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

Abstract

Background: The invasive fly Drosophila suzukii has 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 that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing.

Results: We experienced differences in the usability of piggyBac-based germline transformation in different strains of D. 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 an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer.

Conclusion: Our results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline 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 one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

Keywords: Binary expression system; Enhancer/promoter; Insect transgenesis; Molecular entomology; Pest management; Spotted wing Drosophila; Sterile insect technique.

Fig. 1

D. suzukii serendipity α and the use of its promoter/enhancer for directed expression. a Schematic representation of the D. suzukii gene serendipity α. b piggyBac-based transgenic construct HMMA006 [52] to drive tTA during early embryonic development. c-e Whole mount in situ hybridisation to detect Ds_sry α expression in wildtype D. suzukii embryos. f-h Whole mount in situ hybridisation to detect tTA expression in transgenic D. suzukii embryos of line 06_F5M2 (attP#1) carrying construct HMMA006. c,f Syncytial blastoderm embryos before start of cellularization. d,g Syncytial blastoderm embryos during cellularization show expression of sry α or tTA, respectively. e,h Germ band retracting embryos

Fig. 2

Spermatogenesis-specific driver for binary tet-off expression system. a piggyBac-based transgenic construct HMMA389 to generate a testes-specific driver line carrying the β2t promoter [52] fused to tTA. b-d Whole mount in situ hybridisation to detect gene expression in D. suzukii male reproductive organs. b Testes-specific tTA expression driven by the Ds_β2t promoter in line 389_F25M1. c Ds_β2t expression in wildtype testes detected by an antisense probe. d Negative control using a Ds_β2t sense probe on wildtype testes

Fig. 3

φC31-mediated site-specific integration and RMCE. (A) Scheme for site-specific germline transformation. D. suzukii line 06_F5M2 [52] carries construct HMMA006 that contains an attP recombination target sequence, which - in the presence of a helper plasmid providing φC31 integrase (HMMA098) - is targeted by construct HMMA182 carrying the corresponding attB recombination site to integrate the complete plasmid. The integration leads to a modification of the transgenic insert, which can be used for additional integration of transgenes (light green “?”) as well as transgene stabilization by removing part of the transgenic composition by piggyBac excision [39]. (A′-A‴) Integration can be detected by the addition of the EGFP marker. (B) RMCE to generate diverse transgenes at the same genomic position. D. suzukii line carrying construct HMMA185 is targeted by construct HMMA336 in the presence of a helper plasmid (HMMA098) providing φC31 integrase to exchange marker genes and integrate a specific cargo gene (TRE-Cas9). (B′-B‴) RMCE can be detected by the replacement of the DsRed marker with the EGFP marker. Images of a male fly of each indicated line are taken with cold light (A′,B′), RFP filter (A″,B″), or EYFP filter (A‴,B‴)

Fig. 4

D. suzukii nanos and the use of its promoter/enhancer for directed expression. a Schematic representation of the D. suzukii gene nanos. b Whole mount in situ hybridisation to detect nanos expression in wildtype D. suzukii ovaries. c piggyBac-based transgenic construct HMMA223 to generate φC31 integrase RMCE self-docking lines. RMCE in a self-docking line, which provides both the recombination target sequences attP as well as the φC31 integrase driven by the nanos promoter/enhancer providing maternal expression, will result in marker exchange as well as cargo gene (GOI) integration and removal of the integrase source. d Whole mount in situ hybridisation to detect φC31 integrase expression in transgenic D. suzukii ovaries carrying construct HMMA223. Expression of nanos or φC31 integrase, respectively, is detected in the nurse cells of the ovaries