Author
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GENC, HANIFE - Canakkale University |
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SCHETELIG, MARC - Justus-Liebig University |
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XAVIER, NIRMALA - Former ARS Employee |
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Handler, Alfred - Al |
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Submitted to: Turkish Journal of Biology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/7/2015 Publication Date: 12/7/2015 Citation: Genc, H., Schetelig, M.F., Xavier, N., Handler, A.M. 2015. Germline transformation of the olive fruit fly, Bactrocera oleae (Rossi)(Diptera:Tephritidae) with a piggyBac transposon vector. Turkish Journal of Biology. 40:845-855. https://doi.org/10.3906/biy-1510-55. DOI: https://doi.org/10.3906/biy-1510-55 Interpretive Summary: One biocontrol strategy for the population suppression of pest insects is the sterile insect technique (SIT). The primary needs for successful SIT is the ability to mass rear and release large numbers of sterile, though sexually active, adult male insects, and to eliminate females early in development to avoid rearing costs and their subsequent sterilization and release. Scientists at the USDA, Agriculture Research Service, Center for Medical, Veterinary and Agricultural Entomology in Gainesville, Florida, in collaboration with scientists at Çanakkale Onsekiz Mart University, Turkey, work to achieve gene transfer in economically important insects such as olive fruit fly, which is a destructive pest of olives. The ability to genetically manipulate fruit flies allows the creation of transgenic conditional lethal strains that eliminate females during rearing, and sterilize surviving males. As a first step in development of SIT for the olive fruit fly, methods to develop germline transformation using a piggyBac transposon vector that can be immobilized post-integration for strain stability and environmental safety were employed. Successful genetic transformation of a Turkish strain of olive fruit fly was achieved where marker expression and life fitness parameters were comparable to other tephritid transgenic strains. These biotechnologies can be employed to create SIT strains that can be used for biocontrol of these pest insects. Technical Abstract: The olive fruit fly, Bactrocera oleae, is a highly significant pest in olive growing countries whose control may be enhanced by the use of genetically-modified strains, especially for sterile insect technique programs. To improve and expand this technology, piggyBac-mediated germline transformation was achieved in a laboratory-adapted wild olive fruit fly strain. A piggyBac vector was used that is marked with both green (EGFP) and red (DsRed) fluorescent protein genes, with a duplicate piggyBac 5' terminal inverted repeat sequence inserted between the marker genes for subsequent immobilization of vectors integrated into the host genome. Five transformant G1 adults were selected based on marker gene expression, yielding an estimated germline transformation frequency of approximately 0.42% per fertile G0 adult. All transgenic lines expressed DsRed and EGFP, though DsRed was more visible and robust compared to EGFP expression, which remained stable for more than 20 generations. Marker expression and PCR analysis, including an insertion site sequence, was consistent with stable genomic insertions. Several life fitness parameters including egg hatching, larval viability, larval to pupal survival, pupal to adult survival, and fertility were tested for three transgenic lines. Viability of the transgenic lines at all life stages was similar, but overall fitness was significantly lower in the transgenic lines compared to wild type olive flies, but similar to fitness levels previously reported for transgenic Mexican fruit flies. The studies presented here demonstrate the development of marked strains for olive fly using polyubiquitin-regulated fluorescent proteins in transformation vectors that can be stabilized for strain stability and ecological safety. This is the first successful effort to establish transgenic strains for an important agricultural pest in Turkey. |
