Assessment of the susceptibility status of Aedes albopictus (Diptera: Culicidae) from Interior, Sandakan and Tawau divisions of Sabah, Malaysia based on WHO diagnostic doses of larvicides

@# Susceptibility status of Aedes albopictus (Skuse) sampled from residential areas in Interior, Sandakan and Tawau divisions of Sabah, Malaysia, was evaluated based on the WHOrecommended doses of organochlorine and organophosphate larvicides. To determine susceptibility status, larval bioassays were carried out and post 24-hour mortalities based on WHO resistance classifications were adopted. The results demonstrated that Ae. albopictus larvae were resistant toward 5 out of the 8 larvicides tested. Larvae from all populations were resistant against bromophos, fenitrothion, malathion, temephos and dichlorodiphenyltrichloroethane (DDT), with mortalities ranging from 0.00 to 89.33%. Dieldrin, on the other hand, could induce 100.00% mortalities in all populations, followed by fenthion and chlorpyrifos, with mortalities ranging from 97.33 to 100.00% and 81.33 to 100.00% respectively. Despite most populations exhibiting similitude in their resistance status, larvae from Sandakan exhibited the highest resistance level whereas the lowest level was observed in Keningau. In view of the inadequacy of some larvicides in controlling Ae. albopictus in this study, integrated management such as insecticide rotation or combination of interventions is warranted.

PesTrapp mobile app: A trap setting application for real-time entomological field and laboratory study

@#Diseases such as malaria, dengue, Zika and chikungunya remain endemic in many countries. Setting and deploying traps to capture the host/vector species are fundamental to understand their density and distributions. Human effort to manage the trap data accurately and timely is an exhaustive endeavour when the study area expands and period prolongs. One stop mobile app to manage and monitor the process of targeted species trapping, from field to laboratory level is still scarce. Toward this end, we developed a new mobile app named “PesTrapp” to acquire the vector density index based on the mobile updates of ovitraps and species information in field and laboratory. This study aimed to highlight the mobile app’s development and design, elucidate the practical user experiences of using the app and evaluate the preliminary user assessment of the mobile app. The mobile app was developed using mobile framework and database. User evaluation of the mobile app was based on the adjusted Mobile App Rating Scale and Standardized User Experience Percentile Rank Questionnaire. The process flows of system design and detailed screen layouts were described. The user experiences with and without the app in a project to study Aedes surveillance in six study sites in Selangor, Malaysia were elucidated. The overall mean user evaluation score of the mobile app was 4.0 out of 5 (SD=0.6), reflects its acceptability of the users. The PesTrapp, a one-stop solution, is anticipated to improve the entomological surveillance work processes. This new mobile app can contribute as a tool in the vector control countermeasure strategies.

Residual efficacy of deltamethrin applied to foliage against Malaysian Aedes aegypti (L.) and Aedes albopictus (Skuse)

@#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.

Status of pyrethroid resistance in Aedes (Stegomyia) aegypti (Linneaus) from dengue hotspots in Klang Valley, Malaysia

@#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.

Pteridine fluorescence in age-determination of immature Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae)

@#In the practice of forensic entomology, the chronological age of the maggots retrieved from the cadaver is used to determine the minimum post-mortem interval (mPMI) i.e. minimum time of death. The conventional method of aging the maggots is based on measuring the growth rate of these maggots. Although effective, the constraint associated with conventional method necessitates the development of new age determination method, such as pteridine determination. Pteridine, a by-product of protein metabolism in insects is known to correlate with the age of a variety of dipterans. A number of studies were conducted on aging the adults of forensically important flies. In this study, pteridine was extracted from Chrysomya megacephala and Chrysomya rufifacies maggots of known age using established methods and determined by measuring the fluorescence at excitation of 330nm and the emissions between 350nm and 600nm. Results exhibited significant positive linear relationships between the pteridine accumulations and age of the fly immature. Pteridine determination is a potential new age determination tool that can be used to determine mPMI.

A preliminary proteomic study of permethrin resistant and susceptible Aedes aegypti (L.)

@#The mechanism of insecticide resistance is traditionally attributed to detoxification enzymes, target site alteration, decreased penetration of insecticides and behavioural resistance. Other form of mechanisms, such as the role of protein(s) in resistance is unknown. In the present study, the protein profiling of both IMR-PSS strain (permethrin-selected) and IMR-LS strain (laboratory-susceptible) 24 hours post exposure period to permethrin was carried out via 1D-gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/ MS). The bands which appeared in the gel of 1D-electrophoresis revealed an abundance of proteins. The band pattern of both strains looked macroscopically alike and differed only in band intensity. However, LC-MS/MS analysis revealed that the IMR-PSS strain produced extra 388 peptides that were not found in the IMR-LS strain, indicating that IMR-PSS strain reacted differently from IMR-LS strain as a result of persistent exposure to permethrin. Since the complex banding patterns of 1D-gel electrophoresis were difficult to interpret the significance of the protein difference between IMR-PSS and IMR-LS strain, hence LC-MS/MS analysis is ideally suited for better protein resolution and thus will allow more in-depth comparison of the complex pattern. The findings here provide the first preliminary evidence that insecticide resistance in mosquito induces up regulation of proteins that may be protective to mosquitoes against insecticide and proteins could be another mechanism that contributes to development of resistance.

Effects of vitamin B fortified sucrose solution on the longevity and reproductive potentials of laboratory-bred Culex quinquefasciatus Say adult

Laboratory colonised mosquitoes are usually maintained on vitamin B complex fortified sucrose solution, however only few studies were conducted to evaluate the effects of such practice. This study aimed to determine the effects of different concentrations of sucrose solution fortified with and without 1% vitamin B complex on the longevity and reproductive potential of adult female of a local strain of Culex quinquefasciatus Say. Two arms of studies were carried out separately and each arm was triplicated. In one arm, concentrations of sucrose solution at 0%, 1%, 3%, 5% and 10% fortified with 1% Vitamin B complex were fed to the mosquitoes, while in the other arm, similar sucrose concentrations were used but without 1% vitamin B complex. Adult Cx. quinquefasciatus maintained on 5% sucrose solution fortified with 1% vitamin B complex exhibited significant extended vitality and longevity in stimulating ovarian development, compared with other vitamin fortified sucrose concentrations (p<0.05). The vitality and longevity of F0 and F1 males were 76.67±2.19 days and 57.67±8.19 days respectively. The F0 females survived the longest duration of 107.67±5.61 days and the F1 females survived 90.67±12.47 days with higher number of eggs laid, i.e. 1427.67±62.89 eggs at a higher hatchability rate of 57.05±8.39% or 814.49 eggs hatched. Thus, 5% sucrose solution fortified with 1% Vitamin B complex should be used to produce colonies of homogenous mosquitoes as this exerts positive biological effects on laboratory-bred Cx. quinquefasciatus.

Similar vertical transmission rates of dengue and chikungunya viruses in a transgenic and a non-transformed Aedes aegypti (L.) laboratory strain

The increase of the burden of dengue and chikungunya and the relative failure of traditional vector control strategies have highlighted the need to develop new control methods. RIDL-SIT, a vector control method based on the release of engineered male mosquitoes, has shown promising results from field trials conducted in the Cayman Islands and Brazil. In large scale use, a small proportion of females might be released along with the males. Such females are potential virus vectors; here we investigate the vertical transmission of dengue and chikungunya of homozygous OX513A females.We provided females of OX513A-My1 and a wild type comparator strain with blood meals artificially infected with dengue serotype 1, 2, 3, 4 or chikungunya viruses. For 14 days post-feeding, eggs laid by females were collected. Larvae and their mothers were first tested by qRT-PCR, then by inoculation on cell cultures to search for infectious viral particles. We found no significant difference between the minimum infection rate of OX513A-My1 and wild type females. We also discussed the potential number of females being released, a fraction of the female wild population. Consequently, we conclude that there are no evidence that OX513A-My females, if released into the environment, would cause more harm than their wild counterparts.

Evaluation of Sumithion L-40 against Aedes aegypti (L.) and Aedes albopictus Skuse

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 240ml/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. INTRODUCTION Dengue is a serious public health disease in Malaysia since the first nationwide dengue outbreak in 1973; the most prevalent vectors for dengue are Aedes aegypti and Aedes albopictus (Lee et al., 1997). In the continued absence of specific treatment and effective vaccine against dengue virus, dengue control relies on suppressing Aedes populations and subsequent interruption of disease transmission through the use of insecticides, especially during outbreaks (Esu et al., 2010). The organophosphate fenitrothion was applied in space spraying for dengue and malaria control since 1970s (Samutrapongse & Pant, 1973; Pant

Vertical distribution of Aedes mosquitoes in multiple storey buildings in Selangor and Kuala Lumpur, Malaysia

The aim of the present study was to determine the vertical distribution and abundance of Aedes mosquitoes in multiple storey buildings in Selangor and Kuala Lumpur, Malaysia. Ovitrap surveillance was conducted for 4 continuous weeks in multiple storey buildings in 4 residential areas located in Selangor [Kg. Baiduri (KB)] and Kuala Lumpur [Student Hostel of University of Malaya (UM), Kg. Kerinchi (KK) and Hang Tuah (HT)]. The results implied that Aedes mosquitoes could be found from ground floor to highest floor of multiple storey buildings and data from different elevation did not show significant difference. Ovitrap index for UM, KB, HT and KK ranged from 0 – 29.17%, 0 – 55.56%, 8.33 – 83.33% and 0 – 91.17% respectively. Aedes aegypti and Aedes albopictus were found breeding in HT, KK and KB; while only Ae. albopictus was obtained from UM. The results indicate that the invasion of Aedes mosquitoes in high-rise apartments could facilitate the transmission of dengue virus and new approaches to vector control in this type of residential area should be developed.

A new record for Lispe orientalis Wiedemann, 1824 (Diptera: Muscidae) from peninsular Malaysia

Lispe orientalis Wiedemann, 1824 is recorded for the first time in peninsular Malaysia. Specimens were collected from a mushroom cultivation farm in Genting Highlands, Pahang (3°25’18"N 101°47’48"E). Previously, this species had been recorded from Azerbaijin, India, Russia, Tajikistan, Thailand, Turkey and South Korea. The male of Lispe orientalis can be determined by the following characteristics: body non-metallic, ashy gray, third antennal segment black, R5 cell not narrow apically, hind metatarsus normal, legs entirely black, femora with long bristle-like hairs on av and pv surfaces, hind tibia without av and pv seta and the palpi orangish in colour.

Entomological study of chikungunya infections in the State of Kelantan, Malaysia.

Background & objectives: Chikungunya infection has become a public health threat in Malaysia since the 2008 nationwide outbreaks. Aedes albopictus Skuse has been identified as the chikungunya vector in Johor State during the outbreaks. In 2009, several outbreaks had been reported in the State of Kelantan. Entomological studies were conducted in Kelantan in four districts, namely Jeli, Tumpat, Pasir Mas and Tanah Merah to identify the vector responsible for the virus transmission. Methods: CHIKV cases records were obtained from State Health Department, Kelantan and localities involved were identified. Larva survey was conducted to collect the immature mosquito stages. Modified aspirators were used to collect the adult mosquitoes. All samples on dry ice were transferred to laboratory and the presence of the virus was detected using reverse transcriptase PCR. Results: A total of 1,245 mosquito larvae were collected during larval survey and 2,019 adult mosquitoes were collected using aspirator. From these collections, 640 mosquito pools were tested for the presence of CHIKV by RT-PCR but none found positive. Ae. albopictus was the most abundant mosquito collected, followed by Culex sp., Armigeres sp. and Anopheles sp. A total of 2, 814 artificial containers were inspected during the study. Interpretation & conclusions: Since none of the mosquito samples was found to be positive for chikungunya virus, the vector(s) of chikungunya virus in these localities could not be identified.

Insecticide resistance development in Aedes aegypti upon selection pressure with malathion

Bioassay test against malathion had been carried out with larval and adult stages of Aedes aegypti. The mosquitoes were under selection pressure against malathion for fortyfive consecutive generations. The rate of resistance development was measured by LC50 and LT50 values. The larvae and adult females, after subjection to malathion selection for 45 generations, developed high resistance level to malathion, with resistance ratio of 52.7 and 3.24 folds, respectively over control mosquitoes. Cross-resistance towards the same and different groups of insecticides was determined using the F44 and F45 malathion-selected adult females. Insecticides tested were DDT (4.0%), permethrin (0.75%), propoxur (0.1%), fenitrothion (1%), λ-cyhalothrin (0.05%) and cyfluthrin (0.15%). Results indicated that the mosquitoes were highly resistant to DDT and fenitrothion, moderately resistant to propoxur, tolerant to permethrin and λ-cyhalothrin, and very low resistant to cyfluthrin.

Scanning electron microscopic evaluation of the successful sterilization of Lucilia cuprina (Wiedemann) utilized in maggot debridement therapy (mdt)

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.

Aedes larval population dynamics and risk for dengue epidemics in Malaysia

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. Dengueprone 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.

Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

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.

Cross-mating between Malaysian strains of Aedes aegypti and Aedes albopictus in the laboratory.

Reciprocal and homologous mating experiments between Malaysian Aedes aegypti and Aedes albopictus mosquitoes were conducted in the laboratory. Two methods were employed, namely an artificial mating technique and a natural cage mating technique. The study demonstrated there exists a strong reproductive isolation between Ae. aegypti and Ae. albopictus. Insemination occurred in cross-mating experiments between Ae. aegypti females and Ae. albopictus males and also between Ae. albopictus females and Ae. aegypti males. Cross mating between Ae. aegypti females and Ae. albopictus males produced more eggs than that between Ae. albopictus females and Ae. aegypti males with both artificial mating and natural cage mating techniques. The matings did not result in the production of viable eggs by the females. No embryonation of these eggs was observed when the eggs were bleached. With homologous mating Aedes aegypti produced significantly greater numbers of eggs compared to Aedes albopictus mosquitoes, and all the eggs hatched successfully.

Persistency of transovarial dengue virus in Aedes aegypti (Linn.).

A study was conducted to examine the persistency of transovarial dengue virus type 2 (DEN-2) in a Selangor strain of Aedes aegypti mosquitoes. Two hundred 4-5 day old female mosquitoes were fed with blood containing dengue virus. The infected mosquitoes were reared to the 7th generation; each generation was screened for the virus using immunological staining methods. The virus was detectable until the 5th generation but absent in the 6th and the 7th generations. Therefore, dengue virus type 2 can be transmitted transovarially in Aedes aegypti mosquitoes until the fifth generation under laboratory conditions.

Impact of larviciding with a Bacillus thuringiensis israelensis formulation, VectoBac WG, on dengue mosquito vectors in a dengue endemic site in Selangor State, Malaysia.

The field bioefficacy of a wettable granule (WG) formulation of Bacillus thuringiensis israelensis (Bti), VectoBac WG (Bti strain AM65-52) against dengue vectors, Aedes aegypti and Ae albopictus; was evaluated in a suburban residential area (TST) and in a temporary settlement site (KB) in the state of Selangor, Malaysia. Pre-control ovitrap surveillance of the trial sites indicated a high population of both types of Aedes mosquitoes. The populations were monitored continuously by weekly ovitrapping. Bti was sprayed biweekly at a dosage of 500 g/ha by using a mist-blower. The spray application was targeted into outdoor larval habitats. If required, Bti formulation was also applied directly into indoor water-holding containers at 8 g/1,000 l. Based on ovitrap surveillance, a significant reduction in Aedes populations was evident 4 weeks after initiating the first Bti treatment. The ovitrap index (OI) and the larvae density decreased drastically in both trial sites. In TST, the indoor OI was significantly reduced from 57.50 +/- 7.50% to 19.13 +/- 5.49% (p<0.05), while the outdoor OI decreased from 38.89 +/- 11.11% to 15.36 +/- 5.93%. In KB, similarly, the OI was significantly reduced by more than half, from 66.66 +/- 6.67% to 30.26 +/- 2.99% (p< 0.05). In all cases, the reduction in OI was paralleled by reduction in larval density.

Selective cytotoxic activity against leukemic cell lines from mosquitocidal Bacillus thuringiensis parasporal inclusions.

The discovery of parasporin has triggered an interest in examining various Bacillus thuringiensis (Bt) isolates for specific anti-cancer activity. The aim of this study was to determine the potency and specificity of parasporal inclusions from Malaysian mosquitocidal Bt isolates against a leukemic cell line (CEM-SS). The Bt isolates used in this study were identified as having weak to potent larvicidal activity against Aedes aegypti and varying hemolytic activity. The 12 mosquitocidal Bt isolates examined in this study showed low to moderate cytotoxicity when tested against CEM-SS and HeLa. Interestingly the parasporal inclusions of Bt 18 (non-hemolytic isolate), showed therapeutic potential demonstrating specificity for CEM-SS compared to HeLa, whilst being non-cytotoxic to normal T lymphocytes. The mode of cell death by Bt 18 was shown to be initially apoptotic. SDS-PAGE analysis and N-terminal sequencing of the upper and lower bands of Bt 18 showed similarity between Bt 18 parasporal inclusions with Cry 24Aa and 25Aa of Bacillus thuringiensis subsp jegathesan and Cry 15Aa of Bacillus thuringiensis subsp israelensis. Although the BLAST analysis did not show sequence similarity between Bt 18 and Parasporin, we propose that the Bt 18 parasporal inclusions share similar characteristics to Parasporin since Bt 18 is not hemolytic but discriminately cytotoxic towards leukemic cell lines.