Jacobus C. de Roode

WITHIN‐HOST COMPETITION IN GENETICALLY DIVERSE MALARIA INFECTIONS: PARASITE VIRULENCE AND COMPETITIVE SUCCESS

Abstract Humans and animals often become coinfected with pathogen strains that differ in virulence. The ensuing interaction between these strains can, in theory, be a major determinant of the direction of selection on virulence genes in pathogen populations. Many mathematical analyses of this assume that virulent pathogen lineages have a competitive advantage within coinfected hosts and thus predict that pathogens will evolve to become more virulent where genetically diverse infections are common. Although the implications of these studies are relevant to both fundamental biology and medical science, direct empirical tests for relationships between virulence and competitive ability are lacking. Here we use newly developed strain‐specific real‐time quantitative polymerase chain reaction protocols to determine the pairwise competitiveness of genetically divergent Plasmodium chabaudi clones that represent a wide range of innate virulences in their rodent host. We found that even against their background of widely varying genotypic and antigenic properties, virulent clones had a competitive advantage in the acute phase of mixed infections. The more virulent a clone was relative to its competitor, the less it suffered from competition. This result confirms our earlier work with parasite lines derived from a single clonal lineage by serial passage and supports the virulencecompetitive ability assumption of many theoretical models. To the extent that our rodent model captures the essence of the natural history of malaria parasites, public health interventions which reduce the incidence of mixed malaria infections should have beneficial consequences by reducing the selection for high virulence.

Virulence-transmission trade-offs and population divergence in virulence in a naturally occurring butterfly parasite

Why do parasites harm their hosts? Conventional wisdom holds that because parasites depend on their hosts for survival and transmission, they should evolve to become benign, yet many parasites cause harm. Theory predicts that parasites could evolve virulence (i.e., parasite-induced reductions in host fitness) by balancing the transmission benefits of parasite replication with the costs of host death. This idea has led researchers to predict how human interventions—such as vaccines—may alter virulence evolution, yet empirical support is critically lacking. We studied a protozoan parasite of monarch butterflies and found that higher levels of within-host replication resulted in both higher virulence and greater transmission, thus lending support to the idea that selection for parasite transmission can favor parasite genotypes that cause substantial harm. Parasite fitness was maximized at an intermediate level of parasite replication, beyond which the cost of increased host mortality outweighed the benefit of increased transmission. A separate experiment confirmed genetic relationships between parasite replication and virulence, and showed that parasite genotypes from two monarch populations caused different virulence. These results show that selection on parasite transmission can explain why parasites harm their hosts, and suggest that constraints imposed by host ecology can lead to population divergence in parasite virulence.

Dynamics of Multiple Infection and Within‐Host Competition in Genetically Diverse Malaria Infections

Within‐host competition between coinfecting parasite strains shapes the evolution of parasite phenotypes such as virulence and drug resistance. Although this evolution has a strong theoretical basis, within‐host competition has rarely been studied experimentally, particularly in medically relevant pathogens with hosts that have pronounced specific and nonspecific immune responses against coinfecting strains. We investigated multiple infection in malaria, using two pairs of genetically distinct clones of the rodent malaria Plasmodium chabaudi in mice. Clones were inoculated into mice simultaneously or 3 or 11 days apart, and population sizes were tracked using immunofluorescence or quantitative polymerase chain reaction. In all experiments, at least one of the two clones suffered strong competitive suppression, probably through both resource‐ and immune‐mediated (apparent) competition. Clones differed in intrinsic competitive ability, but prior residency was also an important determinant of competitive outcome. When clones infected mice first, they did not suffer from competition, but they did when infecting mice at the same time or after their competitor, more so the later they infected their host. Consequently, clones that are competitively inferior in head‐to‐head competition can be competitively superior if they infect hosts first. These results are discussed in the light of strain‐specific immunity, drug resistance, and virulence evolution theory.

Competitive release and facilitation of drug-resistant parasites after therapeutic chemotherapy in a rodent malaria model

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.

The Role of Immune‐Mediated Apparent Competition in Genetically Diverse Malaria Infections

Competitive interactions between coinfecting genotypes of the same pathogen can impose selection on virulence, but the direction of this selection depends on the mechanisms behind the interactions. Here, we investigate how host immune responses contribute to competition between clones in mixed infections of the rodent malaria parasite Plasmodium chabaudi. We studied single and mixed infections of a virulent and an avirulent clone and compared the extent of competition in immunodeficient and immunocompetent mice (nude mice and T cell–reconstituted nude mice, respectively). In immunocompetent mice, the avirulent clone suffered more from competition than did the virulent clone. The competitive suppression of the avirulent clone was alleviated in immunodeficient mice. Moreover, the relative density of the avirulent clone in mixed infections was higher in immunodeficient than in immunocompetent mice. We conclude that immune‐mediated interactions contributed to competitive suppression of the avirulent clone, although other mechanisms, presumably competition for resources such as red blood cells, must also be important. Because only the avirulent clone suffered from immune‐mediated competition, this mechanism should contribute to selection for increased virulence in mixed infections in this host‐parasite system. As far as we are aware, this is the first direct experimental evidence of immune‐mediated apparent competition in any host‐parasite system.

Self-Medication in Animals

Animal self-medication against parasites is more widespread than previously thought, with profound implications for host-parasite biology. The concept of antiparasite self-medication in animals typically evokes images of chimpanzees seeking out medicinal herbs to treat their diseases (1, 2). These images stem partly from the belief that animals can medicate themselves only when they have high cognitive abilities that allow them to observe, learn, and make conscious decisions (3). However, any concept of self-medication based solely on learning is inadequate. Many animals can use medication through innate rather than learned responses. The growing list of animal pharmacists includes moths (4), ants (5), and fruit flies (6). The fact that these animals self-medicate has profound implications for the ecology and evolution of animal hosts and their parasites.

Non-immunological defense in an evolutionary framework

After parasite infection, invertebrates activate immune system-based defenses such as encapsulation and the signaling pathways of the innate immune system. However, hosts are often able to defend against parasites without using these mechanisms. The non-immunological defenses, such as behaviors that prevent or combat infection, symbiont-mediated defense, and fecundity compensation, are often ignored but can be important in host-parasite dynamics. We review recent studies showing that heritable variation in these traits exists among individuals, and that they are costly to activate and maintain. We also discuss findings from genome annotation and expression studies to show how immune system-based and non-immunological defenses interact. Placing these studies into an evolutionary framework emphasizes their importance for future studies of host-parasite coevolution.

Host heterogeneity is a determinant of competitive exclusion or coexistence in genetically diverse malaria infections.

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.

Why infectious disease research needs community ecology

Bringing ecology to infection The tools we use to investigate infectious diseases tend to focus on specific one-host–one-pathogen relationships, but pathogens often have complex life cycles involving many hosts. Johnson et al. review how such complexity is analyzed by community ecologists. Ecologists have the investigative tools to probe cause and effect relationships that change with spatial scale in multispecies communities. These techniques are used to monitor the ways in which communities change through time and to probe the heterogeneity that characterizes individuals, species, and assemblages—all issues that are also essential for disease specialists to understand. Science, this issue 10.1126/science.1259504 BACKGROUND Despite ongoing advances in biomedicine, infectious diseases remain a major threat to human health, economic sustainability, and wildlife conservation. This is in part a result of the challenges of controlling widespread or persistent infections that involve multiple hosts, vectors, and parasite species. Moreover, many contemporary disease threats involve interactions that manifest across nested scales of biological organization, from disease progression at the within-host level to emergence and spread at the regional level. For many such infections, complete eradication is unlikely to be successful, but a broader understanding of the community in which host-parasite interactions are embedded will facilitate more effective management. Recent advances in community ecology, including findings from traits-based approaches and metacommunity theory, offer the tools and concepts to address the complexities arising from multispecies, multiscale disease threats. ADVANCES Community ecology aims to identify the factors that govern the structure, assembly, and dynamics of ecological communities. We describe how analytical and conceptual approaches from this discipline can be used to address fundamental challenges in disease research, such as (i) managing the ecological complexity of multihost-multiparasite assemblages; (ii) identifying the drivers of heterogeneities among individuals, species, and regions; and (iii) quantifying how processes link across multiple scales of biological organization to drive disease dynamics. We show how a community ecology framework can help to determine whether infection is best controlled through “defensive” approaches that reduce host suitability or through “offensive” approaches that dampen parasite spread. Examples of defensive approaches are the strategic use of wildlife diversity to reduce host and vector transmission, and taking advantage of antagonism between symbionts to suppress within-host growth and pathology. Offensive approaches include the targeted control of superspreading hosts and the reduction of human-wildlife contact rates to mitigate spillover. By identifying the importance of parasite dispersal and establishment, a community ecology framework can offer additional insights about the scale at which disease should be controlled. OUTLOOK Ongoing technological advances are rapidly overcoming previous barriers in data quality and quantity for complex, multispecies systems. The emerging synthesis of “disease community ecology” offers the tools and concepts necessary to interpret these data and use that understanding to inform the development of more effective disease control strategies in humans and wildlife. Looking forward, we emphasize the increasing importance of tight integration among surveillance, community ecology analyses, and public health implementation. Building from the rich legacy of whole-system manipulations in community ecology, we further highlight the value of large-scale experiments for understanding host-pathogen interactions and designing effective control measures. Through this blending of data, theory, and analytical approaches, we can understand how interactions between parasites within hosts, hosts within populations, and host species within ecological communities combine to drive disease dynamics, thereby providing new ways to manage emerging infections. The community ecology of disease. (A) Interactions between parasites can complicate management. Among Tsimane villagers, treatment of hookworms increases infections by Giardia lamblia. (B) Similarly, understanding how ecological communities of hosts assemble can help forecast changes in disease. Biodiversity losses can promote interactions between white-footed mice and deer ticks, leading to an increase in the risk of Lyme disease from Borrelia burgdorferi. [Credits: (A) A. Pisor, CDC, F. Dubs; (B) J. Brunner, T. Shears, NIH] Infectious diseases often emerge from interactions among multiple species and across nested levels of biological organization. Threats as diverse as Ebola virus, human malaria, and bat white-nose syndrome illustrate the need for a mechanistic understanding of the ecological interactions underlying emerging infections. We describe how recent advances in community ecology can be adopted to address contemporary challenges in disease research. These analytical tools can identify the factors governing complex assemblages of multiple hosts, parasites, and vectors, and reveal how processes link across scales from individual hosts to regions. They can also determine the drivers of heterogeneities among individuals, species, and regions to aid targeting of control strategies. We provide examples where these principles have enhanced disease management and illustrate how they can be further extended.

The genetics of monarch butterfly migration and warning colouration

The monarch butterfly, Danaus plexippus, is famous for its spectacular annual migration across North America, recent worldwide dispersal, and orange warning colouration. Despite decades of study and broad public interest, we know little about the genetic basis of these hallmark traits. Here we uncover the history of the monarch’s evolutionary origin and global dispersal, characterize the genes and pathways associated with migratory behaviour, and identify the discrete genetic basis of warning colouration by sequencing 101 Danaus genomes from around the globe. The results rewrite our understanding of this classic system, showing that D. plexippus was ancestrally migratory and dispersed out of North America to occupy its broad distribution. We find the strongest signatures of selection associated with migration centre on flight muscle function, resulting in greater flight efficiency among migratory monarchs, and that variation in monarch warning colouration is controlled by a single myosin gene not previously implicated in insect pigmentation.