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Mechanisms of DDT and Permethrin Resistance in Aedes aegypti from Chiang Mai, Thailand

by La-aied Prapanthadara*#, Nongkran Promtet*, Surangchit Koottathep*, Pradya Somboon**, Wonnapa Suwonkerd***, Lynn McCarroll+ and Janet Hemingway+

*Research Institute for Health Sciences, Chiangmai University, PO Box 80, Chiangmai 50200, Thailand **Faculty of Medicine, Chiangmai University, PO Box 80, Chiangmai 50200, Thailand ***Office of Vector Born Disease Control, Department of Communicable Disease Control, Ministry of Public Health, Thailand +Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

Abstract

Two strains of Aedes aegypti, one resistant to DDT but susceptible to permethrin(RdSp), and the other resistant to both DDT and permethrin (RdRp), were established in mosquitoes collected from Chiang Mai province, northern Thailand. Comparisons with a susceptible reference strain indicated that DDT resistance in both RdSp and RdRp strains was mainly due to an increase in DDTase activity. Similar moderate increases in cytochrome P450 levels were observed in the two resistant strains, hence this enzyme family may also play a role in DDT resistance. Glutathione S-transferase and esterase activities in the two resistant strains were similar and slightly higher than those of the susceptible strain, suggesting that neither enzyme group has a major role in permethrin resistance. The lack of an evident metabolic basis for the resistance in the RdRP strain suggests that nerve insensitivity may be present in this strain. The two mutations at residues reported to produce kdr resistance in other insects were not present, but some individuals from the permethrin resistant strain had an amino acid mutation at position 106 involving a valine to glycine mutation in the same segment 6 of domain II of the para sodium-channel gene, which may confer kdr-like resistance.

Keywords: Aedes aegypti, DDT, permethrin resistance, Chiang Mai, Thailand.

# For correspondence: [email protected]

Dengue Bulletin – Vol 26, 2002 185 Mechanisms of DDT and Permethrin Resistance in Aedes aegypti from Chiang Mai, Thailand

Introduction

Aedes aegypti, the primary vector of yellow females had <1% mortality, whereas those fever, dengue and dengue haemorrhagic exposed to 0.25% permethrin for 60 min. fever (DHF), is -resistant in gave 70% mortality. Single-family selections numerous locations throughout the world(1). for several generations produced two strains DDT resistance is now widely distributed in of Aedes aegypti, one fully resistant to DDT d p Aedes aegypti throughout northern Thailand (R S ) but susceptible to permethrin and the and it is also resistant to the , other resistant to both DDT and permethrin d p permethrin and , and the neo- (R R ). These two strains were maintained pyrethroid in many areas (P. under insecticide pressure for at least 10 Somboon, unpublished data). generations before the resistance ratios were determined and the insects were harvested The major mechanisms involved in for biochemical and molecular assays. Aedes DDT resistance in insects are increased aegypti Rockefeller (Rock) strain was used as metabolism of DDT by the glutathione S- a reference-susceptible for comparisons. transferase (GST) enzyme family and the insensitivity to inhibition of the voltage-gated sodium channel (known as kdr)(2). As both Bioassay and determination DDT and pyrethroids act on the nervous of LT50 system by modifying the gating kinetics of For all bioassay, four replicates (25 females voltage-sensitive sodium channels, mutation per replicate) of 4 and 10 different exposure in specific regions of this sodium channel time periods for 4% DDT and 0.25% can lead to binding failure with DDT and permethrin respectively were undertaken. pyrethroids; therefore; cross-resistance Percentage mortalities were calculated for between DDT and pyrethroids is common. each exposure time and the mortality data In this paper, we applied biochemical and were analysed on a log-time probit mortality molecular assays to elucidate the resistance regression using a computer programme mechanisms in DDT-resistant and provided by Dr C J Schofield, WHO, DDT/permethrin cross-resistant strains of Geneva. Aedes aegypti from Northern Thailand.

Enzyme assays Materials and methods One-day old females of each strain were Mosquitoes used for enzyme or molecular assays. Each batch was homogenized in an appropriate One-day old females, emerging from field- buffer at 4oC and the 10,000 g supernatant collected larvae of Aedes aegypti from Ban was then determined for enzyme activities. Pang Mai Dang, Mae Tang district, Chiang The GST and DDTase activity were Mai province, Thailand, were exposed to 4% determined using the methods of DDT-impregnated papers for 30 minutes in Prapanthadara et al. 1996(3). The esterase the WHO standard exposure tubes. These

186 Dengue Bulletin – Vol 26, 2002 Mechanisms of DDT and Permethrin Resistance in Aedes aegypti from Chiang Mai, Thailand activity was measured as described by Peiris Table 1: Lethal time (minutes) that gave 50% & Hemingway 1990(4) with p-nitrophenyl mortality (LT50) in two insecticide-selected lines acetate as the substrate(4). The method for of Aedes aegypti compared with the susceptible determination of insensitive acetylcho- Rock strain when exposed to 4% DDT or linesterase (IAChE) was as from ffrench- 0.25% permethrin on impregnated filter papers Constant & Bonning 1989(5). Mono- in a standard WHO tarsal contact assay oxygenase activity was indirectly determined DDT Permethrin by measuring the different spectra of Strains LT50 χ2 P LT50 χ2 P cytochrome P450 in microsomal fraction by ROCK 17 36.9 NS 22 34.4 NS using a carbon monoxide trap. (15- (21 – 19) 24) Protein was assayed using the Bio-Rad RdSp 390 27.9 < 18.607 5.8 NS protein reagent with bovine serum albumin (352- 0. (17.41- as the standard protein. 427) 00 19.76) 1

The PCR fragments of the segment 6 RdRp 513 24.5 < 534 2.1 NS domain II region of the sodium channel gene (466- 0. (502 - 559) 00 566) were obtained as described by Martinez- 1 Torres et al. 1998(6). Table 2: Comparison of mean GST, DDTase, Results cytochrome P450 and esterase activities in Aedes d p d p aegypti for the Rock, RdSp and RdRp strains The LT50s of the R S and R R strains for d p d p DDT (Table 1) were 23- and 30-fold higher, Strains Rock R S R R respectively, than the LT of the Rock strain. GST 0.38± 0.37± 50 0.22± d p (µmole/min/mg) 0.09 0.04 Log time-probit mortality lines for both R S 0.05 and RdRp (Figure) were not linear for DDT DDTase 3.23± 31.03± 26.85± (Chi square analysis P<0.005), indicating (nmole/mg) 1.23 13.88 5.12 that resistance had not been selected to EST homogeneity. In contrast, the Chi square 0.2± 0.34± 0.26± (µmole/min/mg) 0.03 0.07 0.04 value for permethrin in both the RdSp and 450 RdRp strain were non-significant which Cytochrome P 0.07± 0.24± 0.28± (nmole/mg) 0.02 0.02 0.04 suggested that resistance to this insecticide had been selected to homogeneity. The enzyme activities are presented in One-day old adult female were used to Table 2. There was an ~10-fold increase in determine all enzyme activities except DDTase activity in the two resistant strains 450 cytochrome P450, where larvae were used in and a 4-fold increase in cytochrome P order to minimize pigment interference. activity compared to the Rock strain. The Activities in each strain were determined GST and esterase activities were slightly using at least 20 batches of 10 mosquitoes increased. There was no change in the and the mean activities ± SD were sensitivity of acetylcholinesterase to calculated. inhibition by , in either resistant

Dengue Bulletin – Vol 26, 2002 187 Mechanisms of DDT and Permethrin Resistance in Aedes aegypti from Chiang Mai, Thailand strain compared to the Rock susceptible. Discussion The levels of increased DDTase and d p cytochrome P450 activities were very similar We found that DDT resistance in both R S d p in the two resistant strains. This is in contrast and R R strains was due to increased 450 to the difference in the DDT-resistance DDTase activity and cytochrome P content d p ratios. Sequencing of the S6 domain II whereas permethrin resistance in the R R region of the para-sodium channel gene strain probably involved a non-metabolic kdr demonstrated that the standard leucine to mechanism. This kdr should also generate phenylalanine or leucine to serine mutation, cross-resistance to DDT and would explain d p which confers kdr-resistance in An. the greater resistance ratio to DDT in R R as d p gambiae(6), was not present in either resistant compared to the R S strain. strain of Aedes aegypti. However, there was In earlier reports of DDT and a valine to glycine mutation at position 106 pyrethroid resistance in Aedes aegypti, d p in this domain in the 4 R R individuals selection of a resistant strain with DDT sequenced. This mutation was not detected generated moderate resistance to d p in the 4 R S or 20 Rock individuals pyrethroids, whereas selection with sequenced. permethrin resulted in strong resistance to both permethrin and DDT(7). Our results Figure: Log time-probit mortality lines for the 450 ROCK, RdSp and RdRp strains of suggest that DDTase/cytochrome P -based Aedes aegypt with DDT and permethrin DDT-resistance does not confer cross-

Permethrin resistance to pyrethroids. However, the kdr- 120 based pyrethroid resistance mechanism ROCK 100 RdSp RdRp probably increases the DDT-resistance

80 levels.

60 In conclusion, our study suggested that Percent mortality 40 DDT resistance was due to multiple factors.

20 DDT selection, as shown by our results and others(8), often generates GST-based 0 10 100 1000 metabolic resistance which does not usually Log time (minutes) confer cross-resistance to other . This should be considered before a policy of 120 DDT abolishing DDT use for susceptible mosquito

100 ROCK d dP p RRS S vectors is implemented. Although the RdRd p p 80 persistence of DDT residues in the environment are well documented, making 60 it unsuitable for large-scale spraying, limited Percent mortality mortality Percent 40 use of this insecticide as an indoor spray

2 0 may still be a good method of mosquito vector control in many countries. 0 10 100 1000

Log time (minutes)

188 Dengue Bulletin – Vol 26, 2002 Mechanisms of DDT and Permethrin Resistance in Aedes aegypti from Chiang Mai, Thailand

Acknowledgements Institute for Health Sciences) for their assistance in statistical analysis. We thank Dr V Ruangyuttikarn of the Department of Toxicology, Faculty of Financial support: This work was Medicine, Chiang Mai University, for her supported by a Thailand Research Fund advice with the cytochrome P450 assays. (TRF) award to La-aided Prapanthadara, and We also thank Mr Chumnong Kingkeaw and a Wellcome Trust Tropical Development Mrs Darika Chittpramodya (Research award to Janet Hemingway.

References

1. Brown AWA. Insecticide resistance in mosquitoes: 6. Martinez-Torres D, Chandre F, Williamson MS, a pragmatic review. J. Am. Mosq. Control Assoc., Darriet F, Bergé J-B, Devonshire AL, Guillet P, 1986, 2: 123-140. Pasteur N and Pauron D. Molecular 2. Hassall KA, 1990. Hassall KA ed. The biochemistry characterization of pyrethroid knockdown and uses of pesticides: Structure, metabolism, resistance (kdr) in the major vector mode of action and uses in crop protection. Anopheles gambiae s.s. Insect Molecular Biology, MacMillan. London. 1989, 7: 179-184. 3. Prapanthadara L, Koottathep S, Promtet N, 7. Grant DF and Matsumura F. Glutathione S- Hemingway J and Ketterman A. Purification and characterization of a major glutathione S- transferase 1 and 2 in susceptible and insecticide transferase from the mosquito Anopheles dirus resistant Aedes aegypti. Pesticide Biochemistry and (species B). Insect Biochemistry and Molecular Physiology, 1989, 33: 132-143. Biology, 1996, 26: 277-285. 8. Ranson H, Jensen B, Vulule JM, Xang X, 4. Peiris HTR and Hemingway J. Temephos Hemingway J and Collins FH. Identification of resistance and the associated cross-resistance mutation in the voltage-gate sodium channel gene spectrum in a strain of Culex quinquefasciatus Say of Kenyan Anopheles gambiae associated with (Diptera: Culicidae) from Peliyagoda, Sri Lanka. resistance to DDT and pyrethroids. Insect Bulletin of Entomological Research, 1990, 80: 49-55. Biochemistry and Molecular Biology, 2000, 30: 5. French-Constant RH and Bonning BC. Rapid 443-453. microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotype in the mosquitoes Anopheles albimanus, An. nigerrimus and Culex pipiens. Medical and Veterinary Entomology, 1989, 3: 9-16.

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