Abstract
Beewolf wasps rely on an ancient defensive symbiosis with Streptomyces bacteria that protect their larvae from fungal infection. Female beewolves apply the bacteria to the brood-cell ceiling, and larvae later transfer the symbionts onto the cocoon surface, where they produce antifungal metabolites. Here, we investigated the mechanism of symbiont transfer from the beewolf brood cell to the larval cocoon and characterized the microbial community dynamics across different beewolf life stages and during larval hibernation. Fluorescence in situ hybridization revealed that the symbionts are transiently taken up into the proximal midgut lumen and then regurgitated onto the cocoon during the spinning process. High-throughput sequencing showed that the bacterial community of beewolf feeding larvae resembles that of the honeybee prey, whereas that of adults and diapausing larvae is dominated by Wolbachia. Moreover, the cocoon bacterial community is initially dominated by the defensive Streptomyces philanthi symbiont, but when larvae excrete the gut content inside the cocoon, other bacterial taxa including Lactobacillus, Gilliamella, and Bartonella shift the community composition toward dominance by Pseudomonadota. Our findings provide new insights into the transmission route of an ancient extracellular symbiont and its potential competition with other bacteria in this long-term defensive symbiosis.