Wolbachia populations across organs of individual Culex pipiens: highly conserved intra-individual core pangenome with inter-individual polymorphisms.

Wolbachia is a maternally inherited intracellular bacterium that infects a wide range of arthropods including mosquitoes. The endosymbiont is widely used in biocontrol strategies due to its capacity to modulate arthropod reproduction and limit pathogen transmission. Wolbachia infections in Culex spp. are generally assumed to be monoclonal but the potential presence of genetically distinct Wolbachia subpopulations within and between individual organs has not been investigated using whole genome sequencing. Here we reconstructed Wolbachia genomes from ovary and midgut metagenomes of single naturally infected Culex pipiens mosquitoes from Southern France to investigate patterns of intra- and inter-individual differences across mosquito organs. Our analyses revealed a remarkable degree of intra-individual conservancy among Wolbachia genomes from distinct organs of the same mosquito both at the level of gene presence-absence signal and single-nucleotide polymorphisms (SNPs). Yet, we identified several synonymous and non-synonymous substitutions between individuals, demonstrating the presence of some level of genomic heterogeneity among Wolbachia that infect the same C. pipiens field population. Overall, the absence of genetic heterogeneity within Wolbachia populations in a single individual confirms the presence of a dominant Wolbachia that is maintained under strong purifying forces of evolution.

pWCP is a widely distributed and highly conserved Wolbachia plasmid in Culex pipiens and Culex quinquefasciatus mosquitoes worldwide.

Mosquitoes represent the most important pathogen vectors and are responsible for the spread of a wide variety of poorly treatable diseases. Wolbachia are obligate intracellular bacteria that are widely distributed among arthropods and collectively represents one of the most promising solutions for vector control. In particular, Wolbachia has been shown to limit the transmission of pathogens, and to dramatically affect the reproductive behavior of their host through its phage WO. While much research has focused on deciphering and exploring the biocontrol applications of these WO-related phenotypes, the extent and potential impact of the Wolbachia mobilome remain poorly appreciated. Notably, several Wolbachia plasmids, carrying WO-like genes and Insertion Sequences (IS), thus possibly interrelated to other genetic units of the endosymbiont, have been recently discovered. Here we investigated the diversity and biogeography of the first described plasmid of Wolbachia in Culex pipiens (pWCP) in several islands and continental countries around the world-including Cambodia, Guadeloupe, Martinique, Thailand, and Mexico-together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. We used PCR and qPCR to determine the presence and copy number of pWCP in individual mosquitoes, and highly accurate Sanger sequencing to evaluate potential variations. Together with earlier observation, our results show that pWCP is omnipresent and strikingly conserved among Wolbachia populations within mosquitoes from distant geographies and environmental conditions. These data suggest a critical role for the plasmid in Wolbachia ecology and evolution, and the potential of a great tool for further genetic dissection and possible manipulation of this endosymbiont.

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.