Transcriptome analysis and RNAi knockdown highlight the complexity of larval epidermis pigmentation in silkworm, Bombyx mori

The color patterns of Lepidoptera larvae play crucial roles in mimicry and predator deterrence, serving as key adaptive factors. They also provide valuable molecular markers for research on evolution and development. Melanin is one of the main pigments contributing to the pigmentation in insect color patterns, and its synthesis is regulated by various spatial and temporal factors. While the pathway of melanogenesis is clearly explained, the regulatory mechanism of complex pattern formation remains incompletely understood. In this study, we performed an association analysis between the color patterns and regulatory factors of the melanogenesis pathway in silkworm larvae with different color patterns by using transcriptomic data. We found that RNAi of the serine protease resulted in lighter pigmentation of normal markings. Additionally, Phenol oxidase expression was downregulated in the non-pigmented strain, suggesting that the Serine protease–Phenol oxidase pathway mediates the formation of the normal markings. Furthermore, we identified the key roles of unreported RR-2 family cuticular proteins and PTB domain proteins in epidermal pigmentation, and RNAi of RR-2 cuticular protein also leads to a pigmented reduction of the markings. Our results provide important insights into the key molecular events involved in epidermal pigmentation in Lepidoptera larvae and offer valuable resource data for deep analysis on regulatory mechanisms of color pattern formation.