Objective 1, investigating the specificity and efficacy of immune priming response in ACP to pathogenic bacteria, is nearing completion and will be analyzed during the next quarter. Subsequent experiments to address the effects of non-pathogen immune priming to pathogenic bacteria are underway, and should be nearly complete by this summer. The second project objective, to determine whether double strand RNA (dsRNA) uptake can result in immune priming in D. citri, we will be conducting RNAi experiments on D. citri adults of a CLas-free laboratory colony. dsRNA of genes that exist (e.g., V-type proton ATPase) or do not exist in D. citri (a bacterial GFP gene) will be used for this experiment. Briefly, primers targeting GFP fragments in plasmid pCMV-GFP (Matsuda and Cepko 2004) or a vATPase gene fragment of D. citri [based on sequences determined in a published work (Vyas et al. 2015)] wereover time. Insect mortality will be recorded every 24 h. Depending on the results, we may also attempt to determine the effects of RNAi-treatments on D. citri transmission of CLas in the coming months. designed. The primers were synthesized with T7 promoters added to the 5′ ends (Table 2). Amplification products obtained by using these primers and their template DNA will be purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). Purified amplicons will be used for dsRNA synthesis using the MEGAscript RNAi kit (Ambion, Austin, TX). RNAi treatments will be conducted by feeding D. citri with 30% sucrose solutions including 100 ng/ul of each type of dsRNA, using previously described methods (Wuriyanghan, Rosa, and Falk 2011) with slight modification. For the negative control, products of a non-template RNAi synthesis reaction will be used. The insects will be kept on the diets for 24 h. RNAi-treated insects will be moved to caged citrus plants (var. “Swingle”) for one week. The insects will then be challenged with Serratia marcescens by microinjection of bacterial suspensions (~107 cells/ml) using a FemtoJet Microinjector (Eppendorf, Inc., Fremont. CA). Effects of RNAi-treatments on Serratia marcescens growth will be determined by qPCR using primers specific to this bacteria (Joyner et al. 2014). Insects will be sampled every 48 h, over two weeks. A separate cohort of the challenged insects will be kept on the citrus plant and monitored for their survivorship