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Aporocotylids from batoid and elopomorph fishes from Moreton Bay, Queensland, Australia, including a new genus and species of blood fluke infecting the Giant shovelnose ray, Glaucostegus typus (Rhinopristiformes: Glaucostegidae)

Scott C. Cutmore, Thomas H. Cribb, Russell Q.-Y. Yong

PII: S1383-5769(18)30188-0 DOI: doi:10.1016/j.parint.2018.08.003 Reference: PARINT 1826 To appear in: Parasitology International Received date: 14 May 2018 Revised date: 8 August 2018 Accepted date: 13 August 2018

Please cite this article as: Scott C. Cutmore, Thomas H. Cribb, Russell Q.-Y. Yong , Aporocotylids from batoid and elopomorph fishes from Moreton Bay, Queensland, Australia, including a new genus and species of blood fluke infecting the Giant shovelnose ray, Glaucostegus typus (Rhinopristiformes: Glaucostegidae). Parint (2018), doi:10.1016/ j.parint.2018.08.003

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Aporocotylids from batoid and elopomorph fishes from Moreton Bay,

Queensland, Australia, including a new genus and species of blood fluke infecting the Giant shovelnose ray, Glaucostegus typus (Rhinopristiformes:

Glaucostegidae)

Scott C. Cutmore* [email protected], Thomas H. Cribb, & Russell Q-Y. Yong

The University of Queensland, School of Biological Sciences, St Lucia, Queensland, 4072,

Australia

*Corresponding author.

ABSTRACT

Fishes of the elasmobranch superorder Batoidea and the basal teleost superorder

Elopomorpha were assessed for blood flukes (Digenea: Aporocotylidae) during a parasitological survey conducted in Moreton Bay, Queensland, Australia. A new blood fluke genus and species, Ogawaia glaucostegi n. gen., n. sp., is described from the Giant shovelnose ray, Glaucostegus typus (Anonymous [Bennett]) (Rhinopristiformes:

Glaucostegidae). Ogawaia glaucostegi differs from species of all other aporocotylid genera in the combination ofACCEPTED the absence of anterior caeca MANUSCRIPT and oral sucker, having a pronounced distal oesophageal chamber, a strongly coiled testis and a common genital pore. The new species most closely resembles Myliobaticola richardheardi Bullard & Jensen, 2008, from which it differs in lacking an oral sucker and in possessing a straight (rather than coiled) oesophagus, longer caeca in proportion to the oesophageal and total body length, and a much longer testis relative to body length. Ogawaia glaucostegi is just the eighth aporocotylid described from

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chondrichthyans, of which four belong to monotypic genera. This is the first description of a blood fluke from the order Rhinopristiformes, and the first of a chondrichthyan-infecting aporocotylid from Australian waters. Elopicola bristowi Orélis-Ribeiro & Bullard, 2017 is reported from Australia for the first time, from the type-host, Elops hawaiensis Regan

(Elopiformes: Elopidae). This species is identified by morphological and molecular data and distinctions between our specimens and those of the original description are discussed.

Keywords: Ogawaia; Elopicola; Glaucostegus; Elops; Batoidea; Elopomorpha; Moreton

Bay; Blood fluke.

1. Introduction

Knowledge of the systematics and taxonomy of the fish blood flukes (Aporocotylidae

Odhner, 1912) from Australian waters has progressed rapidly in the last 15 years, largely due to two intense periods of focused study, the first by Nolan & Cribb [1-5] and the second by

Yong et al. [6-10]. These works have resulted in Australia being the most well-studied region globally for marine aporocotylids [11, 12], with 43 species (approximately 27% of the global fauna) now known from the region. However, there are major gaps in the knowledge; just three aporocotylids are known from basal teleosts in Australian waters (two from ostariophysan gonorynchiforms and one from elopomorph anguilliforms), and those infecting chondrichthyans are completely unknown in the region.

Of the 159 known aporocotylid species, just seven have been reported from ACCEPTED MANUSCRIPT chondrichthyans: two species from lamniforms [13, 14], two from carcharhiniforms [15, 16], two from myliobatiforms [17, 18] and one from a chimaeriform [19]. Recently, Cribb et al.

[20] incorporated novel sequence data for an unidentified aporocotylid from Glaucostegus typus (Anonymous [Bennett]) (Glaucostegidae) in a phylogenetic analysis of the

Aporocotylidae, which they identified as “cf. Myliobaticola sp.”. In this study we formally

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describe this form, which we conclude represents a genus and species new to science. We also record a species of the elopomorph-infecting genus Elopicola Bullard, 2014, from

Australian waters for the first time, in the Hawaiian ladyfish Elops hawaiensis Regan. Only one aporocotylid species was previously known from Australian elopomorph fishes;

Paracardicoloides yamagutii Martin, 1974, which infects three species of freshwater eels

(Anguillidae) [21-23].

2. Materials and Methods

2.1. Specimen collection

Batoid and elopomorph fishes were collected by seine, tunnel netting, spear and hand-nets in various locations throughout Moreton Bay, Queensland, Australia. The heart (including, in the case of batoid specimens, the conus arteriosus and ventral aorta) was removed, placed in saline solution (0.85% NaCl solution) and each section opened separately. Gill filaments were removed and examined for the presence of eggs following Yong et al. [8].

Aporocotylids were washed in vertebrate saline, fixed by pipetting into near-boiling saline, and preserved in 70% ethanol for parallel morphological and molecular characterisation.

Some individual worms were processed for both morphological and molecular analysis

(hologenophores sensu Pleijel et al. [24]).

2.2. Morphological analysis ACCEPTED MANUSCRIPT Specimens for morphological analysis were washed in fresh water, stained in Mayer’s haematoxylin, destained in a solution of 1.0% HCl and neutralised in 1.0% ammonium hydroxide solution. Specimens were then dehydrated through a graded ethanol series, cleared in methyl salicylate and mounted in Canada balsam. Measurements were made using an

Olympus SC50 digital camera mounted on an Olympus BX-53 compound microscope using

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cellSens Standard imaging software. Measurements are in micrometres (µm) and given as a range followed by the mean in parentheses. Where length is followed by breadth, the two measurements are separated by ‘×’. Drawings were made using an Olympus BX-53 compound microscope and drawing tube. Type- and voucher specimens are lodged in the

Queensland Museum (QM), Brisbane.

2.3. Molecular sequencing and phylogenetic analysis

As recommended by Blasco-Costa et al. [25], four genetic markers were sequenced; specimens for molecular analysis were processed according to protocols used by Cutmore et al. [26] for the ITS2 and 28S rDNA regions, Huston et al. [27] for the 18S rDNA region and

Wee et al. [28] for the cox1 mtDNA region. The complete ITS2 rDNA region was amplified and sequenced using the primers 3S [29] and ITS2.2 [30]; the partial D1-D3 28S rDNA region using LSU5 [31], 300F [32], ECD2 [33] and 1500R [34]; complete 18S rDNA using

18S-E [35], 300F [36], 1100F [35], Cestode-1 [35], 1270R [35] and WormB [35]; and partial cox1 mtDNA region using Dig_cox1Fa [28] and Dig_cox1R [28]. Geneious® version 10.2.3

[37] was used to assemble and edit contiguous sequences and the start and end of the ITS2 rDNA region were determined by annotation through the ITS2 Database [38, 39], using the

‘Metazoa’ model.

3. Results ACCEPTED MANUSCRIPT 3.1. General results

Aporocotylids were found in nine of 16 G. typus examined from Moreton Bay; no aporocotylids were found in any of the eight other batoid species assessed (Table 1). Within

G. typus, aporocotylids were typically found intertwined in the connective tissue of the valves of the conus arteriosus, and less commonly in the ventricle. These specimens represent a new

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genus and species, just the seventh blood fluke to be described from an elasmobranch host.

Aporocotylids were found in a single Elops hawaiensis examined from Moreton Bay; these were identified as Elopicola bristowi Orélis-Ribeiro & Bullard, 2017, which is here reported from Australia for the first time. No aporocotylids were found in four other elopomorph species assessed from Moreton Bay (Table 1).

3.2. Ogawaia n. gen.

Diagnosis: Body overall narrowly lanceolate, flat in forebody, becoming more cylindrical and narrow in midbody before broadening and flattening again at level of terminal genitalia, broadest at levels of oesophagus and uterine coils. Tegumental spines absent. Oral sucker absent. Mouth a simple pore, just ventrally subterminal. Oesophagus mostly straight, occasionally slightly sinuous. Oesophageal chamber prominent at distal extremity of oesophagus. Anterior caeca absent. Posterior caeca mostly straight, occasionally with some sinuosity at distal ends, sub-equal to unequal in length, with ends variably swollen. Testis occupying nearly half of total body length, strongly coiled, with smooth margins, entirely post-caecal. Vas deferens originates medially from posterior margin of testis, simple and uncoiled for much of length. Seminal vesicle an indistinctly-defined expansion of vas deferens, leads directly to common genital pore; cirrus-sac and cirrus absent. Common genital pore dorso-sinistral. Ovary broadly trigonal, with margins crenulated to distinctly lobed, medio-dextral, entirely post-testicular. Oviduct originates dextrally from posterior ACCEPTED MANUSCRIPT margin of ovary, passes sinistrally and posteriorly to meet oötype. Oötype well-defined, dextral. Vitelline follicles finely granular, largely confined by nerve cords, evenly distributed from dorsal nerve commissure to level of common genital pore, overlapping oesophagus, caeca, testis and ovary, without division into lateral fields. Vitelline duct runs dextrally parallel to and overlapping lateral nerve cord, straight for most of traceable length,

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convoluted at level of oviduct, dorsal to remainder of genitalia. Uterus strongly convoluted, passing sinistral to oötype anteriorly to parallel sinistral ovarian margin, then posteriorly to meet common genital pore. Distal uterine coils not distinctly muscularised or modified to form metraterm. Confluence of male and female genital systems at site of genital pore not expanded to form genital atrium or similar. Uterine coils often filled with sperm, with one coil consistently expanded to form uterine seminal receptacle. Uterine seminal receptacle oblong, varying in turgidity according to egg and sperm content. Eggs oblong, unspined and anoperculate, often numerous. Excretory system not characterised.

Etymology: The generic name honours Professor Kazuo Ogawa, director of the Meguro

Parasitological Museum, Tokyo, Japan, in recognition of his invaluable contribution to the study of blood flukes.

3.3. Ogawaia glaucostegi n. sp. Fig. 1, 2

Syn. “cf. Myliobaticola sp.” of Cribb et al. [20]

Description [Based on 16 specimens, including 2 hologenophores]: Body 4,418–5,675

(5,086) long, overall narrowly lanceolate, flat and 188–290 (227) broad in forebody to level of testis, narrowing medially to 150–240 (185) and becoming more cylindrical, before flattening again and broadening to 205–308 (241) at level of terminal genitalia, broadest at levels of oesophagus and uterine coils, 16.5–27.9 (21.0) times longer than wide. Dorsal nerve ACCEPTED MANUSCRIPT commissure 120–166 (137) from anterior extremity, 59–106 (81) across. Nerve cords traceable to level of female genitalia, run length of body at 13–23 (18) from body margins.

Tegumental spines absent. Oral sucker absent. Mouth a simple pore, just ventrally subterminal. Oesophagus mostly straight, slightly sinuous distally in some specimens, 936–

1,328 (1,111), occupying 20.1–23.6% (22.1%) of total body length, with prominent

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oesophageal chamber present at distal extremity. Distal oesophageal chamber globose, 38–61

× 34–51 (49 × 40). Intestinal bifurcation medial in anterior half of body. Anterior caeca absent. Posterior caeca mostly straight, occasionally with some sinuosity at distal ends, sub- equal to unequal in length, with ends variably swollen, 47.5–69.4% (55.7%) length of oesophagus, occupying 11.0–14.1% (12.9%) of total body length; left caecum 449–777

(608); right caecum 526–803 (638).

Testis a strongly coiled tube, with smooth margins, entirely post-caecal, occupying

2,042–2,840 (2,505) or 44.4–53.1% (49.2%) of total body length, 121–237 (161) wide, with tubular diameter of 52–79 (62). Post-testicular space 686–827 (785), or 13.6–16.9% (15.4%) of total body length. Vas deferens originates medially from posterior margin of testis, simple and uncoiled for much of length. Seminal vesicle an indistinctly-defined expansion of vas deferens, leads directly to common genital pore, 120–179 × 27–53 (157 × 38). Cirrus-sac and cirrus absent. Common genital pore dorsosinistral, 20–55 (33) from lateral margin, 468–575

(530) from posterior extremity. Ovary broadly trigonal, with margins crenulated to distinctly lobed, medio-dextral, entirely post-testicular, 151–237 × 105–167 (203 × 141). Oviduct originates dextrally from posterior margin of ovary, passes sinistrally and posteriorly to meet oötype. Oötype well-defined, 114–169 (132) from sinistral body margin, 59–118 (81) from dextral body margin, 219–335 (276) from posterior extremity. Vitelline follicles finely granular, largely confined by nerve cords, evenly distributed from dorsal nerve commissure to level of common genital pore, overlapping oesophagus, caeca, testis and ovary, without ACCEPTED MANUSCRIPT division into lateral fields. Vitelline duct runs dextrally parallel to and overlapping lateral nerve cord, straight for most of traceable length, convoluted at level of oviduct, dorsal to remainder of genitalia. Uterine coils strongly convoluted; proximal uterine coils passing sinistrally and anteriorly from oötype to parallel sinistral ovarian margin. Distal uterine coils not distinctly muscularised or modified to form metraterm; descend parallel to sinistral

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margin to meet common genital pore. Confluence of male and female genital systems at site of genital pore not expanded to form genital atrium or similar. Uterine coils often filled with sperm, with one coil consistently expanded to form uterine seminal receptacle. Uterine seminal receptacle 142–275 × 60–94 (203 × 75). Eggs in utero oblong, 12–19 × 9–12 (15 ×

11).

Excretory system not visible in any specimens.

3.4. Taxonomic summary

Type-host: Glaucostegus typus (Anonymous [Bennett]), Giant shovelnose ray

(Rhinopristiformes: Glaucostegidae).

Type-locality: Off Dunwich, North Stradbroke Island, eastern Moreton Bay, Queensland,

Australia (27°29'S, 153°23'E).

Other localities: Off Peel Island, eastern Moreton Bay (27°29'S, 153°20'E); off Wellington

Point, western Moreton Bay (27°28'S, 153°15'E).

Site in host: Primarily intertwined in valves of conus arteriosus, less commonly in ventricle.

Trapped eggs seen in gill lamellar tissue.

Prevalence: nine of 16 fish infected: 8/8 from eastern Moreton Bay, and 1/8 from western

Moreton Bay.

Type-material: Holotype (QM G236234) and 15 paratypes (QM G236235–49).

Representative DNA sequences: ITS2 rDNA, three identical replicates (MF503312, reported ACCEPTED MANUSCRIPT by Cribb et al. [20]); partial 28S rDNA, two identical replicates (MF503308, reported by

Cribb et al. [20]); complete 18S rDNA, 1 sequence (submitted to GenBank MH720954); partial cox1 mtDNA, three identical replicates (one submitted to GenBank MH720956).

Etymology: This species is named for the genus of elasmobranchs it infects, Glaucostegus.

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3.5. Remarks

Ogawaia glaucostegi n. gen., n. sp. is sufficiently different from all other aporocotylids to warrant the proposal of a new genus. The new species is readily differentiated from all other aporocotylids through the combination of lacking an oral sucker and anterior caeca, and possessing a pronounced distal oesophageal chamber, strongly coiled testis and common genital pore. Lacking anterior caeca is a characteristic of the majority of elasmobranch- and basal teleost-infecting aporocotylid species; the trait is seen in species of

Acipensericola Bullard, Snyder, Jensen & Overstreet, 2008, Chimaerohemecus van der Land,

1967, Elopicola Bullard, 2014, Hyperandotrema Maillard & Ktari, 1978, Myliobaticola

Bullard & Jensen, 2008, Orchispirium Madhavi & Hanumantha Rao, 1970 and

Paracardicoloides Martin, 1974. Of these, Ogawaia glaucostegi can be immediately differentiated from species of Acipensericola, Elopicola and Paracardicoloides by its lack of an oral sucker, and from species of Hyperandotrema by the lack of large, recurved tegumental spines covering most of the body. The sole species of Chimaerohemecus, C. trondheimensis van der Land, 1967, differs from Ogawaia glaucostegi in having caeca which extend posteriad as far as the terminal genitalia, a massive, entirely intercaecal testis and separate male and female genital pores.

Ogawaia glaucostegi is most similar to the two other known species of batoid- infecting aporocotylids, the sole species of Orchispirium, Or. heterovitellatum Madhavi &

Hanumantha Rao, 1970, and the sole species of Myliobaticola, M. richardheardi Bullard & ACCEPTED MANUSCRIPT Jensen, 2008. Like Ogawaia glaucostegi, Or. heterovitellatum has a coiled testis, pronounced and globose posterior oesophageal chamber, common genital pore and no tegumental spines.

However, it possesses an oral sucker, and the testis is intercaecal rather than post-caecal. In addition, the testis, rather than strongly coiling as in O. glaucostegi, is instead folded laterally into a zigzagging series of loops. Ogawaia glaucostegi most closely resembles M.

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richardheardi. Like M. richardheardi, it lacks tegumental spines, and possesses a prominent distal oesophageal chamber, strongly coiled testis and a common genital pore. Ogawaia glaucostegi, however, differs from M. richardheardi in lacking an oral sucker. It also differs from M. richardheardi by its much larger body (4,418–5,675 µm vs 365–650 µm body length, 7–8 times larger than M. richardheardi), straight (not distinctly sinuous) oesophagus, caecal arms proportionally longer relative to oesophageal length (47.5–69.4% vs ~20%), a much longer testis relative to body length (> 49% total body length, vs < 20% in M. richardheardi) and an ovary that is entirely posterior to the testis (not overlapping anteriorly with the testis).

3.6. Elopicola bristowi Orélis-Ribeiro & Bullard, 2017 Fig. 3, 4

Type-host: Elops hawaiensis Regan, Hawaiian ladyfish (Elopiformes: Elopidae).

Type-locality: Eastern Sea, off Nha Trang, Vietnam.

Description [based on 2 specimens]: Body broadly fusiform, broadest at level of testis,

1,123–1,318 × 299–399, 3.8–3.9 times longer than wide. Dorsal nerve commissure 92–115 from anterior extremity, 83–88 across. Nerve cords traceable along entire length of body at

45–64 from body margins. Tegumental spines 7–9 long throughout, slightly curved, arranged in transverse marginal rows, with each row bearing 4–5 spines; rows commencing just posterior to oral sucker, not confluent at posterior extremity. Mouth a broad funnel medial in ACCEPTED MANUSCRIPT prominent oral sucker. Oral sucker bowl-shaped, distinctly pedunculate, 32–33 × 47–60.

Oesophagus gently sinuous, divided into three distinct sections; proximal section commences immediately posterior to mouth as a narrowly fusiform chamber showing weak localised muscularisation, 61–77 × 20; midsection comprises majority of length of oesophagus, highly glandularised both internally and externally, 397–453 long with maximal breadth 38–77;

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distal section expanded to form elliptical oesophageal chamber, showing external glandularisation different from oesophageal midsection and with same gastrodermal morphology as caeca, 69–76 × 28–35. Total oesophageal straight length 500–576 or 43.7–

44.5% of total body length; total curved length 521–625. Intestinal bifurcation medial in anterior half of body. Caeca form X-shape. Anterior caeca mostly straight with some diverticulation, sub-equal to unequal in length; left anterior caecum 46–82; right anterior caecum 85–87. Posterior caeca mostly straight with some diverticulation, 4.4–7.3 times longer than anterior caeca; left posterior caecum 319–358; right posterior caecum 338–351.

Testis single, elliptical, with smooth margins, entirely intercaecal, 221–223 × 123–

155, occupying 16.8–19.9% of total body length. Post-testicular space 287–353, or 25.6–

26.8% of total body length. Vas deferens originates medially from near anterior margin of testis, expands greatly to accommodate secondary seminal vesicle. Secondary seminal vesicle dorsal to testis, 84–91 × 48–51, distant from cirrus-sac. Cirrus-sac 91–104 × 28–56, elongate, accommodates primary seminal vesicle. Primary seminal vesicle varying in turgidity according to seminal volume; extrudes via common genital pore. Common genital pore dorsosinistral, 73–82 from sinistral body margin, 145–173 from dextral body margin, 259–

301 from posterior extremity. Ovary trigonal, with margins crenulated to distinctly lobed, medial, entirely post-testicular, 88–119 × 81–86. Oviduct, oötype and uterine coils occupy space between testis and ovary. Oviduct originates medially from ovary, passes anteriorly and dextrally, then sinistrally to meet oötype. Oötype medio-dextral, immediately anterior to ACCEPTED MANUSCRIPT ovary, 123–141 from sinistral body margin, 86–108 from dextral body margin, 258–326 from posterior extremity. Vitelline follicles in irregular clumps, not confined by nerve cords, confluent in midbody, extend from mid-length of oesophagus to level of common genital pore. Vitelline duct not traceable for much of length, meets oötype having passed parallel to

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dextral margin of testis. Uterine coils occupying much of space between testis and ovary, passing mostly sinistrally to meet common genital pore.

Excretory vesicle V-shaped, prominent in all specimens, 116–132 × 85–118; highly turgid and contiguous with lateral margins of ovary. Excretory pore terminal.

3.7. Taxonomic summary

Host: Elops hawaiensis Regan, Hawaiian ladyfish (Elopiformes: Elopidae).

New locality: Wynnum Banks, Moreton Bay, Queensland, Australia (27°24'S, 153°11'E).

Site in host: In vessels of gill arches.

Prevalence: One of one fish infected with five worms.

Voucher material: Three whole-mount voucher specimens (QM G237623–5).

Representative DNA sequences: ITS2 rDNA, two identical replicates (one submitted to

GenBank, MH720953); partial 28S rDNA, one sequence (submitted to GenBank,

MH720952); partial cox1 mtDNA, two identical replicates (one submitted to GenBank,

MH720955).

3.8. Remarks

The new specimens identified as E. bristowi closely match those in the original description of this species by Orélis-Ribeiro et al. [40]. Generally, only minor morphological differences were observed between our specimens and those in the original description. The ACCEPTED MANUSCRIPT posterior oesophageal swelling noted by Orélis-Ribeiro et al. [40] as present in all species of

Elopicola (referred to as a “distal oesophageal chamber” by us here) was indistinct in our specimens, its presence only indicated by a slight proximal constriction and gastrodermal morphology and glandularisation different from the rest of the oesophagus. The gastrodermal morphology of the posterior oesophageal chamber was identical to that of the caeca. This

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structure is therefore best interpreted as a pseudoesophagus, along the lines of those seen in some other digeneans, e.g. some lepocreadiids [41].

Although a pharynx was described for all three species of Elopicola [40, 42], no such structure was observed here. Pearson [43] recognised the digenean pharynx to be “a discrete muscular organ composed largely of radial fibres and set off from the adjacent musculature and parenchyma by a distinct and continuous capsule”. In so doing he interpreted the

“pharynx” variously reported for certain gyliauchenids, microscaphidiids and paramphistomids as independent specialisations of the gut. A slight broadening of the proximal oesophagus was present in our specimens, followed by an immediate slight constriction; no special muscularisation, indicating a discrete organ, was observed. It is our view that this structure represents no more than a broadening of the oesophageal lumen which may function in a manner analogous to a true pharynx. This suggests that Elopicola bristowi

(and presumably all the species in the genus) lack the “distinct and continuous capsule” that identifies the true digenean pharynx. In lacking a pharynx, therefore, the species of Elopicola are consistent with the Schistosomatoidea as a whole, which are diagnosed as lacking this structure [44].

4. Discussion

4.1. Aporocotylids of chondrichthyans

Including the current study, there have been just 14 reports of described blood flukes ACCEPTED MANUSCRIPT in chondrichthyans in the 80 years since the first report. There have been four reports of the single species of Chimaerohemecus [19, 45-47], four reports of the two species of

Hyperandotrema [13, 14, 48, 49], three reports of the two species of Selachohemecus Short,

1954 [15, 16] and one report for each of the single species of Orchispirium [18],

Myliobaticola [17] and Ogawaia. Despite the limited number of records of chondrichthyan-

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infecting aporocotylids, two aspects of their biology are apparent when the reports are considered together: i) known chondrichthyan-infecting aporocotylid species are not closely related; and ii) known hosts of aporocotylids are distantly related.

The eight known species of chondrichthyan-infecting aporocotylids belong to six genera; only Hyperandotrema and Selachohemecus have congeners, each having two species.

These six genera differ greatly in morphology, with key differences in the testicular morphology, the possession or absence of an oral sucker, the possession and structure of tegumental spines, and the structure and extent of the caeca. The few species characterised by molecular data also differ greatly genetically; in the phylogram of Cribb et al. [20], the branch lengths between the chondrichthyan-infecting blood flukes (two adults and the bivalve cercarial infection) are greater than the branch lengths within any actinopterygian-infecting genera (i.e. between species of Ankistromeces Nolan & Cribb, 2004 [9], Aporocotyle Odhner,

1900 [50-52], Cardicola Short, 1953 [7], Elopicola [40], Paradeontacylix McIntosh, 1934

[53, 54], Phthinomita Nolan & Cribb, 2006 [6] and Psettarium Goto & Ozaki, 1930 [6, 55]) and between closely related but distinct actinopterygian-infecting genera (e.g. between

Ankistromeces and Phthinomita [6], Cardicola and Paradeontacylix [6] and Psettarium and

Skoulekia Alama-Bermejo, Montero, Raga & Holzer, 2011 [56]). Thus, although there are molecular data for just two of the six genera, there is little doubt that they are valid and phylogenetically distinct.

This disparity between species of chondrichthyan-infecting aporocotylids is further ACCEPTED MANUSCRIPT supported by the fact that their hosts are so phylogenetically disjunct. Reports of aporocotylids span the entire chondrichthyan phylogeny (Fig. 5), with reports of blood flukes in all three major lineages (chimaeras, sharks and rays). However, aporocotylids have been reported from only two of the four ray orders, and just two of the nine shark orders. This distribution is even more scattered at the family level. Despite the reports from sharks coming

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only from species in the sister orders Carcharhiniformes and Lamniformes, blood flukes have been reported from just three of the 16 families in these two orders. We predict that our knowledge of the chondrichthyan-infecting blood flukes is incomplete, and that the perceived host distribution is an artefact of sampling bias. Due to their unusual infection locations, aporocotylids are seldom searched for in general examinations of fishes and rarely found by accident. The species described in this study was sought specifically and overwhelmingly found intertwined in the valves of the conus arteriosus; such site-specificity means that forms such as Ogawaia glaucostegi will not be discovered without focused examination of all sections of the circulatory system. We doubt that such detailed examinations have been made for many of the chondrichthyan orders yet to be reported as hosts. In this context, we note the recent report of an undescribed aporocotylid from the smalltooth sawfish, Pristis pectinata

Latham, off Florida [57]. Just a single specimen was collected from P. pectinata, which appears to possess many of the key features of Ogawaia and, strikingly, also infects a rhinopristiform.

Our new species is clearly related to other chondrichthyan-infecting aporocotylids and, particularly, the two other batoid-infecting species, M. richardheardi and Or. heterovitellatum. Fundamental similarities exist between the three taxa which indicate a close relationship; all three species share an absence of tegumental spines (the only chondrichthyan-infecting aporocotylids to do so), a lack of anterior caeca, possession of a single coiled testis and common genital pores. Molecular data do not exist for either M. ACCEPTED MANUSCRIPT richardheardi or Or. heterovitellatum; obtaining these data will be crucial for understanding the relationships of these species with each other and with O. glaucostegi. In the absence of such data, we cannot make firm conclusions regarding such relationships. Indeed, the similarities between O. glaucostegi and M. richardheardi, in particular, suggest a closeness in relationship that could be interpreted as congeneric. The disparities in host distribution,

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however, weighed against the relatively conserved nature of the body plan of batoid-infecting aporocotylids, mean we are comfortable in asserting that our new species warrants generic distinction.

4.2. Phylogenetic position of chondrichthyan-infecting aporocotylids

The phylogenetic structure of the Aporocotylidae has been a subject of debate in recent years, with several authors proposing differing constructs. Until recently, the molecular position of chondrichthyan-infecting aporocotylids has been based solely on sequence data of C. trondheimensis. In a phylogenetic analysis of the Trematoda, based on

28S rDNA, Olson et al. [51] found this species to be basal to all other blood flukes; however, this dataset included just six aporocotylids. Bullard et al. [58] also found C. trondheimensis to be basal to marine and freshwater aporocotylids, based on 18S rDNA; however, this dataset was also limited in breadth of taxa and also included just six aporocotylids. Orélis-Ribeiro et al. [59] analysed a larger 28S dataset and found C. trondheimensis to be basal to just the marine aporocotylids, which together were sister to the clade of freshwater aporocotylids, spirorchiids and schistostomatids; this analysis included eight marine teleost-infecting genera, but no identified aporocotylids of freshwater teleosts. Orélis-Ribeiro et al. [59] also found the topology of the group changed dramatically based on the inclusion or exclusion of sequence data for clinostomids and cercariae from freshwater gastropods.

More recently, Orélis-Ribeiro et al. [40] used combined 18S+28S data to show that C. ACCEPTED MANUSCRIPT trondheimensis forms a clade with a basal teleost-infecting species (Acipensericola petersoni

Bullard, Snyder, Jensen & Overstreet, 2008), sister to all other aporocotylids included in the analysis. These two species, however, did not form a clade with the other basal teleost- infecting species included in the phylogeny (species of Elopicola). In the most recent phylogenetic analysis for the group, Cribb et al. [20] generated the first additional genetic

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data for chondrichthyan-infecting aporocotylids beyond C. trondheimensis, incorporating novel sequence data of Ogawaia glaucostegi and of an aporocotylid infection from a bivalve.

Cribb et al. [20] suggested that chondrichthyan-infecting aporocotylids represent an early, divergent lineage, sister to those infecting freshwater fishes and distinct from those infecting marine actinopterygians. Cribb et al. [20] also incorporated intermediate host usage data in their evaluation of the phylogeny, inferring that the aporocotylids infecting chondrichthyans, marine actinopterygians and freshwater actinopterygians represent independent lineages following an ancient diversification with their hosts.

Evidently there is still confusion regarding the phylogeny of the fish blood flukes, and it is likely higher classification changes will be required. This confusion is exemplified by the differences in recent analyses and is likely a result of limited taxa being incorporated in analyses and relationships being inferred on major clades with low support in some analyses.

Some undescribed intermediate freshwater taxa incorporated in recent phylogenetic analyses

(28S GenBank accession AY858880.1 and AY858881.1, relating to infections from planorbid snails in Kenya and Uganda, respectively) introduce extensive alignment difficulties when included in analyses and weaken support for relationships in phylograms generated; their relationship to the rest of the aporocotylids (and even their status as aporocotylids) remains uncertain. Furthermore, differences in gene choice between studies has led to inconsistent and incomplete datasets for complete family and superfamily level analyses. Despite marine actinopterygian-infecting aporocotylids being the best represented ACCEPTED MANUSCRIPT for sequence coverage, 28S data still only exist for only 12 of the 25 known genera.

Ultimately, further genetic characterisation of chondrichthyan-infecting, freshwater actinopterygian-infecting and marine actinopterygian-infecting aporocotylids is essential before any major classification changes can be proposed.

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Funding

SCC and THC acknowledge the Australian Biological Resources Study (ABRS) for their ongoing support. This study was funded by the ABRS National Taxonomy Research Grant

RF215-40. RQY is supported by a Holsworth Wildlife Research Endowment, as well as a

Moreton Bay Research Station (MBRS) research scholarship. RQY also acknowledges the support of the Australian government through an Australian Government Research Training

Program (RTP) Scholarship.

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethical standards

All applicable institutional, national and international guidelines for the care and use of animals were followed.

Acknowledgements

We thank John Page and Dave Thompson for their assistance in the collection of rays in

Moreton Bay. We thank Storm Martin for assistance with both the collection and dissection of rays and Dan Huston for his assistance in the production of SEM images. We acknowledge ACCEPTED MANUSCRIPT the staff of Moreton Bay Research Station for their support in the field and the Australian

Microscopy and Microanalysis Research Facility (AMMRF) at The Centre for Microscopy and Microanalysis (CMM), The University of Queensland, for the facilities and scientific and technical assistance which enabled SEM photography.

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Fig. 1 Ogawaia glaucostegi n. gen., n. sp. A: Whole worm, holotype, dorsal view. B:

Terminal genitalia, paratype, dorsal view. Abbreviations: GP- genital pore; Oo- oötype; Ov- ovary; SD- seminal duct; USR- uterine seminal receptacle; Ut- uterus; VD- vitelline duct.

Scale-bars: A, 500 µm; B, 250 µm. ACCEPTED MANUSCRIPT Fig. 2 Scanning electron micrographs of Ogawaia glaucostegi n. gen., n. sp. A. Anterior extremity, showing mouth. B: Tegument in middle third of body. C: Posterior extremity. D.

Tegument of posterior extremity, showing absence of spines. Scale-bars: A, 5 µm; B, D, 20

µm; C, 50 µm.

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Fig. 3 Elopicola bristowi ex Elops hawaiensis from Moreton Bay, Queensland, Australia,

dorsal view. Scale- Host No. examined bar: 300 µm. (infected) BATOIDEA Fig. 4

Photomicrographs

of Elopicola

bristowi showing

details of the

oesophagus. A:

Proximal

oesophageal region.

B: Medial

oesophageal region.

C: Distal

oesophageal region

including pseudoesophagus. Scale-bars: A-C, 50 µm.

Fig. 5 Distribution of aporocotylids relative to chondrichthyan orders. Orders infected by blood flukes shown in bold, the number of stars represent the number of reports for each order. Phylogram adapted from phylogenies of Naylor et al. [60] and the Chondrichthyan ACCEPTED MANUSCRIPT Tree of Life (https://sharksrays.org/). Abbreviations: C- Chimaeras; H- Holocephalans.

Table 1 Batoid and elopomorph species assessed for blood flukes from Moreton Bay in this study. Those infected by aporocotylids shown in bold

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MYLIOBATIFORMES Dasyatidae Hemitrygon fluviorum (Ogilby) 3 (0) Himantura uarnak (Gmelin) 1 (0) Maculabatis cf. astra 1 (0) Maculabatis toshi (Whitley) 4 (0) Neotrygon trigonoides (Castelnau) 4 (0) Pastinachus ater (Macleay) 1 (0)

Myliobatidae Aetobatus ocellatus (Kuhl) 6 (0)

RHINOPRISTIFORMES Glaucostegidae Glaucostegus typus (Anonymous [Bennett]) 16 (9)

Rhinobatidae Aptychotrema rostrata (Shaw) 6 (0)

ELOPOMORPHA ANGUILLIFORMES Muraenesocidae Muraenesox bagio (Hamilton) 2 (0)

Muraenidae Gymnothorax eurostus (Abbott) 3 (0) Gymnothorax pseudothyrsoideus (Bleeker) 22 (0)

ELOPIFORMES Elopidae Elops hawaiensis Regan 1 (1)

Megalopidae Megalops cyprinoides (Broussonet) 1 (0)

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Figure 1 Figure 2 Figure 3 Figure 4 Figure 5