Humoral immune factors modulated by copper and chitosan in healthy or parasitised carp (Cyprinus carpio L.) by Ptychobothrium sp. (Cestoda)

Aquatic Toxicology 68 (2004) 325–338

Humoral immune factors modulated by copper and chitosan in healthy or parasitised carp (Cyprinus carpio L.) by Ptychobothrium sp. (Cestoda) Claire Dautremepuits a,b,∗ , Stéphane Betoulle a,b , Séverine Paris-Palacios a , Guy Vernet a,b b

a Laboratory of Eco-Toxicology, University of Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France International Research Institute on Metal Ions, University of Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France

Received 26 July 2003; received in revised form 8 April 2004; accepted 18 April 2004

Abstract As an environmental protection point of view, the potential toxicity of chitosan on aquatic animal health, alone or associated with copper must be investigated. Fish possess defence mechanisms to counteract the impact of toxics. The non-cellular and non-specific immune defences (total immunoglobulin, ceruloplasmin, lysozyme and potential killing activity of phagocytic cells) can be modulated by the potential environmental pollutants but also by natural stimulants such as bacteria, viruses or parasites. In this study, we investigate the potential toxicity of copper (0.1 and 0.25 mg/L) or chitosan (75 and 150 mg/L) and the combination copper and chitosan (0.1 and 75 mg/L, respectively) on two groups of carp: healthy or parasitised by Ptychobothrium sp. Fish exposed to water-soluble chitosan for 96 h had significantly high levels of natural antibodies in plasma. Moreover, activities of lysozyme and ceruloplasmin were also increased in plasma after the same treatment. The exposition of fish to copper have shown apparently contradictory effects on the immune parameters measured but, significant increase of this bacteriolytic activity was observed, particularly in head kidney after 4 days of treatment of fish with copper. The two products may induce separately an acute, short and local inflammatory acute phase response by stimulating some components of the innate immune response of healthy fish. The mixture seems to reduce the impact of the each product due to the physical and chemical properties of chitosan to complex with copper. The responses of humoral immune factors of treated carp was modulated by the presence of the parasite, as shown by the high elevation of lysozyme activity observed in parasitised carps after exposition to copper and by increases in natural antibodies levels observed in parasitised carp treated with the copper–chitosan mixture. This could indicate an additive effect on the stress response mediated by parasite. It occurred a greater stress response in the parasitised group than healthy group exposed to the same treatment evoking an additive effect.

∗ Corresponding author. Tel.: +33-4-7546-5810; fax: +33-4-7546-5810. E-mail address: [email protected] (C. Dautremepuits).

0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2004.04.003

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So, it is important to specify the health status of organisms to understand responses of immunological markers in fish. © 2004 Elsevier B.V. All rights reserved. Keywords: Carp; Chitosan; Copper; Immune system; Parasite

1. Introduction In ecotoxicology, evaluation of aquatic systems quality and animal health has required the development of biological markers of environmental pollution during the last few years (Braunbeck and Völkl, 1993; Lagadic et al., 1998). This strategy includes many disciplines and lower levels of biological organisation (Connell et al., 1999). One level of organisation is the innate immune system, which is something common to all multicellular organisms. Innate (natural or non-specific) immunity is the collection of defense mechanisms that protect an organism against infection, without depending upon prior exposure to any particular microorganism. For fish populations, a link between environmental contamination and disease has long been suspected (Sniesko, 1974). An explanation for this connection might lie in impairment of the innate immune system. Therefore, understanding the effect of toxicants on fish innate immunity supports the larger ecotoxicological goal of comprehending the actions of ecotoxicants on fish populations. Nevertheless, these fish immune parameters can also be modulated by natural environmental influences [seasonal variations, e.g. thermoperiod and photoperiod, feeding behaviour of species (food availability. . . ) and obviously pathogens] (Zeeman, 1986; Le Morvan et al., 1997; Alcorn et al., 2002). All the more, development of aquaculture has resulted in much greater attention being paid to problems posed by parasites and the constraints they imposed on the productivity of aquaculture (Kennedy, 1994). Besides direct losses caused by mortality, parasites may have considerable impact on defence mechanisms of fish (Williams and Jones, 1994; Kumaraguru et al., 1995; Woo, 1995). It is well known that parasites may modulate a wide range of physiological processes in fish including, for example, their resistance to other stressing factors. Whenever a fish is injured by a parasite, the host organism will initiate an inflammatory

process aimed at restricting, reducing and ending the damage (Jones, 2001; Ardelli and Woo, 2002). It is then of importance to determine the background occurred by the presence of a parasite in order to evaluate the environmental quality. For this reason, we proposed in this study to compared parasitised and unparasitised fish response in case of aquatic pollution especially in Champagne–Ardenne (France) where the viticulture is developed and necessitate the use of various phytosanitary products released in aquatic ecosystems. High concentrations of copper used as antifungal in vineyards were detected in aquatic ecosystems collecting vineyard runoff water (until 450 ␮g/L in vineyard detention ponds or until 380 ␮g/L in Marne river, Champagne, France) and even copper utilisation is forbidden, it still be highly concentrated in ground (GERBE, 1996; Teisseire, 1999). This heavy metal is already known to be accumulated in aquatic animals and is toxicity has been demonstrated (Mance and Worsfold, 1988; Sorensen, 1991; Betoulle et al., 2002). Copper, as other heavy metals, can affect the immunological status of fish (Zelikoff, 1993; Dethloff and Bailey, 1998; Sanchez-Dardon et al., 1999; Shariff et al., 2001). So, health problems associated to copper uses have prompted the demand for an alternative strategy using low toxic molecules, such as chitosan, in plant protection against fungi pathogens. Water-soluble chitosan is the deacetylated form of chitin and is extracted primarily from shells of crustaceans such as shrimps and crabs, and from squid pens. Chitosan is used as a non-toxic cationic flocculent in treatment of wastewater from sewage, sludge or breweries and as a chelator of heavy metals (Sandford, 1989). Agricultural applications include coating for seed and fruit preservation associated with fungistatic properties (Ravi Kumar, 2000). Chitosan, pulverised on grapevine plant, can be also used as a natural agri-bioactive substance controlling fungal diseases and contributing at the same time to reduce

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the viticultural uses of copper. Moreover, derivatives of chitin are already known to have immunostimulant properties, they increase macrophage activities or cytokines production in mammals (Nishimura et al., 1986, 1987). In fish, chitin or chitosan increase the activity of seabream or brook trout innate immune systems (Anderson and Siwicki, 1994; Esteban et al., 2000, 2001). Actually, this molecule is presented as a copper alternative in vineyard and uses of chitosan are expected to increase its emissions into the aquatic environment. So, related pollution risks need to be considered in terms of environmental risk assessment. The potential viticultural use of chitosan, alone or with copper released in surface water, associated with the immunomodulating properties of the two products; lead us to investigate their impact on humoral immune factors of fish as an ecotoxicological point of view. We studied components of the humoral immunity, belong to the innate immune system, whose modulations by pollutants are an ecotoxicological concern because they have the potential to influence populations by affecting the susceptibility of individuals to disease (Bols et al., 2001). Humoral factors (non-cellular non-specific defense mechanisms), such as total natural antibodies, ceruloplasmin and lysozyme activities, inhibit non-specifically the growth of pathogens and are found in the serum and various organs of fish (Magor and Magor, 2001). In the present study, we had the opportunity to examine a carp population coming from a local fish breeding and constituted by two kinds of fish: healthy or carrying a intestinal parasite, Ptychobothrium sp. (Cestoda). We exposed these two groups to copper and/or chitosan and assessed humoral factors involved in non-cellular and -specific immune defences of fish (total natural antibodies, ceruloplasmin and lysozyme activities). To our knowledge, it was the first time that the effect of a parasitose was examined on the fish immune system ability to respond to environmental pollutants. In this study, levels in humoral immune factors were investigated in two organs (liver and head kidney) and in plasma of healthy or parasitised fish. The liver was examined as a primary role in the metabolism and excretion of xenobiotic compounds where biochemical alterations can obviously occur in some toxic conditions (Rocha and Monteiro, 1999). As the two products have potential immunomodulating activities, we examined the same toxicological

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parameters in head kidney, one of the major organs of the fish immune system and in blood, the circulating fluid of immunological factors. Thus, the immunotoxicological impacts observed in fish exposed to environmental contaminants can be variable in function of the fish health status.

2. Material and methods Common carp (12 ± 2 g and 6 ± 0.7 cm) were purchased from a local hatchery (Ets “Au Vairon”, Reims, France). Fish were maintained in tanks containing aerated springwater (17 ± 1 ◦ C) (Ca2+ , 98 mg/L; Mg2+ , 4 mg/L; Na+ , 4.1 mg/L; K+ , 1.9 mg/L; HCO3 − , 269 mg/L; SO4 2− , 43 mg/L; Cl− , 3.6 mg/L; NO3 − , <2 mg/L) (Aurele, Ardennes, France) with a photoperiod 12 h:12 h. They were acclimatised in laboratory conditions for 10 days before the start of experiments. They were fed once daily with commercial pellets (TetraminTM , Germany). In each tanks, the water quality parameters were evaluated daily according to Standard Methods for Examination of Water and Wastewater (APHA, 1989). 2.1. Experimental procedure Carp were exposed to sublethal concentrations of copper in water (0.1 or 0.25 mg/L) corresponding approximately to 20 and 50% of 96 h-LC50 , respectively, for cyprinidae (Shariff et al., 2001). The LC50 -96 h of chitosan was determined in our laboratory as 300 ± 18 mg/L (Dautremepuits et al., 2002). Carp were then exposed to two sublethal concentrations of chitosan (75 or 150 mg/L) corresponding to 25 and 50% of LC50 -96 h respectively, or to a mixture of copper (0.1 mg/L) + chitosan (75 mg/L). For experiments, copper stock solution getting from copper sulfate (CuSO4 ) (Prolabo, France) or water-soluble chitosan getting from Chitogel® (Aber Tech, France), were added to the dilution springwater in tanks to obtain test concentrations. Control groups were carp maintained in normal springwater. Group of 20 fish were used for each one of the six treatments. Experiments were run under static conditions for 96 h. Fish were then killed by a blow to the head and liver, head kidney were rapidly dissected out in ice-cold conditions, blood samples were simultaneously collected

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on heparin (Sanofi, France). Moreover, intestines were removed to determine the presence or absence of internal parasites and to evaluate the number of parasitised fish. Tissue samples (head kidney and liver) were suspended in phosphate buffer saline (PBS) (pH 7.2) (Gibco-Brl, France). Liver and head kidney samples were homogenised with a potter–servodyne homogeniser (Cole-Palmer Ins. Co., USA). Homogenates were centrifuged at 30,000 × g for 15 min at 4 ◦ C to remove nuclei and cell debris. At the same time, blood samples were centrifuged to obtain plasma (10 min, 18,000×g, 4 ◦ C). The resulting supernatants, after centrifugation of tissular homogenates or blood samples, were collected and stored at −80 ◦ C until examination of immune related parameters (total natural antibodies, ceruloplasmin and lysozyme activities). 2.2. Humoral immune factors Analysis of total natural antibodies (immunoglobulins (Ig)) levels in tissue samples is based on a spectrophotometric technique described by Siwicki and Anderson (1993). The total Ig is separated from the samples (0.1 ml) by precipitation with 0.1 ml polyethylenic glycol 10,000 (PEG) (12%) (Sigma, France). After 2 h of incubation under constant mixing, samples were centrifuged for 10 min at 1000 × g. Protein readings from supernatant give amount of protein taken out by absorption to PEG. To calculate total Ig, readings were substracted from total protein on individual samples. The protein content in fish liver, head kidney and plasma samples was determined by the method of Bradford (1976) using bovine serum albumin (Sigma, France) as standard. Ceruloplasmin activity in samples (liver, head kidney and plasma) was measured as p-phenylene diamine (PPD) oxidase activity (Sigma, France) as described by Pelgröm et al. (1995). Biological sample or standard of ceruloplasmin (Sigma, France) was mixed with 1 ml acetate buffer (1.2 M, pH 5) containing 0.1% PPD as substrate. Concomitantly, each sample was incubated in the presence of 1 ml NaN3 (0.5%) (azide blank) (Sigma, France). The mixtures were incubated for 30 min at 37 ◦ C. The reaction was stopped by the addition of 1 ml NaN3 (0.5%). One unit of ceruloplasmin was defined as the amount of oxidase that catalysed a decrease in absorbance of 0.001/min at 550 nm.

The lysozyme activity in tissue was measured by the turbidimetric assay described by Studnicka et al. (1986). Sample or lysozyme standard (Sigma, France) was added to Micrococcus lysodeikticus suspension (0.2 g/L) and the decrease in absorbance was recorded at 450 nm by spectrophotometry (Spectronic, Genesys 5, France). The unit of lysozyme activity was defined as the amount of enzyme that catalysed a decrease in absorbance of 0.001/min. 2.3. Statistical analysis For each one of the six treatment (chitosan 75 mg/L, chitosan 150 mg/L, copper 0.1 mg/L, copper 0.25 mg/L, copper 0.1 mg/L + chitosan 75 mg/L, or nothing for control), a group of 20 fish was used. At the time of the end of experiment (96 h), the numbers of healthy and parasitised fish were determined for each group as described above in the experimental procedure section. Each value was expressed as mean ± standard error (S.E.) and corresponded to two fish categories (healthy or parasitised) and was obtained from various numbers of individuals (8 < n < 13 for healthy and 7 < n < 12 for parasitised). Differences in means were analysed by the Student t test of significance using SigmaStat 2.03 software (P < 0.05 was considered statistically significant).

3. Results 3.1. Healthy carps The Ig levels detected in livers and head kidneys of healthy carp treated 96 h with chitosan, copper alone were not significantly different from those measured in control, excepted the higher amount of Ig measured in head kidneys of 0.1 mg/L copper treated carps (Fig. 1A and B). Conversely, the plasmatic levels of Ig were higher in carps exposed to sublethal chitosan concentrations as to 0.1 mg/L copper but it was lower in carp exposed to 0.25 mg/L copper compared to control (Fig. 1C). No significant modulation of natural antibodies levels compared to control was observed in organs of healthy carps after 96 h exposition to the mixture copper + chitosan; this treatment has no biological effects on the level of Ig (Fig. 1A–C).

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Fig. 1. Total natural antibodies (Ig) in liver (A), head kidney (B) and plasma (C) of carp parasitised ( ) or not (䊏) by Ptychobothrium sp. and exposed to two sublethal concentration of copper (0.1 or 0.25 mg/L), of chitosan (75 or 150 mg/L) or to the mixture copper (0.1 mg/L) + chitosan (75 mg/L) for 96 h. Data are expressed as mean ± S.E. Significant changes vs. control: *P < 0.05 for healthy fish; # P < 0.05 for parasitised fish. For each treatment, significant changes between healthy and parasitised fish: P < 0.05 in bold type.

1.0 ± 1.4

Data are expressed as mean ± S.E. Significant changes vs. control: *P < 0.05 for healthy fish; # P < 0.05 for parasitised fish. For each treatment, significant changes between healthy and parasitised fish: P < 0.05 in bold type.

9.1 ± 1.4* 3.1 ± 1.4*

22.63 ± 7.84# (P < 0.05)

5.66 ± 0.17# (P < 0.05) 50.53 ± 6.73# P < 0.05 4.55 ± 0.01* 4.88 ± 0.16# 5.38 ± 0.29*

3.26 ± 0.3 (P < 0.05) Undetectable 0.72 ± 0.34*

2.99 ± 0.16 (P < 0.05) Undetectable 1.54 ± 0.44*

3.06 ± 0.16 (P < 0.05) Undetectable 3.93 ± 0.33

Healthy Parasitised Healthy Parasitised Healthy

Parasitised

9.6 ± 1.4*

3.31 ± 0.01# (P < 0.05) Undetectable (P < 0.05) 3.32 ± 0.19*

Parasitised Healthy

Liver (U.I./mg protein) Head kidney (U.I./mg protein) Plasma (U.I./mg protein)

75 mg/L Healthy

Undetectable

Parasitised

Healthy

Parasitised

Copper + Chitosan

0.25 mg/L 0.1 mg/L

Copper

150 mg/L Chitosan Control

The highest tissular ceruloplasmin levels were found in plasma of healthy carp (Fig. 2C). In healthy carp, the amount of ceruloplasmin were significantly reduced in liver of fish exposed to 75 or 150 mg/L chitosan and in head kidney of fish exposed to 150 mg/L chitosan (Fig. 2A and B). In plasma, healthy carps treated with chitosan (75 or 150 mg/L for 96 h) had a significantly high levels of ceruloplasmin all the more since the chitosan concentration used was important (Fig. 2C). The 96 h copper treatment was only associated with a lowest hepatic ceruloplasmin level in healthy fish exposed to 0.25 mg/L copper (Fig. 2A). No modulation in ceruloplasmin activity was observed in liver and head kidney for the other copper treatments (Fig. 2A and B). In plasma of healthy carps, a lowest ceruloplasmin activity was measured after treatment with 0.1 or 0.25 mg/L copper in comparison with fish remained in normal fresh water. Concerning the impact of the mixture copper and chitosan on the ceruloplasmin activity, we noticed a lowest level of this parameter in liver and plasma of healthy carps comparatively to untreated controls (Fig. 2A–C). Conversely, no effect was observed in head kidney (Fig. 2B). If we compare the ceruloplasmin levels evaluated in tissues of fish treated with copper or chitosan alone with those detected in the same compartments in fish treated with the copper–chitosan mixture, we noticed that the exposure of healthy fish to the mixture was associated with a lowest ceruloplasmin activity in liver whereas no modulation was observed in liver of copper-treated fish (Fig. 2A). Moreover, the decrease in liver ceruloplasmin level associated with the mixture treatment of healthy carps was less pronounced than the decrease noted in healthy carps treated with 75 mg/L of chitosan (Fig. 2A). In plasma, the treatment of healthy fish with chitosan for 96 h was associated with a higher ceruloplasmin activity whereas a lower activity was measured in healthy carp treated with the copper–chitosan mixture. The decrease in ceruloplasmin activity noticed in plasma of healthy carp exposed to the mixture was comparable to those measured in plasma of healthy fish exposed to 0.1 mg/L of copper (Fig. 2C). Lysozyme activity was evaluated in the two organs liver, and head kidney and also in plasma of fish. This activity was undetectable in the liver of fish (Table 1).

0.1 mg/L + 75 mg/L

C. Dautremepuits et al. / Aquatic Toxicology 68 (2004) 325–338 Table 1 Lysozyme activity in liver, head kidney and plasma of carp parasitised or not by Ptychobothrium sp. and exposed to two sublethal concentrations of copper or chitosan and to the mixture copper and chitosan for 96 h

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Fig. 2. Ceruloplasmin activity in liver (A), head kidney (B) and plasma (C) of carp parasitised ( ) or not (䊏) by Ptychobothrium sp. and exposed to two sublethal concentration of copper (0.1 or 0.25 mg/L), of chitosan (75 or 150 mg/L) or to the mixture copper (0.1 mg/L) + chitosan (75 mg/L) for 96 h and Data are expressed as mean ± S.E. Significant changes vs. control: *P < 0.05 for healthy fish; # P < 0.05 for parasitised fish. For each treatment, significant changes between healthy and parasitised fish: P < 0.05 in bold type.

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The treatment of fish with sublethal concentrations of chitosan was associated with a lowest lysozyme activity in head kidney (Table 1). The plasmatic lysozyme activity of 75 mg/L chitosan-treated carp was no detectable whereas it appeared for healthy fish contaminated with 150 mg/L chitosan (Table 1). Whereas any lysozyme activity was detectable in control groups, significant increase of this bacteriolytic activity was observed after 4 days in copper-treated fish. In head kidney, the lysozyme activity was also enhanced after the treatment of fish with copper. Healthy fish treated with the mixture copper + chitosan had significant high levels of lysozyme activity in plasma in comparison with control (Table 1). Moreover, the plasmatic lysozyme activity measured in mixture-treated fish was similar to those measured in fish exposed to 0.25 mg/L copper. In head kidney, decreases induced by the mixture (copper and chitosan) in healthy fish were less pronounced than the decrease induced by sublethal chitosan concentrations alone and opposed to the higher lysozyme activity measured in copper exposed carps (Table 1). 3.2. Ptychobothrium-parasitised carps The examination of intestinal contents revealed that approximately half fish of each group of treatment was infected by a cestoda identified as Ptychobothrium sp. Its species is known as parasite of marine and freshwater teleosts characterised by a shape of scolex heart and bothridia oval with having disc (Wongsawad et al., 1998). The presence of Ptychobothrium sp. in the gut of carps induced a decrease of the Ig levels in liver and head kidney whereas we measured a higher Ig amount in the circulating fluid, the plasma in comparison with non-infected fish (Fig. 1). Fig. 1A showed that Ig levels were lowest in liver of parasitised fish treated with sublethal copper concentrations or 150 mg/L chitosan than those measured in non-treated infected fish, and no effect of the mixture was noticed on this parameter (Fig. 1A). In head kidney and plasma of parasitised fish, chitosan and/or copper treatments were associated with higher levels of total Ig compared with no contaminated infected-fish (Fig. 1B and C). For all the treatments, amounts of total Ig measured in the liver and in the plasma of infected-fish were

respectively lowest and highest than levels in contaminated non-infected carps (Fig. 1A–C). In the head kidney, there was no significant difference of the total Ig content between both groups (infected or not), excepted with 75 mg/L chitosan contamination (Fig. 1B). Compared to the healthy group, the presence of the parasite Ptychobothrium in fish was associated to a higher hepatic ceruloplasmin activity and conversely to a lower plasmatic ceruloplasmin activity (Fig. 2A–C). In control groups, no difference between healthy and parasitised fish was observed in levels of ceruloplasmin detected in head kidney (Fig. 2B). In Ptychobothrium-infected carps, copper treatments (0.1 and 0.25 mg/L) have no impact on the ceruloplasmin activity in both organs as in the plasma (Fig. 2). Sublethal chitosan concentration treatments were associated to a lowest ceruloplasmin activity detected in head kidney and plasma whereas a highest activity was measured in liver of infected fish contaminated with 150 mg/L chitosan (Fig. 2). Concerning the mixture impact on ceruloplasmin activity, no significant changes were noticed in liver and plasma, but we noticed a lowest level in infected-carp head kidney after 96 h mixture exposure (Fig. 2). Conversely to results observed in treated infected fish compared to treated uninfected fish, the ceruloplasmin activity was higher in liver and lower in head kidney and plasma of infected carp (Fig. 2). For control groups, the lysozyme activity was significantly lower in head kidney of Ptychobothriuminfected fish than those detected in the same organ of normal carp (Table 1). The treatment of fish with copper or chitosan alone was associated with increases in lysozyme activities measured in head kidney which were more pronounced than those observed in healthy carps (Table 1). A same phenomenon can be noticed in plasma but only with the copper exposure, the highest concentration used (0.25 mg/L) leading to a more important difference between healthy and parasitised organisms (Table 1). While healthy fish treated with the mixture copper + chitosan had significant high levels of lysozyme activity in plasma in comparison with control, this parameter remained undetectable in plasma of Ptychobothrium-parasitised fish as observed in carp exposed to 75 mg/L of chitosan (Table 1). In parasitised fish, no lysozyme activity was measured in plasma of mixture-treated group whereas significantly

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high levels of lysozyme were detected in plasma of fish exposed to 0.1 mg/L of copper (Table 1). The exposition of parasitised fish to the mixture led to an increase in lysozyme activity measured in head kidney whereas a decrease of this activity was observed in head kidney of unparasitised fish after submission to the same polluting exposition (Table 1).

4. Discussion Among the biochemical, cellular and physiological systems of multicellular animals that can be monitored in ecotoxicology, the innate immune system has some uniquely attractive features (Bols et al., 2001). Innate immunity serves as a first line of defense against environmental aggressions. It is found through all animal phyla and well conserved. Modulation of immune parameters appeared for sublethal concentration of toxicants which still are lower than concentrations inducing the acute toxicity and the death of non-target organisms (Fournier et al., 2000). Therefore, alterations of the innate immune responses can be considered as biomarkers against any aggression. In mammals, chitosan has been reported to have immune stimulating activities such as increasing accumulation and activation of macrophages and polymorphonuclear cells, inducing cytokines release, augmenting antibody responses and enhancing cytotoxic T lymphocyte (CTL) response (Nishimura et al., 1985; Muzzarelli et al., 1988; Azuma, 1992; Peluso et al., 1994; Usami et al., 1994; Shimbata et al., 1997; Otterlei et al., 1994; Marcinkiewicz et al., 1991; Calvo et al., 1997). In this study, modulation of plasmatic immune parameters selected as biomarkers (total Ig, lysozyme and ceruloplasmin activities) in fish exposed to water-soluble chitosan for 96 h confirmed previous reports indicating that chitin and its derivatives have immunostimulant properties. For example, Saiki et al. (1992) have reported immunostimulating effect of chitin in rainbow trout (Oncorhynchus mykiss). This property conferred also an increasing resistance against other aggressions. Yellowtail injected with chitin showed increased resistance to Pasteurella piscida (Kawakami et al., 1998) and intraperitoneally injection of chitin particules in gilthead seabream was associated with increases in humoral and cellular immune responses such as increasing natural haemolytic

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complement, phagocyte respiratory burst and natural cytotoxic activities since 3 days post-injection (Esteban et al., 2000). Siwicki et al. (1994) showed that oral administration of chitosan to rainbow trout was found to cause elevated myeloperoxidase activity and phagocytosis in polymorphonuclear leukocytes from blood and the immunostimulated fish seemed to be more resistant to infection with Aeromonas salmonicida. In these studies carried out in fish, chitin or chitosan was administrated by injection or orally in the fish diet. To our knowledge, it is the first time that the effect of chitosan was examined on the fish immune response after exposition of fish by immersion. Then, we noticed that chitosan had immunostimulating activities whatever the administration method might be. Exposition of carps to copper induced higher levels of natural antibodies and in lysozyme activity in plasma and head kidney of fish while ceruloplasmin activity was simultaneously decreased in this second biological compartment. Thus, in this study, copper seemed to induce a contradictory modulation of the humoral immune factors studied at the copper concentrations tested and with the exposure time used (96 h), as previously reported in the literature. In vivo copper expositions of different aquatic organisms have demonstrated apparently contradictory effects on the immune parameters as the inhibition of respiratory burst activity which was inhibited in rainbow trout leukocytes (Elsasser et al., 1986) or enhanced in the goldfish (Muchvich et al., 1995; Jacobson and Reimschuessel, 1998). Although, in fish, more evidence is shown for immunosuppression induced by copper as indicated by decreased antibody titers, phagocyte respiratory burst activity and B-like cell proliferation (Elsasser et al., 1986; Khangarot et al., 1988; Dunier, 1994; Shariff et al., 2001), no changes in immune parameters such as T-like cell proliferation, phagocytosis and total natural antibodies level have also been observed (Carballo et al., 1992; Dethloff and Bailey, 1998). Nevertheless, depending of the immune parameters tested, an immunostimulation has also been demonstrated in fish exposed to low concentrations of metals (Zelikoff et al., 1996). Thus, copper effect of fish immune parameters may vary depending on dose, exposure duration, fish species and age of individuals. . . . This is also true for non-specific humoral parameters such as lysozyme

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activity in fish tissues. Exposure to heavy metals modulates lysozyme levels in organs but the nature of the modulation can be complex (Bols et al., 2001). As the other immune factors studies, lysozyme response may be influenced by exposure route, time of exposure or both tissue explaining the immunostimulating effects observed in carp exposed to copper. Moreover, the exposure of rainbow trout to an acute stressor revealed that plasma lysozyme activity was significantly elevated in stressed fish (Moeck and Peters, 1990; Demers and Bayne, 1997). Thus, in our study, copper or chitosan-exposed carps may live under acute stressful conditions associated with enhancing of physiological parameters such as some immune factors as reported by Demers and Bayne (1997). In this work, we also studied the effects of a mixture, associating the two products copper and chitosan, on the same humoral immune factors. The association of copper and chitosan seems to reduce the impact of the each product on natural antibodies particularly in plasma. Moreover, the stimulatory effect of chitosan on ceruloplasmin activity was inhibited when it is combined with copper, the effect of the mixture being identical to these observed with copper alone. Finally, the stimulatory effect of chitosan on plasmatic lysozyme activity was exacerbated when fish were treated with the mixture. These discrepancies between immunomodulation induced by copper or chitosan alone and those observed after treatment of fish with the mixture can be due to the physical and chemical properties of chitosan. Chitosan has particularly the capacity to form complexes with metal ions such as copper, lead, mercury, cadmium. . . (Findon et al., 1993; Evans et al., 2002). Thus, we can think that the potential complexion of copper with chitosan reduced the availability of the heavy metal to modulate humoral immune factors. Chitosan would then be able to protect carp against immunotoxicological damages induced by copper. Besides, Shon et al. (2002) showed that mice treated with chitosan were protected from dioxin-induced oxidative stress. This dual effect of copper–chitosan combination may depend on the concentration of copper and chitosan and the exposure time. Moreover, the uptake of metal ions by chitosan is profoundly affected by physicochemical conditions, including pH, particle size, agitation rate. . . (Evans et al., 2002). Thus, the biological effects of the associated

two products can depend on the physicochemical conditions measured in the experimental medium. Moreover, further studies have to be carried out to elucidate the mechanism of the potential of chitosan against copper activity and vice versa. In our study, when we have dissected out the fish after 96 h of exposure to the different treatment (copper or chitosan or copper + chitosan), the examination of intestinal contents revealed that approximately half fish of each group of treatment was infected by a cestoda identified as Ptychobothrium sp. Its specie is known as a parasite of marine and freshwater teleosts characterised by a shape of scolex heart and bottridia oval with having disc (Wongsawad et al., 1998). We could then observe how the humoral factors studied were modulated by the parasitose. In the environment, the immunotoxicological response of fish to pollutants can be modulated by abiotic and biotic factors. For example, age appears to be a factor in determining impact of polychlorinated biphenyls on the immune function of Japanese medaka (Oryzias latipes). (Duffy et al., 2002). Moreover, fish natural pathogens can also modify the biological responses of individuals to pollutants. It was then possible to examine the immunotoxicological responses of humoral immune factors in function of fish health status in the different groups. The responses of humoral immune factors of carp to copper, chitosan or copper + chitosan, was modulated by the presence of the parasite Ptychobothrium. This study showed that exposure to an adult form of parasite can modulate the host stress response to a subsequent stressor (copper of chitosan or copper + chitosan). The exposition protocol in this experiment evoked a greater stress response in the parasitised group as indicated particularly by the high elevation of lysozyme activity observed in copper-exposed carp. This could indicate an additive effect on the stress response mediated by parasite as observed for other stress markers (Flos et al., 1988). An identical hypothesis can be done for increases in natural antibodies levels observed in parasitised carp treated with the copper–chitosan mixture. But we must not forget that copper was often used in aquaculture for treatment of fish populations against parasites. Indeed, this immunostimulating activity of copper was observed on lysozyme activity of healthy carps.

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Differences in humoral immune factors responses to pollutants observed between healthy and parasitised carps can be explained by the redistribution of immune cells within lymphoid organs. The movement of circulating lymphocytes in mammals are known to be affecting by circulating corticosteroid during different chronic stress and particularly during parasitic infestation (Chilmonczyk and Monge, 1999; Ruane et al., 1999). Moreover, cellular disturbances such as modulation in receptor expression and cellular markers in parasitised fish could not be excluded. As a matter of fact, lysozyme, ceruloplasmin and natural antibodies are some major components of the fish innate immune system occurring in inflammatory process (Bayne and Gerwick, 2001; Magor and Magor, 2001). Inflammation is a physiological response to numerous stimuli such as infection, tissue trauma or different stressful factors. Generally, this response is considered as an acute, transitory phenomenon, belonging to the flight-or-flight response which prepares an animal for coping with alarming situations and their potential consequences including injury (Barton and Iwama, 1991). The acute inflammation processes are usually associated with a systemic response called the acute phase response (APR). One clear indication of the response is the increase in synthesis and secretion by the liver of several plasma-proteins, with simultaneous decreases in other (Bayne and Gerwick, 2001). This acute phase proteins (such as lysozyme, ceruloplasmin and natural antibodies) function in a variety of defence-related activities such as limiting the dispersal of infectious agents, repair of tissue damage, killing of microbes and other potential pathogens, and restoration of the healthy state. The “pro-inflammatory” process induced by stimuli such as copper or chitosan contamination was associated with modulated rates of plasma-protein synthesis in carps. The proteins whose rates of synthesis increase are involved in repair tissue damage, in restoring the healthy state of organisms. The most responsive of these so called acute phase proteins have been used as indicators of the extent of tissue damage or of incipient septicaemia. So, copper and chitosan may induce separately an inflammatory acute phase response by stimulating some components of this innate immune response. Moreover, we noticed an additional impact of stress occurred by the presence of a parasite and the contamination which lead to a decrease of the

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plasmatic ceruloplasmin activity. This decrease can be transitory and compensate by the resumption of the hepatic synthesis. Thus, the contaminant impacts are often modulated by the persistence of the cestoda in carp which may enhance the pro-inflammatory process or lead to a chronical inflammation. Such acute, short and local inflammatory response must be imperatively regulated to control potential cellular damages and facilitate the tissular repairing mechanisms. In case of the persistence of stimulating agent (parasite or pollutant such as heavy metal) in the organism, immunological activation can lead to a chronical inflammation usually associated with pathological consequences such as important necrosis and tissular lesions (Bayne and Gerwick, 2001). Thus, Molinaro et al. (2002) showed that the injection of chitosan led to an acute inflammatory response in rat. Lindenstrom et al. (2004) have also demonstrated that a ciliate parasite (Ichthyophthirius multifiliis) initiate rapidly an inflammation processes in rainbow trout (O. mykiss) and high continuous infection pressure could prevent to clear the infection. Continuous infection can lead to an exhausting phase leading to the death as the stress is too high. Actually, there was no data on the potential consequences to a long-term exposition of organisms parasitised or not, to copper or chitosan in term of chronic inflammatory response, and our work did not permit to answer to these questions, because of the shorter exposure time of carps to pollutants used here. Such response will have to be considered in future studies.

5. Conclusion It is commonly stated that innate immunity is “non-specific”. By such means, fish can be used as sensitive sentinels capable of indicating the presence of toxins, other pollutants, it is therefore important of reporting other environmental perturbations as parasitism infection to determined the potential impact of such pollutants. It is also important to notice the physiological state of fish, parasitised or not, and the impact of this parasitism before and during all experimentations. Moreover, results of the present study have shown that exposure to copper, at environmentally realistic levels, or chitosan, at sublethal concentrations, may

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significantly influence various aspects of immune functions in infected or not carp such as non-specific humoral immunity. These two products orally administrated, are immunostimulating provoking an acute, short and local inflammation whereas the impact of the mixture is less pronounced. The potential simultaneously presence of these two products in the aquatic environment would be a relatively low ecotoxicological problem in term of immunotoxic impacts on fish populations. Many factors likely influence the susceptibility of fish to pollutant, including the route of entrance, the type of toxic and whether the exposure is made to na¨ıve fish or fish with previous exposure or virus, bacteria, parasitism.

Acknowledgements This study was financed by the French Ministry of Research and Europol’ Agro Foundation. The authors wish to thank Aber Tech Society to provide chitogel® , Dr F. Arnoult for the determination of parasite species and Mrs A. Conreux for her technical assistance.

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