Pestic. Sci. 1997, 49, 252È258

Apparent Tolerance of a Field-Collected Strain ofMyzus nicotianae to Imidacloprid due to StrongAntifeeding Responses

Ralf Nauen* & Alfred Elbert

Bayer AG, Agrochemicals Division, Research Insecticides, Institut fu� r Tierische Scha� dlinge,Landwirtschaftszentrum Monheim, D-51368 Leverkusen, Germany

(Received 29 November 1995 ; revised version received 17 July 1996 ; accepted 14 October 1996)

Abstract : A French strain of the tobacco aphid Myzus nicotianae Blackman(Homoptera : Aphididae), strain FR, showed high tolerance to imidacloprid inshort-term (48-h) oral ingestion bioassays when compared to a susceptible refer-ence strain of Myzus persicae, strain NS. The resulting tolerance factors were[50. Measures of the contact activity of imidacloprid by the FAO dip methodfailed to detect these high factors of tolerance. The tolerance factor was ingeneral \10 when using the dip method. The resulting di†erence between toler-ance factors could be attributed to a behavioural component to Ðtness betweenstrain FR and strain NS as further experiments revealed. When measuring thee†ect of systemically applied imidacloprid on honeydew excretion, a 50%reduction occurred in both strain FR and strain NS at nearly the same concen-tration of imidacloprid, providing evidence for a similar antifeedant response inboth strains. Starvation experiments revealed that the French strain was able tosurvive approximately 24 h longer than a reference laboratory strain of M. per-sicae. This result coincided with the fact that systemically applied imidaclopridshowed the same aphicidal potential against strain FR after three days as againststrain NS after two days, i.e. 24 h later. After rearing in the laboratory for sixmonths the French strain of M. nicotianae lost its hardiness and also its apparentability to tolerate imidacloprid. However, strain FR was a heterogeneous Ðeldstrain and it is possible that a susceptible variant out-reproduced a more hardyvariant.

These Ðndings indicate that the type of bioassay is very important whenassessing aphid populations for resistance against the chloronicotinyl insecticideimidacloprid, because of its distinct mode of action. It is obvious that an aphiddip test, i.e. FAO dip test, produces more reliable results than the di†erent kindsof short-term oral ingestion bioassays, because of the reversible behaviouralchanges induced by imidacloprid after oral uptake. Thus a short-term oral inges-tion bioassay (¹48 h) is not recommended for precise detection of possibleresistance of Myzus sp. to imidacloprid, although this mode of uptake for imid-acloprid might be sometimes more realistic in terms of Ðeld behaviour. The idealtest to generate most accurate data would be a slightly longer (72-h) feedingbioassay, perhaps used in conjunction with a dip test. The possible inÑuence ofthe results on resistance monitoring is discussed.

Key words : imidacloprid, behaviour, Myzus sp., bioassay, hardiness, starvation

* To whom correspondence should be addressed.

252Pestic. Sci. 0031-613X/97/$09.00 1997 SCI. Printed in Great Britain(

Apparent imidacloprid tolerance in tobacco aphids 253

1 INTRODUCTION

One of the major problems in modern agriculture is thegrowing tendency of insect pests to become more andmore resistant to a wide variety of insecticidal classes.The peach-potato aphid, Myzus persicae Sulzer, and thetobacco aphid, Myzus nicotianae Blackman, are twoclosely related homopteran pests and resistance to con-ventional insecticides is now widespread in bothspecies.1,2 They showed the same mechanisms of resist-ance against organophosphates, carbamates and pyre-throids, i.e. elevated levels of carboxylesterase E4 orFE4, depending on karyotype.3 The genes of esteraseE4 and FE4 in the two species are identical.4 Addi-tionally a pirimicarb- and triazamate-insensitive acetyl-cholinesterase has very recently been reported as a newresistance mechanism in strains of M. persicae and M.nicotianae from di†erent parts of the world.5,6

The new chloronicotinyl insecticide, imidacloprid, isnow registered in 60 countries and fulÐls the ambitionof an agrochemical company in developing compoundswith a new or unconventional mode of action tocombat highly resistant insect pests.7 Like nicotine, thecompound acts as an agonist of the nicotinic acetyl-choline receptor.8 In contrast to nicotine, however, it ishighly speciÐc to insect receptors, as studies on verte-brate receptors have revealed.9 The physicochemicalproperties of imidacloprid and the corresponding bio-logical proÐle as well as some peculiarities, e.g. the anti-feedant e†ect on aphids and the ability to decrease thefertility of aphids at extremely low concentrations, arewell documented.10h13 Imidacloprid lacks any bondssusceptible to attack by the common esterase-basedmechanism of resistance in aphids and therefore it is avaluable tool in resistance management strategies.14

The tobacco-feeding form of M. persicae, M. nico-tianae, was Ðrst described using multivariate morpho-metrics by Blackman in 1987.15 Although originallydescribed as anholocyclic it is now well established thata holocycle is possible in certain countries, and that M.nicotianae and M. persicae can interbreed and producefertile eggs.16 M. nicotianae has been described as aserious pest in several crops, in particular on tobaccoplants, and most strains examined have been more orless resistant to conventional insecticides.1,17 In 1988estimated losses of 10È15 million dollars in the NorthCarolina tobacco crop were attributed to this singlepest.17

One study of a red-coloured morph of a highly resist-ant tobacco-feeding form of M. persicae from Japan,closely related to M. nicotianae, revealed only low ratiosof tolerance against imidacloprid, nicotine and cartap inoral ingestion as well as in dip bioassays.6 The results ofa monitoring programme running for several years,using discriminating concentrations of imidacloprid incontact bioassays, revealed only minor di†erencesbetween populations of M. persicae and M. nicotianae

from all over the world.14 Other investigations demon-strated that M. nicotianae from around the worldshowed low levels of resistance to imidacloprid andnicotine, but that some clones seemed to be less suscep-tible to the recently described antifeedant e†ect of imid-acloprid.12,13 M. nicotianae feeds on a plant containingconsiderable amounts of nicotine and one might specu-late that, in the course of evolution, this could have animpact on nicotinic acetylcholine receptor structure,although nicotine is transported in the xylem whileMyzus sp. prefers the phloem as a feeding site.18 Thereis a single publication which reports measurements ofthe affinity of imidacloprid to its receptor in susceptibleand resistant (tobacco-associated) M. persicae fromJapan, but no di†erences could be detected.6

In November 1994 we received a strain of M. nico-tianae collected from a tobacco Ðeld in France withapparent tolerance to imidacloprid when compared to asusceptible reference strain of M. persicae. The tolerancefactor for imidacloprid in this French strain of M. nico-tianae depends on the type of bioassay (applicationmethod) used. The present study was performed toinvestigate possible causes of these di†ering tolerancefactors among contact bioassays (FAO dip), systemictests and an artiÐcial double membrane feeding bio-assay.

2 MATERIALS AND METHODS

2.1 Insecticides

All insecticides used were technical grade. Nicotine waspurchased from Sigma (St. Louis, USA). Pirimicarb wasobtained from Promochem GmbH (Wesel, Germany).All other compounds were from Bayer AG (Leverkusen,Germany), except pymetrozine which was from CibaGeigy (Basel, Switzerland). Insecticidal stock solutionswere prepared in acetone or water, depending on thebioassay, and subsequently diluted with an aqueoussolution of “TritonÏ X-100 (1 g litre~1), when using theFAO dip test, pure water for the systemic test orsucrose solution (150 g litre~1) for the artiÐcial doublemembrane feeding bioassay.

2.2 Aphids

The tobacco aphid, Myzus nicotianae (strain FR), wascollected from a tobacco Ðeld in the Rhone valley inFrance (provided by Yves Bouchery, INRA, Colmar,France). The susceptible strain (NS) of the peach-potatoaphid, Myzus persicae, has been reared in the labor-atory since 1967 under the conditions described below.Both strains were reared on Chinese cabbage at 22È23¡C, 60% RH and a 16 : 8 h light : dark photoperiod.

254 Ralf Nauen, Alfred Elbert

2.3 Bioassays

All the bioassays described below were done in thelaboratory under controlled conditions, i.e. 21È22¡C,45È55% RH and an ambient photoperiod. All insecti-cidal tests were repeated at least three times with two orthree replicates of Ðve to six concentrations (30È45aphids) in each bioassay. Aphids not able to move in acoordinated manner (irreversible symptoms) werescored as dead. The starvation experiment was per-formed three times with 45 aphids in each bioassay.Lethal concentration values were calculated from probitregressions using the POLO computer program (LeOraSoftware, Berkeley, USA).

2.3.1 FAO dip testThe contact activity of the insecticides used was testedusing a modiÐed version of the FAO dip test.19Apterous adults of Myzus sp. were dipped for 5 s ininsecticidal solutions containing “TritonÏ X-100 (0É2 glitre~1). After dipping, the aphids were transferred tofreshly excised cabbage leaves, the petioles of whichwere immersed in a small tube containing pure water.Percentage mortality was scored 24 and 48 h post-dip.

2.3.2 Sachet testThe activity of the insecticidal compounds after oralingestion was tested with a modiÐcation of an artiÐcialdouble-membrane feeding bioassay, called the sachettest.20,21 The insecticides were diluted in an aqueoussolution of sucrose (150 g litre~1). The prepared solu-tion (0É4 ml) was pipetted between two layers ofstretched which formed the sachet. Groups ofParaÐlm}10 to 15 aphids, which had been starved for 4 h prior tothe bioassay, were placed into small Petri dishes(diameter 2É8 cm). These were sealed by stretching theprepared sachets across the top. A piece of yellow cel-lophane was placed over the sachet to enhance thefeeding activity of the aphids. Percentage mortality wasscored after 24 and 48 h.

2.3.3 Systemic test procedureThe systemic test procedure used was recentlydescribed.12 The cut petioles of excised cabbage leaveswere immersed in small tubes of aqueous dilutions ofimidacloprid. After a 4-h equilibration phase, each leafwas infested with 15 aphids. A Ðlter-paper disc wasplaced under the leaves to catch the honeydew excretedover each 24-h period. Mortality was assessed after 24,48 and 72 h. After each assessment the Ðlter paper discwas replaced and stained with a 1 g litre~1 solution ofninhydrin in acetone to make the excreted honeydewdroplets visible as dark purple spots.

2.3.4 Starvation testThe hardiness of adults of the di†erent strains used wasdetermined in starvation experiments. Aphids of similarsize and nutritional status of each strain were placed ingroups of 15 individuals in Petri dishes without a foodsource and the percentage mortality was scored at dif-ferent time intervals.

3 RESULTS AND DISCUSSION

3.1 Sachet test

The insecticidal activity of imidacloprid by oral inges-tion on strain FR was apparently rather poor in com-parison to the very good e†ect against the susceptibleM. persicae strain NS (Table 1). A similar result wasobtained with nicotine, another agonist of the nicotinicacetylcholine receptor. The slopes of the dose responsecurves were less steep for strain FR than for strain NS,especially in the case of imidacloprid, which suggests aconsiderable heterogeneity in the Ðeld-collected popu-lation of strain FR. For this strain, tolerance factors forthe two organophosphates tested, methamidophos andoxydemeton-methyl, were low, thus indicating only alow level of tolerance to organophosphates in strain FR.Apart from the insecticides mentioned above, we also

TABLE 1Activity of Di†erent Insecticides on Myzus persicae (NS) and Myzus nicotianae (FR) using an ArtiÐcial Double

Membrane Feeding Bioassay (Sachet Test)

Strain NS Strain FR

L C50 (48 h) L C50 (48 h)Insecticide (mg litre~1) 95% CL a Slope (mg litre~1) 95% CL Slope T Fb

Imidacloprid 0É073 0É046È0É11 1É24 14 3É9È108 0É495 192Nicotine 4É5 1É7È8É5 1É21 [100 È È [22Methamidophos 1É5 0É73È3É2 1É71 3É4 0É91È12 1É08 2Oxydemeton-methyl 0É41 0É31È0É53 1É71 3É2 0É73È16 1É30 8Pymetrozine 1É6 1É2È2É0 2É13 [100 È È [63

a 95% conÐdence limits.b Tolerance factor, for strain for strain NS.LC50 FR/LC50

Apparent imidacloprid tolerance in tobacco aphids 255

tested pymetrozine, a new aphicide which acts by inter-ruption of the aphid feeding process.22 Using pymetro-zine we also found a high tolerance in strain FR relativeto strain NS when scoring for mortality in the oralingestion bioassay after 48 h.

3.2 FAO dip test

In contrast to the results of the oral ingestion bioassay,the FAO dip test revealed only a tolerance factor of 9 toimidacloprid by strain FR, indicating that there wasindeed a degree of tolerance in this tobacco-feedingstrain. As with the feeding bioassay, the slope of thedose response curve of the FAO dip test was lower forstrain FR than for strain NS. The results in Table 1 andTable 2 revealed that the tolerance factor between aphidstrains against imidacloprid depends strongly on thebioassay procedure used.

3.3 Systemic test

Bioassays with aphids placed on cut cabbage leaveswith their petioles immersed in imidacloprid solutionsrevealed a high degree of tolerance of the French strainFR towards imidacloprid (Fig. 1). This result agreed

TABLE 2Activity of Imidacloprid in Aphid Dip Bioassays (FAO Dip

Test)

L C50 (48 h)Species (mg litre~1) 95% CL a Slope T Fb

M. persicae (NS) 1É7 1É3È2É3 2É10 ÈM. nicotianae (FR) 16 4É8È46 1É21 9

a 95% conÐdence limits.b Tolerance factor, for strain for strain NS.LC50 FR/LC50

with the Ðndings of the sachet test. After 48 h the LC50value of imidacloprid for strain FR was [10 mg litre~1whereas strain NS showed an value of 0É21 mgLC50litre~1, giving a tolerance factor of [47. The resultsfrom either oral ingestion bioassay, i.e. sachet test andsystemic test, suggest that the French strain of M. nico-tianae, FR, is highly tolerant to imidacloprid in di†erenttypes of short-term (¹48 h) oral ingestion bioassays.We know from our previous work that imidaclopridalso acts as an antifeedant on aphids after oral adminis-tration of small amounts of active ingredient.12 Thus wemeasured the e†ect of di†erent concentrations of sys-temically applied imidacloprid on the honeydew excre-tion activity of strain FR relative to the excretionactivity in our susceptible reference strain NS. Theresult of these tests clearly show that imidacloprid has astrong e†ect on the feeding behaviour of both aphidstrains (Table 3). However, there is no signiÐcant di†er-ence in the calculated (48 h) values for theEC50reduction in honeydew excretion between strain FR andstrain NS, indicating that within the 48-h test period,imidacloprid has the same potential as an antifeedantfor both strains. As displayed in Fig. 1 the French strainshowed a similar dose mortality curve for imidaclopridthree days after infestation as the susceptible strain hadafter two days. However, the real end-point in this bio-assay could not be estimated for both strains, becausethe leaves showed Ðrst signs of senescence after fourdays. Abbot-corrected mortality data for strain NSrevealed that values of both strains came closerLC50together, i.e. the tolerance factor dropped considerably,to values between 15 and 20 after three days. Furtherexperiments would be needed to determine the time-course of behavioural e†ects and mortality over alonger period of time using other Ðeld-collected strainsof M. nicotianae. The French strain showed the samedegree of mortality in the concentration range from 0É1to 10 mg litre~1 after 48 h and most of the individuals

Fig. 1. DoseÈresponse relationship for imidacloprid against Myzus persicae (strain NS) and Myzus nicotianae (strain FR) aftersystemic application through cut cabbage leaf petioles. Aphids dead or with irreversible symptoms were scored after one day,(=)

two days and three days. (È È È) Honeydew excretion was evaluated after one day.(K) (+)

256 Ralf Nauen, Alfred Elbert

TABLE 3Reduction in Honeydew Excretion of Myzus persicae (NS) and Myzus nico-tianae (FR) 24 h after Systemic Application of Imidacloprid through Cut

Cabbage Leaf Petioles (Systemic Test)

EC50 (48 h)aSpecies (mg litre~1) 95% CL b Slope T Fc

M. persicae (NS) 0É0157 0É002 94È0É0676 1É25 ÈM. nicotianae (FR) 0É0272 0É008 37È0É0760 0É875 1É7

a E†ective concentration, where honeydew excretion is reduced by 50% fromcontrol.b 95% conÐdence limits.c Tolerance factor, for strain for strain NS.LC50 FR/LC50

were not feeding and were restless. After 72 h, the dose-response curve rises, suggesting that some of the aphids,which showed restless behaviour at the di†erent concen-trations, starved to death. The time after which 50% ofthe aphids of strain FR on cabbage leaves treated sys-temically with 1 mg litre~1 imidacloprid died was 52È56 h.

3.4 Starvation test

In the next experiment we studied the relative hardinessof the French strain compared to strain NS in order toclarify the delayed response of strain FR in systemicbioassays. We performed some simple experimentswhere the aphids were placed in a Petri dish withoutaccess to a food source, to determine how long theFrench strain is able to survive without food or water.The French M. nicotianae strain FR survived starvationbetter than the reference laboratory strain NS (Table 4),thus explaining to some extent the high tolerancefactors of strain FR in short-term oral ingestion bio-assays. Note that the value of 56É9 h (Table 4) forLT50strain FR in starvation experiments reÑects the LT50value determined for the same strain after systemicapplication of 1 mg litre~1 imidacloprid through cutcabbage leaf petioles. Another starvation experimentwas performed Ðve months later and revealed thatstrain FR lost its hardiness when reared on Chinesecabbage in the laboratory. At present it can only be spe-

TABLE 4Survival Times of Di†erent Myzus sp. Strains without Access

to a Food Source (Starvation Bioassay)

Species Strain/clone L T 50 (h)b 95% CL c

M. persicae NS 37É4 35É8È39É0M. nicotianae FR (2 months)a 56É9 55É1È58É9M. nicotianae FR (7 months)a 38É2 35É4È41É0

a Duration of rearing of strain FR in our laboratory.b Time in hours after which 50% of the aphids died.c 95% conÐdence limits.

culated that this loss is associated with suboptimalrearing conditions, i.e. strain FR was maintained onChinese cabbage and not on tobacco, the original hostplant in the Ðeld. The loss of starvation hardiness couldbe due to a phenotypic change, but it is also possiblethat strain FR was merely a heterogeneous populationin which a susceptible variant out-reproduced a hardy,tolerant variant. Further experiments with other hardystrains of M. nicotianae should clarify which of theabove-mentioned reasons is responsible for theobserved loss of starvation hardiness.

4 CONCLUSIONS

The present investigation revealed that with short-termbioassays (¹48 h), the only reliable measure of imid-acloprid tolerance is that made using an aphid dip tech-nique, e.g. the FAO dip test which has been used in thepast.14 At present it remains unclear whether the lowfactor of tolerance of strain FR to imidacloprid in theapplied dip test is a real mechanism-based tolerance ornatural variation between closely related species. Theresults obtained with the organophosphate meth-amidophos indicated that our laboratory strain of M.persicae (reared for approx. 30 years in the laboratory)is not over-susceptible and is therefore a suitable refer-ence. The tolerance factor of imidacloprid determined ina French Ðeld strain of M. nicotianae in the dip bioassayis comparable to those published very recently for someother strains of M. nicotianae and tobacco-associatedforms of M. persicae from other parts of theworld.5,13,14 Short term bioassays, e.g. 24 h or 48 h,where oral ingestion of the active ingredient is necessaryin order to detect possible tolerance, cannot be recom-mended without prior testing of the hardiness of theaphids, because of imidaclopridÏs ability to act as anantifeedant.12 The tolerance factors in such bioassayscould depend more on behavioural characteristics of theconsidered aphid strains than on the actual toxicity ofimidacloprid. If oral ingestion bioassays for tolerancedetection are unavoidable, scoring mortality after 72 h

Apparent imidacloprid tolerance in tobacco aphids 257

or even better after 96 h would be recommended. Incontrast to an aphid dip test, the oral ingestion bioassaymainly measures the reversible behavioural alterationsin aphids which cause them to die due to starvation. Iftolerance is measured in such kinds of bioassay, then itcould be interpreted as behavioural tolerance, inducedby the avoidance of imidacloprid-treated plants or bystarvation tolerance (hardiness) of the aphids as shownin our investigation. Thus it is safer to choose the aphiddip technique for determining possible tolerance,because it focuses more on the fast-acting neuro-toxicological mode of action which causes death withclearly visible symptoms resulting from interferencewith the nervous system. Apart from the lack of possibleinformation on aphid behaviour, the FAO dip test isquick, reliable and produces repeatable results asdemonstrated in other studies.14 In order to build up amore distinct picture of tolerance mechanism, it couldbe useful sometimes to run simple measures of contactefficacy alongside assays which allow the aphid some“choiceÏ in its response. However, because of the factthat we looked at only two strains of Myzus sp. it is notpossible to declare the starvation hardiness as acommon mechanism of tolerance to the e†ects of chlo-ronicotinyls ; it is much more an example of how theexpression of behaviour may decrease the efficacy ofinsecticides and that such behaviour could be lost aftera few months of rearing in the laboratory.

The Ðndings outlined in the present report shouldalso be considered when assessing whiteÑy populationsor other sucking pests for possible resistance againstimidacloprid. Di†erences in the hardiness of di†erentpopulations could strongly a†ect the results of resist-ance monitoring, if imidacloprid has the same distinctmode of action on other homopteran pests as onaphids, i.e. neurointoxication with clearly visible symp-toms after a few hours at recommended Ðeld dose ratesand the more inconspicuous antifeeding response insublethal concentrations, causing death by starvationover a longer period of time.

ACKNOWLEDGEMENTS

The excellent technical assistance of Dagmar Simon andDirk-Uwe Zwitzers is gratefully acknowledged. We arealso grateful to Peter Lo� sel for correcting the Englishgrammar.

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258 Ralf Nauen, Alfred Elbert

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