Nature and distribution of copper in green lagoon crab Scylla serrata (Forskal)

In Scylla serrata (Forskal), copper is present in detectable quantities in all tissues except the central nervous system. 75% of animal's total copper is found in the haemolymph, hepatopancreas, and cuticle. Both the haemolymph and the hepatopancreas share equally ≈ 60% of the animal's total copper. Copper is found in the TCA-soluble and insoluble fractions of water-soluble tissue extracts and in the chloroform : methanol-soluble and insoluble fractions of water-insoluble residue from the hepatopancreas. No other tissue has all the fractions of copper. In haemolymph copper is present only in the TCA-insoluble fraction. The TCA-soluble fraction is also absent in muscles. The absence of any lipid-bound copper in other tissues and the presence of copper in all fractions from the hepatopancreas indicates that this tissue may play a major role in the mobilization, conservation and detoxication of copper.     

Haemolymph protein composition and copper levels in decapod crustaceans

Variations in haemolymph protein composition and concentration, in copper content and copper distribution in the tissue of decapod crustaceans are reviewed. Haemocyanin is the major haemolymph constituent (> 60%); the remaining proteins (in order of concentration) include coagulogen, apohaemocyanin, hormones and antisomes. Moulting, nutritional state, infection, hypoxia and salinity fluctuations are the major factors affecting the relative proportions and total quantities of the haemolymph proteins. With regard to haemocyanin, the changes in concentration during the moult cycle are principally associated with changes in haemolymph volume, rather than with changes in total haemocyanin content due to synthesis or catabolism. The role of the midgut gland in regulating haemolymph copper and haemocyanin concentration has been re-evaluated. More than 50% of the whole body copper load is stored in the haemolymph. In contrast, less than 3% of the copper load resides in the midgut gland. The latter has little potential for regulating haemolymph copper levels, at least in the short term (hours to a few days), though it may be involved in regulating haemocyanin levels over longer periods (weeks to months). The total copper content of the haemolymph remains within a narrow range, except during starvation when levels may decrease. Consequently, variations in the copper content of soft tissues, which constitute only 20% of decapod dry weight, do not significanlty alter whole body copper concentrations. Evidence that copper released following haemocyanin catabolism becomes bound to metallothionein for later use in the resynthesis of haemocyanin is reviewed and found to be inconclusive. The amount of copper that can be stored in this way is trivial compared with the amount of copper required to permit significant changes in haemolymph haemocyanin concentration. Average tissue copper requirements, calculated during the present study, are approx. 4 times higher than previous theoretical estimates.     

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.     

Metal binding compounds in hepatopancreas and haemolymph of porcellio scaber (isopoda) from contaminated and reference areas

1. In order to evaluate the role of metal-binding proteins in the tolerance mechanism of Porcellio scaber to heavy metals, a comparative study was made using isopods from three locations: a zinc-lead mine (Plombiéres), a zinc smelter (Budel) and a reference wood (Spanderswoud). The Cu, Zn and Cd concentrations and the protein composition were determined in the haemolymph and hepatopancreas from the isopods.2. A constant Cu/Zn molar ratio of about 5 was found in the haemolymph of all populations and no correlation was found between hepatopancreas and haemolymph Cu and Zn content.3. Using fast-performance liquid chromatography (FPLC), most of the haemolymph Cu and Zn appeared to be associated with a single UV absorbing peak corresponding with an apparent molecular weight of ± 70 kD; this peak is probably the monomer of hemocyanin.4. The hepatopancreas Zn and Cd concentration were elevated compared to the hepatopancreas of the smelter and mine isopods; after homogenization and centrifugation 70–80% of the metals were found in the supernatant.5. In all populations the hepatopancreas Cu-, Zn- and Cd-binding compounds eluted in separate peaks of low molecular weight, suggesting the absence of an MT-like compound in Porcellio scaber.6. The similarity of the protein profiles in haemolymph, and the similar distribution of the metals over the fractions in haemolymph and hepatopancreas suggests that inducible metal binding compounds are not involved in metal tolerance differences between populations.     

Changes taking place in the electrophoretic haemolymph protein pattern during metamorphosis of Polytela gloriosae (Lepidoptera)

The haemolymph proteins of the larva, pupa and adult of Polytela gloriosae have been fractioned by Polyacrylamide gel disc electrophoresis. In the haemolymph of the fifth instar larval stage a total of ten protein fractions have been detected. The concentration of the protein fractions 2, 3, 4, 9 and 10 shows oscillations in their concentration in the early fifth instar, middle fifth instar and late fifth instar larval stage. In all 11 protein fractionswere detected in the haemolymph of different stages of the pupa. The protein bands 1, 7 and 10 of the pupa appear newly in the haemolymph as these bands were not found in the haemolymph of the larvae. The protein fraction 9 of larva was not found in the pupa. In the haemolymph of adult insect sexual difference was observed in the haemolymph protein pattern. In the haemolymph of adult female a total of 10 protein fractions were detected while from the male haemolymph a total of 8 protein fractions were detected. The pupal band 7 was not found in the adults of both the sexes. In the haemolymph of larva and adult one pigmented protein fraction was observed. No pigmented protein fraction was found in the haemolymph of pupa. Iron - containing protein fraction and the acid mucopolysaccharides were not found in the haemolymph. The protein fractions 3, 4, 5, 6 and 7 of adult haemolymph were darkly stained by the Schiff reagent and, thus, they are the fractions of glycoprotein. One protein fraction of lipoprotein was also found in the haemolymph.     

Haemolymph Defense Capacity of the Neotropical sawfly <Emphasis Type="Italic">Aneugmenus merida</Emphasis> against Ant Predation

The defensive characteristics of the sawflies have received special attention due to the involvement of toxic compounds obtained from host plants. In this context, the haemolymph-based defense is one of the strategies known in sawflies to dissuade the attack of predators. Aneugmenus merida is a neotropical sawfly whose larvae are herbivorous on the toxic bracken fern Pteridium spp. The present study examines the defensive properties of the A. merida larval haemolymph and its possible link with the chemistry of its host plant. We report the behavior of the solitary hunter ant Odontomachus chelifer towards A. merida larvae under laboratory conditions. In addition, we studied the liquid intake behavior of the ants provided with solutions of crude haemolymph, bracken extracts, and its fractions. A. merida larvae showed a marked defensive capacity against the ants. The inhibition of the attack was observed during the stages of antennal contact and mandibular blow, suggesting that larval defensive capacity is due to factors present in the integument and haemolymph. Aqueous and methanolic fractions of haemolymph and bracken also deterred feeding. Although some common compounds were detected in the haemolymph and bracken fractions, they were in very small quantities, suggesting that they are not responsible for the bioactivity. Therefore, the hypothetical connection between the host plant chemistry and larval defensive capacity could not be evidenced. We suggest that the deterrent compounds present in the haemolymph and integument could be jointly acting in the sawfly’s defensive strategy.     

CLOTTING PROCESSES IN CRUSTACEA DECAPODA

1. In Limulidae, all the factors involved in the coagulation processes are located inside the amoebocytes. The cellular coagulogen is a single 20,000-polypeptide-chain protein. It is converted into a non-covalently crosslinked gel by a serine protease enzyme which cleaves a single peptide bond, releasing peptice C.
2. Pro-clotting enzyme can be activated by two independent pathways: coagulation is induced by either LPS or 1,3-β-D-glucan, both of which result in gel formation. The two pathways comprise a complex enzyme cascade with several limited protein proteolyses.
3. In Decapoda, clotting factors are found in both the cell-free plasma and haemocyte compartments. Analogous factors are present in Insecta.
4. Plasma coagulogen is a 400,000 molecular weight protein with both lipid and carbohydrate moieties. Its soluble polymers are converted into covalently crosslinked polymers of coagulin by Ca²⁺-dependent transglutaminase. In crayfish, it is also found in other tissues such as soft integument and calcified cuticle. Its concentration varies greatly with the species investigated. It seems to possess many diversified functions such as plasma coagulation, protein transport of tanning agents, lipid and sugar transport and protein storage, and resembles fibronectin.
5. A type of cellular coagulogen seems to be present in the haemocytes of Decapoda. It can be converted to a gel by a serine protease pro-clotting enzyme. This pro-enzyme can be activated by either LPS or 1,3-β-D-glucans. The mechanism of LPS action is not entirely clear. 1,3-β-D-glucans also activate the prophenoloxidase system and cause phenoloxidase attachment to foreign surfaces of haemocyte lysates. The latter system is restricted to semi-granular and granular haemocytes, and plays an important part in host-defence reactions.
6. The evolutin of clotting processes throughout the phylogenetic tree is discussed.     

Accumulation of manganese in the haemolymph, nerve and muscle tissue of Nephrops norvegicus (L.) and its effect on neuromuscular performance

Exposure of Norway lobsters, Nephrops norvegicus (L.) for 3 weeks to manganese concentrations, (5 & 10 mg Mn l(-1) (90-180 microM)), led to its accumulation in various body tissues. The highest concentration was in nerve tissue (brain and abdominal ganglia) which had up to 6 times (on wet wt. basis) the manganese concentration of the exposure concentration, whereas the haemolymph accumulated 3 times and the muscle tissue only 0.5 times the exposure concentration. In the haemolymph the manganese was bound mainly to protein, predominantly (80-90%) to the respiratory protein haemocyanin, as the concentration was 14 times higher in the protein fraction than in the supernatant. Manganese did not substitute for copper in the haemocyanin, as the copper concentration remained constant despite the manganese exposure. The possibility that manganese exposure induced neurotoxic effects sufficient to reduce neuromuscular performance was assessed from the kinematics of free tail-flip swimming, and from measures of the forces produced by abdominal movements in tethered animals. No significant reduction in tail flip velocity or flexion force, but a significant reduction in the maximum post-flip extension force was found. No correlation was found between the manganese concentration in a single tissue or different fractions of the haemolymph and the post-flip extension, except for a weak negative correlation with the manganese concentration in the abdominal ganglion. The ecophysiological implications of these results are discussed.     

Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.     

Studies on the proteins of Poecilocera picta. I-changes taking place in the haemolymph protein during moulting

The haemolymph proteins of the 6th nymph of P. picta were fractioned by the polyacrylamide gel disc electrophoresis. A total of seven proteins fractions have been detected from the haemolymph. The chemical nature of different protein fractions have been examined by histochemical methods. The changes taking place in the cuticle and epidermal cells have been examined during the transformation of 6th nymph into adult. The fat body proteins have been electrophoretically fractioned and the changes in the concentration of different protein fractions have been examined. It is suggested that the protein fraction 3 of the haemolymph is utilized in the formation of new cuticle. It is concluded by the histochemical observations that proteins of the band 3 are synthesized in the fat body.     

Comparative analysis of two biliproteins, BP1 and BP2, from haemolymph of cabbage white butterfly, Pieris rapae

Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs.     

Partial characterization and amino acid composition of a high molecular weight peptide with salmon calcitonin immunoreactivity in a crustacean: Nephrops norvegicus

Anti-salmon calcitonin antibodies were used to follow the purification of a high molecular weight peptide present both in the haemolymph and in the hepatopancreas of the Norway lobster Nephrops norvegicus. An apparent molecular weight of 22 kDa has been measured in electrophoresis on SDS gels and amino acid composition compared to salmon calcitonin. The amount determined by the immunoreactivity assay corresponds to about 1/40 and 1/140 of that which is based on direct protein measurement for the hepatopancreas and the haemolymph respectively. The total amount of this peptide could be estimated as 3.5 mg/g fresh weight for the hepatopancreas and 140 ug/ml for the haemolymph. The function of this peptide is still unknown.     

The vitellogenin of the ring-legged earwig,Euborellia annulipes (Lucas) (Insecta: Dermaptera)

Using Polyacrylamide gel electrophoresis, labelling procedures and immunological methods, an extraovarian synthesis of vitellogenin has been demonstrated in Euborellia annulipes. Electropherograms of the haemolymph of 1-wk-old mated females show an extra protein fraction on the second day after mating; the concentration of this fraction in the haemolymph falls from the fifth day after mating. The fatbody incorporates [³H]leucine into the proteins synthesised by it during the period of oocyte formation. The proteins extracted from the fatbody and ovary of females of the first reproductive cycle show a high level of radioactivity 3 days after mating, suggesting a simultaneous release of proteins from the fatbody and uptake by the oocytes. On the other hand, TCA-precipitable fractions of the ovary, obtained from females on the pre-ovipositional day, record a high [³H]activity but similar fractions from the fatbody only show minimum radioactivity. Antibodies prepared against the antigens obtained from the crude yolk extract reacted with the proteins from the haemolymph and also with the proteins from fatbody extracts of females of the first reproductive cycle.     

Characterization of red sternum syndrome in mud crab farms from Thailand

Samples of abnormal mud crabs, Scylla serrata (Forsk?l, 1755) (Decapoda: Portunidae), were collected from crab farms in Samutsongkhram Province, Thailand. These crabs had hard carapaces, red chelipeds and joints, pale hepatopancreas, gills, and soft muscles. They were almost immobile and finally died. The haemolymph revealed three stages of the syndrome, namely orange, orange-white, and milky-white in colors. The haemolymph, integument, hepatopancreas, gills, abdominal and claw muscle, stomach, and heart were dissected and histologically examined using transmission and scanning electron microscopy. Closer examinations found infection with rod-, curve rod-, or coccus-shape bacteria with thin and thick cell walls in all investigated organs and haemolymph. Isolation of the microorganisms from the infected tissues of red sternum syndrome crabs resulted in five types of bacteria. No microorganism growth was observed in normal crabs. Interestingly, the types of isolated bacteria can be classified according to the severity of the disease. Additionally, the degree of bacterial infection found was consistent with the stages of the disease. It was postulated that the bacteria entered the crabs via the gills, and then migrated through circulating haemocytes, before reaching the internal organs.     

Haemolymph protein patterns during the spinning stage and metamorphosis of Rhynchosciara americana

An acrylamide gel electrophoretical analysis of the haemolymph proteins of R. americana was carried out at different stages of development. In mature larvae there are about 14 haemolymph protein fractions from which one stains heavily and two others faintly for lipoprotein, while three fractions stain for glycoprotein. The haemolymph protein fraction with Rm 0·25 decreases remarkably in mass during spinning, while the others decrease to a lesser extent. The protein fractions could be used in cocoon spinning since previous work suggests that haemolymph proteins are a major pool of cocoon protein precursors. The finding of a protein fraction in the salivary glands with an electrophoretical mobility similar to that of the haemolymph fraction with Rm 0·25 reinforces our hypothesis.     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

Antibacterial activity in four marine crustacean decapods

A search for antibacterial activity in different body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid, and further extracted and concentrated on C18 cartridges. Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity. Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts. V. anguillarum and C. glutamicum were generally the most sensitive micro-organisms. In P. borealis and P. bernhardus most of the active fractions were not affected by proteinase K treatment, while in H. araneus and P. camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature. In P. bernhardus the active fractions were generally heat labile, whereas in H. araneus the activities were resistant to heat. Differences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested.     

Chronic effect of atrazine on hydromineral balance in the crab

Effect in vivo of atrazine on total body weight, hydration level, haemolymph volume, inorganic electrolytes was studied in the gill, hepatopancreas and haemolymph of the crab. Significant changes were seen in the tissues indicating disturbances in the hydromineral balance of the crab as a consequence of atrazine.     

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.