Timeless-BmaaNAT axis regulates temperature-dependent diapause plasticity in the silkworm, Bombyx mori

The involvement of circadian clock genes in diapause induction has been demonstrated across various insect species, yet the downstream effector molecules mediating this process remain largely uncharacterized. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is regulated through the perception of temperature and photoperiod cues perceived by circadian clock genes during a critical environmental-sensitive window of maternal embryogenesis. In this study, we used a knockout mutant (Tim^-/-) of Timeless, a core circadian clock gene, which disrupts diapause induction, as a model system. Employing label-free quantitative (LFQ) proteomics on embryonic heads from wild-type Dazao eggs incubated at 25°C (diapause) and 15 °C (non-diapause), alongside Tim^-/- embryos, we identified differentially expressed proteins (DEPs) associated with diapause fate determination. Comparative analysis revealed significant downregulation of BmaaNAT (B. mori arylalkylamine N-acetyltransferase) in destined diapause egg producers compared to destined non-diapause egg producers. Dual luciferase assays confirmed direct transcriptional activation of BmaaNAT by the CLOCK/CYCLE (CLK/CYC) heterodimer binding to the fourth E-box element of its promoter. Functional validation demonstrated that BmaaNAT knockout shortened larval duration and disturbed diapause occurrence, while overexpression extended larval development, and partially restored diapause capacity in Tim^-/- mutants. This study establishes BmaaNAT as a critical effector linking circadian clock outputs to diapause regulation, uncovering a novel molecular mechanism underlying seasonal adaptation in insects.