Comparison of Biochemical and Molecular Tests for Detecting Insecticide Resistance Due to Insensitive Acetylcholinesterase in Culex quinquefasciatus¹

Abstract

Insecticide resistance to organophosphates and carbamates can be the result of changes in acetylcholinesterase activity conferred by the ACE-1 mutation. Detection of this altered target site mutation is important in guiding informed decisions for resistance management. In this study we compared a competitive enzyme assay with a polymerase chain reaction assay utilizing a restriction enzyme. Both assays detected the ACE-1 mutation in Culex quinquefasciatus and agreement was 100%. The costs and benefits of each assay are presented. KeywordsCulex quinquefasciatus; acetylcholinesterase; organophosphate resistance; ACE-1 mutation Because of biological fitness costs usually associated with insensitivity of target sites to insecticides, insects carrying modified target site gene(s) generally remain at a low frequency in the absence of selection pressure. Remarkably, it is only minor changes in the gene(s), often a single base change, which make target proteins (enzyme or receptor) insensitive to insecticides (Chaudhry and MacNicoll 1998). The gene, ACE-1, is present worldwide and causes organophosphate (OP) and carbamate resistance. A high level of acetylcholinesterase (AChE)-1 protein insensitivity or resistance displayed in Culex pipiens L. is due to a single amino acid substitution, G119S, a mutation in the 3rd exon of the ACE-1 gene, leading to the replacement of a glycine (GGC, susceptible allele) by a serine (AGC) (Weill et al. 2003(Weill et al. , 2004. This mutation is associated with reduced susceptibility to OP insecticides, modifications of the catalytic properties of AChE-1, and a high fitness cost (Weill et al. 2003). Understanding the underlying resistance mechanisms is important in making informed decisions on alternative control strategies. In our current economic situation, with funding and programs being cut, it is important to have access to a variety of different testing methods for detecting mechanisms of resistance depending on one's budget, personnel, and time. In this study we compare a biochemical assay used in our laboratory HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript with a molecular assay, both of which detect resistance due to insensitive acetylcholinesterase. We evaluated not only the efficacy of each assay, but also calculated the time, cost, and skill level each assay required. Culex quinquefasciatusSay egg rafts were collected in Harris County, TX from 2004 through 2008. Mosquitoes were reared in incubators at 27.5°C, 80-85% RH, and 14 : 10 light : dark cycle. They were aspirated into 1.5-ml microtubes 1-2 wk after they had emerged and stored in a −80°C freezer (Thermo Scientific, Waltham, MA) for future use. Fifteen mosquitoes from each collection site throughout Harris County were tested, for a total sample size of 300. In order to compare the competitive enzyme assay to the polymerase chain reaction (PCR) assay, the same mosquitoes were used for both tests. For the PCR assay and sequencing, all 6 legs were removed from the females,...

Characterization of Pyrethroid Resistance Mechanisms in Aedes aegypti from the Florida Keys

“…Glutathione-S-transferase levels were not significantly different between Key West in general (median 0.0783 μg GST/μg protein) and the ovitrap survivors (median 0.0719 μg GST/μg protein), but both were significantly higher than the colony (median 0.0178 μg GST/μg protein). Using absorbance ranges described in Scott and McAllister 26 for the acetylcholinesterase (ACE-1) mutation, the frequency of the mutation in Key West and ovitrap individuals was 0.036 (95% CI: 0.096) and 0.35 (95% CI: 0.125), respectively. This target site mutation is associated with organophosphate/carbamate resistance.…”

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“…Insensitive acetylcholinesterase (AChE) assay. Modification of Ellman's method described by Brogdon [30] and the protocol of Scott and McAllister [31]. Firstly, 500 μL of 0.25M sodium acetate buffer (pH 5.0) was added into a 1.5mL Eppendorf tube containing an individual mosquito which was homogenized.…”