Primary culture of Pacific oyster,Crassostrea gigas,heart cells

Summary Pacific oyster,Crassostrea gigas, is the most economically important specie to the world shellfish breeding. It is important to note that infectious diseases, particularly viruses, may be hazardous for theC. gigas live-stocks. The study of these viral diseases and the development of diagnosis method need the establishment of in vitro methods for viral multiplication. As no oyster cell line is available actually, we have developed a procedure for primary culture of heart cells which could enable to study molluscan viruses in vitro, and could also provide a diagnosis method based on the search of eventual cytopathogen viral effects. Cells fromC. gigas ventricle of heart were dissociated by trypsin-EDTA treatment and the mechanical action of a Dounce type homogeneizer. The cells were inoculated in previously poly-D-lysin coated flasks. The optimised culture medium was L-15 (Leibovitz) prepared three fold concentrated, then diluted half with sea water, this mixture was supplemented with 10% FCS and 5%C. gigas hemolymph. Different cell types could be identified by transmission electron microscopy analysis, as mostly cardiomyocytes, fibroblast-like cells and pigmented cells, but also haemocytes were present in the cultures.     

A glutamic acid decarboxylase (CgGAD) highly expressed in hemocytes of Pacific oyster Crassostrea gigas

Glutamic acid decarboxylase (GAD), a rate-limiting enzyme to catalyze the reaction converting the excitatory neurotransmitter glutamate to inhibitory neurotransmitter γ-aminobutyric acid (GABA), not only functions in nervous system, but also plays important roles in immunomodulation in vertebrates. However, GAD has rarely been reported in invertebrates, and never in molluscs. In the present study, one GAD homologue (designed as CgGAD) was identified from Pacific oyster Crassostrea gigas. The full length cDNA of CgGAD was 1689 bp encoding a polypeptide of 562 amino acids containing a conserved pyridoxal-dependent decarboxylase domain. CgGAD mRNA and protein could be detected in ganglion and hemocytes of oysters, and their abundance in hemocytes was unexpectedly much higher than those in ganglion. More importantly, CgGAD was mostly located in those granulocytes without phagocytic capacity in oysters, and could dynamically respond to LPS stimulation. Further, after being transfected into HEK293 cells, CgGAD could promote the production of GABA. Collectively, these findings suggested that CgGAD, as a GABA synthase and molecular marker of GABAergic system, was mainly distributed in hemocytes and ganglion and involved in neuroendocrine-immune regulation network in oysters, which also provided a novel insight to the co-evolution between nervous system and immune system.     

Pharmacological evidence that alpha 1-adrenoceptors mediate metamorphosis of the Pacific oyster, Crassostrea gigas

Oyster larvae can be induced to metamorphose by exposure to the natural vertebrate adrenergic agonists, epinephrine and norepinephrine. The larval receptors mediating this induction were pharmacologically characterized by testing the ability of a variety of adrenergic agonists and selected structural analogs of epinephrine and norepinephrine to induce oyster metamorphosis, and by testing the ability of various adrenergic antagonists to block the induction of metamorphosis by epinephrine. Oyster metamorphosis can be induced by vertebrate adrenergic agonists with relative potencies: cirazoline greater than epinephrine greater than phenylephrine greater than or equal to norepinephrine greater than alpha-methylnorepinephrine greater than isoproterenol much greater than methoxamine = clonidine. Other structural analogs of epinephrine and norepinephrine, including dopamine and octopamine, were ineffective at inducing metamorphosis. Induction of metamorphosis by epinephrine can be blocked by vertebrate adrenergic antagonists with relative potencies: chlorpromazine greater than or equal to prazosin greater than phentolamine greater than WB4101 greater than propranolol greater than yohimbine greater than metoprolol. These data demonstrate that receptors similar to vertebrate-type alpha 1-adrenoceptors mediate oyster metamorphosis. This is the first evidence for alpha 1-adrenoceptors in molluscs, and provides an important clue to the control of the complex process of molluscan metamorphosis and to the evolution of vertebrate adrenergic receptors.     

cDNA cloning and molecular identification of the major oyster allergen from the Pacific oyster Crassostrea gigas

BACKGROUND: Shellfish is one of the most common food allergens. Despite the recent cloning and molecular identification of the major heat stable crustacean allergens in shrimp, lobster and crab, there have been no similar studies on molluscs to which a significant portion of populations allergic to shellfish are also hypersensitive. Recent biochemical evidence suggests that tropomyosin is also an allergen in molluscs, but data on the molecular cloning, nucleotide sequencing, expression and IgE binding to mollusc tropomyosin are lacking. OBJECTIVE: This study was undertaken to clone, identify and determine the primary structure of a major IgE-reactive mollusc allergen in oyster at the DNA and protein level. METHODS: We constructed an expression cDNA library from the Pacific oyster Crassostrea gigas. This library was screened for IgE binding clones using sera from 15 subjects with a well-documented history of type I hypersensitivity reactions to oysters. An IgE reactive clone was selected and sub-cloned into plasmids for nucleotide sequence determination and expression in E. coli. RESULTS: We identified a 1.3-kb cDNA designated as Cra g 1.03. Expression of Cra g 1.03 in plasmid vector pGEX produced a 59-kDa recombinant fusion protein reactive to the IgE antibodies from patients with oyster allergies but not non-allergic controls. Cra g 1.03 has an open reading frame of 233 amino acids and demonstrates marked similarity in amino acid composition and peptide sequence with mollusc and crustacean tropomyosins. Absorption of oyster allergic sera with Cra g 1.03 totally removed IgE reactivity to oyster extract. Moreover, absorption of allergic sera with recombinant shrimp tropomyosin (Met e 1), lobster tropomyosin (Pan s 1) and crab tropomyosin (Cha f 1) removed most of the IgE reactivity to Cra g 1.03. CONCLUSION: Cra g 1.03 is the first oyster allergen identified at the molecular level. Nucleotide and amino acid comparison shows that this protein is the oyster tropomyosin.     

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg)

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6 h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with ‘6 h embryo’ and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO–PO system, whereas a purified trypsin triggers proPO–PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as β-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO–PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.     

Apoptosis by RGD-containing peptides observed in hemocytes of the Pacific oyster,Crassostrea gigas

We observed in vitro that after treatment with the Arg-Gly-Asp (RGD) peptide, non-spreading Crassostrea gigas hemocytes underwent cell death. Utilizing a combination of a Hoechst staining method and a DNA fragmentation assay, the typical features of apoptosis were shown, i.e. cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. The hemocyte cell death caused by the RGD peptide appears to be sequence-specific, since no induction was shown in the alanine-substituted control peptide (RAD) treatment. Interestingly, the glutamic acid-substituted control peptide (RGE) also induced hemocytic cell death, but a different type of the death to that induced by the RGD peptide. This is the first report that specific peptides induce cell death in molluscan hemocytes.     

Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas

An antimicrobial polypeptide was purified from an acidified gill extract of Pacific oyster (Crassostrea gigas) by C(18) reversed-phase HPLC. The purified polypeptide had a molecular weight of 8471Da containing 74 amino acid residues. Comparison of the obtained N-terminal sequences with those of others revealed that it was identical to ubiquitin reported from other species and named cgUbiquitin. cgUbiquitin showed broad potent antimicrobial activity against Gram-positive and -negative bacteria including Streptococcus iniae and Vibrio parahemolyticus (minimal effective concentrations, 7.8 and 9.8μg/mL), respectively, without hemolytic activity. The cgUbiquitin cDNA was identified from an expressed sequence tag (EST) library of oyster gill as a precursor form, encoding ubiquitin consisting of 76 amino acids fused to ribosomal protein of S27. Although the cgUbiquitin precursor mRNA was expressed at the intermediate level in the gill, the mRNA was significantly up-regulated at 48h post injection with Vibrio sp. Analysis of the cgUbiquitin C-terminus by carboxypeptidase B treatment and comparison of the retention times revealed that cgUbiquitin lacks the terminal Gly-Gly doublet and ends in an C-terminal Arg residue which might be related to antimicrobial activity. Study of the kinetics of killing and membrane permeabilization showed that this peptide was not membrane permeable and acted through a bacteriostatic process. According to the homology modeling, this peptide is composed of three secondary structural motifs including three α-helices and four β-strands separated by 7 loops regions. Our results indicate that cgUbiquitin might be related to the innate immune defenses in the Pacific oyster and this is the first report for antimicrobial function of ubiquitin isolated from any oyster species.     

Agglutinin activity in Pacific oyster (Crassostrea gigas) hemolymph following in vivo Vibrio anguillarum challenge.

Hemolymph from the Pacific oyster (Crassostrea gigas) contains lectins that agglutinate horse (Gigalin E) and human (Gigalin H) erythrocytes. The gigalins also agglutinate bacteria, including Vibrio anguillarum, and were adsorbed from oyster hemolymph at different temperatures by living, heat-killed, and freeze-dried V. anguillarum cells. Baseline activities of the two gigalins were established by measuring their activities in oyster hemolymph over a period of 4 years. A normal distribution of Gigalin H activity (mean titer 139) was found, whereas the distribution of Gigalin E activity in the same samples was skew (mean titer 512). No covariance was observed between the two agglutinin activities. Increased lectin activity above this baseline was found in oysters exposed for varying time intervals to V. anguillarum at different seasons and temperatures over a period of 2 years. Such exposure resulted in an increase in activity (titer) of four- to nine-fold for Gigalin E and three- to seven-fold for Gigalin H when compared with controls, and in augmentation in the hemolymph of a protein with the same electrophoretic mobility as affinity-purified oyster lectins (gigalins). Challenge with either living or heat-killed bacteria resulted in a significant increase of Gigalin E activity, whereas results for Gigalin H were variable. Oysters challenged with bacteria were observed to filter normally with open shells during the experiments. Also, no increase was found in hemolymph calcium that could indicate anoxia following bacterial challenge (0.49 +/- 0.004 mg mL-1) compared to unexposed oysters (0.50 +/- 0.001 mg mL-1). Increase in the concentration of free amino acids in oyster hemolymph was observed following exposure to bacteria (15.05 mM) and anaerobiosis (13.51 mM) compared to controls (9.06 mM), and changes (in mol %) of individual amino acids differed considerably between hemolymph from animals challenged with bacteria and animals kept anaerobic. The augmented lectin activity in oyster hemolymph, following in vivo exposure to increased bacteria in the seawater, suggests their involvement in enhancing bacterial clearance and defense in the oyster.     

Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010

Since summer 2008, high mortality rates of young Pacific oysters Crassostrea gigas have been recorded in association with the detection of the Ostreid Herpesvirus 1 (OsHV-1). A new variant called μVar has been recently described, characterized mainly by 12 consecutive deletions followed by one deletion of an adenine in the C region. The purpose of this study is to characterize the genotype (variants or OsHV-1 reference) of 300 positive samples of C. gigas analyzed between July 2008 and July 2010 collected along the French, Jersey, and Irish coasts. Samples were quantified by TaqMan PCR, amplified with conventional PCR, targeting the area of the deletion, and then sequenced. Eighty-seven percent of the samples were characterized and the OsHV-1 μVar was detected in 257 oyster samples. The genotype OsHV-1 reference was never detected during the 25 months of the present survey. Thirty-eight samples could not be determined and the majority of them had a low viral load. A novel genotype containing only 9 consecutive deletions named OsHV-1 μVar Δ9 was found in 5 samples. These observations indicate the emergence of different OsHV-1 variants.     

Genetic and evolutionary patterns of innate immune genes in the Pacific oyster Crassostrea gigas

The invertebrate innate immune system functions in immune defence and the stress response. However, knowledge of the genetic and evolutionary patterns of innate immune genes in Mollusca is limited, especially for oysters. Such information would help clarify how oysters adapt to pathogen-rich environments. Here, we characterized the genetic and evolutionary patterns of the innate immune genes in Crassostrea gigas, using population diversity analysis and evolution rates comparison. Innate immune genes have higher median nucleotide diversity than non-immune genes. Nucleotide diversity varied with functional regions and different immune-related gene families. Evolutionary analysis of two Crassostrea species showed that the innate immune genes are less conserved and have higher rates of evolution in C. gigas. We also noted a positive association between nucleotide diversity and selective pressures for genes having orthologues. Our findings will help determine the evolutionary patterns of innate immune genes and the association of these genes with mollusc immunity.     

Differences in integrin-dependent phagocytosis among three hemocyte subpopulations of the Pacific oyster "Crassostrea gigas"

Integrins play a key role in immunoresponses such as attachment, spreading, and phagocytosis in invertebrate hemocytes. This study was designed to identify integrin expression patterns at the hemocyte subpopulation level, and correlate the expression levels with phagocytic ability. First, we cloned a beta integrin from Crassostreagigas hemocytes and used real-time RT-PCR to analyze the quantitative expression level of its encoding mRNA. The expression level in hyalinocytes was significantly higher than that in granulocytes and agranulocytes. Subsequently, we investigated the phagocytic ability of each subpopulation using anti-alpha(5)beta(1) integrin antibody, and found that phagocytosis of hyalinocytes was inhibited by neutralization with the antibody but enhanced against the antibody-conjugated microspheres. In contrast, phagocytic abilities of granulocytes and agranulocytes showed high and zero levels, respectively, regardless of the antibody. These results suggest that phagocytosis of hyalinocytes is regulated by an integrin-dependent mechanism and that of granulocytes is elicited by other functional receptors.     

Suppression substractive hybridisation (SSH) and real time PCR reveal differential gene expression in the Pacific cupped oyster, Crassostrea gigas, challenged with Ostreid herpesvirus 1

Virus-induced genes were identified using suppression subtractive hybridisation (SSH) from Pacific cupped oyster, Crassostrea gigas, haemocytes challenged by OsHV-1. A total of 304 clones from SSH forward library were sequenced. Among these sequences, some homologues corresponded to (i) immune related genes (macrophage express protein, IK cytokine, interferon-induced protein 44 or multicopper oxidase), (ii) apoptosis related genes (Bcl-2) and (iii) cell signalling and virus receptor genes (glypican). Molecular characterization and phylogenic analysis of 3 immune-related genes (macrophage expressed protein, multicopper oxidase and immunoglobulin domain cell adhesion molecule) were performed. Finally, quantitative PCR revealed significant changes in the expression of immune related genes (multicopper oxidase, macrophage expressed protein, myeloid differentiation factor 88 and interferon-induced protein 44) in oysters experimentally challenged with OsHV-1.These findings provide a first basis for studying the role of innate immunity in response to viruses in bivalves and identified genes may serve as markers of interest in breeding programs in order to obtain selected oysters presenting OsHV-1 resistance.     

Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas)

Ostreid Herpesvirus 1 (OsHV-1) was likely detected in Pacific oysters, Crassostrea gigas, at different stages of development. Viral infections were associated with high mortality rates in the spat and larvae. Furthermore, the persistance of OsHV-1 in asymptomatic adults was demonstrated by detection of viral DNA and proteins. In the present study, three successive generations of C. gigas (G0 and G1 parental oysters, G1 and G2 larvae) were screened for OsHV-1 by PCR. Viral DNA was detected in 2-day-old larvae, indicating that infection may take place at very early stages. Although results strengthen the hypothesis of a vertical transmission, it was not possible to predict the issue of a particular type of cross. Indeed, the detection of viral DNA in parental oysters did not systematically correspond to a productive infection or result in a successful transmission to the progeny. However, the infective status of the parents appeared to have an influence on both the infection and the survival rates of the progeny. Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of cross. These results suggest that OsHV-1-infected females may transmit to their offspring some kind of protection or resistance against viral infection.     

The modulation of extracellular superoxide dismutase in the specifically enhanced cellular immune response against secondary challenge of Vibrio splendidus in Pacific oyster (Crassostrea gigas)

Extracellular superoxide dismutase (EcSOD) is a copper-containing glycoprotein playing an important role in antioxidant defense of living cells exposed to oxidative stress, and also participating in microorganism internalization and cell adhesion in invertebrates. EcSOD from oyster (designated CgEcSOD) had been previously reported to bind lipopolysaccharides (LPS) and act as a bridge molecule in Vibrio splendidus internalization. Its mRNA expression pattern, PAMP binding spectrum and microorganism binding capability were examined in the present study. The mRNA expression of CgEcSOD in hemocytes was significantly up-regulated at the initial phase and decreased sharply at 48 h post V. splendidus stimulation. The recombinant CgEcSOD protein (rCgEcSOD) could bind LPS, PGN and poly (I:C), as well as various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica at the presence of divalent metal ions Cu²⁺. After the secondary V. splendidus stimulation, the mRNA and protein of CgEcSOD were both down-regulated significantly. The results collectively indicated that CgEcSOD could not only function in the immune recognition, but also might contribute to the immune priming of oyster by inhibiting the foreign microbe invasion through a specific down-regulation.