Åsa Langefors

Association between major histocompatibility complex class IIB alleles and resistance to Aeromonas salmonicida in Atlantic salmon

We have tested the importance of genetic variation in the major histocompatibility complex (MHC) class IIB in Atlantic salmon (Salmo salar) for survival after challenge with a highly virulent bacterial pathogen. Forty juvenile full siblings from each of 120 families were infected with the bacterium Aeromonas salmonicida, which causes high mortality in salmon due to furunculosis. Fishes from high–resistance (HR, < 35% mortality) and low–resistance (LR, > 80% mortality) families were screened for their MHC class IIB genotypes using the denaturing gradient gel electrophoresis (DGGE) technique. The exon 2 sequences, encoding the major part of the peptide–binding region, were established for each DGGE fragment. One allele, e, containing a missense single base substitution was significantly more prevalent in HR families than in LR families. An odds–ratio test showed that broods carrying this allele had a 12–fold higher chance of being HR than broods without the e allele. A second allele, i, showed significantly higher frequencies in uninfected and surviving individuals than in infected dead individuals. A third allele, j, tended to be more prevalent both in LR families and in individuals that had died of the infection. There was no correlation between MHC heterozygosity and resistance to A. salmonicida. Our results support the hypothesis that MHC polymorphism is maintained through pathogen–driven selection acting by means of frequency–dependent selection rather than heterozygous advantage.

MHC and kin discrimination in juvenile Arctic charr, Salvelinus alpinus (L.)

Kin recognition and discrimination are thought to occur in several species of various taxonomic groups. In salmonid fish, juveniles can discriminate between odours of siblings and nonsiblings from the same population even if the odour donors and the test fish have been reared separately since fertilization. This indicates that some genetic factor is important in the recognition process. The mechanisms behind kin recognition and discrimination have not yet been described. In the present study, we performed fluviarium tests to examine whether kin recognition and discrimination in juvenile Arctic charr are influenced by the major histocompatibility complex (MHC). Prior to the fluviarium tests, exon 2 of an MHC class II B gene in charr was analysed with denaturing gradient gel electrophoresis (DGGE) and individual genotypes were determined. In the fluviarium, when fish had the choice between water scented by an MHC identical sibling and a sibling with a different MHC genotype they preferred water from identical siblings. Moreover, water scented by an MHC different sibling was preferred to water from an MHC different nonsibling. However, we observed no discrimination when the test fish shared one allele with the nonsibling donor but no alleles with the sibling donor. Our results indicate that the MHC has a significant influence on the odours used for kin recognition and discrimination in juvenile Arctic charr.Copyright 1998 The Association for the Study of Animal Behaviour

Experimental evidence for major histocompatibility complex-allele-specific resistance to a bacterial infection

The extreme polymorphism found at some major histocompatibility complex (MHC) loci is believed to be maintained by balancing selection caused by infectious pathogens. Experimental support for this is inconclusive. We have studied the interaction between certain MHC alleles and the bacterium Aeromonas salmonicida, which causes the severe disease furunculosis, in Atlantic salmon (Salmo salar L.). We designed full–sibling broods consisting of combinations of homozygote and heterozygote genotypes with respect to resistance or susceptibility alleles. The juveniles were experimentally infected with A. salmonicida and their individual survival was monitored. By comparing full siblings carrying different MHC genotypes the effects on survival due to other segregating genes were minimized. We show that a pathogen has the potential to cause very intense selection pressure on particular MHC alleles; the relative fitness difference between individuals carrying different MHC alleles was as high as 0.5. A co–dominant pattern of disease resistance/susceptibility was found, indicative of qualitative difference in the immune response between individuals carrying the high– and low–resistance alleles. Rather unexpectedly, survival was not higher among heterozygous individuals as compared with homozygous ones.

Stress, immunocompetence and leukocyte profiles of pied flycatchers in relation to brood size manipulation

The two main trade-offs considered determining reproductive patterns in iteroparous organisms are the one between current and future reproduction, and the one between the number and quality of offspring. Recently, it has been suggested that these trade-offs may be mediated by stress-induced reduction in immunocompetence. To test the hypothesis that stress reduces immune function, we investigated the effects of brood size manipulation on stress hormone levels, leukocyte profiles and immune responses against challenge with novel antigens in nestling and parent male pied flycatchers (Ficedula hypoleuca). In male parents, heterophil (H) and lymphocyte (L) numbers, as well as H/L ratio increased with experimentally enlarged brood size, and corticosterone levels tended to do so, indicating that high parental work load altered their stress level and physiological state. Despite this, we found no effects on humoral immune responsiveness, measured as antibody production against diphtheria-tetanus vaccine. In nestlings, heterophil numbers and H/L ratio increased in enlarged broods, whereas T-cell-mediated immune responsiveness, measured against phytohemagglutinin (PHA), decreased in enlarged broods. The results support the view that growth-stress-induced immunosuppression may be an important physiological pathway mediating the trade-off between the number and viability of offspring. The difference in the observed immune-related responses between nestlings and males may be because we measured different aspects of the immune system (cellular vs humoral). However, it may also be a result of males lowering their own costs by feeding less, (and their mate possibly compensate by feeding more), whereas nestlings cannot escape the costs of increased intra-brood competition.

An experimental test of the immunocompetence handicap hypothesis in a teleost fish: 11-ketotestosterone suppresses innate immunity in three-spined sticklebacks

The immunocompetence handicap hypothesis (ICHH) provides a functional explanation for how sexual ornaments can provide honest signals of male quality. A key aspect of this hypothesis is that testosterone (T) has a bimodal effect: a higher T level enhances the expression of ornaments (increasing mating success and, ultimately, fitness); however, at the same time, it suppresses immune function. Tests of the latter assumption, which have focused mainly on aspects of adaptive immunity in birds, led to equivocal results. We performed a hormone‐implant experiment in male three‐spined sticklebacks (Gasterosteus aculeatus) to test the key assumptions of the ICHH in a fish, where the dominant circulating androgen is 11‐ketotestosterone (11kT) rather than T. Males were implanted with 11‐ketoandrostenedione, which is a natural precursor of 11kT. Each individual’s circulating 11kT level, ornamentation, and immunocompetence were measured 2 weeks later. In addition, we quantified oxidative tissue damage because the ICHH has been hypothesized to work via oxidative stress. We found that the males’ 11kT levels correlated positively with ornamentation but negatively with immunocompetence, in particular, measures of innate immunity. Moreover, there was a trend for fish with high 11kT levels to suffer more from oxidative stress. Thus, our data provide support for the ICHH.

Extra-pair fertilizations in the Sedge Warbler

Parentage of 201 young (from 44 broods) in a population of Sedge Warblers Acrocephalus schoenobaenus in South Central Sweden, 1990-1992, was determined by using multilocus DNA fingerprinting. The frequency of extra-pair young (EPY) was 7.5% and they occurred in 23% of the broods. For 11 out of 15 extra-pair young we could identify the true father; in all of the cases he was a close neighbour. Among ten broods with extra-pair young, nine contained only one extra-pair sire and the remaining brood two extra-pair sires. The frequency of EPF varied among years (1.8-11.8%). The seasonal timing of broods with and without extra-pair young did not differ, and the occurrence of extra-pair fertilizations (EPF) was not related to the length of the pair males mate-guarding period. EPF were not related to breeding synchrony (estimated as the mean number of fertile females per day during a females fertile period). Pairs whose nests contained extra-pair young had more territories within 100 m of their nest than pairs without extra-pair young. Females that engaged in EPF had fewer attractive (i.e. singing) males to choose among the day before and at the day they formed their pair bend. Moreover, in all but one case the extra-pair male was not singing (i.e. not available as pair mate) the day the EPF-female settled. Males that fertilized extra-pair young tended to arrive earlier and to have higher pairing success than both males that were cuckolded and other males. Hence, female Sedge Warblers engaged in extra-pair fertilizations with attractive male neighbours. These results are consistent with the hypotheses that females participating in EPF are seeking genetic benefits to their offspring, but we cannot exclude the alternative explanation that attractive males are more efficient in forcing females to accept EPF.

Allelic variation of Mhc class II in Atlantic salmon; a population genetic analysis

We have studied the degree of genetic variation at a variable Mhc class II β gene in four populations of Atlantic salmon Salmo salar by using RFLPs. The class II β gene encodes the part of the Mhc class II molecule that contains the antigen binding region and is therefore essential for disease resistance. There was extensive genetic variation in all four populations; the expected heterozygosity (HSi) varied between 0.50 and 0.81. Heterozygosity tended to be higher in broods surviving a syndrome causing high mortalities (60–95%) in Swedish salmon hatcheries. Populations that had experienced more incidences of genetic bottlenecks (years when fewer than 60 adults had been used for breeding) and had a lower average effective population size (Ne), had a lower degree of heterozygosity. The four populations differed significantly in allele frequencies as measured by FST=0.13 and Neis genetic distance (D=0.09−∞). Pairwise FST values varied between 0.01 and 0.23, all but one being highly significant, indicating a differentiation between the populations in Mhc class II β. This study shows that the four populations of Atlantic salmon have a high degree of polymorphism in the Mhc class II β gene. However, there was great variation between different hatcheries, both in heterozygosity levels and allele frequencies.

Screening of Mhc variation in Atlantic salmon (Salmo salar): a comparison of restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE) and sequencing

We compared three different molecular methods currently used for screening of Mhc variation in population studies of Atlantic salmon. Restriction fragment length polymorphism (RFLP) of the entire class II gene detected 22 haplotypes. Seventeen exon 2 sequences were obtained from individuals carrying the 22 haplotypes, two of which had not been detected by RFLP. The six alleles (27%) detected by RFLP and not by exon 2 sequencing probably resulted from sequence variation outside exon 2. Within exon 2, RFLP differentiated 88% of the sequences. Alternatively, denaturing gradient gel electrophoresis (DGGE) performed under two run conditions detected 94% of the sequence variation. Both RFLP using different probes, and the two PCR‐based methods using three different primer pairs, suggest that there is only a single Mhc class II B gene in the Baltic populations of Atlantic salmon.

Adaptive and Neutral Genetic Variation and Colonization History of Atlantic Salmon, Salmo salar

SynopsisI combined neutral microsatellite markers with the major histocompatibility complex (MHC) class IIB to study genetic differentiation and colonization history in Atlantic salmon, Salmo salar, in the Baltic Sea and in the north-eastern Atlantic. Baltic salmon populations have lower levels of microsatellite genetic variation, in terms of heterozygosity and allelic richness than Atlantic populations, confirming earlier findings with other genetic markers, suggesting that the Baltic Sea populations have been exposed to genetic bottlenecks, most likely at a founding event. On the other hand, the level of MHC variation was similar in the Baltic and in the north-eastern Atlantic, indicating that positive balancing selection has increased the level of MHC-variation. Both microsatellite and MHC class IIB genetic variation give strong support to the hypothesis that the Baltic salmon are of a biphyletic origin, the southern population in this study is strongly differentiated from both the northern Baltic salmon populations and from the north-eastern Atlantic populations. Salmon may have colonized the northern Baltic Sea either from the south, via the so called “Närke strait” or from the north, via a proposed historical connection between the White Sea and the northern Baltic. At microsatellites, no significant isolation-by distance was found at either colonization route. At the MHC, populations were significantly isolated by distance when assuming that colonization occurred via the “Närke strait”.