Chemosphere 52 (2003) 11471151 www.elsevier.

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Lysosomal membrane stability, phagocytosis and tolerance to emersion in the mussel Perna viridis (Bivalvia: Mytilidae) following exposure to acute, sublethal, copper
S. Nicholson
*
Department of Ecology and Biodiversity, The Swire Institute of Marine Science, The University of Hong Kong, Cape dAguilar, Shek O, Hong Kong Received 4 September 2002; received in revised form 17 February 2003; accepted 23 March 2003

Abstract The mytilid mussel Perna viridis is distributed throughout the Indo-Pacic region and is potentially a suitable candidate for biological eects (biomarker) monitoring in the subtropics. A suite of cytological and physiological responses to acute (4872 h) copper exposures of 50200 lg l1 were assessed in order to determine the suitability of P. viridis for marine pollution monitoring. Copper elicited signicant destabilisation of the haemocyte lysosomal membranes and also impaired phagocytosis. Survival during emersion following exposure to copper was not related to the experimental copper exposures suggesting that higher metal concentrations may be required to interfere with anaerobic enzymes responsible for suppression of metabolism. Based on this preliminary study, cytological biomarkers evaluated in the haemocytes extracted from P. viridis should prove an eective non-destructive means of assessing metal pollution throughout the mussels subtropical range. 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Perna viridis; Haemocyte; Lysosome; Phagocytosis; Biomarker

1. Introduction Mussels are often used to determine marine water quality as they accumulate contaminants such as metals from seawater and food to concentrations signicantly above ambient levels thereby providing an indication of the biologically available fraction. Although the chemical analysis of metals accumulated in the tissues provides a reasonably accurate indication of concentrations present in the ambient environment, due to inherent

* Address: Mouchel Asia Ltd., 12/F MLC Tower, 248 Queens Road East, Wanchai, Hong Kong. E-mail address: shaun_nicholson@mouchel.com.hk (S. Nicholson).

dierences in toxicokinetics and the potential for detoxication, body burden analysis cannot fully determine whether they are a true reection of previous levels present and biological injury associated with metal uptake is not elucidated. In order to determine an individuals condition (health) the eects of excess metal uptake and consequent homeostasis can only be determined through biological responses (biomarkers). Many contaminants including metals are sequestered within mussel lysosomes and accumulation of higher concentrations can alter the permeability of the membrane initiating increased autophagy and also hydrolytic enzyme release. The degree of membrane labilisation is proportional to the magnitude of stress (Moore, 1985; Nicholson, 1999a) and these cytological responses can be used to determine the primary cytotoxicological

0045-6535/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00328-X

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eects of metal exposure that would not be evident from tissue chemical analysis alone. Haemocyte-mediated phagocytosis is the predominant form of internal defence in mussels (Pipe and Coles, 1995) although it is generally suppressed by exposure to a wide spectrum of toxicants including metals (Cheng, 1988). The mussel immune response is comprised of an integrated process of phagocytosis and lysosomal degradation (Sminia, 1980) and pollutioninduced dysfunction of these processes may suppress immunocompetence. Exposure to toxicants can uncouple the anaerobic pathways that mussels utilise during emersion in order to maintain basal metabolism. Suppression of metabolism during emersion relies on the inhibition of anaerobic enzymes which aords an improved potential for survival during aerial exposure (Eertman and de Zwaan, 1994). Toxicant exposure, uptake, storage and detoxication is energetically costly and may induce dysfunction of the anaerobic enzymes and subsequently reduce the ability to withstand emersion. The tolerances of mussels during emersion, assessed through survival during prolonged aerial exposure can, therefore, be used as a simple physiological index of pollutant exposures. The marine mytilid mussel Perna viridis is a widespread Indo-Pacic equivalent of the temperate Mytilus edulis and has been identied as a candidate for pollution monitoring utilising biomarkers in the subtropics (Nicholson, 1999a,b, 2001). Lysosomal membrane labilisation, phagocytic indices and tolerance to emersion following metal exposure have been determined in temperate species such as M. edulis and in order to assess the potential of these biomarkers for water quality monitoring in the subtropics, they were investigated in P. viridis during acute, sublethal exposures to environmentally realistic copper concentrations.

aerated, natural seawater and the appropriate copper solution. Nominal copper solutions were prepared from a 1000 mg l1 stock solution of CuSO4 (BDH, Poole, UK) and consisted of control, 50, 100, 150 and 200 lg l1 (as CuSO4 ). Water and test solutions were renewed daily and individuals were exposed for 48 h during the lysosomal assays and 72 h in the phagocytosis and tolerance to emersion following copper-exposure experiments. In order to determine the copper accumulating capacity of the tissues, an additional experimental treatment was run concurrently to the biological-eects measurements and P. viridis were exposed to a series of copper concentrations (control, 31.6, 56.2, 100.0 and 316.0 lg l1 ) for 72 h. Physico-chemical water parameters over the course of the experiments ranged from 18.0 to 21.0 C, 3032 salinity, pH 8.08.1 and 7.17.5 mg l1 dissolved oxygen. To assess copper accumulated in the mussels, dry weights were established by drying to constant weight (10%) at 80 C and tissues digested in 2 ml concentrated nitric acid (Analar grade, BDH) and dissolved at 100 C until mineralised. Copper concentrations were determined using inductively coupled plasma atomic emission spectrometry (PerkinElmer, Plasma 400). 2.2. Neutral red assay and cell viability Lysosome membrane stability and cell viability were measured using methods described previously (Lowe et al., 1995; Nicholson, 1999a,b, 2001). Following exposure to the experimental treatments, P. viridis valves were prised apart ($5 mm) and 0.5 ml of haemolymph withdrawn from a sinus in the adductor muscle into a 2.5 ml 23 G syringe containing 0.5 ml of temperatureadjusted saline (Nicholson, 1999a). Monocultures of haemocytes are not isolated using this technique and only lysosomal neutral red retention in the larger (>8 lm) haemocytes were analysed. Cell viability was determined using eosin Y exclusion. 2.3. Phagocytosis Phagocytosis was determined through an in vivo technique as it is likely to provide a more realistic indication of cellular physiology than in vitro techniques where haemocyte agglutination may occur. After 48 h exposure to the experimental treatments, individual P. viridis were injected (adductor muscle sinus) with a 200 ll solution (microsphere concentration of $5.5 109 ml1 saline) of uorescent 0.94 lm microspheres (G900, Duke Scientic Corp., California, USA). P. viridis were returned to the appropriate treatment for a further 24 h then 500 ll of haemolymph was extracted from the adductor muscle. Haemocytes were xed im-

2. Methods and materials Two groups of P. viridis of between 86.8 5.9 mm (mean SD, n 40) for biological eect experiments and 55.6 3.3 mm shell length (mean SD, n 30) for the assessment of copper body burden analysis were collected from a reference subtidal population in Kat O, Hong Kong (844050 N; 849150 E). Mussels were transported to the laboratory in a cool-box, cleaned of epibionts and maintained in unltered seawater in a once ow-through tank (363 l, ow rate $ 6 l min1 ) for $24 h. 2.1. Copper exposure and tissue accumulation P. viridis were randomly assigned to 8 l static aquaria (four individuals aquarium1 ) containing unltered,

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mediately with modied Bakers calcium formol (200 mg sodium acetate dissolved in 100 ml of 10% formalin in seawater). Phagocytic indices (% phagocytosis) were evaluated in the larger, granular haemocytes by plating out 40 ll of haemolymph and counting the presence of microsphere inclusions in 100 cells individual1 . 2.4. Tolerance to emersion Following 72 h exposure to the experimental treatments, P. viridis were transferred to a temperaturecontrolled room (20.622.1 C, relative humidity 56.959.6%) and survivorship on emersion monitored (response to tactile stimulus on the mantle edge). Survivorship was determined at 0, 1, 2, 4, 8, 25, 30, 48 h intervals then daily up to 8 d (192 h).

Fig. 1. P. viridis. Mean (+ SEM, n 8) neutral red retention times in haemocyte lysosomes following 48 h exposure to copper.

3. Results Mortality was not evident in P. viridis over the course of the copper exposures although sublethal responses did occur in the individuals subjected to the higher concentrations (150 and 200 lg l1 ) discharged profuse amounts of mucus and secreted fewer byssus threads onto the aquaria walls. Although copper exposure failed to induce mortality, sublethal cytological responses (lysosomal membrane labilisation and reduced phagocytic capacity) were nevertheless evident. 3.1. Neutral red assay and cell viability The haemocyte lysosomal membranes of P. viridis exposed to the copper tended to have lower retention of neutral red with respect to controls. Lysosomes from the individuals exposed to copper showed signicantly lower neutral red retention than controls and the rapid permeation to the cytosol in the metal-exposed individuals was a clear indication of lysosomal membrane labilisation (KruskalWallis test, 4, 39 d.f; P 0:03; SNK multiple comparison test, control > 100.0 50.0 200.0 150.0 lg l1 Cu; Fig. 1). Eosin Y exclusion assays revealed that haemocytes were not perturbed by either extraction or subsequent storage. Haemocytes always had a viability of at least 80% indicating that extraction did not signicantly aect cell physiology. 3.2. Phagocytosis Haemocyte phagocytosis assessed through microsphere uptake was generally low for all treatments although the highest index was recorded in the control treatments (mean of 8% phagocytosis). The low phagocytic indices recorded may have been a consequence of

Fig. 2. P. viridis. Phagocytic indices (mean + SEM, n shown on gure) from individuals following 72 h copper exposure.

the in vivo technique used, as injected microsphere solutions are likely to become highly dilute owing to the relatively high haemolymph volume and large number of circulating haemocytes. Some haemocytes had engulfed several microspheres although only presence or absence in each cell was evaluated. Nevertheless, control haemocytes showed the highest percentage microsphere uptake compared to the copper treatments (one-way ANOVA, 4, 34 d.f.; P 0:04; SNK multiple comparison test unable to determine signicant dierences between treatments; Fig. 2). 3.3. Metabolic arrest (survivorship) Most P. viridis individuals gaped their shell when emersed although adduction was evident following

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Fig. 3. P. viridis. Metabolic arrest (n 8) following 72 h copper-exposure.

Fig. 4. P. viridis. Mean (+ SD, n 6) copper concentrations from the tissues following 72 h exposure.

prolonged aerial exposure. Survivorship was initially high for all treatments during short-term (48 h) emersion but rapid mortality ensued after 96 h. Results were not entirely exposure-concentration dependent and highest survivorship over the emersion period was observed in 50, 100, 200 and 150 lg l1 copper treatments, respectively with controls showing the lowest ability to survive emersion (KruskalWallis test on survivorship following 96 h emersion, 4, 39 d.f; P 0:04; Dunns multiple comparison test unable to resolve signicant dierences; Fig. 3). Handling stresses and haemolymph extraction may have altered the capability of P. viridis to suppress metabolism and survive emersion because controls were least resistant, although survivorship in the copper treatments was generally related to the exposure concentration. 3.4. Copper accumulation in the tissues The control individuals showed relatively low copper concentrations in the tissues whereas P. viridis exposed to copper showed exposure concentration dependent accumulation. The signicant concentration-dependent copper accumulation after 72 h exposure indicated that the metal was taken-up into the tissues by rst order kinetics and physiological regulation was not evident (one-way ANOVA on log-transformed data, 4, 29 d.f., P < 0:0001; SNK multiple comparison test 316.0 > 100.0 > 56.2 31.6 > control; Fig. 4).

4. Discussion The lysosomal membranes and phagocytic capacity of P. viridis haemocytes exposed to copper were impaired at concentrations that did not induce whole animal toxicity. Lysosomes are a major site of accumulation and homeostasis of metals in marine mussels (George, 1983;

Cheung et al., 1998; Nicholson, 1999a, 2001) and exposure to elevated copper concentrations may overload their sequestering capacity thereby rendering the membrane particularly susceptible to excess ambient concentrations (Viarengo et al., 1981; Regoli, 1992). Although there was not an obvious copper exposureresponse in P. viridis, probably due to the acute exposure duration, haemocyte lysosomal membrane injury was evident using the neutral red probe at dissolved metal concentrations that are frequently present in contaminated estuaries (20160 lg l1 Hong Kong, Chan et al., 1974; 10100 lg l1 UK, Bryan et al., 1987) and elevated membrane permeability is likely to increase autophagic events and hydrolytic enzyme activity in the cytosol leading to a greater risk of cytotoxicity. The in vivo phagocytic indices recorded in the present study were generally lower than reported using in vitro techniques although this is likely due to microsphere dilution in the haemolymph and as phagocytosis is inuenced by temperature (Alvarez et al., 1989) the ambient thermal regime may have been below optima for physiological processes in P. viridis (Nicholson, 2002). Phagocytic indices revealed that haemocytes from P. viridis exposed to copper were unable to eciently remove injected microspheres from the haemolymph indicating a modulated immune response. Although relatively low phagocytic indices were recorded for all treatments (including control) it is likely that the signicantly lower phagocytosis in copper exposed individuals is biologically relevant and immune suppression in P. viridis inhabiting contaminated estuaries mediated through both inhibited phagocytosis and dysfunctional lysosomes will be manifested in an inability to ght invasive pathogens and potentially result in disease. The ability of P. viridis to withstand prolonged emersion produced anomalous results as control individuals were the most emersion intolerant although individuals exposed to the copper regime did show some

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relationship between the ambient exposure concentration and survivorship. Previous studies (Eertman and de Zwaan, 1994) have shown that ability to tolerate prolonged emersion is related to the copper exposure concentration although lower levels (20 lg l1 ), as in the present study with P. viridis, did not signicantly impair the survivorship of M. edulis (Weber et al., 1992). The tolerance of P. viridis to emersion following exposure to copper may, therefore, only be sensitive to higher or chronic metal exposures. Copper was accumulated in P. viridis tissues in a concentration-specic manner indicating that physiological regulation at these ambient concentrations was not possible during continued exposure and ecient intralysosomal sequestration of the metal may have prevented mortality. The ability to withstand further copper exposure may, however, be limited and continual metal uptake is likely to induce whole-animal toxicity. In summary, short-term laboratory experiments showed that lysosomal membrane integrity and phagocytic indices utilising living haemocytes were responsive to ambient copper exposures and these cytological markers can serve as cost-eective indicators of water quality degradation prior to the onset of physiological or whole-organism toxicity. Cytological biomarkers will facilitate an integrated assessment of pollutant discharges and given its wide Indo-Pacic range, P. viridis should prove suitable for marine water quality monitoring in the subtropics.

Acknowledgements This work was supported by a studentship from The University of Hong Kong and represents research conducted for the partial fullment of a PhD. Sta of the Agriculture and Fisheries Department are gratefully acknowledged for providing assistance in the collection of P. viridis.

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