Introduction
Members of Stomachicola Yamaguti, Reference Yamaguti1934 represent a group of digeneans included in the family Hemiuridae Looss, 1899 that are commonly found in the stomach of marine teleosts. Yamaguti (Reference Yamaguti1934) erected the genus Stomachicola and recorded Stomachicola muraenesocis Yamaguti, Reference Yamaguti1934 as the type species from the stomach of the daggertooth pike conger Muraenesox cinereus (Forsskål) in the Inland Sea of Japan (South China Sea). Stomachicola was distinguished from Dinurus Looss, 1907 by having an unlobed seminal vesicle and a longer ecsoma (Yamaguti, Reference Yamaguti1934). Tseng (Reference Tseng1935) reported representatives of Stom. muraenesocis collected from M. cinereus as Lecithocladium longicaudum Tseng, Reference Tseng1935. Some years later, Srivastava (Reference Srivastava1939) described Stom. secundus Srivastava, Reference Srivastava1939 from the stomach of Hyporhamphus limbatus (Valenciennes), but this species was later transferred by Yamaguti (Reference Yamaguti1958) to Allostomachicola Yamaguti, Reference Yamaguti1958. Bhalerao (Reference Bhalerao1943) described specimens of Stom. muraenesocis from the Indian region and reported some morphological variations in the type species (number of vitelline tubes and extension of the uterus into the tail). Chauhan (Reference Chauhan1945) and Chauhan (Reference Chauhan1954) transferred Lec. longicaudum to Stomachicola and redescribed Stom. muraenesocis and Stom. secundus from M. cinereus and Congresox talabonoides (Bleeker) in India. Linton (Reference Linton1905) reported ovigerous and immature forms of Distomum tornatum Rudolphi, 1819 from several fish species inhabiting Beaufort, North Carolina, USA. Manter (Reference Manter1931) reevaluated Linton's materials and reidentified them as Dinurus magnus Manter, Reference Manter1931. Later, Manter (Reference Manter1947) considered Stomachicola as a valid genus and transferred Din. magnus and Din. rubeus Linton, Reference Linton1910 to Stomachicola (with the adjectival feminine names Stom. magna and Stom. rubea) based on the shape of the seminal vesicle. Skrjabin and Guschanskaja (Reference Skrjabin, Guschanskaja and Skrjabin1954) established Pseudostomachicola Skrjabin and Guschanskaja, Reference Skrjabin, Guschanskaja and Skrjabin1954 and transferred Stom. magna, Stom. rubea and Stom. secunda to their newly erected genus based on the position of the genital pore and the distribution of vitellaria. Yamaguti (Reference Yamaguti1958) erected the genus Allostomachicola, synonymized Pseudostomachicola with Stomachicola and placed Stomachicola and Allostomachicola in the subfamily Stomachicolinae (Yamaguti, Reference Yamaguti1958). He considered Pseud. magna and Pseud. rubea of Skrjabin and Guschanskaja (Reference Skrjabin, Guschanskaja and Skrjabin1954) as Stom. magnus and Stom. rubeus, and Stom. secunda of Srivastava (Reference Srivastava1939) as Allo. secundus. Sinclair et al. (Reference Sinclair, Smith and Sullivan1972) found no difference between the eggs of Stom. magna and Stom. rubea and considered Stom. magna as junior synonym of Stom. rubea.
Several species were later recorded in the literature as dissimilar types from Stom. muraenesocis. These include Acerointestinecola karachiensis Jahan, Reference Jahan1970, Cameronia octovitellarii Bilqees, Reference Bilqees1971, Cam. pakistani Bilqees, Reference Bilqees1971, Cestodera gastrocecus Bilqees, Reference Bilqees1971, Ces. unicecus Bilqees, Reference Bilqees1971, Segmentatum karachiense Bilqees, Reference Bilqees1971, Seg. qadrii Bilqees, Reference Bilqees1971, Seg. cinereusis Bilqees, Reference Bilqees1971, Seg. magnaesophagustum Bilqees, Reference Bilqees1971, Indostomachicola kinnei Gupta and Sharma, Reference Gupta and Sharma1973, Stom. mastacembeli Verma, Reference Verma1973, Stom. lepturusi Gupta and Gupta, Reference Gupta and Gupta1974, Stom. pelamysi Gupta and Gupta, Reference Gupta and Gupta1974, Stom. polynemi Gupta and Gupta, Reference Gupta and Gupta1974, Stom. bayagbonai Siddiqi and Hafeezullah, Reference Siddiqi and Hafeezullah1975, Linguastomachicola serpentina Srivastava and Sahai, Reference Srivastava and Sahai1978, Stom. singhi Gupta and Ahmad, Reference Gupta and Ahmad1978, Stom. rauschi Gupta and Ahmad, Reference Gupta and Ahmad1978, Stom. chauhani Gupta and Singh, Reference Gupta and Singh1981, Stom. chauhani Pandey and Tewari, Reference Pandey and Tewari1984 and Stom. guptai Gupta and Gupta, Reference Gupta and Gupta1991. Acerointestinecola Jahan, Reference Jahan1970, Cameronia Bilqees, Reference Bilqees1971, Cestodera Bilqees, Reference Bilqees1971, Segmentatum Bilqees, Reference Bilqees1971 and Indostomachicola Gupta and Sharma, Reference Gupta and Sharma1973 were recognized congeneric with Stomachicola (Gibson and Bray, Reference Gibson and Bray1979; Hafeezullah, Reference Hafeezullah1980). The 8 species described by Bilqees (Reference Bilqees1971) were transferred to the genus Stomachicola by Kazmi and Naushaba (Reference Kazmi and Naushaba2013); however, the authors were not aware that these species had been previously considered as junior synonyms of Stom. muraenesocis by Hafeezullah (Reference Hafeezullah1980). Stomachicola mastacembeli, Stom. polynemi, Stom. singhi, Stom. bayagbonai, Stom. pelamysi, Lin. serpentina, Stom. chauhani and Stom. guptai were also synonymized with Stom. muraenesocis (Hafeezullah, Reference Hafeezullah1980, Reference Hafeezullah1985; Gupta and Gupta, Reference Gupta and Gupta1991; Tanzola and Seguel, Reference Tanzola and Seguel2012; Madhavi and Bray, Reference Madhavi and Bray2018). Gupta and Singh (Reference Gupta and Singh1981) transferred Stom. lepturusi and Stom. rauschi to Allostomachicola (Gupta and Singh, Reference Gupta and Singh1981; Gupta and Gupta, Reference Gupta and Gupta1991; Tanzola and Seguel, Reference Tanzola and Seguel2012). Afterwards, 5 new species were described including Stom. hainanensis Shen, Reference Shen1990, Stom. sexaginta Li and Sun, Reference Li and Sun1994, Stom. pritchardae Pande et al., Reference Pande, Neetu and Neela2000, Stom. lycengraulidis Tanzola and Seguel, Reference Tanzola and Seguel2012 and Stom. bengalensis Mishra et al., Reference Mishra, Chandra, Johri, Bajpai and Saxena2014.
The handling and processing of newly collected specimens of Stomachicola, in particular large worms, have been reported to be problematic primarily due to good development of musculature and contraction of the whole body of the parasite immediately after fixation (Sinclair et al., Reference Sinclair, Smith and Sullivan1972; Hafeezullah, Reference Hafeezullah1985). Moreover, several of the previous descriptions of species of Stomachicola were based on morphometric and morphological analyses performed by light microscopy using an inadequate number of specimens (Hafeezullah, Reference Hafeezullah1985; Gupta and Gupta, Reference Gupta and Gupta1991) or inadequate processing methods (e.g. different fixation temperatures, see Sinclair et al., Reference Sinclair, Smith and Sullivan1972), which could influence some characteristics of taxonomic value. In addition, a high intraspecific (individual) variability in most morphological and metrical characters has been reported for Stom. muraenesocis (Bhalerao, Reference Bhalerao1943; Gupta and Gupta, Reference Gupta and Gupta1991). Consequently, the validity of several species of Stomachicola has been questioned by several authors who consider them as synonyms of Stom. muraenesocis (Bhalerao, Reference Bhalerao1943; Hafeezullah, Reference Hafeezullah1985; Gupta and Gupta, Reference Gupta and Gupta1991; Madhavi and Bray, Reference Madhavi and Bray2018).
Species of Stomachicola are characterized by possessing a long ecsoma, long filiform vitelline tubules and a sinuous pars prostatica (Madhavi and Bray, Reference Madhavi and Bray2018). Stomachicola and Allostomachicola are distinguished by the position of the seminal vesicle (restricted to hindbody vs forebody) (Gibson and Bray, Reference Gibson and Bray1979). Species of Stomachicola are distributed worldwide and known to infect several fish species (particularly marine eels) from different families including Acanthuridae (Gupta and Ahmad, Reference Gupta and Ahmad1978; Pande et al., Reference Pande, Neetu and Neela2000), Anguillidae (Sinclair et al., Reference Sinclair, Smith and Sullivan1972, Stunkard, Reference Stunkard1980), Ariidae (Bilqees, Reference Bilqees1971), Carangidae (Pandey and Tewari, Reference Pandey and Tewari1984), Chirocentridae (Shen, Reference Shen1990), Congridae (Gupta and Sharma, Reference Gupta and Sharma1973; Gupta and Singh, Reference Gupta and Singh1981; Gupta and Gupta, Reference Gupta and Gupta1991), Cynoglossidae (Corkum, Reference Corkum1966; Stunkard, Reference Stunkard1973), Engraulidae (Tanzola and Seguel, Reference Tanzola and Seguel2012), Mastacembelidae (Verma, Reference Verma1973), Megalopidae (Sinclair et al., Reference Sinclair, Smith and Sullivan1972), Muraenesocidae (Yamaguti, Reference Yamaguti1934; Tseng, Reference Tseng1935; Bhalerao, Reference Bhalerao1943; Chauhan, Reference Chauhan1945, Reference Chauhan1954; Bilqees, Reference Bilqees1971; Siddiqi and Hafeezullah, Reference Siddiqi and Hafeezullah1975; Srivastava and Sahai, Reference Srivastava and Sahai1978; Hafeezullah, Reference Hafeezullah1980; Tang, Reference Tang1981; Hafeezullah, Reference Hafeezullah1985; Shen, Reference Shen1990; Shen and Qiu, Reference Shen and Qiu1995; Hafeezullah and Dutta, Reference Hafeezullah and Dutta1998; Shaukat, Reference Shaukat2008; Văn Hà et al., Reference Văn Hà, Ngọ, Bính, Hùng and Văn Hiền2012), Muraenidae (Linton, Reference Linton1910; Nahhas and Cable, Reference Nahhas and Cable1964), Paralichthyidae (Corkum, Reference Corkum1966; Sinclair et al., Reference Sinclair, Smith and Sullivan1972), Sciaenidae (Manter, Reference Manter1931; Sinclair et al., Reference Sinclair, Smith and Sullivan1972; Overstreet, Reference Overstreet1983a, Reference Overstreet1983b), Scombridae (Jahan, Reference Jahan1970; Gupta and Gupta, Reference Gupta and Gupta1974), Serranidae (Nahhas and Short, Reference Nahhas and Short1965), Synbranchidae (Gupta and Gupta, Reference Gupta and Gupta1991; Mishra et al., Reference Mishra, Chandra, Johri, Bajpai and Saxena2014) and Synodontidae (Linton, Reference Linton1905; Manter, Reference Manter1931; Corkum, Reference Corkum1959; Sinclair et al., Reference Sinclair, Smith and Sullivan1972; Li and Sun, Reference Li and Sun1994).
In the present study, specimens of Stom. muraenesocis collected from the stomach of M. cinereus off the Persian Gulf of Iran were morphologically and morphometrically characterized using light and scanning electron microscopy (s.e.m.). For the first time, the phylogenetic affinities of Stomachicola within the superfamily Hemiuroidea Looss, 1899 were explored based on molecular sequence data from the small (18S) and large (28S) subunit nuclear ribosomal RNA genes (rDNA).
Materials and methods
Sample collection and preparation
A total of 30 daggertooth pike congers M. cinereus (total mean length ± standard deviation, 87.85 ± 6.50 cm) from Zir Ahak (28°17′N, 51°13′E), Bushehr, Iran, were examined between January and February 2021. The fish had been collected by fishing vessels along the coastal waters of the Persian Gulf before being purchased from local fishermen. The digestive tract of fresh fish was excised, placed in Petri dishes with saline and examined using a stereomicroscope. Specimens of Stom. muraenesocis were washed thoroughly in saline and relaxed following the procedure described by Dailey (Reference Dailey1978). They were then killed with hot (nearly boiling) saline and fixed both in neutral-buffered formalin (10%) and in absolute ethanol. Some specimens were fixed in pure ethanol without being killed in hot saline for subsequent examination by s.e.m. (Cribb and Bray, Reference Cribb and Bray2010).
Morphological examination
Digeneans were stained with Schneider's aceto-carmine solution (Gower, Reference Gower1939), destained using acid ethanol, dehydrated in an ascending series of ethanol concentrations, cleared in glycerin and mounted in pure glycerin (semipermanent slides) or Canada balsam (permanent slides) (Wotton, Reference Wotton1937; Klimpel et al., Reference Klimpel, Kuhn, Münster, Dörge, Klapper and Kochmann2019). Mounted specimens were measured using cellSens imaging software integrated with a digital camera (Olympus SC50 CMOS) installed on a compound microscope (Olympus BX-53). Line drawings were made with the aid of a drawing tube. All measurements are in millimetres and are presented as the range followed by the mean in parentheses. Parasite identification was performed according to the keys and descriptions provided by Yamaguti (Reference Yamaguti1934), Yamaguti (Reference Yamaguti1958), Gibson et al. (Reference Gibson, Jones and Bray2002) and Madhavi and Bray (Reference Madhavi and Bray2018).
Infection parameters (i.e. prevalence, mean intensity and mean abundance) were calculated following Bush et al. (Reference Bush, Lafferty, Lotz and and Shostak1997). To determine whether the ratios associated with soma length to width, soma to ecsoma length, oral sucker to pharynx length and oral sucker to ventral sucker length can be used as stable taxonomic characters, the range, mean ± standard deviation (s.d.) and coefficient of variation (CV, expressed in percent) of the ratios were calculated using the corresponding morphometric data obtained in this study and those reported in previous literature.
Two adult specimens preserved in pure ethanol were transferred into 70% acetone overnight and dehydrated in a series of increasing acetone concentrations. Then, they were treated with a mixture (1:1 v/v) of anhydrous acetone and hexamethyldisilazane (HMDS, Sigma–Aldrich, Germany) and immersed in HMDS (as the final desiccation step). Specimens were air-dried and mounted on metal stubs using conductive double-sided adhesive tape, coated with a thin layer of gold (4 nm) in a sputter coater (Balzers SCD 050) and examined with a tabletop scanning electron microscope (Hitachi TM-1000, operated at an accelerating voltage of 15 kV) equipped with a high-sensitive semiconductor BSE detector. Voucher specimens from the present study are deposited in the Parasite Collection of the Natural History Museum, London, UK (accession numbers 2023.2.27.6-7).
Molecular identification
Two specimens of Stom. muraenesocis were separately placed into a pre-autoclaved laboratory mortar immersed in liquid nitrogen. As soon as most of the liquid nitrogen was evaporated, the trematodes were ground into a fine powder using an autoclaved pestle and placed in 2 mL Eppendorf tubes (Cox et al., Reference Cox, Pratt, Hageman and Boisvenue1990). Total genomic DNA was extracted from the homogeneous powders using the DNeasy Blood and Tissue kit in accordance with the manufacturer's guidelines (Qiagen GmbH, Hilden, Germany). PCR amplification of 28S rDNA gene was performed using the primers LSU5 and 1200R (Table 1) with the following cycling conditions: an initial denaturation at 95°C for 4 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1.5 min and a final extension step at 72°C for 5 min (Littlewood, Reference Littlewood1994; Lockyer et al., Reference Lockyer, Olson and Littlewood2003). The entire 18S rDNA gene was amplified by PCR using the primer sets Worm A and Worm B (Table 1) as described previously (Littlewood and Olson, Reference Littlewood, Olson, Littlewood and Bray2001) with the following profile: an initial denaturation at 94°C for 2 min, followed by 40 cycles of 30 s at 94°C, 30 s at 54°C, 2 min at 72°C; and 7 min extension at 72°C. PCR reactions were carried out on a C1000 Touch Thermal Cycler (Bio-Rad) in a total volume of 50 μL containing 25 μL of DreamTaq Green PCR master mix (Thermo Scientific), 15 μL of nuclease-free water, 2 μL of 10 pmol μL−1 forward and reverse primers, 1 μL of 25 mm MgCl2 (Thermo Scientific) and 5 μL of 50 ng μL−1 DNA. PCR products were electrophoresed on 1% agarose gel (in Tris-acetate-EDTA buffer), excised from the gel and purified using a MinElute Gel Extraction Kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany). Purified DNA samples from 28S rDNA region were sequenced in both orientations using the same primers used in PCR reactions, while those from 18S rDNA region were sequenced using the 2 PCR primers and internal primers 1270R, 18SU467F and 18SL1170R (Table 1) (Littlewood and Olson, Reference Littlewood, Olson, Littlewood and Bray2001; Indaryanto et al., Reference Indaryanto, Abdullah, Wardiatno, Tiuria and Imai2015). Sequence data were generated using an automated sequencer (ABI 3730 XL) at LGC Biosearch™ Technologies (LGC Genomics GmbH, Berlin, Germany). Contiguous sequences were assembled manually, and base-calling were differences resolved using MEGA X and analysed with Chromas v2.6.6 to ensure accuracy (Sokolov et al., Reference Sokolov, Shchenkov, Frolov and Gordeev2022). The newly generated sequences have been deposited in GenBank under accession numbers OR552105-OR552108.
List of primers used in the present study
Phylogenetic analysis
The Basic Local Alignment Search Tool (BLAST) was employed to compare the novel sequences with publicly available sequences from GenBank. Newly generated sequences were aligned with those previously reported for species within the superfamily Hemiuroidea (see Table 2) following the alignments from Louvard et al. (Reference Louvard, Cutmore, Yong, Dang and Cribb2022b) and Pantoja and Kudlai (Reference Pantoja and Kudlai2022). Sequences of Azygia longa (Leidy, 1851) (Azygioidea: Azygiidae), Proterometra sp. (Azygioidea: Azygiidae), Otodistomum cestoides (Van Beneden, 1870) (Azygioidea: Azygiidae) and Paucivitellosus fragilis (Coil, Reid and Kuntz, 1965) (Bivesiculoidea: Bivesiculidae) were used as outgroups following Blair et al. (Reference Blair, Bray and Barker1998) and Sokolov et al. (Reference Sokolov, Atopkin, Urabe and Gordeev2019). Only sequences with approximately similar lengths to our novel sequences were retrieved from GenBank. Sequences were aligned using ClustalW (Thompson et al., Reference Thompson, Higgins and Gibson1994) implemented in MEGA X (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018) with default parameters for the 18S dataset, and gap opening penalty and gap extension penalty values, respectively set at 15.00 and 5.00, for the 28S dataset (Sokolov et al., Reference Sokolov, Atopkin, Urabe and Gordeev2019), and the extremes of both alignments were trimmed to match the shortest sequences (Hall, Reference Hall2013).
Taxa included in the phylogenetic analyses with their host, locality, systematic position and GenBank accession number
Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) analyses on XSEDE (Towns et al., Reference Towns, Cockerill, Dahan, Foster, Gaither, Grimshaw, Hazlewood, Lathrop, Lifka, Peterson and Roskies2014) using the CIPRES Science Gateway (Miller et al., Reference Miller, Pfeiffer and Schwartz2010). Maximum likelihood analyses were performed using IQ-TREE v2.1.2 with 1000 bootstrap replicates (ultrafast bootstrap type) to estimate the nodal support (Minh et al., Reference Minh, Schmidt, Chernomor, Schrempf, Woodhams, Von Haeseler and Lanfear2020). The best nucleotide substitution models GTR + F + I + G4 and TVM + F + R4 were respectively determined for 18S and 28S rDNA datasets in IQ-TREE under the Bayesian Information Criterion. Bayesian inference analyses were implemented using MrBayes v3.2.7a (Huelsenbeck and Ronquist, Reference Huelsenbeck and Ronquist2001). The best nucleotide substitution models (for BI analyses) were predicted with jModelTest v2.1.10 using the Akaike Information Criterion and Bayesian Information Criterion (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012). The best nucleotide substitution models GTR + I + G and TVM + I + G were estimated for the 18S and 28S rDNA sequence data, respectively (Pérez-Ponce de León et al., Reference Pérez-Ponce de León, Garcia-Varela, Pinacho-Pinacho, Sereno-Uribe and Poulin2016). Bayesian analyses were performed using 2 independent 10 million generation runs of 4 simultaneous Markov chain Monte Carlo (MCMC) chains (nchains = 4) with trees sampled every 1000 generations (printfreq = 1000; samplefreq = 1000) and the first 3000 generations being discarded as burn-in (sump burnin = 3000; sumt burnin = 3000). Trees were re-rooted manually with designated outgroups and visualized using FigTree v1.4.4 (Rambaut, Reference Rambaut2007).
Results
Taxonomic summary
Stomachicola Yamaguti, Reference Yamaguti1934
Syn. Pseudostomachicola (Skrjabin and Guschanskaja, Reference Skrjabin, Guschanskaja and Skrjabin1954), Acerointestinecola (Jahan, Reference Jahan1970), Cameronia (Bilqees, Reference Bilqees1971), Cestodera (Bilqees, Reference Bilqees1971), Segmentatum (Bilqees, Reference Bilqees1971), Indostomachicola (Gupta and Sharma, Reference Gupta and Sharma1973) and Linguastomachicola (Srivastava and Sahai, Reference Srivastava and Sahai1978).
Stomachicola muraenesocis Yamaguti, Reference Yamaguti1934 (Figs 1–3)
Syn. Distomum tornatum of Linton, Reference Linton1905, Dinurus rubeus (Linton, Reference Linton1910), Dinurus magnus (Manter, Reference Manter1931), Lecithocladium longicaudum (Tseng, Reference Tseng1935), Stomachicola magna (Manter, Reference Manter1947), Stomachicola rubea (Manter, Reference Manter1947), Pseudostomachicola magna (Skrjabin and Guschanskaja, Reference Skrjabin, Guschanskaja and Skrjabin1954), Pseudostomachicola rubea (Skrjabin and Guschanskaja, Reference Skrjabin, Guschanskaja and Skrjabin1954), Acerointestinecola karachiensis (Jahan, Reference Jahan1970), Cameronia octovitellarii (Bilqees, Reference Bilqees1971), Cameronia pakistani (Bilqees, Reference Bilqees1971), Cestodera gastrocecus (Bilqees, Reference Bilqees1971), Cestodera unicecus (Bilqees, Reference Bilqees1971), Segmentatum karachiense (Bilqees, Reference Bilqees1971), Segmentatum qadrii (Bilqees, Reference Bilqees1971), Segmentatum cinereusis (Bilqees, Reference Bilqees1971), Segmentatum magnaesophagustum (Bilqees, Reference Bilqees1971), Indostomachicola kinnei (Gupta and Sharma, Reference Gupta and Sharma1973), Stomachicola mastacembeli (Verma, Reference Verma1973), Stomachicola polynemi (Gupta and Gupta, Reference Gupta and Gupta1974), Stomachicola bayagbonai (Siddiqi and Hafeezullah, Reference Siddiqi and Hafeezullah1975), Stomachicola pelamysi (Gupta and Gupta, Reference Gupta and Gupta1974), Stomachicola singhi (Gupta and Ahmad, Reference Gupta and Ahmad1978), Linguastomachicola serpentina (Srivastava and Sahai, Reference Srivastava and Sahai1978), Stomachicola chauhani (Gupta and Singh, Reference Gupta and Singh1981), Stomachicola chauhani (Pandey and Tewari, Reference Pandey and Tewari1984) and Stomachicola guptai (Gupta and Gupta, Reference Gupta and Gupta1991).
Microphotographs of the general morphology of Stomachicola muraenesocis from the stomach of Muraenesox cinereus from Zir Ahak, Bushehr, Iran. (A) Adult trematodes attached to the lumen of the stomach; (B) adult worm killed with hot saline, ventral view; (C) soma of an adult worm, ventral view; (D) soma of an adult worm, dorsal view; (E, F) ovigerous worms stained with Schneider's aceto-carmine, dorsal view; (G) soma of a stained adult worm, ventral view; (H) soma of a stained adult worm, dorsal view; (I) anterior end of an adult worm, ventral view; (J) detail of the female reproductive organs, ventral view; (K) ‘Linguiform projection’ arised from the oral sucker, ventral view; (L) posterior end of a stained adult worm, ventral view; (M) detail of the oral sucker, pharynx and oral sucker opening, ventral view; (N) detail of the sinus-sac, ventral view; (O) internal organs at level of ventral sucker, dorsal view and (P) detail of mature eggs inside the metraterm and large glandular cells of the pars prostatica. Abbreviations: ep, excretory pore; ic, intestinal caecum; lp, linguiform projection; m, metraterm; mg, Mehlis' gland; o, ovary; oo, opening of oral sucker; os, oral sucker; ph, pharynx; pp, pars prostatica; ss, sinus-sac; sv, seminal vesicle; t, testis; u, uterus; v, vitellaria; vs, ventral sucker.
Line drawings of Stomachicola muraenesocis from Muraenesox cinereus from Zir Ahak, Bushehr, Iran. (A) Whole worm, ventral view and (B) soma, dorsal view. Abbreviation: gp, gential pore; ic, intestinal caecum; m, metraterm; mg, Mehlis' gland; o, ovary; os, oral sucker; ph, pharynx; pp, pars prostatica; ss, sinus-sac; sr, seminal receptacle; sv, seminal vesicle; t, testis; u, uterus; v, vitellaria; vs, ventral sucker.
Scanning electron micrographs of Stomachicola muraenesocis from Muraenesox cinereus from Zir Ahak, Bushehr, Iran. (A) Soma, ventral view; (B) detail of the soma (ventral view) and the middle region of the ecsoma (ventral view), arrow points to the division between soma and ecsoma; (C) oral sucker, subapical view; (D) posterior end of the ecsoma, lateral view; (E) ventral sucker, ventral view and (F) detail of the ecsoma surface.
Host: Daggertooth pike conger, Muraenesox cinereus (Forsskål, 1775) (Anguilliformes: Muraenesocidae).
Locality: Zir Ahak (28°17′N, 51°13′E), Bushehr, Iran.
Site of infection: Stomach.
Prevalence: 43.3% (in 13 out of 30 fish).
Mean intensity: 18 worms.
Mean abundance: 7.8 worms.
Total number of specimens collected: 234 (213 ovigerous and 21 immature specimens).
Description
Based on whole-mounts of 8 ovigerous adults and 2 specimens examined using s.e.m. (Figs 1–3). Measurements are presented in Table 3. Body (soma) elongated, curved ventrad (Figs 1C, 3A and B), cylindrical, widest at level of ventral sucker, narrower posterior to ventral sucker. Body surface smooth (Figs 1B–D and 3A). Body parenchyma refractive throughout (Figs 1B–D). Tegumental musculature is moderately thick and well-developed. Tegument is slightly to deeply segmented (Figs 1B and 3B), these segmentations do not truly exist because they are not observed in the live specimens. Ecsoma enormous, well developed (Figs 1B, E and F), dorsoventrally flattened. Soma and ecsoma are separated by telescoping demarcated line (Fig. 3B). Oral sucker is small, ventrally subterminal, spherical to subspherical or slightly funnel-shaped (Fig. 1I). Linguiform projection may arise from the lumen of oral sucker (Fig. 1K), connecting to the oral sucker opening near or precisely at the anterior extremity (Figs 1I and 3A). Preoral lobe short. Ventral sucker is large, rounded, and about 3 times larger than the oral sucker (Figs 1C, G, 3A and E). Forebody short relative to soma. Prepharynx absent. Pharynx globular to subglobular, slightly overlapping posterior border of oral sucker dorsally (Fig. 1I and M). Oesophagus very short. ‘Drüsenmagen’ present. Caeca is long, sinuous, filled with black-brown contents and terminates equally or unequally close to posterior end of ecsoma (Figs 1E, F and L). Testes pair, subtriangular to oval, symmetrical or oblique, almost at midlevel of hindbody (Fig. 1G and H). Seminal vesicle thin-walled, oval to elongate-oval, posterior to ventral sucker (Fig. 1G and H). Pars prostatica well-developed, undivided, tubular, convoluted, mostly or completely invested by large glandular cells (Fig. 1O and P), joins the base of the sinus-sac close to posterior margin of pharynx. Sinus-sac short, muscular and oval to pear-shaped (Figs 1N, 4A and B). Sinus-organ short, slightly muscular and permanent (Fig. 4A and B). Hermaphroditic duct short, straight, enclosed within sinus-sac and sinus-organ. Genital atrium short. Genital pore medial or slightly lateral, at the level of anterior pharynx or oral sucker, anterior to caecal bifurcation (Fig. 1N). Ovary reniform to oval, post-testicular or slightly overlapping testes (Fig. 1H and J). Mehlis' gland small, distinct, median and post-ovarian, between ovary and seminal receptacle (Fig. 1J). Juel's organ present. Laurer's canal was not observed. Seminal receptacle oval to transversely oval or irregularly round (Fig. 1J), medial, small to voluminous, at level of posterior soma or anterior ecsoma. Uterus coiled, mostly inter-caecal, usually extends up to two-thirds of the length of ecsoma and passes anteriorly dorsally to gonads and ventral sucker. Metraterm differentiated, thin-walled, joins male duct at sinus-sac base. Eggs are thick-walled, numerous, small and operculate (Fig. 1P). Vitelline lobes are tubular, mostly extra-caecal, formed by 2 main lateral tubes which are subsequently divided into 5–8, typically 7 (4 dextral and 3 sinistral or vice versa) slender tubes (Fig. 1J), extending from the posterior level of testes to anterior ecsoma. Excretory pore terminal on ecsoma. Excretory vesicle Y-shaped. Excretory arms united at the level of anterior soma.
Comparative morphometric data for Stomachicola species from different fish hosts and localities
Measurements are in millimetres unless otherwise indicated.
a L × W: Range of length (L) × width (W) for each character is reported here (if specified by previous workers).
b These measurements were calculated based on the line drawing of the type species.
a Measurements obtained from 50 eggs.
Terminal genitalia of Stomachicola muraenesocis from Muraenesox cinereus from Zir Ahak, Bushehr, Iran. (A) Line drawing of the terminal genitalia, ventral view and (B) microphotograph of the general morphology of the sinus-sac and sinus-organ, ventral view. Abbreviation: ga, genital atrium; gp, gential pore; eg, egg; hd, hermaphroditic duct; m, metraterm; pp, pars prostatica; so, sinus-organ; ss, sinus-sac.
Molecular characterization and phylogenetic analysis
DNA fragments of different sizes were obtained for the 18S (1779–1789 nt) and 28S (980–982 nt) rDNA genes of Stom. muraenesocis. As sequences of Stom. muraenesocis are not available in GenBank, BLAST showed very low identity and low query coverage between the new sequences and the publicly available sequences of the 18S rDNA. However, our novel 28S rDNA sequences showed 94.51% identity and 99% query cover with an unidentified isolate (MK648287) of hemiurids (collected from a freshwater fish, Brycon guatemalensis) from Mexico (Pérez-Ponce de León and Hernández-Mena, Reference Pérez-Ponce de León and Hernández-Mena2019).
Maximum likelihood and BI trees obtained based on 18S rDNA sequences (Fig. 5) revealed the family Gonocercidae Skrjabin and Guschanskaja, 1955 as a basal group and the other families within the Hemiuroidea in 2 main clades with strong nodal support. The first clade includes representatives of the families Hemiuridae, Lecithasteridae Odhner, 1905 and Bunocotylidae Dollfus, 1950, whereas the second clade incorporates members of the families Didymozoidae Monticelli, 1888, Accacoeliidae Odhner, 1911, Sclerodistomidae Odhner, 1927, Syncoeliidae Looss, 1899 and Derogenidae Nicoll, 1910 (Fig. 5). Hemiuridae and Lecithasteridae formed a strongly supported clade in both trees, but none of these families was resolved as monophyletic. A surprising result of our phylogenetic analyses is that the sequences of Stom. muraenesocis, currently placed in the subfamily Dinurinae Looss, 1907 within the Hemiuridae on the basis of morphological characters, appeared with strong support as a sister to a clade formed by 3 Lecithaster species belonging to the Lecithasteridae (Fig. 5). This result shows that the position of Stomachicola within the Hemiuroidea needs to be reevaluated.
Bayesian inference phylogram reconstructed using the 18S rDNA sequences of Stomachicola muraenesocis (newly generated sequences are indicated in red color) and other members of the Hemiuroidea. The posterior probability and bootstrap support values are shown near the branches for Bayesian inference and maximum likelihood analyses, respectively. Red and green bars respectively represent different subfamilies and families to which taxa included in phylogenetic tree belong.
Maximum likelihood and BI trees constructed from the 28S rDNA dataset (Fig. 6) yielded similar topologies. Our phylogenetic analyses resolved members of the Hemiuroidea into 2 distinct clades with strong support. Members of the Hemiuridae, Lecithasteridae, Bunocotylidae and Isoparorchiidae Travassos, 1922 formed a well-supported clade, sister to a clade composed of representatives of the families Didymozoidae, Derogenidae, Sclerodistomidae, Hirudinellidae Dollfus, 1932, Accacoeliidae, Syncoeliidae, Paraccacladiidae Bray and Gibson, 1977 and Gonocercidae with only strong posterior probability support. Phylogenetic analyses of the 28S rDNA dataset agreed with our phylogenetic assessment of the 18S rDNA dataset and resolved the Hemiuridae and Lecithasteridae as non-monophyletic families. Both ML and BI trees (28S rDNA data) recovered Stom. muraenesocis as sister to an undetermined hemiurid from Mexico (MK648287) in a strongly supported clade (Fig. 6). This clade constituted a sister group relationship with the remaining representatives (with the exception of Merlucciotrema praeclarum) of the Hemiuridae forming an ingroup polytomy of 3 clades in the BI tree (resolved as 2 distinct clades with moderate support in the ML tree). Furthermore, the monophyly of the subfamilies Lecithasterinae Odhner, 1905, Lecithochiriinae Lühe, 1901 and Plerurinae Gibson and Bray, Reference Gibson and Bray1979 was not supported.
Bayesian inference phylogram reconstructed based on the 28S rDNA sequences of Stomachicola muraenesocis (newly generated sequences are indicated in red color) and other members of the Hemiuroidea. The posterior probability and bootstrap support values are given near the branches for Bayesian inference and maximum likelihood analyses, respectively. Blue and purple bars respectively indicate different subfamilies and families to which taxa included in phylogenetic tree belong.
Discussion
The genus Stomachicola was proposed within the subfamily Dinurinae by Yamaguti (Reference Yamaguti1934) to accommodate Stom. muraenesocis from the stomach of M. cinereus as the type species. Later, Yamaguti (Reference Yamaguti1958) erected Allostomachicola and considered Stomachicola and Allostomachicola as members of the subfamily Stomachicolinae. The Stomachicolinae was synonymized with the Dinurinae by Gibson and Bray (Reference Gibson and Bray1979), and this synonymy was accepted by Gibson et al. (Reference Gibson, Jones and Bray2002) and Madhavi and Bray (Reference Madhavi and Bray2018). Martin et al. (Reference Martin, De Silva, Pathirana and Rajapakse2023) transferred the previously recognized dinurines without permanent sinus-organ to the Mecoderinae Skrjabin and Guschanskaja, Reference Skrjabin, Guschanskaja and Skrjabin1954 (Mecoderus Manter, Reference Manter1940, Tubulovesicula Yamaguti, Reference Yamaguti1934, Stomachicola and Allostomachicola). A total of 25 species of Stomachicola have been described to date. Of these species, 19 species are considered conspecific to Stom. muraenesocis, including Stom. chauhani, Stom. guptai, Ind. kinnei, Stom. pelamysi, Stom. polynemi, Stom. rubea, Stom. magna, Lin. serpentina, Stom. mastacembeli, Stom. bayagbonai, Stom. singhi, Cam. octovitellarii, Cam. pakistani, Ces. gastrocecus, Ces. unicecus, Seg. karachiense, Seg. qadrii, Seg. cinereusis and Seg. magnaesophagustum (Hafeezullah, Reference Hafeezullah1980, Reference Hafeezullah1985; Gupta and Gupta, Reference Gupta and Gupta1991; Shaukat, Reference Shaukat2008; Madhavi and Bray, Reference Madhavi and Bray2018). Five species, i.e. Stom. hainanensis, Stom. lycengraulidis, Stom. sexaginta, Stom. pritchardae and Stom. bengalensis have not been revised so far and their systematic position and validity require further evaluation. There is currently no accepted key for the assigned species of Stomachicola, and the taxonomic status of some nominal species has never been verified.
Several morphological characters have been used to distinguish species of Stomachicola. For example, Ind. kinnei, Stom. mastacembeli and Stom. singhi were distinguished from other species by the absence of a seminal receptacle (Gupta and Sharma, Reference Gupta and Sharma1973; Verma, Reference Verma1973; Gupta and Ahmad, Reference Gupta and Ahmad1978). Nevertheless, the seminal receptacle could possibly be overlooked as it may contain no sperm in some specimens. In fact, the absence of a seminal receptable was not considered as a suitable character for species delimitation in Stomachicola by Hafeezullah (Reference Hafeezullah1980) and Hafeezullah (Reference Hafeezullah1985), who considered Ind. kinnei, Stom. mastacembeli and Stom. singhi as synonyms of Stom. muraenesocis. Pande et al. (Reference Pande, Neetu and Neela2000) distinguished Stom. pritchardae from other species by having a tetra-lobed ovary. Since the ovary has been described as an unlobed organ in Stomachicola, the validity of such finding is required to be confirmed. Stomachicola bayagbonai is characterized by 2 frontal projections on either side of the preoral lobe (Siddiqi and Hafeezullah, Reference Siddiqi and Hafeezullah1975; Tanzola and Seguel, Reference Tanzola and Seguel2012). In the material examined in the present study; however, the morphology of the anterior part of the body and the appearance of the preoral lobe were found to vary between individuals of Stom. muraenesocis depending on the contraction state of the parasite during killing. In this regard, Sinclair et al. (Reference Sinclair, Smith and Sullivan1972) reported the appearance of the preoral lobe in Stom. rubea is highly influenced by the temperature of the fixative applied to living specimens. The presence of a ‘linguiform projection’ arising from the lumen of the oral sucker was used as a key feature to distinguish Lin. serpentina by Srivastava and Sahai (Reference Srivastava and Sahai1978). The validity of the ‘linguiform projection’ to distinguish Stomachicola species was questioned by Hafeezullah (Reference Hafeezullah1980), and it was considered as a rare structure in this genus (Gibson et al., Reference Gibson, Jones and Bray2002). In the present study, ‘linguiform projection’ was observed in some specimens of Stom. muraenesocis, which suggests that this structure is rather variable. Stomachicola bengalensis, which was differentiated from Stom. muraenesocis by possessing an oral sucker larger than the ventral sucker, was proposed by Mishra et al. (Reference Mishra, Chandra, Johri, Bajpai and Saxena2014), but the authors have never published the description of this species and thus it is invalid according to the International Code of Zoological Nomenclature (Ride, Reference Ride1999). Stomachicola hainanensis collected from Chirocentrus dorab (Shen, Reference Shen1990; Shen and Qiu, Reference Shen and Qiu1995) may be transferred to Allostomachicola by having a trilobed ovary and seminal vesicle located in the forebody.
Stomachicola chauhani of Pandey and Tewari (Reference Pandey and Tewari1984) was distinguished from Stom. muraenesocis mainly on the basis of the position of the genital pore (posterior to caecal bifurcation). Stomachicola pelamysi was also differentiated from Stom. muraenesocis by Gupta and Gupta (Reference Gupta and Gupta1974) due to the genital pore behind the caecal bifurcation and the diagonal testes. Previous observations showed that the position of the genital pore is anterior to the caecal bifurcation between the base of the oral sucker and the post-pharyngeal region in Stom. muraenesocis (Hafeezullah, Reference Hafeezullah1980, Reference Hafeezullah1985; Gupta and Gupta, Reference Gupta and Gupta1991). In this study, the genital pore was observed medially or slightly laterally at the level of the oral sucker or pharynx. Therefore, the position of the genital pore should be used with caution for the identification of Stomachicola species. Stomachicola chauhani of Gupta and Singh (Reference Gupta and Singh1981) was reported as a new species by having a demarcated line distant (twice) from the posterior end of seminal receptacle. On the other hand, Gupta and Gupta (Reference Gupta and Gupta1991) observed that the position of the genital pore, ventral sucker, testes, ovary and seminal vesicle, the extension of the uterus and vitellaria and the shape and size of internal organs are extremely variable among specimens of Stom. muraenesocis. Our findings also suggest the diverse morphological variations among individuals of the type species in terms of position, arrangement and size of reproductive organs. The number of vitelline lobes has been frequently used for distinction of species of Stomachicola. However, the number of these lobes may vary from 2 to 10 in different individuals. Moreover, the degree of expansion of the uterine coils entering the ecsoma was recognized as a largely varied feature among individuals (ranging from 32% to 71% of the total length). Tanzola and Seguel (Reference Tanzola and Seguel2012) distinguished Stom. lycengraulidis from Stom. muraenesocis on the basis of the length of the ecsoma (25–70% of the total length in Stom. lycengraulidis vs about 92% of the total length in the type species reported by Yamaguti, Reference Yamaguti1934) and the development of the pars prostatica, seminal vesicle and hermaphroditic duct (highly developed vs less developed). However, it was found that the relative size of the ecsoma is considerably variable between species of Stomachicola (see below).
In this study, the ratios of soma length to width, soma to ecsoma length, oral sucker to pharynx length and oral sucker to ventral sucker length were calculated to predict possible stable taxonomic characters which could be used for description of species of Stomachicola. This allowed comparison of these ratios between all species of Stomachicola including those reported as Stom. muraenesocis, all species of Stomachicola excluding those reported as Stom. muraenesocis and only species reported as Stom. muraenesocis. The measurements of Stomachicola hainanensis were excluded from calculations because the species is morphologically associated with Allostomachicola. The range, mean ± s.d. and CV of the ratios for all species of Stomachicola were calculated as follows: soma length to width (n = 17, n represents the number of studies that reported the associated measurements) ranging from 1:0.16 to 1:0.59 with mean (±s.d.) 1:0.36 (±0.12) and CV 34.13%; soma to ecsoma length (n = 18) ranging from 1:0.36 to 1:11.07 with mean (±s.d.) 1:4.72 (±3.12) and CV 66.12%; oral sucker to pharynx length (n = 28) ranging from 1:0.36 to 1:1.67 with mean (±s.d.) 1:0.71 (±0.26) and CV 36.46%; oral sucker to ventral sucker length (n = 31) ranging from 1:1.71 to 1:6.22 with mean (±s.d.) 1:3.18 (±0.88) and CV 27.47%. The range, mean ± s.d. and CV of the ratios for all species of Stomachicola except the type species were as follows: soma length to width (n = 13) ranging from 1:0.16 to 1:0.59 with mean (±s.d.) 1:0.37 (±0.13) and CV 35.69%; soma to ecsoma length (n = 13) ranging from 1:0.36 to 1:10.73 with mean (±s.d.) 1:4.39 (±3.00) and CV 68.36%; oral sucker to pharynx length (n = 18) ranging from 1:0.36 to 1:1.67 with mean (±s.d.) 1:0.70 (±0.29) and CV 41.61%; oral sucker to ventral sucker length (n = 21) ranging from 1:1.71 to 1:6.22 with mean (±s.d.) 1:3.25 (±1.01) and CV 31.22%. The range, mean ± s.d. and CV of the ratios for species recorded as Stom. muraenesocis are summarized as follows: soma length to width (n = 4) ranging from 1:0.19 to 1:0.50 with mean (±s.d.) 1:0.32 (±0.08) and CV 29.94%; soma to ecsoma length (n = 5) ranging from 1:1.85 to 1:11.07 with mean (±s.d.) 1:5.56 (±3.61) and CV 65.01%; oral sucker to pharynx length (n = 10) ranging from 1:0.53 to 1:1.21 with mean (±s.d.) 1:0.73 (±0.18) and CV 24.94%; oral sucker to ventral sucker length (n = 10) ranging from 1:1.84 to 1:4.14 with mean (±s.d.) 1:3.04 (±0.48) and CV 15.69%. No specific value is considered low for a CV, but lower values of CV are correlated with less variability around the mean (Pélabon et al., Reference Pélabon, Hilde, Einum and Gamelon2020). Soma to ecsoma length ratio is therefore a variable and inappropriate distinguishing feature. The fact that the ratios calculated for soma length to width, oral sucker to pharynx length and oral sucker to ventral sucker length from all species (without the type species) closely overlap those from Stom. muraenesocis may suggest that the species previously reported are indeed the representatives of the type species. On the other hand, if the previous species are different from the type, these 3 ratios are not suitable for discrimination of different species belonging to the genus Stomachicola.
With respect to uncertainties related to previous synonymies proposed for species of Stomatichola, difficulties associated with acquisition of vouchers from different localities and morphological variations among individuals of the type species, it is practically impossible to provide a valid list of accepted species of the genus until detailed morphological and molecular studies have been carried out on material from a large number of hosts and localities. In the present study, the molecular sequence data associated with 2 popular genetic markers (18S and 28S) were obtained from Stom. muraenesocis, which will constitute the basis for future taxonomic studies of the genus Stomatichola. However, further nucleotide sequence data are required to demonstrate whether the previously recorded species from different hosts and localities represent different species, or they are genetically associated with the type species. Notably, analysis of sequence data associated with mitochondrial genetic markers may help to identify possible morphotypes of the type species that correspond to intraspecific morphological variations among individuals.
The 2 genera Stomachicola and Allostomachicola are characterized by a combination of common features such as muscular body, well-developed ecsoma, smooth tegument, muscular sinus sac, distinct–indistinct preoral lobe, tubular vitelline lobes (usually 7) and anteriorly united excretory arms as well as species-specific characters including the position of seminal vesicle (hindbody vs forebody) and type of pars prostatica (tubular vs vesicular) (Manter, Reference Manter1940, Reference Manter1947; Gibson and Bray, Reference Gibson and Bray1979; Gibson et al., Reference Gibson, Jones and Bray2002; Nahhas and Sey, Reference Nahhas and Sey2002). Although the ecsoma is a variable characteristic in terms of development, Stomachicola and Allostomachicola can be differentiated from other ecsomate species within the Hemiuridae by possessing an extended ecsoma which is typically several times longer than the body proper. Based on gross morphology, Stomachicola/Allostomachicola belong to the Dinurinae (see Gibson and Bray, Reference Gibson and Bray1979). Recently, Martin et al. (Reference Martin, De Silva, Pathirana and Rajapakse2023) resurrected the Mecoderinae to accommodate the dinurines with a temporary sinus-organ (Allostomachicola, Mecoderus, Stomachicola and Tubulovesicula) and restricted the Dinurinae for dinurines representing a permanent sinus-organ (Dinurus, Ectenurus, Erilepturus, Paradinurus and Qadriana). Morphological examination of our specimens, however, revealed that the sinus-organ is of permanent type in Stom. muraenesocis. Permanent sinus-organ has been previously reported to be absent or rudimentary in Stomachicola (Gibson and Bray, Reference Gibson and Bray1979; Hafeezullah, Reference Hafeezullah1985; Gibson et al., Reference Gibson, Jones and Bray2002; Madhavi and Bray, Reference Madhavi and Bray2018). On the other hand, there are discrepancies in the literature about the presence of a permanent sinus-organ in Allostomachicola (Gibson and Bray, Reference Gibson and Bray1979; Hafeezullah, Reference Hafeezullah1985). Moreover, the sinus-organ was found to be permanent and muscular in Stom. lycengraulidis (Tanzola and Seguel, Reference Tanzola and Seguel2012). According to Gibson and Bray (Reference Gibson and Bray1979), the presence/absence and type of sinus-organ are mostly useful taxonomic features up to the subfamily level, and different types of sinus-organ (permanent and temporary) cannot occur in the same species of trematode. Therefore, such variability in the type of sinus-organ of the Dinurinae/Mecoderinae warrants further examination, and preparation of histological sections from the specimens is of utmost importance for definitive discrimination of the type of sinus-organ (Gibson and Bray, Reference Gibson and Bray1979).
Presently, the superfamily Hemiuroidea comprises 16 families among which molecular sequence data have been reported for certain members of the Accacoeliidae, Bunocotylidae, Derogenidae, Didymozoidae, Gonocercidae, Hemiuridae, Hirudinellidae, Isoparorchiidae, Lecithasteridae, Paraccacladiidae, Sclerodistomidae and Syncoeliidae. There are currently no sequences available for the species within the Bathycotylidae Dollfus, 1932, Dictysarcidae Skrjabin and Guschanskaja, 1955, Ptychogonimidae Dollfus, 1937 and Sclerodistomoididae Gibson and Bray, Reference Gibson and Bray1979. In the present study, the phylogenetic relationship of the representatives of the superfamily Hemiuroidea was not highly supported in the ML tree based on 28S rDNA dataset. However, the topologies obtained in the ML and BI trees were in general congruent with those obtained in previous studies (Pankov et al., Reference Pankov, Webster, Blasco-Costa, Gibson, Littlewood, Balbuena and Kostadinova2006; Atopkin et al., Reference Atopkin, Besprozvannykh, Beloded, Ngo, Ha and Tang2017; Sokolov et al., Reference Sokolov, Atopkin, Urabe and Gordeev2019, Reference Sokolov, Atopkin and Gordeev2021; Faltýnková et al., Reference Faltýnková, Kudlai, Salganskiy, Korol and Kuzmina2022; Pantoja and Kudlai, Reference Pantoja and Kudlai2022; Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022b). As the molecular data of members belonging to genera within the subfamilies Dinurinae/Mecoderinae (Dinurus, Ectenurus Looss, 1907, Paradinurus Vigueras, 1958, Erilepturus Woolcock, 1935, Qadriana Bilqees, Reference Bilqees1971, Allostomachicola, Mecoderus, Stomachicola, Tubulovesicula) are largely unknown, only a few available sequences with similar length to those of Stomachicola were retrieved from the GenBank and included in phylogenetic analyses in this study. Bayesian inference and ML trees reconstructed based on 18S and 28S sequences illustrated that Stomachicola is not genetically clustered with the representatives of the subfamilies Dinurinae/Mecoderinae (Dinurus longisinus Looss, 1907, Ectenurus virgula Linton, Reference Linton1910 and Tubulovesicula laticaudi Parukhin, 1969). Phylogenetic analyses based on the 18S rDNA region revealed the sister relationship between Stomachicola and Lecithaster in trees inferred by ML and BI models. The representatives of the genus Lecithaster are mainly found in the intestine of marine and euryhaline fish (Atopkin et al., Reference Atopkin, Nakao, Besprozvannykh, Ha, Nguyen and Sasaki2018). The main difference between Lecithaster and Stomachicola is the presence of ecsoma in the latter, whereas the species of both genera represent the smooth body surface phenotype (Gibson et al., Reference Gibson, Jones and Bray2002). Lecithaster + Stomachicola constituted a distinct clade in both ML and BI trees, suggesting that the presence of ecsoma, which is a fundamental character for morphological differentiation of species within the family Hemiuridae, may not be associated with their molecular discrimination. In this regard, Atopkin et al. (Reference Atopkin, Besprozvannykh, Beloded, Ngo, Ha and Tang2017) highlighted that texture of the body surface corresponds with molecular distinction of the subfamilies of Hemiuridae but the presence of ecsoma is not associated with taxonomic relationships of the representatives of the family. On the other hand, the basal position of Stom. muraenesocis + Hemiuridae gen. sp. (to the Hemiuridae group) on the phylogenetic trees reconstructed on the basis of 28S rDNA sequence data supports the possibility of the recognition of a distinct subfamily/family for representatives of the genus Stomachicola. However, determination of the exact subfamily/family to which Stomachicola belongs, from a molecular standpoint, requires further sequence data from closely related taxa.
Data availability
The datasets used and/or analysed are available from the corresponding author upon reasonable request. Nucleotide sequences of the 18S rDNA (OR552105-OR552106) and 28S rDNA (OR552107-OR552108) of Stom. muraenesocis have been deposited in GenBank.
Acknowledgements
The scanning electron microscopy was performed by the Electron Microscopy Facility at Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter (VBC), Austria. The authors would like to thank Thomas Heuser and Nicole Drexler from the Vienna BioCenter Core Facilities, Vienna, Austria, for their excellent technical support. The authors are indebted to Drs. Rod Bray and Peter Olson (Natural History Museum, London, UK) for their comments on the terminal genitalia and sinus-organ. We would like to express our gratitude to Dr Amin Mirzazadeh (Boehringer Ingelheim, RCV GmbH and Co KG, Vienna, Austria) and Prof. Hatem Soliman (Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt) for their valuable assistance in various phases of this research. The authors would like to thank Dr David Gibson (Natural History Museum, London, UK), Dr Gokhlesh Kumar (Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria), Dr Arun Sudhagar (Peninsular and Marine Fish Genetic Resources Centre, National Bureau of Fish Genetic Resources, Kochi, Kerala, India), Dr Yang Feng (College of Veterinary Medicine, Sichuan Agricultural University, China) and the Robertson Library staff (University of Prince Edward Island, Canada) for providing literature on trematodes from India and China.
Author contributions
M. S., M. E. and R. G. designed the research. R. G. collected the samples and carried out the experiments. R. G. and J. S. H. made the line drawings. R. G., J. S. H. and M. S. drafted the manuscript. J. S. H., M. E., M. D. F. and S. K. W. provided critical comments and edited the final version of manuscript. All authors reviewed and approved the final manuscript.
Financial support
This research was funded by the University of Veterinary Medicine, Vienna, Austria.
Competing interests
None.
Ethical standards
Not applicable.
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