Efficacy of insecticide mixtures against pyrethroid- and organophosphate-resistant populations of Spodoptera litura (Lepidoptera: Noctuidae)

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Research ArticleReceived: 28 March 2008 Revised: 28 June 2008 Accepted: 28 June 2008 Published online in Wiley Interscience: 2 December 2008

(www.interscience.wiley.com) DOI 10.1002/ps.1681

Efficacy of insecticide mixtures againstpyrethroid- and organophosphate-resistantpopulations of Spodoptera litura(Lepidoptera: Noctuidae)†

Munir Ahmad,a Mushtaq Ahmed Saleema and Ali H Sayyedb∗

Abstract

BACKGROUND: Spodoptera litura (F.) is an important pest worldwide, with over 112 host plants, and is exposed to insecticidesthroughout the year, resulting in the rapid development of resistance. Insecticide mixtures can delay the development ofresistance more effectively than sequences or rotations. Cypermethrin, deltamethrin, profenofos, chlorpyrifos and fipronil wereassessed separately and in mixtures against laboratory susceptible S. litura and two field-collected populations.

RESULTS: The field-collected population from Khanewal (KWL) was significantly more resistant to cypermethrin, deltamethrin,chlorpyrifos and profenofos than one collected from Muzaffar Garh (MGH). Mixtures of cypermethrin + chlorpyrifos orprofenofos and of deltamethrin + chlorpyrifos or profenofos at 1 : 1, 1 : 10 and 1 : 20 ratios significantly increased (P < 0.01)toxicity to cypermethrin and deltamethrin in field populations. The combination indices of cypermethrin + chlorpyrifos at 1 : 1and 1 : 10 ratios and cypermethrin + fipronil at 1 : 1, 1 : 10 and 1 : 20 ratios for the KWL strain and of cypermethrin + profenofosor fipronil at 1 : 1, 1 : 10 and 1 : 20 ratios for MGH were significantly below 1, suggesting synergistic interactions. The inhibitorsDEF and PBO largely overcame resistance to deltamethrin, cypermethrin and profenofos, suggesting that resistance to theinsecticides was associated with esterase and monooxygenase detoxification respectively.

CONCLUSION: Chlorpyrifos, profenofos and fipronil could be used in mixtures to restore cypermethrin and deltamethrinsusceptibility. These findings may have considerable practical implications for S. litura resistance management.c© 2008 Society of Chemical Industry

Keywords: Spodoptera litura; antagonism; synergism; pyrethroids; organophosphates; PBO, DEF

1 INTRODUCTIONSpodoptera litura (Fabricius) is an important insect pest of the SouthAsian region with a voracious leaf-feeding habit and a large hostrange of more than 112 plant species.1,2 It is responsible for indirectand direct damage to crops.3 The presence of this pest on differentcrops throughout the year has widely exposed it to insecticidesand resulted in the rapid development of resistance to a range ofthese.4 Resistance management strategies, using insecticide mix-tures aimed primarily at addressing resistance to pyrethroids andorganophosphates, has been widely used in West Africa, so allow-ing continued use of these insecticides.5 In spite of all efforts, casesof resistance and cross-resistance to pyrethroids and organophos-phates are increasing. This phenomenon has become more alarm-ing as there are very few classes of insecticides available, with onlya limited number of modes of action. Furthermore, the availabilityof new products is limited owing, in part, to the rising standardsof environmental and toxicological safety that are required.6 Toavoid selecting for any particular type of resistance, alternativeclasses of insecticides should be sprayed in sequence, in rotationor as mixtures of compounds with different modes of action.

Theoretically, under certain conditions, insecticide mixturescan delay development of resistance more effectively than se-

quences or rotations7 because, if resistance to each compoundis independent and initially rare, the associated probability ofresistance to both compounds is then extremely rare.8 In WestAfrica, for example, the use of organophosphate and pyrethroidmixtures for cotton spraying has apparently prevented the de-velopment of pyrethroid resistance in populations of the cottonbollworm, Helicoverpa armigera Hubner (Lepidoptera: Noctuidae),for more than 20 years.9 Thus, synergistic interactions may occurbetween the different components used in combination, lead-

∗ Correspondence to: Ali H Sayyed, Department of Biochemistry, School of LifeSciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.E-mail: ali [email protected]

† This article was published online on December 2, 2008. An error wassubsequently identified in the authorship of this article. This notice is includedin the online and print versions to indicate that both have been corrected[January 23, 2009].

a Department of Entomology, University College of Agriculture, BahauddinZakariya University, Multan 60800, Pakistan

b Department of Biochemistry, School of Life Sciences, University of Sussex,Falmer, Brighton BN1 9QG, UK

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Rationale of insecticide mixtures for Spodoptera litura www.soci.org

ing to reduced cost and increased efficacy.4,9,10 Synergism theoryis based on the ability of one molecule to interfere with themetabolic detoxification of another.11 The existence of synergisticinteractions between pyrethroids and organophosphates or car-bamates has been reported in dipteran,12,13 lepidopteran4,9,10,14

and hemipteran pests.4 Gunning et al.14 found that synergismbetween pyrethroids and organophosphates was caused by inhi-bition of esterases, thereby preventing degradation of pyrethroids.In such cases, pyrethroid and organophosphate mixtures providea level of synergism by competitive substrate inhibition.

The mixture of pyrethroids and organophosphates has beenwidely studied in a number of lepidopteran insect pests, but tothe best of the authors’ knowledge there is no documented reportof the mixture efficacy against resistant S. litura. The objectivesof this study, therefore, were to determine the effect of mixingdeltamethrin and cypermethrin with either profenofos, chlorpyri-fos or fipronil against a laboratory susceptible population (Lab-PK)and two field populations of S. litura deriving from Khanewaland Muzaffar Garh (Southern Punjab, Pakistan). Previously, itwas reported that S. litura from these areas are highly resistantto pyrethroids and organophosphates.4 To evaluate insecticidemixtures against S. litura, standard bioassay procedures wereused to assess whether mixtures of deltamethrin or cypermethrinand chlorpyrifos, profenofos and fipronil could yield additive, an-tagonistic or synergistic effects. It was also of interest to establishwhether two synergists, piperonyl butoxide (PBO), an inhibitorof cytochrome P450 monooxygenases (microsomal oxidases)and of esterases, and S,S,S-tri-n-butyl phosphorotrithioate (DEF),an esterase-specific inhibitor, could synergise the activities ofthe insecticides under investigation. Such studies could help toformulate a strategy to minimise the development of resistanceto insecticides in S. litura and provide information that is of use inthe broader context of resistance management.

2 MATERIALS AND METHODS2.1 InsectsSpodoptera litura infestations in Pakistan generally start at the endof March and continue until the end of November, dependingupon the cropping pattern.4 The pest is continuously exposed toinsecticides from April to early November, as it receives sprays firston vegetables (cauliflower, arum and okra) and then on fodder(berseem). When cotton emerges in the field, it moves to thiscrop and remains feeding on it throughout the season. Growerscarry out 1–2 sprays per week using a recommended field rateof an organophosphate (chlorpyrifos or profenofos), a pyrethroid(deltamethrin, cypermethrin or alpha-cyahlothrin) and one of theseveral newer insecticides (spinosad, fipronil or indoxacarb) onvegetables, and 2–3 sprays per week of the same insecticideson cotton to control S. litura.5 Spodoptera litura populations fromcotton were collected, as the treatment regimes used provide agreater chance for the generation of resistance than the regimesused in vegetables.

By walking through the crops in each field from two districts,namely Khanewal and Muzaffar Garh, approximately 300–500larvae were collected. The area is under multiple cropping systemswith several cultivated crops such as cotton, maize, sorghum,millet, rice, sugarcane, wheat, potato, vegetables and foddercrops. These crops are grown side by side, depending on theseason. An insecticide-susceptible S. litura population, designatedas Lab-PK, was collected locally from the field and selectedfor susceptibility in the laboratory as described previously.4

Larvae were reared on semi-synthetic chickpea-based diet inthe laboratory at 25 ± 2 ◦C and 60–65% relative humidity witha 14 : 10 h light : dark photoperiod.4 Diet was replaced after 24 h,and pupae were collected on alternate days. The adults thatemerged were kept in Perspex oviposition cages (30×30×30 cm)with two sides sealed with muslin to maintain ventilation andfed on a solution containing sucrose (100 g L−1), vitamin solution(20 mL L−1) and methyl 4-hydroxybenzoate (2 g L−1) presentedon a soaked cotton wool ball.4 Populations were reared in thelaboratory for one generation to obtain sufficient numbers ofinsects for bioassays.

2.2 InsecticidesCommercial formulations of the different insecticides usedin bioassays comprised cypermethrin 100 g L−1 EC (Arrivo

10EC; FM, Philadelphia, PA), deltamethrin 105 g L−1 EC (DecisSuper 10.5EC; Bayer Crop Science, France), profenofos 500 g L−1

EC (Curacron 50EC; Syngenta Crop Protection, Switzerland),chlorpyrifos 400 g L−1 EC (Lorsban 40EC; Dow AgroSciences,UK) and fipronil 360 g L−1 EC (Regent 36EC; Bayer Crop Science,France).

2.3 BioassaysBioassays were conducted using newly moulted second-instarlarvae (3–6 h old) of S. litura from F1 laboratory cultures using astandard leaf disc bioassay method.4,15 The discs of 5 cm diameterwere cut from cotton leaves collected from unsprayed fields.These were washed, dried, immersed in a test solution for 10 s andallowed to dry on corrugated kitchen foil at ambient temperaturefor 1–1.5 h. Test solutions of insecticides were freshly preparedin distilled water containing 5 µg mL−1 surfactant (Stapple;Dupont, Pakistan). Leaf discs immersed in distilled water andstapple only comprised the control treatments. On drying, theleaf discs were placed in individual petri dishes (5 cm diameter)containing moistened filter paper. Each treatment (concentration)was replicated 8 times, including controls. Five second-instarlarvae were placed on each leaf disc (replication), and thus thetotal number of tested larvae per concentration was 40. Thebioassays were kept at a temperature 25 ± 2 ◦C and 65% relativehumidity with a 14 : 10 h light : dark photoperiod. Mortality wasassessed after 72 h exposure to the insecticides.

2.4 Effect of inhibitors on insecticide toxicityThe toxicities of the insecticides alone and in mixtures wereevaluated in the presence of two synergists, piperonyl butox-ide (PBO; Sigma Ltd, UK), an inhibitor of cytochrome P450monooxygenases (microsomal oxidases) and of esterases, andS,S,S-tributylphosphorotrithioate (DEF; Sigma Ltd, UK), an esterase-specific inhibitor. Stock solutions (10 mg mL−1) of PBO and DEFwere prepared in acetone (analytical reagent grade; Fisher Scien-tific, Loughborough, UK). To test the effect of PBO and DEF onthe toxicity of insecticides, 10 mg mL−1 of synergist was added toeach of the various concentrations of insecticide. Mortality datawere recorded after a 48 h exposure period. The synergism ratio(SR) was calculated by dividing the LC50 of the population treatedwith insecticide by the LC50 of a strain treated with insecticide plussynergist.

2.5 Evaluation of mixturesEach given insecticide mixture could give mortality greater(synergism) or less (antagonism) than the expected additive effect

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www.soci.org M Ahmad, MA Saleem and AH Sayyed

(summation). To determine which of these three possibilitiesresulted from a mixture, mixtures of two insecticides were testedat ratios of 1 : 1, 1 : 10 and 1 : 20 with serial concentrations.The synergism or antagonism can be assessed using variousmethods.16 – 18 The present authors used the combination index(CI) method described by Chou and Talalay16 and the comparisonof observed and expected LC50 values. The CI method is based onthe dose effect of each product used in the mixture and alone. TheCI for quantifying synergism was calculated as follows:

CIx = LC(1m)x

LC1x

+ LC(2m)x

LC2x

+(

LC(1m)x

LC1x

× LC(2m)x

LC2x

)

where LC(1m)x and LC(2m)

x are the lethal doses of insecticides 1 and 2,respectively, used in the mixture, giving mortality x, and LC1

x andLC2

x are the lethal doses of insecticides 1 and 2 that are requiredto produce the same mortality as x when used alone. When twoinsecticides in a mixture have an additive effect, the lethal dose ofthe mixture is as expected and CI = 1. When two insecticides aresynergistic, the lethal dose of the mixture is lower than expectedand results in CI < 1. When two insecticides are anatagonistic,toxicity of the mixture is higher than expected and gives CI > 1.The CI values were calculated at the LC50 dose.

The toxicity of a mixture originates from the sum of theinsecticide intrinsic toxicities (%M1 and %M2) and any synergisticeffect. The toxicity because of the synergism of the mixture wasestimated as

%M(1+2) = 100 − (%M1 + %M2).

2.6 Data analysisMortality data were corrected using Abbott’s formula19 wherenecessary, and estimates of the LC50 values and their 95%fiducial limits were obtained by probit analysis20 using POLO-PC software.21 Because of the inherent variability of the bioassays,pairwise comparisons of LC50 values were made at the 1%significance level (where individual 95% FL for two treatmentsdid not overlap).22

3 RESULTS3.1 Toxicity of test insecticides alone or in combinationagainst a susceptible population (Lab-PK)The toxicities of chlorpyrifos and fipronil were significantly higher(P < 0.01; non-overlapping of 95% FL) than those of cypermethrin,deltamethrin and profenofos (Table 1). There was no differencein the toxicities of cypermethrin and deltamethrin towards theLab-PK strain.

The cypermethrin + chlorpyrifos mixture was significantly moretoxic than cypermethrin + profenofos and cypermethrin + fipronilat the 1 : 10 and 1 : 20 ratios. The toxicity of deltamethrin wasalso significantly increased when mixed with chlorpyrifos overthat of mixtures with profenofos or fipronil at 1 : 10 and 1 : 20ratios (Table 1). In contrast, cypermethrin and deltamethrin mixedwith chlorpyrifos, profenofos or fipronil at the 1 : 1 ratio weresignificantly less toxic (P < 0.01) when compared with the 1 : 10or 1 : 20 ratios (Table 1).

Mixtures of cypermethrin with either chlorpyrifos, profenofosor fipronil produced antagonistic effects at the 1 : 1, 1 : 10 and1 : 20 ratios, with combination index (CI) values all greater than 1(Table 2). Similarly, deltamethrin mixed with either profenofos or

fipronil also resulted in antagonistic interactions at the 1 : 1, 1 : 10or 1 : 20 ratios with CI values greater than 1. The deltamethrin andchlorpyrifos mixture was antagonistic at the 1 : 1 and 1 : 20 ratios,but the 1 : 10 ratio produced synergistic results with a CI of 0.77(Table 2).

3.2 The toxicity of insecticides alone and in combinationagainst field populations of Spodoptera lituraThe field population from Khanewal (KWL) was significantlymore resistant to cypermethrin, deltamethrin, chlorpyrifos andprofenofos than the population deriving from Muzaffar Garh(MGH) when compared with the Lab-PK strain (Table 1). Resistanceto fipronil in the KWL and the MGH populations was significantlyhigher than for the other insecticides tested. The slope of theregression line for the insecticides tested was not significantlydifferent from 2, which confirmed the heterogeneity of resistancein the field populations.

The use of chlorpyrifos and profenofos in mixtures withcypermethrin or deltamethrin in ratios of 1 : 1, 1 : 10 and 1 : 20significantly (P < 0.01) reduced resistance to cypermethrin anddeltamethrin in the KWL population. However, the fipronil +deltamethrin mixture at the 1 : 20 ratio had an additive effect,as the LC50 of the deltamethrin and fipronil mixture was notsignificantly different (P < 0.01; non-overlapping of 95% FL) fromthat of deltamethrin alone (Table 1). Similarly, mixing chlorpyrifos,profenofos or fipronil with cypermethrin significantly reducedthe LC50 of cypermethrin against the MGH population (Table 1).The addition of profenofos and fipronil to deltamethrin in ratiosof 1 : 1, 1 : 10 and 1 : 20 produced an additive effect, since theLC50 values of the profenofos + deltamethrin and fipronil +deltamethrin mixtures were similar to the LC50 of deltamethrinalone for the MGH population (Table 1). In contrast, the LC50 valuesof the chlorpyrifos + deltamethrin mixture in 1 : 1, 1 : 10 and 1 : 20ratios were significantly lower than the LC50 of deltamethrin alone(Table 1).

To assess the synergism or antagonism between two insecti-cides, the combination index was calculated for each mixture. TheCIs of cypermethrin + chlorpyrifos in ratios of 1 : 1 and 1 : 10 andof cypermethrin + fipronil in ratios of 1 : 1, 1 : 10 and 1 : 20 againstKWL were significantly below 1, suggesting a synergistic interac-tion between the insecticides. Likewise, the CIs of cypermethrin +profenofos or fipronil at 1 : 1, 1 : 10 and 1 : 20 ratios against the MGHpopulation were also significantly below 1, indicating synergisticinteraction (Table 2). At the LC50 of the cypermethrin + chlorpyri-fos mixture, the toxicity associated with cypermethrin alone was0% and the toxicities of the mixtures were 40, 25 and 52% at the1 : 1, 1 : 10 and 1 : 20 ratios respectively. Thus, the synergistic effectof the mixtures was 60, 75 and 48% of the observed mortality atthe 1 : 1, 1 : 10 and 1 : 20 ratios respectively. The CI for cypermethrin+ profenofos at 1 : 1, 1 : 10 and 1 : 20 ratios was greater than 1,suggesting antagonistic interactions between the insecticides. Incontrast to the CI of cypermethrin mixtures against KWL, the CIsof deltamethrin + chlorpyrifos at 1 : 1, 1 : 10 and 1 : 20 ratios and ofdeltamethrin + profenofos at 1 : 1 ratio were significantly below 1,suggesting synergistic interaction between the insecticides in themixtures (Table 2). The CIs for deltamethrin + profenofos at 1 : 10and 1 : 20 ratios and deltamethrin + fipronil at 1 : 1, 1 : 10 and 1 : 20ratios were significantly greater than 1, suggesting antagonisticinteractions between the insecticides.

Unlike the KWL population, the CIs for cypermethrin +profenofos at 1 : 1, 1 : 10 and 1 : 20 were significantly below1, indicating a synergistic interaction in the MGH population.

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Contrasting results were obtained for deltamethrin + chlorpyrifos,

profenofos or fipronil, with CIs significantly greater than 1 at

all ratios tested for the mixtures. This was also supported by

the fact that, when the insecticides in the mixture were tested

alone, the mortality was less than expected. For example, in the

deltamethrin + chlorpyrifos mixture at 1 : 10 ratio tested against

the MGH population, deltamethrin alone at 1.88 µg AI mL−1 had

0% mortality and chlorpyrifos at 18.82 µg AI mL−1 had 83%

mortality, with the combination of the two insecticides giving only17% mortality, suggesting antagonistic interaction (Table 2).

3.3 Effect of synergists on insecticidesThe KWL field population had 19-, 45-, 36- and 108-fold resistanceratios to deltamethrin, profenofos, cypermethrin and fipronilrespectively by comparison with the Lab-PK strain (Tables 1and 3). Piperonyl butoxide and DEF largely overcame resistance

Table 1. Toxicity of cypermethrin and deltamethrin alone and in combination with chlorpyrifos, profenofos and fipronil to a susceptible, Lab-PKand two resistant (KWL and MGH) strains of Spodoptera litura

Strain Insecticide tested Ratio LC50 (95% FL) (µg AI mL−1) Slope (±SE) χ2 df P n RRa

Lab-PK Cypermethrin 1 : 0 27.0 (19.8–36.4) 2.19 (±0.34) 2.57 5 0.77 280 –

Deltamethrin 1 : 0 45.2 (31.8–66.5) 1.61 (±0.24) 2.12 6 0.91 320 –

Chlorpyrifos 1 : 0 0.51 (0.37–0.71) 1.89 (±0.27) 1.48 6 0.96 320 –

Profenofos 1 : 0 4.66 (3.24–6.73) 1.59 (±0.24) 2.93 6 0.82 320 –

Fipronil 1 : 0 0.66 (0.49–0.90) 2.19 (±0.34) 2.15 5 0.83 280 –

Cypermethrin + chlorpyrifos 1 : 1 14.5 (10.3–19.8) 1.97 (±0.31) 2.13 5 0.83 280 –

1 : 10 1.15 (0.86–1.54) 2.31 (±0.35) 2.13 5 0.83 280 –

1 : 20 2.91 (2.20–3.81) 2.57 (±0.39) 1.65 5 0.90 280 –

Cypermethrin + profenofos 1 : 1 38.1 (27.5–51.3) 2.12 (±0.32) 1.32 5 0.93 280 –

1 : 10 14.0 (10.3–18.5) 2.36 (±0.37) 2.19 5 0.82 280 –

1 : 20 12.4 (8.01–17.8) 1.60 (±0.28) 1.51 5 0.91 280 –

Cypermethrin + fipronil 1 : 1 29.3 (21.0–41.0) 1.79 (±0.26) 1.32 6 0.97 320 –

1 : 10 9.28 (6.36–13.0) 1.67 (±0.25) 1.43 6 0.96 320 –

1 : 20 11.1 (8.08–14.9) 2.17 (±0.34) 1.53 5 0.91 280 –

Deltamethrin + chlorpyrifos 1 : 1 1.46 (1.09–1.93) 2.38 (±0.36) 1.78 5 0.88 280 –

1 : 10 0.43 (0.31–0.58) 2.10 (±0.33) 2.14 5 0.83 280 –

1 : 20 0.68 (0.48–0.94) 1.98 (±0.32) 2.13 5 0.76 280 –

Deltamethrin + profenofos 1 : 1 10.6 (8.03–13.7) 2.83 (±0.47) 1.87 4 0.80 240 –

1 : 10 20.1 (14.9–26.6) 2.72 (±0.54) 1.01 3 0.90 200 –

1 : 20 22.4 (14.7–32.9) 1.44 (±0.23) 2.19 6 0.90 320 –

Deltamethrin + fipronil 1 : 1 7.05 (5.41–9.01) 3.11 (±0.52) 1.04 4 0.85 240 –

1 : 10 11.9 (9.15–15.2) 3.04 (±0.50) 1.34 4 0.86 240 –

1 : 20 14.3 (11.0–18.4) 2.96 (±0.49) 1.32 4 0.92 240 –

KWL Cypermethrin 1 : 0 960 (751–1208) 2.05 (±0.22) 1.43 5 0.83 280 35.6

Deltamethrin 1 : 0 861 (681–1085) 1.78 (±0.18) 2.14 5 0.66 280 19.0

Chlorpyrifos 1 : 0 28.1 (22.6–35.0) 1.93 (±0.19) 3.23 5 0.68 280 55.1

Profenofos 1 : 0 208 (165–260) 1.84 (±0.19) 3.15 5 0.76 280 44.6

Fipronil 1 : 0 71.5 (55.0–90.3) 1.69 (±0.18) 2.61 5 0.76 280 108

Cypermethrin + chlorpyrifos 1 : 1 31.0 (21.9–42.5) 1.98 (±0.32) 1.76 5 0.88 280 2

1 : 10 16.2 (11.2–23.0) 1.57 (±0.21) 1.32 7 0.98 360 14

1 : 20 29.5 (21.6–40.1) 2.27 (±0.39) 1.98 4 0.74 240 10

Cypermethrin + profenofos 1 : 1 307 (244–385) 3.72 (±0.63) 2.13 4 0.71 240 8

1 : 10 372 (304–457) 2.17 (±0.21) 2.14 5 0.83 280 27

1 : 20 402 (328–492) 2.31 (±0.25) 2.16 4 0.71 240 32

Cypermethrin + fipronil 1 : 1 24.9 (18.6–33.1) 2.36 (±0.36) 2.17 5 0.83 280 0.85

1 : 10 31.7 (23.4–41.9) 2.37 (±0.36) 1.95 5 0.86 280 3

1 : 20 46.3 (34.3–62.7) 2.19 (±0.34) 1.99 5 0.85 280 4

Deltamethrin + chlorpyrifos 1 : 1 17.3 (12.3–23.7) 2.14 (±0.38) 2.15 4 0.71 240 12

1 : 10 12.2 (9.89–15.1) 2.07 (±0.21) 2.16 5 0.83 280 28

1 : 20 10.1 (8.25–12.4) 2.15 (±0.21) 2.19 5 0.82 280 15

Deltamethrin + profenofos 1 : 1 258 (183–359) 1.80 (±0.26) 3.12 5 0.68 280 24

1 : 10 375 (300–464) 2.25 (±0.24) 3.13 5 0.68 280 19

1 : 20 405 (323–506) 1.83 (±0.17) 3.14 6 0.79 320 18

Deltamethrin + fipronil 1 : 1 362 (265–491) 2.04 (±0.29) 3.16 5 0.68 280 51

1 : 10 386 (295–492) 1.98 (±0.22) 3.15 5 0.68 280 32

1 : 20 807 (621–1058) 1.42 (±0.14) 1.01 6 0.98 320 56


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Table 1. (Continued)

Strain Insecticide tested Ratio LC50 (95% FL) (µg AI mL−1) Slope (±SE) χ2 df P n RRa

MGH Cypermethrin 1 : 0 287 (218–380) 2.13 (±0.21) 1.05 5 0.96 280 10.6

Deltamethrin 1 : 0 127 (100–158) 2.35 (±0.27) 1.93 5 0.86 280 2.81

Chlorpyrifos 1 : 0 7.70 (5.71–10.4) 2.02 (±0.20) 1.94 5 0.86 280 15.1

Profenofos 1 : 0 67.7 (54.1–84.8) 1.88 (±0.19) 1.43 5 0.92 280 14.5

Fipronil 1 : 0 62.0 (50.2–76.9) 2.16 (±0.24) 1.63 4 0.80 240 93.9

Cypermethrin + chlorpyrifos 1 : 1 17.5 (12.3–24.6) 1.71 (±0.25) 1.67 6 0.95 320 1

1 : 10 22.4 (14.7–32.9) 1.44 (±0.23) 1.98 6 0.92 320 19

1 : 20 59.5 (43.8–80.8) 2.15 (±0.33) 2.17 5 0.83 280 20

Cypermethrin + profenofos 1 : 1 22.4 (14.7–32.9) 1.44 (±0.23) 2.33 6 0.89 320 0.59

1 : 10 54.7 (42.8–68.7) 2.08 (±0.22) 2.14 5 0.83 280 48

1 : 20 40.6 (32.7–49.7) 2.57 (±0.29) 1.53 4 0.71 240 3

Cypermethrin + fipronil 1 : 1 31.0 (21.9–42.5) 1.98 (±0.32) 2.19 5 0.82 280 1

1 : 10 25.9 (17.6–35.6) 2.08 (±0.39) 2.18 4 0.83 240 3

1 : 20 31.6 (23.4–42.8) 2.21 (±0.34) 2.18 5 0.83 280 3

Deltamethrin + chlorpyrifos 1 : 1 15.3 (10.8–21.1) 1.84 (±0.27) 3.12 5 0.68 280 10

1 : 10 20.7 (16.6–25.8) 1.94 (±0.19) 1.43 5 0.92 280 48

1 : 20 9.40 (7.50–11.8) 1.87 (±0.19) 1.54 5 0.91 280 14

Deltamethrin + profenofos 1 : 1 118 (83.7–163) 1.85 (±0.27) 1.53 5 0.91 280 11

1 : 10 146 (116–180) 2.48 (±0.28) 1.56 4 0.82 240 7

1 : 20 131 (105–161) 2.49 (±0.29) 1.76 4 0.78 240 6

Deltamethrin + fipronil 1 : 1 98.7 (68.0–138) 1.72 (±0.25) 1.45 5 0.92 280 14

1 : 10 103 (79.8–131) 2.05 (±0.23) 1.38 5 0.93 280 9

1 : 20 80.1 (62.5–101) 2.05 (±0.22) 1.67 5 0.89 280 6

a RR = resistance ratio, calculated as (LC50 of field population)/(LC50 of Lab-PK).

to deltamethrin, cypermethrin and profenofos, showing 1-,3-, 4-, 3- and 2-fold resistance ratios, with synergistic ratios of32 and 24 for deltamethrin, 12 and 14 for cypermethrin and14 and 11 for profenofos (Table 3). Similarly, PBO and DEF alsosynergised deltamethrin, cypermethrin and profenofos in the MGHpopulation. In both populations, however, PBO and DEF did notshow synergism with fipronil. Piperonyl butoxide and DEF didnot synergise deltamethrin, cypermethrin, profenofos and fipronilagainst the Lab-PK population (Table 3).

4 DISCUSSIONIn the present studies, bioassays were carried out to evaluatethe insecticidal activities of cypermethrin and deltamethrin, aloneand in combination with chlorpyrifos, profenofos and fipronil,against pyrethroid- and organophosphate-resistant populationsof S. litura. In the KWL population, chlorpyrifos showed synergismwith cypermethrin and deltamethrin at all ratios tested. Incontrast, in the MGH population, chlorpyrifos was antagonisticto cypermethrin and deltamethrin, suggesting that the two strainshad different resistance mechanisms. When profenofos was mixedwith cypermethrin or deltamethrin, it showed antagonism in theKWL population at 1 : 1 and 1 : 10, but synergism was observed atthe same ratios in the MGH population. Synergism of cypermethrinand deltamethrin with profenofos has been reported previouslyin H. armigera,14,23 Spodoptera littoralis (Boisduval),24 Bemisiatabaci (Gennadius)25 and Pectinophora gossypiella (Saunders).26

In contrast, antagonism of chlorpyrifos with cypermethrin inH. armigera from Pakistan and West Africa5,9 and S. littoralisfrom Egypt27 has previously been shown. Mixing fipronil withcypermethrin increased the toxicity of the mixture against the

KWL and the MGH populations, but fipronil showed antagonismwhen mixed with deltamethrin. Recently, Ahmad28 found thatethion (an organophosphate) showed a high level of synergismto several insecticides from the pyrethroid group (cypermethrin,lambda-cyhalothrin, bifenthrin) against H. armigera from Pakistan.

The present findings that chlorpyrifos, profenofos and fipronilincrease the toxicity of cypermethrin and deltamethrin in the re-sistant populations but not in the susceptible population indicatethat the insecticides may be counteracting the mechanisms con-ferring insecticide resistance in the present resistant populations.Pyrethroid detoxification in insect species relies mainly on hydrol-ysis and/or oxidation.29 In spite of several reports of insecticidesynergism in insects, the physiological mechanisms by which in-secticides synergise the activities of other compounds still remainunclear. According to Corbett,11 the general theory of synergismresults from the ability of one insecticide to interfere with themetabolic detoxification of another. It has been shown previouslythat synergism between pyrethroids and organophosphates wascaused by inhibition by organophosphates of either esterases14

or oxidases,30 thereby preventing degradation of the pyrethroid.The present synergist studies with PBO, a mixed-function oxidase(MFO)-specific inhibitor, and DEF, an esterase-specific inhibitor,suggest that resistance to cypermethrin, deltamethrin, chlorpyri-fos and profenofos in the KWL and the MGH populations was MFOor esterase based (Table 3). The authors have previously shownin another study that resistance to deltamethrin in S. litura fromPakistan is associated with the activity of monooxygenase andesterases.4 Fipronil resistance has been shown to be associatedwith esterases in Chilo suppressalis (Walker), as triphenyl phos-phate (TPP), DEF and PBO significantly synergised the activityof fipronil.31 Esterase- or monooxygenase-mediated resistance to


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Table 2. Combination index and intrinsic and synergistic mortalities for different insecticide mixtures in laboratory susceptible (Lab-PK) and fieldpopulations of Spodoptera litura

Active ingredient ratios At LC50 level

Strain Active ingredientUsedratios

Calculated valueof insecticide A

Calculated valueof insecticide B CI %M1 %M2 %M(1+2)

Lab-PK Cypermethrin + chlorpyrifos 1 : 1 7.25 7.25 18.30 13 100 −13

Cypermethrin + profenofos 1 : 1 19.05 19.05 7.68 38 95 −33

Cypermethrin + fipronil 1 : 1 14.65 14.65 34.78 30 100 −30

Cypermethrin + chlorpyrifos 1 : 10 0.10 1.04 2.06 0 68 32

Cypermethrin + profenofos 1 : 10 1.27 12.73 2.91 0 75 25

Cypermethrin + fipronil 1 : 10 0.84 8.44 13.21 0 100 0

Cypermethrin + chlorpyrifos 1.20 0.14 2.77 5.47 0 93 7



Deltamethrin + chlorpyrifos 1 : 1 0.73 0.73 1.47 0 55 45

Deltamethrin + profenofos 1 : 1 5.30 5.30 1.39 0 48 52

Deltamethrin + fipronil 1 : 1 3.52 3.53 5.84 0 82 18




Deltamethrin + chlorpyrifos 1.20 0.03 0.65 1.27 0 60 40



KWL Cypermethrin + chlorpyrifos 1 : 1 15.50 15.50 0.58 0 40 60





Cypermethrin + fipronil 1.10 2.88 28.82 0.41 0 25 75





Deltamethrin + profenofos 1 : 1 129 129 0.86 2 100 −2

Deltamethrin + fipronil 1 : 1 181 181 3.27 2 100 −2




Deltamethrin + chlorpyrifos 1.20 0.48 9.62 0.34 0 8 92



MGH Cypermethrin + chlorpyrifos 1 : 1 8.75 8.75 1.20 0 60 40





Cypermethrin + fipronil 1.10 2.35 23.55 0.39 0 28 72





Deltamethrin + profenofos 1 : 1 59 59 1.74 25 45 30









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Table 3. Toxicity of deltamethrin, profenofos, cypermethrin and fipronil with and without PBO and DEF (10 µL mL−1 each) to different strains ofSpodoptera litura

Fit of probit lineLC50 (FL at 95%)

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

Lab-PK Deltamethrin 45.2 (31.8–66.5) 1.61 (±0.24) 1.93 5 0.86 –

Profenofos 4.66 (3.24–6.73) 1.59 (±0.24) 2.20 5 0.82 –

Cypermethrin 27.0 (19.8–36.4) 2.19 (±0.34) 2.19 5 0.83 –

Fipronil 0.66 (0.49–0.90) 2.19 (±0.34) 1.43 5 0.93 –

Deltamethrin + PBO 27.7 (19.3–39.3) 1.65 (±0.24) 0.67 5 0.98 – 2

Deltamethrin + DEF 46.5 (35.6–60.4) 1.90 (±0.30) 1.52 4 0.82 – 1

Profenofos + PBO 4.86 (3.36–6.90) 1.57 (±0.21) 1.35 6 0.96 – 1

Profenofos + DEF 2.95 (2.16–4.01) 2.27 (±0.39) 0.41 3 0.93 – 1

Cypermethrin + PBO 20.7 (15.6–26.4) 2.21 (±0.29) 3.83 4 0.43 – 1

Cypermethrin + DEF 29.2 (23.5–36.7) 2.61 (±0.32) 3.77 4 0.44 – 1

Fipronil + PBO 0.82 (0.61–1.05) 1.89 (±0.22) 2.41 5 0.79 19 1

Fipronil + DEF 0.39 (0.31–0.50) 2.04 (±0.22) 2.32 5 0.80 45 2

Deltamethrin 861 (681–1085) 1.78 (±0.18) 4.17 5 0.53 36

Profenofos 208 (165–260) 1.84 (±0.19) 3.67 5 0.60 108

Cypermethrin 960 (751–1208) 2.05 (±0.22) 3.12 5 0.68 1

Fipronil 71.5 (55.0–90.3) 1.69 (±0.18) 2.14 5 0.82 1

Deltamethrin + PBO 26.7 (17.3–38.3) 1.64 (±0.24) 0.67 5 0.98 3 32

Deltamethrin + DEF 36.5 (25.6–50.4) 1.90 (±0.30) 1.52 4 0.82 4 24

KWL Profenofos + PBO 14.3 (11.0–18.4) 2.96 (±0.49) 1.01 3 0.79 3 14

Profenofos + DEF 18.7 (14.3–24.3) 2.69 (±0.40) 1.40 4 0.84 2 11

Cypermethrin + PBO 78.7 (62.1–97.8) 1.53 (±0.18) 2.30 5 0.80 84 12

Cypermethrin + DEF 66.3 (51.2–84.5) 1.80 (±0.18) 3.68 6 0.72 94 14

Fipronil + PBO 55.7 (46.2–67.2) 2.55 (±0.25) 1.43 5 0.92 3 1

Fipronil + DEF 61.9 (46.2–79.0) 2.23 (±0.28) 2.35 5 0.80 14 1

Deltamethrin 127 (100–158) 2.35 (±0.27) 1.65 5 0.90 11

Profenofos 67.7 (54.1–84.8) 1.88 (±0.19) 4.09 5 0.54 94

Cypermethrin 287 (218–380) 2.13 (±0.21) 3.15 5 0.68 1

Fipronil 62 (50.2–76.9) 2.16 (±0.24) 1.96 6 0.92 1

Deltamethrin + PBO 9.23 (6.43–13.1) 1.65 (±0.24) 0.67 5 0.98 1 14

Deltamethrin + DEF 12.1 (8.53–16.8) 1.90 (±0.30) 1.52 4 0.82 2 6

Profenofos + PBO 5.18 (3.52–7.12) 2.08 (±0.39) 0.81 3 0.85 1 13

Profenofos + DEF 9.48 (7.02–12.8) 2.21 (±0.34) 1.75 4 0.78 1 7

Cypermethrin + PBO 9.86 (7.62–12.5) 1.98 (±0.23) 4.74 5 0.44 59 29

Cypermethrin + DEF 13.5 (10.4–17.2) 2.02 (±0.23) 2.04 5 0.84 69 21

MGH Fipronil + PBO 38.9 (28.8–49.6) 2.35 (±0.28) 3.14 4 0.53 1

Fipronil + DEF 45.8 (34.2–58.9) 2.07 (±0.24) 2.30 5 0.80 1

a RR = resistance ratio, calculated as (LC50 of field population)/(LC50 of Lab-PK).b SR = synergism ratio, calculated as (LC50 of field population)/(LC50 of insecticide + PBO or DEF).

pyrethroids or organophosphates has been reported for a varietyof other insects, including lepidopteran species.4,32,33 The involve-ment of both monooxygenase and esterase enzymes in testedfield populations might have some effect on the toxicity of thesemixtures, exhibiting a potentiating role in most tested mixtures.The present results are similar to previously reported studies withH. armigera from Pakistan using pyrethroid and organophosphateinsecticides.2 The synergism between pyrethroid and organophos-phate insecticides suggests that, in the H. armigera populationfrom Pakistan, organophosphates had inhibited the esterases andthereby increased the toxicity of pyrethroid insecticides. The au-thors have no direct evidence to prove organophosphates haveinhibited the enzymes, but further in vivo and in vitro studiescould provide such evidence where a resistant population couldfirstly be challenged with organophosphate and then exposed to

pyrethroids. The synergist studies with fipronil suggest that resis-tance to fipronil in the field populations was not associated withesterases or monooxygenase. The synergism between fipronil andcypermethrin or deltamethrin might therefore be due to mecha-nisms other than esterases or monooxygenase, but further workbased on molecular studies is required to confirm the mechanismof resistance to fipronil in S. litura from Pakistan.

The high level of insensitivity shown by the MGH and KWL fieldpopulations of S. litura to pyrethroids (cypermethrin, pyrethroids),organophosphates (chlorpyrifos, profenofos) and fipronil suggeststhat multiple resistance mechanisms may be present. No cross-resistance between fipronil and organophosphates or pyrethroidshas been reported in S. litura,4 and these compounds differin their site of action in the insect nervous system.33,34 Mixinginsecticides with different modes of action could allow growers


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to attain the benefits of an antiresistance strategy at reducedcost and lower insecticide input into the environment againstthe insect pest complex in cotton. The present data indicatethat chlorpyrifos, profenofos and fipronil could be used torestore cypermethrin and deltamethrin susceptibility in S. liturawhen they are used in mixtures. However, further studies arerequired to establish whether the mixtures have phytotoxicityor adverse effects to natural enemies. This is of particularconcern, as broad-spectrum mixtures, having multiple modesof actions, could have a detrimental impact on beneficial insectsabove that of the insecticides deployed singly.28 These findingsmay have considerable practical implications for the resistancemanagement of S. litura and other lepidopterans [e.g. Plutellaxylostella (Linnaeus), H. armigera, etc.), which are highly resistantto organophosphate and pyrethroid insecticides.35,36 Mixturescould, however, also give rise to multiple resistance, which mayextend across other chemical classes and thus become difficult tomanage,10 as has occurred in P. xylostella in parts of South EastAsia.37 Nevertheless, there is a need to strengthen research on thesynergistic effects of other insecticides. Alternative strategies suchas mosaics or rotations should also be considered, as they couldhelp to avoid development of multiple resistance in insect pests.The present authors have recently shown that insecticide rotationcould delay the development of resistance in H. armigera.38

ACKNOWLEDGEMENTSSincere thanks to the Higher Education Commission of Pakistanfor granting a Merit Scholarship to Munir Ahmad to carry out PhDresearch at the University College of Agriculture, BZ University,Multan, Pakistan, and an IRSIP Fellowship for part of his PhDresearch at the Department of Biochemistry, University of Sussex,Brighton, UK. AHS was supported by BBSRC project BB/C504927/1.The authors are particularly grateful to Dr Sahar Fazal and PaulJohnston for reading and commenting on an earlier version of themanuscript.

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Efficacy of insecticide mixtures against pyrethroid- and organophosphate-resistant populations of Spodoptera litura (Lepidoptera: Noctuidae)