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The primary olfactory centres of metazoans as diverse as arthropods and mammals consist of an array of fields of dense synaptic neuropil, the olfactory glomeruli. However, the neurochemical structure of crustacean olfactory glomeruli is largely understudied when compared to the insects. We analysed the glomerular architecture in selected species of hermit crabs using immunohistochemistry against presynaptic proteins, the neuropeptides orcokinin, RFamide and allatostatin, and the biogenic amine serotonin. Our study reveals an unexpected level of structural complexity, unmatched by what is found in the insect olfactory glomeruli. Peptidergic and aminergic interneurons provide the structural basis for a regionalization of the crustacean glomeruli into longitudinal and concentric compartments. Our data suggest that local olfactory interneurons take a central computational role in modulating the information transfer from olfactory sensory neurons to projection neurons within the glomeruli. Furthermore, we found yet unknown neuronal elements mediating lateral inhibitory interactions across the glomerular array that may play a central role in modulating the transfer of sensory input to the output neurons through presynaptic inhibition. Our study is another step in understanding the function of crustacean olfactory glomeruli as highly complex units of local olfactory processing. This article is protected by copyright. All rights reserved.

The impact of numerous diseases has been linked to differences in sex between organisms, including various neurological diseases. As neuropeptides are known to be key players in the nervous system, studying the variation of neuropeptidomic profiles between males and females in a crustacean model organism is of interest. By using high-resolution mass spectrometry with two complementary ionization sources in conjunction with quantitative chemical labeling (isotopic reductive dimethylation), differences were observed in five key neural tissues and hemolymph. Interestingly, while males and females possess numerous neuropeptide isoforms that are unique to their sex, the represented families of each sex remain largely consistent. However, some differences in familial isoforms were also observed, such as the relative numbers of neuropeptides belonging to RFamide and allatostatin A-type families. Additionally, >100 neuropeptides detected across five neural tissues and hemolymph were found to have statistically significant differences in abundance between male and female blue crab samples. Also, hundreds of putative peptide sequences were identified by de novo sequencing that may be indicative of previously undiscovered neuropeptides, highlighting the power of using a multifaceted MS approach.

Insect neuropeptide receptors, including allatostatin receptor type C (AstR-C), a G protein-coupled receptor, are among the potential targets for designing next-generation pesticides that despite their importance in offering a new mode-of-action have been overlooked. Focusing on AstR-C of Thaumetopoea pityocampa, a common pest in Mediterranean countries, by employing resonance energy transfer-based methods, we showed Gαi/o coupling and β-arrestin recruitment of the receptor at sub-nanomolar and nanomolar ranges of the endogenous ligand, AST-C, respectively. Molecular docking and molecular dynamics simulation studies revealed the importance of extracellular loop 2 in AstRC/AST-C interaction, and a combination of in silico and in vitro approaches showed the substantial role of Q2716.55 in G protein-dependent activation of AstR-C possibly via contributing to the flexibility of the receptor's structure. The functional and structural insights obtained on T. pit AstR-C positively assist future efforts in developing environmentally friendly pest control agents that are needed urgently.

G protein-coupled receptors (GPCRs) play a pivotal role in regulating key physiological events in all animal species. Recent advances in collective analysis of genes and proteins revealed numerous potential neuro-peptides and GPCRs from insect species, allowing for the characterization of peptide-receptor pairs. In this work, we used fluorescence resonance energy transfer (FRET)-based genetically encoded biosensors in intact mammalian cells to study the pharmacological features of the cognate GPCR of the type-C allatostatin (AST-C) peptide from the stick insect, Carausius morosus. Analysis of multiple downstream pathways revealed that AST-C can activate the human Gi(2) protein, and not Gs or Gq, through AST-C receptor (AIstRC). Activated AlstRC recruits beta-arrestin2 independent of the Gi protein but stimulates ERK phosphorylation in a Gi protein-dependent manner. Identification of G alpha i-, arrestin-, and GRK-like transcripts from C. morosus revealed high evolutionary conservation at the G protein level, while beta-arrestins and GRKs displayed less conservation. In conclusion, our study provides experimental and homology-based evidence on the functionality of vertebrate G proteins and downstream signaling biosensors to characterize early signaling steps of an insect GPCR. These results may serve as a scaffold for developing assays to characterize pharmacological and structural aspects of other insect GPCRs and can be used in deorphanization and pesticide studies.

Oxygen (O2) is a critical component of life; without proper O2 levels, cells are unable to respire, meaning glucose cannot be utilized. Thus, hypoxia (low O2 levels) is a well-documented stressor, especially in aquatic environments. Neuropeptides are a major class of regulators for stress-induced responses; however, their global expression changes during stress are not well characterized due to the natural complexity of the nervous system. Beyond being a neurological model organism, crustaceans are regularly exposed to hypoxia, making them a relevant system for this study. Several neuropeptide families, including orcokinins, RFamides, and allatostatin A-types, show dynamic dysregulation due to hypoxic stress. In particular, the brain showed the most dynamic changes with a survival mechanism "switching" (i.e., significant increase to decrease) of neuropeptide content between moderate and severe hypoxia (e.g., NFDEDRSGFA, FDAFTTGFGHS, NRNFLRFamide, and APSGFLGMRamide). Globally, neuropeptides in different tissues appeared to exhibit unique expression patterns at the various severities of hypoxia, including LSSSNSPSSTPL and NFDEIDRSSFGF. Overall, this study provides clear evidence for the benefits of globally analyzing biomolecules and that neuropeptides play a critical role in how crustaceans adapt due to hypoxic stress.

In Bombyx mori, two dorsolateral neurosecretory cells (NSCs) in each of the two brain lobes have been identified as prothoracicotropic hormone (PTTH) producing cells. This neuropeptide in insects stimulates the prothoracic gland for the synthesis and release of ecdysone, responsible for the molting events. Allatotropin (AT) and allatostatin (AST) are allatoregulatory neuropeptides that regulate juvenile hormone biosynthesis. Here, by using RT-qPCR, we showed that in B. mori, nutritional stress modulates the mRNA expression of AT and AST-C (allatostain type C) in the central nervous system consisting of the brain lobes and all the associated ganglia. Using whole-mount in situ hybridization, we showed that the feeding status of Bombyx larvae also influences the expression of PTTH in the NSCs of the brain. Food deprivation significantly decreased the mRNA expression levels of PTTH in larvae at active or terminal growth period. Further, we showed that insulin modulates the expression level of PTTH. However, its action was dependent on the feeding status of the larvae. At feeding, the insulin decreased the PTTH expression level, while at food deprivation, the insulin increased the PTTH expression level. The data thus indicates that larval feeding status plays an important role in altering the mRNA expression levels of allatoregulatory peptide genes and PTTH.

The intestine is a central regulator of metabolic homeostasis. Dietary inputs are absorbed through the gut, which senses their nutritional value and relays hormonal information to other organs to coordinate systemic energy balance. However, the specific gut hormones that communicate energy availability to target organs to induce appropriate metabolic and behavioral responses are poorly defined. Here we show that the enteroendocrine cells (EECs) of the Drosophila gut sense nutrient stress via the intracellular TOR pathway, and in response secrete the peptide hormone allatostatin C (AstC), a Drosophila Somatostatin homolog. Gut-derived AstC induces secretion of glucagon-like adipokinetic hormone (AKH) via its receptor AstC-R2, a homolog of mammalian somatostatin receptors, to coordinate food intake and energy mobilization. Loss of gut AstC or its receptor in the AKH-producing cells impairs lipid and sugar mobilization during fasting, leading to hypoglycemia. Our findings illustrate a nutrient-responsive endocrine mechanism that maintains energy homeostasis under nutrient-stress conditions, a function that is essential to health and whose failure can lead to metabolic disorders.

An increasingly amount of evidence supports that the evolution of eusociality is accompanies by shifts in ancient molecular and physiological pathways. The juvenile hormone, one of the most important hormones in the post-embryonic development of insects, attracts the most attention in the context of social organization. Allatoregulatory neuropeptides (Allatotropin, Allatostatin-A and Allatostatin-C) are known to regulate juvenile hormone synthesis and release in insects. In order to clarify the transitions of juvenile hormone synthesis involved in eusocial evolution, the substitutions of amino acid residues and the complexity of post-translational modifications in allatoregulatory neuropeptide receptors were characterized. Both allatotropin and allatostatin receptors are identified in all examined bee species regardless if they are solitary or eusocial. Although the amino acid sequences are highly conserved, phylogenetic results are consistent with the eusocial status. The abundance of predicted post-translational modifications correlates with social complexity except for that in allatostatin-C receptors. Even though the consequences of these specific amino acid substitutions and various post-translational modification complexity have not been studied, they likely contribute to the localizing, binding and coupling characteristics of the receptor groups.

C-type allatostatins (C-type ASTs) are a family of structurally related neuropeptides found in a wide range of insects and crustaceans. To date, the C-type allatostatin receptor in crustaceans has not been deorphaned, and little is known about its physiological functions. In this study, we aimed to functionally define a C-type ASTs receptor in the mud crab, Scylla paramamosian. We showed that C-type ASTs receptor can be activated by ScypaAST-C peptide in a dose-independent manner and by ScypaAST-CCC peptide in a dose-dependent manner with an IC50 value of 6.683 nM. Subsequently, in vivo and in vitro experiments were performed to investigate the potential roles of ScypaAST-C and ScypaAST-CCC peptides in the regulation of ecdysone (20E) and methyl farnesoate (MF) biosynthesis. The results indicated that ScypaAST-C inhibited biosynthesis of 20E in the Y-organ, whereas ScypaAST-CCC had no effect on the production of 20E. In addition, qRT-PCR showed that both ScypaAST-C and ScypaAST-CCC significantly decreased the level of expression of the MF biosynthetic enzyme gene in the mandibular organ, suggesting that the two neuropeptides have a negative effect on the MF biosynthesis in mandibular organs. In conclusion, this study provided new insight into the physiological roles of AST-C in inhibiting ecdysone biosynthesis. Furthermore, it was revealed that AST-C family peptides might inhibit MF biosynthesis in crustaceans.

Allatostatins (AST) are neuropeptides originally described as inhibitors of juvenile hormone (JH) synthesis in insects. Consequently, they have been considered as potential lead compounds for the discovery of new insect growth regulators (IGRs). In the present work, receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) was studied with 48 AST analogs, and a general approach for novel potent bioactive AST analogs is proposed. Hence, six novel AST analogs were designed and synthesized. Bioassays indicated that the majority novel analogs exhibited potent JH inhibitory activity, especially analog A6 (IC50: 3.79 nmol/L), which can be used as lead compound to develop new IGRs.

The southern rock lobster, Jasus edwardsii, is a member of the Achelata native to New Zealand and southern Australia that in addition to being the subject of an extensive wild fishery, has been proposed as a target for aquaculture. One obstacle to the farming of this species is its long maturation period, and the development of large-scale commercial aquaculture will require management strategies that reduce the duration of its time in culture. Increasing food intake can result in increased growth rates, and may be one way to reduce the time to market for farmed lobsters. In crustaceans, food intake and growth are regulated by locally released and/or circulating paracrines/hormones, the largest class being peptides. In the study presented here, the native peptides of J. edwardsii, as well as their cognate receptors, were predicted using a publicly accessible transcriptome. Two hundred and seventy distinct peptides were identified (the largest peptidome currently extant for any decapod), 122 of which possess structures that place them into generally recognized arthropod peptide families, i.e., adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, diuretic hormone 44, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone, GSEFLamide, inotocin, insulin-like peptide, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin. Putative receptors for many of the identified peptide groups were also identified via transcriptome mining. This is the first report of a peptidome and peptide receptors for J. edwardsii, and as such, provides a powerful new resource for beginning to investigate the physiological/behavioral roles played by peptidergic signaling systems in this species, including the roles they play in controlling processes that are of importance for improving aquaculture management strategies.

Summary Insect metamorphosis and mammalian puberty exhibit similar design principles, but the conservation of molecular components has not been documented. A new study demonstrates that the insect neuropeptide allatostatin A and its receptor AstAR1 activate a neuroendocrine signaling cascade that initiates metamorphosis, similar to the way in which their mammalian orthologs Kisspeptin and KISS1R induce puberty.

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the "signal transduction mechanisms" category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

The male accessory glands (AG) and gonoducts of moths develop during metamorphosis and are essential for successful fertilization of females. We found that these reproductive organs are innervated by a sex-specific cluster of peptidergic neurons in the posterior 9th neuromere of the terminal abdominal ganglion (TAG). This cluster of ~20 neurons differentiate during metamorphosis to innervate the accessory glands and sperm ducts. Using immunohistochemistry and in situ hybridization (ISH) we showed that these neurons express four neuropeptide precursors encoding calcitonin-like diuretic hormone (CT-DH), allatotropin (AT) and AT-like peptides (ATLI-III), allatostatin C (AST-C), and myoinhibitory peptides (MIPs). We used contraction bioassay in vitro to determine roles of these neuropeptides in the gonoduct and accessory gland activity. Spontaneous contractions of the seminal vesicle and AG were stimulated in a dose depended manner by CT-DH and AT, whereas AST-C and MIP elicited dose dependent inhibition. Using quantitative RT-PCR we confirmed expression of receptors for these neuropeptides in organs innervated by the male specific cluster of neurons. Our results suggest a role of these neuropeptides in regulation of seminal fluid movements during copulation.

Thirst is a motivational state that drives behaviors to obtain water for fluid homeostasis. We identified two types of central brain interneurons that regulate thirsty water seeking in Drosophila, that we term the Janu neurons. Janu-GABA, a local interneuron in the subesophageal zone, is activated by water deprivation and is specific to thirsty seeking. Janu-AstA projects from the subesophageal zone to the superior medial protocerebrum, a higher order processing area. Janu-AstA signals with the neuropeptide Allatostatin A to promote water seeking and to inhibit feeding behavior. NPF (Drosophila NPY) neurons are postsynaptic to Janu-AstA for water seeking and feeding through the AstA-R2 galanin-like receptor. NPF neurons use NPF to regulate thirst and hunger behaviors. Flies choose Janu neuron activation, suggesting that thirsty seeking up a humidity gradient is rewarding. These findings identify novel central brain circuit elements that coordinate internal state drives to selectively control motivated seeking behavior.

Copper is a necessary nutrient but quickly becomes toxic at elevated levels. To properly handle environmental copper influxes and maintain metal homeostasis, organisms utilize various methods to chelate, excrete, and metabolize heavy metals. These mechanisms are believed to involve complex signaling pathways mediated by neuropeptides. This study incorporates custom N,N-dimethyl leucine isobaric tags to characterize the neuropeptidomic changes after different time points (1, 2, and 4 hours) of copper exposure in a model organism, blue crab, Callinectes sapidus. Using a modified simplex optimization strategy, the number of identifiable and quantifiable neuropeptides was increased three-fold to facilitate a deeper understanding of the signaling pathways involved in responding to heavy metal exposure. The time course exposure showed many interesting findings, including upregulation of inhibitory allatostatin peptides in the pericardial organs. Additionally, there was evidence of transport of a pigment dispersing hormone from the sinus glands to the brain. Overall, this study improves the multiplexing capabilities of neuropeptidomic studies to understand the temporal changes associated with copper toxicity.

Significance Metazoan species optimize the timing of reproduction to maximize fitness. To understand how biological clocks direct reproduction, we investigated the neural substrates that produce oogenesis rhythms in the genetically amenable model organism Drosophila melanogaster. The neuropeptide allatostatin C (AstC) is an insect counterpart of the vertebrate neuropeptide somatostatin, which suppresses gonadotropin production. A subset of the brain circadian pacemaker neurons produces AstC. We have uncovered that these clock-associated AstC neurons generate the circadian oogenesis rhythm via brain insulin-producing cells and the insect gonadotropin juvenile hormone. Identification of a conserved neuropeptide pathway that links female reproduction and the biological clock offers insight into the molecular mechanisms that direct reproductive timing., The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated “posterior dorsal neuron 1” (DN1p), which are among the ∼150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). Here, we report that six pairs of AstC-DN1p send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. We show evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms.

sNPF is a small, powerful bioactive peptide with pleiotropic functions. The range of its role extends from feeding and reproductive behavior to the conditioning of olfactory sensing. Interestingly, sNPF has the opposite function of NPF and does not affect feeding term and food preference, although the C-terminal region of sNPF is similar to that of NPF. Besides, sNPF signaling is involved in the regulation of energy homeostasis, such as food search behavior, starvation resistance, and immune response. Although this peptide belongs to the RFamides family, the presence of sNPF must be abundant in the CNS as well as allatostatin, possibly functioning as a neurotransmitter in insects.

Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.

The intestine is a central regulator of metabolic homeostasis. Dietary inputs are absorbed through the gut, which senses their nutritional value and relays hormonal information to other organs to coordinate systemic energy balance. However, the specific gut hormones that communicate energy availability to target organs to induce appropriate metabolic and behavioral responses are poorly defined. Here we show that the enteroendocrine cells (EECs) of theDrosophilagut sense nutrient stress via the intracellular TOR pathway, and in response secrete the peptide hormone allatostatin C (AstC). Gut-derived AstC induces secretion of glucagon-like adipokinetic hormone (AKH) via its receptor AstC-R2, a homolog of mammalian somatostatin receptors, to coordinate food intake and energy mobilization. Loss of gutAstCor its receptor in the AKH-producing cells impairs lipid and sugar mobilization during fasting, leading to hypoglycemia. Our findings illustrate a nutrient-responsive endocrine mechanism that maintains energy homeostasis under nutrient-stress conditions, a function that is essential to health and whose failure can lead to metabolic disorders.

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).

Insect neuropeptide receptors are among the potential targets for designing next-generation pesticides. Activation of allatostatin receptor type C (AstR-C), a G Protein-coupled receptor (GPCR), upon stimulation with its endogenous ligand, allatostatin C (AST-C), leads to the inhibition of juvenile hormone (JH) secretion that consequently regulates physiology of insects. Here we conducted in silico and in vitro approaches to characterize the structure and function of AstR-C of Thaumetopoea pityocampa (T.pit), a well-known pest in Mediterranean countries. The sequence of AstR-C and AST-C were derived from whole genome sequencing (WGS) data. Resonance energy transfer (RET) methods were used to investigate the downstream effectors of the receptor and the temporal kinetics of G protein activation. Three-dimensional (3D) structure of AstR-C constructed via homology modeling methods was subjected to molecular dynamics (MD) simulations and docking studies to identify the orthosteric pocket. Our results showed that T.pit AstR-C couples to Gαi/o subtype of G proteins at sub-nanomolar ranges of the the ligand with the G protein recruitment and activation kinetics of ∼4 and 6 seconds, respectively, when 1 nM AST-C is administered. At the increasing concentration of native ligand, βarrestin was shown to be recruited at nanomolar ranges the ligand. Docking and MD simulation studies revealed the importance of extracellular loop 2 (ECL2) in T.pit AstRC/AST-C interaction, and combination of in silico and in vitro methods supported the accuracy of the built model and the predicted orthosteric pocket. Q2716.55 (Ballesteros-Weinstein generic numbering) was found to have a substantial role in G protein dependent activation of AstR-C possibly via contributing to the flexibility of the structure.

B-type allatostatin (AST-B) is a pleiotropic neuropeptide, widely found in arthropods. However, the information about its immune effect in crustaceans is unknown. In this study, we identified the nervous tissue as the main site for Sp-AST-B expression, while its receptor gene (Sp-AST-BR) is widely expressed in various tissues, including the hepatopancreas. This suggests the peptide's potential role in diverse physiological processes in the mud crab Scylla paramamosain. In situ hybridization revealed that Sp-AST-BR is mainly localized in the F-cell of hepatopancreas. Furthermore, we found a significant up-regulation of Sp-AST-BR transcripts in the hepatopancreas following exposure to lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (Poly (I:C)). Results from in vitro and in vivo experiments revealed that treatment with a synthetic AST-B peptide mediated significant upregulation in expression of AST-BR, nuclear factor-κB (NF-κB) pathway components (Dorsal and Relish), pro-inflammatory cytokine (IL-16) and antimicrobial peptides (AMPs) in the hepatopancreas. In addition, AST-B treatment mediated significant elevation of nitric oxide (NO) production and enhanced the bacteriostasis capacity of the hepatopancreas tissue in vitro. Taken together, these findings reveal the existence of a basic neuroendocrine-immune (NEI) network in crabs, and indicate that AST-B could couple with its receptor to trigger downstream signaling pathways and induce immune responses in the hepatopancreas.

The sesquiterpenoid juvenile hormone(s) (JHs) of insects are the primary regulators of growth, metamorphosis, and reproduction in most insect species. As a consequence, it is essential that JH production be precisely regulated so that it is present only during appropriate periods necessary for the control of these processes. The presence of JH at inappropriate times results in disruption to metamorphosis and development and, in some cases, to disturbances in female reproduction. Neuropeptides regulate the timing and production of JH by the corpora allata. Allatostatin and allatotropin were the names coined for neuropeptides that serve as inhibitors or stimulators of JH biosynthesis, respectively. Three different allatostatin neuropeptide families are capable of inhibiting juvenile hormone but only one family is utilized for that purpose dependent on the insect studied. The function of allatotropin also varies in different insects. These neuropeptides are pleiotropic in function acting on diverse physiological processes in different insects such as muscle contraction, sleep and neuromodulation. Genome projects and expression studies have assigned individual neuropeptide families to their respective receptors. An understanding of the localization of these receptors is providing clues as to how numerous peptide families might be integrated in regulating physiological functions. In recent years microRNAs have been identified that down-regulate enzymes and transcription factors that are involved in the biosynthesis and action of juvenile hormone.

Members of the decapod infraorder Achelata, specifically species from the genus Panulirus, have storied histories as models for investigating the basic principles governing the generation, maintenance, and modulation of rhythmic motor behavior, including modulation by locally released and circulating peptides. Despite their contributions to our understanding of peptidergic neuromodulation, little is known about the identity of the native neuropeptides and neuronal peptide receptors present in these crustaceans. Here, a Panulirus argus nervous system-specific transcriptome was used to help fill this void, providing insight into the neuropeptidome and neuronal peptide receptome of this species. A neuropeptidome consisting of 266 distinct peptides was predicted using the P. argus assembly, 128 having structures placing them into a generally recognized arthropod peptide family: agatoxin-like peptide, allatostatin A (AST-A), allatostatin B, allatostatin C, bursicon, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31 (DH31), ecdysis-triggering hormone (ETH), FMRFamide-like peptide (FLP), glycoprotein hormone (GPH), GSEFLamide, inotocin, leucokinin, myosuppressin, natalisin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, periviscerokinin, pigment-dispersing hormone, pyrokinin, red pigment-concentrating hormone, RYamide, short neuropeptide F (sNPF), SIFamide, sulfakinin, tachykinin-related peptide (TRP), and trissin. Twenty-five putative neuronal receptors, encompassing 15 peptide groups, were also identified from the P. argus transcriptome: AST-A, bursicon, CCHamide, DH31, diuretic hormone 44, ETH, FLP, GPH, inotocin, insulin-like peptide, myosuppressin, natalisin, periviscerokinin, sNPF, and TRP. Collectively, the reported data provide a powerful resource for expanding studies of neuropeptidergic control of physiology and behavior in members of the genus Panulirus specifically, and decapods generally.

Insects utilize an open circulatory system to transport nutrients, waste, hormones and immune factors throughout the hemocoel. The primary organ that drives hemolymph circulation is the dorsal vessel, which is a muscular tube that traverses the length of the body and is divided into an aorta in the head and thorax, and a heart in the abdomen. The dorsal vessel is myogenic, but its rhythmicity is modulated by neuropeptides and neurotransmitters. This review summarizes how neuropeptides such as crustacean cardioactive peptide (CCAP), FMRFamide-like peptides, proctolin, allatotropin and allatostatin modulate the heart contraction rate and the directionality of heart contractions. Likewise, it discusses how neurotransmitters such as serotonin, octopamine, glutamate and nitric oxide influence the heart rate, and how transcriptomic and proteomic approaches are advancing our understanding of insect circulatory physiology. Finally, this review argues that the immune system may modulate heart rhythmicity, and discusses how the myotropic activity of cardioactive factors extends to the accessory pulsatile organs, such as the auxiliary hearts of the antennae.

Allatostatins (ASTs), a family of insect neuropeptide, can inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA) in Diploptera punctata, and therefore be regarded as potential leads for the discovery of new insect growth regulators (IGRs). But several shortcomings, such as their sensitivity to peptidases and high cost, impeded their practical application in pest management. In order to discover new IGRs, one AST analog B1 possessing non-peptide group was discovered with high ability to inhibit JH biosynthesis in vitro (IC50: 0.09 μmol/L) in our previous studies. In the present work, two series of B1 analogs with different substituents on the N-terminus region were designed and synthesized. The result suggested that benzene showed better activity than other heterocycles, and the para-substitution on the benzene was beneficial for activity. Moreover, analogs with logP value over 2.0 exhibited good activity, which indicated the hydrophobicity is important to the bioactivity. Three dimension quantitative structure-activity relationship (3D-QSAR) studies were performed to highlight the structural requirements of AST analogs, which demonstrated introduction of bulkier substituents on the N-terminus would increase the activity. Analog II12 (IC50: 0.08 μmol/L) exhibited similar inhibitory activity to the lead B1, but its synthetic route was simpler than B1. Therefore, II12 could be used as a new lead compound for the discovery eco-friendly IGRs.

Peptides are the largest/most diverse class of molecules used by animals for chemical communication, and with their cognate receptors, are key players in modulating physiological/behavioral control systems, including those involved in adaptation to environmental change. Crustaceans have long served as models for investigating peptidergic control of physiology/behavior, and members of Notostraca, an ancient branchiopod order, have recently been proposed as models for investigating the genetic/physiological underpinnings of ecoresponsiveness; nothing is currently known about the genes/proteins underlying peptidergic signaling in any member of this crustacean taxon. Transcriptome mining is a powerful tool for peptidome prediction in crustaceans, and all large-scale discovery of crustacean peptide receptors has been achieved via transcriptomics. Here, in silico transcriptome mining was used to elucidate the peptidergic signaling systems of the tadpole shrimp Triops newberryi, a member of Notostraca. Transcripts encoding putative precursor proteins and/or receptors for 28 peptide families were identified within the T. newberryi dataset. The deduced precursor proteins included those for allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone, diuretic hormone 44, ecdysis-triggering hormone, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone, GSEFLamide, inotocin, insulin-like peptide, neuroparsin, neuropeptide F, orcokinin/orcomyotropin, proctolin, pyrokinin/periviscerokinin, SIFamide, sulfakinin and tachykinin-related peptide; 117 distinct mature peptides were predicted from the collective set of deduced pre/preprohormones. Transcripts encoding putative receptors for most of the abovementioned peptide groups were also identified from the T. newberryi assembly, as were those for several families for which no precursors were found, i.e., corazonin, RYamide and short neuropeptide F. This is the first description of a peptidome and peptide receptors from any member of the Notostraca, and as such, provide a foundation for beginning to investigate the roles played by peptidergic signaling systems in T. newberryi and other notostracans, including how they may contribute to modulating organism-environment interactions.

Due to its sensitivity to many environmental and anthropogenic stressors, including a wide range of chemical compounds, Hyalella azteca, a freshwater amphipod, has emerged as one of the most commonly used invertebrates for ecotoxicological assessment.Peptidergic signaling systems are key components in the control of organism-environment interactions, and there is a growing literature suggesting that they are targets of a number of aquatic toxicants.Interestingly, and despite its model species status in the field of ecotoxicology, little is known about the peptide hormones of H. azteca.Here, a transcriptome was produced for this species using the de novo assembler Trinity and mined for sequences encoding putative peptide precursors; the transcriptome was assembled from 460,291,636 raw reads and consists of 133,486 unique transcripts.Seventy-six sequences encoding peptide pre/preprohormones were identified from this transcriptome, allowing for the prediction of 202 distinct peptides, which included members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, ecdysis-triggering hormone, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone, GSEFLamide, inotocin, leucokinin, myosuppressin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families.These peptides expand the known peptidome for H. azteca approximately nine-fold, forming a strong foundation for future studies of peptidergic control, including disruption by aquatic toxicants, in this important ecotoxicological model.