THE EFFECT OF ELEVATED MUTATION RATES ON THE EVOLUTION OF COOPERATION AND VIRULENCE OF PSEUDOMONAS AERUGINOSA

Within-host competition between parasite genotypes can play an important role in the evolution of parasite virulence. For example, competition can increase virulence by imposing selection for parasites that replicate at a faster absolute rate within the host, but may also decrease virulence by selecting for faster relative growth rates through social exploitation of conspecifics. For many parasites, both outcomes are possible. We investigated how competition affected the evolution of virulence of the opportunistic pathogen Pseudomonas aeruginosa in caterpillar hosts, over the course of an approximately 60 generation selection experiment. We initiated infections with clonal populations of either wild-type bacteria or an isogenic mutant with an approximately 100-fold higher mutation rate, resulting in low and high between-genotype competition, respectively. We observed the evolution of increased virulence, growth rate, and public goods cheating (exploitation of extracellular iron scavenging siderophores produced by ancestral populations) in mutator but not wild-type, populations. We conclude increases in absolute within-host growth rates appear to be more important than social cheating in driving virulence evolution in this experimental context.     

From within-host interactions to epidemiological competition: a general model for multiple infections

Many hosts are infected by several parasite genotypes at a time. In these co-infected hosts, parasites can interact in various ways thus creating diverse within-host dynamics, making it difficult to predict the expression and the evolution of virulence. Moreover, multiple infections generate a combinatorial diversity of cotransmission routes at the host population level, which complicates the epidemiology and may lead to non-trivial outcomes. We introduce a new model for multiple infections, which allows any number of parasite genotypes to infect hosts and potentially coexist in the population. In our model, parasites affect one another''s within-host growth through density-dependent interactions and by means of public goods and spite. These within-host interactions determine virulence, recovery and transmission rates, which are then integrated in a transmission network. We use analytical solutions and numerical simulations to investigate epidemiological feedbacks in host populations infected by several parasite genotypes. Finally, we discuss general perspectives on multiple infections.     

VIRULENCE OF MIXED-CLONE AND SINGLE-CLONE INFECTIONS OF THE RODENT MALARIA PLASMODIUM CHABAUDI

Most evolutionary models treat virulence as an unavoidable consequence of microparasite replication and have predicted that in mixed-genotype infections, natural selection should favor higher levels of virulence than is optimal in genetically uniform infections. Increased virulence may evolve as a genetically fixed strategy, appropriate for the frequency of mixed infections in the population, or may occur as a conditional response to mixed infection, that is, a facultative strategy. Here we test whether facultative alterations in replication rates in the presence of competing genotypes occur and generate greater virulence. An important alternative, not currently incorporated in models of the evolution of virulence, is that host responses mounted against genetically diverse parasites may be more costly or less effective than those against genetically uniform parasites. If so, mixed clone infections will be more virulent for a given parasite replication rate. Two groups of mice were infected with one of two clones of Plasmodium chabaudi parasites, and three groups of mice were infected with 1:9, 5:5, or 9:1 mixtures of the same two clones. Virulence was assessed by monitoring mouse body weight and red blood cell density. Transmission stage densities were significantly higher in mixed- than in single-clone infections. Within treatment groups, transmission stage production increased with the virulence of the infection, a phenotypic correlation consistent with the genetic correlation assumed by much of the theoretical work on the evolution of virulence. Consistent with theoretical predictions of facultative alterations in virulence, we found that mice infected with both parasite clones lost more weight and had on average lower blood counts than those infected with single-clone infections. However, there was no consistent evidence of the mechanism invoked by evolutionary models that predict this effect. Replication rates and parasite densities were not always higher in ???mixed-clone infections, and for a given replication rate or parasite density, mixed-clone infections were still more virulent. Instead, prolonged anemia and increased transmission may have occured because genetically diverse infections are less rapidly cleared by hosts. Differences in maximum weight loss occured even when there were comparable parasite densities in mixed- and single-clone infections. We suggest that mounting an immune response against more that one parasite genotype is more costly for hosts, which therefore suffer higher virulence.     

Trade-offs and the evolution of virulence of microparasites: do details matter?

Models of the within-host dynamics of parasites have been used to consider the evolution of microparasites causing acute infections in vertebrate hosts. In this paper, we use these models to examine how the level of virulence to which a parasite evolves, depends on factors such as the relationship between parasite density and its rate of transmission from infected hosts, and the mechanism of parasite-induced pathogenesis. We show that changes in the terms describing transmissibility and pathogenesis may lead to dramatic differences in the level of virulence to which a parasite evolves. This suggests that no single factor is likely to be responsible for the differences in virulence of different parasites, and that understanding of the evolution of virulence of parasites will require a detailed quantitative understanding of the interaction between the parasite and its host.     

Low migration decreases interference competition among parasites and increases virulence

Competition among different parasite genotypes within a host is predicted to affect virulence. The direction of this effect, however, depends critically on the mechanisms that parasites use to compete or to cooperate with each other. One mechanism that bacteria use to compete with each other is via the production of bacteria-killing toxins, called bacteriocins. This warfare among parasites within a host is predicted to reduce the rate of host exploitation, resulting in lower virulence. By contrast, if parasites within a host are highly related, there could be a reduction in within-host conflict, increasing virulence. We examined this idea by allowing an insect-parasitic nematode (Steinernema carpocapsae) and its symbiotic bacteria (Xenorhabdus nematophila) to evolve for 20 passages under two different migration treatments (low and high). We found that host mortality rates were higher in the low-migration treatment when compared with the high-migration treatment. In addition, bacteria isolated from the same insect host inhibited each other's growth, but only in the high-migration treatment. These results show that population structure and interactions among parasites within hosts can be critical to understanding virulence.     

Parasite variation and the evolution of virulence in a Daphnia-microparasite system

Understanding genetic relationships amongst the life-history traits of parasites is crucial for testing hypotheses on the evolution of virulence. This study therefore examined variation between parasite isolates (the bacterium Pasteuria ramosa) from the crustacean Daphnia magna. From a single wild-caught infected host we obtained 2 P. ramosa isolates that differed substantially in the mortality they caused. Surprisingly, the isolate causing higher early mortality was, on average, less successful at establishing infections and had a slower growth rate within hosts. The observation that within-host replication rate was negatively correlated with mortality could violate a central assumption of the trade-off hypothesis for the evolution of virulence, but we discuss a number of caveats which caution against premature rejection of the trade-off hypothesis. We sought to test if the characteristics of these parasite isolates were constant across host genotypes in a second experiment that included 2 Daphnia host clones. The relative growth rates of the two parasite isolates did indeed depend on the host genotype (although the rank order did not change). We suggest that testing evolutionary hypotheses for virulence may require substantial sampling of both host and parasite genetic variation, and discuss how selection for virulence may change with the epidemiological state of natural populations and how this can promote genetic variation for virulence.     

Within-host dynamics of multi-species infections: facilitation, competition and virulence

Host individuals are often infected with more than one parasite species (parasites defined broadly, to include viruses and bacteria). Yet, research in infection biology is dominated by studies on single-parasite infections. A focus on single-parasite infections is justified if the interactions among parasites are additive, however increasing evidence points to non-additive interactions being the norm. Here we review this evidence and theoretically explore the implications of non-additive interactions between co-infecting parasites. We use classic Lotka-Volterra two-species competition equations to investigate the within-host dynamical consequences of various mixes of competition and facilitation between a pair of co-infecting species. We then consider the implications of these dynamics for the virulence (damage to host) of co-infections and consequent evolution of parasite strategies of exploitation. We find that whereas one-way facilitation poses some increased virulence risk, reciprocal facilitation presents a qualitatively distinct destabilization of within-host dynamics and the greatest risk of severe disease.     

Of mice and worms: are co-infections with unrelated parasite strains more damaging to definitive hosts?

Intraspecific competition between co-infecting parasites can influence the amount of virulence, or damage, they do to their host. Kin selection theory dictates that infections with related parasite individuals should have lower virulence than infections with unrelated individuals, because they benefit from inclusive fitness and increased host longevity. These predictions have been tested in a variety of microparasite systems, and in larval stage macroparasites within intermediate hosts, but the influence of adult macroparasite relatedness on virulence has not been investigated in definitive hosts. This study used the human parasite Schistosoma mansoni to determine whether definitive hosts infected with related parasites experience lower virulence than hosts infected with unrelated parasites, and to compare the results from intermediate host studies in this system. The presence of unrelated parasites in an infection decreased parasite infectivity, the ability of a parasite to infect a definitive host, and total worm establishment in hosts, impacting the less virulent parasite strain more severely. Unrelated parasite co-infections had similar virulence to the more virulent of the two parasite strains. We combine these findings with complementary studies of the intermediate snail host and describe trade-offs in virulence and selection within the life cycle. Damage to the host by the dominant strain was muted by the presence of a competitor in the intermediate host, but was largely unaffected in the definitive host. Our results in this host–parasite system suggest that unrelated infections may select for higher virulence in definitive hosts while selecting for lower virulence in intermediate hosts.     

HOST–PARASITE GENETIC INTERACTIONS AND VIRULENCE-TRANSMISSION RELATIONSHIPS IN NATURAL POPULATIONS OF MONARCH BUTTERFLIES

Evolutionary models predict that parasite virulence (parasite-induced host mortality) can evolve as a consequence of natural selection operating on between-host parasite transmission. Two major assumptions are that virulence and transmission are genetically related and that the relative virulence and transmission of parasite genotypes remain similar across host genotypes. We conducted a cross-infection experiment using monarch butterflies and their protozoan parasites from two populations in eastern and western North America. We tested each of 10 host family lines against each of 18 parasite genotypes and measured virulence (host life span) and parasite transmission potential (spore load). Consistent with virulence evolution theory, we found a positive relationship between virulence and transmission across parasite genotypes. However, the absolute values of virulence and transmission differed among host family lines, as did the rank order of parasite clones along the virulence-transmission relationship. Population-level analyses showed that parasites from western North America caused higher infection levels and virulence, but there was no evidence of local adaptation of parasites on sympatric hosts. Collectively, our results suggest that host genotypes can affect the strength and direction of selection on virulence in natural populations, and that predicting virulence evolution may require building genotype-specific interactions into simpler trade-off models.     

Multiple infections, immune dynamics, and the evolution of virulence

Understanding the effect of multiple infections is essential for the prediction (and eventual control) of virulence evolution. Some theoretical studies have considered the possibility that several strains coexist in the same host (coinfection), but few have taken their within-host dynamics explicitly into account. Here, we develop a nested approach based on a simple model for the interaction of parasite strains with their host's immune system. We study virulence evolution by linking the within-host dynamics to an epidemiological framework that incorporates multiple infections. Our model suggests that antigenically similar parasite strains cannot coexist in the long term inside a host. We also find that the optimal level of virulence increases with the efficiency of multiple infections. Finally, we notice that coinfections create heterogeneity in the host population (with susceptible hosts and infected hosts), which can lead to evolutionary branching in the parasite population and the emergence of a hypervirulent parasite strategy. We interpret this result as a parasite specialization to the infectious state of the hosts. Our study has experimental and theoretical implications in a virulence management perspective.     

Virulence evolution in response to anti-infection resistance: toxic food plants can select for virulent parasites of monarch butterflies

Host resistance to parasites can come in two main forms: hosts may either reduce the probability of parasite infection (anti-infection resistance) or reduce parasite growth after infection has occurred (anti-growth resistance). Both resistance mechanisms are often imperfect, meaning that they do not fully prevent or clear infections. Theoretical work has suggested that imperfect anti-growth resistance can select for higher parasite virulence by favouring faster-growing and more virulent parasites that overcome this resistance. In contrast, imperfect anti-infection resistance is thought not to select for increased parasite virulence, because it is assumed that it reduces the number of hosts that become infected, but not the fitness of parasites in successfully infected hosts. Here, we develop a theoretical model to show that anti-infection resistance can in fact select for higher virulence when such resistance reduces the effective parasite dose that enters a host. Our model is based on a monarch butterfly-parasite system in which larval food plants confer resistance to the monarch host. We carried out an experiment and showed that this environmental resistance is most likely a form of anti-infection resistance, through which toxic food plants reduce the effective dose of parasites that initiates an infection. We used these results to build a mathematical model to investigate the evolutionary consequences of food plant-induced resistance. Our model shows that when the effective infectious dose is reduced, parasites can compensate by evolving a higher per-parasite growth rate, and consequently a higher intrinsic virulence. Our results are relevant to many insect host-parasite systems, in which larval food plants often confer imperfect anti-infection resistance. Our results also suggest that - for parasites where the infectious dose affects the within-host dynamics - vaccines that reduce the effective infectious dose can select for increased parasite virulence.     

The expression of virulence during double infections by different parasites with conflicting host exploitation and transmission strategies

In many natural populations, hosts are found to be infected by more than one parasite species. When these parasites have different host exploitation strategies and transmission modes, a conflict among them may arise. Such a conflict may reduce the success of both parasites, but could work to the benefit of the host. For example, the less‐virulent parasite may protect the host against the more‐virulent competitor. We examine this conflict using the waterflea Daphnia magna and two of its sympatric parasites: the blood‐infecting bacterium Pasteuria ramosa that transmits horizontally and the intracellular microsporidium Octosporea bayeri that can concurrently transmit horizontally and vertically after infecting ovaries and fat tissues of the host. We quantified host and parasite fitness after exposing Daphnia to one or both parasites, both simultaneously and sequentially. Under conditions of strict horizontal transmission, Pasteuria competitively excluded Octosporea in both simultaneous and sequential double infections, regardless of the order of exposure. Host lifespan, host reproduction and parasite spore production in double infections resembled those of single infection by Pasteuria. When hosts became first vertically (transovarilly) infected with O. bayeri, Octosporea was able to withstand competition with P. ramosa to some degree, but both parasites produced less transmission stages than they did in single infections. At the same time, the host suffered from reduced fecundity and longevity. Our study demonstrates that even when competing parasite species utilize different host tissues to proliferate, double infections lead to the expression of higher virulence and ultimately may select for higher virulence. Furthermore, we found no evidence that the less‐virulent and vertically transmitting O. bayeri protects its host against the highly virulent P. ramosa.     

Cooperation and virulence in acute Pseudomonas aeruginosa infections

Background  

Efficient host exploitation by parasites is frequently likely to depend on cooperative behaviour. Under these conditions, mixed-strain infections are predicted to show lower virulence (host mortality) than are single-clone infections, due to competition favouring non-contributing social 'cheats' whose presence will reduce within-host growth. We tested this hypothesis using the cooperative production of iron-scavenging siderophores by the pathogenic bacterium Pseudomonas aeruginosa in an insect host.     

Virulence and competitive ability in an obligately killing parasite

Mixed infections are thought to have a major influence on the evolution of parasite virulence. During a mixed infection, higher within‐host parasite growth is favored under the assumption that it is critical to the competitive success of the parasite. As within‐host parasite growth may also increase damage to the host, a positive correlation is predicted between virulence and competitive success. However, when parasites must kill their hosts in order be transmitted, parasites may spend energy on directly attacking their host, even at the cost of their within‐host growth. In such systems, a negative correlation between virulence and competitive success may arise. We examined virulence and competitive ability in three sympatric species of obligately killing nematode parasites in the genus Steinernema. These nematodes exist in a mutualistic symbiosis with bacteria in the genus Xenorhabdus. Together the nematodes and their bacteria kill the insect host soon after infection, with reproduction of both species occurring mainly after host death. We found significant differences among the three nematode species in the speed of host killing. The nematode species with the lowest and highest levels of virulence were associated with the same species of Xenorhabdus, indicating that nematode traits, rather than the bacterial symbionts, may be responsible for the differences in virulence. In mixed infections, host mortality rate closely matched that associated with the more virulent species, and the more virulent species was found to be exclusively transmitted from the majority of coinfected hosts. Thus, despite the requirement of rapid host death, virulence appears to be positively correlated with competitive success in this system. These findings support a mechanistic link between parasite growth and both anti‐competitor and anti‐host factors.     

Host condition as a constraint for parasite reproduction

Environmental stress has been suggested to increase host susceptibility to infections and reduce host ability to resist parasite growth and reproduction, thus benefiting parasites. This prediction stems from expected costs of immune defence; hosts in poor condition should have less resources to be allocated to immune function. However, the alternative hypothesis for response to environmental stress is that hosts in poor condition provide less resources for parasites and/or suffer higher mortality, leading to reduced parasite growth, reproduction and survival. We contrasted these alternative hypotheses in a trematode–snail ( Diplostomum spathaceum – Lymnaea stagnalis ) system by asking: (1) how host condition affects parasite reproduction (amount and quality of produced transmission stages) and (2) how host condition affects the survival of infected host individuals. We experimentally manipulated host condition by starving the snails, and found that parasites produced fewer and poorer quality transmission stages in stressed hosts. Furthermore, starvation increased snail mortality. These findings indicate that in well-established trematode infections, reduced ability of immune allocation has no effect on host exploitation by parasites. Instead, deteriorating resources for the snail host can directly limit the amount of resources available for the parasite. This, together with increased host mortality, may have negative effects on parasite populations in the wild.     

VIRULENCE AND COMPETITIVENESS: TESTING THE RELATIONSHIP DURING INTER‐ AND INTRASPECIFIC MIXED INFECTIONS

Understanding the reasons why different parasites cause different degrees of harm to their hosts is an important objective in evolutionary biology. One group of models predicts that if hosts are infected with more than one strain or species of parasite, then competition between the parasites will select for higher virulence. While this idea makes intuitive sense, empirical data to support it are rare and equivocal. We investigated the relationship between fitness and virulence during both inter‐ and intraspecific competition for a fungal parasite of insects, Metarhizium anisopliae. Contrary to theoretical expectations, competition favored parasite strains with either a lower or a higher virulence depending on the competitor: when in interspecific competition with an entomopathogenic nematode, Steinernema feltiae, less virulent strains of the fungus were more successful, but when competing against conspecific fungi, more virulent strains were better competitors. We suggest that the nature of competition (direct via toxin production when competing against the nematode, indirect via exploitation of the host when competing against conspecific fungal strains) determines the relationship between virulence and competitive ability.     

The evolution of parasites in response to tolerance in their hosts: the good, the bad, and apparent commensalism

Tolerance to parasites reduces the harm that infection causes the host (virulence). Here we investigate the evolution of parasites in response to host tolerance. We show that parasites may evolve either higher or lower within-host growth rates depending on the nature of the tolerance mechanism. If tolerance reduces virulence by a constant factor, the parasite is always selected to increase its growth rate. Alternatively, if tolerance reduces virulence in a nonlinear manner such that it is less effective at reducing the damage caused by higher growth rates, this may select for faster or slower replicating parasites. If the host is able to completely tolerate pathogen damage up to a certain replication rate, this may result in apparent commensalism, whereby infection causes no apparent virulence but the original evolution of tolerance has been costly. Tolerance tends to increase disease prevalence and may therefore lead to more, rather than less, disease-induced mortality. If the parasite is selected, even a highly efficient tolerance mechanism may result in more individuals in total dying from disease. However, the evolution of tolerance often, although not always, reduces the individual risk of dying from infection.     

Selection for high and low virulence in the malaria parasite Plasmodium chabaudi.

What stops parasites becoming ever more virulent? Conventional wisdom and most parasite-centred models of the evolution of virulence suppose that risk of host (and, hence, parasite) death imposes selection against more virulent strains. Here we selected for high and low virulence within each of two clones of the rodent malaria parasite Plasmodium chabaudi on the basis of between-host differences in a surrogate measure of virulence--loss of live weight post-infection. Despite imposing strong selection for low virulence which mimicked 50-75% host mortality, the low virulence lines increased in virulence as much as the high virulence lines. Thus, artificial selection on between-host differences in virulence was unable to counteract natural selection for increased virulence caused by within-host selection processes. The parasite''s asexual replication rate and number of sexual transmission forms also increased in all lines, consistent with evolutionary models explaining high virulence. An upper bound to virulence, though not the asexual replication rate, was apparent, but this bound was not imposed by host mortality. Thus, we found evidence of the factors assumed to drive evolution of increased virulence, but not those thought to counter this selection.     

A meta-analysis of parasite virulence in nestling birds

Parasitism is a common cause of host mortality, but little is known about the ecological factors affecting parasite virulence (the rate of mortality among infected hosts). We reviewed 117 field estimates of parasite-induced nestling mortality in birds, showing that there was significant consistency in mortality among host and parasite taxa. Virulence increased towards the tropics in analyses of both species-specific data and phylogenetic analyses. We found evidence of greater parasite prevalence being associated with reduced virulence. Furthermore, bird species breeding in open nest sites suffered from greater parasite-induced mortality than hole-nesting species. By contrast, parasite specialization and generation time of parasites relative to that of hosts explained little variation in virulence. Likewise, there were little or no significant effects of host genetic variability, host sociality, host migration, host insular distribution or host survival on parasite virulence. These findings suggest that parasite-induced nestling mortality in birds is mainly determined by geographical location and to a smaller extent nest site and prevalence.     

Co-occurrences of parasite clones and altered host phenotype in a snail-trematode system

The frequent co-occurrence of two or more genotypes of the same parasite species in the same individual hosts has often been predicted to select for higher levels of virulence. Thus, if parasites can adjust their level of host exploitation in response to competition for resources, mixed-clone infections should have more profound impacts on the host. Trematode parasites are known to induce a wide range of modifications in the morphology (size, shell shape or ornamentation) of their snail intermediate host. Still, whether mixed-clone trematode infections have additive effects on the phenotypic alterations of the host remains to be tested. Here, we used the snail Potamopyrgus antipodarum-infected by the trematode Coitocaecum parvum to test for both the general effect of the parasite on host phenotype and possible increased host exploitation in multi-clone infections. Significant differences in size, shell shape and spinosity were found between infected and uninfected snails, and we determined that one quarter of naturally infected snails supported mixed-clone infections of C. parvum. From the parasite perspective, this meant that almost half of the clones identified in this study shared their snail host with at least one other clone. Intra-host competition may be intense, with each clone in a mixed-clone infection experiencing major reductions in volume and number of sporocysts (and consequently multiplication rate and cercarial production) compared with single-clone infections. However, there was no significant difference in the intensity of host phenotype modifications between single and multiple-clone infections. These results demonstrate that competition between parasite genotypes may be strong, and suggest that the frequency of mixed-clone infections in this system may have selected for an increased level of host exploitation in the parasite population, such that a single-clone is associated with a high degree of host phenotypic alteration.