Ana Trabanino. ENT. [email protected]
Andy Michel. ENT. [email protected]
Yosra Helmy. FAHRP. [email protected] Rajashekara. FAHRP.
[email protected]
CFAES provides research and related educational programs to
clientele on a nondiscriminatory basis. For more information, visit
go.osu.edu/cfaesdiversity.
INTRODUCTION
Fall armyworm, Spodoptera frugiperda (Lepidoptera:Noctuidae), is
considered one of the most detrimental pests of agriculture4, due
to its ability to feed on a broad range of economically important
crops and its adaptive potential to several insecticides2,4.
For the past decades, fall armyworm (FAW) control has heavily
relied on transgenic plants expressing Bacillus thuringiensis (Bt)
traits4. However, there are now existing FAW populations exhibiting
resistance to Bt, which threatens food security
worldwide2,3,4,8.
RNA interference (RNAi) is a post-transcriptional, gene
silencing mechanism that has shown great potential in insect pest
control by targeting biologically important genes that cause insect
mortality13,14. This technique has been successful in many
important insect pests such as mosquitos, aphids, bed bugs and
beetles14. Yet, current approaches in lepidopteran pests remain
ineffective10,13.
The use of nanoparticles has been an emerging tactic to overcome
obstacles in RNAi15. The use of nanoparticles can improve RNAi mode
of action by increasing the bioavailability of siRNA as well as
stabilizing and protecting siRNA from degradation15.
Therefore, the goal of this study is to determine if RNAi can be
achieved in FAW using nanoparticles coupled with siRNA targeting
Vacuolar protein sorting associating protein 4 (VPS4) gene, a key
regulator for vesicle trafficking7.
We hypothesize that gene expression will be significantly lower
or knocked-down on FAW larvae treated with nanoparticles coupled
and the targeted siRNA (VPS4).
METHODS
siRNA and nanoparticle preparationsiRNA targeting the gene of
interest VPS4 “siVPS" (GenBank accession number JN584642.1) and
siRNA used as negative control SCRAMBLE ”siSCRAM” (randomized
sequence sequence with no matches to the target gene) were designed
using ThermoFisher® BLOCK-iT RNAi and synthesized through Sigma
Aldrich®.Three different type of nanoparticles were prepared by the
Rajashekara Laboratory from the Department of Food Animal Health
Research Program (CFAES/OSU, Wooster). All nanoparticle were
prepared with siVPS and siSCRAM separately following established
protocols. Nanoparticles used: perfluorocarbon-based (PFC)5,
chitosan6, and poly-lactic-glycolic acid (PLGA)1. Every solution
had varying siRNA and nanoparticle concentrations depending on
nanoparticle type based on each protocol. Having a total of 6
treatments.
Nanoparticle and siRNA deliveryNanoparticle and siRNA solution
was delivered to 1st instar FAW larvae using a nebulizer
compressor12.For every treatment, 30 FAW larvae were aerosolized
with the respective treatment (nanoparticle and siRNA) solution for
10 minutes. After aerosolizing, every larva was placed in a labeled
individual cup with artificial diet.
Gene expression and statistical analysesFollowing 24 hours after
aerosolization, 8 larvae from every treatment were collected for
RNA isolation and cDNA synthesis. Gene expression was measured
through RT-qPCR.All analyses were performed in R (R Core Team
2019). VPS4 gene expression was normalized using the Delta Ct
method9. Differences in VPS4 gene expression in the three different
nanoparticle type were evaluated using Welch two sample T-Test.
RESULTS CONCLUSIONS
Our results suggest that delivering siRNA and nanoparticle
complex through aerosol spray to FAW, specifically with PLGA
nanoparticle, show potential for RNAi as a pest control strategy in
lepidopteran pests. PLGA nanoparticle was the only treatment that
showed a significant decrease in gene expression. PFC and Chitosan
nanoparticles also showed a slight but not significant decrease,
which may be due to differences in nanoparticle and siRNA
concentrations.
SIGNIFICANCEThis study can be used as baseline for RNAi as an
alternative, species-specific pest management strategy. This
technique can be used as a tactic to mitigate insect resistance and
control pests in an environmental conscious, different mode of
action than common insecticides.
BIBLIOGRAPHY1.Aldayel, A. M., Naguib, Y. W., O’Mary, H. L., Li,
X., Niu, M., Ruwona, T. B., & Cui, Z. (2016). Acid-Sensitive
Sheddable
PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α
siRNA in Chronic Inflammation Sites. Molecular Therapy -Nucleic
Acids, 5(July), e340. https://doi.org/10.1038/mtna.2016.39
2.Bernardi, D., Salmeron, E., Horikoshi, R. J., Bernardi, O.,
Dourado, P. M., Carvalho, R. A., … Omoto, C. (2015).
Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera
frugiperda) May Affect the Durability of Current Pyramided Bt Maize
Hybrids in Brazil. PLOS ONE, 10(10), e0140130.
https://doi.org/10.1371/journal.pone.0140130
3.Carvalho, R. A., Omoto, C., Field, L. M., Williamson, M. S.,
& Bass, C. (2013). Investigating the Molecular Mechanisms of
Organophosphate and Pyrethroid Resistance in the Fall Armyworm
Spodoptera frugiperda. PLoS ONE, 8(4).
https://doi.org/10.1371/journal.pone.0062268
4.Fatoretto, J. C., Michel, A. P., Silva Filho, M. C., &
Silva, N. (2017). Adaptive Potential of Fall Armyworm (Lepidoptera:
Noctuidae) Limits Bt Trait Durability in Brazil. Journal of
Integrated Pest Management, 8(1), 17.
https://doi.org/10.1093/jipm/pmx011
5.Kaneda, M. M., Sasaki, Y., Lanza, G. M., Milbrandt, J., &
Wickline, S. A. (2010). Mechanisms of nucleotide trafficking during
siRNA delivery to endothelial cells using perfluorocarbon
nanoemulsions. Biomaterials, 31(11), 3079–3086.
https://doi.org/10.1016/j.biomaterials.2010.01.006
6.Katas, H., & Alpar, H. O. (2006). Development and
characterisation of chitosan nanoparticles for siRNA delivery.
Journal of Controlled Release, 115(2), 216–225.
https://doi.org/10.1016/j.jconrel.2006.07.021
7.Li, Z., & Blissard, G. W. (2012). Cellular VPS4 Is
Required for Efficient Entry and Egress of Budded Virions of
Autographacalifornica Multiple Nucleopolyhedrovirus. Journal of
Virology, 86(1), 459–472. https://doi.org/10.1128/jvi.06049-11
8.Mamta, B., & Rajam, M. V. (2017). RNAi technology: a new
platform for crop pest control. Physiology and Molecular Biology of
Plants, 23(3), 487–501.
https://doi.org/10.1007/s12298-017-0443-x
9.Schmittgen, T. D., & Livak, K. J. (2008). Analyzing
real-time PCR data by the comparative CT method. Nature Protocols,
3(6), 1101–1108. https://doi.org/10.1038/nprot.2008.73
10.Singh, I. K., Singh, S., Mogilicherla, K., Shukla, J. N.,
& Palli, S. R. (2017). Comparative analysis of double-stranded
RNA degradation and processing in insects. Scientific Reports,
7(1), 17059. https://doi.org/10.1038/s41598-017-17134-2
11.Storer, N. P., Babcock, J. M., Schlenz, M., Meade, T.,
Thompson, G. D., Bing, J. W., & Huckaba, R. M. (2010).
Discovery and Characterization of Field Resistance to Bt Maize:
Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico.
Journal of Economic Entomology, 103(4), 1031–1038.
https://doi.org/10.1603/EC10040
12.Thairu, M. W., Skidmore, I. H., Bansal, R., Nováková, E.,
Hansen, T. E., Li-Byarlay, H., … Hansen, A. K. (2017). Efficacy of
RNA interference knockdown using aerosolized short interfering RNAs
bound to nanoparticles in three diverse aphid species. Insect
Molecular Biology, 26(3), 356–368.
https://doi.org/10.1111/imb.12301
13.Yoon, J.-S., Gurusamy, D., & Palli, S. R. (2017).
Accumulation of dsRNA in endosomes contributes to inefficient RNA
interference in the fall armyworm, Spodoptera frugiperda. Insect
Biochemistry and Molecular Biology, 90, 53–60.
https://doi.org/10.1016/j.ibmb.2017.09.011
14.Zhang, J., Khan, S. A., Heckel, D. G., & Bock, R. (2017).
Next-Generation Insect-Resistant Plants: RNAi-Mediated Crop
Protection. Trends in Biotechnology, 35(9), 871–882.
https://doi.org/10.1016/j.tibtech.2017.04.009
15.Zhou, J., Shum, K. T., Burnett, J. C., & Rossi, J. J.
(2013). Nanoparticle-based delivery of RNAi therapeutics: Progress
and challenges. Pharmaceuticals, 6(1), 85–107.
https://doi.org/10.3390/ph6010085
ACKNOWLEDGEMENTSWe thank the Center of Applied Plant Sciences
(CAPS) for the funding for this project.
Optimizing RNA interference in fall armyworm,
Spodopterafrugiperda using improved nanoparticles
Ana Trabanino1, Yosra A. Helmy2, Gireesh Rajashekara2 & Andy
Michel11The Ohio State University, Department of Entomology 2 The
Ohio State University, Food Animal Health Research Program
DEPARTMENT OF ENTOMOLOGY
Nanoparticles+ siRNA
FAW larvaeMesh
Fig. 2. The technique of “Nanoparticle + siRNA complex” delivery
to the FAW larvae with the nebulizer compressor.
0.010
0.014
0.018
0.022
PFCScram PFCVPSNP
Expressio
n
siSCRAM siVPS
Nor
mal
ized
expr
essi
on
A
Treatments
0.007
0.009
0.011
0.013
Chscram CHVPSNP
Expressio
nsiSCRAM siVPS
Nor
mal
ized
Expr
essi
on
B
TreatmentsN
orm
aliz
ed E
xpre
ssio
n
0.003
0.004
0.005
0.006
PLGAScram PLGAVPSNP
Expressio
n
siSCRAM siVPS
*
Treatments
C
Fig. 3. mRNA expression of VPS gene in FAW larvae aerosolized
with A) PFC nanoparticle (P=0.2738), B) Chitosan nanoparticle
(P=0.3102) and C) PLGA nanoparticle (P=0.004). All samples were
normalized to reference gene RPL11 using Delta Ct method. Samples
per treatment=8. Black points represent mRNA normalized expression
per larva, red point represents the mean of the mRNA expression in
each treatment and red line the standard deviation. NS =not
significant. Asterisk represents treatments significantly
different. Statistical analysis used Welch two sample t-test.
Fig. 1. A) Severe damage of one FAW larva in a corn ear (Picture
by John C. French 2019. B) Corn foliage damage caused by FAW
(Picture by Plantix 2018).
A B
