Biomarkers of environmental stress in gills of Pinna nobilis (Linnaeus 1758) from Balearic Island

Biomarkers of environmental stress in gills of Pinna nobilis (Linnaeus 1758) from Balearic Island

Ecotoxicology and Environmental Safety 122 (2015) 9–16 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal home...

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Ecotoxicology and Environmental Safety 122 (2015) 9–16

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Biomarkers of environmental stress in gills of Pinna nobilis (Linnaeus 1758) from Balearic Island Antonino Natalotto a, Antoni Sureda b,c,n, Maria Maisano a, Nunziacarla Spanò a, Angela Mauceri a, Salud Deudero d a

Biological and Environmental Sciences Department, University of Messina, Viale Stagno D’Alcontres 31, 98166 Messina, Italy Research Group on Community Nutrition and Oxidative Stress, University of Balearic Islands, Ctra. Valldemossa, km 7.5, E-07122 Palma de Mallorca, Spain c CIBEROBN: Fisiopatología de la Obesidad y la Nutrición, Instituto de Salud Carlos III (ISCIII), Spain d Centro Oceanográfico de Baleares, Instituto Español de Oceanografía, Moll de Ponent s/n, 07015 Palma, Spain b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 March 2015 Received in revised form 23 June 2015 Accepted 24 June 2015

In aquatic environments, bivalve molluscs are used as sentinel species for environmental biomonitoring. In this study Pinna nobilis specimens, the biggest Mediterranean bivalve, were collected in the Magaluf bay (Mallorca), a touristic location and in a pristine area of the Cabrera National Park as the control location. Histological and histochemical analysis in gills of specimens sampled from Magaluf exhibited evident tissue alterations with high presence of haemocytes. Lower acetylcholinesterase (AChE) activity and protein expression were also found in the gills of specimens collected from Magaluf compared with the control area. The determination of antioxidant enzyme activities, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, showed a higher activities of these antioxidant enzymes and total glutathione content in samples from Magaluf bay than in Cabrera. In conclusion, the present study demonstrated that human activities result in morphological tissue alterations and a reduced AChE activity in gills of P. nobilis. Moreover, these stressful environmental conditions induced an adaptive response in P. nobilis as evidenced by increased antioxidant defences and a decreased AChE activity. Capsule: The human activities induce oxidative stress in P. nobilis as evidenced by increased antioxidant defences and a decreased acetylcholinesterase activity. & 2015 Elsevier Inc. All rights reserved.

Keywords: Pinna nobilis AChE Antioxidant enzymes GSH Oxidative stress

1. Introduction The increase of persistent organic pollutants and metals in the seawater results in a serious concern to marine organisms causing several biological effects from molecular dysfunctions to ecological degradation depending on the time of exposure and concentration levels (De Domenico et al., 2013, 2011; Fasulo et al., 2010a, 2010b; Jebali et al., 2014). Aquatic organisms are potentially exposed to anthropogenic contaminants that may strongly affect their performance and survival. The exposure of bivalves to pollutants can result in oxidative stress throughout the formation of reactive species, which can alter biomolecules and induce cell damage. Bivalves are commonly used as sentinel organisms to monitor the pollution in the marine environment because they have a n Corresponding author at: Research Group on Community Nutrition and Oxidative Stress, University of Balearic Islands, Ctra. Valldemossa, km 7.5, E-07122Palma de Mallorca, Balearic Islands, Spain. Fax: þ34 971173184. E-mail address: [email protected] (A. Sureda).

http://dx.doi.org/10.1016/j.ecoenv.2015.06.035 0147-6513/& 2015 Elsevier Inc. All rights reserved.

sedentary state, wide distribution, easy of collection, and ability to accumulate high concentrations of organic and inorganic contaminants (Cappello et al., 2013a; D’Agata et al., 2014a; Deudero et al., 2011, 2009). The bivalve Pinna nobilis L. is the largest endemic bivalve living in the Mediterranean Sea with a shell size reaching 120 cm (Garcia-March et al., 2007; Zavodnik et al., 1991). This species appears in coastal areas between 0.5 and 60 m depth, mostly in soft-sediments overgrown by seagrass meadows of Posidonia oceanica or Cymodocea nodosa (Sureda et al., 2013b; Vazquez-Luis et al., 2014b). Although P. nobilis is nowadays under strict protection and all forms of deliberate capture or killing them are prohibited (EEC, 1992) and its populations have been greatly reduced during the last decades (Vicente and Moreteau, 1991) due to recreational and commercial fishing for food, decorative purposes, incidental killing by trawling and anchoring and anthropogenic pollution, and possible interactions with invasive species (Vazquez-Luis et al., 2014a). Previous studies have showed that the P. nobilis colonized by the invasive algae Lophocladia lallemandii as well as the

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individuals affected by anthropogenic activities showed increased markers of oxidative stress (Box et al., 2009; Sureda et al., 2013a). The gills of the bivalves play a central role in gas exchange but also present several mechanisms to control the functions involved in the maintenance of homoeostasis (Fasulo et al., 2012a; GomezMendikute et al., 2005; Scarfì et al., 2006). The gills are constantly in contact with the surrounding aquatic environment, and thus, highly exposed to environmental factors and contaminants due to their large surface and their main role in gas exchange and feeding (David et al., 2008). Therefore, the structural pattern of the gill tissues and their biochemical profiles reflect the adverse environmental impact on the animal (Cappello et al., 2015, 2013a; Ciacci et al., 2012; Gregory et al., 2002). Haemolymph is rich in haemocytes which are involved in a great number of physiological and pathological functions, mainly including those related with immune response and internal defence. Moreover, these cells also play a role in shell repair and wound, digestion and nutrient transport, and excretion of exogenous and endogenous material (Bayne, 1983; Fisher, 1986). The enzyme acetylcholinesterase (AChE) acts over acetycholine, a neurotransmitter used in efferent systems and also in some central circuits (Woolf, 1991). AChE is involved in the transmission of nerve impulses and has been widely used as an indicator of potential neurotoxicity, both in invertebrates and lower vertebrates (Ciacci et al., 2012; Mora et al., 1999a; 1999b; Peakall, 1992). This enzyme is involved in the control of main branchial functions, such as modulation of ciliary activity, indispensable for the filtration and the feeding, valve opening and embryo development (Corsi et al., 2007). The presence of pollutants results in an increased free radicals and reactive oxygen species (ROS) generation which can damage cellular macromolecules (Lobo et al., 2010). The analysis of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR) or enzymes implicated in detoxification processes such as glutathione-S-transferase (GST) and reduced glutathione (GSH) are also useful biomarkers to monitor highly anthropized aquatic environments (Livingstone, 2003). Therefore the aim of the present work was to evaluate the affection induced by anthropogenic activities on the enzymatic activities of AChE and detoxifying systems in the gills of P. nobilis from two different natural sites.

2. Materials and methods 2.1. Study area and experimental design P. nobilis were sampled from two locations along Mallorca Island and Cabrera waters during July 2011 (Fig. 1). The clean control area was located in a protected area of the Cabrera Archipelago National Park (Western Mediterranean) where human activities are not allowed so that it was considered as a non-polluted area. The exact sampling site is Sta. Maria, a bay in the archipelago of Cabrera, where access by visitors is prohibited and free of sewage or organic inputs. The National park (Act 14/1991) is likewise among the ranks of the Specially Protected Areas of Mediterranean Importance (SPAMI) under the protocols for protected marine areas established by the Barcelona Convention. The second site was located near Magaluf harbour with high touristic impact linked to intense urbanisation and coastal modification. In Magaluf area, there are sewage pipelines discharging from urban zones near the sampling area and high boat traffic. Both areas are in protected bays with similar low wind (with a mean of 10.5 m/s) avoiding the dislodgement of individuals through the enhancement of waves or wind-induced currents (Hendriks et al., 2013, pp. 105–113), and similar values of temperature and salinity (26.6 70.4 °C and 37.9 70.2‰ salinity) have been reported. For this study, P. nobilis (20 individuals, 10 for each location) were collected and immediately processed. Fan mussels were collected at 8–10 m depth by SCUBA diving, measured and carefully dissected according to the procedure described by Yonge (1953). All individuals had similar dimensions (40.6 7 3.2 length and 16.9 714 cm width in Cabrera and 39.9 73.0 length and 15.6 7 13 cm width in Magaluf) and were collected in the same area and period. The work has been carried out in accordance with the EU Directive 2010/63/EU for animal experiments and following the recommendations from the Ethics Committee of the University of the Balearic Islands (Spain). A portion of gill tissue of each individual was frozen in liquid nitrogen for biochemical assays and another portion was fixed in paraformaldehyde for histological, histochemical and immunohistochemical analysis. In the laboratory, gill samples for biochemical analysis were stored at  80 °C until processing and those for histological, histochemical and immunohistochemical analysis were processed and embedded in paraffin.

Fig. 1. Map of sampling locations in Magaluf (Mallorca Island) and Cabrera Island.

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2.2. Histology and histochemical analyses Gills for histological assessment were fixed in 4% paraformaldehyde in 0.1 M phosphate buffered solution (pH 7.4) at 4 °C, dehydrated in ethanol and embedded in Paraplast (Bio-Optica, Italy). Histological sections (4 mm thick) were cut with a rotary automatic microtome (Leica Microsystems, Wetzlar, Germany). Sections were glass-slide mounted and stained with Hematoxylin/ Eosin (Bio-Optica, Italy) (Mazzi, 1977) to visualise typical morphological features. A combined method, using AB/PAS staining (pH 2.5), was used to detect neutral and acid mucous cells in gill epithelium.

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protein) was measured by a modification of the Goldberg and Spooner method (Sureda et al., 2013a). This assay required oxidised glutathione as the substrate. The reaction mixture contained sodium phosphate buffer, pH 7, and 13.5 mM guaiacol. The reaction was initiated by adding H2O2, and changes at 470 nm were monitored. Glutathione S-transferase (GST) activity was measured by the method of Habig et al. (1974). All activities were determined with a Shimadzu UV-2100 spectrophotometer at 25 °C and performed in duplicate. All results were referred to the total protein content of the samples determined with the Bradford method (Biorads Protein Assay) using bovine serum albumin as standard. 2.6. GSH concentration

2.3. Immunohistochemical analyses Sections from the same samples were also used for immunodetection of biomarkers with an immunofluorescence method (Mauceri et al., 1999). The incubations for 1 h into Normal goat serum (NGS) in PBS (1:5) was use to block the non-specific binding sites for immunoglobulins. The sections were incubated overnight in a humid chamber at 4 °C with the primary antibodies anti-AChE developed in mouse and diluted 1:50 (Chemicon). After a rinse in PBS for 10 min, the sections were incubated for 2 h at room temperature with fluorescein isothiocyanate (TRITC) conjugated goat anti-mouse IgG (Sigma). Labelling specificity of the peptide was verified by incubating sections with antiserum preabsorbed with the respective antigen (10–100 g/ml). The pre-absorption procedures were carried out overnight at 4 °C. Negative control for immunohistochemical labeling was performed by substitution of blank sera (without antibodies) for the primary antisera. In order to identify five fields with the largest number of immunostained cells, a first observation of the tissue sections under a 20  objective was carried out. Then using 40  oil-immersion objective, the counting of the immunopositive cells was performed in each one of these fields. All observations were made with a Zeiss epifluorescence microscope (Carl Zeiss AG, Germany), provided of AxioCam camera (Zeiss, Jena, Germany) for the acquisition of images. 2.4. Preparation of gills for biochemical analyses Gills fractions form each samples were homogenised in ten volumes (w/v) of 100 mM Tris–HCl buffer pH 7.5. Each homogenate was sonicated briefly (2–3 s) using ultrasonic processor and centrifuged at 9000g at 4 °C for 15 min (Manduzio et al., 2004). After centrifugation, supernatants were collected and immediately used for the determination of enzymatic activities and GSH levels. 2.5. Enzymatic activities The activity of AChE (mmol/min/mg) was determined in gills using the method of Ellman et al. (1961) with slight modifications. Briefly, thiocholine derivatives are hydrolysed by acetylcholineterase to yield thiocholine. Subsequent combination with 5,5 dithiobis-2-dinitrobenzoic acid (DTNB) forms the yellow anion 5-thio-2-nitrobenzoic acid, which absorbs strongly at 412 nm. Catalase (CAT) activity (K (s  1)/mg protein) was measured by the spectrophotometric method of Aebi (1984) based on the decomposition of H2O2. Superoxide dismutase (SOD) activity (pmol/min/ mg protein) was measured by the xanthine/xanthine oxidase system to generate the superoxide anion (Sureda et al., 2011). This anion produced the reduction of cytochrome C, which was monitored at 550 nm. Glutathione peroxidase (GPX) activity (nmol/ min/mg protein) was measured using an adaptation of the spectrophotometric method of Flohe and Gunzler (1984) using H2O2 as substrate. Glutathione reductase (GR) activity (nmol/min/mg

Gill homogenates were deproteinised with 30% trichloroacetic acid containing 2 mM EDTA. After complete precipitation of the proteins, samples were centrifuged for 5 min at 15,000  g at 4 °C. Supernanant aliquots were placed in a 96-well microtiter plate. GSH was measured by the formation of coloured adducts upon reaction with 0.62 mM 5,5′-dithio-bis(2-nitrobenzoic acid) solution (DTNB). The absorbance of DTNB-treated samples at 412 nm was calculated using GSH as standard and reported as nmol GSH/ mg protein. (Sureda et al., 2013a). 2.7. Statistical analysis For histochemical and immonohistochemical analysis the average of labeling and immunopositive cells was performed using Axio Vision release 4.5 (Zeiss) software. Statistical differences between locations were analysed by one-way analysis of variance (ANOVA) using GraphPad (InStat) software. For biochemical parameters, statistical analyses were carried out using a statistical package (SPSS 17.0 for Windowss). The statistical significance was compared by ANOVA. Results are expressed as mean 7 S.D. and po 0.05 was considered statistically significant.

3. Results 3.1. Histology and histochemical analyses Histomorphological analysis of branchial tissue using the H/E staining revealed a regular morphological organisation in the specimens of P. nobilis collected in Cabrera control site (Fig. 2a), while specimens from Magaluf exhibited evident alterations with a thickening of the apices and thinning of lamellae next to the filaments and a notable haemocytic infiltration (Fig. 2b). The mucous cells were revealed by the Alcian Blue/PAS reaction (Figs. 3a–b). In the branchial epithelium of specimens from Magaluf, a high number of Blue-stained mucous acid cells were evidenced respect to the control site; on the contrary, the PAS reaction revealed minor pink-stained neutral polysaccharides in Magaluf site. The average of AB/PAS staining cells is reported in Fig. 3c. 3.2. Immunohistochemical analysis The immunohistochemical investigation of AChE in the gills of P. nobilis showed a high presence of immunopositive cells in the specimens collected from Cabrera, distributed along the filaments, and varicosities along the gill epithelium (Fig.4a). On the contrary, in the specimens collected from the Magaluf, a drastic reduction of positive cells to AChE was clearly noticed (Fig.4b). Statistical analyses of the mean of immunoreactive cells are represented in Fig.4c (p o0.0001).

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Fig. 2. Haematoxylin and eosin (H/E) staining of 4 μm thick sections of gills of P. nobilis showing the histological structure of mussels from the control (a) and polluted (b) sites. Scale: bar 20 μm. Arrows indicate the haemocytes and the arrowheads indicate the haemolymph vessels.

3.3. Enzymatic activities AChE activity was significantly lower in the gills of fan mussels collected from Magaluf site respect to the activities measured in the organisms of Cabrera (p ¼0.0006), with a mean value of 0.61 mmol/min/mg in Cabrera and 0.24 mmol/min/mg in Magaluf (Fig.5). All antioxidant enzymes activities – CAT, SOD, GPx, GR-, and GST as marker of the detoxification capabilities were increased in Magaluf (p ¼0.0004). Total GSH, measured in gills of P. nobilis, showed higher levels in Magaluf station respect to the Cabrera values. All data obtained from the individual enzymatic analyses are summarised in Table 1.

4. Discussion It is well established that mussels are suitable organisms for environmental quality assessment since they are able to provide cellular and physiological responses to general contamination (Cappello et al., 2015, 2013a, 2013b; Ciacci et al., 2012; Fasulo et al., 2012a; Sureda et al., 2011; Widdows J, 1992). The contamination levels in Cabrera Island are low in contrast to the surroundings of Magaluf that is catalogued as a medium-polluted site. These results were obtained from transplanted mussels in this area within the context of the MYTILOS project. This multicentre project aimed to evaluate the contamination levels by means of caged mussels Mytilus galloprovincialis in the western basin of the Mediterranean Sea (Andral et al., 2011; Galgani et al., 2011; Scarpato et al., 2010). It has been also reported that the coastal waters of Magaluf presented rose levels of dissolved inorganic nitrogen (Alomar et al., 2015). This rise is related to the anthropogenic impacts that

resulted in blooms of phytoplankton (Garcés et al., 2011). Moreover, it has been indicated that submarine groundwater discharge to waters around Magaluf could be an important source of nutrients and CO2 (Basterretxea et al., 2010). Taking into account these previous studies, P. nobilis could be under higher basal stress in the Magaluf site respect to Cabrera. The data reported in this paper show that P. nobilis is an organism sensitive to possible chemical and physical imbalances in the environment derived from anthropogenic activities. In a previous study, haemocytes of P. nobilis collected at the same sites showed increased antioxidant enzyme activities, but there were not changes in malondialdehyde and carbonyl indexes; these results suggest that the antioxidant response was capable to avoid the oxidative damage to lipids and proteins (Sureda et al., 2013a). Histological alterations observed in the gills of the specimens living in the polluted area of Magaluf showed that the branchial epithelium was highly damaged as it was evidenced by epithelial detachment, alteration of ciliated cells and a considerable presence of haemocytes. The histomorphological alterations observed in the gills of P. nobilis may be attributable to two major categories, inflammatory and structural. In the specimens from the control location Cabrera, the acid mucocytes appeared mostly in the frontal zone; whereas neutral mucocytes were present mainly in the intermediate zone. In the gills of specimens collected in the Magaluf area, there was an overproduction of acid mucosubstances, which take part in the transport of food material and in the formation of a protective coat and these also act as a lubricant (D’Agata et al., 2014b). The strong presence of haemocytes and the overproduction of acid mucosubstances attest an activation of the defences by the organisms in response to the environmental changes and possible state of stress (Fasulo et al., 2012b). In fact,

Fig. 3. Alcian Blue/PAS staining of 4-μm sections of P. nobilis gills showing neutral (pink) and acid (blue) mucous cells in the samples from the control (a) and polluted (b) sites. Scale bar: 20 mm. Statistical values are mean 7 SD (n¼ 6). Significance at p o 0.0001 (c).

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Fig. 4. Immunolocalization of AChE on gills. Specimens from Cabrera displayed numerous AChE immunopositive cells along the gill epithelium (a), while samples from Magaluf site showed a drastic inhibition of immunopositivity (b). Mean and standard deviation (SD) of AChE positive cells. Significance at p o0.0001 (c).

Fig. 5. AChE activity inhibited in the gills of sample collected from Magaluf site respect to the sample from Cabrera (p ¼ 0.0006).

Table 1 Antioxidant enzyme activities in gills of Pinna nobilis from a not impacted area (Cabrera) and impacted area (Magaluf). Significant differences were analysed with one-way ANOVA. po 0.05 was considered statistically significant. Values are expressed as mean 7(SD).

CAT (mK/mg prot) SOD (pKat/mg prot) GPX (nKat/mg prot) Gr (nKat/mg prot) GST (nKat/mg prot) GSH (nmol/mg prot)

CABRERA

MAGALUF

p VALUE

7.81 7 2.01 0.067 0.007 0.577 0.06 0.217 0.03 20.81 7 1.47 11.317 1.32

20.2 7 3.9 1.067 0.05 3.86 7 0.37 0.79 7 0.15 63.197 9.2 22.95 7 2.04

p ¼0.015 p o0.0001 p o0.0001 p ¼0.0031 p ¼0.0004 p ¼0.0003

the circulating haemocytes play an important role in immune defence of mussels, via phagocytosis and various cytotoxic reactions, including the release of lysosomal enzymes and the respiratory burst which involves production of oxygen metabolities (Pruzzo et al., 2005). Furthermore, the results presented in this manuscript show, both at enzymatic and immunohistochemical level, a clear effect on AChE in the gills of the specimens collected from Magaluf in respect to the control, suggesting the presence of xenobiotics in this area. Although the AChE activity is inhibited by the presence of neurotoxic compounds (Day and Scott, 1990; Galgani and Bocquené, 1998), the responsiveness of AChE to many other pollutants has also been acknowledged (Ciacci et al., 2012; Guilhermino et al., 1998; Nunes et al., 2014). Consequently, lower

AChE activity is interpreted as a signal, very reliable, of exposure and effect. The inhibition of AChE results in the accumulation of acetylcholine and subsequently on the functionality of cholinergic system at the branchial level, compromising both the filtration capacity as the ciliary motility (Cappello et al., 2015, 2013b; D’Agata et al., 2014b). All antioxidant enzymes from P. nobilis sampled in Magaluf presented higher activities respect to the Cabrera values indicating a more stressful environment in the touristic, urbanised and highly anthropogenic area. These results are in accordance with previous studies reporting similar data (Sureda et al., 2013b). High levels of intracellular ROS are mainly derived from redox reactions occurring during Phase I biotransformation of xenobiotics, leading to the activation of the antioxidant machinery including antioxidant enzymes and scavengers, such as GSH, in order to avoid cellular damage. (Sureda et al., 2013b; Viarengo et al., 1989). The impacted area could induce an increase in basal levels of ROS derived from detoxification processes or by contact with microorganisms which could also be related with the increase of the antioxidant activities (Canesi et al., 2010; Ciacci et al., 2010; Sureda et al., 2013a). Because of gills are in direct contact with the water, it would be very sensitive to the presence of pollutants. Accordingly, this tissue is greatly influenced by external factors, being necessary an excellent enzymatic mechanism against the high free oxygen radicals due to the large volumes of filtered water to cover the oxygen requirements (Sureda et al., 2011). CAT activity, in the organisms, is considered as the primary defence against oxidative damage (Pellerin-Massicotte, 1997), due to its ability to convert hydrogen peroxide into water (Dailianis, 2010) and it responds to a wide range of contaminants capable of ROS production such as PAHs, PCBs, heavy metals, pesticides (Akcha et al., 2000; Khessiba et al., 2005; Regoli and Principato, 1995; Romeo et al., 2003), SOD activity, because of its relatively short time response to environmental stressors, is usually considered as a good biomarker of pollution (Nasci et al., 2002). In the polluted areas, SOD activity increased significantly in gills and it could be related to the fact that gills are directly exposed to pollutants enhancing the cellular antioxidant adaptive response (Box et al., 2009). The GSTs are a group detoxifying enzymes that convert endogenous and xenobiotic electrophilic compounds to water-

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soluble intermediates that may be eliminated. The GST activity is regulated by a diverse range of xenobiotics and in bivalve molluscs have been identified at least 100 chemicals, including organochlorine compounds, inducing the above activities (Dailianis, 2010; Fitzpatrick et al., 1997; Hoarau et al., 2004, 2001). In the present study, the GST activity was significantly higher in the area of Magaluf, which was in accordance with previous studies reporting increased enzyme activities in polluted areas (Viarengo et al., 2007; Bebianno et al., 2007; Bocquene et al., 2004). All together, suggests that GST induction represents an adaptive response to chemical stress caused by electrophiles. GSH is very important in detoxification processes of ROS, which can be generated during the biotransformation of endogenous and exogenous substances. The maintenance of the ratio between reduced GSH and oxidised glutathione (GSSG) is indispensable for the correct functioning of cell metabolism (Jos et al., 2005). The role of GR is important for the recycling of GSH in order to maintain the cellular redox status (Jozefczak et al., 2012). GR protects cells from oxidative damage by catalyzing the reduction of glutathione disulphide in an NADPH-dependant reaction. The high activity of GR in the gills in anthropogenic areas could reflect a high GSSG reduction in order to recycle GSH in the reduced form. GR recycling process is sensitive to pollution as indicated the high activities in the stations with higher anthropic impact (Box et al., 2007). The obtained results, in accordance with the data present in literature (Sureda et al., 2013a), show that the bivalve P. nobilis can be also considered a good and sensitive bioindicator. Furthermore, the selected biomarkers are sensitive enough to monitor the impact induced by anthropogenic activities. Moreover, it has been described that the benthic P. nobilis exhibited increased concentrations for metals (between 30 and 40-fold for Cd, Cu and Zn, up to 4 times for Hg, and 7 times for Pb) in the Balearic Archipelago, when compared to a standard bioindicator of the coastal trace metal pollution, the mussel M. galloprovincialis (Deudero et al., 2007, 2009). It was also determined in a previous studies, that polycyclic aromatic hydrocarbons (PAHs) levels in the soft tissues of P. nobilis were more than 20-fold higher compared to the ones found in M. galloprovincialis (Sureda et al., 2011; Sureda et al., 2013b). In accordance, it has been reported that fish eggs, larvae and juveniles have been affected by combustion derived products from boat engines and nutrients increase due to wastewaters (Whitfield and Becker, 2014; Leon and Warnken, 2008). In a previous study, it has been evidenced that P. nobilis individuals in eutrophic environments showed higher growth rates than those in oligotrophic environments, but had lower survival rates with a density ranging from 5.2 to 8.8 ind/100 m2 in Santa Maria Bay (Cabrera), whereas mean density was about 1.8 ind/100 m2 in Magaluf (Vázquez-Luis et al., 2014; Hendriks et al., 2013). Altogether supports the fact that P. nobilis is clearly affected by the anthropogenic activities as it was evidenced in the histological alterations, the inhibition of AChE and in the activation of the antioxidant defences.

5. Conclusion In conclusion, the present results clearly evidence that the P. nobilis individuals from Magaluf (an anthropogenic affected area) are suffering from physiological stress as it was evidenced by histochemical and immunohistochemical analysis and by a reduced AChE activity. Moreover, antioxidant enzyme activities and GSH content were also elevated in the touristic area supporting the existence of an adaptative response to environmental stress. Further investigations to determine the long-term effects on reproduction and physiological or immunological impairments should be assessed.

Acknowledgements The study was supported by the research program in the National Park Network, Ministry of Agriculture, Food and Environment (024/2010), the Spanish Ministry of Health and Consumer Affairs, CIBEROBN CB12/03/30038 and Balearic Island Government (23/2012) and FEDER funds.

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