Intra-lineage microevolution of Wolbachia leads to the emergence of new cytoplasmic incompatibility patterns.

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.

Nanopore sequencing of PCR products enables multicopy gene family reconstruction.

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 wPip, 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.

Emergence and clonal expansion of Vibrio aestuarianus lineages pathogenic for oysters in Europe.

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.

The mosquito microbiome includes habitat-specific but rare symbionts.

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.

Paternal transmission of the Wolbachia CidB toxin underlies cytoplasmic incompatibility.

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.

From Wolbachia genomics to phenotype: molecular models of cytoplasmic incompatibility must account for the multiplicity of compatibility types.

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.

Experimental evidence of Wolbachia introgressive acquisition between terrestrial isopod subspecies.

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 wPet 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.

Symbiotic Interactions Between Mosquitoes and Mosquito Viruses.

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.

Cytoplasmic Incompatibility Variations in Relation with Wolbachia cid Genes Divergence in Culex pipiens.

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 wPip. 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 wPip groups and exhibiting distinct cid gene repertoires. We showed, as expected, that crosses involving wPip from the same group were mostly compatible. In contrast, only 22% of the crosses involving different wPip 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 wPip-IV strains exhibiting cid variants markedly divergent from other wPip 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.IMPORTANCECulex pipiens mosquitoes are infected with wPip. 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 wPip phylogenetic groups at play. Here, we studied CI variations in relation to wPip groups and cid genes divergence. We showed, as expected, that the crosses involving wPip from the same group were compatible. In contrast, 78% of the crosses involving different wPip 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 wPip-IV strains exhibiting cid variants, which markedly diverge from those of other wPip groups.

Wolbachia modulates prevalence and viral load of Culex pipiens densoviruses in natural populations.

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.

RNA interference identifies domesticated viral genes involved in assembly and trafficking of virus-derived particles in ichneumonid wasps.

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.

Evolution and phylogeography of Culex pipiens densovirus.

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.

Variation in Wolbachia cidB gene, but not cidA, is associated with cytoplasmic incompatibility mod phenotype diversity in Culex pipiens.

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.

Wolbachia prevalence, diversity, and ability to induce cytoplasmic incompatibility in mosquitoes.

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.

The Toxin-Antidote Model of Cytoplasmic Incompatibility: Genetics and Evolutionary Implications.

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.

Sharing cells with Wolbachia: the transovarian vertical transmission of Culex pipiens densovirus.

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.

The cellular phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB diversity.

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.

Author Correction: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia.

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.

Wolbachia diversity and cytoplasmic incompatibility patterns in Culex pipiens populations in Turkey.

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.