School of BioSciences - Theses
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ItemUnderstanding and incorporating aphid parasitoids within IPM strategies in Australian grain crops( 2020)Aphids (Hemiptera: Aphididae) can be particularly devastating to grain crops, with their economic importance weighted on their ability to cause significant yield losses through a variety of methods. From feeding damage alone in 2012, cereal aphids caused an average annual loss of $14 million in Australian wheat crops. For over a century, growers have relied upon host plant resistance and chemical treatments to control invertebrate pests, however suppression of beneficial organisms and increased resistance within targeted species has created an ongoing battle with pest control. For example, the polyphagous green peach aphid (Myzus persicae (Sulzer)), often a pest of canola crops, has developed resistance to over 74 insecticides including carbamates, pyrethroids, and organophosphates around the world. Due to these issues, control of agricultural pests is now focussed on Integrated Pest Management (IPM) strategies, within which natural enemies can play a role as biological controls. Parasitoid wasps have had the most success as biological control organisms in the past, likely due to their host specificity. I spent three years collecting data on grain aphid pests and their associated natural enemies, paying particular attention to the hymenopteran parasitoids. I determined the distribution of grain aphids and their associated Aphidiinae within grain production landscapes around Australia, utilising historic data and citizen science. Additionally, I determined how aphid abundance and diversity, along with their associated parasitoids changed throughout the growing seasons. I created a key of aphid parasitoids (Hymenoptera: Aphidiinae) parasitizing aphids in Australian grain production landscapes. Finally, I determined the effects of seed treatments on specific natural enemies associated with M. persicae, identifying the difference between parasitoid and predator effects. My findings are informative for developing strategies to conserve those Aphidiinae species of particular importance in controlling aphid pests. Additionally, these results can assist with pest management decisions, enabling growers to implement IPM based on a greater breadth of knowledge.
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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.
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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.