Marc F. SHETLIG, Xavier NIRMALA, Alfred M. HUNDLER, Hanife GENÇ

Germline transformation of the olive fruit fly, Bactrocera oleae(Rossi) (Diptera: Tephritidae), with a piggyBac transposon vector

The olive fruit fly, Bactrocera oleae, is a highly significant pest in olive growing countries, and controlling it may be enhanced by using 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 minimum germline transformation frequency of approximately 1.8% per G0 adult. All transgenic lines expressed DsRed and EGFP, although 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. This is the first study of B. Oleae transformant lines to assess life fitness parameters, including egg hatching, larval survival, larval-to-pupal survival, pupal-to-adult survival, and fertility. In three transgenic lines, survival at all biological stages was similar; overall fitness was significantly lower 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.

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Ant T, Koukidou M, Rempoulakis P, Gong HF, Economopoulos A, Vontas J, Alphey L (2012). Control of the olive fruit fly using genetics-enhanced sterile insect technique. BMC Biol10: 51.Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, Fraser MJ (1989). Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172: 156-169.Cosmidis N, Luokas M, Peppa V, Goulielmos G, Zouros E (2002). Effect of acetone feeding on alcohol dehydrogenase activity in the olive fruit fly, Bactroceraoleae. Heredity 89: 453-459. Condon KC, Condon GC, Dafaalla TH, Forrester OT, Phillips CE, Scaife S, Alphey L (2007). Germ-line transformation of the Mexican fruit fly. Insect Mol Biol16: 573-580.Daane KM, Johnson MW (2010). Olive fruit fly: managing an ancient pest in modern times. Annu Rev Entomol 55: 155-169.Dafaalla TH, Condon GC, Condon KC, Phillips CE, Morrison NI, Jin L, Epton MJ, Fu G, Alphey L (2006). Transposon-free insertions for insect genetic engineering. Nature Biotechnology24: 820-821.Das G, Henning D, Reddy R (1987). Structure, organization, and transcription of Drosophila U6 small nuclear RNA genes. J Biol Chem 262: 1187-1193.Dyck VA, Hendrichs J, Robinson AS (2005). Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management. Dordrecht, the Netherlands: Springer.Economopoulos AP (1980). SIRM against the olive fruit fly: differences between wild and lab-reared (normal or sterilized) insects. In:Coyama J, editor. Proceedings of the XVII International Congress Entomology/Symposium on the Fruit Fly Problems, Naha, Kyoto, Japan, pp. 17-26.Economopoulos AP (2001). The Olive Fly, Bactrocera (Dacus) oleae(Gmelin) (Diptera: Tephritidae): Its Importance and Control; Previous SIT Research and Pilot Testing. Vienna, Austria: International Atomic Energy Agency.Economopoulos AP (2002). The Olive Fruit Fly, Bactrocera (Dacus) oleae (Gmelin) (Diptera: Tephritidae): ItsImportance and Control; Review SIT Research and PilotTesting. Vienna, Austria: International Atomic Energy Agency.Economopoulos AP, Avtzis N, Zervas G, Tsitsipis J, Haniotakis G, Tsiropoulos G, Manoukas A (1977). Control of the olive fly, Dacus oleae (Gmelin), by the combined effects of insecticides and release of gamma sterilized insects. J Appl Entomol 83: 201-215.Enkerlin W, Munford J (1997). Economic evaluation of three alternative methods for control of the Mediterranean fruit fly (Diptera: Tephritidae) in Israel, Palestinian territories, and Jordan. J Econ Entomol 90: 1066-1072.Fraser MJ, Smith GE, Summers MD (1983). Acquisition of host cell DNA sequences by baculoviruses: relationship between host DNA insertions and FP mutants of Autographacalifornica and Galleria mellonella nuclear polyhedrosis viruses. J Virol47: 287-300.Genc H (2008). Modified agar based diet for laboratory rearing of olive fruit fly Bactrocera oleae Gmelin (Diptera: Tephritidae). Florida Entomol 9: 651-656.Genç H (2012). Possible residual effect of pesticide on Bactrocera oleae Rossi (Diptera: Tephritidae) adults. Florida Entomol 95: 777-778.Genc H, Nation JL (2008a). Maintaining of Bactrocera oleae (Gmelin.) (Diptera: Tephritidae) colony onits natural host in the laboratory. J Pest Sci 81:167-174.Genc H, Nation JL (2008b). Survival and development of Bactrocera oleae Gmelin (Diptera: Tephritidae) immature stages at four temperatures in the laboratory. African J Biotech 7: 2495-2500.Gong P, Epton M, Fu G, Scaife S, Hiscox A, Condon K, Condon G, Morrison N, Kelly D, Dafaalla T et al. (2005). A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly. Nat Biotech 23: 453-456.Hagen KS (1966). Dependence of the olive fly Dacus oleae larvae on symbiosis with Pseudomonas savastanoi for the utilization of olive. Nat (Lond) 209: 423-424.Hagen KS, Santas L, Tsecouras A (1963). A technique of culturing the olive fruit fly, Dacus oleae Gmel. on synthetic media under xenic conditions. In: Proceedings of the Symposium on the Use and Application of Radioisotopes and Radiation in the Control of Plant and Animal Insect Pests, Vienna, Austria, pp. 333-356.Handler AM (2000). An introduction to the history and methodology of insect gene transfer. In: Handler AM, James AA, editors.Insect Transgenesis: Methods and Applications. Boca Raton, FL, USA: CRC Press, pp. 3-26.Handler AM (2002a). Prospects for using genetic transformation for improved SIT and new biocontrol methods. Genetica116: 137-149.Handler AM (2002b). Use of the piggyBac transposon for germ-line transformation ofinsects. Insect Biochem Mol Biol32: 1211-1220.
Handler AM, Harrell RA (1999). Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol Biol8: 449-457.Handler AM, Harrell RA (2001a). Polyubiquitin-regulated DsRed marker for transgenic insects. Biotechniques31: 824-828.Handler AM, Harrell RA (2001b). Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP. Insect Biochem Mol Biol31: 199-205.Handler AM, McCombs SD (2000). The piggyBac transposon mediates germ-line transformation in the Oriental fruit fly and closely related elements exist in its genome. Insect Mol Biol9: 605-612.Handler AM, McCombs SD, Fraser MJ, Saul SH (1998). The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly. P Natl Acad SciUSA 95: 7520-7525.Handler AM, OBrochta DA (2012). Transposable elements for insect transformation. In: Gilbert LI, editor. Insect Biochemistry and Molecular Biology. London, UK: Academic Press, pp. 90-133.Handler A, Schetelig M (2014). Tephritid fruit fly transgenesis and applications. In: Benedict MQ, editor. Transgenic Insect: Techniques and Applications. Wallingford, UK: CABI, pp. 117-137.Handler AM, Zimowska GJ, Horn C (2004). Post-integration stabilization of a transposon vector by terminal sequence deletion in Drosophila melanogaster. Nature Biotech 22: 1150-1154.Hendrichs J, Robinson AS, Cayol JP, Enkerlin W (2002). Medfly areawide sterile insect technique programmes for prevention, suppression or eradication: the importance of mating behavior studies. Florida Entomol 85: 1-13.Koukidou M, Klinakis A, Reboulakis C, Zagoraiou L, Tavernarakis N, Livadaras I, Economopoulos A, Savakis C (2006). Germ line transformation of the olive fly Bactrocera oleae using a versatile transgenesis marker. Insect Mol Biol15: 95-103.Knipling E (1955). Possibilities of insect control or eradication through the use of sexually sterile males. J Econ Entomol 48: 459-462.Koyama J, Kakinohana H, Miyatake T (2004). Eradication of the melon fly Bactrocera cucurbitae in Japan: importance of behaviour, ecology, genetics and evolution. Ann Rev Entomol 49: 331-349.Lee HS, Simon JA, Lis JT (1988). Structure and expression of ubiquitin genes of Drosophilamelanogaster. Mol Cell Biol8: 4727-4735.Loukas M, Economopoulos AP, Zouros E, Vergini Y (1985). Genetic changes in artificially reared colonies of the olive fruit fly (Diptera: Tephritidae). Ann Entomol Soc Am 78: 159-165.McCombs SD, Lee SG, Saul SH (1993).Translocation-based genetic sexing system to enhance the sterile insect technique against the melon fly (Diptera: Tephritidae). Ann Entomol Soc Am 86: 651-654. Meza JS, Nirmala X, Zimowska GJ, Zepeda-Cisneros CS, Handler AM (2011). Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens. Genetica 139: 53-62.Morel V, Lepicard S, Rey AN, Parmentier ML, Schaeffer L (2014). DrosophilaNesprin1 controls glutamate receptor density at neuromuscular junctions. Cell Mol Life Sci 71: 3363-3379.Nardi F, Carapelli A, Dallai R, Roderick GK, Frati F (2005). Population structure and colonization history of the olive fly, Bactrocera oleae (Diptera, Tephritidae). Mol Ecol 14: 2729-2738.Nirmala X, Olson SR, Holler TC, Cho KH, Handler AM (2011). A DsRed fluorescent protein marker under polyubiquitin promoter regulation allows visual and amplified gene detection of transgenic Caribbean fruit flies in field traps. BioControl 56: 333-340.Raphael KA, Shearman DC, Streamer K, Morrow JL, Handler AM, Frommer M (2011). Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements. Genetica139: 91-97.Schetelig MF, Caceres C, Zacharopoulou A, Franz G, Wimmer EA (2009a). Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae). BMC Biology7: 4.Schetelig MF, Handler AM (2013a). A functional comparison of the 3xP3 promoter by recombinase-mediated cassette exchange in Drosophila and a tephritid fly, Anastrephasuspensa. Genes Genomes Genetics3: 687-693.Schetelig MF, Handler AM (2013b). Y-linked markers for improved population control of the tephritid fruit fly pest, Anastrepha suspensa. In: Vilcinskas A, editor. Insect Biotechnology. Dordrecht, the Netherlands: Springer, pp. 169-194.Schetelig MF, Scolari F, Handler AM, Kittelmann S, Gasperi G, Wimmer EA (2009b). Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata. P Natl Acad Sci USA106: 18171-18176.Scolari F, Schetelig MF, Bertin S, Malacrida AR, Gasperi G, Wimmer EA (2008). Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae). New Biotech 25: 76-84.Silva GM (1970). Sterile-male technique for control of the olive fly. Review of work on rearing and radiation. In:Sterile-Male Technique for Control of the Fruit Flies. Vienna, Austria: International Atomic Energy Agency, pp. 119-130.Steller H, Pirrotta V (1985). A transposable P vector that confers selectable G418 resistance to Drosophila larvae. EMBO J 4: 167-171.Tsitsipis JA, Kontos A (1983). Improved solid adult diet for the olive fruit fly Dacus oleae. Entomol Hellenica 1: 24-29.Tzanakakis ME (1989). Small scale rearing. In: Robinson AS, Hooper G, editors. Fruit Flies: Their Biology, Natural Enemies and Control, Vol. 3B. Amsterdam, the Netherlands: Elsevier, pp. 105-118.
Tzanakakis ME (1971). Rearing methods for the olive fruit fly Dacus oleae (Gmelin). Annals of the School of Agriculture and Forestry, University of Thessaloniki 14: 293-326.Vontas JG, Hejazi MJ, Hawkes NJ, Cosmidis N, Loukas M, Hemingway J (2002). Resistance-associatedpoint mutations of organophosphate insensitive acetylcholinesterasein the olive fruit fly Bactrocera oleae. InsectMol Biol 11: 329-336.Vontas J, Hernandez-Crespo P, Margaritopoulos JT, Ortego F, Feng HT, Mathiopoulos KD, Hsu JC (2011).Insecticide resistance in tephritid flies. Pestic Biochem Physiol 100: 199-205.Zervas GA, Economopoulos AP (1982). Mating frequency in caged populations of wild and artificially reared (normal or gamma-sterilized) olive fruit flies, Dacus oleae (Gmelin) (Diptera: Tepritidae). Environ Entomol 11: 17-20.Zimowska GJ, Nirmala X, Handler AM (2009). The beta2-tubulin gene from three tephritid fruit fly species and use of its promoter for sperm marking. Insect Biochem Mol Biol 39: 508-515.Weens HV, Nation JL (2003). Olive Fruit Fly, Bactrocera oleae (Gmelin) (Insecta: Diptera: Tephritidae).University of Florida Extension, IFAS, EENY-113Document (Originally Published As: DPI EntomologyCircular No. 44). Gainesville, FL, USA: University of Florida.