Responses of diploid and triploid Pacific oysters Crassostrea gigas to Vibrio infection in relation to their reproductive status

Several Vibrio species are known to be pathogenic to the Pacific oyster Crassostrea gigas. Survival varies according to pathogen exposure and high mortality events usually occur in summer during gametogenesis. In order to study the effects of gametogenetic status and ploidy (a factor known to affect reproduction allocation in oysters) on vibriosis survival, we conducted two successive experiments. Our results demonstrate that a common bath challenge with pathogenic Vibriosplendidus and Vibrio aestuarianus on a mixture of mature, spawning and non-mature oysters can lead to significant mortality. Previous bath challenges, which were done using only non-mature oysters, had not produced mortality. Immunohistochemical analyses showed the affinity of Vibrio for gonadic tissues, highlighting the importance of sexual maturity for vibriosis infection processes in oysters. Mortality rate results showed poor repeatability between tanks, however, in this bath challenge. We then tested a standardized and repeatable injection protocol using two different doses of the same combination of two Vibrio species on related diploid and triploid oysters at four different times over a year. Statistical analyses of mortality kinetics over a 6-day period after injection revealed that active gametogenesis periods correspond to higher susceptibility to vibriosis and that there is a significant interaction of this seasonal effect with ploidy. However, no significant advantage of triploidy was observed. Triploid oysters even showed lower survival than diploid counterparts in winter. Results are discussed in relation to differing energy allocation patterns between diploid and triploid Pacific oysters.     

A Persistent, Productive, and Seasonally Dynamic Vibriophage Population within Pacific Oysters (Crassostrea gigas)

In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 × 10⁴ to 11 × 10⁴ viruses cm⁻³) occurred year round in oysters, even when V. parahaemolyticus was undetectable (<3 cells cm⁻³). Viruses were not detected (<10 ml⁻¹) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by ~50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.     

Hemolymph microbiome of Pacific oysters in response to temperature,temperature stress and infection

Microbiota provide their hosts with a range of beneficial services, including defense from external pathogens. However, host-associated microbial communities themselves can act as a source of opportunistic pathogens depending on the environment. Marine poikilotherms and their microbiota are strongly influenced by temperature, but experimental studies exploring how temperature affects the interactions between both parties are rare. To assess the effects of temperature, temperature stress and infection on diversity, composition and dynamics of the hemolymph microbiota of Pacific oysters (Crassostrea gigas), we conducted an experiment in a fully-crossed, three-factorial design, in which the temperature acclimated oysters (8 or 22 °C) were exposed to temperature stress and to experimental challenge with a virulent Vibrio sp. strain. We monitored oyster survival and repeatedly collected hemolymph of dead and alive animals to determine the microbiome composition by 16s rRNA gene amplicon pyrosequencing. We found that the microbial dynamics and composition of communities in healthy animals (including infection survivors) were significantly affected by temperature and temperature stress, but not by infection. The response was mediated by changes in the incidence and abundance of operational taxonomic units (OTUs) and accompanied by little change at higher taxonomic levels, indicating dynamic stability of the hemolymph microbiome. Dead and moribund oysters, on the contrary, displayed signs of community structure disruption, characterized by very low diversity and proliferation of few OTUs. We can therefore link short-term responses of host-associated microbial communities to abiotic and biotic factors and assess the potential feedback between microbiota dynamics and host survival during disease.     

Populations,not clones,are the unit of vibrio pathogenesis in naturally infected oysters

Disease in oysters has been steadily rising over the past decade, threatening the long-term survival of commercial and natural stocks. Our understanding and management of such diseases are of critical importance as aquaculture is an important aspect of dealing with the approaching worldwide food shortage. Although some bacteria of the Vibrio genus isolated from diseased oysters have been demonstrated to be pathogenic by experimental infection, direct causality has not been established. Little is known about the dynamics of how the bacterial population hosted by oysters changes during disease progression. Combining experimental ecology, a high-throughput infection assay and genome sequencing, we show that the onset of disease in oysters is associated with progressive replacement of diverse benign colonizers by members of a phylogenetically coherent virulent population. Although the virulent population is genetically diverse, all members of that population can cause disease. Comparative genomics across virulent and nonvirulent populations identified candidate virulence factors that were clustered in population-specific genomic regions. Genetic analyses revealed that one gene for a candidate virulent factor, a putative outer membrane protein, is necessary for infection of oysters. Finally, analyses of oyster mortality following experimental infection suggest that disease onset can be facilitated by the presence of nonvirulent strains. This is a new form of polymicrobial disease, in which nonpathogenic strains contribute to increase mortality.     

Increases in the Amounts of Vibrio spp. in Oysters upon Addition of Exogenous Bacteria

The bacterial pathogen Vibrio vulnificus is found naturally in brackish coastal waters but can be greatly concentrated by filter-feeding organisms such as shellfish. Numerous experiments in which exogenous V. vulnificus cells are added to oysters in an attempt to measure uptake and depuration have been performed. In nearly all cases, results have shown that laboratory-grown bacteria are rapidly taken up by the oysters but ultimately eliminated, while naturally present Vibrio populations in oysters are resistant to depuration. In this study, oysters harvested during winter months, with low culturable Vibrio concentrations, were incubated in aquaria supplemented with strains of V. vulnificus that were either genotypically or phenotypically distinct from the background bacteria. These exogenous cells were eliminated from the oysters, as previously seen, but other vibrios already inhabiting the oysters responded to the V. vulnificus inoculum by rapidly increasing in number and maintaining a large stable population. The presence of such an oyster-adapted Vibrio population would be expected to prevent colonization by exogenous V. vulnificus cells, thus explaining the rapid depuration of these added bacteria.     

Intraspecific Diversity of Vibrio vulnificus in Galveston Bay Water and Oysters as Determined by Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Dynamics of Seawater Bacterial Communities in a Shellfish Hatchery

Bacterial disease is a significant issue for larviculture of several species of shellfish, including oysters. One source of bacteria is the seawater used throughout the hatchery. In this study carried out at a commercial oyster hatchery in Tasmania, Australia, the diversity of the bacterial community and its relationship with larval production outcomes were studied over a 2-year period using terminal restriction fragment length polymorphism and tag-encoded pyrosequencing. The bacterial communities were very diverse, dominated by the Alphaproteobacteria, Gammaproteobacteria, Flavobacteria and Cyanobacteria. The communities were highly variable on scales of days, weeks and seasons. The difference between the intake seawater and treated clean seawater used in the hatchery was smaller than the observed temporal differences in the seawater throughout the year. No clear correlation was observed between production outcomes and the overall bacterial community structure. However, one group of Cyanobacterial sequences was more abundant when mass mortality events occurred than when healthy spat were produced although they were always present.     

A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.     

Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD₅₀) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10⁴ to 3.0 × 10⁴ CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD₅₀s between 1.2 × 10⁴ and 4.0 × 10⁴ CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10⁴ CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.     

Characterization of the Bacterial Diversity in Indo-West Pacific Loliginid and Sepiolid Squid Light Organs

Loliginid and sepiolid squid light organs are known to host a variety of bacterial species from the family Vibrionaceae, yet little is known about the species diversity and characteristics among different host squids. Here we present a broad-ranging molecular and physiological analysis of the bacteria colonizing light organs in loliginid and sepiolid squids from various field locations of the Indo-West Pacific (Australia and Thailand). Our PCR-RFLP analysis, physiological characterization, carbon utilization profiling, and electron microscopy data indicate that loliginid squid in the Indo-West Pacific carry a consortium of bacterial species from the families Vibrionaceae and Photobacteriaceae. This research also confirms our previous report of the presence of Vibrio harveyi as a member of the bacterial population colonizing light organs in loliginid squid. pyrH sequence data were used to confirm isolate identity, and indicates that Vibrio and Photobacterium comprise most of the light organ colonizers of squids from Australia, confirming previous reports for Australian loliginid and sepiolid squids. In addition, combined phylogenetic analysis of PCR-RFLP and 16S rDNA data from Australian and Thai isolates associated both Photobacterium and Vibrio clades with both loliginid and sepiolid strains, providing support that geographical origin does not correlate with their relatedness. These results indicate that both loliginid and sepiolid squids demonstrate symbiont specificity (Vibrionaceae), but their distribution is more likely due to environmental factors that are present during the infection process. This study adds significantly to the growing evidence for complex and dynamic associations in nature and highlights the importance of exploring symbiotic relationships in which non-virulent strains of pathogenic Vibrio species could establish associations with marine invertebrates.     

Autophagy plays an important role in protecting Pacific oysters from OsHV-1 and Vibrio aestuarianus infections

Recent mass mortality outbreaks around the world in Pacific oysters, Crassostrea gigas, have seriously affected the aquaculture economy. Although the causes for these mortality outbreaks appear complex, infectious agents are involved. Two pathogens are associated with mass mortality outbreaks, the virus ostreid herpesvirus 1 (OsHV-1) and the bacterium Vibrio aestuarianus. Here we describe the interactions between these 2 pathogens and autophagy, a conserved intracellular pathway playing a key role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters.     

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 10² to 5.6 × 10² bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 10⁴), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 10⁴ bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10⁵ bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 × 10⁷ was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8°C. Increased bacterial numbers in seawater at 4°C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.     

Molecular analysis of the bacterial communities in the live Pacific oyster (Crassostrea gigas) and the influence of postharvest temperature on its structure

Aims: To evaluate the effect of postharvest temperature on bacterial communities in live Pacific oysters (Crassostrea gigas) using nonculture‐based methods. Methods and Results: Live oysters were compared before and after storage at 4, 6, 15, 20 and 30°C using terminal restriction fragment length polymorphism (T‐RFLP). Bacterial communities in freshly harvested (control) vs stored oysters were significantly different. Changes in bacterial communities at 4, 15 and 30°C observed by T‐RFLP were further investigated by clone library analysis. Members of the Proteobacteria predominated (43·0–57·0% of clones) in control oysters, while storage altered the bacterial profile. At 4°C, Psychrilyobacter spp. (phylum Fusobacteria) predominated (43·8% of clones), while at 15 and 30°C, members of the phylum Bacteroidetes represented 63·0 and 60·2% of clones, respectively. High microbial diversity in oysters was observed, with at least 73 different genera‐related clones among all samples. Conclusions: Changes in the overall bacterial community of Pacific oysters were influenced by storage temperature and would likely not be detected by standard culture‐based methods currently used to assess oyster quality. Certain dominant genera, such as Psychrilyobacter, Polynucleobacter and a bacterial group related to Alkaliflexus, should be further studied as possible indicators for postharvest temperature control. Significance and Impact of the Study: This work is the first report describing the effect of different storage temperatures on bacterial diversity in postharvest live Pacific oysters using molecular‐based methods.     

Adult Pacific Oyster (Crassostrea gigas) May Have Light Sensitivity

Light-sensitivity is an important aspect of mollusk survival as it plays a vital role in reproduction and predator avoidance. In the Pacific oyster Crassostrea gigas light sensitivity has been demonstrated in the larval stage but has not yet been conclusively demonstrated in adult oysters. In this paper we describe an experiment which was undertaken to determine if adult Pacific oysters were sensitive to light. One LED flashlight was used to shine light onto adult oysters while they were filtering seawater through their shell openings. We found that the degree of opening increased gradually during the light period but rapidly decreased when the flashlight was turned off in the treated group but not in the control group. These results suggest that adult Pacific oyster may be sensitive to light.     

Kanagawa-Negative, <Emphasis Type="Italic">tdh</Emphasis>- and <Emphasis Type="Italic">trh</Emphasis>-Positive <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> Isolated from Fresh Oysters Marketed in Fortaleza,Brazil

Between October 2008 and June 2009, 15 samples of 10 live oysters each (Crassostrea rhizophorae) measuring 8.31–10.71 cm were purchased from a restaurant on the seashore of Fortaleza, Brazil. The Vibrio count ranged from 75 (estimated) to 43,500 CFU/g. Fourteen species were identified among the 56 isolated Vibrio strains, with V. parahaemolyticus as the most prevalent. Two of the 17 V. parahaemolyticus strains were urease-positive and tdh- and trh-positive on multiplex PCR, but neither produced β-hemolysis halos in Wagatsuma agar. Thus, fresh oysters served in natura in Fortaleza, Brazil, were found to contain Vibrio strains known to cause gastroenteritis in humans.     

Mass mortality in Pacific oysters is associated with a specific gene expression signature

Mass mortality events occur in natural and cultured communities of bivalve molluscs. The Pacific oyster, Crassostrea gigas, is a dominant species in many intertidal locations as well as an important aquacultured bivalve species, and for the last 50 years, adult oysters have suffered frequent and extreme mass mortality events during summer months. To investigate the molecular changes that precede these mortality events, we employed a novel nonlethal sampling approach to collect haemolymph samples from individual oysters during the period that preceded a mortality event. Microarray-based gene expression screening of the collected haemolymph was used to identify a mortality gene expression signature that distinguished oysters that survived the mortality event from those individuals that died during the event. The signature was cross-validated by comparing two separate episodes of mortality. Here, we report that near-mortality oysters can be distinguished from longer-lived oysters by the elevated expression of genes associated with cell death, lysosomal proteolysis, and cellular assembly and organization. These results show the potential utility of nonlethal sampling approaches for investigating the environmental causes of mortality in natural populations in the field, and for predicting when such events could occur and which individuals will be affected.     

Adaptation to enemy shifts: rapid resistance evolution to local Vibrio spp. in invasive Pacific oysters

One hypothesis for the success of invasive species is reduced pathogen burden, resulting from a release from infections or high immunological fitness of invaders. Despite strong selection exerted on the host, the evolutionary response of invaders to newly acquired pathogens has rarely been considered. The two independent and genetically distinct invasions of the Pacific oyster Crassostrea gigas into the North Sea represent an ideal model system to study fast evolutionary responses of invasive populations. By exposing both invasion sources to ubiquitous and phylogenetically diverse pathogens (Vibrio spp.), we demonstrate that within a few generations hosts adapted to newly encountered pathogen communities. However, local adaptation only became apparent in selective environments, i.e. at elevated temperatures reflecting patterns of disease outbreaks in natural populations. Resistance against sympatric and allopatric Vibrio spp. strains was dominantly inherited in crosses between both invasion sources, resulting in an overall higher resistance of admixed individuals than pure lines. Therefore, we suggest that a simple genetic resistance mechanism of the host is matched to a common virulence mechanism shared by local Vibrio strains. This combination might have facilitated a fast evolutionary response that can explain another dimension of why invasive species can be so successful in newly invaded ranges.     

Noninvasive Analysis of Microbiome Dynamics in the Fruit Fly Drosophila melanogaster

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.