Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae

Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 x 10(4) colony forming units (CFU) ml(-1) after 24 h and between 3.2 x 102 and 1.9 x 10(3) CFU ml(-1) after 48 h.     

High genetic load in the Pacific oyster Crassostrea gigas

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2-3 months postfertilization in F(2) or F(3) hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8-14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena.     

Infectious diseases of the Pacific oyster,Crassostrea gigas

The Pacific oyster, Crassostrea gigas, is known for not having been affected by major epizootics of infectious diseases, unlike many other commercially important oysters worldwide. Nonetheless, review of the scientific literature reveals more than ten infectious diseases of this species including those with viral, bacterial, protozoan, and metazoan etiologies. These include diseases of larval, juvenile, and adult oysters. Diseases such as oyster velar virus disease, herpes-like infection, and ligament disease are known because of their importance in intensive husbandry systems of this bivalve. Nocardiosis, Marteilioides infection, haplosporidiosis, Denman Island disease, and others are primarily known from their effect on extensively cultured populations of the Pacific oyster. These diseases are reviewed in terms of their disease manifestations, etilogy, epizootiology and economic importance, prevention, and management and diagnosis.     

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).     

A tolloid homologue from the Pacific oyster Crassostrea gigas

The genes governing mesoderm specification have been extensively studied in vertebrates, arthropods and nematodes. The latter two phyla belong to the Ecdysozoan clade but little is understood of the role that these genes might play in the development of the other major protostomal clade, the Lophotrochozoa. As part of a wider project to analyze the functions associated with transforming growth factor beta superfamily members in Lophotrochozoa, we have cloned a gene encoding a tolloid homologue from the bivalve mollusc Crassostrea gigas. Tolloid is a key developmental protein that regulates the activity of bone morphogenetic proteins (BMPs). We have determined the intron-exon structure of the gene encoding C. gigas tolloid and have compared it with those of homologous genes from both protostomes and deuterostomes. In order to analyze the functionality of oyster tolloid the zebrafish embryo has been employed as a reporter organism and we show that over-expression of this protein results in the ventralization of zebrafish embryos at 24h post fertilization. The expression of the C. gigas tolloid gene during embryonic and larval development as well as in adult tissues is also explored.     

Reproductive patterns of the Pacific oyster Crassostrea gigas in France

Summary

In France, national management programs focus research on understanding reproductive factors in Crassostrea gigas to confront problems of the oyster industry. However, little information has been documented in which reproductive patterns include sexual changes. The reproductive cycle of oysters at three sites of the Atlantic coast of France was examined from 1996 to 1998, and the seasonal variations in oocyte size-frequencies, and sex ratio were described. The results showed a synchronism within the population concerning reproductive behavior. Young oocytes are generated after spawning and show no apparent changes during winter. Growth of oocytes begins in spring and cells reach maturity in April-May and are ready for a single spawning season in June-July. Oocytes that were not released during spawning are reabsorbed within the gonad. The significant difference between sites is that spawning occurred 1 month later in the southern area. A modal analysis showed that oocyte populations in the sample individuals are primordially bimodal, but with polymodal occurrences in June-July, in some cases. Irregular alternative sexuality was detected at all sites, and hermaphrodites appear to be a transition phase that allows changes from male to female during early spring. Previous observations, together with the study of the development of oocyte cohorts over time, permit a hypothetical model concerning the kinetics of gametogenesis in C. gigas. The model suggests that primary oocytes are generated from energy supplied from degenerating, as well as young oocytes that do not reach the mature stage within the gonad during autumn-winter. It seems that, during vitellogenesis, there is disintegration of smaller cells coupled with transfer of energy to the larger oocytes, which continue to grow and mature.     

Chromosome inheritance in triploid Pacific oyster Crassostrea gigas Thunberg

Reproduction and chromosome inheritance in triploid Pacific oyster (Crassostrea gigas Thunberg) were studied in diploid female x triploid male (DT) and reciprocal (TD) crosses. Relative fecundity of triploid females was 13.4% of normal diploids. Cumulative survival from fertilized eggs to spat stage was 0.007% for DT crosses and 0.314% for TD crosses. Chromosome number analysis was conducted on surviving progeny from DT and TD crosses at 1 and 4 years of age. At Year 1, oysters from DT crosses consisted of 15% diploids (2n=20) and 85% aneuploids. In contrast, oysters from TD crosses consisted of 57.2% diploids, 30.9% triploids (3n=30) and only 11.9% aneuploids, suggesting that triploid females produced more euploid gametes and viable progeny than triploid males. Viable aneuploid chromosome numbers included 2n+1, 2n+2, 2n+3, 3n-2 and 3n-1. There was little change over time in the overall frequency of diploids, triploids and aneuploids. Among aneuploids, oysters with 2n+3 and 3n-2 chromosomes were observed at Year 1, but absent at Year 4. Triploid progeny were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at 4 years of age. Aneuploids were significantly smaller than normal diploids. This study suggests that triploid Pacific oyster is not completely sterile and cannot offer complete containment of cultured populations.     

Xenobiotic biotransformation in the Pacific oyster (Crassostrea gigas)

1. Oyster visceral mass and gill tissues possessed measurable flavin-containing monooxygenase (FMO) activity. 2. FMO activity was confirmed in visceral mass microsomes by oxygen uptake experiments utilizing various nitrogen and sulfur-containing chemicals along with measurement of N,N-dimethylaniline (DMA) N-oxidase and methimazole oxidation activities. DMA N-oxidase and methimazole oxidation activities also were present in gill microsomes. 3. Excluding oyster gill methimazole oxidation, there were no consistent seasonal differences in FMO activity in oyster gill or visceral mass microsomes. 4. Although lacking spectral evidence for cytochrome P-450, a peak at 418 nm was observed along with NADPH-cytochrome c reductase activity in visceral mass and gill microsomes suggesting the presence of a denatured cytochrome P-450 system. 5. NADPH-independent benzo(a)pyrene hydroxylase (BPH) activity was observed in both oyster visceral mass and gill microsomes suggesting a co-oxidation pathway possibly involving a one electron transfer of oxygen from a lipid hydroperoxide.     

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.     

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.     

Assessment of female gamete quality in the Pacific oyster Crassostrea gigas

Summary

The possible relationship between certain oocyte and embryo characteristics and larvae viability was investigated with reference to the following aspects: (1) morphological—oocyte diameter and shape; (2) cytological—overall ultrastructure and membrane integrity; (3) biochemical—content of lipids, proteins and carbohydrates; and (4) physiological—respiration. The rate of survival and incidence of abnormality were estimated 24 h after fertilization. The first results showed that 80–90% of oocytes were cytologically viable before fertilization. Eighty to 90% of oocytes are apparently viable before fertilization on the basis of staining with Trypan blue, but this parameter shows little correlation with larval viability. However, Trypan blue staining is of value in allowing the recognition of oocytes with damaged membranes. Respiration was measured for unfertilized oocytes 5 min after stripping, after 6 h, and for 3-h embryos. Positive correlations were found between the O₂-consumption of embryos and both the rate of fertilization and the hatching rate of 24-h larvae. In contrast, no correlation was found between hatching parameters and the O₂-consumption of unfertilized oocytes. These results suggest that embryos possess quality indicators, relating to metabolic characteristics, which can be quantified more easily than those of oocytes.     

Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas

Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.