School of Botany - Theses

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Characterisation of cyanogenic glucoside synthesis in Eucalyptus

Neilson, Elizabeth H. (University of Melbourne, 2012)

Cyanogenic glycosides are nitrogen-containing defensive metabolites, releasing toxic hydrogen cyanide following tissue disruption and contact with catabolic enzymes. Within the ecologically and commercially important genus Eucalyptus, approximately 4% of species are cyanogenic, possessing the phenylalanine-derived cyanogenic glucoside prunasin. Cyanogenic Eucalyptus species have been identified as an excellent experimental system in which to investigate defensive ontogenetic regulation as different species have shown variability in the onset and regulation of cyanogenic glucoside biosynthesis through plant development. The overall aim of this PhD thesis is to characterise cyanogenic glucoside synthesis in cyanogenic Eucalytpus species throughout leaf and plant ontogeny by investigating the synthesis, turnover and regulation of cyanogenic glucoside/(s) in E. yarraensis and E. camphora. Using various PCR techniques, four cytochrome P450 genes encoding CYP79 enzymes were detected and isolated from E. yarraensis: CYP79A34, CYP79A35, CYP79A36 and CYP79A37. The CYP79A34 gene was functionally cloned and expressed in Saccharomyces cerevisiae using the USER� cloning technique and a modified pYeDP60 vector, respectively. The recombinantly expressed CYP79A34 was shown to catalyze the conversion of L- phenylalanine into phenylacetaldoxime, verifying its involvement in the first step of prunasin biosynthesis. Using quantitative real-time PCR, CYP79A34 mRNA levels were found to be positively correlated with an increase in prunasin concentration during E. yarraensis ontogeny, suggesting that prunasin synthesis is regulated at the transcript level. Interestingly, CYP79A34 transcript levels also significantly increased in leaves subject to cold temperature and nitrogen application. To further investigate how leaf ontogeny affects the synthesis and possible turnover of prunasin, E. yarraensis coppice branches were pulse-labeled with 14C (via 14C02) and the incorporation of 14C into the nitrile constituent of the cyanogenic glucoside was measured in individual leaves over a 72 h period. Despite the high similarity observed in physical parameters and overall patterns of prunasin allocation across different leaf classes, a surprisingly high level of variability in 14C incorporation was encountered. Prunasin synthesis and turnover was observed in both expanding and fully expanded E. yarraensis leaves, although an accurate measure of turnover was not achieved. Interestingly, diurnal variation in specific activity suggests that the synthesis of prunasin may be influenced by light-dependent plant processes. Whole plant ontogenetic effects on cyanogenic glucoside synthesis were investigated in E. camphora, by identifying, tracking and comparing the concentration and composition of multiple cyanogenic glucosides throughout different ontogenetic stages. Using different analytical methods (LC-MS/MS, GC-MS and NMR) six phenylalanine-derived cyanogenic glucosides were identified, including three cyanogenic diglucosides characterised by unique linkage positions between the two glucose moieties. Two of these cyanogenic glucosides have not previously been reported and were named eucalyptosin B and eucalyptosin C. Interestingly, the different cyanogenic diglucosides could be distinguished by unique MS/MS fragmentation patterns. This knowledge should facilitate analysis and structural elucidation of cyanogenic diglucosides from other plant species. Quantitative and qualitative differences in total cyanogenic glucoside content were observed across different stages of whole plant and tissue ontogeny, as well as within different tissue types. Seedlings of E. camphora were found to produce only the cyanogenic monoglucoside prunasin, but "switch on" prunasin biosynthesis at different times. Once initiated, total cyanogenic glucoside concentration increased throughout plant ontogeny with cyanogenic diglucoside production initiated in saplings and reaching a maximum in flower buds of adult trees. Overall this work uncovered many novel aspects of cyanogenic glucoside synthesis in E. camphora and E. yarraensis, particularly in terms of ontogenetic regulation, and provides a solid platform to further investigate these species and their unique control over cyanogenic glucoside synthesis.
Grape and botrytis cinerea proteases : characterization and utilization in winemaking

Van Sluyter, Steven C (University of Melbourne, 2012)

Grape berries contain high levels of pathogenesis-related (PR) proteins that persist through winemaking and cause commercially unacceptable heat-induced hazes if not removed before bottling. Nearly every commercial winery uses the clay cation exchanger bentonite to remove PR-proteins from wine, but the process results in wine losses, is laborious, and can negatively affect wine sensory properties. An alternative to protein removal by bentonite is hydrolysis of PR-proteins by proteases. However, PR-proteins are highly resistant to enzymatic proteolysis and no commercially viable enzymatic method has been discovered in at least 60 years of attempts. The purpose of the research presented in this thesis was to discover grape and Botrytis cinerea enzymes that could be utilized during winemaking to reduce or eliminate the need for bentonite by degrading PR?proteins. Grapes infected with B. cinerea have been observed previously to have lower levels of PR?proteins than healthy grapes or grapes infected with other pathogens. To investigate Botrytis proteases, knock-out mutants were screened on grape berries and against purified PR-proteins on agar plates. The mutants were no more inhibited by PR?proteins than the wild type, suggesting that either the aspartic proteases serve redundant functions, or their function in vivo is unrelated to grape PR-proteins. A Botrytis protease previously found to be the most highly secreted protein in liquid culture was heterologously produced in Pichia pastoris and used in winemaking trials. The Botrytis protease, BcAP8, significantly reduced the PR-proteins most prone to aggregation, chitinases, but did not significantly reduce,thaumatin?like proteins. BcAP8 could be an effective treatment for reducing bentonite in wines with unstable,chitinases. Using activity-based probes to enrich for proteases followed by mass spectrometry, grape papain-like cysteine proteases were characterized for the first time. One of the proteases is highly glycosylated, contains an extra disulfide bond and several other novel sequence features, and represents a new sub?family of plant proteases. Serine and nepenthesin?like aspartic grape proteases were also discovered by mass spectrometry. The cysteine protease activity of grape berries, most likely attributable to the newly discovered highly glycosylated protease, was associated with the insoluble material of grape juice and was highly heat stable. Those two attributes were exploited to develop a flash pasteurization method that effectively eliminated the need for bentonite in the tested juices.
Evaluating the ecological and operational basis of vegetation condition assessments

Gorrod, EmMA (University of Melbourne, 2011)

Globally, market based policies for biodiversity conservation are emerging that aim to achieve better and more cost efficient biodiversity outcomes than traditional command and control mechanisms. Investment accounting, reverse auctions and biodiversity offsets aim to achieve No Net Loss or Net Gain of biodiversity by incorporating biodiversity into the financial decisions of landholders. Market based policies require a proxy commodity for biodiversity that numerically express the capacity of a given site to support biodiversity, and Australia has pioneered the development of vegetation condition indices for this purpose. For every market based decision, there is a chance that the actual biodiversity outcome will be better or worse than expected. Risks of worse than expected outcomes may have significant financial implications and irreversible repercussions for biodiversity. Uncertainty in estimates of current and future vegetation condition may increase these risks. Despite this, market based policies have not been developed within a risk management framework and no systematic analysis of factors that may contribute to uncertainty in biodiversity valuation has previously been conducted. This thesis evaluates the nature of uncertainties in the ecological and operational basis of vegetation condition assessments, and considers the potential effects of uncertainty on the biodiversity outcomes of market based policies. Chapter 2 proposes a framework for identifying epistemic and linguistic uncertainties in current and predicted future estimates of vegetation condition. It contends that uncertainty may arise due to: i) conceptual uncertainty about the factors and processes that are causally related to the definition of biodiversity value; ii) quantitative uncertainty concerning misrepresentation of causal factors and processes by the mathematical constructs of the index; and iii) data uncertainty arising from errors in the input data. Specific occurrences of these uncertainties are discussed for two Australian vegetation condition indices, BioMetric and Habitat Hectares. It is concluded that the nature of uncertainties in estimates of current and future vegetation condition may increase the risk that any given decision will fail to achieve No Net Loss of biodiversity. Chapters 3 to 5 evaluate sources of uncertainty in current estimates of vegetation condition. Chapter 3 uses empirical biodiversity data to evaluate whether, on average, sites with higher vegetation condition scores support a greater diversity of native species than sites with lower scores. The strength of relationships between species composition and condition scores was primarily driven by plant species, which is included as a predictor variable in both BioMetric and Habitat Hectares. Vegetation condition was very poorly correlated with the richness and diversity of native butterflies and native ants, with the possible exception of very low scoring sites for butterflies. The results suggest that patterns in species composition were driven by factors other than the predictor variables included in the indices, and therefore that conceptual uncertainty may be a substantial cause of poor performance of vegetation condition indices. Chapter 4 empirically investigates the magnitude of uncertainty in estimates of current vegetation condition due to observer error in field estimates of predictor variables. Average coefficients of variation in total vegetation condition scores amongst ten independent observers were 15-18%. All observers estimated vegetation condition scores that were substantially different from the group mean on at least some sites. The results indicate that measurement error in field estimates of site attributes may cause vegetation condition to be under- or over-estimated on all but highly degraded sites. Chapter 5 examines the sensitivity of vegetation condition indices to observer error via simulated scenarios. Larger observer errors caused less precision and greater bias in total scores, although compensatory errors generally led to smaller coefficients of variation in total scores than predictor variables. Unexpectedly, unbiased observer errors in the predictor variables underestimated the true vegetation condition of most sites. The underestimation effect was more significant for sites with intact woody or herbaceous features, depending on the index used. Chapters 4 and 5 highlight the fact that quantitative aspects of vegetation condition indices are as important as the spread and bias of raw observer estimates in determining accuracy of vegetation condition assessments. These operational limitations of vegetation condition indices may significantly increase the risk of worse than expected biodiversity outcomes. Chapter 6 examines the implications of uncertainty in estimates of predicted future vegetation condition for achieving No Net Loss in biodiversity offsets. Expert models of change in 12 vegetation attributes were used to simulate change over time for five states of a grassy woodland ecosystem under six (gain and loss) management scenarios. The quantity of gains that would be required to compensate for each loss scenario (i.e. the offset ratio) was calculated using one method that ignored uncertainty and another that accounted for it. Uncertainty increased the offset ratio up to 1400% where the magnitude of both gains and losses were uncertain, but increased the ratio only marginally where minimal losses were incurred on highly degraded site types and offset with maximal gains. Both vegetation condition indices predicted greatest gains in woody vegetation attributes, which may result in landscape wide decline in herbaceous components of vegetation and their associated biodiversity if policies aim to maximise gains in vegetation condition. The results of this research indicate that aspects of the conceptual, quantitative and data models that underpin biodiversity valuation indices are likely to cause estimates of current and future biodiversity value to be unreliable. Outcomes of market based biodiversity conservation policies would greatly benefit from more rigorous procedures for developing and evaluating biodiversity valuation indices, which systematically identify and minimise uncertainties in the underpinning models. Information about uncertainty should be used in a risk-based approach to decision making in market based policies, whereby only low risk decisions are approved. It will be a shame if the pioneering work Australia has conducted in developing market based instruments for biodiversity conservation, and associated vegetation condition indices, is not used to fully evaluate the potential for overcoming market externalities and achieving optimal biodiversity outcomes for society.
The function and evolution of the dinoflagellate mitochondrion

Ackland, Jillian Clare (University of Melbourne, 2010)

The mitochondrion is a highly evolved, indispensable organelle found in all eukaryotes. This compartment has undergone metabolic and functional changes as cell lineages have diversified and specialised throughout evolution. Alveolata is a well-defined group of single-celled eukaryotes that encompasses related Phyla with extremely diverse lifestyles. Most alveolates belong to one of three main subgroups: predatory ciliates, endoparasitic apicomplexans, and heterotrophic or autotrophic dinoflagellates. Little is known about the biology of the dinoflagellate mitochondrion and studying this compartment offers an opportunity to examine organelle evolution within the alveolate lineage. In this study, I have used the dinoflagellate Karlodinium micrum, as a model to examine mitochondrial evolution. I have investigated the evolution of (1) genes, (2) biochemical functions and (3) protein targeting mechanisms of this organelle. Organelles can replace and gain genes by endosymbiotic gene transfer (EGT, genes derived from an endosymbiont) and lateral gene transfer (LOT, genes derived from an external source). Dinoflagellates have shown a unique propensity to replace their plastids with plastids of other algae during evolution and K. micrum represents a dinoflagellate lineage that has replaced its ancestral plastid with an endosymbiont derived from a haptophyte. In this case, haptophyte endosymbiont plastid genes are located in the dinoflagellate nucleus, providing evidence of EGT in this system. I have assessed if the mitochondrial proteome of K. micrum has been remodelled by EGT and/or LTG. Genes encoding mitochondrial proteins have been identified from a K. micrum expressed sequence tag library and their evolutionary origins inferred by phylogenetics. Several mitochondrial genes are derived from an external source but none originate from the haptophyte endosymbiont, indicating that the K. micrum mitochondrial proteome has been minimally impacted by this endosymbiotic event, but is nevertheless genetically dynamic. Plasmodium falciparum, the disease agent that causes malaria, is a member of Apicomplexa. This protist has a mitochondrion that has been described as metabolically reduced compared to canonical mitochondria, change that has been attributed to the parasitic lifestyle this organism leads. In this thesis, I test whether or not perceived reduction in apicomplexan mitochondrial metabolism is a result of parasitism. A putative metabolic map of the dinoflagellate mitochondrion has been constructed and compared to what is currently known about the mitochondrial biochemistry of closely related apicomplexan parasites and a free living basal alveolate, the ciliate Tetrahymena thermophila. This is the first report of a broad analysis of the mitochondrial metabolism of a dinoflagellate. The mitochondrion of K. micrum shows broad metabolic conservation, having retained pathways implicated in ATP generation by oxidative phosphorylation. Several changes in the metabolism of the P. falciparum mitochondrion were also observed in K. micrum and/or T. thermophila, suggesting that these modifications are not due to parasitism. The presence of most components of the tricarboxylic acid cycle, in addition to what is most likely a functional electron transport chain and ATP synthase complex in both dinoflagellates and P. falciparum indicates that the mitochondrion of the Plasmodium parasite is probably implicated in ATP generation by oxidative phosphorylation. The diversification of dinoflagellates has been accompanied by considerable changes in plastid protein targeting signals, but it is unclear whether or not mitochondrial protein targeting in this lineage has also been modified. In the final experimental chapter of this thesis I have assessed the conservation of mitochondrial protein import mechanisms in dinoflagellates. Genes for K. micrum mitochondrial proteins have been analysed for mitochondrial protein targeting signals using bioinformatic tools, and the function of these signals has been tested using the reporter molecule green fluorescent protein (GFP) and a heterologous yeast expression system. Amino-terminal and internal mitochondrial targeting signals of K. micrum mitochondrial precursors are sufficiently conserved for recognition and import into yeast mitochondria, indicating that dinoflagellate mitochondrial protein targeting signals have been highly conserved, since early in eukaryotic diversification. Overall, my investigations of the dinoflagellate mitochondrion, and this broad comparative analysis of alveolate mitochondria has shown that aspects of mitochondrial biology (ie. mitochondrial gene compliment, mitochondrial biochemistry and function, and mitochondrial protein targeting) have evolved differently during the diversification of alveolates. Alveolate mitochondria are genetically flexible, having experienced gene gains and gene losses. This variation is not always accompanied by functional divergence, and does not necessarily reflect the lifestyle/nutritional requirements of the host. Thus while the mitochondrion is clearly an innovative compartment, its evolutionary behaviour cannot be characterised based on any one aspect of its cell biology alone.
Quantifying search and control performance during marine invasive surveys: a case study from Asterias amurensis

Millers, Kimberley ( 2015)

Marine invasive species are a global threat to marine biodiversity. Effective management of invasive species depends on accurate population information. To best inform management, surveys of abundance, occupancy and detectability must be carefully designed, and account for uncertainty. However, many marine invasive management programs currently do not record and account for the uncertainty of detectability. In this thesis, I document the limitations of searching for a marine invasive species during eradication programs and examine ways to improve detectability in future survey designs. Specifically, I undertake a series of empirical surveys to test how effective observers are at detecting the northern Pacific seastar, Asterias amurensis; this species poses a serious threat to native and commercial species in southern Australia. I use artificial silicone replicas of A. amurensis during empirical surveys so as to eliminate the risk of spreading the marine invasive species. I use data combined with Bayesian methods to develop a population catch-effort model, which provides insights into what influences detectability and whether eradication at these sites was a viable management goal. Finally, I take a novel approach to testing optimal search theory under field conditions. Optimal search theory has been used to support resource allocation when managing invasive species. This is the first time, to my knowledge, environmental decision theory has been tested empirically by examining applications of search theory for any species in an ecological setting. I found that animal size, target distance from the transect line and group clustering size all affect detectability. I also found that pre-survey training reduced the frequency of incorrect detections of A. amurensis for two native co-occurring species by up to 16.1%. I also demonstrate the amount of search effort required to eradicate populations at a site is often considerably higher than the effort actually invested to completely remove a population of A. amurensis. Lastly, I found that using an optimal search strategy compared to three other routinely used strategies during surveys for A. amurensis can improve the number of seastars removed by upwards of 12% for a 20 minute search budget. Evaluating how well previous removal efforts eliminate marine invasive species from a site, and understanding the uncertainty of survey design are critical to improving future post-border management responses. The northern Pacific seastar A. amurensis will continue to threaten the marine environment in its non-native distribution. Understanding how to improve survey design will continue to be essential for active and successful management.
Novel methods to account for individual heterogeneity in capture-recapture studies

BIRD, TOMAS JODA ( 2015)

A major challenge in ecological analyses is estimating population-level parameters such as survival, births and population size when most individuals are not observed by sampling. Capture-mark-recapture (CMR) models provide the ability to understand what proportion of the population is missing in sampling and to account for this missingness in inference. Yet one of the standard assumptions behind CMR analyses is that the probability of sampling an individual is either constant across all individuals or can be modelled as a function of fully-observed covariates. However, in many cases this is not true, a factor that can seriously bias estimates of population-level demographic processes. This thesis develops novel methods to account for individual heterogeneity in capture probabilities. I first provide a general review of CMR studies and history of their development, as well as the application of Bayesian state-space models to CMR studies. I discuss the motivating sampling scenario in which a number of common sources of bias are present, then describe how CMR sampling approaches were deployed to try and account for these biases. In chapter two, I propose a solution to the problem of bias due to temporary migration. I describe a capture-recapture sampling scenario on a population that is closed to births and deaths but in which capture probabilities are confounded by migration, then develop a model to account for migration using auxiliary radio telemetry data. The telemetry data provide a means to estimate migration rates, which can then be used to account for the bias in estimated capture probabilities. Simulation studies show that this approach allows for unbiased estimates of population sizes and should be applicable to a range of situations. Chapter three then considers the problem of state misclassification in data where mortality of animals can be inferred via remote observations of movement patterns. I show that in some cases, misclassification of individuals as dead can result in biased estimates of survival rates. I employ a state misclassification model in order to correct for such errors and show that in wild populations of native fish such bias can result in a reduction in estimated survival rates of up to 50%. Next I describe a means of estimating age-specific capture and survival rates in CMR scenarios involving animals with well-de ned growth patterns. The approach involves estimating growth parameters from length interval data in a capture-recapture context or from other ageing methods, then using these growth as prior information in a CMR model in order to estimate age at first capture. We show how otolith and CMR-based estimates of age correspond well, and simulation studies show how estimate survival rates closely match true values. Finally, I consider a large-scale sampling scenario in which multiple sources of bias are possible and multiple sources of data are available to help account for these errors. I develop a state-space modelling approach to incorporating multiple sources of data in this context. I show how it is possible to evaluate the relative fit of models with differing types of data on the same population through a combination of root-mean-square error evaluation, simulation modelling and comparison of the magnitude of various model parameters. I show how single sources of CMR data in this context are likely influenced by various kinds of individual heterogeneity, but show through simulations how it is possible to help correct for this source of bias by incorporating multiple data sources. I also show how incorporating latent state data can help correct parameter estimates that would otherwise be heavily biased in a model that otherwise appears to have a relatively good fit.
Decision analysis for threatened species management across the captive-wild spectrum

CANESSA, STEFANO ( 2015)

Programs for the recovery of threatened species increasingly involve active management of variable intensity, such as captive breeding, reintroduction and translocation. Managers of such programs thus need to make decisions about whether and how to implement a given type of management, usually in the face of uncertainty and constraints. Structured decision making provides decision makers with a theoretical framework and practical methods to make rational decisions under uncertainty. In this thesis, I investigate how different principles and tools of structured decision making can assist decisions in threatened species management. The fundamental decision problems in threatened species management concern whether and how to implement a given type of management for the target species. In Chapter 2, I examine the case of whether the conservation of a species should incorporate ex-situ management, using the recovery plan for an endangered frog species in south-eastern Australia as an example. I demonstrate that this question can only be answered rationally after determining how the ex-situ component will benefit the overarching objectives of the recovery plan. This logical sequence of decisions (how before whether) is however followed only infrequently in real-world conservation. I illustrate how managers can implement it with the aid of decision trees and multi-criteria decision analysis. Since decisions are aimed at achieving objectives, they necessarily reflect the values and preferences of stakeholders. Using value functions, in Chapter 2 I demonstrate how the optimal decision depends on the relative importance attributed to different objectives (for example, maximising the probability of persistence of the target species and meeting budget limitations). In Chapter 3, I expand this analysis to demonstrate how to account for the attitude of decision makers towards the risk of negative outcomes. I use two case studies of recovery plans for threatened frog species to illustrate the application of stochastic dominance, a useful method to rank alternative actions in the face of uncertainty and risk. Structured decision making provides methods to make decisions under uncertainty. However, in some instances reducing the existing uncertainty by collecting additional information can allow more robust decisions. As a result, managers of threatened species programs almost always advocate further research, in the expectation that it will improve the outcomes of management. In Chapter 4, I apply a formal method to calculate the expected benefit of additional information to two examples in threatened species management. I demonstrate how the value of information is a function of the current knowledge, by the potential to react to new information, and by the effectiveness of the learning process. Value of information analysis can help managers determine whether and how to implement experiments and monitoring programs to improve the ultimate outcomes of management. For several threatened species programs, the scale and speed of the threatening processes often require decisions to be made immediately, leaving no time for formal experimental learning. Adaptive management describes a specific case of structured decision making in which managers learn by monitoring the outcomes of management and adjust actions accordingly. In Chapter 5, I analyse the conditions and challenges that exist to the application of adaptive management in threatened species programs. The temporal scale of such programs is often sufficient to allow managers to collect information and react by updating actions in subsequent time steps. Particularly for programs toward the captive end of the management spectrum, controlled conditions are also favourable for effective learning. Adaptive management requires the ability to clearly structure uncertainty into formal hypotheses, to allow effective and focused monitoring that addresses the most important sources of uncertainty. Most importantly, institutions and stakeholders must be committed and capable of implementing learning. For clearly defined decision problems, structured decision making can draw upon a range of technical approaches to determine optimal management strategies. In Chapter 6, I consider a reintroduction program in which management decisions are complicated by the complex life history of the target species and budget constraints. I combine demographic modelling and cost-effectiveness analysis to identify the optimal rates of translocation between captive and wild populations. This thesis illustrates how the iterative cycle of structured decision making can benefit all stages of the design of management strategies for threatened species conservation. First, it can help managers in thinking clearly about the decision problem, allowing a transparent assessment of subjective preferences and value. It can then ensure an objective evaluation of the available management alternatives, using qualitative or quantitative predictive approaches that explicitly recognise uncertainty. Finally, it can assist in finding solutions to trade-offs and incorporating additional knowledge to allow better decisions.
The shared habitat: understanding and linking the needs of insectivorous bats and people at urban wetlands

STRAKA, TANJA ( 2015)

Urban wetlands can be hotspots for biodiversity but are often managed for human benefits. Understanding and linking the potentially conflicting needs of people and wildlife in urban wetlands will allow us to create and manage wetlands that maintain biodiversity in human-dominated areas. Biodiverse urban wetlands may also encourage human contact with nature contributing to people’s health and well-being. However, for many faunal taxa, little is known about the role that wetlands play in supporting urban biodiversity. For example, insectivorous bats often occur in urban areas, but the importance of urban wetlands as a source of food, and the characteristics of ‘bat-friendly’ wetlands are largely unknown. The aims of this interdisciplinary PhD research were to i) understand the role of wetlands for insectivorous bats in an urban environment, ii) identify the landscape- and local-scale attributes of wetlands that drive the occurrence of insectivorous bats and noturnal flying insects, and iii) determine the aesthetic preferences of local residents for wetlands and the effect of ecological information on these preferences. Insectivorous bats and nocturnal flying insects were investigated simultaneously using acoustic monitoring and light traps at urban wetlands and non-wetland habitats (ecologically similar sites with open grassy areas instead of water) in the greater Melbourne area. To understand the contribution of aquatic-dependent insects in the diet of urban bats, a molecular dietary study using a state-of-the-art next generation sequencing technique (Illumina platform) was undertaken for three insectivorous bat species (Gould’s wattled bat Chalinolobus gouldii, little forest bat Vespadelus vulturnus and large-footed myotis Myotis macropus) captured at wetlands in the greater Melbourne region. A quantitative survey with an experimental design was applied to understand preferences of nearby residents of urban wetlands and if these preferences could be influenced through ecological information. Two theories from social psychology (ecological aesthetics and biased assimilation) were tested as this part of the research, to gain a better understanding of the kind of ecological information that is effective in influencing people’s preferences for wetland landscapes. Wetlands were found to be important drivers of bat species richness in urban areas and supported a higher richness and abundance of nocturnal flying insect orders (predominantly Trichoptera and Diptera). Aquatic-dependent insects were found in the diet of all three bat species (C. gouldii, V. vulturnus and M. macropus). While M. macropus had the highest proportion of aquatic-dependent insects in its diet of all three species, C. gouldii had the smallest proportion. Dipterans were found to be the second most dominant insect prey of all three bat species and Trichopterans were detected in the diet of all three, but in smaller propotions. A range of local and landscape attributes influenced insectivorous bats and nocturnal flying insects at urban wetlands. Wetland vegetation (primarily riparian trees) and low heavy-metal pollution were important predictors for richness and activity of bat species as well as the richness and abundance of insect orders. On a landscape scale, high levels of artificial nocturnal light were found to decrease bat species richness and more urban greeness enhanced the richness and abundance of noturnal flying insects. People had high aesthetic preferences for wetlands with high vegetation complexity (more trees, understorey and emergent aquatic vegetation) which were also found to support a higher number of bat species and several orders of nocturnal flying insects. The provision of ecological information influenced preferences when the information was congruent with people’s existing values for urban wildlife. Given the joint benefits of wetland vegetation for insectivorous bats, nocturnal flying insects and people’s preferences, restoration and conservation efforts may help reconcile the needs of biodiversity and society at urban wetlands. Information might not always be required to influence people’s preferences and to gain their appreciation for a landscape. However, in cases where it is required (e.g. landscapes that have high ecological benefits, but are not aesthetically pleasing to people) ecological information is more effective if messages are value-congruent. This research contributes knowledge to the two theories of ecological aesthetics and biased assimilation, and provides practical recommendations on the management and restoration of urban wetlands to encourage the conservation of insectivorous bats and nocturnal flying insects in urban areas.
Exploring how fatty acids synthesized by malaria parasites are incorporated into lipids: Characterization of the Plasmodium apicoplast glycerol-3-phosphate acyltransferase

SHEARS, MELANIE ( 2015)

The Plasmodium parasites responsible for malaria synthesize fatty acids in a reduced plastid organelle known as the apicoplast. There has been enormous interest in the apicoplast fatty acid synthesis pathway as a potential drug target, but comparatively little research into its role in parasite lipid metabolism. The fatty acid synthesis pathway is essential only at certain stages of the parasite life cycle, and appears to be required to support membrane lipid biosynthesis. However, the nature of the lipid species reliant on the apicoplast pathway and the intermediate steps in their synthesis have largely not been explored. The majority of parasite membrane lipid species can be synthesized from a phosphatidic acid precursor. Phosphatidic acid is composed of two fatty acids linked to glycerol-3-phosphate, and it is synthesized in two steps by a pair of acyltransferases. Plasmodium parasites were initially predicted to have two complete phosphatidic acid synthesis pathways located in the apicoplast and endoplasmic reticulum. Recent discoveries in the P. yoelii rodent model have demonstrated the apicoplast pathway is essential for parasites in the liver stage, consistent with the requirement for fatty acid synthesis in that species. However, it also indicated the apicoplast pathway may be incomplete, suggesting its function was instead to incorporate newly-made fatty acids into the intermediate lysophosphatidic acid. This thesis investigates the glycerol-3-phosphate acyltransferase of the P. falciparum and P. berghei apicoplast lysophosphatidic acid synthesis pathway to gain greater insight its role in lipid metabolism. The localization of the P. falciparum enzyme is confirmed and its activity demonstrated and explored through complementation, site-directed mutagenesis and structure homology modeling. The P. falciparum glycerol-3-phosphate acyltransferase is shown to be non-essential in the blood stage in standard culturing conditions, consistent with the dispensability of fatty acid synthesis at this stage. Unexpectedly, the P. falciparum enzyme is also found to be dispensable in lipid-depleted media conditions that induce fatty acid synthesis, suggesting the fatty acids made by parasites in this environment are used via other pathways. The P. berghei homologue is demonstrated to be essential in the liver stage, and its loss to closely mirror the phenotype of fatty acid synthesis knockouts in rodent models, implicating the pathway as a major route for newly-synthesized fatty acids to be incorporated into membrane lipid precursors. However, loss of the P. berghei enzyme does not impact expression of a key merozoite surface protein to the extent seen in the fatty acid synthesis knockouts, providing novel insight into how newly-made fatty acids might contribute to the synthesis of the lipid anchor of this protein. These findings provide information about the apicoplast lysophosphatidic acid synthesis pathway in two further Plasmodium species, and contribute towards understanding how newly-synthesized fatty acids are incorporated into precursors for membrane lipid synthesis.
The role of fire in the distribution and persistence of the great desert skink, Liopholis kintorei, under climate change

CADENHEAD, NATASHA ( 2013)

Fire regimes are predicted to change under climate change, with associated impacts on species and ecosystems. However, the magnitude and direction of regime changes are uncertain, as are species’ responses to those changes. For many species, how they respond will determine their medium-long-term viability. We propagate fire regime and species’ response uncertainties through a 50-year viability analysis of the great desert skink in Newhaven Sanctuary, central Australia. We characterise fire change with three scenarios. Species’ response uncertainty was characterised as three competing models based on fire and habitat variables fitted to 11 years of occupancy data. We evaluate fire management options for conserving the species, based on their robustness to uncertainty about fire and species’ response. Efforts to minimise the frequency and size of fires provides the most consistent improvements to species’ persistence. We show that disentangling important from unimportant uncertainties enables conservation managers to make more efficient, defensible decisions.

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