Parasitism drives host genome evolution: Insights from the Pasteuria ramosa–Daphnia magna system

Because parasitism is thought to play a major role in shaping host genomes, it has been predicted that genomic regions associated with resistance to parasites should stand out in genome scans, revealing signals of selection above the genomic background. To test whether parasitism is indeed such a major factor in host evolution and to better understand host–parasite interaction at the molecular level, we studied genome‐wide polymorphisms in 97 genotypes of the planktonic crustacean Daphnia magna originating from three localities across Europe. Daphnia magna is known to coevolve with the bacterial pathogen Pasteuria ramosa for which host genotypes (clonal lines) are either resistant or susceptible. Using association mapping, we identified two genomic regions involved in resistance to P. ramosa, one of which was already known from a previous QTL analysis. We then performed a naïve genome scan to test for signatures of positive selection and found that the two regions identified with the association mapping further stood out as outliers. Several other regions with evidence for selection were also found, but no link between these regions and phenotypic variation could be established. Our results are consistent with the hypothesis that parasitism is driving host genome evolution.     

CROSS‐SPECIES INFECTION TRIALS REVEAL CRYPTIC PARASITE VARIETIES AND A PUTATIVE POLYMORPHISM SHARED AMONG HOST SPECIES

A parasite's host range can have important consequences for ecological and evolutionary processes but can be difficult to infer. Successful infection depends on the outcome of multiple steps and only some steps of the infection process may be critical in determining a parasites host range. To test this hypothesis, we investigated the host range of the bacterium Pasteuria ramosa, a Daphnia parasite, and determined the parasites success in different stages of the infection process. Multiple genotypes of Daphnia pulex, Daphnia longispina and Daphnia magna were tested with four Pasteuria genotypes using infection trials and an assay that determines the ability of the parasite to attach to the hosts esophagus. We find that attachment is not specific to host species but is specific to host genotype. This may suggest that alleles on the locus controlling attachment are shared among different host species that diverged 100 million year. However, in our trials, Pasteuria was never able to reproduce in nonnative host species, suggesting that Pasteuria infecting different host species are different varieties, each with a narrow host range. Our approach highlights the explanatory power of dissecting the steps of the infection process and resolves potentially conflicting reports on parasite host ranges.     

The expression of virulence during double infections by different parasites with conflicting host exploitation and transmission strategies

In many natural populations, hosts are found to be infected by more than one parasite species. When these parasites have different host exploitation strategies and transmission modes, a conflict among them may arise. Such a conflict may reduce the success of both parasites, but could work to the benefit of the host. For example, the less‐virulent parasite may protect the host against the more‐virulent competitor. We examine this conflict using the waterflea Daphnia magna and two of its sympatric parasites: the blood‐infecting bacterium Pasteuria ramosa that transmits horizontally and the intracellular microsporidium Octosporea bayeri that can concurrently transmit horizontally and vertically after infecting ovaries and fat tissues of the host. We quantified host and parasite fitness after exposing Daphnia to one or both parasites, both simultaneously and sequentially. Under conditions of strict horizontal transmission, Pasteuria competitively excluded Octosporea in both simultaneous and sequential double infections, regardless of the order of exposure. Host lifespan, host reproduction and parasite spore production in double infections resembled those of single infection by Pasteuria. When hosts became first vertically (transovarilly) infected with O. bayeri, Octosporea was able to withstand competition with P. ramosa to some degree, but both parasites produced less transmission stages than they did in single infections. At the same time, the host suffered from reduced fecundity and longevity. Our study demonstrates that even when competing parasite species utilize different host tissues to proliferate, double infections lead to the expression of higher virulence and ultimately may select for higher virulence. Furthermore, we found no evidence that the less‐virulent and vertically transmitting O. bayeri protects its host against the highly virulent P. ramosa.     

THE CELLULAR IMMUNE RESPONSE OF DAPHNIA MAGNA UNDER HOST–PARASITE GENETIC VARIATION AND VARIATION IN INITIAL DOSE

In invertebrate–parasite systems, the likelihood of infection following parasite exposure is often dependent on the specific combination of host and parasite genotypes (termed genetic specificity). Genetic specificity can maintain diversity in host and parasite populations and is a major component of the Red Queen hypothesis. However, invertebrate immune systems are thought to only distinguish between broad classes of parasite. Using a natural host–parasite system with a well‐established pattern of genetic specificity, the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa, we found that only hosts from susceptible host–parasite genetic combinations mounted a cellular response following exposure to the parasite. These data are compatible with the hypothesis that genetic specificity is attributable to barrier defenses at the site of infection (the gut), and that the systemic immune response is general, reporting the number of parasite spores entering the hemocoel. Further supporting this, we found that larger cellular responses occurred at higher initial parasite doses. By studying the natural infection route, where parasites must pass barrier defenses before interacting with systemic immune responses, these data shed light on which components of invertebrate defense underlie genetic specificity.     

Host sexual dimorphism affects the outcome of within‐host pathogen competition

Natural infections often consist of multiple pathogens of the same or different species. When coinfections occur, pathogens compete for access to host resources and fitness is determined by how well a pathogen can reproduce compared to its competitors. Yet not all hosts provide the same resource pool. Males and females, in particular, commonly vary in both their acquisition of resources and investment in immunity, but their ability to modify any competition between different pathogens remains unknown. Using the Daphnia magna–Pasteuria ramosa model system, we exposed male and female hosts to either a single genotype infection or coinfections consisting of two pathogen genotypes of varying levels of virulence. We found that coinfections within females favored the transmission of the more virulent pathogen genotype, whereas coinfections within male hosts resulted in equal transmission of competing pathogen genotypes. This contrast became less pronounced when the least virulent pathogen was able to establish an infection first, suggesting that the influence of host sex is shaped by priority effects. We suggest that sex is a form of host heterogeneity that may influence the evolution of virulence within coinfection contexts and that one sex may be a reservoir for pathogen genetic diversity in nature.     

Evidence for strong host clone-parasite species interactions in the Daphnia microparasite system

Abstract Organisms are often confronted with multiple enemy species. Defenses against different parasite species may be traded off against each other. However, if resistance is based on (potentially costly) general defense mechanisms, it may be positively correlated among parasites. In an experimental study, we confronted 19 clones from one Daphnia magna population with two bacterial and three microsporidian parasite species. All parasites were isolated from the same pond as the hosts. Host clones were specific in their susceptibility towards different parasite species, and parasite species were host-clone specific in their infectivity, spore production, and virulence, resulting in highly significant host-parasite interactions. Since the Daphnia 's resistance to different parasite species showed no obvious correlation, neither general defense mechanisms nor trade-offs in resistance explain our findings. None of the Daphnia clones were resistant to all parasite species, and the average level of resistance was quite similar among clones. This may reflect a cost of defense, so that the cumulative cost of being resistant to all parasite species might be too high.     

The effects of multiple infections on the expression and evolution of virulence in a Daphnia-endoparasite system

Multiple infections of a host by different strains of the same microparasite are common in nature. Although numerous models have been developed in an attempt to predict the evolutionary effects of intrahost competition, tests of the assumptions of these models are rare and the outcome is diverse. In the present study we examined the outcome of mixed-isolate infections in individual hosts, using a single clone of the waterflea Daphnia magna and three isolates of its semelparous endoparasite Pasteuria ramosa . We exposed individual Daphnia to single- and mixed-isolate infection treatments, both simultaneously and sequentially. Virulence was assessed by monitoring host mortality and fecundity, and parasite spore production was used as a measure of parasite fitness. Consistent with most assumptions, in multiply infected hosts we found that the virulence of mixed infections resembled that of the more virulent competitor, both in simultaneous multiple infections and in sequential multiple infections in which the virulent isolate was first to infect. The more virulent competitor also produced the vast majority of transmission stages. Only when the less virulent isolate was first to infect, the intrahost contest resembled scramble competition, whereby both isolates suffered by producing fewer transmission stages. Surprisingly, mixed-isolate infections resulted in lower fecundity-costs for the hosts, suggesting that parasite competition comes with an advantage for the host relative to single infections. Finally, spore production correlated positively with time-to-host-death. Thus, early-killing of more competitive isolates produces less transmission stages than less virulent, inferior isolates. Our results are consistent with the idea that less virulent parasite lines may be replaced by more virulent strains under conditions with high rates of multiple infections.     

Evidence for a cost of immunity when the crustacean Daphnia magna is exposed to the bacterial pathogen Pasteuria ramosa

The deployment of the immune system has the obvious potential to ameliorate infection outcomes, but immune responses can also harm hosts by either damaging host tissues or monopolizing resources, leading to enhanced mortality. To gain insight into such a 'cost of immunity' when the crustacean Daphnia magna is challenged with the bacterium Pasteuria ramosa, we measured survivorship among hosts that resisted infection following exposure to various strains and doses of the parasite. In the first of two experiments, these exposures were: single exposures with relatively non-aggressive strains, double exposures with non-aggressive strains, and exposure to aggressive strains. Mortality increased across this gradient of exposure. In a second experiment, we varied the dose of the most aggressive P. ramosa strain and found that resisting infection when a large dose was applied resulted in greater mortality than when a medium or low dose was applied. Assuming that resistance is accomplished with an immune response, and that more aggressive parasites and/or larger doses of parasites are more immunostimulatory, these data are compatible with a cost of immunity. Indeed, in terms of survival, resisting parasites can be more harmful than infection.     

Local adaptation of a parasite to solar radiation impacts disease transmission potential,spore yield,and host fecundity*

Environmentally transmitted parasites spend time in the abiotic environment, where they are subjected to a variety of stressors. Learning how they face this challenge is essential if we are to understand how host–parasite interactions may vary across environmental gradients. We used a zooplankton–bacteria host–parasite system where availability of sunlight (solar radiation) influences disease dynamics to look for evidence of parasite local adaptation to sunlight exposure. We also examined how variation in sunlight tolerance among parasite strains impacted host reproduction. Parasite strains collected from clearer lakes (with greater sunlight penetration) were most tolerant of the negative impacts of sunlight exposure, suggesting local adaptation to sunlight conditions. This adaptation came with both a cost and a benefit for parasites: parasite strains from clearer lakes produced relatively fewer transmission stages (spores) but these strains were more infective. After experimental sunlight exposure, the most sunlight-tolerant parasite strains reduced host fecundity just as much as spores that were never exposed to sunlight. Sunlight availability varies greatly among lakes around the world. Our results suggest that the selective pressure sunlight exposure exerts on parasites may impact both parasite and host fitness, potentially driving variation in disease epidemics and host population dynamics across sunlight availability gradients.     

The Red Queen lives: Epistasis between linked resistance loci

A popular theory explaining the maintenance of genetic recombination (sex) is the Red Queen Theory. This theory revolves around the idea that time‐lagged negative frequency‐dependent selection by parasites favors rare host genotypes generated through recombination. Although the Red Queen has been studied for decades, one of its key assumptions has remained unsupported. The signature host‐parasite specificity underlying the Red Queen, where infection depends on a match between host and parasite genotypes, relies on epistasis between linked resistance loci for which no empirical evidence exists. We performed 13 genetic crosses and tested over 7000 Daphnia magna genotypes for resistance to two strains of the bacterial pathogen Pasteuria ramosa. Results reveal the presence of strong epistasis between three closely linked resistance loci. One locus masks the expression of the other two, while these two interact to produce a single resistance phenotype. Changing a single allele on one of these interacting loci can reverse resistance against the tested parasites. Such a genetic mechanism is consistent with host and parasite specificity assumed by the Red Queen Theory. These results thus provide evidence for a fundamental assumption of this theory and provide a genetic basis for understanding the Red Queen dynamics in the Daphnia–Pasteuria system.     

Diet quality determines interspecific parasite interactions in host populations

The widespread occurrence of multiple infections and the often vast range of nutritional resources for their hosts allow that interspecific parasite interactions in natural host populations might be determined by host diet quality. Nevertheless, the role of diet quality with respect to multispecies parasite interactions on host population level is not clear. We here tested the effect of host population diet quality on the parasite community in an experimental study using Daphnia populations. We studied the effect of diet quality on Daphnia population demography and the interactions in multispecies parasite infections of this freshwater crustacean host. The results of our experiment show that the fitness of a low‐virulent microsporidian parasite decreased in low, but not in high‐host‐diet quality conditions. Interestingly, infections with the microsporidium protected Daphnia populations against a more virulent bacterial parasite. The observed interspecific parasite interactions are discussed with respect to the role of diet quality‐dependent changes in host fecundity. This study reflects that exploitation competition in multispecies parasite infections is environmentally dependent, more in particular it shows that diet quality affects interspecific parasite competition within a single host and that this can be mediated by host population‐level effects.     

Resistance to a bacterial parasite in the crustacean Daphnia magna shows Mendelian segregation with dominance

The influence of host and parasite genetic background on infection outcome is a topic of great interest because of its pertinence to theoretical issues in evolutionary biology. In the present study, we use a classical genetics approach to examine the mode of inheritance of infection outcome in the crustacean Daphnia magna when exposed to the bacterial parasite Pasteuria ramosa. In contrast to previous studies in this system, we use a clone of P. ramosa, not field isolates, which allows for a more definitive interpretation of results. We test parental, F1, F2, backcross and selfed parental clones (total 284 genotypes) for susceptibility against a clone of P. ramosa using two different methods, infection trials and the recently developed attachment test. We find that D. magna clones reliably exhibit either complete resistance or complete susceptibility to P. ramosa clone C1 and that resistance is dominant, and inherited in a pattern consistent with Mendelian segregation of a single-locus with two alleles. The finding of a single host locus controlling susceptibility to P. ramosa suggests that the previously observed genotype-genotype interactions in this system have a simple genetic basis. This has important implications for the outcome of host-parasite co-evolution. Our results add to the growing body of evidence that resistance to parasites in invertebrates is mostly coded by one or few loci with dominance.     

Temperature-dependent costs of parasitism and maintenance of polymorphism under genotype-by-environment interactions

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.     

Collateral damage: rapid exposure-induced evolution of pesticide resistance leads to increased susceptibility to parasites

Although natural populations may evolve resistance to anthropogenic stressors such as pollutants, this evolved resistance may carry costs. Using an experimental evolution approach, we exposed different Daphnia magna populations in outdoor containers to the carbamate pesticide carbaryl and control conditions, and assessed the resulting populations for both their resistance to carbaryl as well as their susceptibility to infection by the widespread bacterial microparasite Pasteuria ramosa. Our results show that carbaryl selection led to rapid evolution of carbaryl resistance with seemingly no cost when assessed in a benign environment. However, carbaryl-resistant populations were more susceptible to parasite infection than control populations. Exposure to both stressors reveals a synergistic effect on sterilization rate by P. ramosa, but this synergism did not evolve under pesticide selection. Assessing costs of rapid adaptive evolution to anthropogenic stress in a semi-natural context may be crucial to avoid too optimistic predictions for the fitness of the evolving populations.     

Climate,host phylogeny and the connectivity of host communities govern regional parasite assembly

Aim

Identifying barriers that govern parasite community assembly and parasite invasion risk is critical to understand how shifting host ranges impact disease emergence. We studied regional variation in the phylogenetic compositions of bird species and their blood parasites (Plasmodium and Haemoproteus spp.) to identify barriers that shape parasite community assembly.

Location

Australasia and Oceania.

Methods

We used a data set of parasite infections from >10,000 host individuals sampled across 29 bioregions. Hierarchical models and matrix regressions were used to assess the relative influences of interspecies (host community connectivity and local phylogenetic distinctiveness), climate and geographic barriers on parasite local distinctiveness and composition.

Results

Parasites were more locally distinct (co‐occurred with distantly related parasites) when infecting locally distinct hosts, but less distinct (co‐occurred with closely related parasites) in areas with increased host diversity and community connectivity (a proxy for parasite dispersal potential). Turnover and the phylogenetic symmetry of parasite communities were jointly driven by host turnover, climate similarity and geographic distance.

Main conclusions

Interspecies barriers linked to host phylogeny and dispersal shape parasite assembly, perhaps by limiting parasite establishment or local diversification. Infecting hosts that co‐occur with few related species decreases a parasite's likelihood of encountering related competitors, perhaps increasing invasion potential but decreasing diversification opportunity. While climate partially constrains parasite distributions, future host range expansions that spread distinct parasites and diminish barriers to host shifting will likely be key drivers of parasite invasions.     

Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores

The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.     

Balancing Selection for Pathogen Resistance Reveals an Intercontinental Signature of Red Queen Coevolution

The link between long-term host–parasite coevolution and genetic diversity is key to understanding genetic epidemiology and the evolution of resistance. The model of Red Queen host–parasite coevolution posits that high genetic diversity is maintained when rare host resistance variants have a selective advantage, which is believed to be the mechanistic basis for the extraordinarily high levels of diversity at disease-related genes such as the major histocompatibility complex in jawed vertebrates and R-genes in plants. The parasites that drive long-term coevolution are, however, often elusive. Here we present evidence for long-term balancing selection at the phenotypic (variation in resistance) and genomic (resistance locus) level in a particular host–parasite system: the planktonic crustacean Daphnia magna and the bacterium Pasteuria ramosa. The host shows widespread polymorphisms for pathogen resistance regardless of geographic distance, even though there is a clear genome-wide pattern of isolation by distance at other sites. In the genomic region of a previously identified resistance supergene, we observed consistent molecular signals of balancing selection, including higher genetic diversity, older coalescence times, and lower differentiation between populations, which set this region apart from the rest of the genome. We propose that specific long-term coevolution by negative-frequency-dependent selection drives this elevated diversity at the host''s resistance loci on an intercontinental scale and provide an example of a direct link between the host’s resistance to a virulent pathogen and the large-scale diversity of its underlying genes.     

Disentangling the influence of parasite genotype, host genotype and maternal environment on different stages of bacterial infection in Daphnia magna

Individuals naturally vary in the severity of infectious disease when exposed to a parasite. Dissecting this variation into genetic and environmental components can reveal whether or not this variation depends on the host genotype, parasite genotype or a range of environmental conditions. Complicating this task, however, is that the symptoms of disease result from the combined effect of a series of events, from the initial encounter between a host and parasite, through to the activation of the host immune system and the exploitation of host resources. Here, we use the crustacean Daphnia magna and its parasite Pasteuria ramosa to show how disentangling genetic and environmental factors at different stages of infection improves our understanding of the processes shaping infectious disease. Using compatible host-parasite combinations, we experimentally exclude variation in the ability of a parasite to penetrate the host, from measures of parasite clearance, the reduction in host fecundity and the proliferation of the parasite. We show how parasite resistance consists of two components that vary in environmental sensitivity, how the maternal environment influences all measured aspects of the within-host infection process and how host-parasite interactions following the penetration of the parasite into the host have a distinct temporal component.     

A Two-Locus System with Strong Epistasis Underlies Rapid Parasite-Mediated Evolution of Host Resistance

Parasites are a major evolutionary force, driving adaptive responses in host populations. Although the link between phenotypic response to parasite-mediated natural selection and the underlying genetic architecture often remains obscure, this link is crucial for understanding the evolution of resistance and predicting associated allele frequency changes in the population. To close this gap, we monitored the response to selection during epidemics of a virulent bacterial pathogen, Pasteuria ramosa, in a natural host population of Daphnia magna. Across two epidemics, we observed a strong increase in the proportion of resistant phenotypes as the epidemics progressed. Field and laboratory experiments confirmed that this increase in resistance was caused by selection from the local parasite. Using a genome-wide association study, we built a genetic model in which two genomic regions with dominance and epistasis control resistance polymorphism in the host. We verified this model by selfing host genotypes with different resistance phenotypes and scoring their F1 for segregation of resistance and associated genetic markers. Such epistatic effects with strong fitness consequences in host–parasite coevolution are believed to be crucial in the Red Queen model for the evolution of genetic recombination.     

Pathogen exposure reduces sexual dimorphism in a host’s upper thermal limits

The climate is warming at an unprecedented rate, pushing many species toward and beyond the upper temperatures at which they can survive. Global change is also leading to dramatic shifts in the distribution of pathogens. As a result, upper thermal limits and susceptibility to infection should be key determinants of whether populations continue to persist, or instead go extinct. Within a population, however, individuals vary in both their resistance to both heat stress and infection, and their contributions to vital growth rates. No more so is this true than for males and females. Each sex often varies in their response to pathogen exposure, thermal tolerances, and particularly their influence on population growth, owing to the higher parental investment that females typically make in their offspring. To date, the interplay between host sex, infection, and upper thermal limits has been neglected. Here, we explore the response of male and female Daphnia to bacterial infection and static heat stress. We find that female Daphnia, when uninfected, are much more resistant to static heat stress than males, but that infection negates any advantage that females are afforded. We discuss how the capacity of a population to cope with multiple stressors may be underestimated unless both sexes are considered simultaneously.