cryptochrome genes form an oscillatory loop independent of the per/tim loop in the circadian clockwork of the cricket Gryllus bimaculatus

Background: Animals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood. Results: In the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb ' cry1) and mammalian-type cry2 (Gb'cry2). Gb ' cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb ' cry1(RNAi) treatment had only a limited effect at the behavioral and molecular levels, while Gb ' cry2(RNAi) had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb'cry1RNAi. In Gb ' cry1/ Gb ' cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb ' cry1 and Gb ' cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells. Conclusion: Based on these results, we propose a new model of the cricket's circadian clock, including a molecular oscillatory loop for Gb'cry2, which can operate independent of the Gb ' per/Gb ' tim loop.