This review briefly summarizes 50 years of research on insect neuropeptide and peptide hormone (collectively abbreviated NPH) signaling, starting with the sequencing of proctolin in 1975. The first 25 years, before the sequencing of the Drosophila genome, were characterized by efforts to identify novel NPHs by biochemical means, mapping of their distribution in neurons, neurosecretory cells, and endocrine cells of the intestine. Functional studies of NPHs were predominantly dealing with hormonal aspects of peptides and many employed ex vivo assays. With the annotation of the Drosophila genome, and more specifically of the NPHs and their receptors in Drosophila and other insects, a new era followed. This started with matching of NPH ligands to orphan receptors, and studies to localize NPHs with improved detection methods. Important advances were made with introduction of a rich repertoire of innovative molecular genetic approaches to localize and interfere with expression or function of NPHs and their receptors. These methods enabled cell- or circuit-specific interference with NPH signaling for in vivo assays to determine roles in behavior and physiology, imaging of neuronal activity, and analysis of connectivity in peptidergic circuits. Recent years have seen a dramatic increase in reports on the multiple functions of NPHs in development, physiology and behavior. Importantly, we can now appreciate the pleiotropic functions of NPHs, as well as the functional peptidergic “networks” where state dependent NPH signaling ensures behavioral plasticity and systemic homeostasis.

Mating in insects commonly induces a profound change in the physiology and behavior of the female that serves to secure numerous and viable offspring, and to ensure paternity for the male by reducing receptivity of the female to further mating attempts. Here, we set out to characterize the post-mating response (PMR) in a pest insect, the brown planthopper Nilaparvata lugens and to identify a functional analog of sex peptide and/or other seminal fluid factors that contribute to the PMR in Drosophila. We find that N. lugens display a distinct PMR that lasts for about 4 days and includes a change in female behavior with decreased receptivity to males and increased oviposition. Extract from male accessory glands (MAG) injected into virgin females triggers a similar PMR, lasting about 24h. Since sex peptide does not exist in N. lugens, we screened for candidate mediators by performing a transcriptional and proteomics analysis of MAG extract. We identified a novel 51 amino acid peptide present only in the MAG and not in female N. lugens. This peptide, that we designate maccessin (macc), affects the female PMR. Females mated by males with macc knockdown display receptivity to wild type males in a second mating, which does not occur in controls. However, oviposition is not affected. Injection of recombinant macc reduces female receptivity, with no effect on oviposition. Thus, macc is an important seminal fluid peptide that affects the PMR of N. lugens. Our analysis suggests that the gene encoding the macc precursor is restricted to species closely related to N. lugens.

Neuropeptides and peptide hormones are the most diverse messenger molecules in animals and play important roles in the regulation of daily physiology and a multitude of behaviors. Many of these peptides and their cognate receptors are structurally and functionally conserved over evolution in bilaterians. Prime examples of this are peptides related to cholecystokinin (CCK) and gastrin. Here, we discuss CCK signaling in mammals and several key vertebrate groups, as well as in invertebrates. In mammals, CCK is primarily produced by intestinal endocrine cells and brain neurons and regulates gall bladder contractions, pancreatic enzyme secretion, gut functions, and food intake, as well as playing important signaling roles in the brain. Brain-derived CCK regulates circuits regulating reward, anxiety, aggression, and sexual behavior. In invertebrates, CCK-like peptides (sulfakinins, SKs) are, with a few exceptions, produced by brain neurons only. Invertebrate SKs regulate food ingestion by a variety of mechanisms. Also, regulation of digestive enzymes has been reported. The genetically tractable fly Drosophila melanogaster has been extensively investigated with respect to neuropeptide signaling at the cellular level. These studies have also advanced our understanding of SK signaling mechanisms in regulation of feeding, but also in gustatory sensitivity, locomotor activity, aggression, and reproductive behavior. In Drosophila, a set of only eight SK-expressing brain neurons plays critical roles in regulation of these competing behaviors. In male flies, they integrate internal state and external stimuli to diminish sex drive and increase aggression. The same neurons also diminish sugar gustation and reduce food intake. Although several functional roles of CCK/SK signaling are conserved between Drosophila and mammals, available data suggest that the underlying neuronal systems and mechanisms differ.

Plasticity in animal behavior and physiology is largely due to modulatory and regulatory signaling with neuropeptides and peptide hormones (collectively abbreviated NPHs). The NPHs constitute a very large and versatile group of signaling substances that partake at different regulatory levels in most daily activities of an organism. This review summarizes key principles in NPH actions in the brain and in interorgan signaling, with focus on Drosophila. NPHs are produced by neurons, neurosecretory cells (NSCs) and other endocrine cells in NPH-specific and stereotypic patterns. Most of the NPHs have multiple (pleiotropic) functions and target several different neuronal circuits and/or peripheral tissues. Such divergent NPH signaling ensures orchestration of behavior and physiology in state-dependent manners. Conversely, many neurons, circuits, NSCs, or other cells, are targeted by multiple NPHs. This convergent signaling commonly conveys various signals reporting changes in the external and internal environment to central neurons/circuits. As an example of wider functional convergence, 26 different Drosophila NPHs act at many different levels to regulate food search and feeding. Convergence is also seen in hormonal regulation of peripheral functions. For instance, multiple NPHs target renal tubules to ensure osmotic homeostasis. Interestingly, several of the same osmoregulatory NPHs also regulate feeding, metabolism and stress. However, for some NPHs the cellular distribution and functions suggests multiple unrelated functions that are restricted to specific circuits. Thus, NPH signaling follows distinct patterns for each specific NPH, but taken together they form overlapping networks that modulate behavior and physiology.

Living in dynamic environments such as the social domain, where interaction with others determines the reproductive success of individuals, requires the ability to recognize opportunities to obtain natural rewards and cope with challenges that are associated with achieving them. As such, actions that promote survival and reproduction are reinforced by the brain reward system, whereas coping with the challenges associated with obtaining these rewards is mediated by stress-response pathways, the activation of which can impair health and shorten lifespan. While much research has been devoted to understanding mechanisms underlying the way by which natural rewards are processed by the reward system, less attention has been given to the consequences of failure to obtain a desirable reward. As a model system to study the impact of failure to obtain a natural reward, we used the well-established courtship suppression paradigm in Drosophila melanogaster as means to induce repeated failures to obtain sexual reward in male flies. We discovered that beyond the known reduction in courtship actions caused by interaction with non-receptive females, repeated failures to mate induce a stress response characterized by persistent motivation to obtain the sexual reward, reduced male-male social interaction, and enhanced aggression. This frustrative-like state caused by the conflict between high motivation to obtain sexual reward and the inability to fulfill their mating drive impairs the capacity of rejected males to tolerate stressors such as starvation and oxidative stress. We further show that sensitivity to starvation and enhanced social arousal is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y. Our findings demonstrate for the first time the existence of social stress in flies and offers a framework to study mechanisms underlying the crosstalk between reward, stress, and reproduction in a simple nervous system that is highly amenable to genetic manipulation. In this study we investigated the effects of failure to obtain reward on the behavioral actions and physiology of male flies. We exposed Drosophila males to repeated sexual encounters with non-receptive females that rejected their courtship efforts and tested the effect on their behavioral responses using a collection of behavioral paradigms. These responses encompass alterations in social behavior, increased aggression, heightened motivation to mate, and a reduced capacity to cope with stressors. We further show that the high motivational state and sensitivity to stress is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y.

Tachykinins (TKs) are evolutionarily ancient neuropeptides found throughout most bilaterians, characterized by a conserved FX1GX2Ramide carboxy-terminus in protostomes and FXGLMamide in deuterostomes. In mammals, substance P and other TKs have 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. In invertebrates, the TKs also have multiple functions in the central nervous system and intestine, which have been investigated in detail in the fly Drosophila. This chapter reviews the organization and evolution of TK precursors, peptides, and their receptors in protostomes and deuterostomes. It provides a comparative description of the distribution and functions of TKs in bilaterian organisms. Several TK functions appear to be partly conserved across these animals. Thus, in Drosophila, recent studies indicate roles of TKs in early olfactory and gustatory processing, neuromodulation in circuits controlling locomotion and food search, nociception, aggression, metabolic stress, and hormone release. TK signaling also regulates motility and lipid metabolism in the Drosophila intestine, and during the development TKs play roles in growth, regeneration, and inflammation. In general, TKs are widely distributed and act in neuronal circuits at short range as neuromodulators or co-transmitters. A curiosity is that separate genes encode mimetic TKs in frog skin and in the salivary glands or venom glands of insects. The insect gland TKs are injected into prey animals to cause vasodilation and paralysis, respectively.

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into daily oscillations of Ca²⁺ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contributes to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2) plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert and could serve as a paradigm to understand how circadian clocks are coordinated.

Neuropeptides are the most diverse messenger molecules in metazoans and are involved in regulation of daily physiology and a wide array of behaviors. Some neuropeptides and their cognate receptors are structurally and functionally well conserved over evolution in bilaterian animals. Among these are peptides related to gastrin and cholecystokinin (CCK). In mammals, CCK is produced by intestinal endocrine cells and brain neurons, and regulates gall bladder contractions, pancreatic enzyme secretion, gut functions, satiety and food intake. Additionally, CCK plays important roles in neuromodulation in several brain circuits that regulate reward, anxiety, aggression and sexual behavior. In invertebrates, CCK-type peptides (sulfakinins, SKs) are, with a few exceptions, produced by brain neurons only. Common among invertebrates is that SKs mediate satiety and regulate food ingestion by a variety of mechanisms. Also regulation of secretion of digestive enzymes has been reported. Studies of the genetically tractable fly Drosophila have advanced our understanding of SK signaling mechanisms in regulation of satiety and feeding, but also in gustatory sensitivity, locomotor activity, aggression and reproductive behavior. A set of eight SK-expressing brain neurons plays important roles in regulation of these competing behaviors. In males, they integrate internal state and external stimuli to diminish sex drive and increase aggression. The same neurons also diminish sugar gustation, induce satiety and reduce feeding. Although several functional roles of CCK/SK signaling appear conserved between Drosophila and mammals, available data suggest that the underlying mechanisms differ.

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.

Diapause, a general shutdown of developmental pathways, is a vital adaptation allowing insects to adjust their life cycle to adverse environmental conditions such as winter. Diapause in the pupal stage is regulated by the major developmental hormones prothoracicotropic hormone (PTTH) and ecdysone. Termination of pupal diapause in the butterfly Pieris napi depends on low temperatures; therefore, we study the temperature-dependence of PTTH secretion and ecdysone sensitivity dynamics throughout diapause, with a focus on diapause termination. While PTTH is present throughout diapause in the cell bodies of two pairs of neurosecretory cells in the brain, it is absent in the axons, and the PTTH concentration in the haemolymph is significantly lower during diapause than during post diapause development, indicating that the PTTH signaling is reduced during diapause. The sensitivity of pupae to ecdysone injections is dependent on diapause stage. While pupae are sensitive to ecdysone during early diapause initiation, they gradually lose this sensitivity and become insensitive to non-lethal concentrations of ecdysone about 30 days into diapause. At low temperatures, reflecting natural overwintering conditions, diapause termination propensity after ecdysone injection is precocious compared to controls. In stark contrast, at high temperatures reflecting late summer and early autumn conditions, sensitivity to ecdysone does not return. Thus, here we show that PTTH secretion is reduced during diapause, and additionally, that the low ecdysone sensitivity of early diapause maintenance is lost during termination in a temperature dependent manner. The link between ecdysone sensitivity and low-temperature dependence reveals a putative mechanism of how diapause termination operates in insects that is in line with adaptive expectations for diapause.