An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

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Antisera raised against the denatured polypeptide of two organophosphate-detoxifying esterases (Bi and Al) of Culex mosquitoes were used in an immunoblot method to quantify esterase production in resistant versus susceptible strains and to detect the presence of immunologically related proteins in other insects. It was demonstrated that esterase Bi of Culex quinquefasciatus and esterase Al of Culex pipiens are overproduced in resistant strains by factors of at least 500-fold and 70-fold, respectively, as compared with the corresponding susceptible strains. These factors approximate the levels of resistance to the organophosphate chlorpyrifos determined by bioassay-i.e., about 800-fold and 100-fold, respectively. Antiesterase Bi antiserum was found to react with other type B esterases (B2 of C. quinquefasciatus and B3 of Culex tarsalis) but not with type A esterases (Al of C. pipiens, A2 of C. quinquefasciatus, or A3 of C. tarsalis); similarly, antiesterase Al antiserum was found to react with other type A esterases (A2 and A3) but not with type B esterases (Bi, B2, and B3). Proteins immunologically related to esterase Bi were detected in Aedes aegypti L., Myzus persicae Sultzer, and Musca domestica L., although they were not overproduced in the organophosphate-resistant strains of these species. In none of these species were proteins immunologically related to esterase Al found.In many insect species, resistance to organophosphate insecticides is due to increased detoxification activity (1) resulting either from structural modification of the detoxifying enzymes involved or from enhancement of their synthesis. At present, there is no simple method for distinguishing between these two types of mechanisms, although it is clear that such knowledge would improve our understanding of the evolutionary dynamics of resistance and could have important implications in the formulation of strategies for resistance management.In the present study we have developed immunological techniques for quantifying the synthesis of organophosphatedetoxifying esterases and have demonstrated that in organophosphate-resistant Culex mosquitoes these enzymes may be produced in markedly greater amounts, to 500-fold greater, than in susceptible strains.In mosquitoes, as well as in many other insects (2-13), MATERIALS AND METHODSPurification of Esterases and Preparation of Specific Antibodies. Esterases Al and B1 were purified from strain S54 of C. pipiens and strain Tem-R of C. quinquefasciatus, respectively. These strains are homozygous for the presence of the respective highly active enzymes (15). Mass homogenates of adults were prepared in a 0.025 M imidazole-saline buffer at pH 7.4 and subjected to two consecutive ultracentrifugations at 10,000 and 100,000 x g. Supernatant proteins were then submitted to chromatofocusing between pH 4.0 and pH 7.4 (16). The fractions containing esterase activity were pooled, and their proteins were precipitated by the addition of trichloroacetic acid to a final concentration of 10% (wt/vol). The p...

Esterase activity is monitored in mosquitoes and other arthropod species because high levels of these enzymes can be associated with pesticide resistance. In the 1950s, G. Gomori devised a colorimetric method to detect esterase activity based on their capacity to hydrolyze aryl-esters. We modified this method for use in microtiter plates. Mosquito homogenates (Culex quinquefasciatus Say and C. pipiens L.) from strains susceptible and resistant to insecticides were allowed to hydrolyze alpha-naphthyl acetate in the presence of Triton X-100 and a specific acetylcholinesterase inhibitor. The alpha-naphthol product was detected colorimetrically by a diazo-coupling reaction with Fast Garnet GBC salt. Triton X-100 improved the extraction of esterases and maintained the azo compound in solution. The linear range of the method was 2-20 nmoles of alpha-naphthol; this high sensitivity permitted accurate determinations in 1/30 portions of single adult mosquitoes from the strain with the lowest esterase activity. To avoid variations due to changes in temperature and duration of assay, results were normalized to equivalent enzyme activity units obtained in a spectrophotometer at 25 degrees C. Depending on the number of homogenate dilutions required, performance of the assay in microplates allowed the simultaneous analysis of 20-80 samples. Female mosquitoes showed higher enzyme activity than males when expressed in nmoles/min per mosquito, but differences were reduced when results were expressed as specific activity (nmoles/min per mg protein). A mosquito strain resistant to organophosphates due to the presence of high levels of esterases showed about 200 times more esterase activity than a susceptible strain or a strain resistant due to insensitive acetylcholinesterase.

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