School of BioSciences - Theses
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Computational biology methods for identifying leaderless secretory proteins in Arabidopsis thaliana and other plant species
Leaderless secretory proteins (LSPs) are proteins that are secreted yet lack the classical canonical signal peptide sequences and, therefore, by definition are undergoing unconventional protein secretion (UPS). We cannot necessarily assume all such proteins found in plant secretomes are LSPs due to the high possibility of cellular contamination arising from cellular disruption, leading to a mix of LSPs and contaminant proteins that are both characterised by the absence of signal peptides. The aims of this Thesis are to use computational biology methods to identify LSPs. In Chapter 1 the current knowledge of secretory pathways (LSPs and UPS) in plants, motifs for secretion, the difficulties of isolating the plant secretome (cell wall/apoplastic fluids) without contamination, and the lack of appropriate bioinformatics tools for plants to distinguish them from LSPs is reviewed. Chapter 2 evaluates a commonly used computational tool, SecretomeP which was trained on mammalian data and is the most widely used prediction tool for LSPs, including in plant studies. Exploring whether this tool is applicable to plants required using conventionally secreted proteins as a proxy, and evaluations were made on SecretomePs premise that conventional and unconventional secretory proteins will share properties. By removing the signal peptide from sequence data, the research shows a bias in scores due to the signal peptide, and only a marginally higher true positive rate compared to false positives. The use of the tool on further plant studies was not recommended and suggested previous inferences of plant LSP status based solely on SecretomeP predictions needed to be re-evaluated. This work was published as ”Better Than Nothing? Limitations of the Prediction Tool SecretomeP in the Search for Leaderless Secretory Proteins (LSPs) in Plants” (Lonsdale et al., 2016) Chapter 3 details the creation of a putative LSP database for Arabidopsis thaliana by taking the entire proteome and applying a workflow of collating annotation, literature observation and experimental relationships between proteins. This results in a framework to identify proteins without signal peptides that have been observed (unclassified set) or unobserved (non-secreted set) and compare them to conventionally secreted proteins that have been observed (secreted set) or unobserved (theoretically secreted sets). Protein-protein interactions, GO term and PFAM distributions that are similar between the secreted and unclassified protein sets are used to create confidence lists of putative LSPs. In Chapter 4 new prediction tools were created. Firstly, a SecretomeP-like tool is created using the properties of the observed secreted set, with signal peptides removed. Lessons from Chapter 2 on SecretomeP allowed the bias to be minimized. Secondly, tools were created based on the candidate list from the putative LSP database. Each tool is based on a Random Forest (RF) using protein-derived features and trained on subsets of the LSP database. Consensus predictions between them on new data was used to identify further LSP candidates. Additional reporting tools provide a convenient way to map data from the LSPDB onto new sequences, and the results of these tools on LSPs from plants and other organisms shown. In Chapter 5 additional putative candidates for LSPs in Arabidopsis and other species were identified by applying the tools and databases to additional data excluded from the original database created in Chapter 3, including recent cell wall proteomes, secretory pathway experiments and analysis of extracellular vesicles. In conclusion, Chapter 6 discusses the limitations of the computational methods developed, and suggestions for improvement to predict LSPs in plants for further experimental investigation and confirmation of location.
Predicting pest issues from common and curious pests of crop seedlings in south-eastern Australian
Over fourty species of invertebrate pests can be pests of emerging crop seedlings in south-eastern Australia. Some species are a common and widespread issue for farmers, while others only sporadically cause damage. The inability to predict potential pest problems is a key reason why insecticides are commonly applied to fields prior to sowing (applied as seed treatments or bare-earth sprays) and/or post-sowing (foliar sprays) in a prophylactic manner. This thesis investigates factors which predict the risk of pest problems arising from two common pests and two emerging sporadic pests of crop seedlings. The common pests are the earth mites, the redlegged earth mite, Halotydeus destructor and the blue oat mite, Penthaleus destructor. The emerging pests are the Portuguese millipede, Ommatoiulus moreleti and the common pillbug, Armadillidium vulgare. These species are a curious problem as they generally feed on decaying plant matter and have only recently been recognised as emerging pests. Sampling was conducted to assess the capacity to predict the abundance of the damaging autumn generation of the major pest mites, H. destructor and P. major, at a field level. Across the fields sampled, abundances of both mites showed limited association with a range of common agronomic and environmental field variables of known biological importance. The abundance of H. destructor could however be divided in to risk categories when fields were grouped in to categories of field type. These categories were valid across two years and two farming regions. The abundance of P. major, by contrast showed a regional association. The first pest step in understanding the pest status of O. moreleti and A. vulgare was to understand their ability to damage common crop seedlings. Under shadehouse conditions, this study found O. moreleti has a limited ability to feed on a range of crop seedlings. A. vulgare can damage a wide range of crops, although some crops are only susceptible in the first days of seedling establishment. This finding was supported by pest reports from fields across southern Australia over the past decade. Lupinus angustinus was the most susceptible crop seedlings for O. moreleti. This appears to be due to the removal of deterrent chemicals in commercial breeding as well the thickness or strength of leaf tissue. The fitness benefits of feeding on seedlings appear to vary between crop seedlings. In controlled studies, the level damage was found to be influenced by the lifestage and sex of the individual, while the presence of crop stubbles as alternate food sources were not found to influence the level of damage in controlled studies. To understand the role of water stress on the pest status of A. vulgare, I conducted controlled trials on the influence of dry environmental conditions on the level of feeding damage A. vulgare caused to canola. I hypothesised water stress would result in individuals increasing their feeding on plants, as they searched for water. Conversely, I found individuals kept in environments with low ambient humidity caused less damage. Field monitoring observations were made over the course of the thesis to provide the basis for further research on the population dynamics of O. moreleti. While stubble is known to be important in influencing population sizes, I also found evidence that soil-type has an important effect on O. moreleti numbers. Interestingly, abundances were found to vary greatly between years in some crop fields. This appears to be due to seasonal population dynamics, with O. moreleti leaving fields and, in some cases, not returning. The research conducted in this thesis points to ways to guide monitoring and sustainable management practices. Farm managers should use the association of damaging abundances of H. destructor with field-type to predict pest risk at a farm level. The research on A. vulgare and O. moreleti helps to explain why these species are a sporadic pest risk, given they are limited in their ability to damage crop seedlings. This knowledge will assist in the development of pest management programs for O. moreleti and A. vulgare. The studies on the association of environmental and agronomic factors should be used to guide future research to understand the many remaining knowledge gaps.
Bioremediation of nutrient-enriched coastal ecosystems using macroalgae
Nutrient excess derived from anthropic impacts on coastal ecosystems can have harmful consequences at the economic and ecosystem levels. Traditionally, companies in charge of wastewater treatment in coastal cities have implemented infrastructure improvements to try and reduce eutrophic conditions that arise due to an imbalance between their nutrient inputs and the subsequent consumption within the coastline ecosystems. In this thesis I explored the potential of macroalgae as bioremediation agents as an alternative to engineering solutions. Chapter 2 is focused on the selection of candidates for bioremediation before the wastewater discharge reaches the coastline. Chapter 3 explores the evaluation of macroalgae species and biomass amount present at zones with nutrient excess as an essential step to assess the possibilities that seaweed cultivation can entail at an economic level. Chapters 4 and 5 delve into the consequences that the drift algae cultivation has for the associated fauna and nutrient cycles. In Chapter 2, I investigated the competitiveness and production properties of three species of filamentous macroalgae easily found in temperate environments. The results found competitiveness to be the preferable feature when selecting a candidate species for freshwater bioremediation. In addition, I suggested two different bioremediation systems. The first option is the use of monocultures of two different species, one selected for warm seasons (Oedogonium) and another for cold seasons (Stigeoclonium). The second option is the establishment of bi-cultures of both species with varying dominances depending on the light and temperature conditions of temperate environments. In Chapter 3, I estimated the biomass and seaweed macroalgae composition at three locations in a semi-enclosed Bay in southern Australia. The results showed a 13-times decrease in drift macroalgae density compared to what was recorded 20 years ago, in 1996. However, the biomass retained the red algae dominance and remained abundant, especially during conditions that favoured the appearance of blooms. Isotope analyses showed that these macroalgae fed on anthropogenic nitrogen in areas close to nutrient sources, making them a preferable alternative for nutrient removal through harvest. This chapter also explored the potential co-benefits of the encountered seaweed. Chapter 4 showed how seaweed drifting mats gave shelter to a large number of invertebrates and ichthyofauna; however no individual was noxious/invasive or belonged to a special protection category. Drift macroalgae was not a key habitat for the fauna, since all individuals were abundant in sandy or reef habitats in the area. Finally, in Chapter 5 I studied how the drift mats affected the process of denitrification in the bay. The results obtained from benthic chamber experiments showed how perennial mats located in areas close to the nutrient loadings gave a buffering effect that helped maintain high denitrification rates under unfavourable seasonal periods (staying up to 70% higher in winter compared to controls). Overall, my findings provide a novel approach to select freshwater macroalgae candidates for bioremediation in temperate environments and give information regarding the variable effects on associated organisms and nutrient cycles derived from marine drift algae harvest. The findings of my thesis will be widely applicable to decision-making processes involving bioremediation mediated by fresh- and saltwater macroalgae.
Interaction of a pathogen with its host: The Talaromyces marneffei cell wall and its role in pathogenicity
Fungal pathogens are ubiquitous and are equally proficient in infecting both plants and animals raising the constant concern for public health as well as the economic impacts on agricultural production. Among the huge cohort of human fungal pathogens currently present, one is Talaromyces marneffei, an opportunistic fungus that mainly infects immunocompromised people. This pathogen is unique from others in its order as it is the only species that has the capacity to switch between two different cellular morphologies (known as dimorphism). At 25oC, it grows in a saprophytic multinucleate hyphal form which under favourable conditions undergoes asexual development to produce conidia, through specialised structures called conidiophores. These conidia are the infectious agents of this pathogen. Upon inhalation of these conidia by the host, these are phagocytosed by alveolar macrophages resulting in a temperature shift to 37oC. This shift in temperature induces the dimorphic switch and the conidia germinate into a uninucleate yeast form which is the pathogenic state of this fungus. The aim of this study is to investigate how T. marneffei survives and replicates inside the hostile environment of the host after being phagocytosed and to determine the importance of cell wall in this process. For this purpose, we developed a phagocytic model to mimic the early events in the host-pathogen interaction while analysing the host response to the invading pathogen and the counterattack mechanisms adopted by the pathogen to survive within the hostile environment. This study showcased the ultimate fate of T. marneffei inside the host cell, focussing on important events like phagocytosis, onset of germination, neutralization of phagolysosomes and replication inside the macrophages. This process of T. marneffei survival inside host also highlighted the importance of cell wall for a successful infection as difference in the cell wall architecture led to modulation of host responses against it. The overall cell wall architecture of T. marneffei in different cellular morphologies was determined with focus on beta-1,3;1,4 glucans (Mix linkage beta glucan) and alpha-(1,3) glucan with their respective role in pathogenicity. Given the importance of cell wall in survival of the pathogen inside the host, deletion mutants were also generated for genes involved in the biosynthesis of these cell wall components. This study revealed how disruption in the cell wall layering pattern rendered them susceptible against the applied stress signifying the importance of cell wall in survival and as a potential antifungal drug target. Overcompensation by upregulation of other cell wall genes on loss of a particular cell wall component also displayed the importance of intact cell wall for growth and replication. Overall this study has revealed how T. marneffei survives, germinates and replicates inside the host macrophages while highlighting the significance of the cell wall in its growth and pathogenicity with focus on two essential cell wall components beta-1,3;1,4 glucans (Mix linkage beta glucan) and alpha-(1,3) glucan.
Knockdown resistance in the dengue vector Aedes aegypti and its implications for Wolbachia Aedes aegypti trials
Dengue is a major public heath disease with over 390 million people at risk annually across 110 countries. Dengue control options mainly rely on vector control strategies, mostly through the application of insecticides for adult control and source reduction for larvae. As traditional control methods have limited success, the development of new control methods is gaining importance. One possibility involves the use of some strains of Wolbachia that have been intentionally transinfected into Aedes aegypti with the goal of reducing dengue transmission. Spread to high frequencies of Wolbachia in Yogyakarta has been challenged by insecticide resistance in local populations since existing control has substantially relied on insecticide-based interventions. It is important to measure insecticide susceptibility status and underlying mechanisms in the local population as this may hinder Wolbachia establishment. I developed an RT-PCR HRM (real time-PCR high-resolution melt) assay to detect the kdr mutations F1534C, V1016G, and S989P, in the voltage-sensitive sodium channel gene of Ae. aegypti, that has been associated with pyrethroid resistance. All of these kdr mutations were confirmed in mosquito samples from the outer rim and inner city of Yogyakarta, with the V1016G occurring at the highest frequency, followed by S989P, whereas F1534C had the lowest frequency. A correlation was observed between the V1016G mutation and resistance to deltamethrin and permethrin. In contrast, I observed a weak association between F1534C and permethrin. The S989P mutation, always found with V1016G, showed some association with deltamethrin resistance. The resistance status of the laboratory strain destined for release in Yogyakarta was equivalent to the mosquitoes from the field sites where the original backcross mosquitoes had been collected, as well as having the same resistance allele frequency. I also observed co-occurrence between kdr mutations: V1016G/F1534C; V1016G/S989P; and V1016G/F1534C/S989P. I used the HRM assay to try and detect the kdr mutations in Ae. aegypti populations in Queensland, and there was no evidence of the kdr mutations there. In a separate experiment, a series of laboratory selection procedures was carried out to generate a strain of Wolbachia-infected mosquitoes with some degree of resistance to pesticides, based on Queensland Ae. aegypti nuclear background material. This attempt proved unsuccessful, emphasizing low resistance in the Queensland population likely maintained by low selection pressure. In summary, these data provide information relevant to Wolbachia releases that can help ensure that released material has an appropriate level of resistance to pesticides to allow the released mosquitoes to be competitive with those from natural populations.
The effects of artificial light at night on the behaviour and physiology of Drosophila melanogaster
Artificial light at night is one of the most pervasive and least recognised anthropogenic pollutants. Its extent and intensity is expanding globally, at an estimated rate of 2.2% per annum, such that many urban and peri-urban animals no longer experience natural darkness. The majority of animal species have evolved under bright days and comparatively dark nights, with their physiology and behaviour synchronised to this rhythm. Accordingly, disruption to this cycle is linked to a suite of behavioural and physiological shifts across taxa. Experimental and correlational evidence documenting phenotypic responses to the presence of artificial light at night are accumulating but the underlying mechanisms driving the observed negative impacts associated with artificial light at night are not resolved. A likely reason is that light at night disrupts circadian rhythms and contributes to perturbed oxidative status through its interaction with the indolamine, melatonin, a key driver of circadian rhythm and powerful antioxidant. A major and currently untested gap in the literature is whether light at night can act as a driver of evolutionary change. In this thesis, I investigated the short-term phenotypic impacts of ecologically relevant levels of artificial light at night on life history traits (throughout Chapters 2 to 5) and oxidative status (Chapters 2 and 4), using the model organism Drosophila melanogaster. I then used experimental evolution to explore whether artificial light at night can induce local adaptation in populations evolved over 15 to 25 generations (Chapter 4). Finally, using a melatonin dietary supplementation experiment, I attempted to investigate the relationship between artificial light at night and the melatonin pathway, thus hoping to demonstrate disruption to the melatonin pathway as a causal mechanism underpinning the observed phenotypic responses (Chapter 5). My results demonstrated experimentally, that individuals exposed to artificial light at night have short-term phenotypic changes, with disrupted mating behaviour, reduced fecundity and size, altered development patterns and reduced longevity in D. melanogaster (Chapters 2 to 5). Additionally, I present novel (and somewhat counter intuitive) evidence, that artificial light at night is associated with lower levels of reactive oxygen species in ovaries (Chapter 3) and, aligned with this, reduced oxidative DNA damage in female flies (Chapter 4). In contrast, I found no evidence for such effects in males (Chapters 3 and 4). Despite potential for evolutionary change, I found little evidence for adaptation in most fitness traits after evolution under artificial light at night (Chapter 4), with mating propensity the only life history trait to exhibit limited local adaptation. This suggests that the selective pressure of artificial light at night in the laboratory, may be weaker than anticipated. However, the fact that some level of adaptation was evidenced in relatively benign conditions, suggests that, in a more natural competitive environment, the selection pressure of light at night may be stronger. I was unable to conclude whether melatonin is a potential mechanism driving phenotypic variation following exposure to light at night (Chapter 5), as the melatonin doses and design used, despite replicating a previous study, resulted in negative fitness consequences (reduced longevity under dark nights and disrupted eclosion regardless of light treatment), suggesting the melatonin supplementation was potentially toxic. Nonetheless, these data potentially support the importance of circadian disruption under artificial light at night as a driver of the observed negative effects. I propose that further research is warranted to test different melatonin concentrations and dosage duration at different life stages and at different times relative to circadian rhythm. This variation in dosage regimens may be able to better define its role (and the relevant contributions of circadian disruption and oxidative status) in the effects of artificial light at night. This thesis demonstrates the negative impacts of artificial light at night in the model species, D. melanogaster and adds to the growing body of literature documenting taxa-wide damaging effects of artificial light at night on animal behaviour and physiology. As we continue to light our nights and reduce natural darkness globally, understanding the consequences and mechanisms behind the effects of artificial light at night is paramount to informed decision making around urban lighting strategies. Moreover, understanding the role of artificial light at night in driving evolutionary change, is of the utmost importance in maintaining wildlife stability in an increasingly urbanised world.
Effects of elevated atmospheric carbon dioxide on iron metabolism in bread wheat (Tritium aestivum L.)
Atmospheric carbon dioxide concentrations have increased from pre-industrial levels of 280 ppm to a current level of 406 ppm and are predicted to reach 550 ppm by the year 2050. The rise in atmospheric carbon dioxide is known to directly alter the photosynthetic activity of C3 crops resulting in enhanced photosynthetic carbon fixation and, as a consequence, an increase in water use efficiency, biomass and grain yield. Yet, there is considerable evidence indicating a concomitant reduction in the content of essential mineral micronutrients such as iron in cereal grain. Wheat is the second most consumed staple crop in the world and it is an important source of calories for the human population, however it contains low grain iron concentration. Under rising levels of carbon dioxide wheat grain will likely contain even lower iron concentration and thus, intensify the already existing acute problem of human iron malnutrition, which currently affects over two billion people worldwide. This project aims to study the reasons underlying decreased iron concentration in wheat grain under elevated carbon dioxide concentrations by investigating wheat iron metabolism in relation to its uptake and remobilisation in field settings and to assess the feasibility of lessening the decreased iron concentration in wheat grain using a transgenic biofortification approach. Field trials were conducted over two growing seasons at the Australian Grains Free Air Carbon Dioxide Enrichment facility to investigate changes in iron distribution of bread wheat grown under ambient and elevated carbon dioxide concentration. At maturity, grain iron concentration decreased under elevated carbon dioxide concentration by 25% in the first season and by 26% in the second season. Iron distribution analysis revealed that an increased proportion of iron remained in the lower leaf, flag leaf and bracts during grain filling under elevated carbon dioxide concentration, resulting in a decrease in the iron remobilisation from those organs to the grain. Iron is an essential micronutrient, not only for humans, but for all plants and is involved in several important biological processes, including photosynthesis, respiration and chlorophyll biosynthesis. Iron possesses chemical properties that make it suitable to associate with proteins as a cofactor in the form of heme and iron-sulphur cluster. In order to decipher whether altered iron distribution between organs under elevated carbon dioxide concentration was related to changes in metabolic processes, in which iron is involved, an untargeted and targeted metabolite analysis was performed in flag leaf, bracts and grain at grain filling under ambient and elevated carbon dioxide concentration. In addition, the expression of genes involved in iron long-distance transport, iron influx and efflux transport, iron chelation biosynthesis and iron storage were investigated. The results showed decreases in the levels of compounds involved in most metabolic processes related to iron, including those involved in photosynthesis, nitrogen assimilation and oxidative stress in all three organs under elevated carbon dioxide concentration, with increases in the compartmentalisation of iron in chloroplasts and vacuoles. The iron chelators nicotianamine and deoxymugineic acid; showed decreased concentration in the grain under elevated carbon dioxide concentration, with a decreased expression of the genes involved in their biosynthesis. Furthermore, a decreased expression of genes involved in iron long-distance transport from flag leaf and bracts into the grain was shown. Biofortification is the enrichment of staple crops with essential micronutrients through agricultural practices, conventional breeding and/or genetic engineering. Constitutive expression of nicotianamine synthase genes has been an effective genetic engineering strategy to increase iron concentration in cereals such as wheat. In order to investigate the effects of elevated carbon dioxide concentration on a transgenic iron biofortification trait in wheat, transgenic wheat constitutively overexpressing the nicotianamine synthase gene and corresponding null segregants were grown under ambient and elevated carbon dioxide concentration in a glasshouse setting. The analysis revealed that the transgenic iron biofortified wheat plants grown under elevated carbon dioxide concentration increased grain iron concentration as well as nicotianamine and deoxymugineic acid chelators compared to the null segregants. Overall the results of this PhD project indicate that the decreased iron concentration in wheat grain under elevated carbon dioxide concentration is associated with altered disruption of iron within the plant during grain filling, with a greater proportion of iron remaining in flag leaf and bracts at the expense of the grain. This study suggests that the iron surplus in flag leaf and bracts under elevated carbon dioxide concentration is related to a decreased expression of the long-distance iron transporters and chelator genes responsible for the loading of iron from flag leaf and bracts into the grain, where the iron is compartmentalised in chloroplasts and vacuoles to avoid toxicity. Iron biofortified wheat plants constitutively expressing the OsNAS gene show potential to counteract low grain iron concentration under elevated carbon dioxide concentration.
Dinoflagellate endosymbionts of corals (Symbiodiniaceae) closely associate with a diversity of bacteria
The ecological success of coral reef ecosystems is dependant on their obligate endosymbiosis with dinoflagellates in the family Symbiodiniaceae. This symbiosis does not occur in isolation; a diverse community of bacteria contribute to coral health and functioning. Though microbial-coral relationships are well studied, and dinoflagellate-bacterial associations are abundant in the marine environment, limited research focuses on Symbiodiniaceae-bacterial interactions. We combined autofluorescence quenching of Symbiodiniaceae with fluorescence in situ hybridisation to create three-dimensional reconstructions. These results present the first conclusive evidence that six species of Symbiodiniaceae harbour intracellular bacteria as well as cell surface associated extracellular bacteria. Hybridisation of class specific probes (Gammaproteobacteria, Alphaproteobacteria and Flavobacteriia) showed that taxonomic affiliation of intracellular bacteria differed between Symbiodiniaceae species. Furthermore, 147 members from the phyla Proteobacteria, Bacteroidetes and Firmicutes were isolated from ex hospite Symbiodiniaceae. Low diversity of cultured bacterial symbionts suggest that Symbiodiniaceae are selective in their association with bacteria. Based on the diversity and functional potential of Symbiodiniaceae-bacterial associations microbial interactions should no longer be ignored as potentially contributing to the coral holobiont health and functioning.
Coral-associated bacterial communities in early coral life stages: transmission mode and scope for manipulation
Global impacts of climate change and other anthropogenic disturbances are causing massive declines in coral reef ecosystems. As reef-forming scleractinian corals provide essential resources to a large part of the population, their degradation has severe ecological, social and economic consequences. Efforts are therefore urgently required to limit human impacts (e.g., by drastically reducing emissions of greenhouse gases), and also to assist coral adaptation to climate change. In this context, favourably adjusting coral-associated microbial communities could potentially benefit the host, as microbial symbionts are known to play critical roles in coral health. Probiotics have already proven effective in other organisms such as plants to increase crop yields, and humans to treat various bowel conditions. The successful application of probiotics in corals is contingent on the feasibility to manipulate the coral microbiome. Therefore, a central question of this thesis is whether the coral microbiome can be influenced by targeted bacterial inoculation in the laboratory. Initially, understanding how corals acquire and maintain their bacterial associates will assist in predicting whether probiotics could be taken up and retained across generations. The transmission mode of bacteria in corals with different reproductive strategies therefore constitutes an additional focus of this thesis. I start the thesis by providing information on the ecological importance of coral reefs, the threats that they are currently facing in the context of climate change and approaches that have been proposed to accelerate the adaptation of corals to environmental disturbances (Chapters 1 and 2). Within that scope, I focus on coral-associated microorganisms, highlight their roles for coral health and discuss the potential of microbial biotechnology to mitigate coral reef degradation. Chapter 2 includes experimental data providing proof-of-concept that the bacterial microbiome of juvenile corals can be influenced by exposing coral larvae to the mucus (which is known to contain a diverse microbiome) extracted from adult colonies of different coral species (Chapter 2). In Chapters 3 and 4, I investigate the transmission mode of coral-associated bacteria using 16S rRNA gene metabarcoding and fluorescence in situ hybridisation (FISH) microscopy. No evidence of direct vertical transmission of internalised bacteria was observed in the broadcast spawner Acropora tenuis (Chapter 3). However, metabarcoding shows that the gametes were already associated with diverse bacteria upon release and that early coral life stages successively associated with different bacterial communities, probably acquired from the environment. It is possible that the coral-associated mucus is removed during the FISH fixation procedure and therefore bacteria in the mucus would not be visualised by FISH microscopy. Parental colonies may thus drive the transfer of certain bacteria by releasing them to the water column upon spawning (horizontal transmission) and/or through the mucus layer coating the gametes while these are still inside the coral polyp (vertical transmission). Clear evidence for vertical transmission is present in the brooder, Pocillopora acuta, where newly released larvae contained internalised bacterial aggregates (Chapter 4). DNA metabarcoding provides evidence for vertical transmission as well as horizontal uptake of bacteria in this coral species. In Chapters 5 and 6, I explore the possibility of manipulating coral-associated bacterial communities by exposing coral recruits to fragments of adult corals (Chapter 5), as well as to a cocktail comprising a small number of pure bacterial cultures (Chapter 6). In the former approach, rearing P. acuta coral recruits in the vicinity of adult fragments of P. acuta and Platygyra daedalea under a flow-through system did not result in different bacterial associates developing in juveniles compared to control corals. The temporal changes of bacterial assemblages in early recruits suggest that their microbiome is dynamic, which may facilitate uptake of bacterial strains in an inoculum but also challenge their retention over time. In Chapter 6, I rear A. tenuis and P. daedalea recruits in the same aquaria and repeatedly inoculate them with a bacterial consortium generated in the laboratory. The seven bacterial strains present in the inoculum were enriched in inoculated recruits of the two coral species compared to the no-inoculum controls, and there is a significant effect of the inoculum on the coral-associated bacterial communities. Additionally, some of the structuring in the bacterial microbiomes is explained by host species, highlighting the role of host factors in shaping bacterial community composition. These results support proof-of- concept for the feasibility of coral microbiome manipulation as a first step towards developing probiotics aimed at enhancing coral climate resilience. In Chapter 7, the general discussion, characteristics of microbial inoculation strategies that could be implemented to corals are discussed, as well as their advantages and shortcomings. Emphasis is also drawn to current knowledge gaps and research priorities for the field of microbiome engineering in corals.
Impacts of streetlights on sleep in urban birds
Over the past century, artificial light has dramatically transformed our environment. Light at night is increasing globally, to the extent that in many places, true darkness no longer exists. As the timing of light can influence almost all aspects of biology, the alteration of natural light cycles could pose a severe threat to wildlife. One particularly harmful impact could be the disruption of sleep. In this dissertation, I investigate the impacts of artificial light at night on sleep. Despite the importance and prevalence of sleep across the animal kingdom, sleep is arguably underappreciated in studies of ecology and conservation. After providing a general introduction (Chapter 1), I begin by giving a broad perspective of sleep research, including current methods, opportunities, and the significance of sleep for issues such as artificial light at night (Chapter 2). I then provide a review of the evidence for impacts of artificial light at night, in both humans and wildlife (Chapter 3). Finally, I explore the effects of artificial light at night on two diurnal bird species: pigeons (Columba livia) and black swans (Cygnus atratus). I focus on the effects of one of the most common sources of outdoor lighting: streetlights. Light at night from LED streetlights caused pigeons to have less rapid eye movement (REM) sleep and non-REM sleep, have more fragmented sleep, and sleep less intensely than during darkness (Chapter 4). Some of these effects persisted for more than a day after exposure to light at night. In black swans, light at night in a naturalistic environment reduced night-time rest, which we demonstrate reflects reduced sleep (Chapter 5). This research provides the first direct evidence that exposure to environmentally-realistic artificial light at night can disrupt sleep in birds. One possible strategy for reducing disruption of sleep could be to alter the colour of lighting. To test this idea, I compare the effects of two different lighting colours: white (blue-rich) and amber (blue-reduced) light. Previous research has shown that blue wavelengths of light have the greatest effect on melatonin, a hormone important for sleep regulation. However, contrary to my predictions, amber and white light had very similar effects on sleep in both pigeons (Chapter 4) and swans (Chapter 5). Together, these findings will help councils and other land managers to make more informed decisions about lighting, particularly for areas that might offer important refuges for wildlife.
Bioinformatics methods and approaches to discover disease variants from DNA sequencing data
Next-generation sequencing is increasingly used to diagnose patients with suspected genetic disease. Yet, even after exome or whole genome sequencing, many patients remain undiagnosed. In many cases a genetic diagnosis is not made because we either failed to detect the causal variant, or succeeded in detecting it, but failed to identify it as causative. There is a clear need to develop novel bioinformatics methods and sequencing strategies to address these shortcomings and to increase diagnostic rates. In this thesis I develop several strategies to address these issues. I propose a pooled-parent exome sequencing approach to prioritise de novo variants for genetic disease diagnosis. In this strategy, a set of probands have individual exome sequencing, while the DNA from all the parents of the probands are pooled, exome captured and sequenced together. The variants called in this pool are used to filter out inherited variants in the probands so the remaining list is enriched for de novo variants. Short Tandem Repeat (STR) expansions are a class of disease-causing variants that are frequently missed in short read sequencing data. Here I develop and validate STRetch, a new bioinformatics method to detect STR expansions using STR decoy chromosomes. I show that STRetch can be used to detect both known pathogenic STR expansions, and novel expansions at other annotated STR loci across the genome. I further use STRetch to explore variation across hundreds of individuals to inform our understanding of what is common variation and what is potentially pathogenic, to aid in prioritising STR variants in a gene-discovery setting. Some of the methods that I have developed and describe within this thesis have already been used to help patients receive a genetic diagnosis.
Seagrass perform critical provisioning, regulating, cultural and supporting ecological functions and services humans rely on worldwide. Unfortunately, many seagrass ecosystems are vulnerable to disturbance and are being lost at alarming rates. Some seagrass species have been listed by the International Union for the Conservation of Nature as threatened and endangered where population sizes are small or are highly restricted in geographic distribution. For several other species, there is not enough data to determine whether a species is at risk or not. When ecological resilience thresholds for species survival and reproduction are exceeded, declines in seagrass may occur. Declines over time and space may be episodic or ongoing and occur as a result of impacts from both natural and anthropogenic stressors, such as turbidity, eutrophication, hypersalinity and urbanization. Increasing global change phenomena are expected to exacerbate declines of seagrass in many parts of the world, and vast losses of seagrass have functional implications for other ecosystems and organisms. Heterozostera are unique seagrass broadly distributed throughout coastal southern latitudes especially in Australia. Only three populations of Heterozostera are found outside of Australia, in a small region of the eastern Pacific in Chile. The populations are made up of 2 nonflowering clones. Therefore, Heterozostera of Australia may be functionally endemic to southern Australia, including Tasmania. Substantial loss of Heterozostera has been reported from the middle of Heterozostera's range in several locations of Victoria, Australia. Subsequent recovery of diminished populations has been slow in some sites, especially in Western Port, Victoria. Few published accounts related to resilience, including seagrass population monitoring for conservation and restoration of Heterozostera in the southern hemisphere have been completed. The aim of this thesis is to advance basic botanical research on Heterozostera in support of taxonomic resolution and the development of conservation strategies, especially those focused on modeling and applied seagrass resilience and restoration in Australia. More studies are needed to contribute to the development of restoration and resilience management strategies for lesser studied southern hemisphere seagrass ecosystems, and this work supports the generation of additional discoveries. Specifically, the objective of this dissertation is to identify and examine the autoecology and resilience Heterozostera, including sexual and clonal reproductive success under variable environmental cues. In all, five comprehensive studies of Heterozostera biogeography and biology are included in this dissertation, along with a theoretical framework and summary exploring the roles of environmental parameters in the autoecology of resilience for temperate Australian Heterozostera. Chapter 1 presents a review of research in support of Australian Zosteraceae, including trends and gaps in our understanding of seagrass resilience mechanisms, including both resistance and recovery pathways in Heterozostera. Chapter 2 details the results of an extensive survey of existing sediment and nutrient conditions for Heterozostera populations across 15 seagrass meadows located in southeastern Australia's Port Phillip Bay. Chapter 3 summarizes a series of experiments aimed at discovering effective Heterozostera seed storage and collection protocols for use in land based aquaculture, and specifically examining the potential roles of seed colour, sterilisation and refrigeration with the goal of improving long term seed viability. Chapter 4 contains a fully factorial germination assay undertaken to identify potential cues to Heterozostera germination, including sediment type and sterilization, nutrient loads, and refrigeration. Chapter 5 details a germination experiment using a 2 ppm copper sulphate (CuSO4) solution to cue germination of Heterozostera seed. Lastly, Chapter 6 explores the rates of asexual growth and survival of three types of clonal reproductive Heterozostera propagules (rhizome, plantlet and shoot). This work provides novel information about several topics of research with regard to the autoecology of Heterozostera including: (1) species identification, (2) species ecology and biogeography, (3) reproductive material collection and storage, (4) seed germination, and (5) clonal transplanting. This research demonstrates Heterozostera is capable of widespread recovery across a range of sediment and nutrient conditions. Continued work on lesser known Heterozostera species (H. tasmanica and H. polychlamys), and other rarer seagrasses world-wide are critical for understanding the potential loss of resilience and increased vulnerability (or conversely, the potential for recovery, adaptation and survival) of threatened and endangered seagrass populations with limited global dispersal. Collectively these studies details methods to support future Heterozostera research in both laboratory and field settings where culture of reproductive materials are required. In addition, the outcomes from this research provides novel information in support of progressing insitu seeding and transplanting efforts aimed to improve operation of land based seagrass nurseries and research studies. Novel evidence builds support that Heterozostera nigricaulis as a functionally resilient species of seagrass, largely due to its ability to inhabit variable ecological conditions and to utilize multiple sexual and asexual reproductive strategies to recover populations following disturbances.