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

Abstract

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 precise molecular mechanisms in this context remain 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, includingperiod(per),timeless(tim),Clock(Clk) andcycle(cyc), in photoperiodic diapause induction inB. mori. In this study, we focused on another clock gene,cryptochrome 1(cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species. Phylogenetic analysis and conserved domain identification confirmed the presence of bothDrosophila-typecry(cry1) and mammalian-typecry(cry2) genes in theB. morigenome, akin to other lepidopterans. Temporal expression analysis revealed highercry1gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per,tim,Clkandcyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established acry1knockout 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, thecry1knockout strain exhibited arrhythmic eclosion, implicatingB. mori cry1in the circadian clock feedback loop governing behavior rhythms. Females of thecry1knockout strain failed to induce photoperiodic diapause during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating thatB. moriCRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in acry1/timdouble-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence ofcry1involvement in insect photoperiodism, specifically in diapause induction.