Genome-Wide Allele Frequency Changes Reveal That Dynamic Metapopulations Evolve Differently.

Two important characteristics of metapopulations are extinction-(re)colonization dynamics and gene flow between subpopulations. These processes can cause strong shifts in genome-wide allele frequencies that are generally not observed in "classical" (large, stable, and panmictic) populations. Subpopulations founded by one or a few individuals, the so-called propagule model, are initially expected to show intermediate allele frequencies at polymorphic sites until natural selection and genetic drift drive allele frequencies toward a mutation-selection-drift equilibrium characterized by a negative exponential-like distribution of the site frequency spectrum. We followed changes in site frequency spectrum distribution in a natural metapopulation of the cyclically parthenogenetic pond-dwelling microcrustacean Daphnia magna using biannual pool-seq samples collected over a 5-yr period from 118 ponds occupied by subpopulations of known age. As expected under the propagule model, site frequency spectra in newly founded subpopulations trended toward intermediate allele frequencies and shifted toward right-skewed distributions as the populations aged. Immigration and subsequent hybrid vigor altered this dynamic. We show that the analysis of site frequency spectrum dynamics is a powerful approach to understand evolution in metapopulations. It allowed us to disentangle evolutionary processes occurring in a natural metapopulation, where many subpopulations evolve in parallel. Thereby, stochastic processes like founder and immigration events lead to a pattern of subpopulation divergence, while genetic drift leads to converging site frequency spectrum distributions in the persisting subpopulations. The observed processes are well explained by the propagule model and highlight that metapopulations evolve differently from classical populations.

Genetic Drift Shapes the Evolution of a Highly Dynamic Metapopulation.

The dynamics of extinction and (re)colonization in habitat patches are characterizing features of dynamic metapopulations, causing them to evolve differently than large, stable populations. The propagule model, which assumes genetic bottlenecks during colonization, posits that newly founded subpopulations have low genetic diversity and are genetically highly differentiated from each other. Immigration may then increase diversity and decrease differentiation between subpopulations. Thus, older and/or less isolated subpopulations are expected to have higher genetic diversity and less genetic differentiation. We tested this theory using whole-genome pool-sequencing to characterize nucleotide diversity and differentiation in 60 subpopulations of a natural metapopulation of the cyclical parthenogen Daphnia magna. For comparison, we characterized diversity in a single, large, and stable D. magna population. We found reduced (synonymous) genomic diversity, a proxy for effective population size, weak purifying selection, and low rates of adaptive evolution in the metapopulation compared with the large, stable population. These differences suggest that genetic bottlenecks during colonization reduce effective population sizes, which leads to strong genetic drift and reduced selection efficacy in the metapopulation. Consistent with the propagule model, we found lower diversity and increased differentiation in younger and also in more isolated subpopulations. Our study sheds light on the genomic consequences of extinction-(re)colonization dynamics to an unprecedented degree, giving strong support for the propagule model. We demonstrate that the metapopulation evolves differently from a large, stable population and that evolution is largely driven by genetic drift.

Age Structure Eliminates the Impact of Coinfection on Epidemic Dynamics in a Freshwater Zooplankton System.

AbstractParasites often coinfect host populations and, by interacting within hosts, might change the trajectory of multiparasite epidemics. However, host-parasite interactions often change with host age, raising the possibility that within-host interactions between parasites might also change, influencing the spread of disease. We measured how heterospecific parasites interacted within zooplankton hosts and how host age changed these interactions. We then parameterized an epidemiological model to explore how age effects altered the impact of coinfection on epidemic dynamics. In our model, we found that in populations where epidemiologically relevant parameters did not change with age, the presence of a second parasite altered epidemic dynamics. In contrast, when parameters varied with host age (based on our empirical measures), there was no longer a difference in epidemic dynamics between singly infected and coinfected populations, indicating that variable age structure within a population eliminates the impact of coinfection on epidemic dynamics. Moreover, infection prevalence of both parasites was lower in populations where epidemiologically relevant parameters changed with age. Given that host population age structure changes over time and space, these results indicate that age effects are important for understanding epidemiological processes in coinfected systems and that studies focused on a single age group could yield inaccurate insights.

A new microsporidian parasite, Ordospora pajunii sp. nov (Ordosporidae), of Daphnia longispina highlights the value of genomic data for delineating species boundaries.

Speciation is a complex and continuous process that makes the delineation of species boundaries a challenging task in particular in species with little morphological differentiation, such as parasites. In this case, the use of genomic data is often necessary, such as for the intracellular Microsporidian parasites. Here, we characterize the genome of a gut parasite of the cladoceran Daphnia longispina (isolate FI-F-10), which we propose as a new species within the genus Ordospora: Ordospora pajunii sp. nov (Ordosporidae). FI-F-10 closest relative, Ordospora colligata is only found in D. magna. Both microsporidian species share several morphological features. Although it is not possible to estimate divergence times for Microsporidia due to the lack of fossil records and accelerated evolutionary rates, we base our proposal on the phylogenomic and genomic distances between both microsporidian lineages. The phylogenomic reconstruction shows that FI-F-10 forms an early diverging branch basal to the cluster that contains all known O. colligata strains. Whole-genome comparisons show that FI-F-10 presents a greater divergence at the sequence level than observed among O. colligata strains, and its genomic average nucleotide identity (ANI) values against O. colligata are beyond the intraspecific range previously established for yeast and prokaryotes. Our data confirm that the ANI metrics are useful for fine genetic divergence calibration across Microsporidia taxa. In combination with phylogenetic and ecological data, genome-based metrics provide a powerful approach to delimitate species boundaries.

Parasite resistance and parasite tolerance: insights into transgenerational immune priming in an invertebrate host.

Parasites impose different selection regimes on their hosts, which respond by increasing their resistance and/or tolerance. Parental challenge with parasites can enhance the immune response of their offspring, a phenomenon documented in invertebrates and termed transgenerational immune priming. We exposed two parental generations of the model organism Daphnia magna to the horizontally transmitted parasitic yeast Metschnikowia bicuspidata and recorded resistance- and tolerance-related traits in the offspring generation. We hypothesized that parentally primed offspring will increase either their resistance or their tolerance to the parasite. Our susceptibility assays revealed no impact of parental exposure on offspring resistance. Nonetheless, different fitness-related traits, which are indicative of tolerance, were altered. Specifically, maternal priming increased offspring production and decreased survival. Grandmaternal priming positively affected age at first reproduction and negatively affected brood size at first reproduction. Interestingly, both maternal and grandmaternal priming significantly reduced within-host-parasite proliferation. Nevertheless, Daphnia primed for two consecutive generations had no competitive advantage in comparison to unprimed ones, implying additive maternal and grandmaternal effects. Our findings do not support evidence of transgenerational immune priming from bacterial infections in the same host species, thus, emphasizing that transgenerational immune responses may not be consistent even within the same host species.

Causation without correlation: parasite-mediated frequency-dependent selection and infection prevalence.

Parasite-mediated selection is thought to maintain host genetic diversity for resistance. We might thus expect to find a strong positive correlation between host genetic diversity and infection prevalence across natural populations. Here, we used computer simulations to examine host-parasite coevolution in 20 simi-isolated clonal populations across a broad range of values for both parasite virulence and parasite fecundity. We found that the correlation between host genetic diversity and infection prevalence can be significantly positive for intermediate values of parasite virulence and fecundity. But the correlation can also be weak and statistically non-significant, even when parasite-mediated frequency-dependent selection is the sole force maintaining host diversity. Hence correlational analyses of field populations, while useful, might underestimate the role of parasites in maintaining host diversity.

Sequential infection of Daphnia magna by a gut microsporidium followed by a haemolymph yeast decreases transmission of both parasites.

Over the course of seasonal epidemics, populations of susceptible hosts may encounter a wide variety of parasites. Parasite phenology affects the order in which these species encounter their hosts, leading to sequential infections, with potentially strong effects on within-host growth and host population dynamics. Here, the cladoceran Daphnia magna was exposed sequentially to a haemolymph-infecting yeast (Metschnikowia bicuspidata) and a gut microsporidium (Ordospora colligata), with experimental treatments reflecting two possible scenarios of parasite succession. The effects of single and co-exposure were compared on parasite infectivity, spore production and the overall virulence experienced by the host. We show that neither parasite benefited from coinfection; instead, when hosts encountered Ordospora, followed by Metschnikowia, higher levels of host mortality contributed to an overall decrease in the transmission of both parasites. These results showcase an example of sequential infections generating unilateral priority effects, in which antagonistic interactions between parasites can alleviate the intensity of infection and coincide with maladaptive levels of damage inflicted on the host.

Disentangling non-specific and specific transgenerational immune priming components in host-parasite interactions.

Exposure to a pathogen primes many organisms to respond faster or more efficiently to subsequent exposures. Such priming can be non-specific or specific, and has been found to extend across generations. Disentangling and quantifying specific and non-specific effects is essential for understanding the genetic epidemiology of a system. By combining a large infection experiment and mathematical modelling, we disentangle different transgenerational effects in the crustacean model Daphnia magna exposed to different strains of the bacterial parasite Pasteuria ramosa. In the experiment, we exposed hosts to a high dose of one of three parasite strains, and subsequently challenged their offspring with multiple doses of the same (homologous) or a different (heterologous) strain. We find that exposure of Daphnia to Pasteuria decreases the susceptibility of their offspring by approximately 50%. This transgenerational protection is not larger for homologous than for heterologous parasite challenges. Methodologically, our work represents an important contribution not only to the analysis of immune priming in ecological systems but also to the experimental assessment of vaccines. We present, for the first time, an inference framework to investigate specific and non-specific effects of immune priming on the susceptibility distribution of hosts-effects that are central to understanding immunity and the effect of vaccines.

The role of spore morphology in horizontal transmission of a microsporidium of Daphnia.

The microsporidian parasite Hamiltosporidium tvaerminnensis can infect Daphnia magna both horizontally (through environmental spores) and vertically (through parthenogenetic and sexually produced eggs). The spores of H. tvaerminnensis come in three distinguishable morphologies, which are thought to have different roles in the transmission of the parasite. In this study, we examined the role of the two most common spore morphologies (i.e. oval-shaped spores and pear-shaped spores) in horizontal transmission of H. tvaerminnensis. To this end, we infected hosts with solutions consisting of either mostly oval- or mostly pear-shaped spores, and quantified infection rates, parasite-induced host mortality and mean number of parasite spores produced per host. We found that spore morphology by itself did not influence infection rates and parasite-induced host mortality. Instead, host clone and parasite isolate interacted with spore morphology in shaping infection outcome and mortality. Thus, there appear to be strong genotype-by-genotype (G × G) interactions in this system. While there is no dispute that H. tvaerminnensis can transmit both vertically and horizontally, our findings do not support theoretical predictions that different spore morphologies hold different roles in horizontal transmission of H. tvaerminnensis.

Cost of resistance to trematodes in freshwater snail populations with low clonal diversity.

BACKGROUND: The persistence of high genetic variability in natural populations garners considerable interest among ecologists and evolutionary biologists. One proposed hypothesis for the maintenance of high levels of genetic diversity relies on frequency-dependent selection imposed by parasites on host populations (Red Queen hypothesis). A complementary hypothesis suggests that a trade-off between fitness costs associated with tolerance to stress factors and fitness costs associated with resistance to parasites is responsible for the maintenance of host genetic diversity. RESULTS: The present study investigated whether host resistance to parasites is traded off with tolerance to environmental stress factors (high/low temperatures, high salinity), by comparing populations of the freshwater snail Melanoides tuberculata with low vs. high clonal diversity. Since polyclonal populations were found to be more parasitized than populations with low clonal diversity, we expected them to be tolerant to environmental stress factors. We found that clonal diversity explained most of the variation in snail survival under high temperature, thereby suggesting that tolerance to high temperatures of clonally diverse populations is higher than that of populations with low clonal diversity. CONCLUSIONS: Our results suggest that resistance to parasites may come at a cost of reduced tolerance to certain environmental stress factors.

Host age modulates parasite infectivity, virulence and reproduction.

Host age is one of the most striking differences among hosts within most populations, but there is very little data on how age-dependent effects impact ecological and evolutionary dynamics of both the host and the parasite. Here, we examined the influence of host age (juveniles, young and old adults) at parasite exposure on host susceptibility, fecundity and survival as well as parasite transmission, using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa. Younger D. magna were more susceptible to infection than older ones, regardless of host or parasite clone. Also, younger-infected D. magna became castrated faster than older hosts, but host and parasite clone effects contributed to this trait as well. Furthermore, the early-infected D. magna produced considerably more parasite transmission stages than late-infected ones, while host age at exposure did not affect virulence as it is defined in models (host mortality). When virulence is defined more broadly as the negative effects of infection on host fitness, by integrating the parasitic effects on host fecundity and mortality, then host age at exposure seems to slide along a negative relationship between host and parasite fitness. Thus, the virulence-transmission trade-off differs strongly among age classes, which in turn affects predictions of optimal virulence. Age-dependent effects on host susceptibility, virulence and parasite transmission could pose an important challenge for experimental and theoretical studies of infectious disease dynamics and disease ecology. Our results present a call for a more explicit stage-structured theory for disease, which will incorporate age-dependent epidemiological parameters.

Host age modulates within-host parasite competition.

In many host populations, one of the most striking differences among hosts is their age. While parasite prevalence differences in relation to host age are well known, little is known on how host age impacts ecological and evolutionary dynamics of diseases. Using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa, we examined how host age at exposure influences within-host parasite competition and virulence. We found that multiply-exposed hosts were more susceptible to infection and suffered higher mortality than singly-exposed hosts. Hosts oldest at exposure were least often infected and vice versa. Furthermore, we found that in young multiply-exposed hosts competition was weak, allowing coexistence and transmission of both parasite clones, whereas in older multiply-exposed hosts competitive exclusion was observed. Thus, age-dependent parasite exposure and host demography (age structure) could together play an important role in mediating parasite evolution. At the individual level, our results demonstrate a previously unnoticed interaction of the host's immune system with host age, suggesting that the specificity of immune function changes as hosts mature. Therefore, evolutionary models of parasite virulence might benefit from incorporating age-dependent epidemiological parameters.

The expression of virulence for a mixed-mode transmitted parasite in a diapausing host.

Many parasites survive harsh periods together with their hosts. Without the possibility of horizontal transmission during host diapause, parasite persistence depends entirely on host survival. We therefore hypothesize that a parasite should be avirulent during its host's diapausing stage. In contrast, the parasite may express higher virulence, i.e. parasite-induced fitness reduction of the host, during host life stages with good opportunities for horizontal transmission. Here we study the effects of a vertically and horizontally transmitted microsporidium parasite, Hamiltosporidium tvaerminnensis, on the quantity and survival of resting eggs of its host Daphnia magna. We find that the parasite did not affect egg volume, hatching success and time to hatching of the Daphnia's resting eggs, although it did strongly reduce the number of resting eggs produced by infected females, revealing high virulence during the non-diapause phase of the host's life cycle. These results also explain another aspect of this system - namely the strong decline in natural population prevalence across diapause. This decline is not caused by mortality in infected resting stages, as was previously hypothesized, but because infected female hosts produce lower rates of resting eggs. Together, these results help explain the epidemiological dynamics of a microsporidian disease and highlight the adaptive nature of life stage-dependent parasite virulence.

Coinfection frequency in water flea populations is a mere reflection of parasite diversity.

In nature, parasite species often coinfect the same host. Yet, it is not clear what drives the natural dynamics of coinfection prevalence. The prevalence of coinfections might be affected by interactions among coinfecting species, or simply derive from parasite diversity. Identifying the relative impact of these parameters is crucial for understanding patterns of coinfections. We studied the occurrence and likelihood of coinfections in natural populations of water fleas (Daphnia magna). Coinfection prevalence was within the bounds expected by chance and parasite diversity had a strong positive effect on the likelihood of coinfections. Additionally, coinfection prevalence increased over the season and became as common as a single infection. Our results demonstrate how patterns of coinfection, and particularly their temporal variation, are affected by overlapping epidemics of different parasites. We suggest that monitoring parasite diversity can help predict where and when coinfection prevalence will be high, potentially leading to increased health risks to their hosts.

The expression and evolution of virulence in multiple infections: the role of specificity, relative virulence and relative dose.

BACKGROUND: Multiple infections of the same host by different strains of the same microparasite species are believed to play a crucial role during the evolution of parasite virulence. We investigated the role of specificity, relative virulence and relative dose in determining the competitive outcome of multiple infections in the Daphnia magna-Pasteuria ramosa host-parasite system. RESULTS: We found that infections by P. ramosa clones (single genotype) were less virulent and produced more spores than infections by P. ramosa isolates (possibly containing multiple genotypes). We also found that two similarly virulent isolates of P. ramosa differed considerably in their within-host competitiveness and their effects on host offspring production when faced with coinfecting P. ramosa isolates and clones. Although the relative virulence of a P. ramosa isolate/clone appears to be a good indicator of its competitiveness during multiple infections, the relative dose may alter the competitive outcome. Moreover, spore counts on day 20 post-infection indicate that the competitive outcome is largely decided early in the parasite's growth phase, possibly mediated by direct interference or apparent competition. CONCLUSIONS: Our results emphasize the importance of epidemiology as well as of various parasite traits in determining the outcome of within-host competition. Incorporating realistic epidemiological and ecological conditions when testing theoretical models of multiple infections, as well as using a wider range of host and parasite genotypes, will enable us to better understand the course of virulence evolution.

The parasites of my rival are my friends.

The competitive exclusion principle asserts that two species cannot stably coexist in the same habitat. However, the presence of a parasite can facilitate temporary coexistence between two host species occupying the same habitat. Studies of parasite-mediated interspecific competition typically use two host species that are both susceptible to a single parasite species, as it is rare to find a resistant host species that requires a parasite to enable coexistence with a competitively superior susceptible host. We therefore investigated how two host species characterized by different susceptibility profiles affect each other when they coexist in the same habitat, by conducting two long-term mesocosm experiments in the laboratory. We followed populations of Daphnia similis coexisting with Daphnia magna, in either the presence or absence of the microsporidium Hamiltosporidium tvaerminnensis and then the bacterium Pasteuria ramosa. We found that in the absence of parasites, D. magna competitively excluded D. similis within a short period of time. However, in the presence of either parasites, the competitive ability of D. magna decreased dramatically. Our results emphasize the importance of parasites in shaping community structure and composition, by allowing coexistence of a resistant host species that would otherwise become extinct.

Out of the 'host' box: extreme off-host conditions alter the infectivity and virulence of a parasitic bacterium.

Disease agents play an important role in the ecology and life history of wild and cultivated populations and communities. While most studies focus on the adaptation of parasites to their hosts, the adaptation of free-living parasite stages to their external (off-host) environment may tell us a lot about the factors that shape the distribution of parasites. Pasteuria ramosa is an endoparasitic bacterium of the water flea Daphnia with a wide geographical distribution. Its transmission stages rest outside of the host and thus experience varying environmental regimes. We examined the life history of P. ramosa populations from four environmental conditions (i.e. groups of habitats): the factorial combinations of summer-dry water bodies or not, and winter-freeze water bodies or not. Our goal was to examine how the combination of winter temperature and summer dryness affects the parasite's ability to attach to its host and to infect it. We subjected samples of the four groups of habitats to temperatures of 20, 33, 46 and 60°C in dry and wet conditions, and exposed a susceptible clone of Daphnia magna to the treated spores. We found that spores which had undergone desiccation endured higher temperatures better than spores kept wet, both regarding attachment and subsequent infection. Furthermore, spores treated with heightened temperatures were much less infective and virulent. Even under high temperatures (60°C), exposed spores from all populations were able to attach to the host cuticle, albeit they were unable to establish infection. Our work highlights the sensitivity of a host-free resting stage of a bacterial parasite to the external environment. Long heatwaves and harsh summers, which are becoming more frequent owing to recent climate changes, may therefore pose a problem for parasite survival. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Resolving the infection process reveals striking differences in the contribution of environment, genetics and phylogeny to host-parasite interactions.

BACKGROUND: Infection processes consist of a sequence of steps, each critical for the interaction between host and parasite. Studies of host-parasite interactions rarely take into account the fact that different steps might be influenced by different factors and might, therefore, make different contributions to shaping coevolution. We designed a new method using the Daphnia magna - Pasteuria ramosa system, one of the rare examples where coevolution has been documented, in order to resolve the steps of the infection and analyse the factors that influence each of them. RESULTS: Using the transparent Daphnia hosts and fluorescently-labelled spores of the bacterium P. ramosa, we identified a sequence of infection steps: encounter between parasite and host; activation of parasite dormant spores; attachment of spores to the host; and parasite proliferation inside the host. The chances of encounter had been shown to depend on host genotype and environment. We tested the role of genetic and environmental factors in the newly described activation and attachment steps. Hosts of different genotypes, gender and species were all able to activate endospores of all parasite clones tested in different environments; suggesting that the activation cue is phylogenetically conserved. We next established that parasite attachment occurs onto the host oesophagus independently of host species, gender and environmental conditions. In contrast to spore activation, attachment depended strongly on the combination of host and parasite genotypes. CONCLUSIONS: Our results show that different steps are influenced by different factors. Host-type-independent spore activation suggests that this step can be ruled out as a major factor in Daphnia-Pasteuria coevolution. On the other hand, we show that the attachment step is crucial for the pronounced genetic specificities of this system. We suggest that this one step can explain host population structure and could be a key force behind coevolutionary cycles. We discuss how different steps can explain different aspects of the coevolutionary dynamics of the system: the properties of the attachment step, explaining the rapid evolution of infectivity and the properties of later parasite proliferation explaining the evolution of virulence. Our study underlines the importance of resolving the infection process in order to better understand host-parasite interactions.

Cloning of the unculturable parasite Pasteuria ramosa and its Daphnia host reveals extreme genotype-genotype interactions.

The degree of specificity in host-parasite interactions has important implications for ecology and evolution. Unfortunately, specificity can be difficult to determine when parasites cannot be cultured. In such cases, studies often use isolates of unknown genetic composition, which may lead to an underestimation of specificity. We obtained the first clones of the unculturable bacterium Pasteuria ramosa, a parasite of Daphnia magna. Clonal genotypes of the parasite exhibited much more specific interactions with host genotypes than previous studies using isolates. Clones of P. ramosa infected fewer D. magna genotypes than isolates and host clones were either fully susceptible or fully resistant to the parasite. Our finding enhances our understanding of the evolution of virulence and coevolutionary dynamics in this system. We recommend caution when using P. ramosa isolates as the presence of multiple genotypes may influence the outcome and interpretation of some experiments.

Pathogen dose infectivity curves as a method to analyze the distribution of host susceptibility: a quantitative assessment of maternal effects after food stress and pathogen exposure.

Stress conditions have been found to change the susceptibility of hosts or their offspring to infection. The usual method of testing at just one parasite dose level does not allow conclusions on the distribution of susceptibility. To better understand the epidemiology and evolution of host-parasite systems, however, knowledge about the distribution of host susceptibility, the parameters that characterize it, and how it changes in response to environmental conditions is required. We investigated transgenerational effects of different stress factors by exposing Daphnia magna to standard conditions, to low food levels, or to a high dose of the bacterial pathogen Pasteuria ramosa and then measuring the susceptibility of the offspring to different spore doses of the parasite. For the analysis we used a mathematical model that predicts the fraction of infected hosts at different parasite doses, allowing us to estimate the mean and variance of host susceptibility. We find that low food levels reduce both the mean and the variance of offspring susceptibility. Parasite exposure, on the other hand, widens the offspring's susceptibility distribution without affecting its mean. Our analysis uncovered previously unknown transgenerational effects on the distribution of susceptibilities. The finding of an alteration in the variance of susceptibility to infection has implications for host and parasite dynamics and can contribute to our understanding of the stability of host-parasite interactions.