Blood Flukes of Asiatic Softshell Turtles: Revision of Coeuritrema Mehra, 1933

Research Article Coeuritrema !" # $ % Pelodiscus sinensis &'" ! (!

Jackson R. Roberts1, Raphael Orélis-Ribeiro1, Binh T. Dang2, Kenneth M. Halanych3 and Stephen A. Bullard1

1 Auburn University, School of Fisheries, Aquaculture & Aquatic Sciences and Aquatic Parasitology Laboratory, Auburn, AL, USA; 2 Nha Trang University, Department of Biological Sciences, Institute for Biotechnology and Environment, Nha Trang, Vietnam; 3 Auburn University, Department of Biological Sciences and Molette Biology Laboratory for Environmental & Climate Change Studies, Auburn, AL, USA

Abstract: Coeuritrema Mehra, 1933, previously regarded as a junior subjective synonym of Hapalorhynchus Stunkard, 1922, herein is revised to include Coeuritrema lyssimus Mehra, 1933 (type species), Coeuritrema rugatus (Brooks et Sullivan, 1981) comb. n., and Coeuritrema platti Roberts et Bullard sp. n. These genera are morphologically similar by having a ventral sucker, non-fused caeca, two testes, a pre-testicular cirrus sac, an intertesticular ovary, and a common genital pore that opens dorsally and in the sinistral half of the body. Phylogenetic analysis of the D1–D3 domains of the nuclear large subunit ribosomal DNA (28S) suggested that Coeuritrema and Hapalorhynchus share a recent common ancestor. Coeuritrema Hapalorhynchus by Coeuritrema comprises species that reportedly infect Asiatic softshell turtles (Testudines: Trionychidae) only, whereas Hapalorhynchus (as current- \"#$"%Sternotherus Bell in Gray) and mud turtles (Kinosternon '*+*#$%<*$= (Geoemydidae) and African pleurodirans (Pelomedusidae). Coeuritrema platti sp. n. infects the blood of Chinese softshell turtles, Pel- odiscus sinensis %>*?KKQ%WXW*Y[C. lyssimus by having \%]^_\**%]^`x ***\=%{^`x *[C. rugatus by having small ventrolateral tegumental papillae, testes without deep lobes, and a Laurer’s canal pore that opens posterior to the vitelline reservoir and dorsal to the oviducal seminal receptacle. |\\"Y Keywords: *'*}~'?#$**Griphobilharzia

|\"%|QQ'- P. parviformis Tang, P. sinensis [Wiegmann]) are commer- dae’) comprise 84 accepted species (54 freshwater; 30 ma- cially prized and cultured for food and magic throughout rine) assigned to 20 genera and that infect 37 freshwater Asia (Alves et al. 2008, Fritz et al. 2010, Stuckas and Fritz and three marine turtle species (Platt 1993, 2002, Smith 2011). A survey of Chinese aquaculture facilities indicated 1997a,b, Tkach et al. 2009, Platt and Sharma 2012, Oré- that > 300 million Chinese softshell turtles are in captivi- lis-Ribeiro et al. 2014, Roberts et al. 2016). Seventeen ty now and that ~ 125 million individuals are available for |Q %Hapalorhynchus Stunkard, 1922; sale now in China alone (Haitao et al. 2008). Even though Vasotrema Stunkard, 1926; Coeuritrema Mehra, 1933; they are abundant in southeast Asian aquaculture, several Enterohaematotrema Mehra, 1940, Cardiotrema Dwivedi, of these species have become scarce in the wild (van Dijk 1967) infect softshell turtles (Testudines: Trionychidae) et al. 2000). ?*- %|^*^%}x|Q* suggesting that perhaps numerous innominate TBFs infect ƿ- this turtle lineage. Softshell turtles of Pelodiscus Fitzinger culture. (P. axenaria [Zhou, Zhang et Fang], P. maackii [Brandt],

Address for correspondence: J. Roberts, Auburn University, 203 Swingle Hall, Auburn, AL, 36849, USA. Phone: 334-844-9278; E-mail: [email protected] Zoobank number for article: urn:lsid:zoobank.org:pub:D060C500-3A11-4FC7-8ED9-9523EE402E63

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp.

?Y* \=[$"| we encountered a new TBF species infecting Chinese +%*Y*<*'$\- softshell turtles, which led to a consideration of all TBFs turer’s protocol. DNA sequencing was performed by GENEWIZ infecting softshell turtles. Herein, we revise Coeuritrema, \$Q[W`?#$%#>[*[*' emend its diagnosis, redescribe Coeuritrema rugatus W*#\*'$W}~' (Brooks et Sullivan, 1981) comb. n. (formerly Hapalo- rDNA included the PCR primers and the internal forward prim- rhynchus) based on museum specimens, describe a new ``%=<$$|$<<|$$$$||=`` species, and provide an updated phylogeny for the TBFs %=<<|<||$$$<$<$<<$$= \||Q ^}``K%=<$|$||<$<<$|<|||<=%" reported from Vietnam. 2003). Sequence assembling and analysis of chromatograms were conducted using BioNumerics version 7.0 (Applied Maths, Sint- MATERIALS AND METHODS Martens-Latem, Belgium). Turtles were purchased from commercial turtle trappers in Nha The sequence data for nuclear 28S rDNA generated during this Trang, Vietnam and from a turtle farm in Phu Yen Province dur- \\\"%'- ^^_}`^|\"#- matoidea) available on GenBank. Outgroups were selected from sies were performed using 7.0 g/l sodium citrate saline solution. \"* $ * ^^} \"\=\"\ (see Bullard et al. 2009). The ingroup comprised newly-generated hand lighter under little or no coverslip pressure and transferred \%+^}}Hapalorhy- x % |Q \ nchus foliorchisQ"*^%+^}}}- x $ \ \ - ly available sequences from Baracktrema obamai Roberts, Platt tilled water, specimens intended as whole mounts were stained in Q*}`^_%+`_^``*Griphobilharzia amoena Platt et Y<R\- Blair, 1991 (AY899914), and ten other TBFs (Orélis-Ribeiro et R** al. 2014). The specimens of H. foliorchis were collected in Ala- clove oil, and permanently mounted in Canada balsam. bama from Chelydra serpentina (Linnaeus) during October 2014 >\?}``- (Roberts et al. 2016). '\$|%+- \ toh and Toh 2010) with default settings implemented in the CIP- Parasite measurements are reported in micrometres (μm) followed RES Science Gateway V.3.3 (Miller et al. 2010). The resulting by the mean and number of specimens measured in parentheses. \$}}%| |\?" et al. 2011) and the ends of each fragment were trimmed to match %}`^<|Q\ $\ Luhman (1935 – genital sucker), Byrd (1939 – uterine pouch [see removed using the Gblocks server (Castresana 2000) with set- also Roberts et al. 2016]), Yamaguti (1971 – Manter’s organ), and tings for a less stringent selection. Bayesian inference (BI) was Platt (1993 – median oesophageal pouch; 1998, 2002 – genital performed using the Metropolis-coupled Markov chain Monte spines, plicate organ and most other terms). Carlo method (MC3) in MrBayes version 3.2.6 (Huelsenbeck et Specimens for molecular analyses were handled with cam- al. 2001, Ronquist and Huelsenbeck 2003, Huelsenbeck and Ron- = * * - quist 2005) and run on CIPRES (Miller et al. 2010). The model served in a vial of absolute EtOH and stored at -20 °C. Total of nucleotide substitution was selected based on the Akaike In- ?#$%?#$\?#|Q formation Criterion (Posada and Buckley 2004) as implement- | + %* Y*<* '$ ed in the jModelTest version 2.1.4 (Guindon and Gascuel 2003, R* Darriba et al. 2012). The GTR + I + G (proportion of invariable \=+\* sites = 0.385 and gamma distribution = 1.393) model was inferred \^``)\ as the best estimator. Therefore, BI used the following param- ?#$ eters: nst = 6, rates = invgamma, ngammacat = 4, and default Partial 28S rDNA (domains D1–D3; ~ 1 400 bp) was ampli- priors. Analyses were run in duplicate each containing 4 inde- \Q^~R%=<$<<<<|$$X- pendent chains (three heated and one cold chain) (nchains = 4) for ||$$= Q^_}R %=<<$<<- ^`^`%¡^``````` <$|<<$||||<$=%"}``W

\^*}<2 (New England Biolabs, Ipswich, lihood values of the sample points against generation time using *'$*^#|W`)|- Tracer version 1.5 (Rambaut and Drummond 2009). All retained %)%#\Q| trees were used to estimate posterior probability of each node. consisted of an initial 4 min at 94 °C for denaturation, followed A majority rule consensus tree with average branch lengths was by 40 repeating cycles of 94 °C for 30 s for denaturation, 50 °C QR ` * } < } * - (sumt) in MrBayes. Resulting phylogenetic trees were visualised \} <$W**'$W

Folia Parasitologica 2016, 63: 031 Page 2 of 15 doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp.

RESULTS R[ of Asiatic trionychids. Coeuritrema Mehra, 1933 emended ^ Q \* Diagnosis. Body \%- aspinous; ventrolateral tegumental papillae present. Ventral *\* sucker present. Oesophagus < 1/4 of body length, lacking "* ^ * - diverticula, plicate organ, or median oesophageal pouch. pinous, papillate; ventrolateral tegumental papillae pres- Intestine comprising non-fused caeca. Testes comprising ent, distributing from oral sucker posteriad or from ven- - tral sucker posteriad. Dorsolateral and ventrolateral nerve chords present. of common genital pore. Ovary intertesticular. Oviducal Oral sucker robust, aspinous, demarcated from body by seminal receptacle comprising middle portion of oviduct posterior constriction. Ventral sucker present, aspinous. between ovary and posterior testis. Laurer’s canal intercae- W * - cal, intertesticular, post-ovarian. Vitelline reservoir intert- ]^ esticular. Uterine pouch absent. Egg single, occupying fe- length, ventral to anterior nerve commissure, lacking diverticula, plicate organ, or median oesophageal pouch, ** straight or slightly sinuous; oesophageal gland surrounding storing egg(s). Common genital pore dorsal, sinistral, pre- - dominantly anterior to genitalia. geal-intestinal junction, strongly basophilic, widest at level Type species: Coeuritrema lyssimus Mehra, 1933 from of caecal bifurcation. Intestine comprising non-fused cae- [ \ * Lissemys punctata ca, inverse U-shaped, smooth (lacking diverticula or sec- (Bonnaterre) (Testudines: Trionychidae), in the Ganges River, * ^} India. ** Remarks. Mehra (1933) proposed Coeuritrema for "* C. lyssimus and Hapalorhynchus odhnerensis (Mehra, Testes comprising one anterior testis and one posterior 1933) Byrd, 1939. That diagnosis included “body wall with testis, in posterior half of body, intercaecal, having deep or without small papillae” (= ventrolateral tegumental pa- Y pillae) such that H. odhnerensis, which lacks ventrolateral directly anteriad and ventral to cirrus sac before turning tegumental papillae, could be included. As emended herein, however, Coeuritrema includes papillate TBFs only. "*- This genus has long been regarded as a junior subjective ** synonym of Hapalorhynchus (see Byrd 1939, Hughes et of common genital pore. Cirrus sac robust, pre-testicular, al. 1942, Brooks and Mayes 1976, Platt 1988, 2002, Bour- containing variously-sized secretory cells and large pars gat 1990), which lacks ventrolateral tegumental papillae. prostatica. Coeuritrema Hapalorhynchus by hav- Ovary single, wider than long, triangular, lacking lobes, * margin of ovary, directing posteriad; oviducal seminal receptacle comprising middle portion of oviduct between The ootype and uterus are both relatively short and to- ovary and posterior testis. Laurer’s canal intercaecal, intert- gether, rather than the metraterm, may store the egg in spe- *=* cies of Coeuritrema. If a large egg, presumably one that is oviduct at level of vitelline reservoir, opening dorsally. Vi- developed and ready for passage to the metraterm and/or tellarium follicular, occupying space from caecal bifurca- * tion to caecal termination; vitelline reservoir intercaecal, reproductive tract, the egg occupies the luminal spaces of intertesticular, ventral to ovary. Ootype a weakly glandular both the ootype and uterus (Fig. 4). In that case, the ootype and thin-walled chamber, intercaecal, occupying space be- ƾ%> tween anterior testis and ovary, posterior to cirrus sac. egg, the clear distinction between the ootype and the uter- Uterus intercaecal, intertesticular, straight (not coiled); §= uterine pouch absent. Egg single, occupying female repro- chamber immediately distal to the ovo-vitelline duct and * the uterus is the narrow, short duct connecting it with the *% metraterm (Fig. 6). If our understanding is correct, it may anteriad in parallel with body margin), between ventral be useful to consider the ootype and uterus functioning to- sucker and vitelline reservoir, sinistral to anterior testis, QR#\ having an obviously muscular thick wall, apparently not also is that we did not observe an egg in the metraterm storing egg(s). Common genital pore dorsal, sinistral, pos- of any specimen, indicating that perhaps the metraterm of terior to ventral sucker, predominantly anterior to genitalia, Coeuritrema spp. does not retain the egg. aspinous, lacking suckers. These observations match those of previous workers ** handling specimens of the type species and Coeuritrema posteriad from distal ends of caeca to posterior body end. rugatus. %^-

Folia Parasitologica 2016, 63: 031 Page 3 of 15 doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp. served more than a single egg (“ovum”) in the female re- Tremarhynchus as a senior synonym of Coeuritrema by productive tract of C. lyssimus (type species) and that each . This decision was probably based upon O *P the International Code of Zoological Nomenclature’s Ar- \*%^*^- _^^}%O'W|- ra 1933) could be interpreted as the putative egg chamber. P*\O= Brooks and Sullivan (1981) likewise reported that only is the nominal genus on which its name is based.” (Inter- a single egg occurred in the uterus of ovigerous specimens. national Commission on Zoological Nomenclature 2000). Perhaps this is related to the gestation time for the large Hence, because post-1967 Coeuritrema was correctly un- egg. Tremarhynchus, | \= \" the subfamily name had to be changed. does hold eggs (Bullard and Overstreet 2006, Bullard et Yamaguti (1971) accepted Coeuritrematinae Dwivedi, al. 2006, Bullard and Jensen 2008); however, eggs in those 1967 (as “1968”) and included Coeuritrema, Enterohae- species are smaller, much more numerous in the uterus and matotrema, and Cardiotrema based on the presence of metraterm, and seemingly less developed. Perhaps this a ventral sucker, paired caeca not united posteriorly, two is because they must infect minute branchial vessels be- testes, a cirrus sac “more or less strongly developed” before transiting to the gill epithelium and hatching (Bullard tween the ventral sucker and anterior testis, an intertesticu- }``~|\ lar ovary, a vitellarium as long as caeca, and a genital pore serves as a storage organ for TBF eggs is indeterminate, between the ventral sucker and testes. These genera plus "QR Hapalorhynchus and Vasotrema include species infecting %¡«*"- softshell turtles (Trionychidae) (Table 1). No molecular se- agnostic feature relating genera. Resin-based ultramicroto- Enterohaematotrema \- or Cardiotrema, and Vasotrema does not group with the ductive tract could reveal cellular changes to the putative Coeuritrema + Hapalorhynchus clade (Fig. 7). Hence, egg chamber wall in the presence/absence of the large egg. * Coeuritrema and Hapalorhynchus are similar morpho- TBFs, suggesting that TBFs have repeatedly, independent- logically by having a ventral sucker, non-fused caeca, two ly colonised softshell turtles. testes, a pre-testicular cirrus sac, an intertesticular ova- Coeuritrema Enterohaematotrema by hav- ry, and a common genital pore that opens dorsally and ing a dorsal, sinistral genital pore (rather than a ventral, in the sinistral half of the body. Phylogenetic analysis of medial genital pore), from Cardiotrema by having an inter- the D1–D3 domains of the nuclear large subunit riboso- testicular ovary (rather than a sinistral ovary) and a large mal DNA (28S) suggests that Coeuritrema and Hapalo- ventral sucker >1/2 body width (rather than <1/5 body rhynchus indeed share a recent, common ancestor (Fig. 7; width for Cardiotrema), and from Vasotrema by having see below). We concur with Platt (2002) in that several two testes and a dorsal genital pore (rather than one testis \" posterior to the ovary and a ventral genital pore). Mehra to Coeuritrema should be assigned to Hapalorhynchus: %^ " |Q H. odhnerensis, Hapalorhynchus ocadiae (Takeuti, 1942), Coeuritrema and Hapalorhynchus by the presence or ab- Hapalorhynchus oschmarini (Belous, 1963), Hapalorhyn- sence of a cirrus sac and cirrus. These features have been chus macrotesticularis (Rohde, Lee et Lim, 1968), Hapalo- the focus of intense study because of their stated value as rhynchus mica (Oshmarin, 1971), Hapalorhynchus sheilae (Mehrotra, 1973) and Hapalorhynchus sutlejensis (Mehro- the presence of an obvious cirrus sac among the accepted tra, 1973) (see Takeuti 1942, Rohde et al. 1968, Oshmarin species of Coeuritrema, but this feature may or may not be 1971, Mehrotra 1973). This brings the total number of ac- |Q* cepted species of Coeuritrema and Hapalorhynchus to 3 within the closely-related Hapalorhynchus the presence or and 19, respectively. absence of the cirrus sac and cirrus have been accepted as Yamaguti (1958) considered Coeuritrema a junior syn- generic features by some (Mehra 1939, 1940, Yamaguti onym of Tremarhynchus Thapar, 1933; however, the diag- 1958, 1971) and rejected by others (Byrd 1939, Brooks and nosis of Coeuritrema was published in May 1933 and that Mayes 1976, Platt 1988, 2002, Bourgat 1990). of Tremarhynchus was published immediately thereafter Brooks and Mayes (1976) provided supplemental ob- ¬* servations of the male genitalia of Hapalorhynchus stunk- (Dwivedi 1967). Byrd (1939) and Platt (2002) considered ardi Byrd, 1939 and emended Hapalorhynchus to include Tremarhynchus (and Coeuritrema) a junior subjective syn- “well-developed or poorly-developed cirrus.” Platt (1988) onym of Hapalorhynchus. No author has treated Tremar- observed, in histological sections, prostate cells not bound hynchus since 2002. by a limiting membrane but scattered in the surrounding Tremarhynchinae Yamaguti, 1958 included Enterohae- parenchyma in Hapalorhynchus gracilis Stunkard, 1922, matotrema and Tremarhynchus (type genus) (see Yamagu- H. foliorchis and H. stunkardi. He also observed that the ti 1958). Dwivedi (1967) commented that the subfamily Hapalorhynchus albertoi Lam- should be renamed Coeuritrematinae Dwivedi, 1967 (type othe-Argumedo, 1978 and Hapalorhynchus brooksi Platt, genus Coeuritrema) to correct Yamaguti’s (1958) name 1988 was membrane-bound (= cirrus sac) and that the dis- for the subfamily based on his erroneous acceptance of tal portion of the ejaculatory duct (= cirrus) was eversi-

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Table 1. Q\"%|§|

Turtle Fluke Site in host Locality Museum nos. Reference Amyda cartilaginea Coeuritrema rugatus mesenteric blood Sungei Jempol, Ulu Jempol, State of Negeri HWML 21339 Brooks and Sullivan (Boddaert), Asiatic (Brooks et Sullivan, 1981) vessels Sembilan, West Malaysia (1981) softshell comb. n. Apalone ferox (Schneider), Vasotrema amydae Stunk- blood * AMNH 791 Stunkard (1926) Florida softshell ard, 1926 (type species) Florida, USA blood * AMNH 791 Stunkard (1928) Florida, USA Vasotrema attenuatum blood * AMNH 806, 807 Stunkard (1928) Stunkard, 1928 Florida, USA Vasotrema sp. Apalachicola River, Franklin County, Loftin (1960) Florida, USA Apalone mutica (LeSueur), Vasotrema attenuatum blood Missouri River Drainage, Nebraska, USA Brooks and Mayes (1975) smooth softshell Vasotrema brevitestis blood Missouri River, 2.4 km south of Blair, USNM Brooks and Mayes (1975) Brooks et Mayes, 1975 Nebraska, USA ~^~^ HWML 20077 Vasotrema robustum blood Missouri River Drainage, Nebraska, USA Brooks and Mayes (1975) Apalone spinifera Vasotrema amydae blood Ohio River Drainage, Indiana, USA AMNH 791 Stunkard (1926) (LeSueur), spiny softshell (type species) blood Ohio River Drainage, Indiana, USA AMNH 791 Stunkard (1928) Vasotrema attenuatum blood Ohio River Drainage, Indiana, USA AMNH 806, 807 Stunkard (1928) mesenteric blood Reelfoot Lake, Tennessee, USA HWML9227 Byrd (1939) (see also vessels Platt and Snyder 2007) Missouri River Drainage, Nebraska, USA Brooks and Mayes (1975) Vasotrema brevitestis Atkinson Lake, Atkinson Recreation Area, USNM Brooks and Mayes (1975) 0.8 km west of Atkinson, Nebraska, USA ~^~^ HWML 20076 Vasotrema longitestis arterial circulation Reelfoot Lake, Tennessee, USA USNM 9229, Byrd (1939) (see Platt and Byrd, 1939 80656-8; HWML Prestwood 1990) 31121-8 Vasotrema robustum blood Ohio River Drainage, Indiana, USA AMNH 808, 809 Stunkard (1928) arterial system Reelfoot Lake, Tennessee, USA USNM 80657-2; Byrd (1939) (see Platt and HWML 31122-2 Prestwood 1990) heart, large blood Huron River, Washtenaw County, Michigan, USNM 37306 Wall (1951) vessels USA Missouri River Drainage, Nebraska, USA HWML 20075 Brooks and Mayes (1975) Nishnabotna River, Floyd County, Iowa, USA HWML 45795 Snyder (2004) Dogania subplana Hapalorhynchus heart, arteries near "\*+ Helm. Coll. No. Rohde et al. (1968) (Saint-Hilaire), macrotesticularis (Rohde, heart (purchased from Chinese merchant), R. 769, Zoology Malayan softshell turtle Lee et Lim, 1968) Malaysia Dept., University *+ Lumpur Lissemys punctata (Bonna- Cardiotrema roparensis Sutlej River, Ropar, Punjab State, India Mehrotra (1973) *[\ (Mehrotra, 1973) body wash, hepatic Sutlej River, Ropar, Punjab State; Yamuna Tandon and Gupta (1985) blood vessels K*+*¬'*[ Enterohaematotrema small intestine Ganges River, Allahabad, Uttar Pradesh Mehra (1940) palaeorticum Mehra, 1940 (probably mesenter- State, India (type species) ic blood vessels) Coeuritrema lyssimus ventricle of heart Ganges River, Allahabad, Uttar Pradesh Mehra (1933) Mehra, 1940 (type species) (adult) State, India heart (adult) Ganges River Drainage, Rudrapur, Tandon and Gupta (1982) Uttarakhand State, India Hapalorhynchus ventricle of heart Ganges River, Allahabad, Uttar Pradesh Mehra (1933) odhnerensis (Mehra, 1940) State, India Hapalorhynchus sheilae Patiala River (Ghaggar River Drainage), Mehrotra (1973) (Mehrotra, 1973) Patiala, Punjab State; Sutlej River, Ropar, Punjab State; Ghaggar River, Sangrur, Punjab State; Ganges River Drainage, Rudrapur, Uttarakhand State, India heart, blood, liver Ganges River Drainage, Rudrapur, Uttara- Tandon and Gupta (1982) khand State; Patiala River (Ghaggar River Drainage), Patiala, Punjab State; Ghaggar River, Sangrur, Punjab State, India Hapalorhynchus sutlejensis Sutlej River, Ropar, Punjab State; Ghaggar Mehrotra (1973) (Mehrotra, 1973) River, Sangrur, Punjab State, India ventricle of heart Gomti River, Lucknow, Uttar Pradesh State; Tandon and Gupta (1982) Ghaggar River, Sangrur, Punjab State, India Nilssonia gangetica (Cuvi- Hapalorhynchus indicus heart, large blood Gomti River, Lucknow, Uttar Pradesh Thapar (1933) er), Indian softshell (Thapar, 1933) vessels State, India Nilssonia hurum (Gray), Enterohaematotrema intestine (probably +*K*W Shrivastava (1959) Indian peacock softshell palaeorticum (type species) mesenteric blood State, India vessels) Pelodiscus sinensis (Wieg- Coeuritrema platti sp. n. heart, mesentery, Da Rang River, Phuyen Province, Vietnam USNM 1411790, Present study mann), Chinese softshell lung 1411791 Hapalorhynchus mica hepatic blood Duong River, Hai Phong, Vietnam Oshmarin (1971) (Oshmarin, 1971) vessels Hapalorhynchus hepatic blood "+"*K Belous (1963) oschmarini (Belous, 1963) vessels

Folia Parasitologica 2016, 63: 031 Page 5 of 15 doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp. ble (Lamothe-Argumedo 1978, Platt 1988). As a result, he %x\*}``%}_~\ emended the diagnosis as “cirrus sac present or absent.” As ^x%^}x~\*}%^~\- additional species of Coeuritrema are documented, special ^}x%^~x~\- care should be taken to assess the cirrus sac and cirrus. ~}`^^``%~_x%_~x * _` %x ~ \ ~`x Coeuritrema rugatus (Brooks et Sullivan, 1981) comb. n. %x~\*^`%}~\ Figs. 1, 2 ^x%^^x~\*}%}~\- ^}x%^~x~\- ZooBank number for species: ^^^%^`~~^`x%x urn:lsid:zoobank.org:act:36DE9E3C-2809-476B-978A-67B2CC3B122F end. Anterior testis lobed, intensely follicular marginally, Redescription (based on light microscopy of eight ^^~^~~%^~^x%^`x* whole-mounted adult specimens, including paratypes): `^^%~~\x%`x~\ Q ^}`^` %^} } %_ _`%~~x%_x \*^`}``%^~\^}^x%^}x 7) of body length (Figs. 1, 2). Posterior testis as anterior *}``}_ *^~}`~%^_~^`^x%^}x %}^~\^^x%^x *`^%^~\^x%^x %\*^`}`%^~ \=``%^~ \^^^x%^}x- }_`x%}~x$- nation; forebody (middle of ventral sucker to anterior end) " ``%}x%^x* *}`}~%}}~]^x lacking ventrolateral tegumental papillae; hindbody (mid- %*%~\}x%}x~ "~`^``%_~~ \%}" ___x%_x*- *- eral tegumental papillae (Fig. 1). Ventrolateral tegumental }``}%}~^_^x%^ ^}`%^~*^~}` *%~\^x%}x~\* %^ ~ * %~ ~ \" % ^ ~ %}` ~ - anterior portion of anterior testis (Fig. 2). ®*\^~%}~\* Y~~^%^^^~_^`x%~x `_}%^~\} *~%~\* %}~*\%~\*^`^~%^ `~` %_` ~ ~\~~%}~* _x%x*}^^%_~\* ^%}~\*`}%^~\ `}`%^~\*\ "_`%_x%x %^*}- *~^``%\~`x%x- ~~%~`~^`}^%^ \%^Y"^`^}%^^ ~*}%^~\ ~x%~x*^`^%^}`~ }}^%}_~\}}^}x%~^x~ \x%_~x~\- \%^*}Pars prostat- *^^%^\"%^ ica surrounding distal portion of internal seminal vesicle, #^}}%}`}^^_x%^x ~~%}~*`%}~\^^%^_ of body length from anterior body end. ~\%}<^`%~~* W _~ % }`}x %}x (4; 8) wide; cirrus sac enveloping most of internal semi- *_`~%\`~^%^^ nal vesicle and entirety of pars prostatica*~~^_`%^^~ \ % ^ _^^x%~x*%_~\ }}}%}_^^}^x%^~x* }x%^x~\* ^}%^\*\\^`^~ large putative secretory cells (Figs. 1, 2). %^"*%~\}x Ovary triangular, with broadest portion in sinistral half %^x ~ \ * \ \ *}_`%^`~}^}x%}}x ^``%}~"%^*\^}%}`~ length from middle of ventral sucker buttressing caeca, \^x%^^x~\- ~^`` % ~ _x %_x * *\\^`}`%^~" ^`%^^}~\~x%^x\* }`}%}}~^^x%^_x ^^~ %^ ~ \ = ~^x %~_x * ^``^ _` %~^ ~ `x %^x %^}^~\_~x%`x~\ (Fig. 1); oocytes largest and most basophobic near oviduct bifurcation. % } * * [ }_}` %}~ ^}x "\~_%~x %}`x- %x*^`%~~\* ~`^``%_}~_}x%_x * ^`` %~ \ _x ~`

Folia Parasitologica 2016, 63: 031 Page 6 of 15 doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp.

1 os 2 ec ph esv pp cgp oe pp nc isvisv dln vd og cs cb mt

ave sc dc

vs at

esv cgp cs isv egg vd lvdlvd mt ave ut at lvd lvd oo vlp ov ov lvd ov Lc vt od osr tvd pve vrs od osr lvdlvd pt lvdlvd

pve vr vr pt 250 μm 50 μm ev 250 μm 50 μm ep

Coeuritrema rugatus (Brooks et Sullivan, 1981) comb. n. (Digenea: Schistosomatoidea) from the mesenteric vessels of Amyda cartilaginea (Boddaert) (Testudines: Trionychidae) from Malaysia, paratype (HWML Coll. No. 21339). Fig. 1. Total view, ventrally. Genitalia, ventral view. Abbreviations§" R "pars prostatica "- "

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%_~~^^}x%~x~\*}%}~~ - \^_x%^x~- \*\*- the mouth (Fig. 1). Coeuritrema and Baracktrema Roberts, ~%~}~x%_x Platt et Bullard, 2016 are the only accepted TBF genera *^`^%^}~\x%x~ *- \%}R\- ately posterior to the mouth and muscular oral sucker. Sev- }%^~ eral species of Hapalorhynchus, Spirorchis MacCallum, *~^%^^~\* 1918 and Vasotrema\ middle portion of ovary (Fig. 2). \"%KK*'$QM- Vitellarium comprising a series of interconnected sphe- publ. data). roid masses of follicles, distributing from level of caecal Regarding the oviduct, Brooks and Sullivan (1981) il- bifurcation to distal ends of caeca, lateral collecting ducts lustrated it as originating from the posteromedial aspect of coalescing at level of posterior margin of ovary to form *\ transverse vitelline duct; transverse vitelline duct ventral K *^^%_~}}x%}x the oviducal seminal receptacle, they illustrated a sac-like length from middle of ventral sucker (Figs. 1, 2); vitelline \ reservoir sac-like, ventral to oviducal seminal receptacle; %Q"'^~^* ~ p. 1336). No paratype had such a structure. They stated %_~_x%x that the vitelline reservoir was immediately postovarian \%}ƾ and ventral to the ovary, but we observed that the reservoir * ` %_ ~ * }~ % ~ coalesces sinistro-dorsal to the ovary (Fig. 2). wide, dorsal to anterior margin of ovary (Fig. 2). Coeuritrema platti Roberts et Bullard sp. n. (Fig. 2), with a single egg in seven of eight specimens; ZooBank number for species: * }~` % ~ }x %x urn:lsid:zoobank.org:act:539BDF18-65AD-40E6-B0F9-AB82923967B1 *^~`%}~\^x%^}x~- \ * Description (based on light microscopy of seven ^~}^~%^~^^^x%^}x* \= § Q ^`^^` \}%~^}}^x%^_x~ %^}^~ _ _ %_` _ \* \*}__%~ ^^`^`%^_\^^^x%^}x_ *_~%}x%_ * ^`}} %}` _ \ *}%}\^_x%^}x ^^~x%^x_%- \*^%}\ \ * ^}`^~` %^_ _ \ %}<^`%^}~~^`x ^}^_x %^x _ %x"- `^%_}x%_x_ er (Fig. 1). *__``%~__^_~x ^`^_ %^ ~ ^`^}x %_x_* %^`x*~^``%~~\x %*Y- %`x~\\~^ tending posteriad from middle of ventral sucker to posterior (10; 8) thick (Fig. 1). ^~^%^~*^}`%^~ Type and only known host: Asiatic softshell turtle, *%_% Amyda cartilaginea (Boddaert) (Testudines: Trionychidae). *~}%^*\^` Type locality: Sungei Jempol, Ulu Jempol, State of Negeri %}^ \* `} %^} \ Sembilan, Malaysia. ^`}%^*\^~%}_\* Site in host: Mesenteric blood vessels. ^`}~%^\} '§ Hapalorhynchus rugatus – Har- %^*\~}~%^\*`_}`%^ old W. Manter Laboratory (HWML) of Parasitology Coll. No. 3) wider than long. 21339, paratypes, eight slides comprising eight whole-mount- "`%~_x%x ed specimens, seven of the eight slides labeled as Hapalo- *`^``%~`_\x%x_- rhynchus rugosus*Amyda cartilaginea (as Trionyx carti- \%*Y"^~%^^ lageneus) from Sungei Jempol, Ulu Jempol, State of Negeri _^x%^`x_*_^~%^^ Sembilan, Malaysia (Brooks and Sullivan 1981). \x%x\- *^^_%^\"%* Remarks. Our interpretation of some features associat- #^`}~%^~~_^^x%^x_ ed with type materials (see above) of C. rugatus contradict- of body length from anterior body end. ed those of Brooks and Sullivan (1981), who did not detail W _ %~ _ }`}x %}x _ **R *~%__\^_}}%^ They reported that C. rugatus "\* _\%*

Folia Parasitologica 2016, 63: 031 Page 8 of 15 doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp.

3 os 4 cgp ec

pp ph

esv

oe nc vln mt

og cs isvisv cb

dc vd sc ave at vs egg cgp esv pve mt isv ov cs vlp at tvd egg ov egg od vtvt vt tvd lvd osr vrs od osr pt

vr vr

pt 250 μm 50 μm

ev 250 μm 50 μm

Figs. 3, 4. Coeuritrema platti sp. n. (Digenea: Schistosomatoidea) from viscera of Pelodiscus sinensis (Wiegmann) (Testudines: Tri- onychidae) from Vietnam, holotype (USNM Coll. No. 1411790). Fig. 3. Total view, dorsally. Fig. 4. Genitalia, dorsal view. Abbrevi- ations§" - R " pars prostatica" "

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5 6 ec os pp ph cgp esv

nc oe

og isvisv cs mt cb vln vd sc ave dc vs

at esv cgp vd isv mt ut cs ave vlp at lvdlvd oo ov

lvdlvd lvd ov tvd od vt tvd vt osr lvdlvd pve vrs lvdlvd pt od osr vr vr Lc

250 μm pve pt 50 μm ev 250 μm 50 μm

Figs. 5, 6. Coeuritrema platti sp. n. (Digenea: Schistosomatoidea) from viscera of Pelodiscus sinensis (Wiegmann) (Testudines: Tri- onychidae) from Vietnam, paratype (USNM 1411791). Fig. 5. Total view, ventrally. Fig. 6. Genitalia, ventral view. Abbreviations: " R " pars prostatica" "

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* }}`^ %} _ ^~}x %}^x _ \* *^`^%^_\*\ %_ \^`%_"*}`%_\ Ovary triangular, with broadest portion in sinistral half of %}_x _ \ * \ \ *^_`}`%}__^}x%}`x_ ^`}%}`_"*^^~%^__\ "**^^ ~^_x %^^x _ \ * %~_x%x_*_^%^^^ \\^`^%^}_"^~}~~ _\}`x%x_\*^^^%^ %} _ ^}x %}^x _ _\=}^`%~_ `^^ %^` _ * ^} %^` _ x%}~x_%_ \_~x%^x_\- uniform in size and basophilic throughout ovary (Figs. 4, %*[}_%}~_ _**- ^}x%}x_- *- ~`%_^_~x%_}x_ ^`%_^_x%x_* *%_\}}x%}x_ ^`%~_\* \*^`%}`_\_^x oviducal seminal receptacle; oviducal seminal receptacle %^`x_\*}`%}_\ _~`%^_~x%x ^}^x %^_x _ \ \*}`%\ ~` % _ }_x %_^x _ ^^}`x%^_x\*\ *~%^_\}_x%}x_ *~~`% \*^~%}_\^`^~x _x%x*^`^%^^- %^}x_\*^~%}_\ \_x%x\%_ ^^}`x%^_x_\- R\^}%}^- }^`%^}`_~^x%^`x_- * ~ %_ rior end. \*- Anterior testis follicular throughout, lacking lobes, cal seminal receptacle (Figs. 4, 6). _`^%^`__^^x%x_* Vitellarium comprising a series of interconnected sphe- ` %^ _ \ }x %x _ \ roid masses of follicles, distributing from level of caecal `^` % _ x bifurcation to distal ends of caeca, lateral collecting ducts %_x_%_W- coalescing at level of posterior margin of ovary to form *`^~%^`_~^x%^^x_ transverse vitelline duct; transverse vitelline duct ventral to * ^} %~ _ \ }~x %x _ *}~%}}^}x%}x \ = } "*%_ %^`_}}~x%}x_- vitelline reservoir sac-like, ventral to oviducal seminal re- $" *^` `~%}x%x- %}_^x%}x*% \%_ƾ \^}x%^x\%_- *}`%}* " %~\*%_ *^~~}%}} ^}^x%^~x*%\^x %_*\ %}x\*\" *}%_}x%x* %_ }~ %} \ ^}^~x %^x Y~`^~%^`^_^`x%~x \*^}~^_ *%\*- %^^^^x%^}x*_ _~~% %\^}x%}x\* _x%*}~%\*^} }~~ % (2.3; 5) longer than wide, constricting posteriorly prior to *~`%~__~x%x* %_- `~%\^^x%^_x ~~^^~%^``^^^_%^- \*}}%}\%<- *^%}_\_~% _^~%^`_ _^^x %~x_ \}}x%x body length posterior to middle of ventral sucker. \%_Pars prostatica surrounding dis- ^}`^~` %^_ _ ^}^x %*_* %^x_*~`%_\}x %*}``%}\^~}%}} %x_ \*\\ \<~%_*%\ ^%"%* enveloping most of internal seminal vesicle and entirety of Type and only known host: Chinese softshell turtle, pars prostatica*~^}~%^``_x%~x_ Pelodiscus sinensis (Wiegmann) (Testudines: Trionychidae). *_%}_\}}`x%}_x_

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7 1 AY604707 Learedius learedi 1 AY604708 Hapalotrema mehrai AY604709 Carettacola hawaiiensis 1 KX712242 Hapalorhynchus foliorchis 1 0.85 AY604710 Hapalorhynchus gracilis 1 KX712243 Coeuritrema platti sp. n. AY899914 Griphobilharzia amoena 1 FJ481164 Spirorchis haematobius 1 1 AY222174 Spirorchis scripta 1 FJ481166 Spirhapalum siamensis 1 AY604705 Spirhapalum polesianum AY604706 Vasotrema robustum 0.98 1 KX061500 Baracktrema obamai AY604711 Unicaecum sp. AY222177 Aporocotyle spinosicanalis 1 1 AY222178 Plethorchis acanthus AY222179 Neoparacardicola nasonis AY157239 Chimaerohemecus trondheimensis 0.05 substitutions per site

Fig. 7. W\"Q?^?}~' ^~%^|Q*\"*\"# ?

T y p e l o c a l i t y : Da Rang River Basin, Phu Yen Province, Vietnam. \" Site in host: Heart, mesentery, lung. Remarks. Coeuritrema platti is most similar to C. rug- Prevalence and intensity of infection: Two atus by the combination of having ventrolateral tegumental %^``xP. sinensis from an aquaculture facility in the Da Rang papillae restricted to the hindbody as well as a hindbody KQC. platti. None of two P. sinensis collected from the Cai River Basin in Nha ]^_\|\ is most easily dis- Trang yielded specimens. tinguished from C. rugatus by having small ventrolateral Specimens/materials deposited: Holotype, United % ) \* - States National Museum (USNM) Coll. No. USNM 1411790; tes without deep lobes, and a Laurer’s canal pore open- one paratype, United States National Museum (USNM) Coll. ing posterior to the vitelline reservoir and dorsal to the No. USNM 1411791; one paratype (Institute of Parasitology, oviducal seminal receptacle. Coeuritrema rugatus has $ ' <± K* 5" QD- %) jovice (IPCAS) Coll. No. D-727; GenBank Accession No. base width), deeply lobed testes, and a Laurer’s canal pore +^}} opening anterior to the vitelline reservoir and oviducal § Hapalorhynchus gracilis – Ameri- seminal receptacle. Coeuritrema platti C. lys- can Museum of Natural History (AMNH) Coll. No. 125, holo- simus \%^^^ *\=* width), ventrolateral tegumental papillae restricted to Chelydra serpentina from North Judson, Indiana (Stunkard *x 1922); AMNH 1269, paratypes, four slides (three of the four la- length, a transverse ovary buttressing the caeca, a short, beled Hapalorhychus gracilis) comprising four whole-mount- * Chelydra serpentina from North Judson, \=%^^M^x* Indiana (Stunkard 1922); Vasotrema attenuatum $#¬ and a submarginal genital pore. Coeuritrema lyssimus 806, syntype, 17 slides comprising 25 whole-mounted spec- \%^\* * Apalone ferox and A. spinifera from Florida and tegumental papillae distributing from the oral sucker to Indiana respectively (Stunkard 1928). Hapalorhynchus rug- *^`^x atus¬>}^ of body length, a sinistral ovary that does not buttress the §| platti honours our friend *- and colleague Thomas R. Platt (Professor Emeritus, Saint ^^~x* R<*#?*[*- genital pore.

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Molecular phylogenetic results Fig. 7 Coeuritrema are required to further test the phylogenetic The results of our phylogenetic analysis of the D1–D3 pattern we recovered herein (Fig. 7). domains of the nuclear large subunit ribosomal DNA (28S) Especially noteworthy herein is the sister relationship § \" between Griphobilharzia amoena and the Coeuritrema + Chimaerohemecus trondheimensis van der Land, 1967, Hapalorhynchus clade. Morphologically, G. amoena ap- \"%Aporocotyle spinosican- pears as a schistosome (although having a single testis and alis Williams, 1958, Plethorchis acanthus Martin, 1975 lacking fused caeca), with markedly distinctive morpho- and Neoparacardicola nasonis Yamaguti, 1970), and the logical features that do not intuitively align it with Coeuri- |Q*\"Gri- trema nor Hapalorhynchus or other TBFs for that matter. phobilharzia amoena (Fig. 7). Within TBFs, and as already Griphobilharzia Platt et Blair, 1991 resembles schistoso- reported elsewhere (Snyder 2004, Orélis-Ribeiro et al. matids by dioecity and having a ventral sucker (Platt et al. 2014, Roberts et al. 2016), the marine (Carettacola Manter 1991, 2013). Further, by having a well-developed gynaeco- et Larson, 1950 clade) and freshwater TBFs clustered sep- %+}``}* arately and with high nodal support. Within the freshwater of Schistosomatinae Stiles et Hassall, 1898: Schistosoma TBFs, we recovered separate Hapalorhynchus and Spiror- Weinland, 1858, Ornithobilharzia Odhner, 1912, Austro- chis clades. The new species was sister to Hapalorhynchus bilharzia Johnston, 1917, Macrobilharzia Travassos, 1922, spp., and that clade was sister to G. amoena. The phyloge- Schistosomatium Tanabe, 1923, Heterobilharzia Price, netic distance (branch lengths) within the Hapalorhynchus 1929, Bivitellobilharzia Vogel et Minning, 1940 and Ori- clade supported our conclusions from morphology in that entobilharzia Dutt et Srivastava, 1955. Coeuritrema is distinctive from other accepted TBF gen- The stark discordance between comparative morphol- era. Additional sequences from Enterohaematotrema and ogy and gene sequence analysis reiterate the need for ad- Cardiotrema are required to test monophyly of Coeuri- ditional molecular gene sequence data from specimens trematinae (see above). G. amoena. Griphobilharzia amoena from all other TBFs by being dioecious and by maturing DISCUSSION in the freshwater crocodile, Crocodylus johnstoni + Our morphological descriptions and comparisons cou- Griphobilharzia |Q*Baracktrema pled with our molecular phylogenetic results indicated that (single caecum), Neospirorchis Price, 1934 (fused caeca) Coeuritrema shares a recent common ancestor with Hap- and Unicaecum Stunkard, 1925 (single caecum), by hav- alorhynchus (Fig. 7). ¬\* * ing two non-fused caeca. Griphobilharzia resembles Ba- ƾ racktrema, Neospirorchis, Unicaecum, Uterotrema Platt et = [ - Pichelin, 1994, and Vasotrema by having a single testis. ic, both H. gracilis (type species) and H. foliorchis infect K \" sensu lato, the common snapping turtle, Chelydra serpentina (Testu- for now, this clade as well as the marine TBF clade (Caret- dines: Chelydridae), whereas C. platti infects P. sinensis tacola, Hapalotrema Looss, 1899 and Learedius Price, (Trionychidae). Chelydra serpentina and P. sinensis are as- 1934), must continue to remain in systematic limbo, with- - out a familial assignment (Looss 1899, Price 1934, Manter logenetically closely related (Guillon et al. 2012, Crawford and Larson 1950, Orélis-Ribeiro et al. 2014, Roberts et al. et al. 2015). Moreover, none of these TBFs nor their turtle 2016). hosts have overlapping geographic distributions: H. gracilis and H. foliorchis range in North America only, where- Acknowledgements. We thank Dang Nguyen Anh Tuan and | ' %# | \ \ as C. platti Y%|^ R. Womble (National Oceanic and Atmospheric Administration, *\ >*?<\" knowledge of the life cycles of species of Hapalorhynchus, Vietnam, and Gabor Racz (HWML) and Estefania Rodriguez Coeuritrema and Griphobilharzia (AMNH) for loaning museum specimens. The present study is observed topology. Unfortunately, no life cycle is known a contribution of the Southeastern Cooperative Fish Parasite and for any species of the Hapalorhynchus clade, precluding Disease Project (Auburn University) and was supported in part a deeper discussion of this matter herein. Orélis-Ribeiro et by a grant from the York International Scholars Program (Auburn %}`^\"- University) awarded to JRR, ROR and SAB and by the National *\ Science Foundation Division of Environmental Biology via grant \" \ ^^^}}*^`^^`_%\+¬^`~}\ \"$*- to SAB. We thank and are indebted to Tom Platt for his dona- tion of his library and helminthological collection to SAB, which tory and sequence data sourced from the other 17 accept- made possible the revisionary systematics work presented herein ed species of Hapalorhynchus and additional species of and that which is forthcoming.

Folia Parasitologica 2016, 63: 031 Page 13 of 15 doi: 10.14411/fp.2016.031 Roberts et al.: Coeuritrema spp.

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Received 13 May 2016 Accepted 5 July 2016 Published online 6 September 2016

Cite this article as: KK* =KK*?Q|*¬+*Q'$}`^_§Q\"$- shell turtles: revision of Coeuritrema Mehra, 1933 (Digenea: Schistosomatoidea) and a new species infecting Chinese softshell turtles, Pelodiscus sinensis, (Trionychidae) from Vietnam. Folia Parasitol. 63: 031.

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