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.

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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.

IntroductionEntrainment 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.ResultsA 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.ConclusionThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40851-015-0011-6) contains supplementary material, which is available to authorized users.

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.