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Litopenaeus vannamei, with an annual production of 5–6 million tons and a value of USD 50–60 billion, is a cornerstone of global aquaculture. However, molting-related losses of 5–20% significantly impact this industry, and the physiological mechanisms of molting remain unclear. This study aims to elucidate the role of eclosion hormone (EH) in molting regulation and enhances the understanding of molting physiology in L. vannamei. This study investigated the role of (EH) in L. vannamei molting regulation. Two EH cDNAs, LvEH I and LvEH II, were identified, and their expression patterns across tissues and seven molting stages (A, B, C, D0, D1, D2, and D3) were analyzed. LvEH I was predominantly expressed in the gill, epidermis, and eyestalk, while LvEH II was mainly expressed in the eyestalk and brain. LvEH I was highly expressed in the eyestalk, epidermis, and gills at the D2 and D3 stages of molting, whereas LvEH II was highly expressed in both the D2 (brain) and D3 (eyestalk) stages. RNA interference (RNAi) targeting LvEH I revealed its critical role in molting, as silencing LvEH I disrupted the expression of molting-regulation genes, ETH, CCAP, CHH, EH II, CDA, and bursicon (Burs), significantly delaying the molting process. These findings highlight both LvEH I and LvEH II as indispensable for normal molting in L. vannamei and provide a foundation for developing effective molting management strategies to reduce industry losses. [ABSTRACT FROM AUTHOR]

The process of molting represents a critical phase in the life cycle of arthropods, marking periods of growth and development. Central to this process is the eclosion hormone (EH), a neurohormone that plays a pivotal role in initiating and regulating the complex sequence of events leading to successful molting in holometabolan species. Very little information is available in Hemimetabola, which display a different kind of development characterized by gradual changes. This paper reports on the identification of the two EH precursors and the EH receptor (EHR), a guanylyl cyclase, in a hemimetabolan pest species, the desert locust, Schistocerca gregaria. Using qRT-PCR, an in-depth profiling study of Schgr-EH-1, -2 and Schgr-EHR transcripts was performed. Silencing of Schgr-EH-1, -2 and Schgr-EHR resulted in lethality at the expected time of ecdysis, thereby showing their crucial role during this process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Eclosion hormone (EH) is not only a key trigger of insect ecdysis, but is also involved in the regulation of important physiological processes such as development, diapause, metamorphosis, and reproduction. EH is an ideal target for RNAi treatment and prevention of the Tribolium castaneum. However, two EH genes in T. castaneum demonstrate distinct replication and functional conversion relationships, and the mechanisms of transcriptional regulation of EH remain largely unexplored and poorly understood. In this study, the activity of highly active promoter fragments and potential transcription factors of TcEH and TcEHL were identified using the Dual-Luciferase reporter system and TANSFAC. TcSlbo and TcCAD were revealed to be important transcription factors for TcEH and TcEHL, respectively. Knockdown of TcSlbo failed to slough off the old epidermis of T. castaneum and prevented them from developing into adults. Furthermore, we demonstrated for the first time that 20-hydroxyecdysone affects the expression of TcEH and TcEHL by regulating the transcriptional activities of TcSlbo and TcCAD. This study provides new insights into the transcription regulation of TcEH and TcEHL, their roles in insect growth and development, and the involvement of 20-hydroxyecdysone in eclosion regulation, offering potential molecular targets for future pest management strategies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Molting behavior in insects is controlled by the ecdysis triggering hormone (ETH), eclosion hormone (EH) and the crustacean cardioactive peptide (CCAP). At present, the regulation of molting behavior in crustaceans remains unclear. Here, we studied the roles of ETH, EH, and CCAP in the molt regulation of the crab, Scylla paramamosain from their expression pattern and in vivo assays. The results showed that transcripts of ETH, EH, and CCAP were mainly localized in thoracic ganglia and fluctuated periodically with the molting cycle. When ETH or CCAP was knockdown at early premolt stage (D0), molting of crabs was interrupted and all animals died at late premolt stage (D2). While the EH gene was knock-down, most crabs were dead before D2. Injection of synthetic peptide for ETH or CCAP rescued ETH- or CCAP-gene knock-down crabs separately. However, none of peptides could rescue dsEH-injected crabs. At D0 stage, knockdown of ETH down-regulated the transcriptions of EH and CCAP; while ETH was up-regulated when EH was knockdown. At D2 stage, ETH transcripts levels were reduced with the injection of dsEH but increased with the same dose of dsETH as crabs at D0 stage. Co-injection of dsETH and dsEH down-regulated ETH at D2 stage. Results showed that ETH, EH, and CCAP play essential but different roles in molt regulation in mud crab. In summary, the result of this study contributes to the discovery of different molecular mechanisms between Insecta and Crustacea and may provide insight to develop fishery drugs that helps aquacultured crustaceans to molt successfully.

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline.

[Display omitted] • 20E triggers the expression of Ldeh. • Knockdown of Ldeh at the fourth instar stage impairs pupation. • Silencing Ldeh impacts trachea clearance and wing expansion. In Drosophila melanogaster , ecdysis-triggering hormone (ETH) and eclosion hormone (EH) mutually enhance the release of each other through a positive feedback loop. Both are involved in the stimulation of ecdysis behavior series and molting-related physiological preparations. However, whether 20-hydroxyecdysone (20E) controls eh expression and whether EH plays a similar role in non-Drosophilid insects remain to be determined. In Leptinotarsa decemlineata , we found that Ldeh mRNA levels were high in the early and/or late stages and low in the intermediate period within each larval instar, indicating that the mRNA peaks positively correlate with 20E pulses. Consistent with the indication, we confirmed that 20E signaling stimulated the transcription of Ldeh. RNA interference (RNAi)-aided knockdown of Ldeh at the final instar stage slightly impaired pupation and significantly affected wing expansion. Around 20% of the Ldeh RNAi larvae remained as prepupae, completely wrapped in the old larval cuticles. These prepupae gradually darkened, dried and withered, and finally died. The remaining (about 80%) Ldeh hypomorphs became pupae and emerged as abnormal adults, bearing smaller and wrinkle elytra and hindwings. The adult tracheae in the Ldeh hypomorphs were full of liquid. In addition, the low pupation rate and high deformed adult proportion cannot be rescued by ingestion of either 20E or juvenile hormone. Thus, EH is critical for ecdysis and trachea clearance in L. decemlineata. The failure of trachea clearance may disenable air-swallowing during larva-pupa-adult transition and thus impacts wing growth and expansion. [ABSTRACT FROM AUTHOR]

The Chinese oak silkworm is commonly used in pupal diapause research. In this study, a long photoperiod was used to trigger pupal diapause termination. Genes encoding three hormones, namely prothoracicotropic hormone (PTTH), ecdysis triggering hormone (ETH), and eclosion hormone (EH), were studied. Additionally, ecdysteroids (mainly 20-hydroxyecdysone, 20E) were quantified by HPLC. Pupal diapause stage was determined by measuring respiratory intensity. The pupae enter a low metabolic rate, which starts approximately 1 month after pupal emergence. ApPTTH expression showed a small increase at 14 days and then a larger increase from 35 days under the long photoperiod treatment. A similar pattern was observed for the titer of 20E in the hemolymph. However, ApETH expression later increased under the long photoperiod treatment (42 days) just before eclosion. Moreover, ApEH expression increased from 21 to 35 days, and then decreased before ecdysis. These results suggest that hormone-related gene expression is closely related to pupal development. Our study lays a foundation for future diapause studies in A. pernyi.

Ecdysis is a vital process for insects, during which they shed the old cuticle in order to emerge as the following developmental stage. Given its relevance for survival and reproduction, ecdysis is tightly regulated by peptidic hormones that conform an interrelated neuromodulatory network. This network was studied in species that undergo a complete metamorphosis, but not in hemimetabola. In a recent work, we demonstrated that orcokinin neuropeptides are essential for ecdysis to occur in the kissing bug Rhodnius prolixus. Here we performed gene silencing, quantitative PCR and in vitro treatments in order to study the interrelationships between RhoprOKs and hormones such as ecdysis triggering hormone, corazonin, eclosion hormone, crustacean cardioactive peptide and ecdysone. Our results suggest that RhoprOKs directly or indirectly regulate the expression of other genes. Whereas RhoprOKA is centrally involved in the regulation of gene expression, RhoprOKB is implicated in processes related to midgut physiology. Therefore, we propose that the different transcripts encoded in RhoprOK gene could integrate signaling cues, in order to coordinate the nutritional state with development and ecdysis. Given the emerging data that point to OKs as important factors for survival and reproduction, they could be candidates in the search for new insect management strategies based on neuroendocrine targets. [ABSTRACT FROM AUTHOR]

Abstract: The peptidergic Pigment‐dispersing factor (PDF)‐Tri neurons are a group of non‐clock neurons that appear transiently around the time of adult ecdysis (=eclosion) in the fruit fly Drosophila melanogaster. This specific developmental pattern points to a function of these neurons in eclosion or other processes that are active around pupal–adult transition. As a first step to understand the role of these neurons, we here characterize the anatomy of the PDF‐Tri neurons. In addition, we describe a further set of peptidergic neurons that have been associated with eclosion behavior, eclosion hormone (EH), and crustacean cardioactive peptide (CCAP) neurons, to single cell level in the pharate adult brain. PDF‐Tri neurons as well as CCAP neurons co‐express a classical transmitter indicated by the occurrence of small clear vesicles in addition to dense‐core vesicles containing the peptides. In the tritocerebrum, gnathal ganglion and the superior protocerebrum PDF‐Tri neurites contain peptidergic varicosities and both pre‐ and postsynaptic sites, suggesting that the PDF‐Tri neurons represent modulatory rather than pure interneurons that connect the subesophageal zone with the superior protocerebrum. The extensive overlap of PDF‐Tri arborizations with neurites of CCAP‐ and EH‐expressing neurons in distinct brain regions provides anatomical evidence for a possible function of the PDF‐Tri neurons in eclosion behavior. [ABSTRACT FROM AUTHOR]

[Display omitted] • The EH , ETH and CCAP gene was cloned and characterized from Sogatella furcifera. • The SfEH , SfETH are hightly expressed in 3-day-old 5th instar nymph while the SfCCAP in 40 min adult. • The SfCCAP gene would down-regulated expressed in response to sublethal concentration of methylaminoavermectin benzoate. Insect neuropeptides are involved in many important developmental processes. Here, we cloned three neuropeptide genes (SfEH , SfETH , and SfCCAP) from Sogatella furcifera (Horváth) and analyzed the relative expression levels of these genes through Real-time Quantitative polymerase chain reaction (RT-qPCR) during different developmental stages and in different wing types of S. furcifera following exposure to sublethal concentrations (LC 10 and LC 25) of methylaminoavermectin benzoate. The results revealed that the three neuropeptide genes (SfEH , SfETH , and SfCCAP) of S. furcifera contained open reading frame sequences of 222, 636, and 408 bp, encoding 73, 211, and 135 amino acids, respectively. Moreover, the expression of SfEH and SfETH reached its peak on day 3 of the 5th instar nymph stage, while the SfCCAP expressed pinnacle on 40 min after eclosion. Furthermore, the relative expression levels of the three genes were in the following order: SfEH , short-winged female > long-winged female > long-winged male; SfETH , long-winged female > short-winged female > long-winged male; and SfCCAP , short-winged female > long-winged male > short-winged female. Upon calculating the relative expression of the three peptide genes after 24 and 48 h of treatment with sublethal concentrations of methylaminoavermectin benzoate, it was found that the relative mRNA expression levels of SfEH and SfETH declined upon LC 10 treatment but increased upon LC 25 treatment. Meanwhile, the relative mRNA expression level of SfCCAP declined upon both LC 10 and LC 25 treatments. This result will help us understand the effects of pesticides on insect growth and development at the molecular level. [ABSTRACT FROM AUTHOR]

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales.

Molting behavior is an important physiological process related to metamorphosis, growth, and reproduction in crustaceans. Previous studies indicated that the molting process was controlled by 20-hydroxyecdysone (20E) and upstream hormones, peptides, and environmental factors, which regulate 20E function. Eclosion hormone (EH) in insect is a kind of neuropeptide that is regulated by 20E and triggers ecdysis behavior at the end of molting process. However, the function of eclosion hormone gene during the molting process in crustaceans is still largely unknown. In the present study, an eclosion hormone-like gene EcEHL was identified from Exopalaemon carinicauda. The deduced amino acid sequence of EcEHL contained a signal peptide, a typical eclosion domain, and six conserved cysteine residues forming three putative disulfide bonds. EcEHL was predominantly expressed in the epidermis, gill, and eyestalk of shrimp. In situ hybridization analysis showed that EcEHL transcripts were localized in gill cells and in medulla externa X-organ, medulla terminalis X-organ, sinus gland, and lamina ganglionaris of eyestalks. During the molting process of shrimp, EcEHL showed the highest expression level in shrimp at the premolt stage. The expression level of EcEHL in shrimp at mid premolt stage was up-regulated by injection of exogenous 20E. Silencing of EcEHL using double-stranded RNA delayed both the molting process and ecdysis rate of E. carinicauda. Furthermore, injection of exogenous 20E to shrimp at mid premolt stage (D2) could remarkably speed up the molting process and also raise the ecdysis rate of E. carinicauda. The results revealed that EcEHL might participate in the molting process of shrimp and its expression was regulated by 20E. These data will help us to understand the molecular mechanism of molting in crustacean. [ABSTRACT FROM AUTHOR]

Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori ( B. mori), and expressed in Escherichia coli ( E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion. [ABSTRACT FROM AUTHOR]

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects. [ABSTRACT FROM AUTHOR]

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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

Abstract: ESTs have been generated for many crustacean species, providing an invaluable resource for peptide discovery in members of this arthropod subphylum. Here, these data were mined for novel peptide-encoding transcripts, with the mature peptides encoded by them predicted using a combination of online peptide prediction programs and homology to known arthropod sequences. In total, 70 mature full-length/partial peptides representing members of 16 families/subfamilies were predicted, the vast majority being novel; the species from which the peptides were identified included members of the Branchiopoda (Daphnia carinata and Triops cancriformis), Maxillopoda (Caligus clemensi, Caligus rogercresseyi, Lepeophtheirus salmonis and Lernaeocera branchialis) and Malacostraca (Euphausia superba, Marsupenaeus japonicus, Penaeus monodon, Homarus americanus, Petrolisthes cinctipes, Callinectes sapidus and Portunus trituberculatus). Of particular note were the identifications of an intermediate between the insect adipokinetic hormones and crustacean red pigment concentrating hormone and a modified crustacean cardioactive peptide from the daphnid D. carinata; Arg7-corazonin was also deduced from this species, the first identification of a corazonin from a non-decapod crustacean. Our data also include the first reports of members of the calcitonin-like diuretic hormone, FMRFamide-related peptide (neuropeptide F subfamily) and orcokinin families from members of the Copepoda. Moreover, the prediction of a bursicon α from the euphausid E. superba represents the first peptide identified from any member of the basal eucaridean order Euphausiacea. In addition, large collections of insect eclosion hormone- and neuroparsin-like peptides were identified from a variety of species, greatly expanding the number of known members of these families in crustaceans. [Copyright &y& Elsevier]

Abstract: Ticks are important vectors of numerous pathogens that impact human and animal health. The tick central nervous system represents an understudied area in tick biology and no tick synganglion-specific transcriptome has been described to date. Here we characterize whole or partial cDNA sequences of fourteen putative neuropeptides (allatostatin, insulin-like peptide, ion-transport peptide, sulfakinin, bursicon alpha/beta, eclosion hormone, glycoprotein hormone alpha/beta, corazonin, four orcokinins) and five neuropeptide receptors (gonadotropin receptor, leucokinin-like receptor, sulfakinin receptor, calcitonin receptor, pyrokinin receptor) translated from cDNA synthesized from the synganglion of unfed, partially fed and replete female American dog ticks, Dermacentor variabilis. Their homology to the same neuropeptides in other taxa is discussed. Many of these neuropeptides such as an allatostatin, insulin-like peptide, eclosion hormone, bursicon alpha and beta and glycoprotein hormone alpha and beta have not been previously described in the Chelicerata. An insulin-receptor substrate protein was also found indicating that an insulin signaling network is present in ticks. A putative type-2 proprotein processing convertase was also sequenced that may be involved in cleavage at monobasic and dibasic endoproteolytic cleavage sites in prohormones. The possible physiological role of the proteins discovered in adult tick blood feeding and reproduction will be discussed. [Copyright &y& Elsevier]

Using rapid amplification of cDNA ends (RACE), the cDNA encoding eclosion hormone (EH) was cloned from the brain of Ostrinia furnacalis. The full Osf-EH cDNA is 986 bp and contains a 267 bp open reading frame encoding an 88 amino acid preprohormone, which including a hydrophobic 26 amino acid signal peptide and a 62 amino acid mature peptide. The mature Osf-EH shows high identity with Manduca sexta (95.2%), Helicoverpa armigera (91.9%) and Bombyx mori (85.5%), but low identify with Tribolium castaneum (63.6%), Drosophila melanogaster (56.5%) and Apis mellifera (54.8%). Using the HMMSTR Prediction Server, the 3D structure of Osf-EH was modeled. There are four β-turns and three α-helixes predicted in Osf-EH, with the pattern of β-β-α-α-β-β-α. Northern blot analysis indicated a 1.0 kb transcript present only in the brain. The Osf-EH mRNA can not be detected in other neural tissues, such as the suboesophageal ganglion, thoracic ganglion, abdominal ganglion and other non-neural tissues, such as the midgut, fat body and epidermis. The Osf-EH mRNA content in the brain was measured using the combined method of quantitative RT-PCR and Southern blotting, which reached its highest level the day before the molt. [ABSTRACT FROM AUTHOR]

Abstract: A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42°C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal–adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages. [Copyright &y& Elsevier]

Abstract: The cDNA encoding eclosion hormone (EH), which plays an integral role in triggering ecdysis behavior at the end of each molt, was cloned from the cotton bollworm, Helicoverpa armigera (Har) (Lepidoptera: Noctuidae). The EH polyprotein precursor contains a 26-amino acid signal peptide and a single 62-amino acid mature EH. Compared the mature Har-EH with other known EHs, it shows 94%, 84%, and 59% identities to Manduca sexta, Bombyx mori, and Drosophila melanogaster, respectively. Har-EH mRNA is expressed only in the brain by Northern blot and RT-PCR, but not in other tissues. By in situ hybridization and immunocytochemistry, both Har-EH mRNA and protein are localized in two pairs of neurosecretory cells of the brain. Prior to a molt, expression level of Har-EH gene reaches the highest point, and then drops after molt. EH release is detected both centrally, within the ganglia, and peripherally, into the hemolymph. A peak of the EH titer in hemolymph measured by ELISA presents at ecdysis. These results are consistent with the biological function of Har-EH associated with ecdysis. Furthermore, Har-EH gene is expressed throughout all of the developmental stages examined, implicating that the EH gene may possess other biological functions in post-embryonic development other than triggering ecdysis behavior. [Copyright &y& Elsevier]

This review deals with those neuropeptidic hormones that are completely known in their primary structure together with the control processes that are linked directly or indirectly to development and reproduction. Actions are assessed for neuropeptides that regulate ecdysteroid and Juvenile Hormone production; oocyte growth or yolk deposition; and ecdysis and courtship behaviour. The combined data on the primary sequence, gene expression, biosynthesis, release, interaction with the receptor and mode of action provide an accurate account of the current knowledge on how these peptides function. [ABSTRACT FROM AUTHOR]

To complete each molting cycle, insects display a stereotyped sequence of behaviors to shed the remains of the old cuticle. These behavioral routines, as well as other related physiological events, are critical for proper development and are under the control of several neuropeptides. Their correct deployment and concatenation depends on the complex actions and interactions among several peptide hormones: ecdysis triggering hormone (ETH), eclosion hormone (EH), and crustacean cardioactive peptide (CCAP). Numerous theories, some in conflict, have been proposed to define the functional hierarchies by which these regulatory factors operate. Here we use wild-type Drosophila and transgenic flies bearing targeted ablations of either EH or CCAP neurons, or ablations of both together, to reevaluate their roles. Consistent with findings in moths, our results suggest that EH and ETH affect the release of each other via a positive feedback, although ETH can also be released in the absence of EH. We show that EH and ETH both contribute to the air firing of the air ducts (trachea) of the next stage but that EH may play a primary role in this process. We present evidence that EH, whose actions have always been placed upstream of CCAP, may also regulate ecdysis independently of CCAP. Finally, we confirm that flies lacking EH neurons do not ecdyse prematurely when injected with ETH peptides. These findings are surprising and not easily explained by currently available hypotheses. We propose that important additional neuropeptides, and additional interactions between known regulators, contribute to the mechanisms underlying insect ecdysis behaviors. [ABSTRACT FROM AUTHOR]

The cephalic nervous system of the firebrat contains antigens recognized by antisera to the clock protein period (PER), the prothoracicotropic hormone (PTTH) and the eclosion hormone (EH). The content of the 115 kDa PER-like antigen visualized on the western blots fluctuates in diurnal rhythm with a maximum in the night. The oscillations entrained in a 12:12 h light/dark (LD) cycle persist in the darkness and disappear in continuous light. They are detected by immunostaining in 14 pairs of the protocerebral neurons and are extreme in four suboesophageal neurons and two cells in each corpus cardiacum that contain PER only during the night phase. No circadian fluctuations occur in three lightly stained perikarya of the optic lobe. Five cell bodies located in each brain hemisphere between the deuto-and the tritocerebrum retain weak immunoreactivity under constant illumination. In all cells, the staining is confined to the cytoplasm and never occurs in the cell nuclei. The cells containing PER-like material do not react with the anti-PTTH and anti-EH antisera, which recognize antigens of about 50 and 20 kDa, respectively. The anti-PTTH antiserum stains in each brain hemisphere seven neurons in the protocerebrum, eight in the optic lobe, and 3–5 in the posterior region of the deutocerebrum. The antiserum to EH reacts in each hemisphere with just two cells located medially to the mushroom bodies. No cycling of the PTTH-like and EH-like antigens was detected. [Copyright &y& Elsevier]

The extremely large number of insects and members of allied groups alive today suggests that molting—shedding of an old cuticle—may be one of the most commonly performed behaviors on our planet. Removal of an old cuticle in insects is associated with stereotyped, species-specific patterns of behavior referred to as ecdysis. It has been recognized for decades that the initiation of ecdysis is under hormonal control, but until recently many of the key peptides that regulate ecdysis were unknown. The report in 1996 of a new ecdysis-triggering hormone (ETH) sparked an era of significant advances in our understanding of the regulation of molting. This article summarizes the current model of peptide regulation of ecdysis, a model that is based on a positive feedback loop between ETH and a brain peptide, eclosion hormone. Then the relationship of these regulatory peptides to the neural circuitry that is the ultimate driver of the behavior are described. Because insects can undergo both status quo (larval–larval) and metamorphic (larval–pupal and pupal–adult) molts, differences in ecdysis behavior at different life stages are described and potential sources of these differences are identified. Most of the work described is based on studies of ecdysis in the hawkmoth, Manduca sexta, but results from studies of ecdysis in the fruit fly Drosophila melanogaster are also discussed. [Copyright &y& Elsevier]

Eclosion hormone (EH) is a 62 amino acid neuropeptide that plays an integral role in triggering ecdysis behavior at the end of each molt. At least three populations of cells are thought to be targets for EH, each of which show an EH-stimulated increase in the intracellular messenger guanosine 3′, 5′ cyclic monophosphate (cGMP). These EH target cells are believed to include two pairs of neurons in each of the ganglia of the ventral nerve cord (VNC) that contain the neuropeptide crustacean cardioactive peptide (CCAP), the Inka cells of the peripheral epitracheal glands and intrinsic non-neuronal cells in the abdominal transverse nerves. This review describes likely signaling cascades that result in the EH-stimulated cGMP increase. Several lines of evidence suggest the involvement of a novel nitric oxide insensitive soluble guanylyl cyclase (GC). A novel GC with these properties has recently been identified and we also present evidence to suggest that it is activated by EH and describe possible pathways for its activation. In addition, we review our current knowledge on the cellular and molecular events that take place downstream of the increase in cGMP. [ABSTRACT FROM AUTHOR]

Eclosion hormone is an insect neuropeptide that consists of 62 amino acid residues including three disulfide bonds. We have previously reported its hypothetical 3D structure consisting mainly of three α-helices. In this paper, we report the effects of chaper-one proteins on the refolding of denatured eclosion hormone in a redox buffer containing reduced and oxidized glutathione. Urea-denatured eclosion hormone was spontaneously reactivated within 1 min with a yield of more than 90%, while β-mercap-toethanol-denatured eclosion hormone was reactivated in a few minutes with a yield of 75%. Under the same experimental conditions, eclosion hormone treated with β-mercap-toethanol and urea was reactivated slowly with a yield of 47% over a period of 2 h. Protein disuLfide isomerase, a eucaryotic chaperone protein, markedly increased the reactivation yield and rate of the totally denatured hormone. GroE oligomers slightly improved the reactivation yield but peptidyl prolyl isomerase had no influence on yield or rate. We propose that the folding pathway of eclosion hormone involves at least two rate-limiting steps, and that protein disulfide isomerase is likely to be involved in the folding in insect neuronal cells. [ABSTRACT FROM AUTHOR]

Simple Summary: In insects, the precise timing of metamorphosis and diapause is regulated by hormones. The Chinese oak silkworm, Antheraea pernyi, is a typical pupal diapause insect. Bivoltine species enter diapause in winter and terminate it under suitable environmental conditions in the following year; they produce 70% of total cocoons, whereas univoltine species in lower-latitude areas enter diapause in summer and contribute just one generation a year. A long photoperiod can trigger termination of pupal diapause. It is not clear how photoperiod influences hormone gene expression. Here, hormone-related genes were cloned, and their expression patterns were studied under different photoperiod treatments. The results will help us to understand the molecular changes during diapause termination under long photoperiods and improve breeding of multi-generation tussah pupae in areas where they are naturally univoltine. The Chinese oak silkworm is commonly used in pupal diapause research. In this study, a long photoperiod was used to trigger pupal diapause termination. Genes encoding three hormones, namely prothoracicotropic hormone (PTTH), ecdysis triggering hormone (ETH), and eclosion hormone (EH), were studied. Additionally, ecdysteroids (mainly 20-hydroxyecdysone, 20E) were quantified by HPLC. Pupal diapause stage was determined by measuring respiratory intensity. The pupae enter a low metabolic rate, which starts approximately 1 month after pupal emergence. ApPTTH expression showed a small increase at 14 days and then a larger increase from 35 days under the long photoperiod treatment. A similar pattern was observed for the titer of 20E in the hemolymph. However, ApETH expression later increased under the long photoperiod treatment (42 days) just before eclosion. Moreover, ApEH expression increased from 21 to 35 days, and then decreased before ecdysis. These results suggest that hormone-related gene expression is closely related to pupal development. Our study lays a foundation for future diapause studies in A. pernyi. [ABSTRACT FROM AUTHOR]

In the moth, Manduca sexta, the neuropeptide, eclosion hormone, triggers a dramatic rise in the levels of intracellular cGMP within a group of 50 neurons. The cells within this group include the segmentally repeated neurosecretory cell, Cell 27. In this study the effect of cGMP on the excitability of Cell 27 was investigated using intracellular recordings. Prior to its normal elevation in cGMP, Cell 27 exhibited a high spike threshold, but this was lowered dramatically when intracellular cGMP levels increased. The latter was also associated with spontaneous action potentials. This change in excitability did not correspond with changes in either resting potential, input resistance, or action potential amplitude. A similar lowering of threshold was induced by perfusion of 8-bromo-cGMP, whereas 8-bromo-cAMP caused the threshold to increase. Intracellular recordings using various ion substitution paradigms and channel blockers provided evidence which suggests indirectly that Ca2+ is mostly responsible for the depolarizing phase of the action potential while a Ca2+-activated K+ current contributes to the hyperpolarization. The results of these manipulations are consistent with the hypothesis that cGMP may partially increase excitability in Cell 27 by enhancing an inward Ca2+ current. [ABSTRACT FROM AUTHOR]

The tobacco hornworm, Manduca sexta, undergoes several larval molts before transforming into a pupa and then an adult moth. Each molt culminates in ecdysis, when the old cuticle is shed. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which consists of rhythmic compressions that are synchronous along the abdomen and on both body sides, and rhythmic retractions of the abdominal prolegs. Both pre-ecdysis and ecdysis behaviors are triggered by a peptide, eclosion hormone. The aim of the present study was to investigate the neural circuitry underlying larval preecdysis behavior. The pre-ecdysis motor pattern was recorded in isolated nerve cords from eclosion hormone-treated larvae, and the effects of connective transections and ionic manipulations were tested. Our results suggest that the larval pre-ecdysis compression motor pattern is coordinated and maintained by interneurons in the terminal abdominal ganglion that ascend the nerve cord without chemical synaptic relays; these interneurons make bilateral, probably monosynaptic, excitatory connections with identified pre-ecdysis motor neurons throughout the abdominal nerve cord. This model of the organization of the larval pre-ecdysis motor pattern should facilitate identification of the relevant interneurons, allowing future investigation of the neural basis of the developmental weakening of the pre-ecdysis motor pattern that accompanies the larval-pupal transformation. [ABSTRACT FROM AUTHOR]

Eclosion hormone (EH) is an important neuropeptide that regulates growth and development. This study predicted the EH gene (HvEH) of Heortia vitessoides Moore (Lepidoptera: Crambidae) from the transcriptome database and its expression patterns were determined using quantitative real-time polymerase chain reaction. HvEH was expressed in all developmental stages and especially in the head area. RNA interference-mediated silencing of HvEH (2 μg/individual) with double-stranded HvEH RNA (dsHvEH) was achieved within 48 hr. Abnormal phenotypes appeared in the pupa and adult stages. dsHvEH injection suppressed pupation and eclosion rates. HvEH expression increased upon treatment with 20-hydroxyecdysone but decreased at extreme temperatures. These results suggest that HvEH plays an essential role in ecdysis and wing formation in H. vitessoides., (© 2020 Wiley Periodicals LLC.)