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 U.S. Livestock Insects Research Laboratory in Kerrville, TX, including interactions between the ARS research laboratory at Pacora and USDA-APHIS. In this grant proposal, we seek funds to begin evaluating potential risks associated with field release of a male-only transgenic C. hominivorax into the environment as part of the Screwworm Eradication Program. Three female conditional, lethal vectors have been constructed for use in C. hominivorax, which are based on a tetracycline-repressible transgene system. These vectors are ready to begin transformation experiments to create the male-only strain. We have a cryopreserved transgenic C. hominivorax strain that can be used to begin this study.
1b.Approach (from AD-416):
There are a number of documents that are available on risk assessment of transgenic insects. The report from the IAEA/FAO meeting in 2002 is widely known and often cited. More recently, many in the field have been involved in making a report for the European Food Safety Authority (EFSA), which was published in 2010. This is probably the most thorough document in the area. Also relevant is the USDA environmental risk assessment for field release of transgenic pink bollworm and fruit flies. Some of the potential risks identified in the EFSA document were not addressed in the USDA report, e.g., the risk of horizontal gene transfer of piggyBac elements and the likelihood that a transgenic insect would have increased fitness. We will address the following potential risks in this proposal:
1) Risk: as RIDL is based on a single-lethal gene, there is the potential for development of early resistance. This is analogous to an insecticide that has a single mode of action. Evaluation approach: Raise transgenic strain in relatively large numbers without tetracycline and measure the frequency of resistant females. Determine if resistance is heritable.
We also will propose to make a transgenic strain that has two independent lethal genes.
2) Risk: The transgene is unstable and can jump to other species (i.e., horizontal gene transfer). In practice, it has proved very difficult to remobilize piggyBac transgenes. Evaluation approach: Raise the strain for several generations and determine if transgene is lost or remobilizes, i.e., perform Southern blot hybridization and inverse PCR. We also propose to deliberately try to remobilize the transgene by injection of DNA that encodes transposase.
We propose to engineer a strain that has only one end of a piggyBac transposon. That would essentially make it impossible for the transgene to jump.
3) Risk: the transgenic strain may mate with a related species and thus transfer the transgene (i.e., hybridization). Evaluation: Analyze mating behavior of transgenic males compared to wild type males. Determine if transgenic males can compete effectively with wild type males.
Determine if transgenic males will mate with females from a related species: Cochliomyia macellaria.
4) Risk: Transgenic screwworm strain shows abnormal environmental interaction with predators and parasitoids. Evaluation: Compare interaction of transgenic and non-transgenic screwworm with known predators and parasitoids.
This is the final report for this project. A transgenic strain of New World Screwworm was produced and reared to homogeneity for use in this collaborative project. Several DNA-based molecules were created for use in various New World Screwworm transformation protocols. Each molecule imparts different characteristics to putative transgenic strains.