Parasitology International 62 (2013) 300–308

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Parasitology International

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Trichobilharzia mergi sp. nov. (: : ), a visceral schistosome of Mergus serrator (L.) (Aves: )

Libuše Kolářová a,b,⁎, Karl Skírnisson c, Hubert Ferté d, Damien Jouet d a Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague, Czech Republic b General Hospital in Prague, Czech Republic c Institute for Experimental Pathology, University of , Keldur, Reykjavík, Iceland d EA 4688, USC-VECPAR, UFR de Pharmacie, Université de Reims Champagne-Ardenne, article info abstract

Article history: Parasitological investigations on red-breasted mergansers (Mergus serrator L.) in Iceland revealed digenean Received 16 September 2012 flukes of the family Schistosomatidae. Adult worms were detected in blood vessels of the large intestine and Received in revised form 6 February 2013 eggs were deposited in the mucosa and surrounded by granulomatous reactions. Traditional morphological Accepted 4 March 2013 methods showed that the flukes have very slender filiform bodies, males are equipped with a short Available online 13 March 2013 gynaecophoric canal and both suckers and spatulate ends are present on each sex. Among characteristics of the flukes which render them morphologically distinct from other species are: i) males—well Keywords: fi Trichobilharzia mergi sp. n. developed vesicula seminalis (v.s.) consisting of a short v.s. externa and a signi cantly longer (approx. 3 Description times) v.s. interna, unusually well developed genital papilla and localization of the first testis a relatively long Morphology distance posterior to the gynaecophoric canal; ii) eggs—small and elongated with slightly rounded poles and Sequence a short terminal spine. DNA taxonomic techniques confirmed that a new species had been identified, Phylogenetic analysis Trichobilharzia mergi sp. n. The sequence data were deposited in GenBank under the accession numbers Life-cycle JX456151 to JX456172. Comparison of the results with our previously published data on characterization of Mergus serrator DNA of cercariae isolated from freshwater lymnaeid snails showed that larval development of T. mergi is Iceland associated with balthica L. (= Müller, 1774; = Radix ovata Draparnaud, 1805). Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction 50%, but usually no more than 5% of examined snails are found to be infected by Trichobilharzia larvae [4]. Avian schistosome flukes are characterized by a two- life cycle. Cercariae of Trichobilharzia spp.arealsoabletopenetratetheskinof The larval stadia (cercariae) develop in aquatic snails (intermediate non-compatible hosts (), but their further development is in- hosts) from which they are released into surrounding water. Free swim- complete and the parasites die early after the infection. In skin in- ming cercariae actively seek hosts and soon after penetration fection by schistosomes manifests as an allergic skin reaction known into the skin of specificdefinitive (compatible) hosts they transform to as cercarial dermatitis or swimmer's itch. The causative agents of the dis- schistosomula which migrate throughout the hosts' organs and develop ease are reputed to be mostly cercariae of Trichobilharzia spp. [5]. to adult flukes producing eggs. Depending on the species, adults localize Identification of Trichobilharzia adults and larvae is difficult. Tradi- either in the visceral organs or in the tissue of nasal area [1]. tional methods do not always allow adults and eggs from and The most commonly reported avian schistosomes are flukes of the cercariae from snails to be easily differentiated; the species-specific Trichobilharzia which represent the largest genus of the family morphological features are minute and their recognition is frequently Schistosomatidae. The adult flukes have been found all over the influenced by personal experiences of the examiner [6].Taxonomic world and waterfowl have been described as the most common definition of Trichobilharzia adult flukes is based mainly on the relative definitive hosts [1,2].However,theywerealsodetectedinpasserine positions of internal organs, surface structures of the tegument and the birds and in birds of the orders Ciconiiformes and Coraciiformes [1,3]. location of the parasites in their definitive hosts [1];onthemorphology Pulmonate snails of the families and Physidae were most of eggs [7]; on the distribution of sensory papillae on cercariae [8];or often reported as Trichobilharzia intermediate hosts [4]. The prevalence on the character of parasite life cycle, including information on the rates of adult schistosomes in birds was frequently found to exceed species of intermediate host [2]. Molecular techniques have become eminently suitable tools for identification of adult and larval stages of ⁎ Corresponding author. Tel.: +420 271 019 254; fax.: +420 272 140 458. Trichobilharzia species. Among these are sequences of gene regions E-mail address: [email protected] (L. Kolářová). such as the 28S and the internal transcribed spacers (ITS) of ribosomal

1383-5769/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.03.002 L. Kolářová et al. / Parasitology International 62 (2013) 300–308 301

DNA and the mitochondrial cox1. These have suggested great diversity 2. Material and methods in avian schistosomes and enabled new causative agents of cercarial dermatitis to be discovered [9–15]. Genetic techniques have also sug- 2.1. Examination of birds gested novel explanations of details of parasite life cycles, and thereby help our understanding the epizootology and epidemiology of bird Eighteen ducklings of three species—14 M. serrator, three Anas schistosomes [12,16,17]. Although these techniques are very specific platyrhynchos and one Anas penelope, obtained from Lake Botnsvatn and sensitive methods for identification of particular schistosomes, in early September 2005, 2006 and 2009, and one adult Gavia immer DNA sequencing alone is not sufficient for discrimination between found dead in July 2005—were examined for the presence of visceral species and need supplementing by traditional methods [18]. and nasal schistosomes according to Kolářová et al. [6]. Birds collected We have in the past examined Icelandic snails and birds for the in 2005 (3 specimens) and 2006 (9) were kept frozen at −20 °C until presence of larval and adult schistosomes and our results revealed examination; birds from 2009 (6) were examined fresh. that several taxonomically undefined schistosome species are present Scrapings from intestinal mucosa of the large intestine, rectum in that country [7,10,19]. In the present study we applied both tradi- and posterior part of the small intestine were compressed between tional and molecular techniques in an examination of the taxonomic two slides and examined under a microscope for the presence of status of adult flukes isolated from the red-breasted merganser schistosome eggs. For each bird the shape and size of several dozen (Mergus serrator L.) originating from Lake Botnsvatn. eggs (Fig. 1a,b) were examined morphologically and their dimensions Lake Botnsvatn is an oligotrophic body of water of area approxi- measured. mately 1 km2 in the northern part of Iceland. In both late July 2004 Adult schistosomes were excised from blood vessels of the colon and August 2005 dozens of people acquired swimmer's itch after and small intestine as described by Kolářová et al. [6]. The parasite wading in the lake [20]. Investigations on Radix balthica L. (=Radix fragments and eggs so obtained were examined under a stereomicro- peregra Müller, 1774; = Radix ovata Draparnaud, 1805) collected in scope. Slender fragments of the smaller flukes were separated from a the lake in August 2004 and 2005 confirmed an unusually high considerably larger Trichobilharzia species, the latter being flukes that number of snails that shed ocellate furcocercariae (10% out of 100 the authors aim to deal with in a separate paper. and 19.5% out of 133 snails on the two occasions, respectively). Sequencing data from a part of this material confirmed the presence 2.2. Description of schistosomes of three different species of Trichobilharzia cercariae in the lake: the haplotype Trichobilharzia sp. “haplotype peregra” [19]; Trichobilharzia Measurements of eggs and adults were performed on native fresh regenti [10] and the species that we describe in the present article—a mounts prior to fixation in 95% ethanol and subsequent freezing for mo- species designated as Trichobilharzia sp. V by Skírnisson and Kolářová lecular investigation. The morphological description of the adults was [7] and Trichobilharzia sp. 2 by Aldhoun et al. [10]. made on fresh, separately mounted fragments of 15 males and 11 fe- Only few species breed and raise ducklings at Lake Botnsvatn males, including 6 males and 1 female that were fixed (4% formalde- during summer. In order to find out which of them might act as final hyde) and stained by borax carmine (Tables 1, 2). Morphological hosts for Trichobilharzia spp. in the ecosystem, individuals of the most details were photographed frequently using a digital camera (Leica DC commonly breeding duck species—M. serrator were shot at the onset 300) attached to a microscope (Leica DMLB or Olympus BX50) equipped of the hunting season and examined for the presence of nasal and vis- for differential interference contrast microscopy (DIC; Nomarski). ceral schistosomes. It was observed that all the birds were infected with two different-sized flukes (including eggs), the morphology of 2.3. DNA and phylogenetic analysis which was similar to that of Trichobilharzia spp. One of these was however clearly distinct from all described species within the genus. Eggs and adults were preserved in 95% ethanol and kept frozen The present article describes the adult stages of the smaller of these (−20 °C) until DNA analysis (Table 3). After removing all ethanol two species, eggs of which were described by Skírnisson and Kolářová from samples, DNA was extracted using the Qiamp DNA Mini Kit [7]. We use molecular methods to help define its life cycle in this area. (Qiagen, ) following manufacturer's instructions. The DNA

a b

_____50__ _μ_m______50______μ_m_ __

Fig. 1. a,b Light microscope photographs showing two different-sized egg types in a fresh scraping from the rectal mucosa of Mergus serrator from Lake Botnsvatn. The small egg (arrows) is that of Trichobilharzia mergi; the larger eggs are those of Trichobilharzia sp. “haplotype peregra” [see 19]. 302 L. Kolářová et al. / Parasitology International 62 (2013) 300–308

Table 1 Table 3 Measurements of male Trichobilharzia mergi. Isolates of bird schistosomes from Mergus serrator originating from the Lake Botnsvatn (Iceland) used for molecular analysis. No. Mean ± SD (mm) measured Accession numbers

Maximum width at posterior end 7 0.051 ± 0.006 Taxa Developmental stage D2 ITS2 ITS1 cox1 Width before spatulate end 6 0.042 ± 0.009 MER9A Egg JX456151 Width at posterior end 6 0.096 ± 0.008 MER13 Egg JX456152 Length of spatulate end 6 0.184 ± 0.015 MER23 Adult ♂ JX456153 JX456164 Oral sucker 7 0.031 ± 0.007 × 0.038 ± 0.006 MER24 Adult ♂ JX456154 Oesophagus 6 0.410 ± 0.117 MER26 Adult ♂ JX456155 JX456165 JX456167 JX456171 Acetabulum to anterior end 6 0.652 ± 0.173 MER34 Adult ♂ JX456156 Acetabulum 10 0.037 ± 0.006 × 0.038 ± 0.006 MER42 Adult ♂ JX456157 Caecal reunion to gynaecohoric 6 0.047 ± 0.017 MER44 Adult ♀ JX456158 JX456166 JX456168 JX456172 canal MER46B Egg JX456159 JX456169 Length of gynaecophoric canal 10 0.330 ± 0.055 MER53 Adult ♀ JX456160 Width of gynaecophoric canal 10 0.100 ± 0.018 MER66 Adult ♂ JX456161 Caeca bifurcation to acetabulum 8 0.203 ± 0.042 MER67 Adult ♂ JX456162 Seminal vesicle 8 0.876 ± 0.102 MER68 Adult ♂ JX456163 Vesicula seminalis externa 8 0.200 ± 0.048 × 0.19 ± 0.04 Vesicula seminalis interna 8 0.602 ± 0.105 × 0.018 ± 0.03 Genital papilla 6 0.027 ± 0.004 × 0.018 ± 0.002 Size of testes 22 0.033 ± 0.009 × 0.028 ± 0.006 sequences of avian schistosomes available in GenBank: Trichobilharzia Number of testes 1 >110 franki (FJ174530; FJ793813–14; FJ793874–5; HM131137–39; 1st testis to gynaecophoric canal 6 0.316 ± 0.052 HM131162–64; HM131197–202), T. regenti (AY157190; DQ859919; Last testis to posterior end 3 0.191 ± 0.012 EU413960; EU413967–70; EU413977–79; FJ793829; FJ793831; FJ793887–88; HM439499–504; NC_009680), Trichobilharzia szidati (AY157191; FJ174495–96; FJ793848–49; FJ793896–97), Trichobilharzia was eluted in 50 μl of the buffer provided. The polymerase chain querquedulae (FJ174468–70; FJ174555; FJ174556; FJ174558; FJ174497– reaction (PCR) was performed in a 50 μl volume using 5 μlofDNA 500), Trichobilharzia physellae (FJ174474; FJ174561; FJ174512–515), (40 ng/μl), and 50 pmol of each of the primers. The PCR mix contained Trichobilharzia stagnicolae (FJ174478–79; FJ174544–46; FJ174488–91),

(final concentrations) 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2,50mM Trichobilharzia brantae (FJ174482; FJ174484; FJ793868–70; FJ793917– KCl, 0.01% Triton X 100, 200 μM dNTP of each base, and 1.25 units of Taq 19) and Allobilharzia visceralis (EF114222–23; EF071989–91). Phyloge- polymerase (Eppendorf, Germany). netic trees were constructed using the Neighbor-Joining (NJ), the Max- Sequences of the D2 domain of the 28S subunit and internal imum Likelihood (ML), and Minimum Evolution (ME) methods using transcribed spacers 1 (ITS-1) and 2 (ITS-2) of ribosomal DNA and the the MEGA5 software. For all NJ, ML and ME analyses, the most appropri- cox1 domain of the mitochondrial DNA were used for molecular iden- ate nucleotide substitution model was determined (HKY + G for com- tification of these avian schistosomes [14,21–23]; PCR was performed bined D2–ITS2 and TN + G + I for cox1), gaps were treated as missing under conditions that have been published previously [14].DNA data and internal node support was assessed by bootstrapping over 500 regions were treated separately according to the level of specificity of replicates. each one and in order to allow a comparison with the various se- quences deposited in GenBank for each one of these domains. 2.4. Life cycle description PCR products were directly sequenced in both directions with the primers used for DNA amplification (QIAGEN, Germany). The sequences We were unable to complete a life cycle of the parasites under labo- are deposited in GenBank under the accession numbers JX456151 to ratory conditions in this study. Therefore, the DNA sequences of eggs JX456172. andadultsobtainedinourstudywerecomparedwithsequencesofcer- Sequences were aligned using the ClustalW routine included in the cariae isolated from R. balthica collected in Lake Botnsvatn (Iceland) and MEGA version 5 software [21] and checked by eye. The combined D2 do- Annecy Lake (France) obtained in our former studies [10,19,24].Thedata main and ITS2 of the rDNA, and the cox1 domain of the mDNA were used on cercariae collected from the Icelandic and French snails and reported for tree construction using sequences obtained during this study and as Is5, Is6, Is17, Is18, Is20, Is24, Is27 (Accs. Ns. FJ469792–6; FJ469807) by Aldhoun et al. [10], and referred as EAN17 (Accs. Ns. EU413961; Table 2 EU413971) by Jouet et al. [24], complete the life-cycle description. Measurements of female Trichobilharzia mergi. 3. Results No. measured Mean ± SD (mm)

Width at ovarian region 12 0.059 ± 0.007 3.1. Bird infections Width before spatulate end 7 0.039 ± 0.004 Width at posterior end 7 0.066 ± 0.006 Length of spatulate end 7 0.120 ± 0.007 The examination of all birds revealed that only M. serrator were Oral sucker 9 0.031 ± 0.004 × 0.041 ± 0.01 infected by schistosomes resident in the blood system of the hosts' Acetabulum 10 0.034 ± 0.004 × 0.039 ± 0.004 visceral organs. Moreover, in all red-breasted mergansers we noted Acetabulum to anterior end 9 0.535 ± 0.118 concurrent infection by two different species of visceral flukes of Oesophagus 8 0.453 ± 0.122 Caecal reunion to acetabulum 10 0.125 ± 0.032 Trichobilharzia spp. the bodies of which were easily visually distin- Ovary 6 0.460 ± 0.136 × 0.032 ± 0.008 guishable by size. Ovary to acetabulum 6 0.367 ± 0.119 In addition, two differently-sized eggs of visceral Trichobilharzia Seminal receptacle 5 0.187 ± 0.028 × 0.039 ± 0.003 species were found in all M. serrator:i)Trichobilharzia mergi, previously Vitellaria to seminal receptacle 6 0.164 ± 0.037 ř Vitellaria 19 0.033 ± 0.04 × 0.039 ± 0.004 referred to as Trichobilharzia sp. V by Skírnisson and Kolá ová [7]; ii) a Vitellaria to posterior end 3 0.102 ± 0.008 markedly larger species, producing considerably larger eggs of different Egg in utero 5 0.095 ± 0.044 shapes (Fig. 1a,b), previously referred to as Trichobilharzia sp. IV by a Mature egg in the intestine 15 0.130 ± 0.006 × 0.053 ± 0.001 Skírnisson and Kolářová [7] and Trichobilharzia sp. “haplotype peregra” a Eggs with fully developed miracidia. described by Jouet et al. [19]. L. Kolářová et al. / Parasitology International 62 (2013) 300–308 303

3.2. Adult schistosomes grove). The reunited intestine continued in the testes area in a sinuous course and ended before a slightly dilated tail. In fresh worms, the intes- No intact worms were recovered in the large intestine and sur- tine was filled by dark brown granulae, probably hematin formed as a re- rounding mesenterium and total length could not therefore be mea- sult of digestion of host blood. sured. The longest fragments of anterior and posterior parts of males The reproductive system consisted of v.s., genital papilla, numerous were 2.729 mm and 2.625 mm, respectively. In females, the longest testes (more than 120) and a collecting duct. By comparison with body fragments of anterior and posterior ends were 1.783 mm and length there was a relatively short canalis gynaecophorus which was 2.660 mm, respectively. From examination of body parts that were equipped by spines (2–4 μm in length) situated on its ventral surface. found, the total length of males was estimated to be a minimum of An elongated v.s. is well developed and practically fills the area about 6 mm, that of females a minimum of approx. 5 mm. between acetabulum and ventral grove; it is distinctly divided into Males (Fig. 2, Table 1)hadaslenderfiliform body of almost uniform external (v.s.e.) and internal (v.s.i.) sections; the v.s.e. is markedly width. On the anterior part of the body pigmented spots were located on shorter (approx. 3 times) than the v.s.i. The ejaculatory duct arises at the surface of a pseudostriated tegument. The oral sucker encompassed a the posterior end of the v.s.i. and transverses the prostatic region. The subterminally-situated oral opening which was equipped with short genital opening is situated on the left side of the anterior end of the spines (length of approx. 2–3 μm). The mouth led to a long oesophagus ventral grove; prominent genital papilla (Fig. 3) epitomizes a well- whichinturnbecameabifurcatedcaecum. The caecal bifurcation does developed sexual organ. Testes are spherical or elliptical and com- not directly encompass acetabulum equipped by small spines of approx. mence a relatively long distance posterior to the terminal end of 6 μm in length. Reunion of the caecum occurred posterior to the vesicula gynaecophoric canal. Adjacent testes are situated one behind another seminalis (v.s.) interna, just before the canalis gynaecophorus (ventral and continue nearly to the end of the body; common caecum interlocks

OS ⎯⎯ FEMALE ⎯⎯ OS

OE ⎯⎯ ⎯⎯ OE

⎯⎯ CB ⎯⎯ CB

⎯⎯I ⎯⎯ I

_ ⎯ _ m ⎯⎯ VI _ A μ _

A ⎯⎯ _ ⎯⎯ T ⎯⎯ _ GO _ 200 ______⎯⎯ U ⎯⎯ VSE

T ⎯⎯ VD ⎯⎯

_ ⎯⎯ m E _ μ _ _

_ ⎯⎯

_ CR 100 _

______⎯⎯ OT ⎯⎯ VSI ⎯⎯ OD RS ⎯⎯

O ⎯⎯

⎯⎯ ED GP ⎯⎯ M CR ⎯⎯

⎯⎯ 50 μm MALE CG ______EGG

Fig. 2. Anterior and posterior ends of a male (left) and a female (right) of Trichobilharzia mergi. A, acetabulum; CB, caecal bifurcation; CG, canalis gynaecophorus; CR, caecal reunion; E, egg; ED, ejaculatory duct; GO, genital opening; GP, genital papilla; I, intestine; M, miracidium; O, ovary; OD, oviduct; OE, oesophagus; OS, oral sucker; OT, ootype; RS, receptaculum seminis; T, testes; U, uterus; VD, vitelloduct; VI, vitellaria; VSE, vesicula seminalis externa; VSI, vesicula seminalis interna. 304 L. Kolářová et al. / Parasitology International 62 (2013) 300–308

Trichobilharzia, the flukes originating from red-breasted mergansers were named T. mergi.

3.4. Intermediate hosts

The DNA sequence information obtained from the eggs and adults of T. mergi correspond to the sequences of cercariae previously isolated from R. balthica, not only in Iceland [10], but also in Lake Annecy, μ ______50__ _m___ France [24],thusallowingustoconfirm the involvement of this snail species in the life cycle of T. mergi. Fig. 3. Light microscope photograph showing the prominent genital papilla (arrow) situ- ated on the left side on the anterior end of canalis gynecophorus of male Trichobilharzia 3.5. Pathology mergi. In the large intestines of some of the red-breasted mergansers between them. Due to the fact that only dead flukes could be exam- infected by T. mergi, pathological lesions developed as a consequence ined, flame cells, as well as the arrangement of excretory system, of inflammatory reactions around the fluke eggs (Fig. 6)andgranulo- were not observed. mas were observed in the mucosa. The severity of the pathology was The female (Fig. 2, Table 2) was slightly thinner than the male. dependent on the intensity of infection; severe infections were charac- Similarly to the males, the body was filiform with a slightly spatulated terized by polyposis in the large intestine. posterior end (tail). The morphology of suckers and the digestive tract was generally the same as in males. Bifurcation of the caecum 3.6. Systematic summary occurred anterior to acetabulum and reunion of the caeca was located posterior to the receptaculum seminis. Similarly to males, a broad di- Species: T. mergi sp. n. ameter intestine was filled by dark brown pigment which allowed its Type host (natural infection): red-breasted merganser (M. serrator L.). sinusoidal course to be traced. The intestine was the most dominant Intermediate host: R. balthica L. (=R. peregra Müller, 1774; = R. organ of the female. ovata Draparnaud, 1805). The genital opening of the reproductive system was situated just pos- Site of infection: blood vessels of the large intestine and mesenterium, terior to acetabulum. The uterus lay posterior to the acetabulum. A long sometimes blood vessels of the posterior end of the small intestine. tubular ovary was followed by a seminal receptacle; both of them were Type locality: Lake Botnsvatn, Iceland. clearly visible in the anterior part of the body. The oviduct by-passed Deposition of types: Natural History Museum, Vienna, Austria: the ovaries area, joined the seminal receptacle and turned back. Between allotype—NHMW ZooEV mikro 5590 (anterior end of a female); the seminal receptacle and the posterior body end numerous vitelline holotype—NHMW ZooEV mikro 5591 (anterior end of a male); paratype follicles (more than 130) were found, interlocked by the vitelloduct. Sim- —NHMW ZooEV mikro 5592 (posterior part of a male); paratypes— ilarly to males, flame cells and an excretory system were not seen. NHMW ZooEV mikro 5593 (anterior parts of males). Eggs (Fig. 2, Table 2) in utero were slightly smaller than eggs found Deposition of the nucleotide sequences: D2.sqn—GenBank in the intestinal mucosa. Eggs were elongated without distinct poles. under the accession numbers: JX456151, JX456152, JX456153, Both ends were slightly rounded, the slightly broader end terminating JX456154, JX456155, JX456156, JX456157, JX456158, JX456159, in a slender, curved process (spine) at top. In fresh eggs, well-developed JX456160, JX456161, JX456162 and JX456163; ITS2.sqn—GenBank miracidia were present. under the accession numbers: JX456164, JX456165, JX456166, JX456167, JX456168, and JX456169; cox1.sqn—GenBank under the accession numbers: JX456171 and JX456172. 3.3. Sequencing and phylogenetic analysis Etymology: The nomen “mergi” is derived from the scientific name of the definitive host. The molecular analysis of the combined D2–ITS2 and the cox1 do- mains confirmed that these small flukes from red-breasted mergansers 4. Discussion belonged to the Trichobilharzia genus (Figs. 4, 5). Moreover, our analy- ses clearly showed that DNA sequences of the eggs and adults belonged The morphological description of the flukes described here and to the same clade, thus separating our sequences in only one species comparison with other schistosome genera/species [1,2] revealed a distinct from the other species within the genus. similarity with Trichobilharzia spp. To justify the establishment of a For the combined D2–ITS2 domain (900 positions), sequences of new species a comparison of our samples with other Trichobilharzia eggs and adults were 100% homologous, and with the haplotype of species was performed. furcocercariae (ACC. Ns. EU413961) isolated from R. balthica in France Morphology of adult flukes is the most valuable characteristic for (Fig. 4). species determination, especially the position of the caecal reunion With respect to the partial mitochondrial cox1 (824 bp) fragment, in males [2]. Additionally the size and shape of eggs represent a valu- molecular analyses showed only one intraspecific variation between able tool for quick discrimination between flukes that occur the sequences from this study when compared with the 91 to 112 var- concurrently within the same bird host [7]. Studies on miracidia and iations which separate them from other species of the genus (Fig. 5). cercariae can also sometimes provide taxonomic characteristics Despite the variability of this region, alignment of sequences for ITS-1 [2,8], and as with life-cycle characteristics for adults and larvae [1], confirmed that our haplotypes belonged to the same species, with some non-morphological criteria, e.g. the site where adult flukes re- only one variation observed out of 948 bp. This homology is shared side within the avian host [1] or behaviour of cercariae in the water with sequences FJ469792–99 and FJ469807 corresponding to cercariae column can be used for the description [e.g., 26]. Contrary to adult and adults isolated from R. peregra and M. serrator in Iceland in the flukes, however, species-specific characteristics of Trichobilharzia past [10]. These values are consistent with a new species status, as cercariae are difficult to detect or even absent, and the allocation of already described for other Trematodes in the past [11,25]. cercariae to a particular species is thus usually impossible [1]. In view of the above-mentioned molecular data and morphology Therefore, the use of molecular techniques as a taxonomic aid has be- that is clearly distinct from that of other schistosomes of the genus come very important and they can add valuable information about L. Kolářová et al. / Parasitology International 62 (2013) 300–308 305

* Trichobilharzia franki

Trichobilharzia querquedulae * * * Trichobilharzia physellae * * Trichobilharzia szidati * MER23 MER26 Trichobilharzia mergi * * MER44 EU413961

Trichobilharzia stagnicolae * Trichobilharzia brantae * Allobilharzia visceralis *

0.005

Fig. 4. Phylogenetic tree based on the combined D2 domain of the 28S and internal transcribed spacer 2 of rDNA, constructed using the Neighbor-Joining method (NJ). The scale shows the number of nucleotide substitutions per site between DNA sequences. Allobilharzia visceralis and Trichobilharzia brantae were set as outgroup. The node support is given in NJ, Maximum Likelihood (ML) and Minimum Evolution (ME) bootstraps. The “*” indicates node support of >90% bootstrap for NJ, ML and ME. the biology of the parasites, the epidemiology of particular species furcocercariae originated, showed that molluscs of these species and pathogenicity in snail and vertebrate hosts, including man [18]. collected in France and Iceland belong in fact to R. balthica L., the syno- Larval development of Trichobilharzia spp. occurs mostly in fresh- nym of which is also R. ovata Draparnaud, 1805. water snails of the families Lymnaeidae and Physidae. R. balthica is prob- In the present study, we were unable to obtain larval stages from ex- ably the only representative of the genus Radix in Iceland and it has been perimentally infected snails and, therefore, detailed description of reported that this snail is the intermediate host of many Trichobilharzia T. mergi cercariae is not presented. However, pictures (Fig. 7)and flukes [10,19,24]. An examination of two other snail species, Gyraulus preliminary results could be obtained on morphology and size of the spp. and Physa acuta, which also occur in a few lakes in this country cercariae examined fresh in Iceland and France. We found out that mor- has not yet revealed any infection by ocellate furcocercariae [20,27]. phology of the cercariae corresponds to Trichobilharzia spp. and T. mergi ThecompletelifecycleofT. mergi seems to occur in Botnsvatn Lake in cercariae are, therefore, microscopically undistinguishable from other Iceland as both larval and adult stages were identified here in our recent cercariae of the genus [1]. Total length of the cercariae is about as well as former studies [7,10,24]. However, it seems that the life cycle 750 μm; size of the tail stem is 310 μm and of furca 185 μm. Eye spots can also occur in other countries. Molecular studies on cercariae isolated and acetabulum are situated approx. 119 μm and 174 μm to the anteri- from snails of Radix spp. in France and Belarus performed by Jouet et al. or end, respectively; size of the head organ is about 256 × 81 μmandof [24], Chrisanfova et al. [28] and Rizevsky et al. [29] resulted in the detec- acetabulum 27 μm in diameter. tion of an ITS2 rDNA sequence of a new schistosome species identical to Most of the T. mergi adults were found in the vessels of the large intes- the Icelandic T. mergi. The DNA-based study of Jouet et al. [19] on tine and thus our parasites belong to the group of visceral schistosomes. R. peregra Müller, comprising 1774 snails from which the ocellate Comparison of our schistosomes with all known flukes assigned to the

* Trichobilharzia franki

Trichobilharzia querquedulae * * Trichobilharzia physellae * MER26 * Trichobilharzia mergi MER44

* * Trichobilharzia szidati

Trichobilharzia stagnicolae * Trichobilharzia regenti * Trichobilharzia brantae

0.02

Fig. 5. Phylogenetic tree based on the partial cox1 domain of mtDNA, constructed using the Neighbor-Joining method (NJ). The scale shows the number of nucleotide substitutions per site between DNA sequences. Trichobilharzia brantae was set as outgroup. The node support is given in NJ, Maximum Likelihood (ML) and Minimum Evolution (ME) bootstraps. The “*” indicates node support of >90% bootstrap for NJ, ML and ME. 306 L. Kolářová et al. / Parasitology International 62 (2013) 300–308

the position of caecal reunion in males and the shape of eggs. Contrary to T. mergi, caecal reunion is just posterior to acetabulum in males of Trichobilharzia elvae (Miller, 1923) McMullen and Beaver, 1945, Trichobilharzia limnaeae Yamaguti, 1971, Trichobilharzia ocellata de- scribed by Chikami [30], T. physellae (Talbot, 1936) McMullen and Beaver, 1945, T. stagnicolae (Talbot, 1936) McMullen and Beaver, 1945, T. szidati Neuhaus, 1952, and Trichobilharzia corvi (Yamaguti, 1941) McMullen and Beaver, 1945. In T. querquedulae (McLeod, 1937) McMullen and Beaver, 1945, the reunion of caeca takes place behind the genital open- ing. In T. corvi, moreover, the single caecum is displaced to one side by the mass of testes [31–33]. The morphology and size of T. mergi eggs determined here were the same as already described by Skírnisson and Kolářová [7].Theirshape mostly resembled those of T. corvi. However, as well as the differences in morphology of males in the two species, larval development of fl Fig. 6. Polyposis (arrows) in the large intestine of Mergus serrator caused by T. corvi ukes takes place in pleurocerid snails [see J. Ito, 1960, cited by Trichobilharzia mergi eggs. 2]. Females of other Trichobilharzia species produce eggs with a different shape to those of T. mergi: i.e., Trichobilharzia brevis Basch, 1966, Trichobilharzia cameroni Wu, 1953, T. elvae, T. franki Müller et Kimmig, 1994, Trichobilharzia jianensis Liu, Chen, Jin, Tan et Yang, 1977, genus Trichobilharzia was difficult mainly due to incomplete descriptions Trichobilharzia jequitibaensis Leite, Costa et Costa, 1978, Trichobilharzia. of many species; i.e., frequently either only males or females were known maegraithi Kruatrachue, Bhaibulaya, Chedapan et Harinasuta, 1967, about and information about the life cycle was usually not available. At T. ocellata described by Chikami [30], Trichobilharzia oregonensis present the genus Trichobilharzia comprises more than 40 species [1], (Macfarlane and Macy, 1946) Macy and Moore, 1955, Trichobilharzia though some others that need more detailed characterization are consid- paoi (Kung, Wang et Chen, 1960) Tang et Tang, 1962, Trichobilharzia ered to be species inquirendae [2]. parocellata (Johnston and Simpson, 1939) Islam and Copeman, 1980, The intestinal residence of the species described here made it dis- T. physellae, T. querquedulae (McLeod, 1937) McMullen et Beaver, 1945, tinct from other Trichobilharzia spp. flukes occupying the nasal area of T. stagnicolae (Talbot, 1936) McMullen and Beaver, 1945, T. szidati, birds, but there were also no similarities of morphology between our T. oregonensis (Macfarlane and Macy, 1946) Macy and More, 1955, and isolate and those of Trichobilharzia aureliani Fain, 1956, Trichobilharzia Trichobilharzia salmanticensis Simon–Martin et Simon–Vicente, 1999. In arcuata Islam, 1986, Trichobilharzia australis Blair et Islam, 1983, these species, one egg pole is blunter than the other and the second Trichobilharzia duboisi Fain, 1959, Trichobilharzia nasicola Fain, 1955, pole possesses a long, usually almost straight axis or both poles of the T. regenti Horák, Kolářová et Dvořák, 1998, Trichobilharzia rodhaini eggs are long and sharp and one of the poles forms a curved process or Fain, 1955, and Trichobilharzia spinulata Fain, 1955. spine. The comparison between our flukes and other visceral Trichobilharzia T. mergi also differs in the position of its caecal reunion or in the species with known developmental stages and life cycles revealed that shape of eggs from those Trichobilharzia species which are regarded T. mergi had distinct morphological features; the differences were in as species inquirendae [2]: Trichobilharzia adamsi Edwards et Jansch, 1955; Trichobilharzia anatina Fain, 1955; Trichobilharzia alaskensis Harkema, McKeever et Becker, 1957; Trichobilharzia burnetti (Brackett, 1942) McMullen and Beaver, 1945; Trichobilharzia cerylei,Fain1955; a Trichobilharzia filiformis (Szidat, 1938), McMullen and Beaver, 1945; Trichobilharzia horiconensis (Brackett, 1942) McMullen and Beaver, 1945; Trichobilharzia indica Baugh, 1963; Trichobilharzia kegonsensis FURCA BODY (Brackett, 1942) McMullen and Beaver, 1945; Trichobilharzia kossarewi Skrjabin et Zakharov, 1920; Trichobilharzia littlebi (Byrd, 1956) Farley, 1971; Trichobilharzia kowalewskii (Ejsmont, 1929) McMullen and Beaver, 1945; Trichobilharzia lonchurae (Fischtal and Kuntz, 1973) Tsai et al., 1979; T. ocellata (La Valette, 1855) Brumpt, 1931; TAIL Trichobilharzia waubesensis (Brackett, 1942) McMullen and Beaver, 1945; Trichobilharzia zongshani Tsai et al., 1979. In males of b HO Trichobilharzia berghei Fain, 1955 and Trichobilharzia shoutedeni Fain, 1955, females and eggs of which are unknown, the caecal reunion CPG seems to be at the same position as in T. mergi. However, Fain's drawings [34] of T. berghei and T. shoutedeni show other morphological AC features which are different from T. mergi: there are no differences in PPG the size between v.s.e. and v.s.i., the genital papilla is not unusually well developed, the first testes appear a longer distance posterior to ventral grove, and the last testis does not reach the posterior end of the flukes. Similarly to the study by Aldhoun's et al. [10] on the characterization ES of fragments of adult schistosome flukes isolated from M. serrator,and based on molecular analysis, the sequences from our study belong to the BTGD clade [12] and to the Trichobilharzia genus. Our results confirm Fig. 7. Light microscope photograph of cercaria of Trichobilharzia mergi originated from that the cercariae isolated from R. balthica, and the eggs and adults isolat- Annecy Lake, France. a, ocellata furcocercaria, scale bar: 150 μm; b, details of the body: AC, acetabulum; CPG, circumacetabular penetration glands; PPG, postacetabular pene- ed from M. serrator belong to a new unique species T. mergi, different tration glands; ES, eyespots. HO, head organ. from all the other species of the genus. In the phylogenetic tree, the L. Kolářová et al. / Parasitology International 62 (2013) 300–308 307 basal position of T. mergi compared with the other species of Acknowledgements Trichobilharzia is similar to that of T. stagnicolae. Despite the close phylogenetical relationship between these two species, however, We thank Prof. Michael Doenhoff for reviewing the English lan- T. mergi and T. stagnicolae differ in their morphology and some life-cycle guage of this article. In the Czech Republic, the studies were financially characteristics. Whereas adults and larvae of T. mergi were found in M. supported by the Charles University in Prague (Research Programs serrator and Radix spp., respectively, eggs and cercariae of T. stagnicolae PRVOUK No. P25/LF1/2 and UNCE–Grant No. 204017) and the Grant were detected in Mergus merganser and Stagnicola emarginata,respec- Agency of the Ministry of Health (IGA MZ CR NT 13108-4/2012). In Ice- tively [12]. As for various species of bird schistosomes (e.g., [1]) hosts of land, the authors would like to thank Thorkell Lindberg Thórarinsson different avian species may also participate in the parasite life cycle of and Adalsteinn O. Snaethorsson, North Iceland Nature History Center T. mergi. But conditions (e.g., host specificity, infection dose) under for invaluable help by collecting snails and birds. Financial support which birds become infected remain to be explained. For example, was provided by the Research Fund of the University of Iceland and Brant and Locker [11]werenotabletofind adult flukes of T. stagnicolae the Jules Verne Program for French–Icelandic scientific cooperation. (eggs of which they detected in faeces of naturally infected M. merganser) In France, financial support for this study was provided by the ONCFS in experimentally infected young and peking , i.e. in birds (Office National de la Chasse et de la Faune Sauvage). of the same order as for mergansers (Anseriformes), whereas McMullen and Beaver [35] detected adults of these flukes in experimentally infected canaries (Passeriformes). References The occurrence of bird schistosomes is delineated by distribution of definitive and intermediate hosts. In the case of T. mergi, the definitive [1] Horák P, Kolářová L, Adema CA. Biology of the schistosome genus Trichobilharzia. 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