Mobilization of copper among tissues in the estuarine crab Scylla serrata (Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.     

Susceptibility of testicular cell cultures of crab, Scylla serrata (Forskal) to white spot syndrome virus

Testicular cell culture of crab, Scylla serrata (Forskal) was used to study the effects of White spot syndrome virus (WSSV). We are showing the susceptibility of cell culture of crabs to WSSV. The proliferating cell culture of testes were maintained for more than 4 months in a medium prepared from L15 and crab saline supplemented with epidermal growth factor. The cell cultures inoculated with different concentrations of virus showed distinct cytopathic effects such as change in cell appearance, shrinkage and cell lysis. WSSV infection of cultured cells was confirmed by Nested PCR technique. The incorporation of viral DNA in cultured cells was shown by RAPD profile generated using 10-mer primers. The controls that were not exposed to WSSV did not show cytopathic effects. This work shows the usefulness of proliferating testicular cell culture for studying WSSV infection using molecular tools. Thus, this report gains significance as it opens new vistas for diagnostics and drugs for WSSV.     

Histochemical characteristics of spermatophore layers of Scylla serrata (Forskal) (Decapoda: Portunidae)

The spermatophores of S. serrata are protected by an outer thick chitinous layer and an inner thin non-chitinous one. Both layers are rich in acid mucopolysaccharides containing sulphated (outer layer) and carboxylic groups (inner layer). The proteins of the two layers show much tryptophan, but lack tyrosyl, sulfhydryl and disulphide groups. No phenols or phenol oxidases could be detected histochemically in either layer, suggesting the absence of phenolic tanning in the spermatophore. The physical properties, as revealed from treatment with acids and alkali, indicate the resistant nature of the outer layer; the inner layer easily shrinks or disrupts under such treatment. The outer layer, though resistant, is readily permeable to low molecular weight dye substances employed in permeability experiments. The mechanism of sperm release is recorded and discussed. It is suggested that, in S. serrata, the dehiscence of spermatophore may be caused by imbibing of low molecular weight substances by the sperm mass substances of the spermatophore while the latter is inside the spermatheca.     

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50–200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ?OmpA and ?OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.     

Immunotoxic effects of nickel in the mud crab Scylla serrata

The presence of xenobiotic contaminants especially metals in coastal waters is a major concern as they are immunotoxic to aquatic animals even at low concentrations. In our present study, mud crab Scylla serrata was exposed to three sublethal concentrations (0.4, 0.6 and 0.8 mg/L) of nickel for 30 days under laboratory conditions and the alterations of hematological parameters like haemocyte count, clotting time, haemocyte viability, protein content and immunomodulatory components like phenoloxidase, phagocytosis and superoxide anion generation were measured. In addition, the accumulation patterns of nickel were measured in gills, hepatopancreas and ovary. The accumulation was more in gills when compared to hepatopancreas and ovary of crabs exposed to nickel and was not detected in the control crabs. The results revealed a significant (P < 0.05) induction of superoxide anion generation and phagocytosis activity in the haemolymph of the crabs exposed to nickel when compared to control. On the contrary, the rest of the parameters were significantly (P < 0.05) reduced in the experimental groups when compared to the control. All the studied parameters exhibited a concentration dependent response.     

The breeding cycle of Scylla serrata (Forskal, 1755) at Ramisi River estuary,Kenya

The mud crab Scylla serrata was collected from Ramisi river estuaryfor a period of 14 months (January 1990–February 1991) using madema traps. For each crab, the carapace width (mm), sex and weightwere noted, and the number of ovigerous female crabs was recorded. Both ovigerous and non-ovigerous females were dissected in order to observeof the maturity stages of ovaries. The ovarian maturity stages weredescribed as stage zero – virgin/resting, stage one – developing, stage two– well developed and stage thre e– ripe. The most abundant was stage twothroughout the study period. The smallest ovigerous crab had a carapacewidth of 139 mm. A test of homogeneity of the binomial distributionof the sex ratio showed homoscedasticity (² = 14.615; d.f. = 13; p> 0.05) and the overall sex ratio did not differ from 1:1 (² = 0.776; d.f. = 1; p> 0.05). The variance test ofhomogeneity of the binomial distribution of sex in relation to size showeda very significant heterogeneity (² = 32.83; d.f. = 9; p< 0.05). There was no significant difference when the overall mean sizesfor males and females were compared using t-test (t = 4.26; d.f. = 18;p< 0.001). The relatively high numbers of females with stage two ovariesindicated that spawning took place throughout the year with a possible peakin the second half of the year.     

Variants in the histochemical patterns of lipids in the hepatopancreas of Scylla serrata (Forskal) (Brachyura, Decapoda).

The highest concentrations of phospholipid, neutral lipid and fatty acids were observed in the R cells and connective tissue of the hepatopancreas of Scylla serrata. The basal parts of the B cells and apical parts of the cells lining the main duct also showed moderate presence of these substances. The E cells however, except at their cell membranes were found to be devoid of lipids. F cells on the other hand exhibited lipoid complexes. Considerable reduction in the staining intensity of fatty acids were noticed 4 h after the bilateral ablation of eyestalks, neutral lipid undergo depletion 24 h after the operation whereas phospholipid reserves increase 48 h after the eyestalk removal. A fall in the quantity of neutral lipid and phospholipid was conspicuous when eyestalk extract was injected into normal or destalked crabs. From the present data it appears that R cells and connective tissue form major sites of lipid storage and in an intermolt animal eyestalk factor(s) may have an important role in the control of lipid metabolism.     

Lagenidium infection in eggs and larvae of mangrove crab (Scylla serrata) produced in Indonesia

A fungal infection occurred in the eggs and larvae of mangrove crab (Scylla serrata) in seed production in Bali, Indonesia. The causative fungus was classified as a member of the genusLagenidium (Oomycetes, Lagenidiales). After comparison of its biological and physiological characteristics with those ofL. callinectes ATCC 24973, a known parasite of various crustaceans, was concluded that the isolate is a new species ofLagenidium, L. thermophilum, because of its rapid and thermotolerant growth and unique discharge process. Fungal growth was observed on PYG agar containing 0–5.0% (w/v) NaCl and 0–2.5% (w/v) KCI. Similar pathogenicity toward the zoeae of swimming crab (Portunus trituberculatus) was demonstrated.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by vanadate

Green crab (Scylla serrata) alkaline phosphatase is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by vanadate has been studied. The time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the enzyme in the presence of different Na3VO4 concentrations showed that, at each Na3VO4 concentration, the rate decreased with increasing time until a straight line was approached, the slopes of the straight lines being the same for all concentrations. The results suggest that the inhibition of the enzyme by Na3VO4 is a slow, reversible reaction with fractional residual activity. The microscopic rate constants were determined for the reaction of the inhibitor with the enzyme. As compared with Na2HPO4 (Ki = 0.95 mM), Na2HAsO4 (Ki = 1.10 mM), and Na2WO4 (Ki = 1.55 mM), the results suggest that Na3VO4 (Ki = 0.135 mM) is a considerably more potent inhibitor than other inhibitors.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Purification and some properties of beta-N-acetyl-D-glucosaminidase from viscera of green crab (Scylla serrata)

Beta-N-acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50 degrees C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45 degrees C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 +/- 0.012 mM and Vmax of 17.65 +/- 0.32 micromol/min at pH 5.8 and 37 degrees C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na+ and K+ have no effects on the enzyme activity; Mg2+ and Ca2+ slightly activate the enzyme, while Ba2+, Zn2+, Mn2+, Hg2+, Pb2+, Cu2+, and Al3+ inhibit the enzyme to different extents.     

Effects of controlled pH on organic and inorganic composition in haemolymph,epidermal tissue and cuticle of mud crab Scylla serrata

Analysis of organic and inorganic compounds in plasma, epidermal tissue and cuticle were accomplished in the intermolt (C3 stage) of crab Scylla serrata incubated in different pH media. Significant changes with similar trends for protein, carbohydrates, glycosaminoglycans (GAG), sulphur, calcium, magnesium, potassium, phosphorus and copper in the plasma suggested higher dissolution in an acidic medium while the deposition increased in alkaline medium. Similar decreases in protein, carbohydrate and GAG in the epidermal compartment were observed from pH 4 to pH 12. However, significantly higher contents of sodium, chloride, potassium, phosphorus, magnesium, sulphur and copper were measured at pH 7.5 with a symmetrical decrease profile in both acidic and alkaline media, resulting from synergistic effects in the osmotic regulation. Clear changes in calcium concentrations were observed with a sharp increase from lower contents at pH 7.5 to higher at pH 12. In the cuticle, the acidic condition induced a significant dissolution of HCl-protein, GAG, calcium and magnesium contents. On the other hand, the alkaline condition induced a significant decrease in carbohydrate, calcium, chloride, sulphur and potassium. A reduction trend is seen for NaOH and H(2)O-protein contents in the cuticle. These observations suggest that GAG and HCl-protein might constitute the most soluble fraction with high affinity for calcium binding and easily removed in acidic conditions. Additionally, it is possible to speculate that the carbohydrates associated with the NaOH and H(2)O-proteins may form an interface between the soluble matrix fraction and the chitin framework. Sulphur groups seem to present a strong linkage role in this interface fraction, maybe only broken by a specific enzyme in extreme alkaline conditions with subsequent release of significant calcium from the shell.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.     

Histochemical observations on the occurrence of glycolytic and pentose phosphate cycle enzymes in the hepatopancreas and their possible relation to eyestalk factor(s) in the crab Scylla serrata (Forskal).

Histochemical studies were carried out on some of the glycolytic enzymes viz. phosphorylase, aldose, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and lactic dehydrogenase (LDH) and a key enzyme of the pentose phosphatase cycle, glucose-6-phosphate dehydrogenase (G-6-PDH), in the hepatopancreas of Scylla serrata (Forskal). 1. Weak activities of phosphorylase and aldolase and strong-activities of alpha-GPDH and LDH were noticed mainly in the brush border of the tubules and R-cell cytoplasm. A trace activity of G-6-PDH was noticed in the brush border. 2. Bilateral eyestalk removal results in inhibition of both phosphorylase and aldolase. However, enhanced activities of alpha-GPDH and LDH were noticeable 4 h after the operation. The G-6-PDH activity remained unaltered till 24 h. 3. Injection of eyestalk extract into both intact and destalked crabs activated all the enzymes.     

Inhibitory kinetics of mercuric ion on the activity of beta-N-acetyl-D-glucosaminidase from green crab (Scylla serrata)

Chemical modification of p-chloromercuribenzoate (PCMB) on beta-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) from green crab (Scylla serrata) has been studied. The results show that sulfhydryl group is essential for the activity of the enzyme. Inhibitory kinetics of the enzyme by mercuric chloride (HgCl2) has been studied using the kinetic method of the substrate reaction during inhibitor of enzyme. The kinetic results show that the inhibition of the enzyme by mercuric ion (Hg2+) at lower than 1.0 microM is a reversible reaction with residual activity and the inhibition belongs to be competitive. The inhibition kinetics model of Hg2+ on the enzyme was set up and the microscopic rate constants were determined and the data obtained were well fitted with the model. It was also turned out that only one molecule of HgCl2 binds to the enzyme molecule to lead the enzyme lose its activity. The above results suggest that the cysteine residue is essential for activity and is situated at the active site of the enzyme.     

Inhibitory kinetics of phenol on the enzyme activity of beta-N-acetyl-D-glucosaminidase from green crab (Scylla serrata)

Chemical pollution such as chromium and phenol in the sea water has been increasing in recent years in China sea. At the same time, marine shellfish such as prawn and crab are sensitive to this pollution. beta-N-acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52) catalyzes the cleavage the oligomers of N-acetylglucosamine (NAG) into the monomer. In this paper, the effects of phenol on the enzyme activity from green crab (Scylla serrata) for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) have been studied. The results showed that appropriate concentrations of phenol could lead to reversible inhibition on the enzyme and the inhibitor concentration leading to 50% activity lost, IC(50), was estimated to be 75.0+/-2.0 mM. The inhibitory kinetics of phenol on the enzyme in the appropriate concentrations of phenol has been studied using the kinetic method of substrate reaction. The time course of the enzyme for the hydrolysis of pNP-NAG in the presence of different concentrations of phenol showed that at each phenol concentration, the rate decreased with increasing time until a straight line was approached. The results show that the inhibition of the enzyme by phenol is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction on phenol with the enzyme.