Metagenomics of culture isolates and insect tissue illuminate the evolution ofWolbachia,RickettsiaandBartonellasymbionts inCtenocephalidesspp. fleas

Abstract

While fleas are often perceived simply as a biting nuisance and 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 (Ctenocephalidesfelis) and dog (C. canis) fleas, as well asC. orientis(restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused byBartonellaspp.) andRickettsiaspp. of the so-called "transitional group". The latter includesR. felis(agent of flea-borne spotted fever) andR. asembonensis, an emerging pathogen. The relatively depauperate flea microbiome can also contain arthropod-specific endosymbionts, including a diverse range ofWolbachiastrains. Here, we present circularized genome assemblies for twoC. orientis-associated pathogens (Bartonella clarridgeiaeandR. asembonensis) from Malaysia, a novelWolbachiastrain (wCori), and theC. orientismitochondrion; all obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated twoWolbachiastrains from MalaysianC. felisinto tick cell culture and recovered circularized genome assemblies for both, one of which (wCfeF) is newly sequenced. We demonstrate that the threeWolbachiastrains are representatives of different major clades ("supergroups"), two of which appear to be flea-specific. TheseWolbachiagenomes 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 forR. asembonensisincludes 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 US. Analysis of loci under positive selection in the transitional group revealed genes involved in host-pathogen interactions that may facilitate host switching. Finally, the firstB. clarridgeiaegenome from Asia exhibited largescale 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 ofCtenocephalides-associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.

The evolution of intramitochondriality in Midichloria bacteria

“…The adaptive branch‐site relative effects likelihood model (aBSREL) (Kosakovsky Pond & Frost, 2005) was used to evaluate possible signal of episodic diversifying positive selection using HyPhy v2.5.8 (Smith et al, 2015) on the alignment of each gene, as performed in Beliavskaia et al (2023). The aBSREL model tests for each branch in the phylogenetic tree whether a proportion of sites have evolved under positive diversifying selection.…”

Establishment and partial characterisation of a new cell line derived from adult tissues of the tsetse fly Glossina morsitans morsitans

“…Culture supernatant containing cell-free Wolbachia of each test strain was passed through a 0.45-µm filter to remove any host cells, 0.3-0.5 ml of the filtrate was inoculated into GMA/LULS61 cells and the tubes were returned to 28 °C. Medium was changed weekly and cultures were examined by inverted microscope for signs of cytopathic wPap Phlebotomus papatasi [12] P. papatasi PPL/LULS49 [12] wCfeJ Ctenocephalides felis [42,43] Ixodes scapularis IDE8 [44] wAlbB Aedes albopictus [45] Ae. albopictus C6/36 1 [46] wBol1-b Hypolimnas bolina [47] Spodoptera frugiperda Sf9 2 [48] wStr1 Laodelphax striatellus [49] S. frugiperda Sf9 3 [48] wMelPop-CLA Drosophila melanogaster [50] Ae.…”

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

“…1 Metagenomics sequencing from tick cell cultures infected with Wolbachia isolated from C. felis[37]. Two separate assemblies were obtained from combined nanopore sequencing reads from infected IDE8 (a parent and a P4 subculture) and BME/CTVM23 (a single P1 subculture) cultures and polishing with a combined Illumina dataset from a single subculture for each of Wolbachia-infected IDE8 and BME/CTVM23 cells 2.…”