Dietary risk assessment of v-ATPase A dsRNAs on monarch butterfly larvae

Authors: PAN, HUIPENG - University Of Kentucky; YANG, XIAOWEI - University Of Kentucky; Bidne, Keith; Hellmich Ii, Richard; SIEGFRIED, BLAIR - University Of Nebraska; ZHOU, XUGUO - University Of Kentucky

Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2017
Publication Date: 2/22/2017
Citation: Pan, H., Yang, X., Bidne, K.G., Hellmich II, R.L., Siegfried, B., Zhou, X. 2017. Dietary risk assessment of v-ATPase A dsRNAs on monarch butterfly larvae. Plant Biotechnology Journal. 8:242. https://doi:10.3389/fpls.2017.00242.

Interpretive Summary: RNA interference (RNAi)-based genetically engineered plants targeting insect pests have been developed and offer a new mode of action to complement existing pest control practices. By suppressing the expression of genes with essential biological functions, this transgenic plant can negatively affect the survival and fitness of the targeted pests. The goal of this study is to assess the risks of RNAi transgenic crops on a non-target arthropod, monarch butterfly, Danaus plexippus. We hypothesize that an insecticidal double-stranded (ds) RNA targeting western corn rootworm, Diabrotica virgifera virgifera, has no adverse impacts on D. plexippus. Following a tiered approach, we tested this risk hypothesis using a newly developed dietary RNAi toxicity assay. For RNAi toxicity assay, newly hatch neonates were provisioned with leaf disks surface-coated with dsRNAs derived from D. v. virgifera and D. plexippus, respectively, a control dsRNA generated from a plant transcript, and H2O. On average, each neonate larva ingested approximately 16 µg of dsRNA during the assay, which is equivalent to an exposure of 1,600 times higher than the LC50 reported for D. v. virgifera larvae. The combined results from both temporal RNAi effect study and dietary RNAi toxicity assay support the risk hypothesis, suggesting the negligible impacts of ingested dsRNA on D. plexippus. This work will be useful to all scientists interested in genetically engineered plants that target insect pests.

Technical Abstract: The goal of this study is to assess the risks of RNA interference (RNAi)-based genetically engineered crops on a non-target arthropod, monarch butterfly, Danaus plexippus. We hypothesize that an insecticidal double-stranded (ds) RNA targeting western corn rootworm, Diabrotica virgifera virgifera, has no adverse impacts on D. plexippus. Following a tiered approach, we tested this risk hypothesis using a newly developed dietary RNAi toxicity assay. To create the worst case scenario, full-length v-ATPase A cDNAs from D. v. virgifera and D. plexippus were obtained and a 400 bp fragment with the highest sequence similarity was selected as the template to synthesize dsRNAs. For RNAi toxicity assay, newly hatch neonates were provisioned with leaf disks surface-coated with v-ATPase A dsRNAs derived from D. v. virgifera and D. plexippus, respectively, a control dsRNA generated from a plant transcript, and H2O. On average, each neonate larva ingested approximately 16 µg of dsRNA during the assay, which is equivalent to an exposure of 1,600 times higher than the LC50 reported for D. v. virgifera larvae. The combined results from both temporal RNAi effect study and dietary RNAi toxicity assay support the risk hypothesis, suggesting the negligible impacts of ingested dsRNA on D. plexippus. This study provides a road map for future investigations on the risk assessment of RNAi crops. As a sequence-specific gene silencing technology that has a wide range of pest control potentials, additional surrogate species representing diverse ecosystem services should be tested in a similar fashion before moving RNAi-based gene silencing technology from the bench top to the table top.