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.

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.

Single and co-breeding of different mosquito species in fogging-free and dengue risk areas in West Malaysia

@#Ovitrap surveillance was carried out in fifteen localities encompassing foggingfree and dengue risk areas in West Malaysia to determine on the dispersal and prevalence of single and co-breeding of mosquito larvae particularly Aedes. Ovitraps were sited randomly indoors and outdoors within human settlements in all study areas. All the localities exhibited positive ovitraps with single breeding of Ae. albopictus that ranged between 64.29% and 100.00%. These findings indicated Ae. albopictus as the predominant container-breeding species in all study areas. The co-breeding of Ae. aegypti with Ae. albopictus larvae (34 ovitraps), Ae. albopictus with Culex quinquefasciatus larvae (32 ovitraps) as well as Cx. quinquefasciatus with Armigeres subalbatus larvae (1 ovitrap) were also detected in certain study localities. Interestingly, co-breeding of Ae. albopictus with Ar. subalbatus larvae as well as Ae. albopictus with Uranotaenia sp. larvae in Malaysia is reported for the first time in the present study. Better understanding of the co-breeding scenario involving different species of mosquito larvae is needed to ensure the efficacy of vector control actions to be conducted.

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.

Detection of Wolbachia from field collected Aedes albopictus Skuse in Malaysia.

Background & objectives: Wolbachia-based vector control strategies have been proposed as a mean to augment the existing measures for controlling dengue vector. Prior to utilizing Wolbachia in novel vector control strategies, it is crucial to understand the Wolbachia-mosquito interactions. Many studies have only focused on the prevalence of Wolbachia in female Aedes albopictus with lack of attention on Wolbachia infection on the male Ae. albopictus which also affects the effective expression of Wolbachia induced- cytoplasmic incompatibility (CI). In this study, field surveys were conducted to screen for the infection status of Wolbachia in female and male Ae. albopictus from various habitats including housing areas, islands and seashore. Methods: Adult Ae. albopictus (n=104) were collected using human landing catches and hand aspirator. Standard ovitraps were also set in the selected areas for five days and the larvae were identified to species level. All the collected Ae. albopictus were screened for the presence of Wolbachia using multiplex polymerase chain reaction (PCR) and gene sequencing of Wolbachia surface protein (wsp) gene. Results: A 100 per cent positivity of Wolbachia infection was observed for individual Ae. albopictus screened. For pooled mosquitoes, 73 of the 76 pools (female) and 83 of the 87 pools (male) were positive with Wolbachia infection. The wsp gene sequence of the Wolbachia strain isolated from individual and pooled mosquitoes showed a 100 per cent homology with Wolbachia sp. of Ae. albopictus isolated from various geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the isolates were clustered into groups A and B, respectively. Interpretation & conclusions: The results indicated that Wolbachia infection was widespread in Ae. albopictus population both in female and male Ae. albopictus. All the infected females were superinfected with both A and B strains while the infected males showed a combination of superinfection of A and B strains and single infection of B strain.

Morphological descriptions on the larvae of Hypopygiopsis fumipennis (Walker, 1856) (Diptera: Calliphoridae)

The study on biodiversity of forensically important Diptera in the tropical rain forest in Malaysia is scarce. Thus, a preliminary survey was conducted at a jungle fringe near Kampung Bahagia Bukit Lagong, Sungai Buloh, Selangor. A rat carcass was offered to attract carrion flies and we collected an adult female calliphorid, Hypopygiopsis fumipennis (Walker, 1856) during the fresh stage of carcass decomposition. The female fly was allowed to oviposit on chicken liver in a container and the resulting larvae were reared to the adult stage. Along the developmental process, several individuals from each instar were collected and preserved in 70% ethanol and then processed on the slides. We recorded the duration of development for each instar and described its larval features for the first time. The third instar larvae of H. fumipennis showed accessory oral sclerite present, anterior spiracle with 13-15 papillae, intersegmental spines mostly unicuspid with pointed end, and posterior spiracles heavily sclerotized with inter-slit projections. Some larval differences between H. fumipennis and Hypopygiopsis violacea were noted.

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.

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.

Rate of resistance development in wild Culex quinquefasciatus (Say) selected by malathion and permethrin.

Wild caught female Culex quinquefasciatus (Say) from Kuala Lumpur were blood fed and reared in the insectarium. The late third stage of the F1 larvae which survived the high selection pressure of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected in the subsequent 9 generations to higher selection pressure. The rate of resistance development were measured by LC50 value of larval bioassay, LT50 value of adult bioassay and the frequency of the elevated esterase levels. In another set of experiments using the same batch of Culex mosquitos, the larvae were not exposed to any insecticides and the decrease in resistance rate was monitored in each subsequent 9 generations by using similar methods. The heterozygous standard laboratory strain was selected for susceptibility using the single raft sib-selection method. The result showed that the field collected F1 generation was 96.0 and 6.3 fold more resistant to malathion and permethrin, respectively. After selection for about 9 generations the resistance ratio to malathion and permethrin was 6.2 and 767.3 fold more compared to the LC50 values of F1 generations, respectively. Esterase in F1 larvae was 6.0 fold more than the standard laboratory strain.

Microdroplet application of mosquitocidal Bacillus thuringiensis using ultra-low-volume generator for the control of mosquitos.

In an effort to develop a more effective technique in dispersing a microbial control agent, Bacillus thuringiensis (Bt), a truck-mounted ultra low volume (ULV) generator (Scorpion) was used to disperse B. thuringiensis israelensis (Bti) and Bti with malathion. Complete larval and adult mortalities for all tested mosquito species within the first 70-80 feet from the ULV generator were achieved. Beyond that distance less than 50% mortality was achieved as insufficient sprayed particles reached the area. A minimum of 10(3) Bti colony forming units per ml is required to cause 100% larval mortality. The sprayed Bti larvicidal toxins were persistent in the test water 7 days post ULV. The effectiveness of B. thuringiensis jegathesan (Btj), a new mosquitocidal Bt serotype was also evaluated. Similar mortality results as Bti were achieved except that the Btj toxins underwent degradation in the test water, since less than 50% less in larval mortality was observed in 7 days post ULV samples. This ULV method has the potential to disperse Bt and malathion effectively for a simultaneous control of mosquito adults and larvae.