# Extensive mobilome dynamics in a widespread endosymbiont: long read metagenomics reveal dimeric plasmids and highly fragmented prophages in Wolbachia from Culex pipiens

## TL;DR

This study uses long-read sequencing to reveal new insights into the complex mobile genetic elements in Wolbachia bacteria found in Culex pipiens mosquitoes.

## Contribution

The first near-complete Wolbachia genome with dimeric plasmid and fragmented prophage structures is assembled using long-read sequencing.

## Key findings

- A dimeric form of the pWCP plasmid was assembled, indicating it is a replicating and active molecule.
- Prophage WO regions showed extensive fragmentation and structural complexity despite long-read sequencing.
- Multiple alternative gene arrangements in WO regions suggest heterogeneous prophage architectures and diversity in wPip strains.

## Abstract

The obligate, intracellular bacteria Wolbachia have gained increasing interest due to their selfish modifications of host arthropod reproduction, impacts on host evolution, and utility in vector control efforts to reduce arbovirus transmission. Despite their highly reduced genomes, Wolbachia harbor a rich global mobilome that includes phages and plasmids in mosquito vectors. However, these mobile genetic elements are structurally complex, and standard genome assemblies often fail to resolve their organization and their functional relationships, leaving gaps in our understanding of how they evolve, mobilize, and influence host genomes.

Here, we present the first near-complete genome of Wolbachia and its mobile elements from the vector Culex pipiens molestus in Montpellier (France), reconstructed from Oxford Nanopore long read sequencing of single female ovaries without prior DNA amplification. Additional short reads from individuals of the same strain were used to assess and validate candidate mutations, particularly in repetitive regions. We report the assembly of a new dimeric form of the pWCP plasmid, providing evidence that the element is a replicating molecule and functionally active. We also observed extensive fragmentation of prophage WO regions despite long read sequencing, underscoring their structural complexity. Raw long read analyses recovered multiple alternative gene syntenies within WO regions, pointing to heterogeneous prophage architectures missed by the assembly and marked diversity of WO elements in Wolbachia of Culex pipiens (wPip) group strains.

Taken together, our results show the high dynamism of the endosymbiont genome that is shaped by integrated and episomal active mobile elements.