Experimental feeding scheme and quantitation of actin expression levels after RNAi treatment in Tethya wilhelma. A. After pulse feeding with induced HT115(DE3)/L4440-Tw-actb or non-induced controls for 4.5 hours and subsequent wash out by fresh ASW, sponges were cultivated in ASW for 19.5 hours. Every day, after the pulse feed, some of the sponges were separated out, cultivated for 5 hours, and washed in seawater until they were harvested for RNA preparation and fixation. B - C. qRT-PCR assay for gene expression levels of Tw-actb (purple) after dsRNA treatment with induced HT115(DE3)/L4440-Tw-actb. Levels were normalized to RPS5 and gene expression levels are given as relative levels of RNAi-treated sponges against control sponges . B. Short-term actin knockdown: relative actin-mRNA levels in T. wilhelma specimens sampled at 33 hours (N = 3) and 57 hours (N = 3) after initial pulse-feeding. Bars represent standard deviation (N = 3). C. Independent long-term actin knockdown experiment: actin expression in single T. wilhelma specimens after daily 4.5 hour pulse-feeding with dsRNA-expressing HT115(DE3)/L4440-Tw-ctb E. Coli over 8 days. Levels of single specimens were normalized as above (purple; each bar represents a single specimen); average relative actin expression (green; with N = 5).