Ecdysis Triggering Hormone (ETH) Is Vital for Pupation in Henosepilachna vigintioctomaculata

The initiation of pupal ecdysis behavior by ecdysis-triggering hormone (ETH) has been established in two Lepidopterans and two Dipterans. In Coleoptera Leptinotarsa decemlineata, RNA interference (RNAi) targeting eth impairs pupation. However, whether the RNAi phenotypes result from defects in organ formation or disruption of pupal ecdysis behavior has not been established. Here, we characterized ETH physiological functions during pupation in a potato defoliating ladybird, Henosepilachna vigintioctomaculata. Hveth levels were high just before or right after each molting, it was highly expressed in the trachea-rich tissues, such as dissected epidermis, fat body, and head. RNAi against Hveth impaired pupation of more than 80% of the resultant larvae. They were partially enveloped by the old larval cuticle until death. HE stained slices displayed that new cuticles were well formed under the old ones at the head, pleuron, and pronotum of prothorax. Moreover, adult organs such as compound eyes, mouthparts, and legs were well developed in both the control and the Hveth RNAi groups. Conversely, the freezing sections revealed that no clear separation between the old and the newly formed cuticles in the Hveth RNAi head, in contrast to the control head that showed a visible gap. In addition, the pupal cuticle under the old larval integument was not pigmented. Our findings suggest that ETH initiates pupal ecdysis behavior to shed off old larval exoskeleton and activates pigmentation of pupal cuticle during pupation in H. vigintioctomaculata.