Hydromineral regulation in the hydrothermal vent crab Bythograea thermydron.

This study investigates the salinity tolerance and the pattern of osmotic and ionic regulation of Bythograea thermydron Williams, 1980, a brachyuran crab endemic to the deep-sea hydrothermal vent habitat. Salinities of 33 per thousand-35 per thousand were measured in the seawater surrounding the captured specimens. B. thermydron is a marine stenohaline osmoconformer, which tolerates salinities ranging between about 31 per thousand and 42 per thousand. The time of osmotic adaptation after a sudden decrease in external salinity is about 15-24 h, which is relatively short for a brachyuran crab. In the range of tolerable salinities, it exhibits an iso-osmotic regulation, which is not affected by changes in hydrostatic pressure, and an iso-ionic regulation for Na(+) and Cl(-). The hemolymph Ca(2+) concentration is slightly hyper-regulated, K(+) concentration is slightly hyper-hypo-regulated, and Mg(2+) concentration is strongly hypo-regulated. These findings probably reflect a high permeability of the teguments to water and ions. In addition to limited information about salinity around hydrothermal vents, these results lead to the hypothesis that B. thermydron lives in a habitat of stable seawater salinity. The osmoconformity of this species is briefly discussed in relation to its potential phylogeny.     

Quadrupole time-of-flight mass spectrometry of the native hemocyanin of the deep-sea crab Bythograea thermydron

Since electrospray ionization mass spectrometry (ESI-MS) has demonstrated capabilities for observing intact, weak interactions, there has been increasing interest in studying by this method noncovalently bound complexes. In this communication, we report for the first time the structure obtained by a commercial ESI quadrupole time-of-flight spectrometer on a native hemocyanin of deep-sea crab Bythograea thermydron with a molecular mass of 1.3 MDa. ESI-MS analysis of the native hemocyanin revealed the formation of a 18-mer noncovalent assembly with a measured molecular mass of 1354940 +/- 480 Da. ESI-MS data also revealed that this huge structure is an equilibrium with several assemblages, dodecamer (measured molecular weight = 902570 +/- 110 Da), hexamer (measured molecular weight = 450310 +/- 260 Da), and monomeric structures (measured molecular weight = 74999 +/- 85 Da).     

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.     

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.     

Lipid composition of deep-sea hydrothermal vent tubeworm Riftia pachyptila, crabs Munidopsis subsquamosa and Bythograea thermydron, mussels Bathymodiolus sp. and limpets Lepetodrilus spp

Lipid composition was determined for hydrothermal vent species collected by the Deep Submergence Vehicle ALVIN from chimneys at 2,500 m depth on the East Pacific Rise. These are the first lipid biomarker studies for most of these species. Lipid content was low and dominated by polar lipid in the vestimentiferan tubeworm Riftia pachyptila, mussels Bathymodiolus sp. and limpets Lepetodrilus spp. The galatheid (Munidopsis subsquamosa) and most brachyuran adult (Bythograea thermydron) crabs were characterized by higher storage lipid (triacylglycerol). Total polyunsaturated fatty acids were similar in R. pachyptila plume and body, but higher in the posterior part of the soft body, which had more docosahexaenoic acid (2-5% of total FA) compared to the anterior and plume (< or =0.3%). Two sulphur-oxidizing bacterial markers, 16:1(n-7)c and 18:1(n-7)c, were high in R. pachyptila and mussel (up to 23%), but lower in both crab species (4-17%). R. pachyptila had greater nonmethylene interrupted diunsaturated fatty acids (8-13%) than all other species (2-8%). R. pachyptila may desaturate and elongate 18:1(n-7)c to obtain essential polyunsaturated fatty acids 20:5(n-3) and 20:4(n-6). The sterol composition of R. pachyptila included similar amounts of cholesterol and desmosterol, whereas the other species had a more diverse sterol composition. These differences in lipids, fatty acids and sterols reflect diverse nutritional strategies and possibly temperature regimes in these species.     

The crustacean hyperglycemic hormones from an euryhaline crab Pachygrapsus marmoratus and a fresh water crab Potamon ibericum: eyestalk and pericardial isoforms

The structures of crustacean hyperglycemic hormones (CHH) were investigated in two crabs, the coastal euryhaline crab Pachygrapsus marmoratus and the fresh water crab Potamon ibericum. The neuropeptide mRNAs were extracted from pericardial and X-organs (PO and XO), and the sequences of the cDNA encoding the hormones' precursors were determined. The X-organ preprohormones are composed of 29 and 28 amino acid signal peptides in P. marmoratus and P. ibericum respectively, followed by 43 and 41 amino acid crustacean hyperglycemic hormone precursor related peptide (CPRP) flanking the 72 amino acid crustacean hyperglycemic hormones. A similar organization is reported for pericardial preprohormones with identical sequences for the signal peptide, the CPRP and the N-terminal sequences of CHH (1-40), but remaining sequences (41-72 and 41-71) differing considerably. In P. marmoratus two CHH cDNAs were characterized from XO and evidences were obtained for the existence of at least two forms in the PO. From our results and by comparison with other known sequences, a consensus pattern for crab pericardial CHH could be pointed out. Analysis of the data presented in this article using phylogenetic methods reveals that the two crab species studied are much closer than previously predicted.     

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 9 kDa 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.     

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.     

Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus

Crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) are produced during the proteolytic processing of CHH preprohormones. Currently, the physiological roles played by CPRPs are unknown. Due to their large size, direct mass spectrometric sequencing of intact CPRPs is difficult. Here, we describe a novel strategy for sequencing Cancer productus CPRPs directly from a tissue extract using nanoflow liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Four novel CPRPs were characterized with the aid of MS/MS de novo sequencing of 27 truncated CPRP peptides. Extensive modifications (methionine oxidation and carboxy-terminal methylation) were identified in both the full-length and truncated peptides. To investigate the origin of the modifications and truncations, a full-length CPRP was synthesized and subjected to the same storage and extraction protocols used for the characterization of the native peptides. Here, some methionine oxidation was seen, however, no methylation or truncation was evident suggesting much of the chemical complexity seen in the native CPRPs is unlikely due to a sample preparation artifact. Collectively, our study represents the most complete characterization of CPRPs to date and provides a foundation for future investigation of CPRP function in C. productus.     

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.     

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.     

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.     

Structure of the exopolysaccharide of Vibrio diabolicus isolated from a deep-sea hydrothermal vent

The structure of the exopolysaccharide produced under laboratory conditions by Vibrio diabolicus, a bacterium recovered from a deep-sea hydrothermal vent, has been investigated using sugar and methylation analysis and NMR spectroscopy. The polysaccharide consists of a linear tetrasaccharide repeating unit with the following structure. -->3)-beta-D-Glcp Nac-(1-->4)-beta-D-Glcp A-(1-->4)-beta-D-Glcp A-(1-->4)-alpha-D-Galp NAc-(1-->     

Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii

Heptapeptide (YANAVQV-NH2 = T-) and octapeptide (YANAVQTV-NH2 = T+), the putative C-terminus of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, was synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization in swiss mice. Specificity of the antisera against both peptides was determined by indirect immunoperoxidase ELISA. The best response of antiserum against each peptide was used to determine the presence of the natural CHH in the eyestalk extract after separation by one step of RP-HPLC using dot-ELISA. The peptide immunoreactive substances were found in fraction 30 using anti-T- antiserum and in fraction 38 using anti-T+ antiserum. However, the CHH activity was found only in fractions 37-39. Immunocytochemical localization of peptide immunoreactive substances in the eyestalk of M. rosenbergii using the anti-T- antiserum did not show any specific staining. In contrast, the anti-T+ antiserum revealed specific staining on a group of 24 +/- 5 neurons in medulla terminalis ganglionic x-organ and their processes through the sinus gland. Similar results were also obtained using the eyestalk of another species, the giant tiger prawn Penaeus monodon, in which 34 +/- 4 neuronal cells were recognized. These results strongly indicate that the anti-T+ antibody can bind to the natural CHH while the anti-T- antibody can not; therefore, this isoform of CHH in M. rosenbergii should consist of 72 residues and threonine is predicted to be present at position 71.     

Food sources for the early life history stages of the hydrothermal vent crab <Emphasis Type="Italic">Bythograea thermydron</Emphasis>: a stable isotope approach

We used stable isotope analysis to examine food sources for early life-history stages of the vent crab Bythograea thermydron. During two cruises to a hydrothermal vent site along the East Pacific Rise, we collected a variety of endemic organisms, including approximately 300 specimens of B. thermydron. The crab collection consisted mainly of megalopae and early juveniles, but also included adults and a single zoea. As expected, the carbon-isotope composition of newly released zoeae (−12.2%) was similar to the female (−11.1%) and clearly different from megalopae (21.7%). Because we were unsuccessful in culturing the zoea larvae, we were not able to conduct experiments to determine the effect of diet on isotopic composition. The tissue of megalopae was depleted in ¹³C (−21.7%) when compared to indigenous prey species, but was similar to that expected for carnivorous zooplankton dependent on surface primary production. The nitrogen-isotope composition of megalopae was enriched in ¹⁵N (10%) relative to potential prey species, again suggesting a photosynthetic source of primary production. The mean carbon-isotope composition of newly metamorphosed juvenile crabs (−19.9%) resembled megalopae, while tissue from subsequent juvenile stages was enriched in ¹³C (−10.9%) with values similar to those measured in co-occurring prey species. These results imply that megalopae are dependent on a source of primary production exogenous to the vents, but switch to a vent-based food web soon after metamorphosis to the juvenile stage.     

The crustacean hyperglycemic hormone (CHH) releases amylase from the crayfish midgut gland

The present study aimed to investigate the role of eyestalk factors in the neuroendocrine control of the crustacean midgut gland concerning the release of amylase. The crustacean hyperglycemic hormone (CHH) is considered to be a candidate for this role. An optimum concentration (1.05 nM) CHH increased the in vitro release of amylase about 13-fold. CHH from Carcinus only slightly increased amylase release from Orconectes midgut glands, suggesting a species- or group-specificity. Studies on the possible mechanism of action concentrated on the role of Ca2+, cAMP and cGMP. Extracellular Ca2+ seems to be necessary to produce the amylase-releasing effect of CHH. Addition of dibutyryl derivatives of the cyclic nucleotides evoked the same effect as CHH. Additionally, the presence of forskolin in the incubation medium had an amylase-releasing effect, which points to a role of cAMP in the mode of action.     

Effect of a glycine residue insertion into crustacean hyperglycemic hormone on hormonal activity

Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family. All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs. In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed. This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations. The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude. These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.     

An extremely thermophilic Methanococcus from a deep sea hydrothermal vent and its plasmid

An extremely thermophilic methanogen was isolated from a hydrothermal vent core sample from Guaymas Basin, Gulf of California, at a depth of 2003 m. The isolate, designated strain AG86, was a coccoid autotroph using H₂-CO₂ as energy and carbon source with a growth temperature range of 48 to 92°C, optimum, 85°C. AG86 required NaCl and Mg²⁺ and trace amounts of selenite and tungstate. Vitamins were not required. However, yeast extract, Casamino acids and Trypticase stimulated growth significantly. When grown in the presence of these stimulants and at the optimal growth temperature and pH 6.5, the minimum doubling time was 20 min. Cells were fragile and readily lysed by detergents. The mol% G+C was 33%. These results and partial 16S rRNA sequencing indicated that AG86 belonged to the genus Methanococcus and closely resembled Methanococcus jannaschii. Tests for extrachromosomal DNA revealed a plasmid in AG86 and two plasmids in M. jannaschii. Different patterns were obtained from restriction endonuclease digestion of the three plasmids, and no homology was observed with DNA-DNA hybridization.Abbreviations CCC DNA covalently close circular DNA - DM defined marine medium - G+C Guanine plus cytosine - MPN most probable number     

Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.