Knockdown of BmorCPR67 gene disrupts prepupal–pupal transition of silkworm Bombyx mori by thinning the endocuticle

The cuticle of insects serves as a crucial organ for preserving body composition, protecting against pathogen invasion, and retaining moisture in their bodies. Cuticular proteins (CPs) are the main constituents of insect cuticles and interact with chitin to form the cuticle's structural framework and mechanical properties. In this study, we investigated the role of a cuticular protein with R&R consensus (CPR), BmorCPR67, a member of the RR-2 subfamily, during the prepupal-to-pupal transition in Bombyx mori. The BmorCPR67 gene exhibited high expression levels during the prepupal stage, with the highest expression detected in the epidermis of the day-1 pupa in B. mori. The expression of the BmorCPR67 gene was induced by 20-hydroxyecdysone (20E). Chitin-binding assays indicated that the BmorCPR67 protein selectively binds to crystalline chitin and chitosan but not to amorphous chitin. Silencing the BmorCPR67 gene disrupted the moulting process from prepupa to pupa, resulting in silkworm mortality. Furthermore, the knockdown of BmorCPR67 altered the expression profiles of key genes involved in chitin metabolism. Notably, significant thinning of the endocuticle was observed 48-96 h after siRNA injection in BmorCPR67-silenced silkworms. These findings highlight the critical role of BmorCPR67 in cuticle development during the prepupal-to-pupal transition in B. mori, contributing to our understanding of the functions of CPs in insect metamorphosis.