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Identifier 000446371
Title Molecular characterization of pesticide resistance in Bemisia tabaci and Tetranychus urticae and the development of diagnostics
Alternative Title Μοριακός χαρακτηρισμός της ανθεκτικότητας του αλευρώδη (Bemisia tabaci) και του τετρανύχου (Tetranychus urticae) στα εντομοκτόνα και ανάπτυξη διαγνωστικών
Author Παπαποστόλου, Κυριακή Μαρία Ι
Thesis advisor Χαλεπάκης, Γεώργιος
Βόντας, Ιωάννης
Reviewer Λάμπρου, Νικόλαος
Τσαγκαράκου, Αναστασία
Ροδιτάκης, Εμμανουήλ
Δούρης, Βασίλης
Καλαντίδης, Κρίτων
Abstract The ability of Bemisia tabaci (Hemiptera: Aleyrodidae) to transmit viral diseases, combined with the development of insecticide resistance and the rapid spread of new biotypes in new areas, make this species one of the most important agricultural pests. The spider mite Tetranychus urticae is one of the most important agricultural pests, feeding on more than 1000 different plant species, creating serious problems in rural crops in areas with temperate climate such as in the Mediterranean countries. Many active ingredients are approved for their control in our country, however, due to wrong and/or intensive use of insecticides, resistance has occurred. The main goal of this PhD study is to gain a deeper understanding of the mechanisms of insecticide resistance, to elucidate and functionally validate resistant phenotypes observed, as well as to develop a diagnostic tool to assist Insecticide Resistance Management (IRM). In the 2nd chapter of this Thesis, a T. urticae field population was collected from Peloponnese and after toxicity and synergistic assays, extremely high resistance levels to abamectin, etoxazole, clofentezine, cyflumetofen, fenpyroximate and spirodiclofen, were revealed. In addition, transcriptomic analyses unveiled P450s and UGTs that were over-expressed, on top of known target-site mutations associated with resistance. Several interesting SNPs were also uncovered in genes targeted by cyflumetofen [succinate dehydrogenase subunit D (SdhD)], abamectin [glutamate chloride channel 3 (GluCl3) and ketoenols [acetyl-CoA carboxylase (ACC)] genes that were subsequently validated by employing different approaches. In the 3rd chapter, four substitutions were functionally validated, using the CRISPR-Cas9 genome editing technique in the model organism Drosophila melanogaster, to elucidate their role in resistance to different insecticides/ acarides. The first substitution was detected in a ketoenol resistant population of B. tabaci, in the ACC gene in position 2083, where an alanine was substituted by a valine. The second substitution was identified in the ACC gene of spiromesifen (ketoenol) resistant Trialeurodes vaporariorum strains, with a glutamic acid substitution with lysine in position 645. The last two substitutions were identified in T. urticae strains. After selection pressure with ketoenols, a substitution was revealed in the ACC gene, in position 1079 with an alanine substituted by threonine while T. urticae field collected populations were abamectin resistant and after sequencing of GluCls, a substitution in GluCl3 was detected in position 321 where isoleucine was substituted by threonine. Different fly lines were generated, bearing the A2083V, A1079T and I321T mutations, except E645K, where the mutation was lethal at homozygous state. Toxicity bioassays using flies bearing the A2083V mutation, confirmed its very crucial role in ketoenol resistance with the genome modified flies being resistant to all three available ketoenol compounds (spirodiclofen, spiromesifen and spirotetramat) with Resistant Ratios (RR) >870-fold, when compared to the parental flies. Drosophila lines bearing I321T revealed moderate resistance to abamectin, and along with further experiments, it was concluded that I321T mutation has a complex role in abamectin resistance. In contrast, A1079T mutation found in a T. urticae ketoenol resistant strain, could not be associated with ketoenol resistance, using the Drosophila system. In the 4th chapter, the observed resistance in the T. urticae field collected population from Peloponnese was further investigated concerning resistance to ketoenols (spirodiclofen) and METI-II (cyflumetofen) acaricides. The already multi-resistant population was selected with spirodiclofen until resistance levels were very high (>5000 mgL-1) and was further crossed with a susceptible population. The progeny was divided in nine unsprayed (control), nine spirodiclofen-selected and nine cyflumetofen-selected populations and subsequently, Bulk Segregant Analysis (BSA) was performed. After the selection process, which lasted many generations, DNA and RNA analysis will be performed as well as the BSA genetic mapping, to identify genomic loci associated with the observed resistant phenotype. Resistance to abamectin in T. urticae has been associated with a P450, CYP392A16, that has been functionally validated in vitro showing that it metabolizes abamectin to a less toxic compound. In the 5th chapter, CYP392A16 from an abamectin resistant T. urticae population was introgressed in a susceptible genetic background by marker-assisted backcrosses and via toxicity assays and qPCR, it was unveiled that the CYP392A16 allele deriving from the resistant population provides 3.6-folds abamectin resistance and CYP392A16 is also overexpressed, at the same levels as in the resistant population, following the introgression. In addition, RNAi experiments against CYP392A16, showed that the expression levels of CYP392A16 are downregulated by almost 50% in the resistant population, on top of reducing the insecticide resistance levels from 3400- to 1900- fold, when compared to the susceptible strain. Furthermore, functional analysis of the putative promoter region from the resistant and susceptible strains revealed a higher reporter gene expression in the resistant strain, indicating the presence of cis-acting regulatory mechanisms, which is in line with the over-expression of this P450, when introgressed in a susceptible background. A specific antibody against CYP392A16 was raised and localization experiments indicated that CYP392A16 is primarily localized in the midgut epithelial cells, also supported by the comparison between feeding and contact toxicity bioassays. In the 6th chapter, six T. urticae and eleven B. tabaci field populations collected from different sites of Crete were used for the development of diagnostic assays, to manage field observed resistance. More specifically, known target-site mutations associated with resistance to pesticides with different Mode of Action (MoA) were employed, to monitor the frequency of resistant and susceptible alleles in field populations. We developed a panel of eleven TaqMan assays for T. urticae and four TaqMan assays for B. tabaci where individuals can be screened in addition to a novel, high-tech and highly sensitive version of PCR, the droplet digital PCR (ddPCR), with very high sensitivity and accuracy in bulk (pooled) samples, that can detect resistant alleles even in very low frequency (1 mutant allele in 999 susceptible alleles). The outcome revealed both multi-resistant and susceptible populations of T. urticae and B. tabaci in Crete.
Language English
Subject Molecular diagnostics
Resistance mechanisms
Μηχανισμοί ανθεκτικότητας
Μοριακά διαγνωστικά
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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