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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.

Oysters are keystone species in estuarine ecosystems and are of substantial economic value to fisheries and aquaculture worldwide. Contending with disease and environmental stress are considerable challenges to oyster culture. Here we report a draft genome of the Sydney Rock Oyster, Saccostrea glomerata, an iconic and commercially important species of edible oyster in Australia known for its enhanced resilience to harsh environmental conditions. This is the second reference genome to be reported from the family Ostreidae enabling a genus-level study of lophotrochozoan genome evolution. Our analysis of the 784-megabase S. glomerata genome shows extensive expansions of gene families associated with immunological non-self-recognition. Transcriptomic analysis revealed highly tissue-specific patterns of expression among these genes, suggesting a complex assortment of immune receptors provide this oyster with a unique capacity to recognize invading microbes. Several gene families involved in stress response are notably expanded in Saccostrea compared with other oysters, and likely key to this species’ adaptations for improved survival higher in the intertidal zone. The Sydney Rock Oyster genome provides a valuable resource for future research in molluscan biology, evolution and environmental resilience. Its close relatedness to Crassostrea will further comparative studies, advancing the means for improved oyster agriculture and conservation.

Marine heat waves are predicted to become more frequent and intense due to anthropogenically induced climate change, which will impact global production of seafood. Links between rising seawater temperature and disease have been documented for many aquaculture species, including the Pacific oyster Crassostrea gigas. The oyster harbours a diverse microbial community that may act as a source of opportunistic pathogens during temperature stress. We rapidly raised the seawater temperature from 20 °C to 25 °C resulting in an oyster mortality rate of 77.4%. Under the same temperature conditions and with the addition of antibiotics, the mortality rate was only 4.3%, strongly indicating a role for bacteria in temperature-induced mortality. 16S rRNA amplicon sequencing revealed a change in the oyster microbiome when the temperature was increased to 25 °C, with a notable increase in the proportion of Vibrio sequences. This pattern was confirmed by qPCR, which revealed heat stress increased the abundance of Vibrio harveyi and Vibrio fortis by 324-fold and 10-fold, respectively. Our findings indicate that heat stress-induced mortality of C. gigas coincides with an increase in the abundance of putative bacterial pathogens in the oyster microbiome and highlights the negative consequences of marine heat waves on food production from aquaculture.

Bivalve molluscs, such as oysters, are threatened by shifts in seawater chemistry resulting from climate change. However, a few species and populations within a species stand out for their capacity to cope with the impacts of climate change‐associated stressors. Understanding the intracellular basis of such differential responses can contribute to the development of strategies to minimise the pervasive effects of a changing ocean on marine organisms. In this study, we explored the intracellular responses to ocean acidification in two genetically distinct populations of Sydney rock oysters (Saccostrea glomerata). Selectively bred and wild type oysters exhibited markedly different mitochondrial integrities (mitochondrial membrane potential) and levels of reactive oxygen species (ROS) in their hemocytes under CO2 stress. Analysis of these cellular parameters after 4 and 15 days of exposure to elevated CO2 indicated that the onset of intracellular responses occurred earlier in the selectively bred oysters when compared to the wild type population. This may be due to an inherent capacity for increased intracellular energy production or adaptive energy reallocation in the selectively bred population. The differences observed in mitochondrial integrity and in ROS formation between oyster breeding lines reveal candidate biological processes that may underlie resilience or susceptibility to ocean acidification. Such processes can be targeted in breeding programs aiming to mitigate the impacts of climate change on threatened species.

Sydney rock oysters (Saccostrea glomerata) are an important part of Australian cuisine and are considered to be a gourmet delicacy for their distinctive flavor. The species is native to Australian estuaries where it faces challenges from environmental stressors and human impacts. In addition, the Sydney rock oyster aquaculture industry has been periodically affected by outbreaks of diseases such as Queensland Unknown (QX) disease and Winter Mortality, a protozoan borne disease greatly impacting the production of the Sydney rock oyster industry. These diseases limit the production of Sydney rock oysters but do not affect other major oyster species farmed in New South Wales. Winter Mortality is a protozoan born disease affecting the production of the Sydney rock oyster industry greatly. Over the past few decades, breeding lines of Sydney rock oysters have been selected for resistance to Winter Mortality and Queensland Unknown disease. In this study, we report a proteomic comparison between the Winter Mortality selective breeding line and hatchery control oysters. We identified 22 protein features showing differential intensities between the two proteomes. The identities of 19 of these features were established using nanoflow liquid chromatography-tandem mass spectrometry. The differential proteome identified in the current study coincides well with previous analyses of stress responses in oysters, suggesting commonalities between intracellular reactions to disease and stress. For instance, cytoskeletal effects found in the current study to be associated with Winter Mortality resistance have been previously linked to intracellular stress responses. In addition, we identified novel proteins such as proteasome subunit alpha type-6 and calcium-dependent protein kinase 31, both of which are reported for the first time in disease resistant Sydney rock oysters. Results of this study will add to our understanding of stress responses and provide the first proteomic survey of Winter Mortality resistance in Sydney rock oysters.

Background This study characterises the molecular processes altered by both elevated CO2 and increasing temperature in oysters. Differences in resilience of marine organisms against the environmental stressors associated with climate change will have significant implications for the sustainability of coastal ecosystems worldwide. Some evidence suggests that climate change resilience can differ between populations within a species. B2 oysters represent a unique genetic resource because of their capacity to better withstand the impacts of elevated CO2 at the physiological level, compared to non-selected oysters from the same species (Saccostrea glomerata). Here, we used proteomic and transcriptomic analysis of gill tissue to evaluate whether the differential response of B2 oysters to elevated CO2 also extends to increased temperature. Results Substantial and distinctive effects on protein concentrations and gene expression were evident among B2 oysters responding to elevated CO2 or elevated temperature. The combination of both stressors also altered oyster gill proteomes and gene expression. However, the impacts of elevated CO2 and temperature were not additive or synergistic, and may be antagonistic. Conclusions The data suggest that the simultaneous exposure of CO2-resilient oysters to near-future projected ocean pH and temperature results in complex changes in molecular processes in order to prevent stress-induced cellular damage. The differential response of B2 oysters to the combined stressors also indicates that the addition of thermal stress may impair the resilience of these oysters to decreased pH. Overall, this study reveals the intracellular mechanisms that might enable marine calcifiers to endure the emergent, adverse seawater conditions resulting from climate change. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3818-z) contains supplementary material, which is available to authorized users.

Environmental contamination by metals is a serious threat to the biological sustainability of coastal ecosystems. Our current understanding of the potential biological effects of metals in these ecosystems is limited. This study tested the transcriptional expression of immune- and stress-response genes in Sydney Rock oysters (Saccostrea glomerata). Oysters were exposed to four metals (cadmium, copper, lead and zinc) commonly associated with anthropogenic pollution in coastal waterways. Seven target genes (superoxide dismutase, ferritin, ficolin, defensin, HSP70, HSP90 and metallothionein) were selected. Quantitative (real-time) PCR analyses of the transcript expression of these genes showed that each of the different metals elicited unique transcriptional profiles. Significant changes in transcription were found for 18 of the 28 combinations tested (4 metals × 7 genes). Of these, 16 reflected down-regulation of gene transcription. HSP90 was the only gene significantly up-regulated by metal contamination (cadmium and zinc only), while defensin expression was significantly down-regulated by exposure to all four metals. This inhibition could have a significant negative effect on the oyster immune system, promoting susceptibility to opportunistic infections and disease.

In this chapter we discuss the role of proteomics in bivalves with primary focus on Sydney rock oysters, which are a local delicacy of Australia much loved by oyster connoisseurs and are farmed predominantly along the eastern coast of NSW. A large part of the aquaculture industry in Australia is contributed by oysters and since Sydney rock oysters are native to Australia, they are an indispensable part of the industry. The Sydney rock oyster differs from its counterpart, the Pacific oyster, in many ways, and the uniqueness of this oyster variety makes it even more valuable for the Australian economy. Recently, environmental and anthropogenic impact coupled with certain diseases have negatively impacted the oyster industry heavily. In recent decades, scientists have relied on selective breeding to keep the impact of such diseases under control. However, with advancements in proteomic and genomic sequencing technologies, efforts are now being made to understand the underlying mechanisms of disease resistance. This chapter discusses ways in which different stressors affect the proteome of oysters, and the different proteomics approaches that have been used in investigations undertaken to address these issues.

Many ecosystems around the world are rapidly deteriorating due to both local and global pressures, and perhaps none so precipitously as coral reefs. Management of coral reefs through maintenance (e.g., marine-protected areas, catchment management to improve water quality), restoration, as well as global and national governmental agreements to reduce greenhouse gas emissions (e.g., the 2015 Paris Agreement) is critical for the persistence of coral reefs. Despite these initiatives, the health and abundance of corals reefs are rapidly declining and other solutions will soon be required. We have recently discussed options for using assisted evolution (i.e., selective breeding, assisted gene flow, conditioning or epigenetic programming, and the manipulation of the coral microbiome) as a means to enhance environmental stress tolerance of corals and the success of coral reef restoration efforts. The 2014–2016 global coral bleaching event has sharpened the focus on such interventionist approaches. We highlight the necessity for consideration of alternative (e.g., hybrid) ecosystem states, discuss traits of resilient corals and coral reef ecosystems, and propose a decision tree for incorporating assisted evolution into restoration initiatives to enhance climate resilience of coral reefs.

Pinctada fucata Gould 1850 was first commercially cultivated in Japan in the early 1920s. Japan dominated this market until the proliferation of Akoya viral disease (AVD) in 1996. Since that time the Japanese industry has struggled. In 2000, Japanese production of Akoya pearls represented only 13% of the total world market value. A number of investigations into this downturn have concluded that the proliferation of AVD was a result of stress associated with increasing urbanization and industrialization. This relationship between environmental stress and altered immunological activity is well documented in a number of oyster species. The decline in Japanese pearl production has led to interest in Akoya cultivation in Australia. Commercial Akoya pearl oyster ventures are now in operation in the Australian states of New South Wales, Queensland and Western Australia. The development of a robust and lucrative Akoya pearl industry in Australia will rely on the cultivation and maintenance of healthy oysters. Oyster health is associated with immunological status, which can be jeopardized by both stress and disease. This review details the relationship between the oyster immune system, environmental stress and disease outbreaks in the genus Pinctada, with implications for P. fucata and the fledgling Australian industry.

The Sydney rock oyster (Saccostrea glomerata) forms the basis of an important aquaculture industry on the east coast of Australia. During the 1970s, production of S. glomerata began to decline, in part as a result of mortalities arising from Queensland unknown (QX) disease. Histological studies implicated the paramyxean parasite Marteilia sydneyi in the disease outbreaks. Disease zoning was implemented to prevent the spread of M. sydneyi-infected oysters. This control measure hindered rock oyster farming, which historically has relied on transferring wild-caught spat between estuaries for on-growing to market size and has not prevented the subsequent occurrence of QX disease in the Georges and Hawkesbury rivers in central New South Wales. Management of QX disease has been hampered by the complicated life cycle of M. sydneyi, with outbreaks of QX disease likely to be regulated by a combination of the abundance of intermediate host of M. sydneyi, environmental stressors, and the immunocompetence of S. glomerata. The future of the Sydney rock oyster industry relies on understanding these factors and progressing the industry from relying on farming wild-caught seed to the successful commercialization of hatchery-produced QX-resistant S. glomerata.

The acidification of oceans is predicted to fundamentally alter marine ecosystems. Previous studies have found that elevated CO2 has an effect on adult calcification, fertilisation and larval development, perhaps because of the organisms' inability to regulate acid-base status, but little is known about the mechanisms that underlie such responses. This study investigated the growth response of larvae of a wild and selectively bred line of the Sydney rock oyster, Saccostrea glomerata, to elevated CO2 and measured the pattern of expression of proteins. Overall exposure to elevated CO2 caused a significant reduction in the shell length of D-veliger larvae of the wild, but not in the selectively bred line. Prior to this study, differences in growth between selectively bred and wild oysters have only been found following settlement. Proteome analysis of D-veliger larvae using two-dimensional gel electrophoresis detected a significantly greater number of protein spots in selectively bred compared to wild oyster...

Sydney rock oysters (Saccostrea glomerata) were exposed to environmental stressors at contaminated field sites or in a controlled laboratory setting. RNA seq transcriptome data were generated for the gill and digestive gland using Roche's 454 pyrosequencing technology. 28,685 contigs were de novo assembled which encoded 11,671 different protein products. The data will act as a reference for future studies in ecology, immunology and environmental toxicology.

QX disease causes mass mortalities among Sydney rock oysters (Saccostrea glomerata). To overcome commercial production losses, Industry & Investment NSW has been developing mass selected QX disease-resistant breeding lines since 1997. This breeding programme has significantly reduced QX-associated mortality in the Lime Kiln Bar (LKB) breeding line relative to non-selected, wild-type (WT) oysters. The current study assessed mortality in families produced by single-pair mating between LKB and WT oysters. When these families were grown in a QX disease-prone area, the progeny of LKB × LKB crosses had significantly lower mortality compared with LKB × WT or WT × WT families. Mortality in the different crosses was associated with infection by sporulating Marteilia sydneyi, the parasite responsible for QX disease. Overall, the study identified a strong association between parentage and mortality resulting from QX disease.

The Sydney rock oyster, Saccostrea glomerata, is susceptible to infection by the protozoan parasite, Marteilia sydneyi, the causative agent of QX disease. M. sydneyi infection peaks during summer when QX disease can cause up to 95% mortality. The current study takes a proteomic approach using 2-dimensional electrophoresis and mass spectrometry to identify markers of QX disease resistance among Sydney rock oysters. Proteome maps were developed for QX disease-resistant and -susceptible oysters. Six proteins in those maps were clearly associated with resistance and so were characterized by mass spectrometry. Two of the proteins (p9 and p11) were homologous to superoxide dismutase-like molecules from the Pacific oyster, Crassostrea gigas, and the Eastern oyster, Crassostrea virginica. The remaining S. glomerata proteins had no obvious similarities to known molecules in sequence databases. p9 and p11 are currently being investigated as potential markers for the selective breeding of QX disease-resistant oysters.

The effects of the muscle relaxant magnesium chloride (MgCl 2 ) were monitored in the Sydney rock oyster, Saccostrea glomerata , under laboratory conditions. The relaxant was first tested for efficiency and then for potential deleterious effects on immunological and spawning activity. A range of immunological parameters were tested including, total haemocyte frequencies, acid phosphatase, superoxide and phenoloxidase activities in the haemolymph, as well as total haemolymph protein levels. All of the oysters exposed to MgCl 2 demonstrated total relaxation within 6h of the initial exposure. Total haemocyte frequencies, acid phosphatase activity and superoxide activity all increased significantly during the first 48h after exposure to the relaxant but declined later. Phenoloxidase activity increased immediately after relaxation but then decreased consistently until 48h after exposure. Overall, the data indicate that MgCl 2 is an effective relaxant for use on S. glomerata , and that any potentially deleterious effects of the relaxant do not persist for more than 96h.

This study tested the proteomic responses of three spatially distinct Sydney rock oyster populations to elevated pCO2. Oysters were collected from environmentally different sites, two chronically affected by acid sulfate soil. Oysters from each of the three populations were exposed to ambient (380µatm) or elevated (856 and 1500µatm) pCO2 for 4 weeks. Subsequent proteomic analysis from haemolymph revealed that (1) there were differences between the proteomes of the three populations after exposure to ambient pCO2, and (2) the different oyster populations mounted significantly different responses to elevated pCO2. Proteins that differed significantly in concentration between pCO2 treatments fell into five broad functional categories: energy metabolism, cellular stress responses, the cytoskeleton, protein synthesis and the extracellular matrix. This is consistent with the hypothesis that environmental stress in oysters leads to a generic response involving increased mitochondrial energy production to maintain cellular homeostasis. Proteins involved in the cytoskeleton and energy metabolism were the most differentially expressed and were seen in all three oyster populations. Differences between populations in their proteomic responses suggested that the local environments from which oysters originate may affect their capacity to respond to ocean acidification.

The effects of starvation on various factors related to immunological defence in Sydney rock oysters (Saccostrea glomerata) were tested in controlled laboratory experiments. A range of parameters was assessed, including total haemocyte counts, and phenoloxidase (PO), superoxide, acid phosphatase and peroxidase activities. Condition indices were also monitored as an indicator of oyster fitness. We found that the frequency of haemocytes and phenoloxidase activity decreased by 25% and 14% respectively when oysters were fed half-satiation rations for three weeks. These decreases became statistically significant when oysters were starved for three weeks. Superoxide and peroxidase activities also decreased significantly when oysters were starved for two to four weeks, while phenoloxidase and acid phosphatase activities were significantly inhibited after oysters had been starved for at least three weeks. All of the immunological parameters returned to at least their original levels after starved oysters were fed to satiation for six days. Phenoloxidase activities over-compensated during the recovery response, so that levels post-recovery were substantially higher than those evident before starvation. There were no significant changes in condition indices during any of the starvation treatments. These data indicate that starvation compromises immunological activity, and that the duration and intensity of starvation differentially affect immune responses.

Exposure to prolonged environmental stress can have impacts on the cellular homeostasis of aquatic organisms. The current study employed two-dimensional electrophoresis (2-DE) to test whether exposure to impaired water quality conditions in the Sydney Harbour estuary has significantly altered the proteomes of the resident Sydney rock oyster (Saccostrea glomerata). Adult S. glomerata were sampled from four bays in the estuary. Each bay consisted of a "high-impact" site adjacent to point sources of chemical contamination (e.g., storm drains/canals or legacy hotspots) and a "low-impact" site located ∼5km away from point sources. A mixture of environmental stressors differed significantly between high- and low-impact sites. Specifically, PAHs, PCBs, tributyltin, Pb, and Zn were significantly elevated in oyster tissues from high-impact sites, together with depleted dissolved oxygen and low pH in the water column. A 2-DE proteomics analysis subsequently identified 238 protein spots across 24 2-DE gels, of which 27-50 spots differed significantly in relative intensity between high- and low-impact sites per bay. Twenty-five percent of the differential spots were identified in more than one bay. The identities of 80 protein spots were determined by mass spectrometry. HSP 70, PPIB, and radixin were the three most highly expressed differential proteins. Despite the largely unique proteomes evident in each bay, functional annotations revealed that half of the differentially expressed proteins fell into just two subcellular functional categories-energy metabolism and the cytoskeleton. These findings provide a framework to further investigate adaptation of cellular mechanisms to prolonged stress in S. glomerata.

Ocean acidification (OA) is predicted to have widespread implications for marine organisms, yet the capacity for species to acclimate or adapt over this century remains unknown. Recent transgenerational studies have shown that for some marine species, exposure of adults to OA can facilitate positive carryover effects to their larval and juvenile offspring that help them to survive in acidifying oceanic conditions. But whether these positive carryover effects can persist into adulthood or the next generation is unknown. Here we tested whether positive carryover effects found in larvae of the oyster, Saccostrea glomerata following transgenerational exposure to elevated CO2, could persist into adulthood and whether subsequent transgenerational exposure of adults to elevated CO2 would facilitate similar adaptive responses in the next generation of larvae and juveniles. Following our previous transgenerational exposure of parental adults and first generation (F1) larvae to ambient (385 μatm) and elevated (856 μatm) CO2, newly settled F1 juveniles were transferred to the field at ambient CO2 for 14 months, until they reached reproductive maturity. At this time, the F1 adults were returned to the laboratory and the previous transgenerational CO2 exposure was repeated to produce F2 offspring. We found that the capacity of adults to regulate extracellular pH at elevated CO2 was improved if they had a prior history of transgenerational exposure to elevated CO2. In addition, subsequent transgenerational exposure of these adults led to an increase in the resilience of their larval and juvenile offspring. Offspring with a history of transgenerational exposure to elevated CO2 had a lower percentage abnormality, faster development rate, faster shell growth and increased heart rate at elevated CO2 compared with F2 offspring with no prior history of exposure to elevated CO2. Our results suggest that positive carryover effects originating during parental and larval exposure will be important in mediating some of the impacts of OA for later life-history stages and generations.

This study tested the effects of low salinity on phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata). Phenoloxidase is a key component of the immune system in S. glomerata. Previously, we have shown that decreased phenoloxidase activity is associated with susceptibility to fatal QX disease. In the current study, laboratory-based experiments were used to identify factors that contribute to decreased phenoloxidase activity. We found that exposing oysters to water collected from a QX-prone oyster growing area after heavy rain significantly inhibited their phenoloxidase activities. Similar inhibition was evident when oysters were held in oceanic water with artificially lowered salinities. These results suggest that exposure to low salinity decreases phenoloxidase activity. Field trials that exploited a natural salinity gradient in the Georges River, Sydney, supported this conclusion. The phenoloxidase activities of oysters transplanted to up-river sites that had low salinities were significantly lower than those of oysters held at seaward sites with higher salinities. All of these data implicate low salinity as a key environmental stressor that is associated with inhibition of the S. glomerata immune system.

The paramyxean protozoan, Marteilia sydneyi , is the etiological agent of QX disease in Sydney rock oysters ( Saccostrea glomerata ). QX disease affects the farming of oysters in Queensland and on five rivers in New South Wales (NSW), Australia. Disease outbreaks occur during summer months (January to April) and are associated with high mortality rates (up to 98%), which limit the growing out period for oyster production. This study investigated the relationship between oyster host defense systems and QX disease. Oysters from the same brood stock were harvested from QX prone and QX free growing areas over the course of the 2000–2001 outbreak. A variety of parameters—infection intensity in the digestive diverticulum, total hemolymph protein content, condition index and phenoloxidase (PO) activity—were measured in these oysters. Phenoloxidase activity, which is often associated with host defense, was significantly suppressed in oysters from the QX prone area when compared to those from the QX free river. There was also a strict negative correlation between phenoloxidase activity and the intensity of parasitic infection ( p =0.0038). In contrast, neither total protein content nor condition indexes differed between oysters from the QX prone and QX free rivers. These data suggest that inhibition of prophenoloxidase cascade may facilitate lethal infection by M. sydneyi .

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.

Aquaculture has long been seen as a sustainable solution to some of the world's growing food shortages. However, experience over the past 50 years indicates that infectious diseases caused by viruses, bacteria, and eukaryotes limit the productivity of aquaculture. In extreme cases, these types of infectious agents threaten the viability of entire aquaculture industries. This article describes the threats from infectious diseases in aquaculture and then focuses on one example (QX disease in Sydney rock oysters) as a case study. QX appears to be typical of many emerging diseases in aquaculture, particularly because environmental factors seem to play a crucial role in disease outbreaks. Evidence is presented that modulation of a generic subcellular stress response pathway in oysters is responsible for both resistance and susceptibility to infectious microbes. Understanding and being able to manipulate this pathway may be the key to sustainable aquaculture.

Tunicates are filter feeding marine invertebrates that are susceptible to environmental contamination by toxic metals and polyaromatic hydrocarbons. Recently, we have shown that tunicate immune reactions are profoundly affected by exposure to tributyltin (TBT) and copper, both of which are components of marine antifouling paints. This study tests the effects of those pollutants on the hemocytes of tunicates. Immunofluorescence labeling with an anti-hemocyte monoclonal antibody demonstrated that the antigenic structure of the circulating hemocyte population was substantially affected by TBT and copper. Antigen-positive hemocytes were also found to accumulate in the pharyngeal papillae of TBT-exposed tunicates. Histological analyses indicated that this cellular accumulation in pharyngeal papillae involved refractile vacuolated hemocytes. Refractile vacuolated cells from TBT-exposed tunicates also occurred at greater frequencies in the circulating hemolymph, and had altered morphologies, compared to cells from nontreated controls. These data confirm that exogenous metals can have profound effects on the hemocytes of tunicates.

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

This study assessed the effects of mechanical agitation, hypo-saline conditions, and exposure to the air on the Akoya pearl oyster, Pinctada imbricata, focusing specifically on the immunological activity of haemocytes. Both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to all three stressors. Transient decreases were also evident in total haemocyte counts after mechanical stress and exposure to air, while significant increases in total haemocyte counts were evident after exposure to low salinity. Acid phosphatase activity increased significantly when oysters were exposed to air. The frequency of granulocytes in the haemolymph increased significantly when oysters were stressed by hypo-saline conditions, whilst the relative frequency of granulocytes did not differ significantly after mechanical agitation or exposure to air. The total protein content of haemolymph increased significantly when oysters were stressed by mechanical agitation and low salinity. These results suggest that fluctuations in environmental conditions affect circulating haemocytes and their cytochemistry, and that the different immunological parameters tested were influenced uniquely according to the type of stressor.

There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.

185/333 genes and transcripts from the purple sea urchin, Strongylocentrotus purpuratus, predict high levels of amino acid diversity within the encoded proteins. Based on their expression patterns, 185/333 proteins appear to be involved in immune responses. In the present study, one- and two-dimensional Western blots show that 185/333 proteins exhibit high levels of molecular diversity within and between individual sea urchins. The molecular masses of 185/333-positive bands or spots range from 30 to 250 kDa with a broad array of isoelectric points. The observed molecular masses are higher than those predicted from mRNAs, suggesting that 185/333 proteins form strong associations with other molecules or with each other. Some sea urchins expressed >200 distinct 185/333 proteins, and each animal had a unique suite of the proteins that differed from all other individuals. When sea urchins were challenged in vivo with pathogen-associated molecular patterns (PAMPs; bacterial LPS and peptidoglycan), the expression of 185/333 proteins increased. More importantly, different suites of 185/333 proteins were expressed in response to different PAMPs. This suggests that the expression of 185/333 proteins can be tailored toward different PAMPs in a form of pathogen-specific immune response.

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.

Tunicates (ascidians or sea squirts) are a large group of invertebrate chordates that are closely related to vertebrates. Their critical phylogenetic position has stimulated substantial interest in their host defense (“immune”) responses. Whilst this interest has generated a wealth of knowledge regarding the humoral and cellular mechanisms that undertake defensive responses, there is less known about the regulation of those reactions. This chapter focuses on three cellular responses (cell proliferation, phagocytosis and chemotaxis) that are known to be regulated by cytophilic humoral molecules. Some of the humoral factors that affect these responses have functional and physicochemical similarities to vertebrate cytokines, like interleukin-1. However, the only regulatory molecules that have been characterized at a molecular level bear far greater similarity to C-type lectins or complement components.

Toxic metals, such as tributyltin (TBT), contribute substantially to anthropogenic pollution in many estuarine environments. Animals that live in those environments, particularly invertebrate filter feeders like tunicates, are likely to be exposed to substantial metal contamination. This study investigates the effects of TBT and other metals on the phenoloxidase activity of the estuarine tunicate, Styela plicata, in an effort to identify a biochemical marker of metal pollution. Hemocytes harvested from S. plicata that were exposed to tributyltin or copper in aquaria had significantly enhanced phenoloxidase activities relative to non-exposed controls. This enhanced phenoloxidase activity could be explained by an increased frequency of morula cells, which contain high levels of phenoloxidase's proenzyme, prophenoloxidase. Unlike those from tunicates exposed to metals in aquaria, the phenoloxidase activities of hemocytes incubated with tributyltin in vitro were significantly reduced when compared with hemocytes cultured without tributyltin. The ability of tributyltin to decrease phenoloxidase activity in tissue culture may reflect its known inhibitory effects on calcium-dependent signaling systems such as those involved in the exocytosis of prophenoloxidase from morula cells.

Runoff from acid sulfate soils (ASS) has severe environmental and economic impacts on estuarine ecosystems. Oysters display reduced abundance, growth rate and shell thickness when exposed to ASS runoff, yet the molecular underpinnings of their responses have not been explored. We hypothesised that the proteomes of wild Sydney rock oysters, Saccostrea glomerata, would differ between populations recurrently exposed to ASS compared with those unaffected by runoff from ASS. We used two-dimensional electrophoresis to compare protein abundances in the gills of S. glomerata collected from two sites close to (acidified) and two sites away from (reference) major ASS outflow drains in a south-east Australian estuary. Approximately 5% of the proteome was differentially expressed between oysters from acidified and reference sites, with five protein spots more abundant and one less abundant at the sites close to drains. Another protein spot was present only in oysters from reference sites. This study is the first screening of spatial variation in the protein expression of S. glomerata with respect to discharge from ASS. Altered protein expression may underpin short-term inducible responses to ASS runoff, or genetic resistance acquired through recurrent exposure of populations to the stressor.

Phagocytosis is the predominant defense mechanism of invertebrates. Here we show that phagocytosis by echinoderm bladder amoebocytes and tunicate granular amoebocytes can be enhanced by invertebrate interleukin-1-like molecules. As little as 5 ng/ml of invertebrate interleukin-1 produced a significant stimulation of echinoderm and tunicate amoebocyte phagocytosis. Stimulation of phagocytosis by echinoderm interleukin-1-like molecules was inhibited by antisera to vertebrate interleukin-1. Invertebrate interleukin-1 also acted as an opsonin when preincubated with erythrocytes or yeast. In addition, the cellular mechanisms of invertebrate phagocytosis were studied using pharmacologic agents to inhibit echinoderm amoebocyte phagocytosis. The energy requirements and involvement of cellular cytoskeletal elements in phagocytosis by bladder amoebocytes were similar to those of mammalian macrophages. These results demonstrate a role for interleukin-1 in invertebrate host defense mechanisms.

Spatial and temporal patterns of variability in ascidian assemblages were investigated on horizontal subtidal rocky reefs at Port Stephens, New South Wales (NSW). The study was designed to provide a baseline dataset on ascidian diversity and distribution patterns for an area destined to become a marine park (the Port Stephens–Great Lakes Marine Park: PSGLMP). Differences in ascidian assemblages between exposed oceanic island reefs and sheltered reefs within Port Stephens, and between two depth zones within each subtidal reef, were quantified using non-parametric multivariate techniques coupled with analysis of variance (ANOVA). Ascidian assemblages were highly variable between reef sites, reef exposures and particularly between depth zones within each reef surveyed. However, temporal variation was only observed for a few ascidian species. These highly variable spatial patterns in diversity indicate that numerous subtidal reefs may need to be protected within PSGLMP if the aim of the marine park is to adequately represent the entire array of marine biodiversity in the area.

This study assessed links between host fitness, environmental change and opportunistic parasite infections in a dynamic estuary system. The Hawkesbury River in New South Wales is the most recent Sydney rock oyster growing area to experience outbreaks of infectious QX disease. This area was used to examine a relationship between the intensity of QX disease and inhibition of the oyster immune system. Oysters were grown at various sites along the river and periodically monitored for general condition, total haemolymph protein content, antibacterial capacity and phenoloxidase activity. Phenoloxidase activity was significantly inhibited during a key period of Marteilia sydneyi infectivity in late summer 2005. The degree to which phenoloxidase was inhibited strongly correlated with the intensity of M. sydneyi infection. The data suggest that the presence of some transient environmental stressor may have affected phenoloxidase activity during a key period of infection and increased the susceptibility of oysters to disease. These results provide further evidence for a specific relationship between decreased phenoloxidase activity and susceptibility to QX infection.