Comprehensive reverse genetic analysis of core clock genes underlying the eclosion rhythm in the silkworm, Bombyx mori

In insects, adult eclosion represents a classic circadian-regulated event, yet the roles of core clock genes in lepidopteran eclosion rhythms remain incompletely defined. In the silkworm Bombyx mori, previous studies have analyzed individual clock gene knockouts across different genetic backgrounds, which has hindered an integrated understanding of their roles in eclosion rhythm. Here, we systematically examined the roles of core clock genes in eclosion rhythm using knockout strains of period (per), timeless (tim), Clock (Clk) and cycle (cyc) generated in a single genetic background, the p50T strain. Under constant darkness, knockout of any core clock gene abolished endogenous eclosion rhythm, demonstrating a conserved role of these genes for rhythmic eclosion. In contrast, under light–dark conditions, distinct eclosion patterns emerged between knockouts of negative elements (per and tim) and positive elements (Clk and cyc), particularly with respect to their responses to lights-on. These results indicate that although core clock genes are universally required for eclosion rhythm in B. mori, the manifestation of knockout phenotypes depends on clock gene function and genetic background. Together, our findings provide a unified framework for understanding circadian regulation of eclosion in lepidopteran insects.