A transgenic female killing system for the genetic control of Drosophila suzukii

Abstract

The spotted wing Drosophila (Drosophila suzukii) is an invasive pest of soft-skinned fruit crops. It is rapidly transmitted in Europe and North America, causing widespread agricultural losses. Genetic control strategies such as the sterile insect technique (SIT) have been proposed as environment-friendly and species-restricted approaches for this pest. However, females are inefficient agents in SIT programs. Here we report a conditional female-killing (FK) strategy based on the tetracycline-off system. We assembled sixteen genetic constructs for testing in vitro and in vivo. Twenty-four independent transgenic strains of D. suzukii were generated and tested for female-specific lethality. The strongest FK effect in the absence of tetracycline was achieved by the construct containing D. suzukii nullo promoter for early gene expression, D. suzukii pro-apoptotic gene hid^Ala4 for lethality, and the transformer gene intron from the Mediterranean fruit fly Ceratitis capitata for female-specific splicing. One strain carrying this construct eliminated 100% of the female offspring during embryogenesis and produced only males. However, homozygous females from these FK strains were not viable on a tetracycline-supplemented diet, possibly due to the basal expression of hid^Ala4. Potential improvements to the gene constructs and the use of such FK strains in an SIT program are discussed.

Figure 1

Female-killing genetic constructs and cell culture analysis. Schematic map of the all-in-one (AIO) piggyBac vectors, all of which comprise driver and effector cassettes based on the tetracycline-off system. The AIO constructs contain the female-specific transformer (tra) intron from (a) C. capitata (CctraF), or (b) D. suzukii (DstraF). All constructs harbor the AmCyan marker gene controlled by the constitutive D. melanogaster polyubiquitin (PUb) promoter and an attP recombination site. (c) AsE01 cells were co-transfected with pIE4-EGFP and one of the AIO constructs containing DstraF. The number of cells showing green fluorescence was counted using Image J. The cell survival (%) was calculated by dividing the number of EGFP positive cells from each construct by the number of EGFP positive cells from the control construct (V132). Each bar presents the mean ± SE of n = 3 experiments. Different letters in (c) indicate significant differences at P < 0.05 (one-way ANOVA, Holm-Šidák method).

Figure 2

Determination of the lethal stage in transgenic D. suzukii lines. Homozygous males from lines V229_M4f1 or V229_M44m1 were crossed with wild-type (WT) virgin females on tetracycline-free diet, 500 embryos were collected, and we recorded the numbers of first-instar (L1) and third-instar (L3) larvae, pupae, adult males and adult females. Similar tests were conducted by crossing WT males and females as controls. The survival rate was calculated by dividing the number of flies in the corresponding stage or sex by the number of embryos. Each bar presents the mean ± SE of n = 7 experiments. Different letters indicate significant differences at P < 0.001 (one-way ANOVA, Holm-Šidák method).

Figure 3

The tTA expression level in early embryos (E) and adult females (F) of different transgenic D. suzukii lines determined by quantitative real-time PCR. Homozygous (Ho) flies from lines V185, V215 and V227, and heterozygous (He) flies from lines V188 and V209 were compared. Gene expression was normalized to the reference gene TBP and is presented as a relative quantity based the tTA expression level in the embryos of line V185_M1m1. Data are presented as the mean ± SE from three replicate experiments. Different letters indicate significant differences at P < 0.05 (one-way ANOVA, Holm-Šidák method).