Exploring the co-exposure effects of environmentally relevant microplastics and an estrogenic mixture on the metabolome of the Sydney rock oyster.

In aquatic environments the concurrent exposure of molluscs to microplastics (MPs) and estrogens is common, as these pollutants are frequently released by wastewater treatment plants into estuaries. Therefore, this study aimed to evaluate the independent and co-exposure impacts of polyethylene microplastics (PE-MPs) and estrogenic endocrine-disrupting chemicals (EEDCs) at environmentally relevant concentrations on polar metabolites and morphological parameters of the Sydney rock oyster. A seven-day acute exposure revealed no discernible differences in morphology; however, significant variations in polar metabolites were observed across oyster tissues. The altered metabolites were mostly amino acids, carbohydrates and intermediates of the Kreb's cycle. The perturbation of metabolites were tissue and sex-specific. All treatments generally showed an increase of metabolites relative to controls - a possible stimulatory and/or a potential hormetic response. The presence of MPs impeded the exposure of adsorbed and free EEDCs potentially due to the selective feeding behaviour of oysters to microplastics, favouring algae over similar-sized PE-MPs, and the formation of an eco/bio-corona involving faeces, pseudo-faeces, natural organic matter, and algae.

Exposure to estrogenic mixtures results in tissue-specific alterations to the metabolome of oysters.

The current study investigated the effect of environmentally relevant mixtures of estrogens at levels representative of receiving waters on the metabolome of the Sydney rock oyster, Saccostrea glomerata. Oysters were exposed to a "low" and a "high" mixture of (xeno) estrogens (representative of Australian and global receiving waters respectively) for 7 days and digestive gland, gill, and gonad tissue were sampled for quantification of polar metabolites by 1H NMR spectroscopy. Exposure to both mixtures lowered body mass and altered the metabolite profile in the digestive glands. Comparatively, gills, and ovaries demonstrated lesser sensitivity to the mixtures, with significant metabolomic alterations observed only for the high mixture. The male gonad did not respond to either estrogenic exposure. In the responsive tissues, major metabolites including amino acids, carbohydrates, intermediates of the tricarboxylic acid cycle and ATP were all down-regulated and exhibited tissue-specific patterns of down-regulation with the greatest proportion of metabolites down-regulated due to estrogenic exposure in the digestive gland. Exposure to (xeno) estrogen mixtures representative of concentrations reported in receiving waters in Australia and globally can impact the metabolome and associated energy metabolism, especially in the digestive gland, translating to lower pools of available ATP energy for potential cellular homeostasis, somatic maintenance and growth, reproduction and fitness.

Chitin/chitosan derivatives and their interactions with microorganisms: a comprehensive review and future perspectives.

Chitosan, obtained as a result of the deacetylation of chitin, one of the most important naturally occurring polymers, has antimicrobial properties against fungi, and bacteria. It is also useful in other fields, including: food, biomedicine, biotechnology, agriculture, and the pharmaceutical industries. A literature survey shows that its antimicrobial activity depends upon several factors such as: the pH, temperature, molecular weight, ability to chelate metals, degree of deacetylation, source of chitosan, and the type of microorganism involved. This review will focus on the in vitro and in vivo antimicrobial properties of chitosan and its derivatives, along with a discussion on its mechanism of action during the treatment of infectious animal diseases, as well as its importance in food safety. We conclude with a summary of the challenges associated with the uses of chitosan and its derivatives.

Characterisation of the metallothionein gene in the Sydney rock oyster and its expression upon metal exposure in oysters with different prior metal exposure histories.

The metal-binding protein metallothionein (MT) is widely used as a biomarker of metal contamination. In this study, we cloned a MT gene (sgMT) from the Sydney rock oyster Saccostrea glomerata. The gene encodes a MT-I protein with a classical αß domain structure and is expressed as two transcripts resulting from alternative polyadenylation. The gene promoter contains two putative metal-responsive elements (MREs) which are known to be required for metal-inducible transcription. A specific and efficient qPCR assay was developed to quantify sgMT mRNA expression. Further, we assessed whether prior metal exposure history influences sgMT mRNA expression upon subsequent metal exposure. Oysters with varying prior metal exposure histories (contaminated and reference) were exposed to Cu, Cd and Zn. Expression of sgMT generally increased with metal dose, and oysters with an elevated past metal exposure history exhibited higher sgMT expression under Cd and Zn stress, representing a potential acclimatory response to prior metal exposure.

Effects of chronic pesticide exposure on an epibenthic oyster reef community.

In recent decades, oyster reefs have been deteriorating throughout North America as a result of multiple interacting anthropogenic stressors, including pesticide pollution. Here we elucidated the potential chronic effects of the commonly utilized pesticide, carbaryl, on oyster reef communities in the Loxahatchee River Estuary in southeast Florida. Though carbaryl had a limited effect on total epifaunal community diversity, species richness and evenness, the results of this experiment indicate that carbaryl significantly shifted crustacean community composition, resulting in a substantial loss in total crustacean abundance. One crustacean in particular, Americorophium spp. (tube building amphipod), was significantly less abundant within the carbaryl treatment, driving the shift in crustacean community composition. Ultimately, our results signal that pesticide pollution in estuaries will negatively impact crustaceans. Over time, this may shift benthic community composition, potentially disrupting species interactions and threatening valuable economic and ecosystem services.

Bioavailability and sequential extraction of mercury in soils and organisms of a mangrove contaminated by a chlor-alkali plant.

Botafogo river estuary poses the highest Hg concentration reported for mangrove soils in Brazil. Such high contamination took place owing to the improper waste disposal for 24 years from a chlor-alkali plant nearby the estuary. Here we determined Hg concentrations in soils, mangrove plants (Rhizophora mangle and Laguncularia racemosa), and an aquatic organism (the oyster species Crassostrea rhizophorae) to assess Hg bioavailability. Besides, a sequential extraction procedure was used to separate soil Hg into five fractions: water-soluble; 'human stomach acid' soluble; organically bound; elemental Hg; mercuric sulfide. Results showed that environmentally available concentration of Hg in the mangrove soils were up to 150-fold higher than a pristine mangrove under the same geological context used as a reference. Additionally, Hg concentration in soils was also above sediment quality guidelines and Hg adverse effects towards sensitive estuarine organisms are likely. Mercury concentrations in oysters were the highest reported in Brazil, but within safety standards according to the country food security agency. It seems that Hg stocks in the studied soils are governed by organic matter and redox conditions, but changing on environmental conditions and land use can alter this balance and convert these mangrove areas from sink to source of Hg to the environment.

Imidacloprid and formulated product impacts the fatty acids and enzymatic activities in tissues of Sydney rock oysters, Saccostrea glomerata.

The use of imidacloprid (IMI) and its formulated products in agriculture is a risk to aquatic organisms due to deposition into waterways from runoff and aerial spraying. However, there is limited information on the potential effects of this pesticide on commercially important shellfish, such as oysters. We investigated the impacts of IMI and Spectrum 200SC (IMI formulation) on the activity of the enzymes Glutathione-S-transferase (GST), Catalase (CAT) and Acetylcholinesterase (AChE), in different oyster tissues including the gill, adductor muscle and digestive gland. We also investigated the condition index and fatty acid composition of the flesh of oysters after 2 weeks exposure. The concentrations of IMI in the different tissues was assessed using Liquid Chromatography-Mass Spectrometry (LC-MS) after QuEChERS extraction. Higher concentrations of IMI residues were detected in the adductor muscle of the oysters, followed by the gills and with the lowest amounts recovered from the digestive gland across all the concentrations tested. IMI and Spectrum 200SC significantly affected the gill AChE activity at 2 mg/L, but digestive gland CAT, and gill and digestive gland GST were impacted at environmentally relevant concentrations (0.01 and 0.05 mg/L). In the whole oyster, 2 weeks exposure to IMI (≥0.01 mg/L) resulted in a proportional increase in saturated fatty acids (SFA), altered the polyunsaturated fatty acid (PUFA) to SFA ratio and altered the omega 3 fatty acids (n-3) to omega 6 fatty acids (n-6) ratio, but there were no effects on the condition index of the oyster. Although the oysters responded differently to the formulated product, there was no consistent difference in the sublethal effects of analytical IMI and Spectrum 200SC. This study showed that exposure to IMI and Spectrum 200SC can significantly affect the biochemical processes and metabolites in oysters, with implications for food quality and safety.

Bioaccumulation and toxic effects of nanoparticulate and ionic silver in Saccostrea glomerata (rock oyster).

The increasing production of Ag nanoparticle (AgNP) containing products has inevitably led to a growing concern about their release into the aquatic environment, along with their potential behaviour, toxicity, and bioaccumulation in marine organisms exposed to NPs released from these products. Hence, this study is focused on the effects of AgNPs in Saccostrea glomerata (rock oyster) in artificial seawater (ASW); evaluating the NP's stability, dissolution, and bioaccumulation rate. AgNPs NM300K (20 ±â€¯5 nm) in concentrations of 12.5 µgL-1 and 125 µgL-1 were used to conduct the experiments, and were compared to a blank and a positive control of 12.5 µgL-1 AgNO3. Dissolution in ASW was measured by ICP-OES and stability was assessed by TEM after 1 h and 3, 5, and 7 days of exposure. Bioaccumulation in gills and digestive glands was measured after 7 days of exposure. The higher concentration of AgNPs induced more aggregation, underwent less dissolution, and showed less bioaccumulation, while the lower concentration showed less aggregation, more dissolution and higher bioaccumulation. Five biomarkers (EROD: ethoxyresorufin-o-deethylase, DNA strand breaks, LPO: lipid peroxidation, GST: glutathione S-transferase and GR: glutathione reductase) were analysed at 0, 3, 5 and 7 days. Significant differences compared to the initial day of exposure (day 0) were reported in DNA strand breaks after 5 and 7 days of exposure, GST, from the third day of exposure, in all the Ag samples, and in some samples for LPO and GR biomarkers, while no significant induction of EROD was observed. A combined effect for each type of treatment and time of exposure was also reported for DNA strand breaks and GST biomarkers measured at the digestive glands. In general, the significant inductions measured showed the following trend: 125 µgL-1 AgNPs >12.5 µgL-1 AgNPs ∼12.5 µgL-1 AgNO3 even though bioaccumulation followed the opposite trend.

Copper and ocean acidification interact to lower maternal investment, but have little effect on adult physiology of the Sydney rock oyster Saccostrea glomerata.

It remains unknown how molluscs will respond to oceans which are increasingly predicted to be warmer, more acidic, and heavily polluted. Ocean acidification and trace metals will likely interact to increase the energy demands of marine organisms, especially oysters. This study tested the interactive effect of exposure to elevated pCO2 and copper on the energetic demands of the Sydney rock oyster (Saccostrea glomerata) during reproductive conditioning and determined whether there were any positive or negative effects on their offspring. Oysters were exposed to elevated pCO2 (1000 µatm) and elevated copper (Cu 50 µg L-1 [0.787 µM]) in an orthogonal design for eight weeks during reproductive conditioning. After eight weeks, energetic demands on oysters were measured including standard metabolic rate (SMR), nitrogen excretion, molar oxygen to nitrogen (O:N) ratio, and pHe of adult oysters as well as the size and total lipid content of their eggs. To determine egg viability, the gametes were collected and fertilised from adult oysters, the percentage of embryos that had reached the trochophore stage after 24 h was recorded. Elevated pCO2 caused a lower extracellular pH and there was a greater O:N ratio in adult oysters exposed to copper. While the two stressors did not interact to cause significant effects on adult physiology, they did interact to reduce the size and lipid content of eggs indicating that energy demand on adult oysters was greater when both elevated pCO2 and copper were combined. Despite the lower energy, there were no negative effects on early embryonic development. In conclusion, elevated pCO2 can interact with metals and cause greater energetic demands on oysters; in response oysters may lower maternal investment to offspring.

DNA damage in marine rock oyster (Saccostrea Cucullata) exposed to environmentally available PAHs and heavy metals along the Arabian Sea coast.

Molecular biomarkers are used world wide for quick assessment of the immediate effect of environmental pollution on marine ecosystems. Recently, we evaluated oxidative stress responses of marine rock oyster, Saccostrea cucullata impacted due to polycyclic aromatic hydrocarbons (PAHs) accumulated in their tissues at a few sampling sites along the coast of Goa around the region of the Arabian sea coast, India (Sarkar et al., 2017). Using a combination of partial alkaline unwinding and comet assays, we now report a comprehensive study on the impairment of DNA integrity (DI) in S. cucullata due to exposure to environmentally available PAHs and also heavy metals (Pb, Cd, Cu, Fe and Mn) along the Arabian Sea coast, Goa, India exclusively around the entire coast of Goa. First, we determined significant correlation between DI in S. cucullata and the extent of exposure to and bioaccumulation of different PAH compounds including 2-3 aromatic ring PAHs (R2, 0.95), 4-6 aromatic ring PAHs (R2, 0.85), oxygenated-PAHs (oxy-PAHs, R2, 0.84) and total PAHs (t-PAHs, R2, 0.98). Second, we observed dose-dependent decrease in DI in S. cucullata with increasing concentrations of different PAH components in oyster tissues. We substantiated our field observations with appropriate laboratory controls using benzo[a]pyrene (BaP). Third, we performed stepwise multiple regression analyses of different water quality parameters including pH, salinity, temperature, dissolved oxygen (DO), biochemical oxygen demand (BOD), nitrite (NO2), nitrate (NO3), phosphate (PO4), turbidity and also t-PAH-biota, t-PAH-water with DI as the dependent variable. Among all these parameters, only four parameters such as t-PAH-biota in combination with DO, BOD and NO2 showed significant correlation (R¯2 = 0.95) with loss in DI in S. cucullata. Based on these results, we created a map indicating the percentage of DNA damage in S. cucullata exposed to PAHs and heavy metals at each sampling location along the west coast of India around Goa, India.

Cholinesterase activity in the cup oyster Saccostrea sp. exposed to chlorpyrifos, imidacloprid, cadmium and copper.

In the present study, the sensitivity and concentration dependence of three functionally-defined components of cholinesterase activity (total: T-ChE; eserine-sensitive: Es-ChE; and eserine-resistant: Er-ChE) were quantified in the gill, digestive gland and adductor muscle of the tropical cup oyster Saccostrea sp., following acute (96h) aqueous exposure to commercial formulations of the organophosphate (OP) insecticide chlorpyrifos and the neonicotinoid (NN) imidacloprid (concentration range: 0.1-100mg/L), as well as to dissolved cadmium and copper (concentration range: 1-1000µg/L). Oysters (1.5-5.0cm shell length), field-collected from a boating marina in Santa Marta, Colombia (Caribbean Sea) were exposed in the laboratory to each substance at five concentrations. T-ChE, Es-ChE, and Er-ChE activity were quantified in the three tissues in pools of 5 individuals (3 replicates per concentration), before and after inhibition with the total cholinesterase inhibitor eserine (physostigmine, 100µM). Oysters exposed to chlorpyrifos, imidacloprid and Cd showed reduced T-ChE and Es-ChE activity in gills at highest exposure concentrations, with Es-ChE activity being inhibited proportionally more so than T-ChE, whereas Er-ChE activity showed no significant concentration-response. Digestive gland also showed diminished T-ChE, Es-ChE and Er-ChE activity for highest chlorpyrifos and Cd concentrations relative to controls, but an increase of T-ChE and Er-ChE activity at the highest imidacloprid concentration (100mg/L). For Cu, T-ChE, Es-ChE and Er-ChE activities in gills and digestive gland were elevated relative to controls in oysters exposed to Cu concentrations > 100µg/L. In adductor muscle, T-ChE, Es-ChE and Er-ChE activity showed no apparent pattern for any of the four xenobiotics and concentration levels tested. Although this study confirms acute (96h) concentration-dependent reduction of tissue T-ChE and Es-ChE activity in gills and digestive glands of Saccostrea sp. exposed to high concentrations of chlorpyrifos (100mg/L), significant changes in T-ChE, Es-ChE and Er-ChE were also caused by exposure to Cd and Cu at concentrations > 100µg/L and by exposure to imidacloprid (100mg/L), indicating that cholinesterase activity is not a specific biomarker of organophosphate exposure in this species, but, rather, a biomarker of diverse xenobiotic exposure.

Dissolution and bandgap paradigms for predicting the toxicity of metal oxide nanoparticles in the marine environment: an in vivo study with oyster embryos.

Dissolution and bandgap paradigms have been proposed for predicting the ability of metal oxide nanoparticles (NPs) to induce oxidative stress in different in vitro and in vivo models. Here, we addressed the effectiveness of these paradigms in vivo and under conditions typical of the marine environment, a final sink for many NPs released through aquatic systems. We used ZnO and MnO2 NPs as models for dissolution and bandgap paradigms, respectively, and CeO2 NPs to assess reactive oxygen radical (ROS) production via Fenton-like reactions in vivo. Oyster embryos were exposed to 0.5-500 µM of each test NP over 24 h and oxidative stress was determined as a primary toxicity pathway across successive levels of biological complexity, with arrested development as the main pathological outcome. NPs were actively ingested by oyster larvae and entered cells. Dissolution was a viable paradigm for predicting the toxicity of NPs in the marine environment, whereas the surface reactivity based paradigms (i.e. bandgap and ROS generation via Fenton-like reaction) were not supported under seawater conditions. Bio-imaging identified potential cellular storage-disposal sites of solid particles that could ameliorate the toxicological behavior of non-dissolving NPs, whilst abiotic screening of surface reactivity suggested that the adsorption-complexation of surface active sites by seawater ions could provide a valuable hypothesis to explain the quenching of the intrinsic oxidation potential of MnO2 NPs in seawater.

How Does Oyster Shell Immobilize Cadmium?

The exact mechanism of cadmium (Cd) immobilization by oyster shell (OS) has not been reported. The effect of OS on Cd immobilization and the exact mechanism should be known before applying remediation technology using OS to Cd contaminated soils. Therefore, the objective of this study was to elucidate the mechanism of Cd immobilization by OS. Three grams of OS (< 0.84 mm) was reacted with 30 mL of 0-3.56 mg Cd L-1 solution at 25 °C for 48 h. Cadmium adsorption increased with increasing initial concentration of Cd in solution. The X-ray diffraction patterns clearly demonstrated that precipitation of CdCO3 did not take place in suspensions of OS after reacting with up to 3.56 mol Cd L-1. Interestingly, we found formation of Ca0.67Cd0.33CO3 crystalline in suspension of OS after reacting with maximum initial Cd concentrations. Precipitation and chemisorption might contribute to Cd immobilization together. However, we feel confident that chemisorption is the major mechanism by which Cd immobilization occurs with OS. In conclusion, OS could be an effective bioadsorbent to immobilize Cd through formation of geochemically stable Cd mineral.

Contrasting impacts of ocean acidification and warming on the molecular responses of CO2-resilient oysters.

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

Adult exposure to ocean acidification is maladaptive for larvae of the Sydney rock oyster Saccostrea glomerata in the presence of multiple stressors.

Parental effects passed from adults to their offspring have been identified as a source of rapid acclimation that may allow marine populations to persist as our surface oceans continue to decrease in pH. Little is known, however, whether parental effects are beneficial for offspring in the presence of multiple stressors. We exposed adults of the oyster Saccostrea glomerata to elevated CO2 and examined the impacts of elevated CO2 (control = 392; 856 µatm) combined with elevated temperature (control = 24; 28°C), reduced salinity (control = 35; 25) and reduced food concentration (control = full; half diet) on their larvae. Adult exposure to elevated CO2 had a positive impact on larvae reared at elevated CO2 as a sole stressor, which were 8% larger and developed faster at elevated CO2 compared with larvae from adults exposed to ambient CO2 These larvae, however, had significantly reduced survival in all multistressor treatments. This was particularly evident for larvae reared at elevated CO2 combined with elevated temperature or reduced food concentration, with no larvae surviving in some treatment combinations. Larvae from CO2-exposed adults had a higher standard metabolic rate. Our results provide evidence that parental exposure to ocean acidification may be maladaptive when larvae experience multiple stressors.

Potential mechanisms underlying estrogen-induced expression of the molluscan estrogen receptor (ER) gene.

In vertebrates, estrogens and estrogen mimicking chemicals modulate gene expression mainly through a genomic pathway mediated by the estrogen receptors (ERs). Although the existence of an ER orthologue in the mollusc genome has been known for some time, its role in estrogen signalling has yet to be deciphered. This is largely due to its constitutive (ligand-independent) activation and a limited mechanistic understanding of its regulation. To fill this knowledge gap, we cloned and characterised an ER cDNA (sgER) and the 5'-flanking region of the gene from the Sydney rock oyster Saccostrea glomerata. The sgER cDNA is predicted to encode a 477-amino acid protein that contains a DNA-binding domain (DBD) and a ligand-binding domain (LBD) typically conserved among both vertebrate and invertebrate ERs. A comparison of the sgER LBD sequence with those of other ligand-dependent ERs revealed that the sgER LBD is variable at several conserved residues known to be critical for ligand binding and receptor activation. Ligand binding assays using fluorescent-labelled E2 and purified sgER protein confirmed that sgER is devoid of estrogen binding. In silico analysis of the sgER 5'-flanking sequence indicated the presence of three putative estrogen responsive element (ERE) half-sites and several putative sites for ER-interacting transcription factors, suggesting that the sgER promoter may be autoregulated by its own gene product. sgER mRNA is ubiquitously expressed in adult oyster tissues, with the highest expression found in the ovary. Ovarian expression of sgER mRNA was significantly upregulated following in vitro and in vivo exposure to 17ß-estradiol (E2). Notably, the activation of sgER expression by E2 in vitro was abolished by the specific ER antagonist ICI 182, 780. To determine whether sgER expression is epigenetically regulated, the in vivo DNA methylation status of the putative proximal promoter in ovarian tissues was assessed using bisulfite genomic sequencing. The results showed that the promoter is predominantly hypomethylated (with 0-3.3% methylcytosines) regardless of sgER mRNA levels. Overall, our investigations suggest that the estrogen responsiveness of sgER is regulated by a novel ligand-dependent receptor, presumably via a non-genomic pathway(s) of estrogen signalling.

Histopathological effect and stress response of mantle proteome following TBT exposure in the Hooded oyster Saccostrea cucullata.

Tributyltin (TBT), an environmental pollutant in marine ecosystems, is toxic to organisms. Although contamination by and bioaccumulation and toxicity of this compound have been widely reported, its underlying molecular mechanisms remain unclear. In the present study, we exposed the Hooded oyster Saccostrea cucullata to TBT to investigate histopathological effects and proteome stress response. Animals were exposed to three TBT sub-lethal concentrations, 10, 50 and 150 µg/l for 48 h. TBT produced stress leading to histopathological changes in oyster tissues including mantle, gill, stomach and digestive diverticula. TBT induced mucocyte production in epithelia and hemocyte aggregation in connective tissue. Cell necrosis occurred when exposure dosages were high. Comparative proteome analyses of mantle protein of oysters exposed to 10 µg/l and control animals were analyzed by a 2-DE based proteomic approach. In total, 32 protein spots were found to differ (p < 0.05). Of these, 17 proteins were identified which included 14 up-regulated and 3 down-regulated proteins. TBT induced the expression of proteins involved in defensive mechanisms (HSP-78, HSP-70, aldehyde dehydrogenase and catalase), calcium homeostasis (VDAC-3), cytoskeleton and cytoskeleton-associated proteins, energy metabolism and amino acid metabolism. Our study revealed that TBT disturbs calcium homeostasis via VDAC-3 protein in mantle and this probably is the key molecular mechanism of TBT acting to distort shell calcification. Moreover, proteins involved in cell structure (tubulin-alpha and tubulin-beta) and protein synthesis were reduced after TBT exposure. Additionally, differential proteins obtained from this work will be useful as potential TBT biomarkers.

Is there a direct relationship between stress biomarkers in oysters and the amount of metals in the sediments where they inhabit?

The effects exerted by metals in oysters are still a matter of debate and require more detailed studies. In this work we have investigated whether the health status of oysters are affected by the amount of metals present in the sediments of their habitat. Sediments and oysters were collected in the tidal part of the estuary of the Oka River (Basque Country), representative of other mesotidal, well mixed and short estuaries of the European Atlantic coast. The concentrations of 14 elements were determined in all the samples. Several biomarkers were also measured in the soft tissues of oysters. According to the concentrations found, the sediments were classified as non-toxic or slightly toxic. In good agreement, the histological alterations observed in oysters were not severe. Interestingly, in those sampling sites where the sediments showed relatively high metal concentrations, the metallic content in oysters was lower, and vice versa.

Combined exposure to pyrene and fluoranthene and their molecular effects on the Sydney rock oyster, Saccostrea glomerata.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously detected in the water column, associated with particulate matter or in the tissue of marine organisms such as molluscs. PAH exposure and their resultant bioaccumulation in molluscs can cause a range of serious physiological effects in the affected animals. To examine the molecular response of these xenobiotics in bivalves, Sydney rock oysters (Saccostrea glomerata) were exposed to pyrene and fluoranthene for seven days. Chemical analysis of the soft-tissue of PAH stressed S. glomerata confirmed that pyrene and fluoranthene could be bioaccumulated by these oysters. RNA-Seq analysis of PAH-exposed S. glomerata showed a total of 765 transcripts differentially expressed between control and PAH-stressed oysters. Closer examination of the transcripts revealed a range genes encoding enzymes involved in PAH detoxification (e.g. cytochrome P450), innate immune responses (e.g. pathogen recognition, phagocytosis) and protein synthesis. Overall, pyrene and fluoranthene exposure appears to have resulted in a suppression of pathogen recognition and some protein synthesis processes, whereas transcripts of genes encoding proteins involved in clearance of cell debris and some transcripts of genes involved in PAH detoxification were induced in response to the stressors. Pyrene and fluoranthene exposure thus invoked a complex molecular response in S. glomerata, with results suggesting that oysters focus on removing the stressors from their system and dealing with the downstream effects of PAH exposure, potentially at the exclusion of other, less immediate concerns (e.g. protection from infection).

Photodynamic effect of curcumin on Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is currently a major cause of bacterial diarrhoea associated with seafood consumption. The objective of this study was to determine the inactivation effect of curcumin-mediated photodynamic action on V. parahaemolyticus. First of all, V. parahaemolyticus suspended in PBS buffer was irradiated by a visible light from a LED light source with an energy density of 3.6J/cm(2). Colony forming units (CFU) were counted and the viability of V. parahaemolyticus cells was calculated after treatment. Singlet oxygen ((1)O2) production after photodynamic action of curcumin was evaluated using 9,10-Anthracenediyl-bis (methylene) dimalonic acid (ADMA). Bacterial outer membrane protein was extracted and analyzed using electrophoresis SDS-PAGE. DNA and RNA of V. parahaemolyticus were also extracted and analyzed using agarose gel electrophoresis after photodynamic treatment. Finally, the efficacy of photodynamic action of curcumin was preliminarily evaluated in the decontamination of V. parahaemolyticus in oyster. Results showed that the viability of V. parahaemolyticus was significantly decreased to non-detectable levels over 6.5-log reductions with the curcumin concentration of 10 and 20µM. Photodynamic action of curcumin significantly increased the singlet oxygen level with the curcumin concentration of 10µM. Notable damage was found to bacterial outer membrane proteins and genetic materials after photodynamic treatment. Photodynamic action of curcumin reduced the number of V. parahaemolyticus contaminating in oyster to non-detectable level. Our findings demonstrated that photodynamic action of curcumin could be a potentially good method to inactivate Vibrio parahaemolyticus contaminating in oyster.