2012 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 transformation of C. hominivorax with the female conditional-lethal vectors are necessary to evaluate their potential for implementation in the Screwworm Eradication Program's facility at Pacora, Panama. These transformation must be done in the biological containment facilities at Pacora. This agreement will be used to provide research and technical needs to carry out the female lethal vector evaluations and develop a second vector as a contingency in the event the current vectors do not meet the Program's needs.
To meet these project needs, the first half of the project will be conducted at the ARS-Kerrville facility, while the second half of the project will be conducted at the ARS-Panama laboratory in Pacora, Panama.
Year 1 Approach: (at Knipling-Bushland U.S. Livestock Insects Research Laboratory)
1. Develop tetracycline-repressible female lethal conditional lethal cloning vector based on piggybac transposable element 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. Isolate the transformer sex-specific intron from C. hominivorax.
c. Create new candidate female-lethal vector using the optimized tTAV coding region and C. hominivorax transformer intron.
2. Evaluate the new vector in Drosophila melanogaster. Test for gene expression and female lethality.
Year 2 Approach: (at ARS Screwworm Research Unit laboratory in Pacora, Panama)
1. Perform microinjections of all available female conditional lethal vector constructs into C. hominivorax and evaluate for female-specific lethality.
2. Provide technical advice and participate in transgenic strain rearing and maintenance.
The researcher assigned to work at the New World Screwworm Production Facility in Pacora, Panama, created several transgenic strains of New World Screwworm. These strains are being evaluated as candidates to replace the currently used strain to reduce costs of production in the Pacora facility.