The Wolbachia mobilome in Culex pipiens includes a putative plasmid

Julie Reveillaud,Patrick Makoundou,Özcan C Esen,Alon Shaiber,Seth R Bordenstein,A Murat Eren,Mylène Weill,Sarah R Bordenstein,Ignace Rakotoarivony,Corinne Cruaud,Andrea R Watson,Karen Lolans

doi:10.1038/s41467-019-08973-w

Abstract

Wolbachia is a genus of obligate intracellular bacteria found in nematodes and arthropods worldwide, including insect vectors that transmit dengue, West Nile, and Zika viruses. Wolbachia’s unique ability to alter host reproductive behavior through its temperate bacteriophage WO has enabled the development of new vector control strategies. However, our understanding of Wolbachia’s mobilome beyond its bacteriophages is incomplete. Here, we reconstruct near-complete Wolbachia genomes from individual ovary metagenomes of four wild Culex pipiens mosquitoes captured in France. In addition to viral genes missing from the Wolbachia reference genome, we identify a putative plasmid (pWCP), consisting of a 9.23-kbp circular element with 14 genes. We validate its presence in additional Culex pipiens mosquitoes using PCR, long-read sequencing, and screening of existing metagenomes. The discovery of this previously unrecognized extrachromosomal element opens additional possibilities for genetic manipulation of Wolbachia.

Highlights

Wolbachia transinfections into mosquito species (i.e., Aedes aegypti, Ae. albopictus, Ae. polynesienses, and Anopheles stephensi) result in mosquito lines refractory to various types of pathogenic RNA viruses[11,12,13,14]
Shotgun metagenomes from individual C. pipiens ovary samples allowed us to de novo reconstruct Wolbachia genomes from single mosquitoes and compare these metagenome-assembled genome (MAG) to each other as well as to the reference wPip Pel genome through pangenomic strategies
Our data reveal an extensive diversity of previously undetected Wolbachia phage WO and other viral genes and notably the first indication that Wolbachia harbours a candidate plasmid, shedding new light on the richness of the Wolbachia mobilome

Summary

Introduction

Wolbachia transinfections into mosquito species (i.e., Aedes aegypti, Ae. albopictus, Ae. polynesienses, and Anopheles stephensi) result in mosquito lines refractory to various types of pathogenic RNA viruses[11,12,13,14]. The genes responsible for CI24–26 occur in the eukaryotic association module of Wolbachia’s temperate phage WO25–27, which laterally transfers between Wolbachia coinfections and evolves rapidly[28,29,30,31,32] Overall, these findings emphasize the importance of further investigating mobile genetic elements in Wolbachia. Most metagenomic approaches far rely on pooled individuals grown in the laboratory environment due to low infection densities[40,41] Since these limitations can conceal naturally occurring genomic diversity among Wolbachia populations, highly resolved analyses of individual mosquitoes may reveal additional insights into the Wolbachia ‘mobilome’, the pool of all mobile genetic elements associated with Wolbachia populations. Besides a diverse set of virus-associated genes that were missing or absent in the reference Wolbachia genome wPip Pel, our data reveal the first lines of evidence for an extrachromosomal circular element with genetic and functional hallmarks of a plasmid that we tentatively name pWCP

Methods

Results

Conclusion