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...

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Resistance of Plasmodium falciparum to drugs such as chloroquine and sulfadoxine-pyrimethamine is a major problem in malaria control. Artemisinin (ART) derivatives, particularly in combination with other drugs, are thus increasingly used to treat malaria, reducing the probability that parasites resistant to the components will emerge. Although stable resistance to artemisinin has yet to be reported from laboratory or field studies, its emergence would be disastrous because of the lack of alternative treatments. Here, we report for the first time, to our knowledge, genetically stable and transmissible ART and artesunate (ATN)-resistant malaria parasites. Each of two lines of the rodent malaria parasite Plosmodium chabaudi chabaudi, grown in the presence of increasing concentrations of ART or ATN, showed 15-fold and 6-fold increased resistance to ART and ATN, respectively. Resistance remained stable after cloning, freeze-thawing, after passage in the absence of drug, and transmission through mosquitoes. The nucleotide sequences of the possible genetic modulators of ART resistance (mdr1, cg10, tctp, and atp6) of sensitive and resistant parasites were compared. No mutations in these genes were identified. In addition we investigated whether changes in the copy number of these genes could account for resistance but found that resistant parasites retained the same number of copies as their sensitive progenitors. We believe that this is the first report of a malaria parasite with genetically stable and transmissible resistance to artemisinin or its derivatives.

During an infection, malaria parasites compete for limited amounts of food and enemy-free space. Competition affects parasite growth rate, transmission and virulence, and is thus important for parasite evolution. Much evolutionary theory assumes that virulent clones outgrow avirulent ones, favouring the evolution of higher virulence. We infected laboratory mice with a mixture of two Plasmodium chabaudi clones: one virulent, the other avirulent. Using real-time quantitative PCR to track the two parasite clones over the course of the infection, we found that the virulent clone overgrew the avirulent clone. However, host genotype had a major effect on the outcome of competition. In a relatively resistant mouse genotype (C57Bl/6J), the avirulent clone was suppressed below detectable levels after 10 days, and apparently lost from the infection. By contrast, in more susceptible mice (CBA/Ca), the avirulent clone was initially suppressed, but it persisted, and during the chronic phase of infection it did better than it did in single infections. Thus, the qualitative outcome of competition depended on host genotype. We suggest that these differences may be explained by different immune responses in the two mouse strains. Host genotype and resistance could therefore play a key role in the outcome of within-host competition between parasite clones and in the evolution of parasite virulence.

Vaccine research in malaria has a high priority. However, identification of specific antigens as candidates for vaccines against asexual blood stages of malaria parasites has been based on largely circumstantial evidence. We describe here how genes encoding target antigens of strain-specific immunity in malaria can be directly located in the parasite's genome without prior information concerning their identity, by the method we call linkage group selection. Two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi, each of which induces an immunity in laboratory mice that is more protective against challenge with itself than with the heterologous strain, were genetically crossed, and the uncloned cross progeny selected in mice that had been made partially immune by infection and drug cure with one or the other parental strain. Proportions of parental alleles in the selected and unselected cross progeny were compared by using quantitative genome-wide molecular markers. A small number, including groups of linked markers forming socalled selection valleys, were markedly reduced under strainspecific immune pressure. A very prominent selection valley was found to contain the gene for merozoite surface protein-1, a major candidate antigen for malaria vaccine development, at the locus at which the strongest reduction under strain-specific immune selection was detected. Closely linked to the merozoite surface protein-1 gene was a gene containing the signature motif of the ring-infected erythrocyte surface antigen family. Another affected locus, unlinked to this selection valley, contained a member of the serine repeat antigen gene family.amplified fragment length polymorphism ͉ merozoite surface protein