School of BioSciences - Theses
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ItemDefining new methods to assess the impacts of endocrine disrupting chemicals to reproductive development( 2020)Hypospadias is the most common persisting congenital abnormality in males, affecting one in 125 live male births in Australia and the U.S.A. Characterised by the abnormal placement of the urethra in the ventral surface of the penis, hypospadias is associated with a reduced fertility and other differences in sexual development (DSDs). Alarmingly, numerous countries have observed a 50% increase in the incidence of hypospadias over the last 50 years. The development of the penis is hormone dependent, and exposure to endocrine disrupting chemicals (EDCs) which alter the hormonal signalling in the male reproductive tract has been implicated in driving the increase in hypospadias. Despite its high incidence, significant gaps in our understanding of normal penis development and the aetiology of hypospadias are apparent. In this thesis I examine the hormonal control of normal penis development and the induction of hypospadias by EDCs. Since oestrogenic EDCs are among the most pervasive in our environment, I first examined the endogenous and exogenous role for oestrogen in the development of the penis and hypospadias. Using oestrogen signalling deficient mouse lines I demonstrate that endogenous oestrogen is required for normal penis development and that any disruption to oestrogen signalling results in mild hypospadias. Additionally, I developed a novel in vitro culturing method to isolate the embryonic penis and demonstrate that exogenous oestrogen and oestrogenic EDCs can directly impact the penis to cause hypospadias. This mechanism of action was previously undescribed and it was assumed that oestrogens impacted androgen output from the testis to cause hypospadias. Together these data are a significant shift in our understanding of the hormonal control of penis development and the mechanisms and range of EDCs which may induce hypospadias. Further exploiting my genital tubercle culture system I sought to define the molecular targets downstream of altered hormonal signalling in the penis. The findings from this work have helped to define some of the molecular drivers of hypospadias during early development and identified several critical signalling pathways which seem to be very sensitive to an altered hormonal environment. We have also been able to define genes which seem particularly sensitive to certain EDCs which can be used in future studies to develop high through-put assays to classify and assess potential EDCs. Together the data from this thesis have improved our understanding of penis development and how this process is disrupted in the presence of EDCs. This thesis has provided a valuable first step in describing new mechanisms through which EDCs can induce hypospadias which is vital to reform current policies and produce new guidelines on the usage of EDCs.
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ItemThe role of cell wall related genes of plant pathogenic fungus Leptosphaeria maculans in causing blackleg disease of canola( 2020)Canola (Brassica napus) is the second largest oilseed crop globally, providing 10-15% of the world’s supply of vegetable oils. In Australia, canola is the largest oil crop and represents over half of total oilseed production in the last 10 years. Blackleg is the major disease of canola, caused by the plant pathogenic fungus Leptosphaeria maculans, restricting canola production worldwide. Yield losses of 50 per cent or even higher have been recorded in some seasons due to blackleg disease. There is an urgent need to reduce the economic damage caused by blackleg disease. The fungal cell wall is the outermost cellular component, thus regulates the host-pathogen interactions like adhesion or phagocytosis, which influences the infection process. The role of cell wall composition in pathogenicity of L. maculans towards canola is unidentified. This study takes a genetics-based analysis of key components in the synthesis of the L. maculans cell wall, by creating mutants of L. maculans with altered cell wall composition to identify pathogenicity genes. The cell wall composition and its role in virulence of L. maculans is unknown. Therefore, based on current information in the literature I selected cell wall genes in other fungi which have shown a role in pathogenicity to identify L. maculans homologs and then utilised a novel CRISPR-Cas9 tool to mutate these genes. Ten cell wall related genes were mutated, including the solo hydrophobin gene (Lema_T007750.1) present in L. maculans and all nine predicted nucleotide sugar transporter genes. The hydrophobin gene mutant exhibited a severe impact on pathogenicity of L. maculans as the mutant could hardly cause any lesion both in canola cotyledon (Brassica napus cv. Westar and Brassica juncea cv. Aurea) and stem (Brassica napus cv. Westar). The hydrophobin mutants had poor conidiation, poor spore germination rate and highly reduced spore attachment capability which likely led to perturbed pathogenicity. It is also hypothesised that hydrophobin plays a role in masking L. maculans from host recognition and to survive under stressed conditions. The substrate specificity of Lema_T023100.1 as GDP-mannose transporter, Lema_T022990.1 as UDP-galactopyranose/UDP-glucose transporter, Lema_T018380.1 as UDP-galactopyranose transporter and Lema_T030160.1 as UDP-galactofuranose transporter were confirmed by performing heterologous expression in a Saccharomyces cerevisiae assay. The substrate of the remaining five proteins could not be determined. Among the nucleotide sugar transporter genes mutants of GDP mannose transporter (Lema_T023100.1), UDP-galactopyranose/UDP-glucose transporter (Lema_T022990.1), Lema_T099510.1 (unknown substrate), Lema_T016830.1 (unknown substrate), Lema_T029030.1 (unknown substrate) and Lema_T030160.1 (UDP-galactofuranose transporter) perturbed the pathogenicity of L. maculans in causing blackleg disease. Mutation of the other genes did not exhibit any impact on L. maculans pathogenicity. In vitro characterisation was performed for all the gene mutants compared to wild type and their complemented strains, that includes growth assay, sporulation, spore germination competency, spore attachment proficiency, ability to endure under stressed conditions as these criterions were considered fundamental pathogenicity factors by mycologists. Though the nucleotide sugar transporter mutants varied in in vitro phenotypes, among all the nucleotide sugar transporter mutants that showed reduced pathogenicity a common feature was highly reduced spore attachment capability. That indicates that in plant pathogenic fungus L. maculans, which does not produce specialised infections structures but directly invades into the host by natural openings, adhesion is the commonly shared mechanism for pathogenesis that is lost in all the non-pathogenic strains. Thus, targeting different stages of the cell wall biosynthesis process in plant pathogenic fungi can be motive for developing novel anti-fungal agents. The outcomes of this research contribute fundamental new knowledge towards the protection against blackleg disease of canola, and potentially other plant diseases caused by fungi.
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ItemPersistence of marine species of the Australian South-east Marine Region under alternative fisheries management strategies( 2020)The marine environment is heavily affected by fishing pressure and knowing how different management strategies affect the persistence of marine species is crucial for ensuring sustainable fishing. Models used within the fisheries management strategies evaluation context have been implemented in the past decades from traditional single species methods to an ecosystem-based approach, but there are still uncertainties on the effectiveness of these models. This thesis aims to understand the drivers of marine species persistence coupling network-based methods and spatially explicit population viability analyses (PVA). This thesis focuses on marine species of south-eastern Australian waters, a region characterised by rich biodiversity and oceanographic complexity, supporting important Australian commercial fisheries sectors. PVA is an approach not yet widely investigated in the context of fisheries management strategies evaluation, however understanding species viability and ensuring metapopulation persistence is fundamental to avoid overexploitation of the stocks. In this thesis is examined how spatially explicit PVA compares to other commonly used models, demonstrating that PVA is a powerful alternative, suitable to inform fisheries management. PVA is applied to estimate metapopulation persistence in a spatial context, analysing how persistence changes when modelling alternative fisheries management scenarios for important commercial fisheries species. The knowledge of species distribution is very important for any spatially explicit population modelling and is needed when studying population viability and dynamics. A recent challenge in understanding and predicting species’ distributions has been focused on the influence of population connectivity. This thesis explores how to integrate graphbased metrics, representative of seascape connectivity, into marine-based species distribution models (SDMs) to understand the contribution of connectivity to predict marine species spatial distribution. Connectivity is a key determinant of metapopulation persistence of species inhabiting fragmented habitats. Larval connectivity is quantified using a biophysical model. Connectivity is visualized as networks, where habitat patches are represented by nodes and dispersal connections are represented by linkages. In this thesis graph-based metrics are calculated because of their significant role as indicator of metapopulation persistence, identifying key habitat patches for the overall persistence of the species. The results of this thesis demonstrate how PVA is a valuable tool helping to inform fisheries managers on the effects of fishing on marine species. Habitat heterogeneity and movement ecology demonstrate to be critical model parameters, strongly influencing metapopulation persistence, suggesting that fisheries managers might benefit from better understanding of movement behaviour and habitat characteristics. This thesis results also provide insight on the role of connectivity to determine marine populations persistence and distribution. Hotspots of connectivity reveal to be key habitats, often enhancing habitat suitability, and strongly influencing metapopulation persistence. Managers and ecologists would benefit by employing similar approaches in making more efficient and more ecologically informed decisions and focusing more on local connectivity patterns to better understand and protect marine species.
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ItemQuantifying biodiversity and maximising species persistence during offsetting( 2020)Biodiversity is declining globally, with habitat clearing playing a major role in perpetuating these declines. Offsets aim to achieve no net loss of biodiversity by implementing conservation actions that deliver equal biodiversity gains to those lost through development. Broad habitat-based biodiversity metrics are commonly used to quantify development impacts and assign offset requirements. However, it is unclear what reliance on habitat-based offset metrics means for biodiversity long-term. In this thesis I explore the current state of biodiversity metrics within the scientific literature and how these metrics are applied in policies around the world. I also quantitatively test nine biodiversity metrics and explore the long-term ramifications of their use in offsetting on species persistence. In a sample of 255 publications from ecology (n = 158), conservation planning (n = 54), and offsetting (n = 43), I identify 24 categories of metrics used to quantify biodiversity. The offsetting literature focused largely on habitat attributes and area-based approaches for measuring biodiversity. Similarly, consistent use of habitat attributes, habitat condition, and area in 21 offset policies demonstrates that offsets do not adequately capture individual species or communities. Many offset policies measure species presence, however, less than half of those I reviewed measured any species-specific attributes which could be used to help inform conservation actions for those species. These two reviews demonstrate that the metrics currently used to measure biodiversity don’t reflect some of the key biodiversity attributes offsets aim to protect. To understand the long-term consequences of relying on such habitat-based metrics for offsetting I developed a spatial simulation tool and present a methodological framework that quantitatively tests the effects of metric choice on long-term persistence. The R-based simulation tool uses raster data to simulate development impacts and restoration efforts. I tested nine biodiversity metrics spanning an axis of pattern versus process and capturing different levels of biodiversity: area of habitat, condition of habitat, vegetation area, vegetation condition, area * habitat suitability, condition * habitat suitability, abundance, metapopulation connectivity and rarity-weighted richness. The resulting raster outputs are used in population viability analyses for five species. Most metrics achieved no net loss or net gains in the metric values, species’ habitat suitability or abundance, but only when impacts avoided high suitability areas. When impacts cannot avoid high suitability areas, species-specific metrics deliver higher gains in abundance per hectare restored than habitat-based metrics alone. Failing to improve current offset practices ensures species will continue to decline under development-offset trading programs and should provide an incentive for policy makers to properly enforce impact avoidance measures where possible.
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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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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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ItemSpatial and temporal drivers of mammal and reptile distributions in fire-prone landscapes( 2020)Fire is a key disturbance process in >50% of terrestrial ecosystems where species are often adapted to specific fire regimes. However, ongoing changes to fire regimes now represent a considerable threat to biodiversity, including mammals and reptiles in Australia. Despite this, there are large knowledge gaps around how different types of fire – or ‘pyrodiversity’ – affect biodiversity and how fire interacts with other key drivers of animal populations. This thesis explored the drivers of mammal and reptile distributions in fire-prone mallee woodlands, to answer key questions of wider relevance to fire-prone ecosystems. First, I explored the influence of daily meteorological conditions on small mammal capture rates during field surveys. I built regression models of the daily capture rates of different species and families against six meteorological variables to determine favourable conditions for ecological surveys. Capture rates of all species were influenced by a least one meteorological variable, although responses to specific conditions varied between taxa. Small mammal surveys should be conducted in a variety of weather conditions to ensure a representative sample of the local community and my results can be used to determine conditions favourable to a single species or family. Second, I explored how pyrodiversity influences the distributions of whole animal assemblages in conjunction with other environmental gradients. I built species distribution models for 17 mammals and 54 reptiles against different measures of fire and mapped the distribution of each species across a >100 000 km2 region. Native mammals showed a variety of responses. Microbats were more likely to occur as time since fire increased, whereas rodent occurrence was positively correlated with recently burned areas. Occurrence of small dasyurid marsupials was positively correlated with the area and configuration of older post-fire age classes, showing the importance of spatial context in understanding the fire responses of animals. Reptile responses to pyrodiversity were more consistent, however there was variation within taxonomic groups regarding the importance of different fire elements. Many reptiles occurred most frequently between 0-50 years post-fire, coinciding with peaks in hummock grass growth. However, configuration of this key habitat component was important to reptiles, and planning for appropriate configurations of fire-derived habitat should be incorporated into fire management for reptile conservation. Maps of species distributions can be used to better incorporate the needs of individual species into fire and conservation planning. Lastly, I used a field experiment to test different mechanisms for how fine-scale patterns of fire influence animal populations in recently burnt environments. Small mammal, reptile and invasive predator activity was monitored in the year following a large planned burn to explore how the amount and configuration of burnt and unburnt habitat affects recovery. Reptile assemblages varied between burnt and unburnt sites, and reptile species richness increased at sites with higher amounts and connectivity of unburnt vegetation. Mammals did not have clear relationships with spatial patterns of planned fire, however fox activity increased after the fire at all sites. Small mammals appear resilient to this scale of planned fire and retaining well-connected unburnt refuges is important for maintaining reptile diversity. Overall, my work helps to define the types of fire that benefit mallee mammal and reptile assemblages, and this thesis highlights how this knowledge can be used to improve fire management for biodiversity conservation.
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ItemAdaptive evolution of Annual ryegrass (Lolium rigidum, L.) to herbicides in agroecosystems( 2020)Significance: The rapid evolution of herbicide resistance in weeds provides a tractable system to study adaptive evolution. Investigations into the dynamics of herbicide resistance development in agroecosystems will advance our understanding of evolutionary biology, as well as guide the formulation of recommendations to control herbicide-resistant weeds. Materials and Methods: Accurate and efficient strategies to characterise and monitor weed populations are required. Populations can be monitored directly by gathering phenotype and genotype data; as well as indirectly by predicting phenotypes using genomic prediction. This thesis investigated the adaptive evolution of weed populations with respect to herbicides. We used Lolium rigidum (rigid/annual ryegrass) as the model weed species because of its ability to evolve resistance to many herbicides with different modes of action. The herbicides used were clethodim (fatty acid biosynthesis inhibitor), glyphosate (aromatic amino acid biosynthesis inhibitor), sulfometuron (branched-chain amino acid biosynthesis inhibitor), terbuthylazine (photosystem II disruptor), and trifluralin (tubulin disruptor). Results: The results of this research can be summarised as: 1. Characterising a landscape by sampling more populations using low-resolution low-cost pool sequencing (Pool-seq) will yield better QTL detection and genomic prediction accuracies than sampling less populations with high-resolution high-cost individual sequencing. 2. L. rigidum populations were collected across South-East Australia, and a colorimetric phenotyping method (instaGraminoid) was developed to cost-effectively quantify herbicide resistance using photographs. 3. Pool-sequencing coupled with genome complexity reduction (Pool-ddRADseq) with MseI and NsiI restriction enzymes, followed by filtering at 99% read and mapping accuracies, 227X minimum depth, and 0.001 minor allele frequency were predicted to result in accurate allele frequency estimates while retaining 600,000 SNPs. 4. Genome-wide association experiments using instaGraminoid-derived phenotype data and Pool-seq-derived genotype data show evidence of highly polygenic herbicide resistance traits and the genetic basis of cross-resistance. 5. The putative resistance alleles are more likely to have originated from standing genetic variation than from de novo mutations. 6. Genomic prediction using population-level data can be useful in herbicide resistance monitoring and management by identifying potentially resistant populations at the early stages of resistance evolution. Conclusion and recommendations: Herbicide resistance is a polygenic trait with many loci conferring cross-resistance. It is rapidly evolving from the rich reservoir of standing genetic variation. Herbicide application remains to be a cost-effective approach to control weeds, but it will inevitably become ineffective with the evolution of cross-resistance. I propose a genomically-informed rotation of chemical and non-chemical control strategies. This involves using genomic prediction to cost-effectively and rapidly monitor herbicide resistances. Depending on the predicted levels of resistance, different herbicide mixtures or non-chemical weed control methods can be recommended.
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ItemThe evolutionary and functional characterisation of the ecdysteroid kinase-like (EcKL) gene family in insects( 2020)Many thousands of gene families across the tree of life still lack robust functional characterisation, and thousands more may be under-characterised, with additional unknown functions not represented in official annotations. Here, I aim to characterise the evolution and functions of the poorly characterised ecdysteroid kinase-like (EcKL) gene family, which has a peculiar taxonomic distribution and is largely known for containing an ecdysteroid 22-kinase gene in the silkworm, Bombyx mori. I hypothesised that EcKLs may also be responsible for insect-specific ‘detoxification-by-phosphorylation’, as well as ecdysteroid hormone metabolism. My first approach was to explore the evolution of the EcKLs in the genus Drosophila (Diptera: Drosophilidae), which contains the well-studied model insect Drosophila melanogaster. Drosophila EcKLs have evolutionary and transcriptional similarities to the cytochrome P450s, a classical detoxification family, and an integrative ‘detoxification score’, benchmarked against the known functions of P450 genes, predicted nearly half of D. melanogaster EcKLs are candidate detoxification genes. A targeted PheWAS approach in D. melanogaster also identified novel toxic stress phenotypes associated with genomic and transcriptomic variation in EcKL and P450 genes. These results suggest many Drosophila EcKLs function in detoxification, or at least have key functions in the metabolism of xenobiotics, and additionally identify a number of novel P450 detoxification candidate genes in D. melanogaster. I then broadened the phylogenomic analysis of EcKLs to a manually annotated dataset containing an additional 128 insect genomes and three other arthropod genomes, as well as a number of transcriptome assemblies. Phylogenetic inference suggested insect EcKLs can be grouped into 13 subfamilies that are differentially conserved between insect lineages, and order-specific analyses for Diptera, Lepidoptera and Hymenoptera revealed both highly conserved and highly variable EcKL clades within these taxa. Using phylogenetic comparative methods, EcKL gene family size was found to vary with detoxification-related traits, such as the sizes of classical detoxification gene families, insect diet, and two estimations of ‘detoxification breadth’ (DB), one qualitative and one quantitative. Additionally, the rate of EcKL duplication was found to be low in lineages with small DB—bees and tsetse flies. These results suggest the EcKL gene family functions in detoxification across insects. Building on my previous ‘detoxification score’ analysis, I used the powerful genetic toolkit in D. melanogaster and developmental toxicology assays to test the hypothesis that EcKL genes in the highly dynamic Dro5 clade are involved in the detoxification of selected plant and fungal toxins. Knockout or misexpression of Dro5 genes, particularly CG13659 (Dro5-7), modulated susceptibility to the methylxanthine alkaloid caffeine, and Dro5 knockout also increased susceptibility to kojic acid, a fungal secondary metabolite. These results validate my evolutionary and integrative analyses, and provide the first experimental evidence for the involvement of EcKLs in detoxification processes. Finally, I aimed to find genes encoding ecdysteroid kinases in D. melanogaster, focusing on Wallflower (Wall/CG13813) and Pinkman (pkm/CG1561), orthologs of a known ecdysteroid 22-kinase gene. Wall and pkm null mutant animals developed normally, but misexpression of Wall caused tissue-specific developmental defects, albeit not those consistent with inactivation of the main ecdysteroid hormones, ecdysone and 20-hydroxyecdysone. In addition, my hypothesis that Wall encodes an ecdysteroid 26-kinase was not supported by hypostasis experiments with a loss-of-function allele of the ecdysteroid 26-hydroxylase/carboxylase gene Cyp18a1. Combined with existing expression and regulatory data, these results suggest Wall encodes an ecdysteroid kinase with an unknown substrate, and hint at previously unknown complexity in ecdysteroid signalling and metabolism in D. melanogaster. Overall, this thesis provides a detailed exploration of the functions of the EcKL gene family in insects, showing that these genes comprise a novel detoxification gene family in multiple taxa, and that they may also contribute to understudied aspects of ecdysteroid metabolism in a model insect. This work also demonstrates the power and potential of integrating evolutionary, genomic, transcriptomic and experimental data when characterising genes of unknown function.
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ItemDissecting the establishment of symbiotic partnership between algae and animals that powers coral reefs( 2020)All organisms on Earth engage in microbial symbioses. These alliances between animals and microbes are situated along a continuum of benefits versus costs with mutualism at one end and parasitism at the other. Even within one group of microbes—sub-Kingdom Alveolata—we find classic mutualists such as the Symbiodiniaceae (Dinoflagellata) that are endosymbionts of reef-building corals, and the parasitic Apicomplexa such as the malaria-causing Plasmodium. Both Symbiodiniaceae and Plasmodium are intracellular symbionts. Contact, recognition and ingress of the symbionts into their host cells are pivotal for commencement of their symbioses. These mechanisms are mediated by the innate immune system, and eukaryotic pattern recognition receptors (PRRs) recognize microbial-associated molecular patterns (MAMPs). These molecules, traditionally known to trigger an inflammatory response during microbial infections, also mediate the cross-talk between host and symbiont. Glycan-lectin interaction is a common MAMP-PRR pathway that apparently acts as a lock-and-key mechanism in both mutualistic and parasitic symbioses. The mechanism used by Plasmodium to invade the host is well understood, whereas the ingress of dinoflagellates into their cnidarian hosts remains enigmatic. In Chapter 2, I compare our knowledge of the Plasmodium-human parasitism to explore whether it could perhaps inform the understanding of how cnidarian Symbiodiniaceae mutualisms are initially established. To explore establishment of symbiosis between corals and their symbiotic algae (Chapter 3), I created a matrix of symbiotic compatibilities between a wide range of Symbiodiniaceae and three genotypes of Exaiptasia diaphana—a model organism for the cnidarian dinoflagellate symbiosis—from the Great Barrier Reef. This study permitted the selection of Symbiodiniaceae types with various level of affinity to the cnidarian host: Breviolum minutum is the homologous species (i.e., native), Cladocopium goreaui is the compatible heterologous species (i.e., non-native), and Fugacium kawagutii is the heterologous-incompatible species. Symbiont species and host genotype influenced colonization dynamics, which is consistent with selectivity roles for both host and symbiont at the onset of symbiosis. This matrix of different host–symbiont compatibilities was used in Chapter 4 to explore the molecular mechanisms of recognition and establishment of the cnidarian Symbiodiniaceae symbiosis. I used anion-exchange chromatography, lectin array technology and confocal microscopy to develop an inventory of sugars potentially acting as MAMPS on the dinoflagellate surface. The Symbiodiniaceae cell surface glycome was diverse and varied among algal species. By comparing the glycan inventories of the homologous symbiont B. minutum, the heterologous, compatible symbiont C. goreaui, and the incompatible species (F. kawagutii), I was able to focus on selected sugars implicated in recognition and then attempt to perturb recognition by modifying or masking epitopes on either the host or symbiont. D-galactose (especially methyl-b-D-galactose), xylose and fucose seem to regulate the host invasion by homologous or heterologous algae. In Chapter 5, I further explored Symbiodiniaceae cell surface with a proteomic analysis of algal cell wall. Several proteins with transmembrane translocating activity were identified, and a large proportion of the amino acid residues are still uncharacterized. Intriguingly, the reticulocyte-binding-like (RBL) protein was identified on the surface of Symbiodiniaceae used here. RBL proteins are involved in the recognition of sialic acid-containing receptors of host cells during malaria infection. This thesis describes in detail the Symbiodiniaceae cell surface and contributes to the knowledge of how this structure could mediate the symbiosis with cnidarians. Furthermore, my work highlights important similarities between Symbiodiniaceae and their relatives in the Phylum Apicomplexa. If, in the Alveolata, mutualism did lead to parasitism, Symbiodiniaceae and Plasmodium may have conserved symbiont/host recognition mechanisms, perhaps sialic acid mediated, to access the host and start symbioses.