Double-stranded RNA (dsRNA)-mediated gene silencing, or RNA interference (RNAi), is an emerging biotechnology that has been a breakthrough tool for crop protection. Exogenous dsRNA triggers the RNAi pathway, silences genes, disrupts protein function, and can cause insect mortality. However, effective delivery of the dsRNA is problematic, particularly in systems with long-lived, endophagous insects such as the emerald ash borer (EAB), Agrilus planipennis, a tree-killing nonnative invader that attacks ash, Fraxinus spp. Larvae feed on cambial tissue causing rapid tree death. EAB is susceptible to RNAi, but we lack a practical means of delivery. Here we evaluated delivery of dsRNA to green, F. pennsylvanica, and tropical ash, F. uhdei, through root and/or petiole absorption, and also demonstrated dsRNA absorption through the EAB egg chorion. We labeled exogenous dsRNA using a fluorescing label and then used confocal microscopy and RT-qPCR to evaluate its distribution in plant and insect tissues. Labeled dsRNAs are detectable in root, stem, and leaf tissues 48-h postapplication. In excised ash branches, labeled dsRNA is detectable in the inner bark and in recovered EAB neonates 8-day postapplication. Eggs and larvae emerging from treated eggs also presented fluorescing dsRNA under confocal imaging. Adult EAB-fed tropical ash leaves treated with in vitro synthesized EAB-specific dsSHI through petiole absorption experience a significant knockdown of the shi gene and a significant mortality. Our findings provide a proof of concept that delivery of dsRNAs through topical or systemic application methods is a feasible means of suppressing EAB, providing hope for future tree protection.

RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest.