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., 1990). 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, 1994; Lockyer et al., 2003). 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, 2001) 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, 2001; Indaryanto et al., 2015). 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., 2022). The newly generated sequences have been deposited in GenBank under accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"OR552105-OR552108","start_term":"OR552105","end_term":"OR552108","start_term_id":"2576978061","end_term_id":"2576978064"}}OR552105-OR552108.

List of primers used in the present study