Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.     

Seasonality in the infection and invasion of Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

The protozoan parasite Marteilioides chungmuensis causes irregular enlargement of the ovary in the Pacific oyster Crassostrea gigas. The parasite invades the oyster through the epithelial tissue of the labial palp, replicates in the connective tissue, and then moves to the gonad, producing spores inside the oocytes. In this study the seasonality and invasion period of the parasite into the host was investigated over a 1 yr cycle. Uninfected 1 and 0 yr old (spat) oysters were placed in an epizootic area every month from July 2004 to July 2005 and September 2005 to March 2006, respectively, and left for 1 mo. Labial palps and gonad were sampled monthly and examined for infection by nested PCR and histological observations. Prevalence of infection detected by PCR was 70% or higher from August to October, but declined sharply in November and reached 7% or lower from February to April. To explain the low detection rate in winter, 1 yr old uninfected oysters were placed in an epizootic area in winter (water temperature: 8 to 10 degrees C) for 2 wk and then transferred to M. chungmuensis-free seawater at 24 degrees C. Although prevalence of infection was ca. 7% before transfer to heated seawater, levels of 87% were detected after 1 wk. After a 3 wk exposure to heated seawater, parasites were found in host oocytes by histological observation. It was concluded that the low prevalence in winter was due to insufficient replication of M. chungmuensis at low seawater temperatures, resulting in levels not detectable by nested PCR, and not to the absence of invasion.     

Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.     

Isolation and 18S ribosomal DNA gene sequences of Marteilioides chungmuensis (Paramyxea), an ovarian parasite of the Pacific oyster Crassostrea gigas

To develop sensitive detection techniques with the aim of elucidating the life cycle of Marteilioides chungmuensis, an intracellular paramyxean infecting the ovary of the Pacific oyster Crassostrea gigas, we isolated the parasite at the sporont stage from infected oysters using a freeze-thaw procedure at -20 degrees C and differential centrifugations in discontinuous sucrose and Percoll gradients. DNA was extracted from the isolated sporonts, and a PCR amplicon of 18S small subunit ribosomal RNA gene DNA was partially sequenced. In situ hybridization using 3 parasite-specific probes designed from the obtained sequence successfully detected parasite cells in infected oysters, and confirmed that the sequenced DNA was derived from M. chungmuensis.     

Non-specific defensive factors of the Pacific oyster Crassostrea gigas against infection with Marteilioides chungmuensis: a flow-cytometric study

In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.     

A study of diagnostic methods for Marteilioides chungmuensis infections in the Pacific oyster Crassostrea gigas

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a lumpy appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

Characterization of a gonad-specific transforming growth factor-beta superfamily member differentially expressed during the reproductive cycle of the oyster Crassostrea gigas

Through differential screening between oyster families selected for high and low summer survival, we have characterized a new transforming growth factor-beta (TGF-beta) superfamily member. This novel factor, named oyster-gonadal-TGFbeta-like (og-TGFbeta-like), is synthesized as a 307 amino acid precursor and displays 6 of the 7 characteristic cysteine residues of the C-terminal, mature peptide. Sequence comparison revealed that og-TGFbeta-like has a low percentage of identity with other known TGF-beta superfamily members, suggesting that og-TGFbeta-like is a derived member of this large superfamily. Real-time PCR (RT-PCR) analysis in different oyster tissues showed that og-TGFbeta-like is specifically expressed in both male and female gonads, at distinct levels according to the reproductive stage. Og-TGFbeta-like relative expression was the lowest at the initiation of the reproductive cycle and increased as maturation proceeded to achieve a maximal level in fully mature female and male oysters. In situ hybridisation demonstrated that expression was exclusively detected in the somatic cells surrounding oocytes and spermatocytes. The role of this newly-characterized TGFbeta member in the reproduction of cupped oyster is discussed in regard to the specificity and the localization of its expression, which singularly contrasts with the pleiotropic roles in a variety of physiological processes commonly ascribed to most TGF-beta family members identified so far.     

Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas

In an attempt to develop a reproducible experimental model of bacterial infection in Crassostrea gigas, oysters taken from very localised sub-populations suffering natural mortality outbreaks were used in cohabitation trials under laboratory conditions. From these trials, a collection of Vibrio strains was isolated from moribund and healthy oysters. In a second step, strains were experimentally tested for virulence by means of injection into healthy oysters. This screening revealed a span of virulence among isolated strains from none to medium. When pooling injected strains, results suggest increased virulence. Vibrio strains may have additive/synergistic action leading to higher C. gigas mortality rates in experimental challenges. Although the study initially aimed to develop a simple experimental model, a complex of interactions emerged between several bacterial strains during the pathogenic process in their molluscan host. Selected strains provide a suitable model of experimental disease for further studies and better understanding of bacterial interaction and pathogenesis in C. gigas.     

Haplosporidiosis of the Pacific oyster, Crassostrea gigas.

Haplosporidan parasites were observed in 10/100 spat and 1/171 adult Pacific oysters, Crassostrea gigas, reared in Matsushima Bay, Japan. Eight of the infected spat contained mild to severe plasmodial infections. The multinucleated plasmodia were 6-12 microm x 7-15 microm and were associated with an infiltration of hemocytes that occurred throughout the vesicular connective tissues of all infected oysters. Two oysters, one adult and one spat, contained advanced sporogonic infections. These were characterized by the presence of sporocysts and immature and mature operculated spores that measured 5.6-6.0 microm x 6.0-8.0 microm and were found exclusively within the digestive tubule epithelium. Electron microscopic examination revealed that mature spores contained a hinge operculum, striated and layered wall, spherule, single nucleus, and haplosporosome formative regions. Parasite morphology and infection pattern closely resemble that of Haplosporidium nelsoni, a pathogen of American oysters (C. virginica).     

Characterization of pathogenic bacteria of the cupped oyster Crassostrea gigas

The French mollusc production is mainly based on the Pacific cupped oyster, Crassostrea gigas. Since 1991, outbreaks of mass mortality of juveniles are reported during the summer period. These outbreaks are a major concern of oyster industry. Several studies have established given bacterial strains to be pathogenic for bivalve species, including oysters. Here we present a study of mortality outbreaks of C. gigas, as initiated in 1995. In a first step, bacterial strains were isolated during mass mortality outbreak and were biochemically characterised. Among the isolated strains, some strains of Vibrio splendidus biovar II were found to be pathogenic by means of experimental challenge of oyster juveniles. In the second step, a genotypical identification of the pathogenic strain was undertaken, based on 16S RNA sequences and phylogenetic analysis. It confirmed that the pathogenetic strain belonged to Vibrio splendidus biovar II.     

Storage metabolism in the Pacific oyster (Crassostrea gigas) in relation to summer mortalities and reproductive cycle (west coast of France)

We describe seasonal changes in the biochemical composition of digestive gland, adductor muscle and gonad and surrounding mantle area in Crassostrea gigas from the Western Atlantic coast of France. Seasonality in histology of storage tissues and glycogen storage capacity in isolated vesicular cells were also studied. Proteins, the main muscle components did not contribute to the gametogenetic effort. Glycogen and lipids were stored in the digestive gland, gonad and surrounding mantle area during the wintering period and the gonad and surrounding mantle area represented the main storage compartment supplying the reproductive effort. Gametogenesis in spring and summer was associated with an increase in lipid and protein contents and took place at the expense of glycogen reserves. Histological study of storage tissue in the gonad led us to define four seasonal stages of storage tissue development. In vitro, glycogen storage capacity in isolated vesicular cells was high from November to March and markedly reduced during gametogenesis, decreasing below detectable levels after spawning. This physiological state should be taken into account with relation to summer mortalities occurring in commercial growing areas.     

First evidence of laccase activity in the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas.     

Isolation of sperm bindin from the oyster (Crassostrea gigas)

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring-shaped and is 84% protein by weight. SDS-polyacrylamide gel electro-phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco-protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.