Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

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.

Evolution of a Cockroach Allergen into the Major Protein of Termite Royal Jelly.

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.

Differential expression of some termite neuropeptides and insulin/IGF-related hormones and their plausible functions in growth, reproduction and caste determination.

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.

Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila.

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.

Different neuroendocrine cell types in the pars intercerebralis of Periplaneta americana produce their own specific IGF-related peptides.

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.

Neuropeptides in Rhipicephalus microplus and other hard ticks.

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.

Identification of cells expressing Calcitonins A and B, PDF and ACP in Locusta migratoria using cross-reacting antisera and in situ hybridization.

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.

Progress in the characterization of insulin-like peptides in aphids: Immunohistochemical mapping of ILP4.

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.

Ambulacrarian insulin-related peptides and their putative receptors suggest how insulin and similar peptides may have evolved from insulin-like growth factor.

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.

Identification of Gonadulin and Insulin-Like Growth Factor From Migratory Locusts and Their Importance in Reproduction in Locusta migratoria.

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.

The neuropeptide SMYamide, a SIFamide paralog, is expressed by salivary gland innervating neurons in the American cockroach and likely functions as a hormone.

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.

Genomics- and Peptidomics-Based Discovery of Conserved and Novel Neuropeptides in the American Cockroach.

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).

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Genome-enabled insights into the biology of thrips as crop pests.

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.

Arthropod IGF, relaxin and gonadulin, putative orthologs of Drosophila insulin-like peptides 6, 7 and 8, likely originated from an ancient gene triplication.

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.

Coupling Neuropeptide Levels to Structural Plasticity in Drosophila Clock Neurons.

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.

Gonadulins, the fourth type of insulin-related peptides in decapods.

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.

Regulatory Roles of Drosophila Insulin-Like Peptide 1 (DILP1) in Metabolism Differ in Pupal and Adult Stages.

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.

A new neuropeptide insect parathyroid hormone iPTH in the red flour beetle Tribolium castaneum.

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.