Triaenophorus nodulosus subsp. molecular

Taube, Konrad, Noreikiene, Kristina, Kahar, Siim, Gross, Riho, Ozerov, Mikhail, Vasem, Anti & agi, 2023, Subtle transcriptomic response of Eurasian perch (Perca fluviatilis) associated with Triaenophorus nodulosus plerocercoid infection, International Journal for Parasitology: Parasites and Wildlife 22, pp. 146-154 : 147

2.2. T. nodulosus molecular identification

Infection status was determined by visual identification of plerocercoids in fresh P. fluviatilis livers. Between 2 and 5 plerocercoids were detected on each infected fish liver (data not shown) and a single plerocercoid was dissected from each of the infected specimens for molecular conformation of the species. Plerocercoid samples were fixed in 96% ethanol and stored at – 20 ◦ C. DNA was extracted from whole plerocercoids using a Dneasy Tissue Kit (Qiagen) according to the manufacturer’ s instructions. The concentration of isolated DNA was measured with a NanoDrop ND-2000 spectrophotometer (Thermo Scientific). Amplification of the rrnL ribosomal subunit gene ( Brabec et al., 2015) was performed in 10 μL reactions to confirm the occurrence of T. nodulosus . Each 10 μL polymerase chain reaction (PCR) consisted of 5 μL of 2 x Typeit Buffer (Qiagen); 0.5 μL of each primer (500 nM); 2 μL of DNA (100–150 ng total) and 2 μL of nuclease-free water. The primer sequences used for rrnL amplification were following: Cest16Sfgen (5′-TRCCTTTTGCATCATG-3′) and Cest_16SRc (5′- AATAGATAAGAAC CGACCTGGC-3′) ( Scholz et al., 2013). The thermocycler amplification protocol consisted of initial denaturation at 95 ◦ C for 15 min, followed by 40 cycles of 30 s at 94 ◦ C, 30 s at 54 ◦ C and 90 s at 72 ◦ C; the final extension at 72 ◦ C was 10 min. Sequencing of PCR product using Sanger method was performed at the Institute of Genomics Core Facility, University of Tartu ( Estonia) from both directions, and the 22 sequences (forward and reverse) were successfully merged manually. BLAST analysis of 11 consensus sequences indicated that the infecting worms were T. nodulosus (GenBank ID: KR780832.1, highest sequence similarity: 97%). Sequences produced in this study can be accessed at GenBank (OR065063-OR065071).

2.3. RNA extraction

Frozen liver tissue samples were mechanically crushed in liquid nitrogen using a steel mortar and pestle to produce a homogenized pow- der, while frozen spleen tissue was mechanically crushed using a Retsch Mixer Mill MM 400 (Retsch) (the tissue consistency of spleen samples was not conducive to mortar and pestle homogenization). Total RNA was extracted using a NucleoSpin RNA extraction kit (MACHEREY- NAGEL, Duren, Germany). RNA sample concentrations were measured with a NanoDrop 2000 (ThermoFisher), and sample quality was evaluated using a TapeStation 2200 (Agilent Technologies).

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