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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.


Effects of Gb’cry1RNAi (a), Gb’cry2RNAi (b), and Gb’cry1RNAi/Gb’cry2RNAi (c) on daily clock gene expression. Relative abundance of Gb’per, Gb’tim, Gb’cry1, and Gb’cry2 mRNA in the optic lobes are shown for intact (blue) and Gb’cry1RNAi (yellow) (a), Gb’cry2RNAi (green) (b), and Gb’cry1RNAi/Gb’cry2RNAi (pink) adult male crickets (Gryllus bimaculatus) (c). The data for intact Gb’cry1 and Gb’cry2 mRNA are replotted from Fig. 2. In dsRNA-injected crickets, the optic lobes were collected about seven days after the dsRNA injection. The abundance of mRNA was measured by quantitative real-time RT-PCR with total RNA extracted from the optic lobes. The data collected from 3 and 5 independent experiments were averaged and plotted as mean ± SEM for dsRNA-injected and intact crickets, respectively. The abundance of Gb’rpl18a mRNA was used as an internal reference. Asterisks indicate significant difference between intact and treated crickets (*P < 0.05, **P < 0.01, t-test). Different letters indicate that values are significantly different with each other (ANOVA followed by Tukey-test, P < 0.05). For further explanations, see the text
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Fig5: Effects of Gb’cry1RNAi (a), Gb’cry2RNAi (b), and Gb’cry1RNAi/Gb’cry2RNAi (c) on daily clock gene expression. Relative abundance of Gb’per, Gb’tim, Gb’cry1, and Gb’cry2 mRNA in the optic lobes are shown for intact (blue) and Gb’cry1RNAi (yellow) (a), Gb’cry2RNAi (green) (b), and Gb’cry1RNAi/Gb’cry2RNAi (pink) adult male crickets (Gryllus bimaculatus) (c). The data for intact Gb’cry1 and Gb’cry2 mRNA are replotted from Fig. 2. In dsRNA-injected crickets, the optic lobes were collected about seven days after the dsRNA injection. The abundance of mRNA was measured by quantitative real-time RT-PCR with total RNA extracted from the optic lobes. The data collected from 3 and 5 independent experiments were averaged and plotted as mean ± SEM for dsRNA-injected and intact crickets, respectively. The abundance of Gb’rpl18a mRNA was used as an internal reference. Asterisks indicate significant difference between intact and treated crickets (*P < 0.05, **P < 0.01, t-test). Different letters indicate that values are significantly different with each other (ANOVA followed by Tukey-test, P < 0.05). For further explanations, see the text

Mentions: We then examined the effects of RNAi of Gb’cry1 and Gb’cry2 on Gb’per and Gb’tim genes, using dsGb’cry1#d1 and dsGb’cry2#d1. In intact crickets, transcripts of Gb’per and Gb’tim showed a rhythmic expression to peak around midnight (ZT18). These expression profiles were similar to those reported previously [9–11]. When Gb’cry1 was knocked-down by RNAi, Gb’cry2 and Gb’tim maintained a rhythmic expression (ANOVA, F5,24 = 15.10, P < 0.01; cosinor, P < 0.05 for Gb’cry2 and ANOVA, F5,23 = 21.38, P < 0.01; cosinor, P < 0.05 for Gb’tim) with a peak similar to that of untreated crickets (Gb’cry2) or advanced by about 4–8 h to peak during late day to early night (Gb’tim); Gb’per also showed a similar rhythmic pattern to Gb’tim, showing a significant fluctuation (ANOVA, F5,21 = 3.59, P < 0.05), but no daily rhythm was detected by the single cosinor method (P > 0.05) (Fig. 5a). The results of Gb’cry2 RNAi are shown in Fig. 5b. No clear effect was observed on Gb’cry1 mRNA levels. Gb’tim showed a rhythm (ANOVA, F5,20 = 3.90, P < 0.05; cosinor, P < 0.05) with a peak at late day (ZT10), while Gb’per fluctuated around a medium range of the control, with no significant daily rhythm (ANOVA, F5,21 = 1.54, P > 0.23; cosinor, P > 0.05).Fig. 5


cryptochrome genes form an oscillatory loop independent of the per / tim loop in the circadian clockwork of the cricket Gryllus bimaculatus
Effects of Gb’cry1RNAi (a), Gb’cry2RNAi (b), and Gb’cry1RNAi/Gb’cry2RNAi (c) on daily clock gene expression. Relative abundance of Gb’per, Gb’tim, Gb’cry1, and Gb’cry2 mRNA in the optic lobes are shown for intact (blue) and Gb’cry1RNAi (yellow) (a), Gb’cry2RNAi (green) (b), and Gb’cry1RNAi/Gb’cry2RNAi (pink) adult male crickets (Gryllus bimaculatus) (c). The data for intact Gb’cry1 and Gb’cry2 mRNA are replotted from Fig. 2. In dsRNA-injected crickets, the optic lobes were collected about seven days after the dsRNA injection. The abundance of mRNA was measured by quantitative real-time RT-PCR with total RNA extracted from the optic lobes. The data collected from 3 and 5 independent experiments were averaged and plotted as mean ± SEM for dsRNA-injected and intact crickets, respectively. The abundance of Gb’rpl18a mRNA was used as an internal reference. Asterisks indicate significant difference between intact and treated crickets (*P < 0.05, **P < 0.01, t-test). Different letters indicate that values are significantly different with each other (ANOVA followed by Tukey-test, P < 0.05). For further explanations, see the text
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Fig5: Effects of Gb’cry1RNAi (a), Gb’cry2RNAi (b), and Gb’cry1RNAi/Gb’cry2RNAi (c) on daily clock gene expression. Relative abundance of Gb’per, Gb’tim, Gb’cry1, and Gb’cry2 mRNA in the optic lobes are shown for intact (blue) and Gb’cry1RNAi (yellow) (a), Gb’cry2RNAi (green) (b), and Gb’cry1RNAi/Gb’cry2RNAi (pink) adult male crickets (Gryllus bimaculatus) (c). The data for intact Gb’cry1 and Gb’cry2 mRNA are replotted from Fig. 2. In dsRNA-injected crickets, the optic lobes were collected about seven days after the dsRNA injection. The abundance of mRNA was measured by quantitative real-time RT-PCR with total RNA extracted from the optic lobes. The data collected from 3 and 5 independent experiments were averaged and plotted as mean ± SEM for dsRNA-injected and intact crickets, respectively. The abundance of Gb’rpl18a mRNA was used as an internal reference. Asterisks indicate significant difference between intact and treated crickets (*P < 0.05, **P < 0.01, t-test). Different letters indicate that values are significantly different with each other (ANOVA followed by Tukey-test, P < 0.05). For further explanations, see the text
Mentions: We then examined the effects of RNAi of Gb’cry1 and Gb’cry2 on Gb’per and Gb’tim genes, using dsGb’cry1#d1 and dsGb’cry2#d1. In intact crickets, transcripts of Gb’per and Gb’tim showed a rhythmic expression to peak around midnight (ZT18). These expression profiles were similar to those reported previously [9–11]. When Gb’cry1 was knocked-down by RNAi, Gb’cry2 and Gb’tim maintained a rhythmic expression (ANOVA, F5,24 = 15.10, P < 0.01; cosinor, P < 0.05 for Gb’cry2 and ANOVA, F5,23 = 21.38, P < 0.01; cosinor, P < 0.05 for Gb’tim) with a peak similar to that of untreated crickets (Gb’cry2) or advanced by about 4–8 h to peak during late day to early night (Gb’tim); Gb’per also showed a similar rhythmic pattern to Gb’tim, showing a significant fluctuation (ANOVA, F5,21 = 3.59, P < 0.05), but no daily rhythm was detected by the single cosinor method (P > 0.05) (Fig. 5a). The results of Gb’cry2 RNAi are shown in Fig. 5b. No clear effect was observed on Gb’cry1 mRNA levels. Gb’tim showed a rhythm (ANOVA, F5,20 = 3.90, P < 0.05; cosinor, P < 0.05) with a peak at late day (ZT10), while Gb’per fluctuated around a medium range of the control, with no significant daily rhythm (ANOVA, F5,21 = 1.54, P > 0.23; cosinor, P > 0.05).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: Animals exhibit circadian rhythms with a period of approximately 24&nbsp;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&rsquo;cry1) and mammalian-type cry2 (Gb&rsquo;cry2). Gb&rsquo;cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb&rsquo;cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb&rsquo;cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb&rsquo;cry1RNAi. In Gb&rsquo;cry1/Gb&rsquo;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&rsquo;cry1 and Gb&rsquo;cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells.

Conclusion: Based on these results, we propose a new model of the cricket&rsquo;s circadian clock, including a molecular oscillatory loop for Gb&rsquo;cry2, which can operate independent of the Gb&rsquo;per/Gb&rsquo;tim loop.

No MeSH data available.