Recent advances on the complement system of teleost fish

Fish & Shellfish Immunology 20 (2006) 239e262 www.elsevier.com/locate/fsi

Recent advances on the complement system of teleost fish H. Boshra 1, J. Li 1, J.O. Sunyer *,1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 413 Rosenthal, 3800 Spruce St., Philadelphia, PA 19104, USA Received 8 March 2005; accepted 4 April 2005 Available online 13 June 2005

Abstract The complement system plays an essential role in alerting the host of the presence of potential pathogens, as well as in their clearing. In addition, activation of the complement system contributes significantly in the orchestration and development of an acquired immune response. Although the complement system has been studied extensively in mammals, considerably less is known about complement in lower vertebrates, in particular teleost fish. Here we review our current understanding of the role of fish complement in phagocytosis, respiratory burst, chemotaxis and cell lysis. We also thoroughly review the specific complement components characterized thus far in various teleost fish species. In addition, we provide a comprehensive compilation on complement hostepathogen interactions, in which we analyze the role of fish complement in host defense against bacteria, viruses, fungi and parasites. From a more physiological perspective, we evaluate the knowledge accumulated on the influence of stress, nutrition and environmental factors on levels of complement activity and components, and how the use of this knowledge can benefit the aquaculture industry. Finally, we propose future directions that are likely to advance our understanding of the molecular evolution, structure and function of complement proteins in teleosts. Such studies will be pivotal in providing new insights into complementrelated mechanisms of recognition and defense that are essential to maintaining fish homeostasis. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Complement; Teleost; Evolution; Fish; Components; Host-pathogen interactions; Stress; Nutrition

1. The complement system The complement system is composed of more than 35 soluble plasma proteins that play key roles in innate and adaptive immunity [1,2]. Complement is initiated by one or a combination of three pathways,

* Corresponding author. Tel.: C1 215 573 8592; fax: C1 215 898 7887. E-mail address: [email protected] (J.O. Sunyer). 1 The three authors contributed equally to the writing of this review. 1050-4648/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2005.04.004

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the alternative, lectin and classical (Fig. 1). The classical pathway is initiated by a complex between an antigen and an antibody [2]. Activation of this pathway is triggered by binding of the Fc portion of the IgG to the C1q component of the C1 complex [3]. This initiates the activation and cleavage of C1r and C1s molecules. C1s is then able to cleave the C4 component into C4b and C4a, enabling the C4b fragment to covalently bind to the activating surface via its exposed thioester bond. Thereafter, the C2 component binds to C4b, leading to its cleavage into C2a and C2b by the C1s component. The C2a fragment remains bound

Fig. 1. Complement activation pathways and functions. Activation of the complement system through any of the three existing pathways (classical, alternative or lectin) leads to the activation of C3 into C3b and C3a. C3b covalently binds to complement activating surfaces (i.e., bacteria, fungi, viruses). Bound C3b can be degraded into iC3b by factor I in the presence of factor H. C3b and iC3b bind to complement receptors (CR1, CR3) and promote phagocytosis, respiratory burst, and antigen-uptake processes. C4 activated through the classical or lectin pathways can also bind to an activating surface and promote its uptake, however the number of C4 molecules binding to a surface is always many fold less than that of C3 molecules. Antigen containing covalently bound C3b or C4b molecules (or their degradation fragments) can be further processed and presented to T-lymphocytes. Antigen containing bound Ig and C3d lead to the coligation of the B cells receptor (BCR or mIg) and complement receptor type 2 (CR2/CD21) on B cells, which in turn lowers the threshold for B cell activation. In addition, C3b/C4b bound to a microorganism can lead to the formation of the membrane attack complex (MAC) which results in cell lysis. C5a and C3a anaphylatoxins generated during complement activation play a key role in inflammatory processes. (Modified from figure 1 of Ref. [32])

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to C4b thereby forming an enzymatic unit referred to as the classical C3 convertase (C4b2a). This complex can then cleave surrounding native C3 into C3a and C3b fragments, and with the association of an additional molecule of C3b to the classical C3 convertase, leads to the formation of the classical C5 convertase (C4b2a3b) [4]. This convertase is responsible for cleaving C5 into C5a and C5b, which allows for the assembly of the membrane attack complex or MAC (C5b-C9) [5]. In the alternative pathway, the spontaneous activation of C3 (referred to as ‘‘tick over’’ activation) is amplified upon the covalent binding of C3(H2O) to various microbial surfaces (i.e., viruses, bacteria, fungi, parasites). This newly bound C3(H2O) is then capable of interacting noncovalently with Factor B (Bf). Thereafter, factor D (Df) present in plasma, cleaves Bf into Ba and Bb fragments, resulting in the formation of the alternative C3 convertase C3(H2O)Bb [6]. This convertase, while short-lived, is capable of cleaving many surrounding C3 molecules into C3b and C3a. If C3b is within proximity of an activating surface, C3b will be able to covalently bind to the surface via its exposed and highly reactive thioester bond. Bound C3b can then form additional C3 convertases, resulting in the amplification of C3 cleavage (amplification loop), which rapidly leads to a massive deposition of C3b molecules onto the activating surface. Should newly generated C3b bind to an existing C3 convertase, the resulting enzyme (C3bBb3b) forms the alternative C5 convertase. Like the classical C5 convertase (C4b2a3b), the alternative C5 convertase is capable of cleaving C5, which leads to the subsequent assembly of the MAC [5]. The lectin pathway requires the interaction of lectins such as mannose-binding lectin (MBL) and ficolins, with sugar moieties found on the surface of microbes [7,8]. Upon binding of the lectins to the microbial sugars, the enzymes associated with these lectins (MBL-associated serine proteases, MASPs) are activated. These proteases have been shown to be structurally homologous to C1r and C1s [9]. There are three types of MASP molecules, MASP-1,2,3 all of which have been shown to be associated with MBL and ficolins [10]. While it is unequivocal that MASP-2 has the ability to cleave C4 and C2, it remains controversial whether MASP-1 can cleave C3 [11]. It is clear however that MASP-1 does not cleave C4, although it appears to have the ability to cleave C2 [12]. It has been proposed therefore that MASP-1, enhances complement activation triggered by MASP-2 [12]. Interestingly, a recent study has shown that MASP-3 is unable to cleave C2, C3 or C4 and its role in the lectin pathway remains a mystery [13]. The functions mediated by complement activation products in innate immunity include phagocytosis and cytolysis of pathogens, solubilization of immune complexes, and inflammation [1,2]. Complement also plays an important role in mediating and enhancing humoral immunity [14e16]. In this regard, it has been shown that C3/C4 fragments bound to antigen or immune complexes enhance uptake and processing of antigen by antigen-presenting cells (APCs), and this leads to more effective primary and secondary antibody responses to the antigen [17e19]. In addition, it has been demonstrated that antigen linked to the C3d portion of C3 lowers the threshold for B cell activation, and it becomes 1000e10,000 times more immunogenic when compared to the antigen alone [20].

2. Functional aspects of the complement system in teleosts The complement system is a very old mechanism of defense that has been recently identified in primitive protostome species [21], as well as in invertebrate deuterostome species (including echinoderms [22] and tunicates [23,24]). Complement components participating in the alternative and lectin pathways have also been demonstrated in the most ancient group of vertebrates, the jawless fish [25e27]. The cartilaginous fish (sharks), the most primitive species containing immunoglobulins [28], have been shown to have molecules involved in all three pathways of complement activation [29,30], although the exact mechanisms and components implicated in their activation sequences remain to be investigated. The features of the complement system of the next more evolved animal group, the teleost fish, are reviewed below. For more extensive reviews on complement evolution, the reader is directed to the following references [31,32].

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2.1. General properties of teleost complement The complement system of teleost fish, like that of higher vertebrates, can be activated through all three pathways of complement. While the activation sequence and components involved in the alternative and classical pathways have been characterized to a significant degree [33e35], we know very little about the mechanisms and molecules involved in the activation of the lectin pathway (the specific involvement of fish complement components in the three pathways is reviewed in Section 3). In contrast to mammals, complement in teleosts is active at very low temperatures, and their alternative complement pathway titers are several orders of magnitude higher [34,36]. It is also interesting to note that through the alternative pathway, fish complement mediates the lysis of erythrocytes (RBCs) from a variety of animal species, whereas in humans this activity is mainly restricted to the lysis of rabbit RBCs [36]. This property of fish complement may suggest that this system allows for a wider recognition of foreign surfaces when compared to that of mammals. This broader recognition of nonself particles may be due to the fact that in contrast to mammals, some complement components in fish are present in multiple isoforms. In this respect, one of the most unusual and significant properties of the complement system of teleost fish, is that its key component, the protein C3, is present in several isoforms that are the products of different genes [37e40]. A particularly fascinating feature of these C3 isoforms is the fact that they show important differences in their binding efficiencies to complement-activating surfaces. It has been hypothesized that this unique structural and functional diversity in teleost C3 provides a mechanism for recognizing a broader range of microorganisms, thereby allowing fish to expand their innate immune recognition capabilities [41]. As we will see later, diversification of fish complement components is not restricted to the C3 molecule (see Section 3). The combination of high titers and activation of this system at a very wide range of temperatures, along with the diversity of some of its key components, makes complement a very powerful defense system in fish. This system is likely to have evolved into its present form in fish in order to meet the survival demands enforced by the aquatic environment in which they live, which in many cases, is characterized by waters filled with an extremely high concentration of countless types of microbes. Below we review the involvement of fish complement in important immune functions that are pivotal to the recognition and clearance of microbes.

2.2. Opsonization and phagocytosis Complement-mediated opsonization involves the covalent binding of activated C3 and/or C4 to microbes, which allows, in turn, the recognition and phagocytosis of these microbes by phagocytes bearing complement receptors on their surface. Thus, complement-dependent phagocytosis is mediated by C3b/ iC3b and C4b/iC4b activation products and C3 receptors. It should be stressed that since many more C3b than C4b molecules are generated during complement activation, C3 plays a much more relevant role than C4 in complement-mediated phagocytic processes. In mammals, two major receptors are involved in complement-mediated phagocytosis, complement receptor one (CR1) and three (CR3) [42,43]. CR1 promotes the binding and phagocytosis of C3b/C4b-coated particles by phagocytes only in the presence of co-stimulatory signals such as IFN-g or C5a. In addition, synergy between CR1 and Fc receptors has been shown to be important in promoting phagocytosis. Unlike CR1, complement receptor three (CR3, CD11b/ CD18) binds specifically to the iC3b or the iC4b fragment in a divalent cation-dependent manner [43]. Furthermore, CR3 mediates opsonization and phagocytosis of microorganisms without any co-stimulatory signals and promotes natural killer (NK) cell activity for iC3b-coated targets [44]. All phagocytes, including neutrophils, macrophages, and monocytes, express both complement receptors on their surface. From an evolutionary perspective, a CR3 homolog in tunicates has been characterized, and it has been shown to play

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an important role in phagocytosis as well [45]. No other complement receptors have been identified in species other than mammals. The knowledge we have on complement-mediated phagocytosis in fish is very limited, and it is usually restricted to general activities, not to specific components. Thus, opsonization that appears to be dependent on complement has been reported in a number of teleost species [46,47]. In addition, it has been shown that fish complement and Ig can have a synergistic effect in the opsonization of microbes [47,48]. To better understand the role of fish complement in opsonization and phagocytosis, future studies will require evaluating the specific contributions of C3 and C4 activation fragments in such processes. While fixation of C3b or C4b on microbes can lead to their opsonization, it can also result in the assembly of the MAC which will result into their lysis. Bactericidal and lytic effects of fish complement are discussed below in Section 4. 2.3. Inflammation 2.3.1. Complement-mediated inflammation in mammals Activation and cleavage of C3, C4 and C5 molecules results in the generation of C3a, C4a and C5a anaphylatoxins, respectively. Since the C3, C4 and C5 molecules are evolutionarily related to each other, the C3a, C4a and C5a anaphylatoxins share a relatively high degree of structural and functional homology. Nevertheless, the C5a anaphylatoxin has been shown to be considerably more potent than C3a and C4a in eliciting biologically relevant responses [49,50]. In contrast, C4a is the weakest, and physiologically less relevant of the three anaphylatoxins. Some of the roles shared by C3a and C5a in mammals include: (1) their ability to induce migration of leukocytes at the site of infection; (2) their role in stimulating respiratory burst [51,52] and production of prostaglandins in various types of leukocytes [53,54]; and (3) their ability to induce IL-1b expression in monocytes [55,56]. Most of the actions mediated by the C3a anaphylatoxin are suppressed by removal of its carboxy-terminal arginine, through cleavage by serum carboxypeptidase N [50]. However, the elimination of this residue from C5a has a much lesser impact on its function [57e59]. C3a and C5a anaphylatoxins exert their functions through their respective C3a and C5a receptors (reviewed below in Section 3). 2.3.2. Anaphylatoxin molecules in teleost fish Up until very recently, virtually nothing was known about the structure, function and evolution of C3a and C5a molecules and their receptors in non-mammalian species. Two recent reports, have shown the presence of a chemotactic C3a-like molecule in tunicates [60,61]. Studies performed in hagfish and sharks appear to indicate that these species contain chemotactic receptors that act in response to mammalian C5a [30,62]. Recent reports have described for the first time in non-mammalian vertebrate species (teleost fish), the presence of bona fide C3a and C5a anaphylatoxins and their receptors. With regard to the C3a anaphylatoxin, a recent study described the purification and functional characterization of three C3a anaphylatoxins from rainbow trout, a species containing three C3 isoforms. All three C3a isoforms strongly stimulated the respiratory burst of head kidney leukocytes in a dosedependent manner [63]. In contrast, none of them induced chemotaxis in the same cells. Removal of the carboxy-terminal Arg from trout C3a molecules destroyed their ability to induce superoxide production in leukocytes. These studies demonstrated for the first time not only the presence of bona fide C3a molecules in teleost fish, but also provided evidence for the existence of three functional C3a molecules in a single animal species. In agreement with the previous study, a more recent report showed that C3a derived from the carp C3-H1 isoform was not able to stimulate leukocyte migration [64]. The same study demonstrated that carp C4a derived from the C4-2 isotype lacked the same activity. Significant advances have also been made in the characterization of C5a in teleosts. To evaluate the role of C5a in these animals, rainbow trout C5a was recombinantly produced and shown to stimulate peripheral blood and head kidney leukocyte chemotaxis [65,66]. In addition, similar to the action of C5a in mammals,

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trout C5a also triggered the respiratory burst of leukocytes from the same organs [65]. Interestingly, human C5a was unable to induce any of the latter activities in trout leukocytes, strongly suggesting that C5amediated functions are, in this case, species-specific [65]. More recent studies have shown that desArginated carp C5a (derived from the carp C5-I isotype) acted as a strong chemoattractant [64]. In line with this observation, desArginated trout C5a retained its biological activities [65,66] suggesting that, like in mammals, removal of the carboxy-terminal arginine in fish C5a does not have a major impact on the activities tested thus far. A C5a receptor has been cloned and characterized in rainbow trout (described in Section 3). While teleost fish C3a and C5a have shown thus far a significant degree of functional conservation relative to their mammalian counterparts, a recent study appears to indicate a novel role of fish anaphylatoxins in the stimulation of phagocytic processes. Recently, our group identified and purified from complement-activated trout serum a protein fraction composed almost entirely of C3a, C4a and C5a molecules. This fraction (named as PUEF-8) had an unexpected capacity to greatly enhance particle uptake in trout head-kidney leukocytes [67]. To better evaluate the role of PUEF-8 in inducing phagocytosis, we developed a flow cytometric assay that measured the capacity of the leukocytes to ingest fluorescent beads (Fig. 2). With this assay it was demonstrated that PUEF-8 increased the number of phagocytic cells by three- to four-fold when compared to the negative control values (PBS-treated cells). Moreover, PUEF-8 acted as a strong chemoattractant for trout peripheral blood and head kidney leukocytes. Since mammalian anaphylatoxins are not known to exert such an influence in phagocytosis, these findings appear to imply a novel role of anaphylatoxins in fish innate immunity. Future studies are needed to address the molecular mechanisms and cells involved in the actions of fish anaphylatoxins in promoting phagocytic processes.

3. Complement components 3.1. C1q/MBL family The lectin pathway is initiated through the interaction of MBL and ficolins with sugar moieties expressed on the surface of many microorganisms [68]. This pathway has been found to be functionally active in species as primitive as ascidians, where individual components have already been characterized [69]. It has been proposed that the classical pathway arose as a result of the gene duplication of components of the lectin pathway, following the appearance of antibodies in cartilaginous fish [35]. In this regard, MBL and C1q are collectins, both sharing a high degree of structural homology. Recently, a GlcNAc-binding lectin was purified from lamprey, and its primary sequence demonstrated a high degree of homology with mammalian C1q molecules. The authors of that study proposed that C1q may have emerged initially as a lectin and participated in innate immunity in species lacking Ig molecules [70]. In teleost fish full-length MBL-like molecules have been cloned in carp, zebrafish and goldfish [71]. Mannose binding lectins have been partially purified and characterized in Atlantic salmon, although the exact molecular identity and primary sequence of these proteins was unresolved [72]. In teleosts, cDNAs displaying homology to the A, B or C chains of mammalian C1q have been cloned in channel catfish I. punctatus [73] and killifish F. heteroclitus [74] (Table 1). It should be pointed out that the structural and functional characterization of teleost MBL or C1q proteins is currently lacking, therefore, the role of these molecules in the lectin and/or classical pathways in these animals is still speculative. While no MBL-like proteins have been shown to play a role in complement activation in fish thus far, a lectin purified from blue gourami (Trichogaster trichopterus) serum (BGL) was found to mediate the killing of virulent A. hydrophila in the presence of complement from naive fish serum [75]. Interestingly, the agglutinating activity of this lectin was primarily inhibited by N-acetyl-D-glucosamine and to a lesser extent

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by D-(C)-mannose [76], suggesting perhaps that lectins with specificities other than mannose may also activate complement in fish.

3.2. The C1r/C1s/MASP family In the classical pathway, the binding of C1q to immunoglobulins results in the autoactivation of C1r, which in turn, cleaves C1s into its active form. This assembled C1 complex then enables the cleavage of C2 and C4 into their respective ‘‘a’’ and ‘‘b’’ fragments (Fig. 1). In the lectin pathway, interaction of sugars with MBL or ficolins lead to the activation of MBL-associated serine proteases (MASPs) which in turn cleave C4 and C2 [68]. Shared domain structures and primary sequences between C1r, C1s and MASPs are indicative of a common ancestor [77]. In this respect, it should be noted that MASP molecules have been cloned and characterized in species as primitive as tunicates [78]. In teleosts, a C1r-like molecule has been cloned in rainbow trout [79], while in carp, two MASP-like molecules (a MASP-3 like and a truncated product named MRP) have been cloned [80], along with two C1r/C1s/MASP-like molecules designated as C1r/s-A and C1r/s-B [81]. Although the latter two sequences share a near equal homology to mammalian C1r and C1s, recent studies suggest a stronger phylogenetic relationship with C1r [79]. Since the primary sequences of C1r/C1s/MASP-like molecules in fish cannot predict their true identities, functional characterization of their gene products will be required to resolve their roles in the lectin and/or classical pathways and thus, draw their definitive assignment. In this regard, a recent study has shown that rainbow trout possesses a C1s-like molecule capable of cleaving trout C4 into C4a and C4b fragments [33]. This molecule however did not have the capacity to cleave any of the trout C3 or Bf/C2 isoforms. To elucidate the identity of this molecule, its primary sequence awaits further characterization.

3.3. C2/Bf and factor D molecules In mammals, Bf and C2 reside both within the MHC III region [82]. While Bf plays a key role in the alternative pathway, C2 is required for the activation of classical and lectin pathways [83]. In teleost fish, molecules equally homologous in sequence to Bf and C2 have been cloned in trout [84], carp [85], medaka [86] and zebrafish [87]. Thus far Bf/C2 proteins have only been purified and functionally characterized in rainbow trout [84]. The two Bf/C2 molecules identified in this species were shown to be cleaved into Ba and Bb fragments in the presence of any of three trout C3 isoforms, in combination with trout factor D and magnesium. Interestingly the carp Bf/C2 molecules showed different expression patterns and the B/C2-A3 isoform showed an extrahepatic inducible expression, perhaps indicating a specialized role of this molecule as an acute-phase reactant. It is worth mentioning that the sequence of the carp B/C2-B isoform was to some extent more similar to mammalian C2 than to Bf sequences. In any case, the true identity of these molecules will probably come only after and the functions of their gene products are defined in the alternative and/or classical pathway. The presence of multiple Bf/C2 and C3 molecules in fish poses some fascinating questions: [1] Will the different C3 isoforms associate to a specific C2/Bf molecule in forming the alternative C3 convertase, or will any combination of C3 and C2/Bf isoform yield an active C3 convertase? [2] With regard to the specificity of the newly formed C3 convertase, can the C3 convertase cleave any C3 isoform, or is the convertase C3 specific? Answering these questions is key to understanding the role of complement diversity in teleost immunity. Factor D in teleosts has been purified in trout and carp [84,88], although its role in cleaving a Bf/C2 molecule has only been demonstrated in rainbow trout [84]. It appears that a kallikrein-like molecule that was cloned in brook trout (Salvelinus fontinalis) is highly homologous to the primary sequence factor D, and probably represents the only cDNA available thus far for Df in teleosts [89].

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Fig. 2. Development of a flow cytometric assay to assess phagocytosis of fluorescent beads by trout leukocytes. Trout head kidney leukocytes were incubated with fluorescent beads (2.0-m diameter) at a cell/bead ratio of 1:10. Prior to collection, the cells were treated with trypsin-EDTA for 5 min at room temperature. Non-ingested beads were removed by centrifuging the cell suspension (100!g, 10 min at 4  C) over a cushion of 3% BSA in PBS supplemented with 4.5% D-glucose. The cell pellets were then resuspended in PBS, and the flow cytometric analyses were carried out using a standard fluorescence-activated cell analyzer (FACScan, Becton Dickinson). A total of 20,000 cells/sample were analyzed. (A) Histogram of cell number (y-axis) vs. fluorescence intensity (x-axis) representative of phagocytic activity of trout leukocytes incubated with beads in the presence (green) or absence (blue) of PUEF-8 (4 mg/ml). Increased peak fluorescence indicates an increased number of ingested fluorescent beads. Picture insets show the leukocytes that have ingested one, two, three or more beads. The right side of (A) shows the cells incubated in the presence of PUEF-8, before being analyzed by flow cytometry (non-phagocyticCphagocytic cells). (B) Histogram (left) of cell number (y-axis) vs. fluorescence intensity (x-axis) of trout leukocytes incubated with beads in the presence (green) of PUEF-8 (4 mg). The dot plot (middle) shows the cells of regions 1, 2, and 3 of the histogram; cells with a higher number of ingested beads have higher granularity (SSC-H values). The dot plot in the right side of the figure shows the forward (FSC-H) and side scatter (SSC-H) properties of HKLs incubated with PUEF-8 in the absence of beads.

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3.4. C3/C4/C5 family 3.4.1. C3 Complement components C3, C4 and C5 belong to the alpha-2 macroglobulin superfamily of thioester containing proteins [90]. C3 is the central component of the complement system, being activated into its respective cleavage products C3a and C3b through any of the three pathways (Fig. 1). While mammalian C3 is encoded by a single gene, almost all teleost fish studied thus far have been found to possess multiple forms of C3 that are the products of different genes [37e40,77,87,91,92]. In contrast only a single isotype of C3 has recently been isolated in Atlantic cod [93], halibut [93], and wolffish [94], although the presence of additional C3 isoforms in these species was not intensively explored either at the protein or cDNA level. It appears that this C3 diversity is not due to the tetraploid nature of some teleost fish. This is supported by two important facts: first, that tetraploidization in teleost fish has only occurred in some species; second, molecular analysis carried out in the Medaka fish (a diploid fish) has recently demonstrated that this fish species possesses two C3 genes that are closely linked, indicating that the mechanism for C3 multiplication in this fish was tandem gene duplication [92]. Furthermore phylogenetic analysis of the multiple C3 genes cloned from several teleosts has shown that C3 genes from each fish species form their own clusters, implying that many independent C3 gene duplications occurred in each of these species [95]. Functional studies in trout, carp and seabream have shown that these C3 isoforms demonstrate different binding efficiencies to several complement activating surfaces suggesting that teleost fish may have developed a novel strategy to augment the innate recognition and destruction of microbes [91]. In human C3, residues His1126 and Glu1128 have been shown to be critical in determining the binding specificity of C3 to its target surface. In trout, the His residue is conserved in the C3-1 and C3-3 isoforms, whereas in C3-4 this residue is substituted by Thr. Moreover, the Glu1128 is only present in trout C3-1, while in C3-3 and C3-4, these residues are replaced by Thr and Ser, respectively. It has been suggested that differences in these residues account, at least in part, for the variations in the binding efficiencies of the various trout C3 isoforms [96]. The situation in the carp C3 isoforms is similar although the residue differences appear to occur only at the position equivalent to human His1126 [40]. It will be important in the future to determine whether the variations in binding are solely due to changes in the aforementioned residues, or whether other residues and domains in the C3 protein are also involved. It is also possible that the differences in C3 binding could be the result of a variation in the affinity of a specific C3 isoform for either factor B/C2 (leading to more convertase formation and more C3 binding to the surface) or for factor H-like molecules (leading to less convertase formation, and less C3 binding to the surface). In this regard, and as shown in mammals, it is likely that the architecture of the microbial surface would be an important factor in determining the binding of Hf or Bf/C2 to the C3b isoform newly bound into that surface [97]. In addition to being present in multiple isoforms, C3 has also been found to be polymorphic in carp [40]. Although it is tempting to speculate about possible functional differences among the polymorphic C3 forms, this intriguing idea remains to be further investigated in carp and other teleosts. In mammals, C3 has been found to be primarily expressed in liver, with secondary sites spanning a variety of tissues including the central nervous system, the gastrointestinal, reproductive and the lymphoid organs [98]. In fish, there are conflicting reports pertaining to the extent of C3 tissue distribution. Studies in cod and halibut [99,100] have shown that C3 is expressed in a wide assortment of organs, with their presence detected at different stages of larval development. These results are in conflict with studies on (C) The effect of PUEF-8 in enhancing the phagocytic activity of leukocytes from one representative trout. Histograms of cell number (y-axis) vs. fluorescence intensity (x-axis) representative of uptake activity by head kidney leukocytes (HKLs) and peripheral blood leukocytes (PBLs) incubated with beads in the presence (green) or absence (blue) of PUEF-8 (4 mg/ml). The phagocytic activity was measured as the percentage of cells that had ingested three or more beads (Region 1[R1]). Results are presented here as the percentage of the analyzed cells ingesting beads comprised in Region 1. It can be observed that PUEF-8 enhances the phagocytic activity of HKLs, whereas it has very little effect on PBLs. (Modified from figure 3 of Ref. [67])

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Table 1 Summary of complement components characterized in teleost fish Component

Species (cDNA-N/protein purification-P)a

Functional characterization

C1q/MBL

A. salmon (Nÿ/PC) [72], B. gourami (NC/Pÿ) [75], carp (NC/Pÿ) [71], catfish (NC/Pÿ) [73], goldfish (NC/Pÿ) [71], killifish (NCp/Pÿ) [74], zebrafish (NC/Pÿ) [71]

Not characterized

C1r/C1s/MASP

Carp (Nÿ/PC) [80], R. trout (NC/PCp) [79]

Cleavage of C4 [79]

C2/Bf

Carp (NC/Pÿ) [85], medaka (NC/Pÿ) [86], R. trout (NC/PC) [84], zebrafish (NC/Pÿ) [87]

Cleavage of C3 [84]; C3 convertase formation [84]

C3

Carp (NC/PC) [40], cod (Nÿ/PC) [93], halibut (Nÿ/PC) [93], medaka (NC/Pÿ) [92], seabream (Nÿ/PC) [38], R. trout (NC/PC) [37,96], wolffish (NC/PC) [94], zebrafish (NC/Pÿ) [87]

Convertase formation [84]; covalent binding of C3b to activating surfaces [37,38,40]; generation of C3a anaphylatoxin

C4

Carp (NC/Pÿ) [77], catfish (Nÿ/PCp) [103], R. trout (NC/PC) [33,79], zebrafish (NC/Pÿ) [104]

Cleavage by immune complexes [33]; essential role in activation of CCP [33]

C5

Carp (NC/PCp) [108,114], seabream (Nÿ/PC) [38], R. trout (NCp/PC) [106,107]

Initiation of MAC [109,114]; generation of C5a anaphylatoxin [64,67]

C6

Carp (Nÿ/PC) [114], R. trout (NC/PCp) [109,110]

MAC formation [109,114]

p

C7

Carp (Nÿ/PC) [114], R. trout (NC/PC ) [109,112]

MAC formation [109,114]

C8

Carp (Nÿ/PC) [114], J. flounder (NCp/Pÿ) [116], R. trout (NCp/PCp) [109,111]

MAC formation [109,114]

C9

Carp (NC/PC) [114], J. flounder (NC/Pÿ) [116], pufferfish (NC/Pÿ) [115], R. trout (NC/PCp) [109,113]

MAC formation [109,114]

Factor D

Carp (Nÿ/PC) [88], trout (NC/PC) [84]

Proteolytic cleavage of Bf/C2 [84]

p

C3aR

R. trout (NC/PC ) [124]

Not characterized

C5aR

R. trout (NC/Pÿ) [124]

Interaction with C5a [124]

C3a anaphylatoxin

Carp (NC/PC) [64], trout (NC/PC) [63] p

Stimulates respiratory burst [63]

C5a anaphylatoxin

Carp (NC/PC) [64], trout (NC/PC ) [67]

Induction of chemotaxis [64,65]; stimulates respiratory burst [65]

SBP1

S. bass (NC/PC) [118]

Complement regulation [118]

C1-like inhibitor

R. trout (NC/Pÿ) [79]

Not characterized

Factor I

Carp (NC/Pÿ) [119]

Not characterized

a

Superscript p denotes partial cloning/protein purification.

C3 expression in the spotted wolffish [94], where C3 expression appeared to be limited only to the liver. While unlikely, it is possible that in teleosts, the pattern of C3 expression is species specific. Another possibility is that perhaps more C3 isoforms exist in these teleosts and the authors of these studies only determined the expression of one isoform. Thus, in the future, it will be of great interest to determine the expression patterns of the multiple C3 isoforms shown to be present in other teleosts. In this regard, it will be fundamental to study whether these C3 isoforms show a differential expression pattern in tissues and/or during ontogenic development of fish from eggs to adults. 3.4.2. C4 C4 plays an integral role in the activation of the classical and lectin pathways. Like factor B and C2, C4 also resides within the MHC III region [101]. In some mammals (human, sheep and cattle), two isotypes of C4 have

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been characterized, C4A and C4B; while the former binds to amino- and hydroxyl-groups, the latter prefers to bind to amino groups [102]. C4 molecules have been cloned in several teleost species although the functional characterization of the C4 protein has only been carried out in rainbow trout so far. Thus, trout C4 has recently been cloned and its primary sequence was found to possess the catalytic His residue within the PNPVIH motif, a characteristic attributed to the C4B isotype in mammals [33,79]. While a single gene was found to encode trout C4, purification of the gene product resulted in the isolation of two C4 isoforms that appeared to differ only in their glycosylation pattern. Thus, the two C4 proteins bound in a similar manner to trout IgM-sensitized sheep erythrocytes only in the presence of Ca2C and Mg2C. In addition both molecules equally restored the classical pathway-mediated hemolytic activity of trout serum depleted of C3 and C4. Reconstitution of this hemolytic activity was dependent on the presence of both trout C3-1 and C4-1/C4-2, as well as on the presence of IgM bound to the target cells. We have recently isolated a cDNA encoding an additional trout C4 molecule that appears to be homologous to the mammalian C4A isotype (non-published results). C4 has also been partially purified from the channel catfish although its functional characterization and primary sequence remain to be elucidated [103]. Recently, carp C4 has been cloned in two different forms, C4-1 and C4-2 [77]. Since the catalytic His residue is conserved only in C4-2, this molecule could represent the ortholog for mammalian C4B. Two partial sequences encoding for C4-like molecules have also been reported in zebrafish [104]. It appears that these molecules are more similar to carp C4-1 than C4-2. Since teleosts appear to have multiple C4 isoforms, it will be interesting to see whether binding differences exist among them, and whether these potential differences in binding parallel those described between the mammalian C4B and C4A isotypes. 3.4.3. C5 Activation of C3 by any of the three pathways leads to the assembly of the MAC, in which the initial step involves the C5 convertase cleaving C5 into C5a and C5b fragments [105]. In teleost fish, C5 has been partially cloned and purified in trout [106,107] and seabream [38], both displaying noticeable size and structural homology to its mammalian counterpart. In carps two different full-length C5-like cDNA sequences (C5-I and C5-II) have recently been cloned [108]. Interestingly carp C5-I appeared to be in multiple copies as indicated by Southern hybridization. 3.5. Membrane attack complex (MAC) assembly The membrane attack complex consists of C5b, C6, C7, C8 and C9 components [5]. Rainbow trout was the first teleost species in which molecules very similar to the C5b-C9 components were biochemically characterized and isolated [109]. At the molecular level trout C6, C7, C9 and the beta chain of C8 have been demonstrated [110e113]. In carp, MAC complexes extracted from lysed rabbit erythrocytes were found to possess all terminal components in a ratio similar to that found in humans [114]. In addition, C8 and C9 were purified and biochemically characterized in this species [114]. It is also worth noting that individual components have been cloned in other species; an example of this is the C9 molecule in pufferfish [115], and the C8b chain and C9 components in Japanese flounder [116]. 3.6. Complement regulatory proteins In mammals, activation of the complement system is regulated by several soluble and surface-bound complement regulatory proteins. Some of these molecules act as cofactors for factor I, an enzyme that is capable of specifically cleaving and inactivating C3b and C4b molecules. These complement regulators play a pivotal role in protecting self-tissue from potential damage derived from autologous complement activation [117]. In mammals these molecules include complement receptor type 1 (CR1), decay

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acceleration factor (DAF), membrane cofactor protein (MCP), factor H, C4-binding protein (C4bp), C1 inhibitor, CD59 and clusterin. The two latter regulatory molecules are known to block the formation of the membrane attack complex [2]. CR1, MCP, C4bp and factor H have all been shown to act as cofactors for factor I-mediated proteolytic inactivation of C3b and C4b [117]. The genes for C4bp, CR1, DAF, and MCP have been mapped to the so-called regulator of C# activation (RCA) locus, and they are all composed of tandemly arranged short consensus repeats (SCRs). SCRs are typically composed of 60e70 amino acids that include four highly conserved cysteines. In addition, all SCRs share a 3D configuration that is characterized by a rigid triple-loop structure. Factor H- and I-like activities have been shown in the plasma of various teleosts. For example the five different isoforms of seabream C3 have been demonstrated to be cleaved in serum to iC3b and C3d-like fragments similar to those generated in mammalian C3 molecules. Interestingly, the cleaving activity required calcium and magnesium, whereas in mammals, factor I-mediated cleavage occurs in the absence of these metals [34,38]. In teleosts, the only instance of a functionally characterized complement regulator is in barred sand bass Paralabrax nebulifer, where SBP1, a soluble protein of 110 kDa, has been shown to combine the functional activities of C4bp and factor H. SBP1 has also been shown to act as a cofactor for trout C3 and human C4. Thus it was suggested that this molecule could represent the ancestral regulatory component from which C4bp and factor H were derived [118]. In order to confirm this hypothesis, further studies must be conducted using other teleost species. In addition, it should be pointed out that successive studies with complement regulatory proteins in teleosts will require homologous sources of C3 and C4 in order to draw definitive conclusions on the function and identity of these regulatory molecules. Moreover, future studies will also have to address whether a RCA-like region containing the corresponding mammalian complement regulatory orthologs is present in teleost fish. Evidence of the existence of other complement regulatory molecules include the recent cloning of a C1-like inhibitor in trout [79], and two factor I isotypes in carp [119].

3.7. Complement receptors Upon the covalent binding of C3b to an activating surface, subsequent factor I-mediated cleavage results in the generation of C3d and iC3b. In mammals, it has been shown that C3b binds to complement receptor 1 (CR1), while C3d and iC3b can interact with complement receptor 2 (CR2). Complement receptors 3 and 4 (CR3 and CR4) have also been demonstrated to bind iC3b. In mammals, these interactions are critical for phagocytic uptake and antigen processing, linking the innate and adaptive immune response [117]. The more recently described C1q receptor appears to have an important role in the regulation of phagocytosis and immune complex clearance. While little is known about the functional or structural existence of these receptors in bony fish, it has recently been illustrated that C3d of two carp C3 isoforms (C3d-H1 and to a lesser extent, C3d-S) are capable of binding to carp peripheral lymphocytes [120]. Clearly more studies are required in this important area of complement biology to better understand the evolution and function of these receptors in innate and adaptive immune processes of teleost fish. In this regard, it will be important to elucidate whether C3d-like receptors in these animals have, like in mammals, the ability to influence the generation of antibody responses. If such receptors are present in teleosts, this could lead to the potential use of C3d as a molecular adjuvant in fish. By interacting with CR2 present on B cells and follicular dendritic cells, C3d has already been shown to act as a very potent molecular adjuvant in mammals [20]. Other important complement receptors include the C3a and C5a anaphylatoxin receptors. These molecules are members of the rhodopsin family of seven-transmembrane G-protein-coupled receptors. C3a and C5a bind almost exclusively to their receptors, C3aR and C5aR, respectively [50]. However, a second C5a receptor, C5L2, has recently been characterized in humans and mice [121,122]. C5L2 shows the highest sequence identity to C5aR. In contrast to C5aR, C5L2 seems to lack the capacity to transduce signals, and

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it has been suggested that it may serve as a decoy receptor, thereby modulating the concentration of both C5a and C5adesArg [122]. Until recently, nothing was known about the presence of a C5a or C3a-specific receptor in non-mammals. Two recent studies have reported the cloning of the same rainbow trout cDNA encoding for a 350-amino acid protein that shows the highest sequence similarity to C5aR from other species [123]. This represents the only complement receptor cloned thus far in teleosts. Trout C5aR (TC5aR) shared several conserved domains with its mammalian counterpart with regards to its secondary structure; this includes an acidic N-terminus, a serine-rich C-terminus, the presence of seven transmembrane regions and the presence of the C5aR-specific DRF motif. This motif is absent in mammalian C5L2, and its presence in C5aR is required for G-protein coupled signaling. Northern blot analysis showed significant expression of the TC5aR message in peripheral blood leukocytes and in the kidney. One of the studies [124] using flow cytometry, demonstrated that antibodies generated against two different areas of the extracellular N-terminal region of TC5aR stained principally B lymphocytes and granulocytes, whereas thrombocytes were negatively stained. Recent data obtained in our laboratory has confirmed by RT-PCR the presence of the full length C5aR cDNA in presorted trout B-lymphocytes (unpublished results). In disagreement with these data, a different study reported that recombinant trout C5a bound only to the granulocyte fraction of trout peripheral blood cells [66]. The presence of C5aR in fish B cells may have important implications in the generation or development of an immune response. Therefore more studies are needed to further our understanding on the presence and roles of C5aR in fish B lymphocytes. From a functional perspective, it was also shown that the anti-trout C5aR antibodies had the capacity to inhibit chemotaxis of peripheral blood cells toward an anaphylatoxincontaining fraction purified from complement-activated trout serum [124]. This indicates that the role of C5aR in leukocyte migration has been conserved for more than 300 million years.

4. Complement and host-pathogen interactions Below we summarize the current knowledge on the role of teleost complement in host defense against bacteria, viruses, fungi and parasites. 4.1. Fungi While the role of complement in clearing fungal infections is well established in mammals, whether this is the case in fish remains to be demonstrated. One study has investigated the inhibition effects of fish sera on fungal germination and germling growth in some susceptible and resistant fish species against epizootic ulcerative syndrome (EUS) [125]. EUS is a serious fungal disease of fish caused by Aphanomyces invadans. It was found that fungal germination could be inhibited by serum in all susceptible fish, and that heatinactivated serum was not inhibitory. This suggests a role of complement in the inhibition of A. invadans germination. Clearly many more studies are needed to understand the involvement of complement in fighting fungal diseases in teleost fish. 4.2. Viruses The involvement of fish complement in clearing viral infections has been ill defined thus far [126]. It has been shown that neutralization of some fish viruses is complement independent, and their neutralization depends solely on the presence of specific antibodies against the virus [127]. However, other studies have shown that the neutralizing ability of rainbow trout antibodies to rhabdoviruses such as viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) is dependent on the presence of complement [127e129]. Rainbow trout complement-mediated IHNV neutralization requires the

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activation of the classical pathway, and is dependent on the presence of trout Bf-2 [130]. Interestingly, it has been reported that trout and masu salmon fry, whose serum did not appear to possess complement activity, were more susceptible to IHNV and IPNV than were chum salmon fry, whose serum did seem to have complement activity [131]. Whether susceptibility was due to the lack of complement in trout and salmon fry will require further work. However, the latter study emphasizes the importance of understanding the ontogenic development of complement activity and specific components in teleosts. While the available evidence has demonstrated the involvement of complement in the neutralization of viruses in teleost species, the specific pathways and components implied await further characterization. More particularly, future studies will have to address whether viral neutralization requires the action of the lytic pathway (i.e., assembly of the membrane attack complex), or whether C3/C4 fixation on the surface of the virus is sufficient for its neutralization. In addition, since teleosts contain multiple C3 isoforms, it will be of interest to determine which particular isoforms are involved in the neutralization of viruses. 4.3. Parasites The involvement of complement, present in skin or in serum, of various fish in the killing mechanisms against parasites has been well established [132e135]. Gyrodactylids were found to be rapidly killed by the activation of the alternative pathway (ACP). It was shown however that C3 bound to the surface of the parasite, particularly in carbohydrate-rich areas, which could also suggest a role of the lectin pathway in the activation of trout complement [136,137]. Further studies will have to determine the specific trout C3 isoforms involved in the activation and binding to the parasite surface. An enhancement of expression of C3 genes, as well as the production of nitric oxide synthase (iNOS), MHC-II and IgM antibodies were evident in the skin and lymphoid organs of rainbow trout infected by the parasitic ciliate Ichthyophthirius multifiliis [138]. In this study, the primer sequence used to amplify C3 was common to trout C3-3 and C3-4 sequences, but not to C3-1; therefore, it will be of interest to address which of the C3 isoforms was in fact up-regulated. Evidence for ACP-mediated killing of Discocotyle sagittata oncomiracidia has been reported in both rainbow trout and brown trout, where scanning electron microscopy (SEM) analyses showed varying degrees of surface disruption in the parasitic larvae after exposure to fish plasma [139]. It has been shown that the hemoflagellate Trypanoplasma borreli can be effectively killed by resistant carp sera, but cannot be killed by either the sera from susceptible carp or heat-treated sera from the resistant carp, suggesting the involvement of antibody-mediated classical pathway (CCP), and not ACP, in the killing mechanism of carp against the hemoflagellate parasite [140]. A recent report showed that cultured trypanosomes (Trypanosoma danilewskyi) were resistant to lysis by either immunized or non-immunized goldfish serum. Interestingly, trypsinized trypanosomes became susceptible to lysis by goldfish ACP, suggesting the presence of surface proteins on the parasite with the capacity to inhibit the activation of goldfish complement [141]. 4.4. Bacteria The bactericidal activity of complement has been well recognized as one of the key killing mechanisms of clearing bacteria in teleosts [126,133,142]. Alternative pathway activity, which is antibody independent, can be directly activated by the lipopolysaccharide (LPS) of Gram-negative bacteria, and can result in lysis of the bacterial cell [36]. In mammals, various studies indicate that only avirulent Gram-negative bacteria are highly susceptible to the lytic effect of the ACP, while virulent Gram-negative bacteria or Gram-positive bacteria are resistant to being killed by this mechanism, due to structural differences of their cell surface components [143,144]. Studies performed with channel catfish pathogenic and non-pathogenic bacteria have shown that the presence of sialic acid in the pathogenic bacteria rendered them more resistant to ACP

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attack, whereas non-pathogenic bacteria showed very little amount of sialic acid on their surface [145]. Similarly, the presence of sialic acid in some mammalian bacterial pathogens has been shown to negatively modulate ACP activation [146]. In fish, representative isolates of capsulated and non-capsulated Lactococcus garvieae from Japan and Europe were evaluated for their ability to fix complement. Results indicated that all non-capsulated isolates fixed complement regardless of origin, and specific antibodies did not markedly enhance complement fixation. The capsulated isolates were less efficient at fixing complement, but complement fixation was markedly increased by the addition of homologous antibody. This suggests the involvement of the ACP in killing the non-capsulated isolates, while activation of the CCP was involved only with the capsulated isolates [147]. In contrast, various Streptococcus iniae isolates from different geographical and fish host origins, all of which possess polysaccharide capsules, were demonstrated to be resistant to either normal or immunized serum, and grew well in the presence of fresh normal serum [148]. A different study has shown that Vibrio anguillarum O1 is resistant to normal rainbow trout serum, but highly susceptible to serum from immunized animals [149] implying the involvement of the CCP in the clearance of the bacterium. However, the same authors reported that 80% of the Vibrio anguillarum O2a strains analyzed could not be killed by trout CCP [150]. Interestingly, it was shown that the high-molecularweight O-antigen side chains of the O2a strains were responsible for protecting bacteria from complementmediated killing [150]. It should be pointed out, that when studying the bactericidal action of fish complement, analysis of complement consumption may not necessarily indicate susceptibility of bacteria to complement attack. For example, it has been shown that both virulent and non-virulent strains of Flavobacterium psychrophilum can consume complement (in this case, ACP was reported to be consumed). However, the survival and growth of the same strains were affected very little when grown in the presence of non-immunized and immunized trout serum in vitro, indicating that these bacteria were resistant to both the ACP and the CCP [144]. While the bactericidal action of fish complement has been well documented, future studies will be needed to further address the specific pathways and complement components implied in the complement-mediated killing of bacteria.

5. Factors influencing complement activity in fish 5.1. Stress and complement It is well established that the immune system of fish can be severely depressed by various stress conditions [151e153]. High stocking density is one of the most common causes of stress for cultured fish in modern intensive aquaculture. The immunosuppressive effect on serum alternative complement activity, as well on other non-specific immune responses has been well documented in various fish species following crowding stress [152e154]. For example, fish densities of 25g/l in fancy carp (Cyprinus carpio L.) resulted in a significant drop of complement-mediated bactericidal activity on day 1, and low activity remained throughout the remaining time intervals [152]. Similarly, a situation of short-term crowding stress (100 kg/m3 for 2 h) was found to induce an immediate depressive effect on serum complement activity in the gilthead seabream (Sparus aurata), although normal complement activity was recovered after 3 days [153]. Besides crowding stress, situations of acute stress or daily acute stress (i.e., handling, transportation, anesthesia treatment) can also dramatically decrease complement activity in fish [155,156]. Since complement is down-regulated in many situations of stress, it has been proposed that complement activity could be a good indicator of fish immunocompetence in stressed animals [157]. While it seems apparent that complement levels decrease when fish are subjected to variety of stressful conditions, it remains to be determined which specific pathways and components are the most affected. Moreover, future studies should address the neuroendocrine immune interactions leading to the depression of complement under such conditions.

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5.2. Nutrition and complement Various immunostimulants (i.e., glucans, vitamins, essential fatty acids) have been widely used in aquaculture, and their stimulating effects on disease and stress resistance have been documented [158e160]. Here we will briefly review recent progress made towards the understanding of the effects of some of these immunostimulants on fish complement. The complement activity of fish serum has been reported to be significantly enhanced by oral administration of vitamin E- or C-supplemented diets [161e165]. Diets deficient in vitamin E depleted alternative complement pathway activity in the gilthead seabream [166] and rainbow trout [164,167]. However, excessively higher dosages (1800 mg/kg) of vitamin E were not effective for enhancing the hemolytic activity of gilthead seabream, probably due to the imbalance of vitamin E with that of other antioxidants [168]. Therefore, moderate doses of vitamin E (i.e., 100 mg/kg diet for rainbow trout and 1200 mg/kg for the gilthead seabream) have been found optimal in upregulating the complement activity of fish [153,167,168]. Similar stimulating effects of vitamin C on complement activity have been demonstrated in various fish species [161,169]. However, fish fed with vitamin C-supplemented diets (3 g/kg) only exhibited an enhancement of respiratory burst activity, but not complement activity [153]. This was possibly due to the low intake doses used in that report. It has been shown that gilthead seabream cultured in high density stocks and fed with a vitamin E and C-supplemented diet, exhibited similar complement activity as fish held in low stocking conditions and fed with unsupplemented control diets. These findings suggest a stressresistant role of vitamins E and C in fish [170]. Similar observations have been reported in other fish species fed with vitamin-supplemented diets [161,163]. Complement-mediated hemolytic activity has also been increased in fish fed with immunostimulants other than vitamins, including glucans [158], N-3 highly unsaturated fatty acids [171], carotenoids from natural products [172], as well as probiotic bacteria (e.g. Lactobacillus rhamnosus) [173]. In contrast the feeding of whole yeast cells was found to be ineffective in augmenting serum complement activity in seabream [174].

5.3. Season and temperature influence Due to their poikilothermic character, the modulation effect of seasons and in vitro temperatures on fish immune function has been well established [175]. It has been demonstrated that complement-mediated lytic activity of trout is affected by acclimation of fish at different temperatures [176,177]. These studies showed that higher temperatures increased the complement-mediated lytic activities tested. The lowest values of complement activity in the serum of gilthead seabream were recorded in the coldest months (January), and the highest complement titers were seen in the beginning of autumn when water temperatures reach the maximum values [178]. In contrast, a different study showed the highest ACP titers of tinca (Tinca tinca) in winter [179]. Thus, further research using different species of fish will be required to establish whether seasonal changes have similar effects on the complement activity.

6. Future directions During the last few years considerable progress has been made in understanding the complement system of teleosts. However the knowledge we have from most aspects of fish complement is just a fraction of what we know about the complement system of mammals. Throughout this review, we have suggested, in each

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section, potential future directions that will advance our knowledge in that particular area. Overall, the following areas require special attention: (1) The development of molecular tools is a major priority for advancing our understanding of complement biology. Thus far very few reagents exist for the evaluation of specific teleost complement-related activities or components. Development of such reagents should include the generation of speciesspecific antibodies against fish complement components, the production of recombinant proteins, the development of cDNA probes, the standardization of protocols for in situ hybridization, and other techniques to assess and quantify complement expression. Equally important, we also need to develop functional assays to better assess the role of fish complement in immunity. Such assays should include: the standardization of flow cytometric or other powerful analytic techniques to assess phagocytosis; respiratory burst activation as well as phenotypic analysis of leukocytes; assays to evaluate alternative, lectin or classical complement pathway-mediated hemolytic or bactericidal activities; and assays to evaluate complement fixation on microbial surfaces. (2) We have to clarify the mechanisms and specific components involved in the activation of alternative, classical and lectin pathways of complement activation in teleosts. The recent assembly of the genome of three different teleost fish (zebrafish, fugu and tetraodon), opens a new era in the study of fish complement, and fish immunity in general. These genomes will be extremely instrumental in filling in the gaps we have about the occurrence of many complement components in fish. (3) We have to further study the role of complement in host-pathogen interactions in order to learn how fish complement can eliminate microbes, and how pathogens evade complement attack. It is important to understand whether opsonization is enough for the clearing of the microbe, or whether the participation of the lytic pathway is required for its killing. Thus, it will be important to address which specific C3 (or C4) fragments (C3b/iC3b/C3d) play a role in the opsonization of microbes. Special emphasis should be placed on understanding how complement clears fungal and viral infections, an area of study that has been thus far neglected. Given the diversity of C3 in most analyzed teleosts, it will be of great interest to determine which particular isoforms are more efficient in the opsonization or clearing of a particular microbe. (4) Another area that needs much attention is the study of the regulation of complement activities and components in response to infection and other endogenous/exogenous stimuli (i.e., viral, bacterial or parasitic infections, stress, cytokines, hormones). (5) Ontogenic studies on complement activities and specific components are also missing, although recent progress has been made in regards to the ontogeny of the C3 molecule in several teleosts. As suggested in these studies, complement may not only fulfill a defense function in the early stages of fish development, but it may also play a role in development processes [99,100]. (6) It is well established that in mammals, the complement system heavily contributes to the generation and development of an acquired immune response. In fact, this ancient mechanism of defense has evolved from a primitive mechanism of innate immune-recognition in invertebrate species to that of an effector system that bridges the innate with the adaptive immune response in vertebrate species. A key question that remains to be answered is whether complement in fish has already evolved into a system capable of influencing adaptive immunity, or whether its role is restricted to innate immunity. (7) We should not overlook potential important applications that may arise from the study of fish complement. With complement being a key innate immune mechanism in fish, it would be desirable to search for immunostimulants (i.e., probiotics, vitamins, pathogen-associated molecular patterns (PAMPS) that have the capacity to up-regulate complement activities and components in fish. Such compounds would be administered orally or by injection, and could make fish more resistant to potential pathogens in situations in which these animals may be subjected to stress (i.e., vaccine delivery, transport of fish, overcrowding conditions). In addition, if fish complement is found to

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enhance antibody or cell-mediated immune responses, one could think of potential applications of specific complement components in the area of adjuvant technology. Finally, the future directions proposed above should expand our knowledge of the molecular evolution, structure and function of complement proteins in teleost fish; this will, at the same time, provide insights into novel complement-related mechanisms of recognition and defense that are essential to both fish and human health. From an applied perspective, it should be pointed out that aquaculture is the fastest growing animal food sector worldwide. However, disease and health management problems are one of the major hurdles for the developing aquaculture industry. Research into the area of fish complement is likely to lead to the discovery of molecules that can be employed, in the future, as immunostimulants or can be used as immunoadjuvants.

Acknowledgements This work was supported by National Science Foundation grant MCB-0417078 and by the National Research Initiative of the USDA Cooperative State Research; Education and Extension Service, grant number 2004-01599.

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