Knockouts of positive and negative elements of the circadian clock disrupt photoperiodic diapause induction in the silkworm,Bombyx mori

Diapause is one of the most important traits that have sustained insects to thrive. Insects can prospectively arrest their development and increase environmental stress-resistance by entering diapause. The photoperiod is the signal that indicates insects the proper timing to enter diapause. Circadian clock genes are shown to be involved in photoperiodic diapause induction in various insect species. The silkworm, Bombyx mori , enters diapause at the embryonic stage. In bivoltine strains, diapause determination is affected by embryonic temperature and photoperiodic conditions during embryonic and larval stages. Two independent studies showed that knocking out the core clock gene, period , perturb photoperiodic diapause induction. However, whether the circadian clock as whole or individual clock genes are responsible for the photoperiodic diapause induction remains unknown. In this study, using CRISPR/Cas9 we knocked out negative ( period and timeless ) and positive elements ( Clock and cycle ) in p50T, a bivoltine strain which exhibits photoperiodic diapause induction during both embryonic and larval stages. The temporal expression patterns of clock genes changed in each core clock gene knockout strain, suggesting disruption of normal feedback loops produced by circadian clock genes. Furthermore, photoperiodic diapause induction during both embryonic and larval stages was lost in all knockout strains. Our results indicate the involvement of circadian clock in photoperiodic diapause induction in B. mori . Highlights We established knockout Bombyx mori strains of four core clock genes The temporal expression patterns of clock genes changed in knockout strains Photoperiodic diapause induction was not observed in any knockout strains Graphical abstract