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Bursicon is the main regulator of post molting and post eclosion processes during arthropod development. The active Bursicon hormone is a heterodimer of Burs-α and Burs-β. However, adult midguts express Burs-α to regulate the intestinal stem cell niche. Here, we examined the potential expression and function of its heterodimeric partner, Burs-β in the adult midgut. Unexpectedly, our evidence suggests that Burs-β is not significantly expressed in the adult midgut. burs-β mutants displayed the characteristic developmental defects but showed wild type-like adult midguts, thus uncoupling the developmental and adult phenotypes seen in burs-α mutants. Gain of function data and ex vivo experiments using a cAMP biosensor, demonstrated that Burs-α is sufficient to drive stem cell quiescence and to activate dLGR2 in the adult midgut. Our evidence suggests that the post developmental transactivation of dLGR2 in the adult midgut is mediated by Burs-α and that the β subunit of Bursicon is dispensable for these activities.

The crustacean hyperglycaemic hormone (CHH), a pleiotropic neuropeptide, belongs to a family of structurally related peptides, having six cysteine residues in conserved positions forming three disulphide bridges, and regulating several physiological processes in crustaceans and insects. Structure-activity studies have shown that amidation of the C-terminus is important to confer biological activity to CHH. In this study we investigated the function of the d-Phe(3) of the N-terminal motif of Pontastacus leptodactylus CHH by a mutational analysis. The d-Phe in position 3 was substituted by a d-Ala and the functionality of the mutated analogue (Glp-d-A-CHH) was tested by in vivo biological assays. The mutated analogue resulted far less active than its wild-type counterparts, either in d- (Glp-d-CHH) or l- (Glp-l-CHH) configuration. These results suggest that Phe(3) is essential for the biological activity of P. leptodactylus CHH, demonstrating that also the N-terminus is involved in the binding with the receptor, and identifying in the Phe(3) a hot spot for the peptide-receptor binding.

Molting is induced in decapod crustaceans via multiple leg autotomy (MLA) or eyestalk ablation (ESA). MLA removes five or more walking legs, which are regenerated and become functional appendages at ecdysis. ESA eliminates the primary source of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), which suppress the production of molting hormones (ecdysteroids) from the molting gland or Y-organ (YO). Both MLA and ESA are effective methods for molt induction in Gecarcinus lateralis. However, some G. lateralis individuals are refractory to MLA, as they fail to complete ecdysis by 12weeks post-MLA; these animals are in the "blocked" condition. Quantitative polymerase chain reaction was used to quantify mRNA levels of neuropeptide and mechanistic target of rapamycin (mTOR) signaling genes in YO, eyestalk ganglia (ESG), thoracic ganglion (TG), and brain of intact and blocked animals. Six of the seven neuropeptide signaling genes, three of four mTOR signaling genes, and Gl-elongation factor 2 (EF2) mRNA levels were significantly higher in the ESG of blocked animals. Gl-MIH and Gl-CHH mRNA levels were higher in the TG and brain of blocked animals and levels increased in both control and blocked animals in response to ESA. By contrast, mRNA levels of Gl-EF2 and five of the 10 MIH signaling pathway genes in the YO were two to four orders of magnitude higher in blocked animals compared to controls. These data suggest that increased MIH and CHH synthesis in the ESG contributes to the prevention of molt induction by MLA in blocked animals. The up-regulation of MIH signaling genes in the YO of blocked animals suggests that the YO is more sensitive to MIH produced in the ESG, as well as MIH produced in brain and TG of ESA animals. Both the up-regulation of MIH signaling genes in the YO and of Gl-MIH and Gl-CHH in the ESG, TG, and brain appear to contribute to some G. lateralis individuals being refractory to MLA and ESA.

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO2) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab−1) or 2 mgO2·L−1 (200 pmol·crab−1). Since luzindole (200 nmol·crab−1) did not significantly (p &gt, 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab−1) increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L−1, and luzindole (200 nmol·crab−1) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L−1 significantly (p &lt, 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab−1), 2 (200 and 20,000 pmol·crab−1) and 0.7 (200 or 20,000 pmol·crab−1) mgO2·L−1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.

Echinoderms are of special interest for studies in comparative endocrinology because of their phylogenetic position in the animal kingdom as deuterostomian invertebrates. Furthermore, their pentaradial symmetry as adult animals provides a unique context for analysis of the physiological and behavioral roles of peptide signaling systems. Here we report the first extensive survey of neuropeptide and peptide hormone precursors in a species belonging to the class Holothuroidea. Transcriptome sequence data obtained from the sea cucumber Apostichopus japonicus were analyzed to identify homologs of precursor proteins that have recently been identified in the sea urchin Strongylocentrotus purpuratus (class Echinoidea). A total of 17 precursor proteins have been identified in A. japonicus, including precursors of peptides related to thyrotropin-releasing hormone, pedal peptide/orcokinin-type peptides, AN peptides/tachykinins, luqins, corticotropin-releasing hormone (CRH), GPA2-type glycoprotein hormone subunits and bursicon. In addition, an unusual finding was an A. japonicus calcitonin-type precursor protein (AjCTLPP), the first to be discovered that comprises two calcitonin-like peptides; this contrasts with the products of the alternatively-spliced calcitonin/CGRP gene in vertebrates, which comprise either calcitonin or CGRP. Collectively, the data obtained provide new insights on the evolution and diversity of neuropeptides and polypeptide hormones. Furthermore, because A. japonicus is one of several sea cucumber species that are used for human consumption, our findings may have practical and economic impact by providing a basis for neuroendocrine-based strategies to improve methods of aquaculture.

Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.

Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral-ventral pacemaker neurons (LN(v)s) that secrete the neuropeptide PDF (pigment dispersing factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. Although LN(v)s also express functional PDFR, the role of these autoreceptors has remained enigmatic. Here, we show that (1) PDFR activation in LN(v)s shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions, and (2) this shift is mediated by stimulation of the Gα,s-cAMP pathway and a consequent change in PDF/neurotransmitter corelease from the LN(v)s. These results suggest another mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits.

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, we purified GSS from the radial nerves of the starfish Asterina pectinifera and identified the chemical structure as a relaxin-like peptide. This study examined the hormonal action of GSS on ovaries in the growing (stage IV) and fully grown states (stage V) of the starfish. The sensitivity of oocytes to 1-methyladenine (1-MeAde) as starfish maturation-inducing hormone was enhanced as oocytes enlarged in stage V. GSS-stimulated 1-MeAde production by ovarian follicle cells was also correlated with the size of oocytes. Although 1-MeAde production was observed in whole ovaries in stage V, GSS failed to induce 1-MeAde production in young ovaries (stage IV). This suggests that follicle cells in ovaries in a growing state (stage IV) are still unresponsive to the hormonal action of GSS. According to competitive experiments using radioiodinated and radioinert GSS, however, dissociation constant (K(d)) values and the number of binding sites for GSS were mostly constant in the ovaries from stages IV to V. These results strongly suggest that GSS receptors are expressed in follicle cells of ovaries in the growing state. The failure of GSS to induce 1-MeAde production in young ovaries may be due to the uncoupling of signal transduction from the receptor to 1-MeAde biosynthesis in follicle cells.

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.

The crustaceans produce several related peptides that belong to the crustacean hyperglycemic hormone (CHH) family. While these peptides have similar amino acid sequences, they have diverse biological functions that must arise, in part, from differences in the 3D shape of these peptides. However, it is generally accepted that peptides with a high degree of sequence similarity also have a similar 3-D structure. We used the solution structure of one peptide in the crustacean hyperglycemic hormone family, the molt-inhibiting hormone of the kuruma prawn (Marsupenaeus japonicus), to predict the shape of the five known peptides related to CHH in the Pacific white shrimp, Litopenaeus vannamei. The high similarity of the 3-D structures of these peptides suggests a common fold for the entire family. Nevertheless, minor differences in the shape of these peptides were observed, which may be the basis for their different biological properties.

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera , from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin.

SUMMARYCrustacean hyperglycaemic hormone from the pericardial organ (PO-CHH) is a CHH-related neuropeptide but its function and target tissues are not known in crustaceans. To investigate this issue, we employed radiolabelled ligand binding and cGMP assays, using eyestalk-CHH (ES-CHH) as a reference neuropeptide. The membranes were prepared from various tissues of Callinectes sapidus: hepatopancreas, hindgut, midgut, gills, heart,abdominal muscles and scaphognathites. Like ES-CHH, recombinant PO-CHH(rPO-CHH) specifically bound to the membranes of scaphognathites=abdominal muscles>midgut>gills> heart>hindgut and hepatopancreas (list order corresponds to the number of binding sites). The specific binding sites of 125I-ES-CHH in hepatopancreas and gills were saturable and displaceable. The abdominal muscle membrane binding sites were specific and saturable to both CHHs. These binding sites were displaced by homologous neuropeptides, but poorly displaced by the heterologous counterpart. As for the second messenger, the expected increment (3- to >20-fold) in the amount of cGMP produced by ES-CHH was noted in most tissues tested except midgut. Recombinant PO-CHH increased cGMP production 1.5- to 4-fold in scaphognathites, heart, midgut, hindgut and abdominal muscles. The results obtained from the binding study suggest that PO-CHH also has multiple target tissues of which abdominal muscles and scaphognathites are the primary ones. The differences in the primary amino acid sequences of PO-CHH and ES-CHH,particularly in the C-terminal region and in the amidation at C-terminus, may contribute to the truncated responses of hyperglycaemia, cGMP stimulation and binding affinity.

The neuropeptides of the crustacean hyperglycaemic hormone (CHH) family are encoded by a multigene family and are involved in a wide spectrum of essential functions. In order to characterize CHH family peptides in one of the last groups of decapods not yet investigated, CHH was studied in two anomurans: the hermit crab Pagurus bernhardus and the squat lobster Galathea strigosa. Using RT-PCR and 3' and 5' RACE methods, a preproCHH cDNA was cloned from the major neuroendocrine organs (X-organs) of these two species. Hormone precursors deduced from these cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of 29 and 31 amino acids, respectively, and CHH precursor-related peptides (CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hormone of 72 amino acids. The presence of these predicted CHHs and their related CPRPs was confirmed by performing MALDI-TOF mass spectrometry on sinus glands, the main neurohaemal organs of decapods. These analyses also suggest the presence, in sinus glands of both species, of a peptide related to the moult-inhibiting hormone (MIH), another member of the CHH family. Accordingly, immunostaining of the X-organ/sinus gland complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera showed the presence of distinct cells producing CHH and MIH-like proteins. A phylogenetic analysis of CHHs, including anomuran sequences, based on maximum-likelihood methods, was performed. The phylogenetic position of this taxon, as a sister group to Brachyura, is in agreement with previously reported results, and confirms the utility of CHH as a molecular model for understanding inter-taxa relationships. Finally, the paraphyly of penaeid CHHs and the structural diversity of CPRPs are discussed.

The aquaculture of crabs from the genus Scylla is of increasing economic importance for many Southeast Asian countries. Expansion of Scylla farming has led to increased efforts to understand the physiology and behavior of these crabs, and as such, there are growing molecular resources for them. Here, publicly accessible Scylla olivacea transcriptomic data were mined for putative peptide-encoding transcripts; the proteins deduced from the identified sequences were then used to predict the structures of mature peptide hormones. Forty-nine pre/preprohormone-encoding transcripts were identified, allowing for the prediction of 187 distinct mature peptides. The identified peptides included isoforms of adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon β, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, eclosion hormone, FMRFamide-like peptide, HIGSLYRamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide and tachykinin-related peptide, all well-known neuropeptide families. Surprisingly, the tissue used to generate the transcriptome mined here is reported to be testis. Whether or not the testis samples had neural contamination is unknown. However, if the peptides are truly produced by this reproductive organ, it could have far reaching consequences for the study of crustacean endocrinology, particularly in the area of reproductive control. Regardless, this peptidome is the largest thus far predicted for any brachyuran (true crab) species, and will serve as a foundation for future studies of peptidergic control in members of the commercially important genus Scylla.

During posteclosion, insects undergo sequential processes of wing expansion and cuticle tanning. Bursicon, a highly conserved neurohormone implicated in regulation of these processes, was characterized recently as a heterodimeric cystine knot protein in Drosophila melanogaster. Here we report the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the moth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit-specific protein labeling of burs and pburs as well as crustacean cardioactive peptide in neurons of the ventral nervous system were observed in pharate larval, pupal, and adult stages. A subset of bursicon neurons located in thoracic ganglia of larvae expresses ecdysis-triggering hormone (ETH) receptors, suggesting that they are direct targets of ETH. Projections of bursicon neurons within the CNS and to neurohemal secretory sites are consistent with both central signaling and circulatory hormone functions. Intrinsic cells of the corpora cardiaca contain pburs transcripts and pburs-like immunoreactivity, whereas burs transcripts and burs-like immunoreactivity were absent in these cells. Recombinant bursicon induces both wing expansion and tanning, whereas synthetic eclosion hormone induces only wing expansion.

Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal.

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in an X-organ sinus gland complex of crustacean eyestalks. GIH inhibits crustacean ovarian maturation by suppressing vitellogenin (Vtg) synthesis, whereas estrogen is responsible for the stimulation of vitellogenesis (not established). In this study, the effects of 17β-estradiol (E2, 10(-6) M), estrogen receptor antagonist tamoxifen (TAM, 10(-6), 10(-7), and 10(-8) M), and the environmental estrogen nonylphenol (NP, 1 μg/L and 100 μg/L) on LvGIH expression in the eyestalks of shrimp were determined by quantitative real-time PCR. Results showed that LvGIH expression decreased significantly during the L. vannamei ovarian maturation cycle. E2 and NP significantly reduced LvGIH transcripts in vivo, but TAM neutralized the inhibitory action of E2 in a dose-dependent manner (P < 0.05). In addition, the LvGIH expression levels decreased significantly in a time-dependent manner (P < 0.05) when ovary fragments were cultured in vitro with E2. The results of this study suggested that estrogen regulates GIH expression in L. vannamei eyestalks. E2 promoted ovarian development not only by directly upregulating vitellogenesis in the hepatopancreas, but it was also capable of downregulating LvGIH expression, which indirectly resulted in the stimulation of L. vannamei vitellogenesis.

Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed.

Serotonin, a highly conserved neurotransmitter, controls many biological functions in vertebrates, but also in invertebrates. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are commonly used in human medication to ease depression by affecting serotonin levels. Their residues and metabolites can be detected in the aquatic environment and its biota. They may also alter serotonin levels in aquatic invertebrates, thereby perturbing physiological functions. To investigate whether such perturbations can indeed be expected, shore crabs (Carcinus maenas) were injected either with serotonin, fluoxetine or a combination of both. Dose-dependent effects of fluoxetine ranging from 250 to 750nM were investigated. Gene expression of crustacean hyperglycemic hormone (chh) as well as moult inhibiting hormone (mih) was assessed by RT-qPCR at 2h and 12h after injection. Glucose and ecdysteroid levels in the haemolymph were monitored in regular intervals until 12h. Serotonin led to a rapid increase of chh and mih expression. On the contrary, fluoxetine only affected chh and mih expression after several hours, but kept expression levels significantly elevated. Correspondingly, serotonin rapidly increased glycaemia, which returned to normal or below normal levels after 12h. Fluoxetine, however, resulted in a persistent low-level increase of glycaemia, notably during the period when negative feedback regulation reduced glycaemia in the serotonin treated animals. Ecdysteroid levels were significantly decreased by serotonin and fluoxetine, with the latter showing less pronounced and less rapid, but longer lasting effects. Impacts of fluoxetine on glycaemia and ecdysteroids were mostly observed at higher doses (500 and 750nM) and affected principally the response dynamics, but not the amplitude of glycaemia and ecdysteroid-levels. These results suggest that psychoactive drugs are able to disrupt neuroendocrine control in decapod crustaceans, as they interfere with the normal regulation of the serotonergic system.

The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in controlling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript sequences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C-terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the peptide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males.

This study was aimed at determining the role of the crustacean hyperglycemic hormone (CHH) in the physiological compensation to both saline and thermal stress, in the freshwater crayfish Cherax quadricarinatus. By determining the expression of the CHH gene in the eyestalk of juvenile crayfish, we found that maximal induction of CHH was induced at high salinity (10 g/L) and low temperature (20 °C). In order to investigate the role of CHH in the physiological compensation to such stressful conditions, recombinant CHH was supplied to stressed animals. CHH-injected crayfish showed increased hemolymphatic levels of glucose, in accordance with a significant utilization of glycogen reserves from the hepatopancreas. Furthermore, CHH administration allowed stressed animals to regulate hemolymphatic sodium and potassium at more constant levels than controls. Taken together, these results suggest a relevant role of CHH in increasing the energy available intended for processes involved in the physiological compensation of C. quadricarinatus to both saline and thermal stress. Fil: Prymaczok, Natalia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Pasqualino, Valeria M.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Viau, Veronica Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Rodriguez, Enrique Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Medesani, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

Recombinant peptides related to vitellogenesis-inhibiting hormone (VIH) of the American lobster Homarus americanus were expressed in bacterial cells, and then purified after being allowed to refold. Biological activities of the recombinant VIHs having an amidated C-terminus (rHoa-VIH-amide) and a free carboxyl-terminus (rHoa-VIH-OH) were examined using an ovarian fragment incubation system derived from the kuruma prawn, Marsupenaeus japonicus. The rHoa-VIH-amide significantly reduced vitellogenin mRNA levels in the ovary, while rHoa-VIH-OH had no effect. This is the first report that describes the production of a crustacean VIH having biological activity and the importance of the C-terminal amidation for its vitellogenesis-inhibiting activity.

In crustaceans, eyestalk ablation induces gonadal maturation of which vitellogenin gene expression is an essential step. However, the molecular mechanisms by which the hormones produced by the X-organ/sinus gland complex in the eyestalk regulate vitellogenesis remain poorly understood. We therefore investigated the effects of sinus gland extracts and certain sinus gland peptides belonging to the crustacean hyperglycemic hormone peptide family on vitellogenin gene expression in ovarian fragments of immature kuruma prawn, Marsupenaeus japonicus. Vitellogenin mRNA levels in incubated ovarian fragments were significantly higher than those in unincubated ovarian fragments prepared from the same animal. Sinus gland extracts and sinus gland peptide-III (type I peptide) both reduced vitellogenin mRNA levels in a dose-dependent manner. In contrast, neither molt-inhibiting hormone (sinus gland peptide-IV) nor molt-inhibiting hormone B, both of which are type II peptides, exerted significant effects on vitellogenin mRNA levels. These results suggest that, in the immature ovary, sinus gland peptide-III is involved in the suppression of vitellogenin gene expression. The existence of such a peptide in the X-organ/sinus gland complex provides a rationale for the significant increase in vitellogenin mRNA levels in the ovaries of eyestalk-ablated prawns.

SUMMARYThis study investigates (by means of bioassays and ELISA using an antibody against recombinant cHH) the variation of cHH levels in the eyestalks and haemolymph of Palaemon elegans (Decapoda, Caridea) following exposure to various stresses (heavy metals and lipopolysaccharide), and correlates them with the variation in amount and time course of blood glucose. The dose-relationship between exposure to copper and quick release of cHH from the eyestalk into haemolymph was confirmed by variation of blood glucose with a dose-related hyperglycaemia, that peaked 2 h after immersion in contaminated seawater. Animals exposed to a sublethal concentration of mercury showed the same dose relation between toxicant, release of cHH from the eyestalk,increment of circulating hormone level and subsequent hyperglycaemia as observed for copper contamination. It is of note that although the highest lethal mercury concentration induced the release of cHH from the eyestalk into the haemolymph, it was not followed by a significant variation of blood glucose. Step doses of a bacterial contaminant [such as lipopolysaccharide(LPS) from E. coli injected into shrimps] confirmed the dose-relationship and convergent chain of events that bring about hyperglycaemia. These are the first data that relate the release of cHH from the eyestalk, the circulating hormone level and the consequent glycaemic response to stress. Moreover, they confirm the dose-related pathway that leads to variation of blood glucose as a quantitative biomarker of environmental quality, even at sublethal toxicant concentrations.

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.

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, 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 finding highlight the utility of membrane tethered inhibitors as important genetic/pharmacological tools to dissect the tissue specific roles of GPCRs in vivo.

Summary Background Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. Results Here we report a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts—beyond its known roles in development—as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2, the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. Conclusions We therefore identify a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells., Graphical Abstract, Highlights • Enteroendocrine cells control the local stem cell niche via Bursicon • Enteroendocrine cells express the Bursicon ligand, and the muscle its receptor, DLGR2 • Bursicon/DLGR2 signaling increases cAMP in visceral muscle to control EGF-like Vein • Bursicon signaling in the adult midgut instructs intestinal stem cell quiescence, Scopelliti et al. show that in the adult Drosophila midgut, enteroendocrine cells act locally—using Bursicon/DLGR2 signaling—to restrain stem cell proliferation via paracrine signaling to the mesenchymal stem cell niche.

The effects of a dinoflagellate parasite (Hematodinium sp.) on carbohydrate metabolism were examined in the Norway lobster, Nephrops norvegicus. Five stages of infection were observed. These included uninfected (Stage 0), subpatently infected (SP), and patently infected (Stage 1-4) lobsters. During patent infection, the concentration of glucose in the hemolymph was reduced significantly from its value of 180 microg ml(-1) in uninfected (Stage 0) lobsters to 25.3 microg ml(-1) in Stage 3-4. These changes were accompanied by significantly lower levels of hepatopancreatic glycogen in lobsters at Stage 2 (2.01 mg g(-1)) and Stage 3-4 (0.84 mg g(-1)) of infection than in those at Stage 0 (16.19 mg g(-1)) and Stage 1 (14.71 mg g(-1)). Due to disruption of the normal feedback loops which control the release of crustacean hyperglycemic hormone (CHH), plasma concentrations increased with the severity of infection from 32.2 fmol ml(-1) in Stage 0 to 106.6 fmol ml(-1) in Stage 3-4. The increased CHH concentrations occurred concomitantly with reduced concentrations of plasma glucose and tissue glycogen. A significantly increased hemolymph CHH titer (107.7 fmol ml(-1)) was also observed during SP infection. It is concluded that the parasite places a heavy metabolic load on the host lobster.

Injection of Ascaris FMRFamide-like (AF) peptides and peptides encoded by genes in Caenorhabditis elegans were analyzed for effects on locomotion, body waveforms, and cAMP concentrations in adult female Ascaris suum. Injection of AF1 (KNEFIRFamide) or AF2 (KHEYLRFamide) inhibited the propagation of locomotory waves and reduced the number of waveforms, decreased the body length, and caused a large, long-lasting increase in cAMP. Muscle tissue was identified as a major source of the cAMP response induced by AF1. The AF1 analog AF1R6A did not affect cAMP levels by itself, but inhibited the cAMP response produced by AF1. AF8 (KSAYMRFamide) produced ventral coiling in the behavioral assay, and AF10 (GFGDEMSMPGVLRFamide) decreased the body length and increased the number of body waveforms. In dorsal muscle strips, AF10 produced a long-lasting contraction. Neither AF8 nor AF10 changed cAMP concentrations. AF17 (FDRDFMHFamide) increased body length and decreased cAMP. The neuropeptides encoded by C. elegans genes flp-4, flp-7, flp-9, and flp-13 produced paralysis and loss of waveforms, increased body length and, like AF17, decreased cAMP. Three new predicted peptides from C. elegans genome sequences were synthesized and tested. One produced ventral coiling but no change in cAMP; the other two gave no detectable responses. The fact that C. elegans neuropeptides produce behavioral and physiological effects in A. suum suggests that structurally related peptides may exist in A. suum. The profound changes in cAMP produced by some neuropeptides has important implications for understanding cAMP signaling and shows that neuropeptide-mediated signal transduction pathways are potential targets for anthelmintic drug development.

Total extracts of sinus glands (SG) of the euryhaline grapsid crab Pachygrapsus marmoratus contain peptidic factor(s) that stimulate osmoregulatory processes in isolated and perfused posterior gills from crabs acclimated to dilute seawater. This study investigated the nature of the active factor(s). Separation of P. marmoratus SG peptides by reverse-phase HPLC, followed by a direct enzyme-linked immunosorbent assay using an anti-Carcinus maenas crustacean hyperglycemic hormone (CHH) antiserum, identified a major immunoreactive chromatographic peak. A glucose quantification bioassay demonstrated a strong and specific hyperglycemic activity following injection of the immunoreactive peak, therefore defined as the CHH of P. marmoratus. Isolated posterior gills were then perfused with HPLC fractions using a dose of 4 SG equivalents/assay. The CHH fraction consistently and significantly increased the transepithelial potential difference and Na(+) influx by about 50%. The effect was rapid and reversible. Another substance of unknown nature (eluted earlier than CHH in the HPLC gradient) caused a small increase in Na(+) influx (14%) but had no effect on the transepithelial potential difference. No other peptidic product from the SG had significant effect on the measured osmoregulatory parameters. These results indicate that CHH, in addition to its hyperglycemic activity, is also implicated in the control of branchial ionic transport. This neuropeptide may thus constitute a major factor involved in the control of osmoregulation in decapod crustaceans.

Molting or ecdysis is the most fundamentally important process in arthropod life history, because shedding of the exoskeleton is an absolute prerequisite for growth and metamorphosis. Although the hormonal mechanisms driving ecdysis in insects have been studied extensively, nothing is known about these processes in crustaceans. During late premolt and during ecdysis in the crab Carcinus maenas , we observed a precise and reproducible surge in hemolymph hyperglycemic hormone (CHH) levels, which was over 100-fold greater than levels seen in intermolt animals. The source of this hormone surge was not from the eyestalk neurosecretory tissues but from previously undescribed endocrine cells (paraneurons), in defined areas of the foregut and hindgut. During premolt (the only time when CHH is expressed by these tissues), the gut is the largest endocrine tissue in the crab. The CHH surge, which is a result of an unusual, almost complete discharge of the contents of the gut endocrine cell, regulates water and ion uptake during molting, thus allowing the swelling necessary for successful ecdysis and the subsequent increase in size during postmolt. This study defines an endocrine brain/gut axis in the arthropods. We propose that the ionoregulatory process controlled by CHH may be common to arthropods, in that, for insects, a similar mechanism seems to be involved in antidiuresis. It also seems likely that a cascade of very precisely coordinated release of (neuro) hormones controls ecdysis.

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

While much is known about the neural and endocrine mechanisms that control egg laying in the gastropod mollusk Aplysia, relatively little is known about the regulation of male reproductive activity in this simultaneous hermaphrodite. In the present study, we have cloned and sequenced a cDNA that encodes a precursor protein, the predicted posttranslational processing of which presumably generates nine copies of the neuropeptide Ala-Pro-Gly-Trp-NH2 (APGWamide), five connecting peptide sequences, and a C-terminal peptide. The sequence of one connecting peptide is identical to the previously characterized cerebral peptide 1. Northern blot analysis identified two major APGWamide mRNA transcripts (approximately 1.3 kb, approximately 2.4 kb), which were present in central nervous system ganglia, but were most abundant in the right cerebral and right pedal ganglia. Immunohistochemical studies using sexually mature Aplysia demonstrated that the vast majority of APGWamide-like immunoreactivity was localized in 30-40 neurons along the anterior and medial margins of the right cerebral ganglion and in a cluster of 15-20 neurons in the right pedal ganglion. A total of only about ten immunoreactive neurons were located in other ganglia. Immunohistochemistry also demonstrated that APGWamide was present in the reproductive organs that participate in the storage or transport of sperm, including the small hermaphroditic duct (site of sperm storage before mating), the white hemiduct (also known as the copulatory duct), and penial complex. As a group, these data suggest that APGWamide may play a role in regulating male reproductive function in Aplysia, as it does in other gastropods.

Eight vertebrate and invertebrate hormones were screened for their effect on population growth, mictic female production, and body size of the marine rotifer Brachionus plicatilis. Growth hormone (GH) or human chorionic gonadotropin (HCG) at 0.0025–25 I.U. ml−1 and estradiol-17s (E2), triiodothyronine (T3), 20-hydroxyecdysone (20-HE), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) or juvenile hormone (JH) at 0.05–50 mg 1−1 were added to 5-ml of Nannochloropsis oculata suspension (7 × 106 cells m1−1). From an initial density of 1 individual m1−1, rotifers were cultured with hormones for 48 hours in 22 ppt seawater at 25 °C, in darkness. Rotifers were counted and classified into female types and transferred to a new algal food suspension without hormone every other day until day 8 when body size was measured.

Blood ingestion by the female mosquito initiates a series of endocrine events that is dominated by juvenile hormone, ecdysteroids, and several peptide hormones, resulting in the maturation of a batch of eggs. The mechanisms of hormone release and their roles during the previtellogenic, vitellogenic, and postvitellogenic phases are discussed. Arch. Insect Biochem. Physiol. 35:491–512, 1997. © 1997 Wiley-Liss, Inc.

Reich, G., K. E. Doble, D. A. Price and M. J. Greenberg. Effects of cardioactive peptides on myocardial cAMP levels in the snail Helix aspersa. Peptides 18 (3) 355–360, 1997.—Several cardioactive peptides from the pulmonate snail Helix aspersa were tested for their effects on myocardial cAMP levels, but only the family of small cardioactive peptides (SCPs) were clearly effective. SCP increased cAMP in a dose dependent manner; the time course was phasic. The structure-activity relations of this effect were examined with a set of 3 synthetic analogs having characteristics, at the carboxyterminal, of both the SCPs and FMRFamide-related peptides. The adenylate cyclase activator forskolin mimicked the mechanical effect of SCPs on the heartbeat. We conclude that the effect of SCPs on the Helix heart may be mediated by cAMP.

Hyperglycemia that is caused by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland to hemolymph is one of the hallmark physiological phenomena, occurring in decapod crustaceans experiencing stressful conditions. However, the mechanism(s) by which such elevated glucose levels return to resting levels is still unknown. Interestingly, noted is a difference in the clearance rate of hemolymph glucose between adult females and adult males of the blue crab, Callinectes sapidus: the former with more rapid clearance than the latter. The presence of an endogenous-insulin-like molecule is suggested in C. sapidus because an injection of bovine insulin, significantly reduces the levels of hemolymph glucose that were previously elevated by emersion stress or the glucose injection. Using 5' and 3' RACE, the full-length cDNA of an insulin-like molecule is isolated from the hepatopancreas of an adult female C. sapidus and shows the same putative sequence of an insulin-like androgenic gland factor (IAG) but differs in 5' and 3' UTR sequences. A knock-down study using five injections of double-stranded RNA of CasIAG-hep (dsRNA-CasIAG-hep, 10μg/injection) over a 10-day period reduces CasIAG-hep expression by ∼50%. The levels of hemolymph glucose are also kept higher in dsRNA-CasIAG-hep injected group than those treated with dsRNA-green fluorescent protein (dsRNA-IAG-hep) or saline. Most importantly, the hepatopancreas of dsRNA-CasIAG-hep injected animals contains amounts of carbohydrate (glucose, trehalose, and glycogen) significantly lower than those of control groups, indicating that the function of CasIAG-hep in carbohydrate metabolism in crustaceans is similar to carbohydrate metabolism in vertebrates.

1. Modulatory effects of the four molluscan neuroactive peptides. FMRFamide (Phe-Met-Arg-Phe-NH2), APGW-amide (Ala-Pro-Gly-Trp-NH2), oxytocin and [SER2]-Mytilus inhibitory peptide ([SER2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH2) were examined on the inward current (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), which has been isolated from the Achatina ganglia. 2. Two Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone), were used. Achatin-I was applied locally to the neurone tested by brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfused around the ganglia. 3. FMRFamide, perfused at 3 microM, suppressed markedly the Iin elicited by the achatin-I of both v-RCDN and PON. APGW-amide at 3 microM also suppressed the Iin of v-RCDN, but did not affect that of PON. Oxytocin at 1 microM suppressed the Iin of PON, but did not affect that of v-RCDN. [Ser2]-MIP at 3 microM did not affect the Iin of v-RCDN. 4. The dose-response curves of FMRFamide, APGW-amide and oxytocin, indicated that their respective suppressive effects on the Iin of achatin-I were dose-dependent, and that APGW-amide was slightly more potent than the other peptides. The dose (pressure duration)-response curves of achatin-I (1 kg/cm2, 10(-3) M, 5 min interval), obtained by varying the duration of the achatin-I pressure ejection, were measured in the presence and absence of each of the three peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

When a larva of the red flour beetle Tribolium freemani (Hinton) was introduced into a vial, the larva pupated within 6 days, whereas when two larvae were introduced, the larvae pupated in 12 days. Crowded larvae delayed production of juvenile-hormone esterase (JHE) and release of ecdysteroids, as measured by radioimmunoassay, compared with the isolated larvae. The highest JHE activity was observed at pupal ecdysis in both groups and there were increases in whole-body ecdysteroid levels during the last part of intermoult before pupation and adult emergence, which coincided with decreases in ecdysteroid titres in both isolated and crowded groups. Cold shock, heat stress, the JHE inhibitor methamidophos, and juvenile hormone (JH) I delayed pupation in isolated larvae. Cold shock, heat stress and methamidophos reduced the JHE activity, and cold shock and JH I reduced ecdysteroid levels in isolated larvae. Light, vibration and a nonsteroidal ecdysteroid agonist RH 5849 stimulated pupation in crowded T. freemani larvae, which was antagonized by JH I. The JHE activity and ecdysteroid levels were higher in crowded larvae reared under 24 h light or treated with vibration, octopamine agonists and precocene II than those control larvae kept in constant darkness.

The objectives of the present study were to characterize the changes in crustacean hyperglycemic hormone (CHH) transcript and peptide levels in response to infection of white spot syndrome virus (WSSV) in a crustacean, Procambarus clarkii. After viral challenge, significant increase in virus load began at 24 h post injection (hpi) and the increase was much more substantial at 48 and 72 hpi. The hemolymph CHH levels rapidly increased after viral challenge; the increase started as early as 3 hpi and lasted for at least 2 d after the challenge. In contrast, the hemolymph glucose levels did not significantly changed over a 2 d period in the WSSV-infected animals. The CHH transcript and peptide levels in tissues were also determined. The CHH transcript levels in the eyestalk ganglia (the major site of CHH synthesis) of the virus-infected animals did not significantly change over a 2 d period and those in 2 extra-eyestalk tissues (the thoracic ganglia and cerebral ganglia) significantly increased at 24 and 48 hpi. The CHH peptide levels in the eyestalk ganglia of the virus-infected animals significantly decreased at 24 and 48 hpi and those in the thoracic ganglia and cerebral ganglia remained unchanged over a 2 d period. These data demonstrated a WSSV-induced increase in the release of CHH into hemolymph that is rapid in onset and lasting in duration. Changes in the CHH transcript and peptide levels implied that the WSSV-induced increase in hemolymph CHH levels primarily resulted from an enhanced release from the eyestalk ganglia, but the contribution of the 2 extra-eyestalk tissues to hemolymph pool of CHH increased as viral infection progressed. The combined patterns of change in the hemolymph glucose and CHH levels further suggest that the virus-enhanced CHH release would lead to higher glycolytic activity and elevated glucose mobilization presumably favorable for viral replication.

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.

The bag cell neurons of the marine mollusk Aplysia have been used extensively for investigating the electrophysiology and molecular biology of neurosecretory cells. However, there has been little attempt to carefully describe the pattern of secretion of the 36-amino acid peptide hormone that controls egg-laying behavior. This egg-laying hormone (ELH) is secreted in response to a highly characteristic pattern of action potential firing called the afterdischarge. The purpose of this study was to document the pattern of ELH secretion in response to the bag cell afterdischarge. To accomplish this goal, we developed the first RIA for measurement of ELH. The study was conducted in vitro in excised neural preparations that included the bilateral bag cell clusters; preparations were maintained in static culture of filtered artificial sea water (ASW) at 21-22 C. Afterdischarges were electrically stimulated, and action potentials were monitored via extracellular electrodes. The entire volume of ASW was collected every 5 min and immediately replaced with fresh ASW. In all 20 preparations, ELH levels were low to undetectable before stimulation of the afterdischarge; ELH rose above baseline within 5-10 min of the start of the afterdischarge, reached peak levels near the end or after the end of the afterdischarge, and then remained elevated long after action potentials had ceased firing. These results indicate a temporal dissociation between action potential firing and secretion. We suggest that the afterdischarge is important for triggering intracellular events that culminate in peptide release. Importantly, once these secretion-inducing events have been initiated, they can continue in the absence of further action potential stimulation.

Some old and recent views concerning the mode of action of insect juvenile hormone (JH) have been reviewed. The original, 60-year-old hormone concepts were based on the qualitative, inhibitory role of JH in insect metamorphosis. After 1950, insect endocrinologists created a model of quantitative JH mode of action, on the basis of findings on epidermal transplantations in Galleria. According to this model, a single epidermal cell would produce larval characters at high concentrations of JH, pupal characters at intermediate concentrations, whereas in the absence of JH it would give rise to adult epidermal patterns. This simple model of JH action became the leading concept of insect endocrinology until this time. However, direct experimental proof that qualitative structural changes between the juvenile and adult stage are controlled by the JH concentration has never been provided. Moreover, essential aspects of the concept, such as 'reversal of metamorphosis', appear to be completely illusive. Alternative views, explaining the mode of action ofJH by inhibition of morphogenesis in some genetically determined ontogenetic periods have been presented. According to these views JH cannot cause the 'reversal of metamorphosis' and its action at the cellular level would proceed according to the common 'all-or-none' rule.

Double labelling experiments were performed on the same tissue section at the electron microscopic level, in order to show the involvement of the opioid leucine-enkephalin (Leu-enk) in the modulation of crustacean hyperglycaemic hormone (CHH) mobilization. Both neuropeptides were stored in distinct axon terminals of the sinus gland of Carcinus maenas. A post-embedding immunogold cytochemical technique for Leu-enk, CHH and the CHH neurohormone related moult inhibiting hormone (MIH) was combined with a scintillator intensified autoradiographic method to demonstrate binding of the opioid antagonist [3H] naloxone. Ultrathin sections were successively incubated with antisera against Leu-enk, CHH or MIH, and the corresponding colloidal gold labelled antisera, followed by autoradiographic processing. At the ultrastructural level [3H] naloxone binding sites were easily recognized by their silver tracks after development. Opioid binding sites for [3H] naloxone were visualized only at membranes of CHH-containing axon terminals. These results provide morphological evidence for direct enkephalinergic control of CHH containing neurons in the sinus gland of C. maenas and are furthermore the first autoradiographic demonstration of opioid binding sites in the nervous system of invertebrates.

The binding properties of the crustacean hyperglycemic hormone (CHH) of two species, the shore crab, Carcinus maenas (Brachyura) and the crayfish, Orconectes limosus (Astacura), were investigated using purified plasma membranes of one of the main target organs, the hepatopancreas. Assays were performed under equilibrium binding conditions with 125I-CHH as labeled ligand and unlabeled CHH in increasing concentrations as displacing ligand. In both cases, comparable binding characteristics were observed for the homologous CHH to the respective hepatopancreatic membrane source, indicating saturable and displaceable binding with nonlinear dependence on monovalent cations and a dissociation constant (Kd) of 4 to 6 x 10(-10) M. Binding capacity was in the range of 200-400 fmol/mg membrane protein. In heterologous displacement studies a certain degree of species specificity was found, particularly with regard to selective binding by the Orconectes receptor, suggesting that the group specificity of biological activity of CHH reflects coevolution of both hormone and receptor.

HPLC separation of corpus cardiacum homogenates from Tabanus lineola, T. proximus and T. calens all produced two peaks whose retention times corresponded to the retention times of the synthetic Tabanus atratus adipokinetic and T. atratus hypotrehalosaemic hormones. Corpus cardiacum extracts from T. proximus and from T. lineola produced hyperlipaemic and hyperglycaemic responses when injection into sucrose fed T. lineola. Synthetic T. atratus hypotrehalosaemic hormone induced strong hyperlipaemic and hyperglycaemic responses in T. lineola, but synthetic T. atratus adipokinetic hormone produced only a weak hyperglycaemic response. The current names of the tabanid metabolic peptides do not correspond to their action in T. lineola. Octopamine also elevated both lipids and sugars in the blood of sucrose fed T. lineola, indicating the sensitivity of storage tissues to both biogenic amines and to metabolic peptides. Lack of response to synthetic T. atratus hypotrehalosaemic by starved T. lineola suggests an inhibition of the hyperlipaemic/glycaemic response when the available tissue reserves falls below a given level.

Prothoracic glands of the locus, Locusta migratoria, incubated in vitro converted tritiated 3-dehydrocetodiol (22,23,24,25 3H4-14 alpha-hydroxy-5 beta-cholest-7-en-3,6-dione) into ecdysteroids and 3-dehydroecdysteroids as far as the final products of the two series, ecdysone and 3-dehydroecdysone. In the two series, the different compounds are formed in the same quantities, except for 2,22-desoxy-products, the nature of which could not have been determined. Converted 3-dehydroecdysone issued from 3-dehydrocetodiol is transformed into ecdysone after several hours incubation with Locusta last instar larvae hemolymph. Till now is has been impossible to determine if the reduction of 3-dehydroecdysteroids took place into the prothoracic glands or in the incubation medium. In no case is 3-dehydrocetodiol converted into cetodiol. Conversion rates of the different compounds, either issued from cetodiol or from 3-dehydrocetodiol as precursors, are of same importance, so that a weak specificity of the hydroxylation enzymes must be considered.

Crustacean hyperglycemic hormone (CHH) is a polypeptide neurohormone involved in regulation of multiple physiological processes. We report here the cloning from thoracic ganglia of the blue crab (Callinectes sapidus) a cDNA (CsCHH-2) encoding a putative CHH isoform (CsCHH-2). CsCHH-2 is structurally similar to a putative preproCHH (CsCHH-1) previously cloned from eyestalk ganglia of C. sapidus. The two preprohormones possess an identical signal peptide and CHH precursor related peptide, but differ in the mature CHH polypeptide. An analysis by RT-PCR of the tissue distribution of CsCHH-1 and CsCHH-2 revealed the former is restricted to eyestalk neural ganglia, while the latter is widely distributed among tissues. The type of CHH transcript present in eyestalk and thoracic ganglia did not vary as a function of the molt cycle. An assessment of transcript abundance in tissues of intermolt crabs showed the abundance of the CsCHH-1 transcript in eyestalk ganglia far exceeds the abundance of the CsCHH-2 transcript in extra-eyestalk tissue. An assessment of transcript abundance during a molt cycle showed CsCHH-1 transcript abundance in eyestalk ganglia was low during intermolt, rose during premolt, reaching a peak in D(3), then fell prior to molting, and remained low during postmolt. By contrast, CsCHH-2 transcript abundance in thoracic ganglia was low during intermolt, rose sharply during D(2), then dropped in D(3) and remained low during postmolt. The results are consistent with the hypothesis that CsCHH-1 and CsCHH-2 differ with respect to physiological function.

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 BAG), 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 BAGand located within the subesophageal ganglion inDrosophila(the BSEG) is involved in controlling wing expansion behaviors. Unlike the BAG, the BSEGarborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the BAG.Indeed, we show that animals lacking bursicon receptor function have deficits both in the humoral release of bursicon and in posteclosion apoptosis of the BAG. Our results reveal novel neuromodulatory functions for bursicon and support the hypothesis that the BSEGare essential for orchestrating both the behavioral and somatic processes underlying wing expansion.