The mechanism of insecticide resistance is traditionally attributed to detoxification enzymes, target site alteration, decreased penetration of insecticides and behavioural resistance. Other form of mechanisms, such as the role of protein(s) in resistance is unknown. In the present study, the protein profiling of both IMR-PSS strain (permethrin-selected) and IMR-LS strain (laboratory-susceptible) 24 hours post exposure period to permethrin was carried out via 1D-gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/ MS). The bands which appeared in the gel of 1D-electrophoresis revealed an abundance of proteins. The band pattern of both strains looked macroscopically alike and differed only in band intensity. However, LC-MS/MS analysis revealed that the IMR-PSS strain produced extra 388 peptides that were not found in the IMR-LS strain, indicating that IMR-PSS strain reacted differently from IMR-LS strain as a result of persistent exposure to permethrin. Since the complex banding patterns of 1D-gel electrophoresis were difficult to interpret the significance of the protein difference between IMR-PSS and IMR-LS strain, hence LC-MS/MS analysis is ideally suited for better protein resolution and thus will allow more in-depth comparison of the complex pattern. The findings here provide the first preliminary evidence that insecticide resistance in mosquito induces up regulation of proteins that may be protective to mosquitoes against insecticide and proteins could be another mechanism that contributes to development of resistance.
Routine surveillance on resistant status of field mosquito populations is important to implement suitable strategies in order to prevent pest outbreaks. WHO test kit bioassay is the most frequent bioassay used to investigate the susceptibility status of field-collected mosquitoes, as it is relatively convenient to be carried out in the field. In contrast, the topical application of active ingredient is less popular in investigating the susceptibility status of mosquitoes. In this study, we accessed the susceptibility status of Aedes albopictus Skuse collected from two dengue hotspots on Penang Island: Sungai Dua and Persiaran Mayang Pasir. Two active ingredients: permethrin and deltamethrin, were used. WHO test kit bioassay showed that both wild strains collected were susceptible to the two active ingredients; while topical application assay showed that they were resistant. This indicated that WHO test kit bioassay less sensitive to low level of resistance compared to topical application assay. Hence, topical application is expected to be more indicative when used in a resistance surveillance programme.
Toxicities of three pyrethroids, d-phenothrin, decamethrin, and permethrin, were evaluated in the laboratory against Leptotrombidium fletcheri (Womersley & Heaslip). The susceptibilities between populations of the species infected and noninfected with scrub typhus were investigated. The three pesticides exhibited different toxicities to the chiggers. D- phenothrin was the most toxic, followed by decamethrin, then permethrin. There were no significant differences between susceptibilities of the infected and noninfected populations. Log-probit regression lines indicated that the species was most sensitive to increasing concentrations of d-phenothrin and least sensitive to permethrin. The results show that the three pesticides are potential candidates for chemical control of L. fletcheri. It may be possible in the future to conduct similar bioassays only with the noninfected population, thus reducing risk of infection to workers conducting the bioassays. Similarly, there may not be a need to separate field-collected chiggers into the two populations before performing the bioassays.
Three pyrethroids were evaluated in the laboratory against Aedes albopictus females by exposure to insecticide impregnated papers, and to 4th instar Ae. albopictus larvae as insecticide solutions. Lambda-cyhalothrin was found to be the most effective pyrethroid when tested against Aedes albopictus adult females and larvae compared with that of deltamethrin and permethrin.
Studies on persistence, mobility and the effect of repeated application of permethrin on its half-life were carried out under field conditions. The half-life of permethrin in the top 20 cm of the soil increased from 11.5 to 23.6 days as the application rates increased from 35 to 140 g ha(-1). Induced by heavier rainfall, more residues moved downward in trial 2 than in trial 1. Repeated applications enhanced degradation rates and mobility of permethrin in the soil. The residue level in the 0-5-cm layer was reduced at day 28 after 17 consecutive applications to a level lower than after 5 applications. The half-life of permethrin was reduced from 15.9 days to 11.2 days after 5 and 17 applications, respectively. The residue reached the 15-20 cm layer much earlier (approximately 3 days after treatment) in soil that received 17 applications as compared to those with two applications.
The resistance status towards permethrin among the laboratory strain, the permethrin-selected strain and four field strains of Culex quinquefasciatus collected in Kuala Lumpur, Malaysia was determined using three standard laboratory methods: WHO larval bioassay, WHO adult bioassay and biochemical microplate assay. Cx. quinquefasciatus permethrin-selected strain larvae were the least susceptible to permethrin with a resistance ratio of 47.28-folds, whereas all field strain larvae of the same species were tolerant to permethrin with resistance ratios of more than 3-folds. In contrast, in adult stage, the permethrin exposed permethrin-selected strain (resistance ratio = 1.27) was found to be more susceptible to permethrin than all permethrin-exposed field strains (resistance ratios = 2.23-2.48). Complete mortalities for all strains of Cx. quinquefasciatus adults proved the effectiveness of the synergist; piperonyl butoxide (PBO). For the biochemical microplate assay, the reduction of the mean optical density of elevated oxidase activity of three field strains upon exposure to PBO confirmed the association between oxidase activity and permethrin tolerance. On the other hand, irregular patterns of the mean optical density of elevated oxidase activity in the laboratory strain, permethrin-selected strain and Jalan Fletcher strain illustrated the gene variation within these mosquito colonies as well as the involvement of other enzyme activities in the permethrin resistance occurred.
The characteristics of a potentiometric biosensor for the determination of permethrin in treated wood based on immobilised cells of the fungus Lentinus sajor-caju on a potentiometric transducer are reported this paper. The potentiometric biosensor was prepared by immobilisation of the fungus in alginate gel deposited on a pH-sensitive transducer employing a photocurable acrylic matrix. The biosensor gave a good response in detecting permethrin over the range of 1.0-100.0 µM. The slope of the calibration curve was 56.10 mV/decade with detection limit of 1.00 µM. The relative standard deviation for the sensor reproducibility was 4.86%. The response time of the sensor was 5 min at optimum pH 8.0 with 1.00 mg/electrode of fungus L. sajor-caju. The permethrin biosensor performance was compared with the conventional method for permethrin analysis using high performance liquid chromatography (HPLC), and the analytical results agreed well with the HPLC method (at 95% confidence limit). There was no interference from commonly used organophosphorus pesticides such as diazinon, parathion, paraoxon, and methyl parathion.
Insecticide resistance has become a serious issue in vector management programs. Information on insecticidal resistance and its associated mechanisms is important for successful insecticide resistance management. The selection of a colony of permethrin-resistant Aedes albopictus (Skuse) (Diptera: Culicidae), originating from Penang Island, Malaysia, yielded high larval-specific resistance to permethrin and cross-resistance to deltamethrin. Synergism assays showed that the major mechanism underlying this resistance involves cytochrome P450 monooxygenase. The resistance is autosomal, polygenically inherited and incompletely dominant (D = 0.26). Resistant larvae were reared under different conditions to assess the fitness costs. Under high larval density, larval development time of the resistant SGI strain was significantly longer than the susceptible VCRU strain. In both high- and low-density conditions SGI showed a lower rate of emergence and survival compared with the VCRU strain. Resistant larvae were more susceptible to predation by Toxorhynchites splendens (Wiedemann) (Diptera: Culicidae) larvae. The body size of SGI females reared under high-density conditions was larger compared with females of the susceptible strain. SGI females survived longer when starved than did VCRU females. The energy reserve upon eclosion was positively correlated with the size of the adults.
The adsorption, desorption, and mobility of permethrin in six tropical soils was determined under laboratory and greenhouse conditions. The six soils were selected from vegetable growing areas in Malaysia. Soil organic matter (OM) was positively correlated (r2 = 0.97) with the adsorption of permethrin. The two soils, namely, Teringkap 1 and Lating series with the highest OM (3.2 and 2.9%) released 32.5 and 30.8% of the adsorbed permethrin after four consecutive repetitions of the desorption process, respectively, compared to approximately 75.4% of the Gunung Berinchang soil with the lowest OM (1.0%) under the same conditions. The mobility of permethrin down the soil column was inversely correlated to the organic matter content of the soil. Permethrin residue penetrated only to the 10-15 cm zone in the Teringkap 1 soil with 3.2% OM but penetrated to a depth of more than 20 cm in the other soils. The Berinchang series soil with the lowest OM (1.0%) yielded leachate with 14.8% permethrin, the highest level in leachates from all the soils tested. Therefore, the possibility for permethrin to contaminate underground water may be greater in the presence of low organic matter content, which subsequently allows a higher percentage of permethrin to move downwards through the soil column.
Insecticide-based vector control approaches are facing challenges due to the development of resistance in vector mosquitoes. Therefore, a proper resistance surveillance program using baseline lethal concentrations is crucial for resistance management strategies. Currently, the World Health Organization's (WHO) diagnostic doses established for Aedes aegypti and Anopheles species are being used to study the resistance status of Aedes albopictus. In this study, we established the diagnostic doses for permethrin, deltamethrin, and malathion using a known susceptible reference strain. Five field-collected populations were screened against these doses, following the WHO protocol. This study established the diagnostic dose of malathion at 2.4%, permethrin at 0.95%, and deltamethrin at 0.28%, which differ from the WHO doses for Aedes aegypti and Anopheles spp. Among the insecticides tested on the 5 wild populations, only deltamethrin showed high effectiveness. Different susceptibility and resistance patterns were observed with permethrin, malathion, and dichloro-diphenyl-trichloroethane (DDT) at 4%. This study may assist the health authorities to improve future chemical-based vector control operations in dengue-endemic areas.
A 14-months survey was carried out to identify the species composition of Anopheles mosquitoes from Kampung Bongor, Grik, Perak. Adding to that, a preliminary one month mosquito population screening was done at Kampung Tepin, Serian, Sarawak. Consequently, the insecticide susceptibility status of a pyrethroid was tested against two selected species of Anopheles collected from these two locations in Malaysia. A total of 4,497 Anopheles from 11 species were identified from collections in Kampung Bongor, whereas 2,654 An. letifer were collected from Kampung Tepin. The An. maculatus of Kampung Bongor and An. letifer of Kampung Tepin were then selected and tested using WHO standard diagnostic test kits and impregnated papers with 0.75% permethrin. The response values of KT50 and KT95 for An. maculatus were recorded at 28.09 minutes and 62.98 minutes respectively. Anopheles letifer recorded much slower response values of KT50 and KT95, which was at 35.09 minutes and 73.03 minutes respectively. Both An. maculatus and An. letifer showed 100% mortality after 24 hours holding period. The results indicate that both species were still susceptible to the tested pyrethroid. For effective vector control and resistance management, accurate and periodic insecticide resistance monitoring should be undertaken especially in rural areas with agricultural usage of insecticides.
Larvae and adults of Culex quinquefasciatus were used for the test undertaken for malathion resistant strain (F61 - F65) and permethrin resistant strain (F54 - F58). The results showed that the LC50 for both malathion (F61 - F65) and permethrin (F54 - F58) resistant Cx. quinquefasciatus increased steadily throughout the subsequent five generations, indicating a marked development of resistance. The adult female malathion resistant strain have developed a high resistance level to malathion diagnostic dosage with a resistance ratio of 9.3 to 17.9 folds of resistance compared with the susceptible Cx. quinquefasciatus. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developed at a higher rate in adult females compared to permethrin. Enzyme-based metabolic mechanisms of insecticide resistance were investigated based on the biochemical assay principle. From the results obtained obviously shows that there is a significant difference (p < 0.05) in esterase level in both malathion and permethrin selected strains. Female malathion selected strain has the higher level of esterase activity compared to the female permethrin selected strain at (0.8 to 1.04) alpha-Na micromol/min/mg protein versus (0.15 to 0.24) alpha-Na micromol/min/mg protein respectively. This indicated increased level of non-specific esterase is playing an important role in resistance mechanism in female malathion selected strain. Permethrin selected strain exhibited non-specific esterase activity at a very low level throughout the different life stages compared to malathion selected strain. This study suggests that life stages play a predominant role in conferring malathion and permethrin resistance in Cx. quinquefasciatus.
Laboratory-bred females of Culex quinquefasciatus, Aedes aegypti and Aedes albopictus from the insectarium, Unit of Medical Entomology, Institute for Medical Research were used in the experiment. The late third stage of the F0 larvae which survived the high selection pressure of malathion, permethrin and temephos were reared and colonies were established from adults that emerged. Cx. quinquefasciatus larvae were subjected to selection by malathion and permethrin for 40 generations, Ae. aegypti larvae to malathion, permethrin and temephos for 32 generations and Ae. albopictus larvae were selected against malathion and permethrin for 32 generations and 20 generations against temephos. The rate of resistance development was measured by LC50 value. Cx. quinquefasciatus larvae developed higher resistance to malathion and permethrin compared to Ae. aegypti and Ae. albopictus. On the whole, permethrin resistance developed at a faster rate than malathion and temephos.
To determine resistance level and characterize malathion and permethrin resistance in Culex quinquefasciatus, two methods were used namely: WHO procedures of larval bioassay to determine the susceptibility of lethal concentration (LC) and adult bioassay to determine the lethal time (LT) which are resistant to malathion and permethrin. These mosquito strains were bred in the Insectarium, Division of Medical Entomology, IMR. Thousands of late fourth instar larvae which survived the selection pressure to yield 50% mortality of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected to the subsequent 10 generations in the test undertaken for malathion resistant strain (F61 - F70) and permethrin resistant strain (F54 - F63). Selection pressure at 50% - 70% mortality level was applied to the larvae of each successive generation. The rate of resistance development and resistance ratio (RR) were calculated by LC5 0 for larval bioassay and LT50 value for adult bioassay. The lab bred Cx. quinquefasciatus was used as a susceptible strain for comparison purpose. The adult bioassay test was carried out by using diagnostic dosages of malathion 5.0%, permethrin 0.75% and with propoxur 0.1%. All bioassay results were subjected to probit analysis. The results showed that LC5 0 for both malathion (F61 - F70) and permethrin (F54 - F63) resistant Cx. quinquefasciatus increased steadily to the subsequent 10 generations indicating a marked development of resistance. The adult female malathion resistant strain have developed high resistance level to malathion diagnostic dosage with resistance ratio 9.3 to 9.6 folds of resistance. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developing at a higher rate in adult females compared to permethrin. Female adults exposed to 2 hours of exposure period for propoxur 0.1% showed presence of cross-resistance among the both strains of mosquitoes towards propoxur and it was indicated by 70%-100% mortality at 24 hours post-recovery period.
Observational and survey methodologies were used to probe human behavioral factors influencing the use of insecticide-impregnated bednets to control malaria in rural Sabah, Malaysia. One aim was to investigate why a field trial of such nets in an interior area yielded disappointing results. A second aim was to gather baseline data prior to a field trial proposed for a coastal area. Interior villagers reported a significantly higher net usage rate than that observed directly, suggesting that subject self-reports need to be validated in some way. The poor results of the interior field trial appeared related to reluctance to regularly use nets, which were not in wide use previously. Prospects for reducing malaria transmission through bednets appeared better for the coastal area since nearly half of observed villagers were sleeping in them. However, significantly more coastal than interior villagers were observed watching television at night, an activity that may increase malaria risk by keeping villagers awake and out of bednets.
Field tests were conducted to compare the degree of protection from bites of Mansonia species and Anopheles maculatus by applying two repellent/insecticidal bars, MOSBAR and MOSKIL, to exposed arms and legs. Human test subjects were exposed to natural populations of mosquitos for an 8-hour night time period while using the repellent/insecticidal bars. MOSBAR gave good protection against the bites of Mansonia and An. maculatus. MOSKIL was effective against An. maculatus but not against Mansonia. High mortality was observed among the mosquitos collected from human test subjects treated with the repellent/insecticidal bars. Use of MOSBAR in terms of cost-effectiveness and safety by field and health workers entering into malaria and filariasis endemic areas is discussed.
Vector control is still the principal method to control dengue and chemical insecticides, especially the
pyrethroids such as permethrin are the forerunners of mosquito control agent. Intensive and extensive use
of pyrethroids often result in resistance, thereby hampering control efforts. The present study was
conducted to evaluate the susceptible status of Aedes aegypti, the primary vector of dengue against
permethrin. A nationwide mosquito sampling via ovitrapping was conducted in 12 dengue hotspots across 5
states in Peninsular Malaysia. Field collected Aedes eggs were hatched and reared until L3 larval and further
identified it species. Adult F0 Aedes aegypti were reared until F1 progeny and the female were used in
adult assay, performed according to World Health Organization (WHO) protocol as to determine the
resistance level. The laboratory strain maintained for more than 1000 generations that were susceptible to
permethrin served as the control strain. Evaluation of resistance ratio was assessed by comparing the
knockdown rate with laboratory susceptible strain. In this present study, 70% ofAe. aegypti population from
dengue hotspots was highly resistance to permethrin. The study clearly demonstrated that widespread of
permethrin resistant Ae. aegypti in Malaysian mosquito’s population, indicating the need of implementing
an efficient pyrethroid resistance management.