School of BioSciences - Theses
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ItemThe effects of bisphenol A and atrazine on male development in the tammar wallaby (Macropus eugenii)( 2024-03)The mammalian gonad originates as a bipotential gonadal primordium before it differentiates into either a testis or ovary. During the process of differentiation, the gonad can differentiate either into a testis or ovary under the influence of genetic, hormonal and environmental influences. Both androgens and estrogens are critical to this process. Perturbations of these signals at critical stages of development by environmental endocrine disruptors (EEDs) can lead to gonadal sex reversal or gonadal dysgenesis. Xenoestrogens are EEDs that can bind to estrogen receptors, acting via multiple pathways, including the metabolism of reproductive steroids of the androgen synthesis pathway. EEDs are believed to contribute to the increasing incidence of disorders of sex development (DSDs) in vertebrates, including humans. Two of the commonest EEDs are bisphenol A (BPA), used in the production of plastics) and atrazine (the most commonly used herbicide in Australia). This study investigated the effects of bisphenol A and atrazine on testis and phallus differentiation in the tammar wallaby (Macropus eugenii). The tammar is an ideal mammalian model to study sexual development, because all sexual differentiation takes place after birth, when the developing young animals can be handled and treated while still in their mother’s pouches at stages that reflect the in utero stages of eutherian mammals. Developing tammar young were treated daily with BPA (50 microgram/kg/day), from day 0 postpartum (pp) to day 10 pp or from day 20 pp to day 40 pp, and atrazine (5 mg/kg/day), from day 20 pp to day 50 pp, during the male programming window (MPW). The BPA group was analysed immediately after the last treatment on day 10 pp and day 40 pp. Half of the atrazine group were analysed after treatment from day 20 to day 50 pp and the remainder at day 150 pp to assess long term effects. To analyse the role of estrogen on normal male differentiation, an additional group was treated daily with an estrogen receptor degrader, fulvestrant (1 mg/kg) between days 20 to 90 pp and analysed at day 150 pp. BPA treatment significantly downregulated the expression of SOX9 at day 10 pp, reduced the number of Sertoli cells at day 10 pp and downregulated androgen receptor (AR), SOX9, DHH, and androgen pathway genes STAR, CYP17A1, POR, CYP11A1 and PTCH1 at day 40 pp in testes. Atrazine treatment significantly downregulated the expression of SOX9 and NR2F2 and upregulated POR expression at day 50pp in testes. Atrazine treatment also significantly upregulated DHH expression at day 150 pp. Fulvestrant treatment significantly downregulated AR expression in testis. The phallus at day 150 pp was significantly shorter than control phalluses after atrazine and fulvestrant treatments. The results from this research using an alternative model species to examine the effects of EEDs on sexual development highlight the importance of androgen-oestrogen balance for normal sexual male development of the pouch young as it does in developing eutherian mammals (Hess et al, 2021). Disturbance this balance after exposure to EEDs affects the key male sexual differentiation genes and interferes with normal testis and phallus development in the developing tammar.
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ItemAdaptations for temperature control in bird eggs: the role of reflectance( 2021)Early life stages are highly vulnerable in many animals. In birds, egg temperature must be maintained within an optimal range for successful development and survival. Chilling and especially overheating can lead to abnormal embryos and even be lethal. Bird embryos are effectively ectotherms because heat from incubation by parents is essential for embryo development. Therefore, parental behaviours are crucial for egg temperature regulation during the incubation period. Parents, however, face tradeoffs between incubation and self-maintenance. There are often periods of time when bird parents will leave the nest for foraging or territory defense etc., and eggs will be then left unattended. During this time, egg temperature will not be regulated by parents and will be affected by both extrinsic factors such as environmental conditions (air temperature, solar radiation and humidity) and the architecture of the nest; and intrinsic factors such as eggshell reflectivity and egg size. In this thesis I focused on studying the relationship between these factors and egg thermoregulation. Eggshell reflectivity is one of the most important intrinsic properties that may affect egg temperature. Wisocki et al. (2020) and Gomez et al. (2018) provided evidence that eggs tend to be darker in colder climates and higher latitudes. They suggested that darker colours are adaptive for eggs to retain heat in colder environments for thermoregulation. These studies, however, looked only at ultraviolet-visible wavelengths (300 – 700 nm). These wavelengths correspond to egg colour patterns and may play a role in camouflage, egg recognition, protection from DNA damaging light, in addition to temperature regulation. Due to the multiple potential functions of egg coloration, it can be difficult to isolate the role of specific selective pressures. By contrast, reflectance of near infrared (NIR) wavelengths (700 – 2500 nm) primarily affects temperature. NIR wavelengths account for more than half of the solar energy. In 1978 Bakken et al. (1978) found a uniformly high near infrared reflectance among 25 species (19 of them are ground nesting birds with cryptic eggs) and pointed out the potential importance of NIR reflectance for egg thermoregulation. However, since then there have been no subsequent studies examining the importance of NIR reflectance on bird eggs on a broader scale. In this thesis, I first aimed to study the macro-evolutionary relationships between eggshell reflectivity, climate and nest type in birds. We predicted that eggs would have higher eggshell reflectivity in hot climates, and species with eggs that are exposed would have higher eggshell reflectivity. Using a phylogenetic comparative approach among 186 species of Australian birds we found that eggs tend to have higher total reflectivity in hotter climates with higher solar radiation. Moreover, species with open nests (i.e. with potentially higher exposure to direct sunlight) had higher NIR reflectivity than species with closed nests. This suggests that eggshell reflectivity, especially NIR reflectivity, may play an important role in egg thermoregulation. Second, we investigated how egg size, reflectivity and exposure interact to affect egg temperature. We predicted that high eggshell reflectivity and shade can protect eggs from reaching lethal temperature compared to eggs with low eggshell reflectivity and without shade. We used a biophysical model to predict egg temperatures in a hot environment (Kimberley, Western Australia). The results showed that eggs with higher reflectivity exceeded the critical thermal limit for less time and did not reach temperatures as high as eggs with lower reflectivity. The amount of sunlight eggs are exposed to (shade levels in the model) also strongly affected egg temperature. These results indicate that eggshell reflectivity and exposure to sunlight are likely to be important for egg temperature regulation during incubation, particularly in hot environments. Reflectivity had a greater effect on the temperature reached by large eggs than small eggs, even though small eggs had a higher heating rate. Together, this thesis highlights the importance of eggshell reflectivity on egg thermal control and contributes to our understanding of the macro-evolution of reflectivity in bird eggs.
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ItemSeasonality and community composition of parasitoid wasps of four agromyzid leafminer species (Diptera: Agromyzidae) in Victoria( 2020)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.
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ItemModeling the Distribution of a Widely Distributed butVulnerable Marsupial: Where and How to Fit Useful Models?( 2020)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.
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ItemThe effect of cardiovascular diseases on resistance vasculature and the efficacy of different treatments( 2019)Diabetes and hypertension are conditions that contribute to cardiovascular disease, which is a major cause of morbidity and mortality in the developed world. As the prevalence of obesity rises, the health burden will only increase. Vascular dysfunction, characterised by endothelial dysfunction and increased arterial stiffness, is a hallmark of cardiovascular disease and a major risk factor for the development of further cardiovascular events. Animal models are a necessary tool for diabetes research to elucidate disease pathologies and investigate potential treatment options. However, the precise disease pathology in different animal models of diabetes remains unknown. Further characterisation of vascular dysfunction in cardiovascular disease will help determine treatment options. Annexin-A1, an anti-inflammatory molecule that acts as a second messenger of the glucocorticoid pathway, has beneficial effects in diseases like diabetes, ischaemia-reperfusion injury and stroke. However, its effects on the vasculature remain unknown. The peptide hormone relaxin has shown some promise as a vasoprotective agent. My thesis first aimed to increase understanding of the pathogenesis of vascular disease in different animal models of diabetes. I then investigated the effects of annexin-A1 deficiency on the vasculature to determine the potential therapeutic role of annexin-A1. Finally, I explored the efficacy of relaxin treatment in a hypertensive model of vascular dysfunction. In Chapter 3, I describe the mesenteric vascular phenotype in moderate hyperglycaemia (20mM blood glucose) compared to severe hyperglycaemia (< 30mM blood glucose). While both models showed increased arterial stiffness, only severe hyperglycaemia caused endothelial dysfunction, indicating that arterial wall mechanics are more sensitive than endothelial function to increased blood glucose. Chapter 4 further investigated the effects of type 2 diabetes on the mesenteric vasculature. This study utilised a high fat diet with low dose STZ in mice to induce insulin resistance. Furthermore, this study characterised the effects of annexin-A1 deficiency on arterial remodelling in both insulin resistance and insulin deficiency. The main findings were that insulin resistance induced significant outward remodelling but had no effect on passive stiffness. Interestingly, insulin resistant annexin-A1 gene knockout mice had significantly increased vascular stiffness. Insulin deficiency induced outward remodelling and increased volume compliance in the mesenteric artery, regardless of genotype. Relaxin treatment reverses endothelial dysfunction in the mesenteric artery in hypertension and diabetes over a short treatment duration (<72 hours). However, the effect of a longer treatment duration in hypertension has not been investigated. Chapter 5 demonstrated that 10 days of continuous relaxin treatment reversed mesenteric artery endothelial dysfunction in hypertension by augmenting NO and EDH-mediated relaxation. Overall, my thesis further characterised the vascular phenotype in animal models of type 2 diabetes, which is important in understanding the disease process and for testing potential treatments. Additionally, I showed that both annexin-A1 and relaxin have the potential to be used as successful treatments in vascular dysfunction in the setting of diabetes and hypertension.
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ItemThe physiological effects of artificial light at night on the Australian black field cricket( 2018)The presence of artificial light at night (ALAN) is one of the fastest growing, most pervasive and, until recently, under-appreciated forms of global pollution. Current ALAN levels in urban environments are associated with changes to animal behaviour, dramatic shifts in the timing of life history events, reductions in individual fitness and disrupted physiological processes, including immune function. This thesis explores the physiological effects of ecologically relevant levels of ALAN on a model invertebrate species, the Australian black field cricket, Teleogryllus commodus. In Chapter 1, I reviewed the literature with a particular emphasis on the physiological effects of ALAN, including growth, survival, reproductive success, and immune function. I also speculate as to the potential mechanistic links behind these ALAN induced biological effects. In Chapter 2, I explored experimentally the effects of ecologically relevant levels of ALAN (1, 10 and 100 lux) on life history and fitness traits of the black field cricket. Under controlled laboratory conditions, I reared crickets from egg to adult in an environment with either no ALAN (0 lux) or one of the above dim-ALAN intensities and assessed the consequences of ALAN for growth, survival and reproductive success. I demonstrated that egg hatch, adult survival and reproductive measures were largely unaffected by the presence of ALAN, however juvenile development time was longer and adults were larger when crickets were exposed to any light at night (1, 10 or 100 lux). In Chapter 3, I examined the effects of ALAN (1, 10 and 100 lux) on three key measures of adult immune function (haemocyte concentration, lytic activity, and phenoloxidase activity). The presence of any ALAN (1, 10 or 100 lux) had a clear negative effect on the cellular immune response. Specifically, individuals exposed to any ALAN were unable to increase their haemocyte concentration in response to a stressor challenge. In Chapter 4, I investigated a novel method for the measurement of circulating melatonin in small samples of cricket haemolymph using high-performance liquid chromatography tandem mass spectrometry, with methyl tert-butyl ether (MTBE)/ethyl acetate as an extraction agent. The calibration curve for melatonin was linear in the range of 0.25 and 10 pM (R2 = 0.999), and the limit of detection was 0.25pM. When applied to a set of pilot data from crickets reared under different ALAN environments (0, 1, 10, and 100 lux), the results were however inconclusive, due to small sample sizes. In Chapter 5, I discuss the significance of these findings and their ecological implications. My thesis advances our understanding of the biological ef fects of ALAN for invertebrates, a key taxon contributing to ecological community structure and composition. It is one of the first set of studies to simultaneously investigate multiple traits in the same individuals exposed to lifelong ALAN, and to assess changes in immune function throughout their adult life. Combined, the results presented demonstrate a disruption to physiological processes, and highlight the potential for ALAN to alter the phenology of communities and reduce the overall fitness of individuals.
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ItemEvaluating uncertainty when applying the trait-based protocol for climate-change vulnerability in freshwater crayfish( 2018)Climate change has been recognized as one of the greatest threats to the persistence of biodiversity. Several approaches have been used to assess species’ vulnerability to climate change such as correlative niche models, mechanistic models, trait-based models, and combination of these model outputs. The trait-based protocol for climate-change vulnerability assessment (TVA) is increasingly used in a variety of taxa due to its suitability for assessing data-poor species. Yet, TVA has thus far remained unevaluated for potential uncertainties. In TVA, climate change-relevant traits are selected and scored against three dimensions: sensitivity, adaptive capacity, and exposure to climate change. In this thesis, I applied TVA to assess climate-change vulnerability in a data-poor invertebrate taxon (freshwater crayfish; 574 species) and explored the potential sources of uncertainty in TVA. I found that climate-change vulnerable crayfish are distributed globally with high concentrations in the USA and Australia, reflecting global pattern of crayfish richness. Ninety-one species are already identified as vulnerable to climate change in the IUCN Red List. I identified hotspots of species vulnerable to climate change that require additional conservation action. I assessed multiple sources of uncertainty including trait selection, the use of arbitrary thresholds for quantitative traits, and climate model choices. I quantified that in TVA, it is likely that as more trait variables are included in the study, more species are identified as vulnerable to climate change. The use of arbitrary thresholds in TVA was relatively robust to produce species’ vulnerability ranking. However, I found that the number of species identified as vulnerable to climate change varied greatly (79-156) depending on which individual climate model was used. TVAs are an effective tool to understand climate change vulnerabilities of data-poor species, however, assessors applying the protocol should be aware of these uncertainty sources and perform sensitivity analyses to better understand their impact on TVA results.
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ItemCharacterisation of seed germination in sea rockets (Cakile spp.)( 2017)The sea rocket genus (Cakile) includes several species; two of these are C. edentula and C. maritima. Both are invasive and have become widely distributed throughout the world. C. edentula was introduced earlier than C. maritima in Australia, and quickly expanded. However, after C. maritima was introduced to Australia, C. edentula disappeared from many regions. This replacement of C. edentula by C. maritima has occurred in other areas of the world. An exception also exists where the two species show coexistence in climates where the winters are cold and summers are wet. Several hypotheses have been raised to explain this replacement. In this thesis, a difference in the germination timing of the two species was hypothesised to be of importance in different climates. In order to work towards understanding and predicting germination of Cakile species, the germination of C. edentula from two different climates was examined in this thesis. A hydrothermal time model was used to describe seed germination. The results showed that populations from different climates differ in seed germination behaviour. The C. edentula population from temperate area showed lower base temperature and smaller hydrothermal time accumulation requirement than that from sub-tropical area. Overall, the sub-tropical population germinated more readily than the temperate population across almost all experimental conditions. Dormancy was found in C. edentula seeds during the germination experiment. Further study therefore investigated methods in relieving dormancy of the two Cakile seeds to see whether they responded differently. Two treatments (i.e. damage on seed coat and cold stratification) were applied. The results indicate that C. edentula generally had a higher percentage germination than its counterpart in each treatment (including the control group) while C. maritima germinated faster than C. edentula under most damage levels. The data generated by this thesis could be used as the basis of comparing germination timing between the two Cakile species. For predicting the germination timing more effectively, populations of both species from more climates are required, and dormancy needs to be studied under field conditions.