The prokaryotic and eukaryotic microbiome of Pacific oyster spat is shaped by ocean warming but not acidification.

Pacific oysters (Magallana gigas, a.k.a. Crassostrea gigas), the most widely farmed oysters, are under threat from climate change and emerging pathogens. In part, their resilience may be affected by their microbiome, which, in turn, may be influenced by ocean warming and acidification. To understand these impacts, we exposed early-development Pacific oyster spat to different temperatures (18°C and 24°C) and pCO2 levels (800, 1,600, and 2,800 µatm) in a fully crossed design for 3 weeks. Under all conditions, the microbiome changed over time, with a large decrease in the relative abundance of potentially pathogenic ciliates (Uronema marinum) in all treatments with time. The microbiome composition differed significantly with temperature, but not acidification, indicating that Pacific oyster spat microbiomes can be altered by ocean warming but is resilient to ocean acidification in our experiments. Microbial taxa differed in relative abundance with temperature, implying different adaptive strategies and ecological specializations among microorganisms. Additionally, a small proportion (~0.2% of the total taxa) of the relatively abundant microbial taxa were core constituents (>50% occurrence among samples) across different temperatures, pCO2 levels, or time. Some taxa, including A4b bacteria and members of the family Saprospiraceae in the phyla Chloroflexi (syn. Chloroflexota) and Bacteroidetes (syn. Bacteroidota), respectively, as well as protists in the genera Labyrinthula and Aplanochytrium in the class Labyrinthulomycetes, and Pseudoperkinsus tapetis in the class Ichthyosporea were core constituents across temperatures, pCO2 levels, and time, suggesting that they play an important, albeit unknown, role in maintaining the structural and functional stability of the Pacific oyster spat microbiome in response to ocean warming and acidification. These findings highlight the flexibility of the spat microbiome to environmental changes.IMPORTANCEPacific oysters are the most economically important and widely farmed species of oyster, and their production depends on healthy oyster spat. In turn, spat health and productivity are affected by the associated microbiota; yet, studies have not scrutinized the effects of temperature and pCO2 on the prokaryotic and eukaryotic microbiomes of spat. Here, we show that both the prokaryotic and, for the first time, eukaryotic microbiome of Pacific oyster spat are surprisingly resilient to changes in acidification, but sensitive to ocean warming. The findings have potential implications for oyster survival amid climate change and underscore the need to understand temperature and pCO2 effects on the microbiome and the cascading effects on oyster health and productivity.

Identification, diversity, and evolution analysis of thioester-containing protein family in Pacific oyster (Crassostrea gigas) and immune response to biotic and abiotic stresses.

Thioester-containing proteins (TEPs) play a vital role in the innate immune response to biotic and abiotic stresses. In this study, the TEPs in C. gigas were identified, and their gene structure, phylogenetic relationships, collinearity relationships, expression profiles, sequence diversity, and alternative splicing were analyzed. Eight Tep genes were identified in C. gigas genome. Functional analysis and evolutionary relationships indicated a high level of homology to other mollusks TEPs. The transcriptome quantitative analysis results showed that the Tep genes in C. gigas respond to heat stress and Vibrio stress. Alternative splicing analysis revealed four Tep genes (designated A2M_1, CD109_3, CD109_5, complement C3) encode multiple alternative splice variants. Analysis of gene structure and multiple alignments revealed that seven CD109_5 variants are produced through the alternative splicing of the 19th exon, which encodes the highly variable central region. Sequence diversity analysis revealed thirteen missense variants within the 19th exon region of these seven CD109_5 alternative splice variants. Furthermore, the differential alternative splicing analysis showed significant induction of CD109_5, A2M_1 and A2M_2 variants after infection with V. parahaemolyticus. This study explores the Tep genes of C. gigas, providing insights into the molecular mechanisms underlying the involvement of C. gigas TEPs in innate immunity.

Host and Water Microbiota Are Differentially Linked to Potential Human Pathogen Accumulation in Oysters.

Oysters play an important role in coastal ecology and are a globally popular seafood source. However, their filter-feeding lifestyle enables coastal pathogens, toxins, and pollutants to accumulate in their tissues, potentially endangering human health. While pathogen concentrations in coastal waters are often linked to environmental conditions and runoff events, these do not always correlate with pathogen concentrations in oysters. Additional factors related to the microbial ecology of pathogenic bacteria and their relationship with oyster hosts likely play a role in accumulation but are poorly understood. In this study, we investigated whether microbial communities in water and oysters were linked to accumulation of Vibrio parahaemolyticus, Vibrio vulnificus, or fecal indicator bacteria. Site-specific environmental conditions significantly influenced microbial communities and potential pathogen concentrations in water. Oyster microbial communities, however, exhibited less variability in microbial community diversity and accumulation of target bacteria overall and were less impacted by environmental differences between sites. Instead, changes in specific microbial taxa in oyster and water samples, particularly in oyster digestive glands, were linked to elevated levels of potential pathogens. For example, increased levels of V. parahaemolyticus were associated with higher relative abundances of cyanobacteria, which could represent an environmental vector for Vibrio spp. transport, and with decreased relative abundance of Mycoplasma and other key members of the oyster digestive gland microbiota. These findings suggest that host and microbial factors, in addition to environmental variables, may influence pathogen accumulation in oysters. IMPORTANCE Bacteria in the marine environment cause thousands of human illnesses annually. Bivalves are a popular seafood source and are important in coastal ecology, but their ability to concentrate pathogens from the water can cause human illness, threatening seafood safety and security. To predict and prevent disease, it is critical to understand what causes pathogenic bacteria to accumulate in bivalves. In this study, we examined how environmental factors and host and water microbial communities were linked to potential human pathogen accumulation in oysters. Oyster microbial communities were more stable than water communities, and both contained the highest concentrations of Vibrio parahaemolyticus at sites with warmer temperatures and lower salinities. High oyster V. parahaemolyticus concentrations corresponded with abundant cyanobacteria, a potential vector for transmission, and a decrease in potentially beneficial oyster microbes. Our study suggests that poorly understood factors, including host and water microbiota, likely play a role in pathogen distribution and pathogen transmission.

Application of multi-omics combined with bioinformatics techniques to assess salinity stress response and tolerance mechanisms of Pacific oyster (Crassostrea gigas) during depuration.

Depuration is a vital stage to ensure the safety of oyster consumption, and salinity had a great impact on the environmental adaptability of oysters, but the underlying molecular mechanism was poorly understood during depuration stage. Here, Crassostrea gigas was depurated for 72 h at different salinity (26, 29, 32, 35, 38 g/L, corresponding to ±20%, ±10% salinity fluctuation away from oyster's production area) and then analyzed by using transcriptome, proteome, and metabolome combined with bioinformatics techniques. The transcriptome showed that the salinity stress led to 3185 differentially expressed genes and mainly enriched in amino acid metabolism, carbohydrate metabolism, lipid metabolism, etc. A total of 464 differentially expressed proteins were screened by the proteome, and the number of up-regulated expression proteins was less than the down-regulated, indicating that the salinity stress would affect the regulation of metabolism and immunity in oysters. 248 metabolites significantly changed in response to depuration salinity stress in oysters, including phosphate organic acids and their derivatives, lipids, etc. The results of integrated omics analysis indicated that the depuration salinity stress induced abnormal metabolism of the citrate cycle (TCA cycle), lipid metabolism, glycolysis, nucleotide metabolism, ribosome, ATP-binding cassette (ABC) transport pathway, etc. By contrast with Pro-depuration, more radical responses were observed in the S38 group. Based on the results, we suggested that the 10% salinity fluctuation was suitable for oyster depuration and the combination of multi-omics analysis could provide a new perspective for the analysis of the mechanism changes.

Marine mollusc extracts-Potential source of SARS-CoV-2 antivirals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus and the causative agent of coronavirus disease 2019 (Covid-19). There is an urgent need for effective antivirals to treat current Covid-19 cases and protect those unable to be vaccinated against SARS-CoV-2. Marine molluscs live in an environment containing high virus densities (>107 virus particles per ml), and there are an estimated 100,000 species in the phylum Mollusca, demonstrating the success of their innate immune system. Mollusc-derived antivirals are yet to be used clinically despite the activity of many extracts, including against human viruses, being demonstrated in vitro. Hemolymph of the Pacific oyster (Crassostrea gigas) has in vitro antiviral activity against herpes simplex virus and human adenovirus, while antiviral action against SARS-CoV-2 has been proposed by in silico studies. Such evidence suggests that molluscs, and in particular C. gigas hemolymph, may represent a source of antivirals for human coronaviruses.

The Effects of Larval Cryopreservation on the Epigenetics of the Pacific Oyster Crassostrea gigas.

High mortalities and highly variable results during the subsequent development of post-thaw larvae have been widely considered as key issues restricting the application of cryopreservation techniques to support genetic improvement programs and hatchery production in farmed marine bivalve species. To date, few studies have been undertaken to investigate the effects of cryodamage at the molecular level in bivalves. This study is the first to evaluate the effect of larval cryopreservation on the epigenetics of the resultant progenies of the Pacific oyster Crassostrea gigas. The results show that the level of DNA methylation was significantly (p < 0.05) higher and lower than that of the control when the trochophore larvae were revived and when they developed to D-stage larvae (day 1 post-fertilization), respectively, but the level returned to the control level from day 8 post-fertilization onwards. The expression of the epigenetic regulator genes DNMT3b, MeCP2, JmjCA, KDM2 and OSA changed significantly (p < 0.05) when the trochophore larvae were thawed, and then they reverted to the control levels at the D- and later larval developmental stages. However, the expression of other epigenetic regulator genes, namely, MBD2, DNMT1, CXXC1 and JmjD6, did not change at any post-thaw larval developmental stage. For the newly thawed trochophore larvae, the amount of methylated H3K4Me1 and H3K27Me1 significantly changed, and the expression of all Jumonji orthologs, except that of Jumonji5, significantly (p < 0.05) decreased. These epigenetic results agree with the data collected on larval performances (e.g., survival rate), suggesting that the effect period of the published cryopreservation technique on post-thaw larvae is short in C. gigas.

Differential DNA methylation in Pacific oyster reproductive tissue in response to ocean acidification.

BACKGROUND: There is a need to investigate mechanisms of phenotypic plasticity in marine invertebrates as negative effects of climate change, like ocean acidification, are experienced by coastal ecosystems. Environmentally-induced changes to the methylome may regulate gene expression, but methylome responses can be species- and tissue-specific. Tissue-specificity has implications for gonad tissue, as gonad-specific methylation patterns may be inherited by offspring. We used the Pacific oyster (Crassostrea gigas) - a model for understanding pH impacts on bivalve molecular physiology due to its genomic resources and importance in global aquaculture- to assess how low pH could impact the gonad methylome. Oysters were exposed to either low pH (7.31 ± 0.02) or ambient pH (7.82 ± 0.02) conditions for 7 weeks. Whole genome bisulfite sequencing was used to identify methylated regions in female oyster gonad samples. C- > T single nucleotide polymorphisms were identified and removed to ensure accurate methylation characterization. RESULTS: Analysis of gonad methylomes revealed a total of 1284 differentially methylated loci (DML) found primarily in genes, with several genes containing multiple DML. Gene ontologies for genes containing DML were involved in development and stress response, suggesting methylation may promote gonad growth homeostasis in low pH conditions. Additionally, several of these genes were associated with cytoskeletal structure regulation, metabolism, and protein ubiquitination - commonly-observed responses to ocean acidification. Comparison of these DML with other Crassostrea spp. exposed to ocean acidification demonstrates that similar pathways, but not identical genes, are impacted by methylation. CONCLUSIONS: Our work suggests DNA methylation may have a regulatory role in gonad and larval development, which would shape adult and offspring responses to low pH stress. Combined with existing molluscan methylome research, our work further supports the need for tissue- and species-specific studies to understand the potential regulatory role of DNA methylation.

Pathogenic Impacts of Bacillus cereus Strains on Crassostrea gigas.

Regarding the economic importance of bivalve farming, a great deal of interest has recently been devoted to studying the pathogenesis of infectious diseases of these mollusks to prepare for public health emergencies. Bacillus cereus is one of these pathogens; it is a ubiquitous soil bacterium responsible for many types of gastrointestinal diseases associated with food. This study was conducted to determine the pathogenic effect of B. cereus on Crassostrea gigas. This effect was studied by assessing hemocytes death using flow cytometry analysis. The results showed that only ∼15% of C. gigas were able to survive after B. cereus artificial infection with 108 CFU (colony-forming unit)/oyster. Evenly, the percentage of nonviable hemocytes gradually increased with the concentration of B. cereus, with a peak value of ∼40% after infection. Indeed, findings showed that this strain is harmful to C. gigas.

Distribution of Tetrodotoxin in Pacific Oysters (Crassostrea gigas).

A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.

Regulatory Role of Sugars on the Settlement Inducing Activity of a Conspecific Cue in Pacific Oyster Crassostrea gigas.

This study evaluated the larval settlement inducing effect of sugars and a conspecific cue from adult shell extract of Crassostrea gigas. To understand how the presence of different chemical cues regulate settlement behavior, oyster larvae were exposed to 12 types of sugars, shell extract-coated and non-coated surfaces, and under varied sugar exposure times. Lectin-glycan interaction effects on settlement and its localization on oyster larval tissues were investigated. The results showed that the conspecific cue elicited a positive concentration dependent settlement inducing trend. Sugars in the absence of a conspecific cue, C. gigas adult shell extract, did not promote settlement. Whereas, in the presence of the cue, showed varied effects, most of which were found inhibitory at different concentrations. Sugar treated larvae exposed for 2 h showed significant settlement inhibition in the presence of a conspecific cue. Neu5Ac, as well as GlcNAc sugars, showed a similar interaction trend with wheat germ agglutinin (WGA) lectin. WGA-FITC conjugate showed positive binding on the foot, velum, and mantle when exposed to GlcNAc sugars. This study suggests that a WGA lectin-like receptor and its endogenous ligand are both found in the larval chemoreceptors and the shell Ethylenediaminetetraacetic acid (EDTA) extract that may complementarily work together to allow the oyster larva greater selectivity during site selection.

Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas.

BACKGROUND: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish. RESULTS: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation. CONCLUSION: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome.

BACKGROUND: As a major threat to the oyster industry, Pacific Oyster Mortality Syndrome (POMS) is a polymicrobial disease affecting the main oyster species farmed across the world. POMS affects oyster juveniles and became panzootic this last decade, but POMS resistance in some oyster genotypes has emerged. While we know some genetic loci associated with resistance, the underlying mechanisms remained uncharacterized. So, we developed a comparative transcriptomic approach using basal gene expression profiles between different oyster biparental families with contrasted phenotypes when confronted to POMS (resistant or susceptible). RESULTS: We showed that POMS resistant oysters show differential expression of genes involved in stress responses, protein modifications, maintenance of DNA integrity and repair, and immune and antiviral pathways. We found similarities and clear differences among different molecular pathways in the different resistant families. These results suggest that the resistance process is polygenic and partially varies according to the oyster genotype. CONCLUSIONS: We found differences in basal expression levels of genes related to TLR-NFκB, JAK-STAT and STING-RLR pathways. These differences could explain the best antiviral response, as well as the robustness of resistant oysters when confronted to POMS. As some of these genes represent valuable candidates for selective breeding, we propose future studies should further examine their function.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

A new member of the runt domain family from Pacific oyster Crassostrea gigas (CgRunx) potentially involved in immune response and larvae hematopoiesis.

The Runx family is a kind of heteromeric transcription factors, which is defined by the presence of a runt domain. As transcriptional regulator during development and cell fate specification, Runx is best known for its critical roles in hematopoiesis. In the present study, a Runx transcription factor (designed as CgRunx) was identified and characterized from the oyster Crassostrea gigas. The complete coding sequence of CgRunx was of 1638 bp encoding a predicted polypeptide of 545 amino acids with one conserved runt domain, which shared high similarity with other reported Runx proteins. CgRunx was highly expressed in hemocytes, gill and mantle both at the protein and nucleic acid levels. CgRunx protein was localized specifically in the cell nuclei of hemocytes, and distributed at the tubule lumen of gill filament. During the larval developmental stages, the mRNA transcripts of CgRunx gradually increased after fertilization, reached to a relative high level at the 8 cell embryos and the blastula stage of 2-4 hpf (hours post fertilization) (about 40-fold), and peaked at early trochophore larvae (10 hpf) (about 60-fold). Whole-mount immunofluorescence assay further revealed that the abundant immunofluorescence signals of CgRunx distributed through the whole embryo at blastula stage (5 hpf), and progressively reduced with the development to a ring structure around the dorsal region in trochophore larvae (10 hpf). Scattered positive immunoreactivity signals finally appeared in the velum region of D-veliger larvae. After LPS and Vibrio splendidus stimulations, the expression levels of CgRunx mRNA in hemocytes were up-regulated significantly compared with that in the control (0 h), which were 2.98- and 2.46-fold (p < 0.05), 2.67- and 1.5-fold (p < 0.05), 2.36- and 1.38-fold (p < 0.05) at 3 h, 6 h and 12 h, respectively. These results collectively suggested that CgRunx involved in immune response and might participate in larvae hematopoiesis in oyster.

Multiplex PCR for the rapid detection of insulin-like growth factor in the Pacific oyster, Crassostrea gigas: a useful indicator for growth assessment.

Insulin-like growth factor (IGF) expression plays a critical role in the endocrine regulation of proliferation, differentiation, and growth in shellfish as well as in fish. The Pacific oyster, Crassostrea gigas, is a significant aquaculture species that comprises a large percentage of the Korean shellfish industry; moreover, its growth is economically important in aquaculture. However, when measuring the growth rate in shellfish, the soft tissue weight is difficult to determine because of the shell weight. In the present study, we describe an indirect method of measuring the growth rate using multiplex polymerase chain reaction (PCR) and analyzing levels of molluscan insulin-related peptide (MIP), the acid labile subunit of the IGF-binding protein complex (IGFBP ALS), and insulin-related peptide receptor (CIR) in Pacific oysters. The predicted sizes of amplicons were 776, 537, 380, and 198 bp, and the detection limit of the annealing temperatures was confirmed to be 65 °C. The annual expression of MIP and IGFBP ALS in tissues reached high levels in the winter following the condition index (CI). MIP and IGFBP ALS in male gonads and CIR in female gonads were related to the CI. This newly improved multiplex PCR provides an indirect measure of the growth rate; thus, it can be used to rapidly assess the growth rate. In addition, this method can supplement traditional growth data from oyster farms.

The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster (Crassostrea gigas) microbiome.

AIMS: This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. METHODS AND RESULTS: Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. CONCLUSIONS: Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.

First detection of Ostreid herpesvirus 1 in wild Crassostrea gigas in Argentina.

Ostreid herpesvirus 1 (OsHV-1) is a DNA virus of the genus Ostreavirus (Malacoherpesviridae family, Herpesvirales order). Worldwide, OsHV-1 and its microvariants have been associated with increased mortality of Pacific oysters, Crassostrea gigas. Adult asymptomatic oysters also have shown a high prevalence of viral infection. As a consequence, surveillance is needed to better describe OsHV-1 diversity, pathogenicity, clinical signs, and geographical distribution. We examined Crassostrea gigas sampled in October 2017 from the inner zone of the Bahía Blanca Estuary, Argentina, and found that 8 of 30 specimens (26.7%) presented macroscopic lesions in mantle tissues. Histological analysis revealed abnormal presentation of mantle epithelial cells and connective tissues. Conventional and real-time PCR conducted on the oyster samples revealed 70% to be positive for presence of OsHV-1 DNA. The nucleotide sequence of the amplicon obtained from one sample using the primer pair IA1/IA2 (targeting ORF 42/43) was 99% identical to OsHV-1 reference as well as µVar strains B and A (KY271630, KY242785.1), sequenced from France and Ireland. This finding represents the first detection of OsHV-1 DNA in a wild population of C. gigas in Argentina in association with gross mantle lesions.

Application of the bacteriophage pVco-14 to prevent Vibrio coralliilyticus infection in Pacific oyster (Crassostrea gigas) larvae.

Vibrio coralliilyticus infects a variety of shellfish larvae, including Pacific oyster (Crassostrea gigas) larvae worldwide, and remains a major constraint in marine bivalve aquaculture practice, especially in artificial seed production facilities. In this study, we isolated and characterized the bacteriophage (phage) that specifically infects V. coralliilyticus. The phage was designated pVco-14 and classified as Siphoviridae. We also investigated the potential efficacy of the isolated phage against V. coralliilyticus infection. We conducted a survey to replace the overuse of antibiotics, which generate multi-antibiotic-resistant strains and causes environmental pollution. The latent period of pVco-14 was estimated to be approximately 30 min, whereas the burst size was 13.3 PFU/cell. The phage was found to infect four strains of tested V. coralliilyticus. pVco-14 was stable at wide temperature (4-37 °C) and pH (5.0-9.0) ranges. Eighty-one percent of oyster larvae died in an immersion challenge at a dose 1.32 × 105 CFU/ml of virulent V. coralliilyticus (strain 58) within 24 h. When oyster larvae were pre-treated with the phage before the bacterial challenge (bacterial conc.: 1.32 × 104 and 1.32 × 105 CFU/ml), mortality of the phage-treated oyster larvae was lower than that of the untreated control. These results suggest that pVco-14 has potential to be used as a prophylactic agent for preventing V. coralliilyticus infection in marine bivalve hatcheries and can reduce the overuse of antibiotics.

Fine-scale temporal dynamics of herpes virus and vibrios in seawater during a polymicrobial infection in the Pacific oyster Crassostrea gigas.

The Pacific oyster Crassostrea gigas is currently being impacted by a polymicrobial disease that involves early viral infection by ostreid herpesvirus-1 (OsHV-1) followed by a secondary bacterial infection leading to death. A widely used method of inducing infection consists of placing specific pathogen-free oysters ('recipients') in cohabitation in the laboratory with diseased oysters that were naturally infected in the field ('donors'). With this method, we evaluated the temporal dynamics of pathogen release in seawater and the cohabitation time necessary for disease transmission and expression. We showed that OsHV-1 and Vibrio spp. in the seawater peaked concomitantly during the first 48 h and decreased thereafter. We found that 1.5 h of cohabitation with donors was enough time to transmit pathogens to recipients and to induce mortality later, reflecting the highly contagious nature of the disease. Finally, mortality of recipients was associated with increasing cohabitation time with donors until reaching a plateau at 20%. This reflects the cumulative effect of exposure to pathogens. The optimal cohabitation time was 5-6 d, the mortality of recipients occurring 1-2 d earlier.

Anti-Inflammatory Activity of ß-thymosin Peptide Derived from Pacific Oyster (Crassostrea gigas) on NO and PGE2 Production by Down-Regulating NF-κB in LPS-Induced RAW264.7 Macrophage Cells.

ß-thymosin is known for having 43 amino acids, being water-soluble, having a light molecular weight and ubiquitous polypeptide. The biological activities of ß-thymosin are diverse and include the promotion of wound healing, reduction of inflammation, differentiation of T cells and inhibition of apoptosis. Our previous studies showed that oyster ß-thymosin originated from the mantle of the Pacific oyster, Crassostrea gigas and had antimicrobial activity. In this study, we investigated the anti-inflammatory effects of oyster ß-thymosin in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells using human ß-thymosin as a control. Oyster ß-thymosin inhibited the nitric oxide (NO) production as much as human ß-thymosin in LPS-induced RAW264.7 cells. It also showed that oyster ß-thymosin suppressed the expression of prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, oyster ß-thymosin reduced inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Oyster ß-thymosin also suppressed the nuclear translocation of phosphorylated nuclear factor-κB (NF-κB) and degradation of inhibitory κB (IκB) in LPS-induced RAW264.7 cells. These results suggest that oyster ß-thymosin, which is derived from the mantle of the Pacific oyster, has as much anti-inflammatory effects as human ß-thymosin. Additionally, oyster ß-thymosin suppressed NO production, PGE2 production and inflammatory cytokines expression via NF-κB in LPS-induced RAW264.7 cells.