Sci Parasitol 15(1-4):19-26, December 2014 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE

First description of lutzi from Peru using SEM

Omar M. Amin1, Richard A. Heckmann2

1 – Institute of Parasitic Diseases, 11445 E. Via Linda, # 2-419, Scottsdale, AZ 85259, USA. 2 – Brigham Young University, Department of Biology, 4102 LSB, Provo, UT 84602, USA. Correspondence: Tel. 480-767-2522, Fax 480-767-5855, E-mail [email protected]

Abstract. Our SEM and microscopic studies of specimens of Pseudoacanthocephalus lutzi (Hamann, 1891) collected from the Peru coast toad Chaunus limensis Werner, 1901 (Bufonidae) in Peru provide the first description of Peruvian material and shed new light on its external morphology and internal structures. The dermal micropores pitted proboscis hooks and anterior trunk collar are described for the first time. Pseudoacanthocephalus lutzi [syns. lutzi Hamann, 1891; lutzi (Hamann, 1891) Meyer, 1932; Acanthocephalus saopaulensis Smales, 2007; Pseudoacanthocephalus saopaulensis (Smales, 2007) Arredondo and Gil de Pertierra, 2009] has undergone a number of taxonomic name changes. Descriptions varied especially in the proboscis armature, cement gland number, extension of lemnisci, and egg size. They were, however, within the acceptable range of intraspecific variations of P. lutzi. These variations were at the root of describing conspecific taxa as new species or relegation to other genera. Anuracanthorhynchus lutzi Bursey and Goldberg, 2007 from Costa Rica is not P. lutzi but is another species. Comparisons with specimens from Argentina, Brazil, and Paraguay are also made.

Keywords: ; Pseudoacanthocephalus lutzi; SEM; Chaunus limensis; Peru. Received 05/09/2014. Accepted 09/12/2014.

Introduction and Peru (Barrera et al., 1988; Naupay, 1973; Tantaleán, 1976; Tantaleán et al., 2005). Pseudoacanthocephalus lutzi Hamann, 1891 (Arredondo and Gil de Pertierra, 2009) was Our specimens were obtained from the Peru originally described as Echinorhynchus lutzi coast toad Chaunus limensis Werner, 1901 from the Rhinella marina (Linnaeus, (Bufonidae). Other hosts from Peru include 1758) in Brazil. Meyer (1932) transferred it to Bufo arequipensis Vellard, 1959, Bufo Acanthocephalus Koelreuther. It has been flavolineatus Vellard, 1959, the cane toad reported from various and Rhinella marina and Bufo trifolium Tschudi, in Argentina (Arredondo and Gil de Pertierra, 1845 in Arquibpa, Huánuco, Chincha, Ica, 2009; Gutierrez et al., 2005; Lajmanovich and Nasca, Palpa, Ica, Lima, and Ucayali (Tantaleán Martinez de Ferrato, 1995), Uruguay (Cordero, et al., 2005). The assignment of “B. limensis, B. 1933), Brazil and Paraguay – Smales, 2007, arequipensis, B. flavolineatus, and B. trifolium to who also included a description of the the Bufo spinulosa” Weigmann, 1834 group is conspecific A. saopaulensis (Smales, 2007) – based on the proposals of some including those

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of Córdova (1999). Acanthocephalans were not Samples were then mounted on SEM sample found in 9 other species of amphibians from mounts, sputter coated with 15 nm AuPd (gold- Peru (McAllister et al., 2010). pallidium), and observed in an FEI Helios Dual Beam Nanolab 600 (FEI, Hillsboro, Oregon) Chaunus limensis is endemic to Peru. Its natural Scanning Electron Microscope with digital habitats are rivers deserts, sandy shores, images obtained in the Nanolab system (FEI X arable land, and rural gardens (Angulo et al., L30 ESEMFEG) and then transferred to a USB. 2004). None of the reports of P. lutzi from Peru by Barrera et al. (1988; a symposium abstract), Measurements are in micrometers, unless Naupay (1973; unpublished bachelor’s thesis), otherwise stated. Range values are followed by Tantaleán (1976), and Tantaleán et al. (2005) the mean in parentheses. Length included a taxonomic description of Peruvian measurements are given before the width; the specimens. We describe Peruvian specimens of latter refers to maximum width. Trunk length P. lutzi for the first time. does not include the neck, proboscis, or bursa. Eggs refer only to fully developed eggs. For illustrations of internal anatomy, see Voucher specimens were deposited at the Arredondo and Gil de Pertierra (2009, figures Harold W. Manter Laboratory (HWML) 2, 4) as illustrative presentation of our collection nos. 74991-74993, at the University Peruvian material. Their figures 3 A-F are poor of Lincoln, Nebraska. SEM depictions of contracted and distorted structures: 2 proboscides, posterior trunk, Results bursa, and egg. The following results are based on a Morphological variations in proboscis microscopic study of 7 sexually mature whole armature, cement glands, and egg size were mounted specimens (3 males and 4 females) noted in specimens from above host species with eggs and sperms, and an SEM study of a and those from Argentina, Brazil, Paraguay, few more collected from the Peru coast toad C. Uruguay, and partially attributed to host limensis. factors (Arredondo and Gil de Pertierra, 2009). Variations observed with microscopy and SEM Description of our Peruvian material in our specimens were noted herein. We provide the first description of P. lutzi from General. Echinorhynchidae, with characters of Peru with new features reported for the first the . Shared structures larger in females time. than males; trunk about twice as large in females. Trunk cylindrical, aspinose, somewhat Materials and methods wider anteriorly and tapering towards anterior trunk collar, rounded posteriorly in males and Dr. José Iannacone, President of the Asociación females (figure 1). Collar longitudinally Peruana de Helmintologia e Invertebrados furrowed, more demonstrable in worms with Afines (APHIA), Lima, Peru kindly provided in partially retracted proboscis (figure 2). Body 2009 1 vial containing 70% ethanol preserved wall distinctly and uniformly very thick but specimens used for SEM studies and 7 carmine narrowing at extremities, with reticular stained whole mounted specimens of P. lutzi on lacunar system and micropores of different 7 slides collected from the Peru coast toad. diameter and distribution in various trunk Specimens were collected in the Lima area, regions (figures 6, 7). Neck unremarkable. Peru (12°02′35″ S, 77°01′41″ W) in July, 1987 Proboscis cylindrical, progressively widening and January, 1988. posteriorly, with 16-17 (rarely 14) longitudinal and regularly alternating rows of 6-7 rooted For SEM, a few specimens of P. lutzi previously hooks each. Hooks thin (figures 2-4), pitted fixed in 70% ethanol were placed in CPD (figure 5), progressively increasing in size baskets and dehydrated using ETOH series of posteriorly figure 3). Roots about half as long 95% and 100% for at least 10 minutes per soak as blades, simple, spatulate, directed followed by critical point drying (Lee, 1992). posteriorly, with no manubria. Proboscis

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receptacle double-walled, simple, with cerebral Discussion ganglion at its posterior end and cellular elements associated with retractor muscles Specimens of P. lutzi show marked exterior to its posterior tip. Lemnisci claviform intraspecific morphological variations in hook extending shortly past posterior end of number and size, size and extension of receptacle. lemnisci, number of cement glands, and size of eggs in various host species and geographical Male (based on 3 adults with sperm). Trunk locations. These variations in our Peruvian 6.80-7.75 (7.00) mm long by 1.25-1,95 (1.50) and other specimens are summarized below. mm wide at middle. Proboscis 437-603 (513) We also report 3 new features for the first long by 312-395 (343) wide posteriorly, with time: pitted proboscis hooks, anterior trunk 16-17 rows of 6-7 rooted hooks each. Hook collar, and dermal micropores of variable length from anterior 57-72 (68), 62-77 (70), diameter and distribution. 70-85 (77), 75-85 (80), 75-87 (82), 87-90 (85). Proboscis receptacle 0.83-1.19 (1.00) Trunk and body wall mm long by 0.28-0.42 (0.34) mm wide. Lemnisci 0.77-1.18 (0.86) mm long by 0.16- Trunk with anterior collar clearly 0.26 (0.21) mm wide posteriorly. Whole demonstrable in specimens with partially reproductive system post-equatorial. Testes retracted proboscis. Such a collar was rounded, contiguous; anterior testis 624-800 sketched in figure 1B of Arredondo and Gil de (700) long by 458-800 (593) wide, slightly Pertierra (2009), but not discussed. The body larger than posterior testis 572-875 (676) wall in our Peruvian specimens was very thick long by 406-775 (560) wide. Cement glands with notable differences in micropore pyriform 4-6, 400-575 (498) long by 275-281 diameter and distribution in various trunk (327) wide, in compact cluster of 2 sets regions reflecting differential absorption of ducted to 2 posterior cement reservoirs 572- nutrients as has been previously described in 650 (599) long by 218-300 (258) wide. other genera and species including Gonopore terminal. Papillated bursa (figure Leptorhynchoides polycristatus Amin, 10), 676 long by 957 wide (n=1). Heckmann, Halajian, El-Naggar, Tavakol, 2013, Neoechinorhynchus zabensis Amin, Female (based on 4 mature specimens, some Abdullah and Mhaisen 2003, Acanthosentis gravid). Trunk 11.80-16.50 (14.12) mm long tilapiae (Baylis, 1948) in Amin and Heckmann by 1.75-2.32 (2.02) mm wide at middle. Body (2012), (Müller, 1776) wall 275-450 (367) and 300-575 (429) thick Lühe, 1911 in Amin et al. (2011), and dorsally and ventrally, respectively. Proboscis Acanthocephalus ranae (Schrank, 1788) Lühe, 510-676 (593) long by 385-421 (402) wide at 1911 in Heckmann et al. (2011). Wright and base with 16-17 (rarely 14) rows of 6-7 Lumsden (1970) and Byram and Fisher rooted hooks each. Hook length from anterior (1973) reported that these peripheral pore 67-80 (72), 72-92 (78), 74-90 (80), 75-95 are continuous with canalicular crypts. These (83), 75-95 (84), 80-100 (88), 92 (n=1). crypts appear to “constitute a huge increase in Proboscis receptacle 1.15-1.35 (1.26) mm external surface area… implicated in nutrient long by 0.21-0.52 (0.44) wide. Lemnisci 1.29- uptake.” Whitfield (1979) estimated a 44–fold 1.53 (1.37) mm long by 0.29-0.42 (0.34) wide increase at a surface density of 15 posteriorly. One specimens with 1 branched invaginations per 1 μm2 of the tegumental lemniscus 750 long by 124 wide and 750 long surface of Moniliformis moniliformis (see by 177 wide. Reproductive system 1.23 long Byram and Fisher, 1973). (9% of trunk length) (n=1). Gonopore subterminal-ventral, 775-785 (817) anterior Hooks to posterior end of trunk (figures 8, 9). Eggs fusiform elongate without polar prolongation The observation that all hooks of specimens of of fertilization membrane and with fibrillar P. lutzi from Peru are pitted is noted here for coat (figure 11), 70-77 (75) long by 22-27 the first time. We suspect that a close study of (25) wide. specimens from other locations and hosts will

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demonstrate the same. Our Peruvian (Van Cleave, 1931), with a wide host and specimens are similar to geographical distribution in North America, Pseudoacanthocephalus nguyenthileae Amin, the number of cement glands ranged from 0- Ha, Heckmann, 2008; in the pattern of hook 12 (normally 6) (Amin, 1984). Thirty-seven size gradation being progressively longer percent of 211 males and 10% of 1,801 males posteriorly. In Argentinean specimens, hook of A. dirus from New England and from length increases from apex to mid proboscis Wisconsin had other than 6 cement glands (4- then “decreases slightly or not towards base” 5, 7-11, and 0-5, 7-12, respectively) (Amin, (Arredondo and Gil de Pertierra, 2009). The 1975). For a detailed discussion of the cement maximum length of posterior hooks from our glands numbers and patterns in the Peruvian female specimens was 100 but Acanthocephala, see Amin et al. (2008). reached as long as 115 and 154 in specimens from Argentinean and Brazilian females It is apparent from above comparisons that (Arredondo and Gil de Pertierra, 2009 and these features fall within the normal range of Smales, 2007, respectively). The number of intraspecific variations for this species. The rows and number of proboscis hooks per row relegation of P. lutzi to Anuracanthorhynchus were reported to be 14-18 and 5-7 in Bursey, Vrcibradic, Hatano, Rocha, 2006 by specimens from Argentina (Arredondo and Gil Bursey and Goldberg (2007) based on having de Pertierra, 2009), 14 and 6 from Argentina 4 cement glands is invalid. (Lajmanovich and Martinez de Ferrato, 1995), 16 and 5-6 from Brazil, and 12-14 and 5-6 Eggs from Paraguay (Smales, 2007), 14-17 and 6-7 from Peru (this paper). The eggs in the body cavity of our Peruvian specimens were elongated at the poles but not Lemnisci so much so in SEM images, 70-77 (75) X 22-27 (25). Eggs were more ovoid 54-88 (77) X 19- In our Peruvian specimens, the lemnisci 39 (31) in specimens from Argentina invariably extended a short distance posterior (Arredondo and Gil de Pertierra, 2009; figures to the posterior end of the receptacle. In 2E, 3C). Other specimens from Argentina also specimens from Argentina, the lemnisci were had “huevos ovoides” (Lajmanovich and slightly shorter, longer or the same length as Martinez de Ferrato, 1995) but no the receptacle (Arredondo and Gil de measurements were provided. Eggs were Pertierra, 2009). In specimens from Brazil, the comparable in size in specimens from lemnisci were slightly shorter than the Paraguay (75-86 X 26-30) but markedly larger receptacle in males and longer in females, but in specimens originally designated as shorter than the receptacle in both sexes in Acanthocephalus saopaulensis from Brazil: 89- specimens from Paraguay (Smales, 2007). 102 (95) X 26-33 (30) also described as ellipsoidal (ovoid) by Smales (2007; figure 6). Cement glands Specimens from Costa Rica Our Peruvian specimens had 4-6 cement glands each. Four to six and 4 glands were Bursey and Goldberg (2007) described reported in specimens from Argentina by specimens of presumed P. lutzi from other Arredondo and Gil de Pertierra (2009) and species of frogs in Costa Rica and assigned Lajmanovich and Martinez de Ferrato (1995), them to the genus Anuracanthorhynchus respectively, 6 from Brazil (Smales, 2007), 4 Bursey, Vrcibradic, Hatano, Rocha, 2006 based from Paraguay (Smales, 2007). Meyer (1932) on having 4 cement glands, oval testis, reported 4 glands (with an exclamation mark) absence of trunk spination, and hooks of equal as did Golvan (1969) but Hartwich (1956) length. reported 5. A wide range of intraspecific variation in the number of cement glands is not uncommon, e.g., in

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Figures 1-5. SEM of specimens of Pseudoacanthocephalus lutzi from Chaunus limensis 1. A female specimen showing the gradual widening of the trunk toward the anterior end and the rounded posterior end. 2. A partially retracted proboscis and anterior trunk collar (left). 3. A near apical perspective of a proboscis showing the smaller anterior hooks. 4. A lateral perspective of thin hooks near the anterior and of the proboscis. 5. A greater magnification of the surface of one hook showing diffuse pits; all hooks were pitted.

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Figures 6-11. SEM of specimens of Pseudoacanthocephalus lutzi from Chaunus limensis. 6, 7. Micropores of different diameter and density on the cuticle of the mid-trunk and the hind trunk of a male specimen, respectively. 8. A ventral view of the female gonopore showing detail of the genital orifice. 9. A near lateral view of the posterior end of the same female specimen showing the definate subterminal-ventral position of the gonopore and the rounded posterior end. 10. A terminal perspective of a partially retracted bursa. 11. An egg. Note the pattern of the fibrillar coat through the outer membrane.

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We find that these specimens are of a different to species of Pseudoacanthocephalus species than P. lutzi because, based on Bursey Petrochenko, 1956 (Echinorhynchidae) and and Goldberg (2007): (1) the hook roots were Sphaerechinorhynchus Johnston and Deland, shown to be pointed posteriorly and not 1929 (Plagiorhynchidae). J. Parasitol. 94:181- spatulate with blunt end (see figure 13 – 189. Amin O.M., Heckmann R.A., El-Naggar A.M. 2011. Bursey and Goldberg, 2007); (2) the neck is Revisiting the morphology of Acanthocephalus long and narrower than proboscis (see figure lucii (Acanthocephala: Echinorhynchidae) in 12 – Bursey and Goldberg, 2007); (3) the testes Europe, using SEM. Sci. Parasitol. 12:185-189. are noncontiguous (see figure 11 – Bursey and Angulo A., Sinsch U., Aguillar Puntriano C., Arizabal Goldberg, 2007); and (4) most importantly, the W., Lehr E. 2004. Bufo limensis female gonopore appears definitely terminal (http://www.iucnredlist.org/search/details. (see figure 15 – Bursey and Goldberg, 2007) php/54691/all). 2006 IUCN Red List of despite the text reference to a “subterminal” Threatened Species. position. The gonopore of female P. lutzi is Arredondo N.J., Gil de Pertierra A. 2009. positioned a marked distance anterior to the Pseudoacanthocephalus lutzi (Hamann, 1891) comb. n. (Acanthocephala: Echinorhynchidae) posterior end of the trunk. We therefore, for Acanthocephalus lutzi (Hamann, 1891), conclude that the Costa Rican specimens are parasite of South American amphibians. Folia not P. lutzi and belong in a different taxon. We Parasitol. 56:295-304. know what the Costa Rican specimens are not Barrera J., Maldonado M., Solis M.I. 1988. but a generic assignment will have to await the Hemlmintos paràsitos de Bufo spinulosus examination of reference material. limensis del department de Ica. Lib. Res. IX Cong. Nac. Biol. y IV Simpos. Nac. Educ. Cien. Biol. Acknowledgments Piura. Res. 179. Bursey C.R., Goldberg S.R. 2007. New species of We are grateful to Dr. José Iannacone, Hedruris (Nematoda: Hedruridae), Anuracanthorhynchus lutzi (Hamann, 1891) n. President of the Asociación Peruana de comb., and other helminths in Lithobates Helmintologia e Invertebrados Afines (APHIA), warszewitschii (Anura: Ranidae) from Costa Lima, Peru for providing the research material Rica. Carib. J. Sci. 43:1-10. from Peru. We are also grateful to Mr. Michael Byram J.E., Fisher F.M. 1973. The absorptive surface D. Standing and Miss Kyrie Carpenter, of Moniliformis dubius (Acanthocephala). J. Fine Microscopy lab., College of Life Sciences, Struct. Tiss. Cell 5:553-579. Brigham Young University, Provo, Utah, USA Cordero E.-H. 1933. Sur quelques acanthocéphales for their help with the production and editing de l’Amerique Méridionale. I. Ann. Parasitol. the SEM plates. 11:271-279. Córdova J.H. 1999. On karyomorphs, cladistics and taxonomic status of the Bufo spinulosus species group (Amphibia: Anura) in Peru. Stuttgarter References Beitr. Naturk. Ser. A. (600):1-28. Golvan Y.J. 1969. Systematique des Acanthocephales Amin O.M. 1975. Variability in Acanthocephalus (Acanthocephala Rudolphi, 1801), L’ordre des partksidei Amin, 1974 (Acanthocephala: Meyer, 1931, La Echinorhynchidae). J. Parasitol. 61:307-317. superfamille des Echinorhynchidea (Cobbold, Amin O.M. 1984. Variability and redescription of 1876) Golvan et Houin 1963. Mém. Mus. Nat. Acanthocephalus dirus (Acanthocephala: d’Hist. Nat. 47:1-371. Echinorhynchidae) from freshwater fishes in Gutierrez C., Attademo A., Guerrero S., Peltzer P., North America. Proc. Helminthol. Soc. Wash. Lajmanovich R. 2005. Physalaemus biligonigerus 51:225-237. (False-eyed frog) endoparasites. Herpetol. Rev. Amin O.M., Heckmann R.A. 2012. An SEM study of 36:161-162. Acanthogyrus (Acanthosentis) tilapiae Hartwich G. 1956. Südamerikanische (Acanthocephala: Quadrigyridae) from Africa Acanthocephalen aus der Zoologischen documenting previously unreported features Sammlung des Bayerischen Staates. Zoolog. and host parasite interface. Sci. Parasitol. 13:57- Anzeig. 156:299-308. 63. Heckmann R.A., Amin O.M., Tepe Y., Dusen S., Oguz Amin O.M., Ha N.V., Heckmann R.A. 2008. New and M.C. 2011. Acanthocephalus ranae already known acanthocephalans from (Acanthocephala: Echinorhynchidae) from amphibians and reptiles in Vietnam, with keys amphibians in Turkey, with special reference to

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new morphological features revealed by SEM, Smales L.R. 2007. Acanthocephala in amphibians and histopathology. Sci. Parasitol. 12:23-32. (Anura) and reptiles (Squamata) from Brazil Lajmanovich R.C., Martinez de Ferrato A. 1995. and Paraguay with description of a new species. Acanthocephalus lutzi (Hamann, 1891) parasite J. Parasitol. 93:392-398. de Bufo arenarum en El Rio Parana, Argentina. Tantaleán M. 1976. Contribución al conocimiento de Rev. Asoc. Cienc. Nat. Litoral 26:19-23. los helmintos de vertebrados del Perú. Biota Lee R.E. 1992. Scanning electron microscopy and X- 10:437-443. ray microanalysis. Prentice Hall. Englewood Tantaleán M., Sánchez L., Gómez L., Huiza A. 2005. Cliffs, New Jersey, 458 pp. Acanthocephalan from Peru. Rev. Peru. Biol. McAllister C.T., Bursey C.R., Freed P.S. 2010. 12:83-92. Helminth parasites of amphibians and reptiles Whitfield P.J. 1979. The biology of parasitism: an from the Ucayali region, Peru. J. Parasitol. introduction to the study of associating 96:444-447. organisms. Univ. Park Press, Baltimore, 277 pp. Meyer A. 1932. Acanthocephala. In: Dr. H. G. Bronn’s Wright R.D., Lumsden R.D. 1970. The acanthor Klassen und Ordnungen des Tier-Reichs, Vol. 4, tegument of Moniliformis dubius. J. Parasitol. Leipzig: Akad. Verlag. MBH, pp. 1-332. 56:727-735. Naupay I.A. 1973. Helmintos paràsitos de Bufo spinulosus trifolium (Tschudi) de la localidad de Huànuco. Con la descripción de dos nuevas especies y una nueva combinación. Tesis Bachiller Biologia. Universidad Particular “Ricardo Palma”, Lima.

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