Biotechnological approaches in entomology


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Biotechnological approaches in entomology

  1. 1. Role of Biotechnological Approaches in Entomological Research Speaker : Kamaldeep Singh (A-2010-40-01)
  3. 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. 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. 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.
  6. 6. Central Dogma DNA mRNA Protein
  7. 7. Gene transfer in plants Microprojectile bombardment
  8. 8. Gene transfer in Insect Transposon have left and right terminal inverted repeats (TIR). Most employed transposon: piggy-bac. Stable transformation with high frequency.
  9. 9. 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.
  10. 10. 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
  11. 11. 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
  12. 12. 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)
  13. 13. 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
  14. 14. 26 MARCH 2002 Govt. of India approved Mahyco’s Bt-cotton to control bollworms India’s first transgenic crop 15
  15. 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)
  16. 16. 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)
  17. 17. 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.
  18. 18. 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
  19. 19. 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
  20. 20. Behaviour modifying chemicals (BMC) in crop protection • Alter the behaviour of the insect. • It includes pheromone, allomone and Kairomone. • Second generation GM crop.
  21. 21. 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.
  22. 22. 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.
  23. 23. 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)
  24. 24. 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)
  25. 25. 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.
  26. 26. 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)
  27. 27. 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)
  28. 28. 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)
  29. 29. 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)
  30. 30. 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.
  31. 31. 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)
  32. 32. 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
  33. 33. 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
  34. 34. 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)
  35. 35. 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)
  36. 36. 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)
  37. 37. 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
  38. 38. Miscellaneous Insect-Plant Interaction Insect-Pathogen Interaction Insecticide Research Genetic Diversity Genetic Map Insect Behaviour Study
  39. 39. 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
  40. 40. Insect-Pathogen Interaction Mapping of quantitative trait loci (QTL). Species would transmit dengue-2 virus by Aedes aegypti. Bosio et al. 2000
  41. 41. 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
  42. 42. 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
  43. 43. 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
  44. 44. Insect Behaviour  Stinging behaviour.  Body size.  Pheromone alarm level.  Hygienic behaviour.
  45. 45. Limitation of Biotechnological approaches Mirid bug out break Lu et al. 2010
  46. 46. 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
  47. 47. 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.
  48. 48. 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