Physiological and biochemical responses of three Veneridae clams exposed to salinity changes

Comparative Biochemistry and Physiology, Part B 177–178 (2014) 1–9

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Physiological and biochemical responses of three Veneridae clams exposed to salinity changes Vanessa Carregosa a, Cátia Velez b, Amadeu M.V.M. Soares b, Etelvina Figueira b, Rosa Freitas b,⁎ a b

Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e

i n f o

Article history: Received 14 May 2014 Received in revised form 31 July 2014 Accepted 5 August 2014 Available online 13 August 2014 Keywords: Venerupis Invasive and native species Climate change Antioxidant defenses Salinity stress

a b s t r a c t Given their global importance, coastal marine environments are a major focus of concern regarding the potential impacts of climate change, namely due to alterations in seawater salinity. It is known that environmental characteristics, such as salinity, affect immune and physiological parameters of bivalves. Nevertheless, scarce information is available concerning the biochemical alterations associated with salinity changes. For this reason, the present work aimed to evaluate the biochemical responses of three venerid clam species (Venerupis decussata, Venerupis corrugata, Venerupis philippinarum) submitted to salinity changes. The effects on the native (V. decussata and V. corrugata) and invasive (V. philippinarum) species collected from the same sampling site and submitted to the same salinity gradient (0 to 42 g/L) were compared. The results obtained demonstrated that V. corrugata is the most sensitive species to salinity changes and V. decussata is the species that can tolerate a wider range of salinities. Furthermore, our work showed that clams under salinity associated stress can alter their biochemical mechanisms, such as increasing their antioxidant defenses, to cope with the higher oxidative stress resulting from hypo and hypersaline conditions. Among the physiological and biochemical parameters analyzed (glycogen and protein content; lipid peroxidation levels, antioxidant enzymes activity; total, reduced and oxidized glutathione) Catalase (CAT) and especially superoxide dismutase (SOD) showed to be useful biomarkers to assess salinity impacts in clams. © 2014 Elsevier Inc. All rights reserved.

1. Introduction According to recent reports, increases in temperature, water acidification and changes in seawater salinity are predicted to occur in the next 100 years (IPCC, 2013). Therefore, identifying the effects of predicted climate changes in aquatic ecosystems must be a priority in order to maintain their biodiversity. Among climate changes there is an increasing concern about future alterations in seawater salinity values, mainly in estuarine and coastal areas (Booij, 2005; Kay et al., 2006), which will affect the performance of native and invasive species. When different stressors act together, namely biological invasions associated with salinity changes, they may have unexpected and irreversible consequences for the native communities (Occhipinti-Ambrogi and Savini, 2003; Whitfield et al., 2007). Salinity is one of the dominant environmental factors controlling species distribution and influencing physiological processes in marine and estuarine organisms (Navarro and Gonzalez, 1998). In bivalves the impact of salinity on host–pathogen interactions (Hauton et al., 2000; Reid et al., 2003; Malagoli and Ottaviani, 2005; Gagnaire et al., 2006; ⁎ Corresponding author at: Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. Tel.: +351 234370782; fax: +351 234372587. E-mail address: [email protected] (R. Freitas).

http://dx.doi.org/10.1016/j.cbpb.2014.08.001 1096-4959/© 2014 Elsevier Inc. All rights reserved.

Matozzo et al., 2007; Bussell et al., 2008; Dang et al., 2010; Kuchel et al., 2010; Perrigault et al., 2012), immune responses (Reid et al., 2003; Matozzo et al., 2007), metabolic and physiological alterations (Sarà et al., 2008; Coughlan et al., 2009; Carregosa et al., 2014), and endogenous rhythm (Kim et al., 2001) has been also documented. Nevertheless few studies have looked at the biochemical alterations on bivalves due to salinity changes (Pfeifer et al., 2005; Hamer et al., 2008). It is well known that estuaries and coastal lagoons are subjected to wide variations in salinity under the impact of tidal and seasonal changes. The ebb and flood of the tide, combined with freshwater inputs from rivers and climate changes can dramatically alter the salinity of these aquatic systems. Since bivalves are mostly estuarine or nearshore in nature, they are highly influenced by these salinity variations. Furthermore, bivalves are good aquatic bioindicators because they have a wide geographical distribution, sedentary behavior, easy to sample, tolerance to a wide range of environmental conditions, and high capacity to bioaccumulate contaminants. The typically euryhaline clams Venerupis philippinarum, Venerupis decussata and Venerupis corrugata have been widely used as biomonitor or ecotoxicological test organisms, since they are found world-wide (Flassch and Leborgne, 1992; Usero et al., 1997; Allam et al., 2000; Kim et al., 2001; Elston et al., 2003; Jensen et al., 2004; Pranovi et al., 2006; Delgado and Pérez-Camacho,

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2007; Bebianno and Barreira, 2009; Coughlan et al., 2009; Dang et al., 2010; Ramos-Gómez et al., 2011; Figueira et al., 2012; Moschino et al., 2012; Anacleto et al., 2013; Figueira and Freitas, 2013; FAO, 2014). Apart from their ecological relevance, these species are economically important worldwide, namely in Portugal where the most recent statistics shows that the national annual production of clams represents 42% of the total shellfish production (INE, 2013), being fundamental to the national socioeconomic framework. Different works showed that stress related biomarkers are powerful tools to assess the biological effects of contamination in bivalves, both under environmental and laboratory conditions. Biomarkers such as antioxidant enzyme activity (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase) or oxidative stress parameters (lipid peroxidation, DNA strand breaks) have shown to be useful to assess organisms impacts due to stress conditions (among others, Dellali et al., 2004; Geret and Bebianno, 2004; Bebianno and Barreira, 2009; Ramos-Gómez et al., 2011; Wang et al., 2011, 2012; Freitas et al., 2012a; Silva et al., 2012). Thus, the present work aims to study the biochemical alterations resulting from impacts of salinity changes in three Veneridae clams (V. philippinarum, V. decussata and V. corrugata) and assess how oxidative stress markers are correlated with these alterations. Furthermore, the present study aims to compare the performance of native and invasive species, to evaluate the potential spatial distribution of these species under the predicted climate change scenario. Since little information is available about V. corrugata, the present work will further enrich the knowledge on this species that inhabits coastal areas along the Atlantic. 2. Materials and methods 2.1. Sampling and experimental conditions In the present study, the invasive clam V. philippinarum (Adams and Reeve, 1850), and the native species V. decussata (Linnaeus, 1758) and V. corrugata (Gmelin, 1791), formerly known as Venerupis pullastra (Montague, 1983) were collected in October from a sampling site at the Ria de Aveiro, a shallow coastal system located in the northwest of Portugal. In order to minimize the effect of body size on biochemical and physiological responses to salinity changes, organisms with similar size were used in the laboratory experiments. Thus, the V. decussata individuals presented an average length of 49 ± 2 mm and 38 ± 2 mm width. V. philippinarum clams showed an average length of 50 ± 2.7 mm and 39 ± 3 mm width. V. corrugata specimens presented, in average, 38 ± 3 mm length and 25 ± 2 mm width. In the laboratory, organisms were acclimated for 48 h (Freitas et al., 2012b), by placing individuals in plastic tanks with artificial seawater (salinity of 28 g/L), under continuous aeration. According to measures in the sampling site, the salinity of 28 g/L was selected as representing control conditions. After depuration, the organisms were exposed for 144 h to salinity assays, consisting on the exposure of 9 organisms/salinity (3 replicates, 3 individuals/replicate) to different salinities (0, 7, 14, 21, 28, 35, 42 g/L). Salinity was set up by the addition of artificial sea salt to deionized water. The range of salinity used was selected taking into account the salinity range found in the Ria de Aveiro, where the 3 species were harvested. In summer and winter periods these values easily reach salinities of 10 and 38 g/L, respectively (Santos et al., 2007). A plastic container with 1 L of water was used for each replicate. A temperature of 18 ± 1 °C was maintained during depuration and experimental period and the photoperiod was fixed at 12 h. During the experiment, clams were not fed, the water of each container was continuously aerated and renewed every other day and dead organisms were removed from the containers whenever the water was changed. Organisms were considered dead when their shells gaped and failed to shut again after external stimulus. At the end of the experiment, surviving organisms were frozen at −80 °C for further analysis.

2.2. Laboratory analysis For all analyses, shells of frozen organisms were removed and the soft tissues were mechanically pulverized, in a mill, with liquid nitrogen. For each organism, the pulverized tissue was distributed in aliquots of 0.5 g. Extraction was performed using 0.5 g of pulverized clam tissues and the specific buffer for each biochemical analysis. Before biomarker quantification, samples were sonicated for 15 s at 4 °C and centrifuged for 10 min at 10,000 g at 4 °C. Supernatants were stored at − 80°C or used immediately to determine: glycogen (Gly) content, total protein content, lipid peroxidation (LPO) levels, superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione S-transferase (GST) activity, total glutathione (GSHt) content, and reduced glutathione (GSH) content. All the biochemical parameters were performed twice, using five replicates (n = 5). 2.2.1. Physiological analysis For protein and glycogen content determination, supernatants were obtained using 0.5 g of pulverized clams and sodium phosphate buffer (1:2 w/v), pH 7.0 (sodium dihydrogen phosphate monohydrate 50 mM, disodium hydrogen phosphate dihydrate 50 mM, 0.1% Triton X-100). Total protein content was determined according to the spectrophotometric method of Biuret (Robinson and Hogden, 1940), using bovine serum albumin (BSA) as standards (0–40 mg/mL). Biuret reagent (600 μL) was added to the samples (50 μL) and the mixture was shaken and let to incubate at 30 °C for 10 min. At the end of this time absorbance was read at 540 nm. Results were expressed in mg per g of fresh tissue. Following the procedure described by Yoshikawa (1959), glycogen was quantified by the sulphuric acid method and glucose standards (0–5 mg/mL) were used as comparison with glycogen concentrations. Samples were previously diluted 25 times with the same sodium phosphate buffer, using 50 μL of supernatant for V. philippinarum and 10 μL for V. decussata and V. corrugata. For every sample, 100 μL of phenol (5%) and 600 μL of H2SO4 (96%) were added, shaken and then incubated at room temperature for 30 min. For V. decussata and V. corrugata the volume was made up to 750 μL with sodium phosphate buffer. Absorbance was measured at 492 nm and results were expressed as mg per g of fresh tissue. 2.2.2. Biochemical analysis Lipid peroxidation (LPO) was measured by the quantification of TBARS (thiobarbituric acid reactive substances), according to the protocol described by Ohkawa et al. (1979). The supernatants were extracted using 0.5 g of pulverized clams and 1 mL trichloroacetic acid (TCA; 20% (1:2 w/v)) and then 300 μL of thiobarbituric acid (TBA) were added (0.5% in 20% (v/v) TCA). 100 μL of each sample was incubated at 96 °C for 25 min with 400 μL of thiobarbituric acid and 300 μL of TCA. The reaction was stopped by transferring samples to ice. Lipid peroxidation levels were expressed in nmol of MDA formed per g of fresh tissue. The amount of malondialdehyde (MDA) was quantified spectrophotometrically at a wavelength of 532 nm. The calculation of MDA concentration was made using its extinction coefficient (ε = 1.56 × 105 M−1 cm−1). Superoxide dismutase activity (SOD, EC 1.15.1.1) was determined based on the method of Beauchamp and Fridovich (1971), with some modifications to the microplate method. Supernatants were extracted using 0.5 g of pulverized clams and 50 mM potassium phosphate buffer (1:2 w/v), pH 7.0 (dipotassium phosphate 50 mM; potassium dihydrogen phosphate 50 mM; EDTA 1 mM; Triton X-100 1% (v/v); PVP 1% (v/v); DTT 1 mM). The standard curve was performed with SOD standards (0.25–60 U/mL). In a microplate, 25 μL of each sample (previously diluted 4 times) was incubated, at room temperature for 10 min, with 25 μL of xanthine oxidase, 56.1 mU/mL and 250 μL of reaction buffer (Tris–HCl 50 mM, pH 8.0); diethylene triamine pentaacetic acid (DTPA) 0.1 mM; hypoxanthine 0.1 mM and nitro blue tetrazolium

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(NBT) 68.4 μM. Standards (25 μL) were incubated with 25 μL of xanthine oxidase, 25 μL of extraction buffer and 225 μL of reaction buffer. SOD activity was measured spectrophotometrically at 560 nm in a microplate reader, and expressed in U per g of fresh tissue. One unit (U) of enzyme activity corresponds to a reduction of 50% of nitroblue tetrazolium (NBT). Catalase activity (CAT, EC 1.11.1.6) was quantified according to Lars et al. (1988). Supernatants were extracted using 0.5 g of pulverized clams and potassium phosphate buffer 50 mM (1:2 w/v), pH 7.0 (the same used for SOD determination). The standard curve was determined using formaldehyde standards (0–150 μM). In a microplate, samples (diluted 2 times) and standards (25 μL) were incubated with 125 μL of potassium phosphate buffer 50 mM (pH 7.0), 37.5 μL of methanol and 25 μL of hydrogen peroxide 35.28 mM. To perform the reaction, the microplate was incubated at room temperature for 20 min in a shaker. To stop this reaction it was added with 37.5 μL of KOH 10 M and 37.5 μL of Purpald 34.2 mM. Again, the microplate was incubated for 10 min at room temperature in a shaker. At the end of this time, it was incubated with 12.5 μL of potassium periodate 65.2 mM, at room temperature for a period of 5 min in a shaker. Then, the absorbance was read at 540 nm in a microplate reader. Catalase activity was expressed in U per g of fresh tissue. One unit (U) is defined as the amount of enzyme that caused the formation of 1.0 nmol formaldehyde, per min, under the assay conditions. The activity of glutathione S-transferase (GST, EC 2.5.1.18) was determined following an adaptation of the method described by Habig et al. (1974). Supernatants analyzed were obtained using 0.5 g of pulverized clams and potassium phosphate buffer (1:2 w/v), pH 7.0 (potassium dihydrogen phosphate 50 mM, dipotassium phosphate 50 mM, Triton X-100 0.1% (v/v); EDTA 1 mM; Polyvinylpyrrolidone (PVP) 1% (v/v); Dithiothreitol (DTT) 1 mM). In a microplate, samples (50 μL of supernatant, previously diluted 4 times) were incubated with 200 μL of a reaction solution consisting of 1-chloro-2,4-dinitrobenzene (CDNB) 60 mM, reduced glutathione (GSH) 10 mM and potassium phosphate buffer 0.1 M, pH 6.5 (dipotassium phosphate 0.1 M, potassium dihydrogen phosphate 0.1 M). After the addition of the reaction solution, GST activity was measured spectrophotometrically at 340 nm in a microplate reader. Absorbance values were read at intervals of 10 s for 5 min. For enzyme activity quantification it was selected at a time interval (5 min) during which the activity was linear. The enzymatic activity was expressed in U per g of fresh tissue, where U represents the quantity of the enzyme that catalyzes the conversion of 1 μmol of substrate per min. The activity of GSTs was determined using extinction coefficient of 9.6 mM−1 cm−1 for CDNB. Following the method of Anderson (1985), total glutathione content (GSHt) was determined and expressed as SH equivalents. Supernatants were extracted using 0.5 g of pulverized clams and potassium phosphate buffer 50 mM (1:2 w/v), pH 7.0 (dipotassium phosphate 50 mM; potassium dihydrogen phosphate 50 mM; EDTA 1 mM; Triton X-100 1% (v/v); polyvinylpyrrolidone (PVP) 1% (v/v); DTT 1 mM). The procedure was adapted to a microplate method and 23 μL of glutathione standards (0–500 μmol/L) and samples (diluted 2 times) were incubated for 5 min at room temperature with 240 μL of potassium phosphate buffer, pH 7.0 (dipotassium phosphate 50 mM; potassium dihydrogen phosphate 50 mM); 9.23 μL of NADPH 30 mM; 23 μL of 5,5′-dithiobis2-nitrobenzoic acid (DTNB) 10 mM and 4.62 μL of glutathione reductase (GR) 10 U/mL. Absorbance was measured at 412 nm in a microplate reader and GSHt was expressed as μmol per g of fresh tissue. Reduced glutathione content (GSH) was determined according to Moron et al. (1979) with some modifications. Supernatants were extracted using 0.5 g of pulverized clams and trichloroacetic acid (TCA) 20% (1:2 w/v). Glutathione standards (0–500 μmol/L) were also prepared in TCA 20%. 50 μL of supernatant and standards were neutralized with 20 μL of 2 M NaOH. Then 500 μL of potassium phosphate buffer 50 mM (pH 7.0) and 50 μL of 10 mM DTNB (final volume of 620 μL) were added. Samples and standards were incubated for 5 min at room temperature and after this time absorbance was

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measured spectrophotometrically at 412 nm. GSH was expressed as μmol per g of fresh tissue. Oxidized glutathione (GSSG) was calculated as being the difference between total glutathione (GSHt) and reduced glutathione (GSH) and expressed as μmol per g of fresh tissue. 2.3. Data analysis The GSH/GSSG ratio was considered to be an index of cellular redox status (e.g. Ault and Lawrence, 2003) and was obtained by the relationship between the reduced and oxidized state of glutathione. The biochemical and physiological descriptors (protein content, glycogen, LPO, CAT, SOD, GSTs, GSHt, GSH, GSH/GSSG) were submitted to hypothesis testing using permutation multivariate analysis of variance, employing the PERMANOVA+ add-on in PRIMER v6 (Anderson et al., 2008). These descriptors were analyzed following a one-way hierarchical design, with areas as the main fixed factor. The null hypotheses tested were: i) for each species and for each parameter analyzed, no significant differences exist among salinities; ii) for each salinity and for each parameter analyzed, no significant differences exist among species. The pseudo-F values in the PERMANOVA main tests were evaluated in terms of significance among different areas. Significance levels (p ≤ 0.05) among areas are presented with letters. The matrix gathering the biochemical and physiological descriptors, for each species, per salinity was used to calculate the Euclidean distance similarity matrix. This similarity matrix was simplified through the calculation of the distance among centroids matrix based on the species condition, which was then submitted to ordination analysis, performed by Principal Coordinates Analysis (PCO). Pearson correlation vectors of physiological and biochemical descriptors (correlation N 0.5) were provided as supplementary variables being superimposed at the top of the PCO graph. 3. Results 3.1. Physiological responses 3.1.1. Mortality The results obtained showed that when exposed to different salinity values V. philippinarum and V. decussata can tolerate a wider range of salinities than V. corrugata (Fig. 1). In fact, V. corrugata is the most sensitive species, showing 100% of mortality at the lowest (0 g/L) and the highest

Fig. 1. Mortality of V. decussata, V. philippinarum and V. corrugata when exposed to different salinities (0, 7, 14, 21, 28, 35, 42 g/L). Values are the mean of six replicates ± standard deviation. For each species, significance levels (p ≤ 0.05) among salinities are presented with letters (a–c).

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(35 and 42 g/L) salinities. V. philippinarum revealed high percentage of mortality at salinities 0 and 7 g/L (77.8 and 66.7%, respectively) although 100% of the individuals survived at the remaining salinities. V. decussata specimens survived at all salinities except 0 g/L (33% of mortality at this condition).

3.1.2. Glycogen and protein content Along the increasing salinity gradient V. decussata and V. philippinarum tend to increase the protein content, specially noticed at salinities higher than 21 g/L (Fig. 2A). Furthermore, along the salinity gradient no significant differences were found between these two species. On the contrary, at salinities 14 and 21 g/L V. corrugata presented significantly higher protein content than V. decussata and V. philippinarum. From the results obtained it was also possible to observe that V. corrugata presented significantly higher protein content at salinities 14 and 21 g/L (cf. Fig. 2A). The glycogen determination revealed that V. corrugata was the species with the highest content, followed by V. decussata (Fig. 2B). While V. corrugata presented similar glycogen content in all the salinities tested, V. decussata showed a significant increase in the glycogen at a salinity of 42 g/L, when comparing to the remaining salinities, including a salinity of 28 g/L (cf. Fig. 2B). V. philippinarum was the species with the lowest glycogen content, presenting significantly lower glycogen content at salinities lower than 28 g/L and at a salinity of 42 g/L (cf. Fig. 2B).

Fig. 2. Protein (A) and glycogen (B) content in V. decussata, V. philippinarum and V. corrugata when exposed to different salinities (0, 7, 14, 21, 28, 35, 42 g/L). Values are the mean of six replicates ± standard deviation. For each species, significance levels (p ≤ 0.05) among salinities are presented with letters (a–c).

3.2. Biochemical responses The results obtained showed that V. philippinarum and V. decussata presented higher LPO levels at salinities 0, 7, 14, 35 and 42 g/L, when compared to salinities 21 and 28 g/L (Fig. 3A). For all salinities tested, V. corrugata presented higher LPO values than V. decussata and V. philippinarum, with the lowest LPO value at a salinity of 21 g/L (cf. Fig. 3A). Regarding the total glutathione (GSHt), V. corrugata was the species with the lowest content, with the minimum values found at salinities 21 and 28 g/L (Fig. 3B). Also V. decussata presented the lowest GHSt content at a salinity of 28 g/L, with significantly higher values at salinities 0, 7, 14 and 42 g/L (cf. Fig. 3B). V. philippinarum tended to maintain the GSHt content along the salinity gradient, despite having shown significantly lower and higher values at salinities 0 and 42 g/L, respectively, when comparing to a salinity of 28 g/L (cf. Fig. 3B). The three studied species revealed that GSH values were higher at all salinities tested than at the physiological one (28 g/L) (Fig. 3C). Nevertheless, for V. philippinarum no significant differences were found at salinities higher than 28 g/L (Fig. 3C). V. decussata and V. corrugata presented a similar pattern although the former species showed a sharper and significant decrease between the lowest (0, 7 and 14 g/L) and the highest (35 and 42 g/L) salinities (cf. Fig. 3C). Nevertheless, both species presented the lowest GSH values at a salinity of 28 g/L (cf. Fig. 3C). The GSH/GSSG ratio (Fig. 3D) demonstrated that V. decussata and V. philippinarum showed significantly higher values at a salinity of 14 g/L, with no significant differences between the remaining salinities. On the other hand, V. corrugata showed significantly higher GSH/GSSG values at salinities 7, 14 and 21 g/L (cf. Fig. 3D) when comparing to a salinity of 28 g/L. Regarding SOD (Fig. 4A), the three species revealed the highest enzyme activity at a salinity of 14 g/L. For V. decussata, except at salinities 14 and 21 g/L, this species showed significantly lower SOD values at salinities 0, 7, 35 and 42 g/L than at a salinity of 28 g/L (cf. Fig. 4A). Although with lower values, a similar trend was noticed for V. philippinarum, showing that the activity of this enzyme was significantly higher at a salinity of 14 g/L, when comparing to the remaining salinities (cf. Fig. 4A). V. corrugata was the species with the lowest SOD activity, with significantly lower values at a salinity of 7 g/L. The three species showed higher CAT activity at salinities 0, 7 and 14 g/L (Fig. 4B). V. corrugata revealed the highest CAT values at the salinities 7 and 14 g/L, comparing to salinities 21 and 28 g/L (cf. Fig. 4B). V. decussata and V. philippinarum demonstrated significantly lower CAT activity at salinities higher than 21 and 28 g/L, respectively (cf. Fig. 4B). Concerning the activity of GST (Fig. 4C), V. corrugata was the species with the highest values, presenting the lowest and the highest enzyme activity at salinities 21 and 28 g/L, respectively (cf. Fig. 4C). For V. decussata, when comparing to a salinity of 28 g/L, GST activity was significantly higher at salinities 14 and 21 g/L, and significantly lower at salinities 0, 35 and 42 g/L (cf. Fig. 4C). Along the salinity gradient V. philippinarum was the species with the less pronounced differences in the GST activity (cf. Fig. 4C), with no significant differences among salinities. The results from the PCO analysis (Fig. 5) revealed that the first principal component (axis 1) explained 45.6% of the total variation among conditions, clearly separating V. corrugata in the negative axis and V. philippinarum in the positive axis. Axis 2 described 18.6% of the total variation separating the lower (negative axis) from the higher salinity conditions (positive axis) (cf. Fig. 5). The physiological and biochemical descriptors superimposed on the PCO showed that glycogen content presented a high positive correlation with V. corrugata, which is also characterized by the higher LPO levels (cf. Fig. 5). The protein content was significantly correlated with V. decussata exposed to higher

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Fig. 3. Indicators of cellular damage (A-LPO, B-GSHt, C-GSH, D-GSH/GSSG) in V. decussata, V. philippinarum and V. corrugata when exposed to different salinities (0, 7, 14, 21, 28, 35, 42 g/L). Values are the mean of six replicates ± standard deviation. For each species, significance levels (p ≤ 0.05) among salinities are presented with letters (a–d).

salinities, especially 28 and 35 g/L. Lower salinities, for both species (V. philippinarum and V. decussata), presented a strong correlation with the activity of the antioxidant enzymes (SOD and CAT), with the content of the total (GSHt) and reduced (GSH) glutathione (cf. Fig. 5).

On the other hand, the oxidized glutathione (GSSG) was strongly correlated with V. philippinarum exposed to the highest salinity. The ratio GSH/GSSG revealed to be correlated with V. decussata exposed to a salinity of 21 g/L.

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Fig. 4. Enzymatic activity (A-SOD, B-CAT, C-GSTs) in V. decussata, V. philippinarum and V. corrugata when exposed to different salinities (0, 7, 14, 21, 28, 35, 42 g/L). Values are the mean of six replicates ± standard deviation. For each species, significance levels (p ≤ 0.05) among salinities are presented with letters (a–g).

4. Discussion Bivalves in estuaries periodically experience hyposaline stress, especially during extreme precipitation that dilutes seawater and creates an upper layer of low-density brackish water (Levinton et al., 2011). Weather conditions leading to major precipitation and hyposaline stress have increased over the past decade and are predicted to become even more frequent in a warmer scenario (Karl and Trenberth, 2003; Durack et al., 2012). Generally, when an organism is subjected to stressful conditions it can modify its physiological, biochemical and behavioral responses.

Typical responses in bivalves exposed to low salinities include physiological alterations such as valve closure, reducing feeding activity, slower growth rates, and alterations on endogenous rhythm (measured in oxygen consumption) (among others, Navarro and Gonzalez, 1998; Hamer et al., 2008; Sarà et al., 2008). Previous studies also demonstrated that immunosuppression due to changes in environmental factors (e.g. temperature, salinity and dissolved oxygen) is one of the most important causes of mortality in bivalves (Ford and Tripp, 1996; Matozzo et al., 2007; Munari et al., 2011). Furthermore, while several studies showed that bivalves, and namely clams, may alter their biochemical performance when exposed to pollutants (among others, Pranovi et al., 2006; Delgado and Pérez-Camacho, 2007; Bebianno and Barreira, 2009; Ramos-Gómez et al., 2011; Figueira et al., 2012; Moschino et al., 2012; Figueira and Freitas, 2013), a revision over the literature revealed limited information on the biochemical alterations of bivalves due to salinity changes. In this way, the present study compared the survival capacity and the biochemical alterations of three bivalve species, the clams V. corrugata, V. decussata and V. philippinarum, when under different salinities. The results obtained showed that one of the native species V. corrugata was the species with lower survival capacity, presenting significantly higher mortality rates at the lowest (0 and 7 g/L) and the highest (35 and 42 g/L) salinities. The other native species, V. decussata, was the clam with the wider salinity range of tolerance presenting 100% of survival at all salinities, expect for 0 g/L (with 33% of mortality). The invasive species, V. philippinarum, has been described as an euryhaline species with the capacity to tolerate salinities ranging from 20 g/L to full strength seawater (Coughlan et al., 2009). The present work corroborates this information, showing that this species was able to tolerate most of the tested salinities, with 100% of survival for salinities higher than 7 g/L. Since V. philippinarum is an invasive species in the Ria de Aveiro, the capacity to tolerate a wider range of salinities might give an advantage for species to occupy different areas in the environment, namely in the Ria de Aveiro. Thus, these results may indicate a threat to the native species, especially V. corrugata which tolerates a narrower range of salinities. According to several authors (Burrel, 1977; Akberali, 1978; Elston et al., 2003; Gosling, 2003), when the surrounding salinity is changed bivalves may immediately close their valves as a strategy of protection against osmotic stress. Widdows and Shick (1985) demonstrated that salinity values lower than 20 g/L caused reductions in filtration and respiration rates in the mussel Mytilus edulis. However, at salinities 7 and 14 g/L, which are lower than the range of salinities where the studied species are normally found (Coughlan et al., 2009; Freitas et al., 2012b; Anacleto et al., 2013; Carregosa et al., 2014), the three clams significantly increased their survival rate, when compared to a salinity of 0 g/L which may be related to clams ability to develop anaerobic pathways during shell closure (Livingston, 1983). The ability of these animals to tolerate prolonged periods of anoxia is linked to a coordinated suppression of many metabolic processes including enzymes, protein synthesis, and the movement of ions across membranes (Elston et al., 2003; Carregosa et al., 2014). Kim et al. (2001) suggested that reduced oxygen consumption rate, due to shell closure in the Manila clam, could function as a way of “energy conservation” to a certain extent, by reducing energy expense on respiration and activity when exposed to lower salinities. This mechanism may explain why the three species used in the present study did not decrease the energy reserves such as glycogen, when exposed to lower salinities compared to glycogen content found at the “optimal salinity conditions” for the studied species (between 21 and 28 g/L). The results obtained evidenced that clams mobilize stored energy (glycogen) and may also use protein breakdown to cope with extreme salinity levels. The valves closure not only induces anoxia but also reduces food intake. At a given limiting situation, energy resources are exhausted and osmotic imbalance may arise, inducing water influx

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Fig. 5. Principal Coordinates diagram (PCO) based on the physiological and biochemical responses V. decussata (D), V. philippinarum (P) and V. corrugata (C) when exposed to different salinities (0, 7, 14, 21, 28, 35, 42 g/L). Pearson correlation vectors are superimposed as supplementary variables, namely physiological and biochemical data (r N 0.5).

into the cells, and causing swelling and cellular rupture. These effects may explain the high mortality of V. philippinarum at salinities 0 and 7 g/L, which could be related with the faster growing capacity of this species, strongly associated with the higher filtration rates of this species when compared to the other ones. Physiologically stressful conditions, such as salinity changes, can increase cellular damage in marine invertebrates due to an increased production of reactive oxygen species (ROS), leading to the oxidation of the lipid membranes (Abele, 2002; Abele and Puntarulo, 2004). The present study also showed that at the salinities outside the optimal values for the studied species (optimal values ranged between 21 and 28 g/L), clams tend to significantly increase lipid peroxidation, which results from the higher ROS production. Monari et al. (2005) showed that anoxia, due to shell closure, significantly decreased total hemocyte count as well as SOD activity (an enzyme scavenging superoxide anion), in the clam Chamelea gallina. Our results are in agreement with such findings since at the lowest tested salinities (0 and 7 g/L) the three species presented the lowest activity of SOD due to their tendency to remain their valves closed at low salinities. Our study further revealed that at a salinity of 14 g/L the three species significantly increased the activity of antioxidant enzymes (CAT and SOD) to cope with the increase of oxidative stress at this salinity. Several authors demonstrated the positive relationship between CAT and SOD (Geret et al., 2003; Geret and Bebianno, 2004; Wang et al., 2012). In the present work the increase in the SOD activity contributed to the strong decrease of the LPO levels, especially at salinities 14 and 21 g/L. At higher salinities (35 and 42 g/L) the activity of these antioxidant enzymes significantly decreased, contributing to the increase in the LPO levels. Also Zaccaron da Silva et al. (2005) showed that CAT activity in the oyster Crassostrea rhizophorae was higher at a salinity of 9 g/L decreasing with the increase of salinity (15, 25 and 35 g/L). Our study also evidenced that the higher GST activity was accompanied by lower LPO levels. GST are a major Phase II detoxification enzymes found mainly in the cytosol and function as a substrate of antioxidant enzymes to eliminate the reactive oxygen induced by xenobiotic

compounds, providing protection against electrophiles and products of oxidative stress (Hoarau et al., 2002). Thus, the elevation of GST activity between salinities 14 and 28 g/L may strongly contributed to the lower LPO levels found at this salinity range. Furthermore, the decrease in GST activity, accompanied by the decrease in the activity of the antioxidant enzymes SOD and CAT, may be responsible for the increase in the LPO levels at the highest tested salinities (35 and 42 g/L). Glutathione (a tripeptide of glutamate, cysteine and glycine) is a detoxification agent and it has been considered important in osmotic and oxidative stresses (Figueira et al., 2005; Manduzio et al., 2005). In the present study, along the salinity increasing gradient, the three studied species tended to decrease the total glutathione content, up to a salinity of 35 g/L. Similar findings were reported by Anthony and Patel (2000) who demonstrated that at higher salinities (32 g/L) glutathione significantly decreased compared to a salinity of 16 g/L. The GSH/GSSG ratio is considered to be an index of cellular redox status, indicating the level of oxidative stress in cell (Ault and Lawrence, 2003). When the levels of GSSG arise due to higher amount of oxyradicals, this ratio decreases, meaning higher oxidative stress in cells (Storey, 1996). V. corrugata showed higher GSH/GSSG values at a salinity of 21 g/L, which may indicate lower oxidative stress in cells, being in agreement with other markers, like LPO and GST. In conclusion, the results here presented revealed that V. corrugata was the most sensitive clam to salinity changes, with high mortality rates at the lowest (0 and 7 g/L) and the highest (35 and 42 g/L) salinities. On the other hand, V. decussata and V. philippinarum were able to tolerate salinities higher than 7 g/L. The present work showed that clams experiencing changes in salinity altered their biochemical mechanisms to cope with these stressful conditions. To cope with hyposaline conditions the three studied species increased their antioxidant defenses, decreasing the oxidative stress shown by lower lipid peroxidation levels. The antioxidant enzymes CAT and especially SOD showed to be useful biomarkers to salinity stress, with a negative correlation with the increasing salinity gradient. The clams used in the present study demonstrated that the optimal salinity range varied between 21

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and 28 g/L, where these species presented lower LPO levels and therefore lower mortality. Overall, this study contributed to gain insight into the effects of salinity alterations on native and invasive clam species, demonstrating the high capacity of V. philippinarum to survive at different salinity levels. This capacity may constitute a threat to the native species in the Ria de Aveiro, namely V. corrugata that presented a narrower range of salinities tolerated. The results obtained reinforce the need to adopt appropriate management measures for the conservation and sustainable exploitation of these valuable resources. Acknowledgments This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEst-C/MAR/LA0017/2013. Cátia Velez benefited from a Ph.D grant (SFRH/BD/86356/2012) given by FCT. The authors would like to thank to Anthony Peter Moreira for the English editing. References Abele, D., 2002. 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