Phase-Resolved Pupal Ecdysis Behavior and Requirement of Ecdysis-Triggering Hormone in Henosepilachna vigintioctomaculata.

In holometabolous insects, the pupal stage enables the transition from larva to adult and provides a useful system for studying the organization and evolution of ecdysis behavior. However, the motor programs underlying the formation of exarate pupae remain less well characterized than those of obtect and coarctate pupae. Here, we describe the pupal ecdysis sequence in Henosepilachna vigintioctomaculata at phase-resolved behavioral resolution. Pupal ecdysis was subdivided into pre-ecdysis, ecdysis, and post-ecdysis. During pre-ecdysis, prepupae exhibited slight dorso-ventral (D-V) contractions followed by progressively strengthened twitch contractions that loosened the old exoskeleton. During ecdysis, successive anteriorly directed peristaltic posterior-anterior (P-A) contractions, together with D-V contractions and lateral swinging, drove exuvial shedding and the freeing and stretching of pupal wings and cephalothoracic appendages. During post-ecdysis, posteriorly directed anterior-posterior (A-P) and D-V movements restored stretched appendages to their normal sizes and positions. RNA interference against the ecdysis-triggering hormone gene Hveth, delivered by microinjection of in vitro synthesized dsRNA or by oral administration of bacterially expressed dsRNA, impaired pupation. Knockdown of Hveth prolonged pre-ecdysis, reduced twitch frequency, and partially or completely disrupted ecdysis and post-ecdysis movements. Together, these results define the pupal ecdysis sequence of an exarate coleopteran pupa and support a requirement for ETH signaling in the proper execution and coordination of this motor program.