Nanoparticle-bound, Aerosolized siRNA Knockdown in Aphids

Mary (Chaffee) Grantham is a graduate student in the laboratory of Dr. Jennifer Brisson in the Department of Biology at the University of Rochester. Her research is focused on the molecular basis of wing polyphenism in aphids.
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Thairu et al. (2017) report in Insect Molecular Biology their use of aerosolized siRNAs bound to nanoparticles to silence genes in the pea aphid, Acyrthosiphon pisum, while circumventing  the problem of rapid dsRNA degradation in the hemolymph of this species.

While whole genome sequencing has become common, a major obstacle for the field has been functionally validating gene annotations. Thus far, most annotations have relied on bioinformatic approaches using ab initio gene predictions and homology. Theoretically, RNA interference (RNAi) is a promising method to address this issue by knocking down gene expression in any eukaryote with short interfering RNAs (siRNAs). Experimentally, successful gene knockdown with RNAi methods have been limited to specific taxa and by inherent characteristics of the target gene, tissue target, and delivery method.

Aphids are a group that has proven difficult to use RNAi methods for gene knockdown, even though their RNAi pathway is conserved. In the pea aphid, Acyrthosiphon pisum, dsRNA has been shown to be quickly degraded in the hemolymph, resulting in inefficient gene knockdown (Christiaens et al. 2014).  Nanoparticles are reported to stabilize siRNAs and increase their uptake by cells (Li-Byarlay et al. 2013).

To test the stability of the siRNAs in aphids, Thairu et al. (2017) treated A. pisum with dsGFP (to avoid phenotypic effects) by three methods: microinjection; unbound, aerosolized dsGFP; and nanoparticles-bound, aerosolized dsGFP. Unbound, aerosolized siRNAs were significantly degraded in the hemolymph of treated A. pisum; however, when the aerosolized siRNAs were bound to nanoparticles they did not show any degradation over a 24 hour period. Microinjection also did not exhibit significant degradation after 24hrs, in contrast to Christiaens et al. (2014).

Perfluorocarbons or PFCs, are organofluorine compounds with the formula CxFy. Thairu et al. used perfluorcarbon nanoparticles.  Image Credit: Chen J, Pan H, Lanza GM, Wickline SA. Perfluorocarbon Nanoparticles for Physiological and Molecular Imaging and Therapy. Advances in chronic kidney disease. 2013;20(6):466-478. doi:10.1053/j.ackd.2013.08.004.

Thus nanoparticle-bound, aerosolized siRNAs appear to be stable in aphids, but the siRNAs need to access cells for gene knockdown. To test for gene knockdown and phenotypic effects with nanoparticle-bound, aerosolized siRNAs, they tested two genes: carotene dehydrogenase (tor) in A. pisum, and branched-chain amino acid transaminase (bcat) in three aphid species (A. pisum, soybean aphid: Aphis glycines, and greenbug: Schizaphis graminum).

Schizaphis graminum oat usda (cropped).jpg

Schizaphis graminum. Image Credit

The tor gene is a carotenoid gene present in A. pisum causing pink pigmentation and successful knockdown is expected to result in a green-yellow phenotype. Significant knockdown of tor was achieved in A. pisum with a 12% gene knockdown at 100nM nanoparticle-bound siRNA and 8% gene knockdown with 200nM nanoparticle-bound siRNA. Results from siRNAs not bound to nanoparticles and higher concentrations of siRNAs with nanoparticles were not significant from control siRNAs. Unfortunately, no phenotypic differences were observed between control and treated aphids even with significant gene knockdown. However, carotenoid proteins are highly stable, so any gene knockdown may not have any effect on pigment that is already present.

Acyrthosiphon pisum with carotenoid pigmentation. Image Credit

The bcat gene is important in the synthesis of leucine, valine, and isoleucine, which the aphid symbiont, Buchnera does not produce. This gene is present in all three aphid species and successful knockdown is expected to cause a reduction in body mass due to dysregulation of these essential amino acids. In A. glycines, a 30% knockdown of bcat was observed along with a significant reduction in body size with a 200nM concentration of bcat siRNA bound to nanoparticles. A knockdown of 19% was achieved with aerosolized siRNA bcat only, but no phenotypic effects were observed indicating a need for nanoparticles in successful cellular uptake. Regardless of the presence of nanoparticles or siRNA concentration, knockdown of bcat was unsuccessful in A. pisum and S. graminum and no phenotypic effects were observed.

Together this data suggests that the nanoparticles are necessary for cellular uptake and the nanoparticle may also stabilize the siRNA, preventing degradation. Aerosolizing the siRNA as a delivery method is also a non-invasive technique that can be optimized for a high-throughput gene knockdown study with greater reproducibility. Overall, the successful use of RNAi in aphids with aerosolized siRNAs bound to nanoparticles is exciting for future uses of RNAi in non-model organisms.

Thairu MW, Skidmore IH, Bansal R, Nováková E, Hansen TE, Li-Byarlay H, Wickline SA, Hansen AK. 2017. Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species. Insect Mol. Biol. [Internet] 0. Available from: http://doi.wiley.com/10.1111/imb.12301

Christiaens O, Swevers L, Smagghe G. 2014. DsRNA degradation in the pea aphid (Acyrthosiphon pisum) associated with lack of response in RNAi feeding and injection assay. Peptides [Internet] 53:307–314. Available from: http://www.sciencedirect.com/science/article/pii/S0196978113004300

Li-Byarlay H, Li Y, Stroud H, Feng S, Newman TC, Kaneda M, Hou KK, Worley KC, Elsik CG, Wickline SA, et al. 2013. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee. Proc. Natl. Acad. Sci. U. S. A. [Internet]. Available from: http://www.pnas.org/content/early/2013/07/11/1310735110.abstract

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