The Nuclear Receptor Genes HR3 and E75 Are Required for the Circadian Rhythm in a Primitive Insect

Insect circadian rhythms are generated by a circadian clock consisting of transcriptional/translational feedback loops, in which CYCLE and CLOCK are the key elements in activating the transcription of various clock genes such as timeless (tim) and period (per). Although the transcriptional regulation of Clock (Clk) has been profoundly studied, little is known about the regulation of cycle (cyc). Here, we identify the orphan nuclear receptor genes HR3 and E75, which are orthologs of mammalian clock genes, Rorα and Rev-erbα, respectively, as factors involved in the rhythmic expression of the cyc gene in a primitive insect, the firebrat Thermobia domestica. Our results show that HR3 and E75 are rhythmically expressed, and their normal, rhythmic expression is required for the persistence of locomotor rhythms. Their RNAi considerably altered the rhythmic transcription of not only cyc but also tim. Surprisingly, the RNAi of HR3 revealed the rhythmic expression of Clk, suggesting that this ancestral insect species possesses the mechanisms for rhythmic expression of both cyc and Clk genes. When either HR3 or E75 was knocked down, tim, cyc, and Clk or tim and cyc, respectively, oscillated in phase, suggesting that the two genes play an important role in the regulation of the phase relationship among the clock genes. Interestingly, HR3 and E75 were also found to be involved in the regulation of ecdysis, suggesting that they interconnect the circadian clock and developmental processes

Expression profiles of <i>Td'HR3</i> and <i>Td'E75</i> mRNA and effects of their dsRNAs on their mRNA levels and the survival rate in firebrats.

<p>Both <i>Td'HR3</i> (A, C) and <i>Td'E75</i> (B, D) were rhythmically expressed in LD (A, B) and DD (C, D), peaking late (subjective) day to early (subjective) night (black symbols). Ds<i>HR3#1,2</i> (pink triangles and squares for #1 and #2, respectively) and ds<i>E75#1,2</i> (green triangles and squares for #1 and #2, respectively) downregulated mRNA levels and disrupted the rhythm of their respective genes (A and B). Treatment with ds<i>DsRed2</i> as a negative control did not disrupt the rhythmic expression in both genes (A and B, blue symbols), but caused a significant decrease in <i>Td'HR3</i> mRNA levels at ZT 2 and ZT 18 (+ <i>P</i><0.05, <i>t</i> test vs intact). White, black, and gray bars above each graph indicate day, night/subjective night, and subjective day, respectively. Total RNA was extracted from firebrats collected at 4-h intervals starting 2 h after lights on or 2 h into subjective day (ZT2 or CT2, respectively). The data collected from 3–4 independent experiments were averaged and plotted as the mean ± SEM values relative to the value of <i>rp49</i> mRNA used as the reference. Values with different letters significantly differ from each other within the same treatment groups (<i>P</i><0.05, ANOVA with Tukey test, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899.s001" target="_blank">S1 Table</a>). *<i>P</i><0.05, **<i>P</i><0.01, <i>t</i> test vs ds<i>DsRed2</i> (exact <i>P</i> values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899.s002" target="_blank">S2 Table</a>). (E) Survival rate after injection of 10.23–13.95 µg/g ds<i>HR3</i>, ds<i>E75</i>, or ds<i>DsRed2</i> into the abdomen. Ds<i>HR3</i> and ds<i>E75</i> significantly reduced the survival rate. For further explanation, see text.</p

Effects of <i>HR3</i> and <i>E75</i> dsRNA on locomotor rhythms in the firebrat.

<p>Double-plotted actograms (left) and Lomb-Scargle periodograms (right) of locomotor rhythms under LD 12∶12 and DD at a constant temperature of 30°C are shown for firebrats injected with <i>HR3</i> (A, B) and <i>E75</i> dsRNA (C, D) or <i>DsRed2</i> dsRNA (E, F). White and black bars above the actograms indicate light (white) and dark (black) conditions. Arrowheads indicate the day when the firebrats were transferred from LD to DD. a and b indicated in the periodogram correspond to the analyzed time span, a and b, indicated in the actogram, respectively. An oblique line in the periodogram indicates a significance level of <i>P</i><0.05; a peak value above the line was designated as significant. (A–D) Some of the <i>HR3</i> and <i>E75</i> RNAi firebrats showed a rhythm in LD, which disappeared upon transfer to DD. (E, F) Control firebrats injected with <i>DsRed2</i> dsRNA showed a significant rhythm throughout the recording period. No activity was recorded during the period indicated by double headed arrows because of molting. For further explanation, see text.</p

PCR primers used for PCR, quantitative RT-PCR, and dsRNA synthesis.

<p>PCR primers used for PCR, quantitative RT-PCR, and dsRNA synthesis.</p

Effects of <i>HR3</i> (ds<i>HR3</i>, pink symbols) and <i>E75</i> dsRNA (ds<i>E75</i>, green symbols) on mRNA levels of <i>cyc</i>, <i>tim</i>, and <i>Clk</i> in firebrats.

<p>Values for intact and ds<i>DsRed2</i> injected control are shown in black and blue symbols, respectively, and replotted in lower panels with an expanded ordinate. (A) <i>cyc</i> normally peaked during the day. ds<i>E75</i> significantly upregulated the mRNA levels, while ds<i>HR3</i> shifted the rhythm to peak late at night. (B) <i>tim</i> showed a rhythm with a peak at late day or early night in intact firebrats. Ds<i>E75</i> slightly advanced the rhythm while ds<i>HR3</i> upregulated and shifted the rhythm to peak late at night. (C) <i>Clk</i> showed no significant rhythm in intact firebrats. Ds<i>E75</i> had no significant effect on <i>Clk</i> mRNA levels, while ds<i>HR3</i> induced a rhythm with a peak at mid–late night. *<i>P</i><0.05, **<i>P</i><0.01, <i>t</i> test vs ds<i>DsRed2</i> (exact <i>P</i> values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899.s003" target="_blank">S3 Table</a>). Values with different letters significantly differ from each other (p<0.05, ANOVA with Tukey test, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899.s004" target="_blank">S4 Table</a>). For <i>tim</i> in firebrats treated with ds<i>HR3</i> (B), significant rhythm was confirmed by the single cosinor method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899-Nelson1" target="_blank">[28]</a> and significant difference was found between values at ZT6 and ZT22 (<i>t</i>-test, P<0.05). For further explanation, see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone-0114899-g002" target="_blank">Fig. 2</a>.</p

Effects of <i>cyc</i>, <i>Clk</i>, and <i>tim</i> dsRNA (ds<i>cyc</i>, ds<i>Clk</i>, and ds<i>tim</i>) on mRNA levels of <i>HR3</i> and <i>E75</i> in firebrats.

<p>(A) Ds<i>cyc</i>, ds<i>Clk</i>, and ds<i>tim</i> abolished the rhythmic expression of <i>HR3</i> with significant downregulation of mRNA levels. (B) RNAi of the three genes abolished the rhythm of <i>E75</i>. Ds<i>tim</i> effectively downregulated <i>E75</i> mRNA but ds<i>cyc</i> and ds<i>Clk</i> had no significant effects, except at ZT18 where a significant increase was observed. *<i>P</i><0.05, **<i>P</i><0.01, <i>t</i> test vs ds<i>DsRed2</i>. Data for ds<i>DsRed2</i> firebrats are replotted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone-0114899-g002" target="_blank">Fig. 2</a>. For further explanation, see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone-0114899-g002" target="_blank">Fig. 2</a>.</p

Effects of ds<i>HR3</i>, ds<i>E75</i>, and ds<i>DsRed2</i> on the locomotor rhythm of the firebrat <i>Thermobia domestica</i>.

a<p><i>P</i><0.05, ^b<i>P</i><0.001 vs <i>DsRed2</i> dsRNA, Chi-square test; SD, standard deviation; LD, light–dark cycle; DD, constant darkness.</p><p>Effects of ds<i>HR3</i>, ds<i>E75</i>, and ds<i>DsRed2</i> on the locomotor rhythm of the firebrat <i>Thermobia domestica</i>.</p

Effects of ecdysone on mRNA levels of <i>Td'HR3</i> (A) and <i>Td'E75</i> (B) in firebrats.

<p>White and blue columns indicate the mRNA levels of EtOH (negative control) and ecdysone injected firebrats, respectively. 100% EtOH (69 nl) or 34.5 ng ecdysone (dissolved in 69 nl of EtOH) was injected and firebrats were collected at ZT 10 (10 h after light on) one week after the injection. The abundance of mRNA was measured by qPCR. The data collected from 4 independent experiments were averaged and plotted as mean ± SEM values relative to the value of <i>rp49</i> mRNA used as reference.*<i>P</i><0.05, <i>t</i>-test.</p

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

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’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. 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