Medical and Veterinary Entomology (2009) 23, 122–131

Insecticide resistance and detoxifying enzyme activityin the principal bancroftian filariasis vector, Culexquinquefasciatus, in northeastern India

M. S A R K A R1, I. K. B H A T T A C H A R Y Y A2, A. B O R K O T O K I3,D. G O S W A M I1, B. R A B H A1, I. B A R U A H1 and R. B. S R I V A S T A V A1

1Medical Entomology Division, Defence Research Laboratory (DRDO), Tezpur, India, 2Department of Zoology, Cotton College,

Guwahati, India and 3Department of Zoology, Gauhati University, Guwahati, India

Abstract. The insecticide resistance status of Culex quinquefasciatus Say (Diptera:Culicidae) to DDT and deltamethrin across army cantonments and neighbouringvillages in northeastern India was investigated. In India, DDT is still the insecticideof choice for public health programmes. In military stations, pyrethroids, especiallydeltamethrins, are used for insecticide-treated nets (ITNs). Recent information onthe levels of resistance to DDT and deltamethrin in mosquito populations ofnortheastern India is scare. Continued monitoring of insecticide resistance status,identification of the underlying mechanisms of resistance in local mosquito populationsand the establishment of a baseline data bank of this information are of primeimportance. Insecticide susceptibility assays were performed on wild-caught adultfemale Cx. quinquefasciatus mosquitoes to the discriminating doses recommendedby the World Health Organisation (WHO) to DDT (4%) and deltamethrin (0.05%).Across all study sites, mortality as a result of DDT varied from 11.9 to 50.0%,as compared with 91.2% in the susceptible laboratory strain (S-Lab), indicatingthat Cx. quinquefasciatus is resistant to DDT. The species was found to be 100%susceptible to deltamethrin in all study sites except Benganajuli and Rikamari. Knock-down times (KDT) in response to deltamethrin varied significantly between studysites (P < 0.01) from 8.3 to 17.8 min for KDT50 and 37.4 to 69.5 min for KDT90.All populations exceeded the threshold level of alpha-esterase, beta-esterase andglutathion S-transferase (GST) established for the S-Lab susceptible strain, and allpopulations had 100% elevated esterase and GST activity, except Missamari andSolmara. Beta-esterase activity in Field Unit II (96.9%) was less than in any ofthe other populations. Benganajuli had the highest activity level for all the enzymestested. There was a significant correlation between all enzyme activity levels andinsecticide resistance phenotype by populations (P < 0.05). The results presentedhere provide the first report and baseline information of the insecticide resistancestatus of Cx. quinquefasciatus in northeastern India, and associated informationabout biochemical mechanisms that are essential for monitoring the development ofinsecticide resistance in the area.

Key words. DDT, deltamethrin, esterase, glutathion S-transferases, insecticideresistance, knock-down, Assam, India.

Correspondence: Manas Sarkar, Medical Entomology Division, Defence Research Laboratory (DRDO); Post Bag No. 2, Tezpur 784001, Assam,India. Tel: +91-9435 185664; Fax: +91-3712-258534; E-mail: manas_sarkar54491@yahoo.com

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Insecticide resistance in Culex quinquefasciatus 123

Introduction

Culex quinquefasciatus (Say) is the most abundant mosquitospecies reported in the vector data bank of the Armed Forcesof India (Tilak et al., 2008). It is also one of the mostimportant and abundant mosquito species throughout world.It is a continuous biting nuisance, mostly for those living closeto larval habitats, and poses a threat to human health where ittransmits parasites which cause diseases such as bancroftianfilariasis. In a tea agro-ecosystem in Assam, the averageinfection rate of Cx. quinquefasciatus with filarial worms is4.6% and the overall prevalence of infective mosquitoes is0.8%, with an average load of third larval-stage parasites (L3load) of 2.0 per mosquito. It has been estimated that a totalof over 22 000 mosquito bites are received per person/year in‘tea gardens’ (commercial tea plantations alternatively knownas tea estates owned by national and international agenciesor private parties), of which 182 bites/person/year (0.81%)are infective (Mahanta et al., 2001). In India, vector controlprogrammes are designed and implemented mainly to targetmalaria vectors. The strategy of the national vector controlprogramme is based on effective management of the use ofinsecticides for indoor residual spraying (IRS) and insecticide-treated nets (ITNs) with deltamethrin and permethrin. Highlevels of insecticide resistance in Cx. quinquefasciatus alsopresent an obstacle to malaria prevention, as people may notperceive the personal protective effect of ITNs if the netsfail to kill biting mosquitoes, which are actually resistantCx. quinquefasciatus (Corbel et al., 2007).

Several factors other than frequency of insecticide use serveto influence the intensity and development of resistance in apopulation. The most important factors include the frequencyof the resistance gene in a population, number of genes inter-acting to produce the resistant character, size of the populationand the dominance status of the gene (cf Chareonviriyaphapet al., 2002). In addition, several physiological mechanismsare involved in insecticide resistance, including reduced sen-sitivity of sodium channels to insecticides, over-production ofdetoxifying enzymes, such as esterases, mixed function oxi-dases (MFOs) and glutathione S-transferases (GSTs), which areresponsible for detoxification of toxic substances (Georghiou,1986; Roberts & Andre, 1994; Nelson et al., 1996; Brogdon &McAllister, 1998; Scott et al., 1998; Feyereisen, 1999). Amongthe different mechanisms of insecticide resistance, knock-downresistance (kdr), which results from a mutation in the para-type voltage-gated sodium channel (the target site for DDTand pyrethroids), and metabolic resistance are equally impor-tant in this part of the world. In India, the kdr mechanismin Cx. quinquefasciatus has been reported only by researchersinvolved in the current study (Sarkar et al., 2008).

In India, DDT is still the insecticide of choice for publichealth programmes. In military establishments, pyrethroids,especially deltamethrins, are used for ITNs (Joshi et al., 2003).Pyrethroids are also used in tea agro-ecosystems in Assamalong with malathion. During 2003, deltamethrin was alsoused to impregnate ‘patches of clothes’ or ‘bands’, which canbe worn over an army uniform, in Assam on a trial basis(Bhatnagar & Mehta, 2007). Recent information on the DDT

and deltamethrin susceptibility/resistance status of mosquitospecies in Assam is scare. There is concern that this updatedinformation is needed to ensure that the pattern of insecticideuse in this agro-ecosystem is optimized to avoid increasedresistance that could threaten the sustainability of the vectorcontrol strategy. Thus, continued monitoring of insecticidesusceptibility/resistance status and establishment of a baselinedata bank for the area is of prime importance.

The current study presents the first report of the insec-ticide resistance status of Cx. quinquefasciatus to DDT anddeltamethrin in northeastern India, plus an assessment of theunderlying resistance mechanisms present in these vector pop-ulations. The results are of importance to the development offuture insecticide resistance management strategies and willinform the selection of insecticides for mosquito control inthese areas.

Materials and methods

Eco-environment and pattern of insecticide use in the studyarea

The study was carried out in army cantonments and sur-rounding villages in Assam, India (Fig. 1). The size of theentire study area was approximately 248 km2. The study siteswere located approximately 1–10 km apart from each other.The locations were selected for mosquito collection on thebasis of use of insecticides and/or the eco-environmental set-tings. The localities were: (1) Benganajuli village, a malaria-prone area where DDT is applied regularly by public healthworkers, (2 and 3) two army animal transport Field Units(I and II) which are mainly surrounded by rice fields, withdeep forest and tea gardens to the east of Field Unit II, (4)Rikamari village, which is surrounded by forest, (5) the Mis-samari cantonment areas, where there is little or no use ofDDT inside the cantonment, but pyrethroids are used regularlyand some organophosphates are used occasionally, and (6 and7) the Solmara cantonment areas (I and II) in Tezpur, the townwhere the Defence Research Laboratory is situated, which ismainly surrounded by rice fields. Meteorologically, these areasare characterized by a long rainy season and humid climate.Global Positioning System (GPS) coordinates of the study siteare presented in Table 1. A Geographical Information System(GIS)-based map of the study area is presented in Fig. 1, whichwas created with ArcGIS 9.2 software (ESRI®ArcMapTM9.2,Environmental Systems Research Institute, Redlands, CA).

Mosquito collections

Adult mosquitoes for testing the resistance/susceptibilitystatus of each population were collected by aspiration fromhuman dwellings in villages and barracks in cantonmentsin the evening from 18.30 to 20.30 hours and early in themorning from 05.00 to 07.00 hours. Mosquitoes were iden-tified as Cx. quinquefasciatus on the basis of morphologicalcharacteristics after bioassays were performed. Insecticide-susceptibility tests were performed on wild-caught adult female

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124 M. Sarkar et al.

Fig. 1. Study sites and its eco-environmental settings.

Table 1. GPS coordinates of field sites where a study wasconducted.

Site GPS coordinates

Benganajuli 26◦51′48.4′′ N 92◦32′27.6′′ EField unit–I 26◦51′47.4′′ N 92◦33′48.7′′ EField unit–II 26◦51′14.1′′ N 92◦35′16.2′′ ERikamari 26◦50′45.3′′ N 92◦35′33.2′′ EMissamari 26◦48′43.0′′ N 92◦35′39.3′′ ESolmara I 26◦41′24.7′′ N 92◦46′59.8′′ ESolmara II 26◦40′58.2′′ N 92◦46′51.9′′ E

mosquitoes and from a mosquito colony maintained in theDefence Research Laboratory, at Tezpur, as a ‘susceptible’reference strain (S-Lab), to compare against the susceptibil-ity levels of field populations. This susceptible strain was

collected from Udmari village near Tezpur town in the 1996and colonized in the insectary of the Medical Entomology Divi-sion of Defence Research Laboratory at Tezpur.

A sample of 25–50 adult female mosquitoes (depending onthe number of mosquito collected in a particular site) fromthe collections described above were directly stored in liquidnitrogen and carried back to the laboratory for the biochemicalassays.

Insecticide susceptibility bioassays

Insecticide susceptibility assays were performed on wild-caught adult female mosquitoes. Mortality and knock-downresulting from tarsal contact with insecticide-treated filterpapers were measured using WHO test kits (WHO, 1998). Thetests were carried out using 4% DDT and 0.05% deltamethrin,

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Insecticide resistance in Culex quinquefasciatus 125

Table 2. Susceptibility and/or resistance status of Culex quinquefasciatus to diagnostic dose of DDT (4%) and deltamethrin (0.05%).

DDT (4%) Deltamethrin (0.05%)

% Mortality % MortalityStudy sites (sample size) Mean (±SD) (sample size) KDT50 (95% CI) KDT90(95% CI) χ2 (df)

1. Benganajuli 11.9 (160) 2.38 (1.302) 96.2 (80) 17.8 (12.05–23.57) 69.5 (36.26–102.83) 2.333 (4)2. Field Unit I 17.5 (80) 3.50 (1.29) 100 (80) 10.09 (4.34–15.82) 44.2 (22.06–66.33) 1.235 (3)3. Field Unit II 30.63 (160) 6.12 (2.031) 100 (80) 10.3 (4.8–15.89) 41.4 (18.44–64.45) 0.116 (2)4. Rikamari 41.25 (80) 8.25 (1.50) 98.7 (80) 14.5 (9.07–20.01) 56.1 (32.26–80.13) 1.052 (4)5. Missamari 50.0 (80) 10.0 (2.16) 100 (80) 8.4 (2.50–14.39) 37.4 (15.47–59.42) 1.150 (2)6. Solmara I 30.63 (160) 6.12 (0.835) 100 (80) 8.3 (2.30–14.28) 40.3 (19.84–60.88) 0.742 (3)7. Solmara II 38.75 (160) 7.75 (1.388) 100 (80) 9.4 (3.91–14.99) 39.7 (21.70–57.60) 0.414 (3)S–Lab 91.2 (160) 18.25 (1.282) 100 (80) 5.1 (−1.20–11.54) 27.5 (12.36–42.72) 0.051 (2)

Table shows the mean mortality values (replicate mean) and other descriptive statistical parameters of the probability distribution of mortality ratesacross study sites. Table also shows 50% and 90% knock-down time (KDT50 & KDT90) in minutes and Chi-square (χ2) value for deltamethrin(0.05%).

the diagnostic doses recommended by WHO. For each of theinsecticides tested, mosquitoes were divided into batches of 20per test and exposed to insecticide-treated papers for 4 h inthe case of DDT (4%) and 1 h for deltamethrin (0.05%). Theeffects of papers treated only with carrier oils were assayedin parallel as a control. At the end of the exposure period,mosquitoes were transferred into tubes with untreated whitefilter papers (known as holding tubes) and allowed a 24-hrecovery period.

For mosquitoes exposed to DDT, mortality rates wererecorded after the recovery period, and for mosquitoes exposedto deltamethrin, the numbers knocked down were recordedevery 10 min for up to 1 h during exposure. The samebioassays were carried out on the laboratory-reared susceptiblestrain (S-Lab) to compare with the susceptibility levels ofthe field populations. During the 24-h recovery period, allmosquitoes were provided with 10% sugar water.

Biochemical assays

Sample preparation. Adult non-blood fed femaleCx. quinquefasciatus, collected from the field and stored inliquid nitrogen, were homogenized individually in 1.5-mLmicrofuge tubes in 30 μL Milli-Q water, obtained from Direct-QTM5 (Millipore India Pvt. Ltd., Bangalore, India) and thendiluted with an additional 270 μL Milli-Q water. Tubes werekept in ice during the whole homogenization procedure. Thehomogenates were spun at 10 000 g for 2 min at 4◦C in anultracentrifuge. The supernatant was used as a crude enzymeextract for the biochemical assays. Three microplates withduplicate mosquito homogenates were used for three enzymesand one microplate for total protein. Each biochemical assaywas replicated twice with new individuals from the samemosquito population on two different days. Sample sizes foreach biochemical assay ranged from 24 to 48 mosquitoes perlocation, depending on the availability in the field population.A minimum of three positive and three negative controls wereused per plate. Absorbance was measured using a Bio-RadMicroplate Reader (Bio-Rad Laboratories, Philadelphia, PA).

Total protein assay. The total protein content of individualCx. quinquefasciatus mosquitoes was determined to correct forsize variation among the specimens (Brogdon, 1984) using acommercial protein assay kit (Bangalore GENEI, Bangalore,India) according to the user’s guide. The results were comparedwith a bovine serum albumin (BSA) standard curve. The plateswere read at 550 nm wavelength.

Non-specific esterase assay. The method of Peiris & Hem-ingway (1990) was used with alpha- and beta-naphthyl acetateas the substrate. The plates were read at 550 nm wavelength.

Glutathion S-transferase assay. Glutathion S-transferaseassay (GST) activity was assayed according the methodof Brogdon & Barber (1990) with some minor modifica-tions. Reduced glutathione was used as the substrate and theplate was read at 340 nm after a 20-min incubation as theend point.

Statistical analysis

Mean mortality was determined across all batches ofmosquitoes tested for a particular insecticide, and the WHOcriteria for evaluating resistance or susceptibility in a mosquitopopulation was used (WHO, 1992); mortality rates of less than80% indicate resistance, whereas those greater than 98% indi-cate susceptibility. Mortality rates between 80 and 98% suggestthe possibility of resistance that needs to be verified. Anal-ysis of variance (ANOVA) was used to compare knock-downrates after 10-min intervals between mosquitoes from differentstudy sites. The times to 50 and 90% knock-down (KDT50 andKDT90) were estimated by regression analysis between per centknock down and exposure time, using the log-probit method(Finney, 1971). Descriptive statistical analysis was used to cal-culate means and standard deviations, for the samples collectedfrom different study sites and exposed to the diagnostic dose ofDDT (4%).

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126 M. Sarkar et al.

Table 3. Susceptibility threshold based on maximum absorbance of different detoxifying enzymes in the laboratory susceptible strain (S-Lab) andpercentage of adult Culex quinquefasciatus that exceeded the susceptibility threshold established in S-Lab.

Susceptible threshold Percentage of adult mosquitoes exceeding the threshold(maximum absorbance

Enzymes value of S–Lab) Benganajuli Rikamari FU–I FU–II Missamari Solmara

α-esterase 1.5825 100 100 100 100 41.66 20.83B -esterase 1.5235 100 100 100 96.9 41.66 54.16GST 0.10335 100 100 100 100 16.66 95.83

Table 4. Enzyme activity level (mean and standard deviation of mean absorbance) of adult Culex quinquefasciatus as measured by absorbance indifferent study sites.

Alpha esterase Beta esterase Glutathion S–tranferase

95% Confidence 95% Confidence 95% ConfidenceStudy sites Mean (n) SD intervals Mean (n) SD intervals Mean SD intervals

Benganajuli 3.5 (48) 0.39 3.39–3.63 3.6 (48) 0.25 3.47–3.70 0.47 (48) 0.033 0.46–0.47Rikamari 2.5 (24) 0.32 2.32–2.66 2.4 (24) 0.29 2.26–2.59 0.14 (24) 0.029 0.13–0.16FU–I 2.7 (36) 0.61 2.53–2.81 2.8 (36) 0.56 2.63–2.90 0.34 (36) 0.03 0.33–0.36FU–II 2.2 (32) 0.22 2.13–2.43 2.4 (32) 0.47 2.25–2.53 0.17 (32) 0.028 0.16–0.18Missamari 1.6 (24) 0.45 1.41–1.75 1.4 (24) 0.41 1.27–1.60 0.09 (24) 0.022 0.08–0.10Solmara 1.3 (24) 0.49 1.20–1.55 1.6 (24) 0.47 1.40–1.72 0.22 (24) 0.035 0.20–0.23S–Lab 0.7 (24) 0.33 0.54–0.88 0.8 (24) 0.32 0.59–0.92 0.05 (24) 0.02 0.04–0.07

n = number of mosquitoes tested .

The results of the biochemical analyses of levels of detox-ifying enzymes in different populations were expressed asabsorbance values. The maximum absorbance value for the lab-oratory susceptible strain (S-Lab) was used as the susceptibilitythreshold. ANOVA was used to compare the protein content andenzyme expression levels between populations from differentstudy sites.

Results

Susceptibility bioassay

The susceptibility status of Cx. quinquefasciatus to a diag-nostic dose of DDT (4%) is shown in Table 2. The datashow that Cx. quinquefasciatus is resistant to DDT in allthe study sites. The per cent mortality range was 11.9–50.0%, compared with 91.2% for the S-Lab control strain. Thehighest degree of resistance to DDT was found in Bengana-juli, and the lowest in Missamari. Adult bioassays were per-formed in batches of 20 mosquitoes per test, with replicates,and the mean mortality values of replicates are presented inTable 2. The probability distribution of mortality rates in Ben-ganajuli, Field Unit I and II, Rikamari, Missamari, SolmaraI and II differ significantly (P < 0.05) from a normal dis-tribution. In contrast, Field Unit I displayed a true normaldistribution.

Table 2 shows the 50 and 90% knock-down time, i.e.KDT50 and KDT90, of Cx. quinquefasciatus after continuousexposure to 0.05% deltamethrin for up to 1 h and the percent mortality after the 24-h post-exposure holding period.

The species was found to be 100% susceptible to deltamethrinin Field Unit I, Field Unit II, Missamari, Solmara I andSolmara II, and the ranges of KDT50 and KDT90 values were8.3–10.3 min and 37.4–41.4 min, respectively. In Benganajuliand Rikamari, the per cent mortalities were 96.2 and 98.7%,respectively; and the ranges of KDT50 and KDT90 were17.8–14.5 min and 69.5–56.1 min, respectively. The resultsfor knock-down time in Field Unit II (KDT50 = 10.3 min andKDT90 = 41.4 min) and Rikamari (KDT50 = 14.5 min andKDT90 = 56.1 min) are interesting, because these two areasare nearest each other but surprisingly exhibited contrastingresults. The KDT50 and KDT90 values of all study sitesare much higher than for the known susceptible strain (S-Lab) of Cx. quinquefasciatus, whereas KDT50 and KDT90

were 5.1 and 27.5 min, respectively. Knock-down rates fordeltamethrin were significantly different between differentstudy sites (ANOVA, F = 2.0583, d.f. = 36, P = 0.00458).

Biochemical assay

The susceptibility threshold, as measured by the maximumabsorbance value of the susceptible laboratory strain (S-Lab),and the percentage of adult Cx. quinquefasciatus that exceededthat established threshold are presented in Table 3. Results ofthe biochemical assays of detoxifying enzymes from differ-ent study sites are presented in Table 4 and Fig. 2(a–c). Totalprotein was measured in each mosquito to control for sizedifferences between mosquitoes. Protein assay results basedon total protein in individual mosquitoes revealed that theaverage size of individuals from different study sites was not

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Insecticide resistance in Culex quinquefasciatus 127

(a)

(c)

(b)

Fig. 2. Activities of detoxifying enzymes, as measured by absorbance, in field populations of Culex quinquefasciatus across different studysites. Box plot distributions of the mean, standard deviation and standard error of absorbance values for (a) alpha-esterase, (b) beta-esterase and(c) glutathion S-transferase.

significantly different (P > 0.05), and therefore no adjustmentwas needed for the enzyme analysis to take into account dif-ferences in the size of mosquitoes. The enzyme activity resultsare presented here as absorbance (OD value), rather than con-centration, so the details of the protein assay results from eachmosquito are not presented. Activities of different enzymestested are shown in Fig. 2(a–c) as absorbance. Box plot dis-tributions add additional information not apparent in tables ofthe mean and range data because they reveal the distributionof activity levels of enzymes and why significant differencesexist between different populations (Zayed et al., 2006). All thepopulations exceeded the S-Lab strain threshold for alpha- andbeta-esterase, and GST (Table 3). Enzyme activity levels aresignificantly different among different populations includingthe S-Lab strain (P < 0.001). All populations except Mis-samari and Solmara had 100% elevated esterase and GST activ-ity. Beta-esterase activity in FU-II is slightly less (96.9%) thanfor the other populations. Benganajuli had the highest activitylevel of all the enzymes tested (Fig. 2). Benganajuli and FieldUnit I had the maximum number of individuals with higherlevels of GST activity (Fig. 5). There is a significant correlation

between all enzyme activity levels and insecticide resistancephenotype by population (P < 0.05). The distribution patterns(number of individuals observed) of elevated enzyme activi-ties, as measured by absorbance in different populations, aredisplayed in Figures 3, 4 and 5.

Discussion

The resistance status of Cx. quinquefasciatus mosquitoes toDDT and deltamethrin was investigated in and around armycantonments in Assam, northeastern India. Based on the WHOcriteria for characterizing insecticide resistance/susceptibility,where susceptibility is defined by mortality rates greater than98% after 24-h post-exposure, evidence for resistance to DDTwas found at all the study sites (Table 2). The per cent mortalityafter 24-h post exposure obtained in knock-down bioassays fordeltamethrin suggests complete susceptibility to this insecticidein all study sites, except in Benganajuli (Table 2), where furtherverification data are needed.

In all the study areas, deltamethrin-treated bednets wererecently introduced by the public health department and

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128 M. Sarkar et al.

Fig. 3. Frequency distribution of alpha-esterase enzyme activity, measured by absorbance in field population of Culex quinquefasciatus amongdifferent study sites.

army authority, but in villages such as Benganajuli andRikamari bednets were not regularly retreated after pur-chase. However, the KDT50 and KDT90 for deltamethrin forCx. quinquefasciatus mosquitoes in the different study sitesare comparatively higher than for the susceptible S-Lab lab-oratory strain. Knock-down rates at 10-min intervals weresignificantly different (P < 0.01) between study sites. The con-trasting results for knock-down times in two adjacent areas suchas Field Unit II and Rikamari (1 km apart) suggests that themosquito population in these areas seems to be physiologicallydistinct. In Benganajuli and Rikamari, higher values of knock-down times (KDT50 and KDT90) possibly indicate the develop-ment of incipient resistance to deltamethrin in these populationsof Cx. quinquefasciatus. But unfortunately we could not corre-late this incipient resistance to the use of deltamethrin in theseareas. There are similar reports for Anopheles fluviatilis Jamesfrom Orissa (Sharma et al., 2004) and An. culicifacies Gilesfrom Tamil Nadu, India (Mittal et al., 2002), where delayedknock-down effects were observed, although there was 100%mortality 24-h post-exposure against 0.05% deltamethrin.

In this study, a high level of DDT resistance was observed inCx. quinquefasciatus, which may be correlated with the use of

DDT for vector control in these areas for many years. In armycantonments (i.e. Missamari, Solmara) and field units (i.e. FUI & II), the use of DDT was discontinued in the past few years,but persistence of DDT in the environment may have resulted incontinued selection for resistance. A similar study conducted inPatna, Bihar in India, demonstrated that this species is resistantto DDT and dieldrin, but susceptible to organophosphatesand pyrethroid insecticides (Mukhopadhyay et al., 1993).Insecticide susceptibility tests on adults and larvae conductedin Panaji, Goa, revealed that Cx. quinquefasciatus adults wereresistant to DDT, dieldrin, malathion and fenitrothion, andlarvae were highly resistant to DDT but showed low resistanceto malathion and fenitrothion (Thavaselvam et al., 1993).There are other reports of high levels of DDT resistance inCx. quinquefasciatus in different parts of the world (Duran etal., 1983; Majori et al., 1986; Somboon et al., 2003).

A simple measure of the degree of resistance is the pro-portion of adults sampled that have enzyme activity levelsgreater than those of susceptible controls. Based on the resultsof the biochemical assays presented here, it is most evident thatinsecticide resistance, especially high DDT resistance, in these

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Insecticide resistance in Culex quinquefasciatus 129

Fig. 4. Frequency distribution of beta-esterase enzyme activity, measured by absorbance in field population of Culex quinquefasciatus amongdifferent study sites.

populations is mediated by metabolic sequestration or detoxi-fication. This is because the biochemical profiling of candidatedetoxification enzyme systems from all of the study sites showsevidence of alpha-esterase, beta–esterase and GST elevationand the presence of clear correlations between enzyme levelsand resistance phenotypes across study sites, which amountsto a definite identification of the mechanisms controlling resis-tance. Elevated esterase activity accounts for resistance toorganophosphates, carbamate and pyrethroid insecticides (Ter-riere, 1984; Brogdon, 1989; Hemingway & Karunaratne, 1998).Elevated GST activity often accounts for DDT and organophos-phate resistance (Hemingway et al., 1985; Penilla et al., 1998;Chen et al., 2003). The high levels of GST activity in all thestudy site populations may explain the high levels of DDTresistance observed in these areas. Pyrethroid resistance is oftena result of elevated esterase activity, as observed in Bengana-juli, where incipient tolerance to deltamethrin was recorded.However, high esterase activity, as observed in other study sitesbut without accompanying pyrethroid resistance, indicates thatthis high esterase activity may also be contributing to observedDDT resistance as suggested by Hemingway & Ranson (2000).High esterase activity as discussed above also suggests the pos-sibility of organophosphate resistance in these areas; althoughcarbamates are not generally used in these areas, malathion is

used. Further study on malathion susceptibility may reveal thehidden reasons for such a high level of esterase activity in thesepopulations.

Effective vector control can only be achieved by propermanagement of insecticide resistance in field populations.There are several methods to delay the onset of resistancethat are based on the strategic use of available insecticides,such as the avoidance of using insecticides that simultaneouslyselect resistance to other chemically related insecticides, andthe use of a number of insecticides in rotation (Raghavendra& Subbarao, 2002). Thus, there is a need, not only forcontinuous monitoring of the status of insecticide resistanceand its possible mechanisms in different settings, but also forthe assessment of the impact of any observed resistance onthe effectiveness of vector control programmes. The data alsoprovide baseline information that is essential for monitoringthe development of insecticide resistance in northeastern areasof India.

Acknowledgements

We thank the Army authorities for providing permission towork in Cantonment areas. We also acknowledge Mr Manash

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130 M. Sarkar et al.

Fig. 5. Frequency distribution of glutathion S-transferase enzyme activity, measured by absorbance in field population of Culex quinquefasciatusamong different study sites.

Jyoti Nath for his kind help in preparing the GIS-based mapof the study sites.

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Accepted 8 February 2009

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