Knockout of cryptochrome 1 disrupts circadian rhythm and photoperiodic diapause induction in the silkworm, Bombyx mori

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species, remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on the involvement of cry1 gene in B. mori photoperiodism. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction.