KANERIA PRASHANT BHARATBHAI Ph. D. Scholar Junagadh Agriculture University

2. MANAGEMENT OF INSECT PESTS BY RNAi – A NEW TOOL FOR CROP PROTECTION.
ENT 691
SEMINAR PRESENTATION
ON
Major Guide
Dr. M. F. ACHARYA
PROFESSOR & HEAD
DEPARTMENT OF ENTOMOLOGY
JUNAGADH AGRICULTURAL UNIVERSITY
JUNAGADH.
Minor Guide
Dr. L. F. AKBARI
PROFESSOR & HEAD
DEPARTMENT OF PLANT PATHOLOGY
JUNAGADH AGRICULTURAL UNIVERSITY
JUNAGADH.
Speaker
Mr. PRASHANT B. KANERIA
Ph. D. (Agri.) Agril. Entomology
DEPARTMENT OF ENTOMOLOGY
JUNAGADH AGRICULTURAL UNIVERSITY
JUNAGADH.

3. 3
 Introduction
 RNA interference (RNAi)
 Mechanism of RNA interference (RNAi)
 Diagrammatic representation of RNAi Mechanism
 Cellular mechanism of RNAi pathway
 Methodology of dsRNA uptake in insects
 Possible methods for mass-production of dsRNA for pest control
 The basic levels of RNAi from an insect control
 Case Studies
 Conclusion

4. INTRODUCTI

5.  Agriculture is the backbone of Indian economy. In India around 70% of the
population earns its livelihood from agriculture. In India, the crop losses due
to insect pests have declined from 23.3 % in post-Green Revolution era to
17.5 % at present.
 Chemical pesticides are still the major approach for controlling insect pests,
but they are associated with significant hazards to the environment and
human health.
 The alternative commercial biotechnological system relies mostly on the
expression of Bt (Cry toxins). Its effectiveness is threatened by the
development of resistance in some species insects.
 As a result, there is an urgent need to develop economically and ecologically
sound alternatives for pest control.
 Gene silencing has been suggested as one of the new alternatives to reduce
damage from insect pests

6. RNA interference
(RNAi)

7.  RNA interference (RNAi) refers to the ability of double-stranded RNAs to
shut down the expression of a messenger RNA with which they have
sequence in common.
 RNA interference (RNAi) is a biological process in
which RNA molecules inhibit gene expression or translation, by neutralizing
targeted mRNA molecules.
 Historically, it was known by other names, including co-suppression, post-
transcriptional gene silencing (PTGS), and quelling.
 Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology
or Medicine for their work on RNA interference in
the nematode worm Caenorhabditis elegans, which they published in 1998.
Since the discovery of RNAi and its regulatory potentials, it has become
evident that RNAi has immense potential in suppression of desired genes.

8. 8

9.  RNAi is an important and natural anti defense mechanism.
 Two types of small ribonucleic acid (RNA) molecules – micro
RNA (miRNA) and small interfering RNA (siRNA) – are central
to RNA interference. RNAs are the direct products of genes, and
these small RNAs can bind to other specific messenger
RNA (mRNA) molecules and either increase or decrease their
activity.
 Dicer is one of the enzymes involved in RNAi mechanism that is
encoded by a variable number of genes and presents distinct
specificity among organisms
 The most recognized RNAi pathways are the siRNA and
miRNA; despite being triggered by different molecules, both
precursors are long double-stranded RNAs (dsRNAs).

10.  An siRNA-containing effector complex is referred to as an “RNA-induced
silencing complex” (RISC), and an miRNA-containing effector complex is
referred to as an miRNP(microinterferanceribonucleoprotein).
 In these complexes, the regulation is at a post transcriptional level and every
RISC or miRNP contains a member of the Argonaute (Ago) protein family .
For the regulation at the transcriptional level as guided siRNAs, a
specialized nuclear Argonaute-containing complex, known as the RNA-
Induced Transcriptional Silencing complex (RITS) mediates gene silencing
 In general, one strand of the short-RNA duplex (the guide strand) is loaded
onto an Argonaute protein at the core of the effectors complexes.

14. 1. siRNAs
Endogenous dsRNA initiates RNAi by activating
the ribonuclease protein Dicer.
Produce double-stranded fragments of 20–25 base
pairs with a 2-nucleotide overhang at the 3' end.
These short double-stranded fragments are called
small interfering RNAs (siRNAs).
These siRNAs are then separated into single
strands and integrated into an active RNA induced
silencing complex (RISC).
After integration into the RISC, siRNAs base-pair
to their target mRNA and induce cleavage of the
mRNA, thereby preventing it from being used as a
translation template.

16. 2. MicroRNAs
 MicroRNAs (miRNAs) are genomically encoded non-coding RNAs
that help in regulating gene expression, particularly during
development.
 A miRNA is expressed from a much longer RNA-coding gene as a
primary transcript known as a pri-miRNA which is processed, in the cell
nucleus, to a 70-nucleotide stem-loop structure called a pre-miRNA by
the microprocessor complex.
 The dsRNA portion of this pre-miRNA is bound and cleaved by Dicer
to produce the mature miRNA molecule that can be integrated into the
RISC complex

18. Methodology of dsRNA
uptake in insects

19. 1. Microinjection: Microinjection, i.e. the direct injection of dsRNA into
the body of insects.
2. Soaking: dsRNA solution can inhibit gene expression, and its
effectiveness is comparable to the injection method in that it requires a
higher concentration of dsRNA.
3. Feeding of artificial diet: dsRNA feeding is the most attractive
primarily because it is convenient and easy to manipulate.
4. Developing transgenic insects: The using transgenic insects that carry
the dsRNA is that as it is inheritable, the expression can be stable and
continuous. The technique has been proposed to help either reduce
population through introduction of sterile insects or for population
replacement.
5. Virus-mediated uptake: Virus-mediated RNAi methods involve the
infection of the host with viruses carrying dsRNA formed during viral
replication and targeting the gene of interest in the host.

21. The left panel: Feeding habits of the target insect is important in planning the (delivery) strategy.
The middle panel: Illustrates the dsRNA path/uptake by the microvilli of the columnar cells (MCC) in the insect
midgut, as well as its environmental and systemic properties.
The right panel: Shows the cellular siRNA mechanism of gene silencing.

22. https://www.youtube.com/watch?v=p8KhRhqYnkY

23. Case Studies

24. F1 plants expressing a V-ATPase A dsRNA
are protected from Western Corn Rootworm
feeding damage.
(a) Map of the expression cassette.
(b) Mean root damage ratings for eight F1
populations, the parental inbred line
(negative control) and the corn rootworm–
protected Cry3Bb event MON863; NIS,
nodal injury score (Iowa State ranking
system).
(c) The plant on left is a non-transgenic
control with average root damage, whereas
the plant on the right shows the average
root protection seen when the transgene is
expressed.
Belgium Baum et al., (2007)

25. CYP6AE14 gene is highly
expressed in the midgut of the
H. armigera and its expression
correlates with larval growth
when gossypol is included in the
diet. When larvae are fed plant
material expressing double-
stranded RNA (dsRNA) specific
to CYP6AE14, levels of this
transcript in the midgut decrease
and larval growth is retarded.
Mao et al., (2007)China

26. Chemically synthesized siRNA
molecules were directly fed to
H. armigera larvae along with
the artificial diet. The siRNA
treatment resulted in specific
gene silencing of AChE and
consequently brought about
mortality, growth inhibition of
larvae, reduction in the pupal
weight, malformation and
drastically reduced fecundity as
compared to control larvae.
India Kumar et al., (2009)
siRNA-mediated inhibition of
larval growth in a dose-dependent
manner 1.control, 2. unrelated
siRNA, 3. 25 nM AChE siRNA,
4. 37.5 nM AChE-siRNA and 5.
50 nM AChE-siRNA.

27. M. persicae when fed on the transgenic plants for different time intervals under controlled
growth conditions resulted in a significant attenuation of the expression of MySP and a
commensurate decline in gut protease activity. Although the survivability of these aphids was
not affected, there was a noticeable decline in their fecundity resulting in a significant
reduction in parthenogenetic population.
India Bhatia et al., (2012)

28. Quantitative Real-time PCR was used to confirm silencing and detected that the transcript
levels of P. xylostella AChE2 (PxAChE2) were reduced by 90 % mortality compared to the
control group. Finally, effects of the siRNAs on treated plants of Brassica oleracea and
Brassica alboglabra were investigated with different siRNA doses. Our results showed that
Si-ace2_001 had no negative effects on plant morphology, color and growth of vein under
experimental conditions.
China Gong et al., (2013)

29. The results found that Planococcus citri showed lower fecundity and pronounced death of
crawlers after feeding on recombinant TMV-infected plants. Taken together, our data show
that actin, chitin synthase 1 and V-ATPase mRNAs are potential targets for RNAi
against P. citri, and that recombinant TMV is an effective tool for evaluating candidate
RNAi effectors in plants.
China Khan et al., (2013)

30. To manage the pest population of Spodoptera exigua knockdown of eight genes
including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and
arf1 caused a significantly high level of mortality compared to the negative control.
Li et al., (2013)China

31. Last larval injection of the EYFP dsRNA results in a reduction of EYFP, expression or lack there of
in the wing primordial and eyes. (E-H) Penultimate larval RNAi for vg. (E) Wild-type
pupa. (F) vg RNAi pupa. The lack of wing structures is already visible at the pupal stage
(arrow). G) Wild-type adult. (H) vg RNAi adult. Wing related structures are completely
USA David et al., (2014)

32. RNA interference response against cathepsin-L gene in the pea aphid, Molting or gut
phenotypes specifically induced by injection
Phenotypes morphological external defects (on the right), is compared with a healthy
injected aphid (on the left). The arrows indicate the regions in the aphid body where the
defects are the most evident. S: sick aphid, m: melanization point.
France Panagiotis et al., (2014)

33. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in
Citrus Psyllid: Diaphorina citri
I. Images of D. citri adults developed from nymphs after exposure to Citrus tristeza virus wild
type (CTV) CTV-wt
II. Abnormal wing disc gene (tAwd) expressing CTV vector (CTV-tAwd )
USA Subhas et al., (2014)

34. Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and
knock down the V-ATPase mRNA in whitefly. Molecular analysis and insecticidal properties of the
transgenic plants established the formation of siRNA targeting the whitefly V-ATPase, in the leaves. The
transcript level of V-ATPase in whiteflies was reduced up to 62% after feeding on the transgenic plants.
India Thakur et al., (2014)

35. The dsRNA expression was driven by T7
RNA polymerase over expressed by an
inducer in the transformed E. coli. The
transformed bacteria gave a significant
oral toxicity to S. exigua larvae with a
significant reduction of the SeINT
expression. The larvae treated with the
transformed bacteria suffered tissue
damage in the midgut epithelium, which
exhibited a marked loss of cell-cell
contacts and underwent a remarkable
cell death. Moreover, these treated larvae
became significantly susceptible to a
Cry toxin.
South Korea Kim et al., (2015)

36. Chitinase (HaCHI) gene, critically
required for insect molting and
metamorphosis was selected as a
potential target. Continuous feeding on
leaves of RNAi lines drastically reduced
the target gene transcripts and
consequently, affected the overall growth
and survival of H. armigera. Various
developmental deformities were also
manifested in H. armigera larvae after
feeding on the leaves of RNAi lines.
These results demonstrated the role of
chitinase in insect development and
potential of HI-RNAi for effective
management of H. armigera.
Mamta et al., (2015)India

37. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes
(Vacuolar ATPase-A and Arginine kinase) showed 60% reduction in target
gene transcript accumulation, an increase in larval mortality and less leaf
damage.
Brazil Camargo et al., (2016)

38. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their
homologous double stranded RNAs in plants has great potential for management of whiteflies
to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta
transient expression of double stranded RNA (dsRNA) homologous to the acetyl
cholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in approx.
90 % significant adult whitefly mortality.
Malik et al., (2016)Pakistan

39. Image of pink bollworm larvae were injected with V-ATPase specific-dsRNA (dsRNA); “i” dead larvae and
“ii” retardation of larval development. Control larvae were injected with buffer showing normal
development of control larvae
Mohammed et al., (2016)Egypt

40. The potential of RNAi is Rhynchophorus
ferrugineus has been investigated through the silencing of
three different genes (α-amylase, V-ATPase, Ecdysone
receptor). Results show that RNAi mediated gene silencing
in R. ferrugineus varies from gene to gene, and that the
response is dose-dependent, with stronger effects when
dsRNA was administered by injection.
Italy Laudani et al. (2017)

41. Feeding double-stranded RNA effectively silenced TcCHIT transcription in Tetranychus
cinnabarinus. Meanwhile, TcCHIT silencing in larvae and adult resulted in an extremely high
mortality rate (53.3%), respectively, compared with those in the control group.
China Zhou et al. (2017)

42. Leaf disc feeding assays in two spotted spider mite revealed that knockdown in the
expression genes coding for proteins involved in the biosynthesis and action of juvenile
hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor β,
farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35–56% mortality.
China Yoon et al. (2018)

43. Chitin is a vital part of the insect
exoskeleton and peritrophic
membrane, synthesized by chitin
synthase enzymes (CHS). Results
revealed that the expression level of
CHS1 gene significantly decreased
after the oral delivery of dsRNA-
CHS1. The knockdown of CHS1 gene
caused up to 43%, 47%, and 59%
mortality in third-instar nymph of A.
gossypii after feeding of dsCHS1 for
24, 48, and 72 h, respectively, as
compared to the control.
China Ullah et al. (2019)

44. In this study, Scientists tested the expression of dsRNA in RNaseIII-deficient Escherichia coli HT115 which was then
fed to Plagiodera versicolora larvae, an insect pest of Salicaceae plants worldwide. By targeting six potential genes,
including (ACT), (SRP54), (HSC70), (SHI), (CACT), and (SNAP), we found that feeding bacteria-expressed dsRNA
successfully triggered the silencing of the five target genes tested and the suppression of ACT and SRP54 genes
caused significant mortality.
China Zhang et al. (2019)

45. Double-stranded RNA targeting these
two dsRNases, AAEL008858 and
AAEL004103 when fed to the larvae,
effectively reduced gut dsRNease
activity. When these dsRNases-
specific dsRNAs were co-delivered
with dsRNA targeting a cyan
fluorescent protein (CFP) reporter
gene, greater knockdown of CFP
fluorescence was observed. These
results suggest that inhibiting
dsRNases activity could enable the
implementation of RNAi-based
mosquito control methods.
Canada Giesbrecht et al. (2020)

46. The salivary gland transcript dsAkC002 and the gap gene hunchback
dsAkhbd, which resulted in aphid mortality when knocked down by injected
targeted siRNA or feeding-based dsRNA, respectively to control blue green
aphid.
Jacques et al. (2020)Australia

47.  RNAi has great potential to contribute toward development of modern pest
management methods
 RNAi, the sequence-specific suppression of gene expression, offers great
opportunities for insect science, especially to analyse gene function, to
manage pest populations
 The success rate of RNAi could be enhanced by selecting a biologically
crucial target gene and refining dsRNA molecule and its delivery method.
 Further, the development of transplastomic in order to express high amount of
dsRNA opens the new avenue for effective controlling of agricultural pests.
 Supply of dsRNA solution through irrigation water, root drench, or trunk
injection would be a great strategy for pest control method and low
environmental risks.