Fbxl4 Regulates the Photic Entrainment of Circadian Locomotor Rhythms in the Cricket Gryllus bimaculatus.

Photic entrainment is an essential property of the circadian clock that sets the appropriate timing of daily behavioral and physiological events. However, the molecular mechanisms underlying the entrainment remain largely unknown. In the cricket Gryllus bimaculatus, the immediate early gene c-fosB plays an important role in photic entrainment, followed by a mechanism involving cryptochromes (crys). However, the association between c-fosB expression and crys remains unclear. In the present study, using RNA-sequencing analysis, we found that five Fbxl family genes (Fbxl4, Fbxl5, Fbxl16, Fbxl-like1, and Fbxl-like2) encoding F-box and leucine-rich repeat proteins are likely involved in the mechanism following light-dependent c-fosB induction. RNA interference (RNAi) of c-fosA/B significantly downregulated Fbxls expression, whereas RNAi of the Fbxl genes exerted no effect on c-fosB expression. The Fbxl genes showed rhythmic expression under light-dark cycles (LDs) with higher expression levels in early day (Fbxl16), whole day (Fbxl-like1), or day-to-early night (Fbxl4, Fbxl5, and Fbxl-like2), whereas their expression was reduced in the dark. We then examined the effect of their RNAi on the photic entrainment of the locomotor rhythm and found that RNAi of Fbxl4 either disrupted or significantly delayed the re-entrainment of the locomotor rhythm to shifted LDs. These results suggest that light-induced c-fosB expression stimulates Fbxl4 expression to reset the circadian clock.

Temperature Entrainment of Circadian Locomotor and Transcriptional Rhythms in the Cricket, Gryllus bimaculatus.

Most animals exhibit circadian rhythms in various physiological and behavioral functions regulated by circadian clock that resides in brain and in many peripheral tissues. Temperature cycle is an important time cue for entrainment, even in mammals, since the daily change in body temperature is thought to be used for phase regulation of clocks in peripheral tissues. However, little is known about the mechanisms by which temperature resets the clock. In the present study, we investigated the effect of temperature on circadian activity rhythm and clock gene transcription by using the cricket, Gryllus bimaculatus. We show that temperature cycle can entrain both behavioral and transcriptional rhythms of clock genes, such as period, timeless, cryptochrome2 and cycle in the circadian pacemaker tissue, optic lobe. Under temperature cycle, phase of evening peak of locomotor activity occurred 1 h before the warm-to-cold phase transition, which is associated with earlier peaks of mRNA expression rhythm of the clock genes than that under light/dark cycles. When the temperature cycle was advanced by 6 h, behavioral rhythms re-entrained to newly phased temperature cycle after ∼16 transient cycles. The mRNA oscillation of period and timeless gained stable rhythm under phase advanced temperature cycles with a lesser number of transient cycles than cryptochrome2 and cycle. These results suggest that temperature cycle can entrain behavioral and molecular rhythms in cricket and clock genes vary in sensitivity to temperature. It is thus likely that clock genes play differential roles in resetting the clock with environmental temperature changes.

Functional analysis of Pdp1 and vrille in the circadian system of a cricket.

Circadian rhythms are generated by a circadian clock for which oscillations are based on the rhythmic expression of the so-called clock genes. The present study investigated the role of Gryllus bimaculatus vrille (Gb'vri) and Par domain protein 1 (Gb'Pdp1) in the circadian clock of the cricket Gryllus bimaculatus. Structural analysis of Gb'vri and Gb'Pdp1 cDNAs revealed that they are a member of the bZIP transcription factors. Under light/dark cycles (LD) both genes were rhythmically expressed in the clock tissue, the optic lobes, whereas the rhythm diminished under constant darkness (DD). Gb'vri and Gb'Pdp1 mRNA levels were significantly reduced by RNA interference (RNAi) of Gb'Clk and Gb'cyc, suggesting they are controlled by Gb'CLK/Gb'CYC. RNAi of Gb'vri and Gb'Pdp1 had little effect on locomotor rhythms, although their effects became visible when treated together with Gb'cycRNAi. The average free-running period of Gb'vriRNAi/Gb'cycRNAi crickets was significantly shorter than that of Gb'cycRNAi crickets. A similar period shortening was observed also when treated with Gb'Pdp1RNAi/Gb'cycRNAi. Some Gb'Pdp1RNAi/Gb'cycRNAi crickets showed rhythm splitting into two free-running components with different periods. Gb'vriRNAi and Gb'Pdp1RNAi treatments significantly altered the expression of Gb'Clk, Gb'cyc, and Gb'tim in LD. These results suggest that Gb'vri and Gb'Pdp1 play important roles in cricket circadian clocks.

The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus.

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.

A novel photic entrainment mechanism for the circadian clock in an insect: involvement of c-fos and cryptochromes.

BACKGROUND: Entrainment to the environmental light cycle is an essential property of the circadian clock. Although the compound eye is known to be the major photoreceptor necessary for entrainment in many insects, the molecular mechanisms of photic entrainment remain to be explored. RESULTS: We found  that cryptochromes (crys) and c-fos mediate photic entrainment of the circadian clock in a hemimetabolous insect, the cricket Gryllus bimaculatus. We examined the effects of RNA interference (RNAi)-mediated knockdown of the cry genes, Gb'cry1 and Gb'cry2, on photic entrainment, and light-induced resetting of the circadian locomotor rhythm. Gb'cry2 RNAi accelerated entrainment for delay shifts, while Gb'cry1/ Gb'cry2 double RNAi resulted in significant lengthening of transient cycles in both advance and delay shifts, and even in entrainment failure in some crickets. Double RNAi also strongly suppressed light induced resetting. The Gb'cry-mediated phase shift or resetting of the rhythm was preceded by light-induced Gb'c-fosB expression. We also found that Gb'c-fosB, Gb'cry2 and Gb'period (Gb'per) were likely co-expressed in some optic lobe neurons. CONCLUSION: Based on these results, we propose a novel model for photic entrainment of the insect circadian clock, which relies on the light information perceived by the compound eye.