BACKGROUND
symbioses between the entomopathogenic nematodes steinernema spp.
and the enterobacteriacae xenorhabdus spp.
are associations in which both partners receive benefits from each other  <cit> .
in the soil, the infective juveniles  of the nematodes act as vectors dispersing the bacteria from insect host to insect-host and in turn, the bacteria increase the nematode's fitness within the insects hosts  <cit> .
previous studies showed that these symbioses were highly specific and that no steinernema spp.
was able to associate with a xenorhabdus spp.
genetically distant from its natural one  <cit> .
as the bacterial dispersion is totally dependent upon the fitness of the nematode within the insect-host, it is possible that xenorhabdus spp.
might select special traits in order to enhance their vector's fitness.
it is known that xenorhabdus spp.
are beneficial to their nematodes in providing the latter with a better ability to kill the insect and feed on it  <cit> .
previous studies that focused on two different steinernema species  have provided us with insights into the association characteristics  <cit> .
although the two nematode species demonstrated increased fitness when they parasitized insect-hosts with their own native symbiont, s. scapterisci appeared less dependent upon its native symbiont  than s. carpocapsae .
thus, s. scapterisci's symbiont increased the reproductive rate of its naturally associated nematode by a factor of  <dig> , whereas x. nematophila increased the reproductive rate of its naturally associated nematode by sevenfold   <cit> .
moreover, s. scapterisci, transported 700-fold fewer cells of its xenorhabdus than s. carpocapsae   <cit> .
these two steinernema species also differed in their ability to deal with non-native xenorhabdus strains in case of co-infection in an insect  <cit> .
while s. scapterisci reproduced in co-infection situations with all the tested xenorhabdus strains , s. carpocapsae could not reproduce at all with most of them in the same situation  <cit> .
despite these specific differences, the global trend emerging from these previous experiments was that non-naturally associated xenorhabdus strains tend to be antagonist against nematodes species which cannot disperse them.
one can easily think that this antagonistic effect of xenorhabdus strains on the fitness of nematodes naturally associated with others xenorhabdus strains could be selected in case of frequent interspecific competition.
in such an evolutionary context, we can postulate that each xenorhabdus strain should try to provide its own nematode-vector with competitive advantages by producing antagonistic molecules against foreign nematodes.
indeed, a previous study has shown with the association s. carpocapsae-x.
nematophila as a model-system that, in insects co-infected by antagonistic xenorhabdus, x. nematophila partly counteracted their antagonistic effect on the nematodes fitness most probably by the mean of bacteriocins  <cit> .
the goal of the present study was to address the question of the impact of xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different steinernema species.
for this, we monitored experimental interspecific competition between  two nematode species: s. carpocapsae and s. scapterisci and  their respective symbionts: x. nematophila and x. innexi within an insect-host .
in this study, three conditions of competition between nematodes were tested:  infection of insects with aposymbiotic ijs  of both species  infection of insects with aposymbiotic ijs of both species in presence of variable proportion of their two xenorhabdus symbionts and  infection of insects with symbiotic ijs  of both species.
RESULTS
proportion of each xenorhabdus in insect's hemolymph  <dig> h post-infection with ijs
we observed that both bacterial strains were able to multiply and co-exist within the hemolymph of the insect.
nevertheless, x. nematophila was clearly less represented within the hemolymph  <dig> h post-infection with nematodes when an initial injection of a suspension containing 50% of each bacterium in the insect was performed .
when 70% and 90% of x. nematophila were injected, the two bacteria were found in a very variable proportion and co-existed .
in the competition resulting from infection of insects with symbiotic ijs of both nematode species, as well as when 100% of x. nematophila were injected into insects infected with aposymbiotic ijs of both nematode species, no x. innexi were detected within the hemolymph .
assessment of nematodes maturation in competition
to know if gfp labelling of x. nematophila, employed to discriminate the two nematode species within the global offspring, triggered differences on the progress of competition between nematodes, the data obtained with or without gfp labelling of x. nematophila in each competition situation were compared with a mann withney test.
we showed that gfp labelling had no statistically significant effect on both  the ratio of s. carpocapsae among all females found in the insect  <dig> h after infection  and  the ratio of s. carpocapsae among all males found in the insect  <dig> h after infection .
because of the non-significant differences observed in these comparisons, the results obtained in each situation with and without gfp labelled x. nematophila were pooled in further analyses.
the ratios of s. carpocapsae among all males found in the insects were highly heterogeneous for the different competition conditions .
the noether test showed that no significant difference in the ratio of s. carpocapsae males occurred under four competition conditions:  competition resulting from infection of insects with aposymbiotic ijs of both nematode species without injection of bacterium,  competition resulting from infection with aposymbiotic ijs of both nematode species in insects injected with 100% of x. innexi,  competition resulting from infection with aposymbiotic ijs of both nematode species with 50% of each symbiont injected in the insects and  competition resulting from infection with aposymbiotic nematodes in insects injected with 70% of x. nematophila and 30% of x. innexi .
in these four conditions, the ratios of s. carpocapsae among males were significantly lower than in the three other conditions of competition:  competition resulting from infection with aposymbiotic ijs in insects injected with 90% of x. nematophila and 10% of x. innexi,  competition resulting from infection between aposymbiotic ijs in insects injected with 100% of x. nematophila and  competition resulting from infection with symbiotic ijs  .
these results showed that an increase in the proportion of x. nematophila within bacteria injected into the insect triggered an increase of s. carpocapsae within adult worms found in the insects.
the ratios of s. carpocapsae among females found in the insects were also highly heterogeneous for the different competition conditions .
the noether test showed that the ratios of s. carpocapsae within females were lower in competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. innexi than  in competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. nematophila and  in competition resulting from infection with symbiotic ijs .
in competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. innexi, no s. carpocapsae females were observed.
assessment of the nematode offspring
total number of infective juveniles
the total number of ijs emerging from insects in the different competition situations for s. carpocapsae and s. scapterisci was heterogeneous under the different competition conditions .
the noether test showed that competition resulting from infection with aposymbiotic ijs without injection of bacteria into the insect resulted in the production of significantly fewer ijs than  competition resulting from infection with symbiotic ijs ,  competition resulting from infection with aposymbiotic ijs in insects injected with 50% of each symbiont ,  competition resulting from infection with aposymbiotic ijs in insects injected with 70% of x. nematophila and 30% of x. innexi  and  competition resulting from infection between aposymbiotic ijs in insects injected with 90% of x. nematophila and 10% of x. innexi .
proportion of each nematode species in the total nematode offspring
in the competition situation resulting from infection with aposymbiotic ijs of both nematode species without injection of bacteria into the insect, the proportion of each nematode species in the total offspring emerging from four insects was assessed with pcr-rflp made after global extraction of dna from pools of  <dig> ijs.
in control samples, the presence of 25% and 10% of s. carpocapsae ijs within pools of  <dig> ijs containing respectively 75% and 90% of s. scapterisci were easily detected  .
in all the four pools of  <dig> ijs tested here, only one band was observed after incubation of the pcr product with hind iii .
this result suggested that after competition between aposymbiotic nematodes without injection of bacteria, more than 90% of all ijs emerging from the insect belonged to s. scapterisci.
in other competition situations, the proportion of each nematode species was evaluated by counting the proportion of nematodes harbouring gfp labelled x. nematophila within the global offspring.
gfp-fluorescence of x. nematophila is a good indicator of s. carpocapsae's ijs because pilots experiments showed that gfp labelled bacteria were still observable in ijs that were stored during  <dig> months at 8°c and that after several generations within different insects, at each generation, 95% of the ijs emerging from the insects habored gfp labelled bacteria.
nevertheless, in order to check that the gfp expression was not lost all along our experiments, we checked gfp expression for all the x. nematophila isolated from pools of  <dig> ijs and confirmed that none of them did not express gfp.
this result confirmed the perfect stability of gfp labelled x. nematophila all along our experiments.
competition resulting from infection with aposymbiotic ijs in insects injected with  50% of each symbiont,  70% of x. nematophila and 30% of x. innexi and  90% of x. nematophila and 10% of x. innexi gave offspring in which almost no ijs harboured gfp labelled x. nematophila .
these observations showed that s. scapterisci was highly dominant in the total offspring emerging from insects after such competitions.
for competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. nematophila, the proportion of ijs harbouring gfp within the total offspring was very variable from  <dig> to 100% with almost all the combinations .
for competition resulting from infection with symbiotic ijs, most of the ijs emerging from the insects  harboured gfp labelled x. nematophila .
the latter result suggested that in competition resulting from infection with symbiotic ijs, most of the offspring emerging from the insects belonged to the species s. carpocapsae.
discussion
many invertebrates are associated with prokaryotes that provide them with the ability to live in extreme environments  <cit> .
the most documented symbioses are those where symbionts provide their hosts with new abilities to take advantage of poorly nutritive resources  <cit> .
nevertheless, symbionts can play other important roles on diverse life history traits of their hosts, for example, the bacterium vibrio which provides its sepiolids host with a better discretion towards its predators  <cit> .
we already knew that xenorhabdus symbionts provided steinernema nematodes with diverse adaptations to their environment:  they produce virulence factors in order to kill the insect  <cit> ,  they lead to a better access to the insect biomass and are themselves food for the nematodes  <cit> ,  they produce antibiotics which counteract the multiplication of both distant and closely related bacteria  <cit> .
the goal of this study was to infer the abilities of xenorhabdus to modulate the progression and outcome of interspecific competition between steinernema species.
for this, we made experimental competitions between s. carpocapsae and s. scapterisci and their bacterial symbionts.
we assessed several parameters of these competitions:  the proportion of each symbiont  within the hemolymph of the insect after delay of multiplication,  the ratio of s. carpocapsae among the adults found in the insects and  the offspring of each nematode species after competition.
the assessment of bacterial multiplication within insect hemolymph showed that x. innexi appeared more competitive than x. nematophila.
moreover, in insects infected with aposymbiotic ijs and in which 50% of each xenorhabdus strains were initially injected, we found x. innexi highly dominant within the hemolymph after  <dig> h of multiplication .
nevertheless, the ability of x. innexi to outcompete x. nematophila was not sufficient enough in the symbiotic competition where s. scapterisci ijs brought 700-fold less of their symbionts than s. carpocapsae in the insects  <cit> .
in competition resulting from infection of insects with symbiotic ijs, our results showed that such a competition took place within the insects where only x. nematophila proliferated.
in this study, we studied both  the development of nematodes within the insects and  their ability to reproduce in them.
regarding the first aspect, we showed that the nematode's development into adults differed significantly for  competition resulting from infection with aposymbiotic ijs of both nematode species without injection of bacteria and  competition resulting from infection between symbiotic ijs of both nematode species.
the competition between aposymbiotic nematodes  allowed evaluation of the intrinsic ability of each nematode species to outcompete the other one without the help of its symbiotic xenorhabdus.
in such a situation, we showed that  significantly fewer nematodes developed into adults and that  s. scapterisci was highly dominant in both males and females found in the insects.
these results showed that s. scapterisci had a better competitive ability without its xenorhabdus symbiont than did s. carpocapsae .
on the contrary, in the competition between symbiotic nematodes, s. carpocapsae was highly dominant in adults .
experiments in which the amount of each xenorhabdus sp.
initially introduced in the insect was experimentally modified showed that the proportion of adults from each nematode species in the insects was directly linked to the proportion of their bacterial symbionts within the insect hemolymph.
.
the more x. nematophila was dominant within the insect hemolymph, the more the proportion of s. carpocapsae in adult worms found in the insects increased.
the analyses of the offspring emerging from the insects showed that competition resulting from infection with aposymbiotic ijs without injection of bacteria almost only produced ijs belonging to the species s. scapterisci.
on the contrary, and in accordance with the results obtained on adults development, almost all the ijs emerging from the competition resulting from infection with symbiotic ijs belonged to the species s. carpocapsae.
regarding the offspring production of each nematode species, the experimental modification of the bacterial symbiotic environment within the insects led to surprising results.
in all insects where x. innexi was injected [even at the lowest dose , the association s. carpocapse-x.
nematophila totally outcompeted the association s. scapterisci-x.
innexi.
the outcome of the competition between symbiotic nematodes in favour of s. carpocapsae is quite understandable since this competition occurred in a bacterial environment constituted exclusively of x. nematophila.
this dominance of x. nematophila within the insect hemolymph is due to the fact that s. scapterisci is poorly associated with its symbiont compared to s. carpocapsae   <cit> .
thus, dramatically less cells of x. innexi than x. nematophila were released by the worms within the insect hemolymph.
CONCLUSIONS
this study shows that the bacterial symbionts xenorhabdus modify the progress and outcome of interspecific competitions between their steinernema associated nematodes.
this suggests that xenorhabdus not only provides steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors.
thus, the evolution of the specific association between steinernema and xenorhabdus in environments where interspecific competitions occurred frequently should lead to the selection for an increased number of bacterial cells retained by each nematode in order to outcompete other specific nematodes-bacteria complexes.
a previous study has reported that there is dramatically different bacterial retention between some species of steinernema  <cit> .
this bacterial retention variability could be linked to differences in the biotic environments where each symbiotic association evolves.
indeed, s. carpocapsae is a worldwide distributed nematode contrary to s. scapterisci which is only found in south-america  <cit> .
moreover, s. carpocapsae is supposed to have a wider insect-host range than s. scapterisci  <cit> .
these specific ecological traits of each steinernema species suggest that s. carpocapsae could have evolved under a higher competition pressure than s. scapterisci.
this evolutionary constraint could have induced a selection toward a tighter relationship  in the s. carpocapsae-x.
nematophila association than in the s. scapterisci-x.
innexi association.

 symbioses between the entomopathogenic nematodes steinernema spp. and the enterobacteriacae xenorhabdus spp. are associations in which both partners receive benefits from each other. in the soil, the infective juveniles of the nematodes act as vectors dispersing the bacteria from insect host to insect-host and in turn, the bacteria increase the nematode's fitness within the insects hosts. previous studies showed that these symbioses were highly specific and that no steinernema spp. was able to associate with a xenorhabdus spp. genetically distant from its natural one. as the bacterial dispersion is totally dependent upon the fitness of the nematode within the insect-host, it is possible that xenorhabdus spp. might select special traits in order to enhance their vector's fitness. it is known that xenorhabdus spp. are beneficial to their nematodes in providing the latter with a better ability to kill the insect and feed on it. previous studies that focused on two different steinernema species have provided us with insights into the association characteristics. although the two nematode species demonstrated increased fitness when they parasitized insect-hosts with their own native symbiont, s. scapterisci appeared less dependent upon its native symbiont than s. carpocapsae. thus, s. scapterisci's symbiont increased the reproductive rate of its naturally associated nematode by a factor of, whereas x. nematophila increased the reproductive rate of its naturally associated nematode by sevenfold. moreover, s. scapterisci, transported 700-fold fewer cells of its xenorhabdus than s. carpocapsae. these two steinernema species also differed in their ability to deal with non-native xenorhabdus strains in case of co-infection in an insect. while s. scapterisci reproduced in co-infection situations with all the tested xenorhabdus strains, s. carpocapsae could not reproduce at all with most of them in the same situation. despite these specific differences, the global trend emerging from these previous experiments was that non-naturally associated xenorhabdus strains tend to be antagonist against nematodes species which cannot disperse them. one can easily think that this antagonistic effect of xenorhabdus strains on the fitness of nematodes naturally associated with others xenorhabdus strains could be selected in case of frequent interspecific competition. in such an evolutionary context, we can postulate that each xenorhabdus strain should try to provide its own nematode-vector with competitive advantages by producing antagonistic molecules against foreign nematodes. indeed, a previous study has shown with the association s. carpocapsae-x. nematophila as a model-system that, in insects co-infected by antagonistic xenorhabdus, x. nematophila partly counteracted their antagonistic effect on the nematodes fitness most probably by the mean of bacteriocins. the goal of the present study was to address the question of the impact of xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different steinernema species. for this, we monitored experimental interspecific competition between two nematode species: s. carpocapsae and s. scapterisci and their respective symbionts: x. nematophila and x. innexi within an insect-host. in this study, three conditions of competition between nematodes were tested: infection of insects with aposymbiotic ijs of both species infection of insects with aposymbiotic ijs of both species in presence of variable proportion of their two xenorhabdus symbionts and infection of insects with symbiotic ijs of both species.  proportion of each xenorhabdus in insect's hemolymph h post-infection with ijs we observed that both bacterial strains were able to multiply and co-exist within the hemolymph of the insect. nevertheless, x. nematophila was clearly less represented within the hemolymph h post-infection with nematodes when an initial injection of a suspension containing 50% of each bacterium in the insect was performed. when 70% and 90% of x. nematophila were injected, the two bacteria were found in a very variable proportion and co-existed. in the competition resulting from infection of insects with symbiotic ijs of both nematode species, as well as when 100% of x. nematophila were injected into insects infected with aposymbiotic ijs of both nematode species, no x. innexi were detected within the hemolymph. assessment of nematodes maturation in competition to know if gfp labelling of x. nematophila, employed to discriminate the two nematode species within the global offspring, triggered differences on the progress of competition between nematodes, the data obtained with or without gfp labelling of x. nematophila in each competition situation were compared with a mann withney test. we showed that gfp labelling had no statistically significant effect on both the ratio of s. carpocapsae among all females found in the insect h after infection and the ratio of s. carpocapsae among all males found in the insect h after infection. because of the non-significant differences observed in these comparisons, the results obtained in each situation with and without gfp labelled x. nematophila were pooled in further analyses. the ratios of s. carpocapsae among all males found in the insects were highly heterogeneous for the different competition conditions. the noether test showed that no significant difference in the ratio of s. carpocapsae males occurred under four competition conditions: competition resulting from infection of insects with aposymbiotic ijs of both nematode species without injection of bacterium, competition resulting from infection with aposymbiotic ijs of both nematode species in insects injected with 100% of x. innexi, competition resulting from infection with aposymbiotic ijs of both nematode species with 50% of each symbiont injected in the insects and competition resulting from infection with aposymbiotic nematodes in insects injected with 70% of x. nematophila and 30% of x. innexi. in these four conditions, the ratios of s. carpocapsae among males were significantly lower than in the three other conditions of competition: competition resulting from infection with aposymbiotic ijs in insects injected with 90% of x. nematophila and 10% of x. innexi, competition resulting from infection between aposymbiotic ijs in insects injected with 100% of x. nematophila and competition resulting from infection with symbiotic ijs. these results showed that an increase in the proportion of x. nematophila within bacteria injected into the insect triggered an increase of s. carpocapsae within adult worms found in the insects. the ratios of s. carpocapsae among females found in the insects were also highly heterogeneous for the different competition conditions. the noether test showed that the ratios of s. carpocapsae within females were lower in competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. innexi than in competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. nematophila and in competition resulting from infection with symbiotic ijs. in competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. innexi, no s. carpocapsae females were observed. assessment of the nematode offspring total number of infective juveniles the total number of ijs emerging from insects in the different competition situations for s. carpocapsae and s. scapterisci was heterogeneous under the different competition conditions. the noether test showed that competition resulting from infection with aposymbiotic ijs without injection of bacteria into the insect resulted in the production of significantly fewer ijs than competition resulting from infection with symbiotic ijs, competition resulting from infection with aposymbiotic ijs in insects injected with 50% of each symbiont, competition resulting from infection with aposymbiotic ijs in insects injected with 70% of x. nematophila and 30% of x. innexi and competition resulting from infection between aposymbiotic ijs in insects injected with 90% of x. nematophila and 10% of x. innexi. proportion of each nematode species in the total nematode offspring in the competition situation resulting from infection with aposymbiotic ijs of both nematode species without injection of bacteria into the insect, the proportion of each nematode species in the total offspring emerging from four insects was assessed with pcr-rflp made after global extraction of dna from pools of ijs. in control samples, the presence of 25% and 10% of s. carpocapsae ijs within pools of ijs containing respectively 75% and 90% of s. scapterisci were easily detected. in all the four pools of ijs tested here, only one band was observed after incubation of the pcr product with hind iii. this result suggested that after competition between aposymbiotic nematodes without injection of bacteria, more than 90% of all ijs emerging from the insect belonged to s. scapterisci. in other competition situations, the proportion of each nematode species was evaluated by counting the proportion of nematodes harbouring gfp labelled x. nematophila within the global offspring. gfp-fluorescence of x. nematophila is a good indicator of s. carpocapsae's ijs because pilots experiments showed that gfp labelled bacteria were still observable in ijs that were stored during months at 8°c and that after several generations within different insects, at each generation, 95% of the ijs emerging from the insects habored gfp labelled bacteria. nevertheless, in order to check that the gfp expression was not lost all along our experiments, we checked gfp expression for all the x. nematophila isolated from pools of ijs and confirmed that none of them did not express gfp. this result confirmed the perfect stability of gfp labelled x. nematophila all along our experiments. competition resulting from infection with aposymbiotic ijs in insects injected with 50% of each symbiont, 70% of x. nematophila and 30% of x. innexi and 90% of x. nematophila and 10% of x. innexi gave offspring in which almost no ijs harboured gfp labelled x. nematophila. these observations showed that s. scapterisci was highly dominant in the total offspring emerging from insects after such competitions. for competition resulting from infection with aposymbiotic ijs in insects injected with 100% of x. nematophila, the proportion of ijs harbouring gfp within the total offspring was very variable from to 100% with almost all the combinations. for competition resulting from infection with symbiotic ijs, most of the ijs emerging from the insects harboured gfp labelled x. nematophila. the latter result suggested that in competition resulting from infection with symbiotic ijs, most of the offspring emerging from the insects belonged to the species s. carpocapsae. discussion many invertebrates are associated with prokaryotes that provide them with the ability to live in extreme environments. the most documented symbioses are those where symbionts provide their hosts with new abilities to take advantage of poorly nutritive resources. nevertheless, symbionts can play other important roles on diverse life history traits of their hosts, for example, the bacterium vibrio which provides its sepiolids host with a better discretion towards its predators. we already knew that xenorhabdus symbionts provided steinernema nematodes with diverse adaptations to their environment: they produce virulence factors in order to kill the insect, they lead to a better access to the insect biomass and are themselves food for the nematodes, they produce antibiotics which counteract the multiplication of both distant and closely related bacteria. the goal of this study was to infer the abilities of xenorhabdus to modulate the progression and outcome of interspecific competition between steinernema species. for this, we made experimental competitions between s. carpocapsae and s. scapterisci and their bacterial symbionts. we assessed several parameters of these competitions: the proportion of each symbiont within the hemolymph of the insect after delay of multiplication, the ratio of s. carpocapsae among the adults found in the insects and the offspring of each nematode species after competition. the assessment of bacterial multiplication within insect hemolymph showed that x. innexi appeared more competitive than x. nematophila. moreover, in insects infected with aposymbiotic ijs and in which 50% of each xenorhabdus strains were initially injected, we found x. innexi highly dominant within the hemolymph after h of multiplication. nevertheless, the ability of x. innexi to outcompete x. nematophila was not sufficient enough in the symbiotic competition where s. scapterisci ijs brought 700-fold less of their symbionts than s. carpocapsae in the insects. in competition resulting from infection of insects with symbiotic ijs, our results showed that such a competition took place within the insects where only x. nematophila proliferated. in this study, we studied both the development of nematodes within the insects and their ability to reproduce in them. regarding the first aspect, we showed that the nematode's development into adults differed significantly for competition resulting from infection with aposymbiotic ijs of both nematode species without injection of bacteria and competition resulting from infection between symbiotic ijs of both nematode species. the competition between aposymbiotic nematodes allowed evaluation of the intrinsic ability of each nematode species to outcompete the other one without the help of its symbiotic xenorhabdus. in such a situation, we showed that significantly fewer nematodes developed into adults and that s. scapterisci was highly dominant in both males and females found in the insects. these results showed that s. scapterisci had a better competitive ability without its xenorhabdus symbiont than did s. carpocapsae. on the contrary, in the competition between symbiotic nematodes, s. carpocapsae was highly dominant in adults. experiments in which the amount of each xenorhabdus sp. initially introduced in the insect was experimentally modified showed that the proportion of adults from each nematode species in the insects was directly linked to the proportion of their bacterial symbionts within the insect hemolymph.. the more x. nematophila was dominant within the insect hemolymph, the more the proportion of s. carpocapsae in adult worms found in the insects increased. the analyses of the offspring emerging from the insects showed that competition resulting from infection with aposymbiotic ijs without injection of bacteria almost only produced ijs belonging to the species s. scapterisci. on the contrary, and in accordance with the results obtained on adults development, almost all the ijs emerging from the competition resulting from infection with symbiotic ijs belonged to the species s. carpocapsae. regarding the offspring production of each nematode species, the experimental modification of the bacterial symbiotic environment within the insects led to surprising results. in all insects where x. innexi was injected [even at the lowest dose, the association s. carpocapse-x. nematophila totally outcompeted the association s. scapterisci-x. innexi. the outcome of the competition between symbiotic nematodes in favour of s. carpocapsae is quite understandable since this competition occurred in a bacterial environment constituted exclusively of x. nematophila. this dominance of x. nematophila within the insect hemolymph is due to the fact that s. scapterisci is poorly associated with its symbiont compared to s. carpocapsae. thus, dramatically less cells of x. innexi than x. nematophila were released by the worms within the insect hemolymph.  this study shows that the bacterial symbionts xenorhabdus modify the progress and outcome of interspecific competitions between their steinernema associated nematodes. this suggests that xenorhabdus not only provides steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors. thus, the evolution of the specific association between steinernema and xenorhabdus in environments where interspecific competitions occurred frequently should lead to the selection for an increased number of bacterial cells retained by each nematode in order to outcompete other specific nematodes-bacteria complexes. a previous study has reported that there is dramatically different bacterial retention between some species of steinernema. this bacterial retention variability could be linked to differences in the biotic environments where each symbiotic association evolves. indeed, s. carpocapsae is a worldwide distributed nematode contrary to s. scapterisci which is only found in south-america. moreover, s. carpocapsae is supposed to have a wider insect-host range than s. scapterisci. these specific ecological traits of each steinernema species suggest that s. carpocapsae could have evolved under a higher competition pressure than s. scapterisci. this evolutionary constraint could have induced a selection toward a tighter relationship in the s. carpocapsae-x. nematophila association than in the s. scapterisci-x. innexi association. 
three conditions of competition between nematodes were tested: infection of insects with aposymbiotic ijs of both species infection of insects with aposymbiotic ijs of both species in presence of variable proportion of their two xenorhabdus symbionts and infection of insects with symbiotic ijs of both species. this suggests that xenorhabdus not only provides steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors. thus, the results obtained with aposymbiotic nematodes were totally opposite to those obtained with symbiotic nematodes. the goal of the present study was to address the question of the impact of xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different steinernema species. for this, we monitored experimental interspecific competition between two nematode species: s. carpocapsae and s. scapterisci and their respective symbionts: x. nematophila and x. innexi within an experimental insect-host. we found that both the progression and the outcome of interspecific competition between entomopathogenic nematodes were influenced by their bacterial symbionts. previous studies demonstrated a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and that mutualism plays a role in several important life history traits of each partner such as access to insect host resources, dispersal and protection against various biotic and abiotic factors. moreover, the experimental introduction of different ratios of xenorhabdus symbionts in the insect-host during competition between steinernema modified the proportion of each species in the adults and in the global offspring. we showed that xenorhabdus symbionts modified the competition between their steinernema associates.
 symbioses between invertebrates and prokaryotes are biological systems of particular interest in order to study the evolution of mutualism. the symbioses between the entomopathogenic nematodes steinernema and their bacterial symbiont xenorhabdus are very tractable model systems. previous studies demonstrated a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and that mutualism plays a role in several important life history traits of each partner such as access to insect host resources, dispersal and protection against various biotic and abiotic factors. the goal of the present study was to address the question of the impact of xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different steinernema species. for this, we monitored experimental interspecific competition between two nematode species: s. carpocapsae and s. scapterisci and their respective symbionts: x. nematophila and x. innexi within an experimental insect-host. three conditions of competition between nematodes were tested: infection of insects with aposymbiotic ijs of both species infection of insects with aposymbiotic ijs of both species in presence of variable proportion of their two xenorhabdus symbionts and infection of insects with symbiotic ijs of both species.  we found that both the progression and the outcome of interspecific competition between entomopathogenic nematodes were influenced by their bacterial symbionts. thus, the results obtained with aposymbiotic nematodes were totally opposite to those obtained with symbiotic nematodes. moreover, the experimental introduction of different ratios of xenorhabdus symbionts in the insect-host during competition between steinernema modified the proportion of each species in the adults and in the global offspring.  we showed that xenorhabdus symbionts modified the competition between their steinernema associates. this suggests that xenorhabdus not only provides steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors. 