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Introduction: Entrainment to light cycle is a prerequisite for circadian rhythms to set daily physiological events to occur at an appropriate time of day. In hemimetabolous insects, the photoreceptor molecule for photic entrainment is still unknown. Since the compound eyes are the only circadian photoreceptor in the cricket Gryllus bimaculatus, we have investigated the role of three opsin genes expressed there, opsin-Ultraviolet (opUV), opsin-Blue (opB), and opsin-Long Wave (opLW) encoding a green-sensitive opsin in photic entrainment., Results: A daily rhythm was detected in mRNA expressions of opB and opLW but not of opUV gene. When photic entrainment of circadian locomotor rhythms was tested after injection of double-stranded RNA (dsRNA) of three opsin genes, no noticeable effects were found in opUV RNAi and opB RNAi crickets. In opLW RNAi crickets, however, some crickets lost photic entrainability and the remaining crickets re-entrained with significantly longer transient cycles to a phase-advanced light-dark cycle as compared to control crickets. Crickets often lost entrainability when treated doubly with dsRNAs of two opsin genes including opLW., Conclusion: These results show that green-sensitive OpLW is the major circadian photoreceptor molecule for photic entrainment of locomotor rhythms in the cricket G. bimaculatus. Our finding will lead to further investigation of the photic entrainment mechanism at molecular and cellular levels, which still remains largely unknown.

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.

RNA interference (RNAi) strategy, which enables gene-specific knock-down of transcripts, has been spread across a wide area of insect studies for investigating gene function without regard to model and non-model insects. This technique is of particular benefit to promote molecular studies on non-model insects. However, the optimal conditions for RNAi are still not well understood because of its variable efficiency depending on the species, target genes, and experimental conditions. To apply RNAi technique to long-running experiments such as chronobiological studies, the effects of RNAi have to persist throughout the experiment. In this study, we attempted to determine the optimal concentration of double-stranded RNA (dsRNA) for systemic RNAi and its effective period in two different insect species, the cricket Gryllus bimaculatus and the firebrat Thermobia domestica. In both species, higher concentrations of dsRNA principally yielded a more efficient knock-down of mRNA levels of tested clock genes, although the effect depended on the gene and the species. Surprisingly, the effect of the RNAi reached its maximum effect 1-2 weeks and 1 month after the injection of dsRNA in the crickets and the firebrats, respectively, suggesting a slow but long-term effect of RNAi. Our study provides fundamental information for utilizing RNAi technique in any long-running experiment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Reverse genetic studies have revealed that common clock genes, such as period (per), timeless (tim), cycle (cyc), and Clock (Clk), are involved in the circadian clock mechanism among a wide variety of insects. However, to what degree the molecular oscillatory mechanism is conserved is still to be elucidated. In this study, cDNA of the clock gene Clk was cloned in the cricket Gryllus bimaculatus, and its function was analyzed using RNA interference (RNAi). In adult optic lobes, the Clk mRNA level showed no significant rhythmic changes both under light-dark cycle (LD) and constant darkness (DD). A single injection of Clk double-stranded RNA (dsRNA) resulted in a knockdown of the mRNA level to about 25% of the peak level of control animals. The injected crickets lost their locomotor rhythms in DD. The arrhythmicity in locomotor activity persisted for up to 50 days after the Clk dsRNA injection. Control animals injected with DsRed2 dsRNA showed a clear locomotor rhythm like intact animals. Injection of Clk dsRNA not only suppressed the mRNA levels of both per and tim but also abolished their rhythmic expression. per RNAi down-regulates the Clk mRNA levels, suggesting that per is required for sufficient expression of Clk. These results suggest that Clk is an essential component and plays an important role in the cricket's circadian clock machinery like in Drosophila, but regulation of its expression is probably different from regulation in Drosophila.

Many physiological functions of insects show a rhythmic change to adapt to daily environmental cycles. These rhythms are controlled by a multi-clock system. A principal clock located in the brain usually organizes the overall behavioral rhythms, so that it is called the "central clock". However, the rhythms observed in a variety of peripheral tissues are often driven by clocks that reside in those tissues. Such autonomous rhythms can be found in sensory organs, digestive and reproductive systems. Using Drosophila melanogaster as a model organism, researchers have revealed that the peripheral clocks are self-sustained oscillators with a molecular machinery slightly different from that of the central clock. However, individual clocks normally run in harmony with each other to keep a coordinated temporal structure within an animal. How can this be achieved? What is the molecular mechanism underlying the oscillation? Also how are the peripheral clocks entrained by light-dark cycles? There are still many questions remaining in this research field. In the last several years, molecular techniques have become available in non-model insects so that the molecular oscillatory mechanisms are comparatively investigated among different insects, which give us more hints to understand the essential regulatory mechanism of the multi-oscillatory system across insects and other arthropods. Here we review current knowledge on arthropod's peripheral clocks and discuss their physiological roles and molecular mechanisms.

Recent studies show that the timeless (tim) gene is not an essential component of the circadian clock in some insects. In the present study, we have investigated whether the tim gene was originally involved in the insect clock or acquired as a clock component later during the course of evolution using an apterygote insect, Thermobia domestica. A cDNA of the clock gene tim (Td'tim) was cloned, and its structural analysis showed that Td'TIM includes 4 defined functional domains, that is, 2 regions for dimerization with PERIOD (PER-1, PER-2), nuclear localization signal (NLS), and cytoplasmic localization domain (CLD), like Drosophila TIM. Td'tim exhibited rhythmic expression in its mRNA levels with a peak during late day to early night in LD, and the rhythm persisted in DD. A single injection of double-stranded RNA (dsRNA) of Td'tim (dstim) into the abdomen of adult firebrats effectively knocked down mRNA levels of Td'tim and abolished its rhythmic expression. Most dsRNA-injected firebrats lost their circadian locomotor rhythm in DD up to 30 days after injection. DsRNA of cycle (cyc) and Clock genes also abolished the rhythmic expression of Td'tim mRNA by knocking down Td'tim mRNA to its basal level of intact firebrats, suggesting that the underlying molecular clock of firebrats resembles that of Drosophila. Interestingly, however, dstim also reduced cyc mRNA to its basal level of intact animals and eliminated its rhythmic expression, suggesting the involvement of Td'tim in the regulation of cyc expression. These results suggest that tim is an essential component of the circadian clock of the primitive insect T. domestica; thus, it might have been involved in the clock machinery from a very early stage of insect evolution, but its role might be different from that in Drosophila.

Comparative molecular analysis reveals a wide variation of clock mechanisms among insects. In this study, the clock gene homologues of Clock (Td'Clk) and cycle (Td'cyc) were cloned from an apterygote insect, Thermobia domestica. Structural analysis showed that Td'CLK includes bHLH, PAS-A, PAS-B domains but lacks a polyglutamine repeat in the C terminal region that is implicated for transcriptional activity in Drosophila CLK. Td'CYC contains a BCTR domain in its C terminal in addition to the common domains found in Drosophila CYC, i.e. bHLH, PAS-A, PAS-B domains. Unlike in Drosophila, Td'Clk mRNA levels showed no significant daily fluctuation, while Td'cyc exhibited rhythmic expression. A single injection of double-stranded RNA (dsRNA) of Td'Clk or Td'cyc into the abdomen of adult firebrats effectively knocked down respective mRNA levels and abolished the rhythmic expression of Td'cyc. Most Td'Clk or Td'cyc dsRNA-injected firebrats lost their circadian locomotor rhythm in constant darkness up to 30 days after injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. From these results, it is likely that Td'Clk and Td'cyc are involved in the circadian clock machinery in the firebrat. However, the structure and expression profile of Td'Clk and Td'cyc more closely resembles those of mammals than Drosophila.