Pestic. Sci. 1998, 52, 53È57

Efficacy of Plant Metabolites of Imidaclopridagainst Myzus persicae and Aphis gossypii(Homoptera: Aphididae)Ralf Nauen,1,* Klaus Tietjen,2 Klaus Wagner3 & Alfred Elbert11 Bayer AG, Agrochemicals Division, Research Insecticides, Institute for Insect Control, D-51368Leverkusen, Germany2 Bayer AG, Agrochemicals Division, Molecular Target Research/Biotechnology, D-51368 Leverkusen,Germany3 Bayer AG, Agrochemicals Division, Chemical Technology and Analytical Services, D-51368 Leverkusen,Germany

(Received 30 September 1996 ; revised version received 20 March 1997 ; accepted 19 June 1997)

Abstract : The metabolism of the chloronicotinyl insecticide imidacloprid isstrongly inÑuenced by the method of application. Whilst in foliar applicationmost of the residues on the leaf surface display unchanged parent compound,most of the imidacloprid administered to plants by soil application or seed treat-ment is metabolized more or less completely, depending on plant species andtime. The present study revealed that certain metabolites of imidacloprid whichhave been described in crop plants are highly active against aphid pests in di†er-ent types of bioassays. Some of these metabolites showed a high oral activityagainst the green peach aphid (Myzus persicae), and the cotton aphid (Aphisgossypii). The aphicidal potency of the metabolites investigated was weaker inaphid dip tests than in oral ingestion bioassays using artiÐcial double mem-branes. The most active plant metabolite was the imidazoline derivative of imid-acloprid. The values of this metabolite for M. persicae and A. gossypii inLC50oral ingestion bioassays were in the lower ppb-range, i.e. 0É0044 and 0É0068 mglitre~1, respectively. Most of the other reported metabolites showed muchweaker activity. Compared to imidacloprid, the imidazoline derivative showedsuperior affinity to houseÑy (Musca domestica) head nicotinic acetylcholinereceptors, while all other metabolites were less speciÐc than imidacloprid. Itseems possible that, after seed treatment or soil application, a few of the bio-logically active metabolites arising are acting in concert with remaining levels ofthe parent compound imidacloprid, thus providing good control and long-lastingresidual activity against plant-sucking pests in certain crops. 1998 SCI.(

Pestic. Sci., 52, 53È57, 1998

Key words : imidacloprid ; metabolites ; Myzus persicae ; Aphis gossypii ; sachet ;FAO-Dip ; nicotinic acetylcholine receptor

1 INTRODUCTION

The chloronicotinyl insecticide imidacloprid is highlyactive against homopteran pests such as aphids, plant-

* To whom correspondence should be addressed at : BayerAG, PF-F/IB, D-51368 Leverkusen, Germany.Email : RALF.NAUEN.RN=bayer-ag.de

hoppers and whiteÑies, but is also excellent in control-ling some chewing pests, e.g. certain beetles.1h3Imidacloprid acts as an agonist of the nicotinic acetyl-choline receptor (nAChR).4 However, in contrast toother insecticides acting on nAChR, e.g. nicotine, imid-acloprid is highly speciÐc to insect receptors, as studieson di†erent nAChR preparations from insects and ver-tebrates have revealed.5h7 Insecticides acting on nAChRare not common and have not been used as heavily as

531998 SCI. Pestic. Sci. 0031-613X/98/$17.50. Printed in Great Britain(

54 Ralf Nauen et al.

organophosphates, carbamates and pyrethroids in thepast. Thus imidacloprid is a valuable tool in resistancemanagement strategies to control homopteran pestsresistant to the above-mentioned conventional insecti-cides.8,9

Owing to its systemic properties, imidacloprid isoften used in soil (drench) application and seed treat-ment in a variety of Ðeld crops, though its efficacytowards di†erent plant-sucking pests after foliar appli-cation is also very good. In most cases a soil applicationor seed treatment is recommended, thus providing goodresidual activity for several weeks.10,11 In contrast tofoliar application, where most of the remaining residueon the leaf surface is imidacloprid, the active ingredientis metabolized more or less completely after soil appli-cation or seed treatment, depending on plant speciesand time. The general metabolic pathway of imid-acloprid in plants after spray and granular applicationhas been described earlier.12,13 Some of these metabo-lites are closely related to imidacloprid, and the aim ofour study was to elucidate the aphicidal potency ofthese closely related metabolites in oral ingestion bio-assays using two main aphid pests, the cotton aphid(Aphis gossypii Glover), and the green peach aphid(Myzus persicae Sulzer), and to measure the affinity ofplant metabolites of imidacloprid to nAChR in headmembrane preparations from the houseÑy (Muscadomestica L.). Biologically active metabolites could actin concert with remaining levels of imidacloprid in theplant and may provide residual activity as long as orlonger than would imidacloprid residues alone.

2 MATERIALS AND METHODS

2.1 Insecticides, metabolites and chemicals

Imidacloprid was technical grade of the highest purityavailable. The purity of the imidacloprid metabolites,numbered 1–7 (Fig. 1), was at least 97%. Imidaclopridand the plant metabolites of imidacloprid were pro-vided by Bayer AG (Leverkusen, Germany).[3H]Imidacloprid (1É25 ] 1015 Bq mol~1) for receptorbinding studies was synthesized and labelled as hasbeen described elsewhere.5 All other chemicals andorganic solvents used were of analytical grade. Stocksolutions of imidacloprid and metabolites were pre-pared in acetone, and diluted with sucrose solutions(150 g litre~1) or aqueous “TritonÏ X-100 (1 g litre~1)when oral or contact activity was bioassayed, respec-tively.

2.2 Aphids

The green peach aphid (M. persicae), an insecticide-susceptible strain and reared in the laboratory since1967 was maintained on chinese cabbage (Brassicapekinensis (Lour) Rapr) at 22È23¡C, 60% relativehumidity and L16 : D8 photoperiod. An insecticide-susceptible strain of the cotton aphid (A. gosspyii) wasmaintained in the greenhouse on cotton plants(Gossypium hirsutum L.) at 24¡C, 60% relative humid-ity and ambient photoperiod.

Fig. 1. Selected plant metabolites of imidacloprid investigated in this study.

Aphicidal activity of plant metabolites of imidacloprid 55

2.3 Bioassays

All bioassays were performed in the laboratory at 21È22¡C, 45È55% relative humidity and ambient photo-period. All bioassays were repeated at least three timeswith two or three replicates of Ðve or six di†erent con-centrations. The number of aphids in each bioassayranged from 100 to 180. Lethal concentration valueswere computed from probit regressions using the com-puter program POLO PC (LeOra, Software, Berkeley,USA).

2.3.1 Feeding bioassay (sachet test)The aphicidal potency of imidacloprid and its plantmetabolites after oral ingestion was tested using a modi-Ðcation of the so-called sachet test described in detailelsewhere.9,14 Acetonic stock solutions of the com-pounds were diluted in aqueous sucrose (150 g litre~1) ;the acetone concentration in the bioassayed dilutionswas less than 10 ml litre~1. The prepared solution(0É4 ml) was pipetted between two layers of stretched

which formed the sachet. Groups of 10È15“ParaÐlmÏ'aphids, which had been starved for 4 h prior the bio-assay, were placed into small Petri dishes (diameter2É8 cm) including an appropriate Ðlter paper disc. Theholding containers were sealed by stretching the pre-pared sachets across the top. A piece of yellow cellop-hane was placed over the artiÐcial double membrane toenhance the feeding activity of aphids. Percentage mor-tality was determined after 48 h.

2.3.2 FAO dip testThe contact activity of some of the investigated metabo-lites was tested using a modiÐed version of the FAO diptest.15 Apterous adults of Myzus sp. were dipped for10 s in insecticidal solutions containing “TritonÏ X-100(0É2 g litre~1). After dipping, the aphids were transferredonto freshly excised cabbage leaves and the petioleswere immersed in a small tube containing pure water.The leaves were placed in a plastic container of appro-priate size and covered with a ventilated lid. Percentagemortality was scored 48 h post-dip.

2.4 Receptor binding assay

Binding of imidacloprid to houseÑy nAChRs was deter-mined as described by Liu & Casida5 with some minormodiÐcations. HouseÑy heads stored frozen in liquidnitrogen were homogenized in a blender in sucrosesolution (320 mM ; 20 ml). After centrifugation for10 min at 1200g the supernatant was Ðltered throughÐve layers of cheesecloth and used directly for bindingassays. The assay (total volume 1 ml) consisted of pot-assium phosphate bu†er (0É1 M, pH 7É4 ; 0É7 ml) con-taining bovine serum albumin (binding bu†er ; 1 glitre~1), homogenate (0É25 ml) and [3H]imidacloprid

(0É05 ml ; 333 Bq \ 0É266 pmol) in water containingmethanol (16 ml litre~1). Unlabeled imidacloprid wasadded in binding bu†er containing up to 0É02 kldimethylsulfoxide. After incubation for 60 min at 22¡C,ice-cold binding bu†er (3 ml) was added, followedimmediately by Ðltration through prewetted WhatmanGF/C glass Ðbre Ðlters and rinsing with ice-coldbinding bu†er (2] 3 ml). Bound radioactively wasdetermined by scintillation counting of the Ðlters. Allvalues were measured in duplicates. valuespI50([1 g M of the concentration of cold ligand displacing50% of bound [3H]imidacloprid) were calculated usinga four-parameter logistic curve Ðtting program (GraFit,Erithacus Software Ltd.).

3 RESULTS AND DISCUSSION

The aphicidal potency of selected plant metabolitesafter oral ingestion was investigated using artiÐcialdouble membranes. The test system produced reliableresults and control mortality was in general less than10%. All of the metabolites (2 to 6) which showed activ-ity in the feeding bioassay described induced symptomstypical for compounds interfering with the insectnervous system, i.e. uncoordinated movement andtremor. The results revealed two plant metabolites (5and 6) which were more active against M. persicae andA. gossypii than the parent compound imidaclopriditself (Tables 1 and 2). The most active metabolite wascompound 5, the imidazoline derivative (oleÐnemetabolite) of imidacloprid. This metabolite showed a16 times higher activity (based on values) thanLC50(48 h)imidacloprid on both aphid species after oral ingestion.The 95% Ðducial limits indicate that the di†erencein efficacy between imidacloprid and metabolite 5 wassigniÐcant. Another metabolite, the nitroso-derivative 6,was also slightly more efficacious than imidacloprid,though not signiÐcantly so by considering the 95%Ðducial limits which overlap with those for imidacloprid(Table 2). The hydroxy-substituted derivatives, com-pounds 2 and 3, showed also considerable activity

TABLE 1Efficacy of Plant Metabolites of Imidacloprid against Myzus

persicae in Oral Ingestion Bioassays (48 h)

L C50Compound (mg litre~1) FL 95% Slope

Imidacloprid 0É073 0É046È0É11 1É241 [10 È È2 3É2 0É42È20 0É853 0É53 0É20È0É97 1É14 5É9 1É1È37 1É25 0É0044 0É000 87È0É018 0É726 0É013 0É0090È0É017 2É17 [10 È È

56 Ralf Nauen et al.

TABLE 2Efficacy of Plant Metabolites of Imidacloprid against Aphis

gossypii in Oral Ingestion Bioassays (48 h)

L C50Compound (mg litre~1) FL 95% Slope

Imidacloprid 0É11 0É048È0É27 1É01 [10 È È2 1É1 0É37È3É1 0É943 0É85 0É24È2É8 1É24 9É3 1É4È54 1É05 0É0068 0É0031È0É014 0É706 0É048 0É021È0É11 0É847 [10 È È

against M. persicae and A. gossypii. The 4-hydroxy-sub-stituted compound 3 seems to be slightly more activeagainst both species than compound 2 which ishydroxylated in position 5, though not signiÐcantly so.Even the dihydroxy metabolite 4 showed an valueLC50below 10 mg litre~1 for both species. Metabolites 1(guanidine compound) and 7 (urea compound) wereboth inactive at the concentrations tested. In order toÐnd out possible di†erences between oral activity andcontact efficacy against M. persicae, some of the metab-olites were also bioassayed using an aphid dip test. Theaphid dip test revealed that, in contrast, to the results ofthe feeding bioassay, imidacloprid was superior to theoleÐne compound 5 (Table 3), perhaps indicating phar-

TABLE 3Contact Activity (Dip Test) of Some Plant Metabolites of

Imidacloprid against Myzus persicae (48 h)

L C50Compound (mg litre~1) FL 95% Slope

Imidacloprid 0É22 0É17È0É26 3É772 9É3 6É4È13 1É243 6É9 5É3È8É9 1É825 0É84 0É67È1É0 2É53

TABLE 4Activity of Imidacloprid Metabolites as Inhibitors of[3H]Imidacloprid binding to Nicotinic Acetylcholine Recep-

tors in HouseÑy Head Membranes

Compound IC50 (nM)

Imidacloprid 0É791 50002 5É03 254 6305 0É256 n.d.7 [10000

macokinetic di†erences due to di†erent physicochemicalproperties, e.g. the lipophilicity of compound 5 is lowerand the water solubility higher than those of imid-acloprid. The two monohydroxylated metabolites 2 and3 were approximately 40 and 30 times less active incontact bioassays, respectively. This is in contrast to theresults in the feeding bioassay with M. persicae wherethe 4-hydroxy-substituted metabolite 3 was just seventimes less active than imidacloprid, indicating its highintrinsic potential, whereas the 5-hydroxylated com-pound 2 was more than 40 times less efficacious in thefeeding bioassay than the parent compound, whichcoincides well with the results from the aphid dip bio-assay.

The biological efficacy in feeding bioassays on aphidscorrelates with the relative affinities of the metabolitestowards the houseÑy nAChR (Table 4). The oleÐnemetabolite 5 showed a somewhat higher affinity to thenAChR than imidacloprid, which was perhaps not sur-prising judging from its superior action in the feedingbioassay on both aphid species tested. In contrast to thebioassay results on M. persicae and A. gossypii wherethe 4-hydroxy metabolite 3 was slightly more activethan metabolite 2 which is hydroxylated in position 5 ofthe imidazolidine ring, the receptor binding assayrevealed the opposite, i.e. metabolite 2 showed a lower

value than metabolite 3. It is not clear at present ifI50nAChR preparations from other insect species such asaphids will show the same speciÐcity as the houseÑyreceptor. The lowest binding affinities, i.e. values ofIC505 kM, and [10 kM were measured with the biologicallyleast-active guanidine metabolite 1 and the cyclic ureacompound, metabolite 7, respectively. The 4,5-dihy-droxy metabolite 4 showed a poor affinity to the house-Ñy nAChR, i.e. its value was more than 2500 timesIC50lower than the value for the most active metabo-IC50lite, 5. However, the biological efficacy of metabolite 4on both aphid species was also 1000 times lower thanthat of metabolite 5, indicating a rather good corre-lation between the binding data using tritiated imid-acloprid as a radioligand in houseÑies and insecticidalpotency on aphids.

4 CONCLUSIONS

The conclusions which can be drawn from the results ofthis study are that perhaps in several cases of goodlong-lasting residual activity of imidacloprid after soilapplication or seed treatment, imidacloprid is not byany means the only compound which is responsible forthe aphicidal e†ects observed. It is much more likelythat imidacloprid and the orally active metabolitesdescribed here are acting in concert, i.e. a mixture ofdi†erent metabolites including unchanged parent com-pound guarantees long-lasting insecticidal potency. It isunresolved so far if certain combinations of imid-

Aphicidal activity of plant metabolites of imidacloprid 57

acloprid and its metabolites may act in a synergisticmanner. Experiments with seed-treated cotton plantsrevealed that only 5% of the applied imidacloprid wastaken up by the young plant and that, 27 days aftersowing, approximately 95% of the parent compoundwas metabolized.16 However, the residual activity isÈdepending on the aphid species consideredÈhigherthan 49 days on such plants, even though not all aphidsshow typical intoxication symptoms, but most of theindividuals walk o† treated plants due to the reportedantifeedant action of imidacloprid.11,17,18 Thus wecould conclude that either the remaining amount ofimidacloprid in such plants is still sufficient to controlthe aphids by virtue of its sub-lethal e†ects or that theappearance of certain metabolites in conjunction withlow amounts of imidacloprid may provide the excellentcontrol properties after seed treatment or soil applica-tion.

ACKNOWLEDGEMENTS

The excellent technical assistance of Christiane Hoff-mann, Heike Hungenberg, Dagmar Simon, TanjaNepute, Claudia Wehr and Dagmar Brattig is gratefullyacknowledged.

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