Detection and population genetic analysis of kdr L1014F variant in eastern Ethiopian Anopheles stephensi

Abstract

Anopheles stephensi is a malaria vector that has been recently introduced into East Africa, where it threatens to increase malaria disease burden. The use of insecticides, especially pyrethroids, is still one of the primary malaria vector control strategies worldwide. The knockdown resistance (kdr) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel (vgsc) is one of the main molecular mechanisms of pyrethroid resistance in Anopheles. Extensive pyrethroid resistance in An. stephensi has been previously reported in Ethiopia. Thus, it is important to determine whether or not the kdr mutation is present in An. stephensi populations in Ethiopia to inform vector control strategies. In the present study, the kdr locus was analyzed in An. stephensi collected from ten urban sites (Awash Sebat Kilo, Bati, Dire Dawa, Degehabur, Erer Gota, Godey, Gewane, Jigjiga, Semera, and Kebridehar) situated in Somali, Afar, and Amhara regions, and Dire Dawa Administrative City, to evaluate the frequency and evolution of kdr mutations and the association of the mutation with permethrin resistance phenotypes. Permethrin is one of the pyrethroid insecticides used for vector control in eastern Ethiopia. DNA extractions were performed on adult mosquitoes from CDC light trap collections and those raised from larval and pupal collections. PCR and targeted sequencing were used to analyze the IIS6 transmembrane segment of the vgsc gene. Of 159 An. stephensi specimens analyzed from the population survey, nine (5.7%) carried the kdr mutation (L1014F). An. stephensi with kdr mutations were only observed from Bati, Degehabur, Dire Dawa, Gewane, and Semera. We further selected randomly twenty resistant and twenty susceptible An. stephensi mosquitoes from Dire Dawa post-exposure to permethrin and investigated the role of kdr in pyrethroid resistance by comparing the vgsc gene in the two populations. We found no kdr mutations in the permethrin-resistant mosquitoes. Population genetic analysis of the sequences, including neighboring introns, revealed limited evidence of non-neutral evolution (e.g., selection) at this locus. The low kdr mutation frequency detected and the lack of kdr mutation in the permethrin-resistant mosquitoes suggest the existence of other molecular mechanisms of pyrethroid resistance in eastern Ethiopian An. stephensi.

Keywords: An. Stephensi; Insecticide resistance; Invasive mosquito; Kdr mutation; L1014F; Malaria.

Fig. 1.

Portions of the An. stephensi vgsc gene amplified.

Fig. 2.

Distribution of An. stephensi with kdr mutation (kdrL1014F) haplotype per site in eastern Ethiopia.The blue color in the pie charts represents the wild type kdrL1014 haplotype and the orange color represents the mutant kdrL1014F haplotype.

Fig. 3.

Phylogenetic tree of kdr exon and downstream neighboring intron in eastern Ethiopian An. stephensi and available global An. stephensi. The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model. The tree with final ML optimization likelihood (−173.70) and bootsrap values >70 are shown.

Fig. 4.

Minimum spanning network of eastern Ethiopian An. stephensi kdr exon and downstream intron haplotypes. Colors represent Ethiopian regions. Each node represents a haplotype and the proportion of that haplotype contributed by each Ethiopian region. The size of the nodes is proportional to the sample size. The ticks between nodes represent the number of nucleotide differences. The node with the red star represents the kdr mutation haplotype.