Genetic manipulation of the circadian clock's timing of sexual behaviour in the Queensland fruit flies, Dacus tryoni and Dacus neohumeralis

ABSTRACT. Mating in Dacus tryoni is restricted to dusk, whereas that of a sibling species, Dacus neohumeralis , occurs in the middle of the day. The timing of sexual behaviour in both species is determined by an interaction between a circadian clock and light intensity. In D. tryoni peak mating responsiveness is at the time of dusk, and the optimal light intensity for mating is approximately 91x. In D.neohumeralis peak responsiveness is in the middle of the day, and the optimal light intensity for mating is greater than 10 000 lx. The two species were crossed and the time of mating and response to light intensity of F₁, F₂ and backcross progeny determined. The circadian clock set a mating phase ('gate') as narrow in F₁ flies as in their parents, suggesting the circadian timing mechanism to be common between the two species. The results indicate that the genetic mechanism controlling timing is independent of that controlling response to light intensity, and that both genetic mechanisms are complex.     

Circadian timekeeping during early Arabidopsis development

The circadian coordination of organismal biology with the local temporal environment has consequences for fitness that may become manifest early in development. We directly explored the development of the Arabidopsis (Arabidopsis thaliana) clock in germinating seedlings by monitoring expression of clock genes. Clock function is detected within 2 d of imbibition (hydration of the dried seed). Imbibition is sufficient to synchronize individuals in a population in the absence of entraining cycles of light-dark or temperature, although light-dark and temperature cycles accelerate the appearance of rhythmicity and improve synchrony among individuals. Oscillations seen during the first 2 d following imbibition are dependent on the clock genes LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, ZEITLUPE, GIGANTEA, PSEUDO-RESPONSE REGULATOR7 (PRR7), and PRR9, although later circadian oscillations develop in mutants defective in each of these genes. In contrast to circadian rhythmicity, which developed under all conditions, amplitude was the only circadian parameter that demonstrated a clear response to the light environment; clock amplitude is low in the dark and high in the light. A circadian clock entrainable by temperature cycles in germinating etiolated seedlings may synchronize the buried seedling with the local daily cycles before emergence from the soil and exposure to light.     

Circadian rhythms in the mating behavior of the cockroach, Leucophaea maderae

Mating behavior of small populations of virgin males and females of the cockroach Leucophaea maderae were continuously monitored via time-lapse video recording in controlled laboratory conditions. The time of onset of copulation was found to be rhythmic in a light cycle of 12 h light alternated with 12 h of darkness, with the peak of mating behavior occurring near the light to dark transition. This rhythm persisted in constant dim red illumination and constant temperature. In constant conditions, the period of the rhythm was slightly less than 24 h, with a peak of copulation during the late subjective day. These data demonstrated that mating behavior is gated by a circadian clock. When males and females were taken from light cycles that were 12 h out of phase, a bimodal rhythm was observed with one peak in the males' late subjective day and a second peak of equal amplitude in the late subjective day of females. The results indicated that circadian systems in both males and females contribute to the circadian rhythm in copulation. Bilateral section of the optic tracts (OTX) of both males and females abolished the rhythm, but the rhythm persisted when OTX females were paired with intact males or when OTX males were paired with intact females. Furthermore, when OTX males or OTX females were paired with intact animals that were 12 h out of phase, a bimodal rhythm was still observed. These results suggested that the circadian pacemaker in the optic lobes of both male and female cockroaches participates in the control of mating, but that a pacemaker outside the optic lobes is also likely involved. Finally, it was shown that the female's olfactory response (measured by electroantennogram) to components of the male sex pheromone exhibited a circadian rhythm, but the data suggested the peripheral olfactory rhythm is not likely to be involved in the rhythm of mating behavior.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

The period gene and allochronic reproductive isolation in Bactrocera cucurbitae

Clock genes that pleiotropically control circadian rhythm and the time of mating may cause allochronic reproductive isolation in the melon fly Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Flies with a shorter circadian period (ca. 22 h of locomotor activity rhythm) mated 5 h earlier in the day than those with a longer circadian period (ca. 30 h). Mate-choice tests demonstrated significant pre-mating isolation between populations with short and long circadian periods. Pre-mating isolation did not occur when the mating time was synchronized between the two populations by photoperiodic controls, indicating that reproductive isolation is due to variations in the time of mating and not any unidentified ethological difference between the two populations. We cloned the period (per) gene of B. cucurbitae that is homologous to the per gene in Drosophila. The relative level of per mRNA in the melon fly exhibited a robust daily fluctuation under light : dark conditions. The fluctuation of per expression under dark : dark conditions is closely correlated to the locomotor rhythm in B. cucurbitae. These results suggest that clock genes can cause reproductive isolation via the pleiotropic effect as a change of mating time.     

Data-driven modelling captures dynamics of the circadian clock of Neurospora crassa

Eukaryotic circadian clocks are based on self-sustaining, cell-autonomous oscillatory feedback loops that can synchronize with the environment via recurrent stimuli (zeitgebers) such as light. The components of biological clocks and their network interactions are becoming increasingly known, calling for a quantitative understanding of their role for clock function. However, the development of data-driven mathematical clock models has remained limited by the lack of sufficiently accurate data. Here we present a comprehensive model of the circadian clock of Neurospora crassa that describe free-running oscillations in constant darkness and entrainment in light-dark cycles. To parameterize the model, we measured high-resolution time courses of luciferase reporters of morning and evening specific clock genes in WT and a mutant strain. Fitting the model to such comprehensive data allowed estimating parameters governing circadian phase, period length and amplitude, and the response of genes to light cues. Our model suggests that functional maturation of the core clock protein Frequency causes a delay in negative feedback that is critical for generating circadian rhythms.     

Extracellular pH is under circadian control in Gonyaulax polyedra and forms a metabolic feedback loop

This study investigates the relationship between the circadian clock and metabolism based on recordings of the extracellular pH in cultures of the marine dinoflagellate, Gonyaulax polyedra. In light-dark cycles, pH of the medium rises during the light phase and declines in the dark. The amplitude of this pH-rhythm correlates with light intensity, indicating photosynthesis (and respiration) as the driving force. The recorded extracellular pH changes probably reflect the need to control intracellular pH in spite of pH-modifying reactions. The daily pH-changes are under control of the circadian clock because they continue to oscillate with a circa-24 h period in constant light, albeit with a smaller amplitude. Similar to other circadian output rhythms, the pH rhythm depends (amplitude and phase) on nitrate levels in the medium. Both the bioluminescence and the pH rhythm can also be shifted by extracellular pH-changes although Gonyaulax is rarely exposed to significant pH changes in its marine ecosystems (except for highly dense algal blooms). Because intracellular proton levels are both affecting circadian input and output they form a feedback loop with the Gonyaulax circadian system indicating complex interactions between metabolism and the circadian clock.     

Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis

We recorded the eclosion time of the flesh fly, Sarcophaga crassipalpis, at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory, to examine the timing adjustment of eclosion in response to temperature cycles and their amplitudes in the pupal stage. In the soil, most eclosions occurred in the late morning, which was consistent with the eclosion time under pseudo-sinusoidal temperature cycles in the laboratory. The circadian clock controlling eclosion was reset by temperature cycles and free-ran with a period close to 24 h. This clock likely helps pupae eclose at an optimal time even when the soil temperature does not show clear daily fluctuations. The eclosion phase of the circadian clock progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased. This response allows pupae located at any depth in the soil to eclose at the appropriate time despite the depth-dependent phase delay of the temperature change. In contrast, the abrupt temperature increase in square-wave temperature cycles reset the phase of the circadian clock to the increasing time, regardless of the temperature amplitude. The rapid temperature increase may act as the late-morning signal for the eclosion clock.     

The circadian clock within the cardiomyocyte is essential for responsiveness of the heart to fatty acids

Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreatic beta-cell dysfunction, and myocardial contractile dysfunction. Here we tested the hypothesis that the intrinsic circadian clock within the cardiomyocytes of the heart allows rapid and appropriate adaptation of this organ to fatty acids by investigating the following: 1) whether circadian rhythms in fatty acid responsiveness persist in isolated adult rat cardiomyocytes, and 2) whether manipulation of the circadian clock within the heart, either through light/dark (L/D) cycle or genetic disruptions, impairs responsiveness of the heart to fasting in vivo. We report that both the intramyocellular circadian clock and diurnal variations in fatty acid responsiveness observed in the intact rat heart in vivo persist in adult rat cardiomyocytes. Reversal of the 12-h/12-h L/D cycle was associated with a re-entrainment of the circadian clock within the rat heart, which required 5-8 days for completion. Fasting rats resulted in the induction of fatty acid-responsive genes, an effect that was dramatically attenuated 2 days after L/D cycle reversal. Similarly, a targeted disruption of the circadian clock within the heart, through overexpression of a dominant negative CLOCK mutant, severely attenuated induction of myocardial fatty acid-responsive genes during fasting. These studies expose a causal relationship between the circadian clock within the cardiomyocyte with responsiveness of the heart to fatty acids and myocardial triglyceride metabolism.     

Human cone light sensitivity and melatonin rhythms following 24-hour continuous illumination

This study investigates the possibility of an endogenous circadian rhythm in retinal cone function in humans. A full-field cone electroretinogram (ERG) was performed every 2 h for 24 h under continuous rod-saturating ambient white light (53 ± 30 lux; pupils dilated) in nine healthy subjects. Distinct circadian variations were superimposed upon a gradual decrease in cone responsiveness to light, demonstrated most reliably in the implicit times of b-wave and oscillatory potentials, and to a lesser extent in amplitude and a-wave implicit times. After mathematical correction of the linear trend, the cone response was found to be greatest around 20:00 h and least around 06:00 h. The phase of the ERG circadian rhythm was not synchronized with the phase of the salivary melatonin rhythm measured the previous evening. Melatonin levels measured under constant light on the day of ERG assessments were suppressed by 53% on average compared to melatonin profiles obtained previously under near-total darkness in seven participants. The progressive decline in cone responsiveness to light over the 24 h may reflect an adaptation of the cone-driven retinal system to constant light, although the mechanism is unclear. The endogenous rhythm of cone responsiveness to light may be used as an additional index of central or retinal circadian clock time.     

Light and temperature entrainment of a locomotor rhythm in honeybees

Abstract. The circadian locomotor (walking) rhythms of forager honeybees (Apis mellifera ligustica L.) were entrained to eight different 24 h light-dark cycles. The phases of activity onset, peak activity, and offset were correlated with the lights-off transition, suggesting lights-off as the primary zeitgeber for the rhythm. Further support for this hypothesis was provided by LD 1:23 experiments, in which entrainment occurred when the light pulse was situated at the end, but not at the beginning, of the subjective photophase. Steady-state entrainment of the locomotor rhythm was achieved with square-wave temperature cycles of 10^oC amplitude under constant dark: most of the activity occurred within the early thermophase. Smaller amplitude temperature cycles yielded relative coordination of the rhythm. Interactions of temperature and light-dark cycles resulted in entrainment patterns different from those elicited in response to either cycle alone or those formed by a simple combination of the two separate responses. Furthermore, temperature cycles having amplitudes insufficient for entrainment of the rhythm nevertheless modified the pattern of entrainment to light - dark cycles, suggesting a synergism of light and temperature effects on the underlying circadian clock system.     

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT_1B receptors, decreasing responsiveness to light. Consequently, 5-HT_1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT_1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.     

Temperature synchronization of the Drosophila circadian clock

BACKGROUND: Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS: We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS: We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.     

RFI2, a RING-domain zinc finger protein, negatively regulates CONSTANS expression and photoperiodic flowering

The red and far-red light-absorbing phytochromes interact with the circadian clock, a central oscillator that sustains a 24-h period, to measure accurately seasonal changes in day-length and regulate the expression of several key flowering genes. The interactions and subsequent signalling steps upstream of the flowering genes such as CONSTANS (CO) and FLOWERING LOCUS T (FT) remain largely unknown. We report here that a photomorphogenic mutant, red and far-red insensitive 2-1 ( rfi2-1), flowered early particularly under long days. The rfi2-1 mutation also enhanced the expression of CO and FT under day/night cycles or constant light. Both co-2 and gigantea-2 (gi-2) were epistatic to rfi2-1 in their flowering responses. The gi-2 mutation was also epistatic to the rfi2-1 mutation in the expression of CO and hypocotyl elongation. However, the rfi2-1 mutation did not affect the expression of GI, a gene that mediates between the circadian clock and the expression of CO. Like many other flowering genes, the expression of RFI2 oscillated under day/night cycles and was rhythmic under constant light. The amplitude of the rhythmic expression of RFI2 was significantly reduced in phyB-9 or lhy-20 plants, and was also affected by the gi-2 mutation. As previously reported, the gi-2 mutation affects the period length and amplitude of CCA1 and LHY expression, and GI may act through a feedback loop to maintain a proper circadian function. We propose a regulatory step in which RFI2 represses the expression of CO, whereas GI may maintain the proper expression of RFI2 through its positive action on the circadian clock. The regulatory step serves to tune the circadian outputs that control the expression of CO and photoperiodic flowering.     

Circadian dependency of nocturnal immediate-early protein induction in rat retina

Daily rhythms in the mammalian retina are regulated by an endogenous circadian clock. Previously it was found that neuronal elements of the rat retina respond to light:dark (L:D) transitions with cell-specific changes in expression of the c-fos gene. Using a pan-Fos antibody to probe Western blots of rat retina, we have now shown that darkness is associated with a 60-fold increase in c-Fos protein, whereas levels of FosB and Fos-related antigens are invariant. The induction of c-Fos exhibits circadian dependency; accumulation of c-Fos protein was significantly enhanced, by a factor of 2.5-fold, when darkness onset was coincident with the established L:D transition. c-Fos exhibited only a low amplitude circadian rhythm in the absence of L:D cycles. Similar results were obtained for another immediate early gene (IEG) protein, Egr-1. These findings show that IEG induction in the rodent retina exhibits circadian clock dependency.