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Mosquitoes of the Culex pipiens complex are worldwide vectors of arbovirus, filarial nematodes, and avian malaria agents. In these hosts, the endosymbiotic bacteria Wolbachia induce cytoplasmic incompatibility (CI), i.e., reduced embryo viability in so-called incompatible crosses. Wolbachia infecting Culex pipiens (wPip) cause CI patterns of unparalleled complexity, associated with the amplification and diversification of cidA and cidB genes, with up to 6 different gene copies described in a single wPip genome. In wPip, CI is thought to function as a toxin-antidote (TA) system where compatibility relies on having the right antidotes (CidA) in the female to bind and neutralize the male's toxins (CidB). By repeating crosses between Culex isofemale lines over a 17 years period, we documented the emergence of a new compatibility type in real time and linked it to a change in cid genes genotype. We showed that loss of specific cidA gene copies in some wPip genomes results in a loss of compatibility. More precisely, we found that this lost antidote had an original sequence at its binding interface, corresponding to the original sequence at the toxin's binding interface. We showed that these original cid variants are recombinant, supporting a role for recombination rather than point mutations in rapid CI evolution. These results strongly support the TA model in natura, adding to all previous data acquired with transgenes expression., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Namias et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This protocol describes approaches to qualify Wolbachia-induced cytoplasmic incompatibility (CI) patterns (compatible, uni or bidirectional) in crosses between two or more Culex pipiens isofemale lines, hosting different Wolbachia (wPip) strains. Here, we describe how to (1) collect the larvae in the field and grow them to the adult stage in the insectary, (2) set up isofemale lines in the insectary, (3) genetically characterize the wPip group of these lines, and (4) perform reciprocal crosses to qualify CI., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The importance of gene amplifications in evolution is more and more recognized. Yet, tools to study multi-copy gene families are still scarce, and many such families are overlooked using common sequencing methods. Haplotype reconstruction is even harder for polymorphic multi-copy gene families. Here, we show that all variants (or haplotypes) of a multi-copy gene family present in a single genome, can be obtained using Oxford Nanopore Technologies sequencing of PCR products, followed by steps of mapping, SNP calling and haplotyping. As a proof of concept, we acquired the sequences of highly similar variants of the cidA and cidB genes present in the genome of the Wolbachia w Pip, a bacterium infecting Culex pipiens mosquitoes . Our method relies on a wide database of cid genes, previously acquired by cloning and Sanger sequencing. We addressed problems commonly faced when using mapping approaches for multi-copy gene families with highly similar variants. In addition, we confirmed that PCR amplification causes frequent chimeras which have to be carefully considered when working on families of recombinant genes. We tested the robustness of the method using a combination of bioinformatics (read simulations) and molecular biology approaches (sequence acquisitions through cloning and Sanger sequencing, specific PCRs and digital droplet PCR). When different haplotypes present within a single genome cannot be reconstructed from short reads sequencing, this pipeline confers a high throughput acquisition, gives reliable results as well as insights of the relative copy numbers of the different variants., Competing Interests: The authors have declared no competing interests., (© 2023 The Authors.)

Crassostrea gigas oysters represent a significant global food source with 4.7 million tons harvested per year. In 2001, the bacterium Vibrio aestuarianus subsp. francensis emerged as a pathogen that causes adult oyster mortality in France and Ireland. Its impact on oyster aquaculture has increased in Europe since its re-emergence in 2012. To better understand the evolutionary mechanisms leading to the emergence and persistence over time of this pathogen, we conducted a survey of mollusc diseases through national reference laboratories across Europe. We analysed 54 new genomes of Vibrio aestuarianus (Va) isolated from multiple environmental compartments since 2001, in areas with and without bivalve mortalities. We used a combination of comparative genomics and population genetics approaches and show that Va has a classical epidemic population structure from which the pathogenic Va francensis subspecies emerged and clonally expanded. Furthermore, we identified a specific cus-cop-containing island conferring copper resistance to Va francensis whose acquisition may have favoured the emergence of pathogenic lineages adapted and specialized to oysters., (© 2023 Crown copyright. Molecular Ecology © 2023 John Wiley & Sons Ltd. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.)

Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction of arthropods through a diversity of cellular mechanisms. In cytoplasmic incompatibility (CI), a sterility syndrome originally discovered in the mosquito Culex pipiens, uninfected eggs fertilized by sperm from infected males are selectively killed during embryo development following the abortive segregation of paternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CI factor (cif) genes, the mechanism by which they control the fate of paternal chromosomes at fertilization remains unknown. Here, we have analyzed the cytological distribution and cellular impact of CidA and CidB, a pair of Cif proteins from the Culex-infecting Wolbachia strain wPip. We show that expression of CidB in Drosophila S2R+ cells induces apoptosis unless CidA is co-expressed and associated with its partner. In transgenic Drosophila testes, both effectors colocalize in germ cells until the histone-to-protamine transition in which only CidB is retained in maturing spermatid nuclei. We further show that CidB is similarly targeted to maturing sperm of naturally infected Culex mosquitoes. At fertilization, CidB associates with paternal DNA regions exhibiting DNA replication stress, as a likely cause of incomplete replication of paternal chromosomes at the onset of the first mitosis. Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidB does not abolish its cell toxicity or its ability to induce CI in Drosophila. Our study thus demonstrates that CI functions as a transgenerational toxin-antidote system and suggests that CidB acts by poisoning paternal DNA replication in incompatible crosses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Wolbachia endosymbionts commonly induce cytoplasmic incompatibility, making infected males' sperm lethal to the embryos unless these are rescued by the same bacterium, inherited from their mother. Causal genes were recently identified but two families of mechanistic models are still opposed. In the toxin-antidote model, interaction between the toxin and the antidote is required for rescuing the embryos. In host modification models, a host factor is misregulated in sperm and rescue occurs through compensation or withdrawal of this modification. While these models have been thoroughly discussed, the multiplicity of compatibility types, that is, the existence of many mutually incompatible strains, as seen in Culex mosquitoes, has not received sufficient attention. To explain such a fact, host modification models must posit that the same embryonic defects can be induced and rescued through a large variety of host targets. Conversely, the toxin-antidote model simply accommodates this pattern in a lock-key fashion, through variations in the toxin-antidote interaction sites., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microbial communities are known to influence mosquito lifestyles by modifying essential metabolic and behavioral processes that affect reproduction, development, immunity, digestion, egg survival, and the ability to transmit pathogens. Many studies have used 16S rRNA gene amplicons to characterize mosquito microbiota and investigate factors that influence host-microbiota dynamics. However, a relatively low taxonomic resolution due to clustering methods based on arbitrary threshold and the overall dominance of Wolbachia or Asaia symbionts obscured the investigation of rare members of mosquito microbiota in previous studies. Here, we used high resolution Shannon entropy-based oligotyping approach to analyze the microbiota of Culex pipiens , Culex quinquefasciatus and Aedes individuals from continental Southern France and overseas Guadeloupe as well as from laboratories with or without antibiotics treatment. Our experimental design that resulted in a series of mosquito samples with a gradient of Wolbachia density and relative abundance along with high-resolution analyses of amplicon sequences enabled the recovery of a robust signal from typically less accessible bacterial taxa. Our data confirm species-specific mosquito-bacteria associations with geography as a primary factor that influences bacterial community structure. But interestingly, they also reveal co-occurring symbiotic bacterial variants within single individuals for both Elizabethkingia and Erwinia genera, distinct and specific Asaia and Chryseobacterium in continental and overseas territories, and a putative rare Wolbachia variant. Overall, our study reveals the presence of previously overlooked microdiversity and multiple closely related symbiotic strains within mosquito individuals with a remarkable habitat-specificity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Mosquitoes not only transmit human and veterinary pathogens called arboviruses (arthropod-borne viruses) but also harbor mosquito-associated insect-specific viruses (mosquito viruses) that cannot infect vertebrates. In the past, studies investigating mosquito viruses mainly focused on highly pathogenic interactions that were easier to detect than those without visible symptoms. However, the recent advances in viral metagenomics have highlighted the abundance and diversity of viruses which do not generate mass mortality in host populations. Over the last decade, this has facilitated the rapid growth of virus discovery in mosquitoes. The circumstances around the discovery of mosquito viruses greatly affected how they have been studied so far. While earlier research mainly focused on the pathogenesis caused by DNA and some double-stranded RNA viruses during larval stages, more recently discovered single-stranded RNA mosquito viruses were heavily studied for their putative interference with arboviruses in female adults. Thus, many aspects of mosquito virus interactions with their hosts and host-microbiota are still unknown. In this context, considering mosquito viruses as endosymbionts can help to identify novel research areas, in particular in relation to their long-term interactions with their hosts (e.g. relationships during all life stages, the stability of the associations at evolutionary scales, transmission routes and virulence evolution) and the possible context-dependent range of interactions (i.e. beneficial to antagonistic). Here, we review the symbiotic interactions of mosquito viruses considering different aspects of their ecology, such as transmission, host specificity, host immune system and interactions with other symbionts within the host cellular arena. Finally, we highlight related research gaps in mosquito virus research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Altinli, Schnettler and Sicard.)

In arthropods, Wolbachia endosymbionts induce conditional sterility, called cytoplasmic incompatibility (CI), resulting from embryonic lethality. CI penetrance (i.e., embryonic death rate) varies depending on host species and Wolbachia strains involved. All Culex pipiens mosquitoes are infected by the endosymbiotic alphaproteobacteria Wolbachia w Pip. CI in Culex , characterized as a binary "compatible/incompatible" phenomenon, revealed an unparalleled diversity of patterns linked to the amplification-diversification of cidA and cidB genes. Here, we accurately studied CI penetrance variations in the light of cid genes divergence by generating a C. pipiens compatibility matrix between 11 lines hosting different phylogenetic w Pip groups and exhibiting distinct cid gene repertoires. We showed, as expected, that crosses involving w Pip from the same group were mostly compatible. In contrast, only 22% of the crosses involving different w Pip groups were compatible, while 54% were fully incompatible. For the remaining 24% of the crosses, "intermediate" compatibilities were reported, and a cytological observation of the first zygotic division confirmed the occurrence of "canonical" CI phenotypes in a fraction of the eggs. Backcross experiments demonstrated that intermediate compatibilities were not linked to host genetic background but to the Wolbachia strains involved. This previously unstudied intermediate penetrance CI was more severe and frequent in crosses involving w Pip-IV strains exhibiting cid variants markedly divergent from other w Pip groups. Our data demonstrate that CI is not always a binary compatible/incompatible phenomenon in C. pipiens but that intermediate compatibilities putatively resulting from partial mismatch due to Cid proteins divergence exist in this species complex. IMPORTANCE Culex pipiens mosquitoes are infected with w Pip. These endosymbionts induce a conditional sterility called CI resulting from embryonic deaths, which constitutes a cornerstone for Wolbachia antivectorial methods. Recent studies revealed that (i) two genes, cidA and cidB , are central in Wolbachia -CI mechanisms, and (ii) compatibility versus incompatibility between mosquito lines depends on the w Pip phylogenetic groups at play. Here, we studied CI variations in relation to w Pip groups and cid genes divergence. We showed, as expected, that the crosses involving w Pip from the same group were compatible. In contrast, 78% of the crosses involving different w Pip groups were partially or fully incompatible. In such crosses, we reported defects during the first zygotic division, a hallmark of CI. We showed that CI was more severe and frequent in crosses involving w Pip-IV strains exhibiting cid variants, which markedly diverge from those of other w Pip groups., (Copyright © 2021 Sicard et al.)

Wolbachia are the most widespread endosymbiotic bacteria in animals. In many arthropod host species, they manipulate reproduction via several mechanisms that favor their maternal transmission to offspring. Among them, cytoplasmic incompatibility (CI) promotes the spread of the symbiont by specifically decreasing the fertility of crosses involving infected males and uninfected females, via embryo mortality. These differences in reproductive efficiency may select for the avoidance of incompatible mating, a process called reinforcement, and thus contribute to population divergence. In the terrestrial isopod Porcellio dilatatus , the Wolbachia w Pet strain infecting the subspecies P. d. petiti induces unidirectional CI with uninfected individuals of the subspecies P. d. dilatatus . To study the consequences of CI on P. d. dilatatus and P. d. petiti hybridization, mitochondrial haplotypes and Wolbachia infection dynamics, we used population cages seeded with different proportions of the 2 subspecies in which we monitored these genetic parameters 5 and 7 years after the initial setup. Analysis of microsatellite markers allowed evaluating the degree of hybridization between individuals of the 2 subspecies. These markers revealed an increase in P. d. dilatatus nuclear genetic signature in all mixed cages, reflecting an asymmetry in hybridization. Hybridization led to the introgressive acquisition of Wolbachia and mitochondrial haplotype from P. d. petiti into nuclear genomes dominated by alleles of P. d. dilatatus . We discuss these results with regards to Wolbachia effects on their host (CI and putative fitness cost), and to a possible reinforcement that may have led to assortative mating, as possible factors contributing to the observed results., (© The Author(s) (2020). Published by Oxford University Press on behalf of Editorial Office, Current Zoology.)

The inadequacy of standard mosquito control strategies calls for ecologically safe novel approaches, for example the use of biological agents such as the endosymbiotic α-proteobacteria Wolbachia or insect-specific viruses (ISVs). Understanding the ecological interactions between these "biocontrol endosymbionts" is thus a fundamental step. Wolbachia are transmitted vertically from mother to offspring and modify their hosts' phenotypes, including reproduction (e.g., cytoplasmic incompatibility) and survival (e.g., viral interference). In nature, Culex pipiens (sensu lato) mosquitoes are always found infected with genetically diverse Wolbachia called wPip that belong to five phylogenetic groups. In recent years, ISVs have also been discovered in these mosquito species, although their interactions with Wolbachia in nature are unknown. Here, we studied the interactions between a widely prevalent ISV, the Culex pipiens densovirus (CpDV, Densovirinae), and Wolbachia in northern Tunisian C. pipiens populations. We showed an influence of different Wolbachia groups on CpDV prevalence and a general positive correlation between Wolbachia and CpDV loads. By investigating the putative relationship between CpDV diversification and wPip groups in the different sites, we detected a signal linked to wPip groups in CpDV phylogeny in sites where all larvae were infected by the same wPip group. However, no such signal was detected where the wPip groups coexisted, suggesting CpDV horizontal transfer between hosts. Overall, our results provide good evidence for an ecological influence of Wolbachia on an ISV, CpDV, in natural populations and highlight the importance of integrating Wolbachia in our understanding of ISV ecology in nature., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

There are many documented examples of viral genes retained in the genomes of multicellular organisms that may in some cases bring new beneficial functions to the receivers. The ability of certain ichneumonid parasitic wasps to produce virus-derived particles, the so-called ichnoviruses (IVs), not only results from the capture and domestication of single viral genes but of almost entire ancestral virus genome(s). Indeed, following integration into wasp chromosomal DNA, the putative and still undetermined IV ancestor(s) evolved into encoding a 'virulence gene delivery vehicle' that is now required for successful infestation of wasp hosts. Several putative viral genes, which are clustered in distinct regions of wasp genomes referred to as IVSPERs (Ichnovirus Structural Protein Encoding Regions), have been assumed to be involved in virus-derived particles morphogenesis, but this question has not been previously functionally addressed. In the present study, we have successfully combined RNA interference and transmission electron microscopy to specifically identify IVSPER genes that are responsible for the morphogenesis and trafficking of the virus-derived particles in ovarian cells of the ichneumonid wasp Hyposoter didymator. We suggest that ancestral viral genes retained within the genomes of certain ichneumonid parasitoids possess conserved functions which were domesticated for the purpose of assembling viral vectors for the delivery of virulence genes to parasitized host animals., Competing Interests: The authors have declared that no competing interests exist.

Viruses of the Parvoviridae family infect a wide range of animals including vertebrates and invertebrates. So far, our understanding of parvovirus diversity is biased towards medically or economically important viruses mainly infecting vertebrate hosts, while invertebrate infecting parvoviruses-namely densoviruses-have been largely neglected. Here, we investigated the prevalence and the evolution of the only mosquito-infecting ambidensovirus, Culex pipiens densovirus (CpDV), from laboratory mosquito lines and natural populations collected worldwide. CpDV diversity generally grouped in two clades, here named CpDV-1 and -2. The incongruence of the different gene trees for some samples suggested the possibility of recombination events between strains from different clades. We further investigated the role of selection on the evolution of CpDV genome and detected many individual sites under purifying selection both in non-structural and structural genes. However, some sites in structural genes were under diversifying selection, especially during the divergence of CpDV-1 and -2 clades. These substitutions between CpDV-1 and -2 clades were mostly located in the capsid protein encoding region and might cause changes in host specificity or pathogenicity of CpDV strains from the two clades. However, additional functional and experimental studies are necessary to fully understand the protein conformations and the resulting phenotype of these substitutions between clades of CpDV., (© The Author(s) 2019. Published by Oxford University Press.)

Endosymbiotic Wolbachia bacteria are, to date, considered the most widespread symbionts in arthropods and are the cornerstone of major biological control strategies. Such a high prevalence is based on the ability of Wolbachia to manipulate their hosts' reproduction. One manipulation called cytoplasmic incompatibility (CI) is based on the death of the embryos generated by crosses between infected males and uninfected females or between individuals infected with incompatible Wolbachia strains. CI can be seen as a modification-rescue system (or mod-resc) in which paternal Wolbachia produce mod factors, inducing embryonic defects, unless the maternal Wolbachia produce compatible resc factors. Transgenic experiments in Drosophila melanogaster and Saccharomyces cerevisiae converged towards a model where the cidB Wolbachia gene is involved in the mod function while cidA is involved in the resc function. However, as cidA expression in Drosophila males was required to observe CI, it has been proposed that cidA could be involved in both resc and mod functions. A recent correlative study in natural Culex pipiens mosquito populations has revealed an association between specific cidA and cidB variations and changes in mod phenotype, also suggesting a role for both these genes in mod diversity. Here, by studying cidA and cidB genomic repertoires of individuals from newly sampled natural C. pipiens populations harbouring wPipIV strains from North Italy, we reinforce the link between cidB variation and mod phenotype variation fostering the involvement of cidB in the mod phenotype diversity. However, no association between any cidA variants or combination of cidA variants and mod phenotype variation was observed. Taken together our results in natural C. pipiens populations do not support the involvement of cidA in mod phenotype variation., (© 2019 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

To protect humans and domestic animals from mosquito borne diseases, alternative methods to chemical insecticides have to be found. Pilot studies using the vertically transmitted bacterial endosymbiont Wolbachia were already launched in different parts of the world. Wolbachia can be used either in Incompatible Insect Technique (IIT), to decrease mosquito population, or to decrease the ability of mosquitoes to transmit pathogens. Not all mosquito species are naturally infected with Wolbachia: while in Culex pipiens and Aedes albopictus almost all individuals harbor Wolbachia, putative infections have to be further investigated in Anopheles species and in Aedes aegypti. All Wolbachia-based control methods rely on the ability of Wolbachia to induce cytoplasmic incompatibility (CI) resulting in embryonic death in incompatible crossings. Knowledge on CI diversity in mosquito is required to find the better Wolbachia-mosquito associations to optimize the success of both 'sterile insect' and 'pathogen blocking' Wolbachia-based methods., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Wolbachia bacteria inhabit the cells of about half of all arthropod species, an unparalleled success stemming in large part from selfish invasive strategies. Cytoplasmic incompatibility (CI), whereby the symbiont makes itself essential to embryo viability, is the most common of these and constitutes a promising weapon against vector-borne diseases. After decades of theoretical and experimental struggle, major recent advances have been made toward a molecular understanding of this phenomenon. As pieces of the puzzle come together, from yeast and Drosophila fly transgenesis to CI diversity patterns in natural mosquito populations, it becomes clearer than ever that the CI induction and rescue stem from a toxin-antidote (TA) system. Further, the tight association of the CI genes with prophages provides clues to the possible evolutionary origin of this phenomenon and the levels of selection at play., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Culex pipiens densovirus (CpDV), a single stranded DNA virus, has been isolated from Culex pipiens mosquitoes but differs from other mosquito densoviruses in terms of genome structure and sequence identity. Its transmission from host to host, the nature of its interactions with both its host and host's endosymbiotic bacteria Wolbachia are not known. Here, we report the presence of CpDV in the ovaries and eggs of Cx. pipiens mosquitoes in close encounters with Wolbachia. In the ovaries, CpDV amount significantly differed between mosquito lines harbouring different strains of Wolbachia and these differences were not linked to variations in Wolbachia densities. CpDV was vertically transmitted in all laboratory lines to 17%-20% of the offspring. For some females, however, the vertical transmission reached 90%. Antibiotic treatment that cured the host from Wolbachia significantly decreased both CpDV quantity and vertical transmission suggesting an impact of host microbiota, including Wolbachia, on CpDV transmission. Overall our results show that CpDV is transmitted vertically via transovarian path along with Wolbachia with which it shares the same cells. Our results are primordial to understand the dynamics of densovirus infection, their persistence and spread in populations considering their potential use in the regulation of mosquito vector populations., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Wolbachia are maternally inherited endosymbiotic bacteria, widespread among arthropods thanks to host reproductive manipulations that increase their prevalence into host populations. The most commonly observed manipulation is cytoplasmic incompatibility (CI). CI leads to embryonic death in crosses between i) infected males and uninfected females and ii) individuals infected with incompatible Wolbachia strains. CI can be conceptualized as a toxin-antidote system where a toxin deposited by Wolbachia in the sperm would induce embryonic death unless countered by an antidote produced by Wolbachia present in the eggs. In Drosophila melanogaster, transgenic expression of Wolbachia effector cidB revealed its function of CI-inducing toxin. Moreover in Culex pipiens, the diversity of cidB variants present in wPip strains accounts for the diversity in crossing-types. We conducted cytological analyses to determine the CI mechanisms that lead to embryonic death in C. pipiens, and assess whether diversity in crossing-types could be based on variations in these mechanisms. We revealed that paternal chromatin condensation and segregation defects during the first embryonic division are always responsible for embryonic death. The strongest observed defects lead to an exclusion of the paternal chromatin from the first zygotic division, resulting in haploid embryos unable to hatch. The proportion of unhatched haploid embryos, developing with only maternal chromatin, which reflects the frequency of strong defects can be considered as a proxy of CI intensity at the cellular level. We thus studied the putative effect of variations in crossing types and cidB diversification on CI defects intensity. Incompatible crosses involving distinct wPip strains revealed that CI defects intensity depends on the Wolbachia strains hosted by the males and is linked to the diversity of cidB genes harbored in their genomes. These results support that, additionally to its implication in C. pipiens crossing type variability, cidB diversification also influences the strength of CI embryonic defects., Competing Interests: The authors have declared that no competing interests exist.

In the originally published HTML and PDF versions of this Article, gel images in Figures 7c and 8c were not prepared as per the Nature journal policy. These figure panels have now been corrected in both the PDF and HTML versions of the Article.In Fig. 7c, the lane labelled 'Ha' was inappropriately duplicated to represent the lane labelled 'Ich13'. The corrected version of Fig. 7c includes PCR-RFLP on DNA from the Ichkeul 13 line, which had been run on a separate gel. The original unprocessed gel images are provided in Supplementary Figure 1 associated with this correction, with the relevant corresponding bands denoted. A repeat experiment of the PCR-RFLP test is also presented as Supplementary Figure 2.In Fig. 8c, the image was assembled from two separate gels without clear demarcation. The corrected Fig. 8c clearly separates lanes from the two gels, and the original unprocessed gel images are provided in the Supplementary Information associated with this correction.These corrections do not alter the original meaning of the experiments, their results, their interpretation, or the conclusions of the paper. We apologize for any confusion this may have caused to the readers of Nature Communications.

Background: Wolbachia are maternally transmitted bacteria that can manipulate their hosts' reproduction causing cytoplasmic incompatibility (CI). CI is a sperm-egg incompatibility resulting in embryonic death. Due to this sterilising effect on mosquitoes, Wolbachia are considered for vector control strategies. Important vectors for arboviruses, filarial nematodes and avian malaria, mosquitoes of Culex pipiens complex are suitable for Wolbachia-based vector control. They are infected with Wolbachia wPip strains belonging to five genetically distinct groups (wPip-I to V) within the Wolbachia B supergroup. CI properties of wPip strongly correlate with this genetic diversity: mosquitoes infected with wPip strains from a different wPip group are more likely to be incompatible with each other. Turkey is a critical spot for vector-borne diseases due to its unique geographical position as a natural bridge between Asia, Europe and Africa. However, general wPip diversity, distribution and CI patterns in natural Cx. pipiens (s.l.) populations in the region are unknown. In this study, we first identified wPip diversity in Turkish Cx. pipiens (s.l.) populations, by assigning them to one of the five groups within wPip (wPip-Ito V). We further investigated CI properties between different wPip strains from this region., Results: We showed a wPip fixation in Cx. pipiens (s.l.) populations in Turkey by analysing 753 samples from 59 sampling sites. Three wPip groups were detected in the region: wPip-I, wPip-II and wPip-IV. The most dominant group was wPip-II. While wPip-IV was restricted to only two locations, wPip-I and wPip-II had wider distributions. Individuals infected with wPip-II were found co-existing with individuals infected with wPip-I or wPip-IV in some sampling sites. Two mosquito isofemale lines harbouring either a wPip-I or a wPip-II strain were established from a population in northwestern Turkey. Reciprocal crosses between these lines showed that they were fully compatible with each other but bidirectionally incompatible with wPip-IV Istanbul infected line., Conclusion: Our findings reveal a high diversity of wPip and CI properties in Cx. pipiens (s.l.) populations in Turkey. Knowledge on naturally occurring CI patterns caused by wPip diversity in Turkey might be useful for Cx. pipiens (s.l.) control in the region.

Culex pipiens mosquitoes are infected with Wolbachia (wPip) that cause an important diversity of cytoplasmic incompatibilities (CIs). Functional transgenic studies have implicated the cidA-cidB operon from wPip and its homolog in wMel in CI between infected Drosophila males and uninfected females. However, the genetic basis of the CI diversity induced by different Wolbachia strains was unknown. We show here that the remarkable diversity of CI in the C. pipiens complex is due to the presence, in all tested wPip genomes, of several copies of the cidA-cidB operon, which undergoes diversification through recombination events. In 183 isofemale lines of C. pipiens collected worldwide, specific variations of the cidA-cidB gene repertoires are found to match crossing types. The diversification of cidA-cidB is consistent with the hypothesis of a toxin-antitoxin system in which the gene cidB co-diversifies with the gene cidA, particularly in putative domains of reciprocal interactions.

Vertical transmission mode is predicted to decrease the virulence of symbionts. However, Wolbachia , a widespread vertically transmitted endosymbiont, exhibits both negative and beneficial effects on arthropod fitness. This 'Jekyll and Hyde' behaviour, as well as its ability to live transiently outside host cells and to establish new infections via horizontal transmission, may reflect the capacity of Wolbachia to exhibit various phenotypes depending on the prevailing environmental constraints. To study the ability of Wolbachia to readily cope with new constraints, we forced this endosymbiont to spread only via horizontal transmission. To achieve this, we performed serial horizontal transfers of haemolymph from Wolbachia -infected to naive individuals of the isopod Armadillidium vulgare. Across passages, we observed phenotypic changes in the symbiotic relationship: (i) The Wolbachia titre increased in both haemolymph and nerve cord but remained stable in ovaries; (ii) Wolbachia infection was benign at the beginning of the experiment, but highly virulent, killing most hosts after only a few passages. Such a phenotypic shift after recurrent horizontal passages demonstrates that Wolbachia can rapidly change its virulence when facing new environmental constraints. We thoroughly discuss the potential mechanism(s) underlying this phenotypic change, which are likely to be crucial for the ongoing radiation of Wolbachia in arthropods., (© 2017 The Author(s).)

Several microsatellite markers have already been developed for different terrestrial isopod species such as Armadillidium vulgare, A. nasatum and Porcellionides pruinosus. In all these species, the endosymbiont Wolbachia has a feminizing effect that generates a female bias in sex ratio and reduces the number of reproductive males. Thus this can potentially decrease the genetic diversity of host populations. However, in some other isopod species, Wolbachia induces cytoplasmic incompatibility (CI); the most commonly described effect of Wolbachia in arthropods. The CI by rendering some crossings incompatible can reduce the gene flow and strengthen genetic differentiation between isopod populations. To date, the influence of Wolbachia inducing CI on population structure of terrestrial isopods has never been investigated. In this study, we developed 10 polymorphic microsatellite markers shared by two sub-species of Porcellio dilatatus. Crossings between the two sub-species are partially incompatible due to two CI-inducing Wolbachia strains. These new microsatellite markers will allow us to investigate the effect of CI on host genetic differentiation in this species complex.

Wolbachia is an intracellular α-proteobacterium which is transmitted vertically from mother to offspring but also frequently switches horizontally from one host to another. Our hypothesis is based on the role of immune cells and the organs that produce them, the hematopoietic organs (HOs), as primordial niches for the propagation of Wolbachia via hemocytes both (i) within hosts: to initiate and maintain the systemic infection and (ii) between hosts: to promote both vertical and horizontal transmission of Wolbachia. Therefore, we review some fundamental ideas underlying this hypothesis and go further with new empirical data that lead to a first close-up analysis of the potential role of HOs in Wolbachia propagation. The monitoring of the first steps of Wolbachia infection in horizontally infected host organs by transmission electron microscopy and qPCR suggests that (i) HOs are colonized early and extensively as soon as they are in contact with Wolbachia which find in these cells a favorable niche to multiply and (ii) infected HOs which expel hemocytes all lifelong can generate and maintain a systemic infection that could contribute to increase both vertical and horizontal propagation of these symbionts.

Wolbachia is a vertically transmitted endosymbiont whose radiative success is mainly related to various host reproductive manipulations that led to consider this symbiont as a conflictual reproductive parasite. However, lately, some Wolbachia have been shown to act as beneficial symbionts by protecting hosts against a broad range of parasites. Still, this protection has been mostly demonstrated in artificial Wolbachia-host associations between partners that did not co-evolved together. Here, we tested in two terrestrial isopod species Armadillidium vulgare and Porcellio dilatatus whether resident Wolbachia (native or non-native) could confer protection during infections with Listeria ivanovii and Salmonella typhimurium and also during a transinfection with a Wolbachia strain that kills the recipient host (i.e., wVulC in P. dilatatus). Survival analyses showed that (i) A. vulgare lines hosting their native Wolbachia (wVulC) always exhibited higher survival than asymbiotic ones when infected with pathogenic bacteria (ii) P. dilatatus lines hosting their native wDil Wolbachia strain survived the S. typhimurium infection better, while lines hosting non-native wCon Wolbachia strain survived the L. ivanovii and also the transinfection with wVulC from A. vulgare better. By studying L. ivanovii and S. typhimurium loads in the hemolymph of the different host-Wolbachia systems, we showed that (i) the difference in survival between lines after L. ivanovii infections were not linked to the difference between their pathogenic bacterial loads, and (ii) the difference in survival after S. typhimurium infections corresponds to lower loads of pathogenic bacteria. Overall, our results demonstrate a beneficial effect of Wolbachia on survival of terrestrial isopods when infected with pathogenic intracellular bacteria. This protective effect may rely on different mechanisms depending on the resident symbiont and the invasive bacteria interacting together within the hosts.

The Wolbachia are intracellular endosymbionts widely distributed among invertebrates. These primarily vertically transmitted α-proteobacteria have been intensively studied during the last decades because of their intriguing interactions with hosts, ranging from reproductive manipulations to mutualism. To optimize their vertical transmission from mother to offspring, the Wolbachia have developed fine-tuned strategies. However, the Wolbachia are not restricted to the female gonads and frequently exhibit wide intra-host distributions. This extensive colonization of somatic organs might be necessary for Wolbachia to develop their diverse extended phenotypes. From an endosymbiont's perspective, the within-host environment potentially presents different environmental constraints. Hence, the Wolbachia have to face different intracellular habitats, their host's immune system as well as other microorganisms co-occurring in the same host individual and sometimes even in the same cell. A means for the Wolbachia to protect themselves from these environmental constraints may be to live 'hidden' in vacuoles within host cells. In this review, we summarize the current knowledge regarding the extent of the Wolbachia pandemic and discuss the various environmental constraints these bacteria may have to face within their 'host ecosystem'. Finally, we identify new avenues for future research to better understand the complexity of Wolbachia's interactions with their intracellular environment., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Wolbachia are symbiotic bacteria vertically transmitted from one host generation to another. However, a growing amount of data shows that horizontal transfers of Wolbachia also frequently occur within and between host species. The consequences of the arrival of new symbionts on host physiology can be studied by their experimental introduction in asymbiotic hosts. After experimental transfers of the eight major isopod Wolbachia strains in the isopod Porcellio dilatatus only two of them (wCon and wDil) were found to (1) have no pathogenic effect on the host and (2) be able to pass vertically to the host offspring. In the present work, we studied the influence of these two strains, able to complete an horizontal transfer, on immunity and reproduction of P. dilatatus at two stages of the transfer: (1) in recipient hosts that encounter the symbionts: to test the influence of symbiont when acquired during host life and (2) in vertically infected offspring: to test the influence of a symbiotic interaction occurring all lifelong. The impact of Wolbachia varied depending on the stage: there were clearer effects in vertically infected individuals than in those that acquired the symbionts during their lives. Moreover, the two Wolbachia strains showed contrasted effects: the strain wCon tended to reduce the reproductive investment but to maintain or increase immune parameters whilst wDil had positive effects on reproductive investment but decreased the investment in some immune parameters. These results suggest that horizontally acquisition of Wolbachia can influence the balance between host immune and reproductive traits., (Copyright © 2014. Published by Elsevier Ltd.)

In the terrestrial isopod species Porcellio dilatatus, unidirectional Cytoplasmic Incompatibility (CI) between two morphs (P. d. dilatatus and P. d. petiti) caused by a Wolbachia strain (wPet) infecting the morph P. d. petiti has been previously described by experiments initiated four decades ago. Here, we studied another Wolbachia that has been recently detected in a population of the morph P. d. dilatatus. The MLST markers reveal that this Wolbachia is a new strain called wDil distinct from wPet also belonging to the isopod clade of Wolbachia. Quantifications of both Wolbachia strains in the gonads of the two P. dilatatus morphs revealed that all males exhibit similar Wolbachia titers while the titers in females depend on the Wolbachia strain they host. Crossing experiments showed that both wDil and wPet induced partial unidirectional CI with different intensities. Moreover, these two strains induced bidirectional CI when individuals were both infected with one of the two different Wolbachia strains. This way, we demonstrated that P. dilatatus can be infected by two closely related Wolbachia strains (wDil and wPet), that seem to have different modification-rescue systems., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The alphaproteobacteria Wolbachia pipientis are among the most common and widespread symbionts in the animal world. Their vertical transmission mode is predicted to favour genotypes with low virulence. On the contrary, horizontal transfers of Wolbachia from one host to another have been shown to possibly increase the symbiont virulence. This situation has been previously described when two feminizing Wolbachia strains, wVulC and wVulM, from the ovaries of the woodlouse Armadillidium vulgare were introduced into another woodlouse named Porcellio dilatatus. These two Wolbachia strains induced severe symptoms and eventually caused the death of the recipient host. However, symptoms and death appeared sooner with wVulC than with wVulM. To know whether this difference was due to variation in the dose of infection or a difference in virulence between the two Wolbachia strains, we performed controlled and gradual doses of injection with wVulC and wVulM in P. dilatatus. We showed that the two strains differed intrinsically in their virulence against P. dilatatus and that their virulence is related to the injection dose. Moreover, we showed that wVulC reached higher concentrations in the recipient host than wVulM suggesting a potential link between the bacterial titers and the levels of virulence. We also addressed the impact of the tissue source of the Wolbachia used for the transinfection and demonstrated that Wolbachia transinfected via hemolymph colonized the body of the recipient more quickly and caused accelerated symptoms compared to Wolbachia introduced via a crushed ovaries suspension., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The endosymbiont Wolbachia pipientis infects various hosts in which it navigates vertically from mothers to offspring. However, horizontal transfers of Wolbachia can occur between hosts. The virulence of the horizontally acquired Wolbachia can change in the new host as it has been illustrated by the case of the feminizing strain wVulC from the woodlouse Armadillidium vulgare that turns to a pathogen when introduced into Porcellio dilatatus dilatatus. In the present study, we aim to show whether symbiotic traits, such as (i) host sex manipulation and (ii) colonization patterns, which differ between eight isopod Wolbachia strains, are connected to their virulence towards the recipient host P. d. dilatatus. Among the transferred Wolbachia, some feminizing strains gradually differing in feminizing intensity in their native hosts induced different levels of pathogenicity to P. d. dilatatus. Not a single feminizing strain passed vertically with high titres to the next generation. The non-feminizing Wolbachia strains, even if they reached high densities in the host, did not impact host life-history traits and some vertically passed with high titres to the offspring. These results suggest that a potential link between the manners Wolbachia manipulates its native host reproduction, its virulence and its ability to vertically infect the offspring., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The alpha-proteobacteria Wolbachia are the most widespread endosymbionts in arthropods and nematodes. Mainly maternally inherited, these so-called sex parasites have selected several strategies that increase their vertical dispersion in host populations. However, the lack of congruence between the Wolbachia and their host phylogenies suggests frequent horizontal transfers. One way that could be used for horizontal Wolbachia transfers between individuals is predation. The aim of this study was to test whether horizontal passage of Wolbachia is possible when an uninfected terrestrial isopod eats an infected one. After having eaten Armadillidium vulgare harbouring Wolbachia, the predator-recipients (the two woodlice A. vulgare and Porcellio dilatatus dilatatus) that were initially Wolbachia-free were tested positive for the presence of Wolbachia both by quantitative PCR and Fluorescence in situ Hybridization (FISH). Even if the titers were low compared to vertically infected individuals, this constitutes the first demonstration of Wolbachia occurrence in various organs of an initially uninfected host after eating an infected one.

Wolbachia are widespread endosymbionts found in a large variety of arthropods. While these bacteria are generally transmitted vertically and exhibit weak virulence in their native hosts, a growing number of studies suggests that horizontal transfers of Wolbachia to new host species also occur frequently in nature. In transfer situations, virulence variations can be predicted since hosts and symbionts are not adapted to each other. Here, we describe a situation where a Wolbachia strain (wVulC) becomes a pathogen when transfected from its native terrestrial isopod host species (Armadillidium vulgare) to another species (Porcellio d. dilatatus). Such transfer of wVulC kills all recipient animals within 75 days. Before death, animals suffer symptoms such as growth slowdown and nervous system disorders. Neither those symptoms nor mortalities were observed after injection of wVulC into its native host A. vulgare. Analyses of wVulC's densities in main organs including Central Nervous System (CNS) of both naturally infected A. vulgare and transfected P. d. dilatatus and A. vulgare individuals revealed a similar pattern of host colonization suggesting an overall similar resistance of both host species towards this bacterium. However, for only P. d. dilatatus, we observed drastic accumulations of autophagic vesicles and vacuoles in the nerve cells and adipocytes of the CNS from individuals infected by wVulC. The symptoms and mortalities could therefore be explained by this huge autophagic response against wVulC in P. d. dilatatus cells that is not triggered in A. vulgare. Our results show that Wolbachia (wVulC) can lead to a pathogenic interaction when transferred horizontally into species that are phylogenetically close to their native hosts. This change in virulence likely results from the autophagic response of the host, strongly altering its tolerance to the symbiont and turning it into a deadly pathogen.

While parasites are likely to encounter several potential intermediate hosts in natural communities, a parasite's actual range of compatible hosts is limited by numerous biological factors ranging from behaviour to immunology. In crustaceans, two major components of immunity are haemocytes and the prophenoloxidase system involved in the melanisation of foreign particles. Here, we analysed metazoan parasite prevalence and loads in the two sympatric crab species Hemigrapsus crenulatus and Macrophthalmus hirtipes at two sites. In parallel, we analysed the variation in haemocyte concentration and amount of circulating phenoloxidase (PO) in the haemolymph of the same individuals in an attempt to (a) explain differences in parasite prevalence and loads in the two species at two sites and (b) assess the impact of parasites on these immune parameters. M. hirtipes harboured more parasites but also exhibited higher haemocyte concentrations than H. crenulatus independent of the study site. Thus, higher investment in haemocyte production for M. hirtipes does not seem to result in higher resistance to parasites. Analyses of variation in immune parameters for the two crab species between the two sites that differed in parasite prevalence showed common trends. (a) In general, haemocyte concentrations were higher at the site experiencing higher parasitic pressure while circulating PO activity was lower and (b) haemocyte concentrations were influenced by microphallid trematode metacercariae in individuals from the site with higher parasitic pressure. We suggest that the higher haemocyte concentrations observed in both crab species exposed to higher parasitic pressure may represent an adaptive response to the impact of parasites on this immune parameter.

Ecological factors modulate animal immunocompetence and potentially shape the evolution of their immune systems. Not only environmental parameters impact on immunocompetence: Aging is one major cause of variability of immunocompetence between individuals, and sex-specific levels of immunocompetence have also been frequently described. Moreover, a growing core of data put in light that vertically transmitted symbionts can dramatically modulate the immunocompetence of their hosts. In this study, we addressed the influence of gender, age and the feminising endosymbiont Wolbachia (wVulC) on variations in haemocyte density, total PO activity and bacterial load in the haemolymph of the terrestrial isopod Armadillidium vulgare. This host-symbiont system is of particular interest to address this question since: (1) wVulC was previously shown as immunosuppressive in middle-aged females and (2) wVulC influences sex determination. We show that age, gender and Wolbachia modulate together immune parameters in A. vulgare. However, wVulC, which interacts with aging, appears to be the prominent factor interfering with both PO activity and haemocyte density. This interference with immune parameters is not the only aspect of wVulC virulence on its host, as reproduction and survival are also altered.

A new entomopathogenic nematode, Steinernema boemarei n. sp., is described from southern France. Morphological, molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. Both molecular and morphological data indicate that the new species belongs to the 'glaseri-group' of Steinernema spp. Key morphological diagnostic traits for S. boemarei n. sp. include the presence of prominent deirids (cervical papillae) on adult males, the morphology of the spicules and gubernaculum, and the arrangement of the 23 genital papillae of the first generation males. Additionally, morphometric traits of the third-stage infective juvenile, including total body length (mean 1,103 micron), tail length (mean 86 micron), location of the excretory pore (mean 91 micron), and D% (mean 63), E% (mean 106) and H% (mean 41) values are definitive. In addition to these morphological characters, analysis of both 28S and ITS rDNA sequences depict this Steinernema species as a distinct and unique entity.

Background: After decades during which endosymbionts were considered as silent in their hosts, in particular concerning the immune system, recent studies have revealed the contrary. In the present paper, we addressed the effect of Wolbachia, the most prevalent endosymbiont in arthropods, on host immunocompetence. To this end, we chose the A. vulgare-Wolbachia symbiosis as a model system because it leads to compare consequences of two Wolbachia strains (wVulC and wVulM) on hosts from the same population. Moreover, A. vulgare is the only host-species in which Wolbachia have been directly observed within haemocytes which are responsible for both humoral and cellular immune responses., Methodology/principal Findings: We sampled gravid females from the same population that were either asymbiotic, infected with wVulC, or infected with wVulM. The offspring from these females were tested and it was revealed that individuals harbouring wVulC exhibited: (i) lower haemocyte densities, (ii) more intense septicaemia in their haemolymph and (iii) a reduced lifespan as compared to individuals habouring wVulM or asymbiotic ones. Therefore, individuals in this population of A. vulgare appeared to suffer more from wVulC than from wVulM. Symbiotic titer and location in the haemocytes did not differ for the two Wolbachia strains showing that these two parameters were not responsible for differences observed in their extended phenotypes in A. vulgare., Conclusion/significance: The two Wolbachia strains infecting A. vulgare in the same population induced variation in immunocompetence and survival of their hosts. Such variation should highly influence the dynamics of this host-symbiont system. We propose in accordance with previous population genetic works, that wVulM is a local strain that has attenuated its virulence through a long term adaptation process towards local A. vulgare genotypes whereas wVulC, which is a widespread and invasive strain, is not locally adapted.

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.

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.

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.

The free-living soil nematode Caenorhabditis elegans interacts with diverse microorganisms in its natural habitat. These microorganisms may serve as a food source or represent a harmful threat. As such, they constitute one of the most important ecological factors of the worm's natural environment. In this study, we examined the interaction between two natural isolates of C. elegans and three natural isolates of the entomopathogenic bacterium Photorhabdus luminescens. Two of the tested P. luminescens strains were clearly pathogenic. They consistently reduced the worm's developmental, survival and reproductive rate, most likely through the production of nematicidal compounds. The third natural isolate tested did not decrease worm fitness. These differences could be associated with a deletion of one gene (tcdA4) ( approximately 7kb) of the toxin complex D pathogenicity island, which was found in only the non-pathogenic strain. Our study additionally highlights the importance of behaviour in defence. Caenorhabditis elegans showed two distinct avoidance behaviours towards the pathogenic but not the non-pathogenic P. luminescens strains. In particular, nematodes physically escaped and also reduced the ingestion of pathogenic bacteria. Our results strongly suggest that C. elegans possesses the capacity to distinguish between and subsequently respond to harmful and non-harmful strains of the same bacterial species. The underlying mechanisms of such interactions are currently unknown. Their dissection will represent a major challenge for future research and should enhance our knowledge of the ecology of this important model organism in biological research.

Background: 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 (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., Results: 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., Conclusion: 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.

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 nematode's 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. scapterisci's 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. scapterisci's 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.

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

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.