Transcriptomic landscape and chromatin accessibility uncover pivotal regulators driving programmed larval-larval molting in the domesticated silkworm.

Insects undergo periodic ecdysis to shed their old chitinous exoskeleton and form a new cuticular layer. The steroid hormone 20-hydroxyecdysone (20E) is widely recognized as a central regulator of insect molting. Acting as a signaling molecule, 20E pulses orchestrate gene expression in a concentration- and time-dependent fashion. However, investigations into the transcriptomic and epigenomic alterations linked to dynamic 20E fluctuations remain limited. In this study, we explored the temporal dynamics of epidermal transcriptomes and genome-wide chromatin accessibility during the larval-larval molting cycle of the silkworm, Bombyx mori. Our results unveiled pronounced shifts in gene expression and chromatin architecture between early and late molting stages, correlating with ascending and descending 20E titers, respectively. Chromatin footprint analysis identified the Ecdysone receptor (EcR) and Grainy head (GRH) as early-stage regulators. Strikingly, during late molting phases, we uncovered a novel regulatory axis involving CCAAT/enhancer-binding protein (C/EBP) alongside the established factor Fushi-tarazu f1 (βFTZ-F1). Moreover, decline of the 20E titer triggers the expression of C/EBP, which subsequently regulates βFtz-f1 expression through promoter binding. Furthermore, epidermal-specific knockout of C/EBP and βFtz-f1 genes led to dysregulation of cuticular protein and chitin biosynthesis genes, impairing new cuticle formation. Collectively, our multi-omics dissection illuminates the dynamic regulatory circuitry coordinating epidermal remodeling and establishes a hierarchical transcriptional cascade governing cuticular renewal. These findings advance our understanding of hormone-driven developmental transitions in insects.