Characterization of bacterial microbiome and molecular detection of rickettsiosis in free-living ticks (Amblyomma sculptum Berlese, 1888).

Amblyomma sculptum is a major tick species in southeastern Brazil and an important vector in the epidemiology of Brazilian Spotted Fever (BSF). This study characterized the bacterial microbiome of free-living A. sculptum ticks in a BSF-endemic area, focusing on differences among developmental stages and sexes, and investigated rickettsial agents using 16S rRNA gene (V3-V4) sequencing. A total of 154 ticks were collected and analysed as 13 pooled samples grouped by stage and sex. Sequencing identified a diverse bacterial community of 180 genera, dominated by Sphingomonas, Nocardioides, Actinomycetospora and Methylobacterium, alongside genera of potential zoonotic relevance such as Rickettsia, Anaplasma, Ehrlichia and Coxiella, mainly in nymph pools. Alpha and beta diversity analysis showed that microbial community composition differed among stages and sexes, with adult males exhibiting higher richness and compositional dispersion, whereas nymphs showed reduced diversity and tighter clustering. Because 16S sequencing does not allow species-level identification of rickettsiae, positive samples were further analysed by PCR amplification and sequencing of the gltA gene, which identified Rickettsia bellii. Together, these results highlight stage- and sex-associated patterns in the microbiome of free-living A. sculptum and support the use of integrated microbiome profiling and targeted rickettsial detection in ecological and epidemiological studies of tick-borne diseases.