In Situ Hybridization of Monogenean Genes

EnCL1/EnCL3 gene-specific primers (Additional file 3) and first-strand cDNA of adult E. nipponicum were used for the PCR. The amplified products were ligated into pGEM®-T Easy vector (Promega, Madison, Wisconsin) and verified by DNA sequencing. The constructs were linearised and used as a template for RNA probes. Both sense and anti-sense RNA probes were synthesised in vitro (Dig RNA Labelling Kit (SP6/T7); Roche, Basel, Switzerland). In situ hybridisation was performed using a modified protocol [44 (link)]. Briefly, the sections of adult worms on slides were incubated for 19 h at 41 °C (EnCL1) and at 37 °C (EnCL3) with specific RNA probes diluted 1:100 in a hybridisation mixture (5× SSC, 1× PBS, 50% deionised formamide, 1% Tween-20, 10% dextran sulphate Mw 500×, 1 mg/ml Torula yeast RNA). Detection was achieved with alkaline phosphatase-conjugated anti-digoxigenin antibodies (1:500, Roche) and Fast Red TR substrate (Sigma-Aldrich). Negative controls were incubated under the same conditions but with an anti-sense probe or without a probe. Specific strand RT-PCR was used for the detection of natural anti-sense transcripts occurring in the monogenean cells [45 (link)].