Pestic. Sci. 1997, 49, 90È96

Communication to the Editor

Insecticide Resistance in a Strain of Aphis gossypiifrom Southern FranceRobert Delorme,* Danielle Auge� , Marie-The� rese Be� thenod & FrancÓ ois Villatte

Unite� de Phytopharmacie et Me� diateurs chimiques, INRA, Route de St Cyr, 78026 Versailles Cedex,France

(Received 23 October 1995 ; revised version received 26 June 1996 ; accepted 16 August 1996)

Abstract : A strain (R) of Aphis gossypii from Southern France was found to beresistant to several insecticides, particularly to pirimicarb, as compared to a sus-ceptible strain (S). Resistance levels were determined by biological tests, and thehighest resistance factor (1350) was for pirimicarb. Resistance was mainlyrestricted to anticholinesterase inhibitors. Use of synergists, DEF and PB, sug-gested that resistance mechanisms based on detoxiÐcation were involved to aminor extent, since a good correlation was observed between values andI50 kivalues of AChE and in-vivo bioassay data. The two strains di†ered in esteraseactivity, with a 27É7-fold increase in the R strain. Resolution of esterases by poly-acrylamide gel electrophoresis showed di†erent patterns in the S and R strains,and two isozymes were less sensitive to pirimicarb in the S strain ; however, noin-vitro degradation of [14C]pirimicarb was observed. These data suggest thatthe main mechanism of resistance was through a decrease in the sensitivity of thetarget, AChE, to the insecticides.

Key words : Aphis gossypii, resistance, pirimicarb, acetylcholinesterase, carb-oxylesterases

1 INTRODUCTION

The cotton or melon aphid Aphis gossypii (Glover) is ahighly polyphagous pest able to develop on a broadrange of host plants, with a preference for cucurbits,1,2where it causes serious damage.3 In cotton, this pest candecrease yield and quality.4 Chemical protection has ledto resistance of A. gossypii in many countries. The Ðrstreport concerned pirimicarb, twenty years ago inEngland in chrysanthemum crops.5 Resistance toorganophosphates and carbamates has been reported inthe Soviet Union,6 China,7 Israel,8 Japan,9,10 UK,11,12Hawaii13 and the USA.14,15 Pyrethroid resistance wasmentioned in China,16 Israel8 and the USA.14 Oncotton crops in Sudan, this aphid is resistant to all themain insecticide groups.17 Mechanisms underlying

* To whom correspondence should be addressed.

these resistances are varied : acetylcholinesterase (AChE)insensitivity,5,17h20 enhancement of carboxyl-esterases7,9,15 and enhanced oxidative metabolism.7,21In France, some failures of pirimicarb were reported in1987 on melon, and resistance was conÐrmed on astrain collected at St Martin-de-Crau (Provence).18 Theaims of the present work were to study the resistance toinsecticides of a resistant strain of A. gossypii fromSouthern France in comparison with a susceptible refer-ence strain and to determine the underlying biochemicalmechanisms.

2 MATERIALS AND METHODS

2.1 Aphid populations

The resistant strain (R) was collected in 1992 at Pernes-les-Fontaines (Provence) from a sweet pepper (Capsidum

90Pestic. Sci. 0031-613X/97/$09.00 1997 SCI. Printed in Great Britain(

Insecticide resistance of a strain of A. gossypii 91

annuum L.) Ðeld. A susceptible clone (S) was obtainedfrom Dr G. Labonne (INRA-ENSA Montpellier) whocollected it in 1985 at Navacelles (Provence) in a non-treated area. The R and S strains were kept and rearedanholocyclically on cucumber seedlings (Cucumis sativusL., CV. Marketer), in controlled-environment chamberswith a temperature of 20(^1)¡C, 70(^10)% relativehumidity and a 16 : 8 h light : dark photoperiod. Noselection pressure on the R strain has been applied sincethe strain was collected in 1992 and the susceptibilityand resistance level of each strain was measured by bio-assays performed twice per year.

2.2 Bioassays

Sixteen insecticides were tested for contact toxicity(Table 1). Formulations were gifts from commercialsources. The choice of compounds was based on theirpractical use and their chemical structures. The pro-cedure was the same as described previously.17 Testswere repeated three times and resistance factors (RF)were determined as the of the R ofLC50 strain/LC50the S strain.

2.3 Synergism

The action of two synergists was investigated to deter-mine whether metabolism was involved in resistance.The synergists were piperonyl butoxide (PB), an MFO(Mixed Function Oxidase) inhibitor and S,S,S-tributylphosphorotrithioate (DEF), an inhibitor of both ester-ases and glutathione transferases. They were applied 1 hprior to insecticide application by spraying as describedpreviously for the insecticides. Doses (1000 mg litre~1for PB and 200 mg litre~1 for DEF) were the highestconcentration inducing no observable toxicity on the Sand R strains. Synergism ratios insecticide(LC50

insecticide] synergist) were calculated foralone/LC50both strains.

2.4 Acetylcholinesterase assay

Among insecticides tested in vivo, Ðve AChE inhibitorswere studied for and paraoxon-ethyl, (E)-I50 ki :mevinphos, methomyl, pirimicarb and triazamate. Allwere technical grade of the highest purity available andwere gifts from industrial producers. Paraoxon-ethylwas used instead of parathion-ethyl, the thiol analogue

TABLE 1of Insecticides against Two Strains of Aphis gossypii and Resistance FactorsLC50

L C50 (g hl~1) (95% conÐdence limits) Slope

Compounds S strain R strain S strain R strain RFa

OrganochlorinesEndosulfan 2É07 [1É93È2É23] 2É30 [2É15È2É45] 3É35 4É64 1É11c-BHC 4É57 [3É97È5É27] 3É52 [3É22È3.84] 2É30 3É17 0É770

OrganophosphatesAcephate 1É95 [1É79È2É11] 9É36 [8É12È10É8] 3É58 3É77 4É80Dimethoate 0É228 [0É199È0É262] 18É0 [15É7È20É6] 3É53 2É29* 78É9Ethyl-parathion 0É694 [0É632È0É763] 7É57 [6É82È8É41] 4É35 3É62 10É9Methidation 0É471 [0É406È0É547] 1É60 [1É50È1É72] 4É16 4É85 3É40Mevinphos 0É343 [0É316È0É373] 0É882 [0É803È0É968] 4É91 5É00 2É57

CarbamatesMethomyl 0É383 [0É325È0É451] 1É13 [1É02È1É26] 1É96 2É06 2É95Pirimicarb 0É152 [0É142È0É163] 205 [156È270] 4É61 1É87* 1350

PyrethroidsBifenthrin 0É003 32 [0É002 82È0É003 91] 0É006 41 [0É005 71È0É007 20] 2É25 2É20 1É93Deltamethrin 0É0107 [0É009 53È0É0120] 0É0239 [0É0211È0É0271] 2É22 2É37 2É23Fenvalerate 0É140 [0É120È0É164] 0É119 [0É107È0É134] 1É62 2É06 0É850Lambda-cyhalothrin 0É004 09 [0É003 71È0É004 52] 0É004 10 [0É003 40È0É004 95] 3É16 1É97* 1É00Tau-Fluvalinate 0É740 [0É0650È0É0843] 0É0915 [0É0800È0É105] 2É18 2É06 1É24

ChlornicotinylesImidacloprid 0É165 [0É151È0É180] 0É168 [0É151È0É188] 2É67 2É54 1É02

TriazolesTriazamate 0É168 [0É159È0É177] 5É49 [4É91È6É14] 4É99 4É78 32É7

R S strain (mean of three replicates).a RF\ LC50 strain/LC50b * SigniÐcantly di†erent from S strain (Student test, P\ 0É05).

92 Robert Delorme et al.

that is a poor inhibitor in vitro. We could not determinethe or for dimethoate because the was greaterI50 ki I50than 10~3 M and dimethoxon was not available. (E)-Mevinphos was chosen because it is the more potentisomer. AChE activity was measured by the Ellmanmethod22 using 10~5 M DTNB. Ten apterous adultswere homogenised in ice-cold phosphate bu†er (1 ml ;25 mM, pH 7) containing “TritonÏ X-100 (1É0 ml litre~1)in an Eppendorf tube with a TeÑon pestle and centri-fuged at 10 000g at 4¡C for 7 min. The supernatant wasthe enzyme source. For the determination of anticholin-esterase activity, phosphate bu†er (870 kl) homogenate(100 kl) and the appropriate amount of inhibitor dis-solved in acetone (10 kl) were preincubated for 5 min at25¡C. In the control tubes, the inhibitor was replaced byacetone and in the blank by a 10~3 M eserineacetone] methanol (90] 10 by volume). Acetyl-thiocholine (10 kl ; 100 mM) and DTNB (10 kl, 1 mM)were added and tubes were incubated for 5 min at 25¡C.The absorbance was measured by a Hitachi U1100spectrophotometer at 412 nm. The value was calcu-I50lated from Ðve inhibitor concentrations by computedlinear regression. For determination of bimolecular rateconstants, insecticide was incubated with homoge-ki ,nate at a concentration equal to the at 25¡C. Ali-I50quots were taken and residual activity of AChE wasrecorded over time by the method described above, theslope was calculated by linear regression on a micro-computer and the value was calculated as describedkiby Main and Iverson.23

2.5 Carboxylesterase assay

The carboxylesterase activity was measured colorimet-rically as described by Devonshire.24 Individual aphidswere weighed on a Sartorius micro-balance and homog-enised in phosphate bu†er (1 ml ; 0É4 M, pH 7). Sixtymicrolitres of the homogenate were used for the assay.Under these conditions, increase in colour density wasproportional to enzyme activity and measured at605 nm on a U1100 Hitachi spectrophotometer.

2.6 Polyacrylamide gel electrophoresis

Electrophoresis was performed using a vertical Bio-Radelectrophoresis unit with a 7É5% acrylamide gel. Aphidswere homogenised in Tris HCl bu†er (pH 8É8) contain-ing “TritonÏ X-100 (1É0 ml litre~1) : 300 kl bu†er mg~1aphid for the R strain and 20 kl bu†er mg~1 aphid forthe S strain. The homogenate was centrifuged at 15 000gfor 10 min at 4¡C. Samples of supernatant were electro-phoresed for 2 h at 200 V in a continuous system at4¡C, using Tris glycine (pH 8É8) as the bu†er. Totalesterases were strained for 20 min with shaking in 2 mlTris HCl, 94 ml water, 50 mg Fast Blue RR and 2 ml of10 g litre~1 a-naphthyl acetate in acetone. For the inhi-

bition assay, gels were preincubated at 4¡C in a TrisKCl bu†er (0É2 M) containing pirimicarb (1É7 ] 10~4 M)for 30 min.

2.7 In-vitro metabolism of [14C ]pirimicarb

[14C]Pirimicarb (speciÐc activity 2É044 GBq mmol~1)was provided by Zeneca Agrochemicals Company.Enzyme was prepared by homogenising 60 mg aphidsin ice-cold phosphate bu†er (2 ml ; 50 mM, pH 7É2), con-taining EDTA, 1 ; DTT 0É1 and PMSF 0É4 mM, in aPotter homogeniser. Homogenates were centrifuged at12 000g for 10 min at 4¡C. Supernatant (1 ml) was incu-bated with an acetonic [14C]pirimicarb solution (10 kl ;Ðnal concentration 10~5 M, 1É2 ] 106 dpm) and anaqueous NADPH solution (26 kl ; 0É04 M). After incu-bation for 24 h in the dark at 25¡C, 50 kl were spottedon the TLC plater (Merck 60 and chromato-SiO2)graphed with hexane] ethyl acetate] methanol(5] 4 ] 1 by volume). Results were obtained by directcounting with a linear counter (Bertold) and auto-radiography (HyperÐlm b Max) for seven days at[ 10¡C. Other measurements were obtained by HPLC(reverse phase), coupled with a solid scintillationcounter.

3 RESULTS

3.1 Bioassays

Table 1 summarises the toxicological data for the S andR strains for the 16 insecticides tested. The susceptibleclone from Navacelles was killed at low doses of theinsecticides tested, with the exception of c-BHC, whichis considered an inadequate aphicide. This high suscep-tibility conÐrms the value of the Navacelles clone as areference strain. The R strain from Pernes-les-Fontainespossessed a high level of resistance to pirimicarb(RF\ 1350) and a weaker resistance to the other car-bamate methomyl (RF\ 2É95). Resistance factors fororganophosphorus compounds ranged from 2É57 formevinphos to 78É9 for dimethoate. Moderate (RF \ 33)resistance was also found for another AChE inhibitor,triazamate. No resistance was found to organochlorines,imidacloprid or pyrethroids. Only a small signiÐcantdi†erence was seen between the S and R strains forbifenthrin and deltamethrin. Since resistance seemed tobe restricted to compounds acting as anticholinesteraseinhibitors, these data suggested an insensitive AChE inthe R strain, although this insensitivity was clearly notexpressed to all inhibitors.

3.2 Synergism experiments

Synergism experiments were conducted with the twoinsecticides having the highest resistance factors in theR strain : pirimicarb and dimethoate. PB reduced the

Insecticide resistance of a strain of A. gossypii 93

TABLE 2Toxicity of Insecticides tested against S and R Strains of Aphis gossypii in Absence and in Presence of PB or

DEF. Synergism Ratio (SR) and Resistance Factor (RF) in Absence and in Presence of Synergist

S strain R strain

L C50 (g hl~1) L C50 (g hl~1)Product (95% CL ) SR (95% CL ) SR RF

Pyrimicarb 0É152 (0É142È0É163) 205 (156È270) 1350]PB 0É0176 (0É0153È0É0203) 8É64 29É1 (26É7È31É8) 7É04 1650]DEF 0É0465 (0É0350È0É0619) 3É27 26É3 (23É3È29É8) 7É79 566

Dimethoate 0É228 (0É199È0É262) 18É0 (15É7È20É6) 78É9]PB 0É155 (0É138È0É174) 1É47 12É1 (11É3È12É9) 1É49 78É1]DEF 0É174 (0É158È0É191) 1É31 4É70 (4É45È4É97) 3É83 27É0

of pirimicarb by a synergism ratio (SR) of 8É64LC50and 7É04 in the S and R strains respectively (Table 2).For dimethoate, the SR values were, respectively, 1É47and 1É49 for the S and R strains. This suggests thatMFOs were probably involved in the metabolism of atleast pirimicarb. As the SR values were not di†erent for

the two strains, MFOs seemed not to be involved as amechanism of resistance. DEF reduced the of piri-LC50micarb, with SR values 3É27 and 7É80 for the S and Rstrains, respectively. For dimethoate the SR values wererespectively 1É31 and 3É83 for the S and R strains. DEFsynergised 2- to 3-fold more pirimicarb and dimethoate

TABLE 3Anticholinesterase Activity of Five Insecticides for the S and R Strains of Aphis gossypii

Parametera/pesticide S strain R strain Ratio R/S

I50 (M) (95% CL)Paraoxon-ethyl 5É52 (2É10È14É50)] 10~7 2É55 (2É24È2É91) ] 10~6 4É62(E)-Mevinphos 3É71 (2É64È5É19) ] 10~9 7É18 (5É52È9É34) ] 10~8 19É4Methomyl 4É51 (2É92È6É94) ] 10~6 2É91 (2É02È4É16) ] 10~6 0É645Pirimicarb 1É01 (0É501È2É02) ] 10~6 2É13 (1É72È2É64) ] 10~4 210Triazamate 9É66 (8É55È10É92)] 10~8 4É95 (3É55È6É91) ] 10~7 5É12

ki (M~1 min~1)(^SE)Paraoxon-ethyl. 3É04 (^0É20) ] 105 4É81 (^0É26) ] 104 6É32(E)-Mevinphos 6É45 (^0É64) ] 107 2É59 (^0É15) ] 106 24É9Methomyl 2É42 (^0É16) ] 104 1É75 (^0É63) ] 104 1É32Pirimicarb 3É07 (^0É20) ] 105 2É55 (^0É10) ] 102 1200Triazamate 1É57 (^0É20) ] 106 2É36 (^0É76) ] 105 6É65

a Inhibitory concentration for 50% of AChE activity Bimolecular rate constant Means of(I50). (ki).three replicates.

Fig. 1. Frequency distribution of carboxylesterase activities on individual insects.

94 Robert Delorme et al.

Fig. 2. Native electrophoresis of general esterases (2 h, 200 V),with Fast Blue RR staining. A : S strain, B : R strain (15-folddiluted), C : R strain (15-fold diluted) after 30 min incubationwith pirimicarb 1É7 ] 10~4 M, D : S strain after 30 min incu-

bation with pirimicarb 1É7 ] 10~4 M.

in the R strain than in the S strain. This could indicatethat carboxylesterases played a minor role in resistance.

3.3 AChE assay

The method used to determine did not involve anyI50dilution of the insecticide after addition of substrate, butno inhibition was noticed during the reaction time

(linear reaction). The values and in-vivo toxicologicalI50data seemed to be correlated (Table 3), with the excep-tion of mevinphos. The S ratio for pirimicarbI50 R/I50was 210 and the in-vivo resistance factor (RF) was 1350.For paraoxon-ethyl and triazamate, the value forI50the R strain showed a 5-fold increase, which corre-sponded to RF values of 10É9 and 32É7 respectively. NosigniÐcant di†erence was found for methomyl(RF\ 2É95). On the other hand, the 19É4-fold increaseof the value for (E)-mevinphos was not correlatedI50with a high level of resistance. These results suggestedthat decrease in the sensitivity of the target was themain mechanism underlying resistance.

The calculation of which is a measure of theki ,overall inhibitor potency of a compound, was possibledue to the linearity of the metabolism curves. For piri-micarb, there were 1200 units of di†erence between theR and S strains, possibly reÑecting a change in the cata-lytic centre of the enzyme.

3.4 Frequency distributions of carboxylesteraseactivities on individuals

Distributions of individual carboxylesterase activitiesshowed no overlap between the S and R strains (Fig. 1).When activities were expressed as their log, the distribu-tions could be Ðtted to Gaussian curves with a highcorrelation factor (0É87 and 0É97 for S and R strains,respectively) demonstrating the homogeneity of thestrains. The ratio between the mean activities within the

Fig. 3. In-vitro degradation of [14C]pirimicarb. QuantiÐcation by linear counting of radioactivity on TLC. A: S strain, B : R strain,C : reference (homogenate replaced by phosphate bu†er).

Insecticide resistance of a strain of A. gossypii 95

two strains (1É99 ] 10~8 mole 100 kg~1 30 min~1 and5É52 ] 10~7 mole 100 kg~1 30 min~1 for S and Rstrains respectively) was 27É7.

3.5 Native PAGE

The electrophoretic patterns of the esterases from S andR strains were di†erent (Fig. 2), indicating a di†erencein the electrophoretic mobility of the enzymes, i.e. qual-itative di†erences in the esterases from the two strains.These electrophoretic patterns were similar to thoseobserved in A. gossypii by other authors.5,9 Homoge-nate of R strain was diluted 15-fold in order to improveband resolution and conÐrm the quantitative changesmentioned previously. Inhibition assay by pirimicarbrevealed di†erences in the sensitivities of two isozymes.All bands were inhibited in the R strain, whereas twobands remained visible in the S strain.

3.6 In-vitro degradation of [14C ]pirimicarb

QuantiÐcation obtained by direct counting (Fig. 3) aswell with HPLC and autoradiography (results notshown) failed to show any noticeable di†erencesbetween the control, the S strain and the R strain. Themajor peak corresponds to [14C]pirimicarb and theminor ones to impurities present in the initial solution.To check for a weak metabolism masked by too much[14C]pirimicarb, the experiment was repeated with ten-fold less substrate. Results were similar. A third experi-ment using the same conditions as for thecarboxylesterase assay (phosphate bu†er pH 7, 25 mM)gave the same results. Thus, in our conditions, we didnot observe any noticeable breakdown of pirimicarb invitro.

4 DISCUSSION

The bioassay in A. gossypii showed that the R strainfrom Pernes-les-Fontaines was highly resistant to piri-micarb, the main insecticide used in Southern Franceagainst A. gossypii. Similarly to observations by othersauthors,5 it was found that resistance to pirimicarb wasstable in the absence of selection pressure, with an RFthat was essentially unchanged over several years ofrearing in captivity. Probit plots issued from bioassaysand data from determination experiments suggestedI50that the R strain is homogenous. Slopes of theconcentrationÈmortality lines were signiÐcantly lower inthe R strain concerning pirimicarb, dimethoate andlambda cyhalothrin. In general, the slope is correlatedwith genetic variability, but some authors have recentlyshown that this linkage is doubtful.25 The resistance

level to pirimicarb was not found for the other car-bamate insecticides tested ; nevertheless, lower resistancewas detected to some organophosphorus compounds(dimethoate, parathion-ethyl) and triazamate. All theseinsecticides are acetylcholinesterase inhibitors. Thecorrelation observed in the AChE assay between the I50value and the in-vivo bioassay points to a decrease inthe sensitivity of the target as the main mechanismunderlying resistance. The slight resistance tomevinphos, opposed to the higher insensitivity of theAChE from the R strain, could be due to the presence ofthe two isomers in the commercial formulation used inbioassays, whereas only the (E) isomer has been testedin AChE inhibition experiments. Synergist experimentsusing a combination of pirimicarb and dimethoate onone hand, PBO and DEF on the other hand, suggestedthat resistance mechanisms based on detoxiÐcationwere only very slightly involved in resistance. The slighte†ect of DEF, an esterase inhibitor, on the resistancefactor for both pirimicarb and dimethoate led us tostudy the carboxylesterase activity of the S and Rstrains.

Surprisingly, we found important quantitative andqualitative di†erences between the two strains. Quanti-tatively, the ratio for carboxylesterase activity was 27É7and the level of activity for R strain was of the sameorder of magnitude as for Myzus persicae resistantclones,24,26 in which carboxylesterases are known to beresponsible for resistance against some OP and car-bamates. Moreover, electrophoretic patterns of esterasesshowed bands with important di†erences in electro-phoretic mobilities between the two strains. These pat-terns were similar to those obtained by otherauthors.5,9 We have no precise idea of the possiblechange(s) leading to these di†erences, but we couldsuggest an esterase glycosylation in the R strain, asobserved on a resistant strain of L aodelphax striatellus(Fall.).27 In our case, the susceptibility of the two iso-zymes to pirimicarb was di†erent between the twostrains : two bands remained visible in the S strain afterincubation with the insecticide, whereas all bands disap-peared in the R strain. Esterase modiÐcations in resist-ant strains of A. gossypii have been noted by severalauthors15,28 and have been claimed to be responsiblefor resistance, but no other mechanisms have beeninvestigated. Thus, in our case, despite esterasicchanges, no noticeable di†erences were found in in-vitrodegradation of [14C]pirimicarb, even in optimal condi-tions for esterases. In consequence, we could not estab-lish a link between esterase changes and resistance,since the hypothesis of a phylogenetic inter-strain varia-tion could not be deÐnitively excluded.

The above combined data suggested a minor inci-dence of carboxylesterases in the resistance to thestudied compounds, acting probably by a weak seque-stration of toxicants, as observed for other species.24,29Thus, the main mechanism of resistance in this strain of

96 Robert Delorme et al.

A. gossypii was the modiÐcation(s) of the target, as is thecase in an increasing number of species. Character-isation of AChE in this aphid, and determination of thedi†erent alleles in natural populations may contributeto a better knowledge of its resistance status andprovide tools for an improved monitoring and manage-ment.

ACKNOWLEDGEMENTS

We thank agrochemical companies for gifts of insecti-cide formulations and technical grade products andZeneca Agrochemicals Company for providing the[14C]pirimicarb.

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