Dengue 2 and 4 viruses obtained from dengue-infected patients were maintained in a C6/36 Aedes albopictus Skuse cell line and used to infect adult female Aedes aegypti mosquitoes. Each serotype was mixed separately with fresh human erythrocytes and fed to adult female mosquitoes using an artificial membrane feeding technique. Fully engorged mosquitoes were selected and retained at 26 degrees C, 28 degrees C and 30 degrees C to observe dengue virus development in Aedes vectors. Virus detection was carried out by reverse-transcriptase polymerase chain reaction (RT-PCR). The virus was first detected on Day 9 at 26 degrees C and 28 degrees C and on Day 5 at 30 degrees C for both dengue 2 and 4. The study shows the incubation period of the viruses decreased when the extrinsic incubation temperature increases.
Toxorhynchites splendens larvae are a natural predator of dengue vector mosquito larvae, Aedes albopictus. This study was carried out to evaluate the predation rate of Tx. splendens third instar larvae on Ae. albopictus larvae in 24 h. Each predator was offered prey at a density between 10 to 50 individuals. Predation rate of Tx. splendens were also tested with two manipulated factors; various types of container and different water volumes. The experiment was evaluated in man-made containers (tin cans, plastic drinking glasses and rubber tires) and natural container (bamboo stumps) which were filled with different water volumes (full, half full, 1/4 full, and 1/8 full). The prey density and the characteristics of the container were found as significant factors which influence the predation rate of Tx. splendens. The predator consumed significantly more prey at higher prey densities (40 and 50 preys) compared to the lowest density (10 preys) (F=3.935, df=4, p=0.008). The results showed significantly higher consumption in horizontal shaped container of rubber tire than in vertical shape of bamboo stumps (F=3.100, df=3, p=0.029). However, the water volume had no significant effect on predation rate of Tx. splendens (F=1.736, df=3, p=0.162). We generally suggest that Tx. splendens is best to be released in discarded tires or any other containers with horizontal shape design with wide opening since Tx. splendens can become more effective in searching prey in this type of container design. This predator is also a suitable biocontrol candidates to be introduced either in wet and dry seasons in Malaysia.
The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA--random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.
To understand the effects of fish predator’s kairomones on Aedes mosquitoes’
oviposition, we established an experiment using gravid Aedes females. Kairomones
concentrations were established using Hampala macrolepidota. One individual fish was
placed inside containers with varying water levels (1 L, 5 L, and 10 L of water). The fish
were kept in the containers for 24 hours and were removed immediately at the start of
each trial in order to have the kairomones remnants. Twenty gravid adult females of
Aedes aegypti and Aedes albopictus were allowed to lay eggs on oviposition site with
various treatments: (1) control without any kairomones; (2) kairomone remnant in 1 L of
water; (3) kairomone remnant in 5 L of water; and (4) kairomone remnant in 10 L of water.
There are significant differences between the numbers of eggs laid by both Aedes species
for each different treatment (F = 9.131, df = 16, p
Insecticide resistance has become a serious issue in vector management programs. Information on insecticidal resistance and its associated mechanisms is important for successful insecticide resistance management. The selection of a colony of permethrin-resistant Aedes albopictus (Skuse) (Diptera: Culicidae), originating from Penang Island, Malaysia, yielded high larval-specific resistance to permethrin and cross-resistance to deltamethrin. Synergism assays showed that the major mechanism underlying this resistance involves cytochrome P450 monooxygenase. The resistance is autosomal, polygenically inherited and incompletely dominant (D = 0.26). Resistant larvae were reared under different conditions to assess the fitness costs. Under high larval density, larval development time of the resistant SGI strain was significantly longer than the susceptible VCRU strain. In both high- and low-density conditions SGI showed a lower rate of emergence and survival compared with the VCRU strain. Resistant larvae were more susceptible to predation by Toxorhynchites splendens (Wiedemann) (Diptera: Culicidae) larvae. The body size of SGI females reared under high-density conditions was larger compared with females of the susceptible strain. SGI females survived longer when starved than did VCRU females. The energy reserve upon eclosion was positively correlated with the size of the adults.
Four insect repellent products (RPs) (RP 1, Experimental Repellent Lotion [Bayrepel 12%]; RP 2, Experimental Repellent Cream [Bayrepel 5%]; RP 3, Off! Insect Repellent II Aerosol [deet 15%]; and RP 4, Off! Skintastic II Cream [deet 7.5%]) were evaluated simultaneously for their efficacy against vector and nuisance mosquitoes. The aim of this study was to compare the relative efficacy of RPs based on a new repellent compound, Bayrepel (1-piperidinecarboxylic acid, 2-(2-hydroxyethyl)-1-methylpropylester), with deet (N,N-diethyl-m-toluamide)-based RPs. An 8-h field efficacy of above repellents was evaluated against the day-biting mosquito (Aedes albopictus) and night-biting mosquitoes (Culex quinquefasciatus and Anopheles spp.). Evaluation was carried out by exposing humans with repellent-treated bare limbs to mosquitoes landing and to mosquitoes landing and biting. Repellent product 1 or 2 was applied on the left arm and leg, whereas RP 3 or 4 was applied on the right arm and leg, respectively. Application of these 4 RPs significantly reduced (P < 0.05) the landing and the landing and biting of day-biting and night-biting mosquitoes. All 4 RPs were found to be equally effective (P < 0.05) against Ae. albopictus and Cx. quinquefasciatus. However, for protection against Anopheles spp., RPs 1 and 3 exhibited significantly (P < 0.05) better repellency effect than RPs 2 and 4.
Susceptibility levels of a few laboratory-cultured and dengue-endemic area field-collected strains of Aedes aegypti and Aedes albopictus to Bacillus thuringiensis israelensis (Bti) at different storage ages were studied. The susceptibility of laboratory-cultured World Health Organization (WHO) Bora Bora reference, Vector Control Research Unit (VCRU), and Fumakilla Malaysia Berhad (FMB) strains of Ae. aegypti to Bti was examined. The sensitivity to Bti decreased with storage age. The median lethal concentration (LC50) for Bti increased by 2-3 times after 2 years compared to a fresh sample (3-6 months of storage). However, after the 2-year storage period, Bti still provided very good efficacy against all laboratory-cultured susceptible strains of Ae. aegypti and Ae. albopictus. The observed 95% lethal concentration values were about 20 times lower than the recommended concentration (6,000 international toxic units (ITU)/liter). Results obtained from the study against the dengue-endemic area field-collected strains of Ae. aegypti and Ae. albopictus confirmed the effectiveness of the Bti after storage for 2 years (18-24 months). For Ae. aegypti, the Ujung Batu strain was the most susceptible to Bti, whereas the Sungai Nibong strain showed the most tolerance. Susceptibility of laboratory-cultured strains varied; the Air Itam strain of Ae. albopictus was the most susceptible to Bti, whereas the Kampung Serani strain was the most tolerant among the field strains. However, the laboratory strain of Ae. albopictus was more susceptible than all the field strains.
Bioefficacy of thermal fogging application of 2 Bacillus thuringiensis israelensis formulations, Vectobac ABG 6511 water-dispersible granules (3,000 international toxic unit [ITU]/mg) and Vectobac 12AS liquid (1,200 ITU/mg), was assessed for larvicidal activities against Aedes aegypti, Aedes albopictus, Anopheles dirus, and Culex quinquefasciatus. Portable Agrofog AF35 sprayers were used to apply the 2 formulations indoors in residential premises on Penang Island, Malaysia. Vectobac ABG 6511 showed good larvicidal effect against all 4 mosquito species at 3 of the higher doses tested (2.91 x 10(9), 1.45 x 10(9), and 0.71 x 10(9) ITU/ha), with more than 96% mortality at 48 h after spraying. As a comparative formulation, Vectobac 12AS also showed good larvicidal activity against all 4 mosquito species at 2 of the higher doses tested (2.87 x 10(9) and 1.46 x 10(9) ITU/ha), with more than 92.5% mortality at 48 h after spraying. Larvae of An. dirus were significantly more susceptible to both water-based Vectobac formulations when compared to the other 3 mosquito species. Both microbial formulations showed better efficacy at higher doses. However, even at the lowest dose tested, Vectobac ABG 6511 and Vectobac 12AS (both at 0.36 x 10(9) ITU/ha) showed larvicidal properties, with more than 66% mortality at 48 h after spraying. Overall, for this bacterial agent, the water-dispersible granule formulation has better prospects than the liquid formulation for the control of larvae of vector mosquitoes.
Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450.
Routine surveillance on resistant status of field mosquito populations is important to implement suitable strategies in order to prevent pest outbreaks. WHO test kit bioassay is the most frequent bioassay used to investigate the susceptibility status of field-collected mosquitoes, as it is relatively convenient to be carried out in the field. In contrast, the topical application of active ingredient is less popular in investigating the susceptibility status of mosquitoes. In this study, we accessed the susceptibility status of Aedes albopictus Skuse collected from two dengue hotspots on Penang Island: Sungai Dua and Persiaran Mayang Pasir. Two active ingredients: permethrin and deltamethrin, were used. WHO test kit bioassay showed that both wild strains collected were susceptible to the two active ingredients; while topical application assay showed that they were resistant. This indicated that WHO test kit bioassay less sensitive to low level of resistance compared to topical application assay. Hence, topical application is expected to be more indicative when used in a resistance surveillance programme.
Studies were carried out on the residual efficacy of Bacillus thuringiensis H-14 (water dispersible granule, VectoBac ABG 6511) as direct application in the control of Aedes larvae in the field. Field Aedes sp populations in the earthen and glass jars were predetermined before initiation of the trial. On confirmation of the presence of Aedes species in the designated area, Sungai Nibong Kecil, Penang Island, Malaysia, Bti was introduced in the 55L earthen and 3L glass jars). Two test designs were carried out. The first design had treated water replenished daily with 6L of seasoned water and the second design is without the replenishment of water but evaporated water was replenished. Bti was effective in the field for at least 35 days with more than 80% reduction in the Aedes larvae in the treated containers. For earthen jars with daily replenishment of water, 100% reduction was recorded for the first 3 days, while more than 80% reduction was recorded up to day 40. At day 60, Bti still provided an efficacy of 54.32 +/- 4.61 (%) of reduction. Whilst for earthen jars without daily replenishment of water, 100% reduction was recorded for the first 5 days, while more than 80% of reduction was recorded up to day 40. For the glass jars studied, similar efficacy was observed. In jars with daily replenishment of water a better larval control was observed. Percentage of reduction from day 50 to 60 for replenishment of water was between 50 to 70% compared to without replenishment of water with less than 40%.
Laboratory efficacy and residual activity of a water dispersible granule formulation of Bacillus thuringiensis israelensis (Bti) at the dosages of 3000, 6000 and 15000 ITU/L were conducted in this study. The study was conducted in two different size containers, earthen jar (45 L) and glass jar (3 L) with or without daily replenishment of 6 L and 0.3 L of water in the earthen and glass jars, respectively. Results indicate that for both earthen jar and glass jar evaluations, Bti at the tested dosages, performed effectively against Aedes aegypti, giving a minimum of 42 days effective killing activity. When the dosage was increased from 3000 ITU/L to 6000 ITU/L or 15000 ITU/L, the effective periods of the Bti increased by an additional one to three weeks. The Bti water dispersible granule provided better larvicidal activity with replenishment of water compared with non-replenishment of water especially for the higher dosage (15000 ITU/L).
Dengue is a disease propagated by vectors namely Aedes spp. mosquitoes. One of the effective approaches to control dengue is through integrated vector management and intervention programs. COMBI or Communication for Behavioral Impact is a strategic approach to control the Aedes population as well as dengue cases. This study was conducted at Taman Desa Kolej, Nilai, Negeri Sembilan, an suburban residential area to determine the effectiveness of COMBI, by using questionnaire and entomological survey as well as implementation of an intervention program. We carried out activities such as the establishment of COMBI promoter team, clean up events, talk shows with the residential community and distributed pamphlets containing information about dengue. Results indicated significant difference (p
Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation of the viral vectors, Aedes aegypti and Aedes albopictus mosquitoes. Therefore, the absence of effective anti-CHIKV drugs to combat chikungunya outbreaks often leads to a significant impact on public health care. In this study, we investigated the antiviral activity of drugs that are used to alleviate infection symptoms, namely, the non-steroidal anti-inflammatory drugs (NSAIDs), on the premise that active compounds with potential antiviral and anti-inflammatory activities could be directly subjected for human use to treat CHIKV infections. Amongst the various NSAID compounds, Mefenamic acid (MEFE) and Meclofenamic acid (MECLO) showed considerable antiviral activity against viral replication individually or in combination with the common antiviral drug, Ribavirin (RIBA). The 50% effective concentration (EC50) was estimated to be 13 μM for MEFE, 18 μM for MECLO and 10 μM for RIBA, while MEFE + RIBA (1:1) exhibited an EC50 of 3 μM, and MECLO + RIBA (1:1) was 5 μM. Because MEFE is commercially available and its synthesis is easier compared with MECLO, MEFE was selected for further in vivo antiviral activity analysis. Treatment with MEFE + RIBA resulted in a significant reduction of hypertrophic effects by CHIKV on the mouse liver and spleen. Viral titre quantification in the blood of CHIKV-infected mice through the plaque formation assay revealed that treatment with MEFE + RIBA exhibited a 6.5-fold reduction compared with untreated controls. In conclusion, our study demonstrated that MEFE in combination with RIBA exhibited significant anti-CHIKV activity by impairing viral replication in vitro and in vivo. Indeed, this finding may lead to an even broader application of these combinatorial treatments against other viral infections.
We compared climatic distribution ranges between Aedes albopictus (Skuse) (Diptera: Culicidae) and the five wild (nondomesticated) species of Albopictus Subgroup of Scutellaris Group of Aedes (Stegomyia) in southern Asia. Distribution sites of the wild species concentrate in seasonal forest and savannah climate zones in India, Indochina, and southern China. The distribution of Ae. albopictus is broader than the wild species under 1) tropical rain-forest climate, 2) steppe and temperate savannah climate, and 3) continental climate with large seasonal temperature variation (hot summer and cold winter) at temperate lowlands (northernmost sites 40°N in Ae. albopictus vs 32°N in the wild species). However, the distribution of Ae. albopictus is more limited at tropical and subtropical highlands where the climate is cool but less continental (small seasonal variation, mild summer, and winter). We discuss a possibility that the broader climate ranges of Ae. albopictus are ecological or eco-evolutionary consequences of adaptation to human habitats. We also propose a general scenario for the origin, dispersal, and adaptation of Ae. albopictus in Asia as a hypothesis for future research.
Knowledge of the interrelationship of mosquito communities and land use changes is of paramount importance to understand the potential risk of mosquito disease transmission. This study examined the effects of land use types in urban, peri-urban and natural landscapes on mosquito community structure to test whether the urban landscape is implicated in increased prevalence of potentially harmful mosquitoes. Three land use types (park, farm, and forest nested in urban, peri-urban and natural landscapes, respectively) in Klang Valley, Malaysia, were surveyed for mosquito larval habitat, mosquito abundance and diversity. We found that the nature of human activities in land use types can increase artificial larval habitats, supporting container-breeding vector specialists such as Aedes albopictus, a dengue vector. In addition, we observed a pattern of lower mosquito richness but higher mosquito abundance, characterised by the high prevalence of Ae. albopictus in the urban landscape. This was also reflected in the mosquito community structure whereby urban and peri-urban landscapes were composed of mainly vector species compared to a more diverse mosquito composition in natural landscape. This study suggested that good environmental management practices in the tropical urban landscape are of key importance for effective mosquito-borne disease management.
Fogging with insecticides is one of the main control measures for adult mosquito populations employed in countries that are affected by dengue. In many such countries, urban communities are increasingly characterised by high-density residence in high-rise condominia. Although fogging is typically applied at the ground level, its efficacy in three-dimensional urban environments is poorly understood. Here, we investigated the effect of fogging on vector mosquito distribution and abundance in high-rise condominia by conducting a before-after fogging survey. We showed that although mosquitoes were significantly concentrated at the lower levels in high-rise condominia, they were found throughout the three-dimensional environments. Fogging did not significantly alter this distribution or abundance pattern across any floor level. Thus, any fogging effect was short-lived as mosquito populations recovered within a few days before the subsequent scheduled treatment. In addition, increasing fogging frequency within practicable limits did not prolong the intended control effect. As urban mosquitoes are increasingly insusceptible to fogging due to insecticide resistance and vertical avoidance, this study demonstrates the need to implement other mosquito control strategies for high-rise condominia to manage mosquito populations.