Point Pelee National Park in Ontario, Canada has been affected by a long history of human activity. This activity has encouraged the establishment of approximately 276 exotic invasive plant species. These plants decrease biodiversity and effective function of ecosystems within the Park. A 5 Year Exotic Plant Species Management Plan for the Park was written in 1990. Since that time inventories have been done on some of the species but a monitoring of all the high priority exotic invasive plants has not. As part of this study an inventory of the high priority exotic invasive plants and their spatial extent in the Park, was created. From May to September, 2011 a comprehensive inventory took place within a 5.5km stretch in the terrestrial area at the southern end of the Park. Systematic belt transects were performed, on foot, from west to east at 100 m intervals. Within the quadrats percent cover of each plant species was determined. The data collected in the field were compiled using geographic information systems (GIS), resulting in maps of the extent of the most abundant species studied. Analysis as part of this study included using the data to determine which plants and areas are higher priorities for management within the Park. Quadrats were analysed for diversity using the Simpson Index and, since the data was non-parametric, comparisons were made across diversity and native richness using the Kruskal Wallis test. Alliaria officinalis is the only non-native species that is widespread within the study area. Other non-native species with a high potential for invasiveness were observed but only consisted of a few individuals along roads and paths. Some non-native species were present along or near roads and paths in denser patches (17-100% plot coverage). Recommendations include removal of some non-native species deemed to be a potential threat to native richness and diversity, followed by re-vegetation with native species, and continued monitoring. Future restoration efforts are best directed at the area around DeLaurier, along west beach and at the Tip. These areas have the lowest diversity and native richness and therefore need the most improvement.

This study focused on an invasive riparian reed grass, Arundo donax L., a clonal plant of the family Poaceae that is widely distributed in North America. Water availability, including water taken up from the roots locally or transported from the neighboring ramets, may affect the performance of A. donax in riparian zones. The first objective was to find out how moisture gradients affected the performance of A. donax in riparian zones. I measured leaf photosynthetic rate, leaf δ13C ratio, and plant growth-related parameters across two summer growing seasons at four transects perpendicular to the water course on the Rio Grande in South Texas. The second objective was to find out whether physiological integration existed in A. donax and how resource sharing, if any, affected plant growth. A rhizome severing experiment was conducted on five paired plots to compare growth-related parameters between plots with rhizomes severed and intact at 3, 7, and 11 weeks after treatment. Heavy water (δ 2H ~1800‰) was applied on three 1-m2 area over 3 successive days and rhizome samples were collected beyond the watering zone after 5, 24, and 48 hours of last watering. At short-term scales, A. donax performance was adversely affected by both drought and inundated conditions; over longer time scales, plant performance decreased as water availability declined in general, but biomass and stem density were similar across moisture gradients. I also found evidence of physiological integration in A. donax. Water was transported through interconnected rhizomes at least 3.5 m; transport distances averaged 1.67 m. Rhizome severing stimulated higher ramet production initially but over longer periods produced shorter thinner stems with lower flood tolerance. However, after 11 weeks of re-growth, plot-level biomass was similar between plots with severed and intact rhizomes. These results suggest that performance of A. donax is affected by water availability in riparian zones; however, clonal plant plasticity, water use efficiency, and clonal integration ameliorate impacts of water stress on the performance of A. donax. Such traits enhance its resource use, which could potentially increase competitive ability rate of establishment, and extent of this invasive species in heterogeneous riparian environments.

Abstract Introduction: Physiotherapy in the human intensive care setting is in common use and supported by Level 1 evidence. In the canine intensive care setting, however, physiotherapy is novel, despite many studies published to support aspects of human physiotherapy being conducted on experimental dogs and there being 83 canine intensive care beds in Sydney alone. As veterinary care progresses and people place more importance on their pet’s health care, an opportunity arises to examine the potential for physiotherapy to augment the veterinary care of client-owned dogs in a veterinary referral hospital setting. The first objective of this thesis was therefore to determine which common conditions cause dogs to present recumbent to veterinary referral hospitals, and then to conduct a literature review on these conditions and ways in which physiotherapy has the potential to add to the treatment of them. The next objective was to describe the short-term effects of recumbency on a number of key body systems in clinically unwell dogs, as concern was that recumbency itself may contribute to pathogenesis and/or poorer recovery, however this could not be investigated before preliminary studies like this were performed. The next objective was to conduct clinical trials of the use of two well-proven techniques in human physiotherapy on dogs with conditions commonly reported in the earlier studies of this thesis. The chosen techniques were continuous positive airway pressure (CPAP) to assist in the recovery of dogs with brachycephalic airway obstructive syndrome (BAOS) from general anaesthetic, and the use of manual chest physiotherapy in dogs with aspiration pneumonia following tick (Ixodes holocyclus) poisoning. Design: Retrospective epidemiological study; Literature review; Cross-sectional descriptive study; Randomised controlled trial; Clinical trial. Results: Dogs admitted recumbent represented 3.18% of total admissions, however had a confirmed survival rate of only 43.5%. Sydney canine intensive care units have a combined capacity of 83 beds, so many other dogs experience recumbency as part of their veterinary management, despite being admitted ambulant, and therefore also have the potential to benefit from physiotherapy. Diagnoses were split into categories, with suspected intervertebral disc disease the most common neurological diagnosis, hit by car the most common orthopaedic diagnosis, collapse the most common diagnosis in the ‘otherwise unwell’ category and tick (Ixodes holocyclus) poisoning and poison (e.g. metaldehyde) ingestion the most common toxicology diagnosis, all of which have the potential to be just as amenable to physiotherapy in dogs as they are in people. However, interspecies comparisons revealed that although the dogs studied had broadly comparable diagnoses to humans, they had a higher mortality rate and received much less physiotherapy than humans. Key body systems were therefore measured in recumbent dogs, with the finding that the dogs included under the strict definition of recumbency in this study maintained a fairly consistent clinical state in the short-term of their period of recumbency (median days 3 to 5 of hospitalization). This finding supported the investigation of physiotherapy as a potential to not only reduce the mortality rate but to speed return to function, which has the potential to also lower the economic burden of prolonged hospitalization on owners. Continuous positive airway pressure was therefore investigated for its potential to improve the safety of recovery from general anaesthetic in dogs with BAOS. Tolerance and effect of CPAP were investigated with positive results demonstrated to improve time to recovery, with no additional distress, in dogs with BAOS recovering from a general anaesthetic. An evidence-based protocol of manual chest physiotherapy was then tested on dogs with aspiration pneumonia following tick (Ixodes holocyclus) poisoning. Demeanour, saturation of peripheral oxygen and auscultation all improved following the first manual chest physiotherapy session, demonstrating tolerance and effect. Conclusions: Primary recumbency is an uncommon but serious condition in dogs that warrants attention by physiotherapy researchers, as dogs admitted ambulant but who spend part of their hospitalization recumbent also have the potential to benefit. This thesis determined the common causes of recumbency in dogs and determined that key body systems of recumbent dogs are clinically stable without the assistance of physiotherapy to improve their short-term rate of recovery. Positive short-term results were yielded from the use of CPAP and manual chest physiotherapy that may benefit other clinically unwell dogs with similar respiratory conditions. Further research should be conducted into other ways in which physiotherapy can augment the care of recumbent dogs, as well as lower the mortality rate and speed the return to function in dogs in the intensive care setting, as it is possible that not all positive findings in human research were replicated in these studies due to Type II error resulting from small sample sizes due to abnormal weather patterns that reduced the number of presentations of dogs with BAOS or tick poisoning during the study period.

Invasive species are problematic but their control can be difficult. When the history and dynamics of invasive populations are unknown, analytical techniques utilising genetic data can provide information to management. The application of these techniques has typically been limited to species with restricted dispersal or to localised geographic scales. Here these techniques are used in conjunction with traditional approaches to study the invasion of a highly vagile species over a continental scale. Originally introduced to eastern Australia, the common starling (Sturnus vulgaris) is expanding westward and has reached the southeast agricultural region of Western Australia (WA). Spatial, temporal and museum samples representing historical incursions into WA were used in genetic analyses to answer questions important to WA management agencies. Where possible, multiple marker classes were used to evaluate their relative ability to address these questions, including microsatellites, mitochondrial DNA (mtDNA), and a nuclear gene. Between two and four genetic groups were identified in Australia, and the localities on the edge of the range expansion (ERE) were consistently genetically differentiated from all others. Levels of genetic exchange between groups appeared to be low, suggesting that localised control may be effective. Analyses of historical samples indicated patterns of genetic exchange are temporally stable. This suggests that starlings will continue to arrive from the east, requiring vigilance from management. Consistently, a negative relationship was found between genetic diversity and date of first record, which may help in the future to distinguish ineffective surveillance from recent colonisations. Genetic evidence of female-biased dispersal indicated that control strategies preferentially targeting females may more efficiently limit dispersal than those targeting both sexes equally. Variants private to particular genetic groups were identified; tracking the spread of these variants may assist future monitoring programs in understanding ongoing exchange between groups. One mtDNA variant private to ERE localities showed dramatic temporal change, which could not be adequately explained by genetic drift or admixture, but may be explained by selection. Microsatellites were most useful in determining origins and identifying dispersers, mtDNA sequences provided a unique tool for ongoing monitoring of dispersal, and nuclear sequences had high levels of gene diversity and resolution of population structure.

Information on the reproductive biology and behavioural ecology of southern hairy-nosed wombats (Lasiorhinus latifrons) is limited. Field reproductive and behavioural studies have been hindered by the difficulties associated with the routine recapturing and direct observation of wombats in the wild. Additionally, due to their extremely poor breeding success within captivity and the intrinsic complications associated with the monitoring of nocturnal and semi-fossorial activity, little formal research has been conducted on captive-held individuals. The overarching objectives of this research were to gain a better understanding of (1) both male and female L. latifrons reproductive physiology/behaviour that will lead to improved captive breeding program outcomes and (2) captive L. latifrons activity rhythms and behavioural time budgets in order to identify the impacts of captivity on wombat behaviour and wellbeing. The primary experiments of this research were centred around the development of ‘non-invasive methodologies’ for the collection of biological samples (Chapter 2), direct observation of behaviour and activity (Chapter 3), assessment of male reproductive function (Chapter 4), detection of female oestrus and cyclicity (Chapter 5), characterisation of activity patterns (Chapter 6) and monitoring of stress (Chapter 7). The last two experiments also tested the efficacy of gentling (Chapter 7) and enrichment (Chapter 8) to improve the wellbeing of the captive wombats. Faecal steroid analysis is a non-invasive tool that allows the stress-free monitoring of steroid hormones and has been used on a wide range of animal species to examine their reproductive physiology and adrenal function. The usefulness of faecal hormone analysis, however, is directly related to the reliable collection and identification of individual faecal samples. In group-housed animals, the identification of faecal samples can be difficult and time consuming and is generally only accurate if a marker is incorporated into the animal’s diets. Hence, Chapter 2 examined the usefulness of non-toxic plastic glitter as a faecal marker in group-housed L. latifrons. Forty-two food treats were tested as vehicles for the oral delivery of glitter; of these, vehicle palatability (> 75% consumed) and consistency of intake (eaten > 80% of times offered) was high for six treats: (1) golden syrup with horse pellets, (2) golden syrup with weetbix, (3) pitted-dates, (4) honey with kangaroo pellets, (5) nutrigel with rolled-oats and (6) strawberry sauce with rolled-oats. Marker mean rate of passage was 2.9 ± 0.5 d, with maximal output occurring 4.2 ± 0.3 d after oral administration. A minimum marker dose of 1.6 g was necessary to achieve high labelling consistency (> 2 flecks of glitter were defaecated in > 90% of pellets) and this dosage was required every 3 days to maintain a steady and detectable state of marker output. Twelve glitter colours were tested and optimum labelling results were obtained with gold, metallic red, metallic green and metallic blue. Once established, this technique was then used to facilitate the long-term collection of faecal samples in order to characterize patterns of reproduction (Chapter 4), monitor ovarian events (Chapter 5) and to quantify stress-responses (Chapter 7) via faecal steroid analysis. The direct observation of wombats is difficult; individuals are not easily identified and the animals are often out-of-view (residing in burrows) or obscured (low-light conditions) during sampling. Published behavioural data available on L. latifrons is largely restricted to visual observations made during dawn and dusk only, whilst published activity data pertains to time spent in or out of the burrows rather than actual physical activity. Hence, Chapter 3 tested the effectiveness of two electronic monitoring systems to remotely observe wombat behaviour and physical activity. Digital video-surveillance proved to be an effective technique for the recording and monitoring of captive wombat behaviour. Animal visibility was good, behavioural events unambiguous and the system enabled the long-term, concurrent recording of behaviour with no direct human presence. Similarly, radio-telemetry proved to be an effective way of recording captive wombat physical activity. The system was reliable, removed observer error and enabled the continuous and concurrent recording of wombat activity. Once established, these two remote monitoring systems were then used to describe wombat behaviour elements and activity patterns (Chapters 3 and 6). After a year of continuous monitoring it was established that the wombats spent, on average, 69.9% sleeping, 8.8% lying resting, 5.2% feeding, 5.2% exploring, 4.3% stereotyping, 2.5% sitting-at-rest, 1.7% digging, 1.4% foraging, 0.4% being handled, 0.3% sun-basking, 0.2% grooming and 0.1% courtship/mating. Temporal patterns were bimodal for 8/12 of the wombat behaviours and unimodal for the remaining four. The mean proportion of total daily time spent active was 18.2 ± 1.8%. Daily activity patterns were characterized by a strong circadian cycle, with high nocturnal activity and low diurnal activity. Daily onset (18:19 h) and cessation (04:34 h) of activity was seasonally constant and strongly associated (P < 0.01) with sunrise/set, but not influenced by either temperature or humidity (P ≥ 0.09). At night there was an alternating rhythm of active and rest periods, with activity peaking at the beginning (19:00 h) and end (03:00 h) of each night. Activity was seasonal with annual changes in temperature, humidity and night-time length being the triggers of variation. Mean daily activity was greater during winter (19.7%) and spring (18.9%), than during summer (16.3%) and autumn (17.2%), with the degree of activity being largely governed by ambient temperature. Feeding, sleeping and stereotyping varied significantly with season. Feeding and stereotyping were negatively associated with ambient temperature and humidity, whilst being positively associated with night-time length; the inverse relationship was true for sleeping. Ambient temperature exerted its largest effect on time spent feeding; feeding times decreased by 3.1 min / 1ºC above 13ºC and compared to spring, feeding times were reduced by 41% during summer. There is, at present, very little data published on male and female wombat reproduction. The reticence of wombats to breed in captivity makes it difficult to study reproduction in captive animals and their cautious, nocturnal nature makes field studies challenging. Non-invasive techniques to monitor reproductive status/function will assist in improving the general knowledge of wombat reproduction and the development of new captive breeding management strategies, by allowing the easy monitoring of captive animals. Thus, the series of experiments in Chapters 4 and 5 explored the efficacy of a number of non-invasive methodologies to assess male reproductive function, monitor female cyclicity and predict the timing of oestrus. To address the pulsatile nature of testosterone, a GnRH agonist stimulation test was developed in the male. IM injection of 4 μg of buserelin (a GnRH agonist) resulted in an increase (P < 0.05) in plasma testosterone concentrations, with maximum secretion occurring at 90 min. Thereafter, plasma testosterone concentrations remained near maximum for 150 min. There was a strong, positive correlation (r = 0.73, P < 0.01), between pre-stimulation testosterone concentrations and the maximal concentrations achieved post-stimulation with post-stimulation concentrations varying between individuals (P < 0.01). These findings indicate that individual male wombats can show large fluctuations in plasma testosterone concentrations over time and that a GnRH agonist can be used to obtain a diagnostic index of the prevailing testosterone biosynthetic capacity of the testes. This technique was then used as part of a larger experiment (Chapter 4) to investigate seasonal changes in male wombat reproduction. To date, the effects of breeding season on captive male wombat fertility have yet to be examined and a better understanding of this phenomenon will pinpoint the most favourable times for mating. Seasonal changes in a series of male reproductive parameters were non-invasively examined over a 18 month period: (1) testosterone concentration, both plasmic and faecal, was monitored using enzyme-immunoassays (EIA), (2) testicular volume was measured manually using digital vernier callipers, (3) sperm production was evaluated by way of spermatorrhoea, whilst (4) prostate and bulbourethral gland cross-sectional areas were assessed by ultrasonography. Plasma testosterone secretion increased in early-winter, peaked late-winter and declined in early-spring (P < 0.01). No seasonal variation (P = 0.22) in faecal testosterone metabolite concentrations was apparent. Testicular volume showed no significant variation (P = 0.29) over the sampling period and spermatozoa were found in the urine throughout the year; these two observations suggest that the captive male wombats remain spermatogenically active year round. While there was no significant seasonal change (P = 0.20) in prostate size, bulbourethral gland size increased in late-autumn, peaked in mid-winter and declined in early-summer (P < 0.01). Ultimately, captive male reproductive function was influenced by seasonality, with a peak in plasma testosterone and bulbourethral gland size occurring in winter (Jun-Aug). In an attempt to characterize oestrus-specific behaviour and develop a reliable method of oestrus detection in L. latifrons, the reproductive physiology and behaviour of eight adult females was monitored for a period of 12 months (Chapter 5). The reproductive behaviour of both sexes (4♂: 8♀) was monitored using 24-h video surveillance, whilst female physical activity was remotely measured using radio-telemetry. A faecal sample was collected every three days, from each female to assess changes in faecal progesterone and oestradiol-17β metabolite concentrations. Each female also received an injection of 0.01, 0.1 or 0.2 mg/kg of oestradiol benzoate (OB) in one of two hormone trials. Video surveillance revealed that the courtship (n = 426) and mating (n = 46) ritual of L. latifrons consisted of 13 distinctive behaviours expressed over six obvious phases: investigation, attraction, chase, restraint, copulation and recovery. Reproductive behaviour was observed in five (2♂, 3♀) wombats, with female receptivity occurring at night and lasting for only 13-h. Faecal progesterone metabolite analysis proved to be a reliable method for mapping oestrous cycle activity, but was not useful for the prediction of oestrus. Six out of the eight female wombats displayed periods of elevated progesterone secretion. From these six individuals, 23 luteal phases, 12 follicular phases and 12 oestrous cycles were recorded, with a mean (± SE) length of 20.9 ± 1.1 d, 11.6 ± 0.6 d and 31.8 ± 1.1 d, respectively. In contrast, changes in the secretion of faecal oestradiol-17β metabolites provided little instructive information on oestrous cycle activity and were not associated with oestrus. Administration of OB resulted in a spike of oestradiol-17β metabolites in the faeces 3-4 d later, but was not dose dependent nor did it elicit oestrus-behaviour. Activity monitoring does not appear to be a useful method for detecting oestrus in L. latifrons, as changes were not associated with key events in the oestrous cycle. However, 24-h video surveillance proved to be a reliable method for oestrus detection in the captive L. latifrons. Threatening or aversive stimulation is experienced in wild and captive conditions alike and evokes similar physiological responses. If an animal, wild or captive, cannot cope with this stimulation it may experience stress. An uncontrollable source of stress for all captive animals lies in their interactions with their human caretakers. A high or persistent fear of people can be a source of psychological stress for animals in captivity. Positive handling is a potent method of reducing the specific fear of human beings through desensitisation. The response of animals to handling by humans has been extensively evaluated in domesticated species, but rarely assessed in wild animals. Hence, Chapter 7 examined the usefulness of a regular handling program to lower the behavioural fear and physiological stress responses of L. latifrons to human interaction. Adult L. latifrons (n = 12) were exposed to two different treatments in a replicated design: (1) daily handling: 15 min of tactile contact from a human handling 5 d/wk for 12 wk and (2) no-handling: no contact apart from that received during routine husbandry. The effect of handling was assessed using overt response, human approach, stressor and novel stimulus tests. Synthetic ACTH was used to validate a method for monitoring faecal cortisol in L. latifrons by EIA. IM injection of 250 μg of Synacthen resulted in a significant (P < 0.05) increase in plasma and faecal cortisol concentration 30 min and 2 d after administration, respectively. Handling positively affected the behavioural responses of the wombats to human approach and contact in two ways: (1) a significant reduction (P < 0.01) in the wombat’s mean flight distances to human approach and (2) a significant (P < 0.01) decline in the strength of the wombat’s behaviour-based fear reactions (i.e. fear scores) to human proximity and contact. Handling had no discernable effect on the wombat’s physiological stress responses to human contact or on their reactions to novelty. While faecal cortisol secretion increased in response to a stressor test involving human contact, it was not alleviated by regular handling (P = 0.84). Similarly, the wombat’s reactions to unfamiliar objects during novel stimulus testing were also unaffected (P = 0.17) by the handling treatment. Therefore, handling exerted its main effect on the behavioural responses of the wombats, representing fear responses to human handlers, rather than reducing their anxiety. The main difference between wild and captive environments lies in the differential ability of control, i.e. a free-living animal is able to control its amount of incoming stimulation whereas a captive animal has a limited capacity to alter the external stimulation to which it is exposed. Without natural behaviour outlets, captive animals have to rely on abnormal behaviour patterns to modify their expectations of the captive environment and exert some control over incoming stimulation. A ‘stereotypy’, defined as a repetitive movement pattern with no apparent goal or function, is a common abnormal behavioural pattern expressed by zoo animals. Stereotypies are of concern because of their association with poor welfare. A previous behavioural study revealed that common wombats (Vombatus ursinus) in captivity are very susceptible to the development of stereotypic behaviour. For that reason, the final experiment (Chapter 8) was designed to further examine stereotypic behaviour in wombats and tested whether environmental enrichment could be used to reduce its prevalence. Adult L. latifrons (n = 12) were subjected to two different treatments in a replicated design with 12 week periods: (1) Enrichment – animals received feed and olfactory enrichment, 5 d/wk and (2) No-enrichment - animals received the standard captive diet only. Wombat behaviour was remotely observed via 24-h video surveillance. Eight out of the 12 wombats displayed one of four stereotyped movements (straight-line pacing, boundary pacing, figure-8 pacing or wall-climbing), with time spent stereotyping ranging from 61-129 min/d (mean 87 ± 7 min/d); time devoted to stereotyping took up 4-9% of the daily budget. There was a significant increase in foraging (333%) and exploration (13%) in response to enrichment. Enrichment also encouraged the expression of a wider range of naturalistic foraging behaviours. Despite these positive effects, enrichment had no discernable effect on the time spent stereotyping (P = 0.87) or inactive (P = 0.13). Despite the fact that stereotypies and inactivity were not reduced by enrichment, animal welfare was still enhanced as there was a notable improvement in natural wombat-specific behavioural expression and diversity.

Flaviviruses are a group of medically relevant pathogens, known to cause serious disease in animals and humans. The previously defined roles of the flavivirus non-structural protein 2A (NS2A) in RNA replication, and modulation of the host antiviral response, has recently been extended to include virus assembly and secretion. In West Nile virus subtype Kunjin (KUN), an Isoleucine (I)-to-Asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. In this study, prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59, but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59, or the compensatory T149P mutation into NS2A-I59N replicon RNA, restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. These results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for these induced membranes in virus assembly. To further our understanding of how mutations within NS2A are able to affect the induction of virus-induced membranes, leading to a block in virus assembly, the membrane topology of KUN NS2A was investigated. Using a plasmid encoding NS1 and NS2A proteins with C-terminal c-myc and FLAG epitopes, NS2A proteins containing N-linked acceptor sites and C-terminal truncations were generated. Assays were performed to identify the subcellular localization of specific sequences within NS2A by Western blot and immunofluorescence analyses. While the membrane topology could not be determined experimentally, the findings of this study support the assertion that cleavage at the NS1/NS2A junction requires the majority, if not all of the NS2A protein for proper processing to occur, and suggests that the interaction between hydrophilic loops and -helical transmembrane segments plays an important role in the formation and stability of the flavivirus NS2A protein topology. Based on the knowledge of polyprotein processing events, and utilizing a range of software packages, a topology model of NS2A was predicted. The likelihood of additional sequences within NS2A affecting the ability to induce virus-specific membranes, and facilitate virion assembly, has led to the development of an invasive bacterial screening system, as a delivery vehicle to screen libraries of mutated KUN replicon clones. Using these invasive bacteria to deliver mutated KUN replicons into BHK-derived packaging cells, mutations causing a deficiency in either RNA replication or encapsidation can be identified by performing -gal assays on cells maintained in the presence, or absence of Doxycycline (suppressing the expression of structural proteins), respectively. Furthermore, this system was adapted for use in a 96-well plate format, allowing for high-throughput screening. Thus, KUN replicon clones capable of RNA replication, but unable to assemble and secrete virus-like particles can be identified and further analyzed, in the hope of mapping amino acid residues and motifs involved in encapsidation of flavivirus RNA. Finally, a range of hypotheses are discussed, explaining the possible mechanisms by which NS2A is involved in flavivirus assembly. A number of future directions and applications are also presented.

Invasive plants pose serious threats to economic, social and environmental interests throughout the world. Developing strategies for their management requires a range of information that is often impractical to collect from ground based surveys. In other cases, such as retrospective analyses of historical invasion rates and patterns, data is rarely, if ever, available from such surveys. Instead, historical archives of remotely sensed imagery provides one of the only existing records, and are used in this research to determine invasion rates and reconstruct invasion patterns of a ca 70 year old exotic mesquite population (Leguminoseae: Prosopis spp.) in the Pilbara Region of Western Australia, thereby helping to identify ways to reduce spread and infill. A model was then developed using this, and other, information to predict which parts of the Pilbara are most a risk. This information can assist in identifying areas requiring the most vigilant intervention and pre-emptive measures. Precise information of the location and areal extent of an invasive species is also crucial for land managers and policy makers for crafting management strategies aimed at control, confinement or eradication of some or all of the population. Therefore, the third component of this research was to develop and test high spectral and spatial resolution airborne imagery as a potential monitoring tool for tracking changes at various intervals and quantifying the effectiveness of management strategies adopted. To this end, high spatial resolution digital multispectral imagery (4 channels, 1 m spatial resolution) and hyperspectral imagery (126 channels, 3 m spatial resolution) was acquired and compared for its potential for distinguishing mesquite from coexisting species and land covers. / These three modules of research are summarised hereafter. To examine the rates and patterns of mesquite invasion through space and time, canopies were extracted from a temporal series of panchromatic aerial photography over an area of 450 ha using unsupervised classification. Non-mesquite trees and shrubs were not discernible from mesquite using this imagery (or technique) and so were masked out using an image acquired prior to invasion. The accuracy of the mesquite extractions were corroborated in the field and found to be high (R2 = 0.98, P<0.001); however, accuracy varied between classes (R2 = 0.55 to 0.95). Additional sampling may be required in some of the wider class intervals, particularly the moderate density class (30 to 90%) as sampling frequency was poor within the range of 60 to 90%. This is a direct result of there being relatively few quadrats available to be randomly selected in this class. That is, quadrats with between 60-90% cover were only evident in 4% of the test area. A more robust approach would, therefore, be to split this class into two (e.g. 30-60% and 60-90%) and select an additional 15 quadrats in the 60-90% range. The resolution of the imagery (1.4 m) precluded mapping shrubs smaller than 3 m2. Rates and patterns were compared to mesquite invasions in its native range. / It was determined that: (i) the shift from grass to mesquite domination had been rapid, with rates of increase in canopy cover comparable to invasive populations where it is native; (ii) rate of patch recruitment was high in all land types (stony flats, red-loamy soils and the riparian zone), but patch expansion and coalescence primarily occurred over the riparian zone and redloamy soils; (iii) mesquite had been spread by sheep and macropods and the recent switch to cattle is likely to exacerbate spread as it is a far more effective dispersal vector; and (iv) early successional patterns, such as high patch initiation followed by coalescence of existing stands are similar to where mesquite is native, but patch mortality did not occur. A knowledge based model was used to predict which parts of the Pilbara region are most at risk. Several limitations of models often employed in predicting suitability ranges of invasive plants were identified and include: (i) an inability to incorporate the notion that within a suitability range there is likely to be a scale of favourability; (ii) an inability to assign greater importance to evidence that is likely to have more importance in defining the areas suitable for invasion; and (iii) an inability to control the level of conservatism in the final results. These three shortcomings were mitigated through the use of: (i) fuzzy membership functions to derive a range of favourability from poor to best; (ii) pairwise comparison to derive higher weights for layers perceived to be more important and vice versa; and (iii) the use of ordered weighted averaging to directly control the level of conservatism (or risk) inherent in the models produced. / Based on the outcomes of the historical reconstruction of spatial rates and patterns, data sources included land types, land use, and the derivation of a steady state wetness index from spot height data. Model outputs were evaluated using two methods: the area under the curves (AUC) produced from relative operating characteristic (ROC) plots and by the maximum Kappa procedure. Both techniques agreed that the model most representative of the validation data was the one assuming the most risk. To create a Boolean output representing areas suitable/not suitable for invasion, optimal cut-points were derived using the point closest to the top left hand corner of the ROC plot and by the maximum Kappa method. Both methods obtained identical cut-points, but it is argued that the coefficient produced by the maximum Kappa method is more easily interpreted. The highest AUC was found to be 0.87 and, based on the maximum Kappa method, can be described as good to very good agreement with the validation records used. Digital multispectral imagery (DMSI), acquired in the visible and near infrared portions of the spectrum (3 visible bands, 1 near infrared) with a spatial resolution of 1 m and hyperspectral imagery (126 bands, 3 m spatial resolution) was acquired to assess the potential of developing a reliable and repeatable mapping tool to facilitate the monitoring of spread and the effects of control efforts. Woody vegetation was extracted from the images using unsupervised classification and grouped into patches based on contiguity. Various statistics (e.g. maximum, minimum, median, mean, standard deviation, majority and variety) were assigned to these patches to garner more information for species separation. / These statistics were explored for their ability to separate mesquite from coexisting species using Tukey’s Honestly Significantly Different (HSD) test and, to reduce redundancy, followed by linear discriminant analysis. Two approaches were taken to select the patch statistics offering the best discrimination. The first approach selected patch statistics that best discriminated all species (named “overall separation”). This was compared to a second approach, which selected the best patch statistics that separated each species from mesquite on a pairwise basis (named “pairwise separation”). The statistics offering the best discrimination were used as input in an Artificial Neural Network (ANN) to assign class labels. An incremental cover evaluation, whereby producer’s accuracy was computed from mesquite patches grouped into various size-classes, showed that identification of mesquite patches smaller than 36 m2 was relatively low (43-51%) regardless of the method used for choosing between the patch statistics or image type. Accuracy improved for patches >36 m2 (66-94%) with both approaches and image types. However, both approaches used on the hyperspectral imagery were more reliable at capturing patches >36 m2 than the DMSI using either approach. The lowest omission and commission rates were obtained using pairwise separation on the hyperspectral imagery, which was significantly more accurate than DMSI using an overall separation approach (Z=2.78, P<0.05), but no significant differences were found between pairwise separation used on either media. / Consequently, all methods and imagery types, except for DMSI processed using overall separation, are capable of accurately mapping mesquite patches >36 m2. However, hyperspectral imagery processed using pairwise separation appears to be superior, even though not statistically different to hyperspectral imagery processed using overall separation or DMSI processed using pairwise separation at the 95% confidence level. Mapping smaller patches may require the use of very high spatial resolution imagery, such as that achievable from unmanned airborne vehicles, coupled with a hyperspectral instrument. Alternatively, management may continue to rely on visual airborne surveys flown at low altitude and speed, which have proven to be capable at mapping small and isolated mesquite shrubs in the study area used in this research.

The introduction of trout has been implicated in the declines in native fish fauna in New Zealand and worldwide. Since the introduction of brown (Salmo trutta) and rainbow (Oncorhynchus mykiss) trout to New Zealand in 1867, their distribution has spread and they have been implicated in the fragmentation of native fish distributions, particularly native non-migratory galaxiids. However, in the Upper Waimakariri basin the co-occurrence of trout and galaxiid populations is relatively common, even in streams where trout reach sizes known to be piscivorous. To investigate mechanisms that may regulate trout and galaxiid co-occurrence, I investigated differences in antipredator behaviour and growth rate between stream types with varying levels of trout presence. Using quantitative survey data collected between 1997 and 2006, I found that trout abundance was low and varied annually in frequently disturbed sites compared their high abundance in stable streams. This finding was used to classify streams into three population types, barrier (trout absent), disturbed (trout presence intermittent) and sympatric (constant trout presence). Using this classification, I tested the effects of trout chemical cues on galaxiid activity and refuge use in artificial channels. There were no differences in activity or refuge use between trout odour and there were no effects of population type or galaxiid size during both the day and the night. Using otolith weight-fish length relationships in galaxiids collected from each population type, I found that galaxiid growth rate was higher in disturbed streams than in stable streams either with or without trout. An experiment manipulating trout size and presence, over two months in a natural stream, found galaxiids from treatments without trout grew slower than those with trout. Slow growth rates in galaxiids above trout-migration barriers and in sympatry, combined with low growth rates in treatments without trout suggest that the mechanisms that regulate galaxiid growth are more complex than previously thought.

This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

Thesis (M. Sc.)--University of Canterbury, 2007. / Typescript (photocopy). Includes bibliographical references (leaves 80-90). Also available via the World Wide Web.