3 ISJ 6: 44-48, 2009 ISSN 1824-307X SHORT COMMUNICATION Seasonal changes in functional parameters of the hemolymph of Mytilus galloprovincialis C Ciaccia, R Fabbrib, M Bettia, P Rochc, L Canesib aDISUAN, Dipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Urbino, Italy bDipartimento di Biologia, Universita` di Genova, Italy cJRU Ecosystèmes Lagunaires, CNRS-Université de Montpellier 2-IFREMER, France Accepted April 7, 2009 Abstract In bivalves, many functional parameters show seasonal changes in relation to both abiotic (such as temperature and salinity) and biotic factors (such as gonad maturation, food availability). Available data indicate that also immune parameters can show seasonal fluctuations in the marine mussel Mytilus spp.. In this work we report data on hemocyte lysosomal membrane stability (LMS) and phagocytic activity, as well as on soluble lysozyme activity, in the hemolymph of mussels (Mytilus galloprovincialis) collected over a 24 month period in the Adriatic Sea (2006-2007). The results indicate that all the parameters measured show seasonal fluctuations over the year, with lysozyme activity showing the largest changes. Lowest LMS values were observed in early winter and early autumn, whereas maximal values of phagocytic activity were observed in winter and increasing serum lysozyme activities were recorded in autumn. The observed seasonal fluctuations are discussed in relation to both abiotic (temperature) and biotic (changes in endogenous modulators) factors. Key Words: Mytilus; hemocytes; lysosomal membrane stability; phagocytosis; lysozyme; immune parameters; seasonal variation Introduction Bivalves (such are mussels, clams and oysters) possess both cellular and humoral defence mechanisms that co-operate to kill and eliminate invading bacteria (Mitta et al., 2000; Canesi et al., 2002). Hemocytes are responsible for cell-mediated immunity through phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes and antimicrobial peptides, and the production of oxygen metabolites (Mitta et al., 2000, Canesi et al., 2002). In the recent years, we have investigated the immune responses of Mytilus galloprovincialis to different stimuli, from bacterial challenge to exposure to endogenous modulators and heterologous cytokines, in both in vitro and in vivo studies (Canesi et al., 2001, 2003, 2004, 2005, 2006a, b; Betti et al., 2006). Although a number of assays can be utilized in order to evaluate the immune function in invertebrates (Ballarin et al., 2008), the phagocytic activity is generally considered ______________________________________________________________________ Corresponding author: Laura Canesi Department of Biology University of Genoa Corso Europa 26, 16132, Genoa, Italy E-mail: Laura.Canesi@unige.it as one of the most important parameters, especially in the bivalve Mytilus spp. where active phagocytes represent the main circulating cell type (Carballal et al., 1998; Ottaviani et al., 1998; Wottoon et al., 2003). However, another hemocyte parameter, lysosomal membrane stability, evaluated in live hemocytes by the Neutral Red Retention time (NRR) assay, has emerged as an extremely sensitive indicator not only of cellular stress due to environmental perturbations (Lowe et al., 1995), but also of the functional status of immunocytes (Hauton et al., 2001; Pruzzo et al., 2005). In fact, the majority of Mytilus hemocytes are endowed with an extremely developed lysosomal vacuolar system which is involved not only in digestion of engulfed foreign particles, but that also contains a number of hydrolases that are secreted for extracellular degradation of components from invading microorganisms, such as bacterial cell wall (Canesi et al., 2002). In addition, lysosomal production of oxygen radicals has been demonstrated (Winston et al., 1996). In mussels, LMS response to a variety of extracellular stimuli has been long widely investigated in a number of studies so that recorded changes can be related to a different functional status of the cell; in particular, moderate lysosomal   44 mailto:Laura.Canesi@unige.it destabilisation indicates activation of lysosomal membrane fusion processes related to endo/exocytosis, whereas larger decreases in LMS correspond to increasing cellular stress conditions that can lead to irreversible cellular damage and autophagy (Moore et al., 2006). Changes in both LMS and phagocytosis are induced by immune stimuli, like bacterial challenge or exposure to cytokines (Canesi et al., 2001, 2003; 2005; Betti et al., 2006). Moreover, we have identified the natural estrogen 17β-estradiol as an endogenous modulator of these parameters, as well as of lysosomal enzyme release and oxyradical production (Canesi et al., 2004, 2006b). In bivalves, many functional parameters show seasonal changes in relation to both abiotic (such as temperature and salinity) and biotic factors (such as gonad maturation or food availability). These factors may affect also the immune function. In Mytilus spp., only a few studies have been focused on seasonal variations in immune parameters (Carballal et al., 1997, 1998; Malagoli et al., 2006, 2007; Novas et al., 2007). In this work, we report data on hemocyte LMS and phagocytic activity, as well as on soluble lysozyme activity in mussels, Mytilus galloprovincialis, collected over a 24 month period in the Adriatic Sea in 2006-2007. Materials and Methods Chemicals All reagents were of analytical grade and were purchased by Sigma (St. Louis, MO). Animals Specimens of the bivalve mollusc Mytilus galloprovincialis, collected in the Cesenatico area (RN, Italy) were purchased monthly from a local fishing company (SEA, Gabicce Mare, PU) for two years (from January to December 2006 and 2007). Analysis were carried out on individuals of 4-5 cm size. After collection, animals (30 mussels) were taken immediately to the laboratory where they were kept in an aquarium for 24 h in static tanks containing aerated artificial sea water (ASW) (1 l/mussel), 36 % PSU, at different temperatures (from 15 to 20 °C, depending on the sampling period to minimize the effect of laboratory conditions). Hemolymph was sampled from the posterior adductor muscle using a sterile 1 ml syringe with an 18 G1/2” needle. With the needle removed, hemolymph was filtered through sterile gauze and pooled in 50 ml Falcon tubes at 18°C. Hemolymph samples from 8-10 mussels were pooled and utilised for subsequent analyses. Hemolymph serum was obtained by centrifugation of whole hemolymph at 200xg and the supernatant was sterilised through a 0.22 µm pore size filter. All analyses were performed in quadruplicate. Lysosomal membrane stability (LMS) LMS was evaluated by the NRR (Neutral Red Retention time) assay as previously described (Canesi et al., 2005) according to Lowe et al. (1995). Hemocyte monolayers on glass slides were incubated with 30 µl of a neutral red (NR) solution (final concentration 40 mg/ml from a stock solution of NR 40 µg/ml in DMSO); after 15 min excess dye was washed out, 30 µl of ASW was added, and slides were sealed with a coverslip. Every 15 min, slides were examined under an optical microscope and the percentage of cells showing loss of the dye from lysosomes in each field was evaluated. For each time point 10 fields were randomly observed, each containing 8-10 cells. The end point of the assay was defined as the time at which 50 % of the cells showed sign of lysosomal leaking (the cytosol becoming red and the cells rounded). All incubations were carried out at 18 °C. Phagocytosis assay Phagocytosis of Neutral Red-stained zymosan was used to assess the phagocytic ability of hemocytes as previously described (Canesi et al., 2006b) according to Pipe et al. (1995). Neutral Red- stained zymosan in 0.05M Tris-HCl buffer (TBS), pH 7.8, containing 2 % NaCl was added to each monolayer at a concentration of about 1:50 hemocytes:zymosan diluted in ASW, and allowed to incubate for 30 and 60 min at 18 °C. Monolayers were then washed three times with TBS, fixed with Baker’s formol calcium (4 %, v/v, formaldehyde, 2 % NaCl, 1 % calcium acetate) for 30 min and mounted in Kaiser’s medium for microscopical examination with a Vanox (Olympus Italy 1.2.1, MI) optical microscope. For each slide, the percentage of phagocytic hemocytes was calculated from a minimum of 200 cells. Data are expressed as % of phagocytizing cells. Serum lysozyme activity Lysozyme activity in aliquots of serum was determined as previously described (Pruzzo et al., 2005) following Chu and La Peyre (1989). Briefly, lysozyme activity was determined as the ability to lyse a standard suspension of M. lysodeikticus (15 mg/100 ml in 66 mM phosphate buffer, pH 6.4) and measured as decrease in absorbance at 450 nm at room temperature. Hen egg-white (HEW) lysozyme was used to construct a standard curve and lysozyme activity was expressed as HEW lysozyme equivalents/mg protein/ml. Protein content was determined according to the Lowry method using bovine serum albumin (BSA) as a standard. Data analysis Results are presented as the arithmetical mean ± SD of experiments carried out in quadruplicate. Statistical analysis was performed by using the Mann-Whitney U-test with significance at p<0.05. Results and Discussion Mussels were sampled for 24 months during 2006 and 2007. Since no significant differences in the results obtained were recorded between the two years, only data from Jan-Dec 2007 are reported in Fig. 1. Figure 1A shows the results obtained for hemocyte LMS: mean annual values of NR Retention times were 117.75 ± 13.6 min. Lowest LMS (about 100 min) were recorded in winter (Jan- Feb) and early autumn (Sept-Oct). With respect to these values, significantly higher LMS were   45 recorded in spring, with a maximum in May (+39 %; p<0.05) and December + 31 %; p < 0.05). However, both minima and maxima did not significantly differ from mean annual values. Data on hemocyte phagocytic activity are reported in Fig. 1B as % of phagocytosing cells. Mean values (%) were 53.87 ± 5.55. Higher values were observed in winter, with a maximum in January (64 %), followed by a slow decrease in spring-early summer, that reached lowest values in June and September (-27 % and 26 %, respectively, with respect to January; p<0.05). However, as observed for LMS data, also for phagocytosis neither minima nor maxima were significantly different from mean annual values. In Figure 1C data on soluble lysozyme activity are reported. Mean annual values were 147.25 ± 68 mU/mg protein. In this case, larger seasonal differences were observed, with lower values in late winter-early spring (less than 100 mU/mg protein from February to April, with a minimum in March of 84±15 mU/mg protein.). A large, progressive rise was observed from late summer to autumn (up to a +150 % increase in October and November with respect to July; p<0.05). Maximal values recorded in October and November were also significantly different from mean annual values (+83 % and +100 %, respectively; p<0.05). Taken together, the results indicate seasonal fluctuations in the parameters measured in the hemolymph of mussels from the Adriatic Sea. Lowest LMS values were observed in early winter and early autumn. On the other hand, maximal values of phagocytic activity were observed in winter and increasing serum lysozyme activities were recorded in autumn. These observations are in line with the fact that decreases in LMS are associated with membrane fusion processes during both endo/phagocytosis and release of lysosomal enzymes by exocytosis. However, when data were analysed by the Spearman rank correlation test, no significant correlation was observed among the different parameters measured (data not shown). The observed changes in hemolymph functional parameters can be related to both abiotic (such as temperature) and biotic factors (such as reproductive stage and also food availability). In the Adriatic Sea, fluctuations in hemolymph cytotoxicity of M. galloprovincialis were reported (Malagoli et al., 2006, 2007), with two peaks at the end of spring and of summer. However, the temperature apparently was not the main parameter affecting cytotoxicity (Malagoli et al., 2007). Moreover, no significant correlation was found between changes in immune parameters recorded in the present work and environmental temperature (data not shown). Although circulating hemocyte concentration did not show seasonal variations in M. galloprovincialis (Carballal et al., 1998), the proportion of different hemocyte subpopulations endowed with different phagocytic activity due to different hemocyte maturation stages may change at different times of the year. Since phagocytosis represents one of the main components of the innate immune response, lower phagocytic activities observed in this work during the summer may be Fig. 1 Seasonal trend of lysosomal membrane stability (LMS) (A) and phagocytic activity (B) in hemocytes and of serum lysozyme activity (C) in hemolymph of mussels from Adriatic Sea. Data, representing the mean ± SD of four replicates were analysed by the Mann-Whitney U test. A) * = p<0.05: May vs January and February; December vs September and October B) * = p<0.05: January vs June and September C) * = p<0.05: July vs October and November responsible for lower immune defence against invading micro-organisms during this period. Moreover, the effect of seasonal changes in endogenous modulators (such as neuropeptides, cytokines and sex steroids) should be taken into account. In the hemocytes of M. galloprovincialis, physiological concentrations of the natural estrogen 17β-estradiol can stimulate immune parameters, including phagocytosis, both in vitro and in vivo (Canesi et al., 2004, 2006b). In bivalves, 17β- estradiol has a role in reproduction: seasonal changes in estrogen content have been observed in   46 different bivalve species in relation to gametogenesis, with increases during gonad maturation and decreases at spawning (Osada et al., 2004; Gauthier-Clerc et al., 2006). However, in the present study, different extents of gonad ripening were observed in different individuals (males and females) throughout the year; although during acclimation in the laboratory a major gamete emission was observed in early spring, minor spawnings were also recorded at different times of the year. The absence of marked seasonal changes in gametogenesis may be partly related to a relatively constant food availability throughout the year in the Adriatic Sea. In M. galloprovincialis from the northern Spanish coast, basal hemocyte NO production showed lowest values in winter. However, only in mussels collected in winter IL-2 greatly induced NO production probably through an immunoreactive e- NOS protein that was expressed only in this period by the hemocytes (Novas et al., 2007). These data suggest that, in addition to fluctuations in basal values of immune parameters, changes in the inducibility of the immune response over the year should be considered. 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