Addressing the challenges of symbiont-mediated RNAi in aphids.

Because aphids are global agricultural pests and models for bacterial endosymbiosis, there is a need for reliable methods to study and control their gene function. However, current methods available for aphid gene knockout and knockdown of gene expression are often unreliable and time consuming. Techniques like CRISPR-Cas genome editing can take several months to achieve a single gene knockout because they rely on aphids going through a cycle of sexual reproduction, and aphids often lack strong, consistent levels of knockdown when fed or injected with molecules that induce an RNA interference (RNAi) response. In the hopes of addressing these challenges, we attempted to adapt a new method called symbiont-mediated RNAi (smRNAi) for use in aphids. smRNAi involves engineering a bacterial symbiont of the insect to continuously supply double-stranded RNA (dsRNA) inside the insect body. This approach has been successful in thrips, kissing bugs, and honeybees. We engineered the laboratory strain HT115 and the native aphid symbiont CWBI-2.3 to produce dsRNA inside the gut of the pea aphid () targeting salivary effector protein (C002) or ecdysone receptor genes. For C002 assays, we also tested co-knockdown with an aphid nuclease (Nuc1) to reduce RNA degradation. However, we found that smRNAi was not a reliable method for aphid gene knockdown under our conditions. We were unable to consistently achieve the expected phenotypic changes with either target. However, we did see indications that elements of the RNAi pathway were modestly upregulated, and expression of some targeted genes appeared to be somewhat reduced in some trials. We conclude with a discussion of the possible avenues through which smRNAi, and aphid RNAi in general, could be improved in the future.