R.J.M. Stet

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005

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 Göttingen, 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.

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles

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.

Natural selection acts on Atlantic salmon major histocompatibility (MH) variability in the wild

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.

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44.

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.

Patterns of variability at the major histocompatibility class II alpha locus in Atlantic salmon contrast with those at the class I locus

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.

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different 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.

Rainbow trout (Oncorhynchus mykiss) possess multiple novel immunoglobulin-like transcripts containing either an ITAM or ITIMs.

The recognition of pathogens by the innate immune system relies on a wide range of inhibitory and activating receptors. Some of these non-rearranging receptors belong to the immunoglobulin superfamily (IgSF) and in teleost fish the novel immune-type receptor (NITR) and the novel immunoglobulin-like transcript (NILT) have been reported. Here we describe the identification and characterisation of three new NILTs from rainbow trout (Oncorhynchus mykiss), with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain. The cytoplasmic regions contain either immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or an immunoreceptor tyrosine-based activating motif (ITAM) for downstream signalling. Alignment of the various NILT Ig domains revealed a high similarity, especially between Ig domains from NILTs found in this study. Furthermore, a phylogenetic tree showed that NILTs are more closely related to the triggering receptor expressed on myeloid (TREM) cells and NKp44 than to NITRs. The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues. Expression of NILTs was also shown to be an early event in development, with both eyed eggs and embryos expressing all four genes. The results obtained in this study and future experiments will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in rainbow trout.

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.)

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.

Varying disease-mediated selection at different life-history stages of Atlantic salmon in fresh water

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.

Contrasting responses to selection in class I and class IIα major histocompatibility-linked markers in salmon.

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.