Material and methods

One specimen of ocean sunfish was caught off Iturup Island (Kuril Islands, Russia) on 10 September 2021. The specimen (TL 60 cm, weigh 22 kg) was collected from the fixed seine installed by RUSKOR Ltd. for Pacific salmon fishing in the Odessky Bay (‘Odesskiy Zaliv’) at the coordinates 44°48′24″N&147°13′37″E. In the laboratory, the specimen was examined for parasites using standard techniques of parasitological dissection (Bykhovskaya-Pavlovskaya 1985; Klimpel et al. Reference Klimpel, Kuhn, Münster, Dörge, Klapper and Kochmann2019). We found the body fragment of Accacladocoelium sp. (anterior extremity from the oral to the ventral suckers) and seven whole specimens of O. calyptrocotyle (Figure 1) in the intestine. The digenean worms were washed in fresh water and subsequently fixed in 70% ethanol. A small fragment (the ventral sucker) of Accacladocoelium sp. and one whole specimen of O. calyptrocotyle were examined using molecular techniques. Remaining Accacladocoelium sp. fragment and other O. calyptrocotyle specimens were stained with acetocarmine, dehydrated in a graded ethanol series, cleared with dimethyl phthalate, and finally mounted in Canada balsam. The drawings were made with the help of the camera lucida. Parasite species were identified according to Linton (Reference Linton1898), Timon-David and Musso (Reference Timon-David and Musso1971), and Bray and Gibson (Reference Bray and Gibson1977). Voucher of O. calyptrocotyle was deposited in the Museum of Helminthological Collections of the Centre of Parasitology of the A.N. Severtsov Institute of Ecology and Evolution, RAS (IEE RAS; Moscow, Russia) numbers # 14363.

Figure 1.

Paragenophore of Odhnerium calyptrocotyle ex Mola mola off Iturup Island, lateral view. Scale bar 3 mm.

DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen, Toronto, ON, Canada) as per the manufacturer’s instructions. The polymerase chain reaction (PCR) amplification volume amounted of 10 μl containing 5 μl GoTaq® Green Master Mix, 1 μl each primer, 1 μl DNA template, and 3 μl sterile deionised water. Fragment of the gene encoding 28S ribosomal DNA (28S rRNA gene) was amplified with the primers 28SA (5′-TCG ATT CGA GCG TGA WTA CCC GC-3′) (Matejusova and Cunningham Reference Matejusova and Cunningham2004) and 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003) with an annealing temperature of 55°C. A ribosomal ITS2 fragment was amplified with primers BD1 (5’-GTC GTA ACA AGG TTT CCG TA-3’) and BD2 (5’-TAT GCT TAA ATT CAG CGG GT-3’) (Luton et al. Reference Luton, Walker and Blair1992) with an annealing temperature of 54°C. Mitochondrial cox1 gene partial sequences were amplified with PCR primers JB3 (5’-TTT TTT GGG CAT CCT GAG GTT TAT-3’) and COI-R1_Trema (5’-CAA CAA ATC ATG ATG CAA AAG G-3’) (Bowles et al. Reference Bowles, Blair and McManus1995; Miura et al. Reference Miura, Uematsu, Sakaki and Ito2005). Negative and positive controls using both primers were included. Polymerase chain reaction was performed in a GeneAmp 9700, Applied Biosystems, USA.

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 rRNA gene, and Luton et al. (Reference Luton, Walker and Blair1992) for the ITS2 rDNA fragment. The cox1 gene fragments were sequenced with PCR primers, described above. PCR products were analysed using a GA3500 genetic analyser (Life Technologies, USA) at the FSC of Biodiversity FEB RAS. Sequences were submitted to GenBank of the NCBI database with the accession numbers described in Table 1.

Table 1.

List of nucleotide sequences of hemiuroidean trematodes incorporated into molecular analysis. New material marked with bold

The phylogenetic relationships among species of the Accacoeliidae were inferred from our data and the sequences of the other Hemiurata species obtained from the NCBI GenBank database (Table 1). Ribosomal 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 7.0 (Kumar et al. Reference Kumar, Stecher and Tamura2016). Phylogenetic analyses of the nucleotide sequences were performed using the Bayesian inference algorithm with MrBayes v. 3.2.6 software (Huelsenbeck et al. Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001). The best nucleotide substitution models, TIM3+G and TPM1uf+G for ribosomal 28S rRNA and mitochondrial cox1 gene sequence fragments, respectively, were estimated with jModeltest v. 2.1.5 software (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012). The HKY+G nucleotide substitution model was estimated as optimal for concatenated ITS2 rDNA and cox1 gene of mtDNA sequence dataset. Bayesian analysis was performed using 10,000,000 generations, with two independent runs. Summary parameters and the phylogenetic tree were calculated with a burnin of 2,500,000 generations. The significance of the phylogenetic relationships was estimated using posterior probabilities (Huelsenbeck et al. Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001).