Crustacean cardioactive peptide activates pupal ecdysial behavior in the 28-spotted larger potato ladybird

During larval development, pupation, and adult eclosion, holometabolous insects must shed the old exoskeleton and expand, harden, and pigment the newly formed cuticle in a process termed ecdysis. Although ecdysis is regulated by multiple neuropeptides, the specific role of crustacean cardioactive peptide (CCAP) in coleopteran pupal ecdysis remains incompletely understood. Here, we investigated the function of CCAP in pupal ecdysis behavior in the 28-spotted larger potato ladybird, Henosepilachna vigintioctomaculata. Injection of in vitro-synthesized double-stranded RNA targeting Hvccap (dsccap) or oral delivery of bacterially expressed dsccap at the final larval instar caused severe pupation failure by disrupting pupal ecdysis behavior. Hvccap RNAi prepupae showed only a slight reduction in twitching, whereas the ecdysis and postecdysis periods were markedly prolonged and the old larval cuticle was not shed. Despite this failure, the pupal cuticle formed beneath the retained larval exoskeleton still underwent tanning and pigmentation. These results demonstrate that CCAP is required for the proper execution of pupal ecdysis behavior in H. vigintioctomaculata and identify Hvccap as a potential molecular target for the management of this pest.