genetics and mechanism of resistance to deltamethrin in a field population of spodoptera litura...

Pest Management Science Pest Manag Sci 63:1002–1010 (2007)

Genetics and mechanism of resistanceto deltamethrin in a field population ofSpodoptera litura (Lepidoptera: Noctuidae)Munir Ahmad,1,2 Ali H Sayyed,2∗ Neil Crickmore2 and Mushtaq A Saleem1

1Department of Entomology, University College of Agriculture, Bahauddin Zakariya University, Multan, Pakistan2Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK

Abstract

BACKGROUND: Spodoptera litura (F.) causes enormous losses in many economically important crops. Thegenetics of insecticide resistance has been extensively studied in several insect pests, but there is a lack ofinformation on S. litura. Therefore, the genetics and mechanisms of the resistance of S. litura to deltamethrinwere investigated.

RESULTS: Bioassays at generation G1 gave resistance ratios of 9, 5, 41, 52 and 49 for deltamethrin, cypermethrin,profenofos, chlorpyrifos and triazofos respectively, when compared with the susceptible Lab-PK strain. Bioassaysat G4 with a deltamethrin-selected population (Delta-SEL) showed that selection gave resistance ratios of 63 and7 for deltamethrin when compared with the Lab-PK and UNSEL strains respectively. Cross-resistance to otherinsecticides tested was observed in the selected population. A notable feature of the Delta-SEL strain was thatresistance to deltamethrin, cypermethrin, profenofos and chlorpyrifos did not decline over the course of fivegenerations. Synergism tests with microsomal oxidase (MO) and esterase-specific inhibitors indicated that thedeltamethrin resistance was associated with MO and, possibly, esterase activity. Reciprocal crosses between theDelta-SEL and Lab-PK strains indicated that resistance was autosomal and incompletely dominant. A direct testof monogenic inheritance suggested that resistance to deltamethrin was controlled by more than one locus.

CONCLUSION: Stability and dominance of resistance and cross-resistance suggest that insecticides with differentmodes of action should be recommended to reduce pyrethroid selection pressure. 2007 Society of Chemical Industry

Keywords: Spodoptera litura; microsomal oxidases; esterases; deltamethrin; incompletely dominant; autosomal;organophosphates; multiple loci

1 INTRODUCTIONThe common cutworm, Spodoptera litura (Fabricius),is a polyphagous and serious pest causing enormouslosses to many economically important crops in SouthAsia1–3 and has been found to cause 26–100% yieldloss in the field.4 The extensive use of organochlorine,organophosphates and pyrethroids against S. lituraprovides an ideal environment for the evolution ofresistance in the Indo-Pakistan subcontinent.5,6 Inrecent years, S. litura outbreaks have been more com-mon in South Asia because of the development ofresistance to various groups of insecticides used againstthe pest.5–8 A high level of resistance to various insec-ticides including organochlorines, organophosphates,carbamates, pyrethroids and Bacillus thuringiensisproducts has been reported in S. litura from variousparts of the World, including Pakistan.5–12 The majorresistance mechanisms involve elevated detoxification,increased pyrethroid sequestration capacities, struc-tural changes in the voltage-gated sodium channels,

owing to some well-defined point mutations, or a com-bination of these mechanisms.13 Reduced insecticidepenetration through the cuticle may also occur as aminor mechanism.14

The genetic basis of insecticide resistance in naturalinsect populations has been extensively studied.15

The types of resistance that evolve to insecticidescan be quite distinct, depending upon the speciesand geographical origin of the colony.16 However,once field exposure has occurred and low levels ofresistance are detected, there appears to be littledifference in the type of resistance that evolves amongcolonies that are further selected in the laboratory.17

The successful management of insecticide resistancedepends ultimately on a thorough knowledge of itsgenetic basis and the mechanisms involved. Themode of inheritance helps in resistance detection,monitoring, modelling and risk assessment.17 Somemanagement strategies are particularly effective whenresistance is inherited as a recessive trait.

∗ Correspondence to: Ali H Sayyed, Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UKE-mail: [email protected](Received 28 November 2006; revised version received 3 April 2007; accepted 11 April 2007)Published online 3 August 2007; DOI: 10.1002/ps.1430

2007 Society of Chemical Industry. Pest Manag Sci 1526–498X/2007/$30.00

Genetics and cross-resistance to deltamethrin in S. litura

In the present study, a field population ofS. litura from the Multan region in the Punjabprovince of Pakistan was examined with the aimof investigating the genetics and mechanisms ofresistance to deltamethrin and the degree of cross-resistance to a range of other insecticides. Suchknowledge can provide the basis for managementprogrammes aimed at minimizing the developmentof resistance.

2 MATERIALS AND METHODS2.1 InsectsFifth-instar larvae of S. litura were collected from theMultan region of Punjab, Pakistan, in April 2006.Larvae were reared on arum leaves in the labo-ratory at 25 ± 2 ◦C and 60–65% relative humiditywith a 14:10 h light:dark photoperiod. Fresh arumleaves were replaced after 24 h and pupae were col-lected on alternate days. The emerged moths wereprovided with 10% vitamin solution (nicotinic acid3.0 g L−1, calcium pantothenate 3.0 g L−1, riboflavine1.52 g L−1, aneurine hydrochloride 0.76 g L−1, pyri-doxine hydrochloride 0.76 g L−1, folic acid 0.76 g L−1,D-biotin 0.60 g L−1, cyanocobalamine 0.006 g L−1).Adults were kept in Perspex oviposition cages withmesh sides to maintain ventilation and were fedon a solution containing sucrose 100 g L−1, vitaminsolution 20 mL L−1 and methyl 4-hydroxybenzoate2 g L−1) in a soaked cotton wool ball.

2.2 InsecticidesThe commercial formulations of insecticides usedin the bioassays were: deltamethrin 105 g L−1 EC(Decis Super; Bayer Crop Science), cypermethrin100 g L−1 EC (Arrivo; FMC), profenofos 500 g L−1

EC (Curacron; Syngenta), chlorpyrifos 400 g L−1 EC(Lorsban; Bayer Crop Science), triazofos 400 g L−1

EC (Hostathion; Bayer Crop Science), DDT 500 gkg−1 WP (Hindustand Chemicals Ltd, India), phoxim400 g L−1 EC (Volaton; Bayer Crop Science),spinosad 240 g L−1 SC (Tracer; Dow Agro Sciences),fipronil 36 g L−1 EC (Regent; Bayer Crop Science)and chlorfenapyr 360 g L−1 SC (Pirate; BASF).Stapple (DuPont, USA), a non-ionic surfactant, wasused at 5 mg L−1 to enhance the adhesiveness of theinsecticides.18

2.3 Generation of a susceptible strainSingle-pair crosses were set using individuals fromthe heterogeneous field population. Bioassays wereused to identify families in which the F1 progeny ofthese crosses were susceptible to a given insecticide.Those in which 100% mortality was observed using aconcentration of insecticide equal to the LC20 of thefield population were then used in a second round ofcrossing. F2 progeny showing 100% mortality at LC10

levels of insecticide were then used to propagate thesusceptible strain, which was designated Lab-PK.

2.4 Selection of insecticide-resistant populationF1 progeny from single-pair crosses of the fieldpopulation were tested with insecticide levels equatingto LC90 for the field population. Those families thatshowed 0% mortality were then exposed to 1 mg mL−1

of deltamethrin, and the survivors were reared toadults. The adults were crossed in single-pair matings,and the second-instar larvae were exposed to 1.5 mgmL−1 deltamethrin. In the third selection, the larvaeof single-pair matings were again exposed to 1.5 mgmL−1, and a family with 100% survival was chosenand designated Delta-SEL.

2.5 Leaf-disc bioassaysBioassays were conducted on second-instar larvae (L2)of S. litura using a standard leaf-disc bioassay.17

Each disc (4.8 cm diameter) from arum leaves wasimmersed in a test solution for 10 s and allowed todry at ambient temperature for 1–1.5 h. Control leafdiscs were immersed in distilled water containing 5 mgL−1 Stapple. The leaf discs were placed in individualpetri dishes (5 cm diameter) containing moistenedfilter paper. Five L2 larvae were placed in each dish,and each treatment was repeated 5–8 times, includingcontrols. Mortality was assessed after 48 h exposure toinsecticides.

2.6 Stability of resistance in Delta-SELpopulationApproximately 100 adult S.litura were kept in aPerspex oviposition cage with mesh sides to maintainventilation. Insects were fed with 10% vitamin solutionon a soaked cotton wool ball ad libitum. Larvae weremaintained on arum leaves which were replaced dailywith fresh ones until all the larvae had pupated.The pupae were then transferred to ovipositioncages. In order to avoid excessive insect numbers,adult insects were culled at random when theirnumber exceeded 100. The experiment continued for5 months, equivalent to five generations of S. litura.

2.7 Test for synergismThe toxicities of deltamethrin and endosulfan werealso evaluated in the presence of two synergists, piper-onyl butoxide (PBO; Sigma Ltd, UK), an inhibitorof cytochrome P450 monooxygenases (microsomaloxidases) and of esterases, and S,S,S-tri-n-butyl phos-phorotrithioate (DEF; Sigma Ltd, UK), an esterase-specific inhibitor. Stock solutions (10 mg mL−1) ofPBO and DEF were prepared in acetone (analyticalreagent; Fisher Scientific, Loughborough, UK). Totest the effect of PBO and DEF on the efficacy ofinsecticide, 10 mg L−1 of synergist was added to eachof the various concentrations of insecticide. Mortalitydata were taken after 48 h exposure. The synergismratio (SR) was calculated by dividing the LC50 of thepopulation treated with insecticide alone by the LC50

of the strain treated with insecticide plus synergist.

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2.8 Genetic studiesThe response of F1 and backcross (BC) progeny todeltamethrin was evaluated as describedpreviously.17,19 Mass reciprocal crosses betweenDelta-SEL and Lab-PK populations produced the F1

progeny. Mass crosses with Lab-PK then producedthe F2 progeny. The pupae of both sexes were sepa-rated on the basis of distinct abdominal characteristicsby viewing the ventral abdomen under 10× magnifi-cation. For mass crosses, 20 Delta-SEL females werepooled with 20 susceptible males, and 20 suscepti-ble females were pooled with 20 Delta-SEL males.Mass crosses provided enough offspring for the calcu-lation of LC50 values. The degree of dominance forLC50 (DLC) was calculated as described by Bourguetet al.20

The standard error of dominance was estimated bytaking the square root of variance of D:21

σ 2D = 4

(X1 − X3)2 ×

{σ 2

X2

(X2 − X3)2

(X1 − X3)2

σ 2X1

+ (X2 − X1)2

(X1 − X3)2 σ 2

X3

}

where X1, X2 and X3 are the logarithms of the LC50

values for Delta-SEL, F1 and susceptible respectively,and σ 2

X1, σ 2

X2and σ 2

X3are the phenotypic variances of

Delta-SEL, F1 and susceptible respectively. Variancewas estimated as the inverse of the slope squared(standard deviation).

2.9 Data analysisWhere necessary, bioassay data were corrected forcontrol mortality by Abbott’s method,22 and estimatesof LC50 values and their 95% fiducial limits (FL)were obtained by probit analysis using Polo.23

Because of the inherent variability of bioassays,pairwise comparisons of LC50 values were at the1% significance level (where individual 95% FL fortwo treatments do not overlap).24 The number ofloci influencing resistance was estimated using twoapproaches. Firstly, by analysing the response ofbackcross progeny to deltamethrin and applying adirect test for a monogenic model of resistance. Ifthe resistance is monogenic, a backcross of F1 (Lab-PK SS × Delta-SEL RR) × Lab-PK will produceprogeny that are 50% RR and 50% RS. To testthis hypothesis, expected mortality was calculatedas mortality at concentration x = 0.5 (proportion ofF1 dead at x concentration + proportion of Lab-PKdead at x concentration). A contingency test was usedto test for independence between the expected andobserved number of dead at each concentration.25 Thesecond method involved comparison of the slopes andvariance, and the calculation of the effective numberof factors using Lande’s method:26

nE = (µP2 − µP1)2

8σ 2s

where µP2 and µP1 are the logarithms of the LC50

values for Delta-SEL and Lab-PK respectively, andwhere σ 2

s is estimated as

σ 2s = σ 2

B1+ σ 2

B2− [σ 2

F1+ 1/2σ 2

P1+ 1/2σ 2

P2]

where σ 2B2

, σ 2B1

, σ 2P1

and σ 2P2

are the phenotypicvariances of the backcross (F1 × Lab-PK), F1, Lab-PKand Delta-SEL respectively. Variance was estimated asthe inverse of the slope squared (standard deviation).As the F1 × Delta-SEL backcross was not done, σ 2

B1

could not be estimated. Therefore, it was assumedthat σ 2

B1= 0. This will inflate the estimation of nE

because σ 2B1

contributes positively to σ 2s which is in the

denominator of the estimate of nE.27

3 RESULTS3.1 Susceptibility of Lab-PK and the fieldpopulation to various insecticidesThe susceptible Lab-PK strain was derived asdescribed in Section 2.3. Among the 60 F1 families,mortality in response to 40 µg mL−1 deltamethrinranged from 0 to 100% after 2 days (data not shown).The only F1 family with 100% mortality was used tocreate the F2 generation. Among the 60 F2 families,ten families showed 100% mortality at 20 µg mL−1,and these were used to start the susceptible straindesignated Lab-PK.

Results of bioassays with Lab-PK (Table 1) showedthat this population was significantly less suscepti-ble to the pyrethoid insecticides deltamethrin andcypermethrin than to the other insecticides tested(profenofos, chlorpyrifos, triazofos, endosulfan, DDT,phoxim, spinosad, fipronil and chlorfenapyr). Lab-PKwas particularly susceptible to chlorfenapyr.

The field population at G1 was tested against fiveof these insecticides, and the results are shown inTable 2. Compared with Lab-PK, this population hadresistance ratios of 9 and 5 towards deltamethrin andcypermetharin respectively and significantly higherratios towards profenofos (41) chlorpyrifos (52) andtriazofos (49).

3.2 Response to selection with deltamethrinSelection (G1 to G3) of the field population withdeltamethrin increased the resistance ratio for thisinsecticide to 63 compared with Lab-PK and to 7compared with the field population at G1 (Table 3).There was a significant (P < 0.05) increase in the slopefor Delta-SEL compared with the field population atG1 (Tables 2 and 3).

The Delta-SEL population showed resistance ratiosof 83, 97, 104 and 86 for cypermethrin, profenofos,chlorpyrifos and triazofos respectively compared withLab-PK, and 17, 2, 2 and 2 compared with the fieldpopulation at G1 (Table 3). Resistance ratios of 86 and187 were also observed against endosulfan and DDTrespectively in comparison with Lab-PK. There was noLC50 for the field population at G1 against these latter

1004 Pest Manag Sci 63:1002–1010 (2007)DOI: 10.1002/ps


Table 1. Response (mortality) of Lab-PK population of Spodoptera litura to representative pyrethroids, organophosphates and other insecticides

Fit of probit line

Insecticide LC50 (95%FL) (µg AI mL−1) Slope (± SE) χ2 df P na

Deltamethrin 39.3 (31.8–47.9) 2.60 (±0.29) 2.67 5 0.75 280Cypermethrin 28.5 (23.2–35.1) 2.13 (±0.21) 2.32 5 0.80 280Profenofos 4.63 (3.79–5.69) 2.36 (±0.25) 1.78 4 0.77 240Chlorpyrifos 0.43 (0.34–0.55) 2.02 (±0.21) 3.93 5 0.56 280Triazofos 13.8 (11.1–16.9) 2.09 (±0.21) 2.65 5 0.75 280Endosulfan 1.33 (1.01–1.69) 1.89 (±0.21) 2.49 5 0.78 280DDT 9.15 (7.24–11.3) 2.34 (±0.27) 3.06 4 0.55 240Phoxim 0.63 (0.49–0.78) 2.59 (±0.32) 1.69 4 0.79 240Spinosad 1.06 (0.87–1.29) 2.36 (±0.26) 2.17 4 0.70 240Fipronil 0.54 (0.43–0.66) 2.26 (±0.25) 2.22 4 0.70 240Chlorfenapyr 0.03 (0.02–0.04) 2.22 (±0.21) 1.79 5 0.88 280

a Number of larvae used in bioassay, including controls.

Table 2. Response (mortality) of the field population of Spodoptera litura to representative pyrethroids and organophosphate

Fit of probit line

Insecticide LC50 (95% FL) (µg AI mL−1) Slope (± SE) χ2 df P na RRb

Deltamethrin 353 (269–451) 1.84 (±0.20) 3.01 5 0.70 240 9Cypermethrin 138 (102–185) 2.31 (±0.27) 3.10 4 0.54 320 5Profenofos 191 (146–243) 1.89 (±0.20) 3.00 6 0.81 320 41Chlorpyrifos 22.3 (18.0–27.1) 2.65 (±0.31) 2.27 4 0.68 280 52Triazofos 678 (496–897) 1.61 (±0.18) 4.45 6 0.62 240 49

a Number of larvae used in bioassay, including control.b Resistance ratio = LC50 of field population/LC50 of Lab-PK.

Table 3. Cross-resistance between deltamethrin and other insecticides in the deltamethrin-selected (Delta-SEL) population of Spodoptera litura at

G4

Fit of probit line

Insecticide LC50 (95% FL) (µg AI mL−1) Slope (± SE) χ2 df P na RRb RRc

Deltamethrin 2464 (2012–3051) 2.33 (±0.25) 3.97 4 0.41 240 63 7Cypermethrin 2374 (1946–2886) 2.39 (±0.26) 1.78 4 0.77 240 83 17Profenofos 448 (353–566) 1.75 (±0.18) 3.96 5 0.55 280 97 2Chlorpyrifos 44.7 (37.3–53.4) 2.80 (±0.30) 2.87 4 0.56 280 104 2Triazofos 1183 (951–1477) 1.94 (±0.19) 3.67 5 0.60 280 86 2Endosulfan 114 (93.5–139) 2.32 (±0.23) 3.80 5 0.58 280 86 –DDT 1708 (1549–1891) 2.03 (±0.22) 3.37 5 0.64 240 187 –Phoxim 1.2 (0.99–1.49) 2.19 (±0.24) 3.29 4 0.51 240 2 –Spinosad 2.36 (1.94–2.87) 2.39 (±0.26) 3.59 4 0.46 240 2 –Fipronil 1.2 (0.99–1.49) 2.19 (±0.24) 3.29 4 0.51 240 2 –Chlorfenapyr 0.05 (0.04–0.06) 1.94 (±0.19) 3.19 5 0.67 280 2 –

a Number of larvae used in bioassay, including controls.b Resistance ratio = LC50 of Delta-SEL population/LC50 of Lab-PK.c Resistance ratio = LC50 of Delta-SEL population/LC50 of field population at G1.

two compounds. There was little or no cross-resistanceto phoxim, spinosad, fipronil or chlorfenapyr in Delta-SEL when compared with Lab-PK.

3.3 Stability of resistance in the Delta-SELpopulationOver five generations without exposure to delta-methrin, resistance in Delta-SEL was stable withno significant decline (P > 0.01) (Table 4) in LC50

values to this compound when tested at G8. Thelogit mortality regression slope for Delta-SEL was alsosimilar. There was also no rate of decline in resistancetowards cypermethrin, profenofos or chlorpyrifos.

3.4 Maternal effects, sex linkage anddominanceMale and female adults of Delta-SEL were separatelycrossed with the Lab-PK population. The F1


M Ahmad et al.

Table 4. Stability of resistance to deltamethrin, cypermethrin, profenofos and chlorpyrifos in the Delta-SEL population of Spodoptera litura between

G3 and G8

Fit of probit line

Insecticide LC50 (95% FL) (µg AI mL−1) Slope (± SE) χ2 df P na RRb Rate of decreasec

Deltamethrin (G3) 2464 (2012–3051) 2.33 (±0.25) 3.97 4 0.41 240 63 –Deltamethrin (G8) 2364 (1926–2888) 2.29 (±0.25) 2.58 4 0.63 240 60 −0.004Cypermethrin (G3) 2374 (1946–2886) 2.39 (±0.26) 1.78 4 0.77 280 83 –Cypermethrin (G8) 2268 (1874–2733) 2.58 (±0.28) 2.87 4 0.56 280 80 −0.004Profenofos (G3) 448 (353–566) 1.75 (±0.18) 3.96 5 0.55 280 97 –Profenofos (G8) 465 (388–558) 2.83 (±0.33) 2.96 3 0.39 200 100 0.004Chlorpyrifos (G3) 44.7 (37.3–53.4) 2.80 (±0.30) 2.87 4 0.56 280 104 –Chlorpyrifos (G8) 44.5 (35.8–55.0) 2.03 (±0.20) 2.15 5 0.83 280 103 0.000

a Number of larvae used in bioassay, including controls.b Resistance ratio = LC50 of Delta-SEL population/LC50 of Lab-PK.c Rate of decrease in LC50 = log (final LC50 – initial LC50)/N, where N is the number of generations for which the population was reared withoutinsecticide exposure.

Table 5. Response (mortality) of Delta-SEL and Lab-PK populations of Spodoptera litura and their hybrid F1 progeny to deltamethrin

Strain LC50 (95% FL) (µg AI mL−1) Slope (± SE) DLCa RRb

Delta-SEL 2670 (2036–3409) 1.87 (±0.20) – 68Lab-PK 39.3 (31.8–47.9) 2.60 (±0.29) – –Delta-SEL female × Lab-PK male 1217 (943–1530) 2.20 (±0.27) 0.82 31Lab-PK female × Delta-SEL male 953 (728–1212) 2.04 (±0.23) 0.76 24F1 pooled 1125 (951–1341) 3.17 (±0.31) 0.79 29F1 × Lab-PK 217 (188–252) 3.79 (±0.28) – 6

a DLC = (log LCRS − log LCss)/(log LCRR − log LCss) (DLC varies from 0 to 1, where 0 = completely recessive and 1 = complete dominance).b Resistance ratio = LC50 of Delta-SEL or F1 progeny/LC50 of Lab-PK.

generation was then tested with deltamethrin. TheLC50 values did not differ significantly between theF1 progeny of the reciprocal crosses between thesusceptible and resistant strains, and neither didthe mean slope of the concentration–mortality lines(Table 5). Thus, inheritance was autosomal, showingneither maternal effect nor sex linkage.

The resistance ratio for deltamethrin was reducedfrom 68 in Delta-SEL to 31 and 24 for Delta-SELfemale × Lab-PK male and Lab-PK female × Delta-SEL male respectively. The degree of dominance(DLC) for reciprocal crosses was 0.82 (±0.42) for F1

(Delta-SEL female × Lab-PK male) and 0.76 (±0.48)for F1 (Lab-PK female × Delta-SEL male), whichindicates an incompletely dominant inheritance.

3.5 Slope, variance and minimum number ofgenesThe pooled F1 generation from the Delta-Sel ×Lab-PK cross was backcrossed to Lab-PK. Theestimated slope of the concentration–mortality line forbackcrossed progeny was 3.79, about 1.5-fold steeperthan the estimated slope for Lab-PK (2.60), twofoldsteeper than for Delta-SEL (1.87) and about 1.2-foldsteeper than the estimated slope for the F1 generation(3.17) (Table 5). This pattern indicates decreasedgenetic variance in the backcross progeny comparedwith the parental and F1 strains and suggests thatthe number of loci with major effects on resistance to

deltamethrin was high. The direct test for a monogenicmode of inheritance of resistance was based on thegoodness of fit χ2 between the F1 backcross and theLab-PK expected values, calculated as described bySokal and Rohlf:25

χ2 = (F1 − pn)2

pqn

where F1 is the observed number of dead in thebackcross generation at dose x, p is the expectedproportion dead, n is the number of backcross progenyexposed to dose x and q = 1 − p. The null hypothesisis rejected if the test results in a probability < 0.05,comparable with a χ2 distribution with one degree offreedom. The numbers of classes used in the test arelimited (one degree of freedom), and in this case thepotential of committing a type-II error accepting H0

(lack of differences between observed and expectedoutcome) is high. One way to minimize this would beto test different crosses and pesticide concentrations.

The direct test of a monogenic model showedsignificant deviation (P < 0.05) between observedand expected mortality at five concentrations outof six tested (Table 6). Calculation of the minimumnumber of independently segregating loci with equaland additive contributions26 to resistance yielded anestimate of 1.67. This estimate also supports theconclusion that resistance was conferred primarily bymore than one locus.



Table 6. Direct test of monogenic inheritance for resistance to deltamethrin by comparing expected and observed mortality of the backcross

(F1 × Lab-PK) of Spodoptera litura

Concentration (µg mL−1) Number of larvae tested Observed mortality Expected mortalitya χ2 (df = 1) Pb

32 80 3 19.2 17.99 <0.00164 80 16 36.0 20.20 <0.001128 80 30 56.4 22.31 <0.001256 80 45 62.4 22.05 <0.001512 80 61 70.4 10.46 0.0011024 80 75 77.6 2.47 0.12

a Expected number of larvae dead at given dose = 0.5 (number of F1 larvae that die + number of Delta-SEL larvae that die).b Probability values were considered significantly different at P < 0.05.

Table 7. Toxicity of deltamethrin with and without PBO or DEF to laboratory susceptible and Delta-SEL population of Spodoptera litura

Fit of probit line

Strain Treatment LC50 (95%FL) (µg AI mL−1) Slope (± SE) χ2 df P RRa SRb

Lab-PK Deltamethrin 38.2 (31.1–46.9) 2.25 (±0.24) 2.83 4 0.58 – –Lab-PK Deltamethrin + PBO 28.6 (23.2–35.3) 2.08 (±0.20) 2.01 5 0.85 – 1Lab-PK Deltamethrin + DEF 34.3 (27.7–42.1) 2.20 (±0.24) 2.20 4 0.70 – 1Lab-PK Endosulfan 1.33 (1.01–1.69) 1.89 (±0.21) 2.49 5 0.78 – –Lab-PK Endosulfan + PBO 0.13 (0.10–0.15) 2.11 (±0.20) 2.36 5 0.80 – 10Lab-PK Endosulfan + DEF 0.31 (0.25–0.38) 2.17 (±0.23) 3.53 4 0.47 – 4Delta-SEL Deltamethrin 2364 (1926–2888) 2.29 (±0.25) 2.58 4 0.63 62 –Delta-SEL Deltamethrin + PBO 220 (178–271) 2.09 (±0.21) 2.65 5 0.75 8 10Delta-SEL Deltamethrin + DEF 1312 (1065–1614) 2.19 (±0.24) 3.42 4 0.49 38 1.8Delta-SEL Endosulfan 114 (93.5–139) 2.32 (±0.23) 3.80 5 0.58 86Delta-SEL Endosulfan + PBO 9.83 (7.68–12.4) 2.09 (±0.24) 2.67 4 0.61 – 12Delta-SEL Endosulfan + DEF 22.4 (17.5–28.1) 2.25 (±0.28) 3.89 4 0.42 – 5

a Resistance ratio was calculated as LC50 of Delta-SEL/LC50 of Lab-PK.b Synergism ratio was calculated as LC50 of insecticide/LC50 of insecticide + PBO or of insecticide + DEF.

3.6 Effect of inhibitors on resistanceThe effect of the microsomal oxidase and esteraseinhibitors, PBO and DEF respectively, on the efficacyof deltamethrin and endosulfan was tested (Table 7).Against Lab-PK these inhibitors had no effect onthe LC50 values obtained for deltamethrin. Withendosulfan both inhibitors synergised the activity ofthe insecticide. Against Delta-SEL PBO reduced theLC50 values for both deltamethrin and endosulfanmore than tenfold. DEF also resulted in a significant,albeit much smaller, reduction in LC50 values for bothinsecticides.

4 DISCUSSIONA high level of resistance to two representativepyrethroids (deltamethrin and cypermethrin) andthree organophosphates (profenofos, chlorpyrifos andtriazofos) was found in a field population of S. litura.In recent years, both of these insecticide groups havebeen used extensively to control S. litura on vegeta-bles and other field crops in Pakistan. Selection ofthe field population with deltamethrin significantlyincreased its resistance to this insecticide from anLC50 of 353 µg mL−1 to an LC50 of 2464 µg mL−1,a sevenfold increase. However, the slopes of thedose–mortality curves at G1 in the field-collected pop-ulation and at G4 in the Delta-SEL population were

not significantly different, which indicates low geneticvariation in the population collected from the field.It has been reported that the slope of dose–mortalitycurves represents the phenotypic variation in suscepti-bility in a population comprising both environmentaland genetic components.28 However, Chilcutt andTabashnik29 suggest that the slope of the concentra-tion–mortality line is not a good indicator of geneticvariation in susceptibility owing to being confoundedby the environmental component of variation. Analy-sis of resistant populations relies on comparisons withstandard susceptible laboratory strains. The authorshave used single-pair crosses to remove resistant allelesfrom a heterogeneous strain to generate a suscepti-ble strain in their laboratory.30 By including severalindividuals from each F1 family identified as suscep-tible in bioassays, the odds of incorrectly identifyinga heterogeneous family as a homozygous susceptiblefamily were greatly reduced. The resulting Lab-PKwas significantly more susceptible to chlorpyrifos thananother laboratory population of S. litura from Chinawith identical bioassay systems to the present studies,31

but, by comparison, deltamethrin and cypermethrinwere less toxic.

Cross-resistance can result from non-specificenzymes, such as microsomal oxidases, mutationat an insecticidal target site and factors such as


M Ahmad et al.

delayed cuticular penetration. The high level ofinsensitivity shown by the Delta-SEL population tocypermethrin, profenofos, chlorpyrifos and triazo-fos suggests either a common mechanism affectingthese insecticides or genetically linked independentmechanisms for pyrethroids and organophosphates.Microsomal oxidase (MO) and esterases have beenshown to be associated with organophosphate andpyrethroid resistance.32 Both MO and esterases havemany isoenzymes,33 and, if an insecticide selectssome isoenzymes that can act on different insecti-cides, cross-resistance might be possible. The resultsare in agreement with previously reported cross-resistance between pyrethroids and organophosphatein S. litura31 and Helicoverpa armigera (Hubner).32 Thepresent synergist studies with PBO (an MO-specificinhibitor) suggested that resistance to deltamethrin inthe Delta-SEL population was MO associated. DEF(an esterase-specific inhibitor) had a much reducedeffect, suggesting the possibility of an esterase-basedmechanism or of a non-specific effect of this inhibitor.The lack of cross-resistance of Delta-SEL to chlor-fenapyr would be consistent with an MO-mediatedmechanism, since MOs are believed to be involvedmore in the activation rather than the detoxification ofchlorfenapyr. Piperonyl butoxide is generally regardedas a specific inhibitor of MO.34 It has been shownpreviously that PBO can also inhibit esterase activityin H. armigera35,36 and Plutella xylostella L.19 Micro-somal oxidase- and esterase-mediated resistance topyrethroids has also been reported for a variety ofother insects, including lepidopteran species.19,32,37,38

Since PBO could not completely abolish the resis-tance phenotype, and there was evidence of multi-ple resistance alleles, we tested for cross-resistancebetween deltamethrin and DDT – a classical indica-tor of a knockdown resistance (kdr) mechanism. Thehigh degree of cross-resistance to DDT in Delta-SEL indicated the presence of a kdr mutation in thevoltage-gated sodium channel.

The cross-resistance between deltamethrin andendosulfan was not unexpected. Although the syn-thetic pyrethroids act principally on the voltage-sensitive sodium channels,39 whereas the cyclodienes,including endosulfan, specifically attack the picrotox-inin receptor site,40 both insecticides are believed to besusceptible to metabolic detoxification by microsomaloxidases.

Apart from the selection of resistance, the stabilityof resistance in the absence of exposure to insecticidesis of interest in pest management. In the presentstudy, resistance to deltamethrin in Delta-SEL overfive generations in the absence of selection pressureappeared to be stable. Typically, resistance todeltamethrin in lepidoptera has been reported to beunstable41 irrespective of whether the population hasbeen selected in laboratory or field. The stability ofresistance in Delta-SEL has at least two non-mutuallyexclusive explanations. Firstly, fitness costs expressedby resistant strains can be environmentally dependent

and may not occur under ordinary laboratory cultureconditions.41,42 Alternatively, resistance in the Delta-SEL population may have been near fixation, leadingto a very slow increase in heterozygosity. The variationin resistance between cages and sampling datesdoes, however, lend more support to the formerexplanation. In similar microcosm experiments overseven generations with a P. xylostella population fromPakistan that had been reselected with deltamethrin inthe laboratory, resistance declined rapidly.16 There arediverging opinions on the impact of fitness costs on thereversal of resistance. Tabashnik et al.43 suggested thatfitness costs caused directly by resistance alleles wouldhave an important effect in the field, but Roush44

considered that even strong fitness costs would have aminimal impact.

Crosses between Delta-SEL and Lab-PK indicatedthat resistance to deltamethrin was inherited auto-somally. Unlike resistance to deltamethrin, which isincompletely recessive in some other lepidopteranspecies (e.g. Cydia pomonella L.), in the S. liturapopulation it showed incomplete dominance. In thisrespect, it was similar to pyrethroid resistance reportedin H. armigera45 and deltamethrin resistance in P.xylostella from Pakistan.16 These results are also inbroad agreement with those for a Bacillus thuringien-sis Cry1Ac toxin resistant population of P xylostellafrom lowland Malaysia.17,46 The significant deviationsbetween observed and expected dose–responses sug-gested that resistance to deltamethrin in delta-SEL isnot due to a single major gene, and that it adheresto the general pattern described by McKenzie andBatterham47 for laboratory-selected polygenic resis-tance. Polygenic resistance has been found to beequally distributed among field and laboratory res-elected populations.46 It seems, therefore, that bothmajor and minor genes can contribute to field andlaboratory selected resistance, as theoretical findingspredict,48 although major genes, which by definitionhave a larger effect on fitness, will tend to respond toany kind of selection more rapidly than minor genes.If resistance had been controlled entirely by one locus,then the sole allele would have been fixed and furtherincrease in resistance would not have occurred in selec-tion experiments. However, the observed increased inresistance indicates that the Delta-SEL had more thanone locus affecting resistance. Stability of resistancein the absence of selection pressure, dominance ofresistant genes to deltamethrin and cross-resistancebetween pyrethroids and organophosphates suggestthat alternative insecticides with different modes ofaction should be recommended to reduce pyrethroidselection pressure. Avoiding pyrethroid use on crops,other than major cash crops, would also enhance resis-tant management, as S. litura is a polyphagous pest.Alternative insecticides such as spinosad, fipronil,phoxim and chlorfenapyr, with different modes ofaction, can be included in the management plan.These insecticides have demonstrated effectiveness



and no cross-resistance with pyrethroids against resis-tant S. litura in the authors’ laboratory studies. Theestablishment and spread of pyrethroid-resistant S.litura could also have serious implications for thesuccessful use of Bt-transgenic crops; therefore, thissituation must be closely monitored.

ACKNOWLEDGEMENTSThese studies were part of PhD work by the firstauthor, and sincere thanks are due to the HigherEducation Commission of Pakistan for granting aPhD Merit Scholarship to carry out studies at BZU,Pakistan, and an IRSIP fellowship for studies at theUniversity of Sussex, UK. The authors are extremelygrateful to the editor for his guidance on improvingthe text of the paper.

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