RNA Interference of the Clock GeneperiodDisrupts Circadian Rhythms in the CricketGryllus bimaculatus

Abstract

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.

Roles of PER immunoreactive neurons in circadian rhythms and photoperiodism in the blow fly, Protophormia terraenovae

“…per is an important circadian clock gene in D. melanogaster (Konopka and Benzer, 1971;Baylies et al, 1987), and circadian oscillation of per mRNA or distribution of per-expressing neurons has been shown in different insect orders (Frisch et al, 1996;Sauman and Reppert, 1996;Goto and Denlinger, 2002;Wise et al, 2002;Hodková et al, 2003;Závodská et al, 2005;Iwai et al, 2006;Codd et al, 2007;Moriyama et al, 2008). However, few studies have demonstrated that per or per-expressing neurons are functionally important for circadian behavioural rhythms or photoperiodism.…”

“…However, few studies have demonstrated that per or per-expressing neurons are functionally important for circadian behavioural rhythms or photoperiodism. In G. bimaculatus, it has been reported that per knockdown by RNAi suppresses circadian rhythms in locomotor activities and electrical activities of optic lobe neurons (Moriyama et al, 2008). In M. sexta, surgical ablation of per expressing neurons from larval brains caused loss of photoperiodic control of pupal diapause (Wise et al, 2002;Shiga et al, 2003).…”

RNAi of the circadian clock geneperioddisrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket

“…Moreover, a circadian rhythm modifying the circatidal rhythm by inhibiting activity during subjective day free-runs under constant darkness and can be entrained by light -dark cycles [9]. In insects, the molecular basis of the circadian clock has been extensively studied [10], and the predominant role of the gene period (per) is confirmed in some species, including a cricket, Gryllus bimaculatus [11]. Thus, the mangrove cricket is a suitable subject for examining the molecular basis of the circatidal rhythm.…”

A comparative view of insect circadian clock systems

“…Thus, there seem to be considerable differences in the molecular oscillatory mechanism among insects. In crickets, the role of clock genes has been examined in a more direct way using RNA interference (RNAi) [61][62][63]. Like other insects, the expression of cricket's per gene cycles in a daily fashion in both LD and DD, and peaks at early night.…”

“…Like other insects, the expression of cricket's per gene cycles in a daily fashion in both LD and DD, and peaks at early night. Double stranded RNA (dsRNA) of per knocks down per mRNA levels, and disrupts both its cycling and the locomotor activity rhythm [61]. Functional analysis with RNAi of other clock genes will clarify the molecular oscillatory mechanism in the cricket.…”