|Meza, J Salvador|
|Zepeda, C Silvia|
|Handler, Alfred - Al|
Submitted to: Genetica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/25/2010
Publication Date: 1/2/2011
Citation: Meza, J., Xavier, N., Zimowska, G.J., Zepeda, C., Handler, A.M. 2011. Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens. Genetica. 139:53-62. Interpretive Summary: The creation of transgenic strains of economically important insects for the development of more effective biological control programs is a major goal of the USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida. This article describes the development of new transformant strains in the Mexican fruit fly, which is a highly significant agricultural pest species in Mexico and the southern United States. Genes were integrated into the mexfly, having potential importance to biological control, the Sterile Insect Technique in particular. The first transformation prevents other new genes from moving again. Gene stability is highly important to minimizing strain breakdown and ecological risks related to unintended movement of the genes to other organisms. The second transformation inserts a fluorescent protein gene that makes sperm fluoresce. Fluorescent sperm were observed in the spermathecae of non-transgenic females mated to transgenic males, allowing the reliable identification of females that have mated to transgenic males. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.
Technical Abstract: The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac¬-based transposon vector system using independent vector and transposase helper plasmids. Estimated germ-line transformation frequencies were approximately 26 to 39% per fertile G0 individual, which is significantly higher than previous reports using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control, the Sterile Insect Technique in particular. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was efficiently integrated into the Chiapas wild type mexfly strain, with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3’ terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5 to 10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the Anastrepha suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.