Circadian dysfunction reduces lifespan in Drosophila melanogaster

Circadian clocks regulate physiological and behavioral processes in a wide variety of organisms, and any malfunction in these clocks can cause significant health problems. In this paper, we report the results of our study on the physiological consequences of circadian dysfunction (malfunctioning of circadian clocks) in two wild-type populations of fruit flies (Drosophila melanogaster). We assayed locomotor activity behavior and lifespan among adult flies kept under constant dark (DD) conditions of the laboratory, wherein they were categorized as rhythmic if their activity/rest schedules followed circadian (approximately 24 h) patterns, and as arrhythmic if their activity/rest schedules did not display any pattern. The rhythmic flies from both populations lived significantly longer than the arrhythmic ones. Based on these results, we conclude that circadian dysfunction is deleterious, and proper functioning of circadian clocks is essential for the physiological well being of D. melanogaster.     

Robustness of Circadian Timing Systems Evolves in the Fruit Fly Drosophila melanogaster as a Correlated Response to Selection for Adult Emergence in a Narrow Window of Time

Robustness is a fundamental property of biological timing systems that is likely to ensure their efficient functioning under a wide range of environmental conditions. Here we report the findings of our study aimed at examining robustness of circadian clocks in fruit fly Drosophila melanogaster populations selected to emerge as adults within a narrow window of time. Previously, we have reported that such flies display enhanced synchrony, accuracy, and precision in their adult emergence and activity/rest rhythms. Since it is expected that accurate and precise circadian clocks may confer enhanced stability in circadian time-keeping, we decided to examine robustness in circadian rhythms of flies from the selected populations by subjecting them to a variety of environmental conditions comprising of a range of photoperiods, light intensities, ambient temperatures, and constant darkness. The results revealed that adult emergence and activity/rest rhythms of flies from the selected stocks were more robust than controls, as they displayed enhanced stability under a wide variety of environmental conditions. These results suggest that selection for adult emergence within a narrow window of time results in the evolution of robustness in circadian timing systems of the fruit fly D. melanogaster. (Author correspondence: vsharma@jncasr.ac.in or vksharmas@gmail.com)     

Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants

Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons – the dorsal and ventral lateral neurons – are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LN_vs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LN_vs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LN_vs stained. However, these flies lacked the LN_v fibers in the superior protocerebrum. The results suggest that the presence of single LN_vs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LN_v terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LN_vs indicates that also other cells contribute to the rhythmic control of locomotor activity. Accepted: 17 September 1997     

Egg-laying rhythm in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Extensive research has been carried out to understand how circadian clocks regulate various physiological processes in organisms. The discovery of clock genes and the molecular clockwork has helped researchers to understand the possible role of these genes in regulating various metabolic processes. In Drosophila melanogaster, many studies have shown that the basic architecture of circadian clocks is multi-oscillatory. In nature, different neuronal subgroups in the brain of D. melanogaster have been demonstrated to control different circadian behavioural rhythms or different aspects of the same circadian rhythm. Among the circadian phenomena that have been studied so far in Drosophila, the egg-laying rhythm is unique, and relatively less explored. Unlike most other circadian rhythms, the egg-laying rhythm is rhythmic under constant light conditions, and the endogenous or free-running period of the rhythm is greater than those of most other rhythms. Although the clock genes and neurons required for the persistence of adult emergence and activity/rest rhythms have been studied extensively, those underlying the circadian egg-laying rhythm still remain largely unknown. In this review, we discuss our current understanding of the circadian egg-laying rhythm in D. melanogaster, and the possible molecular and physiological mechanisms that control the rhythmic output of the egg-laying process.     

Temperature sensitivity of circadian clocks is conserved across Drosophila species melanogaster,malerkotliana and ananassae

Light and temperature are the major environmental cycles that can synchronize circadian rhythms in a variety of organisms. Previously, we have shown that under light/dark cycles of various photoperiods, the Drosophila species ananassae exhibits unimodal activity pattern with a prominent morning activity peak in contrast with Drosophila melanogaster and Drosophila malerkotliana, which show bimodal activity pattern with morning and evening activity peaks. Here we report that circadian clocks controlling activity/rest rhythm of these two less-studied species D. malerkotliana and D. ananassae can be synchronized by temperature cycles and that even under temperature cycles D. ananassae exhibits only a pronounced morning (thermophase onset) activity peak. Although D. melanogaster and D. ananassae exhibit differences in the phase of activity/rest rhythm under temperature cycles, circadian clocks of both show similar sensitivity to warm temperature pulses. Circadian period of activity/rest rhythm of D. ananassae differs from the other two species at some moderate-range temperatures; however, in conditions that are more extreme, circadian clocks of D. melanogaster, D. malerkotliana and D. ananassae appear to be largely temperature compensated.     

The 28th Conference of the International Society for Chronobiology (ISC) will be held in Bucharest (Romania), at Ramada Parc Hotel on 4–8 June 2014

The eclosion rhythm of a laboratory population of Drosophila melanogaster was studied under 12h light, 12h dark (LD 12:12) cycles. Although most of the flies were found to eclose just after “lights on” in LD 12:12, termed within gate (WG) flies, a few flies were found to eclose nearly 10h after peak eclosion, termed outside gate (OG) flies. The circadian parameters of the clocks controlling oviposition rhythms in the WG and the OG flies were estimated to understand the cause of such differences in the timing of eclosion. The distribution of the fraction of individual flies exhibiting single, multiple, and no significant period in the WG flies was significantly different from distribution in the OG flies. Compared to the WG flies, more OG flies were found to exhibit oviposition rhythm with multiple periodicity, whereas more WG flies exhibited an oviposition rhythm with a single significant period. The fraction of flies with arrhythmic oviposition was similar in both the WG and the OG flies. Free-running period τ in constant darkness (DD) and the phase angle difference ψ in LD 12:12 for the oviposition rhythm of WG and OG flies were significantly different. These results suggest that the differences in the time of eclosion between the flies eclosing within the gate and outside the gate of eclosion are probably due to differences in the circadian system controlling eclosion, which is reflected by the differences in their oviposition rhythm. (Chronobiology International, 18(4), 601–612, 2001)     

Correlated changes in circadian clocks in response to selection for faster pre-adult development in fruit flies Drosophila melanogaster

Although, circadian clocks are believed to be involved in the regulation of life-history traits such as pre-adult development time and lifespan in fruit flies Drosophila melanogaster, there is very little unequivocal evidence either to support or refute this. Here we report the results of a long-term study aimed at examining the role of circadian clocks in the temporal regulation of pre-adult development in D. melanogaster. We employed laboratory selection protocol for faster pre-adult development on four large, outbred, random mating populations of Drosophila. We assayed pre-adult development time and circadian period of locomotor activity rhythm of these flies at regular intervals of 5–10 generations. After 50 generations of selection, the overall egg-to-adult duration in the selected stocks was reduced by ~29 h (~12.5 %) relative to controls, with the selected populations showing a concurrent reduction in time taken to hatching, pupation and wing pigmentation, by ~2, ~16, and ~25.2 h, respectively. Furthermore, selected populations showed a concomitant reduction in the circadian period of locomotor activity rhythm, implying that circadian clocks and development time are correlated. Thus, our study provides the first ever unequivocal evidence for the evolution of circadian clocks as a correlated response to selection for faster pre-adult development, suggesting that circadian clocks and development are linked in fruit flies D. melanogaster.     

Cryptochrome restores dampened circadian rhythms and promotes healthspan in aging Drosophila

Circadian clocks generate daily rhythms in molecular, cellular, and physiological functions providing temporal dimension to organismal homeostasis. Recent evidence suggests two‐way relationship between circadian clocks and aging. While disruption of the circadian clock leads to premature aging in animals, there is also age‐related dampening of output rhythms such as sleep/wake cycles and hormonal fluctuations. Decay in the oscillations of several clock genes was recently reported in aged fruit flies, but mechanisms underlying these age‐related changes are not understood. We report that the circadian light–sensitive protein CRYPTOCHROME (CRY) is significantly reduced at both mRNA and protein levels in heads of old Drosophila melanogaster. Restoration of CRY using the binary GAL4/UAS system in old flies significantly enhanced the mRNA oscillatory amplitude of several genes involved in the clock mechanism. Flies with CRY overexpressed in all clock cells maintained strong rest/activity rhythms in constant darkness late in life when rhythms were disrupted in most control flies. We also observed a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY‐deficient mutants showed accelerated functional decline and accumulated greater oxidative damage. Interestingly, overexpression of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. Together, these data suggest novel anti‐aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging.     

Cyclic Presence and Absence of Conspecifics Alters Circadian Clock Phase But Does Not Entrain the Locomotor Activity Rhythm of the Fruit Fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms. (Author correspondence: vsharma@jncasr.ac.in, vksharmas@gmail.com)     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Pigment-dispersing hormone-immunoreactive neurons in the cockroach Leucophaea maderae share properties with circadian pacemaker neurons

Neurons immunoreactive with antisera against the crustacean peptide -pigment dispersing hormone fullfill several anatomical criteria proposed for circadian pacemakers in the brain of the cockroach Leucophaea maderae. These include position of somata, projections to the lamina and midbrain and possible coupling pathways between the two pacemakers through commissural fibers. In behavioral experiments combined with lesion studies and immunocytochemical investigations we examined whether the presence of pigment-dispersing hormone-immunoreactive arborizations in the midbrain of the cockroach correlates with the presence of circadian locomotor activity. No rhythm was detected after severing both optic stalks in any animal for at least 12 days. Within the same time pigment-dispersing hormone-immunoreactive fibers in the midbrain disappeared. Two to seven weeks after the operation some of the cockroaches regained circadian locomotor activity, while others remained arrhythmic. In all cockroaches which regained rhythmic behavior pigment-dispersing hormone-immunoreactive fibers had regenerated and had largely found their original targets within the brain. In all arrhythmic cockroaches either none or very little regeneration had occurred. The period of the regained circadian activity inversely correlated with the number of regenerated immunoreactive commissural fibers. These data provide further evidence for the involvement of pigment-dispersing hormone-immunoreactive neurons in circadian clocks of orthopteroid insects.Abbreviations DD constant darkness - PDH pigment-dispersing hormone - PDHLI pigmentdispersing hormone-like immunoreactivity - PDFL a pigment-dispersing factor containing cells in the lamina - PDFMe pigment-dispersing factor containing cells in the medulla - QV quantification value     

Early- and Late-Emerging Drosophila melanogaster Fruit Flies Differ in Their Sensitivity to Light During Morning and Evening

The authors derived early and late populations of fruit flies showing increased incidence of emergence during morning or evening hours by imposing selection for timing of emergence under 12:12?h light/dark (LD) cycles. From previous studies, it was clear that the increased incidence of adult emergence during morning and evening hours in early and late populations was a result of evolution of divergent and characteristic emergence waveforms in these populations. Such characteristic waveforms are henceforth referred to as “evolved emergence waveforms” (EEWs). The early and late populations also evolved different circadian clocks, which is evident from the divergence in their clock period (τ) and photic phase response curve (PRC). Although correlation between emergence waveforms and clock properties suggests functional significance of circadian clocks, τ and PRCs do not satisfactorily explain the early and late emergence phenotypes. In order to understand the functional significance of the PRC for early and late emergence phenotypes, the authors investigated whether circadian clocks of these flies exhibit any difference in photosensitivity under entrained conditions. Such differences would suggest that the light requirement for circadian entrainment of the emergence rhythm in early and late populations is different. To test this, they examined if early and late flies differ in their light utilization behavior, first by assaying their emergence rhythm under complete photoperiod and then in three different skeleton photoperiods. The results showed that early and late populations require different durations of light during the morning and evening to achieve their EEWs, suggesting that for the circadian entrainment of the emergence rhythm, early and late flies utilize light from different parts of the day. (Author correspondence: vsharma@jncasr.ac.in or vksharmas@gmail.com)     

Entrainment of Eclosion Rhythm in Drosophila melanogaster Populations Reared for More Than 700 Generations in Constant Light Environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light‐dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free‐running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights‐on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Evolution of circadian rhythms in Drosophila melanogaster populations reared in constant light and dark regimes for over 330 generations

Organisms are believed to have evolved circadian clocks as adaptations to deal with cyclic environmental changes, and therefore it has been hypothesized that evolution in constant environments would lead to regression of such clocks. However, previous studies have yielded mixed results, and evolution of circadian clocks under constant conditions has remained an unsettled topic of debate in circadian biology. In continuation of our previous studies, which reported persistence of circadian rhythms in Drosophila melanogaster populations evolving under constant light, here we intended to examine whether circadian clocks and the associated properties evolve differently under constant light and constant darkness. In this regard, we assayed activity-rest, adult emergence and oviposition rhythms of D. melanogaster populations which have been maintained for over 19 years (~330 generations) under three different light regimes – constant light (LL), light–dark cycles of 12:12 h (LD) and constant darkness (DD). We observed that while circadian rhythms in all the three behaviors persist in both LL and DD stocks with no differences in circadian period, they differed in certain aspects of the entrained rhythms when compared to controls reared in rhythmic environment (LD). Interestingly, we also observed that DD stocks have evolved significantly higher robustness or power of free-running activity-rest and adult emergence rhythms compared to LL stocks. Thus, our study, in addition to corroborating previous results of circadian clock evolution in constant light, also highlights that, contrary to the expected regression of circadian clocks, rearing in constant darkness leads to the evolution of more robust circadian clocks which may be attributed to an intrinsic adaptive advantage of circadian clocks and/or pleiotropic functions of clock genes in other traits.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.     

The circadian rhythm controls telomeres and telomerase activity

Circadian clocks are fundamental machinery in organisms ranging from archaea to humans. Disruption of the circadian system is associated with premature aging in mice, but the molecular basis underlying this phenomenon is still unclear. In this study, we found that telomerase activity exhibits endogenous circadian rhythmicity in humans and mice. Human and mouse TERT mRNA expression oscillates with circadian rhythms and are under the control of CLOCK–BMAL1 heterodimers. CLOCK deficiency in mice causes loss of rhythmic telomerase activities, TERT mRNA oscillation, and shortened telomere length. Physicians with regular work schedules have circadian oscillation of telomerase activity while emergency physicians working in shifts lose the circadian rhythms of telomerase activity. These findings identify the circadian rhythm as a mechanism underlying telomere and telomerase activity control that serve as interconnections between circadian systems and aging.     

Circadian regulation of response to oxidative stress in Drosophila melanogaster

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism’s internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H₂O₂), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H₂O₂ compared to wild-type flies, coinciding with enhanced generation of mitochondrial H₂O₂ and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.