Honey bee functional genomics using symbiont-mediated RNAi.

Honey 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. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA 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 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.