2.4.3 Antimicrobial peptides
Antimicrobial peptides (AMPs) are small peptides (less than 100 amino acids) with low molecular weight and direct microbicidal activities. They are mostly cationic and amphipathic, although some anionic peptides are included among them. AMPs are synthesized as pre-pro-peptides, and, once the active peptides are released by cleavage, they interact directly with pathogens, leading to rapid destruction of the microorganism, either bacteria, virus, protozoa, fungi or tumor cells (Cuesta et al., 2008; Chia et al., 2010; Mihajlovic and Lazaridis, 2010).
Although the main function of AMPs is the direct lytic activity on pathogens, other relevant functions have been described for them such as endotoxin neutralization, chemotactic activity, and immunomodulation. A large number of AMPs have been isolated from fish, including cationic peptides, linear amphipathic a-helical peptides (piscidins, moronecidins, gaduscidins, epinecidin, pleurocidin, chrysophsins, cathelicidins), cationic peptides derived from larger proteins (histone-derived, hemoglobin-derived, hemocyanin-derived peptides) or cationic peptides with cysteines that form intramolecular bonding (defensins, hepcidins) (reviewed in Rajanbabu and Chen, 2011; Valero et al., 2013). Anionic peptides have also been reported in the case of Setipinna taty (Song et al., 2012).
AMPs are mainly expressed in the skin and mucosal epithelia, but constitutive and inducible expression of different AMPs have been described in many tissues, including liver, kidney, blood, spleen, gills, eyes, gonads, or pituitary gland (Nam et al., 2010; Rajanbabu and Chen, 2011; Valero et al., 2013). The activity of many of these fish AMPs have been demonstrated against a wide number of bacterial, viral, and fungal pathogens (Casadei et al., 2009; Chang et al., 2005; Chia et al., 2010; Cuesta et al., 2008; Jin et al., 2010; Rajanbabu and Chen, 2011; Zhao et al., 2009).
2.4.4 Lectins
Lectins are oligomeric carbohydrate-binding proteins characterized by the presence of a carbohydrate recognition domain that is highly specific for sugar moieties. They are ubiquitous and have been found in animals, plants, and microorganisms. This diversified group of proteins has different roles in animal biology, including the mediation of cell-to-cell interactions, homeostatic regulation, and immune recognition of foreign carbohydrates. Animal lectins are classified in several families, depending on their amino acid sequences and biochemical properties (Zelensky and Gready, 2005). Fish present a greatly diversified lectin repertoire with representatives from most lectin families described so far (C-type lectins, galectins, pentraxins, X-type lectins/intelectins, calnexin, and calreticulin) and also with members of lectin families described for the first time in fish (F-type lectins, rhamnose-binding lectins, and pufflectins) (reviewed in Vasta et al., 2011).
The tissue-specific expression of the diverse lectin repertoires in fish suggests distinct biological roles in innate and adaptive immunity. Some lectins bind endogenous ligands, whilst others bind sugars on the surface of potential pathogens; therefore, in addition to pathogen recognition and opsonization, some lectins display additional effector roles such as complement activation and regulation of immune functions. Furthermore, some fish lectins mediate nonimmune processes through the recognition of exogenous ligands, acting, for example, as antifreeze proteins or preventing polyspermy during fertilization (reviewed in Vasta et al., 2011).
2.4.5 Other humoral factors
Lysozyme is a mucolytic enzyme that acts on the peptidoglycan of bacterial walls, catalyzing the hydrolysis of the linkages between the N-acetylmuramic acid and the N-acetyl-D-glucosamine. It is produced mainly by monocyte–macrophages and neutrophils and, consequently, is abundant in lymphoid tissues, serum, mucus, and eggs. Serum lysozyme has been identified in practically all aquacultured species and its modulation, in response to infection and physiological stress, has been demonstrated (Fast et al., 2002; Grinde, 1989; Lie et al., 1989; Saurabh and Sahoo, 2008).
Transferrins are iron-binding proteins that control the level of free iron in biological fluids. They are associated with innate immunity by chelating free iron and thus blocking the survival of many classes of bacteria. Ferritin is another protein related with iron storage that can also have a role in immunity. Both proteins have been identified in several fish species and, surprisingly, are mainly expressed in the liver and brain (Bayne and Gerwick, 2001; Neves et al., 2009). During bacterial infections, these proteins can act as negative or positive acute phase proteins, and, consequently, their protein expression can be either up- or down-regulated. Additionally, fish transferrin exerts a novel role as macrophage activator, which may represent a primitive and evolutionary conserved mechanism for the induction of NO response in macrophages (Stafford and Belosevic, 2003). There are other mucosal factors, such as a2-macroglobulin, a protease inhibitor that can act against pathogen proteases (Armstrong and Quigley, 1999), or lytic enzymes like the hydrolases chitinase and cathepsin, which have been also described in fish, but their role in the immune system remains essentially unexplored (Magnadottir, 2006).
2.4.6 The mucus
Mucus represents the biochemical interface between fish and the aqueous external environment and is continuously exposed to microbes and stressors. All fish surfaces in contact with the external environment are covered by a mucosal layer, including the skin, the gills, the gut, and the eggs. Mucus composition, structure, and thickness of the mucosal layer can vary depending on the mucosal territory and the physiological, immunological, or environmental conditions. The functions of this mucosal barrier include mechanical, physiological, and immunological aspects. Mucus is produced by secretory cells and is composed of a matrix of glycoproteins (mucins) that confer a gel structure and contain diverse humoral immune factors with biostatic or biocidal activities against pathogen infiltration, including Igs, complement factors, C-reactive protein, lectins, lysozyme, proteolytic enzymes, antimicrobial peptides, phosphatases, and esterases (Alexander and Ingram, 1992a; Jones, 2001; Palaksha et al., 2008).