DOI: 10.2478/s11686-014-0217-3 © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(1), 115–121; ISSN 1230-2821
New species of Orientatractis (Nematoda: Atractidae), new species of Rondonia (Nematoda: Atractidae) and other helminths in Austrochaperina basipalmata (Anura: Microhylidae) from Papua New Guinea Charles R. Bursey1*, Stephen R. Goldberg2 and Fred Kraus3 1
Department of Biology, Pennsylvania State University, Shenango Campus, Sharon, Pennsylvania 16146, USA; 2 Department of Biology, Whittier College, Whittier, California 90608, USA; 3 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109, USA
Abstract Two new nematode species, Orientatractis hamabatrachos sp. nov. and Rondonia batrachogena sp. nov. (Nematoda: Atractidae), from the gastrointestinal tract of Austrochaperina basipalmata (Anura: Microhylidae) collected in Papua New Guinea are described. Orientatractis hamabatrachos sp. nov. is characterized by the presence of the cephalic end armed with 4 wellsclerotized structures, consisting of 2 “horns” extending outward and downward and immediately below a single well-sclerotized spine. It differs from 5 congeners in spicule lengths and caudal papillae arrangements. Rondonia batrachogena sp. nov. is characterized by the presence of a female cloaca. It differs from 2 congeners primarily in body size. Orientatractis hamabatrachos sp. nov. and Rondonia batrachogena sp. nov. represent the first species assigned to either genus found to infect anurans or to occur in the Australo-Papuan region.
Keywords Nematoda, Atractidae, Orientatractis hamabatrachos sp. nov., Rondonia batrachogena sp. nov., Anura, Microhylidae, Austrochaperina basipalmata, Australo-Papuan region.
Introduction The microhylid frog, Austrochaperina basipalmata (Van Kampen, 1906), (no common name) is endemic to the island of New Guinea, ranging from the Tawarin River in Papua (Indonesia) to the Prince Alexander Mountains southwest of Wewak, Papua New Guinea, from sea level to 1550 m (Zweifel 2000; Kraus 2013). To our knowledge, there are no helminthological reports for this species. The purpose of this paper is to describe 2 new species of Nematoda harbored by A. basipalmata and to provide an initial helminth list for this host.
imens were fixed in neutral buffered 10% formalin. The body cavity was opened by a longitudinal incision and the gastrointestinal tract was removed by cutting across the oesophagus and rectum. The stomach, small intestine and large intestine were examined separately for endoparasites. Only nematodes were found which were placed in lactophenol, allowed to clear and examined under a light microscope. Illustrations were made with the aid of a microprojector. Measurements are in µm with mean ± SD and range in parenthesis unless otherwise stated. Frogs are deposited in the Bernice P. Bishop Museum (BPBM), Honolulu, Hawaii as BPBM 35470, 35472, 35474–35478, 35480–35486.
Materials and Methods Results Fourteen Austrochaperina basipalmata were collected by hand by FK in the Prince Alexander Range, East Sepik Province, Papua New Guinea (15–23 September 2009). Spec-
Four species of Nematoda, Aplectana macintoshii (Stewart, 1914), Cosmocercella phrynomantisi Moravec 1990, an un-
*Corresponding author: [email protected]
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Table I. Site of infection, number of helminthes, prevalence, range of infection and USNPC accession numbers for 4 helminth species in Austrochaperina basipalmata from Papua New Guinea Helminth Aplectana macintoshii Cosmocercella phrynomantisi Orientatractis hamabatrachos sp. nov. Rondonia batrachogena sp. nov.
Site
Number
Large intestine Large intestine Intestine Stomach
11 185 2816 344
described species of Orientatractis Petter, 1966 and an undescribed species of Rondonia Travassos, 1920 were found. Site of infection, number of helminths, prevalence, mean intensity and range are given in Table I. Selected helminths were deposited in the United States National Parasite Collection (USNPC), Beltsville, Maryland (Table I). Description of the 2 new species follows. Orientatractis hamabatrachos sp. nov. (Figs 1–10) Description: Small fusiform nematodes, body more or less cylindrical along its length; both males and females with relatively long, thin tails. Cuticle with longitudinal striations. Lateral alae present beginning just posterior to lips and terminating near tail midpoint in both males and females. Anterior extremity bluntly rounded; mouth terminal, funnel shaped, with 6 lips, 4 submedian lips each lip with a 1 papilla and a bicornuate, sclerotized structure (2 recurved pointed spines with fused root), and 2 lateral lips, each supporting 1 large amphid with a pair of small sclerotized spines lateral to the amphidial pore. Oesophagus divided into anterior muscular portion and posterior glandular portion, terminating in a prominent valved bulb. Nerve ring just posterior to junction of oesophageal regions. Excretory pore at mid point of glandular esophageal portion. Spicules unequal, pointed distal tips. Vulva opens close to anus. Viviparous. Male (holotype and 11 paratypes): Length 3.76 ± 0.37 mm (3.20–4.35 mm); width at mid-body 86 ± 10 (73–104). Oesophagus 393 ± 20 (372–433) in length, divisible into 3 regions: anterior muscular portion 125 ± 7 (110–134) long; middle glandular portion 202 ± 16 (189–244) long; valved bulb 66 ± 9 (55–79) long by 46 ± 5 (40–55) wide. Nerve ring 190 ± 11 (177–214) and excretory pore 275 ± 17 (244–317) from anterior end, respectively. Gubernaculum cylindrical, approximately 6 in width, slight expansion in proximal third, 61 ± 5 (55–67) in length. Spicules similar, transversely striated, unequal, left spicule 155 ± 6 (146–165) long, right spicule 89 ± 8 (79–98) long, distal ends pointed. Posterior half of body most often in 3–4 tight coils. Tail 363 ± 28 (306–408) in length; abrupt narrowing at midpoint of tail. Eight pairs of caudal papillae plus 1 unpaired papilla on midline just anterior to cloaca: 2 pairs precloacal, anterior-most pair approximately 75 anterior of cloaca, second pair approximately 10 anterior of cloaca; 1 pair adcloacal; 5 pairs post cloacal, 2 pairs approximately 10 and 20 posterior to cloaca, 2 ventrolateral pairs and
Prevalence (%) 5/14 5/14 11/14 7/14
36% 36% 79% 50%
x ± SD
Range
USNPC number
2.2 ± 1.3 37.0 ± 27.9 256 ± 241 49.1 ± 24.4
1–4 1–146 4–639 22–82
106961 106962 106956 106960
1 dorsolateral pair just anterior to narrowing at tail midpoint. Phasmids open near posterior end of lateral alae. Female (allotype and 11 paratypes): Length 4.24 ± 0.39 mm (3.39–4.86 mm); body cylindrical throughout. Anterior half of body more narrow, width at level of oesophagus 60 ± 6 (52–73); posterior half of body wider, width at level of ovary 116 ± 12 (98–134). Oesophagus 452 ± 62 (402–621) in length, divisible into 3 parts: anterior muscular portion 140 ± 9 (128– 159) long; middle glandular portion 246 ± 58 (195–396) long; valved bulb 66 ± 8 (55–79) long by 48 ± 4 (43–55) wide. Nerve ring 202 ± 16 (183–232) and excretory pore 297 ± 32 (223–366) from anterior end, respectively. Tail 538 ± 35 (510– 612) in length, slender and sharply pointed. Vulva 655 ± 43 (612–740) from posterior end of tail filament, 118 ± 17 (102– 153) anterior of anus. Vagina short; monodelphic, 1 to 5 thinshelled eggs 198 ± 12 (183–214) × 79 ± 6 (73–92) in various stages of development present in anterior portion of uterus; hatched larvae present often filling uterus; single ovary near mid-body. Phasmids open near posterior end of lateral alae. Type host: Austrochaperina basipalmata (Van Kampen, 1906); BPBM 35484 collected 21 September 2009. Type locality: Mindangua Stream, Prince Alexander Range, East Sepik Province, Papua New Guinea (3°21´48˝S, 143°17´43˝E; 410 m elevation). Site of infection: Small and large intestine. Type specimens: Holotype male USNPC 106953; allotype female USNPC 106954; paratypes USNPC 106955; voucher specimens USNPC 106956. Etymology: The new species is named for its parasitism of a host group: hama, G. “at the same time” and batrachos, G. “frog”. Remarks Currently Orientatractis Petter, 1966 is comprised of O. levanhoai Petter, 1966 from the tortoise Indotestudo elongata (reported as Testudo elongata) collected at the Botanical Gardens of the Pasteur Institute of Saigon, Vietnam; O. leiperi Buckley, 1969 from the tortoise Podocnemis vogli collected in Colombia, South America; O. campechensis Gonzalez-Solis and Moravec, 2004 from the fishes Paraneetroplus bifasciatus (reported as Vieja bifasciata) and Cichlasoma pearsei collected in Mexico; O. chiapasensis Gonzalez-Solis and Moravec, 2004 from the fishes Theraps intermedius (reported as Vieja intermedia) and Tomocichla tuba collected in Mexico;
Two new species of atractid nematodes
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Figs 1–10. Orientatractis hamabatrachos sp. nov. 1. Female anterior end, lateral view. 2. Female, en face view. 3. Female, anterior end, lateral view. 4. Male, spicules and gubernaculum. 5. Male, posterior end, lateral view. 6. Female, posterior end. 7. Female, ovarian region. 8. Female, entire, lateral view. 9. Male, entire, lateral view. 10. Male, semidiagrammatic, posterior end, ventral view
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Table II. Selected characters of species of Orientatractis Orientatractis spp. (male) O. asymmetrica O. campechensis O. chiapasensis O. hamabatrachos sp. nov. O. levanhoai O. leiperi Orientatractis spp. (female) O. asymmetrica O. campechensis O. chiapasensis O. hamabatrachos sp. nov. O. levanhoai O. leiperi
Length (mm)
Oesophagus
Right spicule
Left spicule
Gubernaculum
Caudal papillae*
4.1–4.3 3.0–3.5 2.7–1.9 3.2–4.4 3.3 3.0–3.9
524–558 388–469 361–459 372–433 390 570
86–104 84–106 60–68 79–98 90 160
220–239 430–506 204–238 146–165 170 470
56–66 62–74 44–49 55–67 40 76
8:4:6 + 1 6:2:10 + 1 6:2:10 + 1 4:2:10 + 1 6:0:10 6:2:10 + 1
Length (mm)
Oesophagus
Nerve ring from anterior end
Excretory pore from anterior end
Tail
Vulva from posterior end
3.7–5.5 3.3–4.3 2.0–3.8 3.4–4.9 3.4 2.7–4.3
532–596 417–491 360–475 402–621 390 660
264–292 209–243 178–254 183–232 100 266
424–484 327–437 281–410 223–366 350 480
820–1,080 866–1,246 616–851 510–612 1,000 880
920–1,192 946–1,322 380–722 612–740 1,050 960
*precloacal:adcloacal:postcloacal + median
O. asymmetrica Gibbons and Platt, 2006 from the turtle Rhinoclemmys pulcherrima collected in Costa Rica (Petter, 1966; Buckley, 1969; Gonzalez-Solis and Moravec, 2004; Gibbons and Platt, 2006). Orientatractis hamabatrachos sp. nov. is the first species found in an amphibian or in the Australo-Papuan region and is assigned to Orientatractis based upon the presence of specialized cuticular formations around the mouth. The 6 species can easily be separated based upon spicule length and caudal papillae arrangement (Table II). Rondonia batrachogena sp. nov. (Figs 11–22) Description: Small nematodes, body more or less cylindrical throughout its length; males fusiform; females fusiform but developing truncated posterior at maturity. Depending upon microscope focus, both transverse and longitudinal striations can be seen in cuticle. Narrow lateral alae present beginning just posterior to lips and terminating near tail midpoint in both males and females. Anterior extremity bluntly rounded; terminal mouth, opening triangular surrounded by 3 lips, each lip with 2 simple, pedunculate papillae; 1 amphid on each ventrolateral lip. Oesophagus divided into anterior muscular portion and posterior glandular portion, terminating in a prominent valved bulb. Nerve ring just posterior to junction of oesophageal parts. Excretory pore posterior to oesophageal bulb; pore surrounded by circular striated cuticle approximately 30 in diameter. Spicules unequal, pointed distal tips. Both males and females with cloaca. Viviparous. Male (based on holotype and 11 paratypes): Length 2.13 ± 0.11 mm (2.04–2.42 mm); width at mid-body 94 ± 16 (67– 113). Oesophagus 380 ± 13 (360–411) in length, divisible into 3 parts: anterior muscular portion 242 ± 10 (232–268) long; middle glandular portion 75 ± 5 (67–85) long; valved bulb 63
± 3 (58–67) long by 56 ± 4 (49–61) wide. Nerve ring 256 ± 10 (244–274) and excretory pore 572 ± 21 (549–616) from anterior end, respectively. Gubernaculum with cylindrical body, approximately 6 in width, 61 ± 4 (55–67) in length, supporting at midpoint lateral projections approximately 15 in length. Spicules similar, transversely striated, unequal, left spicule 238 ± 8 (220–250) long, right spicule 78 ± 7 (73–98) long, distal ends pointed. Tail 150 ± 8 (134–159) in length; thick anterior portion 86 ± 8 (73–98) long supporting thin terminal filament 64 ± 9 (49–79) in length. Tail bent ventrally to form a “shepherd’s crook”. Eight pairs of caudal papillae: 3 pairs precloacal: 5 pairs post cloacal, 3 pairs ventrolateral in position, 2 lateral pairs, 1 pair just posterior to cloaca, 1 pair just anterior to tail filament. Phasmids open near posterior end of lateral alae. Female (based on allotype and 11 paratypes): Length 1.77 ± 0.14 mm (1.58–1.99 mm); width at level of ovary 116 ± 16 (92–153). Body cylindrical throughout, immature females with elongate posterior that changes with maturity to form a truncate posterior in which the base of the tail is drawn into body. Cloaca empties into crease caused by tail withdrawal. Oesophagus 384 ± 14 (363–405) in length, divisible into 3 parts: anterior muscular portion 243 ± 10 (229–256) long; middle glandular portion 77 ± 5 (73–85) long; valved bulb 64 ± 5 (55–73) long by 53 ± 5 (43–61) wide. Nerve ring 258 ± 14 (244–284) and excretory pore 551 ± 38 (488–653) from anterior end, respectively. Tail 214 ± 14 (189–232) in length. Vulva opening into rectum; vagina short; monodelphic, 1 to 3 thin-shelled eggs 169 ± 9 (158–183) × 81 ± 7 (73–99) that are in various stages of development in anterior portion of uterus; hatched larvae present and often filling uterus; single ovary near middle of body. Phasmids open near posterior end of lateral alae.
Two new species of atractid nematodes
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Figs 11–22. Rondonia batrachogena sp. nov. 11. Female, mature, entire, lateral view. 12. Female, early gravid stage, lateral view. 13. Female, larval development stage, lateral view. 14. Female, hatched larval stage, lateral view. 15. Male, entire, lateral view. 16. Female, en face view. 17. Female, anterior end, dorsal view. 18. Female, posterior end, lateral view. 19. Male, posterior end, lateral view. 20. Male, posterior end, ventral view. 21. Male, anterior end, lateral view. 22. Female, excretory pore
Type host: Austrochaperina basipalmata (Van Kampen, 1906); BPBM 35475 collected 17 September 2009. Type locality: Imuk, Prince Alexander Range, East Sepik Province, Papua New Guinea (3°21´4˝S, 143°17´49˝E, 184 m elevation).
Site of infection: Stomach Type specimens: Holotype male USNPC 106957; allotype female USNPC 106958; paratypes USNPC 106959; voucher specimens USNPC 106960. Etymology: The new species is named for life history: batrachos G. “frog” and gena, L. “born in”.
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Table III. Selected characters of Rondonia Rondonia spp. (male)
Length (mm)
2.0–2.4 R. batrachogena sp. nov. 3.9–8.1 R. rondoni Males unknown R. lophii
Oesophagus
Right spicule
Left spicule
Gubernaculum
Caudal papillae*
360–411 524–993 –
73–98 100–130 –
220–250 240–290 –
55–67 60–70 –
6:0:10 6:2:12 –
Tail
Egg
Nerve ring from Excretory pore anterior end from anterior end
Rondonia spp. (female)
Length (mm)
Oesophagus
R..batrachogena sp. nov.
1.6–2.0
363–405
244–284
488–653
189–232
158–183 × 73–99
R. rondoni
6.8–9.9
1003–1035
530–585
1120–1220
116–1320
480–560 × 220–260
2.7
480
260
700
310
42 × 29
R. lophii
*precloacal:adcloacal:postcloacal + median
Remarks Currently Rondonia Travassos, 1919 is comprised of R. rondoni Travassos, 1920 from various fish species, Piaractus brachypomus (type host), Acanthodoras spinosissimus, Doras granulosus, Luciopimelodus pati, Myeletes torquatus, Myleus asterias, Synodontis clarias, Trachydoras paraguayensis and Zungaro zungaro, collected in Mexico and South America (Travassos, 1923; Travassos et al., 1928; Baylis, 1936; Costa, 1962, 1963; Santos et al., 1979; Hamann, 1982; Moravec et al., 1992), and R. lophii Gallego Berenguer, 1947 also from a fish species, Lophius piscatorius, collected on the Cantabrico Coast, Spain (Gallego Berenguer, 1947). Rondonia batrachogena sp. nov. is assigned to Rondonia based upon the presence of a cloaca in the female and is the first species to be reported from an amphibian or from the Australo-Papuan region. The 3 species can easily be separated on body size (Table III).
Discussion We have assigned the 2 new species to the Atractidae because the oesophagus is clearly divided into an inflated muscular anterior region, a posterior glandular region and a bulb with valve, the male lacks a precloacal sucker, and the female is viviparous (Chabaud 1978). Adamson and Baccam (1988) listed 14 genera for the Atractidae, which were divided into 2 groups: those characterized by didelphy, Fitzsimmonsnema Petter, 1966, and Probstmayria Ransom, 1917, and those characterized by monodelphy, Atractis Dujardin, 1945, Cobboldina Leiper, 1911, Crossocephalus Railliet, 1909, Cyrtosomum Gedoelst, 1919, Grassenema Petter, 1969, Labiduris Schneider, 1866, Leiperenia Khalil, 1922, Monhysterides Baylis and Daubney, 1922, Orientatractis Petter, 1966, Paratractis Sarmiento, 1959, Proatractis Caballero, 1971, and Rondonia Travassos, 1919. To the list of didelphic atractid genera should be added Nouvelnema Petter, 1959, and to the list of monodelphic atractid genera should be added Buckleyatractis Khalil and Gibbons, 1988, Diceronema Gibbons,
Knapp and Drecek, 1966; Klossinemella Costa, 1961, Paraorientatractis Gibbons, Khalil and Marinkelle, 1997, Podocnematractis Gibbons, Khalil and Marinkelle, 1995, Rhinoclemmysnema Gibbons and Platt, 2006, and Pneumoatractis Bursey, Reavill and Greiner, 2009 (Bursey et al. 2009). It should be noted that Moravec and Thatcher (1997) moved Proatractis to synonymy with Klossonemella. Members of the Atractidae are unusual in that eggs hatch and larvae develop to third stage in utero to autoinfect the current host (Anderson 2000). Their transmission from host to host is not understood. The life cycles of Orientatractis and Rondonia have not been determined, however Costa (1962) believed that larvae of Rondonia (previously known only from fish) pass from the host to infect other fish directly. Petter (1966) claimed that tortoises became infected only after attaining sexual maturity and wondered if transmission occurred during mating.
Acknowledgements. We thank Ezra Teodoro and Jeanette Arreola for help with dissections; Peggy Firth for preparing the illustrations constituting Figs 1–22; Justin Jiramori, Agustin Nakung, Charlie Wintawa, Damien Yaripma, and Ben Yawi for field or logistical assistance. We thank the PNG National Museum and Art Gallery for providing in-country collaborative assistance and the Department of Environment and Conservation; National Research Institute; and the East Sepik Provincial Government for permission to conduct this research. This research was supported by National Science Foundation grant EEB-0743890.
References Adamson M.L., Baccam D. 1988. Systematic revision of the Atractidae sensu Chabaud (1978) (Nematoda; Cosmocercoidea): Maracaya belemensis n. sp. and Aplectana albae n. sp. from Amphisbaena alba in Brazil. Canadian Journal of Zoology, 66, 1857–1864. DOI: 10.1139/z88×268. Anderson R.C. 2000. Nematode Parasites of Vertebrates: Their development and transmission. 2nd Ed. CABI Publishing, Wallingford, Oxon, UK, 650 pp. Baylis H.A. 1936. A nematode genus Rondonia Travassos. Annals and Magazine of Natural History, Series 10, 17, 606–609.
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Buckley J.J.C. 1969. On a remarkable oxyurid nematode, Orientatractis leiperi n. sp., (Atractidae) from a South American tortoise, Podocnemis vogli. Journal of Helminthology, 43, 218–286. DOI: 10.1017/S0022149X0000482X. Bursey C.R., Reavill D., Greiner E. 2009. Pneumoatractis podocnemis n. gen., n. sp. (Nematoda: Atractidae) from the yellowspotted Amazon River turtle, Podocnemis unifilis (Testudines: Pelomedusidae). Comparative Parasitology, 76, 149–153. DOI: 10.1654/4379.1. Chabaud A.G. 1978. CIH Keys to the Nematode Parasites of Vertebrates. No. 6, Keys to genera of the superfamilies Cosmocercoidea, Seuratoidea, Heterakoidea and Subuluroidea. Commonwealth Agricultural Bureaux, Farnham Royal, Buckinghamshire, UK, 71 pp. Costa S.C.G.da. 1962. Aspectos biológicos do género Rondonia Travassos 1920 (Nematoda, Atractidae). Arquivo do Museu Nacional, 52, 75–78. Costa S.C.G.da. 1963. Rondonia rondoni Travassos, 1920 (Nematoda, Atractidae). Memorias do Instituto Oswaldo Cruz, 61, 75–88. Gállego Berénguer J.G. 1947. Revision de la famila Atractidae Travassos, 1920, con descripcion de dos neuvas especies. Revista Ibérica de Parasitologia, 7, 3–90. Gibbons L.M., Platt T.R. 2006. Rhinoclemmysnema n. g. and three new species of nematodes of the family Atractidae (Cosmocercoidea), with notes on the helminth fauna of Rhinoclemmys pulcherrima (Testudines: Bataguridae) in Costa Rica. Journal of Helminthology, 80–333–340. DOI: 10.1017/JOH 2006360. González-Solis D., Moravec F. 2004. Two new nematode species, Orientatractis campechensis n. sp. and Orientatractis chia-
Received: July 8, 2013 Revised: October 15, 2013 Accepted for publication: December 31, 2013
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pasensis n. sp. (Nematoda: Atractidae) from cichlid fishes in southern Mexico and Nicaragua. Journal of Parasitology, 90, 1443–1449. DOI: 10.1645/GE-3366. Hamann M.I. 1982. Parasitos del Pacu (Colossoma mitrei) del rio Parana Medio, Republica Argentina (Pisces, Serrasalmidae). Historia Natural, 2, 153–160. Kraus F. 2013. Further range extensions for reptiles and amphibians from Papua New Guinea. Herpetological Review, 44, 277– 280. Menzies J. 2006. The Frogs of New Guinea and the Solomon Islands. Pensoft, Sofia-Moscow, 345 pp. Moravec F., Kohn A., Fernandes B.M.M. 1992. Nematode parasites of fishes of the Paraná River, Brazil. Part 1. Trichuroidea, Oxyuroidea and Cosmocercoidea. Folia Parasitologica, 39, 327–353. Petter A.J. 1966. Équilibre des espèces dans les populations de nématodes parasites du colon des tortues terrestres. Mémoires du Muséum national d’Histoire naturelle, Series A., Zoologie, 39, 1–252. Santos, E. dos, Vicente J.J., Jardim C.R. 1979. Helmintos de peixes de rios Amazônicos da coleção helmintologica do Instituto Oswaldo Cruz. II. Nematoda. Atas da Sociedade de Biologia do Rio de Janeiro, 20, 11–19. Travassos L. 1923. Informações sôbre a fauna helmintholôgica de Matto Grosso. Fôlha Médica, Rio de Janeiro, 5, 29–30. Travassos L., Artigas P., Pereira C. 1928. Fauna helminthologica dos peixes de agua doce do Brazil. Arquivos do Instituto Biológico (São Paulo), 1, 5–68. Zweifel R.G. 2000. Partition of the Australopapuan microhylid genus Sphenophryne with descriptions of new species. Bulletin of the American Museum of Natural History, 253, 1–130.
New species of Orientatractis (Nematoda: Atractidae), new species of Rondonia (Nematoda: Atractidae) and other helminths in Austrochaperina basipalmata (Anura: Microhylidae) from Papua New Guinea Charles R. Bursey1*, Stephen R. Goldberg2 and Fred Kraus3 1
Department of Biology, Pennsylvania State University, Shenango Campus, Sharon, Pennsylvania 16146, USA; 2 Department of Biology, Whittier College, Whittier, California 90608, USA; 3 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109, USA
Abstract Two new nematode species, Orientatractis hamabatrachos sp. nov. and Rondonia batrachogena sp. nov. (Nematoda: Atractidae), from the gastrointestinal tract of Austrochaperina basipalmata (Anura: Microhylidae) collected in Papua New Guinea are described. Orientatractis hamabatrachos sp. nov. is characterized by the presence of the cephalic end armed with 4 wellsclerotized structures, consisting of 2 “horns” extending outward and downward and immediately below a single well-sclerotized spine. It differs from 5 congeners in spicule lengths and caudal papillae arrangements. Rondonia batrachogena sp. nov. is characterized by the presence of a female cloaca. It differs from 2 congeners primarily in body size. Orientatractis hamabatrachos sp. nov. and Rondonia batrachogena sp. nov. represent the first species assigned to either genus found to infect anurans or to occur in the Australo-Papuan region.
Keywords Nematoda, Atractidae, Orientatractis hamabatrachos sp. nov., Rondonia batrachogena sp. nov., Anura, Microhylidae, Austrochaperina basipalmata, Australo-Papuan region.
Introduction The microhylid frog, Austrochaperina basipalmata (Van Kampen, 1906), (no common name) is endemic to the island of New Guinea, ranging from the Tawarin River in Papua (Indonesia) to the Prince Alexander Mountains southwest of Wewak, Papua New Guinea, from sea level to 1550 m (Zweifel 2000; Kraus 2013). To our knowledge, there are no helminthological reports for this species. The purpose of this paper is to describe 2 new species of Nematoda harbored by A. basipalmata and to provide an initial helminth list for this host.
imens were fixed in neutral buffered 10% formalin. The body cavity was opened by a longitudinal incision and the gastrointestinal tract was removed by cutting across the oesophagus and rectum. The stomach, small intestine and large intestine were examined separately for endoparasites. Only nematodes were found which were placed in lactophenol, allowed to clear and examined under a light microscope. Illustrations were made with the aid of a microprojector. Measurements are in µm with mean ± SD and range in parenthesis unless otherwise stated. Frogs are deposited in the Bernice P. Bishop Museum (BPBM), Honolulu, Hawaii as BPBM 35470, 35472, 35474–35478, 35480–35486.
Materials and Methods Results Fourteen Austrochaperina basipalmata were collected by hand by FK in the Prince Alexander Range, East Sepik Province, Papua New Guinea (15–23 September 2009). Spec-
Four species of Nematoda, Aplectana macintoshii (Stewart, 1914), Cosmocercella phrynomantisi Moravec 1990, an un-
*Corresponding author: [email protected]
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Table I. Site of infection, number of helminthes, prevalence, range of infection and USNPC accession numbers for 4 helminth species in Austrochaperina basipalmata from Papua New Guinea Helminth Aplectana macintoshii Cosmocercella phrynomantisi Orientatractis hamabatrachos sp. nov. Rondonia batrachogena sp. nov.
Site
Number
Large intestine Large intestine Intestine Stomach
11 185 2816 344
described species of Orientatractis Petter, 1966 and an undescribed species of Rondonia Travassos, 1920 were found. Site of infection, number of helminths, prevalence, mean intensity and range are given in Table I. Selected helminths were deposited in the United States National Parasite Collection (USNPC), Beltsville, Maryland (Table I). Description of the 2 new species follows. Orientatractis hamabatrachos sp. nov. (Figs 1–10) Description: Small fusiform nematodes, body more or less cylindrical along its length; both males and females with relatively long, thin tails. Cuticle with longitudinal striations. Lateral alae present beginning just posterior to lips and terminating near tail midpoint in both males and females. Anterior extremity bluntly rounded; mouth terminal, funnel shaped, with 6 lips, 4 submedian lips each lip with a 1 papilla and a bicornuate, sclerotized structure (2 recurved pointed spines with fused root), and 2 lateral lips, each supporting 1 large amphid with a pair of small sclerotized spines lateral to the amphidial pore. Oesophagus divided into anterior muscular portion and posterior glandular portion, terminating in a prominent valved bulb. Nerve ring just posterior to junction of oesophageal regions. Excretory pore at mid point of glandular esophageal portion. Spicules unequal, pointed distal tips. Vulva opens close to anus. Viviparous. Male (holotype and 11 paratypes): Length 3.76 ± 0.37 mm (3.20–4.35 mm); width at mid-body 86 ± 10 (73–104). Oesophagus 393 ± 20 (372–433) in length, divisible into 3 regions: anterior muscular portion 125 ± 7 (110–134) long; middle glandular portion 202 ± 16 (189–244) long; valved bulb 66 ± 9 (55–79) long by 46 ± 5 (40–55) wide. Nerve ring 190 ± 11 (177–214) and excretory pore 275 ± 17 (244–317) from anterior end, respectively. Gubernaculum cylindrical, approximately 6 in width, slight expansion in proximal third, 61 ± 5 (55–67) in length. Spicules similar, transversely striated, unequal, left spicule 155 ± 6 (146–165) long, right spicule 89 ± 8 (79–98) long, distal ends pointed. Posterior half of body most often in 3–4 tight coils. Tail 363 ± 28 (306–408) in length; abrupt narrowing at midpoint of tail. Eight pairs of caudal papillae plus 1 unpaired papilla on midline just anterior to cloaca: 2 pairs precloacal, anterior-most pair approximately 75 anterior of cloaca, second pair approximately 10 anterior of cloaca; 1 pair adcloacal; 5 pairs post cloacal, 2 pairs approximately 10 and 20 posterior to cloaca, 2 ventrolateral pairs and
Prevalence (%) 5/14 5/14 11/14 7/14
36% 36% 79% 50%
x ± SD
Range
USNPC number
2.2 ± 1.3 37.0 ± 27.9 256 ± 241 49.1 ± 24.4
1–4 1–146 4–639 22–82
106961 106962 106956 106960
1 dorsolateral pair just anterior to narrowing at tail midpoint. Phasmids open near posterior end of lateral alae. Female (allotype and 11 paratypes): Length 4.24 ± 0.39 mm (3.39–4.86 mm); body cylindrical throughout. Anterior half of body more narrow, width at level of oesophagus 60 ± 6 (52–73); posterior half of body wider, width at level of ovary 116 ± 12 (98–134). Oesophagus 452 ± 62 (402–621) in length, divisible into 3 parts: anterior muscular portion 140 ± 9 (128– 159) long; middle glandular portion 246 ± 58 (195–396) long; valved bulb 66 ± 8 (55–79) long by 48 ± 4 (43–55) wide. Nerve ring 202 ± 16 (183–232) and excretory pore 297 ± 32 (223–366) from anterior end, respectively. Tail 538 ± 35 (510– 612) in length, slender and sharply pointed. Vulva 655 ± 43 (612–740) from posterior end of tail filament, 118 ± 17 (102– 153) anterior of anus. Vagina short; monodelphic, 1 to 5 thinshelled eggs 198 ± 12 (183–214) × 79 ± 6 (73–92) in various stages of development present in anterior portion of uterus; hatched larvae present often filling uterus; single ovary near mid-body. Phasmids open near posterior end of lateral alae. Type host: Austrochaperina basipalmata (Van Kampen, 1906); BPBM 35484 collected 21 September 2009. Type locality: Mindangua Stream, Prince Alexander Range, East Sepik Province, Papua New Guinea (3°21´48˝S, 143°17´43˝E; 410 m elevation). Site of infection: Small and large intestine. Type specimens: Holotype male USNPC 106953; allotype female USNPC 106954; paratypes USNPC 106955; voucher specimens USNPC 106956. Etymology: The new species is named for its parasitism of a host group: hama, G. “at the same time” and batrachos, G. “frog”. Remarks Currently Orientatractis Petter, 1966 is comprised of O. levanhoai Petter, 1966 from the tortoise Indotestudo elongata (reported as Testudo elongata) collected at the Botanical Gardens of the Pasteur Institute of Saigon, Vietnam; O. leiperi Buckley, 1969 from the tortoise Podocnemis vogli collected in Colombia, South America; O. campechensis Gonzalez-Solis and Moravec, 2004 from the fishes Paraneetroplus bifasciatus (reported as Vieja bifasciata) and Cichlasoma pearsei collected in Mexico; O. chiapasensis Gonzalez-Solis and Moravec, 2004 from the fishes Theraps intermedius (reported as Vieja intermedia) and Tomocichla tuba collected in Mexico;
Two new species of atractid nematodes
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Figs 1–10. Orientatractis hamabatrachos sp. nov. 1. Female anterior end, lateral view. 2. Female, en face view. 3. Female, anterior end, lateral view. 4. Male, spicules and gubernaculum. 5. Male, posterior end, lateral view. 6. Female, posterior end. 7. Female, ovarian region. 8. Female, entire, lateral view. 9. Male, entire, lateral view. 10. Male, semidiagrammatic, posterior end, ventral view
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Table II. Selected characters of species of Orientatractis Orientatractis spp. (male) O. asymmetrica O. campechensis O. chiapasensis O. hamabatrachos sp. nov. O. levanhoai O. leiperi Orientatractis spp. (female) O. asymmetrica O. campechensis O. chiapasensis O. hamabatrachos sp. nov. O. levanhoai O. leiperi
Length (mm)
Oesophagus
Right spicule
Left spicule
Gubernaculum
Caudal papillae*
4.1–4.3 3.0–3.5 2.7–1.9 3.2–4.4 3.3 3.0–3.9
524–558 388–469 361–459 372–433 390 570
86–104 84–106 60–68 79–98 90 160
220–239 430–506 204–238 146–165 170 470
56–66 62–74 44–49 55–67 40 76
8:4:6 + 1 6:2:10 + 1 6:2:10 + 1 4:2:10 + 1 6:0:10 6:2:10 + 1
Length (mm)
Oesophagus
Nerve ring from anterior end
Excretory pore from anterior end
Tail
Vulva from posterior end
3.7–5.5 3.3–4.3 2.0–3.8 3.4–4.9 3.4 2.7–4.3
532–596 417–491 360–475 402–621 390 660
264–292 209–243 178–254 183–232 100 266
424–484 327–437 281–410 223–366 350 480
820–1,080 866–1,246 616–851 510–612 1,000 880
920–1,192 946–1,322 380–722 612–740 1,050 960
*precloacal:adcloacal:postcloacal + median
O. asymmetrica Gibbons and Platt, 2006 from the turtle Rhinoclemmys pulcherrima collected in Costa Rica (Petter, 1966; Buckley, 1969; Gonzalez-Solis and Moravec, 2004; Gibbons and Platt, 2006). Orientatractis hamabatrachos sp. nov. is the first species found in an amphibian or in the Australo-Papuan region and is assigned to Orientatractis based upon the presence of specialized cuticular formations around the mouth. The 6 species can easily be separated based upon spicule length and caudal papillae arrangement (Table II). Rondonia batrachogena sp. nov. (Figs 11–22) Description: Small nematodes, body more or less cylindrical throughout its length; males fusiform; females fusiform but developing truncated posterior at maturity. Depending upon microscope focus, both transverse and longitudinal striations can be seen in cuticle. Narrow lateral alae present beginning just posterior to lips and terminating near tail midpoint in both males and females. Anterior extremity bluntly rounded; terminal mouth, opening triangular surrounded by 3 lips, each lip with 2 simple, pedunculate papillae; 1 amphid on each ventrolateral lip. Oesophagus divided into anterior muscular portion and posterior glandular portion, terminating in a prominent valved bulb. Nerve ring just posterior to junction of oesophageal parts. Excretory pore posterior to oesophageal bulb; pore surrounded by circular striated cuticle approximately 30 in diameter. Spicules unequal, pointed distal tips. Both males and females with cloaca. Viviparous. Male (based on holotype and 11 paratypes): Length 2.13 ± 0.11 mm (2.04–2.42 mm); width at mid-body 94 ± 16 (67– 113). Oesophagus 380 ± 13 (360–411) in length, divisible into 3 parts: anterior muscular portion 242 ± 10 (232–268) long; middle glandular portion 75 ± 5 (67–85) long; valved bulb 63
± 3 (58–67) long by 56 ± 4 (49–61) wide. Nerve ring 256 ± 10 (244–274) and excretory pore 572 ± 21 (549–616) from anterior end, respectively. Gubernaculum with cylindrical body, approximately 6 in width, 61 ± 4 (55–67) in length, supporting at midpoint lateral projections approximately 15 in length. Spicules similar, transversely striated, unequal, left spicule 238 ± 8 (220–250) long, right spicule 78 ± 7 (73–98) long, distal ends pointed. Tail 150 ± 8 (134–159) in length; thick anterior portion 86 ± 8 (73–98) long supporting thin terminal filament 64 ± 9 (49–79) in length. Tail bent ventrally to form a “shepherd’s crook”. Eight pairs of caudal papillae: 3 pairs precloacal: 5 pairs post cloacal, 3 pairs ventrolateral in position, 2 lateral pairs, 1 pair just posterior to cloaca, 1 pair just anterior to tail filament. Phasmids open near posterior end of lateral alae. Female (based on allotype and 11 paratypes): Length 1.77 ± 0.14 mm (1.58–1.99 mm); width at level of ovary 116 ± 16 (92–153). Body cylindrical throughout, immature females with elongate posterior that changes with maturity to form a truncate posterior in which the base of the tail is drawn into body. Cloaca empties into crease caused by tail withdrawal. Oesophagus 384 ± 14 (363–405) in length, divisible into 3 parts: anterior muscular portion 243 ± 10 (229–256) long; middle glandular portion 77 ± 5 (73–85) long; valved bulb 64 ± 5 (55–73) long by 53 ± 5 (43–61) wide. Nerve ring 258 ± 14 (244–284) and excretory pore 551 ± 38 (488–653) from anterior end, respectively. Tail 214 ± 14 (189–232) in length. Vulva opening into rectum; vagina short; monodelphic, 1 to 3 thin-shelled eggs 169 ± 9 (158–183) × 81 ± 7 (73–99) that are in various stages of development in anterior portion of uterus; hatched larvae present and often filling uterus; single ovary near middle of body. Phasmids open near posterior end of lateral alae.
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Figs 11–22. Rondonia batrachogena sp. nov. 11. Female, mature, entire, lateral view. 12. Female, early gravid stage, lateral view. 13. Female, larval development stage, lateral view. 14. Female, hatched larval stage, lateral view. 15. Male, entire, lateral view. 16. Female, en face view. 17. Female, anterior end, dorsal view. 18. Female, posterior end, lateral view. 19. Male, posterior end, lateral view. 20. Male, posterior end, ventral view. 21. Male, anterior end, lateral view. 22. Female, excretory pore
Type host: Austrochaperina basipalmata (Van Kampen, 1906); BPBM 35475 collected 17 September 2009. Type locality: Imuk, Prince Alexander Range, East Sepik Province, Papua New Guinea (3°21´4˝S, 143°17´49˝E, 184 m elevation).
Site of infection: Stomach Type specimens: Holotype male USNPC 106957; allotype female USNPC 106958; paratypes USNPC 106959; voucher specimens USNPC 106960. Etymology: The new species is named for life history: batrachos G. “frog” and gena, L. “born in”.
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Table III. Selected characters of Rondonia Rondonia spp. (male)
Length (mm)
2.0–2.4 R. batrachogena sp. nov. 3.9–8.1 R. rondoni Males unknown R. lophii
Oesophagus
Right spicule
Left spicule
Gubernaculum
Caudal papillae*
360–411 524–993 –
73–98 100–130 –
220–250 240–290 –
55–67 60–70 –
6:0:10 6:2:12 –
Tail
Egg
Nerve ring from Excretory pore anterior end from anterior end
Rondonia spp. (female)
Length (mm)
Oesophagus
R..batrachogena sp. nov.
1.6–2.0
363–405
244–284
488–653
189–232
158–183 × 73–99
R. rondoni
6.8–9.9
1003–1035
530–585
1120–1220
116–1320
480–560 × 220–260
2.7
480
260
700
310
42 × 29
R. lophii
*precloacal:adcloacal:postcloacal + median
Remarks Currently Rondonia Travassos, 1919 is comprised of R. rondoni Travassos, 1920 from various fish species, Piaractus brachypomus (type host), Acanthodoras spinosissimus, Doras granulosus, Luciopimelodus pati, Myeletes torquatus, Myleus asterias, Synodontis clarias, Trachydoras paraguayensis and Zungaro zungaro, collected in Mexico and South America (Travassos, 1923; Travassos et al., 1928; Baylis, 1936; Costa, 1962, 1963; Santos et al., 1979; Hamann, 1982; Moravec et al., 1992), and R. lophii Gallego Berenguer, 1947 also from a fish species, Lophius piscatorius, collected on the Cantabrico Coast, Spain (Gallego Berenguer, 1947). Rondonia batrachogena sp. nov. is assigned to Rondonia based upon the presence of a cloaca in the female and is the first species to be reported from an amphibian or from the Australo-Papuan region. The 3 species can easily be separated on body size (Table III).
Discussion We have assigned the 2 new species to the Atractidae because the oesophagus is clearly divided into an inflated muscular anterior region, a posterior glandular region and a bulb with valve, the male lacks a precloacal sucker, and the female is viviparous (Chabaud 1978). Adamson and Baccam (1988) listed 14 genera for the Atractidae, which were divided into 2 groups: those characterized by didelphy, Fitzsimmonsnema Petter, 1966, and Probstmayria Ransom, 1917, and those characterized by monodelphy, Atractis Dujardin, 1945, Cobboldina Leiper, 1911, Crossocephalus Railliet, 1909, Cyrtosomum Gedoelst, 1919, Grassenema Petter, 1969, Labiduris Schneider, 1866, Leiperenia Khalil, 1922, Monhysterides Baylis and Daubney, 1922, Orientatractis Petter, 1966, Paratractis Sarmiento, 1959, Proatractis Caballero, 1971, and Rondonia Travassos, 1919. To the list of didelphic atractid genera should be added Nouvelnema Petter, 1959, and to the list of monodelphic atractid genera should be added Buckleyatractis Khalil and Gibbons, 1988, Diceronema Gibbons,
Knapp and Drecek, 1966; Klossinemella Costa, 1961, Paraorientatractis Gibbons, Khalil and Marinkelle, 1997, Podocnematractis Gibbons, Khalil and Marinkelle, 1995, Rhinoclemmysnema Gibbons and Platt, 2006, and Pneumoatractis Bursey, Reavill and Greiner, 2009 (Bursey et al. 2009). It should be noted that Moravec and Thatcher (1997) moved Proatractis to synonymy with Klossonemella. Members of the Atractidae are unusual in that eggs hatch and larvae develop to third stage in utero to autoinfect the current host (Anderson 2000). Their transmission from host to host is not understood. The life cycles of Orientatractis and Rondonia have not been determined, however Costa (1962) believed that larvae of Rondonia (previously known only from fish) pass from the host to infect other fish directly. Petter (1966) claimed that tortoises became infected only after attaining sexual maturity and wondered if transmission occurred during mating.
Acknowledgements. We thank Ezra Teodoro and Jeanette Arreola for help with dissections; Peggy Firth for preparing the illustrations constituting Figs 1–22; Justin Jiramori, Agustin Nakung, Charlie Wintawa, Damien Yaripma, and Ben Yawi for field or logistical assistance. We thank the PNG National Museum and Art Gallery for providing in-country collaborative assistance and the Department of Environment and Conservation; National Research Institute; and the East Sepik Provincial Government for permission to conduct this research. This research was supported by National Science Foundation grant EEB-0743890.
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Buckley J.J.C. 1969. On a remarkable oxyurid nematode, Orientatractis leiperi n. sp., (Atractidae) from a South American tortoise, Podocnemis vogli. Journal of Helminthology, 43, 218–286. DOI: 10.1017/S0022149X0000482X. Bursey C.R., Reavill D., Greiner E. 2009. Pneumoatractis podocnemis n. gen., n. sp. (Nematoda: Atractidae) from the yellowspotted Amazon River turtle, Podocnemis unifilis (Testudines: Pelomedusidae). Comparative Parasitology, 76, 149–153. DOI: 10.1654/4379.1. Chabaud A.G. 1978. CIH Keys to the Nematode Parasites of Vertebrates. No. 6, Keys to genera of the superfamilies Cosmocercoidea, Seuratoidea, Heterakoidea and Subuluroidea. Commonwealth Agricultural Bureaux, Farnham Royal, Buckinghamshire, UK, 71 pp. Costa S.C.G.da. 1962. Aspectos biológicos do género Rondonia Travassos 1920 (Nematoda, Atractidae). Arquivo do Museu Nacional, 52, 75–78. Costa S.C.G.da. 1963. Rondonia rondoni Travassos, 1920 (Nematoda, Atractidae). Memorias do Instituto Oswaldo Cruz, 61, 75–88. Gállego Berénguer J.G. 1947. Revision de la famila Atractidae Travassos, 1920, con descripcion de dos neuvas especies. Revista Ibérica de Parasitologia, 7, 3–90. Gibbons L.M., Platt T.R. 2006. Rhinoclemmysnema n. g. and three new species of nematodes of the family Atractidae (Cosmocercoidea), with notes on the helminth fauna of Rhinoclemmys pulcherrima (Testudines: Bataguridae) in Costa Rica. Journal of Helminthology, 80–333–340. DOI: 10.1017/JOH 2006360. González-Solis D., Moravec F. 2004. Two new nematode species, Orientatractis campechensis n. sp. and Orientatractis chia-
Received: July 8, 2013 Revised: October 15, 2013 Accepted for publication: December 31, 2013
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