Syst Parasitol https://doi.org/10.1007/s11230-017-9772-y A new genus and species of turtle blood fluke (Digenea: Schistosomatoidea) from the Mekong snail-eating turtle, Malayemys subtrijuga (Schlegel & Mu¨ller) (Testudines: Geoemydidae) in Vietnam, with a reassessment of related Asiatic turtle blood flukes and molecular phylogeny Jackson R. Roberts . Cova R. Arias . Kenneth M. Halanych . Binh T. Dang . Stephen A. Bullard Received: 23 August 2017 / Accepted: 9 December 2017 Ó Springer Science+Business Media B.V., part of Springer Nature 2018 Abstract Platt sinuosus Roberts & Bullard n. g., n. previously of Hapalorhynchus Stunkard, 1922 are sp. (type-species) infects the kidney and mesenteric reassigned herein to Platt: P. odhnerensis (Mehra, blood vessels of Mekong snail-eating turtles, Malay- 1933) Roberts & Bullard n. comb.; P. yoshidai (Ozaki, emys subtrijuga (Schlegel & Mu¨ller), in the Mekong 1939) Roberts & Bullard n. comb.; P. ocadiae; P. River Basin. Species of Platt Roberts & Bullard n. g. oschmarini (Belous, 1963) Roberts & Bullard n. are unique by the combination of having a papillate comb.; P. sutlejensis (Mehrotra, 1973) Roberts & ventral sucker, vasa efferentia that are dorsal to the Bullard n. comb.; P. synderi (Platt & Sharma, 2012) gonads, a massive cirrus-sac that is directed anteriad Roberts & Bullard n. comb.; and P. tkachi (Platt & or laterad, and a vitellarium that surrounds the Sharma, 2012) Roberts & Bullard n. comb. A intestinal caeca. The new species resembles Platt dichotomous key to Platt spp. is provided. Ha- ocadiae (Takeuti, 1942) Roberts & Bullard n. comb. palorhynchus sheilae (Mehrotra, 1973) Bourgat, but differs from it by having an external seminal 1990 and Hapalorhynchus mica (Oshmarin, 1971) vesicle that overlaps with or is immediately posterior Bourgat, 1990 are considered as species inquirendae, to the level of the ventral sucker. Seven species and Hapalorhynchus indicus (Thapar, 1933) Price, 1934 and Hapalorhynchus macrotesticularis (Rohde, Lee, & Lim, 1968) Brooks & Sullivan, 1981 are This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as EA5233B0-DCF8-4890-8762- considered as species incertae sedis. Phylogenetic 37EBFEB5C14E. This article was published as an Online First analysis of the large subunit rDNA (28S) showed P. article on the online publication date shown on this page. The sinuosus and P. snyderi to be sister taxa distinct from a article should be cited by using the doi number. This is the monophyletic Hapalorhynchus and Coeuritrema Version of Record. platti Roberts & Bullard, 2016. This article is part of the Topical Collection Digenea. J. R. Roberts Á S. A. Bullard (&) K. M. Halanych Aquaculture & Aquatic Sciences and Aquatic Department of Biological Sciences, Molette Biology Parasitology Laboratory, School of Fisheries, Auburn Laboratory for Environmental & Climate Change Studies, University, 203 Swingle Hall, Auburn, AL 36849, USA Auburn University, 101 Rouse Life Sciences Building, e-mail: [email protected] Auburn, AL 36830, USA C. R. Arias B. T. Dang Aquaculture & Aquatic Sciences and Aquatic Department of Biology, Institute for Biotechnology and Microbiology Laboratory, School of Fisheries, Auburn Environment, Nha Trang University, 02 Nguyen Dinh University, 559 Duvall Drive, Auburn, AL 36832, USA Chieu Street, Nha Trang City, Vietnam 123 Syst Parasitol Introduction Turtle scientific and common names follow van Dijk et al. (2014). Classification and anatomical terms for Hapalorhynchus Stunkard, 1922 (Digenea: Schistoso- TBFs follow Roberts et al. (2016a, b, c) and Roberts matoidea) currently includes 20 nominal species that et al. (2017) except that ‘‘ventrolateral tegumental collectively infect freshwater turtles of North America papillae’’ of Roberts et al. (2016b) is replaced by (Cryptodira: Chelydridae, Kinosternidae), Asia (Tri- ‘‘ventrolateral tegumental mamillae’’. onychidae, Geoemydidae), and Africa (Pleurodira: Griphobilharzia amoena Platt, Blair, Purdie & Pelomedusidae). As such, it is the only genus of Melville, 1991 served as an outgroup with which to freshwater turtle blood fluke (TBF) that includes root the phylogeny. Specimens intended for collection species infecting turtles on more than one continent of molecular data were placed directly in 95% non- (Smith, 1997a, b; Platt, 2002; Platt & Sharma 2012; denatured ethanol. Total genomic DNA (gDNA) was Roberts et al., 2016a, b, 2017). Eleven of those 20 extracted using DNeasyTM Blood and Tissue Kit species were described from Asiatic softshell turtles (Qiagen, Valencia, California, USA) according to the (Trionychidae) or pond turtles (Geoemydidae); none manufacturer’s protocol except that the incubation have been reported since their original description. An period with proteinase-K was extended to overnight opportunistic examination of two Mekong snail-eating and that the final elution step was performed using turtles, Malayemys subtrijuga (Schlegel & Mu¨ller), only 100 ll of elution buffer to increase the final DNA (Testudines: Geoemydidae) from a market in Can Tho, concentration. The partial 28S rDNA (domains D1- Vietnam, revealed infections by two turtle blood D3; c.1,400 bp) was amplified using the forward flukes: Hapalorhynchus snyderi Platt & Sharma, 2012 primer ‘‘U178’’ (50-GCA CCC GCT GAA YTT AAG- and a new species. Herein, we propose a new genus to 30) and the reverse primer ‘‘L1642’’ (50-CCA GCG accommodate the new species and seven species CCA TCC ATT TTC A-30) (Lockyer et al., 2003). The formerly of Hapalorhynchus, provide a dichotomous internal transcribed region 2 (ITS2 rDNA) was key to all species of the new genus, and provide both a amplified using the forward primer ‘‘GA1’’ (50-AGA phylogenetic reconstruction based on the large subunit ACA TCG ACA TCT TGA AC-3; Anderson & ribosomal DNA (28S rDNA) only and additional Barker, 1998) and the reverse primer ‘‘ITS2.2’’ (50- sequence data for the internal transcribed region 2 CCT GGT TAG TTT CTT TTC CTC CGC-30; Cribb (ITS2 rDNA) for species of Hapalorhynchus and et al., 1998). The PCR amplifications were performed Coeuritrema platti Roberts & Bullard, 2016. using a total volume of 50 ll with 2 llofDNA template, 0.4 lM of each primer along with 19 buffer, 2.5 mM MgCl2, 1 mM dNTP mixture, and 0.3 ll Taq Materials and methods polymerase (5 U/ll) (Promega, Madison, Wisconsin, USA). The thermocycling profile for both genes In November 2015, during a parasitological survey of comprised an initial 5 min at 95°C for denaturation, aquatic vertebrates in the Mekong River Basin, two followed by 40 cycles of 94°C for 30 s for denatura- individuals of the Mekong snail-eating turtle Malay- tion, 63°C for 30 s for annealing, and 72°C for 2 min emys subtrijuga were opportunistically sampled from for extension, followed by a final five min at 72°C for a market in Can Tho, Vietnam (10°01042.1500N, extension. All PCR reactions were carried out in a MJ 105°47014.1500E). Flukes intended for morphology Research PTC-200 (BioRad, Hercules, California, were fixed, stained, and whole-mounted as per Roberts USA). PCR products (10 ll) were verified on a 1% et al. (2017); illustrated with the aid of Leica DM 2500 agarose gel and stained with ethidium bromide. PCR and Leica DMR (Leica, Wetzler, Germany) micro- products were purified by microcentrifuge with the scopes each equipped with differential interference QIAquick PCR Purification Kit (Qiagen, Valencia, contrast (DIC) optical components, an ocular microm- California, USA) according to the manufacturer’s eter, and a drawing tube; and compared with morpho- protocol, except that the last elution step was per- logically similar species. Measurements of turtle formed with autoclaved nanopure H2O rather than the blood fluke (TBF) specimens herein are reported in provided buffer. DNA sequencing was performed by micrometres (lm) as the range followed by the mean ACGT, Incorporated (Wheeling, Illinois, USA). Reac- and number of specimens measured in parentheses. tions were sequenced using BigDye terminator version 123 Syst Parasitol 3.1, cleaned-up with magnetic beads (CleanSeq dye from pharynx to caecal bifurcation. Intestine compris- terminator removal kit), and analyzed using ABI 3730 ing non-fused posterior caeca bifurcating immediately XL or 3730 Genetic Analyzer. Primers used in anterior to ventral sucker, smooth, lacking diverticula; sequencing of 28S rDNA included the PCR primers anterior caeca absent; posterior caeca inverse and the reverse primer 1200R (50-GCA TAG TTC U-shaped, extending 1/2–3/4 of body length directly ACC ATC TTT CGG-30) (Lockyer et al., 2003). posteriad, not extensively convoluted, terminating in Sequence assembly and analysis of chromatograms posterior body extremity. Testes 2, comprising one were performed with BioNumerics version 7.0 (Ap- anterior and one posterior testis in posterior 2/3 of plied Maths, Saint-Martens-Latem, Belgium). body, intercaecal, smooth or lobed. Male terminal Analysis of sequences, which followed Roberts genitalia pre-gonadal. Vas deferens ventral to cirrus- et al. (2017), is briefly described here. Assembled sac; anterior and posterior trunks of vasa efferentia sequences were aligned with MAFFT 7.310 (Katoh & dorsal to gonads; external seminal vesicle posterior to Standley, 2013) and subsequently corrected by eye in ventral sucker, intercaecal, extending anteriad to level Mesquite 3.2 (Maddison & Maddison, 2017). Regions of genital pore or level of ventral sucker; internal that could not be unambiguously aligned were seminal vesicle present;