2.1 Mosquitoes
Wild Ae. aegypti mosquitoes were collected from the University of Sri Jayewardenepura (USJ) premises, situated in Gangodawila, Nugegoda, Sri Lanka, a highly urbanized environment and thermal fogging is practiced frequently. Laboratory-reared susceptible Ae. aegypti New Orleans strain was used as a reference strain while USJ collected mosquitoes as field strain. Mosquitoes were reared in an insectary and the temperature within the insectary was 27°C, relative humidity was between 70-80% and natural light periods (12:12 hours light: dark periods). During rearing, larvae were fed on fish feed and adults were fed on 10% glucose solution. Only sugar-fed, reference strain and F1 generation of field strains’ females between 3- 5 days old were used in experiments.
2.2 Tested household insecticides
A preliminary survey was conducted to identify commonly used mosquito coils and liquid vaporizers in Sri Lanka. Three brands of mosquito coils and liquid vaporizers were identified to be commonly available in the Sri Lankan market and their active ingredients and concentrations are listed in Table 1.
2.3 Household insecticide bioassay
Bio-efficacy of household insecticides were evaluated in the WHO standard Peet-Grady chamber (180 cm x 180 cm x 180 cm) [11]. Four cages (20 cm x 20 cm x 20 cm) were hung in the chamber’s four corners, each containing 25 Ae. aegypti female mosquitoes. Cages were made from a 0.5mm hole opening nylon mesh net and hung at 80 cm from the ceiling and 10 cm from the side walls that allow the cages positioning in front of the observation windows. A 30-cm diameter fan (wind speed 4.8 m/s) was used to circulate the air in the chamber and a 30 cm diameter flat dish was attached on top of the fan rail guard. The fan was placed facing upwards on the center of the chamber’s floor and the test product, either mosquito coil or liquid vaporizer was placed in the middle of the flat dish.
The Relative Humidity (RH) and the temperature within the chamber were measured before each test. The coil was lit, and the liquid vaporizer device was pre-heated away from the test chamber 5 minutes before the test. A control test was done before testing each brand of mosquito coil and liquid vaporizer in which mosquitoes were kept inside the chamber without household insecticides.
The stopwatch was started after closing the chamber door and a number of knock-down mosquitoes count were taken through observation windows by three observers at a time. A number of knock-down mosquitoes were counted at regular intervals for 60 minutes and the count was taken for every minute of the first 10 minutes and subsequently at 10 minutes intervals (Ex: 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes) for a total of 60 minutes.
After 60 minutes cages were quickly removed from the chamber, and knock-down observations were completed. Exposed mosquitoes were transferred into clean holding cups by using an aspirator. They were provided 10% sugar solution on cotton wool and were kept in the insectary for 24 hours at 27 ± 5°C temperature and 80% ± 15% RH to take the post 24-hour mortality observations. After each test, the chamber was ventilated by an exhaust fan and was washed with detergent to make sure that no residues were remaining in the wall of the chamber and the cages. Three replicates were done for each brand and the results were pooled.
2.4 kdr Genotyping
Genomic DNA was extracted from 40 mosquitoes that survived the bio-efficacy tests of mosquito coils and vaporizers including all three brands and all dead mosquitoes. Genomic DNA is extracted from mosquitoes by using the phenol-chloroform extraction method [18]. Extracted DNA was stored in 100 µl TE buffer (10 Mm Tris, 0.1 mM EDTA, pH 7.5-8.0) [16].
F1534C AS-PCR genotyping assay was done by adopting the protocol of one of the previous studies [19] and the PCR products visualized by agarose gel electrophoresis. Each reaction was performed in a 25 µl volume with final concentrations of 1x PCR reaction buffer, 1.5mM MgCl2, 200 µM dNTP mixture, 0.5 µM common reverse primer (5′-TCTGCTCGTTGAAGTT GTCGAT-3′), 0.5 µM forward primer (5′-GCGGGCTCTACTTTGTGTT CTTCATCATATT-3′), 0.5 µl Cys forward primer (5′- GCGGGCAGGGCGGCGGG GGCGGGGCCTCTACTTTG TGTTCTTCATCATGTG-3′), 0.2 U Taq DNA polymerase (Promega, USA) and 2 ng template DNA from extracted 100 µl DNA stock solution. The thermal cycling condition begins with an initial DNA denaturation step for two minutes at 95 °C, followed by 35 cycles of 30 sec at 95 °C (denature), 30 sec at 60 °C (anneal), and 30 sec at 72 °C (extension). This is then followed by two minutes at 72°C for a final extension. Amplified PCR products were loaded onto 3.5% agarose gel and electrophoresis was conducted at 100v for 45 minutes [15].
The V1016G mutation was analyzed with the use of AS-PCR [20]. Each reaction was performed in a 25 µl volume with final concentrations of 1x PCR reaction buffer, 1.5 mM MgCl2, 200 µM dNTP mixture, 0.25 µM forward primer (5′-ACCGACAAATTGTTTCCC-3′), 0.125 µM Gly reverse primer (5′-GCGGGCAGGGCGGCGGGGGCGGGGCCAGCAAGGCTAAGAAAAGGTTAACTC-3′), 0.125 µM Val reverse primer (5′-GCGGGCAGCAAGGCTAAGAAAAGGTTAATTA-3′), 0.2 U Taq DNA polymerase (Promega, USA) and 2 ng template DNA from extracted 100 µl DNA stock solution to detect V1016G genotyping. Amplification was done at 94 °C for 2 min initial activation stage and followed by 35 cycles of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 30 sec, in turn, followed by a final extension at 72 °C for 2 min [20]. PCR products of V1016G genotyping were loaded onto 4% agarose gels and the electrophoresis was conducted at 100v for 50 minutes.
2.5 Statistical analysis
Knock-down data in the range of 5-95% of the bioassay tests were undergone to probit analysis [21] using SPSS (Version 20) software to calculate KT50 and KT95 (time taken to knock-down time 50% and 95% respectively). KT50 values of field strains were divided by the KT50 value of the reference strain to calculate the resistance ratio (RR) which exhibits cross-resistance. RR values <5 indicate low resistance, medium resistance with RR values within 5-10 and high resistance with RR values >10 [22].
According to WHO resistance indicators, if the mortality percentage was more or equal to 98% then the tested mosquitoes were susceptible to the particular insecticide, possible development of resistance was indicated with 98 - 90% mortality and the confirmed resistance of a mosquito population to a particular insecticide indicated by less than 90% mortality [23]. If the control’s mortality was between 5–20%, the mortality percentage would be corrected according to Abbott’s formula. The mean mortality percentage and knock-down percentage were calculated, and bar plots were prepared using R statistical software (version 4.2.0) (R Foundation for Statistical Computing, Vienna, Austria).
The mosquito mortality data were analyzed with a one-way analysis of variance (ANOVA) using Minitab-version 2017 analysis and the p ≤ 0.05 was taken as a statistically significant association. RR values 10 are indicative of high resistance [22]. The mortality rate at 24-h posttreatment was used to assess the susceptibility status of the mosquitoes, where 98–100% indicate susceptible, and 80–97% indicate possible development of resistance. The mosquito mortality data were analyzed with a one-way analysis of variance (ANOVA) using Minitab-version 2017.