Genetic variation in MHC class II expression and interactions with MHC sequence polymorphism in three-spined sticklebacks

Genes of the major histocompatibility complex (MHC) have been studied for several decades because of their pronounced allelic polymorphism. Structural allelic polymorphism is, however, not the only source of variability subjected to natural selection. Genetic variation may also exist in gene expression patterns. Here, we show that in a natural population of three-spined sticklebacks (Gasterosteus aculeatus) the expression of MHC class IIB genes was positively correlated with parasite load, which indicates increased immune activation of the MHC when infections are frequent. To experimentally study MHC expression, we used laboratory-bred sticklebacks that were exposed to three naturally occurring species of parasite. We found strong differences in MHC class IIB expression patterns among fish families, which were consistent over two generations, thus demonstrating a genetic component. The average number of MHC class IIB sequence variants within families was negatively correlated to the MHC expression level suggesting compensatory up-regulation in fish with a low (i.e. suboptimal) MHC sequence variability. The observed differences among families and the negative correlation with individual sequence diversity imply that MHC expression is evolutionary relevant for the onset and control of the immune response in natural populations.     

Multiple parasites are driving major histocompatibility complex polymorphism in the wild

Parasite mediated selection may result in arms races between host defence and parasite virulence. In particular, simultaneous infections from multiple parasite species should cause diversification (i.e. balancing selection) in resistance genes both at the population and the individual level. Here, we tested these ideas in highly polymorphic major histocompatibility complex (MHC) genes from three-spined sticklebacks (Gasterosteus aculeatus L.). In eight natural populations, parasite diversity (15 different species), and MHC class IIB diversity varied strongly between habitat types (lakes vs. rivers vs. estuaries) with lowest values in rivers. Partial correlation analysis revealed an influence of parasite diversity on MHC class IIB variation whereas general genetic diversity assessed at seven microsatellite loci was not significantly correlated with parasite diversity. Within individual fish, intermediate, rather than maximal allele numbers were associated with minimal parasite load, supporting theoretical models of self-reactive T-cell elimination. The optimal individual diversity matched those values female fish try to achieve in their offspring by mate choice. We thus present correlative evidence supporting the 'allele counting' strategy for optimizing the immunocompetence in stickleback offspring.     

Habitat-specific adaptation of immune responses of stickleback (Gasterosteus aculeatus) lake and river ecotypes

Freshwater populations of three-spined sticklebacks (Gasterosteus aculeatus) in northern Germany are found as distinct lake and river ecotypes. Adaptation to habitat-specific parasites might influence immune capabilities of stickleback ecotypes. Here, naive laboratory-bred sticklebacks from lake and river populations were exposed reciprocally to parasite environments in a lake and a river habitat. Sticklebacks exposed to lake conditions were infected with higher numbers of parasite species when compared with the river. River sticklebacks in the lake had higher parasite loads than lake sticklebacks in the same habitat. Respiratory burst, granulocyte counts and lymphocyte proliferation of head kidney leucocytes were increased in river sticklebacks exposed to lake when compared with river conditions. Although river sticklebacks exposed to lake conditions showed elevated activation of their immune system, parasites could not be diminished as effectively as by lake sticklebacks in their native habitat. River sticklebacks seem to have reduced their immune-competence potential due to lower parasite diversity in rivers.     

Innate versus adaptive immunity in sticklebacks: evidence for trade-offs from a selection experiment

In vertebrates, the immune system consists of two arms of different characteristics: the innate and the acquired immune response. Parasites that are only shortly exposed to the immune system are most efficiently attacked by fast, constitutive innate immune mechanisms. Here, we experimentally selected within four fish families for high innate resistance versus susceptibility of three-spined sticklebacks (Gasterosteus aculeatus) against infection with the eye-fluke (Diplostomum pseudospathacaeum), a parasite whose metacercariae are protected from the immune system within the eye lens. We predicted that in families with high susceptibility, the adaptive immune system would be upregulated when challenged with infection. In accordance, we found that MHC class IIB expression is increased by approximately 50% in those lines selected for higher parasite load (i.e. low innate response). This suggests extensive genetic correlations between innate and adaptive immune system and/or crosstalk between both lines of defense. An efficient, specific innate immune response might reduce overall activation of the immune system and potentially alleviate associated effects of immunopathology.     

Who is in control of the stickleback immune system: interactions between Schistocephalus solidus and its specific vertebrate host

The cestode Schistocephalus solidus is a frequent parasite of three-spined sticklebacks and has a large impact on its host's fitness. Selection pressure should therefore be high on stickleback defence mechanisms, like an efficient immune system, and also on parasite strategies to overcome these. Even though there are indications for manipulation of the immune system of its specific second intermediate host by the cestode, nothing is yet known about the chronology of specific interactions of S. solidus with the stickleback immune system. We here expected sticklebacks to first mount an innate immune response directly post-exposure to the parasite to clear the infection at an early stage and after an initial lag phase to upregulate adaptive immunity. Most interestingly, we did not find any upregulation of the specific lymphocyte-mediated immune response. Also, the pattern of activation of the innate immune system did not match our expectations: the proliferation of monocytes followed fluctuating kinetics suggesting that the parasite repeatedly installs a new surface coat not immunogenic to the host. Furthermore, the respiratory burst activity, which has the potential to clear an early S. solidus infection, was upregulated very late during infection, when the parasite was too big to be cleared but ready for transmission to its final host. We here suggest that the late activation of the innate immune system interferes with the neuroendocrine system, which mediates reduced predation avoidance behaviour and so facilitates the transmission to the final host.     

Specific Gene Expression Responses to Parasite Genotypes Reveal Redundancy of Innate Immunity in Vertebrates

Vertebrate innate immunity is the first line of defense against an invading pathogen and has long been assumed to be largely unspecific with respect to parasite/pathogen species. However, recent phenotypic evidence suggests that immunogenetic variation, i.e. allelic variability in genes associated with the immune system, results in host-parasite genotype-by-genotype interactions and thus specific innate immune responses. Immunogenetic variation is common in all vertebrate taxa and this reflects an effective immunological function in complex environments. However, the underlying variability in host gene expression patterns as response of innate immunity to within-species genetic diversity of macroparasites in vertebrates is unknown. We hypothesized that intra-specific variation among parasite genotypes must be reflected in host gene expression patterns. Here we used high-throughput RNA-sequencing to examine the effect of parasite genotypes on gene expression patterns of a vertebrate host, the three-spined stickleback (Gasterosteus aculeatus). By infecting naïve fish with distinct trematode genotypes of the species Diplostomum pseudospathaceum we show that gene activity of innate immunity in three-spined sticklebacks depended on the identity of an infecting macroparasite genotype. In addition to a suite of genes indicative for a general response against the trematode we also find parasite-strain specific gene expression, in particular in the complement system genes, despite similar infection rates of single clone treatments. The observed discrepancy between infection rates and gene expression indicates the presence of alternative pathways which execute similar functions. This suggests that the innate immune system can induce redundant responses specific to parasite genotypes.     

MHC-based mate choice combines good genes and maintenance of MHC polymorphism

Polymorphic genes of the major histocompatibility complex (MHC) are regarded as essential genes for individual fitness under conditions of natural and sexual selection. To test this hypothesis, we investigated the ultimate individual fitness trait — that of reproductive success. We used three-spined sticklebacks ( Gasterosteus aculeatus ) in seminatural enclosures, located in natural breeding areas where the experimental fish had been caught. During their reproductive period, fish were exposed continuously to their natural sympatric parasites. By genotyping almost 4000 eggs with nine microsatellites, we determined parenthood and inferred female mating decision. We found that with reference to their own MHC profile, female sticklebacks preferred to mate with males sharing an intermediate MHC diversity. In addition, males with a specific MHC haplotype were bigger and better at fighting a common parasite ( Gyrodactylus sp.). This translated directly into Darwinian fitness since fish harbouring this specific MHC haplotype were more likely to be chosen and had a higher reproductive output. We conclude that females also based their mating decision on a specific MHC haplotype conferring resistance against a common parasite. This identifies and supports 'good genes'. We argue that such an interaction between host and parasite driving assortative mating is not only a prerequisite for negative frequency-dependent selection — a potential mechanism to explain the maintenance of MHC polymorphism, but also potentially speciation.     

Disentangling the role of MHC‐dependent ‘good genes' and ‘compatible genes' in mate‐choice decisions of three‐spined sticklebacks Gasterosteus aculeatus under semi‐natural conditions

To investigate and disentangle the role of major histocompatibility complex (MHC)‐based ‘good genes' and ‘compatible genes' in mate choice, three‐spined sticklebacks Gasterosteus aculeatus with specific MHC IIB genotypes were allowed to reproduce in an outdoor enclosure system. Here, fish were protected from predators but encountered their natural parasites. Mate choice for an intermediate genetic distance between parental MHC genotypes was observed, which would result in intermediate diversity in the offspring, but no mate choice based on good genes was found under the current semi‐natural conditions. Investigation of immunological variables revealed that the less‐specific innate immune system was more active in individuals with a genetically more divergent MHC allele repertoire. This suggests the need to compensate for an MHC‐diminished T‐cell repertoire and potentially explains the observed mate choice for intermediate MHC genetic distance. The present findings support a general pattern of mate choice for intermediate MHC diversity (i.e. compatible genes). In addition, the potentially dynamic role of MHC good genes in mate choice under different parasite pressures is discussed in the light of present and previous results.     

Individual MHC class I and MHC class IIB diversities are associated with male and female reproductive traits in the three-spined stickleback

Genes of the major histocompatibility complex (MHC) are indispensable for pathogen defence in vertebrates. With wild-caught three-spined sticklebacks (Gasterosteus aculeatus) we conducted the first study to relate individual reproductive parameters to both MHC class I and II diversities. An optimal MHC class IIB diversity was found for male nest quality. However, male breeding colouration was most intense at a maximal MHC class I diversity. One MHC class I allele was associated with male redness. Similarly, one MHC class IIB allele was associated with continuous rather than early female reproduction, possibly extending the reproductive period. Both alleles occurred more frequently with increasing individual allele diversity. We suggest that if an allele is currently not part of the optimum, it had not been propagated by choosy females. The parasite against which this allele provides resistance is therefore unlikely to have been predominant the previous year - a step to negative frequency-dependent selection.     

Costly major histocompatibility complex signals produced only by reproductively active males,but not females,must be validated by a ‘maleness signal’ in three-spined sticklebacks

Olfactory information about individual major histocompatibility complex (MHC) immune genotypes is important for mate choice in several species. For example, during the mate choice decisions of three-spined sticklebacks, females assess males on the basis of odour cues that convey information about their MHC diversity. Here, we show that an additional ‘maleness’ signal is needed to validate the MHC signal. Furthermore, using interaction between natural odour of sticklebacks and synthetic MHC-ligand peptides, we show that MHC signals are conditional on the reproductive state in males. By contrast, we find that gravid females do not produce such signals. Since MHC olfactory signals relevant to mate choice decisions are conditional upon gender and reproductive state, we suggest that their manufacture is likely to be costly to senders, and therefore, potentially conditional on the health/parasitization status of the sender. We hypothesize that shedding of peptide–MHC complexes compromises immune function, selecting against unconditional use of these signals.     

Sex, parasites and resistance--an evolutionary approach

Immune systems are among the most diverse biological systems. An evolutionary arms race between hosts and rapidly evolving pathogens is supposed to be a reason for this diversity, and might explain why most eukaryotic hosts and parasites reproduce sexually. In this review, I will focus on possible benefits of sexual reproduction in hosts and parasites, using a model system consisting of a tapeworm and its two intermediate hosts, copepods and sticklebacks. We found that the hermaphroditic tapeworms can increase their infection success by reproducing sexually with a partner (outcrossing), instead of reproducing alone. The defence system of the copepods provides highly specific discrimination of antigenic characteristics of the tapeworms. This supports the finding that tapeworms benefit from outcrossing, but contradicts the conventional notion that the immune system of invertebrates, in contrast to vertebrates, is not able to react with specificity. Finally, sticklebacks seem to benefit from optimal diversity in their specific immune system. Previous studies showed that female sticklebacks prefer mates, which sire offspring with an optimal diversity in the MHC (genes involved in antigen presentation). We now found that these individuals suffer less from tapeworm infection. Furthermore, they are able to reduce the expression of an unspecific immune trait, thereby possibly avoiding harmful side effects of a highly activated, unspecific immune system.     

MHC genes and oxidative stress in sticklebacks: an immuno-ecological approach

Individual variation in the susceptibility to infection may result from the varying ability of hosts to specifically recognize different parasite strains. Alternatively, there could be individual host differences in fitness costs of immune defence. Although, these two explanations are not mutually exclusive, they have so far been treated in separate experimental approaches. To analyse potential relationships, we studied body condition and oxidative stress, which may reflect costs of immunity, in three-spined sticklebacks that had been experimentally exposed to three species of naturally occurring parasite. These sticklebacks differed in a trait, which is crucial to specific parasite defence, i.e. individual genetic diversity at major histocompatibility complex (MHC) class IIB loci. Oxidative stress was quantified as tissue acrolein, a technique that has been applied to questions of immuno-ecology for the first time. We measured gene expression at the MHC and other estimates of immune activation. We found that fish with high levels of MHC expression had poor condition and elevated oxidative stress. These results indicate that MHC-based specific immunity is connected with oxidative stress. They could, thus, also be relevant in the broader context of the evolution of sexually selected signals that are based on carotenoids and are, thus supposed to reflect oxidative stress resistance.     

Female sticklebacks transfer information via eggs: effects of maternal experience with predators on offspring

There is growing evidence that maternal experience influences offspring via non-genetic mechanisms. When female three-spined sticklebacks (Gasterosteus aculeatus) were exposed to the threat of predation, they produced larger eggs with higher cortisol content, which consumed more oxygen shortly after fertilization compared with a control group. As juveniles, the offspring of predator-exposed mothers exhibited tighter shoaling behaviour, an antipredator defence. We did not detect an effect of maternal exposure to predation risk on the somatic growth of fry. Altogether, we found that exposure to an ecologically relevant stressor during egg formation had several long-lasting consequences for offspring, some of which might be mediated by exposure to maternally derived cortisol. These results support the hypothesis that female sticklebacks might influence the development, growth and behaviour of their offspring via eggs to match their future environment.     

Evolutionary implications of the adaptation to different immune systems in a parasite with a complex life cycle

Many diseases are caused by parasites with complex life cycles that involve several hosts. If parasites cope better with only one of the different types of immune systems of their host species, we might expect a trade-off in parasite performance in the different hosts, that likely influences the evolution of virulence. We tested this hypothesis in a naturally co-evolving host-parasite system consisting of the tapeworm Schistocephalus solidus and its intermediate hosts, a copepod, Macrocyclops albidus, and the three-spined stickleback Gasterosteus aculeatus. We did not find a trade-off between infection success in the two hosts. Rather, tapeworms seem to trade-off adaptation towards different parts of their hosts' immune systems. Worm sibships that performed better in the invertebrate host also seem to be able to evade detection by the fish innate defence systems, i.e. induce lower levels of activation of innate immune components. These worm variants were less harmful for the fish host likely due to reduced costs of an activated innate immune system. These findings substantiate the impact of both hosts' immune systems on parasite performance and virulence.     

Parasite fauna in sticklebacks (Gasterosteidae) from bodies of water of the Kola region

The results of a parasitological study of the two species of sticklebacks (Pungitius pungitius and Gasterosteus aculeatus) inhabiting the Kola region are presented. The 42 and 14 species of parasite were found in the nine-spined and three-spined stickleback, respectively. The paucity of the parasite fauna in the three-spined stickleback is observed. Distribution of parasites in Kola waterbodies and ecological peculiarities in system "parasite-host" are presented.     

Dispersion patterns of parasites in 0+ year three-spined sticklebacks: a cross population comparison

Two ciliates and 16 metazoan parasites were identified in 434 0+ year three-spined sticklebacks Gasterosteus aculeatus collected from two small rivers and four lakes located in Schleswig-Holstein, Germany. By repeated sampling and analysis of dispersion patterns of six frequently occurring parasites no consistent evidence was found for mortality induced by a single parasite species. Linear log-variance to log-mean abundance ratios with slopes of c. 2 indicated negative binomial distributions for five of the six parasites. The numbers of these six parasites were combined as multiples of S.D. of each parasite species over all samples to form an 'individual parasitation index' ( I _PI), which showed that only in one locality a slight decrease in parasite burden occurred between September and April. In two of the lake populations, however, there was a distinct decline in the degree of dispersion in spring samples. This indicates that a combination of different species might cause parasite-induced host mortality, undetectable by patterns obtained from single species. There were differences in parasite diversity and intensity of infection among river compared to lake populations suggesting a role for parasites as selective agents in the ecological divergence of three-spined sticklebacks.     

Complexity and context of MHC-correlated mating preferences in wild populations

There is now substantial and growing evidence for a role of the major histocompatibility complex (MHC) in shaping individual mate preferences. In view of both its codominant expression and its function in immune response, it is often expected that females aim to avoid inbreeding or maximize offspring MHC-heterozygosity by selecting as mates those males which share fewest or no MHC alleles with themselves. However, it is becoming increasingly clear that this view is over-simplistic: not only is MHC dissimilarity just one of several (perhaps many) criteria important in mate choice decision-making, extremely MHC-dissimilar males may be avoided, and furthermore, specific alleles or combinations might be preferred if they bestow particular advantages. These points are raised in two papers in this Molecular Ecology issue, in which patterns of reproductive success in tiger salamanders ( Ambystoma tigrinum ) and three-spined sticklebacks ( Gasterosteus aculeatus ) are each inconsistent with a generalized preference for MHC dissimilarity. Together, these studies demonstrate several adaptive reasons for decision rules that do not necessarily result in maximizing mate dissimilarity.     

Maintaining functional major histocompatibility complex diversity under inbreeding: the case of a selfing vertebrate

Major histocompatibility complex (MHC) genes encode proteins that present pathogen-derived antigens to T-cells, initiating the adaptive immune response in vertebrates. Although populations with low MHC diversity tend to be more susceptible to pathogens, some bottlenecked populations persist and even increase in numbers despite low MHC diversity. Thus, the relative importance of MHC diversity versus genome-wide variability for the long-term viability of populations after bottlenecks and/or under high inbreeding is controversial. We tested the hypothesis that genome-wide inbreeding (estimated using microsatellites) should be more critical than MHC diversity alone in determining pathogen resistance in the self-fertilizing fish Kryptolebias marmoratus by analysing MHC diversity and parasite loads in natural and laboratory populations with different degrees of inbreeding. Both MHC and neutral diversities were lost after several generations of selfing, but we also found evidence of parasite selection acting on MHC diversity and of non-random loss of alleles, suggesting a possible selective advantage of those individuals with functionally divergent MHC, in accordance with the hypothesis of divergent allele advantage. Moreover, we found that parasite loads were better explained by including MHC diversity in the model than by genome-wide (microsatellites) heterozygosity alone. Our results suggest that immune-related overdominance could be the key in maintaining variables rates of selfing and outcrossing in K. marmoratus and other mixed-mating species.     

Observations on the host-parasite relations of Thersitina gasterostei (Crustacea: Copepoda)

This study seeks to analyse the degree of infection of a euryhaline fish with the parasitic copepod Thersitina gasterostei in an extensive brackish-water system. For this purpose, a survey of the occurrence of the parasite on three-spined sticklebacks in the Norfolk Broads was conducted in Spring 1969. An attempt was made to correlate the incidence levels of T. gasterostei with variations in salinity. In addition, observations were made upon the location of the parasite on its host, the changes in maturation of the parasite during the sampling period and the differences in infection between three- and nine-spined sticklebacks.     

Wild cyclic voles maintain high neutral and MHC diversity without strong evidence for parasite-mediated selection

The major histocompatibility complex (MHC) is an important component of vertebrate immune defense involved with self/nonself recognition and disease susceptibility. The high variability of genes of the MHC is thought to arise from both parasite-mediated and sexual selection. An outstanding question involves the degree to which balancing selection can oppose genetic drift to maintain high MHC diversity in the face of population bottlenecks. To address this question we examined genetic diversity and population structure at neutral (microsatellite) and MHC genes in montane voles [Microtus montanus (Peale, 1848)] subject to high amplitude population fluctuations, and compared these to measures of infection by common gastrointestinal parasites. We found high neutral and MHC allelic variability, indicating low impacts of genetic drift despite large fluctuations in population size. Greater MHC diversity did not predict lower parasite richness or infection by the two most common endoparasites (cestodes and coccidian protozoa), as might be expected if genotypic composition confers resistance to infection. One specific MHC allele predicted lower cestode intensity, but we found no other associations between MHC and infection measures. Neutral heterozygosity was positively associated with total parasite richness, possibly owing to greater parasite tolerance among heterozygous relative to more inbred hosts. Overall, these results suggest that factors beyond the parasites examined here, such as high inter-patch migration, mate choice, gene conversion or other infectious agents, are likely maintaining the high levels of MHC diversity observed in wild montane voles.