Histopathology and stress biomarkers in the clam Venerupis philippinarum from the Venice Lagoon (Italy)

Fish & Shellfish Immunology 39 (2014) 42e50

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Histopathology and stress biomarkers in the clam Venerupis philippinarum from the Venice Lagoon (Italy) Michele Boscolo Papo a, Daniela Bertotto a, Francesco Quaglio a, Marta Vascellari b, Francesco Pascoli b, Elena Negrato a, Giovanni Binato b, Giuseppe Radaelli a, * a b

Dipartimento di Biomedicina Comparata e Alimentazione, Università degli Studi di Padova, Agripolis, Viale dell’Università 16 e 35020 Legnaro (PD), Italy Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10 e 35020 Legnaro (PD), Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 January 2014 Received in revised form 9 March 2014 Accepted 23 April 2014 Available online 2 May 2014

The aim of this study was to evaluate the histomorphology and the stress response in the bivalve Venerupis philippinarum sampled in four differently polluted sites of the Venice Lagoon (Palude del Monte, Marghera, Ca’ Roman and Val di Brenta). This species is often used as bioindicator of environmental pollution since it can bioaccumulate a large variety of pollutants because of its filter feeding. Chemical analyses for heavy metals (Cd, Cu, Hg and Pb) and polycyclic aromatic hydrocarbons (PAHs) were performed on whole soft tissues of V. philippinarum. The histological evaluation of clams revealed the presence of Perkinsus sp. infection in animals from all sites, although a very high prevalence of parasites was evidenced in clams from Ca’ Roman. Perkinsus sp. were systemically distributed in the mantle, in the intestine and digestive gland, in gonads and gills. The trophozoites of Perkinsus sp. were found isolated or in cluster surrounded by a heavy hemocitical response. Haemocytes always exhibited an immunopositivity to cytochrome P4501A (CYP1A), heat shock protein 70 (HSP70), 4-hydroxy-2-nonenal (HNE) and nitrotyrosine (NT) antibodies. The digestive gland of animals from Palude del Monte showed the highest malondialdehyde (MDA) concentration, whereas clams from Ca’ Roman exhibited the highest quantity of metallothioneins. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Clam Histology Perkinsosis Haemocytes Stress biomarkers

1. Introduction The Venice Lagoon is a transitional environment on the Adriatic coast influenced by such human activities as agriculture, industry and tourism. The sludge of Venice and the rivers from the hinterland pour into the lagoon, where sediments trap pollution. For this reason, in recent years numerous national and international projects have been carried out in order to evaluate the quality status of the lagoon environment. Biomonitoring programmes usually involve the use of biomarkers, which represent biochemical, physiological or behavioural variations measured in tissues, biological fluids or whole organisms [1,2]. Several vertebrate and invertebrate species are employed in marine monitoring programmes as sentinel models to evaluate environmental quality. Among invertebrates, the Manila clam Venerupis philippinarum has largely been used to investigate the water/sediment pollution in coastal lagoon ecosystems since it is a filter-feeding bivalve living in

* Corresponding author. E-mail address: [email protected] (G. Radaelli). http://dx.doi.org/10.1016/j.fsi.2014.04.016 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.

soft bottoms [3e8]. Moreover, it represents an important economic resource for fisheries in North Adriatic lagoons, where this species is fished and farmed. At the cellular level, the metabolism of environmental stressors frequently results in the formation of reactive oxygen species (ROS) [9]. They are produced naturally during metabolism and their toxic effects are usually prevented by antioxidants, both molecular and enzymatic ones. During oxidative stress conditions, the production of ROS is greater than the ability of cells to remove them, leading to lipid peroxidation, protein carbonils formation and cell death [10,11]. Lipid peroxides are unstable indicators of oxidative stress in cells that decompose to form more complex and reactive compounds such as malondialdehyde (MDA), which is a natural byproducts of lipid peroxidation [12]. MDA originates from the oxidative degradation of PUFAs and represents a highly toxic aldehyde with a specific affinity to proteins and DNA [12]. 4-hydroxy-2-nonenal (HNE), the most abundant and toxic a,bunsaturated aldehyde, originates from the b-cleavage of hydroperoxides from u-6 PUFAs and is mainly involved in the inhibition of protein and DNA synthesis, in the inactivation of enzymes, and is also a potent mutagen agent [12]. Moreover, one of the most

M. Boscolo Papo et al. / Fish & Shellfish Immunology 39 (2014) 42e50

important ROS is the superoxide radical, which reacts with nitric oxide giving rise to peroxynitrite, a potent oxidant that may oxidize proteins, lipids and DNA [13]. Nitrotyrosine (NT) is a relatively stable marker for peroxynitrite production [13]. Heat shock proteins (HSPs), also called stress proteins, are a family of highly conserved cellular proteins that are present in all cells in all life forms [14e16]. An evident biomarker role was shown to be played by Heath Shock Protein 70 (HSP70) that protects cells against harmful conditions by binding and refolding damaged proteins. There are constitutive members (HSC70) of the heat shock proteins, which play important chaperoning role in unstressed cells, and inducible (HSP70) forms, which are expressed in detectable levels after acute stressor insults [14,17]. In aquatic species, the expression of HSP70 has been studied in fish after exposure to heat shock, pesticides, virus, metals and other toxic compounds [18e21]. In mussel, an increased expression of HSP70 has been detected after exposure to contaminants [22,23]. Moreover, an upper regulation of HSP70 has been observed in the clam Venerupis decussata upon Perkinsus olseni infection [24]. The cytochrome P4501A (CYP1A) subfamily is involved in the biotransformation of a variety of contaminants like polychlorodibenzo dioxins (PCDDs), polycyclic aromatic hydrocarbons (PAHs), polyhalogenated aromatic hydrocarbons (PHAHs) and polychlotobiphenils (PCBs). Its induction plays a central role in transforming pesticides in aquatic organisms [25]. Metallothioneins (MTs) are ubiquitary proteins that have been found in a very wide range of organisms, including vertebrates, invertebrates, plants and bacteria [26]. MTs exhibit high affinity for metals, giving rise to their important role in metal metabolism, detoxification of heavy metals, immune response and the antioxidant process [27,28]. The use of (MTs) seems to be recognized as the most valid method to indicate metal exposure. In fact MTs are induced by metals residues and measurements of their levels are at present used in both vertebrates and invertebrates. The aim of this study was to evaluate the histomorphology of the different tissues and organs of the bivalve V. philippinarum sampled in four differently polluted sites of the Venice Lagoon: Palude del Monte, Marghera, Ca’ Roman and Val di Brenta [29]. We also used an immunohistochemical approach to detect the localization of the following oxidative stress biomarkers: CYP1A, HSP70, HNE and NT. Chemical analyses for heavy metals (Cd, Cu, Hg and Pb) and PAHs were performed on whole soft tissues of clams. Moreover, to test the amount of lipid peroxidation, we evaluated the MDA concentration in digestive gland using the thiobarbituric acid reactive substances (TBARS), a well-established assay for screening and monitoring lipid peroxidation. The amount of metallothioneins, as a biomarker of heavy metal exposure, was estimated in digestive gland by a spectrophotometric method.

43

a graded series of ethanol and embedded in paraffin. Consecutive sections were cut at a thickness of 4 mm using a microtome. For the chemical analysis the whole soft body from 40 animals (10 clams/site) was immediately frozen in liquid nitrogen and stored at 80  C until analysis. For the TBARS assay and metallothionein determination, digestive glands were grouped in three pools of 10 clams per site and stored at 80  C. 2.2. Chemical analysis 2.2.1. PAH determination Chemical analysis was performed on whole soft tissues of V. philippinarum. Extraction was carried out using SampliQ Buffered QuEChERS AOAC Extraction kit (Agilent, CA, USA). An amount of 3 g of homogenized clam sample was weighed in a 50 ml centrifuge tube; before extraction an Agilent Ceramic Bar Homogenizer was added. To sample 10 ml deionized water and 12 ml acetonitrile were added; after each solvent addition the sample was shaked for 15 min. After addition of salts (6 g anhydrous MgSO4 and 1.5 g anhydrous NaOAc) tubes were vigorously shaked for 1 min, and then centrifuged at 4000 rpm for 10 min. Clean-up was performed by means of Agilent Dispersive 15 ml SPE Fatty Sample AOAC kit. A 4 ml aliquot of the previous upper organic extraction layer was transferred into a dispersive SPE 15 ml tube containing salts (400 mg PSA/400 mg C18 EC/1200 mg anhydrous MgSO4). Subsequently tubes were vigorously shaked for 1 min and then centrifuged at 4000 rpm for 10 min. After filtration with disposable syringe filter (Millipore) the extract is ready for analytical determination. HPLC-FLD analyses were carried out by means of an Alliance Empower HPLC system equipped with a 2475 Multi l fluorimetric detector a sample manager and a quaternary solvent manager (Waters, MA, USA). The column was a Supelcosil LC-PAH 150  3 mm 5 mm HPLC column with a Supelguard LC-18 20  3mm guard column (Supelco, PA, USA), kept at 30  C. The flow rate was 0.5 ml min1 with an injection volume of 30 ml. The sample manager was maintained at 30  C.

2. Materials and methods 2.1. Organisms The clams V. philippinarum were collected at four sites of Venice Lagoon (northern terminus of the Adriatic Sea) characterized by different pollution levels. All the clams were adult organisms with a shell size of 4.03
0.3 cm. The four sites were: Palude del Monte [45.28.59 N; 12.21.15 E], Marghera [45.25.55 N; 12.16.09 E], Ca’ Roman [45.14.28 N; 12.16.55 E] and Val di Brenta [45.11.50 N; 12.15.38 E] (Fig. 1). For each site, 50 animals were caught, immediately transferred in portable fridge at 4  C to the laboratory and processed for the analysis. For histology and immunohistochemistry, the whole body from 40 animals (10 clams/site) was fixed in 4% paraformaldehyde prepared in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) at 4  C overnight, washed in PBS, dehydrated through

Fig. 1. Map of the Venice Lagoon indicating the location of sampling station: Palude del Monte [45.28.59 N; 12.21.15 E], Marghera [45.25.55 N; 12.16.09 E], Ca’ Roman [45.14.28 N; 12.16.55 E] and Val di Brenta [45.11.50 N; 12.15.38 E].

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M. Boscolo Papo et al. / Fish & Shellfish Immunology 39 (2014) 42e50

Two spiked (2 mg kg1) blank samples and one blank sample were included in each analytical batch. The quantification of analytes was carried out using a matrix-matched calibration curve in the concentration range of 0.225e7.5 mg L1. 2.2.2. Metal determination (Cd, Cu, Hg and Pb) The concentration of selected metal elements was determined in the whole soft tissues of clams. Following homogenization and acid digestion (HNO3) in Teflon liners by means of a CEM (Matthews, NC, USA) Mars Xpress microwave oven, the metal elements were determined by Atomic Absorption Spectrometry (AAS) with flame atomisation (F-AAS) for Cu (wavelength 324.8 nm) and electrothermal atomisation (graphite furnace GF-AAS) for Cd (wavelength 228.8 nm) and Pb (wavelength 283.3 nm). Analyses were performed by means of a Thermo Electron M6 mkII Atomic Absorption Spectrometer, with D2 and Zeeman background correction, equipped with flame burner and GF95 Graphite Furnace atomiser (Thermo Scientific, Waltham, MA, USA). For Hg determination an Altec (Prague, Czech Republic, EU) TDA AAS direct analyser AMA254 was employed at the following analytical conditions: wavelength 253.6 nm, accumulation time 200 s, drying time 150 s and decomposition time 45 s. Limits of quantification (LOQs) of the employed analytical methods are hereby reported: Pb 0.03 mg/kg; Cd 0.01 mg/kg; Cu 0.10 mg/kg and Hg 0.002 mg/kg. Trueness of analytical data was verified by means of Certified Reference Materials (BCR185R, NRCC DORM3) analysed concurrently with samples in each analytical batch. 2.3. Histology Dewaxed sections including all internal organs from each clam were stained with Haematoxylin and Eosin (H&E) and Periodic Acid Shiff (PAS). Clams histological sections were examined by light microscopy for general morphology, parasite presence and histological alteration. 2.4. Immunohistochemistry Immunohistochemistry was carried out by an automated immunostainer (Autostainer link 48 Dako, Italy). Sections were deparaffinized in xylene, rehydrated in graded ethanol and rinsed in distilled water. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH ¼ 6.0) at 97  C for 15 min. Endogenous peroxidases were neutralized by incubating the sections with the EnVision FLEX Peroxidase-Blocking Reagent (SM801, Dako), for 10 min at RT; serial sections were incubated overnight at þ4  C with the following primary antibodies: mouse polyclonal HSP70 antiserum, dilution 1:200 (Stressgen Biotechnologies, USA), rabbit polyclonal CYP1A antiserum, dilution 1:500 (Biosense laboratories, Norway), mouse monoclonal NT antiserum, dilution 1:1000 (GeneTex, Inc, USA), mouse monoclonal HNE antiserum, dilution 1:100 (Abcam, UK). Sections were then incubated with the detection system EnVision FLEX/HRP (Dako), whereas the EnVision FLEX Substrate Buffer EnVision FLEX DAB (Dako) was used as chromogen. The sections were then counterstained with the EnVision FLEX Haematoxylin (Dako). The specificity of the immunostaining was verified by incubating the sections with PBS instead of the specific primary antibody. 2.5. TBARS assay MDA content was evaluated in three pools of 10 digestive glands of V. philippinarum. MDA is the most abundant reactive carbonyl compounds derived from polyunsaturated fatty acid peroxidation.

The amount of lipid peroxidation in the digestive gland of the clams was assessed by measuring thiobarbituric acid-reactive substances (TBARS) according to Yoshida et al. (2005) [30]. Thiobarbituric acid reaction was carried out by mixing 0.2 ml sodium dodecyl sulphate solution (8.1%, w/v), 1.5 ml acetic acid buffer (20%, v/v, pH 3.5), 1.5 ml thiobarbituric acid (1%, v/v), 0.775 ml water and 0.05 ml ethanol containing butylated hydroxytoluene (0.8 wt.%, w/v) with 200 mg digestive gland. The reaction mixture was incubated at 100  C for 60 min and then cooled in ice followed by mixing vigorously with 1 ml water and 5 ml of n-butyl alcohol and pyridine (15/1, by volume). Then, the mixture was centrifuged (1400 g, 0  C) for 10 min and then supernatant was measured spectrophotometrically at 535 nm. Tetramethoxypropane was used as a standard to estimate TBARS formation as nanomoles of malondialdehyde (MDA) equivalents per g of digestive gland. 2.6. Metallothionein The amount of metallothionein was estimated in digestive gland homogenates, according to a spectrophotometric method [31]. The pools of 10 digestive glands were homogenized in Tris Buffer (Tris 20 mM, 0.5 M sucrose and pH 8,6) containing anti-proteolitic substances, then centrifugated at 15,000 g for 30 min at 4  C. The supernatant was treated with ethanol and chloroform to obtain a pellet with the metallothionein. The pellet was washed with the homogenizing buffer adding chloroform and ethanol, centrifuged at 6000 g for 10 min and dried using nitrogen gas stream. MT concentration was quantified spectrophotometrically at 412 nm using the Ellman’s reagent [32] by evaluating the SH residue content. The GSH (Sigma G4251) was used as standard and the results expressed as mg/g of tissue. 2.7. Statistical analysis Statistical analysis was carried out with STATISTICA 9.1 (StatSoft, Inc.). Differences among sites on pollutants concentrations were assessed by ANOVA and Tuckey HSD post hoc. Differences among sites on MDA and metallothionein were assessed by nonparametric ANOVA. In all analyses a p < 0.05 value was accepted as significant. All data are reported as mean
standard error of the mean (SEM). 3. Results 3.1. Chemical analysis Heavy metals and PAH quantity estimated in the whole body of the clams are presented in Table 1. Maximum concentration of all the heavy metals and PAH was found in animals sampled at Marghera. In the same animals, concentrations of Pb (0.57
0.14 mg/ kg) and PAH (6.17
0.77 mg/kg) were significantly higher than those observed in animals sampled at the other sites of the Lagoon. 3.2. Histology The examination of the histological sections revealed the occurrence of the parasite Perkinsus sp. (Fig. 2AeF) which was detected in 100% of the clams sampled at Ca’ Roman site, 90% of the clams sampled at Val di Brenta and Marghera and 80% of the clams sampled at Palude del Monte. Animals from Ca’ Roman also exhibited the highest number of trophozoites. Perkinsus sp. were systemically found in a variety of host tissues including mantle, intestine, digestive gland, gills, kidney, heart, testis and ovary (Fig. 2AeF). The presence of parasite in the different tissues and a

M. Boscolo Papo et al. / Fish & Shellfish Immunology 39 (2014) 42e50

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Table 1 Top of the table: heavy metals and polycyclic aromatic hydrocarbons (PAHs) concentrations in Venerupis philippinarum sampled in different sites of Venice Lagoon. Values are expressed as means
SEM. n ¼ 10. Significant differences are indicated by different letters. Bottom of the table: Perkinsus sp. presence in the various organs of V. philippinarum collected at different areas of Venice Lagoon. Scale of parasite infestation: - absence; þ low; þþ medium; þþþ high. n ¼ 10. Sites: 1 Palude del Monte; 2 Marghera, 3 Ca’ Roman and 4 Val di Brenta. Sites

1

Heavy metals and PAH concentrations

Cd mg/kg Cu mg/kg Hg mg/kg Pb mg/kg PAHc mg/kg

0.16
1.25
0.05
0.29
<2.5a

Perkinsus sp. presence

Gills Mantle Intestine Digestive gland Gonads Kidney Heart

þþ þ þ þ þ þ -

c

2 a

0.03 0.14a 0.02a 0.09a

0.19 2.06 0.07 0.57 6.17

3






a

0.05 0.30a 0.01a 0.14b 0.77b

þþ þþ þþ þþ þþ þ þþ

0.09
1.54
0.05
0.11
<2.5a þþþ þþ þþþ þþ þþþ þþ þþþ

4 a

0.02 0.11a 0.01a 0.02a

0.17
1.53
0.05
0.18
<2.5a

0.02a 0.11a 0.01a 0.03a

þ þ þ þ þ þ -

PAH concentrations were determined as the sum of benzo(a)antracene, benzo(b)fluoranthene, benzo(a)pyrene and crisene.

scale of parasite infestation are reported in Table 1. The trophozoites of Perkinsus sp. were found isolated or in cluster of trophozoites surrounded by a heavy hemocitical response (Fig. 2B, C, E and F). The hemocitical response was composed of granular eosinophilic haemocytes with PAS-positive granules (inset Fig. 2B and D), hyaline (agranular) haemocytes and rare brown cells. A few number of encapsulated trophozoites appeared degraded.

In the mantle, the cellular infiltration around the parasites presented various degrees of intensity independently of the amount of trophozoites (Fig. 2A). Occasionally, the haemocytical reaction was surrounded by an eosinophilic, strongly PAS-positive, amorphous matrix without parasites. In the digestive gland, trophozoites were observed in the interstitial space between the tubules of the gland (Fig. 2B) and

Fig. 2. Histological identification of Perkinsus sp. in different tissues of Venerupis philippinarum. A, C and E: animals sampled at Ca’ Roman site; B, D and F: animals sampled at Palude del Monte. A) Numerous trophozoites (arrows) are detectable in the mantle (H&E staining). B) Trophozoites of Perkinsus sp. (arrow) in the interstitial space of the digestive gland. Massive inflammatory reaction with granular eosinophilic haemocytes (asterisks) is visible around the parasites (H&E staining). Insert in B shows PAS positivity of trophozoites. C) Perkinsus sp. (arrows) with massive cellular infiltration (asterisks) in the epithelium and connective tissue of the intestinal tract (arrow) (H&E staining). D) Detection of single trophozoites (arrow) in the gills, which show sloughing of filament epithelial cells (PAS reaction). E) Testis parenchyma showing a massive haemocytic infiltration (asterisks) around the parasites (arrow) (H&E). F) Ovary showing parasites (arrow) surrounded by a haemocytic infiltration (asterisks) (H&E). Scale bars: A, C, E and F 40 mm; B, insert in B and D 20 mm.

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rarely in the glandular epithelium. Degeneration and necrosis were noticed in digestive diverticula in case of severe infestation by Perkinsus sp. Encapsulations were observed in the digestive tract, particularly in the connective tissue, in the muscular wall and less frequently in the intestinal epithelium (Fig. 2C). The connective layer and the muscular wall resulted thicker and with abundant inflammatory response. Numerous clusters of trophozoites or single parasites were found distributed in the connective tissues of the gills surrounded by a granulocytic reaction at different level of intensity (Fig. 2D). The highly infected gills showed erosion, or sloughing off of the surface epithelial layer. In the gonads of both genders the haemocytic infiltration around the parasites invaded the interfollicular connective tissue and the follicular walls reducing the reproductive area (Fig. 2E and F). Perkinsus sp. were focally distributed in the parenchyma of kidney and heart although the intensity of infestation was lower than that observed in the other tissues and organs. Rare clams with a severe Perkinsus sp. infestation exhibited isolated trophozoites in the adductor muscles, foot and siphones. Colonies of intracellular prokaryotic rickettsia-like microorganisms (Fig. 3A) were observed within the epithelial cells of gills, mantle and digestive gland tubules and muscular fibres of the mantle and intestine in 3% of animals from Val di Brenta and 2% from Marghera. Trichodinid ectoparasites (Fig. 3B) and other sessile peritrich ciliates (Fig. 3C) were occasionally found on the surface of the mantle without host response. Unidentified helminths (Fig. 3D) were observed with low prevalence into the lumen of intestine. 3.3. Immunohistochemistry All tested antibodies gave an immunoreactivity at the level of the mantle, intestine, digestive gland and gills of animals from all

sites (Fig. 4AeF). The reactivity was localized in the cytoplasm of haemocytes, which were often organized in cluster of cells distributed throughout the epithelium and the underlying connective tissue. No differences in terms of intensity and distribution of immunoreactivity were detected among sites. 3.4. TBARS assay Malondialdehyde (MDA) concentration varied from 13.95
1.68 to 36.80
5.98 nmol/g (Fig. 5). MDA was significantly higher in the digestive gland of clams sampled at Palude del Monte than in those collected at the other sites (Marghera 24.46
0.92 nmol/g and Val di Brenta 22.14
0.87 nmol/g). Animals from Ca’ Roman showed the lowest quantity of MDA. 3.5. Metallothionein content Fig. 6 illustrates the metallothionein (MT) content in the digestive gland of V. philippinarum at four sites of the Lagoon. The clams from Ca’ Roman exhibited the highest quantity of MT (76.77
3.42 mg/g). The lowest MT levels were detected in animals from Val di Brenta (41.43
5.58 mg/g). Marghera exhibited a concentration of 54.39
12.19 mg/g whereas Palude del Monte a quantity of 67.66
10.68 mg/g. 4. Discussion The study of the biological responses of organisms to different environmental conditions and the quantitative evaluation of their physiological status is being considered as a successful approach for the assessment of environmental quality [33e35]. Among invertebrates, bivalve molluscs are widely used as sentinel models in monitoring programmes due to their wide distribution, sedentary lifestyle and their ability to bioaccumulate several chemicals in

Fig. 3. Histological identification of other parasites in different tissues of Venerupis philippinarum. All sections are stained with H&E. AeC: animals sampled at Val di Brenta; D: animals sampled at Ca’ Roman. A) Colonies of intracellular prokaryotic rickettsia-like microorganisms (arrows) in the gills. B) Trichodinid ectoparasite in the mantle. C) Sessile peritrich ciliates located on the surface of the mantle. D) Unidentified helminth into the lumen of intestine. Scale bars: A and C 20 mm; B 10 mm; D 40 mm.

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Fig. 4. Immunohistochemical localization of CYP1A, NT, HNE and HSP70 in different tissues of Vernerupis philippinarum. All sections are counterstained with haematoxylin. A) Digestive gland of a clam from Val di Brenta site showing an immunoreactivity to HSP70 antibody in the cytoplasm of haemocytes. B) Gills of a clam from Ca’ Roman site showing the presence of Perkinsus sp. (arrow). The parasite is surrounded by haemocytes with an intense immunopositivity to CYP1A antibody (asterisks). C) Intestine of a clam from Palude del Monte showing an intense immunopositivity to NT antiserum in haemocytes distributed in the epithelium as well as connective tissue. D) Haemocytes immunopositive to NT (arrows) distributed in the gills of a clam from Val di Brenta site. E) Intestine of a clam from Val di Brenta site showing haemocytes immunopositive to HNE antiserum (arrows). F) Gills of a clam from Marghera site. Arrow indicates a trophozoite surround by haemocytes immunopositive to HNE antiserum. Scale bars: AeF 20 mm.

their tissues, thus reflecting the contamination state of their habitat [36e39]. The aim of this study was to evaluate the general morphology and the oxidative stress response in the bivalve V. philippinarum sampled in four different sites of the Venice Lagoon: Palude del Monte, Marghera, Ca’ Roman and Val di Brenta. The oxidative stress response was evaluated both by determining the levels of MDA and metallothioneins in the digestive gland and by an immunohistochemical analysis aimed to detect the localization in

different tissues of some oxidative stress biomarkers: CYP1A, HSP70, HNE and NT. Chemical analyses performed to detect heavy metals and PAHs on whole soft tissues of the clams sampled at the different sites evidenced the maximum concentrations in animals from Marghera. This site is one of the most important chemical industrial area in Italy and our results confirm that its industrial activities affect the surrounding environment by contamination of soil groundwater and inner tidal canals.

Fig. 5. Malondialdehyde content (nmol/g) in digestive gland of clams (Venerupis philippinarum). Data are presented as means
SEM. Different letters indicate significant differences. n ¼ 3 pools of 10 animals.

Fig. 6. Metallothioneins quantity, expressed as mg/g, in digestive glands of Venerupis philippinarum collected at four stations (means
SEM). Letters indicate significant difference. n ¼ 3 pools of 10 animals.

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Since many environmental pollutants such as heavy metals and PAHs are known to be strongly pro-oxidant, we focused our research on the study of biomarkers reflecting oxidative stress. Bivalves have evolved an extensive battery of antioxidant defences [10], including heat shock proteins [40,41], components of the cytochrome P450 (CYP)-dependent monooxygenase system [42], metallothioneins [43,44] and variations in these biomarkers were found to be useful in environmental monitoring. In our study, an immunopositivity to CYP1A, HSP70, HNE and NT antibodies was detected by immunohistochemistry in various tissues: mantle, intestine, digestive gland and gills. No differences in terms of intensity and distribution of immunoreactivity were detected among sites. The reactivity was localized in the cytoplasm of haemocytes, which were often organized in cluster of cells distributed throughout the epithelium and the underlying connective tissue. In bivalves, haemocytes constitute the primary line of defence against materials recognized as non-self [45]. The presence of non-self materials in tissues initiates a complex molecular signalling cascade to stimulate cell-mediated immune responses, mainly involving phagocytosis or encapsulation of foreign materials, and the production of reactive oxygen species (ROS) [46,47]. In the present work, oxidative stress status was also evaluated in digestive gland by the measure of markers of lipid peroxidation: the thiobarbituric acid reactive substances (TBARS). Although the specificity of TBARS towards compounds other than MDA has been controversial, the assay continues to be the most widely employed format for monitoring lipid peroxidation [29,48e50]. In this study, MDA was significantly higher in the digestive gland of the molluscs sampled at Palude del Monte than in those collected at the other sites, whereas animals from Ca’ Roman showed the lowest quantity of MDA. In accordance with our results, a high concentration of MDA was observed in the mussel Mytilus galloprovincialis sampled in an area closed to Palude del Monte [51]. Although the clams from Ca’ Roman exhibited the highest quantity of MT, the results is contrast with the highest concentration of all the heavy metals detected in animals sampled at Marghera. In a previous work a high concentration of MT was detected in the mussel M. galloprovincialis sampled at the site of Ca’ Roman [51]. The general morphology of clams revealed a massive presence of Perkinsus species in animals from all examined sites. Its presence has been previously described in clams sampled in the North Adriatic Sea by Abollo et al. (2006) [52] who classified it as P. olseni by a species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Perkinsus sp. infestation is known to cause a reduction of growth, gametogenesis inhibition, defence system reduction and it is correlated to mortality of clams as well as reduction of semen in nature [53e56]. In the present study, the level of infestation was particularly intense in clams from Ca’ Roman and Val di Brenta, in accordance with previous results [57,58]. It is noteworthy that the annual temperature cycle and salinity determine a seasonal pattern of variation of prevalence and intensity [57,59]. Most epizootiological studies on perkinsosis have focused on the geographical range of the disease and the effects of salinity on parasite and disease dynamics; prevalence and intensity of perkinsosis in the natural environment increase with increasing salinity [59]. Among the examined lagoon sites, Ca’ Roman is the nearest to the sea which exhibits a salinity higher than that of lagoon environment. Animals from Ca’ Roman also exhibited the highest number of trophozoites if compared with clams from other sites. The parasite was detected in 90% of the clams sampled at Val di Brenta and Marghera and 80% of the clams sampled at Palude del Monte. Since Perkinsus is eliminated by the fecal emission [60], the level of the infestation could be related to the animal population density in the sampled areas. Moreover,

Perkinsus sp. were detected in all the examined tissues with the exception of the nervous tissue. The presence of Perkinsus sp. induced a strong host response, with an infiltration of numerous haemocytes into the surrounding parasitized tissues. In the present study, trichodinid ectoparasites and other sessile peritrich ciliates were occasionally found on the surface of the mantle without host response. Peritrich ciliates remain a poorly studied group in molluscs and only a few publications had reported the occurrence of these ectoparasites in marine bivalves [61e63]. There was no evidence of pathology by Trichodynia species as reported by Bower et al. (1992) [64]. In the samples examined, the presence of protozoa epibionts was not correlated to lesions of disease and these ciliates could be considered ectocommensals. In conclusion, our histopathological examination evidenced that clam populations from the Venice lagoon were affected by serious perkinsosis, which under stressful conditions could induce severe mortality. Although perkinsosis has been observed to increase with exposure to heavy metals and PAHs, with the hypothesis that pollutants impair the host defence capacities [65], we described the parasite presence of parasitism in all examined sites. There is evidence that immunological functions are extremely sensitive to anthropogenic chemicals at exposure concentrations that are not toxic [66e69]. Although immune response is affected by contaminants it is difficult to identify the degree of pollution that can reduce the immune system defence. All our sampling sites exhibit a basal level of pollution; this condition could influence the presence of Perkinsus and suggests that other factors, such as salinity and temperature, could modulate replication and development of Perkinsus sp. [70]. Interestingly, parasites were surrounded by a severe haemocitical response, which always showed a strong immunopositivity to CYP1A, HSP70, HNE and NT, indicating an active defence response of the host in combating parasitic infection. Acknowledgements Authors wish to thank Giovanni Caporale and Erica Melchiotti for technical assistance. Special thanks to Laguna Project snc team that provided the clams. This research was supported by grants from the University of Padua (Progetto ex 60%) and Istituto Zooprofilattico delle Venezie (Ricerca Finalizzata Sanitaria 2006, Drg. n. 3094, 3 October 2006) to G. Radaelli and D. Bertotto. Moreover, the work of Michele Boscolo Papo was financially supported by a grant from Veneto Agricoltura (GABASDOT11). References [1] Depledge MH, Fossi MC. The role of biomarker in environmental assessment (2). Invertebrates. Ecotoxicology 1994;3:161e72. [2] Cajaraville MP, Bebianno MJ, Blasco J, Porte C, Sarasquete C, Viarengo A. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci Total Environ 2000;247:295e 311. [3] Matozzo V, Tomei A, Marin MG. Acetylcholinesterase as a biomarker of exposure to neurotoxic compounds in the clam Tapes philippinarum from the Lagoon of Venice. Mar Pollut Bull 2005;50:1686e93. [4] Matozzo V, Marin MG. First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice. Mar Pollut Bull 2007;55:494e504. [5] Matozzo V, Monari M, Foschi J, Cattani O, Serrazanetti GP, Marin MG. First evidence of altered immune responses and resistance to air exposure in the clam Chamelea gallina exposed to benzo(a)pyrene. Arch Environ Contam Toxicol 2007;56:479e88. [6] Matozzo V, Binelli A, Parolini M, Locatello L, Marin MG. Biomarker responses and contamination levels in the clam Ruditapes philippinarum for biomonitoring the Lagoon of Venice (Italy). J Environ Monit 2010;12:776e86. [7] Matozzo V, Binelli A, Parolini M, Previato M, Masiero L, Finos L, et al. Biomarker responses in the clam Ruditapes philippinarum and contamination levels in sediments from seaward and landward sites in the Lagoon of Venice. Ecol Indicat 2012;19:191e205.

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