Crustacean Hyperglycemic Hormone Research Articles

Bursicon and neuropeptide cascades during the ecdysis program of the shore crab, Carcinus maenas

Very little is known regarding the release patterns of neuropeptides involved in ecdysis of crustaceans compared to insects. In particular, the dynamics of release of the insect cuticle hardening hormone bursicon, which has only recently been discovered in crustaceans, is unknown. Bursicon has not previously been identified as a circulating neurohormone in these animals. Since patterns of release were likely to be ephemeral, bursicon, as well as two other neurohormones involved in the ecdysis program in crustaceans, crustacean cardioactive peptide (CCAP) and crustacean hyperglycaemic hormone (CHH) were measured in single haemolymph samples in Carcinus maenas. For bursicon, an ultrasensitive time resolved-fluoroimmunoassay (TR-FIA) was developed, which firstly involved its characterisation by HPLC, bioassay and immunoassay. Simultaneous measurement of three neurohormones was performed at unparalleled levels of resolution, which has not previously been reported in any invertebrate. Additionally, expression patterns and architecture of neurones expressing both bursicon and CCAP were determined in the CNS during the moult cycle. Bursicon and CCAP are released in a massive surge, likely a single global exocytotic event on emergence, just after release of CHH. Despite co-localisation of CCAP and bursicon in neurones of the CNS, observations suggest that differential packaging of CCAP can occur in the pericardial organs in a small population of secretory boutons, thus accounting for observations showing release of some CCAP during the penultimate stages of the ecdysis program. The results obtained vividly illustrate the dynamism of neuropeptide cascades occurring during crustacean ecdysis, and also allow proposal of a hypothesis of its endocrine control.

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8-9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.

Neuroendocrine responses of a crustacean host to viral infection: Effects of infection of white spot syndrome virus on the expression and release of crustacean hyperglycemic hormone in the crayfish Procambarus clarkii

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 24h post injection (hpi) and the increase was much more substantial at 48 and 72hpi. The hemolymph CHH levels rapidly increased after viral challenge; the increase started as early as 3hpi and lasted for at least 2d 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 48hpi. The CHH peptide levels in the eyestalk ganglia of the virus-infected animals significantly decreased at 24 and 48hpi 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.

Crustacean hyperglycemic hormone (cHH) as a modulator of aggression in crustacean decapods.

Biogenic amines, particularly serotonin, are recognised to play an important role in controlling the aggression of invertebrates, whereas the effect of neurohormones is still underexplored. The crustacean Hyperglycemic Hormone (cHH) is a multifunctional member of the eyestalk neuropeptide family. We expect that this neuropeptide influences aggression either directly, by controlling its expression, or indirectly, by mobilizing the energetic stores needed for the increased activity of an animal. Our study aims at testing such an influence and the possible reversion of hierarchies in the red swamp crayfish, Procambarus clarkii, as a model organism. Three types of pairs of similarly sized males were formed: (1) ‘control pairs’ (CP, n = 8): both individuals were injected with a phosphate saline solution (PBS); (2) ‘reinforced pairs’ (RP, n = 9): the alpha alone was injected with native cHH, and the beta with PBS; (3) ‘inverted pairs’ (IP, n = 9): the opposite of (2). We found that, independently of the crayfish’s prior social experience, cHH injections induced (i) the expression of dominance behaviour, (ii) higher glycemic levels, and (iii) lower time spent motionless. In CP and RP, fight intensity decreased with the establishment of dominance. On the contrary, in IP, betas became increasingly likely to initiate and escalate fights and, consequently, increased their dominance till a temporary reversal of the hierarchy. Our results demonstrate, for the first time, that, similarly to serotonin, cHH enhances individual aggression, up to reverse, although transitorily, the hierarchical rank. New research perspectives are thus opened in our intriguing effort of understanding the role of cHH in the modulation of agonistic behaviour in crustaceans.

The ex vivo effects of eyestalk peptides on ovarian vitellogenin gene expression in the kuruma prawn Marsupenaeus japonicus

Seven major peptides belonging to the crustacean hyperglycemic hormone family were purified from the sinus gland located in the eyestalk of the kuruma prawn Marsupenaeus japonicus, and their effects on vitellogenin gene expression were examined using the ex vivo ovary incubation system. Six molecular species of crustacean hyperglycemic hormone, Pej-SGP-I, -II, -III, -V, VI, and VII, displayed significant inhibitory effects on vg expression with almost the same efficacies, whereas Pej-SGP-IV (known as molt-inhibiting hormone) did not. Two chromatophorotropic peptides, red pigment-concentrating hormone and pigment-dispersing hormone, which were also present in the sinus glands, did not have a clear effect on the gene expression levels in this incubation system. These results suggest that the six crustacean hyperglycemic hormones are potentially capable of acting as vitellogenesis-inhibiting hormones in M. japonicus.

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.

Changes in crustacean hyperglycemic hormones in Pacific whiteleg shrimp Litopenaeus vannamei subjected to air-exposure and low-salinity stresses

Changes in crustacean hyperglycemic hormone (CHH)-family peptides in response to stress were investigated in Litopenaeus vannamei. Stress treatments consisted of air exposure and low salinity. High-performance liquid chromatography was used to quantify CHH-family peptides in the X-organ–sinus gland complex (XO–SG) in the eyestalks. Among the CHH-family peptides analyzed, only the level of sinus gland peptide-G (SGP-G) in the XO–SG was decreased. SGP-G was also detectable by Western blotting analysis in the hemolymph of animals subjected to stress. These results suggest that SGP-G was secreted from the XO–SG into the hemolymph during stress. Glucose levels in the hemolymph increased under conditions during which SGP-G was detected in the hemolymph. Hyperglycemia was also observed when SGP-G was injected. SGP-G may function to shift energy use to deal with stress.

Decreased level of crustacean hyperglycemic hormone (CHH) in black tiger shrimp Penaeus monodon suffering from Monodon Slow-Growth Syndrome (MSGS)

Monodon Slow-Growth Syndrome (MSGS), a pathological condition in the black tiger shrimp Penaeus monodon, is associated with Laem–Singh virus (LSNV) infection. Infected shrimp grow slowly when the virus invades the part of the retina called the zona fasciculata. Since molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) are synthesized in the optic lobe and are related to molting activity and growth of the shrimp, the purpose of this study is therefore to determine the levels of these hormones and related parameters in MSGS shrimp. P. monodon juveniles were sampled from normal and MSGS ponds and were divided into small-negative, large-negative, small-positive and large-positive groups, depending on the size of the shrimp and whether they were LSNV-negative or LSNV-positive. Individual shrimp were measured for duration of each molt stage and molt interval. Levels of MIH1 and CHH1 transcripts were determined from the optic lobe, using real-time reverse transcriptase–polymerase chain reaction. The results revealed that the small-positive and the small-negative shrimp did not differ in durations of molt stages or molt intervals, as well as on the levels of MIH1 transcript in the optic lobe, MIH1 peptide in the optic lobe and hemolymph, or ecdysteroids in the hemolymph. Differences in molting activities and related transcript and hormones were observed between the small- and large-sized shrimp, but not in the status of LSNV infection. While levels of CHH1 transcript in the optic lobe of both small-negative and small-positive shrimp did not differ, levels of CHH1 peptide, as well glucose, in the hemolymph of the small-positive shrimp were significantly lower than those of the small-negative group. Levels of glycogen in the hepatopancreas of the small-positive shrimp were also significantly higher than that of the small-negative ones. The results suggest that growth retardation in MSGS shrimp is related to the suppression of the release of CHH1 peptide by LSNV invasion in the zona fasciculata, consequently causing decreased hepatopancreatic glycogenolysis and persistent hypoglycemia, resulting in growth stunting.

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): Cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.

A chimeric recombinant crustacean hyperglycemic hormone fromLitopenaeus schmitti(Burkenroad) obtained as C-terminus fusion protein boost hemolymph glucose concentration inLitopenaeus vannamei(Boone)

To date, no hormonal treatments are available for control of shrimp reproduction and only eyestalk ablation is of practical use. The crustacean hyperglycemic hormone (CHH) is the most abundant neuropeptide of the eyestalk CHH family. It plays an important role in the regulation of hemolymph glucose levels, as its principal function, but it is also implicated in additional physiological processes such as moulting and reproduction. In the present study, the cDNA encoding Litopenaeus schmitti (Burkenroad) mature CHH was cloned into the Escherichia coli pTYB2 expression vector. Using this strategy we have obtained, for the first time, the recombinant CHH from L. schmitti with its C-terminus fused to an intein tag. The expected fused protein of about 63 KDa was expressed in E. coli forming inclusion bodies. It was purified in a soluble form by electroelution following molecular size fractionation in sodium dodecil sulphate polyacrylamide gel electrophoresis. The ability of the chimeric CHH protein to elevate glucose levels in the hemolymph of the eyestalk-ablated Litopenaeus vannamei (Boone) shrimps indicates that its biological activity as hyperglycemic protein is preserved. The results provide an alternative tool to obtain soluble recombinant proteins from the CHH family of neuropeptides to get a better understanding of shrimp endocrinology.

Natural and synthetic chiral isoforms of crustacean hyperglycemic hormone from the crayfish Astacus leptodactylus: Hyperglycemic activity and hemolymphatic clearance

In the crayfish Astacus leptodactylus, as in several crustacean species, the crustacean hyperglycemic hormone is present as two isoforms differing by the chirality of the third residue, a phenylalanine. In the present work, isoforms synthesized full length by solid-phase peptide synthesis have been purified, refolded, the location of the disulfide bridges has been checked, their immunoreactivity against different antibodies have been analyzed and their hyperglycemic activity tested, to ensure the identity of the synthetic peptides with their natural homologs. Different parameters of the hyperglycemic activity of both isoforms were studied. In addition to a difference in the kinetics of hyperglycemia, already known from other studies, it was observed that the dose–response was different depending on the season where experiments were performed, the response being stronger in spring than in autumn, especially for the d-Phe containing isoform. A dosage method based on sandwich enzyme linked immunosorbent assay (ELISA) has been developed to measure hemolymphatic levels of the isoforms after spiking of the animals with one isoform or the other. It was found that hemolymphatic clearance was identical for both isoforms, indicating that their differential effect is not linked to their different lifetime in the hemolymph but may rather rely on other mechanisms such as their binding to different target tissues.

Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes

Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8±86.7fmol/mg protein) and SGCs (497.8±49.4fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed.

Novel Protocol for the Chemical Synthesis of Crustacean Hyperglycemic Hormone Analogues — An Efficient Experimental Tool for Studying Their Functions

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe3 and (Glp1, D-Phe3) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe3 modifies the cHH functionality, contributing to the diversification of the hormone pool.

Roles of crustacean hyperglycaemic hormone in ionic and metabolic homeostasis in the Christmas Island Blue crabDiscoplax celeste

There is a growing body of evidence implicating the involvement of crustacean hyperglycaemic hormone (CHH) in ionic homeostasis in decapod crustaceans. However, little is known regarding hormonally influenced osmoregulatory processes in terrestrial decapods. As many terrestrial decapods experience opposing seasonal demands upon ionoregulatory physiologies, we reasoned that these would make interesting models in which to study the effect of CHH upon these phenomena. In particular, those (tropical) species that also undergo seasonal migrations might be especially informative, as we know relatively little regarding the nature of CHHs in terrestrial decapods, and hormonally mediated responses to seasonal changes in metabolic demands might also be superimposed or otherwise integrated with those associated with ionic homeostasis. Using Discoplax celeste as a model crab that experiences seasonal extremes in water availability, and exhibits diurnal and migratory activity patterns, we identified two CHHs in the sinus gland. We biochemically characterised (cDNA cloning) one CHH and functionally characterised (in terms of dose-dependent hyperglycaemic responses and glucose-dependent negative feedback loops) both CHHs. Whole-animal in situ branchial chamber (22)NaCl perfusion experiments showed that injection of both CHHs increased gill Na(+) uptake in a seasonally dependent manner, and (51)Cr-EDTA clearance experiments demonstrated that CHH increased urine production by the antennal gland. Seasonal and salinity-dependent differences in haemolymph CHH titre further implicated CHH in osmoregulatory processes. Intriguingly, CHH appeared to have no effect on gill Na(+)/K(+)-ATPase or V-ATPase activity, suggesting unknown mechanisms of this hormone's action on Na(+) transport across gill epithelia.

Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-β-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction.

Crystallization and preliminary X-ray analysis of crustacean hyperglycaemic hormone from the kuruma prawn Marsupenaeus japonicus in its weakly active precursor form.

Crustacean hyperglycaemic hormone (CHH) plays a pivotal role in the regulation of glucose metabolism in crustaceans. Pej-SGP-I, one of the six known CHHs in the kuruma prawn Marsupenaeus japonicus, was heterologously expressed in Escherichia coli as an N-terminally His-tagged and Nus-tagged protein in its weakly active precursor form, Pej-SGP-I-Gly, which has an extra glycine residue at the C-terminus. The recombinant peptide was subjected to affinity purification, tag removal, further purification and crystallization by the sitting-drop vapour-diffusion method using NaCl as the main precipitant. The crystals diffracted to 1.95 Å resolution and the space group was assigned as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 40.19, b = 53.65, c = 53.63 Å. The Matthews coefficient (V(M) = 1.73 Å(3) Da(-1)) indicated that the crystal contained two Pej-SGP-I-Gly molecules per asymmetric unit, with a solvent content of 29.0%.

Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques.

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules-neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.

Molt-inhibiting hormone from Chinese mitten crab (Eriocheir sinensis): Cloning, tissue expression and effects of recombinant peptide on ecdysteroid secretion of YOs

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 1457bp with a 330bp 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 0.01–1nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01–100nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation.

GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons

In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones.

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography–tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.