Plankton and marine aggregates as transmission vectors for V. aestuarianus 02/041 infecting the pacific oyster Crassostrea gigas.

Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks.

2D Collagen Membranes from Marine Demosponge Chondrosia reniformis (Nardo, 1847) for Skin-Regenerative Medicine Applications: An In Vitro Evaluation.

Research in tissue engineering and regenerative medicine has an ever-increasing need for innovative biomaterials suitable for the production of wound-dressing devices and artificial skin-like substitutes. Marine collagen is one of the most promising biomaterials for the production of such devices. In this study, for the first time, 2D collagen membranes (2D-CMs) created from the extracellular matrix extract of the marine demosponge Chondrosia reniformis have been evaluated in vitro as possible tools for wound healing. Fibrillar collagen was extracted from a pool of fresh animals and used for the creation of 2D-CMs, in which permeability to water, proteins, and bacteria, and cellular response in the L929 fibroblast cell line were evaluated. The biodegradability of the 2D-CMs was also assessed by following their degradation in PBS and collagenase solutions for up to 21 days. Results showed that C. reniformis-derived membranes avoided liquid and protein loss in the regeneration region and also functioned as a strong barrier against bacteria infiltration into a wound. Gene expression analyses on fibroblasts stated that their interaction with 2D-CMs is able to improve fibronectin production without interfering with the regular extracellular matrix remodeling processes. These findings, combined with the high extraction yield of fibrillar collagen obtained from C. reniformis with a solvent-free approach, underline how important further studies on the aquaculture of this sponge could be for the sustainable production and biotechnological exploitation of this potentially promising and peculiar biopolymer of marine origin.

Large-scale impact of the 2016 Marine Heatwave on the plankton-associated microbial communities of the Great Barrier Reef (Australia).

The Great Barrier Reef (GBR) is the world's largest coral ecosystem and is threatened by climate change. This study investigated the impact of the 2016 Marine Heatwave (MHW) on plankton associated microbial communities along a ∼800 km transect in the GBR. 16S rRNA gene metabarcoding of archived plankton samples collected from November 2014 to August 2016 in this region showed a significant increase in Planctomycetes and bacteria belonging to the genus Vibrio and Synechococcus during and after the heatwave. Notably, Droplet Digital PCR and targeted metagenomic analysis applied on samples collected four months after the MHW event revealed the presence of several potential pathogenic Vibrio species previously associated with diseases in aquatic animals. Overall, the 2016 MHW significantly impacted the surface picoplankton community and fostered the spread of potentially pathogenic bacteria across the GBR providing an additional threat for marine biodiversity in this area.

Responses of Mytilus galloprovincialis to challenge with environmental isolates of the potential emerging pathogen Malaciobacter marinus.

Bacteria of the Arcobacter-like spp. represent emerging foodborne zoonotic pathogens in humans and animals. Their increasing presence in seafood, suggesting higher occurrence in seawater due to marine pollution, is raising some environmental concern. Although Arcobacter is frequently detected in diseased oysters and stressed bivalve species, no data are available so far on its potential pathogenicity or interactions with the immune system of the bivalve host. In this work, responses to challenge with two strains of Malaciobacter marinus IRTA-19-131 and IRTA-19-132, R1 and R2), isolated from adult Crassostrea gigas during a mortality event in 2019 in Spain, were investigated in the mussel Mytilus galloprovincialis. In vivo experiments were performed in larvae (48 h post-fertilization), and in adult mussels at 24 h post-injection, in order to evaluate the pathogenicity for early developmental stages, and the hemolymph immune responses, respectively. Both R1 and R2 were moderately pathogenic to early larvae, with significant decreases in the development of normal D-veligers from 104 and 103 CFU/mL, respectively. In adults, both strains decreased hemocyte lysosomal membrane stability (LMS), and stimulated extracellular defense responses (ROS production and lysozyme activity). The interactions between mussel hemocytes and M. marinus were investigated in in vitro short-term experiments (30-90 min) using the R1 strain (106-108 CFU/mL). R1 decreased LMS and induced lysosomal enlargement, but not cell detachment or death, and stimulated extracellular ROS production and lysozyme release, confirming in vivo data. Moreover, lysosomal internalization and degradation of bacteria were observed, together with changes in levels of activated mTor and LC3, indicating phagocytic activity. Overall, the results indicate the activation of both extracellular and intracellular immune defenses against M. marinus R1. Accordingly, these responses resulted in a significant hemolymph bactericidal activity, with a large contribution of hemolymph serum. The results represent the first data on the potential pathogenicity of Arcobacter isolated from a shellfish mortality to bivalve larvae and adults, and on their interactions with the immune system of the host.

A deep-sea bacterium related to coastal marine pathogens.

Evolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here, we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309 m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptation to the deep-sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc-metalloprotease, previously associated with Vibrio infections in farmed oysters. The results of functional in vitro assays on immunocytes (haemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, which might have played an evolutionary role in the origin of coastal marine pathogens.

Aquatic reservoir of Vibrio cholerae in an African Great Lake assessed by large scale plankton sampling and ultrasensitive molecular methods.

The significance of large tropical lakes as environmental reservoirs of Vibrio cholerae in cholera endemic countries has yet to be established. By combining large scale plankton sampling, microbial culture and ultrasensitive molecular methods, namely Droplet Digital PCR (ddPCR) and targeted genomics, the presence of Vibrio cholerae was investigated in a 96,600 L volume of surface water collected on a 322 nautical mile (596 km) transect in Lake Tanganyika. V. cholerae was detected and identified in a large area of the lake. In contrast, toxigenic strains of V. cholerae O1 or O139 were not detected in plankton samples possibly in relation to environmental conditions of the lake ecosystem, namely very low salinity compared to marine brackish and coastal environments. This represents to our knowledge, the largest environmental study to determine the role of tropical lakes as a reservoir of V. cholerae.

Identification, Purification and Molecular Characterization of Chondrosin, a New Protein with Anti-tumoral Activity from the Marine Sponge Chondrosia Reniformis Nardo 1847.

: Chondrosia reniformis is a common marine demosponge showing many peculiarities, lacking silica spicules and with a body entirely formed by a dense collagenous matrix. In this paper, we have described the identification of a new cytotoxic protein (chondrosin) with selective activity against specific tumor cell lines, from C. reniformis, collected from the Liguria Sea. Chondrosin was extracted and purified using a salting out approach and molecular weight size exclusion chromatography. The cytotoxic fractions were then characterized by two-dimensional gel electrophoresis and mass spectrometry analysis and matched the results with C. reniformis transcriptome database. The procedure allowed for identifying a full-length cDNA encoding for a 199-amino acids (aa) polypeptide, with a signal peptide of 21 amino acids. The mature protein has a theoretical molecular weight of 19611.12 and an IP of 5.11. Cell toxicity assays showed a selective action against some tumor cell lines (RAW 264.7 murine leukemia cells in particular). Cell death was determined by extracellular calcium intake, followed by cytoplasmic reactive oxygen species overproduction. The in silico modelling of chondrosin showed a high structural homology with the N-terminal region of the ryanodine receptor/channel and a short identity with defensin. The results are discussed suggesting a possible specific interaction of chondrosin with the Cav 1.3 ion voltage calcium channel expressed on the target cell membranes.

New insights into the possible multiple roles of histidine-rich glycoprotein in blue mussels.

Histidine-rich Glycoprotein (HRG) is the most abundant protein in mussel haemolymph plasma. In this study, we determined by qRT-PCR and FISH analysis the tissues involved in HRG synthesis in Mytilus galloprovincialis. The relative HRG mRNA abundance in haemocytes, digestive gland, gills, gonads, posterior adductor muscle, and mantle edge was evaluated. Immunofluorescence analysis of HRG protein distribution in the whole mussel body was performed by a specific antibody. Our data showed the highest gene expression level of HRG in the mantle edge. In particular the outer fold of the mantle edge was shown to be the site that produced the highest amount of the protein. These data indicate a possible role of this Ca2++-binding protein in shell growth. HRG was also found in many other tissues and cells in contact with the haemolymph. This may be related to the immuno-responsive role of this protein. The presence of HRG in tissues related to the feeding pathways and mucous production could indicate the potential significance of this protein into mucus associated antimicrobial action. Overall, the results demonstrate that numerous mussel tissues are involved in HRG production, some of which can release the protein into the haemolymph and others into the extrapallial fluid. These data indicate that extrapallial (EP) protein and HRG are the same protein. An annual cycle survey showed a maximum HRG mRNA as well HRG protein production in mussel tissues in summer, a season in which the animals show the greatest growth, but are more likely to be exposed to microbial pathogens.

Effects of fullerene C60 in blue mussels: Role of mTOR in autophagy related cellular/tissue alterations.

The effects of C60 on mTOR (mechanistic Target of Rapamycin) activity in mussel digestive gland were investigated. mTOR is a kinase that senses physiological and environmental signals to control eukaryotic cell growth. mTOR is present in two complexes: the phosphorylated mTORC1 regulates cell growth by activating anabolic processes, and by inhibiting catabolic processes (i.e. autophagy); mTORC2 also modulates actin cytoskeleton organization. Mussels were exposed to C60 (0.01, 0.1 and 1 mg/L) for 72 h. Immunocytochemical analysis using a specific antibody revealed the cellular distribution of C60 in mussel digestive gland, already at the lowest concentration. In exposed mussels, the dephosphorylation of mTORC1 and mTORC2 may explain the C60 effects, i.e. the reduction of lysosomal membrane stability, the enhancement of LC3B protein, and the increase of lysosomal/cytoplasmic volume ratio; as well the cytoskeletal alterations. No oxidative stress was observed. Multivariate analysis was used to facilitate the interpretation of the biomarker data. Finally, a low density oligo-microarray was used to understand the cellular responses to fullerene. Transcriptomics identified a number of differentially expressed genes (DEGs) showing a maximum in animals exposed to 0.1 mg/L C60. The most affected processes are associated with energy metabolism, lysosomal activity and cytoskeleton organization. In this study, we report the first data on the subcellular distribution of C60 in mussel's cells; and on the involvement of mTOR inhibition in the alterations due to nanoparticle accumulation. Overall, mTOR deregulation, by affecting protein synthesis, energy metabolism and autophagy, may reduce the capacity of the organisms to effectively grow and reproduce.

Molecular mechanisms underlying the effects of temperature increase on Mytilus sp. and their hybrids at early larval stages.

The present work aims to investigate the effects of water temperature increase on Mytilus galloprovincilis and Mytilus edulis pure larvae (PG, PE) and their hybrids (HFG, HFE). D-larvae were maintained at 18 °C or exposed to a higher temperature of 22 °C for 48 h. Initially, Embryotoxicity test was evaluated. Second, a transcriptomic analysis using a recently developed microarray platform was applied to determine the main biological processes involved in early life stages responses to temperature increase. Finally, an immunofluorescence investigation was performed to bridge the gap between transcriptomic regulation and the real changes at cellular/tissue levels. Embryotoxicity test revealed a higher sensitivity of M. edulis (PE) D-larvae as well as hybrids from females M. edulis (HFE) to temperature increase, with the highest rate of larval malformations. Transcriptomic results indicated a lack of an adequate heat shock protein (Hsp) response in PE and HFE larvae (the high expression was observed in PG larvae); the differential expression of gene involved in translation, energy metabolism and oxidative stress response may contribute to explain the observed complex alterations in the studied conditions. As revealed by immunohistochemistry, cytoskeleton proteins changes associated with a drastic decrease of Histidine-Rich Glycoprotein (HRG) may elucidate the larval abnormalities in shell development observed for PE and HFE larvae. Overall, the results indicate that each type of pure larva (PG and PE) and their respective female hybrid (HFG and HFE) react similarly to the temperature increase. Our data should be carefully considered in view of the water temperature increase in marine ecosystems and especially for the mussel's species in confluence zones.

Elicited ROS Scavenging Activity, Photoprotective, and Wound-Healing Properties of Collagen-Derived Peptides from the Marine Sponge Chondrosia reniformis.

Recently, the bioactive properties of marine collagen and marine collagen hydrolysates have been demonstrated. Although there is some literature assessing the general chemical features and biocompatibility of collagen extracts from marine sponges, no data are available on the biological effects of sponge collagen hydrolysates for biomedical and/or cosmetic purposes. Here, we studied the in vitro toxicity, antioxidant, wound-healing, and photoprotective properties of four HPLC-purified fractions of trypsin-digested collagen extracts-marine collagen hydrolysates (MCHs)-from the marine sponge C. reniformis. The results showed that the four MCHs have no degree of toxicity on the cell lines analyzed; conversely, they were able to stimulate cell growth. They showed a significant antioxidant activity both in cell-free assays as well as in H2O2 or quartz-stimulated macrophages, going from 23% to 60% of reactive oxygen species (ROS) scavenging activity for the four MCHs. Finally, an in vitro wound-healing test was performed with fibroblasts and keratinocytes, and the survival of both cells was evaluated after UV radiation. In both experiments, MCHs showed significant results, increasing the proliferation speed and protecting from UV-induced cell death. Overall, these data open the way to the use of C. reniformis MCHs in drug and cosmetic formulations for damaged or photoaged skin repair.

Application of a new targeted low density microarray and conventional biomarkers to evaluate the health status of marine mussels: A field study in Sardinian coast, Italy.

In the present study, we investigated the health status of marine mussels (Mytilus galloprovincialis) caged and deployed at three different sites on the Sardinian coastline characterized by different levels of contamination: Fornelli (F, the reference site), Cala Real (CR), and Porto Torres (PT). A new low density oligonucleotide microarray was used to investigate global gene expression in the digestive gland of mussels. Target genes were selected to cover most of the biological processes involved in the stress response in bivalve mollusks (e.g. DNA metabolism, translation, immune response, cytoskeleton organization). A battery of classical biomarkers was also employed to complement the gene expression analyses. Chemical analysis revealed higher loads of heavy metals (Pb and Cu) and total polycyclic aromatic hydrocarbons (PAHs) at PT compared to the other sites. In mussels deployed at CR, functional genomics analysis of the microarray data rendered 78 differentially expressed genes (DEGs) involved in 11 biological processes. Animals exposed at PT had 105 DEGs that were characterized by the regulation of 14 biological processes, including mitochondrial activity, adhesion to substrate, DNA metabolism, translation, metal resistance, and cytoskeleton organization. Biomarker data (lysosomal membrane stability, lysosomal/cytoplasm volume ratio, lipofuscin accumulation, metallothionein content, micronucleus frequency, and cytoskeleton alteration) were in trend with transcriptomic output. Biomarker data were integrated using the Mussel Expert System (MES), allowing defining the area in which the presence of chemicals is toxic for mussels. Our study provides the opportunity to adopt a new approach of integrating transcriptomic (microarray) results with classical biomarkers to assess the impact of pollutants on marine mussels in biomonitoring programs.

Role of mTOR in autophagic and lysosomal reactions to environmental stressors in molluscs.

Lysosomal membrane stability (LMS) has been used in various organisms as a very sensitive biomarker of stress. However, despite the abundance of data about regulation of the autophagic process in mammals, in the invertebrates there is only limited mechanistic understanding. Marine mussels (Mytilus galloprovincialis Lam.) are bivalve molluscs, widely used as models in ecotoxicology and as environmental bioindicators of sea water quality. In order to elucidate this fundamental process, in the present study, mussels were exposed for 3 days to a "priority", ubiquitous environmental contaminant, benzo[a]pyrene (B[a]P) at different concentrations (i.e. 5, 50, 100 µg/L seawater). B[a]P accumulated in lysosomes of digestive tubule epithelial cells (digestive cells) and in enlarged lipid-rich lysosomes (autolysosomes) as detected by immunofluorescence and UV-fluorescence. B[a]P also activated the autophagic process with a marked decrease of LMS and concurrent increase in lysosomal/cytoplasmic volume ratio. Dephosphorylation of mTOR contributes to increased lysosomal membrane permeability and induced autophagy. B[a]P induced a decrease in phosphorylated (active form) mTOR. The probable role of mTOR in cell signalling and the regulation of the cellular responses to the contaminants has been also confirmed in a field study, where there was significant inactivation of mTOR in stressed animals. Statistical and network modelling supported the empirical investigations of autophagy and mTOR; and was used to integrate the mechanistic biomarker data with chemical analysis and DNA damage.

Effects of Cr(VI) on Ca2+-ATPase activity in the earthworm Eisenia andrei.

The effect of Cr(VI) as a soil contaminant on the edaphic worm Eisenia andrei was studied by evaluating the activity of Ca2+-ATPase in the intestinal mucosa. In eukaryotes, Ca2+-ATPase is a key mediator of cell signaling although comparatively little is known about its activity in earthworms. Size and anatomical constraints (i.e. small and complex) led us to develop and optimize a cyto-biochemical method to measure Ca2+-ATPase activity in earthworms. The principal site of enzyme activity was found to be the post clitellar intestinal tract; immunohistochemistry then identified plasma membrane Ca2+-ATPase (PMCA ATPase) in the apical area of the intestinal epithelium. Earthworms exposed for 28days to OECD soil contaminated with 1, 2, and 15mg/Kg Cr(VI) demonstrated about 70% inhibition of Ca2+-ATPase activity at the low Cr (VI) concentration (the half of the Italian law limit for residential areas), rising to approximately 84% inhibition at the highest concentration. Reduced enzyme activity was accompanied by decreased enzyme content and reduced lysosomal membrane stability (LMS), which is a well established early warning biomarker of stress. These data demonstrate the potential utility of Ca2+-ATPase activity as a sensitive parameter with which to detect environmental stress in earthworms.

Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses.

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new 'STressREsponse Microarray' (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 µg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota.

Haemolymph from Mytilus galloprovincialis: Response to copper and temperature challenges studied by (1)H-NMR metabonomics.

Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16 °C) or high temperature (24 °C) and in the absence or presence (5 µg/L, 20 µg/L, or 40 µg/L) of sublethal copper (Cu(2+)). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the (1)H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16 °C became detectable at a dose of 20 µg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24 °C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.

Molecular and Cellular Effects Induced in Mytilus galloprovincialis Treated with Oxytetracycline at Different Temperatures.

The present study evaluated the interactive effects of temperature (16°C and 24°C) and a 4-day treatment with the antibiotic oxytetracycline (OTC) at 1 and 100 µg/L on cellular and molecular parameters in the mussel Mytilus galloprovincialis. Lysosomal membrane stability (LMS), a sensitive biomarker of impaired health status in this organism, was assessed in the digestive glands. In addition, oxidative stress markers and the expression of mRNAs encoding proteins involved in antioxidant defense (catalase (cat) and glutathione-S-transferase (gst)) and the heat shock response (hsp90, hsp70, and hsp27) were evaluated in the gills, the target tissue of soluble chemicals. Finally, cAMP levels, which represent an important cell signaling pathway related to oxidative stress and the response to temperature challenges, were also determined in the gills. Exposure to heat stress as well as to OTC rendered a decrease in LMS and an increase in malonedialdehyde accumulation (MDA). CAT activity was not significantly modified, whereas GST activity decreased at 24°C. Cat and gst expression levels were reduced in animals kept at 24°C compared to 16°C in the presence or absence of OTC. At 16°C, treatment with OTC caused a significant increase in cat and gst transcript levels. Hsp27 mRNA was significantly up-regulated at all conditions compared to controls at 16°C. cAMP levels were increased at 24°C independent of the presence of OTC. PCA analysis showed that 37.21% and 25.89% of the total variance was explained by temperature and OTC treatment, respectively. Interestingly, a clear interaction was observed in animals exposed to both stressors increasing LMS and MDA accumulation and reducing hsp27 gene expression regulation. These interactions may suggest a risk for the organisms due to temperature increases in contaminated seawaters.

Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis).

Mollusc haemolymph proteins are known to play several important physiological roles in the immune system, heavy metal transport and the tissue distribution of lipophilic compounds. In this study, we analysed acetone-extracted proteins from mussel haemolymph by one- and two-dimensional gel electrophoresis. The proteins were identified by comparing mass spectrometry data with the invertebrate EST database, allowing us to establish the mussel haemolymph serum proteome. Extrapallial protein (EP) precursor represents the most abundant serum protein; astacin and CuZn superoxide dismutase were also detected. Slight contamination from muscle proteins, due to the sampling method, was also found. No differences were observed in the profiles obtained for male and female serum proteins. One aspect of interest was the previously reported finding that alkali-labile phosphate (ALP) from haemolymph serum may be representative of vitellogenin (vtg)-like protein content in the circulatory fluid of molluscs. In our analysis of mussel haemolymph serum, vitellogenin-like proteins were never found. To confirm these data, a typical methyl-tert-butyl-ether (MTBE) extraction, which is specific for vtg-like proteins, was performed, and the results of the electrophoretic analyses were compared with those obtained by acetonic precipitation. The results showed that the electrophoretic profiles are similar and that vtg-like proteins cannot be identified. Moreover, the main phosphoprotein present in female and male extracts is EP protein precursor. In addition, agarose gel electrophoresis demonstrates that high-molecular-weight forms of vtg-like proteins are not detectable.

Transcriptional expression levels and biochemical markers of oxidative stress in Mytilus galloprovincialis exposed to nickel and heat stress.

The present study aims to evaluate transcriptional expression levels and biochemical markers of oxidative stress responses to nickel (Ni) exposure along with heat stress gradient in a mussel (Mytilus galloprovincialis). For this purpose, we investigated the response of oxidative stress markers, metallothionein accumulation and gene expression in digestive gland of mussels exposed to a sublethal concentration of Ni (2.5µM) along with a temperature gradient (18°C, 22°C, and 26°C) for 24h and 72h. Ni digestive gland uptake was evaluated after the exposure periods. Co-exposure to Ni and higher temperature (26°C) for 72h significantly decreased the antioxidant enzyme activities termed as catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) and caused a pronounced increase of lipofuscin and neutral lipid (NL) accumulation. Ni-uptake was different with respect to the exposure periods and temperatures in Ni-exposed mussels. Sod, cat, gst, mt-10 and mt20 gene expression levels showed a substantial increased pattern in animals exposed for one day to heat stress compared to the control condition (18°C). The same pattern but with highest level was registered in animals co-exposed to Ni and temperatures within one day. Three days exposure to 18°C, 22°C and 26°C, resulted in a significant decrease in mRNA abundance of cat, gst and sod and a significant down-regulation of mts targets (22°C and 26°C). Our data provide new insights into the importance of the early protective response of oxidative stress related-gene expression and regulation in mussels challenging heat stress and sublethal Ni concentration.

Development and application of an innovative expert decision support system to manage sediments and to assess environmental risk in freshwater ecosystems.

With the aim of supporting decision makers to manage contamination in freshwater environments, an innovative expert decision support system (EDSS) was developed. The EDSS was applied in a sediment quality assessment along the Bormida river (NW, Italy) which has been heavily contaminated by an upstream industrial site for more than a century. Sampling sites were classified by means of comparing chemical concentrations with effect-based target values (threshold and probable effect concentrations). The level of each contaminant and the combined toxic pressure were used to rank sites into three categories: (i) uncontaminated (8 sites), (ii) mildly contaminated (4) and (iii) heavily contaminated (19). In heavily contaminated sediments, an environmental risk index (EnvRI) was determined by means of integrating chemical data with ecotoxicological and ecological parameters (triad approach). In addition a sediment risk index (SedRI) was computed from combining chemical and ecotoxicological data. Eight sites exhibited EnvRI values ≥0.25, the safety threshold level (range of EnvRI values: 0.14-0.31) whereas SedRI exceeded the safety threshold level at 6 sites (range of SedRI values: 0.16-0.36). At sites classified as mildly contaminated, sublethal biomarkers were integrated with chemical data into a biological vulnerability index (BVI), which exceeded the safety threshold level at one site (BVI value: 0.28). Finally, potential human risk was assessed in selected stations (11 sites) by integrating genotoxicity biomarkers (GTI index falling in the range 0.00-0.53). General conclusions drawn from the EDSS data include: (i) in sites classified as heavily contaminated, only a few exhibited some significant, yet limited, effects on biodiversity; (ii) restrictions in re-using sediments from heavily contaminated sites found little support in ecotoxicological data; (iii) in the majority of the sites classified as mildly contaminated, tested organisms exhibited low response levels; (iv) preliminary results on genotoxicity biomarkers indicate possible negative consequences for humans if exposed to river sediments from target areas.