Figure 4

Expression of mimicry candidate genes in three different S. solidus developmental stages confirmed by retrotranscription polymerase chain reaction (RT-PCR). RT-PCR was performed on 17 different S. solidus worms from three life stages (non-infective, infective and post-reproduction adults) to confirm that the four best mimicry candidate genes, i.e. secreted proteins, are expressed in at least one developmental stage of the parasite. A) Gel electrophoresis for each gene, wnt4 (WNT4), zinc transporter (ZIP12), lysyl oxidase (LOXL2B) and palmitoyltransferase (ZDHCC17). Wells 1–7 contain cDNA from non-infective worms (mass < 50 mg), wells 8–14 contain cDNA from infective worms (mass > 100 mg) and wells 15–17 contain cDNA from adult worms (mass > 350 mg, after egg production outside the fish). Well 18 = positive control with elongation factor 1 alpha (EF1-α), a gene commonly expressed in all tissues and developmental stages. Well 19 = negative control (no template). Well 20 = negative control (no primers). B) Additional controls to confirm that our positive control gene (EF1-α) is expressed in all worms and all life stages and to confirm that RNA samples used to perform the RT-PCR reactions are DNA-contamination free. Positive control: non-infective stage (wells 1–7), infective stage (wells 8–14), adult stage (wells 15–17) and controls (well 18 = no template, well 19 = no primers). Negative control: wells 1–7 (RNA samples from all three life stages, i.e. two non-infective, two infective and one adult worms respectively), wells 6–7 (positive controls with cDNA from one infective worm and one non-infective worm respectively), wells 8–9 (negative control, no template and no primers respectively).