Identification of factors responsible for insecticide resistance in Helicoverpa armigera

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Abstract

Moth larvae (Helicoverpa armigera Hübner) collected from field crops were tested for resistance to cypermethrin, fenvalerate, endosulfan, monocrotophos and quinolphos. Larvae were treated with a dose of the pesticide that would kill 99% of the susceptible insects. The percent survival of the resistant strains was determined. Highest seasonal average percentage survival was recorded by fenvalerate (65.0%) followed by cypermethrin (62.4%). Acetylcholinesterase of resistant larvae was less sensitive to monocrotophos and methyl paraoxon. Resistant larvae showed higher activities of esterases, phosphatases and methyl paraoxon hydrolase compared with susceptible larvae. The presence of high activity of esterases was attributed to appearance of extra bands of esterases in native PAGE. The presence of P-glycoprotein expression was detected in resistant larvae using P-gp antibodies; this was not detected in the susceptible larvae. Our results indicate that the high level of resistance detected in the field pests could be because of a combined effect of decreased sensitivity to AChE, higher levels of esterases, phosphatases and the expression of P-gp.

Introduction

American bollworm (Helicoverpa armigera Hübner; Lepidoptera; Noctuidae) is a major pest of cotton, legumes, and more than 100 other plant species (Bhatnagar et al., 1982). In India, crop losses due to H. armigera are commonly more than half the yield, and annual losses to cotton and pulses alone have been estimated at US $300–500 million (King, 1994). This pest has developed resistance to all the major insecticide classes and it has become increasingly difficult to control their population in India (Dhingra et al., 1988, Armes et al., 1992, Kranthi et al., 1997, McCaffery et al., 1998), Australia (Gunning et al., 1991, Forrester et al., 1993), Indonesia (McCaffery et al., 1991) and Thailand (Ahmad et al., 1989).

Resistance to organophosphate and carbamate insecticides has been reported in H. armigera and the leaf worm, Spodoptera letura (Fabricius) in India (Armes et al., 1996, Armes et al., 1997, Kranthi et al., 2001a). Insensitive acetylcholinesterase (AChE) has been implicated as one of the mechanisms of resistance to organophosphorous (OP) and carbamate insecticides in tobacco white fly, Bemisia tabaci (Genn.); (Dittrich et al., 1985, Dittrich et al., 1990) and Heliothis virescens (Brown and Bryson, 1992). Similar insensitive AChE variants have now been detected in heterogenous populations of many important pest species; in housefly, the cotton aphid (Aphis gossypiella) (Moores et al., 1988, Moores et al., 1989) and several mosquito species (ffrench-Constant and Bonning, 1989).

Model substrates such as naphthyl esters are commonly used in preference to insecticidal esters, which are normally more difficult to use as assaying agents. Insecticide breakdown by metabolism is the common mechanism that has evolved to protect insects (Scott, 1991, Price, 1991). A correlation between high naphthyl esterase activities and resistance to organophosphate and pyrethroid resistance was reported from B. tabaci from Sudanese cotton (Dittrich et al., 1985). Many more cases of esterase mediated OP resistance have been reported for example in mosquito, Culex tarselis (Whyard et al., 1995), tobacco budworm, H. virescens F.; (Goh et al., 1995, Harold and Offea, 2000), Colarado potato beetle, Leptinotarsa decemlineata (Say) (Anspaugh et al., 1995) and Tritoma infestans (Casabe and Zerba, 1981). Increased malathion carboxylesterase activity is responsible for malathion resistance in insects such as Lucilia cuprina (Raftos, 1986) and the green rice leaf hopper, Nephotettix cincticeps (Miyata and Saito, 1976).

Lanning et al. (1996) reported that H. virescens is capable of over expressing P-glycoprotein, primarily localized in the cuticle which is one of the proteins responsible for mediating resistance. This glycoprotein was an analogue of the multidrug resistance (MDR) protein expressed in mammalian cancer cells. It has been shown that a primary function of this protein is the efflux of drugs resulting in a decrease in intracellular drug accumulation (Gottesman and Pastan, 1993, Prasad et al., 1996).

Several mechanisms of resistance have been identified in H. armigera populations in various parts of the world. These include reduced penetration (Gunning et al., 1991, Armes et al., 1992, Kennaugh et al., 1993, Kranthi et al., 1997, Kranthi et al., 2001b), decreased nerve sensitivity (West and McCaffery, 1992) and enhanced metabolism (Ahmad and McCaffery, 1991).

It is clear from these studies that esterases and P-glycoprotein are involved in mediating the resistance in several pests. However, such information is lacking in H. armigera of Indian populations. Here we report the activities of different esterases and the presence of P-gp in the resistant strain of H. armigera in an attempt to explain the mechanism of pesticide resistance in field strain.

Section snippets

Chemicals

C219 antibodies were purchased from Signet Laboratories (Dedham, MA, USA) and ECL luminescence kit from Amersham. Methyl paraoxon was supplied by Sigma-Aldrich (St. Louis, MO, USA). Technical grades of cypermethrin, fenvalerate, endosulfan, monocrotophos and quinolphos were donated by Dr B.V. Patil, Agriculture Research Station, Raichur, India. All other chemicals used were of reagent grade.

Assessment of survival of field insects at a single lethal dose

Adult pod borer larvae were collected from fields cultivating pigeonpea and chickpea around Gulbarga

Assessment of resistance levels

Our studies showed that during 1999–2000 season, H. armigera in the Gulbarga region had high resistance to insecticides. Among the different insecticides tested, the resistance level was high for pyrethroides (fenvalerate and cypermethrin) followed by quinolphos, monocrotophos and endosulfan. The rate of survival was approximately 40.8% during October and as the season advanced the survival rate increased to 75.4% (Table 1). This was true with respect to individual insecticides as well. There

Discussion

High levels of pyrethroid resistance were recorded in intensive cotton and pulse growing regions of Central and Southern India where excessive application of insecticide is common (Armes et al., 1996). H. armigera had developed high resistance as the season advanced and reached the highest level between January and February (Table 1). A similar seasonal pattern of cypermethrin resistance frequencies was reported in the discriminating dose monitoring studies conducted by ICRISAT Asia Form (Armes

Acknowledgments

We thank Dr J.V. Rao, IICT, Hyderabad for carrying part of this work in his lab. We also thank Dr C.L. Christian, A. Stephan and Dr P. Werner, Medical University, Lübeck, Germany for their help in performing the immunoblots. We thank Dr V.H. Mulimani and Dr T.B. Karegoudar, Department of Biochemistry, Gulbarga University, Gulbarga for their support during this study and Dr Cletus D'Souza, Mysore University, Mysore for critical comments.

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