Metagenomics of culture isolates and insect tissue illuminate the evolution of , and symbionts in spp. fleas.

While fleas are often perceived simply as a biting nuisance and a cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat () and dog () fleas, as well as (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by spp.) and spp., including (agent of flea-borne spotted fever) and , a suspected pathogen. These spp. are members of a phylogenetic clade known as the ‘transitional group’, which includes both human pathogens and arthropod-specific endosymbionts. The relatively depauperate flea microbiome can also contain other endosymbionts, including a diverse range of strains. Here, we present circularized genome assemblies for two -derived pathogens ( and ) from Malaysia, a novel strain (Cori), and the mitochondrion; all were obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two strains from Malaysian into tick cell culture and recovered circularized genome assemblies for both, one of which (CfeF) is newly sequenced. We demonstrate that the three strains are representatives of different major clades (‘supergroups’), two of which appear to be flea-specific. These genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors and the biotin operon of obligate intracellular microbes. The first circularized assembly for includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the USA. Analysis of loci under positive selection in the transitional group revealed genes involved in host–pathogen interactions that may facilitate host switching. Finally, the first genome from Asia exhibited large-scale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of -associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.