10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.

1. Potential for dsRNA-
based management of
insect pests
Steve Whyard
Department of Biological Sciences
University of Manitoba
Winnipeg, Canada
Presentation to: Regulation of Externally-Applied dsRNA-based products for Management of Pests
Apr 10, 2019

2. The problem: Insect Pests
Pests of food and fiber
Despite using pesticides, ~20% crops lost
Disease vectors cause > 1 million deaths/year

3. The compounded problems: controlling pest insects
Our most commonly used method of insect control:
chemical pesticides
Two main problems:
Increasing incidences of resistance
Off-target effects Sparks & Nauen 2015 Pestic Biochem Physiol 121: 112-128

4. A new ideal: species-limited dsRNA-based pesticides
Species-specific
dsRNAs
fly-specific
mortality
Harnessing the
sequence-specificity
of RNAi
mosquito-specific
mortality
No impacts on beneficial
species

5. Increasing interest in dsRNA-mediated insect control
0
100
200
300
400
500
600
700
1999 2001 2003 2005 2007 2009 2011 2013 2015 2017
Publications
Year
RNAi in insects
RNAi - control of pest insects

6. Overview of RNAi in insects
• DsRNA uptake by insect cells
• SID-like transporters – in a subset of insects
• Endocytosis - widely prevalent
• Specific receptors? Long (> 60 nt) dsRNA >> siRNAs
• DsRNA access to target mRNAs
• Escape of dsRNA from endosomes maybe be limiting in some species
• No apparent amplification of dsRNA in insects
• No endogenous RdRp has been confirmed in insects
• Systemic RNAi
• Exosomes, nanotubes, or other carriers?
dsRNA
endosome
SIL channel
Exosome?
Environmental dsRNA
Relevance: Uptake, release, and export could all affect RNAi efficacy and resistance

7. How can dsRNA pesticides be delivered to insects?
Topically?
 Can a naked dsRNA penetrate an insect cuticle?
 Rare examples cited – entering through spiracles, or through gut
via preening?
Ingestion
 Many examples – once ingested, dsRNA can move systemically
European corn borer
Wang et al 2011 Plos One

8. DsRNA specificity
Con: dsRNA is slow acting
Pro: dsRNA can be species-limited
Ex. Corn rootworm killed by vATPase-A-dsRNA
Baum et al 2007 Nature 25: 1322-26
Transgenic corn
expressing dsRNA
targeting vATPase
are protected from
rootworms
Family
Chrysomelidae
Genus Diabrotica
Lethality
of dsRNA
++++
++++
++
-
Shared
21mers
Many
few
none
Anthonomous grandis
Leptinotarsa decemlineata

9. Lack of shared siRNAs can ensure specificity
Even highly conserved
genes can be targeted if
no shared siRNAs found
Targeted γ-tubulin mRNA
(3’UTR)
But are these targets the
best choices for general
use?
%
mortality
Dm Ds Dy Dp
Larvae fed on
D. melanogaster dsRNA
Dm Ds Dy Dp
Larvae fed on
D. pseudoobscura dsRNA
40
80
Drosophila species
Dm – D. melanogaster Ds – D. sechellia
Dy – D. yakuba Dp – D. pseudoobscura

10. How to ensure dsRNA specificity?
1. Bioinformatics analyses
 Can predict off-target effects
 Bioinformatics screens not enough
 Many off-target species lack genomic data
2. Empirical testing
 10 x highest exposure
 How many species is considered enough?
 How to deliver dsRNA?
 Natural feeding route
 Force-feeding
 Injection
Droplet feeding
starved insects
Hemocoel injection

11. Choice of dsRNA targets
• Resistance factors
• Targeting genes associated with resistance to existing pesticides
DsRNA targeting sodium channel increased
sensitivity to pyrethoids in Aedes aegypti
Bona et al., 2016 Parasit Vectors. 9: 397.
Asian citrus psyllid, Cyp genes
Topically-applied dsRNA increased sensitivity to imidacloprid;
Killiny et al. 2014 PLoS One 9(10): e110536.
Resistant controls 20 % mortality
dsRNA treated 65 % mortality

12. Choice of dsRNA targets - II
• Developmental and/or arthropod genes
• Many insect-specific genes worth considering - will reduce risk of off-target
effects in vertebrates
EcR KD in grain aphids
Yan et al. 2016 Int J Mol Sci. 17(12): 2098.
CHS KD in potato beetles
Shi et al 2016, Int J Biol Sci. 12(11): 1319–1331.
40% reduced
consumption
90% mortality

13. Choice of dsRNA targets - III
Many of the most potent dsRNAs target intracellular trafficking pathways
 ESCRT proteins including Snf7
 vATPases
 COP pathway proteins
 Endocytosis proteins
Velez & Fishilevich, 2018. Pestic Biochem Physiol 151: 25-31
Doubly important to explore these targets:
 Widely applicable?
 Overcome current or future resistance?

14. Flea beetles
• Pest species in Manitoba:
• Phyllotreta cruciferae (crucifer flea beetle)
• Phyllotreta striolata (striped flea beetle)
• Feed on cruciferous plants
• including canola
P. cruciferae P. striolata

15. DEADBUG
Developing RNAi pesticides vs flea beetles
Field collections
Lab colonies
RNA sequencing
Identify flea beetle-specific
gene sequences
DsRNA on
leaf discs
Feeding bioassays
Assess mRNA knockdown
Assess mortality
Assess leaf damage

16. 10 20 30 40 50
> 85% mortality
No effect
control
dsRNA-targeted genes
Identifying ideal RNAi insecticide target genes%survival
100

17. RNAi-based crop protection
Different dsRNA delivery approaches:
• In-planta (transgenic approach)
• Foliar sprays
• Soil dredges / Root delivery
• Stem injections
• Baits / traps
Hairpin dsRNA
GM plant
dsRNA mixed with carriers/adjuvants

18. Transgenic plants
• Cytoplasmic expression of dsRNA
• First EPA-approved insecticidal dsRNA: SmartStax
Pro corn expressing DvSnf7-dsRNA (Bayer-Dow) vs
corn rootworm
• Plastid expression of dsRNA

19. Foliar dsRNA pesticides
• Foliar dsRNAs (in simple formulations) will likely have variable
efficacies, dependent on species’ sensitivities to dsRNA:
• Coleoptera – highly sensitive to dsRNAs
• Diptera - moderately sensitive to dsRNAs
• Lepidoptera
• Hemiptera
Weakly sensitive to dsRNAs
Baum & Roberts, 2014 Adv. Insect Physiol. 47
LC50 1-10 ppb
LC50 10-500 ppm
LC50 >1000 ppm

20. Foliar pesticides: DsRNA persistence on plant leaves
DsRNA is stable on leaves for
> 14 days
Testing new formulations:
• improve dsRNA stability
• leaf adhesion
• Leaf penetration -0hISPG 0h -24hISPG 24h -48hISPG 48h -72hISPG 72h ISPG 7d - 7d ISPG 10d - 10d - 14dISPG 14d
310
438
575
Roche MW1
ladder
BA
310
438
575
Roche MW1
ladder
ISPG
dsRNA
Northern blot analysis: RNA from treated and un-treated leaves
0 h 24 h 48 h 72 h 7 d 10 d 14 d
+ - + - + - + - + - + - + -
Spreaders, stickers, penetrants and other
dispersal formulations could improve dsRNA
efficacy for some pests …. but could influence off-
target or non-specific effects

21. Other dsRNA-based insect control technologies
Sterile insect technique
• Non-radiation, non-GM method of
sterilizing males
• Potential method to sex-sort males from
females
• For SIT breeding factories
Transformed yeast
or
Agar feeding cubes
>90 % sterility
Variable efficacy for sex sorting

22. Limiting factors - I
1. Insect nucleases
• Some species have potent nucleases in the
gut, hemolymph, and/or saliva
• Counter-measures include:
• Chemically-modified, nuclease-resistant
nucleotides
• Cationic lipids/liposomes
• Nanoparticles
• Carbon quantum dots
• Chitosan
• Silica
• Co-delivery of nuclease-specific dsRNA
Asian corn borer
(Guan et al., 2018)
Locusts
Song et al. 2017; Spit et al. 2017
Liposomes - German cockroach
Lin et al. 2016
Nanoparticles – Aedes aegypti
Das et al. 2015
Nuclease protection assay
Neg G RNAi-nuclease

23. Limiting factors - II
2. DsRNA uptake
• Evidence in Lepidopteran insects
that dsRNA can remained entrapped
in endosomal compartment
• Counter-measures to use alternative
receptors or cell-entry pathways
include:
• Alternative dsRNA structures
• Alternative microcarriers
• Alternative dsRNA packaging
Yeast expressing siRNA kill mosquitoes
Hapairai et al. 2017 Sci Reports 7: 13223
Velez & Fishilevich, 2018. Pestic Biochem Physiol 151: 25-31

24. Summary of key points
1. dsRNA pesticides have potential to be species-limited
2. Delivery methods depend on target pest
3. More bioinformatics databases are required
4. More bioassay data are needed
5. Some insects (e.g. beetles) are highly susceptible to dsRNA
– will be the first applications tested in the field
6. More understanding of cellular mechanisms will aid in
development of second generation dsRNA pesticides, which
may include:
• Nanoparticles and other microcarriers
• Nuclease inhibition formulations
• Modified dsRNAs