The Dual-Oscillator System of Drosophila melanogaster Under Natural-Like Temperature Cycles

Dual-oscillator systems that control morning and evening activities can be found in a wide range of animals. The two coupled oscillators track dawn and dusk and flexibly adapt their phase relationship to seasonal changes. This is also true for the fruit fly Drosophila melanogaster that serves as model organism to understand the molecular and anatomical bases of the dual-oscillator system. In the present study, the authors investigated which temperature parameters are crucial for timing morning and evening activity peaks by applying natural-like temperature cycles with different daylengths. The authors found that the morning peak synchronizes to the temperature increase in the morning and the evening peak to the temperature decrease in the afternoon. The two peaks did not occur at fixed absolute temperatures, but responded flexibly to daylength and overall temperature level. Especially, the phase of the evening peak clearly depended on the absolute temperature level: it was delayed at high temperatures, whereas the phase of the M peak was less influenced. This suggests that the two oscillators have different temperature sensitivities. The bimodal activity rhythm was absent in the circadian clock mutants Clk^Jrk and cyc⁰¹ and reduced in per⁰¹ and tim⁰¹ mutants. Whereas the activity of Clk^Jrk mutants just followed the temperature cycles, that of per⁰¹ and tim⁰¹ mutants did not, suggesting that these mutants are not completely clockless. This study revealed new characteristics of the dual-oscillator system in Drosophila that were not detected under different photoperiods. (Author correspondence: yoshii@cc.okayama-u.ac.jp)     

The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage

Many insects survive seasonal adversities during diapause, a form of programmed developmental and metabolic arrest. Photoperiodically regulated entry into diapause allows multivoltine insect species to optimize the number of generations. The molecular mechanism of the photoperiodic timer is unknown in insects. In the present study, we take advantage of the robust reproductive diapause response in the linden bug Pyrrhocoris apterus and explore the fifth‐instar nymphal stage, which is the most photoperiod‐sensitive stage. The nymphs display daily changes in locomotor activity during short days; this differs from the activity observed during long days. We find evidence of cyclical expression of the circadian clock genes, per and cyc, in nymphal heads; in addition, per expression is also photoperiod‐dependent. The RNA interference‐mediated knockdown of the two circadian clock genes, Clk and cyc, during the nymphal stage results in reproductive arrest in adult females. Furthermore, Clk and cyc knockdown induces the expression of the storage protein hexamerin in the fat body, whereas the expression of vitellogenin diminishes. Taken together, these data support the involvement of circadian clock genes in photoperiodic timer and/or diapause induction.     

模拟微重力效应对果蝇运动及睡眠的影响

本研究旨在探讨利用模拟微重力效应研究微重力对果蝇运动及睡眠影响的可行性.通过研制能够在模拟微重力环境下实时监测果蝇行为的随机定位仪,监测短时间(3 d)模拟微重力处理过程中,及长时间(10 d、20 d、30 d)处理后雄蝇运动和睡眠的变化;选取受影响较显著的短时间处理组,研究模拟微重力效应对生物钟核心基因(period (per)、timeless(tim)、clock (clk)、cycle (cyc)、cryptochrome (cry))、神经递质多巴胺(dopamine,DA)和5-羟色胺(5-hydroxytryptamine,5-HT)关键合成酶(多巴脱羧酶、酪氨酸羟化酶、色氨酸羟化酶)的编码基因ddc、pale和trh表达水平及DA和5-HT含量的影响.结果显示:短时间暴露下,雄蝇夜晚的运动量增加、单位时间运动次数增加、睡眠时间和次数减少、生物钟基因tim、clk、cyc、cry及神经递质合成相关编码基因ddc、pale和trh的表达水平均显著上升;长时间处理后对雄蝇运动和睡眠的影响较小.本研究认为利用模拟微重力效应研究微重力对果蝇运动及睡眠的影响是可行的,相关研究结果对航天医学研究具有借鉴意义.     

Role for Circadian Clock Genes in Seasonal Timing: Testing the Bünning Hypothesis

A major question in chronobiology focuses around the “Bünning hypothesis” which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. Here, we used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. We have corroborated previous observations that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Here, we show that arrhythmic mutant strains, per⁰¹, tim⁰¹ and Clk^Jrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. Our results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements.     

Circadian expression of the presynaptic active zone protein bruchpilot in the lamina of Drosophila melanogaster

In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild‐type Canton‐S flies in constant darkness (DD), nor in norpA⁷ phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per⁰ arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton‐S flies in DD, as well as in the norpA⁷ mutant in LD, but is absent in per⁰¹, tim,⁰¹ and cry⁰¹ mutants in LD. In addition both peaks seem to depend on clock gene‐expressing photoreceptors and glial cells of the visual system. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

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.     

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.     

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.     

Novel masking effects of light are revealed in Drosophila by skeleton photoperiods

Here, we show that in a skeleton photoperiod where all midday light is removed from a standard laboratory 12:12 LD photoperiod, a large diurnal peak of activity is revealed that is continuous with the E peak seen in constant dark (DD). We further show that the circadian clock gene tim regulates light-dependent masking of daytime activity, but the clock gene per does not. Finally, relative to wild-type flies, mutants for both of these clock genes showed increased nighttime activity in the skeleton photoperiod but not in the standard photoperiod. This result suggests that nighttime activity is suppressed by the intact circadian clock, and in its absence, by exposure to a standard photoperiod. These results support and extend the literature addressing the complex interactions between masking and clock-controlled components of overt circadian rhythms.     

Scotopic illumination enhances entrainment of circadian rhythms to lengthening light:dark cycles

Endogenously generated circadian rhythms are synchronized with the environment through phase-resetting actions of light. Starlight and dim moonlight are of insufficient intensity to reset the phase of the clock directly, but recent studies have indicated that dim nocturnal illumination may otherwise substantially alter entrainment to bright lighting regimes. In this article, the authors demonstrate that, compared to total darkness, dim illumination at night (< 0.010 lux) alters the entrainment of male Syrian hamsters to bright-light T cycles, gradually increasing in cycle length (T) from 24 h to 30 h. Only 1 of 18 hamsters exposed to complete darkness at night entrained to cycles of T > 26 h. In the presence of dim nocturnal illumination, however, a majority of hamsters entrained to Ts of 28 h or longer. The presence or absence of a running wheel had only minor effects on entrainment to lengthening light cycles. The results further establish the potent effects of scotopic illumination on circadian entrainment and suggest that naturalistic ambient lighting at night may enhance the plasticity of the circadian pacemaker.     

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.     

Insights into the Molecular Mechanisms of Temperature Compensation from the Drosophila Period and Timeless Mutants

The relative constancy of the circadian period over a wide range of temperatures is a general property of circadian rhythms. Insights into the molecular mechanisms of temperature compensation are emerging from genetic and molecular genetic studies of the period (per) and timeless (tim) genes in Drosophila. These genes encode proteins that are thought to be part of a negative feedback cycle, which results in circadian oscillations of both per and tim mRNA, as well as a complex of the two proteins. Complex formation is temporally regulated and apparently necessary for nuclear localization of both per and tim proteins. While insights into the roles of per and tim in temperature compensation have been intriguing, they have also been somewhat perplexing. For instance, the interaction of wild-type per peptides is relatively insensitive to temperature in the yeast two-hybrid assay or in assays employing in-vitro-translated peptides, while the interaction of per^L mutant peptides is reduced at a high temperature. Apparently, the per^L mutation increases an intramolecular interaction between different parts of the per peptide in these assays, and this interaction reduces the amount of per homodimer. On the other hand, the same assays show that the intermolecular interaction between the per and tim peptides is reduced at a high temperature by the per^L mutation; this reduction does not require the competing intramolecular interaction. Despite this difference, in all of the experiments employing these assays the per^L mutation has rendered per-per and per-tim peptide interactions sensitive to high temperature, so it is likely that one or both of these reduced interactions contribute to the longer circadian periods at high temperature in per^L mutant flies. However, the tim^SL and per^S mutations, as well as deletion of the Thr-Gly repeats from per, affect temperature compensation but have not been shown to affect these molecular interactions of per and tim. Finally, a recent report of oscillating per and tim proteins in the cytoplasm (rather than the nuclei) of silk moth neurons may suggest an alternative mechanism for per and tim function in these cells. (Chronobiology International 14(5), 455–468, 1997)     

Illuminance preferences of nocturnal primates

Laboratory tests reveal a preference for illuminances in a broad range of night light by individuals belonging to four species of nocturnal primates (Aotus trivirgatus, Galago crassicaudatus, Galago senegalensis, andNycticebus coucang). In volitional tests the animals altered the light level very frequently, in one case as often as every 7 sec. In these tests the animals tended to avoid total darkness and extremely dim light as well as moderately bright light. These avoided levels, particularly extremely dim light and darkness, inhibited locomotor activity. The greater bush babies preferred dimmer light for manipulative activities than for locomotion. Nocturnal primates differ from nocturnal rodents in being much more highly motivated to seek variety and frequent stimulus change when in deprivation conditions, and in their avoiding and being markedly inhibited by darkness.     

Circadian Control of Dendrite Morphology in the Visual System of Drosophila melanogaster

Background

In the first optic neuropil (lamina) of the fly''s visual system, monopolar cells L1 and L2 and glia show circadian rhythms in morphological plasticity. They change their size and shape during the day and night. The most pronounced changes have been detected in circadian size of the L2 axons. Looking for a functional significance of the circadian plasticity observed in axons, we examined the morphological plasticity of the L2 dendrites. They extend from axons and harbor postsynaptic sites of tetrad synaptic contacts from the photoreceptor terminals.

Methodology/Principal Findings

The plasticity of L2 dendrites was evaluated by measuring an outline of the L2 dendritic trees. These were from confocal images of cross sections of L2 cells labeled with GFP. They were in wild-type and clock mutant flies held under different light conditions and sacrified at different time points. We found that the L2 dendrites are longest at the beginning of the day in both males and females. This rhythm observed under a day/night regime (LD) was maintained in constant darkness (DD) but not in continuous light (LL). This rhythm was not present in the arrhythmic per⁰¹ mutant in LD or in DD. In the clock photoreceptor cry^b mutant the rhythm was maintained but its pattern was different than that observed in wild-type flies.

Conclusions/Significance

The results obtained showed that the L2 dendrites exhibit circadian structural plasticity. Their morphology is controlled by the per gene-dependent circadian clock. The L2 dendrites are longest at the beginning of the day when the daytime tetrad presynaptic sites are most numerous and L2 axons are swollen. The presence of the rhythm, but with a different pattern in cry^b mutants in LD and DD indicates a new role of cry in the visual system. The new role is in maintaining the circadian pattern of changes of the L2 dendrite length and shape.     

Interaction between sexes and between different circadian phenotypes affects lifespan in the fruit fly Drosophila melanogaster

Circadian clocks regulate the daily temporal structure of physiological and behavioural functions. In the fruit fly Drosophila melanogaster Meigen, disruption of daily rhythms is suggested to reduce the fly's lifespan. In the present study, because pairs of mixed‐sex flies are known to show an activity pattern different from that of individual flies, this hypothesis is tested by measuring the lifespan of flies housed same‐sexually or mixed‐sexually under an LD 12 : 12 h photocycle at a constant temperature of 25 °C. The effect of housing wild‐type (Canton‐S) flies with period (per) circadian clock mutant flies is also examined because the mutant flies have different daily activity patterns. When males and females of wild‐type flies are housed together, their lifespan is substantially lengthened (males) or shortened (females) compared with same‐sex housed flies. The shortening of the lifespan in females is significantly enhanced when mated with per mutant males. The shortening effects are significantly reduced when the mixed‐sex interaction is limited for the first 5 days after emergence. A slight elongation in lifespan, rather than a reduction, occurs when wild‐type females are housed same‐sexually with per⁰ or per^L mutant flies. In male flies, the elongation of lifespan occurs not only when wild‐type males are housed with wild‐type, per⁰ or per^L females, but also when housed with per⁰ or per^S mutant males. Mixed‐sex couples always show altered daily locomotor rhythms with an enhanced night‐time activity, whereas same‐sex couples show daily behavioural profiles slightly altered but essentially similar to a sum of the respective two flies. No significant correlation is found between the lifespan and reproductive capacity. These results suggest that the alteration of daily activity rhythms and sexual interaction may have significant impact on the fly's lifespan.