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.

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

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.

Life History Traits, Protein Evolution, and the Nearly Neutral Theory in Amniotes.

The nearly neutral theory of molecular evolution predicts that small populations should accumulate deleterious mutations at a faster rate than large populations. The analysis of nonsynonymous (dN) versus synonymous (dS) substitution rates in birds versus mammals, however, has provided contradictory results, questioning the generality of the nearly neutral theory. Here we analyzed the impact of life history traits, taken as proxies of the effective population size, on molecular evolutionary and population genetic processes in amniotes, including the so far neglected reptiles. We report a strong effect of species body mass, longevity, and age of sexual maturity on genome-wide patterns of polymorphism and divergence across the major groups of amniotes, in agreement with the nearly neutral theory. Our results indicate that the rate of protein evolution in amniotes is determined in the first place by the efficiency of purifying selection against deleterious mutations-and this is true of both radical and conservative amino acid changes. Interestingly, the among-species distribution of dN/dS in birds did not follow this general trend: dN/dS was not higher in large, long-lived than in small, short-lived species of birds. We show that this unexpected pattern is not due to a more narrow range of life history traits, a lack of correlation between traits and Ne, or a peculiar distribution of fitness effects of mutations in birds. Our analysis therefore highlights the bird dN/dS ratio as a molecular evolutionary paradox and a challenge for future research.