Fig. 3

RNA interference (RNAi) of calmodulin (FhCaM) transcripts in juvenile Fasciola hepatica, as measured by relative quantitative PCR (qPCR). Juvenile fluke exposed to 50 ng/μl short interfering (si)RNA (27 nt) or 100 ng/μl long (~200 nt) double stranded (ds)RNA for 4 h were maintained for a further 68 h (a–c) or 7 (d), 14 (e) or 21 days (f) before analysis of transcript abundance by qPCR. Over a 72 h time course, impacts of individual CaM dsRNA, a cocktail of FhCaM1-3 dsRNAs, and siRNAs were tested for impact on abundance of FhCaM1 (a), FhCaM2 (b) and FhCaM3 (c) transcripts, alongside negative control treatments (dsCTRL and siCTRL). Effective combinatorial dsRNA treatments were then tested over longer timeframes (d–f). Data represent mean ± SEM of percentage changes in target transcript abundance relative to a GAPDH reference transcript, normalised against the abundance of those transcripts in an untreated control group [27]. Each bar represents data from at least three RNAi treatment replicates, 20 worms per replicate. Statistical analyses were performed using One Way ANOVA with Dunnett’s post hoc test, or Student’s t-test (siRNA vs dsRNA comparisons in b, c). *, P < 0.05; **, P < 0.01; ***, P < 0.001