Low efficacy of delthamethrin-treated net against Singapore Aedes aegypti is associated with kdr-type resistance.

There has been a worldwide surge in the number and severity of dengue in the past decades. In Singapore, relentless vector control efforts have been put in to control the disease since the 1960's. Space spraying, fogging, chemical treatment and source reduction are some commonly used methodologies for controlling its vectors, particularly Aedes aegypti. Here, as we explored the use of a commercially available delthamethrin-treated net as an alternative strategy and the efficacy of the treated net was found to be limited. Through bioassays and molecular studies, the failure of the treated net to render high mortality rate was found to be associated with the knockdown resistance (kdr) mutation. This is the first report of kdr- mutations in Singapore's Ae. aegypti. At least one point mutation, either homozygous or heterozygous, at amino acid residue V1016G of DIIS6 or F1269C of DIIIS6 was detected in 93% of field strains of Ae. aegypti. Various permutations of wild type and mutant amino acids of the four alleles were found to result in varying degree of survival rate among local field Ae. aegypti when exposed to the deltamethrin treated net. Together with the association of higher survival rate with the presence of both V1016G and F1269C, the data suggest the role of these mutations in the resistance to the deltamethrin. The high prevalence of these mutations were confirmed in a country wide survey where 70% and 72% of the 201 Ae. aegypti analysed possessed the mutations at residues 1016 and 1269 respectively. The highest mutated frequency combination was found to be heterozygous alleles (VG/FC) at both residues 1016 and 1269 (37.8%), followed by homozygous mutation at allele 1269 (24.4%) and homozygous mutation at allele 1016 (22.9%). The kdr- type of resistance among the vector is likely to undermine the effectiveness of pyrethroids treated materials against these mosquitoes.

Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing.

Virologic surveillance for dengue through the detection of the prevalent serotype(s) circulating in the human population during inter- and intra-epidemic periods constitutes a reliable sentinel system for dengue outbreaks. We have applied a rapid and sensitive, semi-nested, reverse transcription-polymerase chain reaction (RT-PCR) assay using nonstructural protein 3 gene primers for the type-specific-detection of dengue viruses in artificially infected and in field-caught adult Aedes mosquitoes. In laboratory experiments, the assay was sensitive enough to detect one virus-infected mosquito head in pools of up to 59 uninfected heads. In a prospective field study conducted from April 1995 to July 1996, female adult Ae. aegypti and Ae. albopictus mosquitoes were caught from selected dengue-sensitive areas in Singapore and assayed by RT-PCR. Approximately 20% of 309 mosquito pools were positive for dengue viruses. Of the 23 RT-PCR-positive Ae. aegypti pools (containing 1-17 mosquitoes each), 18 pools (78.3%) were positive for dengue 1 virus. There were 40 RT-PCR-positive Ae. albopictus pools (containing 1-33 mosquitoes each) of which 31 (77.5%) were positive for dengue 1 virus. The predominant virus type responsible for the current dengue epidemic since 1995 was also dengue 1. The geographic locations of the virus-infected mosquitoes correlated with the residences or workplaces of patients within dengue outbreak areas. A total of 43.5% of the positive Ae. aegypti pools and 25.0% of the positive Ae. albopictus pools contained only a single mosquito. Both Aedes species showed similar overall minimum infection rates of 57.6 and 50 per 1,000 mosquitoes. Infected Ae. aegypti were detected as early as six weeks before the start of the dengue outbreaks in 1995 and 1996. However, infected Ae. albopictus appeared later, when the number of cases was increasing. Virologic surveillance by RT-PCR for detecting dengue virus-infected Aedes mosquitoes in the field may serve as an early warning monitoring system for dengue outbreaks.

Bioassay and biochemical studies of the status of pirimiphos-methyl and cypermethrin resistance in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Singapore.

Aedes (Stegomyia) aegypti (Linnaeus) and Ae. (Stegomyia) albopictus (Skuse) were sampled from five regions of Singapore (Central, North East, North West, South East and South West) and tested with diagnostic concentrations of the technical grade insecticides, pirimiphos-methyl and cypermethrin. Biochemical assays were performed on the same populations of Ae. aegypti and Ae. albopictus to determine activities of detoxifying enzymes, including non-specific esterase (EST), monooxygenase (MFO) and acetylcholinesterase (AChE). The diagnostic test showed that all Ae. aegypti populations were susceptible to pirimiphos-methyl (mortality = 99 to 100%), but resistant to cypermethrin (mortality = 11 to 76%). Resistance to pirimiphos-methyl was observed in all Ae. albopictus populations (mortality = 49 to 74%) while cypermethrin resistance was detected in most Ae. albopictus populations (mortality = 40 to 75%), except those from Central (mortality = 86%) and South East (mortality = 94%) showing incipient resistance. The biochemical assays showed that there was significant enhancement (P < 0.001) of MFO activity in pyrethroid-resistant Ae. albopictus populations and most Ae. aegypti populations. The biochemical assay results suggested that AChE could play a role in pirimiphos-methyl resistance of Ae. albopictus in South West, South East and North East regions. The small but significant increase in EST activities in Ae. aegypti from all regions suggest that it may play a role in the observed cypermethrin resistance.

A novel mosquito feeding system for routine blood-feeding of Aedes aegypti and Aedes albopictus.

A novel mosquito feeding system for routine blood-feeding of Aedes aegypti and Aedes albopictus was developed and evaluated. The system consisted of a collagen membrane casing filled with specific pathogen free (SPF) mini-pig blood, which is warmed by a simple in-house designed heating device. Blood feeding rate, fecundity, survival rate and hatchability of Ae. aegypti and Ae. albopictus colonies maintained by the feeding system were compared with those raised by conventional guinea pig feeding method. Aedes aegypti, displayed a significant difference in the feeding rate when offered blood meal using the membrane feeding (85.3%) and the guinea pig feeding (96.2%) methods (P=0.012). Though the feeding rate was reduced, the level was acceptable for maintenance of laboratory colonies. There was no significant difference in the fecundity (P=0.556), survival rate (P=0.715), and hatchability (P=0.932) between the two methods. For Ae. albopictus, the two feeding methods yielded no significant difference for the three parameters (fecundity, survival rate and hatchability=0.887, 0.580 and 0.564, respectively). Hence, we conclude that this simple collagen based membrane blood feeding system can be used for routine colonization of laboratory strains of Ae. aegypti and Ae. albopictus.

New mosquito species records (Diptera: Culicidae) from Singapore.

Nine species of mosquitoes in eight genera are recorded for the first time in Singapore. An additional two species were overlooked in a 1986 checklist for mosquitoes in Singapore, and one was described after 1986. Location and habitat data are provided for the nine new records. With the inclusion of these new records the number of species reported from Singapore is 137.

Evaluation of biological and chemical insecticide mixture against Aedes aegypti larvae and adults by thermal fogging in Singapore.

To improve the operational efficiency of dengue vector control in Singapore, larvicide and adulticide were applied together by thermal fog generator (Agrofog AF40). The mixture consisted of Bacillus thuringiensis var. israelensis (Vectobac 12 AS) as biological larvicide at 1.5 L/ha and pirimiphos-methyl (Actellic 50 EC) as adulticide at 100 g ai/ha, diluted 10-fold with water. Aerosol of this mixture was evaluated against the mosquito Aedes aegypti (L.) (Diptera: Culicidae) in bioassays using cages of 10 adult females exposed at heights of 0.3-2.4 m and distances of 3-12 m from the hand-held generator. Cups containing 200 mL water were treated at ground level by exposure to the aerosol application at the same distances from the generator. Subsequent larval bioassays on days 1, 7, 14, 21 and 28 post-spray involved exposing 20 larvae/cup for 48 h. Droplets had VMD 57 microm and female mosquitoes were killed by 2 s exposure to the aerosol at 3 m. We obtained 92-100% mortality of the adult mosquitoes and 100% control of larvae at 3 m distance, but only 10-13% mortality at 12 m from the fogger. In treated cups, larvae showed high mortality (92%) when exposed for 48 h even 1 month post-treatment. Results demonstrate the practical advantage of using this mixture of Vectobac 12AS and Actellic 50 EC for simultaneous control of Aedes adults and larvae, with prolonged larvicidal efficacy in treated containers.