1. Role of Biotechnological Approaches in
Entomological Research
Speaker : Kamaldeep Singh
(A-2010-40-01)

2. INTRODUCTION

3.  The
world population will increase to 7.5 billion by 2020.
 Out
of which 97% living in developing countries.
 Nearly
30-50% crop yield lost due to ravages of Insect-Pest
and Diseases.
 Biotechnology
may help to increase resistance to Insect-pest
and diagnosis of their natural enemies.

4. Biotechnology
The use of biological means to develop processes and
products by studying organisms and their components.
Biological means
Bioreactors
Immunolocalisation
Gene transfer
Recombinant DNA technology (rDNA)
RNA interference (RNAi)
DNA fingerprinting

5. Biotechnological approaches
 Development of transgenic insecticidal crops through rDNA
technology.
 Genetic modification of insects and biocontol agents
 DNA fingerprinting of insects to study insect population
structure, distinguish biotypes, monitor genetic changes in
the insect population and spread of insecticidal resistance.

7. Central Dogma
DNA
mRNA
Protein

8. Gene transfer in plants
Microprojectile bombardment

9. Gene transfer in Insect
Transposon have left and right terminal inverted repeats (TIR).
Most employed transposon: piggy-bac.
Stable transformation with high frequency.

10. Genetic engineering of Plants for Insect
Resistance
 Cry toxin Bt: Cry1Ab, Cry1Ac, Cry2a, Cry9c, Cry2B, Vip I,
VipII etc.
 Plant metabolites: Flavonoids, aklaloids, terpenoids.
 Enzyme inhibitors: SbTi, CpTi.
 Enzymes: Chitinase, Lipoxigenase.
 Plant lectins: GNA.
 Toxin from predators: Scorpion, spiders.
 Insect hormones: Neuropeptides and peptidic hormones.

11. Bacillus thuringiensis (Bt)
 Common soil bacterium.
 Present in nature in a variety of forms (species &
strains).
 Produces proteins that are toxic to insects.
 Commonly
commercial
farming.
used
in
garden
agriculture,
sprays
including
&
for
organic

12. Crystal protein of Bacillus thuringiensis and their
Crystal protein of specificitythuringiensis and
Bacillus
their specificity
Crystal proteins
Order(s) specific
Cry-I
Lepidoptera
Cry-II
Lepidoptera & Diptera
Cry-III
Coleoptera
Cry-IV
Diptera
Cry-V
Lepidoptera & Coleoptera

13. Crystal protein of Bacillus thuringiensis gene for
Transgenic plants expressing foreignand their
specificity
insect resistance
Crop
Foreign gene
Origin of gene
Target Insect Pest (s)
Cotton
Cry1Ab,
Cry1Ac,
Cry2Ab
Bacillus thuringiensis
Helicoverpa zea (Boddie)
Spodoptera exigua (Hubner)
Trichoplusia ni (Hubner)
Brinjal
CryIIIb
B. thuringiensis
Leptinotarsa decemlineata (say)
Maize
Cry1Ab
B. thuringiensis
Ostrinia nubilalis (Hubner)
Rice
Corn cystatin
(cc)
Corn
Sitophilus zeamais (Motschulsky)
Pin 2
Potato
Chilo suppressalis (Walker)
CpTi
Cowpea
C. suppressalis
Cry1Ab
B. thuringiensis
C. suppressalis, Cnaphalocrosis
medinalis (Guenee), Scirpophaga
incertulas (Walker)

14. Contd..
Crystal
Crop
Potato
protein of Bacillus thuringiensis and their
Foreign gene specificity
Origin of gene
Target Insect Pest (s)
Cry1Ab
B. thuringiensis
Phthorimaea operculella
(Zeller)
Oryza cystatin 1 (oc1)
Rice
L. decemlineata
Sugarcane
Cry1Ab
B. thuringiensis
Diatraea sachharalis
(Fabricius)
Tobacco
Cry1Ab
B. thuringiensis
Heliothis virescens
(Fabricius)
α-ai
Pea
Tenebrio molitor
(Linnaeus)
CpTi
Cowpea
H. virescens, Manduca
sexta (L.)
Cry1Ac
B. thuringiensis
M.Sexta
B.t. (k)
B.thuringiensis
H.zea, M.sexta, Keifera
lycopersicella
(Walsingham)
Tomato

15. 26 MARCH 2002
Govt. of India approved Mahyco’s
Bt-cotton
to control bollworms
India’s first transgenic crop
15

16. Response of Helicoverpa armigera (Hübner) larvae on
different genetically engineered cotton hybrids

NCEH 6 (Fusion Bt: cry1Ac+cry1Ab), JK 1947 (cry1Ac Modified), NCS
913 (cry1Ac) and RCH 134 (cry1Ac) against Helicoverpa armigera.

Mortality was more on dual toxin as compare to modified cry1Ac and
alone cry1Ac genotypes.

Maximum mortality was observed on leaves, squares of hybrid NCEH 6
at 90 days old plant followed by 120 and 150 days old plants.

However in case of bolls maximum mortality was observed on 120 days
old plant.
Matharu and Singh (2009)

17. Corrected mortality of S. litura neonates (%) on
Corrected mortality of Spodoptera litura (one-day-old larvae)
different plant parts
on different plant parts in BGII cotton genotypes
100
RCH 134 BG II
80
MRC 7031
60
MRC 7017
40
Tulsi 4
20
Ankur Jassi
RCH 134 BG
0
Leaves
Squares
Bolls
(Saini 2009)

18. Bt Brinjal
 Mahyco (Mumbai), TNAU (Coimbatore), IVRI
(Varanasi), UAS (Dharwad), IARI (New delhi) and
Sungro Seeds Ltd. (New delhi).
 cry1Aa, cry1Ac.
 Recommended for commercialization by GEAC in Oct,
2009.
 70% less incidence for BSFB.
 42% less incidence for others insects.

19. Impact of rDNA Technology
•
•
•
•
•
Direct exposure of pest species to toxins
Reduced environmental contamination by
pesticides
Reduced operative exposure to pesticides
Effective pest control throughout the plant
Compatible
with natural enemies and
pesticides in IPM programmes

20. Some resistance genes against Nilaparvata lugens (Stal)
Gene
Source
Marker
Reference
Bph9
Kaharamana pokki
RFLP and RAPD
Murata et al. 2001
Bph13
Oryza eichingeri derived
line acc 105159
SSR and RFLP
Lui et al. 2001
Qbp1 (Bph14)
B5 (O. officinalis)
Linkage analysis
Quantitative trait loci (QTL)
analysis; RFLP
Huang et al. 2001
Qbp2 (Bph15)
B5 (O. officinalis)
Linkage analysis
Quantitative trait loci (QTL)
analysis; RFLP
Huang et al. 2001
Bph12 (t)
B14 (O. latifolia)
SSR and RFLP
Yang et al. 2002
Bph13 (t)
IR 54745-2-21-12-17-6
RAPD
Renganayaki et al.
2002
Bph18 (t)
O. australi derived line IR
65482-7-216-1-2
SSR and STS
Jena et al. 2005
Bph19 (t)
Indica cv AS 20-1
SSR, STS & CAPS
Chen et al. 2006

21. Behaviour modifying chemicals (BMC) in
crop protection
• Alter the behaviour of the insect.
• It includes pheromone, allomone and Kairomone.
• Second generation GM crop.

22. Second Generation GM Crops
 Use an alarm pheromone, (E)-β-farnesene.
 Aphids produce chemicals to alert other.
 Also attracts the natural enemies of aphids, eg. ladybirds.

23. Genetic engineering of Insects
 Genetic engineering can be achieved rapidly, without rearing
several generation.
 Gene from any species can be used for genetic improvement.
 Desirable characters:
Cold Hardiness.
Pesticide resistance.

24. Genetic engineering of Predator and
Parasitoids
 Transgenic strain of Metaseilus occidentalis Predator of
spider mite
 Maternal microinjection
 Transgenic strain can be used routinely in applied pest
management programme.
(Hoy 2000)

25. Genetically modified Trichogramma sp
Gene
Source
Against
Parathion hyrdolase gene
Pseudomonas diminuta
& Flavobacterium
Organophosphate
Acetylcholine estrase gene
Drosophila melanogaster
& Anopheles strephansi
Organophosphate
Esterase B1 gene
Culex sp.
Organophosphate
Rechcigl and Rechcigl (2000)

26. Genetic engineering of Biocontrol
agents (fungi)
Limiting factors:
 Solar
UV radiation
 Temperature
 Humidity
Molecular techniques:
1)
Identified and characterized genes involved in infection.
2)
Manipulated the genes of the pathogen to improve biocontrol performance.

27. Role of tryrosinase gene in UV Resistance &
Virulence
 Yellowish pigment: UV resistance.
 tryrosinase gene inserted into Beauveria bassiana
which increase UV radiation.
 Virulence of the transgenic isolate increases against
the Tenebrio molitor
(Shang 2011)

28. Recombinant fungal pathogens
 Gene encoding: cuticle-degrading protease Pr1
inserted into the
genome of the Metarhizium
anisopliae.
 Virulence of recombinant pathogen increases
 The resultant strain showed a 25 per cent mean
reduced survival times (LT50) toward the Manduca
sexta.
(Leger 2010)

29. Genetic engineering of Nematode
o Susceptibility to environmental stress
o Temperature extremes
o Solar radiation and desiccation
Gene
Source
Inserted
Hsp70A
Caenorhabd Heterorhabditis
itis elegans bacteriophora
HP88
C. elegans
effect
90 per cent transformed
nematode survive exposure
to 40º C
H. bacteriophora Heat tolerant
Rechcigl and Rechcigl (2000)

30. Recently reported toxins from bacteria
• Photorhabdus luminescens, contain a toxin effective
against Cockroaches and boll weevils.
• Bacteria of Yersinia genus encodes homologues of
insect toxin.
• Photorhabdus, Xenorhabdus and Serratia entomophila
contain toxin complexes.
• Y.
enterocolitica
8081
genes
involved
in
insect
pathgenicity, secreate lipases and protesases.
(Sikka 2008)

31. Viruses
 Through Genetic engineering foreign genes encoding
insect
specific
toxins
or
hormones
or
enzymes
incorporated.
 Reduce the time to kill the pest and less feeding
damage.

32. Genetic engineering of Baculoviruses
Gene
Source
Effect
BeIT
Scorpion
Neurotoxin and effect
feeding
HD73
Bacillus thuringiensis kurstaki
Feeding deterrent
JHE gene
Heliothis virescence
Cessation feeding
VEF gene
Trichoplusia ni
10 fold reduction in LD50
(Kaushik 2008)

33. Role of Cecropin gene for disease resistance in
Honey bees

Cecropin
genes
coding
for
proteins

That
have
bactericidal
very
and
strong
fungicidal
effects.

AFB
Resistant to American foul brood
(AFB) and European foul brood
(EFB
EFB

34. Role of rDNA technology for disease
resistance in Apis cerana
 Thai sac brood is a virus disease of Apis cerana
 Gene in A. mellifera which conferred resistance to this sac
brood virus.
Humberto FB et al. 2009

35. Application in Sericulture
 Ecdysteroid UDP-glucosyltransferase
(EGT) gene :silkworm, Bombyx mori.
 Egt gene from B. mori
nucleopolyhedrovirus (BmNPV), and a
green fluorescent protein gene (gfp)
 The vector was transferred into silkworm
eggs by sperm-mediated gene transfer.
 EGT suppressed transgenic silkworm
molting, and arrest of metamorphosis
from pupae to moths.
(Zhang 2012)

36. Application in study of Phylogenetic
Relationship
o Using a combination of
o Nuclear (28S ) and
o Mitochondrial (12S, 16S, ND1, and CO1)
o Etc.
o It can be used to study phylogenetic relations among different
genera and species.
(Smith 2008)

37. Biodiversity of fruit flies
• Eight species of fruit flies: mtCOI gene
• Genes of Bactrocra nigrofemoralis, Dacus
longicornis and D. sphaeroidalis totally new to
gene bank, NCBI.
• Genetic diversity of B.cucurbitae and B.tau is
low
(Prabhakar 2011)

38. RNA interferance
 fru gene expressed in adult locust
 Expression sites: testes, brain and accessory glands
 fru specific RNAi injected into 3rd and 4th instar
 Effects:
Lower cumulative copulation frequency
Less tested weight, less egg pod from female
Less fertilized eggs.
Boerjan et al. 2011

39. Miscellaneous
Insect-Plant
Interaction
Insect-Pathogen
Interaction
Insecticide
Research
Genetic Diversity
Genetic Map
Insect Behaviour
Study

40. Insect-Plant Interaction
 Sitobion avenae feed on Different host Grasses and Cereals.
 RAPD band pattern correlate with host adaptation.
Lushai et al. 2002
 Bemisia tabaci genotype holding specificity to specific host plant.
Gupta et al. 2010

41. Insect-Pathogen Interaction
Mapping of quantitative trait loci (QTL).
Species would transmit dengue-2 virus
by Aedes aegypti.
Bosio et al. 2000

42. Contd..
Host specificity of
white fly
No. of whitefly individuals
showing amplification of CLCuV
DNA
CLCuV acquesition efficiency (%)
Cotton
10
100
Potato
6
60
Tomato
2
20
Soybean
8
80
Brinjal
4
40
Sida Sp
6
60
Gupta et al. 2010

43. Insecticide Research
 Mapping of insecticide resistance genes in insect.
 RAPD genetic loci have been mapped in lesser grain
borer (Rhyzopertha dominica).
 High level resistance to phosphine.
Schlipalius et al. 2002

44. Prey-predator relationship
• Trialeurodes vaporariorum and Helicoverpa armigera.
• Found in gut of Dicyphus tamaninii.
• Better understanding of prey-predator-parasite trophic
interaction.
Agusti et al. 2000

45. Insect Behaviour
 Stinging behaviour.
 Body size.
 Pheromone alarm level.
 Hygienic behaviour.

46. Limitation of Biotechnological approaches
Mirid bug out break
Lu et al. 2010

47. Risk associated with Biotechnological
approaches
 Human and Animal Health: Toxicity, food quality, allergenicity.
 Risk for Agriculture: Loss of biodiversity, alternation in
nutritional level, development of resistance.
 Risk for environment: Persistence of gene, unpredictable
gene expression, impact on non target organisms.
 Risk for horizontal transfer: Interaction among different
genetically modified organisms, genetic pollution through
pollen or seed dispersal, transfer of gene to microorganism

48. Conclusion
 Biotechnological approaches play important role in
insect-pest management.
 The efficacy of bio-control agents
can be increased
through rDNA technology.
 DNA barcoding can help in quick and accurate
identification.
 DNA fingerprinting helps for identification of biotypes and
genetic changes in Insect-pest.

49. Future prospects
• The impact of genetically modified organism must be
assessed on the ground level, taking into account the
ecological input of different organisms.
• Benefits of pesticide reductions need to be examined
• Acceptance of work demonstrating negative impacts has
been poor and need to be well inferred