Qualitative profiling of the gut-specific chlamydial population in Ixodes ricinus ticks

Members of the phylum Chlamydiota are obligate intracellular bacteria increasingly recognized across a wide range of arthropod hosts, including ticks. In this study, we investigated the diversity and distribution of chlamydiae in Ixodes ricinus ticks and their potential association with Lyme borreliosis spirochetes. A total of 250 questing nymphal and female I. ricinus ticks were collected from three recreational sites in Vienna, Austria. Individual tick guts were screened for chlamydiae using pan-Chlamydiota PCR assays targeting the 16S rRNA gene, followed by sequencing for taxonomic identification. The presence and abundance of Borrelia burgdorferi sensu lato were quantified by specific qPCR to evaluate potential co-occurrence patterns. Chlamydiota DNA was detected in ticks from all investigated areas, with prevalence varying according to geography and developmental stage. Phylogenetic analyzes revealed high chlamydial diversity within the gut microbiome, predominantly comprising members of the metagenomic family MCF-D, followed by Parachlamydiaceae, Endochlamydiaceae, and Parasimkaniaceae. A positive, albeit not statistically significant, association between Chlamydiota and Borrelia was also observed. These findings indicate that the I. ricinus gut microbiome harbours a diverse assemblage of chlamydiae, suggesting potential ecological and functional relevance. Overall, our study highlights the importance of tissue-specific, single-tick analyzes for elucidating microbiome complexity and advances current understanding of Chlamydiota diversity in the tick vector. Further experimental and multi-omics studies are warranted to elucidate the biological roles of these bacteria in tick physiology and pathogen infection dynamics.