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Esterase Inhibitors Restore Pyrethroid Susceptibility in Australian Helicoverpa armigera

R.V. Gunning1, G.D. Moores2 and A.L. Devonshire2 1 NSW , Tamworth Centre for Crop Improvement, RMB 944 Calala Lane, Tamworth, NSW, Australia 2340. 2 IACR Rothamstead, Harpenden, Herts., AL5 2JQ, UK.

ABSTRACT Pyrethroid resistance in Australian Helicoverpa armigera field populations is primarily a consequence of the over production of esterase isoenzymes that metabolize and possibly sequester pyrethroid . Biochemical studies show that organophosphorous compounds inhibit pyrethroid resistance associated esterases in H. armigera. Esterase inhibition by does not occur immediately after dosage, but occurs rapidly, with maximum inhibition from 2 to 24h after dosage, depending on the inhibitor used. These enzyme inhibition studies are supported by pyrethroid bioassays using synergists, showing excellent levels of pyrethroid synergism against resistant H. armigera. One hundred percent mortality could be achieved when H. armigera were dosed with pyrethroid, during the time of maximum organophosphate induced enzyme inhibition. Use of organophosphate synergists in the field may have the potential to restore some pyrethroid susceptibility in Australian H. armigera.

R.V. Gunning et al.

Introduction experiments. Larvae (2 - 3 mg), were topically applied with 0.5 l of acetone alone, or acetone The bollworm Helicoverpa armigera containing non-toxic doses of , , (Hübner), is a serious of cotton and other , chlorpyrifos- or . The summer crops in Australia, where it has a long larvae were provided with food and left at 25 ± history of resistance. Pyrethroid 1oC. At time intervals from 0.25 - 72 h. after resistance in field populations of Australian H. dosage, samples of larvae (3 - 4 mg), were placed armigera is largely due to overproduction of in the deep freeze and stored in at -15oC until the specific esterase isoenzymes (Rm 0.24 - 0.33), conclusion of the experiments. Polyacrylamide which are thought to sequester and hydrolyze gel electrophoresis preparation methods were pyrethroids (Gunning et al., 1996). However, the similar to those used by Devonshire and Moores degree of resistance to all pyrethroids is not (1982). Total esterase activity of the larval identical and some compounds are more resisted homogenates was detected using 1-naphthyl than others. acetate as a substrate, using kinetic assays, as Previous studies with esterase inhibitors, such as previously described (Gunning et al., 1996). profenofos, have shown little pyrethroid Results synergism against resistant H. armigera (Forester et al., 1993; Gunning et al., 1991). Inhibition of esterase activity by However, these studies used only a very short organophosphates organophosphate pre-treatment period and Gel electrophoresis and assays of total esterase utilized only one highly resisted pyrethroid activity indicated that all organophosphates used () and it is possible that synergism in these experiments bound to H. armigera may have been more effective with longer pre- esterases, and in particular, those bands treatment times and with other pyrethroids. implicated in pyrethroid resistance (Gunning et Indeed, in vivo inhibition of esterase by al., 1996). organophosphates, such as profenofos in Bemisia tabaci, has been shown to take up to 24 hours Acephate rapidly bound to H. armigera (Byrne and Devonshire, 1991). Thus, it seemed esterases, (0.5 - 1h. after treatment), however this logical to consider the possibility that esterase effect was short lived and esterase activity in inhibition (and pyrethoid synergism) in resistant larvae, was completely recovered 8h after H. armigera, might also be enhanced by a longer treatment. Chlorpyrifos and its oxon also rapidly pre-treatment period. inhibited H. armigera esterases, however, recovery of activity was slow, with full enzyme Materials and Methods activity not recovered until 36 h after treatment. Insects and Bioassay Inhibition of esterase activity by profenofos occurred primarily between 4 and 8 h after Pyrethroid resistant H. armigera obtained from treatment (at 8 h, some 70% of esterase activity field collections, was used in these studies. The had been inhibited). However, recovery of H. armigera were susceptible to enzyme activity was rapid. By contrast, ethion organophosphates. Standard 3rd instar larval caused almost complete inhibition of all esterase

bioassays (Gunning et al., 1984) were used to activity, between 1 to 72 h. after application. determine pyrethroid resistance status. Pyrethroid synergism studies were carried out Organophosphate /Pyrethroid Synergism using a non-toxic dose of organophosphate and a standard single discriminating dose pyrethroid. All the organophosphates used synergised Pyrethroids used were fenvalerate (which is an pyrethroids during the time of maximum enzyme example of a highly resisted pyrethroid) and two inhibition, however, the greatest effects were isomers of that are much less obtained with the less resisted pyrethroids. Pre- resisted (alpha and zeta cypermethrin). Resistant treatment with ethion, greatly increased H. armigera larvae (30 mg) were pre-treated with pyrethroid mortality and the increased period of non-toxic doses of ethion, acephate, profenofos, susceptibility persisted up to 24 hours after pre- chlorpyrifos and chlorpyrifos-oxon. Larvae were treatment with the synergist. The synergistic dosed with pyrethroid 0 - 72 h later. Mortality effect declined over longer time intervals. was assessed 48 h after pyrethroid application. Acephate also showed a strong, but short lived, synergistic effect with pyrethroids. These effects Electrophoresis and total esterase assays rapidly declined with time intervals greater than Small larvae, which are the most suitable weight 3h between acephate and pyrethroid applications. range for electrophoretic and biochemical Profenofos was also synergistic with pyrethroids. analysis of esterase isoenzymes in H. armigera However, these effects were not rapid, a 4h time (Gunning et al., 1996) were used in these interval between application of profenofos and Proceedings of the World Cotton Research Conference -2. Athens, Greece, September 6-12, 1998. pp.753-755.

Esterase inhibitors restore pyrethroid susceptibility in Australian Helicoverpa armigera pyrethroid was required to reach maximum The present results support previous findings mortality and the effects rapidly declined (Gunning et al., 1996) that pyrethroid resistance thereafter. Chlorpyrifos was also shown to be a in field populations of Australian H. armigera is reasonably effective pyrethroid synergist. largely a consequence of enhanced esterase Maximum mortality was achieved after a 4 h, activity. pre-treatment time but this level of mortality Evidence that organophosphate esterase rapidly decreased. inhibitors make excellent pyrethroid synergists Discussion has provided additional evidence that pyrethroid resistance in H. armigera is due to esterase These experiments showed that detoxification and the increased production of organophosphates most effectively inhibited these enzymes. These findings may have esterases, which have been linked to pyrethroid considerable practical application for H. resistance in Australian H. armigera (Gunning et armigera resistance management in Australia al., 1996). However, this did not happen and may greatly assist in restoring some measure immediately after application and the timing of of pyrethroid susceptibility in field populations and recovery from inhibition varied according to of H. armigera. organophosphate. While the inhibitory effects of most organophosphates on H. armigera esterases Acknowledgements were generally not long lived, the effects of The H. armigera work was initiated at IACR- ethion lasted at least 72 h. Rothamstead, UK whilst R.V. Gunning was a These data, showing organophosphate inhibition visiting scientist in 1989 and 1992. It was of resistant H. armigera esterases, are supported partially funded by the Cotton Research and by bioassays, in which organophosphates proved Development Corporation, Australia, the to be effective pyrethroid synergists, especially Winston Churchill Memorial Trust of Australia with more effective, (less resisted), pyrethroids. and the Australian Academy of Science/Royal For each organophosphate inhibitor, pyrethroid Society Scientific Exchange IACR-Rothamstead synergism occurred at pre-treatment times that receives grant aided support from the corresponded to maximum vivo esterase Biotechnology and Biological Sciences Research inhibition in the larvae used for biochemical Council of the United Kingdom. studies. Ethion (which is non-toxic and therefore could be applied in larger doses) was a more References effective synergist than any other and was Byrne, F.J. and A.L. Devonshire. (1991): In vivo capable of producing 100% mortality. Had it inhibition of esterase and been possible to increase the synergistic doses of acetylcholinesterase activities by profenofos acephate, chlorpyrifos, and profenofos, (which treatments in the tobacco whitefly Bemisia can be toxic to H. armigera), better synergism tabaci (genn.): Implications for routine would have been expected. biochemical monitoring of these enzymes. These data open up the possibility that Pestic. Biochem. Physiol. 40:198-204. organophosphate synergists could be used in Devonshire, A.L. and G.D. Moores. (1982): A Australia to restore some pyrethroid carboxylesterase with broad substrate susceptibility in H. armigera. Obviously, it specificity causes organophosphorous, would be best to treat with organophosphates and pyrethroid resistance in prior to pyrethroid spray but this approach would peach-potato (Myzus persicae) Pestic require two insecticide applications instead of Biochem. Physiol. 18:235. one and would probably be rejected on economic grounds. However, the better, less resisted Forrester, N.W., M. Cahill, L.J. and J.K. pyrethroids combined with organophosphate Layland. (1993): Management of pyrethroid synergists, could still produce high mortality and resistance in Helicoverpa when applied as a mix with no pre-treatment armigera (Lepidoptera: Noctuidae) in time. The pyrethroids that were most easily Australia, Bull. Ent. Res. Supplement No. 1. synergized were those less resisted. The potential Gunning, R.V., C.S. Easton, M.E. Balfe and I.G. use of organophosphates as pyrethroid synergists Ferris. (1991): Pyrethroid resistance would need to be carefully incorporated into a mechanism in Australian Helicoverpa resistance management strategy because armigera. Pestic. Sci. 33:473. uncontrolled use of synergists designed to eliminate a major, metabolic pyrethroid Gunning, R.V., C.S. Easton, L.R. Greenup and resistance mechanism, may well result in the V. E. Edge. (1984): Pyrethroid resistance in selection of other resistance mechanisms, such as Heliothis armiger (Hübner) Kdr. (Lepidoptera:Noctuidae) in Australia, J. Econ. Ent. 77:1283. Gunning, R.V., G.D. Moores and A.L. Devonshire. (1996): Esterases and fenvalerate resistance in Australian Helicoverpa armigera (Hübner) Lepidoptera:Noctuidae. Pestic. Biochem. and Physiol. 54:12-23.