Resistance: Cotton bollworms and Bt toxins

1. Resistance:Cotton bollworms and Bt toxins Derek Russell Natural Resources Institute (UK) Genetics Dept, University of Melbourne (Australia) With thanks to Dr Keshav Kranthi Central Institute for Cotton Research, Nagpur

2. Resistance • A genetic change in a population as a result of mortality caused by a chemical or other toxin (e.g.Bt) • Those organisms with more natural tolerance to the toxin survive and breed and pass on the ability to survive • In time the whole population becomes able to survive the toxin – they have become resistant!

3. Antibiotic Resistance • You have multiplying bacteria in a cut. • The doctor gives you an antibiotic • If you don’t take the full dose – the most tolerant bacteria survive and breed quickly • If you do take the full dose but only once, it is possible that a few bacteria survive to breed • Therefore you are told to take the full 5 day course at full rates to kill even the tolerant bacteria

4. Killed by Bt cotton Bollworms - cotton bollworm - pink bollworm - spiny bollworm Not affected Leafworms (mostly) Aphids Jassids Mites Pests of cotton in India

5. Bollworms African/American - Helicoverpa armigera Spiny/Spotted - Earias insulana/biplaga Pink - Pectinophora gossypiella Leafworms Cotton leafworms – Spodoptera sp. Main pest groups

6. Bt cotton targets • Cotton bollworm Helicoverpa armigera 181 host plants - 69 crop species losses - US $ 540 mill annually • Spiny bollworms -Earias insulanaandE. vitella • Pink bollworm -Pectinophora gossypiella

8. A key pest of cotton and vegetables: Cotton bollworm – Helicoverpa armigera Photos: CIRAD

9. Commercialised transgenic plants resistant to insects • Cotton:1st commercialised in 1996 • 2004: 9 countries; • 9.0 mill ha • 28% global cotton area • 11% of global GMO area • Insect resistance and herbicide tolerance traits • 25% increase in area from 2003-2004 Potato: first commercialised in 1996, withdrawn in 2001 • Maize:1st commercialised in 1996 • 2004 : 8 countries • 19.3 mill ha • 14% global maize area • 23% of global GMO area • Insect resistance and herbicide tolerance traits • also 25% increase in area from 2003-4 Modified from Giband 2004

10. Genetically Modified Bt plants Bt plants have: • Gene (DNA) for producing the insecticidal toxin from a soil bacterium • Promotor ‘switches’ on either side of the gene to turn it on and off • Other regulatory genes or introns • May have ‘Marker’ gene used only in the selection process (used to be an antibiotic - kanamycin) Note: • The plant itself makes the toxin in its tissues using the bacterial gene • The step to turn the pro-toxin into the toxin in the insect gut is not necessary • The gene may be ‘turned on’ more in some tissues than others.

11. Bacillus thuringiensis (Bt): More than a century of history … • 1901 : Discovered in silkworm by a Japanese • bacteriologist Shigetane ISHIWATA “Sottokin”. • 1911 : A new isolation by Ernst BERLINER on Ephestia • kuehniella (Zeller) larvae from Thuringe (Germany) Bacillus thuringiensis Berliner • 1938 : First commercial preparation (Sporéine) by Libec Laboratories in France, used • against meal moth Ephestia sp. in flour. • : Bt has been used for more than 65 years as biological insecticide (>90% of the • total biopesticides market). • 1981: first cloning of a Cry gene • 1985: First insect resistant transgenic plant • 1990: First commercialization of a transgenic plant (in China : virus resistant tobacco). • 1995: First Bt transgenic plant commercialised in USA • 2005: more than 81 mill ha of transgenic crops all over the world.

12. Mode of action of Cry toxins J-M Vassal - CIRAD

13. Mode of action of Cry toxins Domain III Domain I Domain II Aminopeptidases N Cadherins R. A. de Maagd, 2001

14. Resist Resist Resist (b) Change solubilisation of the toxin(c) prevent passage through the peritrophic membrane (e) Prevent membrane insertion and pore formation Resistance mechanisms for Bt

15. Insects controlled by transgenic plants: Cotton Modified from Giband 2004

16. World Bt cotton adoption – 2003(2004)Countrieswith pest complex like India (esp.Helicoverpa armigera)

17. Bt Cotton in China% of total cotton area

18. GM cotton worldwide in 2005 Area: 28% of global cotton is GM (9.5mill ha) (herbicide tolerant and insecticidal) Countries: 9 countries with 59% of world cotton area Farmers:c 7.2 million (>85% in developing countries – mainly China)

19. Expected yield improvements with Bt crops Why?: Pest losses in non-Bt: USA 12%, China 15% India 60%, Uganda ???? Qaim and Zilberman - Science (299 p 901) 2003

20. Efficacy: Spiny bollworms (Earias sps) Pink Bollworm (Pectinophora gossypiella) Cotton bollworm (Helicoverpa armigera) Insecticide use: Global experience gives an average reduction of c.60% in insecticide applications (80% for bollworms) - Very effective - Very effective - Good mid-season - Poor in late season (reduction in bio-availability of toxin) Target – bollwormsCry1Ac efficacy in current Bt varieties

21. Questions? If toxin expression in Bt plants is not always sufficient to kill bollworms it will select for resistant insects. • Does Bt cotton give season-long control of caterpillars? • Are all the parts of the plant equally lethal? • Are all Bt varieties equally effective? • What is the chance of resistance to Bt developing?

22. H. armigera larval mortality on Bt-cotton plant parts Data from an Indian variety 2003 Kranthi et al 2003

23. Variability in expression of Cry1Ac in plant parts of commercial Indian hybrids Bt Hybrids

24. Variability in expression of Cry1Ac in plant parts of commercial Indian hybrids Expression < 1.8ppm is not sufficient for H.armigera control Bt Hybrids _____________________________1.8ppm__

25. Bt-Quant

26. Bt-Express

27. IS THERE RESISTANCE TO Bt COTTON?

28. Insects resistant to sprayed Bt • 1985 : McGaughey • -1st case of resistance to Bt - in Meal moth Plodia interpunctella (stock grain population) • 1990 : Tabashnick et al. • Diamond back moth Plutella xylostella on cabbagein Hawaï • Firstcase of Bt resistance in the field following intensive Bt treatments. Since then cases of resistance have been ONLY in the lab. Photo - CIRAD

29. Bt resistance in cotton ? We have Bt resistance in the lab by : Heliothis virescens (Tobacco Bollworm) - USA Pectinophora gossypiella (Pink Bollworm) - USA Helicoverpa armigera (Cotton Bollworm) – India, China

30. Helicoverpa armigera : (only in the lab.) Completely recessive Sex influenced • In India : Kalia et Gujar, 2004 • In Australia : Akhurst et al, 2003 • In China : Xu et al, 2005 • In China : Liang et al, 2000 • In India : Kranthi et al. 2000 • In Africa : Uraichuen 2002 Incompletely recessive Autosomal Incompletely dominant Autosomal Completely dominant, Autosomal In all that works : several cross resistances with the Cry1A family toxins

31. In the case of Bt resistance One resistance gene with two alleles S and R SS RS RR 3 genotypes Dominance describe the RS phenotype compare to SS and RR phenotype When RS = SS Resistance is recessive WhenRS = RR Resistance is dominant

32. Implications of the Indian resistance inheritance system • Survival on Mech 184 - Bt plants (75-85 days old) RR two copies of the resistant gene 75% RS one “ “ “ “ “ 33% SS no “ “ “ “ “ 5% • Inheritance -Semi-dominance(0.42 and 0.55 in two populations)

33. Monitoring of Bt resistance • Survey for the presence of the Bt resistance gene • Screen for possible resistant caterpillars in the cotton This will warn us before resistance is a major problem and give us time to do something about it.

34. Is there field resistance to Bt?(field collections of H.armigera from around the country) LC50 µgCry1Ac/ml diet Regional variability India 2001 Baseline* 0.01 - 0.67 67 fold 2002-3 32 fold 2003-4 0.04 - 0.38 10 fold China 1999 Baseline+0.091 - 9.093 100 fold IC50µgCry1Ac/ml diet Regional variability 1998 & 0.015 – 0.20 13 fold 1999 $ 0.016 – 0.099 6 fold 2000 $ 0.016 – 0.080 5 fold Answer - No definite resistance yet - but it will be difficult to detect by screening for increased survivorship. - If the truncated cadherin is the major mechanism in China and India we can now screen directly using molecular tools* *Kranthi et al 2001; +K.Wu et al 1999; * Y Wu et al. 2005; $K Wu et al 2002

35. Resistance gene frequency in the field(using F2 screens on field collected insects) • At least one resistance allele in each group • Frequency is worryingly high for a semi-dominant resistance Data from K.Kranthi – pers com

36. Resistance gene frequency calculated from offspring of field collections - China Resistance Resistant Allele Phenotype Frequency Frequency K.Wu 2002 Hebei 0.001 0.004 (moths at traps) Shandong 0.0006 0.002 Y.Wu 2004 Jiangsu 0.004-0.01 0.003 (Bt field survivors)

37. What is the Bt Resistance Mechanism in H.armigera?All from survivors in Bt fieldsadditionally laboratory selected • Chinese H.armigera – cadherin truncation demonstrated (Wu et al 2004) • Australian H.armigera – unknown binding site mutation (Akhurst et al 2003) • Indian H.armigera – suggestions of an aminopeptidase involvement and possibly a second mechanism (Kranthi et al 2004) • Binding affinity reduced in 6 fold in trypsin activated toxins and 10 fold in protease activated toxins • Major portion of APN-1 cDNA of Cry1Ac resistant strain sequenced • 16 base substitutions, 6 additions in 2766 nucleotides leading to 17 a.a. differences • One of the these is related to glycosylation (Ser911 to Phe911)

38. Cadherin disruption Normal cadherin Truncated cadherin Putative Bt toxin binding site Outside Membrane Inside

39. How can we manage Resistance?

40. IRM in the USA

41. Refuge strategy for India • 5 Rows of non-Bt around Bt cotton • Influenced by USA • Arbitrary • Inherent weaknesses

42. Potential strategies for conservation of susceptibility • 1. Crop manipulationA=Bt cotton • Rotation:A:B:C:D:E:A:B:C…… • Alternation:AAA:BBB:CCC:DDD…. • Mosaic:A BCD AC • DAB CD • Refuge:AAAA:00: AAAA:00….. • 2.Conserve & encourage natural control • 3. Cultural control; Biopesticides; Biological control. • 4. ETL based pesticide optimization with selective Insecticides

43. Conditions for effective refugia strategy

45. Bt-Adapt*Factors affecting resistance developmentData from EU China work Genetic factors: • Initial resistant allele frequency • Dominance • Mode of inheritance • Relative survival rate of RR, RS and SS genotypes on Bt and non-Bt plants • Other factors influencing Hardy-Wienberg *Kranthi K.R. and Kranthi N.R. (2004) : Modelling adaptability of cotton bollworm Helicoverpa armigera (Hubner) to Bt-cotton in India. Current Science 87(8): 1096-1107

46. Bt-Adapt (2)Data from published literature Ecological factors: • Relative host oviposition preference • Host plant availability in the agro-ecosystem • Natural survival on the various hosts • Synchrony between resistant and susceptible genotypes • Relative fitness of the different genotypes on the different hosts • Level and distance of in-season and between-season migration Control Factors: • Survival after insecticides on Bt and non-Bt hosts • Survival after other mortality on Bt and non-Bt hosts

47. BT-AdaptStochastic model in Visual Basic • Recursion equations expressing net increases in population density as a function of: • Fecundity • Natural survival of life stages • Survival after insecticide • Survival after Bt exposure • With survival rates of the 3 genotypes as: RR – L; RS -Lh+(1-h)K; SS – K Where h is dominance

48. Default Parameters for ‘Bt Adapt’ *Kranthi et al 2002, 2005 +Zhang and Tang 2002; Y.Wu et al 2005; K.Wu et al 2002 & 2003

49. Default parameters for H.armigera in Shandong areas in ‘000 ha

50. Initial frequency of r allele at 1:10,000