During the past quarter, we have optimized dsRNA targets and have conducted immune priming assays to investigate the specificity of immune priming in ACP following exposure to dsRNA. The yield of redesigned dsRNA using MEGAscript RNAi Kit transcription was initially low, possibly because the QG buffer in the Zymoclean Gel DNA Recovery Kit may have decreased transcription. In order to avoid using QG buffer in gel purification and increased PCR concentration,gel purification method was modified. PCR products were analyzed on 1% agarose gel and the band was excised, and transferred to a filter tip and centrifuged at 5000 rpm for 10 min. Three �l of purified gel product were run on 1% agarose gel which showed a high concentration (based on the intensity of the band) of the product. The remaining purified gel product was precipitated and resuspended in 30 �l of RNase-DNase free water, and 2 �l of the product were analyzed on agarose gel and directly amplified. PCR products obtained from the modified purification protocol for dsRNA synthesis yielded significantly higher dsRNA concentrations to compared to the kit. Bioassays were conducted with adult uninfected ACP. After 3 hrs of starvation, insects were fed on dsRNA for 24hrs and 5 days. After 24 hrs, insects were starved for 3 hrs and then transferred to a lethal dose of S. marcescens for 4 days. For each dsRNA concentration, two cages were exposed to dsRNA; then either fed on S. marcescens in diet (20% sucrose, and 0.5 green food coloring dye) or diet only (control). Diet containing the pGEM-T easy vector without a T7 tail, a blank sucrose + buffer diet, and container without food were also included as controls in the feeding experiment. The stability of different concentrations of dsRNA on diet solution were confirmed with agarose gel electrophoresis at the end of the 24hr and 5 d feeding periods. DsRNA of 1000 ng/�l and 100 ng/�l were present in agarose gel. Also the survival of S. marcescens after 4 days feeding was checked on nutrient agar plate. S. marcescens from feeding cage was streaked on nutrient agar and kept O/N at 30�C. Result confirmed that bacteria were viable during the feeding assay. Following exposure to bacteria, insect were placed on Citrus macrophylla in the greenhouse, and mortality was recorded daily. After 14 d, live insects from the 24hr and 5 d feeding treatments were collected and laterally bisected. Half of the insects were examined for the presence of S. marcescens, and the remaining insects were used for RNA extraction. To quantify S. marcescens, single insects were smashed in tube with 20 �l of nuclease-free water and plated on nutrient agar, and kept at 30�C O/N. Colonies were obtained from most of the insects on nutrient agar. No colonies were obtained from control insects. Conventional PCR confirmed the presence S. marcescens in insects. To study the effect of dsRNA on mRNA levels, total RNA was extracted from single ACP using TRIzol (Invitrogen) and cDNA was prepared. The survival rate of insects after feeding on S. marcescens and from 14 days on C. macrophylla will be analyzed and presented when all replicates are completed; however, initial results indicate thigh mortality after feeding on S. marcescens as compared with those feeding on sucrose, suggesting that dsRNA exposure does not modulate ACP immune responses to S. marcenscens e.g. does not prime ACP to tolerate or mitigate lethal doses of pathogenic bacteria, although it must still be determined whether this response is similar following exposure to sublethal doses of bacteria. Whether this is responses is specific or is generalizable to other Gram negative bacteria, such as Liberibacter, will be determined in the next quarter.