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

Abstract

BackgroundAnimals 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.ResultsIn 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’cry1 RNAi. 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.ConclusionBased 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.

cryptochrome genes mediate photoperiodic responses in the cricket Modicogryllus siamensis

“…The present study shows that the cricket M. siamensis also has two cry genes: a Drosophila ‐type Ms'cry1 and a mammalian‐type Ms'cry2 . As reported for the cricket G. bimaculatus and the brown leafhopper N. lugens (Xu et al ., ; Tokuoka et al ., ), Ms'cry1 is rather constitutively and Ms'cry2 is rhythmically expressed (Fig. ).…”

“…Therefore, the most parsimonious hypothesis is that CRY1 mediates the photoperiodic responses by mediating the photic entrainment of the circadian clock. However, this is unlikely in M. siamensis because, in the cricket G. bimaculatus , cry s do not encode the circadian photoreceptor but instead play a role in circadian molecular oscillatory mechanism of the clock (Tokuoka et al ., ). This view is also supported by Ms'cry1 and Ms'cry2 double pRNAi crickets showing an adult emergence pattern and moult numbers similar to those of DsRed2 pRNAi control crickets under long‐day conditions (Figs A and A).…”

“…cry2 is suggested to be a component of the feedback loop of insect circadian clocks (Yuan et al, 2007), acting with PER to repress CLK/CYC transcriptional activity (Zhu et al, 2005(Zhu et al, , 2008. In the cricket G. bimaculatus, cry2 has several splicing variants and forms a feedback loop with a combination of cry2 variant product proteins or cry1 protein (Tokuoka et al, 2017). If a similar oscillatory system operates in M. siamensis, Ms'cry2 and Ms'cry1 would be expected to contribute differently to the photoperiodic time-measurement via their role in the circadian oscillatory mechanism.…”

“…Accordingly, in the present study, we examine the potential involvement of cry genes in the cricket's photoperiodic responses because CRYs are known to be involved in the light-dependent phase setting or core oscillatory mechanism of the circadian clock in insects (Stanewsky et al, 1998;Zhu et al, 2005Zhu et al, , 2008, as well as in photoperiodic responses in plants (Mockler et al, 2003). Most insects, including the cricket Gryllus bimaculatus (Tokuoka et al, 2017), possess two cry genes: cry1 and cry2. cry1 encodes the CRY1 protein that acts as a blue light photoreceptor to synchronize the clock with the light cycle in Drosophila cerebral clock neurones (Stanewsky et al, 1998).…”

“…cry1 encodes the CRY1 protein that acts as a blue light photoreceptor to synchronize the clock with the light cycle in Drosophila cerebral clock neurones (Stanewsky et al, 1998). CRY1 is also known to act as a transcriptional repressor in the circadian clock in the peripheral tissues of Drosophila (Krishnan et al, 2001;Levine et al, 2002;Collins et al, 2006) and probably in the central clock of G. bimaculatus (Tokuoka et al, 2017). On the other hand, CRY2 is involved in the central clock mechanism as a transcriptional repressor in the butterfly (Zhu et al, 2005), the honeybee (Rubin et al, 2006) and the cricket (Tokuoka et al, 2017).…”