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.

Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France).

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the "glaseri-group". The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.

A survival-reproduction trade-off in entomopathogenic nematodes mediated by their bacterial symbionts.

In 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.

Effect of bacterial symbionts Xenorhabdus on mortality of infective juveniles of two Steinernema species.

Steinernema species are entomopathogenic nematodes associated with Xenorhabdus bacteria. The life cycle of these associations is composed of two stages: (1) a free stage in the soil, where infective juveniles (IJs), which carry bacteria in their guts, search for new insect hosts; and (2) a parasitic stage, where the IJs infect insects, release their Xenorhabdus symbionts and reproduce in order to produce new IJs. Previous studies clearly showed benefits to the association for several Steinernema species during the parasitic stage. Nevertheless, no study has so far explored, during the free stage, the existence of costs or benefits to the association for different Steinernema. Here, we compared the survival of both symbiotic and aposymbiotic IJs in two nematode species: (1) Steinernema carpocapsae-exhibiting IJs that carry a high number of Xenorhabdus cells in their guts; and (2) its closely relative species, S. scapterisci-exhibiting IJs, that carry very few Xenorhabdus cells in their guts. We showed that the bacterial symbionts were costly for S. carpocapsae by increasing IJs' mortality but not for S. scapterisci. This difference in cost induced by bacteria to IJs during the free stage could be correlated with the difference in the numbers of bacteria carried by IJs of each nematode species.