Differential response of biomarkers in the native European flat oyster Ostrea edulis and the non-indigenous Pacific oyster Crassostrea gigas co-exposed to cadmium and copper

Journal of Experimental Marine Biology and Ecology 523 (2020) 151271

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Differential response of biomarkers in the native European flat oyster Ostrea edulis and the non-indigenous Pacific oyster Crassostrea gigas co-exposed to cadmium and copper

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Lorena Perića, , Victor Stinga Peruscob, Vedrana Nerlovićc a

Ruđer Bošković Institute, Division for Marine and Environmental Research, Bijenička cesta 54, 10000 Zagreb, Croatia University of Dubrovnik, Department of Aquaculture, Ćira Carića 4, 20000 Dubrovnik, Croatia c University of Split, University Department of Marine Studies, Ruđera Boškovića 37, 21000 Split, Croatia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Crassostrea gigas Ostrea edulis Metals Stress Co-exposure Biomarker response

Marine pollution favours the invasion and spreading of non-indigenous species in their new habitats. Comparative advantages of introduced and potentially invasive bivalves over their native counterparts might be reflected in better ability to cope with chemical stressors. The objective of the present study was to investigate differences in the biochemical stress response to Cd and Cu between the flat oyster Ostrea edulis (Linnaeus, 1758), native to European coasts, and its non-indigenous congener, the Pacific oyster Crassostrea gigas (Thunberg, 1793), recently detected in the eastern Adriatic. Oysters were co-exposed in vivo to sub lethal concentrations of metals. The stress response of two oyster species was evaluated using biomarkers of neurotoxicity (acetylcholinesterase, AChE), detoxification (metallothioneins, MTs) and oxidative stress (glutathione S-transferase, GST; lipid peroxidation, LPO). Biomarkers related to oxidative stress response were the most informative and suggested comparably lower capability of the non-indigenous C. gigas for handling pro-oxidant conditions after Cu exposure. Overall, the species-specific biomarker alterations displayed by oysters exposed simultaneously to the same experimental conditions represent the first evidence of differences between these two bivalves in the ability to overcome the chemically induced stress. The present research highlights the need for monitoring of biochemical features that might determine the behaviour of oysters in newly colonised habitats under environmental challenges foreseen in the upcoming years.

1. Introduction Marine ecosystems have been seriously threatened by unintentional or deliberate introduction of species that could interact and compete with their native counterparts, and eventually become invasive. Among them, the non-indigenous bivalves are likely to have significant potential for changing the ecosystem structure and functioning in future years in case they successfully invade new habitats (Sousa et al., 2009). Owing to their filter-feeder lifestyle and reef building abilities, the bivalves have major influence on various ecosystem functions including the abundance and composition of suspension particles in water column, biogeochemical cycles, hydrodynamic features and shape of local benthic communities (Dame and Olenin, 2005). The impact of invasive bivalves in their new habitats is difficult to predict as it depends on factors specific for the invaded ecosystem including settlement substrate types, local biodiversity and interactions with native ⁎

communities (Padilla, 2010). Potentially detrimental effects of non-indigenous bivalves commonly range from competition for habitat and food resources with native species, to changes in biodiversity and impact on recruitment of other species (Cavaleiro et al., 2019; Craeymeersch et al., 2019; Guy et al., 2018; Troost, 2010). The effects of colonisation by invasive bivalves is not necessarily negative, as it reportedly contributed to biodiversity increase within degraded habitats and formation of complex three dimensional shelters from predators (Christianen et al., 2018). The European flat oyster Ostrea edulis L. is native to Europe and formerly occupied a wide coastal range from Norway to Morocco in the Atlantic, the entire Mediterranean basin and the Black Sea, but the excessive exploitation, habitat deterioration and massive mortality caused by parasitic infection led to an overall decline of its wild and cultivated populations (Pogoda, 2019 and references therein). To restore the decimated flat oysters' production, the Pacific oyster

Corresponding author. E-mail address: [email protected] (L. Perić).

https://doi.org/10.1016/j.jembe.2019.151271 Received 28 February 2019; Received in revised form 25 September 2019; Accepted 10 November 2019 0022-0981/ © 2019 Elsevier B.V. All rights reserved.

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AChE, MTs, GST and LPO in O. edulis and C. gigas, exposed to Cd and Cu under identical experimental design to obtain an insight into differential sensitivity of these two closely related oyster species to acute metal stress. The main hypothesis of this work was that the non-indigenous C. gigas, known for its successful invasion of many coastal ecosystems, would show better ability to activate its defence against metal toxicity than the native O.edulis. Biochemical responses to these common pollutants could be helpful in understanding of C. gigas spreading and distribution features within its receiving environments. This knowledge could also promote the need for prevention of new C. gigas invasions, as well as for management and conservation of important natural O. edulis beds (Zwerschke et al., 2018a).

Crassostrea gigas, originating from East Asia, was introduced to different European aquaculture sites (Miossec et al., 2009). This species has since displayed high phenotypic adaptability, high fecundity, rapid growth and considerable potential for colonisation and natural spread (Troost, 2010 and references therein) and its newly established wild populations have been replacing now almost extinct O. edulis in particular wide across European coasts of Atlantic (Anglès D'Auriac et al., 2017; Dutertre et al., 2010; Miossec et al., 2009). However, in areas where natural O. edulis beds have persisted to this time, such as the eastern Adriatic, farming of O. edulis based on wild-spat recruitment still represents an essential segment of income for local communities and is largely supported by steady market demand. Since the first record of the non-indigenous C. gigas larvae occurrence (Filić and Krajnović-Ozretić, 1978), wild self-sustaining populations, fragmented patches or individuals were found at several sites along the eastern part of Adriatic coast (Šegvić-Bubić et al., 2016). Evidence of the non-indigenous C. gigas expansion along the eastern Adriatic coast consequently added to concerns over danger of local displacement and gradual extinction of the native O. edulis (ŠegvićBubić et al., 2016). Yet, the potential of C. gigas to become dominant and outcompete the native O. edulis populations is currently insufficiently understood. It seems that the range of environmental conditions that sustain both C. gigas and O. edulis overlap, in particular temperature, salinity and feeding traits (Nielsen et al., 2017). Co-occurrence of native and non-indigenous oysters recently detected in the NE Atlantic (Zwerschke et al., 2018a) might be dependent on the severity of abiotic stress and in particular on settlement substrate surface (Zwerschke et al., 2018b). Recent laboratory simulations of increasing temperature and CO2 indicated that population of C. gigas could be more affected in future than O. edulis by global climate changes (Lemasson et al., 2018). Pollution of marine coastal environment might facilitate the introduction, spreading and invasion of non-indigenous species in particular within urbanised bays or harbours (Guarnieri et al., 2017). Thus, the approach based on the use of biomarkers, i.e. molecular, biochemical and physiological changes at the sub-individual level that indicate an early adverse effects of various stressors (Lagadic, 2002), has become widely applied in comparative studies of non-indigenous vs native bivalves under likely scenario of increasing marine pollution. For example, differences of biochemical biomarkers responses between invasive and closely related indigenous bivalves revealed better ability of the first to cope with chemical stress (Bielen et al., 2016; Nogueira et al., 2018). In contrast, some native bivalves displayed better tolerance to pollutants than their non-indigenous counterparts (Figueira et al., 2012; Velez et al., 2015, 2016). Studies on direct comparison of susceptibility to chemical stress between O. edulis and C. gigas are currently not available. Considering the ever-increasing anthropogenic pressure from land-based and marine sources, better understanding of pollutant-induced biochemical stress response could be useful for identification of conditions that might favour or limit further spreading of the introduced C. gigas within the receiving habitats. Metals are known as widespread pollutants, released from various traditional and new sources in a broad range of ways. Some metals may pose a serious threat for marine ecosystems owing to their ubiquity, long-term persistence in the sediment and accumulation in the tissues of benthic organisms (Komar et al., 2018; Qian et al., 2015). Cadmium (Cd), a toxic metal mainly associated to industrial activity and copper (Cu), a component of antifouling paints and pesticides, are two such metals. The biochemical biomarkers involved in nerve impulse transmission, xenobiotic detoxification and antioxidant defence, namely, acetylcholinesterase activity (AChE), metallothioneins content (MTs), glutathione S-transferase activity (GST) and lipid peroxidation level (LPO), are reportedly affected in bivalves exposed to metals (Figueira et al., 2012; Maria and Bebianno, 2011; Moncaleano-Niño et al., 2018; Perić et al., 2017). The goal of this comparative study was to evaluate the responses of

2. Materials and methods 2.1. Oyster collection and experimental setup Adult specimens of flat oyster O. edulis and Pacific oyster C. gigas were obtained from a commercial aquaculture facility located within the semi-enclosed Medulin Bay (Pula, Croatia). Oysters O. edulis were produced by traditional technique starting from wild spat capture on artificial substrates from surrounding natural environment. There have been no records of C. gigas aquaculture anywhere in this area, but patches of C. gigas adults emerged typically on bivalve growing installations within the same aquaculture facility following successful attachment of wild spat originating from natural beds in the vicinity. Thus, for the purpose of the current study, individual adult specimens of both O. edulis and C. gigas were manually collected from their respective substrates and carefully placed in separate lanterns. Oysters selected for the experiments were then pre-adapted for the next six months in the lanterns installed one next to another in a fixed position. That procedure was carried out in order to minimise the influence of temperature, food availability or other hydrographic parameters' variations that might occur locally within the farming site and modulate oysters' physiology. Prior to experiments, oysters were further acclimated to laboratory conditions in tanks supplied by running seawater delivered from an uncontaminated site far from anthropogenic activities, at a distance of 2 km offshore the coastline, for a week. The concentrations of Cd and Cu detected in the seawater from Adriatic off-shore areas were found to be below values equal to 0.02 and 0.3 μg/l, respectively (Cindrić et al., 2015; Cuculić et al., 2009; Illuminati et al., 2019). The reproductive activity of O. edulis and C. gigas is related to environmental parameters, and is generally not synchronised between these two oyster species in the Mediterranean waters. To reduce the influence of reproductive cycle, the sampling of pre-adapted individuals for laboratory experiments was performed during resting gametogenesis period (late November). The mean shell height of oysters was 11.1 ± 2.8 cm for O. edulis and 13.4 ± 3.3 cm for C. gigas. The experiments were performed by simultaneous exposure of both oyster species to each metal, separately. After acclimation to laboratory conditions, oysters were randomly distributed into experimental 50 l polypropylene tanks containing sea water (1 l / specimen). Tanks (each containing twenty organisms) were continuously aerated and maintained at constant temperature (18 °C) with natural light dark conditions. Oysters were submitted to short 96 h - exposure with daily change of water and renewal of toxicants with appropriate stock solutions to achieve the nominal exposure concentrations (Cd: control, 0.2 μg/l, 1 μg/l, 10 μg/l and 100 μg/l; Cu: control, 1 μg/l, 5 μg/l, 10 μg/l and 50 μg/l). The lowest exposure concentrations for Cd and Cu were adjusted in accordance to EC environmental quality standards (European Commission, 2008) and water quality criteria derived from ecotoxicological tests (Durán and Beiras, 2013). Intermediate exposure concentrations included those detected over moderately and highly contaminated marine coastal areas under various degrees of anthropogenic 2

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Fig. 1. Box-Whiskers plots of acetylcholinesterase activity (nmol min−1 mg prot.−1) in the gills of O. edulis ( ) and C. gigas (■) after exposure to a) Cd and b) Cu for 96 h. Hashes indicate significant difference (#p < .0001) with respect to control (C). For further details see Section 2.3.

were assessed following the protocol for thiobarbituric reactive species assay (TBARS) based on the reaction of malondialdehyde (MDA) as peroxidation breakdown product with thiobarbituric acid (TBA) (Buege and Aust, 1978), and with the use of standard curve of 1,1,3,3-tetramethoxypropane. Samples were incubated in a reaction mixture consisting of 0.5% (w/v) TBA and 20% (w/v) trichloroacetic acid using glass cuvettes at 95 °C for 1 h and then cooled on ice. The absorbance was determined at a wavelength of 530 nm and for turbidity correction at 630 nm. The values for LPO content were expressed as nmol of MDA equivalents per mg of proteins. The amount of proteins in each sample for enzymatic analyses was determined using the Bradford assay (Bradford, 1976) with bovine serum albumin as standard.

impacts (Manfra and Accornero, 2005; Cindrić et al., 2015). The maximal selected concentrations of Cd and Cu were sufficient to elicit observable response of molecular, biochemical and cellular biomarkers following previous in vivo acute exposures of bivalves (Conners and Ringwood, 2000; Géret et al., 2002; Jo et al., 2008; Maria and Bebianno, 2011; Meng et al., 2017; Perić et al., 2017). At the end of the experiment, gill and digestive gland tissues of each oyster were excised, snap-frozen in liquid nitrogen and stored as individual samples at −80 °C until further analyses.

2.2. Biochemical analyses For enzymatic activity and lipid peroxidation (LPO) analyses, the gill tissue of each specimen was individually processed using homogenizing buffers suitable for each protocol (see below) and the resulting suspension was centrifuged for 30 min at 10,000g and 4 °C to obtain cytosolic fraction for subsequent analyses. These samples were individually analyzed. Acetylcholinesterase activity (AChE) in the gill tissue was determined by the method of Bocquené and Galgani (1998) using 0.02 M sodium phosphate buffer (pH 7.0) as homogenization buffer. The reaction mixtures contained 5.5′-dithiobis-2-dinitrobenzoic acid (0.5 mM final) and the appropriate amount of gill tissue sample. Enzyme activity was determined by measurement of absorbance at 412 nm after addition of substrate acetylthiocholine (2.6 mM final). The results were expressed as nmol of thiocholine produced per min and per mg of proteins. Concentration of metallothioneins (MTs) was determined in a partially purified low molecular weight metalloproteins fraction following acidic ethanol/chloroform extraction of the digestive gland tissue homogenate (Viarengo et al., 1999). Prior to processing, each sample was prepared by pooling the digestive gland tissue from three individuals. Quantification of MTs after spectrophotometric measurement of absorbance at 412 nm was performed using standard curve of reduced glutathione (GSH). The results were expressed as μg MTs per g of tissue (wet weight). Samples for determination of glutathione S-transferase (GST) and LPO in the gill issue were homogenized in 50 mM K-phosphate buffer containing 2 mM EDTA (pH 7.5). Activity of GST in the gill tissue was determined by using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (Habig et al., 1974). The reaction mixture consisted of 0.1 M K-phosphate buffer pH 6.5, 1 mM CDNB and 1 mM GSH. The enzymatic reaction was monitored at 340 nm and the activity was expressed as nmol of substrate converted per min and per mg of proteins. Levels of LPO

2.3. Data analysis Biomarker data were displayed as Box-Whiskers plots. Square boxes represent lower and upper quartile and whiskers indicate minimum and maximum values (1.5 interquartile range). Median and outliers are presented as solid line and small circles, respectively. Statistical analyses were performed using the RStudio software, version 1.0.153 (RStudio Team, 2017). The Levene's and Shapiro Wilk tests were applied to test the homoscedasticity and normality of data, respectively. Significant differences between biomarker responses for all groups were assessed using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc test. Otherwise, the non-parametric Kruskal-Wallis test was used for data that did not meet the assumptions of homogeneity of variance and normality. When significant, the Dunn's post hoc test was applied to determine the differences with respect to control. The significance level was set to p < .05. 3. Results No mortalities of any of the two oyster species were recorded during acute 96-h exposure. A significant increase of AChE activity when compared to control occurred in O. edulis after exposure to all concentrations of Cd (ANOVA, F4,83 = 8.66, p < .0001; Bonferroni, p < .0001) (Fig. 1a). The AChE activity in the gills of C. gigas displayed slight but gradual decrease with increasing concentration of Cd, although the effect was not significant (ANOVA, F4,58 = 2.973, p > .05). Exposure to Cu failed to induce changes of AChE activity in O. edulis or C. gigas (ANOVA, F4,80 = 0.987, F4,70 = 1.724, respectively, p > .05) (Fig. 1b). While Cd provoked only minor alterations of MTs content in 3

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Fig. 2. Box-Whiskers plots of metallothionein content (μg g−1 w.w.) in the digestive gland of O. edulis ( ) and C. gigas (■) after exposure to a) Cd and b) Cu for 96 h. Asterisks indicate significant difference (*p < .05, **p < .01) with respect to control (C). For further details see Section 2.3.

increased, with significant difference with respect to control recorded at 1, 10 and 50 μg/l (KW, H (4, N = 80) = 22.73, p < .001, Dunn, p < .05, p < .05 and p < .01, respectively) (Fig. 4b).

digestive gland of O. edulis (KW, H (4, N = 30) = 3.25, p > .05) (Fig. 2a), the significant increase in comparison to control value was observed after exposure of this oyster species to 10 and 50 μg/l of Cu (KW, H(4, N = 40) = 21.79, p < .001, Dunn, p < .05 and p < .01, respectively) (Fig. 2b). The effect of Cd and Cu on MTs content in C. gigas digestive gland could not be determined given the large variability of data. As shown in Fig. 3a there was no evidence of changes in GST activity after Cd exposure of both O. edulis and C. gigas (ANOVA, F4,84 = 1.81, F4,79 = 1.91, respectively, p > .05). In O. edulis, a significant increase of GST activity when comparing to control was observed in response to intermediate concentrations of 5 and 10 μg/l of Cu (ANOVA, F4,93 = 6.01, p < .001, Bonferroni, p < .01), whereas C. gigas did not display any GST activity differences across the whole range of Cu concentrations (ANOVA, F4,94 = 1.24, p > .05) (Fig. 3b). The GST activity was generally higher in C. gigas than in O. edulis. After exposure to Cd, both oyster species displayed a gradual decrease of LPO with increasing concentrations of metal, but the values were significantly different with respect to control only at 10 μg/l for O. edulis and 100 μg/l for C. gigas (ANOVA, F4,53 = 4.46, F4,50 = 4.18, respectively, p < .01, Bonferroni p < .01 and p < .05, respectively) (Fig. 4a). Exposure to Cu did not result in a significant effect on LPO level in O. edulis (KW, H (4, N = 78) = 4.55, p > .05). A gradual increase of LPO in C. gigas occurred as Cu exposure concentrations

4. Discussion This study reports for the first time the assessment of biochemical biomarkers related to neurotoxicity, detoxification and oxidative stress in two oyster species exposed to metals under identical experimental design. The results indicate different ability of O. edulis and C. gigas to handle metal stress and some potential implications for the spreading success of introduced oysters in the new habitat. Reduction of AChE activity, an enzyme crucial for transmission of nerve impulse, has mainly been associated to organophosphorus pesticide and carbamates exposure in several aquatic organisms. In addition, bivalves and other invertebrates have often displayed a marked diversity of AChE responses to metals, depending on the species, tissue, cation type and duration of exposure (Brown et al., 2004; Cunha et al., 2007; Moncaleano-Niño et al., 2018; Najimi et al., 1997; Perić et al., 2017). Rather unexpectedly, the acute Cd exposure resulted in a significant increase of AChE activity in O. edulis, which was not observed in C. gigas. Similar induction of AChE activity after exposure to Cd was reported for marine gastropod Nucella lapillus (Cunha et al., 2007) and bivalve Perna perna (Bainy et al., 2006; Najimi et al., 1997). It was

Fig. 3. Box-Whiskers plots of glutathione S-transferase activity (nmol min−1 mg prot.−1) in the gills of O. edulis ( ) and C. gigas (■) after exposure to a) Cd and b) Cu for 96 h. Asterisks indicate significant difference (*p < .05, **p < .01) with respect to control (C). For further details see Section 2.3. 4

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Fig. 4. Box-Whiskers plots of lipid peroxidation (nmol of MDA equivalents per mg of protein) in the gills of O. edulis ( ) and C. gigas (■) after exposure to a) Cd and b) Cu for 96 h. Asterisks indicate significant difference (*p < .05, **p < .01) with respect to control (C). For further details see Section 2.3.

previously for O. edulis digestive gland tissue (Alonso and MartinMateo, 1996; Tanguy et al., 2003). The results of these studies are clearly in contrast with the increase of MTs content in O. edulis after exposure to Cu reported here, which nevertheless suggests the contribution of MTs in counteracting the toxicity of this metal. An unexpected feature of the present study was a substantial interindividual variability of MTs content in the digestive gland of C. gigas which hindered the comparison of metal detoxification response between these two species. The inability to accurately measure MTs content in some C. gigas samples was surprising, considering that a marked inducibility of MTs in C. gigas by Cd and Cu was previously reported both for laboratory and field exposure conditions (Boutet et al., 2002; Damiens et al., 2006; David et al., 2012). It is plausible that significant MTs content changes in C. gigas could have been masked by large fluctuations of data related to contamination of some digestive gland tissue samples by surrounding storage and gonadal tissue during dissection. As redox-stable and non-essential metal, Cd generate free radical species and induce oxidative stress in bivalves after in vitro and in vivo exposures (Jo et al., 2008; Katsumiti et al., 2014; Meng et al., 2017). When present in excess quantities, ROS interact with membrane lipids, thereby initiating the excessive production of lipid peroxides (LPO) and disruption of normal cellular function (Regoli and Giuliani, 2014). However, the negative effect of Cd in terms of increased LPO concentration was not observed neither in O. edulis nor C. gigas. This apparent lack of change and even occasional decrease of LPO below the control level may indicate an efficient action of cellular antioxidants in prompt alleviation of the negative effect of ROS following a short term exposure of both oyster species to Cd. In addition to a classic role as phase II detoxification enzyme, GST exerts an important antioxidant function by reducing ROS induced LPO to alcohol, concomitantly with the oxidation of glutathione (Hayes et al., 2005; Regoli and Giuliani, 2014). While induction of GST as an important response against metal induced oxidative stress was observed in bivalves (Buffet et al., 2015; Piscopo et al., 2016) and other invertebrates (Lee et al., 2008; Øverjordet et al., 2014), the GST activity was not altered in O.edulis or C. gigas after Cd exposure. One possible explanation for the lack of GST activity increase is that the potentially harmful effect of Cd that could have resulted in lipid peroxidation was prevented by an early and efficient action of ROS scavenging enzymes. In particular, decomposition of superoxide anion to H2O2 catalyzed by superoxide dismutase (SOD) and subsequent reduction of H2O2 to H2O by catalase (CAT) and glutathione peroxidase (GPx), are triggered upon the onset of oxidative stress (Regoli and Giuliani, 2014). Although these antioxidant response features were not measured here, alterations of

previously hypothesised that metals could interact with acetylcholine receptor during acute exposure, leading to an early de novo synthesis of AChE needed to degrade the neurotransmitter accumulated in the synaptic cleft (Bainy et al., 2006). Therefore, this finding indicates that O. edulis, but not C. gigas, may have been able to display a compensatory response by increasing AChE activity to overcome the potential neurotoxicity of Cd during short acute exposure. Further experimental evidence is required to ensure the validity of the above hypothesis since a progressive decrease of AChE activity would be expected after prolonged exposure (Bainy et al., 2006). Different and sometimes contrasting effects of Cu on AChE activity in bivalves were previously reported, such as a clear concentration dependent reduction in mussel Mytilus galloprovincialis (Perić et al., 2017) or increase in cup oyster Saccostrea sp. (Moncaleano-Niño et al., 2018). The ability to cope with presumably neurotoxic Cu effect remained obscured for both oyster species, since AChE activity did not change during acute exposure. Similarly, a significant effect of Cu on AChE activity could not be discerned in clam Scrobicularia plana (Bonnard et al., 2009) and mussel Mytilus edulis (Brown et al., 2004). Oysters might be capable of relatively rapid recovery of AChE activity after metal inhibition, but fundamental knowledge on the mechanism underlying metal interaction with AChE in bivalves is currently not sufficient to explain the lack of Cu effect on enzyme activity. Homeostasis of essential and detoxification of toxic metals are mainly regulated by metallothioneins (MTs), the cysteine rich low molecular weight cytosolic proteins with high affinity to bind metals (Amiard et al., 2006). Generally, detoxification of metals by MTs was extensively reported for bivalves (Amiard et al., 2006; MoncaleanoNiño et al., 2017; Weng and Wang, 2014), yet only few studies have investigated changes of MTs content in oyster O. edulis after exposures to metals. The induction of MTs was not recorded in O. edulis digestive gland after exposure to Cd, in accordance with the results obtained by Tanguy et al. (2003). Furthermore, Piano et al. (2004) detected an increase of MTs content in the same tissue only after exposure to relatively high Cd concentrations (500 μg/l). Recently, Moncaleano-Niño et al. (2017) reported a 2-fold elevation of MTs content in the digestive gland of cup oyster Saccostrea sp. exposed for short period of 96 h to as high as 1000 μg/l of Cd. In addition, these authors reported no induction of MTs for concentrations lower than 100 μg/l which corresponds to the highest Cd concentration used for experimental exposures in the present study. The inhibition of protein metabolism could be one possible reason for the absence of O. edulis MTs induction at least at higher concentrations of metals (Tanguy et al., 2003). Moreover, only a minor role of MTs in metal detoxification in O. edulis was suggested. Likewise, the absence of significant MTs induction after Cu exposure was reported 5

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and their native counterparts. It is evident that in future, such investigations will require an upgraded analysis using multiple endpoints, due to the complexity of biological response elicited by toxic pollutants in vivo. For instance, genotoxicity indicators have repeatedly been used in studies addressing the susceptibility of closely related bivalves to pollutants (Potet et al., 2016; Schäfer et al., 2012) and acute abiotic stress (Yao and Somero, 2013). Extension of the current study by integration of biochemical biomarkers with physiological responses (Bielen et al., 2016; Louis et al., 2019; Velez et al., 2016) is also needed to enable more detailed evaluation of stress tolerance and increase the utility of direct comparison between native oysters and their potentially competing congeners. Bioaccumulation kinetics and the capacity of metal sequestration by formation of insoluble metal-rich granules or aggregates with cellular proteins determine the level of metabolically available metals that ultimately exert a toxic effect, and these processes may vary between different oyster species (Wang et al., 2018 and references therein). Mostly complex and inconsistent relationships between bioaccumulation of metals in the tissues of aquatic invertebrates and metal toxicity have been recently addressed by using a fairly promising theory-based approach (Rainbow and Luoma, 2011), but more knowledge on the ability of oysters to handle metals is still needed to substantially improve the interpretation of Cd and Cu toxicity in these species. Nevertheless, data on accumulated body Cd and Cu concentrations, which were not measured in this study, could have been useful to at least partially disclose some aspects of interspecific difference in biomarker responses. Moreover, a relatively simply designed experimental setup carried out in controlled laboratory conditions only partially illustrates the conditions typical for natural environment. In fact, marine organisms are constantly subjected to fluctuating abiotic factors such as salinity, temperature and nutrients that also affect biological functions by inducing biochemical alterations (Moreira et al., 2017) and by modulating the biomarker response of bivalves to toxic compounds (Freitas et al., 2017; Velez et al., 2016).

SOD, CAT and GPx as a part of antioxidant defence pathway were evidenced in bivalves challenged by metals (De Almeida et al., 2004; Fang et al., 2010; Rocha et al., 2016). Besides, it cannot be ruled out that the oxidative stress and concomitant lipid peroxidation might take longer than 96 h of exposure to Cd to become apparent and thus could not be observed under acute conditions. For instance, an LPO increase was reported previously for C. virginica only after weeks of exposure to 50 μg/l Cd (Lannig et al., 2006). Transition metals, such as Cu, are potent catalysts of Fenton/HaberWeiss reactions, which generate highly reactive toxic hydroxyl radicals (●OH) capable of reaction with lipids that leads to formation of LPO (Halliwell and Gutteridge, 2006). Despite an intense prooxidant activity of Cu exerted in bivalves (Jorge et al., 2018; Katsumiti et al., 2018), the LPO level in the gills of O. edulis exposed to Cu was constant across the whole concentration range indicating a highly efficient antioxidant capacities of this oyster species during acute exposure. The significant increase of GST activity in the gills of O. edulis was also observed possibly suggesting its important role in protection against oxidative damage caused by Cu exposure. Moreover, it was recently pointed out that induction of GST may be a compensatory mechanism of LPO elimination when ROS reducing enzymes fail to prevent the oxidative damage of lipid membrane (Rocha et al., 2016). For instance, only GST was activated to counteract the oxidative stress associated to Hg accumulation in the tissues of clam Ruditapes philippinarum (Velez et al., 2015). Noteworthy, the increased content of MTs in O. edulis might be in agreement with the protective antioxidant function of these proteins acting as scavengers of Cu-generated oxiradicals. Similar effect was also observed in other studies with Cu exposed oysters (Conners and Ringwood, 2000; Géret et al., 2002; Ringwood et al., 1998). Conversely, it seems that the antioxidative defence mechanism in C. gigas was less efficient than in O. edulis during Cu exposure, considering the significant LPO increase that occurred in C. gigas even at relatively low and environmentally realistic Cu concentrations. In agreement with the present results, similar relatively rapid increase (as early as within few days of exposure) of LPO level in oysters was also previously induced by Cu (Ringwood et al., 1998). However, that report also showed a somewhat delayed increase of MTs needed to detoxify Cu that prevented further accumulation of LPO at later stages of exposure. It is also important to note the insensitivity of GST to Cu in C. gigas, possibly indicating little efficiency in LPO elimination. A relatively quick but opposing antioxidative response pattern when simultaneously submitted to the same range of metals concentrations and under identical laboratory conditions might indicate the existence of different mechanisms for preventing oxidative stress in two oyster species. Taken together, the occurrence of Cu-induced oxidative stress in C. gigas suggests that these non-indigenous oysters might experience more severe consequences of exposure to Cu in receiving marine habitats than the native O. edulis, at least under circumstances of accidental discharges or massive runoffs. O. edulis seems to be more tolerant to short-term Cu exposure, possibly by allocating more energy to activation of antioxidant defence. The inefficiency of C. gigas antioxidant system might as well indicate higher demands of energy in the processes that support growth and reproductive activity at the expense of metal defence mechanisms (Potet et al., 2016). Such energy metabolism adjustments under shortterm metal stress were recently reported for two sympatric species of dreissenid mussels currently undergoing a dominance shift of their populations all over the world (Louis et al., 2019). However, when it comes to chronic exposure of low to moderate intensity, frequently encountered along the coastline, the ability of O. edulis and C. gigas to endure pollutant-induced stress and eventually outperform one another, remains open to question and should be addressed through experiments over longer timescales. Evaluation of well-established biochemical biomarkers, in particular of oxidative stress response, was confirmed as a useful approach for investigation of tolerance to chemical stress in non-indigenous bivalves

5. Conclusions A snapshot of biomarker signals in the native O. edulis and the introduced C. gigas recorded following simultaneous exposure to metals brings the first insight into different patterns of stress response in two closely related and potentially competing oyster species. Of all examined toxicity endpoints, those associated to oxidative stress appeared as the most sensitive, even at concentrations meaningful for marine coastal systems. These findings however do not support the notion that the non-indigenous oyster C. gigas might possess better ability to endure metal stress than O. edulis during short acute exposure. In that respect, studies simulating various environmentally realistic exposure setups are needed to develop a more detailed and comprehensive picture of marine habitat conditions that might affect the survival, spreading and eventually the invasive success of C. gigas outside its native range (Dutertre et al., 2010; Rinde et al., 2017). However, regular monitoring of biochemical biomarkers in the native O. edulis and the non-indigenous C. gigas residing in natural and newly invaded habitats, respectively, could represent a useful approach that might provide new pieces of information on potential behaviour of oysters facing the increasing marine pollution. Declarations of Compeitng Interest None. Acknowledgements This study was supported by the Croatian Academy for Science and Arts [grant number 0252Z06]. The authors would like to thank the owner and the employees of hatchery enterprise Rak d.o.o. (Pula, 6

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Croatia) for providing the specimens of O. edulis and C. gigas.

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