The goal of this project is to identify insecticidal peptides with efficacy against phloem-feeding citrus pests. Our first objective is to evaluate a range of known insecticidal peptides against the Asian Citrus Psyllid (ACP) in artifical feeding assays. We have procured candidate peptides for screening and are optimizing artificial bioassays for evaluation of their effect on ACP mortality. In a second objective, we have proposed to utilize Citrus tristeza virus (CTV) for delivery of insecticidal peptides into citrus plants for management of the ACP, Brown Citrus Aphid (BCA), and Citrus Leafminer (CLM). Development of CTV as a vector for foreign peptides was previously conducted in a separate project by Dawson et al. Using the CTV expression vector, we have elected to examine four peptides known to have insecticidal activity against phloem-feeding agricultural pests. To facilitate bioassays for screening individual insecticidal peptides, we have recently established laboratory colonies of BCA and CLM. BCA were obtained from a colony currently housed at the Citrus Research and Education Center in the laboratory of Dr. Bill Dawson. The CLM colony was established by collecting insects from citrus groves located in Polk County, Florida. In addition, we have procured plants for use in experiments evaluating CTV-expressed insecticidal peptides. Prior to the inception of the current project, one of the peptides proposed for evaluation, snow drop lectin, Galanthus nivalis agglutinin (GNA), was successfully cloned into the CTV-expression vector and inoculated into citrus plants. At the present time we are conducting experiments to evaluate the efficacy of CTV-vectored GNA against ACP and BCA. These bioassays will evaluate the effect of GNA on insect feeding, survival, development, and fecundity. Concurrently, we are conducting bioassays using synthetic GNA in an artificial feeding system to evaluate the effect of GNA concentration on ACP mortality. In addition to GNA, we have proposed to evaluate three CTV-vectored insecticidal peptides against ACP, BCA, and CLM. Indolicidin, which was previously cloned into the CTV vector, has been inoculated into citrus and should be available for screening in bioassays with the target insects in the next few months. Allium sativum leaf agglutinin (ASAL) and Pinellia ternate agglutinin (PTA) peptide genes have been sequenced and are currently being cloned into CTV-vectors. This process should be completed within two months, after which the CTV-peptide constructs will be bark flap inoculated into citrus. Following successful establishment of CTV vectors into initial citrus hosts, stems from plants exhibiting a high level of CTV will be harvested and graft-inoculated onto subsequent ‘Valencia’ sweet orange plants for use in bioassays. This process is expected to be completed in the next four months. To quantify peptide expression in CTV-inoculated plants, we are developing enzyme-linked immunosorbant (ELISA) assays specific to the peptides under investigation. At the present time, an ELISA assay for GNA is being optimized. Identifying the concentration of expressed peptides should facilitate comparisons between the efficacy observed in artificial bioassays with synthetic peptides and in planta assays with CTV-expressed peptides.