Sylvie Pagès

When mutualists are pathogens: an experimental study of the symbioses between Steinernema (entomopathogenic nematodes) and Xenorhabdus (bacteria)

In this paper, we investigate the level of specialization of the symbiotic association between an entomopathogenic nematode (Steinernema carpocapsae) and its mutualistic native bacterium (Xenorhabdus nematophila). We made experimental combinations on an insect host where nematodes were associated with non‐native symbionts belonging to the same species as the native symbiont, to the same genus or even to a different genus of bacteria. All non‐native strains are mutualistically associated with congeneric entomopathogenic nematode species in nature. We show that some of the non‐native bacterial strains are pathogenic for S. carpocapsae. When the phylogenetic relationships between the bacterial strains was evaluated, we found a clear negative correlation between the effect a bacterium has on nematode fitness and its phylogenetic distance to the native bacteria of this nematode. Moreover, only symbionts that were phylogenetically closely related to the native bacterial strain were transmitted. These results suggest that co‐evolution between the partners has led to a high level of specialization in this mutualism, which effectively prevents horizontal transmission. The pathogenicity of some non‐native bacterial strains against S. carpocapsae could result from the incapacity of the nematode to resist specific virulence factors produced by these bacteria.

Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction

Abstract Steinernema species are entomopathogenic nematodes. They are symbiotically associated with Enterobacteriaceae of the genus Xenorhabdus. These nematode–bacteria symbioses are extremely diversified and constitute an important new model in ecology and evolution to investigate symbioses between microbes and invertebrates. However, no study has so far adequately evaluated either the outcome of the interactions or the obligate nature of interactions in different Steinernema species in the same way. Studying three different species of Steinernema, we showed that symbiotic nematodes are always fitter than aposymbiotic ones. Nevertheless, we revealed contrasting types of interaction in terms of outcome and obligate nature of the interaction. Bacterial analyses showed that nematode species differed dramatically in the number of symbiotic Xenorhabdus they carried. We suggested that when the interaction appeared more facultative for a nematode species, the nematodes carried fewer Xenorhabdus cells than strongly dependent worm species. Thus, the symbiont transmission appeared to become more efficient as the relationship between the nematode and the bacteria became tighter.

Interspecific competition between entomopathogenic nematodes (Steinernema) is modified by their bacterial symbionts (Xenorhabdus)

BackgroundSymbioses 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 (i) a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and (ii) 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 (i) two nematode species: S. carpocapsae and S. scapterisci and (ii) their respective symbionts: X. nematophila and X. innexi within an experimental insect-host (Galleria mellonella). Three conditions of competition between nematodes were tested: (i) infection of insects with aposymbiotic IJs (i.e. without symbiont) of both species (ii) infection of insects with aposymbiotic IJs of both species in presence of variable proportion of their two Xenorhabdus symbionts and (iii) infection of insects with symbiotic IJs (i.e. naturally associated with their symbionts) of both species.ResultsWe 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.ConclusionWe 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.

Studies of the dynamic expression of the Xenorhabdus FliAZ regulon reveal atypical iron‐dependent regulation of the flagellin and haemolysin genes during insect infection

Xenorhabdus nematophila engages in complex interactions with invertebrates, through its symbiosis with soil nematodes and its pathogenicity to a broad range of insect larvae. Among the regulatory proteins of Xenorhabdus involved in host interactions, the sigma factor FliA and the regulator FliZ, expressed from the fliAZ operon, play a key role in mediating the production of exoenzymes, motility and full virulence in insects (Lanois et al., 2008). In this study, we investigated the dynamics of the FliA-dependent flagellin gene fliC and FliZ-dependent haemolysin genes xaxAB during insect infection and nematode association by carrying out real-time expression analysis using an unstable GFP monitoring system. We showed that expression of the FliAZ-dependent genes in infected insects is not restricted to a specific tissue but increases significantly just prior to host death and reaches a maximal level in larvae cadaver. Using an iron availability reporter construct, we also showed that iron starvation conditions inhibit expression of FliAZ-dependent genes in vitro, as well as during the first steps of the infectious process. These findings shed further light on the role of the FliAZ regulon in the Xenorhabdus life cycle and suggest that iron may constitute a signal governing Xenorhabdus adaptation to shifting host environments.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).

Specialization of the entomopathogenic nematode Steinernema scapterisci with its mutualistic Xenorhabdus symbiont

The level of specialization of the entomopathogenic nematode Steinernema scapterisci with its native Xenorhabdus symbiont was investigated by testing (1) the influence of non-native bacterial strains on nematode fitness within an insect-host (Galleria mellonella) and (2) specificity of the association between the nematode infective juveniles and non-native bacteria. All non-native Xenorhabdus spp. or Photorhabdus spp. strains tested were mutualistically associated with other entomopathogenic nematodes in nature. We showed that most of the Xenorhabdus spp. strains tested led to an insignificant difference of the nematodes fitness compared to the one obtained with the native bacterium. Conversely, Photorhabdus spp. strains almost entirely abolished nematode reproduction. The phylogenetic analysis of bacterial strains tested, showed that there was a negative correlation between S. scapteriscis reproduction rate with a bacterial strain and the genetic distance of this bacterial strain from the native one. We also showed that the native bacterium was the only one which was transmitted by S. scapteriscis infective juveniles. All these results, suggested a specialization between S. scapterisci and its native Xenorhabdus. As the same phenomenon was already demonstrated in the association between S. carpocapsae and X. nematophila, specialization between partners would not be an exception in entomopathogenic nematode-bacteria interactions. Nevertheless, S. scapterisci showed a dramatically higher compatibility with non-native Xenorhabdus spp. strains than did S. carpocapsae, suggesting differences in the co-evolutionary processes between nematodes and bacteria in these two model systems.Table 1List of the bacterial strains, native nematode species with their geographical origin, accession numbers of bacterial 16S rDNA partial sequences and number of combination experiments for each bacterium testedBacterial species and strainsNative nematode speciesAccession no. of the 16S rDNA sequenceGeographical originNo. of combination experimentsX. innexi UY61S. scapterisciAY521243Uruguay80X. poinarii SK72S. glaseriAY521239USA40X. beddingii Q58Steinernema sp.D78006Australia40X. bovienii FR10S. feltiaeAY521240France40Xenorhabdus sp. USTX62S. riobraveAY521244USA40X. nematophila F1S. carpocapsaeAY521241France40P. luminescens TT01H. bacteriophoraAJ007404Trinidad40P. temperata XLNACHH. megidisAJ007405Russia40

Manifold aspects of specificity in a nematode–bacterium mutualism

Coevolution in mutualistic symbiosis can yield, because the interacting partners share common interests, to coadaptation: hosts perform better when associated with symbionts of their own locality than with others coming from more distant places. However, as the two partners of a symbiosis might also experience conflicts over part of their life cycle, coadaptation might not occur for all life‐history traits. We investigated this issue in symbiotic systems where nematodes (Steinernema) and bacteria (Xenorhabdus) reproduce in insects they have both contributed to kill. Newborn infective juveniles (IJs) that carry bacteria in their intestine then disperse from the insect cadaver in search of a new host to infect. We ran experiments where nematodes coinfect insects with bacteria that differ from their native symbiont. In both Steinernema carpocapsae/Xenorhabdus nematophila and Steinernema feltiae/Xenorhabdus bovienii symbioses, we detected an overall specificity which favours the hypothesis of a fine‐tuned co‐adaptation process. However, we also found that the life‐history traits involved in specificity strongly differ between the two model systems: when associated with strains that differ too much from their native symbionts, S. carpocapsae has low parasitic success, whereas S. feltiae has low survival in dispersal stage.

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica

The bacterial symbiont AM7(T), isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7(T) within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7(T) revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97 % proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) ( = ATCC BAA-2407(T)  = DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov.

Description of Xenorhabdus magdalenensis sp. nov., the symbiotic bacterium associated with Steinernema australe

A symbiotic bacterium, strain IMI 397775(T), was isolated from the insect-pathogenic nematode Steinernema australe. On the basis of 16S rRNA gene sequence similarity, this bacterial isolate was shown to belong to the genus Xenorhabdus, in agreement with the genus of its nematode host. The accurate phylogenetic position of this new isolate was defined using a multigene approach and showed that isolate IMI 397775(T) shares a common ancestor with Xenorhabdus doucetiae FRM16(T) and Xenorhabdus romanii PR06-A(T), the symbiotic bacteria associated with Steinernema diaprepesi and Steinernema puertoricense, respectively. The nucleotide identity (less than 97%) between isolate IMI 397775(T), X. doucetiae FRM16(T) and X. romanii PR06-A(T) calculated for the concatenated sequences of five gene fragments encompassing 4275 nt, several phenotypic traits and the difference between the upper temperatures that limit growth of these three bacteria allowed genetic and phenotypic differentiation of isolate IMI 397775(T) from the two closely related species. Strain IMI 397775(T) therefore represents a novel species, for which the name Xenorhabdus magdalenensis sp. nov. is proposed, with the type strain IMI 397775(T) ( = DSM 24915(T)).

Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.