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 U.S. 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 conditionallethal 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.
Currently one male-only line of screwworms has been established and is under laboratory evaluation. About 99% are males. For this line, females perish in the 3rd larval stage. General fitness parameters of this line, measures for several generations in the laboratory, are similar/equivalent to the strain (Jamaica - 06) from which it was derived.
A post-doctorate researcher funded by a BRAG grant arrived in Panama in December 2012. Several transgenic lines with the FL11 and FL12 gene constructs have been made and are currently being bred to homozygosity. These single-component constructs have been optimied for high expression in blow flies. The post-doc is performing molecular analyses to determine the nature of the genetic insertions.
Another post-doctorate researcher began a 1-year fellowship at NCSU on June 1, 2013. His goal is to develop and test two-component tetracycline-repressible carly-acting female lethal gene constructs. The "driver" component makes iTA in early embryos and the "effector" component is switched on by tTA in females, causing death. The post-doc has made several "effector" and "driver" gene constructs in piggyBac vectors. Additional constructs are in progress. The plan is to test constructs in Lucillia cuprina at the NSCU laboratory. The best combination of tTA driver/lethal effector wll be transferred to Pacora, Panama, for injection and screening/testing Cochliomyia nominivorax. Currently several transgenic L. cuprina lines have been obtained with one "effector" and one "driver" gene construct.
Protocols are established to determine if transgenic, genetic sexing, screwworm males mate with secondary screwworm females. This is part of the 'environmental safety' requirements necessary before GMOs could be used under standard field release conditions. Once the ARS Field Laboratory is 'reconditioned' to rear secondary screwworms, in the next 2-3 months, these tests will commence.