The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. �l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 �L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80�C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/�l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.