The holobiont's extracellular microbiome modulates the adaptation of an intracellular endosymbiont

Animal holobionts contain two broad classes of microorganisms, intra- and extracellular microbes, that are largely studied in symbiotic siloes. Here we demonstrate the impact of the extracellular gut microbiome on an intracellular reproductive endosymbiont using the Drosophila melanogaster holobiont and its endosymbiont Wolbachia that adaptively cause a paternal-effect lethality of embryos termed cytoplasmic incompatibility (CI). We report the endosymbiont's adaptation is not only self-sufficient but stronger in a host without an extracellular microbiome. Indeed, host transcripts associated with the CI adaptation are enhanced, while cell biological hallmarks exhibit complex quantitative and qualitative differences in microbiome-free hosts. Decreases in the gut bacteria Lactobacillus specifically link with high penetrance of the CI adaptation. Notably, both the extracellular microbiome and endosymbiont harbor markedly higher cellular densities in a complete holobiont setting than when either constituent is reared alone. Altogether, this study uncovers a novel interaction whereby the extracellular microbiome modulates the phenotype of an intracellular endosymbiont, revealing intricate symbiotic networks within the holobiont.