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Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems.
We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.
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 Canton-S wildtype (WT\(_{CS}\)) flies in an isogenic and non-isogenic background using LC–MS. In the non-isogenic background, metabo-lites 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 WTCS 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
Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.
Behavioral flexibility is an important cornerstone for the ecological success of animals. Social Cataglyphis nodus ants with their age‐related polyethism characterized by age‐related behavioral phenotypes represent a prime example for behavioral flexibility. We propose neuropeptides as powerful candidates for the flexible modulation of age‐related behavioral transitions in individual ants. As the neuropeptidome of C. nodus was unknown, we collected a comprehensive peptidomic data set obtained by transcriptome analysis of the ants’ central nervous system combined with brain extract analysis by Q‐Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different regions of the brain by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. In total, we identified 71 peptides with likely bioactive function, encoded on 49 neuropeptide‐, neuropeptide‐like, and protein hormone prepropeptide genes, including a novel neuropeptide‐like gene (fliktin). We next characterized the spatial distribution of a subset of peptides encoded on 16 precursor proteins with high resolution by MALDI MS imaging (MALDI MSI) on 14 µm brain sections. The accuracy of our MSI data were confirmed by matching the immunostaining patterns for tachykinins with MSI ion images from consecutive brain sections. Our data provide a solid framework for future research into spatially resolved qualitative and quantitative peptidomic changes associated with stage‐specific behavioral transitions and the functional role of neuropeptides in Cataglyphis ants.
Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.