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Poulton, Beth (2021) Characterizing insecticide resistance mechanisms in mosquitoes using genetic modification and a rapid automated larval resistance detection assay, Thesis (Doctoral), Liverpool School of Tropical Medicine.
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B Poulton PhD - Thesis Final.pdf - Accepted Version Download (9MB) | Preview |
Abstract
Insecticide resistance is a threat to malaria and arbovirus control programmes targeting mosquito vectors. Integrated control programmes which include control of larval stages are becoming more important for Anopheles control as urbanisation in malaria endemic areas increases and remain crucial in Aedes control. However, the success of control programmes is threatened by the evolution of molecular mechanisms which confer insecticide resistance. Potential resistance mechanisms are identified by screening the genome, transcriptome and proteome for mutations or gene upregulation that correlate with resistance phenotypes. Once candidate mechanisms have been identified they need to be functionally characterised in isolation to determine their role, as in field and lab insecticide selected mosquitoes many mutations may co-occur which complicate the analysis. This functional characterisation is best conducted using genetically modified mosquitoes, which has been realised for members of several gene families thought to be involved in adulticide resistance. However, very little has been conducted in relation to larvicide resistance.
One reason for the lack of research on larval resistance is that the existing WHO recommended mortality-based larval resistance assay is low-throughput and subject to investigator bias. To address these issues, a novel assay was developed in collaboration with the Sattelle group at UCL using the Invertebrate Automated Phenotyping Platform (INVAPP) and analysis algorithm (Vectorgon). The INVAPP assay provides automated quantification of larval motility after insecticide exposure. In this project, three statistical methods, based in R and python, were trialled to analyse a complex data set collected by exposing a set of transgenic Anopheles gambiae larvae to a range of insecticides. The transgenic larvae each ubiquitously overexpressed a single gene, which had previously demonstrated roles in adult resistance. The drc package showed some promise in defining larval resistance status, but ultimately more data is needed draw conclusions with confidence. Further data collection and optimisation is required before this assay can be reliably used for such relative resistance analysis.
A second project aimed to functionally characterise the carboxylesterase, CCEae3A, which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae, using a GAL4 UAS expression system in An. gambiae. Insecticide resistance profiling in larvae indicated significant increases in resistance ratio compared to a strain which does not express CCEae3A, for three organophosphate insecticides, temephos (5.98), chloropyrifos (6.64) and fenthion (3.18). Cross resistance to adulticides from four insecticide classes: malathion and fenitrothion (organophosphates), bendiocarb and propoxur (carbamates), pirimiphos methyl (phosphorothioate) and alpha cypermethrin (pyrethroid) was also detected. Pirimiphos methyl and alphacypermethrin resistance had not previously been associated with CCEae3A, despite previously occurring in strains where this gene was upregulated. This highlights the importance of characterising mechanisms in isolation to ensure accurate information is used for guiding vector control strategies.
The final project aimed to localise transcription of ace1 (the neuronal target for organophosphate and carbamate insecticides) and characterise the insecticide resistance and fitness cost profiles associated with the ACE1-G280S single nucleotide polymorphism. These aims were approached by genome modification using CRISPR-Cas9 based homology directed repair. An F2A protospacer-fluorescent protein was used to tag the ace1 gene in An. gambiae and confirmed that ace1 transcription is highest in larval and adult nerve cord and ganglia but failed to detect embryonic expression. An. gambiae carrying the G280S mutation in an otherwise insecticide susceptible background were also created with high efficiency. Mosquitoes homozygous for 280S displayed decreased susceptibility to propoxur, fenitrothion and malathion, but surprisingly not to temephos, the most common organophosphate larvicide. However, the significant reductions in longevity and fecundity observed in the 280S transgenics may explain the absence of single copy ace1 mutant homozygotes in field mosquitoes. This project reports the first use of genetically modified An. gambiae to study mosquito larval resistance mechanisms and the first use of a 2A protospacer to tag an endogenous gene in mosquitoes
| Item Type: | Thesis (Doctoral) | ||||||||
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| Subjects: | QU Biochemistry > Genetics > QU 470 Genetic structures QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes QX Parasitology > Insects. Other Parasites > QX 600 Insect control. Tick control QX Parasitology > Insects. Other Parasites > QX 650 Insect vectors |
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| Faculty: Department: | Biological Sciences > Vector Biology Department | ||||||||
| Depositing User: | Lynn Roberts-Maloney | ||||||||
| Date Deposited: | 22 Jun 2022 13:41 | ||||||||
| Last Modified: | 22 Sep 2022 01:02 | ||||||||
| URI: | https://archive.lstmed.ac.uk/id/eprint/20634 |
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