2010 Annual Report 1a.Objectives (from AD-416) Produce and test transgenic Mexican fruit fly (A. ludens) strains that incorporate the tetracycline-suppressible embryonic lethality system developed for medfly. A female-specific component for embryonic lethality will also be developed that should result in male only strains based on female-specific embryonic lethlity. 1b.Approach (from AD-416) The genetic components for these systems will be first identified and isolated from either A. ludens or A. suspensa. These will include regulatory DNA sequences from embryonic genes such as serendipity, slam, and mdg88; sex-specific intron splicing cassettes isolated from the A. ludens sex-determination gene transformer; and the lethal effector gene, hid. To test the Mediterranean fruit fly embryonic lethality system in Anastrepha species existing driver (srya-tTA) and effector (TRE-hidala5) transgene constructs shown to be functional in the Mediterranean fruit fly will be integrated into a wild A. ludens host strain. Endogenous early and late embryonic genes will be isolated from the Anastrepha species to test their promoters in driver constructs, and efforts will be made to isolate the hid lethal-effector from Anastrepha. Transgenic A. suspensa and A. ludens will be created using the strongest early and late promoter-tra intron-tTA constructs (based on tTA transcription) inserted into DsRed-marked piggyBac vector plasmids having the TRE-tra intron-hidala5 driver and phiC31 attP integrase recombination sites. Transformation experiments will be initiated in A. ludens using transgene vectors yielding the most efficient female-lethality in A. suspensa. If no homozygous line yields 100% female-lethality, the transgenes will be re-mobilized by transposase injection to create new insertion sites that will be tested for more optimal parameters. Optimal homozygous lines will be expanded for large scale sexing, fitness, and mating competitiveness tests in Tapachula and Petapa. 3.Progress Report This research relates to inhouse objective 1: 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. Transgenetic strains have been created in the Mexican fruit fly that incorporate flourescent protein marker genes that allow monitoring of released males and facilitate genetic sexing, and sperm-specific markers to identify females that have mated with released males. New trangene vectors for female-specific conditional lethality that allow genetic-sexing in embroys, are now being developed. Cooperator activities for this project have been monitored by site visits (June, 2010) and routine voice and email correspondence. Activities were monitored via email, calls, reports and meetings.