Primers used in the experiments.

<div><p>Holometabolous insects undergo morphological remodeling from larvae to pupae and to adults with typical changes in the cuticle; however, the mechanism is unclear. Using the lepidopteran agricultural insect <i>Helicoverpa armigera</i>, cotton bollworm, as a model, we revealed that the transcription factor RUNT-like (encoded by <i>Runt-like</i>) regulates the development of the pupal cuticle via promoting a pupal cuticle protein gene (<i>HaPcp</i>) expression. The <i>HaPcp</i> was highly expressed in the epidermis and wing during metamorphosis and was found being involved in pupal cuticle development by RNA interference (RNAi) analysis in larvae. <i>Runt-like</i> was also strongly upregulated in the epidermis and wing during metamorphosis. Knockdown of <i>Runt-like</i> produced similar phenomena, a failure of abdomen yellow envelope and wing formation, to those following <i>HaPcp</i> knockdown. The insect molting hormone 20-hydroxyecdysonen (20E) upregulated <i>HaPcp</i> transcription via RUNT-like. 20E upregulated <i>Runt-like</i> transcription via nuclear receptor EcR and the transcription factor FOXO. Together, RUNT-like and HaPCP are involved in pupal cuticle development during metamorphosis under 20E regulation.</p></div>