Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

Molecular effects on spermatozoa of Mytilus galloprovincialis exposed to hyposaline conditions.

Salinity represents a critical environmental and an ecological factor in the reproduction of marine species. As global climate changes and anthropogenic factors affect salinity, in this study, we have analyzed the responses of Mytilus galloprovincialis spermatozoa to hyposaline stress. We exposed mussels, in laboratory tanks, for 24 hr at 18°C to control (35.9 psu) and three hyposaline (17.1, 22.6, and 26.2 psu) conditions, and evaluated the expression of sperm hsp70 and protamine-like proteins genes. Further we analyzed the electrophoretic pattern, the DNA binding and the release from sperm nuclei of protamine-like proteins. For all experimental approaches used, the results obtained at 17.1 psu condition were very similar to those obtained in the control condition, while alterations were always recorded at 22.6 and 26.2 psu conditions. Particularly, at 22.6 and 26.2 psu, was observed: 42.5- and 17.1-fold increase in hsp70 expression, respectively, and hypoexpression of PL-II/PLIV protamine-like proteins genes. Further, electrophoretic mobility shift assays and salt-induced release of nuclear proteins from sperm nuclei, revealed alterations in the PL proteins/DNA binding, in these two hyposaline conditions. The similarity between the results obtained in control and in the more severe hyposaline condition (17.1 psu) could indicate a phenomenon of fertility preservation strategy due to gamete plasticity.

Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.

Flow cytometry and micro-Raman spectroscopy: Identification of hemocyte populations in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) from Faro Lake and Tyrrhenian Sea (Sicily, Italy).

Immunological and structural characteristics of hemocyte populations in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae), going from two different Sicilian habitats (Faro Lake and Tyrrhenian sea), was investigated by means of two different techniques (flow cytometric and micro-Raman spectroscopy analyses). For this purpose, three hundred and sixty mussels Mytilus galloprovincialis were analyzed during November 2017. They were divided into two equal groups (triplicate sample) on the basis of the site of collection (n = 60 caught in Faro Lake - group A, and n = 60 caught in Tyrrhenian Sea - group B). Some several differences between the species of Faro Lake and Tyrrhenian Sea are observed and ascribed to the disruption of immune parameters induced by the variations of some qualitative water parameters (temperature, salinity, dissolved oxygen, pH, ammonium 10, free chlorine, total chlorine, total phosphate, orthofhosphate) recorded in the two habitats. This study is relevant for monitoring the conditions of the sea and Faro Lake, which is strongly influenced by the currents of the Tyrrhenian Sea. Faro lake is well known for the cultivation of mussels and this is part of a coastal habitat of particular interest, consisted of a peculiar biocenotic complex. Further, for the first time, significant different arrangement in the mussels cell structural organization was evidenced by simply following their highly reproducible Raman biomolecular signatures.

Effects of nanosilver on Mytilus galloprovincialis hemocytes and early embryo development.

Silver nanoparticles (AgNP), one of the main nanomaterials for production and use, are expected to reach the aquatic environment, representing a potential threat to aquatic organisms. In this study, the effects of bare AgNPs (47 nm) on the marine mussel Mytilus galloprovincialis were evaluated at the cellular and whole organism level utilizing both immune cells (hemocytes) and developing embryos. The effects were compared with those of ionic Ag+(AgNO3). In vitro short-term exposure (30 min) of hemocytes to AgNPs induced small lysosomal membrane destabilization (LMS EC50 = 273.1 µg/mL) and did not affect other immune parameters (phagocytosis and ROS production). Responses were little affected by hemolymph serum (HS) as exposure medium in comparison to ASW. However, AgNPs significantly affected mitochondrial membrane potential and actin cytoskeleton at lower concentrations. AgNO3 showed much higher toxicity, with an EC50 = 1.23 µg/mL for LMS, decreased phagocytosis and induced mitochondrial and cytoskeletal damage at similar concentrations. Both AgNPs and AgNO3 significantly affected Mytilus embryo development, with EC50 = 23.7 and 1 µg/L, respectively. AgNPs caused malformations and developmental delay, but no mortality, whereas AgNO3 mainly induced shell malformations followed by developmental arrest or death. Overall, the results indicate little toxicity of AgNPs compared with AgNO3; moreover, the mechanisms of action of AgNP appeared to be distinct from those of Ag+. The results indicate little contribution of released Ag+ in our experimental conditions. These data provide a further insight into potential impact of AgNPs in marine invertebrates.

[omim][BF4]-mediated toxicity in mussel hemocytes includes its interaction with cellular membrane proteins.

The current study is based on the increasing demand for the assessment of ionic liquid (IL)-mediated aquatic toxicity. Specifically, although a lot of studies have been performed so far, investigating IL-mediated adverse effects on numerous aquatic organisms, little is known about their mode of action. Given that the use of in vitro models is considered as a reliable tool for determining the mediated biological effects, the modulation of specific biochemical pathways and the onset of various forms of damage with great precision and reproducibility, mixed primary cultures of mussel Mytilus galloprovincialis hemocytes were used for investigating whether 1-octyl-3-methylimidazolium tetrafluoroborate ([omim][BF4]) mediated toxicity is related to its interaction with cellular membrane proteins. Specifically, [omim][BF4]-mediated cytotoxic, oxidative and genotoxic effects were investigated in mussel hemocytes before and after pre-treatment of cells with non-toxic concentration of guanidine hydrochloride (1 mM GndHCl). The results showed that [omim][BF4] at concentrations ranging from 0.7 to 1.75 µM can induce cytotoxic (almost <50% reduction of cell viability), oxidative (increased levels of O2•- production and lipid peroxidation by-products) and genotoxic (increased levels of DNA damage) effects, while cells pre-treated with 1 mM GndHCl showed a significant attenuation of IL's toxic potency in all cases. According to the latter, the current study showed that [omim][BF4]-mediated toxicity could be related not only to its well-known interaction with membrane lipid bilayers, but also to its interference with membrane proteins. Using GndHCl, a chaotropic agent that disrupts the hydrogen bonding network and the stability of membrane proteins via its interference with the intramolecular interactions mediated by non-covalent forces on cellular membranes, it was firstly shown that altering the membrane integrity as well as the native state of cellular membrane proteins, by weakening the hydrophobic effect, could attenuate the possible interaction of [omim][BF4] with cellular membranes and the concomitant induction of protein-based intracellular processes, commonly linked with the induction of severe cellular damage.

Evaluation of biomarkers in Mytilus galloprovincialis as an integrated measure of biofilm-membrane bioreactor (BF-MBR) system efficiency in mitigating the impact of oily wastewater discharge to marine environment: a microcosm approach.

The large volumes of oily wastewater discharged to marine environment cause heavy impacts on the coastal marine ecosystem. The selection of an appropriate technology to reduce these impacts should be based on the respect of the discharge limits and on the effective assessment and monitoring of its effects on biological organism preservation. To this aim, we set up a controlled microcosm-scale system to compare the effects of a treated and untreated oily wastewater discharge in which the restore process is performed through a Membrane Bio-Reactor. The system is completed by other three microcosms to control and isolate any possible concurrent effect on the Mytilus galloprovincialis, used as sentinel organism. Mytilus galloprovincialis have been kept in all these microcosms, and then mRNA expression and morphology were evaluated on gills and digestive gland. The genes considered in this work are Heat Shock Protein 70 and Metallothionein 10, involved in response to physicochemical sublethal stressors, Superoxide dismutase 1, Catalase, and Cytochrome P450 involved in oxidative stress response. Our results evidenced a significant overexpression, both in gills and digestive gland, of HSP70 in samples maintained in the microcosm receiving the untreated effluent, and of MT10 in those animals kept in microcosm where the effluent was treated. Even though the mRNA modifications are considered "primary" and transient responses which do not always correspond to protein content, the study of these modifications can help to gain insights into the mechanisms of action of xenobiotic exposure. Morphological analysis suggested that, although different, depending on the microcosm, the most serious damages were found in the gill epithelium accompanied with severe haemocyte infiltration, whilst in digestive gland the tissue architecture alterations and the haemocyte infiltration were less pronounced. These observations suggest that the immune system was activated as a general response to stressful stimuli such as the presence of toxic compounds. Moreover, the results indicate that the treatment process is useful. In fact, samples derived from the microcosm receiving the treated effluent, even though presenting some signs of stress, seemed to partially recover the normal structure, although their mRNA expression indicated some cellular suffering.

Cytotoxicity and cellular mechanisms of toxicity of CuO NPs in mussel cells in vitro and comparative sensitivity with human cells.

There is a need to assess human and ecosystem health effects of copper oxide nanoparticles (CuO NPs), extensively used in many industrial products. Here, we aimed to determine the cytotoxicity and cellular mechanisms involved in the toxicity of CuO NPs in mussel cells (hemocytes and gill cells) in parallel with exposures to ionic Cu and bulk CuO, and to compare the sensitivity of mussel primary cells with a well-established human cell line (pulmonary TT1 cells). At similar doses, CuO NPs promoted dose-dependent cytotoxicity and increased reactive oxygen species (ROS) production in mussel and human cells. In mussel cells, ionic Cu was more toxic than CuO NPs and the latter more than bulk CuO. Ionic Cu and CuO NPs increased catalase and acid phosphatase activities in both mussel cells and decreased gill cells Na-K-ATPase activity. All Cu forms produced DNA damage in hemocytes, whereas in gill cells only ionic Cu and CuO NPs were genotoxic. Induction of the MXR transport activity was found in gill cells exposed to all forms of Cu and in hemocytes exposed to ionic Cu and CuO NPs. Phagocytosis increased only in hemocytes exposed to CuO NPs, indicating a nanoparticle-specific immunostimulatory effect. In conclusion, toxicity of CuO NPs is driven by ROS in human and mussel cells. Mussel cells respond to CuO NP exposure by triggering an array of defensive mechanisms.

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.

Rapid Assessment of Genotoxicity by Flow Cytometric Detection of Cell Cycle Alterations.

Flow cytometry is a convenient method for the determination of genotoxic effects of environmental pollution and can reveal genotoxic compounds in unknown environmental mixtures. It is especially suitable for the analyses of large numbers of samples during monitoring programs. The speed of detection is one of the advantages of this technique which permits the acquisition of 104-105 cells per sample in 5 min. This method can rapidly detect cell cycle alterations resulting from DNA damage. The outcome of such an analysis is a diagram of DNA content across the cell cycle which indicates cell proliferation, G2 arrests, G1 delays, apoptosis, and ploidy.Here, we present the flow cytometric procedure for rapid assessment of genotoxicity via detection of cell cycle alterations. The described protocol simplifies the analysis of genotoxic effects in marine environments and is suitable for monitoring purposes. It uses marine mussel cells in the analysis and can be adapted to investigations on a broad range of marine invertebrates.

Lipid unsaturation per se does not explain the physical state of mitochondrial membranes in Mytilus galloprovincialis.

Through a multiple approach, the present study on the mitochondrial membranes from mussel gills and swine heart combines some biochemical information on fatty acid composition, sterol pattern, and temperature dependence of the F1FO-ATPase activity (EC 3.6.3.14.) with fluorescence data on mitochondrial membranes and on liposomes obtained from lipid extracts of mitochondria. The physical state of mussel gills and swine heart was investigated by Laurdan steady state fluorescence. Quite surprisingly, the similar temperature dependence of the F1FO complex, illustrated as Arrhenius plot which in both mitochondria exhibits the same discontinuity at approximately 21°C and overlapping activation energies above and below the discontinuity, is apparently compatible with a different composition and physical state of mitochondrial membranes. Accordingly, mussel membranes contain highly unsaturated fatty acids, abundant sterols, including phytosterols, while mammalian membranes only contain cholesterol and in prevalence shorter and less unsaturated fatty acids, leading to a lower membrane unsaturation with respect to mussel mitochondria. As suggested by fluorescence data, the likely formation of peculiar microdomains interacting with the membrane-bound enzyme complex in mussel mitochondria could produce an environment which somehow approaches the physical state of mammalian mitochondrial membranes. Thus, as an adaptive strategy, the interaction between sterols, highly unsaturated phospholipids and proteins in mussel gill mitochondria could allow the F1FO-ATPase activity to maintain the same activation energy as the mammalian enzyme.

An integrated proteomic and metabolomic study on the gender-specific responses of mussels Mytilus galloprovincialis to tetrabromobisphenol A (TBBPA).

Tetrabromobisphenol A (TBBPA), accounting for the largest production of brominated flame-retardants (BFRs) along the Laizhou Bay in China, is of great concern due to its diverse toxicities. In this study, we focused on the gender-specific responses of TBBPA in mussel Mytilus galloprovincialis using an integrated proteomic and metabolomic approach. After exposure of TBBPA (10 µg L(-1)) for one month, a total of 9 metabolites and 67 proteins were altered in mussel gills from exposed group. The significant changes of metabolites in female mussel gills from exposed group exhibited the disturbances in energy metabolism and osmotic regulation, while in male samples only be found the variation of metabolites related to osmotic regulation. iTRAQ-based proteomic analysis showed biological differences between male and female mussel gills from solvent control group. The higher levels of proteins related to primary and energy metabolism and defense mechanisms in male mussel gills meant a greater anti-stress capability of male mussels. Further analysis revealed that TBBPA exposure affected multiple biological processes consisting of production and development, material and energy metabolism, signal transduction, gene expression, defense mechanisms and apoptosis in both male and female mussels with different mechanisms. Specially, the responsive proteins of TBBPA in male mussels signified higher tolerance limits than those in female individuals, which was consistent with the biological differences between male and female mussel gills from solvent control group. This work suggested that the gender differences should be considered in ecotoxicology.

Comet assay with gill cells of Mytilus galloprovincialis end point tools for biomonitoring of water antibiotic contamination: Biological treatment is a reliable process for detoxification.

This article investigates the ability of Pseudomonas peli to treat industrial pharmaceuticals wastewater (PW). Liquid chromatography-mass spectrometry (MS)/MS analysis revealed the presence, in this PW, of a variety of antibiotics such as sulfathiazole, sulfamoxole, norfloxacine, cloxacilline, doxycycline, and cefquinome.P. peli was very effective to be grown in PW and inducts a remarkable increase in chemical oxygen demand and biochemical oxygen demand (140.31 and 148.51%, respectively). On the other hand, genotoxicity of the studied effluent, before and after 24 h of shaking incubation with P. peli, was evaluated in vivo in the Mediterranean wild mussels Mytilus galloprovincialis using comet assay for quantification of DNA fragmentation. Results show that PW exhibited a statistically significant (p< 0.001) genotoxic effect in a dose-dependent manner; indeed, the percentage of genotoxicity was 122.6 and 49.5% after exposure to 0.66 ml/kg body weight (b.w.); 0.33 ml/kg b.w. of PW, respectively. However, genotoxicity decreased strongly when tested with the PW obtained after incubation with P. peli We can conclude that using comet assay genotoxicity end points are useful tools to biomonitor the physicochemical and biological quality of water. Also, it could be concluded that P. peli can treat and detoxify the studied PW.

Identification of ß integrin-like- and fibronectin-like proteins in the bivalve mollusk Mytilus trossulus.

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the ß integrin-like protein and its presumptive ligand, fibronectin-like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that ß integrin-like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of ß integrin-like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin-like protein was detected firstly at the blastula stage and later, the FN-LP-immunoreactive cells were scattered in the trochophore larvae. The fibronectin-like protein was not expressed in the ß integrin-positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the ß integrin- and fibronectin-like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.

ß Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

In this study, we focus on the specific contribution of ß integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of ß integrin-positive cells. The experiments, carried out on selected substrates, found that ß integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by ß integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel ß integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that ß integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of ß integrin-like protein depends on the cell type and the factors influencing the adhesion.

Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: Influence of synthesis method, crystalline structure, size and additive.

Increasing the production and applications of TiO2 nanoparticles (NPs) has led to grow concerns about the consequences for the environment. In this study, we investigated the effects of a set of TiO2 NPs on the viability of mussel hemocytes and gill cells using neutral red and thiazolyl tetrazolium bromide assays. For this, we compared the cytotoxicity of TiO2 NPs (0.1-100 mg Ti/L) produced by different techniques: rutile NPs (60 nm) produced by milling and containing disodium laureth sulfosuccinate (DSLS), rutile NPs (10, 40 and 60 nm) produced by wet chemistry and anatase/rutile NPs (∼100 nm) produced by plasma synthesis. The commercially available P25 anatase/rutile NPs (10-20 nm) were also tested. Exposures were performed in parallel with their respective bulk forms and the cytotoxicity of the additive DSLS was also tested. Z potential values in distilled water indicated different stabilities depending on the NP type and all NPs tested formed agglomerates/aggregates in cell culture media. In general, TiO2 NPs showed a relatively low and dose-dependent toxicity for both cell models with the two assays tested. NPs produced by milling showed the highest effects, probably due to the toxicity of DSLS. Size-dependent toxicity was found for NPs produced by wet chemistry (10 nm > 40 nm and 60 nm). All TiO2 NPs tested were more toxic than bulk forms excepting for plasma produced ones, which were the least toxic TiO2 tested. The mixture bulk anatase/rutile TiO2 was more toxic than bulk rutile TiO2. In conclusion, the toxicity of TiO2 NPs varied with the mode of synthesis, crystalline structure and size of NPs and can also be influenced by the presence of additives in the suspensions.

Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32.

Marine bivalves can accumulate large numbers of bacteria, in particular Vibrio species, whose persistence in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors. The interactions between vibrios and hemolymph have been investigated, in particular in bivalve species susceptible to infection by certain Vibrio spp. and strains. In this work, the effects of two bivalve pathogens, Vibrio splendidus LGP32 (V.s.) and Vibrio aestuarianus 01/032 (V.a.), isolated from oyster mortality outbreaks, on the hemocytes of Mytilus galloprovincialis were investigated. In vitro, V.s., but not V.a., induced a dramatic decrease in lysosomal membrane stability-LMS in the hemocytes; both vibrios induced a moderate lysozyme release, with V.s. > V.a.. The V.s.-induced decrease in LMS was mediated by activation of PI-3Kinase, as shown by use of different kinase inhibitors. TEM analysis showed rapid internalization of both vibrios; however, V.s. lead to cellular and lysosomal damage and was able to survive within the hemocytes, whereas significant killing of V.a. was observed. In vivo, in mussels challenged with either vibrio and sampled at 6, 24 and 96 h post-injection, transient decreases in hemocyte LMS and progressive increases in serum lysozyme activity were observed, with V.s. > V.a.. Moreover, whereas V.a. was efficiently cleared from hemolymph, V.s. showed significant growth, that was maximal at 24 h p.i. when lowest LMS values were recorded in the hemocytes. Both vibrios also induced significant decreases in LMS in the digestive gland, again with V.s. > V.a.. The results indicate distinct interactions between mussel hemocytes and the two vibrio strains tested. The effects of V.s. may be due to the capacity of this strain to interfere with the signaling pathways involved in hemocyte function, thus escaping the bactericidal activity of the host cell, as observed for certain mammalian pathogens. Although V.s. is considered not pathogenic to Mytilus, this vibrio strain can affect the lysosomal function at the cellular and tissue level, thus leading to stressful conditions.

Transcriptional response of the mussel Mytilus galloprovincialis (Lam.) following exposure to heat stress and copper.

Global warming is a major factor that may affect biological organization, especially in marine ecosystems and in coastal areas that are particularly subject to anthropogenic pollution. We evaluated the effects of simultaneous changes in temperature and copper concentrations on lysosomal membrane stability (N-acetyl-hexosaminidase activity) and malondialdehyde accumulation (MDA) in the gill of the blue mussel Mytilus galloprovincialis (Lam.). Temperature and copper exerted additive effects on lysosomal membrane stability, exacerbating the toxic effects of metal cations present in non-physiological concentrations. Mussel lysosomal membrane stability is known to be positively related to scope for growth, indicating possible effects of increasing temperature on mussel populations in metal-polluted areas. To clarify the molecular response to environmental stressors, we used a cDNA microarray with 1,673 sequences to measure the relative transcript abundances in the gills of mussels exposed to copper (40 µg/L) and a temperature gradient (16°C, 20°C, and 24°C). In animals exposed only to heat stress, hierarchical clustering of the microarray data revealed three main clusters, which were largely dominated by down-regulation of translation-related differentially expressed genes, drastic up-regulation of protein folding related genes, and genes involved in chitin metabolism. The response of mussels exposed to copper at 24°C was characterized by an opposite pattern of the genes involved in translation, most of which were up-regulated, as well as the down-regulation of genes encoding heat shock proteins and "microtubule-based movement" proteins. Our data provide novel information on the transcriptomic modulations in mussels facing temperature increases and high copper concentrations; these data highlight the risk of marine life exposed to toxic chemicals in the presence of temperature increases due to climate change.

Thermal stress and cellular signaling processes in hemocytes of native (Mytilus californianus) and invasive (M. galloprovincialis) mussels: cell cycle regulation and DNA repair.

In a previous study using hemocytes from native and invasive congeners of Mytilus (Mytilus californianus and Mytilus galloprovincialis, respectively) we showed that DNA damage and cell signaling transduction processes related to the cellular stress response and apoptosis were induced by acute temperature stress. The present study extends this work by examining effects of acute heat- and cold stress on total hemocyte counts (THCs) and expression of key regulatory molecules involved in responding to stress: tumor suppressor factor (p53), cell cycle arrest activator (p21), and a DNA base excision repair enzyme (apurinic/apyrimidinic endonuclease (APE)). Hyperthermia (28 °C, 32 °C) led to significant decreases of THCs in both species. The extent of decrease in THC was temperature-, time-, and species-dependent; lower THC values were found in M. californianus, the more cold-adapted species. Western blot analyses of hemocyte extracts with antibodies specific for p53 protein, several site-specific phosphorylation states of p53, p21 protein, and APE indicated that heat- and cold (2 °C) stress induced a time-dependent activation of stress-related proteins in response to DNA damage; these stress-induced changes could govern cell cycle arrest or DNA damage repair. Our results show that the downstream regulatory response to temperature-induced cell damage may play an important role in deciding cellular fate following heat- and cold stress. Compared to M. californianus, the more warm-adapted M. galloprovincialis appears to have a higher temperature tolerance due to a lesser reduction in THC, faster signaling activation and transduction, and stronger DNA repair ability following heat stress.