Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. We show that the mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant TIMELESS protein isoform to CRYPTOCHROME, the circadian photoreceptor. This interaction results in a more stable TIMELESS product. These findings reveal a molecular link between diapause and circadian photoreception.

With the approach of winter, many insects switch to an alternative protective developmental program called diapause. Drosophila melanogaster females overwinter as adults by inducing a reproductive arrest that is characterized by inhibition of ovarian development at previtellogenic stages. The insulin producing cells (IPCs) are key regulators of this process, since they produce and release insulin-like peptides that act as diapause-antagonizing hormones. Here we show that in D. melanogaster two neuropeptides, Pigment Dispersing Factor (PDF) and short Neuropeptide F (sNPF) inhibit reproductive arrest, likely through modulation of the IPCs. In particular, genetic manipulations of the PDF-expressing neurons, which include the sNPF-producing small ventral Lateral Neurons (s-LN_vs), modulated the levels of reproductive dormancy, suggesting the involvement of both neuropeptides. We expressed a genetically encoded cAMP sensor in the IPCs and challenged brain explants with synthetic PDF and sNPF. Bath applications of both neuropeptides increased cAMP levels in the IPCs, even more so when they were applied together, suggesting a synergistic effect. Bath application of sNPF additionally increased Ca²⁺ levels in the IPCs. Our results indicate that PDF and sNPF inhibit reproductive dormancy by maintaining the IPCs in an active state. Diapause is a hormonally mediated process that allows insects to predict and respond to unfavourable conditions by altering their metabolism and behavior to resist the oncoming environmental challenges. In Drosophila melanogaster females a protective state of reproductive dormancy is induced by lower temperatures and shorter photoperiods that mimic the approach of winter. By genetically manipulating the circadian pacemaker s-LN_vs cells, which express two neuropeptides, Pigment dispersing factor (PDF) and short Neuropeptide F (sNPF), we were able to modulate levels of gonadal arrest. PDF and sNPF appear to act as antagonists to dormancy, as do the Drosophila insulin-like peptides (dILPs) that are expressed in the insulin producing cells (IPCs). Indeed, we observe that the axonal projections from the s-LNvs appear to overlap with those from the IPCs implying that the clock cells signal to the IPCs. We confirm this possible communication by applying the two synthetic peptides to the IPCs and detecting a response in the IPC signal transduction pathway. We conclude that the clock neurons activate the IPCs via PDF and sNPF, which in turn release the dILPs, antagonise dormancy and lead to reproductive growth, thereby uncovering a neurogenetic circadian-overwintering axis.

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific “regulators” that bind the C terminus of the protein. This C-terminal region harbors several protein–protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visual-signaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY–Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.

In response to adverse environmental conditions many organisms from nematodes to mammals deploy a dormancy strategy, causing states of developmental or reproductive arrest that enhance somatic maintenance and survival ability at the expense of growth or reproduction. Dormancy regulation has been studied in C. elegans and in several insects, but how neurosensory mechanisms act to relay environmental cues to the endocrine system in order to induce dormancy remains unclear. Here we examine this fundamental question by genetically manipulating aminergic neurotransmitter signaling in Drosophila melanogaster. We find that both serotonin and dopamine enhance adult ovarian dormancy, while the downregulation of their respective signaling pathways in endocrine cells or tissues (insulin producing cells, fat body, corpus allatum) reduces dormancy. In contrast, octopamine signaling antagonizes dormancy. Our findings enhance our understanding of the ability of organisms to cope with unfavorable environments and illuminate some of the relevant signaling pathways.

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 per^spliced 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 tim^spliced 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 TIM^unspliced 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.

Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database “KrillDB” allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment’s challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.