Species-Specific Bacterial Associations Emerge From Stochastically Assembled Microbiomes in Northeastern American...

Many insects harbour microbial communities that can profoundly influence the biology of their host. Yet, the relative contribution of random exposure (i.e., stochastic) events and deterministic ecological factors in shaping these communities remains unclear for most taxa. We examined microbiome assembly across 344 firefly (Coleoptera: Lampyridae) specimens from the Northeastern United States, spanning 12 species and species groups, and generating a high-resolution dataset through deep 16S rRNA gene amplicon sequencing and quantitative PCR. To formally assess the balance between stochastic and deterministic forces, we applied integrative statistical approaches, including an innovative null-modelling framework based on the normalized stochasticity ratio (NST) index. We hypothesized that firefly microbiome assembly is dominated by stochastic processes driven by unpredictable microbial exposures. Consistent with this, we observed elevated NST values for most bacteria, coupled with high intraspecific variability in bacterial abundance and composition. However, microbiomes were more similar among closely related fireflies and unusually prevalent mollicute strains showed low NST values, species-specific associations and retention across geography and host development. While adult bioluminescence and diet could not be directly linked to microbiome abundance or composition, considering seasonal factors and intra-host anatomy within host species revealed patterns explaining some of the intraspecific microbiome variation. These results show that deterministic processes, likely arising from host-specific microbial filtering mechanisms, act alongside stochastic forces to shape firefly-microbe associations. By integrating broad field sampling with quantitative bacterial load estimates and comprehensive microbiome analyses, this study clarifies how evolutionary history, ecology and chance jointly govern microbiome assembly in a diverse insect lineage.