School of BioSciences - Theses
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The evolutionary and functional characterisation of the ecdysteroid kinase-like (EcKL) gene family in insects
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.
Dissecting the establishment of symbiotic partnership between algae and animals that powers coral reefs
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.
Devising computational tools to quantify the actin cytoskeleton and pavement cell shape using network-based approaches
Recent advances in microscopy have led to an improved visualization of different cell processes. Yet, this also leads to a higher demand of tools which can process images in an automated and quantitative fashion. Here, we present two applications that were developed to quantify different processes in eukaryotic cells which rely on the organization and dynamics of the cytoskeleton. In plant cells, microtubules and actin filaments form the backbone of the cytoskeleton. These structures support cytoplasmic streaming, cell wall organization and tracking of cellular material to and from the plasma membrane. To better understand the underlying mechanisms of cytoskeletal organization, dynamics and coordination, frameworks for the quantification are needed. While this is fairly well established for the microtubules, the actin cytoskeleton has remained difficult to study due to its highly dynamic behaviour. One aim of this thesis was therefore to provide an automated framework to quantify and describe actin organization and dynamics. We used the framework to represent actin structures as networks and examined the transport efficiency in Arabidopsis thaliana hypocotyl cells. Furthermore, we applied the framework to determine the growth mode of cotton fibers and compared the actin organization in wild-type and mutant cells of rice. Finally, we developed a graphical user interface for easy usage. Microtubules and the actin cytoskeleton also play a major role in the morphogenesis of epidermal leaf pavement cells. These cells have highly complex and interdigitated shapes which are hard to describe in a quantitative way. While the relationship between microtubules, the actin cytoskeleton and shape formation is the object of many studies, it is still not clear how and if the cytoskeletal components predefine indentations and protrusions in pavement cell shapes. To understand the underlying cell processes which coordinate cell morphogenesis, a quantitative shape descriptor is needed. Therefore, the second aim of this thesis was the development of a network-based shape descriptor which captures global and local shape features, facilitates shape comparison and can be used to evaluate shape complexity. We demonstrated that our framework can be used to describe and compare shapes from various domains. In addition, we showed that the framework accurately detects local shape features of pavement cells and outperform contending approaches. In the third part of the thesis, we extended the shape description framework to describe pavement cell shape features on tissue-level by proposing different network representations of the underlying imaging data.
Defining the impacts of oestrogen on cell fate decisions in the gonad
The increasing incidence of testicular dysgenesis syndrome-related conditions and decline in fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact the latter’s function and development in both males and females, but little is understood about the processes behind this. In non-mammalian vertebrates, oestrogen is the critical driver of ovarian differentiation, while in mammals, oestrogen is not required for ovarian determination but is essential for its maintenance. In marsupials, exogenous oestrogen signalling causes cytoplasmic retention of the key testis factor SOX9, leading to a switch from testicular to ovarian developmental programs. SOX9 nuclear import is critical for testis differentiation; several key processes regulate its subcellular localisation, including the microtubule cytoskeleton, post-translational modifications of SOX9, and binding to importin-b and calmodulin. In this thesis, I demonstrated that exogenous oestrogen similarly causes cytoplasmic retention of SOX9, suppression of pro-testis genes and activation of pro-ovarian genes in the human testis-derived cell line NT2/D1. Utilising this established system, I investigated the effects of oestrogen on SOX9 nuclear import requirements. Immunofluorescence studies showed that exogenous oestrogen treatment of NT2/D1 cells led to the nuclear accumulation of importin-b, decreased phosphorylation of SOX9, and promoted the stabilisation of the microtubule network. ERK1/2 has a role in mediating gonad developmental pathways, is responsive to oestrogen and is known to affect the microtubule network—thus, ERK1/2 presented as a candidate for regulating the microtubule network and SOX9 in response to oestrogen. To further understand the non-genomic regulation of microtubules and role of ERK1/2 in this process, I assessed the rapid response of these factors to brief oestrogen treatment. Oestrogen rapidly stabilised microtubules and caused the cytoplasmic retention of SOX9, while treatment with an ERK1/2 inhibitor prevented these effects of oestrogen. From these experiments, I demonstrated that oestrogen rapidly activates ERK1/2 to facilitate stabilisation of microtubules and the cytoplasmic retention of SOX9. This established that oestrogen acts in a non-genomic manner to influence SOX9 bioavailability, which is reinforced later by genomic changes that promote a switch to ovarian somatic cell fate. Using proteomic and phosphoproteomic analyses, I examined the response of the MAP3K1 and MAP3K4 cascades—which have a role in mediating gonad differentiation and act upstream of ERK1/2—to brief or prolonged oestrogen treatment. The MAP3K1 cascade appeared upregulated following EE2 treatment and b-catenin was activated by phosphorylation at Ser552, while the MAP3K4 pathway remained unchanged. This demonstrated that oestrogen can target this crucial pathway to promote a shift from testicular (pro-SOX9) to ovarian (pro-b-catenin) fate. From these analyses, I further showed that oestrogen treatment can impact proteins involved in SOX9 post-translational modification, the nuclear import pathway and the mTOR pathway. These studies also demonstrated that oestrogen can impact microtubule regulation through hypophosphorylation of MAPs, remodel the actin cytoskeleton through ARP2/3- and mDia2-mediated actin polymerisation, and increase the abundance of the intermediate filament vimentin. Together, these analyses provide an unparalleled investigation of the mechanisms behind the cytoplasmic retention of SOX9 and the widespread impacts of oestrogen on a testis-derived cell line. Finally, I assessed the impact of exogenous oestrogen on the localisation of SOX9 in mouse gonads. In an in vitro gonad culture system, exogenous oestrogen caused cytoplasmic retention of SOX9, suppression of the testis gene Dhh and activation of the ovarian gene Fst. Treatment of mice in utero to the oestrogenic EDC diethylstilbestrol at a concentration known to cause hypospadias and reduced anogenital distance similarly increased cytoplasmic SOX9, demonstrating an association between the suppression of SOX9 and development of hypospadias and reduced anogenital distance. In this thesis, I have demonstrated that the cytoplasmic retention of SOX9 is a conserved effect of oestrogen in mammals, suggesting this mechanism may be a remnant of oestrogen-driven sex determination in non-mammalian vertebrates. Furthermore, I have revealed some of the processes involved in the cytoplasmic retention of SOX9, which are directly relevant for our understanding of how exogenous oestrogen can regulate gonad somatic cell fate. These findings are not only important for understanding the impact of oestrogenic EDCs on male fertility, but also ovarian regulation and other diseases associated with misregulation of SOX9.
Genomic analysis of the metabolic and microbial basis of human disease
Genetics influences the phenotype and behavior of living organism. Human genetic variants have been widely linked to diseases, while the biological pathways through which genetic variants affect the physiology remain unclear. Genetic associations with molecular traits, for example levels of plasma metabolites, may aid the interpretation of genetic associations on higher-order phenotypes. The human body is a symbiont with a large number of microbial organisms, which have a close relationship with human health. In particular, the gut microbiota can influence various organs of human host through the synthesis of bioactive metabolites. Understanding the microbial organisms living in the human body holds great promise for increasing our understanding of human health and disease. This thesis focuses on genetic aspects of metabolic and microbial traits and has four general aims: 1. To identify genetic determinants of human plasma metabolite levels and prioritize metabolites with putative causality on complex diseases. 2. To identify genetic associations on gut microbiota and determine how these are relevant to host-microbe interactions. 3. To identify causal associations between gut microbes and plasma metabolites and characterize how these relate to diseases. 4. To identify novel genetic determinants of drug resistance in Mycobacterium tuberculosis and characterize the lineage-specific profiles of drug-resistant mutations. Through this research, I uncovered novel insights into communicable and non-communicable diseases in humans. In addition to identifying novel genetic loci associated with metabolism, my work prioritized plasma metabolites with putative causal effects on complex diseases. The robust genetic associations with gut microbiota expand our understanding of host-microbe interactions and may facilitate precision intervention of the gut microbiota. Causal relationships between gut microbes and plasma metabolites suggest that plasma metabolites may be involved in the microbial effects on human diseases. In infectious diseases, distinct lineage-specific profiles on drug-resistant associated mutations may guide public health decisions on tuberculosis control and treatment. In summary, this thesis demonstrates how genomic information can be leveraged to generate hypotheses, prioritize biomarkers, and uncover disease causality. The results expand our understanding of the human body as a complex symbiont.
Preimplantation embryo metabolism as a biomarker of embryonic viability and health
Worldwide, 12% of couples suffer from infertility and therefore rely on assisted reproductive technologies to conceive. The current success rate of an initial IVF cycle is ~20%, leaving ~80% of couples unsuccessful after their first cycle. Although success rates increase with subsequent cycles, represented by an increase in cumulative pregnancy rates, IVF treatment is expensive and can have a significant impact on the psychological wellbeing of couples. It is therefore imperative that research is focused on increasing the success rate of initial IVF cycles to reduce the time to pregnancy. The success of an IVF cycle significantly relies on the ability to select the most competent embryo from a patient’s cohort that has the greatest chance of establishing a viable pregnancy. Current embryo selection methods focus on the morphological and/or the morphokinetic development of the preimplantation embryo and preimplantation genetic testing can be utilized to ensure the embryo transferred is genetically normal. However, despite an embryo being regarded as high quality based on these selection methods, its success post-transfer is not guaranteed. Blastocyst metabolism is a key regulator of embryo development and through metaboloepigenetic interactions, embryonic health, and its assessment represents an additional biomarker that may improve the accuracy of embryo selection. A comparison between blastocyst metabolism, morphology, time-lapse annotations, artificial intelligence, chromosomal status and transfer success was conducted. High glucose uptake and high amino acid consumption were found to be associated with human blastocysts of high viability according to current methods of selection. Further, glucose uptake was significantly higher in human blastocysts that established a viable pregnancy. Genetically abnormal, or aneuploid, human embryos developed slower and were assigned lower viability scores. Additionally, blastocyst amino acid utilization appeared to be perturbed due to aneuploid associated stress. An analysis of vitrified mouse and human blastocyst pyruvate and/or glucose uptake post-warm was unable to provide a measure of viability. However, a morphological assessment of human blastocyst reexpansion post-warm revealed blastocysts with a greater degree of re-expansion were associated with higher live birth rates. Finally, using a mouse model to demonstrate how changes in embryo culture media can impact blastocyst metabolism and health, the addition of antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and alpha-lipoic acid) to embryo culture media, individually and in combination, was investigated. A reduction in oxidative stress, regulation of blastocyst carbohydrate metabolism, lower NADH levels and improved blastocyst development were identified. Together the data presented in this thesis provides a comprehensive analysis of human blastocyst physiology and lay the foundation for the development of an algorithm incorporating morphological, morphokinetic and metabolic biomarkers which could assist in the identification of the most viable and healthy blastocyst for transfer. Such an algorithm may also be used to validate future advances in embryo culture conditions. Therefore, these data provide an opportunity to significantly improve human IVF success rates and reduce the time to pregnancy.
Seasonality and community composition of parasitoid wasps of four agromyzid leafminer species (Diptera: Agromyzidae) in Victoria
The vegetable leafminer, Liriomyza sativae Blanchard (Diptera: Agromyzidae), was first detected in the Torres Straits in 2008, crossed over to the tip of Cape York in 2015 and now poses a threat to Australian crops. This species is an economically important secondary pest in many parts of the world; populations readily evolve resistance to insecticides and benefit from the use of chemical controls against other pests, which kill their natural enemies. Liriomyza sativae is expected to spread into Australia’s agricultural production areas and it is therefore valuable to investigate the local biological agents that could contribute to future integrated pest management (IPM). I assessed the abundance and diversity of the hymenopteran parasitoid wasps of four common agromyzid flies in southern Victoria (Liriomyza brassicae (Riley), Liriomyza chenopodii (Watt), Phytomyza plantaginis Goureau, and Phytomyza syngenesiae (Hardy)) to evaluate their potential as candidates for control of L. sativae. I monitored six sites in Melbourne over a period of 18 months by collecting mined leaves from a range of plant species and rearing adult flies and their parasitoids. Additional sites around Victoria were also sampled to offer a snapshot of the agromyzid presence across the state. Eleven wasp species were identified in total, two of which were only classified to morphospecies level, including known parasitoids of L. sativae overseas and species reared from L. sativae populations from far north Queensland. Ninety percent of the parasitoids were eulophids (Hymenoptera: Eulophidae), with the rest consisting of opiines (Braconidae) and Pteromalinae (Pteromalidae). The adventive Chrysocharis pubicornis (Zetterstedt) was the most abundant parasitoid, reared almost entirely from Phytomyza hosts, followed by an Asecodes sp. and the introduced Diglyphus isaea (Walker). We recorded the first male Ph. plantaginis for Australia. Males were only found at Melbourne locations and these populations tended to have a female-biased sex ratio. Variation in sex ratio may be host plant dependent, as the female:male ratio was 4:1 from the host Plantago lanceolata L. while it was 1:1 in more limited samples from Plantago major L. The four common local agromyzids reached peak abundance at different times of the year and together supported a stable community of both adventive and native parasitoids. Of the species reared, several are known to attack L. sativae including D. isaea, Hemiptarsenus varicornis (Girault) and Neochrysocharis formosa (Westwood). The wasps reared include candidates for augmentative or conservation biological control that should be further considered in the event that L. sativae becomes a widespread pest of Australian crops.
Modeling the Distribution of a Widely Distributed butVulnerable Marsupial: Where and How to Fit Useful Models?
The greater glider (Petauroides volans) is the largest of the Australian gliding marsupials. Once abundant, it is now nationally listed as vulnerable because evidence of population decline exists across its distributional range. This decline and its likely relation with wildfire and logging, has prompted focus on conservation of this species. Species Distribution Models (SDMs) relate species occurrences to environmental variables at observation sites to predict distributions or make inferences about their key drivers. Conservation planning and land management use SDMs to deliver predictions of species distributions across landscapes. When modelling a broadly distributed species like the greater glider, data are often gathered from sources that vary in quality. In such cases, accounting for sampling biases and selection of geographic extent for model fitting are two key methodological steps that can largely influence models results. In this thesis I tested the effect of taking alternative decisions regarding occurrence data processing, modelling method and geographic extent on models’ predictive performance and how different decisions might (or not) provide different information for conservation and land management actions in a region subject to commercial logging. In the first research chapter, I tested different methods for dealing with sampling biases when modelling the distribution of the greater glider across its entire range. I compiled a dataset of occurrence data of the greater glider and other arboreal marsupials and tested alternative ways to use this large but biased dataset. I used modelling methods that utilize different types of occurrence data, namely, presence-background and presence-absence methods. I found that using presence-absence models fitted to an expanded presence-absence dataset in which some data were inferred provided the best performing models. In the second research chapter, I compared range-wide and local SDMs to predict the distribution of the greater glider in East Gippsland, Victoria. I found that two models: a range-wide one, and a local model fitted with higher quality variables, were the best performing. Models delivered somewhat different spatial predictions but broadly agreed on the largest patches of high predicted probability and gave similar estimates of the proportion of habitat across different land uses in the East Gippsland Regional Forest Agreement. I also completed a preliminary assessment of the extent of greater glider habitat burnt during the 2019-2020 wildfires that affected eastern Australia. I found that a large proportion of habitat was affected, including recently established protected areas. Throughout this thesis I show that decisions regarding data processing, selection of modelling method and geographic extent can lead to substantially different distribution predictions. In a context of local conservation planning such as the East Gippsland Regional Forest Agreement, different models, nevertheless, provided similar information on the implications that forest management and logging restrictions may have on the conservation of greater glider habitat in this region. Although the solutions we implemented relied on the broad availability of biodiversity data in Australia, we advocate for modellers and users to undertake thorough assessments of the data available in their regions and think carefully on how to make the best use of it.
Feasibility of bacterial probiotics for mitigating coral bleaching
Given the increasing frequency of climate change driven coral mass bleaching and mass mortality events, intervention strategies aimed at enhancing coral thermal tolerance (assisted evolution) are urgently needed in addition to strong action to reduce carbon emissions. Without such interventions, coral reefs will not survive. The seven chapters in my thesis explore the feasibility of using a host-sourced bacterial probiotic to mitigate bleaching starting with a history of reactive oxygen species (ROS) as a biological explanation for bleaching (Chapter 1). In part because of the difficulty to experimentally manipulate corals post-bleaching, I use Great Barrier Reef (GBR)-sourced Exaiptasia diaphana as a model organism for this system, which I describe in Chapter 2. The comparatively high levels of physiological and genetic variability among GBR anemone genotypes make these animals representatives of global E. diaphana diversity and thus excellent model organisms. The ‘oxidative stress theory for coral bleaching’ provides rationale for the development of a probiotic with a high free radical scavenging ability. In Chapter 3, I construct a probiotic comprised of E. diaphana-associated bacteria able to reduce oxidative stress by neutralizing free radicals such as ROS. I identified six strains with high free radical scavenging ability belonging to the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae, and Micrococcaceae. In parallel, I established a “negative” probiotic consisting of closely related strains with poor free radical scavenging capacities. The application of this probiotic to mitigate the negative impacts of exposure to a simulated heat wave was tested in Chapter 4. There was no evidence for improved thermal tolerance in E. diaphana. Changes in the relative abundance of anemone-sourced Labrenzia provided evidence for its integration in the E. diaphana microbiome. Uptake of other probiotic members was inconsistent and probiotic members did not persist in the anemone microbiome over time. Consequently, the failure of the probiotic inoculation to confer improved thermal tolerance may have been due to the absence of probiotic bacteria for the full duration of the experiment. Importantly, there were no apparent physiological impacts on the holobiont following inoculation, thus showing that shifting the abundance of native anemone microbiome members was not detrimental to holobiont health. Further, I found no evidence for an increase in ROS in the E. diaphana holobiont when it was exposed to heat. Some of the most compelling evidence in support of the ‘oxidative stress theory of coral bleaching’ comes from three published studies that expose corals, cultures of their algal endosymbiont, or E. diaphana to exogenous antioxidants during thermal stress. To confirm that ROS is the main driver behind thermal bleaching in E. diaphana, I replicated these previous experiments with novel methods that allowed a more accurate quantitation of ROS, and found that dosing with exogenous antioxidants (mannitol and ascorbate plus catalase) mitigates bleaching in E. diaphana, with no correlation between bleaching and increased ROS (Chapter 5). A serendipitous finding was that the E. diaphana bacterial community diversity can be rapidly reduced when anemones are reared in sterile seawater, making this model suitable for testing the efficacy of microbial restructuring strategies (Chapter 6). Taken together, the work from my PhD has shown that ROS scavenging varies among anemone-associated bacteria and that a high ROS-scavenging probiotic can be developed. Further, my findings have unveiled several main knowledge gaps that need to be filled before probiotics can be implemented, including administration strategies and choice of probiotic bacteria that maximise the maintenance of probiotic communities over time and a direct measurements of ROS in bleaching corals (Chapter 7).
The effect of sub-optimal temperature on the cellular metabolism of wheat and Arabidopsis thaliana
Low or suboptimal temperature stress is one of the primary abiotic conditions limiting the growth and productivity of economic crops in many regions of the world. Wheat is one of the major crops in Australia, it is grown during winter to avoid hot summers and they flower in early spring. The sensitive flowering stage of wheat is therefore frequently exposed to spring frost. In Australia, the frequency of spring frosts during the flowering stage has increased significantly since 1960, and the reoccurrence of frost events led to an estimated $360 million of losses in the Australian wheat industry per annum. It is therefore important for breeders to minimize the loss via the development of more chilling/frost-tolerant wheat varieties, especially during their reproductive stages. Two approaches could be employed to achieve this goal. The first one is by employing metabolomics approaches to understand the underlying molecular mechanisms involved in cold responses of wheat upon cold stress. The second approach is via bioengineering of cold responsive genes into wheat to create chilling/frost-tolerant varieties. With this in mind, my PhD study was carried out with three main objectives. The first objective was to investigate and understand metabolic traits involved in the cold acclimation of two Australian wheat varieties with contrasting cold tolerance using targeted metabolomics and lipidomics approaches. The cold-sensitive variety used in this study was Wyalkatchem and the cold tolerant variety used was Young. The second objective of this study was to identify potential metabolite and lipid responsible for chilling tolerance in the two studied wheat varieties. The third objective was to evaluate the potential of REIL (Required for isotropic bud growth1 – like) protein as cold acclimation factor in Arabidopsis thaliana for potentially enhancing wheat cold tolerance. Chapter 1 consists of a review of the recent literature covering cold stress responses (physiologically and metabolically) of plants and how plants adopt to cold stress. It describes how metabolomics and lipidomics can be used as promising tools to decipher cold stress responses in wheat and discuss the role of cold-induced genes to increase cold tolerance in plants. The targeted protein in this study, REIL, as a new potential cold acclimation factor in Arabidopsis thaliana and wheat is also reviewed. To achieve the first and second objectives of this study, work described in Chapter 2 was conducted to investigate the cold acclimation of two Australian wheat varieties with contrasting cold tolerance using targeted metabolomics and lipidomics approaches. The selected cold-sensitive spring wheat variety used in this study was Wyalkatchem and the selected cold-tolerant spring wheat variety was Young. Samples of flag leaves and spikes at the young microspore stage were collected and analysed in this study. The results obtained provide us with a better understanding of the cold responses of wheat, and pointed out the potential of several sugars, amino acids, amines and glycerolipids to confer cold-tolerance to the Young variety. The outcomes gained from this study have been published in Cheong et al., (2019) for the study on flag leaves, and in Cheong et al., (2020) for the study on spikes. The outcomes also pointed out the profound potential of lipid species as biomarkers that can be explored to distinguish the two varieties. This further motivated us to expand the lipidomics study on the underground part of wheat, the roots (Chapter 3). There are limited cold stress studies on the lipidome of whole roots and to the best of our knowledge, no data are available on responses of specific root developmental zones. In Chapter 3, the lipid profiles of the spatial root zones derived from young seedlings of Wyalkatchem and Young grown at optimal, chilling and freezing temperatures were investigated. The outcomes indicate the involvement of not only glycerolipids in discriminating Young from Wyalkatchem, but sphingolipids are also involved in conferring cold-tolerance of Young. Next, to fulfil the third objective of this study, REIL, a protein that has been postulated to act as a potential cold acclimation factor in the mature leaves of Arabidopsis thaliana, was evaluated in roots in Chapter 4, followed by the evaluation of its potential in wheat in Chapter 5. REIL proteins have been postulated to be involved in late ribosomal biogenesis and affect the accumulation of 60S large subunits in the mature leaves of A. thaliana upon cold stress. To validate these roles in A. thaliana, a systematic analysis of roots grown at optimized and cold temperatures was conducted in Chapter 4. The outcomes substantiate the role of REIL proteins as a cold acclimation factor in Arabidopsis by being involved in ribosomal biogenesis during cold acclimation. In Chapter 5, three REIL homologs are found to be expressed in wheat. Evaluation of the REIL expressions in wheat subjected to cold stress through the re-analyses of published transcriptomics datasets show the potential cold and heat responsiveness of REILs. A real-time PCR analysis was then performed to evaluate the REIL expressions in Wyalkatchem and Young under cold stress, but no significant changes of expressions were observed in both varieties upon cold stress. It is then yet-to-be-known whether the cold acclimation function of REIL is conserved among dicots (A. thaliana) and monocots (wheat). Therefore, more in-depth investigation such as overexpression or silencing of the REIL expression in Australian spring wheat varieties is needed. The last chapter of this thesis (Chapter 6) summarizes the key results from each research chapter (Chapter 2 to 5) and also discusses the future directions and perspectives.
Landscape connectivity and keystone frugivores: implications for the rare rainforests tree Ryparosa kurrangiiLandscape connectivity and keystone frugivores: implications for the rare rainforests tree Ryparosa kurrangii B. L. Webber (Achariaceae)
Tropical rainforests (TRFs), contain the most structurally complex and biodiverse plant communities of any terrestrial biome. Within these communities there are a variety of physical, temporal and physiological barriers that create unique challenges for effective long-distance seed dispersal. Endozoochorous seed dispersal by frugivorous animals is the most effective means of seed dispersal in these environments, particularly over long distances. The largest of these frugivores play a disproportionately important role as keystone seed dispersal vectors, processing large quantities of seed, transporting them over long distances, and swallowing seeds across the full spectrum of size allometries. For plant species with the largest of seed sizes, large-bodied frugivores constitute the only viable means of long-distance seed dispersal. In an era of unprecedented rates of TRF deforestation, fragmentation, and defaunation, the erosion of faunal diversity generally starts with the largest species. The extirpation of large-bodied frugivores from TRF creates a plethora of negative impacts for plants that ultimately impacts on their fitness and resilience. These impacts include shrinking mean seed dispersal distances, increases in the spatial clustering of young plant cohorts and a reduction in the genetic variability of plant populations as a whole. At the dawn of the 21st century, the vast majority of the world’s TRF exist in a fragmented and partially defaunated state. However, we have a very limited understanding of how defaunation, particularly for large-bodied animals, impacts the seed dispersal and population dynamics of tropical tree species. An ideal study system in which to generate broader insight for these knowledge shortfalls exists in Australia, in populations of the rare TRF tree, Ryparosa kurrangii B.L. Webber (Achariaceae). Restricted in range to just three lowland valleys of the Daintree region of Northern Queensland, R. kurrangii has many traits that make it amenable to addressing questions on plant-animal seed dispersal mutualisms. First, due to the large seed size and fleshy fruit of R. kurrangii, it was theorised that the species was adapted to seed dispersal by large-bodied frugivores, and therefore would be sensitive to changes in the population structures and foraging habits of frugivorous fauna. Second, the only functional long-distance seed disperser of R. kurrangii is the southern cassowary (Casuarius casuarius johnsonii). Cassowaries are the only extant frugivores with a gape-width large enough to swallow R. kurrangii seeds and pass them intact. Third, earlier work conducted in three fully-mapped study populations of R. kurrangii identified a gradient of recent anthropogenic disturbance between sites. These study populations have a history of regular measurement and provide an ideal basis for demographic studies of tree populations across their entire ontogeny. To better understand the links between anthropogenic disturbance, seed dispersal, resource availability and plant demography, research was conducted simultaneously on a number of facets of R. kurrangii life-history. To understand the basic population dynamics of the species, studies of demographic changes, recruitment rates, mortality, growth rates and maximum lifespan were conducted. Ryparosa kurrangii were found to grow and recruit exceptionally slowly, and could have lifespans of up to c. 600 years. High mortality within mature tree cohorts over the monitoring period make it seem likely that the number of reproductive trees will decline and will not be offset by growth of small individuals into taller height cohorts. Using growth models from repeated measures of R. kurrangii it was determined that almost all reproductively mature R. kurrangii are likely to have recruited prior to European settlement of the region (c. 85 years of age). To understand how the life-history processes of R. kurrangii populations were influenced by within-population and environmental conditions, the patterning of tree distributions and population attributes were analysed using spatial point processes. The spatial aspects of population recruitment, growth, reproduction and mortality, as well as their interactions, were assessed and compared between populations. Hot-spots of seedling recruitment were identified, but were not found in areas of high average sapling growth rate. It was therefore unlikely that superior site conditions were a strong spatial determinant for recruitment likelihood. Instead, recruitment hot-spots occurred in areas where high concentrations of seedlings had been previously recorded, and, that were also close to fruiting trees. This observation, coupled with the landscape scale clustering of mature stands of R. kurrangii trees, suggest that long-distance seed dispersal was uncommon in all populations, and had been so for many centuries. To understand how the visitation rates of vertebrate fauna, particularly those of cassowaries and introduced pigs (Sus scrofa; a known seedling antagonist) varied between populations, a camera trapping program using passive infra-red cameras was conducted over three years. Cassowary visitation was not consistent throughout the year, and varied in frequency between R. kurrangii populations, with visitation rate scaling with the ranking of anthropogenic disturbance. Moreover, cassowaries were not frequently recorded during peak R. kurrangii fruiting periods, indicating that prolific fruiting of R. kurrangii trees were not strong incentives for site visitation. Feral pigs were the most commonly detected animal species in the study, and were consistently detected throughout the year. Finally, to understand whether there was a ‘smoking gun’ signature of dispersal limitation in any R. kurrangii stand, population genetic analyses using microsatellite markers were conducted on c. 1300 plants across all ontogenetic stages. FST estimates suggested that 7% of genetic variation was partitioned between populations, indicating moderate between-population differentiation. The finding was in close agreement with an earlier study that used different genetic markers. FIS estimates were calculated to be between 0.39 and 0.47 across populations, revealing some of the most extreme inbreeding reported for outcrossing tropical trees. Strong spatial genetic structuring (SGS) was also detected in all R. kurrangii populations and in all stages of tree ontogeny. Significant SGS implied that closely spaced plants in R. kurrangii stands were close genetic relatives. The extreme population genetic indices reported for this species strongly indicate that effective long-distance seed dispersal has been severely limited in R. kurrangii populations for thousands of years. Evidence from the spatial clustering of trees at all stages of ontogeny, the lack of linkage between cassowary visitation and R. kurrangii fruiting season, and, the extreme inbreeding and spatial genetic structure in all populations points to a similar story. Instead of measuring a recent disruption to frugivore visitation from the disturbance caused by European settlement of the Daintree, the magnitude of observed patterns are more readily explained by the loss of one or more large-bodied seed dispersal agents in the distant past. Despite the apparent capability of extant cassowaries to be efficient and high-quality dispersal agents to R. kurrangii, it appears that currently they are not effective in consuming and dispersing their seed. These findings have significant implications not only for the conservation of R. kurrangii, but also for the broader conservation of rainforest trees in systems where it is assumed generalist frugivores are providing adequate seed dispersal services. Ryparosa kurrangii could join a growing list of plant species with anachronistic adaptations for dispersal by extinct frugivores. It is postulated that the original co-evolved dispersal agent(s) may have been driven to extinction through a much older anthropogenic disturbance, that of the arrival of the first Aboriginal peoples to North Queensland more than 40,000 years ago. The implication that extant large-bodied frugivores that are apparently capable of dispersing large-seeded TRF plants, but that are not adequately fulfilling a keystone dispersal role, is a cause for concern. Many plant species across the world that are presumed to be adequately serviced by local fauna may similarly be under-dispersed to the point of inbreeding depression and population collapse. If the species diversity and community complexity of tropical rainforests are to be maintained beyond the next few generations of trees, a shift in conservation tactics is likely needed. Future conservation management will need to more strongly focus on identifying and retaining key plant-animal interactions, as well as mitigating for those that are now long gone.
Effects of fishing and climate change on the Chondrichthyan species in the Gulf of California region
This thesis is composed of three data chapters and a general introduction and discussion. Each chapter, except for the general introduction and the general discussion is composed of an introduction, materials and methods, results, discussion, and conclusions. The aim of this study was to assess the vulnerability risk to fishing and climate change stressors of 106 species of chondrichthyans with ≥10% of their distribution within the EEZ off western Mexico. For my analysis, I determine the vulnerability of the chondrichthyan species inside the Gulf (GCI) and compare these results with those for two other contiguous broad regions with different oceanographic conditions, the region around the entrance to the Gulf of California (GCE) and Mexico’s remaining Pacific waters (MPW). I have built on existing approaches to provide, in a single framework, a vulnerability analysis and risk assessment of the Mexican chondrichthyan (sharks, rays, skates and chimaeras) fauna by combining three components of vulnerability risk to climate change (exposure, sensitivity and adaptive capacity) (ESA), together with three components of vulnerability risk to fishing stressors (exposure, productivity, and susceptibility) (EPS). Here, vulnerability is expressed as the risk of marked reduction of the population of chondrichthyans based on the knowledge of its biology and its exposure to stressors associated with fishing and climate change. For fishing stressors, I use the productivity of the chondrichthyan species, which is related to the maximum age of the species, and susceptibility, which derives from four parameters; availability, encounterability, selectivity, and post-encounter mortality. For climate change stressors, I use sensitivity, which has two parameters; rarity and habitat specificity as species attributes that contribute to this, and adaptive capacity. Adaptive capacity involves distributional flexibility and trophic level as relevant attributes. I assigned each species to one of six ecological groups (EGs), which is a flexible and novel way to allocate a large number of species based on habitat use, depth strata (shelf-inshore and shelf-offshore), habitat dependence (freshwater, reef substrate, and sandy substrate), and lifestyle (demersal or pelagic). For fishing stressors, I analyzed data sets from 2006 to 2017 for the prawn trawl fishery, the elasmobranch fishery (artisanal and semi-industrial) and for the sardine fishery, and published information on the sport-recreational fishing. These fisheries have the potential to reduce the size of the population of a chondrichthyan species by altering the mortality rate in the regions where the fisheries operate. I then characterised the fishing stressors in terms of fishing methods and the bathymetric range of deployment of the fishing gear (Chapters 1, 3 and 4). For climate change stressors, I obtained data sets from several sources to show trends in the past oceanographic conditions and how they may vary in response to climate change. I then characterised the oceanography of the Gulf of California and adjacent waters. My assessment is based on observed changes from 1960 to 2017, and projected changes. In the period from 1960 to 2017 two important phenomena that warm the sea surface water occurred; “El Nino” and “El Blob”. The latter is a phenomenon related to a warm mass of water as a result of high levels of atmospheric pressure and of which origin is detected in the Gulf of Alaska in 2013. The name “Blob” echos the 1958 horror film which describes a character that keeps growing as it consumes everything in its path just as this warming event did (Cornwall, 2019). The “El Blob” was detected until several months later 2013, so it is unknown whether “El Blob” can occur again with the same or higher intensity. The other timescales are based on projected changes by 2055 and by 2099 using low (2.6), medium (4.5) and high (8.5) emissions scenarios from the RCP (representative concentration pathway) family. Because of a temperature gradient in coastal waters increasing from north-western Mexico to southwestern Mexico (Chapter 2), I established ten contiguous ‘subregions’ in these waters (Chapter 4). This allowed me to evaluate the risk associated with the attribute 'distributional flexibility' of the chondrichthyan species and to determine thermal tolerance range categories as follows: all waters (AW), cool waters (CW), warm waters (WW) or Gulf of California water (GoCw). These categories provided a basis for projecting how chondrichthyan distributions might change in response to climate change. I identified a total of 54 species of sharks, 48 species of rays and 4 species of chimaeras, which belong to 3 superorders, 12 orders, and 33 families. Based on the thermal tolerance range indicated by the current presence-absence of each species in the subregions, a total of 35 chondrichthyan species are distributed in all Mexican waters (AW), suggesting the species are adapted to the full range of temperatures currently occurring in Mexican waters. The majority of these are commercial shark species and these are the least likely species to redistribute out of Mexican waters as waters warm progressively northward as climate change progresses. A total of 31 species were classed CW (i.e., favouring cooler waters) and likely to reduce their distributional range northwards as Mexican waters warm in response to climate change. The majority of these are also commercial shark species. On the other hand, 34 species of chondrichthyans were classed WW (i.e., favouring warm waters), and are likely to expand their distribution northwards within Mexican waters. The majority of these are ray species, some of them of commercial importance. One species of shark, one species of ray and one species of chimaera are distributed only in the GoC waters, and another species of shark and two species of rays are distributed in only inside and outside the GoC in the adjacent MP-C subregion. The ecological groups (developed for all three regions) are shelf-inshore, shelf-reef, shelf-sand (<75 m), shelf-sand (75–150 m), pelagic waters and bathyal (>150 m). A total of 46 species were allocated to the ecological group ‘shelf-sand (<75m)’, 14 species were allocated to the ecological group ‘shelf-sand (75–150 m)’, and 22 species to the ecological group ‘pelagic waters’. Some of these species are demersal and others swim near the bottom or may swim up in the water column. A total of 19 species of chondrichthyans are in the ecological group ‘bathyal (>150 m)’, one species is in the ecological group ‘shelfinshore’, and 4 species were allocated to the ecological group ‘shelf-reef’. Vulnerability risk varies among the current chondrichthyan species, among ecological groups and among fishing and climate change stressors. For total vulnerability to fishing stressors, there were 10 species in the GCI and GCE regions, and 40 species in the MPW region at medium vulnerability risk. I determined 33 species in the GCI and GCE regions, and one species in the MPW region were at high vulnerability risk. For climate change (CC) stressors in the whole of western Mexico, a total of 15 and 10 species were at medium vulnerability risk under the medium and high emissions scenarios, respectively, and 10 species were at high vulnerability risk under the high emission scenario. The species allocated in the EG shelf-sand (<75 m) are highly vulnerable to the combination of fishing and CC stressors in all three regions for all the CC scenarios. In contrast, the species allocated in the EG bathyal (>150 m) are at low vulnerability but varies for species allocated to the other EGs.