Correction: The biology of environmental stress: molecular biomarkers in Sydney rock oysters (Saccostrea glomerata).

Correction for 'The biology of environmental stress: molecular biomarkers in Sydney rock oysters (Saccostrea glomerata)' by D. A. Raftos et al., Environ. Sci.: Processes Impacts, 2016, 18, 1129-1139.

The biology of environmental stress: molecular biomarkers in Sydney rock oysters (Saccostrea glomerata).

This review describes our recent work on environmental stress in Sydney rock oysters, focusing on the identification of molecular biomarkers for ecotoxicological analysis. We begin by describing the environmental pressures facing coastal estuaries in Australia, with particular reference to Sydney Harbour. After providing that context, we summarise our transcriptional and proteomic analyses of Sydney rock oysters responding to chemical contamination and other forms of environmental stress. This work has shown that the intracellular processes of oysters are highly responsive to environmental threats. Our data agree with the broader literature, which suggests that there is a highly conserved intracellular stress response in oysters involving a limited number of biological processes. We conclude that many effective molecular markers for environmental biomonitoring are likely to lie within these biological pathways.

Differential proteomic response of Sydney rock oysters (Saccostrea glomerata) to prolonged environmental stress.

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.

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

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

Protein markers of Marteilia sydneyi infection in Sydney rock oysters, Saccostrea glomerata.

Marteilia sydneyi is the causative agent of QX disease in Sydney rock oyster, Saccostrea glomerata. It is responsible for disease outbreaks among oysters that occur during summer and can result in up to 95% mortality. QX disease has significantly decreased S. glomerata production in some areas of Australia's eastern seaboard over the past 30 years. Marteilia sydneyi sporulates in the digestive gland of oysters leading to complete disorganization of the infected tissues. The current study used proteomics to identify potential molecular markers of sporulating M. sydneyi infection during a field trial undertaken in the Georges River, Sydney, between December 2006 and May 2007. Early stages of M. sydneyi infection were detected by polymerase chain reaction, whilst cytological examination was used to identify sporulating M. sydneyi in the gut. Protein expression in oyster haemolymph was assessed during the M. sydneyi infection period by two dimensional electrophoresis. Proteome maps identified significant differences in the expression of four proteins in oysters with sporulating M. sydneyi infections.

Anatomy and cytology of the thymus in juvenile Australian lungfish, Neoceratodus forsteri.

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.

Tunicate cytokine-like molecules and their involvement in host defense responses.

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

A complement component C3-like protein from the tunicate, Styela plicata.

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 170kDa heterodimer composed of polypeptides (116 and 80kDa) that have molecular weights comparable to those of C3alpha and C3beta 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.

Effects of tributyltin and other metals on the phenoloxidase activating system of the tunicate, Styela plicata.

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

Humoral opsonins of the tunicate, Pyura stolonifera.

This study characterizes humoral opsonins from the tunicate, Pyura stolonifera. The predominant opsonic components in P. stolonifera hemolymph were found to be calcium-dependent lectins with broad carbohydrate specificities. The opsonic lectins were purified by carbohydrate affinity chromatography which eluted a complex pattern of proteins ranging in molecular mass from 80 to >200kDa. Reducing and two dimensional SDS-PAGE indicated that the diversity of mature lectins evident under non-reducing conditions resulted from the differential oligomerization of two polypeptide sub-units (35 and 22kDa). In addition to lectin-mediated opsonic activity, hemolymph was also found to contain proteolytically activated opsonins. These data suggest that multiple, possibly interactive opsonic systems co-exist in P. stolonifera.

A C-type lectin from the tunicate, Styela plicata, that modulates cellular activity.

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.

Effects of metal-based environmental pollutants on tunicate hemocytes.

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

A collectin-like protein from tunicates.

Collectins are a sub-family of C-type lectins from mammals and birds that are characterized by their collagen-like domains. The mammalian collectin, mannose binding lectin, has attracted considerable interest because it can activate complement components via a lectin-mediated complement pathway that is independent of immunoglobulins. In this study, we have identified a calcium-dependent lectin from the invertebrate (tunicate), Styela plicata, that bears substantial similarities to mammalian collectins. The tunicate lectin, which was isolated by carbohydrate affinity chromatography, has a reduced apparent molecular mass of 43 kDa. The 43 kDa reduced polypeptide appeared as dimers, trimers and hexamers when analyzed by non-reducing and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while gel filtration suggested that the native form of the protein was a nonamer. Amino acid sequence and amino acid composition analysis revealed obvious similarities between the tunicate lectin and mammalian collectins, notably the inclusion of a collagenous domain and a short, cysteine bearing N-terminal domain. The identification of a collectin-like protein in an invertebrate such as S. plicata, which does not express immunoglobulin, indicates that lectin-mediated complement pathways may predate the origin of antibodies.

Chemotactic responses of tunicate (Urochordata, Ascidiacea) hemocytes in vitro.

A number of molecules were found to alter the motility of tunicate hemocytes. Bacterial lipopolysaccharide (LPS) significantly enhanced cell mobility relative to non-stimulated controls. Responses to LPS were not directional and so represented chemokinesis. In contrast, checkerboard analyses indicated that two tunicate hemolymph proteins, tunIL1-alpha and -beta, stimulated truly directional chemotaxis by hemocytes. The data suggest that tunIL1 proteins may contribute to defense by altering the localization of immunocompetent cells.

Effects of Common Estuarine Pollutants on the Immune Reactions of Tunicates.

Tunicates are filter-feeding estuarine and marine animals that are frequently exposed to chronic environmental pollution. This study demonstrates that exposure to low-level (i.e., below the threshold of acute lethality) contamination with tributyltin, creosote, and copper can have substantial effects on natural immune reactions in tunicates. Sublethal doses of toxicants administered either in vitro or in vivo profoundly affected phagocytosis, cellular cytotoxicity, and hematopoietic cell proliferation. Effects were not always inhibitory, and responses often varied depending on the route of toxicant administration. The data suggest that pollutants can activate cascades of cellular processes and compensatory mechanisms, as well as directly inhibiting some of the responses tested. Some evidence indicates that toxicants exert their effects by altering the relative frequencies of circulatory hemocytes.

Interactions of tunicate immunomodulatory proteins with mammalian cells.

The ability of a tunicate immunomodulatory protein, tunIL1, to interact with mammalian cells was investigated. TunIL1 is known to regulate inflammatory defence reactions in tunicates and to stimulate the proliferation of mammalian thymocytes. In the current study, tunIL1 was shown to enchance L929 fibroblast proliferation, induce IL-2 secretion from human mononuclear cells and enhance IL-2 receptor expression by EL-4 murine lymphoma cells. These biological activities are comparable with those of the mammalian inflammatory cytokine, IL-1. However, tunIL1 does not appear to stimulate its effects on mammalian cells by interacting with cell surface receptors in a manner analogous to mammalian IL-1. TunIL1 cannot block the binding of anti-IL-1 receptor antibodies to EL-4 cells, nor can anti-IL-1 receptor antibodies inhibit the capacity of tunIL1 to stimulate thymocyte proliferation. This indicates that tunIL1 does not induce its IL-1-like activities via structural homology to mammalian IL-1.

Cytotoxicity reactions in the solitary tunicate Styela plicata.

Hemocytes from the tunicate, Styela plicata, undertake cytotoxic responses toward human K562 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.

A cell-surface opsonic receptor on leucocytes from the phylogenetically primitive vertebrate, Eptatretus stouti.

A humoral recognition molecule that is homologous to the mammalian complement components C3, C4 and C5 has recently been identified in the Pacific hagfish, Eptatretus stouti. One function of this complement-like protein (CLP) is to opsonize foreign material for phagocytosis by hagfish leucocytes. Here, we demonstrate that CLP's opsonic activity can be abrogated by pre-incubating phagocytes with an anti-hagfish leucocyte mAb (1B1). Moreover, antigen-activated CLP can block the binding of the 1B1 antibody to hagfish leucocytes. Flow cytometry and immunoprecipitation indicate that 1B1 recognizes a 105 kDa cell-surface, monomeric protein that is expressed exclusively on phagocytic hagfish leucocytes. It is concluded that this 105 kDa protein represents the cell surface receptor by which CLP mediates the phagocytosis of opsonized targets.