Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

Chlorfenapyr is a widely used insecticide known to harm non-target insects, but its effects on reproductive development in the silkworm (Bombyx mori L.) remain incompletely understood. In this study, we investigated the histopathological and transcriptional changes in the gonads (ovaries and testes) of newly molted fifth-instar silkworm larvae exposed to chlorfenapyr. Histopathological analysis revealed delayed gonadal development, a reduction in oogonia and oocytes in the ovaries, and decreased numbers of spermatocytes in the testes. Transcriptome analysis identified significant differentially expressed genes (DEGs), mainly enriched in pathways such as “Drug metabolism—cytochrome P450”, “Insect hormone biosynthesis”, and “Ribosome”. Key up-regulated genes included members of the cytochrome P450 family (CYP6B5, CYP9f2, CYP6B6), glutathione S-transferases (GSTT1, GST1), and juvenile hormone-related enzymes (JHAMT, JHEH), indicating active detoxification and hormonal regulation responses. Several transcription factor families, particularly C2H2, HB-other, and TRAF, exhibited altered expression, suggesting roles in stress adaptation. Protein–protein interaction (PPI) network analysis identified hub genes such as EcR, Kr-h1, and various ribosomal proteins, highlighting their potential involvement in reproductive development. Quantitative PCR (qPCR) validated the transcriptomic data, confirming the reliability of the results. Overall, these findings enhance our understanding of chlorfenapyr’s impact on silkworm reproductive development and the underlying molecular mechanisms, providing valuable insights for sustainable pest management and ecological risk assessment of insecticides.