RR-1 cuticular protein TcCPR69 is required for growth and metamorphosis in Tribolium castaneum

Cuticle is not only critical for protecting insects from noxious stimuli but is also involved in a variety of metabolic activities. Cuticular proteins (CPs) affect cuticle structure and mechanical properties during insect growth, reproduction, and environmental adaptation. Here, we describe the identification and characterization of a member of the RR-1 subfamily of CPs with an R&R consensus (CPR) in Tribolium castaneum (TcCPR69). Although it was previously reported to be highly expressed in the wings, we found that knocking down TcCPR69 by RNA interference (RNAi) did not cause obvious wing abnormalities but markedly disrupted the growth and metamorphosis of beetles with 100% cumulative mortality; additionally, the chitin content of the pharate adult was decreased and the new abdominal cuticle was significantly thinner before molting. TcCPR69 showed chitin-binding ability and the expression levels of key genes involved in chitin metabolism (trehalase [TcTRE], chitin synthase [TcCHSA and TcCHSB], and chitinase [TcCHT5 and TcCHT10]) were also decreased by TcCPR69 knockdown. TcCPR69 gene expression peaked shortly after molting and was increased 2.61 fold at 12 h after 20-hydroxyecdysone (20E) injection. This was reversed by RNAi of the ecdysone-related genes ecdysone receptor (TcECR) and fushi tarazu transcription factor 1 (TcFTZ-F1). These results indicate that TcCPR69 is positively regulated by 20E signaling to contribute to cuticle formation and maintain chitin accumulation during the growth and metamorphosis of beetles.