Zoology - Theses
Permanent URI for this collection
Search Results
1 - 4 of 4
-
ItemBiochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research( 2018)All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry.
-
ItemNovel gene therapy for the treatment of diabetes-induced heart failure( 2018)People with diabetes are at risk of developing myocardial abnormalities known as diabetic cardiomyopathy. This is characterised by diabetes-induced left-ventricular (LV) impairment, which develops independent of hypertension, coronary artery or valvular heart disease, leading to an increased risk of heart failure. To date, there is still no effective or specific treatment for diabetic cardiomyopathy. Hence, the overall aim of my thesis was to investigate the therapeutic potential of targeting two distinct novel pathways, the phosphoinositide 3-kinase (PI3K)p110α axis and the hexosamine biosynthesis pathway (HBP)/O-GlcNAcylation, in the setting of diabetic cardiomyopathy. PI3K(p110α) is a lipid kinase that regulates several physiological functions, including membrane trafficking, adhesion, actin rearrangement, cell growth, and survival. Recent findings from our laboratory and others have highlighted that PI3K(p110α) is cardioprotective in a range of different cardiac pathologies. In Chapter 3, I investigated whether cardiac-directed PI3K(p110α) gene therapy ameliorates diabetic cardiomyopathy in a mouse model of type-1 diabetes (T1D) in vivo. I revealed that administration of recombinant adeno-associated virus-6 constitutively-active PI3K(p110α) (rAAV6-caPI3K) attenuated diabetic cardiomyopathy, even when administered after the initial manifestation of LV diastolic dysfunction. I then proceeded to investigate the cardioprotective effects of rAAV6-caPI3K in the more clinically-prevalent type-2 diabetes (T2D) setting. In Chapter 4, I elucidated that restoration of cardiac PI3K(p110α) activity, through the administration of rAAV6-caPI3K gene therapy, attenuates T2D-induced cardiomyopathy, together with limiting ROS generation. In comparison to the cardioprotective nature of PI3K(p110α), the HBP and subsequent protein O-GlcNAcylation have been implicated in the development of diabetic cardiomyopathy. The generation of β-N-acetylglucosamine (O-GlcNAc) from HBP is a substrate for the post-translational protein modification (PTM) called O-GlcNAcylation. Two specific enzymes regulate the addition and removal of O-GlcNAc modification; O-GlcNAc transferase (OGT) catalyses the addition of GlcNAc to proteins and O-GlcNAc-ase (OGA) facilitates its removal. In Chapter 5, I aimed to elucidate the effect of cardiac manipulation of O-GlcNAcylation in the setting of diabetes-induced cardiac dysfunction in vivo. I demonstrated here that a cardiac-selective increase in OGT (via rAAV6-OGT gene delivery), the enzyme responsible for O-GlcNAcylation, is sufficient to drive cardiac dysfunction and remodelling, resembling that seen in diabetic cardiomyopathy. In contrast, increasing cardiac OGA (via rAAV6-OGA gene delivery), the enzyme responsible for the removal of the O-GlcNAc moiety, attenuates several characteristics of diabetic cardiomyopathy, likely at least in part through the improvement of mitochondrial function. Finally, in Chapter 6 I investigated the impact of O-GlcNAcylation on the PI3K(p110α), pathway and the effect of PI3K(p110α) gene therapy on HBP signalling. I elucidated that PI3K(p110α) can negatively regulate consequences of the HBP and O-GlcNAcylation as part of its cardioprotective actions in diabetic cardiomyopathy, while HBP/O-GlcNAcylation inhibits PI3K(p110α)-mediated signalling, likely contributing to the ability for this pathway to exert cardiac impairments. In conclusion, data from this thesis reveal that gene therapies targeting PI3K(p110α) and HBP/O-GlcNAcylation are viable therapeutic targets for diabetic cardiomyopathy. These results hence provide a basis for pursuing gene therapy for the treatment of diabetes-induced heart failure.
-
ItemTaxonomy, ecology and conservation genomics of North-Eastern Australian Earless Dragons (Agamidae: Tympanocryptis spp.)( 2018)Land clearing and modification of natural habitats is threatening biodiversity globally. In Australia, most native grassland habitats have been heavily modified for agriculture, including cropping and grazing. Grassland specialist species, including earless dragon lizards (Tympanocryptis spp.) in north-eastern Australia, are of conservation concern due to this continued habitat loss and fragmentation. However, the north-eastern Australian group of earless dragons (including the recently described T. condaminensis, T. wilsoni and T. pentalineata) are at significant risk, due to the presence of multiple undescribed cryptic Tympanocryptis lineages within this region. It is imperative that the taxonomy is resolved for these cryptic lineages of conservation concern, so conservation of these species may occur. One of the major challenges for taxonomists in recent times has been the species delimitation of morphologically cryptic taxa. The detection of distinct molecular lineages within cryptic genera has increased exponentially over the past decades with advances in genetic techniques. However, there are discrepancies in the rate and success of detection of cryptic taxa between studies using genetic methods and those using classic external morphology analyses. Therefore, novel integrative methods for species delimitation of cryptic taxa provide an avenue to incorporate multiple lines of evidence, including the application of osteological variation assessment where external morphological assessment fails to distinguish species. I develop a new pipeline integrating genomic data using single nucleotide polymorphisms (SNPs) and osteological geometric morphometric evidence from micro X-ray computed tomography (CT) imagery to assess variation between cryptic lineages for confident species delimitation. Here, I use this novel integrative pipeline to delimit cryptic lineages of earless dragons in north-eastern Australia. Prior to this study, there was evidence of three undescribed species of Tympanocryptis in this region. Using single mitochondrial and nuclear genes along with >8500 SNPs, I assess the evolutionary independence of the three target lineages and several closely related species. I then integrate these phylogenomic data with osteological cranial variation from CT imagery between lineages. I find that the very high levels of genomic differentiation between the three target lineages is also supported by significant osteological differences between lineages. By incorporating multiple lines of evidence for species delimitation, I provide strong support that the three cryptic lineages of Tympanocryptis in north-eastern Australia warrant taxonomic review. Earless dragons are found in most environments across the Australian continent, including a variety of ecological niches, from stony desert to tropical woodland or cracking clay savannah, although each species is often restricted to s certain habitat-type. I investigate the phylogenetic relationships among currently described earless dragons and newly delimited putative species with an assessment of broad biogeographic divisions, focussing on the north-eastern Australian Tympanocryptis group. I found significant structure across the north-eastern Australian lineages, with deep divergence between lineages occurring in the inland Great Artesian Basin region and more coastal Great Dividing Range. Regional diversification is estimated to have occurred in the late Miocene with subsequent Plio-Pleistocene speciations, and divergence and distributions of these species may therefore be reflective of the climate induced grassland-rainforest oscillations during this time. Based on these phylogenetic geographic relationships and the species delimitation from the integrative taxonomy approach, I describe three new species of Tympanocryptis from the cracking clay grasslands of the Darling Riverine Basin (T. darlingensis sp. nov.) and Queensland Central Highlands (T. hobsoni sp. nov.), and the stony open eucalypt woodlands on the Einasleigh Uplands (T. einasleighensis sp. nov.). The revision of these species provides further taxonomic clarity within the Tympanocryptis genus, and is an imperative step in the conservation of the north-eastern Australian earless dragons. These three putative Tympanocryptis species and the other three recently described earless dragons in north-eastern Australia inhabit restricted niches and areas with varying levels of habitat fragmentation and modification, and are therefore of significant conservation concern. However, little is known about these six north-eastern Australian earless dragon species. I utilise genomic methods to investigate population connectivity and genetic structure to determine management units. I then use species distribution modelling (SDM) to assess habitat suitability and fragmentation of each species. I integrate results of these analyses to form conclusions on the distribution and population structure of these earless dragons. I then discuss the major threatening processes and potential conservation strategies. This thesis uses several integrative approaches in resolving the taxonomy and forming conclusions on the conservation management of the north-eastern Australian Tympanocryptis species. This study successfully delimits cryptic lineages, explores the phylogenetic and geographic relationships between species, and provides baseline population genomics and ecological data to be used for conservation assessments and management decisions of earless dragons in north-eastern Australia.
-
ItemBreeding biology of the platypus (Ornithorhynchus anatinus)( 2018)This thesis examines the different behavioural stages of the reproductive cycle in the platypus, Ornithorhynchus anatinus, the time and energy investment of the female in breeding, and use of burrows by wild juveniles during the period after they first emerge. Many aspects of platypus reproduction are poorly understood due to their cryptic, nocturnal, semi-fossorial and semi-aquatic behaviour, which makes studies in the wild difficult. I studied a group of captive platypuses at Healesville Sanctuary and newly emerged juveniles from the wild population within Badger Creek, Victoria. My aims were to examine prey selection and seasonal energy intake, quantify and describe courtship, mating, nesting behaviour and maternal care given to nestlings, and describe how juvenile platypuses use the habitat in their natal home range. In captivity, platypuses consumed the fewest kilojoules during the breeding season and most kilojoules during the post-breeding breeding season. They showed a preference for less-mobile prey (mealworms, earthworms and fly pupae). Crayfish formed the largest quantity of food in the diet and was highly nutritious for energy (kJ), vitamins and minerals. The platypus diet was influenced by nutritional content, the stage of the breeding season and the behaviour of the prey species. Female platypuses controlled breeding encounters with males via three strategies; avoidance, by having lower activity levels and changing their activity pattern to partially diurnal; flight, by leaving the area immediately upon encountering the male; and resistance, terminating breeding encounters with the male and using a non-contact courtship behaviour prior to contact courtship behaviours. These strategies are likely to protect females from injury and coercion. After mating, females invested 8 ± 1.5 hours over 3 nights collecting wet vegetation for their nesting burrow. The morphology of burrows varied each year, but contained the same structural features: narrow tunnels, dead ends, ‘pugs’ of backfilled earth and multiple entrances that lead to a nesting chamber containing a spherical vegetation nest. The female’s energy intake increased to twice that of a non-lactating female in the final month of lactation, indicating the high cost of milk production. The length of lactation dependence for platypus nestlings was 128 ± 1 days. Females spent less time in the nest with twins compared with a single nestling. I developed an infra-red camera technique which allowed platypus nestling behaviour, growth and development to be observed in their burrow. Weaning occurred as an instantaneous event when the nestlings emerged into the water. Newly emerged wild juvenile platypuses each used multiple burrows for single or multiple nights within their natal home range. There was no significant correlation with vegetation communities along the bank at burrow sites, indicating burrow site selection was not driven by vegetation structure. No juveniles dispersed, suggesting they persist in the natal home range until the sub-adult stage which may assist their survival as they develop their skills and complete their growth in high quality habitat. My study demonstrates that female platypuses invest a high amount of time and energy in breeding, from avoiding the male platypus, through courtship and mating, creating the nesting burrow, maternal care during lactation, and while juveniles persist in her home range after weaning. I have provided captive management recommendations based on my research to advance the animal welfare and captive breeding success of the platypus.