Extensive mobilome heterogeneity in a widespread endosymbiont: A candidate dimeric plasmid and highly fragmented prophages in Wolbachia from Culex pipiens

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 worldwide. Despite their highly reduced genomes, Wolbachia harbor a rich mobilome that includes phages, insertion sequences and plasmids in mosquito vectors. However, these repeat-rich mobile genetic elements are notoriously difficult to assemble and can generate artificial, chimeric sequences that obscure their true diversity. As a result, conventional genome sequencing and assembly approaches often fail to resolve the complexity, leaving critical gaps of knowledge on how these elements evolve, mobilize, and shape both symbiont and host genomes. To address these challenges, we established a novel Oxford Nanopore long-read sequencing strategy that avoids prior PCR amplification and leverages single-dissected, female ovaries. Using this approach, we report and characterize-directly from native long-read molecules-the first near-complete Wolbachia genome and its mobile elements from individual ovaries of the mosquito vector Culex pipiens molestus. Additional short reads from individuals of the same strain were used to assess and validate candidate mutations, particularly in repetitive regions. We report a new dimeric structure of the pWCP plasmid, suggesting the existence of multimeric plasmid forms potentially associated with active replication. 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 illuminate the structural heterogeneity of the endosymbiont genome that is shaped by integrated and episomal mobile elements.