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 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.
摘要：
大多数昆虫进入滞育期，生理休眠状态对于持久的严酷季节至关重要，光周期作为其诱导的主要线索，确保过程的适当季节性时机。尽管通过敲除或敲除时钟基因已经证明了昼夜节律时钟在光周期时间测量中的参与，时钟基因隐色素1(cry1)的参与，它作为光感受器，涉及各种昆虫物种的昼夜节律时钟的光夹带，尚不清楚。在家蚕的双伏特菌株中，家蚕,胚胎滞育受母蛾在胚胎和幼虫阶段经历的环境条件的控制和影响。先前的研究强调了核心时钟基因的作用，包括期间(每)，永恒（tim），时钟(Clk)和周期(cyc),在B.mori的光周期滞育诱导中。在这项研究中,我们重点研究了cry1基因在B.mori光周期中的参与。系统发育分析和保守域鉴定证实了果蝇型cry（cry1）和哺乳动物型cry（cry2）基因在B.mori基因组中的存在，类似于其他鳞翅目。时间表达分析显示，在光相期间cry1基因表达较高，而在阴相期期间表达较低。核心时钟基因敲除(每，Tim,Clk和cyc）破坏了这种时间表达模式。使用CRISPR/Cas9介导的基因组编辑，我们在p50T中建立了cry1敲除菌株，在胚胎和幼虫阶段都表现出清晰的光周期的双伏特菌株。尽管野生型菌株在持续的黑暗中表现出昼夜节律，cry1敲除应变表现出心律失常性羽化，将B.moricry1包含在控制行为节奏的昼夜节律时钟反馈回路中。cry1敲除品系的雌性在胚胎和幼虫阶段均未能控制光周期滞育诱导，反映了在持续黑暗中饲养的野生型个体的滞育表型，表明B.moriCRY1作为光感受器有助于光周期时间测量。此外，在cry1/tim双敲除菌株中，幼虫期的光周期滞育诱导被废除，表明CRY1接收到的光信息被中继到昼夜节律时钟。总的来说,这项研究代表了cry1参与昆虫光周期的第一个证据，特别是在滞育诱导中。
