Slide 1

10/14/20141WELCOME

UNIVERSITY OF AGRICULTURAL & HORTICULTURAL SCIENCES, SHIMOGASeminar- ISACHIN, U. SSr. M. Sc. (Hort. )Dept. of Horticultural Entomology COLLEGE OF HORTICULTURE, MUDIGERE10/14/20142

2

10/14/20143Mechanisms of Insecticide Resistance in insects and its Management

3

Topic division10/14/20144I. Introduction History Insecticide resistance Terms used Genetics of resistanceII. Mechanisms of Resistance with case studiesIII. Factors favorable to rapid development of resistanceIV. Management of Insecticide Resistance with case studiesV. Conclusion

Fig. 1: Dynamics of increase of the resistant species of arthropods in the world.

10/14/20145Sokolyanskaya, 2010

5/8/20146Table.1: Resistant insect and mite species according to economic importanceEconomic importanceNumber of speciesPercentage of totalAgricultural28356.1Medical/ Veterinary19839.3Beneficial 234.6Total 504

Simon, 2008

6

Table.2: Occurrence of resistance in insects and mites according to pesticide chemical group10/14/20147Chemical groupNumber of speciesPercentage of totalCyclodiene 291 57.7DDT26352.2Organophosphate26051.6Carbamate 8516.9Pyrethroid 489.5Fumigant 122.4Others 407.9

(Simon, 2008)

OrderChemical groupCyclodienesDDTOPCarbamatesPyrethroidsFumigantOther Diptera 108107621110-1Lepidoptera 4141341410-2Coleoptera5724269485Hemiptera31223614531Others2321931-2Acarina161845132-27Total27623321264321138%66524714729

10/14/20148Table.3: Number of species of insects and mites reported resistant to insecticides till 1984 (chemical groupwise)(Gour and Sridevi, 2012)

8

Insecticide ResistanceAn added ability to withstand an insecticide acquired by breeding of those individuals which survive exposures to that particular toxicant insufficient to wipe out the whole colony.

(Hoskins and Gordon, 1956)

An inherited ability to tolerate dosage of insecticide that would be lethal to the majority of individuals in a normal wild populations of same species. (WHO, 1957).

10/14/20149

Table.4: Terms usedInsecticideInsecticide is a toxic substance that kills insects or eliminates disease-transmitting pests/vectors Cross Resistance It refers to a type of resistance in which a pest population develops resistance to more than one pesticide within a chemical family (e.g., organophosphate insecticides, etc.). Multiple Resistance It involves multiple, independent resistance mechanisms, which often lead to resistance to chemicals from different families (i.e., organophosphate and carbamate insecticides)

10/14/201410Karunamurthy and Sabesan, 2013

Insecticide Resistance Management It is an effort to slow down or prevent the development of resistance.Insecticide CombinationThe use of two or more insecticide applications within a field, instead of single.Insecticide MixtureTwo or more compounds are mixed within a single product or formulationSynergist A substance which does not itself have insecticidal properties, but which, when mixed or applied with insecticides of a particular class, considerably enhances their potency.

10/14/201411Karunamurthy and Sabesan, 2013

Resistance is BiphasicPhase I- Due to selection of variants in the population according to genetic principle, the resistance which is initially present in the population is expressed.Phase II- Acceleration of resistance takes place by induction of pre-existing detoxifying enzymes towards enhanced activity, resulting in faster breakdown of the chemicals.10/14/201412Saxena, 1996

10/14/201413

Fig.2: Pesticide resistance can build up in the pest population when a change in the genetic characteristic of the pest population is inherited from one generation to the next. Increased or frequent use of pesticides often hastens resistance(Goodell et al. 2001)

13

Genetics of resistance 10/14/201414Preadaptation : Resistance is preadaptiveEg: Resistance to DDT in House flies was eight times higher than in original strain2. Gene frequency : Low in original natural population (0.0001 to 0.01%) High in wild & resistance population Eg.: Mosquito in Nigeria Dieldrin-R gene @ 0.4-0.6%(Simon, 2008)

3. Dominance and number of genes 10/14/201415Resistant gene can be dominant, recessive, incomplete dominant or incomplete recessive. Carbmates & OPs Dominant or incomplete dominant DDT, Bt & Spinosyns Recessive Dieldrin Incomplete dominant Pyrethroid Incomplete recessive

Single gene High resistance

(Simon, 2008)

Table 5: Rate development of resistance to fenvalerate and Deltamethrin in the parental field strain of Spodoptera litoralis10/14/201416Strain and generation testedFenvalerate (R-FN strain)Deltamethrin (R-DM strain)Selecting concentration (mg/lit.)LD50aRRbSelecting concentration (mg/lit.)LD50aRRbS-FM strain0.4610.0211P- strain50.651.40.360.0371.8F150.541.20.360.0321.5F250.611.30.360.0311.5F360.841.80.360.0321.5F460.801.70.540.0422.0F560.881.40.540.0452.1F680.751.60.540.0391.8F7121.352.90.720.0452.1F8161.493.20.900.0472.2F9202.274.91.440.0884.2

(Riskallah et al. 1983, Egypt)a-As g/g body weight. b-Resistance ratio=LD50 of tested generation/ LD50 of S-FM strain

16

Cont .10/14/201417F10302.315.01.800.0914.3F11402.435.32.160.1306.2F12502.495.42.520.1406.7F13602.735.92.880.1507.1F14703.186.93.240.1808.6F15804.249.23.600.1909.0F16904.329.33.960.21010.0F171004.259.24.320.27012.8F181205.9312.94.680.32015.2F191407.0015.25.040.35016.7F2016010.2022.25.400.41019.5F2118012.1026.45.760.42020.1F2220013.2028.86.120.44021.0F2315.1032.80.57027.1

Riskallah, et al. 1983

10/14/201418Mechanisms of Resistance

10/14/201419I. Behavioral Resistance

(Simon, 2008)

10/14/201420I. Behavioral ResistanceDevelopment of ability to avoid a dose that would prove lethal.Stimulus dependent & matter of Hypersensitivity or Hyperirritability

Avoid lethal dose or treated surface

Leg Autotomy

(Simon, 2008)

10/14/201421Legs droppedNo. moths % Moths in each activity stateDead Knocked downActive 09431125714620107023020773350208041001001-482181072

Table.5:Leg-drop and activity status of diamondback adults at 48 h after walking on fenvalrate residues (1,000 mg/cm2) for 5 min(Moore et al., 1989)

10/14/201422Fig.3: Scheme of potential Behavioral and physiological changes associated with Resistance in insect (a) Susceptible insect, (b) Resistant insect(Corbell and Guessan, 2013)

10/14/201423Reduced penetration Target site insensitivity Enhanced detoxification II Physiological Resistance(Simon, 2008)

10/14/201424

A. Reduced penetrationCuticle contains more protein and Lipid.Increased sclerotization.Binding protein and Lipid reservoir traps insecticide in the cuticle.Slight resistance.

(Simon, 2008)

10/14/201425Fig.4: Means and variation in cuticle thicknesses (with 95% limits) of two samples of An. funestus.(Wood, et al. 2010, South Africa)

Mean cuticle thickness of intolerant specimen was 2.12m & tolerant was 2.33m giving mean diff. of 0.20m 25

Fig.5: Time-to-knockdown (KDT) during exposure to permethrin vs. mean cuticle thickness (microns). 10/14/201426

(Wood, et al. 2010, South Africa)

Lenior regration of KDT & thickness shown signi. Trend c. thickness increasing length to KDT, there by inducing physiological R26

10/14/201427

Reduced penetration alone- low level of Resistance

Reduced penetration + Other mechanism- high level of resistance(Simon, 2008)

10/14/201428B. Target site insensitivity

(Simon, 2008)

Types 10/14/201429Nerve insensitivity

AchE insensitivity

Reduction in midgut target site binding(Simon, 2008)

10/14/201430Structure of Neuron and Nerve transmission

Vesicles with AChAChEReceptor site axon

30

Mode of action of different chemicals 10/14/201431

10/14/201432 Nerve insensitivity DDT and Pyrethroid Resistant strain Insensitive Na+ channelCyclodiene- Point mutation in GABA receptor protein

(Simon, 2008)

10/14/201433AChE insensitivity OP and carbamates resistant strains Insensitive AChE enzyme

Point mutation in receptor protein

(Simon, 2008)

Reduction in midgut target site binding10/14/201434Reduced binding of toxinDisruption of Cadherin superfamily gene.Tobacco bud worms-showed high levels of resistance to Cry1AcPink boll worm- alteration in BtR-4 gene Alteration of sugar structure- affects Bt toxin attachment

Fig.6: The resistance development to chlorantraniliprole in the S strain of P. xylostella.10/14/201435

(Gong, et al. 2014)

resistance ratio of F18 was 52.5 folds Reason: Rhynodene receptor transcript level lower in R strain than S strain.35

10/14/201436C. Enhanced detoxification

insecticideDetoxification of insecticides by enzymesTarget site(Simon, 2008)

10/14/201437

Detoxifying Enzymes a. Hydrolases Carboxyl Esterase (CarE) i. Esterase gene amplification ii. Esterase mutation

b. Mixed fuction oxidases (MFOs) Cytochrome P450 monoxygenases (P450)

c. Glutathion-S-transferases (GST)(Simon, 2008)

H OPs, carbamates, &pyrethroidsCy. P450-OPs, carbamates, Pyrethroides,neonicotinoides, etc.GST- OPs37

10/14/201438

Fig. 7: The three Principal types of insecticide resistance mechanisms in cross section through susceptible and resistant insects.(Karunamurthy and Sabesan, 2013)

10/14/201439Resistance profileOPsCarbaryl, methomyl & Propoxur Carbofuran Synthetic pyrethroidsIGRsResistance mechanism involved Multifactorial Monofactorial Monofactorial Monofactorial Monofactorial Responsible biochemical entities Insensitivity, AChE,CarboxylesteraseMFOsMFOsMFOsMFOsResistance amplitude mediumhigh high high high

Table.7: Important mechanisms in DBM insecticide resistance (Cheng et al. 1998)

10/14/201440 EnzymeStrainEnzyme activityRateP450S33.12 4.48 a1.00GDLZ- R35.94 1.77 a1.08CarES37.52 2.16 a1.00GDLZ- R42.74 10.06 a1.14GSTS10.20 0.39 a1.00GDLZ- R34.12 9.69 b3.34

Table.8: Activities of detoxification enzymes in different strains of P. xylostella L. against chlorantraniliprole(Zhen-di et al., 2014, China)

10/14/201441

Fig.8a: P450 activity of control and chlorantraniliprole exposed P.xylostella L. larvae after 6 h, 12h and 24h exposure. Data represent the mean SE of three replicates. * indicate significant difference from control and chlorantraniliprole exposure for 6 h, 12 h in S strain where *=0.05. (Zhen-di, et al. 2014)

10/14/201442

Fig. 8b: CarE activity of control and chlorantraniliprole exposed P.xylostella L. larvae after 6 h, 12 h and 24 h exposure. Data represent the mean SE of three replicates. *indicate significant difference from control and chlorantraniliprole exposure for 6 h in S strain where *=0.05. (Zhen-di, et al. 2014)

10/14/201443

Fig.8c : GST activity of control and chlorantraniliprole exposed P.xylostella L. larvae after 6 h, 12 h and 24 h exposure. Data represent the mean SE of three replicates. * indicate significant difference from control and chlorantraniliprole exposure for 6 h in susceptible strain and 12 h, 24 h in GDLZ-R strain where *=0.05.(Zhen-di, et al. 2014)

43

Table.9: Mode of resistance and number of cytochrome P450s, carboxylesterases, esterases and transferases among the genomes of blood-feeding insects10/14/201444Mode of resistancePediculus humanusAnopheles gambiaeCulex quinquefasciatusAedes aegyptiCimex lectularius *TSRMR & TSRMR & TSRMR & TSRMR & TSRP4503710617215873#CESNot reported254730-EST17151719-TRA1331373214#

MR = metabolic resistance; TSR=target sensitivity resistance; P450 = cytochrome P450s; CES = carboxylesterases; EST = esterases; TRA = transferases; # = occurrences; * as per 454 pyrosequencing data, not complete genome of the bed bug (Mamidala, et al. 2011)

Table.10: Comparison of EST, GST and AChE activities in nine different populations of Oxya chinensis (Orthoptera: Acrididae)10/14/201445PopulationEST specific activitya ( mol/min/mg protein) GST specific activitya ( mol/min/mg protein) AChE specific activityb ( mol/min/mg protein) XY0.170.04e79.1941.22bcd9.343.48aJY0.360.10ab83.6846.11bcd9.261.61aBDG0.300.07c96.3745.40b8.321.77aYC0.100.01f52.649.44d4.830.78bcXX0.250.06d89.2855.14bc9.023.30aHZ0.330.03bc67.1115.43bcd2.800.70cBD0.320.04bc166.9531.88a2.180.46cCA0.120.04f81.4232.52bcd7.001.54abJN0.370.08a73.8348.67bcd3.490.60c

(Haihua, et al. 2007, China)a Average of 64 individual thoraxes, each with triplicate analyses. b Average of four groups of heads, each with triplicate analyses.

Table.11: Metabolic enzyme activities of fourth instars from the SZ strain and the Fipronil-resistant SZ-F strain of Plutella xylostella.10/14/201446Detoxification enzymeSpecific activity (Mean SE)SZ strain (susceptible)SZ-F strain (resistant)Ratio (SZ-F/SZ)Oxidases (pmol/min/mg protein)PNOD0.260.060.280.041.07ECOD1.630.511.830.341.12MCOD4.521.14.710.591.04Esterases (nmol/min/mg protein)-NA esterase102.212108.54.11.06GSTs (nmol/min/mg protein)CDNB conjugation179.158191.67.71.07DCNB conjugation5.060.35.740.311.13

P > 0.05; t- test(Ageng et al. 2006, China)

Table.12:Resistance levels of Plutella xylostella strains to Acephate10/14/201447StrainnLC50(mg/L)95%CLRRSS28816.814.0-20.31OR2153316.72683.3-4253.6197.4KU-10165581.5439.6-787.534.6SMN43255.051.1-754.415.2WH25290.259.3-129.15.4BJ1-10333308.0195.5-488.318.3BJ3911307.2894.0-2092.877.8

RR: resistant ratio= LC50 of strains tested/LC50 of SS strain.(Sonoda, et al. 2014)

Reason : due to Acetyl choline esterase1 gene activity & extensive duplication of strain of DBM47

Table.13: Mean resistance frequency * (%) to different chemistries in H. armigera population of Tamilnadu (a comparision among the three locations)10/14/201448Insecticides Coimbatore Poluvampatty Madukarai Fenvalerate 95.097.293.4Cypermethrin 96.797.493.3Deltamethrin 91.995.488.9Lamdacyhalothrin 91.591.986.3Betacyfluthrin 81.889.481.4Quinalphos 49.234.438.9Chlorpyrifos 49.346.644.0Profenofos 35.424.527.8Endosulfan 36.526.029.4Thiodicarb 36.232.133.3Spinosad 0.00.00.0

*Mean of resistance frequency data obtained for 49 weeks (June 2002- April 2003)(Ramasubramaniam and Ragupathy, 2004, Coimbatore)

Reason: Profenofos is a specific synergist recommended to inhibit the activity of CE so R was lower Spinosad mode of action is entirely different than that of already exiting chemicals48

10/14/201449Factors favorable to rapid development of resistance Prolonged exposure to a single insecticide

High selection pressure

Large coverage area

Immigration or Migratory

Insects multiplying by asexual means

Short life cycle of insect

Selection at every stage of insect life cycle

Table.14: Biological, genetic, and operational factors in resistance development. 10/14/201450Factor Potential for resistance development LowerHigher Biological factors Population size SmallLargeReproductive potential LowHighGeneration turnoverOne or less generations per yearMany generations per yearPesticide metabolismDifficult EasyNumber of target sites of the pesticideMultiple sitesSingleSpecific Pest host rangeNarrow Wide

International Code of Conduct on the Distribution and Use of Pesticides, 2012.

10/14/201451Factor Potential for resistance development LowerHigher Genetic factors Occurrence of resistance genesAbsentPresent Number of resistance mechanismsOneSeveral Gene frequencyLow High Dominance of resistance genesRecessiveDominant Fitness of R individualsPoor Good Cross resistanceNegative or nonePositive Modifying genesAbsent Present

International Code of Conduct on the Distribution and Use of Pesticides, 2012.

10/14/201452Factor Potential for resistance development LowerHigher Operational factors Activity spectrum of the pesticideNarrow spectrum Broad spectrum Pesticide application rate LessMorePresence of secondary pests Absent (only the target pest is treated) Present (non targeted (potential) pests are also treated) Pest control tactics Multiple control tactics (chemical, biological, cultural)Continuous use of single method or compound

International Code of Conduct on the Distribution and Use of Pesticides, 2012.

10/14/201453Management of Insecticide ResistanceIntegrated pest managementGrow trap cropsInundative release of Biological agentsSwabbing & stem bandingJudicious use of insecticides (Need based & Recommended dose)The use of insecticide synergistsWindow system of pesticide applicationInsecticide rotationArea wide management

53

Table.15: Phases of resistance monitoring and management for a new pesticide 10/14/201454TimingResistance detection and monitoring activities Other management activities 1-2 years before start of sales Establish sampling and testing methods & Survey for initial sensitivity data Assess riskDuring years of use Monitor randomly in treated areas for resistance, if justified by risk assessment of special importance of crop/pestImplement the RMP; watch practical performance of the pesticide closely

International Code of Conduct on the Distribution and Use of Pesticides, 2012.

10/14/201455As soon as signs of resistance have been detected Monitor to determine the extent and practical significance of resistance

Study cross resistance, fitness of variants of resistant organisms, assess other factors affecting the development of resistance If resistance problem is confirmed, review and modify RMP Subsequently Monitor rate of spread or decline of resistance Watch pesticide performance; review RMPs

International Code of Conduct on the Distribution and Use of Pesticides, 2012.

Figure. 11: Swabbing with mineral oil & 10/14/201456

(Ayyasamy & Regupathy, 2010, Tamil Nadu)

Stem banding with black polythene sheet

Need and scope for insecticide resistance management for the invasive papaya mealy bug Paracoccus marginatus Williams and Granara de Willink in small scale papaya farming system in Tamil Nadu, India.

56

Table.16: Mean synergistic suppression (%) of insecticide resistance by MFO and CE inhibitorsInsecticides Piperonyl butoxide (50 g)Pungam oil (50 g)Profenofos (0.1 g)Fenvalerate 57.946.024.4Cypermethrin 52.245.819.7Endosulfan 14.22.119.5Thiodicarb 0.6--------2.9

10/14/201457(Ramasubramaniam and Ragupathy, 2004, Coimbatore)

Table.17: Combination products in pest management in different crop eco-system10/14/201458Combination productsDose (per ha)Target pestBhendi Polytrin C 44EC (40% profenofos+ 4% cypermethrin)2lit.Erias spp., Aphis gossypii Glover, Tetranychus cinnabarinus Boisdual, Amrasca devastans (Dist.)Cypermethrin+Chlorpyrifos100+1000g a.iErias vittella and Helicoverpa armigeraBrinjal Polytrin C 44EC0.044%Bemisia tabaciSpark 36 EC (35% of Triazophos+ 1% of Deltamethrin)1.25 lit.Leucinodus orbonalis GueneeCarbosulfan + Quinalphos1ml each/lit.Leucinodus orbonalis

(Ragupathy, et al. 2004, Tamilnadu.)

Cont10/14/201459TomatoPolytrin C 44 EC (40% profenofos+ 4% cypermethrin)1 lit.Helicoverpa armigeraSpark 36 EC (35% of Triazophos+ 1% of Deltamethrin)1 lit.Helicoverpa armigera

Lethal super2 lit.Helicoverpa armigera

Mango Nurelle D 5051.5 ml/litAmritodes atkinsoni L.Sugar beetNagata 45 EC675 g a.iSpodoptera litura F.Cabbage Spark 36 EC1.25 lit.Plutella xylostella (L.)

(Ragupathy, et al. 2004)

Table.18: Insecticide resistance management guidelines for beet army-worm (March 2001).10/14/201460Insecticide classEarly season(April to mid-june)Mid season (Mid-June through July)Late season (August and September)Bacillus thuringiensisVarious productsVarious productsOrganophosphateLorsban or CuracronLorsbanCarbamateLannateLannateMiscellaneousStewardSuccessConformSuccessStewardConform

PyrethroidCaptureAsana

Goodell, et al. 2001

Table.19: Insecticide resistance management guidelines for Silver leaf whitefly (March 2001).10/14/201461Chemical class Initial build up Gradual invasion Heavy migration Insect growth regulator Chitin synthesis inhibitor Applaud Insect growth regulator Metamorphosis inhibitorKnack Chloronicotinyl Provado Amidene Ovasyn Pyrethroid Capture Pyrethroid+ organochlorinPyrethroid+ Endosulfan Pyrethroid+ organophosphate/carbamate Denitol+Orthene/Curacron/Lannate/Vydate

Goodell, et al. 2001

10/14/201462RotationNumber of WFT adults per plantCumulative number of WFT adult2DAA13DAA22DAA32DAA4Untreated14.1a23.0a44.8a123.6a205.5aRadiant/ Movento0.8b11.3b40.4abc16.8bcd69.3bcRadiant/ Assail1.5b 7.3b31.7abcd17.9bcd 58.4cdRadiant/ Venom1.1b8.6b26.0cd11.1cd46.8deRadiant/ Beleaf0.9b13.1b43.3ab26.2b83.5bRadiant / Requiem1.2b10.8b36.5abc15.5bcd64.0cdRadiant/ Eco+ Req1.4b13.6b30.7abcd13.0cd58.7cdRadiant/ Agri- Mek 1.4b6.5b18.9d8.2d35.0eRadiant/M- Pede 1.4b12.2b26.6bcd22.7bc62.9cd

Table.20: Effect of several insecticide rotations on WFT on adult numbers (onion trial; Fresno, CA), 2009(James et al., 2010)2DAA1 = 2 days after the first application

10/14/201463RotationNumber of WFT larvae per plantCumulative number of WFT larvae2DAA13DAA22DAA32DAA4Untreated72.6 a118.0 a127.1 a305.1 a624.0 aRadiant/ Movento9.0 b16.1 b106.0 ab36.3 b168.0 bcdRadiant/ Assail16.9 b7.7 b50.5 c45.8 b120.0 deRadiant/ Venom11.1 b12.7 b43.4 c62.0 b128.0 cdRadiant/ Beleaf10.2 b11.5 b83.5 b50.3 b156.0 bcdRadiant / Requiem10.1 b18.2 b113.2 ab63.3 b204.0 bRadiant/ Eco+ Req15.8 b18.1 b110.7 ab38.2 b184.0 bcRadiant/ Agri+ Mek 13.6 b9.4 b14.9 d29.6 b68.0 eRadiant- Pede 14.0 b21.6 b104.5 ab51.1 b192.0 b

Table.21: Effect of several insecticide rotations on WFT on larval numbers (onion trial; Fresno, CA), 2009.(James et al., 2010)2DAA1 = 2 days after the first application

10/14/201464conclusion

10/14/201465