Polymorphism in a serine protease inhibitor gene and its association with disease resistance in the eastern oyster (Crassostrea virginica Gmelin)

Serine protease inhibitors (SPIs) are a superfamily of structurally related but functionally diverse proteins found in almost all organisms ranging from viruses to humans. Some of them play important roles in host defense. A recently identified SPI from the eastern oyster (Crassostrea virginica), cvSI-1, has been shown to inhibit the proliferation of the Dermo pathogen Perkinsus marinus in vitro, although direct evidence linking it to disease resistance is lacking. In this study, we identified polymorphism in the cvSI-1 gene and studied its association with improved survival after disease-caused mortalities and in disease-resistant eastern oyster strains. Full-cDNA sequence of cvSI-1 was sequenced in a diverse panel of oysters, revealing 12 single-nucleotide polymorphisms (SNPs) in the 273 bp coding region: five were synonymous and seven non-synonymous. The Dn/Ds ratio, 1.4, suggests that cvSI-1 is under positive selection. Selected SNPs were genotyped in families before and after disease-caused mortalities as well as in disease-resistant and susceptible strains. At SNP198, the C allele consistently increased in frequency after mortalities that are caused primarily by Dermo and possibly also by MSX. Its frequency in the disease-resistant strain is significantly higher than that in the susceptible strains and the base population from which the selected strains were derived. These results indicate that polymorphism at cvSI-1 is associated with Dermo (possibly also MSX) resistance in the eastern oyster. SNP198 is a synonymous mutation, and its association with disease resistance may be due to its close linkage to a functional polymorphism nearby.     

Sensitivity of a digoxigenin-labelled DNA probe in detecting Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), in oysters

The protistan parasite Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), induces mortality and reduces marketability in the Pacific oyster, Crassostrea gigas, in British Columbia, Canada. This parasite is a pathogen of international concern because it infects a range of oyster species, and because its life cycle and mode of transmission are unknown. A digoxigenin-labelled DNA probe in situ hybridisation technique (DIG-ISH) was developed, and its detection sensitivity was compared to standard histological sections stained with haematoxylin and eosin stain (H&E-histo). In H&E-histo preparations, the detection of M. mackini was certain only when the parasite occurred within the vesicular connective tissue of adult oysters. However, the DIG-ISH technique clearly demonstrated the presence of infection in all other host tissues as well as in juvenile oysters with poorly developed vesicular connective tissue. The probe hybridised strongly to M. mackini, did not hybridise to oyster tissues or with the other shellfish parasites tested, and was more sensitive for detecting infections when compared to H&E-histo.     

HMGB in Mollusk Crassostrea ariakensis Gould: Structure,Pro-Inflammatory Cytokine Function Characterization and Anti-Infection Role of Its Antibody

Background

Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO). Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box) can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB’s antibody (anti-HMGB) has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB) and anti-CaHMGB (Ca-HMGB’s antibody) in oyster RLO/LPS (RLO or LPS)-induced disease or inflammation.

Methodology/Principal Findings

Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4–12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4–12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates.

Conclusions/Significance

Ca-HMGB can be released extracellularly and its subcellular localization varies when stimulated with RLO. Ca-HMGB is involved in oyster immune reactions and functions as a pro-inflammatory cytokine. Anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory responses and hemocyte necrosis and apoptosis, suggesting that Ca-HMGB is a potential target to prevent and control RLO/LPS-induced disease or inflammation.     

A restoration suitability index model for the eastern oyster (Crassostrea virginica) in the Mission-Aransas Estuary, TX, USA

Oyster reefs are one of the most threatened marine habitats on earth, with habitat loss resulting from water quality degradation, coastal development, destructive fishing practices, overfishing, and storm impacts. For successful and sustainable oyster reef restoration efforts, it is necessary to choose sites that support long-term growth and survival of oysters. Selection of suitable sites is critically important as it can greatly influence mortality factors and may largely determine the ultimate success of the restoration project. The application of Geographic Information Systems (GIS) provides an effective methodology for identifying suitable sites for oyster reef restoration and removes much of the uncertainty involved in the sometimes trial and error selection process. This approach also provides an objective and quantitative tool for planning future oyster reef restoration efforts. The aim of this study was to develop a restoration suitability index model and reef quality index model to characterize locations based on their potential for successful reef restoration within the Mission-Aransas Estuary, Texas, USA. The restoration suitability index model focuses on salinity, temperature, turbidity, dissolved oxygen, and depth, while the reef quality index model focuses on abundance of live oysters, dead shell, and spat. Size-specific Perkinsus marinus infection levels were mapped to illustrate general disease trends. This application was effective in identifying suitable sites for oyster reef restoration, is flexible in its use, and provides a mechanism for considering alternative approaches. The end product is a practical decision-support tool that can be used by coastal resource managers to improve oyster restoration efforts. As oyster reef restoration activities continue at small and large-scales, site selection criteria are critical for assisting stakeholders and managers and for maximizing long-term sustainability of oyster resources.     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD.     

Survival of eastern oysters Crassostrea virginica from three lines following experimental challenge with bacterial pathogens

Shellfish production is often affected by bacterial pathogens that cause high losses in hatcheries and nurseries. We evaluated the relative survival of larvae and juveniles of 3 Crassostrea virginica oyster lines: (1) GHP, a Rhode Island line; (2) NEHY, a line resistant to dermo and multinucleated sphere X diseases; and (3) FLOWERS, a line resistant to Roseovarius oyster disease, experimental challenge with Vibrio spp. isolates RE22 and RE101, causative agents of bacillary necrosis in Pacific oyster larvae, and the type strain of Roseovarius crassostreae, causative agent of Roseovarius oyster disease. All of the isolates were able to induce significant mortalities in oyster larvae and juveniles. Susceptibility to bacterial challenge in larvae was significantly higher at 25 degrees C than at 20 degrees C. Susceptibility decreased with oyster age; mean survival time ranged from 24 h in oyster larvae to more than 6 wk in juveniles. Significant differences in susceptibility to bacterial challenge were observed between oyster lines; NEHY was the most resistant line overall. Extracellular products (ECPs) from Vibrio sp. RE22 and R. crassostreae, as well as viable bacteria, were toxic to hemocytes from the 3 oyster lines, suggesting that ECPs are involved in pathogenesis and that external and mucosal barriers to infection are major contributors to resistance to bacterial challenge. These protocols will be useful in the elucidation of mechanisms of bacterial pathogenesis and resistance to infection in oysters.     

Evidence in oyster of a plasma extracellular superoxide dismutase which binds LPS

We have characterized in the oyster Crassostrea gigas an extracellular superoxide dismutase (Cg-EcSOD) which appears to bind lipopolysaccharides (LPS). The protein has been purified from the oyster plasma and identified as a Cu/ZnSOD according to its N-terminal sequencing and biological activity. Cg-EcSOD expression and synthesis are restricted to hemocytes as revealed by in situ hybridization and immunocytochemistry. Cg-EcSOD-expressing hemocytes were seen in blood circulation, in connective tissues, and closely associated to endothelium blood vessels. Cg-EcSOD presents in its amino acid sequence a LPS-binding motif found in the endotoxin receptor CD14 and we show that the protein displays an affinity to Escherichia coli bacteria and with LPS and Lipid A. Additionally, an RGD motif known to be implicated in the association to membrane integrin receptor is present in the amino acid sequence. The purified Cg-EcSOD was shown to bind to oyster hemocytes and to be immunocolocalized with a beta-integrin-like receptor.     

The ecology of Bonamia and decline of bivalve molluscs

Bonamia is a protozoan parasite of the haemocytes of oysters (Tiostrea chilensis), in which it has an annual developmental cycle between November and August each year. The parasite transmits directly, oyster to oyster, and therefore disease spread is related to host stock density. The Foveaux Strait oyster population experiences large mortalities every 20-30 years, and these may be attributable to Bonamia. The parasite appears to become less pathogenic at the end of, and probably between, mass mortalities, and some oysters appear more tolerant of infection than others. On the basis of these observations, and considering other protist pathogen:oyster models, the apparently reduced pathogenicity of Bonamia is discussed in terms of parasite kinetics. The population dynamics and selection of parasite tolerant host stocks, and kinetics of parasite transmission, may explain the cyclic nature of large-scale mortalities in Foveaux Strait, without change in parasite pathogenicity.     

Immune parameters of QX-resistant and wild caught Saccostrea glomerata hemocytes in relation to Marteilia sydneyi infection

Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters.     

Landscape-Level Variation in Disease Susceptibility Related to Shallow-Water Hypoxia

Diel-cycling hypoxia is widespread in shallow portions of estuaries and lagoons, especially in systems with high nutrient loads resulting from human activities. Far less is known about the effects of this form of hypoxia than deeper-water seasonal or persistent low dissolved oxygen. We examined field patterns of diel-cycling hypoxia and used field and laboratory experiments to test its effects on acquisition and progression of Perkinsus marinus infections in the eastern oyster, Crassostrea virginica, as well as on oyster growth and filtration. P. marinus infections cause the disease known as Dermo, have been responsible for declines in oyster populations, and have limited success of oyster restoration efforts. The severity of diel-cycling hypoxia varied among shallow monitored sites in Chesapeake Bay, and average daily minimum dissolved oxygen was positively correlated with average daily minimum pH. In both field and laboratory experiments, diel-cycling hypoxia increased acquisition and progression of infections, with stronger results found for younger (1-year-old) than older (2-3-year-old) oysters, and more pronounced effects on both infections and growth found in the field than in the laboratory. Filtration by oysters was reduced during brief periods of exposure to severe hypoxia. This should have reduced exposure to waterborne P. marinus, and contributed to the negative relationship found between hypoxia frequency and oyster growth. Negative effects of hypoxia on the host immune response is, therefore, the likely mechanism leading to elevated infections in oysters exposed to hypoxia relative to control treatments. Because there is considerable spatial variation in the frequency and severity of hypoxia, diel-cycling hypoxia may contribute to landscape-level spatial variation in disease dynamics within and among estuarine systems.     

Perkinsus marinus in coastal Georgia, USA, following a prolonged drought

Oysters Crassostrea virginica are 'keystone' estuarine species in the southeastern USA, providing essential fish habitat, food for human consumption, filtration of water bodies, and protection against shoreline erosion. Relatively few oyster pathology studies have been conducted in Georgia. The parasitic protozoan Perkinsus marinus was first observed here in the 1960s, but has not been investigated since the late 1990s, when increasing oyster infection levels were apparent. In the late 1990s and early 2000s, the region suffered a prolonged drought, resulting in elevated salinities and the proliferation of various diseases in the marine environment. By 2003, salinities had returned to normal levels, but the effect of the drought on oysters was unknown. In June 2003, oyster reefs throughout Chatham County were sampled to evaluate the prevalence and intensity of P. marinus. The disease appears to have remained prevalent in the coastal waters of Georgia (100% prevalence at some sites), but the intensity was low, ranging from 0 to 1.83 on a scale where heavy infections rated a score of 5. While the disease did not occur at levels high enough to cause oyster mortalities, further monitoring, particularly on a temporal scale, is warranted.     

A genetic test for recruitment enhancement in Chesapeake Bay oysters, Crassostrea virginica, after population supplementation with a disease tolerant strain

Many of the methods currently employed to restore Chesapeake Bay populations of the eastern oyster, Crassostrea virginica, assume closed recruitment in certain sub-estuaries despite planktonic larval durations of 2–3 weeks. In addition, to combat parasitic disease, artificially selected disease tolerant oyster strains are being used for population supplementation. It has been impossible to fully evaluate these unconventional tactics because offspring from wild and selected broodstock are phenotypically indistinguishable. This study provides the first direct measurement of oyster recruitment enhancement by using genetic assignment tests to discriminate locally produced progeny of a selected oyster strain from progeny of wild parents. Artificially selected oysters (DEBY strain) were planted on a single reef in each of two Chesapeake Bay tributaries in 2002, but only in the Great Wicomico River (GWR) were they large enough to potentially reproduce the same year. Assignment tests based on eight microsatellite loci and mitochondrial DNA markers were applied to 1579 juvenile oysters collected throughout the GWR during the summer of 2002. Only one juvenile oyster was positively identified as an offspring of the 0.75 million DEBY oysters that were planted in the GWR, but 153 individuals (9.7%) had DEBY ×wild F1 multilocus genotypes. Because oyster recruitment was high across the region in 2002, the proportionately low enhancement measured in the GWR would not otherwise have been recognized. Possible causes for low enhancement success are discussed, each bearing on untested assumptions underlying the restoration methods, and all arguing for more intensive evaluation of each component of the restoration strategy.     

Climate change, precipitation and impacts on an estuarine refuge from disease

Background

Oysters play important roles in estuarine ecosystems but have suffered recently due to overfishing, pollution, and habitat loss. A tradeoff between growth rate and disease prevalence as a function of salinity makes the estuarine salinity transition of special concern for oyster survival and restoration. Estuarine salinity varies with discharge, so increases or decreases in precipitation with climate change may shift regions of low salinity and disease refuge away from optimal oyster bottom habitat, negatively impacting reproduction and survival. Temperature is an additional factor for oyster survival, and recent temperature increases have increased vulnerability to disease in higher salinity regions.

Methodology/Principal Findings

We examined growth, reproduction, and survival of oysters in the New York Harbor-Hudson River region, focusing on a low-salinity refuge in the estuary. Observations were during two years when rainfall was above average and comparable to projected future increases in precipitation in the region and a past period of about 15 years with high precipitation. We found a clear tradeoff between oyster growth and vulnerability to disease. Oysters survived well when exposed to intermediate salinities during two summers (2008, 2010) with moderate discharge conditions. However, increased precipitation and discharge in 2009 reduced salinities in the region with suitable benthic habitat, greatly increasing oyster mortality. To evaluate the estuarine conditions over longer periods, we applied a numerical model of the Hudson to simulate salinities over the past century. Model results suggest that much of the region with suitable benthic habitat that historically had been a low salinity refuge region may be vulnerable to higher mortality under projected increases in precipitation and discharge.

Conclusions/Significance

Predicted increases in precipitation in the northeastern United States due to climate change may lower salinities past important thresholds for oyster survival in estuarine regions with appropriate substrate, potentially disrupting metapopulation dynamics and impeding oyster restoration efforts, especially in the Hudson estuary where a large basin constitutes an excellent refuge from disease.     

Copper exposure affects hemocyte apoptosis and Perkinsus marinus infection in eastern oysters Crassostrea virginica (Gmelin)

Dermo disease in the eastern oyster (Crassostrea virginica) is caused by an intracellular protistan parasite Perkinsus marinus. The progression and outcome of this disease is determined by a complex interplay between the host's immunity and parasite's escape mechanisms, both of which can be influenced by environmental pollutants including heavy metals such as copper (Cu). The goal of the present study was to determine the effects of Cu on the levels of apoptosis (which can serve as an important host defense mechanism) in oyster immune cells (hemocytes) in?vitro and in?vivo as well as on the establishment of P.?marinus infections in?vivo. Surprisingly, Cu exerted opposing effects on apoptosis levels of hemocytes in?vitro and in?vivo, stimulating apoptosis in isolated hemocytes but suppressing it during Cu exposure of whole oysters. The mechanisms of this effect are presently unknown and may be related to the different bioavailability of the metal in?vitro and in?vivo. As expected, Cu accumulated in oyster soft tissues during in?vitro exposure. Unexpectedly, this metal also strongly accumulated in hemolymph plasma which is classically considered isoionic with the surrounding seawater, likely reflecting the presence of soluble Cu-binding proteins in oyster plasma. Cu reduced growth of P.?marinus in?vitro and greatly reduced infection levels of hemocytes in?vivo, presumably by direct toxic effects on the parasite. As a possible parasitic counterbalance, Cu accumulation in the hemocytes was reduced by P.?marinus infection, although this reduction was not sufficient to prevent the parasiticidal effects of the heavy metal in?vivo. This effect of Cu may be useful as a potential therapeutic against Dermo disease in aquaculture conditions. Overall, this study provides important new insights into the potential role of environmental metals in host-parasite relationships and disease dynamics in C.?virginica.     

Infectious diseases of the Pacific oyster,Crassostrea gigas

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

Microsatellite markers for the Sydney rock oyster,Saccostrea glomerata,a commercially important bivalve in southeastern Australia

The Sydney rock oyster (Saccostrea glomerata) is a commercially important bivalve in southeastern Australian. We describe the isolation and characterization of nine microsatellite markers for S. glomerata. The loci are highly polymorphic, with between five and 20 alleles identified among 30 individuals. Expected heterozygosity levels ranged from 0.608 to 0.936. The markers will be used to study natural dispersal, translocations and population structure. We will also use the microsatellites to test the genetic effects of QX disease on oyster populations. This infectious parasitic disease has decimated S. glomerata productivity in a number of areas over the past few decades.