{ "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 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. Author summary: Intracellular bacteria that live within the cells of animals are widespread and can profoundly influence their hosts’ biology. Among them, Wolbachia are one of the most influential and extensively studied. It occurs in numerous insect species, manipulates host reproduction, reduces viral replication, and is widely used in mosquito control programs. A hallmark of intracellular bacteria such as Wolbachia is the abundance of mobile genetic elements in their genomes, notably prophage-derived regions. These elements are major contributors to genome evolution and functional innovation. However, they are highly variable and notoriously difficult to assemble accurately, particularly when DNA amplification introduces artificial rearrangements. To better resolve this complexity, we developed a long-range sequencing approach that avoids prior PCR amplification and applied it at the scale of a single mosquito ovary. Using this strategy, we generated a near-complete Wolbachia genome and uncovered unexpected structural variation in its mobile elements. Notably, we provide evidence for a previously unrecognized dimeric structure of the pWCP Wolbachia plasmid, that could indicate active replication, as well as multiple alternative phage-associated configurations. Together, these findings reveal hidden genomic diversity and underscore the dynamic nature of intracellular bacterial genomes, despite the constraints imposed by life within host cells.", "abstract_shown": true, "affiliations": [ "MIVEGEC, University of Montpellier, INRAe, CNRS, IRD, Montpellier, France", "Departments of Biology and Entomology, One Health Microbiome Center, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, United States of America" ], "article_type": "Research Article", "arxiv": null, "author_total": 9, "authors": [ { "affs": [ 0 ], "name": "Alice Brunner" }, { "affs": [ 1 ], "name": "Maxime Mahout" }, { "name": "Julien Amoros" }, { "name": "Massilva Rahmoun" }, { "name": "Mateo Jarry" }, { "name": "Seth R. Bordenstein" }, { "name": "Sarah R. Bordenstein" }, { "name": "Blandine Trouche" }, { "name": "Julie Reveillaud" } ], "bibtex": "https://research.draconitic.com/paper/bw5y19cm6seqx.bib", "cited_by": 0, "doi": "10.1371/journal.pgen.1012347", "fulltext_url": "https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012347", "id": "bw5y19cm6seqx", "issns": [ "1553-7404" ], "landing_url": "https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012347", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "1109|Insect symbiosis and bacterial influences" ], [ "plos", "/Biology and life sciences/Anatomy/Reproductive system/Ovaries" ], [ "plos", "/Biology and life sciences/Genetics/Genomics" ], [ "plos", "/Biology and life sciences/Genetics/Genomics/Animal genomics/Invertebrate genomics" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Sequencing techniques/Genome sequencing" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Wolbachia" ], [ "plos", "/Biology and life sciences/Organisms/Eukaryota/Animals/Invertebrates/Arthropoda/Insects/Mosquitoes" ], [ "plos", "/Biology and life sciences/Organisms/Viruses/Bacteriophages" ], [ "plos", "/Biology and life sciences/Species interactions/Disease vectors/Insect vectors/Mosquitoes" ], [ "plos", "/Biology and life sciences/Zoology/Animals/Invertebrates/Arthropoda/Insects/Mosquitoes" ], [ "plos", "/Biology and life sciences/Zoology/Entomology/Insects/Mosquitoes" ], [ "plos", "/Medicine and health sciences/Anatomy/Reproductive system/Ovaries" ], [ "plos", "/Medicine and health sciences/Medical conditions/Infectious diseases/Disease vectors/Insect vectors/Mosquitoes" ], [ "plos", "/Research and analysis methods/Database and informatics methods/Bioinformatics/Sequence analysis/Sequence alignment" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Sequencing techniques/Genome sequencing" ] ], "openalex": "W7223688184", "pdf_url": "https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1012347&type=printable", "pmcid": null, "pmid": null, "published": "2026-10-26", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/bw5y19cm6seqx.ris", "sources": [ { "first_seen": "2026-10-30T12:17:17Z", "native_id": "10.1371/journal.pgen.1012347", "source": "plos", "url": "https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012347" } ], "subjects": [ { "field": "biology", "origin": "plos:genomics", "subfield": "genetics-genomics" }, { "field": "biology", "origin": "plos:zoology", "subfield": "zoology-behaviour" }, { "field": "medicine", "origin": "plos:anatomy", "subfield": "physiology" } ], "title": "Extensive mobilome heterogeneity in a widespread endosymbiont: A candidate dimeric plasmid and highly fragmented prophages in Wolbachia from Culex pipiens", "title_text": "Extensive mobilome heterogeneity in a widespread endosymbiont: A candidate dimeric plasmid and highly fragmented prophages in Wolbachia from Culex pipiens", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:17Z", "url": "https://research.draconitic.com/paper/bw5y19cm6seqx", "venue": "PLOS Genetics", "versions": [] }