Differential Impact of Simultaneous or Sequential Coinfections With <i>Borrelia afzelii</i> and Tick‐Borne Encephalitis Virus on the <i>Ixodes ricinus</i> Microbiota

Ticks, particularly <i>Ixodes ricinus</i>, are significant vectors of pathogens such as <i>Borrelia</i> spp. and tick-borne encephalitis virus (TBEV), which cause Lyme borreliosis (LB) and tick-borne encephalitis (TBE), respectively. Understanding how these pathogens interact within the tick microbiome is essential for developing vector control strategies. This study investigates the impact of <i>Borrelia afzelii</i> and TBEV, as well as their coinfection, on the microbiota composition and structure of <i>I. ricinus</i> nymphs. Using a network-based approach, we analyzed the microbial communities of ticks exposed to infected or coinfected mice. DNA extracted from newly molted nymphs was sequenced for the bacterial 16S rRNA gene, and microbial diversity metrics (alpha and beta diversity) were calculated. Our results showed that TBEV infection increased microbiome diversity compared to the uninfected and <i>Borrelia</i> groups. Co-occurrence network analyses revealed that while microbial structures remained consistent across conditions, TBEV-infected networks exhibited higher robustness to perturbations, indicating a stabilizing effect on the tick microbiome. Furthermore, the hierarchical position and associations of <i>Borrelia</i> varied significantly depending on the infection scenario, highlighting its adaptive role within the tick microbiota. The study demonstrates that pathogen presence alters tick microbial dynamics, with TBEV enhancing stability, suggesting virus-mediated modifications of the microbiome. These findings advance our understanding of pathogen-tick-microbiome interactions and provide insights into the ecological mechanisms underlying pathogen coexistence within ticks. This research underscores the importance of microbial networks in ticks and offers new perspectives for targeted approaches in managing tick-borne diseases.