Medical and Veterinary Entomology (2009) 23, 122–131
Insecticide resistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, Culex quinquefasciatus, 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 1 Medical
Entomology Division, Defence Research Laboratory (DRDO), Tezpur, India, 2 Department of Zoology, Cotton College, Guwahati, India and 3 Department 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 neighbouring villages in northeastern India was investigated. In India, DDT is still the insecticide of choice for public health programmes. In military stations, pyrethroids, especially deltamethrins, are used for insecticide-treated nets (ITNs). Recent information on the levels of resistance to DDT and deltamethrin in mosquito populations of northeastern India is scare. Continued monitoring of insecticide resistance status, identification of the underlying mechanisms of resistance in local mosquito populations and the establishment of a baseline data bank of this information are of prime importance. Insecticide susceptibility assays were performed on wild-caught adult female Cx. quinquefasciatus mosquitoes to the discriminating doses recommended by 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), indicating that Cx. quinquefasciatus is resistant to DDT. The species was found to be 100% susceptible to deltamethrin in all study sites except Benganajuli and Rikamari. Knockdown times (KDT) in response to deltamethrin varied significantly between study sites (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 and glutathion S-transferase (GST) established for the S-Lab susceptible strain, and all populations had 100% elevated esterase and GST activity, except Missamari and Solmara. Beta-esterase activity in Field Unit II (96.9%) was less than in any of the other populations. Benganajuli had the highest activity level for all the enzymes tested. There was a significant correlation between all enzyme activity levels and insecticide resistance phenotype by populations (P < 0.05). The results presented here provide the first report and baseline information of the insecticide resistance status of Cx. quinquefasciatus in northeastern India, and associated information about biochemical mechanisms that are essential for monitoring the development of insecticide resistance in the area. DDT, deltamethrin, esterase, glutathion S-transferases, insecticide resistance, knock-down, Assam, India.
Key words.
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:
[email protected] © 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
Insecticide resistance in Culex quinquefasciatus 123 Introduction Culex quinquefasciatus (Say) is the most abundant mosquito species reported in the vector data bank of the Armed Forces of India (Tilak et al., 2008). It is also one of the most important and abundant mosquito species throughout world. It is a continuous biting nuisance, mostly for those living close to larval habitats, and poses a threat to human health where it transmits parasites which cause diseases such as bancroftian filariasis. In a tea agro-ecosystem in Assam, the average infection rate of Cx. quinquefasciatus with filarial worms is 4.6% and the overall prevalence of infective mosquitoes is 0.8%, with an average load of third larval-stage parasites (L3 load) of 2.0 per mosquito. It has been estimated that a total of over 22 000 mosquito bites are received per person/year in ‘tea gardens’ (commercial tea plantations alternatively known as tea estates owned by national and international agencies or private parties), of which 182 bites/person/year (0.81%) are infective (Mahanta et al., 2001). In India, vector control programmes are designed and implemented mainly to target malaria vectors. The strategy of the national vector control programme is based on effective management of the use of insecticides for indoor residual spraying (IRS) and insecticidetreated nets (ITNs) with deltamethrin and permethrin. High levels of insecticide resistance in Cx. quinquefasciatus also present an obstacle to malaria prevention, as people may not perceive the personal protective effect of ITNs if the nets fail to kill biting mosquitoes, which are actually resistant Cx. quinquefasciatus (Corbel et al., 2007). Several factors other than frequency of insecticide use serve to influence the intensity and development of resistance in a population. The most important factors include the frequency of the resistance gene in a population, number of genes interacting to produce the resistant character, size of the population and the dominance status of the gene (cf Chareonviriyaphap et al., 2002). In addition, several physiological mechanisms are involved in insecticide resistance, including reduced sensitivity of sodium channels to insecticides, over-production of detoxifying enzymes, such as esterases, mixed function oxidases (MFOs) and glutathione S-transferases (GSTs), which are responsible for detoxification of toxic substances (Georghiou, 1986; Roberts & Andre, 1994; Nelson et al., 1996; Brogdon & McAllister, 1998; Scott et al., 1998; Feyereisen, 1999). Among the different mechanisms of insecticide resistance, knock-down resistance (kdr), which results from a mutation in the paratype voltage-gated sodium channel (the target site for DDT and pyrethroids), and metabolic resistance are equally important in this part of the world. In India, the kdr mechanism in Cx. quinquefasciatus has been reported only by researchers involved in the current study (Sarkar et al., 2008). In India, DDT is still the insecticide of choice for public health programmes. In military establishments, pyrethroids, especially deltamethrins, are used for ITNs (Joshi et al., 2003). Pyrethroids are also used in tea agro-ecosystems in Assam along with malathion. During 2003, deltamethrin was also used to impregnate ‘patches of clothes’ or ‘bands’, which can be 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 mosquito species in Assam is scare. There is concern that this updated information is needed to ensure that the pattern of insecticide use in this agro-ecosystem is optimized to avoid increased resistance that could threaten the sustainability of the vector control strategy. Thus, continued monitoring of insecticide susceptibility/resistance status and establishment of a baseline data bank for the area is of prime importance. The current study presents the first report of the insecticide resistance status of Cx. quinquefasciatus to DDT and deltamethrin in northeastern India, plus an assessment of the underlying resistance mechanisms present in these vector populations. The results are of importance to the development of future insecticide resistance management strategies and will inform the selection of insecticides for mosquito control in these areas. Materials and methods Eco-environment and pattern of insecticide use in the study area The study was carried out in army cantonments and surrounding villages in Assam, India (Fig. 1). The size of the entire study area was approximately 248 km2 . The study sites were located approximately 1–10 km apart from each other. The locations were selected for mosquito collection on the basis of use of insecticides and/or the eco-environmental settings. The localities were: (1) Benganajuli village, a malariaprone area where DDT is applied regularly by public health workers, (2 and 3) two army animal transport Field Units (I and II) which are mainly surrounded by rice fields, with deep forest and tea gardens to the east of Field Unit II, (4) Rikamari village, which is surrounded by forest, (5) the Missamari cantonment areas, where there is little or no use of DDT inside the cantonment, but pyrethroids are used regularly and some organophosphates are used occasionally, and (6 and 7) the Solmara cantonment areas (I and II) in Tezpur, the town where the Defence Research Laboratory is situated, which is mainly surrounded by rice fields. Meteorologically, these areas are characterized by a long rainy season and humid climate. Global Positioning System (GPS) coordinates of the study site are presented in Table 1. A Geographical Information System (GIS)-based map of the study area is presented in Fig. 1, which was created with ArcGIS 9.2 software (ESRI®ArcMapTM 9.2, Environmental Systems Research Institute, Redlands, CA). Mosquito collections Adult mosquitoes for testing the resistance/susceptibility status of each population were collected by aspiration from human dwellings in villages and barracks in cantonments in the evening from 18.30 to 20.30 hours and early in the morning from 05.00 to 07.00 hours. Mosquitoes were identified as Cx. quinquefasciatus on the basis of morphological characteristics after bioassays were performed. Insecticidesusceptibility tests were performed on wild-caught adult female
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
124 M. Sarkar et al.
Fig. 1. Study sites and its eco-environmental settings. Table 1. GPS coordinates of field sites where a study was conducted. Site Benganajuli Field unit–I Field unit–II Rikamari Missamari Solmara I Solmara II
GPS coordinates 26◦ 51 48.4 26◦ 51 47.4 26◦ 51 14.1 26◦ 50 45.3 26◦ 48 43.0 26◦ 41 24.7 26◦ 40 58.2
N N N N N N N
92◦ 32 27.6 92◦ 33 48.7 92◦ 35 16.2 92◦ 35 33.2 92◦ 35 39.3 92◦ 46 59.8 92◦ 46 51.9
E E E E E E E
mosquitoes and from a mosquito colony maintained in the Defence Research Laboratory, at Tezpur, as a ‘susceptible’ reference strain (S-Lab), to compare against the susceptibility levels of field populations. This susceptible strain was
collected from Udmari village near Tezpur town in the 1996 and colonized in the insectary of the Medical Entomology Division of Defence Research Laboratory at Tezpur. A sample of 25–50 adult female mosquitoes (depending on the number of mosquito collected in a particular site) from the collections described above were directly stored in liquid nitrogen and carried back to the laboratory for the biochemical assays.
Insecticide susceptibility bioassays Insecticide susceptibility assays were performed on wildcaught adult female mosquitoes. Mortality and knock-down resulting from tarsal contact with insecticide-treated filter papers were measured using WHO test kits (WHO, 1998). The tests were carried out using 4% DDT and 0.05% deltamethrin,
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
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%)
Study sites
% Mortality (sample size)
Mean (±SD)
% Mortality (sample size)
1. Benganajuli 2. Field Unit I 3. Field Unit II 4. Rikamari 5. Missamari 6. Solmara I 7. Solmara II S–Lab
11.9 (160) 17.5 (80) 30.63 (160) 41.25 (80) 50.0 (80) 30.63 (160) 38.75 (160) 91.2 (160)
2.38 (1.302) 3.50 (1.29) 6.12 (2.031) 8.25 (1.50) 10.0 (2.16) 6.12 (0.835) 7.75 (1.388) 18.25 (1.282)
96.2 (80) 100 (80) 100 (80) 98.7 (80) 100 (80) 100 (80) 100 (80) 100 (80)
KDT50 (95% CI) 17.8 (12.05–23.57) 10.09 (4.34–15.82) 10.3 (4.8–15.89) 14.5 (9.07–20.01) 8.4 (2.50–14.39) 8.3 (2.30–14.28) 9.4 (3.91–14.99) 5.1 (−1.20–11.54)
KDT90 (95% CI) 69.5 44.2 41.4 56.1 37.4 40.3 39.7 27.5
(36.26–102.83) (22.06–66.33) (18.44–64.45) (32.26–80.13) (15.47–59.42) (19.84–60.88) (21.70–57.60) (12.36–42.72)
χ 2 (df) 2.333 1.235 0.116 1.052 1.150 0.742 0.414 0.051
(4) (3) (2) (4) (2) (3) (3) (2)
Table shows the mean mortality values (replicate mean) and other descriptive statistical parameters of the probability distribution of mortality rates across study sites. Table also shows 50% and 90% knock-down time (KDT50 & KDT90 ) in minutes and Chi-square (χ 2 ) value for deltamethrin (0.05%).
the diagnostic doses recommended by WHO. For each of the insecticides tested, mosquitoes were divided into batches of 20 per test and exposed to insecticide-treated papers for 4 h in the case of DDT (4%) and 1 h for deltamethrin (0.05%). The effects of papers treated only with carrier oils were assayed in parallel as a control. At the end of the exposure period, mosquitoes were transferred into tubes with untreated white filter papers (known as holding tubes) and allowed a 24-h recovery period. For mosquitoes exposed to DDT, mortality rates were recorded after the recovery period, and for mosquitoes exposed to deltamethrin, the numbers knocked down were recorded every 10 min for up to 1 h during exposure. The same bioassays were carried out on the laboratory-reared susceptible strain (S-Lab) to compare with the susceptibility levels of the field populations. During the 24-h recovery period, all mosquitoes were provided with 10% sugar water. Biochemical assays Sample preparation. Adult non-blood fed female Cx. quinquefasciatus, collected from the field and stored in liquid nitrogen, were homogenized individually in 1.5-mL microfuge tubes in 30 μL Milli-Q water, obtained from DirectQTM 5 (Millipore India Pvt. Ltd., Bangalore, India) and then diluted with an additional 270 μL Milli-Q water. Tubes were kept in ice during the whole homogenization procedure. The homogenates were spun at 10 000 g for 2 min at 4◦ C in an ultracentrifuge. The supernatant was used as a crude enzyme extract for the biochemical assays. Three microplates with duplicate mosquito homogenates were used for three enzymes and one microplate for total protein. Each biochemical assay was replicated twice with new individuals from the same mosquito population on two different days. Sample sizes for each biochemical assay ranged from 24 to 48 mosquitoes per location, depending on the availability in the field population. A minimum of three positive and three negative controls were used per plate. Absorbance was measured using a Bio-Rad Microplate Reader (Bio-Rad Laboratories, Philadelphia, PA).
Total protein assay. The total protein content of individual Cx. quinquefasciatus mosquitoes was determined to correct for size variation among the specimens (Brogdon, 1984) using a commercial protein assay kit (Bangalore GENEI, Bangalore, India) according to the user’s guide. The results were compared with a bovine serum albumin (BSA) standard curve. The plates were read at 550 nm wavelength. Non-specific esterase assay. The method of Peiris & Hemingway (1990) was used with alpha- and beta-naphthyl acetate as the substrate. The plates were read at 550 nm wavelength. Glutathion S-transferase assay. Glutathion S-transferase assay (GST) activity was assayed according the method of Brogdon & Barber (1990) with some minor modifications. Reduced glutathione was used as the substrate and the plate was read at 340 nm after a 20-min incubation as the end point. Statistical analysis Mean mortality was determined across all batches of mosquitoes tested for a particular insecticide, and the WHO criteria for evaluating resistance or susceptibility in a mosquito population was used (WHO, 1992); mortality rates of less than 80% indicate resistance, whereas those greater than 98% indicate susceptibility. Mortality rates between 80 and 98% suggest the possibility of resistance that needs to be verified. Analysis of variance (ANOVA) was used to compare knock-down rates after 10-min intervals between mosquitoes from different study sites. The times to 50 and 90% knock-down (KDT50 and KDT90 ) were estimated by regression analysis between per cent knock down and exposure time, using the log-probit method (Finney, 1971). Descriptive statistical analysis was used to calculate means and standard deviations, for the samples collected from different study sites and exposed to the diagnostic dose of DDT (4%).
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
126 M. Sarkar et al. Table 3. Susceptibility threshold based on maximum absorbance of different detoxifying enzymes in the laboratory susceptible strain (S-Lab) and percentage of adult Culex quinquefasciatus that exceeded the susceptibility threshold established in S-Lab.
Enzymes
Susceptible threshold (maximum absorbance value of S–Lab)
Percentage of adult mosquitoes exceeding the threshold Benganajuli
Rikamari
FU–I
100 100 100
100 100 100
100 100 100
1.5825 1.5235 0.10335
α-esterase B -esterase GST
FU–II
Missamari
100 96.9 100
41.66 41.66 16.66
Solmara 20.83 54.16 95.83
Table 4. Enzyme activity level (mean and standard deviation of mean absorbance) of adult Culex quinquefasciatus as measured by absorbance in different study sites. Alpha esterase
Study sites Benganajuli Rikamari FU–I FU–II Missamari Solmara S–Lab
Mean (n) 3.5 2.5 2.7 2.2 1.6 1.3 0.7
(48) (24) (36) (32) (24) (24) (24)
SD
95% Confidence intervals
0.39 0.32 0.61 0.22 0.45 0.49 0.33
3.39–3.63 2.32–2.66 2.53–2.81 2.13–2.43 1.41–1.75 1.20–1.55 0.54–0.88
Beta esterase
Glutathion S–tranferase
Mean (n)
SD
95% Confidence intervals
Mean
SD
3.6 2.4 2.8 2.4 1.4 1.6 0.8
0.25 0.29 0.56 0.47 0.41 0.47 0.32
3.47–3.70 2.26–2.59 2.63–2.90 2.25–2.53 1.27–1.60 1.40–1.72 0.59–0.92
0.47 0.14 0.34 0.17 0.09 0.22 0.05
0.033 0.029 0.03 0.028 0.022 0.035 0.02
(48) (24) (36) (32) (24) (24) (24)
(48) (24) (36) (32) (24) (24) (24)
95% Confidence intervals 0.46–0.47 0.13–0.16 0.33–0.36 0.16–0.18 0.08–0.10 0.20–0.23 0.04–0.07
n = number of mosquitoes tested .
The results of the biochemical analyses of levels of detoxifying enzymes in different populations were expressed as absorbance values. The maximum absorbance value for the laboratory susceptible strain (S-Lab) was used as the susceptibility threshold. ANOVA was used to compare the protein content and enzyme expression levels between populations from different study sites.
Results Susceptibility bioassay The susceptibility status of Cx. quinquefasciatus to a diagnostic dose of DDT (4%) is shown in Table 2. The data show that Cx. quinquefasciatus is resistant to DDT in all the study sites. The per cent mortality range was 11.9–50.0 %, compared with 91.2% for the S-Lab control strain. The highest degree of resistance to DDT was found in Benganajuli, and the lowest in Missamari. Adult bioassays were performed in batches of 20 mosquitoes per test, with replicates, and the mean mortality values of replicates are presented in Table 2. The probability distribution of mortality rates in Benganajuli, Field Unit I and II, Rikamari, Missamari, Solmara I and II differ significantly (P < 0.05) from a normal distribution. In contrast, Field Unit I displayed a true normal distribution. Table 2 shows the 50 and 90% knock-down time, i.e. KDT50 and KDT90, of Cx. quinquefasciatus after continuous exposure to 0.05% deltamethrin for up to 1 h and the per cent mortality after the 24-h post-exposure holding period.
The species was found to be 100% susceptible to deltamethrin in Field Unit I, Field Unit II, Missamari, Solmara I and Solmara II, and the ranges of KDT50 and KDT90 values were 8.3–10.3 min and 37.4–41.4 min, respectively. In Benganajuli and Rikamari, the per cent mortalities were 96.2 and 98.7%, respectively; and the ranges of KDT50 and KDT90 were 17.8–14.5 min and 69.5–56.1 min, respectively. The results for knock-down time in Field Unit II (KDT50 = 10.3 min and KDT90 = 41.4 min) and Rikamari (KDT50 = 14.5 min and KDT90 = 56.1 min) are interesting, because these two areas are nearest each other but surprisingly exhibited contrasting results. The KDT50 and KDT90 values of all study sites are much higher than for the known susceptible strain (SLab) of Cx. quinquefasciatus, whereas KDT50 and KDT90 were 5.1 and 27.5 min, respectively. Knock-down rates for deltamethrin were significantly different between different study sites (ANOVA, F = 2.0583, d.f. = 36, P = 0.00458).
Biochemical assay The susceptibility threshold, as measured by the maximum absorbance value of the susceptible laboratory strain (S-Lab), and the percentage of adult Cx. quinquefasciatus that exceeded that established threshold are presented in Table 3. Results of the biochemical assays of detoxifying enzymes from different study sites are presented in Table 4 and Fig. 2(a–c). Total protein was measured in each mosquito to control for size differences between mosquitoes. Protein assay results based on total protein in individual mosquitoes revealed that the average size of individuals from different study sites was not
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
Insecticide resistance in Culex quinquefasciatus 127 (a)
(b)
(c)
Fig. 2. Activities of detoxifying enzymes, as measured by absorbance, in field populations of Culex quinquefasciatus across different study sites. 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 adjustment was needed for the enzyme analysis to take into account differences in the size of mosquitoes. The enzyme activity results are presented here as absorbance (OD value), rather than concentration, so the details of the protein assay results from each mosquito are not presented. Activities of different enzymes tested are shown in Fig. 2(a–c) as absorbance. Box plot distributions add additional information not apparent in tables of the mean and range data because they reveal the distribution of activity levels of enzymes and why significant differences exist between different populations (Zayed et al., 2006). All the populations exceeded the S-Lab strain threshold for alpha- and beta-esterase, and GST (Table 3). Enzyme activity levels are significantly different among different populations including the S-Lab strain (P < 0.001). All populations except Missamari and Solmara had 100% elevated esterase and GST activity. Beta-esterase activity in FU-II is slightly less (96.9%) than for the other populations. Benganajuli had the highest activity level of all the enzymes tested (Fig. 2). Benganajuli and Field Unit I had the maximum number of individuals with higher levels of GST activity (Fig. 5). There is a significant correlation
between all enzyme activity levels and insecticide resistance phenotype by population (P < 0.05). The distribution patterns (number of individuals observed) of elevated enzyme activities, as measured by absorbance in different populations, are displayed in Figures 3, 4 and 5. Discussion The resistance status of Cx. quinquefasciatus mosquitoes to DDT and deltamethrin was investigated in and around army cantonments in Assam, northeastern India. Based on the WHO criteria for characterizing insecticide resistance/susceptibility, where susceptibility is defined by mortality rates greater than 98% after 24-h post-exposure, evidence for resistance to DDT was found at all the study sites (Table 2). The per cent mortality after 24-h post exposure obtained in knock-down bioassays for deltamethrin suggests complete susceptibility to this insecticide in all study sites, except in Benganajuli (Table 2), where further verification data are needed. In all the study areas, deltamethrin-treated bednets were recently introduced by the public health department and
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
128 M. Sarkar et al.
Fig. 3. Frequency distribution of alpha-esterase enzyme activity, measured by absorbance in field population of Culex quinquefasciatus among different study sites.
army authority, but in villages such as Benganajuli and Rikamari bednets were not regularly retreated after purchase. However, the KDT50 and KDT90 for deltamethrin for Cx. quinquefasciatus mosquitoes in the different study sites are comparatively higher than for the susceptible S-Lab laboratory strain. Knock-down rates at 10-min intervals were significantly different (P < 0.01) between study sites. The contrasting results for knock-down times in two adjacent areas such as Field Unit II and Rikamari (1 km apart) suggests that the mosquito population in these areas seems to be physiologically distinct. In Benganajuli and Rikamari, higher values of knockdown times (KDT50 and KDT90 ) possibly indicate the development of incipient resistance to deltamethrin in these populations of Cx. quinquefasciatus. But unfortunately we could not correlate this incipient resistance to the use of deltamethrin in these areas. There are similar reports for Anopheles fluviatilis James from Orissa (Sharma et al., 2004) and An. culicifacies Giles from Tamil Nadu, India (Mittal et al., 2002), where delayed knock-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 in Cx. quinquefasciatus, which may be correlated with the use of
DDT for vector control in these areas for many years. In army cantonments (i.e. Missamari, Solmara) and field units (i.e. FU I & II), the use of DDT was discontinued in the past few years, but persistence of DDT in the environment may have resulted in continued selection for resistance. A similar study conducted in Patna, Bihar in India, demonstrated that this species is resistant to DDT and dieldrin, but susceptible to organophosphates and pyrethroid insecticides (Mukhopadhyay et al., 1993). Insecticide susceptibility tests on adults and larvae conducted in Panaji, Goa, revealed that Cx. quinquefasciatus adults were resistant to DDT, dieldrin, malathion and fenitrothion, and larvae were highly resistant to DDT but showed low resistance to malathion and fenitrothion (Thavaselvam et al., 1993). There are other reports of high levels of DDT resistance in Cx. quinquefasciatus in different parts of the world (Duran et al., 1983; Majori et al., 1986; Somboon et al., 2003). A simple measure of the degree of resistance is the proportion of adults sampled that have enzyme activity levels greater than those of susceptible controls. Based on the results of the biochemical assays presented here, it is most evident that insecticide resistance, especially high DDT resistance, in these
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
Insecticide resistance in Culex quinquefasciatus 129
Fig. 4. Frequency distribution of beta-esterase enzyme activity, measured by absorbance in field population of Culex quinquefasciatus among different study sites.
populations is mediated by metabolic sequestration or detoxification. This is because the biochemical profiling of candidate detoxification enzyme systems from all of the study sites shows evidence of alpha-esterase, beta–esterase and GST elevation and the presence of clear correlations between enzyme levels and resistance phenotypes across study sites, which amounts to a definite identification of the mechanisms controlling resistance. Elevated esterase activity accounts for resistance to organophosphates, carbamate and pyrethroid insecticides (Terriere, 1984; Brogdon, 1989; Hemingway & Karunaratne, 1998). Elevated GST activity often accounts for DDT and organophosphate resistance (Hemingway et al., 1985; Penilla et al., 1998; Chen et al., 2003). The high levels of GST activity in all the study site populations may explain the high levels of DDT resistance observed in these areas. Pyrethroid resistance is often a result of elevated esterase activity, as observed in Benganajuli, where incipient tolerance to deltamethrin was recorded. However, high esterase activity, as observed in other study sites but without accompanying pyrethroid resistance, indicates that this high esterase activity may also be contributing to observed DDT resistance as suggested by Hemingway & Ranson (2000). High esterase activity as discussed above also suggests the possibility of organophosphate resistance in these areas; although carbamates are not generally used in these areas, malathion is
used. Further study on malathion susceptibility may reveal the hidden reasons for such a high level of esterase activity in these populations. Effective vector control can only be achieved by proper management of insecticide resistance in field populations. There are several methods to delay the onset of resistance that are based on the strategic use of available insecticides, such as the avoidance of using insecticides that simultaneously select resistance to other chemically related insecticides, and the use of a number of insecticides in rotation (Raghavendra & Subbarao, 2002). Thus, there is a need, not only for continuous monitoring of the status of insecticide resistance and its possible mechanisms in different settings, but also for the assessment of the impact of any observed resistance on the effectiveness of vector control programmes. The data also provide baseline information that is essential for monitoring the development of insecticide resistance in northeastern areas of India.
Acknowledgements We thank the Army authorities for providing permission to work in Cantonment areas. We also acknowledge Mr Manash
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
130 M. Sarkar et al.
Fig. 5. Frequency distribution of glutathion S-transferase enzyme activity, measured by absorbance in field population of Culex quinquefasciatus among different study sites.
Jyoti Nath for his kind help in preparing the GIS-based map of the study sites. References Bhatnagar, A. & Mehta, V.K. (2007) Efficacy of deltamethrin and cyfluthrin impregnated cloth over uniform against mosquito bites. Medical Journal Armed Forces India, 63, 120–122. Brogdon, W.G. (1984) Mosquito protein microassay. I. protein determinations from small portions of single mosquito homogenates. Comparative Biochemistry and Physiology, 79, 457–459. Brogdon W.G. (1989) Biochemical resistance detection: An alternative bioassay. Parasitology Today, 5, 56–60. Brogdon, B.D & Barber, A.M. (1990) Microplate assay of glutathione s—transferase activity for resistance detection in single mosquito triturates. Comparative Biochemistry and Physiology B, 96, 339–342. Brogdon, W.G & McAllister, J.C. (1998) Insecticide resistance and vector control. Emerging Infectious Diseases, 4(4). Chareonviriyaphap, T., Rongnoparut, P & Juntarumporn, P. (2002) Selection for pyrethroid resistance in a colony of Anopheles minimus species A, malaria vector in Thailand. Journal of Vector. Ecology, 27, 222–229. Chen, L., Hall, P.R., Zhou, X.E., Ranson, H., Hemingway, J & Meehan, E.J. (2003) Structure of an insect delta class GlutathioneS-Transferase from a DDT—resistant strain of malaria vector
Anopheles gambiae. Acta Crystallographica Section D: Biological Crystallography, 59, 2211–2217. Corbel, V., Guessan, R.N., Brengues, C. et al . (2007) Multiple insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus from Benin, West Africa. Acta Tropica, 101, 207–216. Duran, M & Stevenson, H.R. (1983) Insecticide resistance in adult Culex quinquefasciatus mosquitoes from Olongapo City, Philippines. Southeast Asian Journal of Tropical Medicine and Public Health, 14, 403–406. Feyereisen, R. (1999) Insect P450 enzymes. Annual Review of Entomology, 44, 507–533. Finney, J.D. (1971) Probit Analysis, 3rd edn. Cambridge University Press, Cambridge, United Kingdom. Georghiou, G.P. (1986) The Magnitude of Resistance Problem. Pesticide Resistance: Strategies and Tactics for Management, pp. 14–43. National Academy Press: Washington, DC. Hemingway, J & Karunaratne S.H. (1998) Mosquito carboxylesterase: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism. Medical and Veterinary Entomology, 12, 1–12. Hemingway, J & Ranson, H. (2000) Insecticide resistance in insect vectors of human diseases. Annual Review of Entomology, 45, 371–391. Hemingway, J, Malcolm, C.A., Kisson, K.E., Boddington, R.G., Curtis, C.F & Hill, N. (1985) The biochemistry of insecticide resistance in Anopheles sacharovi : comparative studies with a range of insecticide
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
Insecticide resistance in Culex quinquefasciatus 131 susceptible and resistance Anopheles and Culex species. Pesticide Biochemistry Physiology, 24, 68–76. Joshi, R.M., Ghose, G., Som, T.K & Bala, S. (2003) Study of the impact of deltamethrin impregnated mosquito nets on malaria incidence at a military station. Medical Journal Armed Forces India, 59, 12–14. Mahanta, B., Handique, R., Narain, K., Dutta, P & Mahanta, J. (2001) Transmission of bancroftian filariasis in tea agro-ecosystem of Assam, India. Southeast Asian Journal of Tropical Medicine and Public Health, 31, 581–584. Majori, G., Sabatineli, G., Villani, F & Petrarca, V. (1986) Studies on insecticide susceptibility of Anopheles gambiae s.l. and Culex quinquefasciatus in the area of Ouagadougou, Burkina Faso (West Africa). Journal of American Mosquito Control Association, 2, 305–309. Mittal, P.K., Adak, T., Singh, O.P., Raghavendra, K & Subbarao, S.K. (2002) Reduced susceptibility to deltamethrin in Anopheles culcifacies sensu lato, in Ramnathapuram district, Tamil Nadu—Selection of a pyrethroid—resistance strain. Current Science, 82, 185–188. Mukhopadhyay, A.K., Sinha, S.N., Yadav, R.L & Narasimham, M.V. (1993) Susceptibility status of Culex quinquefasciatus in Patna to insecticides. Indian Journal of Public Health, 37, 57–60. Nelson, D.R, Koymans, L., Kamataki, T. et al . (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 6, 1–42. Peiris, H.T.R & Hemingway, J. (1990) Temephos resistance and the associated cross-resistance spectrum in a strain of Cx. quinquefasciatus from Peliyagoda, Sri Lanka. Bulletin Entomological Research, 80, 49–55. Penilla, P.R., Rodrigues, A.D., Hemingway, J., Torres, J.T., Jimenez, J.A & Rodrigues, M.H. (1998) Resistance management strategies in malaria vector mosquito control. Baseline data for a large-scale field trial against Anopheles albimanus in Mexico. Medical and Veterinary Entomology, 12, 217–223. Raghavendra, K & Subbarao, S.K. (2002) Chemical insecticides in malaria vector control in India. Indian Council of Medical Research Bulletin, 32, 93–99. Roberts, D.R & Andre, R.G. (1994) Insecticide resistant issues in vector-borne disease control. American Journal of Tropical Medicine and Hygiene, 50, (Suppl. 6), 21–34. Sarkar, M., Baruah, I., Bhattacharyay, I.K., Borkotoki, A & Srivastava, R.B. (2008) Molecular study on knock-down resistance
(kdr ) allele in relation to pyrethroid and DDT resistance in Culex quinquefasciatus from India. Proceeding of International Conference on Science and Technology: Application in Industry & Education, pp. 2087–2093. Universiti Teknologi MARA, Pulau Pinang, Malaysia. Scott, J.G., Liu, N & Wen, Z. (1998) Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins. Comparative Biochemistry and Physiology C, 121, 147–155. Sharma, S.K., Upadhyay, A.K., Haque, M.A., Singh, O.P., Adak, T & Subbarao, S.K. (2004) Insecticide susceptibility status of malaria vectors in some hyper endemic tribal districts of Orissa. Current Science, 87, 1722–1726. Somboon, P., Prapanthadara, L.A & Suwonkerd, W. (2003) Insecticide susceptibility tests of Anopheles minimus s.l., Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus in northern Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 34, 87–93. Terriere, L.C. (1984) Introduction of detoxification enzymes in insects. Annual Review of Entomology, 29, 71–88. Thavaselvam, D., Kumar, A & Sumodan, P.K. (1993) Insecticide susceptibility status of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti in Panaji, Goa. Indian Journal of Malariology, 30, 182. Tilak, R., Duttagupta, K.K & Verma, A.K. (2008) Vector data bank in the Indian armed forces. Medical Journal Armed Forces India, 64, 36–39. WHO (1992) Vector Resistance to Insecticides. 15th Report of the WHO Expert Committee on Vector Biology and Control . World Health Organization Technical Report Series, 818, pp. 1–62. WHO, Geneva, Switzerland. WHO (1998) Test Procedure for Insecticide Resistance Monitoring in Malaria Vector, Bio-efficacy and Persistence of Insecticides on Treated Surfaces. Document WHO/CDS/CPC/Mal/98.12. Geneva Switzerland. Zayed, A.B.B., Szumlas, D.E., Hanafi, H.A., Fryauff, D.J., Mostafa, A.A., Allam, K.M & Brogdon, W.G. (2006) Use of bioassay and microplate assay to detect and measure insecticide resistance in field population of Culex pipiens from Filariasis endemic areas of Egypt. Journal of American Mosquito Control Association, 22, 473–482. Accepted 8 February 2009
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 122–131
