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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.

The Sydney Rock Oyster (SRO) ( Saccostrea glomerata ) industry of Australia has faced repeated challenges from both anthropogenic and natural threats. One important factor that has caused substantial loss to the industry is a protozoan-borne disease known as QX disease. To ensure sustainable production of SRO, a selective breeding programme has been employed to develop a line of QX disease resistant oysters. In this study, we have performed a label-free quantitative shotgun proteomic analysis of control and selectively bred SRO to identify potential protein biomarkers of disease resistance. We extracted proteins from the gills of two different oyster populations (QX resistant and non-selected wild type, n = 20 per population) sampled at two different time points, to gain insights into disease resistance during the oyster growth cycle. The current study identifies a pattern of protein changes over the two time points, which includes some results coincident with previous published reports, as well as some novel findings. We have identified 21 differentially expressed proteins between the two lines of oysters at the first time point, and 22 differentially expressed proteins at the second time point. These include proteins involved in cytoskeletal remodelling, immune response, energy metabolism, and numerous other functions. In particular, we report the increased abundance in selectively bred oysters of High Mobility Group Box 1 protein (HMGB1), which has been reported to play an important role in the oyster immune response. In addition, we have identified changes in abundance levels of Major Vault Protein (MVP), Histone H2A, dscam, Ras related Rab11B, proteasome subunits, and numerous other proteins that have previously been reported to be relevant in the context of molluscan stress responses. These proteins have potential for use as disease resistance biomarkers within a population, and also help build our understanding of the biological phenomena underlying selection for disease resistance.

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.

Some populations of marine organisms appear to have inherent tolerance or the capacity for acclimation to stressful environmental conditions, including those associated with climate change. Sydney rock oysters from the B2 breeding line exhibit resilience to ocean acidification (OA) at the physiological level. To understand the molecular basis of this physiological resilience, we analysed the gill transcriptome of B2 oysters that had been exposed to near-future projected ocean pH over two consecutive generations. Our results suggest that the distinctive performance of B2 oysters in the face of OA is mediated by the selective expression of genes involved in multiple cellular processes. Subsequent high-throughput qPCR revealed that some of these transcriptional changes are exclusive to B2 oysters and so may be associated with their resilience to OA. The intracellular processes mediated by the differentially abundant genes primarily involve control of the cell cycle and maintenance of cellular homeostasis. These changes may enable B2 oysters to prevent apoptosis resulting from oxidative damage or to alleviate the effects of apoptosis through regulation of the cell cycle. Comparative analysis of the OA conditioning effects across sequential generations supported the contention that B2 and wild-type oysters have different trajectories of changing gene expression and responding to OA. Our findings reveal the broad set of molecular processes underlying transgenerational conditioning and potential resilience to OA in a marine calcifier. Identifying the mechanisms of stress resilience can uncover the intracellular basis for these organisms to survive and thrive in a rapidly changing ocean.

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.

The Pacific oyster (Crassostrea gigas) is farmed globally. Ostreid herpesvirus (OsHV-1) causes severe mortalities of farmed C. gigas. Management of OsHV-1 has proven difficult. Oysters treated with poly(I:C) exhibit enhanced protection (EP) against OsHV-1. This chemical treatment is highly effective, but it is not feasible to treat every oyster on a farm. To circumvent this practical limitation, previous studies on arthropods have suggested that EP can be transferred from parents to their offspring (trans-generational EP, TGEP). This suggests that the treatment of relatively few parents could be used to produce large numbers of offspring with TGEP. Here, we investigated TGEP in oysters to test whether it might be used as a cost effective management tool to control OsHV-1. We found that offspring (D-veliger larvae) produced from poly(I:C)-treated parents had double the chance of surviving exposure to OsHV-1 compared to controls. Furthermore, the larvae of poly(I:C)-treated parents contained elevated levels of mRNA encoding a key transcription factor that regulates antiviral immunity (IRF2). Poly(I:C) treatment had no effect on the survival of oyster parents. Hence, the enhanced immunity of their offspring could not be explained by genetic selection, and instead may reflect epigenetic reprogramming or maternal provisioning.

International audience; The oyster's immune system is capable of adapting upon exposure to a pathogen-associated molecular pattern (PAMP) to have an enhanced secondary response against the same type of pathogen. This has been demonstrated using poly(I:C) to elicit an antiviral response in the Pacific oyster (Crassostrea gigas) against Ostreid herpesvirus (OsHV-1). Improved survival following exposure to poly(I:C) has been found in later life stages (within-generational immune priming) and in the next generation (transgenerational immune priming). The mechanism that the oyster uses to transfer immunity to the next generation is unknown. Here we show that oyster larvae have higher survival to OsHV-1 when their mothers, but not their fathers, are exposed to poly(I:C) prior to spawning. RNA-seq provided no evidence to suggest that parental exposure to poly(I:C) reconfigures antiviral gene expression in unchallenged larvae. We conclude that the improved survival of larvae might occur via maternal provisioning of antiviral compounds in the eggs.

Many species of bivalve mollusks (phylum Mollusca, class Bivalvia) are important in fisheries and aquaculture, whilst others are critical to ecosystem structure and function. These crucial roles mean that considerable attention has been paid to the immune responses of bivalves such as oysters, clams and mussels against infectious diseases that can threaten the viability of entire populations. As with many invertebrates, bivalves have a comprehensive repertoire of immune cells, genes and proteins. Hemocytes represent the backbone of the bivalve immune system. However, it is clear that mucosal tissues at the interface with the environment also play a critical role in host defense. Bivalve immune cells express a range of pattern recognition receptors and are highly responsive to the recognition of microbe-associated molecular patterns. Their responses to infection include chemotaxis, phagolysosomal activity, encapsulation, complex intracellular signaling and transcriptional activity, apoptosis, and the induction of anti-viral states. Bivalves also express a range of inducible extracellular recognition and effector proteins, such as lectins, peptidoglycan-recognition proteins, thioester bearing proteins, lipopolysaccharide and β1,3-glucan-binding proteins, fibrinogen-related proteins (FREPs) and antimicrobial proteins. The identification of FREPs and other highly diversified gene families in bivalves leaves open the possibility that some of their responses to infection may involve a high degree of pathogen specificity and immune priming. The current review article provides a comprehensive, but not exhaustive, description of these factors and how they are regulated by infectious agents. It concludes that one of the remaining challenges is to use new "omics" technologies to understand how this diverse array of factors is integrated and controlled during infection.

Previous transplantation analysis has identified a sensitive histocompatibility system in the solitary urochordate Styela plicata that obeys genetic transplantation "rules" identical to those of vertebrates. The current study demonstrates that histocompatibility acts to prevent fusion between conspecifics in sedentary aggregations of this species. Pairs of naturally fused individuals were present at low frequencies (0.06%) in natural populations. Comparative transplantation analysis, in which grafts were transferred from fused donors to panels of single (non-paired) recipients, confirmed that such fusion occurs only between individuals of identical histocompatibility tissue type. Ninety-four percent of recipients yielded identical responses to allografts from both members of fused pairs, suggesting that fused individuals expressed concordant histocompatibility tissue types. In contrast, only 69% of hosts receiving allografts from pairs of randomly selected unfused individuals yielded identical responses to both donor tissues. Allozyme analysis confirmed the specificity of this correlation between fusion and shared histocompatibility type. Fused individuals retained distinct genetic identities for electrophoretically resolved loci independent of tissue type. In light of these results, it is argued that the role of histocompatibility systems in preventing fusion may have been a strong selective force in the evolution of allogeneic recognition.

View references (418) The Echinodermata are an ancient phylum of benthic marine invertebrates with a dispersal-stage planktonic larva. These animals have innate immune systems characterized initially by clearance of foreign particles, including microbes, from the body cavity of both larvae and adults, and allograft tissue rejection in adults. Immune responsiveness is mediated by a variety of adult coelomocytes and larval mesenchyme cells. Echinoderm diseases from a range of pathogens can lead to mass die-offs and impact aquaculture, but some individuals can recover. Genome sequences of several echinoderms have identified genes with immune function, including expanded families of Toll-like receptors, NOD-like receptors, and scavenger receptors with cysteine-rich domains, plus signaling pathways and cytokines. The set of transcription factors that regulate proliferation and differentiation of the cellular immune system are conserved and indicate the ancestral origins of hematopoiesis. Both larval and adult echinoderms are in constant contact with potential pathogens in seawater, and they respond to infection by phagocytosis and encapsulation, and employ proteins that function in immune detection and response. Antipathogen responses include activation of the SpTransformer genes, a complement system, and the production of many types of antimicrobial peptides. Echinoderms have homologues of the recombinase activating genes plus all associated genes that function in vertebrates for immunoglobulin gene family rearrangement, although their gene targets are unknown. The echinoderm immune system has been characterized as unexpectedly complex, robust, and flexible. Many echinoderms have very long life-spans that correlate with an excellent capacity for cell damage repair. In many marine ecosystems, echinoderms are keystone predators and herbivores, and therefore are species that can serve as optimal sentinels of environmental health. Coelomocytes can be employed in sensor systems to test for the presence of marine pollutants. When Elie Metchnikoff inserted a rose prickle into a larval sea star and observed chemotaxis, phagocytosis, and encapsulation by the mesenchyme cells, he initiated not only the field of immunology but also that of comparative immunology, of which the echinoderms have been an important part.

In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p

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.

Ostreid herpes virus causes serious disease in the Pacific oyster (Crassostrea gigas), but not in the Sydney Rock Oyster (Saccostrea glomerata). To investigate differences in disease progression, we injected oysters with double stranded RNA (dsRNA). dsRNA is known to mimic viral infection, and can evoke immune responses when Toll-like receptors detect the dsRNA, leading to the production of type 1 interferon and inflammation cytokines. The uptake and processing of dsRNA was tracked in gill and mantle tissue of Crassostrea gigas and Saccostrea glomerata after injection of fluorochrome labelled poly (I:C) dsRNA. The two species showed significant differences in tissue uptake and clearance, and differences in immune responses confirmed by real time PCR. These results showed that S. glomerata was more efficient in processing dsRNA than C. gigas, and that the gill tissue is an important site of dsRNA processing and response.

Viral diseases are a significant cause of mortality and morbidity in oysters, resulting in significant economic losses. We investigated the proteomic responses of these two species of oysters to generic double-stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data using isobaric tags for relative and absolute quantitaion (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. Gene ontology analysis showed that several biological processes, cellular components, and molecular function were unique to the different data sets. For example, a number of proteins implicated in the TLR signaling pathway were associated with the Saccostrea glomerata data set but were absent in the Crassostra gigas data set. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility. Molecular targets previously shown to be expressed in C. gigas in response to OsHV1 infections were not present in our proteomic data sets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge, and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.

This study used proteomics to assess the impacts of metal contamination in the field on Sydney Rock oysters. Oysters were transplanted into Lake Macquarie, NSW, for two weeks in both 2009 and 2010. Two-dimensional electrophoresis identified changes in protein expression profiles of oyster haemolymph between control and metal contaminated sites. There were unique protein expression profiles for each field trial. Principal components analysis attributed these differences in oyster proteomes to the different combinations and concentrations of metals and other environmental variables present during the three field trials. Identification of differentially expressed proteins showed that proteins associated with cytoskeletal activity and stress responses were the most commonly affected biological functions in the Sydney Rock oyster. Overall, the data show that proteomics combined with multivariate analysis has the potential to link the effects of contaminants with biological consequences.

Aquaculture necessitates a range of husbandry and handling procedures that induce or exacerbate stress. The relationship between environmental stress and suppression of immune responses has been defined in a range of invertebrate models. In the current study, we investigated the effects of antifouling practices on PO (phenoloxidase activity), which is associated with host defense in mature pearl oysters. The stressors included the use of a cleaning machine (high pressure water jets), being struck with a chisel to remove fouling, a combination of the cleaning machine and chiseling, and exposure to air. We also investigated the effects of exposure to air on PO activity in juvenile pearl oysters. PO activity declined significantly (by 56%, relative to the control) 48 h after oysters were chiseled (p

The Akoya pearl oyster is an economically important species farmed throughout Japan and China, and on a small scale in Australia. Recent proliferation of Akoya viral disease (AVD) has dramatically reduced both wild and farmed populations in Asia. It is thought that AVD is exacerbated by anthropogenic pressures associated with over-crowding and stress caused by fluctuations in the environment, as well as by husbandry and handling. To test the effects of these stressors, the differential expression of genes encoding three antioxidant enzymes (peroxiredoxin, PRx; glutathione-S-transferase, GST; glutathione peroxidase, GPx) and the generic stress biomarker heat shock protein 70 (HSP-70) was investigated using quantitative real-time PCR on mRNA extracted from gill tissues of Pinctada fucata that had been exposed to either mechanical agitation (MA, a combination of air exposure and shaking) or air alone for 1 h and 6 h. We observed a significant decline (up to 50%; p

Currently, there are few predictive biomarkers in key biomonitoring species, such as oysters, that can detect heavy metal pollution in coastal waterways. Several attributes make oysters superior to other organisms for positive biomonitoring of heavy metal pollution. In particular, they are filter feeders with a high capacity for bioaccumulation. In this study, we used two proteomics approaches, namely label-free shotgun proteomics based on SDS-PAGE gel separation and gas phase fractionation, to investigate the heavy metal stress responses of Sydney rock oysters. Protein samples were prepared from haemolymph of oysters exposed to 100 μg/L of PbCl(2), CuCl(2), or ZnCl(2) for 4 days in closed aquaria. Peptides were identified using a Bivalvia protein sequence database, due to the unavailability of a complete oyster genome sequence. Statistical analysis revealed 56 potential biomarker proteins, as well as several protein biosynthetic pathways to be greatly impacted by metal stress. These have the potential to be incorporated into bioassays for prevention and monitoring of heavy metal pollution in Australian oyster beds. The study confirms that proteomic analysis of biomonitoring species is a promising approach for assessing the effects of environmental pollution, and our experiments have provided insights into the molecular mechanisms underlying oyster stress responses.

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an 'inducer' bacterium stimulated the production of antimicrobial molecules by 'producer' bacteria that were active against the 'inducer' bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.

Exposure to fluctuating environmental conditions in bivalve molluscs can lead to physiological stress and up-regulated production of stress-associated hormones, such as noradenaline (NA). Since environmental stressors have been found to have an immunosuppressive effect on Pinctada imbricata, we investigated the in vitro affects of NA exposure on their defensive haemocytes, focussing specifically on markers of apoptosis. Terminal dUTP nick-end (TUNEL) labelling was used to detect cells displaying DNA fragmentation within tissue exposed to NA. DNA fragmentation was most significant when haemocytes were exposed to 10.0 ng NA/μg protein relative to non-treated controls. Similarly, Annexin V-FITC staining, a marker of early apoptotic events, was evident in cells exposed to 5.0 and 10.0 ng NA/μg protein after 120 min (p0.05), and haemocyte adhesion to glass slides declined significantly when cells were exposed to 10.0 ng NA/μg protein (p0.05). A number of morphological and ultrastructural changes in NA-exposed haemocytes were also identified using transmission and scanning electron microscopy. These alterations included chromatin and cytoplasmic condensation, the formation of apoptotic bodies, vacuolisation and blebbing. In NA-treated cells, polymerisation of F-actin was observed around the periphery of the cytoplasm. All of these data suggest that NA induces apoptosis in P. imbricata haemocytes.

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 current study uses proteomics to assess the effects of metal contamination on Sydney Rock oyster haemolymph. Saccostrea glomerata were exposed in aquaria for four days to three environmentally relevant metals (copper, lead or zinc). Oyster haemolymph proteins from metal-exposed oysters were then compared to haemolymph from non-exposed controls using 2-dimensional electrophoresis to identify proteins that differed significantly in intensity. These proteins were then subjected to tandem mass spectrometry so that putative protein identities could be assigned. The data suggest that there are unique protein expression profiles for each metal. Exposure to 100 μg/l of copper, lead or zinc yielded a total of 25 differentially expressed proteins. However, only one of these protein spots exhibited altered intensities in response to all three metals. Eighteen of the 25 spots were significantly affected by just one of the three metals. Differentially expressed proteins were assigned to five different categories of biological function. Proteins affecting shell properties were the most common functional group accounting for 34% of the identified proteins. Cytoskeletal activities and metabolism/stress responses each accounted for a further 25% of the proteins.

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 antibacterial activities of essential oils and herbal extracts have been demonstrated against a range of bacterial species. In this study, the antibacterial effects of a new combination of essential oils from the herbs Thymus vulgaris, Salvia officinalis, Eucalyptus globulus and Mentha piperita were assayed against common bacterial isolates (Staphylococcus aureus, Escherichia coli and Pseudomonas aeroginosa) and fifteen novel marine bacteria isolates. The sensitivity of different isolates to antibacterial activity of the essential oils was determined using well diffusion assays. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were quantified by micro dilution assays. The data demonstrate that the combination of essential oils had potent antibacterial effects and marine bacteria were more sensitive to growth inhibition (P < 0.05). MIC rates were 0.77–6.18 mg/ml, and MBC rates were 1.67–12.30 mg/ml. This indicates that the combined essential oils (CEO) can be a new source of antibacterial agents for use in marine aquaculture.

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.

Our prior work has shown that the catecholamine hormone, noradrenaline, mediates environmental stress responses in Sydney rock oysters, resulting in impaired immunological function. In the current study, we tested the cellular basis of this stress response. Hemocytes were exposed to noradrenaline in vitro before cell morphology and viability were analyzed. Noradrenaline was shown to induce apoptotic markers, including the loss of mitochondrial membrane potential, DNA fragmentation and plasma membrane blebbing. F-actin appeared to play an important role in the changes observed in hemocytes, being concentrated mostly in the plasma membrane blebs of noradrenaline-treated hemocytes. This may explain why hemocyte adhesion and pseudopodia formation were inhibited by noradrenaline. Cellular dysfunction induced by norarenaline mainly affected the hyalinocyte sub-population of hemocytes, whilst the other major cell type, granulocytes, remained unaffected. Given that hyalinocytes are important immunological effectors, the results of this study help to explain why immunosuppression accompanies noradrenaline-mediated stress responses in oysters.

Insect seminal fluid commonly comprises a complex cocktail of proteins and other biochemical components that migrate away from the female reproductive tract to sites elsewhere in the female body and elicit changes in female reproductive behaviour. The transfer of male seminal fluid molecules to reproductive and somatic tissues of the female Queensland fruit fly ('Q-fly') Bactrocera tryoni is examined and some putative target sites identified. Male Q-flies are fed a diet containing radiolabelled ( 35 S) amino acids, which are incorporated into male accessory gland products. Radioactivity diminishes within the accessory glands and increases in all assessed parts of the female body during copulation, indicating the transfer of these products into the female soma via the reproductive tract. There are significant changes in the absolute and proportional radioactivity profiles among female tissues over the next 22 h, with substantial reductions in the thorax and increases in the head. This is consistent with accumulation of behaviour-modifying male products at binding sites in the female head. Parallels can be drawn between the data in the present study and seminal fluid distribution profiles and receptor binding docu- mented in other insects.

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 investigates the effects of noradrenaline injection on a range of immunological activities in Sydney rock oysters (Saccostrea glomerata). Noradrenaline caused a decrease in most of the immunological parameters tested. Phenoloxidase activities in both whole hemolymph and serum decreased significantly within the first 60 min of noradrenaline injection, as did the total frequency of hemocytes in hemolymph, differential hemocyte frequencies, the frequency of phenoloxidase positive cells and phagocytic activity. All of these parameters started to return to normal levels within 120 min. In contrast, the total protein content of hemolymph, which also decreased after noradrenaline injection continued to decline throughout the experimental period. In vitro studies found that superoxide and peroxide production by hemocytes increased in the presence of noradrenaline, but acid phosphatase activity decreased significantly. Additional experiments showed that noradrenaline secretion was stimulated by altered salinity, altered temperature and physical agitation. This suggests that stressors commonly associated with oyster farming may result in noradrenaline-based stress responses that suppress immunological activity.

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.

This study investigates the effects of two pathogen-associated molecular patterns (PAMPs), LPS and zymosan, on the Sydney rock oyster ( Saccostrea glomerata ) immune system. Phenoloxidase and phagocytic activities, total and differential haemocyte frequencies, as well as peroxide and superoxide concentrations were measured after the injection of lipopolysaccharide and zymosan. All of the immunological parameters were induced by both PAMPs. Phenoloxidase (monophenolase and diphenolase) and phagocytic activities, as well as the frequencies of phenoloxidase-positive haemocytes, hyalinocytes and granulocytes in the haemolymph, increased within 24 h of PAMP injection. Values for all of these parameters peaked within 48 h of challenge and began to decrease to levels that were indistinguishable from those of controls within 96 h. The only exception to this pattern was diphenolase activity, which remained elevated for at least 96 h. Control saline injections that lacked PAMPs also induced responses in most of the parameters measured. However, reactions to saline injections were of far lower magnitude compared to those induced by PAMPs. All of the data suggest that the phenoloxidase and phagocytic systems of oysters are inducible components of the Sydney rock oyster immune system, and that induction is primarily due to increased frequencies of specialised haemocytes in the haemolymph.

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca2+ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (Km=4.45+/-1.46 mM) than for the diphenol, l-DOPA (Km=10.27+/-1.33 mM). Maximum enzyme activity was evident at 37 degrees C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.

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.

Little is known about the response of non-model invertebrates, such as oysters, to virus infection. The vertebrate innate immune system detects virus-derived nucleic acids to trigger the type I IFN pathway, leading to the transcription of hundreds of IFN-stimulated genes (ISGs) that exert antiviral functions. Invertebrates were thought to lack the IFN pathway based on the absence of IFN or ISGs encoded in model invertebrate genomes. However, the oyster genome encodes many ISGs, including the well-described antiviral protein viperin. In this study, we characterized oyster viperin and showed that it localizes to caveolin-1 and inhibits dengue virus replication in a heterologous model. In a second set of experiments, we have provided evidence that the haemolymph from poly(I : C)-injected oysters contains a heat-stable, protease-susceptible factor that induces haemocyte transcription of viperin mRNA in conjunction with upregulation of IFN regulatory factor. Collectively, these results support the concept that oysters have antiviral systems that are homologous to the vertebrate IFN pathway.

International audience; Marine molluscs, like all living organisms, are constantly exposed to viruses and have evolved efficient antiviral defences. We review here recent developments in molluscan antiviral immunity against viruses belonging to the order Herpesvirales. Emerging results suggest an interferon-like response and autophagy are involved in the antiviral defence of bivalves to viral infection. Multi-functional plasma proteins from gastropods and bivalves have been identified to have broad-spectrum antiviral activity against mammalian viruses. The antiviral defences present in molluscs can be enhanced by genetic selection, as shown by the presence of oyster strains specifically resistant to ostreid herpesvirus type 1. Whether varying amounts or different isoforms of these antiviral plasma proteins contributes to genetic resistance is worthy of further research. Other evolutionarily conserved antiviral mechanisms, such as RNA interference and apoptosis, still need further characterization

Synthetic double stranded RNA (Poly(I:C)) injection of Crassostrea gigas results in a systemic antiviral response involving many evolutionary conserved antiviral effectors (ISGs). Compared to mammals, the timing of C. gigas ISG expression to viral or poly(I:C) injection is delayed (>12 h p.i.). It could be interpreted that a cytokine is responsible for the systemic, but delayed expression of C. gigas ISGs. We therefore analysed the acellular fraction of C. gigas hemolymph by two-dimensional electrophoresis (2-DE) to identify hemolymph proteins induced by poly(I:C). Poly(I:C) injection increased the relative intensity of four protein spots. These protein spots were identified by tandem mass spectrometry (LC-MS/MS) as a small heat shock protein (sHSP), poly(I:C)-inducible protein 1 (PIP1) and two isoforms of C1q-domain containing protein (C1qDC). RT-qPCR analysis confirmed that the genes encoding these proteins are induced in hemocytes of C. gigas injected with poly(I:C) (p

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.