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Role of Biotechnological Approaches in Entomological Research
Speaker : Kamaldeep Singh (A-2010-40-01)
INTRODUCTION
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.
Biotechnology
The use of biological means to develop processes and
products by studying organisms and their components.
Bioreactors
Immunolocalisation
Gene transfer
Recombinant DNA technology (rDNA)
RNA interference (RNAi)
DNA fingerprinting
Biological means
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.
DNA Structure
Central Dogma
DNA
mRNA
Protein
Gene transfer in plants
Microprojectile bombardment
Gene transfer in Insect
Transposon have left and right terminal inverted repeats (TIR).
Most employed transposon: piggy-bac.
Stable transformation with high frequency.
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.
Bacillus thuringiensis (Bt)
Common soil bacterium.
Present in nature in a variety of forms (species &
strains).
Produces proteins that are toxic to insects.
Commonly used in garden sprays & for
commercial agriculture, including organic
farming.
Crystal protein of Bacillus thuringiensis and their specificityCrystal protein of Bacillus thuringiensis and
their specificityCrystal proteins Order(s) specific
Cry-I Lepidoptera
Cry-II Lepidoptera & Diptera
Cry-III Coleoptera
Cry-IV Diptera
Cry-V Lepidoptera & Coleoptera
Crystal protein of Bacillus thuringiensis and their specificity
Transgenic plants expressing foreign gene for 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)
Crystal protein of Bacillus thuringiensis and their specificity
Contd..
Crop Foreign gene Origin of gene Target Insect Pest (s)
Potato 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.)
Tomato Cry1Ac B. thuringiensis M.Sexta
B.t. (k) B.thuringiensis H.zea, M.sexta, Keifera lycopersicella (Walsingham)
26 MARCH 200226 MARCH 2002
Govt. of India approved Mahyco’s
Bt-cottonto control bollworms
India’s first transgenic
crop 15
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)
0
20
40
60
80
100
Leaves Squares Bolls
RCH 134 BG II
MRC 7031
MRC 7017
Tulsi 4
Ankur J assi
RCH 134 BG
Corrected mortality of Spodoptera litura (one-day-old larvae) on different plant parts in BGII cotton genotypes
(Saini 2009)
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.
Bt Brinjal
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
Some resistance genes against Nilaparvata lugens (Stal)
Gene Source Marker ReferenceBph9 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 analysisQuantitative trait loci (QTL) analysis; RFLP
Huang et al. 2001
Qbp2 (Bph15) B5 (O. officinalis) Linkage analysisQuantitative 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
Behaviour modifying chemicals (BMC) in crop protection
• Alter the behaviour of the insect.
• It includes pheromone, allomone and Kairomone.
• Second generation GM crop.
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.
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.
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)
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)
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 bio-
control performance.
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)
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)
Genetic engineering of Nematode
Gene Source Inserted effect
Hsp70A Caenorhabditis elegans
Heterorhabditisbacteriophora
90 per cent transformed nematode survive exposure to 40º C
HP88 C. elegans H. bacteriophora Heat tolerant
o Susceptibility to environmental stress
o Temperature extremes
o Solar radiation and desiccation
Rechcigl and Rechcigl (2000)
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)
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.
Genetic engineering of Baculoviruses
Gene Source EffectBeIT 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)
Role of Cecropin gene for disease resistance in Honey bees
Cecropin genes coding for
proteins
That have very strong bactericidal
and fungicidal effects.
Resistant to American foul brood
(AFB) and European foul brood
(EFB
AFB
EFB
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
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)
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)
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)
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
Miscellaneous
Insect-Plant Interaction
Insect-Pathogen
Interaction
Insecticide Research
Genetic Diversity
Genetic Map
Insect Behaviour
Study
Insect-Plant Interaction
Sitobion avenae feed on Different host Grasses and Cereals.
RAPD band pattern correlate with host adaptation.
Bemisia tabaci genotype holding specificity to specific host plant.
Lushai et al. 2002
Gupta et al. 2010
Insect-Pathogen Interaction
Bosio et al. 2000
Mapping of quantitative trait loci (QTL).
Species would transmit dengue-2 virus by Aedes aegypti.
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
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
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
Insect Behaviour
Stinging behaviour.
Body size.
Pheromone alarm level.
Hygienic behaviour.
Limitation of Biotechnological approaches Mirid bug out break
Lu et al. 2010
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
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.
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