INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Survey of Resistance to Four Insecticides and their Associated Mechanisms in Different Genotypes of the Green Peach Aphid (Hemiptera: Aphididae) From Chile EDUARDO FUENTES-CONTRERAS,1,2 CHRISTIAN C. FIGUEROA,3 ANDREA X. SILVA,3 LEONARDO D. BACIGALUPE,3 LUCI´A M. BRIONES,3 STEPHEN P. FOSTER,4 5 AND THOMAS R. UNRUH

J. Econ. Entomol. 106(1): 400Ð407 (2013); DOI: http://dx.doi.org/10.1603/EC12176

ABSTRACT The green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) is a major pest of agriculture worldwide that has proved to be particularly adept at evolving insecticide resistance. Several mechanisms that confer resistance to many insecticide types have been described in M. persicae. We measured the resistance status of nine multilocus genotypes (MLGs) of this aphid species collected in Chile. MLGs were identiÞed using microsatellite markers, and these MLG clonal populations were measured for the presence of modiÞed acetylcholinesterase (MACE), kdr and super kdr mutations, and enhanced carboxyl esterase activity. Toxicological bioassays were used to estimate aphid LC50 when treated with metamidophos (organophosphate), pirimicarb (dimethyl carbamate), cyßuthrin (pyrethroid), and imidacloprid (neonicotinoid). Two MLGs presented !20-fold resistance to pirimicarb, which was associated with the MACE mutation in the heterozygous condition. The kdr mutation was found in only four MLGs in the heterozygous condition and they showed resistance ratios (RR) to cyßuthrin of less than sevenfold. The super kdr mutation was not detected. Enhanced carboxyl esterase activity was predominantly found in the susceptible (S) to Þrst level of resistance (R1) with RR to metamidophos less than eight-fold. Finally, RR to imidacloprid was also less than eight-fold in all MLGs tested. A few MLGs with resistance to pirimicarb were found, while susceptibility to cyßuthrin, metamidophos and imidacloprid was still predominant. A signiÞcant positive correlation between imidacloprid tolerance with pirimicarb resistance was detected, as well as between imidacloprid and metamidophos tolerance. With the increase in the use of neonicotinoid insecticides, better rotation of insecticides with different modes of action will be necessary to prevent further development of M. persicae insecticide resistance in Chile. KEY WORDS Myzus persicae, cyßuthrin, metamidophos, pirimicarb, imidacloprid

The green peach aphid, Myzus persicae (Sulzer) is an exceptional pest, based on its cosmopolitan distribution, highly polyphagous habits, tremendous reproductive rate, and ability to vector many viral diseases (Blackman and Eastop 2007). High levels of variability in color, life cycle, hostÐplant relationships, and insecticide resistance reßect its ability to adapt as an agricultural pest (Blackman and Eastop 2007). Management of M. persicae is based mainly on chemical control, although a long record of insecticide resistance has been described for this pest (Foster et al. 2007a). Resistance to organophosphates Þrst was reported in this pest in the 1950s (Anthon 1955, 1 Departamento de Produccio ´ n Agrõ´cola, Facultad de Ciencias Agrarias, Universidad de Talca, Casilla 747, Talca, Chile. 2 Corresponding author,e-mail: [email protected]. 3 Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. 4 Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom. 5 USDAÐARS, 5230 Konnowac Pass Rd., Yakima, WA 98951.

Georghiou 1963), and later to cyclodienes (Bauernfeind and Chapman 1985, Unruh et al. 1996), carbamates (Hurkova 1973, McClanahan and Founk 1983), and pyrethroids (Attia and Hamilton 1978, McClanahan and Founk 1983). More recently, resistance to neonicotinoid insecticides has been described as well (Philippou et al. 2010, Puinean et al. 2010, Bass et al. 2011). Seven different molecular mechanisms of insecticide resistance are known in M. persicae, which include target site insensitivity and gene ampliÞcation of detoxifying enzymes, showing the remarkable capacity for microevolution of this species in agroecosystems (Fenton et al. 2010). Organophosphate and partial carbamate and pyrethroid resistance is based on gene ampliÞcation resulting in elevated production of carboxylesterase enzymes, identiÞed as E4 and FE4, that detoxify and sequester these ester insecticides before they affect their target sites (Field and Blackman 2003). Genotypes of M. persicae can be classiÞed as susceptible (S), mildly resistant (R1), highly resistant (R2), or ex-

0022-0493/13/0400Ð0407$04.00/0 ! 2013 Entomological Society of America

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FUENTES-CONTRERAS ET AL.: RESISTANCE OF M. persicae FROM CHILE

tremely resistant (R3) based on levels of carboxylesterase activity (Devonshire et al. 1992). A modiÞed acetylcholinesterase (MACE) is insensitive to dimethyl-carbamates, associated with the S431P mutation in the active site of this enzyme (Moores et al. 1988, Nabeshima et al. 2003). Cyclodiene resistance is produced by the mutation A302G (Rdl) in the !-aminobutyric acid (GABA) receptor (Anthony et al. 1998), whereas the mutations L1014 F (kdr) and M918T (super kdr) in the voltage gated sodium channel confer resistance to DDT and pyrethroids (Martinez-Torres et al. 1999, Eleftherianos et al. 2008). Neonicotinoid resistance recently has been found based on an increase in oxidase activity associated with the ampliÞcation of the gene CYP6CY3 coding a cytochrome P450 (Philippou et al. 2010, Puinean et al. 2010) and a mutation in the nicotinic acetyl choline receptor (Bass et al. 2011). Multiple insecticide resistance mechanisms carried by a single genotype is not infrequent; indeed, such linkage disequilibrium makes rotation of insecticides with different modes of action more difÞcult (Field et al. 1996, Foster et al. 2000, Field and Foster 2002). The insecticide resistance status of this M. persicae can be inßuenced by its life cycle characteristics (Margaritopoulos et al. 2007, Criniti et al. 2008). This species has holocyclic mode of reproduction wherein one generation of sexual reproduction and oviposition occurs during autumn on peach (Prunus persica L.), its primary host. Offspring from this sexual stage reproduce by parthenogenesis and disperse from peach to secondary host plants consisting of herbaceous crops and weeds during spring and summer (Blackman and Eastop 2007). Under mild winter conditions, asexual reproduction on secondary host plants may persist throughout the year corresponding with partial or complete loss of the holocyclic life cycle. In many agricultural areas, M. persicae frequently are subjected to insecticide sprays on both primary and secondary host plants, which selects for insecticide resistance. Asexual reproduction during the growing season maintains these resistant genotypes as long as insecticide sprays provide them a Þtness advantage over susceptible genotypes (Foster et al. 2002). However, signiÞcant Þtness costs, suffered by some forms of insecticide resistance at least, can lead to a reduction of resistant genotypes in the population when insecticide selection pressure is relaxed (Foster et al. 1997, 2003b, 2005, 2007b, 2011). In Chile, M. persicae historically has been controlled with organophosphate, carbamate, and pyrethroid insecticides (Casals and Silva 1999). More recently, neonicotinoid insecticides have been included in control programs, often used in combination with pyrethroids to enhance the aphid knock-down effect (FuentesContreras et al. 2007). Resistance to organophosphate insecticides of M. persicae in Chile has been reported on sugar beet (Beta vulgaris L.) (Casals and Silva 1999), peach (Casals and Silva 2000), and with lower intensity in the specialized subspecies M. persicae nicotianae on tobacco (Fuentes-Contreras et al. 2004). Few detailed studies on the resistance mechanisms in

401

Table 1. Collection site and host plant for different multilocus genotypes (MLGs) of Myzus persicae in Chile, and obtained as insecticide resistant (4824J) and susceptible (4255A) genotypes from Rothamsted Research, United Kingdom MLGs

Region, country

N 50Ð1 N 30-A1 N 36Ð1 13 A

Atacama, Chile Coquimbo, Chile Coquimbo, Chile Valparaõ´so, Chile

16 A

Valparaõ´so, Chile Los Rõ´os, Chile OÕHiggins, Chile Maule, Chile Los Rõ´os, Chile Atacama, Chile Los Rõ´os, Chile Los Lagos, Chile Brittany, France Worcestershire, United Kingdom

26 A Teno 7B S 74Ð1 S 25-A3 4824J 4255A

a

Host plant Brassica rapa (w)a Brassica rapa (w) Malva nicaensis (w) Solanum esculentum (c)a Anoda cristata (w) Anoda cristata (w) Brassica rapa (w) Brassica rapa (w) Capsicum annuum (c) Brassica rapa (w) Raphanus raphanistrum (w) Brassica rapa (w) Brassica rapa (w) Prunus persica (c) Brassica napus (c)

W $ weed, C $ crop.

genotypes collected from different areas or crops have been performed (Castan˜ eda et al. 2011). No genetic diversity was observed in clones of M. persicae nicotianae sampled in tobacco Þelds in Chile; the single asexual lineage detected to date and was probably introduced from the northern hemisphere in recent decades (Fuentes-Contreras et al. 2004, Zepeda-Paulo et al. 2010). In contrast, higher genetic diversity and no deviation from HardyÐWeinberg equilibrium was found in samples of M. persicae from potatoes (Solanum tuberosum L.,) which suggests that a signiÞcant level of sexual reproduction in M. persicae occurs in Chile (Fenton et al. 2010). Herein we measure the insecticide resistance status of nine M. persicae genotypes through toxicological bioassays and biochemical and molecular tests to establish the main insecticide resistance mechanisms present. Comparative bioassays with reference asexual lineages of known levels of resistance and susceptibility also were performed. Materials and Methods Sampling. During spring of 2008 and summer of 2009, aphids were sampled from different crops and weeds to obtain a sample accounting for the genetic diversity that has been subjected to different insecticide managements (Table 1). To limit the chance of collecting individuals from the same parthenogenetic colony, aphids were collected on plants separated by at least 20 m. Parthenogenetic colonies were established from individual wingless adult females collected in the Þeld. Colonies were reared on pepper seedlings (Capsicum annuum L. variety grossum ÔResistantÕ) in Blackman boxes (Blackman 1971) under conditions that ensure parthenogenetic reproduction (20 " 1#C and a photoperiod of 16:8 [L:D] h). Colonies were maintained by transferring Þve wingless adults on new 7-d-old pepper seedlings every 10 d, and for at least 10 generations before bioassay experiments. Two colonies of M. persicae MLGs originally collected in the

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Table 2. Allele combinations and main resistance mechanisms described for different multilocus genotypes (MLGs) of Myzus persicae in Chile, and obtained as insecticide resistance references from Rothamsted Research, United Kingdom MLGs

Myz 2

Myz 3

Myz 25

M35

M37

M40

Fa

kdr

super kdr

MACE

Esterase

N 50Ð1 N 36Ð1 N 30-A1 13 A 16 A 26 A Teno 7B S 25-A3 S 74Ð1 4824J 4255A

177193 177177 177177 193193 191193 191191 177203 177193 191191 191193 179207

107107 107125 125125 125125 125125 125125 125125 107107 107125 103121 103121

121123 121121 121123 123125 121121 123125 123125 121123 121121 119121 119121

186186 186186 186186 186203 186186 186186 186186 186186 186186 186186 186186

157163 155155 155157 155163 155155 155157 155157 155163 155155 155155 155155

128130 128130 128130 132132 128130 128130 124126 124126 128130 130130 128130

S S S ML MW S S MW ML -

ss ss rs ss rs rs ss ss rs rr ss

ss ss ss ss ss ss ss ss ss rr ss

ss ss ss ss rs ss ss ss rs ss ss

R1-R2 S S-R1 R1 S-R1 S-R1 S S-R1 S R3 S

a F $ frequency of MLG detection in other samples. S $ single copy, ML $ multiple copies in different host plants in the same locality, MW $ multiple copies widespread in different host plants in different localities.

United Kingdom and France were provided by Rothamsted Research (in the United Kingdom) and represented insecticide resistant (4824J) and susceptible (4255A) reference genotypes. The reference resistant MLG 4824J has been described as resistant to organophosphates, dimethyl carbamates, and pyrethroids, but not to neonicotinoids. Both reference MLGs were maintained in Chile for at least 20 generations, under the same conditions listed above for Chilean samples. Microsatellite and Insecticide Resistance Mechanisms Analyses. Each M. persicae colony was genotyped using six previously described microsatellite loci (Myz2, Myz3, Myz25, M35, M37, M40) (Wilson et al. 2004, Zepeda-Paulo et al. 2010). Multilocus genotypes (MLGs) were deÞned as the different genotypes obtained after combining alleles from each ampliÞed locus in the whole sample (Zepeda-Paulo et al. 2010). Redundant colonies (i.e., colonies with the same MLGs) were discarded. The presence of insecticide resistance mutations was screened for MLGs by using allelic discrimination based on quantitative (real time) polymerase chain reaction assays for kdr (L1014 F) and super kdr (M918T) (Anstead et al. 2004), and for MACE (Anstead et al. 2008). Carboxylesterase activity was measured using 1-naphthyl acetate as a substrate in a microplate bioassay (Devonshire et al. 1992), with Þve biological replicates and three technical replicates per measurement. Nine genotypes that included the complete combination of genetic conÞgurations for insecticide resistance mechanisms were selected for the toxicological experiments. The results of the insecticide resistance mechanisms of the remaining MLGs in Chile have been published in Castan˜ eda et al. (2011). Toxicological Measurements. Nine parthenogenetic colonies originating from Chile and the two reference parthenogenetic colonies from the Rothamsted collection (Table 1) were screened in toxicity bioassays. These bioassays were performed using insecticides representative of the main functional groups used for M. persicae control in Chile. In particular, the following active ingredients were selected: metamidophos (organophosphate), pirimicarb (dimethyl-carbamate), cyßuthrin (pyrethroid), and imida-

cloprid (neonicotinoid). All insecticides were analytic standards (Supelco, Bellefonte, PA) with purity levels above 98%. The bioassay was performed using the leaf-dip technique described by (Nauen and Elbert 2003) with a few modiÞcations. Five insecticide concentrations and a control treatment were used, with four replicates of each concentration and with control mortality lower than 20%. Insecticide solutions were prepared in distilled water with 0.02% vol:vol Triton X-100 (Sigma-Aldrich, St. Louis, MO), which also was used as a control treatment. Pepper leaf-discs of the same cultivar used for rearing were dipped into each insecticide solution. Then the leaf-discs were left to dry and placed in petri dishes with an agar layer to maintain humidity. Thirty adult wingless aphids were placed on the leaf-discs on each petri dish. Because of the large number of individual aphids necessary for the bioassays, it was not possible to synchronize the age of the adults, therefore our sample was composed by newly molted adults up to %10-d-old adults. All bioassays were scored 48 h after treatment, counting the survivors that reacted to contact with a Þne brush. Lethal concentration at 50% (LC50) and their 95% CL were computed with the Probit regression (SPSS 2004). Resistance ratios (RR) were calculated for each insecticide as the ratio between the LC50 of each MLG and the most susceptible MLG found. LC50 values were considered signiÞcantly different if their 95% FL did not overlap (Finney 1971). Because normality assumption of the data were not achieved Spearman rank correlations were calculated between LC50 obtained for each insecticide and MLG to measure crossresistance between different insecticides. Bonferroni correction was used to reduce the probability of type I error (Sokal and Rohlf 1995). Results From the nine Chilean MLGs selected for this study, the voltage-gated sodium channel mutation kdr was found only in the heterozygote condition in four MLGs: N 30-A1, 16 A, 26 A, and S 74Ð1. Homozygote kdr and super kdr mutations were present only in the reference genotype 4824J (Table 2). MACE was de-

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FUENTES-CONTRERAS ET AL.: RESISTANCE OF M. persicae FROM CHILE

403

Table 3. Statistics from concn-mortality bioassays for four insecticides in different multilocus genotypes (MLGs) of Myzus persicae collected in Chile and two reference genotypes MLGs Metamidophos N 36Ð1 S 25-A3 13 A N 50Ð1 S 74Ð1 16 A Teno 7B N 30-A1 26 A 4824J 4255A Pirimicarb S 25-A3 N 36Ð1 13 A N 30-A1 26 A Teno 7B N 50Ð1 S 74Ð1 16 A 4824J 4255A Cyßuthrin S 74Ð1 N 50Ð1 N 30-A1 N 36Ð1 Teno 7B S 25-A3 13 A 16 A 26 A 4824J 4255A Imidacloprid S 25-A3 N 36Ð1 N 50Ð1 13 A N 30-A1 26 A 16 A S 74Ð1 Teno 7B 4824J 4255A a

Slope (SE)a

x2

P

LC50

95% FL

RR

3.41 (0.22) 2.33 (0.19) 2.29 (0.17) 2.98 (0.23) 2.70 (0.20) 2.25 (0.15) 2.33 (0.15) 2.03 (0.13) 1.68 (0.11) 2.14 (0.14) 2.94 (0.31)

46.4 39.1 86.9 57.6 37.0 72.5 56.6 48.7 69.3 58.5 7.3

&0.001 0.03 &0.001 &0.001 0.005 &0.001 &0.001 &0.001 &0.001 &0.001 0.09

4.47 13.57 15.71 17.89 19.24 22.33 27.46 27.82 34.12 65.91 7.41

3.80Ð5.26 10.98Ð16.84 11.29Ð22.05 14.36Ð23.04 15.95Ð23.68 16.62Ð30.92 21.21Ð36.14 21.47Ð36.33 23.95Ð49.70 49.44Ð90.16 6.50Ð8.30

3.0 3.5 4.0 4.3 5.0 6.1 6.2 7.6 14.8 1.7

1.82 (0.14) 2.13 (0.15) 1.69 (0.13) 3.08 (0.22) 3.11 (0.22) 2.08 (0.14) 1.91 (0.13) 1.02 (0.13) 0.85 (0.13) 2.57 (0.17) 2.56 (0.17)

38.2 44.7 35.5 35.8 54.1 42.5 106.1 29.9 31.0 51.6 44.7

0.04 &0.001 0.008 0.008 &0.001 0.001 &0.001 0.04 0.03 &0.001 &0.001

7.34 9.12 9.27 10.9 11.44 14.37 15.23 160,74 407.45 26.90 10.38

5.70Ð9.29 7.22Ð11.50 7.23Ð11.80 9.25Ð13.0 9.35Ð14.12 11.45Ð18.32 10.37Ð23.64 87.38Ð482.05 153.63Ð3965.05 21.28Ð34.9 8.84Ð13.46

1.2 1.3 1.5 1.6 2.0 2.1 21.9 55.5 3.7 1.4

3.35 (0.39) 4.74 (0.42) 1.37 (0.10) 2.14 (0.17) 3.58 (0.25) 2.21 (0.16) 1.99 (0.15) 2.15 (0.16) 1.95 (0.16) 3.57 (0.25) 2.17 (0.17)

66.0 31.3 32.5 35.1 29.0 40.7 49.8 27.7 39.1 43.3 88.9

&0.001 0.03 0.02 0.009 0.05 0.02 &0.001 0.07 0.03 0.01 &0.001

1.20 1.94 2.23 2.92 3.34 3.49 3.94 6.66 7.57 33.54 3.92

0.74Ð1.54 1.72Ð2.17 1.67Ð3.00 2.34Ð3.53 2.95Ð3.78 2.80Ð4.26 3.04Ð4.98 5.64Ð7.95 6.12Ð9.65 28.69Ð39.06 2.94Ð5.18

1.6 1.9 2.4 2.8 2.9 3.3 5.6 6.3 28.0 3.3

1.45 (0.12) 1.39 (0.11) 1.49 (0.11) 1.37 (0.10) 2.56 (0.21) 1.70 (0.11) 1.63 (0.11) 1.45 (0.10) 1.38 (0.10) 1.91 (0.13) 1.65 (0.11)

33.1 64.1 33.2 22.5 198.9 57.6 24.7 100.7 72.5 66.7 46.0

0.02 &0.001 0.02 0.21 &0.001 &0.001 0.14 &0.001 &0.001 &0.001 &0.001

0.31 0.33 0.36 0.77 0.86 1.69 1.73 1.79 2.48 3.5 0.78

0.22Ð0.42 0.19Ð0.51 0.26Ð0.49 0.62Ð0.94 0.48Ð1.55 1.20Ð2.39 1.38Ð2.17 1.08Ð3.09 1.60Ð4.11 2.47Ð5.13 0.57Ð1.07

1.1 1.2 2.5 2.8 5.5 5.6 5.8 8.0 11.3 2.5

Sample size, N $ 4 for all experiments.

tected in the heterozygote condition in MLGs 16 A and S 74 Ð1 (Table 2). Finally, three MLGs (N 36 Ð1, Teno 7B, and S 74 Ð1) showed low activity levels of carboxylesterase (mean $ 0.122, SD $ 0.07), within the ranges reported for the susceptible reference S genotype (4255A) (Table 2). Another four MLGs showed levels intermediate between S and R1 (N 30-A1, 16 A, 26 A, and S 25-A3; mean $ 0.334, SD $ 0.12), whereas only 13A (mean $ 0.446, SD $ 0.08) reached R1 and N 50-A1 (mean $ 0.538, SD $ 0.09) an intermediate level between R1 and R2. No MLGs with R2 or R3 resistance levels were found; therefore, the higher carboxylesterase activity level was the R3 reference genotype 4824J (Table 2). Multiple copies of some MLGs on different host plants and localities (MW) were detected for 16 A and S 25-A3, whereas

on different host plants in the same locality (ML) they were found for 13 A and S 74Ð1. However, single copy (S) ones were more common with Þve MLGs (N 50Ð1, N 36Ð1, N 30-A1, 26 A, and Teno 7B) (Table 2). Metamidophos LC50 values ranged between 4.47 ppm for N 36Ð1 and 34.12 ppm for 26 A. The susceptible reference genotype (4255A) showed a LC50 of 7.41 ppm, therefore RRs were calculated with the most susceptible MLG N 36Ð1 (Table 3). The high RR of 7.6 for MLG 26 A was almost half of the RR of 14.8 found for the R3 reference genotype 4824J (Table 3). Metamidophos and imidacloprid LC50 values were significantly correlated (Spearman rank correlation $ 0.77, P & 0.05). Mortality for pirimicarb showed a higher variation with LC50 from 7.34 ppm for S 25-A3Ð407.45 ppm for

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JOURNAL OF ECONOMIC ENTOMOLOGY

16 A (Table 3). The higher RRs were 55.5-fold for 16 A and 21.9-fold resistance for S 74 Ð1 (Table 3). Both of these MLGs carry the MACE mutation in the heterozygous condition (Table 2). The resistant reference genotype (4824J) did not have this mutation and showed an intermediate RR for pirimicarb possibly related to its higher carboxylesterase activity (Table 2). Pirimicarb and imidacloprid LC50 values were also signiÞcantly correlated (Spearman rank correlation $ 0.76, P & 0.05). Cyßuthrin LC50 values were less variable ranging from 1.2 ppm for S 74 Ð1 and 7.57 for 26 A and an RR of 6.3-fold (Table 3). In contrast, the reference genotype (4824J) was 28-fold resistant to cyßuthrin, which is probably associated with the kdr and super kdr mutations in this homozygous resistant genotype (Table 2). No association between cyßuthrin LC50 values and kdr mutation in the heterozygous condition was evident with the MLGs from Chile (Table 2). LC50 values with imidacloprid ranged from 0.31 ppm for S 25-A3Ð2.48 ppm for Teno 7B (Table 3). The reference genotype (4824J) was 11.3-fold resistant to imidacloprid, but all other MLGs exhibited RR lower than 10-fold (Table 3). Discussion SigniÞcant resistance to pirimicarb was present in two analyzed M. persicae MLGs, associated with the presence of the MACE mutation. Low levels of resistance to metamidophos also were found, probably mediated by enhanced carboxylesterase activity in few MLGs. The super kdr mutation was not detected and the kdr mutation was found only in the heterozygous condition, with rather low levels of cyßuthrin resistance (&10-fold). Studied MLGs were also susceptible to imidacloprid, although a signiÞcant correlation with toxicity toward pirimicarb and metamidophos was detected. The pirimicarb resistant MLGs were collected in multiple copies on different host plants in the same locality (S 74 Ð1) or from different host plants on different distant localities (16 A). This suggests that MLG presence on different host-plant species (or host-plant families) and geographic locations are not related with the historical or current use of dimethylcarbamate sprays, and that parthenogenetic reproduction and dispersal during spring and summer could allow a wide distribution of some MLGs in Chile. Another widespread MLG was S 25-A3, which was fully susceptible to the insecticides tested. Single copy MLGs were more frequent with predominant susceptible phenotypes. Castan˜ eda et al. (2011), by using a more extensive MLG collection from which our group of nine genotypes were selected, found that insecticide resistance mutations (kdr and MACE) and enhanced carboxylesterase activity were present in linkage disequilibrium and not associated with metabolic rate or reproductive Þtness. Several ecological factors can interact to produce a dynamic variation of insecticide resistance in the Þeld. Among them, predominant crops or weeds as host

Vol. 106, no. 1

plants, climatic conditions that favor reproductive life cycles, and insecticide management can impose selection pressures that result in substantial variation in insecticide resistance frequency between regions, countries, and seasons (Foster et al. 2002, Fenton et al. 2010). Surveys from European countries in Mediterranean areas with extensive cultivation of peach and therefore with sexual reproduction, have shown a high frequency of M. persicae carrying high and extreme carboxylesterase activity (mainly FE4), MACE, kdr and super kdr as resistance mechanisms (Mazzoni and Cravedi 2002, Guillemaud et al. 2003, Margaritopoulos et al. 2007, Criniti et al. 2008). Unruh et al. (1996, 2008) in peach growing areas from the western United States detected signiÞcant levels of endosulfan resistance associated with an rdl mutation (Anthony et al. 1998) that later was found in southern France (Guillemaud et al. 2003). Colder areas without signiÞcant peach cultivation, like England, show substantial variation in insecticide resistance frequency with temporal changes probably mediated by the predominant insecticide sprays regimes (Foster et al. 2000, 2002; Field and Foster 2002; Parker et al. 2006; Anstead et al. 2007). Similar results with susceptible and resistant MLG dynamics after insecticide sprays have been shown for M. persicae on potatoes in Scotland (Fenton et al. 2005, Kasprowicz et al. 2008), New Zealand (van Toor et al. 2008), and oilseed rape from northern France (Zamoum et al. 2005). Toxicological surveys made one decade ago reported resistance to organophosphate insecticides on sugar beet and peach (Casals and Silva 1999, 2000) and lower levels on tobacco (Fuentes-Contreras et al. 2004). Now we Þnd resistance to dimethyl carbamates, but lower levels of resistance to organophosphates. Such pirimicarb resistance results may be associated with the progressive replacement of organophosphate insecticides by other chemical groups, among them pirimicarb. We do not have reports of Þeld failure of sprays of pirimicarb in Chile, but recommended Þeld rates in crops with M. persicae infestation are in the range of 250Ð1,500 ppm. Such values are close to the LC50 of resistant MLG 16 A and S 74Ð1 calculated in our bioassay, therefore Þeld failure of pirimicarb sprays in crops with predominant abundance of MLGs carrying MACE mutation is likely to occur. Recent reports of pirimicarb resistance from Myzus persicae ssp. nicotianae Blackman on tobacco crops in the United States (Srigiriraju et al. 2010) and the great invasive potential of aphid pests (Zepeda-Paulo et al. 2010) makes the insecticide resistance mitigation strategies a priority for aphid pest management. In tobacco crops in the United States, mainly neonicotinoids and pymetrozine are used (Srigiriraju et al. 2010), whereas neonicotinoid insecticides and neonicotinoid mixtures with pyrethroids are used widely in Chile (Fuentes-Contreras et al. 2007). The aphid MLGs that we tested were all susceptible to both of these insecticide groups (neonicotinoids and pyrethroids), although a signiÞcant correlation of pirimicarb resistance ratios and those for imidacloprid was detected. These results suggest that perhaps further

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FUENTES-CONTRERAS ET AL.: RESISTANCE OF M. persicae FROM CHILE

insecticide resistance mechanisms based on broad spectrum detoxiÞcation enzymes not studied to date in M. persicae could be present (Figueroa et al. 2007; Silva et al. 2012a,b). Similar levels of variation in RR in imidacloprid and correlation with other mechanisms of insecticide resistance have been reported in New Zealand (van Toor et al. 2008), in Europe (Foster et al. 2003a, 2008; Margaritopoulos et al. 2007), and North America (Unruh and Willett 2008), before the development of resistance to imidacloprid was seen in Europe (Philippou et al. 2010, Puinean et al. 2010, Slater et al. 2012). The increasing use of neonicotinoids in Chile should be considered a warning for the possible evolution of resistance to this class of insecticides in this country. Therefore, a careful rotation strategy with pyrethroids and organophosphates should be followed and other insecticides with different modes of action such as pymetrozine (pyridine azomethine), spirotetramat (keto-enol), sulfoxaßor (sulfoximine), and cyantraniliprole (anthranilic diamide) should be measured for use against M. persicae to help mitigate the development of insecticide resistance in this important aphid pest in Chile.

Acknowledgments Technical support form Carlos Cavieres is greatly acknowledged. This work was funded by Fondecyt 1090378 to C.C.F. and E.F.C. and Fondecyt 1080085 to L.D.B. and E.F.C.

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