Partitioning the mechanisms by which genetic diversity of parasite infections affects total parasite load

Genetically-diverse parasite infections are common in nature, however what mechanisms influence parasite load are still under debate. Rauch et al. found consistently lower parasite loads in genetically-mixed infections compared to uniform infections. Using the additive partition of Loreau and Hector they demonstrated that this lower parasite load was due to negative complementarity effects, but they only found weak selection effects. Complementarity effects arise from differentiation among genotypes that accrue equally to all genotypes, while selection effects arise from unexpectedly high performance of certain genotypes in mixed infections. However, selection effects might arise either because genotypes with certain traits perform unexpectedly well in mixed infections at the expense of other genotypes ('dominance effects', DEs), or because genotypes with certain traits perform unexpectedly well, but not at the expense of others genotypes ('trait dependent complementarity effects', TDCEs). Here, we reanalyze the data of Rauch et al. using the tripartite partition of Fox to separate DEs, TDCEs and trait-independent complementarity effects (TICEs, corresponding to the complementarity effect of Loreau and Hector). We found significantly negative TDCEs that contribute strongly to the low parasite loads in mixed infections. We suggest novel, testable hypotheses to explain negative TDCEs. Ours is the first study to demonstrate consistently-strong TDCEs, which are rare in studies of the productivity of plant mixtures. Our results highlight the importance of testing for TDCEs, rather than assuming them to be small. We discuss the interpretation and value of the tripartite partition as an analytical tool complementary to more mechanistic approaches.     

Synchronous attack is advantageous: mixed genotype infections lead to higher infection success in trematode parasites

Co-infecting parasite genotypes typically compete for host resources limiting their fitness. The intensity of such competition depends on whether parasites are reproducing in a host, or using it primarily as a transmission vehicle while not multiplying in host tissues (referred to as 'competition hypothesis'). Alternatively, simultaneous attack and co-infection by several parasite genotypes might facilitate parasite infection because such a diverse attack could present an additional challenge to host immune defence (referred to as 'facilitation hypothesis'). We tested the competition hypothesis by comparing the production of transmission stages (cercariae) from snails infected with one or two genotypes of the trematode Diplostomum pseudospathaceum. We found that cercarial production did not differ between the two groups of snails, suggesting lower per genotype production in double infections, and competition for host resources. Second, we tested the facilitation hypothesis by comparing parasite infection success on fishes (proportion of parasites establishing in the host) using cercariae originating from single-infected snails, double-infected snails and artificial mixtures of the single genotypes. In both cases, we found higher infection success when fishes were challenged with two parasite genotypes instead of one, supporting the facilitation hypothesis. Our results suggest that constraints defining the success of multiple genotype infections in parasites with multiple host life cycles include both between-genotype resource competition in the host and performance of host immune defences against a diverse parasite challenge.     

Relatedness affects competitive performance of a parasitic plant (Cuscuta europaea) in multiple infections

Theoretical models predict that parasite relatedness affects the outcome of competition between parasites, and the evolution of parasite virulence. We examined whether parasite relatedness affects competition between parasitic plants (Cuscuta europaea) that share common host plants (Urtica dioica). We infected hosts with two parasitic plants that were either half-siblings or nonrelated. Relative size asymmetry between the competing parasites was significantly higher in the nonrelated infections compared to infections with siblings. This higher asymmetry was caused by the fact that the performance of some parasite genotypes decreased and that of others increased when grown in multiple infections with nonrelated parasites. This result agrees with the predictions of theories on the evolution of parasite virulence: to enhance parasite transmission, selection may favour reduced competition with genetically related parasites in hosts infected by several genotypes. However, in contrast to the most common predictions, nonrelated infections were not more virulent than the sibling infections.     

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.     

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.     

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.     

Plasmodium falciparum genotype population dynamics in asymptomatic children from Senegal

In areas where malaria is endemic, infected individuals generally harbor a mixture of genetically distinct Plasmodium falciparum parasite populations. For the first time, we studied temporal variations of blood parasite densities and circulating genotypes in asymptomatic Senegalese children, at time intervals as short as 4-12 h. Twenty-one Senegalese children, presenting with an asymptomatic P. falciparum infection, were sampled eight times within three days. Parasite density was assessed by thick blood smears, and all infecting genotypes were quantified by the fragment-analysis method. Parasite densities showed dramatic fluctuations up to a 1 to 1,000 ratio, with at least one peak of parasite density. Polyclonal infections were detected in all children, with a multiplicity of infection of 5.2-6.8 genotypes per child. A single sample never reflected the full complexity of the parasite populations hosted by a given individual. Genotypes with different behaviors were detected in all children, some genotypes undergoing major fluctuations, while others were highly stable during the follow-up. A single peripheral blood sampling does not reflect the total parasite load. Repeated sampling is needed to have a more detailed scheme of parasite population dynamics and a better knowledge of the true complexity of an infection.     

Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.     

Fecundity compensation and tolerance to a sterilizing pathogen in Daphnia

Hosts are armed with several lines of defence in the battle against parasites: they may prevent the establishment of infection, reduce parasite growth once infected or persevere through mechanisms that reduce the damage caused by infection, called tolerance. Studies on tolerance in animals have focused on mortality, and sterility tolerance has not been investigated experimentally. Here, we tested for genetic variation in the multiple steps of defence when the invertebrate Daphnia magna is infected with the sterilizing bacterial pathogen Pasteuria ramosa: anti-infection resistance, anti-growth resistance and the ability to tolerate sterilization once infected. When exposed to nine doses of a genetically diverse pathogen inoculum, six host genotypes varied in their average susceptibility to infection and in their parasite loads once infected. How host fecundity changed with increasing parasite loads did not vary between genotypes, indicating that there was no genetic variation for this measure of fecundity tolerance. However, genotypes differed in their level of fecundity compensation under infection, and we discuss how, by increasing host fitness without targeting parasite densities, fecundity compensation is consistent with the functional definition of tolerance. Such infection-induced life-history shifts are not traditionally considered to be part of the immune response, but may crucially reduce harm (in terms of fitness loss) caused by disease, and are a distinct source of selection on pathogens.     

Genotype-Specific vs. Cross-Reactive Host Immunity against a Macroparasite

Vertebrate hosts often defend themselves against several co-infecting parasite genotypes simultaneously. This has important implications for the ecological dynamics and the evolution of host defence systems and parasite strategies. For example, it can drive the specificity of the adaptive immune system towards high genotype-specificity or cross-reactivity against several parasite genotypes depending on the sequence and probability of re-infections. However, to date, there is very little evidence on these interactions outside mammalian disease literature. In this study we asked whether genotype-specific or cross-reactive responses dominate in the adaptive immune system of a fish host towards a common macroparasite. In other words, we investigated if the infection success of a parasite genotype is influenced by the immunization genotype. We reciprocally immunized and re-exposed rainbow trout (Oncorhynchus mykiss) to a range of genotypes of the trematode eye fluke Diplostomum pseudospathaceum, and measured infection success of the parasite. We found that the infection success of the parasite genotypes in the re-exposure did not depend on the immunization genotype. While immunization reduced average infection success by 31%, the reduction was not larger against the initial immunization genotype. Our results suggest significant cross-reactivity, which may be advantageous for the host in genetically diverse re-exposures and have significant evolutionary implications for parasite strategies. Overall, our study is among the first to demonstrate cross-reactivity of adaptive immunity against genetically diverse macroparasites with complex life cycles.     

Within‐host interactions shape virulence‐related traits of trematode genotypes

Within‐host interactions between co‐infecting parasites can significantly influence the evolution of key parasite traits, such as virulence (pathogenicity of infection). The type of interaction is expected to predict the direction of selection, with antagonistic interactions favouring more virulent genotypes and synergistic interactions less virulent genotypes. Recently, it has been suggested that virulence can further be affected by the genetic identity of co‐infecting partners (G × G interactions), complicating predictions on disease dynamics. Here, we used a natural host–parasite system including a fish host and a trematode parasite to study the effects of G × G interactions on infection virulence. We exposed rainbow trout (Oncorhynchus mykiss) either to single genotypes or to mixtures of two genotypes of the eye fluke Diplostomum pseudospathaceum and estimated parasite infectivity (linearly related to pathogenicity of infection, measured as coverage of eye cataracts) and relative cataract coverage (controlled for infectivity). We found that both traits were associated with complex G × G interactions, including both increases and decreases from single infection to co‐infection, depending on the genotype combination. In particular, combinations where both genotypes had low average infectivity and relative cataract coverage in single infections benefited from co‐infection, while the pattern was opposite for genotypes with higher performance. Together, our results show that infection outcomes vary considerably between single and co‐infections and with the genetic identity of the co‐infecting parasites. This can result in variation in parasite fitness and consequently impact evolutionary dynamics of host–parasite interactions.     

Reciprocal Interaction Matrix Reveals Complex Genetic and Dose-Dependent Specificity among Coinfecting Parasites

Abstract Understanding genetic specificity in factors determining the outcome of host-parasite interactions is especially important as it contributes to parasite epidemiology, virulence, and maintenance of genetic variation. Such specificity, however, is still generally poorly understood. We examined genetic specificity in interactions among coinfecting parasites. In natural populations, individual hosts are often simultaneously infected by multiple parasite species and genotypes that interact. Such interactions could maintain genetic variation in parasite populations if they are genetically specific so that the relative fitness of parasite genotypes varies across host individuals depending on (1) the presence/absence of coinfections and/or (2) the genetic composition of the coinfecting parasite community. We tested these predictions using clones of fish eye flukes Diplostomum pseudospathaceum and Diplostomum gasterostei. We found that interactions among parasites had a strong genetic basis and that this modified genetic variation in infection success of D. pseudospathaceum between single and multiple infections as well as across multiply infected host individuals depending on the genetic identity of the coinfecting D. gasterostei. The relative magnitude of these effects, however, depended on the exposure dose, suggesting that ecological factors can modify genetic interactions between parasites.     

Interactions among bacterial strains and fluke genotypes shape virulence of co-infection

Most studies of virulence of infection focus on pairwise host–parasite interactions. However, hosts are almost universally co-infected by several parasite strains and/or genotypes of the same or different species. While theory predicts that co-infection favours more virulent parasite genotypes through intensified competition for host resources, knowledge of the effects of genotype by genotype (G × G) interactions between unrelated parasite species on virulence of co-infection is limited. Here, we tested such a relationship by challenging rainbow trout with replicated bacterial strains and fluke genotypes both singly and in all possible pairwise combinations. We found that virulence (host mortality) was higher in co-infections compared with single infections. Importantly, we also found that the overall virulence was dependent on the genetic identity of the co-infecting partners so that the outcome of co-infection could not be predicted from the respective virulence of single infections. Our results imply that G × G interactions among co-infecting parasites may significantly affect host health, add to variance in parasite fitness and thus influence evolutionary dynamics and ecology of disease in unexpected ways.     

Host genetic architecture in single and multiple infections

Hosts are often target to multiple simultaneous infections by genetically diverse parasite strains. The interaction among these strains and the interaction of each strain with the host was shown to have profound effects on the evolution of parasite traits. Host factors like genetic architecture of resistance have so far been largely neglected. To see whether genetic architecture differs between different kinds of infections we used joint scaling analysis to compare the genetic components of resistance in the red flour beetle Tribolium castaneum exposed to single and multiple strains of the microsporidian Nosema whitei. Our results indicate that additive, dominance and epistatic components were more important in single infections whereas maternal components play a decisive role in multiple infections. In detail, parameter estimates of additive, dominance and epistatic components correlated positively between single and multiple infections, whereas maternal components correlated negatively. These findings may suggest that specificity of host–parasite interactions are mediated by genetic and especially epistatic components whereas maternal effects constitute a more general form of resistance.     

Population genetic structure and adaptation of malaria parasites on the edge of endemic distribution

To determine whether the major human malaria parasite Plasmodium falciparum exhibits fragmented population structure or local adaptation at the northern limit of its African distribution where the dry Sahel zone meets the Sahara, samples were collected from diverse locations within Mauritania over a range of ~1000 km. Microsatellite genotypes were obtained for 203 clinical infection samples from eight locations, and Illumina paired‐end sequences were obtained to yield high coverage genomewide single nucleotide polymorphism (SNP) data for 65 clinical infection samples from four locations. Most infections contained single parasite genotypes, reflecting low rates of transmission and superinfection locally, in contrast to the situation seen in population samples from countries further south. A minority of infections shared related or identical genotypes locally, indicating some repeated transmission of parasite clones without recombination. This caused some multilocus linkage disequilibrium and local divergence, but aside from the effect of repeated genotypes there was minimal differentiation between locations. Several chromosomal regions had elevated integrated haplotype scores (|iHS|) indicating recent selection, including those containing drug resistance genes. A genomewide F_ST scan comparison with previous sequence data from an area in West Africa with higher infection endemicity indicates that regional gene flow prevents genetic isolation, but revealed allele frequency differentiation at three drug resistance loci and an erythrocyte invasion ligand gene. Contrast of extended haplotype signatures revealed none to be unique to Mauritania. Discrete foci of infection on the edge of the Sahara are genetically highly connected to the wider continental parasite population, and local elimination would be difficult to achieve without very substantial reduction in malaria throughout the region.     

Distribution of schistosome infections in molluscan hosts at different levels of parasite prevalence

Biomphalaria glabrata snails infected with Schistosoma mansoni were collected during consecutive seasons from a site in Brazil known to have a very high percentage of infected snails. Schistosoma mansoni cercariae from single snails were used to infect individual mice, and the recovered adult worms were genetically assessed using a mtVNTR marker. The number of unique parasite genotypes found per snail was compared to expected abundance values, based on the infection prevalence at the site, to determine the distribution of S. mansoni infections within the snail population. The observed distributions and those from previous studies were used to examine the relationship between schistosome prevalence and aggregation across a wide range of prevalence values. Our analysis showed that prevalence was inversely related to the degree of parasite overdispersion, and at high prevalence, S. mansoni infections were randomly distributed among snails.     

ON THE EVOLUTION OF SEXUAL REPRODUCTION IN HOSTS COEVOLVING WITH MULTIPLE PARASITES

Host–parasite coevolution has been studied extensively in the context of the evolution of sex. Although hosts typically coevolve with several parasites, most studies considered one‐host/one‐parasite interactions. Here, we study population‐genetic models in which hosts interact with two parasites. We find that host/multiple‐parasite models differ nontrivially from host/single‐parasite models. Selection for sex resulting from interactions with a single parasite is often outweighed by detrimental effects due to the interaction between parasites if coinfection affects the host more severely than expected based on single infections, and/or if double infections are more common than expected based on single infections. The resulting selection against sex is caused by strong linkage‐disequilibria of constant sign that arise between host loci interacting with different parasites. In contrast, if coinfection affects hosts less severely than expected and double infections are less common than expected, selection for sex due to interactions with individual parasites can now be reinforced by additional rapid linkage‐disequilibrium oscillations with changing sign. Thus, our findings indicate that the presence of an additional parasite can strongly affect the evolution of sex in ways that cannot be predicted from single‐parasite models, and that thus host/multiparasite models are an important extension of the Red Queen Hypothesis.     

Consequences of multiple infection with Plasmodium falciparum in an area of high endemicity

Most Plasmodium falciparum infections occur in partially immune hosts in highly endemic areas. In such situations, many hosts are simultaneously infected with multiple parasite genotypes, which must lead to intense competition between different parasite populations. We here summarise a series of studies of multiple infection, mostly using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) genotyping of the highly polymorphic msp-2 gene. These indicate that chronic infections, characteristic of the partially immune host, appear to protect against super-infecting parasites. This protection is not seen in infants. A consequence is that selection for fast-growing (virulent) parasites, occurs mainly in the youngest, immunologically na?ve, hosts. The normal situation for P. falciparum is one in which the host is partially immune, and competition between parasite genotypes in this situation is not expected to result in selection for virulence.     

Population‐level dynamics in experimental mixed infections: evidence for competitive exclusion among bacterial parasites of Paramecium caudatum

Parasites frequently share their host populations with other parasites. However, little is known about how different parasites respond to competition with diverse competitor species in the within‐host and between‐host environments. We explored the repeatability of competition by simultaneously exposing microcosm populations of the ciliate Paramecium caudatum to pairs of parasites from the Holospora species complex (H. undulata, H. caryophila and H. obtusa). We measured how competition affected the persistence and prevalence of each compared to single infections, across three host genotypes. Three weeks post‐inoculation we identified the presence of each parasite using fluorescence in situ hybridisation (FISH). Competitive exclusion (62/72) was more common than co‐existence (10/72) in populations inoculated with two parasites. There was a clear pattern of competitive superiority, with H. caryophila persisting in all doubly inoculated populations (with either H. undulata or H. obtusa), and H. undulata tending to exclude H. obtusa. This mirrored infection success in single infections, with H. caryophila having a higher infection prevalence in single inoculations, followed by H. undulata then H. obtusa. The probability of persistence in co‐inoculations did not change across the different host genotypes, and prevalence was the same as in single infections. Our results are consistent with superinfection models, which assume the competitive exclusion of parasites upon contact within the same host. Furthermore, such non‐random competitive epidemiological dynamics, where one parasite always wins, may be of interest for public health management, especially if the winning parasite is avirulent, as is seemingly the case here.     

Spatial variation in parasite-induced mortality in an amphipod: shore height versus exposure history

Characterizing the causes of spatial and temporal variation in parasite-induced mortality under natural conditions is crucial to better understanding the factors driving host population dynamics. Our goal was to quantify this variation in the amphipod Paracalliope novizealandiae, a second intermediate host of the trematode, Maritrema novaezealandensis. If infection and development of trematode metacercariae are benign, we expected mature metacercariae to accumulate within amphipods inhabiting high infestation areas. In field samples, intensity levels of mature metacercariae decreased linearly when amphipods harbored >5 immature metacercariae. This finding is consistent with the hypothesis that the parasite can be detrimental at high intensities of infection. Short-term field experiments showed that host survival also declines with the intensity of new infections and drops below 80% when early stage metacercariae reach 10 amphipod^?1. However, parasite effects varied over space and time. High-shore amphipods suffered an increased risk of infection in the summer and a lower likelihood of survival: there was a 10–30% decrease in survivorship for any given infection intensity at high- versus low-shore locations. We also tested for differences in the susceptibility of naive and exposed populations using transplant experiments, and found that naive amphipods acquired greater parasite loads (on average, 4.7 vs. 2.8 metacercariae amphipod^?1). Because survival decreases rapidly with infection intensity of both early- and late-stage metacercariae, naive populations would suffer considerably if the parasite were to increase its range. Our results indicate that trematode infections cause high mortality in amphipods during summer months under natural conditions, and emphasize that the effects of parasitism vary at local spatial scales and with exposure history.