CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.

CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and interaction with metabolic enzymes

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.Author SummaryEscalation of pyrethroid resistance in Anopheles mosquitoes threatens to reduce the effectiveness of our most important tools in malaria control. Studying the mechanisms underlying insecticide resistance is critical to design mitigation strategies. Here, using genome modified mosquitoes, we functionally characterize the most prevalent mutation in resistant mosquitoes, showing that it confers substantial levels of resistance to all tested pyrethroids and undermines the performance of pyrethroid-treated nets. Furthermore, we show that combining this mutation with elevated levels of a detoxification enzyme further increases resistance. The pipeline we have developed provides a robust approach to quantifying the contribution of different combinations of resistance mechanisms to the overall phenotype, providing the missing link between resistance monitoring and predictions of resistance impact.

The discovery of a novel knockdown resistance (kdr) mutation A1007G in the Voltage-Gated Sodium Channel (vgsc) on Aedes aegypti (Diptera: Culicidae) from Malaysia

Background: The usage of insecticide rendered the successful vector control program with the high usage of the pyrethroid. However, the intensive and extensive use of pyrethroid, causing resistance in Aedes aegypti and hampered the control program. Knockdown resistance (kdr) resulting from the Voltage-Gated Sodium Channel (vgsc) is one of the mechanisms of resistance in pyrethroid group insecticide. Investigating the phenotypic status of Ae. aegypti mosquitoes is a lead in knowing the current resistance status and as an indicator of the genotypic resistance. In this study, we investigate the resistance in phenotypic and genotypic of Ae. aegypti with a new kdr mutation point, A1007G was detected. Methods: Using the adult bioassay, we tested the phenotypic resistance from the Selangor state against 0.75% permethrin, 0.05% deltamethrin with and without the addition of PBO synergist. Permethrin-resistant and deltamethrin-resistant, including susceptible samples, were subjected to genotyping analysis on mutation point in domain II and domain III of Voltage-Gated Sodium Channel (vgsc). Results: Adult bioassay revealed that the Ae. aegypti was highly resistance toward 0.75% permethrin and 0.05% deltamethrin. The bioassay with the presence of PBO synergist showed an increment of mortality rate, but the status of Ae. aegypti is still resistance towards both insecticides. Genotyping result showed that three common kdr mutations (S989P, V1016G, and F1534C) have existed in the Ae. aegypti population. Luckily, a new mutation on A1007G was also detected in this population, and this is the first time reported. Conclusions: This study has brought a piece of information on the current resistance status in Ae. aegypti in Malaysia. The detection of new mutation point of A1007G has added the knowledge on the resistance in mosquitoes. Thus, this study will aid the decision in using the insecticide usage in vector control program before this invaluable insecticide rendered ineffective in killing mosquitoes.

Novel CONCOMITANT mutations L932F and I936V in the Voltage-Gated Sodium Channel and Its Association With Pyrethroid Resistance in Culex quinquefasciatus (Diptera: Culicidae)

Highly residual pyrethroids such as permethrin have been used for controlling mosquitoes that transmit infectious diseases. However, the selective pressure from such insecticides may result in cross-resistance against other pyrethroids used for household insecticides. In this study, we investigated the susceptibility of Culex quinquefasciatus Say collected from Brazil and Myanmar to permethrin in addition to four types of household pyrethroids. Both strains exhibited high resistance against all pyrethroids tested, indicating cross-resistance. Furthermore, we detected the knockdown resistance (kdr) mutations L932F+I936V in the voltage-gated sodium channel gene (VGSC) in the Brazilian strain. Notably, the L932F+I936V haplotype has previously been observed in in silico data, but it should be detected not directly from living insects. In comparison, a common kdr mutation, L1014F, was detected from the Myanmar strain. Although L1014F was also detected from the Brazilian strain, the allele frequency was too low to affect resistance. Both strains harbored the resistance-associated haplotypes of the cytochrome P450 gene, CYP9M10. The Brazilian strain demonstrated comparable resistance against pyrethroids as that of the Myanmar strain even when a cytochrome P450 inhibitor, piperonyl butoxide was added to the bioassay. Our results suggested that the L932F+I936V mutations confer the Brazilian strain of Cx. Quiquefasciatus with resistance at a comparable level to that conferred by the well-recognized kdr mutation L1014F in the Myanmar strain. The identification of unexplored mutations may improve the diagnosis and understanding of resistance of this medically important species.

Susceptibility to Pyrethroids and the First Report of L1014F kdr Mutation in Culex quinquefasciatus (Diptera: Culicidae) in Colombia

The use of insecticides for the control of Aedes aegypti (L.) (Diptera: Culicidae) in Colombia has indirectly influenced the susceptibility status of Culex quinquefasciatus Say populations. We evaluated pyrethroid susceptibility in two populations of Cx. quinquefasciatus in the Atlantico Department of Colombia and its possible resistance mechanism (kdr mutation). Bottle bioassays were performed for permethrin, deltamethrin, and λ-cyhalothrin in female mosquitoes of Cx. quinquefasciatus. The resistance ratios (RRs) for KC50 and LC50 for each insecticide in the field populations examined were determined, using the Cartagena strain as the susceptible control. The L1014F kdr mutation was identified in the para gene of the voltage-gated sodium channel (vgsc), along with its allelic and genotypic frequency. Low knockdown resistance (RRKC50) to deltamethrin was found in Puerto Colombia and Soledad populations as well as low resistance to λ-cyalothrin in this latter population. Moderate knockdown resistance to permethrin was found in both populations. At 24 h post-exposure on the other hand, there was low resistance (RRLC50) to permethrin in Puerto Colombia and moderate resistance in Soledad. Moderate resistance to deltamethrin was found in Puerto Colombia and low resistance in Soledad. Low resistance to λ-cyhalothrin was seen in Puerto Colombia and moderate resistance in Soledad. Variability was found in the susceptibility to the pyrethroids in the populations of Cx. quinquefasciatus evaluated, and the L1014F kdr mutation is reported for the first time as a possible pyrethroid resistance mechanism in this species in Colombia.