Demonstration of the cellular expression of genes encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in the eyestalk of the shore crab Carcinus maenas

Based on the amino acid sequence of the molt-inhibiting hormone of Carcinus maenas, two degenerated oligonucleotide primers were synthesized and used in the polymerase chain reaction. By use of complementary DNA of a library constructed from medulla terminalis-X-organ RNA of C. maenas as template, the specific complementary DNA between the primers was amplified, cloned and sequenced. This strategy revealed a DNA sequence for which the deduced amino acid sequence is identical to the recently published C. maenas molt-inhibiting hormone sequence as determined by Edman degradation. Visualization of messenger RNAs encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in different perikarya of the X-organ was obtained using digoxigenin-labelled complementary RNA probes. Combination of immunocytochemical staining using polyclonal antisera against the native C. maenas neuropeptides and in situ hybridization performed on alternating sections confirmed the specificity of the reaction. The results show that there is no co-localization of molt-inhibiting hormone and crustacean hyperglycemic hormone at the messenger RNA and the protein level.     

Structure of the male reproductive system of the blue swimmer crab Portunus pelagicus (Decapoda: Portunidae)

An attempt was made here to study the structure of the male reproductive system of Portunus pelagicus, which would improve the knowledge base on the reproductive biology of the species and also help in the maintenance of broodstock under controlled conditions. Male P. pelagicus of different sizes were collected from the Palk Bay off Mandapam (9°17′ N, 79°9′ E) and maintained under controlled conditions for the study. Tissues from testis, anterior vas deferens (AVD), median vas deferens (MVD), posterior vas deferens (PVD), ejaculatory duct and penis were fixed in Bouin's fluid and 2.5% buffered glutaraldehyde separately and processed for light and electron microscopic studies, respectively. The reproductive system consisted of testis, commissure, vas deferens, ejaculatory duct and penis. The vas deferens was divided based on the morphology and/or histology into AVD, MVD and PVD. The AVD was further divided based on histology into proximal and distal regions, and the MVD, based on diameter into major and minor coils. The testicular lobe had several lobules with a central seminiferous tubule, which continued till the penis. The seminiferous tubule was lined by a layer of cuboidal or columnar epithelium. The lining of the central tubule of the vas deferens formed several ‘folds’, which at times formed ‘pouches’. High incidence of cell organelles in the columnar epithelial cells, aggregations of vesicles and occurrence of blebs at the luminal periphery and the projection of numerous microvilli containing electron‐dense materials into the lumen from the cell lining denoted high secretory activity of the epithelial cells.     

Induced thermal stress on serotonin levels in the blue swimmer crab,Portunus pelagicus

The temperature of habitat water has a drastic influence on the behavioral, physiological and biochemical mechanisms of crustaceans. Hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. In crustaceans increased circulating crustacean hyperglycemic hormone (CHH) and hyperglycemia are reported to occur following exposure to several environmental stress. The biogenic amine, serotonin has been found to modulate the CHH levels and oxidation of serotonin into its metabolites is catalysed by monoamine oxidase. The blue swimmer crab, Portunus pelagicus is a dominant intertidal species utilized throughout the indo-pacific region and is a particularly important species of Palk bay. It has high nutritional value and delicious taste and hence their requirements of capture and cultivation of this species are constantly increasing. This species experiences varying and increasing temperature levels as it resides in an higher intertidal zone of Thondi coast. The present study examines the effect of thermal stress on the levels of serotonin and crustacean hyperglycemic hormone in the hemolymph of P. pelagicus and analyzes the effect of the monoamine oxidase inhibitor, pargyline on serotonin and CHH level after thermal stress. The results showed increased levels of glucose, CHH and serotonin on exposure to 26 °C in control animals. Pargyline injected crabs showed highly significant increase in the levels of CHH and serotonin on every 2 °C increase or decrease in temperature. A greater CHH level of 268.86±2.87 fmol/ml and a greater serotonin level of 177.69±10.10 ng/ml was observed at 24 °C. This could be due to the effect of in maintaining the level of serotonin in the hemolymph and preventing its oxidation, which in turn induces hyperglycemia by releasing CHH into hemolymph. Thus, the study demonstrates the effect of thermal stress on the hemolymph metabolites studied and the role of pargyline in elevating the levels of serotonin and CHH on thermal stress in the blue swimmer crab, P. pelagicus.     

Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone

Crustacean hyperglycemic hormone (CHH) from Carcinus maenas, a 72 amino acid neuropeptide, originates in neurosecretory perikarya in the eyestalk ganglia. Poly (A)RNA was isolated from these perikarya and a cDNA library was prepared. Screening of 20,000 clones with a 26-mer oligonucleotide, corresponding to a partial sequence of CHH, yielded one positive clone with an insert of approximately 2,000 bp, which contained the complete coding sequence for a pre-pro CHH. This precursor consists of a putative 26 amino acid signal sequence, a 38 amino acid peptide of unknown function (Peptide C), and the CHH sequence at the carboxyl end. The CHH-sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by the tetrapeptide Gly-Arg-Lys-Lys which is followed by the stop codon.     

Ovarian maturation and oogenesis in the blue swimmer crab,Portunus pelagicus (Decapoda: Portunidae)

The study was aimed at understanding the process of reproduction and the changes happening in the ovary of Portunus pelagicus during maturation, which would be useful for its broodstock development for hatchery purposes. For that, tissue samples from different regions of the ovary at various stages of maturation were subjected to light and electron microscopy, and based on the changes revealed and the differences in ovarian morphology, the ovary was divided into five stages such as immature (previtellogenic oocytes), early maturing (early vitellogenic oocytes), late maturing (late vitellogenic oocytes), mature (vitellogenic oocytes), and spent (resorbing oocytes). The ovarian wall comprised of an outermost thin pavement epithelium, a middle layer of connective tissue, and an innermost layer of germinal epithelium. The oocytes matured as they moved from the centrally placed germinal zone toward the ovarian wall. The peripheral arrangement of nucleolar materials and the high incidence of cell organelles during the initial stages indicated vitellogenesis I. Movement of follicle cells toward oocytes in the early maturing stage and low incidence of mitochondria and endoplasmic reticulum in the ooplasm during late vitellogenic stage marked the commencement and end of vitellogenesis II, respectively. Yolk granules at various stages of development were seen in the ooplasm from late vitellogenic stage onwards. The spent ovary had an area with resorbing oocytes and empty follicle cells denoting the end of one reproductive cycle and another area with oogonial cells and previtellogenic oocytes indicating the beginning of the next.     

Regulation of circulating levels of the crustacean hyperglycemic hormone: evidence for a dual feedback control system

The effects of glutamate, aspartate, glycine, proline, alanine, taurine, glycerol, glucose and lactate injections on the haemolymph levels of the crustancean hyperglycemic hormone and/or glucose and lactate in the shore crab, Carcinus maenas, were investigated. Only glucose and lactate caused significant changes of hyperglycaemic hormone levels. Glucose injections resulted in a drop of both hormone and lactate, while lactate had an opposite effect, i.e. it raised both crustacean hormone and glucose levels. The results suggest that during increases in glycolytic flux, lactate may cause a release of hormone by a positive feedback mechanism. The hormone would then stimulate glycogenolysis, thus increasing glucose availability. If more glucose is released than is metabolized, excess glucose may leak from the cells and suppress crustancean hyperglycemic hormone release from the X-organ/sinus gland complex by negative feedback.Abbreviations ABTS 2,2-azino-bis (3-ethylbenzthiazoline sulphonic acid) - ANOVA one-way analysis of variance - BSA bovine serum albumin - BW body weight - CHH crustacean hyperglycemic hormone - ELISA cnzyme-liked immunosorbent assay - GIH gonadinhibiting hormone - IgG immunoglobin G - MIH moult-inhibiting hormone - MTGXO medulla terminalis X-organ - PB sodium phosphate buffer - PBS phosphate buffered saline - Pi inorganic phosphate - XO-SG X-organ-sinus gland complex     

Structure and phylogeny of the crustacean hyperglycemic hormone and its precursor from a hydrothermal vent crustacean: the crab Bythograea thermydron

The structure of a well-known neurohormone involved in homeostasis regulation and stress response, the crustacean hyperglycemic hormone, was investigated in the deep-sea hydrothermal vent crab Bythograea thermydron. The neuropeptide was isolated from neurohemal organs (sinus glands) and its biological activity checked using an homologous bioassay. Partial amino acid sequence was established by a combination of Edman chemistry and mass spectrometry. Then, the sequence of the cDNA encoding the hormone precursor was determined. The preprohormone is composed of a 29 amino acid signal peptide, followed by a 41 amino acid associated peptide flanking the 72 amino acid hyperglycemic hormone. Comparison of these data with other known crab hyperglycemic hormone and prohormone sequences was performed using phylogenetic analysis methods.     

Molt cycle related changes and effect of short term starvation on the biochemical constituents of the blue swimmer crab Portunus pelagicus

Synthesis and hardening of a new exoskeleton are essential to the arthropod molting process. The present study emphasizes the variations in the levels of hemolymph total free sugars, hepatopancreas glycogen and cuticular proteins during the molting stages of Portunus pelagicus. It also reports the effect of short-term starvation conditions on the biochemical constituents of the hemolymph. Intermolt crabs were subjected to 6 days of starvation and hemolymph samples were taken. Standard biochemical procedures were followed toward the quantification of total proteins, total free sugars and total lipids. The total free sugar level in the hemolymph of P. pelagicus was observed to increase during early premolt D₀ (3.108 ± 0.032 g/ml) and a gradual decrease till late postmolt B stage (0.552 ± 0.124 g/ml), suggesting the need for total free sugars to provide energy for the apolysis process. Increase in the levels of hepatopancreas glycogen was observed from 1225 ± 0.04 μg/mg in early premolt D₀ to 1700 ± 0.3 μg/mg in late premolt D_2–3. This is in correlation with the decreased levels of free sugars during premolt stages, suggesting an increase in the storage of glycogen reserves in the hepatopancreas. Cuticular proteins increased during stage B (2.702 ± 0.093 g/ml) and stage C (3.065 ± 0.012 g/ml), indicating exoskeleton hardening and mineralization. Results of the starvation studies clearly showed a steady decline in the level of total free sugars till day 6 (0.099 ± 0.00 g/ml) when compared to the control (8.646 ± 0.08 g/ml). Gradual decrease of total lipids was also observed from the first day of the experiment (6.088 ± 2.44 g/ml) to the last day of the study (0.401 ± 0.20 g/ml) which was 85% lesser than the control (8.450 ± 0.49 g/ml)suggesting the efficient usage of total sugars to consolidate the loss of energy reserves during starvation. The knowledge of Molt-cycle events can be used as a tool for the evaluation of the developmental state providing a morphological reference system for physiological and biochemical studies related to crab aquaculture. Starvation studies enlightens that increasing carbohydrate levels in crab feed together with good protein content could alleviate the natural effects of starvation, improve farm productivity and reduce the deleterious impact of nitrogen pollution generated by rich-protein feeds used in crab farming.     

Functional relations of crab molt-inhibiting hormone and neurohypophysial peptides

Biological and immunological relationships between molt-inhibiting hormone (MIH) activity in eyestalk ganglia extracts of the crab, Cancer antennarius Stimpson, and peptides of the vasopressin-oxytocin family were assessed. Lysine vasopressin (LVP), arginine vasopressin (AVP), vasotocin (VT), and oxytocin (OT) mimicked MIH action by inhibiting ecdysteroid production of Y-organ segments in vitro with the relative potencies LVP greater than AVP greater than VT much much greater than OT. The inhibitory effect was reversible and specific (6 other peptides did not alter Y-organ activity). MIH and LVP increased Y-organ cyclic adenosine 3',5' monophosphate (cAMP) levels dose-dependently and with identical time course in which the rise in cAMP preceded inhibition of ecdysteroid production. The synthetic vasopressin antidiuretic agonist 1-deamino-8-D-AVP (dDAVP) inhibited Y-organ steroidogenesis dose-dependently; the vasopressin analog ([1(B-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine[AVP) (d(CH2)5Tyr(Me)AVP), a vasopressor antagonist, had no effect on basal or MIH-suppressed steroidogenesis. AVP antiserum abolished the inhibitory action of MIH, LVP, and AVP. Competitive binding curves for MIH, LVP, AVP, VT, and OT with the AVP antiserum suggested that MIH is most closely related to LVP. MIH may be structurally related to the vasopressins and act on Y-organ cells via type V2 (cAMP-linked) receptors.     

Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.     

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background  

Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton.     

Positive Darwinian selection on crustacean hyperglycemic hormone (CHH) of the green shore crab, Carcinus maenas

The tissue-specific expression and differential function of the crustacean hyperglycemic hormone (CHH) in Carcinus maenas indicate an interesting evolutionary history. Previous studies have shown that CHH from the sinus gland X-organ (XO-type) has hyperglycemic activity, whereas the CHH from the pericardial organ (PO-type) neither shows hyperglycemic activity nor it inhibits Y-organ ecdysteroid synthesis. Here we examined the types of selective pressures operating on the variants of CHH in Carcinus maenas. Maximum likelihood-based codon substitution analyses revealed that the variants of this neuropeptide in C. maenas have been subjected to positive Darwinian selection indicating adaptive evolution and functional divergence among the CHH variants leading to two unique groups (PO and XO-type). Although the average ratio of nonsynonymous to synonymous substitution (omega) for the entire coding region is 0.5096, few codon sites showed significantly higher omega (10.95). Comparison of models that incorporate positive selection (omega > 1) with models not incorporating positive selection (omega <1) at certain codon sites failed to reject (p=0) evidence of positive Darwinian selection.     

Structure-activity relationship of crustacean molt-inhibiting hormone from the kuruma prawn Marsupenaeus japonicus

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

三疣梭子蟹蜕皮抑制激素cDNA的克隆与序列分析

甲壳动物的蜕皮是由位于头胸部前鳃腔的一对Y-器通过分泌蜕皮激素（Molting hormone）来控制的（Lachaise et al．,1993）,而蜕皮激素的分泌又受到蜕皮抑制激素（Molt-inhibiting hormone,MIH）的调控（Watson et al．,2001）。MIH和性腺抑制激素（Gonad-inhibiting hormone,GIH）、甲壳动物高血糖激素（Crustacean hyperglycemic hormone,CHH）、     

Molecular evolution of the crustacean hyperglycemic hormone family in ecdysozoans

Background  

Crustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated.     

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect,Carausius morosus

Summary Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.