Effect of dsRNA dose on feeding RNAi efficiency.

<p>(A) wild-type, (B) KA dsRNA, (C–F) <i>lac2</i> RNAi with various doses of dsRNA, (G–J) <i>ebony</i> RNAi with various doses of dsRNA. Note that feeding 500 ng/mL or more of dsRNA induced clear pigmentation defects both in <i>lac2</i> and <i>ebony</i> RNAi. (K, L) Reduction of mRNA by various doses of <i>lac2</i> dsRNA (K) or <i>ebony</i> dsRNA (L). The Y-axis indicates relative expression levels compared to the control (KA dsRNA). (M, N) quantification of larval head pigmentation by ImageJ. The Y-axis indicates the pigmentation index with 1 being the wild-type mean gray value. The lower the value, the darker the head pigmentation. (M) ImageJ analysis for <i>lac2</i> RNAi with various doses of dsRNA. Larvae fed with 5 µg/mL and 500 ng/mL of dsRNA had significantly higher pigmentation values compared to the KA dsRNA control larvae (P***<0.001, n = 10). For an unknown reason, the larvae fed with 5 ng/mL of dsRNA had a lower pigmentation value than that of control. (N) ImageJ analysis for <i>ebony</i> RNAi with various doses of dsRNA. All four doses of dsRNA had significantly lower pigmentation indices compared to the KA dsRNA control (P***<0.001, n = 10). Larvae fed with 5 µg/mL and 500 ng/mL of dsRNA had significantly lower pigmentation values compared to the larvae fed with 50 ng/mL of dsRNA (P***<0.001, n = 10).