Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, Snodgrassella alvi, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered S. alvi can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered S. alvi can kill parasitic Varroa mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.

The gut microbiome plays a critical role in the health of many animals. Honeybees are no exception, as they host a core microbiome that affects their nutrition and immune function. However, the relationship between the honeybee immune system and its gut symbionts is poorly understood. Here, we explore how the beneficial symbiont Snodgrassella alvi affects honeybee immune gene expression. We show that both live and heat-killed S. alvi protect honeybees from the opportunistic pathogen Serratia marcescens and lead to the expression of host antimicrobial peptides . Honeybee immune genes respond differently to live S. alvi compared to heat-killed S. alvi, the latter causing a more extensive immune expression response. We show a preference for Toll pathway upregulation over the Imd pathway in the presence of both live and heat-killed S. alvi . Finally, we find that live S. alvi aids in clearance of S. marcescens from the honeybee gut, supporting a potential role for the symbiont in colonization resistance. Our results show that colonization by the beneficial symbiont S. alvi triggers a replicable honeybee immune response. These responses may benefit the host and the symbiont, by helping to regulate gut microbial members and preventing overgrowth or invasion by opportunists.

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Bees are indispensable pollinators and model organisms for studying social behavior, development, and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a host RNA interference response that reduces expression of a targeted gene. To employ this FUGUES (FUnctional Genomics Using Engineered Symbionts) procedure, a double-stranded RNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by five days after colonization. This protocol can be accomplished in four weeks by bee researchers with microbiology and molecular cloning skills.