Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Summary

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Abstract

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

Introduction

Hemipteran insects comprise some of the most economically significant pests of agriculturebecause of their ability to attain elevated population growth and spread diseases in plants. The BMSB, H. halys Stål, is an invasive pest that was accidentally introduced in the Western hemisphere in Allentown, Pennsylvania from Asia (China, Taiwan, Korea, and Japan) with the first sighting reported in 1996¹. Since its introduction, BMSB has been detected in 43 states, with the highest populations in the Mid-Atlantic (DE, MD, PA, NJ, VA, and WV), as well as in Canada and Europe, and represents a potential threat to agriculture

Protocol

1. BMSB Rearing

1. Rear BMSB insects as per standard lab practice and previously described⁶³.
2. Raise ACP (D. citri) insects on Citrus macrophylla in a glasshouse (22 °C) and natural light. Use adult ACP, at approximately 5-7 days post eclosion.

2. Selection of Gene Regions and In Vitro Synthesis of dsRNA

1. Select genes specific to BMSB from previously published transcriptome profiles³².
2. Ensure the regions of interest selected vary between 200 to 500 base pairs.
3. Perform polymerase chain reaction (PCR) using t....

Results

Vegetable mediated dsRNA delivery through feeding in BMSB 4^th instar nymphs was tested for the development of molecular biopesticides using RNAi for invasive insect pests. BMSBs feed using their needle-like stylets by a mechanism known as lacerate and flush, which causes considerable damage to crops. Slender organic green beans, P. vulgaris L., were used to test if nutrients or dsRNA could be delivered in vivo to BMSB through feeding³. .......

Discussion

RNAi has proven to be an important tool for exploring gene biological function and regulation, with great potential to be utilized for management of insect pests^{19,68,69,70,71}. The design and selection of an appropriate gene(s) for silencing in a given insect species and the method of delivery of the corresponding dsRNA(s) to the insect are both of utmost impo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BMSB (H. halys) insects	USDA
ACP (D. citri) insects	USDA
organic green beans	N/A
Citrus plants	USDA
sodium hypochlorite solution	J.T. Baker
green food coloring	McCormick & Co., Inc
Thermo Forma chambers	Thermo Fisher Scientific
Magenta vessel (Culture)	Sigma
Primers	IDT DNA
SensiMix SYBR	Bioline
qPCR ABI 7500	Applied Biosystems
Spray bottle	N/A
Parafilm	American Can Company
TaKaRa Ex Taq	Clontech
QIAquick	Qiagen