Introduction
The family Orientocreadiidae Yamaguti 1958 consists of the single genus Orientocreadium Tubangui 1931, which comprises intestinal trematodes of freshwater fish and reptile species of Eurasia, Africa, and India (Jones and Bray Reference Jones, Bray, Bray, Gibson and Jones2008). The type species of the genus, Orientocreadium batrachoides Tubangi 1931, was found in the Philippines; specimens of this trematode were also reported from Africa and Turkey (Tepe et al. Reference Tepe, Oguz, Belk and Ozgen2013; Dumbo et al. Reference Dumbo, Santos and Avenant-Oldewage2019). In addition, a number of Orientocreadium species, described from India, Egypt, and Pakistan, were synonymised with O. batrachoides in the study of Hafeezullah (Reference Hafeezullah and Jairajpuri1989). Besides O. batrachoides, several Orientocreadium species were described in the East Asian region, including, namely, Orientocreadium pseudobagri Yamaguti, 1934, from Japan, China, Korea, and the Russian Far East; Orientocreadium chaenogobii Shimazu 1990, from Japan; and Orientocreadium koreanum Kim and Rim 1995 from Korea, Orientocreadium elegans Besprozvannykh, Ermolenko, and Deveney 2009 from the Russian Far East, and Orientocreadium fulvidraconis Li, Li, and Fan 2020 from China (Yamaguti Reference Yamaguti1934; Besprozvannykh Reference Besprozvannykh1984; Wang et al. Reference Wang, Sun, Zhao, Zhang and Wang1985; Kim and Rim Reference Kim and H-J1995; Shimazu Reference Shimazu2014; Sokolov, Shchenkov Reference Sokolov and Shchenkov2017; Li et al. Reference Li, Li and Fan2020; Moravec and Nie Reference Moravec and Nie2025). Life cycle data are known only for O. pseudobagri and O. elegans from the Russian Far East (Besprozvannykh Reference Besprozvannykh1984; Besprozvannykh et al. Reference Besprozvannykh, Ermolenko and Deveney2009). Lymnaeid snails were revealed as first intermediate hosts for these trematode species, and second intermediate hosts are freshwater gastropods, fish, and amphibians. The taxonomy of these trematode species is based on the morphology of adults in most cases. Molecular characteristics, 28S rDNA sequence data, were provided for trematodes denoted as O. batrachoides from Africa, Orientocreadium indicum Pande 1934, O. fulvidraconis from China, and O. pseudobagri from China and the Russian Far East (Sokolov and Shchenkov Reference Sokolov and Shchenkov2017; Dumbo et al. Reference Dumbo, Santos and Avenant-Oldewage2019; Li et al. Reference Li, Li and Fan2020). These data confirmed the validity of Orientocreadium batrachoides.
In the present study we provide morphological and molecular data for adults of Orientocreadium trematodes from freshwater fish species Perccottus glenii Dybowski 1877, Tachysurus fulvidraco (Richardson 1846), and Silurus asotus Linnaeus 1758, and cercariae from gastropods Radix ussuriensis Kruglov and Starobogatov 1989 from different locations in the south of the Russian Far East. The main goal of our study is species identification and reconstruction of phylogenetic relationships of these trematodes.
Material and methods
Collection of trematodes
Adult worms Orientocreadium were collected from fishes P. glenii from isolated water bodies located in suburb of Vladivostok city and denoted as the Lake ‘LX1’ and the Lake ‘LX2’ (15 fish specimens were dissected, eight specimens were infected, intensity of infection—2 to 7 worms per fish) (Figure 1). Orientocreadium cercariae were collected from snails R. ussuriensis from the same location. Adult trematodes form the same fish species were collected from the Lake Soldatskoe (2 of five dissected fishes were infected, intensity of infection was 1–2 worms per fish) and dead channel of the River Ananyevka, (5 of 13 dissected fishes were infected; intensity of infection was 1–5 per fish); both locations belong to the River Razdolnaya basin, and from the dead channel of the River Ilistaya of the Lake Khanka basin (2 of 3 fishes were infected, intensity of infection—1 to 2 worms per fish).
Map of material collection localities: 1—the Lake Magdykovoe, the River Bolshaya Ussurka basin (45o57′45′′N, 133o56′06′′E); 2—the River Komissarovka (44o56′47′′N, 131o39′32′′E); 3—the River Arsenyevka (45o43′05′′N, 133o33′34′′E); 4—the River Ilistaya(44o24′55′′N, 132o26′22′′E); 5—the Lake Soldatskoe (43o46′44′′N, 131o56′02′′); 6—The Lake X1-2 (43o23′10′′N, 132o03′10′′); 7—The River Ananyevka (43o24′43′′N, 131o34′42′′). Locations of detection of O. cf. pseudobagri and O. cf. elegans by Besprozvannykh (Reference Besprozvannykh1984) and Besprozvannykh et al. (Reference Besprozvannykh, Ermolenko and Deveney2009) are marked with black points.
Trematodes were also collected from T. fulvidraco from the Lake Magdykovoe near the bottomland of the River Ussuri (13 of 17 dissected fishes were infected, intensity of infection was 3–7 worms per fish), the pond near bottomland of the river Arsenyevka of the River Ussuri basin (6 of 7 dissected fishes were infected, intensity of infection was 3–5 worms per fish), and the dead channel of the River Komissarovka of the Lake Khanka basin (4 of 10 dissected fishes were infected, intensity of infection was 2–5 worms per fish). Additionally, adults of Orientocreadium were collected from S. asotus from the pond near bottomland of the river Arsenyevka (3 of 16 dissected fishes were infected; intensity of infection was 1–2 worms per fish).
All adults and cercariae were firstly placed into 70% ethanol, and then replaced into 96% ethanol. For whole mounts adult worms were soaked in distilled water and stained in alum carmine, dehydrated in an ethanol series, cleared in clove oil, and mounted in Canada balsam. All sizes are given in micrometres.
For easier interpretations of our results, all trematodes were artificially sorted into five groups according to water basins where the infected fish and snail specimens were collected: the group G1—trematodes from the suburb isolated the Lake ‘LX1’ and ‘LX2’, suburb of Vladivostok city, the group G2—from fishes of basin of the River Razdolnaya; the groups G3 and G4—from fishes of basin of the River Ussuri (the Magdykovoe Lake and the pond near the River Arsenyevka), the group G5—form fishes of basin of the Lake Khanka (ponds near the River Komissarovka and the River Ilistaya).
For comparative morphological studies we used holotype 17-Tr and paratypes 18-Tr and 19-Tr of O. elegans, and ethanol-preserved exemplars of Orientocreadium trematodes collected by Besprozvannykh (Reference Besprozvannykh1984) from small pond of Vladivostok city (Figure 1); all trematode exemplars stored in the Bioresource Collection of the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657).
DNA extraction, amplification, and sequencing
Overall, 24 mature worms and two cercariae of Orientocreadium from seven locations of Primorye Region were collected for this study; a number of specimens, hosts, locations, and voucher numbers for studied trematodes are provided in Table 1. Total DNA was extracted from flukes using a “hot shot” technique (Truett Reference Truett and Kieleczawa2006).
List of original material incorporated into molecular phylogenetic analyses of Orientocreadiidae N—Number of collected samples; n/a—not available information; *—unpublished data (information available only in GenBank)
The V4 domain of 18S rRNA gene was amplified with the following primers: 18S-8 (5’-GCA GCC GCG GTA ACT CCA GC-3’) and 18S-A27 (5’-CCA TAC AAA TGC CCC CGT TTG-3’) with an annealing temperature of 58°C as previously described (Littlewood and Olson Reference Littlewood, Olson, Littlewood and Bray2001). Ribosomal fragments of 28S rRNA gene 1200bp in length were successfully amplified with the primers DIG12 (5’-AAG CAT ATC ACT AAG CGG -3’) and 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3’) with annealing temperature 55°C (Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003).
Mitochondrial cox1 and nd1 gene fragments were amplified and directly sequenced with primers Trema-cox1/F (5’-TTCGGTCATCCTGAGGTTTATGTT-3’) and Trema-cox1/R (5’-CAGCAAATCATGATGCAAAAGGTA-3’) (Bowles et al. Reference Bowles, Blair and McManus1995; Miura et al. Reference Miura, Kuris, Torchin, Hechinger, Dunham and Chiba2005), and NDJ1/NDJ2A (Morgan and Blair Reference Morgan and Blair1998), respectively.
The amplification volume of polymerase chain reaction (PCR) amounted to 10 μl containing 5 μl GoTaq® Green Master Mix, 1 μl each primer, 1 μl DNA template, and 3 μl sterile deionised water. Amplification was performed using GeneAmp 9700 Thermocycler (Applied Biosystems, USA). Negative and positive controls for amplification of all molecular markers were included.
PCR products were directly sequenced using the ABI Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, USA), as recommended by the manufacturer, with the internal sequencing primers described by Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003) for 28S rDNA; all other nucleotide sequences were generated with primers for amplification, described above. PCR products were analysed using an ABI 3500 genetic analyser (Applied Biosystems, USA) at the Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences. Sequences were submitted to GenBank of the NCBI database with the accession numbers described in Table 1.
The DNA sequences were assembled with SeqScape v. 2.6 software. Alignments and estimation of the number of variable sites and sequence differences were performed using MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018). Phylogenetic analyses of the nucleotide sequences were performed using the Bayesian algorithm with MrBayes v. 3.2.6 software (Ronquist et al. Reference Ronquist, Teslenko, Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). The best nucleotide substitution models, TIM3+G for nuclear 28S rRNA gene and cox1+nd1 mitochondrial genes datasets, and TPM1uf+I for mitochondrial cox1 gene sequence dataset were estimated with jModeltest v. 2.1.5 software (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012) based on Akaike’s information criterion (AIC) (Akaike Reference Akaike1974). Phylogenetic analysis based on V4 domain of nuclear 18S rRNA gene was performed using reversible jump MCMC algorithm (Green Reference Green1995) without evolutionary model estimation. Bayesian analysis was performed using 10000000 generations, with two independent runs. Summary parameters and the phylogenetic tree were calculated with a burnin of 1000000 generations. The significance of the phylogenetic relationships was estimated using posterior probabilities (Huelsenbeck et al. Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001). The phylogenetic relationships among the species of Orientocreadiidae were inferred from our data, along with the partial 28S rRNA gene and mitochondrial genes of other trematode specimens obtained from the NCBI GenBank database (Table 1). Additionally, a Median-Joining network was reconstructed for specimens of Orientocreadium on the basis of concatenated mitochondrial cox1 and nd1 gene sequence dataset with Network v.10.2.0.0 software, developed by Flexus Technology Ltd., Suffolk, UK (https://www.fluxus-engineering.com/sharenet.htm).
Results
Results of mitochondrial DNA variation analysis show that trematodes from ponds ‘LX1’ and ‘LX2’ are representatives of the same species, whereas specimens from other localities belong to another species. In the study of Besprozvannykh et al. (Reference Besprozvannykh, Ermolenko and Deveney2009), trematodes from P. glenii from isolated ponds of the suburb of Vladivostok city were denoted as O. pseudobagri, and trematodes from fishes of the Lake Khanka basin were designated as O. elegans. Accepting these results, specimens from P. glenii and from snails R. ussuriensis from the Lake ‘LX1’ and the Lake ‘LX2’ (group G1) from our study we designated as O. cf. pseudobagri. All other trematodes from our material, obtained from T. fulvidraco, P. glenii, and S. asotus from different locations (groups G2–G5), we designated as O. cf. elegans.
Morphological characteristics of Orientocreadium trematodes
Orientocreadium cf. pseudobagri
Host: Perccottus glenii (Odontobutidae).
Site: intestine.
Locality: the Lake ‘LX1’ (43°21′34.7″N; 131°59′55.4″E), the Lake ‘LX2’ (43°21′23.5″N 131°59′20.4″sE), suburb of the Vladivostok city, and small isolated ponds out of river basins.
This material is held in the Bioresource Collection of the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657); Email: petrova@ibss.dvo.ru.
Adult worm (material examined: 9 specimens) (Figure 2a and Table 2). Body elongate. Tegument with spines. Oral sucker subterminal, elongate oval or round. Prepharynx long. Pharynx transversally oval, with 4 (1 ventral, 1 dorsal, and 2 lateral) anterior muscular protuberances. Oesophagus equal or shorter prepharynx. Intestine bifurcation anterior to ventral sucker. Caeca reach near posterior extremity of body. Ventral sucker round or transversally oval, equal, or smaller than oral sucker, occupies posterior part of anterior third of body or anterior part middle thirds of body. Testes round or oval, tandem or slightly diagonal, at small distance or adjacent to each other in middle of posterior half body. Cirrus-sac clavate, curved, mostly covered ventral sucker. External seminal vesicle present. Internal seminal vesicle oval. Prostatic cells well developed. Ejaculatory duct with spines. Genital pore median, opening into shallow genital atrium, between intestine bifurcation and ventral sucker. Ovary entire, round or oval at midline, pre-testicular at distance to anterior testis and ventral sucker. Mehlis’ gland posterior to ovary. Uterus coiled, extends between ventral sucker and posterior end of body. Seminal receptacle uterine. Metraterm long, with spines, covered ventral sucker. Metraterm opens into genital atrium. Eggs operculate. Vitellarium, extends in lateral fields from level of ovary, or immediately anterior to ovary, to midlevel or posterior third of post-testicular space. Excretory vesicle Y-shaped, excretory pore terminal.
Adult worms of Orientocreadium: a—Orientocreadium cf. pseudobagri ex Perccottus glenii, location: the Lake ‘LX1-2’, Vladivostok; b—Orientocreadium cf. elegans ex P. glenii, location: the River Ananyevka basin; c—Orientocreadium cf. elegans ex Tachysurus fulvidraco, location: the River Arsenyevka basin; d—O. cf. elegans ex Silurus asotus, location: the River Arsenyevka basin; e—O. cf. elegans ex T. fulvidraco, location: the river Komissarovka basin.
Measurements (μm) of adult worms Orientocreadium
*G1: host—Perccottus glenii, location— “Lake X” Vladivostok city, Russian Far East; G2: host—P. glenii, location—the River Ananyevka, basin Razdolnaya, River Russian Far East; G3 and G4: hosts—Tachysurus fulvidraco and Silurus asotus, location—the Arsenyevka River, basin Ussuri River, Russian Far East; G5: host—T. fulvidraco, location—the River Komissarovka, basin Khanka Lake, Russian Far East; **Hosts—T. nudiceps and S. lithophilus, location—Japan; ***Host—T. fulvidraco, the River Ilistaya, basin Khanka Lake, measurements given with inclusion of our data; ****Host—P. glenii, location—Vladivostok city, Russian Far East.
Orientocreadium cf. elegans
Host: Tachysurus fulvidraco (Bagridae), Perccottus glenii (Odontobutidae), Silurus asotus (Siluridae).
Site: intestine.
Localities: Lakes in the basins of Ussuri River (45°57’45"N, 133°56’06"E; 45°43’05"N, 133°33’34"E), the Lake Khanka (44°56’47"N, 131°39’32"E; 44°24’48.90", 132°25’47.48"), the River Razdolnaya (43°46’42.34"N, 131°56’4.72"E; 43°24’42.13"N, 131°40’55.23"E) of Primorsky Region, south of the Russian Far East.
Type-deposition: The sample material is stored in the Bioresource Collection of the Federal Scientific Centre of East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657); email: petrova@ibss.dvo.ru. Deposited 22 November 2018.
Adult worm (material examined: 13 specimens) (Figures 2b–e and Table 2). Body narrow or wide elongate. Tegument with spines. Oral sucker subterminal, elongate oval or round. Prepharynx long or short. Pharynx transversally oval, with 4 (1 ventral, 1 dorsal, and 2 lateral) anterior muscular protuberances. Oesophagus equal or shorter than prepharynx. Intestine bifurcation anterior to ventral sucker. Caeca reaching near posterior extremity of body. Ventral sucker elongate oval or round or transversally oval, equal or smaller of oral sucker occupies posterior part of anterior third of body or anterior part middle thirds of body or at border anterior and middle thirds of body. Testes round or oval, tandem, separated, or adjacent to each other in posterior half of body. Cirrus-sac clavate, partly covered ventral sucker or around ventral sucker sinistrally. External seminal vesicle present. Internal seminal vesicle oval. Prostatic cells well developed. Ejaculatory duct long, with spines. Genital pore median, opening into shallow genital atrium, between intestine bifurcation and ventral sucker. Ovary entire, round or oval at midline, pre-testicular at different distances to ventral sucker and anterior testis or adjacent to anterior testis. Mehlis’ gland posterior to ovary. Uterus coiled, extends between ventral sucker and posterior end of body. Seminal receptacle uterine. Metraterm long, with spines, covered ventral sucker opens into genital atrium. Eggs operculate. Vitellarium, extends in lateral fields from level of ovary, or immediately anterior to ovary, to posterior half of post-testicular field or level posterior end posterior testis. Excretory vesicle Y-shaped, excretory pore terminal.
Molecular characteristics of Orientocreadium trematodes
The ribosomal V4 region of the 18S rRNA gene complete sequences, 421 bp in length, and the 28S rRNA gene partial sequences, 1200 bp in length, were successfully amplified for 26 and 24 specimens of Orientocreadium trematodes, respectively. After assembling, alignment, and Gblocks processing, 421 bp and 855 bp fragments for the V4 domain of the 18S rRNA gene and the 28S rRNA gene, respectively, were available for analysis.
Results of 28S rRNA gene sequence analysis show the presence of four variable sites. Of these, three singletons were detected for one specimen of O. cf. elegans ex T. fulvidraco from the River Arsenyevka, and one fixed substitution, transition A/G, at the #262 position, was detected for specimens of O. cf. elegans ex T. fulvidraco from the River Komissarovka and O. cf. elegans ex P. glenii from the River Ananyevka and the River Ilistaya. Genetic p-distances between all newly studied trematodes ranged from 0 to 0.35±0.2%. The p-distance values between specimens from our study and trematodes from GenBank, denoted as O. pseudobagri and O. indicum, ranged from 0 to 0.23±0.16%; between these specimens and O. fulvidraconis, they ranged from 0.47±0.23% to 0.7±0.27%. Genetic p-distance values between our samples, O. pseudobagri, O. indicum, and O. fulvidraconis from GenBank, and O. batrachoides ranged from 2.93±0.56% to 3.77±0.64%.
Results of phylogenetic analysis based on the 28S rRNA gene sequence dataset show highly supported monophyly of the genus Orientocreadium (Figure 3). Within the genus, O. batrachoides from the GenBank database formed distinct, highly supported lineages. All trematodes from our study were gathered within a large, highly supported clade together with several Orientocreadium specimens from the GenBank database, denoted as O. pseudobagri, O. indicum, and O. fulvidraconis. Several specimens, including O. cf. elegans ex T. fulvidrao from the River Komissarovka and O. cf. elegans ex P. glenii from the River Ananyevka and the River Ilistaya, were within a separate highly supported subclade. Partial nucleotide sequences of the 28S rRNA gene of these specimens differ from others by a single fixed substitution, mentioned above.
Bayesian phylogenetic tree of the genus Orientocreadium based on 28S rRNA gene partial sequence dataset. Nodal numbers—posterior probabilities for statistically significant relationships.
In the V4 domain of the 18S rRNA gene, there were three variable sites, #1, #402, and #421, that contain substitutions A/T, G/A, and C/A, respectively. Based on the existence of these three variable sites, all trematodes divide into two groups for which these substitutions can be considered as fixed. The first group includes all trematodes, firstly identified as O. cf. elegans from Lake Magdykovoe ex T. fulvidraco, worms from Lake Soldatskoe ex P. glenii, and three specimens from the River Arsenyevka ex T. fulvidraco, collected in June 2014. All other trematodes of Orientocreadium were within another group, including three specimens from the River Arsenyevka ex T. fulvidraco, collected in June 2020. Results of phylogenetic analysis show that trematodes that possess different nucleotides at the three above-mentioned sites slightly differ from each other (Supplementary Figure S1). Trematodes from most of the localities and different hosts were within the separate clade, whereas 8 trematodes from Lake Magdykovoe, Lake Soldatskoe, and the Arsenyevka River each appear as distinct lineages and create strong polytomy, indicating that trematodes from these locations possess ancestral sequence variants of the V4 domain of the 18S rRNA gene. Genetic p-distance values between trematodes with different variants of the V4 domain of 18S rRNA gene sequences were 0.71 ± 0.4%.
Mitochondrial cox1 and nd1 gene partial sequences of 867 bp and 431 bp, respectively, were generated for 34 and 29 specimens of Orientocreadium trematodes. Results of cox1 gene sequence analysis show the presence of 13 variable nucleotide positions, including 10 parsimony-informative ones. Some of these 10 sites contain nucleotides specific for trematodes from certain locations. The sequences of all specimens from the River Ananyevka and the River Ilistaya differ from others by transversions G/C and G/T, respectively, at site #21. The same G/C and G/T transversions at the same nucleotide position were detected in cox1 gene sequences of all specimens of O. cf. elegans ex P. glenii from Lake Soldatskoe and in a single specimen of O. cf. elegans ex T. fulvidraco from the River Komissarovka, respectively. Consequently, specimens from the River Ananyevka and the River Ilistaya differ from each other by a single fixed transition T/C at this nucleotide position. Additionally, cox1 gene sequences of trematodes from the River Ananyevka differ from all other trematodes from our material by the fixed transition G/A at the nucleotide position #783. The same substitution was observed for one specimen ex T. fulvidraco from the River Arsenyevka (Table 3). Mitochondrial cox1 gene sequences of trematodes from the Lake ‘LX1’ contain highly specific bases at nucleotide positions #207, #537, #684, #789, and #863 (Table 3).
Variable nucleotide positions of mitochondrial cox1 gene sequences of Orientocreadium trematodes from different hosts and locations in the south of Russian Far East. Nucleotide positions with fixed substitutions associated with geographical locations are marked in grey
Results of phylogenetic analysis based on the mitochondrial cox1 gene sequence dataset show strongly supported monophyly for the genus Orientocreadium. Trematodes O. cf. pseudobagri ex P. glenii from the Lakes ‘LX1-2’ formed a separate, highly supported clade, whereas other trematodes denoted as O. cf. elegans were within a poorly supported large clade and distributed chaotically, with no concordance to either host species or location (Figure 4). Genetic p-distance values between trematodes O. cf. pseudobagri and O. cf. elegans ranged from 0.58 ± 0.25% to 1.04 ± 0.45%, whereas genetic distances within O. cf. elegans ranged from 0 to 0.58 ± 0.26%.
Bayesian phylogenetic tree of the genus Orientocreadium based on mitochondrial cox1 gene partial sequence dataset. Specimens ex Perccottus glenii from the Lakes ‘LX1’ and ‘LX2’ are at grey background. Nodal numbers—posterior probabilities for statistically significant relationships.
Mitochondrial nd1 gene sequences contained 14 variable sites, including 6 parsimony-informative and 8 singletons. There were two sites with fixed nucleotide substitutions of trematodes from isolated lakes ‘LX1’ and ‘LX2’: #28 (A/G) and #366 (C/T). Genetic p-distances between Orientocreadium specimens ranged from 0 to 1.39 ± 0.54%. The phylogenetic tree based on the 431 bp fragment of the nd1 gene was poorly informative, demonstrating hard polytomy for almost all studied specimens of Orientocreadium (data not shown).
We carried out phylogenetic analysis of Orientocreadium based on concatenated mitochondrial cox1 and nd1 gene sequences 1280 bp in length (Figure 5). Results show strongly supported monophyly for the genus Orientocreadium, show Chinese Orientocreadium sp. (OK662969) from the GenBank formed a distinct lineage, and show separation of all specimens of Orientocreadium from our study into three lineages. The first highly supported lineage includes trematodes of O. cf. pseudobagri; the second lineage was represented by a single specimen of O. cf. elegans ex S. asotus from the Arsenyevka River, and the third lineage represented a highly supported large clade that includes all other trematodes of O. cf. elegans (Figure 5).
Bayesian phylogenetic tree of the genus Orientocreadium based on concatenated mitochondrial cox1 and nd1 gene partial sequence dataset. Specimens ex Perccottus glenii from the Lakes ‘LX1’ and ‘LX2’ are at grey background. Nodal numbers—posterior probabilities for statistically significant relationships.
Discussion
Orientocreadium pseudobagri was first described from Pseudobagrus aurantiacus (Temminck and Schlegel 1846) in Japan; in this territory fish species of Bagridae Bleeker 1858 and Siluridae Cuvier 1816 are shown to be exploited by this parasite as definitive hosts (Shimazu Reference Shimazu2014). These trematodes were detected in China and Korea in Siluriformes fish species (Wang et al. Reference Wang, Sun, Zhao, Zhang and Wang1985; Kim and Rim Reference Kim and H-J1995; Li et al. Reference Li, Li and Fan2020; Moravec and Nie Reference Moravec and Nie2025). In the Russian Far East this species was found in the intestines of Siluriformes Cuvier 1816 and P. glenii (Strelkov Reference Strelkov1971; Besrozvannykh 1984; Besprozvannykh et al., Reference Besprozvannykh, Ermolenko and Deveney2009; Sokolov, Shchenkov Reference Sokolov and Shchenkov2017). Results of comparative analysis of morphological and metric parameters (Table 2) of adults of O. cf. pseudobagri ex P. glenii from the Lake ‘LX1’ and the Lake ‘LX2’ and the trematode specimen extracted from P. glenii from the study of Besprozvannykh et al. (Reference Besprozvannykh, Ermolenko and Deveney2009) show that these flukes are identical. Alongside this, these trematodes agree with the data on the first description of O. pseudobagri (Yamaguti Reference Yamaguti1934). Particularly, Yamaguti (Reference Yamaguti1934) notes that the body length of the worms does not exceed 1470 μm. At the same time, O. pseudobagri from Korean, Chinese, and Japanese Bagridae and Siluridae fishes (Kim and Rim Reference Kim and H-J1995; Shimazu Reference Shimazu2014; Li et al. Reference Li, Li and Fan2020; Moravec and Nie Reference Moravec and Nie2025) reported maximal values of body length of mature worms that are 1.5-2.5 times higher than values provided by Yamaguti (Reference Yamaguti1934), Besprozvannykh et al. (Reference Besprozvannykh, Ermolenko and Deveney2009), and our data. All specimens from our study, extracted from fishes of both Odontobutidae Hoese and Gill 1993 and Siluriformes, have significantly smaller values of the maximum body length parameter (Table 2). Based on these results, we propose that Korean, Chinese, and Japanese worms denoted as O. pseudobagri can be members of different species; this hypothesis can be confirmed after generating and analysing molecular markers of nuclear and mitochondrial nucleotide sequences for these worms. Moravec and Nie (Reference Moravec and Nie2025), in their paper, noted that the characters used by Besprozvannykh et al. (Reference Besprozvannykh, Ermolenko and Deveney2009) for differentiating O. pseudobagri and O. elegans are doubtful, and these two species are conspecific. Morphologically, species of Orientocreadium in the East Asian region have no considerable morphological differences. Specimens of O. cf. pseudobagri are most similar to worms O. cf. elegans ex T. fulvidraco from the River Komissarovka basin and ex P. glenii from the River Ananyevka but differ from them by maximal size of ventral sucker, metraterm length, and by maximal size of most organs and body length/width ratio (Table 2). Other specimens of O. cf. elegans from Tachysurus fulvidraco and Silurus asotus from the River Arsenyevka basin differ from O. cf. pseudobagri by maximal values of the body and forebody length and body length and width ratio, as well as ventral sucker and pharynx width ratio. Moreover, there was one trematode specimen ex S. asotus, which differs from others by the length of the cirrus sac and metraterm. Thus, morphologically similar trematodes of O. cf. pseudobagri and O. cf. elegans possess expressed differences in metric parameters that can be considered as interspecific criteria. Molecular results support that O. cf. pseudobagri and O. cf. elegans from our material belong to different species.
The phylogenetic tree based on 28S rDNA sequences was poorly efficient for delimitation of O. cf. pseudobagri, O. cf. elegans, O. indicum, and O. fulvidraconis (Figure 3). Genetic distance values between members of these species ranged from 0 to 0.7 ± 0.27%, whereas genetic distance values between these trematodes and O. batrachoides reached 3.77 ± 0.64%. Unfortunately, there are only 28S rDNA sequences available for species O. indicum, O. fulvidraconis, and O. batrachoides. For this reason, it is impossible to delimit O. cf. pseudobagri and O. cf. elegans from our study. Results of phylogenetic analysis based on mitochondrial cox1 gene sequences revealed differentiation between trematodes from isolated ponds ‘LX1-2’, which we defined as O. cf. pseudobagri, and all other trematodes from basins of the River Razdolnaya, the River Ussuri, and the Lake Khanka, defined as O. cf. elegans. These trematodes formed two highly supported clades sister to each other (Figure 4). Nucleotide sequences from these two clades differ by several fixed substitutions (Table 3), and genetic distance values between them ranged from 0.58 ± 0.25% to 1.04 ± 0.45%; within O. cf. elegans genetic distance values do not exceed 0.58 ± 0.26%. Thus, metric and molecular differences found between trematodes from our study represent evidence for delimitation of O. cf. pseudobagri and O. cf. elegans. We assign for these trematodes the status “confer” because of the absence of molecular data for O. pseudobagri from the type location (Lake Biwa, Japan) and for O. koreanum described by Kim and Rim (1995) from Korean fishes. Moreover, mitochondrial DNA sequences for Orientocreadium indicum Pande 1934, O. fulvidraconis, and O. pseudobagri from China are absent as well. These data, alongside morphological and metric characteristics, are necessary to clarify the taxonomical status of O. cf. pseudobagri and O. cf. elegans from fishes of the south of the Russian Far East, as well as O. fulvidraconis and O. koreanum. It is important to pay attention to the results of a number of taxonomical studies of trematodes, where morphologically similar worm specimens are recognised as different species by molecular, ecological, or any other criteria, and representatives of the same species can possess morphological and molecular variation (Jousson et al. Reference Jousson, Bartoli and Pawlowski2000; Atopkin et al. Reference Atopkin, Ivashko, Besprozvannykh and Zhokhov2023; Izrailskaia et al. Reference Izrailskaia, Tatonova and Belousova2024; Shumenko et al. Reference Shumenko, Tatonova and Shchelkanov2024; Ivashko et al. Reference Ivashko, Atopkin and Besprozvannykh2025). These features can be applied to both O. cf. pseudobagri and O. cf. elegans from our study.
Trematodes of O. cf. elegans are characterised by a wide range of morphometrical variation. For example, mature worms detected in T. fulvidraco from the River Ilistaya (the Lake Khanka basin) and the River Arsenyevka (the River Ussuri basin) possess both maximal values of the body length and short bodies (Figure 2 and Table 2). These worms have similar values of body length/width ratio but differ by prepharynx and oesophagus length, by distance between ovary and anterior testis, and by distance between testes. Both ‘morphotypes’ were found in the same fish specimens. The exemplar of O. cf. elegans with maximal body size was detected in S. asotus from the River Arsenyevka (Table 2). Infection intensity of S. asotus with Orientocreadium is approximately ten times lower in comparison with that for T. fulvidraco and P. glenii; commonly, trematodes of the genus Orientocreadium can be found in S. asotus rarely, 1–2 specimens per fish, compared to T. fulvidraco from the same water body. The catfish seems to not be an obligate definitive host of Orientocreadium trematodes. Most likely, catfish can be classified as accidental hosts, where these trematodes are able to survive during reinvasion as a result of their feeding on infected P. glenii and T. fulvidraco. Except for one individual of O. cf. elegans with a body length of 2202 μm that was found in catfish, trematodes of this species ex P. glenii from the Ananyevka River have the greatest differences from other individuals of O. cf. elegans in the maximum values of organ sizes and body width, the ratio of body length to width, an extremely short oesophagus, and the absence of distances between the ovary and the anterior of the testis, as well as between the testes (Figure 2 and Table 2). Thus, among the individuals designated as O. cf. elegans, 4 morphotypes (Figure 2) can be distinguished; each could be recognised as a separate species morphometrically. Accepting the high molecular similarity of O. cf. elegans, the morphometrical variations of specimens of this species are possibly related to low specificity to definitive hosts and can be interpreted as a phenotypic display of adaptation, which is not associated with molecular variation.
Results of phylogenetic analysis based on the 28S rRNA gene sequence dataset show that several specimens of O. cf. elegans from certain locations, namely the River Komissarovka, the River Ananyevka, and the River Ilistaya, differ from other worms of this species by a single fixed transition (Figures 2b, e) and some specific differences with trematodes from the basins of the River Razdolnaya, the River Ussuri, and Lake Khanka by the mitochondrial cox1 gene sequence dataset (Figure 4 and Table 3). Alongside this, nucleotide sequences of the 28S rRNA gene of trematodes O. cf. elegans ex P. glenii from Lake Soldatskoe, which is connected with the River Razdolnaya, were similar to those of trematodes from the basin of the River Ussuri. Based on the 28S rRNA gene sequence dataset, trematodes of Orientocreadium from the south of the Russian Far East can be subdivided into two groups related to complexes of different basins: the ‘Khanka-Razdolnaya’ related group and the ‘Ussuri-Razdolnaya’ related group; the first group includes both O. cf. elegans and O. cf. pseudobagri, and the second group includes only O. cf. elegans (Figure 3). On the other hand, results of the V4 domain of the 18S rRNA gene-based phylogenetic analysis (Figure S1) show relationships of trematodes from the basin of the River Razdolnaya with trematodes from both the Lake Khanka basin and the River Ussuri basin. This molecular marker highlighted molecular differentiation between trematodes O. cf. elegans from the River Arsenyevka ex T. fulvidraco. Three of six specimens were identical to worms from Lake Soldatskoe and Lake Magdykovoe, whereas another three, together with specimens of O. cf. elegans ex S. asotus from the River Arsenyevka, were identical to trematodes from other locations in the River Razdolnaya and the Lake Khanka basins and isolated Lakes ‘LX1’ and ‘LX2’, which combined within a separate highly supported clade. This result indicates the existence of a common nearest ancestor for trematodes from this separate clade, which hypothetically existed in the River Ussuri basin. In our opinion, all these molecular differences revealed for trematodes reflect the evolutionary history of their hosts, freshwater fish species, which possibly was associated with partial or complete isolation of different locations and with fish species introduction into water bodies of the south of the Russian Far East. The basin of the River Razdolnaya, which extends through Russian and Chinese territories and is geographically isolated from the River Ussuri and the Lake Khanka basins, was not a native area of T. fulvidraco, a main host of Orientocreadium trematodes (Taranets Reference Taranets1936a, Reference Taranetsb). Tachysurus fulvidraco is a representative of the fish species complex of the Lake Khanka basin. Most probably, this fish species was introduced into the River Razdolnaya from reservoirs located in China or from the basin of Lake Khanka. The River Ussuri and the Lake Khanka basins are incompletely isolated from each other, connecting to each other with the low-water River Song’acha (Figure 1). In these conditions, slight molecular differentiation between parasite communities, observed in our study, can occur. In light of this proposition, to clarify molecular differentiation and relationships between local samples of Orientocreadium trematodes, we applied the median-joining network analysis based on concatenated mitochondrial cox1 and nd1 gene sequences. Results show that all Orientocreadium specimens are subdivided into four groups, representing the River Ussuri basin, the River Razdolnaya basin, the Lake Khanka basin, and the Lakes ‘LX1’ and ‘LX2’ (Figure 6). Of these, trematodes from the Lakes ‘LX1’ and ‘LX2’ have a unique single haplotype that differs from the other three groups by seven mutational steps. These trematodes were directly related to worms from the River Ananyevka (the River Razdolnaya basin), and one specimen with a unique haplotype from the River Arsenyevka (the River Ussuri basin) ex S. asotus through median vectors. Other groups possess variable levels of haplotype diversity and differ from each other by 1–3 mutational steps. These results indicate deep molecular differentiation of trematodes from the Lakes ‘LX1’ and ‘LX2’ from specimens from other locations.
Median-Joining network of Orientocreadium trematodes based on concatenated mitochondrial cox1 and nd1 gene sequences.
Conclusion
Based on results of our study, the existence of two trematode species, Orientocreadium cf. pseudobagri and O. cf. elegans, in the south of the Russian Far East was confirmed. We revealed morphological and molecular variation of trematode specimens of these two species. These variations are most possibly associated with population-forming processes of these trematodes and their definitive hosts—freshwater fishes in the investigated territory. The evolutionary history of the populations of both trematode species in the Russian Far East seems to be a complex process that occurred during several stages. Our molecular data show that the trematode assemblages studied here can be subdivided for 28S rDNA markers into two large tentative groups related to basins of different water bodies: the ‘Khanka-Razdolnaya’ related group and the ‘Ussuri-Razdolnaya’ related group. The trematodes from each of these groups are not strongly separated from each other; some of the sequence variants (for rDNA markers) or haplotypes (for mtDNA markers) from different groups are closely related to each other. On the other hand, within each group there are specific nucleotide substitutions that are unique for several geographical local samples of trematodes that reflect local processes of divergence of these worms. Results of the median-joining network analysis based on concatenated mitochondrial cox1 and nd1 gene sequences support this inference, showing the existence of four groups of haplotypes of Orientocreadium trematodes that are associated with geographical locations.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25100825.
Acknowledgments
Authors are thankful to senior researcher of laboratory parasitology, FSC of Biodiversity FEB RAS, PhD Yulia V. Tatonova for help with dissection of fishes P. glenii from Lake Soldatskoe.
Financial support
The research was carried out within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (themes No. 121031000154-4).
Competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
