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Termites live in colonies, and their members belong to different castes that each have their specific role within the termite society. In well-established colonies of higher termites, the only food the founding female, the queen, receives is saliva from workers; such queens can live for many years and produce up to 10,000 eggs per day. In higher termites, worker saliva must thus constitute a complete diet and therein resembles royal jelly produced by the hypopharyngeal glands of honeybee workers that serves as food for their queens; indeed, it might as well be called termite royal jelly. However, whereas the composition of honeybee royal jelly is well established, that of worker termite saliva in higher termites remains largely unknown. In lower termites, cellulose-digesting enzymes constitute the major proteins in worker saliva, but these enzymes are absent in higher termites. Others identified a partial protein sequence of the major saliva protein of a higher termite and identified it as a homolog of a cockroach allergen. Publicly available genome and transcriptome sequences from termites make it possible to study this protein in more detail. The gene coding the termite ortholog was duplicated, and the new paralog was preferentially expressed in the salivary gland. The amino acid sequence of the original allergen lacks the essential amino acids methionine, cysteine and tryptophan, but the salivary paralog incorporated these amino acids, thus allowing it to become more nutritionally balanced. The gene is found in both lower and higher termites, but it is in the latter that the salivary paralog gene got reamplified, facilitating an even higher expression of the allergen. This protein is not expressed in soldiers, and, like the major royal jelly proteins in honeybees, it is expressed in young but not old workers.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far . In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

Severe sleep deprivation (SD) has been highly associated with systemic energy wasting, such as lipid loss and glycogen depletion. Despite immune dysregulation and neurotoxicity observed in SD animals, whether and how the gut-secreted hormones participate in SD-induced disruption of energy homeostasis remains largely unknown. Using Drosophila as a conserved model organism, we characterize that production of intestinal Allatostatin A (AstA), a major gut-peptide hormone, is robustly increased in adult flies bearing severe SD. Interestingly, the removal of AstA production in the gut using specific drivers significantly improves lipid loss and glycogen depletion in SD flies without affecting sleep homeostasis. We reveal the molecular mechanisms whereby gut AstA promotes the release of an adipokinetic hormone (Akh), an insulin counter-regulatory hormone functionally equivalent to mammalian glucagon, to mobilize systemic energy reserves by remotely targeting its receptor AstA-R2 in Akh-producing cells. Similar regulation of glucagon secretion and energy wasting by AstA/galanin is also observed in SD mice. Further, integrating single-cell RNA sequencing and genetic validation, we uncover that severe SD results in ROS accumulation in the gut to augment AstA production via TrpA1. Altogether, our results demonstrate the essential roles of the gut-peptide hormone AstA in mediating SD-associated energy wasting., (© 2023. The Author(s).)

Of the nine genes of the American cockroach, Periplaneta americana, coding for peptides related to insulin and insulin-like growth factor, seven show significant expression in the central nervous system as demonstrated by the polymerase chain reaction on reverse transcribed RNA. In situ hybridisation shows that five of those are expressed by cells in the pars intercerebralis. Antisera raised to the predicted peptides show that these cells are neuroendocrine in nature and project to the corpora cardiaca. Interestingly, there are at least three cell types that each express different genes. This contrasts with Drosophila where a single cell type expresses a number of genes expressing several such peptides. Whereas in Drosophila the neuroendocrine cells producing insulin-like peptides also express sulfakinins, the arthropod orthologs of gastrin and cholecystokinin, in Periplaneta the sulfakinins are produced by different cells. Other neuropeptides known to be produced by the pars intercerebralis in Periplaneta and other insect species, such as the CRF-like diuretic hormone, neuroparsin, leucokinin or myosuppressin, neither colocalize with an insulin-related peptide. The separate cellular localization of these peptides and the existence of multiple insulin receptors in this species implies a more complex regulation by insulin and IGF-related peptides in cockroaches than in the fruit fly., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Insulin-like growth factor (IGF) and other insulin-like peptides (ilps) are important hormones regulating growth and development in animals. Whereas most animals have a single female and male adult phenotype, in some insect species the same genome may lead to different final forms. Perhaps the best known example is the honeybee where females can either develop into queens or workers. More extreme forms of such polyphenism occur in termites, where queens, kings, workers and soldiers coexist. Both juvenile hormone and insulin-like peptides are known to regulate growth and reproduction as well as polyphenism. In termites the role of juvenile hormone in reproduction and the induction of the soldier caste is well known, but the role of IGF and other ilps in these processes remains largely unknown. Here the various termite ilps are identified and hypotheses regarding their functions suggested., Methods: Genome assemblies and transcriptome short read archives (SRAs) were used to identify insulin-like peptides and neuropeptides in termites and to determine their expression in different species, tissues and castes., Results and Discussion: Termites have seven different ilps, i.e. gonadulin, IGF and an ortholog of Drosophila insulin-like peptide 7 (dilp7), which are commonly present in insects, and four smaller peptides, that have collectively been called short IGF-related peptides (sirps) and individually atirpin, birpin, cirpin and brovirpin. Gonadulin is lost from the higher termites which have however amplified the brovirpin gene, of which they often have two or three paralogs. Based on differential expression of these genes it seems likely that IGF is a growth hormone and atirpin an autocrine tissue factor that is released when a tissue faces metabolic stress. Birpin seems to be responsible for growth and in the absence of juvenile hormone this may lead to reproductive adults or, when juvenile hormone is present, to soldiers. Brovirpin is expressed both by the brain and the ovary and likely stimulates vitellogenesis, while the function of cirpin is less clear., Competing Interests: The author declares that he has no competing interests., (©2023 Veenstra.)

The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

This work was initiated because an old publication suggested that electrocoagulation of four paraldehyde fuchsin positive cells in the brain of Locusta migratoria might produce a diuretic hormone, the identity of which remains unknown, since none of the antisera to the various putative Locusta diuretic hormones recognizes these cells. The paraldehyde fuchsin positive staining suggests a peptide with a disulfide bridge and the recently identified Locusta calcitonins have both a disulfide bridge and are structurally similar to calcitonin-like diuretic hormone. In situ hybridization and antisera raised to calcitonin-A and -B were used to show where these peptides are expressed in Locusta. Calcitonin-A is produced by neurons and neuroendocrine cells that were previously shown to be immunoreactive to an antiserum to pigment dispersing factor (PDF). The apparent PDF-immunoreactivity in these neurons and neuroendocrine cells is due to crossreactivity with the calcitonin-A precursor. As confirmed by both an PDF-precursor specific antiserum and in situ hybridisation, those calcitonin-A expressing cells do not express PDF. Calcitonin B is expressed by numerous enteroendocrine cells in the midgut as well as the midgut caeca. A guinea pig antiserum to calcitonin A seemed quite specific as it recognized only the calcitonin A expressing cells. However, rabbit antisera to calcitonin-A and-B both crossreacted with neuroendocrine cells in the brain that produce ACP (AKH/corazonin-related peptide), this is almost certainly due to the common C-terminal dipeptide SPamide that is shared between Locusta calcitonin-A, calcitonin-B and ACP., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aphids were the first animals described as photoperiodic due to their seasonal switch from viviparous parthenogenesis to sexual reproduction (cyclical parthenogenesis) caused by the shortening of the photoperiod in autumn. This switch produces a single sexual generation of oviparous females and males that mate and lay diapausing cold-resistant eggs that can overcome the unfavourable environmental conditions typical of winter in temperate regions. Previous studies have hinted at a possible implication of two insulin-like peptides (ILP1 and ILP4) in the aphid seasonal response, changing their expression levels between different photoperiodic conditions. Moreover, in situ localization of their transcripts in particular neurosecretory cells (NSCs) in the aphid brain supported the idea that these neuropeptides could correspond to the formerly called virginoparin, an uncharacterized factor originally proposed to be transported directly to the aphid embryos to promote their development as parthenogenetic individuals. To further investigate the fate of these ILPs, we raised a specific antiserum against one of them (ILP4) and mapped this neuropeptide by immunohistochemistry (IHC) in Acyrthosiphon pisum and Megoura viciae aphids. Coincident with in situ localization, our results show that ILP4 is synthesized in two groups (one in each brain hemisphere) of four neurosecretory cells in the pars intercerebralis (NSC group I) and then it is transported outside the brain to the corpora cardiaca. From there, three nerves (two laterals and one medial) transport it to the abdomen. Although no precise site of release has been found, the terminations of these nerves near the germaria would be compatible with the proposal of a direct connection between group I of NSCs and the reproductive system by localized release. In addition, we detected some collateral arborizations originating from the eight NSCs going to the pars lateralis, where clock neurons and some photoreceptors have been previously localized, suggesting a possible communication between the circadian and photoperiodic systems., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Some insulin/IGF-related peptides (irps) stimulate a receptor tyrosine kinase (RTK) that transfers the extracellular hormonal signal into an intracellular response. Other irps, such as relaxin, do not use an RTK, but a G-protein coupled receptor (GPCR). This is unusual since evolutionarily related hormones typically either use the same or paralogous receptors. In arthropods three different irps, i.e. arthropod IGF, gonadulin and Drosophila insulin-like peptide 7 (dilp7), likely evolved from a gene triplication, as in several species genes encoding these three peptides are located next to one another on the same chromosomal fragment. These arthropod irps have homologs in vertebrates, suggesting that the initial gene triplication was perhaps already present in the last common ancestor of deuterostomes and protostomes. It would be interesting to know whether this is indeed so and how insulin might be related to this trio of irps., Methodology: Genes encoding irps as well as their putative receptors were identified in genomes and transcriptomes from echinoderms and hemichordates., Results: A similar triplet of genes coding for irps also occurs in some ambulacrarians. Two of these are orthologs of arthropod IGF and dilp7 and the third is likely a gonadulin ortholog. In echinoderms, two novel irps emerged, gonad stimulating substance (GSS) and multinsulin, likely from gene duplications of the IGF and dilp7-like genes respectively. The structures of GSS diverged considerably from IGF, which would suggest they use different receptors from IGF, but no novel irp receptors evolved. If IGF and GSS use different receptors, and the evolution of GSS from a gene duplication of IGF is not associated with the appearance of a novel receptor, while irps are known to use two different types of receptors, the ancestor of GSS and IGF might have acted on both types of receptors while one or both of its descendants act on only one. There are three ambulacrarian GPCRs that have amino acid sequences suggestive of being irp GPCRs, two of these are orthologs of the gonadulin and dilp7 receptors. This suggests that the third might be an IGF receptor, and that by deduction, GSS only acts on the RTK. The evolution of GSS from IGF may represent a pattern, where IGF gene duplications lead to novel genes coding for shorter peptides that activate an RTK. It is likely this is how insulin and the insect neuroendocrine irps evolved independently from IGF., Conclusion: The local gene triplication described from arthropods that yielded three genes encoding irps was already present in the last common ancestor of protostomes and deuterostomes. It seems plausible that irps, such as those produced by neuroendocrine cells in the brain of insects and echinoderm GSS evolved independently from IGF and, thus, are not true orthologs, but the result of convergent evolution., Competing Interests: The author declares that he has no competing interests., (©2021 Veenstra.)

Many insect species have several genes coding for insulin-related peptides (IRPs), but so far only a single IRP gene has been identified in migratory locusts. Here, we report and characterize two other genes coding for peptides that are related to insulin, namely gonadulin and arthropod insulin-like growth factor (aIGF); peptides postulated to be orthologs of Drosophila melanogaster insulin-like peptides 8 and 6 respectively. In Locusta migratoria the aIGF transcript is expressed in multiple tissues as was previously reported for IRP in both L. migratoria and Schistocerca gregaria , but there are significant differences in expression patterns between the two species. The gonadulin transcript, however, seems specific to the ovary, whereas its putative receptor transcript is expressed most abundantly in the ovary, fat body and the central nervous system. Since the central nervous system-fat body-ovary axis is essential for successful reproduction, we studied the influence of gonadulin on vitellogenesis and oocyte growth. A reduction in the gonadulin transcript ( via RNA interference) led to a significant reduction in vitellogenin mRNA levels in the fat body and a strong oocyte growth inhibition, thus suggesting an important role for gonadulin in reproduction in this species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Veenstra, Leyria, Orchard and Lange.)

As a model hemimetabolous insect species and an invasive urban pest that is globally distributed, the American cockroach, Periplaneta americana , is of great interest in both basic and applied research. Previous studies on P. americana neuropeptide identification have been based on biochemical isolation and molecular cloning. In the present study, an integrated approach of genomics- and peptidomics-based discovery was performed for neuropeptide identification in this insect species. First, 67 conserved neuropeptide or neurohormone precursor genes were predicted via an in silico analysis of the P. americana genome and transcriptome. Using a large-scale peptidomic analysis of peptide extracts from four different tissues (the central nervous system, corpora cardiac and corpora allata complex, midgut, and male accessory gland), 35 conserved (predicted) neuropeptides and a potential (novel) neuropeptide were then identified. Subsequent experiments revealed the tissue distribution, sex difference, and developmental patterns of two conserved neuropeptides (allatostatin B and short neuropeptide F) and a novel neuropeptide (PaOGS36577). Our study shows a comprehensive neuropeptidome and detailed spatiotemporal distribution patterns, providing a solid basis for future functional studies of neuropeptides in the American cockroach (data are available via ProteomeXchange with identifier PXD021660).

The SMYamide genes are paralogs of the SIFamide genes and code for neuropeptides that are structurally similar to SIFamide. In the American cockroach, Periplanea americana, the SMYamide gene is specifically expressed in the SN2 neurons that innervate the salivary glands and are known to produce action potentials during feeding. The SN2 axon terminals surround rather than directly innervate the salivary gland acini. Therefore one may expect that on activation of these neurons significant amounts of SMYamide will be released into the hemolymph, thus suggesting that SMYamide may also have a hormonal function. In the Periplaneta genome there are two putative SIFamide receptors and these are both expressed not only in the central nervous system and the salivary gland, but also in the gonads and other peripheral tissues. This reinforces the hypothesis that SMYamide also has an endocrine function in this species., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set., Results: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta., Conclusions: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Insulin and related peptides play important roles in the regulation of growth and reproduction. Until recently three different types of insulin-related peptides had been identified from decapod crustaceans. The identification of two novel insulin-related peptides from Sagmariasus verreauxi and Cherax quadricarinatus suggested that there might a fourth type. Publicly available short read archives show that orthologs of these peptides are commonly present in these animals. Most decapods have two genes coding such peptides, but Penaeus species have likely only one and some palaemonids have three. Interestingly, expression levels can vary more than thousand-fold in the gonads of Portunus trituberculatus, where gonadulin 1 is expressed by the testis and gonadulin 2 by the ovary. Although these peptides are also expressed in other tissues, the occasionally very high expression in the gonads led to them being called gonadulins., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We have previously reported that pigment dispersing factor (PDF) neurons, which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections, a phenomenon called circadian structural plasticity. Axonal arborizations display higher complexity during the day and become simpler at night, and this remodeling involves changes in the degree of connectivity. This phenomenon depends on the clock present within the ventrolateral neurons (LNvs) as well as in glia. In this work, we characterize in detail the contribution of the PDF neuropeptide to structural plasticity at different times across the day. Using diverse genetic strategies to temporally restrict its downregulation, we demonstrate that even subtle alterations to PDF cycling at the dorsal protocerebrum correlate with impaired remodeling, underscoring its relevance for the characteristic morning spread; PDF released from the small LNvs (sLNvs) and the large LNvs (lLNvs) contribute to the process. Moreover, forced depolarization recruits activity-dependent mechanisms to mediate growth only at night, overcoming the restriction imposed by the clock on membrane excitability. Interestingly, the active process of terminal remodeling requires PDF receptor (PDFR) signaling acting locally through the cyclic-nucleotide-gated channel ion channel subunit A (CNGA). Thus, clock-dependent PDF signaling shapes the connectivity of these essential clock neurons on daily basis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Insects have several genes coding for insulin-like peptides and they have been particularly well studied in Drosophila . Some of these hormones function as growth hormones and are produced by the fat body and the brain. These act through a typical insulin receptor tyrosine kinase. Two other Drosophila insulin-like hormones are either known or suspected to act through a G-protein coupled receptor. Although insulin-related peptides are known from other insect species, Drosophila insulin-like peptide 8, one that uses a G-protein coupled receptor, has so far only been identified from Drosophila and other flies. However, its receptor is widespread within arthropods and hence it should have orthologs. Such putative orthologs were recently identified in decapods and have been called gonadulins., Methodology: In an effort to identify gonadulins in other arthropods public genome assemblies and short-read archives from insects and other arthropods were explored for the presence of genes and transcripts coding insulin-like peptides and their putative receptors., Results: Gonadulins were detected in a number of arthropods. In those species for which transcriptome data from the gonads is available insect gonadulin genes are expressed in the ovaries and at least in some species also in the testes. In some insects differences in gonadulin expression in the ovary between actively reproducing and non-reproducing females differs more than 100-fold. Putative orthologs of Drosophila ilp 6 were also identified. In several non-Dipteran insects these peptides have C-terminally extensions that are alternatively spliced. The predicted peptides have been called arthropod insulin-like growth factors. In cockroaches, termites and stick insects genes coding for the arthropod insulin-like growth factors, gonadulin and relaxin, a third insulin-like peptide, are encoded by genes that are next to one another suggesting that they are the result of a local gene triplication. Such a close chromosomal association was also found for the arthropod insulin-like growth factor and gonadulin genes in spiders. Phylogenetic tree analysis of the typical insulin receptor tyrosine kinases from insects, decapods and chelicerates shows that the insulin signaling pathway evolved differently in these three groups. The G-protein coupled receptors that are related to the Drosophila ilp 8 receptor similarly show significant differences between those groups., Conclusion: A local gene triplication in an early ancestor likely yielded three genes coding gonadulin, arthropod insulin-like growth factor and relaxin. Orthologs of these genes are now commonly present in arthropods and almost certainly include the Drosophila insulin-like peptides 6, 7 and 8., Competing Interests: The authors declare there are no competing interests., (©2020 Veenstra.)

In the postgenomics era, comparative genomics have advanced the understanding of evolutionary processes of neuropeptidergic signaling systems. The evolutionary origin of many neuropeptidergic signaling systems can be traced date back to early metazoan evolution based on the conserved sequences. Insect parathyroid hormone receptor (iPTHR) was previously described as an ortholog of vertebrate PTHR that has a well-known function in controlling bone remodeling. However, there was no sequence homologous to PTH sequence in insect genomes, leaving the iPTHR as an orphan receptor. Here, we identified the authentic ligand insect PTH (iPTH) for the iPTHR. The taxonomic distribution of iPTHR, which is lacking in Diptera and Lepidoptera, provided a lead for identifying the authentic ligand. We found that a previously described orphan ligand known as PXXXamide (where X is any amino acid) described in the cuttlefish Sepia officinalis has a similar taxonomic distribution pattern as iPTHR. Tests of this peptide, iPTH, in functional reporter assays confirmed the interaction of the ligand-receptor pair. Study of a model beetle, Tribolium castaneum, was used to investigate the function of the iPTH signaling system by RNA interference followed by RNA sequencing and phenotyping. The results suggested that the iPTH system is likely involved in the regulation of cuticle formation that culminates with a phenotype of defects in wing exoskeleton maturation at the time of adult eclosion. Moreover, RNAi of iPTHRs also led to significant reductions in egg numbers and hatching rates after parental RNAi., Competing Interests: The authors have declared that no competing interests exist.

The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. Eight insulin-like peptides (DILP1-8) have been identified in Drosophila , and several of these are known to regulate growth, metabolism, reproduction, stress responses, and lifespan. However, the functional role of DILP1 is far from understood. Previous work has shown that dilp1 /DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. Here, we study the role of dilp1 in the pupa, as well as in the first week of adult life, and make some comparisons to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. We show that mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. We next show that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1 / dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but our data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage., (Copyright © 2020 Liao, Post, Lehmann, Veenstra, Tatar and Nässel.)

The primary sequence of the Arthropod neurohormone neuroparsin is so variable that so far no orthologs from moths and butterflies have been characterized, even though classical neurosecretory stains identify cells that are homologous to those producing this hormone in other insect species. Here Lepidopteran cDNAs showing limited sequence similarity to other insect neuroparsins are described. That these cDNAs do indeed code for authentic neuroparsins was confirmed by in situ hybridization in the wax moth, Galleria mellonella, which labeled the neuroparsin neuroendocrine cells. Although in virtually all genome assemblies from Lepidoptera a neuroparsin gene could be identified, the genome assembly from the silkworm, Bombyx mori, has a neuroparsin gene containing a 16 nucleotide deletion that renders this gene nonfunctional. Although only a small number of all silkworm strains carry this deletion, it suggests that the domestication of the silkworm has rendered the function of this neurohormone dispensable., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Arthropod EFLamide genes in chelicerates, myriapods, decapods and non pterygote hexapods encode various EFLamide paracopies on a single precursor. However, in more advanced insect species such multiple EFLamide paracopies encoding genes are absent. In some Hemiptera putative exons of an EFLamide gene coding for a single EFLamide have been identified, while in the migratory locust a similar exon could potentially code for two EFLamide peptides. The recent identification of an EFLGamide from Platynereis dumerilii as the ligand for an ortholog of the TRH GPCR, suggested that the arthropod EFLamides might similarly activate TRH GPCR orthologs. We here identify the TRH GPCR ortholog from Locusta migratoria and show that it is activated in nanomolar concentrations by the two EFLamides previously predicted from this species. We also show that in the central nervous system there seems to be only a single bilateral neuron in the protocerebrum expressing this peptide. Given this very limited expression of EFLamide in locusts, it is perhaps not surprising that this gene and its receptor have been lost in many other insect species. This shows again that although neuropeptides and their receptors may persist in different evoltionary lineages, their functions can change dramatically., (Copyright © 2019. Published by Elsevier Ltd.)

Phasmid neuropeptide genes were identified in the genomes of two phasmids, Timema cristinae and Clitarchus hookeri. The two species belong to two sisters groups, the Timematodea and Euphasmatodea respectively. Neuropeptide genes were identified using the BLAST+ program on the genome assemblies and the absence of some neuropeptides was confirmed by the concomitant absence of their G-protein coupled receptors. Both genomes were assembled using short reads and the average coverage of the genome is more than 166 times for both species. This makes it virtually impossible that there would not be a single short read for at least one of the conserved transmembrane regions of a GPCR coded by such a genome. Hence, when not a single read can be found for a specific GPCR, it can be concluded that the particular gene is absent from that species. Most previously identified insect neuropeptides are used by these two species. Of the three arthropod allatostatin C related peptides, only allatostatins CC and CCC are present. Both species lack leucokinin, while sulfakinin and dilp8 signaling is absent from Clitarchus, but present in Timema. Interestingly, whereas Timema has lost a vasopressin-related peptide, the gene coding such a peptide is amplified in the Clitarchus genome. Furthermore, while Clitarchus has a specific tryptopyrokinin gene, Timema does not and in this species tryptopyrokinin is coded only by the pyrokinin and periviscerokinin genes. Finally, both species have genes coding EFLamide and its GPCR; in phasmids these genes codes for one (Clitarchus) or two (Timema) EFLamide paracopies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Insect neuropeptides are interesting for the potential their receptors hold as plausible targets for a novel generation of pesticides. Neuropeptide genes have been identified in a number of different species belonging to a variety of insects. Results suggest significant neuropeptide variation between different orders, but much less is known of neuropeptidome variability within an insect order. I therefore compared the neuropeptidomes of a number of Coleoptera., Methodology: Publicly available genome sequences, transcriptomes and the original sequence data in the form of short sequence read archives were analyzed for the presence or absence of genes coding neuropeptides as well as some neuropeptide receptors in seventeen beetle species., Results: Significant differences exist between the Coleoptera analyzed here, while many neuropeptides that were previously characterized from Tribolium castaneum appear very similar in all species, some are not and others are lacking in one or more species. On the other hand, leucokinin, which was presumed to be universally absent from Coleoptera, is still present in non-Polyphaga beetles., Conclusion: The variability in neuropeptidome composition between species from the same insect order may be as large as the one that exists between species from different orders., Competing Interests: The author declares that he has no competing interests.

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Introduction: Malnutrition at the time of ICU admission is associated with an increased morbidity and mortality. Malnutrition is most often assessed by a questionnaire but can also be determined with bio-impedance and measurement of phase angle. In a single-centre observational study we compared the percentage of malnutrition in patients admitted to our ICU, according to the Short Nutritional Assessment Questionnaire (SNAQ) with the phase angle measured with bio-impedance. Furthermore, we questioned whether malnutrition is related to outcome parameters., Methods: In a 15 week period consecutive patients admitted to the ICU were included. Exclusion criteria included age <18 years, anatomical and/or neurological abnormalities of the limbs and an ICU stay less than 6 h. Bio-impedance measurements were performed shortly after admission, the SNAQ was obtained from the patient or legal representative. Malnutrition is considered with a SNAQ ≥2 or a phase angle of <5° for men and <4.6° for women. The study was approved by our local ethical committee (MCL, nWMO 77, April 18, 2015)., Results: 299 Patients were included (66% male, age 66 ± 12 year, BMI 27 ± 4 kg/m 2 and APACHE II score 14 [11-17]), of which 64% elective and 36% acute admissions. Hospital mortality was 7.4%. Malnutrition was present in 16% according to the SNAQ and in 36% according to phase angle. There was a fair accordance between the SNAQ and phase angle. Phase angle was significantly higher in patients with a SNAQ score of 0-1 (5.5°±1.3) in comparison with patients with a SNAQ score ≥2 (4.4°± 1.1) (p < 0.001). Logistic regression analysis showed a significant relation between SNAQ and BMI, malignant disease and hospital length of stay and between phase angle and age, sex, BMI, malignant disease, hospital length of stay and hospital mortality., Conclusion: Malnutrition was present in 16% according to the SNAQ and in 36% according to phase angle in our IC population. Malnutrition was associated with prolonged hospital length of stay. In this small population of mixed ICU patients, a low phase angle was found to independently predict hospital mortality. CLINICAL TRIALS., Gov Number: NCT02911181., (Copyright © 2017 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

The salivary gland salivation stimulating peptide was identified from the salivary glands of the migratory locust by its ability to stimulate cAMP production in the same tissue. The gene coding for this peptide has recently been identified and been shown to code for a precursor consisting of a signal peptide, several copies of the peptide separated by Lys-Arg doublets and a few other peptides. These data are consistent with it being a neuropeptide. However, antiserum raised to this peptide labels the acini of the salivary glands while RT-PCR only gives positive results in the salivary gland, but not in any ganglion of the central nervous system. Thus, this peptide is not a typical neuropeptide as previously assumed., Competing Interests: The author declares that he has no competing interests.

RYamides are arthropod neuropeptides with unknown function. In 2011 two RYamides were isolated from D. melanogaster as the ligands for the G-protein coupled receptor CG5811. The D. melanogaster gene encoding these neuropeptides is highly unusual, as there are four RYamide encoding exons in the current genome assembly, but an exon encoding a signal peptide is absent. Comparing the D. melanogaster gene structure with those from other species, including D. virilis, suggests that the gene is degenerating. RNAseq data from 1634 short sequence read archives at NCBI containing more than 34 billion spots yielded numerous individual spots that correspond to the RYamide encoding exons, of which a large number include the intron-exon boundary at the start of this exon. Although 72 different sequences have been spliced onto this RYamide encoding exon, none codes for the signal peptide of this gene. Thus, the RNAseq data for this gene reveal only noise and no signal. The very small quantities of peptide recovered during isolation and the absence of credible RNAseq data, indicates that the gene is very little expressed, while the RYamide gene structure in D. melanogaster suggests that it might be evolving into a pseudogene. Yet, the identification of the peptides it encodes clearly shows it is still functional. Using region specific antisera, we could localize numerous neurons and enteroendocrine cells in D. willistoni, D. virilis and D. pseudoobscura, but only two adult abdominal neurons in D. melanogaster. Those two neurons project to and innervate the rectal papillae, suggesting that RYamides may be involved in the regulation of water homeostasis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Four genomes and two transcriptomes from six Chelicerate species were analyzed for the presence of neuropeptide and neurohormone precursors and their GPCRs. The genome from the spider Stegodyphus mimosarum yielded 87 neuropeptide precursors and 101 neuropeptide GPCRs. High neuropeptide transcripts were also found in the trancriptomes of three other spiders, Latrodectus hesperus, Parasteatoda tepidariorum and Acanthoscurria geniculata. For the scorpion Mesobuthus martensii the numbers are 79 and 74 respectively. The very small genome of the house dust mite, Dermatophagoides farinae, on the other hand contains a much smaller number of such genes. A few new putative Arthropod neuropeptide genes were discovered. Thus, both spiders and the scorpion have an achatin gene and in spiders there are two different genes encoding myosuppressin-like peptides while spiders also have two genes encoding novel LGamides. Another finding is the presence of trissin in spiders and scorpions, while neuropeptide genes that seem to be orthologs of Lottia LFRYamide and Platynereis CCRFamide were also found. Such genes were also found in various insect species, but seem to be lacking from the Holometabola. The Chelicerate neuropeptide and neuropeptide GPCR genes often have paralogs. As the large majority of these are probably not due to local gene duplications, is not impossible that they reflect the effects of one or more ancient whole genome duplications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF., Competing Interests: The authors have declared that no competing interests exist. CW is an AE of PLOS ONE.

Background. Neuropeptides are important regulators of physiological processes and behavior. Although they tend to be generally well conserved, recent results using trancriptome sequencing on decapod crustaceans give the impression of significant differences between species, raising the question whether such differences are real or artefacts. Methods. The BLAST+ program was used to find short reads coding neuropeptides and neurohormons in publicly available short read archives. Such reads were then used to find similar reads in the same archives, and the DNA assembly program Trinity was employed to construct contigs encoding the neuropeptide precursors as completely as possible. Results. The seven decapod species analyzed in this fashion, the crabs Eriocheir sinensis, Carcinus maenas and Scylla paramamosain, the shrimp Litopenaeus vannamei, the lobster Homarus americanus, the fresh water prawn Macrobrachium rosenbergii and the crayfish Procambarus clarkii had remarkably similar neuropeptidomes. Although some neuropeptide precursors could not be assembled, in many cases individual reads pertaining to the missing precursors show unambiguously that these neuropeptides are present in these species. In other cases, the tissues that express those neuropeptides were not used in the construction of the cDNA libraries. One novel neuropeptide was identified: elongated PDH (pigment dispersing hormone), a variation on PDH that has a two-amino-acid insertion in its core sequence. Hyrg is another peptide that is ubiquitously present in decapods and is likely a novel neuropeptide precursor. Discussion. Many insect species have lost one or more neuropeptide genes, but apart from elongated PDH and hyrg all other decapod neuropeptides are present in at least some insect species, and allatotropin is the only insect neuropeptide missing from decapods. This strong similarity between insect and decapod neuropeptidomes makes it possible to predict the receptors for decapod neuropeptides that have been deorphanized in insects. This includes the androgenic insulin-like peptide that seems to be homologous to drosophila insulin-like peptide 8.

The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0-10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles.

Allatostatin C is the arthropod homolog of vertebrate somatostatin. The gene went through a local gene triplification leading to the existence of three genes coding such peptides, allatostatins C, CC and CCC. All three genes are still present in several chelicerates, such as the horseshoe crab Limulus polyphemus, several spiders and the scorpion Mesobuthus martensii, the myriapod Strigamia maritima, as well as at least two insect species, Locusta migratoria and Athalia rosae, a sawfly. All three peptides have well conserved primary structures and peptides can easily be classified as either allatostatin C, CC or CCC. In most insect species only two of the genes have been preserved. In many species, these are CC and CCC, but in Diptera, Lepidoptera and Coleoptera it are allatostatins C and CC that are still present. In some arthropod species two or even all three genes can still be found closely associated in the genome and are present on the same scaffold showing that a local amplification was at the origin of these genes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Drosophila gene fruitless expresses male and female specific transcription factors which are responsible for the generation of male specific neuronal circuitry for courtship behavior. Mutations in this gene may lead to bisexual behavior in males. Bisexual behavior in males also occurs in the absence of the neuropeptide SIFamide. We show here that the SIFamide neurons do not express fruitless. However, when fruitless neurons are made to express RNAi specific for the SIFamide receptor, male flies engage in bisexual behavior, showing that SIFamide acts on fruitless neurons. If neurons expressing a SIFaR-gal4 transgene are killed by the apoptotic protein reaper or when these neurons express SIFamide receptor RNAi, males also show male-male courtship behavior. We next used this transgene to localize neurons that express the SIFamide receptor. Such neurons are ubiquitously present in the central nervous and we also found two neurons in the uterus that project into the central nervous system., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Transcriptomes of the crayfish Procambarus clarkii were analyzed for the presence of transcripts encoding neurohormones, neuropeptides and their receptors. A total of 58 different transcripts were found to encode such ligands and another 82 for their receptors. A very large number of the neuropeptide transcripts appeared to be complete and for those that were not only small parts seemed to be lacking. Transcripts for the neuropeptide GPCRs as well as for the putative receptors for insulin, neuroparsin and eclosion hormone were often also complete or almost so. Of particular interest is the presence of three different neuroparsin genes and two putative neuroparsin receptors. There are also three pigment dispersing hormones as well three likely receptors for these neuropeptides. CNMamide, calcitonin, CCRFamide, natalisin, trissin and relaxin appear to be new crustacean neuropeptides. The recently identified crustacean female sex hormone was also found and in the crayfish appears to be not only expressed in the eyestalk, but in the ovary as well (though not in the testis). Interestingly, there are two other proteins in the crayfish with a structure similar to crustacean female sex hormone, that could be precursors of neurohormones, but these are not expressed by the ovary. The ovary also appears to contain significant numbers of transcripts encoding pigment dispersing hormones, CNMamide as well as glycoprotein B5, but not glycoprotein A2., (Copyright © 2015 Elsevier Inc. All rights reserved.)

How animals allocate energy and metabolic decisions are coordinated is a fundamental physiological question. Metabolic research is strongly driven by an increasing obesity rate in humans. For insects-which contain many pest species and disease vectors-the control of feeding is of agroeconomical and medical importance. Regulatory peptides have since long been in focus of metabolic research. In insects, major advances have been made recently, mostly due to research in the genetically tractable Drosophila melanogaster with focus on the central nervous system as a source of neuropeptides. Research on peptides produced by enteroendocrine cells remained peripheral, but this situation is about to change. This review highlights current knowledge and advances on the identity and role of enteroendocrine insect peptides., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1-5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The genomes of the migratory locust Locusta migratoria and the termite Zootermopsis nevadensis were mined for the presence of genes encoding neuropeptides, neurohormones, and their G-protein coupled receptors (GPCRs). Both species have retained a larger number of neuropeptide and neuropeptide GPCRs than the better known holometabolous insect species, while other genes that in holometabolous species appear to have a single transcript produce two different precursors in the locust, the termite or both. Thus, the recently discovered CNMa neuropeptide gene has two transcripts predicted to produce two structurally different CNMa peptides in the termite, while the locust produces two different myosuppressin peptides in the same fashion. Both these species also have a calcitonin gene, which is different from the gene encoding the calcitonin-like insect diuretic hormone. This gene produces two types of calcitonins, calcitonins A and B. It is also present in Lepidoptera and Coleoptera and some Diptera, but absent from mosquitoes and Drosophila. However, in holometabolous insect species, only the B transcript is produced. Their putative receptors were also identified. In contrast, Locusta has a highly unusual gene that codes for a salivation stimulatory peptide. The Locusta genes for neuroparsin and vasopressin are particularly interesting. The neuroparsin gene produces five different transcripts, of which only one codes for the neurohormone identified from the corpora cardiaca. The other four transcripts code for neuroparsin-like proteins, which lack four amino acid residues, and that for that reason we called neoneuroparsins. The number of transcripts for the neoneuroparsins is about 200 times larger than the number of neuroparsin transcripts. The first exon and the putative promoter of the vasopressin genes, of which there are about seven copies in the genome, is very well-conserved, but the remainder of these genes is not. The relevance of these findings is discussed.

The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. We demonstrate that tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the fruitfly Drosophila melanogaster and its larvae. Although the antisera to CCHamide 1 and 2 are mutually cross-reactive, polyclonal mouse antisera raised to the C-terminals of their respective precursors allowed the identification of the two different peptides. In both larva and adult, CCHamide 2 immunoreactive endocrine cells are large and abundant in the anterior midgut and are also present in the anterior part of the posterior midgut. The CCHamide 2 immunoreactive endocrine cells in the posterior midgut are also immunoreactive with antiserum to allatostatin C. CCHamide 1 immunoreactivity is localized in endocrine cells in different regions of the midgut; those in the caudal part of the posterior midgut are identical with the allatostatin A cells. In the larva, CCHamide 1 enteroendocrine cells are also present in the endocrine junction and in the anterior part of the posterior midgut. Like in other insect species, the Drosophila orcokinin gene produces two different transcripts, A and B. Antiserum to the predicted biologically active peptide from the B-transcript recognizes enteroendocrine cells in both larva and adult. These are the same cells as those expressing β-galactosidase in transgenic flies in which the promoter of the orcokinin gene drives expression of this enzyme. In the larva, a variable number of orcokinin-expressing enteroendocrine cells are found at the end of the middle midgut, while in the adult, those cells are most abundant in the middle midgut, while smaller numbers are present in the anterior midgut. In both larva and adult, these cells also express allatostatin C. We also made a specific polyclonal antiserum to the NPF precursor in order to determine more precisely the expression of this peptide in the midgut. Using this antiserum, we find expression in the midgut to be the same as described previously using transgenic flies, while in the adult, midgut expression appears to be concentrated in the middle midgut, thus suggesting that in the anterior midgut only minor quantities of NPF are produced.

We have examined the distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus. These neuropeptides, adipokinetic hormone (RhoprAKH), corazonin (CRZ) and adipokinetic hormone/corazonin-related peptide (RhoprACP) are present in distinct, non-overlapping neuronal subsets in the central nervous system (CNS), as determined by immunohistochemistry. Corazonin-like immunoreactive cell bodies are present in the brain and ventral nerve cord, whereas ACP-like immunoreactive cell bodies are only present in the brain, and AKH-like immunoreactive cell bodies only present in the corpus cardiacum (CC). The immunoreactivity to ACP, CRZ and AKH in R. prolixus suggests that ACP and CRZ are released within the CNS, and that CRZ and AKH are released as neurohormones from the CC. Injection of RhoprAKH into adult males elevated haemolymph lipid levels, but injection of CRZ or RhoprACP failed to have any effect on haemolymph lipid levels. Corazonin stimulated an increase in heart-beat frequency in vitro, but RhoprAKH and RhoprACP failed to do so. Thus, although all three neuropeptides share sequence similarity, the AKH and CRZ receptors only respond to their own ligand., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We have examined the distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus. These neuropeptides, adipokinetic hormone (RhoprAKH), corazonin (CRZ) and adipokinetic hormone/corazonin-related peptide (RhoprACP) are present in distinct, non-overlapping neuronal subsets in the central nervous system (CNS), as determined by immunohistochemistry. Corazonin-like immunoreactive cell bodies are present in the brain and ventral nerve cord, whereas ACP-like immunoreactive cell bodies are only present in the brain, and AKH-like immunoreactive cell bodies only present in the corpus cardiacum (CC). The immunoreactivity to ACP, CRZ and AKH in R. prolixus suggests that ACP and CRZ are released within the CNS, and that CRZ and AKH are released as neurohormones from the CC. Injection of RhoprAKH into adult males elevated haemolymph lipid levels, but injection of CRZ or RhoprACP failed to have any effect on haemolymph lipid levels. Corazonin stimulated an increase in heart-beat frequency in vitro, but RhoprAKH and RhoprACP failed to do so. Thus, although all three neuropeptides share sequence similarity, the AKH and CRZ receptors only respond to their own ligand., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Drosophila ATP7 copper transporter has sequence homology to the human copper transporters ATP7A and ATP7B, which are defective in Menkes and Wilson disease, respectively. We show here that in Drosophila ATP7 is expressed by many peptidergic neurons. As C-terminal amidation of neuropeptides depends on the copper-containing enzyme PHM, it seemed likely that in the absence of ATP7 the activity of PHM might be compromised. Indeed, inhibition of ATP7 expression by RNAi led to a decrease in mature amidated neuropeptides and the appearance of C-terminally Gly-extended neuropeptides. The strength of this effect differed from one cell type to another; it was very pronounced for AKH and corazonin, but much less so for SIFamide and myosuppressin. Nevertheless, down-regulation of ATP7 specifically in the SIFamide-expressing neurons resulted in male-male courtship behavior., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In the honey bee no allatotropin gene has been found, even though allatotropin stimulates the synthesis of juvenile hormone in this species. We report here that honey bees and other Hymenoptera do have a typical allatotropin gene, although the peptides predicted have a somewhat different structure from that of other insect allatotropins. Polyclonal antisera to honey bee allatotropin reacted with material in the neurohemal organs of the segmental nerves of abdominal ganglia. We were unable to find the allatotropin peptide using mass spectrometry in extracts from these tissues. Thus the expression of this gene in honey bees is less important than in other insect species. We also characterized the leucokinin gene which similarly appears to be very weakly expressed in worker honey bees. Unlike the allatotropin gene, which is conserved within Hymenoptera, the leucokinin gene is much more variable in structure and was not found in ants nor the parasitic wasp Nasonia vitripennis. The absence of significant expression of adipokinetic hormone (AKH) in the honey bee may be due to the existence of a second TATA box in the promotor region of the gene, which explains the production of an mRNA encoding a putative peptide precursor from which no AKH should be released. Such a second TATA box was not found in other Hymenoptera, and may therefore be specific for the two Apis species. It is suggested that functional disintegration of this important metabolic gene became possible in Apis because of the highly evolved social nature of the species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies., (©2011 Macmillan Publishers Limited. All rights reserved)

Genes encoding neurohormones and neuropeptide precursors were identified in the genomes of two annelids, the leech Helobdella robusta and the polychaete worm Capitella teleta. Although no neuropeptides have been identified from these two species and relatively few neuropeptides from annelids in general, 43 and 35 such genes were found in Capitella and Helobdella, respectively. The predicted peptidomes of these two species are similar to one another and also similar to those of mollusks, particular in the case of Capitella. Helobdella seems to have less neuropeptide genes than Capitella and it lacks the glycoprotein hormones bursicon and GPA2/GPB5; in both cases the genes coding the two subunits as well as the genes coding their receptors are absent from its genome. In Helobdella several neuropeptide genes are duplicated, thus it has five NPY genes, including one pseudogene, as well as four genes coding Wwamides (allatostatin B). Genes coding achatin, allatotropin, allatostatin C, conopressin, FFamide, FLamide, FMRFamide, GGRFamide, GnRH, myomodulin, NPY, pedal peptides, RGWamide (a likely APGWamide homolog), RXDLamide, VR(F/I)amide, WWamide were found in both species, while genes coding cerebrin, elevenin, GGNG, LFRWamide, LRFYamide, luqin, lymnokinin and tachykinin were only found in Capitella., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Thyrostimulin is a dimer hormone formed from glycoprotein A2 (GPA2) and glycoprotein B5 (GPB5) that activates the TSH receptor in vertebrates. A Drosophila GPA2/GPB5 homolog has recently been characterized. Cells producing this novel hormone were localized by in situ hybridization using both the GPA2 and GPB5 DNA sequences and by making transgenic flies in which the GPB5 promoter drives the expression of gal4. Endocrine cells producing GPA2/GPB5 were found in the abdominal neuromeres and are different from the endocrine cells producing crustacean cardioactive peptide or those making leucokinin. They are also not immunoreactive to antisera to the CRF- or calcitonin-like diuretic hormones. Their axons leave the central nervous system through the segmental nerves and project to the periphery were they likely release GPA2/GPB5 into the hemolymph. As has been described for the leucokinin endocrine cells their axons run over the surface of the abdominal musculature, however, the projection patterns of the leucokinin and GPA2/GPB5 neuroendocrine cells are not identical. The chances of adult eclosion of insects from which the GPA2/GPB5 cells have been genetically ablated or have been made to express GPB5-RNAi are severely compromised, demonstrating the physiological importance of the cells producing this hormone. As the receptor for GPA2/GPB5 stimulates the production of cyclic AMP (cAMP) and is highly expressed in the hindgut, where cAMP stimulates water reabsorption in locusts, it is suggested that GPA2/GPB5 may be an insect anti-diuretic hormone., (Copyright © 2010 Elsevier Inc. All rights reserved.)