Infected Oysters Research Articles

Characterizing the Epigenetic and Transcriptomic Responses to Perkinsus marinus Infection in the Eastern Oyster Crassostrea virginica

Eastern oyster populations in the northern Gulf of Mexico are routinely infected with the protistan parasite Perkinsus marinus, the cause of the disease commonly known as dermo. Recent experimental challenges among Atlantic coast populations have identified both resistant and susceptible genotypes using comparative transcriptomics. While controlled experimental challenges are essential first assessments, expanding this analysis to field reared individuals provides an opportunity to identify key genomic signatures of infection that appear both in the laboratory and in the field. In this study we combined reduced representation bisulfite sequencing with 3’ RNA sequencing (Tag-seq) to describe two molecular phenotypes associated with infection in oysters outplanted at a common garden field site. These combined approaches allowed us to examine changes in DNA methylation and gene expression for a large number of individuals (n=40) that developed infections during the course of a common garden outplant experiment. Our epigenetic analysis of DNA methylation identified significant changes in gene body methylation associated with increasing infection intensity, across genes associated with immune responses. There was a smaller transcriptomic response to increasing infection intensities with 32 genes showing differential expression, and 40 % of these genes were found to also be differentially methylated. While there was no clear pattern between direction of differential methylation and gene expression, there was a significant effect of percent methylation on gene-by-gene expression levels and the coefficient of variation in gene body methylation between treatments. These results show that heavily methylated genes have high levels of gene expression with low levels of variation. Comparing our differential expression results with previously published experimental P. marinus challenges identified overlapping expression patterns for genes associated with C1q-domain-containing and V-type proton ATPase proteins. Through our comparative transcriptomic approach using field reared individuals and co-expression network analysis we have also been able to identify a network of genes that change in expression in response to infection. These combined analyses provide evidence for a conserved response to P. marinus infections and suggest that gene body DNA methylation is a better predictor of gene expression variation than it is of the overall magnitude of gene expression.

A universal oyster infection model demonstrates that Vibrio vulnificus Type 6 secretion systems have antibacterial activity in vivo.

With the rapid increase of aquaculture contributing to sustainable food security, comes the need to better understand seafood associated diseases. One of the major aquatic bacterial genera responsible for human infections from seafood is Vibrio, especially from oysters. Currently, in vivo study of bacterial interactions within oysters is limited by the inability to promote high-level uptake of bacteria by oysters. This study has therefore evolved current natural marine snow protocols to generate 'artificial' marine snow, into which bacteria can be incorporated to facilitate extensive uptake by oysters. This presents an adaptable model for bacterial study within filter-feeding shellfish. Using this model, we demonstrate for the first time the antibacterial activity of Vibrio vulnificus Type 6 secretion systems in vivo, revealing an important role for the T6SS in V. vulnificus ecology.

Novel peptides functionalized gold nanoparticles decorated tungsten disulfide nanoflowers as the electrochemical sensing platforms for the norovirus in an oyster

Human norovirus is one of the potential foodborne pathogens causing acute gastroenteritis, diarrhea, and food poisoning. For norovirus detection, we developed the norovirus specifically capturable peptides functionalized gold nanoparticles (AuNP) decorated tungsten disulfide nanoflower (WS2NF/AuNP). Based on the 3D-structured WS2NF acting as a supporting material, AuNP were immobilized on its surface to assist the conductivity of WS2NF and provide effective immobilization sites for bioreceptors. Since norovirus is composed of protein that functions as an insulant, as the virus bind with WS2NF/AuNP, the impedance is elevated by hindering the charge transfer between the working electrode and redox species ([Fe(CN)6]3-/4-). The prepared nano-bio hybrid sensor was assessed by electrochemical impedance spectroscopic technique with spiked samples and deliberately infected oyster samples, respectively. In the case of the former, the electrochemical sensor represents a low detection limit (2.37 copies/mL) with a detection range from negative control to 104 copies/mL, and it was shown a detection limit of 6.21 copies/mL even in the oyster samples. Besides, when it applies to the different types of viruses such as rotavirus and dengue virus to verify the selectivity of this system, there were no effective signals except the norovirus. Therefore, it revealed that combining WS2NF and AuNP enhances electrochemical activity and stability via mutual synergetic effects.

Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite

Perkinsus marinus, a World Organisation for Animal Health (OIE) notifiable parasite, infects several species of oyster, including Crassostrea virginica and Crassostrea corteziensis. There is little information on possible treatments for this parasite, but the biocidal properties of silver nanoparticles (AgNP) suggest their potential use. The lethal effects of the Argovit™ formulation of AgNP was evaluated for the first time against hypnospores of P. marinus, a particularly resistant stage of the parasite that persists in the environment until favorable conditions occur for zoosporulation to be induced. Hypnospores were exposed to 1, 10 and 100 µg/mL of silver compounded in Argovit™ (corresponding to 0.009, 0.093 and 0.927 mM of Ag), to 157.47 µg/mL (0.927 mM) of silver nitrate (AgNO3) used as a positive control, and to polyvinylpyrrolidone (PVP, 1570 µg/mL) used as a vehicle control. Hypnospores in culture medium without treatment served as a negative control. Dose-dependence after 24 h of exposure to AgNP was observed. A concentration of 0.093 mM AgNP resulted in 50% mortality of P. marinus. Treatment with 0.927 mM of silver, as AgNP or AgNO3, was highly lethal, with greater than 90% mortality. Silver nanoparticles were implicated in the deformation of hypnospores. Transmission electron microscopy (TEM) revealed AgNP within the hypnospore wall and involved in the degradation of lipid droplets in the cytoplasm. AgNP were effective in a saline medium, suggesting the utility of detailed studies of the physicochemical interactions of AgNP under these conditions. These results suggest investigations of possible effect of Argovit™ formulation of AgNP against stages of the parasite like trophozoites and tomonts that develop in tissues or hemolymph of infected oysters as well as studies on its effects in the host and environment.

Long-term temporal and spatial patterns of Ostreid herpesvirus 1 (OsHV-1) infection and mortality in sentinel Pacific oyster spat (Crassostrea gigas) inform farm management

Disease due to Ostreid herpes virus − 1 (OsHV-1) is a major constraint to farming the Pacific oyster Crassotrea gigas in Europe and Australasia, requiring new management strategies. The objectives of this study were to determine the seasonal limits of transmission of OsHV-1, the spatial distribution of the virus, the seasonal water temperatures associated with OsHV-1 transmission and disease and the suitability of active surveillance for OsHV-1 using sentinel oysters. Spat were deployed and sampled regularly between 2012 and 2017 at 15 sites in two large estuaries and the times of OsHV-1 exposure and mortality were identified at weekly to monthly intervals. Mortality due to OsHV-1 was observed each summer season but was significantly more likely in the Georges than the Hawkesbury River estuary. Mortality was most widespread and frequent between December and April and was not observed prior to late October or after May. However, subclinical infections with OsHV-1 that did not progress to mortality were detected from early October until late June. There was a significant reduction in mortality due to OsHV-1 as well as subclinical infection with OsHV-1 between 2012–13 and 2016–17, and viral loads in infected oysters also appeared to decline after 2012–13. Mortality due to OsHV-1 was observed in sentinel spat at 14 of 15 sites but not at every site in every season. Different outcomes were sometimes seen between spat in paired baskets that were 1 to 100 m apart at the same site, attributable to differential exposure to OsHV-1 (clustering). Fifteen different batches of spat were used and mortality due to OsHV-1 occurred in all but three; both triploid and diploid spat were affected. In 2016–17, there was a trend of better survival in selected spat with potential genetic resistance to OsHV-1 compared to unselected spat. There were no biologically significant differences in the growth of spat that subsequently died due to OsHV-1 infection and those that survived. Mortality due to OsHV-1 commenced when the mean water temperature rose above approximately 20 °C in spring, which is 4–5 °C warmer than the initiation threshold in Europe. These consistent epidemiological patterns of infection and mortality will enable strategic placement of susceptible oysters in these estuaries at certain locations and within specific time windows with predicted low risk of mortality due to OsHV-1; water temperature monitoring may be used to predict this risk. Use of sentinel oysters for surveillance for OsHV-1 can be effective, but the clustering of exposure at all scales renders this more reliable at estuary level rather than at bay or site level and it requires an intensive sampling program. Non-specific mortality of spat was relatively common, therefore tests for OsHV-1 should be applied in field trials to distinguish mortality due to OsHV-1 from other causes of mortality.

Influence of environment on the pathogenesis of Ostreid herpesvirus-1 (OsHV-1) infections in Pacific oysters (Crassostrea gigas) through differential microbiome responses

The oyster microbiome is thought to contribute to the pathogenesis of mass mortality disease in Pacific oysters, associated with OsHV-1. As filter-feeders, oysters host a microbiota that can be influenced by the estuarine environment. This may alter susceptibility to OsHV-1 infections, causing variable mortality. This study aimed at: (1) differences in the microbiome of Pacific oysters with a common origin but grown in geographically distinct estuaries; (2) evaluating changes occurring in the microbiota, especially in Vibrio, and (3) differential responses of the oyster microbiome, in response to an OsHV-1 infection. Pacific oysters sourced from a single hatchery but raised separately in Patonga Creek, Shoalhaven River and Clyde River of NSW, Australia, were used and challenged with OsHV-1. The initial microbiome composition was different in the three batches and changed further, post-injection (p < 0.05). The Patonga oysters with the highest mortality also had higher OsHV-1 and Vibrio quantities compared to the other two batches (p < 0.05). The higher initial bacterial diversity in Patonga oysters decreased in moribund oysters which was not observed in the other two batches (p < 0.05). The microbiome of survivors of OsHV-1 infection and negative control oysters of two batches, did not show any changes with the relevant pre-challenged microbiome. A strong correlation was observed between the OsHV-1 and Vibrio quantities in OsHV-1 infected oysters (r = 0.6; p < 0.001). In conclusion, the Pacific oyster microbiome differed in different batches despite a common hatchery origin. Different microbiomes responded differently with a differential outcome of OsHV-1 challenge. The higher Vibrio load in oysters with higher OsHV-1 content and higher mortality, suggests a role in Vibrio in the pathogenesis of this mortality disease. This study provided insights of the potential of different estuarine environments to shape the Pacific oyster microbiome and how different microbiomes are associated with different outcomes of OsHV-1 infection.

Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

First report of black-heart disease in Kumamoto oyster Crassostrea sikamea spat caused by Polydora lingshuiensis in China.

A black-heart disease caused by polydorid infestation is reported for the first time in Kumamoto oyster Crassostrea sikamea Amemiya, 1928 spat in a pond at Beihai city, Guangxi province, China, with a prevalence of 100% and a cumulative mortality rate of 50% within 2 mo. In heavily infected oyster spat, blisters extended toward the center of the inner shell surface, around the adductor muscle scar area to form a large black area occupying approximately 50% of the area of the inner shell surface. Morphological analysis identified the pathogen as Polydora lingshuiensis Ye et al., 2015, which was reconfirmed by comparison of its corresponding 18S rRNA and mitochondrial CO1 gene sequences with those in the GenBank database. The mean abundance of mud blisters was significantly higher in live spat than in dead spat, suggesting that P. lingshuiensis preferentially infests live oyster spat. Additionally, P. lingshuiensis larvae were detected in the inlet near the dam, which suggests that the source of P. lingshuiensis larvae infecting the spat may be larvae entering the ponds through the water current from the sea.

Effect of emersion on the mortality of Pacific oysters (Crassostrea gigas) infected with Ostreid herpesvirus-1 (OsHV-1)

Microvariant genotypes of Ostreid herpesvirus-1 (OsHV-1) have been responsible for mass mortalities in farmed Pacific oyster (Crassostrea gigas) populations in Europe, New Zealand and Australia since its first detection in France in 2008. Previous studies conducted in the Georges River estuary, New South Wales, Australia demonstrated a significant protective effect of increased emersion time for adult oysters, when oysters were grown 300 mm above the standard intertidal growing height used by local farmers. The aim of this study was to investigate the protective effect of tidal emersion on infection by OsHV-1 in a controlled laboratory environment. Adult C. gigas (24 months old 70–90 mm) and C. gigas spat (5 months old, 20–40 mm) were infected with OsHV-1 by intramuscular injection and housed with either twice daily emersion or constant immersion. Oysters were monitored daily for gaping of the valves, as an indicator of clinical disease. Contrary to observations in prior field studies, adult oysters subjected to emersion in this study had significantly higher mortalities (67.2%) compared to the constantly immersed adults (11.3%). No significant difference was observed between treatments for the spat. Real-time quantitative PCR confirmed that all mortality was associated with a high concentration of OsHV-1 DNA. Constant immersion appeared to have a protective effect on infected adult oysters, as many gaping oysters recovered. These results suggest that the beneficial effect of high growing height on adult oysters in the field is due to avoidance of infection with OsHV-1, rather than an effect on oyster physiology. Furthermore, it suggests that if infected oysters can be immersed in such a way that predators and secondary infections are avoided, then many may survive the infection.

Regulation of apoptosis-related genes during interactions between oyster hemocytes and the alveolate parasite Perkinsus marinus

The alveolate Perkinsus marinus is the most devastating parasite of the eastern oyster Crassostrea virginica. The parasite is readily phagocytosed by oyster hemocytes, but instead of intracellular killing and digestion, P. marinus can survive phagocytosis and divide in host cells. This intracellular parasitism is accompanied by a regulation of host cell apoptosis. This study was designed to gain a better understanding of the molecular mechanisms of apoptosis regulation in oyster hemocytes following exposure to P. marinus. Regulation of apoptosis-related genes in C. virginica, and apoptosis-regulatory genes in P. marinus, were investigated via qPCR to assess the possible pathways involved during these interactions. In vitro experiments were also carried out to evaluate the effect of chemical inhibitors of P. marinus antioxidant processes on hemocyte apoptosis. Results indicate the involvement of the mitochondrial pathway (Bcl-2, anamorsin) of apoptosis in C. virginica exposed to P. marinus. In parallel, the antioxidants peroxiredoxin and superoxide dismutase were regulated in P. marinus exposed to C. virginica hemocytes suggesting that apoptosis regulation in infected oysters may be mediated by anti-oxidative processes. Chemical inhibition of P. marinus superoxide dismutase resulted in a marked increase of reactive oxygen species production and apoptosis in infected hemocytes. The implication of oxygen-dependent apoptosis during P. marinus infection and disease development in C. virginica is discussed.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

Seawater detection and biological assessments regarding transmission of the oyster parasite Mikrocytos mackini using qPCR.

Mikrocytos mackini is an intracellular parasite of oysters and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Although M. mackini has been investigated for decades, its natural mode of transmission, mechanism for host entry, and environmental stability are largely unknown. We explored these biological characteristics of M. mackini using a recently described quantitative PCR (qPCR) assay. We detected M. mackini in the flow-through tank water of experimentally infected oysters and during disease remission in host tissues following 6 wk of elevated water temperature. Waterborne exposure of oysters to M. mackini further confirmed the potential for extracellular seawater transmission of this parasite and also identified host gill to have the highest early and continued prevalence for M. mackini DNA compared to stomach, mantle, labial palps, or adductor muscle samples. However, infections following waterborne challenge were slow to develop despite a substantial exposure (>106 M. mackini l-1 for 24 h), and further investigation demonstrated that M. mackini occurrence and infectivity severely declined following extracellular seawater incubation of more than 24 h. This study demonstrates a potential for using qPCR to monitor M. mackini in wild or farmed oyster populations during periods of disease remission or from environmental seawater samples. This work also suggests that gill tissues may provide a primary site for waterborne entry and possibly shedding of M. mackini in oysters. Further, although extracellular seawater transmission of M. mackini was possible, poor environmental stability and infection efficiency likely restricts the geographic transmission of M. mackini between oysters in natural environs and may help to explain localized areas of infection.

Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality outbreaks in European flat oyster stocks in Europe. As eradication and treatment are not possible, the control of the disease mainly relies on transfer restriction. Moreover, selection has been applied to produce resistant flat oyster families, which present better survival and lower prevalence than non-selected oysters. In order to better understand the mechanisms involved in resistance to bonamiosis, cellular and molecular responses of 2 oyster groups (selected oysters and wild-type oysters) were analyzed in the context of experimental injection and cohabitation infections. Cellular responses including non-specific esterases detection, ROS production and phagocytosis activity were analyzed by flow cytometry. Four genes homologous to those shown to be involved in immunity were selected (Inhibitor of apotosis OeIAP, Fas ligand OeFas-ligand, Oe-SOD, and OeEc-SOD) and monitored by quantitative reverse-transcription PCR (qRT-PCR). Infected oysters showed higher phagocytosis activity than controls. Infected selected oyster show a lower phagocytosis activity which might be a protection against the parasite infection. The expression of OeIAP and OeFas-ligand gene was significantly increased in selected oysters at 5 days post-injection. OeIAP gene expression appeared to be significantly increased in wild-type oysters at 8 days post-injection. Our results suggest that resistance to bonamiosis partly relies on the ability of the oysters to modulate apoptosis.

Perkinsus infection is associated with alterations in the level of global DNA methylation of gills and gastrointestinal tract of the oyster Crassostrea gasar.

Gills (G) and gastrointestinal tract (GT), as well as cultured P. marinus trophozoites were preserved in 95% ethanol for DNA extractions. DNA methylation levels were estimated from G and GT tissues of uninfected (n=60) and infected oysters (n=60), and from P. marinus trophozoites, by ELISA assays. Results showed that the mean prevalence of Perkinsus sp. infections was high (87.3%) in 2015 and moderate (59.6%) in 2016. DNA methylation levels of G and GT tissues were significantly lower in infected oyster than in uninfected oysters, suggesting that infections are associated with hypomethylation. Methylation level was significantly higher in G than in GT tissues, indicating a likely tissue-specific mechanism. P. marinus trophozoites showed 33% methylation. This was the first study that confirms alterations of DNA methylation in two tissues of C. gasar oysters in association with Perkinsus sp. infections.

Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays.

Pooled testing of samples is a common laboratory practice to increase efficiency and reduce expenses. We investigated the efficacy of 2 published SYBR Green real-time PCR assays when used to detect the haplosporidian parasite Bonamia ostreae in pooled samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene or the 18S rRNA gene. Tissue homogenates (150 mg) of the New Zealand flat oyster Ostrea chilensis were spiked with ~1.5 × 103 purified B. ostreae cells to create experimental pools of 3, 5, and 10. Ten positive replicates of each pool size were assayed twice with each PCR and at 2 different amounts of DNA template. The PCR targeting the actin 1 gene was unable to reproducibly detect B. ostreae in any pool size. Conversely, the 18S rRNA gene PCR could reproducibly detect B. ostreae in pools of up to 5. Using a general linear model, there was a significant difference in the number of pools that correctly detected B. ostreae between each PCR ( p < 0.01) and each pool size ( p < 0.01). It is likely that the single copy actin 1 gene is more likely to be diluted and not detected by pooling than the multi-copy 18S rRNA gene. Our study highlights that validation data are necessary for pooled sample testing because detection efficacy may not be comparable to individual sample testing.

Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas

This study investigated oyster infection dynamics by different strains of Vibrio aestuarianus isolated before and after the apparent re-emergence of this pathogen observed in France in 2011. We conducted experiments to compare minimal infective dose, lethal dose 50 and bacterial shedding for six V. aestuarianus strains. Whatever the strain used, mortality was induced in juvenile oysters by intramuscular injection and reached 90–100% of mortality within 5 days. Moreover, bacterial shedding was comparable among strains and reached its maximum after 20 h (≈10 EXP5 bacteria/mL/animal). Similarly, our first estimations of lethal dose 50 were comparable among strains (minimal infective dose around 0.4 × 10EXP5 bacteria/mL and LD50 around 10EXP5 bacteria/mL) by using seawater containing freshly shed bacteria. These results indicate that, at least with these criteria, despite V. aestuarianus strains genetic diversity, the disease process is similar. The strains isolated after the apparent re-emergence of the bacteria in 2011, do not present a more acute virulence phenotype than the reference strains isolated between 2002 and 2007. Finally, our study provides original and noteworthy data indicating that infected oysters shed bacteria at a level above the threshold of LD50 a few days before they die, meaning that infection is expected to spread in a susceptible population.

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

Selection of lactic acid bacteria as candidate probiotics forVibrio parahaemolyticusdepuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre-formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 106 UFC to 104 UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non-toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection.Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level.

Unusually abundant and large ciliate xenomas in oysters, Crassostrea virginica, from Great Bay, New Hampshire, USA

During routine histological examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, USA, a high prevalence and intensity of ciliate xenomas has been noted since sampling began in 1997. Xenomas are hypertrophic lesions on the gills of bivalve molluscs caused by intracellular ciliates, likely Sphenophrya sp. Although not known to cause mortality in oysters, xenomas have not previously been reported at this high abundance. The objectives of this study were to characterize the xenomas, describe the ciliates, and gather baseline epizootiological data with correlations to environmental and biological parameters.Upon gross examination, xenomas appeared as white nodules, up to 3mm in diameter, located in the gill tissue and occasionally fusing into large masses along the gill filaments. Light microscopy of histological sections revealed xenomas located in the gill water tubes, which they often completely blocked. Higher magnification revealed dual nuclei, eight kineties, and conjugation of the ciliates. Transmission electron microscopy revealed dual nuclei that varied in density, a maximum of twenty cilia in each kinety radiating from the oral apparatus to the posterior, and a 9+2 axoneme structure within the cilia. These traits place the ciliates into the Order Rhynchodida, but insufficient molecular data exist to confirm classification of this ciliate to the Genus Sphenophrya. Since 1997, xenoma prevalence has fluctuated with peaks in 2000, 2004, and 2011. Infected oysters generally contained <30 xenomas, but 2.1% contained >100, sharply contrasting the rare prevalence and low intensity reported elsewhere. Prevalence increased with oyster size, leveling off near 50% in oysters >60mm. Infection intensity peaked in 70–90mm oysters and declined in larger oysters. Individual oyster condition was not associated with xenoma intensity, but sites with oysters in higher condition generally had a greater prevalence and intensity of xenoma infections. Seasonal data indicated an infection cycle increasing from summer to fall, peaking at 55–65% in November and dropping to <10% by spring. The oyster population in Great Bay, NH warrants further examination to understand the mechanisms and conditions controlling xenoma formation.