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.
Detail chemical investigations on the stem bark of Mesua daphnifolia gave three triterpenoids and four xanthones. They are friedelin (1), friedelan-1,3-dione (2), lup-20(29)- en-3ss-ol (3), cudraxanthone G (4), ananixanthone (5), 1,3,5-trihydroxy-4-methoxyxanthone (6) and euxanthone (7). These chemical constituents were tested in vitro for their cytotoxic activities against four cell lines, MDA-MB-231 (human estrogen receptor negative breast cancer), HeLa (cervical carcinoma), CEM-SS (T-lymphoblastic leukemia) and CaOV3 (human ovarian cancer). Compound 4 showed a broad spectrum of activity against the MDA-MB-231, HeLa and CEM-SS cell lines with IC5 0 values of 1.3, 4.0 and 6.7 microg/ml respectively. Meanwhile, the other compounds 1, 2, 3, 5, 6 and 7 gave only selective activities against the cell lines.
The rapid detection of dengue infection in mosquito vectors is important for early warning to forestall an outbreak. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) provides a rapid method for dengue detection in man and mosquitoes. An RT-PCR kit developed by the Medical Entomology Unit, Institute for Medical Research to detect dengue infection in mosquitoes, was tested for its shelf life at 3 storage temperatures: room temperature, refrigerator and freezer. Test kits were tested once every 3 days for kits stored at room temperature, and once every week for those stored at refrigerator and freezer temperatures. The results showed that the test kit could only be stored above its recommended storage temperature of -20 degrees C for not more than 3 days. DNA 100 bp markers in the kits appeared to be stable at the tested temperatures and were usable up to the 20th day when stored at 2 degrees C and below.
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.
Phlebotomine sand flies were collected using CO2 baited CDC light trap in 2000 and 2001 in limestone areas and caves of western Malaysia. A total of 1,548 specimens were collected comprising 18 species from two genera: Phlebotomus (6 spp) and Sergentomyia (12 spp). Phlebotomus major major (38.9%) was the predominant species followed by Sergentomyia perturbans (20.1%), P. stantoni (15.3%) and others. Biting activity of the sand flies at the Gua Senyum caves, Gua Kota Gelanggi, Batu caves and Gua Kelam were observed using the bare leg landing catch (BLC) technique. Four Phlebotomus spp at Gua Senyum were found to bite humans with a unimodal biting peak (between 01:00 and 04:00 AM). At Gua Kota Gelanggi P. major major was observed to bite humans, but at Batu Caves and Gua Kelam no sand flies were observed to bite humans. Sergentomyia spp did not feed on humans even though high numbers were caught in light traps. The populations of phleobotomine sand flies fluctuated, with several peaks especially among P. major major which peaked in December and was low in February and August. Phlebotomus stantoni was abundant throughout 2001. Most species populations were weakly related to rainfall because they inhabited caves.
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.
This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.
A simulated field study on the efficacy of commercial household aerosol insecticides was conducted. The bioefficacy of three pyrethroid aerosols, designated as PA1, PA2 and PA3, was tested in cabins furnished to simulate bedroom conditions. Each aerosol product was tested against lab-bred Aedes aegypti mosquitoes based on the insecticide manufacturers' recommended dosages. Ten cages with mosquitoes were placed in the following locations: one cage in the middle of the room; two each on and underneath the bed; three each placed inside, behind and on top of the wardrobe; and four placed on and in the desk. With the desk, each cage was placed inside each of three drawers (totally closed, partially closed and opened). Prior to the experiments, the discharge rate of each aerosol can was determined. Ten to 20 lab-bred 2-5 day-old sugar-fed Ae. aegypti adult mosquitoes were placed inside the test cages. The aerosol was then discharged into the cabin at the recommended dosage. After 30 minutes, the mosquitoes were transferred into clean paper cups and their mortality recorded after 24 hours. All the aerosols induced complete or very high mortality in the caged Ae. aegypti females, except in the cages hidden completely inside the drawers and wardrobes. Insecticide droplet analysis indicated variable uniformity of the droplets was produced. The aerosol insecticides were effective against mosquitoes provided they were used in accordance with the manufacturers' recommendations.
Photon (light) technology has already been widely used in make-up, medical treatment etc, but repelling mosquitoes by photon technology is an innovation. The objective of this study was to determine the efficacy of a mosquito repelling lamp, E Da under indoor conditions. E Da lamp is a lamp coated with yellow luminous pigment on the inner part of the glass bulb of the lamp which is used to screen out the UV radiation, and when it is turned on, the yellow illuminating wavelength will drive the mosquitoes away. The tests were conducted inside 2 cabins measuring 8' X 8' X 20'. The mosquito population was estimated by using the Bare Leg Catch (BLC) techniques. For treated test, E Da lamp was placed indoor 2 - 3 meters away from a human bait. Another cabin without the lamp was used as untreated control. BLC was conducted in both sites simultaneously. The mosquitoes collected in this study were solely those of Culex quinquefasciatus and Aedes albopictus. There was an 91.34% reduction of Cx. quinquefasciatus population in the treated test compared with the untreated cabin during the 4 hours catches (p < 0.05). E Da mosquito repelling lamp used in this study exerted repellency effect against the mosquitoes especially Cx. quinquefasciatus.
Preservation of larvae retrieved from cadavers is important in ensuring the quality and integrity of entomological specimens used for the estimation of post-mortem interval (PMI). The process of killing and preserving larvae could distort the larvae leading to inaccurate estimation of PMI. In this study, the effects of killing Chrysomya megacephala larvae with hot water at different temperatures and subsequent maintenance in various preservatives were determined. Larvae not killed by hot water but preserved directly were used as control. The types of preservative used were 10% formalin, 70% ethanol and Kahle's solution. The morphological features examined were length, turgidity, curvature and coloration of larvae. Larvae killed in 80ºC hot water have shorter mean length (12.47 ± 2.86 mm) compared to those in 60ºC hot water (12.95 ± 2.69 mm). Increasing the duration of preservation in all types of preservative caused elongations of larvae treated or untreated with hot water. There were no significant changes in larval turgidity preserved in Kahle's solution compared to other two preservatives and were unaffected by the duration of storage. Larvae preserved in Kahle's solution experienced the least changes in coloration and shape compared to other preserved larvae in 70% ethanol or 10% formalin. Larvae directly immersed alive in 70% ethanol experienced the most changes in curvature, coloration and turgidity. This study suggested that killing larvae with hot water at 80ºC and preservation in Kahle's solution is the optimum method resulting in least changes in morphological features of Ch. megacephala larvae.
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.
DNA identification of blow fly species can be a very useful tool in forensic entomology. One of the potential benefits that mitochondrial DNA (mtDNA) has offered in the field of forensic entomology is species determination. Conventional identification methods have limitations for sibling and closely related species of blow fly and stage and quality of the specimen used. This could be overcome by DNA-based identification methods using mitochondrial DNA which does not demand intact or undamaged specimens. Mitochondrial DNA is usually isolated from whole blow fly and legs. Alternate sources for mitochondrial DNA isolation namely, egg, larva, puparium and empty puparium were explored in this study. The sequence of DNA obtained for each sample for every life cycle stage was 100% identical for a particular species, indicating that the egg, 1st instar, 2nd instar, 3rd instar, pupa, empty puparium and adult from the same species and obtained from same generation will exhibit similar DNA sequences. The present study also highlighted the usefulness of collecting all life cycle stages of blow fly during crime scene investigation with proper preservation and subsequent molecular analysis. Molecular identification provides a strong basis for species identification and will prove an invaluable contribution to forensic entomology as an investigative tool in Malaysia.
Estimation of post-mortem interval (PMI) is crucial for time of death determination. The advent of DNA-based identification techniques forensic entomology saw the beginning of a proliferation of molecular studies into forensically important Calliphoridae (Diptera). The use of DNA to characterise morphologically indistinguishable immature calliphorids was recognised as a valuable molecular tool with enormous practical utility. The local entomofauna in most cases is important for the examination of entomological evidences. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different insect species on corpses. In this study, calliphorids were collected from 13 human corpses recovered from indoors, outdoors and aquatic conditions during the post-mortem examination by pathologists from the government hospitals in Malaysia. Only two species, Chrysomya megacephala and Chrysomya rufifacies were recovered from human corpses. DNA sequencing was performed to study the mitochondrial encoded COI gene and to evaluate the suitability of the 1300 base pairs of COI fragments for identification of blow fly species collected from real crime scene. The COI gene from blow fly specimens were sequenced and deposited in GenBank to expand local databases. The sequenced COI gene was useful in identifying calliphorids retrieved from human corpses.
This study was conducted using the glass chamber method to determine the susceptibility status of the dengue vector, Aedes aegypti (L.) from 11 states in Malaysia to commercial mosquito coils containing four different active ingredients, namely metofluthrin, d-allethrin, d-trans allethrin, and prallethrin. Aedes aegypti exhibited various knockdown rates, ranging from 14.44% to 100.00%, 0.00% to 61.67%, 0.00% to 90.00%, and 0.00% to 13.33% for metofluthrin, d-allethrin, d-trans allethrin, and prallethrin, respectively. Overall, mortality rates ranging from 0.00% to 78.33% were also observed among all populations. Additionally, significant associations were detected between the knockdown rates of metofluthrin and d-allethrin, and between metofluthrin and d-trans allethrin, suggesting the occurrence of cross-resistance within pyrethroid insecticides. Overall, this study revealed low insecticidal activity of mosquito coils against Ae. aegypti populations in Malaysia, and consequently may provide minimal personal protection against mosquito bites.
The present study aims to investigate the susceptibility status of Aedes albopictus (Skuse) collected from residential areas in Sabah, Malaysia towards eight WHO-recommended dosages of larvicides representing the classes of organophosphates and organochlorines. Field and reference strains of Ae. albopictus larvae were bioassayed in accordance to WHO standard methods using diagnostic dosages of bromophos, malathion, fenthion, fenitrothion, temephos, chlorpyrifos, dichlorodiphenyltrichloroethane (DDT), and dieldrin. The results revealed that Ae. albopictus was resistant (mortality < 90%) towards malathion, temephos, and DDT. In addition, most of the Ae. albopictus strains exhibited a wide range of susceptibilities against bromophos, with mortality ranged from 49.33 to 93.33%. On the contrary, only dieldrin was able to induce 100% mortality against all strains of Ae. albopictus. Tolerance to fenitrothion, fenthion, and chlorpyrifos, with mortality ranging from 81.33 to 97.33%, was also observed in this study.
In this paper, we will start off by introducing the classical Ross-Macdonald model for vector-borne diseases which we use to describe the transmission of dengue between humans and Aedes mosquitoes in Shah Alam, which is a city and the state capital of Selangor, Malaysia. We will focus on analysing the effect of using the Mosquito Home System (MHS), which is an example of an autodissemination trap, in reducing the number of dengue cases by changing the Ross-Macdonald model. By using the national dengue data from Malaysia, we are able to estimate λ, which represents the initial growth rate of the dengue epidemic, and this allows us to estimate the number of mosquitoes in Malaysia. A mathematical expression is also constructed which allows us to estimate the potential number of breeding sites of Aedes mosquitoes. By using the data available from the MHS trial carried out in Section 15 of Shah Alam, we included the potential effect of the MHS into the dengue model and thus modelled the impact MHS has on the spread of dengue within the trial area. We then extended our results to analyse the effect of the MHSs on reducing the number of dengue cases in the whole of Malaysia. A new model was constructed with a basic reproduction number, R0,MalaMHS, which allows us to identify the required MHSs coverage needed to achieve extinction in Malaysia. Numerical simulations and tables of results were also produced to illustrate our results.
The spraying of insecticide on foliage to provide an insecticidal barrier may serve as part of the vector control measures to combat the increasing threat of Aedes-borne diseases. The effectiveness of insecticide barrier spraying was evaluated by assessing the residual efficacy of deltamethrin sprayed on foliage against Malaysian Ae. aegypti (L.) and Ae. albopictus (Skuse). In this semi-field study, landscape plants grown within the vicinity of the Institute for Medical Research (IMR), Malaysia, were treated with deltamethrin suspension concentrate (SC) with the dosage of 30 mg/m2 and 50 mg/m2 in three rounds of spraying. Deltamethrin residual activity on treated and untreated leaves was investigated using standard WHO cone bioassays. Wild Aedes populations at both deltamethrin-treated and untreated plant clusters were monitored by ovitrap surveillance. Ovitrap monitoring revealed that the mean number of Ae. albopictus larvae at deltamethrin-treated were significantly lower than the mean number of larvae of the same species at the untreated plant cluster. Cone bioassay results showed that the insecticide remained effective for up to 4 weeks (> 80% mortality), but the insecticide residual activity was affected by rainfall. These results suggest that insecticide barrier spraying is a promising tool and may be used along with other mosquito control tools such as indoor residual spray and space spraying to reduce the dengue burden.
The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.
In Malaysia, maggot debridement therapy (MDT) utilizes maggots of Lucilia cuprina (Wiedemann) to debride necrotic tissue from wound surface, reduce bacterial infection and therefore, enhance wound healing process. To evaluate the sterility of the sterile maggots produced after sterilization process before delivering onto patient wounds. Sterility of sterile maggots is crucial in ensuring the safe usage of MDT and patient's health. Eggs of L. cuprina collected from a laboratory colony were divided into treated group (sterilized) and control group (non-sterilized). Treated group underwent sterilization while eggs from control group were allowed to hatch without sterilization. Sodium hypochlorite and formaldehyde were the main disinfectants used in this sterilization process. Scanning electron microscope (SEM) was used to examine and ascertain the sterility of sterile maggots. SEM results showed that all sterilized L. cuprina eggs and maggots achieved sterility and all were cleared from bacterial contamination. In contrast, all non-sterilized eggs and maggots were found to be colonized by microorganisms. Sterilization method employed to sterilize eggs and maggots used in Malaysia MDT was proven successful and MDT is safe to be used as wound management tools.
Early detection of a dengue outbreak is an important first step towards implementing effective dengue interventions resulting in reduced mortality and morbidity. A dengue mathematical model would be useful for the prediction of an outbreak and evaluation of control measures. However, such a model must be carefully parameterized and validated with epidemiological, ecological and entomological data. A field study was conducted to collect and analyse various parameters to model dengue transmission and outbreak. Dengue prone areas in Kuala Lumpur, Pahang, Kedah and Johor were chosen for this study. Ovitraps were placed outdoor and used to determine the effects of meteorological parameters on vector breeding. Vector population in each area was monitored weekly for 87 weeks. Weather stations, consisting of a temperature and relative humidity data logger and an automated rain gauge, were installed at key locations in each study site. Correlation and Autoregressive Distributed Lag (ADL) model were used to study the relationship among the variables. Previous week rainfall plays a significant role in increasing the mosquito population, followed by maximum humidity and temperature. The secondary data of rainfall, temperature and humidity provided by the meteorological department showed an insignificant relationship with the mosquito population compared to the primary data recorded by the researchers. A well fit model was obtained for each locality to be used as a predictive model to foretell possible outbreak.