Experimental manipulation of population-level MHC diversity controls pathogen virulence evolution in Mus musculus.

The virulence levels attained by serial passage of pathogens through similar host genotypes are much higher than observed in natural systems; however, it is unknown what keeps natural virulence levels below these empirically demonstrated maximum levels. One hypothesis suggests that host diversity impedes pathogen virulence, because adaptation to one host genotype carries trade-offs in the ability to replicate and cause disease in other host genotypes. To test this hypothesis, with the simplest level of population diversity within the loci of the major histocompatibility complex (MHC), we serially passaged Friend virus complex (FVC) through two rounds, in hosts with either the same MHC genotypes (pure passage) or hosts with different MHC genotypes (alternated passage). Alternated passages showed a significant overall reduction in viral titre (31%) and virulence (54%) when compared to pure passages. Furthermore, a resistant host genotype initially dominated any effects due to MHC diversity; however, when FVC was allowed to adapt to the resistant host genotype, predicted MHC effects emerged; that is, alternated lines show reduced virulence. These data indicate serial exposure to diverse MHC genotypes is an impediment to pathogen adaptation, suggesting genetic variation at MHC loci is important for limiting virulence in a rapidly evolving pathogen and supports negative frequency-dependent selection as a force maintaining MHC diversity in host populations.

Protein pheromone expression levels predict and respond to the formation of social dominance networks.

Communication signals are key regulators of social networks and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odours of dominants and nondominants differentially influence behaviour, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analysing expression levels before, during and after competition for reproductive resources over 3 days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species.

Experimental viral evolution reveals major histocompatibility complex polymorphisms as the primary host factors controlling pathogen adaptation and virulence.

Using an experimental evolution approach, we recently demonstrated that the mouse-specific pathogen Friend virus (FV) complex adapted to specific major histocompatibility complex (MHC) genotypes, which resulted in fitness tradeoffs when viruses were exposed to hosts possessing novel MHC polymorphisms. Here we report the analysis of patterns of pathogen adaptation and virulence evolution from viruses adapting to one of three hosts that differ across the entire genome (A/WySn, DBA/2J and BALB/c). We found that serial passage of FV complex through these mouse genotypes resulted in significant increases in pathogen fitness (156-fold) and virulence (11-fold). Adaptive responses by post-passage viruses also resulted in host-genotype-specific patterns of adaptation. To evaluate the relative importance of MHC versus non-MHC polymorphisms as factors influencing pathogen adaptation and virulence, we compared the magnitude of fitness tradeoffs incurred by post-passage viruses when infecting hosts possessing either novel MHC polymorphisms alone or hosts possessing novel MHC and non-MHC polymorphisms. MHC polymorphisms alone accounted for 71% and 83% of the total observed reductions in viral fitness and virulence in unfamiliar host genotypes, respectively. Strikingly, these data suggest that genetic polymorphisms within the MHC, a gene region representing only -0.1% of the genome, are major host factors influencing pathogen adaptation and virulence evolution.

Cooperative display and relatedness among males in a lek-mating bird.

Long-tailed manakins mate in leks and cooperate in multiyear male-male partnerships. An alpha male is responsible for virtually all mating, whereas a beta male assists in the courtship displays. Such altruism by the beta male poses a problem for evolutionary theory because most theoretical treatments and empirical examples of cooperative behavior involve kin selection or reciprocity. Here it is shown that alpha and beta partners are not relatives and that reciprocity is not involved. Instead, direct, though long-delayed benefits to beta males are demonstrated, which include rare copulations, ascension to alpha status, and female lek fidelity. These benefits maintain this unusual form of male-male cooperation.

Experimental infection magnifies inbreeding depression in house mice.

It is often assumed that inbreeding reduces resistance to pathogens, yet there are few experimental tests of this idea in vertebrates, and no tests for the effects of moderate levels of inbreeding more commonly found in nature. We mated wild-derived mice with siblings or first cousins and compared the resistance of their offspring to Salmonella infection with outbred controls under laboratory and seminatural conditions. In the laboratory, full-sib inbreeding reduced resistance to Salmonella and survivorship, whereas first-cousin inbreeding had no detectable effects. In competitive population enclosures, we found that first-cousin inbreeding reduced male fitness by 57% in infected vs. only 34% in noninfected control populations. Our study provides experimental evidence that inbreeding reduces resistance and ability to survive pathogenic infection, and moreover, it shows that even moderate inbreeding can cause significant fitness declines under naturalistic conditions of social stress, and especially with exposure to infectious agents.

Evolution of MHC genetic diversity: a tale of incest, pestilence and sexual preference.

Evidence from the house mouse (Mus) suggests that the extreme diversity of genes of the major histocompatibility complex (MHC) results from three different forms of selection involving infectious disease (pestilence), inbreeding (incest) and MHC-based mating (sexual) preferences. MHC-based disassortative mating preferences are presumed to have evolved because they reduce homozygosity throughout the genome, and particularly within loci linked to the MHC. Progeny derived from such disassortative matings would enjoy increased fitness because of both reduced levels of inbreeding depression and increased resistance to infectious disease arising from their increased MHC heterozygosity.

The nature of selection on the major histocompatibility complex.

Only natural selection can account for the extreme genetic diversity of genes of the major histocompatibility complex (MHC). Although the structure and function of classic MHC genes is well understood at the molecular and cellular levels, there is controversy about how MHC diversity is selectively maintained. The diversifying selection can be driven by pathogen interactions and inbreeding avoidance mechanisms. Pathogen-driven selection can maintain MHC polymorphism based on heterozygote advantage or frequency-dependent selection due to pathogen evasion of MHC-dependent immune recognition. Empirical evidence demonstrates that specific MHC haplotypes are resistant to certain infectious agents, while susceptible to others. These data are consistent with both heterozygote advantage and frequency-dependent models. Additional research is needed to discriminate between these mechanisms. Infectious agents can precipitate autoimmunity and can potentially contribute to MHC diversity through molecular mimicry and by favoring immunodominance. MHC-dependent abortion and mate choice, based on olfaction, can also maintain MHC diversity and probably functions both to avoid genome-wide inbreeding and produce MHC-heterozygous offspring with increased immune responsiveness. Although this diverse set of hypotheses are often treated as competing alternatives, we believe that they all fit into a coherent, internally consistent thesis. It is likely that at least in some species, all of these mechanisms operate, leading to the extreme diversification found in MHC genes.

Size and fluctuating asymmetry of morphometric characters in mice: their associations with inbreeding and t-haplotype.

Fluctuating asymmetry (FA), a ubiquitous type of asymmetry of bilateral characters, often has been used as a measure of developmental instability in populations. FA is expected to increase in populations subjected to genetic stressors such as inbreeding or environmental stressors such as toxins or parasites, although results have not always been consistent. We tested whether FA in four skeletal size characters and mandible shape was greater in a population of wild-derived mice reared in the laboratory and subjected to one generation of inbreeding (F = 0.25) versus that in an outbred group (F = 0.00). FA did not significantly differ between the inbred and outbred groups, despite the fact that these two groups differed dramatically in fitness under seminatural population conditions. As far as we know, this is the first study to evaluate the relationship between FA and inbreeding in wild house mice, and our general conclusion is opposite that of earlier work on laboratory inbred strains of mice and their hybrids. Size for two of the characters was significantly less in inbreds than in outbreds, however, and there was a significant difference between inbreds and outbreds in the signed differences of right and left sides in one character (humerus length). Some of the mice in both groups also were heterozygous or homozygous carriers of the t-complex. Because mice carrying this chromosome 17 variant are known to have reduced fitness, we also tested whether they had greater FA than mice carrying non-t-haplotypes. The overall level of a composite FA index calculated from all four characters was in fact significantly higher in the t-bearing mice. These combined results suggest that FA is not a generally sensitive proxy measure for fitness, but can be associated with fitness reductions for certain genetic stressors.

Accumulated background variation among H2 mutant congenic strains: elimination through PCR-based genotyping of F2 segregants.

Many commercially and privately available congenic strains of laboratory animals were founded decades ago and are likely to differ from one another by dozens of fixed mutational differences at background loci. This problem is often ignored despite growing evidence that such background variation exists. Eliminating this confounding variation can be largely accomplished by crossing congenic strains to produce F2 segregants that are homozygous (or heterozygous) for relevant genes. Discriminating F2 homozygotes can be difficult when strain differences are minor, as are mutant mouse strains differing at single major histocompatibility loci (H2 mutant congenics). Here, we describe a two-step polymerase chain reaction (PCR) method utilizing heteroduplex analysis and sequence specific primers (SSP-PCR) that efficiently discriminates the F2 progeny of two such H2 mutant congenic mice crosses (bm1xB6 and bm1xbm3). A third H2 mutant cross cannot be resolved by heteroduplexing, but is discriminated (albeit less efficiently) with SSP-PCR alone. This sensitive application can be extended to any congenic mutant strains.

Untrained mice discriminate MHC-determined odors.

Immune recognition occurs when foreign antigens are presented to T-lymphocytes by molecules encoded by the highly polymorphic genes of the major histocompatibility complex (MHC). House mice (Mus musculus) prefer to mate with individuals that have dissimilar MHC genes. Numerous studies indicate that mice recognize MHC identity through chemosensory cues; however, it is unclear whether odor is determined by classical, antigen-presenting MHC loci or closely linked genes. Previous studies have relied on training laboratory mice and rats to distinguish MHC-associated odors, but there are several reasons why training experiments may be inappropriate assays for testing if MHC genes affect odor. The aim of this study was to determine whether classical MHC genes affect individual odors and whether wild-derived mice can detect MHC-associated odors without training. In the first experiment, we found that wild-derived mice can be trained in a Y-maze to detect the odors of mice that differ genetically only in the MHC region. In the second and third experiments, we used a naturalistic habituation assay and found that wild-derived mice can, without training, distinguish the odors of mice that differ genetically only at one classical MHC locus (dm2 mutants).

A microsatellite-based MHC genotyping system for house mice (Mus domesticus).

Major histocompatibility complex (MHC) genes are the most polymorphic loci known for vertebrates. Although this has been known for over two decades, the selective forces maintaining this genetic diversity are unclear. Efforts to study selection on these loci in nature have been hampered because no simple MHC typing systems are available. Here, we describe and evaluate a microsatellite-based MHC genotyping system for house mice (Mus domesticus). Thirty-five MHC-linked microsatellite loci were tested for amplification and scoring reliability, and 21 were deemed useful. These 21 loci were efficient at discriminating among nine serologically distinct MHC haplotypes, with 52% of microsatellite pairs providing 100% resolution. Since these microsatellite loci are scattered across the entire MHC region, they will be effective at detecting recombinant haplotypes. The number of alleles is higher for microsatellites inside the MHC than outside it, which presumably reflects genetic hitchhiking with MHC alleles under balancing selection. This microsatellite typing system now allows testing hypotheses about the nature of selection operating on MHC genes in natural populations of M. domesticus and other murid rodent species.

Pathogen-based models favoring MHC genetic diversity.

We present six models that are currently the most likely ways that pathogens might favor the evolution of MHC genetic diversity. Although each model makes one or more unique predictions, the current lack of crucial data prevents distinguishing the relative importance of each model. However, this first-time organization of these models should contribute to the design of critical experiments. This synthetic review yields at least three essentially new ideas. First, MHC-dependent immune recognition may be sufficiently redundant to render it essentially escape-proof by pathogens. Second, the four models based on pathogen escape do not work (or work weakly) for diversifying class II genes, unless class II-restricted cytotoxic T-cells are important, an idea that is controversial. Third, pathogen-escape events have traditionally been thought to result in only frequency-dependent selection but here we show that heterozygote advantage is an inevitable consequence of such pathogen evasion. Therefore, the controversy over the relative importance of these two forms of balancing selection is largely a false dichotomy.

Evolution of diversity at the major histocompatibility complex.

Recent evidence from both population data and DNA sequence analyses indicates that the unprecedented genetic diversity found at MHC loci is selectively maintained in contemporary natural populations, although the strength and nature of this selection are currently unclear. Due to the critical role played by MHC molecules in immune recognition, it is generally assumed that some form of parasite-driven selection is operating. However, the general failure to implicate MHC in the susceptibility to specific infectious diseases has been troubling, and may indicate that selection is too weak to detect directly. Alternatively, strong selection can be reconciled by a variety of factors including the amplification of minor (disease-based) vigor differences into large fitness differences by intraspecific competition, or non-disease-based selection such as mating preferences and selective abortion.

Chemical signals and parasite-mediated sexual selection.

Research into visual and acoustic signals has demonstrated that exaggerated sexual displays often provide an honest indicator of a male's resistance to parasites. Recent studies with rodents and humans now suggest that chemosensory signals also reveal a male's disease resistance and his genetic compatibility. Our understanding of sexual selection has been greatly enriched by considering the mechanisms underlying visual and acoustic displays, and recent advances in chemical communication will help to determine what kind of information is revealed by an individual's scent.

Male-male competition magnifies inbreeding depression in wild house mice.

The detrimental effects of inbreeding on vertebrates are well documented for early stages of the life cycle in the laboratory. However, the consequences of inbreeding on long-term survival and reproductive success (Darwinian fitness) are uncertain for vertebrates in the wild. Here, we report direct experimental evidence for vertebrates that competition increases the harmful effects of inbreeding on offspring survival and reproduction. We compared the fitness of inbred (from full-sib matings) and outbred wild house mice (Mus domesticus) in large, seminatural enclosures. Inbred males sired only one-fifth as many surviving offspring as outbred males because of their poor competitive ability and survivorship. In laboratory conditions, inbreeding had relatively minor effects on male reproductive success and no effect on survivorship. Seminatural conditions did not increase inbreeding depression for females, probably because females were not competing for any critical resources. The overall reduction in fitness from inbreeding was 57%, which is 4.5 times as great as previous estimates from the laboratory. These results have important implications for medicine, conservation, evolutionary biology, and functional genomics.

Mating patterns in seminatural populations of mice influenced by MHC genotype.

Because of the central role of major histocompatibility complex (MHC) genes in immune recognition, it is often assumed that parasite-driven selection maintains the unprecendented genetic diversity of these genes. But associations between MHC genotype and specific infectious diseases have been difficult to identify with a few exceptions such as Marek's disease and malaria. Alternatively, MHC-related reproductive mechanisms such as selective abortion and mating preferences could be responsible for the diversity. To determine both the nature and strength of selection operating on MHC genes by we have studied components of selection in seminatural populations of mice (Mus musculus domesticus). Here we assess MHC-related patterns of reproduction and early (preweaning) mortality by analysing 1,139 progeny born in nine populations, and 662 progeny from laboratory matings. Reproductive mechanisms, primarily mating preferences, result in 27% fewer MHC-homozygous offspring than expected from random mating. MHC genotype had no detectable influence on neonatal (preweaning) mortality. These mating preferences are strong enough to account for most of the MHC genetic diversity found in natural populations of Mus.

Communal nesting patterns in mice implicate MHC genes in kin recognition.

House mice (Mus musculus domesticus) form communal nests and appear to nurse each other's pups indiscriminately. Communal nesting probably functions to reduce infanticide, but it also makes females vulnerable to exploitation if nursing partners fail to provide their fair share of care. Kinship theory predicts that females will preferentially form communal nests with relatives to minimize exploitation and further increase inclusive fitness. Here we provide evidence from seminatural populations that females prefer communal nesting partners that share allelic forms of major histocompatibility complex genes. Such behaviour would lead to the selection of close relatives as communal nesting partners. Although criteria for the demonstration of kin recognition are currently embroiled in controversy, this is the first vertebrate study to meet Grafen's restrictive requirements: discrimination is based on genetic similarity at highly polymorphic loci, incidental correlations due to relatedness are experimentally controlled, and strong reasons exist for expecting the assayed behaviour to be kin-selected.

The role of infectious disease, inbreeding and mating preferences in maintaining MHC genetic diversity: an experimental test.

In house mice, and probably most mammals, major histocompatibility complex (MHC) gene products influence both immune recognition and individual odours in an allele-specific fashion. Although it is generally assumed that some form of pathogen-driven balancing selection is responsible for the unprecedented genetic diversity of MHC genes, the MHC-based mating preferences observed in house mice are sufficient to account for the genetic diversity of MHC genes found in this and other vertebrates. These MHC disassortative mating preferences are completely consistent with the conventional view that pathogen-driven MHC heterozygote advantage operates on MHC genes. This is because such matings preferentially produce MHC-heterozygours progeny, which could enjoy enhanced disease resistance. However, such matings could also function to avoid genome-wide inbreeding. To discriminate between these two hypotheses we measured the fitness consequences of both experimentally manipulated levels of inbreeding and MHC homozygosity and heterozygosity in semi-natural populations of wild-derived house mice. We were able to measure a fitness decline associated with inbreeding, but were unable to detect fitness declines associated with MHC homozygosity. These data suggest that inbreeding avoidance may be the most important function of MHC-based mating preferences and therefore the fundamental selective force diversifying MHC genes in species with such mating patterns. Although controversial, this conclusion is consistent with the majority of the data from the inbreeding and immunological literature.

Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds.

To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution.