Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Bacterial diversity of the digestive gland of Sydney rock oysters,Saccostrea glomerata infected with the paramyxean parasite,Marteilia sydneyi

Aims: To determine whether the infestation by the protozoan paramyxean parasite, Marteilia sydneyi, changes the bacterial community of the digestive gland of Sydney rock oysters, Saccostrea glomerata. Methods and Results: Six 16S rDNA clone libraries were established from three M. sydneyi‐infected and three un‐infected oysters. Restriction enzyme analysis followed by sequencing representative clones revealed a total of 23 different operational taxonomic units (OTUs) in un‐infected oysters, comprising the major phyla: Firmicutes, Proteobacteria, Cyanobacteria and Spirocheates, where the clone distribution was 44, 36, 7 and 5%, respectively. Close to half of the OTUs are not closely related to any other hitherto determined sequence. In contrast, S. glomerata infected by M. sydneyi had only one OTU present in the digestive gland. Phylogenetic analysis of the 16S rDNA sequence reveals that this dominant OTU, belonging to the α‐Proteobacteria, is closely related to a Rickettsiales‐like prokaryote (RLP). Conclusions: The microbiota of the digestive gland of Sydney rock oysters is changed by infection by M. sydneyi, becoming dominated by a RLP, and generally less diverse. The bacterial community of un‐infected S. glomerata differs from previous studies in that we identified the dominant taxa as Firmicutes and α‐Proteobacteria, rather than heterotrophic γ‐Proteobacteria. Significance and Impact of the Study: This is the first culture‐independent study of the microbiota of the digestive glands of edible oysters to the species level. The commercial viability of the Sydney rock oyster industry in Australia is currently threatened by Queensland Unknown disease and the changes in the bacterial community of S. glomerata corresponding with infection by M. sydneyi sheds further light on the link between parasite infection and mortality in this economically damaging disease.     

The density and spatial arrangement of the invasive oyster Crassostrea gigas determines its impact on settlement of native oyster larvae

Understanding how the density and spatial arrangement of invaders is critical to developing management strategies of pest species. The Pacific oyster, Crassostrea gigas, has been translocated around the world for aquaculture and in many instances has established wild populations. Relative to other species of bivalve, it displays rapid suspension feeding, which may cause mortality of pelagic invertebrate larvae. We compared the effect on settlement of Sydney rock oyster, Saccostrea glomerata, larvae of manipulating the spatial arrangement and density of native S. glomerata, and non‐native C. gigas. We hypothesized that while manipulations of dead oysters would reveal the same positive relationship between attachment surface area and S. glomerata settlement between the two species, manipulations of live oysters would reveal differing density‐dependent effects between the native and non‐native oyster. In the field, whether oysters were live or dead, more larvae settled on C. gigas than S. glomerata when substrate was arranged in monospecific clumps. When, however, the two species were interspersed, there were no differences in larval settlement between them. By contrast, in aquaria simulating a higher effective oyster density, more larvae settled on live S. glomerata than C. gigas. When C. gigas was prevented from suspension feeding, settlement of larvae on C. gigas was enhanced. By contrast, settlement was similar between the two species when dead. While the presently low densities of the invasive oyster C. gigas may enhance S. glomerata larval settlement in east Australian estuaries, future increases in densities could produce negative impacts on native oyster settlement. Synthesis and applications: Our study has shown that both the spatial arrangement and density of invaders can influence their impact. Hence, management strategies aimed at preventing invasive populations reaching damaging sizes should not only consider the threshold density at which impacts exceed some acceptable limit, but also how patch formation modifies this.     

Predation by the endemic whelk Tenguella marginalba (Blainville, 1832) on the invasive Pacific oyster Crassostrea gigas (Thunberg, 1793)

The endemic mulberry whelk (Tenguella marginalba) is a common predator on Australian intertidal rocky shores. The introduced Pacific oyster (Crassostrea gigas), found within the natural range of T. marginalba, is potential prey for the whelk. In experiments designed to increase our understanding of predatory behaviour by the whelk on oysters, we found that adult T. marginalba detected C. gigas and increased movement in the presence of oyster prey. Tenguella marginalba showed a preference for smaller C. gigas, but consumed oysters up to 60?mm in shell height. To access oyster flesh, whelks used their radula to drill holes in the oyster’s shell. These holes were on average 0.68?±?0.09?mm in diameter, most frequently located central to the pericardial cavity on the right (upper) valve. Predation was greatest when predator and prey were both submerged, but was unaffected by a diurnal light cycle. When offered a choice among the native Sydney rock oysters (Saccostrea glomerata), mussels (Trichomya hirsuta) or the invasive C. gigas, whelks displayed no preference among prey. We conclude that the invasive oyster C. gigas represents a viable food source for T. marginalba, which may help to slow the spread of this invasive oyster throughout eastern Australia.     

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.     

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.     

Characterization of the major plasma protein of the eastern oyster,Crassostrea virginica,and a proposed role in host defense

The major plasma protein of the eastern oyster, Crassostrea virginica, was purified, characterized and named dominin. SDS-PAGE analyses revealed that dominin consistently made up more than 40% of eastern oyster plasma and extrapallial fluid proteins. Three different forms of dominin were observed under non-reducing conditions. PCR and RACE primers designed from partial amino acid sequences obtained by tandem mass spectrometry of purified dominin identified 720 bp of complete cDNA encoding 192 amino acid residues. Based on the deduced amino acid sequence of mature dominin, its molecular mass was calculated to be 19,389 Da and was lower than the molecular mass of purified dominin measured by MALDI. This difference is likely due to post-translational modifications of dominin as the purified protein was found to be glycolysated, phosphorylated and likely sulfated. The amino acid sequence showed high similarity to the major plasma protein of the Pacific oyster (Crassostrea gigas), cavortin, and of the green-lipped mussel (Perna canaliculus), pernin, and to a recently described protein labeled as an extracellular superoxide dismutase from the Sydney rock oyster Saccostrea glomerata. While dominin was found to possess a Cu/Zn superoxide dismutase (SOD) domain, the domain was not completely conserved which explained why purified dominin lacked SOD activity. Dominin mRNA was detected in hemocytes by in situ hybridization and its expression measured by quantitative real time RT-PCR was significantly higher in winter than summer. Although the function(s) of dominin and homologous proteins is uncertain, the reported ability of cavortin to sequester iron and possibly limit the availability of this essential metal to pathogens suggests a potential role in host defense for this group of dominant plasma proteins. Other possible functions of dominin in antioxidation, wound repair, metal transport and shell mineralization are discussed leading us to conclude that dominin is likely a multifunctional protein.     

Genetic structure and effective population size of Sydney rock oysters in eastern Australia

Oyster reef habitats are critical to coastal biodiversity and their decline has prompted restoration efforts in Australia. Knowledge gaps exist regarding the population structure and diversity of key species in these habitats. This may be critical information for the design of effective restoration programs. Sydney rock oysters (Saccostrea glomerata) are the dominant reef-forming bivalve in eastern Australia. Wild populations of S. glomerata have declined due to overharvesting, disease outbreaks, coastal development and reduced water quality. Here, we use genetic markers identified by genome-wide sequencing to investigate the genetic structure and diversity of wild Sydney rock oysters throughout their distribution in eastern Australia. We examine evidence for past population bottlenecks and spatial genetic structure associated with the East Australian Current. Analysis of 3, 400 neutral single-nucleotide polymorphisms (SNPs) revealed a single population, and an overlap with two other Saccostrea sp. at the northernmost boundary of the distribution. We detected signals of asymmetric gene flow consistent with the direction of the East Australian Current, and spatial structure patterns of limited genetic isolation by distance and spatial autocorrelation in the northern region (which experiences stronger effects of the East Australian Current) but not in the southern region of the distribution. We found no evidence of significant recent bottlenecks, with high effective population size throughout the species’ range. This information will provide a baseline against which to assess the impact of restoration projects, and guide strategies for sourcing stock for the enhancement of wild oyster populations. Our results provide a positive outlook for the resilience and adaptive capacity of Sydney rock oysters, and highlight wild populations as valuable resources for aquaculture and restoration initiatives.

     

Breeding for QX disease resistance negatively selects one form of the defensive enzyme, phenoloxidase, in Sydney rock oysters

QX disease in Sydney rock oysters (Saccostrea glomerata) is caused by the paramyxean protozoan, Marteilia sydneyi. Disease outbreaks occur during summer (January to May) and can result in up to 95% mortality. The New South Wales Department of Primary Industries has been selectively breeding S. glomerata for resistance to QX disease since 1996. Previous work suggests that this breeding program has specifically affected the defensive phenoloxidase enzyme system of oysters. The current study more thoroughly characterises the effect of selection on the different forms of phenoloxidase found in oyster populations. Native polyacrylamide gel electrophoresis (native-PAGE) identified five discrete types of phenoloxidase in non-selected (wild type) and fourth generation QX disease resistant (QXR4) oysters. One electrophoretically distinct form of phenoloxidase, POb, is significantly less frequent in resistant oysters when compared to the wild type population. The frequency of POb also decreased in both the wild type and QXR4 populations over the course of a QX disease outbreak. This suggests that possession of POb makes oysters susceptible to QX disease and that breeding for resistance has resulted in negative selection against this form of phenoloxidase.     

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.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Differential proteomic responses of selectively bred and wild‐type Sydney rock oyster populations exposed to elevated CO2

Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild‐type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO₂ (856 μatm) before their proteomes were compared to those of oysters held under ambient conditions (375 μatm pCO₂). Exposure to elevated pCO₂ resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild‐type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild‐type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade‐off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO₂, whilst being deleterious to adult oysters.     

Supplementation of Perkinsus marinus Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.     

Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster,Crassostrea gigas,in Korea

Polyclonal antibody developed from egg proteins of the Pacific oysters, Crassostrea gigas, was used in quantification of the eggs. Quantity of the egg protein was measured using ELISA and a weight-normalized gonadosomatic index (GSI) was then established. Histology indicated that the oysters collected from Goseong Bay, Korea, in 2000 initiated gametogenesis as early as late February and spawned mostly in late June to early July when water temperature reached 23–25 °C. A second spawning peak was also observed in late August and the spawning activity continued to late October. Oyster egg proteins could be detected by ELISA in all months except March and November; a few oysters collected during December and January contained a measurable amount of eggs. The highest monthly mean GSI, 0.423, was observed in mid-June when the oysters were ready to spawn. The maximum GSI recorded in this study was 0.667. A positive correlation was found between oyster size and fecundity; the number of eggs increased as oyster biomass increased (r²=0.7497). Our data suggest that oysters in Goseong Bay produce 3–196 million eggs during the spawning season. The immunological method developed in this study was sensitive enough to measure a small quantity of eggs and considered to be method of choice for studying bivalve reproduction.     

Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas)

Cadmium, zinc and copper, accumulated from polluted habitats by the oysters Ostrea edulis and Crassostrea gigas, were studied. Three distinct low molecular weight (less than 3000 daltons) zinc complexes were separated from highly contaminated C. gigas, one of which may be a complex with the amino acid taurine. Only one of these zinc complexes was present in less contaminated specimens of O. edulis. On gel permeation chromatography of oyster extracts, copper was eluted together with amino acids (principally taurine) and the betaine homarine. No evidence of metallothionein type proteins was found.     

Selectively bred oysters can alter their biomineralization pathways,promoting resilience to environmental acidification

Commercial shellfish aquaculture is vulnerable to the impacts of ocean acidification driven by increasing carbon dioxide (CO₂) absorption by the ocean as well as to coastal acidification driven by land run off and rising sea level. These drivers of environmental acidification have deleterious effects on biomineralization. We investigated shell biomineralization of selectively bred and wild‐type families of the Sydney rock oyster Saccostrea glomerata in a study of oysters being farmed in estuaries at aquaculture leases differing in environmental acidification. The contrasting estuarine pH regimes enabled us to determine the mechanisms of shell growth and the vulnerability of this species to contemporary environmental acidification. Determination of the source of carbon, the mechanism of carbon uptake and use of carbon in biomineral formation are key to understanding the vulnerability of shellfish aquaculture to contemporary and future environmental acidification. We, therefore, characterized the crystallography and carbon uptake in the shells of S. glomerata, resident in habitats subjected to coastal acidification, using high‐resolution electron backscatter diffraction and carbon isotope analyses (as δ¹³C). We show that oyster families selectively bred for fast growth and families selected for disease resistance can alter their mechanisms of calcite crystal biomineralization, promoting resilience to acidification. The responses of S. glomerata to acidification in their estuarine habitat provide key insights into mechanisms of mollusc shell growth under future climate change conditions. Importantly, we show that selective breeding in oysters is likely to be an important global mitigation strategy for sustainable shellfish aquaculture to withstand future climate‐driven change to habitat acidification.     

Exploring the decline of oyster beds in Atlantic Canada shorelines: potential effects of crab predation on American oysters (<Emphasis Type="Italic">Crassostrea virginica</Emphasis>)

Atlantic Canada’s American oyster (Crassostrea virginica) beds, while economically and ecologically important, have been in decline over the past few decades. Predation by crabs, in particular by the European green crab (Carcinus maenas), has been proposed as one of the potential causes of such decline. Hence, this study examined oyster mortality levels in multiple beds across Prince Edward Island (PEI) and then experimentally assessed the contribution of green crab predation to oyster mortality. Results from surveys conducted in 10 estuaries across PEI in 2014 indicate that the probability of mortality for small oysters was significantly higher when green crabs were present then in areas without green crabs. This probability of mortality was significantly less when there was the presence of alternative prey like natural mussel beds (Mytilus edulis). The odds of oyster mortality were also higher when beds had rock crabs (Cancer irroratus) compared to beds with no rock crabs. Given the potential importance of green crab predation, its influence was assessed in 2015 using two field experiments with tethered oysters. Our results indicate that odds of small oyster mortality occurring were much higher in green crab inclusion cages than in the open environment and the exclusion cages. These results reaffirm that oysters up to ~40 mm SL are vulnerable to predation, and at least some of the mortality affecting these oysters can be causally attributed to green crab predation. Green crab predation rates upon small oysters are relevant given the economic benefits and ecosystem services provided by these bivalves. They highlight the need for the industry to consider mitigation measures and potentially adapt their oyster growing strategies.     

Predictive models for the effect of storage temperature on Vibrio parahaemolyticus viability and counts of total viable bacteria in Pacific oysters (Crassostrea gigas)

Vibrio parahaemolyticus is an indigenous bacterium of marine environments. It accumulates in oysters and may reach levels that cause human illness when postharvest temperatures are not properly controlled and oysters are consumed raw or undercooked. Predictive models were produced by injecting Pacific oysters (Crassostrea gigas) with a cocktail of V. parahaemolyticus strains, measuring viability rates at storage temperatures from 3.6 to 30.4°C, and fitting the data to a model to obtain parameter estimates. The models were evaluated with Pacific and Sydney Rock oysters (Saccostrea glomerata) containing natural populations of V. parahaemolyticus. V. parahaemolyticus viability was measured by direct plating samples on thiosulfate-citrate-bile salts-sucrose (TCBS) agar for injected oysters and by most probable number (MPN)-PCR for oysters containing natural populations. In parallel, total viable bacterial counts (TVC) were measured by direct plating on marine agar. Growth/inactivation rates for V. parahaemolyticus were −0.006, −0.004, −0.005, −0.003, 0.030, 0.075, 0.095, and 0.282 log₁₀ CFU/h at 3.6, 6.2, 9.6, 12.6, 18.4, 20.0, 25.7, and 30.4°C, respectively. The growth rates for TVC were 0.015, 0.023, 0.016, 0.048, 0.055, 0.071, 0.133, and 0.135 log₁₀ CFU/h at 3.6, 6.2, 9.3, 14.9, 18.4, 20.0, 25.7, and 30.4°C, respectively. Square root and Arrhenius-type secondary models were generated for V. parahaemolyticus growth and inactivation kinetic data, respectively. A square root model was produced for TVC growth. Evaluation studies showed that predictive growth for V. parahaemolyticus and TVC were “fail safe.” The models can assist oyster companies and regulators in implementing management strategies to minimize V. parahaemolyticus risk and enhancing product quality in supply chains.