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. Minimum estimated germ-line transformation frequencies were approximately 13–21% per fertile G0 individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. 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 integrated into the Chiapas wild type 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 5–10% 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 A. suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles 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.
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Acknowledgments
Grateful appreciation is extended to the International Atomic Energy Agency (IAEA) for funding though the Technical Co-operation Project MEX/5/027. Additional support was provided by the USDA-NIFA-Agriculture and Food Research Initiative (AMH) and the Campaña Moscas de la Fruta (Mexican Fruit Fly Campaign) DGSV-SAGARPA.
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10709_2010_9484_MOESM2_ESM.doc
Supplementary Figure 1 PCR product sequences for the 5’ and 3’ insertion sites for the pB[L1-EGFP-L2-DsRed-R1] vector in genomic DNA from the 11M3 green/red strain and the L1-EGFP sequence stabilized by post-integration deletion of the L2-DsRed-R1 sub-vector in the 11M3 green strain. PCR products were extracted from indicated lanes (see Fig. 2 and Table 3) and sequenced from subclones using either M13 or internal primers. (A) shows the same sequence for the 5’ insertion site for both the 11M3 green/red (lane 1) and green (lane 5) strains using the 534F and 122R primers; (B) shows the 3’ insertion site sequence for the 11M3 green/red strain using primers 144F and 515R; and (C) shows the 3’ insertion site sequence for the 11M3 green strain (lane 9) using primers 387F and 515R after deletion of the L2-DsRed-R1 sub-vector. Primers sites are indicated by arrows below or above, the TTAA duplicated insertion site is double-underlined, piggyBac terminal sequences are in bold (A, B), and EGFP/SV40 sequences are single-underlined (C)
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Meza, J.S., Nirmala, X., Zimowska, G.J. et al. Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens . Genetica 139, 53–62 (2011). https://doi.org/10.1007/s10709-010-9484-6
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DOI: https://doi.org/10.1007/s10709-010-9484-6