Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

Abstract

AbstractBackgroundThe invasive flyDrosophila suzukiihas become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown thatD. suzukiiis amenable to transgenesis using the transposon-based vectorspiggyBacandMinos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing.ResultsWe experienced differences in the usability ofpiggyBac-based germline transformation in different strains ofD. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used anattPlanding site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with twoattPsequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions ofDs nanosto express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer ofDs serendipity αto drive the heterologoustetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenousbeta-2-tubulin(β2t) promoter/enhancer.ConclusionOur results provide evidence that theD. suzukiistrain AM derived from the French Alps is more suitable forpiggyBacgermline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least oneattPsite in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression systemtet-offto engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.