This thesis describes a comprehensive study of the cellular responses of a number of endangered marsupial species with a principal focus on the tammar wallaby (Macropus eugenii) as a model macropod species. The development of in vitro experimental assays for the assessment of immune responses in this model species are described, which provided a set of benchmarks for comparisons with other members of the Macropodidae and with eutherian mammals. Once this data was collected and protocols were established, the study was extended to include investigations of the immune responses in opportunistic samples obtained from the Rufous Hare-wallaby (Lagorchestes hirsutus), the Long-footed potoroo ( Potorous longipes) and the more common, but nonetheless still vulnerable, Long-nosed potoroo (Potorous tridactylus) with a view to investigating their apparent susceptibility to infection with intracellular pathogens, particularly mycobacterial species. The findings from the application of these assays suggest that the cellular immune responses of these species are relatively complex and involve a level of sophistication that rivals their eutherian counterparts. Specifically peripheral blood and tissue leukocytes were morphologically similar to those of other mammals, with the exception of tammar wallaby monocytes that appeared to contain few lysosomal granules, and the basophils of the Rufous Hare-wallaby that contained very large atypical granules. The overall findings of this study suggest that the immune systems of macropod species possess most of the sophistication associated with that of eutherian mammals. Whilst some differences were apparent in cells and their products in the test species, no single factor common to all macropods was identified as a cause for immune dysfunction. It appears likely that as yet undefined factors related to their confinement rather than an inherent defect in their immunocapacity is responsible for the apparent disease susceptibility of these animals. / Doctor of Philosophy (PhD)

Microsatellites are simple repetitive DNA sequences that are used as genetic markers throughout the biological sciences. The high levels of variation observed at microsatellite loci contribute to their utility in studies at the population and individual levels. This variation is a consequence of mutations that change the length of microsatellite repeat tracts. Current understanding suggests that most mutations are caused by polymerase slippage during DNA replication and lead to changes of a single repeat unit in length, but some changes involving multiple repeats can also occur. Despite this simplistic overview, there is evidence for considerable heterogeneity in mutation processes between species, loci and alleles. Such complex patterns suggest that other mechanisms, including those associated with DNA recombination, are also involved in the generation of microsatellite mutations. Understanding which mutational mechanisms are responsible for variation at microsatellite markers is essential to enable accurate data interpretation in genotyping projects, as many commonly used statistics assume specific mutation models. I developed microsatellite markers specific to the X and Y chromosomes and an autosome in the tammar wallaby, Macropus eugenii, and investigated their evolutionary properties using two approaches: indirectly, as inferred from population data, and directly, from observation of mutation events. First, I found that allelic richness increased with repeat length and that two popular mutation models, the stepwise mutation model and the infinite allele model, were poor at predicting the number of alleles per locus, particularly when gene diversity was high. These results suggest that neither model can account for all mutations at tammar wallaby microsatellites and hint at the involvement of more complex mechanisms than replication slippage. I also determined levels of variation at each locus in two tammar wallaby populations. I found that allelic richness was highest for chromosome 2, intermediate for the X chromosome and lowest for the Y chromosome in both populations. Thus, allelic richness varied between chromosomes in the manner predicted by their relative exposure to recombination, although these results may also be explained by the relative effective population sizes of the chromosomes studied. Second, I used small-pool PCR from sperm DNA to observe de novo mutation events at three of the most polymorphic autosomal markers. To determine the reliability of my observations I developed and applied strict criteria for scoring alleles and mutations at microsatellite loci. I observed mutations at all three markers, with rate variation between loci. Single step mutations could not be distinguished because of the limitations of the approach, but 24 multi-step mutations, involving changes of up to 35 repeat units, were recorded. Many of these mutations involved changes that could not be explained by the gain or loss of whole repeat units. These results imply that a large number of mutations at tammar wallaby microsatellites are caused by mechanisms other than replication slippage and are consistent with a role for recombination in the mutation process. Taken as a whole, my results provide evidence for complex mutation processes at tammar wallaby microsatellites. I conclude that careful characterisation of microsatellite mutation properties should be conducted on a case-by-case basis to determine the most appropriate mutation models and analysis tools for each locus. In addition, my work has provided a set of chromosome-specific markers for use in macropod genetic studies, which includes the first marsupial Y chromosome microsatellites. Sex chromosome microsatellites open a new range of possibilities for population studies, as they provide opportunities to investigate gene flow in a male context, to complement data from autosomal and maternally-inherited mitochondrial markers.

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

[Truncated abstract] Understanding the effect that the disturbance of habitat by humans has on the population dynamics and ecology of wild animals is critical for the management of these populations. By understanding the demographic effects of disturbance the ways in which a population can be managed to increase or decrease its rate of change in size also become apparent. This thesis describes the effect that human disturbance, through the establishment of a large naval base, has had on the population dynamics and ecology of tammar wallabies (Macropus eugenii) on Garden Island, Western Australia. The disturbance of the environment on the HMAS Stirling Naval Base included the establishment of large areas of irrigated and fertilised couch grass (Cynodon dactylon) that increased and made virtually constant the amount of food available to the tammars in that area. In addition, traffic associated with the naval base resulted in large numbers of tammar wallabies being killed by vehicles. The effects of these disturbances were determined by comparing population dynamics, through vital rates of survival and fecundity and population growth rates, and spatial ecology, through the size of the animals' home ranges, in three areas of Garden Island. The three areas were the naval base (highly disturbed), southern bushland (adjacent to the naval base) and the northern bushland (undisturbed). The tammars on the naval base were in better body condition than those living in the two bushland areas of the island. ... When the impact of road-kills was removed, increased to 1.150.101 per year on the naval base and 0.960.076 per year in the southern bushland. Fecundity transitions, defined as the product of the rates of birth and pouch-young survival, and adult survival rates were lower in the bushland areas compared with the naval base in two of the three years, which were the main reasons for the lower estimates. There were no significant differences in the size of the tammars' home ranges between areas with modified or unmodified habitats or between the sexes (P>0.05). In summer the mean size of the home ranges was 3.90.66 ha, which was larger than winter when home ranges were 3.20.54 ha, but this difference failed to reach significance (P=0.058). These results indicate that the modification of the tammars' habitat has probably not caused significant changes in the size of the animals' home ranges. The size of the home ranges of tammar wallabies is likely to be determined by a complex interaction of many factors, and habitat modification alone has not been sufficient to cause substantial changes. The results presented in this thesis demonstrate that the disturbance caused by the establishment of the naval base on Garden Island has altered the population dynamics of the tammars wallabies, through increasing in the amount of food available to the tammars and through high numbers of road-kills. These results also demonstrate how gaining detailed knowledge of population dynamics can have direct application to managing the impact of disturbance on populations of wild animals.