Temperature Entrainment of Circadian Locomotor and Transcriptional Rhythms in the Cricket, Gryllus bimaculatus.

Most animals exhibit circadian rhythms in various physiological and behavioral functions regulated by circadian clock that resides in brain and in many peripheral tissues. Temperature cycle is an important time cue for entrainment, even in mammals, since the daily change in body temperature is thought to be used for phase regulation of clocks in peripheral tissues. However, little is known about the mechanisms by which temperature resets the clock. In the present study, we investigated the effect of temperature on circadian activity rhythm and clock gene transcription by using the cricket, Gryllus bimaculatus. We show that temperature cycle can entrain both behavioral and transcriptional rhythms of clock genes, such as period, timeless, cryptochrome2 and cycle in the circadian pacemaker tissue, optic lobe. Under temperature cycle, phase of evening peak of locomotor activity occurred 1 h before the warm-to-cold phase transition, which is associated with earlier peaks of mRNA expression rhythm of the clock genes than that under light/dark cycles. When the temperature cycle was advanced by 6 h, behavioral rhythms re-entrained to newly phased temperature cycle after ∼16 transient cycles. The mRNA oscillation of period and timeless gained stable rhythm under phase advanced temperature cycles with a lesser number of transient cycles than cryptochrome2 and cycle. These results suggest that temperature cycle can entrain behavioral and molecular rhythms in cricket and clock genes vary in sensitivity to temperature. It is thus likely that clock genes play differential roles in resetting the clock with environmental temperature changes.

The duration of caffeine treatment plays an essential role in its effect on sleep and circadian rhythm.

Sleep is regulated by the homeostatic system and the circadian clock. Caffeine intake promotes wakefulness in Drosophila. In humans, caffeine is consumed on a daily basis and hence it is important to understand the effect of prolonged caffeine intake on both circadian and homeostatic regulation of sleep. Furthermore, sleep changes with age and the impact of caffeine on age-dependent sleep fragmentation are yet to be understood. Hence in the present study, we examined the effect of short exposure to caffeine on homeostatic sleep and age-dependent sleep fragmentation in Drosophila. We further assessed the effect of prolonged exposure to caffeine on homeostatic sleep and circadian clock. The results of our study showed that short exposure to caffeine reduces sleep and food intake in mature flies. It also enhances sleep fragmentation with increasing age. However, we have not assessed the effect of caffeine on food intake in older flies. On the other hand, prolonged caffeine exposure did not exert any significant effect on the duration of sleep and food intake in mature flies. Nevertheless, prolonged caffeine ingestion decreased the morning and evening anticipatory activity in these flies indicating that it affects the circadian rhythm. These flies also exhibited phase delay in the clock gene timeless transcript oscillation and exhibited either behavioral arrhythmicity or a longer free-running period under constant darkness. In summary, the results of our studies showed that short exposure to caffeine increases the sleep fragmentation with age whereas prolonged caffeine exposure disrupts the circadian clock.

Modified Capillary Feeder assay using micro-tips to measure real-time feeding in Drosophila.

Capillary Feeder assay (CAFE) is a real-time feeding assay used in Drosophila that employs micro-capillaries, which are costly. Here, we present a modified version of the assay by replacing micro-capillaries with micro-tips, hence ensuring the same principle with cost reduction by 500 times. We developed a mathematical approach to measure volume for the conical shaped micro-tips. In this protocol, we describe step-by-step procedures of pre-assay setup along with fly rearing; assay setup included with detailed analysis for volume calculations. For further verification and use of this protocol, please refer to Segu and Kannan.1.

miR-277 regulates the phase of circadian activity-rest rhythm in Drosophila melanogaster.

Circadian clocks temporally organize behaviour and physiology of organisms with a rhythmicity of about 24 h. In Drosophila, the circadian clock is composed of mainly four clock genes: period (per), timeless (tim), Clock (Clk) and cycle (cyc) which constitutes the transcription-translation feedback loop. The circadian clock is further regulated via post-transcriptional and post-translational mechanisms among which microRNAs (miRNAs) are well known post-transcriptional regulatory molecules. Here, we identified and characterized the role of miRNA-277 (miR-277) expressed in the clock neurons in regulating the circadian rhythm. Downregulation of miR-277 in the pacemaker neurons expressing circadian neuropeptide, pigment dispersing factor (PDF) advanced the phase of the morning activity peak under 12 h light: 12 h dark cycles (LD) at lower light intensities and these flies exhibited less robust rhythms compared to the controls under constant darkness. In addition, downregulation of miR-277 in the PDF expressing neurons abolished the Clk gene transcript oscillation under LD. Our study points to the potential role of miR-277 in fine tuning the Clk expression and in maintaining the phase of the circadian rhythm in Drosophila.

Clock accuracy and precision evolve as a consequence of selection for adult emergence in a narrow window of time in fruit flies Drosophila melanogaster.

Although circadian clocks are believed to have evolved under the action of periodic selection pressures (selection on phasing) present in the geophysical environment, there is very little rigorous and systematic empirical evidence to support this. In the present study, we examined the effect of selection for adult emergence in a narrow window of time on the circadian rhythms of fruit flies Drosophila melanogaster. Selection was imposed in every generation by choosing flies that emerged during a 1 h window of time close to the emergence peak of baseline/control flies under 12 h:12 h light:dark cycles. To study the effect of selection on circadian clocks we estimated several quantifiable features that reflect inter- and intra-individual variance in adult emergence and locomotor activity rhythms. The results showed that with increasing generations, incidence of adult emergence and activity of adult flies during the 1 h selection window increased gradually in the selected populations. Flies from the selected populations were more homogenous in their clock period, were more coherent in their phase of entrainment, and displayed enhanced accuracy and precision in their emergence and activity rhythms compared with controls. These results thus suggest that circadian clocks in D. melanogaster evolve enhanced accuracy and precision when subjected to selection for emergence in a narrow window of time.

A model based on oscillatory threshold and build-up of a developmental substance explains gating of adult emergence in Drosophila melanogaster.

Adult emergence (eclosion) of fruit flies Drosophila melanogaster under constant laboratory conditions follows a circadian pattern with bouts of eclosion recurring at approximately 24 h intervals. Under periodic light:dark (LD) cycles, adults emerge only during a specific time of the day followed by little or no emergence for the rest of the day. This phenomenon is therefore equated to a gate of emergence that, when open, allows adults to emerge and when closed, no emergence takes place. In this study, we attempt to understand the mechanism underlying adult emergence rhythm in D. melanogaster using a model based on interplay between developmental and circadian clock systems. The model is composed of an oscillatory threshold of a substance that builds up during pre-adult development. Computer simulations based on this model enabled us to make specific predictions about the 'gate width' of the adult emergence rhythm under conditions of fast/slow pre-adult development and short/long circadian periods, which we subsequently tested empirically. The main predictions from the simulations are: (1) flies with faster development have greater gate width and vice versa, and (2) flies with faster circadian clocks have shorter gate width and vice versa. To empirically validate these predictions, we carried out experiments on D. melanogaster populations known to have fast/slow pre-adult development, short/long circadian periods and narrow/wide gate width. Additionally, we manipulated the rate of pre-adult development of the above flies by increasing/decreasing ambient temperature to further examine the influence of developmental rates on gate width of adult emergence rhythm by a complementary approach. The results show that gate width is greatly influenced by the duration of pre-adult development and the length of circadian cycles. This suggests that the adult emergence rhythm of D. melanogaster may be based on mechanisms involving oscillatory threshold and build-up of a developmental substance.

Chronotype differences in Drosophila are enhanced by semi-natural conditions.

Morning and evening chronotypes of sleep/wake cycles in humans are often metaphorically termed as 'larks' and 'owls'. We derived Drosophila populations early and late, displaying lark- and owl-like emergence chronotypes by imposing selection for adult emergence during morning and evening hours. Preference for morning and evening emergence in these populations was accompanied by divergence in their circadian period (τ) and photic phase response curve. To test if lark- and owl-like emergence chronotypes displayed by these flies under weak environmental cycles of the laboratory would also persist in nature where several zeitgebers are present in the strongest form, we examined the emergence rhythm of early and late flies under semi-natural conditions. The early and late flies not only continued to exhibit divergent emergence waveforms under semi-natural conditions, the differences became even more prominent. However, phases of early and late emergence waveforms did not match natural morning and evening transitions, unlike that observed under laboratory conditions. These results thus provide evidence consistent with the notion that chronotypes are the result of interactions between circadian clocks and natural environmental cycles.

Only time will tell: the interplay between circadian clock and metabolism.

In most organisms ranging from cyanobacteria to humans, the endogenous timekeeping system temporally coordinates the behavioral, physiological, and metabolic processes with a periodicity close to 24 h. The timing of these daily rhythms is orchestrated by the synchronized oscillations of both the central pacemaker in the brain and the peripheral clocks located across multiple organs and tissues. A growing body of evidence suggests that the central circadian clock and peripheral clocks residing in the metabolically active tissues are incredibly well coordinated to confer coherent metabolic homeostasis. The interplay between nutrient metabolism and circadian rhythms can occur at various levels supported by the molecular clock network, multiple systemic mechanisms, and the neuroendocrine signaling pathways. While studies suggest the reciprocal regulation between circadian clock and metabolism, it is important to understand the precise mechanisms and the underlying pathways involved in the cross-talk among circadian oscillators and diverse metabolic networks. In addition to the internal synchronization of the metabolic rhythms, feeding time is considered as a potential external synchronization cue that fine tunes the timing of the circadian rhythms in metabolic peripheral clocks. A deeper understanding of how the timing of food intake and the diet composition drive the tissue-specific metabolic rhythms across the body is concomitantly important to develop novel therapeutic strategies for the metabolic disorders arising from circadian misalignment. This review summarizes the recent advancements in the circadian clock regulation of nutrient metabolism and discusses the current understanding of the metabolic feedback signals that link energy metabolism with the circadian clock.

Post-transcriptional modulators and mediators of the circadian clock.

The endogenous circadian timekeeping system drives ~24-h rhythms in gene expression and rhythmically coordinates the physiology, metabolism and behavior in a wide range of organisms. Regulation at various levels is important for the accurate functioning of this circadian timing system. The core circadian oscillator consists of an interlocked transcriptional-translational negative feedback loop (TTFL) that imposes a substantial delay between the accumulation of clock gene mRNA and its protein to generate 24-h oscillations. This TTFL mediated daily oscillation of clock proteins is further fine-tuned by post-translational modifications that regulate the clock protein stability, interaction with other proteins and subcellular localization. Emerging evidence from various studies indicates that besides TTFL and post-translational modifications, post-transcriptional regulation plays a key role in shaping the rhythmicity of mRNAs and to delay the accumulation of clock proteins in relation to their mRNAs. In this review, we summarize the current knowledge on the importance of post-transcriptional regulatory mechanisms such as splicing, polyadenylation, the role of RNA-binding proteins, RNA methylation and microRNAs in the context of shaping the circadian rhythmicity in Drosophila and mammals. In particular, we discuss microRNAs, an important player in post-transcriptional regulation of core-clock machinery, circadian neural circuit, clock input, and output pathways. Furthermore, we provide an overview of the microRNAs that exhibit diurnal rhythm in expression and their role in mediating rhythmic physiological processes.

Mycobacterium smegmatis Vaccine Vector Elicits CD4+ Th17 and CD8+ Tc17 T Cells With Therapeutic Potential to Infections With Mycobacterium avium.

Mycobacterium avium (Mav) complex is increasingly reported to cause non-tuberculous infections in individuals with a compromised immune system. Treatment is complicated and no vaccines are available. Previous studies have shown some potential of using genetically modified Mycobacterium smegmatis (Msm) as a vaccine vector to tuberculosis since it is non-pathogenic and thus would be tolerated by immunocompromised individuals. In this study, we used a mutant strain of Msm disrupted in EspG3, a component of the ESX-3 secretion system. Infection of macrophages and dendritic cells with Msm ΔespG3 showed increased antigen presentation compared to cells infected with wild-type Msm. Vaccination of mice with Msm ΔespG3, expressing the Mav antigen MPT64, provided equal protection against Mav infection as the tuberculosis vaccine, Mycobacterium bovis BCG. However, upon challenge with Mav, we observed a high frequency of IL-17-producing CD4+ (Th17 cells) and CD8+ (Tc17 cells) T cells in mice vaccinated with Msm ΔespG3::mpt64 that was not seen in BCG-vaccinated mice. Adoptive transfer of cells from Msm ΔespG3-vaccinated mice showed that cells from the T cell compartment contributed to protection from Mav infection. Further experiments revealed Tc17-enriched T cells did not provide prophylactic protection against subsequent Mav infection, but a therapeutic effect was observed when Tc17-enriched cells were transferred to mice already infected with Mav. These initial findings are important, as they suggest a previously unknown role of Tc17 cells in mycobacterial infections. Taken together, Msm ΔespG3 shows promise as a vaccine vector against Mav and possibly other (myco)bacterial infections.

Benzoic Acid-Inducible Gene Expression in Mycobacteria.

Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.

Selection for narrow gate of emergence results in correlated sex-specific changes in life history of Drosophila melanogaster.

Since the ability to time rhythmic behaviours in accordance with cyclic environments is likely to confer adaptive advantage to organisms, the underlying clocks are believed to be selected for stability in timekeeping over evolutionary time scales. Here we report the results of a study aimed at assessing fitness consequences of a long-term laboratory selection for tighter circadian organisation using fruit fly Drosophila melanogaster populations. We selected flies emerging in a narrow window of 1 h in the morning for several generations and assayed their life history traits such as pre-adult development time, survivorship, adult lifespan and lifetime fecundity. We chose flies emerging during the selection window (in the morning) and another window (in the evening) to represent adaptive and non-adaptive phenotypes, respectively, and examined the correlation of emergence time with adult fitness traits. Adult lifespan of males from the selected populations does not differ from the controls, whereas females from the selected populations have significantly shorter lifespan and produce more eggs during their mid-life compared to the controls. Although there is no difference in the lifespan of males of the selected populations, whether they emerge in morning or evening window, morning emerging females live slightly shorter and lay more eggs during the mid-life stage compared to those emerging in the evening. Interestingly, such a time of emergence dependent difference in fitness is not seen in flies from the control populations. These results, therefore, suggest reduced lifespan and enhanced mid-life reproductive output in females selected for narrow gate of emergence, and a sex-dependent genetic correlation between the timing of emergence and key fitness traits in these populations.

Strong (type 0) phase resetting of activity-rest rhythm in fruit flies, Drosophila melanogaster, at low temperature.

Amplitude modulation in limit cycle models of circadian clocks has been previously formulated to explain the phenomenon of temperature compensation. These models propose that invariance of clock period (τ) with changing temperature is a result of the system traversing small or large limit cycles such that despite a decrease or an increase in the linear velocity of the clock owing to slowing down or speeding up of the underlying biochemical reactions, respectively, the angular velocity and, thus, the clock period remain constant. In addition, these models predict that phase resetting behavior of circadian clocks described by limit cycles of different amplitudes at low or high temperatures will be drastically different. More specifically, this class of models predicts that at low temperatures, circadian clocks will respond to perturbations by eliciting larger phase shifts by virtue of their smaller amplitude and vice versa. Here, we present the results of our tests of this prediction: We examined the nature of photic phase response curves (PRCs) and phase transition curves (PTCs) for the circadian clocks of 4 wild-type fruit fly Drosophila melanogaster populations at 3 different ambient temperatures (18, 25, and 29 °C). Interestingly, we observed that at the low temperature of 18 °C, fly clocks respond to light perturbations more strongly, eliciting strong (type 0) PRCs and PTCs, while at moderate (25 °C) and high (29 °C) temperatures the same stimuli evoke weak (type 1) responses. This pattern of strong and weak phase resetting at low and high temperatures, respectively, renders support for the limit cycle amplitude modulation model for temperature compensation of circadian clocks.

The Mycobacterium tuberculosis PE proteins Rv0285 and Rv1386 modulate innate immunity and mediate bacillary survival in macrophages.

The unique PE/PPE multigene family of proteins occupies almost 10% of the coding sequence of Mycobacterium tuberculosis (M.tb), the causative agent of human tuberculosis. Although some members of this family have been shown to be involved in pathways essential to M.tb pathogenesis, their precise physiological functions remain largely undefined. Here, we investigate the roles of the conserved members of the 'PE only' subfamily Rv0285 (PE5) and Rv1386 (PE15) in mediating host-pathogen interactions. Recombinant Mycobacterium smegmatis strains expressing PE5 and PE15 showed enhanced survival vs controls in J774.1 and THP-1 macrophages - this increase in viable counts was correlated with a reduction in transcript levels of inducible nitric oxide synthase. An up-regulation of anti- and down-regulation of pro-inflammatory cytokine levels was also observed in infected macrophages implying an immuno-modulatory function for these proteins. Induction of IL-10 production upon infection of THP-1 macrophages was associated with increased phosphorylation of the MAP Kinases p38 and ERK1/2, which was abolished in the presence of the pharmacological inhibitors SB203580 and PD98059. The PE5-PPE4 and PE15-PPE20 gene pairs were observed to be co-operonic in M.tb, hinting at an additional level of complexity in the functioning of these proteins. We conclude that M.tb exploits the PE proteins to evade the host immune response by altering the Th1 and Th2 type balance thereby favouring in vivo bacillary survival.

Stability of adult emergence and activity/rest rhythms in fruit flies Drosophila melanogaster under semi-natural condition.

Here we report the results of a study aimed at examining stability of adult emergence and activity/rest rhythms under semi-natural conditions (henceforth SN), in four large outbred fruit fly Drosophila melanogaster populations, selected for emergence in a narrow window of time under laboratory (henceforth LAB) light/dark (LD) cycles. When assessed under LAB, selected flies display enhanced stability in terms of higher amplitude, synchrony and accuracy in emergence and activity rhythms compared to controls. The present study was conducted to assess whether such differences in stability between selected and control populations, persist under SN where several gradually changing time-cues are present in their strongest form. The study revealed that under SN, emergence waveform of selected flies was modified, with even more enhanced peak and narrower gate-width compared to those observed in the LAB and compared to control populations in SN. Furthermore, flies from selected populations continued to exhibit enhanced synchrony and accuracy in their emergence and activity rhythms under SN compared to controls. Further analysis of zeitgeber effects revealed that enhanced stability in the rhythmicity of selected flies under SN was primarily due to increased sensitivity to light because emergence and activity rhythms of selected flies were as stable as controls under temperature cycles. These results thus suggest that stability of circadian rhythms in fruit flies D. melanogaster, which evolved as a consequence of selection for emergence in a narrow window of time under weak zeitgeber condition of LAB, persists robustly in the face of day-to-day variations in cycling environmental factors of nature.

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.

Temperature can entrain egg laying rhythm of Drosophila but may not be a stronger zeitgeber than light.

In Drosophila multiple circadian oscillators and behavioral rhythms are known to exist, yet most previous studies that attempted to understand circadian entrainment have focused on the activity/rest rhythm and to some extent the adult emergence rhythm. Egg laying behavior of Drosophila females also follows circadian rhythmicity and has been seen to deviate substantially from the better characterized rhythms in a few aspects. Here we report the findings of our study aimed at evaluating how circadian egg laying rhythm in fruit flies Drosophila melanogaster entrains to time cues provided by light and temperature. Previous studies have shown that activity/rest rhythm of flies entrains readily to light/dark (LD) and temperature cycles (TC). Our present study revealed that egg laying rhythm of a greater percentage of females entrains to TC compared to LD cycles. Therefore, in the specific context of our study this result can be taken to suggest that egg laying clocks of D. melanogaster entrains to TC more readily than LD cycles. However, when TC were presented along with out-of-phase LD cycles, the rhythm displayed two peaks, one occurring close to lights-off and the other near the onset of low temperature phase, indicating that upon entrainment by TC, LD cycles may be able to exert a greater influence on the phase of the rhythm. These results suggest that temperature and light associatively entrain circadian egg laying clocks of Drosophila.