The effect of phenobarbital (PB) on the induction of tolerance to the organophosphorous insecticide temephos (TE) was investigated in Aedes aegypti L4 larvae submitted to two different PB-treatments:(1) continuous treatment from the egg to the larval L4 stage and (2) discontinuous treatment in which L4 larvae were exposed for 30 h. Mosquitoes from two Brazilian cities were studied: São José do Rio Preto (SJ) in São Paulo State and Goiânia (GO) in Goiás State. According to criterions established by World Health Organization (WHO) mosquitoes from SJ are organophosphate-susceptible while mosquitoes from GO are organophosphate-resistant. For both SJ and GO larvae the two different PB-treatments resulted in significantly increased tolerance (measured by reduced mortality) to 0.01mg/L TE while for larvae exposed to 0.02 mg/L TE only continuous PB-treatment resulted in significantly increased TE-tolerance. The reduction of mortality rate was greater in SJ larvae than in GO larvae, confirming data from other organisms indicating that the effect of PB is more pronounced in susceptible strains. To test if oxidase enzymes were involved in PB-induced tolerance we treated PB-pretreated SJ and GO larvae with the oxidase inhibitor piperonyl butoxide (PBO) before exposure to TE and observed increased (rather than decreased) tolerance, suggesting that oxidases are not involved in the tolerance process and that PB and PBO can act in concert or synergistically. Esterase patterns of PB-pretreated larvae indicated that the cholinesterases EST-13 and EST-14 are involved in the PB-induced TE-tolerance, reinforcing a previous study carried out in our laboratory which suggested that increased esterase synthesis is the mechanism responsible for the development of insecticide resistance in Aedes aegypti.
Aedes aegypti from the Brazilian cities of São José do Rio Preto (SJ) and Goiânia (GO) were analyzed as to their esterase patterns and the results were compared with data obtained about 5 years before for SJ population. Esterase bands not detected in the previous study were now observed in mosquitoes from both SJ and GO populations, being the last considered a population resistant to insecticides. Other similarities between SJ and GO populations in this study, and some differences in comparison with the previous data on SJ were observed, involving, in addition to changes in band type, changes in frequency of mosquitoes expressing them and differential gene activation during development. As it is generally true for genetic features, changes in the esterase patterns are expected to be the result of factors such as selection by environmental conditions and genetic drift. In the present case, continuous use of insecticides aiming mosquito population size control in SJ by sanitary authorities could be involved in the observed changes. Changed esterases were classified as carboxylesterases and cholinesterases, which are enzymes already shown to take part in the development of resistance in several organisms. In addition, data obtained in the elapsed time by authorities responsible for the mosquito control has shown increasing insecticide resistance of SJ population mosquitoes parallel to increase in the total amount of esterases, reinforcing the mentioned possibility.
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