Comparative DDT-susceptibility status and glutathion s-transferase (GST) activity of Malaysian Anopheles maculatus, Culex quinquefasciatus and Aedes aegypti was investigated to ascertain the role of this enzyme in DDT resistance. The standardised WHO dose-mortality bioassay tests were used to determine DDT susceptibility in these mosquitos, whilst GST microassay (Brogdon and Barber, 1990) was conducted to measure the activity of this enzyme in mosquito homogenate. It appeared that DDT susceptibility status of Malaysian mosquitos was not correlated with GST activity.
Studies were carried out on the residual efficacy of Bacillus thuringiensis H-14 (water dispersible granule, VectoBac ABG 6511) as direct application in the control of Aedes larvae in the field. Field Aedes sp populations in the earthen and glass jars were predetermined before initiation of the trial. On confirmation of the presence of Aedes species in the designated area, Sungai Nibong Kecil, Penang Island, Malaysia, Bti was introduced in the 55L earthen and 3L glass jars). Two test designs were carried out. The first design had treated water replenished daily with 6L of seasoned water and the second design is without the replenishment of water but evaporated water was replenished. Bti was effective in the field for at least 35 days with more than 80% reduction in the Aedes larvae in the treated containers. For earthen jars with daily replenishment of water, 100% reduction was recorded for the first 3 days, while more than 80% reduction was recorded up to day 40. At day 60, Bti still provided an efficacy of 54.32 +/- 4.61 (%) of reduction. Whilst for earthen jars without daily replenishment of water, 100% reduction was recorded for the first 5 days, while more than 80% of reduction was recorded up to day 40. For the glass jars studied, similar efficacy was observed. In jars with daily replenishment of water a better larval control was observed. Percentage of reduction from day 50 to 60 for replenishment of water was between 50 to 70% compared to without replenishment of water with less than 40%.
Laboratory efficacy and residual activity of a water dispersible granule formulation of Bacillus thuringiensis israelensis (Bti) at the dosages of 3000, 6000 and 15000 ITU/L were conducted in this study. The study was conducted in two different size containers, earthen jar (45 L) and glass jar (3 L) with or without daily replenishment of 6 L and 0.3 L of water in the earthen and glass jars, respectively. Results indicate that for both earthen jar and glass jar evaluations, Bti at the tested dosages, performed effectively against Aedes aegypti, giving a minimum of 42 days effective killing activity. When the dosage was increased from 3000 ITU/L to 6000 ITU/L or 15000 ITU/L, the effective periods of the Bti increased by an additional one to three weeks. The Bti water dispersible granule provided better larvicidal activity with replenishment of water compared with non-replenishment of water especially for the higher dosage (15000 ITU/L).
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.
Seasonal population of the fruit-piercing moths Eudocima spp. was monitored throughout the citrus growing seasons in a citrus orchard and in site adjacent to secondary forest from July 2007 to June 2009. The moth was detected practically throughout the year with activity lowest during the wet months (September-February) when fruits are still available and while highest during the dry months (May-June) which also coincided with the main fruiting season. The effects of an nC24 horticultural mineral oil (HMO) on the citrus fruit damage caused by fruit-piecing moths was also determined. The percent fruit damage was significantly lowest (P≤0.05) in HMO-treated plots (8.4), followed by Dimethoate-treated plots (11.6) and untreated plots (22.5). However, there was no significant difference between HMO and Dimethoate treated plots indicating HMO is effective in reducing percent fruit damage.
This study examined the presence of insecticide resistance in different developmental stages (adults, first instars, and eggs) of the tropical bed bug, Cimex hemipterus (F.) using several insecticide formulations. Adults and first instars of five strains (Queensland, Kuala Lumpur, Bukit Mertajam, Saujana, and Krystal Point) were evaluated using the surface contact method and compared with a susceptible strain (Monheim) of the common bed bug Cimex lectularius L. The insecticide formulations were used at their label rates in this study: Tandem (thiamethoxam [11.6%], lambda-cyhalothrin [3.5%]) at 183.96 mg/m2; Temprid SC (imidacloprid [21%], beta-cyfluthrin [10.5%]) at 106.13 mg/m2; Sumithion 20CS (fenitrothion [20%]) at 250 mg/m2; Pesguard FG161 (d-tetramethrin [4.4%], cyphenothrin [13.2%]) at 110 mg/m2; and Sumithrin 10SEC (d-phenothrin [10%]) at 100 mg/m2. Results showed a very high level of resistance to Pesguard FG161 (388.3 to >605.0 times) and Sumithrin (302.9 to >365.5 times) in all adults of the strains tested, whereas low to high levels of resistance were registered for Tandem (1.4-4.7 times), Temprid (7.3-16.7 times), and Sumithion (1.2-14.6 times) for adults of all bed bug strains. For first instars, resistance to the former two formulations were high to very high (31.4-118.1 times). In contrast, they showed lower resistance to Tandem, Temprid, and Sumithion (1.0-10.2 times). An immersion method used to test on bed bug eggs found high to very high resistance toward all tested formulations. Results demonstrate that the resistance level varies between bed bug developmental stages.
Two highly active synthetic pyrethroid insecticides, lambdacyhalothrin and cypermethrin, were evaluated as thermal fogs against houseflies (Musca domestica Linnaeus) and mosquitos (Aedes aegypti Linnaeus). Lambdacyhalothrin (OMS 3021) showed an average of 2.5 times more knockdown activity and over 5 times more adulticidal activity than cypermethrin against Musca domestica and Aedes aegypti. These results demonstrate that lambdacyhalothrin is highly effective at very low rates as a thermal fog against Ae. aegypti and M. domestica. Commercially available formulations of 2.5% and 5% lambdacyhalothrin can be diluted either with water for ULV cold aerosol space-spraying or with diesel/kerosene for thermal fogging at recommended application rates of 0.5-1 g ai/ha for mosquito control and 2 g ai/ha for housefly control. Due to the very low rates of application, formulated products of lambdacyhalothrin are unlikely to present any acute hazards in normal use. The low dosages required to bring about rapid control of houseflies and mosquitos make this new pyrethroid insecticide particularly cost-effective. Coupled with its good residual activity (Jutsum et al, 1984), lambdacyhalothrin can be adopted as a powerful tool in integrated pest management program for the control of medically important pests and vectors.
Two juvenile hormone analogs (JHAs), pyriproxyfen and S-methoprene, were impregnated into dried tuna fish and fed to colonies of Monomorium pharaonis (L.) at very low concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 microg/ml). Its effects on the production of sexuals and colonial growth were observed. Colonies treated with pyriproxyfen yielded sexuals with physical abnormalities. Both female and male sexuals developed bulbous wings, decreased melanization, and died shortly after emergence. Sexuals emerged from colonies treated with S-methoprene did not possess anomalous characteristics. Both pyriproxyfen and S-methoprene did not have significant effects on colonial growth because of the low concentrations of the baits. A commercial bait containing 0.3% S-methoprene (Bioprene-BM) also was evaluated for its efficacy on Pharaoh's ant colonies. Results showed that Pharaoh's ant colonies succumbed to the lethal effects of S-methoprene. Colony members were reduced significantly. Production of queens also decreased significantly in treated colonies and treated queens were unable to lay eggs. JHAs are slow acting and eliminate ant colonies at a relatively slow rate. At low concentrations, pyriproxyfen recorded baffling results, i.e., bulbous wings and demelanized exoskeleton, and it is vital that further studies are initiated to solidify these findings.
Space spraying of chemical insecticides is still an important mean of controlling Aedes mosquitoes and dengue transmission. For this purpose, the bioefficacy of space-sprayed chemical insecticide should be evaluated from time to time. A simulation field trial was conducted outdoor in an open field and indoor in unoccupied flat units in Kuala Lumpur, to evaluate the adulticidal and larvicidal effects of Sumithion L-40, a ULV formulation of fenitrothion. A thermal fogger with a discharge rate of 240 ml/min was used to disperse Sumithion L-40 at 3 different dosages (350 ml/ha, 500 ml/ha, 750 ml/ha) against lab-bred larvae and adult female Aedes aegypti and Aedes albopictus. An average of more than 80% adult mortality was achieved for outdoor space spray, and 100% adult mortality for indoor space spray, in all tested dosages. Outdoor larvicidal effect was noted up to 14 days and 7 days at a dosage of 500 and 750 ml/ha for Ae. aegypti and Ae. albopictus, respectively. Indoor larvicidal effect was up to 21 days (500 ml/ha) and 14 days (750 ml/ha), respectively, after spraying with larval mortality > 50% against Ae. aegypti. This study concluded that the effective dosage of Sumithion L-40 thermally applied against adult Ae. aegypti and Ae. albopictus indoor and outdoor is 500 and 750 ml/ha. Based on these dosages, effective indoor spray volume is 0.4 - 0.6 ml/m³. Additional indoor and outdoor larvicidal effect will be observed at these application dosages, in addition to adult mortality.
The susceptibility status of field-collected Aedes aegypti (L.) from a dengue endemic area to Bacillus thuringiensis israelensis (Bti) and temephos was determined. Since August 2007, biweekly ovitrap surveillance (OS) was conducted for 12 mo in 2 sites, A & B, in Shah Alam, Selangor. Site A was treated with a Bti formulation, VectoBac® WG at 500 g/ha, from December 2007 - June 2008 while Site B was subjected to routine dengue vector control activities conducted by the local municipality. Aedes aegypti larvae collected from OS in both sites were bred until F3 and evaluated for their susceptibility. The larvae were pooled according to 3 time periods, which corresponded to Bti treatment phases in site A: August - November 2007 (Bti pre-treatment phase); December 2007 - June 2008 (Bti treatment phase); and July - September 2008 (Bti post-treatment phase). Larvae were bioassayed against Bti or temephos in accordance with WHO standard methods. Larvae collected from Site A was resistant to temephos, while incipient temephos resistant was detected in Site B throughout the study using WHO diagnostic dosage of 0.02 mg/L. The LC50 of temephos ranged between 0.007040 - 0.03799 mg/L throughout the year in both sites. Resistance ratios (LC50) indicated that temephos resistance increased with time, from 1.2 - 6.7 folds. The LC50 of Ae. aegypti larvae to Bti ranged between 0.08890 - 0.1814 mg/L throughout the year in both sites, showing uniform susceptibility of field larvae to Bti, in spite of Site A receiving 18 Bti treatments over a period of 7 mo. No cross-resistance of Ae. aegypti larvae from temephos to Bti was detected.
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.
Given the lack of molecular evidence in altered target-site insecticide resistance mechanism in Aedes albopictus (Skuse) worldwide, the present study aims to detect the presence of A302S mutation in the gene encoding the gamma aminobutyric acid receptor resistant to dieldrin (Rdl) in Ae. albopictus for the first time from its native range of South East Asia, namely Malaysia. World Health Organization (WHO) adult susceptibility bioassay indicated a relatively low level of dieldrin resistance (two-fold) in Ae. albopictus from Petaling Jaya, Selangor. However, PCR-RFLP and direct sequencing methods revealed the presence of the A302S mutation with the predomination of heterozygous genotype (40 out of 82 individuals), followed by the resistant genotype with 11 individuals. This study represents the first field evolved instance of A302S mutation in Malaysian insect species.
Use of mosquito coils for personal protection against malaria and mosquito nuisance is advocated under mosquito and malaria control programmes. We performed field studies of mosquito coils containing either metofluthrin or esbiothrin in experimental huts situated in Kamhororo village, Gokwe district, Zimbabwe. All tests were performed on 3-5 day old reared female Anopheles gambiae sensu lato mosquitoes. The burning times were 9hr 20min for mosquito coils containing metofluthrin and 8 hr for those containing esbiothrin and the results were significantly different (p = <0.001). The mean knock down rate for mosquito coils containing metofluthrin was 90% and that for esbiothrin was 73.3% and the results were significantly different (p = 0.00). Mosquito coils containing metofluthrin had a mean repellence of 92.7% as compared to 85.4% for esbiothrin and the results were not significantly different (p=0.27). The protection time as required by EPA (1999) was 6 hr for mosquito coils containing metofluthrin and 5 hr for those containing esbiothrin. The mean insecticidal effect of mosquito coils containing metofluthrin was 84% as compared to 83% for those containing esbiothrin and the results were not significantly different (p = 0.56). Both mosquito formulations could not be classified as having insecticidal effect since none of them met the 95% mortality rate criteria.
The bioefficacy of Piper aduncum L. essential oil formulated in aerosol cans was evaluated against Aedes aegypti and Aedes albopictus in a simulated room. The aerosol spray test was based on the Malaysian test standard for aerosol (MS 1221:1991UDC 632.982.2 modified from WHO 2009 methodology) and examined the knockdown effect within 20 minutes of exposure. Mortality rate after 24 hour of holding period was also determined. A commercial aerosol spray (0.09% prallethrin 0.05% d-phenothrin) was also tested as a comparison. Our results showed that the knockdown effect of the commercial aerosol spray and P. aduncum essential oil spray (8% and 10% concentrations) was significantly higher in Ae. albopictus adult females, when compared with that of Ae. aegypti adult females (P<0.05). There was a significant difference in knockdown between commercial aerosol spray and essential oil spray for both Aedes spp. (P<0.05). The essential oil induced significantly higher mortality in Ae. aegypti (80%) than in Ae. albopictus (71.6%) (P<0.05). The commercial aerosol spray caused 97.7% and 86.5% mortality against Ae. aegypti and Ae. albopictus respectively (P<0.05). Based on these data, P. aduncum essential oil has the potential to be used as an aerosol spray against Aedes spp.
Mosquito vectors (Diptera: Culicidae) are responsible for transmission of serious diseases worldwide. Mosquito control is being enhanced in many areas, but there are significant challenges, including increasing resistance to insecticides and lack of alternative, cost-effective, and eco-friendly products. To deal with these crucial issues, recent emphasis has been placed on plant materials with mosquitocidal properties. Furthermore, cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. Nanotechnology is a promising field of research and is expected to give major innovation impulses in a variety of industrial sectors. In this study, we synthesized titanium dioxide (TiO2) nanoparticles using the hydrothermal method. Nanoparticles were subjected to different analysis including UV-Vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), zeta potential, and energy-dispersive spectrometric (EDX). The synthesized TiO2 nanoparticles exhibited dose-dependent cytotoxicity against human breast cancer cells (MCF-7) and normal breast epithelial cells (HBL-100). After 24-h incubation, the inhibitory concentrations (IC50) were found to be 60 and 80 μg/mL on MCF-7 and normal HBL-100 cells, respectively. Induction of apoptosis was evidenced by Acridine Orange (AO)/ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. In larvicidal and pupicidal experiments conducted against the primary dengue mosquito Aedes aegypti, LC50 values of nanoparticles were 4.02 ppm (larva I), 4.962 ppm (larva II), 5.671 ppm (larva III), 6.485 ppm (larva IV), and 7.527 ppm (pupa). Overall, our results suggested that TiO2 nanoparticles may be considered as a safe tool to build newer and safer mosquitocides and chemotherapeutic agents with little systemic toxicity.
Malaria, the most widespread mosquito-borne disease, affects 350-500 million people each year. Eco-friendly control tools against malaria vectors are urgently needed. This research proposed a novel method of plant-mediated synthesis of silver nanoparticles (AgNP) using a cheap seaweed extract of Ulva lactuca, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The U. lactuca extract and the green-synthesized AgNP were tested against larvae and pupae of the malaria vector Anopheles stephensi. In mosquitocidal assays, LC50 values of U. lactuca extract against A. stephensi larvae and pupae were 18.365 ppm (I instar), 23.948 ppm (II), 29.701 ppm (III), 37.517 ppm (IV), and 43.012 ppm (pupae). LC50 values of AgNP against A. stephensi were 2.111 ppm (I), 3.090 ppm (II), 4.629 ppm (III), 5.261 ppm (IV), and 6.860 ppm (pupae). Smoke toxicity experiments conducted against mosquito adults showed that U. lactuca coils evoked mortality rates comparable to the permethrin-based positive control (66, 51, and 41%, respectively). Furthermore, the antiplasmodial activity of U. lactuca extract and U. lactuca-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. Fifty percent inhibitory concentration (IC50) values of U. lactuca were 57.26 μg/ml (CQ-s) and 66.36 μg/ml (CQ-r); U. lactuca-synthesized AgNP IC50 values were 76.33 μg/ml (CQ-s) and 79.13 μg/ml (CQ-r). Overall, our results highlighted out that U. lactuca-synthesized AgNP may be employed to develop newer and safer agents for malaria control.
A main challenge in parasitology is the development of reliable tools to prevent or treat mosquito-borne diseases. We investigated the toxicity of magnetic nanoparticles (MNP) produced by Magnetospirillum gryphiswaldense (strain MSR-1) on chloroquine-resistant (CQ-r) and sensitive (CQ-s) Plasmodium falciparum, dengue virus (DEN-2), and two of their main vectors, Anopheles stephensi and Aedes aegypti, respectively. MNP were studied by Fourier-transform infrared spectroscopy and transmission electron microscopy. They were toxic to larvae and pupae of An. stephensi, LC50 ranged from 2.563 ppm (1st instar larva) to 6.430 ppm (pupa), and Ae. aegypti, LC50 ranged from 3.231 ppm (1st instar larva) to 7.545 ppm (pupa). MNP IC50 on P. falciparum were 83.32 μg ml(-1) (CQ-s) and 87.47 μg ml(-1) (CQ-r). However, the in vivo efficacy of MNP on Plasmodium berghei was low if compared to CQ-based treatments. Moderate cytotoxicity was detected on Vero cells post-treatment with MNP doses lower than 4 μg ml(-1). MNP evaluated at 2-8 μg ml(-1) inhibited DEN-2 replication inhibiting the expression of the envelope (E) protein. In conclusion, our findings represent the first report about the use of MNP in medical and veterinary entomology, proposing them as suitable materials to develop reliable tools to combat mosquito-borne diseases.
We constructed recombinant phage particles displaying the Bacillus thuringiensis Cry1Ba4 active toxin using the pfUSE5 and pComb3X phagemid vectors. The recombinant phage particles were screened and evaluated for displayed biologically active Cry1Ba4 toxin against the target insect larvae. Concurrent expression of Cry1Ba4 protoxin was carried out using the pETBlue -2 plasmid expression vector in Escherichia coli Tuner (DE3)pLacI and the protoxin was successfully expressed at a size of 129 kDa. In the bioassay, 3.30 mg crude extract of Cry1Ba4 protoxin, 9.35 x 10(9) TU and 7.70 x 10(9) TU of induced recombinant phage particles carrying Cry1Ba4 active toxin displayed on pComb3X and pFUSE5, respectively, demonstrated mortality of greater than 85% against Plutella xylostella (third-instar) within 48 hours. Thus, we have successfully displayed the Cry1Ba4 activated toxin on the surface of a phage and demonstrated toxicity towards larvae.
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.
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.