Yellow-b, -c, -d, and -h are required for normal body coloration of Henosepilachna vigintioctopunctata.

The involvement of yellow genes y-b, y-c, y-e, and y-h in cuticle tanning has poorly been clarified. In the present paper, six putative yellow (y-y, y-b, y-c, y-e y-f, and y-h) genes were identified in Henosepilachna vigintioctopunctata. Hvy-b, Hvy-c, Hvy-e, and Hvy-h were abundantly transcribed at early larval and late pupal stages, especially in the epidermis. Accordingly, RNA interference (RNAi) experiments were performed by an injection of dsy-b, dsy-c, dsy-e, or dsy-h into the second instar larvae and 1-day-old pupae. The head capsule, scoli and strumae, and legs in the fourth-instar larvae became blacker; the blackish spots in the pupae were darkened and widened after RNAi of Hvy-b, compared with those of dsegfp-treated controls. Depletion of Hvy-b at the 1-day-old pupal stage expanded two pair of black markings on the sternum of the metathorax, and darkened the black patched on the sterna of the abdomen segments I-VI in the resultant adults. Depletion of Hvy-e caused darker pigmented adult body and elytral cuticles than those of dsegfp-introduced controls. However, there was no obvious difference in pigmentation of the black markings. Hvy-h-deficient larvae displayed dark yellow body color, whereas the body color of the dsegfp-injected control was pale yellow. There was no obvious difference in coloration of larval specific-black markings or pupal cuticle between dsHvy-h- and dsegfp-treated animals. Moreover, silence of Hvy-c at the second instar larval stage lightened black markings in the resulting larvae and pupae, but had no influence on pale yellow body color. Our results demonstrated their different roles of the four yellow genes during body pigmentation: HvY-b and HvY-c, respectively, inhibit and facilitate the coloration within dark markings, whereas HvY-e and HvY-h, respectively, repress the pigmentation in adult and larval body cuticles outside the black patches in H. vigintioctopunctata.