Spatial population genetic structure of a bacterial parasite in close coevolution with its host

Knowledge of a species’ population genetic structure can provide insight into fundamental ecological and evolutionary processes including gene flow, genetic drift and adaptive evolution. Such inference is of particular importance for parasites, as an understanding of their population structure can illuminate epidemiological and coevolutionary dynamics. Here, we describe the population genetic structure of the bacterium Pasteuria ramosa, a parasite that infects planktonic crustaceans of the genus Daphnia. This system has become a model for investigations of host–parasite interactions and represents an example of coevolution via negative frequency‐dependent selection (aka “Red Queen” dynamics). To sample P. ramosa, we experimentally infected a panel of Daphnia hosts with natural spore banks from the sediments of 25 ponds throughout much of the species range in Europe and western Asia. Using 12 polymorphic variable number tandem repeat loci (VNTR loci), we identified substantial genetic diversity, both within and among localities, that was structured geographically among ponds. Genetic diversity was also structured among host genotypes within ponds, although this pattern varied by locality, with P. ramosa at some localities partitioned into distinct host‐specific lineages, and other localities where recombination had shuffled genetic variation among different infection phenotypes. Across the sample range, there was a pattern of isolation by distance, and principal components analysis coupled with Procrustes rotation identified congruence between patterns of genetic variation and geography. Our findings support the hypothesis that Pasteuria is an endemic parasite coevolving closely with its host. These results provide important context for previous studies of this model system and inform hypotheses for future research.     

Rapid change in parasite infection traits over the course of an epidemic in a wild host–parasite population

By combining a field study with controlled laboratory experimentation, we examined how infection traits of the sterilizing bacterium, Pasteuria ramosa, changed over the course of a growing season in a natural population of its crustacean host Daphnia magna. The number of parasite transmission spores per infected host increased ten‐fold over the course of the season, concomitant with a decline in the density of infected hosts. Plausible explanations for this variation include changes in environmental conditions, changes in host quality, or that parasite migration or natural selection caused a genetic change in the parasite population. We sought to distinguish some of these possibilities in a laboratory experiment. Thus, we preserved field‐collected parasite spores throughout the season, and later exposed a set of hosts to a fixed dose of these spores under controlled laboratory conditions. Parasites collected late in the season were more infectious and grew more rapidly than parasites collected early in the season. This result is compatible with the hypothesis that the observed increase in infectivity in the field was due to genetic change, i.e. evolution in the P. ramosa population.     

Genetic variation for maternal effects on parasite susceptibility

The expression of infectious disease is increasingly recognized to be impacted by maternal effects, where the environmental conditions experienced by mothers alter resistance to infection in offspring, independent of heritability. Here, we studied how maternal effects (high or low food availability to mothers) mediated the resistance of the crustacean Daphnia magna to its bacterial parasite Pasteuria ramosa. We sought to disentangle maternal effects from the effects of host genetic background by studying how maternal effects varied across 24 host genotypes sampled from a natural population. Under low‐food conditions, females produced offspring that were relatively resistant, but this maternal effect varied strikingly between host genotypes, i.e. there were genotype by maternal environment interactions. As infection with P. ramosa causes a substantial reduction in host fecundity, this maternal effect had a large effect on host fitness. Maternal effects were also shown to impact parasite fitness, both because they prevented the establishment of the parasites and because even when parasites did establish in the offspring of poorly fed mothers, and they tended to grow more slowly. These effects indicate that food stress in the maternal generation can greatly influence parasite susceptibility and thus perhaps the evolution and coevolution of host–parasite interactions.     

Pesticide exposure strongly enhances parasite virulence in an invertebrate host model

Parasites are a common and constant threat to organisms at all levels of phyla. The virulence of a parasite, defined as the impact on survival and reproduction of its host, depends on the specific host–parasite combination and can also be influenced by environmental conditions. Environmental pollution might be an additional factor influencing host–parasite interactions. We here aimed to test whether the combined stress of pollutant exposure and parasite challenge results in stronger impacts on host organisms than expected from the single stressors applied alone. We used the water flea Daphnia magna and two of its endoparasites, the bacterium Pasteuria ramosa and the microsporidium Flabelliforma magnivora, as invertebrate host–parasite models. For each parasite, we tested in a full‐factorial design for interactions between parasitism and pollution using the neurotoxic pesticide carbaryl as a model substance. Sublethal concentrations of the pesticide synergistically enhanced the virulence of both parasites by increasing host mortality. Furthermore, host castration induced by P. ramosa was accelerated by carbaryl exposure. These effects likely reflect decreased host resistance due to direct or indirect immunosuppressive activity of carbaryl. The present study provides experimental evidence that the in vivo development of infectious diseases can be influenced by a pesticide at environmentally realistic concentrations. This implies that host–parasite interactions and subsequently co‐evolution might be influenced by environmental pollution at toxicant concentrations being sublethal to parasite‐free hosts. Standard toxicity testing as employed in the current way of conducting ecological risk assessments for anthropogenic substances does not consider natural antagonists such as infectious diseases, and thereby likely underestimates the impact these substances may pose to natural populations in the environment.     

The bacterial parasite Pasteuria ramosa is not killed if it fails to infect: implications for coevolution

Strong selection on parasites, as well as on hosts, is crucial for fueling coevolutionary dynamics. Selection will be especially strong if parasites that encounter resistant hosts are destroyed and diluted from the local environment. We tested whether spores of the bacterial parasite Pasteuria ramosa were passed through the gut (the route of infection) of their host, Daphnia magna, and whether passaged spores remained viable for a “second chance” at infecting a new host. In particular, we tested if this viability (estimated via infectivity) depended on host genotype, whether or not the genotype was susceptible, and on initial parasite dose. Our results show that Pasteuria spores generally remain viable after passage through both susceptible and resistant Daphnia. Furthermore, these spores remained infectious even after being frozen for several weeks. If parasites can get a second chance at infecting hosts in the wild, selection for infection success in the first instance will be reduced. This could also weaken reciprocal selection on hosts and slow the coevolutionary process.     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

The virulence–transmission relationship in an obligate killer holds under diverse epidemiological and ecological conditions,but where is the tradeoff?

Parasite virulence is a leading theme in evolutionary biology. Modeling the course of virulence evolution holds the promise of providing practical insights into the management of infectious diseases and the implementation of vaccination strategies. A key element of virulence modeling is a tradeoff between parasite transmission rate and host lifespan. This assumption is crucial for predicting the level of optimal virulence. Here, I test this assumption using the water flea Daphnia magna and its castrating and obligate‐killing bacterium Pasteuria ramosa. I found that the virulence–transmission relationship holds under diverse epidemiological and ecological conditions. In particular, parasite genotype, absolute and relative parasite dose, and within‐host competition in multiple infections did not significantly affect the observed trend. Interestingly, the relationship between virulence and parasite transmission in this system is best explained by a model that includes a cubic term. Under this relationship, parasite transmission initially peaks and saturates at an intermediate level of virulence, but then it further increases as virulence decreases, surpassing the previous peak. My findings also highlight the problem of using parasite‐induced host mortality as a “one‐size‐fits‐all” measure of virulence for horizontally transmitted parasites, without considering the onset and duration of parasite transmission as well as other equally virulent effects of parasites (e.g., host castration). Therefore, mathematical models may be required to predict whether these particular characteristics of horizontally transmitted parasites can direct virulence evolution into directions not envisaged by existing models.     

Ecological implications of parasites in natural <Emphasis Type="Italic">Daphnia</Emphasis> populations

In natural host populations, parasitism is considered to be omnipresent and to play an important role in shaping host life history and population dynamics. Here, we study parasitism in natural populations of the zooplankton host Daphnia magna investigating their individual and population level effects during a 2-year field study. Our results revealed a rich and highly prevalent community of parasites, with eight endoparasite species (four microsporidia, one amoeba, two bacteria and one nematode) and six epibionts (belonging to five different taxa: Chlorophyta, Bacillariophyceae, Ciliata, Fungi and Rotifera). Several of the endoparasites were associated with a severe overall fecundity reduction of the hosts, while such effects were not seen for epibionts. In particular, infections by Pasteuria ramosa, White Fat Cell Disease and Flabelliforma magnivora were strongly associated with a reduction in overall D. magna fecundity. Across the sampling period, average population fecundity of D. magna was negatively associated with overall infection intensity and total endoparasite richness. Population density of D. magna was negatively correlated to overall endoparasite prevalence and positively correlated with epibiont richness. Finally, the reduction in host fecundity caused by different parasite species was negatively correlated to both parasite prevalence and the length of the time period during which the parasite persisted in the host population. Consistent with epidemiological models, these results indicate that parasite mediated host damages influence the population dynamics of both hosts and parasites.     

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.     

Within- and between-population variation for resistance of Daphnia magna to the bacterial endoparasite Pasteuria ramosa

Genetic variation among hosts for resistance to parasites is an important assumption underlying evolutionary theory of host and parasite evolution. Using the castrating bacterial parasite Pasteuria ramosa and its cladoceran host Daphnia magna, we examined both within- and between-population genetic variation for resistance. First, we tested hosts from four populations for genetic variation for resistance to three parasite isolates. Allozyme analysis revealed significant host population divergence and that genetic distance corresponds to geographic distance. Host and parasite fitness components showed strong genetic differences between parasite isolates for host population by parasite interactions and for clones within populations, whereas host population effects were significant for only a few traits. In a second experiment we tested explicitly for within-population differences in variation for resistance by challenging nine host clones from a single population with four different parasite spore doses. Strong clone and dose effects were evident. More susceptible clones also suffered higher costs once infected. The results indicate that within-population variation for resistance is high relative to between-population variation. We speculate that P. ramosa adapts to individual host clones rather than to its host population.     

Genetic structure and host–parasite co‐divergence: evidence for trait‐specific local adaptation

Host–parasite co‐evolution can lead to genetic differentiation among isolated host–parasite populations and local adaptation between parasites and their hosts. However, tests of local adaptation rarely consider multiple fitness‐related traits although focus on a single component of fitness can be misleading. Here, we concomitantly examined genetic structure and co‐divergence patterns of the trematode Coitocaecum parvum and its crustacean host Paracalliope fluviatilis among isolated populations using the mitochondrial cytochrome oxidase I gene (COI). We then performed experimental cross‐infections between two genetically divergent host–parasite populations. Both hosts and parasites displayed genetic differentiation among populations, although genetic structure was less pronounced in the parasite. Data also supported a co‐divergence scenario between C. parvum and P. fluviatilis potentially related to local co‐adaptation. Results from cross‐infections indicated that some parasite lineages seemed to be locally adapted to their sympatric (home) hosts in which they achieved higher infection and survival rates than in allopatric (away) amphipods. However, local, intrinsic host and parasite characteristics (host behavioural or immunological resistance to infections, parasite infectivity or growth rate) also influenced patterns of host–parasite interactions. For example, overall host vulnerability to C. parvum varied between populations, regardless of parasite origin (local vs. foreign), potentially swamping apparent local co‐adaptation effects. Furthermore, local adaptation effects seemed trait specific; different components of parasite fitness (infection and survival rates, growth) responded differently to cross‐infections. Overall, data show that genetic differentiation is not inevitably coupled with local adaptation, and that the latter must be interpreted with caution in a multi‐trait context.     

The potential for arms race and Red Queen coevolution in a protist host–parasite system

The dynamics and consequences of host–parasite coevolution depend on the nature of host genotype‐by‐parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies (“Red Queen” dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps (“arms race” dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host–parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host–parasite system.     

The effect of a pathogen epidemic on the genetic structure and reproductive strategy of the crustacean Daphnia magna

Host–parasite coevolution is potentially of great importance in producing and maintaining biological diversity. However, there is a lack of evidence for parasites directly driving genetic change. We examined the impact of an epidemic of the bacterium Pasteuria ramosa on a natural population of the crustacean Daphnia magna through the use of molecular markers (allozymes) and laboratory experiments to determine the susceptibility of hosts collected during and after the epidemic. Some allozyme genotypes were more heavily infected than others in field samples, and the population genetic structure differed during and after the epidemic, consistent with a response to parasite‐mediated selection. Laboratory studies showed no evidence for the evolution of higher resistance, but did reveal an intriguing life‐history pattern: host genotypes that were more susceptible also showed a greater tendency to engage in sex. In light of this, we suggest a model of host–parasite dynamics that incorporates the cycles of sex and parthenogenesis that Daphnia undergo in the field.     

Simulated climate change,epidemic size,and host evolution across host–parasite populations

Climate change is causing warmer and more variable temperatures as well as physical flux in natural populations, which will affect the ecology and evolution of infectious disease epidemics. Using replicate seminatural populations of a coevolving freshwater invertebrate‐parasite system (host: Daphnia magna, parasite: Pasteuria ramosa), we quantified the effects of ambient temperature and population mixing (physical flux within populations) on epidemic size and population health. Each population was seeded with an identical suite of host genotypes and dose of parasite transmission spores. Biologically reasonable increases in environmental temperature caused larger epidemics, and population mixing reduced overall epidemic size. Mixing also had a detrimental effect on host populations independent of disease. Epidemics drove parasite‐mediated selection, leading to a loss of host genetic diversity, and mixed populations experienced greater evolution due to genetic drift over the season. These findings further our understanding of how diversity loss will reduce the host populations’ capacity to respond to changes in selection, therefore stymying adaptation to further environmental change.     

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.     

Biogeography and host‐related factors trump parasite life history: limited congruence among the genetic structures of specific ectoparasitic lice and their rodent hosts

Parasites and hosts interact across both micro‐ and macroevolutionary scales where congruence among their phylogeographic and phylogenetic structures may be observed. Within southern Africa, the four‐striped mouse genus, Rhabdomys, is parasitized by the ectoparasitic sucking louse, Polyplax arvicanthis. Molecular data recently suggested the presence of two cryptic species within P. arvicanthis that are sympatrically distributed across the distributions of four putative Rhabdomys species. We tested the hypotheses of phylogeographic congruence and cophylogeny among the two parasite lineages and the four host taxa, utilizing mitochondrial and nuclear sequence data. Despite the documented host‐specificity of P. arvicanthis, limited phylogeographic correspondence and nonsignificant cophylogeny was observed. Instead, the parasite–host evolutionary history is characterized by limited codivergence and several duplication, sorting and host‐switching events. Despite the elevated mutational rates found for P. arvicanthis, the spatial genetic structure was not more pronounced in the parasite lineages compared with the hosts. These findings may be partly attributed to larger effective population sizes of the parasite lineages, the vagility and social behaviour of Rhabdomys, and the lack of host‐specificity observed in areas of host sympatry. Further, the patterns of genetic divergence within parasite and host lineages may also be largely attributed to historical biogeographic changes (expansion‐contraction cycles). It is thus evident that the association between P. arvicanthis and Rhabdomys has been shaped by the synergistic effects of parasite traits, host‐related factors and biogeography over evolutionary time.     

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.     

Host and parasite life history interplay to yield divergent population genetic structures in two ectoparasites living on the same bat species

Host–parasite interactions are ubiquitous in nature. However, how parasite population genetic structure is shaped by the interaction between host and parasite life history remains understudied. Studies comparing multiple parasites infecting a single host can be used to investigate how different parasite life history traits interplay with host behaviour and life history. In this study, we used 10 newly developed microsatellite loci to investigate the genetic structure of a parasitic bat fly (Basilia nana). Its host, the Bechstein's bat (Myotis bechsteinii), has a social system and roosting behaviour that restrict opportunities for parasite transmission. We compared fly genetic structure to that of the host and another parasite, the wing‐mite, Spinturnix bechsteini. We found little spatial or temporal genetic structure in B. nana, suggesting a large, stable population with frequent genetic exchange between fly populations from different bat colonies. This contrasts sharply with the genetic structure of the wing‐mite, which is highly substructured between the same bat colonies as well as temporally unstable. Our results suggest that although host and parasite life history interact to yield similar transmission patterns in both parasite species, the level of gene flow and eventual spatiotemporal genetic stability is differentially affected. This can be explained by the differences in generation time and winter survival between the flies and wing‐mites. Our study thus exemplifies that the population genetic structure of parasites on a single host can vary strongly as a result of how their individual life history characteristics interact with host behaviour and life history traits.     

Dissecting the effect of a heterogeneous environment on the interaction between host and parasite fitness traits

Environmental variation can alter the probability of parasitic infection or the fitness consequence of infection, and thus has the potential to dramatically alter the dynamics of host parasite coevolution. Here we investigated the effect of a changing temperature on host-parasite interactions using the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa. By reciprocally varying (1) the temperature at which exposure to parasites occurred and (2) the temperature at which within-host parasite growth occurred, and measuring several fitness-related traits, we show that while there are temperature combinations that favour either host or parasite, there are also conditions that favour neither, that is, negative fitness consequences for the host without fitness benefits for the parasite. This result highlights the importance of considering a heterogeneous rather than static environment in coevolutionary studies, while also showing support for an optimal virulence strategy in castrating parasites.