Pyrethroid resistance in Aedes aegypti from Grand Cayman

Abstract

The Grand Cayman population of Aedes aegypti is highly resistant to DDT and pyrethroid insecticides. Glutathione transferase, cytochrome P450, and esterase levels were increased in the Grand Cayman population relative to a susceptible laboratory strain, but synergist studies did not implicate elevated insecticide detoxification as a major cause of resistance. The role of target site resistance was therefore investigated. Two substitutions in the voltage-gated sodium channel were identified, V1016I in domain II, segment 6 (IIS6) (allele frequency = 0.79) and F1534C in IIIS6 (allele frequency = 0.68). The role of the F1534C mutation in conferring resistance to insecticides has not been previously established and so a tetraplex polymerase chain reaction assay was designed and used to genotype mosquitoes that had been exposed to insecticides. The F1534C mutation was strongly correlated with resistance to DDT and permethrin.

Figure 1.

Diagnostic polymerase chain reaction (PCR) for F534C sodium channel mutation. Panel A: shows the partial sequence of the Aedes aegypti sodium channel with the position of the primers used in the assay marked. Exonic regions are shown in grey with the amino acid translation above the sequence data, boxed text indicates the position of the primers and the mutation detected in the Cayman population is indicated in black. Panel B: shows a schematic of the tetraplex PCR assay indicating the expected product sizes. Panel C: provides an example of the results obtained. Lane 1: contains a 100-bp ladder and lanes 2–7: contain PCR products obtained using template from a single mosquito. The amino acid sequence at position 1534, as deduced by the results of this tetraplex assay and confirmed by sequencing, is indicated above each lane.

Figure 2.

Boxplots of results from biochemical assays. The median activity is shown by a horizontal bar; the box denotes the upper and lower quartiles. The vertical lines show the full range of the data set. Panel A = GST assay using CDNB; B = esterase assay using PNPA; C = esterase assay using α and β naphthol and D = P450 assay using heme peroxidase. Results are expressed as µmole/min/mg protein with the exception of the P450 assay, which is expressed as mg of cytochrome C equivalents/mg protein.

Figure 3.

Histogram showing acetylcholinesterase activity in the presence of propoxur. In both the New Orleans and Grand Cayman populations, remaining AchE activity was less than 30% for all individuals, suggesting that insensitive acetylcholinesterase is not a major resistance mechanism in the Cayman Islands population.