Genotype specificity among hosts,pathogens, and beneficial microbes influences the strength of symbiont‐mediated protection

The microbial symbionts of eukaryotes influence disease resistance in many host‐parasite systems. Symbionts show substantial variation in both genotype and phenotype, but it is unclear how natural selection maintains this variation. It is also unknown whether variable symbiont genotypes show specificity with the genotypes of hosts or parasites in natural populations. Genotype by genotype interactions are a necessary condition for coevolution between interacting species. Uncovering the patterns of genetic specificity among hosts, symbionts, and parasites is therefore critical for determining the role that symbionts play in host‐parasite coevolution. Here, we show that the strength of protection conferred against a fungal pathogen by a vertically transmitted symbiont of an aphid is influenced by both host‐symbiont and symbiont‐pathogen genotype by genotype interactions. Further, we show that certain symbiont phylogenetic clades have evolved to provide stronger protection against particular pathogen genotypes. However, we found no evidence of reciprocal adaptation of co‐occurring host and symbiont lineages. Our results suggest that genetic variation among symbiont strains may be maintained by antagonistic coevolution with their host and/or their host's parasites.     

LOCAL MALADAPTATION IN THE ANTHER-SMUT FUNGUS MICROBOTRYUM VIOLACEUM TO ITS HOST PLANT SILENE LATIFOLIA: EVIDENCE FROM A CROSS-INOCULATION EXPERIMENT

Conventional wisdom holds that parasites evolve more rapidly than their hosts and are therefore locally adapted, that is, better at exploiting sympatric than allopatric hosts. We studied local adaptation in the insect-transmitted fungal pathogen Microbotryum violaceum and its host plant Silene latifolia. Infection success was tested in sympatric (local) and allopatric (foreign) combinations of pathogen and host from 14 natural populations from a metapopulation. Seedlings from up to 10 seed families from each population were exposed to sporidial suspensions from each of four fungal strains derived from the same population, from a near-by population (< 10 km distance), and from two populations at an intermediate (< 30 km) and remote (< 170 km) distance, respectively. We obtained significant pathogen X plant interactions in infection success (proportion of diseased plants) at both fungal population and strain level. There was an overall pattern of local maladaptation of this pathogen: average fungal infection success was significantly lower on sympatric hosts (mean proportion of diseased plants = 0.32 ± 0.03 SE) than on allopatric hosts (0.40 ± 0.02). Five of the 14 fungal populations showed no strong reduction in infection success on sympatric hosts, and three even tended to perform better on sympatric hosts. This pattern is consistent with models of time-lagged cycles predicting patterns of local adaptation in host-parasite systems to emerge only on average. Several factors may restrict the evolutionary potential of this pathogen relative to that of its host. First, a predominantly selfing breeding system may limit its ability to generate new virulence types by sexual recombination, whereas the obligately outcrossing host 5. latifolia may profit from rearrangement of resistance alleles by random mating. Second, populations often harbor only a few infected individuals, so virulence variation may be further reduced by drift. Third, migration rates among host plant populations are much higher than among pathogen populations, possibly because pollinators prefer healthy over diseased plants. Migration among partly isolated populations may therefore introduce novel host plant resistance variants more often than novel parasite virulence variants. That migration contributes to the coevolutionary dynamics in this system is supported by the geographic pattern of infectivity. Infection success increased over the first 10–km range of host-pathogen population distances, which is likely the natural range of gene exchange.     

THE JACK OF ALL TRADES IS MASTER OF NONE: A PATHOGEN'S ABILITY TO INFECT A GREATER NUMBER OF HOST GENOTYPES COMES AT A COST OF DELAYED REPRODUCTION

A trade‐off between a pathogen's ability to infect many hosts and its reproductive capacity on each host genotype is predicted to limit the evolution of an expanded host range, yet few empirical results provide evidence for the magnitude of such trade‐offs. Here, we test the hypothesis for a trade‐off between the number of host genotypes that a fungal pathogen can infect (host genotype range) and its reproductive capacity on susceptible plant hosts. We used strains of the oat crown rust fungus that carried widely varying numbers of virulence (avr) alleles known to determine host genotype range. We quantified total spore production and the expression of four pathogen life‐history stages: infection efficiency, time until reproduction, pustule size, and spore production per pustule. In support of the trade‐off hypothesis, we found that virulence level, the number of avr alleles per pathogen strain, was correlated with significant delays in the onset of reproduction and with smaller pustule sizes. Modeling from our results, we conclude that trade‐offs have the capacity to constrain the evolution of host genotype range in local populations. In contrast, long‐term trends in virulence level suggest that the continued deployment of resistant host lines over wide regions of the United States has generated selection for increased host genotype range.     

Host resistance and pathogen aggressiveness are key determinants of coinfection in the wild

Coinfection, whereby the same host is infected by more than one pathogen strain, may favor faster host exploitation rates as strains compete for the same limited resources. Hence, coinfection is expected to have major consequences for pathogen evolution, virulence, and epidemiology. Theory predicts genetic variation in host resistance and pathogen infectivity to play a key role in how coinfections are formed. The limited number of studies available has demonstrated coinfection to be a common phenomenon, but little is known about how coinfection varies in space, and what its determinants are. Our aim is to understand how variation in host resistance and pathogen infectivity and aggressiveness contribute to how coinfections are formed in the interaction between fungal pathogen Podosphaera plantaginis and Plantago lanceolata. Our phenotyping study reveals that more aggressive strains are more likely to form coinfections than less aggressive strains in the natural populations. In the natural populations most of the variation in coinfection is found at the individual plant level, and results from a common garden study confirm the prevalence of coinfection to vary significantly among host genotypes. These results show that genetic variation in both the host and pathogen populations are key determinants of coinfection in the wild.     

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

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

Local adaptation of a holoparasitic plant,Cuscuta europaea: variation among populations

Locally adapted parasites have higher infectivity and/or fitness on sympatric than on allopatric hosts. We tested local adaptation of a holoparasitic plant, Cuscuta europaea, to its host plant, Urtica dioica. We infected hosts from five sites with holoparasites from the same five sites and measured local adaptation in terms of infectivity and parasite performance (biomass) in a reciprocal cross‐infection experiment. The virulence of the parasite did not differ between sympatric and allopatric hosts. Overall, parasites had higher infectivity on sympatric hosts but infectivity and parasite performance varied among populations. Parasites from one of the populations showed local adaptation in terms of performance, whereas parasites from one of the populations had higher infectivity on allopatric hosts compared with sympatric hosts. This among‐population variation may be explained by random variation in parasite adaptation to host populations or by time‐lagged co‐evolutionary oscillations that lead to fluctuations in the level of local adaptation.     

Ample genetic variation but no evidence for genotype specificity in an all‐parthenogenetic host–parasitoid interaction

Antagonistic coevolution between hosts and parasites can result in negative frequency‐dependent selection and may thus be an important mechanism maintaining genetic variation in populations. Negative frequency‐dependence emerges readily if interactions between hosts and parasites are genotype‐specific such that no host genotype is most resistant to all parasite genotypes, and no parasite genotype is most infective on all hosts. Although there is increasing evidence for genotype specificity in interactions between hosts and pathogens or microparasites, the picture is less clear for insect host–parasitoid interactions. Here, we addressed this question in the black bean aphid (Aphis fabae) and its most important parasitoid Lysiphlebus fabarum. Because both antagonists are capable of parthenogenetic reproduction, this system allows for powerful tests of genotype × genotype interactions. Our test consisted of exposing multiple host clones to different parthenogenetic lines of parasitoids in all combinations, and this experiment was repeated with animals from four different sites. All aphids were free of endosymbiotic bacteria known to increase resistance to parasitoids. We observed ample genetic variation for host resistance and parasitoid infectivity, but there was no significant host clone × parasitoid line interaction, and this result was consistent across the four sites. Thus, there is no evidence for genotype specificity in the interaction between A. fabae and L. fabarum, suggesting that the observed variation is based on rather general mechanisms of defence and attack.     

VARIATION BETWEEN POPULATIONS AND LOCAL ADAPTATION IN ACANTHOCEPHALAN‐INDUCED PARASITE MANIPULATION

Many trophically transmitted parasites manipulate their intermediate host phenotype, resulting in higher transmission to the final host. However, it is not known if manipulation is a fixed adaptation of the parasite or a dynamic process upon which selection still acts. In particular, local adaptation has never been tested in manipulating parasites. In this study, using experimental infections between six populations of the acanthocephalan parasite Pomphorhynchus laevis and its amphipod host Gammarus pulex, we investigated whether a manipulative parasite may be locally adapted to its host. We compared adaptation patterns for infectivity and manipulative ability. We first found a negative effect of all parasite infections on host survival. Both parasite and host origins influenced infection success. We found a tendency for higher infectivity in sympatric versus allopatric combinations, but detailed analyses revealed significant differences for two populations only. Conversely, no pattern of local adaptation was found for behavioral manipulation, but manipulation ability varied among parasite origins. This suggests that parasites may adapt their investment in behavioral manipulation according to some of their host's characteristics. In addition, all naturally infected host populations were less sensitive to parasite manipulation compared to a naive host population, suggesting that hosts may evolve a general resistance to manipulation.     

The impact of parasite dispersal on antagonistic host-parasite coevolution

Coevolving populations of hosts and parasites are often subdivided into a set of patches connected by dispersal. Higher relative rates of parasite compared with host dispersal are expected to lead to parasite local adaptation. However, we know of no studies that have considered the implications of higher relative rates of parasite dispersal for other aspects of the coevolutionary process, such as the rate of coevolution and extent of evolutionary escalation of resistance and infectivity traits. We investigated the effect of phage dispersal on coevolution in experimental metapopulations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage SBW25Phi2. Both the rate of coevolution and the breadth of evolved infectivity and resistance ranges peaked at intermediate rates of parasite dispersal. These results suggest that parasite dispersal can enhance the evolutionary potential of parasites through provision of novel genetic variation, but that high rates of parasite dispersal can impede the evolution of parasites by homogenizing genetic variation between patches, thereby constraining coevolution.     

Host resistance and pathogen infectivity in host populations with varying connectivity

Theory predicts that hosts and pathogens will evolve higher resistance and aggressiveness in systems where populations are spatially connected than in situations in which populations are isolated and dispersal is more local. In a large cross‐inoculation experiment we surveyed patterns of host resistance and pathogen infectivity in anther‐smut diseased Viscaria alpina populations from three contrasting areas where populations range from continuous, through patchy but spatially connected to highly isolated demes. In agreement with theory, isolated populations of V. alpina were more susceptible on average than either patchily distributed or continuous populations. While increased dispersal in connected systems increases disease spread, it may also increase host gene flow and the potential for greater host resistance to evolve. In the Viscaria–Microbotryum system, pathogen infectivity mirrored patterns of host resistance with strains from the isolated populations being the least infective and strains from the more resistant continuous populations being the most infective on average, suggesting that high resistance selects for high infectivity. To our knowledge this study is the first to characterize the impacts of varying spatial connectivity on patterns of host resistance and pathogen infectivity in a natural system.     

Genetic variation in a host-parasite association: potential for coevolution and frequency-dependent selection

Abstract.— Models of host‐parasite coevolution assume the presence of genetic variation for host resistance and parasite infectivity, as well as genotype‐specific interactions. We used the freshwater crustacean Daphnia magna and its bacterial microparasite Pasteuria ramosa to study genetic variation for host susceptibility and parasite infectivity within each of two populations. We sought to answer the following questions: Do host clones differ in their susceptibility to parasite isolates? Do parasite isolates differ in their ability to infect different host clones? Are there host clone‐parasite isolate interactions? The analysis revealed considerable variation in both host resistance and parasite infectivity. There were significant host clone‐parasite isolate interactions, such that there was no single host clone that was superior to all other clones in the resistance to every parasite isolate. Likewise, there was no parasite isolate that was superior to all other isolates in infectivity to every host clone. This form of host clone‐parasite isolate interaction indicates the potential for coevolution based on frequency‐dependent selection. Infection success of original host clone‐parasite isolate combinations (i.e., those combinations that were isolated together) was significantly higher than infection success of novel host clone‐parasite isolate combinations (i.e., those combinations that were created in the laboratory). This finding is consistent with the idea that parasites track specific host genotypes under natural conditions. In addition, correspondence analysis revealed that some host clones, although distinguishable with neutral genetic markers, were susceptible to the same set of parasite isolates and thus probably shared resistance genes.     

Strong specificity in the interaction between parasitoids and symbiont‐protected hosts

Coevolution between hosts and parasites may promote the maintenance of genetic variation in both antagonists by negative frequency‐dependence if the host–parasite interaction is genotype‐specific. Here we tested for specificity in the interaction between parasitoids (Lysiphlebus fabarum) and aphid hosts (Aphis fabae) that are protected by a heritable defensive endosymbiont, the γ‐proteobacterium Hamiltonella defensa. Previous studies reported a lack of genotype specificity between unprotected aphids and parasitoids, but suggested that symbiont‐conferred resistance might exhibit a higher degree of specificity. Indeed, in addition to ample variation in host resistance as well as parasitoid infectivity, we found a strong aphid clone‐by‐parasitoid line interaction on the rates of successful parasitism. This genotype specificity appears to be mediated by H. defensa, highlighting the important role that endosymbionts can play in host–parasite coevolution.     

Parasite‐mediated selection in a natural metapopulation of Daphnia magna

Parasite‐mediated selection varying across time and space in metapopulations is expected to result in host local adaptation and the maintenance of genetic diversity in disease‐related traits. However, nonadaptive processes like migration and extinction‐(re)colonization dynamics might interfere with adaptive evolution. Understanding how adaptive and nonadaptive processes interact to shape genetic variability in life‐history and disease‐related traits can provide important insights into their evolution in subdivided populations. Here we investigate signatures of spatially fluctuating, parasite‐mediated selection in a natural metapopulation of Daphnia magna. Host genotypes from infected and uninfected populations were genotyped at microsatellite markers, and phenotyped for life‐history and disease traits in common garden experiments. Combining phenotypic and genotypic data a Q_ST–F_ST‐like analysis was conducted to test for signatures of parasite mediated selection. We observed high variation within and among populations for phenotypic traits, but neither an indication of host local adaptation nor a cost of resistance. Infected populations have a higher gene diversity (Hs) than uninfected populations and Hs is strongly positively correlated with fitness. These results suggest a strong parasite effect on reducing population level inbreeding. We discuss how stochastic processes related to frequent extinction‐(re)colonization dynamics as well as host and parasite migration impede the evolution of resistance in the infected populations. We suggest that the genetic and phenotypic patterns of variation are a product of dynamic changes in the host gene pool caused by the interaction of colonization bottlenecks, inbreeding, immigration, hybrid vigor, rare host genotype advantage and parasitism. Our study highlights the effect of the parasite in ameliorating the negative fitness consequences caused by the high drift load in this metapopulation.     

Pathogen and host genotype differently affect pathogen fitness through their effects on different life-history stages

ABSTRACT: BACKGROUND: Adaptation of pathogens to their hosts depends critically on factorsaffecting pathogen reproductive rate. While pathogen reproduction is the end result of an intricate interaction between host and pathogen, the relative contributions of host and pathogen genotype to variation in pathogen life history within the hostare not well understood. Untangling these contributions allows us to identify traits withsufficient genetic variation for selection to act and to identify mechanisms of coevolution between pathogens and their hosts. We investigated the effects of pathogen and host genotype on three life-history components of pathogen fitness; infection efficiency, latent period, and sporulation capacity, in the oat crown rust fungus, Puccinia coronata f.sp. avenae, as it infects oats (Avena sativa). RESULTS: We show that both pathogen and host genotype significantly affect total spore production butdo so through their effects on different life-history stages. Pathogen genotype has the strongest effect on the early stage of infection efficiency, while host genotype most strongly affects the later life-history stages of latent period and sporulation capacity.In addition, host genotype affected the relationship between pathogen density and the later life-history traits oflatent period and sporulation capacity. We did not find evidence of pathogen-by-host genotypic (GxG) interactions. CONCLUSION: Our results illustrate mechanisms by which variation in host populationswill affect the evolution of pathogen lifehistory. Results show that differentpathogen life-history stages have the potential to respond differently to selection by host or pathogen genotypeand suggest mechanisms of antagonistic coevolution. Pathogen populations may adapt tohost genotype through increased infection efficiency while their plant hosts may adapt by limiting the later stages ofpathogen growthand spore production within the host.     

Pathogen fitness components and genotypes differ in their sensitivity to nutrient and temperature variation in a wild plant-pathogen association

Understanding processes maintaining variation in pathogen life-history stages affecting infectivity and reproduction is a key challenge in evolutionary ecology. Models of host-parasite coevolution are based on the assumption that genetic variation for host-parasite interactions is a significant cause of variation in infection, and that variation in environmental conditions does not overwhelm the genetic basis. However, surprisingly little is known about the stability of genotype-genotype interactions under variable environmental conditions. Here, using a naturally occurring plant-pathogen interaction, I tested whether the two distinct aspects of the infection process - infectivity and transmission potential - vary over realistic nutrient and temperature gradients. I show that the initial pathogen infectivity and host resistance responses are robust over the environmental gradients. However, for compatible responses there were striking differences in how different pathogen life-history stages and host and pathogen genotypes responded to environmental variation. For some pathogen genotypes even slight changes in temperature arrested spore production, rendering the developing infection ineffectual. The response of pathogen genotypes to environmental gradients varied in magnitude and even direction, so that their rankings changed across the abiotic gradients. Hence, the variable environment of spatially structured host-parasite interactions may strongly influence the maintenance of polymorphism in pathogen life-history stages governing transmission, whereas evolutionary trajectories of infectivity may be unaffected by the surrounding environment.     

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.     

Investigating the production of sexual resting structures in a plant pathogen reveals unexpected self‐fertility and genotype‐by‐environment effects

The sexual stage of pathogens governs recombination patterns and often also provides means of surviving the off‐season. Despite its importance for evolutionary potential and between‐season epidemiology, sexual systems have not been carefully investigated for many important pathogens, and what generates variation in successful sexual reproduction of pathogens remains unexplored. We surveyed the sexually produced resting structures (chasmothecia) across 86 natural populations of fungal pathogen Podosphaera plantaginis (Ascomycota) naturally infecting Plantago lanceolata in the Åland archipelago, southwestern Finland. For this pathosystem, these resting structures are a key life‐history stage, as more than half of the local pathogen populations go extinct every winter. We uncovered substantial variation in the level of chasmothecia produced among populations, ranging from complete absence to presence on all infected leaves. We found that chasmothecia developed within clonal isolates (single‐strain cultures). Additionally, these clonal isolates all contained both MAT1‐1‐1 and MAT1‐2‐1 genes that characterize mating types in Ascomycetes. Hence, contrary to expectations, we conclude that this species is capable of haploid selfing. In controlled inoculations, we discovered that pathogen genotypes varied in their tendency to produce chasmothecia. Production of chasmothecia was also affected by ambient temperature (E) and by the interaction between temperature and pathogen genotype (G × E). These G, E and G × E effects found both at a European scale and within the Åland archipelago may partly explain the high variability observed among populations in chasmothecia levels. Consequently, they may be key drivers of the evolutionary potential and epidemiology of this highly dynamic pathosystem.     

PATHOGEN LIFE‐HISTORY TRADE‐OFFS REVEALED IN ALLOPATRY

Trade‐offs in life‐history traits is a central tenet in evolutionary biology, yet their ubiquity and relevance to realized fitness in natural populations remains questioned. Trade‐offs in pathogens are of particular interest because they may constrain the evolution and epidemiology of diseases. Here, we studied life‐history traits determining transmission in the obligate fungal pathogen, Podosphaera plantaginis, infecting Plantago lanceolata. We find that although traits are positively associated on sympatric host genotypes, on allopatric host genotypes relationships between infectivity and subsequent transmission traits change shape, becoming even negative. The epidemiological prediction of this change in life‐history relationships in allopatry is lower disease prevalence in newly established pathogen populations. An analysis of the natural pathogen metapopulation confirms that disease prevalence is lower in newly established pathogen populations and they are more prone to go extinct during winter than older pathogen populations. Hence, life‐history trade‐offs mediated by pathogen local adaptation may influence epidemiological dynamics at both population and metapopulation levels.     

Temperature checks the Red Queen? Resistance and virulence in a fluctuating environment

Numerous studies have revealed genetic variation in resistance and susceptibility in host–parasite interactions and therefore the potential for frequency‐dependent selection (Red Queen dynamics). Few studies, if any, have considered the abiotic environment as a mediating factor in these interactions. Using the pea aphid, Acyrthosiphon pisum, and its fungal pathogen, Erynia neoaphidis, as a model host–parasite system, we demonstrate how temperature can mediate the expression of genotypic variation for susceptibility and virulence. Whilst previous studies have revealed among‐clone variation in aphid resistance to this pathogen, we show that resistance rankings derived from assessments at one temperature, are not conserved across differing temperature regimes. We suggest that variation in environmental temperature, through its nonlinear impact on parasite virulence and host defence, may contribute to the general lack of evidence for frequency‐dependent selection in field systems.     

Mixed inoculations of a microsporidian parasite with horizontal and vertical infections

Mixed infections, where more than one parasite genotype is present in a single host, have been suggested to be an important factor in host–parasite interactions. As the host represents a limited resource, co-infecting parasite genotypes are expected to be under resource competition. Competition will not only modify the survival of the co-infecting genotypes, but is also likely to affect total within-host parasite growth as well as host survival and reproduction. We measured parasite infectivity and spore production of seven isolates of Octosporea bayeri (Microsporidia) and their effect on the reproduction and longevity of its host Daphnia magna (Cladocera), after single- or double-isolate inoculations through vertical and horizontal transmission. Double-isolate inoculations increased parasite infectivity and total spore production in horizontal infections, but had no significant effect on host reproduction or longevity. The increase in spore production in double-isolate inoculations was not found when infections occurred vertically. Our results suggest that, depending on the way the infection was acquired, within-host reproduction can increase as a result of parasite genetic diversity, without necessarily affecting the hosts fitness. Whether this challenges the current views of virulence evolution depends on the definitions used.