Mercury induced haemocyte alterations in the terrestrial snail Cantareus apertus as novel biomarker

Comparative Biochemistry and Physiology, Part C 183–184 (2016) 20–27

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Mercury induced haemocyte alterations in the terrestrial snail Cantareus apertus as novel biomarker Alessandro Leomanni, Trifone Schettino, Antonio Calisi, Maria Giulia Lionetto ⁎ Dept. of Biological and Environmental Science and Technologies (DiSTeBa), Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy

a r t i c l e

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Article history: Received 11 September 2015 Received in revised form 18 December 2015 Accepted 21 January 2016 Available online 23 January 2016 Keywords: Haemocyte Heavy metal Biomarker Cantareus apertus Metallothionein Acetylcholinesterase

a b s t r a c t The aim of the present work was to study the response of a suite of cellular and biochemical markers in the terrestrial snail Cantareus apertus exposed to mercury in view of future use as sensitive tool suitable for mercury polluted soil monitoring and assessment. Besides standardized biomarkers (metallothionein, acetylcholinesterase, and lysosomal membrane stability) novel cellular biomarkers on haemolymph cells were analyzed, including changes in the spread cells/round cells ratio and haemocyte morphometric alterations. The animals were exposed for 14 days to Lactuca sativa soaked for 1 h in HgCl2 solutions (0.5 e 1 μM). The temporal dynamics of the responses were assessed by measurements at 3, 7 and 14 days. Following exposure to HgCl2 a significant alteration in the relative frequencies of round cells and spread cells was evident, with a time and dose-dependent increase of the frequencies of round cells with respect to spread cells. These changes were accompanied by cellular morphometric alterations. Concomitantly, a high correspondence between these cellular responses and metallothionein tissutal concentration, lysosomal membrane stability and inhibition of AChE was evident. The study highlights the usefulness of the terrestrial snail C. apertus as bioindicator organism for mercury pollution biomonitoring and, in particular, the use of haemocyte alterations as a suitable biomarker of pollutant effect to be included in a multibiomarker strategy. © 2016 Published by Elsevier Inc.

1. Introduction Expanding of human activities has produced an increase of soil pollution during the last decades due to the intensive use of fertilizers and biocides in agriculture, industrial activities, urban waste and atmospheric deposition. Some of the most diffusive chemicals occurring in soil are heavy metals. They can enter in the soil from different sources, such as organic and inorganic amendants, pesticides and fertilizers, mining, wastes and sludge residues, and deposition from atmospheric transport following coal and oil burning (Capri and Trevisan, 2002). Unlike to the harmful organic compounds, heavy metals do not decompose and do not disappear from soil even if their release to the environment can be restricted (Chabikovsky et al., 2004). Therefore, the effects of heavy metal contamination on soil organisms and on decomposition processes persist for many years and pose serious problems for living organisms, as well as being difficult to be evaluated. Mercury is one of the highly toxic metals in soil pollution because of its multiple natural and anthropogenic sources, the high volatility of its elemental metallic form and the high atmospheric persistence (UNEP, 2013). Burning of fossil fuels, waste incinerator and mining activity are some of the major source of this metal. ⁎ Corresponding author. E-mail address: [email protected] (M.G. Lionetto).

http://dx.doi.org/10.1016/j.cbpc.2016.01.004 1532-0456/© 2016 Published by Elsevier Inc.

Due to the increasing concern about soil chemical contamination there is an increasing interest in the scientific community and international agencies for soil pollution monitoring and assessment. The traditional approach, based on chemical analysis in order to establish the presence and concentration of specific toxicants, does not provide alone indication about the deleterious effects of contaminants on the biota (Calisi et al., 2011; De Vaufleury and Pihan, 2000). Therefore, the development of new biological tools based on the use of biological indicators has become of great importance for the assessment of the quality of this environmental compartment (Kammenga et al., 2000). The detection of pollutant concentrations in the tissues of bioaccumulator organisms has been identified as an indirect measure of the abundance and availability of metals in the soil. Moreover, the measurement of biochemical, cellular and physiological responses (i.e. biomarkers) developed by bioindicator organisms to soil pollutants are considered early warning signals helpful for gaining insight regarding the exposure and the mechanisms causing observed toxic effects. Gastropods are among the most successful invertebrates in terrestrial ecosystems (Dallinger et al., 2001). Most terrestrial Gastropods are detritivorous animals, feeding on decaying litter, which exhibits a high capacity for retaining trace elements and organic pollutants on its surface (Dallinger et al., 2001). Therefore, terrestrial gastropods feeding on this material can absorb pollutants from their food sources. These organisms exhibit very high capacities for metal accumulation (Dallinger,

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1993; Dallinger et al., 2001; De Vaufleury and Pihan, 2000). In doing so they play an important role in directly transferring pollutants to higher trophic levels of terrestrial food chains being prey or hosts for a variety of other animals (Oehlmann and Shulte-Oehlmann, 2002). The main site of accumulation is represented by the digestive gland (Dallinger, 1993), followed by foot sole, mantle, and intestine. The high capacity for metal accumulation and storage is attributed to the induction of metallothioneins which constitute an efficient tool for metal detoxification (Höckner et al., 2011). Previous studies have demonstrated the useful use of snails as bioindicator organisms for heavy metal soil pollution monitoring (Dallinger et al., 2004; Rabitsch, 1996). Snail species such as Achatina marginata (Achuba Fidelis, 2008), Eobania vermiculata (Bertani et al., 1994), Helix aspersa (Courdassier et al., 2001; Beeby and Richmond, 2002), Helix pomatia (Dallinger et al., 2005), Helicella candicans (Honek, 1993), Monacha cartusiana (Ismail et al., 2013) have been validated as quantitative indicators of environmental metal pollution. Although some information is available about metal bioaccumulation and metallothionein induction in terrestrial gastropods, less knowledge is available about other responses to heavy metal exposure in these organisms. A particularly interesting tissue from a toxicological point of view is represented by the haemolymph, which transports pollutants throughout the exposed organism and its cells are involved in the internal defense system. For this reason any alteration of haemocyte functioning can compromise the health of the entire organism. As previously reported by several authors (Calisi et al., 2008, 2009; Nigro et al., 2006) haemocytes represent one of the first targets of toxic action in invertebrates, so their alterations following pollutant exposure can provide useful information for a more complete understanding of the biological effects associated with exposure to metals. Recently, mollusk haemolymph cells (haemocytes) have received a growing interest for pollutant biomarker development (Calisi et al., 2008; Da Ros and Nesto, 2005). From a morphological point of view two types of cells are recognized in snail haemolymph: round cells and spread cells (Adamowicz and Bolaczek, 2003). Round cells are small, with a high nucleus-cytoplasm ratio. Spread cells are polymorphic cells, with large pseudopodia, polymorphic nucleus and numerous granules in the cytoplasm. The aim of the present study was to analyze the response of a suite of cellular and biochemical biomarkers in the snail Cantareus apertus exposed to mercury through contaminated food. Novel cellular biomarker on heamolymph cells were analyzed, including changes in the spread cell/ round cell ratio and haemocyte morphometric alteration. In parallel standardized biomarkers were analyzed such as 1) tissutal metallothioneins (MT) concentration, specific biomarker of exposure to heavy metals, used to establish the activation of detoxification responses against mercury in the studied animals and thus as analytical confirmation of exposure, 2) acetylcholinesterase (AChE) inhibition, which recently has been demonstrated to be sensitive to some metallic ions (for a review see Lionetto et al., 2010), and 3) lysosomal membrane stability, which is routinely used as an early indicator of the adverse effects of contaminants across a wide range of animal species. C. apertus is a central Mediterranean species that is recorded from France (maritime influenced areas), Italy, and Greece. Outside Europe it is also known from Algeria, Tunisia, and western Libya, and the Mediterranean coast of Turkey, inhabiting all types of maritime influenced Mediterranean habitats. Previous studies demonstrated the suitability of this organism for environmental assessment in in-situ heavy metal pollution (Fritsch et al., 2011) and ex-situ pesticide exposure studies. C. apertus is also a commercial value species (Avagnina, 2011; Novelli et al., 2002). As reported by Avagnina (2011) only in the year 2011, the quantitative of C. apertus eaten was equal to 3.760 tons. Therefore, the assessment of metal induced alterations in this organism could also be of concern for potential effects on human health due to the consumption of this species.

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2. Materials and methods 2.1. Material Adult specimens of C. apertus (mean wt 3.475 ± 0.2 g), obtained from a local dealer, were reared in a plastic box (55 × 39 × 25 cm) under controlled conditions of temperature (20 ± 2 °C), photoperiod (18/6 light/dark regime) and humidity (85%) according to De Vaufleury and Gimbert (2009). The floor of the cage was covered with blotting paper, dampened with tap water. Leaves of Lactuca sativa were administered ad libitum as food. 120 adult snails were randomly chosen for the exposure experiment and starved for 2 days before starting the experiment. All chemicals were reagent grade. Diff-Quick® Kit was purchased from Dade Behring, while the other chemicals were purchased from Sigma-Aldrich (St. Luis, MO, U.S.A.). 2.2. Experimental design A 14 day exposure to HgCl2 through contaminated L. sativa was carried out. Three times a week the animals were exposed to L. sativa, which had been soaked for 1 h in HgCl2-solutions, according to the method described by Dallinger et al. (2005).Two concentrations were utilized, 0.5 and 1 μM, respectively. Foliar consumption of contaminated leaves is one of the main natural pollutant exposure pathways in terrestrial snails. Mercury is known to be easily absorbed by the leaves from air or water (Niu et al., 2013). In gaseous form, Hg metals can be taken up from the air through the stomata (Gaggi et al., 1991), in ionic form it can be taken up through the cuticle (Greger, 1999). The concentrations used were below those considered hazardous to humans (TCLP = 0.2 ppm) and were below the LC50 values for acute exposure of aquatic mollusks (Harry and Aldrich, 1963; Meena and Balakrishnan, 1993). Biomarker responses were monitored in control and treated animals through the time. A three factor experimental design was chosen: factor (A) “mercury exposure” which included three levels (“not exposed” or control animals and “exposed” to 0.5 μM and 1 μM HgCl2), factor (B) “time of exposure” which included four levels (0, 3, 7 and 14 days), and factor (C) “box replication”. Five boxes for each condition were utilized and three animals were added in each box (plastic box dimensions: 14 × 10 × 7 cm). All the groups were held in controlled conditions of temperature, photoperiod, and humidity (see above). At any time three animals per box were sampled. Each specimen underwent to haemolymph sampling, performed by a sterilized hypodermic syringe (needle size 26G½: 0.45 mm × 13 mm) through a small hole created on the shell at the hemocoele level according to Regoli et al. (2006). Haemolymph was immediately utilized for cytological staining of haemocytes and for Neutral Red Retention Assay. Then, each snail underwent cold anesthesia (4 °C for 20 min) and was sacrificed. The hepatopancreas were dissected and stored at −80 °C until utilized for metallothionein measurements. 2.3. Haemocyte morphometric analysis Haemocyte morphometric alterations were determined by image analysis on Diff-Quick® (Dade Behring, Newark, U.S.A.) stained cells, according to the method described by Calisi et al. (2008) and slightly modified. The rapid alcohol-fixed Diff-Quick stain is widely utilized in clinical and veterinary applications for immediate interpretation of histological samples. In recent years it was successfully applied to mussel haemocyte (Calisi et al., 2008) and, also, earthworm coelomocyte staining (Calisi et al., 2009, 2011). In this work, for the first time, the Diff-Quick staining protocol was shown to be suitable for cytological staining of snail haemocytes too. A volume of 40 μl of haemolymph, diluted 1:1 in a snail ringer solution containing 97 mM NaCl, 2 mM KCl, 9 CaCl2, 9mM N-

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[hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), pH 7.5 with NaOH 1 M – was dispensed on a poly-L-lysine coated slide, incubated in a humid chamber (16 °C) for 30 min and stained with the DiffQuick® kit. Samples were fixed and stained on slides by three repeated dips in the reagents of the Diff-Quick® kit, in sequence: Fast Green (fixative), Eosin G in phosphate buffer pH 6.5 and Thiazin Dye in phospate buffer pH 6.5 and, then, subsequently washed with distilled water and airdried. Diff-Quick stained haemocytes were observed by the optic microscope (Eclipse E600, Nikon, Tokio) and the images obtained from video camera (TK-C1381, JVC, Yokoama, Japan) were acquired and digitalized using the LUCIA® image analysis software (Nikon, Tokio, Japan). Approximately 70 cells per sample were analyzed. 2.3.1. Metallothioneins Metallothionein content in the C. apertus hepatopancreas was determined by the spectrophotometric method previously described by Viarengo et al. (1997), suitably modified for this species. Briefly, the single organ was homogenized in three volume of 0.2 M sucrose, 20 mM Tris–HCl buffer, pH 8.6, to which 0.006 mM leupeptine and 0.5 mM phenylmethylsulfonilfluoride were added as antiproteolytic agents and 0.01% β-mercaptoethanol as a reducing agent. Subsequently, the homogenate obtained was treated by ethanol/chloroform precipitation to obtain a partially purified metallothioneins fraction. Finally, the MT concentration was quantified by evaluating the sulfhydryl residues content, according to Ellman's method, with DTNB and reduced glutathione (GSH) as a standard. Data were expressed as μg MT/g of wet weight. 2.3.2. Neutral red retention assay The lysosomal membrane stability was assessed by the neutral red retention assay (NRRA) method according to Lowe and Pipe (1994) with few modifications. Approximately 40 μl of haemolymph collected by single snails (diluted 1:1 as above) were dispensed on a poly-Llysine coated slide, incubated in a humid chamber (16 °C) for 30 min; 40 μl of neutral red solution (995 μl of saline solution and 5 μl of neutral red solution obtained by dissolving 20 mg of neutral red powder in 1 ml of dimethyl sulfoxide) were added. The slides so prepared were left in a humid chamber (16 °C) for 15 min and then observed under the microscope: every 15 min – for the first hour – and every 30 min for the next 2 h thereafter. The time required for 50% of the cell lysosomes to leach the neutral red into the cytosol was determined. 2.3.3. Acetylcholinesterase activity For the measurement of AChE activity an aliquot (10 μl) of the single snail haemolymph was utilized for the spectrophotometrically determination — according to the method described by Ellman et al. (1961) by means of the measure of sample's absorbance increasing at 412 nm in the presence of 1 mM acetylthiocoline as substrate and 0.1 mM 5,5′dithiobis-2-dinitrobenzoicacid (DTNB). The reaction rate was quantified against a blank, without any type of substrate, for each measurement of activity; moreover, in order to subtract the natural hydrolysis of the substrate, a second blank was performed without a sample. The enzyme's activity was expressed as nmole of product, developed per minute per milligram of proteins. Protein concentration was determined by the Bradford assay using NanoDrop ND-1000 UV–Vis (Thermo Scientific) (Desjardins et al., 2009). 2.4. Data analysis Data were analyzed by three-way ANOVA, using the WinGmav 5 software (designed, coded and complied by A.J. Underwood and M.G. Chapman, Institute of Marine Ecology, University of Sidney, Australia). The homogeneity of a variance was tested by Cochran's test, prior to the application of the ANOVA.

3. Results 3.1. Haemocytes morphometric alteration Diff-Quick-stained snail haemocytes appeared well defined when observed by optical microscopy. (Fig. 1A and B). According to Adamowicz and Bolaczek (2003), two cell types were recognized in snail haemolymph: round cells and spread cells. The round cells appeared as small cells with a high nucleuscytoplasm ratio, while spread cells appeared as large cells with pseudopodia and granules in the cytoplasm. In basal physiological conditions spread cells represented about the 60% of the haemocyte population (Fig. 2). When the animals were exposed to HgCl2 for two weeks, a time and dose-dependent increase of the percentage of round cells with respect to spread cells was evident with a significant effect (P b 0.01, χ2 test) observed already at the seventh day of exposure. Besides changes in the relative cell type frequency, also morphometric alterations occurred in snail haemocytes observed following mercury exposure. Cell dimension was quantified as the area of twodimensional digitized haemocyte images and is reported in Fig. 3 for spread cells (A) and round cells (B) respectively. A considerable cell enlargement was observed for the two cell types with a more pronounced effect in spread cells with respect to round cells. The effect appeared dose and time-dependent for both cell types. The effect was maximal because no further increase was observed after another week of exposure for both concentrations. The statistical analysis of data by three way ANOVA is reported in Table 1. The two variability factors, “toxicant exposure” and “time of exposure”, exerted a highly significant effect on the dimension of both haemocyte types, while the “box replication” factor was not significant. In the case of spread cells the interaction between the two variability factors was significant suggesting that the intensity of the response changed with the time of exposure. In Fig. 4 the representative image of spread cells from control animals and from animals exposed to 1 μM HgCl2 for 14 days is shown. Although control animal spread cells typically appeared with the characteristic pseudopodial projection, the treated animal spread cells showed an increase in their dimensions, cell rounding, and loss of pseudopods.

3.2. 2.3 Metallothionein content The two Hg concentrations used were able to evoke a significant MT induction response in the exposed animals as shown in Fig.5A. A maximal increase of about 100% was already observed after three days of exposure to both HgCl2 concentrations tested. No further increase was observed following 7 day, but a decrease in the response was evident following 14 days of exposure. The statistical analysis of data by three way ANOVA (Table 1) revealed that the two variability factors, “toxicant exposure” and “time of exposure”, exerted a highly significant effect with a response changing with the time of exposure (significant interaction between the two variability factors).

3.3. Neutral Red Retention Assay Lysosomal membrane stability of spread cells showed a significant decrease in treated animals during the course of the experiment (Fig.5B). The effect was evident at both exposure concentrations already at the third day of exposure and increased with the time. The statistical analysis of data by three-way ANOVA showed that HgCl2 exposure had a very significant (P b 0.001) effect on the lysosomal membrane stability and that the toxicant effect was time-dependent, as indicated by the significant interaction between the two variability factors “exposure” and “time” (Table 1).

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A

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B

10 µm

Fig. 1. (A,B). Representative images of snail (A) spread and (B) round cell, respectively. The cells were stained with Diff Quick (Dade Behring, Newark, NJ, USA) and visualized by optical microscopy. Objective utilized: 100 oil immersion.

3.4. Acetylcholinesterase activity AChE activity in haemolymph appeared highly sensitive to HgCl2 exposure (Fig.5C). AChE activity progressively decreased starting from the 3th day of exposure for both HgCl2 exposure concentrations. The two variability factors, “toxicant exposure” and “time of exposure” exerted a significant effect on the AChE activity, with a significant interaction between them.

focusing on vertebrates for mercury biomonitoring in terrestrial environments. A few works studied terrestrial invertebrate indicator organisms for Hg pollution (Colacevich et al., 2011; Gimbert et al., 2015). In the present work the effects of HgCl2 exposure through contaminated food on the snail C. apertus was investigated by the analysis of haemocyte alterations integrated with the measure of standardized biomarkers such as, tissutal metallothionein concentration, haemocyte lysosomal membrane stability and acetylcholinesterase inhibition. Snails are profoundly affected by pollution, coming from several anthropogenic sources (Colacevich et al., 2011; Leomanni et al., 2015). These

4. Discussion Mercury is highly toxic, persistent and bio-accumulative and therefore it is of special concern for wild life and humans. Early warning systems, including specific indicator organisms, have been studied in different environmental compartments (Harris et al., 2007) mostly t=0

3 day exposure

7 day exposure

A

14 day exposure

100% 90% 80% 70% 60% 50% 40%

B

30% 20% 10%

0% C

C

C

Spread cells

C

Round cells

Fig. 2. Relative frequency of spread cells and round cells in C. apertus hemolymph following HgCl2 exposure. (7 day exposure: P b 0.01; 14 day exposure: P b 0.001, χ2 test).

Fig. 3. (A,B) Area of two-dimensional digitized images of spread cells (A) and round cells (B). The cells were stained with Diff Quick and visualized by optical microscopy. Data are reported as mean ± standard error. The statistical significance of data was determined by three-way ANOVA and is reported in Table 1.

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Table 1 Statistical analysis of mercury exposure experiment by three way analysis of variance. A three factor experimental design was utilized: factor (A) “mercury exposure” which included three levels (“not exposed” or control animals and “exposed” to 0.5 μM and 1 μM Hg2+ respectively), factor (B) “time of exposure” which included four levels (0, 3, 7 and 14 days), and factor (C) “box replication”. Five boxes for each condition were utilized and three animals were added in each box.

Area of 2D spread cell images

Area of 2D round cell images

Lysosomal membrane stability

MT

AChE

Sources of variation

Significativity

Exposure (A) Time of exposure (B) Box replication (C) Interaction (AXB) Exposure (A) Time of exposure (B) Box replication (C) Interaction (AXB) Exposure (A) Time of exposure (B) Box replication (C) Interaction (AXB) Exposure (A) Time of exposure (B) Box replication (C) Interaction (AXB) Exposure (A) Time of exposure (B) Box replication (C) Interaction (AXB)

P b 0.0001 P b 0.0001 n.s. P b 0.0001 P b 0.01 P b 0.01 n.s. n.s. P b 0.001 P b 0.001 n.s. P b 0.001 P b 0.001 P b 0.001 n.s. P b 0.001 P b 0.001. P b 0.001. n.s. P b 0.001.

organisms can absorb pollutants by dermal contact with soil, ingestion of soil, vegetation, water, and inhalation of air. Therefore, they play an important role in directly transferring pollutants to higher trophic levels of terrestrial food chains being prey or hosts for a variety of other animals. Snails are known to be good metal bioaccumulators and bioindicators. Metal accumulation in snail is favored by the efficient physiological detoxification mechanisms of these animals, such as chelation by binding to metallothioneins and intracellular compartmentalization. In the last decade the interest for terrestrial molluscs as bioindicator organisms has increased. The present work focuses on the study of haemolymph, which is becoming very interesting from the toxicological perspective for the development of novel cellular biomarkers. Several contaminants are known to alter mollusk haemocyte functions, which are known to play an important role in the internal defense of invertebrates. Total haemocyte count (Dyrynda et al., 1998), phagocytosis and production of reactive oxygen species (Gómez-Mendikute and Cajaraville, 2003; Pipe, 1992) hydrolytic and oxidative enzyme activities (Company et al., 2004), and lysosomal membrane stability (Lowe and Pipe, 1994)

Fig. 5. (A,B,C) Metallothionein level (A), lysosomal membrane stability of the haemocytes (B) and acetylcholinesterase activity (C) measured in control and treated animals during the time-course of the exposure experiments (see Materials and methods). The statistical significance of data was determined by three-way analysis of variance and is reported in Table 1.

Fig. 4. Representative spread cell images from control snail (A) and 1 μM HgCl2 treated snail following 14 days of exposure (B).

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all are recognized as pollutant-induced immunotoxicity alterations in other molluscs species, particularly bivalves, the most investigated mollusk species in ecotoxicological studies. On the other hand in snail very few information are available about the effect of pollutants, in particular metals, on haemolymph cells. Haemocytes play a number of key roles in the immune response of snail (Adamowicz and Bolaczek, 2003; Marigómez et al., 1990; Snyman et al., 2000) such as recognition and phagocytosis of invaders or capsule formation around larger exogenous objects (Reinecke and Reinecke, 2000; Snyman, 1981). Besides immunity, haemocytes serve a variety of functions such as blood hemostasis and wound healing, (Franchini and Ottaviani, 2000) shell formation and repair (Mount et al., 2004). Haemocytes also have been involved in the stress response through releasing vertebrate like endocrine molecules (Ottaviani et al., 1992). Due to the important immunological role of these cells, the adverse effects of pollutants on haemocytes may increase the susceptibility of animals to diseases and reduce their survival capability. Therefore, measurements of pollutant-induced haemocyte alterations represent potentially useful biomarkers of effect, which may be directly linked to organism health. According to previous observation in gastropods haemolymph (Mahilini and Raiendran, 2008), two cell types were morphologically identified by Diff-Quick staining in C. apertus haemolymph: round cells or agranulocytes, which are round, unspread haemocytes and have a large nucleo-cytoplasmic ratio, and spread cells or granulocytes, which appear as spreading haemocytes, forming numerous pseudopodia. Following exposure to HgCl2 a significant alteration in the relative frequencies of the two cell types was evident. In particular a time and dose-dependent increase of the frequencies of round cells with respect to spread cells was recorded. It is reported by Adamowicz and Bolaczek (2003) that snail haemocyte number and appearance change depending on both environmental conditions and physiological status of the animal. This is the first time that the relative percentage of the cells was detected to change following exposure to a metal. During the course of the exposure experiment a percentage decrease of spreading cells in time and a parallel percentage increase of round cells was evident. The observed alteration in the relative frequencies of the two haemocyte types could result in alteration of the immune response of the animal, being spread cells mostly involved in phagocytosis process. Moreover, exposure to HgCl2 for 14 d induced a time-dependent increase in the haemolymph cell dimension. A pollutant-induced increase in the cell size was previously described by Marigómez et al. (1990) in the basophilic cells of the digestive gland of the marine prosobranch Littorina littorea and recently in the granulocytes of Mytilus galloprovincialis (Calisi et al., 2008) and Eisenia fetida (Calisi et al., 2009). In general cell swelling can result from the damage of several cellular mechanisms such as alteration in protein catabolism, amino acid and ion transport across cell membrane (Lang et al., 1998; Pedersen et al., 2001). Inorganic ionic Hg2+ is known from mammalian cell studies to cross the plasma membrane mainly by diffusion, and to a smaller extent by active transport involving exchangers (Endo et al., 1997). Once in cell, Hg2+ is able to disrupt a wide range of metabolic functions and membrane transport mechanisms through oxidative stress induction or direct binding to -SH groups which are essential for the normal function of several proteins. Therefore, the haemocyte enlargement could represent the integrated effect of the impairment of several cellular functions and can be related to manifestations of sublethal injury due to the pollutant exposure, as yet suggested by many authors (Fritsch et al., 2011; Lionetto et al., 1998; Sanchez-Hernandez, 2006; Scott-Fordsmand and Weeks, 2000). It was also observed that in the exposed organism the increase in haemocyte dimension was accompanied by cell rounding with reduction of pseudopod number. These effects suggest Hg2+ induced cytoskeletal alterations. Previous studies on snail hepatopancreas cells demonstrated the impact of metals on actin cytoskeleton (Manzl et al.,

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2004). Mercury is known to induce alterations in a number of cytoskeletal components, such as interference with tubulin assembly (Liliom et al., 2000), decrease of cellular F-actin content (Sweet et al., 2006), and disruption of the balance between the phosphorylated and nonphosphorylated form of cofilin, which regulates actin dynamics and facilitates actin filament turnover (Vendrell et al., 2010). Moreover, in mussel haemocytes Hg2+ has been previously demonstrated to alter the intracellular Ca2+ concentration (Marchi et al., 2004) which is a prominent regulator of the structure and dynamics of cytoskeleton. The cytoskeleton, among its multiple physiological functions, is involved in cell volume regulation (Pedersen et al., 2001), in cell shape determination, and in pseudopod formation (Nabi, 1999). Therefore, the hypothized interference of Hg2+ with the microtubule and/or microfilament cytoskeletal components in C. apertus haemocytes is consistent with the alteration in haemocyte dimension and shape observed in exposed organisms. In parallel to cellular haemocyte alterations MT were determined in control and exposed animals as a tool to establish the activation of a detoxification response against mercury in the studied animals and thus as confirmation of exposure. In mercury exposed organisms, the levels of MT measured in the digestive gland showed a time-dependent increase, which reached the maximum value after 7 days of exposure for both concentrations tested. However, a decrease in the cytoplasmic MT concentration was observed at 14 day exposure, presumably due to the interference of mercury with the protein synthesis apparatus at prolonged time of exposure. In fact, several authors documented the ability of mercury to inhibit protein synthesis in vitro (Nakada et al., 1980). Moreover, lysosomal membrane stability and AChE activity were also determined in control and exposed animals in order to verify the applicability of haemocyte enlargement response in a biomarker battery on the bioindicator organism C. apertus. Lysosomal membrane stability is routinely used as an early indicator of the adverse effects of contaminants across a wide range of animals (Calisi et al., 2011; Scott-Fordsmand et al., 1998; Svendsen et al., 2004). In C. apertus haemocytes the exposure to both concentrations of HgCl2 induced a significant time-dependent decrease in the lysosomal membrane stability. A high correspondence between lysosomal membrane stability and morphometrical alterations was evident by comparative analysis of the obtained results. These observations confirm that in C. apertus haemocytes lysosomes are a subcellular sensitive target for the action of mercury. AChE belongs to a class of serine hydrolases (cholinesterases), which catalyze the lysis of choline-based esters. In snails two forms of cholinesterases were previously demonstrated (Talesa et al., 1995): an AChE, representing the 90% of the total cholinesterase activity, and a butyrylcholinesterase. The former is fully soluble and is present in haemolymph, presumably involved in the hydrolysis of choline esters produced by metabolism, while the latter is detergent soluble and is likely to be functional in the cholinergic synapses. In mercury exposed animals haemolymph AChE was significantly inhibited in a dose-dependence manner: the inhibition was already increased at the 3rd day of exposure and was time dependent. AChE is traditionally considered as biomarker of exposure/effect of organophosphate and carbamate pesticides. However, recently, the potential of some metallic ions, such as Hg2+, Cd2+, Cu2+ and Pb2+ to depress the activity of AChE of fish and invertebrates in vitro and or in vivo conditions has been demonstrated in several studies (for a review see Lionetto et al., 2010). In snail AChE showed a high sensitivity to mercury exposure in agreement with results obtained in other species. 5. Conclusions Changes in the relative cell type frequency occurred in snail haemocytes following mercury exposure with a percentage decrease of spreading cells in time and a parallel percentage increase of round

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cells. This finding suggests possible alteration of the immune response of the animal, been spread cells mostly involved in phagocytosis process. In parallel alterations in cells size and shape occurred in the haemocytes of mercury exposed organisms. These morphometric alterations can represent the integrated effect of the impairment of several cellular functions and can be related to manifestations of mercury induced sublethal injury. Concomitantly, a high correspondence between these cellular responses and MT tissutal concentration, lysosomal membrane stability and inhibition of AChE was evident. According to the important immunological role of haemocytes, the observed adverse effect induced by the heavy metal exposure on these cells may increase the susceptibility of the organisms to diseases and may reduce their survival capability. 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