Hélène Henri

Strain‐specific regulation of intracellular Wolbachia density in multiply infected insects

Vertically transmitted symbionts suffer a severe reduction in numbers when they pass through host generations, resulting in genetic homogeneity or even clonality of their populations. Wolbachia endosymbionts that induce cytoplasmic incompatibility in their hosts depart from this rule, because cytoplasmic incompatibility actively maintains multiple infection within hosts. Hosts and symbionts are thus probably under peculiar selective pressures that must shape the way intracellular bacterial populations are regulated. We studied the density and location of Wolbachia within adult Leptopilina heterotoma, a haplodiploid wasp that is parasitic on Drosophila and that is naturally infected with three Wolbachia strains, but for which we also obtained one simply infected and two doubly infected lines. Comparison of these four lines by quantitative polymerase chain reaction using a real‐time detection system showed that total Wolbachia density varies according to the infection status of individuals, while the specific density of each Wolbachia strain remains constant regardless of the presence of other strains. This suggests that Wolbachia strains do not compete with one another within the same host individual, and that a strain‐specific regulatory mechanism is operating. We discuss the regulatory mechanisms that are involved, and how this process might have evolved as a response to selective pressures acting on both partners.

Interaction between host genotype and environmental conditions affects bacterial density in Wolbachia symbiosis

Regulation of microbial population density is a necessity in stable symbiotic interactions. In Wolbachia symbiosis, both bacterial and host genotypes are involved in density regulation, but environmental factors may also affect bacterial population density. Here, we studied the interaction between three strains of Wolbachia in two divergent homozygous lines of the wasp Leptopilina heterotoma at two different temperatures. Wolbachia density varied between the two host genotypes at only one temperature. Moreover, at this temperature, reciprocal-cross F1 insects displayed identical Wolbachia densities, which were intermediate between the densities in the two parental lines. While these findings confirm that the host genotype plays an important role in Wolbachia density, they also highlight its interaction with environmental conditions, making possible the evolution of local adaptations for the regulation of Wolbachia density.

Effect of temperature on Wolbachia density and impact on cytoplasmic incompatibility

The outcome and the evolution of host-symbiont associations depend on environmental constraints, but responses are difficult to predict since they arise from a complex interaction between the host, the parasite and the environment. The situation can be even more complex when multiple parasite genotypes, with potentially different responses to environmental changes, coexist within a single host. In this paper, we investigated the effect of the temperature (from 14 to 26 degrees C) during the host development on the density of 3 strains of the intracellular bacterium Wolbachia that coexist within the wasp Leptopilina heterotoma. In this species, Wolbachia induces cytoplasmic incompatibility, a sperm-egg incompatibility that allows it to spread and persist in host populations. Using real-time quantitative PCR we found that (i) Wolbachia density is temperature-specific and highest at 26 degrees C; (ii) the order of the abundance of the 3 Wolbachia strains does not vary with temperature changes; (iii) the response of bacterial density to temperature occurs within a single insect generation, during the egg-to-adult developmental period; (iv) in this species, temperature-related changes in Wolbachia density do not influence cytoplasmic incompatibility.

A new case of Wolbachia dependence in the genus Asobara: evidence for parthenogenesis induction in Asobara japonica.

Wolbachia is a maternally inherited bacterium that is widely distributed among arthropods, in which it manipulates the reproduction of its hosts. Although generally facultative for its hosts, Wolbachia has recently become obligatory in Asobara tabida (Hymenoptera: Braconidae) in which it is required for the completion of oogenesis. Here, we describe a new Wolbachia strain (wAjap) that is associated with the genus Asobara and infects Asobara japonica. wAjap was detected in all female-biased populations of A. japonica found in the main islands of Japan, but not in the arrhenotokous populations from the southern islands. Using phylogenetic analyses based on multi-locus sequence typing (MLST), we show that this strain is closely related to wAtab3 (the strain required for oogenesis in A. tabida), even though they differ on Wolbachia surface protein (WSP) and WO phage sequences. Using antibiotic treatments, we show that cured thelytokous females are not dependent on Wolbachia for oogenesis. However, they produced only sons, showing that wAjap induces thelytokous parthenogenesis. Analyses of mating behavior and offspring production of individuals from Wolbachia-infected populations showed that while males were still sexually functional, females no longer attract males, making Wolbachia an obligate partner for daughter production in thelytokous populations. The fact that Wolbachia has become independently obligatory in two species of the same genus tends to show that dependence evolution can be common and swift, although no clear benefit for the parasitoid can be attributed to this dependence. Although dependence should lead to co-divergence between Wolbachia and its hosts, the very few cases of co-speciation observed in host–Wolbachia associations question the stability of these obligatory associations.

Influence of Wolbachia on host gene expression in an obligatory symbiosis

BackgroundWolbachia are intracellular bacteria known to be facultative reproductive parasites of numerous arthropod hosts. Apart from these reproductive manipulations, recent findings indicate that Wolbachia may also modify the host’s physiology, notably its immune function. In the parasitoid wasp, Asobara tabida, Wolbachia is necessary for oogenesis completion, and aposymbiotic females are unable to produce viable offspring. The absence of egg production is also associated with an increase in programmed cell death in the ovaries of aposymbiotic females, suggesting that a mechanism that ensures the maintenance of Wolbachia in the wasp could also be responsible for this dependence. In order to decipher the general mechanisms underlying host-Wolbachia interactions and the origin of the dependence, we developed transcriptomic approaches to compare gene expression in symbiotic and aposymbiotic individuals.ResultsAs no genetic data were available on A. tabida, we constructed several Expressed Sequence Tags (EST) libraries, and obtained 12,551 unigenes from this species. Gene expression was compared between symbiotic and aposymbiotic ovaries through in silico analysis and in vitro subtraction (SSH). As pleiotropic functions involved in immunity and development could play a major role in the establishment of dependence, the expression of genes involved in oogenesis, programmed cell death (PCD) and immunity (broad sense) was analyzed by quantitative RT-PCR. We showed that Wolbachia might interfere with these numerous biological processes, in particular some related to oxidative stress regulation. We also showed that Wolbachia may interact with immune gene expression to ensure its persistence within the host.ConclusionsThis study allowed us to constitute the first major dataset of the transcriptome of A. tabida, a species that is a model system for both host/Wolbachia and host/parasitoid interactions. More specifically, our results highlighted that symbiont infection may interfere with numerous pivotal processes at the individual level, suggesting that the impact of Wolbachia should also be investigated beyond reproductive manipulations.

Biomphalysin, a New β Pore-forming Toxin Involved in Biomphalaria glabrata Immune Defense against Schistosoma mansoni

Aerolysins are virulence factors belonging to the β pore-forming toxin (β-PFT) superfamily that are abundantly distributed in bacteria. More rarely, β-PFTs have been described in eukaryotic organisms. Recently, we identified a putative cytolytic protein in the snail, Biomphalaria glabrata, whose primary structural features suggest that it could belong to this β-PFT superfamily. In the present paper, we report the molecular cloning and functional characterization of this protein, which we call Biomphalysin, and demonstrate that it is indeed a new eukaryotic β-PFT. We show that, despite weak sequence similarities with aerolysins, Biomphalysin shares a common architecture with proteins belonging to this superfamily. A phylogenetic approach revealed that the gene encoding Biomphalysin could have resulted from horizontal transfer. Its expression is restricted to immune-competent cells and is not induced by parasite challenge. Recombinant Biomphalysin showed hemolytic activity that was greatly enhanced by the plasma compartment of B. glabrata. We further demonstrated that Biomphalysin with plasma is highly toxic toward Schistosoma mansoni sporocysts. Using in vitro binding assays in conjunction with Western blot and immunocytochemistry analyses, we also showed that Biomphalysin binds to parasite membranes. Finally, we showed that, in contrast to what has been reported for most other members of the family, lytic activity of Biomphalysin is not dependent on proteolytic processing. These results provide the first functional description of a mollusk immune effector protein involved in killing S. mansoni.

Distribution of Bemisia tabaci (Homoptera: Aleyrodidae) biotypes and their associated symbiotic bacteria on host plants in West Africa

Abstract.  1. The whitefly Bemisia tabaci is a pest of many agricultural and ornamental crops worldwide and particularly in Africa. B. tabaci is a complex of more than 20 biotypes. Effective control of B. tabaci calls for a greater knowledge of the local biological diversity in terms of biotypes or putative species. Information is available about biotype distribution in Northern, Eastern, and Southern Africa, but data for Western Africa remain very scarce. At the time of this study, data were available for only three sampling sites in Burkina Faso, where three biotypes have been detected, the native Sub‐Saharan Africa non‐Silver Leafing (AnSL), the Sub‐Saharan Africa Silverleafing (ASL), and the Mediterranean Q biotypes, but no information is available about their respective distributions on host plant species ( Gueguen et al., 2010 ).

Multiple infections and diversity of cytoplasmic incompatibility in a haplodiploid species

Cytoplasmic incompatibility (CI) is a sperm–egg incompatibility commonly induced by the intracellular endosymbiont bacterium Wolbachia that, in diploid species, results in embryo mortality. In haplodiploid species, two types of CI exist depending on whether the incompatible fertilized eggs develop into males (male development (MD)) or abort (female mortality (FM)). CI allows multiple infections to be maintained in host populations, and thus allows interactions to occur between co-infecting strains. In Leptopilina heterotoma, three Wolbachia strains coexist naturally (wLhet1, wLhet2, wLhet3). When these three strains are all present, they induce a CI of FM type, whereas wLhet1 alone expresses a CI phenotype intermediate between MD and FM. Here, we compare CI effects in crosses involving insect lines sharing the same nuclear background, but harboring different mixtures of strains. Mating experiments showed that: (i) wLhet2 and wLhet3 also induce an intermediate CI when acting alone, and show a bidirectional incompatibility; (ii) there is no interaction between the co-infecting strains in CI expression; (iii) the diversity of Wolbachia present within a male host influences the expression of CI: an increase in the number of strains is correlated with a decrease in the proportion of the MD type, which is also correlated with an increase in bacterial density. All these data suggest that the CI of FM type results from a stronger effect than the MD type, which conflicts with the conventional hypotheses used to explain CI diversity in haplodiploids, and could provide some new information about CI mechanisms in insects.

Epidemiology of asexuality induced by the endosymbiotic Wolbachia across phytophagous wasp species: host plant specialization matters

Among eukaryotes, sexual reproduction is by far the most predominant mode of reproduction. However, some systems maintaining sexuality appear particularly labile and raise intriguing questions on the evolutionary routes to asexuality. Thelytokous parthenogenesis is a form of spontaneous loss of sexuality leading to strong distortion of sex ratio towards females and resulting from mutation, hybridization or infection by bacterial endosymbionts. We investigated whether ecological specialization is a likely mechanism of spread of thelytoky within insect communities. Focusing on the highly specialized genus Megastigmus (Hymenoptera: Torymidae), we first performed a large literature survey to examine the distribution of thelytoky in these wasps across their respective obligate host plant families. Second, we tested for thelytoky caused by endosymbionts by screening in 15 arrhenotokous and 10 thelytokous species for Wolbachia, Cardinium, Arsenophonus and Rickettsia endosymbionts and by performing antibiotic treatments. Finally, we performed phylogenetic reconstructions using multilocus sequence typing (MLST) to examine the evolution of endosymbiont‐mediated thelytoky in Megastigmus and its possible connections to host plant specialization. We demonstrate that thelytoky evolved from ancestral arrhenotoky through the horizontal transmission and the fixation of the parthenogenesis‐inducing Wolbachia. We find that ecological specialization in Wolbachias hosts was probably a critical driving force for Wolbachia infection and spread of thelytoky, but also a constraint. Our work further reinforces the hypothesis that community structure of insects is a major driver of the epidemiology of endosymbionts and that competitive interactions among closely related species may facilitate their horizontal transmission.

Two Host Clades, Two Bacterial Arsenals: Evolution through Gene Losses in Facultative Endosymbionts

Bacterial endosymbiosis is an important evolutionary process in insects, which can harbor both obligate and facultative symbionts. The evolution of these symbionts is driven by evolutionary convergence, and they exhibit among the tiniest genomes in prokaryotes. The large host spectrum of facultative symbionts and the high diversity of strategies they use to infect new hosts probably impact the evolution of their genome and explain why they undergo less severe genomic erosion than obligate symbionts. Candidatus Hamiltonella defensa is suitable for the investigation of the genomic evolution of facultative symbionts because the bacteria are engaged in specific relationships in two clades of insects. In aphids, H. defensa is found in several species with an intermediate prevalence and confers protection against parasitoids. In whiteflies, H. defensa is almost fixed in some species of Bemisia tabaci, which suggests an important role of and a transition toward obligate symbiosis. In this study, comparisons of the genome of H. defensa present in two B. tabaci species (Middle East Asia Minor 1 and Mediterranean) and in the aphid Acyrthosiphon pisum revealed that they belong to two distinct clades and underwent specific gene losses. In aphids, it contains highly virulent factors that could allow protection and horizontal transfers. In whiteflies, the genome lost these factors and seems to have a limited ability to acquire genes. However it contains genes that could be involved in the production of essential nutrients, which is consistent with a primordial role for this symbiont. In conclusion, although both lineages of H. defensa have mutualistic interactions with their hosts, their genomes follow distinct evolutionary trajectories that reflect their phenotype and could have important consequences on their evolvability.