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Identifier 000381598
Title Unraveling the molecular basis of pyrethroid resistance in dengue vector Aedes aegypti
Alternative Title Μοριακή βάση της ανθεκτικότητας του κουνουπιού Aedes aegypti στα πυρεθροειδή
Author Μπαριάμη, Βασιλική
Thesis advisor Βόντας, Ιωάννης
Reviewer Τζαμαρίας, Δημήτρης
Hilary, Ranson
Abstract Dengue is the most rapidly spreading arboviral infection of humans and each year there are 50–100 million cases of dengue fever. There is no vaccine or drug to prevent dengue infection so control of the mosquitoes that transmit this virus is the only option to reduce transmission. Removing mosquito habitats close to human homes can be effective but in reality most dengue control programs rely on a small number of chemical insecticides. Therefore, when the mosquito vectors develop resistance to the available insecticides, dengue control is jeopardized. In this study we examined the causes of resistance to the insecticide class most commonly used in mosquito control, the pyrethroids. Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance. Two previously described target site mutations; 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes). Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification. Another gene group classified as lipid metabolism genes was commonly upregulated in both resistant populations. Overexpression of lipid metabolism genes has been seen in a number of occasions where genomic analysis of insecticide resistant populations has been performed. Yet their roles in insecticide resistant have never been assessed. Seven of these genes two ApoD proteins, a fatty acid synthase, two lysosomal acid lipases sharing 99% sequence identity, a steroid dehydrogenase and their transcriptional regulator SREBP were selected for validation with Real Time PCR. With the exception of SREBP the rest of these genes were indeed highly expressed in both pyrethroid resistant populations compared to a laboratory susceptible strain. Once more gene amplification was found responsible, at least in part, for the elevated levels of LIPA in the resistant populations. Rough dissections on mosquitoes heads and bodies, indicated that expression of the lipid metabolism genes, as well as two of the previously examined P450s (CYP9J26 and CYP9J28), occurred mainly in the insect body. A ubiquitous driver daGAL4 was used to drive expression of ApoD and LIPA and CYP9J26, in Drosophila melanogaster. Flies overexpressing each of these genes separately showed significant levels of resistance. Combination of the metabolizing CYP9J26 with each of the novel genes ApoD and LIPA resulted in an even stronger resistance phenotype, while combination of ApoD and LIPA did not give any higher levels of resistance. This result implies that these genes acting from distinct pathways contribute in an additive manner to the resistance phenotype. Finally targeted expression of these proteins to the main detoxification organs via 6g1(HR) GAL4 is sufficient to confer comparable levels of resistance.
Language English
Issue date 2013-11-07
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
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