Monitoring the biochemical and cellular responses of marine bivalves during thermal stress by using biomarkers

Marine Environmental Research 73 (2012) 70e77

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Monitoring the biochemical and cellular responses of marine bivalves during thermal stress by using biomarkers Vasileios K. Dimitriadis a, Christina Gougoula a, Andreas Anestis b, Hans O. Pörtner c, Basile Michaelidis b, * a

Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece Laboratory of Animal Physiology, Department of Zoology, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece c Alfred-Wegener-Institut für Polar-und Meeresforschung, Ökophysiologie mariner Tiere, Postfach 120161, D-27515 Bremerhaven, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 July 2011 Received in revised form 27 September 2011 Accepted 4 November 2011

The present work aimed to study the cellular, biochemical and molecular biomarkers in the digestive glands and hemocytes of Modiolus barbatus and whether there is a hierarchy in their response to thermal stress. We determined a) the neutral red retention assay (NRR) in heamotocytes and b) the lysosomal membrane stability (LMS), the levels of second messenger cAMP, the activity of acetylcholinesterase (AChE) in the digestive glands of Modiolus barbatus after acclimation to 18
C, 24
C, 28
C or 30
C for 30 days. Moreover, in order to estimate the threshold of temperature inducing expression of stress proteins we determined the levels of Hsp70 and Hsp90 in the digestive glands. Hsps are expressed at lower temperature than those causing reduction in the LMS and NNR times. The reduction in the LMS and NNR times at high temperatures of acclimation might be related to inability of Modiolus barbatus to gain energy from the ingested food. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Bivalves Modiolus barbatus Thermal stress Hsps Biomarkers

1. Introduction It is well known that several biomarkers could define the biochemical, cellular, physiological or behavioral variations in the tissue, body fluids or of whole marine organisms and they offer an effective early warning system in biomonitoring of aquatic environments (Regoli et al., 2000, 2002; Lam and Gray, 2003; Galloway et al., 2002, 2004). The digestive gland of mussels has been used as a model system for studying the response of biomarkers to several environmental stressful factors. This gland is involved in the intracellular digestion of food materials and possesses a welldeveloped endolysosomal system, whereas basophilic cells are less abundant secretory cells believed to contribute to extracellular digestion and metabolic regulation (Garmendia et al., 2011). In addition, the lysosomes play a central role in innate defense (Cheng, 1981) and they release lysosomal enzymes, reactive oxygen species (ROS) (Austin and Paynter, 1995; Cheng, 1983) to destroy foreign invaders such as bacteria and viruses. Moreover, lysosomes have been used as a model tissue for studying the defense of mussels against oxidative stress (Moore et al., 2006b), which is known to be induced during extreme thermal exposure (Abele et al., 1998, 2001, 2002).

* Corresponding author. Tel.: þ30 2310 998401; fax: þ30 2310 998269. E-mail address: [email protected] (B. Michaelidis). 0141-1136/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2011.11.004

Among the cellular biomarkers studied in the digestive glands is the lysosomal membrane stability (LMS). LMS is the main lysosomal response to a wide range of pollutants (Moore, 1985; Viarengo et al., 1987), thus considered as a very reliable biomarker of general stress in biomonitoring studies (Regoli, 1992; Krishnakumar et al., 1994; Domouhtsidou and Dimitriadis, 2001). The stability of the lysosomal membranes is evaluated using the lysosomal membrane stability test of the digestive gland (Domouhtsidou and Dimitriadis, 2001; Krishnakumar et al., 1994; Petrovic et al., 2001; Regoli, 1992; UNEP, 1997), as well as in vitro using the neutral red retention assay (NRR) of the hemocyte lysosomes. NRR measures the lysosomal content efflux into the cytosol which, in stressed mussels, reflects a physiological process after membrane damage and comparatively measures the capacity of cellular processes to adapt to stress conditions (Lowe and Pipe, 1994; Zhang and Li, 2006). The LMS and NRR are influenced in marine bivalves by a wide range of stressors including temperature changes (Moore, 1976; Hole et al., 1995; Hauton et al., 1998; Tremblay et al., 1998a; Kagley et al., 2003; Petrovic et al., 2004; Bocchetti and Regoli, 2006; Moore et al., 2006a; 2007; Wang et al., 2006; Zhang et al., 2006). Many field studies have demonstrated the interest of the measurement of acetylcholinesterase (AChE) activity in invertebrates as an exposure biochemical biomarker in coastal waters and rivers (Moulton et al., 1996; Stien et al., 1998). AChE activity has been often tested in the marine environment because of its

V.K. Dimitriadis et al. / Marine Environmental Research 73 (2012) 70e77

potential application in screening for the effects of pesticides and other pollutants in a range of vertebrate and invertebrate species. Its activity is also inhibited in the presence of organophosphorus compounds and carbamates (Day and Scott, 1990; Weiss, 1965; Weiss and Gakstatter, 1964; Williams and Sova, 1966; Zinckl et al., 1987). In addition, AChE activity in bivalves is modified by abiotic factors such as salinity and temperature (Leiniö and Lehtonen, 2005; Pfeifer et al., 2005) and it may thus be proved to be a useful biomarker of general physiological stress in aquatic organisms. Moreover, recent publications have supported the cAMP as a highly effective biochemical biomarker of heavy metal and organic pollution both in aquatic and terrestrial pollution (Dailianis et al., 2003; Dailianis and Kaloyianni, 2007; Raftopoulou et al., 2006; Itziou and Dimitriadis, 2009). However, limited data exist on the affect of abiotic factors on cAMP, with MacDonald and Storey (1999) to refer to fluctuation of cAMP in mollusks due to temperature influence. Additionally, heat shock (or stress) proteins induction (e.g Hsp60, Hsp70, Hsp90) is proposed as another general biomarker of stress, whose expression can be demonstrated in response to a wide variety of chemical and physical insults (Bauman et al., 1993; Amiard et al., 2006; Fabbri et al., 2008; Sokolova and Lannig, 2008; Franzellitti et al., 2010). During the last decades much attention has been paid to the expression of Hsps and their physiological role in determining the thermal limits of marine organisms since the capacity of these cytoprotective systems can have serious implications in the whole-organism survival under conditions of extreme temperature (Hofmann, 2005; Tomanek, 2010). However, except for Hsps, it is not well known whether all the above mentioned biomarkers, are induced in the tissues of marine bivalves during exposure to thermal stress and especially it is not known whether different threshold temperatures induce them. On the other hand, there is a need in understanding the functional hierarchy in the response of marine organisms to thermal stress, ranging from systemic to tissue to cellular and molecular levels (Pörtner, 2002, 2010; Pörtner and Knust, 2007). In the context of global warming the use of biochemical, cellular, molecular and physiological biomarkers could help in understanding the hierarchy in the cellular damage and dysfunction during thermal stress and in determining the threshold of temperatures inducing cell dysfunction. Also, such data may contribute in understanding of how ‘environmental signals’ (e.g. air, surface and water temperatures) might translate into signals at the scale of the organism or cell (Pörtner and Farrell, 2008; Helmuth, 2009; Helmuth et al., 2010; Hofmann and Todgham, 2010). The present work aimed to contribute in understanding the responses of the above biomarkes in digestive glands and hemocytes of horse-bearded mussel Modiolus barbatus during thermal stress. In contrast to the marine bivalve Mytilus galloprovincialis, M. barbatus lives at larger depths between 8 and 30 m in the sublittoral fringe of the Thermaikos Gulf, where it is exposed to temperatures between 10
C and 25
C (Anestis et al., 2008). Consequently, in an effort to examine the threshold inducing molecular and cellular responses in means of reported biomarkers and whether they are induced in a hierarchy manner, we determined a) the NRR in heamotocytes and b) the LMS, the levels of cAMP, the activity of AChE and the levels of Hsp70 and Hsp90 in the digestive glands of M. barbatus during long term acclimation to increasing sea water temperatures. 2. Materials and methods Adult specimens of Modiolus barbatus (55e60 mm length) were collected during the spring of 2009 (average sea water temperature 17
C) in the area of Halastra in the Thermaikos Gulf and they were

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kept in aquaria of 250 L containing recirculating natural aerated seawater. Water temperature was controlled at 18  0.5
C and salinity at 32  3.5&. Mussels were kept in aquaria under these conditions for 1 week prior to experimentation and were fed with a culture of the flagellate Isochrysis galbana. Seawater pH was 8.05  0.02. 2.1. Animal treatments and experimental procedure In order to determine the threshold temperatures inducing cellular and molecular responses in Modiolus barbatus, four groups of individuals placed correspondingly into four aquaria containing 50 L of natural sea water and they were left to acclimate to 18
C for 2 days. Mussels were randomly distributed among four aquaria to ensure sample independence. Each aquarium contained 40e45 mussels, which were maintained at these conditions for two days fed as described above. Then the water temperature of three aquaria was adjusted to 24
C, 28
C or 30
C respectively by warming of the water at a rate of 0.1
C per minute and mussels were left to acclimate to these temperatures for 30 days. Following acclimation, individuals were drawn out at 10, 20 and 30 days and digestive glands and heamolymph from 10 mussels were sampled for further analysis of Hsps, LMS, NNR, AChE activity and the levels of cAMP. Animals kept at fourth aquarium at 18
C for 30 days were used as controls. In all cases mussels were exposed to 10 h light:14 h dark. 2.2. Preparation of tissue samples for SDS/PAGE For studying the Hsp70 and Hsp90 expression in the digestive glands of M. barbatus during acclimation at increasing temperatures, tissue from six mussels was homogenized in 3 ml/g of cold lysis buffer (20 mM b-glycerophosphate, 50 mM NaF, 2 mM EDTA, 20 mM Hepes, 0.2 mM Na3VO4, 10 mM benzamidine, pH 7, supplemented with 200 mM leupeptin, 10 mM trans-epoxy succinyl-Lleucylamido-(4-guanidino)butane, 5 mM dithiothreitol, 300 mM phenyl methyl sulfonyl fluoride (PMSF), 120 mM pepstatin, 1% v/v Triton X-100) and extracted on ice for 30 min. Samples were centrifuged (10,000 g, 10 min, 4
C), the supernatants were recovered, their volume measured and heated up at 100
C for 3 min, after the addition of 0.33 volumes of sample buffer and the supernatants were boiled when combined with 0.33 volumes of SDS/PAGE sample buffer (330 mM TriseHCl, pH 6.8, 13% v/v glycerol, 133 mM DTT, 10% w/v SDS, 0.2% w/v bromophenol blue). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). 2.3. SDS/PAGE and immunoblot analysis Equal amounts of proteins (100 mg) were separated on 10% (w/v) acrylamide, 0.275% (w/v) bisacrylamide slab gels and transferred electrophoretically onto nitrocellulose membranes (0.45 mm, Schleicher & Schuell, Keene NH.,USA). Non-specific binding sites on the membranes were blocked with 5% (w/v) non fat milk in TBST (20 mM TriseHCl, pH 7.5, 137 mM NaCl, 0.1% (v/v) Tween 20) for 30 min at room temperature. Subsequently, the membranes were incubated overnight with the appropriate primary antibodies. Antibodies used were: monoclonal mouse antieheat shock protein 70 kDa and monoclonal mouse antieheat shock protein 90 kDa (Sigma). After washing in TBST (3  5 min) the blots were incubated with horseradish peroxidase-linked secondary antibodies, washed again in TBST (3  5 min) and the bands were detected using enhanced chemiluminescence (Chemicon) with exposure to Fuji Medical X-ray films. Films were quantified by laser-scanning densitometry (GelPro Analyzer Software, Graphpad).

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2.4. Tissue preparation for determination of LMS Determination of lysosomal membrane stability (LMS) was performed by the cytochemical method according to Moore (1976) and UNEP/RAMOGE (1999) and specifically by measuring the activity of b-N-acetylhexosaminidase (EC 3.2.1.52) in the digestive glands of mussels. For N-acetyl-b-hexosaminidase histochemistry (LMS test), digestive glands from 10 mussels were dissected following acclimation in the laboratory. Small pieces of the digestive gland (approx. 5  5  5 mm) were placed on aluminum cryostat chucks, with five pieces of tissue deriving from the five mussels, in a straight row across the center of the chuck. The chuck was then placed for 40 s in a small bath of n-hexane, which had been precooled by immersion for 2e3 min in liquid nitrogen in order to quench the tissue. Chucks were double wrapped in parafilm and stored at 80
C until required for sectioning. Alternatively, chucks were kept at 30
C and sectioned within 1 week. 2.5. LMS determination The determination of LMS values was based on the time of acid labilization period required to produce the maximum staining intensity (Moore, 1976). The labilization period is the time of acid labilization required to fully labilize the responsive fraction of lysosomal hydrolase in the digestive cells. This was assessed under the light microscope as the first peak or maximum of reaction product associated with lysosomes. Each of the five sections was divided into 4 segments and the labilization period of each segment was assessed. The mean value of the above four determinations corresponded to the labilization period of each animal. The LMS value of each group was calculated as the mean value (n ¼ 10) of the five labilization periods corresponded to 10 animals. 2.6. Evaluation of NRR The procedure was performed according to Lowe and Pipe (1994), with slight modifications. Haemolymph was withdrawn from the posterior adductor muscle of 10 mussels and each haemolymph sample mixed with physiological saline so as to obtain a 50/50 of cell/physiological saline suspension. The physiological saline, pH 7.3 contained 20 mM HEPES, 436 mM NaCl, 53 mM MgSO4, 10 mM KCl, 10 mM CaCl2. Suspensions were spread on slides, transferred to a lightproof humidity chamber, and allowed to attach. Then, 40 ml of the neutral red (NR) probe were added to the cell monolayer. After a 15-min incubation period, slides were examined systematically under a light microscope every 15 min. The time after the NR probe application, where there was evidence of dye loss from the lysosomes to the cytosol or of other lysosomal abnormalities in at least 50% of the examined cells, belonging to the granular hemocytes, represented the NRR time for the mussel. 2.7. Determination of AChE activity For AChE measurements, digestive gland from 10 mussels were rapidly dissected out following acclimation and 1 g was diluted in Tris 0.1 M buffer, pH 8. The tissue were homogenized (1/4 w/v) and centrifuged at 15,000 g for 30 min. Crude supernatants were used as enzyme source and samples were stored at 85
C without significant activity loss. For quantifying the protein concentration the BCA assay protocol, with bovine serum albumin (BSA) as standard, was used (Sorensen and Brodbeck, 1986). For the measurement of AChE activity Ellman’s method (Ellman et al., 1961), a procedure proposed by UNEP (1997), adapted to microplate reading as described by Galgani and Bocquene (1988) was used. Acetylthiocholine iodide (21.67 mg/ml) was added as

substrate to initiate the enzymatic reaction. Specific activity was expressed as Units/mg protein (U/mgr protein). 2.8. cAMP determination cAMP content was measured according to the modification described by Dailianis et al. (2003). Digestive gland of mussels from 10 mussels was dissected out to make 1 g and homogenized with three volumes of 4 mM EDTA, to prevent enzymatic degradation of cAMP. The homogenate boiled for 5 min in a water bath and thereafter centrifuged for 5 min (16,000 g, 4
C). The supernatant stored in liquid nitrogen. The concentration of cAMP from the supernatant estimated with the Amersham, [83H] adenosine 30 e500 cyclic phosphate, radioimmunoassay kit (Cyclic AMP [3H] Biotrak Assay System, No TRK432, Amersham). The assay was based on the competition between unlabeled cAMP and a fixed quantity (0.5 mCi/ml) of the tritium labeled compound for binding to a protein, which has a high specificity and affinity for cAMP. The amount of labeled proteinecAMP complex formed is inversely related to the amount of unlabelled cAMP present in the assay sample. Separation of the protein bound cAMP from the unbound nucleotide achieved by absorption of the free nucleotide on to coated charcoal, followed by centrifugation. An aliquot of the supernatant was then removed for liquid scintillation counting. The concentration of unlabelled cAMP in the sample was then determined from a linear standard curve. The range of labeled cAMP in the calibration curve ranged from 0 to 16 pmol cAMP/incubation tube. The detection limit of the assay is 0.05 pmol cAMP. The reproducibility of the assay method is 95e100%. 2.9. Data analysis The data of the LMS, AChE activity and cAMP was not presented normal distribution and, thus, the statistical analysis was fulfilled with non parametric tests such as ManneWhitney U-test (P < 0.05) according to UNEP (1997). Data on NRR assay were tested using Duncan’s test (P < 0.05, breakdown and one-way ANOVA). Simple linear correlation (Pearson’s test) conducted with the mean values, was used to establish significant relationships between the biological responses. The analyses were carried out using the STATISTICA statistical package (STATISTICA, Microsoft Co.). For Hsps statistical analysis, changes over time were tested for significance at the 5% level by using one-way analysis of variance (ANOVA) and by performing Bonferroni post-hoc tests for group comparisons. 3. Results The expression of Hsps was monitored in digestive glands M. barbatus and regarding the Hsp70 family two main bands were identified during acclimation to any temperature, the Hsp72 (the inducible form) and Hsp73 (the constitutive form). The expression of Hsp72 inceased gradually even from the fifth day of acclimation at 24
C. However, statistical significantly differences observed only at 20 and 30 days. In contrast, such significant differences in the levels of Hsp72 were observed even at first 5 days of acclimation at 28
C and 30
C. The Hsp72 remained at high levels by the 30 days of mussel acclimation at 28
C and 30
C. In contrast to Hsp72, there was no change in the levels of Hsp73 (Fig. 1). In response to acclimation at various temperatures, Hsp90 displayed similar changes as Hsp72, except that increases in its levels were observed even form the fifth day of acclimation at 24
C (Fig. 2). The LMS times were not significantly changed during acclimation of M. barbatus at 24
C for 20 days, while longer acclimation (30 days) at the same temperature caused a non statistically significant decrease in the LMS times compared to 10 and 20 days. An earlier

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Time (days) Fig. 1. Levels of Hsp70 in the digestive glands of M. barbatus during acclimation to different water temperatures. Tissue extracts were subjected to SDS-PAGE and immunoblotted for Hsp70. Representative immunoblots are shown for each acclimation temperature. Blots were quantified by laser-scanning densitometry. Values are expressed as means  SE; n ¼ 6 preparations from different animals. Inducible isoform Hsp72, open circles; constitutive isoform Hsp73, closed circles. *P < 0.05 compared with the control (0 days).

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Time (days) Fig. 2. Levels of Hsp90 in the digestive glands mantle of M. barbatus during acclimation to different water temperatures. Tissue extracts were subjected to SDS-PAGE and immunoblotted for Hsp90. Representative immunoblots are shown for each acclimation temperature. Blots were quantified by laser-scanning densitometry. Values are expressed means  SE; n ¼ 6 preparations from different animals. *P < 0.05 compared with the control (0 days).

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decrease in the LMS times was observed when M. barbatus acclimated at 28
C and 30
C. Specifically the LMS times decreased after the first 20 days of acclimation at 28
C and within the first 10 days of acclimation at 30
C (Fig. 3A). The NRR times were not significantly changed during acclimation at 24
C and during the first days of acclimation at 28
C and 30
C. Longer acclimation at 28
C and 30
C caused a significant reduction in the NMR times (Fig. 3B). The activity of AchE was not significantly changed when M. barbatus acclimated at 24
C for 30 days, at 28
C for 20 days and at 30
C for 10 days. However longer acclimation either at 28
C or at 30
C caused a significant reduction in the activity of AchE (Fig. 4). No significant change observed in the levels of cAMP determined in digestive glands of M. barbatus after 30 days of acclimation either at 24
C or at 28
C. Exposure to 30
C, however, caused a significant reduction in its levels even from the first days of acclimation (Fig. 5).

Fig. 4. Determination of AChE activity in the haemolymph of M. barbatus acclimated to different temperatures. Values (expressed as nmol/mg protein) are means  S.D., n ¼ 10. Asterisks in the triangular matrix represent statistical differences between each value, obtained after ManneWhitney U-test analysis (P < 0.05).

4. Discussion Several works have reported a close relation between thermal stress and the response of several biomarkers in marine molluscs (Regoli, 1992; Tremblay et al., 1998a,b; Domouhtsidou and Dimitriadis, 2001; Kagley et al., 2003; Petrovic et al., 2004; Bocchetti and Regoli, 2006; Moore et al., 2006a, 2007; Wang et al., 2006; Zhang et al., 2006). Moreover, other studies have revealed the physiological role of Hsps in determining the thermotolerance of these organisms (Hofmann, 1999, 2005; Tomanek, 2010). The data obtained in the present work, however, show that the induction of Hsps and the response of the other biomarkers differ in M. barbatus during exposure to increasing temperature. Specifically, the results show that the expression of Hsps proceeds the

Fig. 3. (A). LMS values (min) of the digestive gland of M. barbatus acclimated to different temperatures. The significant differences between pairs of mean values are shown in the triangular matrix by asterisks. ManneWhitney U-test (P < 0.05). (B). NRR values (min) of the haemocytes of M. barbatus acclimated to different temperatures. Values are expressed as mean  S.D. From 10 replicates corresponding to 10 mussels. The significant differences between pairs of mean values are shown in the triangular matrix by asterisks. Duncan’s test (ANOVA, P < 0.05).

Fig. 5. Determination of cAMP content in the digestive glands of mussel M. barbatus. Values (expressed as pmol cAMP/g wet tissue) are expressed as means  S.D., n ¼ 10. Asterisks in the triangular matrix represents statistical differences between each value, obtained after ManneWhitney U-test analysis (P < 0.05).

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reduction in the LMS and NNR times, indicating a cellular hierarchy in response to thermal stress. The expression of Hsp70 and Hsp90 in the digestive glands of horse-bearded mussels, M. barbatus, seems to be in agreement with our previous understanding of the heat-shock response (HSR) in mussels (Hofmann, 1999, 2005; Ioannou et al., 2009; Tomanek, 2010). The heat-shock induction temperature (Ton) for the digestive glands found to be lower than 24
C (Figs. 1 and 2) and it is in line with results from a previous laboratory work on M. barbatus, where heat-shock induction temperature (Ton) determined to be <22
C in the mantle and posterior adductor muscle (PAM) (Anestis et al., 2008). Nevertheless, HSR is not parallel with increases in the mortality of mussels. As shown in a previous work significant mortality of M. barbatus is recording after the 26
C (Anestis et al., 2008). However, the induction of Hsps in digestive glands at lower temperatures of acclimation (Figs. 1 and 2) indicates an earlier need for chaperoning. Such a molecular response should be attributed not only in increase in the levels of irreversible damaged proteins because of thermal stress, but in other physiological process as well taking place in the tissue during warming. As pointed elsewhere maintenance of full aerobic scope is crucial for long term thermal tolerance of marine organisms (cf. Pörtner, 2002; Pörtner and Knust, 2007). According to conceptual model of oxygen- and capacity-limited thermal tolerance (OCLTT) and the resulting thermal window of performance of a species it is suggested that, during exposure of marine organism to thermal stress, the elevated energy demand of defense to stress leads to reduction of oxygen supply to tissues, which is reflected by an earlier onset of the hypoxemia and shift to anaerobic metabolism (cf. Pörtner, 2010). Although the results presented can not show such a relationship, recent publications (Gracey et al., 2008; Anestis et al., 2010a) reported a close relationship between the onset of anaerobiosis and the HSR in bivalves and that, a shift to anaerobic metabolism is observed in the tissues of M. barbatus, after acclimation to temperatures beyond of 26
C (Anestis et al., 2008). The HSR in molluscs during anaerobiosis, might be induced by several cellular factors, including generation of reactive oxygen species (ROS). As reported elsewhere oxygen deficiency during progressive development of anaerobiosis and reduction of aerobic capacity, elicits the transition to passive tolerance (Pörtner, 2010) and leads to a fall of residual mitochondrial oxidative capacity and of metabolic capacity in somatic tissues, enhancing the generation of ROS and oxidative stress (Abele et al., 1998, 2001, 2002; Lockwood et al., 2010; Tomanek and Zuzow, 2010). Antioxidant enzymes are found in all tissues of the mussel, but are present in highest activities in the digestive gland (Livingstone et al., 1990), which is also the major site of xenobiotic uptake and oxyradicalgenerating biotransformation enzymes (Livingstone, 1991; Moore et al., 2006b). Mytilus edulis exposed to higher seawater temperatures showed a significant increase in the expression of Hsp70 and activities of antioxidant enzyme SOD (Lesser et al., 2010). In supporting the close relation between reduction of aerobic capacity and induction of oxidative stress, Lesser and Kruse (2004) reported that the congeneric species Modiolus modiolus maintained the same level of antioxidant protection in summer and winter and it seems to be consistent with high aerobic metabolism in both winter and summer. The fact that the LMS and NRR decreased (Fig. 3) significantly when M. barbatus acclimated at temperatures higher than 26
C strongly support the assumption that M. barbatus faces thermal stress after long term acclimation at these temperatures. In line with our results several publication by using either the LMS or the NRR test showed the negative effects of thermal stress on the lysosomal membrane stability (Regoli, 1992; Hole et al., 1995; Tremblay et al., 1998a,b; Domouhtsidou and Dimitriadis, 2001;

75

Kagley et al., 2003; Petrovic et al., 2004; Bocchetti and Regoli, 2006; Moore et al., 2006a, 2007; Wang et al., 2006; Zhang et al., 2006). Recent investigations reported that lysosomes might be implicated to cell autophagy, a cellular process involving in the remove of oxidative damaged organelles and proteins. This process, which is initiated by several stressors (e.g., restricted nutrients, hyperthermia, hypoxia and salinity increase), is often considered to be primarily a survival strategy in multicellular organisms, protecting thus the cells against oxidative stress (Moore et al., 2006a,b; Moore et al., 2008). By breaking down longer-lived proteins and organelles, and recycling the products into protein-synthesis and energyproduction pathways, this process allows cells to be temporarily self-sustaining during periods when nutrients are restricted (Cuervo, 2004; Levine, 2005; Moore et al., 2006a). As we have shown in an earlier work, the expression of Hsps is continued at temperatures higher than 26
C, without M. barbatus surviving sea water temperatures beyond this temperature over extended periods of time. At 26
C only a small fraction (about 3%) of the mussels died within 30 days of acclimation, while mussels’ mortality increased most drastically during warming to 28
C and 30
C and reached 10% and 20% respectively after 30 days (Anestis et al., 2008). It has been pointed out that the ability of organisms to survive thermal stress is not only a matter of Hsps functioning, but also of the organism’s ability to meet the energy demand for protein repair (Somero, 2002; Hofmann, 2005). Energy is required at several steps of the heat-shock response, including the activation of transcription of heat-shock genes, the synthesis of Hsps, and the ATP-requiring chaperoning by Hsps. Furthermore, energy is required if proteins are irreversibly denatured and need to be replaced and Hawkins (1985) estimated that the cost of proteinsynthesis constitutes 20e25% of the energy budget of mussel, M. edulis. These data highlight strongly the assumption that the ability of an organism to keep positive energy balance during thermal stress might play a crucial role in its thermotolerance. The time course of cellular responses and specifically the reduction in LMS and NRR seems to coincide well with the initiation of mussel’s lost to gain energy from the ingested food and the target of their mortality. It has been recently reported that M. barbatus keeps c et al., a positive scope for growth (SFG) up to 26
C (Ezgeta-Bali 2010) and reduction in its ability to gain energy from the absorbed food is initiated at higher temperatures. The latter might be the main reason for the target of M. barbatus death after acclimation at temperatures higher than 26
C. Several works have related the summer mortality of mussels to drop in SFG and disturbance in energetic balance (Incze et al., 1980; Worrall and Widdows, 1984; Mallet et al., 1990; Tremblay et al., 1998a,b; Anestis et al., 2010b). Drop of SFG indicates depletion of energy reserves at high temperatures and it implies that animals are utilizing energy reserves. The period in which mass mortality of mussels normally occurred (August) was associated with elevated activities of lysosomes, indicating high autophagy in tissues as part of the protein degrading activity. As far as the activity of AchE and the levels of cAMP are concerned the data showed significant changes when M. barbatus acclimated to 30
C (Figs. 4 and 5 respectively). It has been reported that change in enzymatic activities could be a general mechanism by which different tissues try to compensate to the action of several environmental pollutants (Pareschi et al., 1997). Moreover, alteration of cAMP content may be attributed to the different responses of adenyl cyclase or phosphodiesterase activities of each tissue during the exposure to several abiotic factors (Evanson and Van Der Kraak, 2001; Dailianis and Kaloyianni, 2007). Nevertheless their physiological roles in M. barbatus during environmental thermal stress are obscure. M. barbatus never face temperatures higher than 30
C in nature. Consequently it is doubt whether they play any

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physiological role in thermotolerance of M. barbatus under natural conditions. In conclusion, the results of the present work indicate a hierarchy in the cellular responses of M. barbatus to thermal stress. Although M. barbatus lives at larger depths between 8 and 30 m in the sublittoral fringe of the Thermaikos Gulf than M. galloprovincialis it is exposed during summer at ambient temperatures reaching the upper thermal limits (Anestis et al., 2008). In the context of climate change, studies on seawater temperature conditions already show a clear-cut warming trend of coastal waters worldwide including the Mediterranean Sea (Levitus et al., 2000; IPCC, 2007; Vargas-Yánẽz et al., 2008; Gambaiani et al., 2009). Consequently, it is suggested that prolonged exposure of M. barbatus to warming during summer might detrimentally affect them. On the other hand, the energy shortage taken place during summer time in Mediterranean Sea will shorten its thermal limits, causing higher rates of mortality as proposed for several benthic species (Coma et al., 2002, 2009). Based on the obtained data, we could suggest that, except of Hsps, the use of biomarkers could provide useful information about the higher critical lethal ambient temperatures (Tcmax) initiating irreversible cellular damage in the tissues of marine bivalves during perturbation of its ecosystem because of global warming. Also, the results emphasize the need for an in depth study of cellular mechanisms setting thermal limits in marine organisms and for considering a functional hierarchy ranging from systemic to tissue to cellular and molecular levels in analyses of whole-organism sensitivities as reported elsewhere (Pörtner, 2002, 2010; Pörtner and Knust, 2007).

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