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

View Article: PubMed Central - PubMed

ABSTRACT

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’cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb’cry2RNAi 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.

No MeSH data available.


Distribution of free-running periods of locomotor rhythms of individual crickets Gryllus bimaculatus. Crickets were treated with DsRed2RNAi, Gb’cry1RNAi (dsGb’cry1#d1, dsGb’cry1#d2), Gb’cry2RNAi (dsGb’cry2#d1, dsGb’cry2#d2), or both Gb’cry1RNAi and Gb’cry2RNAi and their locomotor rhythms were measured under DD at a constant temperature of 25 °C. Blue and red dots indicate individual and average values, respectively. Gb’cry1RNAi crickets showed a similar distribution to that of control, DsRed2RNAi crickets. The distribution is widely ranging from 23.2 to 25.0 h in Gb’cry2RNAi crickets, and the range is even greater in Gb’cry1/Gb’cry2 double RNAi crickets. For further explanations, see the text
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Fig4: Distribution of free-running periods of locomotor rhythms of individual crickets Gryllus bimaculatus. Crickets were treated with DsRed2RNAi, Gb’cry1RNAi (dsGb’cry1#d1, dsGb’cry1#d2), Gb’cry2RNAi (dsGb’cry2#d1, dsGb’cry2#d2), or both Gb’cry1RNAi and Gb’cry2RNAi and their locomotor rhythms were measured under DD at a constant temperature of 25 °C. Blue and red dots indicate individual and average values, respectively. Gb’cry1RNAi crickets showed a similar distribution to that of control, DsRed2RNAi crickets. The distribution is widely ranging from 23.2 to 25.0 h in Gb’cry2RNAi crickets, and the range is even greater in Gb’cry1/Gb’cry2 double RNAi crickets. For further explanations, see the text

Mentions: To examine the role of Gb’cry1 and Gb’cry2 in circadian locomotor rhythm regulation, locomotor activity was recorded in adult males injected with dsGb’cry1 (dsGb’cry1#d1, N = 19; dsGb’cry1#d2, N = 22) or dsGb’cry2 (dsGb’cry2#d1, N = 37; dsGb’cry2#d2, N = 22). Since similar results were obtained in crickets treated with two different dsRNAs for Gb’cry1 and Gb’cry2, the results were pooled. We used DsRed2RNAi crickets (n = 21) as a negative control. As was the case for DsRed2RNAi crickets, all of the Gb’cry1RNAi and the Gb’cry2RNAi crickets exhibited a nocturnal activity rhythm under LD12:12, with a major peak at lights-off and a minor peak at lights-on (Fig. 3). In the ensuing constant darkness (DD), the rhythm free-ran, except in 4 Gb’cry2RNAi crickets which became arrhythmic, and the free-running periods varied with treatments (Figs. 3 and 4). The Gb’cry1RNAi (n = 16) and DsRed2RNAi (n = 15) crickets showed free-running periods shorter than 24 h, averaging 23.58 ± 0.25 h and 23.71 ± 0.24 h, respectively. However, the free-running period varied widely in Gb’cry2RNAi crickets (n = 33): the average period was 24.12 ± 0.34 h ranging from 23.2 h to 25.0 h. When treated doubly with dsGb’cry1 and dsGb’cry2, the crickets were all rhythmic but showed a wider range of free-running period (22.4–25.8 h) than Gb’cry2RNAi crickets. Notably, some of the Gb’cry2RNAi crickets (dsGb’cry2#d1, n = 3; dsGb’cry2#d2, n = 2) showed a rhythm that split into two components in the free-running condition (as exemplified in Fig. 3d) and that individuals with the free-running periods longer than 24 h often became arrhythmic (dsGb’cry2#d1, N = 4) or only weakly rhythmic (dsGb’cry2#d1, n = 8; dsGb’cry2#d2, n = 10) in due course (as exemplified in Fig. 3d, j).Fig. 3


cryptochrome genes form an oscillatory loop independent of the per / tim loop in the circadian clockwork of the cricket Gryllus bimaculatus
Distribution of free-running periods of locomotor rhythms of individual crickets Gryllus bimaculatus. Crickets were treated with DsRed2RNAi, Gb’cry1RNAi (dsGb’cry1#d1, dsGb’cry1#d2), Gb’cry2RNAi (dsGb’cry2#d1, dsGb’cry2#d2), or both Gb’cry1RNAi and Gb’cry2RNAi and their locomotor rhythms were measured under DD at a constant temperature of 25 °C. Blue and red dots indicate individual and average values, respectively. Gb’cry1RNAi crickets showed a similar distribution to that of control, DsRed2RNAi crickets. The distribution is widely ranging from 23.2 to 25.0 h in Gb’cry2RNAi crickets, and the range is even greater in Gb’cry1/Gb’cry2 double RNAi crickets. For further explanations, see the text
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Fig4: Distribution of free-running periods of locomotor rhythms of individual crickets Gryllus bimaculatus. Crickets were treated with DsRed2RNAi, Gb’cry1RNAi (dsGb’cry1#d1, dsGb’cry1#d2), Gb’cry2RNAi (dsGb’cry2#d1, dsGb’cry2#d2), or both Gb’cry1RNAi and Gb’cry2RNAi and their locomotor rhythms were measured under DD at a constant temperature of 25 °C. Blue and red dots indicate individual and average values, respectively. Gb’cry1RNAi crickets showed a similar distribution to that of control, DsRed2RNAi crickets. The distribution is widely ranging from 23.2 to 25.0 h in Gb’cry2RNAi crickets, and the range is even greater in Gb’cry1/Gb’cry2 double RNAi crickets. For further explanations, see the text
Mentions: To examine the role of Gb’cry1 and Gb’cry2 in circadian locomotor rhythm regulation, locomotor activity was recorded in adult males injected with dsGb’cry1 (dsGb’cry1#d1, N = 19; dsGb’cry1#d2, N = 22) or dsGb’cry2 (dsGb’cry2#d1, N = 37; dsGb’cry2#d2, N = 22). Since similar results were obtained in crickets treated with two different dsRNAs for Gb’cry1 and Gb’cry2, the results were pooled. We used DsRed2RNAi crickets (n = 21) as a negative control. As was the case for DsRed2RNAi crickets, all of the Gb’cry1RNAi and the Gb’cry2RNAi crickets exhibited a nocturnal activity rhythm under LD12:12, with a major peak at lights-off and a minor peak at lights-on (Fig. 3). In the ensuing constant darkness (DD), the rhythm free-ran, except in 4 Gb’cry2RNAi crickets which became arrhythmic, and the free-running periods varied with treatments (Figs. 3 and 4). The Gb’cry1RNAi (n = 16) and DsRed2RNAi (n = 15) crickets showed free-running periods shorter than 24 h, averaging 23.58 ± 0.25 h and 23.71 ± 0.24 h, respectively. However, the free-running period varied widely in Gb’cry2RNAi crickets (n = 33): the average period was 24.12 ± 0.34 h ranging from 23.2 h to 25.0 h. When treated doubly with dsGb’cry1 and dsGb’cry2, the crickets were all rhythmic but showed a wider range of free-running period (22.4–25.8 h) than Gb’cry2RNAi crickets. Notably, some of the Gb’cry2RNAi crickets (dsGb’cry2#d1, n = 3; dsGb’cry2#d2, n = 2) showed a rhythm that split into two components in the free-running condition (as exemplified in Fig. 3d) and that individuals with the free-running periods longer than 24 h often became arrhythmic (dsGb’cry2#d1, N = 4) or only weakly rhythmic (dsGb’cry2#d1, n = 8; dsGb’cry2#d2, n = 10) in due course (as exemplified in Fig. 3d, j).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

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’cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb’cry2RNAi 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.

No MeSH data available.