Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.

Hormones regulate development, as well as many vital processes in the daily life of an animal. Many of these hormones are peptides that act at a higher hierarchical level in the animal with roles as organizers that globally orchestrate metabolism, physiology and behavior. Peptide hormones can act on multiple peripheral targets and simultaneously convey basal states, such as metabolic status and sleep-awake or arousal across many central neuronal circuits. Thereby, they coordinate responses to changing internal and external environments. The activity of neurosecretory cells is controlled either by (1) cell autonomous sensors, or (2) by other neurons that relay signals from sensors in peripheral tissues and (3) by feedback from target cells. Thus, a hormonal signaling axis commonly comprises several components. In mammals and other vertebrates, several hormonal axes are known, such as the hypothalamic-pituitary-gonad axis or the hypothalamic-pituitary-thyroid axis that regulate reproduction and metabolism, respectively. It has been proposed that the basic organization of such hormonal axes is evolutionarily old and that cellular homologs of the hypothalamic-pituitary system can be found for instance in insects. To obtain an appreciation of the similarities between insect and vertebrate neurosecretory axes, we review the organization of neurosecretory cell systems in Drosophila. Our review outlines the major peptidergic hormonal pathways known in Drosophila and presents a set of schemes of hormonal axes and orchestrating peptidergic systems. The detailed organization of the larval and adult Drosophila neurosecretory systems displays only very basic similarities to those in other arthropods and vertebrates.

Dysregulation of sleep and feeding has widespread health consequences. Despite extensive epidemiological evidence for interactions between sleep and metabolic function, little is known about the neural or molecular basis underlying the integration of these processes. D. melanogaster potently suppress sleep in response to starvation, and powerful genetic tools allow for mechanistic investigation of sleep-metabolism interactions. We have previously identified neurons expressing the neuropeptide leucokinin (Lk) as being required for starvation-mediated changes in sleep. Here, we demonstrate an essential role for Lk neuropeptide in metabolic regulation of sleep. The activity of Lk neurons is modulated by feeding, with reduced activity in response to glucose and increased activity under starvation conditions. Both genetic silencing and laser-mediated microablation localize Lk-dependent sleep regulation to a single pair of Lk neurons within the Lateral Horn (LHLK neurons). A targeted screen identified a role for 50 adenosine monophosphate-activated protein kinase (AMPK) in starvation-modulated changes in sleep. Knockdown of AMPK in Lk neurons suppresses sleep and increases LHLK neuron activity in fed flies, phenocopying the starvation state. Further, we find a requirement for the Lk receptor in the insulin-producing cells (IPCs), suggesting LHLK-IPC connectivity is critical for sleep regulation under starved conditions. Taken together, these findings localize feeding-state-dependent regulation of sleep to a single pair of neurons within the fruit fly brain and provide a system for investigating the cellular basis of sleep-metabolism interactions.

This review focuses on neuropeptides and peptide hormones, the largest and most diverse class of neuroactive substances, known in Drosophila and other animals to play roles in almost all aspects of daily life, as w;1;ell as in developmental processes. We provide an update on novel neuropeptides and receptors identified in the last decade, and highlight progress in analysis of neuropeptide signaling in Drosophila. Especially exciting is the huge amount of work published on novel functions of neuropeptides and peptide hormones in Drosophila, largely due to the rapid developments of powerful genetic methods, imaging techniques and innovative assays. We critically discuss the roles of peptides in olfaction, taste, foraging, feeding, clock function/sleep, aggression, mating/reproduction, learning and other behaviors, as well as in regulation of development, growth, metabolic and water homeostasis, stress responses, fecundity, and lifespan. We furthermore provide novel information on neuropeptide distribution and organization of peptidergic systems, as well as the phylogenetic relations between Drosophila neuropeptides and those of other phyla, including mammals. As will be shown, neuropeptide signaling is phylogenetically ancient, and not only are the structures of the peptides, precursors and receptors conserved over evolution, but also many functions of neuropeptide signaling in physiology and behavior.

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. Here, we investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. We also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. Our data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.

Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, we delineated LK circuitry that governs various aspects of post-feeding physiology and behavior. We found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, we sought to define the populations of LK neurons that contribute to the different aspects of this physiology. We find that the calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of Lkr expression, mined a brain single-cell transcriptome dataset for genes coexpressed with Lkr, and identified synaptic partners of LK neurons. Lkrexpression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lkand Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons have different targets, but modulate the establishment of postprandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology.

Gonadotropin-releasing hormone (GnRH) was first discovered in mammals on account of its effect in triggering pituitary release of gonadotropins and the importance of this discovery was recognized forty years ago in the award of the 1977 Nobel Prize for Physiology or Medicine. Investigation of the evolution of GnRH revealed that GnRH-type signaling systems occur throughout the chordates, including agnathans (e.g. lampreys) and urochordates (e.g. sea squirts). Furthermore, the discovery that adipokinetic hormone (AKH) is the ligand for a GnRH-type receptor in the arthropod Drosophila melanogaster provided evidence of the antiquity of GnRH-type signaling. However, the occurrence of other AKH-like peptides in arthropods, which include corazonin and AKH/corazonin-related peptide (ACP), has complicated efforts to reconstruct the evolutionary history of this family of related neuropeptides. Genome/transcriptome sequencing has revealed that both GnRH-type receptors and corazonin-type receptors occur in lophotrochozoan protostomes (annelids, mollusks) and in deuterostomian invertebrates (cephalochordates, hemichordates, echinoderms). Furthermore, peptides that act as ligands for GnRH-type and corazonin-type receptors have been identified in mollusks. However, what has been lacking is experimental evidence that distinct GnRH-type and corazonin-type peptide-receptor signaling pathways occur in deuterostomes. Importantly, we recently reported the identification of two neuropeptides that act as ligands for either a GnRH-type receptor or a corazonin-type receptor in an echinoderm species - the common European starfish Asterias rubens. Discovery of distinct GnRH-type and corazonin-type signaling pathways in this deuterostomian invertebrate has demonstrated for the first time that the evolutionarily origin of these paralogous systems can be traced to the common ancestor of protostomes and deuterostomes. Furthermore, lineage-specific losses of corazonin signaling (in vertebrates, urochordates and nematodes) and duplication of the GnRH signaling system in arthropods (giving rise to the AKH and ACP signaling systems) and quadruplication of the GnRH signaling system in vertebrates (followed by lineage-specific losses or duplications) accounts for the phylogenetic distribution of GnRH/corazonin-type peptide-receptor pathways in extant animals. Informed by these new insights, here we review the history of research on the evolution of GnRH/corazonin-type neuropeptide signaling. Furthermore, we propose a standardized nomenclature for GnRH/corazonin-type neuropeptides wherein peptides are either named GnRH or corazonin, with the exception of the paralogous GnRH-type peptides that have arisen by gene duplication in the arthropod lineage and which are referred to as AKH (or red pigment concentrating hormone, RCPH, in crustaceans) and ACP.

Neuropeptides are responsible for regulating a variety of functions, including development, metabolism, water and ion homeostasis, and as neuromodulators in circuits of the central nervous system. Numerous neuropeptides have been identified and characterized. However, both discovery and functional characterization of neuropeptides across the massive Class Insecta has been sporadic. To leverage advances in post-genomic technologies for this rapidly growing field, insect neuroendocrinology requires a consolidated, comprehensive and standardised resource for managing neuropeptide information. The Database for Insect Neuropeptide Research (DINeR) is a web-based database-application used for search and retrieval of neuropeptide information of various insect species detailing their isoform sequences, physiological functionality and images of their receptor-binding sites, in an intuitive, accessible and user-friendly format. The curated data includes representatives of 50 well described neuropeptide families from over 400 different insect species. Approximately 4700 FASTA formatted, neuropeptide isoform amino acid sequences and over 200 records of physiological functionality have been recorded based on published literature. Also available are images of neuropeptide receptor locations. In addition, the data include comprehensive summaries for each neuropeptide family, including their function, location, known functionality, as well as cladograms, sequence alignments and logos covering most insect orders. Moreover, we have adopted a standardised nomenclature to address inconsistent classification of neuropeptides. As part of the H2020 nEUROSTRESSPEP project, the data will be actively maintained and curated, ensuring a comprehensive and standardised resource for the scientific community. DINeR is publicly available at the project website: http://www.neurostresspep.eu/diner/.

Stress triggers cellular and systemic reactions in organisms to restore homeostasis. For instance, metabolic stress, experienced during starvation, elicits a hormonal response that reallocates resources to enable food search and readjustment of physiology. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. Here we show that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila. The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the fly's resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, our findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. Our study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems.