RNAi‐based functional analysis of yellow‐e in Tribolium castaneum

Abstract

Pigmentation/melanization is an important physiological event in insect development and is involved in cuticle tanning, wound healing and encapsulation as a defensive response as well as in egg chorion hardening. “Yellow” protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway and significantly accelerates pigmentation and melanization reactions. Recent studies have suggested that yellow is a rapidly evolving gene family generating functionally diverse paralogs. However, the exact physiological functions of several yellow genes are still not understood. Tribolium castaneum (Tc) is an excellent experimental insect to determine the roles of individual yellow genes because it is possible to carry out RNA interference (RNAi) experiments by injecting gene‐specific double‐stranded RNAs (dsRNAs) into insects at all developmental stages.Injection of dsRNAs for Tcyellow‐e (TcY‐e) into last instar larvae had no effect on larval‐pupal molting, and the resulting pupae developed normally. The pupal cuticle including the setae, gin traps and urogomphi showed normal tanning. Adult cuticle could also be viewed through the translucent pupal cuticle. Adult cuticle tanning including the head, mandibles and legs was initiated on schedule (pupal day 4‐5), indicating that TcY‐e is not required for pupal or adult cuticle pigmentation in T. castaneum. The subsequent pupal‐adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (∼20%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became completely desiccated. Failure of the pupal‐adult molt and desiccation‐induced mortality were prevented by maintaining dsRNA TcY‐e‐treated insects at 100% relative humidity (rh). When the humidity‐rescued adults were subsequently moved from 100% rh to 50% rh, they quickly desiccated and died (wild‐type insects thrive throughout development at 50% rh). These results suggest that TcY‐e has a “critical waterproofing” function for cuticle rather than cuticle pigmentation.