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.     

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.     

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.     

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.     

Circadian clock properties of fruit flies Drosophila melanogaster exhibiting early and late emergence chronotypes

The role of circadian clocks in timing daily behaviors is widely acknowledged, and while empirical evidence suggests that clock period is correlated with the preferred phase of a rhythmic behavior (chronotype), other clock properties have also been hypothesized to underlie chronotype variation. Here, we report that fruit fly Drosophila melanogaster populations exhibiting evening emergence chronotype (late) are characterized by higher incidence of behavioral arrhythmicity in constant dim light, wider range of entrainment, reduced rates of re-entrainment to simulated jet-lag and higher amplitude of both entrained and free-running rhythms as compared to those exhibiting morning emergence chronotype (early). Our results thus highlight the role of circadian clock properties such as zeitgeber sensitivity, amplitude and coupling in driving chronotype variation.     

Casein kinase 2, circadian clocks, and the flight from mutagenic light

Circadian clocks play a fundamental role in biology and disease. Much has been learned about the molecular underpinnings of these biological clocks from genetic studies in model organisms, such as the fruit fly, Drosophila melanogaster. Here we review the literature from our lab and others that establish a role for the protein kinase CK2 in Drosophila clock timing. Among the clock genes described thus far, CK2 is unique in its involvement in plant, fungal, as well as animal circadian clocks. We propose that this reflects an ancient, conserved function for CK2 in circadian clocks. CK2 and other clock genes have been implicated in cellular responses to DNA damage, particularly those induced by ultraviolet (UV) light. The finding of a dual function of CK2 in clocks and in UV responses supports the notion that clocks evolved to assist organisms in avoiding the mutagenic effects of daily sunlight.     

Circadiane Uhren und Energiemetabolismus

Chrononutrition – circadian clocks and energy metabolism Genetically encoded endogenous clocks regulate 24‐hour rhythms of physiology and behavior. A central pacemaker residing in the suprachiasmatic nucleus synchronizes peripheral clocks found in all tissues with each other and with the external day‐night cycle. One function of circadian clocks is the regulation of energy metabolism via rhythmic activation of tissue‐specific clock‐controlled genes. In the liver, genes involved in glucose and lipid metabolism are regulated in this fashion, while in adipocytes, fatty acid release and adipokine secretion are controlled by the circadian clock. Disruption of circadian rhythms as seen, for example, in shift workers promotes the development of metabolic disorders such as obesity and type‐2 diabetes.     

Interaction of MAGED1 with nuclear receptors affects circadian clock function

The circadian clock has a central role in physiological adaption and anticipation of day/night changes. In a genetic screen for novel regulators of circadian rhythms, we found that mice lacking MAGED1 (Melanoma Antigen Family D1) exhibit a shortened period and altered rest–activity bouts. These circadian phenotypes are proposed to be caused by a direct effect on the core molecular clock network that reduces the robustness of the circadian clock. We provide in vitro and in vivo evidence indicating that MAGED1 binds to RORα to bring about positive and negative effects on core clock genes of Bmal1, Rev‐erbα and E4bp4 expression through the Rev‐Erbα/ROR responsive elements (RORE). Maged1 is a non‐rhythmic gene that, by binding RORα in non‐circadian way, enhances rhythmic input and buffers the circadian system from irrelevant, perturbing stimuli or noise. We have thus identified and defined a novel circadian regulator, Maged1, which is indispensable for the robustness of the circadian clock to better serve the organism.     

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD⁺ levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock^Δ19 mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.     

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)     

Modulating effects of hesperidin on circadian pattern indices of rotenone induced redox homeostasis in clock mutant (cryb) of Drosophila melanogaster

The circadian timing system controls drug metabolism and cellular processes over the 24 h period in every cell. Impaired redox homeostasis is a casual factor for a number of diseases and it is desirable to understand the orchestration of circadian clock under oxidative stress in the model organism, Drosophila melanogaster. This study evaluates the effect of hesperidin on the circadian rhythms of lipid peroxidation products and antioxidants during rotenone-induced oxidative stress in fruit fly. The characteristics of temporal rhythms (acrophase, amplitude, and mesor) of glutathione peroxides (GPx), reduced glutathione (GSH)), were markedly declined in rotenone-treated flies when compared to other groups. Treatment of hesperidin to rotenone-treated flies significantly increased the mesor and modified the amplitudes of antioxidants. Further, delays in acrophase in rotenone-induced flies were reversed by hesperidin treatment. Thus, treatment of hesperidin results in normalization of the altered rhythms of these indices plausibly by its cytoprotective and antioxidant effects. Impairment of 24 h rhythms in oxidative stress markers and antioxidants were observed during rotenone treatment and the impairment is severe in circadian clock mutant cry^b flies. A reversibility of rhythms was prominent consequent to hesperidin treatment in wild-type flies than cry^b flies. These findings revealed a role of circadian clock in redox homeostasis and the use of Drosophila model in screening putative antioxidative phytomedicines earlier to their use in mammalian systems.