Resistance management strategies in malaria vector mosquito

Medical and Veterinary Entomology(1998)12, 217–233

Resistance management strategies in malaria vectormosquito control. Baseline data for a large-scalefield trial against Anopheles albimanus in Mexico

R . PAT R I C I A P E N I L L A , A M E R I C O D . R O D R I´ G U E Z ,J A N E T H E M I N G WAY, * J O S E L . T O R R E S ,J U A N I . A R R E D O N D O - J I M EN E Z and M A R I O H . R O D R I´ G U E ZCentro de Investigacioń de Paludismo, Tapachula, Chiapas, Me´xico, and *School of Pure and Applied Biology, University of

Wales, Cardiff, U.K.

Abstract. A high level of DDT resistance and low levels of resistance toorganophosphorus, carbamate and pyrethroid insecticides were detected bydiscriminating dose assays in field populations ofAnopheles albimanusin Chiapas,southern Mexico, prior to a large-scale resistance management project described byHemingwayet al. (1997). Biochemical assays showed that the DDT resistance wascaused by elevated levels of glutathione S-transferase (GST) activity leading toincreased rates of metabolism of DDT to DDE. The numbers of individuals withelevated GST and DDT resistance were well correlated, suggesting that this is theonly major DDT resistance mechanism in this population.

The carbamate resistance in this population is conferred by an alteredacetylcholinesterase (AChE)-based resistance mechanism. The level of resistanceobserved in the bioassays correlates with the frequency of individuals homozygousfor the altered AChE allele. This suggests that the level of resistance conferred bythis mechanism in its heterozygous state is below the level of detection by the WHOcarbamate discriminating dosage bioassay.

The low levels of organophosphate (OP) and pyrethroid resistance could be conferredby either the elevated esterase or monooxygenase enzymes. The esterases wereelevated only with the substratepNPA, and are unlikely to be causing broad spectrumOP resistance. The altered AChE mechanism may also be contributing to the OP butnot the pyrethroid resistance.

Significant differences in resistance gene frequencies were obtained from the F1

mosquitoes resulting from adults obtained by different collection methods. This maybe caused by different insecticide selection pressures on the insects immediately priorto collection, or may be an indication that the indoor- and outdoor-restingA. albimanuscollections are not from a randomly mating single population. The underlying geneticvariability of the populations is currently being investigated by molecular methods.

Introduction

Insecticide resistance is often a problem in the control of manymedically important insects. In malaria control, spraying ofinsecticides inside human habitation severely restricts thenumber of suitable compounds which can be used by control

Correspondence: Professor J. Hemingway, School of Pure andApplied Biology, University of Wales Cardiff, PO Box 915, Cardiff,CF1 3TL, U.K. E-mail: [email protected]

© 1998 Blackwell Science Ltd 217

programmes. The use of an insecticide until resistance becomesa limiting factor is rapidly eroding the number of suitableinsecticides for malaria control. A better management strategymay be the use of compounds in rotational or mosaic alternation(Mellon & Georghiou, 1984; Curtiset al., 1993). Numerousmathematical models have been produced to determine theoptimal strategies for resistance management (Greever &Georghiou, 1979; Georghiou, 1980; Curtis, 1985; Tabashnik,1989). These models have been tested under laboratory butnot field conditions due to the practical difficulties of accuratelyassessing the changes in resistance gene frequencies associated

218 R. Patricia Penilla et al.

with different patterns of insecticide use in large-scale fieldpopulations of insects (Tayloret al., 1983). With the adventof more sophisticated biochemical and molecular assays forresistance detection, it is now practical to analyse accuratelylarge numbers of insects individually for a range of insecticideresistance genes and to monitor their changes over time(Hemingway, 1989; Hemingwayet al., 1995).

Accordingly, a large-scale field programme has beenestablished in southern Mexico to compare changes ofresistance gene frequencies in the major malaria vectorAnopheles albimanusWiedemann after repeated cycles ofhouse-spraying for 3 years with: (i) a single insecticide, (ii)mosaic of two insecticide classes, or (iii) rotation of threeinsecticide classes, where the rotation is on an annual basis.As the objective of this study is to look at the principles ofresistance managment in vector control, rather than to testspecific products, the insecticides used were randomly chosenexamples representing three classes, i.e. organophosphate,carbamate and pyrethroid, referred to only by their class namesfor the purposes of this investigation.

This paper assesses the resistance levels and mechanisms inAn. albimanuspopulations from the study area during the pre-intervention period. Details ofAn. albimanusbehaviour andbionomics in this part of southern Mexico have beendocumented by Bownet al. (1993), Frederickson (1993),Loyola et al. (1993) and Rodriguezet al. (1996). House-spraying with DDT was discontinued some years ago in allour study villages and malaria cases are rarely dected in thisarea nowadays.

In choosing this region to undertake the project a site wasdeliberately picked where broad spectrum resistance to a rangeof insecticides had already been reported. The project site inthe coastal lowlands of Chiapas State of southern Mexico wasformerly a region where moderate to high levels of agriculturalinsecticide were used. In addition, DDT has been used formalaria control in this region for a period of more than 10 years.The combined use of different classes of insecticides foragricultural spraying and DDT for anti-malaria house-sprayingresulted in high levels of resistance to organochlorines,organophosphates, carbamates and pyrethroids inAn. albimanusin this region in the late 1970s (Table 1). Since then, a reductionin agricultural insecticide use has resulted in regression of theresistances (with the exception of DDT) to a point where theyare barely detectable using standard WHO bioassays (seeTable 1). In contrast, the continued use of DDT for malariacontrol has maintained and increased the level of resistance tothis insecticide.

Materials and Methods

The project area consisted of twenty-four villages, which wereassigned to eight zones of three villages. The zones arephysically well separated (Fig. 1), and village groups werechosen on the basis of communal mosquito breeding sitesbetween villages within a zone, and comparable numbers ofhouses between zones. Adult femaleAn. albimanuswerecollected using a number of indoor and outdoor trapping

© 1998 Blackwell Science Ltd,Medical and Veterinary Entomology, 12, 217–233

Table 1. Historical mortality rates ofAnopheles albimanusfromChiapas, southern Mexico, within 24 h following exposure for 1 h tovarious insecticides (after Hemingwayet al., 1997). Susceptibility testsconduced according to standard WHO (1981) procedure using 150–600 mosquitoes (blood-fed wild-caught females) per insecticide testedin any year.

% Mortality%

Insecticide type Concentration 1982 1983 1990

OrganochlorineChlorphoxim 4 98 99 100DDT 4 38 39 47

OrganophosphateFenitrothion* 1 44 57 99Fenthion 2.5 97 100 –Malathion 5 84 93 99Pirimiphos-methyl 4 – 99 –

CarbamateBendiocarb 0.1 – 87 –Propoxur 0.1 89 95 –

PyrethroidCypermethrin 0.1 – 82 –Deltamethrin 0.025 64 57 86

*Exposure time 2 h for fenitrothion.

methods to ensure that a large sample was collected for eachzone. Mosquitoes from each trapping method were maintainedseparately so that it could be determined whether there wereany differences in resistance gene frequency from collectionsobtained by the different sampling methods.

Collections of blood-fed femaleAn. albimanusfrom twenty-one villages were made from February to June 1995. Thecollection methods used were human bait, indoors and outdoors,light traps and daytime resting collections from cattle stallsand houses (including peridomestic resting sites). It was notpractical to analyse these insects directly for resistance, as pre-exposure of some of the insects to insecticides would haveaffected the analysis. An F1 generation was therefore obtainedfrom the field material. This allowed us to standardize age,physiological state and testing conditions for the bioassays andbiochemical assays. Females were maintained under laboratoryconditions until they oviposited, larvae were reared through toadults. One-day-old males and females of the F1 generationwere either subjected to standard WHO bioassays or werefrozen at – 70°C in Eppendorf tubes and transferred to theUniversity of Cardiff, where biochemical and molecularanalyses were undertaken. Analysis of variance was used todetect any significant differences in resistance gene frequenciesbetween the different collection zones, sexes and collectionmethods. Biochemical assays were undertaken for alteredacetylcholinesterase- (AChE), glutathione S-transferase-(GST), esterase- and monooxygenase-based resistance.Metabolism experiments were undertaken to confirm initialresults from the biochemical assays. Frequency distributionswere plotted for each biochemical assay for the laboratoryreference strains and F1s from wild-caught females fromdifferent collection zones to determine the variability between

Resistance management strategies219

Fig. 1. Outline map of coastal southern Mexico (with inset indicating the national location of the study area) showing numbered clusters of villagesused for the eight treatment zones, as listed in Table 2. The different treatment zones are well separated physically and do not share major breedingsites ofAnopheles albimanus.

populations. Random amplified polymorphic DNA analysis(RAPDS) was undertaken to look at gene flow between villagemosquito populations (Hemingwayet al., 1997) and this isnow being expanded to look at genetic make-up of populationssampled through different collection methods.

Insect strains

The Panama strain ofAn. albimanushas been maintainedin the laboratory without insecticide selection for 20 years andprior to this was collected from an area with no history ofinsecticide use. This was used as the standard susceptiblestrain. The Mexico strain was collected from coastal Chiapasin 1991 and has been maintained in the insectary withoutselection. The Mexico strain still contains a low level ofresistance to a number of insecticides. It is being used as acontrol strain to determine the rates of change in resistancegene frequencies under laboratory conditions in the absence ofselection or migration.


Biochemical assays

Batches of forty-seven one-day-old frozen mosquitoes of theF1 generation were individually homogenised in 200µl ofdistilled water in flat-bottomed microtitre plates. Thehomogenization was carried out on ice. For the AChE assay,two replicates of 25µl of crude homogenate were transferredto a fresh microtitre plate. The remaining homogenates weretransferred to 0.5-µl Eppendorf tubes and spun at maximumspeed for 2 mins in a microfuge. Replicates of 20µl of thesupernatants from each sample were transferred to a freshmicrotitre plate for the elevated esterase naphthyl acetate assaysand for the monooxygenase. This procedure was repeated afurther three times for different plates with 10-µl aliquots ofhomogenate for the GST, esterase and protein assays.

Acetylcholinesterase assay

The AChE in the homogenates was solubilized by adding145µl of Triton phosphate buffer (1% Triton X-100 in 0.1M


phosphate buffer pH 7.8) to each replicate aliquot. Tenµl ofDTNB solution (0.01M dithiobis 2-nitrobenzoic acid in 0.1Mphosphate buffer pH 7.0) and 25µl of the substrate ASCHI(0.01M acetylthiocholine iodide) were added to one replicateto initiate the reaction. The latter solution was substituted by25 µl of the substrate ASCHI containing 0.2% of the inhibitorpropoxur (0.1M) for the second test replicate. Control wellscontained 25µl distilled water, 145µl Triton buffer, 10µlDTNB solution and 25µl ASCHI solution without and withpropoxur, respectively. The kinetics of the enzyme reactionwere monitored continuously at 405 nm for 5 mins in amicrotitre plate reader. The percentages of propoxur inhibitionof acetylcholinesterase activity in the test compared tothe uninhibited wells were calculated. The assay conditionswere preset so that individuals without an alteredacetylcholinesterase-based resistance mechanism had. 60%inhibition of activity. Resistance gene frequencies were thencalculated from the resultant data, assuming Hardy–Weinbergequilibrium.

Esterase assays

Naphthyl acetate assays.Two hundredµl of α-naphthylacetate solution (100µl of 30 mM α-NA in acetone in 10 mlof 0.02M phosphate buffer pH 7.2) and 200µl of β-NA solution(prepared as forα-NA) were added to one replicate ofhomogenate. The enzyme reaction ran for 30 mins at roomtemperature before the addition of 50µl of Fast blue stainsolution (22.5 mg Fast blue in 2.25 ml distilled water, then5.25 ml of 5% sodium lauryl sulphate diluted in 0.1M phosphatebuffer, pH 7.0) was added to each well to stop the reaction.Replicate blanks contained 20µl distilled water, 200µl ofα-NA or β-NA solution and 50µl of stain. Enzyme activitywas read at 570 nm as an end point. Absorbance levels forindividual mosquitoes were compared with standard curvesof absorbance for known concentrations ofα-naphthol andβ-naphthol, respectively. The results were reported asµmolesof the product formed/min/mg protein.

p-Nitrophenyl acetate (pNPA) esterase assay

Two hundredµl of pNPA working solution (100 mM pNPAin acetonitrile : 50 mM sodium phosphate buffer pH 7.4, 1:100)were added to each replicate. Two blanks were prepared foreach plate with 10µl distilled water and 200µl pNPA workingsolution. Enzyme rates were measured at 405 nm for 2 mins.ThepNPA activity per individual was reported asµmol product/min/mg protein.

Glutathione-S-transferase assay

Two hundredµl of GSH/CDNB working solution (10 mMreduced glutathione prepared in 0.1M phosphate buffer pH 6.5and 63 mM chlorodinitrobenzene diluted in methanol) wereadded to each replicate. Two blanks were prepared for each


plate with 10µl distilled water and 200µl GSH/CDNB workingsolution. Enzyme rates were measured at 340 nm for 5 mins.The GST activity per individual was reported as mmol CDNBconjugated/min/mg protein, using published extinctioncoefficients corrected for the path length. The level of GSTactivity was linked to the ability to metabolize DDT as reportedpreviously (Hemingwayet al., 1998).

Monooxygenase assay

The total amount of cytochrome P450 in each mosquitowas titrated using the haem-peroxidase assay as modified by(Brogdon et al., 1998). Eighty µl of 0.625M potassiumphosphate buffer pH 7.2 were added to the aliquots of thehomogenates and 200µl of the solution 3,39,5,59-tetramethylbenzidine diluted in absolute methanol (0.01 g in 5 ml,respectively) and then mixed with 15 ml of 0.25M sodiumacetate buffer pH 5.0 (TMBZ). Twenty-fiveµl of 3% hydrogenperoxide was added and the mixture left for 2 h at roomtemperature. Samples, and two controls per plate, were preparedwith 20 µl of distilled water, 200µl of the solution TMBZ and25 µl of 3% H202. Absorbance was read at 650 nm and valuescompared with a standard curve of absorbance for knownconcentrations of cytochrome C. The values are reported asequivalent units of cytochrome P450/mg protein, correcting forthe known haem content of cytochrome C and P450.

Protein assay

Three hundredµl of BIO Rad protein reagent solution,prepared as a 1:4 dilution in distilled water, were added to10 µl of the crude homogenate. Two blanks were prepared foreach plate with 10µl of distilled water and 300µl of BIO Radsolution. The reaction was read at 570 nm after 5 mins at roomtemperature. Protein values in mg\ml were calculated forindividual mosquitoes from a standard curve of absorbance ofknown concentrations of bovine serum albumin.

Results and discussion

The combined biochemical and bioassay data show that broadspectrum resistance is still present in theAn. albimanusfieldpopulation in southern Mexico. The resistance is conferredby a number of different resistance mechanisms which aresegregating in the field population. The bioassay results usingsingle WHO (1981) discriminating dosages for a range ofinsecticides (Table 2) suggest that the highest resistancefrequency is for DDT, with much lower levels of resistancedetected to carbamate, OP and pyrethroid insecticides. It shouldbe noted that the WHO discriminating dosages are set at doublethe insecticide dose that gives 100% mortality of the leastsusceptibleAnophelesmosquito species. Hence, these assaysare good indicators of the presence of significant (. 2- to 10-fold) resistance in a mosquito population, but they cannot beused to measure resistance gene frequencies accurately, which


Fig. 2. Acetylcholinesterase (AChE) inhibition ranges inAnopheles albimanus(F1 adult progeny from wild-caught females collected in 1995) fromeight study zones in coastal Chiapas, compared to standard susceptible Panama strain and the local reference Chiapas 1991 Mexico strain (see text).



Table 2. Susceptibility* rates ofAnopheles albimanusfrom the eightstudy zones in Chiapas, southern Mexico in 1994–95. The insecticidesused are referred to only by their class names, as the objective of thestudy is to look at the principles of resistance management in vectorcontrol rather than to endorse the use of specific products.

Insecticide and % MortalityCollectionzone DDT OP Carbamate Pyrethroid

1 100 100 1002 28.1 100 100 1003 9.4 99 100 1004 46.9 100 93.5 1005 17.4 100 100 1006 35.5 99 98.5 1007 100 100 1008 28.9 100 86.5 99

*Standard WHO (1981) susceptibility tests usingAnopheles albimanusadults (sugar-fed females and males), laboratory-reared F1 progenyfrom wild-caughtAnopheles albimanusfemales.

Table 3. Inhibition of AChE activity by propoxur in one-day-oldAn.albimanus of Panama and Mexico laboratory strains and the F1generation from 1995 collections.

% inhibitionStrainsand zones Mean6 SD n

Panama 86.66 8.3 94Mexico 816 18.1 1021 51.16 19.2 2402 78.96 18.1 3103 76.56 23.8 2284 82.96 16.9 2005 68.16 22.3 4816 67.36 22.3 1567 64.86 27 1998 74.26 17.8 268

Mean 70.56 21 2082

are likely to be higher than indicated by the bioassay data.This is confirmed by our results with the biochemical assays.

Previous studies onAn. albimanusfrom Central Americahave shown that an altered AChE is the most common OP\carbamate resistance mechanism (Ayad & Georghiou, 1979;Hemingway & Georghiou, 1983). Our results suggest that thisresistance mechanism is present in MexicanAn. albimanus.The mean percentage propoxur inhibition values of AChE forthe two laboratory populations and the eight zone collectionsare given in Table 3 and their frequency distributions in Fig. 2.As expected, the inhibition values in the Panama susceptiblelaboratory strain ranged from 100 to 60%, indicating that noneof the individuals in this population have the altered AChE


Table 4. Gender comparisons of the inhibition of AChE activity bypropoxur in one-day-oldAn. albimanusof the Panama and Mexicolaboratory strains and the F1 generation from 1995 collections.

% inhibition: Mean6 SD GenderStrains diff.and zones Males n Females n P

Panama 86.56 9.0 48 86.76 7.5 46 NS*Mexico 81.66 17.4 54 80.26 19.0 48 NS1 54.76 20.9 121 47.36 16.5 119 0.012 80.66 17.1 148 77.36 18.8 162 NS3 74.46 23.6 101 78.16 24.0 127 NS4 81.46 18.7 95 84.16 15.1 105 NS5 65.06 23.0 228 70.96 21.2 253 0.016 67.56 23.1 74 67.26 21.7 82 NS7 62.76 25.1 95 66.66 28.6 104 NS8 78.96 13.6 117 70.56 19.7 151 0.01

Mean 70.36 22.7 979 70.66 22.9 1103 NS

*NS 5 Not significant.

gene. In contrast, the ranges for the field-collected insects weremuch broader (from 0 to 100% inhibition), demonstrating thatthe altered AChE resistance gene was present throughout thestudy area.

The frequency of the AChE resistance mechanism varied inthe different field-collected samples. The frequencies of theresistance gene in the field samples can be calculated directly.Results reported previously show that the highest altered AChEgene frequencies were in zones 1, 5, 6 and 7, but all valueswere in the range 0.13–0.28. The altered AChE gene was stillpresent in the Mexican laboratory population, but the resistancegene frequency in that colony had declined to 0.09 in theabsence of pesticide selection pressure in the laboratory overa number of years (Hemingwayet al., 1998).

Only three collection zones (1, 5 and 8) showed anysignificant difference in the level of propoxur inhibition ofAChE between the sexes (Table 4). Males had higher meaninhibition levels in zones 1 and 8 than females (P , 0.01), andfemales in zone 5 compared to males (P , 0.01). This is asexpected, as the AChE resistance gene is not sex-linked (Kaiseret al., 1978), although recently sex-linked acetylcholinesterasegenes have been cloned from bothCulexandAedesmosquitoes.Hence, some variation in total AChE content may occurbetween the sexes (Malcolmet al., 1998).

When samples obtained by different collection methods werecompared (Table 5), all zones were significantly different(P , 0.01, P , 0.05). Insects from the cattle collections hadhigher mean inhibition values in four of the collection zones,whereas in zones 4 and 8 the indoor collection methods hadthe higher inhibition values. In zone 5, the human-bait outdoorcollection contained fewer individuals with the altered AChEresistance mechanism than the indoor collection.

Esterase activity

The laboratory strains ofAn. albimanusand the F1 progenyfrom the mosquitoes from the different collection zones all


Fig. 3. Range of esterase activity with the substrateα-naphthyl acetate inAnopheles albimanus(F1 adult progeny from wild-caught femalescollected in 1995) from the eight study zones in coastal Chiapas, compared to the standard susceptible Panama strain and the local referenceChiapas 1991 Mexico strain (see text).



Fig. 4. Range of esterase activity with the substrateβ-naphthyl acetate inAnopheles albimanus(F1 adult progeny from wild-caught femalescollected in 1995) from the eight study zones in coastal Chiapas, compared to the standard susceptible Panama strain and the local referenceChiapas 1991 Mexico strain (see text).



Fig. 5. Range of esterase activity with the subsrate p-nitrophenyl acetate (pNpa) inAnopheles albimanus(F1 adult progeny from wild-caughtfemales collected in 1995) from the eight study zones in coastal Chiapas, compared to the standard susceptible Panama strain and the local referenceChiapas 1991 Mexico strain (see text).



Fig. 6. Ranges of cytochrome P450 representing monooxygenase activity in Anopheles albimanus (F1 adult progeny from wild-caught femalescollected in 1995) from the eight study zones in coastal Chiapas, compared to the standard susceptible Panama strain and the local referenceChiapas 1991 Mexico strain (see text).



Fig. 7. Ranges of glutathione S-transferase (GST) activity with the substrate dichloronitrobenzene inAnopheles albimanus(F1 adult progeny fromwild-caught females collected in 1995) from the eight study zones in coastal Chiapas, compared to the standard susceptible Panama strain and thelocal reference Chiapas 1991 Mexico strain (see text).



Table 5. Acetylcholinesterase inhibition comparisons between samples from five methods of collection: inhibition of AchE activity by proproxurin one-day-oldAn. albimanusof the F1 generation from 1995 collections. Mean6 SD percentage inhibition.

Collection methodSample dif.

Zone Human bait outdoor Human bait indoor Cattle stalls Light trap Indoor-resting P

1 47.115 38.46 18.8 64.16 11.6n 5 48 n 5 90 n 5 102 **

2 78.86 18.1 NSn 5 310

3 77.86 25.4 66.86 24.2 80.36 21.1 Negativen 5 49 n 5 50 n 5 128 n 5 1 **

4 83.56 19.2 89.66 18.8 76.76 9.7n 5 61 n 5 63 n 5 76 **

5 79.66 22.3 65.96 21.7 68.26 21.4 72.46 25.6n 5 21 n 5 227 n 5 179 n 5 54 *

6 54.26 20.2 68.16 23.4 81.06 14.5n 5 69 n 5 22 n 5 65 **

7 81.26 11.1 41.96 18.0 87.36 12.8n 5 21 n 5 96 n 5 82 **

8 99.96 0.2 99.96 0.1 73.16 17.8 84.86 5.6n 5 2 n 5 4 n 5 251 n 5 11 **

Mean 68.26 24.8 60.46 26.2 75.56 18.5 71.16 27.1 84.86 5.6n 5 271 n 5 552 n 5 1193 n 5 55 n 5 11 **

Table 6. Esterases activity in one-day-oldAn. albimanusof thePanama and Mexico laboratory strains and the F1 generation from1995 collections. Activity was measured with the substratesα- andβ-naphthyl acetate.

Means6 SD nmol of product/min/mg protein

Strains and µmolesα-naphthol µmolesβ-naphtholzones produced n produced n

Panama 0.00086 0.0003 94 0.00066 0.0003 94Mexico 0.00076 0.0004 104 0.00046 0.0002 1041 0.00116 0.0010 425 0.00076 0.0007 3672 0.00136 0.0009 325 0.00096 0.0008 3253 0.00176 0.0021 303 0.00146 0.0016 2574 0.00866 0.0751 188 0.00956 0.0722 1935 0.00126 0.0025 753 0.00096 0.0018 7546 0.00196 0.0010 113 0.00136 0.0007 1127 0.00176 0.0048 269 0.00126 0.0026 2718 0.00256 0.0021 304 0.00176 0.0015 309

Mean 0.00206 0.0200 2680 0.00176 0.0198 2588

showed very low esterase activity with the substratesα- andβ-naphthyl acetate (Figs 3 and 4), although the distributionpatterns were significantly different to those of the Panamaand Mexican laboratory strains. Mean values of esterase activityare given in Table 6 Although the values for zone 4 are 10-foldhigher than the baseline susceptible strains, they are still wellbelow the levels seen inCulex mosquitoes, where the mainmechanism of OP resistance is esterase elevation (Peiris &


Table 7. Gender comparisons of esterase activity: amount ofα-naphthol produced per one-day-oldAn. albimanusof the Panama andMexico laboratory strains and the F1 generation from 1995 collections.

Mean6 SD nmol of product/min/mg protein GenderStrains diff.and zones Males n Females n P

Panama 0.00086 0.0003 48 0.00076 0.0002 46 NSMexico 0.00076 0.0004 55 0.00076 0.0003 49 NS1 0.00106 0.0010 218 0.00116 0.0009 207 NS2 0.00136 0.0011 155 0.00126 0.0008 170 NS3 0.00176 0.0019 126 0.00176 0.0023 177 NS4 0.00356 0.0054 91 0.01346 0.1045 97 NS5 0.00136 0.0035 343 0.00116 0.0010 410 NS6 0.00216 0.0013 50 0.00176 0.0007 63 ,0.057 0.00136 0.0014 124 0.00216 0.0065 145 NS8 0.00276 0.0024 121 0.00236 0.0019 183 NS

Mean 0.00166 0.0027 1228 0.00236 0.0272 1452 NS

Hemingway, 1993). Differences in the levels of esterase activitybetween males and females were observed only withα-naphthylacetate in zone 6, where males were significantly higher(P , 0.05) than females (Table 7). However, differences in thelevels of esterase activity between collection methods wereobserved for both substrates (P , 0.01) in 1, 5, 6 and 7collection zones (Tables 8 and 9).

Although there was low activity with both the naphthylacetates, a third substratepNPA was tested, as there are someesterases (for example, those associated with the metabolism


Table 8. Esterase activity comparisons between samples from five methods of collection:µmoles of α-naphthol produced per one-day-oldAn. albimanusof the F1 generation from 1995 collections. Mean6 SD nmol of product/min/mg protein.

Collection methodSample diff.

Zones Human bait outdoor Human bait indoor Cattle stalls Light trap Indoor-resting P

1 0.00076 0.0007 0.00136 0.0011 0.00116 0.0008n 5 134 n 5 153 n 5 138 0.01

2 0.00136 0.0009n 5 325 –

3 0.00176 0.0025 0.00146 0.0028 0.00206 0.0007 0.0008n 5 50 n 5 123 n 5 129 n 5 1 NS

4 0.02206 0.1327 0.00276 0.0021 0.00206 0.0016n 5 60 n 5 61 n 5 67 NS

5 0.00186 0.0010 0.00116 0.0010 0.00116 0.0042 0.00226 0.0006n 5 30 n 5 436 n 5 232 n 5 55 0.01

6 0.00156 0.0005 0.00256 0.0012 0.00276 0.0014n 5 73 n 5 13 n 5 27 0.01

7 0.00146 0.0017 0.00096 0.0005 0.00336 0.0084n 5 44 n 5 141 n 5 84 0.01

8 0.00146 0.0016 0.00216 0.0012 0.00266 0.0021 2.87–5 6 4.10–5 0.00176 0.0017n 5 8 n 5 4 n 5 278 n 5 3 n 5 11 NS

Mean 0.00436 0.0517 0.00136 0.0015 0.00186 0.0031 0.00216 0.0008 0.00176 0.0017n 5 399 n 5 931 n 5 1280 n 5 59 n 5 11 NS

Table 9. Esterase activity comparisons between samples from five methods of collection:µmoles of β-naphthol produced per one-day-oldAn.albimanusof the F1 generation from 1995 collections. Mean6 SD nmol of product/min/mg protein.



1 0.00056 0.0005 0.00106 0.0009 0.00066 0.0005n 5 132 n 5 148 n 5 87 0.01

2 0.00096 0.0008n 5 325 –

3 0.00116 0.0013 0.00176 0.0027 0.00146 0.0005 Negativen 5 50 n 5 77 n 5 129 n 5 1 NS

4 0.02626 0.1276 0.00216 0.0016 0.00156 0.0013n 5 61 n 5 61 n 5 71 NS

5 0.00166 0.0012 0.00086 0.0007 0.00096 0.0030 0.00166 0.0005n 5 30 n 5 437 n 5 232 n 5 55 0.01

6 0.00116 0.0003 0.00166 0.0007 0.00186 0.0009n 5 72 n 5 13 n 5 27 0.01

7 0.00136 0.0018 0.00076 0.0004 0.00206 0.0044n 5 50 n 5 139 n 5 82 0.01

8 0.00156 0.0019 0.00196 0.0012 0.00176 0.0014 0.00166 0.0010 0.00316 0.0024n 5 10 n 5 4 n 5 275 n 5 9 n 5 11 0.05

Mean 0.00486 0.0500 0.00106 0.0012 0.00126 0.0020 0.00166 0.0006 0.00316 0.0024n 5 405 n 5 879 n 5 1228 n 5 65 n 5 11 0.05

of malathion) that have little or no activity with naphthylacetates, but may be detectable with alternative substrates. Themean values forpNPA esterase activity in one-day-oldAn.albimanus in all the collection zones were 2.60–7.66-foldhigher than those for the susceptible strain (Table 10), althoughall values were still significantly lower than the rates seen in


Culexmosquitoes with amplified resistance-associated esterases(Karunaratneet al., 1995). Insects from zone 4 and 6 hadthe highestpNPA activity which was 7.66 and 4.13-fold,respectively, higher than the insecticide-susceptible Panamastrain. Distribution patterns ofpNPA activity are given inFig. 5. These are similar to those with the naphthyl acetates


Table 10. Esterase substrate pNPA activity in one-day-oldAn. albimanusof the Panama and Mexico laboratory strains and theF1 generation from 1995 collections.

µmol Mean6 SDStrains product/min/mg Higher thanand zones n protein Panama strain

Panama 105 0.046 0.02 –Mexico 128 0.046 0.02 01 379 0.126 0.10 3.022 324 0.116 0.15 2.823 300 0.156 0.12 3.804 190 0.316 2.22 7.665 765 0.126 0.39 2.996 256 0.176 0.15 4.137 320 0.116 0.20 2.608 266 0.136 0.10 3.12

Mean 2800 0.146 0.62 3.45

Table 11. Gender comparisons of esterase substrate pNPA-activity inone-day-oldAn. albimanusof the Panama and Mexico laboratorystrains and the F1 generation from 1995 collections.

Means6 SD µmol product/min/mg protein GenderStrains diff.and zones Males n Females n P

Panama 0.0406 02 53 0.046 0.01 52 NSMexico 0.046 0.02 63 0.046 0.02 65 NS1 0.136 0.11 176 0.126 0.08 203 NS2 0.126 0.21 154 0.116 0.07 170 NS3 0.146 0.07 124 0.176 0.15 176 0.054 0.146 0.17 88 0.466 3.03 102 NS5 0.166 0.57 343 0.096 0.08 422 0.056 0.186 0.18 121 0.166 0.12 135 NS7 0.096 0.07 148 0.126 0.27 172 NS8 0.136 0.12 110 0.126 0.07 156 NS

Mean 0.146 0.32 1264 0.146 0.79 1536 NS

and activity levels with all three substrates were positivelycorrelated in individual insects. There was no significantdifference inpNPA activity between the Panama and Mexicostrains, suggesting that either the higher activity has been lostfrom the Mexico strain since its colonization in 1991, or thatthe higher levels of esterase activity have only been selectedin the field populations since 1991. The significance of thisshift in total esterase activity with respect to insecticideresistance is currently under investigation, but as yet there isno indication of a direct role for these enzymes in resistance.

Differences inpNPA activity between males and femaleswere detected in three of the five collection zones,P , 0.05(Table 11). Males showed higherpNPA activity in zone 5 thanfemales, but this was not observed in the zone 3, where femalesshowed higher activity values than males.

With reference to the collection method (Table 12),pNPA-


activity of the F1 mosquitoes from three collection zones weresignificantly different (P , 0.01). Zones 3 and 6 showed thehighest pNPA activity in the human-bait indoor collectionmethod, whereas the zone 1 had the highest activity valuesfrom the human-bait outdoor collection method.

Monooxygenases (MFO)

The assay used here for estimation of haem content in theunfed mosquitoes is currently under evaluation as a methodfor detecting MFO-based resistance. Brodgenet al. (1998)reported that changes in haem titration inAn. albimanuswere well correlated with monooxygenase-based resistance inlaboratory selection experiments. The data reported here forthis assay are an indirect measure of possible changes in P450

content of the individual insects. Further metabolism work isunder way to confirm whether any of the changes seen in theassay are well correlated with resistance in the Mexican fieldpopulation ofAn. albimanus.

The Panama susceptible laboratory strain was homogeneousfor low MFO content, as measured by haem titration. Therewere a small number of individuals from the F1 generation ofthe females from the collection zones with elevated levels ofMFO. Mosquitoes from zones 3 and 8 (Fig. 6) had the highestmean levels of MFO, which were 3.5- and 4-fold, respectively,higher than the insecticide-susceptible Panama strain(Table 13). Differences in MFO content between males andfemales were detected in four of the eight collection zones andthe Panama strain,P , 0.05 andP , 0.01 (Table 15). Femalesalways had higher estimated MFO content than males. For thecollection methods, differences in four of the collection zoneswere observed. Zones 4 and 6 (P , 0.01) had the highest MFOcontent in the human-bait indoor collection method, whereaszone 7 had the highest MFO content from the cattle stallscollection (P , 0.05). In zone 8, the highest MFO content wasfrom the indoor-resting collection (P 5 0.01), but very fewinsects were obtained by this collection method (Table 15).

Glutathione S-transferase activity

Elevated GST is a major mechanism of DDT resistance inAnophelesmosquitoes and a cause of both DDT and OPresistance in houseflies andAnopheles subpictus(Clark et al.,1984; Hemingwayet al., 1991). This resistance mechanismcan be detected simply by assaying the total GST activity ofthe insect with the general GST substrates dichloronitrobenzene(DCNB) or chlorodinitrobenzene (CDNB). Once elevatedactivity levels are detected, the correlation with resistanceneeds to be confirmed by metabolism studies on the relevantinsecticides.

The authors have previously reported that GST activitylevels of one-day-oldAn. albimanus(F1 generation) werehigher in the field collections than in the laboratory strains andsignificantly different between the collection zones(Hemingwayet al., 1998). Increasing the sample sizes for allcollections confirmed these initial observations (Table 16). The


Table 12. Esterase activity comparisons between samples from five methods of collection: substrate pNPA-activity in one-day-oldAn. albimanusof the F1 generation from 1995. Mean6 SD µmol/min/mg protein.



1 0.14006 0.0949 0.13126 0.1073 0.08006 0.0544n 5 132 n 5 162 n 5 85 ,0.01

2 0.11456 0.1549n 5 324 –

3 0.10806 0.0885 0.19546 0.1581 0.13366 0.0708 0.0856n 5 50 n 5 123 n 5 126 n 5 1 ,0.01

4 0.74156 4.2469 0.17706 0.1481 0.12556 0.1077n 5 52 n 5 62 n 5 76 NS

5 0.07986 0.0639 0.11466 0.0939 0.12966 0.6410 0.15266 0.0980n 5 23 n 5 416 n 5 271 n 5 55 NS

6 0.12346 0.0490 0.34136 0.3985 0.16446 0.0946n 5 73 n 5 22 n 5 161 ,0.01

7 0.08456 0.0955 0.10396 0.0649 0.11936 0.3350n 5 58 n 5 158 n 5 104 NS

8 0.13716 0.1137 0.12326 0.0782 0.12736 0.0973 0.10906 0.0663n 5 3 n 5 4 n 5 253 n 5 6 NS

Mean 0.20106 1.5524 0.13556 0.1289 0.12606 0.3121 0.15146 0.0975 0.10906 0.0663n 5 391 n 5 947 n 5 1400 n 5 56 n 5 6 NS

Table 13. Monooxygenase equivalent units of cytochrome P450 (EUC)in one-day-oldAn. albimanusof the Panama and Mexico laboratorystrains and the F1 generation from 1995 collections.

Strains µmol Mean6 SD Higher thanand zones n EUC P450/mg protein Panama strain

Panama 132 0.00026 0.0001Mexico 132 0.00026 0.0002 01 292 0.00036 0.0003 1.52 277 0.00046 0.0004 23 315 0.00076 0.0024 3.54 145 0.00066 0.0011 35 609 0.00036 0.0005 1.56 244 0.00066 0.0026 37 351 0.00056 0.0009 2.58 340 0.00086 0.0020 4

Mean 2573 0.00056 0.0015 2.5

order of magnitude of change in GST activity observed issimilar to that seen inAn. gambiaewith GST-based DDTresistance (Hemingwayet al., 1985; Prapanthadaraet al., 1995).Frequency distributions for the GST-activity of one-day-oldmosquitoes from the Panama and Mexico laboratory strains,as well as the F1 insects from the collection zones are givenin Fig. 7. Differences in the levels of GST activity betweenmales and females were observed in zones 5, 6 and 8. GSTactivity in the males was significantly higher than females inthese zones (P , 0.05, Table 17). With reference to thecollection method, GST-activity of the F1 mosquitoes from fivecollection zones were significantly different (P , 0.01). Zones3 and 5 had the highest GST-activity in the human-bait indoor


Table 14. Gender comparisons of cytochrome P450 equivalent unitsrepresenting monooxygenases in one-day-oldAn. albimanusof thePanama and Mexico laboratory strains and the F1 generation from1995 collections.

Mean6 SD nmol EUC P450/mg protein GenderStrains diff.and zones Males n Females n P

Panama 0.00016 0.00009 60 0.00026 0.0002 72 0.01Mexico 0.00016 0.0002 71 0.00026 0.0002 61 NS1 0.00026 0.0003 157 0.00036 0.0003 135 NS2 0.00026 0.0002 123 0.00066 0.0004 154 0.013 0.00066 0.0023 136 0.00076 0.0025 179 NS4 0.00036 0.0011 71 0.00086 0.0011 74 0.055 0.00026 0.0004 281 0.00046 0.0006 328 0.016 0.00056 0.0026 118 0.00076 0.0025 126 NS7 0.00036 0.0007 165 0.00076 0.0011 186 0.018 0.00076 0.0032 111 0.00096 0.0009 229 NS

Mean 0.00046 0.0016 1162 0.00066 0.0014 1411 0.01

collection method, whereas the zones 1, 6 and 8 had theirhighest activity values from the cattle stall collection method.

DDT metabolism

Experiments confirmed that the high GST levels detected inthe field population ofAn. albimanusare associated with DDTresistance.

Different insecticide-resistance management strategies arenow being employed in the study area. Mosquitoes are being


Table 15. Monooxygenase comparisons between five sampling methods: cytochrome P450 per one-day-oldAn. albimanusof the F1 generationfrom 1995 collections. Mean6 SD nmol equivalent units of cytochrome P450/mg protein.

Collection methodsSample diff.


1 0.00036 0.0003 0.00026 0.0003 0.00036 0.0003n 5 109 n 5 133 n 5 50 NS

2 0.00046 0.0004n 5 277 –

3 0.00066 0.0019 0.00106 0.0033 0.00036 0003 0.00026 0.0001n 5 74 n 5 142 n 5 94 n 5 5 NS

4 0.00046 0.0012 0.00136 0.0017 0.00056 0.0007n 5 62 n 5 18 n 5 65 0.01

5 0.00056 0.0005 0.00036 0.0006 0.00036 0.0002 0.00056 0.0005n 5 12 n 5 401 n 5 150 n 5 46 NS

6 0.00066 0.0004 0.00286 0.0072 0.00026 0.0004n 5 68 n 5 28 n 5 148 0.01

7 0.00036 0.0005 0.00056 0.0007 0.00076 0.0013n 5 55 n 5 164 n 5 132 0.05

8 0.00026 0.0002 0.00026 0.0001 0.00076 0.0009 0.00036 0.0002 0.00486 0.0096n 5 19 n 5 4 n 5 297 n 5 9 n 5 11 0.01

Mean 0.00046 0.0010 0.00056 0.0020 0.00056 0.0007 0.00046 0.0005 0.00486 0.0096n 5 399 n 5 890 n 5 1213 n 5 60 n 5 11 0.01

Table 16. GST activity in one-day-oldAn. albimanusof the Panamaand Mexico laboratory strains and F1 generation from 1995 collections.

Mean6 SD mmol/min/mg proteinStrains Higher thanand zones GST activity n Panama strain

Panama 0.146 0.06 128 –Mexico 0.176 0.17 149 1.211 0.486 0.49 534 3.422 0.346 0.33 287 2.433 0.756 2.15 273 5.334 0.636 5.65 175 4.495 0.436 0.63 831 3.046 0.196 0.14 144 1.367 0.316 0.51 207 2.218 0.296 0.27 384 2.02

Mean 0.436 1.62 2835 3.08

collected routinely by all the above trapping methodsthroughout the spray season. The levels of resistance determinedby bioassay, and the underlying resistance mechanismsdetermined by biochemical assays, will be monitored, andchanges over time compared to this initial baseline data willbe determined. The only resistance mechanism we are notcurrently monitoring is ‘kdr’. This pyrethroid resistancemechanism, conferred by changes in the sodium channel,cannot be measured biochemically. However, it is likely thatPCR molecular methods will soon become available to monitorthis mechanism. We have therefore collected DNA samplesfrom numerous mosquitoes from the baseline collection. These


Table 17. Gender comparisons of GST activity in one-day-oldAn.albimanusof the Panama and Mexico laboratory strains and the F1generation from 1995 collections.

Mean6 SD mmol/min/mg protein GenderStrains diff.and zones Males n Females n P

Panama 0.156 0.07 64 0.146 0.06 64 NSMexico 0.186 0.22 77 0.166 0.09 72 NS1 0.486 0.34 270 0.496 0.60 264 NS2 0.376 0.46 129 0.326 0.16 158 NS3 0.946 2.66 128 0.596 1.57 145 NS4 0.216 0.19 85 1.036 7.87 90 NS5 0.576 0.89 376 0.316 0.23 455 0.016 0.226 0.17 70 0.176 0.10 74 0.057 0.356 0.48 99 0.286 0.54 108 NS8 0.336 0.33 160 0.256 0.20 224 0.01

Mean 0.486 1.01 1317 0.396 2.04 1518 NS

will be analysed retrospectively for ‘kdr’ when the method isavailable (Martinez-Torreset al., 1998).

Acknowledgements

A preliminary version of this paper, with partial data, wasreported by Penillaet al. (1996). This work was sponsored bythe Insecticide Resistance Action Committee of the GlobalCrop Protection Federation, with further contributions fromcompanies participating in the Public Health Working Group


of IRAC/GCPF, viz: Agrevo, Bayer, Cheminova, FMC, MitsuiToatsu, Novartis, Rhone Poulenc, Sumitomo and Zeneca.

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Accepted 31 December 1997

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Resistance management strategies in malaria vector mosquito