Copper induced immunomodulation in the marine mussel, Mytilus edulis

Aquatic Toxicology 46 (1999) 43 – 54

Copper induced immunomodulation in the marine mussel, Mytilus edulis R.K. Pipe *, J.A. Coles, F.M.M. Carissan, K. Ramanathan NERC, Plymouth Marine Laboratory, Citadel Hill, Plymouth, De6on PL1 2PB, UK Received 15 December 1997; received in revised form 19 August 1998; accepted 22 September 1998

Abstract The effects on the immune response of the mussel, Mytilus edulis, of short-term, in-vivo exposure to copper were investigated under laboratory-controlled conditions. Parameters measured concentrated on the ability of the blood cells to destroy invading pathogens and included changes in the number and character of the circulating haemocytes, peroxidase and phenoloxidase enzyme activity in the blood cells, intra- and extracellular superoxide radical production, phagocytosis and uptake of neutral red. Copper concentrations of 0.02 and 0.05 ppm were found to increase significantly the total number of circulating haemocytes, while 0.2 and 0.5 ppm decreased the proportion of eosinophilic to basophilic cells. Intracellular superoxide production significantly decreased on exposure to 0.5 ppm copper, whereas phagocytic activity was stimulated at 0.2 ppm but not at 0.5 ppm. Copper exposures of 0.2 and 0.5 ppm reduced the percentage of haemocytes showing binding of lectins from Galanthus ni6alis and Helix pomatia compared with haemocytes from mussels not dosed with copper. No significant alterations were found in peroxidase and phenoloxidase activity, binding of wheat germ agglutinin or uptake of neutral red. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in aquatic organisms. The benefits of using a multi-assay approach to monitoring environmental pollution using such methods are highlighted. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Copper; Mussel; Mytilus edulis; Immunocompetence; Hemolymph; Blood cell; Phagocytosis

1. Introduction The effects of pollution on fisheries, including shellfisheries, has been a topic for concern and * Corresponding author. Tel.: + 44 752 633233; fax: + 44 752 633102; e-mail: [email protected]

research in Britain for more than a century (Burden, 1893; Philpots, 1893). Of particular concern a century ago was the effect of copper mining on local oyster (Ostrea edulis) fisheries. In samples taken from Falmouth in Cornwall, an area affected by copper mining, concentrations of copper in oyster tissues were reported at between ‘23

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centigrammes (about 3.5 grains) of salt of copper’ per dozen oysters (O’Shaughnessy, 1866) and 0.023 grains of copper per oyster (Bulstrode, 1896). Exposure experiments were also conducted to investigate the effects on oysters of treatment with 0.02 and 0.2% copper sulphate (Herdman, 1896). More recent concerns about the effects of elevated copper levels have arisen due to the French ban of tributyl tin as an antifouling paint on boats under 25 m in length in 1982 (Claisse and Alzieu, 1993), followed by the UK ban in 1987, which has forced a return to the traditional copper-based paints. This return to copper as an antifouling base has been suggested as a reason for a perceptible increase in copper contamination in the Bay of Arcachon since 1982 (Claisse and Alzieu, 1993). Bivalve molluscs including the marine mussel, Mytilus edulis, have been postulated as ideal indicator organisms for assessing levels of environmental pollution, as they are ubiquitous, sedentary, filter-feeders inhabiting coastal and estuarine areas. They filter large volumes of seawater and may therefore concentrate contaminants to a factor of 105 within their tissues (Widdows and Donkin, 1992). Environmental contaminant effects may result from direct toxic action on the tissues or from more subtle alterations in homeostatic mechanisms, such as the immune system. The study of a range of components which comprise an integrated biological system like the immune system, may therefore provide a sensitive and comprehensive measure of the health status of an organism, reflecting the degree of pollutantinduced stress and may thus give an early indication of disease susceptibility and, ultimately, survival. The potential relationship between environmental pollution and disease incidence in aquatic organisms has received increasing attention in recent years. There is accumulating evidence that high concentrations of contaminants in coastal and estuarine habitats may increase disease susceptibility in marine organisms (Pipe and Coles, 1995; Dyrynda et al., 1997a,b). Disease conditions observed in fish and shellfish associated with contaminant exposure include larval abnormalities,

shell and skeletal deformities and tissue damage such as ulceration and fin erosion (Sindermann, 1979, 1982, 1989, 1990; Livingstone and Pipe, 1992; Gardner, 1993). Immunomodulation resulting from copper exposure has been observed previously in fish (Sindermann, 1979; Zelikoff, 1993) and in several shellfish species (Cheng and Sullivan, 1984; Pickwell and Steinert, 1984; Cheng, 1988a,b; Larson et al., 1989; Cheng, 1990; Suresh and Mohandas, 1990; Anderson, 1993; Fagotti et al., 1996). Mussels have an open circulatory system which is continually exposed to fluctuating environmental factors including contaminants. The immune defence is comprised of cell-mediated and humoral mechanisms, in which the haemocytes play a key role (Cheng, 1981). Antigenic challenge stimulates migration of haemocytes, followed by phagocytosis and intracellular degradation of the pathogen by means of lytic enzymes (Pipe, 1990a) or the production of highly reactive oxygen metabolites (Pipe, 1992). Degradation may also occur extracellularly following degranulation of the haemocytes. The aim of the present study was to investigate the effects of exposure to copper, under controlled conditions, on a range of aspects of the immune function of the mussel, M. edulis. Parameters measured concentrated on the ability of the blood cells to destroy invading pathogens and included changes in the number and character of the circulating haemocytes, peroxidase and phenoloxidase enzyme activity in the blood cells, intra- and extracellular superoxide radical production, phagocytosis and uptake of neutral red (Pipe et al., 1995). A multi-assay approach was adopted with a view to monitoring the overall health status of the organisms under contaminant stress.

2. Materials and methods

2.1. Experimental animals M. edulis (60–75 mm shell length) were collected at low tide from Whitsand Bay, Cornwall, an exposed open ocean site. Sixty mussels were allocated to each of five polypropylene tanks

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(46 ×28× 15 cm3) containing 5 l of aerated, filtered seawater at 15°C and were left overnight to acclimatize.

2.2. Copper exposure The seawater was changed every day for 7 days and the tanks were dosed with aqueous copper sulphate solution to final concentrations of 0, 0.02, 0.05, 0.2 and 0.5 ppm. The mussels were fed 300 ml of mixed algal suspension per tank, twice weekly, 1 h prior to changing the water.

2.3. Protein quantification In most microplate assays carried out, each haemolymph sample was pipetted into a duplicate microplate and analysed for protein content using a bicinchoninic acid protein assay (Pierce). The protein was solubilised using 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) at 37°C for 30 min (Pierce), bovine serum albumen was used as a standard and readings were obtained on the microplate reader at 550 nm. All results were then expressed relative to haemocyte protein content.

2.4. Assays Assays were carried out after 7 days of exposure. Haemolymph was extracted from the blood sinus of the posterior adductor muscle (six to 15 mussels per treatment) immediately before each assay, using a 2 ml syringe fitted with a 21 gauge needle.

2.4.1. Total and differential blood cell counts Haemolymph samples were withdrawn from 10 – 15 animals into an equal volume of Baker’s formol calcium, containing 2% sodium chloride. The samples were mixed and allowed to fix. Total cell counts were performed using a haemocytometer (improved Neubauer). Blood cells were prepared for differential counts using a cytocentrifuge (Shandon, UK); 250 ml of haemolymph was spun at 170×g onto glass microscope slides. Cells were then post-fixed in methanol for 10 min, stained with Wright’s stain

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(Gurr, BDH) for 2 min, rinsed in water, air dried and mounted in Canada balsam. The Wright’s stain enables eosinophilic (orange) and basophilic (blue) blood cells to be differentiated (Pipe, 1990b; Pipe et al., 1997). Relative numbers of eosinophils and basophils were calculated by counting 300 blood cells from each animal.

2.4.2. Peroxidase and phenoloxidase acti6ity Haemolymph was extracted into Baker’s formol calcium plus 2% NaCl from six animals from each treatment, and three slides prepared from each sample using a cytocentrifuge. One slide was stained with 0.5 mg ml − 1 of diaminobenzidine (DAB) (Sigma, St Louis, MO) in 0.05 M Tris– HCl buffer, pH 7.6, containing 2% NaCl and 0.02% hydrogen peroxide. Incubations were carried out for 35 min at 20°C. The second slide was incubated for 90 min at 30°C in a coplin jar containing 1 mg ml − 1 of L-3,4-dihydroxy-phenylalanine (Sigma) and 2% NaCl in 0.1 M phosphate buffered saline (PBS), pH 7.4. The final slide was used as a negative control and incubated in buffer only. All slides were then washed in water, dehydrated, mounted in Canada balsam and 300 cells counted from each sample. The percentage of haemocytes showing peroxidase or phenoloxidase activity was calculated. 2.4.3. Lectin binding Haemolymph was extracted, from 12 control and 12 test mussels, into an equal volume of Baker’s formol calcium plus 2% NaCl, and the blood cells were spun onto microscope slides using a cytocentrifuge. The slides were left to dry and then incubated for 1 h in 0.1 M glycine and 0.1 M ammonium chloride in PBS, pH 7.0. The solution was then tipped off and the slides incubated in FITC-labelled lectin solutions (50 mg ml − 1) in PBS, pH 7.0, for 1 h in the dark at room temperature. The lectins used were isolated from, wheat germ, (Triticum 6ulgaris, WGA), snowdrop (Galanthus ni6alis, GNL), and edible snail (Helix pomatia, HPA). Negative control slides were incubated in the lectins dissolved in a blocking solution, consisting of 0.1 M N-acetyl glucosamine, 0.1 M N-acetyl galactosamine, 0.1 M fucose, 0.1 M galactose, 0.1 M mannose, 0.1 M glucose, 1%

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fetuin and 1% chitin. The slides were then washed in PBS and mounted in ultraviolet-free aqueous mountant. The percentage of positively fluorescent blood cells was calculated, counting 100 cells from each sample.

2.4.4. Superoxide generation Haemolymph was extracted into an equal volume of 0.05 M Tris– HCl buffer, pH 7.6, containing 2% NaCl. Aliquots of 100 ml of each were pipetted into microplate wells in replicates of eight. 1. Cytochrome c reduction. Cytochrome c solution (100 ml aliquots of 80 mM cytochrome c in Tris–HCl buffer) was added to four wells for each of six samples and cytochrome c solution containing 10 mg ml − 1 phorbol myristate acetate (PMA) (Sigma) as a stimulant added to the remaining four wells. Wells containing cells in buffer only and cytochrome c solution without cells were prepared as negative controls. The optical density (OD) was read immediately at 20°C on an Anthos HTII microplate reader, using a narrow bandwidth, 550 nm filter and a kinetics programme, taking readings over a 20 min period. The first reading was used as the blank value and the results were expressed as unit changes in OD over 20 min mg − 1 ml − 1 protein. 2. Nitroblue tetrazolium salt (NBT) reduction. NBT solution (2 mg ml − 1) dissolved in Tris – HCl buffer (100 ml aliquots) was added to four replicate microplate wells of each of four haemolymph samples. NBT solution containing 10 mg ml − 1 superoxide dismutase (SOD) (bovine; Sigma), was added to the remaining four wells. Blood cells in buffer only and NBT solution without cells were used as blank samples and cells in buffer only were used as negative controls. The plates, wrapped in foil, were incubated for 30 min at 10°C, after which the cells were centrifuged at 120×g for 10 min, washed twice in buffer and fixed for 10 min in methanol. The plates were then centrifuged at 300 × g for a further 10 min and the cells allowed to air dry. After five further washes in 50% methanol, the cells were solubilised in 140 ml of dimethyl sulfoxide (Sigma),

followed by 120 ml of 2 M KOH. The plates were read using a 620 nm filter and the results expressed as OD mg − 1 ml − 1 protein.

2.4.5. Phagocytosis A suspension of neutral red-stained zymosan was prepared by mixing 10 g of zymosan particles (Sigma) with 100 ml of 1% neutral red solution in boiling, distilled water for 1 h. The suspension was then centrifuged at 300×g for 5 min and resuspended in 1.8% phosphomolybdic acid for 30 min at 4°C, followed by 6% ammonium heptamolybdate for 1 h at 4°C. The particles were then spun six times at 300 × g for 5 min, the supernatant discarded and the zymosan resuspended in Tris buffered saline, pH 7.6, containing 2% NaCl. Clumped particles were eradicated by spinning at 70× g and the final suspension was adjusted to 1×107 particles ml − 1. Haemolymph was withdrawn into an equal volume of Tris buffered saline (pH 7.6, containing 2% NaCl). Aliquots of 50 ml of each sample were pipetted into four replicate microplate wells. An equal volume of the neutral red stained-zymosan suspension was added to each well. Aliquots of zymosan suspension in buffer alone were used as controls and aliquots of formalin-fixed cells in zymosan suspension were used as blanks. The microplates were incubated for 30 min at 10°C, and the process was stopped by adding 100 ml aliquots of Baker’s formol calcium, containing 2% NaCl, to the wells. The cells were allowed to fix for 10 min, after which they were centrifuged at 70× g for 5 min and washed several times in buffer. Suspensions of known zymosan concentrations were aliquoted (50 ml) to duplicate wells just prior to the final centrifugation to provide a standard curve. Acetic acid (1%) in 50% ethanol (100 ml) was added to each well to solubilise the neutral red and the plate allowed to stand for 30 min. The OD was then read at 550 nm and the results expressed as zymosan concentration mg − 1 ml − 1 of haemocyte protein. 2.4.6. Uptake of neutral red Haemolymph was withdrawn into an equal volume of anticoagulant buffer and 100 ml aliquots of each sample pipetted into three replicate mi-

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Fig. 1. The percentage increase in the total number of haemocytes ( ×106 cells ml − 1) in the haemolymph of M. edulis, exposed to different concentrations of copper for 7 days ( 91 standard error; * indicates significant difference from control value, P B0.05).

croplate wells. Aliquots (10 ml) of 0.33% neutral red (Sigma) solution in phosphate buffered saline containing 2% NaCl, were added to each well and the plate incubated for 1 h at 10°C. The cells were then centrifuged at 200×g for 5 min and washed twice in buffer. Aliquots (100 ml) of 1% acetic acid in 50% ethanol were added to all wells. The plates were incubated, covered with foil, for 15 min at 20°C and then read at 550 nm. The results were expressed as optical density per mg − 1 ml − 1 haemocyte protein.

2.5. Copper analysis The digestive gland lysosomal compartments represent the major site of metal accumulation in bivalve molluscs (Livingstone and Pipe, 1992). The digestive glands were removed from six mussels from each treatment and washed in PBS containing 2% NaCl. The tissues were then digested in concentrated nitric acid at 20 ml g − 1 dry weight, heat evaporated and made up in 10% hydrochloric acid. Analysis for copper content was then carried out using flame atomic absorption spectrophotometry, with a deuterium ark background correction for NaCl (Bryan et al., 1985).

2.6. Statistical analysis Results of all assays were analysed statistically using one- or two-way analysis of variance (Systat). Mean values are expressed 91 standard error.

3. Results

3.1. Total and differential blood cell counts The effect of 7 days of copper exposure on the total number of circulating haemocytes in M. edulis is shown in Fig. 1. An increase in the number of circulating haemocytes was observed which was significantly different at 0.02 ppm (P =0.03) and 0.05 ppm (P= 0.002) copper compared to control animals but less significant at 0.2 (P= 0.119) and 0.5 ppm (P= 0.567). An alteration in the relative proportions of haemocyte types was also observed (Fig. 2). The percentage of eosinophilic blood cells showed a notable decrease compared with the basophils, with increasing copper concentration. This effect was significant at 0.2 and 0.5 ppm of copper (PB 0.001) compared with the controls.

3.2. Peroxidase and phenoloxidase acti6ity Although the percentage of circulating blood cells with peroxidase and phenoloxidase activity indicated increases at 0.2 ppm copper and decreases at 0.5 ppm copper, these values were not significantly different from the control values due to the high inter-animal variability. Values ranged from 1 to 17% for peroxidase activity and from 1.3 to 9.5% for phenol oxidase activity.

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Fig. 2. The relative proportions of eosinophilic to basophilic haemocytes circulating in the haemolymph of M. edulis exposed to different concentrations of copper for 7 days ( 9 1 standard error; * indicates significant difference from control value, P B 0.05).

3.3. Lectin binding

3.5. Phagocytosis

Copper exposures of 0.05, 0.2 and 0.5 ppm resulted in a significant (P B0.02) reduction in the percentage of haemocytes showing binding of both GNL and HPA (Fig. 3) compared with haemocytes from mussels not dosed with copper. The WGA binding did not show any significant differences between the treatments. Controls, incubated with the blocking solution, showed a considerable reduction in binding of all lectins.

Copper exposure resulted in a stimulation of phagocytic activity at 0.2 ppm (Fig. 6). This increase in phagocytosis was significantly different from control values (P=0.006). There was an apparent reduction in phagocytosis at 0.5 ppm copper but, due to high variability between individuals, this was not significant.

3.4. Superoxide generation The release of reactive oxygen metabolites from haemocytes (Fig. 4) increased with increasing copper concentration but decreased significantly at the 0.5 ppm exposure (P= 0.001 when compared with 0.2 ppm copper, P =0.056 when compared with the control). Intracellular superoxide production (Fig. 5) also decreased significantly with the highest copper concentration (P =0.013, 0.5 ppm compared with the control). Experimental manipulation was sufficient to produce cytochrome c reducing activity by the haemocytes; PMA increased this slightly but not significantly. Addition of SOD to the incubation medium caused a partial inhibition of NBT reduction.

3.6. Neutral red uptake Exposure of mussels to copper, at all concentrations, did not result in significant differences in neutral red uptake by the haemocytes and the values did not show any correlation with the numbers of eosinophilic haemocytes.

3.7. Copper analysis Analysis of the copper content of the digestive gland tissue of mussels demonstrated increasing tissue levels with increasing copper administered (Table 1). Copper exposure of 0.2 ppm resulted in particularly high tissue levels, possibly because the higher concentration may have affected feeding and thus the rate of copper accumulation.

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Fig. 3. The percentage of haemocytes from M. edulis showing binding of lectins from snowdrop (Galanthus ni6alis, GNL), and edible snail (Helix pomatia, HPA) following exposure to different concentrations of copper for 7 days (9 1 standard error; * indicates significant difference from control value, PB 0.05).

4. Discussion Measurements of environmental levels of copper have recorded concentrations as high as 0.6 ppm in some waters such as the Carnon River, feeding the Fal Estuary, Cornwall, UK (Bryan and Langston, 1992), and 0.3 ppm in coastal water off the Dutch coast, following dumping of copper sulphate solution in 1965 (Clark, 1989). Dissolved copper is readily removed from solution by adsorption on to particles and may accumulate in sediments to levels of more than 3000 ppm as found in Restronguet Creek, Cornwall (Bryan and Langston, 1992). Concentrations of between 5000 and 20000 ppm have been recorded in the haemocytes of bivalve molluscs (Orton, 1924; Clark, 1989). In this study, mussels have been exposed to levels of copper which have resulted in increased tissue burdens and significant alteration in some haematological parameters and aspects of immune function. Exposure to 0.02 and 0.05 ppm copper for 7 days resulted in significant increases in the number of circulating haemocytes in M. edulis. The observed increase was not significant on exposure to higher concentrations of copper, perhaps due to enhanced toxicity of the copper causing cell death or possibly due to movement of haemocytes out of circulation to sites of tissue pathology resulting from the copper exposure. Elevation of total circulating blood cells appears to be a com-

mon response to environmental stressors. Exposure of M. edulis to fluoranthene (Coles et al., 1994a), cadmium (Coles et al., 1994b), phenol or temperature–stress (Renwrantz, 1990), all resulted in increased total blood cell counts, of which the last was found to be reversible on withdrawal of the stressor. Exposure of Crassostrea 6irginica to cadmium (Cheng, 1988a) or to a protozoan parasite (Anderson et al., 1992) also stimulated circulating blood cell numbers and a reversible increase in cells was found to occur 72 h after exposure of two clam species, Ruditapes philippinarum and Ruditapes decussatus, to pathogenic bacteria (Oubella et al., 1993). These relatively rapid, reversible responses suggest that increased total numbers of circulating haemocytes occur by stimulation of migration of the cells from tissues, rather than by blood cell proliferation. This may or may not result in altered relative proportions of haemocyte types, depending on the distribution within the tissues of blood cell types in a particular individual or species. The diverse nature of the stimulants suggests that this is a general response to an environmental stressor rather than a reaction to a specific challenge. The circulating haemocytes of bivalves have been classified as granulocytes (containing granules) or hyalinocytes (without granules) (Cheng, 1981); however, in mussels, two types of granulocyte can be identified, those with small granules which are basophilic and those with larger gran-

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Fig. 4. Extracellular superoxide production (cytochrome c reduction; change in OD mg − 1 ml − 1 haemocyte protein) by haemocytes of M. edulis exposed to different concentrations of copper for 7 days ( 91 standard error).

ules which are eosinophilic (Pipe, 1990b; Pipe et al., 1997). Exposure of mussels to copper resulted in a dose-dependent decrease in the percentage of circulating eosinophilic as compared to basophilic haemocytes, which was significantly different from control values at both 0.2 and 0.5 ppm of copper. This may result from increased blood cell mortality or by diapedesis of metal-laden haemocytes for excretory purposes. Alteration in the relative proportion of blood cell types in bivalves has been observed previously in response to various stressors, including hydrocarbons and metals (Anderson, 1993). Copper exposure was found to reduce the percentage of hyalinocytes (Cheng, 1988a) and stimulate the percentage of granulocytes in oysters (Ruddell and Rains, 1975) and mussels (Pickwell and Steinert, 1984). Little is yet known about the different functions of different blood cell types in M. edulis. The granulocytes are thought to be more actively phagocytic (Foley and Cheng, 1975) and work on Percoll-separated cell types has found that the granular eosinophils account for most of the peroxidase, phenoloxidase and phagocytic activity as well as generation of reactive oxygen metabolites (Pipe et al., 1997). Clearly, a contaminant-induced reduction in circulating eosinophils, as observed in this study, may have a profound effect on immune capability. Peroxidase and phenoloxidase activity has been shown previously to be present in M. edulis haemocytes (Pipe et al., 1993; Coles and Pipe,

1994; Renwrantz et al., 1996; Pipe et al., 1997). These enzymes are associated with cellular defence reactions in vertebrate and invertebrate species and are implicated in a number of processes including microbicidal and anti-parasitic activity (Klebanoff, 1970; Harrison and Schultz, 1976; Pawelek and Lerner, 1978; So¨derha¨ll and Ajaxon, 1982; Odell and Segal, 1988; Aspa´n et al., 1995; Johansson et al., 1995; So¨derha¨ll et al., 1996). In the present study, although exposure to 0.5 ppm copper was found to result in a reduction in the activity of these two enzymes, this was not statistically significant. This is surprising, considering the significant reduction in numbers of eosinophilic blood cells. However, peroxidase and phenoloxidase activities have been found to be very variable between individual mussels and, although phenol oxidase and peroxidase positive haemocytes are always eosinophilic, not all eosinophils show activity for these enzymes (Coles and Pipe, 1994; Pipe et al., 1997). Sami et al. (1992, 1993) found that exposure to polycyclic aromatic hydrocarbons (PAHs) resulted in alterations in the proportions of circulating haemocytes and suppression of cell surface, concanavalin A binding sites in the American oyster, C. 6irginica, and that phagocytic activity was also suppressed in field samples from contaminated sites. The reduced lectin binding following PAH exposure follows the same pattern as observed in the present study, where significant reductions in binding for GNL and HPA were observed at the higher copper concentrations.

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Fig. 5. Intracellular superoxide production (NBT reduction; OD mg − 1 ml − 1 haemocyte protein) by haemocytes of M. edulis exposed to different concentrations of copper for 7 days (9 1 standard error; * indicates significant difference from control value, PB 0.05).

Phagocytosis by haemocytes is an integral aspect of immune defence in mussels, occurring prior to the intracellular killing of an invasive pathogen. Previous research has shown that copper exposure at 5 ppm resulted in enhanced phagocytic uptake of polystyrene spheres in the bivalve C. 6irginica (Cheng and Sullivan, 1984). Further studies on uptake of Escherida coli demonstrated that copper had an inhibitory effect on the percentage of blood cells phagocytosing, but stimulated the endocytotic indices (Cheng, 1988b). The present study showed that acute copper exposure of 0.2 ppm resulted in a significant stimulation in phagocytic activity by mussel haemocytes, an effect which was not observed at the higher copper concentration. Although this indicates stimulation of immune capability, the energetic costs to the organism may be high, with possible detrimental implications for long-term, chronic copper exposure. The dose response suggests a concentration threshold, below which there is stimulation of phagocytosis and above which the copper causes inhibition. The potential for killing of invasive pathogens by the release of reactive oxygen metabolites has been demonstrated previously in the blood cells of M. edulis (Pipe, 1992; Noe¨l et al., 1993). Larson et al. (1989) found that exposure of the oyster, C. 6irginica, to concentrations of copper as low as 0.4 ppm produced a reduction in chemiluminescent activity by the haemocytes. The present

study, showing that in-vivo exposure to 0.5 ppm copper results in a decrease of both cytochrome c and NBT reduction by mussel haemocytes, suggests that a threshold copper concentration between 0.2 and 0.5 ppm causes inhibition of reactive oxygen metabolite generation. It should, however, be mentioned that the variable valency of transition metals, including copper, enables them to be effective catalysts of many oxidation and reduction reactions and so the significant alterations in cytochrome c and NBT reduction could be a direct effect of the elevated copper levels in the tissues. Overall, the results of the present study show that short-term exposure to copper, at environmentally realistic levels, may significantly influence various aspects of immune function in mussels. The variability in the nature and degree of immunomodulation in different aspects of internal defence, emphasises the need for a multi-assay approach to the assessment of immunocompetence. A suite of assays may therefore represent a sensitive means of testing for environmental stressors and assessing their possible effects on disease susceptibility in aquatic organisms. Further research is required to investigate the potentially more harmful effects of long-term copper exposure. The results of controlled experiments may help in establishing precise cause and effect relationships between pollution and disease susceptibility, and will con-

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Fig. 6. Phagocytosis of zymosan (number of particles mg − 1 ml − 1 haemocyte protein) by haemocytes of M. edulis exposed to different concentrations of copper for 7 days ( 91 standard error; * indicates significant difference from control value, P B0.05). Table 1 The percentage increase in copper content in digestive glands of mussels exposed to copper for 7 days as compared to non-exposed animals

work on copper analysis and M. Carr for advice on statistics.

Copper conc. administered (ppm)

% Increase in Cu in digestive glands

References

0.02 0.05 0.2 0.5

7 88 567 122

Data are based on pools of digestive glands from six mussels.

tribute toward a much needed database of information on invertebrate immunology in order to allow more accurate interpretation of field observations and to facilitate the application of a biological monitoring system using this species (Sindermann, 1988; Couch, 1993; Sindermann, 1993).

Acknowledgements The authors would like to acknowledge the financial assistance provided by The Commission of the European Community Research Programme on the Fisheries Sector (FAR); Project Numbers AQ2 419 and AQ3 633 and the UK Department of the Environment (PECD Ref. 7/7/ 386). We would also like to thank G. Burt for his

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