RECENT TRENDS IN INSECT PEST MANAGEMENT

1. 1

2. MINOR GUIDE
Dr. H. J. Kapadiya
Associate Professor
Department of Plant Pathology
College of Agriculture
Junagadh Agricultural University
Junagadh
MAJOR GUIDE
Dr. D. M. Jethva
Associate Research Scientist
Department of Entomology
College of Agriculture
Junagadh Agricultural University
Junagadh
PRESENT STATUS AND ROLE OF BIOTECHNOLOGICAL
APPROACHES IN INSECT PEST MANAGEMENT
SPEAKER
Mr. R. NAGANNA CHENNAIAH
Ph. D (Agricultural Entomology)
II nd Year Ist Semester
Department of Entomology
College of Agriculture
Junagadh Agricultural University
Junagadh

3. 1. Introduction
2. Status of biotech crops
3. Biotechnological approaches in
insect pest management
4. Case studies
5. Conclusion
6. Future thrust
CONTENT

4. ➢ Agriculture is the backbone of Indian economy. In India around 70% of the
population earns their 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.
➢ The Global Report on Food Crises in 2017 revealed that around 108 million
people in 48 food crisis-affected countries are still at risk or in severe acute
food insecurity since 2016.
➢ Chemical pesticides are still the major approach for controlling insect pests,
but they are associated with significant hazards to the environment and human
health.
➢ So, there is an urgent need to develop economically and ecologically sound
alternatives for pest control.
➢ Biotechnology in the context of insect pest management can be defined as the
controlled and deliberate manipulation of biological systems to achieve
efficient insect pest control.
➢ The rapid pace of technology advancement in the field of genetics is giving
rise to approaches for the pest management and biodiversity conservation
4
INTRODUCTION

5. Timeline of Biotechnology in Agriculture
YEAR EVENT
1989 Cry gene classification by Hofte and whiteley
1902 B. thuringiensis was first discovered by Ishiwata from silk worm
1996 Field release of Bt cotton
1915 Bt Berliner isolated from flour moth
2002 Commercial cultivation of Bt cotton in India
2018
Bt-III cotton seeds banned in India and illegally seeds are reported
from Telangana, AP and Gujarat
5

7. 7
Source: ISAAA Briefs, 2017

8. 8
Source: ISAAA Briefs, 2017

9. CONTRIBUTION OF BIOTECH CROPS TO SUSTAINABILITY
Source: ISAAA Briefs, 2017 9

10. 10
Source: ISAAA Briefs, 2017

11. AREA OF INSECT RESISTANT (BT) COTTON IN INDIA
Figure-1: In 2017, the area under IR(Bt) cotton in India increased by 600,000
hectares, from 10.8 million hectares in 2016 to 11.4 million hectares, equivalent to
93% of the total cotton area of 12.24 million hectares grown in the country.
Source: ISAAA Briefs, 2017 11

12. Table-1: STATE WISE AREA OF Bt. COTTON CULTIVATION IN INDIA
STATE 2015-16 2016-17 2017-18
Andhra Pradesh 6.50 4.59 6.41
Telangana 16.61 13.80 18.84
Madhya Pradesh 4.86 5.39 4.82
Gujarat 26.23 20.25 22.49
Maharashtra 34.40 32.35 37.86
Karnataka 4.87 3.03 4.50
Tamil Nadu 0.99 1.08 1.80
Punjab 3.33 2.43 2.86
Haryana 5.27 3.64 6.21
Rajasthan 3.56 2.87 4.96
Total 106.62 89.43 110.75
Source: Press Information Bureau 12
Note: Area in Lakh hectare

13. Present Regulatory Mechanism Of GMO /Crops in India
➢ Genetically modified organisms (GMOs) and crops are regulated
under the Environment (Protection) Act, 1986 and rules notified
under it. The regulatory mechanism to enforce these rules consists
of six committees, which are as follows
1. Genetic Engineering Appraisal Committee (GEAC)
2. Recombinant DNAAdvisory Committee (RDAC)
3. Review Committee on Genetic Manipulation (RCGM)
4. State Biotechnology Coordination Committee (SBCC’s)
5. District Level Committees (DLCs)
6. Institutional Biosafety Committee (IBSC)
13

14. BENEFITS AND CONCERNS OF THE GM CROPS
BENEFITS
✓ Improved resistance to diseases, pests and herbicides
✓ Improved tolerance to cold/heat
✓ Improved tolerance to drought/salinity
✓ Increased nutrients, yields, quality and stress tolerance
✓ Increased food security for growing population
CONCERNS
✓ Potential impact on human health including allergens,
✓ The movement of genes from GM plants into conventional
✓ GM food survive digestive processes and are transferred into the human gut
✓ Transfer of trans genes through cross-pollination effects on loss of flora and fauna
biodiversity.
✓ Domination of world food production by a few companies
✓ Increasing dependence of developing countries on industrialized nations
✓ Biopiracy, or foreign exploitation of natural resources
✓ Violation of natural organisms’ intrinsic values by mixing among species
✓ Objections to consuming animal genes in plants
14

16. 1. Genetic Engineering: Transgenic Plants
2. DNA barcoding
3. Gene Silencing: RNA interference
4. Genome Editing or Genome Engineering
BIOTECHNOLOGICAL APPROACHES IN INSECT
PEST MANAGEMENT
16

17. MOLECULAR MARKERS
➢The precise characterization of insect pests has become an
essential prerequisite for their management.
➢Molecular markers help us to determine the existence of
specific DNA segments/regions/sequences/genes that are either
associated with or control important traits in different pest
species.
➢In general, the molecular analysis or screening involves
investigating whole or part of the nuclear DNA (nDNA) or
mitochondrial DNA (mtDNA).
➢Molecular markers have already been developed and exploited
for rapid and precise identification of both old and newly evolved
strains of diverse insect pest species, genetic diversity and host
specificity in insects, population structure, tri-trophic interactions
and insecticide resistance development.
17

18. IMPORTANT MOLECULAR MARKERS IN
INSECT SCIENCE
✓ Cytochrome C Oxidase Subunit 1 - CO1
✓ NADH Dehydrogenase Subunit 1 - NADH1
✓ Cytochrome b - Cytb
✓ Internal Transcribed Spacers Region-2 - ITS-2
✓ Restriction Fragment Length Polymorphism - RFLP
✓ Randomly Amplified Polymorphic - RAPDs
✓ Sequence Characterized Amplified Regions - SCARs
✓ Sequence Tagged Sites - STS
✓ Expressed Sequence Tags - ESTs,
✓ Simple Sequence Repeats - SSRs,
✓ Single Primer Amplification Reaction - SPAR,
✓ Simple Sequence Length Polymorphism - SSLP,
✓ Inter Sample Sequence Repeats - ISSR
✓ Variable Number Tandem Repeats -VNTR
18

19. Recent Applications of Molecular Markers In Insect Science
➢ Molecular markers developed for rapid and precise
identification of both old and newly evolved strains of
insect pest species, genetic diversity and host specificity in
insects, and insecticide resistance development
➢ Screening of cotton germplasm for resistance to whitefly
transmitted gamin virus
➢ Host associated genetic variations in whitefly and
Trichogramma
➢ Genetic relatedness amongst related species
➢ Prevalence of endosymbiont alpha-proteobacteria
(Rickettsiae) Wolbachia in some agriculturally important
insects
➢ Vectoring efficiency of whitefly for Gemini viruses
➢ Molecular typing of mealy bug Phenococcus solenopsis
populations
19

20. GENETIC ENGINEERING: TRANSGENIC PLANTS
➢ Transgenic plants are those DNA is modified using genetic
engineering techniques. The aim is to introduce a new trait to
the plant which does not occur naturally in the species.
A transgenic plant contains a gene or genes that have been
artificially inserted.
➢ The reliance of a worldwide industry on one insect resistance
trait has led to real concerns about the development of Bt-
resistant insects as field based resistance have already been
documented.
➢ This in turn has led to a search for new insecticidal proteins and
their encoding genes that have commercial potential for plant
protection. They include alpha-amylase inhibitors, vegetative
insecticidal protein, chitinases and protease inhibitors, as
well as several other proteins directed to targets in the insect gut
20

21. Table-2: Biotechnological techniques employed for gene transfer/
alteration in crop improvement programme
Technique Application Examples
Agrobacterium-
based plant
transformation
Ti- plasmid –to carry novel DNA
into plants
Bt insect resistant crop
plants
Particle
acceleration
DNA coated gold particles fired
into growing tissue
Transgenic soybean
Electroporation Electric current used to alter
protoplast membranes permitting
DNA uptake
Transgenic rice
Microinjection DNA injected into the nucleus or
cytoplasm of a protoplast
Transgenic tomato
Gene Silencing/
RNA interference
Blockage of gene function by
inserting short sequences of RNA
Potential for protecting
cotton, rice and maize
against insect pests
21

22. GENERAL PROTOCAL FOR PRODUCTION OF
TRANSGENIC PLANTS
22

23. TRANSGENIC CROPS EXPRESSING INSECTICIDAL GENES
➢Cry toxins Bt: Cry 1 Ab, Cry 1 Ac, Cry IIa, Cry 9c, Cry IIB, Vip I,
and Vip II etc.
➢Plant metabolites : Flavonoids, alkaloids, terpenoid
➢Enzyme inhibitors : SBTi, CpTi
➢Enzymes : Chitinase, Lipoxigenase
➢Plant Lectins : GNA, ACAL, WGA
➢Toxins from predators : Scorpion, Spiders
➢Insect hormones : Neuropeptides and Peptide hormones
Non Bt
Insecticidal
genes
Gene Pyramiding : Gene pyramiding program is to generate an
ideal genotype having all desirable genes brought from various
sources
Ex. Cotton Bollworm Resistance: Cry1Ac and Cry2Ab
23

24. MODE OF ACTION OF Bt ENDOTOXIN
24

25. MODE OF ACTION OF PLANT LECTINS
25

26. Table-3: IMPORTANT TRANSGENE AND THEIR MODE OF ACTION
TRANSGENE MODE OFACTION
Bt endotoxin The pore formation
Vegetative
insecticidal
protein (VIP)
They have similar activity to endotoxins from Bt.
Vip1/Vip2 are toxic to coleopteran insects and Vip3
is toxic to lepidopteran insects
Chitinase
(enzyme)
Chitinase catalyses the hydrolysis of chitin, which
is one of the vital components of the lining of the
digestive tract in insects and is not present in plant
and higher animals.
26

27. Cholesterol
oxidase
(enzyme)
Catalyzes the oxidation of cholesterol and
damaging midgut membranes
Lipoxygenases
(enzyme)
Dioxygenase and catalyse the hydroperoxidation
of cis-cis-pentadiene moieties in unsaturated fatty
acids. Functions by damaging midgut membrans
Alpha-amylase
inhibitors
Alpha-amylase inhibitors block starch digestion.
Trypsin
Modulating
Ostatic Factor
(TMOF)
A peptide that blocks trypsin biosynthesis in
Continue…
27

28. TRANSGENE INSECT
Bt endotoxin Pectinophora gossypiella, Helicoverpa
spp and Spodoptera frugiperda
Vegetative insecticidal
protein
Highly toxic to Agroitis and Spodoptera
species. S. frugiperda, Helicoverpa zea
and Trichoplusia ni
Chitinase (enzyme) Manduca sexta and Plutella maculipenis
Cholesterol oxidase H. virescens, H. zea and Pectinophora
gossypiella
Lipoxygenases (enzyme) Manduca sexta
Alpha-amylase inhibitors Bruchus pisorum
Trypsin Modulating
Ostatic Factor (TMOF)
H. virescens.
28
Table-4: IMPORTANT TRANS GENES AND THEIR TARGET INSECTS

30. ➢ A DNA barcode is a short gene sequence taken from
standardized portions of the genome, used to identify species.
➢ DNA barcoding as a global initiative started by Hebert in
2003. The concept of molecular barcoding - proposed by
Floyd in 2002 and developed into a global biological
identification system that it is possible to rapidly and
accurately identify a species by amplifying and sequencing a
short, standardized region of its genome, specifically the
mitochondrial gene, Cytochrome c Oxidase Subunit 1 (COI)
➢ A DNA barcode depends on DNA and not morphology, it is
applicable to any life stage, from egg to adult.
DNA BARCODING: DEFINITION AND HISTORY
30

31. DNA BARCODING: IT’S APPLICATIONS
➢ DNA barcoding provides an important tool to
improve the quality or speed of floral and faunal
studies
➢ DNA barcode data allows comparison of species
across geographical boundaries
➢ It can be use for species recognition, assessment, and
taxonomic description,
➢ Reconstruction of phylogenetic relationships of
organisms.
➢ Comparing trophic interactions across multiple levels
➢ Identification of immature and dimorphic stages
➢ Studies of population structure, mating systems,
heterozygosity, and individual relatedness.
31

32. DNA barcoding protocol for insect identification
Insect collection and identification
DNA Extraction
COI – Gene specific primer (658 bp)
Forward :5’-TACAATTTATCGCCTAAACTTCAGCC-3’
Reverse: 5’-CATTTCAAGTTGTGTAAGCATC-3’
Polymerase Chain Reaction (PCR) amplification and
Sequencing
https://www.ncbi.nlm.nih.gov
http://www.boldsystems.org/
32

33. 33

34. Figure-1: Estimated total no of insect species and barcoded
insect in the world and India
Total No of Species
INDIA 58977
WORLD 861466
SPECIES BARCODED
INDIS 2008
WORLD 106236
Rakshit and Sushil (2014)
India 34

35. Figure-2: Total No. of insects resources characterized in India
35
Source: NBAIR

36. Figure-3:TOTAL NO OF BARCODE DEVELOPED IN INDIA
36
Source: NBAIR

37. Table-4: Insect Barcoding fauna of India at Zoological Survey of India
Vikas et al. (2017)
India
Faunal system Order No. of DNA Barcodes
Thrips Thysanoptera 604
Butterflies & Moths Lepidoptera 499
Horse flies & Deer
flies
Diptera 59
Other invertebrates
Hymenoptera,
Coleotera,
Spongillida,
Decapoda etc.
120
37

39. RNA INTERFERENCE (RNAi)
➢ 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,
➢ Since the discovery of RNAi and its regulatory potentials, it has
become evident that RNAi has immense potential in suppression of
desired genes.
39

40. Mechanism of RNA interference (RNAi)
➢ 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 (miRNA)
molecules and either increase or decrease their activity, Both miRNAs
and siRNAs share a common RNase-III processing enzyme, Dicer, and
closely related effector complexes and both can regulate gene expression
at the post transcriptional level.
➢ 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).
40

41. ➢ 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
➢ 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. During loading, the non guide strand is cleaved by an
Argonaute protein and ejected. The Argonaute protein then uses the
guide RNA to associate with target RNAs that contain a perfectly
complementary sequence and then catalyzes the slicing of these
targets, either to be cleaved by RISC, to be blocked for translation in
miRNP or by inducing histone modifications in RITS.
CONT…..
41

42. Diagrammatic representation of RNAi Mechanism
42

43. Methodology of ds RNA uptake in insects
1. Microinjection: 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 ds RNA formed during viral
replication and targeting the gene of interest in the host.
43

44. Methods for delivery of ds RNA
1. Expression of dsRNA in plants
2. Feeding in vitro synthesized dsRNA
3. Production of dsRNA in bacteria
4. Artificial diet with dsRNA
5. dsRNA as a commercial insecticide
44

45. Possible methods for mass-production of ds RNA for pest control
45

46. The basic levels of RNAi from an insect control perspective
Figure-4:The left panel: Feeding habits of the target insect is important in planning the (delivery)
strategy and whether a transformative or non-transformative RNAi-plant protection approach might be
preferred.
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.
46

47. Table-5: LIST OF IMPORTANT PEST CONTROL BY RNAi
Insect pests Mode of delivery Gene target
Diabrotica virgifera
virgifera
Artificial diet and
transgenic plant
a-Tubulin, vacuolar
ATPase subunit A
Phyllotreta striolata Plant tissue Arginine kinase
Leptinotarsa decemlineata Artificial diet Vacuolar ATPase subunit
A and E
Hemipterans
Acyrthosiphon pisum Injection and Feeding Calreticulin, cathepsin-L
Diaphorina citri Injection and Feeding Abnormal wing disc
Rhodnius prolixus Injection Nitrophorins1–4
47

49. ➢ Genome editing, or genome engineering is a type of genetic engineering
in which DNA is inserted, deleted, modified or replaced in the
genome of a living organism
➢ Unlike early genetic engineering techniques that randomly inserts
genetic material into a host genome, genome editing targets the
insertions to site specific locations
➢ The common methods for such editing use engineered nucleases, or
"molecular scissors". These nucleases create site-specific double-
strand breaks (DSBs) at desired locations in the genome. The induced
double-strand breaks are repaired through Non Homologous End
Joining (NHEJ) or Homologous Recombination (HR), resulting in
targeted mutations
➢ As of 2015 four families of engineered nucleases were used:
1. Meganucleases,
2. Zinc Finger Nucleases (ZFNs),
3. Transcription Activator Like Effector-based Nucleases (TALEN),
4. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system
GENOME EDITING OR GENOME ENGINEERING
49

50. APPLICATIONS OF GENOME EDITING
1. Gene editing enable to targeted genome
modifications such as sequence insertion, deletion,
repair and replacement in living cells
2. To study gene functions in plants and animals
3. Targeted gene mutation
4. Gene therapy
5. Creating chromosome rearrangement
6. Endogenous gene labeling
7. Targeted trans gene addition
50

51. ROLE ENGINEERED NUCLEASES IN GENOME EDITING
➢ Engineered nucleases: Common restriction enzymes are effective at
cutting DNA, but generally recoginse and cut at multiplte sites. To
overcome this challenge and create site-specific DSB, three distinct
classes of nucleases have been discovered viz., ZFNs, TALEN),
CRISPR/Cas9 system and meganucleases
➢ ZFNs and TALEN technology is based on a non-specific DNA
cutting catalytic domain, which can then be linked to specific DNA
sequence recognizing peptides such as zinc fingers and transcription
activator-like effectors (TALEs).
➢ The first step to this was to find an endonuclease whose DNA
recognition site and cleaving site were separate from each other, a
situation that is not the most common among restriction enzymes.
➢ Once this its cleaving portion could be separated which would be very
non-specific as it would have no recognition ability. This portion
could then be linked to sequence recognizing peptides that could lead
to very high specificity.
51

52. ➢ 1. Zinc finger motifs:
These occur in several transcription factors. The zinc
ion, found in 8% of all proteins, plays an important role
in the organization of their thire dimensional structure.
In transcription factors, it is most often located at the
protein-DNA interaction sites, where it stabilizes the
motif. The C-terminal part of each finger is responsible
for the specific recognition of the DNA sequence
➢ 2.Transcription activator-like effector nucleases
(TALENs):
These are specific DNA-binding proteins that
feature an array of 33 or 34-amino acid repeats. TALENs
are artificial restriction enzymes designed by fusing the
DNA cutting domain of a nuclease to TALE domains,
which can be tailored to specifically recognize a unique
DNA sequence. These fusion proteins serve as readily
targetable "DNA scissors" for gene editing applications
52

53. ➢ 3. CRISPRs (Clustered Regularly Interspaced Short Palindromic
Repeats):
✓ These are genetic elements that bacteria use as a kind of acquired
immunity to protect against viruses.
✓ They consist of short sequences that originate from viral
genomes and have been incorporated into the bacterial
genome. Cas (CRISPR associated proteins) process these
sequences and cut matching viral DNA sequences. By
introducing plasmids containing Cas genes and specifically
constructed CRISPRs into eukaryotic cells, the eukaryotic
genome can be cut at any desired position.
➢ 4. Meganucleases, found commonly in microbial species, have
the unique property of having very long recognition sequences
(>14bp) thus making them naturally very specific
53

55. ➢ Genome editing relies on the concept of DNA double stranded
break (DSB) repair mechanics.
➢ There are two major pathways that repair DSB;
1. Non Homologous End Joining (NHEJ)
2. Homology Directed Repair (HDR).
➢ NHEJ uses a variety of enzymes to directly join the DNA ends
➢ HDR uses a homologous sequence as a template for regeneration
of missing DNA sequences at the break point.
➢ This can be exploited by creating a vector with the desired genetic
elements within a sequence that is homologous to the flanking
sequences of a DSB. This will result in the desired change being
inserted at the site of the DSB.
MECHANISM OF GENOME EDITING
55

56. 56

57. 57

58. COMPARISON OF RNAi AND CRISP MECHANISM
58

61. Insect resistance of transgenic tobacco expressing an
insect chitinase gene
Figure-5: Feeding damage ten days after infestation with neonate hornworm larvae on plants
expressing (left) or not expressing (right) the chitinase gene. (B) Hornworms growth from plants
expressing (left) or not expressing (right) the chitinase.
Xiongfeei et al. (1998)
USA 61

62. Effect of transgenic maize plants expressing a chitinase gene for
the cotton leaf worm, Spodoptera littoralis
Figure-6. Larvae of corn borer (S. cretica) turned black and got dead when reared on
transgenic maize plants expressing insect chitinase (T) when compared with
wild type maize plant (C).
Osman et al. (2015)
Saudi Arabia. 62

64. Recorded of the new genus Euclea (Lepidoptera: Limacoididae)
by using DNA Barcoding from south India.
Kannan, et al. (2015)
India 64

65. New invasive agricultural pest Fall Armyworm, Spodoptera
frugiperda, identified by DNA Barcoding in Karnataka
Swamy and Sharanabasappa (2018)
India 65

67. The physiological roles of the G Protein Coupled Receptors (GPCRs) in T.
castaneum through RNAi (dsRNA) mediated knockdown in the expression of
genes
Figure-9: The G Protein Coupled Receptors (GPCRs) are integral cell membrane proteins
and play crucial roles in physiological processes including behavior, development and
reproduction: (F). Accumulation of the ommatidia (G). RNAi insects with wings attached to
the ventral side of the abdomen. (H). Split in the dorsal thoracic region. (I). Unexpended
pupal wings. (J) improperly folded wings and unshed exuviae.
Bai et al. (2011)
USA 67

68. Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum
Figure-8: Last larval injection of the EYFP dsRNA results in a reduction of EYFP, EYFP expression or
lack thereof in the wing primordia 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
David (2014)
USA 68

69. RNA interference response against cathepsin-L gene in the pea
aphid, Acyrthosiphon pisum
Figure-10: 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.
Panagiotis (2014)
France 69

70. RNAi-mediated pest control of Asian citrus psyllid, Diaphorina
citri
Figure-11: I. Images of D. citri adults, II. truncated abnormal wing
disc gene (tAwd) expressing
Subhas (2014)
Florida 70

71. RNAi-based silencing of genes encoding the vacuolar-ATPase
subunits a and c in pink bollworm (Pectinophora gossypiella)
Figure-12: 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 (2016)
Egypt 71

72. Diabrotica v. virgifera larval development and phenotypic expression of DvvLac2 silencing. (A) Normal pigmentation in
a recently molted, buffer-injected larvae 24 h post-molt; (B) DvvLac2 dsRNA injected larva 24h post-molt; (C) detail of reduced
cuticular tanning observed on anal plate and (D) head capsule. High quality figures are available onlinea
The efficacy of RNA interference (RNAi) as a method for target-
site screening in Diabrotica virgifera virgifera
Figure-7: A) Normal pigmentation in a recently molted, buffer-injected larvae 24 h
post-molt; (B) DvvLac2 ds RNA injected larva 24h post-molt; (C) detail of reduced
cuticular tanning observed on anal plate and (D) head capsule.
Alves et al. (2017)
USA 72

73. RNAi-mediated control of soybean pod borer
infestations in soybean.
73
China
Figure-13: Phenotypes resulting from the Lac2 feeding RNAi. Lac2 RNAi affected
the pigmentation of the larval head. The larvae were fed 5 ug/ml 750-bp dsRNA of
Gfp,Yellow-y (Yy), laccase-2 (Lac2), and ebony (Eb) gene separately.
Meng et al. (2018)

75. CRISPR/Cas9 mediated knockout of the abdominal-A homeotic
gene in diamondback moth, Plutella xylostella
Figure-14: Phenotypes with abnormal prolegs and malformed segments were
visible in hatched larva
Note: Abdominal-A gene which has an important role in determining the identity
and functionality of abdominal in segments P. xylostella
Yuping et al. (2016)
China 75

76. CRISPR/Cas9-mediated mutagenesis of the white gene
in the tephritid pest, Bactrocera tryoni
Figure-15: Comparison of B. tryoni wild-type eye colour and the
CRISPR-/ Cas-induced white-eye mutant phenotype.
Note: White mutations in B. tryoni change the behavioral, physiological
characters that would impact on its mating capabilities.
Choo et al. (2017)
Australia 76

77. CONCLUSION
➢ Sustainable agriculture could be achieved not only through proper agricultural
practices but also through continuous research and development of new
technologies, particularly agricultural biotechnology,
➢ Biotechnology approaches applied to insects provides ample opportunities for
identification of new genes and analysis of their functions to open a new field
for their exploitation in effective insect pest control through transgenic
expression in target plants
➢ Bt and non Bt Transgenic crops are an additional tool to supplement
conventional pest resistance programs.
➢ DNA barcoding provides an important tool to improve the quality or speed
identification, conservation of the evolutionary processes and preserve
biodiversity
➢ RNAi mediated plants resistance offer several advantages over conventional
bio-engineering crops resistance
➢ Genome editing has a potential to crop protection
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78. ➢ Biotechnological approaches are important tools for crop protection but
their complexity in mechanism will needs to reduce by discovery of new
tools
➢ Development of insect resistant to Bt transgenic plant will leads to the
stuck down the pace of transgenic plant. So. It is urgent need to evaluate
the non Bt transgenic plants
➢ Gene silencing technique provide the effective control of the pest though
gene knockdown, it is necessary to evaluate the mass production and
application methods
➢ Future uses of genetic editing /gene drives further advance by introducing
a mechanism that promotes the inheritance of a particular gene to increase
its prevalence in a population
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FUTURE THRUST