Functional divergence of white genes in Henosepilachna vigintioctopunctata revealed by RNA interference

Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae in many Asian countries. RNA interference (RNAi) can effectively reduce transcript abundance in this beetle, offering opportunities to explore the biological function of specific genes. The white gene encodes a half-type ATP-binding cassette transporter that plays an essential role in tryptophan, guanine and uric acid transport across membranes. Mutations that disrupt the function of white are known to cause eye pigmentation phenotypes in many insect species. Here, we found evidence for five white gene paralogues present in H. vigintioctopunctata transcriptome datasets sequenced from a range of developmental stages. We individually knocked down each of the five white genes through the injection of corresponding double-stranded RNAs (dsRNAs) to the fourth-instar larvae to determine whether functional divergence has occurred. We found that injecting 1 μg dswhite3 caused compound eye colour of pupae and adults to develop as red/brown and brown, respectively, compared with black eyes in control beetles. Injection of 2 μg dswhite3 increased RNAi efficacy and produced a clearer eye colour phenotype. At both doses, the ocular diaphragm (a ring of black pigment surrounding each eye) did not change in the white3 RNAi hypomorphs. Moreover, our data revealed that injection of dswhite2 at the fourth-instar larval stage impaired the climbing ability of both male and female adults. Our results confirmed, for the first time, functional divergence of duplicated white genes in an insect species.