Second-Generation Sequencing Supply an Effective Way to Screen RNAi Targets in Large Scale for Potential Application in Pest Insect Control

Abstract

The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.

RNA interference: Applications and advances in insect toxicology and insect pest management

“…Much research is currently focusing on uncovering the molecular and physiological mechanisms governing cellular uptake and spread of dsRNA, as well as function of the RNAi machinery [2][3][4][5][82][83][84]. Novel methods for target gene selection [2,85,86], improved dsRNA construct designs [4,8], transfection reagents [4,5,64], and stabilityenhancing technologies such as chitosan/dsRNA nanoparticles [64,79] are being used to enhance RNAi efficiency in many species. Together, these efforts and numerous other toxicology studies utilizing RNAi, many of which are described in this review, are advancing the development of new RNAi-based tools for better management of insect pests.…”

Phenotypic screen for RNAi effects in the codling moth Cydia pomonella

“…We nevertheless cannot preclude the possibility that dsRNAs and siRNAs could also enter into larval tissues via other routes such as across the unsclerotized and untanned cuticle shortly after hatching from the egg or by infiltration of the tracheal system. Consistent with this idea, an earlier study showed that dsRNA can induce an RNAi effect when sprayed onto larvae of another lepidopteran species, the Asian corn borer Ostrinia furnacalis (Guenée) (Wang et al, 2011).…”

Emerging strategies for RNA interference (RNAi) applications in insects

“…76 Microinjection of dsRNAs against actin, chitin synthase 1 and V-ATPase in the citrus mealybug, Planococcus citri, showed that the corresponding mRNAs are good targets for RNAi. 50 In addition to microinjection, RNAi effects have also been achieved by soaking (C. elegans), 77 spraying (Ostrinia furnalalis), 78 and electroporation (Ixodes scapularis) 79 approaches to deliver RNAi inducers. In a recent study, El-Shesheny, et.…”