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The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling (Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling [ABSTRACT FROM AUTHOR]

Previous analysis of Drosophila circadian behavior under natural conditions has revealed a number of novel and unexpected features. Here we focus on the oscillations of per and tim mRNAs and their posttranscriptional regulation and observe significant differences in molecular cycling under laboratory and natural conditions. In particular, robust per mRNA cycling from fly heads is limited to the summers, whereas tim RNA cycling is observed throughout the year. When both transcripts do cycle, their phases are similar, except for the very warmest summer months. We also study the natural splicing profiles of per and tim transcripts and observe a clear relationship between temperature and splicing. In natural conditions, we confirm the relationship between accumulation of the perspliced variant, low temperature, and the onset of the evening component of locomotor activity, first described in laboratory conditions. Intriguingly, in the case of tim splicing, we detect the opposite relationship, with timspliced expression increasing at higher temperatures. A first characterization of the 4 different TIM protein isoforms (resulting from the combination of the natural N-terminus length polymorphism and the C-terminus alternative splicing) using the 2-hybrid assay showed that the TIMunspliced isoforms have a stronger affinity for CRY, but not for PER, suggesting that the tim 3′ splicing could have physiological significance, possibly in temperature entrainment and/or adaptation to seasonal environments. [ABSTRACT FROM AUTHOR]

The circadian clock modulates the adaptive daily patterns of physiology and behavior and adjusts these rhythms to seasonal changes. Recent studies of seasonal locomotor activity patterns of wild-type and clock mutant fruit flies in quasi-natural conditions have revealed that these behavioral patterns differ considerably from those observed under standard laboratory conditions. To unravel the molecular features accompanying seasonal adaptation of the clock, we investigated Drosophila’s neuronal expression of the canonical clock proteins PERIOD (PER) and TIMELESS (TIM) in nature. We find that the profile of PER dramatically changes in different seasons, whereas that of TIM remains more constant. Unexpectedly, we find that PER and TIM oscillations are decoupled in summer conditions. Moreover, irrespective of season, PER and TIM always peak earlier in the dorsal neurons than in the lateral neurons, suggesting a more rapid molecular oscillation in these cells. We successfully reproduced most of our results under simulated natural conditions in the laboratory and show that although photoperiod is the most important zeitgeber for the molecular clock, the flies’ activity pattern is more strongly affected by temperature. Our results are among the first to systematically compare laboratory and natural studies of Drosophila rhythms. [ABSTRACT FROM PUBLISHER]