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.
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ItemInvestigating freshwater snails Potamopyrgus antipodarum as a suitable test species for ecotoxicological testing of surface water in Australia( 2020)Ecotoxicology studies the fate and effect of chemicals in the ecosystem using scientific approaches. Laboratory tests (e.g. acute and chronic tests) establish dose response relationships between toxicants and the test species. Although laboratory tests provide essential data to understand the potential impact of toxicants in the aquatic environment, they lack environmental realism. In situ tests provide environmental realism by exposing the test organism to toxicants in the aquatic environment. Test species are central in ecotoxicity assays. Molluscs are commonly used in ecotoxicological tests and they are the second largest phylum, have a global distribution, widely abundant in freshwaters, and ecological importance (e.g. as a food source, provides habitat and protection) makes them useful in ecotoxicity tests. This thesis used a freshwater snail, Potamopyrgus antipodarum (J.E. Gray, 1843), for laboratory and field testing. Although native to New Zealand, it is a successful invasive species in many parts of the world, including Australia and has been successfully used as a test species for ecotoxicity tests and as a bioindicator in some countries (e.g. in Europe, and the USA). A few Australian studies have used it in laboratory assays and field studies. There is still less knowledge and information of P. antipodarum as a test species and bioindicator in Australia. Therefore, the overall aim of the thesis is to assess the potential of P. antipodarum as a bioindicator of freshwater pollution in Australia. In Chapter 2, adult P. antipodarum were exposed (96 h) to environmentally relevant concentrations (ERCs) of metals (copper and zinc) and a pesticide (imidacloprid) in water. Mortality (LC50) and behavioural responses (EC50) were assessed to investigate the sensitivity of the snail and the potential use of its behavioural response in the environmental risk assessment of toxicants. The Chapter 3 was an extension of the Chapter 2 to assess their sensitivity in chronic toxicity tests. Adult snails were exposed for 28-d to ERCs of several chemicals (Cu, Zn, bifenthrin, and 17 a-ethynylestradiol), physico-chemical conditions (salinity and temperature), and food limitation in water and sediment bioassays. Endpoints including reproduction, growth, and mortality were measured. This study evaluated the sensitivity of this species during chronic exposure and compared the response of these Australian cultures to those abroad. In Chapter 4, adult snails were deployed in cages in the field for 28-d at 9 sites (i.e. 7 impact sites, and 2 reference sites) in Merri Creek and an additional reference site in Cardinia Creek to evaluate the performance of snails at various points along Merri Creek with different land use. Various endpoints were measured at the organism level (growth, mortality and reproduction), and the sub-organism level (glutathione Stransferase, GST; lipid peroxidation, LPO; and catalase, CAT). The biological response of the snails at each impact site were compared to the 2 reference sites on Merri Creek to show the potential impact of land use on the snails. The additional reference site at Cardiana Creek was compared with the reference sites on Merri Creek to identify any difference in response in a different catchment. This project shows that P. antipodarum is a suitable and sensitive species for acute and chronic assays because it responded to ERCs of toxicants, the response is like populations abroad, it showed a similar response to other test species and LC50 and EC50 value was within the range of other test species in use. There is potential to use acute tests (LC50) and behavioural responses (EC50) in the rapid risk assessment of environmental pollutants. Field data also revealed that P. antipodarum is a suitable bioindicator for Australian environmental conditions because the response of this population was similar to the populations abroad, and other test species used in in situ tests and showed a high tolerance to environmental variations. This research also shows that although laboratory tests can provide us with essential data to understand the potential impact of toxicants in the aquatic environment, they lack environmental realism. And we can achieve environmental realism by exposing the species to toxicants in the aquatic environment during in situ tests.
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ItemSystemic impacts of low dose insecticide exposures in Drosophila: a mechanism centred on oxidative stress( 2020)The plight of insect populations around the world has gained increasing attention. A recent meta-analysis published in Science reported an average decline of terrestrial insect abundance by average 9% per decade since 1925 (van Klink et al. 2020). While this is a lower rate of decline than reported in earlier meta-analyses (Sanchez-Bayo and Wyckhuys 2019) it still suggests that many terrestrial insect species are under threat. The extinction of terrestrial insect species would severely affect agriculture and ecosystems due to the vital role that many species play in pollination, the recycling of organic matter, pest control and other ecosystem services. Insecticide exposure has been proposed to be one of the significant contributing factors for population declines of non-pest species. Insecticide contamination in biomes, resulting from intensive usage on agricultural crops, is likely to lead to exposures for many non-pest insect species. Low doses of insecticides are known to impact the fitness and behaviour of various insect species, but the underlying molecular, cellular, and physiological impacts of such doses in insects are not well defined. The absence of a mechanism that explains how low doses affect insects is an obstacle to ascertaining the extent to which insecticides may contribute to the demise of populations. The aim of this study was to scrutinize the impacts of low insecticide doses on the metabolism and physiology of the model organism Drosophila melanogaster in order to propose a mechanism to explain the impact of such doses on insect biology. Two insecticides were investigated in detail. The first of these is the synthetic neonicotinoid imidacloprid. Having been banned in the EU due to some evidence of a role in collapse in honeybee colonies, imidacloprid remains one of the most widely used insecticides in the world. The second insecticide is spinosad. Composed of two structurally similar natural fermentation products from the soil bacterium Saccharopolyspora spinosa, this insecticide is classified as organic and considered to be less harmful to beneficial insects. Both insecticides target evolutionarily conserved nicotinic acetylcholine receptors (nAChRs) in the Central Nervous System (CNS) of insects. nAChRs are pentameric ligand gated ion channels. Activation by the natural ligand, acetylcholine, leads to a flux of calcium, potassium or sodium ions into neurons, regulating a myriad of responses in the insect brain. The Drosophila genome encodes 10 nAChRs subunits (Dalpha1 to Dalpha7 and Dbeta1 to Dbeta3), meaning that there is a vast number of subunit combinations that could assemble to form functionally distinct receptor subtypes. Imidacloprid targets the Dalpha1, Dalpha2, Dbeta1 and Dbeta2, subunits, whilst spinosad targets the Dalpha6 subunit. Acute exposure to imidacloprid, at doses that do not kill Drosophila larvae, rapidly increased in the levels of reactive oxygen species (ROS) in the brain, most likely due to the sustained calcium flux into neurons caused by the interaction between the insecticide and its nAChR targets. This led to oxidative stress marked by mitochondrial dysfunction that in turn led to a significant decrease in energy (ATP) levels. While this process was initiated in the brain, lipid storage in the metabolic tissues (fat body, Malpighian tubules, and midgut) was affected. Transcriptomic analysis of the larval brain and fat body revealed a significant perturbation in the expression of genes involved in metabolism, oxidative stress, and immune response. Using genetic manipulations to elevate ROS levels exclusively in the brain, lipid storage was shown to be perturbed in the metabolic tissues, indicating that a ROS signal initiated in the brain radiates to other tissues. Severe damage to glial cells and neurons (i.e. neurodegeneration) was observed in the visual system of adults subjected to chronic low-dose exposure to imidacloprid. This precipitated a progressive loss of vision. Spinosad showed a different mode of action, blocking nAChRs and preventing calcium influx. The blocked receptors were shown to be recycled from the neuronal membranes through endocytosis. This mechanism led to an increase in the number and size of lysosomes in the CNS, characteristic of lysosomal storage diseases, which precipitates elevated generation of ROS by impairing mitochondrial activity and neurodegeneration. The high levels of ROS measured in the CNS after spinosad exposure, were associated with a cascade of phenotypes in metabolic tissues similar to the ones observed after imidacloprid exposure. Experiments examining the lipid environment in Dalpha6 knockout mutants (resistant to spinosad) indicated that impacts observed in the metabolic tissues of spinosad-exposed larvae are due to the interaction between Dalpha6 and spinosad. These data corroborate the hypothesis that impairments observed in metabolic tissues are triggered by a chemical signal from the brain, suggested to be a peroxidized lipid. Although there were some differences in the responses observed for the two insecticides (e.g. in transcriptomes and lipidomes), a similar cascade of processes was observed to be initiated following the elevation of ROS levels in the brain. A potent antioxidant, N-Acetylcysteine amide, strongly suppressed a range of phenotypes observed in both larvae and adults, indicating a causal role for ROS and oxidative stress. As the nAChR targets of these insecticides are conserved among insects, it is likely that similar impacts would be precipitated by exposures in other non-pest species, albeit at different doses. As insecticides from a wide range of chemical classes create markers of oxidative damage, the low dose mechanism of action observed for imidacloprid and spinosad may apply more broadly. This requires investigation. Considered together, the low dose impacts of imidacloprid and spinosad severely impair insect biology, without necessarily killing. These impairments could render insect species more vulnerable to the other major threats proposed to contribute to the decline of populations: climate change, habitat loss, pathogens, and parasites.