Pestic. Sci. 1998, 53, 201È208

Proinsecticides Eþective againstInsecticide-Resistant Peach-Potato Aphid(Myzus persicae (Sulzer))Douglas Hedley, Bhupinder P. S. Khambay, Antony M. Hooper,Richard D. Thomas & Alan L. Devonshire*

Biological and Ecological Chemistry Department, IACR-Rothamsted, Harpenden, Herts, AL5 2JQ UK

(Received 15 August 1997 ; revised version received 21 January 1998 ; accepted 4 February 1998)

Abstract : A range of potential proinsecticides was synthesised and tested againstinsecticide-susceptible and -resistant clones of Myzus persicae (Sulzer). They wereall esters of compounds known to be toxic or pharmacologically active, and weredesigned to have increased lipophilicity and to be subject to more rapid activa-tion by hydrolysis in resistant than in susceptible aphids due to the increasedamount of esterase present in the resistant clones. The most potent toxins wereesters of monoÑuoroacetic acid. When applied topically, the toxicity of theseesters to M. persicae was directly proportional to the esterase content of theaphids. Such compounds would not be suitable as commercial insecticides, butthe results serve to illustrate the potential beneÐts of exploiting a resistancemechanism against one class of compounds to render another class more toxic,i.e. to design compounds that show negative cross-resistance. 1998 SCI(

Pestic. Sci., 53, 201È208 (1998)

Key words : Myzus persicae ; insecticide resistance ; esterase ; proinsecticides ;negative cross-resistance ; aphids

1 INTRODUCTION

The peach-potato aphid Myzus persicae (Sulzer), amajor pest on a variety of crops, has developed resist-ance worldwide to organophosphorus, carbamate andpyrethroid insecticides through the increased pro-duction of a carboxylesterase, E4, or its closely relatedvariant FE4. These enzymes inactivate insecticides bysequestration and ester hydrolysis.1 In the most highlyresistant aphids there is approximately 60 times asmuch esterase activity as in susceptible aphids, account-ing for 1È2% of total body protein. Molecular geneticstudies have shown that this increase in esterase pro-duction is primarily due to gene ampliÐcation, i.e. thepresence of multiple copies of the esterase gene in resist-ant aphids.2

Increased esterase production is associated withresistance to insecticides in many species. This has been

* To whom correspondence should be addressed.Contract/grant sponsor : Biotechnology and Biological Sci-ences Research Council, UK.

shown primarily by using 1-naphthyl acetate as achromogenic substrate both in homogeneous solutionassays and for staining electrophoresis gels. Such di†er-ences in esterase levels between susceptible and resistantphenotypes o†er the possibility of using proinsecticidalesters for the preferential control of the resistantvariants. Negative cross-resistance (i.e. a resistancemechanism to one class of insecticides conferringincreased sensitivity to another class) is a major, so farunachieved, goal for incorporation into insecticideresistance-management strategies. The best-documentedinstance of negative cross-resistance is in the green riceleafhopper Nephotettix cincticeps (Uhl), in which thetarget enzyme, acetylcholinesterase, has become insensi-tive to the widely used N-methylcarbamates but, by thesame mutation, has been rendered hypersensitive to N-propylcarbamate insecticides.3

Many commercial insecticides are applied as proin-secticides. For example, many organophosphorus com-pounds are applied as phosphorothionates, requiringoxidative activation to the phosphate to become potent

2011998 SCI. Pestic. Sci. 0031-613X/98/$17.50. Printed in Great Britain(

202 Douglas Hedley et al.

cholinesterase inhibitors. Likewise, a number of car-bamate insecticides have been derivatised (e.g. as sul-fenyl compounds) to render them less toxic to mammalsyet be readily activated by the target insect.4 There areother non-commercialised examples. Kochansky et al.5reported the use of delayed-action Ñuoroalkyl esters inthe control of Ðre-ants, Prestwich et al.6h8 investigatedthe insect-speciÐc metabolism of Ñuorinated lipids in thetermite Regiculitermis Ñavipes (Kollar) and the tobaccohorn-worm Manduca sexta (Joh), and Palmer et al.9,10studied the selective toxicity to houseÑies of substitutedbicyclooctane GABA antagonists. However, theseexamples did not aim to exploit the biochemical mecha-nisms of resistance to other insecticides.

In the present study we have used ester proinsecti-cides to exploit a resistance mechanism based on quan-titative changes in enzyme expression rather thanstructural changes in the target protein as in the case ofthe green rice leafhopper.3

2 MATERIALS AND METHODS

2.1 Aphid clones

Apterous adult virginoparae of three M. persicae cloneswere used throughout. Clonal nomenclatures, US1L(susceptible, S), 794J (highly resistant overproducingR3esterase E4) and 800F overproducing esterase FE4),(R3and origins are given by Sawicki et al.11 and Devon-shire et al.12 In one experiment, aphids of intermediateresistance levels (resulting from intermediate esterasecontent) were used : 405D expressing FE4) and T1V(R1

expressing E4). Broad resistance to established(R2insecticides increases through the series S, toR1, R2 R3 .

All aphid clonal lines were maintained at 21(^1É4)¡Cunder a 16/8 h light/dark cycle on Chinese cabbage(Brassica chinensis Juslen cv. Tip Top (Brassicaceae))either on intact plants or on excised leaves in small box-cages.13 Clonal integrity was checked at regular inter-vals by staining for esterases after non-denaturingpolyacrylamide gel electrophoresis and/or immuno-assay.14

2.2 Test compounds

Fluoroacetamide was obtained from Sigma.

2.2.1 Compounds in initial screen (T able 1)In view of the well-established activity of the M. per-sicae esterases in hydrolysing naphthyl acetate andbutyrate, 1-naphthol was chosen primarily as the ester-atic alcohol. The lead compounds for testing as 1-naphthyl esters were chosen from the literature oninsect and mammalian pharmacology and neurochem-istry.15h17

If any reactive groups were present on an acid inaddition to the intended carboxyl group, they were pro-tected before esteriÐcation by reaction with benzyl-

chloroformate (-NH groups) or tert-butyldimethylsilylchloride (-OH groups) prior to acid chloride formation.The chosen acids (100È400 mg), protected as necessary,were converted to their acid chlorides by reaction with1É1 equivalents of thionyl chloride indichloromethane] pyridine (50 ] 1 by volume). Toeach acid chloride, 1É1È2É2 equivalents of 1-naphtholwere added and the mixture stirred overnight at roomtemperature. Any protecting groups were removedand the products dried over anhydrous magnesiumsulfate, then puriÐed by Ñash column chromatographyor distillation.

Aziridinylethyl-p-nitrobenzoate was prepared by theslow addition of 0É99 equivalents (666 mg) of p-nitrobenzoyl chloride to a 10 ml litre~1 solution of 2-aziridinylethanol in dichloromethane. The product wasdried over anhydrous magnesium sulfate and evapo-rated to a yellow oil.

2.2.2 Fluoroacetyl esters (T able 2a)A slight molar excess (1É1 equivalents) of Ñuoroacetylchloride, prepared as above from monoÑuoroacetic acid(Sigma), was added to a solution of 5È10 mmol of therequired alcohol in diethyl ether] pyridine (50] 1 byvolume) at 0¡C under nitrogen. The reaction was stirredat room temperature overnight, then poured into waterand the mixture extracted into ether. The etherealextract was washed with base, acid and Ðnally waterbefore being dried over anhydrous magnesium sulfateand concentrated in vacuo. The residue was puriÐed byÑash column chromatography or distillation.

2.2.3 2-Fluoroethylcyclobutanecarboxylate (T able 2b)A mixture of 2-Ñuoroethanol (2É0 mmol), cyclobutanecarboxylic acid (2É0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4É0 mmol) andN,N-dimethylaminopyridine (0É2 mmol) in dichloro-methane was stirred overnight at room temperature andthen applied directly to a Ñash chromatographycolumn. 2-Fluoroethylcyclobutanecarboxylate wasrecovered as a colourless oil.

The structures of the synthesised compounds wereconÐrmed by 400 MHz [1H] and [13C] NMR spectros-copy using a Jeol JNM-GX400 FT-NMR spectrometer.

2.3 Bioassay

A topical application bioassay was used based on thatdescribed by Needham and Devonshire.18 Discs(38 mm diameter) cut from Chinese cabbage leaves wereplaced with their adaxial surface in contact with 12 glitre~1 agar in small polyethylene pots coated internallywith Fluon to prevent aphid escape. Ten adult apterousM. persicae were placed on each leaf disc (three discsper treatment for each clone) and allowed to settle over-night. A drop (0É25 ml) of acetone alone (as control) orcontaining test material (10È104 mg litre~1) was placedon the back of each aphid using an all-glass 1-ml

Proinsecticides against Myzus persicae 203

TABLE 1Esters Screened for Proinsecticidal Activity

Compound R T oxophore Action

a 1-naphthyl esters

1 Valproic acid Anticonvulsant inmammals, acting atmultiple sitesincluding GABAreceptors

2 c-Hydroxybutyric GABA antagonistacid

3 Fusaric acid Dopamine-b-hydroxylaseinhibitor

4 Nipecotic acid GABA transportinhibitor

5 Oxalic acid Calcium chelator

6 Malonic acid Competetiveinhibitor ofsuccinatedehydrogenase

7 Fluoroacetic acid Lethal synthesis of2-erythro-Ñuorocitric acidinhibits aconitatedehydratase and themitochondrialcitrate transporter

b Para-nitrobenzoyl ester

8 Aziridinyl ethanol Inhibitor ofcholinergicneurotransmission

204 Douglas Hedley et al.

TABLE 2Fluoro Esters Tested as Proinsecticides

Compound R

a Fluoroacetyl esters

9

10

11

12

b Fluoroethyl ester

13

syringe depressed by an Arnold manually-operatedmicrometer-driven applicator (Burkard, UK).

In experiments to determine the rate of intoxicationby test compounds, aphids were inspected under a bin-ocular microscope at intervals from 1 h up to 24 h andclassiÐed as “not a†ectedÏ, “a†ectedÏ or “deadÏ. ThoseclassiÐed as “a†ectedÏ had lost their righting reÑex, weresupine and twitching or immobile but twitched whentouched. Since most of those “a†ectedÏ had not recov-ered by the end of the experiments the numbers ofa†ected and dead aphids were pooled for analysis.AbbottÏs formula19 was used to account for the e†ect ofthe solvent alone. Only some of the data were suitablefor estimation of (time for 50% to be a†ected)ET50values,20 so the Ðndings are presented as progresscurves, which demonstrate the important trends veryclearly.

Bioassays were also carried out to determine end-point values for the compounds that appeared toLC50be most selective for the resistant clones, in terms ofrate of intoxication. Susceptible (US1L) and resistant(800F) aphids were treated with a range of concentra-tions (from 0 to 3000 mg litre~1 depending on the e†ec-tive range in the rate experiments) of some of the bettercompounds showing rapid e†ects and di†erential tox-icity between strains. Aphid mortality was determined

48 h after treatment. The data were analysed using thePOLO statistical analysis program (LeOra Software,1987) to obtain the 95% conÐdence limits andLC50 ,slope of the regression line.

Table 1 gives details of the compounds tested initiallyon 794J aphids at 1000 mg litre~1 as described.(R3)Once Ñuoroacetate had been identiÐed as the mostpromising toxin, a series of esters of this acid with ali-phatic alcohols was evaluated (Table 2).

3 RESULTS AND DISCUSSION

3.1 Initial screen

Of the compounds shown in Table 1, only the 1-naphthyl esters of valproic acid (1) and mono-Ñuoroacetic acid (7) caused any toxic e†ects at 1000 mglitre~1. However, subsequent tests with the valproateester did not give consistent results and experimentswith this compound were discontinued. Aziridinylethylp-nitrobenzoate (8) was found to have poor solubility inorganic solvents but the reasons for the lack of toxicityof the other compounds in the screen, which were allreadily soluble in acetone, were not investigated. Theintrinsic potency of the potential intoxicants in aphids is

Proinsecticides against Myzus persicae 205

unknown but pharmacokinetic factors will also haveinÑuenced the results.

The e†ectiveness of the Ñuoroacetate ester was notunexpected. Fluoroacetic acid is a well-known respir-atory poison, acting through the “lethal synthesisÏ of 2-Ñuorocitric acid, a “suicide substrateÏ for the Krebs cycleenzyme aconitate hydratase and an inhibitor of themitochondrial citrate transporter.21

1-NaphthylÑuoroacetate was the only compoundfrom the initial screen to be studied further.

3.2 Follow-up study using 1-naphthylÑuoroacetate (7)

Time-courses for compound 7 and subsequent com-pounds are presented (Figs 1È5) using the mean percent-age of intoxicated insects from the three replicates.Preliminary experiments showed that topical applica-tion of 0É25 ml of 1600 mg litre~1 7 to the susceptibleand most resistant clones a†ected 90È100% of the(R3)aphids within 2 h, with indications that the resistantaphids responded faster. Application of 200 mg litre~1had little e†ect on either clone. An intermediate dosewas therefore chosen to determine if the greater e†ecton the resistant aphids could be accentuated. Whenapplied at 600 mg litre~1, 7 was markedly more e†ec-tive against the resistant clones than the susceptible one.The clone showed a response intermediate betweenR1the susceptible (S) and highly resistant and(R2 R3)

Fig. 1. E†ect of topical application of 600 mg litre~1 1-naphthyl Ñuoroacetate (7) on Myzus persicae clones classiÐedinto di†erent resistance groups US1L (S), 405D T1V(R1), (R2)794J according to their esterase content.(R3),

Fig. 2. E†ect of topical application of 100 mg litre~1 n-dodecyl Ñuoroacetate (9) on Myzus persicae clones US1L (S),794J overproducing esterase E4) and 800F over-(R3 (R3producing esterase FE4).

Fig. 3. E†ect of topical application of 500 mg litre~1 1,3-diÑuoroisopropyl Ñuoroacetate (10) on Myzus persicae clonesUS1L (S), 794J overproducing esterase E4) and 800F (R3(R3overproducing esterase FE4).

206 Douglas Hedley et al.

Fig. 4. E†ect of topical application of 1000 mg litre~1 Ñuoro-acetamide (12) on Myzus persicae clones US1L (S), 794J

overproducing esterase E4) and 800F overproducing(R3 (R3esterase FE4).

Fig. 5. E†ect of topical application of 2000 mg litre~1 2-Ñuoroethyl cyclobutanecarboxylate (13) on Myzus persicaeclones US1L (S) and 800F overproducing esterase FE4).(R3

clones, the latter both being a†ected to the same extent(Fig. 1). These results suggest that, above a certain levelof esterase expression, the rate of toxin productionexceeds detoxiÐcation.

3.3 E†ect of the alcohol moiety on proinsecticidalactivity

To investigate whether changing the structure of thesubstrate might lead to compounds with increasedpotency and di†erential activity between high- and low-esterase clones, the alcohol moiety of the proinsecticidewas varied. These compounds (Table 2a) were bio-assayed using one S and two clones, one over-R3producing E4 and the other FE4.

Of the Ñuoroacetate esters, the most e†ective were n-dodecylÑuoroacetate (9) and 1,3-diÑuoroisopropylÑuo-roacetate (10). Treatment with 9 at 100 mg litre~1a†ected both of the resistant clones more than the sus-ceptible (Fig. 2). Treatment with 10 at 500 mg litre~1a†ected clone 800F more than 794J or the susceptibleaphids (Fig. 3).

It is not clear whether the 1,3-diÑuoroisopropanolmoiety contributes to the observed e†ects. This alcoholhas been proposed as a substitute for Ñuoroacetate inthe control of mammalian pests in Australia.22 The Ðnaltoxin is thought to be Ñuorocitrate but the mammalianoral is about 100 times that of Ñuoroacetate. ThisLD50alcohol was therefore chosen in an attempt to increasethe aphicidal activity of the proinsecticide by creating acompound with two potentially toxic hydrolysis pro-ducts. However, subsequent experiments using 1,3-diÑuoroisopropyliodoacetate and 1,3-diÑuoroisopropyl-n-butyrate showed no signiÐcant toxicity in any of thethree clones (data not shown), suggesting that thealcohol moiety did not contribute to the aphicidal activ-ity of the Ñuoroacetate ester.

PentylthioÑuoroacetate (11) (100 mg litre~1) had nogreater e†ect on 800F and 794J than on US1L up to 8 hafter treatment. At 1000 mg litre~1 this thioesterappeared to a†ect the resistant clones more rapidly thanthe susceptible clone, but by 3 h all three clones weresimilarly a†ected. The response to this compoundfollows the general trend seen with the true esters, butthe greater lability of the thioester bond may mask theobservation of any negative cross-resistance.

There was some use of Ñuoroacetamide (12) as anaphicide in the 1950s23 but it was displaced byorganophosphorus compounds. Since its activity isdependent on hydrolytic activation to Ñuoroacetate, itwas evaluated in the present study against susceptibleand resistant M. persicae. At 100 mg litre~1 both resist-ant clones were slightly a†ected after 8 h, while the sus-ceptible aphids were una†ected. All three clones werea†ected by 1000 mg litre~1, the resistant ones again sig-niÐcantly more than the susceptible one (Fig. 4).

In-vitro structureÈactivity experiments using 1-

Proinsecticides against Myzus persicae 207

naphthol esteriÐed with a range of carboxylic acids havesuggested that 1-naphthyl cyclobutanecarboxylate is agood substrate for E4 and FE4 (unpublished data). Inorder to exploit this observation, cyclobutane carbox-ylic acid was esteriÐed with 2-Ñuoroethanol. Uponhydrolysis and oxidation with alcohol dehydrogenaseand aldehyde dehydrogenase, this ester again yields Ñu-oroacetate and thus Ñuorocitrate as the Ðnal toxin.Therefore 2-Ñuoroethyl cyclobutanecarboxylate (13)(Table 2b) should behave as a proinsecticide like the Ñu-oroacetyl esters, although perhaps more delayed inaction because of the increased number of metabolicsteps involved.

As expected, 13 a†ected the resistant clone 800F morethan the susceptible clone US1L. At 2000 mg litre~1(Fig. 5) both clones were a†ected, but the e†ect onUS1L was less and its onset more delayed than that on800F. Compared with the Ñuoroacetyl esters, the onsetof symptoms was delayed by up to 3 h.

Rapid expression of toxicity can be important in pre-venting virus transmission. However, for negative cross-resistance to inÑuence selection pressure as part of aresistance management strategy, the di†erential e†ectsobserved so far must also be expressed in terms of end-point toxicity, rather than just knock-down andrecovery. Bioassays were therefore done to determineend-point (48 h) toxicity.

experiments3.4 LC50

Compounds 7, 10 and 13 were lethal to the resistantclone at a concentration signiÐcantly lower than thatwhich killed the susceptible clone (Table 3). ratios,LC50US1L/800F, for the three compounds were 2É7, 2É8 and1É9, respectively.

The lack of discrimination in by 12 correlatesLC50with the progress-curve data for this compound, sug-gesting that Ñuoroacetamide is not a good substrate forFE4. Hydrolysis of Ñuoroacetamide in M. persicae maytherefore be catalysed by an enzyme or enzymes otherthan the elevated esterases responsible for insecticideresistance.

The data for 9 show no di†erence between theLC50resistant and susceptible clones, whereas there is a cleardi†erence in the time-course shown in Fig. 2. Thisimplies that the toxicity of this compound dependsupon its hydrolysis, some of which is non-enzymic, andin the long term this negates any di†erential e†ect of thehigh esterase content of the resistant insects. Both setsof data, however, show that 9 is toxic at a lower concen-tration than most of the other compounds tested.

4 CONCLUSION

These data demonstrate the potential of using proinsec-ticides to control highly resistant M. persicae prefer-entially. The susceptible clone was consistently a†ectedless by most of the treatments tested than were theresistant clones, especially evident in the rate of action,with the FE4-expressing clone 800F usually being themost sensitive. Whether this di†erence reÑects speciÐcitydi†erences between E4 and FE4 or in other phar-macokinetic factors is unknown. However, we havefound FE4 to have a larger than E4 for some insec-kcatticidal esters and the model substrate 1-naphthylacetate, which may extend to the ester proinsecticidesstudied here. Both forms of esterase were active against7 as judged by the staining of non-denaturing PAGEgels using this substrate (unpublished data). The dosesused in the bioassay (100 mg litre~1 is equivalent to25 ng per aphid) are comparable to those oforganophosphorus compounds required to kill suscep-tible aphids and, as a consequence of negative cross-resistance, considerably less than are needed to killorganophosphorus-resistant aphids. However, for suchcompounds to have wider application, evaluationagainst a range of species that depend on a high ester-ase activity for resistance would be needed.

While recognising that the Ñuoroacetate esters areunlikely to be commercialised because they are prob-ably very toxic to mammals, the work demonstrates thevalidity of an approach that exploits a resistance-conferring enzyme to render another class of insecticidesmore toxic. Whilst the concept of negative cross-

TABLE 348-h Values for a Range of Fluorinated Compounds Applied Topically to Susceptible andLC50 R3

Clones of Myzus persicae

Susceptible (US1L ) Resistant, R3 (800F)

L C50 L C50Compound (g litre~1) L imits Slope (mg litre~1) L imits Slope

7 440 350È520 5É9 (^1É5) 160 129È200 3É5 (^0É7)9a 140 110È170 2É0 (^0É2) 130 110È150 5É5 (^1É1)10a 790 660È920 3É3 (^0É6) 280 220È340 2É7 (^0É4)12 140 90È200 3É3 (^0É7) 170 70È250 3É6 (^1É0)13a 890 580È1260 1É8 (^0É2) 460 270È570 4É0 (^1É1)

a Cumulative data from two experiments.

208 Douglas Hedley et al.

resistance to insecticides is compelling, there are veryfew known examples and none has been widelyexploited commercially. The example presented here isthe Ðrst to take advantage of a metabolic resistancemechanism.

ACKNOWLEDGEMENTS

The authors thank Mr D. Beddie for the Ðrst synthesisof naphthyl Ñuoroacetate esters, Mr G. J. S. Ross for thestatistical treatment of the data and Mrs J. Moir fortyping this manuscript. D.H., A.M.H. and R.D.T. weresupported by ROPA (Realising Our Potential Award)grants from the Biotechnology and Biological SciencesResearch Council of the United Kingdom, which alsoprovides grant-aided support to IACR.

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