Insecticide resistance and underlying targets-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan

Abstract

Insecticide resistant Aedes populations have recently been reported in Pakistan, imposing a threat to their control. We aimed to evaluate the susceptibility of Aedes aegypti and Aedes albopictus populations from Lahore to WHO-recommended insecticides and to investigate metabolic and target-site resistance mechanisms. For this purpose, we first carried out bioassays with the larvicides temephos and pyriproxyfen, and the adulticides malathion, permethrin, deltamethrin, alpha-cypermethrin, and etofenprox. We looked for Knockdown resistance mutations (kdr) by qPCR, High-Resolution Melt (HRM), and sequencing. In order to explore the role of detoxifying enzymes in resistance, we carried out synergist bioassay with both species and then checked the expression of CYP9M6, CYP9J10, CYP9J28, CYP6BB2, CCAe3a, and SAP2 genes in Ae. aegypti. Both species were susceptible to organophosphates and the insect growth regulator, however resistant to all pyrethroids. We are reporting the kdr haplotypes 1520Ile + 1534Cys and T1520 + 1534Cys in high frequencies in Ae. aegypti while Ae. albopictus only exhibited the alteration L882M. PBO increased the sensitivity to permethrin in Ae. aegypti, suggesting the participation of P450 genes in conferring resistance, and indeed, CYP928 was highly expressed. We presume that dengue vectors in Lahore city are resistant to pyrethroids, probably due to multiple mechanisms, such as kdr mutations and P450 overexpression.

Figure 1

Larvicide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. (a) Dose–response bioassay to the organophosphate temephos, with mortality evaluated after 24 h of exposure. (b) Proportional mortality of each life stage to the diagnostic dose of the IGR pyriproxyfen. The adult emergence inhibition (AEI) was 100% in all experimental cases. (c) Susceptibility index (SI) average (with SEM error bars) for the pyrethroids permethrin and deltamethrin. The higher the SI, the less susceptible the population. Ae. aegypti Rockefeller strain (Rock) was used as a reference in all these larvicide bioassays.

Figure 2

Adulticide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. PAg and PAb designate Ae. aegypti and Ae. albopictus populations, respectively. Dose-diagnostic tests with the pyrethroids permethrin (0.25%), etofenprox (0.5%), alpha-cypermethrin (0.03%) and deltamethrin (0.03%) in WHO test tubes, and the organophosphate malathion (20 µg) in bottle assays. Bars and errors indicate the mean mortality ± SEM. Populations below 90% of mortality (indicated by the red dotted line) are resistant. Aedes aegypti Rockefeller reached 100% mortality in all assays run in parallel.

Figure 3

Fold-change expression of genes related to metabolic resistance of insecticides in Aedes aegypti from Lahore. The gene Rps14 was used as a normalizing reference gene, and fold-change was relative to the Rockefeller strain. Means with standard deviations are represented.

Figure 4

Kdr genotyping in the Na_V IIIS6 segment in Aedes aegypti from Lahore, Pakistan. (a) The high-resolution melting analyses (HRM) difference plot, where each line represents a sample, is grouped into three variants. Sequencing of some samples of each variant, summed with TaqMan genotyping of the F1534C SNP indicated their genotypes, which then rendered the genotypic (b) and allelic frequencies (c), considering the 1520 and 1534 sites.

Figure 5

Political map of Pakistan, divided into four provinces, with Lahore magnified in red. The map was generated with a free and open- source software QGIS version 2.18.24 (GNU General Public License), developed by the Open Source Geospatial Foundation Project (http://qgis.org).

Figure 6

Representation of the IIS6 (a) and IIIS6 (b) Na_V segments with their respective amino acid (above) and nucleotide (bellow) sequences. The SNPs at 989, 1016, 1520, and 1534 sites are indicated inside brackets with the mutant alternative in red. The shaded colors indicate the amplified sequences in the HRM reactions, with their respective primer sequences underlined and orientation following the arrows.