Functional characterization of carboxylesterases in pyrethroid resistant house fly, Musca domestica
Type of DegreePhD Dissertation
Entomology and Plant Pathology
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House fly, Musca domestica, is a major sanitary pest which can carry and transmit more than 100 human and animal intestinal diseases. Currently, the chemical control with insecticides is still the most efficient weapon to control its population. However, the intensive and inappropriate use of insecticides will lead to resistance issue. House fly can quickly develop resistance and cross-resistance to multiple insecticide classes. The easily development of resistance, large offspring population, and availability of genome and transcriptome database, all of which made house fly become a model insect for insecticide resistance study. As one of the major detoxification enzymes, carboxylesterases play vital roles in metabolizing insecticides and thereby conferring resistance in insects. Up-regulation of carboxylesterase genes is thought to be a major component of resistance development. In our study, a total of 39 carboxylesterase genes have been identified in house fly, eleven of which were significantly overexpressed in resistant ALHF strain compared with susceptible aabys and wild-type CS strains. Eight up-regulated carboxylesterase genes with their expressions were further induced to higher levels in response to permethrin treatments, indicating that both of the constitutive and inductive overexpression of carboxylesterases is co-responsible for the enhanced detoxification of permethrin. Further spatial expression studies revealed that these carboxylesterases were abundantly distributed in detoxification tissues and genetically mapped on autosome 2 and 3 of house flies, and their expressions could be regulated by factors on autosome 1, 2 and 5. The functions of up-regulated carboxylesterases were further explored through in vitro metabolism studies. Here, the baculovirus-mediated insect cell expression system was employed to large-scale produce interested carboxylesterase proteins. Our results indicated that these carboxylesterase proteins efficiently hydrolyzed esterase substrate α-naphthyl acetate. A cell-based MTT cytotoxicity assay revealed that Sf9 cells expressing targeted carboxylesterases enhanced the tolerance to permethrin, suggesting the important roles of carboxylesterases in metabolizing permethrin. The metabolic functions of carboxylesterases were further characterized by conducting in vitro metabolism toward permethrin, and our results suggested that these carboxylesterases showed significant efficiencies in metabolizing permethrin in vitro. Homology modelling and docking analysis were constructed to illustrate the interaction between carboxylesterases and permethrin, thus confirming the metabolic roles of carboxylesterases against insecticides in house flies. Besides the quantitative overexpressions of carboxylesterases, the qualitative changes of carboxylesterases are also responsible for their enhanced hydrolytic activities toward permethrin and thereby conferring pyrethroid resistance in insects. For carboxylesterase MdαE7 gene, eight mutations have been identified in resistant ALHF strain and four homozygous offspring line A1234, A1245, A1235 and A2345, which not only confirming that the MdαE7 gene is located on autosome 2 of house flies, but also indicating that these mutations have correlated with pyrethroid resistance in house flies. We then introduced these mutations individually into the MdαE7 gene extracted from aabys strain and investigated their functions through cell-based MTT assay and in vitro metabolism studies. Our results showed that three mutations could significantly enhance the hydrolytic activities of MdαE7 to permethrin at the expense of decreasing their carboxylic activities to generic esterase substrate α-NA, indicating that these mutations have similar effects with “mutant ali-esterase hypothesis” and play important roles in conferring pyrethroid resistance in house flies. Taken together, this study firstly comprehensive investigated the carboxylesterases in house flies and emphasized their important roles in conferring pyrethroid resistance in insects, which may facilitate the better understanding of carboxylesterase-mediated resistance and thereby providing novel strategies to efficiently prevent or impede the development of insecticide resistance in insects.