近江牡蛎HSP70基因对溶藻弧菌感染的反应

采用实时荧光定量RT-PCR方法,检测了注射溶藻弧菌(Vibiro alginolyticus)后近江牡蛎鳃,闭壳肌,消化腺,外套膜,心脏以及血细胞中HSP70基因的表达变化。结果显示近江牡蛎这五种器官组织中的HSP70基因表达量均出现显著性高表达,且在鳃、外套膜和血细胞中的HSP70基因表达变化规律表现为典型的时间依赖性。血细胞中,显著高表达的峰值出现在24h,至72h恢复到对照水平,高表达持续时间最长:鳃中表达峰值出现时间较早,在第3h,随后在第12h便恢复到对照水平;外套膜,消化腺以及心脏中的峰值分别出现在6h,6h和3h,而在闭壳肌组织中,没出现显著性高表达。由此可见,近江牡蛎HSP70s可能在机体抗菌免疫过程中起了重要作用。     

Enzymatic activities in European flat oyster, Ostrea edulis, and pacific oyster, Crassostrea gigas, hemolymph

Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.     

In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes

In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.     

Flow cytometric analysis of European flat oyster, Ostrea edulis, haemocytes using a monoclonal antibody specific for granulocytes

The importance of haemocytes in mollusc defence mechanisms can be inferred from their functions. They participate in pathogen elimination by phagocytosis (Cheng, 1981; Fisher, 1986). Hydrolytic enzymes and cytotoxic molecules produced by haemocytes contribute to the destruction of pathogenic organisms (Cheng, 1983; Leippe & Renwrantz, 1988; Charlet et al., 1996; Hubert et al., 1996; Roch et al., 1996). Haemocytes may also be involved in immunity modulation by the production of cytokines and neuropeptides (Hughes et al., 1990; Stefano et al., 1991; Ottaviani et al., 1996). As a result, the literature dealing with bivalve haemocyte studies has increased during the last two decades. Most of these publications use microscopy for morphological analysis (Seiler & Morse, 1988; Auffret, 1989; Hine & Wesney, 1994; Giamberini et al., 1996; Carballal et al., 1997; Lopez et al., 1997; Nakayama et al., 1997), and functional analysis (e.g. phagocytosis) (Hinsch & Hunte, 1990; Tripp, 1992; Mourton et al., 1992; Fryer & Bayne, 1996; Mortensen & Glette, 1996). Flow cytometry represents a rapid technique applicable to both morphological and functional studies of cells in suspension. While the measurements based on autofluorescence provide information on cell morphology, the analyses with fluorescent markers including labelled antibodies, offer data on phenotyping and cell functions. As a result, its application has greatly contributed to the investigation of immunocyte functions and differentiation in vertebrates (Stewart et al., 1986; Rothe & Valet, 1988; Ashmore et al., 1989; Koumans-van Diepen et al., 1994; Rombout et al., 1996; Caruso et al., 1997). Some authors studied oyster haemocyte populations by flow cytometry based on cellular autofluorescence (Friedl et al., 1988; Fisher & Ford, 1988; Ford et al., 1994). However, no analysis using specific monoclonal antibodies has been reported to date. In this study, a protocol for studying European flat oyster, Ostrea edulis, haemocytes by flow cytometry using a monoclonal antibody specific for granulocytes and an indirect immunofluorescence technique have been developed. European flat oysters, Ostrea edulis, 7-9 cm in shell length were obtained from shellfish farms in Marenne Oléron bay (Charente Maritime, France) on the French Atlantic coast. All individuals were purchased just before each experiment and processed without any previous treatment.     

Simultaneous flow cytometric assessment for cellular types and phagocytic abilities of the haemocytes of the hard clam, Meretrix lusoria

The aim of this study was to devise a simple protocol for flow cytometric analysis to separate various haemocytic populations of the hard clam Meretrix lusoria based on the mitochondrial membrane potential diversity detected by the fluorescence probe 3,3-dihexyloxacarbocyanine iodide (DiOC6). Compared with the traditional technique for separation of haemocytic populations, continuous Percoll gradient centrifugation, our novel method was more efficient and yielded a higher ratio in separating the clams' haemocytic populations. Based on fluorescence 1 (FL-1) and side scatter (SSC) analysis for haemocytes stained with various fluorescent densities of DiOC6 using flow cytometer, the data showed that there were three obvious cell regions R1, R2, and R3, identified by hyalinocytes, small granulocytes and large granulocytes, respectively. At the same time our results showed that the percentages of haemocytes in R1, R2, and R3 were 49.71+/-0.65%, 19.35+/-00.74%, 30.94+/-0.69%, respectively. After classifying the haemocytic populations, phagocytic activity of the haemocytes was simultaneously analysed with phycoerythrin (PE)-labeled Vibrio vulnificus and detected by flow cytometry. Our results demonstrated that there were higher percentages of large granulocytes compared with hyalinocytes and the percentage of small granulocytes was related to the mitochondrial membrane potential and phagocytic activities.     

Flow cytometric comparison of haemocytes from three species of bivalve molluscs

Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.     

Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast

Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B. exitiosa was first detected, in 2007, on this continent in flat oysters cultured in Galicia (NW Spain). Since then, the parasite has also been detected in France, Italy and the United Kingdom. The bays of the Ebro Delta in the south of Catalonia represent the main bivalve culture area in the Mediterranean coast of Spain. Previous information from the area includes reports of several flat oyster pathogens, including the notifiable parasite Marteilia refringens. However, the status with regard to Bonamia parasites was uncertain. In the present study, a Bonamia parasite was observed in flat oysters cultured in the Alfacs Bay of the Ebro Delta by histology and real-time PCR. PCR-RFLP and sequencing suggested the presence of B. exitiosa. Finally, phylogenetic analyses of the studied Bonamia isolates corroborated B. exitiosa infection. M. refringens was also observed in the same oyster batch, and co-infection with both parasites was also detected. This is the first detection of B. exitiosa, in Catalonia and the Spanish Mediterranean coast. The impact of the parasite on the Mediterranean flat oyster activity needs to be urgently addressed.     

Changes in circulating and tissue-infiltrating hemocyte parameters of European flat oysters,Ostrea edulis,naturally infected with Bonamia ostreae

We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.     

Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor-shell (Ensis siliqua)

Much of the current knowledge concerning bivalve immunology and immunotoxicology has come from studies on the mussel genus, Mytilus, or from the American oyster, Crassostrea virginica. Following a major oil spill, it was observed that the marine mussel, Mytilus edulis, underwent significant immunosuppression but no oil-induced mortalities, while in contrast, mass mortalities were noted in the edible cockle, Cerastoderma edule, and the razor-shell, Ensis siliqua. A study comparing immune cells and functions in these three species was initiated (i) to assess whether M. edulis was a representative model species and (ii) to provide baseline data on immunity in two common species, which had previously received little or no attention in this respect. While all three species shared similar cell types, their lectin-binding and enzyme cytochemistry differed considerably. M. edulis had significantly different proportions of haemocytes binding with the lectins concanavalin A, wheatgerm agglutinin and Helix pomatia agglutinin and stained positive for eight enzymes, compared with only five in C. edule and three in E. siliqua. In terms of immune function, M. edulis haemocytes were much more active in phagocytosis and superoxide generation than haemocytes of the other two species. The results show that immune cells and functions differed extensively in these three closely related species, with M. edulis showing a much higher level of immunological vigour that may be linked to its considerable resilience to adverse environmental conditions. This suggests that M. edulis may not be particularly representative of the bivalves in terms of immune reactivity and that a wider range of species should be included in studies of molluscan immunotoxicology.     

Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst

The capability of an oyster to respond to environmental stresses, such as periodically high summer temperatures, as well as disease or parasite infections, depends, in large measure, upon the viability and functional capability of haemocytes. Eastern oysters (Crassostrea virginica) were subjected to a sudden increase in temperature from 20 to 28 °C for 1 week, and several haemocyte functions were determined before and after the temperature elevation using the flow cytometer. Previously, we described the characterization of different haemocyte types using new and modified flow cytometric methods. In this report, we provide detailed protocols for flow cytometric methods to: (1) determine haemocyte aggregation using paired samples with or without an antiaggregant solution; (2) assess haemocyte viability using propidium iodide (PI); (3) quantify haemocyte phagocytosis with fluorescent microbeads; and (4) measure the respiratory burst response of individual haemocytes using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and zymosan to activate the release of reactive oxygen species (ROS).The temperature increase caused no significant change in haemocyte aggregation, although there was a trend of increasing aggregation in granulocytes and small granulocytes, but a slight decrease in hyalinocyte aggregation. Phagocytosis of all haemocyte types decreased after the temperature increase. Significantly higher percentages of dead haemocytes in all haemocyte types (attributable to a large increase in mortality of hyalinocytes, the most numerous cells) were found after the temperature increase, suggesting generally less capable immune function. Numbers of dead small granulocytes and granulocytes tended to decrease, but this was not statistically significant. Effects of temperature elevation upon respiratory burst were not statistically significant; however, a trend of increased ROS production after temperature elevation was consistent for all haemocyte types. Granulocytes, hyalinocytes, and small granulocytes showed increased production of ROS in the presence of zymosan; granulocytes showed the highest induced fluorescence.     

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.     

Detection of the oyster parasite Bonamia ostreae by fluorescent in situ hybridization

Bonamia ostreae is an economically significant protistan parasite of the flat oyster Ostrea edulis in Europe and North America. Management of this parasite depends partly upon its reliable identification in wild and aquacultured oyster populations, but B. ostreae is small and difficult to detect by traditional microscopic methods. We designed a fluorescent in situ hybridization (FISH) assay to sensitively detect B. ostreae in standard histopathological sections of B. ostreae-infected oysters using fluorescently labeled DNA oligonucleotide probes. Hybridization using a cocktail of 3 presumptively B. ostreae-specific, fluorescein iso(thio)cyanate (FITC)-labeled oligonucleotides produced an unambiguous staining pattern of small green rings inside infected oyster hemocytes that was easily distinguished from host tissue background. This pattern is diagnostic for B. ostreae. A negative control cocktail of oligonucleotides containing 2 mismatches relative to target sequences, on the other hand, failed to hybridize at all. B. ostreae-specific probes did not cross-react with a related protist, Haplosporidium nelsoni.     

Effect of repeated sampling on haemolymph pH,PO2 and haemocyte activity in the Pacific oyster,Crassostrea gigas (Thunberg)

The effects of repeat bleeding via a novel cannulation technique, on the physiology of the Pacific oyster, Crassostrea gigas (Thunberg), were compared to control animals from which haemolymph was sampled by syringe and needle. The effects of surgery and cannulation were characterized by an initial alkalosis (up to 8 h) in the haemolymph of cannulated animals followed by an acidosis of up to 12 h; which occurred in both cannulated and control oysters. A decline in partial pressure of oxygen of oyster haemolymph, observed in both cannulated and control oysters, was directly related to shell closure following handling. Furthermore, a haemocyte concentration observed at 4 h followed by haemocyte dilution at 12 h post-surgery, suggested a handling-related stress response in both cannulated and control oysters. A heightened phagocytic activity of haemocytes at 4 h also supported the occurrence of handling-related stress response in all animals. The cannulation technique described here provides the investigator with a convenient tool for chronic, and repeated sampling of haemolymph with a transient disturbance to the animal.     

The ecology of Bonamia and decline of bivalve molluscs

Bonamia is a protozoan parasite of the haemocytes of oysters (Tiostrea chilensis), in which it has an annual developmental cycle between November and August each year. The parasite transmits directly, oyster to oyster, and therefore disease spread is related to host stock density. The Foveaux Strait oyster population experiences large mortalities every 20-30 years, and these may be attributable to Bonamia. The parasite appears to become less pathogenic at the end of, and probably between, mass mortalities, and some oysters appear more tolerant of infection than others. On the basis of these observations, and considering other protist pathogen:oyster models, the apparently reduced pathogenicity of Bonamia is discussed in terms of parasite kinetics. The population dynamics and selection of parasite tolerant host stocks, and kinetics of parasite transmission, may explain the cyclic nature of large-scale mortalities in Foveaux Strait, without change in parasite pathogenicity.     

Flow cytometry as a tool to quantify oyster defence mechanisms

The fast growing oyster aquaculture industry is greatly hindered by Perkinsus marinus and Haplosporidium nelsoni which can kill up to 80% of the production. The relationship between parasites and oyster defence mechanisms is unclear. Two defence mechanisms of the Eastern Oyster (Crassostrea virginica) were quantified at the single cell level utilising flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the H2O2-specific increase in dichlorofluorescein-associated fluorescence upon stimulation. These two assays distinguished three populations of haemocytes (granulocytes, hyalinocytes and intermediate cells) with unique functional characteristics. Granulocytes were most active at phagocytosis and H2O2 production while hyalinocytes were relatively inactive. The intermediate cells had moderate phagocytic and respiratory burst activity. Flow cytometry can rapidly, accurately and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system.     

Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis (L.)

Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis were investigated. These biochemical constituents of the haemolymph could be an indication of the physiological condition and vitality of the defence system of an animal. Haemolymph protein and lysozyme in oysters were examined over an 18 month period to determine their relationship with the strain of oyster, the season, the site, and parasitism by Bonamia ostreae. Haemolymph protein concentration exhibited seasonal fluctuations and varied between strains. Levels of protein in oysters highly infected with B. ostreae were slightly depressed but not significantly so. Haemolymph lysozyme varied greatly between individuals but no correlation was found between lysozyme levels and infection of oysters by B. ostreae.     

Discriminatory predation by three invertebrates on eastern oysters (Crassostrea virginica) compared with non-native Suminoe oysters (C. ariakensis)

Abstract. Diminished populations of eastern oysters Crassostrea virginica in Chesapeake Bay have stimulated proposals to introduce Crassostrea ariakensis from Asia to restore oyster stocks. As part of a program evaluating possible ramifications of such an introduction, we studied how invertebrate predators responded to this non-native oyster. We compared predation activity under laboratory conditions by oyster drills ( Urosalpinx cinerea; Eupleura caudata ) that bore through an oyster's shell and by the seastar Asterias forbesi that pulls shell valves apart. These three predators preyed significantly (p<0.05) more on the familiar C. virginica than on the novel C. ariakensis . We previously reported that five crab species preyed significantly more on C. ariakensis than on C. virginica , with predation by polyclad flatworms similar between oyster species. Thus, the drills and the seastar differed from the crabs and the flatworms in their response to novel prey. When Urosalpinx cinerea was placed in a Y-maze after being held for 40 d with oysters of one species or the other, the drills moved toward C. virginica effluent more than toward C. ariakensis effluent. This response did not depend on the species of oyster the drills had been held with, suggesting that the drills were responding to more familiar infochemicals from eastern oysters than from the non-native oysters.     

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy.Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.