School of BioSciences - Theses
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ItemDefining the Imidacloprid Targets in Drosophila melanogaster( 2021)Nicotinic acetylcholine receptors (nAChRs) are essential neuronal receptors that mediate fast synaptic neurotransmission influencing many aspects of behaviour. The insecticide imidacloprid (IMI) bind to nAChRs, disrupting their function and causing insect death. The widespread use of IMI has led to the evolution of resistance in a number of pest species. In most cases resistance has been attributed to elevated levels of cytochrome P450 enzymes that detoxify the insecticide. That target site resistances are rarer suggests that resistant mutations may incur fitness costs. Published studies using mutagenesis in the model insect, the vinegar fly Drosophila melanogaster, have proven to be fruitful in identifying some of the nAChR subunits that may co assemble to form IMI targets. This thesis builds on and extends those studies. This study systematically investigated the targets of IMI in D. melanogaster. The first aim of this study was to identify all of the nAChR subunits that contribute to IMI toxicity. Given the relatively small number of nAChR subunit genes in this species, the capacity for individual subunits to contribute to the IMI targets was tested using mutant generated for each of the genes using CRISPR-Cas9 gene editing. Toxicological assays were performed and identified the Dalpha1, Dalpha2, Dbeta1 and Dbeta2 subunits as the primary targets of IMI in this species. The second aim investigated the fitness costs associated with IMI resistance. Given the potential for functional redundancy among the nAChR genes, this research included an investigation of strains with knockout alleles for more than one gene. Two of the IMI targeted nAChR genes, Dalpha1 and Dbeta2, were selected for this study, given their IMI resistance levels and apparent fitness deficits. Single and double knockout mutant strains were generated for Dalpha1 and Dbeta2, which were tested using toxicological and a range of phenotypic assays to investigate the functional contribution that Dalpha1 and Dbeta2 make as the IMI targets and to the capacity to survive and reproduce. This research provided insights into the function of these subunits and the potential for resistance to evolve via mutations in them. The third aim examined the expression patterns of the IMI targets in the D. melanogaster larval brain. This was achieved by using GAL4 and split GAL4 strains combined with a UAS controlled fluorescent reporter. A 3rd instar larval brain single cell transcriptome atlas was also used to help identify the brain cell types that express combinations of the IMI targeted subunits to provide further insights into the functions that the receptors formed might perform. Given that nAChR genes are highly conserved among insects, the results from this study are likely to be applicable to other insect species and facilitate the rational use and rotation of insecticides targeting members of the nAChR family.