Explaining parasite virulence is a great challenge for evolutionary biology. Intuitively, parasites that depend on their hosts for their survival should be benign to their hosts, yet many parasites cause harm. One explanation for this is that within-host competition favors virulence, with more virulent strains having a competitive advantage in genetically diverse infections. This idea, which is well supported in theory, remains untested empirically. Here we provide evidence that within-host competition does indeed select for high parasite virulence. We examine the rodent malaria Plasmodium chabaudi in laboratory mice, a parasite-host system in which virulence can be easily monitored and competing strains quantified by using strain-specific real-time PCR. As predicted, we found a strong relationship between parasite virulence and competitive ability, so that more virulent strains have a competitive advantage in mixed-strain infections. In transmission experiments, we found that the strain composition of the parasite populations in mosquitoes was directly correlated with the composition of the bloodstage parasite population. Thus, the outcome of within-host competition determined relative transmission success. Our results imply that within-host competition is a major factor driving the evolution of virulence and can explain why many parasites harm their hosts.competition ͉ evolution ͉ parasite ͉ Plasmodium ͉ mixed infection E xplaining virulence is fundamental to understanding the life history of parasites, arguably the most abundant group of creatures on the planet (1). The problem is to explain why parasites, which rely on their hosts for survival and fitness, should cause disease or indeed kill their hosts (2-6). Many explanations of parasite virulence have been put forward (3, 4), but the idea that has received the most attention is that virulence is a consequence of a parasite's efforts to maximize its fitness: parasites require extensive within-host replication to achieve transmission to the next host, but at the same time such replication damages host tissues, increasing the chances of killing the host (2, 3, 7-9). Higher levels of virulence than predicted by this model, however, could arise due to within-host competition between parasite strains (9-14). Many, if not most, parasite infections consist of genetically distinct strains of the same parasite species or contain virulent mutants that have arisen de novo (15). It is generally assumed that parasites that exploit their hosts prudently suffer great fitness losses in hosts simultaneously infected with more aggressive parasites. This is because virulent parasites could kill the host or competitively exclude prudent parasites before the latter have realized transmission. Even though host death also reduces the fitness of virulent parasites, prudent parasites suffer disproportionately and are eliminated by natural selection, a process commonly known as ''the tragedy of the commons'' (16).Several authors (17, 18) have gone so far as to claim that reducing t...

Malaria infections frequently consist of mixtures of drug-resistant and drug-sensitive parasites. If crowding occurs, where clonal population densities are suppressed by the presence of coinfecting clones, removal of susceptible clones by drug treatment could allow resistant clones to expand into the newly vacated niche space within a host. Theoretical models show that, if such competitive release occurs, it can be a potent contributor to the strength of selection, greatly accelerating the rate at which resistance spreads in a population. A variety of correlational field data suggest that competitive release could occur in human malaria populations, but direct evidence cannot be ethically obtained from human infections. Here we show competitive release after pyrimethamine curative chemotherapy of acute infections of the rodent malaria Plasmodium chabaudi in laboratory mice. The expansion of resistant parasite numbers after treatment resulted in enhanced transmission-stage densities. After the elimination or near-elimination of sensitive parasites, the number of resistant parasites increased beyond that achieved when a competitor had never been present. Thus, a substantial competitive release occurred, markedly elevating the fitness advantages of drug resistance above those arising from survival alone. This finding may explain the rapid spread of drug resistance and the subsequently brief useful lifespans of some antimalarial drugs. In a second experiment, where subcurative chemotherapy was administered, the resistant clone was only partly released from competitive suppression and experienced a restriction in the size of its expansion after treatment. This finding raises the prospect of harnessing in-host ecology to slow the spread of drug resistance.competition ͉ evolution of drug resistance ͉ Plasmodium chabaudi ͉ transmission ͉ in-host ecology

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The nature of the association between viral fitness and virulence remains elusive in vertebrate virus systems, partly due to a lack of in vivo experiments using statistically sufficient numbers of replicate hosts. We examined the relationship between virulence and fitness in Infectious hematopoietic necrosis virus (IHNV), in vivo, in intact living rainbow trout. Trout were infected with a high or low virulence genotype of M genogroup IHNV, or a mixture of the two genotypes, so as to calculate relative fitness and the effect of a competition environment on fitness. Fitness was measured as total viral load in the host at time of peak viral density, quantified by genotype-specific quantitative RT-PCR (qRT-PCR). The more virulent IHNV genotype reached higher densities in both single and mixed infections. There was no effect of competition on the performance of either genotype. Our results suggest a positive link between IHNV genotype fitness and virulence.

Conspecific competition occurs in a multitude of organisms, particularly in parasites, where several clones are commonly sharing limited resources inside their host. In theory, increased or decreased transmission investment might maximize parasite fitness in the face of competition, but, to our knowledge, this has not been tested experimentally. We developed and used a clone-specific, stage-specific, quantitative PCR protocol to quantify Plasmodium chabaudi replication and transmission stage densities in mixed-clone infections. We co-infected mice from two strains with an avirulent and virulent parasite clone and found competitive suppression of in-host (blood-stage) parasite densities and generally corresponding reductions in transmission stage production, with the virulent clone obtaining overall competitive superiority. In response to competitive suppression, there was little evidence of any alteration in transmission stage investment, apart from a small reduction by one of the two clones in one of the two host strains. This alteration did not result in a competitive advantage, although it might have reduced the disadvantage. This study supports much of the current literature, which predicts that conspecific in-host competition will result in a competitive advantage and positive selection for virulent clones and thus the evolution of higher virulence.

Infectious diseases are economically detrimental to aquaculture, and with continued expansion and intensification of aquaculture, the importance of managing infectious diseases will likely increase in the future. Here, we use evolution of virulence theory, along with examples, to identify aquaculture practices that might lead to the evolution of increased pathogen virulence. We identify eight practices common in aquaculture that theory predicts may favor evolution toward higher pathogen virulence. Four are related to intensive aquaculture operations, and four others are related specifically to infectious disease control. Our intention is to make aquaculture managers aware of these risks, such that with increased vigilance, they might be able to detect and prevent the emergence and spread of increasingly troublesome pathogen strains in the future.

Viral fitness is an active area of research, with recent work involving an expanded number of human, non-human vertebrate, invertebrate, plant, and bacterial viruses. Many publications deal with RNA viruses associated with major disease emergence events, such as HIV-1, influenza virus, and Dengue virus. Study topics include drug resistance, immune escape, viral emergence, host jumps, mutation effects, quasispecies diversity, and mathematical models of viral fitness. Important recent trends include increasing use of in vivo systems to assess vertebrate virus fitness, and a broadening of research beyond replicative fitness to also investigate transmission fitness and epidemiologic fitness. This is essential for a more integrated understanding of overall viral fitness, with implications for disease management in the future.

FCT, PEst, OE/MAT/UI0209/2011, Award Number U54GM088558 from the National Institute of General Medical Science