School of BioSciences - Theses
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ItemManipulating key developmental and regulatory pathways in the Queensland fruit fly to develop improved strategies for pest control( 2022)Sterile insect technique (SIT) is an efficient and species-specific biocontrol strategy involving mass-releases of factory bred, sterilised males that seek and mate with wild females. Fertilized embryos or zygotes die due to dominant lethal chromosome aberrations caused by sterilization processes, which ultimately leads to localised population suppression. This approach is currently being implemented to help control outbreaks of the invasive Queensland fruit fly (Bactrocera tryoni), which is a major insect pest of fruit and vegetable crops in Australia. Sterilised B. tryoni SIT flies are marked with fluorescent powder dyes to distinguish them from wild flies when recaptured in monitoring traps. However, this powder-based marking strategy can cause dehydration and death, fail to thoroughly coat flies, be washed off or potentially passed on to wild flies through contact. Current B. tryoni SIT practices involve sterile releases of males plus females, although increased efficiency could be achieved if high-throughput sex separation methods were available. Here, targeted CRISPR/Cas9 gene editing and insect transgenesis helped create resources or prioritise genes with desirable qualities, for improving SIT pest control applications. Strains with diagnostic body pigmentation mutations were generated to potentially differentiate between wild flies and SIT flies captured in monitoring traps, or to act as sex-linked pupal markers for mechanical separation of females prior to release. Second, a single amino acid substitution in the gene RNA polymerase II 215 (R977C, RpII215ts) was confirmed as a promising temperature sensitive candidate for developing a male-only SIT strain via conditional removal of female embryos. However, an amino acid substitution in the gene shibire (G268D, shits1) carried strong fitness costs and is not suitable for developing a male selecting strain for SIT, but other shibire temperature sensitive mutations with milder effects in Drosophila melanogaster still hold potential.
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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.
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ItemProfiling the molecular mechanisms underlying negative cross-resistance to insecticides using Drosophila melanogaster( 2020)Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate neurotransmission at cholinergic synapses. The nAChRs are mainly expressed in the central nervous system and are highly conserved across a wide range of insect species. Neonicotinoids and spinosyns are two classes of insecticide that target nAChR subunits to kill pest insects. Mutations in genes encoding several nAChR subunits in various insect species, as well as in Drosophila melanogaster, have been documented as conferring insecticide resistance. Chemical control including insecticides has been a key tool in controlling pest insects. The cycle of insecticide use, resistance evolution and insecticide replacement has been continuing for the past decade, leading to many pest species carrying resistance to multiple classes of insecticides. This thesis examines the interplay between different insecticides and nAChR mutations that are associated with resistance to one insecticide but result in hypersensitivity to another, a phenomenon called negative cross-resistance. The negative cross-resistance relationship presents insecticides that could complement current rotation strategies for resistance management, and this warrants further analysis to understand the mechanism. Examination of loss-of-function mutations on the nAChR subunits in this thesis, confirmed the previous identification of the Dalpha1 and Dbeta2 subunits as targets for neonicotinoids, as well as the Dalpha6 subunit as a target for spinosyns. This study also identifies the Dalpha2 subunit as an additional target for imidacloprid. Importantly, mutations on these subunits were also associated with insecticide hypersensitivity, suggesting negative cross-resistance. The neonicotinoid-resistant, Dalpha1 mutants were hypersensitive to spinosyn, except for a full knockout allele, while the spinosyn-resistant, Dalpha6 mutants were all hypersensitive to neonicotinoids. Additionally, negative cross-resistance was found between two neonicotinoids, nitenpyram and imidacloprid in the Dalpha2 mutants. Analysis of different allelic variations at the gene encoding these subunits indicates that this is not an allele specific phenotype. Combining the negative cross-resistance relationship and analyses of molecular changes induced in the nAChR subunits mutants, our study initiated to characterise the changes at the synapse that underlie the negative cross-resistance phenotype. A mechanism involving nAChR compensatory changes in levels of another receptor subunit/subtype was hypothesised to cause the phenotype. Following measurement of transcriptional changes and subunit protein changes, the study classified few correlations between nAChR subunit expressions and the negative cross-resistance, and these vary between the mutants suggesting other possible route(s) for the insecticide hypersensitivity. A genome-wide differential gene expression analysis in specific neuronal cell types of larval brain revealed differentially expressed genes in the Dalpha1 and Dalpha6 mutants. Interestingly, gene ontology enrichment analysis indicates dysregulation of cellular processes, including oxidative stress, protein trafficking and proteasomal degradation pathways in the mutants, that may contribute to the insecticide hypersensitivity. Dysregulation of oxidative stress may predispose the nAChR mutants to further insecticide-induced increase in oxidative levels. Finally, blocking dynamin-mediated endocytosis and proteasome activity, using chemical inhibitors, showed protection against larval movement reduction following imidacloprid and/or spinosad exposure. These findings indicate that the relatively straightforward phenotypic observation of insecticide hypersensitivity in response to loss of a receptor subunit is most likely underpinned by several complex changes in neurons, altering the sensitivity of their response to insecticides and their capacity to cope with downstream effects of insecticide exposure.