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

Vineeta Bilgi,Michael Jones,John Fosu-Nyarko

doi:10.3390/ijms18010080

Vineeta Bilgi, Michael Jones + Show 1 more

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https://doi.org/10.3390/ijms18010080

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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.

Highlights

Insect pests cause direct and indirect damage to crops and are responsible for millions of dollars of losses in crop production worldwide [1]
Based on these criteria eleven inexpensive dyes were tested for their suitability in the study: these were two fluorescent dyes: fluorescein isothiocyanate (FITC; Sigma Aldrich 46952, Castle Hill, NSW, Australia), and fluorescein diacetate (FDA; Sigma Aldrich F7378, Castle Hill, NSW, Australia), six vital dyes: phloxine B (PB; Sigma Aldrich P2759, Castle Hill, NSW, Australia), neutral red (NR; VWR International, Brisbane, QLD, Australia), congo red (CR; VWR International, Brisbane, QLD, Australia), methylene blue (MB; VWR International, Brisbane, QLD, Australia), acridine orange (AO; VWR International, Brisbane, QLD, Australia) and fast green (FG; VWR International, Brisbane, QLD, Australia), one non-vital dye, acid fuchsin (Sigma Aldrich F8129, Castle Hill, NSW, Australia), and two food colours: Red (RFC) and Yellow (YFC) (Queen Fine Foods, Alderley, QLD, Australia)
We examined the suitability of inexpensive vital dyes as an addition to double-stranded RNA (dsRNA) mixtures orally delivered to aphids during in vitro RNA interference (RNAi) to study gene function

Summary

Introduction

Insect pests cause direct and indirect damage to crops and are responsible for millions of dollars of losses in crop production worldwide [1]. The green peach aphid (GPA), Myzus persicae (Sulzer), is a polyphagous insect which feeds on plants from over 40 different families including Brassicaceae and Solanaceae and is a vector for more than 100 viruses [1,3]. These characteristics, combined with its cyclical parthenogenesis, telescoping of generations and recent outbreaks of resistance to chemical insecticides, make GPA a successful pest [4,5,6]. RNA interference (RNAi) is used widely as a tool to study gene function and is being explored as an alternative strategy for crop protection against several different plant pathogens and pests including nematodes and some insects [7,8,9,10]

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