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Identifier 000446603
Title Innovative methods for insect control and insecticide resistance management
Alternative Title Ανάπτυξη καινοτόμων μεθόδων για την καταπολέμηση βλαβερών εντόμων και την αντιμετώπιση της ανθεκτικότητας τους σε εντομοκτόνα
Author Σαμαντσίδης, Γεώργιος-Ραφαήλ Κ
Thesis advisor Βόντας, Ιωάννης
Δελιδάκης, Χρήστος
Reviewer Σκάβδης, Γεώργιος
Δουρής, Βασίλης
Παυλόπουλος, Αναστάσιος
Swevers, Luc
Van Leeuwen, Thomas
Abstract Insect pests pose devastating consequences in both agriculture and public health. Most of thecontrolstrategies employed rely on the use of chemical insecticides. However, the heavy use of the samechemicalcompounds have led to the natural selection of insecticide resistance phenotypes in the field. Functional validationof insecticide resistance mechanisms is a necessary step to manage and cope with this phenomenon, throughthedevelopment of diagnostic molecular tools and/or generation of efficient insecticidal compounds. In the first two chapters we used Drosophila melanagaster as a tool to functionally validate mechanismsthathave been associated with resistance against Sodium Channel Blocker Insecticides (SCBIs) and pyrethroids, twodifferent classes of neurotoxic insecticides. In the first chapter we dealt with two certain point mutationsinthevoltage gated sodium channel that have been previously associated with resistance against SCBIs. Tovalidateandmeasure the size of the resistance that each of them confers, we employed CRISPR/Cas9 technologytogenerateDrosophila strains harboring each of the two mutations (F1845Y or V1848I) in the voltage gated sodiumchannel(para) gene. Toxicity bioassays have revealed that both of them confer resistance against indoxacarbandmetaflumizone, when compared to wild type control lines, but their effect size was not uniform. Inthesecondchapter we sought to validate a putative synergistic interaction between metabolic and target siteresistancemechanisms against pyrethroids, a class of insecticides that is frequently used for the control of mosquito-vectorssuch as Aedes aegypti. Several resistance loci found in this species have been associated and functionallycorrelated(individually) with pyrethroid resistance. However, the most prevalent are mutations in the para gene(e.g. V1016G)and increased pyrethroid detoxification mediated by overexpressed P450s (e.g. CYP9J28). Herein, wecombinedstandard genetics and CRISPR/Cas9 system, to generate Drosophila strains that combine both of theseresistancealleles in the same genetic background. Toxicity bioassays have revealed that there is a synergistic interactionbetween these pyrethroid resistance alleles, yielding resistance ratios greater than the product of theresistanceratios obtained for the individual mechanisms (RRcombination: 19.85 >> RRCYP9J28:1.78 X RRV1016G: 3.00). Thefirsttwochapters provided essential information to understand novel mechanisms of resistance to insecticides, as well astoevaluate and interpret the validity of the available molecular diagnostics that they are used for monitoringresistancein the field. Considering the impacts of insecticide resistance in pest control management, it is necessarytodevelopalternative methods (such as biotechnology based) for effective and sustainable pest control, andthenexttwochapters are under this framework. In Chapter 3, we tried to develop a genetic toolkit for the non-model speciesHelicoverpa armigera. In particular we have identified and functionally characterized 4 strong RNA-Pol II promoterswith strong activity in the midgut derived cell line RP-HzGUT-AW1(MG). Additionally, we characterized4functionalU6 promoters (RNA-Pol III promoters), able to induce the expression of shRNAs and sgRNAs for RNAi andCRISPRrespectively. In Chapter 4 we opted to functionally characterize the OATP74D of two lepidoptera pests, as aputativeecdysone transporter and insecticide target. First, we generated a mutant clone of the cell line RP-HzGUT-AW1(MG), that harbors a deletion in the Oatp74D. Disruption of this gene lead to inability of the activation of theecdysonepathway, as treatment of the mutant cells with ecdysone did not alter the expression of ecdysone responsivegenes,in regard with the wild type cells. Moreover, we generated cell lines overexpressing the Oatp74Dof H. armigeraandS. frugiperda, which indicated that both of them are sufficient to induce ecdysone dependent genetranscription.Inhibition analysis also denoted that both of these proteins are sensitive to inhibitors of organic aniontransporters.Moreover, CRISPR mediated partial disruption of the gene in S. frugiperda increased the lethality rates, comparedtothe negative control. Taken all together, we conclude that lepidoptera OATP74D is an essential protein, sensitivetoinhibitors that could be used in the future as a potent insecticide target. In this context we have alsogeneratedacell-based platform for screening candidate chemical compounds against these
Language English
Subject 0ΚΙ5ΡΚ
Agricultural pests
Ecdysone importer
Insect biotechnology
Insect cell lines
Insecticide resistance
Insecticide target characterization
Molecular genetic tool-kit
U6 promoters
Έντομα εχθροι αγροτικών καλλιεργειών
Ανθεκτικότητα σε εντομοκτόνα
Βιοτεχνολογία εντόμων
Εντομομεταδιδόμενες ασθένειες
Εργαλείο μοριακής γενετικής
Κυτταρικές σειρές εντόμων
Μεταφορέας εκδυσόνης
Υποκινητές 116
Χαρακτηρισμός στόχων εντομοκτόνων
Issue date 2022-03-30
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
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