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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.

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.

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.

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.

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.

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.

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.

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...

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.

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

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.

The sub-cellular and humoral concentrations of a collectin-like protein from the solitary tunicate, Styela plicata, were measured after in vivo challenge with the inflammatory agent, zymosan. Tunicates were injected with zymosan before hemocytes and serum were harvested, subjected to western blotting and immunostained with an anti-S. plicata collectin antibody to determine the relative titers of collectin-like proteins. Concentrations of the predominant 43 kDa collectin polypeptide were found to decrease in hemocytes immediately after zymosan injection, before rising to levels that were six times higher than controls within 96 h. Similarly, immunohistochemistry showed that the frequency of collectin-positive hemocytes in the circulating hemolymph increased significantly within 96 h of injection. Levels of the 43 kDa polypeptide in serum mirrored those of hemocytes. Humoral collectin concentrations decreased immediately after zymosan injection before rising, within 96 h post-injection, to levels three times higher than controls. This response to an inflammatory stimulus resembles that of mammalian collectins like mannose-binding lectin. The data suggest that, like its mammalian counterparts, the tunicate collectin acts as an acute phase antigen recognition protein.

Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

This study identifies a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170 kDa heterodimer composed of polypeptides (116 and 80 kDa) that have molecular weights comparable to those of C3α and C3β from other species. Amino acid sequencing and amino acid composition analysis confirmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi.

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.

Previous studies have identified proteins from tunicates (invertebrate members of the Phylum Chordata) that have physicochemical and functional properties similar to those of the inflammatory cytokine, interleukin 1 (IL-1). Here we characterize one of those proteins from the tunicate, Styela plicata, that can stimulate tunicate and mammalian cell proliferation, activate phagocytosis, increase interleukin 2 (IL-2) secretion by mammalian peripheral blood mononuclear cells and enhance IL-2 receptor (IL-2R) expression by mammalian EL-4.IL-2 cells. Partial amino acid sequence data showed that the S. plicata protein resembles three C-type lectins (TC14, TC14-1 and TC14-2) from a closely related tunicate species, Polyandrocarpa misakiensis. Its similarity to carbohydrate recognition domains (CRDs) from P. misakiensis lectins suggests that the S. plicata protein modulates the activities of mammalian immunocompetent cells by interacting with carbohydrate moieties of glycosylated cell surface receptors.

International audience; : Next generation sequencing (NGS) allows for the rapid, comprehensive and cost effective analysis of entire genomes and transcriptomes. NGS provides approaches for immune response gene discovery, profiling gene expression over the course of parasitosis, studying mechanisms of diversification of immune receptors and investigating the role of epigenetic mechanisms in regulating immune gene expression and/or diversification. NGS will allow meaningful comparisons to be made between organisms from different taxa in an effort to understand the selection of diverse strategies for host defence under different environmental pathogen pressures. At the same time, it will reveal the shared and unique components of the immunological toolkit and basic functional aspects that are essential for immune defence throughout the living world. In this review, we argue that NGS will revolutionize our understanding of immune responses throughout the animal kingdom because the depth of information it provides will circumvent the need to concentrate on a few "model" species.

International audience; The purple sea urchin has a complex immune system that is likely mediated by gene expression in coelomocytes (blood cells). A broad array of potential immune receptors and immune response proteins has been deduced from their gene models. Here we use shotgun mass spectrometry to describe 307 proteins with possible immune function in sea urchins including proteins involved in the complement pathway and numerous SRCRs. The relative abundance of dual oxidase 1, ceruloplasmin, ferritin and transferrin suggests the production of reactive oxygen species in coelomocytes and the sequestration of iron. Proteins such as selectin, cadherin, talin, galectin, amassin and the Von Willebrand factor may be involved in generating a strong clotting reaction. Cell signaling proteins include a guanine nucleotide binding protein, the Rho GDP dissociation factor, calcium storage molecules and a variety of lipoproteins. However, based on this dataset, the expression of TLRs, NLRs and fibrinogen domain containing proteins in coelomic fluid and coelomocytes could not be verified.

Hemocytes from the tunicate, Styela plicata, undertake cytotoxic responses toward human KS62 tumor cells or rabbit red blood cells (RRBC). Reactions to both target cell types were found to require metabolic activity. However, it was not determined whether killing involves soluble factors or direct cell/cell contact. Differences in the response to varied effector: target ratios indicate that reactions against the K562 cells and RRBC are mediated by separate mechanisms. The existence of two discrete cytotoxic mechanisms was supported by experiments which demonstrated that pharmacological treatments have different effects on the cytotoxic destruction of K562 cells and RRBC. Moreover, cytotoxic reactions toward the different targets cells were shown to be mediated by different hemocyte sub-populations isolated from hemolymph by density gradient centrifugation.

International audience; Genome sequences and high diversity cDNA arrays have provided a detailed molecular understanding of immune responses in a number of invertebrates, including sea urchins. However, complementary analyses have not been undertaken at the level of proteins. Here, we use shotgun proteomics to describe changes in the abundance of proteins from coelomocytes of sea urchins after immunological challenge and wounding. The relative abundance of 345 reproducibly identified proteins were measured 6, 24 and 48 h after injection. Significant changes in the relative abundance of 188 proteins were detected. These included pathogen-binding proteins, such as the complement component C3 and scavenger receptor cysteine rich proteins, as well as proteins responsible for cytoskeletal remodeling, endocytosis and intracellular signaling. An initial systemic reaction to wounding was followed by a more specific response to immunological challenge involving proteins such as apolipophorin, dual oxidase, fibrocystin L, aminopeptidase N and α-2-macroglobulin.

The chemotactic responses of hagfish leucocytes were tested using a variety of chemoattractants. Leucocyte migration was significantly enhanced by purified mammalian complement anaphylotoxin (C5a) and LPS-activated hagfish plasma. Checkerboard analyses confirmed that the responses of leucocytes to both of these chemoattractants were directed along concentration gradients (chemotaxis) and did not result from accelerated random movement (chemokinesis). Chemotaxis was undertaken by leucocyte fractions that were enriched in granulocytes, the predominant phagocytic cells of hagfish. The data suggest that chemotactic mechanisms may have been conserved during evolution to such a degree that mammalian chemoattractants can bind and activate chemotactic receptors on hagfish leucocytes. Moreover, hagfish appear to express plasma proteins that are structurally and functionally homologous to mammalian complement anaphylotoxins.

In the current study we examined the effects of metal contamination on the protein complement of Sydney Rock oysters. Saccostrea glomerata were exposed for 4 days to three environmentally relevant concentrations (100 μg/l, 50 μg/l and 5 μg/l) of cadmium, copper, lead and zinc. Protein abundances in oyster haemolymph from metal-exposed oysters were compared to those from non-exposed controls using two-dimensional electrophoresis to display differentially expressed proteins. Differentially expressed proteins were subsequently identified using tandem mass spectrometry (LC-MS/MS), to assign their putative biological functions. Unique sets of differentially expressed proteins were affected by each metal, in addition to proteins that were affected by more than one metal. The proteins identified included some that are commonly associated with environmental monitoring, such as HSP 70, and other novel proteins not previously considered as candidates for molecular biomonitoring. The most common biological functions of proteins were associated with stress response, cytoskeletal activity and protein synthesis.

Opsonins play a key role in invertebrate humoral immune systems. An opsonin for yeast was identified in the plasma of the tunicate, Styela clava. In vitro cultures of hemocytes with homologous plasma-incubated yeast exhibited significantly higher levels of phagocytosis than controls. Studies indicated that the opsonic activity of Styela clava plasma increased the overall capacity for phagocytosis. Opsonization was inhibited by the carbohydrates mannan, N-acetyl-D-galactosamine, and galactose, and by the divalent cation chelator, EDTA. These data suggest that the Styela clava opsonin may share some functional similarities with a C-type lectin. Incubation of yeast with Styela clava and Styela plicata plasma prior to phagocytosis by hemocytes from both species indicated the Styela clava opsonin is species specific.

Specific immunological recognition is not solely a vertebrate trait. Many invertebrates exhibit adaptive histocompatibility responses that bear considerable similarity to venebrate immune reactions. According to our postulate the adaptive immune systems of higher (vertebrate) animals evolved directly from more primitive systems of recognition that are still expressed among advanced invertebrates. If this hypothesis proves to be true, invertebrates may assume an important role as model systems for the analysis of fundamental immunological mechanisms that remain disguised by the complexity of vertebrate systems. Before such invertebrate models can be applied to the rationalization of complex problems, the evolutionary homology of vertebrate and invertebrate immunorecognition must be confirmed. Past studies have demonstrated substantial physiological similarities between invertebrate and vertebrate immunity. However, such functional similarities might also reflect the convergent evolution of otherwi...

The Sp185/333 system of genes, messages and proteins are expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, and is an extraordinary example of diversification of a putative innate immune response system in an invertebrate. Reviewed here, is the current understanding of this complex system as illustrated by sequence comparisons of the genes, messages and deduced proteins with descriptions of diversity, including preliminary results on genomic organization and descriptions of 185/333 in other echinoids. Sp185/333 gene expression in adults and embryos occurs in response to immune challenge and includes changes in the frequencies of Sp185/333-positive coelomocytes in the adults. The diversity of the Sp185/333 protein repertoire in coelomocytes is far greater than the sequence diversity encoded in the genes, which may be the result of rapid gene recombination, RNA editing and/or low-fidelity transcription, plus post-translational modifications. This review concludes with preliminary results and speculations on protein function.

Marine bacteria are a rich source of potentially useful antimicrobial molecules. However, much of the microbial diversity in marine ecosystems with its potential for uncovering new antimicrobial compounds remains to be discovered. This is particularly true for surface-attached marine bacteria, which comprise microbial communities that are generally unique to a host surface and geographic location. The current study characterises culturable microbial communities on marine surfaces from Sydney Harbour, Australia, and tests their antimicrobial activities. A high proportion (47%) of the 104 marine isolates from Sydney Harbour could not be classified to a known genus based on 16S ribosomal RNA gene sequences. Assays of antimicrobial activity from the 104 isolates showed that antimicrobial production is not widespread throughout the phylogeny of isolates with 8 of the 10 antimicrobial producers clustering into a distinct phylogenetic clade. These 8 closely related antibacterial isolates had potent activity in antibacterial cross-dilution assays, with no growth of target bacteria at supernatant concentrations of less than 6.6% v/v. To gain an insight into the types of molecules responsible for this potent activity, differential polarity extractions were carried out on antibacterial culture supernatants from these 8 isolates. All of the activity fractionated into the most polar phase, suggesting that the antibacterial molecules are highly polar. Proteolytic digestion inhibited activity, indicating that the antibacterial molecules were proteins. This study is the first to link the phylogeny of numerous surface-attached marine bacteria with antimicrobial production.

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.

The vertebrate thymus consists of distinctive subpopulations of epithelial cells that contain a diverse repertoire of cytoskeletal proteins. In this study of the thymus in the Australian lungfish, Neoceratodus forsteri, immunohistochemistry was used to distinguish the cytoskeletal proteins present in each class of thymic epithelial cell. A panel of antibodies (Abs), each specific for a different cytoskeletal polypeptide (keratins, vimentin, desmin, actin and tubulins), was used on paraffin and ultrathin resin sections of thymus. Ab AE I (reactive against human type I cytokeratins (CK) 14, 16 and 19) selectively stained the cytoplasm of capsular, trabecular and the outermost epithelial cells of Hassall's corpuscles. Anti-CK 10 Abs strongly labelled the capsular epithelial cells and less than 20% of cortical and medullary epithelial cells. The anti-50-kDa desmin Ab did not react with any thymic cells, whereas the anti-53-kDa desmin Ab labelled some capsular, cortical and medullary thymic epithelial cells. The anti-vimentin Ab stained most of the capsular and ~60% of the cortical epithelium. Thymic nurse cells and Hassall's corpuscles were found to be devoid of actin, which was strongly detected in medullary and perivascular epithelium. Both alpha and beta tubulins were detected in all thymic cells. This study extends the concept of thymic epithelial heterogeneity. The complexity of thymic epithelium in N. forsteri may indicate a relationship between thymic epithelial subpopulations and the thymic microenvironment. These data identify anti-keratin Abs as a valuable tool for studying differentiation and ontogeny of the thymic epithelium in N. forsteri.

Adaptive immune responses in most vertebrates (the gnathostomes: jawed fishes, amphibians, reptiles, birds and mammals) are mediated by lymphocyte-associated antigen receptors encoded by rearranging genes—immunoglobulin (Ig) on B cells and T-cell receptors on T cells. These hypervariable recognition molecules share commonality in their immunoglobulin superfamily (IgSF) domains and the germ-line organization of their rearranging gene elements.

The current paradigm proposes that the innate immune systems of invertebrates are much more complex than previously thought. The highly diverse 185/333 gene family in the purple sea urchin encodes a family of closely related proteins of varying length and sequence composition. Subsets of small phagocytes and polygonal cells express 185/333 proteins with localization on the surface of the small phagocytes and within perinuclear vesicles in both cell types. In short-term cultures, coelomocytes form small aggregates that progress to syncytia that are thought to be equivalent to encapsulation in vivo. These aggregates were found to be enriched for 185/333-positive (185/333+) small phagocytes. In response to lipopolysaccharide challenge, coelomocytes transiently increased, including frequencies of both 185/333+ and 185/333-negative (185/333–) small phagocytes and 185/333+ polygonal cells. The 185/333 proteins were present in a broad array of sizes, most of which were larger than that predicted from the cDNAs. Recombinant 185/333 proteins expressed in bacteria and insect cells were also larger than expected, suggesting that the proteins dimerize and multimerize. The diversity of the 185/333 proteins, their expression in response to immune challenge, and their cellular localization suggests this protein family and the small phagocytes have an important immunological role in the sea urchin.

Pharyngeal explants and circulatory hemocytes from the tunicate Styela clava were cultured in a medium containing tunicate plasma, artificial seawater, RPMI 1640, and antibiotics. Pharyngeal tissue remained viable and proliferated for up to 72 d in vitro. Proliferative activity maintained the pool of hemocytes within explants and facilitated the migration of pharyngeal hemocytes from explants into culture supernatants. The diversity of morphologically distinct cell types within the hemocyte pool of pharyngeal cultures indicated that cell division was followed by regulated differentiation. In contrast to pharyngeal cultures, suspensions of circulatory hemocytes did not survive for prolonged periods in vitro. Proliferative activity could not be detected in circulatory hemocyte cultures. These results are discussed in terms of the differentiation state of hemocytes and the efficacy of culture conditions.

International audience; The anatomy, histology and ultrastructure of the thymus of a dipnoan, the Australian lungfish, Neoceratodus forsteri , was studied by light and transmission electron microscopy. The thymic tissue showed clear demarcation into a cortex and medulla with ample vascularization. Large cells including foamy and giant multinucleated cells with periodic acid Schiff/Alcian blue positive staining properties were localized mainly in the medulla. The major cellular components were epithelial cells and lymphoid cells. The epithelial cells were classified by location and ultrastructure into six sub-populations: capsular cells, cortical and medullary reticular cells, perivascular endothelial cells, intermediate cells, nurse-like cells and Hassall-like corpuscles. Myoid cells were found mainly in the cortico-medullary boundary and medulla. Macrophages and secretory-like cells were also present. These findings will provide a base of knowledge about the cellular immune system of lungfish.

The enzyme phenoloxidase is a critical component of the immunological defence of invertebrates. Previously, we have shown that the activity of phenoloxidase in Sydney rock oysters (Saccostrea glomerata) correlates with the severity of QX disease outbreaks. The aetiological agent of QX disease is the opportunistic protozoan parasite, Marteilia sydneyi. In this study, we examined the response of oyster haemocytes to challenge with M. sydneyi. Granular haemocytes were able to rapidly phagocytose parasite sporonts. Phagocytosis stimulated intracellular associated phenoloxidase activity that led to the complete melanisation of phagosomes. Significant differences in phagocytic indexes and phenoloxidase activities were observed between oysters selected for resistance to QX disease (QXR) and non-selected wild-type oysters. The data suggest that phagocytosis and cellular melanisation are critical defensive responses of Sydney rock oysters infected with M. sydneyi.

SUMMARYThe purple sea urchin Strongylocentrotus purpuratus expresses a homologue of complement component C3 (SpC3), which acts as a humoral opsonin. Significantly increased phagocytic activity was evident when yeast target cells were opsonized after incubation with coelomic fluid containing SpC3. SpC3 could be detected on the surface of yeast, and phagocytic activity could be inhibited by an anti-SpC3 antibody. This indicates that SpC3 promotes phagocytosis by physically tagging target cells for ingestion. Confocal microscopy showed that opsonized yeast were phagocytosed by a single coelomocyte type (polygonal phagocytes), presumably because these cells express SpC3 receptors. Overall, these data indicate that SpC3 is a major humoral opsonin in S. purpuratus coelomic fluid.