Zoology - Theses
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ItemEvolutionary ecology of Australian alpine skinks (genus Pseudemoia)( 2014)Alpine biomes are particularly threatened by climate change and correspondingly, many endemic alpine taxa are at risk of extinction. Within Australia, alpine areas (> 1100 m above sea level) comprise a mere 0.15% of the landmass yet harbour numerous endemic species. One such species, the endangered alpine bog skink Pseudemoia cryodroma, is sympatric throughout its range with ecologically and morphologically similar congeners P. entrecasteauxii and P. pagenstecheri. In this thesis, I explored the biology, ecology, and genetics of these three species to develop a comprehensive understanding of their evolution, which serves as a baseline for assessing how these species may be impacted by future climate change. In a study incorporating field data and museum specimens of P. cryodroma, P. entrecasteauxii, and P. pagenstecheri, I investigated how elevation (an abiotic factor) influences critical thermal limits and reproduction in alpine and lowland populations. Within alpine populations, I also examined how sympatry with congeners (a biotic factor) influenced habitat preferences and morphology. Individuals from alpine areas had significantly lower critical thermal minima and relatively smaller litter sizes than lowland conspecifics. These relationships may be particularly important in the future, as climatic gradients are expected to shift upwards with climate change. Though multivariate analyses revealed morphological and ecological differentiation between P. cryodroma and P. entrecasteauxii, these differences did not vary consistently between sympatric and allopatric populations. However, future habitat fragmentation and degradation may increase resource competition, which could lead to more consistent differentiation between sympatric populations. Phylogenetic studies provide critical insight into the timing and occurrence of speciation events. Previous molecular work on Pseudemoia has contributed to the suggestion that P. cryodroma may have originated from hybridisation between P. entrecasteauxii and P. pagenstecheri. However, no studies have directly tested this hypothesis. Using a multi-locus approach, I further resolved the relationships within this genus, uncovered cryptic lineages, and addressed the likelihood of hybrid speciation. Sequence data from one mitochondrial locus (ND4) and five nuclear loci show marked incongruence. The most parsimonious explanation of this discordance is historic mitochondrial introgression, although a hybrid origin for P. cryodroma cannot be completely rejected. This was further supported by microsatellite data, which clearly shows each species as a distinct group. Hybridisation provides a window into the evolutionary processes maintaining species boundaries. I investigated hybridisation between three syntopic species: P. cryodroma, P. entrecasteauxii, and P. pagenstecheri. Using this three-species hybrid system, I explored correlations between levels of hybridisation and genetic, ecological and morphological differentiation. Despite strong genetic structure between species, hybridisation was detected between all pairs of species. Genetic differentiation was inversely related to levels of hybridisation, with the greatest amount of hybridisation between the sister species P. cryodroma and P. entrecasteauxii. In contrast, there were no consistent relationships between hybridisation and either morphological or ecological differentiation. Continued monitoring and more detailed genetic analyses of sympatric populations of P. cryodroma, P. entrecasteauxii, and P. pagenstecheri will be key to detecting subtle changes in hybridisation levels and identifying potential causes. Since anthropogenic climate change is predicted to alter the alpine habitat of Pseudemoia spp., it is useful to understand how the biogeographic history of these species has shaped their current genetic structure and diversity. In contrast to P. entrecasteauxii, the mitochondrial DNA from P. cryodroma and P. pagenstecheri displayed high phylogeographic structure. Though strong genetic structure was also observed in the microsatellite markers for P. cryodroma, patterns of population connectivity were not concordant with the mtDNA data. Since mtDNA and microsatellites have different modes of inheritance and provide resolution at different time scales, discordance is likely due to historic changes in alpine habitat connectivity. These patterns of geographic and genetic isolation are mirrored in other alpine endemics, indicating a shared biogeographic history. This thesis reveals the extraordinary evolutionary complexity within the genus Pseudemoia. Through the integration of biological, ecological, and genetic data, I provide a thorough assessment of Pseudemoia’s evolutionary history. These results are strengthened by comparisons to the sympatric, less geographically restricted congeners P. entrecasteauxii and P. pagenstecheri. Additionally, I address the current conservation needs of the endangered alpine endemic P. cryodroma and suggest that conservation efforts should focus on maintaining the extent and quality of existing habitat to reduce the potential for inbreeding and/or loss of genetic diversity. Thus, this work serves as a solid stepping-stone for much needed future research on alpine fauna.
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ItemConservation of the dwarf galaxias, Galaxiella pusilla (Mack 1936) (Teleostei: Galaxiidae), a threatened freshwater fish from south-eastern Australia( 2014)The dwarf galaxias, Galaxiella pusilla, is a small native freshwater fish of national conservation significance from south-eastern Australia. Recovery efforts for this species have been restricted by an absence of fundamental biological and ecological knowledge. The purpose of this research was to address key knowledge gaps required for the development of sound management strategies. Firstly, both nuclear and mitochondrial DNA markers were applied to determine broad-scale patterns of genetic structure and diversity across the species entire geographic range. Substantial genetic differences between two distinct geographic regions were found that suggested G. pusilla, as it is currently recognised, comprises two cryptic species. Lower genetic diversity amongst the east region compared to the west region was also found. Following the initial genetic study, a more comprehensive population genetic assessment was conducted to provide robust information on genetic structure and diversity within and between populations. This revealed a strong inverse relationship between genetic uniqueness and genetic diversity within both geographic regions. Furthermore, differentiation within each region was largely due to a loss of diversity and changes in allele frequencies, rather than the presence of unique alleles. This highlighted the influence of genetic drift as a driver of genetic patterns in G. pusilla and may be a reflection of ongoing extreme wetting and drying events (e.g. severe droughts and major floods) that lead to founder events or bottlenecks. In the case of G. pusilla, and mostly likely other freshwater fish species exposed to similar dynamic environments, there is a potential tradeoff between genetic diversity and genetic uniqueness that needs to be balanced when determining conservation priorities. Both habitat drying and respiration experiments demonstrated that Galaxiella pusilla have adaptations that enable them to survive for several days without surface water and that habitats with a greater ability to retain moisture (e.g. detritus, crayfish burrows) increased survival rates and duration. An ability of G. pusilla to shift their mode of respiration to air-breathing when exposed to air was also observed. Parallel experiments with Gambusia holbrooki, an invasive fish species that is commonly cited as a major threat to Galaxiella pusilla, showed comparatively poor survival of Gambusia holbrooki in the absence of surface water and no apparent shift in their mode of respiration in air. These results indicate the potential application of habitat drying to control Gambusia holbrooki in Galaxiella pusilla habitats, with possible additional benefits for Galaxiella pusilla food resources. Targeted revegetation that increases levels of detritus in Galaxiella pusilla habitats may also favour Galaxiella pusilla over Gambusia holbrooki. Considering adaptation to habitat drying, patterns of genetic structure and diversity, and observations of large inter-annual fluctuations in population densities at some sites, G. pusilla conservation strategies are likely to be particularly successful in the context of managing metapopulations. This places emphasis on protecting and restoring multiple habitats, as well as their interconnections across a landscape, so that localised extinctions are balanced by recolonisation and long-term persistence of the population as a whole is achieved. Given the extent of habitat loss and isolation over much of the distribution of Galaxiella pusilla, the restoration of functional metapopulations within river systems is likely to be critical to the recovery of the species. Finally, morphological and meristic measurements on populations from both the east and west regions identified significant differences in a number of characters including total length, number of vertebrae, length of caudal peduncle and dorsal fin-anal fin setback. Ventral surface markings are also likely to aid visual classification. Based on distinct genetic differences, variation in a number of morphometric and meristic characters, and a clear geographic separation, a new species of Galaxiella is proposed: Galaxiella toourtkoourt sp. nov.. Clearly, the separation of populations into two species for a nationally listed species has significant conservation implications. The description of a new Galaxiella species and re-description of Galaxiella pusilla ensures that independent management strategies can be devised in a way that sufficiently accounts for their threatened status (e.g. eastern populations have much lower genetic diversity and are under more immediate threat from habitat loss and isolation) and potential biological and ecological differences.
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ItemThermal advantages of colour change in bearded dragon lizards, Pogona vitticeps( 2014)Many animals possess the remarkable ability to change colour, and this ability has several potential functions, including communication, social signalling, and camouflage. However, the functional significance and drivers of colour change are still poorly understood in many taxa. In this study, I applied laboratory and field approaches to examine an important, but understudied potential function of colour change in terrestrial ectotherms: thermoregulation. I quantified colour change in response to temperature in wild-caught bearded dragon lizards (Pogona vitticeps), which are known for their marked individual colour change and geographic colour variation. For comparison, I also quantified the extent of non temperature-dependent colour change from dark to light states elicited during social interactions and at different times of the day. I found that lizards showed substantial animal-visible (UV-Vis) and near infrared (NIR) skin reflectance change in response to temperature for dorsal but not ventral body regions. By contrast, lizards showed the greatest capacity for colour change on the ventral body regions under other (non temperature-dependent) circumstances. I then used biophysical models to predict the thermal benefit of colour change to a bearded dragon lizard, and found that in the morning, under environmental conditions characteristic of the field site during the breeding season, the maximum temperature-dependent change in dorsal reflectivity would result in a 9% increase in the rate of heating. Although this temperature-dependent colour change may therefore aid thermoregulation, colour change is likely to be less important as a mechanism for bearded dragons to thermoregulate than other mechanisms such as behavioural changes in posture and shuttling between sun and shade. Therefore, colour change may have primarily evolved to enhance social signalling or camouflage, but subsequently been co-opted for thermoregulation. To further understand the relative importance of the different functions of colour change to a bearded dragon in the wild, and how they accommodate these functions when they conflict, I quantified colour change in 11 free-ranging bearded dragons (Pogona vittceps) over an 8-week period during the breeding season (October 9th– November 28th, 2013). In addition to taking daily snapshots of multiple lizards over the entire field study, I followed a single lizard through an entire day (‘day in the life’), and modelled the effect of different thermoregulatory behaviours on core body temperature. From this, I found that lizards showed a significant and consistent relationship between their dorsal skin coloration and both background coloration and either core body temperature or skin temperature. However, background colour explained the most variation in lizard colour, and there was no evidence that these lizards exhibit temperature-dependent background choice. Furthermore, the focal lizard that was followed through the entire day behaved very similarly to a predicted modelled “thermoregulating” lizard, suggesting that in the wild, behavioural thermoregulation may be more important for regulating body temperature than colour change. Overall, the data presented in this thesis shows that colour change in bearded dragons functions in thermoregulation but that this is likely to be secondary to its use for communication and camouflage. These data contribute significantly to our understanding of the evolutionary drivers of colour change in terrestrial ectotherms.
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ItemResilience of kelp dominated reefs of south-eastern Australia( 2014)How stable is an ecosystem and what causes it to change? These are two of the fundamental questions in ecology and particularly pertinent with the range anthropogenic influences on the natural environment. While systems may be able to absorb certain perturbations, ongoing disturbance events or removal of key species can cause major shifts in ecosystems. The various drivers of ecosystem change can be categorised into top-down (predator or herbivore driven), bottom-up (environmental or producer driven) and disturbance (natural or anthropogenic). While we have a good theoretical base from which to test concepts of ecosystem change, we still need more experimental evidence if we are to try to build generaliseable models of drivers of ecosystem change and how they interact. I decided to test the idea of ecological resilience and the importance of top-down (through changes in herbivore density), bottom-up (through in-situ nutrient additions) and different regimes of disturbance in shifting ecosystem state on the kelp dominated (Ecklonia radiata) temperate sub-tidal reefs in south-eastern Australia. In two manipulative field experiments, increases in herbivore (sea urchin) density were able to drive declines in kelp and other algal species over an 18 month period. However, severe disturbance to the kelp canopy with or without increases in urchin density also showed a similar shift. In sea urchin barrens, exclusions of urchins to a low density did allow recovery of a number of algal species over a 27 month period at one site. However, at another site, algae recovered across the entire reef independent of urchin density treatments. These results demonstrate that while herbivores can drive change or maintain an ecosystem state, similar changes can occur independently of urchin density. The effect of additional nutrients in combination with a severe disturbance to the kelp canopy (Ecklonia radiata) was tested in a manipulative experiment at three different sites. At all three sites, the disturbance resulted in an increased number of native kelp recruits compared to control plots. At two sites, additional nutrients had no effect on kelp recruitment. At the third, where kelp was removed and nutrients were added, the introduced Japanese kelp Undaria pinnatifida had increased recruitment and dominated these plots by 6 months. This resulted in a significantly lower cover of the native kelp Ecklonia in these plots. While additional nutrients can result in changes to species dynamics, this effect was site specific and also relied upon an initial disturbance to the kelp canopy. In another field experiment, I tested how different regimes of disturbance influence kelp recruitment, recovery and the response of other algal species in the community. This work showed an interaction between the pathway (one-large or multiple-small disturbances) and severity of disturbance, with greater recruitment and recovery of kelp (Ecklonia radiata) in plots with the least severe disturbance over multiple-small disturbance events. For other algal species in the community, most showed temporally variable effects of the disturbance treatments, often with an interaction between pathway and severity of disturbance. Finally, I constructed a historical timeline of kelp and sea urchin abundances at three sites in northern Port Phillip Bay, Australia, over a 80 year period. This included data from unpublished theses and technical reports, as well as a collection of aerial photographs revealing the total algal cover on the reefs. Together, this showed relative stability of the three reefs between the 1930s and 1990s, with unprecedented declines occurring in two stages in the 2000s. While urchins seem responsible for some of the changes in the late 2000s, large declines appear to have occurred without increases in urchin density in the early 2000s. This thesis demonstrates how top-down, bottom-up and disturbance processes can influence each other in driving change, but that these changes can be spatially and temporally variable. While we may be able to generalise the effects of some factors across larger spatial scales, how these will unfold at a particular site, at a particular time, may be dependent on the local context of the disturbance and nutrient regime.
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ItemThe potential of shelterbelts to enhance the abundance of natural enemies of agricultural pest arthropods( 2014)Due to past land clearing, the Australian rural landscape faces a range of environmental and economic challenges such as biodiversity loss, soil degradation and higher wind speeds. To combat these effects, land holders often utilise non-crop woody vegetation such as shelterbelts to protect crops and increase the long term sustainability of the site. The value of these shelterbelts is further increased if woody vegetation enhances the abundance of arthropods that are beneficial to agriculture, notably predatory species and parasitoids, acting as an incentive for further revegetation. Despite previous work on vegetation effects, there is limited research as to the specific characteristics of woody vegetation that causes the increased abundance. Therefore this study seeks to determine the specific resources and structure of shelterbelts that enhance natural enemy abundance. To examine this issue, 60 sites throughout the Yarra Valley were selected for assessment of different aspects of their structure regarding their location, canopy, shrub layer, ground cover and floral resources. At each of these sites, insect abundance was monitored using yellow sticky traps placed within each shelterbelt for one week once a month over 5 months. In addition, one of these sites was also selected for a manipulative experiment whereby leaf litter and porosity were increased or decreased. In the manipulative experiment, insect abundance within each treatment was also monitored using yellow sticky traps placed for one week, once a month over three months. A third trial assessed relative effects of shelterbelt resources or wind disruption on the distribution of natural enemies. At each site, insect abundance was measured across transects running perpendicular to a shelterbelt 84 m to the north through to 84 m south. The results of these experiments indicated that there were five aspects of shelterbelts and other non-crop woody vegetation that may influence different natural enemies, particularly parasitoid Hymenoptera and coccinellids. Aspects of shelterbelts that increased some species or group abundance included: being adjacent to riparian areas or perennial crops, ground cover structure such as grass height and ground coverage, the abundance of floral resources particularly canopy flowers, and the shelterbelts’ porosity. Conversely the amount of leaf litter appeared to reduce the abundance of a range of natural enemies. However the influence of these features on natural enemies needs to be considered within the context of a substantial influence of wind direction and deposition in the wake of a shelterbelt. This factor influenced the abundance of a range of natural enemies when shelterbelts were compared.
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ItemTrait evolution as a consequence of sexual conflict over sex allocation( 2014)Widely observed deviations from equal primary sex ratios may be adaptive if sons and daughters differ with respect to their associated costs and benefits to parents. Progeny sex ratios produced by species in the genus Bradysia (Sciaridae: Diptera) range from unbiased to single sex progeny. Their unusual sex-determining system is also associated with paternal genome elimination (PGE), and as a consequence male genes are only transferred via daughters. This produces a potential sexual conflict over sex allocation because males should prefer a more female-biased brood than that of their mate. Consequently, theory predicts that selection will favour heritable male traits that somehow increase the proportion of daughters a male produces. Though Hamilton (1967) recognised that a haplodiploid genetic system would lead to the potential for males and females to be in conflict over the offspring sex ratio, studies into sex allocation have largely overlooked this potential conflict, and both empirical and theoretical data are lacking. If both sexes can influence progeny sex ratios in a system where sexual conflict over sex allocation is likely, this may drive the selection of sexually antagonistic traits. This thesis explores whether certain sexual traits are associated with progeny sex ratio outcomes in species of Bradysia where sex ratio conflict between mates is probable. The key aim is to highlight which sexual behaviours potentially drive antagonistic coevolution between the sexes as a consequence of sexual conflict over sex allocation.
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ItemEnergy and mass budgets of kangaroos and their Implications for behaviour and life history( 2014)Multiple factors affect how species respond to climate change, and predictive approaches need to be based on known cause-and-effect links. Formal energy and mass budgets incorporate physiology and functional traits with a mechanistic understanding of thermodynamic constraints. Thus, they grant powerful insight into the impact of climate and nutrition on organisms. In this thesis, I apply two such models of energy and heat transfer to understand thermoregulatory constraints on kangaroos and benefits of behavioural thermoregulation. By applying principles of biophysics to empirical data, I relate observed shade use of red and western grey kangaroos (Macropus rufus and M. fuliginosus) to measured weather conditions and predict how these species are restricted to shade across Australia (Chapter 2). Red kangaroos appear more adept at dealing with heat than western grey kangaroos and are much less restricted to shade on a continental scale. An activity constraint of ~1800 h/year appears associated with the northern range limit of western grey kangaroos. In Chapter 3, I expand on these ideas by adapting a full biophysical model for kangaroos. Biophysical models describe how climate conditions and a species’ behaviour, morphology, and physiology alter mass and energy balance. By predicting hourly field energy and water requirements for kangaroos, the model allows me to quantify metabolic and hydric benefits of changing activity and seeking shelter. During the heat of the day, reducing activity appears more effective at reducing water loss than seeking shade; in contrast, during cold nights, there is a metabolic cost of being inactive, since metabolic rates nearly equal those if active but the kangaroo cannot obtain food to cover these costs. These results provide a clear physiological mechanism for observed activity patterns and show how energetic and water requirements may help to shape nocturnal/crepuscular activity for these species. In Chapter 4, I incorporate lactation and pregnancy into a dynamic energy budget (DEB) model and apply it for mammals. Dynamic Energy Budget (DEB) theory is a mechanistic metabolic theory of energy and mass balance and provides a uniquely powerful, whole-life-cycle framework from which to predict growth and reproductive output of species in varying nutritional environments. I interpret the parameter estimates for three case studies (a monotreme, a marsupial, and a eutherian mammal) and what they might imply for issues in mammalian energetics. I found that the echidna (Tachyglossus aculeatus) – which is thought to have a relatively ‘slow’ pace of life history – grows faster than the tammar wallaby (Macropus eugenii) but slower than the white-tailed deer (Odocoileus virginianus) after accounting for body temperature and size. In Chapter 5, I apply the DEB model for mammals across the family Macropodidae (and one outgroup) to explore how life history strategies of kangaroos and wallabies are shaped by underlying traits. I identify several DEB parameters (particularly energy conductance and somatic maintenance) that appear to underlie observed life history patterns by controlling how quickly the animal uses energy for different purposes; these parameters may grant empirical-based insight into why some smaller macropodid species are more vulnerable to extinction. Combined, these mechanistic models have the potential to generate robust predictions about how and why climate constrains or impacts the ecological performance of a species across climatic and environmental gradients.
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ItemMetapopulation dynamics of the southern hulafish (Trachinops caudimaculatus) in Port Phillip Bay, Victoria( 2014)Metapopulation theory arose as an aid to understand the dynamics of demographically connected populations in patchy habitats. The existence of widespread dispersal in marine systems suggests that metapopulation theory can be very useful in explaining the demographics of marine populations, particularly for species dwelling on patchy habitats such as coral and rocky reefs. However the large scale of larval dispersal and the challenges of accurately estimating local demographics in demersal species have created significant barriers to the application and empirical testing of metapopulation theory in marine ecosystems. Understanding the metapopulation dynamics of a system requires accurate estimation of both the local demographic processes of individual populations and the regional processes of dispersal that govern the exchange of individuals amongst populations. In this thesis I aim to explore the role of local and regional processes in determining the dynamics and persistence of a metapopulation of a marine fish Trachinops caudimaculatus. Firstly, I empirically quantified local processes of survival, growth and fecundity, and the role of density-dependence on local population regulation. Secondly, I empirically estimated larval dispersal and population connectivity using natural otolith markers. Finally, I characterised the dynamics of the metapopulation, the importance of local and regional processes, and which local populations are critical for metapopulation persistence. Predation is an important established driver of local demographics in marine reef fish. Different predators are often assumed to have independent and additive effects on shared prey survival; however, multiple predator effects can be non-additive if predators foraging together reduce prey survival (risk enhancement) or increase prey survival through interference (risk reduction). I tested whether interacting benthic and pelagic predator guilds result in non-additive prey mortality and whether the detection of such effects change over nine weeks as prey are depleted. I observed changes in the additivity over time, with risk enhancement in the first seven days, as shoaling behaviour placed prey between predator foraging domains with no effective refuge, and additive mortality thereafter. My observation of the changing interaction between predators and prey has important implications for assessing the role of predation in regulating populations in heterogeneous landscapes. Density-dependence is an important factor in regulating populations on a local scale. Species that live in groups, such as shoaling fish, can experience both direct density-dependent mortality through resource limitation, and inverse number-dependent mortality via increased feeding rates and predator evasion in larger groups. I manipulated the density and group size of T. caudimaculatus on artificial patch reefs at two locations with different predator fields. Mortality was strongly directly density-dependent throughout the experiment regardless of the dominant predator group, however the limiting resource driving this effect changed over time from shelter to food competition. Direct density effects were much stronger than those of group size, suggesting little survival advantage to shoaling on isolated patch reefs where resource competition is high. Food competition may also be an important regulator of post-settlement reef fish cohorts after the initial intense effects of refuge limitation and predation. Robust analysis of density-demography relationships on larger spatial scales requires the identification of limiting resources and understanding their distribution across a landscape. Unrecognised heterogeneity in habitat quality on a landscape scale can hamper attempts to detect spatial density-dependence in demographic rates. I examined three demographic rates – survival, growth and condition – of juvenile T. caudimaculatus over a three month period across eighteen populations of variable habitat. Strong positive relationships between habitat complexity and both survival and growth were detected, however landscape-scale variability in all three demographic rates was best explained by the additive or interactive effect of both habitat quality and density. Habitat complexity and density were found to be key drivers of T. caudimaculatus demographics and highlight the importance of quantifying environmental heterogeneity when examining density-dependence on a landscape scale. Understanding of the regional connectivity of populations is critical for understanding metapopulation dynamics. Source-sink metapopulation theory has become an important framework to assist conservation management decisions in marine environments, where source populations are considered conservation targets due to their critical role in enhancing metapopulation capacity and persistence. Using field estimates of local demographics and regional dispersal data derived from natural otolith markers, I identify temporally variable source-sink dynamics in a marine metapopulation. The identity of source populations varied amongst years due to significant spatio-temporal variability in survival rates and larval recruitment, but sources were generally characterized by high local retention rates, high per capita successful recruit production and high levels of connectivity. Asynchrony in population performance, together with evidence of temporal density-dependence, promoted stability in the metapopulation despite large variability in individual population sizes. I observed resilience in marine metapopulation dynamics that may enable persistence in the face of stochastic weather events through a combination of high levels of connectivity, asynchrony in population performance and density-dependent compensation of local demographics. The identification of temporal variability in source-sink dynamics raises important implications for conservation management, and highlights the critical need to understand the role of populations across the full spectrum of climatic variability.
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ItemThe role of L-proline and L-proline activated signalling in the regulation of pluripotency( 2014)The study of pluripotent cell populations in the mouse embryo, and mouse pluripotent cells in culture, has revealed four identifiable pluripotent cell populations, or states, that comprise the pluripotent lineage. The four pluripotent populations are the epiblast precursor cells, epiblast, early primitive ectoderm and late primitive ectoderm in the embryo. They are represented in culture by ground state embryonic stem (ES) cells, primed ES cells, early primitive ectoderm-like (EPL) cells and epiblast stem cells (EpiSCs) respectively. Although these cell populations are relatively well characterised, the mechanisms that control progression between cell states remain poorly understood. Understanding the transition of primed ES cells to EPL cells, representing the progression of epiblast to early primitive ectoderm of the post-implantation embryo, is the focus in this thesis. EPL cells are derived from ES cells in response to factors within the conditioned medium MEDII. The ES to EPL cell transition provides a model to investigate the mechanisms that regulate pluripotent cell lineage progression. Amino acids have been shown to regulate cellular processes through modulation of intracellular cell signalling pathways. The amino acid L-proline has been identified as the bioactive factor of MEDII required for EPL cell formation, and ES cells cultured in L-proline-containing medium form EPL cells. EPL cell formation is accompanied by changes in cell morphology, gene expression and differentiation kinetics. In chapter 3, the main L-proline transporter on ES and EPL cells is determined to be the System A amino acid transporter 2 (SNAT2). Identification of SNAT2 was facilitated by radioactive uptake assays. SNAT2 uptake of L-proline can be inhibited by the addition of excess amounts of other SNAT2 substrates, such as alanine. ES cells cultured in L-proline and excess concentrations of SNAT2 substrates, but not other amino acids, were not able to form EPL cells. This suggests that L-proline uptake through SNAT2 is required for EPL cell formation. A requirement for L-proline uptake by SNAT2 in the regulation of pluripotent cells suggests a role for L-proline in activating intracellular pathways in the formation of EPL cells. In chapter 4, pharmacological inhibitors of cell signalling pathways were used to determine requirements of Src tyrosine kinases, p38 MAPK and Erk in the formation and maintenance of EPL cells. Exposure of ES cells to L-proline increased Src tyrosine kinase and p38 MAPK activity. Chemical inhibition of these pathways prevented the acquisition of many, but not all, features of EPL cells in culture. Although Erk1/2 signalling was not activated in response to L-proline addition, it was required within the cell for EPL cell formation. The involvement of multiple signalling pathways in EPL cell formation and maintenance supports the potential role of L-proline activated pathways in primitive ectoderm formation. Amino acids are critical for the developmental processes of the preimplantation mouse embryo to the blastocyst stage. However, regulatory roles of amino acids beyond the blastocyst stage, specifically in pluripotent lineage progression, are not well understood. In chapter 5, the expression of SNAT1 and SNAT2 transporters was determined in mouse embryos between the 2-cell stage and early post-implantation. Both transporters showed temporal dynamic expression patterns and differences in intracellular localisation within cell types. Changes in transporter expression likely reflect different amino acid needs at different stages of development. Dynamic SNAT2 expression was observed in the pluripotent lineage with up regulation in the epiblast prior to primitive ectoderm formation. Primed ES cells cultured in ground state conditions resulted in reduced Slc38a2 expression suggesting regulation of Slc38a2 in the transition of ground state to primed ES cells. SNAT1 and SNAT2 showed regulated expression in the trophectoderm (TE) lineage. SNAT1 was preferentially expressed on outer cells of the compacted morula that are fated to form TE, while SNAT2 was localised in the nucleus of TE and placenta cells. The unexpected nuclear localisation of SNAT2 suggests a novel role for an amino acid transporter in the nucleus. Localisation of SNAT1, in outer cells may indicate requirements for amino acids in TE formation. Collectively, data presented here support a role for amino acids and amino acid transporters in the progression of pluripotent states in vivo and in vitro. Understanding the role of amino acids in pluripotent cell transitions will significantly contribute to the optimisation of differentiation media and protocols used in regenerative medicine.
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ItemInfection patterns and subclinical effects of gastrointestinal helminth parasites in eastern grey kangaroos (Macropus giganteus)( 2014)Large mammalian herbivores are commonly infected with gastrointestinal parasites. In many host species, these parasites cause clinical disease and may trigger conspicuous mortality events. However, they may also have subclinical impacts, reducing fitness as well as causing complex changes to host growth patterns, body condition and behaviour. The gastrointestinal fauna of the family Macropodidae is extraordinarily diverse, with hosts harboring large communities of helminth parasites. Eastern grey kangaroos (Macropus giganteus) are well studied with respect to their biology, but what is known about their parasite communities is limited to three descriptive surveys and one epidemiological study. In this thesis, I investigated the seasonal patterns of helminth infection in eastern grey kangaroos at three sites in Victoria, Australia, and combined field experiments and observations to construct a generalised life cycle for the community. I also report the first test of efficacy of the anthelmintic drugs moxidectin, ivermectin and albendazole in kangaroos, through field trials and laboratory assays. Effective anthelmintics are valuable tools for conducting manipulative field experiments to examine the effects of parasites on wildlife. I then used these two sets of data in field experiments, manipulating parasite burdens in free-ranging kangaroos. These field experiments allowed me to explore the subclinical impacts of the helminth community on the growth, body condition and blood parameters of juvenile kangaroos, which theoretically should experience significantly greater costs from parasites (being immunologically naive and undergoing significant growth). Ruminant hosts reduce their food intake when heavily parasitised. However no data exists for marsupial hosts and it may be that they increase their food intake to compensate for the effects of parasitism. I also tested this prediction, and report behavioural observations in adult female kangaroos, which may bear particularly high costs, especially during lactation. Kangaroo populations in Victoria had very similar helminth communities, with between 20 and 25 species detected at each site. The pre-patent period of infection of nematodes in eastern grey kangaroo is at least three months, and faecal egg output shows a distinct seasonal pattern, with a peak in egg counts from October to January each year. Data from one site indicates that faecal egg counts were influenced predominantly by the abundance of a single nematode species (Pharyngostrongylus kappa), highlighting the problems associated with using faecal egg counts to estimate nematode burdens. Pasture plots showed that nematode eggs take around 14 days to hatch once deposited, and that autumn rains likely trigger emergence from faecal pellets. The abundance of infective larvae in the environment therefore appears to be closely tied to environmental conditions, with a peak in infection of hosts in the winter months. Unexpectedly, moxidectin and ivermectin had low efficacy, with maximum faecal egg count reductions of 82% and 28% respectively. However, treatment with albendazole reduced faecal egg counts by 100% in all kangaroos, and these remained low for up to three months. Laboratory assays confirmed the results of the field trials. Macrocyclic lactones, at recommended dose rates, were much less effective against strongylid nematodes in kangaroos than has been reported for domestic herbivores. This may be due to pharmacokinetics in the host and/or low susceptibility in some of the nematodes infecting eastern grey kangaroos. Comparisons of growth parameters (body condition, growth rates) and haematological parameters of unparasitised juvenile kangaroos with parasitised controls, revealed surprisingly few subclinical effects of parasitism. Treated juvenile kangaroos had significantly higher levels of plasma protein (albumin) but, contrary to predictions, showed negligible changes in all the other parameters measured, despite adequate statistical power. Similarly, manipulations of parasite burdens in adult females did not result in any changes in their foraging, measured by four key foraging variables, although statistical power was lower. This thesis provides important insights into the host-parasite dynamics of eastern grey kangaroos and their helminth communities in Australia. The results from field experiments suggest that kangaroos are largely unaffected by their gastrointestinal helminth burdens, or may be able to compensate for the costs of parasitism in alternative ways.