Status of insecticide resistance and its biochemical and molecular mechanisms in Anopheles stephensi (Diptera: Culicidae) from Afghanistan

Abstract

Background Insecticide resistance of Anopheles stephensi , the main malaria vector in eastern Afghanistan, has been reported previously. This study describes the biochemical and molecular mechanisms of resistance to facilitate effective vector control and insecticide resistance management. Methods Mosquito larvae were collected from the provinces of Kunar, Laghman and Nangarhar from 2014 to 2017. The susceptibility of the reared 3–4 days old adults was tested with deltamethrin 0.05%, bendiocarb 0.1%, malathion 5%, permethrin 0.75% and DDT 4%. Cytochrome P450 content and general esterase, glutathione S -transferase (GST) and acetylcholinesterase (AChE) activities were measured in the three field populations and the results were compared with those of the laboratory susceptible An. stephensi Beech strain. Two separate allele-specific PCR assays were used to identify L1014, L1014F and L1014S mutations in the voltage gated sodium channel gene of An. stephensi . Probit analysis, ANOVA and Hardy–Weinberg equilibrium were used to analyse bioassay, biochemical assay and gene frequency data respectively. Results The population of An. stephensi from Kunar was susceptible to bendiocarb, apart from this, all populations were resistant to all the other insecticides tested. The differences between all values for cytochrome P450s, general esterases, GSTs and AChE inhibition rates in the Kunar, Laghman and Nangarhar populations were statistically significant when compared to the Beech strain, excluding GST activities between Kunar and Beech due to the high standard deviation in Kunar. The three different sodium channel alleles [L1014 (wild type), L1014F ( kdr west ) and L1014S ( kdr east )] were all segregated in the Afghan populations. The frequencies of kdr east mutation were 22.9%, 32.7% and 35% in Kunar, Laghman and Nangarhar populations respectively. Kdr west was at the lowest frequency of 4.44%. Conclusions Resistance to different groups of insecticides in the field populations of An. stephensi from Kunar, Laghman and Nangarhar Provinces of Afghanistan is caused by a range of metabolic and site insensitivity mechanisms, including esterases, cytochrome P450s and GSTs combined with AChE and sodium channel target site insensitivity. The intensity and frequency of these mechanisms are increasing in these populations, calling for urgent reorientation of vector control programmes and implementation of insecticide resistance management strategies.

Evolution of insecticide resistance and its mechanisms in Anopheles stephensi in the WHO Eastern Mediterranean Region

“…stephensi in Kunar Province in Afghanistan is susceptible to bendiocarb so IRS using this insecticide is prescribed for malaria control in this province. Cytochrome p450, involved in pyrethroid insecticide resistance, is the highest in Laghman and Nangarhar Provinces where PBO-nets can be deployed [28]. The same situation is true in Iran as resistance to certain insecticides is province-specific (Table 1).…”

“…There are two types of kdr alleles in Anopheles mosquitoes, i.e., kdr west [33,39,103] and kdr east [26,104]. However, there is a slight difference in those two alleles in terms of conferring resistance to DDT and pyrethroids [28,105]. Therefore, not only it is important to identify the kdr alleles and its frequency in the mosquito populations under investigation, but their differential impact on phenotypic resistance should also be considered when planning for vector control interventions.…”

“…Years later, several other studies detected kdr west and east mutations in An. stephensi in India [31,38,39,48,106] and Afghanistan [26][27][28]. The presence of kdr genotypes itself in low frequency may not jeopardize the effectiveness of pyrethroid insecticides especially in the form of LLINs [107][108][109][110][111][112].…”

“…A follow-up study in Afghanistan revealed that reported resistance to pyrethroid and organophosphorus insecticides, and tolerance to bendiocarb are likely to be caused by a range of metabolic mechanisms, including esterases, cytochrome p450s and GSTs combined with insensitivity in AChE [27]. In another study in eastern Afghanistan, a combination of metabolic mechanisms and increased frequency of kdr alleles in the mosquito were blamed for elevated resistance [28]. In Pakistan, esterases are involved in malathion resistance in An.…”

“…Almost all malaria-endemic countries outside sub-Saharan Africa embraced the GMEP using the main intervention of indoor residual spraying (IRS) [6,7]. Resistance to several insecticides of all major classes has been reported in Anopheles stephensi in other countries of the WHO Eastern Mediterranean Region (EMR), including Saudi Arabia [21,22], Iraq [23,24], Afghanistan [25][26][27][28], and Pakistan [29,30]. Resistance to DDT, dieldrin, malathion, and recently pyrethroid insecticides, was reported from different states of India [31][32][33][34][35][36][37][38][39][40][41].…”