Using Vital Dyes to Trace Uptake of dsRNA by Green Peach Aphid Allows Effective Assessment of Target Gene Knockdown

Int J Mol Sci. 2017 Jan 3;18(1):80. doi: 10.3390/ijms18010080.

Abstract

RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8). There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs.

Keywords: Myzus persicae; RNA interference (RNAi); acridine orange; double stranded RNA (dsRNA); green peach aphid; neutral red; vacuolar (H+)-ATPase (vha-8); vital dyes.

MeSH terms

  • Acridine Orange / analysis
  • Animal Feed / analysis
  • Animals
  • Aphids / genetics*
  • Coloring Agents / analysis*
  • Gene Knockdown Techniques
  • Genes, Insect*
  • Neutral Red / analysis
  • RNA Interference*
  • RNA, Double-Stranded / administration & dosage*
  • RNA, Double-Stranded / analysis*
  • RNA, Double-Stranded / genetics
  • Tobacco / parasitology

Substances

  • Coloring Agents
  • RNA, Double-Stranded
  • Neutral Red
  • Acridine Orange