Effect of hypoosmotic stress by low salinity acclimation of Mediterranean mussels Mytilus galloprovincialis on biological parameters used for pollution assessment.

In the present study, we investigated the progressive acclimation of the mussel Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27 degrees C, salinity 37.5 psu) and winter (13 degrees C, 37 psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11 psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rate, and condition index were measured. Survival in air (SOS test), as a physiological index of mussel's health and vitality, had significantly lower LT50 values (11 psu) in the summer than in the winter, and it seems to be negatively affected by acclimation in comparison to controls (37 psu and mariculture). Condition indexes (CIs) were not significantly different, but mussel's acclimation resulted in decline (i.e., a negative trend), especially of CI-2 and CI-3 calculated on the basis of mussel tissue weight and shell sizes. Oxygen consumption rate (VO2) of M. galloprovincialis acclimated to reduced salinities was a concentration-dependent process and increased considerably to about 51 and 65% in lower SW concentrations (28 and 18 psu) compared to control mussels (37 psu). DNA integrity, determined by Fast Micromethod, was negatively impacted by salinity acclimation and corresponding physiological stress as well. Some differences in 1D protein expression patterns between control groups and mussels acclimated to 28, 18.5 and 11 psu (SW) were established. Reduced SW salinities (18.5 and 11 psu) resulted in significantly higher p38-MAPK phosphorylation, whereas the SW salinity of 28 psu decreased p-p38 significantly compared to control (37 psu). The concentration of metallothioneins in mussels' gills was reduced at 28 and 18.5 psu, while it was significantly higher at 11 psu. Results indicated that SW salinity variation (i.e., hypoosmotic stress) in the marine environment can affect all investigated parameters. This investigation expands our understanding of multifactorial effects of the physical marine environment on the specificity of investigated biomarkers and biotests, providing insight into the acclimation, adaptive and stress response processes of mussels. Effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results.

[1]  G. Hofmann,et al.  Molecular Chaperones in Ectothermic Marine Animals: Biochemical Function and Gene Expression1 , 2002, Integrative and comparative biology.

[2]  K. Hylland Biological effects in the management of chemicals in the marine environment. , 2006, Marine pollution bulletin.

[3]  W. Müller,et al.  Stress-70 proteins in marine mussel Mytilus galloprovincialis as biomarkers of environmental pollution: a field study. , 2004, Environment international.

[4]  D. Blagojević,et al.  The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea. , 2005, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[5]  Fredo Durand,et al.  The point about oxidative stress in molluscs , 2005 .

[6]  K. Storey,et al.  Identification of a 115kDa MAP-kinase activated by freezing and anoxic stresses in the marine periwinkle, Littorina littorea. , 2006, Archives of biochemistry and biophysics.

[7]  G. Isani,et al.  Seasonal dependence of cadmium accumulation and Cd-binding proteins in Mytilus galloprovincialis exposed to cadmium. , 1999, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[8]  A. Viarengo,et al.  Effects of sublethal copper concentrations, temperature, salinity and oxygen levels on calcium content and on cellular distribution of copper in the gills of Mytilus galloprovincialis lam.: A multifactorial experiment , 1988 .

[9]  U. Hentschel,et al.  Molecular response of the sponge Suberites domuncula to bacterial infection , 2001 .

[10]  P. Parsons Environments and evolution: interactions between stress, resource inadequacy and energetic efficiency , 2005, Biological reviews of the Cambridge Philosophical Society.

[11]  A. Pruski,et al.  Marine invertebrate eco-genotoxicology: a methodological overview. , 2002, Mutagenesis.

[12]  M. Cornet Effects of seawater salinity fluctuations on primary tissue culture from the mussel Mytilus galloprovincialis. Potential application to the detection of seawater genotoxicity. , 2006, Toxicology in vitro : an international journal published in association with BIBRA.

[13]  A. Viarengo,et al.  A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs , 1997 .

[14]  A. Pruski,et al.  MARINE INVERTEBRATE ECOGENOTOXICOLOGY: A METHODOLOGICAL OVERVIEW , 2002 .

[15]  D. Sheehan,et al.  Oxidative stress in response to xenobiotics in the blue mussel Mytilus edulis L.: evidence for variation along a natural salinity gradient of the Baltic Sea. , 2007, Aquatic toxicology.

[16]  C. Holliday,et al.  Salinity adaption of gill Na, K‐ATPase in the blue crab, Callinectes sapidus , 1980 .

[17]  P. Almada-Villela The Effects of Reduced Salinity on the Shell Growth of Small Mytilus Edulis , 1984, Journal of the Marine Biological Association of the United Kingdom.

[18]  Tomislav Smuc,et al.  Enhanced analytical power of SDS‐PAGE using machine learning algorithms , 2008, Proteomics.

[19]  H. Steinhart,et al.  Correlation between the level of the potential biomarker, heat-shock protein, and the occurrence of DNA damage in the dab, Limanda limanda: a field study in the North Sea and the English Channel. , 2000, Marine environmental research.

[20]  S. Nicholson Ecocytological and toxicological responses to copper in Perna viridis (L.) (Bivalvia: Mytilidae) haemocyte lysosomal membranes. , 2001, Chemosphere.

[21]  P. Harris,et al.  Survival in air of Mytilus trossulus following long-term exposure to spilled Exxon Valdez crude oil in Prince William Sound. , 1999, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[22]  J. Bierkens Applications and pitfalls of stress-proteins in biomonitoring. , 2000, Toxicology.

[23]  S. Cristobal,et al.  Identification of Proteomic Signatures of Exposure to Marine Pollutants in Mussels (Mytilus edulis)*S , 2006, Molecular & Cellular Proteomics.

[24]  B. Schierwater,et al.  Molecular biomarkers and adaptation to environmental stress in moon jelly (Aurelia spp.) , 2005, Marine Biotechnology.

[25]  Ž. Jakšić,et al.  Adriatic coast as a microcosm for global genotoxic marine contamination--a long-term field study. , 2005, Marine pollution bulletin.

[26]  A. Viarengo,et al.  Metallothionein as a tool in biomonitoring programmes , 1999 .

[27]  P. Garrigues,et al.  Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts. , 1999 .

[28]  E Nevo,et al.  Evolution of genome–phenome diversity under environmental stress , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Viarengo,et al.  Mussels as biological indicators of pollution , 1991 .

[30]  G. Kullenberg The role of the oceans as a waste disposal option , 1986 .

[31]  C Minier,et al.  Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea. , 2004, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[32]  M. Etxeberria,et al.  Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes , 1996 .

[33]  J. Narbonne,et al.  Seasonal variations of pollution biomarkers in two populations of Corbicula fluminea (Müller). , 2002, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[34]  Borghi,et al.  Seasonal variation of MXR and stress proteins in the common mussel, Mytilus galloprovincialis. , 2000, Aquatic toxicology.

[35]  A. Smaal,et al.  “Survival in air” of the blue mussel Mytilus edulis L. as a sensitive response to pollution-induced environmental stress , 1993 .

[36]  J. Widdows,et al.  Physiological and biochemical responses of bivalve molluscs to exposure to air , 1979 .

[37]  M. Tsuchiya Mass mortality in a population of the mussel Mytilus edulis L. Caused by high temperature on rocky shores , 1983 .

[38]  A. Viarengo,et al.  Stress on stress response: A simple monitoring tool in the assessment of a general stress syndrome in mussels , 1995 .

[39]  A. Viarengo,et al.  Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis. , 1999, American journal of physiology. Regulatory, integrative and comparative physiology.

[40]  B. Olsson,et al.  Protein expression signatures identified in Mytilus edulis exposed to PCBs, copper and salinity stress. , 2000, Marine environmental research.

[41]  H. Pörtner,et al.  Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. , 2007, American journal of physiology. Regulatory, integrative and comparative physiology.

[42]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[43]  S. Lipton,et al.  Crosstalk between Nitric Oxide and Zinc Pathways to Neuronal Cell Death Involving Mitochondrial Dysfunction and p38-Activated K+ Channels , 2004, Neuron.

[44]  Béatrice Rocher,et al.  Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase. , 2004, Aquatic toxicology.

[45]  W. Müller,et al.  A microplate assay for DNA damage determination (fast micromethod). , 1999, Analytical biochemistry.

[46]  E. Sazakli,et al.  Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers. , 2006, Aquatic toxicology.

[47]  B. Hamer,et al.  PAH content, toxicity and genotoxicity of coastal marine sediments from the Rovinj area, Northern Adriatic, Croatia. , 2006, The Science of the total environment.

[48]  R. Tremblay,et al.  Effect of the Tidal Cycle on Lysosomal Membrane Stability in the Digestive Gland of Mya arenaria and Mytilus edulis L. , 1997 .

[49]  D. Sheehan,et al.  Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs. , 1999, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[50]  V. Wadley,et al.  Effect of reduced salinity on adenylate energy charge in three estuarine molluscs , 1979 .

[51]  Werner Müller,et al.  The mitogen‐activated protein kinase p38 pathway is conserved in metazoans: Cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula * , 2000, Biology of the cell.

[52]  E. Gosling The mussel Mytilus: ecology, physiology, genetics and culture , 1992 .

[53]  J. Spotila,et al.  Seasonal variation in heat shock proteins (hsp 70) in stream fish under natural conditions , 1994 .

[54]  P. Parsons Energetic efficiency under stress underlies positive genetic correlations between longevity and other fitness traits in natural populations , 2007, Biogerontology.

[55]  W. Maret Zinc and sulfur: a critical biological partnership. , 2004, Biochemistry.

[56]  B. Helmuth,et al.  Microhabitats, Thermal Heterogeneity, and Patterns of Physiological Stress in the Rocky Intertidal Zone , 2001, The Biological Bulletin.

[57]  J P McNamee,et al.  Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution¶ , 2001, Photochemistry and photobiology.

[58]  Amiard-Triquet,et al.  Changes in metallothionein concentrations in response to variation in natural factors (salinity, sex, weight) and metal contamination in crabs from a metal-rich estuary. , 2000, Journal of experimental marine biology and ecology.

[59]  J. Widdows,et al.  Measurement of stress effects (scope for growth) and contaminant levels in mussels (Mytilus edulis) collected from the Irish Sea. , 2002, Marine environmental research.

[60]  A Viarengo,et al.  Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels. , 1991, Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology.

[61]  E. Gourgou,et al.  Acute thermal stress and various heavy metals induce tissue-specific pro- or anti-apoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.) , 2005, Journal of Experimental Biology.

[62]  A. Akhmedov,et al.  Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells , 2004, Journal of Cell Science.

[63]  M. Erk,et al.  Metal and metallothionein level in the heat-treated cytosol of gills of transplanted mussels Mytilus galloprovincialis Lmk. , 2004, Environment international.

[64]  A. Zwaan,et al.  Anoxic or aerial survival of bivalves and other euryoxic invertebrates as a useful response to environmental stress—A comprehensive review , 1996 .

[65]  A. Viarengo,et al.  Mechanisms of heavy metal cation homeostasis in marine invertebrates , 1993 .

[66]  S. Dailianis,et al.  Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers. Combination with trace metal concentrations. , 2004, Marine pollution bulletin.

[67]  C. Jørgensen Bivalve filter feeding : hydrodynamics, bioenergetics, physiology and Ecology , 1990 .

[68]  A. Koehler,et al.  Regulation of expression of multixenobiotic resistance (MXR) genes by environmental factors in the blue mussel Mytilus edulis. , 2004, Aquatic toxicology.

[69]  B. Bayne Measuring the Effects of Pollution at the Cellular and Organism Level , 1986 .

[70]  L. Giambérini,et al.  Towards a validation of a cellular biomarker suite in native and transplanted zebra mussels: a 2-year integrative field study of seasonal and pollution-induced variations. , 2007, Aquatic toxicology.

[71]  A Viarengo,et al.  The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. , 2000, The Science of the total environment.

[72]  F. Trombetti,et al.  Response to alkyltins of two Na+-dependent ATPase activities in Tapes philippinarum and Mytilus galloprovincialis. , 2006, Toxicology in vitro : an international journal published in association with BIBRA.

[73]  B. Ozretić,et al.  Seasonal variations of physiological and cellular biomarkers and their use in the biomonitoring of north Adriatic coastal waters (Croatia). , 2004, Marine pollution bulletin.

[74]  P. Venier,et al.  Characterization of mussel gill cells in vivo and in vitro , 2005, Cell and Tissue Research.

[75]  P. Venier,et al.  Susceptibility to genetic damage and cell types in Mediterranean mussels. , 2002, Marine environmental research.

[76]  Ž. Jakšić,et al.  DNA integrity determination in marine invertebrates by Fast Micromethod. , 2003, Aquatic toxicology.

[77]  S. Pierce INVERTEBRATE CELL VOLUME CONTROL MECHANISMS: A COORDINATED USE OF INTRACELLULAR AMINO ACIDS AND INORGANIC IONS AS OSMOTIC SOLUTE , 1982 .

[78]  J. Hellou,et al.  Stress on stress response of wild mussels, Mytilus edulis and Mytilus trossulus, as an indicator of ecosystem health. , 2003, Environmental pollution.

[79]  N. Introductio PHYSIOLOGICAL INDICES OF STRESS IN MYTILUS EDULIS , 1978 .

[80]  N. Bihari,et al.  Polycyclic Aromatic Hydrocarbons and Ecotoxicological Characterization of Seawater, Sediment, and Mussel Mytilus galloprovincialis from the Gulf of Rijeka, the Adriatic Sea, Croatia , 2007, Archives of environmental contamination and toxicology.

[81]  A. Viarengo,et al.  Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation. , 2005, Gene.

[82]  P. Campbell,et al.  Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects. , 2005, Environmental pollution.

[83]  C. Hogstrand,et al.  ZINC-mediated gene expression offers protection against H2O2-induced cytotoxicity. , 2005, Toxicology and applied pharmacology.

[84]  B. Raspor,et al.  Metal pollution assessment of the marine environment by determination of metal-binding proteins in Mytilus sp. , 1987 .

[85]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[86]  C. D. Vooys Anaerobic metabolism in sublittoral living Mytilus galloprovincialis in the mediterranean—IV. Role of amino acids in adaptation to low salinities during anaerobiosis and aerobiosis , 1991 .

[87]  D. Fattorini,et al.  An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic Sea. , 2008, Marine environmental research.

[88]  A. Zwaan,et al.  The rate of oxygen consumption and ammonia excretion by Mytilus edulis after various periods of exposure to air , 1978 .

[89]  J. Maes,et al.  Effect of different environmental variables on the synthesis of Hsp70 in Raphidocelis subcapitata. , 1998, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[90]  W. Maret,et al.  Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[91]  A. Geffard,et al.  Do seasonal changes affect metallothionein induction by metals in mussels, Mytilus edulis? , 2005, Ecotoxicology and environmental safety.

[92]  P. D. Abel,et al.  Ecotoxicology and the marine environment , 1991 .

[93]  L. Canesi,et al.  Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAP kinases. , 2002, Developmental and comparative immunology.

[94]  D. Ivanković,et al.  Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation. , 2008, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[95]  M. Depledge,et al.  Stress proteins and condition index as biomarkers of tributyltin exposure and effect in mussels , 1997 .

[96]  Edward D. Goldberg,et al.  The mussel watch — A first step in global marine monitoring , 1975 .

[97]  C. Mouneyrac,et al.  Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary, in and out of the reproductive season. , 2001, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[98]  F. Regoli,et al.  Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea. , 2006, Chemosphere.

[99]  E. Conway de Macario,et al.  Stressors, stress and survival: overview. , 2000, Frontiers in bioscience : a journal and virtual library.

[100]  B. Bøhle Effects of adaptation to reduced salinity on filtration activity and growth of mussels (Mytilus edulis L.) , 1972 .

[101]  I. Beis,et al.  Various stressors rapidly activate the p38-MAPK signaling pathway in Mytilus galloprovincialis (Lam.) , 2004, Molecular and Cellular Biochemistry.

[102]  S. Wright,et al.  Animals Response of Cell Volume in Mytilus Gill to Acute Salinity Change , 2022 .

[103]  D. Bobinac,et al.  Lysosomal membrane stability and metallothioneins in digestive gland of Mussels (Mytilus galloprovincialis Lam.) as biomarkers in a field study. , 2001, Marine pollution bulletin.

[104]  Č. Lucu,et al.  Role of seawater concentration and major ions in oxygen consumption rate of isolated gills of the shore carb Carcinus mediterraneus Csrn , 1995 .

[105]  Juliane Ventura-Lima,et al.  Pollution biomarkers in estuarine animals: critical review and new perspectives. , 2007, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[106]  B. Raspor,et al.  Quantitative determination of metallothionein-like proteins in mussels. Methodological approach and field evaluation , 1993 .

[107]  M. Moore Cellular responses to pollutants , 1985 .

[108]  G. Reifferscheid,et al.  DNA damage susceptibility and repair in correlation to calendric age and longevity , 2000, Mechanisms of Ageing and Development.

[109]  Evaluation of PAH bioaccumulation and DNA damage in mussels (Mytilus galloprovincialis) exposed to spilled Prestige crude oil. , 2004, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[110]  M. Erk,et al.  Evaluation of the Mytilus galloprovincialis Lam. digestive gland metallothionein as a biomarker in a long-term field study: seasonal and spatial variability. , 2005, Marine pollution bulletin.

[111]  M. Zakhartsev,et al.  An in vitro study of the effect of reactive oxygen species on subcellular distribution of deposited cadmium in digestive gland of mussel Crenomytilus grayanus. , 2005, Aquatic toxicology.

[112]  J. Davenport The isolation response of mussels (Mytilus edulis L.) exposed to falling sea-water concentrations , 1979, Journal of the Marine Biological Association of the United Kingdom.