Novel real-time PCR assay detects widespread distribution of knock down resistance (kdr) mutations associated with pyrethroid resistance in the mosquito, Culex quinquefasciatus, in Thailand.

Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.

Efficacy of five commercial household insecticide aerosol sprays against pyrethroid resistant Aedes aegypti and Culex quinquefasciatus mosquitoes in Thailand.

Commercial insecticide aerosol sprays are widely used in households for controlling Aedes aegypti and Culex quinquefasciatus, the primary vectors of dengue virus and filarial worm, respectively. In Thailand, however, both mosquitoes are resistant to pyrethroids conferred by knockdown resistance (kdr) mutations, V1016G and F1534C in Ae. aegypti and L1014F in Cx. quinquefasciatus. This study evaluated the efficacy of five sprays (coded as AS1-AS5) with different formulations of pyrethroids against wild mosquitoes by using a cage bioassay in a furnished bedroom of a house. Five cages containing wild mosquitoes and five cages containing a pyrethroid susceptible strain of Ae. aegypti (25 females each), as a bio-indicator, were allocated in the room and spraying was operated for 15 s. Survivors and dead mosquitoes were genotyped individually for the kdr mutations using allele-specific PCR methods. Both mosquito species showed a high resistance to permethrin and deltamethrin with 12.5-58.0% mortality rates. For controlling Ae. aegypti, the spray AS4 showed the highest efficacy (mortality rates 76.0-100.0%, mean 95.2%), followed by AS2 (73.0-100.0%, mean 93.8%). For controlling Cx. quinquefasciatus, the best result was obtained from AS4 (66.0-98.0% mortality, mean 89.8%), followed by AS2 (73.0-97.0%, mean 84.5%). The sprays (AS4 and AS2) containing both type I and type II pyrethroids were more effective than those containing only type I pyrethroids or pyrethrum with the synergist piperonyl butoxide. The mutant G1016 and F1014 allele frequencies were significantly higher in the survivor groups than the dead groups of Ae. aegypti and Cx. quinquefasciatus, respectively, (P < 0.05). The efficacy of the sprays varied depending on the mosquito species, formulations, nozzles and locations of caged mosquitoes. The V1016G and L1014F mutations are associated with the reduced efficacy of sprays used in households for controlling resistant Ae. aegypti and Cx. quinquefasciatus mosquitoes, respectively.

Insecticides resistance in the Culex quinquefasciatus populations from northern Thailand and possible resistance mechanisms.

The mosquito vector Culex quinquefasciatus is known to be resistant to insecticides worldwide, including Thailand. This study was the first investigation of the insecticide resistance mechanisms, involving metabolic detoxification and target site insensitivity in C. quinquefasciatus from Thailand. Adult females reared from field-caught larvae from six provinces of northern Thailand were determined for resistant status by exposing to 0.05% deltamethrin, 0.75% permethrin and 5% malathion papers using the standard WHO susceptibility test. The overall mortality rates were 45.8%, 11.4% and 80.2%, respectively. A fragment of voltage-gated sodium channel gene was amplified and sequenced to identify the knock down resistance (kdr) mutation. The ace-1 gene mutation was determined by using PCR-RFLP. The L1014F kdr mutation was observed in all populations, but the homozygous mutant F/F1014 genotype was found only in two of the six provinces where the kdr mutation was significantly correlated with deltamethrin resistance. However, none of mosquitoes had the G119S mutation in the ace-1 gene. A laboratory deltamethrin resistant strain, Cq_CM_R, has been established showing a highly resistant level after selection for a few generations. The mutant F1014 allele frequency was significantly increased after one generation of selection. A synergist assay was performed to assess the metabolic detoxifying enzymes. Addition of bis(4-nitrophenyl)-phosphate (BNPP) and diethyl maleate (DEM), inhibitors of esterases and glutathione S-transferases (GST), respectively, into the larval bioassay of the Cq_CM strain with deltamethrin showed no significant reduction. By contrast, addition of piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenases, showed a 9-fold reduction of resistance. Resistance to pyrethroids in C. quinquefasciatus is widely distributed in northern Thailand. This study reports for the first time for the detection of the L1014F kdr mutation in wild populations of C. quinquefasciatus in Thailand. At least two major mechanisms, kdr and cytochrome P450 monooxygenases, confer resistance to deltamethrin in Thai C. quinquefasciatus populations.