2008 Annual Report 1a.Objectives (from AD-416) 1) To test the stabilization vector system in the Mediterranean fruit fly, Ceratitis capitata, by modifying the pBac{L1-PUbDsRed-L2-3xP3ECFP-R1} vector originally used in Drosophila. 2) To create new target site and donor vectors for efficient use in tephritid flies. This will include i) modification of the pBac{3xP3-FRT-ECFP-linotte-FRT3} target acceptor vector and the pSL-FRT-EYFP-linotte-FRT3 donor vector by replacing 3xP3 promoters with polyubiquitin promoters and removal of the linotte sequence, and ii) introducing new heterospecific FRT and loxP recombinations sites for repetitive insertions into target sites. 3) To test the modified RMCE target vector and stabilization system in C. capitata in vivo and in cell culture. 4) To create a series of target site strains in C. capitata and the Mexican fruit fly, Anastrepha ludens, that will be characterized initially in terms of strain fitness and transgene marker expression. Optimal strains will be genetically mapped with the target site molecularly characterized. 1b.Approach (from AD-416) Vectors for transformant stabilization and targeting developed for Drosophila will be modified by promoter sequence replacement and insertion of new recombination sites, with subsequent tested by germ-line transformation in tephritid host strains and transfection into tephritid cell lines. Host strains will be analyzed by insertion site sequencing, marker expression, and strain viability and fitness. Targeting by recombinase-mediated cassette exchange will be testing by providing donor vectors and recombinase helper vectors and testing for recombination events. 3.Progress Report This project is related to Objective 1 of this in-house project: Develop techniques and strategies that utilize molecular gene transfer methods to create transgenic strains of Diptera, Lepidoptera, and Coleoptera that will facilitate genetic-sexing or have novel autocidal properties for use in IPM programs. New vectors allowing recombinase-mediated cassette exchange (RMCE) genomic targeting and subsequent stabilization by post-integration terminal sequence deletion have been created, and experiments to integrate them into the germ-line of the Caribbean fruit fly, Anastrepha suspensa, and the Mediterranean fruit fly, Ceratitis capitata, have been initiated.