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Laboratory studies on associations between disease resistance and susceptibility and major histocompatibility (MH) genes in Atlantic salmon Salmo salar have shown the importance of immunogenetics in understanding the capacity of populations to fight specific diseases. However, the occurrence and virulence of pathogens may vary spatially and temporally in the wild, making it more complicated to predict the overall effect that MH genes exert on fitness of natural populations and over several life-history stages. Here we show that MH variability is a significant determinant of salmon survival in fresh water, by comparing observed and expected genotype frequencies at MH and control microsatellite loci at parr and migrant stages in the wild. We found that additive allelic effects at immunogenetic loci were more likely to determine survival than dominance deviation, and that selection on certain MH alleles varied with life stage, possibly owing to varying pathogen prevalence and/or virulence over time. Our results highlight the importance of preserving genetic diversity (particularly at MH loci) in wild populations, so that they have the best chance of adapting to new and increased disease challenges as a result of projected climate warming and increasing aquaculture.

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm–egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.

Microsatellite DNA loci, when used in population genetic studies, are usually assumed to be neutral (unaffected by natural selection, either directly or as a result of tight linkage), but this assumption is rarely tested. Here, the assumption of neutrality is examined using established methods, principally that based on the expected relationship between F ST and heterozygosity, at 12 putative neutral microsatellite loci utilised in a study of Atlantic herring Clupea harengus in the north east Atlantic (west of Great Britain and around Ireland) and in the Baltic Sea. All but two of these loci demonstrate relationships that suggest that they may be regarded as neutral genetic markers. Of the other two loci, however, one shows a relationship suggestive of the action of directional selection and the other of balancing natural selection, though other locus-specific effects may operate. Thus, the latter two loci may provide inaccurate inference if used in phylogeographic studies and also demonstrate the danger of assuming neutrality at all microsatellite loci without explicit testing. However, such loci, particularly those affected by directional as opposed to balancing selection, may be of great use in stock discrimination studies, and selected loci in general, have considerably potential in studies of adaptation.

Udgivelsesdato: 2008-Apr Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of beta2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasabeta2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.

Udgivelsesdato: 2008-Mar The structure of the peptide-binding specificity of major histocompatibility complex (MHC) class I has been analyzed extensively in human and mouse. For fish, there are no crystallographic models of MHC molecules, neither are there data on the peptide-binding specificity. In this study, we describe for the first time the identification of a fish class I peptide-MHC ligand binding motif. Phage display technology using both 7mer and 12mer libraries enabled us to identify peptide ligands with unique specificity that interacts with the recombinant Salmon MHC class I molecule. The recombinant proteins, beta(2)m/SasaUBA*0301, were produced in Escherichia coli, in which the carboxyl terminus of beta(2)-microglobulin is joined together with a flexible (GGGGS)(3) linker to the amino terminus of the heavy chain. One hundred and seven individual phages bound to beta(2)m/SasaUBA*0301 were isolated after four rounds of panning from the 7mer random-peptide library. The peptide encoding sequences were determined and peptide alignment led to the prediction of position-specific anchor residue. A prominent proline at position 2 was observed and we predict that it might be one of the anchors at the N-terminus. Meanwhile, phage display peptide library encoding random 12mer peptides was also screened against beta(2)m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon class I molecule might have a very similar binding motif at the C-terminus compared with a known mouse class I molecule H2-Kb which has L, or I, V, M at p8. Previous work showed that Atlantic Salmon carrying the allele SasaUBA*0301 are resistant to infectious Salmon aneamia virus and there is a significant association between MHC polymorphism and the disease resistance. Therefore, our study might contribute to designing a peptide vaccine against this viral disease.

Several studies have documented the genetic effects of intraspecific hybridization of cultured and wild Atlantic salmon (Salmo salar L.). However, the effect of salmon aquaculture on wild congeners is not so well understood. Diseases, introduced or increased in incidence by salmon aquaculture activities, may have an impact on co-occurring wild sea trout (Salmo trutta L.), as implied by the steep decline in sea trout numbers in many Irish, Scottish, and Norwegian rivers since the late 1980s, which may be linked to sea lice infestations associated with marine salmonid farming. Our data suggest that salmon farming and ocean ranching can indirectly affect, most likely mediated by disease, the genetics of cohabiting sea trout by reducing variability at major histocompatibility class I genes. We studied samples of DNA extracted from scales of sea trout in the Burrishoole River, in the west of Ireland, before and at intervals during aquaculture activities. In these samples, allelic variation at a microsatellite marker, tightly linked to a locus critical to immune response (Satr-UBA), was compared with variation at six neutral microsatellite loci. A significant decline in allelic richness and gene diversity at the Satr-UBA marker locus, observed since aquaculture started and which may indicate a selective response, was not reflected by similar reductions at neutral loci. Subsequent recovery of variability at the Satr-UBA marker, seen among later samples, may reflect an increased contribution by resident brown trout to the remaining sea trout stock.

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Gottingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.

In order to investigate the mechanisms creating and maintaining variability at the major histocompatibility (MH) class II alpha (DAA) locus we examined patterns of polymorphism in two isolated Atlantic salmon populations which share a common post-glacial origin. As expected from their common origin, but contrary to the observation at the MH class I locus, these populations shared the majority of DAA alleles: out of 17 sequences observed, 11 were common to both populations. Recombination seems to play a more important role in the origin of new alleles at the class II alpha locus than at the class I locus. A greater than expected proportion of sites inferred to be positively selected (potentially peptide binding residues, PBRs) were found to be involved in recombination events, suggesting a mechanism for increasing MH variability through an interaction between recombination and natural selection. Thus it appears that although selection and recombination are important mechanisms for the evolution of both class II alpha and class I loci in the Atlantic salmon, the pattern of variability differs markedly between these classes of MH loci.

Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC class I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some class II B loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed class II MHC genes in a hexaploid African 'large' barb. This prompted us to study the number of MHC genes present in the genome of an African 'large' barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic class Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of class I and II B genes. Our study revealed three beta2-microglobulin, five class Ia, four class II A, and four class II B genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The class Ia and class II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.

Interleukin-1beta (Il-1beta) is a central component in innate immunity and the inflammatory response of mammals. Only recently, the first non-mammalian IL-1beta sequences were published. In this study, we describe a second IL-1beta sequence (IL-1beta2) in carp with 74% amino acid identity to the carp IL-1beta1 sequence. The existence of two IL-1beta copies in the carp genome probably originates from the tetraploid nature of the species. In contrast to the first carp Il-1beta sequence, IL-1beta2 is represented by multiple genes with 95-99% identity. Detection of several IL-1beta2 sequences within individual homozygous fish suggests the presence of multiple copies of the Il-1beta2 gene in the carp genome, possibly as a result of subsequent gene duplication of IL-1beta2. In vivo, constitutive mRNA expression of both IL-1beta genes was found in healthy carp. IL-1beta2 mRNA expression could be up-regulated in head kidney cells similar to carp IL-1beta1, in vivo by infection with Trypanoplasma borreli and in vitro by stimulation with lipopolysaccharide (LPS). Cortisol, the major glucocorticoid in fish, is an endocrine-derived factor mediating Il-1beta expression. Although constitutive IL-1beta expression was inhibited by a physiological dose of cortisol, cortisol synergistically enhanced LPS-induced IL-1beta expression in carp. Involvement of the transcription factor nuclear factor (NF)-kappaB in expression of IL-1beta1 and IL-1beta2 was demonstrated. Ratio of IL-1beta expression was determined and this showed IL-1beta1 mRNA expression to be at least tenfold higher compared with IL-1beta2. The possibilities of IL-1beta2 being a functional gene or approaching pseudogene status are discussed. (C) 2002 Elsevier Science Ltd. All rights reserved.

It has become increasingly clear over the course of the past decade that the immune system genes of teleosts and tetrapods are plainly derived from common ancestral genes. The last 5 years, however, have also made it abundantly clear that in the teleost genome some of these genes are organized in a manner very different from that seen in mammals. These differences are probably the result of differences in life history traits, such as fecundancy, within each group of species when faced with an evolutionary fork in the road shortly after their divergence from each other. One group, the tetrapods, including mammals, chose a highly organized linked major histocompatibility complex, while in teleosts the major histocompatibility genes remained unlinked. In this review we will discuss the structural and functional implications of this different organization, particularly for major histocompatibility genes, but drawing on the current knowledge of some other genes for further support for the hypothesis that each group took a different road, one more traveled and one less taken.

Few studies have yet addressed the functional aspects of MHC molecules in fish. To lay the foundation for this, we evaluated the association between disease resistance and MHC class I and class II polymorphism in Atlantic salmon. Standardized disease challenge trials were performed on a semi-wild Atlantic salmon population with subsequent MHC typing and statistical analysis. The pathogens employed were infectious salmon anaemia virus (ISAV) causing infectious salmon anaemia and the Aeromonas salmonicida bacteria causing furunculosis. The material consisted of 1,182 Atlantic salmon from 33 families challenged with A. salmonicida and 1,031 Atlantic salmon from 25 families challenged with ISAV. We found highly significant associations between resistance towards infectious diseases caused by both pathogens and MH class I and class II polymorphism in Atlantic salmon. The observed associations were detected due to independently segregating MH class I and class II single loci, and inclusion of a large number of fish allowing an extensive statistical analysis.

Regular observation of survival of the carp breeding lines constituting a living gene bank at the Institute of Ichthyobiology and Aquaculture in Golysz (Poland) over a period of at least 15 years showed different survival rates for various lines. In this study, we have examined the polymorphism of the major histocompatibility complex (MHC) gene class II B in nine carp lines. The class II B gene encodes for the part of the MHC class II molecule which presents peptides from pathogens and protein antigens that are present in the extracellular milieu and have been taken up into the endocytic vesicles of antigen-presenting cells. Polymerase chain reaction was used to amplify Cyca-DAB gene fragments comprising part of exon 1, complete intron 1 and almost complete exon 2. Exon 2 encodes for the beta(1) domain which is the most polymorphic fragment of MHC class II molecules. Single-strand conformational polymorphism (SSCP) was applied to detect different MHC class II B haplotypes. The analysis revealed the presence of seven different haplotypes occurring with various frequencies. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

A cDNA library screening using the conserved exon 4 of Atlantic salmon Mhc class I as probe provided the basis for a study on Mhc class I polymorphism in a breeding population. Twelve different alleles were identified in the 82 dams and sires studied. No individual expressed more than two alleles, which corresponded to the diploid segregation patterns of the polymorphic marker residing within the 3'-untranslated tail. Close linkage between the Sasa-UBA and Sasa-TAP2B loci strengthens the claim that Sasa-UBA is the major Mhc class I locus in Atlantic salmon. We found no evidence for a second expressed classical or non-classical Mhc class I locus in Atlantic salmon. A phylogenetic analysis of salmonid Mhc class I sequences showed domains conserved between rainbow trout, brown trout and Atlantic salmon. Evidence for shuffling of the alpha(1) domain was identified and lineages of the remaining alpha(2) through the cytoplasmic tail gene segment can be defined. The coding sequence of one allele was found associated with two different markers, suggesting recombination within the 3'-tail dinucleotide repeat itself. Protein modelling of several Sasa-UBA alleles shows distinct differences in their peptide binding domains and enables a further understanding of the functionality of the high polymorphism.

Studies of the innate immune system have recently shown that, in addition to its role in producing the primary response that slows down pathogens, it may also play an important role in initiating and directing the type of response that the adaptive immune system makes. These discoveries have shown a complex web of control containing new roles for the innate immune system in organizing responses of T-cell to antigens being presented by major histocompatibility receptors, as well as new roles for those receptors in innate immune responses. Both of these activities are managed through feedback networks involving elements of both the innate and adaptive immune system. This paper will discuss these newly discovered interactions and how they are influencing current theories regarding the initiation of adaptive immune responses. In particular, it will highlight the recent progress that is being made towards understanding these relationships in the immune systems of teleost fish.

The mouse Lsh/Ity/Bcg locus regulates natural resistance to intracellular pathogens, and the Nramp1 gene was isolated as its candidate. Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. In the present study, a full-length cDNA for carp NRAMP has been isolated and characterized. Nucleotide and predicted amino acid sequence analysis indicate that the carp NRAMP encodes a 548 amino acid membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionarily conserved consensus transport motif. The peptide sequence identity among carp and human NRAMP2 is 78%, and 65% with human NRAMP1. Reverse transcription-polymerase chain reaction revealed that carp NRAMP is ubiquitously expressed. Phylogenetic analysis, using neighbor-joining, showed that the carp NRAMP protein clustered together with mammalian NRAMP2 proteins.

Characterization of beta2-microglobulin in a primitive fish, the Siberian sturgeon (Acipenser baeri)

Modulation of MHC class II β chain expression on head kidney macrophages has been demonstrated. Followingin vitrostimulation for 24 h, 48 h or 72 h with human recombinant TNFα plus LPS increased numbers of MHC class II positive macrophages were found relative to control cells, by FACS analysis with an anti-trout MHC class II β chain serum. Northern blot analysis confirmed that increased transcription was occurring. That TNFα synergised with LPS was also clearly demonstrated using Northern blots, where TNFα induced no effect whilst LPS alone had a small stimulatory effect. A time course experiment demonstrated that 4 h post-stimulation was an optimal time to isolate macrophage RNA post stimulation with TNFα and LPS, with later times showing less clear modulation of MHC class II β expression. Lastly, a dose response experiment performed with LPS alone revealed that only small (if any) effects were apparent when using 6·25 and 12·5 μg ml−1, whereas 25 and 50 μg ml−1were equally stimulatory. The mechanisms and consequences of modulating MHC class II expression are discussed.

A rainbow trout (Oncorhynchus mykiss) cDNA fragment encoding most of the extracellular part of the MHC class II β chain (MhcOnmy-DAB1) was cloned into a bacterial expression vector. After its purification, the recombinant protein was used for the immunisation of rabbits. The resulting antiserum, which demonstrated specific reactivity with the recombinant Onmy-DAB product, recognised a protein band of 34 kDa apparent molecular weight in western blots of SDS-PAGE-separated cell membrane preparations from spleen of pronephros, but not from muscle tissue. Proteins with similar characteristics in SDS-PAGE analysis under reducing conditions could be immunoprecipitated with the antiserum from trout peripheral blood leukocyte cell membranes. Under non-reducing conditions the precipitate was shown to run with an apparent molecular weight of 48 kDa, whereas under reducing conditions this immunoprecipitate consisted of two protein bands. Finally, deglycosylation resulted in a decrease of 3 kDa of both protein as observed under reducing gel electrophoresis conditions, thus confirming the glyco-protein character as suggested by the analysis of the trout MHC class II α and β nucleotide gene sequences.

A unique species flock of large barbs (Barbus spp.) from Lake Tana is presented, from the level of fish stocks to molecules. Evidence is given for the species status of 14 morphotypes of large barbs. They distinctly differ in: (1) head and body morphometrics, (2) food preferences, (3) distribution patterns, (4) maximal body size, (5) spawning area and period, and (6) molecular genetic characters. Most types show early morphological divergence at small size. Major Histocompatibility Complex (MHC) genes, encoding cell membrane proteins involved in defence against pathogens, were found to be diagnostic for the species' genetic identity. A strong selective pressure on particular amino acid positions in the MHC protein sequence most probably arose in response to different pathogen loads from the newly invaded ecological niches after formation of Lake Tana, ca. two million years ago. Arguments for a sympatric origin of this species flock are discussed. An evolutionary scenario suggests a riverine ancestral Barbus intermedius invading Lake Tana after its formation by volcanic blocking of the Blue Nile river and its isolation by waterfalls. Specialisation for particular food types and disruptive selection on many feeding structures are hypothesized as the evolutionary drive in speciation. The causal relationship between the diversity in feeding structures and food types is explained from experiments and models. As an example, the potential food niche of three barb species is predicted from parameter values measured from a large set of feeding structures and tested against the actual gut contents. The co-occurrence of eight piscivorous barb species is unique for cyprinid fish, which lack oral jaw teeth and a stomach. The significance of this aquatic ecosystem as a multidisciplinary evolutionary laboratory and the need for a wise balance between exploitation and conservation is stressed.

Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues should be greatest during speciation, when organisms move into new environments and their MHC molecules encounter new pathogens. The isolation of MHC genes from teleost fishes, the most diverse group of vertebrates, has created possibilities for testing this hypothesis. The large barbels of Lake Tana have undergone an adaptive radiation within the last 5 million years, producing 14 morphotypes which inhabit different ecological niches within the lake. We studied the variability in class II beta chain-encoding genes of four of these morphotypes using polymerase chain reaction amplification and DNA sequencing. The sequences obtained were orthologous to four of the known class II genes from the common carp, from which barbels diverged approximately 32 million years ago. When subjected to phylogenetic analysis, the 48 sequences clustered into groups which represent allelic lineages. A comparison of nonsynonymous and synonymous substitutions between the peptide binding region codons and non-peptide binding region codons of these sequences revealed that they are under strong selective pressure.

Although Major Histocompatibility Complex (MHC) restricted antigen recognition was first described in the early years of the 1970s (Klein, 1986), it was not until 1987 that it became clear what the precise biochemical nature of this restriction was. Detailed X-ray crystallographic data of the human class I heterodimer (HLA-A2) molecule revealed the presence of a peptide inside a groove formed by alpha helices and antiparallel beta-strands of the class I MHC molecule (Bjorkman et al., 1987; Saper et al., 1991). The peptide/MHC molecule complex forms the ligand for the T cell receptor (TCR), and in this way dictates the restricted recognition of non-self peptides and self MHC molecules (Germain & Margulies, 1993). Similarly, the human MHC class II (HLA-DR1) molecules were also found to be comparably structured to MHC class I molecules (Brown et al., 1993). However, class I molecules use the a1 and a2 domains to shape the peptide binding region (PBR), whereas class II molecules utilise the a1 and â1 domains. Class I molecules can be divided into classical (class I-a) and non-classical (class I-b). Both are non-covalently associated with â2-microglobulin. The di#erences between these two types of class I molecules is that, in general, class I-a molecules are highly polymorphic, but encoded only by a few loci, whereas class I-b are oligomorphic, but are sometimes encoded by numerous loci. However, the similarity between the two types is the fact that they both are able to bind and present peptides, with some exceptions such as the FcRn molecule (Shawar et al., 1994). The three-dimensional structure of class I molecules showed that both ends of the groove are occluded, which restricts the length of the peptide that can be bound in the groove to 8–12 amino acids. The peptide can interact with six binding pockets, of which two serve as conserved anchors to bind the Nand C-terminus of the peptide. The other

Comparison of levels and patterns of genetic variation in natural populations either across loci or against neutral expectation can yield insight into locus-specific differences in the strength and direction of evolutionary forces. We used both approaches to test the hypotheses on patterns of selection on major histocompatibility (MH)-linked markers. We performed temporal analyses of class I and class IIα MH-linked markers and eight microsatellite loci in two Atlantic salmon populations in Ireland on two temporal scales: over six decades and 9 years in the rivers Burrishoole and Delphi, respectively. We also compared contemporary Burrishoole and Delphi samples with nearby populations for the same loci. On comparing patterns of temporal and spatial differentiation among classes of loci, the class IIα MH-linked marker was consistently identified as an outlier compared with patterns at the other microsatellite loci or neutral expectation. We found higher levels of temporal and spatial heterogeneity in heterozygosity (but not in allelic richness) for the class IIα MH-linked marker compared with microsatellites. Tests on both within- and among-population differentiation are consistent with directional selection acting on the class IIα-linked marker in both temporal and spatial comparisons, but only in temporal comparisons for the class I-linked marker. Our results indicate a complex pattern of selection on MH-linked markers in natural populations of Atlantic salmon. These findings highlight the importance of considering selection on MH-linked markers when using these markers for management and conservation purposes.

In cyprinids, two paralogous groups of major histocompatibility (MH) class II B genes, DAB1 and DAB3, have been reported but have not been studied in detail. In our study on MH association with immune responsiveness in common carp (Cyprinus carpio L.) we have taken a long-term approach using divergent selection for antibody production. We report the co-segregation of Cyca-DAB1-like and Cyca-DAB3-like genes with antibody response, in backcrosses to high- and low-responsive parental carp lines. We show that the presence of Cyca-DAB1-like, but not Cyca-DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp. Background genes other than Cyca-DAB genes also influenced the level of antibody response. Our data support the hypothesis of a genetic control by Cyca-DAB genes on the antibody response measured. We could not detect an association of the Cyca-DAB genes with disease resistance to the parasite Trypanoplasma borreli. Sequence information, constitutive transcription levels and our co-segregation data indicate that both paralogous Cyco-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. Previously defined differences in allelic diversity between Cyca-DAB1-like genes, especially, identify Cyca-DAB1 as the most interesting DAB gene for further study in common carp. (C) 2008 Elsevier Ltd. All rights reserved.

Peer-reviewed. This document is the Accepted Manuscript version of a Published Work that appeared in final form in Proc. R. Soc. B. To access the final edited and published work see doi: 10.1098/rspb.2006.0053, Pathogen-driven balancing selection is thought to maintain polymorphism in major histocompatibility (MH) genes. However, there have been few empirical demonstrations of selection acting on MH loci in natural populations. To determine whether natural selection on MH genes has fitness consequences for wild Atlantic salmon in natural conditions, we compared observed genotype frequencies of Atlantic salmon (Salmo salar) surviving in a river six months after their introduction as eggs with frequencies expected from parental crosses. We found significant differences between expected and observed genotype frequencies at the MH class II alpha locus, but not at a MH class I-linked microsatellite or at seven non-MH-linked microsatellite loci. We therefore conclude that selection at the MH class II alpha locus was a result of disease-mediated natural selection, rather than any demographic event. We also show that survival was associated with additive allelic effects at the MH class II alpha locus. Our results have implications for both the conservation of wild salmon stocks and the management of disease in hatchery fish. We conclude that natural or hatchery populations have the best chance of dealing with episodic and variable disease challenges if MH genetic variation is preserved both within and among populations.

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II b chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28 bp 50-UTR and a 478 bp 30-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II b chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4 h showed very little difference in the Dila-DAB expression, but after 24 h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1b (ranging from 0 to 100 ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ This work was supported by the European Commission within the project IMAQUANIM (EC contract number FOOD-CT-2005-007103).

Cells from the myeloid lineage are pluripotent. To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we phenotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synthase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish.

We compared major histocompatibility class I allelic diversity in two currently reproductively isolated Atlantic salmon (Salmo salar) populations (Irish and Norwegian) with a common postglacial origin in order to test for among-population differences in allelic composition and patterns of recombination and point mutation. We also examined the evidence for adaptive molecular divergence at this locus by analyzing the rate of amino acid replacement in relation to a neutral expectation. Contrary to our prediction, and in contrast to the situation for other genetic markers, the two populations have almost nonoverlapping sets of major histocompatibility class I alleles. Although there is a strong signal of point mutation that predates population divergence, recent recombination, acting in similar, but not identical, ways in both populations appears to be a significant force in creating new alleles. Moreover, selection acting on peptide-binding residues seems to favor new recombinant alleles and is likely to be responsible for the rapid divergence between populations. © Society for Molecular Biology and Evolution

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.

The 16 African 'large' barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3' UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.

The mammalian CXC chemokine system comprises 16 ligands and six receptors, and its actions stretch well beyond the immune system. Recent elucidation of the pufferfish genome, a representative of an evolutionary ancient vertebrate class, has enabled analysis of the mammalian CXC chemokine system in a phylogenetic context. Comparison of the phylogenies of vertebrate CXC chemokines revealed that fish and mammals have found different solutions to similar problems, grafted on the same basic structural motif. Phylogenetic analyses showed that the large, highly redundant CXC chemokine family is a very recent phenomenon that is exclusive to higher vertebrates. Moreover, its ancestral role is found within the central nervous system and not within the immune system.

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.

Here we describe two rainbow trout major histocompatibility complex (MHC) class I genes characterized from lambda phage genomic clones prepared from a single fish. Clone GC71 contains all exons except a leader peptide-encoding exon. An open reading frame is maintained, and thus the gene MhcOnmy-U71 could be expressed in this individual. The class I gene found on clone GC41 lacks exons encoding the leader peptide and cytoplasmic domain. This gene, MhcOnmy-U41p, is a pseudogene due to a deletion in the alpha(2) domain-encoding exon causing premature termination. Both the Onmy-U71 and Onmy-U41p genes are distinguished by long introns between the exons encoding the alpha(1) and alpha(2) domains. Clone GC41 also contains the 3' exons of the LMP7/ PSMB8 gene encoding the gamma-interferon-induced proteosome subunit of rainbow trout.

Sequence-based typing of a breeding population (G1) consisting of 84 Atlantic salmon individuals revealed the presence of 7 Sasa-DAA and 7 Sasa-DAB expressed alleles. Subsequent typing of 1,182 individuals belonging to 33 families showed that Sasa-DAA and Sasa-DAB segregate as haplotypes. In total seven unique haplotypes were established, with frequencies in the population studied ranging from 0.01 to 0.49. Each haplotype is characterized by a unique minisatellite marker size embedded in the 3' untranslated region of the Sasa-DAA gene. These data corroborate the fact that Atlantic salmon express a single class II locus, consisting of tightly linked class II A and class B genes. The seven haplotypes give rise to 15 genotypes with frequencies varying between 0.01 and 0.23; 21 class II homozygous individuals were present in the G1 population. We also studied the frequency distribution in another breeding population (G4, n=374) using the minisatellite marker. Only one new marker size was present, suggesting the presence of one new class II haplotype. The marker frequency distribution in the G4 population differed markedly from the G1 population. The genomic organizations of two Sasa-DAA and Sasa-DAB alleles were determined, and supported the notion that these alleles belong to the same locus. In contrast to other studies of salmonid class II sequences, phylogenetic analyses of brown trout and Atlantic class II A and class II B sequences provided support for trans-species polymorphism.

The coding sequence of the sea bass light chain was obtained by sequential anchored PCR on a head kidney cDNA library of a DNP(494)-KLH immunised sea bass. The cDNA sequence obtained codes for a leader peptide of 21aa and a mature IgL chain of 223aa. Both the amino acid sequence comparisons and neighbour-joining trees showed that the IgL chain of sea bass obtained is of the L1/G type. To study the variability of the light chain, additional PCRs on the cDNA library and cDNA from pooled head kidneys were performed. Multiple alignment of unique sequences (N=17) could be performed without introducing gaps, and showed extremely low variability in CDR1, and no variability in CDR2 or CDR3. A possible explanation for this low variability of the IgL1 chain might be the enhanced expression of monospecific anti-DNP antibodies. The isolation and characterisation of partial genomic and cDNA IgL sequences, which showed normal variability, corroborate this explanation.

The intron-exon organisation of the carp IL-1beta gene consists of 2455bp and comprises seven exons. Three IL-1beta RNA transcripts have been found in carp: (1) a fully spliced product; (2) exon 1-7 with introns 5 and 6; and (3) exon 1-7 with intron 5 only. The intron-containing products probably represent partially spliced transcripts. IL-1beta mRNA expression in carp was semi-quantitatively analysed by RT-PCR in multiple organs, including brain and pituitary. Constitutive expression of the IL-1beta mRNA was found in these organs with a predominant expression in the immune organs head kidney and spleen. Furthermore, a scattered distribution of IL-1beta producing cells was shown by in situ hybridisations of head kidney tissue. Administration of phorbol-myristate-acetate (PMA), lipopolysaccharide (LPS) or retinoic acid (RA), to phagocytes isolated from the head kidney, resulted in expression of IL-1beta intron-containing transcripts. Of these, only PMA and LPS were stimulators that induced the fully spliced transcript. A role for the nuclear factor (NF)-kappaB pathway in carp IL-1beta expression was shown with suppression of the LPS-induced IL-1beta expression by NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC). Cortisol was able to inhibit in vitro constitutive expression of IL-1beta transcripts. Addition of cortisol simultaneously with LPS could not substantially inhibit transcription.

In this study the characterisation of the Atlantic salmon (MhcSasa-DAA) and rainbow trout (MhcOnmy-DAA) class II alpha chain cDNA sequences is presented. The DAA sequences from these two salmonid species showed a high degree of similarity, although the Onmy-DAA(*)03 cDNA sequence differed in the cytoplasmic region. Interestingly, the Onmy-DAA(*)02 sequence has lost the second cysteine in the alpha-1 domain. However, another cysteine is present in this sequence 7 positions downstream of the cysteine which is substituted for a leucine. Despite a thorough search, only a single locus of expressed class II alpha chain sequences was identified in both salmonid species. Amplification by PCR and sequencing of the alpha-1 domain from genomic DNA of three Atlantic salmon, identified four different variants assumed to have derived from this single locus. Two of these variants originated from one individual and are likely functional alleles.

Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UAC32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the alpha 3 domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2-6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14-16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene.

Summary: The first teleostean MHC sequences were described for carp. Subsequent studies in a number of cyprinid fishes showed that the class I sequences of these fishes are of particular interest. Two distinct lineages (Cyca-Z and Cyca-U) are found in the common and ginbuna crucian carp, but only the U lineage is present in zebrafish and other non-cyprinid species. The presence of the Z lineage is hypothesised to be the result of an allotetraploidisation event. Both phylogenetic analyses and amino acid sequence comparisons suggest that Cyca-Z sequences are non-classical class I sequences, probably similar to CD I. The comprehensive phylogenetic analyses of these sequences revealed different phylogenetic histories of the exons encoding the extracellular domains. The MHC genes were studied in laboratory and natural models. The natural model addressed the evolution of MHC genes in a Barbus species flock. Sequence analysis of class I and class II supported the species designation of the morphotypes present in the lake, and as a consequence the trans-species hypothesis of MHC polymorphism. The laboratory model involves the generation of gynogenetic clones, which can be divergently selected for traits such as high and low antibody response. The role of MHC molecules can be investigated further by producing a panel of isogenic lines.

Despite impressive progress in fish farming during the last years, the impact of fish diseases is still an important problem in aquaculture. Current methods to control diseases consist, among others, of medication or eradication of diseased animals in combination with disinfection of facilities. Another alternative approach is to prevent diseases by improving the immune capacity of fish by vaccination or genetic selection. It is known from studies in mammals, that a limited number of major genes, including the major histocompatibility complex (MHC), is involved in the regulation of the immune response. The existence of a classic MHC in fish is still a matter of debate. Recently, the dogma of the MHC being a single chromosomal region was challenged by investigations into the segregation of MHC genes in fish and other vertebrates. The observed absence of linkage between class I and class II major histocompatibility genes in fish would allow for an independent segregation of immunological traits associated with cytotoxic responses to virus infection (class I) or humoral responses to bacteria (class II). This could be an explanation why in challenge experiments the resistance to one disease is not necessarily correlated with resistance to another disease.

The study of the genetic regulation of infectious disease resistance depends on the availability of inbred lines or selection lines of the species under investigation. The small numbers of such lines of fish has limited the strategy in teleosts to studies of associations between disease and immune/health traits. Attempts to correlate genetic differences in immune responsiveness with survival after experimental challenge with pathogenic bacteria have failed to define immune parameters that can substantially aid selection for genetic resistance to infectious diseases. Advantages and disadvantages of selection strategies as illustrated by mouse and chicken models are discussed. In this study we summarize the present situation in fish as well as our attempts to develop gynogenetic lines of carp for immunogenetic research.

Summary Antibody production to dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) served as the immune parameter to divergently select carp (Cyprinus carpio L.) to produce high- and low-responder F, hybrid lines. Antibody production to trinitrophenyl-lipopolysaccharide [TNP-LPS) and to DNP-KLH were similar in magnitude. By contrast, some high-responder lines were low responders to DNP-human serum albumin, and vice versa. Low-responder carp were relatively susceptible to infection with the parasite Trypanoplasma borreli. This suggested that at least one gene with a major influence on resistance differed between the two homozygous parents (69, 85) used to generate the high- and low-responder homozygous families, respectively. The isogenic lines showed no within-line variation in DNA fingerprints, but differed with respect to their MhcCyca-DAB genes.

This paper reports on the selection of individual carp with a high or low antibody response, in combination with reproduction by gynogenesis, in order to develop well-characterised inbred carp lines consisting of practically unlimited numbers of carp with the same genotype. Two homozygous progenies, previously characterised as having a high or low immune response to dinitrophenyl keyhole limpet haemocyanin (DNP-KLH), were immunised with either a T-dependent (DNP-human serum albumin (DNP-HSA)) or T-independent (trinitrophenyl lipopolysaccharide (TNP-LPS)) hapten-carrier complex. In comparison with the antibody response after DNP-KLH immunisation, the response to DNP-HSA was observed to be highly variable and did not differ between the divergently selected progenies. This suggests that the divergent selection for antibody production to DNP-KLH has been carrier-specific. Immunisation with T-independent TNP-LPS induced a very rapid response which differed between the high and low responders, and likely measured changes in the DNP-specific precursor pool of B cells caused by the selection. A number of selected individuals with a high immune response to DNP-KLH were infected with Trypanoplasma borreli, a haemoflagellate parasite of carp, to examine a possible relationship between the increase in immune responsiveness and disease resistance, but no change could be detected. However, individual homozygous carp were able to escape inbreeding depression and survive the infection. Such carp would be likely candidates for gynogenetic reproduction to obtain viable inbred carp lines.

Carp with an early/high or a late/low antibody response were reproduced gynogenetically, resulting in homozygous progenies. After an initial test of their immune responsiveness at 6 months of age, we chose to continue the selection procedure with high responder progeny No. 69 and low responder progeny No. 85. The antibody response of these groups, at 12 months of age, was shown to be consistent with the parental responder type. The realized heritability (h 2 ) was estimated at 0.29. Progeny No. 69 showed a higher antibody response than progeny No. 85, both at 21 and at 28 days after immunization with dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH). The antibody response in progeny No. 85 did not significantly increase after 21 days, in accordance with this group's classification as late/low responders. Furthermore, a significantly lower percentage (11%) of the animals of this group were early/high responders, compared with progeny No. 69 (45%). Late/low responders, however, were found in almost equal numbers in both progenies. The possibility to reproduce both females and functional males with a high or a low antibody response, will result in standardized fish lines that might be important for experimental studies on the immunogenetics of disease resistance.

The letter by Denis Shields [1] comments on our article on the molecular evolution of CXC chemokines and their receptors [2], in which we hypothesized that the chemokines originate from the central nervous system (CNS). Shield’s comments hinged mainly on the evolutionary analyses. Clearly, inferences drawn from phylogenetic analyses do have their limitations. In the initial stages of the analyses, we constructed phylogenetic trees using an outgroup for both receptor and ligands. Although in general outgroups are chosen arbitrarily, we constructed a CXC receptor (CXCR) tree with CC chemokine receptors (CCRs) as an outgroup. The topology did not change from what was presented, except for CXCR6, which was located in the CCR cluster, close to the starting point of cluster formation but with low bootstrap values. In general, the tips of a phylogenetic tree are well resolved, whereas the deeper nodes are poorly resolved. ‘Supertrees’, which in theory could combine different types of information, and not merely DNA or protein sequence information, might be an alternative for studying long evolutionary histories [3]. We adopted the philosophy of a ‘supertree’ approach by substantiating our hypothesis using not only phylogenetic analyses but also other information, such as protein function, several CXC ligands (CXCL) sharing the same receptor, the presence of the ELR (glutamic acid, leucine, arginine) motif, and chromosomal localizations. Shields also argues that the absence of CXCR4 in Ciona favours a primordial immunological function of the CXC family. However, in this respect, the presence of CXCR4 in jawless fish is more important. Kuroda et al. [4] sequenced 9312 cDNA clones from a lamprey (Petromyzon marinus). The serendipitous finding of only CXCR4 could be a testimony to its ancestral status. The lamprey CXCR4 receptors cluster, with a high bootstrap value, with all other CXCR4 found in jawed fish, amphibia, birds and mammals. In addition, the architecture and sophistication of the CNS of Ciona differ markedly compared with that found in other more advanced chordates [5]. Although using a small number of amino acids to construct a phylogeny can lead to incorrect topologies, this can be overcome by the bootstrap test [6], which we used extensively to identify statistically reliable orthologues. The use of complete sequences is preferred to detect true orthologues, although when only incomplete sequences are available, the phylogenetic analyses can be performed in two ways, using pair-wise deletion or complete deletion of missing information. We performed both, and the results were essentially identical topologies with only minor deviations, notably the position of the Xenopus CXCR3 and rainbow trout CXCR. Phylogenetic trees are a hypothetical model for the evolutionary history of a gene family. A gene conversion event might result in two paralogous genes clustering closer together in trees than in reality. Gene conversions occur relatively frequently; Shields [7] observed that 45% of a control group of clustered genes showed evidence of gene conversion. However, this should not preclude phylogenetic analyses; we should merely be aware of the possibility of gene conversion imposing a bias on the analyses. The gene conversion events between the clustered CXCR1 and CXCR2 sequences analysed by Shields [7] using human, rabbit and rat sequences probably occurred between ,90 and 110 million years ago [8]. The evolutionary scale on which these gene conversions could bias the phylogenetic tree is therefore limited to the clustering of CXCR1 and CXCR2 in mammals. We believe that putative gene conversion events do not interfere with our conclusions and hypotheses derived from phylogenetic analyses on a larger, vertebrate-wide scale that comprises more CXCRs. Shields also touches upon the controversy surrounding genome duplication. Some studies favour whole-genome duplications, whereas others point to accumulated duplication of blocks or single chromosomes. Most data supporting whole genome duplications are derived from analyses of either the major histocompatibility complex (MHC) [9] or the Hox [10] gene clusters. This issue is still unresolved. Following either mode of duplication, it is apparent that extensive reshuffling and loss of genes or gene regions has occurred. This obscures the reconstruction of events occurring during the ‘birth’ of genomes [11]. Our hypotheses do not refute either the whole-genome duplication or the block duplication hypothesis. Regardless of the pitfalls associated with phylogenetic analyses, this approach has the potential to unravel the evolutionary history of the CXCL/CXCR multigene families. Our opinion, based on a combination of these analyses and additional data, is an attempt to set these gene families in a compelling evolutionary perspective. Corresponding author: B.M. Lidy Verburg-van Kemenade (lidy.vankemenade@ wur.nl). Update TRENDS in Immunology Vol.24 No.7 July 2003 356

Summary A base population (n = 101) of carp, consisting of a single hybrid cross, was immunized with the hapten-carrier complex DNP-KLH. to perform a divergent selection for antibody response. Measurement of the DNP-specific antibody response at 12 and 21 days postimmunization, allowed the classification of a low number of individual carp as early/high (10%) or late/low (13%) responders. Three individuals defined as early/high and three defined as late/low responding, were gynogenetically reproduced to obtain corresponding homozygous progenies within one generation only. Upon immunization with DNP-KLH, the antibody response was found to be significantly higher in the early/high responder homozygous offspring. Although the homozygosity of the offspring apparently caused a (s)lower antibody response (compared with the base population), the differences between the high and low responder offspring do indicate a genetic influence on the antibody response. The realized heritability (h2) for antibody production was estimated at 0.37 ± 0.36. The present study provides the basis for a divergent selection of homozygous inbred carp lines with a genetically controlled difference in antibody response. These inbred lines will allow us to investigate relationship(s) between immune responsiveness and resistance to infectious diseases in fish.