Behavioural responses of deltamethrinand permethrin-resistant strains of Aedes aegypti when exposed to permethrin in an excito-repellency test system P . Paeporna , K. Supaphathoma, S. Sathantriphopa, T. Chareonviriyaphapb and R. Yaicharoenc a
Chemical Control Section, Department of Medical Sciences, Ministry of Public Health, Nonthaburi 11000, Thailand
b c
Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand
Department of Parasitology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
Abstract This study compared the behavioural avoidance responses of the permethrin-resistant and deltamethrinresistant strains of Aedes aegypti, a primary vector of dengue haemorrhagic fever (DHF) in Thailand. The background of biochemical-based resistance mechanism assay of these two strains revealed a significant increase of esterase activity and monooxygenase levels when compared with a laboratory-susceptible strain. Glutathione-S-transferase activity was found to increase only in the permethrin-resistant strain. The DNA sequence of knockdown resistance (kdr) mutation in the voltage-gated sodium channel (IIS6 region) was determined but the leucine to phenylalanine amino acid substitution, which is commonly associated with resistance to pyrethroids in many insect species, was not found in either strain. The behavioural escape response of both contact irritancy and non-contact repellency when exposed to permethrin at standard field dose (0.25 g/m2) was observed by using an excito-repellency test chamber. The results showed that in contact trials, the permethrin-resistant strain showed a lower irritancy response when compared with the deltamethrin-resistant strain. This was probably due to the higher levels of resistance to this insecticide for the permethrin resistance strain. For the repellency test by noncontact trials, the response was not significantly different between the two strains. This may be because the repellency effect was much weaker than that of the irritancy effect. This study indicated that the behavioural response of mosquitoes differs according to different pyrethroid compounds and to the physiological resistance mechanism of the mosquitoes. However, further work is necessary to understand how these responses are mediated. Keywords: Behavioural response; Aedes aegypti; Insecticide resistance; Excito-repellency test chamber.
E-mail: pungasem@dmsc.moph.go.th 153
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Introduction Insecticide resistance in Aedes aegypti, a primary vector of dengue haemorrhagic fever (DHF), has been reported in many parts of the world. In Thailand, fenitrothion, deltamethrin and permethrin are the primary insecticides used in thermal fogging and ultra-low volume sprays for the control of adult Aedes aegypti mosquitoes during DHF outbreaks. The widespread use of insecticides has led to selective insecticide resistance in vector mosquitoes reducing the efficacy of control measures. Insecticide resistance can be grouped within four categories:[1] (i) behavioural resistance where the insect behaviour is modified to avoid contact with the insecticide; (ii) penetration resistance, where the composition of the insect’s exoskeleton becomes modified in a way that inhibits insecticide penetration; (iii) target-site insensitivity, where the chemical site of action for the insecticide is modified, limiting the affinity of the insecticide for the target site and reducing its ability to disrupt the function of the target site; and (iv) metabolic resistance, where detoxification enzymes rapidly break down the insecticide so it is no longer toxic to mosquitoes. Although several reports of insecticide resistance in Ae. aegypti have been published, most of them describe biochemical resistance. The impact of these compounds on Ae. aegypti in terms of behavioural resistance has not been studied. In this paper, permethrin and deltamethrin-resistant strains were examined for target-site insensitivity and for behavioural resistance.
Public Health, Thailand, was used as a laboratory-susceptible strain. Two selected strains of Ae. aegypti, i.e. one deltamethrin-selected strain which showed significant elevation of esterase and monooxygenase activity as compared with laboratory-susceptible strains, and the other the permethrin-resistant strain which had a significant increase in glutathione-S-transferase activity when compared with the deltamethrinresistant strain and the susceptible strain. The details to obtain these two resistant strains were described elsewhere.[2]
PCR and sequencing of partial genomic DNA of sodium channel gene Adult female mosquitoes, from permethrinand deltamethrin-selected strains were used for the detection of point mutation in the segment 6 of domain II (IIS6) of sodium channel gene. [3] The mosquitoes were homogenized individually and total genomic DNA was extracted by phenol chloroform method.[4] The DNA pellet was diluted in TE buffer before using as PCR template. PCR was performed by using AegF, (5’ AAC TTA CTC ATT TCC ATC ATG G3’)[5] and Dg2, (5’ GC (T/ G/A) AT (C/T) TT (A/G) TT(G/A/T/C) GT (G/A) TC (G/A) TT (G/A) TC 3’) primers. Nested PCR was carried out using primers Dg1, (5’ TGG AT (T/C/A) G (A/C) (A/G) (T/A) (C/G) (A/C/T) ATG TGG GA (T/C) TG 3’) and Dip2, (5’TTG GAC AAA AGC AA (G/A) GCT AAG 3’) primers.[6] The PCR products were subjected to DNA sequencing (Bioservice Unit, Thailand).
Materials and methods Test populations A susceptible colony of Ae. aegypti from the Department of Medical Sciences, Ministry of 154
Behavioural resistance test Tests were carried out to compare the behavioural responses of the two strains of Ae. aegypti exposed to 0.25 g/m2 permethrin by Dengue Bulletin – Volume 31, 2007
Behavioural responses of deltamethrin- and permethrin-resistant strains of Aedes aegypti
Figure 1: Excito-repellency test chamber 1 7 6 5 7. Exit portal 6. Front door
chemical-treated surfaces whereas repellency is a response from a distinct distance without physical contact with insecticides. The test system consists of two treated test chambers and two paired control chambers. Prior to the exposure, mosquitoes were starved for at least 24 hours. Twenty-five female mosquitoes were carefully transferred into each of the 4 test chambers (Figure 3). Mosquitoes were allowed a 3-min resting period to permit adjustment to the chamber conditions. Observations for behavioural responses were taken at 1 min intervals for 30 mins. After each test was completed, the number of dead or knockdown specimens was recorded separately for each exposure chamber, paired control chamber and external holding cage, which was the receiving box connected to the exit portal for collecting escaped mosquitoes. Escaped specimens and those remaining inside the chambers, for each treatment, were held
4 5. Outer chamber
3 2 1 4. Screened inner chamber 3. Plexiglass holding frame 2. Plexiglass panel with rubbered door 1. Rear door cover
using excito-repellency test chambers (Figures 1 and 2) developed by Chareonviriyaphap et al.[7] Generally, behavioural responses, or insecticide avoidance, can be categorized as contact irritancy and non-contact repellency. Irritancy results from physical contact with
Figure 2: Excito-repellency test chamber 2
1. Non-contact control 2. Non-contact treatment
3. Contact control 4. Contact treatment
Repellency system
Irritability system
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Figure 3: Excito-repellency test chamber 3
Exposure-tested chamber
External holding cage
separately in small holding containers with cotton pads soaked in 10% sugar solution as food. Mortality counts were made at the end of the 24-hour observation period. The test was replicated 4 times for each insecticide strain and each arm (permethrin treatment, control).
leucine (L, CTC) to phenylalanine (F, TTC) substitution was not found from the mosquitoes sequenced, although all mosquitoes showed the highest resistance against insecticide from each strain.
Results PCR and sequencing of the partial genomic DNA of sodium channel gene To investigate whether alteration of the sodium channel gene was involved in resistance mechanism, a 400 bp of genomic DNA sequence comprising the S6 trans-membrane segment of domain II (IIS6) in the sodium channel gene was PCR-amplified and sequenced. The result revealed that the 156
Behavioural test Even though this was the preliminary study, as the sample size was not sufficient for statistical evaluation, it can be seen that in contact trials the deltamethrin-resistant strain showed higher contact escape response (27.3% within 30 mins) compared with the permethrin-selected strain. The difference in escape response was not observed in non-contact trials. The percentage of the mosquitoes remaining in the exposure chambers under contact and noncontact conditions are shown in Figures 4 and 5 respectively.
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Figure 4: Escape pattern of permethrin- and deltamethrin-resistant populations in contact trial with 0.25 g/m2 permethrin % Mosquitoes remaining in exposure chambers
100 90 80 70 60 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Time (minutes)
Permethrin-resistant
Deltamethrin-resistant
Lab-susceptable
Figure 5: Escape pattern of permethrin- and deltamethrin-resistant populations in non-contact trial with 0.25 g/m2 permethrin % Mosquitoes remaining in exposure chambers 100
90
80
70
60 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Time (minutes) Permethrin-resistant Deltamethrin-resistant Lab-susceptable
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Table: Escape response and percent mortalities of permethrin- and deltamethrin-resistant populations when exposed to 0.25 g/m2 in contact and non-contact trials Treated chamber Test condition Strain Control chamber % Mortality Treatment chamber Escaped Contact Permethrin-resistant Deltamethrin-resistant Lab-susceptible Noncontact 99 99 96 99 Deltamethrin-resistant Lab-susceptible 99 95 11.1 27.3 25.0 6 2 35.8 98 97 93 100 100 95 7.1 10.3 43.0 6 6 26.3 0 1 8.3 1 0 0 Control chamber
No. tested
% escaped
No. tested
% escaped
Did Did Escaped not not escape escape 1 2 44.8 2 3.1 20 0 0 1.1 2 0 4.2 1 3.1 5.4 2 1 6.3
The percentage of mortality of the escaped mosquitoes was a little lower than those remaining in the test chamber for the two strains, both in contact and non-contact trials (Table).
Discussion PCR and sequencing of the partial genomic DNA of sodium channel gene The results suggested that other mechanisms, such as the increase of detoxification enzymes, may be involved in the resistance, or another site of point mutation which is not commonly associated with resistance to pyrethroids in many insect species may be involved in Ae. aegypti resistance. This requires further studies.
From this study the lower irritancy response in the permethrin-selected strain was probably due to its higher levels of resistance to this insecticide, which may have influenced the escape response because of increased duration of exposure time with this insecticide. Similar results were observed with Anopheles gambiae by Chandre et al.[8] These authors demonstrated that resistant mosquitoes could tolerate higherdose permethrin and stayed longer than susceptibles. For a repellency test by non-contact trials, the response was not significantly different between the two strains. This may be due to repellency effect which was much weaker than that of the irritancy effect. Even though the permethrin-resistant strain of Ae. aegypti, which has increased metabolic detoxification enzymes involved in its resistance, does not change the target-site insensitivity but it showed a behavioural resistance to permethrin. This study provides important information because the biochemical resistance in mosquito vectors can mediate their behavioural response to that insecticide when applied as a space spray or on surfacetreated areas for adult control. Dengue Bulletin – Volume 31, 2007
Behavioural test In our experiment, the study was designed to know the contact and non-contact responses of the permethrin-resistant strain and the deltamethrin-resistant strain of mosquitoes to permethrin. So, no bait or similar attractant was used to avoid any confusion in the results. 158
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References [1] Miller TA. Mechanism of resistance to pyrethroids insecticides. Parasitology Today. 1988; 4(7): S8-S12. [2] Paeporn P, Supaphathom K, Srisawat R, Komalamisra N, Deesin V, Ya-umphan P, Leeming Sawat S. Biochemical detection of pyrethroid resistance mechanism in Aedes aegypti in Ratchaburi province, Thailand. Trop Biomed. 2004 Dec; 21(2): 145-51. [3] Soderlund DM, Knipple DC. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochem Mol Biol. 2003 Jun; 33(6): 563-77. [4] Ballinger-Crabtree ME, Black WC 4 , Miller BR. Use of genetic polymorphisms detected by the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations. Am J Trop Med Hyg. 1992 Dec; 47(6): 893-901. th
JC, Hemingway J. Pyrethroid and DDT crossresistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med Vet Entomol. 2003 Mar; 17(1): 87-94. [6] Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Bergé JB, Devonshire AL, Guillet P , Pasteur N, Pauron D. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Mol Biol. 1998 May; 7(2): 179-84. [7] Chareonviriyaphap T, Prabaripai A, Sungvornyothrin S. An improved excitorepellency test chamber for mosquito behavioral tests. J Vector Ecol. 2002 Dec; 27(2): 250-2. [8] Chandre F, Darriet F, Duchon S, Finot L, Manguin S, Carnevale P, Guillet P. Modifications of pyrethroid effects associated with kdr mutation in Anopheles gambiae. Med Vet Entomol. 2000 Mar; 14(1): 81-8.
[5] Brengues C, Hawkes NJ, Chandre F, McCarroll L, Duchon S, Guillet P , Manguin S, Morgan
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