The first evidence of positive selection in peptidoglycan recognition protein (PGRP) genes of Crassostrea gigas

The oyster Crassostrea gigas is thought to have developed effective immunity to potentially harmful pathogens while under continuous exposure to marine microorganisms; however, the evolutionary mechanisms by which such immunity developed has not been understood. To understand the evolution of immunity, we characterized the family of peptidoglycan recognition proteins in the oyster (CgPGRPs). These proteins are crucial pattern recognition receptors for peptidoglycans (PGNs) and thereby, for activating the innate immune response of host. Herein, we identify seven new CgPGRP genes. Phylogenetic analysis of the seven new and five previously reported CgPRGP genes reveals that the CgPRGP gene family can be clustered into two groups, CgPRGPS and CgPRGPL. Moreover, the CgPRGPS group can be further divided into five subgroups. A codon-substitution model and three likelihood ratio tests (LRTs) suggest that seven sites in the CgPGRP family of genes have been subjected to strong positive selection (ω = 3.035–4.143), Three dimensional modeling revealed that these sites are found primarily at the periphery of coils and α-helices rather than in β-strands, perhaps allowing PGRP to adapt to, and recognize, variability of PGN structure. In conclusion, our studies provide the first evidence of positive Darwinian selection in the CgPGRP family, contributing to a better understanding of the adaptive mechanism of host-pathogens interaction in marine mollusks.     

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.     

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.     

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10? CFU g?1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.     

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.     

Viral gametocytic hypertrophy caused by a papova-like virus infection in the Pacific oyster Crassostrea gigas in Korea

During a routine survey of the Pacific oyster Crassostrea gigas in Tongyoung (previously Chungmu) on the southern coast of Korea, basophilic inclusions were observed in the gonadal tissues. They were detected from March to May at a prevalence rate of 3.3 to 7.1%. The inclusion bodies were Feulgen-positive and stained orange-red with phloxine tartrazine. Electron microscopic observation revealed non-enveloped, icosahedral particles 40 to 45 nm in diameter. These morphological characteristics resemble those of papova virus-like inclusions previously described from Pacific and eastern (American) oysters C. virginica in North America. Although many mitochondrial bodies and intact sperm cells were observed around the inclusion body, no host reaction, such as hemocytic infiltration, was detected.     

Characterization of genes involved in ceramide metabolism in the Pacific oyster (Crassostrea gigas)

ABSTRACT: BACKGROUND: The lipid signaling molecule, ceramide, is a key component of the vertebrate stress response, however, there is limited information concerning its role in invertebrate species. In order to identify genes involved in ceramide metabolism in bivalve molluscs, Pacific oyster genomic resources were examined for genes associated with ceramide metabolism and signaling. RESULTS: Several genes were identified including full-length sequences characterized for serine palmitoyltransferase-1, 3-ketodihydrosphingosine reductase, acid ceramidase, and ceramide glucosyltransferase. Genes involved in ceramide synthesis and metabolism are conserved across taxa in both form and function. Expression analysis as assessed by quantitative PCR indicated all genes were expressed at high levels in gill tissue. The role of the ceramide pathway genes in the invertebrate stress response was also explored by measuring expression levels in adult oysters exposed to Vibrio vulnificus. Two genes demonstrated increased expression during the bacterial challenge: a gene involved in hydrolytic breakdown of ceramide (acid ceramidase) and a gene involved in de novo generation of ceramide (3-ketodihydrosphingosine reductase), suggesting a possible role of ceramide in the invertebrate stress and immune responses. CONCLUSIONS: In silico and laboratory results support that Pacific oysters have the basic components of the ceramide metabolism pathway. These results also indicate that ceramide may have analogous functions in vertebrates and invertebrates. The gene expression pattern of acid ceramidase and 3-kethodihydrosphingosine reductase in response to bacterial exposure especially supports that ceramide and sphingolipid metabolism may be involved in the oyster's stress and/or immune responses.     

Cryopreservation of Crassostrea gigas vesicular cells: viability and metabolic activity

Cryopreservation is widely used for long-term conservation of various tissues, embryos or gametes. However, few studies have described cryopreservation of invertebrate primary cell cultures and more particularly of marine invertebrate somatic cells. This technique would however be of great interest to facilitate the study of various metabolic processes which vary seasonally. The aim of the present study was to develop a protocol for cryopreservation of Crassostrea gigas vesicular cells. Different parameters were adjusted to improve recovery of cells after freezing. The most efficient cryoprotectant agent was a mix of Me(2)SO, glycerol, and ethylene glycol (4% each). The optimal cooling rate was -1 degrees Cmin(-1) down to -70 degrees C before transfer into liquid nitrogen. In these conditions the percentage of viable cells reached 70% of the control. The glucose metabolism of thawed cells was evaluated using radioactive glucose as a tracer. Immediately after thawing, glucose uptake involving membrane transporters was greatly reduced (24% of control) whereas glucose incorporation into glycogen was less affected (68% of control).     

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.     

Identification of Conserved and Novel MicroRNAs in the Pacific Oyster Crassostrea gigas by Deep Sequencing

MicroRNAs (miRNAs) play important roles in regulatory processes in various organisms. To date many studies have been performed in the investigation of miRNAs of numerous bilaterians, but limited numbers of miRNAs have been identified in the few species belonging to the clade Lophotrochozoa. In the current study, deep sequencing was conducted to identify the miRNAs of Crassostrea gigas (Lophotrochozoa) at a genomic scale, using 21 libraries that included different developmental stages and adult organs. A total of 100 hairpin precursor loci were predicted to encode miRNAs. Of these, 19 precursors (pre-miRNA) were novel in the oyster. As many as 53 (53%) miRNAs were distributed in clusters and 49 (49%) precursors were intragenic, which suggests two important biogenetic sources of miRNAs. Different developmental stages were characterized with specific miRNA expression patterns that highlighted regulatory variation along a temporal axis. Conserved miRNAs were expressed universally throughout different stages and organs, whereas novel miRNAs tended to be more specific and may be related to the determination of the novel body plan. Furthermore, we developed an index named the miRNA profile age index (miRPAI) to integrate the evolutionary age and expression levels of miRNAs during a particular developmental stage. We found that the swimming stages were characterized by the youngest miRPAIs. Indeed, the large-scale expression of novel miRNAs indicated the importance of these stages during development, particularly from organogenetic and evolutionary perspectives. Some potentially important miRNAs were identified for further study through significant changes between expression patterns in different developmental events, such as metamorphosis. This study broadened the knowledge of miRNAs in animals and indicated the presence of sophisticated miRNA regulatory networks related to the biological processes in lophotrochozoans.     

Identification of a tyrosinase gene potentially involved in early larval shell biogenesis of the Pacific oyster Crassostrea gigas

The larval shell emerges early in embryogenesis of mollusks, but the detailed mechanisms of its biogenesis remain to be determined. In this study, we cloned a tyrosinase gene (cgi-tyr1) that potentially functioned in larval shell biogenesis from the Pacific oyster Crassostrea gigas, a worldwide bivalve species. Sequence analysis of cgi-tyr1 revealed that it had typical copper-binding domains and a signal peptide. Through whole mount in situ hybridization and an electron scanning microscopic observation, we detected the expression of cgi-tyr1 firstly in the saddle-shaped shell field in trochophores, indicating that cgi-tyr1 might participate in the biogenesis of the initial non-calcified shell of trochophores. In the following development to early D-veliger, cells in the central region of shell field exhibited no detectable cgi-tyr1 expression, and cgi-tyr1 expression was sustained only in the edge of the shell field and the hinge region, indicating that cgi-tyr1 might function fundamentally in shell growth from trochophore to early D-veliger. Unexpectedly, cgi-tyr1 expression was not detected after the D-veliger stage. This indicated that other molecules might function in later shell development. Our results suggested a role for a tyrosinase gene that specifically functioned in the initial phase of the larval shell biogenesis of C. gigas. This work would shed light on future studies on larval shell development and might be helpful to understand how the molluscan shell emerged during evolution.     

Androgen metabolism in oyster Crassostrea gigas: evidence for 17beta-HSD activities and characterization of an aromatase-like activity inhibited by pharmacological compounds and a marine pollutant.

The annual reproductive cycle of oyster Crassostrea gigas depends on environmental factors, but its endocrine regulations are still unknown. Sexual steroids play important roles at this level in vertebrates, and some estradiol effects have been described in invertebrates such as bivalve mollusks. To question these roles in invertebrates, we studied androgen metabolism in C. gigas. Incubations of tissue homogenates with 14C-steroids such as androstenedione (A), testosterone (T), estrone (E1) and estradiol (E2), followed by TLC and HPLC, provide evidence for 17beta-hydroxysteroid dehydrogenases (17beta-HSDs, conversions of A into T, T into A, E1 into E2 and E2 into E1) and aromatase-like (A into E1) activities. The latter activity was further characterized by tritiated water release assay; it was time- and temperature-dependent. Furthermore, this oyster aromatase-like activity was inhibited by 4-hydroxyandrostenedione (IC(50) 0.456 microM) and by other pharmacological compounds including specific cytochrome P450 inhibitors (MR20494, miconazole) and a marine pollutant (tributyltin).