Kdr-type Mutations Research Articles

Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach

BackgroundAnopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China.MethodsTo understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China.ResultsNineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F.ConclusionsA cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis.Graphical abstract

Kdr type mutations and enhanced GST based insecticide resistance in dengue vector mosquitoes Aedes aegypti and Aedes albopictus

Pyrethroid resistance has imposed a severe threat to the control programmes of dengue vectors Aedes aegypti and Ae. albopictus in Sri Lanka. Present study investigated the resistance status and underlying mechanisms of these mosquitoes in six provinces of Sri Lanka. Mosquitoes were collected from Badulla (Uva Province), Batticaloa (Eastern), Colombo (Western), Jaffna (Northern), Kandy (Central) and Kurunegala (Northwestern). Adults were exposed to discriminating dosages of permethrin, deltamethrin, λ-cyhalothrin, cyfluthrin and DDT according to WHO guidelines. Survivals from bioassays were tested for kdr type mutations. Elevation of insecticide metabolizing enzymes and insensitivity of acetylcholinesterases, the target site of organophosphates and carbamates, were also tested. A total of 4875 mosquitoes (2687 Ae. aegypti, 2188 Ae. albopictus) were subjected to bioassays. All the populations had high resistance to DDT though it has not been used in the country for more than four decades. In general, Ae. aegypti populations showed more resistance towards pyrethroids. Glutathione S-transferase (GST) activities were enhanced in most of the populations with no enhancement of carboxylesterase and monooxygenase activities. Acetylcholinesterases were not altered in both species. Aedes aegypti showed high resistance to pyrethroids, mainly due to three kdr type mutations; V1016I, S989P and F1534C found in the voltage gated sodium channel which is the target site of pyrethroids and DDT. Both species were highly resistant to DDT mainly through a GST based mechanism. In the absence of elevated esterase-based and insensitive AChE mechanisms, organophosphates and carbamates can be recommended as alternatives for future dengue vector control programmes in Sri Lanka.

Insecticide susceptibility of the sand fly leishmaniasis vector Phlebotomus argentipes in Sri Lanka

BackgroundLeishmania donovani-induced and sand fly-transmitted leishmaniasis is a growing health problem in Sri Lanka. Limited knowledge on biological and behavioral characteristics of probable vector Phlebotomus argentipes hinders disease control. Here, insecticide susceptibility patterns of P. argentipes were investigated with exploration of probable underlying resistance mechanisms.MethodsAdult sand flies were collected using standard cattle baited net traps and CDC light traps from selected sites in four districts. Adult F1 progeny of P. argentipes were exposed to different concentrations of DDT, malathion, deltamethrin and propoxur using WHO susceptibility bioassay kits. Post-1-h knockdown and post-24-h mortality were recorded and analyzed. Metabolic enzyme activity and the sensitivity of the acetylcholinesterase target-site were determined by biochemical assays using wild-caught flies. Extracted fly DNA samples were tested for the presence of knockdown-resistance (kdr) type mutations.ResultsThe LC100 values for DDT, malathion, propoxur and deltamethrin were 0.8–1.5%, 0.9–2.0%, 0.017–0.03% and 0.007% respectively. Insecticide-susceptibility levels were higher than the discriminating dosages established for Aedes mosquitoes, except for malathion. The lowest susceptibility levels (except for deltamethrin) were detected in the Mamadala population, whereas the highest levels were detected in the Mirigama population. The percentage of knocked-down sand flies was < 75% at any tested concentration, including those, which exhibited 100% mortality after 24 h. Elevated activity levels of glutathione S-transferase (3%, 7%, 12.5% and 14%) and esterase (2%, 5%, 5.5% and 6.5%) were detected in flies that originated from Mirigama, Pannala, Thalawa and Mamadala respectively, while monooxygenase quantities remained below the cut-off level. Ten to 34.5% of flies were heterozygous for acetylcholinesterases target-site insensitivity, associated with organophosphate and carbamate resistance. Pyrethroid-resistance-associated L1014F kdr-type mutation in the voltage gated sodium channel gene was detected in 30/53 flies.ConclusionsPopulations of P. argentipes in Sri Lanka are largely susceptible to common insecticides, except for malathion (used extensively in the past for malaria control). Their insecticide susceptibility appears negatively associated with past malaria endemicity of the study sites, with signs of early insecticide tolerance. Presence of insecticide target site insensitivity in a notable proportion of flies and enhanced insecticide metabolizing enzyme activities imply potential future challenges for leishmaniasis control, with a call for urgent proactive measures for its containment.

Insecticide resistance mechanisms with novel \u2018kdr\u2019 type gene mutations in the tropical bed bug Cimex hemipterus

BackgroundThe tropical bed bug, Cimex hemipterus, is a serious indoor public health pest in tropical regions causing intense physical discomfort and mental distress to humans. At present, the application of insecticides is the major control strategy. The present study was designed to evaluate the development of resistance and resistance mechanisms in Cimex hemipterus from Kandy district, Sri Lanka.MethodsThe resistance status of the collected bed bugs was determined against the discriminative dosages of DDT, malathion, propoxur, deltamethrin and permethrin by conducting bioassays according to World Health Organization guidelines. Activities of insecticide metabolizing enzymes, i.e. esterases, glutathione S-transferases (GST) and monooxygenases, and the insensitivity of organophosphate/carbamate target site acetylcholinesterase (AChE), were evaluated by biochemical assays. Regions of the gene of the pyrethroid/DDT target site, the voltage-gated sodium channel regulatory protein (VGSC), were sequenced for possible kdr mutations.ResultsSurvival percentages of bed bug population were 71, 68 and 51% for DDT, malathion and propoxur respectively. KT50 and KT90 values, calculated using log-probit mortality curves for deltamethrin were 62.55 and 123.96 h, respectively. These values were much higher for permethrin where KT50 was 201.10 h and the KT90 was beyond the detectable range. Results were compared with previous values reported for the same population in 2002. Resistance to propoxur has increased significantly from 11 to 51% with about a 20-fold increase in the number of individuals with elevated esterase mechanism. No significant change has occurred in malathion and DDT resistance, in GST and monooxygenase activities, and in AChE sensitivity for the past 14 years. Six kdr associated mutations (Y/L995H, V1010L, I1011F, L1014F, V1016E, L1017F/S) and a non-kdr associated mutation (A1007S mutation) were found from the α-region of the VGSC gene. Out of the kdr type mutations, only L1014F has been reported previously form C. hemipterus while the others have been reported from other insects.ConclusionsThe bed bug population has developed high resistance to propoxur with increased esterase activities. KT50 for deltamethrin and permethrin has increased 125- and 20-fold, respectively, over the period 2002 to 2016. To the authors’ knowledge, this is the first time that the possible involvement of a kdr type mutation in developing pyrethroid resistance in C. hemipterus has been shown in Sri Lanka.

Mechanisms of acaricide resistance in the cattle tick Rhipicephalus (Boophilus) microplus in Sri Lanka

High tolerance of ticks to acaricides is increasingly becoming a problem to cattle farmers. Resistance status of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)from two cattle farms of Sri Lanka were determined against different concentrations of pyrethroid permethrin, organophosphate malathion, organochlorine DDT and carbamate propoxur using Larval Packet Test (LPT) as recommended by Food and Agriculture Organization (FAO). Mechanisms of acaricide resistance were studied by conducting biochemical and PCR assays. Tick larvae were tested for the activity levels of acaricide metabolizing enzymes i.e.esterases, glutathione S-transferases (GSTs) and monooxygenases, and for altered target sites i.e.acetylcholinesterase (target site of organophosphates and carbamates) and sodium channel regulatory proteins (target site of pyrethroids and DDT). According to discriminating dosages specified by FAO for ticks both populations were 24–56% resistant to DDT. LC values showed that the both populations were susceptible to permethrin and resistant to malathion. Moderate insensitivity of AChEs and knock-down resistance (kdr) mutations were found as resistance mechanisms. GSTs and monooxygenases were not elevated. The kdr type mutation G72V (G215T in the gene) found in the sodium channel regulatory protein of R. (B.) microplus samples may be responsible for DDT resistance. Systematic and sophisticated insecticide resistance monitoring programmes and a better understanding on the mechanisms which govern resistance development are vital for future tick control programmes.

Pyrethroid resistance is associated with a kdr-type mutation (L1014F) in the potato tuber moth Tecia solanivora.

The Guatemalan potato tuber moth, Tecia solanivora, has been the most important pest species in Hispanico-American potato fields since its first record on potatoes in 1956 in Guatemala. This insect pest has been spreading to other parts of the world, including the Canary Islands in Europe. Tuber moth control relies heavily on the use of insecticides, including pyrethroids. Here, we assessed the likelihood of control failures and performed concentration-response bioassays in five Colombian strains of T. solanivora to evaluate their susceptibilities to the pyrethroid permethrin. Evidence of control failures was observed in four strains tested, which exhibited moderate resistance levels (i.e. ranging from 5.4- to 24.4-fold). However, no spatial dependence was observed between the permethrin LC50 values and the geographic distances among the tuber moth strains. In order to evaluate whether permethrin resistance was mediated by potential mutations in the para-type sodium channels of T. solanivora, the IIS4-IIS6 region of the para gene was PCR amplified and sequenced from the five strains tested. As demonstrated across a range of different arthropod species that exhibited knockdown resistance (kdr), we observed a single point substitution (L1014F) at high frequencies in the para gene of all four resistant strains. This is the first identification of a target-site-alteration-based resistance in the Guatemalan potato tuber moth T. solanivora, which is widespread and exhibits high frequencies among geographically distant strains, indicating that pyrethroids are probably becoming ineffective for the control of this pest species. © 2016 Society of Chemical Industry.

Expansion of the Knockdown Resistance Frequency Map for Human Head Lice (Phthiraptera: Pediculidae) in the United States Using Quantitative Sequencing.

Pediculosis is a prevalent parasitic infestation of humans, which is increasing due, in part, to the selection of lice resistant to either the pyrethrins or pyrethroid insecticides by the knockdown resistance (kdr) mechanism. To determine the extent and magnitude of the kdr-type mutations responsible for this resistance, lice were collected from 138 collection sites in 48 U.S. states from 22 July 2013 to 11 May 2015 and analyzed by quantitative sequencing. Previously published data were used for comparisons of the changes in the frequency of the kdr-type mutations over time. Mean percent resistance allele frequency (mean % RAF) values across the three mutation loci were determined from each collection site. The overall mean % RAF (±SD) for all analyzed lice was 98.3 ± 10%. 132/138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values, and only a single site had no mutations (0.0%). Forty-two states (88%) had a mean % RAF of 100%. The frequencies of kdr-type mutations did not differ regardless of the human population size that the lice were collected from, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix formulation (Prestige Brand, Tarrytown, NY) from 1998 to 2013 was correlated to the increase in kdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix formulation, which is the parallel increase of kdr-type mutations in lice over time.

Robust cuticular penetration resistance in the common bed bug (Cimex lectularius L.) correlates with increased steady-state transcript levels of CPR-type cuticle protein genes

Bed bug (Cimex lectularius L.) populations are increasing worldwide, with resistance to insecticides thought to be a major contributor. Several recent reports have documented widespread kdr-type mutations at the target site for pyrethroid insecticides, and there is substantial published evidence for metabolic resistance mediated through cytochrome P450-mediated oxidation. Here we report that resistance ratios for a bed bug strain collected in Richmond, VA, increase by three orders of magnitude when the route of insecticide treatment is changed from inoculation to topical. This increase suggests that reduced cuticular penetration plays a powerful role in bed bug resistance to insecticides. We identified 62 putative cuticle protein-encoding contigs from bed bug transcriptome data containing the Rebers and Riddiford consensus sequence. We classified these contigs as to CPR type, and compared the amino acid composition of the different types to that of the entire proteome. Quantitative PCR analysis indicated that many of these transcripts were substantially upregulated in resistant bed bugs, with some more than 20-fold higher than in the susceptible strain. These results suggest the possibility that thickening or remodeling of the bed bug cuticle may contribute to decreased insecticide penetration.

Biochemical and Molecular Analysis of Deltamethrin Resistance in the Common Bed Bug (Hemiptera: Cimicidae)

This study establishes deltamethrin resistance in a common bed bug, Cimex lectularius L., population collected from New York City (NY-BB). The NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared with an insecticide-susceptible population collected in Florida (FL-BB). General esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase activities of NY-BB were not statistically different from those of FL-BB. cDNA fragments that encoded the open reading frame of voltage-sensitive sodium channel alpha-subunit genes from the FL-BB and NY-BB populations, respectively, were obtained by homology probing polymerase chain reaction (PCR) and sequenced. Sequence alignment of the internal and 5' and 3' rapid amplification of cDNA ends (RACE) fragments generated a 6500-bp cDNA sequence contig, which was composed of a 6084-bp open reading frame (ORF) encoding 2027 amino acid residues and 186-bp 5' and 230-bp 3' untranslated regions (5' and 3' UTRs, respectively). Sequence comparisons of the open reading frames of the alpha-subunit genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly (Bemisia tabaci). V419L, located in the IS6 transmembrane segment, is a novel mutation. A Val to Met mutation at the corresponding position of the bed bug V419, however, has been identified in the tobacco budworm as a kdr-type mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB through a knockdown-type nerve insensitivity mechanism.

Characterisation of DDT, pyrethroid and carbamate resistance in Anopheles funestus from Obuasi, Ghana

Indoor-resting anopheline mosquitoes were collected from Obuasi, Ghana, and were identified morphologically and by PCR as Anopheles funestus Giles. Wild-caught females were induced to lay eggs. Samples of F1 progeny from each family were divided into cohorts and were either exposed to DDT and permethrin or were stored for biochemical analysis. Bioassay data by family show evidence of DDT and pyrethroid resistance in the parent A. funestus population. The sodium channel gene of DDT survivors and DDT-susceptible individuals was PCR amplified and sequenced to determine whether any kdr-type mutations were present. Molecular analysis of the IIS5-IIS6 segment of the sodium channel gene gave no indication of any kdr-type mutations associated with resistance phenotypes. Biochemical analysis suggests that DDT and pyrethroid resistance may be metabolically mediated, although there were no clear correlations between enzyme levels/activities and insecticide resistance across families. Furthermore, an altered acetylcholinesterase conferring carbamate resistance was evident. These results can be used to plan an effective malaria control strategy in the Obuasi region.

Insecticide resistance may enhance the response to a host-plant volatile kairomone for the codling moth, Cydia pomonella (L.)

The behavioral and electroantennographic responses of Cydia pomonella (L.) to the ripe pear volatile ethyl (2E,4Z)-2,4-decadienoate (Et-E,Z-DD), were compared in insecticide-susceptible and -resistant populations originating from southern France. A dose-response relationship to this kairomonal attractant was established for antennal activity and did not reveal differences between susceptible and resistant strains. Conversely, males of the laboratory strains expressing metabolic [cytochrome P450-dependent mixed-function oxidases (mfo)] or physiological (kdr-type mutation of the sodium-channel gene) resistance mechanisms exhibited a significantly higher response to Et-E,Z-DD than those of the susceptible strain in a wind tunnel experiment. No response of the females to this kairomone could be obtained in our wind-tunnel conditions. In apple orchards, mfo-resistant male moths were captured at significantly higher rates in kairomone-baited traps than in traps baited with the sex pheromone of C. pomonella. Such a differential phenomenon was not verified for the kdr-resistant insects, which exhibited a similar response to both the sex pheromone and the kairomonal attractant in apple orchards. Considering the widespread distribution of metabolic resistance in European populations of C. pomonella and the enhanced behavioral response to Et-E,Z-DD in resistant moths, the development of control measures based on this kairomonal compound would be of great interest for the management of insecticide resistance in this species.

Resistance and cross-resistance to insecticides in human head lice from Florida and California

Head lice from Florida (SF-HL) and California (SC-HL) were resistant to permethrin compared with colonized susceptible lice from Panama (PA-HL) (5.5- and 3.4-fold, respectively) and Ecuador (EC-HL) (8.5- and 5.3-fold, respectively). Permethrin-resistant lice were cross-resistant to pyrethrum and DDT. DNA sequencing validated presence of kdr-type mutations (T929I and L932F). Permethrin resistance was synergized by piperonyl butoxide (PBO) in SC-HL. Resistance to malathion in SF-HL (1.4–2.2-fold) and SC-HL (2.1–3.6-fold) was detected. Malathion resistance in SF-HL was synergized by S, S, S-tributylphosphorotrithioate (DEF) and by PBO in SC-HL. Malathion/permethrin-resistant lice from the UK (BR-HL) were synergized by DEF but not synerziged by PBO. PBO protected BR-HL from malathion, indicating suppressed desulfuration. Abamectin resistance in SF-HL (1.7–2.5-fold) and SC-HL (1.8–2.3-fold) was detected. No resistance to lindane was found. Thus, multiple resistance mechanisms against commonly available and widely used pediculicides and insecticides are apparently occurring.