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.
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.
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 forty-five consecutive generations. The rate of resistance development was measured by LC(50) and LT(50) 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.
The standard laboratory strain was found to be heterozygous for susceptibility. Hence, an attempt was made to obtain a homozygous susceptible strain in Culex quinquefasciatus (Say) using single raft sib-selection method. Lab-bred females of Cx. quinquefasciatus from insectariums, Unit of Medical Entomology were used in the experiment. After blood feeding Cx. quinquefasciatus mosquitoes laid eggs in raft form, ten rafts selected randomly for the test. Each egg raft was introduced into a plastic tray from number one to number ten. Twenty-five third stage larvae from each tray were exposed to 17.5 microl from 500mg/l malathion in a paper cup label number 1 to number ten. In the bioassay, which had 100% mortality, the respective larva in that particular tray was bred to adult stage for the following generation. Less than 7days old female mosquitoes that emerged from F(0) were used in the test. The F(0) and the subsequent adult and larval stage generations were subjected to adult and larval bioassay. After selection for about 10 generations, a homozygous susceptible strain in Cx. quinquefasciatus was obtained.
The susceptibility of Culex quinquefasciatus to chemical insecticides in two field sites in Kuala Lumpur was evaluated using the WHO standard susceptibility test. Less then 7 days old female mosquitos, reared from wild caught females were exposed to discriminating dosages of insecticides at recommended exposure periods. The larval bioassay were conducted using the multiple concentrations and the LC50 value was determined. The results indicated that cyfluthrin is the most effective among all the insecticides tested with LT50 value of 29.95 min and 28.59 min, for the strain from Ampang Hill and Pantai Dalam, respectively. It was surprisingly to note that both these field strains showed 0% mortality when tested against malathion and DDT. The LC50 value indicated that both strains were highly resistant to malathion with resistance ratio of 17,988 folds and 14,053 folds, respectively. This concludes that resistance at larval stages is extremely high compared to adult stages.
Laboratory strain of the Malaysian Culex quinquefasciatus was susceptible to Wuchereria bancrofti. Thirty three percent of the Cx. quinquefasciatus that fed on W. bancrofti patient were infective after 12-14 days. There is a possibility for W. bancrofti to occur in the urban areas of the Malaysia in the near future.
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.
Field strains of house flies, Musca domestica L., from three different breeding sites-garbage dump (IMR), poultry farm (Kundang, Selangor), and agricultural farm (Kampung Batu, Kuala Lumpur), were evaluated against five insecticides. Resistance status of adult female flies was determined using the modified WHO bioassay methods. The WHO susceptibility strain was used as a reference strain for comparison. Flies from the garbage dump and poultry farm were more resistant to the insecticides than the strain from the agricultural farm. Results obtained from bioassay tests were confirmed by in-vitro microenzyme assays of non-specific esterases and glutathione-S-transferases. Significant differences between the esterase levels of WHO and field strains were observed. Levels of glutathion-S-transferases were approximately the same, which may indicate that other enzymes are involved in house fly resistance.
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.
Chikungunya virus (CHIKV) is maintained in the sylvatic cycle in West Africa and is transmitted by Aedes mosquito species to monkeys. In 2006, four verified CHIKV isolates were obtained during a survey of arboviruses in monkeys (Macaca fascicularis) in Pahang state, Peninsular Malaysia. RNA was extracted from the CHIKV isolates and used in reverse transcription polymerase chain reactions (RT-PCR) to amplify PCR fragments for sequencing. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the whole viral sequence. A total of 11,238 base pairs (bp) corresponding to open reading frames (ORFs) from our isolates and 47 other registered isolates in the National Center for Biotechnology Information (NCBI) were used to elucidate sequences, amino acids, and phylogenetic relationships and to estimate divergence times by using MEGA 7.0 and the Bayesian Markov chain Monte Carlo method. Phylogenetic analysis revealed that all CHIKV isolates could be classified into the Asian genotype and clustered with Bagan Panchor clades, which are associated with the chikungunya outbreak reported in 2006, with sequence and amino acid similarities of 99.9% and 99.7%, respectively. Minor amino acid differences were found between human and non-human primate isolates. Amino acid analysis showed a unique amino acid at position 221 in the nsP1region, at which a glycine (G) was found only in monkey isolates, whereas arginine (R) was found at the same position only in human isolates. The time to the most recent common ancestor (MRCA) estimation indicated that CHIKV probably started to diverge from human to non-human primates in approximately 2004 in Malaysia. The results suggested that CHIKV in non-human primates probably resulted from the spillover of the virus from humans. The study will be helpful in understanding the movement and evolution of CHIKV in Malaysia and globally.
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.
Preservation of larvae retrieved from cadavers is important in ensuring the quality and integrity of entomological specimens used for the estimation of post-mortem interval (PMI). The process of killing and preserving larvae could distort the larvae leading to inaccurate estimation of PMI. In this study, the effects of killing Chrysomya megacephala larvae with hot water at different temperatures and subsequent maintenance in various preservatives were determined. Larvae not killed by hot water but preserved directly were used as control. The types of preservative used were 10% formalin, 70% ethanol and Kahle's solution. The morphological features examined were length, turgidity, curvature and coloration of larvae. Larvae killed in 80ºC hot water have shorter mean length (12.47 ± 2.86 mm) compared to those in 60ºC hot water (12.95 ± 2.69 mm). Increasing the duration of preservation in all types of preservative caused elongations of larvae treated or untreated with hot water. There were no significant changes in larval turgidity preserved in Kahle's solution compared to other two preservatives and were unaffected by the duration of storage. Larvae preserved in Kahle's solution experienced the least changes in coloration and shape compared to other preserved larvae in 70% ethanol or 10% formalin. Larvae directly immersed alive in 70% ethanol experienced the most changes in curvature, coloration and turgidity. This study suggested that killing larvae with hot water at 80ºC and preservation in Kahle's solution is the optimum method resulting in least changes in morphological features of Ch. megacephala larvae.
DNA identification of blow fly species can be a very useful tool in forensic entomology. One of the potential benefits that mitochondrial DNA (mtDNA) has offered in the field of forensic entomology is species determination. Conventional identification methods have limitations for sibling and closely related species of blow fly and stage and quality of the specimen used. This could be overcome by DNA-based identification methods using mitochondrial DNA which does not demand intact or undamaged specimens. Mitochondrial DNA is usually isolated from whole blow fly and legs. Alternate sources for mitochondrial DNA isolation namely, egg, larva, puparium and empty puparium were explored in this study. The sequence of DNA obtained for each sample for every life cycle stage was 100% identical for a particular species, indicating that the egg, 1st instar, 2nd instar, 3rd instar, pupa, empty puparium and adult from the same species and obtained from same generation will exhibit similar DNA sequences. The present study also highlighted the usefulness of collecting all life cycle stages of blow fly during crime scene investigation with proper preservation and subsequent molecular analysis. Molecular identification provides a strong basis for species identification and will prove an invaluable contribution to forensic entomology as an investigative tool in Malaysia.
Estimation of post-mortem interval (PMI) is crucial for time of death determination. The advent of DNA-based identification techniques forensic entomology saw the beginning of a proliferation of molecular studies into forensically important Calliphoridae (Diptera). The use of DNA to characterise morphologically indistinguishable immature calliphorids was recognised as a valuable molecular tool with enormous practical utility. The local entomofauna in most cases is important for the examination of entomological evidences. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different insect species on corpses. In this study, calliphorids were collected from 13 human corpses recovered from indoors, outdoors and aquatic conditions during the post-mortem examination by pathologists from the government hospitals in Malaysia. Only two species, Chrysomya megacephala and Chrysomya rufifacies were recovered from human corpses. DNA sequencing was performed to study the mitochondrial encoded COI gene and to evaluate the suitability of the 1300 base pairs of COI fragments for identification of blow fly species collected from real crime scene. The COI gene from blow fly specimens were sequenced and deposited in GenBank to expand local databases. The sequenced COI gene was useful in identifying calliphorids retrieved from human corpses.
The natural and artificial mating of laboratory bred Aedes albopictus and transgenic Aedes aegypti RIDL-513A-Malaysian strain was conducted. The experiment consisted of crossmating of homologous Ae. aegypti RIDL female symbol X Ae. aegypti RIDL male symbol and reciprocal Ae. aegypti RIDL female symbol X Ae. albopictus WT male symbol. The other set comprised homologous Ae. albopictus WT female symbol X Ae. albopictus WT male symbol and reciprocal Ae. albopictus WT female symbol X Ae. aegypti RIDL male symbol. This study demonstrated that reproductive barriers exist between these two species. Cross insemination occurred between A. albopictus male and Ae. aegypti female and their reciprocals. There was 26.67% and 33.33% insemination rate in Ae. aegypti RIDL female cross-mating with A. albopictus WT male and Ae. albopictus female cross-mating with Ae. aegypti RIDL male, respectively. There was 0% hatchability in both directions of the reciprocals. There was also no embryonation of these eggs which were bleached. Although none of the female Ae. albopictus WT was inseminated in the cross-mating with Ae. albopictus WT female symbol X Ae. aegypti RIDL male symbol, a total of 573 eggs were obtained. The homologous mating was very productive resulting in both high insemination rate and hatchability rates. Generally there was a significantly higher insemination rate with artificial mating insemination of homologous than with artificial mating of reciprocal crosses. Interspecific mating between Ae. aegypti RIDL and Ae. albopictus wild type was not productive and no hybrid was obtained, indicating absence of horizontal transfer of introduced RIDL gene in Ae. aegypti to Ae. albopictus.
Larvae and adults of Culex quinquefasciatus were used for the test undertaken for malathion resistant strain (F61 - F65) and permethrin resistant strain (F54 - F58). The results showed that the LC50 for both malathion (F61 - F65) and permethrin (F54 - F58) resistant Cx. quinquefasciatus increased steadily throughout the subsequent five generations, indicating a marked development of resistance. The adult female malathion resistant strain have developed a high resistance level to malathion diagnostic dosage with a resistance ratio of 9.3 to 17.9 folds of resistance compared with the susceptible Cx. quinquefasciatus. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developed at a higher rate in adult females compared to permethrin. Enzyme-based metabolic mechanisms of insecticide resistance were investigated based on the biochemical assay principle. From the results obtained obviously shows that there is a significant difference (p < 0.05) in esterase level in both malathion and permethrin selected strains. Female malathion selected strain has the higher level of esterase activity compared to the female permethrin selected strain at (0.8 to 1.04) alpha-Na micromol/min/mg protein versus (0.15 to 0.24) alpha-Na micromol/min/mg protein respectively. This indicated increased level of non-specific esterase is playing an important role in resistance mechanism in female malathion selected strain. Permethrin selected strain exhibited non-specific esterase activity at a very low level throughout the different life stages compared to malathion selected strain. This study suggests that life stages play a predominant role in conferring malathion and permethrin resistance in Cx. quinquefasciatus.
Larvae obtained from Taman Samudera (Gombak, Selangor), Kampung Banjar (Gombak, Selangor), Taman Lembah Maju (Cheras, Kuala Lumpur) and Kampung Baru (City centre, Kuala Lumpur) were bioassayed with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos. All strains of Aedes aegypti and Aedes albopictus showed percentage mortality in the range of 16.00 to 59.05 and 6.4 to 59.50 respectively, after 24 hours. LT50 values for the 6 strains of Ae. aegypti and Ae. albopictus were between 41.25 to 54.42 minutes and 52.67 to 141.76 minutes respectively, and the resistance ratio for both Aedes species were in the range of 0.68 to 1.82 when tested with operational dosage, 1 mg/L temephos. These results indicate that Aedes mosquitoes have developed some degree of resistance. However, complete mortality for all strains were achieved after 24 hours when tested against 1 mg/L temephos.
Larvae of Aedes aegypti and Aedes albopictus obtained from 6 consecutive ovitrap surveillance (OS) in Taman Samudera and Kg. Banjar were evaluated for their susceptibility to temephos. Larval bioassays were carried out in accordance with WHO standard methods, with diagnostic dosage (0.012 mg/L) and operational dosage (1 mg/L) of temephos respectively. Aedes aegypti and Ae. albopictus obtained from six OS in Taman Samudera showed resistance to diagnostic dosage of temephos with percentage mortality between 5.3 to 72.0 and 9.3 to 56.0, respectively, while Ae. aegypti and Ae. albopictus obtained from Kg. Banjar showed resistance to temephos with percentage mortality between 16.0 to 72.0 and 0 to 50.6, respectively. Only two strains of Ae. aegypti from Kg. Banjar were susceptible to temephos with 93.3% (OS 2) and 100% (OS 3) mortality. The 50% mortality at lethal time (LT50) for all strains of Ae. aegypti and Ae. albopictus tested against operational dosage of temephos showed range between 36.07 to 75.69 minutes and 58.65 to 112.50 minutes, respectively, and complete mortality was achieved after 24 hours. Our results indicated that there is weekly variations of the resistance status for Ae. aegypti and Ae. albopictus. Aedes susceptibility to temephos is changing from time to time in these two study sites. It is essential to continue monitoring the resistance of this vector to insecticides in order to ensure the efficiency of program aimed at vector control and protection of human health.
To determine resistance level and characterize malathion and permethrin resistance in Culex quinquefasciatus, two methods were used namely: WHO procedures of larval bioassay to determine the susceptibility of lethal concentration (LC) and adult bioassay to determine the lethal time (LT) which are resistant to malathion and permethrin. These mosquito strains were bred in the Insectarium, Division of Medical Entomology, IMR. Thousands of late fourth instar larvae which survived the selection pressure to yield 50% mortality of malathion and permethrin were reared and colonies were established from adults that emerged. Larvae from these colonies were then subjected to the subsequent 10 generations in the test undertaken for malathion resistant strain (F61 - F70) and permethrin resistant strain (F54 - F63). Selection pressure at 50% - 70% mortality level was applied to the larvae of each successive generation. The rate of resistance development and resistance ratio (RR) were calculated by LC5 0 for larval bioassay and LT50 value for adult bioassay. The lab bred Cx. quinquefasciatus was used as a susceptible strain for comparison purpose. The adult bioassay test was carried out by using diagnostic dosages of malathion 5.0%, permethrin 0.75% and with propoxur 0.1%. All bioassay results were subjected to probit analysis. The results showed that LC5 0 for both malathion (F61 - F70) and permethrin (F54 - F63) resistant Cx. quinquefasciatus increased steadily to the subsequent 10 generations indicating a marked development of resistance. The adult female malathion resistant strain have developed high resistance level to malathion diagnostic dosage with resistance ratio 9.3 to 9.6 folds of resistance. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developing at a higher rate in adult females compared to permethrin. Female adults exposed to 2 hours of exposure period for propoxur 0.1% showed presence of cross-resistance among the both strains of mosquitoes towards propoxur and it was indicated by 70%-100% mortality at 24 hours post-recovery period.
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.