Methods for Delivery of dsRNAs for Agricultural Pest Control: The Case of Lepidopteran Pests

Abstract

RNA interference (RNAi) is a natural mechanism of gene regulation, highly conserved in eukaryotes. Since the elucidation of the gene silencing mechanism, RNAi became an important tool used in insect reverse genetics. The demonstration of effective target-gene silencing by ingestion of double-stranded RNA (dsRNA) produced by transgenic plants indicated the RNAi potential to be used in insect pest management, particularly in agriculture. However, the efficiency of gene silencing by RNAi in insects may vary according to the target taxa, and lepidopteran species have been shown to be quite recalcitrant to RNAi. Developing transgenic plants is a time-consuming and labor-intensive process, so alternative oral delivery systems are required to develop and optimize RNAi settings, such as selecting an efficient target gene, and dsRNA design, length, and stability, among other features. We have developed delivery systems to evaluate dsRNAs to silence genes from two important lepidopteran crop pests of tomato (Solanum lycopersicum) and sugarcane (Saccharum × officinarum): Tuta absoluta (Meyrick), the South American Tomato Pinworm, and Diatraea saccharalis (Fabricius), the Sugarcane Borer, respectively. The protocol described here can be used in similar species and includes (a) direct oral delivery by droplets containing dsRNA; (b) oral delivery by tomato leaflets that absorbed dsRNA solution; (c) delivery by Escherichia coli expressing dsRNA; and (d) delivery by transgenic plants expressing dsRNA.