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