2013 Annual Report
1a.Objectives (from AD-416):
The New World screwworm, Cochliomyia hominivorax, was eradicated from North America through a multi-national program led by USDA. The sterile insect techniques developed by ARS scientists were the keystone in the eradication program and the eradication status of North America is maintained by the USDA-APHIS Screwworm Eradication Program working in conjunction with counterparts in Panama. The Screwworm Production Facility in Pacora, Panama is producing flies to maintain the barrier zone at the Panama-Columbia border. To pursue opportunities at optimizing Pacora's production capacity, APHIS funded a research proposal to develop a female conditional-lethal transgenic strain of C. hominivorax led by the Knipling-Bushland US Livestock Insects Research Laboratory in Kerrville, TX, including interactions between the ARS research laboratory at Pacora and USDA-APHIS. In this project, the recombinant vectors and necessary protocols to facilitate the transformation of C. hominivorax would be developed at the Kerrville lab in the secondary screwworm, Cochliomyia macellaria, and transferred to Pacora for application in C. hominivorax. Transformation protocols have been used to successfully transform secondary screwworm and transformation experiments with C. hominivorax have been initiated in Panama. Three female conditional lethal vectors have been constructed for use in C. hominivorax which are based on a tetracycline-repressible transgene system.
1b.Approach (from AD-416):
The project has advanced to the stage where evaluations of female conditional-lethal vectors have been initiated in the Screwworm Eradication Program's facility at Pacora, Panama. This agreement will be used to develop a second vector system as a contingency in the event the current vectors do not meet the Program's needs.
1. Develop tetracycline-repressible female lethal conditional lethal cloning vector based on improved piggybac transposable element/helper system, C. hominivorax transformer sex-specific splicing intron, tTAV coding region, and tetO operon/promoter.
a. Using an available C. hominivorax expressed gene database, optimize codons in the tTAV to reflect C. hominivorax codon usage.
b. Provide the transformer and doublesex genes from Lucilia cuprina and isolate the transformer and doublesex genes from C. hominivorax.
c. Create new candidate female-lethal vector using the improved piggybac transposable element/helper system, optimized tTAV coding region and C. hominivorax or L. cuprina transformer intron.
2. Evaluate the new vector through transformation experiments in the secondary screwworm Cochliomyia macellaria.
The major objective is to develop new DNA molecules to use in creation of strains of New World screwworm that can be selectively induced to produce only male flies. The objectives of this agreement were achieved and the transgenic strains transferred to ARS staff at the Screwworm Production Facility in Pacora, Panama, for further evaluations.