Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus

The circadian clock gene period (Gryllus bimaculatus period, Gb’per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb’per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb’per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb’per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb’per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb’per.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Circadian oscillations outside the optic lobe in the cricket Gryllus bimaculatus

Although circadian rhythms are found in many peripheral tissues in insects, the control mechanism is still to be elucidated. To investigate the central and peripheral relationships in the circadian organization, circadian rhythms outside the optic lobes were examined in the cricket Gryllus bimaculatus by measuring mRNA levels of period (per) and timeless (tim) genes in the brain, terminal abdominal ganglion (TAG), anterior stomach, mid-gut, testis, and Malpighian tubules. Except for Malpighian tubules and testis, the tissues showed a daily rhythmic expression in either both per and tim or tim alone in LD. Under constant darkness, however, the tested tissues exhibited rhythmic expression of per and tim mRNAs, suggesting that they include a circadian oscillator. The amplitude and the levels of the mRNA rhythms varied among those rhythmic tissues. Removal of the optic lobe, the central clock tissue, differentially affected the rhythms: the anterior stomach lost the rhythm of both per and tim; in the mid-gut and TAG, tim expression became arrhythmic but per maintained rhythmic expression; a persistent rhythm with a shifted phase was observed for both per and tim mRNA rhythms in the brain. These data suggest that rhythms outside the optic lobe receive control from the optic lobe to different degrees, and that the oscillatory mechanism may be different from that of Drosophila.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.     

Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide widely distributed in insect brains and plays important roles in the circadian system. In this study, we used RNA interference to study the role of the pigment-dispersing factor (pdf) gene in regulating circadian locomotor rhythms in the cricket, Gryllus bimaculatus. Injections of pdf double-stranded RNA (dspdf) effectively knocked down the pdf mRNA and PDF peptide levels. The treated crickets maintained the rhythm both under light-dark cycles (LD) and constant darkness (DD). However, they showed rhythms with reduced nocturnal activity with prominent peaks at lights-on and lights-off. Entrainability of dspdf-injected crickets was higher than control crickets as they required fewer cycles to resynchronize to the LD cycles shifted by 6 h. The free-running periods of the dspdf-injected crickets were shorter than those of control crickets in DD. These results suggest that PDF is not essential for the rhythm generation but involved in control of the nocturnality, photic entrainment, and fine tuning of the free-running period of the circadian clock.     

Short-day and long-day expression patterns of genes involved in the flesh fly clock mechanism: period,timeless, cycle and cryptochrome

Though our knowledge of the molecular details of the circadian clock has advanced rapidly, the functional elements of the photoperiodic clock remain largely unknown. As a first step to approach this issue, we report here the sequences and expression patterns of period (per), timeless (tim), cycle (cyc) and cryptochrome (cry) mRNAs in the flesh fly Sarcophaga crassipalpis. Nucleotide and deduced amino acid sequences of the genes in S. crassipalpis show high similarity to homologous genes in other insects that have been investigated. S. crassipalpis TIM has a unique C-terminus that contains a poly Q region. A diel rhythmicity of per and tim mRNA abundance was detected in the adult heads (peak during scotophase), while cry and cyc mRNA abundance remained fairly constant throughout. The abundance of cyc mRNA was quite low when compared to per, tim and cry mRNA. Rearing temperature affected the amount of per and tim mRNAs: abundance of per mRNA increased at 20 °C when compared to 25 °C, but that of tim mRNA decreased. Photoperiod influenced the expression patterns of per and tim mRNA: the peak of per mRNA expression shifted in concert with onset of the scotophase, while a shift in tim mRNA expression was less pronounced. The amplitude of tim mRNA was severely dampened under long daylength, but that of per mRNA was not affected. These distinct patterns of expression suggest that this information could be used to determine photoperiodic responses such as diapause.     

Circadian clock genes period and cycle regulate photoperiodic diapause in the bean bug Riptortus pedestris males

The photoperiodic response is crucial for many insects to adapt to seasonal changes in temperate regions. It was recently shown that the circadian clock genes period (per) and cycle (cyc) are involved in the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris females. Here, we investigated the involvement of per and cyc both in the circadian rhythm of cuticle deposition and in the photoperiodic diapause of R. pedestris males using RNA interference (RNAi). RNAi of per and cyc disrupted the cuticle deposition rhythm and resulted in distinct cuticle layers. RNAi of per induced development of the male reproductive organs even under diapause-inducing short-day conditions, whereas RNAi of cyc suppressed development of the reproductive organs even under diapause-averting long-day conditions. Thus, the present study suggests that the circadian clock operated by per and cyc governs photoperiodism of males as that of females.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

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.     

RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.     

Clock genes period and timeless are rhythmically expressed in brains of newly hatched, photosensitive larvae of the fly, Sarcophaga crassipalpis

While roles of the clock genes period (per) and timeless (tim) are relatively well understood in relation to circadian clocks, their potential roles in insect photoperiodism remain enigmatic. In this study, the expression of per and tim genes under two contrasting photoperiods is described in the central nervous system of photoperiodically sensitive, newly hatched first instar larvae of the flesh fly, Sarcophaga crassipalpis. Using qPCR, diel oscillations were observed in the mRNA levels of both genes under long-day (15 h light:9 h dark, promotes direct development) and short-day conditions (11 h light:13 h dark, induces pupal diapause). Peak per and tim mRNA oscillations were closely associated with the light/dark transition. The conspicuous difference between the two photoperiodic conditions was that the sharp increase in per and tim mRNA abundance occurred during the light phase under long days but during the dark phase under short days. The diel oscillations were, at least in part, driven by an endogenous component, as demonstrated by transferring larvae to continuous darkness. The cells displaying Tim- and Per-like immunoreactivities (Tim- and Per-LIRs) were localized using anti-Drosophila-Per and anti-Chymomyza-Tim antibodies. Per-LIR and Tim-LIR co-localized in three groups of cells in each brain hemisphere. Two other groups, one in the brain hemispheres and the other in the fused ventral nerve ganglion, expressed only the Per-LIR.     

Cyclical expression of Na/K-ATPase in the visual system of Drosophila melanogaster

In the first (lamina) and second (medulla) optic neuropils of Drosophila melanogaster, sodium pump subunit expression changes during the day and night, controlled by a circadian clock. We examined α-subunit expression from the intensity of immunolabeling. For the β-subunit, encoded by Nervana 2 (Nrv2), we used Nrv2-GAL4 to drive expression of GFP, and measured the resultant fluorescence in whole heads and specific optic lobe cells. All optic neuropils express the α-subunit, highest at the beginning of night in both lamina and medulla in day/night condition and the oscillation was maintained in constant darkness. This rhythm was lacking in the clock arrhythmic per⁰ mutant. GFP driven by Nrv2 was mostly detected in glial cells, mainly in the medulla. There, GFP expression occurs in medulla neuropil glia (MNGl), which express the clock gene per, and which closely contact the terminals of clock neurons immunoreactive to pigment dispersing factor. GFP fluorescence exhibited circadian oscillation in whole heads from Nrv2-GAL4 + UAS-S65T-GFP flies, although significant GFP oscillations were lacking in MNGl, as they were for both subunit mRNAs in whole-head homogenates. In the dissected brain tissues, however, the mRNA of the α-subunit showed a robust daily rhythm in concentration changes while changes in the β-subunit mRNA were weaker and not statistically significant. Thus in the brain, the genes for the sodium pump subunits, at least the one encoding the α-subunit, seem to be clock-controlled and the abundance of their corresponding proteins mirrors daily changes in mRNA, showing cyclical accumulation in cells.     

Circadian control of permethrin-resistance in the mosquito Aedes aegypti

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT₅₀) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12 h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT₅₀ and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT₅₀ was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.     

Diurnal rhythm in expression and release of yolk protein in the testis of Spodoptera littoralis (Lepidoptera: Noctuidae)

Circadian clocks (oscillators) regulate multiple life functions in insects. The circadian system located in the male reproductive tract of Lepidoptera is one of the best characterized peripheral oscillators in insects. Our previous research on the cotton leafworm, Spodoptera littoralis, demonstrated that this oscillator controls the rhythm of sperm release from the testis and coordinates sperm maturation in the upper vas deferens (UVD). We demonstrated previously that a protein that functions as yolk protein in females is also produced in cyst cells surrounding sperm bundles in the testis, and is released into the UVD. Here, we investigated the temporal expression of the yolk protein 2 (yp2) gene at the mRNA and protein level in the testis of S. littoralis, and inquired whether their expression is regulated by PER-based molecular oscillator. We describe a circadian rhythm of YP2 accumulation in the UVD seminal fluid, where this protein interacts with sperm in a circadian fashion. However, we also demonstrate that yp2 mRNA and YP2 protein levels within cyst cells show only a diurnal rhythm in light/dark (LD) cycles. These rhythms do not persist in constant darkness (DD), suggesting that they are non-circadian. Interestingly, the per gene mRNA and protein levels in cyst cells are rhythmic in LD but not in DD. Nevertheless, per appears to be involved in the diurnal timing of YP2 protein accumulation in cyst cells.     

Constant light disrupts the circadian but not the circatidal rhythm in mangrove crickets

Mangrove crickets have a circatidal activity rhythm (~12.6 h cycles) with a circadian modulation under constant darkness (DD), whereby activity levels are higher during subjective night low tides than subjective day low tides. This study explored the locomotor activity rhythm of mangrove crickets under constant light (LL). Under LL, the crickets also exhibited a clear circatidal activity rhythm with a free-running period of 12.6 ± 0.26 h (mean ± SD, n = 6), which was not significantly different from that observed under DD. In contrast, activity levels were almost the same between subjective day and night, unlike those under DD, which were greater during subjective night. The loss of circadian modulation under LL may be explained by the suspension of the circadian clock in these conditions. These results strongly suggest that the circatidal activity rhythm is driven by its own clock system, distinct from the circadian clock.     

Length variation in a specific region of the period gene correlates with differences in pupal diapause incidence in the flesh fly, Sarcophaga bullata

We report differences in the length of a specific region of the circadian clock gene period (per) that correlate with different capacities for pupal diapause in the flesh fly, Sarcophaga bullata. The conspicuous difference is located in a region we refer to as the putative C-terminal photoperiodic (CP) region. The length of the CP region correlates inversely with the incidence of diapause. A deletion of 33 amino acids in this region correlates with a significant increase in the incidence of diapause, from 78.1% to 93.0%, and an insertion of 9 amino acids in the same area correlates with a drop in the diapause incidence to 4.0%. This correlation suggests a possible functional role for this region of per in photoperiodism.     

Photoreception in decapitated larvae of silkworm Bombyx mori

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.