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    Genomic differentiation of brushtail possum (Trichosurus vulpecula) populations : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Zoology at Massey University, Manawatū, New Zealand
    (Massey University, 2023-10-15) Carmelet-Rescan, David
    Toxin resistance, an ecologically functional trait, has emerged as a result of the coevolutionary arms race between plants and herbivores. This adaptive response allows organisms to counteract the detrimental effects of toxins. Such adaptation occurs within three primary ecological contexts: predator resistance, prey resistance, and auto-resistance. In the context of plant-herbivore interactions, the production of toxic secondary metabolites by plants plays a pivotal role, triggering a dynamic arms race with herbivores. This ongoing battle leads to the development of diverse defensive strategies in plants and corresponding counter-adaptations in herbivores. Toxin resistance studies have not only shed light on coevolutionary dynamics but also provided insights into regional adaptations and population fragmentation. The case of sodium fluoroacetate, a potent toxin found in certain plant species, offers a specific example of toxin resistance in brushtail possums (Trichosurus vulpecula). Deepening our understanding of the underlying mechanisms driving toxin resistance becomes particularly interesting in this case because of the notable variation in sodium fluoroacetate resistance observed among subspecies. The brushtail possum (Trichosurus vulpecula) is protected in its natural range of Australia but as an invasive pest in New Zealand populations are controlled by the application of human made sodium fluoroacetate, providing a strong selective force for the potential parallel evolution of toxin resistance. I first focus on the population fragmentation and past connectivity of brushtail possum subspecies in their native range using ecological niche modelling and genetic analysis. My results suggest that Pliocene and Pleistocene climate oscillations played a significant role in shaping the distribution and structure of these widespread marsupials. The findings highlight the limited gene flow between subspecies that would have facilitated their adaptations to regional plant assemblages. I studied gene expression using RNA sequencing to reveal significant differences in transcription levels between adult and juvenile brushtail possums. These findings provide insights into the mechanisms associated with possum development, tissue development, cell cycle, and extracellular matrix. Furthermore, the downregulation of specific genes, such as Cytochrome P450, in juveniles may indicate their role in possums' dietary adaptations. Differential gene expression among subspecies of brushtail possum shed light on genomic differentiation and identified candidate genes involved in regional adaptations involving toxin tolerance. Further analyses comparing subspecies has identified genes under positive selection and enriched biological pathways that may be associated with sodium fluoroacetate resistance. These findings support the idea of genomic divergence in spatial populations and provide hypotheses on the metabolic pathways involved in toxin resistance. Collectively, these studies offer valuable insights into the coevolutionary dynamics, toxin resistance mechanisms, gene expression and selection patterns in brushtail possums (Trichosurus vulpecula) and formulated strong hypotheses related to sodium fluoroacetate resistance. This study presents a reliable method to use transcriptomics to investigate the expression and genomic differences behind observed phenotypic variation within a single species.
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    Environmental influences on polyphosphate accumulation in microalgae : an investigation into species differences and transcriptional responses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Cliff, Alexander
    Many species of microalgae can store phosphorus (P) as polyphosphate (polyP) granules during the process of ‘luxury uptake’, a term describing intracellular P accumulation above levels required for normal metabolism (0.2 – 1% P by dry weight). Environmental conditions can influence P luxury uptake but it is not known whether the effects of environmental conditions on luxury P uptake are the same for all microalgae or whether all microalgae can in fact store P as polyP. The broad aim of the work described in this thesis was to extend the current knowledge of polyphosphate (polyP) synthesis in microalgae, enabling improved exploitation of their ability to sequester P from water sources and enhance recovery of a vital nutrient. Specifically, the mechanisms by which environmental conditions influence luxury uptake and potential species differences need to be studied to better understand observations at the population level and make informed decisions in the design of treatment processes. The experimental work was therefore divided into two main objectives: Objective 1 sought to determine whether there were in fact differences in luxury uptake ‘abilities’ between species. In Chapter 3, this is explored through the use of genetic database searches, biochemical assays, and protein modelling. Objective 2 examined the effects of environmental factors known to influence luxury uptake. In Chapter 4, the responses of the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris to P repletion were studied in a range of conditions to identify similarities and differences in environmental influences. Chapter 5 sought to determine whether the observed differences could be due to responses at the genetic level, by comparing the gene expression levels of P-related genes in C. reinhardtii under selected sets of conditions from the previous chapter. An additional experiment was conducted, to examine gene expression without inducing ‘noise’ through changes in growth conditions, and this is discussed in Chapter 6. Using protein sequence homology searches, phylogenetic tree generation, protein structure modelling, and biochemical assays (using the chlorophytes C. reinhardtii, C. vulgaris, Desmodesmus cf. armatus, Gonium pectorale, Pediastrum boryanum, and the cyanobacterium Microcystis aeruginosa), it was shown that the ability to store P as polyP is common among microalgae, as implied by the broad conservation of the polyP polymerase VTC4, but luxury uptake abilities vary between species. All six tested microalgae responded to P addition following a period of P depletion by accumulating P as granular polyP. Under the conditions tested, the total P assimilated over 24 hours was similar for five of the microalgae tested (2.6 – 3.6% P by dry weight) but C. vulgaris assimilated considerably less P (~1.2% P) than the others. The effects of environmental conditions on P uptake and polyP accumulation were assessed by triggering luxury uptake in C. vulgaris and C. reinhardtii in different conditions of light supply, temperature, and pH, with different P repletion doses following different P depletion times. P uptake and polyP accumulation were influenced by light supply, P depletion time, and P repletion dose in both microalgae but P dose had the strongest influence in C. reinhardtii versus light supply in C. vulgaris. PolyP was still accumulated by these two species in conditions suppressing growth and severely repressing metabolism (10 °C and darkness), evidencing that P uptake and polyP synthesis do not require light energy. The model alga C. reinhardtii was then used to evaluate, for the first time, whether the differences in P uptake and polyP accumulation observed with respect to differences in environmental conditions were associated with differences in gene expression. Although the genes assessed were downregulated (relative to controls) 24 hours after P repletion, as expected, in all experimental conditions, changing conditions at the start of the experiment also caused changes in gene expression in controls, making it hard to distinguish responses to P repletion from ‘global’ responses to changing conditions. Another experiment was therefore performed where temperature and light intensity were maintained constant before and after P repletion. The results confirmed that increased P repletion dose and P depletion time were associated with increased P uptake and polyP accumulation over 24 hours. The results evidenced the expected downregulation of PSR1, VTC4, VTCX, and PTB5 after 1 hour of P repletion, but this response was much more salient after 7 days of P depletion (compared to 3 days). Changes in gene expression were also associated with P repletion dose, but only after 7 days of P depletion. This showed that the response to P repletion is stronger after a longer P depletion time but the observed expression changes did not support the hypothesis that these changes were the reason for the higher observed P uptake and polyP accumulation. For the first time, it has been systematically shown that the ability to accumulate P as polyP is widespread among microalgae but that the kinetics of P accumulation vary between species and this species-dependence is influenced by environmental factors. These factors engender differences at the level of gene expression, involving both components of the VTC complex and phosphate transporters. However, the differences in P uptake and polyP accumulation may be better understood by investigating the structural differences and changes in activity of relevant proteins.
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    Investigations into the uptake and effects of long-term cadmium and arsenic exposure on the earthworm Eisenia fetida : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Wellington, New Zealand
    (Massey University, 2020) Dharmadasa, Lokugama
    Cadmium (Cd) and arsenic (As) are trace elements that differ in their chemistries but are both highly toxic, and common soil contaminants in agricultural land and contaminated sites. Their potential impacts range from adverse effects on soil-dwelling organisms to uptake into food and leading to human exposures. This study examines the uptake and effects of Cd and As individually and in mixtures primarily on the earthworm Eisenia fetida on up to three consecutive generations, and the potential for recovery when exposure ceases. Exposure ranges were selected to minimise mortality and permit reproduction. Key variables examined were contaminant concentrations and associated trace elements in worm tissue, growth, reproduction, and levels of gene expression. In worms exposed to Cd-spiked soils, Cd accumulation was rapid. Three key factors determining [Cd] in worm tissue were exposure level, time, and the bioconcentration factor (BCF), which increased with decreasing soil [Cd]. Results indicate that for all exposure conditions, and given enough time, Cd accumulation will continue until a lethal tissue level is reached. This point may be either before or after reproduction has occurred (depending on circumstances), but indicates a need to re-examine standardised approaches to toxicity testing for cumulative and biologically persistent contaminants such as Cd. The biological half-life for Cd loss was 6.5 months. This implies that worms that have been exposed to elevated Cd for more than a few weeks would unable to eliminate much of the accumulated burden over their normal lifetimes. Worms exposed to Cd took longer to reach sexual maturation, and at higher exposures, cocoon production progressively decreased from generation to generation. However, there were differences depending on the exposure level. At the lowest level (30 mg/kg), first generation worms returned to clean soil showed a large rebound effect, and by the third generation there was a recovery in cocoon production. By contrast, for higher (90 and 270 mg/kg) and longer (56 d and 84 d) exposures worms performed more poorly, suggesting that there is a tissue Cd threshold beyond which recovery becomes challenging. Evidence from gene expression results are consistent with the idea that this threshold corresponds to a point at which the Cd-sequestering protein metallothionein (MT) has reached saturation, as can also occur in human kidney tissue. Below this point, worms transferred to clean soils will recover. Above it, they will not. As (spiked as arsenate, AsO₄³⁻) also accumulated in worm tissue with exposure concentration and time, but showed some distinct differences compared to Cd. Modest As exposure extended for longer than 28 d had the unusual effect of stimulating growth and causing excessive cocoon production, an effect likely to be missed in most standardised tests. The effects are not thought to be related to parasite suppression, because they were accompanied by large-scale changes to gene expression. Despite appearing beneficial, by the second generation it was clear that effects of the As exposure were overwhelmingly negative, both in terms of extremely low survival rates and the delayed growth of surviving earthworms. Perhaps more notably, results for both the lower exposure condition (10 mg/kg soil As) and As-exposed worms returned to clean soils, suggest there are circumstances where As may promote its own uptake in a positive feedback loop. If correct such an effect may be linked to an increase in uptake of phosphate (PO₄³⁻) for cellular repair, with co-uptake of arsenate (AsO₄³⁻, which is isomorphous). A parallel mechanism is known for marine fish. Remarkably, results suggest that Cd exposure may have also caused an increase in As uptake, from soils that contained only natural [As]. Though a tentative finding, such an effect would be consistent with the idea that any contaminant that causes cellular damage in an invertebrate may trigger a need for more soil phosphate, presumably with some As co-uptake. This would also imply that many (presumed) single contaminant exposures whether in the laboratory or the field may in fact be As co-exposures. Relevant to this, the adverse impacts of As and Cd co-exposure were found to be more severe than effects of exposure to either contaminant alone; despite the fact the lower amounts of each contaminant were taken up under the co-exposure condition. This result supports an argument that soil guideline values derived from single contaminant toxicity experiments may be insufficiently protective for soil invertebrates in many real-life settings. Gene expression results were useful as an interpretive tool, with numbers and overlaps of differentially expressed genes being more useful than knowledge of the subset of named genes and their putative functions. Exposure to Cd or/and As triggered large-scale changes in gene expression, indicating ‘organism-wide’ biochemical responses and providing circumstantial evidence that supported particular interpretations, such as existence of an MT saturation-threshold, and the existence of substantive biochemical changes between lower and higher As exposures. Analysis of differentially expressed genes in common between Cd-only, As-only and co-exposure suggests existence of both similar and different impacts of toxicity under the three conditions.
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    Use of small angle x-ray scattering in investigations of leather and the cornea : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Kelly, S. J. R.
    Collagen is the most abundant protein in the body and the major structural component of skin and the cornea, where it provides strength and is an important physical and chemical barrier against the environment. The biological function of collagen lies predominantly in its mechanical properties where its structural arrangement greatly influences the tissue characteristics. Understanding collagen structure, its properties and how these are affected by processing, is essential for the manufacture of skin products with superior function and when considering collagen in abnormal corneal tissue. Leather is derived from skins of various animals, providing aesthetically pleasing products that are strong and hard wearing because of their collagen structure. Collagen is comprised of fibrils which have been studied here in leather produced from skins of ovine (sheep), bovine (cattle) and cervine (deer) origins. Small angle X-ray scattering (SAXS) was used to evaluate the collagen fibril structure and alignment in leather, processed normally and by stretch-tanning, along with tear and bend testing. The average collagen fibril direction at standard sampling points in all species was perpendicular to the backbone, with the average fibril orientation relative to the backbone being 44° in cervine, 66° in bovine and 79° in ovine. The orientation index (OI) suggests the relative alignment of the fibrils, where 1 is perfectly aligned and 0 is randomly aligned. The OI was lowest in cervine (0.24), suggesting a more mesh-like arrangement, increasing in bovine (0.38) and highest in ovine (0.44) where fibrils lay more parallel to one another. There was considerable and unpredictable variability in collagen arrangements in each species but a significant difference in tear strength with ovine leather (21 N/mm) being weakest, and cervine leather (53 N/mm) stronger than bovine leather (43 N/mm), making ovine leather not suitable for high value applications like footwear. Previous correlations between leather strength and fibril alignment suggest greater alignment led to greater strength. When fibrils were aligned artificially by stretch-tanning, the OI in ovine leather increased from 0.48 to 0.79 as did the strength from 27 to 43 N/mm, making it comparable to bovine leather strength. Measurements of the bend modulus of stretch-tanned ovine leather, which was stiffer than the non-stretch tanned leather (15 vs. 34 kPa), when conditioned under increasing relative humidity environments, during which water was incorporated into leather’s collagen structure, resulted in a 66% reduction in stiffness. Examination of clinically normal sheep corneas were used to determine effects of common preservatives on collagen structures using SAXS. Compared to the control, frozen cornea, there was a significant increase in the fibril diameter and D-spacing of collagen in corneas stored for 5 days in all the preservatives studied (5% glutaraldehyde, 10% formalin, Triton X and phosphate buffered saline). Corneas from cats with corneal opacities (Florida spots) that were studied using histology, transmission electron microscopy (TEM) and SAXS showed that there was less collagen in the stroma of the lesions. Here collagen fibrils had larger and more variable diameters (32 nm vs. the normal 27 nm), and a greater relative alignment (OI) compared to normal corneas (0.43 vs. 0.29, respectively). These changes explain the opacity of the lesions as corneal transparency depends on regular small fibril diameters which are aligned orthogonally. The above studies have demonstrated the usefulness of SAXS in characterizing collagen in natural, pathological, and mechanically and chemically altered collagen-based samples.