RNA-based 16S rRNA amplicon sequencing reveals shifts in microbiome transcript profiles of Ixodes ricinus during Borrelia afzelii infection

Abstract

Ticks harbor relatively complex microbial communities that influence vector competence, pathogen persistence, and host-tick-pathogen interactions. However, the effects of pathogen infection on RNA-derived microbial community profiles within ticks remain poorly understood. Here, we used RNA-based 16S rRNA amplicon sequencing complemented by RT-qPCR validation and in vitro interaction assays to investigate whether Borrelia afzelii infection shapes the resident bacterial community of Ixodes ricinus nymphs. This approach captures relative 16S rRNA transcript abundance, which may reflect differences in bacterial abundance, metabolic activity, or both. Our results show that Borrelia infection is associated with broad shifts in bacterial transcript profiles without causing major alterations in overall taxonomic turnover. Host transcriptional profiling further revealed selective activation of the tick JAK/STAT pathway, with significant upregulation of the stat gene, while other immune and redox pathways remained largely unchanged. This restricted immune response may contribute to microbiome modulation during infection. Collectively, these findings underscore the value of RNA-based amplicon sequencing for profiling changes in bacterial rRNA transcript signals and provide new insights into the interplay between pathogens, the resident microbiome, and vector immunity. However, integration with DNA-based and multi-omics approaches will be necessary to disentangle changes in microbial abundance from shifts in cellular physiology and microbiome function.