Malaria and Dengue Mosquito Vectors from Lao PDR Show a Lack of the rdl Mutant Allele Responsible for Cyclodiene Insecticide Resistance

The gamma-aminobutyric acid (GABA) receptor, RDL, plays important roles in neuronal signaling and is the target of highly effective insecticides. A mutation in RDL, commonly A296S, underlies resistance to several insecticides such as cyclodienes. Even though the use of cyclodienes has been banned, the occurrence of mutations substituting A296 is notably high in mosquitoes from several countries. Here, we report a survey investigating the prevalence of the Rdl mutant allele in mosquitoes from Laos, a country where mosquito-borne diseases such as malaria and dengue fever are health concerns. Anopheles and Aedes mosquitoes were collected from 12 provinces in Laos. Adult bioassays on Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) showed that all the populations tested were susceptible to dieldrin (4%) following WHO protocols. Exon 7 from a total of 791 mosquitoes was sequenced to identify the amino acid encoded for at 296 of RDL. Only one of these mosquitoes, Anopheles maculatus rampae Harbach and Somboon (Diptera: Culicidae) from Attapeu, carried the mutant allele being heterozygous for A296S. We therefore found a general lack of the Rdl mutant allele indicating that mosquitoes from Laos are not exposed to insecticides that act on the GABA receptor compared to mosquitoes in several other countries. Identifying the prevalence of the Rdl mutation may help inform the potential use of alternative insecticides that act on the GABA receptor should there be a need to replace pyrethroids in order to prevent/manage resistance.

PCR based monitoring of specific Drosophila (Diptera: Drosophilidae) cyclodiene resistance alleles in the presence and absence of selection

Cyclodiene insecticide resistance persists in field populations of Drosophila spp. at a frequency of approximately 1% (0.01), despite the withdrawal of most cyclodiene type insecticides except endosulfan. However, we have previously documented that resistance-associated amino acid replacements in the gene Rdl, a γ-aminobutyric acid receptor, can significantly affect several channel functions of the integral chloride ionophore. We were therefore interested in investigating if different resistance-associated replacements confer significant fitness disadvantages and whether the use of endosulfan could be maintaining selection for cyclodiene resistance in the field. Using PCR amplification of specific alleles (PASA) within 3000 individual flies, we report that neither the alanine302 > serine (allele 1) replacement in Drosophila melanogaster Meigen nor the alanine302 > serine (allele 1) or alanine302 > glycine (allele 2) replacements in D. simulans Sturtevant showed any reduction in frequency in cage experiments run for one year in the laboratory in the absence of selection. Further, repeated applications of endosulfan selected significantly for cyclodiene resistance in the field. Thus the apparent absence of fitness cost, combined with the continued use of endosulfan, may maintain cyclodiene resistance at this relatively high frequency in field populations.