Increased virulence due to multiple infection in Daphnia leads to limited growth in 1 of 2 co-infecting microsporidian parasites.

Recent outbreaks of various infectious diseases have highlighted the ever-present need to understand the drivers of the outbreak and spread of disease. Although much of the research investigating diseases focuses on single infections, natural systems are dominated by multiple infections. These infections may occur simultaneously, but are often acquired sequentially, which may alter the outcome of infection. Using waterfleas (Daphnia magna) as a model organism, we examined the outcome of sequential and simultaneous multiple infections with 2 microsporidian parasites (Ordospora colligata and Hamiltosporidium tvaerminnensis) in a fully factorial design with 9 treatments and 30 replicates. We found no differences between simultaneous and sequential infections. However, H. tvaerminnensis fitness was impeded by multiple infection due to increased host mortality, which gave H. tvaerminnensis less time to grow. Host fecundity was also reduced across all treatments, but animals infected with O. colligata at a younger age produced the fewest offspring. As H. tvaerminnensis is both horizontally and vertically transmitted, this reduction in offspring may have further reduced H. tvaerminnensis fitness in co-infected treatments. Our findings suggest that in natural populations where both species co-occur, H. tvaerminnensis may evolve to higher levels of virulence following frequent co-infection by O. colligata.

Experimental evidence of size-selective harvest and environmental stochasticity effects on population demography, fluctuations and non-linearity.

Theory and analyses of fisheries data sets indicate that harvesting can alter population structure and destabilise non-linear processes, which increases population fluctuations. We conducted a factorial experiment on the population dynamics of Daphnia magna in relation to size-selective harvesting and stochasticity of food supply. Harvesting and stochasticity treatments both increased population fluctuations. Timeseries analysis indicated that fluctuations in control populations were non-linear, and non-linearity increased substantially in response to harvesting. Both harvesting and stochasticity induced population juvenescence, but harvesting did so via the depletion of adults, whereas stochasticity increased the abundance of juveniles. A fitted fisheries model indicated that harvesting shifted populations towards higher reproductive rates and larger-magnitude damped oscillations that amplify demographic noise. These findings provide experimental evidence that harvesting increases the non-linearity of population fluctuations and that both harvesting and stochasticity increase population variability and juvenescence.

Alternate patterns of temperature variation bring about very different disease outcomes at different mean temperatures.

The dynamics of host-parasite interactions are highly temperature-dependent and may be modified by increasing frequency and intensity of climate-driven heat events. Here, we show that altered patterns of temperature variance lead to an almost order-of-magnitude shift in thermal performance of host and pathogen life-history traits over and above the effects of mean temperature and, moreover, that different temperature regimes affect these traits differently. We found that diurnal fluctuations of ±3°C lowered infection rates and reduced spore burden compared to constant temperatures in our focal host Daphnia magna exposed to the microsporidium parasite Ordospora colligata. In contrast, a 3-day heatwave (+6°C) did not affect infection rates, but increased spore burden (relative to constant temperatures with the same mean) at 16°C, while reducing burden at higher temperatures. We conclude that changing patterns of climate variation, superimposed on shifts in mean temperatures due to global warming, may have profound and unanticipated effects on disease dynamics.

Global warming is increasing average temperatures and causing extreme temperature fluctuations and heatwaves. These changes may affect when, where, and how often infectious disease outbreaks occur. This could have profound impacts on agriculture, human health, and wildlife. Studying how extreme temperatures or temperature fluctuations alter infections in laboratory animals may help scientists to better understand the impact of climate change on disease. A small aquatic invertebrate, such as a water flea, is one good candidate for such studies. These tiny creatures can be grown in small glass jars in temperature-controlled aquariums. Kunze, Luijckx et al. show that temperature fluctuations and heat waves have complex effects on parasitic infections in water fleas. In the experiments, water fleas housed with a parasite that infects them were exposed to constant temperatures, fluctuating temperatures, or three-day heatwaves, while being kept at a broad range of mean water temperatures. Then, Kunze, Luijckx et al. measured how these conditions affected the water fleas' longevity, reproduction, and parasite infections. This revealed that temperature variations had a unique effect on the life span, and reproduction and infection rates of the water fleas, depending on the average water temperature the animals were kept at. Heatwaves drastically increased the number of parasites in the water fleas at an average water temperature of 16 °C but had no effect at all or decreased the number of parasites at 19 °C and 22 °C, respectively. Similarly, at high average water temperatures (>24 °C), temperature fluctuations reduced the number of water fleas infected with parasites and the number of parasites in each infected flea. Moreover, the maximum temperature at which parasites were able to cause infections was 5 °C lower under fluctuating temperatures than under constant temperatures. Kunze and Luijckx et al. show that consistent high temperatures, temperature changes, extreme weather events, and mean water temperature affect disease outcomes in water fleas. More studies are needed to assess how temperature variations change the course of diseases in other organisms and to understand the underlying mechanisms. Learning more about disease-temperature interactions will help scientists predict climate change-driven disease outbreaks.

Rapid evolution generates synergism between multiple stressors: Linking theory and an evolution experiment.

Global change encompasses many co-occurring anthropogenic stressors. Understanding the interactions between these multiple stressors, whether they be additive, antagonistic or synergistic, is critical for ecosystem managers when prioritizing which stressors to mitigate in the face of global change. While such interactions between stressors appear prevalent, it remains unclear if and how these interactions change over time, as the majority of multiple-stressor studies rarely span multiple generations of study organisms. Although meta-analyses have reported some intriguing temporal trends in stressor interactions, for example that synergism may take time to emerge, the mechanistic basis for such observations is unknown. In this study, by analysing data from an evolution experiment with the rotifer Brachionus calyciflorus (~35 generations and 31,320 observations), we show that adaptation to multiple stressors shifts stressor interactions towards synergism. We show that trade-offs, where populations cannot optimally perform multiple tasks (i.e. adapting to multiple stressors), generate this bias towards synergism. We also show that removal of stressors from evolved populations does not necessarily increase fitness and that there is variation in the evolutionary trajectories of populations that experienced the same stressor regimes. Our results highlight outstanding questions at the interface between evolution and global change biology, and illustrate the importance of considering rapid adaptation when managing or restoring ecosystems subjected to multiple stressors under global change.

Experimental evidence of warming-induced disease emergence and its prediction by a trait-based mechanistic model.

Predicting the effects of seasonality and climate change on the emergence and spread of infectious disease remains difficult, in part because of poorly understood connections between warming and the mechanisms driving disease. Trait-based mechanistic models combined with thermal performance curves arising from the metabolic theory of ecology (MTE) have been highlighted as a promising approach going forward; however, this framework has not been tested under controlled experimental conditions that isolate the role of gradual temporal warming on disease dynamics and emergence. Here, we provide experimental evidence that a slowly warming host-parasite system can be pushed through a critical transition into an epidemic state. We then show that a trait-based mechanistic model with MTE functional forms can predict the critical temperature for disease emergence, subsequent disease dynamics through time and final infection prevalence in an experimentally warmed system of Daphnia and a microsporidian parasite. Our results serve as a proof of principle that trait-based mechanistic models using MTE subfunctions can predict warming-induced disease emergence in data-rich systems-a critical step towards generalizing the approach to other systems.

Predicting the Thermal and Allometric Dependencies of Disease Transmission via the Metabolic Theory of Ecology.

The metabolic theory of ecology (MTE) provides a general framework of allometric and thermal dependence that may be useful for predicting how climate change will affect disease spread. Using Daphnia magna and a microsporidian gut parasite, we conducted two experiments across a wide thermal range and fitted transmission models that utilize MTE submodels for transmission parameters. We decomposed transmission into contact rate and probability of infection and further decomposed probability of infection into a product of gut residence time (GRT) and per-parasite infection rate of gut cells. Contact rate generally increased with temperature and scaled positively with body size, whereas infection rate had a narrow hump-shaped thermal response and scaled negatively with body size. GRT increased with host size and was longest at extreme temperatures. GRT and infection rate inside the gut combined to create a 3.5 times higher probability of infection for the smallest relative to the largest individuals. Small temperature changes caused large differences in transmission. We also fit several alternative transmission models to data at individual temperatures. The more complex models-parasite antagonism or synergism and host heterogeneity-did not substantially improve the fit to the data. Our results show that transmission rate is the product of several distinct thermal and allometric functions that can be predicted continuously across temperature and host size using the MTE.

Mutation accumulation in populations of varying size: large effect mutations cause most mutational decline in the rotifer Brachionus calyciflorus under UV-C radiation.

Theory predicts that fitness decline via mutation accumulation will depend on population size, but there are only a few direct tests of this key idea. To gain a qualitative understanding of the fitness effect of new mutations, we performed a mutation accumulation experiment with the facultative sexual rotifer Brachionus calyciflorus at six different population sizes under UV-C radiation. Lifetime reproduction assays conducted after ten and sixteen UV-C radiations showed that while small populations lost fitness, fitness losses diminished rapidly with increasing population size. Populations kept as low as 10 individuals were able to maintain fitness close to the nonmutagenized populations throughout the experiment indicating that selection was able to remove the majority of large effect mutations in small populations. Although our results also seem to imply that small populations are effectively immune to mutational decay, we caution against this interpretation. Given sufficient time, populations of moderate to large size can experience declines in fitness from accumulating weakly deleterious mutations as demonstrated by fitness estimates from simulations and, tentatively, from a long-term experiment with populations of moderate size. There is mounting evidence to suggest that mutational distributions contain a heavier tail of large effects. Our results suggest that this is also true when the mutational spectrum is altered by UV radiation.

Empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics.

The complexity of host-parasite interactions makes it difficult to predict how host-parasite systems will respond to climate change. In particular, host and parasite traits such as survival and virulence may have distinct temperature dependencies that must be integrated into models of disease dynamics. Using experimental data from Daphnia magna and a microsporidian parasite, we fitted a mechanistic model of the within-host parasite population dynamics. Model parameters comprising host aging and mortality, as well as parasite growth, virulence, and equilibrium abundance, were specified by relationships arising from the metabolic theory of ecology. The model effectively predicts host survival, parasite growth, and the cost of infection across temperature while using less than half the parameters compared to modeling temperatures discretely. Our results serve as a proof of concept that linking simple metabolic models with a mechanistic host-parasite framework can be used to predict temperature responses of parasite population dynamics at the within-host level.

Higher rates of sex evolve during adaptation to more complex environments.

A leading hypothesis for the evolutionary maintenance of sexual reproduction proposes that sex is advantageous because it facilitates adaptation. Changes in the environment stimulate adaptation but not all changes are equivalent; a change may occur along one or multiple environmental dimensions. In two evolution experiments with the facultatively sexual rotifer Brachionus calyciflorus, we test how environmental complexity affects the evolution of sex by adapting replicate populations to various environments that differ from the original along one, two, or three environmental dimensions. Three different estimates of fitness (growth, lifetime reproduction, and population density) confirmed that populations adapted to their new environment. Growth measures revealed an intriguing cost of complex adaptations: populations that adapted to more complex environments lost greater amounts of fitness in the original environment. Furthermore, both experiments showed that B. calyciflorus became more sexual when adapting to a greater number of environmental dimensions. Common garden experiments confirmed that observed changes in sex were heritable. As environments in nature are inherently complex these findings help explain why sex is maintained in natural populations.

A Population Biology Perspective on the Stepwise Infection Process of the Bacterial Pathogen Pasteuria ramosa in Daphnia.

The infection process of many diseases can be divided into series of steps, each one required to successfully complete the parasite's life and transmission cycle. This approach often reveals that the complex phenomenon of infection is composed of a series of more simple mechanisms. Here we demonstrate that a population biology approach, which takes into consideration the natural genetic and environmental variation at each step, can greatly aid our understanding of the evolutionary processes shaping disease traits. We focus in this review on the biology of the bacterial parasite Pasteuria ramosa and its aquatic crustacean host Daphnia, a model system for the evolutionary ecology of infectious disease. Our analysis reveals tremendous differences in the degree to which the environment, host genetics, parasite genetics and their interactions contribute to the expression of disease traits at each of seven different steps. This allows us to predict which steps may respond most readily to selection and which steps are evolutionarily constrained by an absence of variation. We show that the ability of Pasteuria to attach to the host's cuticle (attachment step) stands out as being strongly influenced by the interaction of host and parasite genotypes, but not by environmental factors, making it the prime candidate for coevolutionary interactions. Furthermore, the stepwise approach helps us understanding the evolution of resistance, virulence and host ranges. The population biological approach introduced here is a versatile tool that can be easily transferred to other systems of infectious disease.

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.

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.

A matching-allele model explains host resistance to parasites.

The maintenance of genetic variation and sex despite its costs has long puzzled biologists. A popular idea, the Red Queen Theory, is that under rapid antagonistic coevolution between hosts and their parasites, the formation of new rare host genotypes through sex can be advantageous as it creates host genotypes to which the prevailing parasite is not adapted. For host-parasite coevolution to lead to an ongoing advantage for rare genotypes, parasites should infect specific host genotypes and hosts should resist specific parasite genotypes. The most prominent genetics capturing such specificity are matching-allele models (MAMs), which have the key feature that resistance for two parasite genotypes can reverse by switching one allele at one host locus. Despite the lack of empirical support, MAMs have played a central role in the theoretical development of antagonistic coevolution, local adaptation, speciation, and sexual selection. Using genetic crosses, we show that resistance of the crustacean Daphnia magna against the parasitic bacterium Pasteuria ramosa follows a MAM. Simulation results show that the observed genetics can explain the maintenance of genetic variation and contribute to the maintenance of sex in the facultatively sexual host as predicted by the Red Queen Theory.

Sex-specific effects of a parasite evolving in a female-biased host population.

BACKGROUND: Males and females differ in many ways and might present different opportunities and challenges to their parasites. In the same way that parasites adapt to the most common host type, they may adapt to the characteristics of the host sex they encounter most often. To explore this hypothesis, we characterized host sex-specific effects of the parasite Pasteuria ramosa, a bacterium evolving in naturally, strongly, female-biased populations of its host Daphnia magna. RESULTS: We show that the parasite proliferates more successfully in female hosts than in male hosts, even though males and females are genetically identical. In addition, when exposure occurred when hosts expressed a sexual dimorphism, females were more infected. In both host sexes, the parasite causes a similar reduction in longevity and leads to some level of castration. However, only in females does parasite-induced castration result in the gigantism that increases the carrying capacity for the proliferating parasite. CONCLUSIONS: We show that mature male and female Daphnia represent different environments and reveal one parasite-induced symptom (host castration), which leads to increased carrying capacity for parasite proliferation in female but not male hosts. We propose that parasite induced host castration is a property of parasites that evolved as an adaptation to specifically exploit female hosts.

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.