Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP

Germ-line transformation was achieved in the Caribbean fruit fly, Anastreph a suspensa, using a piggyBac Vector marked with an enhanced green fluoresce nt protein gene regulated by the Drosophila melanogaster polyubiquitin prom oter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP e xpression. Southern hybridization indicated the presence of one to four int egrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was de tectable throughout development, and in adults was most intense from the th oracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric ass ay was developed for GFP fluorescence that indicated differing levels of fl uorescence among the transgenic lines, suggesting some level of position ef fect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is pre sented that has the potential to be universally applicable in insect specie s. Published by Elsevier Science Ltd.