**5. Immunotoxicity in marine bivalves and susceptibilty to infectious diseases**

The impact of contaminants and other environmental factors on the immune system of bivalves is an issue of ecological and economical concern, because it may result in clinical pathology and disease, by increasing the susceptibility of affected organisms to pathogens.

Contaminants known to induce alterations of immune functions including pesticides (Vial et al., 1996; Banerjee et al., 1996; Banerjee et al., 2001) are present in almost all coastal areas. Among physiological processes possibly disturbed by pollutants, the immune system is likely to be one of the more sensitive (Baier-Anderson & Anderson, 2000; Fournier et al., 2000).

In contrast to the vertebrate immune system which consists of innate and acquired mechanisms, invertebrate immunity relies only on innate defence mechanisms. The fact that invertebrates represent more than 90% of the total number of species living on earth demonstrates the efficiency of their «primitive» host defence systems. It becomes more and more obvious that some of these innate mechanisms are conserved in invertebrates and vertebrates (Medzhitov et al., 1997; Means et al., 2000). Thus, the fundamental importance of the toxically-induced modulation of non-specific immune functions has increasingly been perceived.

Bivalve immunity is mainly supported by hemocytes and participate directly in eliminating pathogens by phagocytosis (Cheng, 1981; Feng, 1988). In addition, hemocytes produce compounds including lysosomal enzymes and antimicrobial molecules which contribute to the destruction of pathogens (Coles & Pipe, 1994).

Investigating the effects of pesticides on hemocyte functions and immunity in bivalves has been based on the monitoring of several biomarkers (Pipe & Coles, 1995). As an example, Gagnaire et al. (2006) tested the effect of 23 pollutants on Pacific cupped oyster haemocytes by flow cytometry monitoring different cell parameters and demonstrated that 3 pesticides (2,4D, paraoxon, and chlorothalonil) induced a modulation of hemocyte activities. However, biomarkers used differ very often between published studies.

Triforine, a fungicide, induced decreased hemocyte viability in the eastern oyster, *Crassostrea virginica* (Alvarez & Friedl, 1992). Cytotoxic effects were also observed in adult Pacific cupped oyster, *C. gigas*, hemocytes: the mean cell viability was significantly decreased at 1.0 mg L-1 of lindane (gamma-hexachlorocyclohexane) after 12 day exposure period (Anguiano et al., 2006). Alteration in cell viability was also reported in the blue mussel, *Mytilus edulis*, exposed to 0.1 mg L-1 azamethiphos, an organophosphate pesticide

Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 233

Indeed, a mixture of 8 pesticides reduced phagocytosis on hemocytes and enhanced susceptibility to *Vibrio splendidus* (Gagnaire et al., 2007). Pacific cupped ysters were exposed over a 7 day period to the mixture of pesticides. The pesticides were selected on the basis of spread amounts in the Marennes-Oleron Basin (Charente-Maritime, France), one of the most important oyster producing areas in France (Léonard, 2002; Munaron et al., 2006). Moreover, a down-regulation of the LBPB/BPI, TIMP and lysozyme genes were reported in Pacific

The evaluation of acetylcholinesterase activity in marine organisms has been and is at present time extensively used as a biomarker of exposure to neurotoxic agents such as organophosphorus and carbamate pesticides. Indeed, organophosphorous compounds and carbamates including paraoxon and carbaryl are known to inhibit acetylcholinesterase

Paraoxon inhibited the activity of AChE in the hepatopancreas of the blue mussel, *Mytilus edulis,* in vitro at concentrations ranging from 1 µM to 1 mM (Ozretic and Krajnovic-Ozretic, 1992). Inhibition by carbaryl was less distinct. AChE from *M. edulis* hemocytes was inhibited in vitro by 0.1-3 mM paraoxon, eserine and DFP (Galloway et al., 2002). Cantry et al. (2007) showed that exposure of the blue mussel, *M. edulis*, to 0.1 mg L-1 azamethiphos, an organophosphate pesticide used to combat sea lice infestations in farmed salmonids, for periods of up to 24h caused a significant reduction in acetylcholinesterase activity in both the haemolymph and the gill. However, cholinesterases found in the Pacific cupped oyster, *Crassostrea gigas,* appeared to be insensitive to organophosphorous insecticides (Bocquene et

Anguiano et al. (2006) showed that after a 4 h exposure to lindane (gammahexachlorocyclohexane), filtration rates of adult Pacific cupped oysters, *Crassostrea gigas*, were significantly reduced compared with controls at concentrations of 0.3 and 0.7 mg L-1. However, a short term exposure of the blue mussel, *Mytilus edulis*, to azamethiphos did not change the feeding rate (Chantry et al., 2007). Studies carried out in adult Pacific cupped oysters revealed that diuron induces partial spawning and atrophy of the digestive

Greco et al. (2010) investigated effects of a mixture of herbicides on the physiological status of the soft clam, *Mya arenaria*. Clams were exposed for 28 days to 0.01 mg L-1 of a pesticide mixture: dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy) propionic acid (mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Although a gradual sexual maturation was reported in both sexes during the course of the experiment, females

Favret and Lynn (2010) during the course of a study monitoring sperm viability by flow cytometry in the eastern oyster, *Crassostrea virginica*, after exposure to a pesticide (Bayluscide) reported effects on mitochondrial membrane potential and plasma membrane in the sperm. Buisson et al. (2008) studied impact of pesticides in the cupped Pacific oyster, *C. gigas,* and reported partial spawning and atrophy of the digestive tubule epithelium in

A study with a mix of herbicides containing atrazine, diuron and isoproturon revealed effects on gene expression in the Pacific cupped oyster, *Crassostrea gigas* (Tanguy et al., 2005). Gagnaire et al. (2007) studied also the impact of pesticides on *C. gigas*, monitoring

oysters exposed to the mixture of 8 pesticides (Gagnaire et al., 2007).

epithelium after 1 week of exposure at 1 µg L-1 (Buisson et al., 2008).

demonstrated a higher sensitivity to pesticides compared to males.

**6. Other effects of pesticides on marine bivalves** 

(AChE) and carboxylesterase (CE) (Cooreman et al., 1993).

al., 1997).

relation to pesticides.

(Cantry et al., 2007). Moreover, a mix of herbicides containing atrazine, diuron and isoproturon showed an effect on *C. gigas* hemocyte aggregation (Auffret et Oubella., 1997).

Chlordan, an insecticide, demonstrated effects on *C. virginica* hemocyte phagocytosis at 250 µM *in vitro* (Larson et al., 1989). A decreased phagocytosis activity was observed after a triforine exposure in the eastern oyster, *C. virginica* (Alvarez and Friedl, 1992). A pesticide mixture (alachor, metolachlor, terbutylazine, glyphosate, diuron, atrazine, carbaryl and fosteyl aluminium) representative for surface waters of the Marennes-Oleron Basin (Charente Maritime, France, 0.25 nM to 4 nM) induced a decrease of phagocytic activity (Gagnaire et al., 2007). Moreover, Cantry et al. (2007) reported a decrease in phagocytic index in the blue mussel, *Mytilus edulis*, after a short exposure to 0.1 mg L-1 azamethiphos. This result suggests that azamethiphos can modulate haemocyte function in mussels at environmentally relevant concentrations.

At the contrary, Gagnaire et al. (2003) reported no effect on cell viability, cell cycle and cellular activities except for peroxidase activity for Pacific cupped oyster haemocytes exposed to atrazine in *in vitro* and *in vivo* assays.

Pentachlorophenol decreased the production of ROS by the inhibition of NADPH production in the eastern oyster, *Crassostrea virginica* (Baier-Anderson & Anderson, 1996). Dieldrin, tested *in vitro* on *C. virginica* hemocytes induced a decrease of chemiluminescence at concentrations ranging from 3 to 300 µM (Larson et al., 1989). Hemocytes of *C. virginica* exposed to chlorothalonil (fungicide) for 20 h at concentrations between 4 nM and 2 µM showed no modification of cell mortality and phagocytosis, but a decrease of ROS production (Baier-Anderson & Anderson, 2000).

In the past decades, the emergence of infectious diseases has been reported in marine species and disease outbreaks have also increased (Harvell et al., 1999). According to Snieszko (Snieszko, 1974), 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 presenting 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 (Fournier et al., 1988; Van Levoren et al., 2000; Jepson et al., 2005), a few of studies was carried out in invertebrates (Galloway & Depledge, 2001). Rare studies have attempted to link contaminant presence and susceptibility to infectious diseases in marine molluscs and demonstrated harmful effects of pollutants in bivalves.

Contamination of the eastern oyster, *Crassostrea virginica*, by polluted sediment and tributyltin increased the intensity of *Perkinsus marinus* infection, but no cellular or humoral parameters were modulated (Anderson et al., 1996; Chu et al., 2002). Anderson et al. (1981) demonstrated previously that the hard clam, *Mercenaria mercenaria,* exposed to PCP were unable to kill injected bacteria. Kim et al. (2008) studied the relationship of parasite detection to contaminant body burden in sentinel bivalves through a 'Mussel Watch' Program. These authors showed that correlations between parasites/pathologies and pesticides were frequent in mussels and oysters (Kim et al., 2008).

The Pacific cupped oyster, *Crassostrea gigas*, has been also used to evaluate the impact of a pesticide mixture (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) on some immune-related parameters and to demonstrate a relationship between infectious diseases, defence capacities and pollutants. Indeed, a mixture of 8 pesticides reduced phagocytosis on hemocytes and enhanced susceptibility to *Vibrio splendidus* (Gagnaire et al., 2007). Pacific cupped ysters were exposed over a 7 day period to the mixture of pesticides. The pesticides were selected on the basis of spread amounts in the Marennes-Oleron Basin (Charente-Maritime, France), one of the most important oyster producing areas in France (Léonard, 2002; Munaron et al., 2006). Moreover, a down-regulation of the LBPB/BPI, TIMP and lysozyme genes were reported in Pacific oysters exposed to the mixture of 8 pesticides (Gagnaire et al., 2007).
