Independent Selection of Multiple Mechanisms for Pyrethroid ...

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Independent Selection of Multiple Mechanisms for Pyrethroid Resistance in Guatemalan Anopheles albimanus (Diptera: Culicidae) WILLIAM G. BROGDON, JANET C. MCALLISTER, ALANA M. CORWIN, 1 AND CELIA CORDON-ROSALES Entomology Branch, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA 30341

J. Econ. Entomol. 92(2): 298Ð302 (1999)

ABSTRACT Isofemale lines were established containing either, both, or neither of the elevated esterase and oxidase resistance mechanisms conferring pyrethroid resistance in a Guatemalan strain of Anopheles albimanus (Wiedemann). Plots of esterase and oxidase levels for individual mosquitoes from these single families correlated with data obtained using oxidase and esterase synergists in bioassays run in the bottle format. Mixed populations of pyrethroid-resistant A. albimanus adult females were selected using DDT, permethrin, or malathion; and the esterase and oxidase levels of the individual progeny were plotted. These data showed that the 3 classes of insecticide selected the 2 mechanisms differently. These results are discussed in terms of the problem of multiresistance surveillance in the Þeld, especially concerning pyrethroid insecticides and the interaction of agricultural and public health insecticide application. KEY WORDS Anopheles albimanus, insecticide resistance, pyrethroids, esterases, oxidases, multiresistance

RESISTANCE TO PYRETHROIDS based on both an elevated carboxylesterase and an enhanced P450 oxidase activity have been reported in Anopheles albimanus strains from Guatemala (Beach et al. 1989; Brogdon et al. 1990a, 1998). Although detoxiÞcation mechanisms leading to pyrethroid resistance have been reported in other anophelines (Kumar et al. 1991, Ramsdale 1975), there are no reports of the interaction of the esterase and oxidase and their relative impact on resistance selection. These mechanisms and the target site mechanism (kdr) occur in areas where bed-nets are used (Elissa et al. 1993; Vulule et al. 1994, 1999). Because bed-nets are, at present, potentially the most sustainable vector control measure against anophelines in developing countries (Lengeler and Snow 1996), it is imperative that the dynamics of their selection be understood. Pyrethroid resistance was previously reported in populations heavily selected for the esterase after years of organophosphate insecticide use in cotton (Brogdon et al 1990a). The elevated esterase in A. albimanus confers some portion of fenitrothiondeltamethrin cross-resistance, whereas the oxidase mechanism confers cross-resistance to pyrethroids and DDT (Brogdon et al. 1999). The potential contribution of the esterase to permethrin resistance and of oxidase to deltamethrin resistance has not yet been determined. Moreover, the cross-resistance spectra of 1 Centro de Investigaciones en Enfermedades Tropicales, Universidad del Valle de Guatemala, Apartado Postal 82, Guatemala City, Guatemala.

the 2 mechanisms are different (Brogdon et al. 1990a, 1998). The importance of precisely determining crossresistance relationships is exempliÞed by negative cross-resistance to malathion in strains having oxidasebased pyrethroid resistance (unpublished data). Concepts of resistance management become complex, given the existence of both positive and negative cross-resistance between insecticides of 2 different classes in the same population of mosquitoes. Single family rearings and outcrosses have provided subpopulations containing either, both, or neither mechanism. Our objective was to quantify the relative contribution of these mechanisms using both bioassay methods, including synergists and biochemical assay techniques. Their cross-resistance spectra, together and in isolation, are described as are the details of their independent and combined selection by various insecticide groups. Materials and Methods Mosquitoes. Mosquitoes were from insecticide-susceptible or -resistant strains of A. albimanus maintained or reared at the Centers for Disease Control and Prevention, Atlanta, GA. Mosquitoes used in assays were 3Ð4 d after ecdysis and were not blood-fed before the experiments. Resistant mosquitoes were from a strain of A. albimanus from Guatemala that had previously been reported to contain an esterase conferring cross-resistance to deltamethrin-fenitrothion (Brogdon and Barber 1990a). This colony was periodically selected at the LT50 level to preserve the

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mechanism; this level of selection allowed both susceptible and resistant mosquitoes to be maintained in each test population. Two days after blood feeding, 15 females were conÞned individually in vials containing 5 ml of deionized water and a Þlter paper lining (to prevent drowning). Typically, 3 batches of eggs were obtained from each female over a 2-wk period and pooled to form an isofemale line. Bottle Bioassay. The bioassay method used in these studies was bottle-based (Brogdon et al. 1998), allowing efÞcient collection of data, use of synergists, and generation of more complete cross-resistance spectra. For treatment (coating) of bottle interiors, technical grade solutions of insecticides (standards maintained at CDC, Atlanta or purchased from Chem Service, West Chester, PA) were diluted in acetone. A 1-ml portion of diluted toxicant was transferred to a 125-ml Wheaton bottle (Wheaton Glass, Millville, NJ). The bottle was shaken, rolled, and inverted such that all surfaces were exposed to the solution as the acetone was allowed to evaporate. The bottles (and caps) were inverted on paper toweling overnight in a dark cabinet. Mosquitoes were transferred into bottles at time 5 0, and mortality was scored at regular intervals until 100% mortality, or until the experiment was terminated (generally after 24 h). Some highly resistant mosquitoes survived 24 Ð 48 h of continuous exposure. Mortality was determined when mosquitoes could not right themselves or ßy when the test chamber was slowly rotated. Knockdown could be substituted for mortality. Mosquitoes that survived beyond the upper range limit for time of survival in the reference susceptible population were scored as less susceptible. Bottles with incorporated synergist/insecticide combinations were prepared in a similar manner as the insecticide-impregnated bottles. A series of synergist concentrations was used to verify that the concentration chosen for use in experiments was below toxic levels. Insecticides. Dosages used in this study were 43 mg per bottle permethrin, 100 mg malathion, and 50 mg DDT. In these experiments the oxidase inhibitor piperonyl butoxide (PB) and esterase inhibitor S,S,S,tributyl-phosphorotrithioate (DEF) were used as synergists. Concentrations of PB and DEF used were 125 and 400 mg per bottle, respectively. All insecticides and synergists were analytical standards obtained from ChemService (West Chester, PA). Biochemical Assay Methods. Adult 3- to 4-d-old, nonblood-fed female mosquitoes were immobilized by brief exposure to 220 C8 and homogenized in assay buffer (pH 7.2, 0.01 M potassium phosphate) in plastic microtubes using plastic pestles. Homogenates were diluted from the 100-ml volume for homogenization to 1 ml with additional buffer. Procedures for the elevated esterase, glutathione s-transferase (GST), oxidase, and insensitive acetylcholinesterase assays have been reported previously (Brogdon and Dickinson 1983; Brogdon and Barber 1990b; Brogdon et al. 1997). Each of the speciÞc enzyme assays begins with the transfer of 100 ml diluted mosquito homogenate to

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microtiter plate wells. In the esterase assay, 100 ml b-naphthyl acetate (56 mg/10 ml acetone/90 ml phosphate bufferÑdissolve b-naphthyl acetate in acetone before addition of buffer) is added to each well. After a 10-m incubation at RT, 100 ml dianisidine (100 mg/ 100 ml distilled water) is added and absorbance is read at 570 nm. In the GST assay, 100 Fl CDNB (1-chloro2,4Õ-dinitrobenzene, 20 mg/10 ml acetone/90 ml phosphate bufferÑdissolve CDNB in acetone before addition of buffer) and 100 ml reduced glutathione (61 mg/100 ml phosphate buffer) are added and absorbance is read after 5 m at 340 nm. In the oxidase assay, 200 ml TMBZ solution (50 mg 3,3Õ,5,5Õ-tetramethylbenzidine/25 ml methanol/75 ml pH 5.0, 0.25 M sodium acetate bufferÑdissolve TMBZ in methanol before addition of buffer) is added to each well, followed by 25 ml 3% hydrogen peroxide. After 5 m, absorbance is read at 620 nm. In the insensitive acetylcholinesterase assay control, 100 ml acetylthiocholine iodide (ACTH, 75 mg/10 ml acetone/90 ml phosphate buffer) and 100 ml 5,5Õ- dithio-bis-2-nitrobenzoic acid (DTNB, 13 mg/100 ml phosphate buffer) are added to each well and the absorbance is read after 10 m at 414 nm. To test for the resistance mechanism, 21 mg propoxur is added to the ACTH solutionÑdissolved in acetone before addition of buffer. Mosquito protein was measured on 20-ml aliquots of assay homogenates using the Bradford assay (Brogdon 1984a, b) to determine if individuals in test populations were of similar size. Mosquitoes were conveniently assayed in sample sizes of 32 and 3 replicates of 32 mosquitoes Þll a single microplate. As in bioassays, the resistance threshold was deÞned as the upper range limit of a susceptible population. Results Synergism of Resistance in Isofemale Lines. The oxidase inhibitor PB fully synergized permethrin in isofemale line 1, substantially synergized permethrin in line 7, and had no effect on resistance to permethrin in line 11 (Fig. 1). The esterase inhibitor DEF fully synergized permethrin in line 11, partially synergized permethrin in line 7, and had no effect on line 1 (Fig. 2). Enzyme Level Profiles of Isofemale Lines. The oxidase and esterase levels measured for the same individual mosquitoes in the 3 isofemale lines are in Fig. 3. Isofemale lines 1 and 11 showed oxidase levels higher than the threshold for resistance (resistance frequency 5 1.0, homozygous for high oxidase) and esterase levels below the threshold (resistance frequency 5 0.0, homozygous for low esterase), whereas isofemale line 7 had both esterase and oxidase levels above the resistance threshold (resistance frequency 5 1.0 for both mechanisms, homozygous for both). Other Isofemale Lines. The isofemale lines shown were selected because they were homozygous for either or both of the 2 resistance enzyme genotypes. Of the other isofemale lines, 2 were homozygous for high esterase, low oxidase, 1 was homozygous for low

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Fig. 1. Comparison of permethrin and permethrin 1 piperonyl butoxide time-mortality for females from a susceptible population and from single families derived from deltamethrin-selected A. albimanus. Sample size 5 125 (5 replicates of 25 individuals per bottle) for each population and experimental treatment. Acetone control (25 mosquitoes) showed no mortality.

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Fig. 3. Comparison of esterase and oxidase levels for females from single families 1, 7, and 11 of A. albimanus. Sample size 5 100 mosquitoes for each single family. Each point represents the esterase and oxidase level for an individual mosquito. Single families are shown that are homozygous for either or both of the elevated resistance enzymes.

esterase, high oxidase, and 1 was homozygous for high esterase, high oxidase. The remainder was heterozygous for either or both resistance enzyme levels. Selection Experiments. A single selection of the permethrin-resistant strain with DDT (Fig. 4) significantly (MannÐWhitney rank sum test, P , 0.001) increased oxidase levels in the selected strain F1. The

mean oxidase level increased by a factor of 2. Esterase levels were not signiÞcantly affected (MannÐWhitney test, P 5 0.747). Selection of the permethrin-resistant strain with permethrin (Fig. 5) signiÞcantly increased both es-

Fig. 2. Comparison of permethrin and permethrin 1 DEF time-mortality for females from a susceptible population and from single families of deltamethrin-selected A. albimanus. Sample size 5 125 (5 replicates of 25 individuals per bottle) for each population and experimental treatment. Acetone control (25 mosquitoes) showed no mortality.

Fig. 4. Comparison of frequency distributions of esterase and oxidase levels for a permethrin-resistant population of A. albimanus before and after permethrin selection. Sample size 5 32 mosquitoes for each population. Each point represents the esterase and oxidase level for an individual mosquito.

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strain. Mean esterase levels were 20% higher and oxidase levels were 20% lower in the selected strain. Discussion

Fig. 5. Comparison of frequency distributions of esterase and oxidase levels for a permethrin-resistant population of A. albimanus before and after DDT selection. Sample size 5 32 mosquitoes for each population. Each point represents the esterase and oxidase level for an individual mosquito.

terase (t-test, P , 0.001) and oxidase (Mann-Whitney test, P , 0.001)levels in the selected strain F1. Esterase and oxidase mean levels were increased by 20 and 40%, respectively. Selection of the permethrin-resistant strain with malathion (Fig. 6) signiÞcantly (MannÐWhitney test, P , 0.001) increased esterase levels in the selected strain F1. Oxidase levels were signiÞcantly (MannÐ Whitney test, P , 0.037) reduced in the selected

Fig. 6. Comparison of frequency distributions of esterase and oxidase levels for a permethrin-resistant population of A. albimanus before and after malathion selection. Sample size 5 32 mosquitoes for each population. Each point represents the esterase and oxidase level for an individual mosquito.

Synergists fully cancelled the activity of the individual resistance mechanisms and independently synergized each mechanism when both synergists were present together, indicating that the mechanisms are not linked. The biochemical data on esterase and oxidase from the same individual mosquitoes provided conÞrmation. Piperonyl butoxide more completely synergizes permethrin than does DEF in the oxidase 1 esterase single family. This suggests a greater contribution of oxidase activity to the level of permethrin resistance observed, because both mechanisms are Þxed at a frequency of 1.0. A possible contributing factor is that oxidase levels were higher and esterase levels lower in the dual mechanism family than in the high-oxidase and high-esterase single families, respectively. These observations show the potential contribution that speciÞc mechanism assays may make in studies of the activity of synergists against multi-insecticide resistant insects. These studies show that different insecticide classes may select a multiresistant population in different ways. DDT, an organochlorine, selected for higher levels of oxidase in a mixed population of mosquitoes. Permethrin, a pyrethroid, selected both for higher oxidase and esterase activity. Although the 2 mechanisms are not linked, our bioassay data show a contribution by both mechanisms to permethrin resistance. Malathion selected for higher esterase levels but negatively selected for the oxidase activity. That is, malathion selection removed the high oxidase mechanism from the population. Use of new methods such as the bottle bioassay and speciÞc mechanism-detecting biochemistry allow a new approach to monitoring selection in mixed, multiresistant populations such as those actually encountered in the Þeld. Their role is likely to be the generation of high resolution time- and location-speciÞc descriptions of the susceptibility situation, avoiding time delays generally associated with laboratory work-up of vector populations. Ascertaining the relative contribution to selection development of resistance caused by different classes of insecticides directly affects the solution of resistance problems. For example, the relative contribution of agricultural and public health application of insecticides to development or increases in intensity of insecticide resistance has been debated (Lines 1988). Sometimes, agricultural use of insecticides is clearly the driving force (Hemingway 1983; Lines et al. 1984; Brogdon et al. 1988a, b). It has also been proposed that agricultural insecticides favor selection in larvae, through contamination of breeding sites, and that insecticides used for public health are more likely to select for resistance in adults (Hemingway et al. 1986). However, some mechanisms are expressed in both adults and larvae (Brogdon et al. 1990a). Moni-

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toring the shift in frequencies of mechanisms using our methodology might make it possible to assess the actions of different types of compounds in multiresistant populations. Such methods would be of particular usefulness in a situation such as that which occurs in Sri Lanka, where there are 22 species of Anopheles capable of supporting the sporogenic cycle of Plasmodium vivax and each has, potentially, a unique resistance proÞle (Herath and Joshi 1986). The use of certain insecticides in agriculture has been restricted in Sri Lanka since 1977, based on the threat of crossresistance. In Anopheles culicifacies (Giles), resistance is based on a mechanism selected by malaria spraying, while in Anopheles nigerrimus (Giles), resistance is based on a target-site mechanism selected through crop treatment. Because cross-resistance spectra vary according to the resistance mechanisms selected, the existence of 3 mechanisms for pyrethroid resistance and their potential interaction will require more sophisticated means of monitoring selection of Þeld populations. Acknowledgments This work was supported, in part, by World Health Organization TDR grant 910637 and the Emerging Infections initiative at the Centers for Disease Control and Prevention.

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Received for publication 10 August 1998; accepted 13 January 1999.