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Immunotoxicological effects of environmental contaminants on marine bivalves
Fish & Shellfish Immunology xxx (2015) 1e6
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Immunotoxicological effects of environmental contaminants on marine bivalves T. Renault* Ifremer, D epartement Ressources Biologique et Envrionnement, Rue de l'^Ile d'Yeu, 44300 Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 November 2014 Received in revised form 6 April 2015 Accepted 12 April 2015 Available online xxx
Coastal areas are complex environments frequently contaminated by numerous pollutants that represent a potential threat to marine organisms, especially bivalves. These pollutants may have major ecological consequences. Although effects of different environmental contaminants on the immune system in marine bivalves have been already reported, a few of reviews summarizes these effects. The main purpose of this chapter relies on summarizing recent body of data on immunotoxicity in bivalves subjected to contaminants. Immune effects of heavy metals, pesticides, HAP, PCB and pharmaceuticals are presented and discussed and a particular section is devoted to nanoparticle effects. A large body of literature is now available on this topic. Finally, the urgent need of a better understanding of complex interactions between contaminants, marine bivalves and infectious diseases is noticed. © 2015 Published by Elsevier Ltd.
Keywords: Marine bivalves Pollutants Nanoparticles Immunotoxicology Diseases Pathogens
1. Introduction Coastal areas are complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Contaminants are frequently detected in these areas and represent a potential threat to marine organisms, especially bivalves. Pollutants may have major ecological consequences [4]. They can endanger organism growth, reproduction or survival. Their effects may result from direct toxic actions or from alterations of the homeostatic mechanisms including the immune system [7,20]. Among physiological processes possibly disturbed by pollutants, the immune system is likely to be one of the more sensitive [23]. Bivalves filter large volumes of seawater and their immune capacities can be adversely affected by exposure to contaminants. Therefore, bivalves have to face highly variable environmental conditions. Investigating immunity toxicity can provide relevant information on the quality of the marine environment and facilitate understanding of the occurrence of infectious diseases affecting bivalves in coastal areas. Bivalves in culture may be weakened after contaminant exposure, potentially increasing their susceptibility to
* Tel.: þ33 2 40 37 40 00; fax: þ33 2 40 37 40 01. E-mail address: [email protected].
infectious diseases. It has been reported that a mixture of pesticides could induce a decrease of immune defenses in the Pacific oyster, Crassostrea gigas, rendering animals more vulnerable to an experimental bacterial infection [27]. In bivalves, immunity is mainly supported by cells called hemocytes found in the open hemolymphatic circulatory system. Hemocytes have been widely used to explore pollutant effects on bivalve immunity. The monitoring of health conditions by the assessment of marine bivalve immunocompetence may serve as a criterion for the achievement of the Good Environmental Status as defined in the EU Marine Strategy Framework Directive. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their wide geographical distribution, and sensitivity to environmental pollutants [17]. Moreover, the development of techniques allowing the analysis of pollutant effects on bivalve immunity may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, the effects of different environmental contaminants on the immune system of marine bivalves have been already reported. The condition of the immune system determines partly susceptibility to disease and survival. Measuring endpoints linked to immunity can help to identify sub-lethal effects of exposure to contaminants and provide early warning signals. However, there are at present a few of reviews summarizing these effects. The main purpose of this chapter relies on summarizing recent body of data
http://dx.doi.org/10.1016/j.fsi.2015.04.011 1050-4648/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: T. Renault, Immunotoxicological effects of environmental contaminants on marine bivalves, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.011
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on immunotoxicity in bivalves subjected to contaminants. Immune effects of heavy metals, pesticides, HAP, PCB and pharmaceuticals are presented and discussed and a particular section is devoted to the effects of nanoparticles. A large body of literature is now available on this topic. Finally, the urgent need of a better understanding of complex interactions between contaminants, marine bivalves and their infectious agents is identified. 2. Pesticides Pesticides are used especially in agriculture. They are defined as chemical substances used to kill pests including insects, weeds, parasites or rodents. All pesticides act by interfering with the target species normal metabolism. However, some inadvertently effects may affect other organisms in the environment, either directly by their toxic effects or via elimination of the target organism. Hemocyte activities have been extensively studied in order to investigate the effects of pesticides on bivalve immunity. As an example, 23 pollutants including several pesticides have been tested on Pacific oyster hemocytes maintained in vitro through monitoring of cell parameters by flow cytometry [26]. Several pesticides have been shown to induce a modulation of some hemocyte activities including cell mortality and non specific esterase activities. However [40], reported no significant effect of a mixture of 14 pesticides and methaldehyde alone tested at different concentrations on Pacific oyster hemocytes maintained in vitro for short-term periods. Several cell parameters including dead/alive cells, non specific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. Pacific oyster hemocytes appeared thus resistant to a pesticide exposure in the tested conditions [40]. Developing in vivo assays can be useful to better understand pollutant effects on immune system in bivalves. After 7 days of in vivo exposure to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations, phagocytosis was significantly reduced and 19 genes involved in C. gigas functions were down-regulated in treated animals [27]. Ref. [28]. investigated the effects of the same mixture of 8 pesticides on Pacific oyster, C. gigas, during a 7 day period. Enzyme activities including glutamine synthetase (GS), glutathione S-transferase (GST) and catalase (CAT), hemocyte parameters and DNA damages were assessed as effect biomarkers. GS, GST and CAT activities increased after pesticide exposure as well as DNA adducts. Moreover, the hemocyte phagocytic capacity decreased significantly after 7 days of the exposure. In the context of mass mortality outbreaks affecting the Pacific oyster, C. gigas, in France since 2008, Ref. [37] investigated effects on immune related gene expression, enzyme activities, and hemocyte parameters in animals exposed to diuron alone and to diuron, isoproturon, and ibuprofen as a mixture. Mortality outbreaks were mainly reported in spring and summer suggesting a putative role played by the seasonal use of pesticides and freshwater inputs in estuarine areas where oysters are frequently reared. Following exposure to diuron at 1 mg L(1), a reduction of gene expression and enzyme activities including laccase-type phenoloxidase (PO) activity and superoxide dismutase (SOD) activity was reported [37]. Catecholase-type PO activity and hemocyte phagocytosis were reduced after an exposure to the mixture containing the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen [37]. Effects of metaldehyde on Pacific oyster hemocyte parameters were monitored through in vivo experiments based on a short-term exposure by Ref. [41]. Metaldehyde is used to kill terrestrial gastropods and could be potentially more toxic to oysters than other pesticides (herbicides, fungicides, insecticides, …).
Metaldehyde at 0.1 mg L(1), which corresponds to an average concentration detected in the environment, modulated hemocyte activities of Pacific oysters. Ref. [16] explored the sublethal impact of azamethiphos, an organophosphate pesticide, on the blue mussel, Mytilus edulis, after a short term exposure. Azamethiphos is used to combat sea lice infestations in farmed salmonids. A significant reduction in acetylcholinesterase activity in haemolymph and gills, an alteration in cell viability and a decrease in phagocytic index were reported in mussels exposed to azamethiphos for periods of up to 24 h. These results suggested that hemocyte functions of blue mussels could be actively modulated by azamethiphos at environmentally relevant concentrations after only a few hours. [27] reported higher mortality in pesticide-treated Pacific oysters compared to untreated oysters after a bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Gene expression was also up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. As gene expression was up-regulated in pesticide-treated oysters compared to untreated ones after the bacterial challenge, Ref. [27] suggested that gene over-expression due to an interaction between pesticides and bacteria could be related to an injury of host tissues, resulting in higher mortality rates. Although this study reported an effect of pesticides at environmentally relevant concentrations on C. gigas susceptibility to an experimental bacterial infection, no other studies have been then published on such interactions. 3. Heavy metals Constant increase of industrial wastes, a source of heavy metals, results in pollutant transfer towards estuarine areas. Marine bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. Ref. [25] investigated the effects of cadmium and mercury (Hg) on defence mechanisms in the Pacific oysters, C. gigas. Pollutant effects were tested in vitro on oyster hemocytes. Although no effect of cadmium exposure was reported, mercury caused a significant hemocyte mortality after a 24 h in vitro incubation. The aminopeptidase positive cell percentage was enhanced in presence of this pollutant, and the phenoloxidase-like activity was inhibited. Ref. [44] reported high levels of apoptosis in Eastern oyster hemocytes after cadmium (Cd(2þ)) exposure (10e100 mmol L(-1)). Necrosis was observed at higher concentrations (200e1000 mmol L(1)). Enhanced apoptosis of hemocytes after Cd(2þ) exposure could induce immunosuppression and result in reduced disease resistance. Ref. [45] explored immunomodulation produced by metals in the green mussel, Perna viridis. Mussels were exposed to copper and mercury at 20 mg L(1) and 10 mg L(-1), respectively. Both metals affected adversely immune parameters (phenoloxidase, reactive oxygen species generation, and phagocytosis). Some level of recovery (depuration) from the toxic effects of metals was also observed. Ref. [33] reported significant effects on phagocytic activity and bacterial clearance in the blue mussel, M. edulis, after short-term exposure (1, 7 and 13 days) to low copper concentrations (5, 9 and 16 mg L(1)). These authors compared also immune responses of mussels kept at two salinities (12‰ and 20‰) and showed a significant interaction between salinity and copper exposure in terms of metal accumulation. Mussels kept at the lower salinity accumulated markedly more copper than mussels maintained at the highest one. Mercury (Hg) effects on the immunity of the bivalve Scrobicularia plana inhabiting a contaminated area (Laranjo basin, Ria de Aveiro, Portugal) were monitored by Ref. [2]. Animals collected from both moderately and highly contaminated sites demonstrated higher haemolymph heavy metal load and reduced plasma
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agglutination. The auhtors reported also some significant effects on hemocyte density, phagocytosis and oxidative burst activity [2]. Ref. [43] investigated the immunotoxic effects of sublethal Hg exposure in another bivalve species, the blue mussel, M. edulis. Mussels were exposed to a Hg concentration of 50 mg L(-1) as HgCl2 for up to 11 days. These auhtors subjected also mussels to an injection of lipopolysaccharide (LPS) prior to Hg exposure. Although a transient increase of phagocytosis was reported in mussels exposed to Hg alone, no effect on neutral red retention or hemocyte cytotoxicity was observed [43]. A transient decrease in superoxide dismutase activity in hemocytes was reported after LPS injection and Hg exposure. Tissue injuries were greatly increased by the effect of Hg exposure with an LPS injection compared to either treatment alone suggesting that LPS stimulation could increase Hgdependent immunotoxicity [2]. Chromium (Cr) is an important contaminant released from both domestic and industrial effluents and its in vitro and in vivo effects were recently evaluated on immune parameters of Mytilus galloprovincialis by Ref. [18]. Hemocyte incubation with Cr (0.1, 1, 10 and 100 mM) induced a dose-dependent decrease in the Lysosomal Membrane Stability (LMS). Although decreases in extracellular lysozyme release and phagocytic activity were observed, increases of extracellular superoxide production and nitrite accumulation were reported. Increasing metal concentrations were associated to decreases in hemocyte LMS values and in serum lysozyme activity in mussels exposed to Cr (0.1, 1 and 10 mg L(-1)) for 96 h. Decreased phagocytic activity, increased Nitric Oxide (NO) production and decreased Total Hemocyte Counts (THC) were also noticed. However, hemocyte necrosis/apoptosis were not detected. Ref. [18] reported also changes in the transcription of immune related genes (lysozyme, mytilin C, myticin B, defensin, MgC1q), the serotonin receptor (5-HTR) gene and of the stress protein HSP70 gene. The effects of Cu on apoptosis in hemocytes of the Eastern oyster, Crassostrea virginica, as well as on the establishment of Perkinsus marinus infections have been investigated by Ref. [22]. Perkinsus marinus, an intracellular protistan parasite, is the aetiological agent of the Dermo disease. Complex interactions between the host immunity and the parasite escape mechanisms, both of which can be influenced by environmental pollutants, are involved in the progression and the outcome of the disease. Although the Cu exposure induced an increase of apoptosis levels in hemocytes maintained in vitro, an apoptosis suppression was reported during heavy metal exposure of whole oysters. These opposing effects might rely on differences in terms of Cu bioavailability in vitro and in vivo. Growth of P. marinus as well as infection levels of hemocytes were reduced in vitro in presence of Cu. More recently [29], investigated effects of simultaneous exposure of Pacific oyster, C. gigas, to a harmful dinoflagellate, Alexandrium minutum, and cadmium (Cd) and copper (Cu) after 4 day exposure. Antagonistic effects on immune parameters (i.e. hemocyte concentration and phagocytosis) were reported after shortterm, simultaneous exposure to CdeCu and A. minutum. THC increased dramatically in oysters exposed to CdeCu alone. But when oysters were fed A. minutum, this effect was not observed revealing that exposure to the toxic algae interacted antagonistically with CdeCu exposure. A large decrease in hemocyte phagocytosis was reported after CdeCu exposure which indeed counteracted the stimulating effect of A. minutum on phagocytosis, revealing opposite effects [29]. 4. Polycyclic aromatic hydrocarbons (PAHs) PAHs are organic compounds formed during natural and industrial processes; they are termed “persistent organic pollutants” because they are only slowly degraded by natural processes. The
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current expansion of offshore oil activities in coastal regions supports the need to study the potential effects of these oil-related compounds on marine organisms. Ref. [3] investigated the in vitro effects of different PAHs on hemocyte and haemolymph parameters of the Pacific oyster, C. gigas. The phagocytosis activity was significantly reduced in presence of fluorene, pyrene and HFO. Naphthalene, benzofluoranthene and dibenzoanthracene were associated with an increase of the percentage of non-specific esterase positive cells, the phenoloxidase activity and the lysosome presence, respectively. Laboratory exposures of the Arctic scallop, Chlamys islandica, to sub-lethal dispersed oil concentrations (0.06 and 0.25 mg L(-1)) indicated alteration in the measured immune endpoints [30]. After a 15 day incubation followed by a 7-day recovery period in clean seawater, exposed scallops showed a significant increase of hemocyte counts whilst the cell membrane stability and phagocytosis demonstrated a significant reduction [30]. Ref. [32] explored the potential impact of an acute oil exposure mimicking an accidental spill on C. islandica and reported mortalities among exposed animals. Significant impairment of phagocytosis and cell membrane stability was again observed. Finally, prolonged sublethal effects were suggested as no recovery of immune functions was noticed. Sublethal effects (7-d exposure to 50, 100 and 200 mg L(1)) of phenanthrene, a major component of crude oil and one of the most abundant PAHs in aquatic ecosystems, were studied on immunological parameters and the oxidative stress in the scallop, Pecten maximus [31]. Significant reductions in LMS and phagocytosis, and a significant increase in the THC were noticed after pollutant exposure at 200 mg L(1). A significant decrease in total glutathione and significantly increased levels of lipid peroxidation were also reported in the hemolymph of phenanthrene-exposed scallops. These results suggested a possible link between phenanthreneinduced oxidative stress and the subsequent inhibition in some hemocyte functions. Clams, Mya arenaria, exposed to pyrene via the phytoplankton showed a decrease of phagocytosis [24]. More recently, to determine if dietary accumulation is a route of contaminant exposure [21], studied the transfer of PAH compounds (naphthalene, pyrene, and benzo(a)pyrene) by a microphytobenthic diatom, Nitzschia brevirostris, to the Eastern oyster, C. virginica. Diatom cultures were exposed to a range of PAH concentrations (5, 125, 625, and 1000 mg L(1)). Effects on hemocytes were assessed in oysters exposed to the contaminated diatom cultures. Hemocyte responses to dietary PAH exposure included an increase in circulating hemocytes and an increased production of Reactive Oxygen Species (ROS). Ref. [42] explored putative immunity alteration related to the Prestige oil spill in the mussel M. galloprovincialis. For this purpose, mussels were exposed for a 4 month period to Prestige fuel oil. Hemocyte viability, phagocytic activity, nitric oxide production, and chemiluminescence emission showed no significant difference between fuel-treated and control animals. In addition, no tissue lesions were reported by histology in any of the samples. In contrast, the serum protein concentration and the lysozyme activity were found significantly different between fuel oil-exposed mussels and controls. Ref. [47] developed a study to compare effects of phenanthrene at different concentrations (50, 100, 200 or 400 mg L(1)) on the immunocompetence of three bivalve species, M. edulis, the edible cockle, Cerastoderma edule and the razor shell, Ensis siliqua. After phenanthrene exposure immunomodulation reported in M. edulis was different from the immunological changes reported in the other two species suggesting that several species need to be used as sentinel bivalves to reflect harmful effects of pollutants present in marine waters. As an example, a significant reduction in phagocytic
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hemocytes was only observed in C. edule. In contrast minimal alterations to phagocytosis were observed in M. edulis, suggesting that the process in this bivalve was resilient to the phenanthrene concentrations used. Effects of benzo(a)pyrene (0.5 mg L(-1)) on hemocyte parameters were also investigated in the clam Chamelea gallina [38,39]. After pollutant exposure a significant decrease of phagocytic activity, adhesion capability and lysozyme activity was noted. These results suggested that benzo(a)pyrene induced immunomodulation in C. gallina. 5. Nanoparticles The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. In mammalian cells, nanoparticles have been shown to induce inflammation and oxidative stress, and changes in cell signaling and gene expression. Due to the growing concern over biological effects of NPs in the aquatic environment, a better understanding of their interactions with marine organisms is needed. As sessile filter feeders, marine bivalves are likely to be exposed to NPs suspended in the water column. Several studies have been published recently on the topic, particularly in 2013 and 2014. In 2008, Canesi et al. reported effects of commercial nanosized carbon black (NCB) on mussel hemocytes and suggested that bivalve hemocytes can represent a suitable model for investigating the effects and modes of action of nanoparticles in aquatic invertebrates [11]. They showed that exposure to NCB (1, 5, and 10 mg mL(-1)) was associated with an increase in extracellular lysozyme release, extracellular oxyradical production, and NO release. Flow cytometry analysis showed a decrease of mitochondrial mass/number and membrane potential in NCB-exposed cells. Finally, an activation of the stress-activated Mitogen Activated Protein Kinases (MAPKs) p38 and c-Jun N-terminal kinases (JNKs) was also reported and could explain the NCB effects. The potential effects of other types of commercial NPs (C60 fullerene, TiO(2) and SiO(2) at 1, 5, 10 mg mL(-1)) have been then explored on Mytilus hemocytes [12]. Lysozyme release, extracellular oxyradical and NO production were reported after cell exposure to the NPs tested as well as a rapid activation of the stress-activated p38 MAPK. In vivo exposures to NCB, C60 fullerene, TiO(2) and SiO(2) for 24 h were also conducted in mussels using different concentrations (0.05e0.2e1e5 mg L(1)) [13]. Lysosomal membrane destabilisation in the hemocytes was reported for all NP suspensions. Summarizing existing data [14], reported that isolated mussel hemocytes represent a significant target for NPs and intra-cellular uptake of nanosized materials induces lysosomal perturbations and oxidative stress in the digestive gland after NP agglomerates/ aggregates taken up by the gills. Ref. [19] carried out an additional study to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, and n-CeO(2)) on M. galloprovincialis hemocytes and showed that differential responses of hemocytes were elicited by the different n-oxides. Ref. [1] reported also effects of silver NPs, titanium dioxide (TiO(2)) NPs, and silver nitrate on hemocyte phagocytosis in the Eastern oyster, C. virginica. The in vivo effects of n-TiO2 were investigated in mussels, M. galloprovincialis, exposed for 96 h to different concentrations of NP suspensions (1, 10 and 100 mg L(-1)) [5]. A decrease of LMSand phagocytosis was observed in n-TiO2-exposed hemocytes. The ROSproduction and the transcription of antimicrobial peptides were increased. Different functional and molecular parameters of mussel hemocytes were thus significantly affected by n-TiO2, at concentrations close to predicted environmental levels. Ref. [6] carried out a study to compare effects of cadmium sulfate/cadmium telluride (CdS/CdeTe) mixture quantum dots (QDs) and their dissolved components, cadmium chloride/sodium
telluride salts, and a mixture of these dissolved components. They selected four animal models: one bivalve (M. edulis), one fish (Oncorhynchus mykiss), and two mammals (mice and humans). For blue mussels, results showed that viability and phagocytosis were more affected by QDs than by dissolved metals. These auhtors suggested that aquatic species responded more differently than vertebrates to NP. Ref. [35] investigated cytotoxicity of CdS quantum dots (QDs) at a wide range of concentrations (0.001e100 mg Cd L(1)) in isolated hemocytes and gill cells from the mussel, M. galloprovincialis. A series of functional in vitro assays was carried out in the same cell types to better define the mechanisms of action of CdS QDs (0.31e5 mg Cd L(-1)). Significant increases of ROS production, DNA damage, acid phosphatase (AcP) activity and multixenobiotic resistance (MXR) transport were observed in both cell types exposed to the three forms of Cd (ionic Cd, CdS QDs and bulk CdS). Although an increase of CAT activity was reported in hemocytes exposed to the three forms of Cd, the cytoskeleton integrity of hemocytes was not affected. Exposure to CdS and to CdS QDs at concentrations equal or higher than 1.25 mg Cd L(-1) was associated with effects on hemocyte phagocytosis. However, phagocytosis was not modified in hemocytes exposed to ionic Cd suggesting a particle-specific effect [35]. Ref. [15] explored the possible interactive effects of n-TiO2, one of the most widespread NP in use, and 2,3,7,8-TCDD in M. galloprovincialis based on in vitro and in vivo experiments. For in vivo assays, mussels were exposed to n-TiO2 (100 mg L(-1)) or to 2,3,7,8-TCDD (0.25 mg L(1)), alone and in combination, for 96 h. The authors reported synergistic or antagonistic effects between both contaminants. Although neither contaminant alone was able to induce lysosomal enlargement, exposure to n-TiO2/TCDD resulted in a significant increase in lysosome/cytoplasm volume ratio, indicating a synergistic effect of the two contaminants. Additionally, one of the most important results relies on observation of a significant increase in 2,3,7,8-TCDD accumulation in mussels in the presence of n-TiO2. Recently [46], studied the combined effects of oxygen levels (hypoxia: 1.5 mg O2 L(-1), normoxia: 6.0 mg O2 L(1)) and TiO2 (0, 2.5 mg L(1) and 10 mg L(-1)) for 216 h on hemocytes in the green-lipped mussel, P. viridis. The authors reported synergistic effects between the two stressors on hemocyte functions. Significant interactive effects of the oxygen level and TiO2 were thus observed for the percentage of hemocytes showing non-specific esterase activity, ROS production, lysosomal content and THC. 6. Pharmaceuticals The in vitro effects of individual estrogenic chemicals (17alphaethynyl estradiol, mestranol, nonylphenol, nonylphenol monoethoxylate carboxylate, bisphenol A; benzophenone) on mussel hemocyte parameters were compared [9]. The LMS appeared the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. Ref. [38,39] investigated the in vivo effects of 4-nonylphenol at sublethal concentrations (0.0125, 0.025, 0.05 and 0.1 mg L(-1)) on hemocyte parameters from the cockle Cerastoderma glaucum after 7 days exposure. A significant increased of THC was reported after the exposure of cockles to 0.1 mg L(-1). Significant increases in acid phosphatase activity and lysozyme-like activity were observed for nonyphenolexposed cockles (0.05 and 0.1 mg L (1)). The possible effects of two hypolipidemic pharmaceuticals (fibrates), Bezafibrate (BEZA) and Gemfibrozil (GEM), were investigated in the bivalve mollusc Mytilus spp by Ref. [10]. Isolated hemocytes showed rapid lysosomal membrane destabilization, extracellular lysozyme release, NO production and decreased phagocytic activity in presence of both compounds. Activation of
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extracellularly regulated kinase (ERK) and p38 MAPKs could partly explain the effects of BEZA and GEM on hemocytes. The effects of both pharmaceuticals on hemocyte functions were confirmed in vivo, in the hemocytes of mussels injected with 0.01, 0.1 and 1 nmol/animal and sampled at 24h post-injection. Lysosomal destabilization and extracellular lysozyme release were reported in a concentration-dependent way for both compounds. Phagocytosis was increased at the highest concentration. 7. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) PCBs are industrial chemicals which are released into the environment. PCBs exist as different congeners depending on the chlorine substitution on the biphenyl rings. Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame-retardants (BFRs) that are widely used in industrial products and pose potential risk on the coastal environment. Ref. [8] explored effects of individual ortho-substituted non coplanar PCB congeners on the function of M. galloprovincialis hemocytes and the signal transduction pathways involved in the immune response. Bacterial killing and lysosomal enzyme release were affected by the di-ortho-substituted, non coplanar PCB congeners P47 (2,20 ,4,4'-tetrachlorobiphenyl) and P153 (2,20 ,4,40 ,5,5'hexachlorobiphenyl), respectively. However, the non-ortho, coplanar congener P77 (3,30 ,4,40 -tetrachlorobiphenyl) did not modify these activities. Hemocyte LMS was also significantly reduced in presence of the three molecules. Functional differences between different PCB congeners were thus reported with respect to the hemocyte functions. Tyrosine kinase-mediated cell signaling was affected by P47, P153 and P77 with an increase in the phosphorylation level of the stress activated p38 and JNK MAPKs. The level of phosphorylated ERK MAPKs was also increased in presence of P153. Finally, an increase of tyrosine phosphorylation of the Signal Transducer and Activator of Transcription 5 (STAT5) in presence of non coplanar P47 and P153 was observed. These results suggested that MAPKs, key elements in the response of mussel hemocytes to bacterial infection, are a target for different PCB congeners [36]. reported significant effects of Aroclor 1254 on Chlamys farreri immune system in vivo. Although the THC, the proportion of granulocytes and phagocytosis decreased significantly after the PCB exposure, the proportion of hyalinocytes and the production of O2()increased during the sampling time. The auhtors suggested that a relationship might exist between pollution, immunomodulation and mass mortality events affecting C. farreri in China [36]. In a recent study, it has been reported that BDE 47 induced cell apoptosis and reduced ROS in mussel M. galloprovincialis [34]. 8. Conclusion Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. The presence of diverse pollutants in aquatic ecosystems is a growing issue and, pollution of these ecosystems is alarming. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. Despite the difficulty of working in complex environments such as estuaries, there is a need to understand the effect of xenobiotics in these exposed natural environments where exposure to mixture of substances, biotic and abiotic factor interactions, and seasonal fluctuations occur. Bivalves are commonly used as bio-indicators of marine pollution, and immunomodulation due to toxicants is one of the important bio-markers used. Laboratory experiments remain a necessary step to study regulation mechanisms and to validate
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biomarkers. However, there are a number of uncertainties associated with their use. Although animals are exposed to complex mixtures of pollutants in the environment, most of laboratory experiments are based on the use of a unique pollutant. A natural or endogenous factors that may modify the responses of biomarkers to environmental toxicants, may lead to misinterpretation if their influence is not taken into account. Using suites of biomarkers in combination with the measurement of pertinent environmental parameters is more likely to provide a good assessment of the effects of environmental toxicants. The use of contrasted sample sites regarding environmental conditions may help to discriminate between the influence of anthropogenic inputs and natural variations. Various ways including integrated approaches can be used to improve the accuracy of ecotoxicological studies (i.e., use of passive organic samplers, analysis of physiological status of animals, accurate investigation of the influence of abiotic factors). The development of an infectious disease results from an unbalance between the host and the pathogen due to external factors (including pollutants) and/or internal factors of both protagonists (virulence of the pathogen, susceptibility of the host). Animals with impaired defence mechanisms may be more susceptible to infectious diseases. Demonstration of the relationship between pollution and increase of susceptibility to infectious diseases exist in vertebrates. Marine pollution may be one of the reasons for disease incidence in marine organisms, which may be caused due to adverse effects of pollutants on the immune system. However, few studies have been developed to better define interactions between immunocompetence, disease susceptibility and pollutants in marine bivalves. Thus, there is an urgent need for development of specific research on this topic. New insights into the potential role of environmental pollutants in host-pathogen relationships and disease dynamics in marine bivalves are needed. References [1] T.E. Abbott Chalew, J.F. Galloway, T.K. Graczyk, Pilot study on effects of nanoparticle exposure on Crassostrea virginica hemocyte phagocytosis, Mar. Pollut. Bull. 64 (10) (2012) 2251e2253, http://dx.doi.org/10.1016/ j.marpolbul.2012.06.026. [2] I. Ahmad, J.P. Coelho, I. Mohmood, M. Pacheco, M.A. Santos, A.C. Duarte, E. Pereira, Immunosuppression in the infaunal bivalve Scrobicularia plana environmentally exposed to mercury and association with its accumulation, Chemosphere 82 (11) (2011) 1541e1546, http://dx.doi.org/10.1016/ j.chemosphere.2010.11.064. [3] A. Bado-Nilles, B. Gagnaire, H. Thomas-Guyon, S. Le Floch, T. Renault, Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg), Toxicol. Vitro. 22 (6) (2008) 1610e1617, http://dx.doi.org/10.1016/j.tiv.2008.04.011. [4] B.D. Banerjee, B.C. Koner, A. Ray, Immunotoxicity of pesticides: perspectives and trends, Indian J. Exp. Biol. 34 (1996) 723e733. [5] C. Barmo, C. Ciacci, B. Canonico, R. Fabbri, K. Cortese, T. Balbi, A. Marcomini, G. Pojana, G. Gallo, L. Canesi, In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve Mytilus galloprovincialis, Aquat. Toxicol. 132e133 (2013) 9e18, http://dx.doi.org/10.1016/j.aquatox.2013.01.014. [6] A. Bruneau, M. Fortier, F. Gagne, C. Gagnon, P. Turcotte, A. Tayabali, T.A. Davis, M. Auffret, M. Fournier, In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium, Environ. Toxicol. (2013), http://dx.doi.org/10.1002/tox.21890. [7] M.P. Cajaraville, I. Olabarrieta, I. Marigomez, In vitro activities in mussel hemocytes as biomarkers of environmental quality: a case study in the Abra estuary (Biscay Bay), Ecotox. Environ. Saf 35 (1996) 253e260. [8] L. Canesi, C. Ciacci, M. Betti, A. Scarpato, B. Citterio, C. Pruzzo, G. Gallo, Effects of PCB congeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cell signaling, Aquat. Toxicol. 63 (3) (2003) 293e306. [9] L. Canesi, L.C. Lorusso, C. Ciacci, M. Betti, M. Rocchi, G. Pojana, A. Marcomini, Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies, Aquat. Toxicol. 81 (1) (2007a) 36e44. [10] L. Canesi, L.C. Lorusso, C. Ciacci, M. Betti, F. Regoli, G. Pojana, G. Gallo, A. Marcomini, Effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, Mytilus galloprovincialis, Chemosphere 69 (6) (2007b) 994e1002.
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[11] L. Canesi, C. Ciacci, M. Betti, R. Fabbri, B. Canonico, A. Fantinati, A. Marcomini, G. Pojana, Immunotoxicity of carbon black nanoparticles to blue mussel hemocytes, Environ. Int. 34 (8) (2008) 1114e1119, http://dx.doi.org/10.1016/ j.envint.2008.04.002. [12] L. Canesi, C. Ciacci, D. Vallotto, G. Gallo, A. Marcomini, G. Pojana, In vitro effects of suspensions of selected nanoparticles (C60 fullerene, TiO2, SiO2) on Mytilus hemocytes, Aquat. Toxicol. 96 (2) (2010a) 151e158, http://dx.doi.org/ 10.1016/j.aquatox.2009.10.017. [13] L. Canesi, R. Fabbri, G. Gallo, D. Vallotto, A. Marcomini, G. Pojana, Biomarkers in Mytilus galloprovincialis exposed to suspensions of selected nanoparticles (Nano carbon black, C60 fullerene, Nano-TiO2, Nano-SiO2), Aquat. Toxicol. 100 (2) (2010b) 168e177, http://dx.doi.org/10.1016/j.aquatox.2010.04.009. [14] L. Canesi, C. Ciacci, R. Fabbri, A. Marcomini, G. Pojana, G. Gallo, Bivalve molluscs as a unique target group for nanoparticle toxicity, Mar. Environ. Res. 76 (2012) 16e21, http://dx.doi.org/10.1016/j.marenvres.2011.06.005. [15] L. Canesi, G. Frenzilli, T. Balbi, M. Bernardeschi, C. Ciacci, S. Corsolini, C. Della Torre, R. Fabbri, C. Faleri, S. Focardi, P. Guidi, A. Ko can, A. Marcomini, M. Mariottini, M. Nigro, K. Pozo-Gallardo, L. Rocco, V. Scarcelli, A. Smerilli, I. Corsi, Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis, Aquat. Toxicol. 153 (2014) 53e65, http://dx.doi.org/ 10.1016/j.aquatox.2013.11.002. [16] M.N. Canty, J.A. Hagger, R.T. Moore, L. Cooper, T.S. Galloway, Sublethal impact of short term exposure to the organophosphate pesticide azamethiphos in the marine mollusc Mytilus edulis, Mar. Pollut. Bull. 54 (4) (2007) 396e402. [17] H.G. Choi, H.B. Moon, M. Choi, J. Yu, S.S. Kim, Mussel watch program for organic contaminants along the Korean coast, 2001e2007, Arch. Environ. Contam. Toxicol. 58 (4) (2010) 973e984. [18] C. Ciacci, C. Barmo, R. Fabbri, B. Canonico, G. Gallo, L. Canesi, Immunomodulation in Mytilus galloprovincialis by non-toxic doses of hexavalent chromium, Fish. Shellfish. Immunol. 31 (6) (2011) 1026e1033, http://dx.doi.org/10.1016/ j.fsi.2011.09.002. [19] C. Ciacci, B. Canonico, D. Bilani^ cov a, R. Fabbri, K. Cortese, G. Gallo, A. Marcomini, G. Pojana, L. Canesi, Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis, PLoS One 7 (5) (2012) e36937, http://dx.doi.org/10.1371/journal.pone.0036937. [20] J.A. Coles, R.K. Pipe, Phenoloxidase activity in the hemolymph and hemocytes of the marine mussel Mytilus edulis, Fish Shellfish Immunol. 4 (1994) 337e352. [21] A.N. Croxton, G.H. Wikfors, R.D. Schulterbrandt-Gragg, Immunomodulation in eastern oysters, Crassostrea virginica, exposed to a PAH-contaminated, microphytobenthic diatom, Aquat. Toxicol. 118e119 (2012) 27e36, http:// dx.doi.org/10.1016/j.aquatox.2012.02.023. [22] B. Foster, S. Grewal, O. Graves, F.M. Hughes Jr., I.M. Sokolova, Copper exposure affects hemocyte apoptosis and Perkinsus marinus infection in eastern oysters Crassostrea virginica (Gmelin), Fish Shellfish Immunol. 31 (2) (2011) 341e349, http://dx.doi.org/10.1016/j.fsi.2011.05.024. [23] M. Fournier, D. Cyr, B. Blakley, H. Boermans, P. Brousseau, Phagocytosis as a biomarker of immunotoxicity in wildlife species exposed to environmental xenobiotics, Am. Zoologist 40 (2000) 412e420. [24] H. Frouin, J. Pellerin, M. Fournier, E. Pelletier, P. Richard, N. Pichaud, C. Rouleau, F. Garnerot, Physiological effects of polycyclic aromatic hydrocarbons on soft-shell clam Mya arenaria, Aquat. Toxicol. 82 (2) (2007) 120e134. [25] B. Gagnaire, H. Thomas-Guyon, T. Renault, In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes, Fish Shellfish Immunol. 16 (4) (2004) 501e512. [26] B. Gagnaire, H. Thomas-Guyon, T. Burgeot, T. Renault, Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: screening of 23 molecules using flow cytometry, Cell Biol. Toxicol. 22 (1) (2006) 1e14. [27] B. Gagnaire, M. Gay, A. Huvet, J.Y. Daniel, D. Saulnier, T. Renault, Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg), Aquat. Toxicol. 84 (1) (2007) 92e102. [28] F. Geret, T. Burgeot, J. Haure, B. Gagnaire, T. Renault, P.Y. Communal, J.F. Samain, Effects of low-dose exposure to pesticide mixture on physiological responses of the Pacific oyster, Crassostrea gigas, Environ. Toxicol. 28 (12) (2013) 689e699, http://dx.doi.org/10.1002/tox.20764. r e , A. Bruneau, R. Riso, M. Auffret, [29] H. Haberkorn, C. Lambert, N. Le Goïc, C. Que P. Soudant, Cellular and biochemical responses of the oyster Crassostrea gigas to controlled exposures to metals and Alexandrium minutum, Aquat. Toxicol. 147 (2014) 158e167, http://dx.doi.org/10.1016/j.aquatox.2013.12.012.
[30] M.L. Hannam, S.D. Bamber, J.A. Moody, T.S. Galloway, M.B. Jones, Immune function in the Arctic Scallop, Chlamys islandica, following dispersed oil exposure, Aquat. Toxicol. 92 (3) (2009) 187e194, http://dx.doi.org/10.1016/ j.aquatox.2009.01.010. [31] M.L. Hannam, S.D. Bamber, T.S. Galloway, A. John Moody, M.B. Jones, Effects of the model PAH phenanthrene on immune function and oxidative stress in the haemolymph of the temperate scallop Pecten maximus, Chemosphere 78 (7) (2010a) 779e784, http://dx.doi.org/10.1016/j.chemosphere.2009.12.049. [32] M.L. Hannam, S.D. Bamber, A.J. Moody, T.S. Galloway, M.B. Jones, Immunotoxicity and oxidative stress in the Arctic scallop Chlamys islandica: effects of acute oil exposure, Ecotoxicol. Environ. Saf. 73 (6) (2010b) 1440e1448, http:// dx.doi.org/10.1016/j.ecoenv.2010.06.012. €her, F. Regoli, A. Dissanayake, M. Nagel, M. Kriews, A. Ko € hler, K. Broeg, [33] N. Ho Immunomodulating effects of environmentally realistic copper concentrations in Mytilus edulis adapted to naturally low salinities, Aquat. Toxicol. 140e141 (2013) 185e195, http://dx.doi.org/10.1016/j.aquatox.2013.06.001. [34] C. Ji, H. Wu, L. Wei, J. Zhao, J. Yu, Proteomic and metabolomic analysis reveal gender-specific responses of mussel Mytilus galloprovincialis to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47), Aquat. Toxicol. 140e141 (2013) 449e457, http://dx.doi.org/10.1016/j.aquatox.2013.07.009. [35] A. Katsumiti, D. Gilliland, I. Arostegui, M.P. Cajaraville, Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis, Aquat. Toxicol. 53 (2014) 39e52, http://dx.doi.org/10.1016/j.aquatox.2014.02.003. [36] J. Liu, L.Q. Pan, L. Zhang, J. Miao, J. Wang, Immune responses, ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254, Fish Shellfish Immunol. 26 (3) (2009) 422e428, http://dx.doi.org/ 10.1016/j.fsi.2009.01.002. [37] A. Luna-Acosta, T. Renault, H. Thomas-Guyon, N. Faury, D. Saulnier, H. Budzinski, K. Le Menach, P. Pardon, I. Fruitier-Arnaudin, P. Bustamante, Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat, Chemosphere 87 (11) (2012) 1335e1340, http://dx.doi.org/10.1016/j.chemosphere.2012.02.022. [38] V. Matozzo, G. Rova, F. Ricciardi, M.G. Marin, Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum, Mar. Pollut. Bull. 57 (6e12) (2008a) 453e459, http://dx.doi.org/10.1016/j.marpolbul.2008.02.019. [39] V. Matozzo, M. Monari, J. Foschi, O. Cattani, G.P. Serrazanetti, M.G. Marin, First evidence of altered immune responses and resistance to air exposure in the clam Chamelea gallina exposed to benzo(a)pyrene, Arch. Environ. Contam. Toxicol. 56 (3) (2008b) 479e488, http://dx.doi.org/10.1007/s00244-008-9212-8. [40] P. Moreau, T. Burgeot, T. Renault, Pacific oyster (Crassostrea gigas) hemocytes are not affected by a mixture of pesticides in short-term in vitro assays, Environ. Sci. Pollut. Res. Int. 21 (7) (2014a) 4940e4949, http://dx.doi.org/ 10.1007/s11356-013-1931-3. [41] P. Moreau, T. Burgeot, T. Renault, In vivo effects of metaldehyde on Pacific oyster, Crassostrea gigas: comparing hemocyte parameters in two oyster families, Environ. Sci. Pollut. Res. Int. (2014b) http://dx.doi.org/10.1007/ s11356-014-3162-7. s, J.M. Bayona, A. Ord [42] M.C. Ord as, J. Albaige as, A. Figueras, Assessment of in vivo effects of the prestige fuel oil spill on the mediterranean mussel immune system, Arch. Environ. Contam. Toxicol. 52 (2) (2007) 200e206. [43] S.K. Sheir, R.D. Handy, T.S. Galloway, Tissue injury and cellular immune responses to mercuric chloride exposure in the common mussel Mytilus edulis: modulation by lipopolysaccharide, Ecotoxicol. Environ. Saf. 73 (6) (2010) 1338e1344, http://dx.doi.org/10.1016/j.ecoenv.2010.01.014. [44] I.M. Sokolova, S. Evans, F.M. Hughes, Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition, J. Exp. Biol. 207 (2004) 3369e3380. [45] R. Thiagarajan, S. Gopalakrishnan, H. Thilagam, Immunomodulation the marine green mussel Perna viridis exposed to sub-lethal concentrations of Cu and Hg, Arch. Environ. Contam. Toxicol. 51 (3) (2006) 392e399. [46] Y. Wang, M. Hu, Q. Li, J. Li, D. Lin, W. Lu, Immune toxicity of TiO2 under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters, Sci. Total Environ. 470e471 (2014) 791e799, http://dx.doi.org/10.1016/j.scitotenv.2013.09.060. [47] E.C. Wootton, E.A. Dyrynda, R.K. Pipe, N.A. Ratcliffe, Comparisons of PAHinduced immunomodulation in three bivalve molluscs, Aquat. Toxicol. 65 (1) (2003) 13e25.
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Immunotoxicological effects of environmental contaminants on marine bivalves T. Renault* Ifremer, D epartement Ressources Biologique et Envrionnement, Rue de l'^Ile d'Yeu, 44300 Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 November 2014 Received in revised form 6 April 2015 Accepted 12 April 2015 Available online xxx
Coastal areas are complex environments frequently contaminated by numerous pollutants that represent a potential threat to marine organisms, especially bivalves. These pollutants may have major ecological consequences. Although effects of different environmental contaminants on the immune system in marine bivalves have been already reported, a few of reviews summarizes these effects. The main purpose of this chapter relies on summarizing recent body of data on immunotoxicity in bivalves subjected to contaminants. Immune effects of heavy metals, pesticides, HAP, PCB and pharmaceuticals are presented and discussed and a particular section is devoted to nanoparticle effects. A large body of literature is now available on this topic. Finally, the urgent need of a better understanding of complex interactions between contaminants, marine bivalves and infectious diseases is noticed. © 2015 Published by Elsevier Ltd.
Keywords: Marine bivalves Pollutants Nanoparticles Immunotoxicology Diseases Pathogens
1. Introduction Coastal areas are complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Contaminants are frequently detected in these areas and represent a potential threat to marine organisms, especially bivalves. Pollutants may have major ecological consequences [4]. They can endanger organism growth, reproduction or survival. Their effects may result from direct toxic actions or from alterations of the homeostatic mechanisms including the immune system [7,20]. Among physiological processes possibly disturbed by pollutants, the immune system is likely to be one of the more sensitive [23]. Bivalves filter large volumes of seawater and their immune capacities can be adversely affected by exposure to contaminants. Therefore, bivalves have to face highly variable environmental conditions. Investigating immunity toxicity can provide relevant information on the quality of the marine environment and facilitate understanding of the occurrence of infectious diseases affecting bivalves in coastal areas. Bivalves in culture may be weakened after contaminant exposure, potentially increasing their susceptibility to
* Tel.: þ33 2 40 37 40 00; fax: þ33 2 40 37 40 01. E-mail address: [email protected].
infectious diseases. It has been reported that a mixture of pesticides could induce a decrease of immune defenses in the Pacific oyster, Crassostrea gigas, rendering animals more vulnerable to an experimental bacterial infection [27]. In bivalves, immunity is mainly supported by cells called hemocytes found in the open hemolymphatic circulatory system. Hemocytes have been widely used to explore pollutant effects on bivalve immunity. The monitoring of health conditions by the assessment of marine bivalve immunocompetence may serve as a criterion for the achievement of the Good Environmental Status as defined in the EU Marine Strategy Framework Directive. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their wide geographical distribution, and sensitivity to environmental pollutants [17]. Moreover, the development of techniques allowing the analysis of pollutant effects on bivalve immunity may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, the effects of different environmental contaminants on the immune system of marine bivalves have been already reported. The condition of the immune system determines partly susceptibility to disease and survival. Measuring endpoints linked to immunity can help to identify sub-lethal effects of exposure to contaminants and provide early warning signals. However, there are at present a few of reviews summarizing these effects. The main purpose of this chapter relies on summarizing recent body of data
http://dx.doi.org/10.1016/j.fsi.2015.04.011 1050-4648/© 2015 Published by Elsevier Ltd.
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on immunotoxicity in bivalves subjected to contaminants. Immune effects of heavy metals, pesticides, HAP, PCB and pharmaceuticals are presented and discussed and a particular section is devoted to the effects of nanoparticles. A large body of literature is now available on this topic. Finally, the urgent need of a better understanding of complex interactions between contaminants, marine bivalves and their infectious agents is identified. 2. Pesticides Pesticides are used especially in agriculture. They are defined as chemical substances used to kill pests including insects, weeds, parasites or rodents. All pesticides act by interfering with the target species normal metabolism. However, some inadvertently effects may affect other organisms in the environment, either directly by their toxic effects or via elimination of the target organism. Hemocyte activities have been extensively studied in order to investigate the effects of pesticides on bivalve immunity. As an example, 23 pollutants including several pesticides have been tested on Pacific oyster hemocytes maintained in vitro through monitoring of cell parameters by flow cytometry [26]. Several pesticides have been shown to induce a modulation of some hemocyte activities including cell mortality and non specific esterase activities. However [40], reported no significant effect of a mixture of 14 pesticides and methaldehyde alone tested at different concentrations on Pacific oyster hemocytes maintained in vitro for short-term periods. Several cell parameters including dead/alive cells, non specific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. Pacific oyster hemocytes appeared thus resistant to a pesticide exposure in the tested conditions [40]. Developing in vivo assays can be useful to better understand pollutant effects on immune system in bivalves. After 7 days of in vivo exposure to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations, phagocytosis was significantly reduced and 19 genes involved in C. gigas functions were down-regulated in treated animals [27]. Ref. [28]. investigated the effects of the same mixture of 8 pesticides on Pacific oyster, C. gigas, during a 7 day period. Enzyme activities including glutamine synthetase (GS), glutathione S-transferase (GST) and catalase (CAT), hemocyte parameters and DNA damages were assessed as effect biomarkers. GS, GST and CAT activities increased after pesticide exposure as well as DNA adducts. Moreover, the hemocyte phagocytic capacity decreased significantly after 7 days of the exposure. In the context of mass mortality outbreaks affecting the Pacific oyster, C. gigas, in France since 2008, Ref. [37] investigated effects on immune related gene expression, enzyme activities, and hemocyte parameters in animals exposed to diuron alone and to diuron, isoproturon, and ibuprofen as a mixture. Mortality outbreaks were mainly reported in spring and summer suggesting a putative role played by the seasonal use of pesticides and freshwater inputs in estuarine areas where oysters are frequently reared. Following exposure to diuron at 1 mg L(1), a reduction of gene expression and enzyme activities including laccase-type phenoloxidase (PO) activity and superoxide dismutase (SOD) activity was reported [37]. Catecholase-type PO activity and hemocyte phagocytosis were reduced after an exposure to the mixture containing the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen [37]. Effects of metaldehyde on Pacific oyster hemocyte parameters were monitored through in vivo experiments based on a short-term exposure by Ref. [41]. Metaldehyde is used to kill terrestrial gastropods and could be potentially more toxic to oysters than other pesticides (herbicides, fungicides, insecticides, …).
Metaldehyde at 0.1 mg L(1), which corresponds to an average concentration detected in the environment, modulated hemocyte activities of Pacific oysters. Ref. [16] explored the sublethal impact of azamethiphos, an organophosphate pesticide, on the blue mussel, Mytilus edulis, after a short term exposure. Azamethiphos is used to combat sea lice infestations in farmed salmonids. A significant reduction in acetylcholinesterase activity in haemolymph and gills, an alteration in cell viability and a decrease in phagocytic index were reported in mussels exposed to azamethiphos for periods of up to 24 h. These results suggested that hemocyte functions of blue mussels could be actively modulated by azamethiphos at environmentally relevant concentrations after only a few hours. [27] reported higher mortality in pesticide-treated Pacific oysters compared to untreated oysters after a bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Gene expression was also up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. As gene expression was up-regulated in pesticide-treated oysters compared to untreated ones after the bacterial challenge, Ref. [27] suggested that gene over-expression due to an interaction between pesticides and bacteria could be related to an injury of host tissues, resulting in higher mortality rates. Although this study reported an effect of pesticides at environmentally relevant concentrations on C. gigas susceptibility to an experimental bacterial infection, no other studies have been then published on such interactions. 3. Heavy metals Constant increase of industrial wastes, a source of heavy metals, results in pollutant transfer towards estuarine areas. Marine bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. Ref. [25] investigated the effects of cadmium and mercury (Hg) on defence mechanisms in the Pacific oysters, C. gigas. Pollutant effects were tested in vitro on oyster hemocytes. Although no effect of cadmium exposure was reported, mercury caused a significant hemocyte mortality after a 24 h in vitro incubation. The aminopeptidase positive cell percentage was enhanced in presence of this pollutant, and the phenoloxidase-like activity was inhibited. Ref. [44] reported high levels of apoptosis in Eastern oyster hemocytes after cadmium (Cd(2þ)) exposure (10e100 mmol L(-1)). Necrosis was observed at higher concentrations (200e1000 mmol L(1)). Enhanced apoptosis of hemocytes after Cd(2þ) exposure could induce immunosuppression and result in reduced disease resistance. Ref. [45] explored immunomodulation produced by metals in the green mussel, Perna viridis. Mussels were exposed to copper and mercury at 20 mg L(1) and 10 mg L(-1), respectively. Both metals affected adversely immune parameters (phenoloxidase, reactive oxygen species generation, and phagocytosis). Some level of recovery (depuration) from the toxic effects of metals was also observed. Ref. [33] reported significant effects on phagocytic activity and bacterial clearance in the blue mussel, M. edulis, after short-term exposure (1, 7 and 13 days) to low copper concentrations (5, 9 and 16 mg L(1)). These authors compared also immune responses of mussels kept at two salinities (12‰ and 20‰) and showed a significant interaction between salinity and copper exposure in terms of metal accumulation. Mussels kept at the lower salinity accumulated markedly more copper than mussels maintained at the highest one. Mercury (Hg) effects on the immunity of the bivalve Scrobicularia plana inhabiting a contaminated area (Laranjo basin, Ria de Aveiro, Portugal) were monitored by Ref. [2]. Animals collected from both moderately and highly contaminated sites demonstrated higher haemolymph heavy metal load and reduced plasma
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agglutination. The auhtors reported also some significant effects on hemocyte density, phagocytosis and oxidative burst activity [2]. Ref. [43] investigated the immunotoxic effects of sublethal Hg exposure in another bivalve species, the blue mussel, M. edulis. Mussels were exposed to a Hg concentration of 50 mg L(-1) as HgCl2 for up to 11 days. These auhtors subjected also mussels to an injection of lipopolysaccharide (LPS) prior to Hg exposure. Although a transient increase of phagocytosis was reported in mussels exposed to Hg alone, no effect on neutral red retention or hemocyte cytotoxicity was observed [43]. A transient decrease in superoxide dismutase activity in hemocytes was reported after LPS injection and Hg exposure. Tissue injuries were greatly increased by the effect of Hg exposure with an LPS injection compared to either treatment alone suggesting that LPS stimulation could increase Hgdependent immunotoxicity [2]. Chromium (Cr) is an important contaminant released from both domestic and industrial effluents and its in vitro and in vivo effects were recently evaluated on immune parameters of Mytilus galloprovincialis by Ref. [18]. Hemocyte incubation with Cr (0.1, 1, 10 and 100 mM) induced a dose-dependent decrease in the Lysosomal Membrane Stability (LMS). Although decreases in extracellular lysozyme release and phagocytic activity were observed, increases of extracellular superoxide production and nitrite accumulation were reported. Increasing metal concentrations were associated to decreases in hemocyte LMS values and in serum lysozyme activity in mussels exposed to Cr (0.1, 1 and 10 mg L(-1)) for 96 h. Decreased phagocytic activity, increased Nitric Oxide (NO) production and decreased Total Hemocyte Counts (THC) were also noticed. However, hemocyte necrosis/apoptosis were not detected. Ref. [18] reported also changes in the transcription of immune related genes (lysozyme, mytilin C, myticin B, defensin, MgC1q), the serotonin receptor (5-HTR) gene and of the stress protein HSP70 gene. The effects of Cu on apoptosis in hemocytes of the Eastern oyster, Crassostrea virginica, as well as on the establishment of Perkinsus marinus infections have been investigated by Ref. [22]. Perkinsus marinus, an intracellular protistan parasite, is the aetiological agent of the Dermo disease. Complex interactions between the host immunity and the parasite escape mechanisms, both of which can be influenced by environmental pollutants, are involved in the progression and the outcome of the disease. Although the Cu exposure induced an increase of apoptosis levels in hemocytes maintained in vitro, an apoptosis suppression was reported during heavy metal exposure of whole oysters. These opposing effects might rely on differences in terms of Cu bioavailability in vitro and in vivo. Growth of P. marinus as well as infection levels of hemocytes were reduced in vitro in presence of Cu. More recently [29], investigated effects of simultaneous exposure of Pacific oyster, C. gigas, to a harmful dinoflagellate, Alexandrium minutum, and cadmium (Cd) and copper (Cu) after 4 day exposure. Antagonistic effects on immune parameters (i.e. hemocyte concentration and phagocytosis) were reported after shortterm, simultaneous exposure to CdeCu and A. minutum. THC increased dramatically in oysters exposed to CdeCu alone. But when oysters were fed A. minutum, this effect was not observed revealing that exposure to the toxic algae interacted antagonistically with CdeCu exposure. A large decrease in hemocyte phagocytosis was reported after CdeCu exposure which indeed counteracted the stimulating effect of A. minutum on phagocytosis, revealing opposite effects [29]. 4. Polycyclic aromatic hydrocarbons (PAHs) PAHs are organic compounds formed during natural and industrial processes; they are termed “persistent organic pollutants” because they are only slowly degraded by natural processes. The
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current expansion of offshore oil activities in coastal regions supports the need to study the potential effects of these oil-related compounds on marine organisms. Ref. [3] investigated the in vitro effects of different PAHs on hemocyte and haemolymph parameters of the Pacific oyster, C. gigas. The phagocytosis activity was significantly reduced in presence of fluorene, pyrene and HFO. Naphthalene, benzofluoranthene and dibenzoanthracene were associated with an increase of the percentage of non-specific esterase positive cells, the phenoloxidase activity and the lysosome presence, respectively. Laboratory exposures of the Arctic scallop, Chlamys islandica, to sub-lethal dispersed oil concentrations (0.06 and 0.25 mg L(-1)) indicated alteration in the measured immune endpoints [30]. After a 15 day incubation followed by a 7-day recovery period in clean seawater, exposed scallops showed a significant increase of hemocyte counts whilst the cell membrane stability and phagocytosis demonstrated a significant reduction [30]. Ref. [32] explored the potential impact of an acute oil exposure mimicking an accidental spill on C. islandica and reported mortalities among exposed animals. Significant impairment of phagocytosis and cell membrane stability was again observed. Finally, prolonged sublethal effects were suggested as no recovery of immune functions was noticed. Sublethal effects (7-d exposure to 50, 100 and 200 mg L(1)) of phenanthrene, a major component of crude oil and one of the most abundant PAHs in aquatic ecosystems, were studied on immunological parameters and the oxidative stress in the scallop, Pecten maximus [31]. Significant reductions in LMS and phagocytosis, and a significant increase in the THC were noticed after pollutant exposure at 200 mg L(1). A significant decrease in total glutathione and significantly increased levels of lipid peroxidation were also reported in the hemolymph of phenanthrene-exposed scallops. These results suggested a possible link between phenanthreneinduced oxidative stress and the subsequent inhibition in some hemocyte functions. Clams, Mya arenaria, exposed to pyrene via the phytoplankton showed a decrease of phagocytosis [24]. More recently, to determine if dietary accumulation is a route of contaminant exposure [21], studied the transfer of PAH compounds (naphthalene, pyrene, and benzo(a)pyrene) by a microphytobenthic diatom, Nitzschia brevirostris, to the Eastern oyster, C. virginica. Diatom cultures were exposed to a range of PAH concentrations (5, 125, 625, and 1000 mg L(1)). Effects on hemocytes were assessed in oysters exposed to the contaminated diatom cultures. Hemocyte responses to dietary PAH exposure included an increase in circulating hemocytes and an increased production of Reactive Oxygen Species (ROS). Ref. [42] explored putative immunity alteration related to the Prestige oil spill in the mussel M. galloprovincialis. For this purpose, mussels were exposed for a 4 month period to Prestige fuel oil. Hemocyte viability, phagocytic activity, nitric oxide production, and chemiluminescence emission showed no significant difference between fuel-treated and control animals. In addition, no tissue lesions were reported by histology in any of the samples. In contrast, the serum protein concentration and the lysozyme activity were found significantly different between fuel oil-exposed mussels and controls. Ref. [47] developed a study to compare effects of phenanthrene at different concentrations (50, 100, 200 or 400 mg L(1)) on the immunocompetence of three bivalve species, M. edulis, the edible cockle, Cerastoderma edule and the razor shell, Ensis siliqua. After phenanthrene exposure immunomodulation reported in M. edulis was different from the immunological changes reported in the other two species suggesting that several species need to be used as sentinel bivalves to reflect harmful effects of pollutants present in marine waters. As an example, a significant reduction in phagocytic
Please cite this article in press as: T. Renault, Immunotoxicological effects of environmental contaminants on marine bivalves, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.011
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hemocytes was only observed in C. edule. In contrast minimal alterations to phagocytosis were observed in M. edulis, suggesting that the process in this bivalve was resilient to the phenanthrene concentrations used. Effects of benzo(a)pyrene (0.5 mg L(-1)) on hemocyte parameters were also investigated in the clam Chamelea gallina [38,39]. After pollutant exposure a significant decrease of phagocytic activity, adhesion capability and lysozyme activity was noted. These results suggested that benzo(a)pyrene induced immunomodulation in C. gallina. 5. Nanoparticles The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. In mammalian cells, nanoparticles have been shown to induce inflammation and oxidative stress, and changes in cell signaling and gene expression. Due to the growing concern over biological effects of NPs in the aquatic environment, a better understanding of their interactions with marine organisms is needed. As sessile filter feeders, marine bivalves are likely to be exposed to NPs suspended in the water column. Several studies have been published recently on the topic, particularly in 2013 and 2014. In 2008, Canesi et al. reported effects of commercial nanosized carbon black (NCB) on mussel hemocytes and suggested that bivalve hemocytes can represent a suitable model for investigating the effects and modes of action of nanoparticles in aquatic invertebrates [11]. They showed that exposure to NCB (1, 5, and 10 mg mL(-1)) was associated with an increase in extracellular lysozyme release, extracellular oxyradical production, and NO release. Flow cytometry analysis showed a decrease of mitochondrial mass/number and membrane potential in NCB-exposed cells. Finally, an activation of the stress-activated Mitogen Activated Protein Kinases (MAPKs) p38 and c-Jun N-terminal kinases (JNKs) was also reported and could explain the NCB effects. The potential effects of other types of commercial NPs (C60 fullerene, TiO(2) and SiO(2) at 1, 5, 10 mg mL(-1)) have been then explored on Mytilus hemocytes [12]. Lysozyme release, extracellular oxyradical and NO production were reported after cell exposure to the NPs tested as well as a rapid activation of the stress-activated p38 MAPK. In vivo exposures to NCB, C60 fullerene, TiO(2) and SiO(2) for 24 h were also conducted in mussels using different concentrations (0.05e0.2e1e5 mg L(1)) [13]. Lysosomal membrane destabilisation in the hemocytes was reported for all NP suspensions. Summarizing existing data [14], reported that isolated mussel hemocytes represent a significant target for NPs and intra-cellular uptake of nanosized materials induces lysosomal perturbations and oxidative stress in the digestive gland after NP agglomerates/ aggregates taken up by the gills. Ref. [19] carried out an additional study to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, and n-CeO(2)) on M. galloprovincialis hemocytes and showed that differential responses of hemocytes were elicited by the different n-oxides. Ref. [1] reported also effects of silver NPs, titanium dioxide (TiO(2)) NPs, and silver nitrate on hemocyte phagocytosis in the Eastern oyster, C. virginica. The in vivo effects of n-TiO2 were investigated in mussels, M. galloprovincialis, exposed for 96 h to different concentrations of NP suspensions (1, 10 and 100 mg L(-1)) [5]. A decrease of LMSand phagocytosis was observed in n-TiO2-exposed hemocytes. The ROSproduction and the transcription of antimicrobial peptides were increased. Different functional and molecular parameters of mussel hemocytes were thus significantly affected by n-TiO2, at concentrations close to predicted environmental levels. Ref. [6] carried out a study to compare effects of cadmium sulfate/cadmium telluride (CdS/CdeTe) mixture quantum dots (QDs) and their dissolved components, cadmium chloride/sodium
telluride salts, and a mixture of these dissolved components. They selected four animal models: one bivalve (M. edulis), one fish (Oncorhynchus mykiss), and two mammals (mice and humans). For blue mussels, results showed that viability and phagocytosis were more affected by QDs than by dissolved metals. These auhtors suggested that aquatic species responded more differently than vertebrates to NP. Ref. [35] investigated cytotoxicity of CdS quantum dots (QDs) at a wide range of concentrations (0.001e100 mg Cd L(1)) in isolated hemocytes and gill cells from the mussel, M. galloprovincialis. A series of functional in vitro assays was carried out in the same cell types to better define the mechanisms of action of CdS QDs (0.31e5 mg Cd L(-1)). Significant increases of ROS production, DNA damage, acid phosphatase (AcP) activity and multixenobiotic resistance (MXR) transport were observed in both cell types exposed to the three forms of Cd (ionic Cd, CdS QDs and bulk CdS). Although an increase of CAT activity was reported in hemocytes exposed to the three forms of Cd, the cytoskeleton integrity of hemocytes was not affected. Exposure to CdS and to CdS QDs at concentrations equal or higher than 1.25 mg Cd L(-1) was associated with effects on hemocyte phagocytosis. However, phagocytosis was not modified in hemocytes exposed to ionic Cd suggesting a particle-specific effect [35]. Ref. [15] explored the possible interactive effects of n-TiO2, one of the most widespread NP in use, and 2,3,7,8-TCDD in M. galloprovincialis based on in vitro and in vivo experiments. For in vivo assays, mussels were exposed to n-TiO2 (100 mg L(-1)) or to 2,3,7,8-TCDD (0.25 mg L(1)), alone and in combination, for 96 h. The authors reported synergistic or antagonistic effects between both contaminants. Although neither contaminant alone was able to induce lysosomal enlargement, exposure to n-TiO2/TCDD resulted in a significant increase in lysosome/cytoplasm volume ratio, indicating a synergistic effect of the two contaminants. Additionally, one of the most important results relies on observation of a significant increase in 2,3,7,8-TCDD accumulation in mussels in the presence of n-TiO2. Recently [46], studied the combined effects of oxygen levels (hypoxia: 1.5 mg O2 L(-1), normoxia: 6.0 mg O2 L(1)) and TiO2 (0, 2.5 mg L(1) and 10 mg L(-1)) for 216 h on hemocytes in the green-lipped mussel, P. viridis. The authors reported synergistic effects between the two stressors on hemocyte functions. Significant interactive effects of the oxygen level and TiO2 were thus observed for the percentage of hemocytes showing non-specific esterase activity, ROS production, lysosomal content and THC. 6. Pharmaceuticals The in vitro effects of individual estrogenic chemicals (17alphaethynyl estradiol, mestranol, nonylphenol, nonylphenol monoethoxylate carboxylate, bisphenol A; benzophenone) on mussel hemocyte parameters were compared [9]. The LMS appeared the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. Ref. [38,39] investigated the in vivo effects of 4-nonylphenol at sublethal concentrations (0.0125, 0.025, 0.05 and 0.1 mg L(-1)) on hemocyte parameters from the cockle Cerastoderma glaucum after 7 days exposure. A significant increased of THC was reported after the exposure of cockles to 0.1 mg L(-1). Significant increases in acid phosphatase activity and lysozyme-like activity were observed for nonyphenolexposed cockles (0.05 and 0.1 mg L (1)). The possible effects of two hypolipidemic pharmaceuticals (fibrates), Bezafibrate (BEZA) and Gemfibrozil (GEM), were investigated in the bivalve mollusc Mytilus spp by Ref. [10]. Isolated hemocytes showed rapid lysosomal membrane destabilization, extracellular lysozyme release, NO production and decreased phagocytic activity in presence of both compounds. Activation of
Please cite this article in press as: T. Renault, Immunotoxicological effects of environmental contaminants on marine bivalves, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.011
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extracellularly regulated kinase (ERK) and p38 MAPKs could partly explain the effects of BEZA and GEM on hemocytes. The effects of both pharmaceuticals on hemocyte functions were confirmed in vivo, in the hemocytes of mussels injected with 0.01, 0.1 and 1 nmol/animal and sampled at 24h post-injection. Lysosomal destabilization and extracellular lysozyme release were reported in a concentration-dependent way for both compounds. Phagocytosis was increased at the highest concentration. 7. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) PCBs are industrial chemicals which are released into the environment. PCBs exist as different congeners depending on the chlorine substitution on the biphenyl rings. Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame-retardants (BFRs) that are widely used in industrial products and pose potential risk on the coastal environment. Ref. [8] explored effects of individual ortho-substituted non coplanar PCB congeners on the function of M. galloprovincialis hemocytes and the signal transduction pathways involved in the immune response. Bacterial killing and lysosomal enzyme release were affected by the di-ortho-substituted, non coplanar PCB congeners P47 (2,20 ,4,4'-tetrachlorobiphenyl) and P153 (2,20 ,4,40 ,5,5'hexachlorobiphenyl), respectively. However, the non-ortho, coplanar congener P77 (3,30 ,4,40 -tetrachlorobiphenyl) did not modify these activities. Hemocyte LMS was also significantly reduced in presence of the three molecules. Functional differences between different PCB congeners were thus reported with respect to the hemocyte functions. Tyrosine kinase-mediated cell signaling was affected by P47, P153 and P77 with an increase in the phosphorylation level of the stress activated p38 and JNK MAPKs. The level of phosphorylated ERK MAPKs was also increased in presence of P153. Finally, an increase of tyrosine phosphorylation of the Signal Transducer and Activator of Transcription 5 (STAT5) in presence of non coplanar P47 and P153 was observed. These results suggested that MAPKs, key elements in the response of mussel hemocytes to bacterial infection, are a target for different PCB congeners [36]. reported significant effects of Aroclor 1254 on Chlamys farreri immune system in vivo. Although the THC, the proportion of granulocytes and phagocytosis decreased significantly after the PCB exposure, the proportion of hyalinocytes and the production of O2()increased during the sampling time. The auhtors suggested that a relationship might exist between pollution, immunomodulation and mass mortality events affecting C. farreri in China [36]. In a recent study, it has been reported that BDE 47 induced cell apoptosis and reduced ROS in mussel M. galloprovincialis [34]. 8. Conclusion Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. The presence of diverse pollutants in aquatic ecosystems is a growing issue and, pollution of these ecosystems is alarming. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. Despite the difficulty of working in complex environments such as estuaries, there is a need to understand the effect of xenobiotics in these exposed natural environments where exposure to mixture of substances, biotic and abiotic factor interactions, and seasonal fluctuations occur. Bivalves are commonly used as bio-indicators of marine pollution, and immunomodulation due to toxicants is one of the important bio-markers used. Laboratory experiments remain a necessary step to study regulation mechanisms and to validate
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biomarkers. However, there are a number of uncertainties associated with their use. Although animals are exposed to complex mixtures of pollutants in the environment, most of laboratory experiments are based on the use of a unique pollutant. A natural or endogenous factors that may modify the responses of biomarkers to environmental toxicants, may lead to misinterpretation if their influence is not taken into account. Using suites of biomarkers in combination with the measurement of pertinent environmental parameters is more likely to provide a good assessment of the effects of environmental toxicants. The use of contrasted sample sites regarding environmental conditions may help to discriminate between the influence of anthropogenic inputs and natural variations. Various ways including integrated approaches can be used to improve the accuracy of ecotoxicological studies (i.e., use of passive organic samplers, analysis of physiological status of animals, accurate investigation of the influence of abiotic factors). The development of an infectious disease results from an unbalance between the host and the pathogen due to external factors (including pollutants) and/or internal factors of both protagonists (virulence of the pathogen, susceptibility of the host). Animals with impaired defence mechanisms may be more susceptible to infectious diseases. Demonstration of the relationship between pollution and increase of susceptibility to infectious diseases exist in vertebrates. Marine pollution may be one of the reasons for disease incidence in marine organisms, which may be caused due to adverse effects of pollutants on the immune system. However, few studies have been developed to better define interactions between immunocompetence, disease susceptibility and pollutants in marine bivalves. Thus, there is an urgent need for development of specific research on this topic. New insights into the potential role of environmental pollutants in host-pathogen relationships and disease dynamics in marine bivalves are needed. References [1] T.E. Abbott Chalew, J.F. Galloway, T.K. Graczyk, Pilot study on effects of nanoparticle exposure on Crassostrea virginica hemocyte phagocytosis, Mar. Pollut. Bull. 64 (10) (2012) 2251e2253, http://dx.doi.org/10.1016/ j.marpolbul.2012.06.026. [2] I. Ahmad, J.P. Coelho, I. Mohmood, M. Pacheco, M.A. Santos, A.C. Duarte, E. Pereira, Immunosuppression in the infaunal bivalve Scrobicularia plana environmentally exposed to mercury and association with its accumulation, Chemosphere 82 (11) (2011) 1541e1546, http://dx.doi.org/10.1016/ j.chemosphere.2010.11.064. [3] A. Bado-Nilles, B. Gagnaire, H. Thomas-Guyon, S. Le Floch, T. Renault, Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg), Toxicol. Vitro. 22 (6) (2008) 1610e1617, http://dx.doi.org/10.1016/j.tiv.2008.04.011. [4] B.D. Banerjee, B.C. Koner, A. Ray, Immunotoxicity of pesticides: perspectives and trends, Indian J. Exp. Biol. 34 (1996) 723e733. [5] C. Barmo, C. Ciacci, B. Canonico, R. Fabbri, K. Cortese, T. Balbi, A. Marcomini, G. Pojana, G. Gallo, L. Canesi, In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve Mytilus galloprovincialis, Aquat. Toxicol. 132e133 (2013) 9e18, http://dx.doi.org/10.1016/j.aquatox.2013.01.014. [6] A. Bruneau, M. Fortier, F. Gagne, C. Gagnon, P. Turcotte, A. Tayabali, T.A. Davis, M. Auffret, M. Fournier, In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium, Environ. Toxicol. (2013), http://dx.doi.org/10.1002/tox.21890. [7] M.P. Cajaraville, I. Olabarrieta, I. Marigomez, In vitro activities in mussel hemocytes as biomarkers of environmental quality: a case study in the Abra estuary (Biscay Bay), Ecotox. Environ. Saf 35 (1996) 253e260. [8] L. Canesi, C. Ciacci, M. Betti, A. Scarpato, B. Citterio, C. Pruzzo, G. Gallo, Effects of PCB congeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cell signaling, Aquat. Toxicol. 63 (3) (2003) 293e306. [9] L. Canesi, L.C. Lorusso, C. Ciacci, M. Betti, M. Rocchi, G. Pojana, A. Marcomini, Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies, Aquat. Toxicol. 81 (1) (2007a) 36e44. [10] L. Canesi, L.C. Lorusso, C. Ciacci, M. Betti, F. Regoli, G. Pojana, G. Gallo, A. Marcomini, Effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, Mytilus galloprovincialis, Chemosphere 69 (6) (2007b) 994e1002.
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Please cite this article in press as: T. Renault, Immunotoxicological effects of environmental contaminants on marine bivalves, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.011