The neuropeptide pigment-dispersing factor signals independently of Bruchpilot-labelled active zones in daily remodelled terminals of Drosophila clock neurons.

The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. However, the role of these presynaptic sites for PDF release is ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled puncta in the sLNv terminals was similar while their density differed between Zeitgeber time (ZT) 2 and 14. The relative distance between BRP- and PDF-labelled puncta was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes encoding proteins of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones, while BRP may be redistributed to active zones in a time-dependent manner.

A neuroendocrine pathway modulating osmotic stress in Drosophila.

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.

Receptor deorphanization in an echinoderm reveals kisspeptin evolution and relationship with SALMFamide neuropeptides.

BACKGROUND: Kisspeptins are neuropeptides that regulate reproductive maturation in mammals via G-protein-coupled receptor-mediated stimulation of gonadotropin-releasing hormone secretion from the hypothalamus. Phylogenetic analysis of kisspeptin-type receptors indicates that this neuropeptide signaling system originated in a common ancestor of the Bilateria, but little is known about kisspeptin signaling in invertebrates. RESULTS: Contrasting with the occurrence of a single kisspeptin receptor in mammalian species, here, we report the discovery of an expanded family of eleven kisspeptin-type receptors in a deuterostome invertebrate - the starfish Asterias rubens (phylum Echinodermata). Furthermore, neuropeptides derived from four precursor proteins were identified as ligands for six of these receptors. One or more kisspeptin-like neuropeptides derived from two precursor proteins (ArKPP1, ArKPP2) act as ligands for four A. rubens kisspeptin-type receptors (ArKPR1,3,8,9). Furthermore, a family of neuropeptides that act as muscle relaxants in echinoderms (SALMFamides) are ligands for two A. rubens kisspeptin-type receptors (ArKPR6,7). The SALMFamide neuropeptide S1 (or ArS1.4) and a 'cocktail' of the seven neuropeptides derived from the S1 precursor protein (ArS1.1-ArS1.7) act as ligands for ArKPR7. The SALMFamide neuropeptide S2 (or ArS2.3) and a 'cocktail' of the eight neuropeptides derived from the S2 precursor protein (ArS2.1-ArS2.8) act as ligands for ArKPR6. CONCLUSIONS: Our findings reveal a remarkable diversity of neuropeptides that act as ligands for kisspeptin-type receptors in starfish and provide important new insights into the evolution of kisspeptin signaling. Furthermore, the discovery of the hitherto unknown relationship of kisspeptins with SALMFamides, neuropeptides that were discovered in starfish prior to the identification of kisspeptins in mammals, presents a radical change in perspective for research on kisspeptin signaling.

A single pair of leucokinin neurons are modulated by feeding state and regulate sleep-metabolism interactions.

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 5' 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.

Modulation of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin.

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. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lk and 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 post-prandial 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.

Hormonal axes in Drosophila: regulation of hormone release and multiplicity of actions.

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.

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals.

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila.

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.

The evolution and nomenclature of GnRH-type and corazonin-type neuropeptide signaling systems.

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".

Identification of a neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind Cu(II) and generate metal-linked dimers.

BACKGROUND: Neuropeptides with an Amino Terminal Cu(II), Ni(II) Binding (ATCUN) motif (H2N-xxH) bind Cu(II)/Ni(II) ions. Here we report the novel discovery of a neuropeptide precursor that gives rise to a "cocktail" of peptides that bind Cu(II)/Ni(II) and form ternary complexes--the L-type SALMFamide precursor in the starfish Asterias rubens. METHODS: Echinoderm transcriptome sequence data were analysed to identify transcripts encoding precursors of SALMFamide-type neuropeptides. The sequence of the L-type SALMFamide precursor in the starfish Asterias rubens was confirmed by cDNA sequencing and peptides derived from this precursor (e.g. AYHSALPF-NH2, GYHSGLPF-NH2 and LHSALPF-NH2) were synthesized. The ability of these peptides to bind metals was investigated using UV/Vis, NMR, circular dichroism and EPR spectroscopy. RESULTS: AYHSALPF-NH2 and GYHSGLPF-NH2 bind Cu(II) and Ni(II) and generate metal-linked dimers to form ternary complexes with LHSALPF-NH2. Investigation of the evolutionary history of the histidine residue that confers these properties revealed that it can be traced to the common ancestor of echinoderms, which is estimated to have lived ~500 million years ago. However, L-type precursors comprising multiple SALMFamides with the histidine residue forming an ATCUN motif appears to be a feature that has evolved uniquely in starfish (Asteroidea). GENERAL SIGNIFICANCE: The discovery of a SALMFamide-type neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind metal ions and generate metal-linked dimers provides a new insight on ATCUN motif-containing neuropeptides. This property of L-type SALMFamides in the Asteroidea may be associated with a role in regulation of the unusual extra-oral feeding behaviour of starfish.

Leptin- and cytokine-like unpaired signaling in Drosophila.

Animals have evolved a multitude of signaling pathways that enable them to orchestrate diverse physiological processes to tightly regulate systemic homeostasis. This signaling is mediated by various families of peptide hormones and cytokines that are conserved across the animal kingdom. In this review, we primarily focus on the unpaired (Upd) family of proteins in Drosophila which are evolutionarily related to mammalian leptin and the cytokine interleukin 6. We summarize expression patterns of Upd in Drosophila and discuss the parallels in structure, signaling pathway, and functions between Upd and their mammalian counterparts. In particular, we focus on the roles of Upd in governing metabolic homeostasis, growth and development, and immune responses. We aim to stimulate future studies on leptin-like signaling in other phyla which can help bridge the evolutionary gap between insect Upd and vertebrate leptin and cytokines like interleukin 6.

Post-feeding physiology in Rhodnius prolixus: the possible role of FGLamide-related allatostatins.

Allatostatins (ASTs) are neuropeptides that were first identified as inhibitors of juvenile hormone biosynthesis by the corpora allata of some insect species. The FGLamide-related ASTs (FGLa/ASTs) belong to one of three families of insect ASTs. Previously, we showed that Rhodnius prolixus FGLa/ASTs (Rhopr-FGLa/ASTs) are present throughout the R. prolixus central nervous system and are associated with 5 dorsal unpaired median (DUM) neurons in the mesothoracic ganglionic mass. A similar set of neurons contain serotonin which is a diuretic hormone in R. prolixus. Rhopr-FGLa/ASTs inhibit both spontaneous contractions of the anterior midgut and leucokinin-1-induced hindgut contractions. Since these tissues are involved with post-feeding diuresis, these data suggest a possible role for FGLa/ASTs in events associated with feeding, and a possible interaction with serotonin. To investigate this possibility, we have examined the DUM neurons in more detail with regard to their peptide content, examined the potential release of Rhopr-FGLa/ASTs into the haemolymph following feeding, and further investigated the effects of Rhopr-FGLa/ASTs on feeding-related tissues. There are 10 DUM neurons in the abdominal neuromeres, 5 of which express serotonin-like immunoreactivity and the other 5 express FGLa/AST-like immunoreactivity. FGLa/AST-like immunoreactivity is reduced in the 5 DUM neuron cell bodies and their neurohaemal sites on abdominal nerves at 3-5 h post feeding. Rhopr-FGLa/ASTs do not inhibit serotonin-stimulated anterior midgut absorption or Malpighian tubule secretion but do inhibit hindgut contractions induced by an endogenous kinin, suggesting that they may only indirectly affect post-feeding diuresis in R. prolixus.

Discovery of Paralogous GnRH and Corazonin Signaling Systems in an Invertebrate Chordate.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive function in vertebrates. GnRH is related to the corazonin (CRZ) neuropeptide which influences metabolism and stress responses in insects. Recent evidence suggests that GnRH and CRZ are paralogous and arose by a gene duplication in a common ancestor of bilaterians. Here, we report the identification and complete characterization of the GnRH and CRZ signaling systems in the amphioxus Branchiostoma floridae. We have identified a novel GnRH peptide (YSYSYGFAP-NH2) that specifically activates two GnRH receptors and a CRZ peptide (FTYTHTW-NH2) that activates three CRZ receptors in B. floridae. The latter appear to be promiscuous, as two CRZ receptors can also be activated by GnRH in the physiological range. Hence, there is a potential for cross-talk between these closely related signaling systems. Discovery of both the GnRH and CRZ signaling systems in one of the closest living relatives of vertebrates provides a framework to discover their roles at the transition from invertebrates to vertebrates.

Characterization of clock-related proteins and neuropeptides in Drosophila littoralis and their putative role in diapause.

Insects from high latitudes spend the winter in a state of overwintering diapause, which is characterized by arrested reproduction, reduced food intake and metabolism, and increased life span. The main trigger to enter diapause is the decreasing day length in summer-autumn. It is thus assumed that the circadian clock acts as an internal sensor for measuring photoperiod and orchestrates appropriate seasonal changes in physiology and metabolism through various neurohormones. However, little is known about the neuronal organization of the circadian clock network and the neurosecretory system that controls diapause in high-latitude insects. We addressed this here by mapping the expression of clock proteins and neuropeptides/neurohormones in the high-latitude fly Drosophila littoralis. We found that the principal organization of both systems is similar to that in Drosophila melanogaster, but with some striking differences in neuropeptide expression levels and patterns. The small ventrolateral clock neurons that express pigment-dispersing factor (PDF) and short neuropeptide F (sNPF) and are most important for robust circadian rhythmicity in D. melanogaster virtually lack PDF and sNPF expression in D. littoralis. In contrast, dorsolateral clock neurons that express ion transport peptide in D. melanogaster additionally express allatostatin-C and appear suited to transfer day-length information to the neurosecretory system of D. littoralis. The lateral neurosecretory cells of D. littoralis contain more neuropeptides than D. melanogaster. Among them, the cells that coexpress corazonin, PDF, and diuretic hormone 44 appear most suited to control diapause. Our work sets the stage to investigate the roles of these diverse neuropeptides in regulating insect diapause.

Endocrine cybernetics: neuropeptides as molecular switches in behavioural decisions.

Plasticity in animal behaviour relies on the ability to integrate external and internal cues from the changing environment and hence modulate activity in synaptic circuits of the brain. This context-dependent neuromodulation is largely based on non-synaptic signalling with neuropeptides. Here, we describe select peptidergic systems in the Drosophila brain that act at different levels of a hierarchy to modulate behaviour and associated physiology. These systems modulate circuits in brain regions, such as the central complex and the mushroom bodies, which supervise specific behaviours. At the top level of the hierarchy there are small numbers of large peptidergic neurons that arborize widely in multiple areas of the brain to orchestrate or modulate global activity in a state and context-dependent manner. At the bottom level local peptidergic neurons provide executive neuromodulation of sensory gain and intrinsically in restricted parts of specific neuronal circuits. The orchestrating neurons receive interoceptive signals that mediate energy and sleep homeostasis, metabolic state and circadian timing, as well as external cues that affect food search, aggression or mating. Some of these cues can be triggers of conflicting behaviours such as mating versus aggression, or sleep versus feeding, and peptidergic neurons participate in circuits, enabling behaviour choices and switches.

Recent advances in neuropeptide signaling in Drosophila, from genes to physiology and behavior.

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.

Tachykinins: Neuropeptides That Are Ancient, Diverse, Widespread and Functionally Pleiotropic.

Tachykinins (TKs) are ancient neuropeptides present throughout the bilaterians and are, with some exceptions, characterized by a conserved FX1GX2Ramide carboxy terminus among protostomes and FXGLMamide in deuterostomes. The best-known TK is the vertebrate substance P, which in mammals, together with other TKs, has been implicated in health and disease with important roles in pain, inflammation, cancer, depressive disorder, immune system, gut function, hematopoiesis, sensory processing, and hormone regulation. The invertebrate TKs are also known to have multiple functions in the central nervous system and intestine and these have been investigated in more detail in the fly Drosophila and some other arthropods. Here, we review the protostome and deuterostome organization and evolution of TK precursors, peptides and their receptors, as well as their functions, which appear to be partly conserved across Bilateria. We also outline the distribution of TKs in the brains of representative organisms. In Drosophila, recent studies have revealed roles of TKs in early olfactory processing, neuromodulation in circuits controlling locomotion and food search, nociception, aggression, metabolic stress, and hormone release. TK signaling also regulates lipid metabolism in the Drosophila intestine. In crustaceans, TK is an important neuromodulator in rhythm-generating motor circuits in the stomatogastric nervous system and a presynaptic modulator of photoreceptor cells. Several additional functional roles of invertebrate TKs can be inferred from their distribution in various brain circuits. In addition, there are a few interesting cases where invertebrate TKs are injected into prey animals as vasodilators from salivary glands or paralyzing agents from venom glands. In these cases, the peptides are produced in the glands of the predator with sequences mimicking the prey TKs. Lastly, the TK-signaling system appears to have duplicated in Panarthropoda (comprising arthropods, onychophores, and tardigrades) to give rise to a novel type of peptides, natalisins, with a distinct receptor. The distribution and functions of natalisins are distinct from the TKs. In general, it appears that TKs are widely distributed and act in circuits at short range as neuromodulators or cotransmitters.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms.

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide 'cocktails' is functionally important, but with plasticity over long evolutionary time scales.

DINeR: Database for Insect Neuropeptide Research.

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/.

Isolation and characterization of the corticotropin-releasing factor-related diuretic hormone receptor in Rhodnius prolixus.

Rhodnius prolixus, the vector of human Chagas disease, is a hemipteran insect that undergoes rapid post-feeding diuresis following ingestion of a blood meal that can be up to 10 times its initial body weight. Corticotropin-releasing factor-related diuretic hormone (Rhopr-CRF/DH) and serotonin are neurohormones that are synergistic in increasing rates of fluid secretion by Malpighian tubules during this rapid post-feeding diuresis. A Rhopr-CRF/DH receptor transcript has now been isolated and characterized from fifth instar R. prolixus. The receptor is a family B1 (secretin) G protein-coupled receptor (GPCR) and was deorphaned in a heterologous cellular system using Chinese hamster ovary (CHO) cells stably expressing a promiscuous G-protein (Gα16). This assay was also used to demonstrate the presence of Rhopr-CRF/DH in the haemolymph of R. prolixus in response to blood-gorging. Two additional cell lines were used in this heterologous assay to verify that the cyclic adenosine monophosphate (cAMP) pathway and not the inositol triphosphate (IP3) pathway was stimulated upon activation of the receptor. Lastly, quantitative PCR demonstrated strong receptor expression in digestive tissues, upper Malpighian tubules and reproductive tissues. Identification of the Rhopr-CRF/DH receptor now provides tools for a more detailed understanding into the precise coordination of diuresis and other physiological processes in R. prolixus.