Pre- and post-association barriers to host switching in sympatric mutualists.

Coevolution between mutualists can lead to reciprocal specialization, potentially causing barriers to host switching. Here, we conducted assays to identify pre- and post-association barriers to host switching by endosymbiotic bacteria, both within and between two sympatric nematode clades. In nature, Steinernema nematodes and Xenorhabdus bacteria form an obligate mutualism. Free-living juvenile nematodes carry Xenorhabdus in a specialized intestinal receptacle. When nematodes enter an insect, they release the bacteria into the insect hemocoel. The bacteria aid in killing the insect and facilitate nematode reproduction. Prior to dispersing from the insect, juvenile nematodes must form an association with their symbionts; the bacteria must adhere to the intestinal receptacle. We tested for pre-association barriers by comparing the effects of bacterial strains on native versus non-native nematodes via their virulence towards, nutritional support of, and ability to associate with different nematode species. We then assessed post-association barriers by measuring the relative fitness of nematodes carrying each strain of bacteria. We found evidence for both pre- and post-association barriers between nematode clades. Specifically, some bacteria were highly virulent to non-native hosts, and some nematode hosts carried fewer cells of non-native bacteria, creating pre-association barriers. In addition, reduced infection success and lower nematode reproduction were identified as post-association barriers. No barriers to symbiont switching were detected between nematode species within the same clade. Overall, our study suggests a framework that could be used to generate predictions for the evolution of barriers to host switching in this and other systems.

Evolution of increased virulence is associated with decreased spite in the insect-pathogenic bacterium Xenorhabdus nematophila.

Disease virulence may be strongly influenced by social interactions among pathogens, both during the time course of an infection and evolutionarily. Here, we examine how spiteful bacteriocin production in the insect-pathogenic bacterium Xenorhabdus nematophila is evolutionarily linked to its virulence. We expected a negative correlation between virulence and spite owing to their inverse correlations with growth. We examined bacteriocin production and growth across 14 experimentally evolved lineages that show faster host-killing relative to their ancestral population. Consistent with expectations, these more virulent lineages showed reduced bacteriocin production and faster growth relative to the ancestor. Further, bacteriocin production was negatively correlated with growth across the examined lineages. These results strongly support an evolutionary trade-off between virulence and bacteriocin production and lend credence to the view that disease management can be improved by exploiting pathogen social interactions.

Symbiont-mediated competition: Xenorhabdus bovienii confer an advantage to their nematode host Steinernema affine by killing competitor Steinernema feltiae.

Bacterial symbionts can affect several biotic interactions of their hosts, including their competition with other species. Nematodes in the genus Steinernema utilize Xenorhabdus bacterial symbionts for insect host killing and nutritional bioconversion. Here, we establish that the Xenorhabdus bovienii bacterial symbiont (Xb-Sa-78) of Steinernema affine nematodes can impact competition between S. affine and S. feltiae by a novel mechanism, directly attacking its nematode competitor. Through co-injection and natural infection assays we demonstrate the causal role of Xb-Sa-78 in the superiority of S. affine over S. feltiae nematodes during competition. Survival assays revealed that Xb-Sa-78 bacteria kill reproductive life stages of S. feltiae. Microscopy and timed infection assays indicate that Xb-Sa-78 bacteria colonize S. feltiae nematode intestines, which alters morphology of the intestine. These data suggest that Xb-Sa-78 may be an intestinal pathogen of the non-native S. feltiae nematode, although it is a nonharmful colonizer of the native nematode host, S. affine. Screening additional X. bovienii isolates revealed that intestinal infection and killing of S. feltiae is conserved among isolates from nematodes closely related to S. affine, although the underlying killing mechanisms may vary. Together, these data demonstrate that bacterial symbionts can modulate competition between their hosts, and reinforce specificity in mutualistic interactions.

Host Association and Spatial Proximity Shape but Do Not Constrain Population Structure in the Mutualistic Symbiont Xenorhabdus bovienii.

To what extent are generalist species cohesive evolutionary units rather than a compilation of recently diverged lineages? We examine this question in the context of host specificity and geographic structure in the insect pathogen and nematode mutualist Xenorhabdus bovienii. This bacterial species partners with multiple nematode species across two clades in the genus Steinernema. We sequenced the genomes of 42 X. bovienii strains isolated from four different nematode species and three field sites within a 240-km2 region and compared them to globally available reference genomes. We hypothesized that X. bovienii would comprise several host-specific lineages, such that bacterial and nematode phylogenies would be largely congruent. Alternatively, we hypothesized that spatial proximity might be a dominant signal, as increasing geographic distance might lower shared selective pressures and opportunities for gene flow. We found partial support for both hypotheses. Isolates clustered largely by nematode host species but did not strictly match the nematode phylogeny, indicating that shifts in symbiont associations across nematode species and clades have occurred. Furthermore, both genetic similarity and gene flow decreased with geographic distance across nematode species, suggesting differentiation and constraints on gene flow across both factors, although no absolute barriers to gene flow were observed across the regional isolates. Several genes associated with biotic interactions were found to be undergoing selective sweeps within this regional population. The interactions included several insect toxins and genes implicated in microbial competition. Thus, gene flow maintains cohesiveness across host associations in this symbiont and may facilitate adaptive responses to a multipartite selective environment. IMPORTANCE Microbial populations and species are notoriously hard to delineate. We used a population genomics approach to examine the population structure and the spatial scale of gene flow in Xenorhabdus bovienii, an intriguing species that is both a specialized mutualistic symbiont of nematodes and a broadly virulent insect pathogen. We found a strong signature of nematode host association, as well as evidence for gene flow connecting isolates associated with different nematode host species and collected from distinct study sites. Furthermore, we saw signatures of selective sweeps for genes involved with nematode host associations, insect pathogenicity, and microbial competition. Thus, X. bovienii exemplifies the growing consensus that recombination not only maintains cohesion but can also allow the spread of niche-beneficial alleles.

Post-association barrier to host switching maintained despite strong selection in a novel mutualism.

Following a host shift, repeated co-passaging of a mutualistic pair is expected to increase fitness over time in one or both species. Without adaptation, a novel association may be evolutionarily short-lived as it is likely to be outcompeted by native pairings. Here, we test whether experimental evolution can rescue a low-fitness novel pairing between two sympatric species of Steinernema nematodes and their symbiotic Xenorhabdus bacteria. Despite low mean fitness in the novel association, considerable variation in nematode reproduction was observed across replicate populations. We selected the most productive infections, co-passaging this novel mutualism nine times to determine whether selection could improve the fitness of either or both partners. We found that neither partner showed increased fitness over time. Our results suggest that the variation in association success was not heritable and that mutational input was insufficient to allow evolution to facilitate this host shift. Thus, post-association costs of host switching may represent a formidable barrier to novel partnerships among sympatric mutualists.

Spiteful Interactions in a natural population of the bacterium Xenorhabdus bovienii.

An individual behaves spitefully when it harms itself in the act of harming other individuals. One of the clearest potential examples of spite is the costly production and release of toxins called bacteriocins. Bacteriocins are toxins produced by bacteria that can kill closely related strains of the same species. Theoretical work has predicted that bacteriocin-mediated interactions could play an important role in maintaining local genetic and/or species diversity, but these interactions have not been studied at biologically relevant scales in nature. Here we studied toxin production and among-strain inhibitions in a natural population of Xenorhabdus bovienii. We found genetic differences and inhibitions between colonies that were collected only a few meters apart. These results suggest that spite exists in natural populations of bacteria.

Evolutionary consequences of feedbacks between within-host competition and disease control.

Lay Summary: Competition often occurs among diverse parasites within a single host, but control efforts could change its strength. We examined how the interplay between competition and control could shape the evolution of parasite traits like drug resistance and disease severity.

Suppression of bacteriocin resistance using live, heterospecific competitors.

Rapidly spreading antibiotic resistance has led to the need for novel alternatives and sustainable strategies for antimicrobial use. Bacteriocins are a class of proteinaceous anticompetitor toxins under consideration as novel therapeutic agents. However, bacteriocins, like other antimicrobial agents, are susceptible to resistance evolution and will require the development of sustainable strategies to prevent or decelerate the evolution of resistance. Here, we conduct proof-of-concept experiments to test whether introducing a live, heterospecific competitor along with a bacteriocin dose can effectively suppress the emergence of bacteriocin resistance in vitro. Previous work with conventional chemotherapeutic agents suggests that competition between conspecific sensitive and resistant pathogenic cells can effectively suppress the emergence of resistance in pathogenic populations. However, the threshold of sensitive cells required for such competitive suppression of resistance may often be too high to maintain host health. Therefore, here we aim to ask whether the principle of competitive suppression can be effective if a heterospecific competitor is used. Our results show that a live competitor introduced in conjunction with low bacteriocin dose can effectively control resistance and suppress sensitive cells. Further, this efficacy can be matched by using a bacteriocin-producing competitor without any additional bacteriocin. These results provide strong proof of concept for the effectiveness of competitive suppression using live, heterospecific competitors. Currently used probiotic strains or commensals may provide promising candidates for the therapeutic use of bacteriocin-mediated competitive suppression.

Trade-off between reproductive and anti-competitor abilities in an insect-parasitic nematode-bacteria symbiosis.

Mutualistic symbionts can provide diverse benefits to their hosts and often supply key trait variation for host adaptation. The bacterial symbionts of entomopathogenic nematodes play a crucial role in successful colonization of and reproduction in the insect host. Additionally, these symbionts can produce a diverse array of antimicrobial compounds to deter within-host competitors. Natural isolates of the symbiont, Xenorhabdus bovienii, show considerable variation in their ability to target sympatric competitors via bacteriocins, which can inhibit the growth of sensitive Xenorhabdus strains. Both the bacteria and its nematode partner have been shown to benefit from bacteriocin production when within-host competition with a sensitive competitor occurs. Despite this benefit, several isolates of Xenorhabdus do not inhibit sympatric strains. To understand how this variation in allelopathy could be maintained, we tested the hypothesis that inhibiting isolates face a reproductive cost in the absence of competition. We tested this hypothesis by examining the reproductive success of inhibiting and non-inhibiting isolates coupled with their natural nematode host in a non-competitive context. We found that nematodes carrying non-inhibitors killed the insect host more rapidly and were more likely to successfully reproduce than nematodes carrying inhibitors. Lower reproductive success of inhibiting isolates was repeatable across nematode generations and across insect host species. However, no difference in insect mortality was observed between inhibiting and non-inhibiting isolates when bacteria were injected into insects without their nematode partners. Our results indicate a trade-off between the competitive and reproductive roles of symbionts, such that inhibiting isolates, which are better in the face of within-host competition, pay a reproductive cost in the absence of competition. Furthermore, our results support the hypothesis that symbiont variation within populations can be maintained through context-dependent fitness benefits conferred to their hosts. As such, our study offers novel insights into the selective forces maintaining variation within a single host-symbiont population and highlights the role of competition in mutualism evolution.

Plastic responses to competition: Does bacteriocin production increase in the presence of nonself competitors?

Anticompetitor traits such as the production of allelopathic toxins can confer significant competitive benefits but are often costly to produce. Evolution of these traits may be facilitated by environment-specific induction; however, the extent to which costly anticompetitor traits are induced by competitors is not well explored. Here, we addressed this question using bacteriocins, which are highly specific, proteinaceous anticompetitor toxins, produced by most lineages of bacteria and archaea. We tested the prediction that bacteriocin production is phenotypically plastic and induced by the presence of competitors by examining bacteriocin production in the presence and absence of nonself competitors over the course of growth of a producing strain. Our results show that bacteriocin production is detectable only at high cell densities, when competition for resources is high. However, the amount of bacteriocin activity was not significantly different in the presence vs. the absence of nonself competitors. These results suggest that bacteriocin production is either (a) canalized, constitutively produced by a fixed frequency of cells in the population or (b) induced by generic cues of competition, rather than specific self/nonself discrimination. Such a nonspecific response to competition could be favored in the natural environment where competition is ubiquitous.

Cross-generational environmental effects and the evolution of offspring size in the Trinidadian guppy Poecilia reticulata.

The existence of adaptive phenotypic plasticity demands that we study the evolution of reaction norms, rather than just the evolution of fixed traits. This approach requires the examination of functional relationships among traits not only in a single environment but across environments and between traits and plasticity itself. In this study, I examined the interplay of plasticity and local adaptation of offspring size in the Trinidadian guppy, Poecilia reticulata. Guppies respond to food restriction by growing and reproducing less but also by producing larger offspring. This plastic difference in offspring size is of the same order of magnitude as evolved genetic differences among populations. Larger offspring sizes are thought to have evolved as an adaptation to the competitive environment faced by newborn guppies in some environments. If plastic responses to maternal food limitation can achieve the same fitness benefit, then why has guppy offspring size evolved at all? To explore this question, I examined the plastic response to food level of females from two natural populations that experience different selective environments. My goals were to examine whether the plastic responses to food level varied between populations, test the consequences of maternal manipulation of offspring size for offspring fitness, and assess whether costs of plasticity exist that could account for the evolution of mean offspring size across populations. In each population, full-sib sisters were exposed to either a low- or high-food treatment. Females from both populations produced larger, leaner offspring in response to food limitation. However, the population that was thought to have a history of selection for larger offspring was less plastic in its investment per offspring in response to maternal mass, maternal food level, and fecundity than the population under selection for small offspring size. To test the consequences of maternal manipulation of offspring size for offspring fitness, I raised the offspring of low- and high-food mothers in either low- or high-food environments. No maternal effects were detected at high food levels, supporting the prediction that mothers should increase fecundity rather than offspring size in noncompetitive environments. For offspring raised under low food levels, maternal effects on juvenile size and male size at maturity varied significantly between populations, reflecting their initial differences in maternal manipulation of offspring size; nevertheless, in both populations, increased investment per offspring increased offspring fitness. Several correlates of plasticity in investment per offspring that could affect the evolution of offspring size in guppies were identified. Under low-food conditions, mothers from more plastic families invested more in future reproduction and less in their own soma. Similarly, offspring from more plastic families were smaller as juveniles and female offspring reproduced earlier. These correlations suggest that a fixed, high level of investment per offspring might be favored over a plastic response in a chronically low-resource environment or in an environment that selects for lower reproductive effort.

Aging alters interspecific competition between two sympatric insect-parasitic nematode species.

Interspecific competition can vary depending on the stage, age, or physiological state of the competitors. Competitive ability often increases with age or size; alternatively, senescence can lead to a loss of viability and reduced competitive success. Differences between species in their age-specific competitive abilities can promote coexistence in the face of substantial niche overlap.We examined two sympatric species of nematodes (genus Steinernema) to determine whether their competitive relationship changes as a function of age. These obligately killing insect parasites are known for their broad host ranges and are transmitted from insect to insect via a juvenile stage propagule that is free-living in the soil. Here, we tested whether the two species differed in the effects of age by examining the mortality of insect hosts infected with young or old transmission stage nematodes of each species. We also performed mixed infections, where an equal ratio of both species was simultaneously exposed to a host, to determine the effect of age on competitiveness.One species showed reduced performance with age, as older propagules were slower at inducing host mortality. In contrast, the other species increased in killing speed with age. In competition, insect mortality rate was predictive of competitive outcome, such that if one species induced considerably faster host death in a single-species infection, it was competitively dominant in the coinfection. Accordingly, we found a shift in the competitive relationship between the two species with age.Our work demonstrates that species differences in the effects of aging can lead to dramatic shifts in reproductive success. As these effects are realized solely in a competitive environment, both spatial patchiness and temporal niche partitioning may be important for promoting coexistence.

Nematode-bacteria mutualism: Selection within the mutualism supersedes selection outside of the mutualism.

The coevolution of interacting species can lead to codependent mutualists. Little is known about the effect of selection on partners within verses apart from the association. Here, we determined the effect of selection on bacteria (Xenorhabdus nematophila) both within and apart from its mutualistic partner (a nematode, Steinernema carpocapsae). In nature, the two species cooperatively infect and kill arthropods. We passaged the bacteria either together with (M+), or isolated from (M-), nematodes under two different selection regimes: random selection (S-) and selection for increased virulence against arthropod hosts (S+). We found that the isolated bacteria evolved greater virulence under selection for greater virulence (M-S+) than under random selection (M-S-). In addition, the response to selection in the isolated bacteria (M-S+) caused a breakdown of the mutualism following reintroduction to the nematode. Finally, selection for greater virulence did not alter the evolutionary trajectories of bacteria passaged within the mutualism (M+S+ = M+S-), indicating that selection for the maintenance of the mutualism was stronger than selection for increased virulence. The results show that selection on isolated mutualists can rapidly breakdown beneficial interactions between species, but that selection within a mutualism can supersede external selection, potentially generating codependence over time.

Within-host competitive interactions as a mechanism for the maintenance of parasite diversity.

Variation among parasite strains can affect the progression of disease or the effectiveness of treatment. What maintains parasite diversity? Here I argue that competition among parasites within the host is a major cause of variation among parasites. The competitive environment within the host can vary depending on the parasite genotypes present. For example, parasite strategies that target specific competitors, such as bacteriocins, are dependent on the presence and susceptibility of those competitors for success. Accordingly, which parasite traits are favoured by within-host selection can vary from host to host. Given the fluctuating fitness landscape across hosts, genotype by genotype (G×G) interactions among parasites should be prevalent. Moreover, selection should vary in a frequency-dependent manner, as attacking genotypes select for resistance and genotypes producing public goods select for cheaters. I review competitive coexistence theory with regard to parasites and highlight a few key examples where within-host competition promotes diversity. Finally, I discuss how within-host competition affects host health and our ability to successfully treat infectious diseases.

Alternative paths to success in a parasite community: within-host competition can favor higher virulence or direct interference.

Selection imposed by coinfection may vary with the mechanism of within-host competition between parasites. Exploitative competition is predicted to favor more virulent parasites, whereas interference competition may result in lower virulence. Here, we examine whether exploitative or interference competition determines the outcome of competition between two nematode species (Steinernema spp.), which in combination with their bacterial symbionts (Xenorhabdus spp.), infect and kill insect hosts. Multiple isolates of each nematode species, carrying their naturally associated bacteria, were characterized by (1) the rate at which they killed insect hosts, and by (2) the ability of their bacteria to interfere with each other's growth via bacteriocidal toxins called "bacteriocins." We found that both exploitative and interference abilities were important in predicting which species had a selective advantage in pairwise competition experiments. When nematodes carried bacteria that did not interact via bacteriocins, the faster killing isolate had a competitive advantage. Alternatively, nematodes could gain a competitive advantage when they carried bacteria able to inhibit the bacteria of their competitor. Thus, the combination of nematode/bacterial traits that led to competitive success depended on which isolates were paired, suggesting that variation in competitive interactions may be important for maintaining species diversity in this community.

Bacteriocin-mediated interactions within and between coexisting species.

Bacteriocins are bacteriocidal toxins released by almost all bacteria. They are thought to have a narrow range of killing, but as bacteriocin-mediated interactions have been rarely studied at biologically relevant scales, whether this narrow range of action falls mostly within or mostly between coexisting species in natural communities is an open question with important ecological and evolutionary implications. In a previous study, we systematically sampled Xenorhabdus bacteria along a hillside and found evidence for genotypic variability and bacteriocin-mediated interactions within Xenorhabdus bovienii and X. koppenhoeferi colonies that were collected only a few meters apart. In contrast, colonies that were isolated from the same soil sample were always genetically similar and showed no inhibitions. Here, we conducted pairwise growth-inhibition assays within and between seven X. bovienii and five X. koppenhoeferi colonies that were isolated from different soil samples; all seven X. bovienii colonies and at least three of the X. koppenhoeferi have been distinguished as distinct genotypes based on coarse-grain genomic markers. We found signatures for both conspecific and heterospecific bacteriocin inhibitions in this natural community of Xenorhabdus bacteria, but intraspecific inhibitions were significantly more common than interspecific inhibitions. These results suggest that bacteriocins have a major role in intraspecific competition in nature, but also suggest that bacterocins are important in mediating interspecific interactions among coexisting species in natural communities.

The evolution of spite: population structure and bacteriocin-mediated antagonism in two natural populations of xenorhabdus bacteria.

Spite occurs when an individual harms itself in the act of harming other individuals. Such behaviors were once assumed to be of limited evolutionary importance, as the conditions for the evolution of spite were thought to be too restrictive. Recent theoretical work, however, suggests that spatial population structure, which allows local competition among genotypes, could favor the evolution of spite. One of the clearest examples of spite is the costly production and release by bacteria of toxins (called bacteriocins) that can kill unrelated strains of the same species. Here, we establish the existence of spatial structure in two natural populations of bacteriocin-producing bacteria. Specifically, relatedness decreased with increasing spatial distance between the field isolates. In addition, toxin-mediated inhibitions were found only between isolates that were collected more than 1 m apart and that were generally less than 80% similar in their genomic fingerprints. Taken together, the results suggest that the bacteria are spatially structured, with mixing of genotypes and spiteful interactions at the boundaries between demes.

Group selection on population size affects life-history patterns in the entomopathogenic nematode Steinernema carpocapsae.

Selection is recognized to operate on multiple levels. In disease organisms, selection among hosts is thought to provide an important counterbalance to selection for faster growth within hosts. We performed three experiments, each selecting for a divergence in group size in the entomopathogenic nematode, Steinernema carpocapsae. These nematodes infect and kill insect larvae, reproduce inside the host carcass, and emerge as infective juveniles. We imposed selection on group size by selecting among hosts for either high or low numbers of emerging nematodes. Our goal was to determine whether this trait could respond to selection at the group level, and if so, to examine what other traits would evolve as correlated responses. One of the three experiments showed a significant response to group selection. In that experiment, the high-selected treatment consistently produced more emerging nematodes per host than the low-selected treatment. In addition, nematodes were larger and they emerged later from hosts in the low-selected lines. Despite small effective population sizes, the effects of inbreeding were small in this experiment. Thus, selection among hosts can be effective, leading to both a direct evolutionary response at the population level, as well as to correlated responses in populational and individual traits.