RNA interference: Process and Application to Pest Control

Abstract

Innovation is one of the keys to ensure effective insect pest control in crop protection. A much discussed novelty to insect pest management is represented by RNA interference (RNAi). Employing this conserved virus defense mechanism and diverting it to turn against its own host resulting in insect death has sparked research in many insect pests with regards to susceptibility, suitable target genes and delivery strategies. Finding new lethal genes universal to insect pest management would reduce the work invested in researching putative target genes and simplify the adjustment of dsRNA sequences to target adaptable pest species spectra. A large-scale RNAi screen in Tribolium castaneum identified eleven highly lethal target genes that could serve this purpose. The next step was the transfer to other pest species. To this end, the herbivorous mustard leaf beetle Phaedon cochleariae was selected in this study. The transcriptome of P. cochleariae revealed nine orthologs to the highly lethal genes described in T. castaneum and key players of the RNAi mechanism. Employing a similar procedure as the T. castaneum screen, injection of dsRNA induced high levels of mortality confirming that these target genes can be successfully used for the control of other pest species. However, injection is not a viable option for pest control. Therefore, an experimental set-up as close as possible to a spraying application while still maintaining a small-scale screening procedure was established to test the efficacy of oral RNAi. In this set-up, P. cochleariae could serve as a screening model to test sprayable RNAi applications. Five target genes induced high mortality rates accompanied by feeding cessation and stunted growth across three tested dsRNA concentrations demonstrating a good transfer rate of 56% of highly lethal targets. Previously proposed reference genes for P. cochleariae were confirmed for use in RT-qPCR across developmental stages, facilitating expression data analysis for this and future studies. Employing these reference genes, target gene knockdown was observed for most dsRNAs inducing high mortality rates upon dsRNA injection and feeding confirming the gene specificity of this pest control strategy. In summary, this study supports and validates the value of the highly lethal target genes identified in the T. castaneum screen for the application in pest management. Apart from target gene efficacy, the success of RNAi as an insect pest control measure depends on a uniform RNAi response within a species. Very few studies so far questioned whether different natural populations of a species vary in their response towards dsRNA. Instead, many studies rely on a single laboratory population. This work expands the knowledge on RNAi response variability by comparing fourteen populations of the Colorado potato beetle Leptinotarsa decemlineata collected in nine European countries. Spray application of dsRNA targeting the actin gene in a diagnostic dose based on a dose-response experiment in a German inbred strain was used for comparison of all populations. Overall, only minor variability between European populations was observed in terms of mortality, target gene conservation, growth and developmental retardation. These results denote RNAi as a suitable control measure for this species. Nevertheless, the RNAi responses were marked by significant differences in their temporal onset dependent on the population. Additionally, one Spanish population stood out by its relative tolerance to the oral dsRNA treatment. Neither observation was explained by actin nucleic acid sequence divergence, its knockdown or the differential expression of the target gene or molecular participants of the RNAi response between populations. Therefore, integrated pest management should take these data into consideration for the implementation of the RNAi technology as an insect control strategy. Lastly, strong correlation of gene expression was found between two RNAi core machinery genes, dicer2 and argonaute2a, and the recently identified factor of the RNAi response, staufenC. Evidently, research on the underlying mechanism of RNAi in insects is not complete yet which provides incentive for further basic research.