Species-specific bacterial associations emerge from stochastically assembled microbiomes in Northeastern American fireflies

Many insects harbor diverse microbial communities ("microbiomes") that can profoundly influence the biology of their host. Yet, for most insect taxa, the relative roles of stochastic events versus deterministic forces in shaping these communities remain unknown. We investigated microbiome assembly in fireflies (Coleoptera: Lampyridae) using 16S rRNA gene amplicon sequencing and quantitative PCR from 12 species and species groups collected across the Northeastern United States. We hypothesized that firefly microbiomes are predominantly structured by stochastic ecological processes, such as random microbial exposures. Consistent with this, we observed elevated normalized stochasticity ratio (NST) values for most bacteria, coupled with high intraspecific variability in bacterial abundance and composition. However, microbiomes were more similar among closely related firefly hosts, and several bacterial taxa, including endosymbionts and unusually prevalent mollicute strains, showed lower NST values, species-specific associations, and patterns linked to geography, season, and sex. These results reveal that deterministic processes--likely involving host-driven microbial filtering, microbe-microbe interactions, and reliable transmission modes--act alongside stochastic forces to shape firefly-microbe associations. By disentangling these ecological drivers in a diverse, understudied beetle lineage, our study advances understanding of the ecological and evolutionary processes structuring microbiomes in the wild.