**1. Introduction**

240 Pesticides in the Modern World - Risks and Benefits

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Contamination is one of the major problems associated with the environmental sciences. Many of the environmental pollutants affect to the different aquatic animals to certain degree depending on the toxic substance, concentration, self-life and animal behaviour and biology. Direct ingestion of environmental contaminants and bioaccumulation of toxic substances in bivalves, crustaceans, molluscs or fish for human supply is a serious task to consider in human nutrition. Furthermore, it is known that to provide the necessary proteins that need and will need the world's population must intensify efforts in production of both proteins of plant origin and animal origin. Among the latter is predicted that aquaculture will be one of the fields over the coming years will increase. In this regard, aquaculture is trying for some decades to compensate this negative balance for human consumption. Among the important issues to consider in the aquaculture business the impact of the environmental contaminants in the species produced for humans need to be controlled by the farmer. In this specific field, most of studies have evaluated the toxic effects in terms of fish viability or induction of tumors using different fish models. However, relevant fish species for aquaculture are less used in these experiments. Moreover, the impact of the environmental contaminants in the immune response of these fish, and consequently in the disease resistance, have received much less attention.

#### **2. Overview of the teleost fish immune response**

Fish are the first group of vertebrate animals with both innate and adaptive immune responses and are essential for proper understanding of the immune system and its evolution. The fish adaptive immune responses are less effective than in mammals because they are poikilotherms and completely dependent on the environmental temperature. Therefore, the importance of the innate immune response is more relevant, but not exclusive, in the fish disease resistance to pathogens. Overall, the mechanisms and molecules involved in the immune response are quite well conserved during the immune system evolution. However, there are major differences in terms of haematopoietic organs structure and function as well as in leucocyte distribution and function (Figure 1).

Immunotoxicological Effects of Environmental

population (Cuesta et al., 1999).

Hikima et al., 2011).

Contaminants in Teleost Fish Reared for Aquaculture 243

Moreover, they are mainly localized in kidney and spleen where they concentrate the ingested particles and aggregate in melano-macrophage (MM) centres. Granulocytes can be divided in neutrophils, eosinophils and basophils according to their staining properties but in the case of fish the distribution and functions do not fit well with their mammalian counterparts. Monocyte-macrophages and some granulocytes form the phagocytic cells involved in phagocytosis of particulated antigens and in production of a machinery of lytic enzymes and the respiratory burst reaction, in which very toxic reactive oxygen species (ROS) and nitrogen intermediates (RNI) are produced. Finally, nonspecific cytotoxic cells (NCCs) are involved in the lysis of tumor cells, virus-infected cells and parasites in a similar fashion than the mammalian NK cells (Evans et al., 1984). However, they are a heterogeneous population (lymphocytes, granulocytes and/or monocyte-macrophages) and therefore some authors talk of nonspecific cytotoxic activity more than a cellular type or

The humoral immune response is a compilation of proteins and glycoproteins with defense functions found in the fish plasma and other body fluids such as mucus or sexual products (Kaattari & Piganelli, 1997). The complement system, in plasma and mucus, shows classical, alternative and lectin activation pathways with levels 5-10 times higher than in mammalian species with most of its components detected and characterized (Holland & Lambris, 2002). Direct lytic activity against bacteria, virus and parasites is the most relevant and studied function but it also acts as opsonin, chemotactic and neutralize endotoxins (Boshra & Sunyer, 2006). An important bacteriolytic enzyme is the lysozyme, mainly found in eggs, mucus, plasma and leucocytes (Magnadottir, 2006). There are also other innate immune factors such as acute phase proteins (C-reactive protein CRP), antimicrobial peptides, interferon (IFN), lectins, proteases, protease inhibitors or eicosanoids (Secombes, 1996; Aranishi, 1999; Bayne & Gerwick, 2001; Robertsen, 2006; Cammarata et al., 2007; Cuesta et al., 2008a). Finally, and the most interesting in fish, Ig are the major component of the adaptive humoral immune response. Fish were thought to have only one immunoglobulin isoform, the IgM. The fish IgM is tetrameric instead of pentameric as it occurs in mammals. Both membrane and soluble forms are observed by alternative processing of the mRNA (Wilson et al., 1990). Igs are found in the membrane of the B lymphocytes and this can be used to separate Ig+ and Ig- cells. The Ig functions are antigen neutralization, precipitation, opsonization and activation of the classical pathway of the complement system. In the last years, the presence of other Ig isoforms (IgD, IgZ or IgT) is throwing some light into the repertoire of fish immunoglobulins and their evolution in vertebrates (Hsu et al., 2006;

**3. Immunotoxicological effects of environmental contaminants** 

Environmental contaminants are widely distributed in aquatic environments. Although many of them are prohibited or restricted most of them are very persistent in the nature. Field and semi-field experiments are good to have suspicions about the contaminant presence but the setup of laboratory experiments with controlled parameters and precise and pure compounds are strictly necessary to understand the impact on fish immune response and their potential mechanisms. In line with the immunotoxicological studies in mammals, most of fish studies have evaluated the immune response (Figure 1) by measuring the macrophage functions (i.e. phagocytosis and ROS production), lymphoproliferative responses, host disease resistance, antibodies (circulating antibody

Fig. 1. Fish immune system organization (from Manning, 1998) and representative humoral and cellular immune responses used in immunotoxicological studies.

Firstly, the immune tissues are quite different since fish lack the bone marrow and lymphatic nodules (Manning, 1998). Thus, pronephros (anterior/head-kidney) is the main lympho-haematopoietic tissue in fish, whilst the posterior part or mesonephros is mainly excretory and the first site for development and B cells production. Thymus is the main tissue for T cells development and maturation whilst spleen is the main secondary lymphoid tissue in fish. Other important site for the immune response is the mucosal associated-lymphoid tissue (MALT), disperse in the skin, gill and gut. The leucocyte-types present in fish are quite similar between vertebrates but with some specific differences (Meseguer et al., 1994; Secombes et al., 2005; Miller et al., 1998; Rombout et al., 2005). Thus, fish lymphocytes are responsible for the production of antibodies (B cells) and the specific cellular immune response (T cells). B lymphocytes express and secrete immunoglobulin M (IgM), respond to the mitogen lipopolysaccharide (LPS) and constitute about 30% of the circulating lymphocytes. T lymphocytes are mainly detected in the thymus, express the Tcell receptor (TCR) and proliferate with the mitogens concanavalin A and phytohemagglutinin (PHA). They are responsible for the humoral and cellular immune response against T-dependent antigens by the different populations of CD4+ (Th or helper) and CD8+ (Tc or cytotoxic). Moreover, there are also subpopulations of fish lymphocytes lacking proper cell markers, Ig or TCR, and constitute the natural killer (NK) cells (Shen et al., 2002). By other side, monocyte-macrophages are the leucocytes displaying similar characteristics to both mammalian circulating monocytes and tissular macrophages.

Th, thymus Pr, pronephros

**CD8**

Fig. 1. Fish immune system organization (from Manning, 1998) and representative humoral

Firstly, the immune tissues are quite different since fish lack the bone marrow and lymphatic nodules (Manning, 1998). Thus, pronephros (anterior/head-kidney) is the main lympho-haematopoietic tissue in fish, whilst the posterior part or mesonephros is mainly excretory and the first site for development and B cells production. Thymus is the main tissue for T cells development and maturation whilst spleen is the main secondary lymphoid tissue in fish. Other important site for the immune response is the mucosal associated-lymphoid tissue (MALT), disperse in the skin, gill and gut. The leucocyte-types present in fish are quite similar between vertebrates but with some specific differences (Meseguer et al., 1994; Secombes et al., 2005; Miller et al., 1998; Rombout et al., 2005). Thus, fish lymphocytes are responsible for the production of antibodies (B cells) and the specific cellular immune response (T cells). B lymphocytes express and secrete immunoglobulin M (IgM), respond to the mitogen lipopolysaccharide (LPS) and constitute about 30% of the circulating lymphocytes. T lymphocytes are mainly detected in the thymus, express the Tcell receptor (TCR) and proliferate with the mitogens concanavalin A and phytohemagglutinin (PHA). They are responsible for the humoral and cellular immune response against T-dependent antigens by the different populations of CD4+ (Th or helper) and CD8+ (Tc or cytotoxic). Moreover, there are also subpopulations of fish lymphocytes lacking proper cell markers, Ig or TCR, and constitute the natural killer (NK) cells (Shen et al., 2002). By other side, monocyte-macrophages are the leucocytes displaying similar characteristics to both mammalian circulating monocytes and tissular macrophages.

Kid, mesonephros Spl, spleen

> Specific cytotoxicity Proliferation

Cytotoxic activity (NCCs, NK, Tc), circulating leucocytes, infiltration, distribution, gene expression, cytokines…..

**TCR**

**Cellular immunity (kidney, spleen, thymus, blood, peritoneum)**

**Tc Th**

**CD4**

B, lymphocyte B Tc, cytotoxic T lymphocyte Th, helper T lymphocyte MM, monocyte-macrophage

G, granulocytes Ig, immunoglobulin TCR, T-cell receptor NCC, nonspecific cytotoxic cells NK, natural killer cells

> Phagocytosis Respiratory burst Antigen presentation

**<sup>G</sup> MM**

**Ig**

**Humoral immunity (serum, mucus)**

Antibodies Complement Lysozyme Lectins C-reactive protein Interferons Transferrin Anti-proteases Proteases Eicosanoids Cytokines

**B**

Circulating antibodies Antibody-forming cells Proliferation

and cellular immune responses used in immunotoxicological studies.

Moreover, they are mainly localized in kidney and spleen where they concentrate the ingested particles and aggregate in melano-macrophage (MM) centres. Granulocytes can be divided in neutrophils, eosinophils and basophils according to their staining properties but in the case of fish the distribution and functions do not fit well with their mammalian counterparts. Monocyte-macrophages and some granulocytes form the phagocytic cells involved in phagocytosis of particulated antigens and in production of a machinery of lytic enzymes and the respiratory burst reaction, in which very toxic reactive oxygen species (ROS) and nitrogen intermediates (RNI) are produced. Finally, nonspecific cytotoxic cells (NCCs) are involved in the lysis of tumor cells, virus-infected cells and parasites in a similar fashion than the mammalian NK cells (Evans et al., 1984). However, they are a heterogeneous population (lymphocytes, granulocytes and/or monocyte-macrophages) and therefore some authors talk of nonspecific cytotoxic activity more than a cellular type or population (Cuesta et al., 1999).

The humoral immune response is a compilation of proteins and glycoproteins with defense functions found in the fish plasma and other body fluids such as mucus or sexual products (Kaattari & Piganelli, 1997). The complement system, in plasma and mucus, shows classical, alternative and lectin activation pathways with levels 5-10 times higher than in mammalian species with most of its components detected and characterized (Holland & Lambris, 2002). Direct lytic activity against bacteria, virus and parasites is the most relevant and studied function but it also acts as opsonin, chemotactic and neutralize endotoxins (Boshra & Sunyer, 2006). An important bacteriolytic enzyme is the lysozyme, mainly found in eggs, mucus, plasma and leucocytes (Magnadottir, 2006). There are also other innate immune factors such as acute phase proteins (C-reactive protein CRP), antimicrobial peptides, interferon (IFN), lectins, proteases, protease inhibitors or eicosanoids (Secombes, 1996; Aranishi, 1999; Bayne & Gerwick, 2001; Robertsen, 2006; Cammarata et al., 2007; Cuesta et al., 2008a). Finally, and the most interesting in fish, Ig are the major component of the adaptive humoral immune response. Fish were thought to have only one immunoglobulin isoform, the IgM. The fish IgM is tetrameric instead of pentameric as it occurs in mammals. Both membrane and soluble forms are observed by alternative processing of the mRNA (Wilson et al., 1990). Igs are found in the membrane of the B lymphocytes and this can be used to separate Ig+ and Ig- cells. The Ig functions are antigen neutralization, precipitation, opsonization and activation of the classical pathway of the complement system. In the last years, the presence of other Ig isoforms (IgD, IgZ or IgT) is throwing some light into the repertoire of fish immunoglobulins and their evolution in vertebrates (Hsu et al., 2006; Hikima et al., 2011).
