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.     

Possible evidence for morning and evening oscillators in Drosophila melanogaster populations selected for early and late adult emergence

In this paper, we report the results of our study aimed at a systematic analysis of the circadian phenotypes of fruit flies Drosophila melanogaster selected for early and late adult emergence, in light of the "morning and evening oscillator" (M and E) model for circadian clocks. We monitored adult emergence and activity/rest rhythms in these flies under light/dark (LD) cycles with short (8:16 h), normal (12:12 h) and long (16:8 h) photoperiods, as well as under constant darkness (DD). Across all the three LD cycles, the early populations displayed a morning phenotype with peak of emergence and activity occurring earlier than the controls and greater anticipation to "lights-on" and weak anticipation to "lights-off", while the late populations showed an evening phenotype with peak of emergence and activity occurring later than the controls and greater anticipation to lights-off and weak anticipation to lights-on. The gate of adult emergence and duration of activity in the early populations was narrower than the controls, while those of the late populations were wider than the controls. In addition, the circadian periodicities of adult emergence and activity/rest rhythms of the early flies were significantly shorter than the controls, while those of the late flies were significantly longer than the controls. In summary, the circadian phenotypes indicate that the early populations have evolved a dominant M oscillator, while the late populations have evolved a dominant E oscillator, thus providing an empirical support for the M and E model in Drosophila.     

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)     

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

Feeding mistiming decreases reproductive fitness in flies

The diurnally active fruit flies prefer a major meal in the morning. Feeding the flies in the evening uncouples their metabolic cycle from circadian activity rhythms. A paper by Xu et?al. in this issue of Cell Metabolism found that such uncoupled rhythms reduce egg laying.     

Light Activates Output from Evening Neurons and Inhibits Output from Morning Neurons in the Drosophila Circadian Clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

Socially synchronized circadian oscillators

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian ‘clock’). The alternation of environmental light and darkness synchronizes (entrains) these rhythms to the natural day–night cycle, and underlying mechanisms have been investigated using singly housed animals in the laboratory. But, most species ordinarily would not live out their lives in such seclusion; in their natural habitats, they interact with other individuals, and some live in colonies with highly developed social structures requiring temporal synchronization. Social cues may thus be critical to the adaptive function of the circadian system, but elucidating their role and the responsible mechanisms has proven elusive. Here, we highlight three model systems that are now being applied to understanding the biology of socially synchronized circadian oscillators: the fruitfly, with its powerful array of molecular genetic tools; the honeybee, with its complex natural society and clear division of labour; and, at a different level of biological organization, the rodent suprachiasmatic nucleus, site of the brain''s circadian clock, with its network of mutually coupled single-cell oscillators. Analyses at the ‘group’ level of circadian organization will likely generate a more complex, but ultimately more comprehensive, view of clocks and rhythms and their contribution to fitness in nature.     

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.     

Sexual Interactions Influence the Molecular Oscillations in DN1 Pacemaker Neurons in Drosophila melanogaster

Circadian rhythms can synchronize to environmental time cues, such as light, temperature, humidity, and food availability. Previous studies have suggested that these rhythms can also be entrained by social interactions. Here, we used Drosophila melanogaster as a model to study the influence of socio-sexual interactions on the circadian clock in behavior and pacemaker neurons. If two flies of opposite sex were paired and kept in a small space, the daily activity patterns of the two flies were clearly different from the sum of the activity of single male and female flies. Compared with single flies, paired flies were more active in the night and morning, were more active during females’ active phase, and were less active during males’ active phase. These behavioral phenotypes are related to courtship behavior, but not to the circadian clock. Nevertheless, in male-female pairs of flies with clocks at different speeds (wild-type and per ^S flies), clock protein cycling in the DN1 pacemaker neurons in the male brain were slightly influenced by their partners. These results suggest that sexual interactions between male-female couples can serve as a weak zeitgeber for the DN1 pacemaker neurons, but the effect is not sufficient to alter rhythms of behavioral activity.     

Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster--sex-specific differences suggest a different quality of activity

The rhythms of locomotor activity of male and virgin or mated female flies were compared in the Drosophila melanogaster wild-type strains CantonS, Berlin, and OregonR. Under light-dark conditions, most flies showed a bimodal activity pattern with a morning peak around lights-on and an evening peak before lights-off. For all strains, a distinct sexual dimorphism was observed in the phase of the morning peak. Males had a significantly earlier morning peak than females and consequently a larger phase angle between morning and evening peak (psi(m, e)). Under constant dark conditions, the morning component merged with the evening component to a unimodal activity band in about half of the flies. In those flies who maintained bimodality, the sex-specific difference in psi(m, e) disappeared. Other sex-specific differences were now apparent: Males showed a shorter free-running period than females, and in two of the three strains, females were more active than males. Morning and evening components seem to contribute to the free-running period. Spontaneous or externally provoked change in psi(m, e) were correlated with period changes. In some flies, the morning and the evening components showed splitting, indicating that they are the output of two different oscillators. The sexual dimorphism in the phase of the morning peak under LD-conditions suggests that the function of activity during morning and evening peak might be different, for example, during the morning peak, males are active to find females. Overall, the results underline the multioscillatory nature of Drosophila's circadian system.     

Contribution of Drosophila TRPA1-Expressing Neurons to Circadian Locomotor Activity Patterns

In both vertebrates and invertebrates, Transient Receptor Potential (TRP) channels are expressed in sensory neurons and mediate environmental stimuli such as light, sound, temperature, and taste. Some of these channels, however, are expressed only in the brain and their functions remain incompletely understood. Using the GAL4/UAS binary system with a line in which the GAL4 had been knocked into the trpA1 locus in Drosophila, we recently reported new insights into TRPA1 localization and function, including its expression in approximately 15% of all circadian neurons. TRPA1 is expressed in lateral posterior neurons (LPNs), which are known to be highly sensitive to entrainment by temperature cycles. Here, I used the bacterial sodium channel, NaChBac, to examine the effects of altering the electrical properties of trpA1 neurons on circadian rhythms. My results indicate that circadian activity of the flies in the morning, daytime, and evening was affected in a temperature-dependent manner following TRPA1 neuronal activation. Remarkably, TRPA1 neuron activation in flies kept at 18°C impacted the morning peak of circadian activity even though TRPA1 is not expressed in morning cells. Taken together, these results suggest that the activation of TRPA1-expressing neurons may differentially coordinate light/dark circadian entrainment, depending on the temperature.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster''s clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila''s most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential “clock kinase” phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.     

A Stochastic Burst Follows the Periodic Morning Peak in Individual Drosophila Locomotion

Coupling between cyclically varying external light and an endogenous biochemical oscillator known as the circadian clock, modulates a rhythmic pattern with two prominent peaks in the locomotion of Drosophila melanogaster. A morning peak appears around the time lights turn on and an evening peak appears just before lights turn off. The close association between the peaks and the external 12:12 hour light/dark photoperiod means that respective morning and evening peaks of individual flies are well-synchronized in time and, consequently, feature prominently in population-averaged data. Here, we report on a brief but strong stochastic burst in fly activity that, in contrast to morning and evening peaks, is detectable only in single fly recordings. This burst was observed across 3 wild-type strains of Drosophila melanogaster. In a single fly recording, the burst is likely to appear once randomly within 0.5–5 hours after lights turn on, last for only 2–3 minutes and yet show 5 times greater activity compared to the maximum of morning peak with data binned in 3 minutes. Owing to its variable timing and short duration, the burst is virtually undetectable in population-averaged data. We use a locally-built illumination system to study the burst and find that its incidence in a population correlates with light intensity, with ~85% of control flies showing the behavior at 8000 lux (1942 μW/cm²). Consistent with that finding, several mutant flies with impaired vision show substantially reduced frequency of the burst. Additionally, we find that genetic ablation of the clock has insignificant effect on burst frequency. Together, these data suggest that the pronounced burst is likely generated by a light-activated circuit that is independent of the circadian clock.     

Melatonin Shifts Human Orcadian Rhythms According to a Phase-Response Curve

A physiological dose of orally administered melatonin shifts circadian rhythms in humans according to a phase-response curve (PRC) that is nearly opposite in phase with the PRCs for light exposure: melatonin delays circadian rhythms when administered in the morning and advances them when administered in the afternoon or early evening. The human melatonin PRC provides critical information for using melatonin to treat circadian phase sleep and mood disorders, as well as maladaptation to shift work and transmeridional air travel. The human melatonin PRC also provides the strongest evidence to date for a function of endogenous melatonin and its suppression by light in augmenting entrainment of circadian rhythms by the light-dark cycle.     

Melatonin Shifts Human Orcadian Rhythms According to a Phase-Response Curve

A physiological dose of orally administered melatonin shifts circadian rhythms in humans according to a phase-response curve (PRC) that is nearly opposite in phase with the PRCs for light exposure: melatonin delays circadian rhythms when administered in the morning and advances them when administered in the afternoon or early evening. The human melatonin PRC provides critical information for using melatonin to treat circadian phase sleep and mood disorders, as well as maladaptation to shift work and transmeridional air travel. The human melatonin PRC also provides the strongest evidence to date for a function of endogenous melatonin and its suppression by light in augmenting entrainment of circadian rhythms by the light-dark cycle.     

Circadian rhythms of androgen levels in the blood of male hamadryas baboons

Radioimmunoassay was used to investigate circadian rhythms of blood plasma testosterone, 5 alpha-dihydrotestosterone, androstendione and dehydroepiandrosterone in intact animals and in those preadapted for experimental conditions. Blood plasma of monkey demonstrated monophasic circadian rhythms in the levels of androstendione, dehydroepiandrosterone and 5 alpha-dihydrotestosterone, with the maximal values seen early in the morning and minimal values in the evening. Circadian rhythms of testosterone were opposite in character. The adaptation of monkeys for housing and experimental conditions was marked by an increase in testosterone and 5 alpha-dihydrotestosterone levels in the blood as well as by an insignificant elevation of the levels of androgens of adrenal origin, leading to the changes in circadian rhythms of steroid hormones.     

Phase-shifting the fruit fly clock without cryptochrome

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. Here, we monitored phase response curves of cry(01) mutants and control flies to 1-hour 1000-lux light pulses. We found that cry(01) mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 significantly increased the magnitude of phase advances in cry(01) mutants. At circadian time 15, the mutants exhibited phase advances instead of the expected delays. These complex results are discussed.     

Role of photoperiodicity and circadian rhythm of glucocorticoids in synchronizing the fluctuations in free-radical oxidation of lipids in rats

A biphasic circadian rhythm in the content of liver lipid peroxidation products has been demonstrated in male Wistar rats housed under the conditions of 12L: 12D, with 3 hours of morning and evening twilight. Maxima of the concentration of the products were observed in the morning and early at night. The rhythm of lipid anti-oxidative activity was found in an anti-phase. Inversion of both the L: D cycle and glucocorticoid circadian rhythms (cortisol injections) led after 14-16 days to the same shifts in the rhythm of anti-oxidative activity. The data indicate that glucocorticoids modulate the diurnal rhythms of lipid anti-oxidative activity and may be responsible for the shifts in the rhythms of free radical oxidation, induced by inversion of the L: D cycle.     

Circadian changes in central excitability—the origin of behavioural rhythms in tsetse flies and other animals?

Diel changes in seven behavioural responses of male tsetse flies, G.morsitans, were measured under controlled conditions in the laboratory. These responses involved different motor and sensory modalities, and were evoked by stimuli that were either exogenously applied at regular intervals or were ever-present. Six (possibly seven) of the responses were modulated across the photophase (of LD 12 : 12) in the V-pattern typical of biting behaviour in the field: morning and evening responses were greatest, noon least. Two of these rhythms were shown to persist in constant conditions, indicating that the underlying control is truly circadian. Comparison of the form of an optokinetic response rhythm at high and low stimulus intensities, implied central control of the response modulation. This, plus the close similarity in phase of the different rhythms, is interpreted as indicating circadian changes in central excitatory state (arousal) as the underlying neurophysiological basis of behavioural rhythmicity. It is suggested that the sarrie arousal system controls the comparable, longer and shorter term changes in excitability that result from starvation, sex, etc.