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.

A major question in chronobiology focuses around the “Bünning hypothesis” which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. Here, we used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. We have corroborated previous observations that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Here, we show that arrhythmic mutant strains, per⁰¹, tim⁰¹ and Clk^Jrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. Our results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements. The circadian clock consists of an extensive genetic network that drives daily rhythms of physiological, biochemical and behavioural processes. The network is evolutionary conserved and has been extensively studied in a broad range of organisms. Another genetic network constitutes the photoperiodic clock and monitors the seasonal change in day-length. Here, we address a major and long-standing question in chronobiology: whether the circadian clock is involved in photoperiodic timing, also known as the Bünning hypothesis. Drosophila, as with many other insects in temperate regions, exhibits a photoperiodic response that allows the insect to anticipate and survive the winter. Here we show that the cold-tolerance of the fly is regulated by the photoperiod. We use this phenotype to test day-length timing in various circadian clock mutants and observe that in null clock mutants, the photoperiodic response is abolished, whereas in mutants that exhibit short or long daily cycles, the photoperiodic response is modified, further supporting a circadian-clock function. Overall, these results provide the first evidence in Drosophila that support for the Bünning hypothesis, and pave the way for the genetic dissection of seasonal timing in Drosophila melanogaster.

There is mounting evidence that the sandfly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) (Lutz and Neiva 1912), the main vector of the visceral leishmaniasis in Latin America, is a complex of sibling species (Lanzaro et al. 1993 ; Dujardin et al. 1997 ; Lampo et al. 1999 ; Uribe 1999 ; Arrivillaga and Feliciangeli 2001 ; Soto et al. 2001 ). But, the evidence for Brazilian populations is still contradictory. Crossing experiments, pheromonal (Ward et al. 1983 , 1988 ; Hamilton et al. 1999a , 1999b ), copulation songs (Souza et al. 2002 ) and molecular analyses (Bauzer et al. 2002 ) all support the idea of a species complex and seem to confirm the data obtained for other populations of this species. But work with isoenzyme loci suggests that although it is a highly polymorphic and geographically variable species, L. longipalpis should not be considered a species complex in Brazil (Mukhopadhyay et al. 1998b ; Mutebi et al. 1999 ; Azevedo et al. 2000 ).

Aedes aegypti is a major disease vector in urban habitats, involved in the transmission of dengue, chikungunya and Zika. Despite innumerous attempts to contain disease outbreaks, there are neither efficient vaccines nor definite vector control methods nowadays. In recent years, an innovative strategy to control arboviruses, which exploits the endosymbiotic bacterium Wolbachia pipientis, emerged with great expectations. The success of the method depends on many aspects, including Wolbachia’s cytoplasmic incompatibility and pathogen interference phenotypes, as well as its effect on host fitness. In this work, we investigated the influence the Wolbachia strain wMel exerts on embryo development and egg viability and speculate on its field release use. Wild-type (Br or Rockefeller) and Wolbachia-harboring specimens (wMelBr) were blood-fed and submitted to synchronous egg laying for embryo development assays. Samples were analyzed for morphological markers, developmental endpoint and egg resistance to desiccation (ERD). Quiescent egg viability over time was also assessed. wMelBr samples completed embryogenesis 2–3 hours later than wild-type. This delay was also observed through the onset of both morphological and physiological markers, respectively by the moments of germband extension and ERD acquisition. Following the end of embryonic development, wMelBr eggs were slightly less resistant to desiccation and showed reduced viability levels, which rapidly decayed after 40 days into quiescence, from approximately 75% to virtually 0% in less than a month. Our data revealed that the wMel strain of Wolbachia slightly delays embryogenesis and also affects egg quality, both through reduced viability and desiccation resistance. These findings suggest that, although embryonic fitness is somehow compromised by wMel infection, an efficient host reproductive manipulation through cytoplasmic incompatibility seems sufficient to overcome these effects in nature and promote bacterial invasion, as shown by successful ongoing field implementation.

Mosquito-borne diseases are rapidly spreading to vast territories, putting at risk most of the world’s population. A key player in this scenario is Aedes aegypti, a hematophagous species which hosts and transmits viruses causing dengue and other serious illnesses. Since vector control strategies relying only on insecticides have proven unsustainable, an alternative method involving the release of Wolbachia-harboring individuals has emerged. Its successful implementation vastly depends on how fit the released individuals are in the natural habitat, being able to mate with wild populations and to spread Wolbachia to subsequent generations. In mosquitoes, an important aspect of reproductive fitness is the acoustic communication between males and females, which translates to interactions between harmonic frequencies in close proximity flight. This study aimed to characterize the flight tone produced by individuals harboring Wolbachia, also evaluating their ability to establish stable acoustic interactions. Wild-type (WT) and Wolbachia-harboring specimens (wMelBr) were thorax-tethered to blunt copper wires and placed at close proximity to sensitive microphones. Wing-beat frequencies (WBFs) were characterized at fundamental and harmonic levels, for both single individuals and couples. Harmonic interactions in homogeneous and heterogeneous couples of WT and wMelBr variants were identified, categorized and quantified accordingly. In tethered ‘solo’ flights, individuals harboring Wolbachia developed WBFs, differing slightly, in a sex-dependent way, from those of the WT strain. To test the ability to form harmonic ‘duets’, tethered couples of wMelBr and WT individuals were shuffled in different sex pairs and had their flight tones analyzed. All couple types, with WT and/or wMelBr individuals, were able to interact acoustically in the frequency range of 1300–1500 Hz, which translates to the convergence between male’s second harmonic and female’s third. No significant differences were found in the proportions of interacting couples between the pair types. Surprisingly, spectrograms also revealed the convergence between alternative harmonic frequencies, inside and outside the species putative hearing threshold. Wolbachia infection leads to small sex-dependent changes on the flight tones of Ae. aegypti, but it does not seem to prevent the stereotyped harmonic interaction between males and females. Therefore, when released in the natural habitat to breed with native individuals, Wolbachia-harboring individuals shall be fit enough to meet the criteria of acoustically-related mating behavior and promote bacteria dispersion effectively.