Kinetic and molecular differences in the amplified and non-amplified esterases from insecticide-resistant and susceptible Culex quinquefasciatus mosquitoes.
Two non-amplified esterases were purified from the insecticide-susceptible Pel SS strain of Culex quinquefasciatus. These were the two major esterase activity peaks in this strain. The two corresponding amplified carboxylesterases, Est alpha 2 and Est beta 2, involved in organophosphate sequestration were purified from two resistant C. quinquefasciatus strains. The Pel SS esterases were significantly less reactive with the organophosphates than those from the resistant strains. One of the Pel SS esterases was electrophoretically identical to amplified Culex Est beta 1. However, it differed kinetically, and in its nucleotide and predicted amino acid sequences from the two characterized amplified Est beta 1s, it is classified as Est beta 1(3). Restriction fragment analysis suggested Pel SS has only one Est alpha and one Est beta gene, while the resistant Pel RR has both amplified and non-amplified forms of Est alpha and Est beta. The EcoRI fragments for both Pel SS esterases were distinct from those of the amplified Est alpha 2(1), Est beta 2(1), or Est beta 1(1&2). An esterase with the same size EcoRI fragment as Est beta 1(3) was also present in Pel RR. This and restriction enzyme fragment analysis of C. quinquefasciatus field populations suggest that variability of the susceptible alleles may be lower than previously suggested. A non-amplified Est alpha with a unique EcoRI band was present in Pel RR. The previous esterase purification procedures may not have separated these amplified and non-amplified alleles. Hence, the small differences between the purified esterases from resistant strains may reflect mixtures of identical amplified alleles with different non-amplified alleles, which have significantly different k alpha values.
Department of Pure and Applied Biology, University of Wales Cardiff, Wales.
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Karunaratne
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Hemingway
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Jayawardena
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Dassanayaka
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A
Vaughan
D000818:Animals; D009033:Culicidae / Q000201:enzymology*; D005784:Gene Amplification; D007305:Insecticide Resistance; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed