An Environmentally-Friendly RNAi Yeast-Attractive Targeted Sugar Bait Turns off the Drosophila suzukii Rbfox1 Gene

Abstract

Spotted wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), are invasive vinegar flies of East Asian origin that are an increasingly global threat to the small fruit industry. It is essential that new classes of eco-friendly insecticides and cost-effective strategies for SWD control are developed. Here, we describe the preparation of a strain of RNA interference (RNAi) Saccharomyces cerevisiae expressing shRNA that specifically targets the SWD RNA-binding Fox protein 1 (Rbfox1) gene. The yeast effectively silences the SWD Rbfox1 gene, resulting in significant loss of fly neural activity. Laboratory trials demonstrated that the RNAi yeast can be mixed with soda, which functions as SWD attractive targeted sugar bait (ATSB) that can be delivered in a soda bottle feeder. The ATSB, mixed with yeast that was heat-killed prior to suspension in the ATSB, resulted in 92 ± 1% mortality of SWD flies that consumed it, yet had no impact on non-target dipterans. Rbfox.687 yeast delivered in ATSB feeders may one day be a useful component of integrated SWD control programs.

Keywords: berry; control; eco-friendly; fruit crop; insect; insecticide; integrated pest management; spotted wing drosophila.

Figure 1

Rbfox.687 yeast functions as the active ingredient in an ATSB that silences the Rbfox1 gene and kills SWD. (A) qRT-PCR confirmed that the SWD Rbfox1 gene was silenced in the brains of adult flies that consumed Rbfox.687 yeast (** = p < 0.01 vs. control, Student’s t-test). (B) Laboratory trials demonstrated that consumption of Rbfox.687 yeast resulted in significant mortality of SWD flies (*** = p < 0.001 vs. control, Student’s t-test). (C) The corresponding survival curve for these data are shown. Panels (B,C) were compiled from nine replicate trials for each treatment, each of which contained 25 adults. The error bars represent the standard deviation (SD) in (A) and the standard error of the mean (SEM) in (B). (D) Dose-dependent mortality was observed in D. suzukii, with an LD₅₀ of 166 µg/µL; the data shown were compiled from 10 replicate trials (each with 25 flies) for each of the nine different concentrations of yeast.

Figure 2

Neural defects are detected in SWD that consumed Rbfox.687 yeast ATSB. Adult brains prepared from flies that consumed control (A1–A3) or Rbfox.687 (B1–B3) yeast were labeled with mAbnc82 (marker for active synapses; white in (A1,B1), green in (A3,B3)), anti-HRP (neural marker; white in (A2,B2), red in (A3,B3)) and TO-PRO (nuclear stain; blue in (A3,B3)). Although nc82 and HRP levels were significantly reduced in Rbfox.687-treated brains (C); *** p < 0.001 vs. control), no significant levels of nuclear staining were detected (C, p > 0.05). The data in panel (C) are shown as average mean gray values with error bars denoting the SEM and were analyzed with Student’s t-test. Representative larval brains are oriented dorsal upward and labeled as follows: AL, larval antennal lobe; OF, esophageal foramen; OL, optic lobe; SOG, sub-esophageal ganglion; and SuEG, supraesophageal ganglion. Scale Bar = 100 μm. N = 75 brains/treatment from three replicate trials.

Figure 3

Rbfox.687 yeast and soda delivered in a soda bottle feeder induced significant SWD mortality. (A) The soda bottle feeder system is shown. (B) SWD feeding on the yeast-soda mixture were observed. (C) Significant mortality was observed in D. suzukii that drank from a soda bottle ATSB feeder prepared with Coca-Cola^R and Rbfox.687 yeast (*** p < 0.001 vs. flies that fed on soda alone or soda prepared with control yeast, ANOVA). (D) The flies died over a six day period. Data compiled from nine replicate trials for each treatment, each with 50 adults are shown in (C,D).