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Egg laying in the pond snail, Lymnaea stagnalis is regulated by the photoperiod; long-day conditions (16L8D) promote egg laying whereas medium-day conditions (12L12D) suppress it. In this snail, a caudo-dorsal cell hormone (CDCH) is produced by neurosecretory cells, CDCs in the cerebral ganglion (CG), and its release triggers ovulation and subsequent egg laying. However, the physiological basis for photoperiod-dependent egg laying remains unraveled. Here, we compared electrophysiological properties of CDCs between 16L8D and 12L12D using intracellular recording, and found that CDC excitability is higher in 16L8D than in 12L12D. Striking differences are as follows: (1) a shallower resting membrane potential in 16L8D than in 12L12D, and (2) a smaller threshold voltage (minimum depolarization from rest to elicit action potentials) in 16L8D than in 12L12D. Switching of the excitability can be a physiological basis of a photoperiod-dependent CDCH release. Simultaneous intracellular dye injection identified two morphological subtypes of CDCs, validating a previous report. Both types bear short lateral extensions in CG, some of which probably function as integration sites of photoperiodic inputs. In addition, we found two novel CDCH-immunoreactive cell groups (CDC COM and SC m ) in the CG besides conventional CDCs and small cells expressing CDCH. The CDC COM with cell bodies and fibers in the neurohemal commissure may be involved in triggering ovulation. Notably, the total number of CDCs is larger than that previously reported, the right CDC cluster with more cells than the left. Our findings are instructive in following the neurophysiology of photoperiodism in L. stagnalis., (© 2021 Wiley Periodicals LLC.)

Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in Drosophila . Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution., Competing Interests: AE, AB, MH, SR, RS, CC, HS, BW No competing interests declared

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp., (Copyright © 2021. Published by Elsevier B.V.)

Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs β), and burs α-β is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs β are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The crustacean hyperglycemic hormone (CHH) gene of Portunus trituberculatus (Pt-CHH) consists of four exons and three introns spanning 3849 bp in size and generating two mature mRNA, Pt-CHH1, and Pt-CHH2. The primary gene transcript produces a cDNA encoding for the putative Pt-CHH2 from exons 1, 2, 3, and 4 and an alternative transcript encodes for a putative Pt-CHH1 peptide from exons 1, 2, and 4. A promoter fragment of about 3 kb was obtained by genomic walking. The tissue-specific expression pattern is examined by reverse transcriptase chain reaction, and the results show that Pt-CHH1 is detected in the eyestalk, brain, muscle, and blood. However, Pt-CHH2 is detected in the ganglia thoracalis and gill. The results indicate that the expression of Pt-CHH2 in the gill might suggest a potential role in osmoregulation. The Pt-CHH transcript level in the gill increases when the crab is exposed to low salinity. The injection of dsRNA for Pt-CHH causes a significant reduction in Pt-CHH2 transcript level and the activity of Na + /K + -ATPase, and carbonic anhydrase (CA) show a serious decrease. In conclusion, this study provides molecular evidence to support the osmoregulatory function of Pt-CHH2.

Crustacean hyperglycemic hormone (CHH) plays a crucial role in regulating carbohydrate metabolism in crustaceans. In this study, a new cDNA encoding type I CHH peptide, termed Sp-CHH3, was isolated from the mud crab Scylla paramamosain and its potential functions were investigated. The full length cDNA of Sp-CHH3 was identified as encoding a 127-aa precursor composed of a 27-aa signal peptide, a 23-aa CHH precursor-related peptide and a 75-aa mature peptide with a typical motif of CHH. Phylogenic analysis suggested that, Sp-CHH3 is a previously unreported CHH from S. paramamosain. Tissue distribution analysis showed that Sp-CHH3 was mainly expressed in the eyestalk ganglia, thoracic ganglia, stomach and the ovary. A RNA interference experiments showed that after injection of Sp-CHH3-targeted dsRNA, both the level of Sp-CHH3 expression in the eyestalk ganglia and hemolymph glucose level decreased significantly. A further short-term starvation experiments demonstrated that, the level of Sp-CHH3 detected in the eyestalk ganglia was significantly up-regulated at the 12th h of starvation, it then fell back at the 24th h of starvation and subsequently remained relative stability between the 24th to 96th h of starvation. The hemolymph glucose level decreased significantly (P < .05) at each sampling time during the 96 h starvation duration when compared to that of 0 h (prior to starvation) and the overall trend was largely correlated with the level of Sp-CHH3 expression in the eyestalk ganglia. In summary, the results suggest that Sp-CHH3 plays a functional role in regulating carbohydrate metabolism in S. paramamosain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Synchronous spawning as mass reproduction is well known to occur in many hermatypic corals, which is one of the mysterious life birth events. However, its contributing mechanism has not yet been clarified. This study placed focus on elucidating a neurotransmitter as endocrine signals that contribute to the synchronous spawning. First, the determination method of the neurotransmitters in coral was established by LC/MS in the selective ion mode together with a solid phase extraction method. As a result, the similar contents of the neurotransmitters for dopamine (DA), adrenaline (AD) and noradrenaline (NR) were detected in both the hermatypic corals of Acropora intermedia and Acropora digitifera. More interestingly, these neurotransmitters increased through the reproductive event during the synchronous spawning of A. intermedia, particularly, remarkable changes in the NR and DA were observed. In addition, hydrogen peroxide is known as the spawning stimulant and the metabolic by-product of the neurotransmitters, which was exposed to A. digitifera, then the neurotransmitters increased as well as those of the synchronization of spawning. All of the results suggested that the neurotransmitters contribute to the synchronous spawning in the hermatypic corals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain . This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens ( ChqIAG ), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5' untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3' UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain , which are clustered together phylogenetically., Competing Interests: The authors declare no conflict of interest.

The bryozoan Bugula neritina has a biphasic life cycle that consists of a planktonic larval stage and a sessile juvenile/adult stage. The transition between these two stages is crucial for the development and recruitment of B. neritina. Metamorphosis in B. neritina is mediated by both the nervous system and the release of developmental signals. However, no research has been conducted to investigate the expression of neuropeptides (NP)/peptide hormones in B. neritina larvae. Here, we report a comprehensive study of the NP/peptide hormones in the marine bryozoan B. neritina based on in silico identification methods. We recovered 22 transcripts encompassing 11 NP/peptide hormone precursor transcript sequences. The transcript sequences of the 11 isolated NP precursors were validated by cDNA cloning using gene-specific primers. We also examined the expression of three peptide hormone precursor transcripts (BnFDSIG, BnILP1, BnGPB) in the coronate larvae of B. neritina, demonstrating their distinct expression patterns in the larvae. Overall, our findings serve as an important foundation for subsequent investigations of the peptidergic control of bryozoan larval behavior and settlement.

Cell migration plays crucial roles during development. An excellent model to study coordinated cell movements is provided by the migration of border cell clusters within a developing Drosophila egg chamber. In a mutagenesis screen, we isolated two alleles of the gene rickets (rk) encoding a G-protein-coupled receptor. The rk alleles result in border cell migration defects in a significant fraction of egg chambers. In rk mutants, border cells are properly specified and express the marker Slbo. Yet, analysis of both fixed as well as live samples revealed that some single border cells lag behind the main border cell cluster during migration, or, in other cases, the entire border cell cluster can remain tethered to the anterior epithelium as it migrates. These defects are observed significantly more often in mosaic border cell clusters, than in full mutant clusters. Reduction of the Rk ligand, Bursicon, in the border cell cluster also resulted in migration defects, strongly suggesting that Rk signaling is utilized for communication within the border cell cluster itself. The mutant border cell clusters show defects in localization of the adhesion protein E-cadherin, and apical polarity proteins during migration. E-cadherin mislocalization occurs in mosaic clusters, but not in full mutant clusters, correlating well with the rk border cell migration phenotype. Our work has identified a receptor with a previously unknown role in border cell migration that appears to regulate detachment and polarity of the border cell cluster coordinating processes within the cells of the cluster themselves., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Juvenile hormone (JH) and methyl farnesoate (MF) play essential roles in the development and reproduction of insects and crustaceans respectively. Juvenile hormone acid methyltransferase (JHAMT) catalyzes the methyl esterification in insect JH biosynthesis, while the corresponding step in crustacean MF biosynthesis was long thought to be catalyzed by farnesoic acid O-methyltransferase (FAMeT). However, the new discovery of JHAMT orthologs in crustaceans indicates that JHAMT may also play essential role in the MF biosynthesis in crustaceans. Here we cloned and characterized the full-length cDNA encoding JHAMT in the swimming crab Portunus trituberculatus (PtJHAMT). Sequence and structure analysis of PtJHAMT revealed that it was composed of a 6-stranded β sheet with 9 α helices, and contained a signature Sadenosyl-L-methionine (SAM) binding motif, which is the hallmark in all SAM dependent methyltransferases (SAM-MTs). Several active sites that are critical for the interaction of SAM and JH/FA substrate were also conserved in PtJHAMT. The gene expression of PtJHAMT was highly specific to the mandibular organ, which is the sole site of MF synthesis. PtJHAMT expression significantly increased in the late-vitellogenic stage and mature stage, which suggests a possible role of PtJHAMT in modulating ovarian development. The role of PtJHAMT and PtFAMeT in MF biosynthesis was further investigated by RNA interfering (RNAi). Injection of PtJHAMT and PtFAMeT dsRNA both led to a decrease in hemolymph MF titers. Injection of PtHMGR dsRNA caused the decrease in PtJHAMT expression, but had no effect on mRNA level of PtFAMeT. Together these results suggested that JHAMT and FAMeT are both involved in the MF biosynthesis in crustaceans, while the JHAMT is highly specific to FA substrate, and FAMeT may have more catalytic functions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

Elimination of cells and tissues by apoptosis is a highly conserved and tightly regulated process. In Drosophila, the entire wing epithelium is completely removed shortly after eclosion. The cells that make up this epithelium are collectively eliminated through a highly synchronized form of apoptotic cell death, involving canonical apoptosome genes. Here we present evidence that collective cell death does not require cell-cell contact and show that transcription of the IAP antagonist, head involution defective, is acutely induced in wing epithelial cells prior to this process. hid mRNAs accumulate to levels that exceed a component of the ribosome and likewise, Hid protein becomes highly abundant in these same cells. hid function is required for collective cell death, since loss of function mutants shows persisting wing epithelial cells and, furthermore, silencing of the hormone bursicon in the CNS produced collective cell death defective phenotypes manifested in the wing epithelium. Taken together, our observations suggest that acute induction of Hid primes wing epithelial cells for collective cell death and that Bursicon is a strong candidate to trigger this process, possibly by activating the abundant pool of Hid protein already present., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bursicon is a hormone that modulates wing expansion, cuticle hardening and melanization in Drosophila melanogaster. Bursicon activity is mediated through its cognate G protein-coupled receptor (GPCR), rickets. We have developed a membrane-tethered bursicon construct that enables spatial modulation of rickets-mediated physiology in transgenic flies. Ubiquitous expression of tethered bursicon throughout development results in arrest at the pupal stage. The few organisms that eclose fail to undergo wing expansion. These phenotypes suggest that expression of tethered bursicon inhibits rickets-mediated function. Consistent with this hypothesis, we show in vitro that sustained stimulation of rickets by tethered bursicon leads to receptor desensitization. Furthermore, tissue-specific expression of the tethered bursicon inhibitor unraveled a critical role for rickets in a subset of adult muscles. Taken together, our findings highlight the utility of membrane-tethered inhibitors as important genetic/pharmacological tools to dissect the tissue-specific roles of GPCRs in vivo., (© 2014. Published by The Company of Biologists Ltd.)

It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm-egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ovarian maturation in crustaceans is temporally orchestrated by two processes: oogenesis and vitellogenesis. The peptide hormone vitellogenesis-inhibiting hormone (VIH), by far the most potent negative regulator of crustacean reproduction known, critically modulates crustacean ovarian maturation by suppressing vitellogenin (VTG) synthesis. In this study, cDNA encoding VIH was cloned from the eyestalk of Pacific white shrimp, Litopenaeus vannamei, a highly significant commercial culture species. Phylogenetic analysis suggests that L. vannamei VIH (lvVIH) can be classified as a member of the type II crustacean hyperglycemic hormone family. Northern blot and RT-PCR results reveal that both the brain and eyestalk were the major sources for lvVIH mRNA expression. In in vitro experiments on primary culture of shrimp hepatopancreatic cells, it was confirmed that some endogenous inhibitory factors existed in L. vannamei hemolymph, brain, and eyestalk that suppressed hepatopancreatic VTG gene expression. Purified recombinant lvVIH protein was effective in inhibiting VTG mRNA expression in both in vitro primary hepatopancreatic cell culture and in vivo injection experiments. Injection of recombinant VIH could also reverse ovarian growth induced by eyestalk ablation. Furthermore, unilateral eyestalk ablation reduced the mRNA level of lvVIH in the brain but not in the remaining contralateral eyestalk. Our study, as a whole, provides new insights on VIH regulation of shrimp reproduction: 1) the brain and eyestalk are both important sites of VIH expression and therefore possible coregulators of hepatopancreatic VTG mRNA expression and 2) eyestalk ablation could increase hepatopancreatic VTG expression by transcriptionally abolishing eyestalk-derived VIH and diminishing brain-derived VIH.

To identify and characterize CHH (TJ-CHH) gene in the copepod Tigriopus japonicus, we analyzed the full-length cDNA sequence, genomic structure, and promoter region. The full-length TJ-CHH cDNA was 716 bp in length, encoding 136 amino acid residues. The deduced amino acid sequences of TJ-CHH showed a high similarity of the CHH mature domain to other crustaceans. Six conserved cysteine residues and five conserved structural motifs in the CHH mature peptide domain were also observed. The genomic structure of the TJ-CHH gene contained three exons and two introns in its open reading frame (ORF), and several transcriptional elements were detected in the promoter region of the TJ-CHH gene. To investigate transcriptional change of TJ-CHH under environmental stress, T. japonicus were exposed to heat treatment, UV-B radiation, heavy metals, and water-accommodated fractions (WAFs) of Iranian crude oil. Upon heat stress, TJ-CHH transcripts were elevated at 30 °C and 35 °C for 96 h in a time-course experiment. UV-B radiation led to a decreased pattern of the TJ-CHH transcript 48 h and more after radiation (12 kJ/m(2)). After exposure of a fixed dose (12 kJ/m(2)) in a time-course experiment, TJ-CHH transcript was down-regulated in time-dependent manner with a lowest value at 12h. However, the TJ-CHH transcript level was increased in response to five heavy metal exposures for 96 h. Also, the level of the TJ-CHH transcript was significantly up-regulated at 20% of WAFs after exposure to WAFs for 48 h and then remarkably reduced in a dose-dependent manner. These findings suggest that the enhanced TJ-CHH transcript level is associated with a cellular stress response of the TJ-CHH gene as shown in decapod crustaceans. This study is also helpful for a better understanding of the detrimental effects of environmental changes on the CHH-triggered copepod metabolism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, we have explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or β bursicon subunit. Coexpression of the two complementary bursicon MTLs triggered rk-mediated signaling in vitro. We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-β or β-α. Carboxy-terminal deletion of 32 amino acids in the β-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins.

Over than fifty years starfishes have been widely used as model for studying the mechanisms of cell cycle regulation, oocyte maturation and fertilization. Besides, significant work has been done to investigate the role of nervous system in the control of reproduction and spawning in these animals. Nowadays, sea stars represent one of the most thoroughly studied model for hormonal regulation of reproduction among invertebrates. However, while the general picture of neuroendocrine control of asteroid reproduction can be drawn easily, our knowledge concerning the details of this process still has some gaps. Filling these gaps is essential for studying the diversity of hormonal mechanisms involved in regulation of animal reproduction. The present paper aims to briefly summarize current data on hormonal regulation of reproduction in sea stars and to highlight existing gaps in our knowledge on the details of this process., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ecdysis behavior allows insects to shed their old exoskeleton at the end of every molt. It is controlled by a suite of interacting hormones and neuropeptides, and has served as a useful behavior for understanding how bioactive peptides regulate CNS function. Previous findings suggest that crustacean cardioactive peptide (CCAP) activates the ecdysis motor program; the hormone bursicon is believed to then act downstream of CCAP to inflate, pigment, and harden the exoskeleton of the next stage. However, the exact roles of these signaling molecules in regulating ecdysis remain unclear. Here we use a genetic approach to investigate the functions of CCAP and bursicon in Drosophila ecdysis. We show that null mutants in CCAP express no apparent defects in ecdysis and postecdysis, producing normal adults. By contrast, a substantial fraction of flies genetically null for one of the two subunits of bursicon [encoded by the partner of bursicon gene (pburs)] show severe defects in ecdysis, with escaper adults exhibiting the expected failures in wing expansion and exoskeleton pigmentation and hardening. Furthermore, flies lacking both CCAP and bursicon show much more severe defects at ecdysis than do animals null for either neuropeptide alone. Our results show that the functions thought to be subserved by CCAP are partially effected by bursicon, and that bursicon plays an important and heretofore undescribed role in ecdysis behavior itself. These findings have important implications for understanding the regulation of this vital insect behavior and the mechanisms by which hormones and neuropeptides control the physiology and behavior of animals.

Myoinhibitory peptides (MIPs) are a family of insect W(X(6))Wamides with inhibitory effects on visceral muscles and juvenile hormone synthesis. Although MIPs are widely distributed within the nervous system, a detailed analysis of their distribution and function in insect brains is still missing. We analyzed the distribution of MIPs in the brain of the cockroach Leucophaea maderae. We focused on the accessory medulla (AMe), a small neuropil near the medulla that acts as the master circadian clock. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and Nano-LC electrospray ionization (ESI) mass spectrometry revealed five Lem-MIPs in preparations of the AMe and corpora cardiaca. The complete sequences of two of these peptides were identified. Immunocytochemistry revealed wide distribution of MIP-related peptides in the cockroach brain. The superior median protocerebrum, parts of the central complex, and the tritocerebrum showed particularly dense immunostaining. In contrast, only a few local interneurons were stained in the antennal lobe and a few extrinsic neurons in the mushroom body, including a giant neuron innervating the calyces. The noduli of the AMe showed dense immunostaining, and neurons in all AMe cell groups except the anterior neurons were labeled. Pigment-dispersing factor- (PDF) and MIP immunostaining was colocalized in two neurons of the AMe. No colocalization of MIP- and PDF immunostaining was detected in the anterior optic commissure, but two small PDF-immunoreactive commissural fibers near the posterior optic commissure showed colocalized MIP immunostaining. The results suggest that several MIPs participate in different functional circuits of the circadian system and are involved in multiple brain circuits of the Madeira cockroach., (Copyright © 2011 Wiley-Liss, Inc.)

To help celebrate the 50th anniversary of General and Comparative Endocrinology, the history of only a small portion of crustacean endocrinology is presented here. The field of crustacean endocrinology dates back to the decades prior to the establishment of General and Comparative Endocrinology and the first article about crustacean endocrinology published in this journal was concerned with the anatomy of neurosecretory and neurohemal structures in brachyuran crabs. This review looks at the history of neuroendocrinology in crustaceans during that time and tries to put perspective on the future of this field., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Apart from providing an up-to-date review of the literature, considerable emphasis was placed in this article on the historical development of the field of "crustacean eyestalk hormones". A role of the neurosecretory eyestalk structures of crustaceans in endocrine regulation was recognized about 80 years ago, but it took another half a century until the first peptide hormones were identified. Following the identification of crustacean hyperglycaemic hormone (CHH) and moult-inhibiting hormone (MIH), a large number of homologous peptides have been identified to this date. They comprise a family of multifunctional peptides which can be divided, according to sequences and precursor structure, into two subfamilies, type-I and -II. Recent results on peptide sequences, structure of genes and precursors are described here. The best studied biological activities include metabolic control, moulting, gonad maturation, ionic and osmotic regulation and methyl farnesoate synthesis in mandibular glands. Accordingly, the names CHH, MIH, and GIH/VIH (gonad/vitellogenesis-inhibiting hormone), MOIH (mandibular organ-inhibiting hormone) were coined. The identification of ITP (ion transport peptide) in insects showed, for the first time, that CHH-family peptides are not restricted to crustaceans, and data mining has recently inferred their occurrence in other ecdysozoan clades as well. The long-held tenet of exclusive association with the eyestalk X-organ-sinus gland tract has been challenged by the finding of several extra nervous system sites of expression of CHH-family peptides. Concerning mode of action and the question of target tissues, second messenger mechanisms are discussed, as well as binding sites and receptors. Future challenges are highlighted., (Copyright © 2011. Published by Elsevier Inc.)

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic hormone (CHH) family, inhibits the synthesis of ecdysteroid in Y-organ (YO) and plays a significant role in the regulation of molting and growth of crustaceans. A complete cDNA sequence encoding MIH (Ers-MIH, GenBank Accession No.: DQ341280) was cloned from eyestalk of Chinese mitten crab (Eriocheir sinensis) by 5' and 3' RACEs and PCR cloning. The full-length cDNA consists of 1457 bp with a 330 bp open reading frame, encoding 110 amino acids, containing a 75 amino acid mature peptide. The deduced amino acid sequence contains a typical CHH domain. Transcripts of Ers-MIH mRNA were detected in eyestalk by Northern blotting. The production of purified recombinant Ers-MIH (rErs-MIH) expressed in Escherichia coli was 0.3g/L. The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the deduced amino acid sequence of Ers-MIH. By in vitro assay on E. sinensis YOs, a cGMP mediated suppression of rErs-MIH on ecdysteroidogenesis could be observed. Accumulation of cGMP in YOs showed a concentration-dependent manner within 0.01-1 nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01-100 nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100 nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Steroids play fundamental roles regulating mammalian reproduction and development. Although sex steroids and their receptors are well characterized in vertebrates and several arthropod invertebrates, little is known about the hormones and receptors regulating reproduction in other invertebrate species. Evolutionary insights into ancient endocrine pathways can be gained by elucidating the hormones and receptors functioning in invertebrate reproduction. Using a combination of genomic analyses, receptor imaging, ligand identification, target elucidation, and exploration of function through receptor knockdown, we now show that comparable progesterone chemoreception exists in the invertebrate monogonont rotifer Brachionus manjavacas, suggesting an ancient origin of the signal transduction systems commonly associated with the development and integration of sexual behavior in mammals.

Background: Medicinal leeches (Hirudo spp.) are simultaneous hermaphrodites. Mating occurs after a stereotyped twisting and oral exploration that result in the alignment of the male and/or female gonopores of one leech with the complementary gonopores of a partner. The neural basis of this behavior is presently unknown and currently impossible to study directly because electrophysiological recording techniques disrupt the behavior., Results: Here we report that (Arg(8))-conopressin G and two other members of the oxytocin/vasopressin family of peptide hormones induce in Hirudo verbana a sequence of behaviors that closely mimic elements of spontaneous reproductive behavior. Through a series of progressively more reduced preparations, we show that one of these behaviors, a stereotyped twisting that is instrumental in aligning gonopores in preparation for copulation, is the product of a central pattern generator that consists of oscillators in ganglia M5 and M6 (the ganglia in the reproductive segments of the leech), and also in ganglion M4, which was not previously known to play a role in reproductive behavior. We find that the behavior is periodic, with a remarkably long cycle period of around five minutes, placing it among the slowest behavioral rhythms (other than diurnal and annual rhythms) yet described., Conclusion: These results establish the leech as a new model system for studying aspects of the neuronal basis of reproductive behavior., (2010 Elsevier Ltd. All rights reserved.)

Artemia has attracted much attention for its ability to produce encysted embryos wrapped in a protective shell when subject to extremely harsh environmental conditions. However, what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown. Over 20 oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell to become less compact and pultaceous and to show a marked decrease of iron composition within the shell. The RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness, and organic solvent stresses. In contrast, the natural cyst would provide adequate protection from all such factors. SGEG contains a 345-bp open reading frame, and its consequentially translated peptide consists of a 33-amino acid residue putative signal peptide and an 81-amino acid residue mature peptide. The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. This investigation adds strong insight into the mechanism of cyst shell formation of Artemia and may be applicable to other areas of research in extremophile biology.

Hormones are often responsible for synchronizing somatic physiological changes with changes in behavior. Ecdysis (i.e., the shedding of the exoskeleton) in insects has served as a useful model for elucidating the molecular and cellular mechanisms of this synchronization, and has provided numerous insights into the hormonal coordination of body and behavior. An example in which the mechanisms have remained enigmatic is the neurohormone bursicon, which, after the final molt, coordinates the plasticization and tanning of the initially folded wings with behaviors that drive wing expansion. The somatic effects of the hormone are governed by bursicon that is released into the blood from neurons in the abdominal ganglion (the B(AG)), which die after wing expansion. How bursicon induces the behavioral programs required for wing expansion, however, has remained unknown. Here we show by targeted suppression of excitability that a pair of bursicon-immunoreactive neurons distinct from the B(AG) and located within the subesophageal ganglion in Drosophila (the B(SEG)) is involved in controlling wing expansion behaviors. Unlike the B(AG), the B(SEG) arborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the B(AG.) Indeed, we show that animals lacking bursicon receptor function have deficits both in the humoral release of bursicon and in posteclosion apoptosis of the B(AG). Our results reveal novel neuromodulatory functions for bursicon and support the hypothesis that the B(SEG) are essential for orchestrating both the behavioral and somatic processes underlying wing expansion.

Bursicon was identified in 1965 as a peptide neurohormone that initiates the tanning of the insect cuticle immediately after the shedding of the old one during the final stages of the molting process. Its molecular identity as an approximately 30 kDa bioactive heterodimer consisting of two cystine knot proteins resisted elucidation for 43 years. The sequence of the two bursicon subunits is highly conserved among arthropods, and this conservation extends even to echinoderms. We review the efforts leading to bursicon's characterization, the identification of its leucine-rich repeat-containing, G protein-coupled receptor (LGR2), and the progress towards revealing its various functions. It is now clear that bursicon regulates different aspects of wing inflation in Drosophila melanogaster besides being involved at various points in the cuticle tanning process in different insects. We also describe the current knowledge of the expression of bursicon in the central nervous system of different insects in large homologous neurosecretory cells, and the changes in its expression during the development of Manduca sexta and D. melanogaster. Although much remains to be learned, the elucidation of its molecular identity and that of its receptor has provided the breakthrough needed for investigating the diverse actions of this critical insect neurohormone.

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial-mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation.

In the recent decade, tremendous progress has been realized in insect endocrinology as the result of the application of a variety of advanced methods in neuropeptidome- and receptor research. Hormones of which the existence had been shown by bioassays four decades ago, e.g. bursicon (a member of the glycoprotein hormone family) and pupariation factor (Neb-pyrokinin 2, a myotropin), could be identified, along with their respective receptors. In control of diurnal rhythms, clock genes got company from the neuropeptide Pigment Dispersing Factor (PDF), of which the receptor could also be identified. The discovery of Inka cells and their function in metamorphosis was a true hallmark. Analysis of the genomes of Caenorhabditis elegans, Drosophila melanogaster and Apis mellifera yielded about 75, 100 and 200 genes coding for putative signaling peptides, respectively, corresponding to approximately 57, 100 and 100 peptides of which the expression could already be proven by means of mass spectrometry. The comparative approach invertebrates-vertebrates recently yielded indications for the existence of counterparts in insects for prolactin, atrial natriuretic hormone and Growth Hormone Releasing Hormone (GRH). Substantial progress has been realized in identifying the Halloween genes, a membrane receptor(s) for ecdysteroids, a nuclear receptor for methylfarnesoate, and dozens of GPCRs for insect neuropeptides. The major remaining challenges concern the making match numerous orphan GPCRs with orphan peptidic ligands, and elucidating their functions. Furthermore, the endocrine control of growth, feeding-digestion, and of sexual differentiation, in particular of males, is still poorly understood. The finding that the prothoracic glands produce an autocrine factor with growth factor-like properties and secrete proteins necessitates a reevaluation of their role in development.

The present study was focused on the regulation of ecdysteroidogenesis in the Y-organ of Scylla serrata during molting cycle. A strong expression of molt-inhibiting hormone (MIH) and phosphorylation of ERK was predominantly observed in the postmolt and intermolt stages of Y-organs, whereas protein kinase C, steroidogenic acute regulatory protein (StAR) and cytochrome P450(scc) activity were exclusively seen in the premolt stages. Interestingly, inhibition of ERK phosphorylation by PD98059 in the early postmolt (A), middle postmolt (B) and intermolt (C) stages resulted in the prominent expression of PKC and StAR in the postmolt stages. This result indicates that phosphorylation of ERK is required for suppression of ecdysteroid biosynthesis with the involvement of protein kinase C, and StAR protein.

Endogenous circadian clocks are inherent to all living organisms. They are needed to guarantee successful life since they regulate very important biological processes such as behavior and reproduction. Secretin-like G-protein coupled receptors are very important factors in the signal transduction pathways of circadian clocks. In this review, we will focus on the role of two secretin-like signaling pathways that play an important role in the regulation of the mammalian and the insect clock, respectively: the pituitary adenylate cyclase-activating polypeptide (PACAP) and pigment dispersing factor (PDF) signaling pathways. Both pathways are most likely related although their function in the biological clock differs.

Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species.

Crustaceans are major constituents to aquatic ecosystems that provide a variety of ecological and economic services. Individual crustacean species are adept at occupying diverse niches and their success, in part, stems from neuro-endocrine signaling cascades that regulate physiology in response to environmental and internal cues. Peptide hormones are major signal transducers in crustaceans. The crustacean hyperglycemic hormone family of peptides regulates various aspects of growth, reproduction, and metabolism. These peptides may function as the terminal hormone to regulate some physiological activities or may function as intermediates in a signaling cascade. Ecdysteroids and terpenoids are two major classes of terminal signaling molecules in these cascades. Hormones from these two classes function independently or in concert to regulate various processes. Ecdysteroid signaling is subject to toxicological disruption through disturbances in ecdysteroid synthesis or binding of toxicants to the ecdysteroid receptor. Methyl farnesoate is the major terpenoid hormone of crustaceans and also is susceptible to disruption by environmental chemicals. However, the methyl farnesoate signaling pathway is poorly understood and only limited mechanistic confirmation for disruption of this endocrine signaling pathway exists. Disruption of the ecdysteroid/terpenoid signaling pathways in crustaceans has been associated with aberrations in growth, metamorphosis, reproductive maturation, sex determination, and sex differentiation. Population studies have revealed disruptions in crustacean growth, molting, sexual development, and recruitment that are indicative of environmental endocrine disruption. However, environmental factors other that pollution (i.e., temperature, parasitism) also can elicit these effects and definitive causal relationships between endocrine disruption in field populations of crustaceans and chemical pollution is generally lacking.

The water flea Daphnia magna (Crustacea, Cladocera) is a cyclical parthenogen, which can reproduce both by parthenogenesis and by sexual reproduction. With its ease of handling in the laboratory, several testing methods using D. magna exist for regulatory toxicity testing. Recently, several studies revealed that one of the major hormone groups in insects and crustaceans, juvenile hormones, are involved in the shift of reproductive mode from parthenogenesis to sexual reproduction (production of male neonates). Using offspring sex ratio as a new endpoint has made it possible to identify chemicals with juvenile hormone-like effects on crustaceans. The testing method using D. magna, in which offspring sex ratio is incorporated as a new endpoint, is now being proposed to the OECD as an enhanced version of the existing OECD Test Guideline 211: Daphnia magna reproduction test. No other clear-cut endpoint for identifying juvenile-hormone disrupting effects has ever been found in crustaceans than the induction of male neonates production in cladocerans. In this regard, it is expected that testing methods using D. magna are suitable for screening and risk assessment of chemicals with juvenile-hormone disrupting effects.

Steroid molecules are present in all invertebrates, and some of them have established hormonal roles: this is the case for ecdysteroids in arthropods and, to a lesser extent, for vertebrate-type steroids in molluscs. Steroids are not only hormones, they may also fulfill many other functions in chemical communication, chemical defense or even digestive physiology. The increasing occurrence of endocrine disruption problems caused by environmental pollutants, which interfere in particular with reproductive physiology of vertebrates but also of invertebrates has made necessary to better understand the endocrine physiology of the latter and the role of steroids in these processes. So many attempts are being made to better understand the endocrine roles of steroids in arthropods and molluscs, and to establish whether they also fulfill similar functions in other invertebrate phyla. At the moment, both the precise identification of these steroids, the determination of their origin (endogenous versus exogenous) and of their mechanism of action are under active investigation. This research takes profit of the development of genome sequencing programs on many invertebrate species, which allow the identification of receptors and/or biosynthetic enzymes, when related to their vertebrate counterparts, but the story is not so simple, as will be exemplified by estrogen receptors of molluscs.

In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of ALF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P. leniusculus, quantitative PCR showed that knockdown of ALF by RNAi resulted into WSSV levels that were about 10-fold higher than those treated with control double-stranded RNA (dsRNA). In addition, RNAi experiments with other crayfish genes that had been found to be up-regulated by a WSSV infection did not result in any changes of viral loads. Thus, the cell culture does not respond to dsRNA in a similar manner, as shown earlier for dsRNA injected into shrimp, which gave a higher degree of resistance to WSSV infection. If ALF transcription in whole animals was stimulated by the administration of UV-treated WSSV, a partial protection against a subsequent challenge with the active virus was conferred to the host. This is the first crustacean gene product identified with the capacity to interfere with replication of this important pathogen.

The crustacean hyperglycemic hormone (CHH) family of peptides includes CHH, moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). In the crab Cancer pagurus, isoforms of these peptides, as well as CHH precursor-related peptide (CPRP), have been identified in the X-organ-sinus gland (XO-SG) system. Using peptides isolated from the C. pagurus SG, antibodies to each family member and CPRP were generated. These sera were then used to map the distributions and co-localization patterns of these peptides in the neuroendocrine organs of seven Cancer species: Cancer antennarius, Cancer anthonyi, Cancer borealis, Cancer gracilis, Cancer irroratus, Cancer magister and Cancer productus. In addition to the XO-SG, the pericardial organ (PO) and two other neuroendocrine sites contained within the stomatogastric nervous system, the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), were studied. In all species, the peptides were found to be differentially distributed between the neuroendocrine sites in conserved patterns: i.e. CHH, CPRP, MIH and MOIH in the XO-SG, CHH, CPRP and MOIH in the PO, and MOIH in the ACP (no immunolabeling was found in the ACO). Moreover, in C. productus (and probably in all species), the peptides present in the XO-SG and PO were differentially distributed between the neurons within each of these neuroendocrine organs (e.g. CHH and CPRP in one set of XO somata with MIH and MOIH co-localized in a different set of cell bodies). Taken collectively, the differential distributions of CHH family members and CPRP both between and within the neuroendocrine organs of crabs of the genus Cancer suggests that each of these peptides may be released into the circulatory system in response to varied, tissue-specific cues and that the PO- and/or ACP-derived isoforms may possess functions distinct from those classically ascribed to their release from the SG.

Crustacean molt-inhibiting hormone (MIH), a polypeptide produced by neurosecretory cells in eyestalk ganglia, suppresses the synthesis of ecdysteroid molting hormones by paired Y-organs. Data from several sources indicate the effects of MIH are mediated, at least in part, by a cGMP second messenger. Based on these and related findings, our working hypothesis is that the MIH receptor is a receptor guanylyl cyclase (rGC). In studies reported here, we used a PCR-based cloning strategy (RT-PCR followed by 5'- and 3'-RACE) to clone from blue crab (Callinectes sapidus) Y-organs a cDNA (CsGC-YO1) encoding a putative rGC. DNA sequence analysis revealed a 3807 base pair open reading frame encoding a 56 residue signal peptide and a 1213 residue rGC. Analysis of the deduced amino acid sequence showed that CsGC-YO1 contains the signature domains characteristic of rGCs, including an extracellular ligand-binding domain, a single transmembrane domain, a kinase-like domain, a dimerization domain, and a cyclase catalytic domain. CsGC-YO1 is most closely related to an rGC from the crayfish, Procambarus claikii (PcGC-M2, 58.4% identity), and rGCs from three insect species (33.1-37.5% identity). Conserved cysteine residues are similarly distributed in the extracellular domains of CsGC-YO1, PcGC-M2, and the three insect rGCs. RT-PCR revealed the CsGC-YO1 transcript is expressed in Y-organs and several other tissues. While other interpretations of the data are possible, our working hypothesis is that the cloned cDNA encodes an MIH receptor.

One problem in aquaculture is obtaining brood because many commercially important species are incapable of spontaneous maturation under artificial conditions. Commercial shrimp hatcheries commonly use eyestalk ablation to stimulate gonadal maturation in shrimps. Research has been conducted on the inhibition of reproductive maturation by hormones originating in the eyestalk glands and on other endocrine sources (e.g.,brain, thoracic ganglion, ovary, mandibular organ, androgenic gland and Y-organs) to determine their roles. Alternate techniques for acceleration of gonad maturation through the use of synthetic hormones or neurotransmitters may benefit aquaculture. Neurohormones and neuroregulators have been shown to accelerate gonadal maturation but an effective delivery technique must be developed for use in a large-scale aquaculture operation.

All arthropods periodically molt to replace their exoskeleton (cuticle). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Here we show that bursicon, to our knowledge the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The pburs/burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G protein-coupled receptor DLGR2, leading to the stimulation of cAMP signaling in vitro and tanning in neck-ligated blowflies. Native bursicon from Periplaneta americana is also a heterodimer. In D. melanogaster the levels of pburs, burs, and DLGR2 transcripts are increased before ecdysis, consistent with their role in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species revealed the colocalization of pburs- and burs-immunoreactivity in some of the neurosecretory neurons that also express crustacean cardioactive peptide. Forty-three years after its initial description, the elucidation of the molecular identity of bursicon and the verification of its receptor allow for studies of bursicon actions in regulating cuticle tanning, wing expansion, and as yet unknown functions. Because bursicon subunit genes are homologous to the vertebrate bone morphogenetic protein antagonists, our findings also facilitate investigation on the function of these proteins during vertebrate development.

Peptides form a very versatile class of extracellular messenger molecules that function as chemical communication signals between the cells of an organism. Molecular diversity is created at different levels of the peptide synthesis scheme. Peptide messengers exert their biological functions via specific signal-transducing membrane receptors. The evolutionary origin of several peptide precursor and receptor gene families precedes the divergence of the important animal Phyla. In this chapter, current knowledge is reviewed with respect to the analysis of peptide receptors from insects, incorporating many recent data that result from the sequencing of different insect genomes. Therefore, detailed information is provided on six different peptide receptor families belonging to two distinct receptor categories (i.e., the heptahelical and the single transmembrane receptors). In addition, the remaining problems, the emerging concepts, and the future prospects in this area of research are discussed.

In crustaceans, molt-inhibiting hormone (MIH) controls molting by suppressing the synthesis and/or secretion of molting hormone. In our previous study, which determined the solution structure of MIH by NMR, we hypothesized that the peptide's functional site spanned the region encompassing the N-terminal alpha-helix and a portion of the C-terminus, both of which are located sterically close to each other [Katayama et al. (2003) J. Biol. Chem. 278, 9620-9623]. To confirm this hypothesis, various mutants of MIH were prepared and their molt-inhibiting activities were assessed. All peptides mutated at the putative functional site exhibited circular dichroism spectra similar to the natural MIH, suggesting that the mutants retained their natural conformation regardless of the mutations. As expected, a majority of the mutants, except for Delta12 (a deletion mutant of Gly(12)) and Delta75-77 (a deletion mutant of the last three residues of the C-terminus), were less active than the natural MIH. In particular, I72G exhibited no molt-inhibiting activity even at 200 nM, while N13A and S71Y exhibited low activity at the same concentration. In contrast, the natural and recombinant MIHs exhibited full inhibitory activity at 20 nM. All these results indicate that the functional site of MIH is located in the region containing the C-terminal ends of the N- and C-terminal alpha-helices, and that Asn(13), Ser(71), and Ile(72) are especially significant for conferring molt-inhibiting activity. Furthermore, these findings agree with the results and the proposed hypothesis presented in previous studies on the structure-activity relationship of MIH and its related peptides.

The circadian rhythm of black pigment migration of melanophores of the crab Chasmagnathus granulata and the variation in responsiveness of these cells to pigment-dispersing hormone (beta-PDH), crustacean cardioactive peptide (CCAP), and red pigment-concentrating hormone (RPCH) were investigated. Melanophores of C. granulata possess an endogenous circadian rhythm of pigment migration, with black pigments staying more dispersed during the day period and more aggregated during the night period. This rhythm seems to be largely dependent on an endogenous release of neurohormones from eyestalks, and to a lesser extent on a primary response to illumination. beta-PDH was the most potent PDH isoform to induce pigment dispersion in both in vivo (EC50 = 0.4 pmol/animal) and in vitro (EC50 = 0.18 microM) assays. CCAP also induced pigment dispersion in vivo and in vitro assays (EC50 = 12 microM), but it was less potent than beta-PDH. In vivo, RPCH induced a low and nondose-dependent pigment aggregation, while in vitro, it had no effect on pigment migration. The responsiveness of melanophores of C. granulata to beta-PDH was significantly higher during the day period when compared to the night period in both assays, in vitro and in vivo. These results suggest that the endogenous circadian rhythm of black pigment migration is dependent on both endogenous circadian rhythm of beta-PDH synthesis and/or release from eyestalks and on an endogenous rhythm of responsiveness of melanophores to beta-PDH.

FMRFamide-related peptides are common to a wide variety of invertebrate species, including helminths and arthropods. In arthropods, five distinct FMRFamide-related peptide subfamilies are recognised: the myosuppressins, extended-FLRFamides, -FMRFamides, -RFamides, and sulfakinins, members of which induce potent and diverse myotropic effects. Whilst >80 FMRFamide-related peptides have been identified in nematodes, only four FMRFamide-related peptides have been characterised from flatworms. The Ascaris suum ovijector/body wall bioassay and the Procerodes littoralis muscle fibre bioassay have proved both reliable and sensitive systems for assessing the functional activities of FMRFamide-related peptides in vitro, and data describing the effects of native FMRFamide-related peptides in these systems are rapidly accumulating. This is the first study to determine the cross-phyla activities of non-native FMRFamide-related peptides in both nematode and flatworm species. In the present study, the effects of 10 arthropod FMRFamide-related peptides (leucomyosuppressin [pQDVDHVFLRFamide], schistoFLRFamide [PDVDHVFLRFamide] and truncated analogues [HVFLRFamide and VFLRFamide], lobster peptide I [TNRNFLRFamide], lobster peptide II [SDRNFLRFamide], manducaFLRFamide II [GNSFLRFamide], manducaFLRFamide III [DPSFLRFamide], calliFMRFamide 4 [KPNQDFMRFamide] and perisulfakinin [EQFDDY(SO(3)H)GHMRFamide]), representing the five subfamilies, were examined on the body wall and ovijector of the parasitic porcine nematode, A. suum and dispersed muscle fibres from the free-living turbellarian, P. littoralis. The muscle activity of the ovijector was found to be modulated significantly by each of the arthropod FMRFamide-related peptides tested; the effects were concentration-dependent, reversible and repeatable. All but one (perisulfakinin) of the 10 arthropod FMRFamide-related peptides examined modulated significantly the activity of A. suum body wall muscle. In addition, all of the arthropod FMRFamide-related peptides examined induced potent concentration-dependent contractions of P. littoralis muscle fibres. These results reveal similarities in the ligand requirement(s) between FMRFamide-related peptide receptors within the Phyla Arthropoda, Nematoda and Platyhelminthes, and indicate significant receptor promiscuity, which highlights the potential of FMRFamide-related peptide receptors as legitimate targets for novel endectocidal agents.

We describe the molecular analysis and cellular expression of the insect peptide neurohormone, bursicon. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, we determined the molecular weight of bursicon from different insects to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, we successfully searched the Drosophila genome project for the gene encoding bursicon. With Drosophila as a tool, we can now verify the function of the sequence using transgenic flies. Sequence comparisons also allowed us to verify that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution.

The primitive nervous system in planula larvae of Hydractinia echinata (Cnidaria) has sensory neurons containing LWamide or RFamide neuropeptides. LWamides have been shown to induce metamorphosis of planula larvae into adult polyps. We report here that RFamides act antagonistically to LWamides. RFamides inhibit metamorphosis when applied to planula larvae during metamorphosis induction by treatment with LWamides (or other inducing agents such as CsCl ions, diacylglycerol and bacterial inducers). Our results show further that RFamides act downstream of LWamide release, presumably directly on target cells mediating metamorphosis. These observations support a model in which metamorphosis in H. echinata is regulated by sensory neurons secreting LWamides and RFamides in response to environmental cues.