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.

How does parasite environmental transmission stage concentration change before, during, and after disease outbreaks?

Outbreaks of environmentally transmitted parasites require that susceptible hosts encounter transmission stages in the environment and become infected, but we also know that transmission stages can be in the environment without triggering disease outbreaks. One challenge in understanding the relationship between environmental transmission stages and disease outbreaks is that the distribution and abundance of transmission stages outside of their hosts have been difficult to quantify. Thus, we have limited data about how changes in transmission stage abundance influence disease dynamics; moreover, we do not know whether the relationship between transmission stages and outbreaks differs among parasite species. We used digital PCR to quantify the environmental transmission stages of five parasites in six lakes in southeastern Michigan every 2 weeks from June to November 2021. At the same time, we quantified infection prevalence in hosts and host density. Our study focused on eight zooplankton host species (Daphnia spp. and Ceriodaphnia dubia) and five of their parasites from diverse taxonomic groups (bacteria, yeast, microsporidia, and oomycete) with different infection mechanisms. We found that parasite transmission stage concentration increased prior to disease outbreaks for all parasites. However, parasites differed significantly in the relative timing of peaks in transmission stage concentration and infection outbreaks. The "continuous shedder" parasites had transmission stage peaks at the same time as or slightly after the outbreak peaks. In contrast, parasites relying on host death for transmission ("obligate killers") had transmission stage peaks before outbreak peaks. For most parasites, lakes with outbreaks had higher spore concentrations than those without outbreaks, especially once an outbreak began; the exception was for a parasite, Pasteuria ramosa, with very strong genotypic specificity of infection. Overall, our results show that disease outbreaks are tightly linked to transmission stage concentration; outbreaks were preceded by increases in transmission stage concentration in the environment and then were fueled by the production of more transmission stages during the outbreak itself, with concentrations decreasing to pre-outbreak levels as outbreaks waned. Thus, tracking transmission stages in the environment improves our understanding of the drivers of disease outbreaks and reveals how parasite traits may affect these dynamics.

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.

Genetic slippage after sex maintains diversity for parasite resistance in a natural host population.

Although parasite-mediated selection is a major driver of host evolution, its influence on genetic variation for parasite resistance is not yet well understood. We monitored resistance in a large population of the planktonic crustacean Daphnia magna over 8 years, as it underwent yearly epidemics of the bacterial pathogen Pasteuria ramosa. We observed cyclic dynamics of resistance: Resistance increased throughout the epidemics, but susceptibility was restored each spring when hosts hatched from sexual resting stages. Host resting stages collected across the year showed that largely resistant host populations can produce susceptible sexual offspring. A genetic model of resistance developed for this host-parasite system, based on multiple loci and strong epistasis, is in partial agreement with our findings. Our results reveal that, despite strong selection for resistance in a natural host population, genetic slippage after sexual reproduction can be a strong factor for the maintenance of genetic diversity of host resistance.

Infection by a eukaryotic gut parasite in wild Daphnia sp. associates with a distinct bacterial community.

Host-associated bacterial communities play an important role in host fitness and resistance to diseases. Yet, few studies have investigated tripartite interaction between a host, parasite and host-associated bacterial communities in natural settings. Here, we use 16S rRNA gene amplicon sequencing to compare gut- and body- bacterial communities of wild water fleas belonging to the Daphnia longispina complex, between uninfected hosts and those infected with the common and virulent eukaryotic gut parasite Caullerya mesnili (Family: Ichthyosporea). We report community-level changes in host-associated bacteria with the presence of the parasite infection; namely decreased alpha diversity and increased beta diversity at the site of infection, i.e. host gut (but not host body). We also report decreased abundance of bacterial taxa proposed elsewhere to be beneficial for the host, and an appearance of taxa specifically associated with infected hosts. Our study highlights the host-microbiota-infection link in a natural system and raises questions about the role of host-associated microbiota in natural disease epidemics as well as the functional roles of bacteria specifically associated with infected hosts.

Demographic history shapes genomic variation in an intracellular parasite with a wide geographical distribution.

Analysing variation in a species' genomic diversity can provide insights into its historical demography, biogeography and population structure, and thus its ecology and evolution. Although such studies are rarely undertaken for parasites, they can be highly revealing because of the parasite's co-evolutionary relationships with hosts. Modes of reproduction and transmission are thought to be strong determinants of genomic diversity for parasites and vary widely among microsporidia (fungal-related intracellular parasites), which are known to have high intraspecific genetic diversity and interspecific variation in genome architecture. Here we explore genomic variation in the microsporidium Hamiltosporidium, a parasite of the freshwater crustacean Daphnia magna, looking especially at which factors contribute to nucleotide variation. Genomic samples from 18 Eurasian populations and a new, long-read-based reference genome were used to determine the roles that reproduction mode, transmission mode and geography play in determining population structure and demographic history. We demonstrate two main Hamiltosporidium tvaerminnensis lineages and a pattern of isolation-by-distance, but note an absence of congruence between these two parasite lineages and the two Eurasian host lineages. We suggest a comparatively recent parasite spread through Northern Eurasian host populations after a change from vertical to mixed-mode transmission and the loss of sexual reproduction. While gaining knowledge about the ecology and evolution of this focal parasite, we also identify common features that shape variation in genomic diversity for many parasites, such as distinct modes of reproduction and the intertwining of host-parasite demographies.

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.

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.

The Role of Microbiome and Genotype in Daphnia magna upon Parasite Re-Exposure.

Recently, it has been shown that the community of gut microorganisms plays a crucial role in host performance with respect to parasite tolerance. Knowledge, however, is lacking on the role of the gut microbiome in mediating host tolerance after parasite re-exposure, especially considering multiple parasite infections. We here aimed to fill this knowledge gap by studying the role of the gut microbiome on tolerance in Daphnia magna upon multiple parasite species re-exposure. Additionally, we investigated the role of the host genotype in the interaction between the gut microbiome and the host phenotypic performance. A microbiome transplant experiment was performed in which three germ-free D. magna genotypes were exposed to a gut microbial inoculum and a parasite community treatment. The gut microbiome inocula were pre-exposed to the same parasite communities or a control treatment. Daphnia performance was monitored, and amplicon sequencing was performed to characterize the gut microbial community. Our experimental results showed that the gut microbiome plays no role in Daphnia tolerance upon parasite re-exposure. We did, however, find a main effect of the gut microbiome on Daphnia body size reflecting parasite specific responses. Our results also showed that it is rather the Daphnia genotype, and not the gut microbiome, that affected parasite-induced host mortality. Additionally, we found a role of the genotype in structuring the gut microbial community, both in alpha diversity as in the microbial composition.

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.

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

Genotypic variation in parasite avoidance behaviour and other mechanistic, nonlinear components of transmission.

Traditional epidemiological models assume that transmission increases proportionally to the density of parasites. However, empirical data frequently contradict this assumption. General yet mechanistic models can explain why transmission depends nonlinearly on parasite density and thereby identify potential defensive strategies of hosts. For example, hosts could decrease their exposure rates at higher parasite densities (via behavioural avoidance) or decrease their per-parasite susceptibility when encountering more parasites (e.g. via stronger immune responses). To illustrate, we fitted mechanistic transmission models to 19 genotypes of Daphnia dentifera hosts over gradients of the trophically acquired parasite, Metschnikowia bicuspidata. Exposure rate (foraging, F) frequently decreased with parasite density (Z), and per-parasite susceptibility (U) frequently decreased with parasite encounters (F×Z). Consequently, infection rates (F×U×Z) often peaked at intermediate parasite densities. Moreover, host genotypes varied substantially in these responses. Exposure rates remained constant for some genotypes but decreased sensitively with parasite density for others (up to 78%). Furthermore, genotypes with more sensitive foraging/exposure also foraged faster in the absence of parasites (suggesting 'fast and sensitive' versus 'slow and steady' strategies). These relationships suggest that high densities of parasites can inhibit transmission by decreasing exposure rates and/or per-parasite susceptibility, and identify several intriguing axes for the evolution of host defence.

Toxins or medicines? Phytoplankton diets mediate host and parasite fitness in a freshwater system.

Diets must satisfy the everyday metabolic requirements of organisms and can also serve as medicines to combat disease. Currently, the medicinal role of diets is much better understood in terrestrial than in aquatic ecosystems. This is surprising because phytoplankton species synthesize secondary metabolites with known antimicrobial properties. Here, we investigated the medicinal properties of phytoplankton (including toxin-producing cyanobacteria) against parasites of the dominant freshwater herbivore, Daphnia. We fed Daphnia dentifera on green algae and toxic cyanobacteria diets known to vary in their nutritional quality and toxin production, and an additional diet of Microcystis with added pure microcystin-LR. We then exposed Daphnia to fungal and bacterial parasites. Anabaena, Microcystis and Chlorella diets prevented infection of Daphnia by the fungal parasite Metschnikowia, while Nodularia toxins increased offspring production by infected hosts. In contrast to their medicinal effects against Metschnikowia, toxic phytoplankton generally decreased the fitness of Daphnia infected with the bacterial parasite, Pasteuria. We also measured the amount of toxin produced by phytoplankton over time. Concentrations of anatoxin-a produced by Anabaena increased in the presence of Metschnikowia, suggesting parasite-induced toxin production. Our research illustrates that phytoplankton can serve as toxins or medicines for their consumers, depending upon the identity of their parasites.

Molecular and structural assessment of microsporidia infecting daphnids: The "obtusa-like" microsporidia, a branch of the monophyletic Agglomeratidae clade, with the establishment of a new genus Conglomerata.

Microsporidia (Opisthosporidia, Microsporidia) are frequent parasites of planktonic cladocerans, including Daphnia (Crustacea, Branchiopoda). Analysis of available molecular data (ITS region and partial ssu and lsu rDNA) of these parasites indicates that many microsporidia infecting daphnids have a common ancestor and represent a large clade, which splits during evolution into a number of well supported subclades. These subclades are cytologically different but may be most conveniently characterised by their specific ITS barcode. We have analysed one of these subclades and we describe a new microsporidian genus and species combination, and assemble a large group of structurally indistinguishable microsporidian parasites that infect adipose cells of their hosts and form pyriform spores of a certain type ("obtuse spores"). Obtuse spores are non-infectious by feeding to their crustacean hosts and it is plausible that microsporidia forming them actually are parasites of insects with aquatic larval stages, with an obligate two-host life cycle, analogous to the Amblyospora life cycle involving copepods and mosquitoes.

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.

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.

A new microsporidium Fibrillaspora daphniae g. n. sp. n. infecting Daphnia magna (Crustacea: Cladocera) in Siberia and its taxonomic placing within a new family Fibrillasporidae and new superfamily Tubulinosematoidea (Opisthosporidia: Microsporidia).

Infection with a new microsporidium, Fibrillaspora daphniae g. n. sp. n., was found in a local Daphnia magna population in Tomsk region (Western Siberia, Russia) at the prevalence rate of 52%. Histological sections showed parasite cells entirely encompassing the host haemocoel. Methanol-fixed spores were elongate, oval, 4.8 ± 0.3 µm × 2.3 ± 0.2 µm in size. All developmental stages were in direct contact with the host cell cytoplasm, with single nuclei, and division by binary fission. The sporont surface was covered with an additional outer layer composed of fine tubules. The spores possessed a thick endospore, large posterior vacuole filled with electron-dense granules, and a bipartite polaroplast composed of anterior lamellar and posterior globular elements. The polar tube was slightly anisofilar, with 13-19 coils arranged in one row; the two posterior coils were of lesser diameter. The small subunit ribosomal RNA gene sequence was deposited in Genbank under accession # MF278272. Considering the sister relationship between Fibrillanosema crangonycis and our new isolate described here as Fibrillaspora daphniae, we propose a new family Fibrillasporidae fam. n. to contain these two genera and the descendants of their common ancestor. A new superfamily Tubulinosematoidea superfam. n. is proposed as a monophyletic assemblage of Fibrillasporidae fam. n. and Tubulinosematidae.

Parasites driving host diversity: Incidence of disease correlated with Daphnia clonal turnover.

According to the Red Queen hypothesis, clonal diversity in asexual populations could be maintained by negative frequency-dependant selection by coevolving parasites. If common clones are selected against and rare clones gain a concomitant advantage, we expect that clonal turnover should be faster during parasite epidemics than between them. We tested this hypothesis exploring field data of the Daphnia-Caullerya host-parasite system. The clonal make-up and turnover of the Daphnia host population was tracked with high temporal resolution from 1998 until 2013, using first allozyme and later microsatellite markers. Significant differences in the clonal composition between random and infected subsamples of Daphnia populations were detected on six of seven tested occasions, confirming genetic specificity of the host-parasite interaction in this system. We used time series analysis to compare the rates of host clonal turnover to the incidence of parasitism, and found that Caullerya prevalence was significantly associated with microsatellite-based clonal turnover. As alternate hypotheses, we further tested whether turnover was related to a variety of biotic, abiotic, and host demographic parameters. Other significant correlates of turnover were cyanobacterial biomass and (weakly) temperature. Overall, parasitism seems to be a strong driver of host clonal turnover, in support of the Red Queen hypothesis.

Rapid evolution rescues hosts from competition and disease but-despite a dilution effect-increases the density of infected hosts.

Virulent parasites can depress the densities of their hosts. Taxa that reduce disease via dilution effects might alleviate this burden. However, 'diluter' taxa can also depress host densities through competition for shared resources. The combination of disease and interspecific competition could even drive hosts extinct. Then again, genetically variable host populations can evolve in response to both competitors and parasites. Can rapid evolution rescue host density from the harm caused by these ecological enemies? How might such evolution influence dilution effects or the size of epidemics? In a mesocosm experiment with planktonic hosts, we illustrate the joint harm of competition and disease: hosts with constrained evolutionary ability (limited phenotypic variation) suffered greatly from both. However, populations starting with broader phenotypic variation evolved stronger competitive ability during epidemics. In turn, enhanced competitive ability-driven especially by parasites-rescued host densities from the negative impacts of competition, disease, and especially their combination. Interspecific competitors reduced disease (supporting dilution effects) even when hosts rapidly evolved. However, this evolutionary response also elicited a potential problem. Populations that evolved enhanced competitive ability and maintained robust total densities also supported higher densities of infections. Thus, rapid evolution rescued host densities but also unleashed larger epidemics.

Concentration and timing of application reveal strong fungistatic effect of tebuconazole in a Daphnia-microparasitic yeast model.

Given the importance of pollutant effects on host-parasite relationships and disease spread, the main goal of this study was to assess the influence of different exposure scenarios for the fungicide tebuconazole (concentration×timing of application) on a Daphnia-microparasitic yeast experimental system. Previous results had demonstrated that tebuconazole is able to suppress Metschnikowia bicuspidata infection at ecologically-relevant concentrations; here, we aimed to obtain an understanding of the mechanism underlying the anti-parasitic (fungicidal or fungistatic) action of tebuconazole. We exposed the Daphnia-yeast system to four nominal tebuconazole concentrations at four timings of application (according to the predicted stage of parasite development), replicated on two Daphnia genotypes, in a fully crossed experiment. An "all-or-nothing" effect was observed, with tebuconazole completely suppressing infection from 13.5µgl-1 upwards, independent of the timing of tebuconazole application. A follow-up experiment confirmed that the suppression of infection occurred within a narrow range of tebuconazole concentrations (3.65-13.5µgl-1), although a later application of the fungicide had to be compensated for by a slight increase in concentration to elicit the same anti-parasitic effect. The mechanism behind this anti-parasitic effect seems to be the inhibition of M. bicuspidata sporulation, since tebuconazole was effective in preventing ascospore production even when applied at a later time. However, this fungicide also seemed to affect the vegetative growth of the yeast, as demonstrated by the enhanced negative effect of the parasite (increasing mortality in one of the host genotypes) at a later time of application of tebuconazole, when no signs of infection were observed. Fungicide contamination can thus affect the severity and spread of disease in natural populations, as well as the inherent co-evolutionary dynamics in host-parasite systems.