In a survey of parasites on the killifish, Austrolebias bellottii from Punta Indio and Magdalena, Argentina, the acanthocephalan Neoechinorhynchus villoldoi was found. This parasite had not been recorded since 1992 when it was described for the first time parasitizing Corydoras paleatus (Jenyns, 1842). The type material of N. villoldoi was examined, and with addition of the new specimens, the measurements range was extended. This is the first record of an acanthocephalan in the annual fish, A. bellottii.Facultad de Ciencias Naturales y Muse

Barneche, Jorge Adrián

García, Ignacio Daniel

Martorelli, Sergio Roberto

Montes, Martín Miguel

Preisz, Sebastián

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New data on the acanthocephalan Neoechinorhynchus villоldoi Vizcaíno, 1992 (Neoechinorhynchidae: Acanthocephala), based on specimens found in Austrolebias bellottii (Steindachner, 1881) (Rivulidae: Cyprinodontiformes) from Punta Indio, ArgentinaMartin Miguel Montes,1 Jorge Barneche,1 Ignacio García,2 Sebastian Preisz3, Sergio Roberto Martorelli11 Centro de Estudios Parasitológicos y Vectores (CEPAVE), Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de La Plata (CCT, CONICET-UNLP), Calle 120 s/n e/ 61 y 62, La Plata, Argentina. 2 Instituto de Limnología “Dr. Raúl A. Ringuelet”, CONICET, Boulevard 120 y 62, La Plata, Argentina. 3 Universidad Nacional de La Plata, Facultad de Ciencias Naturales y Museo, Calle 120 entre 60 y 61, La Plata, Argentina.Corresponding author: Martin Miguel Montes, martinmiguelmontes@gmail.com AbstractIn a survey of parasites on the killifish, Austrolebias bellottii from Punta Indio and Magdalena, Argentina, the acan-thocephalan Neoechinorhynchus villoldoi was found. This parasite had not been recorded since 1992 when it was described for the first time parasitizing Corydoras paleatus (Jenyns, 1842). The type material of N. villoldoi was examined, and with addition of the new specimens, the measurements range was extended. This is the first record of an acanthocephalan in the annual fish, A. bellottii.Key wordsMetazoan parasite; new record; annual fishes; pampa; temporary ponds.Academic editor: Simone Cohen | Received 15 June 2016 | Accepted 10 May 2017 | Published 11 July 2017Citation: Montes, MM, Barneche, J, García, I, Preisz, S, Martorelli, SR (2017) New data on the acanthocephalan Neoechinorhynchus villaldoi Vizcaíno, 1992 (Neoechinorhynchidae: Acanthocephala), based on specimens found in Austrolebias bellottii (Steindachner, 1881) (Rivulidae: Cyprinodontiformes) from Punta Indio, Argentina. Check List 13 (4): 53–59. https://doi.org/10.15560/13.4.53Check List 13 (4): 53–59 https://doi.org/10.15560/13.4.5341353–59Date 2017Copyright Montes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.IntroductionData on metazoan parasites of South American annual killifish is scarce. Delgado and Garcia (2015) reported 2 larvae of Contracaecum spp. nematode in Austrolebias spp. from Uruguay, and Luque et al. (2011) mentioned the presence of the nematode Hedruris iheringi Pereira & Vaz, 1933 in Austrolebias bellottii (Steindachner, 1881) from Brazil. Due to a lack of information in Argentina, we began to search for parasites on A. bellottii.Among genera of the Neotropical fish fauna, the South American killifish genus Austrolebias Costa, 1998 are the most numerous annual fishes distributed in southern Bolivia, southern Brazil, Paraguay, northeastern Argen-tina and Uruguay with 44 valid species (Costa 2010, Loureiro et al. 2011, Nielsen and Pillet 2015). The genus Austrolebias inhabits temporary wetlands formed during rainy periods and die when the ponds dry out (Costa 2003, Costa 2006). Most species bury their eggs in the substrate, the embryos survive through dry periods in a diapause inside the resistant eggs and hatch at the beginning of the NOTES ON GEOGRAPHIC DISTRIBUTION54 Check List 13 (4)rainy season (Wourms 1972, Podrabsky and Hand 1999). The fishes grow fast, mostly in the first months of their life, reach the sexual maturity early and begin to spawn (Walford and Liu 1965, Gonzalves et al. 2011).The short life cycle, the ecological importance, the aquarist interest of this fish, and the scant information on metazoan parasites in killifishes moved us to study the acanthocephalan Neoechinorhynchus villoldoi Vizcaíno, 1992. Based on reexamination of the type material from Figures 1, 2. Photography’s of the Austrolebias bellottii 1. Male, SL 3.8 cm. 2: female, SL 3.4 cm.12Figure 3. Map of Buenos Aires province, Argentina, with the 2 sample sites, and Villoldo stream (VS). 45Figures 4, 5. Sample sites. 4. Site 1, Punta Indio. 5. Site 2, Magda-lena.Corydoras paleatus (Jenyns, 1842) and the new speci-mens found parasitizing A. bellottii, we extend the range of measurements of this parasite. This is the first record of N. villoldоi on A. bellottii.MethodsFishes (Figs 1, 2) were sampled between October and November 2015 from 2 temporary puddles separated from each other by 38 km (Fig. 3). The puddles (Figs 4, 5) were selected randomly and named: site 1 (Punta Indio, 35°18.932ʹ S, 057°13.176ʹ W) and site 2 (Mag-dalena, 35° 04.865ʹ S, 057°31.627ʹ W). The collections were made with a frame of 1 × 0.50 m and a net-mesh of 0.5 × 0.5 cm. The specimens were transported to the lab in plastic bags with oxygenated water from the collection sites. The fishes were sacrificed by medullar section. The viscera were examined for parasites under a binocular microscope. The acanthocephalans found were cleared as temporary mounts with Lacto-phenol or stained with dehydrated chlorhidric carmin mounted in Canada balsam, and studied under an Olympus Bx51 microscope (Tokyo, Japan). The drawings were made with a drawing tube. The structures were photographed with an AmScope MU 1000 10 MP digital camera (USA) attached to the microscope and measured using ImageJ software (Schneider et al. 2012). The mean followed by minimum and maximum values in parenthesis are given in micrometers (µm), unless stated otherwise. Figures 1 and 2 were photographed with a Canon 7D camera with a Canon 100 mm 2.8 macro lens and Figures 4 and 5 with a Montes et al. | Neoechinorhynchus villaldoi parasitizing Austrolebias bellottii 55Canon 50 mm 1.8 Macro lens. The vouchers were depos-ited in the Helminthological Collection of the Museo de La Plata, Argentina, under the number MLP-He 7150.Examined material included all the type series of Neoechinorhynchus villoldoi, 1537 C, 1539 C, 1541-44 C and 1547-50 C, in the Helminthological Collection of Museo de la Plata.ResultsThe prevalence, median intensity and abundance of N. villoldoi from A. bellottii are provided in Table 1.Description of N. villoldoi found in A. bellottii. Small, Table 1. Number of specimens of Austrolebias bellottii examined (N). Prevalence of infection, mean intensity and mean abundance of Neoechinorhynchus villoldоi in sample sites.N Prevalence of infection Mean intensity Mean abundancePunta Indio (sample site 1) 10 80% 3.13 2.5Madariaga (sample site 2) 22 0% 0 0cylindrical, with gigantic hypodermic nuclei 1 or 2 dor-sal and 1–3 ventral (not drawn), posterior end rounded. Genital pore subterminal in both sexes. Small proboscis, cylindrical, 18 hooks in 3 circles of 6 hooks each. First 2 circles with similar hooks lengths; third circle of hooks significantly smaller (Figs 6–9). Nerve ganglion posterior to the receptacle of the proboscis. Lemnisci short, sub-equal, barely longer than the receptacle of the proboscis. Male (Figs 10, 11) shorter than female, reproductive system consisting of 2 contiguous testicles, cement gland syncytial, rounded reservoir of cement, elongated Säfftigen’s pouch. Copulatory pouch spade-shaped (Figs 12, 13).Figures 6–9. Proboscis of Neoechinorhynchus villoldоi found in Austrolebias bellottii. 6, 7. Male proboscis. . 8, 9. Female proboscis; 9, stained with chloridric carmin. Scale bars: Figs 6, 7, 9 = 20 µm, Fig. 8 = 16 µm.978656 Check List 13 (4)type locality or (b) the similarity in the host feeding habits.Site 1 at Punta Indio is 7 km away from Villoldo stream (the type locality of N. villoldоi) but 35 km from site 2. The distance between sites could be one of the reasons for the absence of this parasite in fishes from Magdalena (site 1). Corydoras paleatus (the type host) and A. bellottii are fishes with different habitat but perhaps they prey over the same food item. The diets of both species are represented mainly by microcrustaceans (Cladocera, Copepoda, and Ostracoda), and immature aquatic insects (Chironomidae) (Escalante 1983, Grosman 2002, Laufer 2009, Keppeler 2014). Better knowledge of the life cycle of N. villoldоi could clarify if this parasite uses 1, 2 or more intermediate hosts, and if both fishes prey over the same or different intermediate host?”Knowledge of the parasite fauna of A. bellottii is important because this fish forms a link between micro- and macroecosystems. Killifishes mainly feed on microcrustaceans and immature aquatic insects, and kil-lifishes serve as food for aquatic birds (Keppeler et al. 2016). They are also important fishes for aquarists due to the high number of species, bright colors, size and the relatively easy breeding in captivity. There are many species of Austrolebias, and the genus has a wide distribution, and therefore we suspect that our record N. villoldоi in A. bellottii is only the first of a several new records (or new species) of adult metazoan parasites and larval stages waiting for to be discovered in these “killis”.AcknowledgementsWe thank Monica Casciaro and Monica Rodriguez of the Direccion y Subdireccion de Flora y Fauna de la Provincia de Buenos Aires for the permits to collect fishes, Samanta Faiad of the Departamento de Dibujo e Ilustracion Cienti-fica del Museo de La Plata for the line drawings, Marcos Gaston Cavallo for helping in the sample collection, Cris-tian Rodriguez for the construction of the fishing gear, and Cecilia Gabellone and Carlos Romero for reading the manuscript and helping to translate it to English. We also want to thank to the Consejo Nacional de Ciencia y Tec-nologia (CONICET) for financial support of this research. Authors’ ContributionsMMM identified the acanthocephalan, measured them, and wrote the text. JB collected the fishes and parasites and stained the acantocephalan. IG collected the samples, determined the fishes, and wrote the text (the part on the fishes). SP found the sample site, collected the samples, and photographed the male and female of A. bellottii and sample sites. SM identified the acanthocephalan, wrote the text.Figures 10, 11. Entire male specimen of Neoechinorhynchus villoldоi. Abbreviations: AT = anterior testicle, CP = copulatory pouch, CG = cement gland, CR. = cement reservoir, L = lemnisci, Pb = proboscis, PT = posterior testicle, PR = proboscis receptacle, S P = Säfftigen’s pouch. Scale bar = 200 µm.Female (Figs 14–17) 3–4 × larger than the males, reproductive system includes uterine bell, selector of eggs, utero and vagina. Eggs elongated (Fig. 18). Mea-surements of male and female structures provided in Table 2.DiscussionNeoechinorhynchus villoldoi is the first acanthocephalan found in a killifish of the genus Austrolebias. This is the second record for this acanthocephalan since the discov-ery of N. villoldoi by Vizcaíno (1992).Our examination of the type series material deposited by Vizcaíno (1992) of N. villoldоi have similar mea-surements with the acanthocephalans found now in A. bellottii, but some minor differences can be distinguished. The type material included mostly juvenile females (only one mature with eggs) and males in poor conservation stage, and this could account for why some of the original measurements have a wide range. The presence of N. villoldоi in this newly recorded host, A. bellottii, could be attributed to: (a) the proximity to the10 11Montes et al. | Neoechinorhynchus villaldoi parasitizing Austrolebias bellottii 57Figures 14–18. Female reproductive of Neoechinorhynchus villoldоi found in Austrolebias bellottii and eggs. 14, 15 Juvenile female. 16, 17. Reproductive female. 18. Eggs. Abbreviations: CR = cement reservoir, SP = Säfftigen’s pouch., Scale bars: Figs 14–15, 17 = 40 µm, Fig. 16 = 50 µm, Fig. 18 = 8 µm 1214161718131558 Check List 13 (4)ReferencesCosta WJEM (2003) Family Rivulidae (South American annual fishes). In: Reis RE, Kullander SO, Ferraris JC Jr (Eds) Check List of the Freshwater Fishes of the South and Central America. Edipucrs, Porto Alegre, 526–548. Costa WJEM (2006) The South American annual killifish genus Aus-trolebias (Teleostei: Cyprinodontiformes: Rivulidae): phylogenetic relationships, descriptive morphology and taxonomic revision. Zootaxa 1213: 1–162. https://doi.org/10.11646/zootaxa.1067.1.1 Costa WJEM (2010) Historical biogeography of Cynolebiasine annual killifishes inferred from dispersal-vicariance analysis. Journal of Biogeography 37: 1995–2004. https://doi.org/10.1111/j.1365-2699.2010.02339.x Delgado C, Garcia G (2015) Coevolution between Contracaecum (Nematoda, Anisakidae) and Austrolebias (Cyprinodontiformes, Rivulidae) host-parasite complex from SW Atlantic coastal basins. Parasitological Research 114: 913–927. https://doi.org/10.007/s00436-014-4257-2 Escalante A (1983) Contribución al conocimiento de las relaciones tró-ficas de peces de agua dulce del área platense. III. Otras especies. Limnobios 2: 453–463.Gonçalves CS, Souza UP, Volcan MV (2011) The opportunistic feeding and reproduction strategies of the annual fish Cynopoecilus mela-notaenia (Cyprinodontiformes: Rivulidae) inhabiting ephemeral habitats on southern Brazil. Neotropical Ichthyology 9: 191–200. http://doi.org/10.1590/s1679-62252011000100019 Grosman F, Gonzalez G, Sanzano P, Agüería D (2002) Alimentación, nichos tróficos y competencia interespecífica de peces de la laguna de Monte, Argentina. Actas del Congreso Iberoamericano Virtual de Acuicultura CIVA 2002, Zaragoza, 129–140.Keppeler FW, Lanés LEK, Rolon AS (2014) The morphology–diet relationship and its role in the coexistence of two species of annual fishes. Ecology of Freshwater Fishes Journal 24: 77–90. https://doi.org/10.1111/eff.12127 Keppeler FW, Cruz DA, Dalponti G, Mormul RP (2016) The role of deterministic factors and stochasticity on the trophic interactions between birds and fish in temporary floodplain ponds. Hydro-biologia 773: 225–240. https://doi.org/10.1007/s10750-016-2705-yLaufer G, Arim M, Loureiro M, Pineiro-Guerra JM, Clavijo-baquet S, Fagúndez C (2009) Diet of four annual killifishes: an intra and interspecific comparison. Neotropical Ichthyology 7: 77–86. http://doi.org/10.1590/s1679-62252009000100010 Loureiro M, Duarte A, Zarucki M (2011) A new species of Austrolebias Costa (Cyprinodontiformes: Rivulidae) from northeastern Uruguay, Table 2. Comparison of Neoechinorhynchus villoldоi measurements between males and females of Austrolebias bellottii (present study), origi-nal description (Vizcaino 1992) and type material. Abbreviations: Max = maximum L=length, W=width, * not measured, *2 material in poor stage of conservation.Males  FemalesHost A. bellottii C. paleatus C. paleatus A. bellottii C. paleatus C. paleatusSource Present study Vizcaíno 1992 Type Material  (present study) Present study Vizcaíno 1992Type material (present study)Total length 1322 (1043–2897)2071 (895–6013)2044 (938–4831)5543 (4256–7076)3081 (637–7505)4347Max. width 392 (245–558) 215 (95–427) 214 (127–388) 604 (521–673) 251 (95–494) 341Proboscis L 98 (89–114) 111 (95–171) *2 89 (77–101) 121 (94–152) 235W 62 (52–75) 81 (57–105) *2 70 (66–73) 86 (76–105) 247Anterior circle of hooks L 24 (23–26) 27 (25–29) *2 27 (25–29) 31 (27–35) *2W 5 (4–5) 5 (5–7) *2 6 (5–8) 7 (5–8) *2Middle circle of hooks L 25 (16–27) 26 (24–28) *2 26 (25–27) 25 (23–28) *2W 5 (4–5) 6 (5–7) *2 5 (5–6) 5 (5–8) *2Posterior circle of hooks L 18 (15–21) 21 (19–23) *2 15 (14–17) 21 (19–30) *2W 4 (3–4) 5 (5–5) *2 4 (3–5) 5 (4–6) *2Long lemnisci L 254 (157–375) * 217 (142–416) 372 (326418) * 137W 64 (32–89) * 101 (32–223) 66 (51–77) * 98Short lemnisci L 237 (168–311) * 176 (128–307) 287 (159–372) * 88W 54 (33–81) * 87 (38–155) 67 (61–72) * 98Proboscis receptacle L 153 (127–179) 206 (133–295) 100 (62–144) 191 (177–212) 159 (162–285) *2W 74 (66–84) 86 (57–114) 74 (50–94) 88 (83–91) 88 (75–109) *2Anterior testis L 529 (400–777) 255 (75–855) 277 (137–774) — — —W 246 (169–414) 145 (38–295) 155 (95–290) — — —Posterior testis L 410 (281–699) 220 (68–741) 242 (101–652) — — —W 218 (144–307) 144 (41–266) 133 (75–251) — — —Cement gland L 425 (292–673) 184 (53–456) 236 (68–517) — — —W 212 (118–299) 122 (60–219) 131 (82–263) — — —Cement reservoir L 157 (113–229) 102 (23–209) 113 (44–242) — — —W 120 (79–170) 92 (34–190) 99 (55–173) — — —Saefftiggen’s pouch L 342 (241–452) 204 (94–428) 226 (137–488) — — —W 127 (93–162) 70 (23–143) 74 (42–118) — — —Everted copulatory pouch L 165 (115–211) * *2 — — —W 126 (98–178) * *2 — — —Eggs L — — — 28 (22–33) 32 (30–34) 29 (26–31)W — — — 8 (6–10) 15 (14–15) 13 (12–15)Embryo — — — * 26 (25–27) *2Genital complex — — — * 256 (26–428) *2Montes et al. | Neoechinorhynchus villaldoi parasitizing Austrolebias bellottii 59with comments on distribution patterns. Neotropical Ichthyology 9: 335–342. http://doi.org/10.1590/S1679-62252011000200010 Luque JL, Aguiar JC, Vieira FM, Gibson DI, Portes Santos C (2011) Checklist of Nematoda associated with the fishes of Brazil. Zoo-taxa 3082: 1–88.Nielsen DTB, Pillet D (2015) Austrolebias accorsii, a new annual fish (Cyprinodontiformes: Rivulidae: Cynolebiatinae) from the upper Río Grande basin, Amazon basin, Bolivia. Aqua, International Journal of Ichthyology 21: 172–179.Podrabsky JE, Hand S (1999) The bioenergetics of embryonic diapause in annual killifish, Austrofundulus limnaeus. Journal of Experimental Biology 202: 2567–2580.Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nature Methods 9: 671–675. https://doi.org/10.1038/nmeth.2089 Vizcano SL (1992) Especie nueva del genero Neoechinorhynchus (Acan-thocephala–Neoechinorhynchidae) parásita de peces de Argentina. Anales Instituto de Biologia Universidad Nacional de Mexico, Serie Zoologica 63: 179–184.Walford RL, Liu RK (1965) Husbandry, life-span and growth rate of the annual fish, Cynolebias adloffi. Experimental Gerontology 1: 161–171.Wourms JP (1972) The developmental biology of annual fishes. III. Pre-embryonic and embryonic diapause of variable duration in the eggs of annual fishes. Journal of Experimental Zoology 182: 389–414. https://doi.org/10.1002/jez.1401820310 View publication stats

New data on the acanthocephalan <i>Neoechinorhynchus villоldoi</i> Vizcaíno, 1992 (Neoechinorhynchidae: Acanthocephala), based on specimens found in <i>Austrolebias bellottii</i> (Steindachner, 1881) (Rivulidae: Cyprinodontiformes) from Punta Indio, Argentina

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New data on the acanthocephalan <i>Neoechinorhynchus villоldoi</i> Vizcaíno, 1992 (Neoechinorhynchidae: Acanthocephala), based on specimens found in <i>Austrolebias bellottii</i> (Steindachner, 1881) (Rivulidae: Cyprinodontiformes) from Punta Indio, Argentina

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