused g, 660 days old), holes developed in the operculum. Further- to form a large trunk, bearing 3 pairs of lateral outgrowths (middle pair more, 36 of these 84 fish died or were killed when close to largest of three pairs), 1 pair of posterior processes, and 2 outgrowths death, over a 3-wk period. This new species, the first within the along middorsal line; posterior middorsal outgrowth larger than anterior Chondracanthidae to be reported from cultured finfish, is herein middorsal outgrowth, and protrudes either partially or completely over the genitoabdomen in dorsal view. Genitoabdomen (Fig. 1C) divisible described. Moreover, a revised key to the species of Chondra- as 2 tagmata by transverse constriction; anterior tagma bearing a minute canthus is provided. seta near opening of each genital aperture and a sensilla on each posterolateral surface; posterior tagma suboval, narrower than genital somite, with a dorsal pair of sensillae and anteroventral pair of caudal rami. Caudal ramus (Fig. 1D) spiniform, armed with 2 ventral setae, 1 Received 14 November 2006; revised 29 January 2007; accepted 31 dorsal seta, and a medial knob. January 2007. Antennule (Fig. 1E) small, subcylindrical, with an armature of 1-1- * Marine Research Laboratories, Tasmanian Aquaculture and Fisheries 1-3-2-8. Antenna (Fig. 2A) 2-segmented, composed of coxobasis and Institute and Cooperative Research Centre for Sustainable Aquacul- 1-segmented endopod; coxobasis short, unarmed; endopod forming un- ture of Finfish, University of Tasmania, Private Bag 49, Hobart, Tas- cinate claw, with transverse striations near apex. Labrum (Fig. 2B) with mania 7001, Australia. a small protrusion on each lateral margin and patches of minute spinules

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FIGURE 1. Chondracanthus goldsmidi n. sp., adult female. (A) Habitus, dorsal; (B) same, lateral; (C) genitoabdomen, lateral; (D) caudal ramus; (E) antennule. Scale bars: A, B ϭ 1.00 mm; C ϭ 200 ␮m; D ϭ 25 ␮m; E ϭ 50 ␮m.

788 TANG ET AL.—C. GOLDSMIDI N. SP. 789 790 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Infection parameters of Chondracanthus goldsmidi* parasitizing Latris lineata cultured at the Tasmanian Aquaculture and Fisheries Institute, Marine Research Laboratories, Australia.

Parasite No. hosts Mean† host Mean† host Host age Prevalence counts Date examined length (cm) weight (kg) (days) (%) (range)

4 June 2003 38 41.6 Ϯ 4.5 1.08 Ϯ 0.27 1,453 92 0 to Ͼ25 11 March 2005 48 24.3 Ϯ 2.1 0.21 Ϯ 0.05 675 54 0 to Ͼ8 21 April 2005 22 47.5 Ϯ 2.8 1.76 Ϯ 0.34 2,120 100 1 to Ͼ10 15 December 2005 5 28.9 Ϯ 3.1 0.36 Ϯ 0.35 954 80 0 to 8 20 December 2005 6 63.5 Ϯ 5.2 4.03 Ϯ 0.95 9–17 years‡ 33 0 to 13 22 March 2006 154 28.0 Ϯ 4.5 0.36 Ϯ 0.25 683 42 0 to 4 30 March 2006 19 49.6 Ϯ 5.1 2.08 Ϯ 0.89 3,285 100 1 to 25 9 May 2006 58 41.2 Ϯ 4.8 0.95 Ϯ 0.54 1,385 100 5 to Ͼ60 23 May 2006 18 49.6 Ϯ 4.5 2.26 Ϯ 0.68 3,338 94 0 to 15

* Parasites were removed manually and fish treated with Neguvon௡ following each date given. † Data are mean Ϯ SD. ‡ Estimated age of wild-caught fish held in captivity for several years as brood stock. along posterior margin. Mandible (Fig. 2C) 1-segmented, bearing apical Type locality: Crayfish Point, Taroona, Tasmania, Australia (43Њ35ЈS, falcate blade armed with 40–51 teeth on convex margin and 29–38 147Њ35ЈE). teeth on concave margin (counts based on each pair of mandibles from Prevalence and parasite counts: See Table I. 3 specimens). Paragnath (Fig. 2D) trilobate, with spinules on small, Type material: The holotype female (AM P.73329), allotype (AM outer lobe and large, medial lobe. Maxillule (Fig. 2E) lobate, bearing a P.73330), and paratypes (3 females, each with attached male; AM large basal protrusion, a subapical patch of spinules, and 2 terminal, P.73328) are deposited in the Australian Museum, Sydney, Australia. unequal elements. Maxilla (Fig. 2F) 2-segmented, comprised of syncoxa Etymology: The specific name is in honor of Ross Goldsmid for his and basis; syncoxa robust, unarmed; basis forming a claw-like process, continuing work and commitment to brood-stock husbandry and the armed with 2 unequal basal setae and 11–13 marginal teeth (counts culturing of striped trumpeter juveniles, including parasite monitoring based on each pair of maxillae from 3 specimens). Maxilliped (Fig. 2G) and management. 3-segmented, composed of syncoxa, basis, and terminal claw (formed from fused endopod and claw); syncoxa naked, longer than last 2 seg- Remarks ments combined; basis stout, with 2 large patches of minute spinules along inner margin; claw short and robust, bearing 1 accessory tooth. With the absence of an atrophied tip on the antenna and possession Leg 1 (Fig. 2H) fleshy and bilobate, armed with an outer protopodal of a cephalosomic head region, outgrowths on the trunk region, and 2 seta; both rami subequal and naked. Leg 2 (Fig. 3A) similar to leg 1, pairs of modified legs in the transformed adult female, the new species except larger in size. is unequivocally a member of Chondracanthus Delaroche, 1811. Chon- Adult male: Body (Fig. 3B) 465 Ϯ 30 ␮m long and 337 Ϯ 12 ␮m dracanthus goldsmidi n. sp. closely resembles Chondracanthus irre- wide; body segmentation indistinct; cephalothorax globose, comprising gularis Fraser, 1920, in lacking lateral processes on the head and having more than half total body length; urosome flexed ventrally. Genital so- 3 pairs of lateral outgrowths on the trunk and 3 middorsal outgrowths mite completely fused with abdomen (Fig. 3C), bearing paired apertures on the body. However, Ch. goldsmidi can be distinguished from Ch. ventrally; opercula unarmed. Caudal rami (Fig. 3C) spiniform, each irregularis by differences in the shape of the first two middorsal body bearing three basal setae, a small medial knob, and minute spinules outgrowths (rounded in Ch. goldsmidi; digitiform in Ch. irregularis), apically. complexity of the mid-lateral trunk outgrowth (lacks a ventrolateral, Antennule (Fig. 3D) filiform, with an armature of 1-1-2-3-8. Antenna digitiform outgrowth in Ch. goldsmidi; with ventrolateral, digitiform (Fig. 3E) short and stout, with 1 seta on the coxobasis and a medial outgrowth in Ch. irregularis), antennular structure (small and subcy- seta on the claw. Labrum (Fig. 3F) as in female, except with median lindrical in Ch. goldsmidi; large and fleshy in Ch. irregularis), and leg knob. Mandibular blade (Fig. 4A) bearing 13–23 teeth on convex side ornamentation (naked in Ch. goldsmidi; covered with fine denticles in and 9–14 teeth on concave side (counts based on each pair of mandibles Ch. irregularis) of the female, as well as in the shape of the antennule from 3 specimens). Maxillule (Fig. 4B) with 2 subequal, terminal ele- (slender in Ch. goldsmidi; inflated in Ch. irregularis) and structural ments, a small patch of spinules, and a medial lobe bearing an acumi- details of the legs (with an inner basal protrusion in Ch. goldsmidi; nate tip. Maxillary basis (Fig. 4C) lacking teeth; large basal seta uni- lacking inner basal protrusion in Ch. irregularis) of the male. laterally spinulated. Maxilliped (Fig. 4D) as in female, except with All specimens identified to date have been from fish held in land- smaller patches of spinules on basis. Leg 1 (Fig. 4E) with inner basal based tank systems at 1 site, and the effects of the parasite in sea cages protrusion, a long subapical seta (representing the protopodal seta), and at alternative sites are unknown. However, this parasite has the 2–3 apical setae (representing the exopodal elements), and an inner potential to affect striped trumpeter in a sea-cage industry via mortality subtriangular process (representing the endopod). Leg 2 (Fig. 4F) sim- in the event of heavy infection, as has occurred in land-based systems, ilar to leg 1, except smaller in size and bearing 1–2 apical setae. One or possibly through reduced growth rates of infected fish, treatment dissected specimen lacking endopod on leg 2 (Fig. 4G). costs, or reduced quality of harvested fish. Research to culture and describe the developmental stages of the parasite, examine the histo- Taxonomic summary pathology of infection, and test potential control and treatment methods Type host: Latris lineata (Forster, 1801) (Perciformes: Latridae). for Ch. goldsmidi are currently underway and will be dealt with in detail Infection site: Branchial cavity wall, operculum, gills, nares. elsewhere. Furthermore, wild striped trumpeter populations from Tas-

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FIGURE 2. Chondracanthus goldsmidi n. sp., adult female. (A) Antenna, (B) labrum, (C) mandible, (D) paragnath, (E) maxillule, (F) maxilla, (G) maxilliped, (H) leg 1. Scale bars: A, B ϭ 100 ␮m; C, F, G ϭ 50 ␮m; D ϭ 12.5 ␮m; E ϭ 25 ␮m; H ϭ 200 ␮m. TANG ET AL.—C. GOLDSMIDI N. SP. 791 792 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 TANG ET AL.—C. GOLDSMIDI N. SP. 793

FIGURE 4. Chondracanthus goldsmidi n. sp., adult male. (A) Mandible, (B) maxillule, (C) maxilla, (D) maxilliped, (E) leg 1, (F) leg 2, (G) abnormal leg 2. Scale bars: A, B, E, F, G ϭ 12.5 ␮m; C, D ϭ 25 ␮m.

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FIGURE 3. Chondracanthus goldsmidi n. sp., adult female (A) and adult male (B–F). (A) Leg 2; (B) habitus, lateral; (C) genitoabdomen, ventral; (D) antennule; (E) antenna; (F) labrum. Scale bars: A ϭ 200 ␮m; B ϭ 100 ␮m; C, E, F ϭ 25 ␮m; D ϭ 12.5 ␮m. 794 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 manian waters will be sampled and examined to determine whether Ch. Anterior end of head as wide as or slightly wider than its pos- goldsmidi is a natural parasite of this finfish species. terior end; posterior end of trunk without ventral swelling ...... psetti (Ho, 1977, p. 164–165) Key to the species of Chondracanthus 12. Leg 2 long, reaching or passing the posterior end of trunk . . . In his preliminary review of Chondracanthus, Ho (1991a) recognized ...... palpifer (Ho, 1991b, p. 2–4) 37 valid species. Subsequently, Ho and Kim (1995) transferred Acan- Leg 2 short, barely reaching the posterior end of the third pe- thochondria solida Gusev, 1951 to Chondracanthus, and Tang and Ho digerous somite ...... australis (Ho, 1991b, p. 5–8) (2005) transferred Chondracanthus quadratus (Heegaard, 1945) to 13. Trunk region with 2 pairs of lateral outgrowths (knobs, protru- Acanthocanthopsis Heegaard, 1945. Although Ho et al. (2005) estab- sions, or processes) ...... 14 lished 2 new Chondracanthus species, Ch. parvus and Ch. yabei,we Trunk region with more than 2 pairs of lateral outgrowths consider the former species to be synonymous with Chondracanthus (knobs, protrusions, or processes) ...... 26 solidus (Gusev, 1951), as there are no marked differences between the 14. Posterior processes well developed; head with or without lateral descriptions and drawings of Ch. solidus and Ch. parvus given in Ho processes ...... 15 and Kim (1995) and Ho et al. (2005), respectively. Thus, with the es- Posterior processes poorly developed, appearing as a corner tablishment of Ch. goldsmidi, a total of 39 Chondracanthus species are knob; head without lateral processes ...... currently considered valid...... narium (Kabata, 1969, p. 3044–3047) The following key, which utilizes features of the transformed adult 15. Trunk region with outgrowths on middorsal surface ...... 16 female only, is adapted from Ho (1991a). Additionally, the following Trunk region without outgrowths on middorsal surface .... 21 errors from Ho (1991a) have been amended in this revised key: (1) 16. Head with 1 pair of posterolateral processes or lateral expan- Chondracanthus neali Leigh-Sharpe, 1930, which was omitted previ- sions ...... 17 ously, was added; (2) the first option in step 15 should state ‘‘trunk Head without processes or lateral expansions ...... 20 region with outgrowths on middorsal surface’’ rather than ‘‘trunk region 17. First pedigerous somite with an outgrowth on dorsal surface 18 with an outgrowth on midventral surface’’ as previously noted; and (3) First pedigerous somite without an outgrowth on dorsal surface the reference given for Chondracanthus colligens Barnard, 1955, should ...... 19 be Ho (1972b) rather than Ho (1972a), as previously noted. Following 18. Lateral outgrowths on head and trunk short and stubby ..... Ho (1991a), the species identified with this revised key should be con- ...... multituberculatus (Markevich, 1956, p. 161–162) firmed by checking with the reference given following the species name. Lateral outgrowths on head and trunk long and attenuate .... 1. Legs 1 and 2 unilobate ...... pinguis (Kabata, 1968, p. 329–332) ...... angustatus (Raibaut et al., 1971, p. 190–193) Lateral outgrowths on head and trunk medium size and bluntly Legs 1 and 2 trilobate ...... 2 pointed ...... polymixiae (Shiino, 1955, p. 77–79) Legs 1 and 2 bilobate ...... 5 19. Trunk with 2 cylindrical dorsal processes; head with bilobate 2. Trunk region with outgrowths in the form of knobs, processes lateral protrusion ..... heterostichi (Ho, 1972a, p. 527–529) or protrusions on dorsal surface ...... 3 Trunk with 3 rounded dorsal processes; head with lateral ex- Trunk region without outgrowths on dorsal surface ...... 4 pansions ...... yabei (Ho et al., 2005, p. 416–418) 3. Head without processes; trunk region with more than a dozen 20. All outgrowths on body large and massive; protopods of legs 1 processes ...... zei (Ho, 1991a, p. 50–54) and 2 with bulging outer surface ...... Head with a pair of posterolateral processes; trunk region with ...... lotellae (Ho, 1975, p. 308–311) less than a dozen processes ...... All outgrowths on body small and minute; protopods of legs 1 ...... horridus (Heller, 1865, p. 232–233) and 2 without bulging outer surface ...... Head with lateral expansions; trunk region with less than a doz- ...... pusillus (Kabata, 1968, p. 335–339) en processes ...... ornatus (Kabata, 1979, p. 122–123) 21. Head with 1 pair of posterolateral processes ...... 22 4. Antennule extremely small; maxilliped claw with only 1 hook- Head with lateral expansions, but not in form of process .... let; caudal ramus with usual long terminal process ...... yanezi (Ho, 1982, p. 451–455) ...... nodosus (Ho, 1971, p. 27–31) Head with lateral expansions and a knob in the anterior corners Antennule large and fleshy; maxilliped claw bearing numer- ...... cottunculi (Ho, 1971, p. 17–20) ous hooklets; caudal ramus lacking long terminal process Head without expansions, knobs or processes ...... 24 ...... wilsoni (Ho, 1971, p. 31–33) 22. Legs 1 and 2 long and slender ...... 23 5. Trunk region with only 1 pair of lateral outgrowths (knobs, Legs1and2shortandstubby ...... protrusions, or processes) ...... 6 ...... theragrae (Shiino, 1955, p. 74–77) Trunk region with more than 1 pair of lateral outgrowths (knobs, 23. Exopods of legs 1 and 2 nearly twice as long as their respective protrusions or processes) ...... 13 protopods ...... lepophidi (Ho, 1974, p. 870–873) 6. Head with 1 or 2 pairs of small knobs on lateral surface . . . 7 Exopods of legs 1 and 2 as long as their respective protopods Head with a pair of prominent lateral processes ...... 8 ...... triventricosus (Sekerak, 1970, p. 1944–1950) Head without knobs or processes ...... 9 7. Head with 1 pair of small knobs; second pedigerous somite with 24. Oral region separated from antennal area to form the first part pair of lateral processes ...... of neck region; exopod of leg 1 twice as long as endopod ...... solidus (Ho and Kim, 1995, p. 31–34) ...... brotulae (Ho and Rokicki, 1987, p. 1031–1033) Head with 2 pairs of small knobs; second pedigerous somite Oral region set far behind antennal area but not separated into without pair of lateral processes ...... a neck region; exopod of leg 1 as long as endopod ..... 25 ...... deltoideus (Kabata, 1984, p. 1710–1713) 25. Legs 1 and 2 short and blunt; each protopod with prominent 8. Posterodorsal portion of head protruded into a large crestlike outer swelling ...... genypteri (Ho, 1975, p. 306–308) outgrowth; abdomen greatly elongated ...... Legs 1 and 2 long and attenuate; protopods without such swell- ...... janebennettae (Ho, 1971, p. 20–23) ing ...... colligens (Ho, 1972b, p. 151–152) Head without crestlike outgrowth; abdomen smaller than genital 26. Head with 1 pair of lateral processes ...... 27 area ...... merluccii (Ho, 1971, p. 23–27) Head with 2 pairs of lateral processes ...... 29 9. Rami of leg 2 short and stout ...... 10 Head without lateral processes ...... 30 Rami of leg 2 long and slender ...... 11 27. Head large, its length about ¾ oftrunklength ...... 10. Trunk with a knoblike outgrowth on dorsal surface; posterior ...... tuberculatus (Ho, 1972b, p. 155–158) processes long ...... gracilis (Kabata, 1968, p. 332–335) Head small, its length shorter than ¼ of trunk length ...... 28 Trunk without knoblike outgrowth on dorsal surface; posterior 28. Posterior end of trunk with 3 processes (including paired pos- processes short ...... lepidionis (Kabata, 1970, p. 180–182) terior processes) ...... lophii (Kabata, 1979, p. 118–119) 11. Anterior end of head distinctly narrower than its posterior end; Posterior end of trunk with 5 processes (including paired pos- posterior end of trunk with a ventral swelling ...... 12 terior processes) ...... barnardi (Ho, 1972b, p. 149–152) TANG ET AL.—C. GOLDSMIDI N. SP. 795

29. Trunk with dorsal outgrowths; abdomen extended posteriorly fishes of the south eastern Pacific (Crustacea, Copepoda). Steen- ...... neali (Ho, 1972b, p. 152–155) strupia 4: 157–165. Trunk lacking dorsal outgrowths; abdomen not extended pos- ———. 1982. Copepod parasites of Psychrolutes (Pisces: Scorpaeni- teriorly ...... distortus (Shiino, 1955, p. 71–74) formes) from deep water in the eastern Pacific. Parasitology 85: 30. Trunk with 3 pairs of lateral outgrowths ...... 31 451–458. Trunk with 6 pairs of lateral outgrowths ...... ———. 1991a. Redescription of Chondracanthus zei Delaroche (Co- ...... shiinoi (Shiino, 1955, p. 79–83) pepoda, Poecilostomatoida) parasitic on Zeus faber L. in the Sea 31. Midlateral trunk outgrowth with ventrolateral, digitiform out- of Japan, with a preliminary review of the genus. Report of the growth; antennule large and fleshy ...... Sado Marine Biological Station, Niigata University 21: 49–79...... irregularis (Kabata, 1968, p. 323–328) ———. 1991b. Two species of chondracanthid copepods (Poecilosto- Mid-lateral trunk outgrowth without ventrolateral, digitiform matoida) parasitic on commercial fishes in the Pacific. Publications outgrowth; antennule small and subcylindrical ...... of the Seto Marine Biological Laboratory 35: 1–10...... goldsmidi (this report) ———, AND I. H. KIM. 1995. Chondracanthid copepods (Poecilosto- matoida) parasitic on fishes of the Sea of Japan. Report of the Sado Marine Biological Station, Niigata University 25: 31–44. ACKNOWLEDGMENTS ———, ———, AND K. NAGASAWA. 2005. Copepod parasites of the The work formed part of a project of the Aquafin CRC, and received fatheads (Pisces, Psychrolutidae) and their implication on the phy- funds from the Australian Government’s CRC Program and other CRC logenetic relationships of psychrolutid genera. Zoological Science Participants. We thank the Department of Zoology at The University of 22: 411–425. Western Australia for use of laboratory facilities, Stephen Battaglene ———, AND J. ROKICKI. 1987. Poecilostomatoid copepods parasitic on and Barbara Nowak for comments on the draft manuscript, and the fishes off the west coast of Africa. Journal of Natural History 21: striped trumpeter research team of the TAFI, Marine Research Labo- 1025–1034. ratories for animal husbandry, particularly Ross Goldsmid and Anna HUMES,A.G.,AND R. U. GOODING. 1964. A method for studying the Overweter for assistance with parasite collection. We are grateful to external anatomy of copepods. Crustaceana 6: 238–240. George W. Benz and an anonymous reviewer for their comments on the KABATA, Z. 1968. Some Chondracanthidae (Copepoda) from fishes of manuscript. British Columbia. Journal of the Fisheries Research Board of Can- ada 26: 321–345. ———. 1969. Chondracanthus narium sp. n. (Copepoda: Chondracan- LITERATURE CITED thidae), a parasite of nasal cavities of Ophiodon elongatus (Pisces: Teleostei) in British Columbia. Journal of the Fisheries Research BATTAGLENE, S. C., AND J. M. COBCROFT. 2007. Advances in the culture Board of Canada 26: 3043–3047. of striped trumpeter larvae: A review. Aquaculture. doi: 10.1016/ ———. 1970. Three copepods (Crustacea) parasitic on fishes of the j.aquaculture.2007.04.048. genus Lepidion Swainson, 1838 (Pisces: Teleostei). Journal of Par- BOXSHALL,G.A.,AND S. H. HALSEY. 2004. An introduction to copepod asitology 56: 175–184. diversity. The Ray Society, London, U.K., 966 p. ———. 1979. Parasitic Copepoda of British fishes. The Ray Society, BRANSDEN, M. P., S. C. BATTAGLENE,D.T.MOREHEAD,G.A.DUNSTAN, London, 468 p. AND P. D . N ICHOLS. 2005. Effect of dietary 22:6n-3 on growth, ———. 1984. A contribution to the knowledge of Chondracanthidae survival and tissue fatty acid profile of striped trumpeter (Latris (Copepoda: Poecilostomatoida) parasitic on fishes of British Co- lineata) larvae fed enriched Artemia. Aquaculture 243: 331–344. lumbia. Canadian Journal of Zoology 62: 1703–1713. BROWN, M. R., S. C. BATTAGLENE,D.T.MOREHEAD, AND M. BROCK. KAILOLA, P. J., M. J. WILLIAMS,P.C.STEWART,R.E.REICHELT,A. 2005. Ontogenetic changes in amino acid and vitamins during early MCNEE, AND C. GRIEVE. 1993. Australian fisheries resources. Bu- larval stages of striped trumpeter (Latris lineata). Aquaculture 248: reau of Resource Sciences and the Fisheries Research and Devel- 263–274. opment Corporation, Canberra, Australia, 422 p. ` GROSSEL, G. W., I. DYKOVA,J.HANDLINGER, AND B. L. MUNDAY. 2003. MARKEVICH, A. P. 1956. Parasitic Copepoda of fishes of the USSR. Pentacapsula neurophila sp. n. (Multivalvulida) from the central Akademiya Nauk Ukraine SSR, Institut Zoology, Kiev, Russia, 259 p. nervous system of striped trumpeter, Latris lineata (Forster). Jour- MOREHEAD,D.T.,AND P. R. HART. 2003. Effect of temperature on hatch- nal of Fish Diseases 26: 315–320. ing success and size of striped trumpeter (Latris lineata) larvae. HELLER, C. 1865. Crustaceen. Reise der O¨ sterreichischen Fregatte No- Aquaculture 220: 595–606. vara um die Erde in den Jahren 1857, 1858, 1859. Zoologie 2: 1– RAIBAUT, A., O. K. BEN HASSINE, AND K. MAAMOURI. 1971. Cope´podes 280. parasites des poissons de Tunisie (Premie`re se´rie). Bulletin de HO, J. S. 1971. Parasitic copepods of the family Chondracanthidae from l’Institut National Scientifique et Technique d’Oceánographie et de fishes of eastern North America. Smithsonian Contributions to Zo- Peˆche, Salammboˆ 2: 169–197. ology 87: 1–39. SEKERAK, A. D. 1970. Parasitic copepods of Sebastodes alatus, includ- ———. 1972a. Four new parasitic copepods of the family Chondra- ing Chondracanthus triventricosus sp. nov. and Colobomatus ky- canthidae from California inshore fishes. Proceedings of the Bio- phosus sp. nov. Journal of the Fisheries Research Board of Canada logical Society of Washington 85: 523–540. 27: 1943–1960. ———. 1972b. Copepods of the family Chondracanthidae (Cyclopoida) SHIINO, S. M. 1955. Copepods parasitic on Japanese fishes. 9. Family parasitic on South African marine fishes. Parasitology 65: 147–158. Chondracanthidae, subfamily Chondracanthinae. Report of Faculty ———. 1974. A new species of Chondracanthus (Copepoda, Chondra- of Fisheries, Prefectural University of Mie 2: 70–111. canthidae) parasitic on Peruvian cusk eels. Journal of Parasitology TANG,D.,AND J. S. HO. 2005. Resurrection of Acanthocanthopsis Hee- 60: 870–873. gaard, 1945 (Copepoda, Poecilostomatoida, Chondracanthidae), ———. 1975. Cyclopoid copepods of the family Chondracanthidae par- parasitic on tetraodontiform fishes. Crustaceana 78: 809–818. asitic on New Zealand marine fishes. Publications of the Seto Ma- TROTTER, A. J., P. M. PANKHURST, AND P. R . H ART. 2001. Swim bladder rine Biological Laboratory 22: 303–319. malformation in hatchery-reared striped trumpeter Latris lineata ———. 1977. Parasitic copepods of the family Chondracanthidae from (Latridae). Aquaculture 198: 41–54. J. Parasitol., 93(4), 2007, pp. 796–805 ᭧ American Society of Parasitologists 2007

IMPACT OF POPULATION STRUCTURE ON GENETIC DIVERSITY OF A POTENTIAL VACCINE TARGET IN THE CANINE HOOKWORM (ANCYLOSTOMA CANINUM)

Jennifer M. Moser*, Ignazio Carbone†, Prema Arasu‡, and Greg Gibson§ Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695-7614. e-mail: [email protected]

ABSTRACT: Ancylostoma caninum is a globally distributed canine parasitic nematode. To test whether positive selection, population structure, or both affect genetic variation at the candidate vaccine target Ancylostoma secreted protein 1 (asp-1), we have quantified the genetic variation in A. caninum at asp-1 and a mitochondrial gene, cytochrome oxidase subunit 1 (cox-1), using the statistical population analysis tools found in the SNAP Workbench. The mitochondrial gene cox-1 exhibits moderate diversity within 2 North American samples, comparable to the level of variation observed in other parasitic nematodes. The protein coding portion for the C-terminal half of asp-1 shows similar levels of genetic variation in a Wake County, North Carolina, sample as cox-1. Standard tests of neutrality provide little formal evidence for selection acting on this locus, but haplotype networks for 2 of the exon regions have significantly different topologies, consistent with different evolutionary forces shaping variation at either end of a 1.3-kilobase stretch of sequence. Evidence for gene flow among geographically distinct samples suggests that the mobility of hosts of A. caninum is an important contributing factor to the population structure of the parasite.

The canine hookworm, Ancylostoma caninum, is a useful vergence, is influenced by levels of gene flow and by host mo- model for the widespread human hookworms Necator ameri- bility (Blouin et al., 1995, 1999; Hawdon et al., 2001; Braisher canus and Ancylostoma duodenale, but it can cause severe pa- et al., 2004). Hookworms display intermediate levels of gene thologies in its own right. Heavy infestations in puppies can flow and structure compared with the highly diverse tricho- lead to anemia and death. Conventional treatments to reduce strongylids and the less diverse, highly structured plant and in- infections must often be repeated, and there is a growing desire sect nematodes. to develop vaccines that will target specific genes and gene One study comparing the diversity of just 2 sequences of products of parasites and interrupt the infection cycle. To be asp-1 isolated from 2 geographic areas showed 97% DNA se- effective, these reagents must recognize all of the variation ob- quence identity between a Chinese and a Baltimore, Maryland, served in the target loci in natural populations. A gene that has strain of A. caninum in the coding regions of asp-1 (Qiang et been investigated for its potential usefulness as a target of such al., 2000). The study suggested that a reduced level of poly- a treatment is Ancylostoma secreted protein-1 (asp-1), which morphism in the second of the 2 domains in the protein pro- encodes a protein that is secreted into the host during the in- vides evidence for selection against amino acid substitutions fection process by the third-stage larvae (Hawdon et al., 1996). there. We elected to focus on this region of the gene in a broad- The asp-1 gene encodes a 2-domain, 431-amino acid protein er survey of nucleotide diversity that includes both the intron (Fig. 1A), and it is one of several secreted by infective larvae and coding regions of the gene. A mitochondrial gene is often when exposed to host factors and in anticipation of infection used as a neutral baseline for comparison of genetic variation (Moser et al., 2005). Because hookworms have evolved to ef- and population structure in a species. The mitochondrial cyto- fectively avoid detection by host immune systems, there may chrome oxidase I locus (cox-1) has served as such in previous be selection pressure to sustain high levels of variation in some studies (Tarrant et al., 1992; Anderson et al., 1998; Blouin, or all of the secreted or excreted proteins in hookworm popu- 1998; Hawdon et al., 2001; Hu, Chilton et al., 2002; Hu, Hog- lations (Vacher et al., 2005). This hypothesis can be tested by lund et al., 2002; Hu et al., 2003). population genetic surveys of nucleotide diversity, because pos- Here, we describe the level of population structure and di- itive or balancing selection should leave signatures of departure versity at asp-1 and cox-1 within a local sample of A. caninum from neutrality (Kreitman, 2000). Furthermore, identification of from Wake County, North Carolina, and a ‘Maryland’ labora- specific haplotypes that are unique to local populations may tory strain initially isolated in that state. The genomic DNA have implications for implementation of immunotherapy, spe- sequences are analyzed with a comprehensive software package cifically vaccines. (SNAP Workbench; Price and Carbone, 2005; Aylor et al., Evidence from studies of diverse parasitic animal nematodes 2006) and are compared with a previously published population suggests that there are several types of population structure in survey of cox-1 from Townsville, Australia (Hu, Chilton et al., these species (Blouin et al., 1995, 1999; Qiang et al., 2000; 2002), and with the reported asp-1 cDNA sequences from a Picard et al., 2004; Nieberding et al., 2005). Population struc- Chinese A. caninum strain (Qiang et al., 2000). ture in these nematodes, in terms of genetic diversity and di- MATERIALS AND METHODS Received 19 October 2006; revised 19 January 2007; accepted 22 DNA extractions of whole adult male and female A. caninum were January 2007. carried out by homogenizing individual worms in a digestion buffer * Present address: Laboratory of Allergic Diseases, National Institutes consisting of 100 nM NaCl, 10 mM Tris HCl, pH 8.0, 25 mM EDTA, of Health, Bethesda, Maryland 20892. pH 8.0, 0.5% SDS, and 0.1 mg/ml proteinase K. A sample of 118 † Center for Integrated Fungal Research, Department of Plant Pathol- worms from a Wake County, North Carolina, animal shelter (near Ra- ogy, North Carolina State University, Raleigh, North Carolina 27695- leigh) was collected by P.A., from which we obtained 54 useable se- 7244. quences. A Maryland sample was maintained in laboratory dogs at the ‡ Department of Molecular Biomedical Sciences, North Carolina State University of Pennsylvania, Philadelphia, Pennsylvania, and it was orig- University College of Veterinary Medicine, Raleigh, North Carolina inally collected in the 1970s. We used 12 individual worms from this 27695-8401. sample for sequencing. Nematode homogenates were prepared as de- § To whom correspondence should be addressed. scribed by incubating nematodes at 50 C for 4 hr to dissolve the pro-

796 MOSER ET AL.—GENETIC DIVERSITY OF A. CANINUM 797

FIGURE 1. (A) Schematic of sequenced regions of the asp-1 gene. Three distinct regions, labeled asp-1C, asp-1D, and asp-1E are mostly contained within domain 2, and they were sequenced using designed primers described under Materials and Methods. The rectangles connected by lines represent exons and introns, respectively. The dashed line represents the 5Ј domain 1 of asp-1, which was not sequenced. The bold dashed line connecting asp-1D and asp-1E represents an unsequenced region. (B) asp-1 DNA sequence. Nucleotides sequenced in the study are bold. The 7 introns were identiﬁed by aligning sequenced genomic DNA with cDNA sequence (GenBank AF13229) and are underlined. The introns included represent the shortest possible intron sequences observed in the data.

teinaceous and cuticular portions of the hookworms (Hu, Chilton et al., All ampliﬁcation reactions were performed in a volume of 30 ␮lin 2002). DNA was extracted by standard phenol-chloroform extraction an MJ Research (Hercules, California) thermocycler using Taq poly- ␮ and ethanol precipitation, and it was stored in 10–20 l of distilled H2O merase (Promega, Madison, Wisconsin). Polymerase chain reaction at Ϫ20 C. Australian samples reported were collected from dogs in (PCR) and sequencing primers for a 373-bp fragment of cox-1 were Townsville, Queensland, Australia (Hu, Chilton et al., 2002), and the reported in Hu, Chilton et al. (2002) (cox-1 F: 5-TTT TTT GGG CAT sequences were taken directly from that reference. The GenBank hap- CCT GAG GTT TAT-3Ј,R:5Ј-TAA AGA AAG AAC ATA ATG AAA lotype accession numbers are AJ407941, AJ407961, AJ407962, ATG-3Ј). The PCR conditions were 95 C for 5 min, followed by 30 AJ407963, AJ407964, AJ407965, and AJ407966. cycles of 95 C for 30 sec, 45 C for 30 sec, 72 C for 30 sec, with a 798 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. cox-1 population parameters.

Haplotypes ␪/locus Sample Fu and Li’s Sample* Mutations size H diversity ␲ ␪/site Tajima’s D D* Fu and Li’s F* Fu’s F

WC 30 54 14 0.014 Ϯ 0.001 6.145 –0.627 ns –2.556* –2.218 ns –0.149 0.828 Ϯ 0.031 0.017 MD 2 8 2 0.001 Ϯ 0.001 0.771 –1.310 ns –1.410 ns –1.514 ns –0.396 0.250 Ϯ 0.18 0.002 Australia 42 38 7 0.055 Ϯ 0.008 17.96 0.778 ns 0.554 ns 0.669 ns 0.762 1.0 Ϯ 0.076 0.048

* MD, Maryland; WC, Wake County. † ns, not signiﬁcant.

final extension at 72 C for 10 min. Amplified DNA bands were extract- type diversity, and Watterson’s estimation of theta. It also was used to ed from 0.8% agarose gels using gel extraction kits (QIAGEN, Frank- perform Tajima’s, Fu and Li’s, and Fu’s tests of neutrality (Tajima, 1989; furt, Germany) following manufacturer’s specifications, and they were Fu and Li, 1993; Fu, 1997). Molecular Evolutionary Genetics Analysis sequenced using BigDye (Applied Biosystems, Foster City, California) 3 (MEGA3; Kumar et al., 2004) was used to infer gene genealogies. termination on ABI 3700 sequencers at the North Carolina State Uni- We tested for linkage disequilibrium among polymorphisms in asp-1C versity Genome Research Lab. Primer sets were created to amplify three and asp-1E using significance tests implemented in TASSEL (http:// regions of asp-1 as diagrammed in Figure 1A as follows: asp-1E F: 5Ј- www.maizegenetics.net). To address potential errors from amplification AGG CTT CAC AAC TCT GGT GG-3Ј,R:5Ј-ACT GAT TAA GGA and sequencing, the asp-1 regions were reanalyzed after further col- GCA CTG CA-3Ј; asp-1D F: 5Ј-ATG CAC CAA AAG CAG CTA AA- lapsing the haplotype sequences that differed by fewer than 2 mutations. 3Ј,R:5Ј-TCC ATA AAG CAG TGG TAA GG-3Ј; and asp-1C F: 5Ј- ACT CCG AAA CGA AAC T-3Ј,R:5Ј-TCA TAC ACC ATC TGG AT-3Ј. For the asp-1E region, the PCR conditions were 95 C for 5 min, RESULTS followed by 30 cycles of 95 C for 30 sec, 50 C for 30 sec, 72 C for 30 sec, and 72 C for 5 min for final extension. The PCR conditions for Cytochrome oxidase subunit 1 asp-1C and asp-1D were 94 C for 4 min, followed by 36 cycles of 94 C for 30 sec, 50 C for 30 sec, 72 C for 2 min, with a final extension The mitochondrial locus cox-1 is traditionally used in pop- at 72 C for 5 min. ulation genetics studies because it exhibits absence of recom- Due to high levels of indel polymorphism in the asp-1 genomic re- bination and a relatively high mutation rate. Table I summarizes gion, it was not possible to sequence amplified DNA directly; so, PCR products for this gene were cloned into the pGEM-T vectors using the the nucleotide diversity statistics for the Wake County and Promega PCR cloning kit as per manufacturer’s instruction. Plasmids Maryland samples and compares these with the study of Aus- were isolated from bacteria with QIAGEN miniprep kits; restriction tralian samples. The estimate of nucleotide diversity (␲), which digests were performed to confirm the presence of the appropriate represents the proportion of pairwise nucleotide differences length insert. Inserts were sequenced with the vector-specific T7 and among sequences, is 0.014 in the Wake County sample, 0.001 SP6 primers. The number of sequences for regions asp-1E, asp-1D, and asp-1C were 70, 8, and 22, respectively. in the Maryland sample, and 0.055 in the Australian sample. Sequence analysis was performed using the ContigExpress module Because the Maryland population was expanded from a small of Vector NTI (InforMax, MD, Carlsbad, California) to manually edit founder group, there was likely a loss of genetic variation, re- the sequence ABI files. To determine the coding and noncoding portions sulting in the relatively low ␲ (Gasnier and Cabaret, 1998). The of the asp-1 regions, BLASTn (Altschul et al., 1990) was used to com- ␲ pare genomic sequence to the known coding regions of the asp-1 Australian sample has a much higher than either of the Amer- mRNA represented by GenBank AF13229 (Fig. 1B). This figure in- ican samples, but this is mostly attributable to 3 haplotypes that cludes the cDNA sequence along with the shortest sequenced introns. differ by at least 22 nucleotides (out of 373) from the other 4 SNAP Workbench (Price and Carbone, 2005; Aylor et al., 2006), a Australian haplotypes. Diversity among the remaining 4 Aus- platform combining numerous population genetic and phylogenetic soft- tralian haplotypes is similar to that observed in the North Amer- ware programs, was used to create multiple sequence alignments (CLUSTAL W; Thompson et al., 1994) and allowed for transformation ican samples. of sequence data into different formats, including NEXUS (Maddison The 62 sequenced cox-1 amplicons and 38 Australian se- et al., 1997) and PHYLIP (Felsenstein, 2004). Haplotype networks were quences represent 19 haplotypes as shown in Figure 2. Phylo- created with GT Miner, version 1.16, a component of SNAP Workbench genetic analysis in the SNAP Workbench results in 56 equally (D. Brown and I. Carbone, pers. comm.) based on a maximum parsimony approach (Cassens et al., 2005). Most parsimonious trees were most parsimonious trees possible from the 19 haplotypes. Most inferred with the Discrete Character Parsimony Algorithm, version 3.6b, of the trees contain 3 major clusters clearly visible in the hap- implemented in PHYLIP (Felsenstein, 2004). SNAP also was used to lotype network (Fig. 3). The 3 highly divergent Australian hap- test for population subdivision and migration using Hudson’s tests lotypes (H3, H4, and H5) are visible at the top of the network, (Hudson et al., 1992) and MDIV (Nielsen and Wakeley, 2001), respec- whereas 4 less divergent Wake County haplotypes (H2, H6, H7, tively. MDIV tests for gene flow over the entire coalescent history of a pair of populations; therefore, it captures both historical and ongoing and H14, each separated by at least 5 nucleotides from the other migration between the two populations. Specifically, MDIV applies co- haplotypes) group at the bottom of the figure. The majority of alescence simulation methods to estimate divergence time and integrat- the sequences form a network with members from each popu- ed migration rates from population 1 to population 2 and migration from lation sample, in which each haplotype is just 1 or 2 nucleotides population 2 to population 1. Posterior likelihood plots of divergence time and migration were plotted using gnuplot (ftp://ftp.gnuplot.info/ away from its nearest neighbor. The 3 most common haplotypes pub/gnuplot). DnaSP, version 4.0 (Rozas et al., 2003), was used to es- are H9 with 25 sequences, H7 with 14, and H1 with 8, the latter timate population parameters representing nucleotide diversity, haplo- 2 haplotypes consisting entirely of Wake County samples. MOSER ET AL.—GENETIC DIVERSITY OF A. CANINUM 799

FIGURE 2. cox-1 haplotype SNP matrix. Sixty-two A. caninum individuals from Wake County and Maryland were sequenced at the cox-1 locus, and the resultant sequences, along with 38 samples representing 7 haplotypes (Hu, Chilton et al., 2002) were collapsed into 19 new haplotypes using the SNAP Workbench. Indels and inﬁnite sites violations were removed from the analysis. The matrix shows the position of SNPs in the multiple sequence alignment, e.g., the SNP at site 1 is located at position 16 in the alignment, the identity of the SNPs relative to the consensus sequence, and displays whether the nucleotide changes are transversions (v) or transitions (t). The number of individual sequences contained in each haplotype is noted in parentheses.

A version of Hudson’s test (Hudson et al., 1992) imple- populations are as similar as Wake County and Maryland. The mented in the SNAP Workbench was used to test for population data suggest that Australian and U.S. populations descended subdivision between samples. KST values, the statistic of Hud- from a common ancestral population not too distantly in the ϭ son’s test, are based on Wright’s fixation index and indicate past (T 0.4, measured in coalescent time units of Ne gener- differentiation among samples of a species. At the cox-1 locus, ations, where Ne is the effective population size), and a low and the KST value between Maryland and Wake County samples is constant level of gene flow accompanied that population diver- low but significant (0.048; P ϭ 0.04), suggesting low levels of gence. There is evidence for deviation from mutation-drift equi- genetic differentiation and moderate levels of gene flow (Table librium at cox-1 in the Wake County sample of A. caninum,as II). The results from MDIV, a program from the SNAP Work- Fu and Li’s D* test statistic is significantly negative (Table I), bench that tests whether both populations descended from a suggesting an excess of low frequency polymorphisms within panmictic population and were then either connected by migra- the population resulting from background selection (Fu and Li, tion or completely isolated, also support the idea of gene flow 1993). This result must be tempered by the recognition that a between the 2 populations. Figure 4A shows the posterior dissimilar test statistic, Fu and Li’s F*, is not significant. tribution of the point parameter M reaches a maximum at 3.4 for this comparison. Combining the sequences from Maryland Ancylostoma secreted protein-1 and Wake County hookworms into a mid-Atlantic (United States) sample facilitates comparison with the Australian sam- The levels of ␲ across the locus are comparable to the mi- ple. Comparing United States to Australian samples results in tochondrial gene cox-1 when the highly polymorphic intron se- Ͻ a larger KST value (0.177; P 0.001), indicating a very high quences are included, but they are considerably lower when level of genetic differentiation between the 2 populations, con- only considering the coding sequences. The 3 consecutive re- sistent with a low level of gene flow between them (Table II). gions of asp-1 highlighted in Figure 1A designated asp-1C, There is supporting evidence for gene flow between the U.S. asp-1D, and asp-1E have ␲ values of 0.033, 0.004, and 0.016, and Australian samples, as seen from the MDIV data, where respectively (Table III). the maximum value of M along the distribution is 0.60 (Fig. The asp-1E region spans approximately 400 nucleotides, 4B); a maximum M of greater than 0 suggests migration, contains 2 introns, and is located at the 3Ј end of the gene. Due whereas M ϭ 0 supports the hypothesis of isolation. This in- to editing for proper sequence alignment, the multiple sequence ference is strengthened if the 3 highly divergent Australian hap- alignments analyzed are only as long as the highest quality, lotypes are removed from the analysis, in which case the 2 shortest sequence. Seventy asp-1E sequences were obtained, 57 800 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. Unrooted cox-1 haplotype network. In this network, generated by the GT Miner component of the SNAP Workbench, the 19 haplotypes from the cox-1 analysis (calculated by removing both indels and inﬁnite-sites violations) are shown as oval nodes connected by branches whose lengths correspond to the number of nucleotide differences between them. The black nodes represent the Australian haplotypes, the unshaded nodes are Maryland and Wake County haplotypes, and the gray nodes represent unsampled haplotypes. H9 is the largest haplotype, and it contains 25 A. caninum samples, including all 13 of the Maryland samples. from Wake County and 13 from Maryland, and these were con- regions (data not shown). The Maryland samples do not form trasted with the existing Chinese sequence (GenBank a unique cluster, but instead they are a part of the unresolved AAD31839). A strict consensus tree of the asp-1E coding re- portion of the tree. The network of 16 haplotypes shown in gions shows a comblike structure that is the hallmark of a recent Figure 5A includes a large interior haplotype (H12; 30 sequenc- population expansion or an unresolved phylogeny that is attrib- es), which contains both Maryland and Wake County sequences utable to the small number of segregating sites in the coding with several haplotypes radiating from it. The Chinese haplo-

TABLE II. Hudson’s test for subdivision and results of MDIV test for isolation.

Samples Genetic Maximum compared* Locus KS KT KST P value differentiation? M (MDIV)

WC/MD cox-1 4.175 4.841 0.048 0.04 Yes 3.40 U.S.*/AUS cox-1 5.480 6.658 0.177 Ͻ0.001 Yes 0.60 WC/MD asp-1E 4.340 4.308 0.016 0.04 Yes 1.74

* WC, Wake County; MD, Maryland; AUS, Australia. U.S. sample was created by combining WC and MD samples. MOSER ET AL.—GENETIC DIVERSITY OF A. CANINUM 801

FIGURE 4. MDIV results from the SNAP Workbench. The graphs show the posterior probability distribution of migration and divergence time between each of 2 geographic samples for a particular gene region. Ten independent runs were simulated under the assumption of an inﬁnite sites model, with 2,000,000 steps in the chain and a burn-in time of 500,000 using a different starting random number seed for each run. The distributions for migration and time of divergence were similar between runs. The data were plotted with Gnuplot, version 4.6. Posterior probability distribution for migration (M) and divergence time (T) between Wake County and Maryland samples at the cox-1 locus (A), between U.S. and Australia samples at cox-1 (B), and between Wake County and Maryland samples at asp-1E (C). 802 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE III. asp-1 population parameters.

Haplotypes ␪/locus Sample Fu and Fu and Sites¶ Mutations size H diversity ␲ ␪/site Tajima’s D Li’s D* Li’s F* Fu’s F

Entire sequence. asp-1E† 348 43 70 33 0.016 Ϯ 0.001 9.33 Ϫ1.400 ns‡ Ϫ0.976 ns Ϫ1.360 ns Ϫ16.75 0.932 Ϯ 0.017 0.028 asp-1D 286 3 8 3 0.004 Ϯ 0.013 1.15 Ϫ0.431 ns Ϫ0.630 ns Ϫ0.642 ns 0.270 0.679 Ϯ 0.12 0.004 asp-1C 715 129 22 20 0.033 Ϯ 0.004 35.39 Ϫ1.436 ns Ϫ2.011 ns Ϫ2.147 ns Ϫ3.808 0.987 Ϯ 0.020 0.043 Coding regions. asp-1E† 208 16 70 16 0.007 Ϯ 0.001 3.321 Ϫ1.704 ns‡ Ϫ0.781 ns Ϫ1.317 ns Ϫ10.318 0.779 Ϯ 0.043 0.016 asp-1D 191 0 8 1 0 0 n/a§ n/a n/a n/a 0.0 asp-1C 289 31 22 18 0.014 Ϯ 0.002 8.504 Ϫ1.997# Ϫ2.842# Ϫ3.020# Ϫ12.751 0.957 Ϯ 0.037 0.029 Noncoding regions. asp-1E† 125‡ 26 70 22 0.028 Ϯ 0.002 5.412 Ϫ1.704 ns Ϫ1.124 ns Ϫ1.169 ns Ϫ8.551 0.889 Ϯ 0.024 0.044 asp-1D 62 3 8 30 0.017 Ϯ 0.006 1.157 Ϫ0.431 ns Ϫ0.632 ns Ϫ0.642 ns 0.268 679 Ϯ 0.015 0.019 asp-1C 425 97 22 19 0.046 Ϯ 0.006 26.61 Ϫ1.626 ns Ϫ1.751 ns Ϫ3.020 ns Ϫ3.399 0.974 Ϯ 0.028 0.065

¶Includes gaps and missing data. † Contains alleles from both Wake County and Maryland A. caninum. ‡ ns, not signiﬁcant. ‡ Sequence alignment gaps removed. § n/a, no sequence variation. # P Ͻ 0.05.

type H2, the most divergent haplotype in the network, is 3 worms indicated that there is very little linkage disequilibrium nucleotide substitutions away from a nonsampled inferred in- between the asp-1C and asp-1E regions (data not shown). terior node and 4 mutational steps from H12. The internal asp-1D region was difﬁcult to sequence, prob- Hudson’s test was performed using the SNAP Workbench to ably because of the high polymorphism content. Only 8 high- assess population subdivision among Wake County and Mary- quality sequences were available, all from Wake County. This

land A. caninum at asp-1E. The KST value of 0.016 is low but small sample contains only a few segregating sites and corre- significant (P ϭ 0.04) (Table II), suggesting little genetic dif- spondingly exhibits a low level of nucleotide diversity (␲ϭ ferentiation and moderate levels of gene flow among the pop- 0.004). Because of the low ␲ and small number of haplotypes ulation samples. The results from MDIV analysis implemented (h ϭ 3), neither a strict consensus tree nor a haplotype network in SNAP Workbench also support the finding of moderate gene was inferred for this region. Further collapsing haplotypes in flow (M ϭ 1.74) (Fig. 4C). This is consistent with the results asp-1 networks by removing sequences that differ by fewer than from the cox-1 locus. 2 nucleotides from other sequences yielded no substantial dif- The 715-nucleotide asp-1C region contains 3 exons and 3 ferences from when all sequences are considered; so, the po- introns, with the latter being highly polymorphic for both indels tential PCR and sequencing errors are assumed to be minimal. and single-nucleotide polymorphisms (SNPs). In the Wake ␲ County sample, the overall is 0.033, which is twice as high DISCUSSION as the asp-1E and cox-1 loci, and 22 sequences are represented by 20 haplotypes. Despite the large number of segregating sites, Before this study, population genetic analysis of the canine there is only 1 most parsimonious tree with all interior branches hookworm was restricted to a small survey of nucleotide di- showing Ͼ98% bootstrap support (Fig. 6). There are 3 main versity in the mitochondrial gene cox-1 and comparison of just clusters of haplotypes, with a region of unresolved phylogeny 2 cDNA sequences of the asp-1 gene. Our survey of 2 mid- in the center cluster, containing 12 sequences in 10 haplotypes. Atlantic samples has placed these studies in a broader perspec- The haplotype network in Figure 5B displays this region of tive, and it suggests that, for the most part, genetic variation in polytomy surrounding an interior node haplotype (H18) con- the species is globally distributed. Intriguingly, there are 2 di- sisting of 3 sequences. Tests of neutrality summarized in Table vergent groups of cox-1 haplotypes in the Australian and Wake III did not suggest any deviation from neutrality in this region. County samples, mainly due to the relatively large number of Independent analysis of genomic sequences isolated from single nucleotide substitutions among the Australian samples, but MOSER ET AL.—GENETIC DIVERSITY OF A. CANINUM 803

FIGURE 5. asp-1 haplotype networks. Networks were generated using the GT Miner component of SNAP Workbench to display the mutational connections between haploypes. The haplotypes are represented by oval nodes, connected by branches that indicate the number of nucleotide differences between them. Empty nodes represent unsampled haplotypes that are inferred to exist, or have existed at some time, but they have not been sampled. (A) asp-1E coding region haplotype network. H1 contains both Maryland and Wake County samples. H2 represents the Chinese asp-1 (GenBank AAD31839). (B) asp-1C haplotype network contains only Wake County samples. most of the sequences share a recent common ancestor in a vergence within the Australian hookworms relates to sampling central network. At asp-1E, the single Chinese sample is the of worms with different infectivity strategies (Hu, Chilton et most divergent sequence, but it is not significantly different al., 2002). At any given nucleotide site in cox-1, pairs of alleles given the diversity observed in Wake County and Maryland. from the Maryland sample will match more than 99.9% of the The nuclear and mitochondrial analyses are in agreement, sug- time. This percentage decreases to 98.6% for Wake County gesting that there has been migration between the populations worms and to an even lower 94.5% in the Australian A. can- and that there is little evidence for strong population structure. inum. The Maryland sample shows lower diversity because of In mitochondrial cox-1, the average nucleotide diversities its confinement in the laboratory, which has enforced inbreed- range from a low of 0.001 in Maryland worms to a high of ing and prevented the gene flow that occurs in a ‘‘wild’’ pop- 0.055 in the Australian samples. It has been suggested the di- ulation. The levels of nucleotide diversity for Wake County 804 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

surprisingly, that negative selection constrains polymorphism in the exons. An unexpected ﬁnding concerns the independence of haplotype network structure within short segments of the asp-1 locus. The 2 networks in Figure 5 have very different topologies, i.e., asp-1E is a very typical topology for a region with low diversity, whereas asp-1C shows an unusual bipartite topology. One cluster of haplotypes radiates around the most frequent haplotype, whereas another 10 haplotypes spread out as a long tail, with several nucleotides in each haplotype different from the others. Standard tests of neutrality do not imply that selection has shaped this variation, and it is possible that the major cluster on the left side of the network represents a fairly recent expansion. It suggests that more extensive sampling may un- cover local population structure such as that described for N. americanus in China (Hu et al., 2003). A further implication of these results is that there is very little linkage disequilibrium in the A. caninum genome. This inference is conﬁrmed by limited bidirectional sequencing of genomic asp-1 products spanning the locus, because 4 gamete tests imply that recombination breaks up associations between common polymorphisms separated by just 2 kilobases (data not shown). In this respect, the canine hookworm is more like Dro- sophila melanogaster than humans or the soil nematode Cae- norhabditis elegans, both of which show extensive linkage disequilibrium over tens of kilobases (Reich et al., 2001; Gabriel et al., 2002; Cutter, 2006). A further consequence is that asso-

FIGURE 6. Maximum parsimony tree of asp-1C haplotypes inferred ciation mapping of genotypes that correlate with infectivity or using PHYLIP in the SNAP Workbench and displayed with MEGA3. antigenicity must be focused on individual SNPs rather than The 20 distinct haplotypes were inferred by collapsing 22 alleles, ex- tagging of haplotypes. A corollary is that if positive selection cluding inﬁnite sites violations. is shaping variation in the asp-1 locus, its impact on diversity has been restricted to just a few hundred nucleotides surrounding any focal site and must, therefore, necessarily be relatively weak. Nevertheless, the relatively high levels of polymorphism samples are on par with other studies of mitochondrial DNA and absence of linkage disequilibrium are excellent conditions diversity in parasites with vertebrate hosts (Blouin et al., 1995, favoring the use of population genetic strategies to identify re- 1999; Hawdon et al., 2001; Brashier et al., 2004). Despite the gions of the genome that shape the parasite’s response to anti- existence of at least 3 clusters of haplotypes in cox-1 of A. helminthics and immunotherapeutic options such as vaccina- caninum, the nucleotide diversity is mostly consistent with a tion. neutral model of evolution. The other gene of interest, the nuclear asp-1, is a highly ACKNOWLEDGMENTS variable gene. Its average nucleotide diversity across the ana- ␲ The Maryland strain of A. caninum was kindly provided by Thomas lyzed regions is 0.020, and the ranges of in 3 segments of Nolan, University of Pennsylvania. J.M. was supported by an IGERT the gene were from 0.004 to 0.033, representing an almost 10- Training Grant from NSF, and the research was facilitated by NIH grant fold difference. Antigenic diversity may exist, although it is not 1-R01-GM61600 to G.G. known whether this may impact the development of vaccines targeted against the ASP-1 protein. There is no a priori reason LITERATURE CITED why amino acid sites that are conserved because of their con- ALTSCHUL, S. F., W. GISH,W.MILLER,E.W.MYERS, AND D. J. LIPMAN. tribution to function of the secreted protein also should be the 1990. Basic local alignment search tool. Journal of Molecular Bi- primary sites that promote antigenicity of the protein. Given ology 215: 403–410. ANDERSON, T. J., M. S. BLOUIN, AND R. N. BEECH. 1998. Population that there is amino acid diversity, it does become relevant to biology of parasitic nematodes: Applications of genetic markers. determine which, if either, domain is more functional in con- Advances in Parasitology 41: 219–283. ferring resistance to an A. caninum infection, response to vac- AYLOR, D. L., E. PRICE, AND I. CARBONE. 2006. SNAP: Combine and cination, or both. There seems to be a nominal level of variation Map modules for multilocus population genetic analysis. Bioinfor- matics 22: 1399–1401. in asp-1 coding regions between Chinese and Baltimore sam- BLOUIN, M. S. 1998. Mitochondrial DNA diversity in nematodes. Jour- ples of A. caninum, with most of the variation occurring in nal of Helminthology 72: 285–289. domain I (Qiang et al., 2000). Our analysis detected higher ———, J. LIU, AND R. E. BERRY. 1999. Life cycle variation and the levels of variation in the coding portions of Wake County and genetic structure of nematode populations. Heredity 83: 253–259. ———, C. A. YOWELL,C.H.COURTNEY, AND J. B. DAME. 1995. Host Maryland DNA than was previously found. There is consider- movement and the genetic structure of populations of parasitic ably higher nucleotide diversity in the introns, implying, not nematodes. Genetics 141: 1007–1014. MOSER ET AL.—GENETIC DIVERSITY OF A. CANINUM 805

BRAISHER, T. L., N. J. GEMMELL,B.T.GRENFELL, AND W. A MOS. 2004. applications to the human. Annual Review of Genomics and Hu- Host isolation and patterns of genetic variability in three popula- man Genetics 1: 539–559. tions of Teladorsagia from sheep. International Journal for Parasi- KUMAR, S., K. TAMURA, AND M. NEI. 2004. MEGA3: Integrated soft- tology 34: 1197–1204. ware for molecular evolutionary genetics analysis and sequence CASSENS, I., P. MARDULYN, AND M. C. MILINKOVITCH. 2005. Evaluating alignment. Brief Bioinformatics 5: 150–163. intraspecific ‘‘network’’ construction methods using simulated se- MADDISON, D. R., D. L. SWOFFORD, AND W. P. M ADDISON. 1997. NEX- quence data: Do existing algorithms outperform the global maxi- US: An extensible file format for systematic information. Systems mum parsimony approach? Systems Biology 54: 363–372. Biology 46: 590–621. OSER REITAS RASU AND IBSON CUTTER, A. D. 2006. Nucleotide polymorphism and linkage disequilib- M ,J.M.,T.F ,P.A , G. G . 2005. Gene ex- rium in wild populations of the partial selfer Caenorhabditis ele- pression profiles associated with the transition to parasitism in An- cylostoma caninum larvae. Molecular and Biochemical Parasitol- gans. Genetics 172: 171–184. ogy 143: 39–48. FELSENSTEIN, J. 2004. PHYLIP (phylogeny inference package) version NIEBERDING, C., R. LIBOIS,C.J.DOUADY,S.MORAND, AND J. R. MI- 3.6. Software distributed by Department of Genomics Sciences, CHAUX. 2005. Phylogeography of a nematode (Heligmosomoides University of Washington, Seattle, Washington. polygyrus) in the western Palearctic region: Persistence of northern FU, Y. X. 1997. Statistical tests of neutrality of mutations against pop- cryptic populations during ice ages? Molecular Ecology 14: 765– ulation growth, hitchhiking and background selection. Genetics 779. 147: 915–925. NIELSEN, R., AND J. WAKELEY. 2001. Distinguishing migration from iso- ———, AND W. H. L I. 1993. Statistical tests of neutrality of mutations. lation: A Markov chain Monte Carlo approach. Genetics 158: 885– Genetics 133: 693–709. 896. GABRIEL, S. B., S. F. SCHAFFNER,H.NGUYEN,J.M.MOORE,J.ROY,B. PICARD, D., O. PLANTARD,M.SCURRAH, AND D. MUGNIERY. 2004. In- BLUMENSTIEL,J.HIGGINS,M.DEFELICE,A.LOCHNER,M.FAGGART, breeding and population structure of the potato cyst nematode (Glo- ET AL. 2002. The structure of haplotype blocks in the human ge- bodera pallida) in its native area (Peru). Molecular Ecology 13: nome. Science 296: 2225–2229. 2899–2908. GASNIER,N.,AND J. CABARET. 1998. Stable and unstable environments PRICE,E.W.,AND I. CARBONE. 2005. SNAP: Workbench management influence the genetic diversity of the nematode Teladorsagia cir- tool for evolutionary population genetic analysis. Bioinformatics cumcincta, a parasite of small ruminants. Parasitology Research 84: 21: 402–404. QIANG, S., Z. BIN,X.SHU-HUA,F.ZHENG,P.HOTEZ, AND J. M. HAWDON. 676–681. 2000. Variation between ASP-1 molecules from Ancylostoma can- HAWDON, J. M., B. F. JONES,D.R.HOFFMAN, AND P. J. HOTEZ. 1996. inum in China and the United States. Journal of Parasitology 86: Cloning and characterization of Ancylostoma-secreted protein. 181–185. Journal of Biological Chemistry 271: 6672–6678. REICH, D. E., M. CARGILL,S.BOLK,J.IRELAND,P.C.SABETI,D.J. ———, T. LI,B.ZHAN, AND M. S. BLOUIN. 2001. Genetic structure of RICHTER,T.LAVERY,R.KOUYOUMJIAN,S.F.FARHADIAN,R.WARD, populations of the human hookworm, Necator americanus, in Chi- ET AL. 2001. Linkage disequilibrium in the human genome. Nature na. Molecular Ecology 10: 1433–1437. 411: 199–204. HU, M., N. B. CHILTON,Y.G.ABS EL-OSTA, AND R. B. GASSER. 2003. ROZAS, J., J. C. SANCHEZ-DELBARRIO,X.MESSEGUER, AND R. ROZAS. Comparative analysis of mitochondrial genome data for Necator 2003. DnaSP, DNA polymorphism analyses by the coalescent and americanus from two endemic regions reveals substantial genetic other methods. Bioinformatics 19: 2496–2497. variation. International Journal for Parasitology 33: 955–963. TAJIMA, F. 1989. Statistical method for testing the neutral mutation hy- ———, ———, X. ZHU, AND R. B. GASSER. 2002. Single-strand con- pothesis by DNA polymorphism. Genetics 123: 585–595. formation polymorphism-based analysis of mitochondrial cyto- TARRANT, C. A., M. S. BLOUIN,C.A.YOWELL, AND J. B. DAME. 1992. chrome c oxidase subunit 1 reveals significant substructuring in Suitability of mitochondrial DNA for assaying interindividual ge- hookworm populations. Electrophoresis 23: 27–34. netic variation in small helminths. Journal of Parasitology 78: 374– 378. ———, J. HOGLUND,N.B.CHILTON,X.ZHU, AND R. B. GASSER. 2002. THOMPSON, J. D., D. G. HIGGINS, AND T. J. GIBSON. 1994. CLUSTAL Mutation scanning analysis of mitochondrial cytochrome c oxidase W: Improving the sensitivity of progressive multiple sequence subunit 1 reveals limited gene flow among bovine lungworm sub- alignment through sequence weighting, position-specific gap pen- populations in Sweden. Electrophoresis 23: 3357–3363. alties and weight matrix choice. Nucleic Acids Research 22: 4673– HUDSON,R.R.,M.SLATKIN, AND W. P. M ADDISON. 1992. Estimation of 4680. levels of gene flow from DNA sequence data. Genetics 132: 583– VACHER, C., S. P. BROWN, AND M. E. HOCHBERG. 2005. Avoid, attack 589. or do both? Behavioral and physiological adaptations in natural KREITMAN, M. 2000. Methods to detect selection in populations with enemies faced with novel hosts. BMC Evolutionary Biology 5: 60. J. Parasitol., 93(4), 2007, pp. 817–823 ᭧ American Society of Parasitologists 2007

IDENTIFICATION OF FASCIOLA HEPATICA RECOMBINANT 15-KDA FATTY ACID–BINDING PROTEIN T-CELL EPITOPES THAT PROTECT AGAINST EXPERIMENTAL FASCIOLIASIS IN RABBITS AND MICE

Antonio Muro, Patricia Casanueva, Julio Lo´ pez-Abań, Vicente Ramajo*, Antonio R. Martıńez-Fernańdez†, and George V. Hillyer‡ Laboratorio de Parasitologıá, Facultad de Farmacia, Universidad de Salamanca, Avda, Campo Charro s/n 37007 Salamanca, Espanã. e-mail: [email protected]

ABSTRACT: Vaccination with fatty acid–binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. More- over, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Th1/Th2 response to vaccination.

Fascioliasis is caused by the liver flukes Fasciola hepatica important for the determination of respective protection mech- and Fasciola gigantica and represents one of the most wide- anisms. spread parasitic diseases in the world. This infection, mostly In the current study, we define potentially protective peptides occurring in ruminants and occasionally affecting humans, is within the sequence of the rFh15 molecule that are predicted to mainly acquired following ingestion of vegetation contaminated be recognized by T cells. These peptides were used together with metacercariae of the fluke. Adult worms localize in the with the recently described adjuvant-adaptation (ADAD) adju- liver, where they cause extensive damage. Evidence suggesting vant-immunomodulatory system (Martıńez-Fernańdez et al., that natural hosts develop immune responses that could protect 2004) against experimental fascioliasis in 2 different models, against secondary F. hepatica infections provided the first in- i.e., rabbits and mice. The selected peptides showed protection sights into the development of vaccines against this parasite levels comparable to those previously obtained with native or (Haroun and Hillyer, 1986). Several candidate molecules have recombinant F. hepatica FABPs. been obtained and assayed in different experimental fascioliasis models. The fatty acid–binding protein (FABP) family from F. MATERIALS AND METHODS hepatica represents a classic example of this vaccine discovery Animals pathway. An FABP antigen was characterized as the major For the protection experiments, 35 3-mo-old New Zealand rabbits component of the so-called FhSmIII(M) complex. This complex and 64 6-wk-old BALB/c and CD1 mice were used. Animals were isolated from F. hepatica adult worms is recognized by cross- maintained in a temperature- and humidity-controlled environment with reacting and cross-protecting antibodies raised against adult a 12-hr dark/light cycle in the animal facility unit at Salamanca Uni- Schistosoma mansoni (Hillyer, 1984; Hillyer, 2005). versity, with free access to water and food. Vaccination trials with defined F. hepatica FABP fractions Parasite and antigens demonstrated moderate to high protection levels in vaccinated animals experimentally infected with this parasite (Muro et al., Fasciola hepatica metacercariae were obtained from miracidia-infected, laboratory-bred Lymnaea (Galba) truncatula snails, as described 1997; Casanueva et al., 2001; Ramajo et al., 2001; Martıńez- in Muro et al. (1997). Metacercariae were used for the experimental Fernańdez et al., 2004). Thus, identification and characteriza- infection of rabbits and mice as described below. tion of protective epitopes in the above-mentioned molecules is Fasciola hepatica excretory/secretory antigens (FhES) were prepared as described in Casanueva et al. (2001), with minor modifications. Brief- ly, F. hepatica adult worms obtained from experimentally infected Received 5 September 2006; revised 26 January 2007, 16 February sheep were washed 4 times for 1 hr at room temperature in phosphate 2007; accepted 21 February 2007. buffered saline (PBS; pH 7.2). The washed worms were then incubated * Departamento de Patologıá Animal, Instituto de Recursos Naturales y at 37 C for 3 hr in PBS (1 ml/worm) plus 1 mM phenyl methyl sul- Agrobiologıá de Salamanca (CSIC), Salamanca, Spain. phonyl fluoride (PMSF), 1 mM ethylenediamine tetraacetic acid † Departamento de Parasitologıá, Facultad de Farmacia, Universidad (EDTA), 1 mM N-ethylmaleimide (NEM), 0.1 mM pepstatin A, and Complutense, Pza. Ramoń y Cajal s/n, 28040 Madrid, Spain. 0.1 mM N-tosylamide-L-phenylalanine chloromethyl ketone (TPCK). ‡ To whom correspondence should be addressed. Department of Pa- After incubation, supernatants were collected and centrifuged at 2,000 thology and Laboratory Medicine (Suite 617-A), University of Puerto g for 10 min. Corresponding supernatants were concentrated in SK fil- Rico School of Medicine, P.O. Box 365067, San Juan, Puerto Rico ters (Millipore Corporation, Billerica, Massachusetts), dialyzed, and fur- 00936-5067. ther centrifuged at 5,000 g for 10 min. Concentration of proteins in final

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TABLE I. Evaluation of protection elicited by vaccination with pure or GST-fused Fh15Ta and Fh15Tb peptides in rabbits experimentally infected with 30 Fasciola hepatica metacercariae. Worm recovery in the necropsy, percentage of reduction, immature ﬂukes, and hepatic lesions are shown.

Hepatic lesions (no. of rabbits)* Worm recovery Reduction Immature Group n (mean Ϯ SD) (%) ﬂukes (%) (Ϫ)(ϩ)(ϩϩ)(ϩϩϩ)

Uninfected control 3 3 0 0 0 Infected control 6 9.10 Ϯ 2.85 14 0 0 5 1 Immunized with Fh15Ta and challenged 5 8.00 Ϯ 3.39 13.0 46† 0 0 3 2 Immunized with Fh15Tb and challenged 6 9.83 Ϯ 4.99 — 5 0 1 2 3 Uninfected control 3 3 0 0 0 Immunized with GST and challenged 3 14.66 Ϯ 1.52 64 0 0 0 3 Immunized with GST-Fh15Ta and challenged 5 7.60 Ϯ 1.66 48.2† 84† 0 0 2 3 Immunized with GST-Fh15Tb and challenged 4 6.00 Ϯ 2.28 59.1† 41† 0 0 4 0

*(Ϫ) No lesions, (ϩ) moderate, (ϩϩ) mild, and (ϩϩϩ) severe. † P Ͻ 0.05.

supernatants was determined by the BCA technique (Pierce, Rockford, damage evaluated considering the size, consistency, color, dilation of Illinois). the bile ducts, and repletion of the vessels in accordance with Casan- ueva et al. (2001). The number of surviving mice were those surviving Identification and production of T-site peptides from the Fh15 after the death of all animals in the infection control group (40–46 days recombinant protein PI) as in Martıńez-Fernańdez et al. (2004).

Identification of putative T-sites in the recombinant Fh15 protein Evaluation of immune responses (GenBank M95291) was done with the ‘‘Peptidestructure’’ application from the GCG Computer Package (Accelrys GCG, Accelrys Software, Antibody titers (serum IgG and bile IgA in rabbits, serum IgG, IgG1, Inc., Madison, Wisconsin), based on the original method of Chou and and IgG2a in mice) were evaluated by the enzyme-linked immunosor- Fasman (1978). The corresponding synthetic oligonucleotides, obtained bent assay (ELISA) (Muro et al., 1997; Casanueva et al., 2001). For in the 392 DNA/RNA Synthesizer as recommended by the commercial total IgG detection, ELISA plates (Sigma) were coated overnight at 4 ␮ ␮ ␮ suppliers (Applied Biosystems, Chicago, Illinois) and containing cor- C with 4 g/ml FhES, 2 g/ml recombinant GST, or 1 g/ml recom- responding adaptors for subcloning, were subcloned in the BamHI- binant Fh15 antigen in carbonate buffer (45 mM sodium carbonate plus EcoRI sites of the pGEX-2TK vector. Recombinant vectors were used 16 mM sodium bicarbonate; pH 9.6). Plates were then washed 3 times for the production of the corresponding glutathione S-transferase (GST)- with PBS plus 0.05% Tween 20 (pH 7.2) and blocked with 0.05% fused peptides. These were purified in affinity columns (Amersham Tween 20 and 0.5% bovine serum albumin (BSA) in PBS (pH 7.2). Pharmacia Biotech, AB, Uppsala, Sweden) and eluted as GST-fusion Corresponding rabbit or mice sera were diluted 1:100 in blocking buffer ␮ proteins, or obtained as nonfused, pure recombinant peptides in super- (100 l/well) and incubated 1 hr at 37 C per triplicate. After washing, natants from thrombin-cleaved GST-purified molecules. anti-rabbit or anti-mouse peroxidase-conjugated IgG, IgG1, and IgG2a (BioRad, Hercules, California) were diluted at 1:3,000 in blocking buff- ␮ Vaccination trials er and incubated (100 l/well) for 2 hr at 37 C. After final washes, reactions were developed with ortho-phenylene-diamine (OPD) and Rabbits: Vaccination trials were performed in New Zealand rabbits read at 492 nm. For estimation of IgA levels in bile, an ELISA test using recombinant Fh15Ta and Fh15Tb peptides or the corresponding described in Wedrychowicz et al. (1995) was used. Absorbance values GST-fused molecules. Complete (CFA) and incomplete Freund adjuvant were expressed as the mean of the optical density (OD) values from (IFA) were used for the first and second rabbit immunizations, respec- animals of each group plus the standard error (SE). Assays were done tively, plus the corresponding parasite recombinant antigen. In the first in triplicate. experiment (Table I) the rabbits were divided into 4 groups as follows: uninfected, nonvaccinated controls; infection-adjuvant controls; Statistical analysis Fh15Ta-adjuvant infection group; and Fh15Tb-adjuvant infection group. Overall differences among groups in parasite burdens and absor- In the second experiment (Table I), they were divided into 4 groups as bances were compared with an ANOVA test. When global differences follows: uninfected, nonvaccinated controls; GST-adjuvant infected were detected by this method, the Fischer Protected Least Significant group; GST-Fh15Ta infected group; and GST-Fh15Tb infected group. Difference (PLSD) test was applied. Differences were considered and Rabbits were immunized twice separated by 2 wk and were orally chal- referred to as significant when P Ͻ 0.05 unless otherwise stated. The lenged with 20 metacercariae 12 wk later. All rabbits were killed 8 wk analyses were performed using Stattview 4.5 package for a Macintosh postinfection (PI). computer (Abacus Concepts’ Statistical Software, Alameda, California). Mice: For vaccination of mice, the adaptation-adjuvant system of Martıńez-Fernandez et al. (2004) was used with the GST-fused peptides in BALB/c and CD1 mice. A micelle made with saponins of Quillaja RESULTS saponaria (10 ␮g/dose/animal; Sigma, St. Louis, Missouri) and the im- Identification of T-cell peptide epitopes from the Fh15 munomodulator Anapsos (0.6 mg/animal/injection; ASAC Pharmaceu- recombinant protein tical Int., Alicante, Spain) in an emulsion with nonmineral oil, Montan- ide ISA 763 (SEPPIC, Paris, France), was injected subcutaneously to Figure 1 shows the rFh15 amino acid sequence, as well as the animals. After this ‘‘adaptive’’ injection, animals were vaccinated the 12- and 10-mer putative T-cell sequences. From the above, with the above-mentioned emulsion plus the corresponding parasite re- the corresponding synthetic oligonucleotides and GST-fused combinant antigen (20 ␮g/dose/animal). In mice 2 immunizations were done separated by 3 wk, with a challenge 8 wk after the second im- peptides were obtained. munization. Surviving mice were killed when all non–vaccinated-infected mice had died, usually 46 days PI (Figs. 3, 4). Protection levels induced by vaccination with Estimation of protection elicited by vaccination in rabbits included Fh15-derived T-epitopes the following: comparison of the total number of F. hepatica worms in bile ducts; the number of immature worms as evaluated by presence/ Protection levels were measured as the percentage reduction absence of eggs in worm uteri; and the degree of macroscopic hepatic in total number of recovered worms, inhibition of worm mat- MURO ET AL.—F. HEPATICA RECOMBINANT PROTECTION 819

FIGURE 1. Base pair sequence of the predicted to be recognizable by T cells, Fh15Ta and Fh15Tb from the recombinant Fh15 protein from Fasciola hepatica. Adaptors for subcloning are underlined. uration, and hepatic damage for the rabbit model, and as the respectively. With respect to worm maturation, those rabbits percentage of surviving animals for the mouse model (Table I). vaccinated with Fh15Ta presented the highest maturation inhi- The immunization and infection protocols for the rabbits and bition (46%; P Ͻ 0.05). Evaluation of hepatic lesions showed mice are seen in Figure 2. lesser hepatic damage in Fh15Ta-vaccinated animals, compared In the first experiment in rabbits, the adjuvant infection con- with only infected or Fh15Tb-vaccinated rabbits (Table I). In trols yielded 9.1 Ϯ 2.9 flukes, whereas for rabbits vaccinated the second experiment with rabbits, higher protection levels de- with Fh15Ta or Fh15Tb peptides, the flukes recovered Ϯ SD fined by lower fluke recoveries were found when rabbits were were 8.0 Ϯ 3.4 (13% reduction, not significant) and 9.8 Ϯ 5.0, vaccinated with GST-Fh15Ta or GST-Fh15Tb (48.2% and

FIGURE 2. Vaccination schedules of rabbits and mice immunized with peptides predicted to be recognizable by T cells, Fh15Ta and Fh15Tb, and their GST-fused peptides GST-Fh15Ta and GST-Fh15Tb with Freund adjuvant (rabbits) or ADAD system (mice), and challenged with Fasciola hepatica metacercariae. 820 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. Survival of vaccination groups of CD1 and BALB/c mice infected with a lethal dose of 5 metacercariae of Fasciola hepatica. The ADAD system used is delineated in each box. Highest survival (40–43%) at 44ϩ days was found in the group vaccinated with an ADAD system consisting of AϩQsϩGSTϩFh15Ta.

59.1% reductions, respectively), with 14.7 Ϯ 1.5, 7.6 Ϯ 1.7, Immune responses Ϯ and 6.0 2.3 worms in GST-, GST-Fh15Ta-, and GST- In the rabbits immunized with GST-fused peptides, IgG and Fh15Tb–vaccinated animals, respectively. The highest imma- IgA levels against FhES antigens rose after infection, with com- Ͻ turity of worms (84%; P 0.05) was found in GST-Fh15Ta parable ODs in all groups at the end of the experiment. Anti- (Table I). Fh15 IgG antibodies rose after immunization, i.e., comparable In the ﬁrst experiment with mice, all F. hepatica infection levels in all groups were present until the end of the experiment. control CD1 mice died between 30 and 40 days PI. CD1 mice In unimmunized and Fh15Ta- and Fh15Tb-immunized rabbits, vaccinated with GST-Fh15Ta showed the highest protection lev- there were no IgG and IgA antibodies detected against FhES el (43% of animals surviving at 48 days), when compared with and Fh15 antigens (data not shown). GST-Fh14Tb and GST vaccinated mice, with 20% and 12% of In CD1 mice, anti-FhES IgG, IgG1, and IgG2a were studied animals surviving, respectively. The ADAD system with GST at the third wk PI (Fig. 4). The group vaccinated with GST- did not elicit protection, with 12% surviving at 40 days (Fig. Fh15Tb showed the highest IgG and the lowest IgG1 responses 3). (P Ͻ 0.05). Th1-type responses (IgG2a anti-FhES) were higher Potential variation in protection levels elicited by vaccination in GST-Fh15Ta–vaccinated animals (P Ͻ 0.05). When anti- due to changes in genetic background of the mice was studied FhES IgG1 and IgG2a levels were compared between surviving with the GST-Fh15Ta molecule. Thus, in the second murine and nonsurviving GST-Fh15Ta–vaccinated animals, surviving experiment, BALB/c mice were vaccinated with GST-Fh15Ta. mice elicited higher IgG2a responses than those that did not Protection levels were similar to those achieved in CD1 mice, survive (Fig. 5). These differences in anti-FhES IgG2a levels with 40% of mice surviving at 40–46 days, and no survivors were not observed between protected and unprotected BALB/c in the infection control group (Fig. 3). GST-Fh15Ta–vaccinated mice (data not shown). MURO ET AL.—F. HEPATICA RECOMBINANT PROTECTION 821

FIGURE 4. Enzyme-linked immunosorbent assay with FhES antigens for detection of IgG, IgG1, and IgG2a antibody isotypes in CD1 mice at the third week of F. hepatica infection. Groups: nonvaccinated and noninfected (NVI), nonvaccinated and infected (Inf), immunized with GST and infected (GST), immunized with GST-Fh15Ta and infected (GST-Fh15Ta), and immunized with GST-Fh15Tb and infected (GST-Fh15Tb). *P Ͻ 0.05. IgG2a antibody levels were highest in the mouse group with the highest protection levels.

DISCUSSION it was of particular interest to identify epitopes reacting with B and T cells in parasite-derived molecules. Identification of such Fascioliasis caused by F. hepatica affecting domestic rumi- epitopes could lead to the synthesis of peptides, which, when nants throughout the world represents one of the most severe used in conjunction with appropriate carriers or adjuvants, veterinary parasitic diseases, and is becoming an increasingly could potentially induce protective responses against well-char- prevalent parasite in humans (Mas Coma and Bargues, 1997; acterized vaccines. This strategy has been already used by nu- Esteban et al., 2002), with the highest prevalence and intensities of infection found in the Bolivian Altiplano. There, prevalences merous authors working with different pathogens, including detected in some communities were 72% to 100% in coprol- parasites (Drabner et al., 2002). ogical and serological surveys, respectively (Hillyer et al., In the present study, we have identified and constructed 2 1992; Mas Coma et al., 2005). Treatment of fascioliasis relies different T-cell epitopes (Fh15Ta and Fh15Tb) based on the on the use of triclabendazole, although resistance has been re- linear Fh15 sequence and the universal sequence for T-cell epi- ported recently (Moll et al., 2000). Thus, vaccination against F. topes described by Rothbard and Taylor (1988). We used these hepatica has been proposed as an alternative to drug treatment. molecules in a first vaccination trial in rabbits immunized with Several parasite-derived molecules, including proteases, struc- Freund adjuvant plus one of the above-mentioned peptides and tural proteins, and FABPs have been used in protection exper- subsequently challenged with F. hepatica metacercariae. Re- iments. Specifically, a native FABP called Fh12 and the cor- sults showed that only Fh15Ta-vaccinated animals elicited a responding recombinant protein, Fh15, have shown protective partially protective immune response. The low protection levels properties against F. hepatica experimental infections in mice, (13% in Fh15Ta-vaccinated rabbits) achieved in this first ex- rabbits, and sheep (Muro et al., 1997; Casanueva et al., 2001; periment could be attributed to the small size of Fh15Ta (12 Ramajo et al., 2001; Martıńez-Fernańdez et al., 2004). Never- amino acids) and Fh15Tb (10 amino acids) peptides used for theless, differences in protection levels were reported when na- immunization, since low responses against small peptides have tive and recombinant molecules were compared. In this context, been already pointed out by other authors (Hey et al., 1994).

FIGURE 5. Enzyme-linked immunosorbent assay with FhES antigens for detection of IgG, IgG1, and IgG2a at the third week of challenge in GST-Fh15Ta–vaccinated CD1 mice comparing surviving and nonsurviving animals. IgG2a antibody levels were highest in the surviving mice group. 822 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

Because small peptides that were being used as the relevant tected in protected animals, compared with unprotected mice. antigenic determinants were likely to be poor immunogens as Th1 responses have been already pointed out by other investi- such, the augmentation of their immunogenic capacity by a car- gators as potentially protective against F. hepatica (Mulcahy rier or another means seemed to be crucial for the induction of and Dalton, 2001). immunity. Thus, we decided to fuse each peptide to GST and The present study follows a progression of vaccination suc- use them in combination with Freund adjuvant for vaccination cesses against fascioliasis with extracts of whole flukes as well of rabbits. Animals immunized with GST-Fh15Ta or GST- as purified antigens (reviewed in Haroun and Hillyer, 1986; Fh15Tb produced epitope-specific antibodies that recognized Spithill et al., 1999; Hillyer, 2005). We are moving towards the whole Fh15 recombinant protein. In addition, the protection recombinant and synthetic antigens and epitopes with new ad- percentage was much higher when GST-fused peptides instead, juvant formulations in another step toward the development of of peptides alone, were used for vaccination. Protection per- a useful vaccine against fascioliasis and, in some instances, also centages obtained in GST-Fh15Ta- or GST-Fh15Tb–vaccinated against schistosomiasis. rabbits (48.2% and 59.1%, respectively) were higher than those In summary, we have shown that vaccination with a single obtained in similar experiments after vaccination with the whole T-cell epitope, derived from the F. hepatica Fh15 FABP recom- Fh15 recombinant molecule (Rodriguez-Perez et al., 1992), in binant protein fused to GST (GST-Fh15Ta) resulted in compa- which 43% reductions in fluke burdens were obtained (Casan- rable protection levels in 2 different animal models. Thus, pep- ueva et al., 2001). Reduction in hepatic damage showed, as tide vaccines also offer an attractive alternative to whole pro- expected, a good correlation with reduction in worm number in teins for their use against F. hepatica infections. In addition, vaccinated groups. Inhibition of worm maturation was also ob- we have identified a more innocuous vaccine formulation (the served in vaccinated animals, being higher in the GST-Fh15Ta– ADAD system) that elicits similar protection levels against F. vaccinated animals. However, whether these flukes will even- hepatica using recombinant peptides derived of FABP (Fh15) tually mature belatedly, remain immature, or die is not yet from the parasite. known. It is well known that protective properties of vaccine candi- ACKNOWLEDGMENTS dates can vary considerably when tested in different animals or We thank ASAC Pharmaceutical International (Alicante, Spain) for in animal species with variable genetic background. For this supplying us with the immunmodulator Anapsos and to Jose´ Lora (Sep- reason, we extended our vaccination trials to a second model, pic, Spain) for kindly providing Montanide ISA 763A. This research using both CD1 and BALB/c mice for our study. At the same was supported by grants from the Direccioń General de Ensenãnza Su- time, we wanted to test new adjuvants and immunomodulators. perior e Investigacioń Cientı´fica (Ministerio de Educacioń y Cultura, Spain) with references: 1FD 97-1313-CO2-01, AGL 2000 0039, and Because Freund adjuvant’s toxicity has been shown, we wanted AGL2005 02168GAN, as well as the U.S. National Science Foundation to search for a good alternative. Thus, we combined GTS- SPACS Program. A.M. was the recipient of a Fulbright scholarship in Fh15Ta or GTS-FH15Tb recombinant proteins with the so- Molecular Parasitology. The support of Mariano Garcia-Blanco in de- called ADAD system (Martinez-Fernandez et al., 2004) com- veloping the recombinant peptides is warmly acknowledged. posed of a new immunomodulator (Anapsos) and saponines of Quillaja saponaria as an adjuvant in a Montanide emulsion. LITERATURE CITED

Protection levels achieved with our vaccination protocol in both CASANUEVA, P., G. V. HILLYER,V.RAMAJO,A.OLEAGA,E.Y.ESPINOZA, mice strains were very similar (43% and 40% for GST-Fh15Ta AND A. MURO. 2001. Immunoprophylaxis against Fasciola hepatica in CD1 and BALB/c vaccinated animals, respectively). Thus, in rabbits using a recombinant Fh15 fatty acid-binding protein. immunization with the Fh15Ta peptide fused to GST in the Journal of Parasitology 187: 697–700. CHOU,P.Y.,AND G. D. FASMAN. 1978. Prediction of the secondary ADAD system resulted in comparable protection levels with structure of proteins. Advances in Enzymology 47: 45–148. those obtained by immunizing mice with the whole Fh15 pro- DRABNER, B., U. REINEKE,J.SCHNEIDER-MERGENER,R.E.HUMPHREYS, tein in other experiments done by our group. S. HARTMANN, AND R. LUCIUS. 2002. Identification of T helper cell- Several studies have shown that vaccination of experimental recognized epitopes in the chitinase of the filarial nematode On- animals against F. hepatica with parasite-derived (B cell) epi- chocerca volvulus. Vaccine 20: 3685–3694. ESTEBAN, J. G., C. GONZALEZ,M.D.BARGUES,R.ANGLES,C.SANCHEZ, topes from molecules different from the FABPs also resulted in AND C. NAQUIRA. 2002. High fascioliasis infection in children medium to high levels of protection (Harmsen et al., 2004). linked to a man-made irrigation zone in Peru. Tropical Medicine Nevertheless, comparison between protection levels elicited by and International Health 73: 39–48. those peptides and the whole molecule (cathepsin L protease 3) HARMSEN, M. M., J. B. CORNELISSEN,H.E.BUIJ,W.J.BOERSMA,S.H. JEURISSEN, AND F. J . VAN MILLIGEN. 2004. Identification of a novel from which they were derived has not been done to date. At Fasciola hepatica cathepsin L protease containing protective epi- the same time, selection of parasite-derived peptides with po- topes within the propeptide. International Journal for Parasitology tential protective capacities, based on criteria not related with 34: 675–682. their capacity to stimulate B or T cells, has been reported by HAROUN,E.M.,AND G. V. HILLYER. 1986. Resistance to fascioliasis— Vilar et al. (2003). These authors demonstrated that a combi- A review. Veterinary Parasitology 20: 63–93. HEY, A. W., J. I. JOHNSEN,B.JOHANSEN, AND T. T RAAVIK. 1994. A two nation of peptides as small as 12 amino acids long elicited fusion partner system for raising antibodies against small immu- comparable protection levels against S. mansoni and F. hepat- nogens expressed in bacteria. Journal of Immunological Methods ica experimental infection as the whole molecule from which 173: 149–56. those peptides were selected. HILLYER, G. V. 1984. Immunity of schistosomes using heterologous trematode antigens—A review. Veterinary Parasitology 14: 263– Our results suggest that protection levels seem to correlate 283. with a Th1 bias in vaccinated and protected animals, as sug- ———. 2005. Fasciola antigens as vaccines against fascioliasis and gested by the higher levels of anti-FhES IgG2a antibodies de- schistosomiasis. Journal of Helminthology 79: 241–247. MURO ET AL.—F. HEPATICA RECOMBINANT PROTECTION 823

———, M. SOLER DE GALANES,J.RODR´ıGUEZ-PE´ REZ,J.BJORLAND,M. tigens related to fatty acid binding proteins. Veterinary Parasitology SILVA DE LAGRAVA,S.RAMIREZ GUZMAN, AND R. T. BRYAN. 1992. 69: 219–229. Use of the FAST-ELISA and the EITB to determine the prevalence RAMAJO, V., A. OLEAGA,P.CASANUEVA,G.V.HILLYER, AND A. MURO. of human fascioliasis in the Bolivian Altiplano. American Journal 2001. Vaccination of sheep against Fasciola hepatica with homol- of Topical Medicine and Hygiene 46: 603–609. ogous fatty acid binding proteins. Veterinary Parasitology 97: 35– MARTIŃEZ-FERNA´ NDEZ, A. R., J. J. NOGAL-RUIZ,J.LO´ PEZ-ABA´ N,V.RA- 46. ´ ´ ´ ´ MAJO,A.OLEAGA,M.Y.MANGA-GONZA´ LEZ,G.V.HILLYER, AND A. RODRIGUEZ-PEREZ, J., J. R. RODRIGUEZ-MEDINA,M.A.GARCIA-BLANCO, MURO. 2004. Vaccination of mice and sheep with Fh12 FABP from AND G. V. HILLYER. 1992. Fasciola hepatica: Molecular cloning, Fasciola hepatica using the new adjuvant/immunomodulator sys- nucleotide sequence and expression of a gene encoding a polypep- tem ADAD. Veterinary Parasitology 126: 287–298. tide homologous to a Schistosoma mansoni fatty acid binding protein. Experimental Parasitology 74: 400–407. MAS COMA, S., AND M. D. BARGUES. 1997. Human liver flukes: A re- OTHBARD,J.B.,AND W. R. T AYLOR. 1988. A sequence pattern common view. Research and Reviews in Parasitology 87: 145–218. R to T cell epitopes. EMBO Journal 7: 93–100. ———, ———, AND M. A. VALERO. 2005. Fascioliasis and other plant- SPITHILL,T.W.,P.M.SMOOKER,J.L.SEXTON,E.BOZAS,C.S.NIRRUSIB, borne trematode zoonoses. International Journal for Parasitology J. CREANY, AND J. C. PARSONS. 1999. Development of vaccines 35: 1255–1278. against Fasciola hepatica. In Fasciolosis, J. P. Dalton (ed.). CAB MOLL, L., C. P. GAASENBEEK,P.VELLEMA, AND F. H . B ORGSTEEDE. 2000. International, Wallingford, Oxon, U.K., p. 377–410. Resistance of Fasciola hepatica against triclabendazole in cattle VILAR,M.M.,F.BARRIENTOS,M.ALMEIDA,N.THAUMATURGO,A.SIMP- and sheep in The Netherlands. Veterinary Parasitology 91: 153– SON,R.GARRATT, AND M. TENDLER. 2003. An experimental bivalent 158. peptide vaccine against schistosomiasis and fascioliasis. Vaccine MULCAHY,G.,AND J. P. DALTON. 2001. Cathepsin L proteinases as vac- 22: 137–144. cines against infection with Fasciola hepatica (liver fluke) in ru- WEDRYCHOWICZ, H., K. BAIRDEN,E.M.DUNLOP, AND P. H . H OLMES. minants. Research in Veterinary Science 70: 83–86. 1995. Immune response of lambs to vaccination with Ostertagia MURO, A., V. RAMAJO,J.LO´ PEZ, AND G. V. HILLYER. 1997. Fasciola circumcincta surface antigens eliciting bile antibody responses. In- hepatica: Vaccination of rabbits, with native and recombinant an- ternational Journal for Parasitology 25: 1111–1112. J. Parasitol., 93(4), 2007, pp. 824–831 ᭧ American Society of Parasitologists 2007

RENEWED HOPE FOR A VACCINE AGAINST THE INTESTINAL ADULT TAENIA SOLIUM

Edda Sciutto, Gabriela Rosas*, Carmen Cruz-Revilla, Andrea Toledo, Jacquelynne Cervantes, Marisela Hernańdez, Beatrı´z Hernańdez†, Fernando A. Goldbaum‡, Aline S. de Aluja§, Gladis Fragoso, and Carlos Larralde Instituto de Investigaciones Biome´dicas, Universidad Nacional Autońoma de Me´xico, A.P. 70228, Me´xico, D.F., C.P. 04510, Me´xico. e-mail: [email protected]

ABSTRACT: Review of experimental and observational evidence about various cestode infections of mammalian hosts revives hope for the development of an effective vaccine against adult intestinal tapeworms, the central protagonists in their transmission dynamics. As for Taenia solium, there are abundant immunological data regarding cysticercosis in humans and pigs, but information about human taeniasis is scarce. A single publication reporting protection against T. solium taeniasis by experimental primo infection and by vaccination of an experimental foster host, the immunocompetent female hamster, kindles the hope of a vaccine against the tapeworm to be used in humans, its only natural deﬁnitive host.

Neurocysticercosis (NC) is a common parasitic disease of the couragement of open air defecation, detection and treatment of central nervous system that seriously affects human health some tapeworm carriers, and vaccination of rural pig hosts worldwide. The highest prevalences of NC are found in Central (Keilbach et al., 1989; Diaz-Camacho et al., 1991; Allan et al., and South America, Africa, and Asia (Schantz et al., 1994; 1997; Sarti et al., 1997, 2000; Gonzalez et al., 1998; Roman et Garcia et al., 2003; Preux and Druet-Cabanac, 2005; Morales al., 2000; Boa et al., 2003; Eddi et al., 2003; Sciutto et al., et al., 2006; Suroso et al., 2006; Waikagul et al., 2006), and the 2007). Unfortunately, experience has shown that these actions infection is spreading with the migration of humans from un- are usually insufficient to achieve wide and long-term control, derdeveloped to developed countries (Wallin and Kurtzke, resulting in rapid reestablishment of the infection following 2004; Esquivel et al., 2005). these usually transient interventions (Keilbach et al., 1989; En- The adult Taenia solium tapeworm is a hermaphroditic par- gels et al., 2003; Pawlowski et al., 2005; Garcia et al., 2006; asite that lives for years in the intestine of the human (its only Pawlowski, 2006; Sciutto et al., 2007). Limitations are exac- known definitive host), where it daily produces tens of thou- erbated by prevention programs that continue to disregard the sands of eggs. Upon defecation, the proglottids are shed to the growing and massive migration of rural peoples, together with environment, each liberating their microscopic eggs, which are their parasites, into urban areas of endemic and nonendemic then dispersed by wind and running water, and finally via con- countries and then back, thus escaping control, spreading the taminated food and unclean hands. If the proglottids or eggs disease to naı¨ve sites and reestablishing parasite transmission are ingested by pigs or humans, the eggs hatch in their intes- upon their return to their rural towns of origin (Larralde and tines and the embryos transform into oncospheres that penetrate Sciutto, 2006; http://www-lab.biomedicas.unam.mx/cistimex/). the intestinal wall and reach the circulation; eventually, many Cysticercosis by T. solium in modern times is a matter of of them transform into metacestodes (cysticerci) when lodged progressive worldwide concern, since it seems to be a massive in skeletal muscles, brain, and other tissues (Santamarıá et al., and growing problem, and is demanding research attention 2002). Taenia solium cysticerci do not reproduce asexually. To (Montresor and Palmer, 2006). Research efforts currently con- reach the reproductive stage, they must first be ingested by a centrate on the basic biology of the parasite and its mechanisms human on eating incompletely cooked cysticercotic pork, and of disease (Sciutto et al., 2000; Aguilar-Diaz et al., 2006). Ep- then they develop into adult tapeworms capable of sexual re- idemiological studies of the T. solium disease could be im- production (Sciutto et al., 2000). proved in terms of design and techniques in order to assess and Notwithstanding some optimistic opinions regarding its im- predict its impact upon public health, as well as to assess the pending control, the eradication of T. solium will probably re- role of human migration in its transmission dynamics. It is also main a major challenge for quite some time in underdeveloped advisable to develop new and stronger technologies to improve countries, where the parasite’s life cycle is firmly established its diagnosis, treatment, and prevention. within complex social, cultural, political, and economic con- Various efforts to develop effective vaccines against porcine texts, which, together, effectively counteract conventional focal cysticercosis directed to prevention have proved to be effective and unsustained control approaches. In some rural villages, var- in the experimental infection of pigs (Flisser et al., 2004; Guo ious levels of successful control have been obtained by pro- et al., 2004; Manoutcharian et al., 2004; Gonzalez et al., 2005; grams designed to locally improve sanitary conditions and per- Wu et al., 2005); a synthetic vaccine has also shown protective sonal health education, upgrading of rustic pig husbandry, dis- effects against naturally acquired infection, more notably so regarding the intensity of the cysticercosis infection (98% reduc- Received 27 July 2006; revised 16 November 2006; 19 January 2007; tion in the number of cysticerci) than with respect to prevalence accepted 22 January 2007. of the disease (50% reduction of totally protected pigs) (Huerta * Facultad de Medicina, Universidad Autońoma del Estado de Morelos, Cuernavaca, Morelos, C.P. 62210, Me´xico. et al., 2001). A weak point of this approach, however, is that † Facultad de Medicina, Universidad Nacional AutońomadeMe´xico, the effect of a vaccine against porcine cysticercosis only lasts Me´xico D.F., C.P. 04510, Me´xico. while the vaccinated pigs are alive. If the pig vaccination pro- ‡ Fundacioń Instituto Leloir, Universidad de Buenos Aires, Consejo Na- gram does not continue, the prevalence of cysticercosis in the cional de Investigaciones Cientı´ficas y Tećnicas, Buenos Aires, Ar- immunologically naı¨ve newly born pigs quickly returns to orig- gentina. § Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional inal levels, probably because of the steady and lasting supply Autońoma de Me´xico, Me´xico, D.F., Me´xico. of egg delivery by the tapeworm carrier in the locality, a pe-

824 SCIUTTO ET AL.—VACCINES AGAINST TAENIA SOLIUM TAPEWORMS 825 rennial and usually unrecognized human (Sciutto et al., 2007). ratic market availability of niclosamide, which, together with Thus, an additional target of immunotherapy should now also praziquantel, are the safe and effective taeniacides included on be considered, i.e., the adult tapeworm. However, three ques- WHO’s Essential Drug List (Flisser et al., 2005; Pawlowski, tions occur. First, are adult tapeworms vulnerable to the im- 2006). Surprisingly, niclosamide is not recommended by Mex- mune response? And, second, how effective is the host’s im- ico’s guidelines for treatment against taeniasis; albendazol mune response directed at adult tapeworms? Finally, can im- (http://www.salud.gob.mx/unidades/nom/m02ssa294.html) is mune response against adult tapeworms be developed in such the usual treatment recommended (Jagota, 1986). It is worth a way as to have lasting effects? noting that niclosamide is not absorbed from the intestine and Considering the different numbers of individual parasites at thus has fewer side effects than albendazo; it also causes the each developmental stage, i.e., tens of millions of eggs in the expulsion of the tapeworms without affecting egg integrity environment, tens or hundreds of cysticerci per intermediary (Chomicz et al., 1995), possibly increasing contamination of the host, and a single tapeworm per definitive host, the chance of local environment in an explosive transient episode if the ex- a single egg completing the full life cycle appears to be quite pelled parasites are not immediately and properly destroyed. In small, but, when successful, it is richly compensated by mul- contrast, praziquantel and albendazol are claimed to be ‘‘envi- tiplying several log units for the next reproductive cycle, start- ronmentally friendly’’ because they kill both the tapeworm and ing as a single tapeworm, capable of shedding tens of millions its eggs. However, both drugs are absorbed from the intestine of eggs in its lifetime. Thus, lowering the number of adult tape- and may cause complications in persons afflicted with neuro- worms in a locality, or reducing their reproductive capacity, or cysticercosis (Flisser et al., 1993). both, should be additional goals. Difficulties in detecting tapeworm carriers are technically by- There are 2 different strategies to fight the adult tapeworm stage of T. solium. These include (1) treatment of tapeworm passed by the indiscriminate treatment of large segments of the carriers and (2) vaccination of individuals likely to become population, usually referred to as ‘‘mass treatment’’ protocols. tapeworm carriers. From the numbers projected in the above Such an approach was tried with niclosamide in the USSR in paragraph, preventing each cysticercus from becoming an adult the l960s–70s, and was claimed to be thoroughly and perma- tapeworm (Garcia et al., 2001; de Aluja et al., 2005) or drug nently successful (Prokopenko, 1981). The difficulty in drug treatment of cysticercotic pigs (Gonzalez et al., 1998) would availability may also be bypassed by the use of other drugs. A imply a significant reduction in parasite numbers. In contrast, similar indiscriminate, but numerically more modest, mass the destruction of a tapeworm, i.e., treatment of tapeworm car- treatment approach was tried with praziquantel in 2 rural com- rier, would reduce it to a lesser extent, since this implies the munities of Ecuador, but with only transient effects upon local tapeworm had a good chance of producing millions of eggs population prevalence (Cruz et al., 1989). Focal indiscriminate before its detection and treatment. Nonetheless, the strategies treatment trials in highly endemic Mexican rural villages using are not disjunctive; both seem of potentially great value to curb praziquantel had no lasting effects on the prevalence of pig transmission, despite their possible differences in decreasing cysticercosis and, in one instance, treatment seems to have parasite numbers. turned on neurological symptoms in one otherwise clinically Treatment of tapeworm carriers usually aims at killing the silent neurocysticercosis case (Flisser et al., 1993). The huge tapeworm. In Latin America, taenicide treatment has been tried cost of treatment and monitoring, the complicated logistics of using niclosamide (Allan et al., 1997; Jeri et al., 2004), prazi- a nationwide approach, debatable humanitarian procedures, and quantel (Cruz et al., 1989; Keilbach et al., 1989; Diaz-Camacho transient effects make mass treatment an inappropriate choice et al., 1991), natural products [(areca nuts, pumpkin seeds), for control. Chung and Ko, 1976], and benzimidazol derivatives (Chavarria More recently, a growing concern that widespread use of et al., 1977) with varying degrees of success. However, they antihelmintics may lead to the development of drug resistance, have had only transient effects upon endemic patterns of infec- point to the need for control measures to complement or replace tion (Pawlowski, 2006). the use of antihelmintics (Wolstenholme et al., 2004; Coles, Drug treatment of intestinal tapeworms has shown several 2005). In keeping with this line, successful results were ob- difficulties, not the least of which is identifying bona fide T. tained using traditional intestinal antiparasitic treatments, such solium tapeworm carriers, even in highly endemic areas (Mar- as boiled areca nuts or pumpkin seeds (Chung and Ko, 1976). tinez-Maya et al., 2003; Flisser, 2006). In addition, people are However, the side effects related to areca nuts discourage its understandably reticent to be treated for a virtually asymptomatic infection (arguments of risk reduction are too abstract to use in human treatment. Pumpkin seeds, however, seem a fea- move people into action). Thus, the effectiveness of the drug sible alternative since they are effective, of agreeable taste, tra- employed against the tapeworms is difficult to assess with pre- ditionally accepted as snack food, and innocuous at effective cision. For example, in a recent and carefully controlled study doses. Moreover, experimental evidence of effective antihel- conducted in Peru (Jeri et al., 2004), identifiable gravid pro- minthic properties of pumpkin seed treatment has been ob- glottids were obtained in only 62% of individuals who received tained. The minimum effective dose is 23 g of pumpkin seeds treatment (2 L of electrolyte-polyethyleneglycol solution [EPS] in 100 ml of distilled water (equivalent to about 73 pumpkin overa2hrperiod, followed by 2 g of niclosamide and another seeds) (Diaz-Obregon et al., 2004). The small quantity of 2 L of EPS). The remaining 38% of tapeworms were either not pumpkin seeds required for antihelminthic effects appears to present at the time of treatment, or digested beyond expert rec- make them an attractive, cost/effective, and safe approach to be ognition, or remained in the intestines of their hosts, harmed or considered in control programs. Nonetheless, more thorough unharmed. Another difficulty for tapeworm treatment is the er- evaluations are required to assess their efficacy with certainly. 826 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Experiences in vaccination against intestinal tapeworm infections.

Source of vaccine antigens; Intestinal cestode Host route of immunization* Challenge Protective effect Reference

Echinococcus granulo- Dogs Cyst membranes or sco- NF† 50% of reduction in the Turner et al. (1936) sus lices number of worms Echinococcus granulo- Dogs Secretory antigens col- 80,000 protosco- 64% of reduction in the Herd et al. (1975) sus lected from worms leces/dog number of worms cultured in vitro; 4 90% suppression of doses; s.c.‡; FCA§ egg production Taenia hydatigena Dogs Secretory antigens col- 5 cysticer- No protection Heath et al. (1980) lected from worms ci/dog cultured in vitro; 1 dose; s.c. ϩ i.m.࿣; FIA# Taenia hydatigena Dogs Subunit antigens with or 5 cysticer- 66.7% to 83.3% Kandil and Abou- without vitamin E ϩ ci/dog Zeina (2005) selenium Taenia hydatigena Dogs Somatic antigens with or 5 cysticer- 50% to 83.3% Kandil and Abou- without vitamin E ϩ ci/dog Zeina (2005) selenium Hymenolepis diminuta Rats 500 nonviable onco- 3 cysticer- 100% Fan et al. (2004) spheres; 1, 2, 3, or 4 coids/rat doses; s.c.; FCA Hymenolepis micro- Mice 500 nonviable onco- 3 cysticer- No protection Fan et al. (2004) stoma spheres; 1, 2, 3, or 4 coids/mice doses; s.c.; FCA Taenia solium Hamster S3Pvac anticysticercosis 6 cysticer- 71% of reduction in the Cruz-Revilla et al. synthetic peptide vacci/hamster number of worms (2006) cine; 1 dose; s.c.; sa- ponine Taenia solium Hamster KETc1 peptide bound to 6 cysticer- 85% of reduction in the Cruz-Revilla et al. Brucella lumazine ci/hamster number of worms (2006) synthase; 1 dose; oral; no adjuvant

* All sources of antigens used as a vaccine were from the same parasite used for infection except the S3Pvac peptides and BLS-KETc1 that were originally identiﬁed in Taenia crassiceps cysticerci. † NF, not found. ‡ s.c., subcutaneous. § FCA, Freund’s complete adjuvant. ࿣ i.m., intramuscular. # FIA Freund’s incomplete adjuvant.

THE CASE FOR VACCINATION AGAINST VARIOUS their egg production up to 10% (Turner et al., 1936; Herd et TAPEWORMS al., 1975; Zhang et al., 2006). With one exception (Heath et al., 1980), dogs also become resistant to the intestinal infection by Several current anticestode vaccine candidates aim primarily Taenia hydatigena when systemically vaccinated with various to achieve immunity against the larval stages of cestodes, albeit homologous antigens, the more so if vitamin E and selenium with various levels of success, i.e., Taenia ovis (Johnson et al., are used to improve the elicited immune response (Kandil and 1989), Taenia saginata (Lightowlers, Rolfe, and Gauci, 1996; Abou-Zeina, 2005). Homologous, nonviable oncospheres of H. Harrison et al., 2005), Taenia solium (Molinari et al., 1983; nana and Hymenolepis diminuta used as systemic vaccines in- Sciutto et al., 1995; Plancarte et al., 1999; Huerta et al., 2001; duce effective protection against intestinal challenge in rats and Flisser et al., 2004; Gonzalez et al., 2005), Echinococus gran- mice, respectively, but not against Hymenolepis microstoma in ulosus (Lightowlers, Lawrence et al., 1996, Lightowlers et al., mice (Fan et al., 2004). 1999; Muller-Schollenberger et al., 2001; Siles-Lucas et al., 2003), Taenia crassiceps (Sciutto et al., 1990; Manoutcharian THE CASE FOR VACCINATION AGAINST TAENIA SOLIUM et al., 1996; Rosas et al., 1998; Toledo et al., 1999, 2001), and Hymenolepis nana (Gabriele et al., 1995). Fewer efforts have There is now plenty of evidence that T. solium cysticercosis, been made to develop effective vaccines against the adult tape- experimentally or naturally acquired, is signiﬁcantly reduced by worms. Experimental evidence for protective vaccination the vaccination of pigs, one of its major nonhuman intermediate against adult tapeworms is summarized in Table I. For example, hosts (Flisser et al., 2004; Gonzalez et al., 2005; Sciutto et al., systemic vaccination of dogs against the E. granulosus tape- 2007). The case for immunological prevention of the adult in- worm reduces intestinal parasites by up to 64% and diminishes testinal T. solium tapeworm is not that strong; its potential rests SCIUTTO ET AL.—VACCINES AGAINST TAENIA SOLIUM TAPEWORMS 827

TABLE II. Experimental evidence of immunologic involvement in intestinal tapeworm infections.

Intestinal cestode Host Reference

Primary infection protects against a second challenge Echinococus granulosus Dog Gemmell (1962) Taenia hydatigena Dog Gemmell (1972) Hymenolepis diminuta Mice Andreassen and Hopkins (1980) Hymenolepis nana Mice Ito and Smyth (1987) Taenia solium Golden hamster Cruz-Revilla et al. (2006) Concomitant immunity in tapeworm infections Hymenolepis microstoma and Hymenolepis nana Mice Ito et al. (1988) Hymenolepis microstoma Mice Novak and Nombrado (1988) Corticosteroids promote worm installation and prevent worm loss Taenia saginata Golden hamster Verster (1971) Taenia solium Golden hamster, chinchilla, gerbil Verster (1971); Maravilla et al. (1998) Hymenolepis diminuta Mice Hindsbo et al. (1965) Taenia crassiceps Golden hamster Kitaoka et al. (1990); Willms et al. (2004) Echinococcus multilocularis Mongolian gerbil, golden hamster Kamiya et al. (1991) Taenia hydatigena Mongolian gerbil, golden hamster Kamiya et al. (1991) Echinococcus granulosus Mongolian gerbil, golden hamster Kamiya (1991) Taenia saginata asiatica Humans, golden hamsters, gerbils Chang et al. (2005) Intestinal worms induce parasite-specific systemic and local immune responses Echinococcus granulosus Dogs Barriga and Al-Khalidi (1986) Taenia solium Golden hamster Avila et al. (2003) Transfer protective immunity with mesenteric lymph nodes cells or spleen cells Hymenolepis nana Mice Palmas et al. (1986) Decreased destrobillation and worm loss increased in thymus deficient animals Hymenolepis diminuta Mice Andreassen et al. (1978) on some observational data consistent with immunologically exposure conditions may promote an acquired immunological mediated protection. The experimental data supporting this ap- TH1/TH2 cytokine response, which results from occasional oral proach are, however, somewhat limited (Table II). contact with the parasite mainly by way of ingesting cysticer- An unresolved puzzle in T. solium disease is the strikingly cus-infected meat, parasite components, or both (Chavarria et low frequency of intestinal taeniasis in the same areas where al., 2003), as has been shown to occur in other enteric worm high frequency of porcine and human cysticercosis is found. infections (Finkelman et al., 2004). Acquired resistance may Several factors may explain this observation. It could be related also arise as a consequence of an active infection in the central to the differential clinical manifestations of each form of infec- nervous system, immunologically aborted by an effective TH2 tion, cysticercosis being more conspicuous than taeniasis. There cell cytokine response (Chavarria et al., 2005) (a mechanism is also a difference in the extent to which larval and adult stages known to be effective against both intestinal tapeworms and develop in their respective hosts. In addition, pigs are more tissue cysticerci [concomitant immunity]) (Gemmell, 1972; likely to be restricted in their movement, which, in turn, reduces Garcia et al., 2001). their distribution to more localized foci. In contrast, the adult worms disseminate eggs wherever the human host defecates, Experimental evidence and this could be over many square kilometers in rural areas, nearby towns, and distant cities, depending on the travel be- Table II summarizes the research done to identify various havior of the infected human. immunological constraints on tapeworm development. The data However, other causes of the inconsistency may be involved, show that protective acquired immunity due to primo infection i.e., the differential effectiveness of biochemical and/or immu- has been reported in different intestinal tapeworm infections nological constraints acting upon the adult tapeworms in the and that immunodepression by corticosteroids increases suscep- intestine (Diaz-Obregon et al., 2004) as opposed to those acting tibility of potential definitive hosts. upon cysticerci in the tissues (de Aluja et al., 1996). Acquired Considering that acquired immunity can attenuate a second- immunity can be gradually gained by living in endemic areas ary infection, there is hope in the development of an effective in which there is a frequent exposure to eggs of T. solium. This vaccine. The systemic and oral immune response elicited during possibility is supported by the clearly different immunological intestinal taeniasis (Barriga and Al-Khalidi, 1986; Avila et al., profiles for T. solium antigens between nonneurocysticercotic 2003) also argues in favor of the possibility that resistance can subjects living in areas of high (Tepetzezintla) and low expo- be induced through vaccination. In addition, an inhibitory role sure (Ensenada) to T. solium in Mexico. Thus, living under high of antibodies in the development of T. solium toward the adult 828 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 tapeworm was found to be mediated by antibodies against one actions of the Royal Society of Tropical Medicine and Hygiene 91: of the components of the S3Pvac anticysticercosis vaccine 595–598. ANDREASSEN, J., O. HINDSBO, AND E. J. RUITENBERG. 1978. Hymenolepis (GK1). This crippling effect in the transformation of T. solium diminuta infections in congenitally athymic (nude) mice: Worm cysticerci to the tapeworm stage indicates a new form of im- kinetics and intestinal histopathology. Immunology 34: 105–113. munological interference with the parasite biology, rather than ———, AND C. A. HOPKINS. 1980. Immunologically mediated rejection simply killing the parasite (Garcia et al., 2001; Diaz-Orea et of Hymenolepis diminuta by its normal host, the rat. Journal of Parasitology 66: 898–903. al., 2003). AVILA, G., M. BENITEZ,L.AGUILAR-VEGA, AND A. FLISSER. 2003. Ki- In testing the possibility of inducing protective immunity netics of Taenia solium antibodies and antigens in experimental against the adult intestinal T. solium tapeworm, the epitopes taeniosis. Parasitology Research 89: 284–289. included in the synthetic vaccine (S3Pvac) originally developed BARRIGA,O.O.,AND N. W. AL-KHALIDI. 1986. Humoral immunity in against T. solium cysticercosis (Huerta et al., 2001) were con- the prepatent primary infection of dogs with Echinococcus granulosus. Veterinary Immunology and Immunopathology 11: 375– sidered. These epitopes are extensively expressed at the tegu- 389. mental surface of adult T. solium (Toledo et al., 1999, 2001). BOA, M., S. MUKARATIRWA,A.L.WILLINGHAM, AND M. V. JOHANSEN. S3Pvac was tested against the intestinal T. solium tapeworm in 2003. Regional action plan for combating Taenia solium cysticer- the immunocompetent golden hamsters, a ‘‘foster’’ intermediate cosis/taeniosis in Eastern and Southern Africa. Acta Tropica 87: 183–186. host model (Verster, 1971). As shown in Table I, a single dose CHANG, S. L., N. NONAKA,M.KAMIYA,Y.KANAI,H.K.OOI,W.C. of S3Pvac, injected subcutaneously (s.c.), significantly reduced CHUNG, AND Y. O KU. 2005. Development of T. saginata asiatica the number of tapeworms established in the vaccinated ham- metacestodes in SCID mice and its infectivity in human and alter- sters (Cruz-Revilla et al., 2006). native definitive hosts. Parasitology Research 96: 95–101. ´ ´ Moreover, because oncospheres enter the human body CHAVARRIA, A., A. FLEURY,E.GARCIA,C.MARQUEZ,G.FRAGOSO, AND E. SCIUTTO. 2005. Relationship between the clinical heterogeneity through mucosal surfaces and the tapeworm’s scolex burrows of neurocysticercosis and the immune-inflammatory profiles. Clin- deep in the intestinal submucosal connective tissue, mucosal ical Immunology 116: 271–278. immunization could also be a promising strategy to prevent ———, B. ROGER,G.FRAGOSO,G.TAPIA,A.FLEURY,M.DUMAS,A. infection against both the larval and adult tapeworm stages. The DESSEIN,C.LARRALDE, AND E. SCIUTTO. 2003. TH2 profile in asymptomatic Taenia solium human neurocysticercosis. Microbes main limitation for the development of an oral vaccine, how- and Infection 5: 1109–1115. ever, is the rather poor immunogenic properties of most of the ———, V. M. VILLAREJOS, AND R. ZELEDON. 1977. Mebendazole in the recently reported subunit vaccine antigens. Nonetheless, the treatment of taeniasis solium and taeniasis saginata. The American polymeric protein, Brucella lumazine synthase (BLS), was re- Journal of Tropical Medicine and Hygiene 26: 118–120. cently reported as a new effective antigen-delivery system for CHOMICZ, L., B. GRYTNER-ZIECINA, AND Z. SWIDERSKI. 1995. Transmis- sion electron microscope studies on the oncospheral envelopes of oral immunization against experimental murine cysticercosis Taenia saginata after niclosamide treatment. Wiadomosći Parazy- caused by larval T. crassiceps (Rosas et al., 2006). Interesting- tologiczne 41: 391–394. ly, up to 85% of protection against adult T. solium has been CHUNG, W. C., AND B. C. KO. 1976. Treatment of Taenia saginata induced using 1 of the orally administered S3Pvac components infection with mixture of areca nuts and pumpkin seeds. Zhonghua Min Guo Wei Sheng Wu Xue Za Zhi 9: 31–35. (KETc1) bound to BLS (Cruz-Revilla et al., 2006) (Table I). COLES, G. C. 2005. Anthelmintic resistance—looking to the future: A New experimental evidence intensifies the hopes of devel- UK perspective. Research in Veterinary Science 78: 99–108. oping effective oral vaccines against intestinal taeniasis. De- CRUZ, M., A. DAVIS,H.DIXON,Z.S.PAWLOWSKI, AND J. PROAN˜ O. 1989. velopment of a safe and effective oral vaccine would be ideal, Operational studies on the control of Taenia solium taeniasis/cys- since it could be self-administered by potential human hosts and ticercosis in Ecuador. Bulletin of the World Health Organization 67: 401–407. easily boosted to circumvent the possible short duration of its CRUZ-REVILLA, C., A. TOLEDO,G.ROSAS,M.HUERTA,I.FLORES-PEREZ, protection, as well as the formidable cost and logistic problems N. PEN˜ A,J.MORALES,J.CISNEROS-QUIN˜ ONES,G.MENESES,A.DIÁZ- of drug treatment or needle vaccination. Such an oral vaccine OREA, ET AL. 2006. Effective protection against experimental Tae- against taeniasis may be an achievable goal in the near future. nia solium tapeworm infection in hamsters by primo-infection and by vaccination with recombinant or synthetic heterologous antigens. Journal of Parasitology 92: 864–867. ACKNOWLEDGMENTS DE ALUJA, A. S., A. N. VILLALOBOS,G.NAVA,A.TOLEDO,J.J.MAR- The authors thank Gerardo Arrellıń, Georgina Dıáz Herrera, and Mer- TINEZ,A.PLANCARTE,L.F.RODARTE,G.FRAGOSO, AND E. SCIUTTO. cedes Baca for technical support and Isabel Pe´rez Montfort for English 2005. Therapeutic capacity of the synthetic peptide-based vaccine correction of this manuscript. This investigation was partially supported against Taenia solium cysticercosis in pigs. Vaccine 23: 4062– by the International Center for Genetic Engineering and Biotechnology, 4069. the Howard Hughes Medical Institute (55004134), CONACyT 2004- ———, ———, A. PLANCARTE,L.F.RODARTE,M.HERNANDEZ, AND 01-040, 46953-M, the Direccioń General de Personal Acade´mico, Univ- E. SCIUTTO. 1996. Experimental Taenia solium cysticercosis in ersidad Nacional Autońoma de Mexico (IN-221905) and PROMEP433 pigs: Characteristics of the infection and antibody response. Vet- UAEMOR-PTC-87 (103.5/03/2530). erinary Parasitology 61: 49–59. DIAZ-CAMACHO,S.P.,A.CANDIL-RUIZ,V.SUATE-PERAZA,M.L.ZAZUE- TA-RAMOS,M.FELIX-MEDINA,R.LOZANO, AND K. WILLMS. 1991. LITERATURE CITED Epidemiologic study and control of Taenia solium infections with AGUILAR-DIAZ, H., R. J. BOBES,J.C.CARRERO,R.CAMACHO-CARRANZA, praziquantel in a rural village of Mexico. American Journal of C. CERVANTES,M.A.CEVALLOS,G.DAVILA,M.RODRIGUEZ-DOR- Tropical Medicine and Hygiene 45: 522–531. ANTES,G.ESCOBEDO,J.L.FERNANDEZ, ET AL. 2006. The genome DIAZ-OBREGON, D., L. LLOJA-LOZANO, AND V. C ARBAJAL-ZUNIGA. 2004. project of Taenia solium. Parasitology International 55: S127–130. Preclinical studies of Cucurbita maxima (pumpkin seeds) a tradi- ALLAN, J. C., M. VELASQUEZ-TOHOM,C.FLETES,R.TORRES-ALVAREZ, tional intestinal antiparasitic in rural urban areas. Revista de Gas- G. LOPEZ-VIRULA,P.YURRITA,H.SOTO DE ALFARO,A.RIVERA, AND troenterologıá del Peru´ 24: 323–327. J. GARCIA-NOVAL. 1997. Mass chemotherapy for intestinal Taenia DIÁZ-OREA, A., N. VILLALOBOS,A.DE ALUJA,G.ROSAS,E.GOME´ Z- solium infection: Effect on prevalence in humans and pigs. Trans- CONDE,P.HERNA´ NDEZ,C.LARRALDE,E.SCIUTTO, AND G. FRAGOSO. SCIUTTO ET AL.—VACCINES AGAINST TAENIA SOLIUM TAPEWORMS 829

2003. Th1 and Th2 indices of the immune response in pigs vac- S. ZHANG,H.Y.ZHANG,Q.M.WANG,D.WU, AND W. J. ZHU. cinated against Taenia solium cysticercosis suggest various host 2004. Protection of pigs against Taenia solium cysticercosis using immune strategies against the parasite. Veterinary Immunology and recombinant antigen or in combination with DNA vaccine. Vaccine Immunopathology 93: 81–90. 22: 3841–3847. EDDI, C., A. NARI, AND W. A MANFU. 2003. Taenia solium cysticercosis/ HARRISON, L. J., T. GARATE,D.M.BRYCE,L.M.GONZALEZ,M.FOSTER- Taeniasis: Potential linkage with FAO activities; FAO support pos- CUEVAS,L.W.WAMAE,J.A.ONYANGO-ABUJE, AND R. M. E. PARK- sibilities. Acta Tropica 87: 145–148. HOUSE. 2005. Ag-ELISA and PCR for monitoring the vaccination ENGELS, D., C. URBANI,A.BELOTTO,F.MESLIN, AND L. SAVIOLI. 2003. of cattle against Taenia saginata cysticercosis using an oncospheral The control of human (neuro)cysticercosis: Which way forward? adhesion protein (HP6) with surface and secreted localization. Acta Tropica 87: 177–182. Tropical Animal Health and Production 37: 103–120. ESQUIVEL, A., F. DIAZ-OTERO, AND S. GIMENEZ-ROLDAN. 2005. Growing HEATH, D. D., S. N. PARMETER, AND P. J. OSBORN. 1980. An attempt to frequency of neurocysticercosis in Madrid (Spain). Neurologia 20: immunise dogs against Taenia hydatigena. Research in Veterinar- 116–120. ian Science 29: 388–389. FAN, P. C., W. C. CHUNG, AND A. ITO. 2004. Immunization of rodents HERD, R. P., R. J. CHAPPEL, AND D. BIDDELL. 1975. Immunization of against Hymenolepis infections using non-viable homologous on- dogs against Echinococcus granulosus using worm secretory anti- cospheres. The Kaohsiung Journal of Medical Sciences 20: 575– gens. International Journal for Parasitology 5: 395–399. 579. HINDSBO, O., J. ANDREASSEN, AND C. A. A. HESSELBERG. 1965. Immu- FINKELMAN, F., T. SHEA-DONOHUE,C.SUZANNE,C.MORRIS,L.GILDEA, nity to Hymenolepis diminuta in the rat. Norwegian Journal of Zo- R. STRAIT,K.B.MADDEN,L.SCHOPF, AND J. F. URBAN JR. 2004. ology 23: 197. Interleukin-4- and interleukin-13-mediated host protection against HUERTA, M., A. S. DE ALUJA,G.FRAGOSO,A.TOLEDO,N.VILLALOBOS, intestinal nematode parasites. Immunological Reviews 201: 139– M. HERNANDEZ,G.GEVORKIAN,G.ACERO,A.DIAZ,I.ALVAREZ, ET 155. AL. 2001. Synthetic peptide vaccine against Taenia solium pig cys- FLISSER, A. 2006. Where are the tapeworms? Parasitology International ticercosis: Successful vaccination in a controlled field trial in rural 55: S117–120. Mexico. Vaccine 20: 262–266. ———, C. G. GAUCI,A.ZOLI,J.MARTINEZ-OCANA,A.GARZA-RODRI- ITO, A., K. ONITAKE, AND J. ANDREASSEN. 1988. Lumen phase specific GUEZ,J.L.DOMINGUEZ-ALPIZAR,P.MARAVILLA,R.RODRIGUEZ-CAN- cross immunity between Hymenolepis microstoma and H. nana in UL,G.AVILA,L.AGUILAR-VEGA, ET AL. 2004. Induction of protec- mice. International Journal for Parasitology 18: 1019–1027. tion against porcine cysticercosis by vaccination with recombinant ———, AND J. D. SMYTH. 1987. Adult cestodes: Immunology of the oncosphere antigens. Infection and Immunity 72: 5292–5297. lumen dwelling cestode infections. In Immune response in parasitic ———, I. MADRAZO,A.PLANCARTE,P.SCHANTZ,J.ALLAN,P.CRAIG, infections: Immunology, immunopathology and immunoprophylax- AND E. SARTI. 1993. Neurological symptoms in occult neurocysti- is, E. J. L. Soulsby (ed.). CRC Press, Boca Raton, Florida, p. 115– cercosis after single taeniacidal dose of praziquantel. Lancet 342: 163. 748. JAGOTA, S. C. 1986. Albendazole, a broad-spectrum anthelmintic, in the ———, A. VAZQUEZ-MENDOZA,J.MARTINEZ-OCANA,E.GOMEZ-COLIN, treatment of intestinal nematode and cestode infection: A multi- R. S. LEYVA, AND R. MEDINA-SANTILLAN. 2005. Short report: Eval- center study in 480 patients. Clinical Therapeutics 8: 226–231. uation of a self-detection tool for tapeworm carriers for use in pub- JERI, C., R. H. GILMAN,A.G.LESCANO,H.MAYTA,M.E.RAMIREZ,A. lic health. The American Journal of Tropical Medicine and Hygiene E. GONZALEZ,R.NAZERALI, AND H. H. GARCIA. 2004. Species iden- 72: 510–512. tification after treatment for human Taeniasis. Lancet 363: 949– GABRIELE, F., G. BORTOLETTI,M.CONCHEDDA, AND C. PALMAS. 1995. 950. Effect of an intraperitoneal injection with activated Hymenolepis JOHNSON, K. S., G. B. HARRISON,M.W.LIGHTOWLERS,K.L.O’HOY, nana and H. diminuta cysticercoids on homologous challenge. Par- W. G. C OUGLE,R.P.DEMPSTER,S.B.LAWRENCE,J.G.VINTON,D. assitologia 37: 41–44. D. HEATH, AND M. D. RICKARD. 1989. Vaccination against ovine GARCIA, G., E. SCIUTTO,G.FRAGOSO,C.CRUZ-REVILLA,A.TOLEDO,N. cysticercosis using a defined recombinant antigen. Nature 13: 585– VILLALOBOS,I.FLORES,A.ALUJA,M.V.JOSE, AND C. LARRALDE. 587. 2001. Inhibitory role of antibodies in the development of Taenia KAMIYA, M., H. SATO,M.KITAOKA,K.ISHIWATA,Y.OKU,M.ITO, AND solium and Taenia crassiceps toward reproductive and pathogenic P. G ATHURA. 1991. Laboratory rodent models for the tapeworm- stages. Journal of Parasitology 87: 582–586. stage of Taenia saginata and other related taeniid species. South- GARCIA, H. H., R. H. GILMAN,A.E.GONZALEZ,M.VERASTEGUI,S. east Asian Journal of Tropical Medicine and Public Health 22: RODRIGUEZ,C.GAVIDIA,V.C.TSANG,N.FALCON,A.G.LESCANO, S262–267. L. H. MOULTON,T.BERNAL, AND M. TOVAR.CYSTICERCOSIS WORK- KANDIL,O.M.,AND H. A. ABOU-ZEINA. 2005. Effect of parenteral vi- ING GROUP IN PERU. 2003. Hyperendemic human and porcine Tae- tamin E and selenium supplementation on immune status of dogs nia solium infection in Peru. The American Journal of Tropical vaccinated with subunit and somatic antigens against Taenia hy- Medicine and Hygiene 68: 268–275. datigena. Journal of the Egyptian Society of Parasitology 35: 537– ———, A. E. GONZALEZ,R.H.GILMAN,L.H.MOULTON,M.VERAS- 550. TEGUI,S.RODRIGUEZ,C.GAVIDIA, AND V. C. TSANG.CYSTICERCOSIS KEILBACH,N.M.,A.S.DE ALUJA, AND E. SARTI-GUTIERREZ. 1989. A WORKING GROUP IN PERU. 2006. Combined human and porcine program to control taeniasis-cysticercosis (T. solium): Experiences mass chemotherapy for the control of T. solium. The American in a Mexican village. Acta Leidensia 57: 181–189. Journal of Tropical Medicine and Hygiene 74: 850–855. KITAOKA, M., Y. OKU,M.OKAMOTO, AND M. KAMIYA. 1990. Develop- GEMMELL, M. A. 1962. Natural and acquired immunity factors interfer- ment and sexual maturation of Taenia crassiceps (Cestoda) in the ing with development during the rapid growth phase of Echino- golden hamster. Journal of Parasitology 76: 399–402. coccus granulosus in dogs. Immunology 5: 496–503. LARRALDE, C., AND E. SCIUTTO. 2006. El control de la Taenia solium ———. 1972. Hydatidosis and cysticercosis. Acquired resistance to en Me´xico, quinientos anõs despue´s de su llegada al nuevo mundo. Taenia hydatigena under conditions of a strong infection pressure. In Cisticercosis: Guıá para profesionales de la salud, C. Larralde Australian Veterinary Journal 48: 26–28. and A. S de Aluja (eds.). Biblioteca de Salud, Fondo de Cultura GONZALEZ, A. E., N. FALCON,C.GAVIRIA,H.H.GARCIA,V.C.TSANG, Econo´mica, Secretaria de Salubridad, Me´xico, D.F., Me´xico, p. T. B ERNAL,M.ROMERO, AND R. H. GILMAN. 1998. Time-response 182–230. curve of oxfendazole in the treatment of swine cysticercosis. The LIGHTOWLERS,M.W.,O.JENSEN,E.FERNANDEZ,J.A.IRIARTE,D.J. American Journal of Tropical Medicine and Hygiene 59: 832–836. WOOLLARD,C.G.GAUCI,D.J.JENKINS, AND D. D. HEATH. 1999. ———, C. G. GAUCI,D.BARBER,R.H.GILMAN,V.C.TSANG,H.H. Vaccination trials in Australia and Argentina confirm the effective- GARCIA,M.VERASTEGUI, AND M. W. LIGHTOWLERS. 2005. Vacci- ness of the EG95 hydatid vaccine in sheep. International Journal nation of pigs to control human neurocysticercosis. The American for Parasitology 29: 531–534. Journal of Tropical Medicine and Hygiene 72: 837–839. ———, S. B. LAWRENCE,C.G.GAUCI,J.YOUNG,M.J.RALSTON,D. GUO, Y. J., S. H. SUN,Y.ZHANG,Z.H.CHEN,K.Y.WANG,L.HUANG, MAAS, AND D. D. HEALTH. 1996. Vaccination against hydatidosis 830 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

using a defined recombinant antigen. Parasite Immunology 18: VILLA,G.MENESES, ET AL. 2006. Brucella spp. lumazine synthase: 457–462. A novel adjuvant and antigen delivery system to effectively induce ———, R. ROLFE, AND C. G. GAUCI. 1996. Taenia saginata: Vaccina- oral immunity. Microbes and Infection 8: 1277–1286. tion against cysticercosis in cattle with recombinant oncosphere SANTAMARIÁ, E., A. PLANCARTE, AND A. S. DE ALUJA. 2002. The exper- antigens. Experimental Parasitology 84: 330–338. imental infection of pigs with different numbers of Taenia solium MANOUTCHARIAN, K., A. DIAZ-OREA,G.GEVORKIAN,G.FRAGOSO,G. eggs: Immune response and efficiency of establishment. Journal of ACERO,E.GONZALEZ,A.DE ALUJA,N.VILLALOBOS,E.GOMEZ- Parasitology 88: 69–73. CONDE, AND E. SCIUTTO. 2004. Recombinant bacteriophage-based SARTI, E., A. FLISSER,P.M.SCHANTZ,M.GLEIZER,M.LOYA,A.PLAN- multiepitope vaccine against Taenia solium pig cysticercosis. Vet- CARTE,G.AVILA,J.ALLAN,P.CRAIG,M.BRONFMAN, AND P. W I- erinary Immunology and Immunopathology 99: 11–24. JEYARATNE. 1997. Development and evaluation of a health educa- ———, G. ROSAS,M.HERNANDEZ,G.FRAGOSO,A.ALUJA,N.VILLA- tion intervention against Taenia solium in a rural community in LOBOS,L.F.RODARTE, AND E. SCIUTTO. 1996. Cysticercosis: Iden- Mexico. The American Journal of Tropical Medicine and Hygiene tification and cloning of protective recombinant antigens. Journal 56: 127–132. of Parasitology 82: 250–254. ———, P. M. SCHANTZ,G.AVILA,J.AMBROSIO,R.MEDINA-SANTILLAN, MARAVILLA, P., G. AVILA,V.CABRERA,L.AGUILAR, AND A. FLISSER. AND A. FLISSER. 2000. Mass treatment against human taeniasis for 1998. Comparative development of Taenia solium in experimental the control of cysticercosis: A population-based intervention study. models. Journal of Parasitology 84: 882–886. Transactions of the Royal Society of Tropical Medicine and Hy- MARTIŃEZ-MAYA, J. J., A. S. DE ALUJA,G.A´ VILA-RAM´ıREZ,L.AGUILAR- giene 94: 85–89. VEGA,A.PLANCARTE-CRESPO, AND C. J. JARAMILLO-ARANGO. 2003. SCHANTZ, P. M., E. SARTI,A.PLANCARTE,M.WILSON,J.L.CRIALES,J. Taeniasis and detection of antibodies against cysticercus among ROBERTS, AND A. FLISSER. 1994. Community-based epidemiological inhabitants of a rural community in Guerrero State, Mexico. Salud investigations of cysticercosis due to Taenia solium: Comparison Pu´blica Me´xico 45: 84–89. of serological screening tests and clinical findings in two popula- MOLINARI, J. L., R. MEZA,B.SUAREZ,S.PALACIOS,P.TATO, AND A. tions in Mexico. Clinical Infectious Diseases 18: 879–885. RETANA. 1983. Taenia solium: Immunity in hogs to the cysticercus. SCIUTTO, E., A. ALUJA,G.FRAGOSO,L.F.RODARTE,M.HERNANDEZ,M. Experimental Parasitology 55: 340–357. N. VILLALOBOS,A.PADILLA,N.KEILBACH,M.BACA,T.GOVEZEN- MONTRESOR,A.,AND K. PALMER. 2006. Taeniasis/cysticercosis trend SKY,S.DIÁZ, AND C. LARRALDE. 1995. Immunization of pigs worldwide and rationale for control. Parasitology International 55: against Taenia solium cysticercosis: Factors related to effective pro- S301–303. tection. Veterinary Parasitology 60: 53–67. MORALES, J., J. J. MARTINEZ,J.GARCIA-CASTELLA,N.PEN˜ A,V.MAZA, ———, G. FRAGOSO,A.FLEURY,J.P.LACLETTE,J.SOTELO,A.ALUJA, N. VILLALOBOS,A.S.ALUJA,A.FLEURY,G.FRAGOSO,C.LARRAL- L. VARGAS, AND C. LARRALDE. 2000. Taenia solium disease in hu- DE, AND E. SCIUTTO. 2006. Taenia solium: The complex interac- mans and pigs: An ancient parasitosis disease rooted in developing tions, of biological, social, geographical and commercial factors, countries and emerging as a major health problem of global di- involved in the transmission dynamics of pig cysticercosis in highly mensions. Microbes and Infection 2: 1875–1890. endemic areas. Annals of Tropical Medicine and Parasitology 100: ———, ———, L. TRUEBA,D.LEMUS,R.M.MONTOYA,M.L.DIAZ, 123–135. T. G OVEZENSKY,C.LOMELI,G.TAPIA, AND C. LARRALDE. 1990. MULLER-SCHOLLENBERGER, V., W. BEYER,P.SCHNITZLER,A.MERCKEL- Cysticercosis vaccine: Cross protecting immunity with Taenia so- BACH,S.ROTH,B.H.KALINNA, AND R. LUCIUS. 2001. Immunization lium antigens against experimental murine Taenia crassiceps cys- with Salmonella typhimurium-delivered glyceraldehyde-3-phos- ticercosis. Parasite Immunology 12: 687–696. phate dehydrogenase protects mice against challenge infection with ———, J. MORALES,J.J.MARTIŃEZ,A.TOLEDO,M.N.VILLALOBOS, Echinococcus multilocularis eggs. International Journal for Para- C. CRUZ,G.MENESES,M.HERNA´ NDEZ,A.DIÁZ,L.F.RODARTE, ET sitology 31: 1441–1449. AL. 2007. Further evaluation of the synthetic peptide vaccine NOVAK,M.,AND S. NOMBRADO. 1988. Mast cell responses to Hyme- S3Pvac against Taenia solium cysticercosis in pigs in an endemic nolepis microstoma infection in mice. Journal of Parasitology 74: town of Mexico. Parasitology 134: 129–133. 81–88. SILES-LUCAS, M., M. MERLI,U.MACKENSTEDT, AND B. GOTTSTEIN. 2003. PALMAS, C., G. BORTOLETTI,M.CONCHEDDA, AND F. G ABRIELE. 1986. The Echinococcus multilocularis 14-3-3 protein protects mice Immunological memory and lymphoblast-migration in mice in- against primary but not secondary alveolar echinococcosis. Vaccine fected with Hymenolepis nana. Zeitschrift fur Parasitenkunde 72: 21: 431–439. 397–403. SUROSO, T., S. S. MARGONO,T.WANDRA, AND A. ITO. 2006. Challenges PAWLOWSKI, Z. S. 2006. Role of chemotherapy of taeniasis in prevention for control of taeniasis/cysticercosis in Indonesia. Parasitology In- of neurocysticercosis. Parasitology International 55: S105–109. ternational 55: S161–165. ———, J. ALLAN, AND E. SARTI. 2005. Control of Taenia solium tae- TOLEDO, A., G. FRAGOSO,G.ROSAS,M.HERNANDEZ,G.GEVORKIAN,F. niasis/cysticercosis: From research towards implementation. Inter- LOPEZ-CASILLAS,B.HERNANDEZ,G.ACERO,M.HUERTA,C.LAR- national Journal for Parasitology 35: 1221–1232. RALDE, AND E. SCIUTTO. 2001. Two epitopes shared by Taenia cras- PLANCARTE, A., A. FLISSER,C.G.GAUCI, AND M. W. LIGHTOWLERS. siceps and Taenia solium confer protection against murine T. cras- 1999. Vaccination against Taenia solium cysticercosis in pigs using siceps cysticercosis along with a prominent T1 response. Infection native and recombinant oncosphere antigens. International Journal and Immunity 69: 1766–1773. of Parasitology 29: 643–647. ———, C. LARRALDE,G.FRAGOSO,G.GEVORKIAN,K.MANOUTCHARI- PREUX,P.M.,AND M. DRUET-CABANAC. 2005. Epidemiology and aeti- AN,M.HERNANDEZ,G.ACERO,G.ROSAS,F.LOPEZ-CASILLAS,C.K. ology of epilepsy in sub-Saharan Africa. Lancet Neurology 4: 21– GARFIAS, ET AL. 1999. Towards a Taenia solium cysticercosis vac- 31. cine: An epitope shared by Taenia crassiceps and Taenia solium PROKOPENKO, L. I. 1981. Taeniasis and its control in USSR. Medicin- protects mice against experimental cysticercosis. Infection and Im- skaja parazitologija i Parazitarnye Bolezni 35: 652. munity 67: 2522–2530. ROMAN, G., J. SOTELO,O.DEL BRUTTO,A.FLISSER,M.DUMAS,N. TURNER, E. L., D. A. BERBERIAN, AND E. W. DENNIS. 1936. The pro- WADIA,D.BOTERO,M.CRUZ,H.GARCIA,P.R.DE BITTENCOURT, duction of artificial immunity in dogs against Echinococcus gran- ET AL. 2000. A proposal to declare neurocysticercosis an interna- ulosus. Journal of Parasitology 22: 14–28. tional reportable disease. Bulletin of the World Health Organization VERSTER, A. 1971. Preliminary report on the golden hamster as a de- 78: 399–406. finitive host of Taenia solium Linnaeus, 1758 and Taenia saginata ROSAS, G., C. CRUZ-REVILLA,G.FRAGOSO,F.LOPEZ-CASILLAS,A.PEREZ, Goeze, 1782. Onderstepoort Journal of Veterinary Research 38: M. A. BONILLA,R.ROSALES, AND E. SCIUTTO. 1998. Taenia cras- 63–64. siceps cysticercosis: Humoral immune response and protection elic- WAIKAGUL, J., P. DEKUMYOY, AND M. T. ANANTAPHRUTI. 2006. Taeniasis, ited by DNA immunization. Journal of Parasitology 84: 516–523. cysticercosis and echinococcosis in Thailand. Parasitology Inter- ———, G. FRAGOSO,N.AINCIART,F.ESQUIVEL-GUADARRAMA,A.SAN- national 55: S175–180. TANA,R.J.BOBES,O.RAMIREZ-PLIEGO,A.TOLEDO,C.CRUZ-RE- WALLIN,M.T.,AND J. F. KURTZKE. 2004. Neurocysticercosis in the SCIUTTO ET AL.—VACCINES AGAINST TAENIA SOLIUM TAPEWORMS 831

United States: Review of an important emerging infection. Neu- WU, L., Z. DIAO,X.DENG,J.GAO,Z.ZHOU,Y.LIU, AND Y. W ANG. rology 63: 1559–1564. 2005. DNA vaccine against Taenia solium cysticercosis expressed WILLMS, K., L. ROBERT,J.A.JIMENEZ,M.EVERHART, AND R. E. KUHN. as a modiﬁed hepatitis B virus core particle containing three epi- 2004. Ultrastructure of spermiogenesis and the spermatozoon in topes shared by Taenia crassiceps and Taenia solium. Journal of Taenia crassiceps strobilae WFU strain (Cestoda, Cyclophyllidea, Nanoscience and Nanotechnology 5: 1204–1210. Taeniidae) from golden hamsters. Parasitology Research 93: 262– ZHANG, W., Z. ZHANG,B.SHI,J.LI,H.YOU,G.TULSON,X.DANG,Y. 267. SONG,T.YIMITI,J.WANG,M.K.JONES, AND D. P. MCMANUS. 2006. WOLSTENHOLME, A. J., I. FAIRWEATHER,R.PRICHARD,G.VON SAMSON- Vaccination of dogs against Echinococcus granulosus, the cause of HIMMELSTJERNA, AND N. C. SANGSTER. 2004. Drug resistance in cystic hydatid disease in humans. Journal of Infectious Disease veterinary helminths. Trends in Parasitology 20: 469–476. 194: 966–974. J. Parasitol., 93(4), 2007, pp. 832–840 ᭧ American Society of Parasitologists 2007

SURFACE MEMBRANE PROTEINS OF BIOMPHALARIA GLABRATA EMBRYONIC CELLS BIND FUCOSYL DETERMINANTS ON THE TEGUMENTAL SURFACE OF SCHISTOSOMA MANSONI PRIMARY SPOROCYSTS

Maria G. Castillo, Xiao-Jun Wu, Nathalie Dinguirard, A. Kwame Nyame*, Richard D. Cummings†, and Timothy P. Yoshino‡ Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2115 Observatory Drive, Madison, Wisconsin. e-mail: [email protected]

ABSTRACT: Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host–parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval–host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (ϳ35–150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan- reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.

Surface carbohydrates (CHOs) of the blood fluke Schistoso- pecially recognition of nonself glycan structures and regulating ma mansoni, a leading causative agent of hepatosplenic schis- cell–cell interactions, is well established (Feizi, 2000; Gadjeva tosomiasis in humans (WHO/TDR; http://www.who.int/tdr/ et al., 2001). In particular, the role of C- and I-type lectins (Lu, diseases/schisto/default.htm), have been the focus of intense re- 1997; Vasta et al., 2004; Liu, 2005) in innate immunity is now search. Thus, CHOs represent the antigenic determinants re- well documented. sponsible for production of the majority of antibodies induced In the snail Biomphalaria glabrata, one of the principal in- during the humoral response in infected mammals (Nyame et termediate host species for S. mansoni, lectins are thought to al., 2004), and they are involved in host immune modulation play an important role in the recognition of microbial and ma- by promoting T-helper (Th)2 (humoral) over Th1 (cellular) lym- crobial pathogens (reviewed in Horak and van der Knaap, 1997; phocyte responses (Pearce and MacDonald, 2002; Thomas and Zhang et al., 2004). Lectinlike activity has been described in Harn, 2004). Furthermore, CHOs have been implicated in the the plasma that bathes snail tissues (Fryer and Bayne 1989; formation of hepatic granulomas characteristic of chronic in- Monroy et al., 1992; Zelck et al., 1995; Johnston and Yoshino, fections (van de Vijver et al., 2006). Previous studies have 1996) as well as on the surface of snail blood cells or hemo- found that the expression of some of these schistosome-exposed cytes (Fryer and Bayne, 1989; Zelck and Becker, 1990; John- glycan antigens changes during parasite development, which ston and Yoshino, 2001). Host cell binding interactions with has led to the suggestion that glycan determinants present on schistosome-related CHOs (Hahn et al., 2000; Johnston and the schistosome surface may serve important roles in the estab- Yoshino, 2001; Humphries and Yoshino, 2006) further imply lishment of infection, growth, and development of the parasites the involvement of lectins in immune resistance to larval in- within their hosts (Cummings and Nyame, 1999). vasion. However, because lectinlike molecules are widespread Invertebrates, although lacking the characteristic adaptive im- in both schistosome-susceptible and -resistant snails, and they mune responses of vertebrate species (Vasta and Ahmed, 1996; also occur in tissues with little known role in parasite immunity Loker et al., 2004), use lectins in the recognition and removal (such as the albumen gland; Michaelson and Dubois, 1977), it of pathogenic organisms (Adema et al., 1997; Vasta et al., 2001; is thought that lectinlike proteins may be playing multiple func- Endo et al., 2006). Lectins are nonenzymatic, structurally ditional roles in the snail host. However, to date very few lectins verse CHO-reactive proteins that are widely distributed within have been identified and fully characterized at the molecular the plant and animal kingdoms (Gabius et al., 2004; Sharon and level in B. glabrata; these lectins include a diverse family of Lis, 2004). Among their physiological functions in vertebrates secreted proteins known as fibrinogen-related proteins, capable species, their role in mediating innate immune responses, es- of precipitating soluble products from larval trematodes (Ade- ma et al., 1997; Zhang et al., 2004), and a selectinlike protein Received 26 May 2006; revised 12 February 2007; accepted 13 Feb- containing a C-type lectin CHO recognition domain reported in ruary 2007. hemocytes and the B. glabrata embryonic (Bge) cell line (Du- * Department of Natural Sciences, University of Maryland-Eastern clermortier et al., 1999). Shore, Princess Anne, Maryland 21853. Previously, we found that fucoidan, a sulfated polymer of † Department of Biochemistry, Emory University, School of Medicine, Atlanta, Georgia 30322. fucose, tightly bound to Bge cells and strongly inhibited the ‡ To whom correspondence should be addressed. adhesion of Bge cells to the surface of S. mansoni primary

832 CASTILLO ET AL.—FUCOSYL-REACTIVE PROTEINS IN B. GLABRATA CELLS 833

sporocysts (Castillo and Yoshino, 2002), suggesting the in- 1 to 4 (1, no binding; 2, Յ10 cells/sporocyst; 3, Ͼ10 cells [but Ͻ50% Ͼ volvement of lectinlike fucosyl-reactive molecules in this host of the larval surface covered with cells]; and 4, 50% of larval surface covered) was assigned to individual sporocysts according to the number cell–parasite interaction. Because of the paucity of information of cells adhering to the parasite surface (Bayne et al., 1984; Castillo on CHO-binding lectinlike proteins in B. glabrata, in the pres- and Yoshino, 2002). Based on the counts obtained from evaluating 100– ent study we have continued to characterize Bge cell surface 150 sporocysts/treatment, a cell adhesion index (CAI) value was deter- proteins responsible for their fucosyl reactivity, and investigate mined according to the following formula: the potential CHO ligands involved in cell adherence to the ͸ binding values for all sporocysts CAI ϭ sporocyst stage. Using affinity chromatography and glycan-spe- number of sporocysts scored cific monoclonal antibodies, we have identified the presence of Data were analyzed using 1-way analysis of variance and the Bonferroni several Bge cell surface proteins that share an affinity for fu- post-test. Treatments were considered significantly different from con- cosyl determinants and that represent candidates for putative trols at P values Յ0.05. parasite-reactive receptors. Preparation of Bge cell surface membrane extracts MATERIALS AND METHODS The initial step in identifying putative CHO-binding receptors associated with the surface of Bge cells involved first the isolation and Maintenance of Bge cells and biotin labeling of surface extraction of Bge cell membranes as described by Gallager and Smith membrane proteins (1992). Briefly, after Bge cells were harvested and washed as described Bge cells, originally obtained from the American Type Culture Col- in the previous section, the cell pellet was resuspended in ice-cold hy- lection (ATCC CRL 1494; Manassas, Virginia), were maintained in potonic buffer containing 10 mM Tris-HCl, 0.5 mM MgCl2, and pro- 250-ml tissue culture flasks containing Bge cell medium (Hansen, 1976) tease inhibitors (TM Mini EDTA-free protease inhibitor cocktail tablets; supplemented with 10% heat-inactivated fetal bovine serum and anti- Roche Diagnostics GmbH, Mannheim, Germany), pH 7.2, at a density ϳ ϫ 8 biotics (60 ␮g/ml penicillin and 50 ␮g/ml streptomycin sulfate). Cells of 5 10 cells/ml. Bge cells were maintained at 4 C for 30 min, were cultured at 26 C under normoxic conditions, and they were pas- and then they were transferred to a chilled 7-ml Dounce glass homog- saged every 4–6 days. Biotin labeling of live Bge cells was achieved enizer and vigorously disrupted with 25–30 strokes. Cell disruption was by treatment with 4 mM sulfo-NHS-biotin (Pierce Chemical, Rockford, monitored microscopically using propidium iodide to verify cell lysis Illinois), pH 7.4, as described previously by Castillo and Yoshino while retaining nuclear membrane integrity. Buffer isotonicity was re- (2002). After labeling, cells were washed 3 times with ice-cold snail stored to homogenates by adding a hypertonic solution consisting of 10 phosphate-buffered saline (sPBS; Yoshino, 1981) and once with diva- mM Tris-HCl, 0.5 mM MgCl2, 0.18 M NaCl, and protease inhibitors lent cation-fortified snail Tris-buffered saline (sTBSϩ; 20 mM Tris-HCl, (Roche Diagnostics GmbH) in a 1:2 buffer:homogenate ratio. Cell nuclei, intact cells, or any large cellular debris were removed from the 45.34 mM NaCl, and 1 mM each of CaCl2 and MgCl2), pH 7.2. Bge cell viability was monitored before and after biotin labeling by trypan lysate by centrifuging twice at 500 g for 10 min at 4 C. The resulting blue exclusion staining following the manufacturer’s suggested protocol supernatant was centrifuged at 100,000 g for 30 min at 4 C, and the (0.2% trypan blue solution; Sigma-Aldrich, St. Louis, Missouri). Post- pellet, containing enriched membrane components, was then solubilized ␤ labeling viability for Bge cells was consistently Ͼ98% using these in 20 mM Tris-HCl buffer containing 1% n-octyl- -glucopyranoside, 45 methods. mM NaCl, 1 mM each of CaCl2 and MgCl2, and protease inhibitors in a volume equivalent to ϳ2 ϫ 108 cells/ml. Membrane preparations were Isolation and culture of S. mansoni sporocysts kept at 4 C for 1 hr with frequent vortexing followed by centrifugation at 4,000 g for 15 min at 4 C. Protein concentration of the resulting Schistosoma mansoni miracidia of the NMR1 strain (Biomedical Re- supernatants (Bge cell membrane extract) was determined using the search Institute, Rockville, Maryland) were hatched and isolated under micro-BCA protein detection kit (Pierce Chemical). Membrane extracts axenic conditions according to previously described procedures (Yosh- were used immediately or stored in 1-ml aliquots at Ϫ80 C until needed. ino and Laursen, 1995). After isolation, miracidia were washed with cold Chernin’s balanced salt solution (CBSS; Chernin, 1963), pH 7.2, Isolation of putative Bge cell fucosyl-reactive receptors by containing 1 mg/ml each of glucose and trehalose, 60 ␮g/ml penicillin, affinity chromatography and 50 ␮g/ml streptomycin sulfate, transferred to 24-well culture plates at an approximate density of 5,000 miracidia/ml CBSS, and they were Results of previous Bge cell-binding inhibition studies (Castillo and cultured for 48 hr at 26 C under normoxic conditions. During this time, Yoshino, 2002; this study) have suggested the presence of fucosyl-re- Ͼ95% of miracidia transformed to primary (mother) sporocysts. active receptors on the surface of Bge cells. Therefore, a fucoidan affinity column was prepared as a general ‘‘fucosyl’’ matrix for the isolation of putative fucoidan/fucosyl-binding proteins from Bge cells. Anti-CHO antibody blocking experiments Construction of a fucoidan affinity matrix first required the production Monoclonal antibodies (MABs) with binding specificities to 2 S. of an aminated derivative of this polysaccharide via an oxidative reac- mansoni tegumental carbohydrates, LacdiNAc (LDN) and fucosylated tion. To accomplish this step, 20 mg of fucoidan (Sigma-Aldrich) was LacdiNAc (LDNF) (Nyame et al., 2002), were used in Bge cell–spo- dissolved in distilled water at 4 C, filter sterilized, and immediately rocyst binding inhibition assays to test the possible role played by these added to a separate tube containing 35 mg of cyanogen bromide 2 glycans in Bge cell recognition and adhesion to sporocysts. MABs (CNBr)/ml distilled water. The sugar was oxidized for 10 min with SMLDN1.1 (IgM) and SMLDNF1 (IgM and IgG) with binding speci- continuous adjustment of the solution to pH 11, followed by desalting ficities for LDN and LDNF, respectively, were generated from splenic of the CNBr-activated fucoidan over a Sephadex G-50 column previ- lymphocytes of S. mansoni-infected BALB/c mice as described by Ny- ously equilibrated with 0.2 M sodium borate buffer, pH 8. One-milliliter ame et al. (1999, 2000). In addition, to control for nonspecific antibody fractions were collected, and their sugar content was monitored spec- x binding, IgM and IgG MABs to the Lewis X glycan antigen [Le ; trophotometrically at A210. Fractions containing fucoidan (usually 4–5 Gal␤1-4(Fuc␣1-3)GlcNAc-R], previously found to be absent from S. ml) were pooled, and they were immediately mixed with a 3-ml solution mansoni sporocysts (Nyame et al., 2002), also were tested. of diaminohexane (10 mg/ml in borate buffer, pH 8.5) and gently rotated The protocols used in the Bge cell–sporocyst binding assay and cell for 18 hr at 4 C. The resulting aminated fucoidan was transferred to a adhesion scoring system were identical to those outlined in Castillo and dialysis cassette (Slide-A-Lyzer, 10,000 molecular weight [MW] cut- Yoshino (2002). To test for antibody inhibitory effects, 5 ϫ 105 Bge off; Pierce Chemical), and dialyzed at 4 C in the dark for 9 hr in 2 L cells and ϳ200 live sporocysts in CBSS were combined in the presence of sPBS buffer, pH 7.2, with 2 buffer changes, followed by dialysis for or absence of each MAB (at 10, 20, or 50 ␮g/ml) in siliconized 1.5-ml an additional 12 hr against 2 L of sodium carbonate buffer (0.2 M ␮ microcentrifuge tubes at a final volume of 200 l. Assays were incu- NaHCO3, pH 8) with 2 buffer changes. bated for 24 hr at 26 C. The binding of Bge cells to sporocysts was For resin cross-linking, the aminated fucoidan was first equilibrated

measured using a semiquantitative method in which a binding value of by dialysis against 2 L of 0.1 M NaHCO3 buffer containing 0.5 M NaCl, 834 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 pH 8.3–8.5, for 2 hr at 22 C, and then it was mixed with 2 ml of CNBr- activated Sepharose 4B beads (Sigma-Aldrich) and incubated with gentle rotation for 12–16 hr at 4 C. After crosslinking, the mixture was loaded into a 10-ml polypropylene column (Bio-Rad, Hercules, Cali- ϳ fornia), and the resin was washed with 40 ml of NaHCO3/NaCl coupling buffer to remove all unbound sugar. Remaining unreacted groups associated with the activated Sepharose resin were blocked with 2 ml of 1 M ethanolamine, pH 8, for 16 hr at 4 C, followed by washing with 20 ml of coupling buffer at pH 8.5, and then with 80–100 ml of 0.1 M acetate buffer, pH 4. The fucoidan affinity resin was finally re-equilibrated with 40–50 ml of sTBSϩ, pH 7.2, and used immediately in affinity chromatography studies or stored at 4 C in 1 M NaCl with 0.02% sodium azide until needed.

Affinity isolation of putative fucoidan-reactive Bge cell proteins Bge cell membrane extracts containing 1.5–2.0 mg protein/ml sTBSϩ were loaded onto TBSϩ-equilibrated fucoidan affinity columns and allowed to incubate for 2–4 hr at 22 C or 16 hr at 4 C under gentle rotation. After sample/matrix incubation, the column was washed extensively with 50 ml of sTBSϩ, followed by the application of 2 nonspecific elution buffers, the first consisting of 15 ml of 1 mg/ml Dex in sTBSϩ (MW 38,000), serving as a sugar specificity control; and the second, 15 ml of 1 mg/ml DexS in sTBSϩ (MW 10,000), serving as a control for charge-mediated protein elution. After removal of ‘‘nonspecifically-bound’’ Bge cell proteins from the column by Dex and DexS FIGURE 1. Anti-CHO antibody blocking assays. Mean CAI values elutions, the column was eluted with 15 ml of 1 mg/ml fucoidan in Ϯ ϩ ( SE) for Bge cells binding to 2-day-old sporocysts of Schistosoma sTBS . Between each column run, the affinity matrix was cleaned with ϭ ϩ mansoni incubated in the presence of CBSS (buffer-only control; n 15 ml of 1 M NaCl in sTBS , and then it was re-equilibrated with 40– 8), 250 ␮g/ml fucoidan (positive inhibition control; n ϭ 8), and various 50 ml of sTBSϩ . Elution fractions of 0.5 or 1 ml were collected through- MABs with specificity to schistosome tegumental glycans. Test MABs out the multiple elution/washing process, and representative fractions included anti-fucosylated LacdiNAc [LDNF-M (IgM; n ϭ 4), LDNF-G were evaluated by sodium dodecyl sulfate-polyacrylamide gel electro- (IgG, n ϭ 4)]; anti-LacdiNAc [LDN-M (IgM, n ϭ 4]; and anti-Lewis phoresis (SDS-PAGE), Western blot, and far-Western blot analyses. X [Lex-M (IgM; n ϭ 4); Lex-G (IgG, n ϭ 4)], all at 50 ␮g/ml sTBSϩ. SDS-PAGE, Western blot, and far-Western blot analyses of CAI values differing significantly from the buffer control are marked Յ Յ fucoidan-reactive Bge cell receptors with asterisks; **P 0.01, ***P 0.001. Fucoidan affinity column-fractionated samples of the solubilized Bge cell surface membrane preparation were separated by SDS-PAGE fol- Bge cell adhesion inhibition assays to further characterize the lowing standard protocols (Gallagher and Smith, 1992) using 10% polyacrylamide gels. Separated protein bands were silver stained using the ligand binding specificity of Bge cell adhesion receptors. Com- SilverQuest kit (Invitrogen, Carlsbad, California). For visualization of pared with the buffer control, only LDNF-reactive MABs sig- biotin-labeled surface proteins in the Bge cell membrane extract, SDS- nificantly inhibited the binding of Bge cells to the surface of S. PAGE–separated cell proteins were transferred to nitrocellulose (NC) mansoni sporocysts, with a mean cell CAI of 2.46 Ϯ 0.38 for sheets (Hoefer TE-70 semi-dry transfer; Hoefer Scientific Instruments, Ϯ San Francisco, California) (Towbin et al., 1979), blocked overnight at the IgM isotype and 2.24 0.14 for the IgG isotype (Fig. 1). 4 C in TBS (19.9 mM Tris and 148.85 mM NaCl, pH 7.4) containing Although anti-LDN treatment also reduced Bge cell binding to 5% bovine serum albumin (BSA), followed by probing with alkaline sporocysts, the difference compared with control treatment was phosphatase (AP)-conjugated streptavidin at a 1:8,000 dilution in TBS not significant. Likewise, the anti-Lex monoclonals (both IgG containing 0.03% Tween 20 (TBS-T) and 5% BSA. The chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium and IgM isotypes) exhibited no inhibitory effect on larval–Bge (BCIP/NBT) (Pierce Chemical) was used to develop strepavidin-reac- cell adherence, which was comparable with treatment with tive bands. CBSS alone. Because primary sporocysts lack Lex antigen ex- Far-Western blot analyses (Blackwood and Eisenman, 1991) were pression (Nyame et al., 2002), these MABs served as conve- carried out to identify Bge cell proteins capable of interacting with nient controls for nonspecific antibody binding effects. Fucoi- parasite tegumental surface polypeptides. NC sheets containing SDS- PAGE–separated and transferred nonbiotinylated Bge cell membrane dan, which served as a positive inhibition control, as expected, samples were incubated in a solubilized biotin-labeled sporocyst extract significantly inhibited the Bge cell–sporocysts adhesion (Fig. (60 ␮g protein/ml) for 16–18 hr at 4 C with continuous agitation. Prep- 1). aration of biotinylated sporocyst surface proteins were identical to that A series of at least 4 replicate binding inhibition assays were detailed in Johnston and Yoshino (1996), with the modification in the ␮ present study that prebiotinylated larvae were extracted in lysis buffer carried out at 3 antibody concentrations (10, 20, and 50 g/ml) x consisting of 1% n-octyl ␤-D-glucopyranoside, 0.5% Triton X-100, and of anti-LDNF (IgG and IgM), anti-LDN (IgM), and anti-Le protease inhibitors (Roche Diagnostics GmbH) in sPBS. After incuba- (IgG and IgM), to determine the inhibitory dose response for tion in the sporocyst extract, the NC sheets were washed 3 times with each of these antibodies. Based on mean CAI values, none of TBS and 3 times with TBS-T, and then they were incubated for 1 hr in AP-streptavidin (1:8,000 dilution) and developed in the BCIP/NBT the antibodies tested exerted significant inhibitory effects at chromogen substrate (Pierce Chemical). concentrations Ͻ50 ␮g/ml. However, at 50 ␮g/ml, both the IgG and IgM isotypes of the anti-LDNF MABs significantly blocked RESULTS adhesion of Bge cells to sporocysts (Fig. 2). Bge cells bind to sporocysts of S. mansoni via fucosyl determinants Fucosyl-binding surface membrane proteins on Bge cells MABs with binding specificities to 3 structurally similar S. A solid-phase affinity matrix coupled with fucoidan was de- mansoni surface glycans, LDN, LDNF, and Lex, were used in veloped to isolate fucosyl-reactive Bge cell membrane proteins. CASTILLO ET AL.—FUCOSYL-REACTIVE PROTEINS IN B. GLABRATA CELLS 835

the fucoidan column that originally were present at the surface membrane of Bge cells, and therefore, may be serving as potential lectinlike receptors. Western blot analyses of SDS- PAGE-separated biotinylated Bge cell proteins revealed the presence of biotin-labeled proteins in the flow-through and subsequent wash fractions (fractions 1 and 5; Fig. 3C). The fractions collected later in the washing and the Dex elution steps show the absence of any biotinylated proteins (fractions 8, 54, and 56; Fig. 3C). By contrast, the first elution fractions containing DexS (fractions 69–74; Fig. 3C) possessed a number of biotin-labeled polypeptides ranging in molecular mass from 15 to Ͼ250 kDa. Similarly, after DexS washout, subsequent elution with fucoidan-containing buffer released an additional subset of biotinylated proteins (fractions 92–95; Fig. 3D), including several enriched bands in the 50–150-kDa range. Western blot analyses of fucoidan-eluted fractions obtained from the Se- pharose 4B control column revealed no detectable protein bands (data not shown), confirming once again the binding specificity FIGURE 2. Anti-CHO antibody dose response. Mean CAI values (ϮSE) for the Bge cell–sporocyst binding assays carried out in the pres- of the fucoidan-affinity matrix. ence of various concentrations of anti-LDNF, anti-LDN, and anti-Lex MABs. Data were analyzed by 1-way ANOVA with follow-up Bonfer- Bge cell membrane proteins with binding affinity to roni’s means comparisons. Asterisks denote significant differences be- fucoidan also bind S. mansoni sporocyst tegumental Յ tween CAI values for MAB- and CBSS buffer-treated groups; *P proteins 0.01, **P Յ 0.001, n ϭ 3. anti-LDNF (IgM) —Ⅵ—; anti-LDNF (IgG) Ⅵ ᭢ x ⅷ x ------; anti-LDN (IgM) — —; anti-Le (IgM) — —; anti-Le (IgG) Far-Western blot assays were used to test the interaction of ---ⅷ---. fucoidan-binding Bge cell proteins with S. mansoni sporocysts tegumental proteins. Unlabeled (nonbiotinylated) Bge cell membrane proteins released from the fucoidan column after se- The elution profiles obtained from separate column chromato- quential DexS and fucoidan elution were separated by SDS- graphic runs using this matrix were highly reproducible in that PAGE (silver-stained gels; Fig. 4A, B), blotted to NC mem- proteins contained within eluted fractions from the column pro- branes, and probed with a tegumental extract of prebiotinylated duced consistent banding patterns as evidenced by SDS-PAGE sporocysts (Fig. 4C, D). Elution with DexS released a range of analyses of selected fractions collected during repeated affinity Bge cell polypeptides, mainly in the low molecular mass range runs. Figure 3A, B shows a representative of a typical Bge cell (Ͻ37 kDa), that were able to bind biotinylated sporocyst pro- protein elution profile. The column initially is overloaded with teins (fractions 80–83; Fig. 4C). However, in contrast to the protein as indicated by the presence of a complex mixture of DexS elution pattern, subsequent introduction of fucoidan-con- proteins eluting in flow-through and subsequent washes (frac- taining buffer to the column resulted in release of additional tions 1, 5, and 8) (Fig. 3A). Extensive washing of the column sporocyst-reactive Bge cell polypeptides, primarily in the 45- with binding buffer resulted in the removal of essentially all to 150-kDa molecular mass range (fractions 106–108; Fig. 4D). unbound proteins (fraction 54; Fig. 3A). Elution with Dex, a Results of replicate far-Western blot experiments suggest that glucose-based polysaccharide, did not remove any proteins a subset of Bge cell surface membrane proteins that possess from the column (fraction 56; Fig. 3A); in contrast, column fucosyl-binding activity also may interact with sporocyst tegu- elution with DexS resulted in the release of multiple proteins mental glycoproteins. A closer comparison of the fucoidan-elut- ranging from Ͻ14 to Ͼ200 kDa in molecular mass (fractions ed Bge cell surface proteins (Fig. 3D) and those exhibiting spo- 69–90; Fig. 3A). After DexS elution and protein washout, the rocyst protein binding reactivity in the far-Western blots (Fig. addition of fucoidan buffer resulted in the release of another 4D) shows that at least 4 Bge cell surface proteins (54, 74, 85, complex series of Bge cell proteins (fractions 92–94; Fig. 3B). and 125 kDa; Fig. 5A) share similar molecular masses with Bge After fucoidan elution, a high salt wash (1 M NaCl) released a proteins capable of binding sporocyst tegumental glycoproteins final set of proteins (fractions 123–163; Fig. 3B), the pattern of (Fig. 5B). which seemed similar to the fucoidan fractions. To address the possibility that Bge cell proteins may be binding nonspecifically DISCUSSION to the Sepharose 4B resin, a control column was prepared following the same protocol used in generating the affinity matrix, CHOs and their lectin receptors play important roles in a except for addition of fucoidan. No discernible protein bands diversity of biological processes in metazoan organisms, in- were observed in any of the elution fractions, except the initial cluding cellular adhesion and migration, growth, and differen- flow-through fractions (data not shown). These results demon- tiation (Varki, 1993; Maramutsu, 2000; Sharon and Lis, 2004). strate that polypeptides in the Bge extracts are binding to im- Likewise, the recognition of foreign CHOs as constituents of mobilized fucoidan and not to the glycan material making up invading parasites (Guha-Niyogi et al., 2001; Thomas and Harn, the gel matrix itself. 2004) is critical in maintaining the integrity of a host’s internal Because live Bge cells were biotinylated before membrane environment. As a result, both adaptive and innate immune re- extraction, it was possible to identify those proteins binding to sponses frequently are triggered by such parasite-associated 836 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. Silver-stained polyacrylamide gels and Western blots showing the biotinylated Bge cell membrane polypeptide composition for selected groups of column elution fractions. Approximately 30 ␮l from the indicated fractions were separated by SDS-PAGE and stained with silver (A–B) or blotted and probed with alkaline phosphatase-conjugated streptavidin (1:8,000 dilution). M indicates the molecular weight marker lane. (A–B) Specified elution fractions collected from a representative aminated fucoidan affinity column. (C–D) Western blots of SDS-PAGE– separated, biotin-labeled Bge cell surface proteins eluted with sequential treatment of the aminated fucoidan column with Dex, DexS, and fucoidan. Fraction 1 corresponds to the flow-through; 5–54, sTBSϩ wash (buffer); 56, nonspecific wash with sTBSϩ containing 1 mg/ml Dex; 69–90, treatment with sTBSϩ containing 1 mg/ml DexS; 92–98, specific elution buffer (sTBSϩ with 1 mg/ml fucoidan); 125–163, sTBSϩ with 1 M NaCl.

CHOs (van Die and Cummings, 2006). Mammalian stages of cance or role of these glycans in establishing successful infec- the schistosome blood flukes synthesize and express at their tions within the snail host. surface, or in secretions, a variety of unique and complex CHOs Cytoadherence of snail hemocytes to S. mansoni sporocysts that are highly immunogenic influencing both host-protective represents a prerequisite for cell-mediated cytotoxic reactions responses as well as immune-mediated pathology (Cummings (Bayne et al., 1980; Loker et al., 1982; Bayne et al., 1984), and Nyame, 1999; Nyame et al., 2004; Thomas and Harn, 2004; including the production and release of reactive oxygen and van de Vijver et al., 2006). Like their mammalian counterparts, nitrogen intermediates involved in larval killing (Bayne et al., the molluscan stages of larval schistosomes, including the mi- 2001). Because selected CHOs have been shown to trigger pro- racidium and mother (primary) sporocyst also possess complex duction of reactive oxygen species (ROS) in snail hemocytes surface membrane and secreted glycans (Yoshino et al., 1977; (Hahn et al., 2000), it has been suggested that recognition of Zelck and Becker, 1990; Uchikawa and Loker, 1991; Nyame et specific CHOs by host hemocytes may be responsible for di- al., 2002), although very little is known regarding the signifi- recting immune responses. Although the recognition mecha- CASTILLO ET AL.—FUCOSYL-REACTIVE PROTEINS IN B. GLABRATA CELLS 837

FIGURE 4. SDS-PAGE gels and far-Western blots showing the polypeptide composition of representative elution fractions from an aminated fucoidan affinity column preloaded with nonbiotinylated Bge cell surface membrane proteins. (A–B) Silver-stained gels showing the protein profiles for selected column fractions sequentially eluted with sTBSϩ, Dex, DexS, fucoidan, and high salt. (C–D) Corresponding far-Western blots in which identical aliquots of Bge protein eluted from the affinity column were probed with a biotin-labeled S. mansoni sporocyst tegumental extract followed by incubation with AP-conjugated streptavidin. Arrows indicate Bge cell polypeptides binding biotinlylated sporocyst proteins. M indicates the molecular weight marker lane. Fraction 1 represent the sample flow-through; 3–51, sTBSϩ wash; 52, dextran wash; 80–103, dextran sulfate; 105–111, fucoidan elution; 172–188 high salt wash (1 M NaCl); and 201, sTBSϩ wash.

nisms used by host cells are as yet unknown, carbohydrate- reactive lectins are considered to be prime candidates due to their documented capacity for recognizing specific sugars and for initiating cellular interactions (Lis and Sharon, 1998; Dodd and Drickamer, 2001; Vasta et al., 2001). Previously, we reported that the sulfated polyfucose fucoidan strongly inhibited the adherence of snail Bge cells to the surface tegument of S. mansoni sporocysts (Castillo and Yoshino, 2002), prompting the notion that Bge cell–sporocyst binding may be mediated by a cell surface fucosyl-type lectin(s). This idea is supported by the present finding that specific MABs to the LDNF trisaccharide significantly inhibited Bge cell binding to sporocysts, FIGURE 5. Composite figure showing the correspondence (based on whereas no such inhibition occurred using the MAB to LDN, relative molecular masses) of Bge cell proteins with binding affinity to which lacks the ␣-1,3–linked fucose moiety. Because S. man- both fucoidan (A) and Schistosoma mansoni sporocyst tegumental proteins (B). The arrows indicate proteins with putative binding affinity to soni sporocysts express both LDN and LDNF determinants at both fucoidan and larval tegumental proteins as suggested by comigra- the tegumental surface (Nyame et al., 2002), results of our ini- tion of similar molecular weight bands. tial antibody experiments indicated that the blocking effect of 838 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 anti-LDNF MABs was not due simply to steric hindrance, but moieties in their CHO binding interactions. The involvement of likely was related to the antibody’s specific epitope-blocking sulfate moieties in enhancing CHO–lectin interactions is now effect. Moreover, the presence of the LDNF fucosyl moiety as- well recognized. For example, there is now clear documentation sociated with the trisaccharide seems to be a key structural de- of the involvement of sulfate moieties in lectin–glycoconjugate terminant for the inhibitory effect of this particular antibody. interactions during the inflammatory process between the cell That both IgG and IgM isotypes of the Lex MABs exhibited no surface mucin P-selectin glycoprotein ligand-1 of human neu- inhibitory effects on the adhesion of Bge cells to sporocysts trophils and P- and L-selectins on endothelial cells (Lepannen corroborates a reported absence of Lex epitopes at the surface et al., 2000, 2003). This is consistent with the possibility that of S. mansoni sporocysts (Nyame et al., 2002), and, in our ex- sulfate residues may be involved in sporocyst CHO–Bge pro- periments, conveniently served as isotope-matched negative tein interactions, in particular those interactions with epitopes controls, demonstrating the absence of any nonspecific antibody bearing terminal fucosylated LacDiNAc. The availability of the binding and inhibitory activity. putative Bge cell lectin(s) should now allow purification of the To further investigate the putative Bge cell receptor(s) in- cognate binding partner(s) from sporocysts, and eventually pro- volved in the binding of larval fucosyl-type sugars, a fucoidan vide insight into the exact structural organization of the glycan affinity column was constructed to isolate cell surface proteins and SO 2Ϫ moieties mediating binding reactions. with fucosyl reactivity. Justification for focusing on fucose-re- 4 Previously, B. glabrata plasma lectins (Mansour et al., 1995) lated sugars stems from earlier findings that hemocytes of B. and hemocyte surface proteins (Johnston and Yoshino, 2001) glabrata react selectively to carbohydrate stimuli, and fucosyl were shown to bind schistosome fucosyl determinants, provid- residues seem to be important. For example, Hahn et al. (2000) ing a direct molecular linkage between these parasites and host showed that exposure of hemocytes to BSA–fucosyl glycocon- immune elements. To date, however, the fucosyl-reactive recep- jugates induced production of ROS. In addition, fucoidan tors on snail hemocytes have not been identified or character- strongly inhibited the binding of sporocyst excretory-secretory ized at the molecular level. In the present study, results of far- products to snail hemocytes (Johnston and Yoshino, 2001) and Bge cell/hemocyte adhesion to S. mansoni sporocysts (Castillo Western blot analyses have identified several fucosyl-binding and Yoshino, 2002; data not shown). However, hemocyte Bge cell proteins (54, 74, 85, and 125 kDa) that also exhibit phagocytosis of yeast seems to be mediated by laminarin (␤- reactivity to sporocyst tegumental surface protein(s). A molec- 1,3-glucan), or mannan (Fryer et al., 1989), or both, indicating ular mass comparison between biotinylated Bge cell surface that the diversity of cellular immune functions in hemocytes is proteins and those proteins with larval CHO reactivity seems likely regulated by different lectinlike receptors and their ability to represent a common subset of proteins based on size simi- to recognize a variety of CHO ligands. larities (50–125 kDa). Clearly, correlation of molecular masses The experimental use of fucoidan constitutes an important is not proof of shared protein identity. However, it is suggestive tool in the study of receptor biology in vertebrate organisms of the presence of Bge cell surface receptor(s) in those protein (Parish et al., 1988; Rochon et al., 1994; Berteau and Mulloy, bands with putative binding affinity to sporocyst tegumental 2003) and their interactions with various pathogens (e.g., Ma- CHOs, especially those exhibiting fucosyl determinants. ruyama et al., 1998; Ortega-Barria and Boothroyd, 1999). In Whether these or other Bge cell proteins with CHO reactivity our studies, the use of fucoidan as an affinity matrix in chro- may actually be mediating in vitro Bge cell–sporocyst adhesion matography experiments permitted the identification of a group (Castillo and Yoshino, 2002) has yet to be confirmed. of Bge cell membrane proteins (putative fucosyl receptors) that Finally, in the present study, based on morphological and exhibited selective binding to this polysaccharide. We think that molecular similarities (Yoshino et al., 1999, 2001), we have the use of other carbohydrates, including the glucose polymers continued to use the B. glabrata embryonic cell line as a cel- Dex and negatively charged DexS, as components in elution lular model for snail hemocytes. It should be noted that in our buffers, aided in the removal of proteins potentially bound to experiments, fucoidan, a natural polysaccharide from brown al- the fucoidan column in a nonspecific manner or due solely to gae (Killing, 1913), was used as an experimental tool to identify charge interactions. Pre-eluting the Bge cell column with Dex and isolate putative fucosyl-reactive membrane receptors from was done to remove any Bge membrane proteins that may have the Bge cell line. We also have found that fluorescein-labeled bound directly to the Sepharose matrix. Sequential introduction fucoidan is capable of binding to B. glabrata hemocytes and of DexS was then used to release proteins bound to the column blocks hemocyte–sporocyst interactions in vitro (data not via sulfate-mediated charge effects. Because the sulfate content shown), suggesting that Bge cells and hemocytes may be shar- of DexS (4.6 SO4 groups/disaccharide) is more than twice that ing similar fucosyl-reactive proteins. We are currently using a of fucoidan (2.0 SO4 groups/disaccharide) (Parish et al., 1988), proteomics approach to begin identifying individual Bge cell it is assumed that DexS, with this large charge difference, membrane proteins with fucosyl reactivity in hopes of facili- would release most, if not all, column proteins bound via neg- tating future comparisons of Bge cell–hemocyte lectinlike re- ative charge. This seems to be the case, because elution with ceptors. DexS resulted in the release of a large subset of proteins, including many that were biotinylated. However, subsequent elution with fucoidan buffer resulted in the further release of a AKNOWLEDGMENTS subset of proteins, especially in the Ͼ45-kDa range. It is our We thank Kathrine Zachman and Melissa Kreikemeier for technical contention that these proteins, which include a group of bioti- assistance. This work was supported by NIH grant AI15503 to T.P.Y., nylated surface polypeptides in the 50–125-kDa range, repre- AI47214 to R.D.C., and NIH schistosome supply grant AI30026 to Fred sent putative fucosyl-reactive receptors that may use sulfate Lewis. CASTILLO ET AL.—FUCOSYL-REACTIVE PROTEINS IN B. GLABRATA CELLS 839

LITERATURE CITED atode immune interactions: A review. Folia Parasitologica 44: 161– 172. ADEMA, C. M., L. A. HERTEL,R.D.MILLER, AND E. S. LOKER. 1997. HUMPHRIES,J.E.,AND T. P. Y OSHINO. 2006. Schistosoma mansoni ex- A family of fibrinogen-related proteins that precipitates parasite- cretory-secretory products stimulate a p38 signalling pathway in derived molecules is produced by an invertebrate after infection. Biomphalaria glabrata embryonic cells. International Journal for Proceedings of the National Academy of Sciences USA 94: 8691– Parasitology 36: 37–46. 8696. JOHNSTON,L.A.,AND T. P. Y OSHINO. 1996. Analysis of lectin- and snail BAYNE, C. J., P. M. BUCKLEY, AND P. C . D EWAN. 1980. Macrophage-like plasma-binding glycopeptides associated with the tegumental sur- hemocytes of resistant Biomphalaria glabrata are cytotoxic for face of the primary sporocysts of Schistosoma mansoni. Parasitol- sporocysts of Schistosoma mansoni in vitro. Journal of Parasitology ogy 112: 469–479. 66: 413–419. ———, AND ———. 2001. Larval Schistosoma mansoni excretory-se- ———, E. S. LOKER,M.A.YUI, AND J. A. STEPHENS. 1984. Immune- cretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria recognition of Schistosoma mansoni primary sporocysts may re- glabrata (Gastropoda) via surface carbohydrate binding receptors. quire specific receptors on Biomphalaria glabrata hemocytes. Par- Journal of Parasitology 87: 786–793. asite Immunology 6: 519–528. KILLING, H. 1913. Zur biochemie der Meersalgen. Zeitschrift fu¨r Phy- ———, U. K. HAHN, AND R. C. BENDER. 2001. Mechanisms of mol- siologische Chemie 83: 171–197. luscan host resistance and of parasite strategies for survival. Para- LEPPANEN, A., S. P. WHITE,J.HELIN,R.P.MCEVER, AND R. D. CUM- sitology 123: S159–S167. MINGS. 2000. Binding of glycosulfopeptides to P-selectin requires BERTEAU,O.,AND B. MULLOY. 2003. Sulfated fucans, fresh perspectives: stereospecific contributions of individual tyrosine sulfate and sugar Structures, functions, and biological properties of sulfated fucan residues. Journal of Biological Chemistry 275: 39569–39578. and an overview of enzymes active toward this class of polysac- ———, T. YAGO,V.I.OTTO,R.P.MCEVER, AND R. D. CUMMINGS. charide. Glycobiology 13: 29R–40R. 2003. Model glycosulfopeptides from P-selectin glycoprotein li- BLACKWOOD,E.M.,AND R. N. EISENMAN. 1991. Mac: A helix-loop- gand-1 require tyrosine sulfation and core-2-branched O-glycan to helix zipper protein that forms a sequence specificity DNA-binding bind to L-selectin. Journal of Biological Chemistry 278: 26391– complex with Myc. Science 251: 1211–1217. 26400. CASTILLO,M.G.,AND T. P. Y OSHINO. 2002. Carbohydrate inhibition of LIS,H.,AND N. SHARON. 1998. Lectins: Carbohydrate specific proteins Biomphalaria glabrata embryonic (Bge) cell adhesion to primary that mediate cellular recognition. Chemical Reviews 98: 637–674. sporocysts of Schistosoma mansoni. Parasitology 125: 513–525. LIU, F. T. 2005. Regulatory roles of galectins in the immune response. International Archives of Allergy and Immunology 1364: 385–400. CHERNIN, E. 1963. Observations on hearts explanted in vitro from the snail Australorbis glabratus. Journal of Parasitology 49: 353–364. LOKER, E. S., C. M. ADEMA, S-M. ZHANG, AND T. B . K EPLER. 2004. Invertebrate immune systems: Not homogeneous, not simple, not CUMMINGS,R.D.,AND A. K. NYAME. 1999. Schistosome glycoconju- 198: gates. Biochimica et Biophysica Acta 1455: 363–374. well understood. Immunological Reviews 10–24. ———, C. J. BAYNE,P.M.BUCKLEY, AND K. T. KRUSE. 1982. Ultra- DODD,R.B.,AND K. DRICKAMER. 2001. Lectin-like proteins in model structure of encapsulation of Schistosoma mansoni mother sporo- organisms: Implications for evolution of carbohydrate-binding ac- cysts by hemocytes of juveniles of the 10-R2 strain of Biomphal- tivity. Glycobiology 11: 71R–79R. aria glabrata. Journal of Parasitology 68: 84–94. DUCLERMORTIER, P., V. LARDANS,E.SERRA,F.TROTTEIN, AND C. DISSOUS. LU, J. 1997. Collectins: Collectors of microorganisms for the innate 1999. Biomphalaria glabrata embryonic cells express a protein immune system. Bioessays 19: 509–518. with a domain homologous to the lectin domain of mammalian MANSOUR, M. H., H. I. NEGM,A.H.SAAD, AND N. I. TAALAB. 1995. selectins. Parasitology Research 85: 481–486. Characterization of Biomphalaria alexandrina-derived lectins rec- ENDO, Y., M. TAKAHASHI, AND T. F UJITA. 2006. Lectin complement sys- ognizing a fucosyllactose-related determinant on schistosomes. tem and pattern recognition. Immunobiology 211: 283–293. Molecular and Biochemical Parasitology 69: 173–184. FEIZI, T. 2000. Carbohydrate-mediated recognition systems in innate im- MARAMUTSU, T. 2000. Protein-bound carbohydrates on cell-surface as munity. Immunology Reviews 173: 79–88. targets of recognition: An odyssey in understanding them. Glyco- FRYER,S.E.,AND C. J. BAYNE. 1989. Opsonization of yeast by the conjugate Journal 17: 577–595. plasma of Biomphalaria glabrata (Gastropoda): A strain specific, MARUYAMA, H., Y. NAWA, AND N. OHTA. 1998. Strongyloides venezue- time dependant process. Parasite Immunology 11: 269–278. lensis: Binding of orally secreted adhesion substances to sulfated ———, C. J. HULL, AND C. J. BAYNE. 1989. Phagocytosis of yeast by carbohydrates. Experimental Parasitology 89: 16–20. Biomphalaria glabrata: Carbohydrate specificity of hemocyte re- MICHAELSON,E.H.,AND L. DUBOIS. 1977. Agglutinins and lysins in the ceptors and a plasma opsonin. Developmental and Comparative molluscan family Planorbidae: A survey of hemolymph, egg-mas- Immunology 13: 9–16. ses, and albumen-gland extracts. Biological Bulletin 153: 219–227. GABIUS, H. J., H. C. SIEBERT,S.ANDRE,J.JIMENEZ-BARBERO, AND H. MONROY, F., L. A. HERTEL, AND E. S. LOKER. 1992. Carbohydrate-bind- RUDIGER. 2004. Chemical biology of the sugar code. Chembi- ing plasma proteins from the gastropod Biomphalaria glabrata: ochem: A European Journal of Chemical Biology 5: 740–764. Strain specificity and the effects of trematode infection. Current GADJEVA, M., S. THIEL, AND J. JENSENIUS. 2001. The mannan-binding- Opinion in Immunology 16: 355–366. lectin pathway of the innate immune response. Current Opinion in NYAME, A. K., Z. S. KAWAR, AND R. D. CUMMINGS. 2004. Antigenic Immunology 13: 74–78. glycans in parasitic infections: Implications for vaccines and di- GALLAGHER, S. R., AND J. A. SMITH. 1992. Electrophoretic separation agnostics. Archives of Biochemistry and Biophysics 426: 182–200. of proteins. In Current protocols in immunology, A. M. Kruisbeck, ———, A. M. LEPPANEN,B.J.BOGITSH, AND R. D. CUMMINGS. 2000. D. H Margulies, E. M. Shevach, and W. Strober (eds.). Greene Antibody responses to the fucosylated LacdiNAc glycan antigen in Publishing Associates and Wiley-Interscience, New York, New Schistosoma mansoni-infected mice and expression of the glycan York, 8.4.1–8.4.21. among schistosomes. Experimental Parasitology 96: 202–212. GUHA-NIYOGI, A., D. R. SULLIVAN, AND S. J. TURCO. 2001. Glycocon- ———, ———, R. DEBOSE-BOYD, AND R. D. CUMMINGS. 1999. Mice jugate structures of parasitic protozoa. Glycobiology 11: 45R–59R. infected with Schistosoma mansoni generate antibodies to HAHN,U.K.,R.C.BENDER, AND C. J. BAYNE. 2000. Production of LacdiNAc (GalNAc beta 1→4GlcNAc) determinants. Glycobiology reactive oxygen species by hemocytes of Biomphalaria glabrata: 9: 1029–1035. Carbohydrate-specific stimulation. Developmental and Compara- ———, T. P. YOSHINO, AND R. D. CUMMINGS. 2002. Differential ex- tive Immunology 24: 531–541. pression of LacdiNAc, fucosylated LacdiNAc, and Lexis x glycan HANSEN, E. 1976. A cell line from embryos of Biomphalaria glabrata antigens in intramolluscan stages of Schistosoma mansoni. Journal (Pulmonata): Establishment and characteristics. In Invertebrate tis- of Parasitology 88: 890–897. sue culture: Research applications, K. Maramorosch (ed.). Academ- ORTEGA-BARRIA, E., AND J. C. BOOTHROYD. 1999. A Toxoplasma lectin- ic Press, New York, New York, p. 75–99. like activity specific for sulfated polysaccharides is involved in cell HORAK,P.,AND W. P. W. VAN DER KNAAP. 1997. Lectins in snail-trem- host infection. Journal of Biological Chemistry 274: 1267–1276. 840 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

PARISH, C. R., V. MCPHUN, AND H. S. WARREN. 1988. Is a natural ligand fish: Zebrafish (Danio rerio) as a model species to address their of the T lymphocyte CD2 molecule a sulfated carbohydrate? Jour- biological roles in development and innate immunity. Glycocon- nal of Immunology 141: 3498–3504. jugate Journal 21: 503–521. PEARCE,E.J.,AND A. S. MACDONALD. 2002. The immunobiology of ———, M. S. QUESENBERRY,H.AHMED, AND N. O’LEARY. 2001. Lec- schistosomiasis. Nature Reviews Immunology 2: 499–511. tins from tunicates: Structure–function relationships in innate im- ROCHON, Y. P., S. I. SIMON,E.B.LYNAM, AND L. A. SKLAR. 1994. A munity. Advances in Experimental Medicine and Biology 484: role for lectin interactions during human neutrophil aggregation. 275–287. Journal of Immunology 152: 1385–1393. YOSHINO, T. P. 1981. Comparison of Concanavalin A-reactive determi- SHARON,N.,AND H. LIS. 2004. History of lectins: From hemagglutinins nants on hemocytes of two Biomphalaria glabrata snail stocks: to biological recognition molecules. Glycobiology 11: 53R–62R. Receptor binding and distribution. Developmental and Comparative THOMAS,P.G.,AND D. A. HARN,JR. 2004. Immune biasing by helminth Immunology 5: 229–239. glycans. Cellular Microbiology 6: 13–22. ———, J. P. BOYLE, AND J. E. HUMPHRIES. 2001. Receptor–ligand in- TOWBIN, H., T. STAEHELIN, AND J. GORDON. 1979. Electrophoretic trans- teractions and cellular signaling at the host–parasite interface. Par- fer of proteins from polyacrylamide gels to nitrocellulose sheets: asitology 123: S143–S157. Procedure and some applications. Proceedings of the National ———. T. C. CHENG, AND L. R. RENWRANTZ. 1977. Lectin and human Academy of Sciences USA 76: 4350–4354. blood group determinants of Schistosoma mansoni: Alteration fol- in vitro UCHIKAWA, R., AND E. S. LOKER. 1991. Lectin-binding properties of the lowing transformation of miracidium to mother sporocyst. surfaces of in vitro-transformed Schistosoma mansoni and Echi- Journal of Parasitology 63: 818–824. nostoma paraensei sporocysts. Journal of Parasitology 77: 742– ———, C. COUSTAU,S.MODAT, AND M. G. CASTILLO. 1999. The Biom- 748. phalaria glabrata embryonic (Bge) cell line: Establishment of an in vitro cellular model for the study of snail host–parasite interac- VAN DE VIJVER, K. K., A. M. DEELDER,W.JACOBS,E.A.VAN MARCK, tions. Malacologia 41: 331–343. AND C. H. HOKKE. 2006. LacdiNAc- and LacNAc-containing gly- ———, AND J. F. LAURSEN. 1995. Production of Schistosoma mansoni cans induce granulomas in an in vivo model for schistosome egg- daughter sporocysts from mother sporocysts maintained in synxenic 16: induced hepatic granuloma formation. Glycobiology 237–243. culture with Biomphalaria glabrata embryonic (Bge) cells. Journal VAN DIE, I., AND R. D. CUMMINGS. 2006. Glycans modulate immune of Parasitology 81: 714–722. In responses in helminth infections and allergy. Parasites and al- ZELCK, U., AND W. B ECKER. 1990. Lectin binding to cells of Schistosoma lergy, Vol. 90, M. Capron and F. Trottein (eds.). S. Karger, Basel, mansoni sporocysts and surrounding Biomphalaria glabrata tissue. Switzerland, p. 91–112. Journal of Invertebrate Pathology 55: 93–99. VARKI, A. 1993. Biological roles of oligosaccharides: All of the theories ———, ———, AND C. J. BAYNE. 1995. The plasma proteins of Biom- are correct. Glycobiology 3: 97–130. phalaria glabrata in the presence and absence of Schistosoma man- VASTA,G.R.,AND H. AHMED. 1996. Animal lectins as cell surface soni. Developmental and Comparative Immunology 19: 181–94. receptors: Current status for invertebrate species. Progress in Mo- ZHANG, S. M., C. M. ADEMA,T.B.KEPLER, AND E. S. LOKER. 2004. lecular and Subcellular Biology 17: 158–182. Diversification of Ig genes in an invertebrate. Science 305: 251– ———, ———, S. DU, AND D. HENRIKSON. 2004. Galectins in teleost 254. J. Parasitol., 93(4), 2007, pp. 841–849 ᭧ American Society of Parasitologists 2007

REDESCRIPTION OF TWO DICYEMENNEA (PHYLUM: DICYEMIDA) FROM ROSSIA PACIFICA (MOLLUSCA: CEPHALOPODA: DECAPODA)

Hidetaka Furuya Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. e-mail: [email protected]

ABSTRACT: Two species of dicyemid mesozoan are redescribed from Rossia paciﬁca Berry, 1911, collected off Iwase in Toyama Bay, Honshu, Japan. Dicyemennea brevicephaloides Bogolepova-Dobrokhotova, 1962, is a large species that reaches about 4,000 ␮m in length. This species attaches to the surface of the branchial heart appendages. The vermiform stages are characterized as having 23 peripheral cells, a disc-shaped calotte, and an axial cell that extends to the propolar cells. An anterior abortive axial cell is absent in vermiform embryos. Infusoriform embryos consist of 35 cells; a single nucleus is present in each urn cell and the refringent bodies are absent. Dicyemennea rossiae Bogolepova-Dobrokhotova, 1962, is a medium species that reaches about 2,000 ␮m in length. This species lives in folds of the renal appendages. The vermiform stages are characterized as having 29– 34 peripheral cells, a conical calotte, and an axial cell that extends to the middle of the metapolar cells. An anterior abortive axial cell is present in vermiform embryos. Infusoriform embryos consist of 39 cells; 2 nuclei are present in each urn cell and the refringent bodies are solid.

Various dicyemid species appear in the sepiolid cephalopod Measurements and drawings were made with the aid of an ocular mi- Rossia pacifica Berry, 1911. Thus, 10 species of dicyemids crometer and a drawing tube (Olympus U-DA, Olympus, Japan), respectively. were recorded from several localities in the northwestern and The renal organ and branchial heart were also fixed with Bouin’s northeastern Pacific Ocean (Bogolepova, 1953; Bogolepova- fluid. The fixed organs were embedded in paraffin and sectioned trans- Dobrokhotova, 1960, 1962; Hoffman, 1965; Hochberg, 1990). versely. The sections were stained with hematoxylin and eosin. Bogolepova-Dobrokhotova (1960) first reported the dicyemid The terminology for cell names used in the description of infusori- species from R. pacifica in the Sea of Okhotsk and the northern form larvae is based on Nouvel (1948), Short and Damian (1966), Fu- region of the Sea of Japan. Later, Bogolepova-Dobrokhotova ruya et al. (1992, 1997), and Furuya (1999). Specimens of the dicyemids are deposited in the Osaka University (1962) detailed an additional 7 species from R. pacifica in the Museum, Toyonaka, Osaka, Japan (OUM) and in the author’s collection. same localities. Although she worked in the Zoological Institute The octopus specimen harboring the dicyemids is deposited in the in St. Petersburg, unfortunately neither her collections of di- OUM. Abbreviations used in figures are as follows: A, apical cell; AA, cyemid preparations nor the host cephalopods she examined anterior abortive axial cell; AG, agamete; AL, anterior lateral cell; AX, have been located. Hence, it is not possible to reexamine either axial cell; BHC, branchial heart coelom; C, couvercle cell; CA, capsule cell; CL, calotte; D, diapolar cell; DC, dorsal caudal cell; DI, dorsal the parasites or the hosts to confirm identifications (F. G. Hoch- internal cell; E, enveloping cell; EN, endothelium; EP, epithelium of berg, pers. comm.). Hoffman (1965) detailed 2 species from R. branchial heart or renal appendage; G, germinal cell; GR, granule; L, pacifica in Puget Sound, Washington and off Coos Bay, lateral cell; LC, lateral caudal cell; M, metapolar cell; MD, median Oregon. One dicyemid species, Dicyemennea brevicephaloides dorsal cell; NI, nucleus of the axial cell of infusorigen; O, oogonium; Bogolepova-Dobrokhotova, 1962, was detected in the branchial P, propolar cell; PA, parapolar cell; PC, podocyte; PD, paired dorsal heart coelom, but not in the renal coelom. Recently, 2 species cell; PO, primary oocyte; PS, primary spermatocyte; PVL, posteroventral lateral cell; R, refringent body; RC, renal coelom; S, spermato- of dicyemids have been found in sections of the branchial heart gonium; SP, sperm; U, urn cell; UC, urn cavity; UP, uropolar cell; V, coelom and renal coelom of R. pacifica collected off Iwase, vena cava; VC, ventral caudal cell; VI, ventral internal cell; V1, first Toyama Bay, Honshu, Japan (Furuya, Ota et al., 2004). The ventral cell; V2, second ventral cell; V3, third ventral cell. author identified these species as D. brevicephaloides and Di- cyemennea rossiae Bogolepova-Dobrokhotova, 1962. The pre- RESULTS sent paper describes additional details of infection sites and infusoriform embryos of these 2 Dicyemennea species and pro- Histology of the branchial heart and the renal organ in vides a revised, detailed description of vermiform stages. In R. pacifica addition, the occurrence pattern and diversity of dicyemid species in R. pacifica are briefly discussed. Among cephalopod species, only in R. pacifica were dicyemids found in the branchial heart appendages. The branchial heart MATERIALS AND METHODS coelom is occupied by D. brevicephaloides. There are grooves In this study, 2 individuals of R. pacifica were examined for dicyem- and folds in the branchial heart appendage. Dicyemids with a ids on 6 March 2003. Host specimens were obtained from fishermen, slightly inflated, saucer-shaped calotte attach their large heads who collected them off Iwase in Toyama Bay, Honshu, Japan (Fig. 1). Small pieces of the branchial heart and renal organ with attached di- to epithelia of appendages (Fig. 2a). This dicyemid species is cyemids were removed and smeared on glass microscope slides. The restricted to branchial heart appendages. In this cephalopod, smears were fixed immediately in Bouin’s fluid for 24 hr and then stored another dicyemid species, D. rossiae, was found in renal ap- in 70% ethyl alcohol. Most of them were stained in Ehrlich’s hematox- pendages and pancreatic appendages. The renal appendages are ylin and counterstained in eosin. Stained smears were mounted with Entellan (Merck, Rahway, New Jersey). Dicyemids were observed with folded a great deal and consist of a single-layered microvillous a light microscope (Olympus BH-2; up to ϫ2,000 [Olympus, Japan]). columnar epithelium covering the vena cava and its branches (Fig. 4a). Dicyemids with cone-shaped calottes (e.g., D. ros- Received 18 July 2006; revised 16 February 2007; accepted 16 Feb- siae) insert the anterior part of the body into folds or crypts in ruary 2007. the renal appendages (Fig. 4a).

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FIGURE 1. Collection localities of Rossia pacifica obtained for this study and in the previous studies by both Bogolepova (1960, 1962) and Hoffman (1965). Gray areas represent distribution of R. pacifica.(a) The region indicated by the arrow 8 in (b). The dot indicates the collection locality of R. pacifica in this study. (b) Arrows indicate the collection localities of several species of dicyemids (see Table II): 1, off Coos Bay; 2, Puget Sound; 3, 4, the northern Okhotsk Sea; 5, the Gulf of Mordivnov and the coast of Cape Tonin; 6, the northern region of the Sea of Japan (southwestern shore of Sakhalin Island); 7, the Sea of Japan near the Putyatin Island; 8, Toyama Bay.

DESCRIPTION Mean diameter of fertilized eggs, 13.5 ␮m; that of spermatozoa, 3.1 ␮m. Axial cell round or ovoid, range in diameter from 25–35 ␮m. Dicyemennea brevicephaloides Bogolepova-Dobrokhotova, 1962 Infusoriform embryos (Figs. 2f, 2g, 3f–h; n ϭ 50): Full-grown em- (Figs. 2, 3; Table I) bryos large, average 30.9 Ϯ 2.0 ␮m in length (excluding cilia; mean Ϯ Diagnosis: Large dicyemid; body lengths reaching 4,000 ␮m. Calotte SD); length-width-height ratio 1.0:0.86:0.87; shape ovoid, bluntly ␮ disc-shaped. Vermiform stages with 23 peripheral cells: 4 propolars ϩ rounded to pointed posteriorly; cilia at posterior end 10 m long. Re- 5 metapolars ϩ 2 parapolars ϩ 12 trunk cells. Infusoriform embryos fringent bodies present, liquid; occupy anterior 20% of embryo length with 35 cells; refringent bodies absent (possibly liquid); and a single when viewed laterally (Fig. 2g). Cilia project from ventral internal cells nucleus present in each urn cell. into urn cavity (Figs. 2g, 3h). Capsule cells contain granules. Mature ϩ Nematogens (Figs. 2b, 3a, c): Body length from 500–4,000 ␮m, embryos with 35 cells: 31 somatic 4 germinal cells. Somatic cells of width from 80–150 ␮m; widest in region of parapolars; trunk width several types present: external cells that cover large part of anterior and mostly uniform. Peripheral cell number 23 (Table I): 4 propolars ϩ 5 lateral surfaces of embryo (2 enveloping cells); external cells with cilia ϩ ϩ metapolars ϩ 2 parapolars ϩ 10 diapolars ϩ 2 uropolars. Calotte disc- on external surfaces (2 paired dorsal cells 1 median dorsal cell 2 ϩ ϩ ϩ shaped, cilia on calotte about 8 ␮m long, oriented anteriorly. Propolar dorsal caudal cells 2 lateral caudal cells 1 ventral caudal cell 2 ϩ cells and their nuclei smaller than metapolar cells and their nuclei, re- lateral cells 2 posteroventral lateral cells); external cells without cilia ϩ ϩ ϩ spectively (Fig. 2b). Cytoplasm of propolar and metapolar cells more (2 apical cells 1 couvercle cell 2 ﬁrst ventral cells 2 second darkly stained by hematoxylin than cytoplasm of other peripheral cells. ventral cells); internal cells with cilia (2 ventral internal cells); and ϩ ϩ Verruciform cells absent. Axial cell cylindrical, rounded anteriorly; cell internal cells without cilia (2 dorsal internal cells 2 capsule cells extends forward to propolar cells. About 20 vermiform embryos present 4 urn cells). Each urn cell contains 1 germinal cell and 1 nucleus (Fig. in an axial cell of large individuals. Agametes occasionally fusiform in 3h). All somatic nuclei appear pycnotic in mature infusoriform embry- shape. Several distinct large agametes present (Fig. 2d). os. Vermiform embryos (Figs. 2c, 3d, e): Full-grown vermiform embryos range from 40–70 ␮m in length, from 20–25 ␮m in width. Peripheral Taxonomic summary cell number 23 (Table I); trunk cells arranged in opposed pairs. Anterior Specimens examined: Specimens on a total of 3 slides from a single end of calotte rounded. Axial cell pointed anteriorly; extends to middle host (OUM-MO-00011) were distributed as follows: 1 slide deposited of propolar cells; nucleus usually located in the center of axial cell. in Osaka, Japan (OUM-ME-00012) and 2 slides retained in the research Anterior abortive axial cell absent. Axial cell of full-grown embryos collection of the author in Osaka, Japan. often with as many as 2–3 agametes. Localities: Japan, Honshu, Toyama Prefecture, Toyama Bay, off Iwa- Rhombogens (Figs. 2a, 3b): Body similar in length but slightly stock- se, 36Њ45ЈN, 137Њ15ЈE, 380 m (Fig. 1). ier than nematogens, length from 500–4,000 ␮m, width from 90–150 Collection date: 6 March 2003. ␮m. Peripheral cell number typically 23 (Table I). Calotte disc-shaped. Additional localities: Sea of Japan, near the Putyatin Island; Sea of Axial cell shape and anterior extent similar to nematogens. About 10 Okhotsk; Puget Sound, Washington; off Coos Bay, Oregon. infusorigens present in axial cell of each parent individual. About 250 Host: Rossia paciﬁca Berry, 1911 (Mollusca: Cephalopoda: Deca- infusoriform embryos present in an axial cell of large individuals. Ac- podidae). cessory nuclei occasionally present in trunk cells. Symbiotype: Female (mature), 71 mm ML; OUM-MO-00011. Infusorigens (Fig. 2e; n ϭ 20): Mature infusorigens large-sized; com- Additional hosts: Octopus sp. (see Bogolepova-Dobrokhotova, 1962) posed of 39–67 (mode 54) external cells (oogonia and primary oocytes) Site of infection: Anterior ends (calottes) attach to the surface of the ϩ 16–45 (mode 31) internal cells (spermatogonia, primary spermato- brachial heart appendages. cytes, and secondary spermatocytes) ϩ 20–32 (mode 26) spermatozoa. Prevalence: In 2 of 2 hosts examined. FURUYA—REDESCRIPTION OF TWO DICYEMENNEA 843

FIGURE 2. Dicyemennea brevicephaloides.(a) Light micrograph of stained section through the branchial heart appendage of Rossia paciﬁca: arrowheads indicate calotte of dicyemids. The calottes are detached from the branchial heart epithelium due to an artifact. (b) Anterior region of nematogen. (c) Vermiform embryo within axial cell. (d) Two types of agamete. The smaller agamete is a typical type, the larger agamete is a progenitor of infusorigen. (e) Infusorigen. (f, g) Infusoriform embryos within axial cell: (f) optical horizontal section; (g) optical sagittal section. Bars ϭ 20 ␮mina,10␮minb–g. See abbreviations in Materials and Methods. 844 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. Dicyemennea brevicephaloides.(a, b) Vermiform stages, entire: (a) nematogen; (b) rhombogen. (c) Anterior region of nematogen. (d, e) Vermiform embryos within the axial cell: (d) optical section; (e) cilia omitted. (f–h) Infusoriform embryos: (f) dorsal view (cilia omitted); (g) ventral view (cilia omitted); (h) sagittal section. See abbreviations in Materials and Methods.

Dicyemennea rossiae Bogolepova-Dobrokhotova, 1962 (Figs. 4, 5; Table I) TABLE I. Dicyemennea brevicephaloides and Dicyemennea rossiae: Diagnosis: Medium dicyemid; body lengths reaching 2,000 ␮m. Ca- number of peripheral cells. lotte shape conical. Vermiform stages with 29–34 peripheral cells: 4 propolars ϩ 5 metapolars ϩ 2 parapolars ϩ 18–23 trunk cells. Infuso- Number of individuals examined riform embryos with 39 cells; refringent bodies solid; and 2 nuclei present in each urn cell. Vermiform Nematogens (Figs. 4b, 5a, c): Body length from 500–2,000 ␮m, Cell no. Nematogens embryos Rhombogens width from 50–80 ␮m; widest in region of diapolars; trunk width mostly uniform. Peripheral cell number 29–34 (Table I): 4 propolars ϩ 5me- D. brevicephaloides tapolars ϩ 2 parapolars ϩ 16–21 diapolars ϩ 2 uropolars. Calotte con- 23 21 8 13 ical in shape, bluntly pointed anteriorly; cilia on calotte about 4 ␮m long, oriented anteriorly. Propolar cells and their nuclei smaller than D. rossiae metapolar cells and their nuclei, respectively. Propolar cells occupy an- 29 4 3 1 terior 20–30% of calotte length when viewed laterally (Figs. 4b, 5c). 30 1 1 0 Cytoplasm of propolar cells more darkly stained by hematoxylin than 31 1 3 1 cytoplasm of other peripheral cells. Verruciform cells present. Axial cell 32 1 1 1 cylindrical, rounded anteriorly; cell extends forward to middle of me- 33 1 1 1 tapolar cells. About 10 vermiform embryos present in an axial cell of 34 0 2 0 large individuals. Agametes occasionally fusiform in shape. Vermiform embryos (Figs. 4c, d, 5e, f): Full-grown vermiform em- FURUYA—REDESCRIPTION OF TWO DICYEMENNEA 845

FIGURE 4. Dicyemennea rossiae.(a) Light micrograph of stained section through the renal appendage of Rossia paciﬁca: asterisks indicate dicyemids. The calottes are detached from the renal epithelium due to an artifact. (b) Anterior region of nematogen. (c, d) Vermiform embryos within axial cell: (c) developing vermiform embryo; (d) fully developed vermiform embryo. (e) Infusorigen. (f, g) Infusoriform embryos within axial cell: (f) optical horizontal section; (g) optical sagittal section. Bars ϭ 20 ␮mina,10␮minb–g. See abbreviations in Materials and Methods.

bryos range from 120–160 ␮m in length, from 10–13 ␮m in width. Peripheral cell number typically 29–34 (Table I). Calotte conical, blunt- Peripheral cell number 29–34 (Table I); trunk cells arranged in opposed ly pointed anteriorly. Verruciform cells present. Axial cell shape and pairs. Anterior end of calotte bluntly pointed. Axial cell rounded ante- anterior extent similar to nematogens. Either 1 or 2, rarely 3 infusori- riorly; extends to middle of metapolar cells; nucleus usually located in gens present in axial cell of each parent individual. About 50 infuso- anterior half of axial cell. Anterior abortive axial cell present (Figs. 4c, riform embryos present in an axial cell of large individuals. Accessory d, 5e). Axial cell of full-grown embryos often with as many as 2–3 nuclei occasionally present in trunk cells. agametes. Agametes fusiform in shape. Infusorigens (Fig. 4e; n ϭ 20): Mature infusorigens large-sized; com- Rhombogens (Fig. 5b, d): Body similar in length but slightly stockier posed of 34–70 (mode 51) external cells (oogonia and primary oocytes) than nematogens, length from 500–2,000 ␮m, width from 60–80 ␮m. ϩ 18–40 (mode 27) internal cells (spermatogonia, primary spermato- 846 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 5. Dicyemennea rossiae.(a, b) Vermiform stages, entire: (a) nematogen; (b) rhombogen. (c) Anterior region of nematogen. (d) Anterior region of rhombogen. (e, f) Vermiform embryos within the axial cell: (e) optical section; (f) cilia omitted. (g–i) Infusoriform embryos: (g) dorsal view (cilia omitted); (h) ventral view (cilia omitted); (i) sagittal section. See abbreviations in Materials and Methods.

cytes, and secondary spermatocytes) ϩ 19–65 (mode 38) spermatozoa. rounded to pointed posteriorly; cilia at posterior end 9 ␮m long. Re- Mean diameter of fertilized eggs, 12.0 ␮m; that of spermatozoa, 1.8 fringent bodies present, solid; occupy anterior 30% of embryo length ␮m. Axial cell round or ovoid, range in diameter from 19–52 ␮m. when viewed laterally (Fig. 4g). Cilia project from ventral internal cells Infusoriform embryos (Figs. 4f, 4g, 5g–i; n ϭ 50): Full-grown em- into urn cavity (Figs. 4g, 5i). Capsule cells contain large granules. Ma- bryos large, average 30.2 Ϯ 1.7 ␮m in length (excluding cilia; mean Ϯ ture embryos with 39 cells: 35 somatic ϩ 4 germinal cells. Somatic SD); length-width-height ratio 1.0:0.80:0.78; shape ovoid, bluntly cells of several types present: external cells that cover large part of FURUYA—REDESCRIPTION OF TWO DICYEMENNEA 847 anterior and lateral surfaces of embryo (2 enveloping cells); external small number of large-sized infusorigens and produce a relatively large cells with cilia on external surfaces (2 paired dorsal cells ϩ 1 median number of gametes; therefore, it belongs to the first group. dorsal cell ϩ 2 dorsal caudal cells ϩ 2 lateral caudal cells ϩ 1 ventral ϩ ϩ caudal cell 2 lateral cells 2 posteroventral lateral cells), external DISCUSSION cells with refringent bodies (2 apical cells); external cells without cilia (1 couvercle cell ϩ 2 anterior lateral cells ϩ 2 first ventral cells ϩ 2 Three species of Rossia have been reported from the northern second ventral cells ϩ 2 third ventral cells); internal cells with cilia (2 Pacific Ocean (Sasaki, 1929). Rossia pacifica inhabits the lower ventral internal cells); and internal cells without cilia (2 dorsal internal cells ϩ 2 capsule cells ϩ 4 urn cells). Each urn cell contains 1 germinal intertidal region down to 300 m in depth and has relatively wide cell and 2 nuclei. All somatic nuclei appear pycnotic in mature infu- distribution, i.e., the northern Pacific Ocean from Japan to soriform embryos. Southern California. Rossia mollicella Sasaki, 1920, has been recorded from bathyal depths off Kinkazan, Kii Peninsula, Tosa Taxonomic summary Bay, and the Sea of Hyuga-Nada. Finally, Rossia bipapillata Specimens examined: Specimens on a total of 3 slides from a single Sasaki, 1920, is rather common in the lower shelf from Suruga host (OUM-MO-00011) were distributed as follows: 1 slide deposited Bay to Tosa Bay and has been recorded in the Philippines. In in Osaka, Japan (OUM-ME-00013) and 2 slides retained in the research sepiolid species, dicyemid species have been reported only in collection of the author in Osaka, Japan. R. pacifica. Localities: Japan, Honshu, Toyama Prefecture, Toyama Bay, off Iwa- se, 36Њ45ЈN, 137Њ15ЈE, 380 m (Fig. 1). Dicyemids in R. pacifica have 2 significant features. First, Collection date: 6 March 2003. the 2 dicyemid species are spatially isolated within a host Additional localities: Sea of Japan, southwestern shore of Sakhalin (Hoffman, 1965). Second, there is a wide diversity of dicyemid Island; Sea of Okhotsk, the Gulf of Mordivnov; off Coos Bay, Oregon; species in R. pacifica (see Fig. 1; Table II); 10 species of di- Puget Sound and San Juan Islands, Washington. cyemid have been recorded from several localities (Bogolepo- Host: Rossia pacifica Berry, 1911 (Mollusca: Cephalopoda: Deca- podidae). va-Dobrokhotova, 1962; Hoffman, 1965; Hochberg, 1990). As Symbiotype: Female (mature), 71 mm ML; OUM-MO-00011. Hoffman noted (1965), his study also showed that D. brevice- Additional hosts: None. phaloides is restricted to the branchial heart coelom of the host, Site of infection: Anterior ends (calottes) inserted into crypts of the while the other species is restricted to the renal and pancreatic renal appendages within the renal sacs. Prevalence: In 2 of 2 hosts examined. organs. In the original description of D. brevicephaloides, Bo- golepova-Dobrokhotova (1962) did not indicate the site of in- Remarks fection of D. brevicephaloides in the host. It is likely that the mixture of branchial heart appendages and nearly renal ap- Dicyemennea brevicephaloides is easily distinguishable from D. ros- pendages was smeared for preparations. siae in calotte shape (disc-shape vs. conical), peripheral cell number (29–34 vs. 23), cell number of infusoriform embryos (35 vs. 39), and The branchial heart appendages have a single-layered excre- abortive axial cell of vermiform embryos (absent vs. present). In cellular tory epithelium, which shows the same structure and a similar composition and cell number of infusoriform embryos, D. brevicephal- histochemical pattern in octopuses and cuttlefishes (Schipp and oides is of the rare type for dicyemids (Furuya, Hochberg et al., 2004). Boletzky, 1975; Schipp et al., 1975; Furuya, Ota et al., 2004). This species lacks refringent bodies in apical cells. This feature sepa- rates D. brevicephaloides from all other known species of Dicyemennea. The branchial heart appendages produce the urine, as well as Dicyemennea rossiae is easily distinguishable from the 5 other Di- the renal appendages. The branchial heart coelom opens within cyemennea species found in R. pacifica by calotte shape and peripheral the renal coelom, and dicyemids possibly move to the renal cell number. This species is similar to D. californica McConnaughey, coelom. Infusoriform larvae may swim through this route into 1941, D. granularis McConnaughey, 1949, and D. nouveli Mc- the renal coelom and leave the host. Nevertheless, vermiform Connaughey, 1959, in the calotte shape of vermiform stages and the cellular composition and cell number of infusoriform embryos (Mc- stages of D. brevicephaloides were not detected from the renal Connaughey, 1941, 1949, 1959; Furuya, Hochberg et al., 2004). How- coelom. Dicyemennea brevicephaloides seems to prefer the ever, D. rossiae is distinguishable from D. californica and D. nouveli branchial heart coelom. The composition of urine in the bran- in the length of infusoriform embryos (30 ␮m vs. nearly 40 ␮m). Eo- chial heart coelom is similar to that in the renal coelom (Lapan, sinophilic granules in trunk peripheral cells are characteristic of D. 1975). The major organic component of cephalopod urine, kyn- granularis (McConnaughey, 1949); in this respect, D. rossiae differs from D. granularis. In cellular composition and cell number of the urenic acid, is possibly secreted by the branchial heart, not by infusoriform embryos, D. rossiae is of the typical type (Furuya, Hoch- the renal appendage. Therefore, D. brevicephaloides might re- berg et al., 2004). However, its embryo has large capsule cells with quire a particular compound restricted to the branchial heart many large granules. coelom. The size and number of infusorigens are diagnostic characteristics of Dicyemennea brevicephaloides, with a large disc-shaped ca- the dicyemid species (Furuya et al., 1993). There is a negative curvilinear relationship between the number of infusorigens per rhombogen lotte, attaches to the surface of the branchial heart appendages. and the number of gametes (egg-line and sperm-line cells) per infuso- Typically, dicyemids with flat, discoidal calottes also attach to rigen (Furuya et al., 2003a; Furuya, 2005, 2006). Irrespective of genera, the surface of renal or pancreatic appendages (Furuya et al., 4 distinct reproductive strategies are classified within the dicyemid spe- 2003b). cies: (1) rhombogens form a relatively small number of medium- to Rossia pacifica large-sized infusorigens (less than 5) and produce a relatively large provides one of the best examples of a host number of gametes (more than 20) per infusorigen; (2) rhombogens infected by a wide diversity of dicyemids on both sides of the produce a large number of infusorigens (more than 5), each of which northern Pacific Ocean (Hochberg, 1990). Typically, 1 to 3 spe- has at most 20 gametes; (3) rhombogens produce large numbers of cies of dicyemids occur in the host species (Nouvel, 1947; large-sized infusorigens with a large number of gametes; and (4) rhom- McConnaughey, 1949; Hochberg, 1990; Short, 1991; Furuya, bogens form a relatively small number of small-sized infusorigens with a few gametes (at most 10). Rhombogens of D. brevicephaloides have 1999; Furuya et al., 2003b). It is a rare case that a single host a relatively large number of large-sized infusorigens; therefore, this spe- species has 10 dicyemid species. Such a high diversity is very cies belongs to the third type. Those of D. rossiae have a relatively unusual and suggests that a complex of host species may be 848 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE II. Dicyemid species described from Rossia paciﬁca.

Collection localities* Dicyemids References**

Eastern north Paciﬁc Ocean, USA (1, 2) Dicyemennea parva 1, 2 Dicyemennea brevicephaloides 1, 2 Northern region of the Okhotsk Sea, Russia (3, 4) Dicyema caudatum 3 Dicyemennea ﬁliformis 4 Southern region of the Okhotsk Sea, Russia (5) Dicyemennea brevicephala 4 Dicyemennea curta 4 Dicyemennea rossiae 4 Dicyemennea brevicephaloides 4 Northern region of the Sea of Japan, Russia (6) Dicyema acuticephalum 3 Dicyema oligomerum 4 Dicyemennea nouveli 2, 4 Dicyemennea brevicephaloides 4 Dicyemennea rossiae 4 The Sea of Japan near the Putyatin Island, Russia (7) Dicyemennea brevicephaloides 4 Dicyemennea rossiae 4 Toyama Bay, Japan (8) Dicyemennea brevicephaloides 5 Dicyemennea rossiae 5

* See Figure 5. ** 1, Hoffman (1965); 2, Hochberg (1990); 3, Bogolepova-Dobrokhotova (1960); 4, Bogolepova-Dobrokhotova (1962); 5, this study. involved (Hochberg, 1990). Dicyemennea brevicephaloides was identified without their characteristic infusoriform embryos (Fu- reported in both sides of the northern Pacific Ocean, but not ruya et al., 1997; Furuya and Tsuneki, 2003; Furuya, Hochberg the other species (Table II). Only D. brevicephaloides inhabits et al., 2004). Careful taxonomic studies on dicyemids, including branchial heart coela without invasion of other species. Thus, characteristics of infusoriforms, and on the host species com- D. brevicephaloides may extend to both sides of the northern plex, are critically required, not only for understanding the di- Pacific Ocean. In contrast, the renal coelom is the usual habitat cyemid occurrence pattern in R. pacifica in the northern Pacific for dicyemids, and interspecific interactions possibly caused a Ocean, but also for further clarifying general patterns of host diversity of dicyemid species. The wide distribution of R. pa- specificity between dicyemids and cephalopods. cifica is likely correlated to the species diversity. Three dicyemid species, Dicyema acuticephalum Nouvel, ACKNOWLEDGMENTS 1947, Dicyemennea brevicephala McConnaughey, 1941, and I thank Kazuhiko Tsuneki of Osaka University for his critical reading Dicyemennea nouveli McConnaughey, 1959, were reported to of the manuscript and valuable advice. I also thank Masaaki Yoshida occur additionally in R. pacifica in the northeastern Pacific of Osaka University for his assistance in collecting host sepiolids and Ocean (Bogolepova-Dobrokhotova, 1960, 1962). Dicyemennea preparing smears. This study was supported by grants from the Japan acuticephalum was originally reported from Octopus vulgaris Society for the Promotion of Science (research grants 14540645 and 18570087). Lamarck, 1798 in Sagami Bay, Japan, by Nouvel (1947). Di- cyemennea brevicephala was reported from Octopus rubescens Berry, 1953, in the eastern Pacific Ocean off Marine View LITERATURE CITED Rock, California, by McConnaughey (1941). Dicyemennea BOGOLEPOVA, I. I. 1953. Materials on dicyemids of far-eastern seas. nouveli was reported from Octopus sp. (ϭEnteroctopus dofleini Ph.D. Dissertation, Academiya Nauk and Zoologicheskii Institute, (Wu¨lker)), 1910 in Monterey Bay, California by Mc- Leningrad, USSR, p. 288. BOGOLEPOVA-DOBROKHOTOVA, I. I. 1960. [Dicyemidae of the far-eastern Connaughey (1959). Dicyemids typically are narrowly host- seas. I. New species of the genus Dicyema]. Zoologicheskii Zhurnal specific (Furuya, 1999, 2006). However, these species represent 39: 1293–1302. a relatively wide host range. Similar instances of a relatively ———. 1962. [Dicyemidae of the far-eastern seas. II. New species of wide host range have been reported in Europe. For example, the genus Dicyemennea]. Zoologicheskii Zhurnal 41: 503–518. FURUYA, H. 1999. Fourteen new species of dicyemid mesozoans from Dicyema macrocephalum van Beneden, 1876, is reported to in- six Japanese cephalopods, with comments on host specificity. Spe- fect 5 species of sepiolids representing 3 different genera (Nou- cies Diversity 4: 257–319. vel, 1947; Hochberg and Bello, 1995). However, recent studies ———. 2005. Three new species of Dicyema (Phylum Dicyemida) suggest that dicyemids do not parasitize cephalopods of differ- from Amphioctopus kagoshimensis (Mollusca: Cephalopoda: Oc- ent genera or different orders (Furuya, 1999, 2006; Furuya and topodidae). Species Diversity 10: 231–247. ———. 2006. Three new species of dicyemid mesozoans (Phylum Di- Hochberg, 2002). The species with a wide host range have been cyemida) from Amphioctopus fangsiao (Mollusca: Cephalopoda), described mostly on the basis of characters of vermiform stages with comments on the occurrence patterns of dicyemids. Zoological without using characters of the infusoriform embryos. Bogo- Science 23: 105–119. lepova-Dobrokhotova (1960, 1962) lacked the description of ———, AND F. G . H OCHBERG. 2002. New species of Dicyemennea (Phy- lum: Dicyemida) in deep-water Graneledone (Mollusca: Cephalop- cellular composition and cell number of infusoriform embryos. oda: Octopoda) from the Antarctic. Journal of Parasitology 88: Thus, the problem of accurate identification remains in some 119–123. dicyemid species (e.g., D. acuticephalum), which could not be ———, ———, AND K. TSUNEKI. 2003a. Calotte morphology in the FURUYA—REDESCRIPTION OF TWO DICYEMENNEA 849

phylum Dicyemida: Niche separation and convergence. Journal of LAPAN, E. A. 1975. Studies on the chemistry of the octopus renal system Zoology 259: 361–373. and an observation on the symbiotic relationship of the dicyemid ———, ———, AND ———. 2003b. Reproductive traits of dicyemids. Mesozoa. Comparative Biochemistry and Physiology 52: 651–657. Marine Biology 142: 693–706. MCCONNAUGHEY, B. H. 1941. Two new Mesozoa from California, Di- ———, ———, AND ———. 2004. Cell number and cellular compo- cyemennea californica and Dicyemennea brevicephala (Dicyemi- sition in infusoriform larvae of dicyemid mesozoans (Phylum Di- dae). Journal of Parasitology 27: 63–69. cyemida). Zoological Science 21: 877–889. ———. 1949. Mesozoa of the family Dicyemidae from California. Uni- ———, M. OTA,R.KIMURA, AND K. TSUNEKI. 2004. The renal organs versity of California Publications in Zoology 55: 1–34. of cephalopods: A habitat for dicyemids and chromidinids. Journal ———. 1959. Dicyemennea nouveli, a new mesozoan from central Cal- of Morphology 262: 629–643. ifornia. Journal of Parasitology 45: 533–537. ———, AND K. TSUNEKI. 2003. Biology of dicyemid mesozoan. Zoo- NOUVEL, H. 1947. Les Dicye´mides. 1re partie: Syste´matique, geńe´ra- logical Science 20: 519–532. tions, vermiformes, infusorige`ne et sexualite´. Archives de Biologie ———, ———, AND Y. K OSHIDA. 1992. Development of the infusori- 58: 59–220. form embryo of Dicyema japonicum (Mesozoa: Dicyemidae). Bi- ———. 1948. Les Dicye´mids. 2e´ partie: Infusoriforme, te´ratologie, spe´- ological Bulletin 183: 248–257. cificite´, du parasitisme, affinite´s. Archives de Biologie 59: 147– ———, ———, AND ———. 1993. The development of the hermaph- 223. roditic gonad in four species of dicyemid mesozoans. Zoological SASAKI, M. 1929. A monograph of the dibranchiate cephalopods of the Science 10: 455–466. Japanese and adjacent waters. Journal of the College of Agriculture, ———, ———, AND ———. 1997. Fine structure of a dicyemid me- Hokkaido Imperial University 20(Suppl.): 154–160. sozoan, Dicyema acuticephalum, with special reference to cell SCHIPP, R., AND S. VON BOLETZKY. 1975. Morphology and function of junctions. Journal of Morphology 231: 297–305. the excretory organs in dibranchiate cephalopods. Fortschritte der HOCHBERG, F. G. 1990. Diseases of Cephalopoda. Diseases caused by Zoologie 23: 89–110. protistans and mesozoans. In Diseases of marine animals, Vol. III, ———, ———, AND G. DOELL. 1975. Ultrastructual and cytochemical O. Kinne (ed.). Biologische Anstalt Helgoland, Hamburg, Germa- investigations on the renal appendages and their concrements in ny, p. 47–202. dibranchiate cephalopods (Mollusca, Cephalopoda). Zeitschrift fu¨r ———, AND G. BELLO. 1995. Dicyemida. In Checklist delle Specie Morphologie der Tiere 81: 279–304. della Fauna Italiana, Fascicolo 4, ‘Turbellaria’, Gnathostomulida, SHORT, R. B. 1991. Marine flora and fauna of the eastern United States, Orthonectida, Dicyemida, Nemertea, A. Minelli, S. Ruffo, and S. Dicyemida. NOAA Technical Reports NMFS 100: 1–16. La Posta (eds.). Edizioni Calderini, Bologna, Italy, p. 3, 5, 28, 33. ———, AND R. T. DAMIAN. 1966. Morphology of the infusoriform larva HOFFMAN, E. G. 1965. Mesozoa of the sepiolid, Rossia pacifica (Berry). of Dicyema aegira (Mesozoa: Dicyemidae). Journal of Parasitology Journal of Parasitology 51: 313–320. 52: 746–751. J. Parasitol., 93(4), 2007, pp. 850–853 ᭧ American Society of Parasitologists 2007

CHIMPANZEE PINWORM, ENTEROBIUS ANTHROPOPITHECI (NEMATODA: OXYURIDAE), MAINTAINED FOR MORE THAN TWENTY YEARS IN CAPTIVE CHIMPANZEES IN JAPAN

Hideo Hasegawa and Toshifumi Udono* Department of Infectious Diseases, Faculty of Medicine, Oita University, Hasama, Yufu, Oita 879-5593, Japan. e-mail: [email protected]

ABSTRACT: The chimpanzee pinworm, Enterobius anthropopitheci (Gedoelst, 1916), was found in chimpanzees, Pan troglodytes, reared in Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho Co., Ltd., Kumamoto, Japan, in 2006. Because the chimpanzees in this institution originated from chimpanzees imported from Africa before 1984, it is considered that E. anthropopitheci infection has persisted for more than 20 yr in the chimpanzees. Analysis of pinworm specimens preserved in the institution revealed that transition of predominant pinworm species occurred, responding to the change of anthelmintics used for pinworm treatment. Present dominance of E. anthropopitheci is surmised to be caused by fenbendazole, which has been adopted from 2002. Scarcity of mixed infection with E. anthropopitheci and Enterobius vermicularis suggests interspeciﬁc competition between the pinworms.

Chimpanzee pinworm, Enterobius anthropopitheci (Ge- under a stereomicroscope to recover pinworms. Worms collected before doelst, 1916), is a specific parasite of chimpanzees, Pan trog- 2002 were directly picked up from fecal surface and fixed in 10% formalin or 70% ethanol. Additional pinworm specimens were collected lodytes, and bonobos, Pan paniscus (Sandosham, 1950; Hugot, from the lumen of formalin-fixed intestines of 3 chimpanzees, which 1993). This parasite seems to be rather common in chimpanzees died and were necropsied. and bonobos in the fields (cf. Hasegawa, Kano, and Mulavwa, The worms were cleared in a glycerol–alcohol solution by evapora- 1983; Hasegawa, 2005). Curiously, pinworms found from cap- tion of alcohol, mounted on glass slide in a 50% glycerol solution, and tive chimpanzees are usually the human pinworm, Enterobius observed under a light microscope with Nomarski contrast interference. Species identification of pinworms was made based on the morpholog- vermicularis (Linnaeus, 1758), which sometimes fatal in these ical difference in spicule for males and lateral alae and eggs for females hosts (Hasegawa and Kinjo, 1996; Murata et al., 2002; Nakano principally (Figs. 1–6) (cf. Hasegawa et al., 2005). Selected specimens et al., 2003, 2005). However, we recently found that E. anthro- were measured and compared with the previous data. popitheci infection had been maintained for more than 20 yr in a captive chimpanzee group in Japan. Herein, the detail of the RESULTS observation is reported with special reference to possible selection exerted by anthelmintic treatment. Transition of pinworm species in the institution

MATERIALS AND METHODS The first pinworm species identified in the institution was E. vermicularis (Table I). The host was 8-mo-old male infant, Mi- Study site noru, which was emaciated and developed severe diarrhea. Nu- Kumamoto Primates Research Park, Sanwa Kagaku Kenkyusho Co., merous pinworms were found discharged following treatment Ltd., Uki, Kumamoto, Japan, was founded in 1982, and presently hous- es 80 chimpanzees. They consist of individuals originally imported with pyrvinium pamoate, but none of the female worms was mostly from Sierra Leone, West Africa, during the period from 1976 to gravid. During the period from 1986 and 1988 all pinworms 1983, for use in medical research in several institutions in Japan, and found from the chimpanzees were E. anthropopitheci (Table I). then transferred to the present institution from 1981 to 2000, and those However, pinworms preserved during the period from 1989 born thereafter. As in many other institutions and zoos, the chimpanzees were infected with pinworms. In early days, pyrvinium pamoate (Po- to 2001 were all E. vermicularis (Table I). Pinworms collected quil௢, Sankyo CO., Ltd., Tokyo, Japan) was used for treatment, and from the lumen of preserved appendix of Manako, a 2-mo-old then mass treatment with pyrantel pamoate (Combantrin௢, Pfizer Co., female infant who died of enteritis in 1989, were found to be Ltd., New York, New York) was repeated at several monthly intervals E. vermicularis. In 1990, an 8-mo-old infant chimpanzee, no. during the period from 1991 to 1995; self-made syrup containing pyrvinium pamoate was given simultaneously to all chimpanzees several 233, died, and its necropsy revealed intestinal invagination and times per year from 1996 to 2001 (Table I). In 2002, fenbendazole massive pinworm infection. The worms collected from its il- (Sigma-Aldrich Japan K.K., Tokyo, Japan) was introduced for treat- eocecal lumen were also E. vermicularis. In 1992, E. vermi- ment, and pinworm infection was greatly suppressed (Emi et al., 2004). cularis also were found from Juhgo, an 8-mo-old infant, which However, it was observed in May 2006 that some chimpanzees were still infected. Accordingly, they were treated with pyrantel pamoate was treated with pyrantel pamoate. In 2001, an adult chimpan- again (Table I). zee, Ikuzou, died of cerebellar infarction, and its cecum contained only E. vermicularis. No gravid female pinworms were Pinworms found from these chimpanzees except Juhgo, which harbored Pinworms were collected from the chimpanzee feces discharged 1 to many gravid worms. 3 days after anthelmintic treatment or at routine examination (Table I). Because pinworm infection could not be controlled by ad- The feces were fixed in 95% ethanol, and then they were washed with ministration of pyrantel pamoate and pyrvinium pamoate, fen- running tap water on a fine strainer with aperture size of 0.01 mm2. Residues on the strainer were transferred to a Petri dish and observed bendazole was adopted for treatment in 2002. In December 2002, mixed infection with both E. vermicularis and E. anthropopitheci was first observed (Table I). Presence of both pin- Received 28 August 2006; revised 12 February 2007; accepted 12 worm species also was demonstrated in the preserved fixed fe- February 2007. * Kumamoto Primates Research Park, Sanwa Kagaku Kenkyusho Co., ces collected in 2003. Gravid females of the both species were Ltd., Misumi, Uki, Kumamoto 869-3201, Japan. found in the feces at this period. However, only E. anthropo-

850 HASEGAWA AND UDONO—ENTEROBIUS ANTHROPOPITHECI IN CAPTIVE CHIMPANZEE 851

TABLE I. Pinworm species collected from chimpanzees reared in Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho Co., Ltd., Kumamoto, Japan.

Host name Date of Enterobius Enterobius Anthelmintics used (sex and age*) collection† anthropopitheci vermicularis and other remarks

Minoru (M, 8 mo) 14 May 1985 —‡ M2, F117§ Pyrvinium pamoate Izou (M, 8 yr) 3 Jul 1986 F6 — Routine examination Sunny (M, 11 yr) 21 Aug 1986 M5, F105 — Spontaneous discharge Sunny (M, 12 yr) 27 Feb 1987 F7 — No treatment record available Maruku (M, 8 yr) 10 Aug 1988 F28 — Pyrvinium pamoate Manako (F, 2 mo) 24 Nov 1989 — M6, F4 Appendix contents; necropsy No. 233 (M, 8 mo) 25 Feb 1990 — M57, F32 Ileocecal contents; necropsy Juhgo (M, 8 mo) 14 Feb 1992 — M3, F141 Pyrantel pamoate Ikuzou (M, 23 yr) 26 Jul 2001 — M21, F38 Cecal contents; necropsy Miro (F, 27 yr) 19 Dec 2002 M1, F81 M2, F2 Fenbendazole Mixed feces of 10 hosts 6 Jun 2003 Numerous M and F Numerous M and F Fenbendazole Hope (M, 15 yr) 6 Mar 2006 M3, F3 — Fenbendazole Youko (F, 17 yr) 17 Mar 2006 F1 — Fenbendazole Satoru (M, 11 yr) 12 May 2006 Numerous M and F — Spontaneous discharge Satoru (M, 11 yr) 18 May 2006 Numerous M and F — Fenbendazole Bell (M, 10 yr) 18 May 2006 Numerous M and F — Fenbendazole Mizuo (M, 16 yr) 18 May 2006 M9, F11 — Fenbendazole George (M, 27 yr) 25 May 2006 M2, F22 — Fenbendazole George (M, 27 yr) 30 May 2006 F25 — Pyrantel pamoate Bell (M, 10 yr) 30 May 2006 F48 — Pyrantel pamoate Hope (M, 16 yr) 7 Jun 2006 M1, F5 — Pyrantel pamoate

* Age at collection of material; M, male; F, female. † Post-treatment feces were collected 1–3 days after administration. ‡ Negative. § Sex and number of pinworms observed; M, male; F, female.

pitheci was observed in the fecal samples collected in 2006 DISCUSSION (Table I). Maintenance of E. anthropopitheci infection in zoo chimpanzees was recently suggested by the presence of this pinworm Morphology of E. anthropopitheci in an introduced colony of chimpanzee on Rubondo Island in Lake Victoria, Tanzania (Hasegawa et al., 2005). The colony Morphology of the present examples of E. anthropopitheci had derived from individuals reared in various European zoos is generally the same as that reported previously (Hasegawa et for 3.5 mo to 9 yr and who then were released onto the island, al., 2005). Because it is very rare to ﬁnd E. anthropopitheci where endemic chimpanzees were not distributed (Petrzelkova infection in captive chimpanzees, summarized measurements et al., 2006). However, it remains unknown how long E. an- are given herein as follows. thropopitheci infection can persist in chimpanzees under cap- ϭ Male (n 13): Body length 1.34–1.72 (mean 1.53) mm, tive conditions. Because the latest import of chimpanzees into ␮ ␮ width 114–143 (128) m. Cephalic vesicle 53–90 (80) m long Japan was made in 1983 (Shinoda et al., 2003), the presence and 56–85 (74) ␮m wide. Pharynx 16–19 (17) ␮m long; esoph- of E. anthropopitheci indicates that this pinworm has been ageal corpus 264–359 (320) ␮m long, 38–46 (42) ␮m wide; maintained in the chimpanzees in Japan for more than 20 yr. isthmus 11–21 (17) ␮m long; bulb 72–83 (78) ␮m long, 64–78 The present observation indicates that E. anthropopitheci can (71) ␮m wide. Nerve ring 112–138 (126) ␮m, excretory pore infect concomitantly with E. vermicularis. However, only a sin- 517–646 (574) ␮m from anterior extremity. Spicule 46–54 (51) gle case of such a mixed infection was observed in the present ␮m long. material, although numerous worms of both species were ob- Female (n ϭ 10): Body length 4.54–6.60 (5.37) mm, width served in the mixed feces of 10 chimpanzees collected in 2003 338–461 (410) ␮m. Cephalic vesicle 80–106 (94) ␮m long and (Table I). Because both pinworms may occupy the same eco- 107–130 (120) ␮m wide. Nerve ring 138–240 (181) ␮m from logical niche, it is probable that interspeciﬁc competition sup- anterior extremity. Excretory pore barely discernible. Pharynx presses one of the species. Actually, only E. vermicularis has 19–29 (23) ␮m long; esophageal corpus 683–826 (756) ␮m been hitherto demonstrated from chimpanzees in other institu- long, 58–96 (83) ␮m wide; isthmus 10–19 (15) ␮m long; bulb tions/zoos in Japan (Hasegawa and Kinjo, 1996; Murata et al., 112–144 (131) ␮m long, 112–144 (129) ␮m wide. Vulva 1.15– 2002; Nakano et al., 2003, 2005), suggesting that E. vermicu- 1.83 (1.43) mm from anterior extremity. Tail 1.20–1.67 (1.45) laris may out compete E. anthropopitheci under captive con- mm long. Eggs 53–58 (55.0 Ϯ 1.1) by 26–29 (26.8 Ϯ 0.8) ␮m ditions. (n ϭ 50). It is of special interest that E. anthropopitheci seems to have 852 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 1–6. Comparison of key characteristics of 2 pinworm species parasitic in captive chimpanzees. (1–3) Enterobius anthropopitheci: spicule of male showing straight distal end and membranous formation (arrowhead) (1), double-crested lateral ala (arrowheads) in female midbody (2) and egg (3). (4–6) Enterobius vermicularis: spicule of male showing hooked distal end (4), single-crested lateral ala (arrowhead) in female midbody (5) and egg (6). Bar ϭ 50 ␮m. HASEGAWA AND UDONO—ENTEROBIUS ANTHROPOPITHECI IN CAPTIVE CHIMPANZEE 853 become predominant species after repeated treatment with fen- doelst, 1916) (Nematoda: Oxyuridae), collected from chimpanzees, bendazole, which is an unusual anthelmintic for pinworm in- Pan troglodytes, on Rubondo Island, Tanzania. Journal of Parasi- tology 91: 1314–1317. fection. Presumably, fenbendazole exerted negative selective ———, T. KANO, AND M. MULAVWA. 1983. A parasitological survey pressure on E. vermicularis, reducing intensity of infection on the feces of pygmy chimpanzees, Pan paniscus, at Wamba, greatly, whereas its effects on E. anthropopitheci might not be Zaire. Primates 24: 419–423. deleterious. The mechanism of the exchange of predominant ———, AND T. K INJO. 1996. Human pinworms collected from a chimpanzee, Pan troglodytes, in a zoo of Okinawa, Japan. Journal of pinworm species should be studied not only from a pharma- the Helminthological Society of Washington 63: 272–275. cological but also from an ecological perspective. It also is sur- HUGOT, J. P. 1993. Redescription of Enterobius anthropopitheci (Ge- mised that a similar selection may occur when captive chim- doelst, 1916) (Nematoda, Oxyurida), a parasite of chimpanzees. panzees are (re)introduced to the field, because only E. anthro- Systematic Parasitology 26: 201–207. MURATA, K., HASEGAWA, H., T. NAKANO,A.NODA, AND T. Y ANAI. 2002. popitheci was found in such an introduced population (Hase- Fatal infection with human pinworm, Enterobius vermicularis,in gawa et al., 2005). a captive chimpanzee. Journal of Medical Primatology 31: 104– The measurements for the present worms of E. anthropopi- 108. theci coincide with those reported previously (Hasegawa et al., NAKANO, T., K. MURATA,Y.IKEDA, AND H. HASEGAWA. 2003. Growth of Enterobius vermicularis in a chimpanzee after anthelmintic treat- 2005). Although some of the present females had a larger body ment. Journal of Parasitology 89: 439–443. than those reported previously (Sandosham, 1950; Hugot, 1993; ———, D. FUKUI,Y.IKEDA, AND H. HASEGAWA. 2005. Effects of re- Hasegawa et al., 2005), probably due to the fact that only fully peated anthelmintic treatment on Enterobius vermicularis infection gravid females in relaxed condition were selected for morphom- in chimpanzees. Journal of Parasitology 91: 679–682. etry. PETRZELKOVA, K. J., H. HASEGAWA,L.R.MOSCOVICE,T.KAUR,M.ISSA, AND M. A. HUFFMAN. 2006. Three new intestinal nematode records for chimpanzees from the introduced population on Rubondo Is- LITERATURE CITED land, Tanzania. International Journal of Primatology 27: 767–777. SANDOSHAM, A. A. 1950. On Enterobius vermicularis (Linnaeus, 1758) EMI, Y., T. UDONO,H.KOBAYASHI, AND I. HAYASAKA. 2004. Efficacy of and some related species from primates and rodents. Journal of fenbendazole against Enterobius vermicularis in chimpanzees. Pri- Helminthology 24: 171–204. mate Research Supplement 20: 52. SHINODA, K., T. UDONO,K.YOSHIHARA,M.SHIMADA, AND O. TAKENAKA. HASEGAWA, H., Y. IKEDA,A.FUJISAKI,L.R.MOSCOVICE,K.J.PETRZEL- 2002. Molecular identification of subspecies of captive chimpan- KOVA,T.KAUR, AND M. A. HUFFMAN. 2005. Morphology of chim- zees reared in Japan using mitochondrial DNA. Primate Research panzee pinworms, Enterobius (Enterobius) anthropopitheci (Ge- 19: 145–155. J. Parasitol., 93(4), 2007, pp. 854–859 ᭧ American Society of Parasitologists 2007

DEVELOPMENT AND PATHOLOGY OF ECHINOSTOMA CAPRONI IN EXPERIMENTALLY INFECTED MICE

Carla Mun˜ oz-Antoli, Javier Sotillo, Carlos Monteagudo*, Bernard Fried†, Antonio Marcilla, and Rafael Toledo‡ Departamento de Parasitologı´a, Facultad de Farmacia, Universidad de Valencia, Av. Vicente Andre´s Estelle´s s/n, 46100 Burjassot, Valencia, Spain. e-mail: [email protected]

ABSTRACT: In the present article, several parasitological features of mice, each experimentally infected with 75 metacercariae of Echinostoma caproni (Trematoda: Echinostomatidae), were studied during the ﬁrst 12 wk postinfection. Moreover, the early pathological responses also were analyzed and compared with data previously published on other host species of E. caproni to gain further insight into the factors determining worm rejection or establishment of chronic infections. The results obtained show that the pattern of E. caproni infection in mice is consistent with a highly compatible host–parasite system. This combination is characterized by a high worm establishment, high egg output, and long survival of the worms. However, some differences with respect to other highly compatible hosts have been observed, particularly in relation to the survival of the adult worms. Histo- logical studies suggest that the kinetics of goblet cells, mucosal neutrophils, and mononuclear inﬂammatory cells in the mesentery seem to be essential in determining the course of E. caproni infection in mice.

Infections with intestinal trematodes are widespread in hu- Monteagudo et al., 2006). The analysis of the parasitological mans and other animals. However, despite the frequency of features of E. caproni infections in mice combined with the these infections, the relationships between intestinal trematodes pathological features seems to be of great interest for the un- and their final hosts have received little attention in experimen- derstanding of the factors determining the worm rejection or tal parasitology. Echinostoma caproni (Trematoda: Echinosto- the parasite establishment. matidae) is an intestinal fluke that does not undergo tissue mi- The aim of this research was to further elucidate host–para- gration in its definitive host. After infection of the definitive site relationships within the E. caproni-rodent systems. For this host with E. caproni, the metacercariae excyst in the duodenum purpose, we have examined in detail several parasitological fea- and the juvenile parasites migrate to the posterior third of the tures of mice experimentally infected with E. caproni during intestine where they attach to the mucosa by the ventral sucker the first 12 wk postinfection (PI). Moreover, we have studied (Fried and Huffman, 1996). the intestinal lesions induced by E. caproni in mice, and these Although E. caproni has a wide range of definitive hosts, its results are compared with our results previously published on compatibility differs considerably between rodent species. Echi- other rodent hosts with differing degrees of compatibility with nostoma caproni infection shows different patterns depending E. caproni infection (Toledo, Monteagudo et al., 2006). This on the host species, which makes these host–parasite systems information may be useful to determine the factors involved in highly suitable for elucidating aspects of the host-specific com- E. caproni worm expulsion, or, in contrast, the development of ponents that determine the course of infections with intestinal chronic infections. trematodes (Toledo and Fried, 2005). For example, hamsters and mice show a high level of compatibility with this echino- MATERIALS AND METHODS stome species, whereas rats and jirds are the opposite (Odaibo Parasite, host, and experimental infections et al., 1988, 1989; Christensen et al., 1990; Hansen et al., 1991; The strain of E. caproni has been described previously by Fujino and Mahler et al., 1995; Toledo et al., 2004). This classification is Fried (1993). Encysted metacercariae of E. caproni were removed from based mainly on worm establishment and survival observed in the kidneys and pericardial cavities of experimentally infected Biom- each host species. However, the highly compatible hosts (ham- phalaria glabrata snails and used to infect ICR male mice. Fifty-two sters and mice) show a markedly different pattern of infection. mice, weighing 30–35 g, were each infected by gastric gavage with 75 E. caproni In mice, low level infections are rapidly expelled, whereas metacercariae of . Mice were randomly allocated to group A (10 mice), B (36 mice), and C (6 mice). Group A was used to study heavy infections are not rejected and they develop considerable the kinetics of egg release of E. caproni adults in the definitive host. resistance to reinfection (Odaibo et al., 1988, 1989). In contrast, Group B was used to analyze worm recovery and its variations over golden hamsters develop only a limited capacity to expel E. the course of the infection and the morphological features. Group C caproni associated with primary infections and limited resis- was used to examine the intestinal lesions induced by E. caproni in mice. Three mice were left uninfected and used as controls in the study tance to reinfection (Christensen et al., 1990). In this context, of pathology. All the animals were maintained under conventional con- the early local lesions induced by E. caproni in each host spe- ditions with food and water ad libitum. cies seem to be of great importance (Toledo, Esteban et al., 2006). Comparison of the histological features in E. caproni- Worm recovery infected hamsters and rats showed marked differences that may This experiment was designed to compare the worm establishment be related to differences in worm survival in each host (Toledo, of E. caproni adults in mice. Three mice of group B were necropsied each week PI, and the number of worms recovered per host was recorded. Received 6 October 2006; revised 20 January 2007; accepted 23 Jan- Kinetics of egg release uary 2007. * Departamento de Patologıá, Facultad de Medicina Universidad de Va- This experiment was designed to examine the kinetics of egg output lencia, Av. Blasco Ibań˜ez 17, 46010 Valencia, Spain. of E. caproni adults during the course of the infection in mice. For this † Department of Biology, Lafayette College, Easton, Pennsylvania purpose, the egg output was determined in mice in group A for each 18042. week PI. ‡ To whom correspondence should be addressed. Fecal samples were examined to determine the number of eggs re-

854 MUN˜ OZ-ANTOLI ET AL.—DEVELOPMENT AND PATHOLOGY OF E. CAPRONI 855

TABLE I. Range and means and their standard deviations for each metrical variable of Echinostoma caproni analyzed in experimentally infected mice over the complete course of the experiment. See text for explanation of variable abbreviations.

Variable Range Mean SD

BA (mm2) 3.742–16.328 12.263 3.903 CW (␮m) 290.7–554 480.3 23.4 OSA (␮m2) 18,162.2–50,884.8 39,625 10,005.9 PL (␮m) 64.4–181.3 90.1 34.4 PHA (␮m2) 19,600.3–34,279.2 24,678.7 5,490.2 OL (␮m) 169.3–426 301.3 64 CSA (␮m2) 18,461.7–59,541.2 36,229.5 13,493.7 ␮ 2 VSA ( m ) 179,589.4–586,226.9 470,635 123,656.9 FIGURE 1. Echinostoma caproni worm recovery at increasing age OVA (␮m2) 62,091.9–196,281 137,274.9 39,682.9 (weeks) in infections with 75 metacercariae/mouse. Vertical bars rep- ATA (␮m2) 160,027–867,674.7 523,392.3 197,775.7 resent the standard deviation. PTA (␮m2) 168,969–1,144,754.9 651,699.1 269,921.5 ANOVA with interaction was used with the time PI and host species as independent variables, using the data published previously (Toledo, leased per animal (EPA) as described in a previous study (Toledo et al., Monteagudo et al., 2006). Moreover, when a significant time–host spe- 2003). Briefly, 24-hr fecal productions were collected and weighed in- cies interaction was detected, the Bonferroni t-test of the difference dividually from each animal. The individual samples were emulsified between means was performed as a post hoc analysis to determine in the ratio of 1 g/30 ml in a solution of 0.1 M NaOH. The mixture whether there were significant differences due to the host species within was shaken at room temperature for 2 hr, and the sediment was resus- the same time PI. P Ͻ 0.05 was considered as significant. Before anal- pended in 2 ml of the same solution. The eggs contained in 200 ␮l yses, data were log transformed to achieve normality. were counted. Five replicated samples were analyzed each day from each animal to determine the number of eggs released daily, and the average Ϯ standard deviation was considered for each week of the ex- RESULTS periment. Patent period and worm recovery Morphological analysis All mice individually exposed to 75 metacercariae of E. cap- To evaluate the effect of aging on adult worms in experimental in- roni became infected as determined by egg examination. The fections with E. caproni in mice, different morphometrical features were duration of the prepatent period studied on the mice in group studied (Table I). Ten of the adult worms collected each week from A was rather uniform. Egg release began 9–12 (10.1 Ϯ 0.4) group B mice were fixed in Bouin’s fluid under coverslip pressure and mounted in Canada balsam. The following features (variables) were days PI. All the mice remained positive by egg examination subjected to analysis: body area (BA), collar width (CW), oral sucker until the end of the experiment at 12 wk PI. area (OSA), prepharynx length (PL), pharynx area (PHA), esophagus All mice in group B were positive at necropsy, except 1 length (OL), cirrus sac area (CSA), ventral sucker area (VSA), ovarian mouse that was found negative at 12 wk PI. The number of area (OVA), anterior testis area (ATA), and posterior testis area (PTA). Each area was calculated as the product of the maximum length and worms recovered weekly per mouse during the first 12 wk PI width of the respective morphological feature. is shown in Figure 1. Worms were recovered each week from 1 to 12 wk PI, and the number ranged from 5 to 49 (23.3 Ϯ Histology 11.6) worms/mouse. The number of worms recovered weekly Histological responses to E. caproni infections in mice were evalu- progressively increased during the first period of the infection ated at 15 and 30 days PI. At each time interval, 3 mice were necrop- to reach a maximum at 4 wk PI. Thereafter, the number of sied, and intestinal sections of 0.7–1 cm in length from the sites where the worms were located were obtained from each animal and fixed in worms suddenly decreased, and the decrease continued during 4% buffered formalin. After embedding in paraffin, 4-␮m serial sections the last period of the experiment. The minimum worm number were cut. Intestinal sections were stained with hematoxylin and eosin, was observed at 12 wk PI. periodic acid-Schiff, Alcian blue, and toluidine blue. Sloughing of the villi tips was considered as a histological criterion Kinetics of egg release of E. caproni induced mucosal damage as described in a previous study (Bindseil and Christensen, 1984). Ten randomly selected low-magnifi- The egg release was continuous from the first day of the ϫ cation ( 100) fields of each section were examined, and the numbers patent period until the end of the experiment. During this pe- of destroyed or eroded villi and the total number of villi were recorded. Three sections of the intestine from each mouse were examined, and riod, eggs were found in all the fecal samples analyzed. How- the results are expressed as the percentage of destroyed or eroded villi. ever, the egg output was not uniform over time (Fig. 2). The All the cell counts (goblet cells, neutrophils, mast cells, eosinophils, egg output rapidly increased during the first period of the in- and mononuclear cells in the mesentery) were expressed as the number fection to reach a period of high release from 5 to 11 wk PI. of cells per villus-crypt unit (VCU), except those of the mesenteric cells, The egg output declined at 12 wk PI. The maximum egg output which are expressed in number of cells per high-power field (HPF) (ϫ400), studied over 10 selected HPFs. Results are expressed as the was observed at 10 wk PI. mean number of cells per VCU or HPF Ϯ standard deviation. Morphological analysis Statistical analysis The results obtained in this study on the lesions induced by E. cap- The ranges and the means and their standard deviations of roni in mice have been compared with those obtained by Toledo, Mon- each metric variable of E. caproni in mice are shown in Table teagudo et al. (2006) on rats and hamsters. For this purpose, a 2-factor I. The variability of each morphological feature is shown in 856 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

Pathology of the infection

All data reported in this section on the lesions of E. caproni infections in rats and hamsters were obtained from results in Toledo, Monteagudo et al. (2006), and they have been used for comparison with the results in mice as mentioned above. Gross examination of the intestine of mice at necropsy showed a marked dilation of the intestine during E. caproni infection. Associated with these dilations were large groups of worms. Histological examination of the intestines did not include the changes at the worm attachment sites because they are traumatic (Simonsen et al., 1989). The main foci of interest were the areas that surrounded the attachment sites. Echino- FIGURE 2. Weekly egg output of Echinostoma caproni at increasing stoma caproni-infected mice did not have villi destruction. Villi age in infections with 75 metacercariae/mouse. Vertical bars represent erosion was not evaluated due to the high percentage of eroded the standard deviation. villus observed in the control mice. All the cell counts were variable over the course of the infection (Fig. 4). Application of 2-way analysis of variance (AN- Figure 3. All the morphological variables analyzed showed a OVA) showed that all the cell counts were subjected to time- progressive increase during the first period of the infection. related changes. Thus, the number of neutrophils in E. caproni- Thereafter, the values became stable until the end of the exper- infected mice was significantly lower than in hamsters at 15 iment, except for CSA, ATA, and PTA, for which a significant and 30 days PI (P Ͻ 0.001) and higher than in rats at 30 days decrease was observed from 10 wk PI and beyond (Fig. 3). PI (P Ͻ 0.05). The number of goblet cells observed in E. cap-

FIGURE 3. Morphological characteristics of Echinostoma caproni at increasing age in infections with 75 metacercariae/mouse. (A) Mean body area. (B) Ovarian area. (C) Anterior (closed circles) and posterior (opened circles) testis area. (D) Oral sucker (closed circles) and cirrus sac area (opened circles). (E) Ventral sucker area. (F) Pharynx (triangles) area. (G) Collar width (closed circles), esophagus length (opened circles) and prepharynx length (closed triangles).Vertical bars represent the standard deviation. See text for explanation of variable abbreviations. MUN˜ OZ-ANTOLI ET AL.—DEVELOPMENT AND PATHOLOGY OF E. CAPRONI 857

FIGURE 4. Numbers of goblet cells, mast cells, eosinophils and neutrophils in the mucosa of the small intestine (A) and inflammatory cells in the mesentery (B) of mice at 0, 15, and 30 days PI with 75 metacercariae of Echinostoma caproni. Vertical bars represent the standard deviation. roni-infected mice over the course of the infection is shown in cells in the mesentery of the small intestine of E. caproni-in- Figure 4. A progressive increase in the counts was observed, fected mice was investigated by light microscopy, and the reaching a maximum at 30 days PI. The number of goblet cells counts are shown in Figure 4. The values at 15 days PI were was significantly higher than in hamsters at 15 and 30 days PI slightly higher than in control mice. However, an increase was (P Ͻ 0.05). No significant differences were observed with re- observed at 30 days PI. The number of inflammatory cells was spect to rats according to the study by Toledo, Monteagudo et significantly lower than in hamsters at 15 days PI (P Ͻ 0.001) al. (2006). Mast cell counts showed an increase at 15 and 30 and higher than in rats at 30 days PI (P Ͻ 0.001). days PI with respect to control mice. No significant differences with respect to hamsters and rats were detected over the course DISCUSSION of the infection in each host species. Probably the most striking observations of the present study Echinostoma caproni has a wide range of definitive hosts, were in relation to the numbers of eosinophils and the popu- although the compatibility may differ considerably between lations of the mononuclear inflammatory cells in the mesentery host species. The differences are reflected mainly in worm es- of the small intestine of mice. The number of eosinophils rap- tablishment and survival (Odaibo et al., 1988, 1989; Christen- idly increased, reaching a maximum at 15 days PI. At 30 days sen et al., 1990; Hansen et al., 1991; Mahler et al., 1995; Toledo PI, the values decreased, but they were higher than in control et al., 2004; Toledo and Fried, 2005). Highly compatible hosts, mice. Application of 2-way ANOVA and Bonferroni test such as hamsters and mice, develop a limited capacity to expel showed that the number of eosinophils was significantly lower primary infections, resulting in long-lasting infections. In hosts than in hamsters at 15 and 30 days PI (P Ͻ 0.001) and lower of low compatibility, such as rats and jirds, the infection is than in rats at 30 days PI (P Ͻ 0.001). However, the differences rapidly expelled. In our study, the course of the infection of E. with respect to rats were not considered due to the marked caproni in mice was examined by presenting quantitative data variability in the counts of the control rats in the study by To- on worm establishment, egg output, and worm morphology, and ledo, Monteagudo et al. (2006). The presence of inflammatory their respective variations over time. Moreover, the intestinal 858 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 lesions induced by E. caproni in mice and the possible conse- in mice. Infected mice and hamsters develop similar mast cell quences in the course of the infection were analyzed. kinetics compared with rats, although worm survival differs The general pattern of E. caproni infection in mice is con- greatly among these host species. This is supported by previ- sistent with that of a highly compatible host–parasite system. ously published studies on the E. caproni–mouse system that This combination is characterized by a high worm establish- showed that reductions in mast cells counts did not affect the ment and egg output and a long survival of the worms. All kinetics of worm expulsion (Fujino et al., 1998). Eosinophilic mice experimentally exposed each to 75 metacercariae of E. infiltration in E. caproni- and E. trivolvis-infected mice has caproni became infected; only 1 mouse became negative in been reported previously, and their involvement on worm re- terms of worm recovery during the course of the experiment. jection has been suggested (Bindseil and Christensen, 1984; Fu- In contrast, all the worms were expelled in rats at 7–8 wk PI jino et al., 1996). Our results show that eosinophilic counts in (Hansen et al., 1991; Toledo et al., 2004). In ICR mice, the E. caproni-infected mice are higher than in hamsters, suggest- kinetics of egg release and the worm growth curves were also ing a role in worm expulsion. However, the difficulty involved similar to those described in other host species of high com- in the comparison of data obtained from low compatible hosts patibility with E. caproni. The number of eggs released weekly (rats) prevents us from making further suggestions (Toledo, is similar to that in hamsters and markedly higher than that in Monteagudo et al., 2006). rats (Toledo et al., 2004). The first period of the infection was Of considerable interest seems to be the kinetics of goblet characterized by a progressive increase in egg output, probably cells, mucosal neutrophils, and mononuclear inflammatory cells in relation to the maturation of the adult worms, to reach a found in the mesentery. The combination of these features period of high release between 5 and 11 wk PI. Interestingly, seems to be essential to explain the differences on E. caproni a decrease in the egg output was observed by 12 wk PI. This survival in mice with respect to rats and hamsters. Goblet cell decrease may be associated with the reduction in the worm counts in E. caproni-infected mice were higher than in ham- burden, but the contribution of other factors cannot be dis- sters, whereas those of mucosal neutrophils and inflammatory missed. A marked reduction in the areas of the testes was ob- cells in the mesentery were higher than in rats. served in the last period of the experiment. This decrease has Goblet cells and secreted mucus may play a major role in not been observed in other Echinostoma sp.–rodent combina- the expulsion of intestinal helminths (McKay and Khan, 2003; tions (Isaacson et al., 1989; Fried et al., 1997; Kostadinova et Seo et al., 2003). Moreover, development of chronic E. caproni al., 2000; Toledo et al., 2003, 2004; Munõz-Antoli et al., 2004). infections has been often related to reduced numbers of goblet In this sense, the infective dose used in our study should be cells, whereas worm expulsion coincides with hyperplasia of considered. Yao et al. (1991) suggested that the testicular area goblet cells (Bindseil and Christensen, 1984; Weinstein and of E. caproni was subjected to a ‘crowding effect.’ They in- Fried, 1991; Fujino and Fried, 1993; Toledo, Monteagudo et al., fected hamsters with 15, 50, and 200 metacercariae/hamster. In 2006). Our study suggests that the increased goblet cell num- the animals infected with higher doses of metacercariae, a sig- bers in mice and rats with respect to hamsters may be involved nificant reduction in testicular size was observed. in the earlier E. caproni expulsion observed in those hosts. According to the biological features of E. caproni infections, However, other immune mechanisms are likely to operate in mice can be considered as highly compatible hosts. However, mice to explain the higher worm survival than in rats. In this some differences with respect to other hosts of high compati- context, the kinetics of mucosal neutrophils and the mesenteric bility have been observed. For example, the capacity of mice inflammatory cells seem to be essential. In contrast to rats, sig- to expel primary E. caproni infections is greater than hamsters. nificant increases in the number of neutrophils and mononuclear Toledo et al. (2004) showed that the survival of E. caproni inflammatory cells have been detected in mice and hamsters, infection in hamsters is at least 20 wk PI without significant showing the importance of local inflammatory responses in changes in the worm burden during this period. Herein, we have these latter species. This response may be a determinant in the observed that infections in mice show a persistence of at least establishment of chronic infections. In Giardia lamblia-infected 12 wk PI, but a marked decrease in the worm burden was ob- mice, local inflammatory response is a factor that contributes served by 5 wk PI. Factors causing the earlier worm expulsion to increased susceptibility of a host (Mu¨ller and Von Allmen, in mice are difficult to elucidate. In this context, we have ex- 2005; Von Allmen et al., 2006). In E. caproni infections, the amined the intestinal lesions induced by E. caproni in mice to development of chronic infections has been associated with ear- compare the results with those described previously in other ly local inflammatory responses (Toledo, Monteagudo et al., hosts of different compatibility with E. caproni. Our results 2006). It is, therefore, likely that differences in intestinal in- show that the particular kinetics of E. caproni worm burden in flammatory responses are involved in the longer survival of E. mice is determined by a complex set of reactions in which caproni in mice than in rats, although other parameters, such goblet cells, mucosal neutrophils, and mesenteric mononuclear as the goblet cell counts, are similar between both hosts. Inter- inflammatory cells seem to play a major role. estingly, the numbers of mucosal neutrophils were significantly The increase in the number of all the cell types analyzed greater in hamster than in mice. Neutrophils are an important probably reflects on the importance of the local cellular mech- source of proinflammatory cytokines such as interleukin-12 and anisms in the response against E. caproni in mice. However, tumor necrosis factor-␣ as well as free radicals and several che- the contribution of each cell population in worm expulsion or mokines. This raises the possibility that neutrophil infiltration chronic worm establishment does not seem to be the same. Al- and the consequences in the inflammatory response are essen- though the role of mast cells on the course of echinostomiasis tial in the longer survival of E. caproni in hamsters. Accord- is not well understood (Toledo, Esteban et al., 2006), our results ingly, approaches on the relation between neutrophil infiltration suggest that they are not determinants in E caproni infections and the development of chronic Echinostoma spp. infections MUN˜ OZ-ANTOLI ET AL.—DEVELOPMENT AND PATHOLOGY OF E. CAPRONI 859 will provide novel information on the susceptibility of a host the golden hamster. International Journal for Parasitology 19: 943– to intestinal helminths. 944. KOSTADINOVA, A., D. I. GIBSON,V.BISERKOV, AND R. IVANOVA. 2000. In summary, we have analyzed the development and patho- A quantitative approach to the evaluation of the morphological var- logical lesions of E. caproni in mice. The biological features iability of two echinostomes, Echinostoma miyagawai Ishii, 1932 of the infection are consistent with those of a highly compatible and E. revolutum (Frolich, 1802), from Europe. Systematic Para- host–parasite system. However, differences with respect to oth- sitology 45: 1–15. MAHLER, H., N. Ø. CHRISTENSEN, AND O. HINDSBO. 1995. Studies on the er hosts of high compatibility have been observed. These dif- reproductive capacity of Echinostoma caproni (Trematoda) in ham- ferences seem to be related to the local immune response gen- sters and jirds. International Journal for Parasitology 25: 705–710. erated by E. caproni in mice. The kinetics of goblet cells, mu- MCKAY,D.M.,AND W. I. KHAN. 2003. STAT-6 is an absolute require- cosal neutrophils, and mononuclear inflammatory cells in the ment for murine rejection of Hymenolepis diminuta. Journal of Par- asitology 89: 188–189. mesentery seem to be essential in determining the course of E. MU¨ LLER,N.,AND N. VON ALLMEN. 2005. Recent insights into the mu- caproni infection in mice. cosal reactions associated with Giardia lamblia infections. Inter- national Journal for Parasitology 35: 1339–1347. ˜ ACKNOWLEDGMENTS MUNOZ-ANTOLI, C., I. CARPENA,A.ESPERT,J.G.ESTEBAN, AND R. TO- LEDO. 2004. The effect of host species on the development of Echi- This study was supported by the projects CGL2005-02321/BOS from nostoma friedi (Trematoda: Echinostomatidae) adult worms. Revis- the Ministerio de Educacioń y Ciencia (Spain) and GV05/039 from the ta Ibe´rica de Parasitologıá 64: 81–87. Conselleria de Cultura, Educacio´ i Esport de la Generalitat Valenciana ODAIBO, A. B., N. Ø. CHRISTENSEN, AND F. M. A. UKOLI. 1988. Estab- (Spain). This work was carried out while J.S. was a recipient of a pre- lishment survival and fecundity in Echinostoma caproni (Trema- doctoral fellowship from the Ministerio de Educacioń, Cultura y De- toda) infections in NMRI mice. Proceedings of the Helmintholog- portes, Madrid (Spain). This research complies with the current laws ical Society of Washington 55: 265–269. for animal health research in Spain. ———, ———, AND ———. 1989. Further studies on the population regulation in Echinostoma caproni infections in NMRI mice. Pro- ceedings of the Helminthological Society of Washington 56: 192– LITERATURE CITED 198. SEO, M., S. M. GUK,E.T.HAN, AND J. Y. CHAI. 2003. Role of intestinal BINDSEIL, E., AND N. Ø. CHRISTENSEN. 1984. Thymus-independent crypt goblet cells in the expulsion of Gymnophalloides seoi from mice. hyperplasia and villous atrophy in the small intestine of mice in- Journal of Parasitology 89: 1080–1082. fected with the trematode Echinostoma revolutum. Parasitology 88: SIMONSEN, P. E., E. BINDSEIL, AND M. KOIE. 1989. Echinostoma caproni 431–438. in mice: Studies on the attachment site of an intestinal trematode. CHRISTENSEN,N.Ø.,P.SIMONSEN,A.B.ODAIBO, AND H. MAHLER. 1990. International Journal for Parasitology 19: 561–566. Establishment, survival and fecundity in Echinostoma caproni TOLEDO, R., A. ESPERT,I.CARPENA,C.MUN˜ OZ-ANTOLI, AND J. G. ES- (Trematoda) infections in hamsters and jirds. Proceedings of the TEBAN. 2003. An experimental study of the reproductive success of Helminthological Society of Washington 57: 104–107. Echinostoma friedi (Trematoda: Echinostomatidae) in the golden FRIED,B.,AND J. E. HUFFMAN. 1996. The biology of the intestinal trem- hamster. Parasitology 126: 433–441. atode Echinostoma caproni. Advances in Parasitology 38: 311– ———, ———, ———, ———, B. FRIED, AND J. G. ESTEBAN. 2004. 368. The comparative development of Echinostoma caproni (Trematoda: ———, T. J. MUELLER, AND B. A. FRAZER. 1997. Observations on Echi- Echinostomatidae) adults in experimentally infected hamsters and nostoma revolutum and Echinostoma trivolvis in single and con- rats. Parasitology Research 93: 439–444. current infections in domestic chicks. International Journal for Par- ———, J. G. ESTEBAN, AND B. FRIED. 2006. Immunology and pathology asitology 27: 1319–1322. of intestinal trematode infections in their definitive hosts. Advances FUJINO,T.,AND B. FRIED. 1993. Expulsion of Echinostoma trivolvis in Parasitology 63: 285–365. (Cort, 1914) Kanev, 1985 and retention of E. caproni Richard, ———, AND B. FRIED. 2005. Echinostomes as experimental models in 1964 (Trematoda, Echinostomatidae) in C3H mice: Pathological, adult parasite–vertebrate host interactions. Trends in Parasitology ultrastructural, and cytochemical effects on the host intestine. Par- 21: 251–254. asitology Research 79: 286–292. ———, C. MONTEAGUDO,A.ESPERT,B.FRIED,J.G.ESTEBAN, AND A. ———, ———, H. ICHIKAWA, AND I. TADA. 1996. Rapid expulsion of MARCILLA. 2006. Echinostoma caproni: Intestinal pathology in the the intestinal trematodes Echinostoma trivolvis and E. caproni from golden hamster, a highly compatible host, and the Wistar rat, a less C3H mice by trapping with increased goblet cell mucins. Interna- compatible host. Experimental Parasitology 112: 164–171. tional Journal for Parasitology 26: 319–324. VON ALLMEN, N., S. CHRISTEN,U.FORSTER,B.GOTTSTEIN,M.WELLE, ———, H. ICHIKAWA, AND B. FRIED. 1998. The immunosuppressive AND N. MU¨ LLER. 2006. Acute trichinellosis increases susceptibility compound FK506 does not affect expulsion of Echinostoma tri- to Giardia lamblia infection in the mouse model. Parasitology 133: volvis in C3H mice. Parasitology Research 84: 519–521. 139–149. HANSEN, K., J. W. NIELSEN,O.HINDSBO, AND N.Ø. CHRISTENSEN. 1991. WEINSTEIN,M.S.,AND B. FRIED. 1991. The expulsion of Echinostoma Echinostoma caproni in rats: Worm population dynamics and host trivolvis and retention of Echinostoma caproni in the ICR mouse: blood eosinophilia during primary 6, 25 and 50 metacercarial in- Pathological effects. International Journal for Parasitology 21: 255– fections, and resistance to secondary and superimposed infections. 257. Parasitology Research 77: 686–690. YAO, G., J. E. HUFFMAN, AND B. FRIED. 1991. The effects of crowding ISAACSON, A., J. E. HUFFMAN, AND B. FRIED. 1989. Infectivity, growth, on adults of Echinostoma caproni in experimentally infected gold- development and pathology Echinostoma caproni (Trematoda) in en hamsters. Journal of Helminthology 65: 248–254. J. Parasitol., 93(4), 2007, pp. 860–865 ᭧ American Society of Parasitologists 2007

A NEW GENUS AND SPECIES OF MACRODEROIDIDAE, AND OTHER DIGENEANS FROM FISHES OF LAKE MALAWI, AFRICA

Rodney A. Bray and Sherman S. Hendrix* Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom. e-mail: [email protected]

ABSTRACT: Malawitrema staufferi n. gen., n. sp., an unusual digenean, is described from Clarias mossambicus (type host) and Bagrus meridionalis from Lake Malawi. It has a small, pyriform body, with a spinous tegument. The ceca are relative short, not reaching to the testes. The 2 testes are symmetrical in the middle hind body. The cirrus sac is long and narrow, reaching into hind body. The genital pore is median, immediately anterior to the ventral sucker. The ovary is pretesticular, and a canalicular seminal vesicle and Laurer’s canal are present. The uterus usually reaches distinctly posteriorly to testes. The eggs are small. The follicular vitellarium is in 2 small fields just anterior to testes. The ventrally subterminal excretory pore leads to a claviform vesicle. This species does not fit clearly into any known family of digeneans and is placed in the Macroderoididae as a temporary measure. Other digeneans reported from Lake Malawi include Haplorchoides cahirinus (Looss, 1896) in C. mossambicus and B. meridionalis; Astiotrema turneri Bray, van Oosterhout, Blais & Cable, 2006 in Protomelas annectens, P. cf. taeniolatus, Labeo- tropheus fuelleborni, Ctenopharynx (Otopharynx) pictus, and Pseudotropheus zebra; Glossidium pedatum Looss, 1899 in C. mossambicus and B. meridionalis; and an unidentifiable sanguinicolid from Bathyclarias nyasensis.

Little is known of the digenean fauna of Lake Malawi (for- Taxonomic summary merly Lake Nyasa). Prudhoe (1957) reported the metacercaria Type species: Malawitrema staufferi n. sp. of Clinostomoides brieni Dollfus, 1950 in Clarias sp., Taylor Etymology: The genus is named after the lake in which it is found. et al. (1998) mentioned unnamed digeneans in haplochromine cichlids, and Bray et al. (2006) recently described a new Astio- Remarks trema species from mbuna cichlids. The present article adds to This species on which this genus is based is not identifiable as any that small data set by reporting 5 species of adult digeneans, of those in Khalil and Polling (1997). In fact, it is difficult to place this including a puzzling new form from siluriforms. form into a currently recognized family; placing the new species in the Macroderoididae is considered a temporary measure until further evidence is available. Using the fish parasite keys in Yamaguti (1971) MATERIALS AND METHODS positions it in either Fellodistomidae Nicoll, 1909 or the Lecithoden- driidae Odhner, 1910. The Fellodistomidae is a predominantly marine Fishes were captured by hook and line, transported alive to the work family, and is characterized by an unarmed tegument, a uterine seminal area, and necropsied within 4 hr of capture. Individual sections of the receptacle, and a Y- or V-shaped excretory vesicle. Clearly, M. staufferi digestive system were examined separately, and all worms were killed does not belong in this family. As far as the Lecithodendriidae is con- in hot water and fixed in alcohol–formalin–acetic acid (AFA). Whole cerned, a few species of the predominantly amphibian, Ganeo Klein, mounts were stained with Mayer’s paracarmine, cleared in beechwood 1905 and Pleurogenoides Travassos, 1921, are reported in freshwater creosote, and mounted in Canada balsam. Measurements were made fishes in India and Bangladesh, but the present specimen does not look through a drawing tube on an Olympus BH-2 microscope, with the use anything like species in either of these genera with their Y-shaped ex- of a Digicad plus digitizing tablet and Carl Zeiss KS100 software adapt- cretory vesicles and lateral genital pores. Nevertheless, according to ed by Imaging Associates, and are quoted in micrometers. The material Yamaguti (1971), a few lecithodendriids are reported as having cylin- is lodged in the BMNH collection of the Natural History Museum, drical or saccular excretory vesicles, and not all species have lateral London, United Kingdom. genital pores. With the use of the keys and diagnoses in the series by Gibson et al. DESCRIPTION (2002), Jones et al. (2005), and in Schell (1985), it appears that the short ceca suggest affinities with the Brachycoeliidae. Although some brachy- Malawitrema n. gen. coeliids are said to have a Y-shaped excretory system, Brachycoelium Du- jardin, 1845 and Cymatocarpus Looss, 1899 have either an I-shaped ex- Diagnosis cretory vesicle, or a Y-shaped vesicle with a long stem and ‘‘short incon- Macroderoididae McMullen, 1937. Body small, pyriform. Eye spot spicuous branches’’ (Prudhoe and Bray, 1982; T. Pojman´ska, pers. comm.). pigment granules in forebody. Tegument spinous. Oral sucker subglobu- The Brachycoeliidae is reported as occurring only in amphibians, reptiles, lar, subterminal. Ventral sucker rounded, slightly pre-equatorial. Pre- and, rarely, mammals, so the finding of a brachycoeliid in a fish would be pharynx short. Pharynx small, subglobular. Esophagus distinct. Intesti- highly unusual. Malawitrema differs from well-established members of the nal bifurcation in posterior forebody. Caeca blind, narrow, reaching to Brachycoeliidae (see Schell, 1985) by the caeca reaching almost to the level of vitelline fields. Testes 2, rounded to oval, margins smooth, testes, the restricted vitelline distribution in the hind body, and the relatively symmetrical in middle hind body, separated, not contiguous. Cirrus sac short, claviform excretory vesicle. long, narrow, reaching into hind body. Internal seminal vesicle long. When Bray et al. (2006) described a new species of Astiotrema Pars prostatica long, wide. Ejaculatory duct short. Genital atrium dis- Looss, 1900 from freshwater fishes in Lake Malawi, they considered tinct. Genital pore median, immediately anterior to ventral sucker. Ova- that there were 4 nominal plagiorchiid-like genera in freshwater fishes ry oval, pretesticular, overlapping ventral sucker, separated from testes in Africa, i.e., Astiotrema, Glossidium Looss, 1899, Alloglossidium Sim- by uterus. Canalicular seminal vesicle and Laurer’s canal present. Uterus er, 1929, and Afromacroderoides Khali, 1972. Malawitrema is not sim- intercecal usually reaches distinctly posteriorly to testes. Eggs small. ilar to any of them; they all have longer caeca, more extensive vitelline Vitellarium follicular; in 2 small fields laterally, mainly just anterior to fields and tandem to oblique testes. McMullen (1937) erected the family testes. Excretory pore ventrally subterminal. Vesicle claviform, reaching Macroderoididae McMullen, 1937, for the plagiorchioids from fishes just anteriorly to testes. and differentiated it from the Plagiorchiidae by its I-shaped (as opposed to Y-shaped) excretory vesicle. Macroderoidids were, at this time, thought to be only in the digestive tract of fishes. This is still the case Received 4 October 2006; revised 24 January 2007; accepted 25 Jan- for the type genus, Macroderoides Pearse, 1924, but not for all the other uary 2007. genera subsequently placed in the family, such as Alloglossidium, which * To whom correspondence should be addressed. Department of Biol- is also found in leeches and crustaceans (Brooks, 2003). Nevertheless, ogy, Gettysburg College, Gettysburg, Pennsylvania 17325. several plagiorchiid-like genera from fishes have been considered mem-

860 BRAY AND HENDRIX—M. STAUFFERI, N. GEN., N. SP., FROM LAKE MALAWI 861

bers of the Macroderoididae, including the marine form Cirkennedya Gibson & Bray, 1979. The constituent genera of the Macroderoididae, and thus of other plagiorchioid families, are the subject of dispute, particularly as the shape of the excretory vesicle is often difficult to distinguish or is poorly described. Molecular studies are now being utilized to elucidate the relationships of the plagiorchioid genera and families. These results indicate that Macroderoides is a plagiorchioid genus, but not especially close to the Plagiorchiidae (Tkach, Snyder, and Swider- ski, 2001; V. Tkach, pers. comm.). Alloglossidium does not cluster with Macroderoides and, therefore, although a plagiorchioid is not a ma- croderoidid (V. Tkach, pers. comm.). Prudhoe and Bray (1982) considered Astiotrema ‘‘a composite group of genera.’’ This idea has been supported by molecular results (Tkach, Pawlowski et al., 2001; V. Tkach, pers. comm.), which have shown that there are at least 3 groups of species, and that Astiotrema (sensu stricto) is not a plagiorchioid. Because of the unsatisfactory state of understanding of the status of plagiorchioid-like digeneans from freshwater fishes, Malawitrema is compared with some putative members of Macroderoididae McMullen, 1937, including Gauhatiana Gupta, 1955 (see Yamaguti, 1958), which is reported mainly from siluriform catfishes in India, China, Thailand, and the Philippines (Gupta, 1955; Wang, 1981; Wang et al., 1983; Lo- pez, 1988; Wongsawad et al., 2004; Lerssutthichawal and Supamattaya, 2005). The type species, G. batrachii Gupta, 1955, has been reported once in Egypt (El-Gwady et al., 1995). It was considered a synonym of Astiotrema reniferum (Looss, 1898) by Yeh and Fotedar (1958). This synonymy was accepted by Kumari et al. (1973), but not by Agrawal (1966), who distinguished the genus by minor details of vitelline dis- FIGURES 1–4. Malawitrema staufferi n. gen., n. sp. (1) Ventral view of tribution. Whichever of these courses is considered best, Malawitrema holotype from Clarias mossambicus, uterus in bold outline only. (2) Ventral is readily distinguished from Gauhatiana (and indeed Astiotrema)by view of worm from Bagrus meridionalis, uterus in bold outline only. (3) the short caeca, the restricted vitelline distribution in the hind body, the Terminal genitalia, specimen from C. mossambicus.(4) Region around fe- symmetrical testes, and the claviform excretory vesicle. male proximal system, uterus in outline only, eggs obscure detail, specimen One species of Alloglossidium Simer, 1929 is described from Africa, from C. mossambicus. Abbreviations: c, caecum; cs, cirrus sac; Lc, Laurer’s namely A. fatemi Abdel-Maksoud, 1998, from the catfish Clarias lazera canal; ov, ovary; sr, seminal receptacle; t, testis; ut, outline of uterus. Scale in Egypt (Abdel-Maksoud, 1998). As all other members of the genus bars ϭ 1, 200 ␮m; 2, 500 ␮m; 3, 4, 100 ␮m. are reported in freshwater fishes, leeches, and crustaceans in North and Central America, Bray et al. (2006) cast doubt on the generic status of this form. It differs greatly from Malawitrema in its long caeca, more extensive vitellarium with confluent fields at the posterior extremity, at level of esophagus. Tegument spinous, largest spines anterior, de- and virtually tandem testes. creasing in size and density posterior, detected to about level of testes. Macroderoides is reported only from freshwater fishes, and mainly Oral sucker subglobular, subterminal. Ventral sucker rounded slightly in North America. Some worms are similar in shape to Malawitrema, pre-equatorial. Prepharynx short, within posterior concavity of oral whereas others are rather more elongate, e.g., the type species M. spi- sucker. Pharynx small, subglobular. Esophagus distinct, with recurved niferus Pearse, 1924, M. parvus (Hunter, 1932), and M. trilobatus Tay- part anterior. Intestinal bifurcation in posterior forebody. Caeca blind, lor, 1978 (Pearse, 1924; Hunter, 1932; Taylor, 1978). Two species are narrow, reach to level of vitelline fields, not quite reaching to testes. more similar to Malawitrema in shape, although somewhat larger; these Testes 2, rounded to oval, margins smooth, symmetrical in middle are M. flavus Van Cleave & Mueller, 1932 and M. typicus (Winfield, hind body, separated, not contiguous. Cirrus sac long, narrow, reaching 1929) (Van Cleave and Mueller, 1932; Winfield, 1929). They differ in into hind body (Fig. 3). Internal seminal vesicle long, with evidence of the length of the caeca, which in both cases reach almost to the posterior folds. Pars prostatica long, wide, with sparse gland cells. Ejaculatory extremity, certainly well past the testes. The vitellarium is somewhat duct short, not seen to form cirrus. Genital atrium distinct. Genital pore more extensive, reaching to the posterior extent of the testes. The testes median, immediately anterior to ventral sucker. in both species are oblique. Ovary oval, pretesticular, overlapping ventral sucker, separated from One nominal species of Macroderoides is reported outside America, testes by uterus. Canalicular seminal receptacle oval, posterior to uterus. namely M. asiaticus Belouss in Skrjabin and Antipin, 1958, from the Laurer’s canal opens dorsally between seminal receptacle and ovary bagrid siluriforms, the yellow catfish Pseudobagrus fulvidraco (Rich- (Fig. 4). Mehlis’ gland posterior to ovary. Uterus intercecal, usually ardson, 1846), Primorskii Krai, far eastern Russia, and the Ussuri catfish reaches distinctly posteriorly to testes, lies ventrally to ovary and me- Liocassis ussuriensis (Dybowski, 1872), in the River Amur basin dian parts of testes. Eggs small, tanned, operculate. Metraterm narrow; (Skrjabin and Antipin, 1958; Akhmerov, 1961). This species looks rath- reaches to about posterior margin of ventral sucker; walls slightly thicker like an Astiotrema, with its long caeca, tandem testes, and relatively ened. Vitellarium follicular; in 2 small fields laterally, anterior extent extensive vitelline fields distinguishing it from Malawitrema. just posterior to ventral sucker, posterior extent to, or just overlapping, This unusual parasite is placed into the Macroderoididae until further testes. studies are undertaken. Regrettably, these worms were fixed in formalin Excretory pore ventrally subterminal. Vesicle claviform, reaching just and are not amenable to molecular study. anteriorly to testes.

Taxonomic summary DESCRIPTION Type host: Clarias mossambicus Peters, 1852, immature male, Clar- Malawitrema staufferi n. sp. iidae, Siluriformes, SL ϭ 430 mm (syn. of North African catﬁsh Clarias (Figs. 1–4) gariepinus [Burchell, 1822] according to Froese and Pauly, 2006). Other host: Bagrus meridionalis Gu¨nther, 1894 juvenile, Bagridae, Diagnosis (Based on 11 specimens from C. mossambicus and ϭ 3 from B. meridionalis) Siluriformes, kampango, SL 445 mm. Type locality: Deep water of the southeast arm of Lake Malawi Measurements in Table I. Body small, pyriform, narrow, widest in (14Њ06ЈS, 35Њ03ЈE) anterior hind body or, in younger specimens, constricted in anterior hind Site of infection: Clarias mossambicus, in pyloric half of intestine; body (Figs. 1 and 2). Eyespot pigment granules scattered in forebody B. meridionalis, distal half of intestine. 862 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Measurements of Malawitrema staufferi n. gen., n. sp.

Host Clarias mossambicus (n ϭ 11) Bagrus meridionalis (n ϭ 3)

Length 664–799 (734) 581–965 (733) Width 194–273 (235) 224–352 (268) Forebody (%) 246–324 (280) 224–370 (284) Oral sucker 96–118 ϫ 113–132 (107 ϫ 123) 107–146 ϫ 110–152 (121 ϫ 125) Prepharynx 0 0 Pharynx 31–39 ϫ 39–52 (35 ϫ 46) 33–44 ϫ 38–54 (38 ϫ 46) Esophagus 96–133 (118) 88–162 (119) Intestinal bifurcation to ventral sucker 23–52 (35) 19–39 (28) Previtelline distance 364–465 (402) 331–527 (412) Long vitelline field 85–150 (114) 107–175 (137) Short vitelline field 83–133 (104) 84–144 (112) Ventral sucker 93–109 ϫ 99–129 (101 ϫ 113) 89–133 ϫ 97–144 (104 ϫ 114) Cirrus sac 141–207 ϫ 25–31 (173 ϫ 29) 154–237 ϫ 28–50 (187 ϫ 38) Ventral sucker to ovary distance 0 0 Ovary 63–80 ϫ 43–73 (72 ϫ 61) 67–81 ϫ 63–77 (74 ϫ 69) Ovary to anterior testis distance 53–108 (76) 40–152 (84) Testes 76–110 ϫ 57–95 (93 ϫ 75) 87–112 ϫ 72–114 (93 ϫ 86) Intertesticular distance 0–99 (42) 6–55 (28) Posttesticular distance 117–179 (140) 76–180 (121) Postvitelline distance 170–245 (201) 152–262 (191) Postuterine distance 78–129 (103) 21–95 (67) Postcecal distance 247–324 (283) 214–228 (221) Eggs 27–38 ϫ 11–15 (34 ϫ 14) 30–35 ϫ 15–18 (32 ϫ 16) Width (%)* 26.5–38.9 (32.1) 35.2–38.5 (36.7) Forebody (%)* 35.3–41.1 (38.2) 38.4–39.7 (38.9) Intestinal bifurcation to ventral sucker as percent of forebody length 8.21–16.0 (12.4) 7.39–10.9 (9.64) Sucker–length ratio 1:0.90–0.99 (0.95) 1:0.83–0.91 (0.86) Sucker–width ratio 1:0.85–0.99 (0.92) 1:0.88–0.95 (0.90) Oral sucker:pharynx ratio 1:0.34–0.40 (0.37) 1:0.34–0.40 (0.37) Oral sucker length (%)* 13.1–17.1 (14.5) 15.2–18.4 (16.7) Pharynx length (%)* 3.96–5.49 (4.72) 4.54–5.76 (5.30) Ventral sucker length (%)* 12.9–15.4 (13.7) 13.8–15.3 (14.4) Esophagus (%)* 13.8–18.2 (16.1) 15.1–16.8 (16.1) Previtelline distance (%)* 47.7–58.7 (54.8) 54.6–58.0 (56.5) Long vitelline field (%)* 11.3–19.6 (15.6) 18.1–19.9 (18.8) Cirrus sac length (%)* 20.1–25.9 (23.6) 24.6–26.5 (25.7) Ovary length (%)* 8.23–12.1 (9.88) 8.34–11.5 (10.4) Ventral sucker to ovary distance (%) 0 0 Ovary to anterior testis distance (%)* 7.58–14.7 (10.4) 6.90–15.7 (10.6) Testis length (%)* 10.1–14.4 (12.6) 9.24–16.5 (13.2) Intertesticular distance (%)* 0–13.8 (5.85) 0.95–5.67 (3.60) Posttesticular distance (%)* 15.3–23.5 (19.0) 13.1–18.7 (16.0) Postvitelline distance (%)* 22.3–32.1 (27.4) 24.5–27.2 (26.0) Postuterine distance (%)* 9.81–18.0 (14.2) 3.59–14.5 (8.97) Postcecal distance (%)* 19.2–40.0 (34.7) 34.1–37.9 (36.0)

* Percent of body length.

Prevalence: Clarias mossambicus, 1 of 1; B. meridionalis, 1 of 1. iepinus [Burchell, 1822] according to Froese and Pauly, 2006); Bagrus Type and voucher specimens: Clarias mossambicus Holotype BMNH meridionalis Gu¨nther, 1894 juvenile, Bagridae, Siluriformes, kampango, 2006.11.8.94, paratypes 2006.11.8.95–118; B. meridionalis, paratypes SL ϭ 445 mm. BMNH 2006.11.8.91–93. Locality: Deep water of the southeast arm of Lake Malawi. Etymology: The speciﬁc name honors Dr. Jay R. Stauffer, Jr. of Penn- Site of infection: Clarias mossambicus, in pyloric half of intestine; sylvania State University for his kindness in granting S.S.H. the op- B. meridionalis, distal half of intestine. portunity to collect the ﬁsh hosts at his research base in Malawi. Prevalence: Clarias mossambicus, 1 of 1; B. meridionalis, 1 of 1. Voucher specimens: Clarias mossambicus BMNH 2006.11.8.145– OTHER TREMATODA COLLECTED 146; B. meridionalis, BMNH 2006.11.8.129–144.

Heterophyidae Leiper, 1909 Remarks Haplorchoides cahirinus (Looss, 1896) Chen, 1949 This species is reported in Bagrus spp. in Egypt and Uganda (Khalil Hosts: Clarias mossambicus Peters, 1852, immature male, Clariidae, and Polling, 1997; Arafa et al., 2002; Hamada and Abdrabouh, 2005). Siluriformes, SL ϭ 430 mm (syn. of North African catﬁsh Clarias gar- We detected no morphological distinctions to published descriptions BRAY AND HENDRIX—M. STAUFFERI, N. GEN., N. SP., FROM LAKE MALAWI 863

TABLE II. Measurements of Astiotrema turneri and Glossidium pedatum.

Species Astiotrema Astiotrema Astiotrema Astiotrema Glossidium Glossidium turneri turneri turneri turneri pedatum pedatum Host Protomelas Protomelas Labeotropheus Ctenopharynx Clarias Bagrus annectens ?taeniolatus fuelleborni (Otopharynx) mossambicus meridionalis (n ϭ 1) (n ϭ 1) (n ϭ 1) pictus (n ϭ 2) (n ϭ 1) (n ϭ 1)

Length 1,329 1,537 1,850 1,490–1,897 1,214 1,757 Width 580 768 584 574–646 376 438 Forebody (%) 305 412 536 371–575 399 484 Oral sucker 130 ϫ 169 137 ϫ 187 179–192 145–176 ϫ 181–205 143 ϫ 174 170 ϫ 174 Prepharynx 5 7 0 2–22 22 5 Pharynx 61 ϫ 71 88 ϫ 68 93 ϫ 71 78–97 ϫ 83–107 74 ϫ 86 79 ϫ 91 Esophagus 224 193 244 289–374 29 26 Intestinal bifurcation to ventral 34 74 84 33–87 169 206 sucker Previtelline distance 241 358 364 360–566 535 589 Long vitelline field 682 803 1,036 773–838 549 700 Short vitelline field 685 682 779 556–712 388 635 Ventral sucker 150 ϫ 175 187 ϫ 203 183 ϫ 188 187–196 ϫ 199–242 164 ϫ 180 200 ϫ 200 Cirrus sac 265 ϫ 49 ? 228 ϫ 50 255–292 ϫ 48–54 366 ϫ 45 385 ϫ 93 Ventral sucker to ovary distance 0 0 0 0–7 12 19 Ovary 172 ϫ 187 104–191 197 ϫ 176 132–165 ϫ 160–165 114 ϫ 111 135 ϫ 211 Ovary to anterior testis distance 132 112 0 12–59 81 42 Anterior testis 128 ϫ 167 114 ϫ 187 140 ϫ 170 149–172 ϫ 169–187 137 ϫ 163 143 ϫ 186 Intertesticular distance 69 177 136 54–68 27 111 Posterior testis 124 ϫ 192 85 ϫ 214 124 ϫ 194 98–115 ϫ 140–144 126 ϫ 209 149 ϫ 220 Posttesticular distance 462 532 634 461–646 187 508 Postvitelline distance 349 435 466 387–389 168 477 Postuterine distance 73 33 12 226–411 12 19 Postcecal distance 188–189 245 ? 226–368 89–106 ? Eggs 34 ϫ 17 41 ϫ 14 37 ϫ 11 39–40 ϫ 11–13 36 ϫ 18 39 ϫ 18 Width (%)* 43.7 49.9 31.5 34.0–38.5 31.0 24.9 Forebody (%)* 22.9 26.8 29.0 24.9–30.3 32.9 27.5 Intestinal bifurcation to ventral 11.0 17.9 15.6 8.76–15.2 42.3 42.6 sucker as % of forebody length Sucker–length ratio 1:1.54 1:1.37 1:1.02 1:1.11–1.29 1:1.15 1:1.18 Sucker–width ratio 1:1.04 1:1.08 1:0.98 1:1.00–1.18 1:1.03 1:1.15 Oral sucker–pharynx ratio 1:0.42 1:0.36 1:0.37 1:0.42–0.52 1:0.49 1:0.52 Oral sucker length (%)* 9.77 8.89 9.70 9.29–9.74 11.8 9.65 Pharynx length (%)* 4.59 5.70 5.02 5.11–5.26 6.07 4.50 Ventral sucker length (%)* 11.3 12.2 9.90 10.3–12.5 13.5 11.4 Esophagus (%)* 16.8 12.6 13.2 19.4–19.7 2.36 1.46 Previtelline distance (%)* 18.2 23.3 19.7 24.2–29.8 44.1 33.5 Long vitelline field (%)* 51.4 52.2 56.0 44.2–51.9 45.3 39.8 Cirrus sac length (%) 19.9 ? 12.3 15.4–17.1 30.1 21.9 Ovary length (%)* 13.0 13.3 10.7 8.70–8.84 9.37 7.66 Ventral sucker to ovary distance (%) 0 0 0 0–0.38 0.99 1.11 Ovary to anterior testis distance (%)* 10.0 7.27 0 0.62–3.93 6.69 2.42 Anterior testis length (%)* 9.63 7.40 7.55 9.08–10.0 11.3 8.11 Intertesticular distance (%)* 5.17 11.5 7.34 2.86–4.58 2.22 6.31 Posterior testis length (%)* 9.31 5.55 6.72 6.05–6.58 10.34 8.49 Posttesticular distance (%)* 34.7 34.6 34.3 30.9–34.1 15.4 28.9 Postvitelline distance (%)* 26.3 28.3 25.2 20.5–26.0 13.8 27.2 Postuterine distance (%)* 5.47 2.12 0.64 0.39–2.09 0.97 1.10 Postcecal distance (%)† 40.9 23.1 ? 42.0–84.5 52.0 ? Postcecal distance (%)* 14.2 7.98 ? 14.3–26.2 7.99 ?

* Percent of body length. † Percent of posttesticular distance. 864 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

(Looss, 1899; Khalil and Thurston, 1973). Many specimens were found Remarks in B. meridionalis and only 2 in C. mossambicus. This appears to be A single, nonovigerous worm was found in the proximal half of the the first report of this species from Clarias. intestine. It has a large, deeply divided testis, which reaches almost to the nerve ring and envelopes the X-shaped part of the intestine. This Plagiorchiidae Lu¨ he, 1901 feature distinguishes this specimen from members of the freshwater sanguinicolid genus Sanguinicola Plehn, 1905 (see Smith, 2002), in- Astiotrema turneri Bray, van Oosterhout, Blais & Cable, 2006 cluding the only species known from Clarias in Africa, i.e., S. clarias Hosts: Protomelas annectens (Regan, 1922) male?, Cichlidae, Per- Imam, Marzouk, Hassan & Itman, 1984, from C. lazera in the Nile at ciformes, SL ϭ 75 mm, kambuzi; Protomelas cf. taeniolatus (Trewavas, Cairo and Giza, Egypt (Imam et al., 1984). 1935) female, Cichlidae, Perciformes, SL ϭ 78 mm, spindle hap; La- beotropheus fuelleborni Ahl, 1926, male, Cichlidae, Perciformes, blue ACKNOWLEDGMENTS mbuna; Ctenopharynx (Otopharynx) pictus (Trewavas, 1935), Cichlidae, Perciformes, SL ϭ 105 mm; Pseudotropheus zebra (Boulenger, 1899) We would like to acknowledge financial support by Gettysburg Col- male, Cichlidae, Perciformes, SL ϭ 84 mm, zebra mbuna. All cichlid lege for making the collection of hosts possible. A special thanks to hosts were collected alive with the use of SCUBA and maintained alive Jay R. Stauffer, Jr. and Matthew E. Arnegard for their help in host until examined. collection and to the government of Malawi for issuing the collecting Localities: All Lake Malawi, July 1996, P. annectens, Songwe Hill, permit to Jay R. Stauffer. We also thank Teresa Pojman´ska, W. Stefanski between Harbor Island and Kanchedza Island, SE Arm; P. cf. taeniola- Institute of Parasitology, Warsaw, Poland, for permission to cite as-yet- tus, Kanchedza Island; L. fuelleborni, Harbor Island; C. pictus, Kan- unpublished work. Vasyl Tkach, University of North Dakota, Grand chedza Island; P. zebra, Harbor Island. Forks, North Dakota, kindly commented on a draft of the article and Sites of infection: Protomelas annectens, midintestine; P. cf. taeni- gave permission to mention an unpublished finding. olatus, midintestine; L. fuelleborni, intestine 1/3 distance from stomach; C. pictus, intestine; P. zebra, intestine. LITERATURE CITED Prevalence: Protomelas annectens,33%,1of3;P. cf. taeniolatus, ABDEL-MAKSOUD, N. M. 1998. Revision of Alloglossidium Simer 1929 25%, 1 of 4; L. fuelleborni, 33%, 1 of 3; C. pictus, 1of 1; P. zebra, (Trematoda: Macroderoididae) and description of A. fatemi sp. n. 20%, 1 of 5. from a freshwater catfish. Journal of the Egyptian German Society Voucher specimens: Protomelas annectens, BMNH 2006.11.8.126; of Zoology. Invertebrate Zoology & Parasitology 25(D): 115–124. P. cf. taeniolatus, BMNH 2006.11.8.125; L. fuelleborni, BMNH AGRAWAL, V. 1966. Four trematode parasites (Plagiorchiidae Lu¨he, 1901 2006.11.8.127; C. pictus, BMNH 2006.11.8.123–124; P. zebra, BMNH emend. Ward, 1917) from reptiles of Lucknow. Revista de Biologia 2006.11.8.128. Tropical 14: 133–151. AKHMEROV, A. H. 1961. On the trematode fauna of fishes in the basin Remarks of the River Amur. Trudy Gelmintologicheskoi Laboratorii 11: 22– This species was recently described by Bray et al. (2006) from the 31. cichlids Pseudotropheus zebra (type host), P. emmiltos (Stauffer et al. ARAFA,S.Z,E.S.REDA, AND M. M. EL-NAGGAR. 2002. Cholinergic 1997), Labeotropheus trewavasae (Fryer 1956), and Melanochromis components of the nervous system of the digenean parasites, Hap- vermivorus Trewavas, 1935, in Lake Malawi. As most of the hosts re- lorchoides cahirinus and Acanthostomum absconditum from the ported here are new, we have included measurements of the specimens catfish Bagrus bayad in Egypt. Acta Parasitologica 47: 272–279. in Table II. There appear to be no morphological reasons for considering BRAY, R. A., C. V. OOSTERHOUT,J.BLAIS, AND J. CABLE. 2006. Astio- any of these specimens distinct. As noted above, the genus Astiotrema trema turneri n. sp. (Digenea: Plagiorchiidae) from cichlid fishes (sensu stricto) is not a plagiorchioid, but is retained in the Plagiorchiidae (Cichlidae: Perciformes) of Lake Malawi. Zootaxa 1319: 43–58. here pending publication of further evidence on its status. BROOKS, D. R. 2003. Lessons from a quiet classic. Journal of Parasi- tology 89: 878–888. EL-GWADY,H.M.,M.G.HASSAN,A.A.ABDEL-AAL, AND A. S. DIAB. Glossidium pedatum Looss, 1899 1995. Internal helminths of some Egyptian fresh water fishes. In Hosts: Clarias mossambicus Peters, 1852, immature male, Clariidae, Program and book of abstracts. IVth International Symposium of Siluriformes, SL ϭ 430 mm (syn. of North African catfish Clarias gar- Fish Parasitology. Munich, Germany, 101 p. iepinus [Burchell, 1822] according to Froese and Pauly, 2006); Bagrus FROESE, R., AND D. PAULY. 2006. FishBase. http://www.fishbase.org. meridionalis Gu¨nther, 1894 juvenile, Bagridae, Siluriformes, kampango, GIBSON, D. I., A. JONES, AND R. A. BRAY. 2002. Keys to the Trematoda. SL ϭ 445 mm. Volume 1. CABI Publishing and the Natural History Museum, Wal- Locality: Deep water of the southeast arm of Lake Malawi. lingford, U.K., 521 p. Sites of infection: Clarias mossambicus, in pyloric half of intestine; GUPTA, S. P. 1955. Trematode parasites of fresh-water fishes. Indian B. meridionalis, distal half of intestine. Journal of Helminthology 5(1953): 1–80. Prevalence: Clarias mossambicus, 1 of 1; B. meridionalis, 1 of 1. HAMADA,S.F.,AND A. E. S. ABDRABOUTH. 2005. Anatomical and mor- Voucher specimens: Clarias mossambicus BMNH 2006.11.8.122; B. phological studies on the intestinal digenean parasite Haplorchoi- meridionalis, BMNH 2006.11.8.119–121. des cahirinus from Bagrus bajad in Egypt. Egyptian Journal of Zoology 44: 81–97. Remarks HUNTER, G. W., III. 1932. A new trematode (Plesiocreadium parvum, sp. nov.) from fresh water fish. Transactions of the American Mi- This species was discussed by Bray et al. (2006), who pointed out croscopical Society 51: 16–21. that it was originally described in Bagrus spp. and has been reported IMAM, E. A., M. S. M. MARZOUK,A.A.HASSAN, AND R. H. ITMAN. in C. mossambicus, or its putative synonyms, from as widely apart as 1984. Studies of Sanguinicola sp. (Trematoda) of Nile fishes. Vet- Ethiopia, Egypt, and South Africa. Measurements are included in Table erinary Medical Journal, Egypt 32: 1–13. II. JONES, A., R. A. BRAY, AND D. I. GIBSON. 2005. Keys to the Trematoda. Volume 2. CABI Publishing and the Natural History Museum, Wal- Sanguinicolidae von Graff, 1907 lingford, U.K., 745 p. KHALIL,L.F.,AND L. POLLING. 1997. Check list of the helminth parasites Host: Bathyclarias nyasensis (Worthington, 1933), Clariidae, Siluri- of African freshwater fishes. University of the North, Pietersburg, formes, sapuwa. South Africa, 185 p. Locality: Deep water of the southeast arm of Lake Malawi. ———, AND J. P. THURSTON. 1973. Studies on the parasites of fresh- Site of infection: Proximal half of intestine. water fishes of Uganda, including the descriptions of two new spe- Prevalence: One of 1. cies of digeneans. Revue de Zoologie et Botanique Africaines 87: Voucher specimen: BMNH 2006.11.8.147. 209–248. BRAY AND HENDRIX—M. STAUFFERI, N. GEN., N. SP., FROM LAKE MALAWI 865

KUMARI, N. V., C. B. SRIVASTAVA, AND B. S. CHAUHAN. 1973. Rede- TAYLOR, M. I., G. F. TURNER,R.L.ROBINSON, AND J. R. STAUFFER,JR. scription of Astiotrema reniferum (Looss, 1898) Looss, 1900 with 1998. Sexual selection, parasites and bower height skew in a bow- comments on the status of the genus Pseudoparamacroderoides er-building cichlid fish. Animal Behaviour 56: 379–384. Gupta & Agarwal, 1968 (Trematoda: Plagiorchiidae). Records of TAYLOR, P. W. 1978. Macroderoides trilobatus sp. n. (Digenea: Ma- the Zoological Survey of India 67: 315–323. croderoididae) from the bowfin, Amia calva, and emendation of the LERSSUTTHICHAWAL,T.,AND K. SUPAMATTAYA. 2005. Diversity and dis- genus. Journal of Parasitology 64: 393–394. tribution of parasites from potentially cultured freshwater fish in TKACH, V. V., J. PAWLOWSKI,J.MARIAUX, AND Z. SWIDERSKI. 2001. Nakhon Si Thammarat. Songklanakarin Journal of Science and Molecular phylogeny of the suborder Plagiorchiata and its position Technology 27(Suppl. 1): 333–345. in the system of Digenea. In Interrelationships of the Platyhelmin- LOOSS, A. 1899. Weitere Beitra¨ge zur Kenntnis der Trematoden-fauna thes, D. T. J. Littlewood, and R. A. Bray (eds.). Taylor & Francis, Aegyptens, zugleich Versuch einer natu¨rlichen Gliederung des Ge- London, U.K., p. 186–193. nus Distomum Retzius. Zoologische Jahrbu¨cher 12: 521–784. ———, S. D. SNYDER, AND Z. SWIDERSKI. 2001. On the phylogenetic LOPEZ, N. C. 1988. Helminth and arthropod parasites of some freshwater relationships of some members of Macroderoididae and Ocheto- fishes from Laguna Lake and vicinities. In Fish health problems in somatidae (Digenea, Plagiorchioidea). Acta Parasitologica 46: 267– Laguna de Bay and environs, M. R. delos Reyes, and E. H. Belen 275. (eds.). Phillippine Council for Agriculture, Forestry and Natural VAN CLEAVE, H. J., AND J. F. MUELLER. 1932. Parasites of the Oneida Resources Research and Development, Book Series No. 49, Los Lake fishes. Part 1. Descriptions of new genera and new species. Banõs, Philippines, p. 7–14. Roosevelt Wildlife Annals 3: 9–71. WANG, P.-Q. 1981. Notes on some trematodes from freshwater fishes in MCMULLEN, D. B. 1937. A discussion of the taxonomy of the family Plagiorchiidae Lu¨he, 1901, and related trematodes. Journal of Par- Fujian Province. Journal of Fujian Teacher’s University. Natural 11: asitology 23: 244–258. Science Edition 81–90. ———, Y.-R. ZHAO,Q.-G.CHEN, AND J.-Y. TAO. 1983. Notes on some PEARSE, A. S. 1924. Observations on parasitic worms from Wisconsin species of parasitic helminths from freshwater fishes and five new fishes. Transactions of the Wisconsin Academy of Sciences, Arts species in Hongze Lake. Journal of Fujian Teacher’s University. and Letters 21: 147–160. Natural Science Edition 1983: 125–134. PRUDHOE, S. 1957. Trematoda. Exploration du Parc National de WINFIELD, G. F. 1929. Plesiocreadium typicum, a new trematode from l’Upemba 48: 1–28. Amia calva. Journal of Parasitology 16: 81–87. ———, AND R. A. BRAY. 1982. Platyhelminth parasites of the Am- WONGSAWAD, C., J. ROJTINNAKOM,P.WONGSAWAD,A.ROJANAPAIBUL, phibia. British Museum (Natural History) and Oxford University T. M ARAYONG,S.SUWATTANACOUPT,P.SIRIKANCHANA,O.SEY, AND Press, London, U.K., 217 p. B. V. JADHAV. 2004. Helminths of vertebrates in Mae Da Stream, SCHELL, S. C. 1985. Handbook of trematodes of North America north Chiang Mai, Thailand. Southeast Asian Journal of Tropical Medi- of Mexico. University of Idaho Press, Moscow, Idaho, 263 p. cine and Public Health 35(Suppl. 1): 140–146. SKRJABIN, K. I., AND D. N. ANTIPIN. 1958. Superfamily Plagiorchioidea YAMAGUTI, S. 1958. Systema helminthum. Vol. I. The digenetic trem- Dollfus, 1930. Part 2. Family Plagiorchidae [sic]Lu¨he, 1901. Tre- atodes of vertebrates. Interscience Publishers, New York, New matody Zhivotnykh I Cheloveka. Osnovy Trematodologii 14: 73– York, 1575 p. 631. ———. 1971. Synopsis of digenetic trematodes of vertebrates, Volume SMITH, J. W. 2002. Family Sanguinicolidae Graff, 1907. In Keys to the I. Keigaku Publishing Co., Tokyo, Japan, 1074 p. trematode parasites of vertebrates, Volume 1, D. I. Gibson, A. YEH, L.-S., AND D. N. FOTEDAR. 1958. A review of the trematode genus Jones, and R. A. Bray (eds.). CAB International, Wallingford, U.K., Astiotrema in the family Plagiorchiidae. Journal of Helminthology p. 433–452. 32: 17–32. J. Parasitol., 93(4), 2007, pp. 866–869 ᭧ American Society of Parasitologists 2007

NEW SPECIES OF SKRJABINODON (NEMATODA: PHARYNGODONIDAE) IN URACENTRON FLAVICEPS (SQUAMATA: IGUANIDAE) FROM ECUADOR AND PERU

Charles R. Bursey and Stephen R. Goldberg* Department of Biology, Pennsylvania State University, Shenango Campus, Sharon, Pennsylvania 16146. e-mail: [email protected]

ABSTRACT: Skrjabinodon dixoni n. sp. from the large intestine of Uracentron flaviceps (Squamata: Iguanidae) from Peru is described and illustrated. It is also reported in the same host from Ecuador. Skrjabinodon dixoni n. sp. differs from other species assigned to Skrjabinodon by morphology of tail filament and number of tail filament spines.

The thornytail iguana (Uracentron flaviceps (Guichenot, DESCRIPTION 1855)) is known from the western Amazonian regions of Brazil, Skrjabinodon dixoni n. sp. Colombia, Ecuador, and Peru; it is a forest dweller found high (Figs. 1–11) above the ground on the trunks of large trees or in the canopy General: Oxyurida: Pharyngodonidae Travassos, 1919, Skrjabinodon (Avila-Pires, 1995). Its diet consists mainly of tree ants (Dixon (Inglis, 1968). Small cylindrical nematodes, evident sexual dimorphism, and Soini, 1975). To our knowledge, there are no reports of males approximately one-fourth length of female. Anterior extremity tapered, tail narrowing abruptly behind anus to form terminal filamen- helminths from U. flaviceps. tous appendage in male, stiff spike in female. Triangular oral opening Four species of Skrjabinodon Inglis, 1968 are currently surrounded by 3 bilobed lips; one small pedunculate amphid on each known from neotropical lizards (Table I): S. cricosaurae Barus ventrolateral lip; additional lip ornamentation not seen. Lateral alae pre- and Coy Otero, 1974 from the Cuban night lizard (Cricosaura sent in males, absent in females. Females with vulva near esophageal bulb. typical), collected in Cuba; S. heliocostai Vicente, Vrcibradic, Male: Based upon holotype and 5 paratypes. Length (lip to posterior Muniz-Pereira and Pinto, 2000 from Cope’s mabuya (Mabuya pair of papillae; excludes tail filament) 2.32 Ϯ 0.34 mm (1.92–2.80 frenata), collected in Brazil; S. spinulosus Vicente, Vrcibradic, mm); width at level of excretory pore 279 Ϯ 55 (204–357). Cuticle Rocha and Pinto, 2002 from the Paraguay mabuya (Mabuya with longitudinal striations at approximately 1.5-␮m intervals; pseu- dorsivittata S. scelopori doannulations present. Lateral alae beginning midway between lips and ), also collected in Brazil; and (Cabal- nerve ring and ending just posterior to first pair of caudal papillae; lero, 1938) from the crevice swift (Sceloporus torquatus), col- anterior half of alae narrow, posterior half widening reaching approxi- lected in Mexico (Caballero, 1938; Barus and Coy Otero, 1974; mately 30 at level of caudal papillae. Esophageal corpus length 221 Ϯ Vicente et al., 2000, 2002). The purpose of this paper is to 24 (180–250), isthmus 25 Ϯ 6 (18–34) long, bulb length 80 Ϯ 9 (67– Ϯ Ϯ describe a new species of Skrjabinodon. 92), bulb width 79 10 (61–88). Nerve ring 123 16 (92–134) and excretory pore 790 Ϯ 122 (638–995) from anterior end, respectively. Spicule absent. Tail filament 380 Ϯ 23 (357–408) in length with 2 Ϯ MATERIALS AND METHODS 1 (1–3) small cuticular spines. Cloaca and associated papillae raised from body surface on distinct cone. Caudal alae absent, 3 pairs of sessile Fifteen specimens of U. flaviceps were examined for helminths: 2 papillae, 1 pair precloacal, 1 pair postcloacal, third pair occurring at from Napo Province, Ecuador (Oklahoma Museum of Natural History, junction with tail filament. Phasmids slightly posteriolateral to base of OMNH 36374, 36376); 8 from Sucumbios Province, Ecuador (OMNH genital cone. Single tubular testis reflexed posterior to excretory pore. 36398–36405); and 5 from Loreto Department, Peru (Texas A&M Uni- Gravid female: Based upon allotype and 8 paratypes. Length (ex- versity, TCWC 44562, 44563, 44566, 44568, 66569). There were 7 cluding tail spike) 6.67 Ϯ 0.77 mm (5.44–7.87 mm), width at mid-body females and 8 males in the sample, with a mean snout-vent length 84 Ϯ Ϯ 751 100 (638–969). Cuticle with narrow longitudinal striations at 23 mm (41–119 mm). The specimens of Skrjabinodon examined in approximately 1.5-␮m intervals; pseudoannulations present. Lateral alae this study were killed in situ by 10% formalin preservation of the hosts, absent. Esophageal corpus length 445 Ϯ 47 (372–512), isthmus 10 Ϯ which were later stored in 70% ethanol. The body cavity of each lizard 2 (6–12) long, bulb length 131 Ϯ 17 (110–159), bulb width 135 Ϯ 25 was opened by a longitudinal ventral incision. The stomachs of the (92–171). Nerve ring 123 Ϯ 15 (92–140), excretory pore 828 Ϯ 86 Ecuadorian specimens were missing, having been used in an ecological (740–1,020), and vulva 915 Ϯ 106 (816–1,148) from anterior end, re- study of the lizards’ diet (Vitt and Zani, 1996); the gastrointestinal tracts spectively. Distance between anus and base of tail filament not deter- of Peruvian specimens were removed by cutting across the esophagus mined, body cavity filled with eggs, which occlude internal organ de- and rectum. The stomach (when available), small intestine, and large tails. Tail spike 258 Ϯ 32 (204–306) in length with 3 small terminal intestine of each lizard were examined separately. Nematodes were cuticular spines. In fully gravid females, uterus extends from posterior placed in lacto-phenol, allowed to clear, and examined using light mi- end of esophageal bulb to end of body. Egg oval, slightly flattened on croscopy. Drawings were made with the aid of a microprojector. Mea- 1 side, operculum not seen, length 91 Ϯ 3 (88–95), width 41 Ϯ 1 (40– surements are in ␮m with mean Ϯ 1 SD and range in parentheses unless 43). Egg surface finely pitted, development to morula stage at deposi- otherwise stated. Nematodes were deposited in the United States Na- tion. tional Parasite Collection (USNPC), Beltsville, Maryland. Mature female: Based on 10 individuals with 0–6 eggs present. Length (excluding tail spike) 3.51 Ϯ 0.39 mm (3.07–4.16 mm), width RESULTS at mid-body 536 Ϯ 40 (485–612). Cuticle, depending upon level of focus, with both longitudinal and transverse striations; pseudoannula- Forty-six individuals (13 fourth stage, 14 mature females, 12 tions also present. Lateral alae absent. Esophageal corpus length 458 Ϯ Ϯ Ϯ gravid females, 7 males) assignable to Skrjabinodon, but dis- 21 (415–482), isthmus 11 3 (6–15) long, bulb length 110 7 (98– 122), bulb width 140 Ϯ 8 (122–153). Nerve ring 109 Ϯ 6 (98–116), similar to any current species, were found. Eight (53%) of the excretory pore 635 Ϯ 78 (510–727), and vulva 699 Ϯ 76 (574–591) 15 lizards were infected; the mean intensity for infected hosts from anterior end, respectively. Distance from anus to tip of tail spike, was 5.75 Ϯ 3.45 (2–11). 713 Ϯ 57 (587–765); tail spike 197 Ϯ 23 (153–217) terminating in 3 cuticular spines; occasionally, a flexible filament approximately 200 in length attached between cuticular spines. Thick-walled muscular ovi- Received 29 November 2006; revised 4 January 2006; accepted 15 jector extending posteriorly 406 Ϯ 59 (332–510) in length; then con- February 2007. tinuing as thin-walled vagina 334 Ϯ 68 (255–434) in length joining 2 * Department of Biology, Whittier College, Whittier, California 90608. uteri, 1 uterus directed anteriorly, the other posteriorly.

866 BURSEY AND GOLDBERG—NEW SPECIES OF SKRJABINODON 867

TABLE I. Current list and selected characters of species assigned to Skrjabinodon.

Biogeographical Realm Male Female Skrjabinodon sp. Spicule Tail ﬁlament Egg shape Tail ﬁlament Reference

Australian Realm S. leristae Mawson, 1971 absent spinous not stated spinous Mawson, 1971 S. oedurae (Johnston and 110 12–15 spines not stated 16 spines Johnston and Mawson, 1947 Mawson, 1947) S. parasmythi Mawson, 1971 present spinous not stated spinous Mawson, 1971 S. piankai Bursey and Gold- 51–57 0–2 spines truncate ends 4–5 spines Bursey and Goldberg, 1999 berg, 1999 S. poicilandri Ainsworth, 1990 type1: absent 21–33 spines spindleform 30–38 spines Ainsworth, 1990 type2: 43–48 smooth S. smythi Angel and Mawson, absent few spines spindleform 7–9 spines Angel and Mawson, 1968 1968 S. trimorphi Ainsworth, 1990 type1: absent 3–8 spines spindleform 16–24 spines Ainsworth, 1990 type2: 58–63 smooth Ethiopian Realm S. mabuyae (Sandground, 62 smooth truncate ends smooth Hering-Hagenbeck et al., 2002 1936) S. dossae (Caballero, 1968) absent smooth ovate few spines Caballero, 1968 S. hemidactyli Moravec and 60 smooth not stated 4 spines Moravec and Barus, 1990 Barus, 1990 S. mabuiensis (Malan, 1939) absent smooth truncate ends smooth Malan, 1939 S. megalocerca (Skrjabin, absent smooth oblong-oval smooth Skrjabin et al., 1960 1916) S. ovocaudatus (Caballero, absent smooth with ﬁlament spinous Caballero, 1968 1968) Nearctic Realm Neotropical Realm S. cricosaurae Barus and Coy 37 smooth truncate ends 3–7 spines Barus and Coy Otero, 1974 Otero, 1974 S. dixoni n. sp. absent 1–3 spines oval 3 spines This paper S. heliocostai Vicente, Vrci- absent smooth oval spinous Vicente et al., 2002 bradic, Muniz-Preira and Pinto, 2000 S. spinosulus Vicente, Vrci- 43–50 smooth spindleform spinous Vicente et al., 2000 bradic, Rocha and Pinto, 2002 S. scelopori (Caballero, 1938) 57 smooth spindleform 10–12 spines Moravec et al., 1997 Oriental Realm Palaearctic Realm S. aegyptiacus (Moravec, Barus 51–60 smooth females unknown Moravec et al., 1987 and Rysavy, 1987) S. alcaraziensis Lafuente and 48–56 smooth oblong-oval 7–10 spines Lafuente and Roca, 1995 Roca, 1995 S. apapillosus (Koo, 1938) absent 3 spines spindleform spinous Koo, 1938 S. canariensis (Solera Puertas, 60 smooth oblong-oval spinous Solera-Puertas et al., 1987 Zapatero-Ran Castano- Fernandez and Carrera- Moro, 1987) S. mascomai Roca, 1985 64 smooth spindleform smooth Roca, 1985 S. medinae (Calvente, 1948) 54–80 smooth oblong-oval 3–7 spines Hornero and Roca, 1992 S. pigmentatus (Markov and absent smooth not stated smooth Markov and Bogdanov, 1961 Bogdanov, 1961) S. schikhobalova (Annaev, 67 smooth not stated smooth Annaev, 1973 1973) 868 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 1–11. Skrjabinodon dixoni n. sp. (1) Gravid female, anterior end, lateral view. (2) Mature female, anterior end, lateral view. (3) Gravid female, en face view. (4) Gravid female, anterior end, lateral view. (5) Gravid female, posterior end, lateral view. (6) Mature female, posterior end, lateral view. (7) Fourth stage female, entire, lateral view. (8) Male, entire, lateral view. (9) Eggs. (10) Male, posterior end, ventral view. (11) Male, posterior end, lateral view. BURSEY AND GOLDBERG—NEW SPECIES OF SKRJABINODON 869

Fourth stage female: Based on 10 individuals. Length 1.28 Ϯ 0.11 ria: Gekkonidae: Nephrurus spp.) from Australia. Journal of the mm (1.09–1.47 mm), width at mid-body 214 Ϯ 22 (191–255). Esoph- Helminthological Society of Washington 66: 175–179. ageal corpus length 146 Ϯ 10 (134–165), isthmus 18 Ϯ 2 (15–21) long, CABALLERO, E. 1938. Ne´matodes parasites des reptiles du Mexique. An- bulb length 57 Ϯ 5 (52–67), bulb with 62 Ϯ 4 (55–70). Nerve ring 88 nales de Parasitologie 16: 327–333. Ϯ 5 (79–92), excretory pore 390 Ϯ 26 (332–421), and vulva primor- CABALLERO, R. G. 1968. Contribution a` la connaissance des ne´matodes dium 414 Ϯ 25 (357–434) from anterior end, respectively. Distance de sauriens malgaches. Annales de Parasitologie 43: 149–200. from anus to base of tail spike 117 Ϯ 17 (102–140); distance from anus DIXON,J.R.,AND P. S OINI. 1975. The reptiles of the Upper Amazon Basin, to tip of tail spike 456 Ϯ 48 (383–536). Tail spike with 13 Ϯ 1 (12– Iquitos Region, Peru I. Lizards and amphisbaenians. Milwaukee Public 15) cuticular spines. Museum Contributions in Biology and Geology 4: 1–58. HERING-HAGENBECK,S.F.B.N.,A.J.PETTER, AND J. BOOMKER. 2002. Taxonomic summary Redescription of some Spauligodon spp. and Parapharyngodon spp. and of Skrjabinodon mabuyae (Sandground, 1936) Inglis, 1968 Type host: Uracentron flaviceps (Guichenot, 1855), thornytail iguana, (Pharyngodonidae: Oxyuroidea) from insectivorous South African Iguanidae. Symbiotype: TCWC 44566; collection date: 13 September lizards. Onderstepoort Journal of Veterinary Research 69: 7–29. 1973. HORNERO, M. J., AND V. R OCA. 1992. Redescription of Skrjabinodon mediane (Garcia-Calvente, 1948) (Nematoda: Pharyngodonidae) Type locality from the cloaca of Podarcis pityusensis (Bosca, 1883) (Sauria: Lac- Moropon, on Rıó Nanay (3Њ43ЈS, 73Њ14ЈW), Loreto Department, ertidae) of the Balealraic Islands (Spain). Systematic Parasitology Peru. 23: 31–35. Additional locality: Sucumbio´s Province, Ecuador. JOHNSTON,T.H.,AND P. M . M AWSON. 1947. Some nematodes from Aus- Site of infection: Large intestine. tralian lizards. Transactions of the Royal Society of South Australia Type specimens: Holotype male, USNPC 98997; allotype female, 71: 22–27. USNPC 98998; paratypes 3 males, 8 females, USNPC 98999; voucher KOO, S. Y. 1938. A new species of Pharyngodon (Nematoda: Oxyuri- specimens, USNPC 99000. dae) from Canton lizard, Gekko gecko, with remarks on the evo- Etymology: The new species is named in honor of Dr. James R. lution of the group. Lingnan Science Journal 17: 395–400. Dixon, Professor Emeritus, Department of Wildlife and Fisheries Sci- LAFUENTE,M.,AND V. R OCA. 1995. Description of Skrjabinodon alcar- ences, Texas A&M University, College Station, Texas, for his many aziensis sp. n. (Nematoda: Pharyngodonidae), a parasite of Algy- contributions to the herpetofauna of South America. roides marchi (Sauria: Lacaertidae). Folia Parasitologica 42: 130– 134. Remarks MALAN, J. R. 1939. Some helminths of South African lizards. Onder- stepoort Journal of Veterinary Science and Animal Industry 12: Skrjabinodon dixoni n. sp. is most similar to those species lacking a 21–74. spicule (see Table I), namely, S. apapillosus (Koo, 1938), S. dossae MARKOV,G.S.,AND O. P. BOGDANOV. 1961. [Parasites of desert lizards (Caballero, 1968), S. heliocostai Vicente, Vrcibradic, Muniz-Pereira and in Central Asia.] Uchenye Zapiski Stalinradsk Gosudarstve Peda- Pinto, 2000, S. leristae Mawson, 1971, S. mabuiensis (Malan, 1939), S. gogicheskii Instytut 13: 101–123. megalocerca (Skrjabin, 1916), S. ovocaudatus Caballero, 1968, S. pig- MAWSON, P. M. 1971. Pearson Island Expedition 1969. 8. Helminths. mentatus Markov and Bogdanov, 1961, and S. smythi Angel and Maw- Transactions of the Royal Society of South Australia 95: 169–183. son, 1968. Of these, only males of S. dixoni, S. leristae, and S. smythi MORAVEC,F.,AND V. B ARUS. 1990. Some nematode parasites from am- have cuticular spines on the tail filament. Tail processes of females of phibians and reptiles from Zambia and Uganda. Acta Societatis S. leristae and S. smythi are filamentous and have cuticular spines along Zoologicae Bohemoslovacae 54: 177–192. the body of the filament; the tail filament of females of S. dixoni n. sp. ———, ———, AND B. RYSAVY. 1987. On parasitic nematodes of the is a relative short nonflexible spike with 3 terminal spines. Skrjabinodon families Heterakidae and Pharyngodonidae from reptiles in Egypt. dixoni is currently the only species assigned to Skrjabinodon in which Folia Parasitologica 34: 269–280. the tail of the mature female does not end in a flexible filament. ———, G. SALGADO-MALONADO, AND E. MAYEN-PENA. 1997. Thubu- naea ctenosauri sp. n. (Nematoda: Physalopteridae) from the iquan- ACKNOWLEDGMENTS id lizard Ctenosaura pectinata and other lizard helminths from Mexico. Journal of the Helminthological Society of Washington 64: We thank Toby Hibbits (Department of Wildlife and Fisheries, Texas 240–247. A&M University, College Station, Texas), Laurie J. Vitt (Sam Noble ROCA, V. 1985. Skrjabinodon mascomai n. sp. (Nematoda: Pharyngo- Museum of Natural History and Zoology Department, University of donidae), parasite of Tarentola mauritanica (Linnaeus, 1758) Gray, Oklahoma, Norman, Oklahoma) for permission to examine specimens, 1845 (Reptilia: Geckonidae) in Valencia (Spain). Rivista di Paras- and Peggy Firth for illustrations 1–11. Sarah Goldsberry, Tenzing Do- sitologia 46: 27–31. leck, and Lauren Shellard assisted with dissections. SKRJABIN, K. I., N. P. SHIKHOBALOVA, AND E. A. LAGODOVSKAYA. 1960. Oxyurata of animals and man. Part one. Oxyuroidea. Izdatel’stvo LITERATURE CITED Akademii Nauk SSSR Moscow [English translation by Amerind Publishing Co, New Delhi, India 1982], 526 p. AINSWORTH, R. 1990. Male dimorphism in two new species of nematode SOLERA-PUERTAS, M. A., L. M. ZAPATERO-RAMOS,C.CASTANO-FERNAN- (Pharyngodonidae: Oxyurida) from New Zealand lizards. Journal DEZ, AND M. P. CARRERA-MORO. 1987. Parathelandros canariensis of Parasitology 76: 812–822. n. sp. (Nematoda: Pharyngodonidae) parasito de Chalcides viri- ANGEL,L.M.,AND P. M . M AWSON. 1968. Helminths from some lizards danus Boulenger, 1887 (Reptilia: Scincidae). Rivista Iberica de Par- mostly from South Australia. Transactions of the Royal Society of asitologia 47: 57–63. South Australia 92: 59–71. VICENTE, J. J., D. VRCIBRADIC,L.C.MUNIZ-PEREIRA, AND P. M . P INTO. ANNAEV, D. 1973. [Parathelandros schikhobalovi n. sp.—a new oxyurid 2000. Skrjabinodon heliocostai sp. n. (Nematoda, Pharyngodoni- nematoda from Eremias intermedia in Turkmenistan.] Izvestiya dae) parasitizing Mabauya frenata (Cope) (Lacertilia, Scincidae) in Akademia Nauk Turkmenistan SSR 4: 85–89. Brazil and the reallocation of Skrjabinodon capacyupanquii (Frei- AVILA-PIRES, T. C. S. 1995. Lizards of Brazilian Amazonia (Reptilia: tas, Vicente & Ibanez) in the genus Thelandros Wedl. Revista Bras- Squamata). Zoologische Verhandelingen, Nationaal Natuurhisto- ileira de Zoologia 17: 361–367. risch Museum, Leiden, Netherlands, 706 p. ———, ———, C. F. D. ROCHA, AND R. M. PINTO. 2002. Description BARUS,V.,AND A. COY OTERO. 1974. Nematodes of the genera Spau- of Skrjabinodon spinosulus sp. n. (Nematoda, Oxyuroidea, Phar- lingodon, Skrjabinodon, and Pharyngodon (Oxyuridae) parasitizing yngodonidae) from the Brazilian lizard Mabuya dorsivittata Cope, Cuban lizards. Ceskoslovenska Spolecnost Zoologika Vestnik 38: 1862 (Scincidae). Revista Brasileira de Zoologia 19: 157–162. 1–12. VITT, L. J., AND P. A . Z ANI. 1996. Ecology of the elusive tropical lizard BURSEY, C. R., AND S. R. GOLDBERG. 1999. Skrjabinodon piankai sp. n. Tropidurus [ϭ Uracentron] flaviceps (Tropiduridae) in lowland rain (Nematoda: Pharyngodonidae) and other helminths of geckos (Sau- forest of Ecuador. Herpetologica 52: 121–132. J. Parasitol., 93(4), 2007, pp. 870–873 ᭧ American Society of Parasitologists 2007

REDESCRIPTION, SYNONYMY, AND NEW RECORDS OF VEXILLATA NOVIBERIAE (DIKMANS, 1935) (NEMATODA: TRICHOSTRONGYLINA), A PARASITE OF RABBITS SYLVILAGUS SPP. (LEPORIDAE) IN THE UNITED STATES

Marıá Celina Digiani, John M. Kinsella*, Thomas B. Kass†, and Marie-Claude Durette-Desset‡ Divisioń Zoologıá Invertebrados, Museo de La Plata, Paseo del Bosque s/n 1900 La Plata, Argentina, CONICET. e-mail: [email protected]

ABSTRACT: Vexillata noviberiae (Dikmans, 1935) (Trichostrongylina: Heligmosomoidea), originally described as a parasite of Sylvilagus floridanus from Louisiana, is redescribed from material collected from Sylvilagus palustris in Florida and from S. floridanus in Kansas. New morphometric and morphological data are provided. Stunkardionema halla Arnold, 1941, described from S. floridanus from Kansas and New York, is proposed as a junior synonym of V. noviberiae. These findings confirm the occurrence of V. noviberiae as a parasite of rabbits and its wide distribution range in North America.

Species of Vexillata (Nematoda, Trichostrongylina) have usu- situated near esophagointestinal junction. Deirids at same level as ex- ally been reported as parasites of geomyid and heteromyid ro- cretory pore or slightly anterior or posterior (Fig. 1). Head: Cephalic vesicle present. In apical view, triangular buccal dents in North and South America (Guerrero, 1984; Durette- opening surrounded by small ring. Presence of 2 amphids, 6 externo- Desset and Digiani, 2005a; Falcoń-Ordaz et al., 2006). Only 1 labial papillae, and 4 submedian cephalic papillae. Small dorsal tooth species, Vexillata noviberiae (Dikmans, 1935), has been represent (Figs. 5 and 6). ported as parasitic in lagomorphs (Sylvilagus spp.: Leporidae) Synlophe (studied in 1 male, 1 female from S. floridanus and 1 male, from Louisiana. Originally described as Longistriata noviberiae 2 females from S. palustris): In both sexes, body bears continuous cu- Dikmans, 1935, it was transferred to Vexillata (Ornithostron- ticular ridges, appearing at different levels mainly on right side, between cephalic vesicle and nerve ring (Figs. 12–14), and disappearing anterior gylidae) by Durette-Desset and Digiani (2005a) on the basis of to caudal bursa in male. In female, ventral ridges disappearing or fusing type material from Sylvilagus floridanus (Allen, 1890). Further at different levels posterior to sphincter. Carene made up of 2 ridges, findings of specimens identified as V. noviberiae in Sylvilagus ventral one slightly more developed at midbody. Number of ridges at palustris (Bachman, 1837) from Florida and in S. floridanus midbody: 13 (carene, 5 dorsal, 6 ventral). At midbody, double axis of from Kansas allow us to provide a redescription of the species orientation of ridges directed from right ventral quadrant to left dorsal Њ Њ and to confirm its wide distribution as a parasite of lagomorphs. quadrant. Right axis inclined at 80 on sagittal axis in male and 67–71 Stunkardionema halla Arnold, 1941, a species described from in female. Left axis subfrontal in both sexes (Figs. 2 and 3, 10 and 11). In females, synlophe modified at ovejector level, with disappearance or S. floridanus from Kansas and New York, seems to correspond fusion of ridges, mainly on lateral and ventral sides (Figs. 17 and 18, to V. noviberiae and is proposed as a junior synonym. 20–24). Two lateral ridges appear posterior to anus (Fig. 9). Males (average and range of measurements of 9 specimens from S. MATERIALS AND METHODS palustris): Length 5.3 (4.3–6.3) mm and width 64 (50–80) at midbody; Nematode specimens from S. palustris were collected by J.M.K. and cephalic vesicle 61 (53–66) long and 30 (25–33) wide; nerve ring, those from S. floridanus by T.B.K. The material was deposited in the excretory pore, and deirids at 185 (177–210), 306 (270–320) and 311 Helminthological Collections of the Museúm National d’Histoire Na- (270–330) from apex, respectively; esophagus 294 (265–315) long. turelle, Paris, France (MNHN) and the Museo de La Plata, La Plata, Caudal bursa with pattern of type 2-2-1. Rays 2 and 3 in V-formation Argentina (CHMLP). The synlophe was studied following the method and of equivalent size. Rays 4 longer than rays 5. Rays 8 arising from of Durette-Desset (1985). The nomenclature used for the study of the basal third of dorsal ray. Dorsal ray divided into 2 branches at its half, synlophe follows Durette-Desset and Digiani (2005b) and that of the each branch giving rise to 2 branches of equivalent length, rays 9 (ex- caudal bursa Durette-Desset and Chabaud (1981). Cuticular ridges of ternal) and rays 10 (internal) (Fig. 7). Spicules 510 (460–670) long. the synlophe at midbody were numbered 1–6 from left to right for the Ratio spicule length/body length: 9.6 (8.0–10.9)%. Gubernaculum 23 dorsal ridges and as 1Ј–7Ј from left to right for the ventral ridges. Mea- (20–30) long and 14 (12–20) wide. surements are given in micrometers except where stated otherwise. The parasite classification used above the family group level is that of Dur- Females (average and range of measurements of 5 specimens from ette-Desset and Chabaud (1993) and the nomenclature of the hosts at S. palustris): Length 7.6 (5.8–9.2) mm and width 60 (50–80); cephalic the species level follows Wilson and Reeder (1993). vesicle 58 (52–65) long and 33 (28–38) wide; nerve ring, excretory pore, and deirids situated at 190 (170–210), 350 (330–365), and 347 DESCRIPTION (330–365) from apex, respectively; esophagus 330 (300, 375) long. Vul- va situated at 130 (120–140) from caudal extremity. Vagina vera 41 Vexillata noviberiae (Dikmans, 1935) (40–45) long. Vestibule 141 (130–150) long, divided into 2 parts by a Durette-Desset and Digiani, 2005a slight constriction, sphincter 37 (30–40) long and 44 (40–45) wide, Longistriata noviberiae Dikmans, 1935 ؍ infundibulum 133 (115–145) long (Fig. 8). Uterus 1.24 (1.13–1.47) mm ؍ Stunkardionema halla Arnold, 1941 long with 34 (24–56) eggs, 62 (40–72) long and 31 (25–38) wide. Ratio (Heligmostrongylus hallus (Arnold, 1941 ؍ uterus length/body length 17 (15.7–19.7)%. Tail conical, 48 (38–55) Durette-Desset, 1978 (Figs. 1–26) long. Females (average and range of measurements of 4 fragmented spec- General: Small nematodes, loosely coiled on ventral side following imens from S. floridanus): Length (n ϭ 2) 5.35–6.75, width 80 (75– 2–4 (usually 3) spires in males and 4–6 in females. Excretory pore 90); cephalic vesicle (n ϭ 3): 71 (52–80) long and 38 (30–45) wide; nerve ring, excretory pore, and deirids (n ϭ 1) at 210, 365 and 365 Received 12 December 2006; revised 31 January 2007; accepted 31 from apex, respectively; esophagus (n ϭ 2) 345–380 long. Vulva (n ϭ January 2007. 3) situated at 136 (130–142) from caudal extremity. Vagina vera (n ϭ * 2108 Hilda Avenue, Missoula, Montana 59801. 3) 33 (30–40) long. Ovejector (n ϭ 3) with bipartite vestibule 155 † 1732 Highway 71, Wall Township, New Jersey 07719. (140–165) long, sphincter 35 (30–45) long and 44 (38–50) wide, in- ‡De´partement de Syste´matique et Evolution, Museúm national fundibulum 145 (135–160) long. Uterus (n ϭ 3) 892 (585–1270) long d’Histoire naturelle, UMR 7138 associeé au CNRS, CP 52, 61, rue with 34 (24–43) eggs, 49 (45–52) long and 28 (24–30) wide. Tail (n Buffon 75231 Paris Cedex 05, France. ϭ 3) 45 (45–45) long.

870 DIGIANI ET AL.— REDESCRIPTION OF V. NOVIBERIAE 871

FIGURES 1–9. Vexillata noviberiae (Dikmans, 1935). Material from Sylvilagus ﬂoridanus (Kansas). (1) Female, anterior extremity, right lateral view. Transverse sections at midbody; (2) male, (3) female. (4) Male posterior extremity, right laterodorsal view. Material from Sylvilagus palustris (Florida). Female head; (5) apical view, (6) left lateral view. (7) Male caudal bursa, ventral view. (8) Female posterior extremity, left lateral view. (9) Female tail, right lateral view. Abbreviations: R, right side; V, ventral side. All sections orientated as in Figure 2. 872 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 10–26. Vexillata noviberiae (Dikmans, 1935) from Sylvilagus palustris (Florida). Transverse body sections; (10 and 11) at midbody, (10) male, (11) female. Female; (12) just posterior to cephalic vesicle, (13)at40␮m posterior to cephalic vesicle, (14)at80␮m posterior to cephalic vesicle, (15) at esophagointestinal junction, (16) at 2 mm from posterior extremity, (17) at level of proximal vestibule, (18) at level of vagina vera. Another female; (19) at midlength of uterus. At different levels of ovejector; (20) proximal sphincter, (21) distal sphincter, (22) proximal vestibule, (23) distal vestibule, (24) at level of vulva, (25) posterior to vulva, (26) between vulva and anus. Abbreviations: R, right side; V, ventral side. All sections orientated as in Figure 10. Arrows indicate the appearance of ridges. DIGIANI ET AL.— REDESCRIPTION OF V. NOVIBERIAE 873

Taxonomic summary Sylvilagus floridanus, the eastern cottontail, is the North Amer- Hosts: Sylvilagus floridanus (Allen, 1890) (Leporidae) and S. pal- ican species of Sylvilagus with the largest geographical range ustris (Bachman, 1837) (Leporidae). and is probably the most frequently captured species (Wilson Site: Small intestine. and Reeder, 1993). It will be interesting to confirm whether V. Geographic origin: Kansas and Florida. Material studied: One male (posterior extremity), 4 females MNHN noviberiae can be found in the rest of the geographical range 341 MC; 3 males, 2 females CHMLP 5638; and 6 males, 3 females of S. floridanus, which reaches the northern part of South MNHN 407 MQ. America, and, especially, in other species of Sylvilagus with a more restricted distribution but with populations still common Remarks or abundant, such as those inhabiting the western part of the By the characters of the synlophe, caudal bursa, and female ovejector, United States (Sylvilagus audubonii, Sylvilagus nuttallii) (Wil- the specimens studied here correspond undoubtedly to V. noviberiae as son and Reeder, 1993). Parasitological studies cannot be fore- described by Dikmans (1935) and Durette-Desset and Digiani (2005a) seen in several endemic species of Sylvilagus from Mexico, in S. floridanus from Louisiana (types). Only the body measurements show slight differences, these specimens being larger compared to the whose populations are endangered (Sylvilagus graysoni, Sylvi- type material. Rays 9 of the caudal bursa, described as shorter than rays lagus insonus) (Chapman and Flux, 1990). 10 by Durette-Desset and Digiani (2005a) were observed as having equivalent length in these specimens, corresponding to the first obser- ACKNOWLEDGMENTS vations by Dikmans (1935) (Fig. 7). In the females from both hosts, 2 lateral ridges were observed on the tail (Fig. 9), which were probably The authors wish to thank Eric P. Hoberg and Patricia Pilitt, from the overlooked in the examination of the type material. The synlophe at USNPC, for providing information on the types of S. halla. ovejector level is also described for the first time. Stunkardionema Arnold, 1941, and its only species Stunkardionema halla Arnold, 1941, were described for a nematode parasitic in the cot- LITERATURE CITED tontail S. floridanus from Kansas and New York (Arnold, 1941). The ARNOLD,J.G.JR., 1941. A new rabbit nematode, Stunkardionema halla. type material of this species was studied by Durette-Desset (1978), who Transactions of the American Microscopical Society 60: 65–68. provided a description of the female ovejector and some observations CHAPMAN,J.A.,AND J. E. C. FLUX. 1990. Rabbits, hares, and pikas: A on the synlophe. This latter could not be properly studied because the status survey and action plan. IUCN/SSC Lagomorph Specialist material was mounted on slides (the only specimens of S. halla in the Group. IUCN/SSC, Gland, Switzerland and Cambridge, U.K., 168 p. U.S. National Parasite Collection, type and paratype, are both on slides DIKMANS, G. 1935. New nematodes of the genus Longistriata in rodents. [P. Pilitt, pers. comm.]). Durette-Desset (1978) described the synlophe Journal of Washington Academy of Sciences 25: 72–81. of S. halla as having 2 left ridges forming a carene, 5 dorsal and 5 DURETTE-DESSET, M.-C. 1978. Nouvelles donneés morphologiques sur ventral ridges, all continuous, and proposed transferring the species to des Ne´matodes Trichostrongyloides des collections du United Heligmostrongylus (Heligmonellidae: Pudicinae). States National Museum. Bulletin du Museúm National d’Histoire The original description and figures by Arnold (1941), as well as the Naturelle, 3e`me se´r., No. 513, Zoologie 352: 135–147. redescription given by Durette-Desset (1978), seem to correspond well ———. 1985. Trichostrongyloid nematodes and their vertebrate hosts: to the specimens studied by Dikmans (1935), Durette-Desset and Di- Reconstruction of the phylogeny of a parasitic group. Advances in giani (2005a), and in this article. Moreover, part of the material studied Parasitology 24: 239–306. in this article (MNHN 341 MC) comes from the same host species and ———, AND A. G. CHABAUD. 1981. Nouvel essai de classification des geographical area as the type material of S. halla. Similarities are evi- Ne´matodes Trichostrongyloidea. Annales de Parasitologie Humaine dent for the body measurements, female ovejector, and caudal bursa. et Compareé 56: 297–312. The only slight difference is in the number of ridges, described by ———, AND ———. 1993. Note sur la nomenclature suprafamiliale Durette-Desset (1978) as 12. However, it must be taken into account des Strongylida. Annales de Parasitologie Humaine et Compareé that the type specimen was mounted and illustrated in median view. In 68: 11–12. this position, the right ridges 6Ј and 7Ј (Figs. 2 and 3) overlap and ———, AND M. C. DIGIANI. 2005a. Systematic position of some Ne- cannot be differentiated from each other, and then they were probably arctic Heligmosomoidea (Nematoda, Trichostrongylina) from the counted as 1. It is very likely that the species described by Arnold U.S. National Parasite Collection and their description. Journal of (1941) as S. halla and that described by Dikmans (1935) as Longistriata Parasitology 91: 893–899. noviberiae, refer to the same taxon. Stunkardionema halla (ϭHelig- ———, AND ———. 2005b. The axis of orientation of the synlophe in mostrongylus hallus) is thus proposed as a junior synonym of V. novi- the Heligmosomoidea (Nematoda, Trichostrongylina): A new ap- beriae. proach. Parasite 12: 195–202. FALCO´ N-ORDAZ, J., H. CHEN, AND R. LAMOTHE-ARGUMEDO. 2006. A new DISCUSSION species of Vexillata (Nematoda: Ornithostrongylidae) in Attwater’s pocket gopher from Texas. Journal of Parasitology 92: 595–599. The known distribution range of V. noviberiae is enlarged GUERRERO, R. 1984. Nematoda: Trichostrongyloidea parasites of Ve- with these findings to include Kansas, New York, and southern nezuelan mammals. III. The genus Vexillata Travassos, 1937. An- nales de Parasitologie Humaine et Compareé 59: 253–261. Florida, and it seems that the species is a typical parasite of WILSON,D.E,AND D. A. M. REEDER. 1993. Mammal species of the rabbits. Its range is apparently coincident with the geographical world. A taxonomic and geographic reference. Smithsonian Insti- ranges of S. palustris and S. floridanus in the United States. tution Press, Washington, DC, 1207 p. J. Parasitol., 93(4), 2007, pp. 875–883 ᭧ American Society of Parasitologists 2007

A COLLECTION OF FISH LEECHES (HIRUDINIDA: PISCICOLIDAE) FROM JAPAN AND SURROUNDING WATERS, INCLUDING REDESCRIPTIONS OF THREE SPECIES

Sharon Furiness, Julianne I. Williams, Kazuya Nagasawa*, and Eugene M. Burreson† Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062. e-mail: [email protected]

ABSTRACT: Leeches were observed incidentally on fishes in collections made from 1975 to 2006 in Japan and surrounding waters, or from mariculture facilities or public aquaria in Japan. Seven species of leeches in 7 genera were collected—Crango- nobdella maculosa, Johanssonia arctica, Limnotrachelobdella okae, Platybdella olriki, Stibarobdella bimaculata, Taimenobdella amurensis, and Trachelobdella livanovi. The transfer of Calliobdella livanovi to Trachelobdella is supported, and Trachelobdella livanovi and Taimenobdella amurensis are redescribed based on new specimens. Stibarobdella bimaculata is synonymized with Stibarobdella macrothela based on eyes, tubercle patterns, and sucker size ratios. Taimenobdella amurensis, C. maculosa, J. arctica, S. macrothela, and P. olriki are reported for the first time from Japan. New hosts are reported for L. okae, T. livanovi, S. macrothela, C. maculosa, J. arctica, and P. olriki. Stibarobdella moorei was not collected during this study, but a well- preserved specimen collected in Japan was discovered in the Museúm National d’Histoire Naturelle, Paris, France, and it allowed a redescription of this species. Stibarobdella loricata is synonymized with S. moorei based on tubercle patterns and the presence of papillae and a marginal fringe on the oral sucker.

Oka (1910) described 10 new species of marine fish leeches In this article, we report on leeches that were observed in- from Japan. Unfortunately, but typical of the time, most of his cidentally on fishes from collections made from 1975 to 2006 descriptions were brief, lacked illustrations, and were inade- in Japan and surrounding waters, or from mariculture facilities quate for actually distinguishing or identifying the species in or public aquaria in Japan. Emended generic and/or species de- question. Subsequently, Oka (1927a, 1933a) described 2 addi- scriptions are provided for Trachelobdella livanovi (Oka, 1910) tional new species and also provided additional morphological and Taimenobdella amurensis (Epshtein, 1964) based on new data for some species described in 1910 (Oka, 1927b, 1927c, information available from the collections for these species. All 1927d, 1927e, 1928, 1931, 1933b). In a review of the Japanese leeches in the collection are reported, some representing new leeches in 1965, only 7 marine fish leeches were listed (Oka records for Japan or new fish hosts for the species. In addition, and Nagao, 1965). The following species described by Oka a specimen of Stibarobdella moorei (Oka, 1910) from Japan (1910) were not mentioned in the review: Pontobdella tateja- was discovered in the Museúm National d’Histoire Naturelle mensis Oka 1910, Ichthyobdella pagri Oka 1910, Ichthyobdella (MNHN), Paris, France, which allowed a redescription of that virgata Oka 1910, Carcinobdella tigrina Oka 1910, and Car- species. cinobdella bimaculata Oka 1910. It is now known (Epshtein, 1967) that Oka’s I. virgata is a valid species in the genus Hep- MATERIALS AND METHODS tacyclus Vasileyev 1939, but the others are species inquirendae. Table I lists the current status of species Oka described and Seven species of leeches were discovered during numerous fish collections from Japan and surrounding waters, or from fish mariculture other fish leeches reported from Japan before the present study. facilities or public aquaria in Japan. Leeches were not collected as part Trachelobdella okae Moore 1924 was described for a leech of a systematic survey for fish leeches, they were observed incidentally improperly identified by Oka (1910) as Trachelobdella sinensis in collections made for other purposes. Leeches from Tottori Prefectural Blanchard 1896; this species was transferred to Limnotrache- Fisheries Research Center and Sunpiazza Aquarium were collected by staff members and preserved in 10% formalin without relaxation. lobdella Epshtein 1968 by Sawyer (1986). As far as we know, Leeches from other locations in Japan, except for T. amurensis, were no new species of fish leeches were described from Japan for collected by K.N. Specimens of T. amurensis were collected by Tetsuya Ͼ70 yr after L. okae (Moore, 1924). In the late 1990s, Ocean- Itoh, Hokkaido University, and they were preserved in 10% formalin obdella alba Epshtein and Utevsky 1996 was described from or 70% ethanol without relaxation. Leeches from Terpenia Bay, Sakha- marine fishes off the coast of Hokkaido; Cottobdella epshteini lin Island, were found on Icelus cataphractus (Pavlenko, 1910) (Cotti- dae) collected by and housed in the Hokkaido Fisheries Experimental Utevsky 1997 was described from the Sea of Japan; and sub- Station. These fishes had been preserved in 70% ethanol; leeches were sequently, Rhopalobdella japonica Burreson and Kearn 2000 removed from the preserved fish hosts and placed in separate vials of was described from rays collected off the south coast of Honshu 70% ethanol. The crab Chionoecetes japonicus Rathbun 1932 (Majidae) (Epshtein and Utevsky, 1996; Utevsky, 1997; Burreson and was collected by the personnel of the Hokkaido Fisheries Experimental Station in a crab pot off Shikabe, Hokkaido; leeches were removed from Kearn, 2000). The latter species was synonymized with Pter- the carapace and preserved in 10% formalin without relaxation. obdella amara Kaburaki 1921 (Burreson, 2006). Two other spe- Specimens of T. livanovi, T. amurensis, C. maculosa, Platybdella cies, Crangonobdella orientalis Utevsky 1999 and Crangonob- olriki Malm 1865, and Johanssonia arctica (Johansson, 1898) were sec- della maculosa Utevsky 2005, described from the southern Ku- tioned to determine internal anatomy for aid in identification. Specimens ril Islands and the Tatar Strait, respectively (Utevsky, 1999, were infiltrated with paraffin on an automated tissue processor and embedded in paraffin on end in stainless steel tissue molds. Serial trans- 2005), probably can be expected to occur in northern Japan. verse sections were cut at 5 ␮m on a rotary microtome. Usually, only the clitellar region and first few segments of the urosome were sectioned; but in some cases, the entire leech was sectioned. Sections were Received 23 June 2006; revised 2 February 2007; accepted 8 February mounted on glass slides by floating ribbons of sections on a puddle of 2007. 2% formalin on slides coated with Szombathy’s affixative. Slides were * Laboratory of Aquaculture, Graduate School of Biosphere Science, air-dried in an oven at 42 C overnight, and then they were deparaffinized Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739- and stained with hematoxylin and eosin on an automated slide stainer. 8528, Japan. All sectioned material remains in E.M.B.’s personal collection. † To whom correspondence should be addressed. All leech specimens, except where noted, have been deposited in the

875 876 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Fish leeches reported from Japan and surrounding waters prior to the present study.

Accepted name Originally reported name Reference for name change

Trachelobdella livanovi (Oka, 1910) Callobdella livanovi Oka, 1910 Oka and Nagao (1965); this study Trachelobdella livanovi (Oka, 1910) Callobdella hastae Oka, 1910 Oka (1928); this study Species inquirenda Carcinobdella bimaculata Oka, 1933 Notostomum cyclostomum Johansson, 1898 Carcinobdella kanibir Oka, 1910 Vasiliev (1939) Species inquirenda Carcinobdella tigrina Oka, 1910 Cottobdella epshteini Utevsky, 1997 Cottobdella epshteini Utevsky, 1997 Oceanobdella alba (Epshtein and Utevsky, 1996) Heptacyclus albus Epshtein and Utevsky, 1996 Utevsky (2003) Species inquirenda Ichthyobdella pagri Oka, 1910 Beringobdella rectangulata (Levinsen, 1881) Ichthyobdella uobir Oka, 1910 Epshtein (1967) Heptacyclus virgatus (Oka, 1910) Ichthyobdella virgata Oka, 1910 Vasileyev (1939); Epshtein (1967) Ostreobdella kakibir Oka, 1927 Ostreobdella kakibir Oka, 1927 Piscicola geometra L., 1761 Piscicola geometra L., 1761 Stibarobdella macrothela (Schmarda, 1861) Pontobdella bimaculata Oka, 1910 This study Stibarobdella moorei (Oka, 1910) Pontobdella moorei Oka, 1910 This study Species inquirenda Pontobdella tatejamensis Oka, 1910 Pterobdella amara Kaburaki, 1921 Rhopalobdella japonica Burreson and Kearn, 2001 Burreson (2006) Limnotrachelobdella okae (Moore, 1924) Trachelobdella sinensis Blanchard, 1896 Moore (1924); Sawyer (1986)

collection of Annelida (An) at the National Science Museum, Tokyo, examined for this study; all other information was from the literature Japan (NSMT-An). Common and scientific names of fishes follow those (Ingram, 1957; Burreson, 1976; Meyer and Burreson, 1990). recommended by Froese and Pauly (2006). Trachelobdella livanovi (Oka, 1910) DESCRIPTIONS (Figs. 1–6) Trachelobdella Diesing, 1850 ϭCallobdella livanovi Oka 1910 ϭCallobdella hastae Oka 1910 Emended description Diagnosis: Body slightly flattened. Oral sucker large, eccentric. Cau- Emended description dal sucker not wider than widest part of body, terminal. Eyes variable. External morphology: Maximum body length 30 mm; maximum Body distinctly divided into trachelosome and urosome. Urosome with width 4 mm (Fig. 1). Oral sucker eccentrically attached (Fig. 2). Caudal 10–13 pairs of large lateral pulsatile vesicles. Mycetomes present or sucker deeply cupped, facing directly posteriorly (Fig. 3). No eyes or absent. Five to 6 pairs of testisacs. Atrium covered with accessory gland caudal ocelli. Clitellum swollen in region of male gonopore. Urosome cells. Muscular vagina. Conducting tissue absent. Muscular organ be- with 12 pairs large pulsatile vesicles; 3(6) annulate. Pigmentation varies tween atrium and bursa weakly developed or absent. Postceca fused from yellow to brown to dark green. Body translucent in preserved with or without fenestrae. specimens; gland cells visible over entire urosome. Digestive system: Mouthpore located in center of oral sucker. Pro- Remarks boscis extends just anterior to ganglion IX. Mycetomes connect to According to Sawyer (1986), Trachelobdella species are character- esophagus at XI through multiple ducts, extend anteriorly to VII. Crop ized by the absence of conducting tissue, oviducts that unite to form a expands at ganglia, alternating with testisacs. Postceca fused with 3 true muscular vagina before opening as the female pore, gland cells on small fenestrae. Rectum large. the atrium, and a distinct division between the trachelosome and uro- Reproductive system: Five pairs of testisacs intersegmentally from some. However, many other characters of the genus are variable among XIV/XV to XVIII/XIX, alternating with gastric ceca. Vasa deferentia the species, including the number of pulsatile vesicles, the presence of expand in XII and XIII, entering convoluted, tightly coiled epididymis, eyes, and the number of testisacs (Burreson, 1976). The diagnosis of then constrict. Vasa deferentia enlarge as ejaculatory bulbs, bend ven- Trachelobdella from Epshtein (1973) and Sawyer (1986) is changed trally in anterior portion of X, entering atrial cornua. Small, narrow from being constricted to eyes absent to eyes present or absent, the lumina of atrial cornua merge to form common atrium midway through number of pulsatile vesicles to range from 10 to 13 instead of 12 to 13, XI. Thin, long atrium opens into large, eversible bursa with anterior and for mycetomes to be present or absent versus solely absent. Trach- lobe. Accessory gland cells cover ejaculatory ducts, atrial cornua, and elobdella oregonensis Burreson 1976 has 1 pair of eyes, and Trache- project as lobes off posterior end of bursa (Figs. 4, 5). Female pore lobdella leptocephali Ingram 1957 has either 1 or 2 pairs of eyes. Trach- opens to long muscular vagina. Vagina in ventral muscle layer in XII/ elobdella oregonensis has 10 pairs of pulsatile vesicles and Trachelob- XIII, emerging and bifurcating into paired, elongate, multilobed ovisacs della bathyrajae Meyer and Burreson 1990 has 11 pairs. Mycetomes (Figs. 4, 5) that reach XV. Conducting tissue absent. are present in T. bathyrajae, T. livanovi, and T. oregonensis. Trache- Coelomic system: Twelve pairs large pulsatile vesicles. Dorsal, ven- lobdella may, in fact, be a composite genus composed of multiple gen- tral, lateral, and testicular sinuses present. Segmental connection be- era, because some of these variable characters, such as number of tes- tween lateral sinus, ventral sinus, and pulsatile vesicle. Intersegmental tisacs, are invariant in other genera. Only specimens of T. livanovi were connection between dorsal, testicular, and lateral sinuses (Fig. 6).

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FIGURES 1–6. Trachelobdella livanovi.(1) External morphology, dorsal view. (2) Oral sucker, lateral view. (3) Caudal sucker, lateral view. (4) Reconstruction of reproductive systems, dorsal view. (5) Reconstruction of reproductive systems, lateral view. (6) Reconstruction of coelomic system, right side segmental, left side intersegmental. A, atrium; B, bursa; D, dorsal sinus; ED, ejaculatory duct; F, female gonopore; G, accessory gland cells; L, lateral sinus; M, male gonopore; O, ovisac; PV, pulsatile vesicle; T, testisac; V, ventral sinus. FURINESS ET AL.—FISH LEECHES FROM JAPAN 877 878 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

Specimens examined of pulsatile vesicles (Ingram, 1957) and a pair of eyes (Meyer and Burreson, 1990) that separate it from T. livanovi. Trachelobdella lep- NSMT-An 344: Tottori Prefectural Fisheries Research Center, Yuri- tocephali can be differentiated from T. livanovi because it has 13 pairs hama, Tottori, Japan; 6 August 1987; host bastard halibut, Paralichthys of pulsatile vesicles and completely fused postceca with no fenestrae olivaceus (Temminck and Schlegel, 1846) (Paralichthyidae), new host (Ingram, 1957). The remaining species of Trachelobdella, i.e., Trach- record; infection site skin; 78 specimens (4 specimens sectioned remain elobdella muelleri (Diesing, 1850) and Trachelobdella luederitzi (Au- in E.M.B.’s personal collection). gener, 1936), were described from external anatomy only, and they are NSMT-An 345: Toyama Bay in the Sea of Japan, off Namerikawa, inadequately distinguished from T. lubrica. Toyama, Japan; 29 September 1992; host P. olivaceus; infection site caudal fin; 2 specimens. NSMT-An 346: Moroiso Bay in the North Pacific Ocean, Miura, Kan- Taimenobdella Epshtein, 1987 agawa, Japan; 21 December 1979; host puffer, Takifugu pardalis (Tem- Emended description minck and Schlegel, 1850) (Tetraodontidae), new host record; infection site skin; 1 specimen. Diagnosis: Body slightly flattened. Suckers well developed. Pulsatile NSMT-An 347: Exact location in Japan unknown; 7 January 1985; vesicles present. Two pairs of eyes on oral sucker. Segmental and caudal host black porgy, Acanthopagrus schlegelii schlegelii (Bleeker, 1854) ocelli present. Urosomal segments 7(14) annulate. Mycetomes present. (Sparidae), new host record; infection site unknown; 1 specimen. Six pairs of testisacs. Extremely large, muscular organ associated with NSMT-An 348: Seto Inland Sea off Shimojima, Shimokamagari, bursa. Ovisacs long. Conducting tissue present. Kure, Hiroshima, Japan; 28 December 2005; host A. schlegelii schlegelii; infection site gill cavity wall; 1 specimen. Remarks NSMT-An 349: Hiroshima Bay in the Seto Inland Sea, Hiroshima, In the original description, Epshtein (1987) described the genus as Japan; 17 May 2006; host A. schlegelii schlegelii; infection site gill having only 5 pairs of testisacs and no conducting tissue. The genus cavity wall; 1 specimen. diagnosis is emended to include features seen in newly collected specimens. The most notable character of this genus is also included in the Remarks diagnosis—an extremely large, muscular organ associated with the bur- Trachelobdella livanovi was originally described by Oka (1910) as sa that is visible in whole specimens as a bulge in the integument. Callobdella livanovi. This description did not include internal anatomy Taimenobdella is a monotypic genus. and no illustrations were provided. Oka (1928) synonymized Callob- della hastae with C. livanovi and provided a more complete external Taimenobdella amurensis (Epshtein, 1964) description with illustrations, but still no information on internal anat- (Figs. 7–12) omy. This description noted a wide variety of pigmentation from a light ϭPiscicola amurensis Epshtein 1964 yellowish to a dark brownish color (Oka, 1928). Oka (1930) repeated this description, but provided a full-body external drawing. Oka and Emended description Nagao (1965) referred to C. livanovi as T. livanovi in an encyclopedia of the fauna of Japan with no explanation for the transfer to Trache- External morphology: Maximum length 19 mm; maximum width 4 lobdella. Examination of specimens herein from P. olivaceus, T. par- mm. Body not distinctly divided into trachelosome and urosome, slight- dalis, and A. schlegeli schlegeli, from Japanese waters supports the ac- ly flattened dorsoventrally. Light brown pigment band across eyespots curacy of the combination because these specimens do not possess con- on oral sucker. Three light brown transverse bands on dorsal and ventral ducting tissue. Conducting tissue is present in species of Calliobdella surfaces of trachelosome (Fig. 7). Urosomal pigmentation mottled Beneden and Hesse 1863, and it is absent in species of Trachelobdella brown on recently fixed specimens. Two pairs small eyes on oral sucker (Sawyer, 1986). (Fig. 8). Oral sucker medium to large and eccentrically attached, max- Because T. livanovi has never been formally compared with other imum width 1.2 mm. Ocelli on caudal sucker (number undetermined) species in the genus, it is important to do so to confirm its separate and segmentally on urosome (Figs. 7, 9). No ocelli on trachelosome. species status. There are currently 10 described species of Trachelob- Caudal sucker large and eccentrically attached, maximum width, 2.0 della. Trachelobdella livanovi is the only Trachelobdella species re- mm, pigmented mottled brown with unpigmented regions between ocel- ported from Japan. Trachelobdella livanovi can be distinguished from li and lateral edge (Fig. 9). Annulation 7(14). Clitellum often swollen Trachelobdella lubrica (Grube 1840) and T. bathyrajae by the number in region of male gonopore. Body smooth with no tubercles or papillae of testisacs and the number of fenestrae in the postceca. Trachelobdella (Fig. 7). Urosome contains 12 pairs of medium-sized pulsatile vesicles. livanovi has 5 pairs of testisacs and fused postceca with 3 fenestrae; T. Digestive system: Mouthpore located in center of oral sucker. Pro- lubrica and T. bathyrajae have 6 pairs of testisacs and 0 and 4 fenestrae, boscis extends nearly to ganglion X. Mycetomes emerge from esoph- respectively (Burreson, 1976; Meyer and Burreson, 1990). Trachelob- agus at XI/XII and extend anteriorly to ganglion XI. Crop expands at della oregonensis is a unique case because there are no testisacs in the ganglia, alternating with testisacs. Postceca fused, presence of fenestrae larger specimens; however, newly hatched specimens were found to be could not be determined. mature males with 5 pairs of testisacs. Trachelobdella oregonensis is Reproductive system: Six pairs of testisacs intersegmentally at XIII/ further distinguished by a pair of eyes, only 10 pairs of pulsatile vesi- XIV through XVIII/XIX, alternating with gastric ceca. Vasa deferentia cles, and postceca that are fused only at the posterior end forming a expand and enter highly convoluted epididymides in segment XII. Vasa single, large fenestrum (Burreson, 1976). Trachelobdella livanovi has deferentia continue anteriorly before enlarging as highly muscularized no eyes, 12 pairs of pulsatile vesicles, and 3 fenestrae. Trachelobdella ejaculatory bulbs and bending ventrally in segment XI to enter atrial rugosa Moore 1898 and Trachelobdella maculata Moore 1898, species cornua. Lumina of atrial cornua very small. Atrial cornua located dor- that have not been reported since the original descriptions and are poor- sally to very large muscular organ that forms near bend of ejaculatory ly known, differ from T. livanovi by having a smaller caudal sucker. In duct. Atrial cornua merge to form common atrium midway through addition, T. rugosa has an unusual oral sucker that closes to a vertical segment XI. Atrium small, opens into very large bursa flanked laterally slit (Moore, 1898), and T. maculata has 13 pairs of pulsatile vesicles by muscular organ. Bursa opens to extremely large male pore. Acces- (Moore, 1898). Trachelobdella australis Blanchard 1900 has 11 pairs sory gland cells form thin layer on lateral surface of muscular organ,

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FIGURES 7–12. Taimenobdella amurensis.(7) External morphology, dorsal view. (8) Oral sucker, lateral view. (9) Caudal sucker, lateral view, not the same specimen as in 7.(10) Reconstruction of reproductive systems, dorsal view. (11) Reconstruction of reproductive systems, lateral view. Testisacs and left ovisac omitted to show conducting tissue arrangement. (12) Reconstruction of coelomic system, right side segmental, left side intersegmental. A, atrium; B, bursa; C, conducting tissue; D, dorsal sinus; E, epididymus; ED, ejaculatory duct; F, female gonopore; G, accessory gland cells; L, lateral sinus; M, male gonopore; MO, muscular organ; O, ovisac; PV, pulsatile vesicle; T, testisac; V, ventral sinus. FURINESS ET AL.—FISH LEECHES FROM JAPAN 879 880 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 common atrium, and bursa (Figs. 10, 11). Female gonopore small. Com- mon oviduct short, quickly bifurcates into long paired ovisacs that reach ganglion XVI. Conducting tissue present as mass that originates on posterior surface of bursa and extends posteriorly splitting into paired small cords that associate closely with ovisacs (Figs. 10, 11). Coelomic system: Twelve pairs of medium-sized pulsatile vesicles. Dorsal, ventral, lateral, and testicular sinuses present. Ventral sinus expands at ganglia to connect to lateral canal and pulsatile vesicle. Lateral canal connects intersegmentally to testicular sinuses (Fig. 12). Dorsal sinus connections could not be determined.

Specimens examined NSMT-An 350: NEOTYPE; Naibetsu River, Chitose, Hokkaido, Ja- pan; 30 September 2001; host Salmonidae, genus and species unknown: infection site unknown; 1 specimen. NSMT-An 351: Naibetsu River, Chitose, Hokkaido, Japan; 30 Sep- tember 2001; host Salmonidae, genus and species unknown; infection site unknown; 3 specimens (2 specimens sectioned remain in E.M.B.’s personal collection).

Remarks The pigmentation, general body shape, long ovisacs, and large muscular organ associated with the bursa present in these specimens match- es the same features described by Epshtein (1964, 1987) for T. amurensis. Biological features are also consistent with both the specimens examined here and those reported by Epshtein (1964) collected from the same geographic region; the specimens reported here are from freshwater in Japan, east of the Sea of Japan, and Epshtein’s are from freshwater in the nearby Amur basin, west of the Sea of Japan. The host for the specimens reported here was an unidentified salmonid fish, and the host for the specimens described by Epstein was taimen, probably Hu- cho taimen (Pallas, 1773) (Salmonidae), also a salmonid fish. However, some significant morphological discrepancies exist between the specimens reported here and the description of Epshtein (1964), which described T. amurensis with only 5 pairs of testisacs and no conducting tissue. There are 6 pairs of testisacs and conducting tissue present in the specimens examined here. The method used by Epshtein (1964) to examine his specimens was not discussed in his description. Conducting tissue in specimens as small as T. amurensis could easily be overlooked in a dissection, especially because the conducting tissue remains closely associated with the ovisacs. The number of testisacs is not as easy to mistake, but it is not known whether Epshtein (1964) dissected any specimens, because he described features as being visible through the FIGURES 13–17. Photomicrographs of various Stibarobdella sp. Bars ϭ integument. No type specimens were deposited when this genus was 5 mm. (13) Stibarobdella bimaculata from MNHN, MNHN 18 VV. first described, and no specimens could be found in the major museum Note large caudal sucker and large eyes on oral sucker (arrow). (14– collections. Epshtein’s personal collection is currently at the V. N. Kar- 15) Stibarobdella moorei from MNHN, MNHN 19 VV. (14) Dorsal azin Khariv National University in the Ukraine, and no specimens of view. Note small, contracted terminal caudal sucker (arrow). (15) Lat- T. amurensis could be found there (S. Utevsky, pers. comm.). Given eral view of anterior portion of body. Note fringe (arrow) on oral sucker. the similarities in features and biology and the lack of type material, it (16) Stibarobdella loricata, National Museum of New Zealand (Te is presumed that the specimens described here are the same as those Papa) ZW1415. Note small, terminal caudal sucker and fringe (arrow) described by Epshtein (1964, 1987) as T. amurensis, and a neotype is on oral sucker. (17) S. loricata, Tasmania, Munro Ichthyological Col- designated. The present collection represents a new record of the species lection, CSIRO, Hobart, Tasmania (unregistered). Arrow indicates for Japan. fringe on oral sucker.

Stibarobdella Leigh-Sharpe, 1925 Stibarobdella macrothela (Schmarda, 1861) (Fig. 13) April 1988; host gecko catshark, Galeus eastmani (Jordan & Snyder, 1904) (Scyliorhinidae) new host; infection site unknown; 1 specimen. ϭPontobdella bimaculata Oka 1910 ϭ NSMT-An 355: Shimoda Floating Aquarium, Shizuoka, Japan; 1 June Stibarobdella bimaculata (Oka 1910) (new synonym) 1986; host blotchy swell shark, Cephaloscyllium umbratile Jordan & Fowler 1903 (Scyliorhinidae), new host record; infection site unknown; Description 1 specimen. See Llewellyn (1966). NSMT-An 356: Shimoda Floating Aquarium, Shizuoka, Japan; 3 June 1987; host unidentified sharks; infection site unknown; 3 specimens. Specimens examined NSMT-An 357: Suruga Bay in the North Pacific Ocean, off Heta, NSMT-An 352: Shimoda Floating Aquarium, Shizuoka, Japan; 6 Shizuoka, Japan; 18 April 1995; host C. umbratile; infection site clasp- April 1975; host Japanese bullhead shark, Heterodontus japonicus Ma- er; 1 specimen. MNHN, Paris, France, MNHN 18VV (in jar AH 1): clay & Macleay 1884 (Heterodontidae), new host record; infection site Tateyama (Province Awa), Japan; 1925; identified by A. Oka as P. skin at the base of pectoral fin; 3 specimens. bimaculata, host unknown; 1 specimen. NSMT-An 353: Shimoda Floating Aquarium, Shizuoka, Japan; 7 May E.M.B.’s personal collection: S. macrothela from Virginia Beach, 1987; host H. japonicus; infection site unknown; 1 specimen. Virginia, U.S.A., from a sand tiger shark, Carcharias taurus Rafinesque NSMT-An 354: Shimoda Floating Aquarium, Shizuoka, Japan; 16 1810 (Odontaspididae); 1 specimen. FURINESS ET AL.—FISH LEECHES FROM JAPAN 881

Remarks Posterior sucker small, terminal, not wider than greatest width of body, and usually contracted in preserved specimens (Fig. 14). Clitellum con- Stibarobdella macrothela was originally described by Schmarda stricted, with small tubercles. Complete urosomal somite 3 annulate (1861) as Pontobdella macrothela from a specimen collected in Jamai- with a annulus slightly wider than a or a annulus. Urosomal tubercle ca. The species is now known to be distributed worldwide on tropical- 2 1 3 numbers vary, the most consistent pattern being 12 tubercles on a1 an- to-subtropical oceanic sharks, and it is characterized by a pair of large, nulus, 8 on a , and 10 on a . The a annulus consists of 4 dorsal tuber- trumpet-shaped eyes on the oral sucker (Fig. 13); large, square tubercles 2 3 1 cles, 2 lateral tubercles, and 6 ventral tubercles. The a2 annulus consists on the a2 annulus of the urosome; and a relatively large caudal sucker of 4 dorsal tubercles and 4 ventral tubercles. The a3 annulus consists of (Llewellyn, 1966; Sawyer, 1986). Oka (1910) described P. bimaculata 4 dorsal tubercles, 2 lateral tubercles, and 4 ventral tubercles. Dorsal in a very brief and vague description in which the major character noted tubercles on a2 annulus larger and more prominent than those on other was the presence of ‘‘enormous’’ eyes on the oral sucker and the pres- 2 annuli. ence of three pairs of papillae on the oral sucker. Oka (1910) did not compare P. bimaculata to any previously described species of Pontob- Specimens examined della Leach 1815 other than the 2 other new species he described in the same paper, Pontobdella moorei Oka 1910 and P. tatejamensis. MNHN; MNHN 19VV (in jar AH 1): Misaki, (Province Sagami) Ja- Later, Oka (1927c) provided additional data on the external morphology pan; no host or date recorded; identified by A. Oka as S. moorei;1 of P. bimaculata, mainly the annulation pattern, which is not helpful in specimen. distinguishing species of Stibarobdella, because the genus is defined as National Museum of New Zealand (Te Papa), Wellington, New Zea- being triannulate (Llewellyn, 1966). However, Oka (1927c) also men- land, ZW1415: 37Њ51.1ЈS, 177Њ19.4ЈE, off Whale Is., New Zealand; 48 tioned, and illustrated, a fourth pair of papillae on the oral sucker. Pon- m; 17 September 1984; no host recorded; identified by E.M.B. as S. tobdella bimaculata, P. macrothela, and all other triannulate shark loricata; 1 specimen. leeches were transferred to Stibarobdella after it was described (Leigh- Munro Ichthyological Collection, Commonwealth Scientific and In- Sharpe, 1925) as a genus resembling Pontobdella, but with 3 annuli per dustrial Research Organization (CSIRO), Hobart, Tasmania: No col- urosomal segment rather than 4 annuli per urosomal segment as in Pon- lection date recorded; host Raja sp.; Tasmania; identified by E.M.B. as tobdella. S. loricata; 1 specimen (unregistered). There have been questions in the literature as to the validity of S. bimaculata. Cordero (1937), for example, considered S. bimaculata a Remarks junior synonym of S. macrothela, but Llewellyn (1966) provisionally Pontobdella moorei was described by Oka (1910) in a very brief and regarded S. bimaculata as separate from S. macrothela, because of dif- vague report that gave no description of tubercle numbers or arrange- ferences in sucker size ratios and tubercle patterns. Sawyer (1986) listed ment; it also stated that the anterior sucker had a smooth margin. The S. bimaculata under the category of ‘‘possible other valid species.’’ description was unaccompanied by illustrations. It was later reported Upon examination of 10 pontobdellids collected from Japan and ten- that P. moorei lacked papillae and eyes on the oral sucker, which was tatively identified as S. bimaculata by K.N., and 1 specimen specifically described as being hemispherical and perfectly smooth (Oka, 1927c). identified by Oka as S. bimaculata and deposited by him in MNHN Pontobdella moorei was transferred to a new genus, Stibarobdella, that (Fig. 13), we found that all these specimens had tubercle patterns con- included all 3-annulate pontobdellids (Leigh-Sharpe, 1925). Harding sistent with S. macrothela, as described by Llewellyn (1966) as well as (1924) described S. loricata but did not compare it with any other Sti- 2 large eyespots and 4 pairs of papillae on the oral sucker. Most of the barobdella species. A key to 3 Stibarobdella species keyed S. moorei specimens from Japan were small, less than 30 mm in length (including as having a smooth oral sucker with neither a marginal fringe nor pa- the specimen identified by Oka), and they had somewhat variable sucker pillae and S. loricata as having an oral sucker with marginal fringe and ratios ranging from 1:1.5 to 1:2, but this is consistent with S. macroth- 3 pairs of small papillae (Harant, 1929). Cordero (1937) considered S. ela. It seems that the sucker ratio of S. macrothela is more variable moorei to be a junior synonym of S. macrothela, but S. moorei lacks than thought and that it may increase as individuals get larger. In ad- the large, trumpet-shaped eyes of S. macrothela. Llewellyn (1966) re- dition, the dorsal tubercles on the a2 annulus of small specimens did garded S. moorei and S. loricata as separate species, citing the presence not seem square as they do on large specimens. We also examined the of a marginal fringe and papillae on the oral sucker of S. loricata and specimen of S. bimaculata in the British Museum (Natural History, the lack thereof on S. moorei as the 2 main differences between the London, U.K.) (1935.6.28.1), presumed to be the same specimen ex- species. Llewellyn (1966), however, did not actually examine any spec- amined by Llewellyn (1966), because of the locality in Panama. The imens of S. moorei and instead seemed to base his description on those specimen is fully engorged with blood and in poor condition. It was written by Oka (1910) and Harant (1929). not possible to confirm the tubercle pattern illustrated by Llewellyn As part of this study, we examined a specimen that was identified by (1966). The caudal sucker of this specimen did seem somewhat small Oka as S. moorei and deposited by him in the MNHN (Figs. 14, 15). for S. macrothela, but it was contracted and it may have seemed small The specimen was small, only 39 mm in length, but we found it to due to the extended body. It was not possible to determine a sucker have both an obvious marginal fringe and 3 pairs of small papillae on ratio because both the oral and caudal suckers were contracted. Because the oral sucker. Furthermore, when compared with large S. loricata of these findings, we agree with Cordero (1937) that S. bimaculata is specimens deposited in various museums, and the description by Llew- a junior synonym of S. macrothela. elllyn (1966), the tuberculation patterns were found to be identical with Stibarobdella macrothela can be distinguished from all other Stiba- the specimen of S. moorei in the MNHN. On this basis, S. loricata robdella species by the presence of 1 pair of large trumpet-shaped eye- should be considered a junior synonym of S. moorei. Stibarobdella spots on the oral sucker (Fig. 13); 4 pairs of papillae on the oral sucker; moorei can be distinguished from all other Stibarobdella species by the a large caudal sucker, tubercle pattern; and tubercles on the a2 annulus tubercle pattern, small, terminal caudal sucker (Fig. 14); large, very that are large and almost square-shaped in large, relaxed, well-preserved deeply cupped oral sucker with marginal fringe; 3 pairs of small mar- specimens. ginal papillae; and lack of eyes (Figs. 15–17).

Stibarobdella moorei (Oka, 1910) Crangonobdella Selensky, 1914 (Figs. 14–17) Crangonobdella maculosa Utevsky, 2005 ϭPontobdella moorei Oka 1910 Description ϭ Stibarobdella loricata (Harding, 1924) (new synonym) See Utevsky (2005).

Emended description Specimens examined Diagnosis: Body elongate, cylindrical to somewhat flattened, divided NSMT-An 358: Sunpiazza Aquarium, Sapporo, Hokkaido, Japan; 4 into trachelosome, clitellum, and urosome. Oral sucker large, deeply July 1988; host sailfin sandfish, Arctoscopus japonicus (Steindachner cup-shaped, eccentrically attached, with obvious marginal fringe (Figs. 1881) (Trichodontidae) new host record; infection site skin; 44 speci- 15–17), and 3 pairs small submarginal papillae. No eyes on oral sucker. mens. (4 specimens sectioned remain in E.M.B.’s personal collection). 882 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

NSMT-An 359: Sunpiazza Aquarium, Sapporo, Hokkaido, Japan; 4 are reported for C. maculosa, J. arctica, L. okae, P. olriki, S. July 1988; host A. japonicus; infection site skin; 4 specimens. macrothela, and T. livanovi. Eight other species, Beringobdella Remarks rectangulata (Levinsen, 1881), Cottobdella epshteini Utevsky 1997, Heptacyclus virgatus (Oka, 1910), Notostomum cyclos- The present collection of C. maculosa is a new record of this species for Japan. tomum Johansson 1898, Oceanobdella alba (Epshtein and Utevsky, 1996), Ostreobdella kakabir Oka 1927, Piscicola geo- Johanssonia Selensky, 1914 metra L. 1761, and Pterobdella amara Kaburaki 1921, are Johanssonia arctica (Johansson, 1898) known from Japan, but they were not reported in the present Description collections. Their absence is likely because the proper hosts for those leeches were not obtained in the fish collections reported See Meyer and Khan (1979). herein, or the leeches went unnoticed by the fish collectors. As Specimens examined stated above, the fish collections reported here were not made for the purpose of looking for leeches; the leeches were inci- NSMT-An 361: Terpenia Bay in the Sea of Okhotsk, southern Sa- khalin, Russia; 25 October 1984; host sculpin, I. cataphractus; new host dentally collected if observed. record; infection site inner surface of opercula; 3 specimens (2 speci- Some species were collected only, or primarily, from mari- mens sectioned remain in E.M.B.’s personal collection). culture facilities or public aquaria. Crangonobdella maculosa NSMT-An 362: North Pacific Ocean, off Shikabe, Hokkaido, Japan; was collected only from the sailfin sandfish in the Sunpiazza 28 September 1987; host Beni-zuwai crab, C. japonicus, new host rec- Aquarium in Sapporo; P. olriki was collected only from the ord; 36 specimens. Shimane Mariculture Institute, Hokadote, Hokkaido, on barfin Remarks flounder and prowfish; and S. macrothela was reported mainly The present collection of J. arctica is a new record of this species from the Shimoda Floating Aquarium, Shizuoka, on a variety for Japan. of small sharks. In all cases, it is not known where the infected hosts were originally collected or whether infections may have Limnotrachelobdella Epshtein, 1968 been acquired in the facility. Limnotrachelobdella okae (Moore, 1924) The fortuitous discovery in the MNHN of well-preserved Description specimens of S. bimaculata and S. moorei, identified and de- See Epshtein (1987). posited by Asijiro Oka, allowed us to resolve some long-stand- ing questions on the validity of these 2 species. The specimen Specimens examined of S. moorei is the only specimen known to exist, and that of NSMT-An 363: Near the mouth of Sarufutsu River (flowing into the S. bimaculata is the only specimen known that was identified Sea of Okhotsk), Sarufutsu, Hokkaido, Japan; 1 August 1986; host Jap- by Oka, who described the species originally. anese huchen, Hucho perryi (Brevoort 1856) (Salmonidae), new host record; infection site skin at the base of anal fin; 1 specimen. ACKNOWLEDGMENTS

Platybdella Malm, 1863 We thank the following people for assistance in collecting the leech Platybdella olriki Malm, 1865 specimens: Soichi Hagiwara, Shimoda Floating Aquarium, Shimoda, Japan; Shin-ichi Wakabayashi, Toyama Prefectural Fisheries Experi- Description mental Station, Namerikawa, Japan; Naonori Okada, Sunpiazza Aquar- See Meyer and Khan (1979) ium, Sapporo, Japan; Tsutomu Matsumoto, Tottori Prefectural Fisheries Research Center, Yurihama, Tottori, Japan; and Tetsuya Umino, Hiro- Specimens examined shima University, Higashi-Hiroshima, Japan. We thank Tetsuya Itoh, Hokkaido University, Sapporo, Japan, for providing the T. amurensis NSMT-An 364: Hokkaido Fisheries Experimental Station, Hokadote, specimens. This study was supported by Grants-in-Aid for Scientific Hokkaido, Japan; 24 March 1994; host barfin flounder, Verasper moseri Research (B) 18380116 from the Japan Society for the Promotion of Jordan and Gilbert 1898 (Pleuronectidae), new host record; infection Science and NSF PEET DEB 0119329. site skin; 24 specimens (3 specimens sectioned remain in E.M.B.’s personal collection). NSMT-An 365: Hokkaido Fisheries Experimental Station, Hokadote, LITERATURE CITED Hokkaido, Japan; 25 March 1994; host prowfish, Zaprora silenus Jordan BURRESON, E. M. 1976. Trachelobdella oregonensis sp. n. (Hirudinea: 1896 (Zaproridae), new host record; infection site skin; 21 specimens. Piscicolidae), parasitic on the cabezon, Scorpaenichthys marmoratus (Ayres), in Oregon. Journal of Parasitology 62: 793–798. Remarks ———. 2006. A redescription of the fish leech Pterobdella amara ϭ The present finding of P. olriki is a new record of this species for ( Rhopalobdella japonica) (Hirudinida: Piscicolidae) based on Japan. specimens from the type locality in India and from Australia. Jour- nal of Parasitology 92: 677–681. ———, AND G. C. KEARN. 2000. Rhopalobdella japaonica n. gen., n. DISCUSSION sp. (Hirudinea, Piscicolidae) from Dasyatis akajei (Chondrichthyes: Dasyatididae) in the northwestern Pacific. Journal of Parasitology Based on collections reported herein and reports in the lit- 86: 696–699. erature, the fish leech fauna of Japan, especially in the marine CORDERO, E. H. 1937. Hirudineos neotropicales y subantarticos nuevos, environment, seems to be relatively rich. Seven species in 7 criticos o ya conocidos del Museo Argentine de Ciencias naturales. genera were found in these collections—C. maculosa, J. arc- Anales del Museo Argentino de Ciencias Naturales, Buenos Aires 39: 1–78. tica, L. okae, P. olriki, S. macrothela, T. amurensis, and T. EPSHTEIN, V. M. 1964. Towards a zoogeographical characterization of livanovi. Five species are new records for Japan—C. maculosa, the fish leeches of the Amur basin. Doklady Akademii Nauk SSSR J. arctica, P. olriki, S. macrothela, and T. amurensis. New hosts 159: 1179–1182. FURINESS ET AL.—FISH LEECHES FROM JAPAN 883

———. 1967. Regularities of the geographical distribution of the ma- description de cette Hirudineé. Proceedings of the Imperial Acad- rine fish leeches (Hirudinea: Piscicolidae). Zoologcheskii Zhurnal emy of Tokyo 3: 470–473. 46: 680–691. ———. 1927c. Sur la morphologie externe de Pontobdelle. Proceedings ———. 1973. Diagnoses of the genera Calliobdella, Trachelobdella, of the Imperial Academy of Tokyo 3: 90–93. Limnotrachelobdella, and Baicalobdella and estimation of the tax- ———. 1927d. Sur la morohologie externe de Trachelobdella okae. onomic value of characters used for diagnosing. Zoologicheskii Proceedings of the Imperial Academy of Tokyo 3: 239–241. Zhurnal 52: 332–341. ———. 1927e. Sur la morphologie externe de Carcinobdella kanibir. ———. 1987. Tip Kolchatyye chevri – Annelida. Opredelitel’ Parazi- Proceedings of the Imperial Academy of Tokyo 3: 171–174. tov. In Presnovodnykh Ryb Fauny SSSR. Volume 3. Parazitiches- ———. 1928. Sur la morphologie et la variabilite de la Callobdella kie Mnogokletochnye (Part 2). Nauka, Leningrad, Russia, p. 340– livanovi. Proceedings of the Imperial Academy of Tokyo 4: 547– 372. 549. ———, AND S. YU.UTEVSKY. 1996. A new fish leech species (Hiru- ———. 1930. Report of the Biological Survey of Mutsu Bay. 17. Hir- dinea: Piscicolidae) from the Northern Pacific. Vestnik Zoologii 30: udinea. The Scientific Reports of the Tohoku Imperial University, 3–8. Fourth Series (Biology) 5: 615–622. FROESE, R., AND D. PAULY. 2006. FishBase. www.fishbase.org, version ———. 1931. Sur l’anatomie de l’Ichthyobdella uobir. Proceedings of 02/2006. the Imperial Academy of Tokyo 7: 64–66. HARANT, H. 1929. Essai sur les Hirudineés. Archives de la Socie´te´ des ———. 1933a. Sur une Ichthyobdellide nouvelle trouve´ dans le plank- Sciences Me´dicales et Biologiques de Montpellier 10: 615–682. ton. Proceedings of the Imperial Academy of Tokyo 9: 541–543. HARDING, W. A. 1924. Descriptions of some new leeches from India, ———. 1933b. Sur l’organisation inte´rieure de la Carcinobdella kani- Burma and Ceylon. Annal and Magazine of Natural History. Serial bir. Proceedings of the Imperial Academy of Tokyo 9: 188–190. 9 14: 491–493. ———, AND Z. NAGAO. 1965. Class Hirudinea. In New illustrated en- INGRAM, D. M. 1957. Some Tasmanian Hirudinea. Papers and Proceed- cyclopedia of the fauna of Japan, Part 1. Hokuryu-Kan Publishing ings of the Royal Society of Tasmania 91: 191–232. Co., Tokyo, Japan, p. 564–574. LEIGH-SHARPE, W. H. 1925. Stibarobdella superba n. g. et sp. Parasi- SAWYER, R. T. 1986. Leech biology and behaviour. Vol. 2. Feeding tology 17: 417–420. biology, ecology, and systematics. Clarendon Press, Oxford, U.K., LLEWELLYN, L. C. 1966. Pontobdellinae (Piscicolidae: Hirudinea) in the 375 p. British Museum (Natural History) with a review of the subfamily. SCHMARDA, L. K. 1861. Neue Turbellarien, Rotatorien und Anneliden Bulletin of the British Museum (Natural History) Zoology 14: 391– beobachtet und gesammelt auf einer Reise um die Erde 1853. Leip- 439. zig 1: 1–161 MEYER, M. C., AND R. A. KHAN. 1979. Taxonomy, biology, and occur- UTEVSKY,S.YU. 1997. A new marine fish leech Cottobdella epshteini rence of some marine leeches in Newfoundland waters. Proceed- gen. n., sp. n. (Hirudinia, Piscicolidae) from the Sea of Japan. Vest- ings of the Helminthological Society of Washington 46: 254–264. nik zoologii 31: 9–11 ———, AND E. M. BURRESON. 1990. Some leeches (Hirudinea: Pisci- ———. 1999. A new species of Crangonobdella orientalis (Hirudinea, colidae) of the southern oceans. Antarctic Research Series: Biology Piscicolidae) from the Kuril Islands, a parasite of Anarhichas or- of the Antarctic Seas XXI 52: 219–236. ientalis (Osteichthyes, Anarhichadidae). Vestnik zoologii 33: 91– MOORE, J. P. 1898. The leeches of the U. S. National Museum. Pro- 94. ceedings of the U.S. National Museum 21: 543–563. ———. 2003. On the systematic position of the marine fish leech Hep- ———. 1924. Notes on some Asiatic leeches (Hirudinea) principally tacyclus albus (Hirudinea, Piscicolidae). Vestnik Zoologii 37: 65– from China, Kashmir, and British India. Proceedings of the Acad- 70. emy of Natural Sciences of Philadelphia 76: 343–388. ———. 2005. A new species of marine fish leeches Crangonobdella OKA, A. 1910. Synopsis der Japanischen Hirudineen, mit Diagnosen der maculosa sp. n. (Hirudinea, Piscicolidae) from the Tatar Strait and Neuen Species. Annotationes Zoologicae Japonenses 7: 165–183. Coastal waters of the Kuril Islands. Vestnik Zoologii 39: 15–22. ———. 1927a. Sur une nouvelle Ichthyobdellide parasite de l’Huıˆtre. VASILEYEV, E. A. 1939. The Ichthyobdellidae of the Far East. Trudi Proceedings of the Imperial Academy of Tokyo 3: 364–367. Karelskogo Gosudarstvennogo Pedagogicheskogo Instituta, Biolo- ———. 1927b. Sur la pre´sence de l’Ozobranchus margoi au Japon, et gy Series 1: 25–66. J. Parasitol., 93(4), 2007, pp. 884–888 ᭧ American Society of Parasitologists 2007

PARACOSMOCERCELLA ROSETTAE N. GEN. ET N. SP. (NEMATODA: COSMOCERCOIDEA: COSMOCERCIDAE) COLLECTED FROM THE JAPANESE TREE FROG, HYLA JAPONICA (ANURA: HYLIDAE), IN JAPAN

Hideo Hasegawa and Yatsukaho Ikeda Department of Infectious Diseases, Faculty of Medicine, Oita University, Hasama, Yufu, Oita 879-5593, Japan. e-mail: [email protected]

ABSTRACT: A new genus, Paracosmocercella (Nematoda: Cosmocercoidea: Cosmocercidae), is proposed with monotypic Par- acosmocercella rosettae n. sp. from the Japanese tree frog, Hyla japonica Gu¨nther, 1859, in Oita, Kyushu Island, Japan. Para- cosmocercella resembles Cosmocercella and Cosmocercoides by having 2 rows of rosette papillae without plectanes ventrally in preanal region of male, but it is readily distinguished from Cosmocercella by lacking clear vesicle supporting rosette papillae and from Cosmocercoides by lacking rosette papillae in perianal region and having large-sized eggs, which hatch in uterus.

The Japanese tree frog, Hyla japonica Gu¨nther, 1859, is a Akatsuka, Niigata, in June 2003. Finally, 7 H. japonica (SVL 24–29 common small amphibian widely distributed in Japan, Korea, mm) in Sumiyoshi, Ryotsu, Sado, in November 2003, and 4 Rhaco- phorus arboreus Okada et Kawano, 1924 (SVL 52–62 mm), in Moura, China, and Far East Russia (Frost, 2006). Although helmin- Ryotsu, Sado, Sado Island, in June 2003, were examined for nematodes. thological studies of this frog in Japan have been carried out Figures were made with the aid of a Nikon drawing tube attached on since the 1920s (Morishita, 1926), only Cosmocerca japonica a Nikon Optiphot microscope with a Nomarski interference contrast Yamaguti, 1938 and Oswaldocruzia insulae Morishita, 1926, apparatus. Measurements, in micrometers unless otherwise stated, are given for the holotype and allotype, followed by range for paratypes in have been recorded as intestinal nematodes (Goldberg et al., parentheses. Type specimens are deposited in the National Science Mu- 1994; Hasegawa and Asakawa, 2004). Recently, a nematode seum, Tokyo, with accession numbers NSMT-As 3090 to 3092. species belonging to the superfamily Cosmocercoidea was found in this frog in Oita, Kyushu Island, Japan. Close exam- RESULTS ination has revealed that it belongs to an undescribed genus. Herein, it is described as new genus and new species. The new nematodes described below were collected from 2 females (SVL 35 mm and 32 mm) of 6 H. japonica captured MATERIALS AND METHODS at Hasama in June 2006, and only 1 (female, SVL 30 mm) of H. japonica Six tree frogs, H. japonica, with snout–vent lengths (SVL) of 27–37 107 captured in the same locality during the period mm, were captured by students of the Faculty of Medicine, Oita Uni- from 1992 to 1996. This new species was not observed in 95 versity, on the footpaths between paddies near the faculty housing in H. japonica in other localities, or in 13 R. schlegelii (including Hasama, Yufu, Oita, Kyushu Island, Japan, in June 2006. On return to 6 individuals from Hasama) and 4 R. arboreus. the laboratory, the frogs were killed with ether inhalation, and their viscera were examined carefully under a stereomicroscope. Nematodes found were fixed in hot 70% ethanol, cleared in glycerol ethanol solu- DESCRIPTION tion by evaporation, and mounted in 50% glycerol aqueous solution for Paracosmocercella n. gen. light microscopy observation. Some nematodes were fixed in cold 70% ethanol and postfixed in 1% osmium tetroxide, dehydrated in a graded Diagnosis: Nematoda: Cosmocercoidea: Cosmocercidae. Mouth tri- ethanol series, transferred to t-butanol, and dried with a VFD-21 t-bu- angular, encircled by 3 lips each with lamellate margin apically; dorsal tanol freeze dryer (Vacuum Device, Ibaragi, Japan). They were coated lip with 2 cephalic papillae; subventral lips each with 1 cephalic papilla with osmium using a HPS-1S osmium coater (Vacuum Device), coated and 1 minute papilla close to amphidial pore. Somatic musculature pla- with gold using a JUC-5000 magnetron sputter (JEOL, Tokyo, Japan), tymyarian type. Esophagus composed of pharynx, long club-shaped cor- and observed under a S-800 scanning electron microscope (Hitachi Sci- pus, narrow isthmus and esophageal bulb with sclerotized apparatus. ence Systems, Tokyo, Japan). Rosette papillae arranged in 2 rows on ventral surface of preanal region To collect additional specimens and to determine prevalence and lim- of male. None of perianal and postanal papillae in rosette form. Didel- ited geographical distribution in Japan, the alimentary canals of pre- phic, prodelphic. Eggs large, with delicate shell, hatching in utero. Par- served tree frogs and preserved nematode specimens collected from H. asitic in an amphibian. japonica also were examined. Accordingly, the alimentary canals were examined from 107 H. ja- Paracosmocercella rosettae n. sp. ponica (SVL 20–31 mm) and 6 Schlegel’s tree frogs, Rhacophorus (Figs. 1–18) schlegelii (Gu¨nther, 1858) (SVL 31–51mm), captured in Hasama, Yufu, Oita, Kyushu Island, during the period from 1992 to 1996, and they General: Minute worm. With generic characters defined above. Cu- were preserved in 70% ethanol. The alimentary canals of 3 H. japonica ticle with single-crested lateral alae, commencing anterior to nerve ring (SVL 20–24 mm) captured in Oku-Dogo, Matsuyama, Ehime, Shikoku and terminating at anal level in both sexes (Figs. 1, 4, 13, 15, 18). Island, on 3 May 1989, were examined. At necropsy, nematodes were Cephalic papillae round; inner labial papillae in form of small projec- collected from 25, 21, and 22 H. japonica captured in, respectively, tions under cuticle (Figs. 2, 3, 11, 12). Nerve ring anterior to middle Shimotakeishi, Agata, Nagano, on 29 April 1975, Konomasawa, Tsukui, of esophageal corpus; excretory pore near posterior end of corpus (Figs. Kanagawa, on 3 May 1977, and Oyamada, Machida, Tokyo, Honshu 1, 13, 18). Island, on 20 April 1977, and the alimentary canals of 10 H. japonica Male (holotype and 12 paratypes): Body tapered to both extremities, (SVL 30–39 mm) and 7 R. schlegelii (SVL 32–53 mm) captured in posterior end bent ventrally (Figs. 1, 4, 7). Length 1.07 (0.99–1.53) Maki, Niigata, on June 2003, and 7 H. japonica (SVL 27–35 mm) on mm, width 43 (42–74). Pharynx 16 (11–21) long; esophageal corpus with isthmus 205 (161–257) long and 18 (16–24) wide at widest portion of corpus, 11 (10–15) wide at narrowest portion of isthmus; esophageal Received 26 November 2006; revised 4 January 2007; accepted 5 bulb 45 (40–53) long by 34 (31–46) wide. Nerve ring 120 (100–150), January 2007. excretory pore 181 (170–262) from cephalic extremity. Testis running

884 HASEGAWA AND IKEDA—A NEW COSMOCERCID GENUS AND SPECIES 885

FIGURES 1–6. Male of Paracosmocercella rosettae n. gen. et n. sp. from Hyla japonica of Yufu, Oita, Japan. (1) Anterior end of holotype, right lateral view. (2) Cephalic end, left lateral view. (3) Cephalic end, apical view. (4) Posterior end of holotype, right lateral view. (5) Spicules and gubernaculum. (6) Perianal region, ventral view.

anteriorly, then flexed posteriorly (Fig. 4). Two rows each with 24 to Pharynx 18 (18–26) long; esophageal corpus with isthmus 305 (266– 27 rosette papillae present on ventral surface of posterior body (Figs. 326) long, 30 (29–38) wide at widest portion of corpus, and 21 (19– 4, 6, 7). Rosette papillae each composed of central papilla and surround- 26) wide at narrowest portion of isthmus; esophageal bulb 75 (72–83) ing several circles of elements, usually slightly inclining posteriorly long by 63 (62–77) wide. Nerve ring 160 (144–176), excretory pore (Figs. 8–10). Rosette papillae absent in perianal and postanal regions. 259 (256–325), from cephalic extremity. Vulva slightly protruded, pos- Oblique musculature present in preanal region. Anterior anal lip with 1 terior to middle of body, 0.85 (0.73–1.00) mm from posterior extremity unpaired median papilla; 4 pairs of simple papillae arranged in arcs (Fig. 14). Vagina directed anteriorly, then flexed posteriorly, divided surrounding anus; 1 pair of subventral and 1 pair of lateral papillae at into 2 branches at vulval level (Figs. 14, 16, 17). Left branch developed, anal level; 6 irregular pairs of minute papillae on tail (Figs. 4, 6). Spic- extending posteriorly forming uterus, then flexed anteriorly to join ovi- ule simple, weakly sclerotized, 44 (40–55) long (Fig. 4). Gubernaculum duct; left ovary running anteriorly then flexed posteriorly in gravid minute, V-shaped (Fig. 5), hardly discernible in small specimens. Tail worm (Figs. 14, 16, 17). Right branch less developed, nonfunctional, slender, 103 (90–106) long (Figs. 4, 6). extending anteriorly as short rod in worms smaller than 2 mm in length Female (allotype and 18 paratypes): Body tapered to both extremities (Figs. 14, 16, 17), becoming functional in larger worms (Fig. 18). Uter- (Figs. 13, 15, 18). Length 1.98 (1.75–2.46) mm, width 102 (102–156). us containing less than 8 eggs and/or up to 3 hatched larvae (Figs. 14, 886 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 7–10. Scanning electron micrographs of male of Paracosmocercella rosettae n. gen. et n. sp. from Hyla japonica of Yufu, Oita, Japan. (7) Posterior body, right lateral view. (8) Enlarged view of the framed portion of Figure 7 showing 2 rows of rosette papillae. (9) Rosette papilla in the middle of the row. (10) Rosette papillae at the posterior end of the row.

17, 18). Tail conical, 186 (161–208) long (Figs. 15, 18). Eggs large, DISCUSSION ellipsoidal, with delicate, thin shell, 144–176 by 74–118 (n ϭ 18) (Figs. ϭ 14, 18). Hatched larvae 722–806 long by 24–28 wide (n 3) (Figs. Paracosmocercella rosettae might be a rather rare parasite, 17, 18). because it was found in only 1 of 107 H. japonica collected Taxonomic summary from 1992 to 1996 at the type locality. Goldberg et al. (1994) examined 102 H. japonica, 69 of which were collected in 1992 Type host: Hyla japonica Gu¨nther, 1859. at Beppu, Oita, in a neighboring area of the present type lo- Site of infection: Posterior small intestine and large intestine. cality, but they did not ﬁnd this nematode. Moreover, P. roset- Type locality: Hasama (35Њ41ЈN, 139Њ41ЈE), Yufu, Oita, Kyushu Is- land, Japan. tae was not observed in any of H. japonica on other islands of Dates of collection: 13 May 1996, 13 and 15 June 2006. Japan examined in this study. These facts suggest that P. ro- Etymology: This species is named after the rosette papillae in the settae is a geographically restricted parasite. male. It is peculiar that maturation of right ovary lags behind that Specimens deposited: NSMT-As 3090 (holotype male and allotype female), NSMT-As 3091, 3092 (paratypes). of the left ovary. Some amphibian nematodes have been known to possess didelphic reproductive tracts with different functions. Remarks For example, in Gyrinicola batrachiensis (Walton, 1929) (Ox- yuroidea: Pharyngodonidae), 1 ovary produces thin-shelled By having a triradiate mouth, somatic musculature of platymyarian eggs from which autoinfective larvae hatch, whereas the other type, thin-shelled eggs hatching in uterus, an esophagus with pharynx, corpus, isthmus, and valved bulb, the present nematode belongs to the ovary produces thick-shelled eggs, which are passed in the fe- superfamily Cosmocercoidea (Chabaud, 1974). In Cosmocercoidea, it ces of the hosts (Adamson, 1981). However, there is no evi- belongs to the Cosmocercidae by having didelphic reproductive system dence that the 2 ovaries in the present species produce eggs of in the female, an elongated isthmus, and in lacking a preanal sucker in a different nature. Hence, the differential development of the the male (Chabaud, 1978). In Cosmocercidae, only Cosmocercella Steiner, 1924 and Cosmocercoides Wilkie, 1930 have rosette papillae ovaries may have other signiﬁcance. arranged in 2 rows, but not supported by plectanes in male preanal Some cosmocercid genera, such as Cosmocercella and Rail- region as in the present form (Chabaud, 1978). However, Cosmocercella lietnema, also produce large-sized eggs, and the number of eggs has a clear vesicle beneath each rosette papillae, and Cosmcercoides and/or larvae found in the uterus is usually few (Chabaud, has numerous small-sized eggs, well-developed spicules and gubernaculum, and rosette papillae also in the perianal region (Chabaud, 1978), 1978). The reproduction strategy of these forms may differ being readily distinguished from Paracosmocercella. Asynchronous de- from those of other species. Although life history of none of velopment of ovaries is also characteristic of Paracosmocercella. the species that produce few eggs has been elucidated, it may HASEGAWA AND IKEDA—A NEW COSMOCERCID GENUS AND SPECIES 887

FIGURES 11–18. Female of Paracosmocercella rosettae n. gen. et n. sp. from Hyla japonica of Yufu, Oita, Japan. (11) Cephalic end, apical view. (12) Cephalic end, left lateral view. (13) Anterior end of allotype, left lateral view. (14) Genital organs of allotype; left lateral view. (15) Posterior end of allotype, left lateral view. (16) Genital organs of immature worm, ventral view. (17) Genital organs of paratype with hatched larva in uterus, ventral view. (18) Entire paratype worm with both ovaries functioning, right lateral view. Arrows indicate right ovary.

relate with the spawning of the hosts. The asynchronous mat- tion of the frogs and parasites personally collected, to T. Kifune for uration of the ovaries may be advantageous to maintain egg suggestions on a nomenclature rule, and to staff of the Institute of Sci- entiﬁc Research, Oita University, for the technical support on scanning production for a longer period. Presumably, this is an adaptation electron microscopy observation. to the long spawning period of H. japonica, which lasts up to 6 mo (Maeda and Matsui, 1999). LITERATURE CITED

ACKNOWLEDGMENTS ADAMSON, M. L. 1981. Gyrinicola batrachiensis (Walton, 1929) n. comb. (Oxyuroidea; Nematoda) from tadpoles in eastern and cen- Sincere thanks are rendered to the students of the Faculty of Medi- tral Canada. Canadian Journal of Zoology 59: 1344–1350. cine, Oita University, especially Y. Mizuno, N. Kawai, and S. Kanasugi, CHABAUD, A. G. 1974. No. 1. Keys to subclasses, orders and superfam- for help in collecting host and parasite material. Thanks also are ex- ilies. In CIH keys to the nematode parasites of vertebrates, R. C. tended to A. Miyata, A. Uchida, and K. Sekiya for allowing examina- Anderson, A. G. Chabaud, and S. Willmott (eds.). Commonwealth 888 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

Agricultural Bureaux, Farnham Royal, Buckinghamshire, U.K., p. dae), from Japan. Journal of the Helminthological Society of Wash- 6–17. ington 61: 225–227. ———. 1978. Keys to genera of the superfamilies Cosmocercoidea, HASEGAWA,H.,AND M. ASAKAWA. 2004. Parasitic nematodes recorded Seuratoidea, Heterakoidea and Subuluroidea. No. 6. In CIH keys from amphibians and reptiles in Japan. Current Herpetology 23: to the nematode parasites of vertebrates, R. C. Anderson, A. G. 27–35. Chabaud, and S. Willmott (eds.). Commonwealth Agricultural Bu- MAEDA,N.,AND M. MATSUI. 1999. Frogs and toads of Japan, revised reaux, Farnham Royal, Buckinghamshire, U.K., 71 p. ed. Bun-Ichi Sogo Shuppan, Tokyo, Japan, 223 p. FROST, D. R. 2006. Amphibian species of the world 4.0, an online ref- MORISHITA, K. 1926. Studies on some nematodes of frogs and toads in erence. http://research.amnh.org/herpetology/amphibia/index.php. Japan, with notes on their distribution and frequency. Journal of GOLDBERG, S. R., C. R. BURSEY, AND I. RAMOS. 1994. Gastrointestinal Faculty of Science, Imperial University of Tokyo, Section IV, Zo- helminths of the Japanese treefrog, Hyla japonica (Anura: Hyli- ology 1: 1–32. J. Parasitol., 93(4), 2007, pp. 889–896 ᭧ American Society of Parasitologists 2007

DIVERSITY AND PHYLOGENY OF MITOCHONDRIAL CYTOCHROME B LINEAGES FROM SIX MORPHOSPECIES OF AVIAN HAEMOPROTEUS (HAEMOSPORIDA: HAEMOPROTEIDAE)

Olof Hellgren, Asta Krizˇanauskiene*, Gediminas Valkiu¯ nas*, and Staffan Bensch Department of Animal Ecology, Ecology Building, Lund University, SE-22362 Lund, Sweden. e-mail: [email protected]

ABSTRACT: Species of Haemoproteus (Haemosporida: Haemoproteidae), avian haemosporidians, have traditionally been described based on morphology of their gametocytes and on limited experimental information on their vertebrate host specificity. We investigated to what extent the morphological species are represented by monophyletic groups based on DNA sequence data using 2 different fragment lengths of the cytochrome b (cyt. b) gene. Phylogenetic reconstructions of obtained cyt. b lineages from 6 morphospecies of Haemoproteus showed that all lineages formed monophyletic clusters matching the morphospecies. Comparing our data with a recently published study showed that this is not always the case; the morphospecies H. belopolskyi consists of 2 distinct clusters of lineages that apparently have converged in morphology. However, the overall broad congruence between the molecular and morphological clustering of lineages will facilitate the integration of the knowledge obtained by traditional and molecular parasitology. Mean between morphospecies variation was 10-fold higher than the within species variation (5.5% vs. 0.54%), suggesting that Haemoproteus lineages with a genetic differentiation Ͼ5% are expected to be morphologically differentiated in most cases. When investigate the utility of 2 different fragment sizes of the cyt. b gene, the partial, 479-bp, cyt. b protocol picked up all mitochondrial (mt)DNA lineages that are found when using the full cyt. b gene, 1073 bp, suggesting that this protocol is sufficient for identification of most mtDNA lineages. All of the mtDNA lineages were associated with unique alleles when amplification was possible at a nuclear locus, strengthening the hypothesis that the designation of lineages based on mtDNA is largely genome-wide representative. We, therefore, propose the use of a cyt. b fragment of this length as a standard gene fragment for a DNA bar-coding system for avian Haemoproteus species.

Haemoproteus (Haemosporida: Haemoproteidae) includes In a recent study, Martinsen et al. (2006) began the task of over 130 morphologically defined species of avian blood par- integrating the disciplines of morphological and molecular iden- asites (Valkiu¯nas, 2005). Molecular data, however, suggest that tification of avian blood parasites, by investigating how the the number of species might be substantially higher (Bensch et morphology of the parasites corresponds to obtained molecular al., 2004). The morphological characters used to group hae- data. In the present study, we continue this work by asking 3 moproteids into different species (hereafter referred to as mor- questions crucial for developing a molecular-based identifica- phospecies) have been the shape and size of gametocytes, po- tion system of haemosporidian blood parasites. First, to what sition of the gametocytes in infected erythrocytes, form and size extent are the morphological identifications consistent with mo- of pigment granules in the gametocytes, peculiarities of influ- lecular identifications? This question was addressed both by ence of gametocytes on their host cells, and limited experimen- investigating whether the same molecular lineage corresponded tal information on their vertebrate host specificity (Valkiu¯nas, to the same morphospecies and vice versa, and by how the 2005). These are characters that potentially could have evolved morphology was corresponding to the molecular based phylog- in parallel in evolutionarily different parasite lineages due to enies. If the morphological and the molecular phylogenies do adaptations to certain environments in the host organism. To not correspond (morphospecies are often paraphyletic), the some extent, when similar looking parasites have been found morphological traits are carrying few phylogenetic signals. If in hosts belonging to different families, they have been identi- morphospecies instead tend to emerge as monophyletic clusters, fied as different species; however, recent molecular analyses then morphological characters must be relatively stable in the have demonstrated that host identity is an invalid taxonomic course of evolution. For these analyses, we also used morpho- character due to frequent host-shifts and host-sharing of Hae- logical and molecular data published in a recent study by an- moproteus spp. (Bensch et al., 2000; Valkiu¯nas and Ashford, other research group (Martinsen et al., 2006). Second, to what 2002; Waldenstro¨m et al., 2002; Fallon, Bermingham, and Rick- extent is the molecular diversity dependent on the length of the lefs, 2003, 2005; Hellgren, 2005; Szymanski and Lovette, 2005; analyzed DNA sequences? The prevailing methods of molec- Krizˇanauskiene˙ et al., 2006). As molecular methods provide us ular identification of these parasites use protocols that amplify with an independent tool of identification of species, we can a part of the cytochrome b (cyt. b) gene located in the parasites now examine the consistency of the morphological identifica- mitochondrial (mt) genome (mtDNA) (Bensch et al., 2000; Per- tion of this group of parasites. To integrate the rapidly growing kins and Schall, 2002; Fallon, Ricklefs et al., 2003; Hellgren et field of molecular identification of the parasites with morpho- al., 2004; Waldenstro¨m et al., 2004). For molecular analyses, it logically described species, the 2 identification methods must is important to use a technique that is robust, but still yields as be grouping the parasites in a similar way. In other words, mor- much information as possible. We, therefore, analyzed the full- phological identification of parasites must rest on an evolution- length cyt. b and asked whether this increased the number of ary basis that groups the same and closely related molecular lineages detected relative to the partial cyt. b protocol. Third, lineages into the same morphospecies. to what extent is the cyt. b gene diversity representative of the genome-wide diversity of the parasites? A problem with identifying parasites using a single gene, such as the cyt. b gene, Received 6 September 2006; revised 6 December 2006, 9 February 2007; accepted 12 February 2007. would be if the diversity is locus specific and not representative * Institute of Ecology, Vilnius University, Akademijos 2, LT-08412 Vil- for the differentiation of the genome. mtDNA diversity is nius, Lithuania. matched with genome diversity in Haemoproteus spp. as sug-

889 890 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 gested by Bensch et al. (2004). However, this conclusion was prefix), and part of the nuclear gene DHFR-TS (220 bp, lineages de- reached without close morphological identification of the par- noted with n- as prefix). For the amplification of the smaller part of the cyt. b, a nested poly- asite lineages and grouping lineages together that only tenta- merase chain reaction (PCR) protocol was used with the primers tively belonged to the same species, whereas Beadell et al. HaemFNI/HeamR3N//HaemF/HaemR2 as described in Hellgren et al. (2006) found low support for this when investigating closely (2004). The method obtains a 479-bp fragment of the cyt. b, and the related lineages of Plasmodium spp., i.e., cyt. b lineages could sequences obtained are in the same region and comparable with sequences obtained by the methods used in Bensch et al. (2000) and not be determined by its nuclear gene lineage for close related Waldenstro¨m et al. (2004). One negative control was used per every 8 parasites. We, therefore, continued the work by Bensch et al. samples to control for false amplification due to the high sensitivity of (2004) to test, when possible, whether the mtDNA lineages of the nested PCR. In no case were false positives obtained. To test the Haemoproteus spp. are matched to lineages identified at a nu- second question, i.e., whether more discrete parasite lineages are ob- clear gene. This would be the case if cyt. b diversity is repre- tained using a longer part of the investigated gene cyt. b, we first used the primers DW2/DW4 followed by a second PCR and sequencing us- senting gene independent diversity of the parasites, and thus ing the primers DW1/DW6 (Perkins and Schall, 2002). The PCR pro- also likely including differentiation of genes responsible for the tocol and temperature profiles were used according to Perkins and morphology of the parasites. Schall (2002), except for the use of a different DNA polymerase (Taq These 3 questions were investigated using 6 distinct mor- platinum; Invitrogen, Carlsbad, California). To test the third question, i.e., whether cyt. b differentiation represented genome-wide differenti- phospecies of Haemoproteus (H. minutus, H. balmorali, H. be- ation, we amplified the nuclear gene DHFR-TS, using the primers lopolskyi, H. lanii, H. pallidus, and H. payevskyi). Demonstrat- DHFR2F/DHFR1R2, PCR protocol, and temperature profile according ing a consistent result between morphological and molecular to Bensch et al. (2004). Positive or negative amplifications were eval- analyses, and demonstrating that the diversity of obtained mo- uated as presence or absence of bands on 2% agarose gels stained in lecular lineages is sufficiently insensitive to the choice of in- ethidium bromide. Samples that showed positive amplification were sequenced using the procedure described by Bensch et al. (2000). All vestigated gene or gene region, are important first steps in a sequences where aligned by eye using BioEdit 6.0.6 (Hall, 1999). long process of integrating existing traditional morphological taxonomy of haemosporidian parasites into the rapidly expand- Analysis of molecular data ing field of sequence-based identification of the parasites. We To test whether morphologically identical parasites were monophy- hope that such efforts will pave the way for an efficient DNA letic, we first constructed unrooted phylogenies (in this analysis, we are bar-coding program of these parasites in the future (Blaxter, interested in the clustering of lineages rather than the evolutionary relationships) based the full cyt. b lineages obtained in this study. We 2003; Herbert et al., 2003). then included 24 lineages (GenBank DQ451430–DQ451408) from Mar- tinsen et al. (2006), which had been morphologically identified inde- MATERIALS AND METHODS pendently by other researchers to further test whether morphospecies were monophyletic. The sequences of these lineages are located be- Sampling tween the bp 1 and bp 607 of the long sequences obtained in our study. Avian species with known associations with 6 morphologically well- Obtained lineages in our study were truncated to match those of Mar- defined species of Haemoproteus (Fig. 1) were caught at 3 different tinsen et al. (2006). This result was presented in a rooted phylogeny localities during spring migration and early breeding season in 2003 (in using 2 Leucocyozoon lineages (AY099063 and AY099064) as out- Sweden at the Ottenby Bird Observatory, Vombs nestbox population; groups to get the basal evolutionary history of the phylogeny. in Russia at the biological station of the Zoological Institute of the Two unrooted phylogenic trees using the whole cyt. b were con- Russian Academy of Sciences on the Curonian Spit in the Baltic Sea) structed, one tree using Bayesian phylogenetics as implemented in using mistnets, Helgolandtraps, large Rybachy traps, and nest box mrBayes version 3.1.1 (Huelsenbeck and Ronquist, 2001; Ronquist and catching. They were tagged, bled by puncturing the brachial vein, and Huelsenbeck, 2003) and the other tree using the maximum likelihood released after collecting the blood samples. Blood films were prepared as implemented in PAUP* 4.0 (Swofford, 2001) after finding an appro- within 15–30 sec after the venipuncture, air-dried, fixed in methanol, priate model of sequence evolution using the software mrModeltest and stained with Giemsa. Blood films from each infected bird were (Nylander, 2004) and modeltest 3.7 (Posada and Crandall, 1998), re- examined by skilled parasitologists (A.K. and G.V.) for 10–15 min at spectively . Two different models of evolution were used: a General ϩ low magnification (ϫ400), and then at least 100 fields were studied at Time Reversible model including invariable sites (GTR I) for the ϩ ϩ high magnification (ϫ1,000). Intensity of infection was estimated as a Bayesian approach and a GTR I G model for the maximum likeli- percentage by counting the number of parasites per 10,000 erythrocytes hood approach, which also includes rate variation among sites. The examined, as recommended by Godfrey et al. (1987). Parasites were Bayesian phylogeny was obtained with the use of 4 heated and 1 cold identified (by A.K. and G.V.) from blood films based on morphological Markov Chains Monte-Carlo (MCMC), which was sampled every 200 cues described in Valkiu¯nas (2005), without using host species as a generations over 20 million generations generating 100,000 trees. After character of identification (see Fig. 1). In total, 67 birds with a single visualizing the parameters and controlling for a burn-in period using infection of corresponding species of Haemoproteus of 487 tested birds the softwares AWTY (Wilgenbusch et al., 2004) and TRACER (Ram- were used in this study. Bird species, morphospecies of parasites, num- baut and Drummond, 2005), 25% of the trees were discarded as burn- ber of infected individuals, and study sites are presented in Table I. in period . The remaining 75,000 trees were used to construct a majority From each individual, an ϳ20-␮l blood sample also was collected and consensus tree. The phylogenies were visualized using MEGA 3.0 (Ku- stored in SET-buffer (0.015 M NaCl, 0.05 M Tris, and 0.001 M EDTA, mar et al., 2004). Support to internal branches for the maximum like- pH 8.0), and the sample was held at ambient temperature during the lihood tree was estimated by bootstrap analyses using 1,000 replicates. field season and later at Ϫ20 C before DNA extraction. After micro- For the combined data set, we constructed 1 phylogeny using maxi- scopic examination, positive samples for parasites of interest were used mum parsimony implemented in MEGA 3.0 and 1 phylogeny based on in further molecular analysis. Bayesian inference as for the full cyt. b; however, the 100th tree was sampled over 10 million generations. The maximum parsimony con- Molecular-based identification sensus tree based on 1,000 bootstrap replicates was visualized using MEGA 3.0, and when the obtained Bayesian consensus tree showed DNA was extracted using either a chloroform/isoamylalcohol method similar topology, the Bayesian posterior probability is shown together (Sambrook et al., 2002) or standard ammonium acetate protocol. For with the maximum parsimony bootstrap value (Fig. 3). The reason for the sequence-based analysis, 3 different regions were amplified: part of constructing more than 1 phylogeny for each test is to ensure that ob- the cyt. b (479 bp, lineages denoted without prefix), the whole (except tained results is not a product of which phylogenetic method that was 67 bp at the 5Ј end) cyt. b gene (1,073 bp, lineages denoted with L- as used. HELLGREN ET AL.—MOLECULAR IDENTIFICATION OF HAEMOPROTEUS SPP. 891

FIGURE 1. Genetic relationship of 11 different cyt. b lineages of 6 morphospecies of Haemoproteus. Each morphospecies is encircled and microphotographs of macrogametocytes (above) and microgametocytes (below) are shown for each cyt. b lineage. The number of times a speciﬁc lineage was found is shown in brackets. All preparations are Giemsa stained. The unrooted Bayesian consensus tree was constructed by sampling every 200 tree over 20 million generations with 5 MCMC chains in mrBayes and visualized using MEGA 3.0 after discarding 25% of the trees as burn-in period. The topology of the tree was identical with its corresponding maximum likelihood tree constructed in PAUP* 4.0. The node support is shown as posterior probabilities obtained from mrBayes and bootstrap values obtained from PAUP*, and it is displayed as nominator and denominator, respectively. 892 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Associations between lineages of 2 different lengths obtained from the cyt. b gene (mtDNA) and its corresponding DHFR-TS (nuclear lineage) for lineages found in 6 morphospecies of Haemoproteus. mtDNA lineages without a preﬁx are obtained between bp 234 and 713 of the cyt. B, and lineages with L- as preﬁx are obtained between bp –10 and 1,131 (Fig. 1). Number of birds from which a lineage was found on each location and host species is noted before each molecular lineage. GenBank number; full cyt. b/DHFR-TS.

GenBank no. Parasite mtDNA lineages Long fragment/nuclear Host species Study site morphospecies Short fragment/long fragment Nuclear lineages fragment

Red-backed shrike Rybachy H. lanii 1 RB1/1 L-RB1 1 N-RB1 DQ630010/DQ630001 (Lanius collurio) Ottenby 2 RB1/2 L-RB1 2 N-RB1 1 RBS2/1 L 1 N DQ630011/DQ630003 1 RBS4/1 L 1 N DQ630012/DQ630002 Blackbird Rybachy H. minutus 6 TURDUS2/5 L-TURDUS2 4 N-TURDUS2 DQ630013/DQ629998 (Turdus merula) Ottenby 3 TURDUS2/1 L-TURDUS2 2 N-TURDUS2 Thrush nightingale Rybachy H. balmorali 2 LULU1/2 L-LULU1 — DQ630007/– (Luscinia luscinia) Spotted ﬂycatcher Ottenby H. balmorali 5 SFC1/3 L-SFC1 — DQ630008/– (Muscicapa striata) Rybachy 3 SFC1/1 L — 2 COLL3/2 L — DQ630014/– Reed warbler Rybachy H. payevskyi 3 RW1/3 L RW1 — DQ630009/– (Acrocephalus scirpaceus) Melodius warbler Ottenby H. belopolskyi 13 HIICT1/13 L-HIICT1 — DQ630006/– (Hippolais icterina) Pied ﬂycatcher Rybachy H. pallidus 13 PFC1/8 L-PFC1 6 N-PFC1 DQ630004/DQ630000 (Ficedula hypoleuca) 3 COLL2/2 L-COLL2 2 N-COLL2 DQ630005/DQ629999 Ottenby 4 PFC1/3 L-PFC1 3 N-PFC1 5 COLL2/3 L-COLL2 2 N-COLL2 Vomb 1 PFC1/1 L-PFC1 1 N-PFC1 1 COLL2/L-COLL2 1 N-COLL2

To test whether the partial and the full-length cyt. b gene would yield Concordance between molecular and morphological different numbers of lineages and degree of diversification, the sequence identification divergence between and within the different morphospecies was calculated for both data sets using a Jukes–Cantor model of substitution, with all substitutions weighted equally, implemented in the program When using our data set, in all cases where the same lineage MEGA version. 3.1 (Kumar et al., 2004). Differences in mean genetic was found in multiple individuals, it corresponded to the same distances between full and partial cyt. b sequences were tested using a morphospecies (Table II). For morphological species that con- paired sample t-test implemented in SPSS 12.0 (SPSS Inc., Chicago, tain different molecular lineages, these lineages belonged to Illinois). monophyletic clusters in the phylogenies (Fig. 1). The bayesian To investigate whether mtDNA diversity represents genome-wide dif- and maximum likelihood phylogenies based on the long se- ferentiation, we compared the obtained cyt. b lineages (when possible) with its corresponding nuclear gene (DHFR-TS) to examine whether quences showed identical topologies (Fig. 1). the lineages identified at the cyt. b are also corroborated at a nuclear The mean molecular distance (Jukes–Cantor in percentage, gene. using the long cyt. b sequences) between the different morphospecies ranged from 0.7% (H. minutus-H. pallidus) to 7.7% (H. RESULTS lanii-H. balmorali), with a total mean distance of 5.5%, whereas the genetic intramorphospecies distance varied between 0% Variations in morphospecies (H. belopolskyi, H. minutes, and H. payevskyi) and 2.7% (H. balmorali), with a mean distance of 0.5% (Table II; Fig. 2). In total, 67 individual birds were identified as having a single Comparing pairwise genetic distances across all lineages, an infection of the parasite species of interest, both by microscopic overlap in genetic distance between morphological intra- and examination of blood films and PCR. The amplification of the interspecific variation was found (Fig. 2). The largest genetic different cyt. b regions yielded the same amount of variation distance within morphospecies was 2.7% (H. balmorali). In within each morphospecies of parasite, i.e., the different meth- contrast, the morphologically distinct species H. pallidus and ods showed similar resolution (11 different cyt. b lineages; H. H. minutus show a genetic distance of only 0.7% (Table I; Fig. pallidus: COLL2, PFC1; H. payevskyi: RW1; H. lanii: RB1, 2). RBS2, RBS4; H. balmorali: SFC1, LULU1 and COLL3, H. The study by Martinsen et al. (2006) included 4 of the spe- minutus: TURDUS2, H. belopolskyi: HIICT1) (Fig. 1; Table I). cies that we have investigated in this study (H. payevskyi, H. All lineages except RBS2 and RBS4 were found in multiple belopolskyi, H. lanii, and H. balmorali), but the molecular cyt. individuals (Table I). When using the protocol to amplify the b lineages were not the same as those that we have obtained. full cyt. b, we failed to amplify the parasite in 12 infected That makes it possible to extend the test of whether morpho- individuals with low (Ͻ0.001% of red blood cells) parasitemia. species are monophyletic groups. The combined data set cor- HELLGREN ET AL.—MOLECULAR IDENTIFICATION OF HAEMOPROTEUS SPP. 893

TABLE II. Mean sequence divergence between and within different morphological species of Haemoproteus calculated for 2 different fragment lengths of cyt. b using Jukes–Cantor model with uniform substitution rate among sites.

Cyt. b bp (0-1070*/242–722†) Mean between-morphospecies distance No. of found Mean within- (1) (2) (3) (4) (5) lineages morphospecies

(1) H. pallidus 2/2 0.002/0.002 (2) H. minutus 0.007/0.005 1/1 0/0 (3) H. balmorali 0.057/0.051 0.054/0.054 3/3 0.027/0.028 (4) H. payevskyi 0.054/0.056 0.055/0.060 0.064/0.060 1/1 0/0 (5) H. lanai 0.067/0.066 0.066/0.069 0.077/0.063 0.047/0.050 3/3 0.003/0.003 (6) H. belopolskyi 0.053/0.054 0.056/0.057 0.072/0.073 0.045/0.051 0.052/0.049 1/1 0/0

* Full length of cyt. b obtained using protocol of Perkins and Schall (2002). † Obtained part using protocol of Hellgren et al. (2004) (paired sample t-test; mean difference ϭ 0.001, 14 df, t ϭ 0.66, P ϭ 0.52). roborated the previous result; morphospecies consist of mono- Amplifications of the nuclear gene DHFR-TS yielded se- phyletic clusters of lineages in the majority of the cases (Fig. quences from some birds with high parasitemia (Ͼ0.1%) of H. 3), with the exception of 2 different monophyletic clusters that lanii, H. minutes, and H. pallidus. In all cases, different cyt. b both have been assigned to consist of the morphospecies H. lineages corresponded to novel nuclear lineages on the DHFR- belopolskyi. Although the exact relationships of lineages within TS gene, and in no case did a nuclear sequence correspond to the morphospecies, and their relationships to each other, have more than 1 cyt. b lineage (Table I). not been fully resolved, the main results in Figure 3 were supported by both the maximum parsimony and Bayesian phylog- DISCUSSION enies. Phylogenetic support of traditional morphospecies Concordance between observed diversity using different taxonomy protocols and genes Similar molecular lineages were in all cases identified to the No additional lineage variation was found when using the same morphospecies (Fig. 1; Table 1). The investigated mor- full cyt. b (1,070 bp) compared with the shorter amplified frag- phospecies either consisted of a single molecular lineage or a ment (479 bp) in terms of more lineages or differences in ge- cluster of lineages all identified as the same morphospecies. netic divergence (mean difference ϭ 0.001, t ϭ 0.66, P ϭ 0.52; There was, however, a rather low branch support for separating Table II). An important difference between the partial and full H. minutus (TURDUS2) from H. pallidus (PFC1 and COLL2) cyt. b protocol is, however, the much lower amplification suc- (Fig. 1), but this was much improved when adding additional cess of the latter for which 20% of the infected individuals lineages and morphospecies in Figure 3. These results remained failed to amplify parasite DNA (Table I). consistent also when combining them with an independent data set, with the exception of H. belopolskyi, which seemed to consist of 2 different clades of molecular lineages (Fig. 3; also see Martinsen et al., 2006). This is an apparent case of cryptic morphospecies; however, ongoing studies of 1 of the lineages (DQ451428) in 1 of the H. belopolskyi clusters (cluster denoted with * in Fig. 3) is identical to a lineage (SYAT1, AY831750) that has a slight but different morphology, and it is suggested to belong to a new morphospecies named H. parabelopolskyi (Valkiu¯nas et al., 2007). Even though cases of cryptic morphospecies have been found among Haematozoan blood parasites (Perkins, 2000; Seghal, 2006), one should bear in mind that strict cryptic speciation might be prominent in Haemoproteus. In Haemoproteus spp., no case has been found where single cyt. b lineages are showing evidence to recombination with close related lineages (Table I; Bensch et al., 2004). Thus, if applying the biological species concept, i.e., lack of recombination as a criterion for different species, all polymorphic morphospecies of Haemoproteus investigated to date have been FIGURE 2. Pairwise genetic distance (Jukes–Cantor, based on 1,070 found to consist of cryptic species (Bensch et al., 2004; Table bp) between cyt. b lineages found within the same morphospecies (black I). The aim of the present study is, however, not to deal with circles), or in different morphospecies (unfilled circles), ranked on the x-axis with respect to increase in genetic distance. Shaded area repre- cryptic speciation of these organisms, as the degree of cryptic sents a zone where the intraspecific variation overlaps with the inter- speciation might be prominent within most morphospecies specific variation. (Bensch et al., 2004; Beadell et al., 2006), but whether the 894 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 3. Rooted phylogenetic relationship of lineages obtained in this study (marked with arrows) together with named lineages from a previous study by Martinsen et al. (2006). The shown data are compiled from 2 phylogenetic analyses. The visualized phylogeny is based on 607 bp of the cyt. b gene, and it is constructed using a maximum parsimony approach implemented in MEGA 3.0. Bootstrap support above 50% for the maximum parsimony, based on 1,000 replications of the analysis, is shown as the nominator on the branches. When the maximum parsimony tree was congruent with the constructed Bayesian major consensus tree (constructed using mrBayes 3.1), the posterior probability obtained for the Bayesian phylogeny is shown as the denominator. GenBank accession numbers are noted before the lineage names in the phylogenies. * A lineage identical to DQ451428 has recently been found to show signiﬁcantly different morphology, and it has been suggested to be a separate morphospecies (H. parabelopolskyi) compared with H. belopolskyi.

morphology of the parasite has a phylogenetic basis. So, al- Even if the number of species in this study is only a small though 2 clusters with similar morphology were found in dif- fraction of all existing morphospecies of haemosporidian par- ferent parts of the phylogeny representing 2 cryptic species, it asites, our analysis suggests that there is a good correspondence is likely that these clusters each consist of multiple cryptic spe- between the morphological and molecular identification, al- cies. though mismatches are likely to be encountered (Martinsen et These results demonstrate that many of the morphological al., 2006). Accounting for the number of lineages used in this characters used for traditional microscopic identification are study, it is also likely that we only have uncovered a fraction phylogenetically informative and that it will be rather straight of all the lineages that are associated with each of the morpho- forward to integrate the huge body of knowledge based on tra- species investigated. For example, H. balmorali can be found ditional taxonomy with data generated from sequencing of cyt. in more than 40 additional bird species with a wide distribution b lineages. Hence, the morphological characters seem not to in the Palaearctic, Ethiopian, and Oriental zoogeographical re- result from plastic responses to the environment in the host. For gions (Valkiu¯nas, 2005). It is, therefore, likely that more within- example, morphological stability across hosts was demonstrated morphospecies variation should be found, both over the host for H. balmorali. This parasite was found in 3 different host range and over the geographical range in these species. It is, species belonging to 2 subfamilies of the Muscicapidae, bird however, encouraging that when including similar comparisons hosts likely providing different biochemical environments for done by other researchers, the results remained consistent, i.e., the parasites, but the parasites still exhibit the same morpho- molecular lineages associated to certain morphospecies are logical traits. Similar results have been found for the morpho- grouped together in the phylogenies. species H. majoris (Krizˇanauskiene et al., 2006). When assigning molecular lineages to morphospecies, it is HELLGREN ET AL.—MOLECULAR IDENTIFICATION OF HAEMOPROTEUS SPP. 895 of great importance to conduct careful microscopic investiga- have data from longer sequences, because they should contain tion of blood samples to ensure that mixed infections are not more phylogenetic informative sites. However, the robustness biasing the results (Valkiu¯nas et al., 2006), and to not rely only and sensitivity of the PCR does decrease with the length of the on the suggestion that mixed infections would be visible as fragment (in this study, 20% of the infections where not de- double nucleotide peaks on sequence electropherograms (Perez- tected when using the 1,070-bp-long fragment). Tris and Bensch, 2005). It might be that sequences examined originate from a parasite with low intensity rather than from Gene-independent diversity the infection that is present in greater intensity in blood sam- Even in the cases with the lowest degree of differentiation ples. Because mixed infections of blood haemosporidian para- between 2 cyt. b lineages (RB1/RBS4 and PFC1/COLL2 with sites are common in wildlife (Valkiu¯nas, 2005), this may be an 0.2% sequence divergence), these were matched to a similar obstacle for using solely PCR-based methods in molecular sys- difference at the nuclear DHFR-TS gene (Table I). Together tematics studies. In such research, sequences examined are as- with data in Bensch et al. (2004), this strongly supports that the sumed to represent the parasite seen under the microscope, but diversity observed at the mtDNA cyt. b gene is representative there may be light infections of other species that will be pref- of a genome-wide differentiation of Haemoproteus spp. line- erentially amplified (Valkiu¯nas et al., 2006), so it is important ages. to link DNA sequences and morphospecies precisely. Since the introduction of the molecular tools, the number of identified unique sequences of Haemoproteus spp. have already Molecular variation between and within morphospecies markedly exceeded the number of described morphological spe- The overall mean genetic variation between different mor- cies (data not shown). To be able to use these molecular line- phospecies in this study was 5.5%, indicating that when cyt. b ages as a future tool for DNA bar coding (Blaxter, 2003; Hebert distances in this range are obtained, the lineages are likely to et al., 2003, 2004; Stoeckle, 2003) and linking the molecular be morphologically distinct (Fig. 2). It is noteworthy that al- diversity with the traditional morphology-based literature, 2 ini- though morphologically similar, the divergence of lineages tial criteria should be met. First, there must be concordance within H. balmorali is almost 4 times as large as between the between morphological and molecular identification; and, sec- morphologically different species H. minutus and H. pallidus ond, the results should not be sensible to the choice of molec- (Figs. 1, 2). This may indicate a difference in the ‘‘evolutionary ular methods. According to this study and the previous inves- robustness’’ of the morphological traits defining these different tigation by Martinsen et al. (2006), both of these criteria seem groups of parasites. The morphological traits defining H. bal- fulfilled for the cyt. b gene. Based on these initial results we, morali (dark staining of gametocytes because of the well de- therefore, suggest that a fragment of approximately 500 bp of veloped endoplasmic reticulum, numerous ribosomes and other the cyt. b is suitable for such a scheme. organelles, as well as volutin granules in the cytoplasm and a dumbbell-like appearance of the growing parasites) (Valkiu¯nas, ACKNOWLEDGMENTS 2005) seem to be quite conserved, whereas the morphological We are grateful to Javier Pere`z-Tris for scientific input and Tatiana change separating H. pallidus and H. minutus (mainly the dif- Iezhova for help with pictures of the parasites. We would also thank 2 ferences in how they occupy host cells) (Valkiu¯nas, 2005) per- anonymous referees whose comments improved the paper considerably. haps have evolved recently. This study has been generously supported by the Swedish Research Council, the Carl Trygger Foundation, the Helge Ax:sson Foundation, and the Lithuanian State Science and Studies Foundation. Resolution of partial versus full-length cyt. b sequencing Regardless of whether we used a partial or full-length cyt. b LITERATURE CITED sequence of the morphospecies, the same amount of variation BEADELL, J. S., E. GERING,J.AUSTIN,J.P.DUMBACHER,M.A.PEIRCE, (in terms of discrete lineages) was found, and the mean genetic T. K . P RATT,C.T.ATKINSON, AND R. C. FLEISCHER. 2004. Preva- variation between morphospecies variation was the same. Thus, lence and differential host-specificity of two avian blood parasite the commonly used methods by Bensch et al. (2000), Hellgren genera in the Australo-Papuan region. Molecular Ecology 13: 3829–3844. et al. (2004), and Waldenstro¨m et al. (2004) do seem to accu- ———, F. ISHTIAQ,R.COVAS,M.MELO,B.H.WARREN,C.T.ATKIN- rately estimate the molecular diversity of the full cyt. b gene. SON,S.BENSCH,G.R.GRAVES,Y.V.JHALA,M.A.PEIRCE, ET AL. When choosing a molecular method for identification of 2006. Global phylogeographic limits of Hawaii’s avian malaria. cryptic organisms such as parasites, one has to trade between Proceedings of the Royal Society of London B Series 273: 2935– 2944. time, money, robustness of the method, and how much relevant BENSCH, S., J. PEREZ-TRIS,J.WALDENSTRO¨ M, AND O. HELLGREN. 2004. information that can be gained. For molecular identification of Linkage between nuclear and mitochondrial DNA sequences in avi- haemosporidian parasites, most researchers have used methods an malaria parasites: Multiple cases of cryptic speciation? Evolu- that amplify about 500 bp of mtDNA based on reasons of sen- tion 58: 1617–1621. ¨ ¨ sibility (the ability of amplifying low parasitemia) and robust- ———, M. STJERNMAN,D.HASSELQUIST,O.OSTMAN,B.HANSSON,H. WESTERDAHL, AND R. T. PINHEIRO. 2000. Host specificity in avian ness (repeatability of the results; Bensch et al., 2000; Fallon, blood parasites: A study of Plasmodium and Haemoproteus mito- Ricklefs et al., 2003, 2004; Beadell et al., 2004; Hellgren et al., chondrial DNA amplified from birds. Proceedings of the Royal 2004; Waldenstro¨m et al., 2004; Ishtiaq et al., 2006). It is, there- Society of London B Series 267: 1583–1589. fore, an important finding that these methods do not seem to BLAXTER, M. 2003. Molecular systematics—Counting angels with DNA. Nature 421: 122–124. underestimate the diversity found when using full-length se- FALLON, S. M., E. BERMINGHAM, AND R. E. RICKLEFS. 2003. Island and quencing of the cyt. b gene. When asking questions about the taxon effects in parasitism revisited: Avian malaria in the Lesser evolutionary relationship between organisms, it is desirable to Antilles. Evolution 57: 606–615. 896 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

———, ———, AND ———. 2005. Host specialization and geographic parasites recovered from cytochrome b gene sequences. Journal of localization of avian malaria parasites: A regional analysis in the Parasitology 88: 972–978. Lesser Antilles. American Naturalist 165: 466–480. POSADA,D.,AND K. A. CRANDALL. 1998. Modeltest: Testing the model ———, R. E. RICKLEFS,B.L.SWANSON, AND E. BERMINGHAM. 2003. of DNA substitution. Bioinformatics 14: 817–818. Detecting avian malaria: An improved PCR diagnostic. Journal of RAMBAUT,A.,AND A. DRUMMOND. 2005. TRACER: A program for an- Parasitology 89: 1044–1047. alysing results from Bayesian MCMC programs such as BEAST & GODFREY, R. D., A. M. FEDYNICH, AND D. B. PENCE. 1987. Quantifica- MrBayes. Available at http://evolve.zoo.ox.ac.uk. tion of hematozoa in blood smears. Journal of Wildlife Diseases RICKLEFS, R. E., S. M. FALLON, AND E. BERMINGHAM. 2004. Evolution- 23: 558–565. ary relationships, cospeciation, and host switching in avian malaria HALL, T. A. 1999. BioEdit: A user-friendly biological sequence align- parasites. Systematic Biology 53: 111–119. ment editor and analysis program for Windows 95/98/NT, version RONQUIST,F.,AND J. P. HUELSENBECK. 2003. MrBayes 3: Bayesian phy- 6.0.6. Nucleic Acids Symposium Series, p. 95–98. logenetic inference under mixed models. Bioinformatics 19: 1572– HEBERT,P.D.N.,A.CYWINSKA,S.L.BALL, AND J. R. DEWAARD. 2003. 1574. Biological identifications through DNA barcodes. Proceedings of SAMBROOK, J., F. J. FRITCH, AND T. M ANIATIS. 2002. Molecular cloning: the Royal Society of London B Series 270: 313–321. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. ———, M. Y. STOECKLE,T.S.ZEMLAK, AND C. M. FRANCIS. 2004. ¯ Identification of birds through DNA barcodes. PLoS Biology 2: SEHGAL, R. N. M., A. C. HULL,N.L.ANDERSON,G.VALKIUNAS,M.J. MARKOVETS,S.KAWAMURA, AND L. A. TELL. 2006. Evidence for 1657–1663. crypticspeciation of Leucocytozoon spp. (Haemosporida, Leuco- HELLGREN, O. 2005. The occurrence of haemosporidian parasites in the cytozoidae) in diurnal raptors. Journal of Parasitology 92: 375–379. Fennoscandian bluethroat (Luscinia svecica) population. Journal of STOECKLE, M. 2003. Taxonomy, DNA, and the bar code of life. Bio- Ornithology 146: 55–60. science 53: 796–797. ¨ ———, J. WALDENSTROM, AND S. BENSCH. 2004. A new PCR assay for SWOFFORD, D. L. 2001. PAUP*: Phylogenetic analysis using parsimony simultaneous studies of Leucocytozoon, Plasmodium, and Haemo- (*and other methods). Version 4.0b. Sinauer Associates, Sunder- proteus from avian blood. Journal of Parasitology 90: 797–802. land, Massachusetts. HUELSENBECK,J.P.,AND F. R ONQUIST. 2001. MrBayes: Bayesian infer- SZYMANSKI,M.M.,AND I. J. LOVETTE. 2005. High lineage diversity and ence of phylogenetic trees. Bioinformatics 17: 754–755. host sharing of malaria parasites in a local avian assemblage. Jour- ISHTIAQ, F., J. S. BEADELL,A.J.BAKER,A.R.RAHMANI,Y.V.JHALA, nal of Parasitology 91: 768–774. AND R. C. FLEISCHER. 2006. Prevalence and evolutionary relation- VALKIU¯ NAS, G. 2005. Avian malaria parasites and other haemosporidia. ships of haematozoan parasites in native versus introduced popu- CRC Press, Boca Raton, Florida, 932 p. lations of common myna Acridotheres tristis. Proceedings of the ———, AND R. W. ASHFORD. 2002. Natural host range is not a valid Royal Society of London B Series 273: 587–594. taxonomic character. Trends in Parasitology 18: 528–529. KRIZˇ ANAUSKIENE, A., O. HELLGREN,V.KOSAREV,L.SOKOLOV,S. ———, S. BENSCH,T.A.IEZHOVA,A.KRIZˇ ANAUSKIENE˙ ,O.HELLGREN, BENSCH, AND G. VALKIU¯ NAS. 2006. Variation in host specificity be- AND C. BOLSHAKOV. 2006. Nested cytochrome b polymerase chain tween species of avian haemosporidian parasites: Evidence from reaction diagnostics underestimate mixed infections of avian blood parasite morphology and cytochrome b gene sequences. Journal of haemosporidian parasites: Microscopy is still essential. Journal of Parasitology 92: 1319–1324. Parasitology 92: 418–422. RIZˇ ANAUSKIENE˙ EZHOVA ELLGREN AND KUMAR, S., K. TAMURA, AND M. NEI. 2004. MEGA3: Integrated soft- ———, A. K ,T.A.I ,O.H , S. ware for molecular evolutionary genetics analysis and sequence BENSCH. 2007. Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida: Haemoproteidae) of sylviid birds, alignment. Briefings in Bioinformatics 5: 150–163. with description of Haemoproteus parabelopolskyi sp. nov. Journal MARTINSEN, E. S., I. PAPERNA, AND J. J. SCHALL. 2006. Morphological of Parasitology 93: 680–687. versus molecular identification of avian Haemosporidia: An explo- WALDENSTRO¨ M, J., S. BENSCH,D.HASSELQUIST, AND O¨ .O¨ STMAN. 2004. ration of three species concepts. Parasitology 133: 279–288. A new nested polymerase chain reaction method very efficient in NYLANDER, J. A. A. 2004. mrModeltest, version 2.2. Software available detecting Plasmodium and Haemoproteus infections from avian at http://www.ebc.uu.se/systzoo/staff/nylander.html. blood. Journal of Parasitology 90: 191–194. ´ PEREZ-TRIS, J., AND S. BENSCH. 2005. Diagnosing genetically diverse ———, ———, S. KIBOI,D.HASSELQUIST, AND U. OTTOSSON. 2002. avian malarial infections using mixed-sequence analysis and TA- Cross-species infection of blood parasites between resident and mi- cloning. Parasitology 131: 15–23. gratory songbirds in Africa. Molecular Ecology 11: 1545–1554. PERKINS, S. 2000. Species concepts and malaria parasites: Detecting a WILGENBUSCH, J. C., D. L. WARREN, AND D. L. SWOFFORD. 2004. cryptic species of Plasmodium. Proceedings of the Royal Society AWTY: A system for graphical exploration of MCMC convergence of London B Series 267: 2345–2350. in Bayesian phylogenetic inference. Available at http://ceb.csit. ———, AND J. J. SCHALL. 2002. A molecular phylogeny of malarial fsu.edu/awty. J. Parasitol., 93(4), 2007, pp. 897–900 ᭧ American Society of Parasitologists 2007

A NEW SPECIES OF MACROBOTHRIIDAE (CESTODA: DIPHYLLIDEA) FROM THORNBACK RAY PLATYRHINA SINENSIS IN CHINA

Hongtao Li and Yanhai Wang Parasitology Research Laboratory, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361005 People’s Republic of China. e-mail: [email protected]

ABSTRACT: A new cestode, Macrobothridium sinensis n. sp., is described from the spiral intestine of Platyrhina sinensis from coast of Xiamen, China. It is the ﬁrst record of the order Diphyllidea in China. The new species can be distinguished from the 3 previously described species of Macrobothridium by the testes number (16–24 vs. 29–37, 27–46, and 5–6, in M. rhynchobati, M. euterpes, and M. syrtensis, respectively) and a single-row arrangement of testes; M. sinensis is most similar to M. euterpes in strobila length (1.77–6.23 vs. 2–4.5 mm) and number of segments (6–8 vs. 5–9). In addition, M. sinensis differs from M. rhynchobati and M. syrtensis in the strobila length, ovary shape, and number of apical hooks. The uterine pore is the ﬁrst described in the Diphyllidea; thus, the present uterine pore can now be used to distinguish Macrobothridium from Echinobothrium and Ditrachybothridium in the Diphyllidae.

Macrobothridium was erected by Khalil and Abdul-Salam apical hooks arranged in 2 staggered rows forming an anterior row of (1989) with its type species Macrobothridium rhynchobati 17 hooks (Type I) and posterior row of 17 hooks (Type II). Each group of lateral hooklets arranged in 2 staggered rows forming anterior row Rhynchobatus granulatus Khail and Abdul-Salam, 1989, from of 4 hooklets and posterior row of 4 hooklets. Type I hook lengths 45– in Kuwaiti coastal waters (Arabian Gulf). Wojciechowska et al. 65 (52.4 Ϯ 4.8, n ϭ 41), type II hook lengths 35–55 (47.2 Ϯ 5.2, n ϭ (1995) reported a single incomplete specimen of Macroboth- 48). Anterior row hooklets 5–25 (11.5 Ϯ 5.0, n ϭ 30), posterior row ridium sp. from Bathyraja eatonii at Heard Island. Olson and hooklets 5–27.5 (13.4 Ϯ 7.8, n ϭ 21). Bothria oval, 156–192 (170.6 Ϯ Caira (1999) and Olson et al. (2001) discussed the interrela- 11.3, n ϭ 20) long by 124.8–168 (150.2 Ϯ 15.9, n ϭ 5) wide; distal tionships of tapeworms and referred to another species of Ma- surfaces covered with slender pectinate with 3 long digits (Fig. 11). Proximal bothrial surfaces covered with palmate microtriches, each crobothridium from Rhinobatus typus in the Timor Sea, Aus- bearing 6–7 digits (Fig. 13); border between proximal and distal bothrial tralia. Neifar et al. (2001) described 2 species, M. euterpes Nei- surfaces marked abrupt change in microtrich type from 6–7 digits pal- far, 2001 from Rhinobatos rhinobatos and M. syrtensis Neifar, mate microtriches to 3 digits pectinate (Fig. 14). Cephalic peduncle 2001 from Rhinobatos cemiculus in the Gulf of Gabe`s, Tunisia. 37.5–62.5 (49.3 Ϯ 10.0, n ϭ 7) long, 45–70 (57.9 Ϯ 8.9, n ϭ 7) wide, The new species described below was collected from the spiral unarmed, covered with palmate microtriches (Fig. 7). intestine of Platyrhina sinensis at a fish market in Xiamen, Chi- Immature proglottids wider than long, becoming longer than wide on maturation. Mature proglottid acraspedote, 1,584–3,417.6 (2,631.2 Ϯ na in 2005. 485.1, n ϭ 24) long by 201.6–432 (292.6 Ϯ 53.3, n ϭ 21) wide. Testes oval, 16–24 (19 Ϯ 2, n ϭ 26) in number, 74.4–144 (106.5 Ϯ 17.4, n MATERIALS AND METHODS ϭ 43) long by 43.2–120 (82.8 Ϯ 17.2, n ϭ 43) wide, arranged in single The cestodes were collected from the spiral intestine of the thornback column extending from anterior margin of proglottid to cirrus sac. Cir- ray Platyrhina sinensis, which was identified according to Zhu (1984); rus sac ovoid, 127.2–201.6 (172.0 Ϯ 23.7, n ϭ 12) long by 98.4–132 they were washed in saline, killed with hot saline, and transferred im- (119.4 Ϯ 11.7, n ϭ 12) wide in lateral view and 103.2 213.6 (155.6 Ϯ mediately into 5% formalin. Fixed cestodes were stained in alum car- 35.0, n ϭ 6) long by 79.2–132 (98.0 Ϯ 18.2, n ϭ 6) wide in ventral mine, dehydrated in ethanol, cleared in methyl salicylate, and mounted view; cirrus 48–67.2 (56.4 Ϯ 7.4, n ϭ 6) in maximum diameter, without permanently in neutral balsam. Four specimens prepared for scanning obvious spinitriches; vas deferens medial, coiled, entering cirrus sac electron microscopy (SEM) were rinsed in saline, postfixed in 2.5% anteriorly. Ovary H-shaped in ventral view, bilobed in cross section; glutaraldehyde water solution, dehydrated in ethanol series, replaced in ovarian lobes elongate, subequal, 120–240 (184.5 Ϯ 33.5, n ϭ 21) long, isoamyl acetate, dried through the CO2 critical point, and viewed with 67.2–93.6 (79.7 Ϯ 8.1, n ϭ 10) wide in lateral view and 36–72 (53.9 scanning electron microscope (XL 30). Drawings were made with the Ϯ 12.1, n ϭ 11) in ventral view. Genital pore midventral, anterior to aid of a drawing tube. All measurements are present in the text in ovary, 45.6–96 (76.3 Ϯ 19.2, n ϭ 5) by 36–88.8 (61.9 Ϯ 20.5, n ϭ 5), micrometers unless otherwise stated. Measurements are given as ranges 17.66–24.71% (21.61 Ϯ 2.29, n ϭ 14) from posterior end of proglottid. followed by the mean Ϯ standard deviation and sample size in paren- Uterine pore ventral, posterior to genital pore, 79.2–156 (101.4 Ϯ 36.5, theses. Type specimens have been deposited in Parasitology Research n ϭ 4) by 74.4–120 (88.2 Ϯ 21.4, n ϭ 4), 14.56–19.49% (17.50 Ϯ Laboratory, Xiamen University in China. 1.90, n ϭ 9) from posterior end of proglottid. Vagina not coiled, extending anteriorly from junction with uterus duct along midline of pro- DESCRIPTION glottid, joining cirrus at genital pore. Vaginal sphincter present, 12–21.6 (17.6 Ϯ 3.3, n ϭ 12) thick. Receptaculum seminis present, 72–108 (99.1 Macrobothridium sinensis n. sp. Ϯ ϭ Ϯ ϭ (Figs. 1–14) 13.1, n 7) long by 60–93.6 (78.2 12.6, n 7) wide. Vitelline glands lateral, in 2 ventral columns, extending entire length of proglot- Description (based on 9 complete specimens and 15 incomplete spec- tid, interrupted by ovary. Vitellaria follicular, 43.2–84 (56.9 Ϯ 10.6, n Ϯ ϭ imens): Small cestodes; mature specimens 1.77–6.23 (4.87 0.44, n ϭ 17) by 19.2–48 (28.2 Ϯ 7.5, n ϭ 17). Uterine duct, 8.4–24 (14.0 Ϯ Ϯ ϭ 6) mm long, composed of 6–8 (7 1, n 9) proglottids. Scolex 6.5, n ϭ 8) in diameter, running to the anterior part of uterus. Mehlis’ bipartite, consisting of scolex proper and short unarmed cephalic pe- gland posterior to ovarian isthmus, 60–86.4 (72.0 Ϯ 8.1, n ϭ 18) in duncle. Scolex proper 187.5–250 (219.6 Ϯ 18.6, n ϭ 12) long, 124.8– Ϯ Ϯ ϭ diameter. In the vicinity of the ovary vitelline duct 12–14.4 (13.2 168 (150.2 15.9, n 5) wide, consisting of armed rostellum and 2 1.3, n ϭ 6) in diameter, from each side run in the median direction, large bothria, 1 dorsal and 1 ventral. Rostellar armature consisting of 1 uniting into a common vessel that joins the vagina close to the junction dorsal and 1 ventral group of 33 large central apical hooks flanked on each side by 8 smaller lateral hooklets. Each group of large central of the latter with the uterine duct. Detached gravid proglottids 2,361.6–3,888 (3,396.8 Ϯ 583.3, n ϭ 6) long by 336–441.6 (377.6 Ϯ 39.7, n ϭ 6) wide. Eggs ovate, unem- Received 22 March 2006; revised 10 November 2006; accepted 25 bryonated, 24–28.8 (27.4 Ϯ 1.4, n ϭ 26) long, 14.4–21.6 (18.4 Ϯ 2.1, January 2006. n ϭ 26) wide.

897 898 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 1–7. Macrobothridium sinensis n. sp. (1) Ventral view of rostellar armature. (2) Lateral view of rostellar armature. (3) Alternate hook types. (4) Whole worm. (5) Mature proglottid. (6) Detail of genitalia. (7) Eggs. c., cirrus; c.s., cirrus sac; g.p., genital pore; M.g., Mehlis’ gland; o., ovary; r.s., receptaculum seminis; t., testes; u., uterus; u.d., uterine duct; u.p., uterine pore; v., vagina; v.d., vas deferens; vit.d., vitelline duct; vit.f., vitelline follicle. LI AND WANG—A NEW CESTODE SPECIES M. SINENSIS N. SP. 899

FIGURES 8–14. Scanning electron micrographs of Macrobothridium sinensis n. sp. (8) Scolex. (9) Lateral view of scolex. (10) Palmate microtriches on cephalic peduncle. (11) Three digits pectinate on distal bothrial surfaces. (12) Lateral view between 2 bothria. (13) Six–seven digits palmate microtriches on proximal bothrial surfaces. (14) Border between distal and proximal bothrial surfaces marked abrupt change in microtrich type from 3 digits pectinate to 6–7 digits palmate microtriches.

Taxonomic summary vious morphological character of unarmed cephalic peduncle, Type host: Platyrhina sinensis (Bloch et Schneider, 1801) (Rajifor- the authors also support the presence of the Macrobothridium. mes: Platyrhinidae). Macrobothridium sinensis is the first record of Diphyllidea Type locality: Xiamen, Fujian, China. 24Њ28ЈN, 118Њ10ЈE. in China and the first reported cestode parasite in Platyrhina Site of infection: Spiral intestine. sinensis. The discovery of M. sinensis brings the total number Type specimens: Holotype no. C2005121801 and 5 paratypes, Nos. C2005110401–C2005110403 and C2005120101–C2005120102, are de- of species in Macrobothridium to 4. All the species have an posited in Parasitology Research Laboratory, School of Life Sciences, armed apical rostellum and unarmed cephalic peduncle. In the Xiamen University, Xiamen, Fujian, China. lateral surfaces of the M. sinensis rostellum, hooklets form 2 Etymology: This species is named for the type host Platyrhina sinen- staggered rows, thereby joining the hooks in the ventral/dorsal sis. surfaces (Fig. 2). In contrast, hooklets on the lateral surfaces of Remarks other species in Macrobothridium and Echinobothrium are arranged in a single row. The lateral hooks of E. fautleyae Tyler Since the erection of Macrobothridium by Khalil and Abdul-Salam and Caira, 1999 are arranged in 2 staggered rows according to (1989), 3 species and 2 unidentified species have been described. All the species in Macrobothridium have armed apical rostellum and un- the figures in their article. armed cephalic peduncle. Macrobothridium sinensis is most similar to Neifar et al. (2001) described the mode of attachment by M. euterpes in strobila length (1.77–6.23 vs. 2–4.5 mm) and number species of Macrobothridium to the gut mucosa of their respec- of fragments (6–8 vs. 5–9). However, it can be distinguished from M. tive hosts. In M. syrtensis and M. euterpes, only the anterior euterpes by the number (16–24 vs. 27–46) and single-row arrangement part of scolex proper is inserted into the crypt; M. sinensis has of testes. Testes number and arrangement are also differences among M. syrtensis, M. rhynchobati, and M. sinensis. In addition, M. sinensis the same attachment mode with the apical hooks inserted into differs from M. rhynchobati in the number of proglottids (6–8 vs. 82– the gut mucosa of host, which is different from the species of 115), length of strobila (1.77–6.23 vs. 30–40 mm), shape of ovary (H Echinobothrium. vs. V); M. sinensis also differs from M. syrtensis in strobila length According to Khalil (1994), a uterine pore is absent in spe- (1.77–6.23 vs. 1–1.5 mm), number of apical hooks (33 vs. 19–21) and cies of Diphyllidea. However, the uterine pore of a species of ovary shape (H vs. V). Macrobothridium is described here for the first time. The absence of description of other species in Macrobothridium does DISCUSSION not mean the lack of a uterine pore in the other 3 species. Ivanov and Hoberg (1999) suggested that Macrobothridium Neifar et al. (2001) suggested that all 3 known species of (specifically M. rhynchobati) may belong within Echinoboth- Macrobothridium reported are parasites of Rhinobatos sp. The rium Van Beneden, 1894, based on the morphological charac- exception is the host species, P. siensis, for M. siensis. Caira ters. Neifar et al. (2001) pointed out that the Ivanov and Hor- and Tracy (2002) reported that species of Yorkeria are parasites Chiloscyllium Y. southwelli berg (1999) analysis was only preliminary, and that the paucity of bamboo sharks, i.e., sp.; Desh- mukh, 1979, from Ginglymostoma concolor is also an excep- of morphological characters used in their analysis left a robust tion. phylogeny out of reach. Therefore, they thought the phylogenetic analysis by Ivanov and Hoberg (1999) was not sufficient ACKNOWLEDGMENT to synonymize Macrobothridium with Echinobothrium, and This study was supported by the Natural Science Fund of China, maintained the validity of Macrobothridium. Based on the ob- grant 40406031. 900 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

LITERATURE CITED NEIFAR, L., G. A. TYLER, AND L. EUZET. 2001. Two new species of Macrobothridium (Cestoda: Diphyllidea) from Rhinobatid elas- CAIRA,J.N.,AND R. TRACY. 2002. Two new species of Yorkeria (Tet- mobranchs in the Gulf of Gabe`s, Tunisia, with notes on the status raphyllidea: Onchobothriidae) from Chiloscyllium punctatum (Elas- of the genus. Journal of Parasitology 87: 673–680. mobranchii: Hemiscylliidae) in Thailand. Journal of Parasitology OLSON,P.D.,AND J. N. CAIRA. 1999. Evolution of the major lineages 88: 1172–1180. of tapeworms (Platyhelminthes: Cestoidea) inferred from 18S ri- IVANOV,V.A.,AND E. P. HOBERG. 1999. Preliminary comments on a bosomal DNA and Elongation factor-1␣. Journal of Parasitology phylogenetic study of the order Diphyllidea van Beneden in Carus, 85: 1134–1159. 1963. Systematic Parasitology 42: 21–27. ———, D. TIMOTHY,J.LITTLEWOOD,R.A.BRAY, AND J. MARIAUX. KHALIL, L. F. 1994. Order Diphyllidea van Beneden in Carus, 1863. In 2001. Intrerrelationships and evolution of the tapeworms (Platy- Keys to the cestode parasites of vertebrates, L. F. Khalil, A. Jones, helminthes: Cestoda). Molecular Phylogenetics and Evolution 19: and R. A. Bray (eds.). CAB International, Wallingford, Oxon, U.K., 443–467. p. 45–49. WOJCIECHOWSKA, A., E. PISANO, AND K. ZDZITOWIECKI. 1995. Cestodes ———, AND J. ABDUL-SALAM. 1989. Macrobothridium rhynchobati n. in ﬁshes at the Heard Island (Subantarctic). Polish Polar Research g., n. sp. from the elasmobranch Rhynchobatus granulatus, repre- 16: 205–212. senting a new family of diphyllidean cestodes, the Macrobothridi- ZHU, Y. D. 1984. The ﬁshes of Fujian province (Part I). Fujian Science idae. Systematic Parasitology 13: 103–109. and Technology Press, Fujian, China, 528 p. J. Parasitol., 93(4), 2007, pp. 901–906 ᭧ American Society of Parasitologists 2007

A NEW SPECIES OF COMEPHORONEMA (NEMATODA: CYSTIDICOLIDAE) FROM THE STOMACH OF THE ABYSSAL HALOSAUR HALOSAUROPSIS MACROCHIR (TELEOSTEI) FROM THE MID-ATLANTIC RIDGE

Frantisˇek Moravec and Sven Klimpel* Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇ eske´ Budeˇjovice, Czech Republic. e-mail: [email protected]

ABSTRACT: A new species of parasitic nematode Comephoronema macrochiri n. sp. (Cystidicolidae), is described from the stomach of the marine deep-sea fish Halosauropsis macrochir (abyssal halosaur) from the Mid-Atlantic Ridge (MAR). The new species, studied with both light and scanning electron microscopy, is characterized mainly by 6 pairs of preanal papillae, by which it principally differs from members of Ascarophis; the spicules are 297–375 ␮m and 99–120 ␮m long and fully developed eggs possess 2 long filaments on 1 pole. Rhabdochona beatriceinsleyae is transferred to Comephoronema as C. beatriceinsleyae (Holloway and Klewer, 1969) n. comb. Comephoronema macrochiri differs from all other congeners mainly in having eggs with filaments on 1 pole only, and from individual species by some additional features such as the number of preanal papillae, the shape of pseudolabial projections, and the body and organ measurements.

The Halosauridae consists of 15 species that are entirely ma- were washed in physiological saline and then fixed in 70% ethanol. For rine, and have their greatest diversity on the upper continental light microscopy (LM), the nematodes were cleared with glycerine. Drawings were made with the aid of a Zeiss drawing attachment. After and insular slopes, down to at least 5,000 m, but with most the LM examination, 3 specimens were also used for scanning electron species occurring between 1,100 and 3,300 m water depth microscopy (SEM). The specimens were transferred to 4% formalde- (Campbell et al., 1980; Froese and Pauly, 2006). The predom- hyde solution and then postfixed in 1% osmium tetroxide, dehydrated inant species of the family is Halosauropsis macrochir (Gu¨n- through a graded ethanol series, critical point dried, and sputter coated with gold; they were examined with the use of a JEOL JSM-6300 scan- ther, 1878), a relatively large (up to 90 cm long), very active ning electron microscope at an accelerating voltage of 15 kV. All mea- fish species; in combination with a high abundance, it is a po- surements are in micrometers unless otherwise stated. The scientific tentially important apex predator in the deep sea, feeding on names of fishes follow FishBase (Froese and Pauly, 2006). bottom invertebrates (polychaetes, crustaceans, echinoderms, echiurids) (Froese and Pauly, 2006). Halosauropsis macrochir DESCRIPTION is an important inhabitant of the deep-sea fish community at Comephoronema macrochiri n. sp. the Mid-Atlantic Ridge. (Figs. 1 and 2) The parasite fauna of H. macrochir is characterized by 9 General: Small, whitish nematodes. Cuticle thick, with fine trans- different parasite species belonging to the Digenea (6 species), verse striations distinctly visible, particularly in middle part of body Cestoda (1), and Nematoda (2) (Klimpel et al., 2001). The par- (Fig. 2H). Cephalic end rounded, with 2 conspicuous conical pseudo- asite diversity in the deep sea may be quite considerable, be- labial terminal protrusions with rounded tips (Figs. 1A–C, 2B, C). Oral cause approximately 421 of the 3,800–4,200 known deep-sea aperture oval, with lateral extensions (Figs. 1C, 2A). Lateral pseudolabia rather large, each provided with conspicuous conical terminal pro- fish species have been studied for their metazoan parasites so trusion; in apical view, flat inner parts of pseudolabia partly cover far. Therefore, it seems likely that many undescribed parasite mouth and are somewhat dorsoventrally expanded, forming 2 (1 dor- species have yet to be found. In July 2004, during helminthol- solateral and 1 ventrolateral) extensions on each; inner margins of both ogical investigations of marine deep-sea fishes from the Mid- pseudolabia dorsoventrally straight, parallel to each other (Figs. 1C, 2A). Four bilobed submedian sublabia well developed (Figs. 1C, 2A, Atlantic Ridge, a few conspecific cystidicolid nematodes ini- C). Four submedian cephalic papillae and pair of lateral amphids pre- tially identified as species of Comephoronema Layman, 1933 sent. Vestibule (stoma) long, with funnel-shaped anterior prostom visi- were isolated from the stomach of H. macrochir. As their mor- ble in lateral view. Glandular esophagus 4-5 times longer than muscular. phology differs considerably from that of congeneric and other Nerve ring encircles muscular esophagus approximately at border of its related species, they are considered to represent a new species, first and second fourths; excretory pore between nerve ring and anterior end of glandular esophagus; deirids small, simple, with distal end lat- which is described below. erally expanded (Figs. 1D, 2G), situated somewhat anterior to midlength of vestibule. MATERIALS AND METHODS Male (5 specimens; measurements of holotype in parentheses): Length of body 4.45–7.41 (5.47) mm, maximum width 75–109 (109). Fish were sampled in July 2004 on board of the Norwegian research Height of cephalic protrusions 3 (3). Vestibule including prostom 96– vessel G. O. Sars during the field phase of the international project Mar- 177 (162) long; prostom 12 (12) long and 8–9 (9) wide. Length of Eco (Census of Marine Life) along the Mid-Atlantic Ridge (MAR). muscular esophagus 222–366 (240), maximum width 18–24 (24); length Sampling was conducted with a bottom trawl at a trawling speed of of glandular esophagus 0.93–1.29 (1.13) mm, maximum width 39–51 approximately 1.73 knots and a towing time of 59.5 min. At super (39); length ratio of muscular and glandular parts of esophagus 1:3.5– station 40 (position 42Њ54.91ЈN, 30Њ20.37ЈW; trawling depth range 4.7 (1:4.7). Length of entire esophagus and vestibule represents 24–28 2,660–2,670 m; bottom depth ca. 2,960 m), a total of 42 specimens of (28)% of total body length. Nerve ring and excretory pore 174–246 H. macrochir was captured. All fishes were deep-frozen at Ϫ40Cim- (192) and 339–345 (342), respectively, from anterior extremity; deirids mediately for later necropsy in the laboratory. The nematodes recovered indistinct in LM. Posterior end of body spirally coiled, provided with narrow caudal alae. Preanal papillae: 6 pairs of subventral pedunculate papillae present, of which first and second, third and fourth, and fifth Received 22 September 2006; revised 29 January 2007; accepted 31 and sixth are shifted in relation to each other. Postanal papillae: 6 pairs January 2007. present, including 5 pairs of pedunculate subventral papillae and 1 pair * Institute of Zoomorphology, Cell Biology and Parasitology, Heinrich- of minute ventral sessile papillae located approximately at level of last Heine-University, Universita¨tsstrasse 1, 40225 Du¨sseldorf, Germany. pair of subventrals. In addition, a pair of very small ventral papilla-like

901 902 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 phasmids present just posterior to caudal papillae (Fig. 2E). Small me- Beneden, 1871; Spinitectus Fourment, 1884; Capillospirura dian ventral caudal protuberance present between both ventral papillae. Skryabin, 1924; Metabronema Yorke and Maplestone, 1926; Ventral cuticular ridges (area rugosa) anterior to cloaca well developed, consisting of about 13 longitudinal rows of tessellated ornamentations Cystidicoloides Skinker, 1931; Cyclozone Dogiel, 1932; Co- (Fig. 2F). Large (left) spicule 297–375 (297) long, with obtuse distal mephoronema Layman, 1933; Pseudoproleptus Khera, 1953; tip provided with ventral membranous conical outgrowth when extruded Parascarophis Campana-Rouget, 1955; Salvelinema Trofimen- from nematode body (Fig. 1L); its shaft 99–120 (99) long, forming 30– ko, 1962; Ctenascarophis Mamaev, 1968; Spinitectoides Petter, 38% (33%) of overall length of spicule. Small (right) spicule broad, 90–102 (90) long, with rounded distal end provided with small cuticular 1969; Cristitectus Petter, 1970; Neoascarophis Machida, 1976; membrane (Fig. 1J). Length ratio of spicules 1:3.3–3.7 (1:3.3). Tail Caballeronema Margolis, 1977; Prospinitectus Petter, 1979; conical, 90–174 (129) long, with rounded tip. Pseudascarophis Ko, Margolis and Machida, 1985; and Simi- Female (3 gravid specimens with mature eggs; measurements of al- lascarophis Munõz, Gonza´lez and George-Nascimento, 2004 lotype in parentheses; measurements of 1 additional specimen with immature eggs in brackets): Length of body 11.21–14.19 (13.21) [10.07] (see Chabaud, 1975; Appy and Anderson, 1982; Ko et al., 1985; mm, maximum width 136 (136) [95]. Height of cephalic protrusions 3 Moravec and Nagasawa, 1999; Munõz et al., 2004). (3) [3]. Vestibule including prostom 150–204 (189) [216] long; prostom Of these, most genera distinctly differ from nematodes of the 12–15 (15) [indistinct] long and 9–12 (12) [–] wide. Length of muscular present material. In general, the morphology of these nematodes esophagus 291–441 (414) [411], maximum width 24–27 (24) [21]; resemble species of Ascarophis, Caballeronema, Capillospiru- length of glandular esophagus 1.38–1.87 (1.69) (1.38) mm, maximum width 48–60 (48) [33]; length ratio of muscular and glandular parts of ra, Cystidicoloides, Pseudascarophis, and Similascarophis, esophagus 1:4.1–4.7 (1:4.1) [1:3.4]. Length of entire esophagus and which are very similar to each other; minute differentiating fea- vestibule represents 15–18 (17) [20]% of total body length. Nerve ring, tures among them exist only in details of their cephalic struc- excretory pore and deirids 189–294 (246), 321–363 (363) [not located] tures visible by SEM. However, the pattern of these structures and 112–138 (135) [105], respectively, from anterior extremity. Tail conical, short, 63–69 (69) [81] long, with low knoblike terminal for- seems to be more or less identical, with various intermediate mation (Fig. 1I). Vulva slightly postequatorial, situated 6.07–7.55 (7.37) features, showing rather interspecific than intergeneric differ- [5.70] mm from anterior end of body, at 51–56 (56) [57]% of body ences. In fact, most representatives of these genera were, or are, length; vulval lips not elevated. Amphidelphic. Uterus filled with nu- reported within a catchall genus, Ascarophis (see Ferrer et al., merous eggs, occupies major part of body, reaching level of anus posteriorly (Fig. 1I). Mature eggs (containing larvae) elongate–oval, thick- 2005). The same pattern of cephalic structures has recently been walled, size 48–51 ϫ 21–24 (48–51 ϫ 21–24); thickness of egg wall 3 observed in SEM by Moravec (unpubl. obs.) in Comephoro- (3). One pole of egg bears small knob provided with 2 thread-like fil- nema oschmarini Trofimenko, 1974, a stomach parasite of Lota aments up to about 330 long; filaments may differ considerably in lota (L.) in Palaearctic Eurasia (Moravec et al., 1999); there- length (Fig. 1F–H). Immature eggs without filaments or 1 or 2 very fore, the only feature distinguishing Comephoronema from As- short filaments present on 1 pole. carophis is more numerous pairs of male preanal papillae in Taxonomic summary the former. Type host: Abyssal halosaur Halosauropsis macrochir (Halosauridae, The cephalic structure of nematodes from H. macrochir is Notacanthiformes). practically identical (Figs. 1C, 2A) with that of the type species Site of infection: Stomach. of Ascarophis, A. morrhuae van Beneden, 1871, as illustrated Њ Ј Њ Ј Type locality: Mid-Atlantic Ridge (42 54.91 N, 30 20.37 W; depth by Ko (1986); their general morphology (except for the number 2660–2670 m; date 7 July 2004). Prevalence and intensity: Prevalence, 57.1% (24 fishes infected/42 of preanal papillae) agrees well with the diagnosis of this genus fishes examined); intensity, 1–10 (mean 3) nematodes per fish. (Ko, 1986). According to Ferrer et al. (2005), Ascarophis in- Deposition of specimens: Holotype, allotype, and paratypes deposited cludes 53 nominal species, 33 of which are currently considered in the Helminthological Collection of the Institute of Parasitology in valid; 12 of them possess egg filaments on a single pole, as in Cˇ eske´ Budeˇjovice (Cat. No. N-868). Other material: Voucher material deposited in the Helminthological the nematodes of the present material. Of them, only Ascaro- Collection of the Institute of Parasitology, Du¨sseldorf, Germany. phis filiformis Polyansky, 1952, has the lengths of spicules and Etymology: The name macrochiri is derived from the specific name the size of eggs comparable with the new species, but the body of the type host of this nematode. of A. filiformis is much longer (9–15 and 19–36 mm in male and female, respectively) and the male has only 4 pairs of pre- DISCUSSION anal papillae (Appy, 1981; Ko, 1986). Ferrer et al. (2005) and Moravec et al. (2006) mentioned that However, all species reported in Ascarophis, Caballeronema, the present taxonomy of cystidicolid nematodes is rather con- Capillospirura, Cystidicoloides, and Similascarophis possess 4 fused; several genera have been based on details in the cephalic pairs of preanal papillae, whereas Pseudascarophis has 3 (Mar- structures visible only with the aid of SEM, and it will be ev- golis, 1977; Appy and Anderson, 1982; Petter, 1984; Ko, 1986; ident whether or not these tiny features are of generic impor- Munõz et al., 2004), whereas there are 6 pairs of these papillae tance only as more cystidicolids are described with the use of (arranged in couples) in the new species. By this feature, it SEM and comparative molecular data become available. resembles Comephoronema spp., particularly C. oschmarini, At present, the following 19 cystidicolid genera are usually and, therefore, it is assigned to this genus. Chabaud (1975) syn- considered valid: Cystidicola Fischer, 1798; Ascarophis van onymized Comephoronema with Cystidicola, but Moravec and

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FIGURE 1. Comephoronema macrochiri n. sp. (A–B) Anterior end of gravid female, lateral and dorsoventral views. (C) Cephalic end, apical view. (D) Deirid, lateral view. (E) Anterior end of male, lateral view. (F–H) Fully developed eggs. (I) Tail of gravid female, lateral view. (J) Right spicule, lateral view. (K) Caudal end of male (holotype), lateral view. (L) Same of another specimen (paratype). Scale bars: A, B, F–H, J ϭ 30 ␮m; C ϭ 25 ␮m; D ϭ 1 ␮m; E, K, L ϭ 100 ␮m. MORAVEC AND KLIMPEL—C. MACROCHIRI N. SP. 903 904 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 2. Comephoronema macrochiri n. sp. scanning electron microscopy micrographs. (A) Cephalic end, apical view. (B–C) Cephalic end, lateral and sublateral views. (D) Male tail, lateral view (arrows shows subventral postanal papillae). (E) Tail tip of male with last 2 pairs of caudal papillae (h, i), phasmids (arrow) and median ventral caudal protuberance (r), subventral view. (F) Precloacal ventral cuticular ridges (area rugosa) on male caudal end, ventral view. (G) Deirid, lateral view. (H) Transverse striation of cuticle. a ϭ amphid; b ϭ pseudolabial protrusion; d ϭ pseudolabium with anterior protrusion; e ϭ cephalic papilla; f ϭ short spicule; g ϭ cloacal aperture; h ϭ postanal papilla of last subventral pair; i ϭ ventral postanal papilla; l ϭ submedian labium; r ϭ ventral caudal protuberance; s ϭ sublabium. Scale bars: A, C, G ϭ 2 ␮m; B, H ϭ 3 ␮m; D–F ϭ 10 ␮m. MORAVEC AND KLIMPEL—C. MACROCHIRI N. SP. 905

Nagasawa (1999) revalidated this genus, indicating that it Thanks are also due to the staff of the Laboratory of Electron Micros- ˇ shows more affinities to Salvelinema than to Cystidicola. How- copy, Institute of Parasitology, ASCR, in Ceske´ Budeˇjovice for their technical assistance, and to Irena Husa´kova´, a technician of the same ever, it cannot be excluded that, based on a broad taxonomic Institute, for her help with illustrations. This study was supported by revision, Comephoronema will become a junior synonym of grant 524/06/0170 from the Grant Agency of the Czech Republic and Ascarophis in the future. by the research projects of the Institute of Parasitology, ASCR At present, Comephoronema comprises 2 valid species, Co- (Z60220518 and LC522). Furthermore, the study was also supported by mephoronema werestschagini Layman, 1933 (type species), a the German Research Council (DFG KL 2087/1-1) and the Forschungs- und Innovationsfonds of the Heinrich-Heine University Du¨sseldorf parasite of the digestive tract of the endemic fishes of the scor- (Klimpel 10007). paeniform, Abyssocottidae, Comephoridae, and Cottocome- phoridae in the Lake Baikal basin (Siberia, Russia), and Co- LITERATURE CITED mephoronema oschmarini Trofimenko, 1962, a stomach parasite of burbot, Lota lota, in Palaearctic Eurasia (Vismanis et al., APPY, R. G. 1981. Species of Ascarophis van Beneden, 1870 (Nema- 1987; Moravec et al., 1999). An additional 2 species, Come- toda: Cystidicolidae) in North Atlantic fishes. Canadian Journal of Zoology 59: 2193–2205. phoronema johnsoni Arya, 1978 and Comephoronema mack- ———, AND R. C. ANDERSON. 1982. The genus Capillospirura Skrja- iewiczi Malhotra and Rautela, 1984, were poorly described bin, 1924 (Nematoda: Cystidicolidae) of sturgeons. Canadian Jour- from fishes in India (Arya, 1979; Malhotra and Rautela, 1984), nal of Zoology 60: 194–202. but both of them are species inquirendae probably belonging to ARYA, S. N. 1979. On a new nematode, Comephoronema johnsoni sp. other genera. nov. (Family: Rhabdochonidae Skrjabin, 1946) from a marine fish Cybium guttatum. Indian Journal of Helminthology 30: 17–20. Holloway and Klewer (1969) described Rhabdochona bea- CAMPBELL, R. A., R. L. HAEDRICH, AND T. A. MUNROE. 1980. Parasitism triceinsleyae Holloway and Klewer, 1969, a gastrointestinal and ecological relationship among deep-sea benthic fishes. Marine parasite of the zoarcid (eelpout) Lycodichthys (as Rhigophila) Biology 57: 301–313. dearborni (DeWitt) (Zoarcidae, Perciformes) from McMurdo CHABAUD, A. G. 1975. Keys to genera of the order Spirurida. Part 2. Spiruroidea, Habronematoidea and Acuarioidea. In CIH keys to the Sound, Antarctica. Later it was transferred by Moravec (1972) nematode parasites of vertebrates, No. 3, R. C. Anderson, A. G. to Cystidicola. Comephoronema beatriceinsleyae was also re- Chabaud, and S. Willmott (eds.). Commonwealth Agricultural Bu- ported by Rocka (2002, 2004) from Lycodichthys antarcticus reaux, Farnham Royal, U.K., p. 1–58. Pappenheim, from the Weddell Sea, Antarctica. Even though FERRER, E., F. J. AZNAR,J.A.BALBUENA,A.KOSTADINOVA,J.A.RAGA, Holloway and Klewer (1969) did not study this species by AND F. M ORAVEC. 2005. A new cystidicolid nematode from Mullus surmuletus (Perciformes: Mullidae) from the western Mediterra- SEM, they provided a relatively good description. It is now nean. Journal of Parasitology 91: 335–344. clear that its cephalic structure is unlike that among Cystidicola FROESE,R.,AND D. PAULY. 2006. FishBase. World Wide Web electronic spp. (see Moravec and Rokicki, 2002) and that the structures publication, www.fishbase.org, version 8/2006. taken by the authors for anterior prostomal teeth were, in fact, HOLLOWAY, H. L., AND H. L. KLEWER. 1969. Rhabdochona beatriceinsleyae n. sp. (Nematoda: Spiruridea: Rhabdochonidae), from the bilobed sublabia. Considering other features, in particular the Antarctic zoarcid, Rhigophila dearborni. Transactions of the Amer- number of preanal papillae, we transfer this species to Come- ican Microscopical Society 88: 460–471. phoronema as C. beatriceinsleyae (Holloway and Klewer, KLIMPEL, S., A. SEEHAGEN,H.W.PALM, AND H. ROSENTHAL. 2001. 1969) n. comb. Deep-water metazoan fish parasites of the world. Logos Verlag, Of the 3 valid species of Commephoronema, C. macrochiri Berlin, Germany, 316 p. KO, R. C. 1986. A preliminary review of the genus Ascarophis van n. sp. is most similar to C. beatriceinsleyae. However, the latter Beneden, 1871 (Nematoda: Cystidicolidae) of the gastrointestinal has filaments on both egg poles, the left spicule is distinctly tract of fishes. Occasional Publications, Department of Zoology, longer (485–573 ␮m), the pseudolabia are rounded in a lateral University of Hong Kong, China, 54 p. view, the glandular esophagus is approximately a half of that ———, L. MARGOLIS, AND M. MACHIDA. 1985. Pseudascarophis ky- phosi in C. macrochiri, although the body is much longer (8–15 and n. gen., n. sp. (Nematoda: Cystidicolidae) from the stomach of the fish Kyphosus cinerascens (Foska˚l) from Japan. Canadian 16–32 mm in male and female, respectively), and the tail of the Journal of Zoology 63: 2684–2688. gravid female is markedly longer (163–240 vs. 63–69 ␮m). The MALHOTRA,S.K.,AND A. S. RAUTELA. 1984. A new rhabdochonid nem- number of preanal papillae is identical (6 pairs) in both species, atode, Comephronema [sic] mackiewiczi n. sp. from a hill-stream as well as the shape of the distal tip of the left spicule. Co- teleost of a new zoogeographical region. Rivista di Parassitologia 45: 285–291. mephoronema oschmarini also has 6 pairs of preanal papillae, MARGOLIS, L. 1977. Caballeronema gen. nov. for Metabronema wardlei but, in contrast to C. macrochiri, its pseudolabia are more Smedley, 1934 (Nematoda, Spiruroidea) from the marine fish Scor- rounded in a lateral view, the length of the glandular esophagus paenichthys marmoratus from the Pacific coast of Canada. Univ- is nearly doubled, and the eggs, which are somewhat larger ersidad Nacional AutońomadeMe´xico, Instituto de Biologıá. Pub- (56–62 ϫ 24–26 ␮m), have numerous filaments on each pole licaciones Especiales 4: 447–454. MORAVEC, F. 1972. General characterization of the nematode genus and at their equatorial zone. Comephoronema werestschagini is Rhabdochona with a revision of the South American species. Acta easily distinguished by more numerous preanal papillae (10 Societatis Zoologicae Bohemoslovacae 36: 196–208. pairs, or 8 ϩ 11 papillae) and numerous short filaments on ———, V. HANZELOVA´ ,T.SCHOLZ, AND D. GERDEAUX. 1999. Come- either egg pole. In contrast to the new species, both C. osch- phoronema oschmarini (Nematoda: Cystidicolidae), a specific parasite of Lota lota (Pisces), occurs in western Europe. Folia Paras- marini and C. werestschagini are parasitic in freshwater fishes. itologica 46: 159–160. ———, S. KLIMPEL, AND E. KARA. 2006. Neoascarophis macrouri n. ACKNOWLEDGMENTS sp. (Nematoda: Cystidicolidae) from the stomach of Macrourus berglax (Macrouridae) in the eastern Greenland Sea. Systematic The authors thank the scientific staff and the crew of the research Parasitology 63: 231–237. vessel G. O. Sars for their help during the collection of the material. ———, AND K. NAGASAWA. 1999. Morphology and taxonomy of Sal- 906 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

velinema species (Nematoda: Cystidicolidae), swimbladder para- matoidea (Spirurida). Description de 4 espe`ces nouvelles de la fam- sites of Pacific area salmonids. Folia Parasitologica 46: 123–131. ille des Cystidicolidae. Revue Suisse de Zoologie 91: 935–952. ———, AND J. ROKICKI. 2002. Some new data on the morphology of ROCKA, A. 2002. Nematodes of fishes of the Weddell Sea (Antarctic). Cystidicola farionis (Nematoda, Cystidicolidae), a swimbladder Acta Parasitologica 47: 294–299. parasite of salmonids, as revealed by SEM. Acta Parasitologica 47: ———. 2004. Nematodes of the Antarctic fishes. Polish Polar Research 29–33. 25: 135–152. MUN˜ OZ, G., M. T. GONZA´ LEZ, AND M. GEORGE-NASCIMENTO. 2004. Sim- VISMANIS,K.O.,V.V.LOMAKIN,V.D.ROYTMAN,M.K.SEMENOVA, ilascarophis n. gen. n. sp. (Nematoda: Cystidicolidae) parasitizing AND V. Y A.TROFIMENKO. 1987. Nemathelminths–Nemathelminthes. marine fishes off the Chilean coast. Journal of Parasitology 90: In Key to parasites of freshwater fishes of the USSR fauna. Volume 823–834. 3. Parasitic metazoans, Part 2, O. N. Bauer (ed.). Nauka, Leningrad, PETTER, A.-J. 1984. Nematodes de poissons du Paraguay II. Habrone- Russia, p. 199–310. J. Parasitol., 93(4), 2007, pp. 907–909 ᭧ American Society of Parasitologists 2007

A NEW SPECIES OF PARACAPILLARIA (NEMATODA: CAPILLARIIDAE) FROM THE INTESTINE OF THE TOAD DUTTAPHRYNUS MELANOSTICTUS (ANURA) FROM THE MALAYAN PENINSULA

Frantisˇek Moravec, David Modry´*, and Miloslav Jirku˚ Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇ eske´ Budeˇjovice, Czech Republic. e-mail: [email protected]

ABSTRACT: A new species of parasitic nematode, Paracapillaria malayensis n. sp. (Capillariidae), is described from the small intestine of the toad Duttaphrynus melanostictus imported from the Malayan Peninsula to the Czech Republic. The new species differs from the only other congeneric species, Paracapillaria spratti, mainly in the shape and structure of the spicular proximal end (with a lobular rim), smaller eggs (45–51 ϫ 21–24 ␮m), longer spicule (336 ␮m), and the number (37–38) of stichocytes in gravid females; whereas P. spratti parasitizes frogs of the Microhylidae in Papua New Guinea, P. malayensis is a parasite of Bufonidae in the Malayan Peninsula. Other Paracapillaria spp. are parasites of ﬁshes, birds, or mammals and they mostly differ from P. malayensis in the structure of eggs and some other morphological features.

During recent studies of parasites of amphibians carried out verse annuli; nuclei of stichocytes large; some stichocytes darker than by both junior authors, capillariid nematodes were recovered others, this difference being indistinct in cleared specimens. Nerve ring encircling muscular esophagus at about its first quarter. Two small wing- from the small intestine of the toads, Duttaphrynus melanostic- like cells present at esophago-intestinal junction. tus (Schneider) (Anura), imported from Malayan Peninsula into Male (1 specimen, holotype): Length of body 3.90 mm, maximum the Czech Republic. A subsequent examination showed that width 54. Width of lateral bacillary bands 15. Length of entire esoph- they represent a previously undescribed species of Paracapil- agus 2.11 mm (54% of body length). Length of muscular esophagus 306, of stichosome 1.80 mm; number of stichocytes 37; stichocytes at laria Mendonça, 1963. It is described below. posterior part of stichosome 36–70 long and 27–39 wide. Nerve ring situated 78 from anterior extremity. Seminal vesicle elongate. Spicule MATERIALS AND METHODS well sclerotized, with almost smooth surface, 336 long. Proximal end Two adult D. melanostictus were obtained from commercial import of spicule distinctly expanded and provided with folded, lobular rim, (Python spol. s.r.o.) into the Czech Republic in March 2006. The ani- distal end rounded; width of spicule at proximal and distal ends 8 and mals originated from the Malayan Peninsula. After keeping them in the 3, respectively. Spicular canal not developed. Surface of spicular sheath laboratory, the toads were killed and examined for the presence of hel- smooth, without spines. Tail rounded, 12 long, provided with cuticular minth parasites on 6 June 2006. The nematodes recovered were washed membrane forming very short bursa. Bursa supported by 2 wide lateral in physiological saline and then fixed in hot 4% formaldehyde solution. digital projections (rays) curved to median line in ventral view; their For light microscopy, the nematodes were cleared with glycerine. Draw- ends at level of posterior border of membranous bursa. One pair of ings were made with the aid of a Zeiss drawing attachment. Addition- large spherical subventral papillae present at base of lateral projections, ally, 18 adult specimens of D. melanostictus (including the 2 above- at about level of cloacal opening. Length of whole bursa in ventral view mentioned toads) from the same import were repeatedly (2–6 times) 10, its maximum width 22; length of membranous margin of bursa 2. examined coprologically. Fecal samples were collected from individu- Lateral caudal alae absent. ally housed animals, suspended in water, and examined for the presence Female (1 complete and 2 incomplete gravid specimens; measure- of parasites by standard flotation method using Sheather’s sacharose ments of allotype in parentheses): Body length of complete specimen solution (s.g. 1.3). Eggs concentrated by flotation method were exam- (allotype) 5.71 (5.71) mm, of incomplete specimens (paratypes) 4.62 ined using the light microscope Olympus AX 70 equipped with No- and 6.66 mm; maximum width of body 54–82 (68). Width of lateral marski interference-contrast optics (NIC). All measurements are in mi- bacillary bands 21–33 (21). Entire esophagus 2.24–2.27 (2.24) mm, crometers unless otherwise stated. It is worth noting that we use the forming (39%) of body length. Length of muscular esophagus 354–405 recently established generic name, Duttaphrynus Frost, Grant, Faivov- (405), of stichosome 1.84–1.92 (1.84) mm; stichocytes 37–38 (38) in ich, Bain, Haas, Haddad, De Sa, Channing, Wilkinson, Donnellan, Rax- number. Nerve ring 75–87 (75) from anterior extremity. Vulva situated worthy, Campbell, Blotto, Moler, Drewes, Nussbaum, Lynch, Green, at about level of esophago-intestinal junction; vulval lips not elevated. and Wheeler, 2006, for the type host throughout the text, instead of its Eggs arranged in single file in uterus. Eggs oval, sometimes irregular widely known synonym, Bufo melanostictus. in shape, polar plugs slightly protruding; egg wall 2-layered; inner layer hyaline, outer layer of eggs in uterus thin, with fine, almost indistinct DESCRIPTION sculpture on surface (Fig. 1J); outer layer of eggs from host’s feces thicker, with sparse irregular sculpture on surface and, in optical section, Paracapillaria malayensis n. sp. fine transverse canals reaching to inner layer (Fig. 2). Eggs from uterus (Figs. 1, 2) including polar plugs 45–51 ϫ 21–24 (45–51 ϫ 21–23), thickness of General: Small filiform nematodes. Anterior end narrow, rounded; egg wall 2 (2); width of polar plugs 6 (6), their total height 5–6 (5), cephalic papillae indistinct. Two lateral bacillary bands distinct, fairly height of their protruding part 2–3 (2). Content of fully developed eggs wide, extending along almost the whole body length. Muscular esoph- uncleaved. Caudal end rounded, anus subterminal; tail 9 (9) long. Rec- agus relatively long. Stichosome consisting of 37–38 stichocytes sub- tum 175–190 (190) long. divided usually (mainly in posterior part of stichosome) into 5–8 trans- Taxonomic summary

Received 20 November 2006; revised 5 January 2007; accepted 10 Type host: Duttaphrynus melanostictus (Bufonidae, Anura). January 2007. Site of infection: Anterior part of small intestine. * Department of Parasitology, Faculty of Veterinary Medicine, Univer- Type locality: Apparently the Malayan Peninsula (imported into sity of Veterinary and Pharmaceutical Sciences, Palacke´ho 1-3, CZ- Czech Republic in March 2006). 612 42 Brno, Czech Republic. Prevalence and intensity: In both toads examined, intensity 1 and 3

907 908 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 1. Paracapillaria malayensis n. sp. (A) Anterior end of male. (B) Stichocytes in posterior part of stichosome. (C) Region of vulva, lateral view. (D–E) Proximal and distal ends of spicule. (F) Posterior end of male, lateral view. (G) Same, higher magniﬁcation. (H) Stichosome region of female body with distinct lateral bacillary band, lateral view. (I) Caudal end of female, lateral view. (J) Fully developed egg from uterus. (K–L) Caudal end of male, ventral and lateral views. Scale bars: A–C, H, I ϭ 50 ␮m; D, E, J–L ϭ 30 ␮m; F ϭ 200 ␮m.

specimens. Coprology revealed the presence of eggs of P. malayensis asitology, Faculty of Veterinary Medicine, University of Veterinary and sp. n. in feces of 13, out of 18 toads examined (prevalence 72%). Pharmaceutical Sciences, Brno, Czech Republic (Cat. No. R 23/06). Deposition of types: Holotype, allotype, and 2 paratypes in the Hel- minthological Collection of the Institute of Parasitology, Biology Cen- Remarks tre, ASCR, Cˇ eske´ Budeˇjovice (Cat. No. N-870). Symbiotype (sensu According to Moravec (2001), adult capillariid nematodes parasitiz- Frey et al., 1992) deposited in the collection of the Department of Par- ing amphibians are represented by species of 6 genera: Amphibiocap- MORAVEC ET AL.—A NEW SPECIES OF PARACAPILLARIA 909

(45–51 ϫ 21–24 ␮m vs. 66–72 ϫ 27–30 ␮m), the spicule of P. malayensis is somewhat longer (336 ␮m vs. 306 ␮m), and there are differences in the numbers of stichocytes (37–38 vs. 32–33) in gravid females of both species. Paracapillaria spratti and P. malayensis are reported from hosts belonging to different families (Microhylidae vs. Bufonidae) and from different geographical regions (Papua New Guinea vs. the Malayan Peninsula). In contrast to P. malayensis and P. spratti from amphibians, all remaining species of Paracapillaria from ﬁshes, except for Paracapillar- ia parophrysi (Moravec, Margolis, and McDonald, 1981), have fully developed eggs without protruding polar plugs; however, P. parophrysi can be easily distinguished from P. malayensis mainly by its thick- walled eggs. Differences between P. malayensis and all other individual species of the subgenus Paracapillaria are apparent from the key provided by Moravec (2001). Wang (1982) described another capillariid, Capillaria fujianensis Wang, 1982, from the intestine of D. melanostictus (referred to as B. melanostictus) in China. Because this species was inadequately described and little attention was paid to the structure of the male caudal end, it cannot be assigned to any genus of the current taxonomic system of capillariids. Therefore, Moravec (1987) designated it a species inquirenda; later, he (Moravec, 2001) speculated that it might belong to Amphibiocapillaria.

ACKNOWLEDGMENTS

FIGURE 2. Paracapillaria malayensis n. sp., eggs from host’s feces, This study was supported by the research project of the Institute of NIC. (A) Egg surface with sparse irregular sculpture. (B) Same egg; in Parasitology, Biology Centre, ASCR no. Z60220518 (F.M.), and by the optical section, outer layer of egg wall appearing to contain ﬁne trans- research grants 206/03/1544 and 524/03/H133 (M.J., D.M.) from the verse canals reaching to inner layer. A, B in same scale. Scale bar ϭ Grant Agency of the Czech Republic. M.J. is indebted to Jakub Bedna´rˇ 20 ␮m. (Python spol. s.r.o.) for organization of import of animals.

LITERATURE CITED illaria Moravec, 1982; Aonchotheca Lo´pez-Neyra, 1947; Capillaria Zeder, 1800; Paracapillaria Mendonça, 1963; Pseudocapillaria Freitas, FREY, K. F., T. L. YATES,D.W.DUSZYNSKI,W.L.GANNON, AND S. L. 1959; and Pseudocapillaroides Moravec and Cosgrove, 1982. By their GARDNER. 1992. Designation and curatorial management of type general morphology, particularly in having a smooth spicular sheath and host specimens (symbiotypes) for new parasite species. Journal of a caudal bursa supported by 2 lateral fingerlike processes (rays) in the Parasitology 78: 930–932. male, the specimens of the present material belong to Paracapillaria. HASEGAWA, H. 1989. Nematodes of Okinawan amphibians and their This genus includes 3 subgenera, of which 10 species of Ophidiocap- host-parasite relationship. In Current herpetology in east Asia, M. illaria Moravec, 1986, are parasites of reptiles (snakes); the only species Matsui, T. Hikida, and R. C. Goris (eds.). Proceedings of the Sec- of Crossicapillaria Moravec, 2000, is parasitic in birds and mammals ond Japan-China Herpetological Symposium, Kyoto, July 1988. (Moravec, 2001). Species of the nominotypical subgenus Paracapillaria Herpetological Society of Japan, Kyoto, Japan, p. 205–217. are mostly parasites of fishes (10 species), and only 2 are reported from MORAVEC, F. 1987. Review of capillariid nematodes (Capillariinae) par- amphibians, i.e., Paracapillaria spratti (Moravec and Sey, 1986) from asitic in amphibians and reptiles. Part 1. General introduction, gen- the intestine of the microhylid frogs Phrynomantis stictogaster Zweifel era Capillaria, Aonchotheca and Paratrichosoma. Acta Societatis and Phrynomantis humicola Zweifel from Papua New Guinea (Moravec Zoologicae Bohemoslovacae 50: 120–131. and Sey, 1986; Moravec, 1990) and an undescribed species Paracap- ———. 1990. Additional records of nematode parasites from Papua illaria sp. from the intestine of a ranid frog Rana narina Stejneger from New Guinea amphibians with a list of recorded endohelminths by Okinawa Island, Japan (Hasegawa, 1989). Also P. malayensis n. sp. amphibian hosts. Folia Parasitologica 37: 43–58. belongs to the subgenus Paracapillaria. ———. 2001. Trichinelloid nematodes parasitic in cold-blooded ver- Although the morphology of P. malayensis is very similar to that of tebrates. Academia, Prague, Czech Republic, 417 p. P. spratti, both species differ distinctly in the shape and structure of the ———, AND O. SEY. 1986. Three new nematode species from Phry- proximal end of the spicule, i.e., it is only slightly expanded and with nomantis spp. (Amphibia: Microhylidae) from Papua New Guinea. a smooth surface in P. spratti, whereas it is markedly expanded and Folia Parasitologica 33: 343–351. provided with a folded, lobular rim in P. malayensis. Moreover, fully WANG, P. Q. 1982. Studies on nematodes of the family Capillariidae developed eggs of P. malayensis are smaller than those of P. spratti from Fujian. Acta Zootaxonomica Sinica 7: 117–126. J. Parasitol., 93(4), 2007, pp. 910–916 ᭧ American Society of Parasitologists 2007

TWO NEW SPECIES OF COSMOCERCIDS (ASCARIDIDA) IN THE TOAD CHAUNUS ARENARUM (ANURA: BUFONIDAE) FROM ARGENTINA

Geraldine Ramallo, Charles R. Bursey*, and Stephen R. Goldberg† Instituto de Invertebrados, Fundacio´n Miguel Lillo, Miguel Lillo 251 (4000), San Miguel de Tucuma´n, Argentina. e-mail: [email protected]

ABSTRACT: Aplectana tarija n. sp. and Cosmocercoides lilloi n. sp., intestinal parasites of the toad Chaunus arenarum, are described. Of the 41 nominal species of Aplectana, A. tarija n. sp. represents the 22nd Neotropical species and the second species reported from Argentina. Of the 14 nominal species of Cosmocercoides, C. lilloi n. sp. represents the 15th species and the ﬁrst species reported from South America.

Members of the Bufonidae are known from all zoogeograph- DESCRIPTIONS ic realms except the Australo-Papuan (Duellman, 1999). Of the Aplectana tarija n. sp. 43 species (sensu Frost et al., 2006) assigned to Chaunus Wag- (Figs. 1–7) ler 1828, 17 occur in Argentina (Montero and Autino, 2004). General: Small slender nematodes. Cuticle bearing transverse stria- Chaunus arenarum (Hensel, 1867) is found in both arid and tions from lips to tail. Triangular mouth surrounded by 1 dorsal and 2 humid environments, and it occurs in southern Brazil, Argen- subventral lips. Cephalic sense organs consisting of inner circle of 3 tina (south to Chubut Province), Uruguay, and Bolivia, from minute papillae and a circle of 4 submedian papillae, 1 on each subventral lip and 2 on dorsal lip (Fig. 2). Two amphids present. Lateral sea level up to 2,600 m, and possibly Paraguay (Cei, 1980; alae not observed. Anterior part of esophageal corpus narrow compared IUCN, 2006). with posterior part isthmus short, and bulb with valvular apparatus. The Cosmocercoidea are mainly parasites of vertebrates and Excretory pore near posterior end of esophageal bulb (Fig. 1). Tail of presumably evolved in amphibians and reptiles (Vanderburgh both sexes slender and sharply pointed. Female: Based on 3 gravid specimens; measurements of holotype in and Anderson, 1987). Species of both Aplectana Railliet and parentheses: length of body 4.7–5.8 (4.82); maximum width 0.49–0.51 Henry, 1916 and Cosmocercoides Wilkie, 1930 are intestinal (0.44). Esophagus 0.59–0.62 (0.60) long (including bulb), bulb 0.12– parasites of reptiles and amphibians (Baker, 1987). Bursey et 0.15 (0.13) long, 0.15–0.19 (0.15) wide. Nerve ring and excretory pore al. (2006) listed 41 species of Aplectana, of which 22 have 0.20–0.28 (0.21) and 0.47–0.56 (0.43) from anterior extremity, respectively. Didelphic. Amphidelphic. Vulva postequatorial, 2.00–2.50 (2.20) Neotropical distribution. In Argentina, only Aplectana delirae from caudal end (Fig. 3). Vagina runs forward from the vulva. Ovary has been reported (Gonza´lez and Hamann, 2006). Fourteen spe- and uterus not extending anteriorly beyond esophageal bulb. Eggs in cies of Cosmocercoides have been described, but none is known uterus, thin shelled with embryos in different stages of development from Neotropical hosts (Table I). 0.10–0.09 (0.08) long, 0.05–0.08 (0.05) wide (Fig. 4). Tail 0.27–0.40 (0.34) long (Fig. 5). The purpose of this article is to describe a new species of Male: Based on 2 adult specimens; measurements of allotype in pa- Aplectana and a new species of Cosmocercoides, the second, rentheses: length of body 3.11 (3.00), maximum width 0.41 (0.36). and first species of either genus, respectively, recorded for Ar- Esophagus 0.44 (0.45) long (including bulb); bulb 0.10 (0.10) long, 0.10 gentina. (0.11) wide. Nerve ring and excretory pore 0.17 (0.19) and 0.38 (0.31) from anterior extremity, respectively. Spicules 0.12 (0.11) long, equal, pointed at distal end. Gubernaculum absent. Five pairs of caudal papil- MATERIALS AND METHODS lae (1 pair preanal; 4 pairs postanal), 1 unpaired median papilla. Tail 0.27 (0.25) long (Figs. 6, 7). Four C. arenarum, 2 (1 female, 1 male; snout-to-vent length [SVL]: 110 mm and 80 mm, respectively) from Itau´ River (22Њ20Ј51ЉS, Taxonomy summary 64Њ05Ј47.7ЉW), and 2 males (SVL: 83 mm and 81 mm, respectively) from Astilleros (22Њ23Ј01ЉS, 64Њ06Ј14.4ЉW), Department of General San Type specimen: Holotype: female CH-FML # 07442-A; allotype: Martıń, Province of Salta, were collected by hand in September 2005 male CH-FML 07442-B, and paratypes (2 females) CH-FML 07442-C. and examined for helminths. The toads were fixed in neutral buffered Type host: Chaunus arenarum (Hensel, 1867) (Anura, Bufonidae), 10% formalin before preservation in 70% ethanol. The body cavity was Herpetology Collection FML 17187, collected in 2005. Type locality: Itau´ River (22Њ20Ј51ЉS, 64Њ05Ј47.7ЉW), General San opened by a mid-ventral incision, and the digestive tract was removed. Martıń Deparment, Province of Salta, Argentina. The esophagus, stomach, and small and large intestines were longitu- Site of infection: Large intestine. dinally slit and the contents were examined for helminths using a dis- Prevalence: One (25%) of 4 Chaunus arenarum was infected. secting microscope. Nematodes were cleared in lactophenol and ex- Intensity: Seventeen nematodes per frog. amined via light microscopy. Drawings were made with the aid of Leica Etymology: The specific epithet is named for the Tarija River, Bolivia. microscope. Some quantitative descriptors of parasite populations were calculated based on the definition of Bush et al. (1997). All the mea- Remarks surements are given in millimeters unless otherwise stated. Type specimens have been deposited in the Coleccioń Helmintolo´gica Of the 22 species of Aplectana found in the Neotropical realm (Table Fundacioń Miguel Lillo (CH-FML), Miguel Lillo 251, (4000) San Mi- II), 7 lack a gubernaculum, namely, A. artigasi, A. chilensis, A. crucifer, guel de Tucumań, Argentina. A. delirae, A. meridionalis, A. papillifera, and Aplectana tarija n. sp. Of these, the spicules of A. meridionalis and Aplectana tarija n. sp. are similar in length (130–136 ␮m and 120 ␮m, respectively); the other species listed above have spicules greater than 150 ␮m in length. Aplec- Received 25 November 2006; revised 23 January 2007; accepted 23 tana meridionalis is differentiated from A. tarija n. sp. by papillae pat- January 2007. tern: A. meridionalis has a 7:0:5 ϩ 1 pattern compared with the 1:0:4 * Department of Biology, The Pennsylvania State University, Shenango ϩ 1 pattern of A. tarija n. sp. Campus, 147 Shenango Avenue, Sharon, Pennsylvania 16146. Aplectana tarija n. sp. represents the second species of the genus to † Deparment of Biology, Whittier College, Whittier, California 90608. be reported from Argentina.

910 RAMALLO ET AL.—A. TARIJA N. SP., C. LILLOI N. SP. 911 (1978) (1981) (1981) papillae Reference Somatic 1 Present Absent Karve (1944) 0 Absent Absent Wilkie (1930) swellings Lateral alae Prebulbar 1 0 Present Present Wilkie (1930) 1 0 Present Absent Harwood (1930) ϩ ϩ 2 0 Absent Absent This study ϩ Papillae 0:20 14 22-30:0:6; 0:0:16 Rosette*; simple* species. m) ␮ ( Guber- naculum 100–120 26-32:2:12; 0:0:12 2 Present Present Arya (1979) 110–160 24-36:2:6; 0:0:4 m) ␮ Cosmocercoides 134 420 m) Spicule ( ␮ 2.24 157 Equal: 140 Absent 20:0:0, 20:2:18-19 0 Present Absent Wang et al. 2.62 320 Equal: 112 45 16:0:0, 0:0:6 0 Present Absent Wang et al. 2.40 302 Equal: 192 105 20:0:0, 8:0:8 0 Present Absent Wang et al. median. ϩ Khera, 1958 5.8–6.4 250–280 Equal: 200–240 130–140 21-27:2:8 1 Present Present Khera (1958) Arya, 1979 4.9–5.5 230–250 130–151; 112– Rao, 1979 1.35 250 Equal: 210 760 10:2:2; 0:0:6 0 Present Absent Rao (1979) (Wang, Zhao, and Chen, Wang, Sun, Zhao, and Rao, 1979 2.11 200 240; 230 60 16:0:4; 0:0:14 0 Present Absent Rao (1979) (Baylis, 1927) 4.5–5.0 170 Equal: 580 188 44:0:2; 0:0:6 0 Absent Absent Baylis (1927) (Harwood, 1930) 2.2–5.0 125–220 Equal: 250– Wilkie, 1930 6.9 220 Equal: 247 143 28-30:4:2; many:0: Karve, 1944 4.60–4.92 200–240 Equal: 190–260 120–145 18-26:2:6; present: Wilkie, 1930 2.70–4.96 140–210 Equal: 270–530 96–146 Ogden, 1966 3.07 161 Equal: 168 121 28:2:6; 0:0:8 0 Present Present Ogden (1966) (Holl, 1928) 2.55 240 Equal; 290 102 22:0:2; not stated 0 Not stated Absent Holl (1928) Wang, Sun, Zhao, and Zhang, n. sp. 2.02–2.44 160–210 Equal:100–120 Absent 18-19:0:8 Biogeographical region Length (mm) Tail ( I. Biogeographical regions, selected features, and references for males of 1978) Zhang, 1981 1981 C. lilloi C. rickae C. barodensis C. bufonis C. lanceolatus C. multipapillata C. nainitalensis C. dukae C. variabilis C. microhylae C. oligodentis C. pulcher C. ranae C. tibetanum C. tridens ABLE Neotropical Oriental T Nearctic * Preanal:adanal:postanal Palaearctic 912 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 1–7. Aplectana tarija n. sp. (1) Female, anterior end, lateral view. (2) Female, apical view. (3) Female, vulva, lateral view. (4) Egg. (5) Female, posterior end, lateral view. (6) Male, posterior end, ventral view. (7) Male, spicules.

Cosmocercoides lilloi n. sp. ynx, cylindrical corpus, and posterior bulb containing valves. Excretory (Figs. 8–15) pore anterior to esophageal bulb. Anterior part of esophageal corpus General: Small, stout nematodes. Triangular mouth opening sur- narrow compared with posterior part; isthmus small (Fig. 8). Vulva rounded by 1 dorsal and 2 subventral lips. Cephalic sense organs con- equatorial. Males possess complex caudal papillae; each papilla is sur- sisting of inner circle of 6 minute papillae and a circle of 4 submedian rounded by a ring of cuticular tubercles. Lateral alae absent. Tail of papillae, 1 on each subventral lip and 2 on dorsal lip. Two amphids both sexes long and tapering. present (Fig. 9). Lateral alae not observed. Esophagus with short phar- Female: Based on 4 gravid specimens; measurements of holotype in RAMALLO ET AL.—A. TARIJA N. SP., C. LILLOI N. SP. 913

TABLE II. Biogeographical regions, selected features, and references for males of Aplectana species.

Papillae Spicule length Gubernaculum Biogeographical region Type host order (␮m) (␮m) Pattern Reference

Australian A. novaezelandiae Baker and Green, Anura 103–123 23–30 Irregular Baker and Green (1987) 1987 A. zweifeli Moravec and Sey, 1986 Anura 450–456 96–105 8-10: 0: 9 ϩ 1 Moravec and Sey (1986) Ethiopian A. brygoo Baker, 1980 Anura 249 51 8: 0: 6-7 ϩ 1 Baker (1980) A. capensis Baker, 1981 Anura 203 35 Irregular Baker (1981) A. chamaeleonis (Baylis, 1929) Squamata 190–220 75 9: 0: 6 ϩ 1 Baylis (1929) A. courdurieri Chabaud and Brygoo, Anura 365 50 Irregular Chabaud and Brygoo 1958 (1958) A. degraafﬁ Baker, 1981 Anura 173 60 Յ8: 0: 6 ϩ 1 Baker (1981) A. praeptialis (Skrjabin, 1916) Anura 220–270 Absent Irregular Skrjabin et al. (1916) A. vercammeni Le Van Hoa, 1962 Anura 240–250 Absent Irregular Le Van Hoa (1962) Nearctic A. incerta Caballero, 1949 Anura 135–143 52–63 11: 0: 5 ϩ 1 Baker (1985) A. itzocanensis Bravo Hollis, 1943 Anura 172–203 67–80 11: 0: 5 ϩ 1 Baker (1985) Neotropical A. tarija n. sp. Anura 110–120 Absent 2:0:8 ϩ 1 This study A. albae Adamson and Baccam, 1988 Squamata 61–63 41–46 6-7: 1: 5 ϩ 1 Adamson and Baccam (1988) A. artigasi Puga and Torres, 1997 Anura 322–364 Absent Յ14: 1: 10-14 ϩ Puga and Torres (1997) 2 A. chilensis Lent and Freitas, 1948 Anura 260–410 Absent 12: 0: 6 ϩ 1 Lent and Freitas (1948) A. crucifer Travassos, 1925 Anura 330 Absent 6: 1: 4 Travassos (1925) A. delirae (Fabio, 1971) (ϭA. crosso- Anura 150–170 Absent Many: 0:6 Fabio (1971) dactyli Baker, 1980*) A. elenae Baker and Vaucher, 1986 Anura 97 28 8: 1: 5 ϩ 1 Baker and Vaucher (1986) A. hamatospicula Walton, 1940 Anura 236–245 70–72 4: 0: 7 ϩ 1 Walton (1940) A. herediaensis n. sp. Squamata 67–79 43–52 9: 0: 3 Bursey et al. (2006) A. hylambatis (Baylis, 1927) Anura 260–293 90–100 8: 1: 5 ϩ 1 Baker (1980) A. lopesi Silva, 1955 Anura 150 34 Irregular Silva (1955) A. membranosa (Schneider, 1866) Anura 227–234 71 5: 2: 4 ϩ 1 Miranda (1924) A. meridionalis Lent and Freitas, 1948 Anura 130–136 Absent 7: 0: 5 ϩ 1 Lent and Freitas (1948) A. mexicana (Caballero, 1933) Gymnophiona 450 56 3: 0: 5 Caballero (1933) A. micropenis Travassos, 1925 Anura 60 10 4: 0: 1 Travassos (1925) A. papillifera (Araujo, 1977) Squamata 400 Absent Irregular Araujo (1977) A. paraelenae Baker and Vaucher, 1986 Anura 227 53 3: 2: 6 ϩ 1 Baker and Vaucher (1986) A. pusilla Miranda, 1924 Squamata 110 49 3: 2: 6 ϩ 1 Miranda (1924) A. raillieti Travassos, 1925 Squamata 230 32 8: 0: 9 ϩ 1 Travassos (1925) A. rysavi Barus and Coy Otero, 1969 Squamata 72 11 7: 2: 4 ϩ 3 Barus and Coy Otero (1969) A. travassosi (Gomes and Motta, 1967) Anura 230–245 80–95 5: 1-2: 4 ϩ 1 Dyer (1990) A. vellardi Travassos, 1926 Anura 169–192 12 2: 2: 6 ϩ 1 Baker (1980) Oriental A. akhrami (Islam, Farooq, and Khan- Anura 150–180 Absent 8: 0: 5 ϩ 2 Islam et al. (1979) um, 1979) A. macintoshii (Stewart, 1914) Anura 130–320 22–90 Irregular Baker (1980) A. ranae (Walton, 1931) Anura 160 25 6: 2: 2-3 Walton (1931) Palearctic A. acuminata (Schrank, 1788) Anura 295–383 89–118 Irregular Baker (1980) A. brumpti Travassos, 1931 Anura 200–230 40 12: 0: 8 ϩ 1 Travassos (1931) A. hylae Wang, 1980 Anura 180–192 56–60 0: 0: 3 Wang (1980) A. leesi Hristovki and Riggio, 1975 Anura 216–247 53–72 10-13: 1: 5 ϩ 1 Baker (1980) A. linstowi York and Maplestone, 1926 Anura 175–204 53–55 10-12: 0: 9 ϩ 1 Baker (1980) A. paucipapillosa Wang, 1980 Anura 120–122 70 9:0:4 Wang (1980)

* See Boquimpani-Freitas et al. (2001). 914 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURES 8–15. Cosmocercoides lilloi n. sp. (8) Female, anterior end, lateral view. (9) Female, apical view. (10) Female, vulva, lateral view. (11) Egg. (12) Female, posterior end, lateral view. (13) Male, posterior end, ventral view. (14) Caudal papilla. (15) Male, spicules. RAMALLO ET AL.—A. TARIJA N. SP., C. LILLOI N. SP. 915 parentheses: length of body 5.23–6.40 (5.70) maximum width 0.47– ———. 1987. Synopsis of the Nematoda parasitic in amphibians and 0.52 (0.54). Esophagus 0.59–0.67 (0.60) long (including bulb); bulb reptiles. Memorial University of Newfoundland, Occasional Papers 0.13–0.14 (0.15) long, 0.15–0.17 (0.16) wide. Nerve ring and excretory in Biology, St. Johns, Newfoundland, Canada, 325 p. pore 0.24–0.25 (0.20) and 0.46–0.49 (0.47) from anterior extremity, ———, AND D. M. GREEN. 1987. Helminth parasites of native frogs respectively. Didelphic. Amphidelphic. Vulva 2.61–3.22 (2.70) from (Leiopelmatidae) from New Zealand. Canadian Journal of Zoology caudal end. Vagina runs forward from the vulva. Ovary and uterus not 66: 707–713. extending anteriorly beyond esophageal bulb. Eggs in uterus, thin ———, AND C. VAUCHER. 1986. Parasitic helminths from Paraguay XII: shelled with embryos in different stages of development 0.07–0.08 Aplectana Railliet & Henry, 1916 (Nematoda: Cosmocercoidea) (0.07) long, 0.04 (0.05) wide (Figs. 10, 11). Tail 0.38–0.51 (0.40) long from frogs. Revue Suisse de Zoologie 93: 607–616. (Fig. 12). BARUS,V.,AND A. COY OTERO. 1969. Systematic survey of nematodes Male: Based on 2 adult specimens; measurements of allotype in pa- parasitizing lizards (Sauria) in Cuba. Helminthologia 10: 329–346. rentheses: length of body 2.02 (2.44), maximum width 0.16 (0.23). BAYLIS, H. A. 1927. On two new species of Oxysomatium (Nematoda) Esophagus 0.30 (0.37) long (including bulb); bulb 0.06 (0.08) long, 0.06 with some remarks on the genus. The Annals and Magazine of (0.09) wide. Nerve ring and excretory pore 0.14 (0.15) and 0.23 (0.27) Natural History Series 9 19: 279–286. from anterior extremity, respectively. Spicules 0.10 (0.12) long, equal, ———. 1929. Some parasitic nematodes from the Uluguru and Usam- pointed at distal end. Gubernaculum absent. Tail 0.16 (0.21) long. bara Mountains, Tanganyika territory. The Annals and Magazine Fourteen pairs of caudal rosette papillae: 9 pairs preanal and 5 pairs of Natural History Series 10 4: 372–381. postanal: of the postanal papillae, 2 pairs ventral in position, and 3 pairs BOQUIMPANI-FREITAS, L., D. VRCIBRADIC,J.J.VICENTE,C.R.BURSEY, lateral in position (Figs. 13–15). C. F. D. ROCHA, AND M. VAN SLUYS. 2001. Helminths of the horned leaf frog, Proceratophrys appendiculata, from southeastern Brazil. Taxonomy summary Journal of Helminthology 75: 1–5. BURSEY, C. R., S. R. GOLDBERG, AND S. R. TELFORD,JR. 2006. New Type specimen: Holotype: female CH-FML 07447-A; allotype: male species of Aplectana (Nematoda: Cosmocercidae) and Mesocoe- CH-FML 07447-B, and paratypes CH-FML 07446. lium monas (Digenea: Brachycoeliidae) in Lepidophyma flavima- Type host: Chaunus arenarum (Hensel, 1867) (Anura: Bufonidae), culatum (Squamata: Xantusiidae) from Costa Rica. Caribbean Jour- Herpetology Collection FML 17195, collected in 2005. nal of Science 42: 164–170. Type locality: Astilleros (22Њ23Ј01ЉS, 64Њ06Ј14.4ЉW), General San BUSH,A.O.,K.D.LAFFERTY,J.M.LOTZ, AND A. W. SHOSTAK. 1997. Martıń Deparment, Province of Salta, Argentina. Parasitology meets ecology on its own terms: Margolis et al. re- Site of infection: Intestine. visited. Journal of Parasitology 83: 575–583. Prevalence: Two (50%) of 4 Chaunus arenarum were infected. CABALLERO, E. 1933. Nema´todos para´sitos de los batracios de Me´xico. Mean intensity: Four nematodes per frog. II. Oxysomatium mexicanum nov. espec. Anales del Instituto de Etymology: The specific epithet is given in honor of Dr. Miguel Lillo Biologia de la Universidad Nacional Autońoma de Mexico 4: 187– (1862–1931) in recognition of his many studies on the flora and fauna 190. of Argentina. CEI, J. M. 1980. Amphibians of Argentina. Monitore Zoologıá, Italia (N.S.), Monografıá 2, 609 p. Remarks CHABAUD,A.G.,AND E. R. BRYGOO. 1958. Description et cycle evolutif Of the 15 species of Cosmocercoides (Table I), 3 species lack lateral d’Aplectana courdurieri n. sp. (Nematoda, Cosmocercidae). Me- alae, namely, C. lilloi n. sp., C. pulcher, and C. tibetanum. These 3 moires de l’Institut Scientifique de Madgascar, Serie A 12: 159– species are easily separated by spicule length: the spicules of C. lilloi 176. n. sp. are 100–120 ␮m in length, those of C. pulcher are 247 ␮min DUELLMAN,W.E.(ED.). 1999. Patterns of distribution of amphibians. A length, and the spicules of C. tibetanum are 580 ␮m in length. It also global perspective. The Johns Hopkins University Press, Baltimore, should be noted that C. lilloi n. sp. lacks a gubernaculum, which is Maryland, 633 p. present in both C. pulcher and C. tibetanum. Cosmocercoides lilloi n. DYER, W. G. 1990. Augmented description of Aplectana travassosi sp. represents the first species of the genus to be reported from Neo- (Nematoda: Cosmocercidae) from leptodactylid frogs of Ecuador. tropical hosts. Journal of Parasitology 76: 639–640. FABIO, S. P. 1971. Sobre uma nova especie do genero Neyraplectana Ballesteros Marquez, 1945 (Nematoda, Cosmocercoidea). Atas da ACKNOWLEDGMENTS Sociedade de Biologia do Rio de Janeiro 15: 11–13. Francisco Brusquetti and Laura Ponssa provided the specimens of C. FROST, D. R., T. GRANT,J.FAIVOVICH,R.H.BAIN,A.HAAS,C.F.B. arenarum. The Fundacioń Miguel Lillo provided financial support for HADDAD,R.O.DE SA,A.CHANNING,M.WILKINSON,S.C.DON- specimen collection. NELLAN ET AL. 2006. The amphibian tree of life. Bulletin of the American Museum of Natural History 297: 1–370. GONZA´ LEZ, C. E., AND M. I. HAMANN. 2006. Nematodos para´sitos de LITERATURE CITED Chaunus granulosus major (Muller & Hellmich, 1936) (Anura: Bu- ADAMSON,M.L.,AND D. BACCAM. 1988. Systematic revision of the fonidae) en Corrientes, Argentina. Cuadernos de Herpetologıá 20: Atractidae sensu Chabaud (1978) (Nematoda; Cosmocercoidea): 41–47. Maracaya belemensis n. sp. and Aplectana albae n. sp. from Am- HARWOOD, P. D. 1930. A new species of Oxysomatium (Nematoda) with phisbaena alba in Brazil. Canadian Journal of Zoology 66: 1857– some remarks on the genera Oxysomatium and Aplectana, and ob- 1864. servations on the life history. Journal of Parasitology 17: 61–73. ARAUJO, P. 1977. Uma nova especie do genero Neyaplectana (Nema- HOLL, F. J. 1928. Two new nematode parasites. Journal of the Elisha toda: Subuluroidea: Cosmocercidae) encontrada en ofidios. Me- Mitchell Scientific Society 43: 184–186. morias, Sao Paulo, Brazil, Instituto Butantan 40/41: 259–264. ISLAM, A., M. FAROOQ, AND Z. KHANUM. 1979. Two new genera of ARYA, S. N. 1979. A new nematode, Cosmocercoides nainitalensis n. nematode parasites (Oxyuridae: Oxysomitiinae) from toad Bufo an- sp. from the frog, Rana cyanophyctis. Revista Brasileira Biologia dersoni of Pakistan. Pakistan Journal of Zoology 11: 69–73. 39: 823–825. [IUCN] CONSERVATION INTERNATIONAL AND NATURESERVE. 2006. Global BAKER, M. R. 1980. Revision of old world species of the genus Aplec- amphibian assessment. www.globalamphibians.org. Accessed 4 tana Railliet and Henry, 1916 (Nematoda, Cosmocercidae). Mu- May 2006. seum National D’Histore Naturelle 2: 955–998. KARVE, J. N. 1944. On a small collection of parasitic nematodes from ———. 1981. Cosmocercoid nematode parasites from frogs of southern Anura. Proceedings of the Indian Academy of Science 20: 71–77. Africa. Koedoe 24: 25–32. KHERA, S. 1958. On a new species of Cosmocercoides. Indian Journal ———. 1985. Redescription of Aplectana itzocanensis and A. incerta of Helminthology 10: 6–12. (Nematoda: Cosmocercidae) from Amphibians. Transactions of the LE VAN,HOA. 1962. Nematodes parasites de mammiferes, reptiles et American Microscopical Society 104: 272–277. amphibiens du Congo. Phasmidiens. Brussels, Fascicle 65: 5–55. 916 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

LENT,H.,AND J. F. T. FREITAS. 1948. Uma coleçaõ de nematodeos de Africa. Scientific Results of the Zoological Expedition to British vertebrados, do Museu de Historia Natural de Montevideo. Me- East Africa and Uganda 1: 99–157. morias do Instituto Oswaldo Cruz 46: 1–71. TRAVASSOS, L. 1925. Aplectana raillieti, n. sp. Competes Rendus des MIRANDA, C. 1924. Alguns nematodeos do geˆnero Aplectana Railliet & Seances de la Societe de Biologie et de ses Filiales 93: 973–974. Henry, 1916. Memorias do Instituto Oswaldo Cruz 17: 45–54. ———. 1931. Pesquizas helmintholo´gicas realizadas em Hamburgo. MONTERO, R., AND A. G. AUTINO. 2004. Sistema´tica y filogenia de los IX. Ensaio monographico da familia Cosmocercidae Trav., 1925 vertebrados, con eńfasis en la fauna argentina. Universidad Na- (Nematoda). Memorias do Instituto Oswaldo Cruz 25: 237–298. cional de Tucumań, Tucaman, Argentina, 317 p. VANDERBURGH, D. J., AND R. C. ANDERSON. 1987. The relationship be- MORAVEC,F.,AND O. SEY. 1986. Three new nematode species from tween nematodes of the genus Cosmocercoides Wilkie, 1930 (Nem- Phrynomantis spp. (Amphibia: Microhylidae) from Papua New atoda: Cosmocercoidea) in toads (Bufo americanus) and slugs Guinea. Folia Parasitologica 33: 343–351. (Deroceras laeve). Canadian Journal of Zoology 65: 1650–1661. OGDEN, C. G. 1966. On some parasitic nematodes from reptiles, mainly WALTON, A. C. 1931. A new parasite of Philippine Amphibia. Philip- from Ceylon. Journal of Helminthology 15: 81–90. pine Journal of Science 45: 351–353. PUGA, S., AND P. T ORRES. 1997. Aplectana artigasi n. sp. (Nematoda: ———. 1940. Notes on amphibian parasites. Proceedings of the Hel- Cosmocercidae) from the frog Eupsophus calcaratus (Anura: Lep- minthological Society of Washington 7: 87–91. todactylidae) in southern Chile. Memorias do Instituto Oswaldo WANG, P. Q. 1980. Studies on some new nematodes of the suborder Cruz 92: 767–770. Oxyurata from Fujian, China. Acta Zootaxonomica Sinica 5: 242– RAO, R. 1979. On four new species of the genera Paracosmocerca Kung 252. et Wu, 1945, Cosmocerca Diesing, 1861 and Cosmocercoides ———, Y. L. SUN,Y.R.ZHAO, AND W. H. Z HANG. 1981. Notes on five Wilkie, 1930 of the family Cosmocercidae Travassos, 1925. Hel- new species of nematodes from vertebrates in Wuyi, Fujian Prov- minthologia 16: 23–34. ince. Wuyi Science Journal 1: 113–118. SILVA, A. A. J. 1955. Nova espećie do geˆnero Aplectana Raillliet & ———, Y. ZHAO, AND C. CHEN. 1978. On some nematodes from ver- Henry, 1916 (Nematoda, Cosmocercidae). Memo´rias do Instituto tebrates in south China. Fujian Shida Xuebao 2: 75–90. Oswaldo Cruz 52: 415–418. WILKIE, J. S. 1930. Some parasitic nematodes from Japanese amphibia. SKRJABIN, K. I. 1916. Parasitic trematodes and nematodes collected by The Annals and Magazine of Natural History, Series 10 6: 606– the expedition of Prof. V. Dogiel and I. Sokolov in British East 614. J. Parasitol., 93(4), 2007, pp. 917–921 ᭧ American Society of Parasitologists 2007

NEW SPECIES OF HAPLOMETROIDES (DIGENEA: PLAGIORCHIIDAE) FROM PHALOTRIS NASUTUS (GOMES, 1915) (SERPENTES, COLUBRIDAE)

Reinaldo Jose´ da Silva, Vanda Lu´ cia Ferreira*, and Christine Stru¨ ssmann† UNESP—Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biocieˆncias, Distrito de Rubiaõ Juńior, Botucatu, Saõ Paulo, Brazil, CEP 18618-000. e-mail: [email protected]

ABSTRACT: A new species of Haplometroides (Digenea, Plagiorchiidae) is described from a specimen of Phalotris nasutus (Gomes, 1915) (Serpentes, Colubridae). The host snake was obtained in the municipality of Corumba´, Mato Grosso do Sul State, Brazil. Trematodes were recovered from esophagus, stomach, and small intestine of the host. The main characteristic of the new species is the vitellaria, which is intercecal, cecal, and extracecal in the preacetabular region. A key for identiﬁcation of the species in Haplometroides is proposed.

Species of Haplometroides Odhner, 1911 parasitize snakes diagonal position. Ovary postacetabular, ovoid, with regular contours, and amphisbaenians from South America (see Silva et al., 2005, pretesticular, intercecal. Mehlis’ gland median, located below and to the right of ovary. Seminal receptacle elongated, submedian, placed near and references therein). Only 2 species are presently included the ovary and Mehlis’ gland. Vitellaria consisting of numerous follicles in this genus, i.e., Haplometroides buccicola Odhner, 1911 and in lateral position, distributed from area slightly posterior of cecal bi- Haplometroides odhneri Ruiz and Perez, 1959. The aim of the furcation to posterior testis area. Vitellaria intercecal, cecal, and extra- present study is to describe a new species of Haplometroides, cecal in the preacetabular region and cecal or extracecal posterior to found in a specimen of the fossorial snake Phalotris nasutus acetabulum. Laurer’s canal not observed. Well-developed uterus, oc- cupying most of posttesticular region, with branches reaching the ace- (Gomes, 1915) collected in the State of Mato Grosso do Sul, tabular region. Metraterm long and narrow. Eggs ovoid, operculated, Brazil. thick shelled, and with 2 small, apical and lateral protuberances. Ex-

MATERIALS AND METHODS The host snake, an adult male P. nasutus, measuring 420 mm for snout–vent length and 60 mm for tail length, was collected in 23 March 2006, at Nhumirim Farm (Nhecolaˆndia region, Southern Pantanal), municipality of Corumba´, Mato Grosso do Sul State, Brazil. It is deposited in the Coleçaõ Zoolo´gica de Refereˆncia do Campus de Corumba´, Seçaõ Herpetologia (CEUCH), of the Universidade Federal de Mato Grosso do Sul, Brazil. The necropsy of the host snake revealed trematodes in the esophagus (n ϭ 5), stomach (n ϭ 1), and small intestine (n ϭ 1). These trematodes were fixed in AFA solution under slight pressure of coverslip for 10 min, and then transferred to 70% alcohol for further processing. Five of them were stained with carmine, cleared with creosote, and analyzed in a computerized system for image analysis (Qwin Lite 3.1, Leica Microsystems, Wetzlar, Germany). Measurements for these 5 specimens are here reported as the mean and range (minimum– maximum). The holotype was deposited in the Coleçaõ Helmintolo´gica of the Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro State, Brazil. Four paratypes were deposited in the Coleçaõ Helmintolo´gica de Re- fereˆncia (CHIBB) of the Departamento de Parasitologia, Instituto de Biocieˆncias, Universidade Estadual Paulista—UNESP, municipality of Botucatu, Saõ Paulo State, Brazil.

DESCRIPTION Haplometroides intercaecalis n. sp. (Figs. 1–3; Table I) General: Body elongated, ﬂattened, covered with spines. Oral sucker subterminal, muscular, wider than long. Prepharynx short and broad. Pharynx muscular, short, and subspherical. Esophagus long and narrow. Intestinal ceca bifurcated, not very sinuous, slightly unequal, with smooth walls, terminating in the region between the testes or after the posterior testis. Acetabulum pre-equatorial, muscular, wider than long. Genital pore preacetabular, postbifurcal, submedian, opening near the right cecal branch. Cirrus pouch preacetabular, small, with an opposed curvature beside the ovary, presenting seminal vesicle and elongated cirrus. Testes ovoid, postovarian, intercecal, with regular contours, in

Received 21 November 2006; revised 27 January 2007; accepted 27 January 2007. * Departamento de Cieˆncias do Ambiente, Campus de Corumba´, Uni- FIGURE 1. Haplometroides intercaecalis n. sp. (holotype; CHIOC versidade Federal de Mato Grosso do Sul, MS. 36810) (Digenea, Plagiorchiidae) from the esophagus of Phalotris na- † Departamento de Produc¸a˜o Animal, Faculdade de Agronomia e Me- sutus (Gomes, 1915) (Serpentes, Colubridae) (ventral view, bar scale ϭ dicina Veterina´ria, Universidade Federal de Mato Grosso, Cuiaba´, MT. 1 mm).

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FIGURE 2. Detail of the anterior portion of the vitellaria of the paratypes of Haplometroides intercaecalis n. sp. (ventral view). Note the intercecal, cecal, and extracecal distribution of the vitellaria between cecal bifurcation and cirrus pouch. Site of infection: (A–B) esophagus, (C) stomach, and (D) small intestine. Magniﬁcation, ϫ10.

cretory vesicle not observed. Excretory pore terminal, at middle line of Type locality: Nhumirim Farm (18Њ59Ј0ЉS, 56Њ39Ј0ЉW), Nhecolaˆndia posterior end. region, Corumba´ municipality, State of Mato Grosso do Sul, Brazil. Type material: Holotype—CHIOC 36810 (specimen from esopha- Taxonomic summary gus); paratypes—CHIBB 1789 (specimens from esophagus), CHIBB Type host: P. nasutus (Serpentes, Colubridae)—CEUCH 3696. 1790 (specimen from stomach), and CHIBB 1891 (specimen from small Site of infection: Esophagus, stomach, and small intestine. intestine). SILVA ET AL.—NEW SPECIES OF HAPLOMETROIDES 919

TABLE I. Morphometrical data of specimens of Haplometroides intercaecalis n. sp., Haplometroides buccicola*, and Haplometroides odhneri*.

Haplometroides buccicola Haplometroides intercaecalis n. sp. (n ϭ 5) (n ϭ 21) Haplometroides odhneri (n ϭ 9) Variables Type specimen Mean (range) (␮m) Mean (range) (␮m) Mean (range) (␮m)

Length 6,271 6,657 (6,271–7,577) 7,231 (6,069–8,211) 4,902 (4,403–5,236) Width 1,177 958 (796–1,177) 1,586 (1,285–2,047) 981 (714–1,190) Oral sucker Length 326 262 (302–334) 563 (521–638) 377 (330–415) Width 347 340 (321–357) 579 (521–660) 349 (298–393) Acetabulum Length 268 280 (266–307) 391 (319–489) 336 (298–393) Width 328 309 (272–328) 393 (351–489) 298 (191–351) Dist OS-AC 1,937 2,169 (1,937–2,553) 1,991 (1,428–2,451) 1,126 (952–1,428) Prepharynx Length 43 53 (41–74) — — Width 126 122 (102–162) — — Pharynx Length 160 161 (147–171) 203 (180–223) 161 (106–213) Width 154 150 (127–169) 228 (180–277) 163 (117–202) Esophagus Length 660 688 (526–793) 818 (638–1,042) 375 (276–457) Width 84 53 (24–84) — — Dist IC-PE 2,245 1,935 (1,618–2,375) 2,228 (1,666–2,784) 1,874 (1,547–2,047) Anterior testis Length 299 339 (256–426) 397 (340–468) 392 (319–500) Width 308 304 (246–355) 407 (319–500) 330 (266–404) Posterior testis Length 342 404 (342–508) 420 (287–510) 426 (330–564) Width 396 374 (303–423) 428 (329–479) 359 (276–425) Cirrus pouch Length 418 461 (413–524) 918 (851–957) 342 (234–393) Width 70 121 (70–164) 164 (138–191) 93 (53–138) Ovary Length 248 233 (220–248) 319 (277–372) 278 (224–319) Width 264 243 (220–264) 288 (255–319) 217 (159–276) Seminal receptacle Length 111 152 (111–211) 194 (170–277) 173 (117–213) Width 269 305 (206–409) 72 (42–95) 98 (63–170) Metraterm Length — — 517 (436–660) — Width — — 61 (53–74) — Eggs† Length 50.9 50.3 (42.5–57.1) 45.7 (44.6–46.8) 35.6 (33.8–37.2) Width 36.4 35.2 (29.9–41.4) 26.9 (24.3–28.4) 24 (22.7–25)

* Data from Ruiz and Perez (1959); Dist OS-AC, distance between oral sucker and acetabulum; Dist IC-PE, distance between intestinal ceca and posterior end of the body. † Mean of 10 eggs/specimen of H. intercaecalis n. sp.

Etymology: The speciﬁc epithet is derived from the disposition of the Two new Haplometroides species, both from amphibians (Haplo- vitelline follicles, which are intercecal in the preacetabular region. metroides rappiae Szydat, 1932 and Haplometroides eburnense Maeder, 1969), were subsequently included in this genus. Haplometroides rap- Remarks piae was described from the intestine of Rappia concolor Hallowell, Species of Haplometroides belong to the Plagiorchiidae (Yamaguti, 1844 from Liberia (Szydat, 1932). Later, this species was reevaluated, 1971). This genus was originally proposed for a trematode species, H. transferred to Ostioloides, and renamed Ostioloides rappiae (Odening, buccicola (Fig. 4A–C), found in the oral cavity of a coral snake Mi- 1960 apud Gasmann, 1975). crurus sp. (ϭElaps sp. in Odhner, 1911) from Paraguay. Haplometroides eburnense was described from Phrynobatrachus na- 920 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

cecal bifurcation to the posterior testis region and intestinal ceca extending beyond the posterior testis. Haplometroides odhneri (Fig. 4D–F) has vitelline follicles distributed from the esophagus or cecal bifurcation region to the ovarian region and intestinal ceca that extends up to the zone of the anterior testis, never reaching the posterior one. Haplometroides intercaecalis n. sp. differs from the 2 previously described species of the same genus mainly by the position of the vitellaria and shape of the eggs. In H. intercaecalis n. sp., the anterior portion of the vitellaria is distributed through intercecal, cecal and extracecal regions. The eggs are longer and wider than those of H. buccicola and H. odhneri. The esophagus and cirrus pouch have an intermediate length in H. intercaecalis n. sp. in comparison with the situation found in the other 2 species; also, the oral sucker, acetabulum, ovary, and seminal receptacle have smaller dimensions. The intestinal ceca in H. intercaecalis n. sp. ends between the testes (holotype specimen) or after the posterior testis (3 of the paratype specimens), a similar pattern to the one in H. buccicola and distinct from the pattern in H. odhneri, in which the intestinal ceca extends up to the zone of the anterior testis. With the morphological characters presented for the 3 species of Hap- lometroides taken into account, a key for identiﬁcation of the species is proposed below. FIGURE 3. Eggs of Haplometroides intercaecalis n. sp. (bar scale ϭ 50 ␮m). Key to the species of Haplometroides: 1a. Vitellaria mainly extracecal in the preacetabular region ..... 2 1b. Vitellaria intercecal, cecal and extracecal in the preacetabular talensis Smith, 1849 and Rana longirostris Witte, 1921, from the Ivory region ...... H. intercaecalis n. sp. Coast, and in Rana fuscigula angolensis Bocage, 1866 and P. natalensis 2a. Vitellaria distributed from esophagus or cecal bifurcation to in Uganda. The classiﬁcation of this trematode species has been studied ovary region ...... H. odhneri by several authors. Gasmann (1975) reallocated this species to Plagi- 2b. Vitellaria distributed from cecal bifurcation to posterior testis tura, and renamed the species as Plagitura eburnense. Bougart and region ...... H. buccicola Combes (1979) disagreed with the conclusions of Gasmann (1975), erected Maederia for this species, and included Maederia eburnense in Except for the terrestrial boid snake Epicrates cenchria crassus this genus. Finally, Goodman (1986) created a new genus, Phrynoba- (Cope, 1862) parasitized by H. buccicola (Ruiz and Perez, 1959), all trachotrema, which presently includes Phrynobatracotrema eburnense. other taxa parasitized by species of Haplometroides are fossorial or Ruiz and Perez (1959) redescribed H. buccicola and described an- semifossorial. This includes the amphisbaenid Amphisbaena alba Lin- other species of Haplometroides, H. odhneri, from a Brazilian specimen naeus 1758 (Ruiz and Perez, 1959), the elapid Micrurus frontalis (Du- of the coral snake Micrurus lemniscatus Linnaeus, 1758 (Fig. 4D–F). me´ril, Bibron et Dume´ril, 1854) (Silva and Barrella, 2002), the lepto- These authors considered H. odhneri closely related to H. buccicola, typhlopid Leptotyphlops koppesi Amaral, 1955 (Silva et al., 2004), and from which it can be distinguished by the following characters: (1) the colubrid Phalotris lativittatus Ferrarezzi, 1993 (Silva et al., 2005), smaller body; (2) a more robust acetabulum in relation to the oral suck- all parasitized by H. buccicola. The only record of H. odhneri is also er; (3) shorter esophagus; (4) more conspicuous cuticular spines; (5) from a fossorial coral snake, the elapid M. lemniscatus (Ruiz and Perez, compact vitellaria situated in the anterior third of the body; (6) size and 1959), as is the single record of H. intercaecalis n. sp., until now found form of the eggs; and (7) more lateral position of the genital pore. exclusively in the fossorial colubrid P. nasutus. Silva et al. (2005) cited that the main characters distinguishing Hap- No closer phylogenetic relationship exists amongst the squamate set lometroides species are the vitellaria and the intestinal ceca. Haplome- of species recorded as hosts for Haplometroides. On the other hand, troides buccicola (Fig. 4A–C) has vitelline follicles distributed from parasite acquisition from a common dietary item could be a plausible

FIGURE 4. (A–C) Haplometroides buccicola Odhner, 1911; (D–F) Haplometroides odhneri Ruiz and Perez, 1959 (ventral view, original drawings from Ruiz and Perez, 1959) (bar scale ϭ 1 mm). SILVA ET AL.—NEW SPECIES OF HAPLOMETROIDES 921

explanation for the observed infection patterns found in this digenean GOODMAN, J. D. 1986. Phrynobatrachotrema n. g. for Haplometroides parasite. eburnense (Trematoda: Omphalometridae) in Africa frogs and Trematode life cycles usually involve mollusks as primary interme- toads. Transactions of the American Microscopical Society 105: diate hosts and, occasionally, ants, grasshoppers, or other invertebrates 296–299. as secondary intermediate hosts (Yamaguti, 1971). Amphisbaena alba GREENE, H. W. 1997. Snakes: The evolution of mystery in nature. Uni- consumes mainly beetles, ants, and spiders (Colli and Zamboni, 1999); versity of California Press, Berkeley, California, 351 p. leptotyphlopid snakes are specialists on ants and termites (Greene, LEMA, T. 1989. Notas sobre a biologia de duas espećies de Elapomor- 1997), and Phalotris species are specialized in consuming invertebrates phus Wiegmann, 1843 (Serpentes Colubridae, Elapomorphini). (mollusks, annelids, and termites) and amphisbaenians (Lema, 1989; Iheringia 69: 61–69. Greene, 1997). In these species, the infection would follow the ingestion ODHNER, T. 1911. Nordostafrikanische Trematoden go¨sstenteils von of metacercaria present in the secondary intermediate hosts of Haplo- Weissen Nil (von der Schwedischen Zoologischen Expedition ges- metroides species. Coral snakes from the species reported as hosts for ammelt). Results of the Swedish Zoological Expedition to Egypt Haplometroides, however, usually feed on other snakes (Roze, 1996), and the White Nile 4: 1–166. usually fossorial or cryptozoic in habits, thus enhancing the possibility ROZE, J. A. 1996. Coral snakes of the Americas: Biology, identification of an indirect acquisition of the parasite (paratenic hosts or postinfec- and venoms. Krieger Publishing Company, Malabar, Florida, 328 p. tion). RUIZ,J.M.,AND M. D. PEREZ. 1959. Geˆnero Haplometroides. Redes- criçaõ da espećie-tipo e descriçaõ de H. odhneri sp. n. (Trematoda: Plagiorchiidae). Anais da Faculdade de Farmaćia e Odontologia, ACKNOWLEDGMENTS Universidade de Saõ Paulo 16: 87–91. We would like to thank the following organizations for their financial SILVA, R. J., AND T. H . B ARRELLA. 2002. Micrurus frontalis as a new support: Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo— host recorded for Haplometroides odhneri (Trematoda, Digenea, Plagiorchiidae). Revista Brasileira de Parasitologia Veterina´ria 11: FAPESP (04/03628-1), PROBIO/MMA, EMBRAPA-CPAP, and Centro 47–48. de Pesquisa do Pantanal (CPP). ———, P. A. ANDRADE,H.A.MONTEIRO E SILVA,M.ROSSELLINI, AND T. H . B ARRELLA. 2005. Report on the occurrence of Haplometroides LITERATURE CITED buccicola (Trematoda, Digenea, Plagiorchiidae) infecting Phalotris lativittatus (Serpentes, Colubridae) in Brazil. Journal of Venomous BOUGART,R.,AND C. COMBES. 1979. Sur la position taxonomique d’un Animals and Toxins Including Tropical Diseases 11: 372–378. Plagiorchiidae d’amphibiens d’Afrique: Proposition de Maederia ———, E. DE O. P. ZICA,M.CRUZ,J.C.O’REILLY, AND M. C. COSTA. n. gen. Bulletin de la Socie´te´ Neuchaˆteloise des Sciences Naturelles 2004. Occurrence of Haplometroides odhneri (Trematoda, Digenea, 1979: 35–38. Plagiorchiidae) infecting Leptotyphlops koppesi (Serpentes, Lep- COLLI,G.R.,AND D. S. ZAMBONI. 1999. Ecology of the worm-lizard totyphlopidae). Arquivo Brasileiro de Medicina Veterina´ria e Zo- Amphisbaena alba in the Cerrado of Central Brazil. Copeia 1999: otecnica 57: 267–269. 733–742. SZYDAT, L. 1932. Parasiten aus Liberia und Franzo¨sisch-Guinea. II. Teil: GASMANN, M. 1975. Contribution a` l’e´tude des tre´matodes d’amphibiens Trematoden. Zeitschrift fur Parasitenkunde 4: 506–521. du Cameroun. Annales de Parasitolologie Humaine et Comparee, YAMAGUTI, S. 1971. Synopsis of digenetic trematodes of vertebrates, Paris 50: 559–577. Vols. I–II. Keigaku Publishing, Tokyo, Japan, 1074 p. J. Parasitol., 93(4), 2007, pp. 922–924 ᭧ American Society of Parasitologists 2007

A NEW SPECIES OF PARACAPILLARIA (NEMATODA: CAPILLARIIDAE) PARASITIZING THE BRAZILIAN SANDPERCH, PINGUIPES BRASILIANUS (PISCES: PINGUIPEDIDAE), FROM ARGENTINA

Juan T. Timi, Marıá A. Rossin, Ana L. Lanfranchi, and Jorge A. Etchegoin Laboratorio de Parasitologıá, Departamento de Biologıá, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata– Consejo Nacional de Investigaciones Cientı´ficas y Tećnicas (CONICET), Funes 3350, (7600) Mar del Plata, Argentina. e-mail: [email protected]

ABSTRACT: A new species, Paracapillaria argentinensis n. sp., is described from the pinguipedid ﬁsh Pinguipes brasilianus Cuvier, 1829 from waters off Mar del Plata, Argentina (38Њ08ЈS, 57Њ32ЈW) (prevalence 22.2%; mean intensity Ϯ SD, 4.42 Ϯ 5.19). The new species is assigned to the subgenus Paracapillaria Moravec, 1987. Of the 10 species so far known in the subgenus, the new species more closely resembles P. (P.) plectroplites, from which it is distinguished by having a spicule with an expanded anterior end and a slender medial section. A similar spicular morphology is observed in P. (P.) epinepheli; however, it shows shorter spicules and a highly reduced caudal bursa. This is the ﬁrst record of Paracapillaria in the southwestern Atlantic Ocean.

Paracapillaria Mendonça, 1963 includes 22 species of nem- entire oesophagus 2.2 (1.9–2.5) long, representing 54.1 (47.1–63.6)% atodes parasitic on fishes, amphibians, reptiles, birds, and mam- of body length. Testis present from level of esophagus–intestinal junction to 658.7 (512.5–937.5) from caudal end. Seminal vesicle not dif- mals. This genus, at present, includes 10 species parasitizing ferentiated from vas deferens and testis, ejaculatory duct saclike, 277.8 fishes, all of them belonging to the subgenus Paracapillaria (212.5–340.0) long. Intestine joining cloaca immediately posterior to Mendonça, 1963 (Moravec, 2001). ejaculatory duct. Cloaca 457.0 (387.5–502.5) long, representing 11.2 During a parasitological survey carried out on samples of (1.0–13.8)% of body length, anterior cloaca 91.1 (67.5–112.5) long, posterior cloaca 370.0 (320.0–390.0) long. Spicule smooth, well scler- Pinguipes brasilianus Cuvier, 1829, landed by fishermen at Mar otized, with expanded anterior end, posterior end rounded, and slight del Plata Port, Argentina, parasitic nematodes referable to a new constriction at mid-length, 298.7 (250.0–370.0) long, representing 7.3 species of Paracapillaria (Paracapillaria) were found in the (6.0–9.5)% of body length, 6.9 (5.0–8.0) wide at proximal end, 4.7 stomach of fishes; these parasites are herein described, and a (3.8–5.0) wide at mid-length and 7.0 (5.0–7.5) wide near tip, in lateral first record of the genus is reported for the southwestern Atlan- view. Spicular canal, short almost absent. Spicular sheath smooth (maximum length when evaginate, observed in 1 specimen) 340.0 long. Cau- tic Ocean. dal end rounded, bearing a cuticular membranous bursa. Bursa supported by 2 lateral rays, each bearing large papilla at base. Rays not MATERIALS AND METHODS reaching posterior border of bursa. Female (means followed by range in parentheses): Body 6.6 (5.6– In total, 54 specimens of P. brasilianus caught by commercial trawl- 76.2) long and 63.5 (57.5–70.0) maximum wide. Bacillary bands 26.8 ers at the Mar del Plata port (38Њ08ЈS, 57Њ32ЈW), during October 2006, (22.5–32.5) wide at level of vulva. Nerve ring situated at 80.0 (75.0– were examined for nematodes immediately after capture. Fish were dis- 87.5) from apex. Muscular esophagus 238.5 (180.0–300.0) long; sti- sected, and the stomachs were removed and examined under a stereo- chosome 2.5 (1.9–3.1) mm long, composed of 30–36 stichocytes; entire scopic microscope. In total, 53 capillariid nematodes were collected and esophagus 2.7 (2.1–3.4) long, representing 41.7 (28.2–45.7)% of body fixed in 4% formaldehyde, preserved in 70% alcohol, cleared in glyc- length. Vulva with no elevated lips, situated at 25.3 (5.00–47.50) from erin-water for several hours, and then studied and measured by light esophagus end, vagina directed posteriorly from vulva, 130.8 (100.0– microscopy. Drawings were made using a drawing tube. All measure- 177.5) long, containing eggs arranged in 1 row. Eggs elongate, with ments are given in micrometers, unless otherwise indicated. Prevalence slightly protruding polar plugs and uncleaved content, 63.4 (57.5–70.0) and mean intensity were calculated according Bush et al. (1997). The long 25.8 (22.5–30.1) wide; polar plugs 5.0–6.50 long, 6.0 wide; egg studied material was deposited in the Helminthological Collection of wall thick; 2 layers observed, an inner hyaline layer and a thicker outer the Museo de La Plata (CHMLP), La Plata, Argentina. layer with fine longitudinal sculpture; thickness of entire wall 2.0–2.5. Ovary extending to approximately the distal end of intestine, at 65.0 DESCRIPTION (50.0–85.0) from caudal end. Rectum 77.5 (70.0–87.5) long. Caudal Paracapillaria (Paracapillaria) argentinensis n. sp. end rounded, anus subterminal. Tail 6.1 (5.0–8.7) long. (Figs. 1–9) Taxonomic summary General: Small-sized nematodes, males smaller than females. Ce- phalic end narrow and rounded. Cephalic papillae indistinct. Cuticle Type host: Pinguipes brasilianus Cuvier, 1829 (Perciformes: Pingui- smooth. Two lateral bacillary bands extending along whole body. Nerve pedidae). ring in first third of muscular esophagus. Muscular esophagus–sticho- Site: Stomach. some junction oblique. Stichosome a single row of 30–36 stichocytes Type locality: Mar del Plata (Buenos Aires Province, Argentina, bearing a medium-sized, irregular, central nucleus. Anterior part of sti- 38Њ08ЈS, 57Њ32ЈW). chosome long, light in color, stichocytes alternating with short, darker Date of collection: October 2006. stichocytes; posterior part of stichosome formed by uniformly larger Type specimens: Holotype: 1 male (CHMLP 5647); allotype: 1 fe- stichocytes, each subdivided in approximately 6–7 annuli. Two large male (CHMLP 5648); paratypes: 5 males and 5 females (CHMLP glandular cells at esophagus–intestinal junction. 5649). Male (means followed by range in parentheses): Body 4.1 (3.4–4.8) Prevalence: 22.2%. mm long and 40.9 (36.0–45.0) maximum wide. Bacillary bands 16.3 Mean intensity Ϯ SD (range): 4.42 Ϯ 5.19 (1–19). (12.0–20.0) wide at middle region of body. Nerve ring situated at 80.3 Etymology: The specific name refers to the geographic region where (60.0–92.5) from apex. Muscular esophagus 228.1 (212.5–257.50) long; parasites were found, the Argentine Sea. stichosome 2.0 (1.7–2.2) mm long, composed of 30–36 stichocytes; Remarks Received 30 November 2006; revised 26 January 2007; accepted 8 By the presence of elongate stichocytes with medium-sized nuclei, February 2007. by the production of eggs instead of larvae by females, and by having

922 TIMI ET AL.—NEW SPECIES OF PARACAPILLARIA 923

FIGURES 1–9. Paracapillaria (Paracapillaria) argentinensis n. sp. (1) Anterior end, lateral view. (2) Bacillary band, at level of stichosome. (3) Male posterior end, lateral view. (4) Proximal end of spicule. (5) Male tail, ventral view. (6) Male tail, lateral view. (7) Female, region of vulva, lateral view. (8) Tail of female, lateral view. (9) Egg. Bars ϭ 1, 3: 100 ␮m; 2, 6, 7, 8: 50 ␮m; 4: 10 ␮m; 5: 30 ␮m; and 9: 25 ␮m.

a fish as a definitive host, the new species is included in the subgenus (Johnston et Mawson, 1940) Moravec, 1987, a parasite of freshwater Paracapillaria Mendonça, 1963. Ten species, parasites of fish, are so perciform fish from southern Australia (Moravec, 2001). However, the far known in Procapillaria. According to the key provided by Moravec new species is readily distinguished by having eggs with slightly pro- (2001), and by having elongate stichocytes and similar morphometric truding polar plugs and by the spicular morphology, which has a non- features. The new species most closely resembles P. (P.) plectroplites expanded anterior end and lacks the medial constriction in P. (P.) plec- 924 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 troplites. In this case, the spicule of the new species is similar to that LITERATURE CITED of P. (P.) epinepheli, Moravec, Mendoza-Franco et Vargas-Va´zquez, 1996, a parasite of serranid fishes from the Gulf of Mexico; nevertheless, in this species, the spicule is shorter (0.180–0.195 mm) and the BUSH,A.O.,K.D.LAFFERTY,J.M.LOTZ, AND A. W. SHOSTAK. 1997. caudal bursa is highly reduced (Moravec et al., 1996). Therefore, a new Parasitology meets ecology on its own terms: Margolis et al. re- species Paracapillaria (Paracapillaria) argentinensis is proposed. visited. Journal of Parasitology 83: 575–583. MORAVEC, F. 2001. Trichinelloid nematodes parasitic in cold-blooded ACKNOWLEDGMENTS vertebrates. Academia, Praha, Czech Republic, 429 p. We thank Roberto Mazella and Hugo Mazella from the fish market ———, E. MENDOZA-FRANCO, AND J. VARGAS-VA´ ZQUEZ. 1996. Para- Albatros, Mar del Plata, for kindly providing fish samples. The present capillaria epinepheli n. sp. (Nematoda: Capillariidae) from the red study was funded by grants from CONICET (5996) and Universidad grouper Epinephelus morio (Pisces) from Mexico. Systematic Par- Nacional de Mar del Plata (15E/225). asitology 33: 149–153. J. Parasitol., 93(4), 2007, pp. 925–931 ᭧ American Society of Parasitologists 2007

CHEMICAL INACTIVATION OF TOXOPLASMA GONDII OOCYSTS IN WATER

Katlyn E. Wainwright, Melissa A. Miller*, Bradd C. Barr†, Ian A. Gardner‡, Ann C. Melli, Tim Essert§, Andrea E. Packham, Tin Truong§, Manuel Lagunas-Solar§, and Patricia A. Conrad࿣ Department of Pathology, Microbiology and Immunology, 1 Shields Avenue, School of Veterinary Medicine, University of California, Davis, California 95616. e-mail: [email protected]

ABSTRACT: The protozoan parasite Toxoplasma gondii is increasingly recognized as a waterborne pathogen. Infection can be acquired by drinking contaminated water and conventional water treatments may not effectively inactivate tough, environmentally resistant oocysts. The present study was performed to assess the efﬁcacy of 2 commonly used chemicals, sodium hypochlorite and ozone, to inactivate T. gondii oocysts in water. Oocysts were exposed to 100 mg/L of chlorine for 30 min, or for 2, 4, 8, 16, and 24 hr, or to 6 mg/L of ozone for 1, 2, 4, 8, or 12 min. Oocyst viability was determined by mouse bioassay. Serology, immunohistochemistry, and in vitro parasite isolation were used to evaluate mice for infection. Initially, mouse bioassay experiments were conducted to compare the analytical sensitivity of these 3 detection methods prior to completing the chemical inactivation experiments. Toxoplasma gondii infection was conﬁrmed by at least 1 of the 3 detection methods in mice inoculated with all doses (105–100) of oocysts. Results of the chemical exposure experiments indicate that neither sodium hypochlorite nor ozone effectively inactivate T. gondii oocysts, even when used at high concentrations.

Toxoplasma gondii is a protozoan parasite whose worldwide form into invasive, pathogenic tachyzoites and cause severe en- importance as a zoonotic, food-borne pathogen is well docu- cephalitis (Frenkel, 1990; Luft and Remington, 1992; Montoya mented (Choi et al., 1997; Mead et al., 1999). In recent years, and Liesenfeld, 2004). The risk is also great for women who increasing reports of major outbreaks of human toxoplasmosis are infected during pregnancy; transplacental transmission of T. associated with public water supplies in several countries have gondii may result in fetal death, abortion, congenital malfor- been attributed to waterborne transmission (Benenson et al., mations, blindness, or mental retardation (Jones et al., 2001). 1982; Bowie et al., 1997; American Water Works Association At present there are no specific regulations or approved meth- Research Division Microbiological Contaminants Research ods to control T. gondii oocysts if present in public water sup- Committee, 1999; Aramini et al., 1999; Hall et al., 1999; Bahia- plies, and research in this area has been limited (Dubey et al., Oliveira et al., 2003; de Moura et al., 2006). Humans and other 1970; Lindsay et al., 2002, 2003). The purpose of this study warm-blooded mammals and birds all serve as intermediate was to evaluate 2 chemicals that are most commonly used to hosts for T. gondii. However, only wild and domestic felids are treat drinking water, i.e., chlorine (sodium hypochlorite) and definitive hosts (Dubey and Beattie, 1988; Frenkel, 1990; Tenter ozone (Betancourt and Rose, 2004), for their ability to inacti- et al., 2000). In felids, the parasite multiplies sexually in the vate T. gondii oocysts in water. Resistance to chlorine has been intestine, producing millions of environmentally resistant oo- demonstrated with other coccidian organisms. In prior research, cysts that are shed in the feces (Tenter et al., 2000; Dubey, 90% inactivation of Cryptosporidium parvum oocysts was 2004). Once shed, these oocysts may sporulate and become achieved following exposure to 80 mg/L of free chlorine for 90 infectious within 24 hr, are widely dispersed, and may survive min (Korich et al., 1990). Cryptosporidium parvum oocysts in terrestrial and aquatic environments for months or years treated with 5.25% aqueous sodium hypochlorite (undiluted (Dubey and Beattie, 1988; Tenter et al., 2000; Lindsay et al., household bleach) for a maximum of 120 min remained infec- 2003; Dubey, 2004). Humans and livestock can be infected di- tious to neonatal mice (Fayer, 1995). Additionally, oocysts from rectly by drinking water contaminated with T. gondii oocysts both Cryptosporidium and Eimeria species are routinely ex- or by ingesting vegetation irrigated with oocyst-contaminated posed to 1.75–5.25% aqueous sodium hypochlorite for 15 to 30 water (Ortega et al., 1997; Slifko et al., 2000; Kniel et al., 2002; min to purify oocysts from bacteria and debris (Wagenbach et Dubey, 2004). Humans can also acquire an infection by con- al., 1966; Bonnin et al., 1991). Given this high resistance of suming inadequately cooked meat products from infected live- coccidian oocysts to at least chlorine, in the present study T. stock or from contact with infected cat feces (Tenter et al., gondii oocysts were exposed to chlorine and ozone at concen- 2000). In most immunocompetent people, T. gondii infections trations greater than would typically be used for water disin- are mild to asymptomatic and parasites ultimately encyst in the fection. Typical chlorine concentrations used to disinfect drink- host tissues as quiescent bradyzoite cysts. However, during pe- ing water range from 0.2 to 2 mg/L; for ozone disinfection, riods of immunosuppression, encysted bradyzoites can trans- concentrations of ozone range from Ͻ0.1 to 1 mg/L (U.S. En- vironmental Protection Agency, 1999a). Thus, the chemical concentrations used in this study for T. gondii oocyst inacti- Received 20 September 2006; revised 19 December 2006; accepted Ն 23 February 2007. vation are 50 times higher than typical for chlorination and * Marine Wildlife Veterinary Care and Research Center, California De- Ն6 times higher for ozonation of drinking water. Post-treatment partment of Fish and Game, 1451 Shaffer Road, Santa Cruz, Califor- oocyst viability was assessed using the mouse bioassay. Mouse nia 95060. infection was confirmed using 3 methods: serology, immuno- † California Animal Health and Food Safety Laboratory, 1 West Health histochemistry, and in vitro parasite isolation. Sciences Drive, University of California, Davis, California 95616. ‡ Department of Medicine and Epidemiology, 1 Shields Avenue, School of Veterinary Medicine, University of California, Davis, California MATERIALS AND METHODS 95616. Animals § Chemistry and Agriculture Program, Crocker Nuclear Laboratory, University of California, Davis, California 95616. All animal experiments were conducted with the approval and over- ࿣ To whom correspondence should be addressed. sight of the Institutional Animal Care and Use Committee at the Uni-

925 926 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

versity of California, Davis, which is accredited by the Association for Chemical inactivation experiments Assessment and Accreditation of Laboratory Animal Care, Internation- al. Female Swiss-Webster (SW) and C57BL/6 mice (Charles River Lab- Chlorine: For the first set of chlorine experiments, oocysts were ex- oratories, Wilmington, Massachusetts), 20 to 22 g in weight, and 9- to posed to approximately 100 mg/L of free chlorine in a sodium hypo- 12-wk-old specific pathogen free kittens (Nutrition and Pet Care Center, chlorite solution (made daily from 10% sodium hypochlorite stock) for Department of Molecular Biosciences, University of California, Davis, 30 min, or for 1, 2, 4, 8, 16, and 24 hr. Prior to use, the stock sodium California) were used for oocyst production. An indirect fluorescent hypochlorite solution (EMD Chemicals, Gibbstown, New Jersey) was antibody test (IFAT) was used to prescreen mouse and kitten sera at 1: stored at 4 C and the percentage of free chlorine was confirmed with 10 and 1:40 dilutions, respectively, for the presence of antibodies to T. iodometric titration (American Public Health Association, 1992). All ϳ gondii (Arkush et al., 2003; Dabritz, 2006). experiments were performed at room temperature ( 22 C) using ddH2O (pH 7.25) as diluent that was previously determined to be demand-free of chlorine and organic materials (1.37 ppm TDS). All new glassware Oocyst production and magnetic stir rods were made demand-free by being soaked for 24 To produce bradyzoite cysts in mouse brains, 64 female mice (32 SW hr in Decon* Contrad* 70 liquid detergent (Fisher Scientific, Pittsburgh, and 32 C57BL/6) were inoculated with culture-derived tachyzoites from Pennsylvania) and rinsed 3 times with ddH2O. For each experiment, the starting concentration of sodium hypochlorite working solution of ap- a well-characterized type II T. gondii strain isolated from a southern Ϯ sea otter (Enhydra lutris nereis) in California (Miller et al., 2004). Half proximately 100 mg/L ( 5 mg/L) free chlorine was measured 3 times of the mice (16 SW and 16 C57Bl/6) were inoculated subcutaneously using a LaMotte Chlorine/Bromine Titrator kit (model CL-BR, code 3624, LaMotte, Chestertown, Maryland) as directed by manufacturer’s (s.c.) with 106 tachyzoites and the remaining half (16 SW and 16 instructions. C57BL/6) were inoculated with 108 tachyzoites. When tissue cysts were For each time point, 50 ml of the working solution of sodium hy- detected by immunohistochemical examination of brains selected from pochlorite were added to each of 3 125-ml Erlenmeyer flasks containing randomly sampled mice, the remaining mice were killed. Their brains a Teflon-coated magnetic stir rod. The starting temperature of the so- were removed and fed to 2 kittens that were serologically and fecally lution was recorded and 104 sporulated oocysts (in 100 ␮lofddHO) negative for T. gondii. Each kitten received a total of 16 brains from 2 were added to each flask. Each flask was sealed with Parafilm and seropositive mice 48–49 days postinfection (PI). From the same group continuously stirred at low speed by placement on a stir plate. Ten of mice fed to the kittens, 24 half-brain sections were also submitted minutes prior to the end of the designated exposure time, the contents for immunohistochemistry. At the time of death, blood samples were of the flasks were transferred to 50-ml tubes and centrifuged for 10 min collected from all mice and screened with the IFAT at a 1:40 dilution. at 1,250 g to pellet the oocysts and prevent contamination when sam- From the day of acquisition of the kittens until 7 days beyond the last pling the supernatant to evaluate chlorine concentration. Five milliliters day of oocyst detection, feces were collected from the kittens and eval- of the supernatant were collected to determine the end concentration of uated for the presence of oocysts by microscopic examination after a free chlorine and 3 ml of 0.1 N sodium thiosulfate were added into the zinc sulfate flotation. Once oocysts were detected PI, all feces were remaining supernatant and oocyst pellet to quench any remaining free collected and processed daily to recover oocysts. Sodium chloride (1.2 chlorine. The triplicate samples were pooled, mixed, washed 3 times specific gravity) was used to concentrate the oocysts by flotation. Brief- with ddH2O, and evenly divided for s.c. inoculation into 3 mice. Pre- ly, feces were mixed with 0.1% Tween 80 to form a smooth slurry at liminary experiments revealed that replicates of 104 sporulated oocysts approximately 10 ml of Tween per g of feces. This mixture was poured remained viable and infective to mice after exposure to 0.1 N sodium through a fine-mesh tea strainer into 50-ml tubes and centrifuged for thiosulfate for 2 hr (data not shown). 10 min at 1,000 g (Sorvall RT 6000D centrifuge; Rotor H-1000B, Ther- The chlorine experiments were repeated twice but, based on prelim- mo Fisher Scientific, Waltham, Massachusetts). The supernatant was inary results, only the highest (24 hr) exposure time point was retested decanted and sodium chloride solution was added to a final volume of in mice starting with either 4 ϫ 104 or 4 ϫ 105 sporulated oocysts. 25 ml. The fecal pellet was thoroughly resuspended in the salt solution Following 24 hr of sodium hypochlorite exposure, the oocysts from and centrifuged for 10 min at 1,250 g. The top 15 ml of the flotation each flask were washed and processed separately and the entire contents fluid containing the oocysts was aspirated and placed into a clean 50- of each flask were inoculated s.c. into 1 of 4 mice. Positive control mice ml tube. The oocysts were then washed 3 times, once with 0.1% Tween were inoculated with 104 or 105 sporulated oocysts suspended in ddH2O 80 and twice with double-distilled water (ddH2O). Following the final and stirred for 24 hr. In a final experiment, mice were each inoculated wash, the oocyst pellet was resuspended in 5 ml of 2% sulfuric acid. with 104 oocysts exposed to 50 ml of 5.25% aqueous sodium hypo- Oocysts were incubated and aerated for 2 wk at room temperature (ϳ22 chlorite (undiluted household bleach) for 24 hr. The bleach was neu- C) to allow for sporulation. They were then stored in the 2% sulfuric tralized with 50 ml of 0.1 N sodium thiosulfate and the oocysts were acid at 4 C and used within 3.5 mo. Prior to use, the oocysts were washed 3 times and inoculated s.c. into 4 SW mice. washed 3 times, the resultant pellet was resuspended in ddH2O, and the Ozone: The first ozone experiments were conducted to test the ability proportion of sporulated oocysts was determined using a hemocytom- of 6 mg/L of ozone to inactivate oocysts, when exposed for 1, 2, 4, 8, eter. All oocysts used for the chemical inactivation experiments were or 12 min at room temperature. Ozone was generated by passing pure from a single batch of oocysts and oocyst concentrations were deter- oxygen across tungsten points, as part of a high-voltage discharge and mined counting the proportion of sporulated (and presumed viable) oo- analysis system (Ozone Generator BMT 803 and Ozone Analyzer BMT cysts. 964, BMT Messtechnik, Berlin, Germany), and was bubbled continuously at a concentration of 40,000 to 50,000 ppm ozone to oxygen into Mouse bioassay experiments 48 ml of ddH2O in demand-free 125-ml Erlenmeyer flasks. Initial concentrations of ozone in each flask were measured with a test kit (ozone Prior to conducting the oocyst inactivation experiments, the analytical test kit 20644-00, Hach Company, Loveland, Colorado). Following each sensitivity of each method of detecting infections in mice (serology, exposure period, 3 ml of 0.1 N sodium thiosulfate were added to neu- immunohistochemistry, and in vitro parasite isolation) was determined. tralize the ozone. Triplicate samples of 104 sporulated oocysts were Five replicates of each log dilution (105–100) of sporulated oocysts were treated and the oocysts from the 3 flasks were pooled, mixed, washed ␮ suspended in 200 lofddH2O water and inoculated s.c. into SW mice. 3 times, and separated into equal doses for s.c. inoculation into 3 mice. Negative controls included 5 mice inoculated with processed fecal ma- For positive controls, 3 ϫ 104 sporulated oocysts were added to 48 ml terial from an uninfected, seronegative cat and 5 mice inoculated with of ddH2O into which pure oxygen was bubbled for 12 min. sterile phosphate-buffered saline solution (PBS). This experiment was Duplicate ozone experiments were conducted with either 104 or 105 conducted twice, using 2 batches of oocysts from the same T. gondii sporulated oocysts, but only the 8-min ozone exposure was tested in isolate, but produced at different times. All exposed and control mice mice. Concentrations of 4 ϫ 104 or 4 ϫ 105 oocysts were added to each

were held for 43–47 days before being killed and were evaluated for of the 3 ﬂasks of dd H2O into which ozone was continually bubbled the presence of T. gondii infection via serology, immunohistochemistry, for the 8-min period. After 3 ml of 0.1 N sodium thiosulfate were added and parasite isolation. All evaluations were completed by persons who to quench the reaction, the contents of each individual ﬂask were were blinded to the infection status of each mouse. washed and inoculated s.c. into each of 4 mice. Positive controls were WAINWRIGHT ET AL.—CHEMICAL INACTIVATION OF T. GONDII 927

TABLE I. Evaluation of the analytical sensitivity of mouse bioassay (ex- TABLE II. Evaluation of the analytical sensitivity of mouse bioassay periment 1) at 43–47 days post-inoculation of Toxoplasma gondii oo- (experiment 2) at 43–47 days post-inoculation of Toxoplasma gondii cysts or at time of death. oocysts.

Immunohistochemistry Immunohistochemistry No. of Parasite No. of Parasite Oocyst dose mice IFAT* Reader 1 Reader 2 isolation Oocyst dose mice IFAT* Reader 1 Reader 2 isolation

105 54†5 5 5 105 55 5 5 5 104 54†5 5 5 104 55 5 5 5 103 55 1 2 5 103 55 5 5 5 102 55 2 3 5 102 55 5 5 5 101‡54124 101 55 5 5 5 100 50 0 0 0 100†54344 Negative controls Negative controls Feces 3 0 0 0 0 Feces 3 0 0 0 0 Saline 3 0 0 0 0 Saline 3 0 0 0 0

* IFAT: Indirect fluorescent antibody test with positive cut-off at 1:40. * IFAT: Indirect fluorescent antibody test with positive cut-off at 1:40. † One mouse died Յ11 days post inoculation and a serologic response was not † One mouse was negative by all three detection methods. detectable. ‡ One mouse was negative by all 3 detection methods. RESULTS performed as previously described, with each of 4 mice being inoculated Mouse bioassay demonstrated that untreated oocysts re- s.c. with 104 sporulated oocysts. mained viable and infective after inoculation in the 2 selected strains of mice. In experiment 1 (Table I), T. gondii infection Evaluation of oocyst viability was detected by serology, immunohistochemistry, and/or para- 5 2 For all inactivation experiments, the chemically treated oocysts were site isolation in all 20 mice inoculated with 10 –10 oocysts and inoculated s.c. into 20- to 22-g female SW mice (Charles River Labo- in 4 of 5 mice inoculated with 101 oocysts. The fifth mouse ratories) that had been prescreened for the presence of T. gondii anti- tested negative for T. gondii by all 3 detection methods. Parasite bodies using the IFAT (1:10) before use. Prior to initiation of the chem- isolation had high analytical sensitivity in detecting infections ical inactivation experiments, we evaluated oocyst viability and com- in all other mice. Serology was also reliable for detecting in- pared 3 methods (serology, immunohistochemistry, and in vitro culti- 5 vation of parasites from brain tissue) of detection of T. gondii infection fections, except in 2 mice, i.e., 1 mouse inoculated with 10 in mice, when killed 46 to 49 days PI. Samples recovered from mice oocysts (PID ϭ 10) and 1 inoculated with 104 oocysts (PID ϭ that died prematurely were also evaluated using the same methods. Se- 11). Since both of these mice died within 11 days of exposure, rum obtained from blood samples from each mouse at death was eval- the negative results on IFAT are likely due to insufficient time uated for the presence and concentration of antibodies to T. gondii ta- chyzoite-specific antigens by 2 independent readers (K.W. and A.P.) to achieve a measurable antibody response. However, both of using the IFAT at a 1:40 positive cut-off dilution, as previously de- these mice tested positive for T. gondii by immunohistochem- scribed (Miller et al., 2001; Arkush et al., 2003). For histological eval- istry and parasite isolation. For IFAT interpretation, the 2 read- uation, the spleen, 1 lung lobe, the heart, and half of the brain were ers were in agreement within 1 dilution of each other 100% of fixed in 10% phosphate-buffered formalin for at least 24 hr, but no the time and were 100% consistent in determining the positive longer than 72 hr. With the first mouse bioassay experiment, all tissues were cut lengthwise into 2 equal sections for immunohistochemical or negative status of mice based on IFAT results. Immunohis- evaluation. For mouse bioassay experiment 2 and all of the chemical tochemistry was the least sensitive detection method in exper- inactivation experiments, the lung lobe and heart were cut lengthwise iment 1, with 100% of infections, as determined by the other 2 into 2 equal sections; the spleen was cut in half and then lengthwise to tests, detected only in mice inoculated with Ն104 oocysts. The yield 4 equal sections; and the half brain was thinly sliced to yield 6 cross-sections of the cerebrum and cerebellum. These formalin-fixed 2 pathologists had 92% agreement on positive samples and only tissues were submitted to the California Animal Health and Food Safety disagreed on the positive status of 3 mice inoculated with low Laboratory, University of California–Davis, California, for sectioning doses of oocysts (103–101). In experiment 2 (Table II), infec- and preparation for immunohistochemical stains, as previously de- tions were detected by all 3 methods in all of the mice except scribed (Miller et al., 2001; Arkush et al., 2003). Two experienced pa- for 1 mouse inoculated with a single oocyst based on serial thologists (M.M. and B.B.) independently examined 2 slides from each mouse for evidence of positively stained parasites. dilution. The pathologists were in 97% agreement on samples The second halves of these same mice brains (removed aseptically) positive by immunohistochemistry, with 1 disagreement on a were placed in antibiotic saline at 4 C for 24 hr, homogenized, and mouse inoculated with 101 oocysts. For experiment 2, using the incubated in 0.25% trypsin solution at 37 C for 1 hr. The trypsinized more complete tissue trimming protocol, results of immunohis- homogenate was then layered over monkey kidney cells (MA104, BioWhittaker, Walkersville, Maryland) and incubated at 37 C for 2 hr. tochemistry were the same or nearly the same as those for se- After incubation, the homogenate and media were removed and the rology or parasite isolation, depending on which reader’s results feeder layer washed once with fresh media (Dulbecco’s modified essen- were considered. For both bioassay experiments, none of the tial medium supplemented with L-glutamine, penicillin G, streptomycin, negative control mice was found to be infected with T. gondii. HEPES buffer, 2-mercaptoethanol, and fetal bovine serum). Fresh me- All mice inoculated with oocysts exposed to approximately dium was added and replaced every 2 to 3 days and the cultures were microscopically assessed each time for the presence of T. gondii tachy- 100 mg/L of free chlorine for up to 24 hr became infected with zoites. Cultures were considered negative and discarded if no parasites T. gondii, demonstrating that under these experimental condi- were observed after 30 days PI. tions, sodium hypochlorite did not inactivate T. gondii oocysts 928 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE III. Results of mouse bioassay (46–49 days post-inoculation or at time of death) to assess viability of Toxoplasma gondii oocysts after exposure to 100 mg/L chlorine.

Chlorine Immunohistochemistry Oocyst exposure Parasite Average initial Average ﬁnal Ct values dose time No. of mice IFAT* Reader 1 Reader 2 isolation chlorine (mg/L) chlorine (mg/L) (mg ϫ min/L)†

Experiment 1 104 0 min 3 3 3 3 3 NA‡ NA NA 104 30 min 3 3 3 3 3 103 102 3,075 104 2 hr 3 3 3 3 2/2§ 101 99 12,000 104 4 hr 3 3 2 2 3 100 100 24,000 104 8hr࿣ 2 2 2 2 2 101 94 46,800 104 16 hr 3 3 3 3 3 101 81 87,360 104 24 hr 3 3 3 3 3 104 82 133,920 Experiment 2 104 0 min 4 4 4 4 4 NA NA NA 105 0 min 4 4 4 4 4 NA NA NA 104 24 hr 12 12 11 12 11 107.5 39.6 105,912 105 24 hr࿣ 11 10# 11 11 11 108.5 15.3 89,165

* IFAT: Indirect ﬂuorescent antibody test with positive cut-off at 1:40. † mg/L used to calculate Ct (disinfectant concentration ϫ contact time) is the average of initial and ﬁnal chlorine concentration. ‡ NA: not applicable. § One culture became contaminated with bacteria and was discarded. ࿣ Mouse lost because of early death and cannibalism. # One mouse died at PID ϭ 11 and a serologic response was not detectable.

(Table III). The infections were detectable by all 3 methods, maximal test dose of 100 mg/L applied to oocysts in water was with the exception of a single mouse inoculated with 104 oo- 4 times the concentration currently recommended to treat raw cysts exposed to chlorine for 24 hr (Table III, experiment 2) sewage and Ն50 times greater than that used to disinfect drink- and another mouse inoculated with 105 oocysts exposed to chlo- ing water (Palin, 1983; U.S. Environmental Protection Agency, rine for 24 hr (Table III, experiment 2). For the first mouse, no 1999a; Cheremisinoff, 2002). As previously described, aqueous parasites were observed in culture after 30 days but infection sodium hypochlorite solutions are used to purify coocidian oo- was detected with serology and immunohistochemistry. The cysts. However, oocyst exposure to the chlorine is generally second mouse died only 11 days PI before a detectable antibody limited to 15–30 min. In this study, T. gondii oocysts were response developed, but parasites were observed in culture and exposed to 5.25% aqueous sodium hypochlorite for 24 hr with- by immunohistochemistry. For both experiments 1 and 2, the out resulting in oocyst inactivation. To our knowledge, no other pathologists had 98% agreement on positive samples, disagree- coccidian organism has remained viable under comparable ex- ing on only 1 mouse inoculated with 104 oocysts exposed to perimental conditions. Another research group reported expos- sodium hypochlorite for 24 hr (Table III, experiment 2). Oo- ing cat feces containing T. gondii oocysts to 5.25% aqueous cysts also remained viable and infectious to mice following sodium hypochlorite for 24 hr, also with no success in killing exposure to undiluted bleach for 24 hr. the oocysts (Dubey et al., 1970). The present study differs be- Oocysts remained infectious following exposure to 6 mg/L cause the oocysts were purified from the cat feces before the of ozone for up to 12 min (Table IV). Infections were detected exposure to bleach. This would allow for an increased inter- by all 3 detection methods in all of the inoculated mice, show- action to occur between the chlorine and the oocyst, instead of ing that under these conditions ozone was ineffective in inac- chlorine chemically reacting with the organic components of tivating oocysts. The pathologists had 96% agreement on pos- the cat feces itself. Overall, our findings suggest that T. gondii itive samples, with 1 disagreement in each of experiments 1 oocysts present in either sewage or drinking water that is dis- and 2. Toxoplasma gondii infections were also detected in all infected with chlorine at standard dosages will likely remain positive control mice in both the chlorine and ozone experi- viable. ments. Ozone is an allotrope of oxygen that forms a highly unstable gas, which can damage cells by modifying cellular components DISCUSSION (U.S. Environmental Protection Agency, 1999c). Potential ad- Both chlorine and ozone are strong oxidizing agents. Chlo- vantages of using ozone to treat drinking water include high rine can cause cell death through inhibition of enzymatic activ- efficacy for inactivating a range of microbes, low production of ity, alterations in cell permeability, or damage to DNA and toxic byproducts, and ease of treating large volumes of water RNA (U.S. Environmental Protection Agency, 1999b). In gen- (Betancourt and Rose, 2004). For our ozone experiments, a eral, chlorination is cost effective as a water treatment and ef- dose of 6 mg/L of ozone was tested because this was the highest ficiently inactivates a range of microbes, has a residual effect concentration of ozone that could be passively bubbled into the Ն after the initial treatment, and is easy to use in sanitizing high ddH2O. This concentration is 6 times that used at many fa- volumes of water (Moore and Payne, 2004). In our study, the cilities to disinfect drinking water. Previous studies have shown WAINWRIGHT ET AL.—CHEMICAL INACTIVATION OF T. GONDII 929

TABLE IV. Results of mouse bioassay (46–49 days post-inoculation) to assess viability of Toxoplasma gondii oocysts after exposure to 6 mg/L ozone.

Immunohistochemistry Oocyst Ozone Parasite Average initial Ct values dose exposure time No. of mice IFAT* Reader 1 Reader 2 isolation ozone (mg/L) (mg ϫ min/L)†

Experiment 1 104 0 min 3 3 3 3 3 NA‡ NA 104 1 min 3 3 3 3 3 6.00 6.00 104 2 min 3 3 3 3 3 6.00 12.00 104 4 min 3 3 2 3 3 6.33 25.32 104 8 min 3 3 3 3 3 6.17 49.34 104 12 min 3 3 3 3 3 5.83 69.96 Experiment 2 104 0 min 4 4 4 4 4 NA NA 104 8 min 12 12 11 12 12 6.00 48.00 105 8 min 12 11§ 12 12 12 6.00 48.00

* IFAT: Indirect ﬂuorescent antibody test with positive cut-off at 1:40. † mg/L used to calculate Ct (disinfectant concentration ϫ contact time) is the average of initial ozone concentrations. ‡ NA: not applicable. § One serum sample was missing.

that oocysts of a related chlorine-resistant parasitic protozoan, between these experimental approaches and the number of Cryptosporidum parvum, are inactivated (Ն99%) following ex- mouse infections that resulted. posure to 3 or 4 mg ϫ 1 min/L of ozone (Finch et al., 1993). Different properties of water, including pH, temperature, tur- However, our studies revealed that T. gondii oocysts remained bidity, or organic compound concentration, could affect the viable and infectious for mice at all times when subjected to 6 ability of both sodium hypochlorite and ozone to inactivate oo- mg/L of ozone for up to 12 min. Differences in ozone suscep- cysts. Cost and time constraints of oocyst production and ap- tibility between T. gondii and C. parvum may be attributed to plication of the mouse bioassay precluded testing a broader differences in oocyst wall composition. The oocyst wall of T. range of water conditions. Our chlorine and ozone experiments gondii is comprised of 3 distinct layers: a 20- to 50-nm thick, were conducted at room temperature (22 C) with pH neutral electron-dense outer layer, a 8- to 10-nm thick, electron-lucent (7.2) ddH2O. These parameters were selected to limit any ad- middle layer, and a 50- to 90-nm thick moderately electron- ditive effects of temperature and pH on oocyst inactivation, as dense inner layer (Speer et al., 1998). The C. parvum oocyst well as effects on the efficacy of the chemicals tested. The wall is comprised of 2 or 3 layers, ranging from 40 to 49.7 nm disinfection properties of chlorine are affected by both pH and in total width (Reduker et al., 1985; Harris and Petry, 1999). temperature, i.e., lower temperatures and higher pH both reduce In addition to having a thicker oocyst wall, T. gondii also has disinfection efficacy. At pH levels of Ͻ8, free chlorine is pre- a sporocyst wall surrounding the sporocysts that is not present dominately present as hypochlorous acid, the most effective disin C. parvum (Dubey et al., 1998; Harris and Petry, 1999). Both infectant form of chlorine (Cheremisinoff, 2002; American Wa- of these characteristics may contribute additional protection to ter Works Association, 2003). The disinfection rate of ozone is the T. gondii sporozoite. less sensitive to changes in pH and temperature levels than that Potential health risks of human exposure to T. gondii and of chlorine. Ozone is less soluble and stable with increasing reduction in oocyst numbers due to sampling and purification water temperatures, but this does not affect disinfection effi- methods following chemical exposure precluded determination ciency (U.S. Environmental Protection Agency, 1999a). of final ozone concentrations after oocyst treatment. Moreover, The mouse bioassay was selected to evaluate oocyst viability prior to the addition of oocysts, continuous flow of ozone into after chlorine or ozone treatment because of the high analytical the test flasks was shown to maintain a consistent ozone con- sensitivity of this assay in previous studies (Hitt and Filice, centration of 6 mg/L. Since there was no evidence of oocyst 1992; Garcia et al., 2006). Mice were held on average for 45 inactivation at this concentration, no recommendations can be days PI to ensure dissemination of infection and development made for the application of this method to treat T. gondii oo- of T. gondii tissue cysts in the brains of infected mice. Prelim- cysts in drinking water. inary studies (data not shown) demonstrated that the type II T. For both chlorine and ozone experiments, flasks of treated gondii strain selected for oocyst production yielded moderate sporulated oocysts were initially pooled by treatment group pri- to high tissue cyst numbers in the brains of the mice strains or to mouse inoculation to increase the likelihood that each selected for our experiments. In mouse bioassay experiment 1 mouse would be inoculated with an equivalent dose of treated (Table I), T. gondii infections could be detected with IFAT and oocysts. In subsequent experiments, replicates were obtained by parasite isolation in mice inoculated with Ն101 oocysts. Ana- increasing the number of mice and increasing the oocysts per lytical sensitivity increased with both of these detection meth- flask, with the contents of each individual flask being inoculated ods and infections were detected in mice inoculated with Ն100 into 4 mice. However, no significant differences were detected oocysts in the second mouse bioassay experiment (Table II). 930 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

The reason(s) for this increase in the sensitivity of detection has many biological contaminants or pathogens, including T. gondii not been determined. Analytical sensitivity of immunohisto- oocysts, which are 10 ϫ 13 ␮m in size (Dubey et al., 1998). chemistry also increased between the 2 mouse bioassay exper- However, filtration is not always available in water treatment iments: infections were detected in mice inoculated with Ն104 facilities serving small communities or with sources of water oocysts in the experiment 1 (Table I), whereas infections were that do not contain sufficient particulate matter for conventional detected in mice inoculated with Ն100 oocysts in experiment 2 coagulation and sedimentation processes (Bowie et al., 1997; (Table II). Increasing the total surface area of tissue examined Betancourt and Rose, 2004; Goh et al., 2005). Therefore, most on the microscope between the first and second mouse bioas- water supplies are treated with chemicals, most notably chlo- says could have significantly contributed to the increased sen- rine, to inactivate potential waterborne pathogens. Given the sitivity of immunohistochemical evaluation. findings of this study, current chemical water treatment practic- Multiple detection methods were employed to increase the es are insufficient to inactivate T. gondii oocysts, if present, in likelihood of correctly determining the true infection status of public water supplies. The biochemical properties of the oocyst inoculated mice after chemical treatment of the oocysts. Each wall that impart such strong chemical resistance for T. gondii of the 3 detection methods offers different advantages and dis- oocysts remain unknown. Such information would be useful to advantages. The IFAT is rapid and ‘‘user-friendly,’’ with high determine effective means of inactivating oocysts. sensitivity in determining infection. However, this test can result in false negative results if an animal dies prematurely be- ACKNOWLEDGMENTS fore a measurable antibody response can develop and, because The authors would like to thank Karen Shapiro for her assistance it is a fluorescent test, results may vary by 1 or more dilutions with Toxoplasma gondii oocyst production and the staff of the histology when results are evaluated by Ͼ1 independent readers. Immu- laboratory at the California Animal Health and Food Safety Laboratory, nohistochemistry is rapid and sensitive and can also provide University of California–Davis, for their assistance with mouse sample preparation for immunohistochemistry. This work was financially sup- visual evidence of tissue distribution of parasites and inflam- ported by the Western Institute for Food Safety and Security, University mation, as well as pathologic changes resulting from a T. gondii of California, Davis. infection. The drawbacks of this test include potential false neg- atives if insufficient tissue surface area is trimmed, stained, and LITERATURE CITED evaluated; false positives if artifacts from antibody staining are AMERICAN PUBLIC HEALTH ASSOCIATION. 1992. Inorganic nonmetallic present; the requirement for technical expertise to process and constituents. In Standard methods for the examination of water and examine tissue samples; and an inherent level of subjectivity, wastewater, 18th ed., A. E. Greenberg, L. S. Clesceri, and A. D. as seen with the IFAT. For the mouse bioassay experiments, we Eaton (eds.). American Public Health Association, Washington, were able to substantially increase analytical sensitivity of im- D.C., p. 4-36–4-47. AMERICAN WATER WORKS ASSOCIATION. 2003. Water treatment, 3rd ed. munohistochemistry in experiment 2 (Table II) by increasing American Water Works Association, Denver, Colorado, 552 p. the number of tissue sections cut and evaluated from each tissue AMERICAN WATER WORKS ASSOCIATION RESEARCH DIVISION MICROBIO- sample. In both mouse bioassay experiments, the high agree- LOGICAL CONTAMINANTS RESEARCH COMMITTEE. 1999. Committee ment (92% in experiment 1 and 97% in experiment 2) between report: Emerging pathogens—Viruses, protozoa, and algal toxins. the 2 pathologists indicates that individual subjectivity was not Journal - American Water Works Association 91: 110–121. ARAMINI, J. J., C. STEPHEN,J.P.DUBEY,C.ENGELSTOFT,H.SCHWANTJE, problematic in this study. In vitro parasite isolation is also a AND C. S. RIBBLE. 1999. Potential contamination of drinking water sensitive detection method and can be advantageous to defini- with Toxoplasma gondii oocysts. Epidemiology and Infection 122: tively establish the presence and viability of parasites from sam- 305–315. pled tissues. Potential limits include expense and labor in main- ARKUSH, K. D., M. A. MILLER,C.M.LEUTENEGGER,I.A.GARDNER,A. E. PACKHAM,A.R.HECKEROTH,A.M.TENTER,B.C.BARR, AND taining tissue cultures, technical expertise required to safely P. A . C ONRAD. 2003. Molecular and bioassay-based detection of handle and maintain parasite and cell lines, increased time re- Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovi- quired to obtain results, and the potential for bacteria or mold cialis). International Journal for Parasitology 33: 1087–1097. to contaminate and destroy culture samples. BAHIA-OLIVEIRA, L. M. G., J. L. JONES,J.AZEVEDO-SILVA,C.C.F. Partial oocyst inactivation may have occurred with chlorine ALVES,F.OREFICE, AND D. G. ADDISS. 2003. Highly endemic, waterborne toxoplasmosis in northern Rio de Janeiro state, Brazil. or ozone treatments, but would be undetectable with the 3 de- Emerging Infectious Diseases 9: 55–62. tection methods employed. The infectious dose of T. gondii BENENSON, M. W., E. T. TAKAFUGI,S.M.LEMON,R.L.GREENUP, AND oocysts for humans is not known, but animal infectivity studies A. J. SULZER. 1982. Oocyst-transmitted toxoplasmosis associated indicate that such doses could be as low as a single oocyst with ingestion of contaminated water. New England Journal of Medicine 11: 666–669. (Dubey, 1996; Dubey et al., 1996, 1997). Therefore, the aim of BETANCOURT,W.Q.,AND J. B. ROSE. 2004. Drinking water treatment our study was to evaluate the efficacy of sodium hypochlorite processes for removal of Cryptosporidium and Giardia. Veterinary and ozone for complete inactivation of oocysts under our ex- Parasitology 126: 219–234. perimental conditions. BONNIN, A., J. F. DUBREMETZ, AND P. C AMERLYNCK. 1991. Characteriza- Treatment of drinking water for human consumption is a tion and immunolocalization of an oocyst wall antigen of Crypto- sporidium parvum (Protozoa: Apicomplexa). Parasitology 103: safety issue of global concern. In 1995, in Vancouver, British 171–177. Columbia, waterborne transmission of T. gondii infected an es- BOWIE, W. R., A. S. KING,D.H.WERKER,J.L.ISAAC-RENTON,A.BELL, timated 2,894 to 7,718 individuals, including 100 individuals S. B. ENG, AND S. A. MARION. 1997. Outbreak of toxoplasmosis identified with acute cases of toxoplasmosis. In this largest associated with municipal drinking water. The Lancet 350: 173– 177. known outbreak of water-transmitted T. gondii, the contami- CHEREMISINOFF, N. P. 2002. Handbook of water and wastewater treat- nated drinking water was chlorinated, but not filtered (Bowie ment technologies. Butterworth-Heinemann, Boston, Massachu- et al., 1997). Filtering water is ideal for physical removal of setts, 636 p. WAINWRIGHT ET AL.—CHEMICAL INACTIVATION OF T. GONDII 931

CHOI, W.-Y., H.-W. NAM,N.-H.KWAK,W.HUH, Y.-R. KIM, M.-W. survival of Toxoplasma gondii oocysts on raspberries and blueber- KANG, S.-Y. CHO, AND J. P. DUBEY. 1997. Foodborne outbreaks of ries. Journal of Parasitology 88: 790–793. human toxoplasmosis. Journal of Infectious Diseases 175: 1280– KORICH, D. G., J. R. MEAD,M.S.MADORE,N.A.SINCLAIR, AND C. R. 1282. STERLING. 1990. Effects of ozone, chlorine dioxide, chlorine, and DABRITZ, H. 2006. The epidemiology of Toxoplasma gondii in cats and monochloramine on Cryptosporidium parvum oocyst viability. Ap- rodents from Morro Bay area, California. Ph.D. Thesis. University plied and Environmental Microbiology 56: 1423–1428. of California, Davis, California, 189 p. LINDSAY, D. S., B. L. BLAGBURN, AND J. P. DUBEY. 2002. Survival of DE MOURA, L., L. M. G. BAHIA-OLIVEIRA,M.Y.WADA,J.L.JONES,S. nonsporulated Toxoplasma gondii oocysts under refrigerator con- 103: H. TUBOI,E.H.CARMO,W.M.RAMALHO,N.J.CAMARGO,R.TREV- ditions. Veterinary Parasitology 309–313. ———, M. V. COLLINS,S.M.MITCHELL,R.A.COLE,G.J.FLICK,C. ISAN,R.M.T.GRACA, ET AL. 2006. Waterborne toxoplasmosis, Brazil, from field to gene. Emerging Infectious Diseases 12: 326– N. WETCH,A.LINDQUIST, AND J. P. DUBEY. 2003. Sporulation and survival of Toxoplasma gondii oocysts in seawater. Journal of Eu- 329. karyotic Microbiology Supplement 50: 687–688. DUBEY, J. P. 1996. Pathogenicity and infectivity of Toxoplasma gondii LUFT, B. J., AND J. S. REMINGTON. 1992. Toxoplasmic encephalitis in oocysts for rats. Journal of Parasitology 82: 951–956. AIDS. Clinical Infectious Diseases 15: 211–222. ———. 2004. Toxoplasmosis—A waterborne zoonosis. Veterinary Par- MEAD, P. S., L. SLUTSKER,V.DIETZ,L.F.MCCAIG,J.S.BRESEE,C. asitology 126: 57–72. SHAPIRO,P.M.GRIFFIN, AND R. V. TAUXE. 1999. Food-related ill- ———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man. ness and death in the United States. Emerging Infectious Diseases CRC Press, Boca Raton, Florida, 220 p. 5: 607–625. ———, D. S. LINDSAY, AND C. A. SPEER. 1998. Structures of Toxo- MILLER, M. A., M. E. GRIGG,C.KREUDER,E.R.JAMES,A.C.MELLI, plasma gondii tachyzoites, bradyzoites, and sporozoites and biol- P. R. CROSBIE,D.JESSUP,J.C.BOOTHROYD,E.BROWNSTEIN, AND P. ogy and development of tissue cysts. Clinical Microbiology Re- A. CONRAD. 2004. An unusual genotype of Toxoplasma gondii is views 11: 267–299. common in California sea otters (Enhydra lutris nereis) and is a ———, J. K. LUNNEY,S.K.SHEN,O.C.H.KWOK,D.A.ASHFORD, cause of mortality. International Journal for Parasitology 34: 275– AND P. T HULLIEZ. 1996. Infectivity of low numbers of Toxoplasma 284. gondii oocysts to pigs. Journal of Parasitology 82: 438–443. ———, K. SVERLOW,P.R.CROSBIE,L.J.LOWENSTINE,F.M.GULLAND, ———, N. L. MILLER, AND J. K. FRENKEL. 1970. Characterization of A. E. PACKHAM, AND P. A . C ONRAD. 2001. Isolation and character- the new fecal form of Toxoplasma gondii. Journal of Parasitology ization of two parasitic protozoa from a Pacific harbor seal (Phoca 56: 447–456. vitulina richardsi) with meningoencephalomyelitis. Journal of Par- ———, C. A. SPEER,S.K.SHEN,O.C.H.KWOK, AND J. A. BLIXT. asitology 87: 816–822. 1997. Oocyst-induced murine toxoplasmosis: Life cycle, pathoge- MONTOYA,J.G.,AND O. LIESENFELD. 2004. Toxoplasmosis. The Lancet nicity, and stage conversion in mice fed Toxoplasma gondii oo- 363: 1965–1976. cysts. Journal of Parasitology 83: 870–882. MOORE, S. L., AND D. N. PAYNE. 2004. Types of antimicrobial agents. In Russell, Hugo and Ayliffe’s principles and practice of disinfec- FAYER, R. 1995. Effect of sodium hypochlorite exposure on infectivity tion, preservation and sterilization, 4th ed., A. P. Fraise, P. A. Lam- of Cryptosporidium parvum oocysts for neonatal BALB/c mice. bert, and J.-Y. Maillard (eds.). Blackwell Publishing Ltd., Oxford, Applied and Environmental Microbiology 61: 844–846. U.K., p. 8–97. FINCH, G. R., E. K. BLACK,L.GYUREK, AND M. BELOSEVIC. 1993. Ozone ORTEGA, Y. R., C. R. ROXAS,R.H.GILMAN,N.J.MILLER,L.CABRERA, Cryptosporidium parvum inactivation of in demand-free phosphate C. TAQUIRI, AND C. R. STERLING. 1997. Isolation of Cryptosporid- buffer determined by in vitro excystation and animal infectivity. ium parvum and Cyclospora cayetanensis from vegetables collect- Applied and Environmental Microbiology 59: 4203–4210. ed in markets of an endemic region in Peru. American Journal of FRENKEL, J. K. 1990. Toxoplasmosis in human beings. Journal of the Tropical Medicine and Hygiene 57: 683–686. American Veterinary Medical Association 196: 240–248. PALIN, A. T. 1983. Chemistry and control of modern chlorination. GARCIA, J. L., S. M. GENNARI,R.Z.MACHADO, AND I. T. NAVARRO. LaMotte Company, Chestertown, Maryland, 70 p. 2006. Toxoplasma gondii: Detection by mouse bioassay, histopa- REDUKER, D. W., C. A. SPEER, AND J. A. BLIXT. 1985. Ultrastructural thology, and polymerase chain reaction in tissues from experimen- changes in the oocyst wall during excystation of Cryptosporidium tally infected pigs. Experimental Parasitology 113: 267–271. parvum (Apicomplexa; Eucoccidiorida). Canadian Journal of Zo- GOH, S., M. REACHER,D.P.CASEMORE,N.Q.VERLANDER,A.CHARLETT, ology 63: 1892–1896. R. M. CHALMERS,M.KNOWLES,A.PENNINGTON,J.WILLIAMS,K. SLIFKO, T. R., H. V. SMITH, AND J. B. ROSE. 2000. Emerging parasite OSBORN, AND S. RICHARDS. 2005. Sporadic cryptosporidiosis de- zoonoses associated with water and food. International Journal for cline after membrane filtration of public water supplies, England, Parasitology 30: 1379–1393. 1996–2002. Emerging Infectious Diseases 11: 251–259. SPEER, C. A., S. CLARK, AND J. P. DUBEY. 1998. Ultrastructure of the HALL, S. M., A. PANDIT,A.GOLWILKAR, AND T. S. WILLIAMS. 1999. oocysts, sporocysts, and sporozoites of Toxoplasma gondii. Journal How do Jains get toxoplasma infections? The Lancet 354: 486– of Parasitology 84: 505–512. 487. TENTER,A.M.,A.R.HECKEROTH, AND L. M. WEISS. 2000. Toxoplasma gondii: From animals to humans. International Journal for Parasi- HARRIS,J.R.,AND F. P ETRY. 1999. Cryptosporidium parvum: Structural tology 30: 1217–1258. components of the oocyst wall. Journal of Parasitology 85: 839– U.S. ENVIRONMENTAL PROTECTION AGENCY. 1999a. Alternative disinfec- 849. tants and oxidants guidance manual. Office of Water, Washington, HITT,J.A.,AND G. A. FILICE. 1992. Detection of Toxoplasma gondii D.C., EPA 815-R-99-014. parasitemia by gene amplification, cell culture, and mouse inocu- ———. 1999b. Combined sewer overflow technology fact sheet: chlo- lation. Journal of Clinical Microbiology 30: 3181–3184. rine disinfection. Office of Water, EPA-832-F-99-034. JONES, J. L., A. LOPEZ,M.WILSON,J.SCHULKIN, AND R. GIBBS. 2001. ———. 1999c. Wastewater technology fact sheet: ozone disinfection. Congenital toxoplasmosis: A review. Obstetrical and Gynecological Office of Water. EPA-832-F-99-063. Survey 56: 296–305. WAGENBACH, G. E., J. R. CHALLEY, AND W. B URNS. 1966. A method for KNIEL, K. E., D. S. LINDSAY,S.S.SUMNER,C.R.HACKNEY,M.D. purifying coccidian oocysts employing Clorox and sulfuric acid– PIERSON, AND J. P. DUBEY. 2002. Examination of attachment and dichromate solution. Journal of Parasitology 52: 1222. J. Parasitol., 93(4), 2007, pp. 932–933 ᭧ American Society of Parasitologists 2007

Multiplication of Trypanosoma paciﬁca (Euglenozoa: Kinetoplastea) in English Sole, Parophrys vetulus, From Oregon Coastal Waters

Eugene M. Burreson and Egil Karlsbakk* Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062; *Department of Biology, Bergen High Technology Centre, University of Bergen, Postboks 7800, N-5020 Bergen, Norway. e-mail: [email protected]

ABSTRACT: Multiplication of Trypanosoma pacifica was common in km off Newport, Oregon, in depths from 40 to 80 m. Fish were trans- the fish host from observations of live flagellates and Giemsa-stained ported to the Oregon State University Hatfield Marine Science Center blood smears. Multiplication began with the elongation of the kineto- and held alive in tanks of circulating seawater. Fish were examined for plast, thickening of the posterior portion of the body, and appearance hemoflagellates within 5 days of capture by withdrawing blood from of a new flagellum near the kinetoplast. The new flagellum was very the caudal artery with a 2.6-ml syringe and 25-gauge needle. A drop rigid when less than 3 ␮m in length, but it became flexible as it elon- of blood was added to a drop of marine fish saline on a glass slide, gated. When the new flagellum was approximately 12 ␮m in length, covered with a coverglass, and examined for flagellates at ϫ100. Fla- cell division began and the kinetoplast also began to divide. The timing gellates were easily detected by their rapid movement. For infected fish, of nuclear division was variable. Generally, it did not occur until divi- additional blood was withdrawn, streaked on a glass slide, air-dried, sion of the kinetoplast had begun, but occasionally binucleate individ- fixed for 5 min in absolute methanol, and stained with Giemsa’s stain uals were observed before cell or kinetoplast division was apparent. As for 3 hr. division continued, 1 nucleus migrated past the dividing kinetoplast into Division stages of T. pacifica were frequently observed in P. vetulus the future daughter trypanosome. Finally, the kinetoplast completed di- in both fresh and stained blood preparations. Although it was never vision and the trypanosomes separated. Cell division was unequal, with possible to follow the complete course of division in living trypano- the daughter trypanosome being smaller than the parent and with a more somes, the sequence of events was determined by examining individuals weakly developed undulating membrane. fixed at various stages of division (Figs. 1–6). Division began with the obvious enlargement of the kinetoplast to an oval rather than round Very few reports exist of the multiplication of bloodstream forms of form, thickening of the posterior portion of the body in the region of fish trypanosomes, especially in marine fish. In freshwater fish, Try- the kinetoplast, and the appearance of a new flagellum, originating close Њ panosoma carassii (ϭT. danilewskyi), T. granulosum, T. remaki, T. tin- to the kinetoplast and protruding at an angle of about 90 to the axis of cae, T. trichogasteri, and unidentified trypanosomes have been ob- the cell (Fig. 2). Initially, the second flagellum seemed stiff and rigid served to divide (Sabraze`s and Muratet, 1904; Needham, 1969; Lom, based on observation of live flagellates, and flagellar movement was ␮ 1979; Woo, 1981; Paterson and Woo, 1984; Gupta et al., 1987) or to not observed until the flagellum had reached a length of about 3 m. undergo changes suggestive of division (Laveran and Mesnil, 1902; The new flagellum continued to elongate (Fig. 3), but a new cell body ␮ Ogawa and Uegaki, 1927; Orecka-Grabda and Wierzbicka, 1996). The did not start to develop along it until it reached about 12 m. At this latter include duplication of the kinetoplast, flagellum, and nucleus. Di- stage, the new flagellum and the associated growing new cell became vision in fish trypanosomes is best known in detail in some European posteriorly directed, and the onset of kinetoplast division became ap- cyprinid trypanosomes, identified as T. carassii and T. tincae, that show parent as it assumed a bilobed shape (Fig. 4). There was considerable a particularly high frequency of division stages in highly susceptible variation in the timing of nuclear division. Generally, it did not occur hosts (Needham, 1969; Woo, 1981; Paterson and Woo, 1984). In divid- until division of the kinetoplast had begun (Fig. 5), but occasionally ing bloodstream forms of these trypanosomes, the appearance of a short, individuals with 2 nuclei were observed before cell or kinetoplast di- new, anteriorly directed flagellum precedes (T. carassii), or directly fol- vision was apparent. After karyokinesis, a nucleus migrated past the lows (T. tincae), the division of the kinetoplast. In the bikinetoplastic bilobed kinetoplast into the forming daughter trypanosome (Fig. 6). flagellate, the gradual extension of the new flagellum is accompanied Finally, the kinetoplasts segregated and the 2 trypanosomes eventually by a swelling of the posterior end of the body and a transition to a separated by a transverse constriction between the kinetoplasts. Cell posterior direction of the flagellum as a new trypanosome body grad- division was unequal, resulting in a daughter trypanosome that was ually forms alongside it. As this process produces a daughter individual smaller and with a more weakly developed undulating membrane than of a similar size as the parental cell, the nucleus commences division, the parent. with karyokinesis where 1 nucleus migrates past the 2 kinetoplasts to The present observations are the first of the complete sequence of the daughter trypanosome. A constriction is formed between the 2 try- events during the division of a marine fish trypanosome. Compared with panosomes, at this stage recognized as mother and daughter through the the division of T. carassii (Needham, 1969; Woo, 1981), the division respective well-developed and poorly developed undulating membranes of T. pacifica is similar, but it differs in 2 notable aspects. First, the (Needham, 1969; Woo, 1981). new flagellum is anteriorly directed in the freshwater trypanosome, but Among marine fish trypanosomes, evidence for division is scanty. perpendicular to the longitudinal axis in T. pacifica.InT. rajae and T. Trypanosoma myoxocephali was observed dividing in the kidney of a yakimovi, the new flagellum is also anteriorly directed, but further di- marine sculpin (Fantham et al., 1942), but no details or figures were vision in these marine trypanosomes has not been observed. Yeld and provided. Structural changes suggestive of division have been observed Smit (2006) observed a dividing specimen of T. haploblephari, a shark in bloodstream forms of T. rajae (ϭT. variabile) and T. haploblephari parasite, which had commenced cell division along a posteriorly di- from elasmobranchs and in Trypanosoma yakimovi from a pipefish rected new flagellum. Second, the delayed completion of kinetoplast (Neumann, 1909; Yakimoff, 1912; Yeld and Smit, 2006), but the com- division in T. pacifica relative to complete nucleus division seems to plete division process has not been observed. be unique among the fish trypanosomes studied thus far. There are rare Trypanosoma pacifica Burreson and Pratt is still the only marine fish observations of binucleate trypanosomes in T. carassii, T. trichogasteri, trypanosome reported from the north Pacific Ocean. It is unusual for a and T. remaki that show single kinetoplasts and no second flagella, but marine fish trypanosome in that it is small, monomorphic, and produces these species normally show clear kinetoplast duplication before nuclear high parasitemia (Burreson and Pratt, 1972). Here, we report the com- division (Laveran and Mesnil, 1902; Woo, 1981; Gupta et al., 1987). mon presence of dividing stages of T. pacifica in the peripheral blood Such deviating forms may be from rare abnormal divisions (Woo, of English sole, Parophrys vetulus, off the Oregon coast, and we de- 1981). The pattern of kinetoplast migration and cytokinesis in T. tri- scribe the division process. chogasteri deviates from that of other trypanosomes and needs confir- English sole were collected with a 5-m semiballoon otter trawl mation (Karlsbakk, 2006). Differences in the division process among monthly from January 1971 through August 1973 between 7 and 11 freshwater and marine fish trypanosomes is not necessarily surprising

932 RESEARCH NOTES 933

DIAMOND, L. S. 1965. A study of the morphology, biology and taxonomy of the trypanosomes of Anura. Wildlife Diseases 44: 1–82. FANTHAM, H. B., A. PORTER, AND L. R. RICHARDSON. 1942. Some hae- matozoa observed in vertebrates in eastern Canada. Parasitology 34: 194–205. GIBSON, W. C., J. LOM,H.PECKOVA´ ,V.R.FERRIS, AND P. B. HAMILTON. 2005. Phylogenetic analysis of freshwater fish trypanosomes from Europe using SSU rDNA gene sequences and random amplification of polymorphic DNA. Parasitology 130: 405–412. GUPTA, N., D. K. GUPTA, AND R. K. SHARMA. 1987. On the division of trypanosomes in Colisa fasciata (Perciformes) in vivo. Angewand- te Parasitologie 28: 15–19. KARLSBAKK, E. 2006. Aspects of the morphology and ecology of some North Atlantic marine fish trypanosomes. Ph.D. Dissertation. Uni- versity of Bergen, Bergen, Norway, 91 p. ———, AND A. NYLUND. 2006. Trypanosomes infecting cod Gadus morhua L. in the North Atlantic: A resurrection of Trypanosoma pleuronectidium Robertson, 1906 and delimitation of T. murmanense Nikitin, 1927, with a review of other trypanosomes from North Atlantic and Mediterranean teleosts. Systematic Parasitology 65: 175–203. FIGURES 1–6. Division stages of Trypanosoma pacifica. Phase con- KHAN, R. A. 1976. The life cycle of Trypanosoma murmanensis Nikitin. trast optics. (1) Nondividing flagellate showing round kinetoplast. k, Canadian Journal of Zoology 54: 1840–1849. ϭ 12 ␮m and applies to all figures. kinetoplast; n, nucleus. Scale bar ———. 1977. Susceptibility of marine fish to trypanosomes. Canadian (2) New rigid flagellum (arrow) originating near the elongated, oval Journal of Zoology 55: 1235–1241. kinetoplast. (3) New flagellum (arrow) has elongated and become flex- LAVERAN,A.,AND F. M ESNIL. 1902. Sur le mode de multiplication des ible. (4) Division of the cytoplasm and kinetoplast (arrow) has begun. trypanosomes des poissons. Comptes Rendus hebdomadaires des (5) Division of the nucleus (n) has occurred, but kinetoplast division is not complete. (6) One nucleus (n) has migrated past the divided kinet- seánces de l’Acade´mie des Sciences, Paris 134: 1405–1409. oplast. LOM, J. 1979. Biology of the trypanosomes and trypanoplasms of fish. In Biology of the Kinetoplastida, vol. 2, W. H. R. Lumsden and D. A. Evans (eds.). Academic Press, New York, New York, p. 269– given that they are in separate clades in recent molecular phylogenetic 337. analyses (Gibson et al., 2005). NEEDHAM, E. A. 1969. Protozoa parasite in fish. Ph.D. Dissertation. The regular occurrence of division stages in T. pacifica is unusual University of London, London, U.K., 228 p. for a marine fish trypanosome. Division of bloodstream forms of many NEUMANN, R. O. 1909. Studien uber protozoische Parasiten im Blut von marine trypanosomes seems to be absent or a very rare event, because Meeresfischen. Zeitschrift fur Hygiene und Infektionskrankheiten; dividing stages have not been observed despite extensive studies on meidizinische Mikrobiologie, Immunolgie und Virolologie 64: 1– some species such as Trypanosoma murmanense and T. pleuronectidium 112. (Khan, 1976, 1977; Karlsbakk and Nylund, 2006). A possibility that OGAWA,M.,AND J. UEGAKI. 1927. Beobachtungen u¨ber die Blutproto- has received little attention in fish is proliferation in certain tissues such zoen bei Tieren Formosas. Archiv fu¨r Protistenkunde 57: 14–30. as spleen and kidney during prepatency, either intra- or intercellularly, ORECKA-GRABDA,T.,AND J. WIERZBICKA. 1996. Observations on Try- as observed in several amphibian trypanosomes (Diamond, 1965; panosoma granulosum Laveral et Mesnil, 1902 (Protozoa, Kineto- Bardsley and Harmsen, 1973). Similar stages may occur in fish (Davies, plastida), a blood parasite of eel, Anguilla anguilla (L.). Acta Ich- 1995). However, the absence of vertebrate phase trypanosome division thyologica et Piscatoria 26: 39–47. also may be adaptive if trypanosomes are pathogenic to their vectors PATERSON,W.B.,AND P. T. K . W OO. 1984. Ultrastructural studies on in a dose-dependent manner. mitosis in Trypanosoma danilewskyi (Mastigophora: Zoomastigo- We thank Robert E. Olson for providing support and facilities at the phorea). Canadian Journal of Zoology 62: 1167–1171. Hatfield Marine Science Center and for valuable assistance during this SABREZE` S, J., AND L. MURATET. 1904. Trypanosome de l’anguille—Pro- study. This research was supported by the Oregon State University Sea cessus de division. Comptes Rendus des seánces de la Socie´te´de Grant Program grant 04-5-158-2. Biologie, Paris 56: 66–68. WOO, P. T. K. 1981. Trypanosoma danilewskyi: A new multiplication LITERATURE CITED process for Trypanosoma (Protozoa: Kinetoplastida). Journal of BARDSLEY,J.E.,AND R. HARMSEN. 1973. The trypanosomes of Anura. Parasitology 67: 522–526. Advances in Parasitology 11: 1–73. YAKIMOFF, W. L. 1912. Trypanosomes parasites du sang des poissons BURRESON,E.M.,AND I. PRATT. 1972. Trypanosoma pacifica sp. n. from marins. Archiv fu¨r Protistenkunde 27: 1–8. the English sole, Parophrys vetulus Girard from Oregon. Journal YELD,E.M.,AND N. J. SMIT. 2006. A new species of Trypanosoma of Protozoology 19: 555–556. (Kinetoplastida: Trypanosomatidae) infecting catsharks from South DAVIES, A. 1995. The biology of fish haemogregarines. Advances in Africa. Journal of the Marine Biological Association of the United Parasitology 36: 117–203. Kingdom 86: 829–833. 934 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

J. Parasitol., 93(4), 2007, pp. 934–937 ᭧ American Society of Parasitologists 2007

Contribution of NADH Dehydrogenase Subunit I and Cytochrome C Oxidase Subunit I Sequences Toward Identifying a Case of Human Coenuriasis in France

Jocelyne Collomb, Marie Machouart*, Marie-France Biava, Me´lanie Brizion, Karine Montagne†, Franc¸ois Ple´nat†,and Bernard Fortier, Service de Parasitologie-Mycologie, CHU Brabois, 54511 Vandoeuvre-Les-Nancy, France; *To whom correspondence should be addressed; †Service d’Anatomie et de Cytologie Pathologiques, CHU Brabois, 54511 Vandoeuvre-Les-Nancy, France. e-mail: [email protected]

ABSTRACT: Coenuriasis is a parasitic disease induced by larval taeniid indicate the nature of the lesion. A biopsy was performed and a 22 ϫ tapeworms that is rarely observed in humans. In December 2005, a case 13-mm portion, surrounded by fibrotic tissue, was excised. The resected was diagnosed in Nancy, France, after surgical excision of a cyst on a cyst was sent for further investigation to the laboratory of Parasitology- 24-yr-old woman returning from the Coˆte d’Ivoire. Morphological and Mycology and the laboratory of Pathological Anatomy and Cytology, epidemiological criteria suggested that the infection was due to Taenia CHU Nancy, France. The surgical biopsy was routinely processed in serialis. Molecular analysis of NADH dehydrogenase subunit I (NDI) Bouin’s fixative, and embedded in paraffin. For the histopathological and cytochrome c oxidase subunit I (COI) sequences was also in favor examination, a paraffin block was used to generate 5-␮m-thick sections of T. serialis identification, but the absence of available genetic data on from the hematoxylin–eosin–safran stained slides. For the parasitolog- T. brauni and T. glomeratus and the small number of published se- ical observations, several scolices were dissected, compressed between quences for T. serialis and T. multiceps must be considered with cau- 2 glasses and examined by ordinary light microscopy. For genomic tion. The NDI partial sequences presented more variations within spe- analyses, DNA was extracted from the biopsy by using QIAamp௡ DNA cies of Taenia than the COI sequences, which make them more useful Mini Kit (Qiagen, Courtaboeuf, France). The tissue protocol was slight- targets for species identification and analysis of intraspecific polymor- ly modified by prolonging the first incubation times, in order to remove phisms. The present study points to the usefulness of molecular biology all traces of formaldehyde. Briefly, a piece of the bladder with 5 in- tools to help make up for the shortcomings of the commonplace para- verted protoscolices was suspended in 200 ␮l of tissue–lysis buffer and sitological diagnosis for coenuriasis. incubated for 3 hr at 37 C in the presence of lysosyme (10 U/␮l final concentration). DNA was then extracted according to the instructions Ϫ Coenuriasis is the term used for a parasitic infection due to the pres- of the manufacturer. Samples were kept at 20 C until used. Two mi- ence in the tissues of a coenurus, the infective larval stage of the ces- tochondrial DNA regions coding the NADH dehydrogenase subunit I tode. The definitive hosts for the adult stage are canids. After the egg (NDI) and the cytochrome c oxidase subunit I (COI) were, respectively, hatches, the oncosphere penetrates the intestinal wall and is carried in amplified with the previously described primers JB11 (5Ј-AGATTCG the blood to the final site of infection, usually a subcutaneous or intra- TAAGGGGCCTAATA-3Ј) and JB12 (5Ј-ACCACTAACTAATTCAC muscular location. (Brumpt, 1936; Euze´by, 1966). Rarely, humans may TTTC-3Ј), and JB3 (5Ј-TTTTTTGGGCATCCTGAGGTTTAT-3Ј) and become infected. In the United States, sheep and jackrabbits (hares) are JB4.5 (5Ј-TAAAGAAAGAACATAATGAAAATG-3Ј) (Gasser et al., intermediate hosts, although Esch et al. (1964) and Larsh et al. (1964) 1999). The assay was performed with the use of a 50-␮l reaction volume generated cerebral infections in Swiss mice experimentally. Approxi- containing 2 U of FastStart Taq Polymerase (Roche Diagnostics, Mey- ␮ ϫ mately 100 or more coenuri cases have been reported worldwide in- lan, France), 5 lof10 PCR Buffer-MgCl2 (20 mM), 20 pmol of each volving any one of the 4 species, i.e., Taenia multiceps, T. serialis, T. primer, 200 ␮M of dATP, dGTP, dTTP, and dCTP, and 5 ␮l of sample brauni, and T. glomeratus. The first species supposedly affects the cen- DNA. The reaction volume was adjusted to 50 ␮l with ultrapure water. tral nervous system, whereas the other three are mainly found in the PCR amplification was performed on a iCycler IQ System (Biorad, eyes, subcutaneous tissues, and intramuscularly (Esch, 1964; Temple- Marne La Coquette, France) under the following conditions: initial de- ton, 1968; Benger et al., 1981; Menard et al., 1982; Kurtycz et al., 1983; naturation of DNA at 95 C for 3 min, followed by 35 cycles at 95 C Ibechukwu and Onwukeme, 1991). Taenia brauni and T. glomeratus for 1 min, 60 C for 40 sec, and 72 C for 1 min. Those steps were have only been reported from Africa (http://www.cfsph.iastate.edu/ followed by an additional 5-min extension step at 72 C. After a first Factsheets/pdfs/taenia.pdf), whereas T. serialis and T. multiceps are en- amplification step, 5 ␮l of the PCR products were amplified a second countered throughout the world (Fain et al., 1956; Templeton, 1971). time in the same conditions. Finally, 5 ␮l of the second PCR product Traditionally, the diagnosis of the genus, and even the species, is based were electrophoresed in a 2% agarose gel in the presence of ethidium on a combination of clinical symptoms, epidemiological events, histo- bromide and visualized under UV light. The expected PCR product size logical lesions, and morphological features. Hook number, size, and was 550 bp for the NADH dehydrogenase subunit I and 450 bp for the shape are the only criteria for the determination of the species; however, cytochrome c oxidase subunit I. The amplicons were first purified by unfortunately, the coenurus may be sterile, immature, or degenerated. the QIAquick PCR Purification Kit (Qiagen). The 2 strands of amplified It is clear, therefore, that applying these criteria is not always sufficient DNA were then sequenced by using the PCR primers and the BigDye௡ to identify coenuri reliably (Brumpt, 1936; Fain et al., 1956; Benger et Terminator v1.1. Cycle Sequencing Kit according to the manufacturer’s al., 1981). To overcome these limitations, molecular biology has re- instructions (Applied Biosystems, Courtaboeuf, France), on an auto- cently provided tools for parasite characterization and species/strain mated sequencer (ABI Prism௡ 3100 Genetic analyzer). Nucleotide sim- identification. Thus, genomic data have been exploited to infer the phy- ilarity searches were performed by using the BLASTN program on the logenetic relationships of cestodes (McManus, 2006). A few studies Web site at http://www.ncbi.nlm.nih.gov/BLAST/. Sequence alignments indicate also that closely related cestode species can be differentiated were then performed by using the AliBee program on the Web site at by sequence polymorphisms in ribosomal DNA domains, as well as in http://www.genebee.msu.su/genebee.html, with the nucleotide sequenc- mitochondrial regions (Gasser and Chilton, 1995; Gasser et al., 1999). es of T. serialis and T. multiceps referred, respectively, as AJ239105 In this report, a molecular approach using the mitochondrial NADH and AY669089. dehydrogenase subunit I and cytochrome c oxidase subunit I gene se- The morphological observation of the resected cyst identified a blad- quences is described for the first time to confirm the classical morpho- der-like structure resembling a coenurus. It was spherical, unilocular, logical diagnosis of coenuriasis. These targets are also interesting for and filled with opalescent jelly. About 30 scolices were attached to its analyzing genetic variations within and among taeniid populations. thin translucent wall. A histological section showed multiple inverted A 24-yr-old military woman was admitted in December 2005 to the scolices developing internally from the base of an invagination canal. Clinique Jeanne d’Arc in Lune´ville (France), suffering from an intra- An overall irregular distribution of the scolices was observed; some of muscular mass in the right scapula, occurring after a journey in Coˆte them were arranged serially, which is characteristic of T. serialis. The d’Ivoire, Africa, 7 mo previously. The echographic examination did not surrounding muscle showed fibrous tissue with a few clusters of inflam- RESEARCH NOTES 935

FIGURE 1. Microscopical analysis of the cyst. (A) Histological section of the bladder showing numerous inverted protoscolices developing internally from the germinal layer. Notice the linear arrangement of some of them (arrow). (B) Individual mature scolex bearing circular suckers (arrow 1) and the armed rostellum with a double ring of developed hooks (arrow 2). (C) Typical small (arrow 1) and large (arrow 2) hooks. (D) A sideways view of a small hook showing a twin-lobed guard (arrow 1).

matory cells presumed to be the result of the host’s reaction (Fig. 1A). the 2 T. serialis sequences, whereas 44 nucleotide positions varied be- Under microscopic examination, the invaginated scolices in their mature tween the T. serialis and the T. multiceps sequences (Fig. 2). In the form were about 900 ␮m in length, with prominent rounded suckers same way, the analysis of the cytochrome c oxidase sequences showed and an armed rostellum. The latter showed a double ring of 26 to 30 9 polymorphisms between the DQ401138 and the AJ239110 T. serialis hooks in 2 sizes. The larger ones were 115–119 ␮m in length with a sequences, and 19 differences between the T. serialis and the T. mul- slightly undulating handle about as long as the blade. The smaller ones ticeps DQ309769 sequences (data not shown). were 78–92 ␮m in length, with a very short handle and a bilobed guard In conclusion, the cyst was identified as a coenurus of T. serialis for (Fig. 1B–D). These features suggested that the cyst was a coenurus of 2 reasons. First, the location of the coenurus is typical for T. serialis, T. serialis. i.e., intramuscular/subcutaneous tissues (Benger et al., 1981; Menard et For the molecular biology analyses, an initial PCR amplification was al., 1982). Second, the morphology of the coenurus fitted closely to the carried out on the DNA extracted from the biopsy without any result. standard description of T. serialis (Brumpt, 1936). However, as the pa- Electrophoresis of a second amplification of 5 ␮l of the first amplicons tient had spent a period of time in Africa, it is impossible to dismiss revealed single products of approximately 550 bp (ND1) and 450 bp the possibility of T. brauni or T. glomeratus. Taenia brauni, in partic- (COI), respectively, with the JB11–JB12 and JB3–JB4.5 primer pairs. ular, is known to be predominant in that part of the world. It is difficult Sequencing of the NDI and COI products with both sense and antisense to differentiate between the 3 species based on hook morphology. More- primers yielded, respectively, 530-bp and a 378-bp consensus sequenc- over, humans are unusual intermediate hosts, so anomalies in size and es. BLAST analyses were then performed to characterize these sequenc- shape of the hooks are often observed (Brumpt, 1936; Fain et al., 1956), es. For the NDI sequence, best BLAST results corresponded to an align- and this increases the identification problem. Molecular biology tech- ment with a partial T. serialis mitochondrial NADH1 gene (AJ239105), niques seem to be appropriate to confirm such a problematic diagnosis with an E-value of 8 ϫ 10Ϫ151 (the E-value assesses the significance of successfully and rapidly. The only available sequences on databases that an alignment, i.e., small values correspond to sequences having high proved to be useful in epidemiological studies on the larval stages of homology or vice versa). The next BLAST results corresponded to the several cestodes are the NADH dehydrogenase and cytochrome c oxi- NDI of other cestodes (T. asiatica, T. multiceps, and Echinococcus dase subunit I mitochondrial sequences, which were chosen as targets granulosus). For the COI sequence, BLAST results revealed a strong in this study (Gasser et al., 1999). These sequences are available on homology with mitochondrial cytochrome c oxidase subunit I genes of databases for both T. serialis and T. multiceps, whereas ribosomal se- T. serialis. High similarity with T. multiceps, T. saginata, and T. solium quences have only been published for T. serialis and, unfortunately, no was also observed. Then, the 530-bp and 37-bp nucleotide sequences sequence at all are available for T. brauni or T. glomeratus. were confirmed to be T. serialis NADH dehydrogenase subunit 1 and After the first PCR reaction, no pattern was detected, suggesting am- cytochrome c oxidase subunit I mitochondrial genes and deposited at plification inhibition due to the effects of formalin fixation. It is known GenBank under the following accession numbers: DQ401137 and that this fixative procedure lowers the success of polymerase chain re- DQ401138. Intra- and interspecific variations were studied through action amplification because of cross-linking between protein and DNA alignments of the nucleotide sequences of the NADH dehydrogenase (Williams et al., 1999). A report of the failure to generate molecular subunit I gene DQ401137 (T. serialis), AJ239105 (T. serialis), and data from such histological and biopsy samples of cestodes has already AY669089 (T. multiceps). Ten polymorphisms were revealed between been published (Garin et al., 2005). To overcome this problem, 2 suc- 936 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 2. Alignment of ND1 nucleotide sequences of Taenia serialis (T. s) and Taenia multiceps (T. m). Crosses are given for similarities among these species and dots indicate differences. Black shaded letters indicate nucleotide similarity between the both T. serialis sequences and polymorphism with the T. multiceps sequence. Grey shaded letters are given for divergence between one of the T. serialis sequence and the T. multiceps sequence. DQ401137 and AJ239105 indicate the accession numbers of 2 nucleotide sequences of T. serialis. AY669089 represents the accession number of the nucleotide sequence of T. multiceps.

cessive amplifications were run with the same primer sets. These PCR LITERATURE CITED conditions were effective to improve the sensitivity of the reaction, reduce the influence of inhibitors, and detect amplicons of expected BENGER, A., R. P. RENNIE,J.T.ROBERTS,J.H.THORNLEY, AND T. S COL- size. It is also true that formalin fixation could lead to randomly dis- TEN. 1981. A human coenurus infection in Canada. American Jour- tributed artificial mutations during PCR and errors in sequencing (Wil- nal of Tropical Medicine and Hygiene 30: 638–644. liams et al., 1999; Quach et al., 2004). Therefore, the experiments were BRUMPT, E. 1936. Prećis de parasitologie, 5th ed. Manson, Paris, France, repeated several times to confirm the data generated. BLAST analysis 1082 p. and sequences alignment showed highest homology with T. serialis,as ESCH, G. W. 1964. The effects of cortisone and pre-infection starvation expected. These results also supported high homology of the NADH on establishment of larval Multiceps serialis in mice. Journal of dehydrogenase (NDI) and cytochrome c oxidase subunit I (COI) mito- the Elisha Mitchell Scientific Society 80: 114–129. chondrial genes within the cestodes, as previously described (Gasser et EUZE´ BY, J. 1966. Les maladies venimeuses des animaux domestiques et al., 1999). Nevertheless, even if this sequence analysis is in favor of T. leurs incidences sur la pathologie humaine. Vigot, Paris, France, serialis identification, the absence of available genetic data on T. brauni 663 p. and T. glomeratus and the small number of published sequences for T. FAIN, A., N. DENISOFF,L.HOMANS,G.QUESTIAUX,L.VAN LAERE, AND serialis and T. multiceps must be considered with caution. Finally, PCR M. VINCENT. 1956. Ceńurose chez l’homme et les animaux due a` and sequencing techniques applied to DNA isolated from the surgical Taenia brauni Setti au Congo Belge et au Rwanda-Urundi. II. Re- piece, even if preserved in formalin, provides a useful tool for the con- lation de huit cas humains. Annales de la Socie´te´ Belge de Me´- firmation of human cases of coenuriasis. Moreover, the COI sequences decine Tropicale 36: 679–696. present more similarities between T. serialis and T. multiceps than the GARIN, Y. J., M. T. GALAN-PUCHADES,A.MOULIGNIER,G.ROBERT,F. NDI sequences. These latter seem a more powerful target for the species HERAN,M.POLIVKA,P.D.OLSON,F.LORENZO,F.DEROUIN, AND D. identification and to investigate population variations within T. serialis, B. CONN. 2005. Case report: Human brain abscess due to a tetra- as previously suggested by Gasser et al. (1999). acetabulate plerocercoid metacestode (Cyclophyllidea). American The authors are grateful to Jean-François Schwarz, Clinique Jeanne Journal of Tropical Medicine and Hygiene 72: 513–517. d’Arc, 54 300 Lune´ville, France and to G. Valantin, Cabinet d’Anatomie GASSER,R.B.,AND N. B. CHILTON. 1995. Characterisation of taeniid et de Cytologie Pathologiques, 22 rue de Verdun, 54000 Nancy, France, cestode species by PCR-RFLP of ITS2 ribosomal DNA. Acta Tro- who supplied the surgical biopsy and contributed to the discussions on pica 59: 31–40. this case. ———, X. ZHU, AND D. P. MCMANUS. 1999. NADH dehydrogenase RESEARCH NOTES 937

subunit 1 and cytochrome c oxidase subunit I sequences compared 1982. La ceńurose humaine. Premier cas a` localisation intermus- for members of the genus Taenia (Cestoda). International Journal culaire observeáuSeńe´gal. Me´decine Tropicale 42: 617–623. for Parasitology 29: 1965–1970. QUACH, N., M. F. GOODMAN, AND D. SHIBATA. 2004. In vitro mutation IBECHUKWU, B. I., AND K. E. ONWUKEME. 1991. Intraocular coenurosis: artifacts after formalin fixation and error prone translesion synthesis A case report. British Journal of Ophthalmology 75: 430–431. during PCR. BMC Clinical Pathology 4: 1–5. KURTYCZ, D., B. ALT, AND E. MACK. 1983. Incidental coenurosis: Larval TEMPLETON, A. C. 1968. Human coenurus infection. A report of 14 cases cestode presenting as an axillary mass. American Society of Clin- from Uganda. Transactions of the Royal Society of Tropical Med- ical Pathology 80: 735–738. icine and Hygiene 62: 251–255. LARSH, J. E., G. J. RACE, AND G. W. ESCH. 1964. A histopathologic ———. 1971. Anatomical and geographical location of human coe- study of mice infected with the larval stage of Multiceps serialis. nurus infection. Tropical and Geographical Medicine 23: 105–108. Journal of Parasitology 51: 45–52. WILLIAMS, C., F. PONTEN,C.MOBERG,P.SO¨ DERKVIST,M.UHLEN,J. MCMANUS, D. P. 2006. Molecular discrimination of taeniid cestodes. PONTEN,G.SITBON, AND J. LUDEBERG. 1999. A high frequency of Parasitology International 55: 31–37. sequence alterations is due to formalin fixation of archival speci- MENARD, M., J. C. DEBRIE,J.M.FAUGERE,G.BRUNETTI, AND P. A UBRY. mens. American Journal of Pathology 155: 1467–1471.

J. Parasitol., 93(4), 2007, pp. 937–944 ᭧ American Society of Parasitologists 2007

Checklist of Helminth Parasites of the Cane Toad Bufo marinus (Anura: Bufonidae) From Mexico

Arlett Espinoza-Jimeńez, Luis Garcıá-Prieto*, David Osorio-Sarabia, and Virginia Leo´ n-Re`gagnon, Laboratorio de Helmintologıá, Instituto de Biologıá, Universidad Nacional Autońoma de Me´xico, A.P. 70-153, C.P. 04510, Me´xico, Distrito Federal, Me´xico; *To whom correspondence should be addressed. e-mail: [email protected]

ABSTRACT: Thirty-four adult cane toads Bufo marinus L. (12 males and number FAUT 0056, Secretarıá del Medio Ambiente y Recursos Na- 22 females) collected from 2 localities in Mexico (Cerro de Oro and turales, Me´xico). Hosts were killed with an overdose of sodium pen- Temascal Dams, Oaxaca) in September 2003 were examined for hel- tobarbital and necropsied while still fresh. During necropsy, the diges- minth parasites. In total, 14,749 helminths belonging to 14 taxa were tive tract, limbs, body wall, and internal organs were examined for collected. Included were 2 adult digeneans (Choledocystus hepaticus, helminth parasites. Each organ was placed into individual Petri dishes Mesocoelium monas); 1 larval cestode (an unidentified pseudophyllid- and examined using a dissecting microscope. Worms were removed and ean); and 11 nematodes, including 3 species of larvae (Contracaecum washed in 0.65% saline. Platyhelminths were relaxed with hot tap water, sp., Physaloptera sp., Physocephalus sexalatus) and 8 species of adults fixed in 4% formalin, and placed in vials containing 70% alcohol; they (Aplectana itzocanensis, Cosmocerca sp., Cruzia morleyi, Ochoterenel- were stained with Mayer’s paracarmine, Delafield’s hematoxylin, or Go- la digiticauda, Oswaldocruzia sp., Raillietnema sp., Rhabdias ameri- mori’s trichrome and mounted on permanent slides in Canada balsam. canus, and Rhabdias fuelleborni). Higher species richness was recorded Nematodes were fixed with hot 70% alcohol, cleared in Amman’s lac- in B. marinus from Cerro de Oro (12 taxa versus 9 in those from Te- mascal); hosts from both localities shared 7 taxa. There were 25 new locality records, and 2 taxa were registered in Mexico for the first time. TABLE I. Characterization of helminth infections recorded in Bufo mar- To date, 112 helminth species have been recorded parasitizing B. marinus from 2 localities of Oaxaca, Mexico. inus along its native and introduced range of distribution, with 40.5% of them reported from Mexico. Cerro de Oro Temascal Dam Dam (n ϭ 24) (n ϭ 10) The normal geographical distribution of the cane toad, Bufo marinus prevalence/mean prevalence/mean L., extends from southern Texas to Central Brazil; however, B. marinus Helminth species abundance Ϯ SD abundance Ϯ SD has been introduced to Florida, many Caribbean and Pacific islands, New Guinea, and northeastern Australia (Speare, 1990). In Mexico, B. Digenea marinus occurs in almost every region, excluding arid zones of Chi- Choledocystus hepaticus 12.5/0.83 Ϯ 2.9 — huahua, Coahuila, Sonora, San Luis Potosı´, and Jalisco (Flores-Villela, Ϯ 1998). To the best of our knowledge, 2 inventories of B. marinus hel- Mesocoelium monas — 50/21.7 37.7 minth parasites from Mexico have been completed (Galicia-Guerrero et Cestoda al., 2000; Goldberg et al., 2002). In addition, 2 global compilations of Pseudophyllidea gen. sp. — 20/0.4 Ϯ 0.63 B. marinus helminth parasites include some Mexican records (Speare, 1990; Barton, 1997). In these studies, there were 26 taxa summarized Nematoda for Mexico, including 5 digeneans, 1 cestode, 1 acanthocephalan, and Aplectana itzocanensis 29.2/85 Ϯ 254 70/443.6 Ϯ 504.4 19 nematodes. However, there are no comprehensive reviews of hel- Contracaecum sp. 4.2/0.33 Ϯ 1.6 — minth fauna associated with this host species in Mexico. The aim of Cosmocerca sp. 4.7/34 Ϯ 65 30/5.8 Ϯ 12.15 the present study is to provide new information concerning helminth Ϯ Ϯ parasites of B. marinus from Mexico and to compile the existing records Cruzia morleyi 16.6/3.3 13.3 50/1.2 1.6 Ϯ Ϯ for this host, as part of an ongoing project to establish a current inven- Ochoterenella digiticauda 32.5/3.6 4.9 30/0.3 0.5 tory of the helminth parasites of native herpetofauna in this country. Oswaldocruzia sp. 4.2/0.04 Ϯ 0.2 — Thirty-four adult B. marinus (12 males, snout-vent length 5.7–12.6 Physaloptera sp. 8.3/0.21 Ϯ 0.8 — cm, 10.73 Ϯ 2.02; 22 females, snout-vent length 6–15.5 cm, 13.27 Ϯ Physocephalus sexalatus 50/178.2 Ϯ 292 50/179.7 Ϯ 271.2 2.53), collected from 2 localities from Oaxaca state, Mexico, Temascal Raillietnema sp. 8.3/19.2 Ϯ 87.5 — Dam (18Њ14Ј13ЉN, 95Њ25Ј04ЉW, sample size ϭ 10) and Cerro de Oro Rhabdias americanus 12.5/5.54 Ϯ 18 10/0.5 Ϯ 1.6 Њ Ј Љ Њ Ј Љ ϭ Dam (18 00 17 N, 96 16 21 W, sample size 24), were examined for Rhabdias fuelleborni 66.6/12 Ϯ 16.3 40/0.4 Ϯ 0.5 helminths in September 2003. Hosts were captured by hand (permission 938 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 `gagnon (2005)‡ ń-Re ńdez et al. (2000) ńdez et al. (2000) ńdez et al. (2000)‡ ńdez et al. (2000) ´vil (2004) ´vil (2004) ´vil (2004) `gagnon et al. (2005)‡ `gagnon et al. (2005) ´pez and Leo ń-Herna ń-Herna ń-Herna ń-Herna ńez-Salazar (2004) ńez-Salazar (2004) ń-Re ń-Re Other collections Reference ń Coleccio Nacional de City (CNHE) Helmintos, Mexico ń 4883 — Martı ń ń Veracruz Los Tuxtlas — — Guille Laguna (Higueras) 4628 — Leo Temascal†Veracruz CatemacoEl ZacatalLos Tuxtlas 5403 1529 1528 — — — This study Goldberg et al. (2002) Guille Lamothe-Argumedo et al. (1993) Corozal Razo-Mendı CatemacoHuixtlaGuerrero San Pedro las PlayasNI NIOaxaca Paso Canoa 1525Cerro de Oro†Tuxtepec 4074 741–743 — 93,038# 36,894# 4695 5402 Galicia-Guerrero 1166, et — 1357 al. Razo-Mendı (2000); Goldberg et Caballero al. et (2002) al. (1944) — — — 21,705‡‡ Razo-Mendı This study Bravo-Hollis (1948) Not indicated (NI) NI —Oaxaca TehuantepecVeracruz Los Tuxtlas 81,864# Etges (1991) 4879 — Martı Guille Los Tuxtlas — — Guille Tuxtepec 810, 811 — Bravo-Hollis (1948) Paso CanoaCoquimatla Nuevo Leo — — Mata-Lo Laguna (Higueras) 4632 — Leo from Mexico. ** Veracruz * Nuevo Leo ࿣ ¶ Oaxaca †† Chiapas § Bufo marinus * Colima ¶ Oaxaca * Oaxaca Helminths Locality II. Helminth fauna of Clinostomum attenuatum Cephalogonimus americanus Mesocoelium monas Choledocystus hepaticus Langeronia macrocirra Gorgoderina festoni Haematoloechus medioplexus Gorgoderina megalorchis Clinostomidae Cephalogonimidae Brachycoeliidae Macroderoididae Lecithodendriidae Gorgoderiidae Haematoloechidae ABLE T Digenea RESEARCH NOTES 939 `gagnon (2001)‡ ń-Re ńdez (1992) ## ࿣࿣ ´vil and Leo ´vil (2004)‡ ´vil (2004) `gagnon et al. (2005) `gagnon et al. (2005)‡ ń-Herna ńez-Villarreal (1969) ńez-Villarreal (1969) ń-Re ń-Re Other collections Reference ń Coleccio Nacional de City (CNHE) Helmintos, Mexico ´mez 420 — Martı ń á — — Martı Catemaco — — Goldberg et al. (2002)‡ Cerro de Oro† 5416 — This study‡## Colima†Guerrero Tres Palos†Veracruz CatemacoCatemaco 4775 4776 4777 — — — — CNHE‡ — CNHE CNHE Galicia-Guerrero et al. (2000); Goldberg et al. (2002) Temascal† 5404 — This study‡ Catemaco — — Galicia-Guerrero et al. (2000)‡ Escondida — — Galicia-Guerrero et al. (2000)‡ Emiliano ZapataVeracruz CatemacoLos Tuxtlas 3777 — — — Galicia-Guerrero et — al. (2000)‡ — Goldberg et Guille al. (2002) Rodrigo Go Pesquerı Tres Palos 3736 — Razo-Mendı Villarreal (El Carmen) 4637 — Leo San Pedro las Playas 4071 — Razo-Mendı San Pedro las Playas 4066 — Razo-Mendı La Laguna (Higueras) 4638 — Leo Catemaco — — Galicia-Guerrero et al. (2000); Goldberg et al. (2002)‡ §§ Oaxaca * Guerrero ࿣ * Nuevo Leo * Colima ¶¶ Jalisco ࿣ ¶¶ Oaxaca ¶¶ Veracruz ࿣ * Guerrero ¶¶ Veracruz ࿣ gen. sp. ࿣ sp. sp. sp. sp. gen. sp.¶ Veracruz sp.* Veracruz Helminths Locality II. Continued. Ascaroidea Pseudophyllidea Nematotaenia dispar Centrorhynchus Rauschiella poncedeleoni Contracaecum Polymorphus Prostenorchis Telorchis Rauschiella tineri Distoichometra bufonis Pseudophyllidea Centrorhynchidae Anisakidae Polymorphidae Oligacanthorhyncidae Telorchiidae Nematotaeniidae ABLE Acanthocephala Nematoda T Cestoidea 940 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 ńdez (1992) ńdez (1992) ## ࿣࿣ `gagnon et al. (2005) `gagnon et al. (2005) `gagnon et al. (2005) `gagnon et al. (2005)‡ ń-Herna ń-Herna ń-Re ń-Re ń-Re ń-Re Other collections Reference ń Coleccio Nacional de City (CNHE) Helmintos, Mexico ń á 4646 — Leo ń ń Cerro de Oro† 5403 — This study‡ Salinas 4644 — Leo Pesquerı Villarreal (El Carmen) 4643 — Leo La Laguna (Higueras) 4647 — Leo Cerro de Oro†Temascal†Veracruz CatemacoEscondidaLos Tuxtlas 5411 5412 — — — — — This study‡ — — This — study Galicia-Guerrero et al. Galicia-Guerrero (2000) et al. Guille (2000) EscondidaLos Tuxtlas — — — — Galicia-Guerrero et al. Guille (2000)‡ Huixtla 2333–2334 — Esslinger (1987) Huixtla 2384, 2451 — Esslinger (1988a) Xtoloc — 319*** Pearse (1936) Oaxaca Cerro de Oro†Temascal†Cerro de Oro†Temascal†Veracruz CatemacoYucata 5427 5428 5414 5415 — 2228 — — This — study This study — This study This study Caballero-Deloya (1974) HuixtlaCerro de Oro†Temascal†Puebla MatamorosVeracruz Catemaco 2025, 2158 5409 5410 2122 — — 2224 — Caballero (1949) — This study — This study Bravo-Hollis (1943) Caballero-Deloya (1974) Xtoloc — — Pearse (1936)‡ ¶¶††† Chiapas ¶¶ Chiapas ¶¶ Chiapas * Oaxaca * Chiapas sp.* Nuevo Leo ࿣ * Oaxaca * sp.* Oaxaca sp.* Oaxaca sp.* Veracruz sp.¶ Yucata Helminths Locality II. Continued. Raillietnema Cosmocercoides Cosmocerca Ochoterenella chiapensis Ochoterenella digiticauda Cruzia morleyi Cruzia morleyi Aplectana incerta Aplectana itzocanensis Aplectana Ochoterenella caballeroi Icosiella Cruzidae Cosmocercidae Onchocercidae ABLE T RESEARCH NOTES 941 ńdez (1992) `gagnon et al. (2005) `gagnon et al. (2005) `gagnon et al. (2005) ń-Herna ńez-Villarreal (1969) ńez-Villarreal (1969) ń-Re ń-Re ń-Re Other collections Reference ń Coleccio Nacional de City (CNHE) Helmintos, Mexico ´mez — — Martı ń ń ´rdenas — — Esslinger (1986) á — — Martı á 4640 — Leo ´zaro Ca HuixtlaNuevo Leo 1986 — Caballero (1949) Rodrigo Go Oaxaca Cerro de Oro†Temascal†Veracruz Catemaco 5405Pesquerı Veracruz 5406Catemaco — — — This study — This study 2227 Galicia-Guerrero et al. (2000); Goldberg et al. (2002) — Caballero-Deloya (1974) EscondidaLos Tuxtlas — — — — Galicia-Guerrero et al. Guille (2000) Pesquerı Villarreal (El Carmen) 4639 — Leo HuixtlaHuixtlaTuxtepecEscondida 1896 2336–2337 — — — — 80,277# Esslinger (1988b) Esslinger Esslinger — (1988b) (1987) Galicia-Guerrero et al. (2000) Emiliano ZapataOaxaca Cerro de Oro†Veracruz CatemacoCerro de Oro†Temascal† 3774Emiliano ZapataNuevo Leo 5417 — — 5407 — 5408 3776 Galicia-Guerrero et al. (2000)‡ — This study — — — Goldberg et al. (2002) This study##‡ This Galicia-Guerrero et study al. (2000) Laguna (Higueras) 4641 — Leo HuixtlaJalisco Emiliano ZapataOaxaca Cerro de Oro†Temascal†Veracruz CatemacoLa 3775 1906, 2093 5424 5425 — — — — Caballero (1944); Esslinger Galicia-Guerrero (1986) — et al. (2000) This study — This study Esslinger (1986); Galicia-Guerrero et al. (2000) ¶¶ Oaxaca ** Chiapas ¶¶ Chiapas ††† Chiapas ** Oaxaca ** Jalisco ‡‡‡ Jalisco ࿣ sp.††† Veracruz sp. sp.** Veracruz Helminths Locality II. Continued. Rhabdias sphaerocephala Rhabdias Physaloptera Ochoterenella lamothei Ochoterenella nanolarvata Ochoterenella Ochoterenella figueroai Rhabdias americanus Rhabdias fuelleborni Physalopteridae Rhabdiasidae ABLE T 942 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 ńdez (1992) ń-Herna ńez-Villarreal (1969)‡ Other collections Reference ń Coleccio Nacional de City (CNHE) Helmintos, Mexico ń á — — Martı ń Veracruz 2057 — Bravo-Hollis and Caballero (1940) Xtoloc — 239*** Pearse (1936) HuixtlaYucata 2027 — Caballero (1949) Cerro de Oro†Veracruz CatemacoLos Tuxtlas 5426 — — — — — This study Goldberg et Guille al. (2002) Pesquerı Catemaco — — Goldberg et al. (2002)‡ Cerro de Oro†Temascal† 5418–5421 5422 — — This study‡## This study * Chiapas ¶¶¶ Oaxaca ࿣ * * Nuevo Leo ‡‡‡ Veracruz ࿣ sp.*‡‡‡ Oaxaca sp. Helminths Locality II. Continued. Oswaldocruzia subauricularis Oswaldocruzia Physocephalus Physocephalus sexalatus Oswaldocruzia pipiens New record for Mexico. Spirocercidae Thichostrongylidae Larvae. ABLE # Specimens deposited in¶ the Urinary United bladder. States** National Lungs. Parasite Collection,†† Beltsville, Bile Maryland. ducts. ‡‡ Specimens deposited in§§ the Muscle. Harold W. Manter Laboratory of Parasitology, University of Nebraska, Lincoln. T * Intestine. † New locality record. ‡ Not included in§ Barton Eyes. (1997). ࿣ ࿣࿣ ## New host record. ¶¶ Mesentery. *** Specimens deposited in††† the Body Museum cavity. of‡‡‡ Zoology, Stomach. University of Michigan. RESEARCH NOTES 943 tophenol, and studied without the aid of a permanent mount. Host and bibliographical sources, the number of helminth taxa known for this helminth voucher specimens were deposited in the Coleccioń Herpe- host in Mexico has increased to 45; these records are from 26 localities, tolo´gica del Museo de Zoologıá de la Facultad de Ciencias (MZFC), distributed in 9 states. The highest species richness is represented by Universidad Nacional Autońoma de Me´xico (UNAM), Mexico City, nematodes (27 taxa), followed by digeneans (11 taxa); only 10 taxa under accession numbers MZFC-ND833, and in the Coleccioń Nacional have been recorded as larval stages (Table II). de Helmintos (CNHE), Biology Institute, UNAM, Mexico City, respec- The number of helminth taxa parasitizing B. marinus in its native tively. Prevalence, mean intensity, and range of intensity follow the and introduced range of distribution is 76 (see Barton, 1997). However, definitions outlined by Bush et al. (1997). this number is not accurate as a result of several taxonomical changes, From the 34 B. marinus studied in the 2 localities, we collected e.g., synonymies and transfers not considered by Barton (1997). Since 14,749 helminths, representing 14 taxa: 2 digeneans (adults), 1 cestode that time, 37 more taxa have been added to the helminth fauna of this (in larval stage), and 11 nematodes (3 larvae and 8 adults). Hosts from anuran species, with 24 of them collected in Mexico (see species both localities shared 7 taxa (Aplectana itzocanensis Bravo-Hollis, marked with ‡ in Table II); 4 from Peru (Acanthocephalus lutzi [Lin- 1943; Cosmocerca sp.; Cruzia morleyi Pearse, 1936; Ochoterenella dig- stow, 1896], see Tantalean, 1976; Aplectana hylambatis [Baylis, 1927]; iticauda Caballero, 1944; Physocephalus sexalatus [Molin, 1860]; Cosmocerca brasiliensis Travassos, 1925; and Cosmocerca parva Tra- Rhabdias americanus Baker, 1978; and Rhabdias fuelleborni Travassos, vassos, 1925 [see Bursey et al., 2001]); 2 from Australia (Johnpearsonia 1925), while Choledocystus hepaticus Lutz, 1928; Contracaecum sp.; pearsoni Durette-Desset, Ben Slimane, Cassone, Barton, and Chabaud, Physaloptera sp.; Oswaldocruzia sp.; and Raillietnema sp. were found 1994 [see Durette-Desset et al., 1994] and Rhabdias cf. hylae sensu in Cerro de Oro Dam. Mesocoelium monas Teixeira de Freitas, 1958, Barton, 1998, see Barton, 1998); 2 from Bermuda (Abbreviata sp. and and larvae of the pseudophyllidean were found only to parasitize hosts Clinostomum sp. [see Linzey et al., 1998]); 2 from Brazil (Oswaldo- from Temascal Dam. cruzia taranchoni Ben Slimane and Durette-Desset, 1995 [see Ben Sli- Helminth taxa were collected from 8 sites within the hosts, with the mane and Durette-Desset, 1995] and Capillaria petiti Justine and Bain, intestine being the most common (7 taxa). Larvae of P. sexalatus and 1987 [see Justine and Bain, 1987]); 1 from Venezuela (Oswaldocruzia O. digiticauda were the only species found in more than 1 location (4 venezuelensis Ben Slimane, Guerrero, and Durette-Desset, 1996 [Ben and 2, respectively). Slimane et al., 1996]); 1 from Hawaii (Acanthocephalus bufonis [Shi- Hosts from Cerro de Oro Dam harbored 12 taxa, while those from pley, 1903] [see Barton and Pichelin, 1999]); and 1 from Trinidad Temascal Dam were parasitized by 9. In both localities, all hosts were (Glypthelmins quieta [Stafford, 1900] [see Ragoo and Omah-Maharaj, infected by at least 1 helminth species. Nematodes constituted the most 2003]). This results in a total of 113 taxa parasitizing B. marinus abundant parasitic group in the 2 B. marinus populations, i.e., 8,193 throughout its entire distribution range, with 40.5% of them found in individuals were collected in hosts from Cerro de Oro and 6,315 from Mexico. The taxonomic composition of helminths from this host species Temascal. Digeneans and cestodes were represented by 237 and 4 in- along its native and introduced distributional range follows a similar dividuals, respectively. Aplectana itzocanensis reached the highest lev- pattern to that observed in Mexican specimens, i.e., nematodes and els of infection in Temascal (70% and 443.6 individuals by examined digeneans constitute the richest groups (43 and 34 taxa, respectively), host), while in Cerro de Oro, R. fuelleborni parasitized 66.6% of hosts with larval stages being scarcely represented. and occurred with a mean abundance of 12 individuals by examined Of the 112 taxa, only 19 species have been added to the helminth host. Values of prevalence and mean abundance for the remaining hel- fauna of B. marinus in its new distribution range (Barton, 1997; present minth species in both localities varied from 10% to 50%, and 0.3 to study). Although well documented, we consider that the helminthol- 179.7 in hosts from Temascal, and from 4.2% to 62.5%, and 0.042 to ogical record of this host species could increase after further inventory 84.9 in those from Cerro de Oro, respectively (Table I). As indicated, work in poorly sampled regions, e.g., Central America and southeastern 7 helminth species were found in the intestine, with a total of 7,256 Mexico. The colonization of helminth species from local anurans by B. individuals. Distinct sample sizes among the 2 host populations pre- marinus introduced populations that share plesiomorphic traits with the clude us from drawing conclusions about infection level differences; original host should allow for host-switching events (Brooks et al., however, in spite of the reduced number of B. marinus examined in 2006). Temascal Dam, prevalence and mean abundance in 4 of 7 shared hel- We thank Yolanda Pe´rez-A´ lvarez, Elizabeth A. Martıńez-Salazar, Ro- minth species were higher than those observed in Cerro de Oro Dam. sario Mata Lo´pez, and Rogelio Rosas-Valdez for their assistance in field In general, this may be attributed to possible differences in the local trips. Edmundo Pe´rez-Ramos identified the hosts. This study was found- availability of intermediate host species as well as local environmental ed by NSF project DEB 0102383 to V.L.R., and J. Campbell, U.T.A. conditions; nevertheless, additional parasitological surveys of hosts representing both localities will be necessary for confirmation. LITERATURE CITED The most frequent mode of parasite recruitment was via direct ingestion; thus, 64.3% of the species were recruited through the food web. ANDERSON, R. C. 2000. Nematode parasites of vertebrates: Their de- In contrast, 28.6% of helminth species are acquired by skin penetration, velopment and transmission, 2nd ed. CAB International, Walling- with 7.1% transmitted by a mosquito vector (Yamaguti, 1975; Schmidt, ford, Oxon, U.K., 650 p. 1986; Anderson, 2000). BARTON, D. 1997. Introduced animals and their parasites: The cane toad, There were 25 new locality records, but only 2 taxa were found for Bufo marinus in Australia. Australian Journal of Ecology 22: 316– the first time in Mexico, i.e., the pseudophyllidean and Raillietnema sp. 324. These 2 latter taxa, along with Contracaecum sp., R. americanus, and ———. 1998. Dynamics of natural infections of Rhabdias cf. hylae P. sexalatus, constituted new host records (Table II). The larval states (Nematoda) in Bufo marinus (Amphibia) in Australia. Parasitology of the pseudophyllidean, Contracaecum sp., and Physaloptera sp. pre- 117: 505–513. clude species determination. However, larvae of P. sexalatus were de- ———, AND S. PICHELIN. 1999. Acanthocephalus bufonis (Acantho- termined at species level based on Moravec et al. (1995), who estab- cephala) from Bufo marinus (Bufonidae: Amphibia) in Hawaii. Par- lished the presence of transverse striations on the body, 2 conical pro- asite 6: 269–272. jections on the cephalic end, and a round terminal formation with mi- BEN SLIMANE, B., AND M. C. DURETTE-DESSET. 1995. Oswaldocruzia nute papillalike outgrowths on the tail as diagnostic traits of this (Nematoda: Trichostrongylina, Molineoidae) parasitic in Brazilian nematode species in larval stage. Likewise, it was not possible to iden- and Ecuadorian amphibians, with redefinition of the type species tify 3 other adult nematode species because only females were collected, O. subauricularis (Rudolphi, 1819) and O. mazzai Travassos, 1935. i.e., Cosmocerca sp., Oswaldocruzia sp., and Raillietnema sp. Eggs and Revue Suisse Zoologie 102: 635–653. body size, as well as host and geographical distribution, suggest that ———, R. GUERRERO, AND M. C. DURETTE-DESSET. 1996. Oswaldocru- specimens of Raillietnema sp. could represent an undescribed species; zia venezuelensis sp. n. (Nematoda: Trichostrongylina, Molineo- notwithstanding, species determination will depend on the study of male idea) a parasite of Bufo marinus from Venezuela. Folia Parasito- specimens. logica 43: 297–300. Prior to the present study, the helminth fauna of B. marinus in Mexico BRAVO-HOLLIS, M. 1943. Dos nuevos nema´todos para´sitos de anuros was thought to include 26 taxa (Galicia-Guerrero et al., 2000). As a del sur de Puebla. Anales del Instituto de Biologıá, Universidad result of our survey and the compilation of records published in several Nacional Autońoma de Me´xico 14: 69–78. 944 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

———. 1948. Descripcioń de dos especies de trema´todos para´sitos de VALE´ , AND C. CAN˜ EDA-GUZMA´ N. 2002. Helminth parasites of six Bufo marinus L., procedentes de Tuxtepec, Oaxaca. Anales del In- species of anurans from Los Tuxtlas and Catemaco Lake, Veracruz, stituto de Biologıá, Universidad Nacional AutońomadeMe´xico 19: Me´xico. Southwestern Naturalist 47: 293–299. 153–161. GUILLE´ N-HERNA´ NDEZ, S. 1992. Comunidades de helmintos de algunos ———, AND E. CABALLERO C. 1940. Nema´todos para´sitos de los batra- anuros de ‘‘Los Tuxtlas’’, Veracruz. M.S. Thesis. Universidad Na- cios de Me´xico IV. Anales del Instituto de Biologıá, Universidad cional Autońoma de Me´xico, Mexico City, D.F., 90 p. Nacional AutońomadeMe´xico 11: 239–247. ———, G. SALGADO-MALDONADO, AND R. LAMOTHE-ARGUMEDO. 2000. BROOKS, D. R., V. LEO´ N-RE` GAGNON,D.A.MCLENNAN, AND D. ZELMER. Digenean (Platyhelminthes: Trematoda) of seven sympatric species 2006. Ecological fitting as a determinant of the community struc- of anurans from Los Tuxtlas, Veracruz, Mexico. Studies on Neo- ture of platyhelminth parasites of anurans. Ecology 87: S76–S85. tropical Fauna and Environment 35: 10–13. BURSEY, C. R., S. R. GOLDBERG, AND J. R. PARMELEE. 2001. Gastroin- JUSTINE,J.L.,AND O. BAIN. 1987. Capillaria petiti n. sp. (Nematoda, testinal helminths of 51 species of anurans from Reserva Capillariinae) parasite du crapaud Bufo marinus (Amphibia) au CuzcoAmazońico, Peru. Comparative Parasitology 68: 21–25. Bresil. Bulletin du Museum National d’Historie Naturelle, Paris 4th BUSH,A.O.,K.D.LAFFERTY,J.M.LOTZ, AND A. W. SHOSTAK. 1997. Series 9: 815–828. Parasitology meets ecology on its own terms: Margolis et al. re- LAMOTHE-ARGUMEDO, R., G. PE´ REZ-PONCE DE LEO´ N, AND L. GARCIÁ- visited. Journal of Parasitology 83: 575–583. PRIETO. 1993. Helmintos para´sitos de animales silvestres. In His- CABALLERO, C. E. 1944. Estudios helmintolo´gicos de la regioń onco- toria natural de Los Tuxtlas, Veracruz, S. E. Gonza´lez, R. Dirzo, cercosa de Me´xico y de la Repu´blica de Guatemala. Nematoda: 1a and R. C. Vogt (eds.). Universidad Nacional AutońomadeMe´xico, Parte. Filarioidea I. Anales del Instituto de Biologıá, Universidad Instituto de Ecologıá y CONABIO, Mexico City, D.F., p. 387–394. Nacional AutońomadeMe´xico 15: 87–108. LEO´ N-RE` GAGNON, V., E. A. MARTIŃEZ-SALAZAR,D.LAZCANO-VILLAR- ———. 1949. Estudios helmintolo´gicos de la regioń oncocercosa de REAL, AND R. ROSAS-VALDEZ. 2005. Helminth parasites of four spe- Me´xico y de la Repu´blica de Guatemala. Nematoda, 5a Parte. An- cies of anurans from Nuevo Leoń, Mexico. Southwestern Naturalist ales del Instituto de Biologıá, Universidad Nacional Autońoma de 50: 251–258. Me´xico 20: 279–292. LINZEY, D. W., C. R. BURSEY, AND J. B. LINZEY. 1998. Seasonal occur- ———, M. BRAVO-HOLLIS, AND C. CERECERO. 1944. Estudios helmin- rence of helminths of the giant toad Bufo marinus (Amphibia: Bu- tolo´gicos de la regioń oncocercosa de Me´xico y de la Repu´blica fonidae), in Bermuda. Journal of the Helminthological Society of de Guatemala. Trematoda I. Anales del Instituto de Biologıá, Univ- Washington 65: 251–258. ersidad Nacional Autońoma de Me´xico 15: 59–72. MARTIŃEZ-SALAZAR, E. 2004. Estudio taxono´mico de algunas pobla- CABALLERO-DELOYA, J. 1974. Estudios helmintolo´gicos de los animales ciones del geńero Langeronia Caballero y Bravo-Hollis, 1949 silvestres de la Estacioń de Biologıá Tropical ‘‘Los Tuxtlas’’, Ve- (Trematoda: Lecithodendriidae) en Me´xico. M.S. Thesis. Univer- racruz. Nematoda 1. Algunos nema´todos para´sitos de Bufo horri- sidad Nacional Autońoma de Me´xico, Mexico City, D.F., 92 p. bilis Wiegmann, 1833. Anales del Instituto de Biologıá, Universi- MARTIŃEZ-VILLARREAL, J. M. 1969. Para´sitos de algunos anfibios colec- dad Nacional Autońoma de Me´xico 45: 45–50. tados en diferentes a´reas de los municipios de Escobedo, Pesquerıá DURETTE-DESSET, M. C., B. BEN SLIMANE,J.CASSONE,D.P.BARTON, y Santiago, Nuevo Leoń, Me´xico. B.S. Thesis. Universidad Autoń- AND A. G. CHABAUD. 1994. Johnpearsonia gen. nov. and John- oma de Nuevo Leoń, Monterrey, Nuevo Leoń, Mexico, 53 p. pearsoniinae subfam. nov. (Molineoidea, Nematoda) from Bufo MATA-LO´ PEZ, R., AND V. L EO´ N-RE` GAGNON. 2005. Gorgoderina festoni marinus, with comments on the primitive trichostrongyle parasite n. sp. (Digenea: Gorgoderidae) in anurans (Amphibia) from Me´x- of amphibians and reptiles. Parasite 1: 153–160. ico. Systematic Parasitology 60: 185–190. ESSLINGER, J. H. 1986. Redescription of Ochoterenella digiticauda Ca- MORAVEC, F., C. VIVAS-RODRI´GUEZ,T.SCHOLZ,J.VARGAS-VA´ ZQUEZ,E. ballero, 1944 (Nematoda: Filarioidea) from the toad, Bufo marinus, MENDOZA-FRANCO,J.SCHMITTER-SOTO, AND D. GONZA´ LEZ-SOLIS. with a redefinition of the genus Ochoterenella Caballero, 1944. 1995. Nematodes parasites in fishes of cenotes (ϭsinkholes) of the Proceedings of the Helminthological Society of Washington 53: Peninsula of Yucatan, Mexico. Part II. Larvae. Folia Parasitologica 210–217. 42: ———. 1987. Ochoterenella caballeroi sp. n. and O. nanolarvata sp. 199–210. n. (Nematoda: Filarioidea) from the toad Bufo marinus. Proceed- PEARSE, A. S. 1936. Parasites from Yucatan. Carnegie Institution of ings of the Helminthological Society of Washington 54: 126–132. Washington Publication 457: 45–59. ———. 1988a. Ochoterenella chiapensis n. sp. (Nematoda: Filarioidea) RAGOO, R., AND I. OMAH-MAHARAJ. 2003. Helminths of the cane toad from the toad Bufo marinus in Mexico and Guatemala. Transactions Bufo marinus from Trinidad, West Indies. Caribbean Journal of of the American Microscopical Society 107: 203–208. Sciences 39: 242–245. ———. 1988b. Ochoterenella figueroai sp. n. and O. lamothei sp. n. RAZO-MENDI´VIL, U. 2004. Sistema´tica del Geńero Glypthelmins Staf- (Nematoda: Filarioidea) from the toad Bufo marinus. Proceedings ford, 1905 (Platyhelminthes: Digenea). Ph.D. Thesis. Universidad of the Helminthological Society of Washington 55: 146–154. Nacional AutońomadeMe´xico, Mexico City, D.F., 343 p. ´ ` ETGES, F. J. 1991. Clinostomum attenuatum (Digenea) from the eye of ———, AND V. L EON-REGAGNON. 2001. Glypthelmins poncedeleoni n. Bufo marinus. Journal of Parasitology 77: 634–635. sp. (Trematoda: Macroderoididae) of amphibians from the Neo- FLORES-VILLELA, O. 1998. Herpetofauna de Me´xico: distribucioń y en- tropical region of Mexico. Journal of Parasitology 87: 686–691. demismos. In Diversidad biolo´gica de Me´xico, E. Ramamoorthy, SCHMIDT, G. D. 1986. Handbook of tapeworm identification. CRC Press, R. Bye, A. Lot, and J. Fa (eds.). Instituto de Biologıá, Universidad Boca Raton, Florida, 675 p. Nacional AutońomadeMe´xico, Mexico City, D.F., p. 251–278. SPEARE, R. 1990. A review of diseases of the cane toad, Bufo marinus, GALICIA-GUERRERO, S., C. R. BURSEY,S.R.GOLDBERG, AND G. SAL- with comments on biological control. Australian Wildlife Research GADO-MALDONADO. 2000. Helminths of two sympatric toad species, 17: 387–410. Bufo marinus (Linnaeus) and Bufo marmoreus Wiegmann, 1833 TANTALEAN, V. N. 1976. Contribucioń al conocimiento de los helmintos (Anura: Bufonidae) from Chamela, Jalisco, Mexico. Comparative de vertebrados del Peru´. Biota 10: 437–443. Parasitology 67: 129–133. YAMAGUTI, S. 1975. A synoptical review of life histories of digenetic GOLDBERG, S. R., C. R. BURSEY,G.SALGADO-MALDONADO,R.BA´ EZ- trematodes of vertebrates. Keigaku, Tokyo, Japan, 575 p. RESEARCH NOTES 945

J. Parasitol., 93(4), 2007, pp. 945–946 ᭧ American Society of Parasitologists 2007

Parasitic Helminths of Free-Ranging Mink (Neovison vison mink) From Southern Florida

Garry W. Foster, Mark W. Cunningham*, John M. Kinsella†, and Mike Owen‡, P.O. Box 919, Archer, Florida 32618; *Florida Fish and Wildlife Conservation Commission, 4005 South Main Street, Gainesville, Florida 32601; †HelmWest Lab, 2108 Hilda Avenue, Missoula, Montana 59801; ‡Fakahatchee Strand Preserve State Park, P.O. Box 548, Copeland, Florida 34137. e-mail: [email protected]

ABSTRACT: Five free-ranging mink, Neovison vison mink, from in or are currently no estimates of population size or density, and Humphrey near the Fakahatchee Strand Preserve State Park, Collier County, Flor- (1992) lists this disjunct population of N. v. mink as rare. ida (26Њ00ЈN, 81Њ25ЈW), were examined for parasitic helminths. Nine Erickson (1946) gave a review of mink parasite literature from North species of helminths were identified (2 trematodes, 5 nematodes, and 2 America to the date of his publication, and Barber and Lockard (1973) acanthocephalans). The most prevalent parasites were Molineus patens published a worldwide mink parasite checklist. The only major survey (4 of 5 mink), mean intensity 173 (range, 12–342); Strongyloides sp. of mink parasites reported recently is that of Zabiega (1996) from south- (4 of 5), mean intensity 48 (range, 1–170); Macracanthorhynchus in- ern Illinois. There appears to be no published reports on the parasites gens (immature) (4 of 5), mean intensity 2.5 (range, 1–4); and Alaria for the 3 subspecies of mink from Florida. Herein, we report on the mustelae (3 of 5), mean intensity 59.3 (range, 11–127). Polymorphus parasites from mink, Neovison vison mink,(ϭ M. v. evergladensis) col- brevis and Dirofilaria lutrae are reported from the mink for the first lected in southern Florida. time. A total of 5 adult male mink, 2 complete and 3 partial carcasses, were examined for parasites. All mink were collected in or near the The distribution of free-ranging mink, Neovison vison (Schreber, Fakahatchee Strand Preserve State Park, Collier County, Florida 1777), in south Florida is largely limited to the shallow freshwater (26Њ00ЈN, 81Њ25ЈW). Only the body, with intact skin, and gastrointes- marshes in the southern areas of Collier and Dade counties, and parts tinal tract were present for the 3 partial carcasses, which were necrop- of mainland Monroe County (Humphrey, 1992). There are 2 other sub- sied previous to the parasite examination. The hosts for the 2 complete species of mink in Florida that occur in coastal salt marshes along the and 1 partial carcass were collected as road-kills. The hosts for the other northern Gulf and Atlantic coasts (Humphrey, 1992). 2 partial carcasses were from animals killed at a rehabilitation facility, Hamilton (1948) described a new subspecies of mink, Mustela vison with signs of canine distemper, within 24 hr of being received. The evergladensis, from a single road-killed specimen obtained in southern carcasses were individually placed in freezer bags and frozen until ex- Florida. Humphrey and Setzer (1989) compared the morphology of 24 amined in the laboratory. One complete and the 3 partial carcasses were skulls of M. v. evergladensis with skulls of 3 other subspecies of mink collected between May and September, 2004, and the remaining full from the southeastern United States and concluded that M. v. everglad- carcass was collected in January 1995. ensis was not a valid subspecies, but a disjunct population of the sub- Parasite screening techniques used were similar to those described by species M. v. mink. Wozencraft (2005) gave several sources supporting Zabiega (1996), but included, when present, the heart, kidneys, and the placement of the M. vison from North America in the genus Neo- urinary bladder. The intestinal contents were screened with the use of vison rather than in Mustela. a 100-mesh (0.150-mm) screen. The skin was removed to look for sub- The mink in southern Florida are currently listed (as M. v. everglad- cutaneous parasites, and the nasal passages were examined also. Trem- ensis) as Threatened by the Florida Fish and Wildlife Conservation atodes and acanthocephalans were preserved in Roudabush’s AFA, and Commission (FWC) (Sullivan, 2006), but there is no federal listing. nematodes in 70% ethanol. Trematodes were stained with Harris’ he- Some authors consider mink in this region to be rare (Hamilton, 1948; matoxylin and mounted in neutral Canada balsam. Nematodes and acan- Layne, 1974; Brown, 1978); however, others consider them relatively thocephalans were mounted in lactophenol for identification and then common in certain areas of the Everglades and Big Cypress Swamp returned to the preservative. (Allen and Neill, 1952; Smith, 1980; Humphrey and Zinn, 1982). There Terminology used follows Bush et al. (1997). Helminth voucher spec-

TABLE I. Intensities of parasitic helminths in 5 free-ranging mink (Neovison vison mink) from southern Florida.

Intensity USNPC* accession Location Number Parasite number in host† positive Mean (SD) Range

Trematodes Alaria mustelae Bosma, 1931 99437 SI 3 59.3 (60.4) 11–127 Baschkirovitrema incrassatum (Diesing, 1950) 99436 SI 2 3.5 (3.5) 1–6 Nematodes Capillaria putorii (Rudolphi, 1819) 99441 SI 2 4.5 (4.9) 1–8 Diroﬁlaria lutrae Orihel, 1965‡ 99439 BC 1 1 (—) — Molineus patens (Dujardin, 1844) 99438 ST, SI 4 173 (135.3) 12–342 Physaloptera sp. (larvae) 99440 ST, SI 1 2 (—) — Strongyloides sp. 99444 SI 4 48 (81.7) 1–170 Acanthocephalans Macracanthorhynchus ingens (immature) (Linstow, 1879) 99443 SI 4 2.5 (1.3) (1–4) Polymorphus brevis (immature)‡ Van Cleave, 1916 99442 SI 1 2 (—) —

* U.S. National Parasite Collection. †BCϭ body cavity, SI ϭ small intestine, ST ϭ stomach. ‡ New host record. 946 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

imens have been deposited in the U.S. National Parasite Collection, BARBER,D.L.,AND L. L. LOCKARD. 1973. Some helminths from mink Beltsville, Maryland (USNPC Nos. 99436–99444). in southwestern Montana, with a checklist of their internal para- A total of 9 helminth species (2 trematodes, 5 nematodes, and 2 sites. Great Basin Naturalist 33: 53–60. acanthocephalans) was collected (Table I). The most prevalent parasites BROWN, L. N. 1978. Everglades mink. In Rare and endangered biota of were Molineus patens (in 4 of 5 mink), Strongyloides sp. (4 of 5), Florida, Volume 1, Mammals, L. J. Layne (ed.). University Press Macracanthorhynchus ingens (immature) (4 of 5), and Alaria mustelae of Florida, Gainesville, Florida, p. 26–27. (3 of 5). All carcasses were carefully skinned, but no subcutaneous BUSH,A.O.,K.D.LAFFERTY,J.M.LOTZ, AND A. W. SHOSTAK. 1997. parasites were found, and no parasites were seen in the nasal sinuses. Parasitology meets ecology on its own terms: Margolis et al. re- None of the immature M. ingens was encysted, and those with everted visited. Journal of Parasitology 83: 575–583. probosci were attached through the mucosa deep into the intestinal wall. DORNEY, R. S., AND L. H. LAUERMAN. 1969. A helminthological survey The 2 specimens of Polymorphus brevis were also immature and not of wild mink in Wisconsin. Bulletin of the Wildlife Disease As- encysted. Dirofilaria lutrae and P. brevis are reported from the mink sociation 5: 35–36. for the first time. ERICKSON, A. B. 1946. Incidence of worm parasites in Minnesota Mus- Most mink parasite surveys that have been reported previously had telidae and host lists and keys to North American species. The sample sizes from 47 up to 120 or more mink that were usually col- American Midland Naturalist 36: 494–509. lected by commercial trappers. Because N. v. mink in southern Florida FORRESTER, D. J. 1992. Parasites and diseases of wild mammals in Flor- is listed as Threatened by the FWC in 1977, the only way we were able ida. University Press of Florida, Gainesville, Florida, 459 p. to obtain specimens was by collecting road-killed animals. Since 1995 ———, AND M. G. SPALDING. 2003. Parasites and diseases of wild birds we were able to collect only the 5 individuals we report herein. It is in Florida. University Press of Florida, Gainesville, Florida, 1132 p. difficult to compare such a small sample of hosts with the other surveys HAMILTON,JR., W. J. 1948. A new mink from the Florida everglades. and draw any meaningful conclusions. Proceedings of the Biological Society of Washington 61: 139–140. However, it is interesting to note that we found less than half the HUMPHREY, S. R. 1992. Southern Florida population of mink. In Rare number of helminth species reported by Miller and Harkema (1964) (21 and endangered biota of Florida, Volume 1, Mammals, S. R. Hum- species) in mink from North Carolina. The prevalence and intensity of phrey (ed.). University Press of Florida, Gainesville, Florida, p. Molineus patens we report (4 of 5 mink, mean intensity 173, range 12– 319–327. 342) was much greater than those reported in North Carolina (34 of ———, AND H. W. SETZER. 1989. Geographic variation and taxonomic 120, no intensity reported) by Miller and Harkema (1964), Wisconsin revision of mink (Mustela vison) in Florida. Journal of Mammalogy (1 of 47, intensity of 1) by Dorney and Lauerman (1969), and Illinois 70: 241–252. (Molineus sp., 1 of 50, intensity of 1) by Zabiega (1996). None was ———, AND T. L. ZINN. 1982. Seasonal habitat use by river otters and reported from 100 mink sampled in Montana by Barber and Lockard 46: (1973). Everglades mink in Florida. Journal of Wildlife Management Dirofilaria lutrae was described from river otters (Lutra canadensis) 375–381. by Orihel (1965). Some of the specimens were collected in Collier LAYNE, J. N. 1974. The land mammals of south Florida. Miami Geo- County, Florida, which is the same general area of southern Florida logical Society Memoir 2: 386–413. where the mink for this study were collected. His mature specimens of MILLER,G.C.,AND R. HARKEMA. 1964. Studies on helminths of North D. lutrae were found in the subcutaneous tissues; however, our speci- Carolina vertebrates. V. Parasites of the mink, Mustela vison Schre- men was an immature female found in the body cavity on the serosal ber. Journal of Parasitology 50: 717–720. surface of the small intestine. ORIHEL, T. C. 1965. Dirofilaria lutrae sp. n. (Nematoda: Filarioidea) No mature acanthocephalans have been reported from southeastern from otters in the southeastern United States. Journal of Parasitol- mink. Miller and Harkema (1964) found cystacanths of Centrorhynchus ogy 51: 409–413. conspectus, a parasite of the barred owl, Strix varia, encysted in the SMITH, A. T. 1980. An environmental study of the Everglades mink small intestine of 17, and cystacanths of M. ingens encysted on the (Mustela vison). South Florida Research Center, Everglades Na- viscera in 2 of 120 mink in North Carolina. The specimens of M. ingens tional Park, Report T-555, p. 1–17. we found here were attached to the intestinal wall, but immature. Two SULLIVAN, J. D., JR. 2006. Florida’s endangered species, threatened spe- specimens of P. brevis were also excysted, but immature. Forrester cies, and species of special concern. Florida Fish and Wildlife Con- (1992) lists M. ingens as a common parasite of raccoons, Procyon lotor, servation Commission, Tallahassee, Florida, 7 p. in southern Florida, and Forrester and Spalding (2003) list P. brevis as WOZENCRAFT, W. C. 2005. Family Mustelidae. In Mammal species of a common parasite of herons and egrets in southern Florida. Both spe- the world: A taxonomic and geographic reference, 3rd ed., D. E. cies are probably accidental in mink and apparently do not mature. Wilson, and D. M. Reeder (eds.). The Johns Hopkins University Press, Baltimore, Maryland, p. 601–605. LITERATURE CITED ZABIEGA, M. H. 1996. Helminths of mink, Mustela vison, and muskrats, ALLEN,A.W.,AND W. T. N EILL. 1952. Notes on the abundance of the Ondatra zibethicus, in southern Illinois. Journal of the Helmin- Everglades mink. Journal of Mammalogy 33: 113–114. thological Society of Washington 63: 246–250. RESEARCH NOTES 947

J. Parasitol., 93(4), 2007, p. 947 ᭧ American Society of Parasitologists 2007

Viability of the Encysted Metacercariae of Echinostoma caproni Judged by Light Microscopy Versus Chemical Excystation

Bernard Fried and Robert C. Peoples, Department of Biology, Lafayette College, Easton, Pennsylvania 18042. e-mail: [email protected]

ABSTRACT: The purpose of this study was to determine the viability of were used. These cysts were stored in amounts of about 1,000 cysts/15 encysted metacercariae of Echinostoma caproni stored in Locke’s so- ml of Locke’s 1:1 in shell vials at 4 C until tested at the appropriate lution 1:1 at 4 C for 1–24 wk. Viability was judged by light microscopy time poststorage. For LM observations, 23–103 cysts from different (LM) based on morphological characteristics of the encysted metacer- batches were observed as described above with the use of a wet mount cariae versus chemical excystation of the cysts in a trypsin–bile salts preparation. Cyst viability was judged based on morphological char- excystation medium. The percent viability was very similar under both acteristics, i.e., a visible curled organism in the cyst showing suckers, methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk spines, and excretory concretions, and well-formed inner and outer poststorage, viability was found to be about 2ϫ greater based on ex- cysts. Photomicrographs of viable and nonviable cysts are available in cystation than using LM. We concluded that LM alone underestimated Rossi et al. (2001). Cysts were considered questionable if they did not the viability of cysts and that determination of cyst viability was more have the aforementioned characteristics, were embedded in cellular de- accurate under assessment by chemical excystation. bris, or were distinctly granular in appearance. When doubt existed in regard to the status of a cyst, it was classiﬁed as questionable and listed Toledo and Fried (2005) provided information on the number of lab- in the nonviable category as judged by LM. For the excystation studies, oratories worldwide that maintain the life cycle of Echinostoma cap- 17–104 cysts from the same batches used for LM were selected at roni. In many of these laboratories, encysted metacercariae are obtained random and subjected to chemical excystation at 41 C for 2 hr. The and stored for later use following the experimental infection of Biom- number of fully excysted metacercariae was determined at the end of phalaria spp. with cercariae of E. caproni. Cysts are removed from the the 2-hr excystation trial. To do this, excysted metacercariae were re- pericardial/kidney region of the snails within 1–2 wk postinfection and moved from the TB medium to Locke’s and counted on a depression stored for prolonged periods in Locke’s 1:1 solution at 4 C (Fried and slide at 100ϫ. All excysted metacercariae were live and active at re- Huffman, 1996). One of us (B.F.) usually supplies workers around the covery. world with E. caproni cysts for teaching and/or research purposes. The results of our observations are summarized in Table I. Obser- Based on cyst availability, we try to ship cysts that that have been stored vations by LM from week 1 to week 24 showed values of 51% at week in our laboratory at 4 C in the Locke’s 1:1 for 1–16 wk. The ability to 1 and 41% at week 24. Values at the other intervals ranged from 83 to maintain the cysts year round makes this species a valuable one for 94%. When we examined cysts from the same storage periods as used obtaining adults of this echinostome in vertebrate hosts at all times. in the LM studies by chemical excystation, we observed high percent We usually determine viability based on light microscopy (LM) at viability at all time periods from a high of 96% at 8 wk, to a low of 100 or 200ϫ of about 20–100 cysts selected randomly from a given 80% at 24 wk. Previous work with chemically excysted metacercariae cyst batch. Cysts are placed on a slide in a drop of Locke’s solution. A (Chien et al., 1993) showed that these larvae implanted surgically into coverslip coated with Vaseline on 2 edges is placed on the drop of the gut of mice developed into adult worms at a very high rate. Thus, Locke’s solution. Coating the slip with Vaseline avoids crushing and chemical excystation of the encysted metacercariae of E. caproni pro- distorting the cysts during examination. Further details on the micro- vides a good indicator of the viability and infectivity of this species to scopical observations of cyst structures are given below. vertebrate hosts. We assess the viability of certain batches of cysts by subjecting them We also concluded that visual observations by LM can underestimate to chemical excystation in a trypsin–bile salts (TB) medium described the viability of cysts, at least for this species of echinostome, and that in Fried and Roth (1976) for the excystation of Parorchis acanthus. a better method of determining viability is to subject a representative This medium has been used to chemically excyst numerous echinosto- sample to chemical excystation. Results from this note are probably matid and nonechinostomatid cysts (see review in Fried, 1994). Use of applicable to other species of Echinostoma and to other genera in the this medium with viable E. caproni cysts usually results in excystation family Echinostomatidae. values of Ͼ90% at 41 C within 2 hr (Fried and LaTerra, 2002). The purpose of the present study was to compare cyst viability of E. caproni maintained at various periods of storage for 1–24 wk in Locke’s LITERATURE CITED 1:1 at 4 C with the use of LM versus chemical excystation. For this CHIEN, W. Y., D. W. HOSIER, AND B. FRIED. 1993. Surgical implantation study, cysts from different batches stored for 1, 4, 8, 16, and 24 wk of Echinostoma caproni metacercariae and adults into the small intestine of ICR mice. Journal of the Helminthological Society of Washington 60: 122. TABLE I. Viability of the encysted metacercariae of Echinostoma cap- FRIED, B. 1994. Metacercarial excystment of trematodes. Advances in roni judged by light microscopy and chemical excystation. Parasitology 33: 91–144. ———, AND J. E. HUFFMAN. 1996. The biology of the intestinal trem- Viability (judged by Viability (judged by atode Echinostoma caproni. Advances in Parasitology 38: 311– light microscopy) chemical excystation) 368. Time of ———, AND R. LATERRA. 2002. In vitro and in vivo encystment of the cyst Number Number Number Number cercariae of Echinostoma caproni. Journal of Parasitology 86: storage of cysts of viable of cysts of viable 1124–1129. (wk) used cysts % Viable used cysts % Viable ———, AND R. M. ROTH. 1974. In vitro excystment of the metacercariae of Parorchis acanthus. Journal of Parasitology 60: 465. 1 53 27 51 104 95 91 ROSSI, M. L., C. D. BALFOUR, AND B. FRIED. 2001. Excystation and 4 50 47 94 104 93 89 infectivity of Echinostoma caproni metacercariae after prolonged 8 103 96 93 51 49 96 storage. Comparative Parasitology 68: 126–129. 16 23 19 83 17 14 82 TOLEDO, R., AND B. FRIED. 2005. Echinostomes as experimental models 24 74 30 41 97 78 80 for interactions between adult parasites and vertebrate hosts. Trends in Parasitology 21: 251–254. 948 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

J. Parasitol., 93(4), 2007, pp. 948–950 ᭧ American Society of Parasitologists 2007

Coprology of Panthera tigris altaica and Felis bengalensis euptilurus From the Russian Far East

P. Gonza´lez, E. Carbonell*, V. Urios†, and V. V. Rozhnov‡, Fundacioń Terra Natura, Foia del Verdader 1, 03503 Benidorm, Spain; *Laboratorio de Parasitologıá, Departamento de Zoologıá, Facultad de Biolo´gicas. University of Valencia, C/Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain; †Estacioń Biolo´gica, Terra Natura, Foia del Verdader 1, 03503 Benidorm, Spain; ‡A.N. Severtsov Institute of Ecology and Evolution, Academy of Sciences, Moscow 119071, Russia. e-mail: [email protected]

ABSTRACT: Fecal samples from the Siberian tiger (Panthera tigris al- Russia was carried out with the aim of studying the Panthera tigris taica) and the Amur cat (Felis bengalensis euptilurus) from Far Eastern altaica subspecies and its survival perspectives, and to collect data on Russia, were examined for parasites. A natural sedimentation method- another under-studied felid: Felis bengalensis euptilurus. Fecal samples ology was used and a complete examination of all the sediment was were collected by a team of Russian guides and biologists as part of performed. This fecal investigation allowed us to isolate and identify this project. The number of fecal samples found was less than expected several developmental stages of gastrointestinal, hepatic, and respiratory because of several different factors: the rugged orography, the number parasites. Five parasites were found from P. t. altaica: 11 trematodes and dispersion of these hosts, and the difficulty in correctly identifying (Platynosomum fastosum) and 4 nematodes (Strongyloides sp., Ancy- these samples (the expedition team only collected samples that were lostomatidae, Toxascaris leonina, and Toxocara cati). Five parasites perfectly identifiable). In this study, 3 fecal samples of Siberian tiger were found from F. b. euptilurus: 1 cestode (Diplopylidium sp.) and 4 (P. t. altaica) and 2 of Amur cat (F. b. euptilurus) were collected. nematodes (Trichuris sp., Ancylostomatidae, Toxascaris leonina, and Samples were from the reserves of Lazovsky and Sucan, from the south- Aelurostrongylus abstrusus). In addition, trophozoites of the amoeba eastern region of Lake Hanka, and zones near Vladivostok (43Њ09ЈN, Acanthamoeba sp. were detected in tiger feces. 131Њ53ЈE). Each fecal sample, approximately 10 g, was collected and preserved in 2% aqueous (w/v) dichromate potassium. We employed a Although there are several postmortem parasitological studies of free- filtration–sedimentation technique for the diagnosis of the parasites. All ranging and captive felids in different parts of the world, coprologic samples were incubated at room temperature in order to observe the investigations are scarce and limited to a few species, such as Panthera embryonic development of eggs. leo (Bjork et al., 2000), Panthera pardus (Patton and Rabinowitz, Parasitic forms representing 9 taxa were found in the fecal samples. 1994), wild Felis catus (Palumbo et al., 1976), Felis sylvestris (Yama- Each analyzed sample was positive. Table I shows the number and guchi et al., 1996), and Felis sylvestris and Lynx pardinus (Rodriguez distribution of the parasitic forms. Parasites were identified according and Carbonell, 1998). Studies on captive tigers have also been done to size and morphology of the forms present in feces (Hartwich, 1974; (Garner et al., 1996; Jakob and Wesemeier, 1996); however, little is Khalil et al., 1994; Bowman, 1995). In P. t. altaica, Toxocara cati known about parasites of wild Panthera tigris (Mandal and Choudhury, shows the higher prevalence, followed by Platynosomum fastosum, 1985; Patton and Rabinowitz, 1994; Marathe et al., 2002). In the case whereas in F. b. euptilurus the Ancylostomatidae eggs have been the of the subspecies, Panthera tigris altaica, an occurrence of toxoplas- most prevalent, followed by Trichuris sp. mosis was described as a result of a single veterinary examination in a One species of amoeba was found: Acanthamoeba sp. We have only zoo (Dorny and Fransen, 1989). References regarding Felis bengalensis observed trophozoites; cysts have not been seen. Acanthamoeba sp. are poor (Patton and Rabinowitz, 1994) and those dealing with the (family Hartmannellidae Volkonsky, 1931) was observed in one of the subspecies, Felis bengalensis euptilurus, are exclusively from a Czech tiger samples. The trophozoites measured 10–25 ␮m, with hyaline pro- Republic zoo (Lukesova and Literak, 1998) or postmortem studies on jections as spikelike, star-shaped pseudopodia (acanthopodia). This wild specimens (Yasuda et al., 1993, 1994). amoeba belongs to a genus of free-living amoebas inhabiting fresh wa- The understanding of these pathogens found on wild animals allows ters, rivers, pools, lagoons, and ponds. Acanthamoeba spp. also has been us to increase the knowledge about the ecology of wild felids and to isolated from vegetation and from animals including fish, amphibians, establish correct programs of prophylaxis if necessary. reptiles, and mammals (Madrigal Sesma, 1988; Dykova et al., 1999). During September and October 2001, an expedition to Far Eastern This amoeba occasionally infects man and animals, behaving as a pathogen mainly in immunosuppressed individuals (Bussie´ras and Cher- mette, 1992). Acanthamoeba spp. are the causative agents of granulo- TABLE I. Number and distribution of each species of parasite. S. ϭ matous amebic encephalitis (GAE), a fatal disease of the central ner- sample; x/x ϭ number of eggs found in the sample/number of larvae vous system (CNS), and amebic keratitis (AK), a painful sight-threat- found in the sample. ening disease of the eyes that can also cause cutaneous lesions and sinusitis (Martinez and Janitschke, 1985; Martinez and Visvesvara, 1997). Acanthamoeba spp. have been isolated from nasal mucosa, Felis throats, brain, lung tissue, skin lesions, corneal tissue, intestinal con- bengalensis tents, and feces (Cerva et al., 1973; De Jonckheere, 1991; Visvesvara Panthera tigris altaica euptilurus et al., 1990). The works about isolation of amoebas from intestinal contents and feces are more common in humans than in animals (Vel- Parasite name S.1 S.2 S.3 S.1 S.2 losa, Alves et al., 1984; Vellosa, Manzini et al., 1984; Moura et al., 1985; Echandi et al., 1994; Zaman et al., 1999). Because of the predis- Acanthamoeba sp. * position that tigers show for water, the lack of extended behavior in Platynosomum fastosum 10/0 — 105/0 — — felids, and the very important role that water plays in the amoeba dis- Diplopylidium sp. — — — 10/0 — persion, it could be surmised that tigers would be exposed to an infec- Trichuris sp. — — — 12/0 — tion of this type, particularly considering immunodeficiency states and Strongyloides spp. 3/1 — 5/10 — — other factors. It is possible that cysts have not been observed because Ancylostomatidae sp. — 9/4 — — 43/0 excystation may have occurred before the samples came to the labora- Aelurostrongylus abstrusus — — — — 0/7 tory. Toxocara cati 95/0 154/0 943/0 — — Eggs of Platynosomum fastosum Kossack, 1910, a hepatopancreatic Toxascaris leonina — — 23/0 2/0 — fluke of the family Dicrocoeliidae Odhner, 1911 that occurs in the liver, bile and pancreatic ducts of numerous domestic and wild felids, were * Presence of amoeba trophozoites. isolated from 2 tiger samples. These eggs, 44–60 ϫ 24–38 ␮m(nϭ RESEARCH NOTES 949

15), were operculated, brownish, and oval, and contained a fully de- also observe them hatch as larvae of 310–350 ␮m with rhabditiform veloped miracidium. The fact that 30% of these eggs hatched could esophagus and without sheath. Eggs found in the cats were bigger: 80 mean that this is a dicrocoelid with an aquatic cycle where miracidia ϫ 60 ␮m. It was not possible to distinguish the different genera of this escape from eggs and would infect aquatic snails, but certainly in the family via examination of these eggs. Focusing on egg size, the eggs known cycle of this species, eggs are ingested by terrestrial snails, such of Ancylostomatidae found in the tigers are more similar to those of as Subulina octona, in which cercariae develops. Therefore, felids only Ancyclostoma caninum and Ancyclostoma tubaeforme (Melhorn et al., become infected by eating the second hosts (amphibians and reptiles). 1992), whereas those recovered from the cats are more similar to An- Because of the size of the second hosts, the infection could occur when cyclostoma ceylanicum. According to the geographical distribution, tigers are kittens or very young. Subulina octona has a worldwide dis- each of the cited parasites is cosmopolitan in distribution, although A. tribution, having been found in Asia, Europe, West Africa, and both ceylanicum has been cited from Asian felids, including the tiger (Bioc- North and South America, but we cannot affirm the existence in eastern ca, 1951). Russia. Genera of snails involved in Dicrocoelidae cycles in the former Larvae of the parasite Aelurostrongylus abstrusus (Railliet, 1898) USSR are Helicella, Zebrina, Helix, and Bradybaena (Boray, 1985). (family Angiostrongylidae Boehm and Gebauer, 1934) were found in 1 Dicrocoelium dendriticum eggs (a parasite of herbivores) are practically of the cat samples; their measurements varied between 340 and 360 ␮m equal to those of Platynosomum but we discarded such possibility for (n ϭ 5) in length by 15 ␮m wide, which is the reason we think they several reasons. In the first place, Dicrocoelium eggs (38–45 ϫ 22–30 are first-stage larvae. We observed the characteristic kinked tail with a ␮m) are smaller than those of Platynosomum and secondly, if they were spine at the terminal end of these larvae. Aelurostrongylus abstrusus from an ingested herbivore, we would have found more spurious par- displays an indirect cycle and requires different snails and slugs (Helix, asites. Deroceras, Arion, and Agriolimax) as intermediate hosts, where the Diplopylidium sp. (family Dilepididae Raillet & Henry, 1909), found third-stage infective larva is developed. Amphibians, reptiles, birds, in- in cat feces, is a tapeworm of carnivores with characteristic embryo- sectivorous mammals, and rodents could act as paratenic hosts. Presum- nated eggs. Elliptical egg capsules 55–60 ϫ 20–44 ␮m(nϭ 10) contain ably, the main route of infection is the ingestion of these paratenic hosts only 1 oncosphere or hexacanth embryo which is well developed and (Hobmaier and Hobmaier, 1935). surrounded by a gelatinous thin layer and an outer fibrous capsule. Toxocara cati (Schrank, 1788) (family Ascarididae Baird, 1853) is a Therefore each capsule contains 1 egg. Diplopylidium species described worldwide parasite that occurs in the small intestine of felids and ap- in felids are Diplopylidium acanthotetrum and Diplopylidium no¨lleri. pears also in humans, causing the disease referred as visceral or ocular Diplopylidium acanthotetrum has been found in wild felids of Europe larva migrans. Eggs of this ascarid have been found in tiger samples and the Middle East, having amphibians and reptiles as paratenic hosts. and were slightly ovoid or subspherical, 70–75 ϫ 56–72 ␮m(nϭ 20), Diplopylidium no¨lleri is a canid and felid parasite found in Asia, Eu- with a finely pitted outer layer, 2–4 ␮m, reminiscent of a golf ball and rope, and North Africa. We cannot disregard the possibility that these also containing a single cell. In both cases, embryonic development eggs belong to another dilepipid tapeworm, Joyeuxiella sp. Fuhrmann, happened in 10–15 days at room temperature and allowed us to observe 1935, in which the uterine capsule also contains only a single egg. To the different segmentation stages until the formation of an embryo, the distinguish Diplopylidium from Joyeuxiella we would need to have the differentiation of the first and second-stage larvae, and the hatch of this adult forms (Schmidt, 1986). Species described in wild and domestic last stage, which took place in 40% of the observed eggs. Bowman felids are Joyeuxiella chyzeri (Ratz, 1897) syn Joyeuxiella pasqualei (1995) mentions that second-stage larvae of Toxocara cati hatch when (Diamare, 1893) and occur in Europe, Asia, and Palestine (Schmidt, eggs come in contact with gastrointestinal and bile fluids of the host. 1986; Khalil et al., 1994), and Joyeuxiella rossicum (Skrjabin, 1923) in We have detected that a considerable number of larvae have hatched Russia. First intermediate hosts known in both dilepidids are coproph- under laboratory conditions. No explanation has been found for this agous arthropods, adult or larvae or related to animals (fleas and lice). behavior. The low intensity of this parasite seems to indicate that tigers

Reptiles, amphibians, and small mammals are also known as paratenic have acquired the parasite by ingestion of L3 larvae in paratenic hosts. hosts (Rysavy, 1973; Roca et al., 1987). Toxascaris leonina (v. Linstow, 1902) is also a member of the family The eggs of Capillaria sp. (family Trichuridae) (Ransom, 1911) ap- Ascarididae. It occurs in the small intestine and sometimes in the bile peared in the cat samples and measured 62–66 ϫ 30–32 ␮m(nϭ 10). ducts of wild and domestic canids and felids in most parts of the world. The eggs differ from those of Trichuris only in detail. They are ovoid, Eggs were found in 1 sample of each host. These eggs measured 60– lemon-shaped, slightly irregular in symmetry, with a transparent plug 75 ϫ 75–85 ␮m(nϭ 20), were colorless and slightly oval with a at either pole and display a yellowish or orange-brown coloration with smooth shell surface. The outer layer was about 2 ␮m, without striations a pitted ornamentation. They contain an unsegmented embryo (a cell or albuminous coat. These eggs contained 1 or 2 spherical cells, which stage) when laid. First-stage larva developed in about 7 days, then did not occupy the entire interior of the egg. In this case, we were also stopped their development. They are widely distributed and can be able to observe every phase of the embryonic development until the found in numerous domestic and wild carnivores (Bowman, 1995; Os- second-stage larvae, which developed in 4–6 days. However, hatching borne and Delmar, 1995). In dogs and cats, the adult worms have been of these second-stage larvae was never observed. The egg of Toxascaris associated with the intestinal and urinary tract as well as the bronchi. leonina containing a second-stage larva is also the infective stage. Capillaria aerophila, Capillaria feliscati, Capillaria hepatica, Capil- All parasites are cited for the first time on these hosts with the ex- laria plica, and Capillaria putorri are the species of this genus, which ception of Toxocara cati which has been described on P. t. altaica. has felids as definitive hosts (Collins and Charleston, 1972; Holmes and This study was supported by Terra Natura Foundation (Benidorm, Kelly, 1973; Greve and Kung, 1983; Be´dard et al., 2002; Foster et al., Spain). 2004). Embryonated eggs and larvae of Strongyloides spp. (family Stron- LITERATURE CITED gyloididae) (Chitwood & Mcintosch, 1934) were found in 2 tiger samples. These eggs have a very thin shell and measured 48–50 ϫ 30–32 BE´ DARD, C., M. DESNOYERS,M.C.LAVALLE´ E, AND D. POIRIER. 2002. ␮m(nϭ 4); the larvae range was 300–380 ␮m(nϭ 8). The eggs Capillaria in the bladder of an adult cat. Canadian Veterinary Jour- hatched in 2 days. It is possible that they were second-stage larvae nal 43: 973–974. because of the presence of a rhabditiform esophagus and because the BIOCCA, E. 1951. On Ancylostoma brasiliensis (de Faria, 1910) and its filariform larvae of the third stage are longer (600–620 ␮m). Four spe- morphological differentiation from A. ceylanicum (Looss, 1911). cies of Strongyloides have been described in felids: Strongyloides tu- Journal of Helminthology 25: 1–10. mefaciens, Strongyloides felis, Strongyloides stercolaris (these 3 have a BJORK, K. E., G. A. AVERBECK, AND B. E. STROMBERG. 2000. Parasites cosmopolitan distribution) and Strongyloides cati, from Southeast Asia and parasite stages of free-ranging wild lions (Panthera leo)of (Carter, 2001). northern Tanzania. Journal of Zoo and Wildlife Medicine 31: 56– Eggs of Ancylostomatidae Looss, 1905, were found in both hosts’ 61. samples, being smaller in the tigers, 60–80 ϫ 36–60 ␮m(nϭ 15), BORAY, J. C. 1985. Flukes of domestic animals. In Parasites; Pests and ovoid-shaped, with a thin and transparent shell and containing 8–16 predators, S. M. Gaafar, E. Woward Walter, and E. Marsh Rex blastomers. Embryonic development happened before the seventh day (eds.). Elseiver Science Publishers, Amsterdam, The Netherlands, and allowed us to see the first-stage larvae inside the egg. We could p. 179–218. 950 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

BOWMAN, D. D. 1995. Georgis’ parasitology for veterinarians, 6th ed. MARATHE, R. R., S. S. GOEL,S.P.RANADE,M.M.JOG, AND M. G. W.B. Saunders Company, Philadelphia, Pennsylvania, 430 p. WATVE. 2002. Patterns in abundance and diversity of fecally dis- BUSSIE´ RAS, J., AND R. CHERMETTE. 1992. Parasitologie ve´te´rinaire. Pro- persed parasites of tiger in Tadoba National Park, central India. tozoologie. Service de Parasitologie Ecole Nationale Ve´te´rinaire, BMC Ecology 2: 6. Alfort, France, 186 p. MARTINEZ,A.J.,AND K. JANITSCHKE. 1985. Acanthamoeba, an oppor- CARTER, G. R. 2001. Internal parasitic diseases of dogs and cats. In A tunistic microorganism: A review. Infection 13: 251–256. concise guide to infectious and parasitic diseases of dogs and cats, ———, AND G. S. VISVESVARA. 1997. Free-living, amphizoic and op- G. R. Carter (ed.). International Veterinary Information Service, portunistic amebas. Brain Pathology 7: 583–598. ࿞ available at: http://www.ivis.org/special books/carter/toc.asp. MELHORN, H., D. DU¨ WEL, AND W. R AETHER. 1992. Atlas de parasitologia CERVA, L., C. SERBUS, AND V. S KOCIL. 1973. Isolation of limax amoebae veterinaria. Grass Ediciones, Barcelona, Espanã, p. 38–40. from the nasal mucosa of man. Folia Parasitologica 20: 97–103. MOURA,H.DE., H. C. SALAZAR,O.FERNANDES,D.C.LISBOA, AND F. COLLINS,G.H.,AND W. A. G. CHARLESTON. 1972. Ollulanus tricuspis G. DE CARVALHO. 1985. Free-living amoebae in human bowel: Ev- and Capillaria putorii in New Zealand cats. New Zealand Veteri- idence of parasitism. Revista Instituto de Medicina Tropical de Sao nary Journal 20: 82. Paulo 27: 150–156. DE JONCKHEERE, J. F. 1991. Ecology of Acanthamoeba. Reviews of In- OSBORNE,C.A.,AND R. F. DELMAR. 1995. Canine and feline nephrology fectious Diseases 13: S385–S387. and urology. Williams & Wilkins, Baltimore, Maryland, 960 p. DORNY,P.,AND J. FRANSEN. 1989. Toxoplasmosis in a Siberian tiger PALUMBO, N. E., D. TAYLOR, AND S. F. PERRI. 1976. Evaluation of fecal (Panthera tigris altaica). Veterinary Record 125: 647. technics for the diagnosis of cat liver fluke infection. Laboratory DYKOVA, I., J. LOM,J.M.SCHROEDER-DIEDRICH,G.C.BOOTON, AND T. Animals Science 26: 490–493. J. BYERS. 1999. Acanthamoeba strains isolated from organs of freshwater fishes. Journal of Parasitology 85: 1106–1113. PATTON, S., AND A. R. RABINOWITZ. 1994. Parasites of wild felidae in ECHANDI, L., D. L. GONZA´ LEZ, AND R. R. MARIŃ. 1994. Amoeba isola- Thailand—A coprological survey. Journal of Wildlife Diseases 30: tion of free life capacitate to produce meningoencephalitis. Revista 472–475. me´dica de Costa Rica y Centroame´rica 61: 11–14. ROCA, V., G. GARCIÁ-ADELL,E.LO´ PEZ, AND L. M. ZAPATERO-RAMOS. FOSTER,S.F.,P.MARTIN,G.S.ALLAN,V.R.BARRS, AND R. MALIK. 1987. Algunas formas para´sitas adultas y larvarias de platelmintos 2004. Lower respiratory tract infections in cats: 21 Cases (1995– de reptiles de las Islas Canarias. Revista Ibe´rica de Parasitologia 2000). Journal of Feline Medicine and Surgery 6: 167–180. 47: 263–270. GARNER, M. M., N. P. LUNG,S.CITINO,E.C.GREINER,J.W.HARVEY, RODRI´GUEZ,A.,AND E. CARBONELL. 1998. Gastrointestinal parasites of AND B. L. HOMER. 1996. Fatal cytauxzoonosis in a captive-reared the Iberian lynx and other wild carnivores from central Spain. Acta white tiger (Panthera tigris). Veterinary Pathology 33: 82–86. Parasitologica 43: 128–136. GREVE,J.H.,AND F. Y. K UNG. 1983. Capillaria putorii in domestic cats RYSAVY, B. 1973. Cysticercoids of cestodes of the family Dipylididae in Iowa. Journal of American Veterinary Medical Association 182: (Mola, 1929) from Egyptian snakes. Folia Parasitologica 20: 27– 511–513. 28. HARTWICH, G. 1974. Keys to genera of the Ascaridoidea, No. 2. In CIH SKRJABIN, K. I. 1991. Key to parasitic nematodes. Volume 4. Amerind keys to the nematode parasites of vertebrates, R. C. Anderson, A. Publishing Co., New Delhi, India, 1097 p. G. Chabaud, S. Willmott, Commonwealth Agricultural Bureaux. SCHMIDT, G. D. 1986. Handbook of tapeworm identification. CRC Press, Farnham Royal, Bucks, U.K., p. 1–15. Inc., Boca Raton, Florida, 675 p. HOBMAIER,A.,AND M. HOBMAIER. 1935. Intermediate hosts of Aeluros- VELLOSA,S.A.,V.A.F.ALVES,A.G.SCHLODTMANN, AND A. C. S. trongylus abstrusus of the cat. Proceedings of the Society for Ex- MANZINI. 1984. Free-living amebas (Naegleria sp. e Acanthamoeba perimental Biology and Medicine 32: 1641–1647. sp.) in human feces: Pathogenicity for mice. Revista do Instituto HOLMES,P.R.,AND J. D. KELLY. 1973. Capillaria aerophila in the Adolfo Lutz 44: 67–72. domestic cat in Australia. Australian Veterinary Journal 49: 472– ———, A. C. S. MANZINI,L.R.NUNES, AND A. G. SCHLODTMANN. 1984. 473. Frequency free-living amebas in the feces of persons from a nurs- JAKOB,W.,AND H. H. WESEMEIER. 1996. A fatal infection in a Bengal eryinSaö Paulo city. Revista do Instituto Adolfo Lutz 44: 61–65. tiger resembling cytauxzoonosis in domestic cats. Journal of Com- YAMAGUCHI, N., D. W. MACDONALD,W.C.PASSANISI,D.A.HARBOUR, parative Pathology 114: 439–444. AND C. D. HOPPER. 1996. Parasite prevalence in free-ranging farm KHALIL, L. F., A. JONES, AND R. A. BRAY. 1994. Keys to the cestode Felis sylvestris catus 116: parasites of vertebrates. CAB International, Oxon, U.K., 751 p. cats, . Epidemiology and Infection 217– 223. LUKESOVA,D.,AND I. LITERAK. 1998. Shedding of Toxoplasma gondii oocysts by Felidae in zoos in the Czech Republic. Veterinary Par- YASUDA, N., M. AKUZAWA,H.MARUYAMA,M.IZAWA, AND T. D OI. 1993. asitology 74: 1–7. Helminths of the Tsushima leopard cat (Felis bengalensis euptilu- MADRIGAL SESMA, M. J. 1988. Isolation of free-living amoebae, poten- ra). Journal of Wildlife Diseases 29: 153–155. tially pathogenic for humans, from 3 species of saurians from the ———, K. EZAKI,M.AKUZAWA,M.IZAWA,T.DOI,N.SAKAGUCHI, AND western Canary Islands. Revista de Sanidad e Higiene Publica 62: M. TATARA. 1994. Helminth survey of wildcats in Japan. Journal 1405–1409. of Veterinary Medical Science 56: 1069–1073. MANDAL,D.,AND A. CHOUDHURY. 1985. Helminth parasites of wild tiger ZAMAN, V., M. ZAKI, AND M. MANZOOR. 1999. Acanthamoeba in human of Sundarbans Forest, West Bengal, India. Proceedings of the In- faeces from Karachi. Annals of Tropical Medicine and Parasitology ternational Symposium on Diseases of Zoo Animals 27: 499–501. 93: 189–191. RESEARCH NOTES 951

J. Parasitol., 93(4), 2007, pp. 951–952 ᭧ American Society of Parasitologists 2007

Seroprevalence of Toxoplasma gondii in Pigs From Vietnam

Lam Thi Thu Huong and J. P. Dubey*†, Department of Parasitology and Pathobiology, Faculty of Veterinary Medicine, University of Agriculture and Forestry, Ho Chi Min City, Vietnam; *U.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350; †To whom correspondence should be addressed. e-mail: [email protected]

ABSTRACT: Pigs are considered an important source of Toxoplasma (HCMC) area. Cats and rodents were present on each farm. The grow- gondii infection for humans. Antibodies to T. gondii were determined ing pigs were usually housed on a cement floor; the housing of older in serum samples from 587 pigs from Vietnam using the modified ag- pigs varied from total confinement to partial enclosures. The abattoirs glutination test (MAT) and found in 160 of 587 (27.2%) pigs, with MAT were in HCMC and the pigs sampled were reported to be from small titers of 1:25 in 32 pigs, 1:50 in 34 pigs, 1:100 in 33 pigs, 1:200 in 24 farms in TienGiang and Dongnai provinces; no other information was pigs, 1:400 in 21 pigs, 1:800 in 14 pigs, and 1:3,200 in 2 pigs. Anti- available. bodies (MAT 1:20 or higher) were found in 75 of 325 (23%) finishers, Sera of pigs were tested for T. gondii antibodies using 2-fold serum 63 of 207 (32.3%) sows, and 22 of 55 (40%) boars. Results indicate dilutions from 1:25 to 1:3,200 with the modified agglutination test high prevalence of T. gondii infection in pigs in Vietnam. This is the (MAT) as described by Dubey and Desmonts (1987). A cut-off titer of first report of prevalence of T. gondii in pigs from Vietnam. 1:25 was considered specific for T. gondii infection based on previous studies (Dubey, Thulliez et al., 1995; Dubey, 1997). Toxoplasma gondii infections are widely prevalent in humans and Antibodies to T. gondii were found in 160 of 587 (27.2%) pigs, with other animals worldwide (Dubey and Beattie, 1988). Humans become MAT titers of 1:25 in 32 pigs, 1:50 in 34 pigs, 1:100 in 33 pigs, 1:200 infected postnatally by ingesting tissue cysts from undercooked meat, in 24 pigs, 1:400 in 21 pigs, 1:800 in 14 pigs, and 1:3,200 in 2 pigs consuming food or drink contaminated with oocysts, or by accidentally (Table I). Antibodies (MAT 1:20 or higher) were present in 75 of 325 ingesting oocysts from the environment. Pigs are considered to be one (23%) finishers, 63 of 207 (30.4%) sows, and 22 of 55 (40%) boars of the most important sources of T. gondii infection for humans in (Table I). several countries and clinical toxoplasmosis has resulted in humans who The finding of antibodies in 23% of fattening (market-age) pigs in had eaten naturally infected pork (Dubey, Thulliez et al., 1995; Dubey, Vietnam is alarming and is considerably higher than in the United States Weigel et al., 1995; Choi et al., 1997; Dubey et al., 2005). and European countries (Dubey, Weigel et al., 1995; Weigel, Dubey, Little is known of T. gondii infection in humans or animals in Vi- Siegel, Hoefling et al., 1995; Tenter et al., 2000). The seroprevalence etnam. In a small survey in Vietnam, antibodies to T. gondii were found of T. gondii in finisher pigs in the United States has declined drastically in 11.2% of 300 pregnant women and 7.7% of 300 intravascular drug from a national prevalence of 23% in 1983 (Dubey et al., 1991) to less users; both populations are high-risk groups for suffering severe medical than 3% a decade later (Dubey, Weigel et al., 1995; Weigel, Dubey, consequences from toxoplasmosis (Buchy et al., 2003). In one study, antibodies to T. gondii were found in 3% of 200 water buffaloes and Siegel, Hoefling et al., 1995; Patton et al., 1996; Gamble et al., 2005), 10.5% of 200 dairy cattle from Vietnam (Huong et al., 1998); however, probably related to better hygiene. In all of these studies, the same MAT viable T. gondii has never been demonstrated in water buffaloes and was used and thus the results are comparable. The MAT has been found has rarely been isolated from beef (Dubey and Beattie, 1988; Dubey et highly specific for detecting antibodies to T. gondii in pigs (Dubey, al., 2005). Thus, the role of beef in the epidemiology of T. gondii is at Thulliez et al., 1995; Dubey, 1997). The present study was not designed best uncertain (Dubey et al., 2005). Nothing is known of the presence to determine risk factors, but cats were present on swine farms. Oocysts of T. gondii infection in other food animals in Vietnam. Pork is an excreted by cats on these farms might have been the source of infection important part of the diet for humans in Vietnam. In the present paper for the pigs as was found on farms in the United States (Weigel, Dubey, we report prevalence of T. gondii antibodies in pigs from Vietnam for Siegel, Kitron et al., 1995). Additionally, on at least 8 small farms, pigs the first time. were fed restaurant waste that may have included scraps of uncooked In total, 587 pigs were surveyed from 13 farms and 2 abattoirs during meat infected with T. gondii. 2003 and 2005. Most of these farms were in the Ho Chi Min City Until more information is available on the presence of viable T. gon-

TABLE I. Serological survey for antibodies to Toxoplasma gondii infection in pigs in Vietnam.

No. of pigs with the following MAT titers: Total No. of seropositive Type of pigs samples 1:25 1:50 1:100 1:200 1:400 1:800 1:1,600 1:3,200 (%)

Boarfarm1 202221300 0 10(50) Boarfarm2 201232000 0 8(40) Boarfarm3 152110000 0 4(26.6) Sowfarm1 504543320 0 21(42) Sowfarm2 503523430 0 20(40) Sowfarm3 404323000 0 12(30) Sowfarm4 511000010 1 3(5.8) Sowfarm5 161300200 1 7(43) Growingpigfarm1803533200 0 16(20) Growing pig farms 6–13 80 4 3 94450 0 29(36.2) Growingpigfarm3653221000 0 8(12.3) Finishing pigs (abattoir) 100 4 3 54130 0 22(22) Total 587 32 34 33 24 21 14 0 2 160 (27.2) 952 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 dii in food animals in Vietnam, it is prudent to cook all pork thoroughly fected sows. American Journal of Veterinary Research 56: 1030– to minimize T. gondii infection. 1036. We thank Oliver Kwok and K. Hopkins for technical assistance. ———, R. M. WEIGEL,A.M.SIEGEL,P.THULLIEZ,U.D.KITRON,M. A. MITCHELL,A.MANNELLI,N.E.MATEUS-PINILLA,S.K.SHEN,O. LITERATURE CITED C. H. KWOK, AND K. S. TODD. 1995. Sources and reservoirs of Toxoplasma gondii infection on 47 swine farms in Illinois. Journal BUCHY, P., J. Y. FOLLEZOU,T.X.LIEN,T.T.AN,L.T.TRAM,D.V.TRI, of Parasitology 81: 723–729. N. M. CUONG,P.GLASIOU, AND B. T. CHIEN. 2003. Serological study GAMBLE, H. R., J. P. DUBEY, AND D. N. LAMBILLOTTE. 2005. Comparison of toxoplasmosis in Vietnam in a population of drug users (Ho Chi of a commercial ELISA with the modified agglutination test for Minh City) and pregnant women (Nha Trang). Bulletin de la So- detection of Toxoplasma infection in the domestic pig. Veterinary cie´te´ de Pathologie Exotique 96: 46–47. Parasitology 128: 177–181. CHOI,W.Y.,H.W.NAM,N.H.KWAK,W.HUH,Y.R.KIM,M.W.KANG, HUONG, L. T. T., B. L. LJUNGSTRO¨ M,A.UGGLA, AND C. BJO¨ RKMAN. 1998. S. Y. CHO, AND J. P. DUBEY. 1997. Foodborne outbreaks of human Prevalence of antibodies to Neospora caninum and Toxoplasma toxoplasmosis. Journal of Infectious Diseases 175: 1280–1282. gondii in cattle and water buffaloes in southern Vietnam. Veteri- DUBEY, J. P. 1997. Validation of the specificity of the modified agglu- nary Parasitology 75: 53–57. tination test for toxoplasmosis in pigs. Veterinary Parasitology 71: PATTON, S., J. ZIMMERMAN,T.ROBERTS,C.FAULKNER,V.DIDERRICH,A. 307–310. ASSADI-RAD,P.DAVIES, AND J. KLIEBENSTEIN. 1996. Seroprevalence ———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man. of Toxoplasma gondii in hogs in the National Animal Health Mon- CRC Press, Boca Raton, Florida, 220 p. itoring System (NAHMS). Journal of Eukaryotic Microbiology 43: ———, AND G. DESMONTS. 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337– 121S. 339. TENTER, A. M., A. R. HECKEROTH, AND M. WEISS. 2000. Toxoplasma gondii: From animals to humans. International Journal for Parasi- ———, D. E. HILL,J.L.JONES,A.W.HIGHTOWER,E.KIRKLAND,J.M. ROBERTS,P.L.MARCET,T.LEHMANN,M.C.B.VIANNA,K.MISKA, tology 30: 1217–1258. C. SREEKUMAR,O.C.H.KWOK,S.K.SHEN, AND H. R. GAMBLE. WEIGEL, R. M., J. P. DUBEY,A.M.SIEGEL,D.HOEFLING,D.REYNOLDS, 2005. Prevalence of viable Toxoplasma gondii in beef, chicken and L. HERR,U.D.KITRON,S.K.SHEN,P.THULLIEZ,R.FAYER, AND T. pork from retail meat stores in the United States: Risk assessment S. TODD. 1995. Prevalence of antibodies to Toxoplasma gondii in to consumers. Journal of Parasitology 91: 1082–1093. Illinois swine in 1992. Journal of the American Veterinary Medical ———, J. C. LEIGHTY,V.C.BEAL,W.R.ANDERSON,C.D.ANDREWS, Association 206: 1747–1751. AND P. T HULLIEZ. 1991. National seroprevalence of Toxoplasma ———, J. P. DUBEY,A.M.SIEGEL,U.D.KITRON,A.MANNELLI,M.A. gondii in pigs. Journal of Parasitology 77: 517–521. MITCHELL,N.E.MATEUS-PINILLA,P.THULLIEZ,S.K.SHEN,O.C. ———, P. THULLIEZ,R.M.WEIGEL,C.D.ANDREWS,P.LIND, AND E. H. KWOK, AND K. S. TODD. 1995. Risk factors for transmission of C. POWELL. 1995. Sensitivity and specificity of various serologic Toxoplasma gondii on swine farms in Illinois. Journal of Parasi- tests for detection of Toxoplasma gondii infection in naturally in- tology 81: 736–741.

J. Parasitol., 93(4), 2007, pp. 952–953 ᭧ American Society of Parasitologists 2007

Seroprevalence of Toxoplasma gondii in Cats From St. Kitts, West Indies

L. Moura, P. Kelly*, R. C. Krecek*†,and J. P. Dubey‡§, Department of Pathobiology, Ross University School of Veterinary Medicine, P.O. Box 334, West Farm, St. Kitts, West Indies; *Department of Clinical Sciences, P.O Box 334, Ross University School of Veterinary Medicine, West Farm, St. Kitts, West Indies; †Department of Zoology, Auckland Park Campus, University of Johannesburg, P.O. Box 524, Auckland Park, 2006, South Africa; and ‡U.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350. §To whom correspondence should be addressed. e-mail: [email protected]

ABSTRACT: The prevalence of antibodies to Toxoplasma gondii was recorded) presented to the Ross University Community Practice for var- determined in sera from 106 domestic cats from St. Kitts, West Indies. ious reasons in 2005 and 2006. The sera were transported by courier Using a modified agglutination test, antibodies to this parasite were from St. Kitts to Beltsville, Maryland, where serology was performed. found in 90 (84.9%) of the cats, with titers of 1:20 in 23 cats, 1:40 in Two-fold serial dilutions were made (1:10 to 1:1,280) and tested with 34 cats, 1:80 in 18 cats, 1:160 in 2 cats, 1:320 in 1 cat, and 1:1,280 or a modified agglutination test, as described previously (Dubey and Des- higher in 11 cats. This is the first report of the prevalence of T. gondii monts, 1987). Whole formalin-fixed tachyzoites and mercaptoethanol infections in cats on St. Kitts and suggests widespread contamination were used as antigen and a titer of 1:20 considered indicative of T. of the environment with oocysts. gondii exposure based on experimental studies in cats (Dubey and Thul- liez, 1989; Dubey et al., 1995a, 1995b). Cats are essential in the life cycle of Toxoplasma gondii because they Antibodies to T. gondii were found in 90 of the 106 sera (84.9%) at are the only hosts that can excrete the environmentally resistant oocysts titers of 1:20 in 23 samples, 1:40 in 34 samples, 1:80 in 18 samples, 1: in nature (Dubey and Beattie, 1988). Prior to this study, nothing was 160 in 2 samples, 1:320 in 1 samples, and 1:1,280 or higher in 11 samples. known of the prevalence of T. gondii in cats in St. Kitts. Recently, Results indicate that a very high percentage of cats on St. Kitts have an- antibodies to T. gondii were reported in women and cats in the neigh- tibodies to T. gondii and undoubtedly shed oocysts into the environment. boring Caribbean island, Grenada. The seroprevalence detected was considerably higher than that found in the St. Kitts Island is located in the eastern Caribbean at 17Њ20ЈN, only other study of cats in the Caribbean, in which 35% of 40 domestic 62Њ45ЈW with habitats ranging from dry areas to rainforests. The area cats from Grenada sampled 10 yr ago were seropositive (Asthana et al., of the island is 179 km2, with a population of approximately 35,000. 2006). Infected cats can release millions of T. gondii oocysts into the en- Sera stored at Ϫ20 C in the Clinical Pathology Laboratory of Ross vironment and people become exposed by ingesting the oocysts or by University School of Veterinary Medicine were used in the study. These eating undercooked meat from animals that have ingested oocysts and de- originated from 106 domestic adult cats (44 females, 56 males, 6 not veloped tissue cysts. Although there are no published data for St. Kitts, our RESEARCH NOTES 953

study suggests that toxoplasmosis might be an important disease on St. DUBEY,J.P.,AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and Kitts and health workers on this and neighboring islands should be alerted man. CRC Press, Boca Raton, Florida, 220 p. to this possibility. Further studies are indicated to further characterize toxo- ———, AND G. DESMONTS. 1987. Serological responses of equids fed plasmosis in the Caribbean. Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337– This project was funded in part by Ross University School of Vet- 339. erinary Medicine. We thank the Ross University Clinical Pathology ———, AND P. T HULLIEZ. 1989. Serologic diagnosis of toxoplasmosis Laboratory for providing the sera and Candita Chapman, Cassandra in cats fed Toxoplasma gondii tissue cysts. Journal of the American Benjamin, and Tiffany Caudill for technical help. Veterinary Medical Association 194: 1297–1299. ———, M. R. LAPPIN, AND P. T HULLIEZ. 1995a. Diagnosis of induced LITERATURE CITED toxoplasmosis in neonatal cats. Journal of the American Veterinary ASTHANA, S. P., C. N. L. MACPHERSON,S.H.WEISS,R.STEPHENS,R. Medical Association 207: 179–185. N. SHARMA, AND J. P. DUBEY. 2006. Seroprevalence of Toxoplasma ———, ———, AND ———. 1995b. Long-term antibody responses of gondii in pregnant women and cats in Grenada, West Indies. Jour- cats fed Toxoplasma gondii tissue cysts. Journal of Parasitology nal of Parasitology 92: 644–645. 81: 887–893.

J. Parasitol., 93(4), 2007, pp. 953–955 ᭧ American Society of Parasitologists 2007

Bartonella and Rickettsia From Fleas (Siphonaptera: Ceratophyllidae) of Prairie Dogs (Cynomys spp.) From the Western United States

Will K. Reeves, Thomas E. Rogers*, and Gregory A. Dasch†, 4757 Habersham Ridge SW, Lilburn, Georgia 30047; *242 Williams Circle, Flowood, Mississippi 39232; †Centers for Disease Control and Prevention, 1600 Clifton Road NE, MS G-13, Atlanta, Georgia 30333. e-mail: [email protected]

ABSTRACT: Fleas of prairie dogs have been implicated in the transmis- deposited in the Georgia Museum of Natural History, University of sion of Bartonella spp. We used PCR to test DNA extracts from 47 Georgia, Athens, Georgia. fleas of prairie dogs from 6 states. We amplified DNA from 5 unique DNA extraction from individual fleas, PCR, and DNA sequencing genotypes of Bartonella spp. and 1 Rickettsia sp. from 12 fleas collected followed the protocols used by Reeves et al. (2005). Control DNA from in North Dakota, Oklahoma, Texas, and Wyoming. Sequences from the Bartonella henselae and Rickettsia sp. genotype Ae-8 and a distilled Bartonella spp. were similar, but not identical, to those from prairie water negative control were used in all assays. All controls performed dogs and their fleas in Colorado. as expected. Sequences with multiple mixed base calls were determined to represent coinfections with more than 1 agent. Bartonella and Rick- Rickettsial agents, such as species of Anaplasma, Bartonella, Coxi- ettsia genotypes were identified based on sequence similarity to those ella, Ehrlichia, Orientia, and Rickettsia, are a polyphyletic group of in the GenBank. primarily vector-borne intracellular bacteria (Dumler et al., 2001). In To control for contamination of the DNA or PCR, no cultures of the United States, prairie dogs (Cynomys spp.) are reservoirs of plague rickettsial agents were in the laboratory where DNA extraction or PCR (Yersinia pestis; e.g., Reed et al., 1970), and the black-tailed prairie dog was performed. DNA extractions were executed in a separate area of (Cynomys ludovicanus) is infected with Bartonella spp. (Stevenson et the laboratory from the PCR and gel electrophoresis. PCR were set up al., 2003). Phylogenetic studies indicated that isolates of Bartonella spp. in a UV-sterilized, regularly cleaned containment hood. Neither the from fleas of C. ludovicanus were closely related to ‘‘Bartonella wash- agents detected nor their DNA had previously been in the laboratory oensis,’’ a cause of myocarditis in humans (Kosoy et al., 2003; Steven- where the research was conducted. son et al., 2003). As a result, Bartonella spp. of C. ludovicanus and its DNA sequences from Bartonella and Rickettsia species were depos- fleas could be human pathogens. Rickettsia spp. have not been described ited in the GenBank with the following accession numbers: Bartonella from fleas of Cynomys spp. (Stevenson et al., 2003), but Rickettsia felis, sp. TR-74 ITS (DQ480759), Bartonella sp. TR-39 ITS (DQ480761), a pathogen of humans, was detected in native fleas (Stevenson et al., Bartonella sp. TR-62 ITS (DQ868319), Bartonella sp. TR-68 ITS 2005). Infections by some Bartonella and Rickettsia species can cause (DQ868318), Bartonella TR-42 ITS (DQ868317), and Rickettsia TR- severe disease or death to vertebrates or their invertebrate vectors (e.g., 39 17-kD antigen gene (DQ480762). Houhamdi et al., 2002; Kosoy et al., 2003). We amplified DNA from Bartonella spp. from 12 of 47 (25%) of the We were interested in elucidating the distribution of the Bartonella fleas, including individuals of all species examined (i.e., O. hirsuta, O. spp. of Cynomys spp. and determining if these fleas harbor Rickettsia tuberculatus cynomuris, and Thrassis fotus; Table I). We did not detect spp., which may be rare in native fleas, as suggested by Stevenson et multiple infections of Bartonella spp. in any fleas. DNA sequences from al. (2005), but further examination could indicate otherwise. The study the Bartonella spp. were unique when compared to those in the by Stevenson et al. (2003) was limited to Colorado, and our goal was GenBank (Table II) with the exception of the conserved tRNA regions to increase the known geographic ranges of these agents. of the ITS sequences, which are conserved for all Bartonella spp. The Fleas were collected by T.E.R. when he encountered prairie dog col- sequences were most similar to Bartonella isolated from squirrels and onies while driving for business. Most were colonies with 10–100 bur- other rodents and fleas. Unfortunately, ‘‘Bartonella washoensis’’ has rows. Methods varied from previous studies (Cully et al., 2000; Ste- never been formally described and the name is not fixed to a type venson et al., 2003) as follows: a white terrycloth wash cloth measuring culture. The original isolate was not deposited in a reference collection. 30.5 ϫ 30.5 cm was folded into pleats and attached to the end of a ‘‘Bartonella washoensis’’ is currently the only moniker used to describe flexible plumber’s cable. The cloth was introduced as far as possible the agent isolated from humans and ground squirrels. A great range of into the burrow, left for 20 sec, then retrieved and placed in a white agents that are genetically similar to each other have been isolated from plastic pan. Fleas were removed and stored in 95% ethanol. ground squirrels (e.g., Stevenson et al., 2003). These could be indepen- Fleas were identified using the keys of Hubbard (1947), Stark (1970), dent species or variations of ‘‘Bartonella washoensis.’’ The eventual and Lewis (2002). In total, 47 fleas representing 3 ceratophyllid species species designation for these isolates will depend on the official naming, (Oropsylla hirsuta (Baker), Oropsylla tuberculatus cynomuris (Jellison), culture deposition, and designation of those taxa. The name ‘‘Bartonella and Thrassis fotus (Jordan)) were collected (Table I). Voucher fleas were washoensis’’ could change if the agent was reisolated or the original 954 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Rickettsial agents and collection data for ﬂeas removed from burrows of Cynomys spp. from 2004 to 2005.

Date of No. tested Agent identified Species of flea Collection locality collection (fleas) (Proportion of infected fleas)

Oropsylla hirsuta (Baker) North Dakota: Sioux Co., Hwy 24, 3 Sep 2004 5 Bartonella sp. TR-74 (1/5), Barton- near Solen ella sp. TR-39 (1/5) O. hirsuta New Mexico: Sante Fe Co., Santa 10 May 2004 3 None (0/3) Fe O. hirsuta Oklahoma: Ellis Co., near May 9 May 2004 13 Bartonella sp. TR-39 (3/13), Bar- tonella sp. TR-42 (2/13), Rickett- sia TR-39 (1/13) O. hirsuta Oklahoma: Canadian Co., Yukon 8 May 2004 4 Bartonella sp. TR-42 (1/4) O. hirsuta South Dakota: Campbell Co., near 3 Sep 2004 2 None (0/2) Mobridge O. hirsuta South Dakota: Meade Co., Sturgis 25 Jun 2004 1 None (0/1) O. hirsuta South Dakota: Pennington Co., near 26 Jun 2004 2 None (0/2) Wall O. hirsuta Texas: Lubbock Co., Lubbock 24 Sep 2005 8 Bartonella sp. TR-68 (1/8), Barton- ella sp. TR-74 (1/8) O. hirsuta Texas: Dallam Co., Conlen 9 May 2004 3 None (0/3) O. hirsuta Wyoming: Park Co., near Meeteetse 19 Jun 2004 1 None (0/1) Oropsylla tuberculatus cynomuris Wyoming: Park Co., near Meeteetse 19 Jun 2004 2 Bartonella sp. TR-42 (1/2) (Jellison) Thrassis fotus (Jordan) North Dakota: Sioux Co., Hwy 24, 14 Jun 2004 2 Bartonella sp. TR-62 (1/2) near Solen

culture rediscovered and formally described. We were unable to com- A single O. hirsuta collected in May (Ellis County, Oklahoma) har- pare the ITS sequences to ‘‘Bartonella washoensis,’’ because this gene bored DNA from both Bartonella sp. TR-39 and Rickettsia sp. TR-39. was not sequenced from ‘‘Bartonella washoensis,’’ and the original iso- We amplified a fragment of the 17-kD antigenic gene of a previously late was not deposited in a culture collection and is, therefore, unavail- unreported spotted fever group of Rickettsia from this flea. The 17-kD able for study. The similarity of the ITS region of Bartonella sp. TR- antigenic gene is highly conserved within species of Rickettsia. The 62 to Bartonella doshiae (Table II) was not a 100% match and does sequence from this flea was unique when compared to those in the not indicate that this flea harbored B. doshiae. GenBank and probably represents a novel Rickettsia sp. The DNA se- There is no specific rule for determining what percentage of genetic quence from Rickettsia sp. TR-39 was 99% similar to Rickettsia sp. AT- similarity defines a species of Bartonella. The best approach is to ex- 1, a spotted fever group of Rickettsia that was implicated as a cause of amine several genes, the morphology, and serologic cross reactivity. fever in humans (Ishikura et al., 2003; Phongmany et al., 2006). We The 16S-23S rRNA ITS is of limited use in identifying Bartonella at conducted no further characterization of the Rickettsia sp. Based on the the species level because of intraspecies sequence polymorphisms in the study by Stevenson et al. (2005), Rickettsia spp. are infrequently de- DNA sequences (Houpikian and Raoult, 2001). There were 6 unique tected in native fleas of ground nesting rodents in the western United ITS sequences, but sequences from Bartonella sp. TR-42, Bartonella States. Our data support this observation. sp. TR-68, and Bartonella sp. TR-62 were 99% similar to each other, The public health significance of Bartonella and Rickettsia species in with the exception of a 1-bp mismatch and a 26-bp insertion in the ITS fleas of prairie dogs is unknown. Stevenson et al. (2003) reported Bar- sequence of Bartonella sp. TR-62 when compared to the other 2 se- tonella Y. pestis quences. These data could indicate polymorphism among strains of Bar- sp. and in fleas of prairie dogs; these fleas are known tonella, or these genotypes might represent a single polymorphic spe- to bite and transmit plague to humans and domestic animals (Reed et cies. Comparison of individual culture isolates is needed to determine al., 1970). Humans are most frequently exposed to Oropsylla spp. when the sequence polymorphism in these agents. Species of Bartonella are plague epizootics kill off prairie dog colonies. Based on the phylogeny diverse, and genetic variability is significant among the hundreds of constructed by Stevenson et al. (2003), the Bartonella spp. of prairie genetic isolates in the GenBank. dogs and their fleas are closely related to ‘‘Bartonella washoensis,’’ a Based on the sequence of the ITS region, Bartonella sp. TR-39 and pathogen of humans (Kosoy et al., 2003). Our study significantly in- Bartonella sp. TR-74 genotypes were 82% and 69% similar, respec- creases the known range of these Bartonella species, which were pre- tively, to the other isolates and were only 75% similar to each other. viously reported only from Colorado. Based on the DNA sequences, we conclude that these agents are dif- This research was partially supported by the American Society for ferent from each other and the other Bartonella genotypes. Microbiology and the Centers for Disease Control and Prevention. The

TABLE II. Bartonella and Rickettsia genotypes from ﬂeas and comparisons of the DNA sequences to those in GenBank.

Bartonella genotype (Gene, bp) DNA sequence similarity (reference when available)

Bartonella sp. TR-39 (ITS, 479 bp) 91% similar to Bartonella sp. clone BT71688 from ﬂeas in Peru (Parola et al., 2002) Bartonella sp. TR-42 (ITS, 603 bp) 92% similar to Bartonella sp. L-2633 Bartonella sp. TR-62 (ITS, 603 bp) 96% similar to Bartonella doshiae Bartonella sp. TR-68 (ITS, 603 bp) 94% similar to Bartonella sp. L-2633 Bartonella sp. TR-74 (ITS, 580 bp) 94% similar to Bartonella sp. clone BT7498 from ﬂeas in Peru (Parola et al., 2002) Rickettsia sp. TR-39 (17 kD, 431 bp) 99% similar to Rickettsia AT-1 (Ishikura et al., 2003) RESEARCH NOTES 955

findings and conclusions in this report are those of the authors and do tonella washoensis isolated from a human patient. Journal of Clin- not necessarily represent the views of the funding agency. ical Microbiology 41: 645–650. LEWIS, R. E. 2002. A review of the North American species of Orop- LITERATURE CITED sylla Wagner and Ioff, 1926 (Siphonaptera: Ceratophyllidae: Cer- atophyllinae). Journal of Vector Ecology 27: 184–206. CULLY,J.F.,JR., L. G. CARTER, AND K. L. GAGE. 2000. New records PAROLA, P., S. SHPYNOV,M.MONTOYA,M.LOPEZ,P.HOUPIKIAN,Z. of sylvatic plague in Kansas. Journal of Wildlife Diseases 36: 389– ZEAITER,H.GUERRA, AND D. RAOULT. 2002. First molecular evi- 392. dence of new Bartonella spp. in fleas and a tick from Peru. Amer- DUMLER, J. S., A. F. BARBET,C.P.J.BEKKER,G.A.DASCH,G.H. ican Journal of Tropical Medicine and Hygiene 67: 135–136. PALMER,S.C.RAY,Y.RIKIHISA, AND F. R. RURANGIRWA. 2001. PHONGMANY, S., J. ROLAIN,R.PHETSOUVANH,S.D.BLACKSELL,V.SOUK- Reorganization of genera in the families Rickettsiaceae and Ana- KHASEUM,B.RASACHACK,K.PHIASAKHA,S.SOUKKHASEUM,K.FRI- plasmataceae in the order Rickettsiales: unification of some species CHITHAVONG,V.CHU, ET AL. 2006. Rickettsial infections and fever, of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehr- Vientiane, Laos. Emerging Infectious Diseases 12: 256–262. lichia with Neorickettsia, descriptions of six new species combi- REED, W. P., D. L. PALMER,R.C.WILLIAMS,JR., AND A. L. KISCH. 1970. nations and designation of Ehrlichia equi and ‘HGE agent’ as sub- Bubonic plague in the southwestern United States. A review of jective synonyms of Ehrlichia phagocytophila. International Jour- recent experience. Medicine 49: 465–486. nal of Systematics, Evolution and Microbiology 51: 2145–2165. REEVES, W. K., A. D. LOFTIS,J.A.GORE, AND G. A. DASCH. 2005. HOUHAMDI, L., P.-E. FOURNIER,R.FANG,H.LEPIDI, AND D. RAOULT. Molecular evidence for novel Bartonella spp. in Trichobius major 2002. An experimental model of human body louse infection with (Diptera: Streblidae) and Cimex adjunctus (Hemiptera: Cimicidae) Rickettsia prowazekii. Journal of Infectious Diseases 186: 1639– from two southeastern bat caves, U.S.A. Journal of Vector Ecology 1646. 30: 339–341. HOUPIKIAN,P.,AND D. RAOULT. 2001. 16S/23S rRNA intergenic spacer STARK, H. E. 1970. A revision of the flea genus Thrassis Jordan 1933 regions for phylogenetic analysis, identification, and subtyping of (Siphonaptera: Ceratophyllidae) with observations on ecology and Bartonella species. Journal of Clinical Microbiology 39: 2768– relationship to plague. University of California Publication in En- 2778. tomology 53: 1–184. HUBBARD, C. A. 1947. Fleas of western North America: Their relation STEVENSON, H. L., M. B. LABRUNA,J.A.MONTENIERI,M.Y.KOSOY,K. to public health. Hafner, New York, New York, 533 p. L. GAGE, AND D. H. WALKER. 2005. Detection of Rickettsia felis in ISHIKURA, M., S. ANDO,Y.SHINAGAWA,K.MATSUURA,S.HASEGAWA, a New World flea species, Anomiopsyllus nudata (Siphonaptera: T. N AKAYAMA,H.FUJITA, AND M. WATANABE. 2003. Phylogenetic Ctenophthalmidae). Journal of Medical Entomology 42: 163–167. analysis of spotted fever group rickettsiae based on gltA, 17-kDa, ———, Y. BAI,M.Y.KOSOY,J.A.MONTENIERI,J.L.LOWELL,M.C. and rOmpA genes amplified by nested PCR from ticks in Japan. CHU, AND K. L. GAGE. 2003. Detection of novel Bartonella strains Microbiology and Immunology 47: 823–832. and Yersinia pestis in prairie dogs and their fleas (Siphonaptera: KOSOY, M., M. MURRAY,R.D.GILMORE,JR., Y. BAI, AND K. L. GAGE. Ceratophyllidae and Pulicidae) using multiplex polymerase chain 2003. Bartonella strains from ground squirrels are identical to Bar- reaction. Journal of Medical Entomology 40: 329–337.

J. Parasitol., 93(4), 2007, pp. 955–957 ᭧ American Society of Parasitologists 2007

Prevalence of Antibodies to Leishmania infantum and Trypanosoma cruzi in Wild Canids From South Carolina

Alexa C. Rosypal, Richard R. Tidwell, and David S. Lindsay*†, Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7525; *Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061-0342; †To whom correspondence should be addressed. e-mail: [email protected]

ABSTRACT: Wild canids are reservoir hosts for Leishmania infantum A sylvatic cycle of T. cruzi exists that involves parasite transmission and Trypanosoma cruzi. The present study examined the prevalence of between reduviid bugs and wildlife reservoir hosts. Little data are avail- antibodies to these zoonotic parasites in a population of wild canids able, however, regarding L. infantum and T. cruzi infections in wild from a nonagricultural setting in South Carolina. Sera from 26 gray canids in the United States. The present study was conducted to deter- foxes (Urocyon cinereoargenteus) and 2 coyotes (Canis latrans) were mine the seroprevalence of antibodies to L. infantum and T. cruzi in a examined for antibodies to L. infantum and T. cruzi using the indirect population of gray foxes (Urocyon cinereoargenteus) and coyotes (Ca- immunofluorescent antibody test and commercially available parasite- nis latrans) from South Carolina. specific immunochromatigraphic strip assays. Antibodies to L. infantum Sera from 26 gray foxes (Lindsay et al., 2001) and 2 coyotes from were not detected by either assay in gray foxes or coyotes. Two (8%) South Carolina were examined for antibodies to L. infantum and T. of 26 gray foxes were positive in both the T. cruzi immunofluorescent cruzi. The wild canids examined in the present study originated from a antibody and strip assays. Antibodies to T. cruzi were not detected in nonagricultural setting in Aiken and Barnwell counties on the Depart- coyotes. Results from this study indicate that wild canids are exposed ment of Energy’s Savannah River site. Other wildlife species are present to T. cruzi, but not L. infantum. in this geographic region. in the study area, including white-tailed deer (Odocoileus virginianus), and they are hunted with domestic dogs only during hunting season; Leishmania infantum and Trypanosoma cruzi are zoonotic parasites otherwise, domestic dogs are not allowed in the study location. The and both are endemic in the United States. Leishmaniasis and Chagas’ animals were collected alive in padded leg-hold traps and sedated using disease caused by infection with L. infantum and T. cruzi, respectively, intramuscular injections of ketamine and xylazine. Animals were bled are potentially fatal diseases in both dogs and humans. Canine leish- while unconscious. After venipuncture, animals were humanely killed maniasis caused by L. infantum is recognized as an important disease for other studies on the population of wild canids in this study site. The in the North American foxhound population, and T. cruzi-infected fox- age of each fox was determined using tooth cementum analysis of an hounds also have been described (Duprey et al., 2006). extracted lower premolar. Ages of coyotes were not available. Sera were 956 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

examined by the quantitative indirect fluorescent antibody test (IFAT) study, 19 of 134 (14.2%) seropositive coyotes were detected in Texas and commercially available immunochromatographic dipstick assays for (Gro¨gl et al., 1984), although only 2 coyotes were tested in the present qualitative antibody detection. work. McKeever et al. (1958) isolated T. cruzi-like parasites from 2 of The IFAT was used to examine sera for IgG antibodies to T. cruzi 118 (1.7%) gray foxes collected in southwestern Georgia and north- and L. infantum as described previously (Dubey et al., 2005; Rosypal western Florida. Our report, however, is apparently the first to document et al., 2005). Briefly, sera were initially screened at a 1:25 dilution in antibodies to T. cruzi in gray foxes from South Carolina. phosphate-buffered saline (PBS). Positive samples were examined at The immunochromatigraphic strip assays used in this study are com- doubling dilutions and titrated to a final dilution of 1:12,800. In the T. mercially available for domestic dogs, but they have not been validated cruzi IFAT, Brazil strain amastigotes and trypomastigotes were washed using wild canine serum. The strip tests are qualitative, and the intensity in PBS, and approximately 40,000 parasites per well were air-dried on of the chromagen in the test region varies depends on the concentration 12-well microscope slides. Parasite antigen was affixed on IFAT slides of antibodies present in the sample. According to the manufacturer, a with acetone. Sera (30 ␮l/well) were diluted to 1:25 in PBS and incu- faint line should be considered a positive result. The positive T. cruzi bated on the slides for 30 min at room temperature in a humidified box. strip assays in 2 gray foxes in the current study had positive IFAT results Slides were washed 2 times for 5 min in PBS. The secondary antibody at a concentration of 1:25. We compared results of the dipstick assays used in the present study was fluorescein isothiocyanate-conjugated goat to IFAT, and we found 100% sensitivity and specificity compared with anti-dog IgG (HϩL) (Kirkegaard and Perry Laboratories, Gaithersburg, the gold standard. To our knowledge, this is the first serological study Maryland). Fluorescent conjugate (30 ␮l/well) at a dilution of 1:10 in using the Leishamania sp. and T. cruzi dipstick assays in wild canids. PBS-Evans blue solution was incubated on the slides for 30 min at room Dipstick assays are simple, rapid tests that are easier to perform than temperature in a humidified box. Slides were washed twice for 5 min IFAT. Results from this work indicate that these tests warrant further in PBS, mounted in Fluoromount-G (Southern Biotechnology Associ- study with additional gray fox samples and possibly other wild canine ates Inc., Birmingham, Alabama), and covered with coverslips (24 ϫ species to validate their usefulness as a serological screening tool in 60 mm). Slides were viewed with an Olympus BX60 epifluorescent wild canids. microscope equipped with differential contrast optics. The procedure We thank J. S. Weston, Savannah River Ecology Laboratory, and S. was the same as described above for the Leishmania IFAT, except E. Little, Center for Veterinary Health Sciences, Oklahoma State Uni- 50,000 promastigotes of L. infantum (ATCC PRA-149) (canine isolate versity, for providing gray fox and coyote sera used in this study. We from a naturally infected Virginia foxhound; Rosypal, Troy, Zajac et are grateful to Stephen Barr from the College of Veterinary Medicine, al., 2003) per well were used as antigen. Canine control sera were used Cornell University, for providing T. cruzi-positive dog sera. The con- on each IFAT slide. Sera from dogs with proven T. cruzi and L. infantum tribution of R.R.T. was supported by a grant from the Bill and Melinda infections were used as positive controls for respective IFAT tests. Neg- Gates Foundation, and the contribution of A.C.R. was supported by the ative control sera used in both IFATs was obtained from a dog proven National Institute of General Medical Sciences GM-00678. A.C.R. is a to be uninfected by both culture and serology. fellow with the Seeding Postdoctoral Innovators in Research and Edu- Sera also were tested qualitatively by commercially available canine cation (SPIRE) postdoctoral training program. immunochromatographic dipstick assays according to the manufacturer’s test procedure. The tests are based on recombinant antigens that LITERATURE CITED previous reports have demonstrated superior performance over traditional serological screening tests based on crude antigens or whole or- BURNS, J. M., JR., W. G. SHREFFLER,D.R.BENSON,H.W.GHALIB,R. ganisms (Houghton et al., 2000; Scalone et al., 2002). Canine sera were BADARO, AND S. G. SEED. 1993. Molecular characterization of a ௢ tested for anti-recombinant K39 (rK39) (Kalazar Detect Canine Rapid kinesin-related antigen of Leishmania chagasi that detects specific Test; InBios International Ltd., Seattle, Washington), which is an amas- antibody in African and American visceral leishmaniasis. Proceed- tigote protein specific to visceralizing Leishmania spp., and it does not ings of the National Academy of Sciences USA 90: 775–779. crossreact T. cruzi (Burns et al., 1993). Anti-T. cruzi antibodies were DUBEY, J. P., A. C. ROSYPAL,V.PIERCE,S.N.SCHEINBERG, AND D. S. ௢ evaluated by a dipstick assay (Trypanosoma Detect MRA Rapid Test; LINDSAY. 2005. Placentitis associated with leishmaniasis in a dog. InBios International Ltd.) based on multiepitope recombinant antigens. Journal of the Veterinary Medical Association 227: 1266–1269. The IFAT was considered the ‘‘gold standard’’ for comparisons of sen- DUPREY, Z. H., F. J. STEURER,J.A.ROONEY,L.V.KIRCHOFF,J.E. sitivity and specificity, because it has been shown to have a high sen- JACKSON,E.D.ROWTON, AND P. M . S CHANTZ. 2006. Canine visceral sitivity for detecting antibodies to T. cruzi in wildlife (Yabsley et al., leishmaniasis: United States and Canada, 2000–2003. Emerging 2001). Infectious Diseases 12: 440–446. Positive T. cruzi IFAT and T. cruzi dipstick assay results were found GRO¨ GL, M., R. E. KUHN,D.S.DAVIS, AND G. E. GREEN. 1984. Anti- in 2 (8%) of 26 gray foxes. One 5-yr-old male fox from Aiken County bodies to Trypanosoma cruzi in coyotes in Texas. Journal of Par- and 1 7-yr-old female fox from Barnwell County were both IFAT pos- asitology 70: 189–191. itive at a final titer of 1:25. None of the coyotes had antibodies to T. HANCOCK, K., A. M. ZAJAC,O.J.PUNG,F.ELVINGER,A.C.ROSYPAL, cruzi by either IFAT or dipstick tests. None of the wild canids tested AND D. S. LINDSAY. 2005. Prevalence of antibodies to Trypanosoma in the present study had evidence of antibodies to L. infantum by either cruzi in raccoons (Procyon lotor) from an urban area of northern serological assay. The sensitivity and specificity of the dipstick assays Virginia. Journal of Parasitology 91: 470–472. were 100% compared with IFAT as the gold standard. HOUGHTON, R. L., D. R. BENSON,L.D.REYNOLDS,P.D.MCNEILL,P. Leishmania infantum and T. cruzi are endemic parasites in the United R. SLEATH,M.J.LODES,Y.A.SKEIKY,R.BADARO,A.U.KRETTLI, States and Canada (Rosypal, Zajac, and Lindsay, 2003; Rosypal et al., AND S. G. REED. 2000. Multiepitope synthetic peptide and recom- 2005; Duprey et al., 2006). Autochthonous canine leishmaniasis caused binant protein for the detection of antibodies to Trypanosoma cruzi by L. infantum is well recognized in the North American foxhound in patients with treated or untreated Chagas’ disease. Journal of population, and Leishmania sp.-infected and T. cruzi-infected fox- Infectious Diseases 181: 325–330. hounds have been detected in South Carolina (Duprey et al., 2006). The JOHN,D.T.,AND K. L. HOPPE. 1986. Trypanosoma cruzi in wild rac- lack of antibodies to L. infantum in wild canids examined in this study coons in Oklahoma. American Journal of Veterinary Research 47: is in agreement with previous findings (Duprey et al., 2006). Serological 1056–1059. surveys conducted by the Centers for Disease Control and Prevention LINDSAY, D. S., J. L. WESTON, AND S. E. LITTLE. 2001. Prevalence of indicated that 2 of 291 (0.7%) wild canid samples collected in the south- antibodies to Neospora caninum and Toxoplasma gondii in gray eastern United States had antibodies to T. cruzi, although both the exact foxes (Urocyon cinereoargenteus) from South Carolina. Veterinary collection location and wild canine species were unclear (Duprey et al., Parasitology 97: 159–164. 2006). MCKEEVER,S.G.,G.W.GORMAN, AND L. NORMAN. 1958. Occurrence Trypanosoma cruzi infection has been described in a variety of wild of Trypanosoma cruzi-like organism in some mammals from south- animals in the southeastern United States (John and Hoppe, 1986; Pung western Georgia and northwestern Florida. Journal of Parasitology et al., 1995). Seropositive raccoons have been reported from both urban 44: 583–587. (Hancock et al., 2005) and nonurban (Yabsley et al., 2002a, 2002b) PUNG, O. J., C. W. BANKS,D.N.JONES, AND M. W. KRISSINGER. 1995. areas in the United States, including South Carolina. In contrast to our Trypanosoma cruzi in wild raccoons, opossums, and triatomine RESEARCH NOTES 957

bugs in southeast Georgia, U.S.A. Journal of Parasitology 81: 324– SCALONE, A., R. DELUNA,G.OLIVA,L.BALDI,G.SATTA,G.VESCO,W. 326. MIGNONE,C.TURILLI,R.R.MONDESIRE,D.SIMPSON, ET AL. 2002. ROSYPAL,A.C.,G.C.TROY,R.B.DUNCAN,JR., A. M. ZAJAC, AND D. Evaluation of the Leishmania recombinant K39 antigen as a diag- S. LINDSAY. 2005. Utility of diagnostic tests used in diagnosis on nostic marker for canine leishmaniasis and validation of a stan- infection in dogs experimentally inoculated with a North American dardized enzyme-linked immunosorbent assay. Veterinary Parasi- isolate of Leishmania infantum infantum. Journal of Veterinary In- tology 104: 275–285. ternal Medicine 19: 802–809. YABLSEY, M. J., AND G. P. NOBLET. 2002a. Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi from ———, ———, A. M. ZAJAC,R.B.DUNCAN,JR.K.WAKI,K.P. South Carolina. Journal of Parasitology 88: 1273–1276. CHANG, AND D. S. LINDSAY. 2003. Emergence of zoonotic canine ———, AND ———. 2002b. Seroprevalence of Trypanosoma cruzi in leishmaniasis in the United States: Isolation and immunohisto- raccoons from South Carolina and Georgia. Journal of Wildlife chemical detection of Leishmania infantum from foxhounds from Diseases 38: 75–83. Virginia. Journal of Eukaryotic Microbiology 50: S691–S693. ———, ———, AND O. J. PUNG. 2001. Comparison of serological ROSYPAL,A.C.,A.M.ZAJAC, AND D. S. LINDSAY. 2003. Canine visceral methods and blood culture for detection of Trypanosoma cruzi in- leishmaniasis and its emergence in the United States. Veterinary fection in raccoons (Procyon lotor). Journal of Parasitology 87: Clinics of North America Small Animal Practice 33: 921–937. 1155–1159.

J. Parasitol., 93(4), 2007, pp. 957–958 ᭧ American Society of Parasitologists 2007

Distribution Pattern of Phthirapterans Infesting Certain Common Indian Birds

A. K. Saxena, Sandeep Kumar, Nidhi Gupta, J. D. Mitra, S. A. Ali, and Roshni Srivastava, Department of Zoology, Government Raza Post Graduate College, Rampur (U.P.) 244 901, India. e-mail: [email protected]

ABSTRACT: The prevalence and frequency distribution patterns of 10 sitizing some common Indian birds: house sparrow (Passer domesticus), phthirapteran species infesting house sparrows, Indian parakeets, com- Indian parakeet (Psittacula eupatria), common myna (Acridotheres tris- mon mynas, and white breasted kingfishers were recorded in the district tis), and white-breasted kingfisher (Halcyon smyrnensis). of Rampur, India, during 2004–05. The sample mean abundances, mean All birds were captured live (with mist nets) in the district of Rampur intensities, range of infestations, variance to mean ratios, values of the (U.P.), India, during 2004 and 2005. After tying the legs, each bird was exponent of the negative binomial distribution, and the indices of dis- thoroughly searched for the presence of lice by visual examination. crepancy were also computed. Frequency distribution patterns of all Infested birds were then subjected to delousing by fumigation (modified phthirapteran species were skewed, but the observed frequencies did ‘‘Fair-Isle’’ method). The efficacy of different methods for delousing not correspond to the negative binomial distribution. Thus, adult–nymph infested birds has been discussed elsewhere (Clayton and Drown, 2001). ratios varied in different species from 1:0.53 to 1:1.25. Sex ratios of Head and body feathers were further examined using a stereozoom trin- different phthirapteran species ranged from 1:1.10 to 1:1.65 and were ocular microscope to remove the remaining lice. The entire louse load female biased. was placed in 70% alcohol and separated according to species, age, and gender. The prevalence (%), mean (X), and variance (s2) were calculat- Certain workers have provided useful information on the prevalence ed. The exponent (k) of negative binomial distribution and index of and frequency distribution pattern of Phthiraptera on selected avian discrepancy (D) (Rozsa et al., 2000) were generated, and the goodness hosts. Work done so far on the subject has been reviewed from time to of fit between observed and expected frequency distribution was deter- time (Marshall, 1981; Price and Graham, 1997; Price et al., 2003). The mined by chi-square tests. patterns of abundance of different lice on their avian hosts have been Three phthirapteran species, Brueelia subtilis (Nitzsch, 1874), Echin- further discussed by Rekasi et al. (1997), Rozsa (1997), and Reiczigel ophilopterus chapini (Ewing, 1927), and Myrsidea quadrifasciata (Pia- et al. (2005). Here, we report information on the prevalence and fre- get, 1880), were recovered from 100 house sparrows (Table I). Fre- quency distribution patterns of 10 different phthirapteran species para- quency distribution patterns of the 3 species were skewed, but were not

TABLE I. Summary of distribution patterns of phthirapterans infesting of house sparrows, Indian parakeets, common myna, and white-breasted kingﬁshers in the district of Rampur, India, during 2004 and 2005.

Sample Mean Variance Exponent of Index of Sample Prevalence mean Range of intensity to mean negative discrepancy Host size Louse species (%) abundance infestation (X) ratio (s2) binomial (k) (D)

House sparrow 100 Brueelia subtilis 31.0 4.1 1–41 13.3 16.0 0.11 0.79 100 Echinophilopterus chapini 14.0 1.1 2–21 7.6 10.6 0.05 0.90 100 Myrsidea quadrifasciata 20.0 1.5 2–28 9.7 12.8 0.07 0.87 Indian parakeet 100 Neopsittaconirmus elbeli 34.0 7.4 2–46 21.8 21.2 0.11 0.76 100 Echinophilopterus chapini 17.0 13.8 1–28 13.8 16.6 0.05 0.88 Common myna 100 Myrsidea invadens 31.0 5.1 2–63 16.3 32.1 0.10 0.86 100 Menacanthus eurysternus 13.0 2.3 2–32 17.5 20.8 0.03 0.90 100 Brueelia chayanh 24.0 6.9 3–82 28.9 41.9 0.06 0.86 100 Sturnidoecus bannoo 42.0 15.6 3–106 37.07 50.3 0.12 0.78 White-breasted kingﬁsher 30 Meropoecus sp. 40.0 7.1 6–44 17.75 20.09 0.15 0.71 958 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE II. The population composition of 10 phthirapteran species infesting house sparrows, Indian parakeets, common mynas, and white-breasted kingﬁshers in the district of Rampur, India, during 2004 and 2005.

Ratios (overall)* Ratio Average (summers) Number of IN:IIN: Host Species hosts Frequency Adults Nymphs M:F III N A:N (n)

House sparrow Brueelia subtilis 100 31 8.7 4.6 1:1.3 1 : 0.7 : 0.5 1:1.7 (6) Echinophilopterus chapini 100 14 4.5 3.1 1:1.3 1 : 0.8 : 0.5 1:1.1 (3) Myrsidea quadrifasciata 100 20 5.5 4.2 1:1.7 1 : 0.8 : 0.6 1:1.1 (5) Indian parakeet Neopsittaconirmus elbeli 100 34 10.5 11.4 1:1.4 1 : 0.8 : 0.8 1:1.3 (17) Echinophilopterus chapini 100 17 6.1 7.7 1:1.4 1 : 0.8 : 0.6 1:1.2 (7) Common myna Myrsidea invadens 100 31 8.6 7.8 1:1.5 1 : 0.7 : 0.6 1:1.3 (5) Menacanthus eurysternus 100 13 9.8 7.7 1:1.1 1 : 0.6 : 0.5 1:0.7 (2) Brueelia chayanh 100 24 15.6 13.3 1:1.2 1 : 0.8 : 0.7 1:1.1 (4) Sturnidoecus bannoo 100 42 20.2 16.8 1:1.3 1 : 0.9 : 0.9 1:1.0 (6) White-breasted kingﬁsher Meropoecus sp. 30 12 11.6 6.2 1:1.5 1 : 0.9 : 1.3 1:2.1 (2)

* Abbreviations: M – male, F – female, A – adult, N – nymph, I N – first instar nymph, II N – second instar nymph, III N – third instar nymph, n – number of birds. described by a negative binomial pattern (␹2 ϭ 82.5, P Ͼ 0.05; ␹2 ϭ females), differential killing by host birds (larger sized females are pre- 33.9, P Ͼ 0.05; and ␹2 ϭ 53.3, P Ͼ 0.05, respectively). sumably more readily killed during preening by host birds), and local Indian parakeets were found infested with 2 ischnoceran phthirapter- mate composition (if female lice disperse more effectively than males) ans, Neopsittaconirmus elbeli (Guimaraes, 1974) and Echinophilopterus have been linked to imbalances in sex ratios of phthirapteran popula- chapini (Ewing, 1927) (Table I). The occurrence of E. chapini on par- tions (Marshall, 1981; Kim, 1985; Rozsa et al., 1996). rots is a new host record. Four specimens of the amblyceran, Keleri- The authors are thankful to 2 anonymous referees for fruitful com- menopon psittaculae, were also recovered from a single parakeet, but ments on the earlier draft of this paper; to the Principal, Govt. Raza excluded from further analysis. The frequency distribution again re- P.G. College, Rampur, for laboratory facilities; to E. Mey (Naturhisto- mained skewed, but could not be described by the negative binomial risches Museum im Thuringer Landesmuseum Heidecksburg, Schlo- model (␹2 ϭ 85.87, P Ͼ 0.05 and ␹2 ϭ 85.49, P Ͼ 0.05, respectively). Bbezirk 1, D-07407 Rudolstadt Bundesrepublik, Germany) for the valu- Common myna (Acridotheres tristis) were found infested with 2 am- able help in identification of lice; and to the Department of Science and blyceran species, Myrsidea invadens (Kellogg and Chapman, 1902) and Technology, New Delhi, India for providing financial support to A.K.S. Menacanthus eurysternus (Burmeister, 1838), and 2 ischnoceran spe- in the form of project SP/SO/AS-30-2002. cies, Brueelia chayanh (Ansari, 1955a) and Sturnidoecus bannoo (An- sari, 1955b) (Table I). The frequency distribution pattern did not conform to the negative binomial model (␹2 ϭ 20.01, P Ͼ 0.05; ␹2 ϭ 14.45, LITERATURE CITED P Ͼ 0.05; ␹2 ϭ 32.28, P Ͼ 0.05; and ␹2 ϭ 66.08, P Ͼ 0.05, respec- CLAYTON,D.H.,AND D. N. DROWN. 2001. Critical evaluation of five tively). methods for quantifying chewing lice (Insecta: Phthiraptera). Jour- Only 1 ischnoceran species, Meropoecus sp., was recovered from 30 nal of Parasitology 87: 1291–1300. white-breasted kingfishers (Table I). A small sample size prevented EVELEIGH, E. S., AND W. T HRELFALL. 1976. Population dynamics of lice curve fitting. (Mallophaga) on auks (alcidae) from Newfoundland. Canadian Table I contains further information regarding the mean intensities Journal of Zoology 54: 1694–1711. (X), values of binomial exponent (k), and the indices of discrepancy KIM, K. C. 1972. Louse populations on the northern fur seal (Callor- (D). Furthermore, the population composition of the lice on 4 avian hinus ursinus). American Journal of Veterinary Research 33: 2027– hosts is shown in Table II. The overall adult–nymph ratio, sex ratio, 2036. and the ratio of 3 nymphal instars are also noted. ———. 1985. Evolution and host associations of Anoplura. In Co- Present studies indicate that prevalence and intensities of different phthirapteran species on 4 Indian birds were not high. Frequency dis- evolution of parasitic arthropods and mammals, K. C. Kim (ed.). tribution patterns were generally skewed but failed to conform to the John Wiley, New York, New York, p. 197–231. negative binomial model. Analysis of the population composition of MARSHALL, A. G. 1981. The ecology of ectoparasitic insects. Academic any species provides clues regarding the temporal stability of the pop- Press, London, U.K., 417 p. ulation (Marshall, 1981). The adult–nymph ratios varied in different PRICE,M.A.,AND O. H. GRAHAM. 1997. Chewing and sucking lice as species from 1:0.53 to 1:1.25. The population structure of bird lice is parasites of mammals and birds. U.S. Department of Agriculture, affected by several factors, including season. Detailed examination of Technical Bulletin No. 1849: 309 p. the data shows that on 9 species (except M. eurysternus), the population PRICE, R. D., R. A. HELLENTHAL,R.L.PALMA,K.P.JOHNSON, AND D. of nymphs was comparatively higher (adult:nymph ranged from 1:0.69 H. CLAYTON. 2003. The chewing lice: World checklist and biolog- to 1:2.06) than the average values shown in Table II on birds examined ical overview, Vol. 24. Illinois Natural History Survey Special Pub- during the summers. The impact of seasons on the population structure lication, Champaign, Illinois, 501 p. of avian lice has rarely been investigated, but the seasonal variations in REICZIGEL, J., Z. LANG,L.ROZSA, AND B. TOTHMERESZ. 2005. Properties age structure among lice have been shown on seals (Kim, 1972). More- of crowding and statistical tools to analyze parasite-crowding data. over, in their study on the population dynamics of lice on 6 species of Journal of Parasitology 91: 245–252. auks (Charadiiformes), Eveleigh and Threlfall (1976) found that REKASI, J., L. ROZSA, AND B. J. KISS. 1997. Patterns in the distribution nymphs did not predominate on adults, though they did on chicks, and of avian lice (Phthiraptera: Amblycera, Ischnocera). Journal of Avi- that nymph populations increased rapidly. Hence, the adult–nymph ratio an Biology 28: 150–156. may vary with time. The sex ratios of different phthirapteran species ROZSA, L. 1997. Patterns in the abundance of avian lice (Phthiraptera: ranged from 1:1.1 to 1:1.65 and were female biased. Sampling errors Amblycera, Ischnocera). Journal of Avian Biology 28: 249–254. (being larger in size, the female lice are easier to collect), unequal ———, J. REICZIGEL, AND G. MAJOROS. 2000. Quantifying parasites in longevity (the lifespan of adult males is generally shorter than that of samples of hosts. Journal of Parasitology 86: 228–232. RESEARCH NOTES 959

J. Parasitol., 93(4), 2007, pp. 959–961 ᭧ American Society of Parasitologists 2007

Feline Cuterebrosis Caused by a Lagomorph-Infesting Cuterebra spp. Larva

Frank Slansky* Department of Entomology and Nematology, Building 970 Natural Area Drive, University of Florida, Gainesville, Florida 32611; *Present address: 14107 NW 61st Lane, Gainesville, Florida 32653. e-mail: fslansky@uﬂ.edu

ABSTRACT: Native species of rodents and lagomorphs in the Americas Lack of knowledge of the particular species causing feline cutere- are the typical hosts of Cuterebra spp. larvae. Although these bot flies brosis stems largely from the difficulty of identifying Cuterebra spp. are relatively host specific, they occasionally parasitize other native and larvae to species without first rearing them to adults (Sabrosky, 1986). introduced mammals (including domestic animals and humans), an af- Typically, live larvae (usually second or third instars) removed from fliction termed cuterebrosis. Cuterebra spp. larvae generally cause be- their hosts are chemically preserved, although in some of these cases nign, subcutaneous lesions (warbles), but when infesting domestic cats, (especially those involving the eyes or brain), living or dead larvae they can invade the eyes, respiratory tract, and cerebral tissues, causing (usually first instars) were only observed in situ (often during necropsy). severe, and in some cases fatal, injury. Despite more than 2 dozen First instars can at best be identified visually as ‘‘Cuterebra spp.’’ How- published reports of feline cuterebrosis, the type (rodent- or lagomorph- ever, to various extents for the later instars, some Cuterebra species infesting) or species parasitizing domestic cats has rarely been deter- may be distinguished based on characters such as cuticular spines and mined. Here, I identify a larva removed from a kitten in southern Wis- platelets, and posterior spiracles (Knipling and Brody, 1940; Bennett, consin as belonging to a lagomorph-infesting Cuterebra species, most 1955; Haas and Dicke, 1958; Capelle, 1970; Baird, 1972). Identification likely C. abdominalis, based especially on features of the cuticular of specimens to species should improve as more Cuterebra species are platelets covering its exterior, and its geographic location. This seems analyzed molecularly (e.g., Otranto et al., 2003; Noel et al., 2004). to be only the third substantiated report of feline cuterebrosis in more At least for third instars, rodent-infesting Cuterebra species, which than 50 yr in which a larva has been identified beyond ‘‘Cuterebra have many flattened, multipointed cuticular platelets, can be distin- spp.’’ In each case, lagomorph-infesting species were involved, sug- guished from those that infest lagomorphs, which are covered with gesting that domestic cats may not be susceptible to rodent-infesting mostly raised, usually single-pointed, spinelike platelets (Knipling and Cuterebra species. However, because these studies are limited in num- Brody, 1940; Bennett, 1955; Haas and Dicke, 1958; Baird and Graham, ber and geographic area, additional research is required to establish the 1973; Slansky and Huckabee, 2006). Here, I provide what seems to be spectrum of Cuterebra species involved in feline cuterebrosis. only the third substantiated, published report of feline cuterebrosis in which the insect(s) responsible was (were) identified beyond ‘‘Cutere- bra Cuterebrosis (or cuterebriasis) is a type of myiasis caused by larvae spp.’’ of Cuterebra spp. These bot flies typically parasitize various native On 5 September 2003, a cream-colored insect larva covered with dark species of either rodents (e.g., mice, rats, tree squirrels) or lagomorphs brown speckles, was removed from a lesion above the right shoulder (e.g., rabbits and hares) in the Americas, but they also can infest a of a domestic shorthair kitten, approximately 8 to 10 wk old, that was variety of ‘atypical’ hosts, including indigenous raccoons and skunks, brought for treatment to a local veterinary clinic in Ripon, Fond du Lac County, Wisconsin. The larva was stored in formalin and subsequently nonindigenous mice and rats, domestic cats, dogs and rabbits, and hu- ௡ mans, among others (Hall, 1925; Sabrosky, 1986; Baird et al., 1989; measured and photographed using an Automontage digital photomi- Suedmeyer et al., 2000; Bradley, 2001; Safdar et al., 2003; Slansky, croscopy system (Synchroscopy, Frederick, Maryland). Portions of the 2006; Slansky and Huckabee, 2006). Infestation occurs when potential specimen were covered by a whitish film that probably is purulent mat- hosts encounter larvae newly hatched from eggs laid on foliage or other ter from the host, as observed with certain other Cuterebra spp. larvae habitat substrates (Catts, 1982). (Slansky and Huckabee, 2006; Slansky, 2007). This coating was par- The most common form of this affliction is subcutaneous infestation, tially removed before the photomicrographs were taken. The specimen which typically is benign, easy to diagnose, and routinely and effec- is deposited as voucher specimen #Cut111 in the Immature Insect Col- tively treated (Fischer, 1983; Bowman et al., 2003; Slansky and Kenyon, lection in the Department of Entomology and Nematology at the Uni- 2003; F. Slansky, unpubl. obs.). More serious manifestations, especially versity of Florida, Gainesville, Florida. in cats, involve the eyes (ophthalmomyiasis; Johnson et al., 1988; Harris The overall appearance of the larva, as well as the features described et al., 2000; Wyman et al., 2005; Stiles and Rankin, 2006), respiratory below, are distinctly characteristic of a third instar of Cuterebra spp. system (nasal/pharyngeal/tracheal myiasis; Thirloway, 1982; Fitzgerald The preserved specimen is 26.5 mm long and 12.0 mm wide (at its et al., 1996; Dvorak et al., 2000), or central nervous system (cerebro- widest). On its anterior segment, the larva possesses a head with 2 spinal myiasis; Cook et al., 1985; Hendrix et al., 1989; Glass et al., black, pointed mouth hooks, but lacking prominent cuticular platelets 1998; King, 2000). In these cases, clinical signs and symptoms can (Fig. 1A). Elsewhere, most of the cuticular platelets are raised with include anorexia, lethargy, inflammation, retinal damage, impaired vi- single points (ranging from blunt to spinelike; Fig. 1B). On the nonter- sion, dyspnea, seizures, paralysis, or a combination, and definitive di- minal segments, platelets in the anterior rows usually have their points agnoses can be difficult (many of the above references; also see Green- facing posteriorly, and those in the posterior rows typically have points berg et al., 2004). Treatments for these conditions are often experimen- directed anteriorly (Fig. 1B), as described for other Cuterebra spp. lar- tal and of uncertain efficacy, and the outcome may be fatal, either be- vae (Knipling and Brody, 1940; Haas and Dicke, 1958; Slansky and cause the animal dies outright or is killed. Huckabee, 2006). Knowledge of which Cuterebra species cause(s) feline cuterebrosis The anal body segment is retracted within the penultimate segment could improve diagnosis in the problematic cases, for example, by com- (Fig. 1C), as is often the case with preserved third instars of Cuterebra paring dates of case presentations with the seasonal activity patterns of species (e.g., Knipling and Brody, 1940; Haas and Dicke, 1958). It the insects responsible for this affliction. In addition, such information possesses a scattering of anterior-pointing, spinelike platelets, with those may allow cat owners to reduce the risk of their pets contracting cu- toward the dorsal being more prominent (Fig. 1C). Also visible on this terebrosis by restricting outdoor exposure during the peak infestation segment are 2, centrally located, kidney-shaped spiracular plates (Fig. period(s) of the causative species of Cuterebra, by limiting access to 1C), each containing serpentine respiratory slits (Fig. 1D). these flies’ habitats, or both. However, of the 20ϩ published cases of Two lagomorph-infesting species of Cuterebra occur in Wisconsin, feline cuterebrosis, I am aware of only 2 substantiated reports in which where the present larva was obtained, i.e., C. abdominalis Swenk (for- Cuterebra spp. larvae were identified to infestation type and likely spe- merly known as C. horripilum Clark) and C. buccata (Fabricius) (Haas cies (Boisvenue, 1955; Haas and Dicke, 1958). Two other studies have and Dicke, 1958; Sabrosky, 1986). Apparently, 1 feature distinguishing reported particular species for Cuterebra sp. larvae infesting domestic between third instars of these 2 species is the presence on C. abdom- cats (Dalmat, 1943; Hatziolos, 1966), but neither of these studies was inalis of light-colored platelets, many with darker, single points, on the substantiated by larval descriptions or other confirmatory information. penultimate body segment (Haas and Dicke, 1958), as is evident in this 960 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

additionally Illinois, Michigan, and Ohio for Boisvenue [1955]), the hypothesis of cat resistance to larvae of rodent-infesting Cuterebra species remains to be adequately evaluated. Larvae of Cuterebra spp. bot ﬂies have been found in a few other species of carnivores (Hall, 1925; Sabrosky, 1986). Of these atypical hosts, substantiated identiﬁcations of larvae beyond ‘‘Cuterebra spp.’’ have been made only for 3 raccoons (all rodent-infesting; Slansky and Huckabee, 2006; F. Slansky, unpubl. obs.), a dog (lagomorph-infesting; F. Slansky, unpubl. obs.), and a mink (lagomorph-infesting; Sabrosky, 1986). Clearly, additional research is required to determine the spectrum of Cuterebra species involved in cuterebrosis in domestic cats as well as in other atypical hosts. I thank Rebecca Hauser for providing the larva and Craig Welch for taking the photomicrographs used in this study. I am grateful to Lou Rea Kenyon for reviewing this manuscript and to Douglas Beckner for assistance in preparing the images. This work was supported in part by an instructional improvement grant from the College of Agricultural and Life Sciences at the University of Florida.

LITERATURE CITED

BAIRD, C. R. 1972. Development of Cuterebra ruficrus (Diptera: Cu- terebridae) in six species of rabbits and rodents with a morphological comparison of C. ruficrus and C. jellisoni third instars. Journal FIGURE 1. Third instar of Cuterebra spp. removed from a kitten in 9: Fond du Lac County, Wisconsin, on 5 September 2003 (dorsal is toward of Medical Entomology 81–85. Cuterebra the top of each image). (A) Anterior end. The head contains 2 black, ———. 1979. Incidence of infection and host specificity of tenebrosa Neotoma cinerea sharply pointed mouth hooks and lacks dark cuticular platelets present in bushy-tailed wood rats ( ) from cen- 65: on the other body segments. (B) Mid-body segments (anterior to left) tral Washington. Journal of Parasitology 639–644. AND RAHAM Cuterebra tenebrosa featuring the spinelike cuticular platelets characteristic of a lagomorph- ———, C. L. G . 1973. : Description of infesting species. (C) Posterior end. The anal segment, with the 2 kid- immature stages and a redescription of the adult (Diptera: Cutereb- 105: ney-shaped spiracular plates, is withdrawn into the penultimate body ridae). Canadian Entomologist 1281–1293. segment, which possesses pale, flat platelets, many with darker, single BAIRD, J. K., C. R. BAIRD, AND C. W. SABROSKY. 1989. North American points (arrow), a feature of Cuterebra abdominalis.(D) Posterior spi- cuterebrid myiasis. Journal of the American Academy of Derma- 21: racular plate with serpentine respiratory slits. The 3 wedge-shaped sec- tology 763–772. ENNETT Cuterebra emasculator tions typical of many Cuterebra species are poorly represented, which B , G. F. 1955. Studies on Fitch 1856 might be a feature of C. abdominalis (see text). (Diptera: Cuterebridae) and a discussion of the status of the genus Cephenemyia Ltr. 1818. Canadian Journal of Zoology 33: 75–98. BOISVENUE, R. J. 1955. Studies on the life history and ecology of Cu- terebra spp. occurring in Michigan cottontails with systematic stud- larva (Fig. 1C). This feature also has been reported from larvae of C. ies on cuterebrine larvae from other mammals. Ph.D. Dissertation. cuniculi Clark, a lagomorph-infesting species found only in Georgia and Michigan State University, East Lansing, Michigan, 218 p. Florida (Knipling and Brody, 1940; Sabrosky, 1986) that also closely BOWMAN, D. D., R. C. LYNN, AND M. L. EBERHARD. 2003. Georgis’ resembles C. abdominalis in the adult stage (Sabrosky, 1986). parasitology for veterinarians, 8th ed. Saunders, St. Louis, Missou- Another characteristic may help differentiate between third instars of ri, 422 p. C. abdominalis and C. buccata. The spaces separating the typically 3, BRADLEY, T. A. 2001. What every veterinarian needs to know about wedge-shaped regions of each spiracular plate apparently are well de- rabbits. Exotic DVM 3.1: 42–46. fined in the latter species (Boisvenue, 1955), as in certain other Cute- CAPELLE, K. J. 1970. Studies on the life history and development of rebra species (Bennett, 1955; Capelle, 1970; Slansky and Huckabee, Cuterebra polita (Diptera: Cuterebridae) in four species of rodents. 2006). In contrast, these divisions seem to be less evident in larvae of Journal of Medical Entomology 7: 320–327. C. abdominalis (Boisvenue, 1955), as is the case in this specimen (Fig. CATTS, E. P. 1982. Biology of New World bot flies: Cuterebridae. An- 1D). However, the taxonomic value of this characteristic is uncertain nual Review of Entomology 27: 313–338. because the extent of these spaces apparently varies with larval age COOK, J. R., JR., D. C. LEVESQUE, AND L. P. NUEHRING. 1985. Intracranial (Knipling and Brody, 1940). cuterebral myiasis causing acute lateralizing meningoencephalitis As in the previous substantiated reports (Boisvenue, 1955; Haas and in two cats. Journal of the American Animal Hospital Association Dicke, 1958) in which larvae infesting cats were identified beyond ‘‘Cu- 21: 279–284. terebra spp.,’’ this case involves a lagomorph-infesting species. Also DALMAT, H. T. 1943. A contribution to the knowledge of the rodent similar to these 2 reports, the larva described here most likely is that warble flies (Cuterebridae). Journal of Parasitology 29: 311–318. of C. abdominalis. Twelve second instars and ‘‘approximately 18’’ third DVORAK, L. D., J. D. BAY,D.T.CROUCH, AND R. M. CORWIN. 2000. instars of this species removed from cats were identified by Boisvenue Successful treatment of intratracheal cuterebrosis in two cats. Jour- (1955), as was the third instar listed in Haas and Dicke (1958). Bois- nal of the American Animal Hospital Association 36: 304–308. venue (1955) also identified 15 third instars of C. buccata involved in FISCHER, K. 1983. Cuterebra larvae in domestic cats. Veterinary Med- cases of feline cuterebrosis. icine/Small Animal Clinician 78: 1231–1233. Feline cuterebrosis has been reported from at least 19 states (primar- FITZGERALD, S. D., C. A. JOHNSON, AND E. J. PECK. 1996. A fatal case ily eastern and midwestern) in the United States and a Canadian prov- of intrathoracic cuterebriasis in a cat. Journal of the American An- ince (F. Slansky, unpubl. obs.), suggesting that domestic cats allowed imal Hospital Association 32: 353–357. outdoors would likely be exposed to many of the 30ϩ species of Cu- GLASS, E. N., A. M. CORNETTA,A.DELAHUNTA,S.A.CENTER, AND M. terebra occurring in North America. Finding only lagomorph-infesting KENT. 1998. Clinical and clinicopathologic features in 11 cats with species involved in this affliction may indicate that cats are not suscep- Cuterebra larvae myiasis of the central nervous system. Journal of tible to parasitization by rodent-infesting species of Cuterebra. Some Veterinary Internal Medicine 12: 365–368. atypical hosts are resistant to infestation by certain Cuterebra species GREENBERG, M. J., S. J. SCHATZBERG,A.DELAHUNTA,T.STOKOL, AND (Baird, 1972, 1979), although the mechanism(s) involved are unknown B. A. SUMMERS. 2004. Intracerebral plasma cell tumor in a cat: A (Slansky, 2007). Nonetheless, given the low number of studies of feline case report and literature review. Journal of Veterinary Internal cuterebrosis in which larvae were identified to infestation type and spe- Medicine 18: 581–585. cies, and their limited geographic area (Wisconsin for all 3 studies, and HAAS,G.E.,AND R. J. DICKE. 1958. On Cuterebra horripilum Clark RESEARCH NOTES 961

(Diptera: Cuterebridae) parasitizing cottontail rabbits in Wisconsin. drial cytochrome I gene of Oestridae species causing obligate my- Journal of Parasitology 44: 527–540. iasis. Medical and Veterinary Entomology 17: 307–315. HALL, M. C. 1925. The occurrence of cuterebrid larvae in dogs and SABROSKY, C. W. 1986. North American species of Cuterebra, the rabbit cats, and the possible modes of infection. Journal of Economic and rodent bot flies (Diptera: Cuterebridae). Entomological Society Entomology 18: 331–335. of America, College Park, Maryland, 240 p. HARRIS, B. P., P. E. MILLER,J.R.BLOSS, AND P. J. PELLITTERI. 2000. SAFDAR, N., D. K. YOUNG, AND D. ANDES. 2003. Autochthonous furun- Ophthalmomyiasis interna anterior associated with Cuterebra spp cular myiasis in the United States: Case report and literature review. in a cat. Journal of the American Veterinary Medical Association Clinical and Infectious Diseases 36: 73–80. 216: 352–355. SLANSKY, F. 2006. Cuterebra bot flies (Diptera: Oestridae) and their HATZIOLOS, B. C. 1966. Cuterebra larva in the brain of a cat. Journal indigenous hosts and potential hosts in Florida. Florida Entomol- of the American Veterinary Medical Association 148: 787–793. ogist 89: 152–160. HENDRIX, C. M., M. N. DIPINTO,N.R.COX,E.SARTIN, AND C. L. ———. 2007. Insect/mammal associations: Effects of cuterebrid bot fly CLEMONS-CHEVIS. 1989. Aberrant intracranial myiasis caused by parasites on their hosts. Annual Review of Entomology 52: 17–36. larval Cuterebra infection. Compendium on Continuing Education ———, AND J. HUCKABEE. 2006. First records of rodent-infesting Cu- for the Practicing Veterinarian 11: 550–562. terebra bot flies parasitizing raccoons (Procyon lotor) in North America. Journal of Parasitology 92: 1369–1373. JOHNSON, B. W., L. C. HELPER, AND M. E. SZAJERSKI. 1988. Intraocular AND L. R. KENYON. 2003. Cuterebra bot fly infestation of ro- Cuterebra in a cat. Journal of the American Veterinary Medical ———, dents and lagomorphs. Journal of Wildlife Rehabilitation 26: 7–16. Association 193: 820–830. STILES, J., AND A. RANKIN. 2006. Ophthalmomyiasis interna anterior in KING, J. M. 2000. Cuterebra species infection in a cat. Veterinary Med- a cat: Surgical resolution. Veterinary Ophthalmology 9: 165–168. icine 95: 291. SUEDMEYER, W. K., E. P. CATTS, AND E. GREINER. 2000. Cuterebra my- KNIPLING,E.F.,AND A. L. BRODY. 1940. Some taxonomic characters of iasis in a group of red kangaroos (Megaleia rufa), a Bennett’s wal- cuterebrine (Diptera) larvae, with larval descriptions of two species laby (Macropus rufogriseus fruticus) and a Gunther’s dik dik (Mal- from Georgia. Journal of Parasitology 26: 33–43. oqua guentheri smithi). J. Zoo and Wildlife Medicine 31: 124–128. NOEL, S., N. TESSIER,B.ANGERS, AND F.-J. LAPOINTE. 2004. Molecular THIRLOWAY, L. 1982. Aberrant migration of a Cuterebra larva in a cat. identification of two species of myiasis-causing flies (Cuterebra) Veterinary Medicine/Small Animal Clinician 77: 619–620. by multiplex PCR and RFLP. Medical and Veterinary Entomology WYMAN, M., R. STARKEY,S.WEISBRODE,D.FILKO,R.GRANDSTAFF, AND 18: 161–166. E. FERREBEE. 2005. Ophthalmomyiasis (interna posterior) of the OTRANTO, D., D. TRAVERSA,B.GUIDA,E.TARSITANO,P.FIORENTE, AND posterior segment and central nervous system myiasis: Cuterebra J. R. STEVENS. 2003. Molecular characterization of the mitochon- spp. in a cat. Veterinary Ophthalmology 8: 77–80.

J. Parasitol., 93(4), 2007, pp. 961–964 ᭧ American Society of Parasitologists 2007

Molecular Identiﬁcation of Two Strains of Third-Stage Larvae of Contracaecum rudolphii Sensu Lato (Nematoda: Anisakidae) From Fish in Poland

Beata Szostakowska and Hans-Peter Fagerholm*, Medical University of Gdan´sk, Department of Tropical Parasitology, Interfaculty Institute of Maritime and Tropical Medicine, Gdynia, Poland; *Laboratory of Aquatic Pathobiology, Department of Biology, A˚ bo Akademi University, BioCity, Artillerigatan 6, FIN-20520 A˚ bo/Turku, Finland. e-mail: hafagerh@abo.ﬁ

ABSTRACT: Contracaecum sp. larvae (L3) from fish were identified us- larvae was earlier deduced from circumstantial (for example, ecological) ing nucleotide sequences of the internal transcribed spacers ITS-1 and evidence, although attempts to obtain adult male worms for identifica- ITS-2 of the ribosomal DNA. The nematode larvae originated from fish tion by culture were also made (Fagerholm, 1988). Recently, larvae in a freshwater situation (crucian carp Carassius carassius, from Sel- noted as Contracaecum rudolphii Hartwich, 1964 have been reported ment Wielki Lake in Mazury, northeastern Poland) and a brackish-water both in marine and freshwater fishes, i.e., Maori chiefs Notothenia an- region (Caspian round goby Neogobius melanostomus from the Baltic gustata and Notothenia cornucola, trout Salmo trutta, wels Silurus glan- Sea, Gdan´sk Bay at the Polish coast). Two strains (Contracaecum ru- is, asp Aspius aspius, perch Perca fluviatilis, ruff Gymnocephalus cer- dolphii A and B) of Contracaecum rudolphii senso lato, a parasite com- nua, eel Anguilla anguilla, and flathead Platycephalus laevigatus (Tor- mon at the adult stage in fish-eating birds, were identified. In fish from res and Cubillos, 1987; Ramallo and Torres, 1995; S. Shamsi, pers. the freshwater site, only the strain temporarily designated C. rudolphii comm.). The life cycle of Contracaecum may include numerous species B was identified; in the brackish-water region, both strains were found, of insect larvae, crustaceans, and fish serving as paratenic hosts, and suggesting that fish serve as paratenic host for both genotypes. Contra- many species of fish-eating birds serving as definitive hosts. It has been caecum rudolphii sensu lato has been recorded in several species of found in birds from Neotropical, Nearctic, Palearctic, Ethiopian, and fish-eating birds in Poland, particularly in the great cormorant, Phala- crocorax carbo, in which the abundance is highest. The results, al- Australian regions (Johnston and Mawson, 1941; Whitfield and Heeg, though based on a restricted number of larvae, suggest that the life 1977; Barus et al., 1978; Torres et al., 1982; Shamsi, pers. comm.). In cycles of both genotypes can be completed in the Polish region and that the Palearctic region, Contracaecum rudolphii sensu lata has been found at least one of them, C. rudolphii B, can develop both in fresh and in 58 bird species from 24 genera (Barus et al., 1978). Anderson (1994) brackish water. and Moravec (1994) review the biology of C. rudolphii s.l. larvae in fishes. Shags and cormorants (Phalacrocorax spp.) may be considered Contracaecum rudolphii Hartwich, 1964 (Nematoda: Ascaridoidea, the main definitive hosts. The distribution of the parasite conforms to Anisakidae) is a common anisakid reported worldwide. However, in that of these bird species (Torres et al., 1983). In Poland, C. rudolphii most studies, the taxonomic status of nematodes designated C. rudolphii s.l. has been noted in small numbers in several birds species of the is not clear. Thus, the large number of reports of the occurrence of C. Gaviidae (Gavia stellata) and Podicipedidae (Podiceps cristatus, Pod- rudolphii in numerous hosts needs to be reconsidered. iceps griseigena, Tachybaptus ruficollis) (Okulewicz, 1997), whereas Identification of anisakid larvae (L3) of Contracaecum sp. has, until mass occurrences have been observed in the great cormorant Phalacro- recently, constituted a considerable problem. The species identity of corax carbo (Z˙ uchowska, 2000; Kijewska et al., 2002; Szostakowska 962 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

TABLE I. Nematode larvae of Contracaecum rudolphii s.l. studied: CrNm ϭ ex. Caspian round goby Neogobius melanostomus (from brackish water); CrCc ϭ ex. crucian carp Carassius carassius (from freshwater); (Roman numerals ϭ ﬁsh ID, Arabic numerals ϭ template number).

Hosts Symbol of template DNA extraction

N. melanostomus no. I CrNmI Single larva N. melanostomus no. II CrNmII* Single larva C. carassius no. I CrCcI-1 Single larva (Ͼ500 Larvae found) CrCcI-2 10 Larvae (pooled) CrCcI-3 20 Larvae (pooled) C. carassius no. II CrCcII-1† Single larva FIGURE 1. Origin of fish examined: northeastern Poland in the Gdan´sk Bay near the Ka˛ty Rybackie (brackish-water site) and Selment CrCcII-2 Single larva Wielki Lake (freshwater site). C. carassius no. III CrCcIII Single larva ⌺ 5 Fish infected ⌺ 8 Templates et al., 2002; Sulgostowska et al., 2004; Szostakowska and Fagerholm, * Sequences deposited in GenBank under accession number DQ316967 (ITS-1) unpubl. data). and DQ316969 (ITS-2). Multilocus enzyme electrophoretic studies have shown that the spe- † Sequences deposited in GenBank under accession number DQ316968 (ITS-1) and DQ316970 (ITS-2). cies C. rudolphii is not genetically homogenous, but contains 2 distinct and reproductively isolated genotypes. These genotypes were denoted ‘‘sibling species’’ and provisionally named Contracaecum rudolphii A and Contracaecum rudolphii B (Bullini et al., 1986; S. Mattiucci, pers. GAA GGA TCA TT 3Ј, and NC13R (reverse), 5Ј GCT GCG TTC TTC comm.). The presence of 2 strains was actually also predicted by Nadler ATC GAT 3Ј, designed to amplify the ITS-1 region, together with flank- et al. (2000); the 2 strains have been confirmed by a study of genomic ing sequences, as well as XZ1 (forward), 5Ј ATT GCG CCA TCG GGT DNA and, specifically, internal transcribed spacers (ITS-1 and ITS-2) TCA TTC C 3Ј, and NC2 (reverse), 5Ј TTA GTT TCT TTT CCT CCG of ribosomal DNA (rDNA) (Li et al., 2005). The authors established CT 3Ј, which amplifies the ITS-2 region together with flanking sequenc- 3% differences in nucleotide sequences of the entire ITS (ϭITS-1 and es (Zhu et al. 2000, 2002; Li et al. 2005). ITS-2) between genotypes C. rudolphii A and B. This result is com- DNA amplification was carried out in 50-␮l reaction mixtures con- parable to the range of nucleotide differences detected within other an- sisting of 5 ␮l of PCR buffer (10ϫ concentrated), 5 ␮l of dNTP mixture isakid species complexes, i.e., Pseudoterranova decipiens complex (concentration of each dNTP was 2.5 mM), 2 ␮l of each primer (con- (0.2–6.8%) and the Contracaecum osculatum complex (0–2.3%) (Zhu centration 10 ␮M), 1 U of RUN polymerase (A&A Biotechnology), 2 et al., 2000, 2002). ␮l of genomic DNA, and 33 ␮l of distilled water. Molecular methods have facilitated the identification of all develop- PCR reaction conditions were as follows: initial activation of RUN mental stages of nematodes and also any sibling forms. In the present polymerase at 94 C for 3 min; 30 cycles of denaturation at 94 C for 30 study, Contracaecum sp. larvae found in fish in 2 localities in north- sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec; eastern Poland were identified using DNA sequence data. followed by a final extension step at 72 C for 5 min and finished with Crucian carp Carassius carassius (n ϭ 120) from Selment Wielki a hold step at 4 C. DNA amplification was performed in the GeneAmp Lake (Mazury, northeastern Poland) and Caspian round goby Neogobius PCR System 2400 or 9700 Gold thermocyclers (Applied Biosystems, melanostomus (n ϭ 82) caught in coastal waters of the Gdan´sk Bay Foster City, California). (southern Baltic Sea) (Fig. 1) were studied. Fish were initially frozen, PCR products were electrophoresed on 1.5% agarose (Sigma, St. and then examined after thawing. Parasites were first searched for on Louis, Missouri), and visualized following ethidium bromide staining. the surface of viscera (Myjak et al., 1994). Then, the intestine was The PCR and sequencing reaction products were purified using the dissected and washed in water and nematodes were searched for in the Clean-Up Kit and the ExTerminator Kit (A&A Biotechnology), respec- debris obtained. Additionally, the intestinal walls of fish were pressed tively. between 2 sheets of glass to find parasites remaining in the intestinal Purified sequencing reaction products were sequenced using the ABI wall. PRISM 310 DNA sequencer (Applied Biosystems) according to the Larvae embedded in the intestinal wall or beneath the liver serosa manufacturer’s protocol. Sequences were analyzed with the use of ABI were isolated by pepsin digestion (1% pepsin in 1% HCl solution). PRISM DNA Sequencing Analysis, version 3.7, for the Windows NT Larvae were washed extensively in distilled water and then frozen at Platform (Applied Biosystems) and GeneStudio Pro Software (Gene- Ϫ20 C or fixed in 75% ethanol prior to DNA isolation. Studio, Inc., Suwanee, Georgia). DNA of nematodes was extracted using the Genomic Mini Kit or Nucleotide sequences of ITS-1 and ITS-2 of rDNA of nematodes Sherlock AX Kit (A&A Biotechnology, Gdynia, Poland) for universal examined were compared with sequences obtained for C. rudolphii A genomic DNA isolation according to manufacturer’s recommendation. and B from great cormorants (Li et al., 2005; Szostakowska and Fa- The Genomic Mini Kit is based on the property of DNA to adsorb to gerholm, unpubl. data). Nucleotide sequence data reported in this paper silica surfaces in the presence of a high concentration of chaotropic are available in the GenBank, EMBL, and DDBJ databases under ac- salts. The Sherlock AX Kit is based on the unique anion exchange cession numbers DQ316767–DQ316770. membrane with a very high affinity to DNA. Pure DNA was eluted in The prevalence of Contracecum sp. L3 larvae in crucian carp from 100 ␮lor30␮l of 10 mM Tris-HCl buffer, pH 8.5. the freshwater lake was 2.4% (range of intensity was 1–500). Of the 3 We have routinely used the Genomic Mini Kit for the extraction of fish infected, the intensity in 2 fish was 1–2 larvae, which in both fish DNA from tissues, and herein it was used to extract DNA from larvae were situated in the intestinal lumen. The third fish specimen was heavi- found in crucian carp. The Sherlock AX Kit is useful when the expected ly infected with some 500 larvae, with worms being localized primarily amount of DNA is small. It was used to extract DNA from larvae from in the muscle layer of the terminal part of intestine and in the liver just round goby because only 2 comparatively small larvae were available beneath the serosa, as well as the mesenteries. The prevalence of Con- for study. tracecum sp. L3 larvae in round goby from the brackish water area was As a rule, DNA templates from nematodes were obtained from in- 2.4% (single larvae were found in each case). These larvae were re- dividual specimens. To secure information on the identity of larvae from covered from debris obtained by washing the intestine in water. They the heavily infected fish, in addition to an individual larva, 10 and 20 were probably localized in the intestinal mesentery because they were larvae were pooled separately before analysis. (Table I). The following encapsulated in fish tissue when inspected. The larvae were microscop- PCR primers were used: NC5 (forward), 5Ј GTA GGT GAA CCT GCG ically identified as Contracaecum sp. third-stage larvae. RESEARCH NOTES 963

TABLE II. Comparison of ITS-1 nucleotide sequences of Contracaecum TABLE III. Comparison of ITS-2 nucleotide sequences of Contracaecum rudolphii strains A and B. rudolphii strains A and B.

Alignment position Alignment position* 8 68 136 155 188 194 376 377 34–42 56 79 92 108 160

C. rudolphii A TTTTAATG C. rudolphii A GTTCGTGTG AACAT C. rudolphii BCGCGGCCAC. rudolphii B ------TGTCG

* According to C. rudolphii A.

The sizes of PCR products were ϳ540 bp and ϳ440 bp for ITS-1 and ITS-2 fragments, respectively (data not shown). On the basis of One possible explanation for this mass infection involves the fish in- ITS-1 and ITS-2 nucleotide sequences obtained it was ascertained that gesting remains of a dead female worm with fully developed eggs. Koie 1 Contracaecum sp. larva from round goby from the brackish water and Fagerholm (1993, 1995) found that Contracaecum osculatum reach site was identical with C. rudolphii A, whereas the other larva from the the third stage in the egg and that hatched larvae have the potential to same fish species conformed with C. rudolphii B. All larvae from cru- infect fish directly. cian carp from the freshwater lake belonged to C. rudolphii B. All C. rudolphii specimens from the freshwater reservoir studied be- The length of the ITS-1 nucleotide sequences of C. rudolphii A and longed to the strain C. rudolphii B and this is in accordance with the B were 451 nucleotides. However, ITS-2 of C. rudolphii A and B dif- results of an isoenzymatic assay (S. Mattiucci, pers. comm.) that sug- fered from each other and were 268 and 277 nucleotides, respectively. gested that this strain is mainly confined to freshwater sites. However, The ITS-1 sequences of specimens belonging to the distinct strains dif- from among 2 larvae obtained from fish caught in the Gdan´sk Bay, only fered in 8 sites (1.77%). There were single base substitutions that rep- 1 was identified as C. rudolphii A, the genotype usually confined to resented 5 transitions (alignment positions 8, 136, 188, 376, and 377) brackish waters (S. Mattiucci, pers. comm.) whereas the other one was and 3 transversions (alignment positions 68, 155, and 194) (Table II). C. rudolphii B. According to the Polish Ringing Center (Institute of The ITS-2 sequences revealed 14 nucleotide differences (5.05%) rep- Ornithology, Gdan´sk, Poland), the cormorants occurring in great num- resenting 1 deletion/insertion event embracing the 9 subsequent nucle- ber in northeastern Poland can migrate between the reservoirs compared otides (alignment positions 34–42 according to C. rudolphii A) and 5 in this study during the breeding season. This certainly facilitates an single base substitutions, i.e., 2 transitions (alignment positions 79 and expansion of the distribution of these parasites. 92) and 3 transversions (alignment positions 56, 108, and 160) (Table Sequence data of C. rudolphii obtained from cormorant caught at III). Together, the sequence difference in the entire ITS between C. Mazury is deposited in GenBank (AF411204) and results of recent stud- rudolphii A and C. rudolphii B was 3.02%. In 1 larva from crucian ies (data not shown) indicate that both C. rudolphii A and B occur in carp (symbol CrCcIII, Table I), 1 polymorphic site (A and G) was de- cormorants inhabiting both the Mazurian lakes and the Gdan´sk Bay. tected in alignment position 237 of ITS-1. Apart from this single ex- This work demonstrates that both C. rudolphii strains occur also in fish ception, the variability in ITS-1 and ITS-2 sequences was not ascer- from brackish-water Gdan´sk Bay. It is worth noting that several typi- tained within the strains studied. However, it cannot be excluded that cally freshwater digenean species present in cormorants in Poland find some polymorphism occurs among specimens included to make DNA suitable conditions for their development in brackish water off Gdan´sk templates from the 10 and 20 individuals pooled originating from the (Sulgostowska et al., 2004). One reason for this could be the low salin- heavy infected crucian carp (samples CrccI-2 and CrccI-3, Table I). ity of these waters (average 7.5‰; Majewski, 1990). The fact that C. The great cormorants, which feed in the Gdan´sk Bay, originate from rudolphii A was not found in fish from the freshwater site studied could the largest breeding colony in Europe, established in the Ka˛ty Rybackie suggest the lack of suitable paratenic hosts to complete its life cycle in village (Vistula Bar; Fig. 1). The Caspian round goby was introduced this site. However, final conclusions can be drawn only after additional to the Baltic Sea in the 1980s. This fish is the main source of food for studies. The occurrence of larvae of the 2 strains of C. rudolphii in fish the great cormorants feeding in Gdan´sk Bay. The crucian carp studied in Poland implies that both can develop in this geographical region and originated from an economically exploited freshwater lake, stocked with that the life cycle of at least one of them (strain B) can be completed fry of different fish species. The basic parameters influencing the cor- in brackish as well as in freshwater situations. morant’s diet include availability of fish of particular species, fish size, The study was supported by the Polish Committee for Scientific Re- and fish shape (Przybysz, 1997). The crucian carp, reaching 12–30 cm search (grant 3 P04C 099 23). A grant from Jubileumsfonden fo¨r A˚ bo in length, and a weight of 0.2–0.5 kg, is an important food item of Akademi is acknowledged. cormorants in stocked lakes in northeastern Poland. For the above reasons, it was decided to search for anisakid larvae in these fish species. LITERATURE CITED The sequence data are largely in conformance with the results of Li et al. (2005) who examined the same DNA regions of C. rudolphii A ANDERSON, R. C. 1994. Nematode parasites of vertebrates, 2nd ed. and B from cormorants. However, although the ITS-1 nucleotide se- CABI Publishing, Wallingford, Oxon, U.K., 650 p. quences of all larvae examined by us were 451 bp in length, the se- BARUS, V., T. P. SERGEEVA,M.D.SONIN, AND K. M. RYZHIKOV. 1978. quences obtained by Li et al. (2005) were 1 nucleotide longer. Thus, Helminths of fish-eating birds of the Palearctic region I. Academia, we found 5 thymidines (T) in alignment positions starting from 103, Publishing House of the Czechoslovak Academy of Science, whereas Li et al. (2005) reported 1 more T in the same DNA region. Prague, Czech Republic, 318 p. Moreover, our results were similar to the corresponding sequence of C. BULLINI, L., G. NASCETTI,L.PAGGI,P.ORECCHIA,S.MATTIUCCI, AND B. rudolphii from cormorant from Poland deposited in GenBank (accession BERLAND. 1986. Genetic variation of ascarid worms with different number AF411204), as well as with our recent results obtained in a life cycles. Evolution 40: 437–440. study on numerous specimens of C. rudolphii from the great cormorants FAGERHOLM, H.-P. 1988. Incubation in rats of nematodal larvae from living in different regions of northeastern Poland (data not shown). The cod to establish its specific identity: Contracaecum osculatum (Ru- difference between specimens from Italy and Poland needs to be re- dolphi). Parasitology Research 75: 57–63. solved. It would thus be useful to compare C. rudolphii A and B from JOHNSTON,T.H.,AND P. M . M AWSON. 1941. Ascaroid nematodes from both geographic regions using other DNA sequences, i.e., specific re- Australian birds. Transactions of the Royal Society of South Aus- gions of mitochondrial DNA. The recent short communication of the tralia 65: 110–115. presence of 2 strains within C. rudolphii s.l. from Australia (Shamsi, KIJEWSKA, A., J. ROKICKI,J.SITKO, AND G. WE˛GRZYN. 2002. Ascaridoi- pers. comm.) should also be considered further. dea: A simple DNA assay for identification of species infecting The prevalence of infection of fish examined was low and, as a rule, marine and freshwater fish, mammals and fish-eating birds 11. Ex- the intensity of infection was low, except for the single crucian carp perimental Parasitology 101: 35–39. found heavily infected with more than 500 larvae of C. rudolphii B. KOIE,M.,AND H.-P. FAGERHOLM. 1993. Third-stage larvae emerge from 964 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

eggs of Contracaecum osculatum (Nematoda, Anisakidae). Journal carbo sinensis (Blumenbach, 1798) from north-eastern Poland. In of Parasitology 79: 777–780. Multidisciplinarity for parasites, vectors and parasitic diseases, S. ———, AND ———. 1995 The life cycle of Contracaecum osculatum Medimond (ed.). Proceedings of the IX European Multicolloquium (Rudolphi, 1802) sensu stricto (Nematoda, Ascaridoidea, Anisaki- of Parasitology, Valencia, Spain, p. 519–523. dae) in view of experimental infections. Parasitology Research 81: SZOSTAKOWSKA, B., P. MYJAK, AND J. KUR. 2002. Identification of ani- 481–489. sakid nematodes from the Southern Baltic using PCR-based meth- LI, A.-X., S. D’AMELIO,L.PAGGI,F.HE,R.B.GASSER, Z.-R. LUN,E. ods. Molecular and Cellular Probes 16: 111–118. ABOLLO,M.TURCHETTO, AND X. Q. ZHU. 2005. Genetic evidence TORRES,P.,AND V. C UBILLOS. 1987. Infeccioń por larvas de Contracae- for the existence of sibling species within Contracaecum rudolphii cum (Nematoda, Anisakidae) en salmońidos introducidos en Chile. (Hartwich, 1964) and the validity of Contracaecum septentrionale Journal of Veterinary Medicine, Series B 34: 177–182. (Kreis, 1955) (Nematoda: Anisakidae). Parasitology Research 96: ———, L. FIGUEROA,A.SALDIVIA, AND J. BARRIENTOS. 1982. Gastro- 361–366. intestinal helminths of fish-eating birds from the Valdivia River, MAJEWSKI, A. 1990. Zatoka Gdan´ska. Wydawnictwo Geologiczne, War- Chile. Journal of Parasitology 68: 1157. saw, Poland, 501 p. ———, V. SIERPE, AND R. SCHLATTER. 1983. Occurrence of Contracae- MORAVEC, F. 1994. Parasitic nematodes of freshwater fishes of Europe, cum rudolphii in new hosts in Chile. Zeitschrift fur Parasitenkunde 2nd ed. Kluwier Academic Publishers, Dordrecht, The Netherlands, 69: 397–399. 473 p. WHITFIELD,A.K.,AND J. HEEG. 1977. On the life cycles of the cestode NADLER, S., S. D’AMELIO, H.-P. FAGERHOLM,B.BERLAND, AND L. PAGGI. Ptychobothrium belones and nematodes of the genus Contracae- 2000. Phylogenetic relationships among species of Contracaecum cum from Lake St. Lucia, Zululand. South African Journal of Sci- Railliet & Henry, 1912 and Phocascaris Host, 1932 (Nematoda: ence 73: 121–122. Ascaridoidea) based on nuclear rDNA sequence data. Parasitology ZHU, X. Q., S. D’AMELIO,L.PAGGI, AND R. B. GASSER. 2000. Assessing 121: 455–463. sequence variation in the internal transcribed spacers of ribosomal OKULEWICZ, A. 1997. Katalog fauny pasoz˙ytniczej Polski. Cz. IV. Pa- DNA within and among members of the Contracaecum osculatum soz˙yty ptako´w. Zeszyt 2B. Nicienie. Polskie Towarzystwio Para- complex (Nematoda: Ascaridoidea: Anisakidae). Parasitology Re- zytologiczne, Komisja Faunistyczna, Warsaw, Poland, 147 p. search 86: 677–683. PRZYBYSZ, J. 1997. Kormoran. Seria ‘‘Monografie przyrodnicze,’’ Lu- ———, ———, H. W. PALM,L.PAGGI,M.GEORGE-NASCIMENTO, AND buski Klub Przyrodniko´w, S´wiebodzin, Poland, 108 p. R. B. GASSER. 2002. SSCP-based identification of members within RAMALLO,G.,AND P. T ORRES. 1995. Contracaecum sp. larvae (Nema- the Pseudoterranova decipiens complex (Nematoda: Ascaridoidea: toda, Anisakidae) infection in Salmius maxillosus (Pisces, Chara- Anisakidae) using genetic markers in the internal transcribed spac- cidae) in the dam of Termas de Raõ Hondo, Argentina. Boletıń ers of ribosomal DNA. Parasitology 124: 615–623. Chileno de Parasitologıá 50: 1–2, 21–23. Z˙ UCHOWSKA, E. 2000. Contracaecum rudolphii Hartwich, 1964 (Nem- SULGOSTOWSKA, T., H.-P. FAGERHOLM, AND B. SZOSTAKOWSKA. 2004. A atoda: Anisakidae) u kormorano´w w Polsce. Wiadomosci Parazy- study on helminth fauna of the great cormorant Phalacrocorax tologiczne 46: 411–412.

J. Parasitol., 93(4), 2007, pp. 964–969 ᭧ American Society of Parasitologists 2007

Genetic and Immunological Characterization of the 14-3-3␨ Molecule From Schistosoma bovis

N. Uribe*, A. Muro, C. Vieira, J. Lopez-Aban, E. del Olmo†, L. Sua´rez‡, A. R. Martıńez-Fernańdez§, and M. Siles-Lucas࿣, Laboratorio de Parasitologıá, Facultad de Farmacia, Universidad de Salamanca, Avda. Campo Charro, s/n, 37007-Salamanca, Spain; *Present address: Universidad de Pamplona, Km. 1 Vıá Bucaramanga, Pamplona, Norte de Santander, Colombia; †Departamento de Quı´mica Farmaceútica, Universidad de Salamanca, Avda. Campo Charro, s/n, 37007-Salamanca, Spain; §Servicio de Citometrıá, Centro de Investigacioń del Cańcer, Universidad de Salamanca, Avda. Campo Charro, s/n, 37007-Salamanca, Spain; ‡Departamento de Parasitologıá, Facultad de Farmacia, Universidad Complutense de Madrid, Avda de la Complutense, s/n, 28040-Madrid, Spain; ࿣To whom correspondence should be addressed e-mail: [email protected]

ABSTRACT: Currently available candidate vaccines against schistoso- obtained to date have been alarmingly disappointing (Hewitson et al., miasis elicit only partial protection. In addition, the type of immune 2005). As a result, it has been suggested that the identification of new, response that could lead to the highest level of protection against schis- biologically essential parasite proteins may provide useful leads for the tosomes has not yet been described. Thus, efforts should be made in development of improved vaccines (El-Ansary and Al-Daihan, 2005). both the identification of novel proteins essential for the parasite cycle In this regard, 14-3-3 proteins have been extensively characterized in and in the modulation of immune responses against these novel can- different eukaryotes, including parasites. The 14-3-3 proteins were the didates through the combined use of immunomodulatory molecules. first signaling molecules to be identified as discrete phosphoserine-thre- Several parasites have 14-3-3 proteins, and these proteins are known to onine binding modules, and they play critical roles in the cell signaling play a key role in parasite biology. In the present work, we report the events that control progress in the cell cycle, transcriptional alterations isolation and characterization of a new 14-3-3 gene from Schistosoma in response to environmental cues, and apoptosis. The general mecha- bovis and offer new information regarding the genetic structure of the nisms of the action of 14-3-3 proteins include changes in the activity gene. In addition, we have produced the corresponding recombinant of bound ligands, an altered association of bound ligands with other protein. Finally, we describe the immune responses elicited by this pro- cellular components, and changes in the intracellular localization of 14- tein when combined with 4 different immunomodulators in immunized 3-3-bound cargo (Yaffe, 2002). In schistosomes, several 14-3-3 proteins mice. have been isolated and characterized, and their interaction with Raf and their role in the TGF response modulation in adult worms described Control campaigns against schistosomiasis could be substantially im- (McGonigle, Beall, and Pearce, 2002; McGonigle, Loschiavo, and Pearce, proved by applying effective prophylactic tools. Nevertheless, despite 2002). These proteins are present in the adult schistosome tegument, in the efforts made to develop a vaccine against schistosomiasis, the results addition to other sites, and are, therefore, potentially accessible to im- RESEARCH NOTES 965

mune recognition (Schechtman et al., 2001). Thus, they are effectively 72 C for 1 min. The corresponding amplicons were subsequently se- recognized by sera from mice immunized with UV-attenuated cercariae quenced. (Zhang et al., 1999). In addition, the ␨ isoform of 14-3-3 proteins pro- Schistosoma bovis genomic DNA and cDNA were used for the am- duces variable protection levels against infections by Schistosoma man- plification of the parasite’s 14-3-3␨ gene and the corresponding coding soni (25–46%; Schechtman et al., 2001), Schistosoma japonicum (34– region, respectively, using the primers SbGFwd (5Ј-ATGACTACGTC 45%; Zhang et al., 2001), and Echinococcus multilocularis (97%; Siles- GTGGGTTTT) and SbGRev (5Ј-TTAGCCGTCATTTTCAACAT) de- Lucas et al., 2003) in experimental murine models (Siles-Lucas and signed on the S. bovis 14-3-3␨ complete cDNA sequence, situated be- Gottstein, 2003). The suboptimal degrees of protection achieved in the tween nucleotides 1 and 20, and 774 and 759 of this sequence, respec- above-mentioned vaccination experiments against schistosomes could tively, giving a 832-bp and a 759-bp length amplicon for genomic DNA be attributed, among others, to the adjuvants, or immunomodulators and cDNA, respectively. Amplification was performed in 30 cycles of used, or both. 94 C for 40 sec, 52 C for 40 sec, and 72 C for 1 min. In this regard, the dissection of putative immune effector mechanisms All the PCR products obtained were electrophoresed in tris-borate- against schistosomiasis from different vaccine candidates, especially at- EDTA (TBE) 1% agarose gels and visualized with ethidium bromide tenuated cercariae (Hewitson et al., 2005), has shown that the immu- under a UV-transilluminator. The corresponding bands were excised nization of experimental animals with defined recombinant antigens from the gel and purified with the agarose gel DNA extraction kit from schistosomes has been consistently less effective in conferring (Roche), following the manufacturer’s instructions. protection than vaccination with attenuated cercariae, regardless of the The Sb14-3-3␨ gene and cDNA were cloned in the TOPO vector specific antigen under study. These differences in protective efficiency (TOPO Cloning kit, Invitrogen, Barcelona, Spain), following the man- may be the result of quantitative and/or qualitative differences in the ufacturer’s instructions. Cloning products were used for the transfor- immune responses generated by attenuated cercariae and those elicited mation of Escherichia coli DH5␣ competent cells (Invitrogen). Trans- by defined vaccines. Thus, maximal protection against schistosome in- formed bacteria were plated in LB-agarose with 50 ␮g/ml of ampicillin fections in mice seems to be dependent on both antibody and cellular and grown overnight at 37 C. Selected colonies were grown and the mechanisms. Accordingly, it seems crucial to characterize new immu- corresponding recombinant plasmids were extracted using the Nucleo- nomodulators that are able to differentially drive molecule-specific re- spin plasmid kit (Macherey-Nagel). The cloned fragments in the ex- sponses to achieve optimum protection levels. tracted plasmids were automatically sequenced with the universal re- Here, we isolated a new cDNA encoding a 14-3-3 protein from Schis- verse and forward primers. ␨ tosoma bovis. We further isolated and characterized the corresponding The recombinant TOPO vector containing the full S. bovis 14-3-3 gene, generated the corresponding recombinant antigen, and combined cDNA was subjected to digestion with EcoRI (Stratagene, Madrid, it with 4 different immunomodulators to immunize BALB/c mice. Fi- Spain) overnight at 37 C. The smallest band was excised and purified nally, we characterized both the humoral and cellular immune responses from an agarose gel as described above. The purified product was resulting from the 4 different vaccination schedules used during our cloned into the EcoRI-digested pGEX4T1 (Stratagene), using the T4 DNA ligase (Roche), and the product was used for the transformation experiments. Schistosoma bovis is considered an immunological ana- of E. coli DH5␣ competent cells (Invitrogen). Transformed bacteria logue for Schistosoma haematobium. In view of the apparent antigenic were plated on LB-agarose with 50 ␮g/ml ampicillin and grown over- similarities between S. haematobium and S. bovis and the relatively night at 37 C. A selected colony was grown and the corresponding greater ease with which the S. bovis life cycle can be maintained in the recombinant plasmid (pGEXSb14␨) was extracted using the Nucleospin laboratory, the animal parasite may be useful for providing information plasmid kit (Macherey-Nagel). The cloned fragment in the extracted S. hae- for further immunological studies that could be extrapolated to plasmid was automatically sequenced with the pGEX forward primer matobium (Agnew et al., 1989). (Stratagene). Subsequently, the purified pGEXSb14␨ plasmid was used Schistosoma bovis adult worms were obtained from experimentally for the transformation of competent BL21 cells (Stratagene). A selected infected mice as described by Abane et al. (2000). Total RNA from the recombinant colony was grown in LB-ampicillin overnight at 37 C. The parasite was extracted using the RNeasy kit (Quiagen, Valencia, Cali- resulting product was diluted 1:10 in LB and further grown until the fornia) according to the manufacturer’s instructions. The same parasite logarithmic phase was reached. Then, 0.1 mM isopropyl-thiogalactoside source was used to extract total genomic DNA with the kit from Nu- (IPTG; Sigma, St. Louis, Missouri) was added to the culture. Purifica- cleospin Tissue (Macherey-Nagel, Du¨ren, Germany), as recommended tion and thrombin digestion of the corresponding GST-fused protein in by the manufacturers. Adult worms and cercariae were also used as GSH-Sepharose 4B columns (Pfizer International, Madrid, Spain) was sources of somatic antigens. PBS-soluble somatic antigens were ob- completed as described elsewhere (Tan et al., 2005). The resulting retained as described elsewhere (Pardo et al., 2004). combinant Sb14␨ polypeptide had high purity, as revealed by SDS- First-strand cDNA was synthesized from S. bovis adult worms RNA PAGE analysis (Fig. 1), and proved to be free of bacterial-derived en- using the first-strand cDNA synthesis kit (Roche, Barcelona, Spain) or dotoxin, or at least below detectable levels, as demonstrated by the lack the 5Ј-3ЈRACE kit (Boehringer Mannheim, Mannheim, Germany), as of reactivity in the Limulus amebocyte lysate (LAL) assay (Biowhit- recommended by the manufacturers. cDNA was then amplified with 2 taker, Walkersville, Maryland), performed as described elsewhere (Es- degenerate primers: SbFwd1 (5Ј-GTGCTCGAAGATCAGCMTGG) pinoza et al., 2002). and SbRev1 (5Ј-CAAGYCGAATWGGATGAGTG). The consensus let- Both the assembly of cDNA sequences obtained by degenerate PCR ters used to indicate degenerate positions in the respective primer se- and 5Ј-3ЈRACE and the sequence obtained with the primers SbGFwd quences are those recommended by the International Union of Pure and and SbGRev on S. bovis cDNA resulted in an identical sequence of 836 Applied Chemistry and the International Union of Biochemistry bp, displaying the start and stop codons as well as a 3Ј-untranslated (IUPAC/IUB). These primers were designed on the consensus sequence region of 80 bp, including a tail of 16 adenine residues (Fig. 1). The of the alignment performed with the Multalign program (at the Institut Sb14-3-3␨ cDNA sequence comprised a continuous open reading frame National de la Recherche Agronomique [INRA], Toulouse, France; of 252 amino acids (Fig. 1). Its 14-3-3 identity was confirmed through http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html; Corpet, the CD-search program (Marchler-Bauer and Bryant, 2004), which as- 1988) of the 14-3-3␨ mRNA sequences from S. mansoni and S. japon- signed the Sb14-3-3␨ protein to this family owing to the presence of all icum (GenBank accession numbers U24281 and AF000369, respective- the very conserved ‘‘14-3-3’’ residues in specific positions (Fig. 1). ly) and were located on the most conserved portions of the above se- Comparison of this protein with homologous sequences in S. mansoni quences. PCR was performed in 35 cycles of 94 C for 40 sec, 46 C for (AAC46893.1) and S. japonicum (AAW24859.1) using the facilities at 40 sec, and 72 C for 1 min for the 5 first cycles, and 94 C for 40 sec, www.expasy.org revealed high similarities between the 3 proteins as 48 C for 40 sec, and 72 C for 1 min for the remaining 30 cycles. regards the percentage of alpha-helix structure (63%, 61%, and 62%), Subsequently, the sequence obtained was used to design 2 S. bovis– isoelectric point (4.72, 4.28, and 4.86), and theoretical molecular weight specific primers: SbFwd2 (5Ј-GTGACTACTACCGGTATCTG) and (28,337, 28,530, and 28,997 Daltons, respectively). Thus, the putative SbRev2 (5Ј-CGGTACTCTTCTCATGGC). These were used for the 3Ј Sb14␨ protein exhibited all the other 14-3-3 characteristics, i.e., more and 5ЈRACE PCR on the S. bovis cDNA, in combination with the than 50% alpha-helix structure, an acidic isoelectric point, and a theo- 3ЈRACE and the 5ЈRACE anchor primers, respectively. Both PCRs retical molecular weight of 28 kDa (Aitken et al., 1992). were performed in 35 cycles of 94 C for 40 sec, 48 C for 40 sec, and Additionally, the 3Ј-untranslated sequence from the Sb14␨ cDNA was 966 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

FIGURE 2. Physical structure of the 14-3-3␨ genes from (a) Schis- tosoma bovis; (b) Echinococcus multilocularis, AF529420; and (c) E. granulosus, DQ631896. Boxes represent exons and lines represent introns. Letters and numbers above boxes indicate amino acids and respective positions at the beginning and the end of each exon. Numbers below lines indicate intron size. Bp, base pairs.

FIGURE 1. (a) Nucleotide sequence of the Schistosoma bovis 14-3- ␨ Ј 3 gene, including 80 terminal base pairs (bp) representing the 3 -un- E. multilocularis and E. granulosus 14-3-3␨ genes, high sequence ho- translated sequence from the corresponding cDNA (italics), terminated mology was found; this proved to be lower for the acceptor regions by a polyA tail of 16 residues (italics and bold). The genomic sequence (Table I). A typical (TC)nNCTAG/G acceptor splice junction consensus includes an open reading frame of 832 bp with start and stop codons sequence (Mount, 1982) was found in all 3 of the sequences studied (upper case), interrupted by 2 introns (underlined) of 35 (intron I) and (Table I); 75% to 67% identity was found between donor and acceptor 38 (intron II) bp. The deduced amino acid (aa) sequence is represented sites from S. bovis, E. multilocularis, and E. granulosus 14-3-3␨ genes, under the corresponding coding regions in the figure. Those aa found while the overall identity in the 2 intron sequences from these 3 genes to be conserved in a 14-3-3 consensus sequence provided by the CD- varied from 60% to 51% (Table I). Thus, the matching intron number Search program are represented in bold. (b) Coomassie-blue polyacryl- and position in the above-mentioned 14-3-3␨ genes, together with a ␨ amide stained gel showing the (A) GST-Sb14 fusion protein and (B) moderate degree of conservation of sequences, indicates that these ␨ the Sb14 recombinant protein after thrombin cleavage, marked with genes probably evolved from a single ancestor gene. ␨ arrows. (c) Immunoblot showing the reactivity of sera from Sb14 -CpG- As mentioned, it has been reported that 14-3-3 proteins from S. man- immunized animals against a soluble extract from S. bovis (C) adult soni and S. japonicum produce suboptimal levels of protection against ␨ worms and (D) cercariae. The position of the Sb14 native antigen is the respective infections in an experimental murine model, i.e., from marked with an arrow. Relative molecular weights are indicated at the 25% to 46% (Siles-Lucas and Gottstein, 2003). Similar results have left of the figure in kilodaltons (kDa). The same results were obtained been obtained with other univalent vaccines against schistosomiasis by ␨ using the sera from Sb14 -AA0029, AA2829, and PAL-immunized different authors (Hewitson et al., 2005). Since protection mechanisms mice (data not shown). Mk, molecular weight markers (molecular against schistosomes depend on both humoral and cellular immune re- weight standard mixture, Sigma). sponses, the use of a defined vaccine candidate in combination with different immunomodulators and the characterization of both the humoral and cellular immune responses elicited by those combinations aligned and compared with the corresponding cDNAs from S. mansoni seem to be essential before ruling out the usefulness of defined mole- and S. japonicum (GenBank accession numbers U24281 and AF000369, cules against schistosomes. Our vaccine formulation includes 4 com- respectively). Both the polyA tail and the whole 3Ј-untranslated cDNA ponents, i.e., the vehicle (Montanide௡ ISA763A -SEPPIC, Paris, France, sequence in Sb14␨ were longer than in the corresponding sequences plus water in a 70/30 emulsion), the adjuvant (saponin from Q. sapon- from S. mansoni and S. japonicum. The overall identity in this sequence aria, QuilA; Sigma), 1 immunomodulator (PAL, AA0029, or AA2829), region was 77.5% and 65% with the S. mansoni and S. japonicum coun- and the recombinant Sb14z protein. This formulation, previously as- terparts, respectively. No evidence of a typical polyadenylation signal sayed by other authors (Martıńez-Fernańdez et al., 2004), is called was found in any of the 3 above-mentioned sequences, although Beau- ADAD (Adaptation ϩ Adjuvant) and is administered in a set of 2 sub- doing et al. (2000) explored alternative variations in human polyade- cutaneous injections. The first, called ‘‘adaptation,’’ contains all the nylation signals, ‘‘ATTAAA’’ being one of the new candidates to signal components except the recombinant protein. The second injection, ad- the onset of polyadenylation. This could also be used by schistosomes, ministered 5 days after the adaptation, contains all the vaccine com- since a similar stretch, i.e., an ‘‘ATTATA’’ motif, was found 42 bases ponents. The first injection should stimulate and ‘‘guide’’ the immune upstream from the Sb14-3-3␨ polyA sequence. system, preparing it for subsequent stimulus with parasite-specific an- PCR with primers Sb14GFwd and Sb14GRev in the S. bovis genomic tigens (Martinez-Fernandez et al., 2004). A fourth immunomodulator, DNA, together with the 3Ј-untranslated region found on the S. bovis CpG ODN 1826, was used with the same vaccine formulation, but cDNA, produced a 909-bp product (Fig. 1). The sequence had initiation administration of the ‘‘adaptation’’ was not done (Kumar et al., 2004). (ATG) and stop (TAA) codons, comprising a coding sequence of 832 CpG ODN 1826 should activate an innate immune response, leading to bp, interrupted by 2 introns of 35 (intron I) and 38 (intron II) bp, re- the nonspecific secretion of Th1 type cytokines, i.e., IFN-␥ and IL-12 spectively. Exon-intron boundaries lay outside functional domains and (Kumar et al., 2004). It has been postulated that IFN-␥ production is were found to be conventional. Comparison of the Sb14␨ gene structure needed to achieve protective responses against both cercariae and schis- with the available 14-3-3␨ genes from Echinococcus multilocularis and tosomulae (Hewitson et al., 2005). PAL, consisting of an extract from Echinococcus granulosus, representing the uniquely available 14-3-3␨ the plant Polypodium leucotomos, has been shown to down-regulate the gene sequences from related organisms in the GenBank (Fig. 2), re- Th2-like response induced by parasitic infections, such as Trichinella vealed the same intron number in the 3 sequences. None of the intron spiralis (Dea-Ayuela et al., 1999) and Trichomonas vaginalis (Nogal- positions relative to the open reading frame of the corresponding pro- Ruiz et al., 2003). Similarly, its use in an antischistosome vaccine is teins interrupted any codon and was identical in the 3 genes under study justified because Th2-type dominant responses have been related to a (Fig. 2). The lengths of both introns were shorter in S. bovis than in E. lack of protection against these parasites (Hewitson et al., 2005). Fi- multilocularis and E. granulosus. Comparative analysis of donor sites nally, both the diamine AA0029 and the aminoalcohol AA2829, de- in both introns revealed the presence of sequences that did not match scribed as synthetic derivates from the immunomodulatory compound the ‘‘MAG/gt’’ consensus shown by several eukaryotes (Brown, 1986). myriocin, have been tested previously for their immunomodulatory On comparing the donor sites found in the present work with those in properties (Olmo et al., 2006), and they have been shown to increase RESEARCH NOTES 967

TABLE I. Splice junction sequences, intron lengths, position, and identity percentage in 14-3-3␨ genes from 3 different platyhelminths.

Identity numbers represent the percentage of identity between introns from S. bovis and introns from E. multilocularis/E. granulosus, respectively. The consensus sequence for the donor and acceptor sites in each intron is indicated. Coding sequences in both introns are represented in uppercase, and noncoding regions are indicated in lowercase. GenBank accession numbers for E. multilocularis and E. granulosus sequences are AF529420 and DQ631896, respectively.

T helper CD4ϩ and CD8ϩ cell numbers and nitric oxide (NO) pro- 5Ј-TCCATGACGTTCCTGACGTT; TIB Molbiol, Berlin, Germany) for duction from mouse immunocompetent cells after in vitro stimulation group 1; 600 ␮g PAL for group 2; 100 ␮g AA0029 for group 3; and (Olmo et al., 2006). To date, nothing is known about the kind of cy- 100 ␮g AA2829 for group 4, in Montanide (70% in aqueous solution; tokine pattern stimulated by these 2 molecules, although their use in a Seppic, Paris, France). A control group of 10 mice was not vaccinated. vaccine against schistosomes could elicit TCD4ϩ and CD8ϩ cell re- The second immunization was performed 2 wk after the ﬁrst. Each sponses as well as the production of NO, immune effectors that have immunization was preceded by preimmunization with all the vaccine been shown to potentially exert deleterious effects on schistosomulae components (excluding the recombinant protein), except for group 1. (Hewitson et al., 2005). Thus, the 4 immunomodulators should afford The doses of the immunomodulators were chosen based on previous immune responses that could play a role in the protection mechanisms work carried out by other authors (Kumar et al., 2004; Martinez-Fer- against schistosomes (Ganley-Leal et al., 2005; Hewitson et al., 2005; nandez et al., 2004; Olmo et al., 2006). The dose of recombinant protein Tran et al., 2006). was sufﬁcient to elicit strong immune responses (see below). Blood was We decided to inject the vaccine subcutaneously because this vacci- taken from each animal on days 0 and 28. Animals were killed on day nation route induces T-cell–mediated immune resistance mechanisms in 28, and spleen cells were recovered. mice that involve mast cells, macrophages, and dendritic cells (James Sera from vaccinated mice recognized a single band with an apparent and Pearce, 1988). In addition, employing similar vaccine components molecular weight of 26 kDa, thus corresponding to the native Sb14-3- this vaccination route has been successfully used against Fasciola he- 3␨ protein, in soluble protein extracts from both adult worms and from patica in mice (Martinez-Fernandez et al., 2004). cercariae separated by SDS-PAGE in 12% acrylamide gels, as demon- For the immunization experiments, 7-wk-old female BALB/c mice strated by immunoblot using the luminol-based ECL detection reagents were used (Criffa S.A., Barcelona, Spain). The animals were maintained (Amersham) described elsewhere (Rodrı´guez-Barbero et al., 2006; Fig. under standard conditions, in an environment with controlled tempera- 1). This suggests that the Sb14␨ protein could be accessible to the im- ture and humidity, a 12-hr light/dark cycle, and free access to water and mune responses against both S. bovis cercariae and adult worms, as food. Fifty mice were vaccinated subcutaneously in groups of 10 ani- demonstrated for the 14-3-3␨ protein from other schistosome species mals with 2 doses of 10 ␮g Sb14z recombinant protein and 20 ␮g QuilA (Zhang et al., 1999; Schechtman et al., 2001). (Sigma) plus 25 ␮g CpG-containing polynucleotide (CpG ODN 1826; For antibody measurement in serum, the ELISA technique was performed basically as described elsewhere (Siles-Lucas et al., 1998). Brief- ly, 96-well polystyrene plates (Costar, New York) were coated with 0.2 ␮g Sb14␨. Serum was then added at 1:100 dilution, followed by the addition of peroxidase-labeled anti-mouse IgG, IgG1, or IgG2a antibodies at a 1:2,000 dilution (Sigma). The reaction was developed with

H2O2 and ortophenylenediamine (Sigma) and measured at 492 nm in an Ear400FT ELISA device (STL Lab Instruments, Grodig, Austria). In comparison with nonvaccinated animals, immunized mice displayed a strong humoral response against the recombinant Sb14␨ protein, with balanced IgG1/IgG2a levels, regardless of the immunomodulator used for immunization, with slightly lower antibody levels in animals receiving PAL (Fig. 3). Cell cytokine production (TNF␣, IFN-␥, IL-2, IL-4, and IL-5) was measured after in vitro stimulation of blood or spleen cells (pooled for FIGURE 3. Antibody responses of (1) BALB/c mice nonimmunized each group; 1.5 ϫ 105 cells/well), prepared as described in Bosze et al. (control group) and mice immunized with the Sb14␨ recombinant pro- (2004), with 2.5 ␮g of Sb14␨ for 6 hr at 37 C in a FACSCalibur cy- tein, plus the immunomodulators (2) AA0029, (3) AA2829, (4) CpG, tometer (BDB) using the kit BA mouse Th1/Th2 cytokine (BD) and and (5) PAL against the recombinant Sb14␨ protein. Bars represent the following the manufacturer’s instructions. We found high TNF␣ pro- mean of the absorbances from all animals in the same group Ϯ standard duction in both blood and spleen in the animals immunized with CpG error. IgG (open bars), IgG1 (gray bars), IgG2a (striped bars). OD, op- plus Sb14␨ (2,502.9 and 104.1 pg/ml from blood and spleen cells, retical density measured at 495 nm. spectively). This production was moderate in blood cells from both 968 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

AA2829- and PAL-immunized mice (74.4 and 117.2 pg/ml, respective- ESPINOZA, E., A. MURO,M.M.MARTIŃ,P.CASANUEVA, AND J. L. PEREZ- ly), and low in the spleens of animals receiving PAL and AA0029 (18.4 ARELLANO. 2002. Toxocara canis antigens stimulate the production and 12.2 pg/ml, respectively). In these latter 2 groups, none of the other of nitric oxide and prostaglandin E2 by rat alveolar macrophages. cytokines (IFN-␥, IL-2, IL-5, and IL-4) studied was detectable either in Parasite Immunology 24: 311–319. blood or in spleen cells. In contrast, CpG-immunized mice displayed GANLEY-LEAL, L. M., J. GUARNER,C.W.TODD,A.A.DA’DARA,G.L. low levels of IFN-␥ in spleen cells (2.3 pg/ml), and AA2829-vaccinated FREEMAN,A.E.BOYER,D.A.HARN, AND W. E. SECOR. 2005. Com- mice showed low levels of both IL-2 and IL-4 in blood cells (4.1 and parison of Schistosoma mansoni irradiated cercariae and Sm23 5.4 pg/ml, respectively). DNA vaccines. Parasite Immunology 27: 341–349. As expected, the 4 immunomodulators used during our experiments HEWITSON, J. P., P. A. HAMBLIN, AND A. P. MOUNTFORD. 2005. Immunity triggered TNF␣ production in immunized animals owing to the rela- induced by the radiation-attenuated schistosome vaccine. Parasite tionship between Th1-type responses and nitric oxide and TNF␣ pro- Immunology 27: 271–280. duction (Lezama-Davila and Isaac-Marquez, 2006). This is important, JAMES, S. L., AND E. J. PEARCE. 1988. The influence of adjuvant on since it is known that TNF␣ induction is essential to protection follow- induction of protective immunity by a non-living vaccine against ing the arrival of challenge larvae in the lungs of mice vaccinated with schistosomiasis. Journal of Immunology 140: 2753–2759. UV-attenuated cercariae (Wilson et al., 1999). Similarly, IFN-␥, IL-2, KUMAR, S., T. R. JONES,M.S.OAKLEY,H.ZHENG,S.P.KUPPUSAMY,A. or both, triggered by the CpG-Sb14␨ and AA2829 immunizations, have TAYE,A.M.KRIEG,A.W.STOWERS,D.C.KASLOW, AND S. L. been related to the expansion of Th1 cells in mice protected after vac- HOFFMAN. 2004. CpG oligodeoxynucleotide and Montanide ISA 51 cination with attenuated cercariae (Wilson et al., 1999). In sum, our adjuvant combination enhanced the protective efficacy of a subunit results show that the immune responses elicited after immunization, and malaria vaccine. Infection and Immunity 72: 949–957. specifically those linked to cell-mediated immunity, largely depend on LEZAMA-DAVILA,C.M.,AND A. P. ISAAC-MARQUEZ. 2006. Systemic cy- the immunomodulator used for vaccination. Thus, while a robust and tokine response in humans with chiclero’s ulcers. Parasitology Re- similar antibody response was obtained with the Sb14␨ regardless of search 99: 546–553. the immunomodulator used in combination with the protein, cytokine MARCHLER-BAUER,A.,AND S. H. BRYANT. 2004. CD-Search: Protein responses were different in the 4 immunized groups. In addition, trig- domain annotations on the fly. Nucleic Acids Research 32: W327– gering of the above-mentioned specific cytokines could facilitate opti- 331. mal activation of the innate immune responses needed to achieve high MARTINEZ-FERNANDEZ, A. R., J. J. NOGAL-RUIZ,J.LOPEZ-ABAN,V.RA- protection levels against schistosomes. In this regard, future work will MAJO,A.OLEAGA,Y.MANGA-GONZALEZ,G.V.HILLYER, AND A. focus on protection levels against S. bovis in vaccinated and infected MURO. 2004. Vaccination of mice and sheep with Fh12 FABP from mice using the 4 vaccine formulations described here. Fasciola hepatica using the new adjuvant/immunomodulator sys- M.S.L. acknowledges financial support from the Ministerio de Edu- tem ADAD. Veterinary Parasitology 126: 287–298. cacioń y Ciencia, Spain (‘‘Ramoń y Cajal’’ contract and project MCGONIGLE, S., M. J. BEALL, AND E. J. PEARCE. 2002. Eukaryotic ini- AGL2002-00644). N.U. acknowledges financial support from the Car- tiation factor 2 alpha subunit associates with TGF beta receptors olina Foundation (Spain). This work was supported by the Red de In- and 14-3-3 epsilon and acts as a modulator of the TGF beta re- vestigacioń de Centros en Enfermedades Tropicales (RICET, FIS, Spain) sponse. Biochemistry 41: 579–587. and the MEC (AGL2005-02168GAN). Thanks are given to A. Rodrı´- ———, M. LOSCHIAVO, AND E. J. PEARCE. 2002. 14-3-3 proteins in guez-Barbero for her help in the immunoblot assays. The nucleotide Schistosoma mansoni: Identification of a second epsilon isoform. sequence data reported in this paper are available in the GenBank, International Journal for Parasitology 32: 685–693. EMBL, and DDBJ databases under accession numbers DQ176433 and MOUNT, S. M. 1982. A catalogue of splice junction sequences. Nucleic AY615728. Acids Research 10: 459–472. NOGAL-RUIZ, J. J., A. GOMEZ-BARRIO,J.A.ESCARIO, AND A. R. MAR- TINEZ-FERNANDEZ. 2003. Effect of Anapsos in a murine model of LITERATURE CITED experimental trichomoniasis. Parasite 10: 303–308. ABANE, J. L., A. OLEAGA,V.RAMAJO,P.CASANUEVA,J.L.PE´ REZ-AR- OLMO, E. D., A. PLAZA,A.MURO,A.R.MARTINEZ-FERNANDEZ,J.J. ELLANO,G.V.HILLYER, AND A. MURO. 2000. Vaccination of mice NOGAL-RUIZ,J.L.LOPEZ-PEREZ, AND A. S. FELICIANO. 2006. Syn- against Schistosoma bovis with a recombinant fatty acid binding thesis and evaluation of some lipidic aminoalcohols and diamines protein from Fasciola hepatica. Veterinary Parasitology 91: 33–42. as immunomodulators. Bioorganic and Medicinal Chemistry Let- AGNEW,A.M.,H.M.MURARE,S.B.LUCAS, AND M. J. DOENHOFF. ters doi:10.1016/j.bmcl.2006.08. 113. 1989. Schistosoma bovis as an immunological analogue of S. hae- PARDO, J., C. CARRANZA,M.C.TURRIENTES,J.L.PEREZ ARELLANO,R. matobium. Parasite Immunology 11: 329–340. LOPEZ VELEZ,V.RAMAJO, AND A. MURO. 2004. Utility of Schisto- AITKEN, A., D. B. COLLINGE,B.P.VAN HEUSDEN,T.ISOBE,P.H.RO- soma bovis adult worm antigens for diagnosis of human schisto- SEBOOM,G.ROSENFELD, AND J. SOLL. 1992. 14-3-3 proteins: A high- somiasis by enzyme-linked immunosorbent assay and electroim- ly conserved, widespread family of eukaryotic proteins. Trends in munotransfer blot techniques. Clinical and Diagnostic Laboratory Biochemical Science 17: 498–501. Immunology 11: 1165–1170. BEAUDOING, E., S. FREIER,J.R.WYATT,J.M.CLAVERIE, AND D. GAUTH- RODRIGUEZ-BARBERO, A., J. OBREO,P.ALVAREZ-MUNOZ,A.PANDIELLA, ERET. 2000. Patterns of variant polyadenylation signal usage in hu- C. BERNABEU, AND J. M. LOPEZ-NOVOA. 2006. Endoglin modulation man genes. Genome Research 10: 1001–1010. of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 BOSZE, S., N. CACCAMO,Z.MAJER,G.MEZO,F.DIELI, AND F. H UDECZ. MAPK activation. Cellular Physiology and Biochemistry 18: 135– 2004. In vitro T-cell immunogenicity of oligopeptides derived from 142. the region 92-110 of the 16-kDa protein of Mycobacterium tuber- SCHECHTMAN, D., R. TARRAB-HAZDAI, AND R. ARNON. 2001. The 14-3- culosis. Biopolymers 76: 467–476. 3 protein as a vaccine candidate against schistosomiasis. Parasite BROWN, J. W. 1986. A catalogue of splice junction and putative branch Immunology 23: 213–217. point sequences from plant introns. Nucleic Acids Research 14: SILES-LUCAS, M., R. S. FELLEISEN,A.HEMPHILL,W.WILSON, AND B. 9549–9559. GOTTSTEIN. 1998. Stage-specific expression of the 14-3-3 gene in CORPET, F. 1988. Multiple sequence alignment with hierarchical clus- Echinococcus multilocularis. Molecular and Biochemical Parasi- tering. Nucleic Acids Research 16: 10881–10890. tology 91: 281–293. DEA-AYUELA, M., M. RODERO,R.RODRIGUEZ-BUENO,F.BOLA´ S-FERNA´ N- ———, AND B. GOTTSTEIN. 2003. The 14-3-3 protein: A key molecule DEZ, AND A. R. MARTIŃEZ-FERNA´ NDEZ. 1999. Modulation by An- in parasites as in other organisms. Trends in Parasitology 19: 575– apsos (Polypodium leucotomos extract) of the antibody responses 581. against the nematode parasite Trichinella spiralis. Phytotherapy ———, M. MERLI,U.MACKENSTEDT, AND B. GOTTSTEIN. 2003. The Research 13: 566–570. Echinococcus multilocularis 14-3-3 protein protects mice against EL-ANSARY,A.,AND S. AL-DAIHAN. 2005. Stage-specifically expressed primary but not secondary alveolar echinococcosis. Vaccine 21: schistosome proteins as potential chemotherapeutic targets. Medical 431–439. Science Monitor 11: RA94–103. TAN, H., G. DAN,H.GONG, AND L. CAO. 2005. Purification and char- RESEARCH NOTES 969

acterization of recombinant truncated human interleukin-11 ex- YAFFE, M. B. 2002. How do 14-3-3 proteins work? Gatekeeper phos- pressed as fusion protein in Escherichia coli. Biotechnological Let- phorylation and the molecular anvil hypothesis. FEBS Letters 513: ters 27: 905–910. 53–57. TRAN, M. H., M. S. PEARSON,J.M.BETHONY,D.J.SMYTH,M.K.JONES, ZHANG, Y., M. G. TAYLOR,M.V.JOHANSEN, AND Q. D. BICKLE. 2001. M. DUKE,T.A.DON,D.P.MCMANUS,R.CORREA-OLIVEIRA, AND Vaccination of mice with a cocktail DNA vaccine induces a Th1- A. LOUKAS. 2006. Tetraspanins on the surface of Schistosoma man- type immune response and partial protection against Schistosoma soni are protective antigens against schistosomiasis. Nature Medi- cine 12: 835–840. japonicum infection. Vaccine 20: 724–730. WILSON, R. A., P. S. COULSON, AND A. P. MOUNTFORD. 1999. Immune ———, ———, M. V. MCCROSSAN, AND Q. D. BICKLE. 1999. Molec- responses to the radiation-attenuated schistosome vaccine: What ular cloning and characterization of a novel Schistosoma japonicum can we learn from knock-out mice? Immunological Letters 65: ‘‘irradiated vaccine-speciﬁc’’ antigen, Sj14-3-3. Molecular and Bio- 117–123. chemical Parasitology 103: 25–34. CRITICAL COMMENT...

J. Parasitol., 93(4), 2007, pp. 970–972 ᭧ American Society of Parasitologists 2007

From the Roots of Parasitology: Hippocrates’ First Scientiﬁc Observations in Helminthology

Constantinos Trompoukis, Vasilios German*, and Matthew E. Falagas†‡, Department of History of Medicine, Faculty of Medicine, University of Crete, P.O. Box 2208, 71003, Heraklion Greece; *Internal Medicine Department, 401 General Army Hospital, 2 Avlonos Street, 16673, Athens, Greece; †Department of Medicine, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02110; and ‡Alfa Institute of Biomedical Sciences (AIBS), 9 Neopoleos Street, 15123 Marousi, Greece. e-mail: [email protected]

ABSTRACT: Although knowledge of intestinal parasites predates Hip- ciples of Hippocratic medicine, as outlined above, which form the ori- pocrates, the Hippocratic Corpus provides the first scientific observa- gins of contemporary Western medicine. tions about the clinical perception and treatment of helminthic diseases. A study of the Hippocratic Corpus, based on the Littre´ edition, was These observations follow the scientific principles of Hippocrates, the made; parts referring to the classification of intestinal parasites, regu- father of modern medicine, who relied on knowledge and observation. larity and period of appearance, and/or patient age, symptoms, and treat- This article is based on a systematic study of the Hippocratic texts, and ment were selected for intense examination. The greatest number of presents observations on diseases caused by intestinal parasites with references to intestinal parasites were concentrated in the following respect to regularity of appearance, patient age, symptoms, and treat- books of the Hippocratic Corpus: Epidemics, the 7 books that have been ment. Three types of helminths are described: ‘‘helmins strongyle’’ considered by some to comprise ‘‘the great medical classics of world (roundworm), ‘‘helmins plateia’’ (flatworm), and ‘‘ascaris’’ (which cor- literature’’ (Sigerist, 1987); the Prognostics, in which valuable details responds to Enterobius vermicularis). Helminthic diseases primarily ap- regarding Hippocratic knowledge of the symptoms of diseases and their pear during childhood, well after teething. The described systemic prognostic value are provided; and the Coan Prognoses, a collection of symptoms include weakness, sickness, discomfort, tiredness, anorexia, 600 brief aphoristic prognoses, most probably the summaries of various and emotional instability; gastrointestinal symptoms include change in books and collections (Sigerist, 1987). We performed a systematic study bowel movements, vomiting, and colic pain in the epigastrium. We and comparative examination of these sources. identified several accounts of cases of helminthic diseases in the Hip- The age at which helminthic diseases usually appear in humans, pocratic texts. Of particular interest are the descriptions of a helminth based on the Hippocratic observations, is determined to be during child- emerging from a fistula in the navel region and the surgical treatment hood, substantially after teething. ‘‘At the approach of dentition, pruritus of helminthic diseases, reinforced by being described on a dedicatory of the gums, fevers, convulsions, diarrhea, especially when cutting the inscription at the Asclepion in Epidaurus. We finally encountered the canine teeth . . . To persons somewhat older, affections of the tonsils, use of powerful purgatives as antihelminthics, which have been widely incurvation of the spine at the vertebra next the occiput, asthma, cal- used, even into the 21st century. culus, round worms [helminths], ascarides, acrochordon, satyriasmus, struma, and other tubercles [phymata], but especially the aforesaid’’ Hippocrates (460–377 BC), the great physician of antiquity, has been (Aphorisms 3, 25–26) (Hippocrates, Littre´, 1839). This description is called the father of medicine. For contemporary Western medicine, Hip- given without excluding the appearance of the disease during adoles- pocrates probably represents the ideal doctor. He embodies all the vir- cence and later ages. The early age is, however, given as the starting tues of the clinical physician, with rational thought and observation, a point, as children did not leave the house prior to teething, and certainly correct approach for the management of clinical problems, and impec- not on their own, so were unlikely to come into contact with the natural cable ethical behavior. causes of the disease. Hippocrates was born in Kos, one of the Dodecanese islands in the As for the season of appearance of diseases due to intestinal parasites, Aegean Sea, in 460 BC, and died in 377 BC in Larissa, a town in according to Hippocrates, they are more prevalent in the autumn. There central mainland Greece. His father, Heraklides, was a physician from are several references to the increased frequency of intestinal parasites a medical family, and taught him medicine. Hippocrates himself, like in the autumn (Epidemics 1.3). Moreover, most of Hippocrates’ clinical other successful physicians of his era, was a traveling physician. Al- descriptions of helminthic diseases date to the autumn (Epidemics 2.3). though we do not know many details about the life of this great phy- Indeed, opinions are also given as to which hour of the day the symp- sician, his methods of thinking were included in the most complete way toms are most pronounced. It is noted that evening fits could be due to in the writings known as the Hippocratic Corpus. The term Hippocratic ascarides (Epidemics 6.11). It is observed, however, that although as- Corpus includes all the works that were collected in the 3rd century carides irritate in the evening, they become more annoying during the BC in the library of Alexandria, Egypt, which contained the medical day, not simply because one becomes tired throughout the day, but thinking of the classical era. These ideas adhered to the following 4 because this is also their nature (Epidemics 2.3). basic principles: (1) etiology: every disease can be attributed to a natural As for locating the helminths, the Hippocratic Corpus contains inter- cause, i.e., the disease is differentiated from every supernatural cause esting relevant information. Roundworms and tapeworms are located in and thus, its natural cause should be sought; (2) diagnosis: the diag- the gastrointestinal tract, and ascarides are more often located in the nostic approach to the patient is based upon clinical examination, which rectum, whereas in women it is also mentioned that they may be located involves careful observation founded on medical history, inspection, in the genitals (Diseases of Women 2.187). Many references to the palpation, auscultation, and percussion (Smith, 1979); (3) prognosis: it symptoms of helminthic diseases can be also found in the Hippocratic is a main tool of the physician; the good physician should be able to Corpus. They relate to general disturbances, gastrointestinal manifes- estimate the evolution of a disease through the clinical findings that he tations, and symptoms of the cardiovascular system, the joints, and the observes according to his medical knowledge; and (4) treatment: a ho- nervous system. listic approach to illness and the patient is advocated; the patient, as a It is noteworthy that in the description of the case of the wife of psychosomatic whole, is located at the center of the physician’s treat- Eumenes, included in Book 4 of the Epidemics, many of the clinical ment approach, and thus, the Hippocratic doctor treats the specific pa- aspects of intestinal parasitic infections are mentioned, i.e., vomiting, a tient and not the illness. characteristic odor, fever with shivers, heartburn, vomiting bile with Knowledge of intestinal parasites can be traced back to much earlier small worms, infrequent bowel movements, loss of appetite, weakness, than the Greek written sources, most specifically to the Ebers Papyrus, and a general unpleasantness (Epidemics 4.16). dated to around 1550 BC. Here, there are references to specific types An account of the case of Thersander’s wife is given in the same of intestinal worms and descriptions of antihelminthic prescriptions book: ‘‘Thersander’s wife, slightly leucophlegmatic, was nursing; she (Bryan and Joachim, 1930). Nevertheless, the Hippocratic Corpus pro- developed acute fever. Her tongue was burned, and she burned all over. vides systematic knowledge that deserves further discussion, especially At that time, her tongue grew quite rough, as though it was covered in when this knowledge is examined through the prism of the main prin- small stones like hail, and worms came out of her mouth’’ (Epidemics

970 CRITICAL COMMENT 971

4.10). In cases of diarrhea due to worms, colic and swelling of the are distinguished: (1) the ‘‘helmins strongyle’’ (␧´␭␮␫␯␵ ␴␶␳␱␥␥␭␩) stomach are also noted (Coan Prognoses 24.459). meaning ‘‘round worm,’’ i.e., Ascaris lumbricoides (Prognostics 11, Symptoms suggestive of a psychological disorder are noted in the Aphorisms 26, Coan Prognosis 589); (2) ‘‘helmins plateia’’ (␧´␭␮␫␯␵ case of a woman who ‘‘had some mania, but when she passed a tape- ␲␭␣␶␧␫´␣) meaning ‘‘flat worm,’’ i.e., the Taenia sp. (Epidemics 4.55); worm that was rather thick and a small amount of feces, her trouble and (3) the ascaris (␣␴␬␣␳␫´␵ ) meaning ‘‘worm,’’ i.e., the Enterobius immediately stopped, she fell asleep and became healthy’’ (Epidemics vermicularis (Epidemics 6.11) (Sigerist, 1987; Grove, 1990). Of the last 4.55). In another description, ‘‘A headache with pain in the seat and category, Galen explains that: ‘‘Ascarides are helminths that are dry the genital organs causes catalepsy and weakness and paralysis of the and small and develop in the rectum.’’ The fact that Galen refers to this voice. These incidents are not unpleasant but the ill are overcome by term in his book Explanation of Hippocrates’ Glossary (Galen et al., sleepiness and hiccups. In the ninth month, after the return of their 1821) reinforces the view that the term was first used by Hippocrates. voices, they are restored to their original condition, after having emitted Through the study of the references to helminthic diseases that have ascarides’’ (Coan Prognoses 4.160). been identified in the Hippocratic Corpus, we can ascertain that there Phenomena of allergic reactions are described in detail in cases of is a systematic description of the abdominal, general, and neurological diarrhea due to intestinal worms: ‘‘when the lienteries disappear with symptoms of helminthic diseases. In addition, the repeated observation colic, they are an advance warning of a swelling of the joints. In this of the increased frequency of illness caused by intestinal parasites dur- case, deep red scales with blisters appear, and these patients turn deep ing the autumn is probably related to the consumption of rotting fruit purple, as though they have been whipped’’ (Coan Prognoses 24.458). and vegetables and to children’s contact with the earth. Elimination of helminths is warned of in advance by heart pains and It is worth noting that the Hippocratic Corpus includes most of the colic (Coan Prognoses 279). symptoms attributed to helminthic infections. For the ‘‘ascarids’’ in In addition to observing the above symptoms, Hippocrates believes particular, which correspond to Enterobius vermicularis, it is significant that clinical examination is important in establishing the diagnosis of a that Hippocrates observed that they may be located in the genital or- helminthic infection (Epidemics 4.56). Of particular interest is the de- gans, as a consequence of the mistaken nocturnal migration of the par- scription of the child of Deinias of Abdera: ‘‘when a medium-sized asite toward the neighboring openings of the urinary system (Deshpan- incision was made in his navel, a fistula was left behind. Then a thick de, 1992; Guiot et al., 1998; Erhan et al., 2000). helminth came out from there, and he said that from then on whenever In the case of the son of Deinias of Abdera, an intestinal parasite he had a fever, bilious material would exit from that opening. The in- exited from a prior incision near the navel. It is not mentioned whether testine had fallen into that fistula, and it had eroded as had the fistula, this incision was the result of an earlier operation to remove an intestinal it would tear, and coughing would not let it stay in its place’’ (Epidemics parasite. It is probable that this account refers to an exceptionally rare 7.117). instance of the location of a helminth in the navel (Surendran et al., Advice for treatment of helminthic infections with medicines is also 1988). Even so, the description of the prolapse and erosion of the in- found in the Hippocratic Corpus: ‘‘Then, having bruised the root of testine corresponds to an enteric intramural abscess caused by a parasite seseli [parsley] to a very fine powder, and poured in some water, let it (Mukhopadhyay et al., 2001). It is not, however, impossible that there macerate for four days, and, mixing the water with honey, let the patient were attempts to remove helminths surgically in antiquity. A dedicatory drink it, fasting, to the amount of three cyathi, and purge away the inscription in the Asclepion at Epidaurus commemorating an operation ascarides’’ (On Fistulae 3). reinforces this view. ‘‘A woman from Pherai with the name of Sostrate In cases of ascarides in women’s genitals or rectum, the use of com- was, to all appearances, pregnant for a year. Being thus in danger, she presses made from greasy hair containing a mixture of fruits or wicker went to the sanctuary transported on a stretcher and slept in the Abaton. leaves kneaded with cedar oil is recommended. The compresses are Yet she was unable to have a dream and so decided to return to her applied ‘‘every three days and left in place for one day and one night. home city. On the return trip, however, she met a handsome young man The woman then washes her hair and eats garlic, cooked and raw. The at a village named Kornoi. Once he had enquired as to her condition, ascarides are emitted and die. The area must be washed with sea water’’ he suggested that they put the stretcher on the ground. He then tore (Diseases of Women 2.187). open the patient’s stomach and removed a large number of worms, Hippocrates contributed significantly to the progress of medicine, in- enough to fill two bowls. When he had stitched her stomach and thus cluding helminthology, as he embodied the great achievements of the cured her, he revealed that he was Asclepius and instructed her to leave physicians of antiquity (Falagas, 2006). Chief among these were di- her fee at Epidaurus’’ (Kavvadias, 1900). recting medical thinking to the natural causes of disease and introducing The treatment of intestinal parasitic infections was done primarily the principle of observation during the clinical examination to medical thinking. through medicines. Because agents with helminthicidal activity were References to intestinal parasites in general predate Hippocrates and not known, treatment was focused on the application of methods to relate to both the name and the description of the parasites themselves remove the parasites from the intestine. The treatment is by itself evi- and the diseases they cause. Homer does not use the term ‘‘helmins’’ dence of how seriously the physicians of antiquity regarded these in- (helminths), as he provides no account of them. By contrast, he refers fections. In Hippocrates Corpus, reference to antihelminthics is limited to earthworms (Il. 13, 654) and worms on rotting meat, on neglected to just a few formulations, such as that with seseli (parsley) as its main wounds (Il. 19, 25), and on corpses (Il. 22, 509), (Il. 24, 414) (Homer, ingredient. 1999). In all these passages he uses the word ‘‘eule’’ (␧␷␭␩´ ), meaning In conclusion, the systematic study of the Hippocratic Corpus pinworm or maggot (Liddell, 1996). The etymology of this word is close points several medical observations regarding the whole range of clin- to that of ‘‘eilo’’ (␧␭␻´␫ ), ‘‘eileomai’’ (␧␫␭␧´ ␱␮␣␫), and ‘‘eliso’’ (␧␭´␫ ␴␴␻), ical manifestations and treatment of enteric helminthic infections, con- meaning to roll or to wind (Liddell, 1996), due to the continuous move- stituting the first thorough scientific discussion in the history of human ment of worms. For this reason, it is probable that the term ‘‘helmins’’ parasitology. (␧´␭␮␫␯␵ ) has the same root as ‘‘eliso’’ ( ␧␭␫´␴␴␻ ). The historian Herodotus used both these terms to refer to the customs LITERATURE CITED of the Persians to cremate their dead and of the Egyptians to embalm them (Herodotus, 1932). In the Ebers Papyrus, reference is made to BRYAN,C.P.,AND H. JOACHIM. 1930. The papyrus Ebers. G. Bles, Lon- worms that can clearly be identified as the roundworm (Ascaris lum- don, U.K., 167 p. bricoides), threadworms (Enterobius vermicularis), and Guinea worms COX, F. E. 2004. History of human parasitic diseases. Infectious Dis- (Dracunculus medinensis). Reference also appears to be made to tape- eases Clinics of North America 18: 171–188. worms, although these are not fully recognizable or definitively iden- DESHPANDE, A. D. 1992. Enterobius vermicularis live adult worms in tifiable (Bryan and Joachim, 1930; Cox, 2004). the high vagina. Postgraduate Medical Journal 68: 690–691. However, it is in the Hippocratic Corpus, i.e., the complete works ERHAN, Y., O. ZEKIOGLU,N.OZDEMIR, AND S. SEN. 2000. Unilateral that are attributed to Hippocrates or those that generally express Hip- salpingitis due to Enterobius vermicularis. International Journal of pocratic views, in which we find the first scientific observations relating Gynecological Pathology 19: 188–189. to the description, epidemiology, symptoms, and treatment of helmin- FALAGAS, M. E., E. A. ZARKADOULIA,I.A.BLIZIOTIS, AND G. SAMONIS. thic diseases. In the Hippocratic Corpus, 3 types of intestinal parasites 2006. Science in Greece: From the age of Hippocrates to the age 972 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007

of the genome. The Federation of American Societies for Experi- KAVVADIAS, P. 1900. The sanctuary of Asclepius at Epidarus and the mental Biology (FASEB) Journal 20: 1946–1950. treatment of the patients. Perris Bros Press, Athens, Greece, 320 p. GALEN, K., G. KU¨ HN, AND F. W. A SSMANN. 1821. Opera omnia. C. Cnob- LIDDELL, H. G. 1996. A Greek–English lexicon, 9th ed. Henry George loch, Lipsiae, Germany, Vol. 19, 781 p. Liddell and Robert Scott (compilers). Clarendon Press and Oxford GROVE, D. I. 1990. A history of human helminthology. C.A.B. Inter- University Press, New York, New York, 320 p. national, Wallingford, Oxon, U.K., 848 p. MUKHOPADHYAY, B., S. SAHA,S.MAITI,D.MITRA,T.J.BANERJEE,M. JHA,M.MUKHOPADHYAY,N.SAMANTA, AND S. DAS. 2001. Clinical GUIOT, F., P. CHARLIER,R.DESSAIVE,J.WALLON, AND A. RAAT. 1998. A case of Enterobius vermicularis salpingitis. Journal de Gynecol- appraisal of Ascaris lumbricoides, with special reference to surgical complications. Pediatric Surgery International 17: 403–405. ogie, Obstetrique et Biologie de La Reproduction (Paris) 27: 822– SIGERIST, H. E. 1987. A history of medicine. II. Early Greek, Hindu, 824. and Persian medicine. Oxford University Press, New York, New HERODOTUS. 1932. P. E. Legrand (translator). He´rodote: Histoires, livre York, 352 p. 1–9. Vol. 3. Belles Lettres, Paris, France, 186 p. SMITH, W. D. 1979. The Hippocratic tradition. Cornell University Press, HIPPOCRATES. 1839. E. Littre´ (translator). Oeuvres comple`tes. Vol. Ithaca, New York, 264 p. 1–10. J. B. Baillie`re, Paris, France. SURENDRAN, N., R. KUMAR, AND A. NASSIR. 1988. Unusual presentation HOMER. 1999. A. T. Murray and W. F. Wyatt (translators). Iliad, 2nd ed. of patent vitello intestinal duct with round worms emerging from Vol. 1 and 2. Harvard University Press, Cambridge, Massachusetts. the umbilicus. Journal of Pediatric Surgery 23: 1061–1062. J. Parasitol., 93(4), 2007, pp. 973–974 ᭧ American Society of Parasitologists 2007

IN MEMORIAM

Wilbur L. Bullock 1922–2007

Dr. Wilbur Lewis Bullock, age 85, passed away on 22 April 2007 at dition to his work on the Acanthocephala, he studied other parasites of Langdon Place, Dover, New Hampshire. He had been a long-time res- both freshwater and marine fishes, including those found in the blood ident of Durham, New Hampshire, where he taught zoology at the Uni- of marine fishes. Professor Bullock’s work was supported by numerous versity of New Hampshire for 39 years before retiring in 1987. He was grants from the National Science Foundation and the National Institutes born in New York, New York, on 8 March 1922 and is survived by his of Health. He published more than 80 scientific articles, book chapters wife of 63 years, Cecilia (Groenewold) Bullock, a brother, four children, (e.g., ‘‘Morphological Features as Tools and as Pitfalls in Acanthoceph- and several grandchildren and great grandchildren. alan Systematics’’), reviews, abstracts, and editorials and presented ma- Wilbur graduated from Queens College in 1942 and attended New terial at several scientific meetings. His work on the histology of the York University before entering the Army, where he served in Europe digestive tract of fishes and the histopathology associated with acantho- during World War II with the U.S. Army Medical Detachment of the cephalan infection was especially noteworthy. He was very proud of Combat Engineers. When the war in Europe ended, he was appointed his meticulous histochemical techniques and staining procedures for fish an Assistant Instructor at the U.S. Army University in France. Upon feces and tissues and parasites. Too few appreciated the many facets of returning home, he earned his M.S. (1947) and Ph.D. (1948) at the his knowledge or the wide range and depth of his scientific contribu- University of Illinois under the direction of Professor Harley J. Van tions. Cleave. His dissertation work involved histochemical studies on the Wilbur developed a course at UNH titled Man, Nature, and Disease Acanthocephala, the distribution of lipase, phosphatase, glycogen, and that was very successful. It was designed to introduce the freshman/ fatty substances. He was appointed a Mountain Lake Biological Station sophomore non–science major to the basic natural history of the infec- Fellow that summer and then joined the faculty of the Department of tious diseases of humans. He published one book, People, Parasites, Zoology at the University of New Hampshire (UNH), where he re- and Pestilence: An Introduction to the Natural History of Infectious mained for his entire career. During his tenure at UNH, he served as Disease, that had its origin in the course he developed. Acting Chair of the Zoology Department on two occasions. He held Throughout his career, Professor Bullock maintained an avid interest appointments as a Research Fellow at Rice Institute (Texas), Visiting in the relationship between science and Christian faith and Christian Research Professor at Florida Presbyterian College, Louisiana State attitudes toward stewardship of the environment. He was active in the University Medical School Fellow in Tropical Medicine and Parasitol- Dover Baptist Church and was a major contributor to the establishment ogy (in Central America), and Research Associate Emeritus at the Har- of the Durham Evangelical Church, serving both churches in various old W. Manter Laboratory of Parasitology at the University of Nebraska. capacities. Everyone who knew him knew that he was a Christian, es- He was a Fellow of the American Association for the Advancement of pecially his students. Science. He was also a member of the American Society of Parasitol- Professor Bullock mentored a number of graduate students (including ogists, the Helminthological Society of Washington, the Wildlife Dis- Patrick Muzzall and David Huffman) who went on to successful careers ease Association, the Society of Systematic Zoology, the American So- in the field of parasitology. He was a great teacher who showed a lot ciety of Tropical Medicine and Hygiene, and Sigma Xi. of personal interest in his students at all levels. He began many of his Wilbur was an internationally respected fish parasitologist who spe- lectures with famous and interesting quotes that involved the lecture cialized in the systematics and biology of the Acanthocephala. In ad- material for that day. David recalls having once written up a short paper

973 974 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 4, AUGUST 2007 about how the acanthocephalan proboscis may have evolved from the eggs and spores in order to see if they could predict in advance what cirrus of cestodes, and then having proudly left it on Wilbur’s desk. parasites might be found. One day, as he carefully inserted a syringe of Later that day, Wilbur provided a reprint of a paper positing the same saline into a ripe and turgid skunk to rinse out a new set of clues, the idea but published years earlier. Wilbur’s succinct comment, obviously hapless animal got its final revenge with 2 jets of mercaptans, which designed to bring David’s pride down a notch, was a quote from Ec- quickly evacuated the entire building. clesiastes: ‘‘Under the sun, nothing is new.’’ After I heard the news about the passing of Wilbur Bullock, I looked I remember that one of Professor Bullock’s favorite fish species to out my window at Michigan State University and thought to myself that examine for parasites was the eel, Anguilla rostrata. These examina- another fish parasitologist had left us. Then I thought a very kind, con- tions took place like clockwork during the afternoons in his office. On siderate, soft-spoken, humble man who never raised his voice will be many an afternoon, I would ask him questions, and he would look over truly missed by all of us who knew him. his glasses and say, ‘‘Pat, you should know the answers to the questions.’’ Many times I did know the answers, but I wanted to talk with him and see what parasites he was finding, so I would ask the questions Patrick M. Muzzall, Department of Zoology, Natural Science Building, anyway. Michigan State University, East Lansing, Michigan 48824; Wilbur and David often delighted in examining road kills in Wilbur’s David G. Huffman, Department of Biology, Texas State University, San office. Wilbur’s favorite game was to do a rectal perfusion to check for Marcos, Texas 78666 J. Parasitol., 93(4), 2007, pp. 975–976 ᭧ American Society of Parasitologists 2007

Editor’s Note . . .

Not very long ago, the book for the following review was received. Although the book is written in Spanish, I decided to go ahead and have it reviewed by someone who is bilingual so that we could see it published in both Spanish and English, a ﬁrst for the Journal of Parasitology. I appreciate the good work of Michael Parkhouse, who graciously accepted the invitation to write the review in both languages.

Gerald W. Esch, Editor

BOOK REVIEW . . .

Cisticercosis: Guıá para Profesionales de la Salud, 2007. Edited by sick. Finally, the presence of T. solium is an immediate, effective, and Carlos Larralde and Aline S. de Aluja. Published by FCE, Secretaria objective indicator of inadequate sanitary and household conditions, de Salud, Instituto Nacional de Salud Publica, Fundacion Mexicana para typically an absence of drains, toilets, and proper water, and cohabita- la Salud, 252 p. ISBN 968-16-8138-X. tion with the intermediate host animals. Thus, improvement in education and basic living conditions would not only control transmission of cys- I first became aware of the tapeworm and its incredible life style ticercosis, but would also bring collateral benefits, for example, reduc- during my entry into puberty. Coincidence? Perhaps yes, but certainly tion in common ‘‘soil transmitted’’ diseases. under the influence of my new and confused hormone balance. At the Cysticercosis, and its associated neurocysticercosis, therefore, merits time, I was reading Miriam Rothschilds marvelous book Fleas, Flukes its status as a ‘‘neglected disease,’’ but one not restricted to the rural and Cuckoos, which provoked my imagination and stimulated a lasting poor, and, as is pointed out in the book, control of cysticercosis requires interest in parasitic worms, later to be rekindled in 1976–1977 when I separate strategies directed at rural, urban, and migrant populations. spent a year in Mexico and met Carlos Larralde, a pioneer in the study There are many more positive aspects and a wealth of little known facts of the tapeworm in Mexico. Now, he and his distinguished co-editor, and curiosities to this very well-conceived and well-presented book. For Dr. Aline Aluja, have assembled an impressive team of contributors to example, Serapio, an ancient Arab author, called the proglottids ‘‘cu- this new book focusing on the human tapeworm Taenia solium in gen- curbitineos’’ for the double reason that proglottids not only resemble eral and in Mexico in particular. pumpkin (Cucurbita peppo) seeds, but also because pumpkin seeds A major reason for my original fascination remains with me still. We, were, and continue to be, a folk remedy for the treatment of Taenia as human beings, go through an elaborate period of psychological stress, spp. sometimes called ‘‘love,’’ to find a partner, then, during a lifetime, pro- As the title indicates, the book is directed at professionals dedicated duce an average of 2 new human beings. In contrast, consider the re- to public and animal health in the laboratory, clinic, and field. A con- productive strategy and almost unimaginable and profligate fecundity siderable proportion of the book, such as the biology, pathogenesis, of the tapeworm. This amazing, and usually solitary, inhabitant of the control, and treatment of cysticercosis, is of sufficient general interest human intestine is a hermaphrodite, and thus has no need to seek a to warrant an English language version of the Spanish original. Indeed, partner. Such a worm may produce 100,000 fertilized, viable eggs a day I would recommend the book to non-ibero-latinamericans solely for the for up to 20 years in order to perpetuate itself, in spite of its wildly information, quality, and immediacy of the tables, figures, and photo- improbable life style. graphs. There is a deliberate decision to focus on cysticercosis in Latin The life cycle of T. solium involves two hosts: humans are the defin- America, in particular Mexico, for example in the chapters dealing with itive host for the adult intestinal worm, and pigs are an intermediate epidemiology and the plausibility of control. Even this, however, has host for a larval form known as the cysticercus or metacestode. The the advantage of acting as a stimulus for the preparation of similar eggs, once voided in human feces, are ingested by the intermediate reviews focusing on national aspects from other endemic countries; porcine host and hatch in the small intestine, liberating a highly motile, these, perhaps, might be published as a series of addenda to this most microscopic oncosphere equipped with three pairs of hooks. The on- scholarly work, which is very balanced in its presentation of the sci- cosphere penetrates the intestinal wall, enters the blood and lymphatics, entific and social aspects of the disease. For example, it is emphasized and then comes to rest within muscle and, sometimes, other tissues. In that laboratory vaccination trials of potential cysticercosis vaccines, this location, and for 2–3 months, the oncosphere develops into a meta- though of course important, will not justify large-scale licensing of a cestode, the causative agent of the disease known as porcine cysticer- vaccine until testing is conducted where the disease is maintained and cosis. The cysts remain viable for months, if not years, and when the transmitted, that is, in the field in rural areas. This is an important point definitive human host eats undercooked, infected pork, the metacestode that is underappreciated. undergoes a developmental transformation into the adult worm in the The book also questions the generally accepted view that improve- intestine of man, and the life cycle is completed. Alarmingly, the eggs ments in public health, sanitation, and meat inspection can be expected of T. solium can also infect man, causing human cysticercosis. The to eradicate T. solium in Latin America, exactly as has been achieved parasite is, therefore, of both medical and veterinary importance, and in Western Europe. Unfortunately, conventional European approaches indeed meat losses are significant in those areas endemic for the para- to control require good infrastructure, are costly, long term, and require site. considerable compliance and are therefore unlikely to be implemented Cysticercosis is a major health threat to the human population of in many of the areas where these parasites are prevalent. In addition, many tropical and subtropical countries. The frequently occurring ce- and as observed, particularly in the last two chapters, effective control rebral metacestode in humans is defined as neurocysticercosis. It is the of cysticercosis is a complex problem requiring an understanding and most common human neurological disease caused by a parasitic worm, integration of scientific, social, and governmental issues. Not for noth- with a spectrum of symptoms, including epileptic seizures, blindness, ing does Mexican black humor joke that ‘‘Un taco callejero es 10% de and death, and may account for over 10% of acute case admissions to carne y 90% de aventura’’ (a taco [cornbread sandwich] bought in the neurological wards in many endemic countries. Indeed, in highly en- street is 10% meat and 90% adventure). This book, with its realistic demic zones, nearly 30% of late-onset seizures are associated with se- appraisal of the problem, gives an excellent start by providing a bal- rological evidence of infection. Figures provided by the World Health anced yet polemical platform for a rational approach towards the even- Organization cite 50,000 deaths annually due to cysticercosis, but the tual elimination of human and porcine cysticercosis. It may not make overall impact is clearly greater because there will obviously be many a lot of money as, fortunately, it will be available online. On the other more infected, and thus disabled, individuals. The latter, being unable hand, the book may focus more efforts towards the control of cysticer- to work, place an extra burden on the rural community, mostly on the cosis, which is surely what the authors wish. women, who conventionally shoulder the responsibility of attending the

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Cisticercosis: Guıá para Profesionales de la Salud, Carlos Larralde objetivo, inmediato y efectivo de condiciones inadecuadas de salubridad y Aline S. de Aluja (Editores). Mexico: FCE, Secretaria de Salud, In- a nivel local y hogarenõ, falta o ausencia de drenaje, sanitarios, agua stituto Nacional de Salud Publica, Fundacion Mexicana para la Salud. potable, y vivir en promiscuidad con los hue´spedes intermediarios. Asi 2007. 252 p. ISBN 968-16-8138-X. pues, una mejorıá en la educacioń y condiciones ba´sicas de vivienda no solamente controlarıá la transmisioń de cisticercosis, sino traerıá be- La primera vez que tuve conciencia de las solitarias y su estilo de neficios colaterales, por ejemplo, la reduccioń de padecimientos ‘‘trans- vida increı´ble fue´ al principio de mi pubertad. ¿Coincidencia? Tal vez; mitidos por el suelo’’. pero lo que si es cierto es que leyendo el maravilloso libro de Miriam La cisticercosis y la neurocisticercosis asociada merecen su sitio Rothschild: ‘‘Fleas, Flukes and Cuckoos’’ bajo la influencia de mi bal- como ‘‘padecimientos descuidados’’, aunque no estań restringidas a la ance hormonal nuevo y confuso, provoco´ mi imaginacioń y estimulo´ poblacioń rural pobre, sino, como esta´ senãlado en el libro, el control un intere´s duradero en para´sitos, que volvio´ a re-avivarse en 1976-7 de la cisticercosis requiere estrategias separadas, una dirigida a la pob- cuando paseúnanõ en Me´xico y conocı´ a Carlos Larralde, un pionero lacioń rural, otra a la poblacioń urbana y otra para la poblacioń emi- en el estudio de solitarias en Me´xico. Hoy dıá, e´l y su distinguida co- grante. Hay muchos mas aspectos positivos y una riqueza de hechos editora, la Dra. Aline Aluja, han conjuntado un grupo impresionante de poco conocidos y curiosidades en este libro muy bien concebido y pre- autores en este nuevo libro enfocado en la solitaria humana Taenia sentado ‘‘Cisticercosis: guıá para profesionales de la salud’’ (‘‘Cysti- solium en general y particularmente en Me´xico. cercosis: A guide for Healthcare Professionals’’). Por ejemplo: Serapio, Mi fascinacioń inicial permanece auń conmigo por una razoń fun- un antiguo escritor a´rabe, llamo´ ‘‘cucurbitineos’’ a los proglo´tidos por damental. Nosotros, como seres humanos, pasamos por perıódos de con- dos razones, una es que los proglo´tidos parecen semillas de calabaza siderable agobio psicolo´gico, a veces llamado ‘‘amor’’, para encontrar (Cucurbita peppo) y otra porque e´stas eran y siguen siendo un remedio una pareja; despue´s, a lo largo de una vida producimos un promedio para tratar solitarias por el pueblo. de dos seres humanos nuevos. Como contraste, consideren la estrategia Como indica su tı´tulo, el libro esta´ dirigido a los profesionales de- reproductiva y la casi inimaginable fecundidad prolı´fica de la solitaria. dicados a la cisticercosis en el laboratorio, en la clıńica y en el campo. Este sorprendente, y comunmente habitante solitario del intestino hu- Una proporcioń considerable, tal como es la biologıá, la patogeńesis, el mano, es hermafrodita, y como tal, no necesita buscar una pareja. Su control y el tratamiento son de intere´s general, suficiente como para forma de vida que es altamente improbable se sutenta en que, para merecer una versioń en ingle´s del original en espanõl. De hecho, yo perpetuar su especie, el para´sito es capaz de producir 100,000 hueve- recomendarıá el libro a no ibero-latinoamericanos, solamente por la cillos fertilizados viables por dia, durante unos veinte anõs. informacioń y la claridad y vitalidad de la presentacioń en las tablas, El ciclo vital de la Taenia solium involucra a dos hue´spedes: el hom- figuras y fotografıás. Hay un enfoque deliberado sobre la cisticercosis bre como el hue´sped definitivo de la forma intestinal adulta del para´sito en Latinoame´rica, en particular en Me´xico, por ejemplo en los capı´tulos y el cerdo como hue´sped intermediario de la forma larval conocida que tratan con la epidemiologıá y la posibilidad de control. Sin embar- como quiste o´ metace´stodo. Asi pues, los huevecillos una vez arrojados go, aun esto tiene la ventaja de ser un estı´mulo para la preparacioń de con las heces humanas, son ingeridos por el hue´sped porcino y eclo- revisiones semejantes enfocadas en aspectos nacionales de otros paises sionan en el intestino delgado liberando larvas oncosferas microsco´picas en donde la enfermedad es ende´mica e´stas tal vez puedan publicarse de alta motilidad y equipadas con tres pares de ganchos. La oncosfera como una serie de adiciones a este trabajo tan escola´stico, sumamente no pierde tiempo, ra´pidamente penetra la pared intestinal y entra al balanceado en su presentacioń de los aspectos cientı´ficos y sociales del torrente circulatorio y vasos linfa´ticos donde es transportada y llega a padecimiento. Asi pues por ejemplo, se enfatiza que las pruebas de los mu´sculos y en algunas ocasiones a otros tejidos. En dicho lugar, vacunacioń en el laboratorio con vacunas con potencial para la cisti- durante los siguientes dos o tres meses, la oncosfera se desarrolla en cercosis, aunque por supuesto sean importantes, no justifican una licen- metace´stodo, que es el agente causante de la enfermedad llamada cis- cia a gran escala de una vacuna hasta que se haya probado en las areas ticercosis porcina. Los quistes permanecen viables entre meses y anõs. donde el padecimiento se mantiene y se transmite, es decir, en el campo Cuando el hue´sped definitivo humano come carne infectada de cerdo y en las a´reas rurales. Este es un punto muy importante que frecuen- mal cocida, ya en el intestino, el metace´stodo se desarrolla y se trans- temente no se aprecia. forma en el adulto y con ello el ciclo vital se completa. Es importante El libro tambień cuestiona la visioń generalmente aceptada de que mencionar que los huevecillos de Taenia solium tambień pueden infec- con el mejoramiento en salud pu´blica, salubridad e inspeccioń de carne tar al hombre, causańdole cisticercosis. Por lo tanto, e´ste para´sito es de se pueda esperar una erradicacioń de Taenia solium en Latinoamerica, importancia me´dica y veterinaria ya que las pe´rdidas de carne son conde la misma manera que se ha logrado en Europa Occidental. Desgra- siderables en las a´reas ende´micas del para´sito. ciadamente, los enfoques europeos convencionales de control, requieren La cisticercosis es una amenaza importante para la salud de la pob- de una buena infraestructura, son costosos y necesitan implementarse a lacioń humana de muchos paises tropicales y subtropicales. Frencuen- largo plazo y, por lo tanto, son improbables de llevarse a cabo en las temente la localizacioń del metace´stodo en el hombre tiene lugar en el a´reas donde estos para´sitos son prevalentes. Adema´s, como se observo´ cerebro y se le define como neurocisticercosis. Esta es la enfermedad antes, en los dos u´ltimos capı´tulos en particular, el control efectivo de neurolo´gica ma´s comuń en el humano causada por un para´sito, con un la cisticercosis es un problema complejo que requiere comprensioń e amplio espectro de sıńtomas que van desde convulsiones parecidas a integracioń de temas cientı´ficos, sociales y gubernamentales. Es por eso las de epilepsia, ceguera y muerte, y representa ma´s del 10% de ad- que el humor negro mexicano dice que ‘‘Un taco callejero es 10% de misiones en estado agudo en las salas de neurologıá en muchos paı´ses carne y 90% de aventura’’. Este libro hace una valoracioń realista del ende´micos. De hecho, en zonas altamente epide´micas, cerca del 30% problema, en el inicio da una excelente plataforma balanceada sin em- de convulsiones desarrolladas en el adulto, estań asociadas con eviden- bargo pole´mica, para un enfoque racional hacia la eliminacioń eventual cia serolo´gica de la infeccioń. De acuerdo con la OMS, 50,000 muertes de las cisticercosis, humana y porcina. Tal vez no se gane mucho dinero anuales se deben a la cisticercosis, sin embargo, el impacto es aun ya que afortunadamente, se podra´ obtener electrońicamente. Por otro mayor ya que obviamente habra´ mas gente infectada e individuos in- lado puede ayudar a enfocar mas esfuerzos para el control de la cisti- capacitados. Estos u´ltimos, incapaces de trabajar, significan una carga cercosis, que seguramente es lo que los autores desean. extra en la comunidad rural, mayormente en las mujeres, ya que son ellas las que por costumbre llevan la responsabilidad de cuidar a los Michael Parkhouse, Instituto Gulbenkian de Ciencia, Rua Quinta enfermos. Finalmente, la presencia de Taenia solium es un indicador Grande 6, 2780-156, Oeiras, Portugal