Microbial network stability, not diversity, drives colonization resistance against Borrelia afzelii in Ixodes ricinus ticks

Most tick-borne pathogens (TBPs) are acquired secondarily, when ticks feed on infected hosts, meaning the pathogen must establish itself within an already assembled microbiota. These scenarios are subject to "priority effects," where the order of microbial arrival influences the success of later colonizers. Microbial interactions within arthropod vectors can therefore shape infection outcomes, producing either infection-refractory states, where resident microbes and their interactions reduce the likelihood of pathogen establishment, or infection-permissive states, where such barriers are absent or weakened and the pathogen establishes infection successfully. Hamilton et al. (2021) assessed larval microbiota before pathogen exposure and sequenced the microbiota of fed nymphs, both exposed or not to Borrelia afzelii, enabling priority-effect hypotheses to be tested. Despite uniform exposure to the highly infectious B. afzelii strain NE4049, only a subset of ticks became Borrelia-positive, suggesting refractory and permissive microbiota states. We reanalyzed the original dataset to test whether differences in microbiome community assembly and co-occurrence network features, beyond diversity metrics, were associated with these states. Refractory nymph networks exhibited higher connectivity and structural resilience, with Staphylococcus emerging as a central taxon already present in unfed larvae. In contrast, permissive networks showed reduced robustness and a marginal role for Staphylococcus. Notably, dysbiosis altered microbial assembly but did not prevent network reconfiguration in refractory ticks. Our findings suggest that colonization resistance is better explained by microbial network integrity than by diversity alone. Methodologically, they show that integrating community assembly theory and network analyses can reveal key features of the tick microbiota associated with vector competence.