Review on the immunology of European sea bass Dicentrarchus labrax

Veterinary Immunology and Immunopathology 117 (2007) 1–16 www.elsevier.com/locate/vetimm

Review

Review on the immunology of European sea bass Dicentrarchus labrax Dimitry A. Chistiakov a,*, B. Hellemans b, F.A.M. Volckaert b a

Department of Pathology, University of Pittsburgh Medical Center, A719 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA b Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Charles Deberiotstraat 32, B-3000 Leuven, Belgium Received 6 December 2006; received in revised form 9 February 2007; accepted 19 February 2007

Abstract European sea bass (Dicentrarchus labrax L.) is a marine species of great economic importance, particularly in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, fungi and parasites affect the species, causing various infectious diseases and thereby leading to the most heavy losses in aquaculture production of sea bass. In this respect, knowledge on molecular and genetic mechanisms of resistance to pathogens and specific features of immune response against various infectious agents should greatly benefit the development of effective vaccines and proper vaccination strategies in marker-assisted selection of fish resistant to a range of infections. To date, genetic knowledge on sea bass immune regulatory genes responsible for resistance to pathogens is relatively poor but tends to accumulate rapidly. In this review, we summarize and update current knowledge on the immune system and immune regulatory genes of the sea bass. # 2007 Elsevier B.V. All rights reserved. Keywords: European sea bass; Immune system organization; Immune response; Immune regulatory genes

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytokines and innate immunity . . . . . . . . . . . . . . . . . Major histocompatibility complex . . . . . . . . . . . . . . . Immunoglobulin gene superfamily and B cell-mediated T cell receptor and T cell-mediated immunity . . . . . . . Development of immune cells . . . . . . . . . . . . . . . . . . Conclusions and future prospects . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: BAC, bacterial artificial chromosome; CDR, complementarity-determining region; CTK, cytotoxic T killer; DNP-KLH, dinitrophenyl-conjugated to keyhole limpet haemocyanin; GALT, gut-associated lymphoid tissue; NCC, non-specific cytotoxic cell; NK, natural killer; MAS, marker-assisted selection; RBR, peptide-binding region; RAG1, recombination activating protein 1; ROS, reactive oxygen species; TNP, trinitrophenyl * Corresponding author. Tel.: +1 412 802 6114; fax: +1 412 802 6799. E-mail address: [email protected] (D.A. Chistiakov). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.02.005

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1. Introduction European sea bass (Dicentrarchus labrax L.; Moronidae; Perciformes) is a marine species of great economic importance, particularly in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, fungi and parasites affect the species, causing various infectious diseases. Among those pathologies, viral encephalopathy and retinopathy (Bovo et al., 1999; Ucko et al., 2004), pasteurellosis and vibriosis (Afonso et al., 2005) caused by D. labrax encephalitis virus and bacterial pathogens Photobacterium damselae subsp. piscicida and Vibrio anguillarum, respectively, lead to the most heavy losses in aquaculture production of sea bass. In this respect, knowledge on molecular and genetic mechanisms of resistance to pathogens and specific features of immune response against various infectious agents should greatly benefit the development of effective vaccines and proper vaccination strategies in marker-assisted selection (MAS) of fish resistant to a range of infections (Bricknell and Dalmo, 2005; Chinabut and Puttinaovarat, 2005). Bony fishes, arising about 300 million years ago, share similar immune system organization with other vertebrates (Litman et al., 2005). The immune components include the presence of non-specific cellmediated cytotoxicity (Vazzana et al., 2003), phagocytes (neutrophils and macrophages; Do Vale et al., 2002), T cell and B cell activity (Scapigliati et al., 2000a, 2003), antigen-presenting cells and major histocompatibility complex (MHC; Venkatesh et al., 1999); T cell receptor (TcR; Scapigliati et al., 2000a) and cytokines (Scapigliati et al., 2000b). Anatomically, lymphoid tissues in teleosts include the thymus, headkidney, spleen, gut-associated lymphoid tissue (GALT) and cellular components, displaying humoral and cell immune responses (Scapigliati et al., 2002). As a typical teleost, European sea bass contains all the above attributes of the immune system. To date, genetic knowledge on sea bass immune regulatory genes responsible for resistance to pathogens is relatively poor but tends to accumulate rapidly. In this review, we summarize and update current knowledge on the immune system and immune regulatory genes of the sea bass. 2. Cytokines and innate immunity Innate immunity is the simplest and non-specific type of immune response. In fish, the innate immune system consists of cell components, such as phagocytes (macrophages, neutrophils) and non-specific cytotoxic

cells (NCCs), and various molecular mediators of inflammation, including antibacterial peptides (dicentracine, hepcidin), complement, transferrin, COX-2, chemokines (CXC and CC chemokines and their receptors), cytokines [interleukins (IL-1, IL-8), interferons (IFN), transforming growth factor b (TGF-b) and tumor necrosis factor a (TNF-a)], acute phase proteins (serum amyloids A and P, C-reactive protein, a2-macroglobulin and complement components), Tolllike receptors and molecules of Toll-like receptormediated signaling pathways (Magor and Magor, 2001). In sea bass, innate immune cells were initially found in the headkidney and described as macrophages, stromal and lymphocyte-like cells (Meseguer et al., 1991). Studies on in vitro cytotoxic activity of leukocytes derived from the head-kidney, blood and peritoneal cavity of D. labrax against mammalian tumor cells allowed to separate cells displaying non-specific cytotoxic response to two subpopulations: monocytelike and lymphocyte-like cells (Mulero et al., 1994). Monocyte-like cells exhibited a broad area of contact with a target cell, had oval or kidney-shaped nucleus and a few granules in cytoplasm, whereas lymphocytelike cells established spot contacts with targets and had a large nucleus and occasional cytoplasmic granules. Similar leukocyte populations, exhibiting non-specific cytotoxicity against mammalian tumor cells, have been found in other teleosts, seawater gilt-head sea bream Sparus aurata and freshwater common carp Cyprinus carpio (Meseguer et al., 1994, 1996; Mulero et al., 1994; Nakayasu et al., 2005). These findings suggested that non-specific cytotoxic response in sea bass is mediated by several populations of leukocytes differed from each other by various mechanisms of target recognition and cytolysis. In teleosts, all NCC have been shown to express on their surface the NCC receptor protein, NCCRP-1, by which a cytotoxic cell contacts to its target (tumor cell or protozoan parasite) to induce the lytic cycle against the target (Jaso-Friedmann et al., 1997). Although sea bass NCCRP-1 is not yet isolated, cDNA for this receptor was recently cloned from gilt-head sea bream, a species closely related to the European sea bass. Molecular characterization of this receptor revealed NCC heterogeneity, showing different types of leukocytes (lymphocytes, monocyte/macrophages and acidophilic granulocytes) that contribute to the non-specific cytotoxic response in sea bream (Cuesta et al., 2005). Similar cell types of leukocytes are likely to mediate NCC activity in sea bass.

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The phagocytic activity of head-kidney adherent cells was shown after stimulation with bacterial (Aeromonas salmonicida, P. damselae subsp. piscicida) and fungal (Candida albicans, Sphaerospora dicentrarchi) pathogens (Bennani et al., 1995; Munoz et al., 2000). Then, phagocytic activity was observed in leukocytes from the peripheral blood and peritoneal cavity (Do Vale et al., 2002). Peritoneal macrophages from sea bass displayed a greater capacity to engulf bacteria than did those isolated from blood, which, in turn, had greater engulfment properties than those isolated from the head-kidney (Esteban and Meseguer, 1997). Killing of bacteria by macrophages was associated with increased release of reactive oxygen species (ROS; Bennani et al., 1995). Low levels of NCC were detected in sea bass headkidney and spleen, but not in blood leukocytes. The NCC was mostly attributed to eosinophilic granule cells isolated from the peritoneal cavity (Cammarata et al., 2000). The eosinophils have abundant cytoplasmic granules positive for peroxidase and arylsulphatase, e.g. enzymes, participating in the generation of ROS (Do Vale et al., 2002). In sea bass, NCCs are shown to kill their targets by inducing necrosis and apoptosis in a similar way to mammalian cytotoxic cells (Meseguer et al., 1996). NCC and phagocyte responses are followed by secretion of a number of proinflammatory molecules that have pleiotropic effects. For example, IL-1 and TNF-a exhibit overlapping activities, including induction of acute phase protein synthesis, proliferation of lymphocytes and stimulation of specific immune reaction (B and T cells; Scapigliati et al., 2000b). In mammals, IL-1 family of molecules consists of several distinct but structurally related compounds: IL-1a, IL1b, IL-18, IL-33, interleukin-1 receptor accessory proteins, interleukin-1 receptor antagonist and around ten more members. IL-1a is predicted to arise from the duplicated Il-1b gene at the estimated time of 270 million years ago (Hughes, 1994). In fish genome, only a single copy of the IL-1b gene presents, except for the duplicated IL-b genes found in rainbow trout Oncorhynchus mykiss and common carp, both are pseudotetrapoloids, having a relatively recent history of tetraploidy (Pleguezuelos et al., 2000; Engelsma et al., 2003). For seabass, the full-length IL-1 b gene was isolated and characterized (GenBank accession AJ269472 and AJ311925; Scapigliati et al., 2001). The IL-1 b gene, encoding a 1292-bp long mRNA and a 29.4-kDa polypeptide, is 2.7-kb long and contains five exons. The highest nucleotide homology was observed between sea

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bass exon 4, trout exon 5, carp, mouse, chicken, pig and human exon 6 (Buonocore et al., 2003). Sequence analysis revealed highest amino acid similarity with rainbow trout (62%), Xenopus (46%) and carp (45.5%) IL-1b sequences. Seven single nucleotide polymorphisms (SNP) were identified in the sea bass IL-1b gene (Chistiakov et al., unpublished data). Four of them T/ C(376), A/G(271), A/G(213) and A/G(118) are located at the promoter, being in strong linkage disequilibrium to each other, whereas the remaining three (C/T +482, C/T +548 and A/C +650) are situated in intronic regions of the genomic sequence (GenBank accession AJ269472). However, these SNPs were uninformative in the Venezia F bis family used to construct a first generation linkage map (Chistiakov et al., 2005). It therefore did not allow for mapping the IL-1 b gene. However, the IL-1b SNPs could be extremely useful for analysis of genetic predisposition or resistance in sea bass challenged with different pathogens. Expression of sea bass IL-1b can be upregulated by bacterial lipopolysaccharide both in vitro and in vivo in leukocytes from blood, head-kidney, spleen, gills and liver, whereas the IL-1b transcript was not detectable in thymus and GALT (Scapigliati et al., 2001). A biologically active recombinant IL-1b from sea bass was produced in bacteria, suggesting for possibility to be used as an immunoadjuvant in sea bass vaccination experiments (Buonocore et al., 2004, 2005a). In mammals, a cleavage of the inactive precursor of IL-1b, proIL-1b, by specific protease, IL-1-converting enzyme (ICE), after the activation of P2X7 receptor by extracellular ATP results in production of the mature and biologically active cytokine (Thornberry et al., 1992; Colomar et al., 2003). Little information is available on the mechanism of processing and release of fish IL-1b, but the IL-1b gene sequences reported to date lack a conserved ICE recognition site (LopezCastejon et al., 2003). In fish, proIL-1b ungergoes posttranslational processing through the cleavage by unknown protease. Caspases are likely to represent alternative peptidesplicing enzymes. For sea bass, cDNAs encoding three different caspases [caspase-1 (Genbank DQ198376 and DQ198377), caspase-3 (DQ45773 and DQ45774) and caspase-9 (DQ345775 and DQ345775)] have been isolated (Reis et al., 2007a, 2007b). Of them, the sequence of the sea bass caspase-9 cDNA displays high similarity with orthologs from other vertebrates, particularly with putative caspases-9 of zebrafish Danio rerio and pufferfish Tetraodon nigroviridis (77.5 and 75.4% homology, respectively). The caspase contains a

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highly conserved caspase recruitment domain (CARD) in the NH2-terminus (Reis et al., 2007a). The CARD domain is important for the formation of so called apoptosome and recruitment of caspase-9 from inactive procaspase-9 through the autoactivation by self-cleavage (Zou et al., 1999a,b). Once activated, caspase-9 disassociates from the apoptosome and becomes available to cleave and activate downstream caspases such as caspase-3. The subsequent activation of downstream caspases transmits the proapoptotic signal to the execution phase. Reis et al. (2007a) observed that expression of caspase-9 in the head-kidney of sea bass infected with the P. damselae subsp. piscicida strain PP3 was increased from 0 to 12 h post-infection and returned to basal levels at 24 h. In addition, caspase-9-like activity was detected in the infected sea bass head-kidney from 18 to 48 h postinfection, when the fish were with advanced septicaemia. Kinetics of the expression profile of the head-kidney caspase-9 was very similar to that of caspase-3 in an experimental sea bass septicaemia infection by P. damselae subsp. piscicida (Reis et al., 2007b). These bacteria are shown to secrete the apoptogenic exotoxin AIP56 that launches apoptosis in sea bass phagocytes, involving the induction of caspase-3 activity (Do Vale et al., 2003, 2005). Caspase-9 seems to be a part of the AIP56-mediated apoptotic pathway but this is needed to be elucidated. Recently, the isolation of sea bass IL-10 cDNA was reported (Pinto et al., 2007). IL-10 regulates growth, differentiation and function of a variety of immune cells including T and B lymphocytes, NCC and macrophages. On the molecular level, this cytokine exhibits a whole spectrum of biological activities including the suppression of expression of various cytokines and downregulation of the expression MHC class II antigens in monocytes and dendtritic cells, while IL-10 inhances it in B cells (Moore et al., 2001). The genomic sequence of the IL-10 gene spans 2.4 kb and comprises 5 exons and 6 introns. The IL-10 fulllength cDNA (GenBank accession DQ821114) consists of a 145-bp 50 untranslated region (UTR), a 564-bp open reading frame encoding a predicted protein of 187 amino acids and a 30 UTR of 223 bp (Pinto et al., 2007). The predicted polypeptide contains 22 amino acids of the signal peptide and remaining 165 amino acids of the mature IL-10 with the predicted molecular weight of 18.9 kDa. Among fish IL-10 with known amino acid sequences, sea bass IL-10 shares the highest homology with IL-10 from Japanese pufferfish Takifugu rubripes (81.8% identity and 65.8% similarity), whereas the lowest homology is observed between the sea bass

protein and IL-10 from zebrafish (only 65.8% similarity and 42.9% identity) (Pinto et al., 2007). In sea bass, IL-10 expression was detected in the head-kidney, intestine and spleen. In sea bass headkidney and spleen, the expression of IL-10 could be induced with bacterial infection and reaches the maximum at 6 h post-infection (Pinto et al., 2007). The expression peak time was very close to that for IL10 expression induced with bacterial lypopolysaccharides in the head-kidney and spleen of rainbow trout (Inoue et al., 2005) and common carp (Savan et al., 2003). Interestingly, the expression of IL-1b could be also induced with bacterial antigens in the same organs but its peak precedes the expression of IL-10 (Savan et al., 2003; Inoue et al., 2005; Pinto et al., 2007) therefore suggesting for the inhibitory effect of IL-10 on IL-1b production. In addition, this observation indicates a primary role of IL-1b in inflammatory response, while IL-10 induction is delayed due to the time required for T cells to initiate the immune response against bacteria. The expression of IL-10 is necessary when B cell should be recruited to start antibody production against recognized bacterial antigens. Genomic and cDNA sequences of D. labrax interleukin-12 (IL-12) peptides p35 (DQ388037 and DQ388038) and p40 (DQ388039 and DQ388040) are also available in Genbank (Nascimento et al., 2007). Interleukin-12 is crucial in inducing the inflammatory response to bacterial, fungal and protozoan pathogens and bridges innate and antigen-specific adaptive immunity. IL-12 is mainly secreted by activated macrophages, neutrophils and dendritic cells, although B lymphocytes are also able to produce this cytokine. In immune response, IL-12 stimulates proliferation and activation of T lymphocytes and natural killer (NK) cells and induces production of IFN-g by these cells as well as mediates differentiation of naı¨ve T cells into T helper type 1 cells. This cytokine also enhances cytotoxic activity of NK cells and cytotoxic T lymphocytes (Stern et al., 1996). Functional IL-12 represents a heterodimer composed of two covalently linked subunits: a 35-kDa chain called IL-12 p35 (or IL-12a) and a 40-kDa chain known as IL12 p40 (or IL-12b; Kobayashi et al., 1989). The sea bass IL-12 p40 gene is 3225-bp long and contains eight exons and seven introns. The IL-12 p40 cDNA of 1380 bp encodes a predicted protein of 349 amino acids. The p40 subunit contains an 18-residue leader peptide and has a molecular weight of 38.3 kDa (Nascimento et al., 2007). As a member of the long hematopoietin receptor family, the IL-12 p40 contains an N-terminal immunoglobulin (Ig) domain and two fibronectin typeIII domains (Merberg et al., 1992). Among the known

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sequences of IL-12 p40, the sea bass protein is the most similar to the pufferfish with 72.7% similarity and 57.1% identity and zebrafish molecule (60.5% and 39.9%, respectively; Nascimento et al., 2007). The sea bass IL-12 p35 gene has the length of 2408 bp and comprises seven exons and six introns, with similar exon –intron junction positions to the pufferfish IL-12 p35. The 976-bp long cDNA for the IL12 p35 subunit of D. labrax encodes a protein of 199 amino acids (with 27 residues of the leader peptide) that has a molecular mass of 18.9 kDa. The sea bass p35 subunit cDNA sequence exhibits the most similarity and identity with the pufferfish (73.4% and 52.0%, respectively; Nascimento et al., 2007). Expression of IL-12 p35 and p40 subunits in the head-kidney and spleen could be induced by exposure to bacterial antigens. In the head-kidney and spleen, expression profile of the p40 subunit is similar to that of IL-1b, reaching the maximum 3 h after stimulation and declining to basal levels by 24 h. In the spleen, expression of the p35 subunit was also maximal by 6 h but decreased to basal levels by 6 h (Nascimento et al., 2007). In contrast to the spleen, IL-12 p35 mRNA was not up-regulated in the head-kidney. Using pyrrolidine dithiocarbamate, a specific inhibitor of NF-kB, a clear role of this transcription factor in the regulation of the IL-12 subunits and IL-1b mRNA expression was shown. In addition, analysis of the promoter region of these genes revealed one and five potential binding sites for NF-kB in mRNA of IL-12 p35 and p40, respectively (Nascimento et al., 2007). TGF-b is a member of a superfamily of proteins, involved in the regulation of embryogenesis, cell differentiation, inflammation and wound repair. Five TGF-b isoforms have been found in vertebrates (Burt and Paton, 1992). A partial sequence of the sea bass TGF-b cDNA was recently deposited to the nucleotide database (Genbank AM421619). Full-length TGF-b1 cDNA was cloned from a closely related moronid, e.g. from hybrid striped bass Morone saxatilis  M. chrysops (Harms et al., 2000) and gilt-head sea bream (Tafalla et al., 2003), and hence could be useful for isolation of the full-length TGF-b cDNA from D. labrax and further comparative analysis. Genomic sequence for the sea bass TNFA gene (DQ200910), encoding tumor necrosis factor a, is available in Genbank. The 2.06-kb gene consists of four exons and three introns and encodes mRNA of 1562 bp. The predicted length of sea bass TNFa is 260 amino acids. This cytokine is secreted by monocytes and macrophages in response to parasitic, bacterial and viral

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infections and plays a key role in immunity and inflammation. In addition, TNFa contributes to antitumor activity and embryonic development (Liu, 2005). TNFa from D. labrax contains a conserved 17-mer transmembrane domain (GAILIVALCGFGVLLFAW, codons 37–54), whose location exactly corresponds to the position of the potential transmembrane domain in the TNFa molecule from gilthead sea bream (GarciaCastillo et al., 2002). In sea bass and sea bream TNFa, the location of the cleavage site (Thr–Leu; codons 86– 87) is also highly conserved. Cleavage at this site results in a mature 174-residue protein. A putative cell attachment sequence (RGD, codons 232–234) is also present in the sea bass molecule, suggesting for its property as an adhesion protein ligand. In sea bass, cDNA for another important component of innate immunity, cyclooxygenase-2 (COX2; GenBank AJ630649), was isolated and characterized (Buonocore et al., 2005b). This enzyme, whose expression in fish macrophages and monocytes could be induced in response to stimulation by bacterial antigens (Brubacher et al., 2000), participates in early events of the proinflammatory reaction, catalyzing two first steps in the synthesis of prostenoids. Prostenoids (prostaglandins, leukotrienes and lipoxines) are metabolites of arachidonic acid, mediating proinflammatory and apoptotic signals. COX-2 is the inducible enzyme, which is distinct from the well-characterized constitutive activity exhibiting by the COX-1 isoform (Zou et al., 1999a,b). COX-2 cDNA of sea bass contained 2350 nucleotides and encoded a protein of 596-amino acids. The COX-2 sequence from D. labrax displayed high nucleotide and amino acid similarity with other fish COX-2 genes (Buonocore et al., 2005b). Production of antimicrobial peptides is controlled trough Toll-like receptor-mediated signaling pathways. These peptides are found in both vertebrates and invertebrates, representing primary and primitive defense mechanisms against bacterial infection (Boman, 1995). The peptides exhibit a wide spectrum of antimicrobial activity of some classical bacterial antibiotics. In bony fish, antibacterial peptides can be secreted by the skin, as shown for winter flounder Pleuronectes americanus (Douglas et al., 2001) and finless sole Pardachirus marmoratus (Oren and Shai, 1996), or be used as a tool in the antibacterial action of phagocytes (Ellis, 2001). To date, two sea bass antimicrobial peptides, dicentracine and hepcidin, have been characterized. Dicentracine was isolated from head-kidney granulocytes (GenBank AY303949; Salerno et al., unpublished data). The sequence and structure of dicentracine

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displays high homology with those of moronecidin, an antimicrobial peptide from other moronids, white bass (Morone chrysops) and striped bass (Morone saxatilis; Lauth et al., 2002). The prepropeptide consists of 79 amino acid residues and subsequently undergoes transformation into the mature 22-residue peptide, which contains a repeating motif (QQDQQDQQYQQDQQDQQA). The repeating motif includes six trimeric submotifs, each containing two positively charged glutamine (Q) residues, which form a cationic channel, penetrating the outer bacterial membrane and then causing death of the bacterial cell. Hepcidin (GenBank DQ131605) was isolated from the liver of D. labrax (Rodrigues et al., 2006). The sequence of hepcidin is highly conserved in vertebrates (Lauth et al., 2005). The prepropeptide comprises 85 amino acids and then undergoes posttranslational processing to the mature 21-residue peptide that includes eight cysteines engaged in four disulfide bonds (Shike et al., 2002). There are several copies of the hepcidin gene in sea bass genome. Hepcidin is expressed at low basal level. This peptide is cationic and adopts an amphipathic structure in solution and thus has the potential to interact with and disrupt bacterial membranes like linear, ahelical peptides. However, hepcidin is less cationic and amphipathic than dicentracine or moronecidin and therefore should exhibit a less potent antimicrobial activity. Experiments with Yersinia enterocolitica demonstrated that in vitro hepcidin kills this bacterium much more slowly than does moronecidin (Lauth et al., 2005). Interestingly, its expression could be stimulated either by bacterial exposure or by excess in iron ions (Rodrigues and Pereira, 2004; Rodrigues et al., 2006). The dual function of hepcidin is preserved in vertebrates: in mammals, except for immune response, this peptide plays an important role in iron homeostasis, and mutations in the human hepcidin gene are shown to be associated with juvenile chemochromatosis, an autosomal recessive disorder (Roetto et al., 2003). Other molecular components of innate immunity of sea bass (chemokines, acute phase proteins, Toll-like receptors) remain unknown and have to be isolated and characterized. Their sequences are available for rainbow trout, Atlantic salmon Salmo salar, zebrafish and some other important fish and therefore could be successfully applied for the isolation of similar genes from sea bass. 3. Major histocompatibility complex In teleosts, the major histocompatibility complex includes classical class I and class II gene clusters,

which are not linked and are even located on different chromosomes (Bingulac-Popovic et al., 1997; Hansen et al., 1999). However, in the genome of tetrapods, class I and class II genes are closely linked (Kasahara, 1998). Class I and class II genes are closely related structurally, genetically and evolutionarily, suggesting for the existence of their common ancestor (Klein and Sato, 1998). Two models have been proposed to explain the different organization of MHC genes in teleost fish and tetrapods (Fig. 1). For teleosts, class I and class II genes were originally in the same chromosome but were separated in a recent teleost ancestor and now lie on different chromosomes. For tetrapods, those genes arose from different paralogous chromosomes in a jawed vertebrate ancestor to come together in a tetrapod ancestor (Klein and Sato, 1998). The function of MHC class I and class II molecules is to display foreign peptides (antigens) to T cells. The interaction between the MHC molecule/antigenic peptide (pMHC) complex and TcR on the surface of T lymphocyte leads to the immune recognition of the peptide and then to the induction of immune response against the presented antigen (Mannie, 2001). Class I molecules are responsible for presentation of endogenously derived antigens to cytotoxic T killers (CTK) and mediate antiviral and anticancer immune responses. Class II molecules interact with exogenous antigenic peptides, presenting them to T helper cells, and participate in the induction of immunity against pathogenic bacteria and parasites (Watts, 1997). Class I and class II proteins consist of four extracellular domains, with two membrane-proximal domains, belonging to the immunoglobulin (Ig) superfamily, and two membrane-distal domains, which form the peptide-binding region (PBR; Bjorkman et al., 1987). The PBR binds the antigenic peptide that then will be presented to the T cell through interaction with the TcR. Class II molecules consist of MHC-encoded a and b chains, each having two extracellular domains (half of the PBR and one Ig domain), while MHCencoded class I heavy chains are composed of three domains, comprising the entire PBR and one Ig domain (Fig. 2). The third domain is constituted of the noncovalently associated b2-microglobulin that contributes the other Ig domain of class I molecules (Ohnishi, 1984). The gene, encoding b2-microglobulin, was probably translocated out of the MHC early in vertebrate evolution (Danchin et al., 2004). Class I and class II genes are highly polymorphic, particularly in the PBR-encoded region. PBR amino acid residues that interact with peptides are under positive selection driven by pathogens (Hughes and Nei,

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Fig. 1. Two evolutionary models, explaining the different organization of the MHC class I and class II genes in teleosts and tetrapods. (A) Class I and class II genes arose on the same chromosome to be further separated in teleosts. (B) Class I and class II loci emerged on a different chromosome and merged in tetrapods.

1989). Class I molecules are expressed ubiquitously and bind peptides generated in the cytoplasm by the multicatalytic proteasome, whereas class II molecules bind to the lysosome membrane to accept lysosomally generated peptides. The expression of class II molecules is restricted only to B cells, antigen-presenting cells (macrophages, dendritic cells, etc.) and the thymus (Margulies, 1999).

Fig. 2. Primary structure of the MHC class I and class II molecule. The class I molecule contains a heavy chain (45 kDa) and a light chain called b2-microglobulin (12 kDa) that contributes to the overall structure of the protein. Class II molecule consists of two (a- and b-) chains of similar size (34 and 30 kDa, respectively). Disulfide bridges (S–S) help to form a secondary structure, very similar for both class I and class II. The extracellular N-terminal portion of the molecule that forms a peptide-binding region is very complex compared to the short cytoplasmic C-terminal tail. Class I and class II molecules reside on the surface of antigen-presenting cells. The transmembrane domain of the MHC molecule penetrates the plasma membrane and serves as anchor for the extracellular portion of the molecule.

To date, only three particular nucleotide sequences for MHC class II of D. labrax (AF134955, AF134956 and AY994059) are publicly available in GenBank. The MHC genes are not mapped yet. However, several studies reported a high level of allelic polymorphism for MHC class II genes in closely related species, such as striped bass (Walker et al., 1994; Hardee et al., 1995) and red sea bream Chrysophrys major (Chen et al., 2006). As expected, the highest variability was detected within the exon, encoding the predicted PBR of known MHC class II molecules (Walker et al., 1994). In addition, the nucleotide sequence of MHC class II of both perciformes was highly similar to each other (75.2% of homology) and to the MHC class II genes of cichlid fishes (74.5%; Chen et al., 2006). Challenge of red sea bream with the pathogenic bacteria, Vibrio anguillarum, resulted in a significant decrease in the expression of MHC class II B mRNA, from 5 to 72 h after infection in liver, spleen, head-kidney and intestine, followed by a recovery to normal level after 96 h (Chen et al., 2006). A similar suppression in the expression of class I and II molecules was observed in the macrophage-like cell line from the head-kidney of the Atlantic salmon treated with lipopolysaccharide from Esherichia coli (Koppang et al., 1999) and in spleen of the rainbow trout infected with infectious hematopoietic necrosis virus (Hansen and La Patra, 2002). These results implied that MHC II plays an important role in the immune response of red sea bream to infection, and head-kidney, spleen, liver, intestine, might be important sites for MHC II function.

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4. Immunoglobulin gene superfamily and B cellmediated immunity The Ig superfamily is an extensively diversified multigene family, whose members share a common structural feature, the Ig fold. Except for MHC class I and class II molecules, the Ig superfamily comprises antigenic receptors, such as immunoglobulins, or B cell receptors (BcR), and TcR. Immunoglobulins are composed of two heavy (H) and two light (L) chains (Fig. 3). Every chain consists of two, three or four constant (C) domains, which specify effector functions, and a variable (V) domain, containing the site of antigenic recognition (Matsunaga et al., 1994). During B cell development, a germline repertoire of fewer than several hundreds gene segments (for example, human contains 250 gene segments in its IgH gene cluster) is used to generate a much larger repertoire of variable domain structure (Tonegawa, 1983). Combinatorial joining of VH, DH and JH gene segments (for H chains) with the joining of VL and JL gene segments (for L chains) yields more than 107 different combinations. Variation in the precise point of segmental joining with insertion of templated (P junctions) and non-templated

Fig. 3. Two-dimensional model of an immunoglobulin (Ig) molecule, containing three constant domains encoded by the CH1, CH2 and CH3 gene segments. The top dimer displays the nucleotide structure of the heavy (H) and light (L) chains. The IgL gene comprises variable (VL), joining (JL) and constant (CL) gene segments, whereas the IgH gene additionally includes 2–4 CH segments and the diversity (DH) segment. VL and VH domains contain three intervals of sequence hypervariability termed complementarity-determining regions (CDRs) that are separated from each other by four intervals of relatively constant sequence termed frameworks (FRs). CDRs 1 and 2, and FRs1, 2 and 3 are encoded entirely by the V gene segment, FR4 by the J, and CDR3 is the product of V(D)J joining. Cleavage with papain or pepsin in the hinge region of the Ig molecule yields two (Fc and Fab) fragments, representing constant and antibody portions of the molecule. Fab fragment is responsible for binding an antigen.

(N regions) nucleotides exponentially amplifies the potential diversity of the pre-immune repertoire (Oka and Kawaichi, 1995). Further somatic diversification can be achieved by a hypermutational process of insertions and single nucleotide substitutions in and around rearranged antibody genes that is associated with exposure to antigen (Tonegawa, 1983). A number of enzymes are involved in V(D)J recombination within Ig genes. One of those, recombination activating 1 protein (RAG1) is a component of RAG1/RAG2 recombinase and is responsible for the initiation of V(D)J recombination by introducing DNA double-strand breaks at specific sites in the genome (Jankovic et al., 2004). The RAG1 gene of the European sea bass was partially sequenced (GenBank accession no. AF137181, AF137202, AF137203 and AH008179; Venkatesh et al., 1999) providing an opportunity to find a polymorphic microsatellite designated DLA0222RAG1 in one of the exons of RAG1 (Chistiakov et al., 2005). However, the microsatellite was non-informative in the mapping family, Venezia Fbis, which therefore failed the possibility to incorporate the RAG1 gene into the linkage map of D. labrax (Chistiakov et al., 2005). Bony fish develop a primary and a secondary humoral response upon antigen administration but, unlike mammals, demonstrate no shift in Ig class (Magor and Magor, 2001). D. labrax is a teleost susceptible to many pathogens, including bacteria V. anguillarum and P. damselae (previously classified as Pasteurella piscicida). Immunization with killed and alive bacteria induced the primary and secondary immune responses in the spleen, head-kidney and gut of sea bass. Higher numbers of specific antibody-producing cells were observed in the secondary responses of the head-kidney and spleen (Dos Santos et al., 2001a). Examination of sea bass sera, using electrophoresis and Western blot analysis, revealed great variability in the molecular weight of bacterial antigens recognized by immune sera (Bakopoulos et al., 1997; Pretti et al., 1999). This suggests that sea bass antibodies are able to recognize variable bacterial antigens, from low-weight lipoproteins to high molecular lipopolysaccharides. Around 30 different clones, each encoding a unique sequence of the Ig light chain, have been isolated from the head-kidney cDNA library of sea bass immunized with hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigen (Dos Santos et al., 2001b). Sequencing of these clones showed their relationship to the IgL1/G isotype but also revealed a low variability in their sequences, indicating that the head-kidney would have mainly a haematopoietic function or that the Ig variability could be generated

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in another organ. However, low variability in VL gene segments also assumes that the VH domain could mainly contribute to the variability of the antibody molecule. This suggestion can be supported by data of Solem et al. (2004), who analyzed a repertoire of antibodies induced by treatment of the Atlantic salmon with trinitrophenyl (TNP) and found the involvement of eight different VH families and only three subgroups of IgL isotype 1 sequences in the formation of anti-TNP antibodies. To study humoral responses of the sea bass, several monoclonal antibodies (Mabs) have been generated against Ig and Ig-bearing cells (Romestand et al., 1995; Dos Santos et al., 1997). Most of these Mabs recognized the heavy chain of Ig, while only a few were developed against the light chain. One of the Mabs, specifically binding to the Ig light chain (DLIg3), was used for flow cytometry analysis of the distribution of antibodyproducing cells in sea bass. The analysis showed that DLIg3 stained 21% of peripheral blood leukocytes, 3% of thymocytes, 30% splenocytes, 33% of head-kidney leukocytes and 2% of GALT (Romano et al., 1997), therefore confirming suggestions of Dos Santos et al. (2001a) on the head-kidney and spleen as major organs for specific antibody production in the sea bass. Immune precipitation with Mab DLIg3 specific to immunoglobulins and B cells was used to enrich immunoreactive cells from the peripheral blood, spleen, and head-kidney with B lymphocytes (up to 77% of leukocytes). The DLIg3-purified cells displayed enhanced expression of the B cell-specific Ig gene and low expression of the T cell-specific TcR, then indicating their higher enrichment with B cells (Scapigliati et al., 2003).

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DLT15, the infiltration of numerous T lymphocytes into the grafted sea bass muscle fibers was observed, suggesting for the important role of these cells in the allograft rejection (Abelli et al., 1999; Romano et al., 2005). Using immune precipitation with DLT15 and magnetic sorting of the precipitated cells, populations of lymphocytes highly enriched by T cells have been isolated from the peripheral blood and GALT of the sea bass (80% and 90% of leukocytes, respectively; Scapigliati et al., 2000a). From the GALT-associated T cells, a partial cDNA for both a and b chains of TcR was isolated (GenBank accession AJ012574, AJ493441 and AY831387). The cDNA, encoding TcRb chain, showed 60% of identity with the nucleotide sequence of TcRVb3 chain of rainbow trout. In addition, a deduced amino acid sequence of one of the TcRb cDNA clones was compared with protein sequences of TcR constant regions from cartilagineous and bony fish, and the highest similarity (51%) was demonstrated with the Atlantic cod Gadus morhua (Scapigliati et al., 2000a). These data strongly suggest that cells recognized by DLT15 are highly likely to be T lymphocytes. The TcR molecule resides on the surface of the T cell and interacts with the pMHC complex of the antigenpresenting cell. The interaction stimulates the T cell and induces immune response against the recognized antigen. The TCRa and TcRb chains each consist of an N-terminal variable (V) and a C-terminal constant (C) region (Fig. 4). The V TNF-a and Vb regions are structurally related to the VL and VH domains of Ig. These regions interact to each other and form a dimeric structure. Peptide loops homologous to Ig complementarity-determining regions (CDRs) protrude at the

5. T cell receptor and T cell-mediated immunity Like other bony fishes, D. labrax exhibits T cell immune responses. The conclusion is based on the induction of the proliferation of some subpopulations of sea bass leukocytes by T cell mitogens (Volpatti et al., 1996) and allograft rejection, a T cell-mediated immune reaction (Abelli et al., 1999). Development of Mab DLT15 specific to thymocytes and peripheral T cells allowed the evaluation of T cell populations in sea bass (Scapigliati et al., 1996). DLT15-positive cells were detected mainly in the thymus (approximately 80% of thymic cells) and intestine (approximately 55%). Fewer DLT15-stained cells were found in the spleen (approximately 7% of cells), head-kidney (approximately 6%) and peripheral blood (approximately 3%, Romano et al., 1997). Using immunocytochemical analysis with

Fig. 4. Schematic representation of the T cell receptor (TcR) molecule, which resides on the surface membrane of the T cell. The TcR is a heterodimer, consisting of a- and b-chains. The chains are bound together with a disulfide bridge in the hinge region. The extracellular portion of the TcR contains two variable and two constant regions. The variable regions are involved in the formation of the peptide–MHCbinding site.

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membrane-distal ends, where they collectively form the pMHC-binding site. The CDR1 and CDR2 equivalents are encoded within the TCR V gene segments, while the CDR3 equivalent is formed during somatic V(D)J recombination, which occurs in the thymus and involves the juxtapostion of Va and Vb gene segments in TCRa genes and of Vb, Db and J TNF-a gene segments in the TcRb chain. During V(D)J recombination, the coding ends of the TcR gene segments are subjected to hypermutation events. The extremely variable CDR3 equivalents are located over the center of the pMHC surface and make contacts with the antigenic peptide as well as with MHC a-helices (Garcia and Adams, 2005). At the 30 UTR of TCRB mRNA, encoding b chain of TcR, a polymorphic cytosine-to-thymine substitution situated at position 757 of the nucleotide sequence (GenBank AJ493441) was found (Chistiakov et al., unpublished data). Using this polymorphic marker, the TCRB gene was mapped to the linkage group 27 (Haley et al., unpublished data). So, the availability of SNP within the TCRB gene allows the evaluation of this important functional candidate gene in genetic analysis of markers responsible for susceptibility/resistance of sea bass to different pathogens. A partial sequence of cDNA, encoding the sea bass T cell membrane co-receptor CD4 (AM418542) is available in Genbank. The CD4 co-receptor is a membrane glycoprotein, which binds to MHC class II molecules. The complex between the CD4 and MHC class II molecules enhances TcR-mediated signaling; the polymorphic region of the MHC molecule interacts with TcR, while the nonpolymorphic regions interact with the co-receptor. Stimulation of the TcR signaling pathway involves a number of signaling molecules, including lymphocyte-specific protein tyrosine kinase (LCK or p56lck; Rudolph et al., 2006). Besides T cell activation, CD4 is also play a role in differentiation of and selection of immature CD4+CD8+ thymocytes into the different mature single-positive T cell populations: cytotoxic CD8+ T cells and CD4+ T helper cells (Parnes, 1989). Interestingly, in the genome of teleosts, two other CD4-like genes have been found. Whereas a classical CD4 molecule contains four Ig domains, a CD4-like molecule comprises only two Ig domains but maintains the canonical LCK association motif. In contrast, the lymphocyte activation 3 protein (LAG-3) contains four Ig domains but lacks the LCK-binding site (Laing et al., 2006). Both CD4 homologs are involved in cellmediated immunity. Recently, a full-length cDNA for another T cellspecific immune-like receptor, CD8a chain, was isolated

from the thymus of sea bass (AJ846849; Buonocore et al., 2006). CD8 is a T cell membrane co-receptor, which binds to MHC I molecules and mediates induction of the antiviral immune response (Garcia et al., 1999). The CD8 molecule includes two subunits, CD8a and CD8b, each encoded by a distinct gene. In CTK, CD8a and CD8b form a heterodimer, whereas in NK cells and intestinal intraepithelial lymphocytes, a CD8 receptor molecule is presented by a homodimer consisted of two a subunits (Parnes, 1989). Both a and b chains constitute an Nterminal extracellular Ig superfamily V domain followed by a transmembrane domain and a C-terminal cytoplasmic tail, containing a binding motif for signaling protein kinase p56lck (Letourneur et al., 1990). The interaction of CD8 and TcR with pMHC class I lead to the phosphorylation of the TcR by p56lck and stimulation of CTK through intracellular signaling mechanisms (Letourneur et al., 1990). The sea bass CD8a cDNA contains 1490 bp and is translated in one reading frame, expressing a protein of 217 amino acids (Buonocore et al., 2006). The highest expression of CD8a cDNA was detected in the thymus, gut and peripheral blood leukocytes of the sea bass. Alignment of the nucleotide sequence of sea bass CD8a with its counterparts in other species showed high homology with Japanese flounder Paralichtys olivaceus (64%), rainbow trout (59%) and brown trout Salmo trutta (59%). The amino acid identity with these fishes was 53%, 48% and 47%, respectively. The conservation of most amino acid residues was involved in the transmembrane and cytoplasmic regions, with conservative cysteine residues, participating in disulfide bonding to form the V domain. However, the binding motif for p56lck kinase, which is present in mammals and birds, is missing in the sea bass CD8a cDNA like in other teleosts (Buonocore et al., 2006). The absence of the binding site for p56lck suggests the involvement of the mechanisms of TcR-mediated T cell activation in bony fish that are different from those in higher vertebrates (Moore et al., 2005). 6. Development of immune cells In teleosts, a unique intraembryonic location for hematopoietic stem cells termed the intermediate cell mass seems responsible for primitive or definitive hematopoiesis (Hansen and Zapata, 1998). After early embryonic development, specialized tissues must assume the role for providing the proper microenvironment for T and B cell development from progenitor stem cells. In all gnathostomes, the thymus is a major site for T cell maturation, as evidenced from the expression of T

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cell-specific markers, such as TcR, T cell-specific lightchain kinase (lck), CD3, CD4, CD8 and several others, in knockout and transgenic zebrafish (Langenau and Zon, 2005; Bowden et al., 2005). In sea bass, the importance of the thymus as a primary organ for differentiation and maturation of T lymphocytes was shown using anti-T cell-specific Mab DLT15 (Dos Santos et al., 2000). DLT15-antigenic determinants became detectable in the thymus at 30th day post hatch (dph), 3 days after the first appearance of lymphoid cells, shortly after in the epithelium of gut mucosa and later in the head-kidney (35 dph) and spleen (44 dph; Abelli et al., 1996; Volpatti et al., 1996). DLT15immunoreactive cells were very numerous in the thymus and increased significantly in the gut mucosa at 44th dph, suggesting for the important role of GALT in the ontogenesis of sea bass T lymphocytes (Abelli et al., 1997). The DLT15-positive cells were infrequent in the developing head-kidney and spleen. In teleosts, the head-kidney is the major source of B cell development based on functional, cellular and molecular indices (Hansen and Zapata, 1998). The major role of the head-kidney in the ontogenesis of sea bass B lymphocytes was demonstrated, using Mabs specific to sea bass Ig heavy (DLIg1) and light (DLIg3) chains (Dos Santos et al., 2000). IgM-bearing cells were first detected by immunochemical analysis in the headkidney at 38th dph, but low numbers of Ig-bearing cells were observed in the head-kidney until 49th dph, suggesting that the immune system of sea bass larvae is competent for antibody production after 50th dph (Volpatti et al., 1996; Breuil et al., 1997). Dos Santos et al. (2001c) observed extremely high numbers of antibody-secreting cells (ASCs) in gills of D. labrax, vaccinating with P.damselae subsp. piscicida by direct immersion. Sufficient numbers of antigenproducing cells have been detected even in fish vaccinated at initial weight of 0.1 g, with peak responses in all organs at day 18 post-immunization, although in the older animals vaccinated at initial weight of 2 and 5 g, the numbers of antigen-producing cells were significantly higher and immune response developed more rapidly, reaching a maximum at day 16 and 8 post-immunization, respectively. There was no response of corresponding magnitude in the gut and only a relatively weak increase in number of ASCs was found in the head-kidney and spleen (Dos Santos et al., 2001c). This therefore suggest that the gills and probably skin presented the major sites of bacterial antigen uptake from the immersed vaccine, with maintaining the antigens on the site of uptake. Only small portion of the antigens was transferred into the

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head-kidney and spleen inducing a weak immune response, with low levels of induced antibody production. Indeed, after immersion vaccination, most antibodies in the mucosal tissue are originated from local production and are not derived from serum transudation (Zilberg and Klesius, 1997). In aquaculture, farmed sea bass is currently vaccinated at the age of 4 months being reared at water temperature of 17–19 8C and having an average weight of 2 g. A 4-month-old fish is immunologically mature (Dos Santos et al., 2000). Immunization of a younger fish, weighting 1 g or even less, is also possible, although the observed immune response is weaker and induction of immunity takes longer time compared to older animals (Gravningen et al., 1998; Dos Santos et al., 2001c; Bakopoulos et al., 2003). For younger fish, the immersion seems to be the most effective way of vaccination. Since pathogens commonly invade fish body through the gills, mouth, intestine, anus and skin injuries, the immersion vaccination represents a natural path of delivery of an antigen to fish (Angelidis, 2006). An efficacy of immersion vaccination, even in case of a bivalent vaccine, of the juvenile sea bass has been already reported (Angelidis, 2006; Angelidis et al., 2006). 7. Conclusions and future prospects To date, significant progress has been made in studying the immunology of sea bass. The generation of T and B cell-specific monoclonal antibodies made it possible to obtain subpopulations of sea bass leukocytes highly enriched with T or B cells and greatly facilitated studying the ontogenesis of the humoral and cellular immune systems. The experimental evidence of the presence of elevated numbers of T cells in sea bass GALT may represent the first step in the evolution of an adaptive mucosal immune system (Litman, 1996). The purification of sea bass T and B lymphocytes provides an opportunity to develop monocultures of sea bass lymphocytes and leukocyte cell lines for in vitro studies of specific proliferative and immune properties of those cells (Munoz et al., 1999; Galeotti et al., 1999). Experimental tools and protocols have been developed for in vitro and in vivo immune cells and immune response in sea bass, including challenge tests with bacterial pathogens in aquaculture. Vaccination trials of farmed sea bass have been performed, using vaccines against vibriosis and pasteurellosis (Bakopoulos et al., 2003; Afonso et al., 2005; Hastein et al., 2005; Angelidis et al., 2006). Identification and characterization of the apoptogenic exotoxin AIP56 of P. damselae

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subsp. piscicida (Do Vale et al., 2005) will allow to unravell the molecular mechanism of the unique virulence strategy of this bacterial pathogen as well as to open very promising prospects towards the development of an effective anti-pasteurellosis vaccine based on AIP56. For nodavirus, causing viral encephalopathy and retinopathy in sea bass, in addition to immunochemical and ELISA detection assays, a PCR-based approach has been recently developed, providing rapid and specific identification of the viral RNA in tissues of affected fish (Ucko et al., 2004). Sea bass (larval, juvenile and adult) was challenged with nodavirus, using various viral strains and different infection models (Skliris and Richards, 1999; Breuil et al., 2001). Construction of a bacterial strain, producing sea bass IL-1b, and purification of the recombinant protein provide opportunities for large-scale production of this important immune modulator and its practical use as a potential immunoadjuvant (Buonocore et al., 2005a). Isolation and characterization of T cell (CD8, TcR) and B cell (Ig light chain) specific molecular markers resulted in the development of PCR-based assays for fast detection and potential quantification of these cells in sea bass tissues and mixed cultures of leukocytes (Scapigliati et al., 2000a, 2003; Buonocore et al., 2006; Meloni et al., 2006). However, current genetic knowledge on sea bass immune regulatory genes responsible for resistance to pathogens is significantly less than that for economically important teleosts, such as rainbow trout, Atlantic salmon, channel catfish Ictalurus punctatus, Japanese medaka Oryzias latipes and zebrafish. However, the availability of advanced genetic knowledge on those species provides an opportunity to apply this information to sea bass through comparative and phylogenetic analyses. For example, using degenerated PCR primers from closely related species help to obtain a molecular probe that then could be used for screening genomic (BAC) and cDNA libraries to isolate a gene of interest. The availability of a medium density linkage map of sea bass (Chistiakov et al., 2005) allows a genome-wide screen for quantitative trait loci linked to resistance to one or several pathogens. Identification and mapping of more immune regulatory genes, finding SNPs and other type I markers within those genes and evaluation of the SNPs in case-control studies will lead to the discovery of genes, truly contributing to susceptibility/resistance to pathogens, and characterization of their diseaseassociated molecular variants. This will greatly benefit large-scale implementation of MAS-based breeding programs in sea bass aquaculture towards the selection

of fish genetically resistant to a wide range of pathogens. Acknowledgements This paper greatly benefited from comments by Chris Secombes and Rene´ Stet of the Scottish Fish Immunology Research Centre, Aberdeen, UK. We acknowledge grants from the European Commission (FP6-STREP project AQUAFIRST and FP6-NoE MARINE GENOMICS EUROPE). References Abelli, L., Baldassini, M.R., Mastrolia, L., Scapigliati, G., 1999. Immundetection of lymphocyte subpopulations involved in allograft rejection in a teleost, Dicentrarchus labrax (L.). Cell Immunol. 191, 152–160. Abelli, L., Picchietti, S., Romano, N., Mastrolia, L., Scapigliati, G., 1996. Immunocytochemical detection of a thymocyte antigenic determinant in developing lymphoid organs of sea bass Dicentrarchus labrax (L.). Fish Shellfish Immunol. 6, 493–505. Abelli, L., Picchietti, S., Romano, S., Mastrolia, L., Scapigliati, G., 1997. Immunochemistry of gut-associated lymphoid tissue of the sea bass Dicentrarchus labrax (L.). Fish Shellfish Immunol. 7, 235–246. Afonso, A., Gomes, S., da Silva, J., Marques, F., Henrique, M., 2005. Side effects in sea bass (Dicentrarchus labrax L.) due to intraperitoneal vaccination against vibriosis and pasteurellosis. Fish Shellfish Immunol. 19, 1–16. Angelidis, P., 2006. Immersion booster vaccination effect on sea bass (Dicentrarchus labrax L.) juveniles. J. Anim. Physiol. Anim. Nutr. (Berl.) 90, 46–49. Angelidis, P., Karagiannidis, D., Crump, E.M., 2006. Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax. Dis. Aquat. Organ. 71, 19–24. Bakopoulos, V., Volpatti, D., Adams, A., Galeotti, M., Richards, R., 1997. Qualitative differences in the immune response of rabbit, mouse and sea bass, Dicentrarchus labrax, L., to Photobacterium damselae subsp piscicida, the causative agent of fish pasteurellosis. Fish Shellfish Immunol. 7, 161–174. Bakopoulos, V., Volpatti, D., Gusmani, L., Galeotti, M., Adams, A., Dimitriadis, G.J., 2003. Vaccination trials of sea bass, Dicentrarchus labrax (L.), against Photobacterium damselae subsp. piscicida, using novel vaccine mixtures. J. Fish Dis. 26, 77–90. Bennani, N., Schmid-Alliana, A., Lafaurie, M., 1995. Evaluation of phagocytic activity in a teleost fish, Dicentrarchus labrax. Fish Shellfish Immunol. 5, 237–246. Bingulac-Popovic, J., Figueroa, F., Sato, A., Talbot, W.S., Johnson, S.L., Gates, M., Postlethwait, J.H., Klein, J., 1997. Mapping of MHC class I and class II regions to different linkage groups in the zebrafish Danio rerio. Immunogenetics 46, 129–134. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., Wiley, D.C., 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329, 512–518. Boman, H.G., 1995. Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 61–92.

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