(a) Malaria Vectors
Habitat Characterization of Anopheline Mosquito Larvae in Pos Senderut, Kuala Lipis, Pahang.
Studies were conducted to characterize larval habitats of anopheline mosquitoes in 18 aboriginal villages located in Pos Senderut, Kuala Lipis, Pahang. A total of more than
200 breeding habitats were examined and 78 aquatic habitats containing Anopheles mosquito larvae were sampled. The habitats were characterized based on habitat type, substrate type, percentage of emergent plant, canopy cover, distance to the nearest house, dissolved oxygen, water temperature, pH, water depth, area size, turbidity and percentage of surface debris coverage. Larval collections were carried out at various locations surrounding the villages and nearby rivers. Pools containing Anopheles larvae were labeled, identified and recorded. Anopheles mosquito larvae were reared to adults and identified to species. A total of 273 anopheline and 1,519 culicine larvae were collected during the survey. Anopheles maculatus Theobaldwere collected from 47 separate sites. The majority of the sites were open. Most of the breeding sites were in clear ground pool with grassy edges. Percentage of debris coverage and substrate type of the breeding habitat showed significant association with the occurrence of Anopheline larvae (Rohani A, Wan Najdah WMA , Zamree I, Ab Malek AA and Lee H L).
Mapping of malaria vectors in a malaria endemic area in Kuala Lipis, Pahang
A geographical information system (GIS) on vector distribution and environment parameters were developed. Surveillance was conducted to identify and characterize the potential habitats of Anopheles maculatus mosquitoes. Ecological parameters, mosquito larvae density and geographical location were used to determine the key ecological factors determining the different densities of Anopheles larvae. A total of 17 villages in Pos Senderot were systematically searched for mosquito breeding sites. All villages, roads and breeding sites were located with a hand held GPS receiver unit and were used to generate a geographical information system (GIS). This GIS allowed the minimum distance of villages to the potential breeding sites to be determined. A total of 86 Anopheles larvae breeding sites were examined. An. maculatus was collected from 47 separate sites. The larvae were reared to adults and identified to species. Water pocket and ground pools with grassy edge along the river margin were associated with high An. maculatus larvae density. Small breeding sites were commonly colonized by An.maculatus larvae. The probability of finding An. maculatus larvae was reduced when water turbidity increased. Larval habitat locality and distance to the nearest house were significantly associated with the relative abundance of An. maculatus
(Rohani A ,Wan Najdah WMA, Mariamni H, Zamree I, Ab Malek AA, Tanrang H, Rahini A and Lee H L).
Biochemical detection of insecticides resistance in field collected anopheline mosquitoes in the State of Selangor.
Human malaria is a global public health problem with millions of death per year. At present, chemical control using pyrethroid is a major method applied for controlling malaria. However, extensive use of chemical insecticides has hastened the development of malaria vector resistance towards insecticides resulting in decreased control effectiveness. This study was therefore conducted to determine the resistance status of field collected anopheline mosquitoes by biochemical determination of resistance enzymes. Field anopheline mosquitoes were captured from several selected locations in Selangor and analysed for non-specific esterases and mixed function oxidases activities by the rapid micro-enzyme assay. The non-specific esterases and mixed function oxidases activities in the field anopheline mosquitoes were elevated compared to a laboratory strain, indicating the presence of resistance. The highest mean optical density of mixed function oxidases (0.78 ± 0.26) was found in An. hyrcanus captured from Sungai Besi Camp. The mean optical density of mixed function oxidases in An. hyrcanus from Puchong (0.52 ± 0.24) and Sungai Besi Camp (0.78 ± 0.26) were significantly different (p<0.05)..The mean optical density of non-specific esterases in An. maculatus of laboratory strain and those captured from Jeram Kedah, were 0.13 ± 0.02 and 0.31 ±0.12, respectively. Based on resistance enzyme activities, Malaysian An. hyrcanus and An. maculatus exhibited resistance against chemical insecticides. (Aziz I, Mya, YY, Rohani, A, Sa'diyah I and Lee, HL)
(b) Dengue Vectors
Comparative life history studies on wild type and transgenic Aedes aegypti (L.)
Release of genetically modified Aedes aegypti males is considered as an effective method of suppressing this vector of dengue. One of the pre-requisites is the capability to mass produce the genetically modified mosquito in the laboratory before release. To do so would necessitate the understanding of the life cycle and parameters of the transformed strain. In this regard, comparative life history parameters of a wild type laboratory strain of Aedes aegypti (WT) and a genetically transformed Aedes aegypti (LA513) were studied in the laboratory. The following parameters were statistically indistinguishable in both strain: number of eggs laid, number of unhatched eggs, egg hatching rate, number of hatched larvae, duration of larval period in all 4 instars, larval survivorship, pupation, adult eclosion rate, gonotrophic cycle, adult fecundity, adult life span and offspring sex ratio. The basic life history and growth rate of the genetically modified Ae aegypti was not affected by genetic manipulation and that its mating competitiveness will remain intact, which is important for the successful use of this technology (Nazni WA, Joko H, Lee HL, Azahari AH, Iswarti A, Norhaida HAT, and Selvi, S).
Mating compatibility and mating competitiveness of Mx and My01a strains of genetically sterile RIDL-513A Aedes aegypti under confined semi-field conditions
RIDL-Sterile Insect Technique (RIDL-SIT) offers a safe and environment-friendly solution to suppress the population of Aedes aegypti mosquitoes below the disease transmission threshold. We report the world"s first evaluation of genetically sterile RIDL Ae aegypti strains under confined semi- field conditions (state-of-the-art, fully-contained field ho use facility, simulating the living space for 2-4 people in Malaysia). Ten genetically sterile RIDL male Ae aegypti mosquitoes competed with ten wild type males inside this field house to mate with ten wild type females. Hatched larvae from mated females were screened under a fluorescent microscope for molecular markers to determine if they were fathered by RIDL male or wild type male, and all results were cross-checked by PCR. Two such experiments were conducted, each repeated sufficient number of times. All strains were on a Malaysian lab strain background for the first experiment, while the RIDL males alone were on a recently-colonised Mexican strain background for the second experiment. 40% of the mating were with RIDL males in the first experiment, while 45% of the mating were with RIDL (Mexican) males in the second experiment. This shows that Ae aegypti RIDL-513A has excellent mating competitiveness under semi- field conditions, verifying earlier trends obtained in small lab cages at the Institute Pasteur. We also observe high mating compatibility between recently-colonised Mexican RIDL males and lab-reared Malaysian wild type females. These results are very promising and suggest that further studies are required in an ecological island, with the view of evaluating RIDL-SIT to combat dengue and chikungunya (Hanum ATN, Norzahira R, Selvi S, Suria MM, Chandru A, Iswarti I, Ummul, Haninah A, Lim KW, Mahathavan M, Nazni WA,. Lee HL, Hidayatul FO,Labbe GMC, Vasan SS)
Field evaluation of RIDL-SIT: Selection of a suitable island site and baseline survey of
Aedes population
Following successful confined semi- field trials of the RIDL-Sterile Insect Technique (RIDL-SIT),the Institute for Medical Research (IMR) plans to field-test this romising Aedes control technology in an island. In 2006, the IMR long-listed several islands off the Melaka Strait, and short- listed Pulau Carey and Pulau Ketam based on logistics. ovitrap surveys conducted during 16.08.2006-04.10.2006 indicated that P.Carey had Aedes albopictus but no Aedes aegypti,whereas P.Ketam had both (with aegypti/albopictus ratio of 2.3:1). Thus P.Ketam, which reports dengue cases regularly, was selected for future trials comprising of an insular test site Ketam Town (pop. 9150) two kilometers away from an insular control site Sungai Lima (pop.1540). The IMR initiated fortnightly surveys in P.Ketam on 26.10.2007. 29 indoor and 31outdoor ovitraps are set in Ketam Town; 10 indoor and 10 outdoor ovitraps are set in Sg.Lima. GPS was used to choose ovitrap locations and parameters like temperature, humidity and rainfall are recorded. During the first five surveys (26.10.2007–26.12.2007), 90-100% of ovitraps were recovered after one week, with typically 35% of the recovered traps Aedes positive in Ketam Town and 55% in Sg.Lima. Ketam Town recorded aegypti/albopictus ratios of
2.5:1 to 9.5:1 for indoor ovitraps and 0.7:1 to 5.9:1 for outdoor ovitraps; Sg.Lima recorded Aedes aegypti but no albopictus. On average, indoor traps in Ketam Town had
7.5±3.4 aegypti and 1.6±0.4 albopictus larvae/trap, while outdoor traps had 5.4±0.8 and
2.9±3.0 larvae/trap respectively. These data indicate that Ketam Town is a good site to test Aedes population suppression using RIDL-SIT (. Lim K.W,. Norzahira R,. Firdaus N.A.R,. Selvi S,. Suria M.M, Iswarti I., Ummul Haninah A.,. Chandru A,. Hanum A.T.N,. Azahari A.H, Mahathavan M., Nazni W.A., Lee H.L.,. Sit N.W, Hidayatul F.O.,. McKemey A.R,. Vasan S.S)
Comparative oviposition preferences of Aedes (Stegomyia) aegypti (L.) to drain water and seasoned tap water
The comparative oviposition preferences of Aedes aegypti to drain water and seasoned tap water were determined in this study. The sampel was collected from concrete drain with stagnant clear water in a dengue endemic site, Taman Samudera, Selangor. Ae. aegypti adults were given a blood meal and released into the cage. Gravid females were given a choice between drain water and seasoned tap water for egg deposition. In a no choice test, there was no significant difference in the numbers of eggs, larvae, pupae and adults colonized from drain water and seasoned tap water (p > 0.05), indicating that Ae. aegypti oviposit their eggs on a substrate which is readily available. In a choice test, the number of eggs laid by Ae. aegypti in drain water (1630.67 ± 204.26) was significantly more than that in seasoned tap water (221.33 ± 53.18) (p < 0.05). The number of eggs was 6 folds higher in drain water compared to seasoned tap water. The oviposition activity index was 0.71, indicating that the drain water was more attractive compared to seasoned tap water as an oviposition substrate. The pH and BOD values of both drain water and seasoned tap water were not significantly different (p > 0.05), indicating that water from the drain did not contain high organic contents. Significant water conductivity (p < 0.05) and the presence of bacteria could have contributed to the site selection for egg laying for Ae. aegypti. The drain water successfully supported the colonization of the immatures, with the emergence of 824.33 ± 13.96 adult mosquitoes. The ratio of male and female mosquitoes was 1. This study concludes that the concrete drainage system with clear stagnant water provides a suitable medium for the colonisation of dengue vector, Ae.aegypti. (Chen, C.D., Nazni, W.A., Seleena, B., Moo, J.Y., Azizah, M., Lee, H.L.)
Surveillance of Aedes mosquitoes in a university campus in Kuala Lumpur, Malaysia
Ovitrap surveillance was initiated for eight continuous weeks to determine the distribution and abundance of Aedes sp. mosquitoes in the University of Malaya campus, Kuala Lumpur and to determine the impact of meteorological conditions on the Aedes populations. Two study areas within the campus were selected: Varsity Lake and Seventh Residential College. The abundance of Aedes population in Varsity Lake was indicated by ovitrap index (OI) which ranged from 60.00% - 90.00%. The mean number of larvae per ovitrap of Ae. albopictus in Varsity Lake ranged from 11.23 ± 2.42 – 43.80 ± 6.22. On the other hand, the outdoor OI for Seventh Residential College ranged from 73.33% - 93.33%, respectively, while the mean number larvae per ovitrap for this area ranged from 19.33 ± 4.55 – 35.27 ± 5.46, respectively. In addition, the indoor OI of Seventh Residential College ranged from 0.00% - 30.00%, while the mean number of larvae per ovitrap for Ae. albopictus ranged from 0 – 5.90 ± 3.55. There was no significant difference (p > 0.05) of Ae. albopictus population between Varsity Lake and Seventh Residential College. The studies showed a correlation between OI and mean number of larvae per ovitrap for outdoor Ae. albopictus populations in Varsity Lake and Seventh Residential College (r = 0.794). There was also a correlation between the mean larvae number per ovitrap of Ae. albopictus obtained from eight weeks indoor ovitrap surveillance in Seventh Residential College with rainfall (r = 0.584). However, there was no correlation between the mean larvae number per ovitrap of Ae. albopictus in both study areas with temperature and relative humidity. Ae. aegypti mosquitoes were found neither indoor nor outdoor in both study areas. This study indicated that the principal dengue vector in the university campus was most likely Ae. albopictus. (Wan-Norafikah, O., Chen, C.D., Soh, H.N., Lee, H.L., Nazni, W.A., Sofian-Azirun, M.)
Distribution and habitat characterisations of container-breeding Aedes larvae collected from dengue prone areas in Kedah, Terengganu, Perak and Kuala Lumpur.
Larval survey method was used to determine the preferred habitats of Aedes larvae and their relative abundance. The studies were carried out in Kedah, Terengganu, Perak and Kuala Lumpur in November 2007. A total of 2024 Aedes albopictus Skuse and 1565 of Aedes aegypti (L) were collected and analyzed by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) for dengue virus detection. Location of breeding sites was recorded using hand-held Geographical Positioning Systems (GPS) device. The biological and chemical characteristics of breeding sources were determined by checking the pH, turbidity, temperature and dissolved oxygen. A total of 112 containers were examined. Plastic container, tyre and aluminium can were found to be major habitats for both Ae. albopictus and Ae. aegypti, representing 48%, 27% and 13% respectively. Ae. albopictus and Ae. aegypti preferred containers containing clear water with debris (77%), followed by turbid (12%), greenish (6%) and brownish (5%). Ae.albopictus larvae coexisting with Ae.aegypti were collected from 20 containers (18%). Overall, Ae.albopictus was the most abundant larvae collected during the survey. (Noor Azleen M.K., Fairuz K., Rohani A., Rozilawati H., Zamree I. and Lee H.L).
Entomological, virological and environmental surveillance of dengue vectors
Dengue, is an Aedes-borne viral infection and is a major public health problem faced by many tropical and subtropical countries. The 2 main vectors are Aedes aegypti (L.) and Aedes albopictus Skuse. This study aims to conduct entomological surveillance of dengue vectors using ovitrap, screening of transovarial dengue virus and to examine the impact of metrological parameters on the vectors. Ovitraps were deployed outside premises in selected locality in dengue prone areas in Selangor, Johor, Kedah and Kuala Lumpur. A total of 5,486 larvae were identified, of which 2,548 were Ae. aegypti and 2,938 were Ae. albopictus. Reverse Transcriptase Polymerase Chain Reactions were used to detect dengue virus from collected Ae. aegypti and Ae. albopictus larvae. Metrological data such as rainfall, humidity and temperature in each locality were collected to correlate the vector population abundance with metrological condition. The short study period showed that rainfall, mean temperature and humidity of each study area are correlated with the Aedes population (Rohani A, Fairuz K., Noor Azleen M.K., Rozilawati H., Lee H. L., Chooi K.H., Mohd Mazlan M.I., Khadijah K., Mohd Anuar I., Maszaitun Salmah M., Topek O. and Suzilah I.
Aedes Pheromone: Isolation, identification and characterisation of pheromones from
Aedes mosquitoes
Extraction of semiochemicals from Aedes aegypti using organic solvents was performed. The method using hexane (HEX) and dichloromethane (DCM) have been established. HEX extract for both male and female of Ae. aegypti were collected and extraction work still going on. Preliminary results shows that semiochemicals from solvent extract containing communication chemicals and yet to be confirmed using behavior responses method. (Khadri MS, Mashitah MY, Nurul Ezzah I, Naim MN).
(c) Chikungunya Vector
Detection of chikungunya virus in field collected Aedes aegypti and Aedes albopictus from selected area.
Three study areas in Terengganu were identified based on the criteria of areas with previous reports of dengue and dengue-liked outbreak; Seberang Takir in Kuala Terengganu, Penarik in Setiu and Kg Gertak Seratus, Besut. Adult collection were conducted from 5.00 p.m to 7.30 pm and 7.00 am to 9.00 am. Larvae were collected between 9-11 am. All samples were identified and brought back to the laboratory. Mosquitoes were pooled in appendorf tubes according to species and RNAs were extracted. RT-PCR was conducted. Results were visualised using the electrophoresis gel. A total of 55 Aedes aegypti; 32 Aedes albopictus;, 5 Aedes niveus; 173 Culex quinquefasciatus; 7 Culex tritaeniorhynchus; 3 Culex vishnui; 6 Armigeres keseli; 3 Armigeres subalbatus, and 1 Anopheles umbrosus adult were collected. Where as a total of 99 Aedes aegypti; 62 Aedes albopictus; 2 Aedes niveus and 1 Culex quinquefasciatus immature were collected. All samples of adult Aedes aegypti and Aedes albopictus collected in Terengganu were found to be negative for chikungunya virus. However more samples might be needed in order to have higher possibility of positive results
(Rozilawati H., Rohani A., Lee H.L., Zamree I., Nor Azlina A.H.).
(d) Other Mosquitoes/Studies
Determination of zoonotic viral infection in non-human primates with special reference to arboviruses
Arthropod–borne viruses (arboviruses) are viruses that can be transmitted to man by arthropod vectors. Transmision cycles of these viruses can be Man – arthropod vector – man cycle or Animal – arthropod vector – man cycle. In the man arthropod man cycle the reservoir may be in either man or arthropod vector whereas in the later cycle the reservoir is in animal and becomes infected incidentally. In many cases the actual reservoir is not known. Rudnick and Lim ( 1986) carried out studies on the ecology of dengue in Malaysia The authors reported the presence of virus in non-human primates and postulated that in future Ae albopictus will be able to pick-up virus from non human primates to human. Much of the work in Malaysia was done in the 1960's and the sylvatic and human dengue viruses were not sequenced to established the relationship between human and syalvatic dengue viruses in Asia. This project was conducted to identifiy arthropod borne viruses in non-human primates and to have a clearer understanding of animal-arthropod-man cycle of viruses. This will assist to develop an early warning system for viral related outbreaks. A total of 54 monkey serum were analysed for virus presence using 3 cell lines such as Vero cells, BHK 21 and C6/36. Our study showed that no virus has been isolated using tissue culture techniques. (Nazni WA, Apandi Y, Norazleen ZA, Indra V, Rozilawati H and Lee HL)
(e) Microbial Control Agents
Efficacy of Bacillus thuringiensis israelensis formulations, VectoBac® WG and VectoBac® 12AS, dispersed by spray application against Aedes aegypti and Culex quinquefasciatus larvae.
Comparison of residual efficacy of two Bti formulations, VectoBac WG (3000 ITU/mg) and VectoBac 12AS (1200 ITU/mg), was evaluated under simulated condition against Aedes aegypti and Culex quinquefasciatus, using spray application. The formulations were tested at equivalent dosages, sprayed with a back-pack mist-blower towards test cups placed at 8 m from the nozzle. The efficacy of the sprays was measured from day 1 to day 28 post spray against 3.5 h, 24 h, 48 h and 7 days larval exposure. Both formulations showed reducing bioefficacy with time after spray. Increasing larval mortality was observed with increased time of exposure. Overall, VectoBac® WG performed better with complete mortality in the Culex sp. and Aedes sp. larvae in the day 1 post spray samples within 3.5 h exposure. There was also no significant difference in the larval mortality between day 1 to day 28 post spray samples against Ae. aegypti larvae (p >0.05). VectoBac® WG also did not show any significant difference in its efficacy against Ae. aegytpi and Cx. quinquefasciatus larvae (p >0.05). However, VectoBac®12AS did not induce complete mortality within 3.5 h exposure. The efficacy of VectoBac® 12AS was significantly different in the day 28 post spray cups, which had significantly lower mortality than day 1 to day 21 post spray samples. The significantly lower mortality was observed for 3.5 h and 24 h exposure in day 28 cups (p < 0.05). VectoBac® 12AS also induced significantly higher mortality in Ae. aegypti than Cx. quinquefasciatus larvae in day 21 post spray test samples (p < 0.05). (Lee, H.L., Andy- Tan, W.A., Loke, S.R., Chiang, Y.F., Lau, K.W., Sokha, L., Tan, S.B., Nazni, W.A., , Seleena, B.)
Susceptibility of Culex quinquefasciatus and Aedes aegypti to sprayed Bacillus thuringiensis israelensis (VectoBac WG) droplets.
In a previous study it was observed that Culex quinquefasciatus larvae exhibited faster susceptibility to sprayed Bti droplets than Aedes aegypti. A preliminary study was then conducted to confirm this observation by placing both species of larvae in the same test cups and mortality was observed over a period of time. A Bti formulation, VectoBac WG was sprayed with a back pack sprayer into cups placed 8 m from the nozzle. Ten larvae of each species were introduced into the same cups at different times of post spray, with 5 replicates of cups for each point of test time. Larval mortality was recorded from 1 to 6 hours of exposure, with a final observation at 24 hours post exposure. This study was replicated over 2 days. Cups holding larvae at the time of spray (0 h) and 1 hour after spray had 90 % mortality in the Cx quinquefasciatus larvae within 1 h exposure and a complete kill within 3 h exposure. In the same cups there was no mortality in the Aedes larvae for 2 h post exposure. Initial Aedes larval mortality was only observed at 3 h exposure with a complete kill after 6 h exposure. The Culex larval mortality reduced with increasing time of post spray, i.e. an average of 55 % Culex sp mortality for the 3 h & 6 h post spray samples, 42 % mortality for days 1 & 2 post spray samples, followed by an average of 6 % mortality for days 3 & 4 post spray samples at 1 h exposure. On the other hand the Aedes larval mortality increased with increased time of post spray, i.e. an 84 % Aedes larval mortality was observed in 2 days post spray cups within 1 h of exposure. Days 2 to 4 post spray samples had significantly higher Aedes larval morality for all observed exposure time in comparison to the days 0 & 1 post spray samples. The declining mortality with increasing time of post spray could be due to the sprayed Bti droplets (71 – 87 um) remaining in the top portion of the water column after the spray with the first hour, i.e. in the zone where Culex sp. larvae which are air breathers remained just below the surface where they filter feed the microscopic suspended organic materials. The reverse in the mortality trend observed on Aedes could be due to Bti droplets settling to the base of the cup, where Aedes sp. larvae were observed to remain and feed. (Lee, H.L, Loke, S.R., Andy-Tan, W.A., Chiang, Y.F., Tan, S.B., Nazni, W.A., Seleena, B.)
(f) Other Insects
Preliminary Study of the Distribution of Phlebotomine Sand Flies (Pyschodidae) in Aborigines Areas in Selangor
Surveillance for sand flies in Malaysia continues. Sand flies surveillance in aborigine villages in two districts of Selangor state viz. Hulu Slangor and Gombak were carried out. Out of 11 localities surveyed in the districts, 40% of the localities were found positive with sand flies. However, the number of sand flies caught at each locality was very low, 1-2 sand flies. Species that were identified are Phlebotomus stantoni, Phlebotomus asperulus and Sergentomyia spp. (Narcissus Henry Sudin, Khadri MS, Mohd Noor I, Azahari AH)
Studies of leishmaniasis among Bangladeshi worker and their vector
Surveillance for sand flies in lime stone areas in Malaysia in relation to Bangladeshi migrant workers continues. Three localities viz. Gua Musang, Kuala Lipis and Cameron Highland, were surveyed in November and December of year 2007. No sand fly was found in Cameron Highland, however 130 Phlebotomus flies were trapped in Gua Musang limestone areas and 181 Phlebotomus flies were trapped in Kuala Lipis limestone areas. However, no sand fly was found positive with Leishmania parasites. (Azian I, Khadri MS,)
Abundance of biting flies collected from selected surra infected animal farm
Trypanosoma evansi is the blood protozoan that causes trypanosomiasis or surra which is an animal disease occurring in Africa, Asia and Latin America. Trypanosoma evansi infections of the cattle and buffalo usually lead to pronounced immunosuppression. Recently, human case of surra has been reported in India.Trypanosoma evansi infection in animals is known in Malaysia. However the insect vector(s) is not known. A collaboration project between IMR and Universiti Putra Malaysia has been conducted in order to incriminate the vector(s). In this early stage of study, the abundance of biting flies in selected cattle farm was determined. Insects were collected using sweep nets early in the morning (8am-12pm), for duration of 5 minutes of every hour. A total of 14 collections have been conducted between June – September 2007. Sweep net is the most suitable method to collect the flies, since the flies only stay for a very short period to blood feed on the cattle body parts and sweep net is easier to handle than modified aspirator. A total of 22, 365 specimens were collected from UPM cattle farm during this period. These flies were identified as Haematobia exigua, 21 878 (97.8%); Musca domestica, 195 (0.87%); Musca crassirostris, 130 (0.58%); Stomoxy sp., 78 ( 0.35%); Fannia sp., 28 (0.12 %); Musca sorbens, 21 (0.09%); Musca stabulans, 19 (0.08%); Orthellia sp., 6 (0.03%); Ophyra sp 4 (0.02%); Sarcophaga sp., 4 (0.02%); Stomorhina sp., 1 (0.005%) and Calliphora sp., 1 ( 0.005%). In addition to this trapping more number of collections will be carried out soon. The flies will be screened to determine the possibility of T. evansi presence in these flies in order to incriminate the possible vectors in Malaysia (Rozilawati H., Rohani A., Lee H.L., Jeffrey J.).
(g) OTHER STUDIES
MAGGOT THERAPHY
Laboratory Upscaling of Sterile Maggots for Therapeutic Use
Maggot therapy has been introduced in Malaysia since 2003 as an alternative way to cure diabetic wounds in diabetic patient. In temperate weather , Lucilia sericata was used as a main fly for maggot therapy. However, in a tropical country such as Malaysia, as an alternative, Institute for Medical Research introduced Lucilia cuprina for use in MDT treatment. A part from the color difference, there is not much different between both species. Lucilia cuprina was also selected because 1) Are easy to rear and sterilize. 2) Prefer to feed on necrotic tissues 3) Develop rapidly and therefore act quickly in the wound. 2) Do not invade internal organs. A prospective case controlled study was conducted to assess the effectiveness of MDT versus conventional debridement for the treatment of diabetic foot ulcers. There was no significant difference in outcome between the two groups. Conclusion made from this sttudy was MDT is as effective as conventional debridement in the treatment of diabetic foot ulcers. Two temperatures that is 4°C and 10 °C were used to study the hatching rate of the flies eggs. Our results indicated that the yield at 10 °C was better than 4 °C. Hence eggs can be kept dormant for 48 hours at 10 °C. ( Nazni WA, Mohd Masri,S, Wan Norjuliana WM and Lee HL.)
Forensic Entomology
Studies on important forensic entomological specimens recovered from monkey carrions exposed to different ecological habitats
There is a knowledge gap especially on the biology and bionomics of the insects recovered from the cadavers and a comprehensive study has yet to be conducted on the wave of succession of arthropod fauna on carrion under different habitats and conditions in Malaysia. Study on succession of fauna on non-human primate carcasses placed outdoor, indoor, in artificial grave and in water at different time period (morning and night) were conducted. There was no activity of flies at night. A total of 6 different species of flies infested the carrion at different decomposing stage. Our study also indicated that when a carrion was placed outdoor, within half an hour flies could locate it and deposited egg within 1.5 h. There was a delay in fly locating the carrion when it was placed indoor.(Nazni WA, Chen Z, Wan Norjuliana WM, Sadiyah I, Abdullah AG, M Nahrawi and Lee HL).
Laboratory study on larval growth of forensically important flies, Hypopygiopsis (Townsend) sp., Chrysomya chani (Kurahashi, 1979) and Chrysomya rufifacies (Macquart, 1842)
Blowfly larvae play an important role on ecological function in decomposition of animal remains. Most commonly, the blowfly larvae are used to estimate the minimum time since death, which is the minimum post-mortem interval (PMI), using growth parameter and larval length as a 'biological clock'. This study examined the larval growth of forensically important fly species, Hypopygiopsis sp. (Diptera: Calliphoridae), Chrysomya chani Kurahashi (Diptera: Calliphoridae) and Chrysomya rufifacies Macquart (Diptera: Calliphoridae) at a temperatures of 28ºC and 70% relative humidity. Detailed observations were made on rate of development of Hypopygiopsis sp., Chrysomya chani and Chrysomya rufifacies by measuring the length of larvae hourly. The larval growth rates of the flies were determined. The growth parameter of egg, first, second and third larval instar, total larval period, pupal period and egg to adult period were also studied and determined. This is the first report on the growth characteristics of Hypopygiopsis sp.. (Chen, C.D., Nazni, W.A., Lee, H.L., Jeffery, J., Wan-Norjuliana, W.M., Sa'diyah, I., Sofian-Azirun, M.)
First study on the insect succession on monkey carrion placed indoor in Malaysia
Medicolegal forensic entomology deals with the utilisation of insects and other arthropods in solving criminal or unexplained death, with the major goal of determining the postmortem interval (PMI). Insects can provide an alternative means of estimating the PMI in cases when other methods are no longer accurate. Insects are used in a complementary ways to estimate PMI, as the succession data can indicate how long a victim has been dead because insects invade a corpse shortly after death in a manner that is predictable and specific to the environment in which it is found. The present studies were conducted on successional fauna using monkey (Macaca fascicularis, Raffles) carcasses in a rural area in Gombak, Selangor State, Malaysia. Comparative patterns of succession on monkey carcasses placed indoor and outdoor, including succession patterns and insect fauna found on the carcasses were studied. Within an hour, Hypopygiopsis (Townsend) sp. (Diptera: Calliphoridae) was observed as the first visitor on the monkey carcass placed outdoor and maggot masses were found on the same day. The maggots found on the carcass placed outdoor (day 1 – day 13) were those of Hypopygiopsis sp., Chrysomya megachephala (Fabricius), Chrysomya villeneuve (Patton), Chrysomya chani (Kurahashi), Chrysomya pinguis (Walker) and Ophyra spinigera (Stein). However, in monkey carcass placed indoor, calliphorid flies were only observed on the fourth day (bloating stage) and maggot masses were found on the fifth day (bloating stage); while for indoor (day 5 – day 41), maggots obtained were those of Chrysomya villeneuve, Chrysomya chani, Chrysomya pinguis, Ophyra spinigera, Musca domestica (Linnaeus) and Sarcophagidae. This is the first study on insect succession on non-human primate carcass placed indoor in Malaysia. (Nazni, W.A., Chen, C.D., Lee, H.L., Jeffery, J., Wan- Norjuliana, W.M., Sa'diyah, I., Sofian-Azirun, M.)
Forensically important fly larvae recovered from human cadavers.
A total of 30 forensic entomological specimens were received from Medical Entomology Unit, Institute for Medical Research, hospitals and the police department in Malaysia. Three species of cyclorrphagic flies were identified consisting of Chrysomya rufifacies (Macquart), Chrysomya megacephala (Fabricius) and Hermetia illucen (Linnaeus). Chrysomya rufifacies and Chrysomya megacephala were the commonest species found in cadavers from different ecological habitats. A total of 29 cases (97.00%) had a single fly infestation and 1 case (3.00%) had a double fly infestation. In conclusion, the predominant fly species recovered from cadavers in Malaysia are still those of Chrysomya. (Kavitha, R., Chen, C.D., Lee, H.L., Nazni, W.A., Sa'diyah, I., Edah, M.A.)
Comparison in Post Mortem Interval (PMI) estimation by forensic pathology and forensic entomology.
A study was conducted on 30 forensic entomological specimens to compare the estimation of PMI by conventional forensic pathology and forensic entomology techniques. Chrysomya rufifacies (Macquart), Chrysomya megacephala (Fabricius) and Hermetia illucen (Linnaeus) specimens were used as indicator of PMI. Statistically, the PMI estimation by entomologist and pathologist was not significantly different in 66.67% of the cases. It is concluded that PMI estimation by entomological techniques is a useful forensic age determination tool. (Kavitha, R., Chen, C.D., Lee, H.L., Nazni, W.A, Sa'diyah, I., Edah, M.A.)
Butterflies associated with pig carrions in Malaysia
An entomological study conducted in Tanjung Sepat, Selangor, Malaysia in May until September 2007 revealed five species of butterflies (all from order Lepidoptera, family Nymphalidae) were attracted to the pig carcasses placed in a palm oil plantation. Euploea mulciber, Hypolimnas bolina, Elymnias hypermnestra, Mycalesis mineus and Ypthima baldus arrived at the carcasses at different stages of decomposition. Adult lepidopterans are frequently attracted to decaying remains and feed on decomposition fluid. (Heo, C.C., Marwi, M.A., Jeffery, J., Sofian Azirun, M., Chen, C.D., Abdullah, W.O., Ishak, I.Y., Omar, O.)
Laboratory & Field Evaluation
Laboratory and simulated field effectiveness of an insect growth regulator, cyromazine against Aedes (Stegomyia) aegypti (Linnaeus)
This study was conducted to evaluate the commercially available cyromazine formulation for its impacts on Ae. aegypti larvae. WHO larval bioassay procedure was performed to obtain the dosage for 50% and 90% inhibition of emergence (IE) of cyromazine against Aedes aegypti. Cyromazine exhibited moderate toxicity against Ae. aegypti, with IE50 and IE90 values of 2.90 mg/L and 4.56 mg?/5L.0(0 mg/L), respectively. Base on the laboratory bioassay, 2 concentrations (5 mg/L and 10 mg/L) of cyromazine were used to treat a set of earthen jars placed outdoor, accordingly. Untreated jars were also set up as controls. Fifty laboratory-bred 2nd instar larvae were introduced into each earthen jar. All earthen jars were observed daily. Number of adults emerged was recorded and the larval mortality were calculated. The indicators of effectiveness of IGRs for these studies were (i) residual activities of each dosage, and (ii) percentage of emergence inhibition (EI). The earthen jars treated with cyromazine showed better residual effect in jars without water replenishment, in comparison to weekly and daily water replenishment. Earthen jars treated with cyromazine without water replenishment showed significant difference on number of adult emergence between both treated and untreated earthen jars up to 21 weeks (p < 0.05). Both concentrations showed > 90% emergence inhibition up to 21 weeks or more and was not significantly different from each other (p > 0.05). Thus, 5 mg/L of cyromazine is recommended to be used in the control of Aedes aegypti. (Chen, C.D., Andy-Tan, W. A., Loke, S.R., Chai, H.W., Lee, H.L.)
Field repellency of Mentha sativa and Cymbopogon citrates essential oil against mosquitoes
Mosquitoes are important vectors of diseases and nuisance pests. Repellents based on essential oils from plant extract are being developed as an alternative personal protection to minimize human contact with mosquitoes. In this study, we evaluated and reported repellent effects of a commercially available essential oil containing 2% Mentha sativa (mint) and 16% Cymbopogon citrates (lemongrass) in a revitalisor against mosquitoes under field conditions using human baits. The tests were conducted inside 2 cabins or rooms in 3 study sites. The mosquito population was estimated by using the Bare Leg Catch (BLC) techniques. For treated test, a revitalisor with essential oil was placed indoor 2 – 3 meters away from human bait. Another cabin or rooms without revitalisor with essential oil was used as untreated control. BLC was conducted in both sites simultaneously. There was an 72.56% - 97.78% reduction of mosquitoes population in the treated test compared with the untreated condition during the 8 hours catches (p < 0.05). Essential oil containing Mentha sativa and Cymbopogon citrates used in this study exerted repellency effect against the mosquitoes. (Chen, C.D., Izzul, A.A., Lee, H.L., Sofian-Azirun, M.)
Larval toxicity of an insect growth regulator, novaluron against Malaysian mosquitoes
The toxicity of an insect growth regulator, novaluron against four species of laboratory bred Malaysian mosquito larvae was determined in the laboratory. The test was performed according to the standard WHO larval bioassay. Based on the 50% inhibition of emergence (IE) values generated by probit analysis computer programme, the most susceptible mosquito was Culex quinquefasciatus and the least was Aedes togoi. The descending order of susceptibility is: Culex quinquefasciatus > Aedes aegypti > Anopheles maculatus > Aedes togoi. (Chen, C.D., Yeong, Y.S., Lee, H.L.)
Field evaluation of the bioefficacy of diflubenzuron (Dimilin®) against container- breeding Aedes sp. mosquitoes
The inhibitory activity of diflubenzuron, a chitin synthesis inhibitor, on the ecdysis of Aedes sp. larvae was evaluated in earthen jars and automobile tires. Two formulations of diflubenzuron were used in this study: Dimilin® WP (wettable powder), 25% and Dimilin® GR (granular), 2%. The equivalent rate of 25 g/ha, 50 g/ha and 100 g/ha active ingredients for both WP and GR formulations were used in this study. Generally, at the higher dosage of 100 g/ha, both formulations were more effective against Aedes mosquitoes. On the whole, the WP formulation appeared to perform better than the GR formulation in terms of residual activity. (Chen, C.D., Seleena, B., Chiang, Y.F., Lee, H.L.)
Laboratory study on the effectiveness of A floor gully with grating to prevent the oviposition of Aedes (Stegomyia) aegypti (Linneaus)
This study was conducted to evaluate the effectiveness of a floor gully come with grating to prevent the oviposition of Aedes aegypti in the floor trap. To conduct the test, two containers were placed into a mosquito cage (30cm x 30cm x 30cm). Both containers were filled with seasoned tap water and covered with floor gully c/w grating and normal floor gully, respectively. A total of 50 gravid Ae. aegypti females were then released into the cage and left for a week. All the eggs obtained from the test were allowed to remain inside the containers for the eggs to hatch. The number of hatched larvae was counted and recorded. Five replicates were conducted concurrently. There was a significant difference of Ae. aegypti larvae obtained between container with floor gully c/w grating and normal floor gully (p < 0.05). A total of 96.41% reduction of Ae. aegypti larvae was obtained in the container with floor gully c/w grating compared with the normal floor gully, indicating that the floor gully c/w grating used in this study was able to prevent oviposition of Ae. aegypti in holding water. (Chen, C.D., Teoh, P.S., Wan-Norafikah, O., Lee, H.L.)
Insecticides Studies
Biochemical detection of resistance in Aedes (Stegomyia) albopictus Skuse from a dengue endemic area in Selangor, Malaysia
Larvae of Aedes albopictus obtained from 6 consecutive ovitrap surveillance in 2 dengue endemic areas, Taman Samudera and Kg. Banjar, Selangor were evaluated for their susceptibility to temephos. Their resistance levels were also detected by biochemical test. Larval bioassays were carried out in accordance to modified WHO standard methods. Biochemical assay of enzymes in Ae. albopictus was conducted to detect the emergence of insecticides resistance and to define the mechanisms involved in temephos resistance. The 50% mortality at lethal time (LT50) for all strains of Ae. albopictus tested against temephos ranged between 58.65 to 112.50 minutes, with resistance ratio ranging from 0.75 – 1.45. Four biochemical enzymes namely non-specific esterases, mixed function oxidases, glutathione-S-transferases and acetylcholinesterases were studied. Esterases played a major role on resistance mechanism in Ae. albopictus to temephos, while mixed function oxidases, glutathione-S-transferases and acetylcholinesterases were not involved in Ae. albopictus resistance to temephos. However, high mixed function oxidases and glutathione-S-transferases levels indicated that Ae. albopictus might be resistant to other groups of insecticides. The resistance studies conducted over a period of time also showed weekly variations in resistance against temephos and variations in enzyme levels in Ae. albopictus, indicating that field populations of Ae. albopictus were heterogenous in susceptibility against insecticides. This study suggests that continuous resistance monitoring should be conducted in all dengue endemic sites in Malaysia regularly to confirm the efficacy of insecticides and to facilitate the selection of most effective insecticides to control dengue infections. (Chen, C.D., Nazni, W.A., Lee, H.L., Seleena, B., Sofian-Azirun, M.)
Comparison between esterase patterns in insecticide resistant and susceptible strains of Culex quinquefasciatus Say and Aedes aegypti (L.)
An electrophoretic analysis of non-specific esterase enzyme system by means of polyacrylamide gel electrophoresis was carried out in malathion and permethrin resistant strains of Culex quinquefasciatus and Aedes aegypti in comparison to respective susceptible strain. Non-specific esterase enzyme activity was assessed at different life stages {egg, 1st instar larva {L1}, 2nd instar larva{L2}, 3rd instar larva{L3}, Pupa stage 1{P1}, Pupa stage 2{P2}, Male & Female) in these populations which were reared consecutively for 10 generations after subjected to selection pressure in the laboratory. The non-specific esterase banding patterns of both populations were characterized using ?- & ?- naphthyl acetate. Each number of esterase bands varied depending on the strains and sex and these bands were designated as E1, E2, E3, E4 and E5. In malathion resistant Cx. quinquefasciatus strain, as many as 4 bands were detected at L3 stage. There was also a noticeable increase of band intensity in esterase activity from one instar to another and the bands appeared to be strong in adult females compared to males. Band E3 appeared consistently as a major band in all the different life stages of malathion resistant Cx. quinquefasciatus strain whereas in permethrin resistant Cx. quinquefasciatus strain, there was variation of esterase bands between sexes and life stages and their intensity was found to be lower compared to malathion resistant strain. In contrast to Cx. quinquefasciatus strains, band E5 was observed in every different life stages of Ae. aegypti and was the major band among the strains. At least one highly active esterase band was present in malathion and permethrin strains of Cx. quinquefasciatus and Ae.aegypti (Selvi Subramaniam, Edah Mohd Aris, Nazni Wasi Ahmad, Lee Han Lim, Azahari Abdul Hadi)
Detection of pyrethroid resistance in mosquitoes of public health importance
A study was conducted to determine the permethrin resistance status, the presence of knock-down resistance (kdr) alleles using the synergist; piperonyl butoxide (PBO), and the level of oxidases in the laboratory susceptible, laboratory permethrin selected and field strains of Aedes albopictus and Culex quinquefasciatus, respectively. Three methods were used for this study; the WHO larval bioassay, the WHO adult bioassay and the enzyme microassay of oxidases. Generally, irregular patterns were presented for the LC50 values, LT50 values, resistance ratio (RR) values and synergistic ratio (SR) values for almost all strains of both Ae. albopictus and Cx. quinquefasciatus including the laboratory strains. These results showed that all strains of Ae. albopictus and Cx. quinquefasciatus tested for this study were heterozygous and permethrin resistance existed in all strains of both mosquito species. The results obtained from the enzyme microassay which showed the correlation between the LC50 values or LT50 values with the oxidases level in all strains of both mosquito species as well as the use of the synergist, PBO confirmed that the knockdown resistance kdr alleles were not yet present in these strains of mosquitoes and only oxidases were involved in the permethrin resistance mechanism in these mosquitoes at this moment. Therefore, permethrin is still effective in vector control. (Wan Norafikah O, Lee HL, Nazni WA, Azahari AH, Wan Norjuliana WM, Sa'diyah I).
