Highlights d Neuronal ADP:ATP is not affected by neurotransmission despite strong Na + pumping d Stimulated mitochondrial pyruvate flux precedes glucose consumption d Energy stability is not explained by conventional homeostasis (ADP:ATP or Ca 2+) d Na + pump activity controls glycolysis and mitochondrial ATP production

It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical Drosophila period mutants for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant’s natural cycle, i.e., a 19-h day in the case of pers mutants and a 29-h day for perl mutants. The arrhythmic per0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but perl mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, pers and per0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and – as revealed for perl mutants – this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that perl and pers persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants.

The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period 01 (per 01) clock mutants and CantonS wildtype (WT CS) flies in an isogenic and non-isogenic background using LC-MS. In the non-isogenic background, metabolites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per 01 mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WT CS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per 01 mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per 01 did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per 01 mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants.

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

The adaptive significance of adjusting behavioural activities to the right time of the day is intuitive.Laboratory studies have implicated an important role of circadian clocks in behavioural 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 semi-natural Zeitgeber conditions.We here report evidence that proper timing of eclosion, a vital behaviour of the fruit fly Drosophila melanogaster, requires a functional molecular clock even under quasi-natural conditions. In contrast to wildtype flies, period 01 mutants with a defective molecular clock eclose mostly arrhythmically in a temperate environment even in the presence of a full complement of abiotic Zeitgebers. Moreover, period 01 mutants eclose during a much larger portion of the day, and peak eclosion time becomes more susceptible to variable day-to-day changes of light and temperature. Under the same conditions, flies with impaired peptidergic inter-clock signalling (pdf 01 and han 5304 mutants) stayed largely rhythmic with normal gate sizes. Our results suggest that the presence of natural Zeitgebers can mitigate a loss of peptide-mediated phasing between central clock neuron groups, but cannot substitute for the lack of a functional molecular clock under natural temperate conditions. BackgroundEndogenous timing via circadian clocks confers adaptive advantages as it allows organisms to anticipate daily changes in the environment (see [1][2][3]). In terms of behaviour, the fitness relevance of being able to schedule locomotor activity, feeding, mating or other actions at the right time of the day is intuitive as it may help maximize success and reduce risks. Many studies under constant laboratory conditions have revealed a key role of the central and peripheral clocks in timing of behaviours across taxa. However, the importance of circadian clocks in daily timing of behaviours under natural conditions or in ecological context has come under debate, as studies in the last decade have assessed the functional importance of endogenous clocks under (semi-) natural conditions in a variety of mostly vertebrate species (see [1,2,4]). One important conclusion derived from these studies is that diel activity rhythms can remarkably differ between seminatural and laboratory conditions, since the phase relationship between behavioural activity and a given Zeitgeber such as light is modulated by other abiotic Zeitgebers, particularly by temperature [5]. Furthermore, intraand interspecific interactions such as predation [6][7][8] or competition for food [9] determine ("mask") activity patterns in the wild. Most strikingly, under semi-natural conditions in an outdoor enclosure, Per2 BRDM1 mice carrying a mutation in a core clock gene showed the same activity pattern as controls, and both showed mostly diurnal feeding, although they are strongly nocturnal in laboratory conditions [10]. In the wild, chipmunks with a lesion in t...