Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

Relationship between differential retention of Escherichia coli and Enterococcus faecalis and variations in enzyme activity in the scallop Patinopecten yessoensis

Uptake of Escherichia coli and Enterococcus faecalis and variations of trypsin amylase activity acid phosphatase and superoxide dismutase in tissue of the scallop Patinopecten yessoensis were detected. The results showed that P. yessoensis accumulated E. faecalis in larger numbers and more rapidly than E. coli, both with the highest concentration in the digestive tract and lowest in hemolymph. Compared to E. coil, all scallops exposed to E. faecalis showed significantly higher trypsin and AMS activity. SOD activity in hemocytes and ACP activity in hemolymph was significantly higher in the treatments with 5 log(10)CFU/ml E. colt than with E. faecalis. But no significant differences in ACP activity of P. yessoensis exposed to a 3 log(10)CFU/ml inoculum of both bacteria were recorded. In conclusion, the mass retention of gut microflora in P. yessoensis is positively correlated with digestive enzymes activity and negatively correlated with ACP activity in the hemocyte. (C) 2010 Published by Elsevier Ltd.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Survival, growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay (Yellow Sea, China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 mu m/d) and 20 m (236.9 mu m/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 mu m/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.

STARVATION-INDUCED CHANGES OF HEMOCYTE PARAMETERS IN THE ZHIKONG SCALLOP CHLAMYS FARRERI

Substantial nutritional and energetic demands m-e associated with immune activation and the maintenance of an efficient immune system. One-year-old Chlamys farreri (Jones and Preston) scallops were maintained ill lantern nets ill different nutritional conditions (satiation and starvation) for 40 days. After the 40-day treatments, the condition index and the total hemocyte count (THC) decreased significantly in the starved group compared with the satiated and initial control groups. The percentage of phagocytic hemocytes also was significantly reduced with starvation. In contrast. no significant effect of starvation was observed oil reactive oxygen species (ROS) production. The acid phosphatase (ACP) activities in cell-free hemolymph increased significantly in scallops in starved and satiated treatments compared with the initial control. whereas ACP activity in hemocyte lysate was significantly lower ill the starved group. These results indicate that starvation stress compromises immunological activities of scallops.

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li+, Na+, NH4+, K+, Mg2+, and Ca2+) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na+, NH4+, K+, Mg2+, and Ca2+ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K+, Mg2+, and Ca2+ significantly, decreased after exposure to Az, except that Na+ and NH4+ remained constant. Li+ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.

A switch in the control of growth of the wing imaginal disks of Manduca sexta.

BACKGROUND: Insulin and ecdysone are the key extrinsic regulators of growth for the wing imaginal disks of insects. In vitro tissue culture studies have shown that these two growth regulators act synergistically: either factor alone stimulates only limited growth, but together they stimulate disks to grow at a rate identical to that observed in situ. It is generally thought that insulin signaling links growth to nutrition, and that starvation stops growth because it inhibits insulin secretion. At the end of larval life feeding stops but the disks continue to grow, so at that time disk growth has become uncoupled from nutrition. We sought to determine at exactly what point in development this uncoupling occurs. METHODOLOGY: Growth and cell proliferation in the wing imaginal disks and hemolymph carbohydrate concentrations were measured at various stages in the last larval instar under experimental conditions of starvation, ligation, rescue, and hormone treatment. PRINCIPAL FINDINGS: Here we show that in the last larval instar of M. sexta, the uncoupling of nutrition and growth occurs as the larva passes the critical weight. Before this time, starvation causes a decline in hemolymph glucose and trehalose and a cessation of wing imaginal disks growth, which can be rescued by injections of trehalose. After the critical weight the trehalose response to starvation disappears, and the expression of insulin becomes decoupled from nutrition. After the critical weight the wing disks loose their sensitivity to repression by juvenile hormone, and factors from the abdomen, but not the brain, are required to drive continued growth. CONCLUSIONS: During the last larval instar imaginal disk growth becomes decoupled from somatic growth at the time that the endocrine events of metamorphosis are initiated. These regulatory changes ensure that disk growth continues uninterrupted when the nutritive and endocrine signals undergo the drastic changes associated with metamorphosis.

Botany origin and protein content of homemade honeys from Galicia hives (NW Spain)

The botanic origin and the protein content of 15 honeys from small bee farms exploitations of Galicia, for family consume, were studied; the aim is to check if the protein wealth and the pollen wealth are dependent parameters. Seven honeys resulted to be Rhamnus frangula unifloral (pollen patterns with low diversity), two Castanea sativa Miller unifloral, other one heather unifloral, and five was multifloral honeys of various pollen patterns (four Castanea predominant and one Rhamnus frangula predominant). Their pollen wealth was low; eight honeys classified in the Maurizio Class I, 3 in Class II, 2 in Class III, and one in Maurizio Class IV. There has been a wide variability in its protein content (0.09- 4.83 mg prot./g honey). The relative amount of pollen from different taxa has a direct or inverse proportionality to wealth protein.

Male morph predicts investment in larval immune function in the dung beetle, Onthophagus taurus

Investment in immunity is costly, so that resource-based trade-offs between immunity and sexually selected ornaments might be expected. The amount of resources that an individual can invest in each trait will be limited by the total resources available to them. It would therefore be informative to investigate how investment in immune function changes during growth or production of the sexual trait as resources are diverted to it. Using the dung beetle, Onthophagus taurus, which displays both sexual and male dimorphism in horn size, we examined changes in one measure of immune function, phenoloxidase (PO) activity, in the hemolymph of larvae prior to and during horn growth. We found that PO levels differed between small- and large-horned males throughout the final instar prior to the point where investment in horn growth was taking place. PO levels in females were intermediate to the 2 male morphs. These differences could not be accounted for by differences in condition, measured as hemolymph protein levels and weight. We suggest that the observed differences might be associated with sex- and morph-specific variation in juvenile hormone levels.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca²⁺ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. © 2008 Elsevier Inc. All rights reserved.

Effects of mixture of nanoparticles under different salinity in the clams Ruditapes philippinarum and Ruditapes decussatus

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidadde do Algarve, 2015

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals

Pathogenicity of the bacterium Xenorhabdus nematophila isolated from the entomopathogenic nematode Steinernema carpocapsae and its secretions against Galleria mellonella larvae

The entomopathogenic bacterium, Xenorhabdus nematophila was isolated from the hemolymph of Galleria mellonella infected with Steinernema carpocapsae. The bacterial cells and its metabolic secretions have been found lethal to the Galleria larvae. Toxic secretion in broth caused 95% mortality within 4 d of application whereas the bacterial cells caused 93% mortality after 6 d. When filter and sand substrates were compared, the later one was observed as appropriate. Similarly, bacterial cells and secretion in broth were more effective at 14% moisture and 25 °C temperature treatments. Maximum insect mortality (100%) was observed when bacterial concentration of 4×106 cells/ml was used. Similarly, maximum bacterial cells in broth (95%) were penetrated into the insect body within 2 h of their application. However, when stored bacterial toxic secretion was applied to the insects its efficacy declined. On the other hand, when the same toxic secretion was dried and then dissolved either in broth or water was proved to be effective. The present study showed that the bacterium, X. nematophila or its toxic secretion can be used as an important component of integrated pest management against Galleria.

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea - Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2-3 times more than the other crabs, whereas G. cruentata ate 1.5-2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs. (C) 2008 Elsevier Inc. All rights reserved.

The importance of dietary calcium consumption in two species of semi-terrestrial grapsoid crabs

Calcium (Ca) is essential for crustaceans, due to calcium carbonate (CaCO(3)) deposition in the new exoskeleton to harden it. The purpose of this work was to study short term Ca balance in terms of dietary Ca ingestion in two phylogenetically related crabs (Superfamily Grapsoidea) showing different degrees of terrestrial adaptations: Sesarma rectum Randall, 1840 and Neohelice granulata (Dana, 1851). Dietary Ca ingestion was studied using purified diets with different Ca concentrations (0, 2.2 and 6.66% Ca), together with measurements of Ca excretion and Ca hemolymph levels. The results showed that both crabs had the same response to foods containing different levels of Ca, with both species eating more of the high Ca diet. However, S. rectum consumed more per mg body mass at all Ca concentrations (6 mg.g(-1) for S. rectum against 3 mg.g(-1) for N. granulata). Both species excreted/egested Ca differently: S. rectum excreted Ca proportionally to ingestion, whereas N. granulata maintained constant faecal Ca output at all dietary Ca levels. Moreover, Ca hemolymph levels for crabs fed the different diets were independent of dietary Ca. In conclusion, both S. rectum and N. granulata seem to regulate the consumption of diets containing more Ca, which suggests a fine balance for Ca intake.