Dissertations / Theses on the topic 'Monotremes'

In this thesis I utilize the recently sequenced genomes of the South American grey short-tailed opossum (Monodelphis domestica), tammar wallaby (Macropus eugeniz') and the platypus (Ornithorhynchus anatinus) to identify and characterize immune genes in order to fill in the gaps in our understanding of evolution of the immune systems in non-eutherian mammals. Many of these genes have proved elusive to identify using conventional lab strategies and automated genome annotation pipelines. I discovered divergent immune genes using bioinformatic protocols that I developed and compiled this sequence information in a publicly available database. I examined species—specific expansions of major immune gene clusters. Using these genes, I developed a comprehensive marsupial immune gene set which is used to compare the expression profiles of the two tammar wallaby thymuses to gain insights into the functional roles of these organs. The availability of these immune sequences allows for analysis of large-scale expression studies and development of marsupial- and monotremespecific reagents.

This thesis began as an investigation into evolution of the platypus family (Ornithorhynchidae, Monotremata), now known from both Australia and South America. The thesis broadened its scope with inclusion of non-ornithorhynchid Mesozoic monotremes from Lightning Ridge, NSW. This change in direction brought an unexpected result: a fossil mammal from Lightning Ridge investigated for this thesis (presumed to be monotreme: Flannery et al., 1995) appears to be a new and unique type of mammal. Specimens were procured through Queensland Museum (Riversleigh material); Australian Museum (Lightning Ridge material); and Museum of Victoria and the South Australian Museum (fossil ornithorhynchids). Specimens were examined under a light microscope and scanning electron microscope; specimens were photographed using light photography and a scanning electron microscope; and illustrations and reconstructions were done with a camera lucida microscope attachment and photographic references. Parsimony analysis utilised the computer programs PAUP and MacClade. Major conclusions: 1) analysis and reconstruction of the skull of the Miocene platypus Obdurodon dicksoni suggest this robust, large-billed platypus was a derived northern offshoot off the main line of ornithorhynchid evolution; 2) the well-preserved skull of Obdurodon dicksoni shows aspects of soft anatomy previously unknown for fossil ornithorhynchids; 3) two upper molars from Mammalon Hill (Etadunna Formation, late Oligocene, central Australia) represent a third species of Obdurodon; 4) the South American ornithorhynchid Monotrematum sudamericanum from the Paleocene of Argentina is very close in form to the Oligocene-Miocene Obdurodon species from Australia and should be considered congeneric; 5) a revised diagnosis of the lower jaw of the Early Cretaceous monotreme Steropodon galmani includes the presence of two previously undescribed archaic features: the probable presence of postdentary bones and a meckelian groove; 6) morphological evidence is presented supporting a separate family Steropodontidae; and 7) analysis of new fossil material for Kollikodon ritchiei suggests that this taxon is not a monotreme mammal as originally identified but is a basal mammal with close relationships to allotherian mammals (Morganucodonta; Haramiyida). Kollikodon is provisionally placed as basal allotherian mammal (Allotheria sensu Butler 2000) and is unique at the ordinal level, being neither haramiyid nor multituberculate. A new allotherian order ??? Kollikodonta ??? is proposed.

Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X-chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals.¶ In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings.¶ The first paper reports a study of the imprinted locus responsible for the human Prader-Willi and Angelman syndromes (PWS and AS). A search for kangaroo and platypus orthologues of PWS-AS genes identified only the putative AS gene UBE3A, and showed it was in a completely different genomic context to that of humans and mice. The only PWS gene found in marsupials (SNRPN) was located in tandem with its ancient paralogue SNRPB, on a different chromosome to UBE3A. Monotremes apparently have no orthologue of SNRPN. The several intronless genes of the PWS-AS domain also have no orthologues in marsupials or monotremes or non-mammal vertebrates, but all have close paralogues scattered about the genome from which they evidently retrotransposed. UBE3A in marsupials and monotremes, and SNRPN in marsupials were found to be expressed from both alleles, so are not imprinted. Thus, the PWA-AS imprinted domain was assembled from many non-imprinted components relatively recently, demonstrating that the evolution of imprinting has been an ongoing process during mammalian radiation.¶ In the second paper, I examine the evolution of the X-inactivation centre, the key regulatory region responsible for X-chromosome inactivation in humans and mice, which is imprinted in mouse extraembryonic membranes. By sequencing and aligning flanking regions across the three mammal groups and non-mammal vertebrates, I discovered that the region homologous to the X-inactivation centre, though intact in birds and frogs, was disrupted independently in marsupial and monotreme mammals. I showed that the key regulatory RNA of this locus (X-inactive specific transcript or XIST) is absent, explaining why a decade-long search for marsupial XIST was unsuccessful. Thus, XIST is eutherian-specific and is therefore not a basic requirement for X-chromosome inactivation in all mammals.¶ The broader significance of the findings reported in these two papers is explored with respect to other current work regarding the evolution and construction of imprinted loci in mammals in the form of a review. This comparison enabled me to conclude that like the PWS-AS domain and the X-inactivation centre, many domains show unexpected construction from disparate genomic elements that correlate with their acquisition of imprinting.¶ The fourth and last paper examines the evolution of CCCTC-binding Factor (CTCF) and its parologue Brother Of Regulator of Imprinted Sites (BORIS) which contribute to the establishment and interpretation of genomic imprinting at the Insulin-Like Growth Factor 2/H19 locus. In this paper I show that the duplication of CTCF giving rise to BORIS occurred much earlier than previously recognised, and demonstrate that a major change in BORIS expression (restriction to the germline) occurred in concert with the evolution of genomic imprinting. The papers that form the bulk of this thesis show that the evolution of epigenetic traits such as genomic imprinting and X-chromosome inactivation is labile and has apparently responded rapidly to different selective pressures during the independent evolution of the three mammal groups. I have introduced these papers, and discussed them generally in terms of current theories of how and why these forms of monoallelic expression have evolved in mammals.

The rise in antimicrobial resistance and paucity of new antimicrobial compounds calls for alternatives to traditional antibiotics. Antimicrobial peptides (AMPs) have emerged as potential candidates. Cathelicidins are a major family of AMPs in mammals which form part of innate immunity through antimicrobial and immunomodulatory functions. Marsupial and monotreme cathelicidins are of particular interest due to their involvement in protecting immunologically naive young during development in the pouch via expression in the pouch lining and milk where they modulate microbial flora and provide passive immunity. As such, the cathelicidin gene family has expanded in marsupials and monotremes, with a high number of cathelicidins in the tammar wallaby, gray short-tailed opossum and platypus. However our knowledge is limited to these species and functional studies involving antimicrobial activity are lacking. This thesis describes the characterisation of cathelicidins in the Tasmanian devil, koala and echidna, and investigates the antimicrobial function of all marsupial and monotreme cathelicidins. As expected, cathelicidins have expanded in the Tasmanian devil and koala, resulting in a high number of cathelicidins which were widely expressed throughout the body, including in pouch lining and milk. Only a single cathelicidin was identified in the echidna due to the quality of the genome. Out of 26 cathelicidins tested, six displayed broad-spectrum antibacterial activity against gram-negative and positive bacteria, including methicillin-resistant Staphylococcus aureus. One koala cathelicidin rapidly inactivated C. pecorum and significantly reduced the number of chlamydial inclusions in vitro. Activity was reduced in the presence of serum and whole blood, and peptides displayed varying levels of haemolytic and cytotoxic activity. Many cathelicidins did not display antimicrobial activity and future work is required to explore their potential immunomodulatory properties. The results presented in this thesis have advanced our understanding of cathelicidins in marsupials and monotremes on a genetic and functional level, and highlights their potential as novel therapeutics in the future.

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Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.A)--University of Adelaide, Dept. of Anatomical Sciences, 1998?

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The evolutionary relationships of the beta-like globin genes were studied by applying maximum parsimony methods to aligned DNA sequences.
Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998?

Genetic sex determination systems are generally based on the presence of differentiated sex chromosomes. Birds have a ZZ/ZW sex chromosome system in which males are ZZ and females ZW, whereas mammals have an XX/XY system with males being XY and females XX. Monotremes have an extraordinary sex chromosome system that consists of multiple sex chromosomes: 5X5Y in platypus and 5X4Y in echidna. Intriguingly, the monotreme sex chromosomes show extensive homology to the bird ZW and not to the therian XY. However, sex determination in monotremes is still a mystery; the Y-specific Sry gene that triggers male sex determination in therian mammals is absent and so far very few genes have been identified on Y chromosomes in monotremes. To gain more insights into the gene content of Y-chromosomes and to identify potential sex determination genes in the platypus a collaborative large scale transcriptomic approach led to the identification of new male specific genes including the anti-Muellerian hormone AMH that I mapped to Y₅, this makes Amhy an exciting new candidate for sex determination in monotremes. Platypus chromosome 6 is largely homologous to the therian X and therefore it represents the therian proto sex chromosome. In addition, this autosome features a large heteromorphic nucleolus organizer region (NOR) and associates with the sex chromosomes during male meiosis (Casey and Daish personal communication). I investigated chromosome 6 heteromorphism in both sexes and found a number of sex-specific characteristics related to the extent of the NOR heteromorphism, DNA methylation, silver staining patterns and interestingly, meiotic segregation bias. This raises the possibility that chromosome 6 may have commenced differentiation prior to monotreme therian divergence. These results led me to investigate the chromosome 6 borne gene Sox3, from which Sry evolved in therian mammals. This revealed a platypus male-specific Sox3 allele, which differs from the alleles observed also in females on the length of one of the Sox3 polyalanine tracts. This raises the possibility that Sox3 may be working differently in males and females. We have used our unique knowledge of monotreme sex chromosomes to determine the sex of captively bred echidnas. I used a PCR based genetic sexing technique that utilizes DNA from small hair samples and primers that amplify male-specific genes. Interestingly, I found that seven out of eight echidnas born in captivity were females. Furthermore, I found a Sox3 deletion in the only male echidna born in captivity. This gives us the unique opportunity to investigate the sexual development of an animal in which this gene is naturally deleted providing an exceptional situation in which to study monotreme sex determination. Furthermore, this sexing technique has the potential of being applied in the wild to investigate sex ratio in natural populations of monotremes, including the critically endangered long-beaked echidna.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2015

Monoallelic expression and replication timing are closely linked fundamental aspects of genome biology, yet their evolutionary trajectory has not been investigated in much detail. The monoallelic expression status of imprinted genes observed in therian species has previously not been found in the earlier-diverged monotreme mammals, or in birds, when measured using molecular techniques. Furthermore, the observation that eutherian imprinted and X-borne genes asynchronously replicate was traditionally thought to be linked to the dissimilar epigenetic states that existed at each allele controlling monoallelic expression. In this study, we use a combination of cytogenetic and molecular techniques to assess the replication status of sex chromosome genes in the platypus and chicken, as well as the replication status and expression pattern of platypus imprinted orthologs. We find that asynchronous replication does occur at specific sex chromosome loci in platypus and chicken, although in chicken the amount of asynchronous replication changes over development. Furthermore, differential chromatin compaction is observed in platypus sex chromosomes, a characteristic observed in therian X-inactivation, suggesting that both asynchronous replication and chromatin compaction are features characteristic of amniote sex chromosomes. Asynchronous replication and monoallelic expression is observed at platypus imprinted orthologs, indicating that a ‘pre-imprinted’ status is observed at these genes in non-therian amniote species. These results show that monoallelic expression predates imprinting at these loci, suggesting that the partial monoallelic expression observed in monotreme mammals has evolved in therian mammals to become parentally-inherited imprinted expression.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Molecular and Biomedical Science, 2015.

Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X-chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals.¶ In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings.¶ ...

The duck-billed platypus and the short-beaked echidna represent the most basal lineage of living mammals and therefore provide important information about mammalian evolution. Monotremes have also undergone remarkable anatomical, physiological and genetic changes. One of the most radical changes involves the monotreme digestive system and genes associated with metabolic control. I have investigated several genes that are vital for metabolic control (specifically genes in the ghrelin and incretin pathways) as well as the histology of the monotreme pancreas. Given the glandless gut in monotremes, I first sought to investigate genes in the appetite regulating ghrelin pathway. Surprisingly, I discovered that genes encoding ghrelin and ghrelin O-acyl transferase (GOAT) are missing in the platypus and echidna genome, whilst, its receptor, growth hormone secretagogue receptor 1a (GHS-R 1a) is present. This is the first report suggesting the loss of ghrelin in a mammal. The conservation of the ghrelin receptor gene despite the lack of the ghrelin and GOAT genes in platypus suggests that another ligand maybe acting via this receptor in monotremes (Chapter 2). Ghrelin is expressed in human pancreatic ε-cells. Hence the lack of ghrelin in platypus led us to investigate in more detail the structure of the monotreme pancreas, another key organ in both metabolic control and digestion (Chapter 3). Generally, the monotreme pancreata share the basic characteristics of other mammalian pancreata, including both endocrine islets and exocrine acini. I performed immunohistochemical analysis to reveal the detailed architecture of the platypus and echidna endocrine islets of Langerhans. The unique phenotypes of the PP-lobe, smaller size of islets, and the abundance of α-cells indicate the monotreme pancreata have more resemblance to that of birds and marsupials than eutherian species. One of the key functions of the pancreas is to release insulin upon food intake. Glucagon-like peptide 1 (GLP-1), a hormone released from the small intestine upon food intake, triggers insulin release via the GLP-1 receptor (GLP-1R) in β-cells of the pancreas. In human, GLP-1 is rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4) and thereby has a very short serum half-life (<2 min). Searching for longacting GLP-1 analogs to improve insulin sensitivity has been a key strategy in Type 2 diabetes (T2D) treatment. We identified and characterised Glp-1, Glp-1r and Dpp-4 and found both Glp-1 and Dpp-4 are expressed in gut and pancreas as expected, and interestingly also in venom. Importantly, evolutionary changes in monotreme GLP-1 sequences led us to predict that it would be resistant to enzymatic degradation. Extensive biochemical analysis of monotreme GLP-1 revealed that this variant is in fact resistant to DDP-4 degradation, however can be degraded by other enzymes (trypsinlike) in their own sera. Moreover, we demonstrated that monotreme GLP-1s can bind and activate both pGLP-1R and hGLP-1R with similar potency and stimulate insulin release in isolated mouse islets (Chapter 4). Together this work provides fundamental new insights into the molecular and anatomical characteristics of the monotreme digestive system, the evolution of metabolic control and potential novel avenues for diabetes treatment based on monotreme GLP-1.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 2015.

Lactation is a specific mammalian adaptation. Milk is the principal source of nutrition for the newborn mammal. Both its needs and the physiology of lactation vary among the different mammalian species. These factors are reflected in the differing milk compositions, in particular the protein compositions. Thus, comparative studies of the protein composition of eutherian, metatherian and prototherian mammals are of great importance in their own right. Furthermore, these proteins exhibit an extraordinary array of conformations and protein-protein interactions. Thus, their study will also throw light on basic aspects of protein structure and physiological function. This thesis is concerned with a study of the milk proteins of the egg-laying mammals (monotremes), the echidna and platypus. At the time of commencement of this study little was known of the protein composition of their milk. Thus, it was necessary to establish initially the overall protein composition of the limited number and volume of milk samples available. This was achieved by electrophoretic and immunological studies, fractionation of the proteins and determination of certain properties, e.g. molecular weight, amino acid composition and partial amino acid sequence. Efforts were concentrated on the whey proteins and where possible they were identified in terms of known proteins, the bovine whey proteins being used as a point of reference. In addition, two groups of whey proteins were chosen for more intensive study: the lysozymes and iron (III) binding proteins. The main echidna whey proteins have been resolved into eleven bands on starch gel electrophoresis at pH 7.G. These were designated A through K on the basis of decreasing electrophoretic mobility. Nine bands, designated 1 through 9, have been resolved in a sodium dodecyl sulphate-gradient polyacrylamide gel system. The relationship between the bands of the two gel systems has been determined and proteins corresponding to Bands A, B, C, E, G-J have been isolated. Band A protein has an apparent molecular weight of ca. 17,800 and appears to have no counterpart in echidna blood serum. Twenty-five residues of the TV-terminal sequence have been determined. It bears no simple relationship to any known protein, but has ca. 30% homology with an acidic whey protein precursor found in rat and mouse milk. Band B protein has an apparent molecular weight of ca. 55,000 and a counterpart is not detected in the blood serum. The sequence of the first fifteen TV-terminal residues was determined. Sequence homology with any known protein is not evident. Band C protein occurs in the milk and blood and is echidna serum albumin. Both milk and blood protein have the. same electrophoretic mobility and apparent molecular weight. The determination of the TV-terminal sequence indicates 63% homology with human and bovine serum albumin over the first 30 residues. Band E protein occurs only in the milk, and has an apparent molecular weight of ca. 21,000. The sequence of the first 68 TV-terminal residues indicates that it is a novel protein. Bands G-J and a minor band (G ) evident in some milk samples are echidna transferrins (milk). The electrophoretic patterns vary depending on the sample, but all exhibit 3-4 of the bands. Bands H-J are evident in the electrophoretic patterns of the blood serum. The milk bands are identified as transferrins on the basis of their electrophoretic mobilities, ,r,^Fe(III) binding capacity, molecular size and A-terminal sequences. The multiple banding reflects differences in the numbers of sialic acid residues (0-4) present. The principal cathodic electrophoretic band in the milk is lysozyme. Echidna milk is relatively rich in this protein, containing ca. 2 mg mf*. Previous observations that there is a lysozyme, echidna lysozyme 1, occurring'in the milk of Tachyglossus aculeatus rnultiaculeatus and another, echidna lysozyme II, of different electrophoretic mobility at pH 5.3, occurring in Tachyglossus aculeatus aculeatus, have been confirmed. Each enzyme has been isolated and the complete amino acid sequences determined. They are both c-type lysozymes, M 13,988 and 13,861 respectively. They differ in three amino acid residues. Both proteins have a number of unusual features, e.g. some residues considered important in a-lactalbumins for lactose synthase activity are conserved. However, contrary to previous findings in this laboratory, echidna lysozyme I isolated during the course of the present study, does not appear to be active in the lactose synthase system. Furthermore, evidence is obtained for the presence of very low concentrations of a conventional o-lactalbumin. Proposals for further work on this particular problem are presented. Seven electrophoretic bands are observed on starch gel electrophoresis (pH 7.6) of platypus whey proteins. These are designated A through G. Their apparent molecular weight range from 19.000-79,000. Proteins corresponding to Bands A, C, D, F and G have been isolated and preliminary characterization made. Bands F and G proteins are shown to be transferrins. The major blood serum iron (III) binding protein has a similar electrophoretic mobility to Band G of the milk. Platypus arid echidna transferrins exhibit 80% homology over the first 30 A-terminal residues. Only trace levels of lysozyme activity and weak lactose synthase activity were detected in the 18 platypus milk samples examined. The significance of the work in the overall framework of comparative studies of milk proteins is discussed.

Extant Monotremata is an egg-laying mammal Order only found in Australia and New Guinea and is comprised of the genera, Ornithorhynchus, Tachyglossus and Zaglossus. Monotremes have been observed to have a large amount of variation as seen in the number of subspecies and species that were previously recognised for the taxa. This thesis studied the factors that may be responsible for this amount of variation. The thesis was broadly divided into five factors hypothesised to affect the variation of the crania; growth and development, sex differences, climate, phylogeny, and other macroecological and ecological factors. Growth and development were analysed using both 3D geometric morphometrics and the timing of suture closure. In all three monotremes the suture closure shows the same patterning as the general pattern for mammals with the skull closing in order from vault, facial, and cranio-facial. The platypus demonstrated an increase in cranial size and shape related to the increase in cranial length and the two echidnas were found to have an increase in cranial vault height independent of cranial length. The mating strategy can be used to indicate size differences between the sexes, with a polygynous mating system predicted to show sexual size dimorphism and a promiscuous mating system predicted to show reduced sexual size dimorphism. To find any differences between the sexes in the crania, a suite of supervised classification techniques were used and as Ornithrohynchus has a polygynous and Tachyglossus has a promiscuous mating system, the results found a large difference between the sexes in the platypus, with the echidna showing little sexual dimorphism in the cranium. When latitude is taken into account some difference between the sexes in Tachyglossus was found. Unfortunately there were not enough specimens to compare the difference between the sexes in Zaglossus. All three monotremes were found to have a relationship between cranial size (platypus) and cranial vault height (echidnas) with latitude and elevation. The platypus was found to have a positive relationship with latitude and a negative relationship with elevation. Tachyglossus and Zaglossus were found to have a weaker positive relationship of cranial vault height with elevation and latitude. In this thesis I used an unsupervised clustering method to find similarities in bioclimatic data to create bioregions of Australia and New Guinea. These bioregions, as well as the Interim Bioregions of Australia, and an Australian vegetation map, were used as classes for a suite of supervised learning analyses, to find whether these classes predict a change in the morphology of the monotreme cranium. The results show that latitude, elevation, basin, isolation and bioclimatic factors were important factors in predicting the size and shape change in Ornithorhynchus crania. In Tachyglossus and Zaglossus, bioclimatic factors, elevation, latitude, isolation, and vegetation were found to be important. From the preceding work several different morphotypes of Ornithorhynchus, Tachyglossus, and Zaglossus are recognised and the thesis suggests populations that may require taxonomic revision.

The echidna is a solitary, seasonally breeding monotreme mammal with a mating system characterized by high levels of intra-male competition for access to receptive females. Throughout Australia the breeding season follows a period of inactivity which ranges from shallow bouts of torpor to prolonged deep hibernation. In this thesis I investigated how the Tasmanian echidna optimizes its reproduction around an obligatory hibernation period and in the presence of intense sexual conflict. The bradymetabolic (slowing of metabolism) effect of hibernation was exploited by both sexes to optimize their reproduction. I found that testes recrudescence (defined as an increase in testes volume and density) was initiated prior to males entering hibernation, a strategy not seen in any other hibernating mammal. This strategy can be linked to the low energy and density diet and requirement to hibernate to maximise energy-savings, and to the large relative size of echidna testes reflecting a mating system with intense levels of intra-male competition. It took approximately two months at euthermic body temperatures from the initiation of recrudescence in December for echidna testes to reach 75% of peak size. Therefore if testes recrudescence did not occur prior to entering hibernation, hibernation would be restricted to a one and a half month period to allow mating in June. Male echidnas initiated mating activity by locating hibernating females and entering their hibernacula. This strategy was common in my study population and males that remained with a female in her hibernaculum for 13 hours or more gained a copulation opportunity. However, all females that mated or were disturbed by males prior to July 27 re-entered hibernation. This indicates that mating often occurred earlier than optimal for female reproductive success. Many of the females that re-entered hibernation after mating were pregnant. Pregnant females entered hibernation only in early pregnancy: hibernation extended the gestation period and hence allowed females to delay egg-laying. Females timed their reproduction so that they emerged from their 37 day period of nursery burrow confinement as ecosystem productivity increased. Hibernation therefore allows successful reproduction in a population where there is asynchronous timing of optimal mating between males and females. This thesis explores the influence of hibernation on sexual conflict, demonstrates the numerous interactions that can occur between hibernation and reproduction and shows that the bradymetabolic property of hibernation is exploited by both male and female echidnas to optimize reproductive fitness.

The evolutionary history of mammals, when including extinct taxa, is mainly reconstructed using tooth morphology and employs terminology based on non-monotreme mammals. Although adult monotremes are edentulous, juvenile platypuses have teeth that can be compared with extinct monotremes, but terminology can be a barrier to efficient comparison to non-monotreme mammals. Deciduous teeth and thickened epithelial plates of the extant platypus, Ornithorhynchus anatinus, are sparsely figured in the literature. New imagery of those teeth and plates from high-resolution x-ray computed tomography and scanning electron microscopy contribute to the understanding of mammal evolution and the unique morphology of platypus teeth. The teeth of the juveniles are highly variable, but early-forming features (major cusps and transverse valleys) are stable enough for comparison. Transverse lophs on monotreme teeth contain complexity not reflected in cusps alone, unlike therian mammals. These differences reinforce the need for caution when applying dental terminology that originally was produced for therian mammals. New imagery highlights potential phylogenetically informative morphology in the pulp cavity and roots. As the roots of the juvenile teeth degenerate, the epithelium below the teeth thickens into epithelial plates. Structures in the epithelial plates are broadly similar to those found in the keratin plates. New images of the epithelial plates offer insight into a series of tubes concentrated under the juvenile teeth. The tubes are a continuous conduit to the plate surface and may serve a sensory function or result from the ever-growing nature of the epithelial plate.
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Communication plays a key role in coordinating all social interactions in an individual’s life, but is particularly crucial for coordinating reproduction. Although chemical (olfactory) signals are ubiquitous in sexual communication in mammals, a disproportionate reliance on laboratory-based studies using a limited range of species, along with several logistical and methodological limitations, limit our broader understanding of their functions in reproduction and as sexually selected traits. I addressed these shortcomings by using an integrated, multidisciplinary approach, combining organic chemical analyses, physiology, genetics and behaviour, to investigate chemical signals, reproduction and sexual selection in a terrestrial, egg-laying mammal (monotreme), the short-beaked echidna (Tachyglossus aculeatus). Although the monotreme and therian lines have been evolutionarily separate for over 150 million years, echidna scent gland secretions from the spur and cloaca showed a typical mammalian pattern in terms of high chemical diversity and complexity. I identified a large number of compounds of varying volatility, molecular weight, functional groups and aromaticity, suggesting a high reliance on olfactory communication. Similarities with other vertebrates can indicate evolutionary convergence on optimal chemicals as signals, although several obscure and even novel compounds were also identified. Consistent with other seasonal breeders, echidna chemical profiles varied between sexes and during the mating season. Profiles also differed between individuals, suggesting they could contain genetic information, although microsatellite markers were inadequate to confirm this hypothesis. Changes in male spur secretions during the mating season coincided with maximum annual plasma testosterone concentrations and could be sexually selected, functioning in mate choice or intra-sexual competition. Males were attracted to female scent, confirming male response to sex-specific scent differences is an important driver of echidna mating behaviour. Consequently, chemical sensory traits that influence the ability to locate mates seem to be favoured by natural and sexual selection.Behavioural responses to chemical signals can be complicated by sexual conflict, although this has rarely been investigated in mammals. Male echidnas locate and mate with hibernating females; however females showed no changes in chemical profiles during hibernation. These results suggest that females do not ‘actively’ signal to males while hibernating, but intense male-male competition for access to females has probably driven earlier male readiness to breed, even before females might otherwise emerge from hibernation and signal to males. In females, chemical cues and reproductive physiology were not closely linked, as females showed continued mating activity during pregnancy and no detectable changes in chemical profiles at the time of fertilization or during pregnancy. Therefore, female reproductive status appears undisclosed to males, and multi-male mating may function to confuse paternity and reduce infanticide risk. These results suggest chemical signals are used differently by males and females to increase their reproductive success, often at the expense of the other sex, resulting in an evolutionary ‘arms race’ between signalers and receivers. Overall, a complex interplay between chemical signals, behaviour, environmental and selective pressures is responsible for the mating behaviour observed in Tasmanian echidnas. My work highlights the benefit of using a comprehensive, multidisciplinary approach based on a free-living, ‘non-model’ mammalian species, representing a significant step towatrds understanding the influences of selective pressures, including selection and conflict, on animal communication.

Meiosis is a specialised form of cell division that occurs specifically in the gonads of sexually reproducing species. It comprises a round of DNA replication followed by two successive rounds of cell division to produce haploid gametes. Each stage is divided into four substages of prophase, metaphase, anaphase and telophase. Prophase I is the longest and most complex stage of meiosis during which homologous chromosomes pair and recombine. The evolution of heteromorphic sex chromosomes has led to a number of changes in meiotic organisation. This includes the non-pairing of sex specific parts of the heteromorphic sex chromosomes and their inactivation in many species. The platypus has a unique set of 10 sex chromosomes with homology to bird sex chromosomes that exist as a chain during meiotic metaphase I. Questions of mode and extent of pairing and the existence of meiotic silencing remained unknown but can inform our understanding of the evolution and mechanisms of meiotic prophase I. Work presented in this thesis provides novel insights into evolution and meiotic organisation of the monotreme sex chromosome complex. The platypus sex chromosome chain forms during zygotene in stepwise manner, with remarkable consistency beginning at the Y5 end of the chain and ending with the X1 (Chapter 1). Synapsis generally relies on 3 main proteins; SYCP1, SYCP2 and SYCP3. Surprisingly platypuses express three different copies of SYCP3 (including a multicopy version on a Y chromosome), genes that generally exist as single isoforms in most other species. Particularly given the SYCP3Y isoform is male specific, this raises the possibility that SYCP3 paralogs may have evolved in relation to the sex chromosome chain during prophase I (Chapter 2). During pachytene, the asynaptic regions of the sex chromosomes adopt a state of folding, similar to that of the avian Z and W chromosomes during synaptic adjustment, albeit without the formation of a central element. During this time the cohesin complex is heavily loaded onto the axial elements of the asynaptic regions of the X and Y regions of the chain. Furthermore at mid-pachytene the asynaptic regions of the chain are pulled to a giant nucleolus at which time the cohesin appears to spread onto the chromosome loops of the asynaptic regions of the chain that are also coincident with DNA condensation (Chapter 3). During platypus pachytene there is global transcriptional downregulation. We observe no localised phosphorylation of the histone H2AX, a hallmark of MSCI but we do observe localised patterns of H2AFY, H3K27me3 and H3K9me3 at a paranucleolar location, however the H2AFY and H3K27me3 showed some colocalisation with sex chromosomes, there was not consistent pattern and H3K9me3 was always associated with a section of chromosome 6 (Chapter 4). Together these results provide novel insights into the meiotic organisation of the monotreme sex chromosome complex and the evolution of MSCI in mammals.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2015

The global prevalence of obesity and type 2 diabetes increases and current treatment options are not satisfactory. This thesis will investigate the efficacy of novel dietary, lifestyle, and pharmaceutical interventions to treat these metabolic disorders. In rodents, acute-isoleucine treatment lowers blood glucose levels and protects from diet-induced weight gain, suggesting chronic-isoleucine may be beneficial in treating obesity and type 2 diabetes. This study aimed to investigate chronic-isoleucine treatment on body weight and glucose tolerance in lean and obese mice. Male C57BL/6 mice, fed a standard-laboratory-diet (SLD) or high-fat-diet (HFD) for 12-weeks, were randomly allocated to 1) Control (water); 2) Acute (0.3 g/ kg isoleucine); 3) Chronic (1.5% isoleucine in drinking water for 6-weeks). At 17-weeks, mice underwent a glucose tolerance test (GTT). In SLD- and HFD-mice, there was no difference in body weight between treatment groups. Acute-isoleucine did not improve glucose tolerance in SLD- or HFD-mice. Chronic-isoleucine impaired glucose tolerance in SLD-mice. In conclusion, chronic-isoleucine supplementation was not effective for weight loss and glucose intolerance in mice. Shift-work increases obesity risk partly through circadian desynchrony. In rodents, time-restricted feeding (TRF) during the dark-phase (DP) reduces weight gain and entrains the circadian rhythms of metabolic genes. Whether TRF during the light-phase (LP) is also effective is unknown. This study aimed to compare TRF-LP and TRF-DP on body weight and circadian rhythmicity of hepatic glucose (Slc2a2, Irβ & Gys2) and lipid (Acc1α) metabolic markers in lean and obese mice. Male C57BL/6 mice were fed a SLD or HFD for 12-weeks. After 4-weeks, mice were randomly fed to 1) ad libitum (AL); 2) during the LP (Zeitgeber (ZT)0-12); 3) during the DP (ZT12-24). In HFD-AL mice, energy intake, weight gain, fat mass, plasma lipids and mean blood glucose levels were elevated compared to SLD-mice. These parameters of obesity were similarly reduced in HFD-LP and HFD-DP mice compared to HFD-AL mice. In general, TRF during the LP or DP aligned circadian rhythms of hepatic markers of glucose and lipid metabolism to the timing of food intake. Human glucagon-like-peptide-1 (GLP-1) is a short-acting, blood glucose-lowering hormone. Comparatively, monotreme GLP-1s are potent, long-acting GLP-1 receptor agonists, resistant to degradation in humans and mice, suggesting monotreme GLP-1s may be beneficial in treating type 2 diabetes. This study aimed to investigate monotreme GLP-1 treatment on glucose tolerance in lean and obese mice. Male C57BL/6 mice were fed a SLD or HFD for 14- weeks. At 13-weeks, mice were randomly allocated to 7-groups and injected twice-daily with either 1) phosphate buffer (PB); 2) exendin-4 (Ex-4); 3) echidna GLP-1 (eGLP-1); 4) F8S; 5) platypus GLP-1 (pGLP-1); 6) N14S; or 7) S26K for 7-days. At 14-weeks, mice underwent a GTT. In SLD-mice, the glucose AUC was reduced in all treatment groups compared to PB controls. In HFD-mice, treatment with Ex-4, pGLP-1, N14S, and S26K reduced the glucose AUC compared to PB controls. In conclusion, monotreme GLP-1s may be effective in treating type 2 diabetes.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2021