Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

Fish & Shellfish Immunology 22 (2007) 98e114 www.elsevier.com/locate/fsi

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina Marta Monari a, Valerio Matozzo b, Jurgen Foschi a, Otello Cattani a, Gian Paolo Serrazanetti a, Maria Gabriella Marin b,* a

Department of Biochemistry ‘‘G. Moruzzi’’, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy b Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy Received 30 January 2006; revised 24 March 2006; accepted 24 March 2006 Available online 25 April 2006

Abstract The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7 days at 20, 25 and 30  C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25  C burrowed completely, whereas at 30  C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30  C showed that the cell population of about 8e10 mm was markedly reduced compared to clams kept at 20 and 25  C. In clams maintained at 25  C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30  C whereas it increased in cell-free haemolymph from clams held at 25  C and 30  C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30  C, whereas the Cu/ Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30  C was shown to decrease significantly, LT50 values fell from 6 days in clams kept at 20  C and 25  C to 4 days in those kept at 30  C. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Bivalve; Immune response; Biomarker; Superoxide dismutase; Survival in air

1. Introduction Circulating haemocytes are involved in immune defence in bivalve molluscs. Generally, non-self materials stimulate cell-mediated immune responses, which include increases in circulating haemocyte number, phagocytosis * Corresponding author. Tel.: þ39 049 827 6200; fax: þ39 049 827 6199. E-mail address: [email protected] (M.G. Marin). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.03.016

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and/or encapsulation of foreign particles, depending on their size. Increases in the total haemocyte count (THC) are commonly considered as a consequence of either proliferation or movement of cells from tissues into the haemolymph, whereas a decrease in THC is thought to be a consequence of cell lysis or reduced movement of cells from tissues to haemolymph [1]. Oubella et al. [2] observed that increases in haemocyte count in the Manila clam, Ruditapes philippinarum, challenged with a pathogenic bacterium, Vibrio P1, were an antibacterial response of the bivalve. After phagocytosis, foreign particles can be degraded by lysosomal hydrolytic enzymes within haemocytes [3]. However, in some cases, lytic enzymes, and lysozyme in particular, are released into the haemolymph during phagocytosis, thus participating in inactivation of invading microbes [4]. In some bivalve species, phagocytosis is associated with increased oxygen consumption (respiratory burst) and the production of reactive oxygen species (ROS), includ ing superoxide anion (O 2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH ) and intermediate compounds with high bactericidal activity [5,6]. The humoral defence reactions of bivalve haemocytes involve mainly the production of agglutinins, opsonins, bactericidins, and cytotoxic substances. It is well known that biotic and abiotic factors may influence haemocyte-dependent defence mechanisms in bivalve molluscs. One of the most important environmental factors involved in bivalve immunomodulation is water temperature [7]. For example, lysosomal and cell membranes of haemocytes from the mussel, Mytilus edulis, appeared destabilised at 0  C with respect to those of haemocytes from mussels acclimated at 10  C [8]. Significant reductions in lysosomal stability were also recorded in haemocytes from the oyster, Ostrea edulis, maintained at 15  C [9]. Important considerations can also be made for seasonal variability (mostly related to natural fluctuations in environmental temperature) in the internal defence of bivalves. Indeed, total haemocyte count (THC) and phagocytic activity of haemocytes from the eastern oyster, Crassostrea virginica, were lower in winter compared to summer [10]. Seasonal changes in free radical metabolism have been reported in many animal species, including fish and invertebrates [11]. Thermal stress is generally characterised by oxidative stress in several marine mollusc species, thus resulting in changes in antioxidant enzyme activity or in malonealdehyde and lipofuscin accumulations [12]. Several studies demonstrate that temperature and salinity modification can affect marine invertebrate defence mechanisms involving the haemocytes that contain hydrolytic enzymes and that produce reactive oxygen species (ROS) implicated  in pathogen degradation [13]. With regards to this, O2 plays an important role in the defence against foreign agents [14,15]. Indeed, molluscan haemocytes, like vertebrate phagocytes [16], respond to stimulation with a respiratory  burst, which produces O2. The oxygen consumed by aerobic organisms generally leads to ROS production, therefore an imbalance of ROS and other pro-oxidant production over antioxidant defences results in increased oxidative damage to unsaturated lipid, DNA and other key molecules [17,18]. The extent of such damage is dependent on the effectiveness of the antioxidant defences [19], including the activity of the antioxidant enzyme superoxide dismutase (SOD; EC 1.15.1.1) [17]. Antioxidant enzyme activity may show strong seasonal variability in molluscs. In the bivalves M. edulis and Macoma balthica from the northern Baltic Sea, seasonal variations of antioxidants catalase (CAT) and glutathione-S-transferase (GST) were related to different phases of the temperature-dependent reproductive cycle of animals [20]. In a recent study, it has been demonstrated that total SOD and CAT activities decreased by 30e40% in hepatopancreas of the snail Helix aspersa during summer-estivation, whereas no changes occurred in the activities of glutathione reductase, GST and glucose-6-phosphate dehydrogenase [11]. In the present study, the effects of high temperatures (20, 25 and 30  C) on some functional responses of haemocytes were evaluated in Chamelea gallina, a commercially important clam species living in sandy bottoms of the Northern Adriatic Sea (Italy). The experimental temperatures were chosen on the basis of the values recorded in the summer period (from June to August) in the Northern Adriatic Sea [21,22]. The occurrence of mortality observed periodically in this area, mostly in early autumn, in the natural beds of C. gallina, led us to investigate how increased temperature can alter immune responses in clams. 2. Materials and methods 2.1. Animals Specimens of C. gallina (about 2.4 cm shell length) were dredged along the west coast of the Northern Adriatic and acclimatised in the laboratory for 5 days before exposure to experimental temperatures. The clams (at least 600 specimens) were maintained in three 75-L aquaria provided with a sandy bottom and aerated seawater (salinity of 35  1&, temperature of 17  0.5  C) and fed with microalgae (Isochrysis galbana).

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2.2. Exposure to experimental temperatures Prior to starting exposure, the clams were acclimatised to experimental conditions by increasing seawater temperature progressively (2  C/day) in the aquaria to 20, 25 and 30  C. The water temperatures in the experimental tanks were then maintained at constant values using electronic thermostats. The clams (at least 200 per experimental condition) were kept for 7 days at the three temperatures, in 75-L aquaria provided with a sandy bottom and well-aerated seawater (salinity of 35  1&, pH 8.2), and fed with microalgae. The seawater was changed every 48 h. Both seawater parameters (temperature, salinity and pH) and clam mortality were checked daily. 2.3. Haemolymph collection For each experimental condition, three pools of haemolymph (from 10 clams each) were used. For immunological indices, haemolymph (about 200 ml per clam) was collected from the anterior adductor muscle with a 1-ml plastic syringe, pooled, and then stored in ice. Haemolymph (500 ml) was immediately used to determine THC, while 300 ml of haemolymph was added to 300 ml of 0.38% sodium citrate (Sigma) in 0.45 mm-filtered sea water (FSW), pH 7.5, to prevent clotting, and then used to prepare short-term haemocyte cultures. Five hundred microlitres of pooled haemolymph (without sodium citrate) was used to measure lysozyme activity in both cell-free haemolymph and haemocyte lysate. For the determination of SOD activity, three additional pools of haemolymph (from 20 clams each) for each experimental condition were used. Haemolymph was centrifuged at 780  g for 10 min. Supernatant was collected, and pelleted cells were re-suspended in distilled water, sonicated with a Braun Labsonic U sonifier at 50% duty cycles for one minute and centrifuged at 12,000  g for 15 min. Enzyme activity was measured both in the haemocyte lysate and in cell-free haemolymph. 2.4. THC and haemocyte volume determination THC and haemocyte volume were determined by a Model Z2 Coulter Counter electronic particle counter/size analyser. Pooled haemolymph (500 ml) was added to 19.5 ml of FSW. THC and haemocyte volume results were expressed as number of haemocytes (106)/ml haemolymph and femtolitre (fl), respectively. 2.5. Haemocyte cultures and phagocytosis assay Short-term haemocyte cultures were prepared according to Ballarin et al. [24]. Haemolymph was centrifuged at 780  g for 10 min and haemocytes were resuspended in FSW at final concentrations of 106 cells/ml. After 30 min, necessary to allow haemocytes to adhere to the slides, FSW was discarded from culture chambers and replaced with equal volumes of a suspension of yeast (Saccharomyces cerevisiae) in FSW (yeast: haemocyte ratio ¼ 10: 1). Monolayers were incubated for 60 min at 25  C, washed several times in FSW to eliminate uningested yeast cells and fixed in a solution of 1% glutaraldehyde (Fluka) and 1% sucrose in FSW at 4  C for 30 min. They were then washed in 0.1 M phosphate buffered saline (PBS: 1.37 M NaCl, 0.03 M KCl, 0.015 M KH2PO4, 0.065 M Na2HPO4), pH 7.2, for 10 min, stained with 10% Giemsa (Fluka) for 5 min, mounted on glass slides in an aqueous medium (Acquovitrex, Carlo Erba) and observed under a Leitz Dialux 22 light microscope at 1250. Two hundred cells per slide were counted, and the phagocytic index was expressed as the percentage of cells containing ingested yeast particles. 2.6. Lysozyme activity assay Lysozyme activity was quantified in both haemocyte lysate and cell-free haemolymph. Pooled haemolymph was centrifuged at 780  g for 10 min. The supernatant, corresponding to cell-free haemolymph, was collected, whereas the haemocytes were resuspended in distilled water, sonicated at 0  C for 1 min, and then centrifuged at 780  g for 30 min to obtain cell lysate (CL). Cell-free haemolymph and CL were frozen and stored at 80  C before analyses. Fifty microlitres of cell-free haemolymph and CL was added to 950 ml of a 0.15% suspension of Micrococcus lysodeikticus (Sigma) in 66 mM phosphate buffer, pH 6.2, and the decrease in absorbance (DA/min) was continuously recorded at 450 nm for 5 min at 20  C. Standard solutions containing 1, 2.5, 5 and 10 mg lysozyme per ml of 66 mM phosphate buffer, pH 6.2, were prepared from crystalline hen egg white lysozyme (Sigma). The average decrease in

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absorbance per minute was determined for each enzyme solution, and a standard curve of enzyme concentration versus DA/min was drawn. One unit of lysozyme was defined as the amount of enzyme producing activity equivalent to 1 mg of lysozyme, in the conditions described above. Results are expressed as mg lysozyme/mg protein. Protein concentrations were quantified according to [25]. 2.7. Superoxide dismutase activity assay The reagents utilised for SOD activity analysis were from Sigma Aldrich S.r.l., Roche Diagnostics and Merck & Co. SOD activity was measured in quadruplicate in a UV/Vis Beckman DU 530 spectrophotometer, in temperaturecontrolled conditions (25  1  C). SOD activity was determined with the xanthine oxidaseecytochrome c method according to Crapo et al. [26]. The cytochrome c reduction by superoxide anions generated by xanthine oxidase/ hypoxanthine reaction was detected at 550 nm. Activity values were expressed in units (U) per mg of protein, 1 U of SOD being defined as the amount of sample producing 50% inhibition under the assay conditions. The final reaction mixture was 46.5 mM KH2PO4/K2HPO4 (pH 8.6), 0.1 mM EDTA, 195 mM hypoxanthine, 16 mM cytochrome c, 2.5 mU xanthine oxidase and 20 mM KCN. The Cu/Zn-containing form of SOD was assayed by the inhibitory effect of 1 mM of KCN on SOD activity [27]. Protein concentrations were quantified according to Bradford [25]. 2.8. Electrophoresis and Western blotting For electrophoresis and Western blotting, minigels, specific buffers, molecular weight markers and chromogenic immunodetection kits were purchased from Invitrogen. Polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad, the primary antibodies were obtained from Stressgen and the Cu/Zn-SOD standard (from bovine erythrocytes) from Sigma Aldrich S.r.l. SDSePAGE of haemocyte lysate and cell-free haemolymph samples were performed in an Invitrogen Xcell SureLock Mini-Cell using minigels (Bis-Tris gels) consisting of 12% running gel with MES [2-(N-morpholino) ethanesulfonic acid] running buffer at pH 7.3, under reducing conditions. Samples were added to LDS (lithium dodecyl sulphate) buffer and reducing agent (0.5 M DTT) and heated for 10 min at 70  C. Aliquots of partially purified Mn-SOD (obtained from the bivalve mollusc Scapharca inaequivalvis after specific extraction of digestive gland samples and followed by gel chromatography using a Sephadex G-75 column, 1  90 cm) and Cu/Zn-SOD standard (from Sigma Aldrich S.r.l.) were loaded as positive controls in gels to identify the two SOD isoforms. Samples were loaded into the gel in volumes corresponding to the protein content of 5 mg and 20.5 mg for haemocyte lysate and cell-free haemolymph, respectively. After electrophoresis, proteins were transferred, for 1 h, to PVDF membranes in an Invitrogen Xcell SureLock Blot Modul using transfer buffer pH 7.2. After blotting, the PVDF membranes were treated with a chromogenic Western blot immunodetection kit. Blots were blocked with concentrated casein solution in buffered saline solution and then incubated with the diluted primary specific antibody for 1 h. Polyclonal rabbit anti-human Cu/Zn-SOD (diluted 1:10,000), and polyclonal rabbit anti-rat Mn-SOD (diluted 1:7000) were used to identify the SOD isoforms. The membranes were washed with a specific antibody wash solution (buffer saline solution containing detergent from immunodetection kit) and incubated with the secondary antibody solution consisting of alkaline phosphatase-conjugated anti-rabbit IgG for 45 min. Then the blots were visualised using a chromogenic substrate containing 5-bromo-4chloro-3-indolyl-1-phosphate (BCIP) and nitro-blue tetrazolium (NBT). Cross-reactivity with the mammalian polyclonal antibodies against Cu/Zn-SOD and with antibodies versus Mn-SOD from molluscs was demonstrated. The SOD expression was indicated by immunoreactive bands, whose apparent molecular weights were determined by comparison with molecular weight markers. Immunopositive bands were semi-quantified by Quantity One Software (Bio-Rad) and results were expressed in arbitrary units. 2.9. Survival in air At the end of the experiment, 30 clams from each experimental temperature were kept in closed plastic boxes in humidity-saturated conditions, at a constant temperature of 17  C, and mortality was recorded daily. Animals were considered dead when their shells gaped and did not shut again after external stimulus. LT50 values were determined.

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2.10. Statistical analysis For THC, phagocytic index and lysozyme activity, data were checked for normal distribution (ShapiroeWilk test) and homogeneity of variances (Bartlett’s test). Results were compared using a one-way ANOVA, followed by a post hoc test (Duncan’s test). SOD activity values and data from densitometric analyses of immunopositive bands were compared by Student’s t-test for independent samples. The results are expressed as means  standard error. In the survival-in-air test, LT50 values were determined according to Kaplan and Meier [28], and the significance of differences between groups was tested using the Gehan and Wilcoxon test [29]. The STATISTICA 5.5 (StatSoft, Tulsa, OK, USA) software package was used for statistical analyses. 3. Results No relevant variations in water pH or salinity values were recorded during the acclimation period and the exposure to the experimental temperatures. Higher mortality was observed in clams kept at 30  C (48%), with respect to the animals at 20  C (2%) and 25  C (3.2%). Moreover, different burrowing behaviour of clams was recorded: animals kept at 20 and 25  C burrowed completely, whereas at 30  C clams progressively emerged from the sediment and then remained on the surface. 3.1. THC and haemocyte volume Exposure of clams to 30  C significantly increased THC (8.9x106 cells/ml), with respect to animals held at 20  C (6.4  106 cells/ml, P < 0.05) and 25  C (5.1  106 cells/ml, P < 0.001) (Fig. 1). No significant differences in THC values were observed between clams kept at 20  C and those at 25  C. Analysis of haemocyte size frequency distributions highlighted that in clams kept at 30  C the cell population of about 8e10 mm (corresponding to 400e500 fl) was mostly affected by temperature, and its presence was greatly lowered (Fig. 2). 3.2. Phagocytosis and lysozyme activity Phagocytic activity significantly decreased in clams held at 30  C with respect to those at 20  C and 25  C (P < 0.01 and P < 0.05, respectively) (Fig. 3). No difference was found between clams kept at 20  C and those at 25  C. Interestingly, a great number of bacteria was detected in haemocyte cultures of animals kept at 30  C (Fig. 4). Lysozyme activity in haemocyte lysate was significantly increased in clams maintained at 25  C with respect to that of bivalves kept at 20  C and 30  C (P < 0.01 and P < 0.05, respectively) (Fig. 5A). Conversely, the lysozyme 20 °C

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temperature (°C) Fig. 1. Effects of temperature on THC, expressed as number of haemocytes (106)/ml haemolymph, in C. gallina. Asterisks: significant difference. Values are means  S.E.M.; n ¼ 3. *P < 0.05; ***P < 0.001. Inset: significance of comparisons between experimental groups.

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Fig. 2. Effects of temperature on haemocyte size frequency distribution in C. gallina. (AeC) Haemocyte diameter, expressed in mm. (DeF) Haemocyte volume, expressed in femtolitres (fl).

activity in cell-free haemolymph of clams kept at 25  C was significantly lower with respect to that of clams maintained at 20  C (P < 0.01) and 30  C (P < 0.001) (Fig. 5B). 3.3. Superoxide dismutase activity Total SOD activity in haemocyte lysate ranged from 72 to 32.14 U/mg protein, the highest value being recorded in clams maintained at 25  C (Fig. 6). No significant difference was observed between clams at 20 and those at 25  C, 35

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temperature (°C) Fig. 3. Effects of temperature on phagocytic activity, expressed as percentage of haemocytes containing ingested yeast particles, in C. gallina. Asterisks: significant difference. Values are means  S.E.M.; n ¼ 3. *P < 0.05; **P < 0.01. Inset: significance of comparisons between experimental groups.

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Fig. 4. Haemocytes of C. gallina held at 20  C and 25  C, containing phagocytosed yeast cells (arrows); haemocytes from clams held at 30  C, showing numerous bacteria surrounding haemocytes (arrows). Scale bar: 3 mm.

whereas 30  C exposure strongly affected SOD activity, which showed a dramatic (P < 0.01) decrease (32.14 U/mg protein) with respect to the other experimental temperatures. SOD isoforms, individually analysed, displayed differing behaviour in response to the experimental temperatures. In particular, Mn-SOD activity (between 49.18 and 20.47 U/mg protein) showed a steady decrease with increasing temperature (Fig. 6), the lowest value observed in 20 °C

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temperature (°C) Fig. 5. Effects of temperature on lysozyme activity, expressed as mg lysozyme/mg protein, in haemocyte-lysate (A) and cell-free haemolymph (B) of C. gallina. Asterisks: significant difference. Values are means  S.E.M.; n ¼ 3. *P < 0.05, **P < 0.01, ***P < 0.001. Inset: significance of comparisons between experimental groups.

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Fig. 6. Superoxide dismutase activity in haemocytes of C. gallina held at experimental temperatures (20, 25, 30  C). Data are presented as mean  S.E.M. (n ¼ 10). The same letters at the top of the bars indicate significant differences: aee ¼ P < 0.01.

haemocytes from clams kept at 30  C, being significantly different than those at 20 and 25  C (P < 0.01). Cu/Zn-SOD activity ranged between 31.57 and 11.67 U/mg protein (Fig. 6). The highest value was observed in haemocytes of clams kept at 25  C and this was significantly different (P < 0.01) from the data recorded at 30  C. The values of total SOD (and isoforms) determined in cell-free haemolymph are shown in Fig. 7. Total SOD activity ranged from 9.92 to 16.10 U/mg protein, showing a significant (P < 0.01) increase in clams maintained at 25 and 30  C with respect to those at 20  C (þ62% and þ50%, respectively). The Mn-SOD activity ranged between 5.84 and 11.33 U/mg protein, the highest value being recorded at 30  C, with a significant increase (in the order of þ94%) in comparison with clams at 20 and 25  C (Fig. 7). On the other hand, no significant increase in Cu/Zn-SOD activity was observed in clams maintained at 25  C with respect to those at 20  C (þ145%), or those kept at 30  C (þ183%) (Fig. 7). 3.4. Superoxide dismutase expression In order to identify SOD isoforms and to evaluate their expression, we performed electrophoretic separation of haemocyte and cell-free haemolymph proteins and immunoblotting. (Figs. 8e11). After Western blotting, Mn-SOD in haemocyte lysate showed an immunoreactive band with an apparent molecular weight of 26 kDa, corresponding to the enzyme monomeric form (Fig. 8A). Values recorded in haemocytes of bivalves exposed to 25 and 30  C were significantly different from those recorded in clams kept at 20  C (P < 0.05 and 20

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Fig. 7. Superoxide dismutase activity in haemolymph of C. gallina held at experimental temperatures (20, 25, 30  C). Data are presented as mean  S.E.M. (n ¼ 9). The same letters at the top of the bars indicate significant differences: aec ¼ P < 0.01; d ¼ P < 0.05.

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Fig. 8. (A) Immunoblotting analyses of Mn-SOD in haemocytes of C. gallina. Lane 1: molecular weight marker; lane 2: Mn-SOD standard; lanes 3e5: 25  C; lanes 6e8: 20  C; lanes 9, 10: 30  C. (B) Densitometric analyses of blot expressed in arbitrary units (Mn-SOD expression in haemocytes samples). Data are presented as mean  S.E.M. The same letters at the top of the bars indicate significant differences: a ¼ P < 0.05; b ¼ P < 0.01.

P < 0.01 respectively); no significant difference was revealed between values found in haemocytes of clams kept at 25 and those kept at 30  C (Fig. 8A,B). From Cu/Zn-SOD expression analysis two immunoreactive bands were obtained, the former showing an apparent molecular weight of 15e16 kDa, and the latter of 29e30 kDa (Fig. 9AeC). Densitometric measures of the 15e 16 kDa band showed only statistically significant differences (P < 0.05) among animals kept at 20 and 30  C (Fig. 9A,B). Concerning expression of the heavier band, values were similar in clams kept at 20 and 25  C, but they were significantly higher (P < 0.05) with respect to that of the animals maintained at 30  C (Fig. 9A,C). After immunoblotting of cell-free haemolymph samples, Mn-SOD showed an immunoreactive band (apparent molecular weight of 26 kDa) similarly expressed in clams at 20 and 25  C, and a significantly higher (P < 0.01) expression peak in clams maintained at 30  C (Fig. 10A,B). As in haemocytes, two different bands (apparent molecular weight 15e16 and 29e30 kDa, respectively) were identified by the antibody versus Cu/Zn-SOD (Fig. 11AeC). A significant decrease in expression (P < 0.01) of the first band (16 kDa) (Fig. 11A,B) was detected in clams at 20 and 25  C (51.15 and 72.29 A.U., respectively) with respect to clams maintained at 30  C (152 A.U.). Conversely, the expression of the second band (31e32 kDa) showed different values in cell-free haemolymph between the clams at 20 and 25  C (P < 0.05), which were, moreover, significantly higher (P < 0.01) than those recorded in the animals kept at 30  C (Fig. 11A,C). 3.5. Survival in air The resistance to air exposure of clams kept at 30  C decreased significantly (P < 0.001) with respect to that of animals held at 20  C and 25  C (Fig. 12). LT50 values fell from 6 days in clams kept at 20  C and 25  C to 4 days in those kept at 30  C. 4. Discussion Numerous studies have demonstrated that defence mechanisms in bivalves are susceptible to changes in environmental factors, such as temperature, salinity, dissolved oxygen, food availability and presence of pollutants

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Fig. 9. (A) Immunoblotting analyses of Cu/Zn-SOD in haemocytes of C. gallina. Lane 1: molecular weight marker; lane 2: Cu/Zn-SOD standard, lanes 3e5: 25  C; lanes 6e8: 20  C; lanes 9, 10: 30  C. (B) Densitometric analyses of blot expressed in arbitrary units (Cu/Zn-SOD expression in haemocyte samples). Data are presented as mean  S.E.M. The same letters at the top of the bars indicate significant differences, a ¼ P < 0.05. (C) Densitometric analyses of blot expressed in arbitrary units (EC-SOD expression in haemocyte samples). Data are presented as mean  S.E.M. The same letters at the top of the bars indicate significant differences: a,b ¼ P < 0.05.

[8,13,30,31,32]. In particular, increases in temperature have been shown to induce mortality in bivalves, probably owing to temperature-induced immunosuppression in these animals [33,34]. In the present study, we investigated the effects of high temperatures on immune responses in the commercially important clam species, C. gallina. The results obtained demonstrated that C. gallina is very sensitive to high temperature, as suggested by alterations in the cell parameters analysed.

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Fig. 10. (A) Immunoblotting analyses of Mn-SOD in haemolymph of C. gallina. Lane 1: molecular weight marker; lane 2: Mn-SOD standard; lanes 3e5: 25  C; lanes 6e8: 20  C; lanes 9, 10: 30  C. (B) Densitometric analyses of blot expressed in arbitrary units (Mn-SOD expression in haemolymph samples). Data are presented as mean  S.E.M. The same letters at the top of the bars indicate significant differences: a,b ¼ P < 0.01.

4.1. THC and haemocyte volume In C. gallina, THC was significantly increased at 30  C. Likewise, significantly higher THC values were found in clams (R. philippinarum) incubated at the highest temperature (21  C) tested [35]. High water temperature has been reported to increase THC also in crustaceans [36]. The molluscan circulatory system is open and haemocytes are distributed in both the vascular system and tissues. It is assumed that increases in THC values can result from either proliferation or movement of cells from tissues into haemolymph, whereas decreases may be due to cell lysis or increased movement of cells from haemolymph to tissues [1]. In the present study, the increased THC recorded in clams maintained at 30  C was presumably a consequence of mobilisation of haemocytes from tissues to haemolymph, in order to respond to the high number of bacteria in the haemolymph. Indeed, a great number of bacteria surrounding haemocytes were observed in haemocyte cultures from 30  C-exposed animals. Analogously, infestation of M. galloprovincialis by the protistan, Marteilia refringens, caused a significant increase of haemocytes in the haemolymph [37]. Oubella et al. [2] suggested that the elevated number of circulating cells of the clam, R. philippinarum, challenged with Vibrio tapetis, resulted from mobilisation and migration of haemocytes from tissues to the haemolymph. Haemocyte migration toward bacterial stimuli has also been demonstrated in vitro in the hard clam, Mercenaria mercenaria [38]. Moreover, seasonal variability (mainly related to changes in water temperature) in circulating haemocyte number was detected in previous studies. Indeed, in M. galloprovincialis, haemocyte number was positively correlated with water temperature, the lowest values being found in winter and the highest in summer [37]. In contrast, THC in C. virginica was lowest in July and August, when the highest water temperatures were recorded [39]. In the present study, analysis of haemocyte size frequency distributions highlighted that in clams kept at 30  C the cell population mostly affected by high temperature was that of about 8e10 mm in diameter. These results are very difficult to explain, mainly due to the few data available in the literature about this aspect. However, apoptosis resulting in cell volume reduction (cell shrinkage is considered to be one of the most peculiar aspect of apoptosis) in clams held at 30  C cannot be excluded. Indeed, the fraction of smaller haemocytes (from 50 to 200 fl) markedly increased, whereas that of larger ones (about 400 fl) considerably decreased. In a recent study, significantly high percentages of dead haemocytes were found in oysters after temperature increase, suggesting a reduced immunosurveillance in stressed animals [40]. However, the relationship between high temperature and apoptotic events in bivalve haemocytes needs to be more fully evaluated in future studies.

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A 32 kDa -

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2

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Fig. 11. (A) Immunoblotting analyses of Cu/Zn-SOD in haemolymph of C. gallina. Lane 1: molecular weight marker; lane 2: Cu/Zn-SOD standard; lanes 3e5: 25  C; lanes 6e8: 20  C; lanes 9, 10: 30  C. (B) Densitometric analyses of blot expressed in arbitrary units (Cu/ZnSOD expression in haemolymph samples). Data are presented as mean  S.E.M. The same letters at the top of the bars indicate significant differences: a,b ¼ P < 0.01. (C) Densitometric analyses of blot expressed in arbitrary units (EC-SOD expression in haemolymph samples). Data are presented as mean  S.E.M. The same letters at the top of the bars indicate significant differences: a ¼ P < 0.05; b,c ¼ P < 0.01.

4.2. Phagocytosis and lysozyme activity Phagocytosis is the main mechanism against non-self materials in bivalve internal defence. It has been reported that phagocytosis is a temperature-dependent process in bivalve molluscs [41]. In the present study, significant inhibition of phagocytic activity was found in clams kept at 30  C. Similarly, phagocytosis decreased significantly in C. virginica subjected to a sudden increase in temperature from 20 to 28  C for 1 week [40]. Conversely, Carballal et al. [42] found that the percentage of phagocytic haemocytes from M. galloprovincialis was lower at 10  C than at 20  C and 30  C.

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20 °C

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60

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0 0

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days Fig. 12. Survival in air response of C. gallina, expressed as percentage of clams surviving, after 7 days at 20, 25 and 30  C. Asterisks: significant difference. Gehan and Wilcoxon test: ***P < 0.001. LT50 values (in days) are reported. Inset: significance of comparisons between experimental groups.

Ordas et al. [43] observed no differences between phagocytosis at 15 and 21  C in both the clam, R. decussatus, and the mussel, M. galloprovincialis. In the present study, increased THC did not correspond to an increase in phagocytic activity in animals kept at 30  C. Presumably, as proposed by Alvarez et al. [44] and Chu and La Peyre [45], temperatures above a certain threshold may result in stress conditions for haemocytes, so that they are less responsive. To support this hypothesis, it is important to highlight that temperature enhanced haemocyte phagocytic activity in eastern oysters (C. virginica) held at 20  C for 68 days compared to those held at 10  C, but activity declined in oysters held at 25  C [23]. Moreover, other physiological responses may be affected by temperature over threshold values: temperatures below 10  C and over 30  C cause a dramatic reduction in the filtering capacities of C. gallina and in its metabolic activities [46]. Temperature also affects lysozyme activity in C. gallina. Lysozyme is the main bacteriolytic agent against several species of Gram-positive and Gram-negative bacteria. It is synthesised in bivalve haemocytes and then secreted into haemolymph during phagocytosis [47]. In the present study, significantly increased lysozyme activity was observed in haemocyte lysate from clams kept at 25  C, and in cell-free haemolymph from animals held at 20 and 30  C. From this, we can infer that 25  C is an optimal temperature for the expression of lysozyme inside haemocytes. Moreover, a relationship between lysozyme activity in haemocyte lysate and cell-free haemolymph was observed at 20  C, enzyme activity being significantly reduced in haemocytes and increased in cell-free haemolymph. This may be due to the high phagocytic activity of clams held at 20  C. Therefore, increased lysozyme secretion from haemocytes into haemolymph probably occurred as a consequence of higher phagocytosis. Interestingly, animals maintained at 30  C also showed increased cell-free haemolymph lysozyme activity, even if their phagocytic activity was low. Clams kept at 30  C might have been induced to release lysozyme in order to respond to the high number of bacteria in the haemolymph. 4.3. SOD activity and expression Results of our study showed that in haemocytes both Mn-SOD and Cu/Zn-SOD had the lowest activity values at 30  C. In mammalian phagocytic cells, the oxygen-dependent defence mechanisms consist of ROS generation with  powerful microbicidal activity [48], and this has also been demonstrated in bivalves [49,50]. In the present study, O2 generation was not directly determined. In several tissues of giant clams (Tridacna), a positive relationship between tissue-specific SOD activity and index of superoxide production was demonstrated [51,52]. We can then hypothesise  that high temperature leads to a decrease in O2 production by clam haemocytes, and consequently to a decrease in both Cu/Zn-SOD and Mn-SOD activities.

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Mn-SOD is specifically located in the mitochondria, which should allow adaptation of cell oxidative metabolism [53,54]. Moreover, the Mn-SOD is expressed in many cell types and tissues at relatively high levels and it is also highly regulated by a variety of intracellular and environmental cues, such as cytokines [55,56]. The expression of Mn-SOD, as measured in immunoblots, by haemocytes was coherent with the enzyme activity, exhibiting a continuous decrease with rising temperature. In the present study, Cu/Zn-SOD expression by haemocytes showed two immunoreactive bands. During the experiment, Cu/Zn-SOD expression was partially coherent with the enzyme activity trend, in fact expression values did not show significant variations between 20 and 25  C, while the expression recorded at 30  C was significantly lower than at 20  C exposure. It seems that Cu/Zn-SOD expression is stable and its activity is considered to be an internal control for SOD1 gene expression in mammals [55]. Indeed, several authors, despite considering this enzyme to be constitutively expressed, have stated that its mRNA levels can be dramatically up- and down-regulated by various physiological conditions [55]. Several stimuli, such as heat shock, UVB, nitric oxide, may be responsible for an up-regulation of Cu/ZnSOD mRNA levels [55]. On the contrary, the results of the present study, show a decrease of Cu/Zn-SOD expression in haemocytes at 30  C with respect to 20  C. After Cu/Zn-SOD immunoblotting, the second band detected showed an apparent molecular weight of 28e30 kDa. From our data, it is not possible to know with certainty whether this band represents a dimer of the cytosolic Cu/Zn-SOD not completely dissociated, or if it corresponds to an extracellular (EC)-SOD monomer; this enzyme in humans is a homotetramer with an apparent molecular weight of 135 kDa [57] and has been found also in invertebrates [58e60]. In our research, EC-SOD like- protein from haemocyte lysate was significantly less expressed at 30  C with respect to 20 and 25  C. In mammals EC-SOD plays a crucial role in working as a scavenger of superoxide anions in the extracellular environment and it is present in extracellular fluids, including plasma and extracellular matrix of tissues [61]. Since EC-SOD contains a heparin-binding tail in the carboxy-terminal region of each of its subunits, most of the enzyme is mainly associated to heparin sulphate proteoglycans of the cell coat [57,61]. It has been proposed that EC-SOD functions by preventing superoxide-mediated inactivation of NO [62]. This is an important intercellular signalling molecule that shows diverse functions in mammalian tissues, including modulation of the vascular tone as well as antiviral and antimicrobial activities. There is evidence that invertebrate haemocytes are able to produce NO, which apparently exerts the same functions [63,64]. Ottaviani et al. [65] had previously suggested a bactericidal activity in invertebrate immunocytes. More difficult is the discussion of data obtained for cell-free haemolymph. Activity values were markedly lower than those of haemocyte lysate, and had a different trend. In fact, total SOD activity values were higher at 25 and 30  C compared to those at 20  C. Moreover, Mn-SOD increased strongly at 30  C exposure while Cu/Zn-SOD did not present statistically significant differences between the experimental groups. Cell-free haemolymph immunoblotting at 30  C showed a strong increase in Cu/Zn-SOD and Mn-SOD expression and a clear decrease in EC-SOD like-protein expression. Uchimura et al. [66] in a study about massive mortality in Japanese pearl oysters found that in the cell extract of low mortality groups, SOD activity (97.7 U/ml) was higher (38.8 U/ml) than in high mortality groups (28.8%). At the same time, DNA damage and oxidative stress products were lower in low mortality groups. Therefore, oysters presenting high SOD activity were better protected against oxidative stress [66]. DNA alterations and malondialdehyde (MDA) presence showed that negative effects of oxyradicals, which determined the destruction of circulatory organs and the cytolysis of haemocytes, were found only in high mortality oysters. Such damage to the haemocyte membrane would explain SOD activity (low but significant) and expression in the haemolymph. Mn-SOD expression, coherent with activity values, might confirm this assumption, even if the EC-SOD expression in the extracellular space was lower with respect to the cytosolic Cu/Zn-SOD. Low levels of EC-SOD like-protein and high expressions of Mn-SOD and Cu/Zn-SOD might indicate high amounts of NO and superoxide anions. Low expression of EC-SOD like-protein in the extracellular matrix could be related to a low defence capability in C. gallina after thermal stress. Further studies may be necessary to define and clarify the role of antioxidant enzyme in the mollusc’s ability to defend itself against pathogen infections. 4.4. Survival in air The survival-in-air test is considered to be one of the simplest, most feasible, sensitive, reproducible and costeffective methods for evaluating the negative effects of environmental stress in euryoxic bivalves [67,68]. In the present study, clams held at 30  C showed reduced tolerance to aerial exposure. LT50 values fell from 6 days in animals

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kept at 20 and 25  C to 4 days in those maintained at 30  C. This indicates that temperatures above 25  C are crucial values and can markedly influence physiological performance of C. gallina, thus confirming the results obtained for the cellular responses here investigated. Critical conditions for clam survival at 30  C are also shown by high mortality values during treatment, and by differing burrowing behaviour of clams that emerged from sediment and then remained on the surface. This observation suggests that in the field elevated temperatures induce the same behaviour, thus rendering stressed clams more vulnerable to predators. Similarly, Blanchet et al. [69] found that in the edible cockle, Cerastoderma edule, mortality rates were significantly correlated with the mean water temperature, and four times higher for unburied cockles (42%) than for buried individuals (10%). 5. Conclusions The functional responses of haemocytes evaluated in this work highlight the strong influence of high temperature on immune responses in C. gallina. In particular, at 30  Cda temperature often recorded on clam beds during summerdimmunosuppression may reduce clams’ resistance to other environmental and endogenous stressors, and enhance susceptibility to pathogens and predators. A general impairment of clam conditions at temperatures higher than 25  C is confirmed in field studies by other physiological measurements, such as scope for growth and condition index [70]. Over 25  C the homeostatic capabilities of clams appear to be heavily compromised. However, clam responsiveness to high temperature in laboratory experiments may be influenced by environmental and endogenous conditions previously experienced by the animals in the field. With regard to this, we have recently found reduced mortality during treatments for 7 days at 30  C (28%) and at 28  C (13%), when using clams collected in March, far from the reproductive phase and the warmest period in the year (unpublished data). As the present study was performed in early autumn, we can hypothesise that clams naturally more stressed, due to summer high temperature and reproductive effort, were used. Therefore, further studies will be carried out far from this period of the year, when clams exhibit higher tolerance to additional stress. Acknowledgements This work was supported by grants from the Italian MiPAF to O.C. (contr. no. 6C66). References [1] Pipe RK, Coles JA. Environmental contaminants influencing immune function in marine bivalve molluscs. Fish and Shellfish Immunology 1995;5:581e95. [2] Oubella R, Paillard C, Maes P, Auffret M. Changes in hemolymph parameters in the manila clam Ruditapes philippinarum (Mollusca, Bivalvia) following bacterial challenge. 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