To understand the mechanisms of insecticide resistance in Anopheles stephensi, DDTand pyrethroid-resistant lines were selected in the laboratory. Selection process showed fixation of L1014S-kdr mutation in DDT- and pyrethroid resistant colonies. Similar fixation was recorded in unselected mosquitoes (control) as well, suggesting low fitness cost of L1014S in laboratory conditions. Synergistic assays using piperonyl butoxide (PBO), a mixed function oxidase and esterase inhibitor, led to reversal of resistance in pyrethroid resistant mosquitoes but no reversal in DDT resistant mosquitoes was recorded. Elevated GST activity in laboratory strains led us to characterize the DDT resistance implicated GST epsilon genes. Quantitative-PCR showed elevated expression of AsGSTe2 and AsGSTe4 in DDT-resistant mosquitoes in both laboratory selected DDT-resistant strain and field caught mosquitoes. These observations prompted further investigations to molecularly characterize these genes examining mutational changes and the possible roles of allelic variation. Cloning and sequencing of the full genes revealed polymorphism which resulted in four variants in AsGSTe2 and three variants in AsGSTe4. Of the four variants of AsGSTe2, two variants (AsGSTe2.1 and AsGSTe2.2) found in DDT-resistant individuals were expressed in vitro in E.coli. Recombinant expression and DDTase assays of AsGSTe2.1 and AsGSTe2.2 showed them to efficiently metabolise DDT. DDTase activity examined for recombinant AsGSTe4 (AsGSTe4.1 and AsGSTe4.2 and AsGSTe4.3) showed that they did not metabolise DDT. Enzyme thermostability tests showed AsGste2 variants to be highly unstable compared to the orthologues in An. gambiae, Aedes aegypti and its corresponding AsGSTe4 variants. Further examination into the GST epsilon array provided evidence of tandem coduplication of AsGSTe2 and AsGSTe4 together in the GST-epsilon array of the laboratory DDT-resistant colony.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:722034 |
| Date | January 2017 |
| Creators | Dykes, C. L. |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/3006097/ |
Page generated in 0.0013 seconds