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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).)

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.)

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.)

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.

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.)

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.)

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.

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.

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.)

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.)

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)

The genomes of the mollusk Lottia gigantea, the leech Helobdella robusta and the polychaete worm Capitella teleta each have a gene encoding an ecdysone receptor homolog. Publicly available genomic and EST sequences also contain evidence for ecdysone receptors in the seahare Aplysia californica, the bobtail squid Euprymna scolopes and the medicinal leech Hirudo medicinalis. Three-dimensional models of the ligand binding domains of these predicted ecdysone receptor homologs suggest that each of them could potentially bind an ecdysone-related steroid. Thus, ecdysone receptors are not limited to arthropods and nematodes., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

The bHLH transcription factor DIMMED has been associated with the differentiation of peptidergic cells in Drosophila. However, whether all Drosophila peptidergic cells express DIMM, and the extent to which all DIMM cells are peptidergic, have not been determined. To address these issues, we have mapped DIMM expression in the central nervous system (CNS) and periphery in the late larval stage Drosophila. At 100 hr after egg-laying, DIMM immunosignals are largely congruent with a dimm-promoter reporter (c929-GAL4) and they present a stereotyped pattern of 306 CNS cells and 52 peripheral cells. We assigned positional values for all DIMM CNS cells with respect to reference gene expression patterns, or to patterns of secondary neuroblast lineages. We could assign provisional peptide identities to 68% of DIMM-expressing CNS cells (207/306) and to 73% of DIMM-expressing peripheral cells (38/52) using a panel of 24 markers for Drosophila neuropeptide genes. Furthermore, we found that DIMM co-expression was a prevalent feature within single neuropeptide marker expression patterns. Of the 24 CNS neuropeptide gene patterns we studied, six patterns are >90% DIMM-positive, while 16 of 22 patterns are >40% DIMM-positive. Thus most or all DIMM cells in Drosophila appear to be peptidergic, and many but not all peptidergic cells express DIMM. The co-incidence of DIMM-expression among peptidergic cells is best explained by a hypothesis that DIMM promotes a specific neurosecretory phenotype we term LEAP. LEAP denotes Large cells that display Episodic release of Amidated Peptides.

Adipokinetic hormone (AKH) is a metabolic neuropeptide principally known for its mobilization of energy substrates, notably lipid and trehalose during energy-requiring activities, such as flight and locomotion. Drosophila melanogaster AKH cell localization in corpora cardiaca, as in other insect species, was confirmed by immunoreactivity and by a genetic approach using the UAS/GAL4 system. To assess AKH general physiological rules, we ablated AKH endocrine cells by specifically driving the expression of apoptosis transgenes in AKH cells. Trehalose levels were decreased in larvae and starved adults, when the stimulation by AKH of the production of trehalose from fat body glycogen is no longer possible. Moreover, we show that these adults without AKH cells become progressively hypoactive. Finally, under starvation conditions, those hypoactive AKH-knockout cell flies survived approximately 50% longer than control wild-type flies, suggesting that the slower rate at which AKH-ablated flies mobilize their energy resources extends their survival.

Previous studies have demonstrated that the synthesis of juvenile hormone (JH) by the isolated corpora allata (CA) complex in vitro as well as the JH titer in the yellow fever mosquito Aedes aegypti are elevated before feeding and low after a blood meal. In the present study, we used an in vitro radiochemical assay to analyze the effect of farnesoic acid (FA) and Aedes allatotropin (Aedes-AT) on the biosynthesis of JH and methyl farnesoate (MF) by the isolated CA complex of A. aegypti adult female. CA complex from day-0 females (0-1 h after emergence) exhibited a low basal juvenile hormone III (JH III) biosynthetic activity and did not respond to either allatotropic or FA stimulation. However, incubation of CA complexes from newly emerged females with Aedes-AT plus FA resulted in very high production of JH III. This is the first report suggesting that allatotropin makes corpora allata in newly emerged females capable for JH biosynthesis. When we studied CA complexes dissected from females 1 day after emergence, the stimulatory action of Aedes-AT was strong and dose-dependent, with maximum stimulation in the range of 10(-8)-10(-9) mol l(-1), suggesting that Aedes-AT is indeed a true allatotropin (a molecule with allatotropic activity) in A. aegypti. The addition to the culture medium of 40 micro mol l(-1) FA, a JH precursor, resulted in a 9-fold increase in JH III biosynthesis in 2-, 4- and 6-day-old sugar-fed females. The two major labeled products synthesized by the stimulated CA complex were identified as JH III and MF by RP-HPLC and GC-MS. Treatment of CA complexes with FA, but not Aedes-AT, resulted in an increase in MF. Application of both Aedes-AT and FA to the CA complexes of 2-, 4- and 6-day-old females resulted in the same effects as FA alone. These data suggest that in sugar-fed females, FA and Aedes-AT exert different effects on the terminal steps in JH biosynthesis.

Dh, the gene that encodes a CRF-like peptide in Drosophila melanogaster, is described. The product of this gene is a 44-amino-acid peptide (Drome-DH(44)) with a sequence almost identical to the Musca domestica and Stomoxys calcitrans diuretic hormones. There are no other similar peptides encoded within the known Drosophila genomic sequence. Functional studies showed that the deduced peptide stimulated fluid production, and that this effect was mediated by cyclic AMP in principal cells only: there was no effect on the levels of either cyclic GMP or intracellular calcium. Stimulation also elevated levels of cyclic AMP (but not cyclic GMP) phosphodiesterase, a new mode of action for this class of hormone. The transcript was localised by in situ hybridisation, and the peptide by immunocytochemistry, to two groups of three neurones in the pars intercerebralis within the brain. These cells also express receptors for leucokinin, another major diuretic peptide, implying that the cells may be important in homeostatic regulation.

A Drosophila gene (capability, capa) at 99D on chromosome 3R potentially encodes three neuropeptides: GANMGLYAFPRV-amide (capa-1), ASGLVAFPRV-amide (capa-2), and TGPSASSGLWGPRL-amide (capa-3). Capa-1 and capa-2 are related to the lepidopteran hormone cardioacceleratory peptide 2b, while capa-3 is a novel member of the pheromone biosynthesis-activating neuropeptide/diapause hormone/pyrokinin family. By immunocytochemistry, we identified four pairs of neuroendocrine cells likely to release the capa peptides into the hemolymph: one pair in the subesophageal ganglion and the other three in the abdominal neuromeres. In the Malpighian (renal) tubule, capa-1 and capa-2 increase fluid secretion rates, stimulate nitric oxide production, and elevate intracellular Ca(2+) and cGMP in principal cells. Capa-stimulated fluid secretion, but not intracellular Ca(2+) concentration rise, is inhibited by the guanylate cyclase inhibitor methylene blue. The actions of capa-1 and capa-2 are not synergistic, implying that both act on the same pathways in tubules. The capa gene is thus the first to be shown to encode neuropeptides that act on renal fluid production through nitric oxide.

The leucokinin (LK) family of neuropeptides has been found widely amongst invertebrates. A member of this family was purified from adults of the fruit fly Drosophila melanogaster. The peptide sequence for Drosophila leucokinin (DLK) was determined as Asn-Ser-Val-Val-Leu-Gly-Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly-amide, making it the longest member of the family characterized to date. Synthetic DLK peptide was shown to act to stimulate fluid secretion in D. melanogaster Malpighian (renal) tubules by approximately threefold, with an EC(50) of approximately 10(-)(10 )mol l(-)(1), and a secondary effect at approximately 10(-)(7 )mol l(-)(1). DLK also acted to elevate intracellular [Ca(2+)] in the Malpighian tubules by approximately threefold, with an EC(50) of 10(-)(10) to 10(-)(9 )mol l(-)(1). Responses were detected in stellate cells and occasionally in principal cells, although at no concentration tested did [Ca(2+)] in the principal cell increase significantly above background. In stellate cells, DLK produced a biphasic rise in intracellular [Ca(2+)] from resting levels of 80-100 nmol l(-)(1), with a transient peak being followed by a slower rise that peaked at 200-300 nmol l(-)(1) after 3 s, then decayed over approximately 10 s. The wide range of concentrations over which DLK acts suggests the involvement of more than one receptor. The genomic sequence encoding the DLK peptide has been identified, and the gene has been named pp. The gene resides at cytological location 70E3-70F4 of chromosome 3L. The localisation of this first Drosophila LK gene in a genetic model permits a genetic analysis of the locus.

A cDNA encoding preproleucokinin was isolated from a cDNA library of the mosquito Aedes aegypti. The deduced amino acid sequence of Aedes preproleucokinin contains a putative signal peptide of 18 amino acid residues and a 210-amino acid residue proleucokinin. Within the proleucokinin are encoded one copy each of the Aedes leucokinins 1, 2, and 3 isolated previously from this species (Veenstra, J. A. (1994) Biochem. Biophys. Res. Commun. 202, 715-719). All three Aedes leucokinins depolarize the transepithelial voltage of the malpighian tubule in concentrations of less than 10(-9) M and increase the frequency of hindgut contractions at concentrations above 10(-8) M. At higher concentrations the Aedes leucokinins 1 and 3 but not Aedes leucokinin 2 are also able to increase the rate of fluid secretion by the malpighian tubules. The differences of the three Aedes leucokinins in their potencies to induce fluid secretion or depolarizations in the malpighian tubules suggest that there may be more than one type of leucokinin receptor in this tissue.

Corazonin, a new cardioaccelerating peptide, has been isolated from the corpora cardiaca of the American cockroach, Periplaneta americana, and its structure determined to be Glp-Thr-Phe-Gln-Tyr-Ser-Arg-Gly-Trp-Thr-Asn-amide. The peptide stimulated heart beat at concentrations as low as 0.2 nM, which makes it the most potent insect cardioactive neuropeptide.