In Lepidoptera, the roles of period ( per) and the negative feedback involving this gene in circadian rhythm are controversial. In the present study, we established a per knockout strain using TALEN in Bombyx mori, and compared eclosion and hatching rhythms between the per-knockout and wild-type strains to examine whether per is actually involved in these rhythms. The generated per knockout allele was considered null, because it encoded an extensively truncated form of PERIOD (198 aa due to a 64-bp deletion in exon 7, in contrast to 1113 aa in the wild-type protein). In this per knockout strain, circadian rhythms in eclosion and hatching were disrupted. Under LD cycles, however, a steep peak existed at 1 h after lights-on in both eclosion and hatching, and was considered to be produced by a masking effect—a direct response to light. In the per-knockout strain, temporal expression changes of per and timeless ( tim) were also lost. The expression levels of tim were continuously high, probably due to the loss of negative feedback by per and tim. In contrast, the expression levels of per were much lower in the per knockout strain than in the wild type at every time point. From these results, we concluded that per is indispensable for circadian rhythms, and we suggest that the negative feedback loop of the circadian rhythm involving per functions for the production of behavioral rhythms in B. mori.

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The bivoltine strain of the domestic silkworm, Bombyx mori, exhibits a facultative diapause phenotype that is determined by maternal environmental conditions during embryonic and larval development. Although a recent study implicated a circadian clock gene period (per) in circadian rhythms and photoperiod-induced diapause, the roles of other core feedback loop genes, including timeless (tim), Clock (Clk), cycle (cyc), and cryptochrome2 (cry2), have to be clarified yet. Therefore, the aim of this study was to elucidate the roles of circadian clock genes in temperature-dependent diapause induction. To achieve this, per, tim, Clk, cyc, and cry2 knockout (KO) mutants were generated, and the percentages of diapause and non-diapause eggs were determined. The results show that per, tim, Clk, cyc, and cry2 regulated temperature-induced diapause by acting upstream of cerebral γ-aminobutyric acid (GABA)ergic and diapause hormone signaling pathways. Moreover, the temporal expression of the clock genes in wild-type (wt) silkworms was significantly different from that of thermosensitive transient receptor potential ankyrin 1 (TRPA1) KO mutants during embryonic development. Overall, the findings of this study provide target genes for regulating temperature-dependent diapause induction in silkworms.