The resistance status of riceland Culex vishnui against four major groups of insecticides (i.e., organochlorines, carbamates, organophosphates and pyrethroids) was investigated. Biochemical assays (ESTα, ESTβ, MFO and GST) were also conducted to detect the resistance levels. Throughout a 12-month study period, multiple insecticide resistance was observed in both larvae and adult Cx vishnui. Culex vishnui larvae exhibited low resistance against malathion, temephos and permethrin with resistance ratio (RR) values < 5. In adult bioassay, Cx. vishnui were highly resistant against all tested adulticides with 24h post-treatment mortality < 70%. Correlations between permethrin and malathion resistance, as well as between deltamethrin and cyfluthrin resistance were found in Cx. vishnui. The results indicated that mixed function oxidases activity of Cx. vishnui was the highest compared to ESTα, ESTβ and GST. Spearman rank-order analysis showed that ESTα, ESTβ and GST were involved in multiple resistances in Cx. vishnui. The findings of this study established a baseline of insecticide susceptibility and revealed the effects of agricultural insecticide pressure on the vectors of Japanese encephalitis in Malaysia.
Specification on residual action of a possible alternative insecticide derived from plant materials is important to determine minimum interval time between applications and the environmental persistence of the biopesticides. The objective of this study is to evaluate crude acethonilic extract of Ipomoea cairica leaves for its residual and persistence effects against Culex quinquefasciatus larvae. Wild strain of Cx. quinquefasciatus larvae were used for the purpose of the study. Two test designs, replenishment of water and without replenishment of water were carried out. For the first design, a total of 10 ml of test solution containing Ip. cairica extracts was replenished daily and replaced with 10 ml of distilled water. For the second design, treatment water was maintained at 1500 ml and only evaporated water was refilled. Larval mortality was recorded at 24 hours post-treatment after each introduction period and trials were terminated when mortality rate falls below 50%. Adult emergences from survived larvae were observed and number of survivals was recorded. For the non-replenishment design, mortality rate significantly reduced to below 50% after 28 days, meanwhile for replenishment of water declined significantly after 21 days (P < 0.05). There was no adult emergence observed up to seven days for non-replenishment and first two days for replenishment of water design. The short period of residual effectiveness of crude acethonilic extract of Ip. cairica leaves with high percentage of larval mortality on the first few days, endorses fewer concerns of having excess residues in the environment which may carry the risk of insecticide resistance and environmental pollution.
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.
The standard laboratory strain was found to be heterozygous for susceptibility. Hence, an attempt was made to obtain a homozygous susceptible strain in Culex quinquefasciatus (Say) using single raft sib-selection method. Lab-bred females of Cx. quinquefasciatus from insectariums, Unit of Medical Entomology were used in the experiment. After blood feeding Cx. quinquefasciatus mosquitoes laid eggs in raft form, ten rafts selected randomly for the test. Each egg raft was introduced into a plastic tray from number one to number ten. Twenty-five third stage larvae from each tray were exposed to 17.5 microl from 500mg/l malathion in a paper cup label number 1 to number ten. In the bioassay, which had 100% mortality, the respective larva in that particular tray was bred to adult stage for the following generation. Less than 7days old female mosquitoes that emerged from F(0) were used in the test. The F(0) and the subsequent adult and larval stage generations were subjected to adult and larval bioassay. After selection for about 10 generations, a homozygous susceptible strain in Cx. quinquefasciatus was obtained.
The susceptibility of Culex quinquefasciatus to chemical insecticides in two field sites in Kuala Lumpur was evaluated using the WHO standard susceptibility test. Less then 7 days old female mosquitos, reared from wild caught females were exposed to discriminating dosages of insecticides at recommended exposure periods. The larval bioassay were conducted using the multiple concentrations and the LC50 value was determined. The results indicated that cyfluthrin is the most effective among all the insecticides tested with LT50 value of 29.95 min and 28.59 min, for the strain from Ampang Hill and Pantai Dalam, respectively. It was surprisingly to note that both these field strains showed 0% mortality when tested against malathion and DDT. The LC50 value indicated that both strains were highly resistant to malathion with resistance ratio of 17,988 folds and 14,053 folds, respectively. This concludes that resistance at larval stages is extremely high compared to adult stages.
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.
The toxic effects of Abate (temephos) on mosquito larvae and non-target organisms were studied in the rice-field and in the laboratory. In the laboratory tests, Culex tritaeniorhychus larvae and cladoceran zooplanktons (predominantly Diaphanosoma and Moinodaphnia species) were found to be highly susceptible to Abate with LC50 values of 0.27 and less than 0.10 parts per billion respectively. Other non-target species in decreasing degree of susceptibility to Abate were copepods (Tropodiaptomus spp.), Aplocheilus panchax and Tubifex worms. In field study, Abate at concentrations 60, 100 and 200 gm hectare-1 is effective in maintaining the rice-fields free of Anopheles and Culex mosquitoes for at least 2 days. No mortality was observed for Aplocheilus panchax and Tubifex worms at the above concentrations; nevertheless, populations of cladoceran zooplanktons and copepods were reduced up to seven days posttreatment.
The efficacy of the larvicidal and pupicidal agent (Agnique) MMF was evaluated against larvae of An. arabiensis and Culex (Diptera: Culicidae) under field conditions in Bahary Locality, Khartoum, Sudan. At an applied dosage of 0.25 ml/m2, MMF resulted in 89.4, 79.8 and 88.2% reductions in L3-L4 instars An. arabiensis and 63.5% in Culex larvae (all stages) 24 to 72 hours post-treatment. Pupae were completely eliminated (100%) within 24 hours posttreatment. The earlier instars (L1-L2) of An. arabiensis were more tolerant with a 62.5% reduction at 72 hours post-treatment compared to (L3-L4) instars and pupae. At 7-days post-treatment Agnique gave a 57.5% reduction in L1-L2 and 92.6% in L3-L4 instar larvae of An. arabiensis and 57.3% and 86.4% in Culex larvae and pupae, respectively. We conclude that Agnique can perform effectively against L3-L4 instars and pupae of An. arabiensis for only 1 week, and 3 to 4 days against L1-L2 instars of Culex spp.
Wild caught female Culex quinquefasciatus (Say) from Kuala Lumpur were blood fed and reared in the insectarium. The late third stage of the F1 larvae which survived the high selection pressure of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected in the subsequent 9 generations to higher selection pressure. The rate of resistance development were measured by LC50 value of larval bioassay, LT50 value of adult bioassay and the frequency of the elevated esterase levels. In another set of experiments using the same batch of Culex mosquitos, the larvae were not exposed to any insecticides and the decrease in resistance rate was monitored in each subsequent 9 generations by using similar methods. The heterozygous standard laboratory strain was selected for susceptibility using the single raft sib-selection method. The result showed that the field collected F1 generation was 96.0 and 6.3 fold more resistant to malathion and permethrin, respectively. After selection for about 9 generations the resistance ratio to malathion and permethrin was 6.2 and 767.3 fold more compared to the LC50 values of F1 generations, respectively. Esterase in F1 larvae was 6.0 fold more than the standard laboratory strain.
Given that there is limited available information on the insensitive acetylcholinesterase in insect species in Malaysia, the present study aims to detect the presence of G119S mutation in the acetylcholinesterase gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia.
Vegetative proteins from Malaysian strains of Bacillus thuringiensis israelensis strains (Bt 11, Bt 12, Bt 15, Bt 16, Bt 17, Bt 21 and Bt 22) and Bacillus sphaericus H-25 strains (Bs 1 and Bs 2) were screened for haemolytic, cytotoxic and larvicidal activity. SDS-PAGE profiles of the Bacillus thuringiensis strains studied consistently showed major bands of 33-37 kDa and 47 kDa. Bt 16 also showed two bands of 66 kDa and 45 kDa similar to the previously reported binary vegetative protein, Vip1Ac (66 kDa) and Vip 2Ac (45 kDa). Both the Bacillus sphaericus strains showed a 35 kDa band that was similiar to a previously reported vegetative protein, the Mtx2 protein. Bs 2 also contains a 37 kDa band, similar to another vegetative protein, the Mtx 3 protein. With the exception of Bt 17 and Bt 21, vegetative proteins from all Bacillus thuringiensis and Bacillus sphaericus strains were highly haemolytic to human erythrocytes, causing more than 75% haemolysis at the highest concentration of 200 microg/ml. High haemolytic activity was associated with high cytotoxic activity with most of the haemolytic strains being indiscriminately cytotoxic to both CEM-SS (human T lymphoblastoid) and HeLa (human uterus cervical cancer) cell lines. Interestingly, the less haemolytic vegetative proteins from Bt 17 and Bt 21 demonstrated cytotoxic activity comparable to that of the highly haemolytic vegetative proteins. Bt 21 displayed toxicity towards both cell lines while Bt 17 was more toxic towards CEM-SS cells. Bioassay against Aedes aegypti and Culex quinquefasciatus larvae revealed that vegetative proteins from the Bacillus thuringiensis strains had activity against both species of larvae but vegetative proteins from Bacillus sphaericus were weakly larvicidal towards Cx. quinquefasciatus only.
This study focuses on the larvicidal, oviposition, and ovicidal effects of a crude extract of Artemisia annua against Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus. Dried cells of Artemisia annua from cell suspension cultures were extracted using hexane. The extract showed moderate larvicidal effects against mosquitoes. At 24-h post treatment, the LC50 values for Anopheles sinensis, Aedes aegypti, and Culex quinquefasciatus were recorded as 244.55, 276.14, and 374.99 ppm, respectively. The percentage mortality of larvae was directly proportional to the tested concentration. Anopheles sinensis was found to be the most susceptible species, whereas Culex quinquefasciatus was the most tolerant to the Artemisia annua extract. The results indicated that the Artemisia annua extract showed concentration-dependent oviposition deterrent activity and had a strong deterrent effect. At 500 ppm, the percentage effective repellency was more than 85% compared with the control group for all the species, with oviposition activity index values of -0.94, -0.95, and -0.78 for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. In the ovicidal assay, the percentage hatchability of eggs after treatment with 500 ppm of Artemisia annua extract was significantly lower than the control, with values of 48.84 ± 4.08, 38.42 ± 3.67, and 79.35 ± 2.09% for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. Artemisia annua was found to be more effective against Aedes aegypti and Anopheles sinensis compared with Culex quinquefasciatus. This study indicated that crude extract of A. annua could be a potential alternative for use in vector management programs.
BACKGROUND & OBJECTIVES: Many essential oils have been reported as natural sources of insect repellents; however, due to high volatility, they present low repellent effect. Formulation technique by using microencapsulation enables to control the volatility of essential oil and thereby extends the duration of repellency. In this study, the effectiveness of microencapsulated essential oils of Alpinia galanga, Citrus grandis and C. aurantifolia in the lotion formulations were evaluated against mosquito bites.
METHODS: Essential oils and N,N-Diethyl-3-methylbenzamide (DEET) were encapsulated by using interfacial pre- cipitation techniques before incorporation into lotion base to form microencapsulated (ME) formulation. The pure essential oil and DEET were also prepared into lotion base to produce non-encapsulated (NE) formulation. All the prepared formulations were assessed for their repellent activity against Culex quinquefasciatus under laboratory condition. Field evaluations also were conducted in three different study sites in Peninsular Malaysia. In addi- tion, Citriodiol® (Mosiquard®) and citronella-based repellents (KAPS®, MozAway® and BioZ Natural®) were also included for comparison.
RESULTS: In laboratory conditions, the ME formulations of the essential oils showed no significant difference with regard to the duration of repellent effect compared to the microencapsulated DEET used at the highest con- centration (20%). It exhibited >98% repellent effect for duration of 4 h (p = 0.06). In the field conditions, these formulations demonstrated comparable repellent effect (100% for a duration of 3 h) to Citriodiol® based repellent (Mosiguard®) (p = 0.07). In both test conditions, the ME formulations of the essential oils presented longer duration of 100% repellent effect (between 1 and 2 h) compared to NE formulations.
INTERPRETATION & CONCLUSION: The findings of the study demonstrate that the application of the microencapsulation technique during the preparation of the formulations significantly increases the duration of the repellent effect of the essential oils, suggesting that the ME formulation of essential oils have potential to be commercialized as an alternative plant-based repellent in the market against the mosquitoes.