Λειτουργική ανάλυση του ρόλου ενζύμων αποτοξικοποίησης και μεταλλαγών στόχου στην ανθεκτικότητα των εντόμων στα εντομοκτόνα

Agricultural pests consist a real threat because they attack crops and cause extensive damage. Crop protection is mainly based on insecticides; however, their extensive use has led to development of high levels of resistance. In the present study, resistance-associated genes are evaluated in vivo, on their ability to confer resistance, using Drosophila melanogaster as a model. The thesis consists of two parts: The first part is referred to metabolic resistance, conferred by increased expression levels of P450s. More specifically, the study focuses on the role of two P450 enzymes, the CYP6BQ23 from the pollen beetle, Meligethes aeneus and the CYP6A51 from the Mediterranean fruit fly, Ceratitis capitata. These genes are overexpressed in resistant populations and have been associated with pyrethroid resistance. In order to validate the contribution of each gene in the resistant phenotype, we attempted to ectopically express both P450s via the GAL4/UAS system in the model organism Drosophila melanogaster. The integration of the UAS transgene was achieved through phiC31 site-specific integrase in the genome of attP background susceptible flies and overexpression was accomplished by crossing the UAS line with a relevant GAL4 driver line. The susceptibility of transgenic flies to pyrethroids was evaluated with contact bioassays. In the case of CYP6BQ23 enzyme, we succeed to generate UAS.CYP6BQ23 transgenic Drosophila lines and indicate that overexpression of CYP6BQ23 in HR-GAL4 x UAS.CYP6BQ23 flies confers significant levels (~ 7-fold) of pyrethroid resistance. Moreover, in the case of CYP6A51, we performed biochemical and functional analyses of bacterially expressed CYP6A51. We achieved isolation of functional CYP6A51 enzyme by heterologous expression in E. coli and showed that it is capable to metabolize lambda-cyhalothrin in vitro, in the presence of a NADPH regenerating system. Additionally, we generated UAS.CYP6A51 flies to be used in future experiments of ectopic expression of CYP6A51 in Drosophila and contact bioassays with lambda-cyhalothrin, in order to investigate its contribution to resistance. The second part is focused on target-site resistance and attempts to study the role of a G4946E mutation in ryanodine receptor (RyR), which has been related to diamide resistance in the diamondback moth, Plutella xylostella. D. melanogaster was used as a model system in order to introduce the G4946E mutation in the ryanodine receptor, using the CRISPR/Cas9 technology, a recently developed tool for targeted genome editing. The evaluation of susceptibility to diamides and the possible effect of this point mutation to resistant phenotype is relied on feeding bioassays. However, although the introduction of the G4946E mutation in D. melanogaster was achieved, the generation of homozygous flies, which was a prerequisite for conducting bioassays considering the recessive character and monogenic inheritance of the relevant mutation, was not feasible. In conclusion, this work as part of a broader research framework, provides a better understanding of insecticide resistance mechanisms and can be used to inform appropriate strategies to manage resistance