Function and heterogeneity of fish lectins

Veterinary Immunology and Immunopathology 108 (2005) 111–120 www.elsevier.com/locate/vetimm Function and heterogeneity of fish lectins Spencer Russell, John S. Lumsden * Fish Pathology Laboratory, Ontario Veterinary College, University of Guelph, Guelph, Ont., Canada N1G 2W1 Abstract Lectins are primordial molecules with multiple known functions. They have been known to exist in fish and other animals for decades and were initially identified as (hem)agglutinins. Demonstration of the importance of vertebrate lectins in innate immunity is a recent effort and is still largely unrealised for fish. This mini-review will tabulate those fish lectins identified since the last major review. In addition, particular lectins for which either functional relevance or functional or structural heterogeneity has been demonstrated are discussed in greater detail. # 2005 Elsevier B.V. All rights reserved. Keywords: Review; Fish; Lectin; Skin; Gill; Plasma; Heterogeneity 1. Introduction The purpose of the present mini-review is to update the recently described defense-related plasma and mucosal lectins of cartilaginous and bony fish. We will focus on evidence for functional relevance and briefly discuss lectin heterogeneity. Those lectins for which physiochemical data are known are summarized in Table 1. Previous reviews should be referred to for fish lectins not covered by Table 1 (Yano, 1996; Alexander and Ingram, 1992). Egg lectins, of which there are numerous examples, are beyond the scope of the present paper and readers should again refer to the previously mentioned reviews. The tabulated information summarizes the lectin class, binding affinity and if known, functional relevance. Table 2 summarizes the * Corresponding author. Tel.: +1 519 824 4120x54519; fax: +1 519 824 5930. E-mail address: jslumsde@uoguelph.ca (J.S. Lumsden). putative fish lectins for which there is cDNA sequence alone. While it may be somewhat premature to include these without evidence of expression, at least one provides tantalizing evidence of a fish orthologue of an important mammalian lectin. The authors of both previous reviews of fish lectins comment on the lack of solid evidence for a significant role of plasma or mucosal lectins in innate defense of fish (Yano, 1996; Alexander and Ingram, 1992). This paucity of functional information has not substantially changed in the intervening period, however there are important exceptions. Defense lectins (those that participate in immunity or inflammation) occur on phagocytes, in plasma or on mucosal surfaces, have rather broad carbohydrate specificity and the ability to bind to surfaces of various infectious agents. There are several relatively well studied lectins that are therefore accepted to be soluble pattern recognition receptors and (potentially in most cases) a key component of the innate immune system. Carbohydrate binding, the 0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.08.004 112 Table 1 Physical, chemical and biological properties of fish defence lectins Species Type HAg activity Cationdependent activity Ligand affinity Tissue expression Microbial binding Biological relevance Reference T-antigen-binding lectin SAP TCBP2 (CRP-homolgue) SL-SAP RT-LL C + 2-Acetomido-2deoxy-D-galactose Agarose Pnuemococcal CPS Serum ND Agglutination Manihar et al. (1991) P P Human A, B, O ND ND Serum Serum ND ND Potential defensive activity Potential defensive activity Murata et al. (1995) Murata et al. (1995) P C ND ND + + Serum Serum ND ND Potential defensive activity Potential defensive activity Jensen et al. (1995) Jensen et al. (1997b) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Atlantic salmon (S. salar) Common wolffish (A. lupus) Cod (G. morhua) Halibut (H. hippoglossus) Atlantic salmon (S. salar) Atlantic salmon (S. salar) TCBP2 RT-SAP RT-LL RT-37 ABP Al-ABP P P C C P P ND ND ND ND ND ND + + + + + + ND Glc, GlcNAc, Man, ManNAc A. salmonicida LPS A. salmonicida LPS A. salmonicida LPS A. salmonicida LPS Agarose Agarose Serum Serum Serum Serum Serum Serum ND ND ND ND ND ND Potential defensive activity Binds A. salmonicida LPS Binds A. salmonicida LPS Binds A. salmonicida LPS Potential defensive activity Potential defensive activity Hoover et al. (1998) Hoover et al. (1998) Hoover et al. (1998) Hoover et al. (1998) Lund and Olafsen (1998b) Lund and Olafsen (1998b) Gm-PCBP Hh-PCBP Ss-PRP MBL P P P C ND ND ND ND + + Phosphoryl-choline Agarose Phosphoryl-choline Mannose Serum Serum Serum Serum ND ND ND V. anguilarum, A. salmonicida Lund and Olafsen (1998b) Lund and Olafsen (1998b) Lund and Olafsen (1998a) Ewart et al. (1999), Ottinger et al. (1999) Japanese eel (A. japonica) Fucolectin F ND L-Fuc ND Sea Bass (D. labrax) DLL1 + DLL2 F DLL2—rabbit D-Galactose Serum, liver(origin), gill, intestine Serum Indian major carp (L. rohita) Blue gourami (T. trichopterus) CRP P ND + Serum ND BGL C Rabbit > chicken, guinea pig mouse or rat + Associated with leukocyte cell surface A. hydrophila, V. anguillarum, yeast Indian major carp (L. rohita) Japanese eel (A. japonica) Snapper (P. auratus) Mannose binding lectin Anguilla anguilla agglutinin (AAA) Snapper serum lectin Carp CRP TAAK LC1q C Guinea pig, chicken, rabbit ND + Man, GlcNAc, glu Serum E. coli L-Fucose, D-galactose Serum ND Potential defensive activity Potential defensive activity Potential defensive activity Binds V. anguilarum, A. salmonicida; " phagocytosis and bactericidal activity Heterogeneity + increased in vitro expression following LPS Agglutination + other potential immune activity Acute phase induction by metal + structural heterogeneity Binds A. hydrophila, V. anguillarum, yeast; " phagocytosis/killing; confer immunity against challenge Agglutination; binds E. coli; increased O2-production Recognition of bacterial LPS Serum ND Cook et al. (2003) Serum Serum Serum ND ND ND Acute phase reactant + activates complement Potential defensive activity Potential defensive activity Component of lectin complement pathway (A) Serum Snakehead murrel (C. leucopunctatus) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Common carp (C. carpio) Common carp (C. carpio) Lamprey (L. japonicus) F + + + + fucose + melibose C-polysaccharide, phosphoryl-choline GalcNac > GlcNac > D-mannose P ND + and H + Le antigens ND P ND GlcNAc-binding lectin ND ND ND + + + ND ND GlcNAc ND Honda et al. (2000) Cammarata et al. (2001) Sinha et al. (2001) Fock et al. (2000, 2001) Mitra and Das (2001) Bianchet et al. (2002) Cartwright et al. (2004) Cartwright et al. (2004) Matsushita et al. (2004) S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 Protein Abbreviations: HAg: haemagglutinin activity; P: pentraxin; G: galectin; C: C-type lectin; F: fucolectin; ND: not determined; CRP: C-reactive protein; SAP: serum amyloid protein; RT-LL: rainbow trout ladder lectin; TCBP2: trout C-polysacharride binding protein 2; SL-SAP: sulfide linked serum amyloid; ABP: agarose binding protein; Al-ABP: A. lupus agarose binding protein; Gm-PCBP: G. morhua phosphorylcholine binding protein; Hh-PCBP: H. hippoglosss phosphorylcholine binding protein; Ss-PRP: S. salar phosphorylcholine reactive protein; TCBP2: trout c-polysaccharide binding protein 2; BGL: blue gourami lectin; AAA: Anguilla anguilla agglutinin; AJL: Anguilla japonica lectin; eCL: eel C-type lectin; DLL: Dicentrarchus labrax lectin; TAAK: pentraxin-like protein; LC1q: lamprey complement component 1q; Glc: glucose; GlcNAc: N-acetyl-glucosamine; Man: mannose; ManNAc: N-acetyl-mannosamine; LPS: lipopolysaccharide; potential immune activity: similar to molecules with demonstrated function. Tsutsui et al. (2003, 2005) Mucus, intestine, esophagus and gill D-Mannose Pufflectin Pufferfish (F. rubripes) Mannose specific Rabbit ND Skin only b-Galactoside specific activity Rabbit G AJL-1 AJL-2 Japanese eel (A. japonica) ND + ND Galactose binding C-type C Japanese eel (A. japonica) Japanese eel (A. japonica) eCL-1 + eCL-2 First fish mucosal lectin demonstrated to bind parasitic metazoans (trematode) Tasumi et al. (2002, 2004) Agglutinates S. dificile, growth regression of E. coli Tasumi et al. (2002, 2004) Binds E. coli K12, growth regression of same Agglutinates Streptococcus dificile, growth regression of E. coli None Lactose Mucous cells of basal lamaelle Skin—club cells ND " expression following i.p. E. coli injection Binds E. coli K12 + restricts growth Paroutaud et al. (1987), Shiomi et al. (1989), Muramoto et al. (1999) Mistry et al. (2001) Activity against marine bacteria and shellfish larvae ND Club cells and skin mucous Lactose and other b-galactoside residues Galactose Rabbit, horse, sheep Congerin 1–4 (B) Skin and gill Conger eel (C. myriaster) G Protein Species Lectin type HAg activity Cation-dependent activity Ligand affinity Tissue expression Bacterial binding activity Biological relevance Reference S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 113 specificity of which is used to determine lectin class, is facilitated by the carbohydrate recognition domain (CRD). The CRD is formed by a pattern of invariant and highly conserved amino acid residues at a characteristic spacing (Drickamer and Taylor, 1993). Most lectins appear to be constitutively produced but a few may be induced as part of the acute phase response to noxious stimuli such as mannose binding lectin (MBL) and C-reactive protein (CRP). In fish, C-type lectins, galectins and pentraxins have been identified from the earliest jawed vertebrate (sharks) to the more advanced teleost species such as salmon and carp (Vasta et al., 2004). In mammals, the best characterized soluble defense lectins are the collagenous lectins. A clearer picture of the functional significance of MBL and ficolin (FCN) to mammals is appearing (reviewed by Turner and Hamvas, 2000; Holmskov et al., 2003; Gadjeva et al., 2004; Fujita et al., 2004a; Lynch et al., 2004; Atkinson et al., 2004). MBL and FCN have been identified from both vertebrates and invertebrates (Vasta et al., 1999; Lu et al., 2002; Janeway and Medzhitov, 2002). MBL are calcium-dependant proteins that form multimeric structures with subunits composed of an N-terminal cysteine-rich domain, a collagen-like domain and a Cterminal CRD (Lu et al., 2002). Ficolins are calcium independant lectins that have a similar multimeric structure and domain organization but possess a fibrinogen-like lectin domain instead of a CRD (Matsushita et al., 2004). During infections, both MBL and ficolin enhance pathogen clearance by opsonizing microbes, activating complement through the lectin complement pathway (LCP) and triggering phagocytosis via receptors on phagocytic cells. In human MBL genes, single nucleotide polymorphisms within the promoter region and exon one, give rise to premature lectin degradation, decreased serum levels, and reduced opsonic abilities (Sumiya et al., 1991; Naito et al., 1999; Summerfield, 2003) resulting in increased susceptibility to bacterial, viral, fungal and protozoal infections (Garred et al., 1997; Hibberd et al., 1999; Luty et al., 1998; Summerfield, 2003; Larsen et al., 2004). Mice with defective MBLA genes are more susceptible to experimental infections while wild type mice develop more severe inflammation (Takahashi et al., 2002). Consequently, a greater understanding of the importance of MBLs, the lectin pathway of comple- 114 Table 2 Putative fish lectins for which mRNA sequence has been identified Protein Lectin type mRNA tissue expression Biological relevance Reference Atlantic salmon (S. salar) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Carp (C. carpio) Carp (C. carpio) Zebrafish (D. rerio) SAP P Liver Potential defensive activity Jensen et al. (1997a) SAP P Liver Potential defensive activity Jensen et al. (1997a) SAA P Liver Major acute phase reactant Jensen et al. (1997a) Clone TO-260 Clone TO-438 MBL C P C " expression after i.p. turpentine injection " expression after i.p. turpentine injection Possible component of complement system Fujiki et al. (2001) Fujiki et al. (2001) Vitved et al. (2003) Carp (C. carpio) MBL C Possible component of complement system Vitved et al. (2003) Goldfish (C. auratus) MBL C Possible component of complement system Vitved et al. (2003) Rainbow trout (O. mykiss) – G " expression following i.p. E. coli injection Inagawa et al. (2001) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Rainbow trout (O. mykiss) Chinook salmon (O. tshawytscha) Chinook salmon (O. tshawytscha) Chinook salmon (O. tshawytscha) Chinook salmon (O. tshawytscha) Chinook salmon (O. tshawytscha) Carp (C. carpio) Carp (C. carpio) C-type 2-1 C – – Spleen > gill > intestine > liver Spleen > gill > intestine > liver Spleen > gill > intestine > liver Spleen, head kidney, thymus peritoneal exudate, ovary, gills heart Liver " expression after i.p. V. aguillarum bacterin injection Bayne et al. (2001) C-type 2-2 C Liver " expression after i.p. V. aguillarum bacterin injection Bayne et al. (2001) Intelectin-like protein 1 SAP I Liver " expression after i.p. V. aguillarum bacterin injection Bayne et al. (2001) P Liver " expression after i.p. V. aguillarum bacterin injection Bayne et al. (2001) TCBP-1 C Liver " expression after i.p. V. aguillarum bacterin injection Bayne et al. (2001) Pentraxin—51S P Liver, kidney, spleen " expression after poly IC injection Alonso and Leong (2002) Pentraxin—60S P Liver, kidney, spleen " expression after poly IC injection Alonso and Leong (2002) Lectin—40S – Liver, kidney, spleen " expression after poly IC injection Alonso and Leong (2002) Lectin—64s – Liver, kidney, spleen " expression after poly IC injection Alonso and Leong (2002) Lectin—71S – Liver, kidney, spleen " expression after poly IC injection Alonso and Leong (2002) C-type l C-type 2 C C Head kidney Head kidney " expression after E. coli LPS and con A stimulation " expression after E. coli LPS and con A stimulation Savan and Sakai (2002) Savan and Sakai (2002) S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 Species Tsoi et al. (2004) Tsoi et al. (2004) Tsoi et al. (2004) Tsoi et al. (2004) " expression after cohabitation exposure to A. salmonicida " expression after cohabitation exposure to A. salmonicida " expression after cohabitation exposure to A. salmonicida Spleen and liver – C C C C Macrophage lectin 2 C-type lectin receptor C-type 2-1 (SL5-0023) C-type 2-1 (SL5-0092) Macrophage lectin 2 Tachylectin Atlantic halibut (H. hippoglossus) Atlantic halibut (H. hippoglossus) Atlantic salmon (S. salar) Atlantic salmon (S. salar) Atlantic salmon (S. salar) Atlantic salmon (S. salar) Abbreviations: SAP: serum amyloid protein; SAA: serum amyloid A; TCBP1: trout C-polysacharride binding protein 1; P: pentraxin; G: galectin; C: C-type lectin; F: fucolectin; I: intelectin; MBL: mannan-binding lectin; ND: not determined; Ca-CTL: carp c-type lectin; Con A: concanavalin A. Park et al. (2005) " expression after i.p. V. anguillarum and A. salmonicida vaccination " expression after i.p. V. anguillarum and A. salmonicida vaccination " expression after cohabitation exposure to A. salmonicida Ca-CTL Carp (C. carpio) C Head kidney, intestine, liver + gill Liver, spleen and kidney Liver, spleen and kidney Kidney, spleen and liver Kidney, spleen and liver Spleen and liver C Park et al. (2005) Savan et al. (2004) " expression after E. coli LPS stimulation S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 115 ment and the pattern recognition receptor/pathogen associated molecular pattern paradigm have refocused efforts to investigate the role of other vertebrate lectins. MBL and other collectins in vertebrates, particularly humans and mice, provide the ‘proof of principle’ that identification and functional characterization of fish plasma and mucosal lectins will reveal useful information regarding innate defense systems and perhaps strain or species variation in resistance/susceptibility. For further information on collagenous lectins refer to Lillie et al. (this issue). 2. Function Several of the lectins summarized in Table 1 deserve further discussion given that there is experimental evidence demonstrating functional relevance or its potential. To illustrate, the largely as yet unrealised, importance of lectins to innate immunity in fish, the lamprey orthologue of mammalian C1q has recently been identified as a GlcNAc-binding lectin that interacts with a serine protease of the mannose binding lectin serine protease (MASP) family to cleave lamprey complement component 3 (C3) (Matsushita et al., 2004). The lectins covered in this section of the review are the mannan-binding lectin of Atlantic salmon (Salmo salar), and the structurally similar ladderlectin of rainbow trout (Oncorhynchus mykiss), the N-acetyl-galactosamine-binding lectin (BGL) of the blue gourami (Trichogaster trichopterus), a pentraxin-like lectin of the snapper (Pagrus auratus) and finally, the skin lectins (AJL-1 & 2) of the Japanese eel (Anguilla japonica). A mannan-binding lectin in the plasma of the Atlantic salmon was shown to bind to both Vibrio anguillarum and Aeromonas salmonicida in a calcium-dependant manner (Ewart et al., 1999) and to increase phagocytosis and killing following incubation with A. salmonicida (Ottinger et al., 1999). This lectin has high homology to the ladderlectin of rainbow trout (Jensen et al., 1997b; Hoover et al., 1998) that binds to Sepharose-based column matrices but also to A. salmonicida (Hoover et al., 1998). Using lipopolysaccharide (LPS) of A. salmonicida these same authors identified three additional plasma lectins 116 S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 that also bound and were eluted with carbohydrates (Hoover et al., 1998). Blue gourami lectin was found to agglutinate strains of Aeromonas hydrophila and at very low concentrations (<1 ng/ml), promoted phagocytosis of the same bacteria. It was also identified by immunohistochemistry to be associated with the cell surface of macrophages. Pre-incubation of A. hydrophila with graded BGL concentrations produced a dose-related increase in survival of blue gourami after i.m. injection of the complex (Fock et al., 2000, 2001). The pentraxins, C-reactive protein (CRP) and CRP homologues and serum amyloid protein (SAP), have been identified in numerous species of fish and all classes of vertebrates (Gewurz et al., 1995) and are characterized by the ability to bind to C-polysaccharide of S. pneumonia (CPR) or to microbial polysaccharides and various matrix components such as heparan (SAP) (Gewurz et al., 1995). In mammals, they are acute phase reactants (Pepys, 1979; Gewurz et al., 1995) that interact with complement thereby activating the classical pathway, and with macrophages acting as opsonins (Mold et al., 2001). Limited functional data are available for fish homologues. Teleost CRP homologues are moderate acute phase reactants (3–10 induction in a variety of models) (Winkelhake et al., 1983). Recently, a pentraxin-like protein isolated from snapper activated complementmediated lysis of ligand-coated sRBC and was minimally induced (2) by i.p. injection of LPS. Interaction with the classical complement pathway was suggested by abrogation of RBC lysis by heat and removal of cations (Cook et al., 2003). The skin lectins of the Japanese eel are produced in the skin, are found within the mucus layer and have been found to agglutinate bacteria. AJL-1 (Tasumi et al., 2004) and -2 (Tasumi et al., 2002) agglutinate Streptococcus difficile and Escherichia coli, respectively, and AJL-2 also inhibits growth of E. coli. One of these skin lections (AJL-1) did not agglutinate four species of gram-negative fish pathogens including Vibrio anguillarum and Aeromonas hydrophila (Tasumi et al., 2002, 2004). While these results indicate that these lectins participate in innate immunity, their relevance to disease resistance in production settings remains to be demonstrated. Further research directed at determining the relevance of these molecules to defense against infectious disease is required. Quantification, in conjunction with individual variability and correlation to resistance/susceptibility to infection, is an indirect method to indicate relevance. Further in vitro experimentation is required to delineate the range of function of these molecules including effector function following carbohydrate binding (e.g. LCP activation). Abrogation of carbohydrate interaction by the addition of the target sugar(s) has been used in in vitro models (Tasumi et al., 2004), but not with in vivo models to date. 3. Heterogeneity Serum and mucus defense lectins possessing multiple structural and functional isoforms (Tables 1 and 2) have been recently described from various fish species. Although the actual mechanisms by which multiple isoforms are produced are still unclear, there are a number of different factors that are known to generate heterogeneity. Single nucleotide polymorphisms within gene sequences can lead to allelic variation and since some fish are tetraploid (e.g. carp), a large number of potential phenotypes could be produced (Allendorf and Throgaard, 1984; Ohno, 1993). Structurally and functionally distinct protein isoforms can be encoded by multiple gene families and differentially expressed in multiple tissues or by alternative pre-mRNA splicing of various sequences from a single gene (Bell, 1998). Post-translational modifications or the unequal cleavage of the C-termini of mature polypeptides may also give rise to protein isoforms with variable downstream functions (Young et al., 1996). In fish, lectin heterogeneity is best demonstrated by the two eel fucolectins. The Japanese eel fucolectin (Honda et al., 2000) exists as at least seven isoforms expressed in the liver, gill and intestine. Northern blots and immunohistochemistry revealed that these lectins were of hepatic origin and were also constituents of gill mucus. A broad range of functionally active lectins with variable specificity for a number of oligosaccharide targets greatly expands the repertoire of patterns recognized on pathogen surfaces (Bianchet et al., 2002). Primary eel hepatocyte cultures exposed to LPS produced an increase in fucolectin expression, suggesting these lectins may be not only an integral S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 part of eels innate immune system by preventing microbial colonization on gill surfaces, but also an acute phase reactant. The Anguilla anguilla agglutinin (AAA), a fucolectin from the serum of the European eel, has both a novel CRD sequence motif and a novel lectin fold (Bianchet et al., 2002) which recognizes polysaccharides (L-fucose and D-galactose) and fucosylated terminals of H and Lewis (a) blood groups (Baldus et al., 1996). Modelling of this lectin complexed to L-fucose revealed interactions of additional residues (structural determinants) surrounding the binding pocket and provided evidence for both the Japanese and European eel possessing fucolectin isoforms that could potentially be specific to various pathogens (Bianchet et al., 2002; Vasta et al., 2004). Moreover, similar to mammalian collectins, the native AAA was shown to possess three-fold cyclic symmetry with spacing and orientation similar to molecular patterns of polysaccharides that appear on the surface of microbial pathogens (Bianchet et al., 2002; Vasta et al., 2004). Studies on the inducibility of CRP from serum of the Indian major carp (Labeo rohita) following exposure to various metal pollutants (Sinha et al., 2001) demonstrated a shift in expression from the normal form of CRP to several structurally different, inducible (up to five-fold) isoforms. These isoforms differed from each other with respect to molecular charge, mass and shape, as well as carbohydrate and amino acid composition. Sequencing of these variants is required to gain insight into the molecular mechanisms that produce the various isoforms and the precise signals that trigger differential expression during inflammation. Molecular cloning and characterization of the Atlantic salmon mannan-binding lectin from kidney revealed four similar cDNA sequences with minor sequence variations and the presence of multiple genes (Richards et al., 2003). It is the only known C-type teleost serum lectin that has been shown to bind and opsonize bacteria (A. salmonicida) (Ottinger et al., 1999; Stratton et al., 2004). Isolation of functional isoforms of this lectin is required to clarify whether sequence variations confer differences in carbohydrate binding, complement activation, multimeric assembly and the ability to bind different types of pathogenic bacteria. Similarly, the full cDNA sequence of a MBL homologue identified from carp (Vitved et al., 2003, 117 Table 2) shows considerable sequence diversity within a single individual and therefore the presence of multiple protein isoforms in plasma is likely. It is well established that genes encoding the molecules of acquired immunity, i.e. immunoglobulins, recombinant activating gene, T-cell receptor and major histocompatibility complex (MHC) appeared within the jawed vertebrates (sharks) and progressively evolved in vertebrates. There is increasing evidence however, to suggest that diversity of fish immunity has also evolved within the innate immune system (Fujita et al., 2004b). For example, like the various isoforms of the eel fucolectins (Honda et al., 2000; Bianchet et al., 2002), the multiple forms of complement component 3 (C3) (Sunyer et al., 1996, 1997, 1998), C4 (Nakao et al., 2003) and C5 (Kato et al., 2003) possess differences in their binding target specificity. The presence of diverse structural and functional innate immune molecules, from either multiple or single genes, would provide the organism with an arsenal of proteins to prevent invasion and multiplication of a range of microbes. Moreover, it is becoming increasingly clear that for ectotherms such as fish, low temperatures are non-permissive for an effective adaptive immune response and they are therefore more reliant on innate immune mechanisms (Bly et al., 1997; Gerwick et al., 2000). For instance, MHC receptor expression is down-regulated in carp at low water temperatures (Rodrigues et al., 1998) suggesting an increased dependence on innate immune proteins including acute phase reactants (Dixon and Stet, 2001). Evidence for multiple forms of other components of the innate teleost humoral proteins have been implicated in resistance to microbial challenge (Arason, 1996; Ellis, 2001). For example, transferrin, which sequesters free iron from plasma and limits bacterial multiplication, exhibits a high degree of genetic polymorphism, with the ‘C’transferrin allele being associated with increased resistance to bacterial kidney disease in coho salmon (O. tshawytsha) (Suzumoto et al., 1977). 4. Conclusions and future research The repertoire of known fish lectins will undoubtably expand as the initial steps of identification, isolation and physiochemical characterization of 118 S. Russell, J.S. Lumsden / Veterinary Immunology and Immunopathology 108 (2005) 111–120 individual lectins continues. Identification of functional properties, particularly those involving downstream effects (LCP activation for example), and more critically, demonstration of their importance in clinically relevant disease is still largely unattained. The lack of particular lectins or isoforms, presence of lectin gene mutations and lectin dysfunction are likely causes of species and/or strain susceptibility/resistance to infectious disease. The basis for lectin heterogeneity and its contribution to functional diversity of innate immunity requires further effort. Future research should concentrate on a few commercially important fish species with developing gene sequence information and should use relatively wellcharacterized infectious agents. References Alexander, J.B., Ingram, G.A., 1992. Noncellular nonspecific defence mechanisms of fish. Ann. Rev. Fish Dis. 249–279. 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