School of BioSciences - Theses
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ItemDeveloping the Dunnart Model: iPSCs and ART as tools for marsupial conservation and genetic manipulation( 2022)With many marsupials at risk of extinction, we need powerful marsupial laboratory models and innovative new tools to buffer conservation efforts for endangered species. However, improved resources for marsupial models are also essential for deepening our understanding of mammalian development and evolution through the power of comparative genetics. We are developing technologies that will establish the fat-tailed dunnart (Sminthopsis crassicaudata) as a robust marsupial model, capable of targeted genetic manipulation. This goal requires stem cell advances, as well as in vitro derived embryos, however marsupial models lack protocols for these. Since the derivation of embryonic stem cells from marsupials is challenging, I reprogrammed dunnart fibroblasts to induced pluripotent stem cells (iPSCs). These iPSCs express core pluripotency genes and are capable of differentiation to all three embryonic germ layers. I, further, used single-cell RNA-sequencing to explore the transcriptome for these cells and discovered enrichment for networks implicated in pluripotency and early development. Dunnart iPSCs offer a route to loss and gain-of-function experimentation via the integration of modified iPSCs into embryos. However, a simpler method would necessarily involve editing zygotes, enabling the direct generation of genetically modified lines. Timing ovulation in marsupials is difficult, however, the use of IVF would allow the generation of many embryos at the right stage, facilitating repeatable high throughput genetic manipulation. Unfortunately, IVF has not yet been successfully applied to Australian marsupials. In vitro, sperm from these animals fail to undergo the maturational changes that enable fertilisation, known as capacitation. The only exception to this, is when they undergo a complex system of co-culture with oviductal secretions. In pursuit of the optimal media conditions for this process, I used liquid chromatography mass spectrometry (LC/MS) to characterise the in vivo environment in the dunnart oviduct - the site of fertilisation. I then tested candidate metabolites and additional factors on dunnart sperm, with the aim of inducing capacitation. This had limited success, however, in the process, I discovered that incubation in bicarbonate buffered media at atmospheric oxygen is sufficient to produce the physiological outcomes associated with sperm capacitation. In these conditions, dunnart sperm bind to oocytes, undergo the acrosome reaction, and display tyrosine phosphorylation. Finally, sperm exhibit the characteristic head realignment that indicates capacitation for marsupials. This is the first time simple in vitro capacitation has been achieved for an Australian marsupial, and paves the way for progress in IVF for these iconic mammals. Importantly, both IVF and iPSC technology will greatly enhance the utility of the dunnart model. Advances in these areas are critical if we are to generate the first genetically modified Australian marsupial-lines. Furthermore, these resources will also have a vast impact on our ability to conserve vulnerable marsupial species, enabling banking of stem cells and embryos, use of in vitro derived embryos and iPSCs to reintroduce genetic diversity to inbred populations, as well as targeted intervention in disease and pathogen susceptibility. Finally, this thesis presents research that can be used to gain a better understanding of conserved developmental pathways in mammalian biology, with a novel role suggested for the oviduct as the signaling niche that regulates the earliest and most fundamental events for mammalian gametes and embryos.
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ItemUnderstanding the patterns of distribution and environmental responses in Australian grasshoppers based on historic field survey notebooks( 2022)What constitutes a species’ environment and distribution is of vital importance in ecology. Our understanding of the patterns of biogeographic distribution and their connection to the environmental responses of insects has been limited due to a lack of baseline data. The baseline distribution data for all species of a given group in a region can provide fundamental insights into biogeographic questions about historic patterns of diversity, endemism, and adaptive strategies to variable environments. Grasshoppers are one major group of insects for which a continent-wide perspective on distribution and response to environmental conditions can be obtained. This is because they were extensively surveyed in Australia for 54 years (1936-1989) as part of Commonwealth scientific expeditions for the Australian National Insect Collection. Field notebooks recorded by Dr Key and associates from those surveys can help fill the hole in our knowledge of invertebrate distribution patterns in Australia. In this thesis, I developed a database of historic occurrences of Australian grasshoppers based on those field notebooks. I transcribed and carefully geocoded field notebooks corresponding to surveys conducted in Western Australia (WA) and Tasmania. I collected 8975 geographic coordinates for 506 species having a confirmed or putative taxonomic name, based on our transcription and geolocation for 47 notebooks containing 590 pages (~24%, out of 2486 pages altogether). Of the 506 species identified from the notebook transcription, only 177 species had complete formal taxonomic names. I analyzed the occurrences that were recorded from WA using species distribution models (SDMs) to provide insights on the patterns of grasshopper diversity in Australia as well as their strategies of adaptation in response to variable climatic conditions. Overall, in this thesis I aimed to achieve the following goals: (i) digitize field notebooks and save them in a digital repository; (ii) transcribe and collect geographical coordinates of survey sites following the description of field notebooks; (iii) develop species richness maps of grasshoppers’ historic distribution; (iv) identify biases in the spatial distribution of survey locations; (v) identify similarities and differences in survey results among the lead surveyors; (vi) identify patterns of species composition; (vii) predict the distribution of grasshopper species richness; (viii) identify centers of endemism; (ix) quantify how well grasshopper centers of endemism are covered by the protected area networks; (x) develop ecological hypotheses of how grasshopper species respond to changing environmental conditions. In this thesis, I have fulfilled all the above aims. The database of historic occurrences created in this study can be regarded as robust because surveyors were consistent in survey methods and what they recorded. This study highlights the in-completeness of taxonomic description of Australian grasshoppers, although the vast majority of species have been given provisional names. The generalized dissimilarity modeling suggested at least four distinct grasshopper assemblages are distributed over the north, north-west, arid-interior, and south-west of WA. Grasshopper species richness varied spatially with higher richness present in the north of WA than in southwest based on both field observation and prediction from SDMs. In addition, the fit of SDMs was negatively correlated with the prevalence of each species and with an index of vagility (the ratio of wing length to body length). Observations from this study provide an important baseline perspective on Australian grasshoppers and highlights the need for more research on their present status, morphology, life-history, and phylogeny.