Virulence and Pathogen Multiplication: A Serial Passage Experiment in the Hypervirulent Bacterial Insect-Pathogen Xenorhabdus nematophila

The trade-off hypothesis proposes that the evolution of pathogens' virulence is shaped by a link between virulence and contagiousness. This link is often assumed to come from the fact that pathogens are contagious only if they can reach high parasitic load in the infected host. In this paper we present an experimental test of the hypothesis that selection on fast replication can affect virulence. In a serial passage experiment, we selected 80 lines of the bacterial insect-pathogen Xenorhabdus nematophila to multiply fast in an artificial culture medium. This selection resulted in shortened lag phase in our selected bacteria. We then injected these bacteria into insects and observed an increase in virulence. This could be taken as a sign that virulence in Xenorhabdus is linked to fast multiplication. But we found, among the selected lineages, either no link or a positive correlation between lag duration and virulence: the most virulent bacteria were the last to start multiplying. We then surveyed phenotypes that are under the control of the flhDC super regulon, which has been shown to be involved in Xenorhabdus virulence. We found that, in one treatment, the flhDC regulon has evolved rapidly, but that the changes we observed were not connected to virulence. All together, these results indicate that virulence is, in Xenorhabdus as in many other pathogens, a multifactorial trait. Being able to grow fast is one way to be virulent. But other ways exist which renders the evolution of virulence hard to predict

Les problèmes de couple dans les symbioses némato-bactériennes parasites d'insecte.

Entomopathogenic nematodes from Steinernema}genus are symbiotically associated with Xenorhabdus bacteria. Their life cycle comprises two phases~: a free stage in the soil, where bacteria are carried inside nematodes' gut and a parasitic stage in the insect, where both partners are separated and multiply in parallel. For nematodes, benefits are clear during the parasitic stage while costs prevail during the free stage. We first tried to measure the balance between costs and benefits in these symbioses to identify selective pressures acting on nematode's symbiotic investment. We showed that nematodes endure costs to the association in free stage in terms of survival, these costs increasing with bacterial load. On the other side, nematodes benefit from the symbiosis in proportion of bacterial load in parasitic stage in terms of reproduction. These two antagonistic effects lead to a trade-off between nematodes' survival and reproduction which is mediated by their bacterial symbionts. Thus, depending on the environment, these correlations could challenge association stability. It could also impact the way the specificity of these associations evolves. In a second part, we addressed the question of symbiosis specificity in natura as well as in laboratory. Field samples confirmed the already-known constancy of association between a nematode species and a bacteria species all over the world. Experimental re-associations between two nematode species and foreign bacteria showed differences between retention and benefit specificities as well as inter-specific variability. Thus the one-to-one association between nematode and bacteria species may not be as strict as previously thought.Les nématodes entomopathogènes Steinernema sont associés symbiotiquement à des bactéries du genre Xenorhabdus. Leur cycle de vie comprend deux phases : une phase libre, dans le sol, où les nématodes portent leurs symbiotes dans le tube digestif, et une phase parasite, dans l'insecte, où les deux partenaires se multiplient côte à côte. Le bilan fin de l'interaction, globalement bénéfique pour les deux partenaires, a été peu étudié pour le moment. Dans un premier temps, nous avons abordé ces symbioses d'un point de vue coûts-bénéfices pour le nématode afin d'identifier les pressions de sélection agissant sur son investissement dans la symbiose. Nous montrons que l'association est à la fois bénéfique (reproduction en phase parasitaire) et coûteuse (mortalité en phase libre) pour le nématode, en proportion de sa charge symbiotique. Ces corrélations engendrent un compromis survie-reproduction pour le nématode, médié par ses symbiotes. Selon les conditions environnementales, elles pourraient éventuellement déstabiliser l'association, et, notamment, altérer sa spécificité. Dans un second temps, nous avons donc exploré la spécificité de ces associations dans la nature et au laboratoire. Lors d'un échantillonage de terrain, nous avons retrouvé la spécificité de ces associations largement décrite par ailleurs. Au laboratoire, la réassociation expérimentale entre nématodes et bactéries non natives montre que le spectre des bactéries retenues est plus étroit que le spectre des bactéries bénéfiques. De plus, des différences de modalités d'association apparaissent entre espèces de nématodes, qui suggèrent que la correspondance nématode - bactérie ne serait pas aussi stricte que prévu

Les problèmes de couple dans les symbioses némato-bactériennes parasites d'insecte

Les nématodes entomopathogènes du genre Steinernema sont associés symbiotiquement à des bactéries du genre Xenorhabdus. Ces associations sont traditionnellement qualifiées de mutualistes car chacun des partenaires bénéficie de la présence de l'autre. Toutefois, l'originalité du cycle de vie de ces associations permet de s'interroger sur la réalité de cette réciprocité des bénéfices. En effet, le cycle de vie de ces associations comprend deux phases : une phase libre, dans le sol, où les nématodes portent leurs symbiotes dans le tube digestif, et une phase parasite, dans l'insecte, où les deux partenaires se multiplient côte à côte. La symbiose est clairement bénéfique pour le nématode en phase parasite, ce qui est moins net en phase libre ; c'est le contraire pour la bactérie. Dans un premier temps, nous avons abordé ces symbioses d'un point de vue coûts-bénéfices pour le nématode afin d'identifier les pressions de sélection agissant sur son investissement dans la symbiose. Nos résultats montrent que l'association est coûteuse pour le nématode en termes de survie en phase libre, et que ce coût augmente avec le nombre de bactéries portées. D'un autre côté, le nématode bénéficie de l'association en termes de reproduction en phase parasitaire, en proportion de sa charge symbiotique. Ces corrélations engendrent un compromis survie-reproduction pour le nématode, médié par ses symbiotes. Selon l'environnement, le bilan de l'association serait donc mitigé pour les nématodes. Ce découplage des coûts et bénéfices peut mener à des pressions de sélection contraires entre les deux phases du cycle de vie. De plus, les coûts à l'association manifestent un conflit d'intérêts entre partenaires, qui pourrait éventuellement déstabiliser l'association, et, notamment, altérer sa spécificité. Dans un second temps, nous avons donc exploré la spécificité de ces associations dans la nature et au laboratoire. Lors d'un échantillonage de terrain, nous avons retrouvé la correspondance entre une espèce de nématode et une espèce de bactérie largement décrite par ailleurs. Au laboratoire, la réassociation expérimentale entre nématodes et bactéries non natives montre que le spectre des bactéries retenues est plus étroit que le spectre des bactéries bénéfiques. De plus, des différences de modalités d'association apparaissent entre espèces de nématodes, qui suggèrent que la correspondance nématode - bactérie ne serait pas aussi stricte que prévuEntomopathogenic nematodes from Steinernema genus are symbiotically associated with Xenorhabdus bacteria. These associations are traditionally considered as mutualistic as both partners benefit from each other. However the original life cycle of these associations raises questions about their real reciprocally-beneficial status. Indeed, the symbiosis life cycle comprises two phases : a free stage in the soil, where bacteria are carried inside nematodes' gut and a parasitic stage in the insect, where both partners are separated and multiply in parallel. Benefits of the association for nematodes are clear in parasitic, but not in free stage, and it is the opposite for bacteria. We first tried to measure the balance between costs and benefits in these symbioses to identify selective pressures acting on nematode's symbiotic investment. We showed that nematodes endure costs to the association in free stage in terms of survival, these costs increasing with bacterial load. On the other side, nematodes benefit from the symbiosis in proportion of bacterial load in parasitic stage in terms of reproduction. These two antagonistic effects lead to a trade-off between nematodes' survival and reproduction which is mediated by their bacterial symbionts. Thus, depending on the environment, symbiosis has mitigated outcomes for nematodes. This decoupling of costs and benefits along the life cycle may lead to contradictory selective pressures between the two stages of the cycle. Moreover, costs of the association for nematodes indicate a conflict of interests between partners, which could challenge association stability, in particular its specificity. In a second part, we addressed the question of symbiosis specificity in natura as well as in laboratory. Field samples confirmed the already-known constancy of association between a nematode species and a bacteria species all over the world. Experimental re-associations between two nematode species and foreign bacteria showed differences between retention and benefit specificities as well as inter-specific variability. Thus the one-to-one association between nematode and bacteria species may not be as strict as previously thoughtMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

A SURVIVAL-REPRODUCTION TRADE-OFF IN ENTOMOPATHOGENIC NEMATODES MEDIATED BY THEIR BACTERIAL SYMBIONTS

International audienceIn this work, we investigate the investment of entomopathogenic Steinernema nematodes (Rhabditidae) in their symbiotic association with Xenorhabdus bacteria (Enterobacteriaceae). Their life cycle comprises two phases: (1) a free stage in the soil, where infective juveniles (IJs) of the nematode carry bacteria in a digestive vesicle and search for insect hosts, and (2) a parasitic stage into the insect where bacterial multiplication, nematode reproduction, and production of new IJs occur. Previous studies clearly showed benefits to the association for the nematode during the parasitic stage, but preliminary data suggest the existence of costs to the association for the nematode in free stage. IJs deprived from their bacteria indeed survive longer than symbiotic ones. Here we show that those bacteria-linked costs and benefits lead to a trade-off between fitness traits of the symbiotic nematodes. Indeed IJs mortality positively correlates with their parasitic success in the insect host for symbiotic IJs and not for aposymbiotic ones. Moreover mortality and parasitic success both positively correlate with the number of bacteria carried per IJ, indicating that the trade-off is induced by symbiosis. Finally, the trade-off intensity depends on parental effects and, more generally, is greater under restrictive environmental conditions

Analysis of insect survival after they are injected with 2000 bacteria cells.

<p>In this analysis, only the 20 lineages we used in the dilution experiment and for phenotypic measurements are present. In addition to the insect weight and the number of CFU present in the injected solution, we considered the median lag, the phenotypic distance to the ancestral lineage, the proportion of non-motile clones and the proportion of clones with the total haemolysis phenotype as explanatory variables. The analysis of all lineages show that the factor that explains the best the highest virulence of selected lineages is the increase in the proportion of total haemolysis, although this effect is marginal. The analysis of HDI lineages demonstrated no significant correlation between the virulence and the phenotypes we have measured. Conversely, in LDI lineages, we found a highly significant correlation between the virulence and the median lag time, i.e. the time that elapses before the absorbance of 50% of the bacterial populations of a lineage overreach that of empty wells. Contrary to our expectations, though, the LDI bacteria that kill insects the fastest are those which take the longest to start growing.</p

Proportion of surviving insects as a function of the time that elapsed since they were injected with bacteria.

<p>PBS injected insects serve here as a negative control, as they were not injected with bacteria. 46 hours after injection, more than 95% of all insects injected with bacteria were dead.</p

Analysis of insect survival after they are injected with 2000 bacteria cells.

<p>This analysis was performed using a non-parametrical Cox proportional hazard model of survival. In this analysis, we controlled for insect weight and for the number of injected CFU which were found to have no significant effect on survival. A total of trials are analyzed here, with the differences between the lots of insects used in this experiment modeled as a random block factor (with an estimated variance of 0.008). The analysis demonstrates that insects injected with selected bacteria die earlier than those injected with the ancestral lineage.</p

Median lethal time (i.e. the time at which 50% of the insects injected with a particular lineage are dead) as a function of median lag time (i.e. the time at which 50% of the populations of a particular lineage have overreached the absorbance of an empty well).

<p>The estimation of these times are performed using a parametric survival regression method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015872#s2" target="_blank">method</a>). Horizontal and vertical bars correspond to the standard error of these estimates. In LDI lineages, the two quantities are negatively correlated, indicating that the fastest growing LDI lineages are the least virulent. No relation could be found between medium lag time and medium lethal time in HDI lineages. The size of symbols indicates the proportion of clones that display the total haemolysis phenotype. When analyzing both ancestral and selected lineages, we found a marginally significant link between this variable and virulence.</p

Distribution of motility halo diameter in the two selection treatments and the ancestral lineage.

<p>Distribution of motility halo diameter in the two selection treatments and the ancestral lineage.</p

Analysis of the time that elapses before absorbance starts increasing.

<p>This analysis was performed using a non-parametrical Cox model of survival. The total number of kinetics that are analyzed here is . The variation between the three replicate experiments is modeled as resulting from a random factor. The possible influence of initial bacterial density is controlled by considering the initial absorbance as a covariate. This analysis shows that selected lines, LDI or HDI, reach the threshold absorbance faster than the ancestral lineage.</p