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Amphibians are undergoing dramatic population declines, with environmental pollution reported as a significant factor in such declines. Technologies are required that are able to monitor populations at risk of deteriorating environmental quality in a rapid, high-throughput and low-cost manner. The application of biospectroscopy in environmental monitoring represents such a scenario. Biospectroscopy is based on the vibrations of functional groups within biological samples and may be used to signature effects induced by chemicals in cells and tissues. Here, attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy in conjunction with multivariate analysis was implemented in order to distinguish between embryos, whole tadpoles at an early stage of development and individual tissues of late-stage tadpoles of the common frog collected from ponds in the UK with varying levels of water quality, due to contamination from both urban and agricultural sources. In addition, a Xenopus laevis cell line was exposed to low-levels of fungicides used in agriculture and assessed with ATR-FTIR spectroscopy. Embryos, in general did not represent a sensitive life stage for discriminating between ponds based on their infrared spectra. In contrast, tadpoles exposed to agricultural and urban pollutants, both at early and late stages of development were readily distinguished on the basis of their infrared spectra. ATR-FTIR spectroscopy also readily detected fungicideinduced changes in X.laevis cells, both as single-agent and binary mixture effects. Data reported in this study confirm the use of ATR-FTIR spectroscopy as a sensitive technique capable of detecting small changes in cellular groups, and as such represents a valuable starting point for its use in the monitoring of amphibian populations. However further research is needed in order to overcome confounding factors existent in natural populations of complex organisms.

Embryonic development involves a variety of cellular processes, including cell growth, cell proliferation, cell differentiation, cell migration and cell death. The precise regulation of these events is essential for morphogenesis and development. Reactive oxygen species (ROS) have been reported as important second messengers to modulate a variety of proteins and signalling pathways, thereby regulating several cellular processes. Previous studies have shown that fertilisation induces an oxidative burst in sea urchins. However, a role for this fertilisation induced ROS during embryonic development in this system has not been shown. Amphibian embryos, such as those from Xenopus laevis (X. laevis), provide an ideal system to study the role of ROS during embryonic development. We previously established a HyPerYFP line in X. laevis, which permits the visualisation of ROS levels in living oocytes, fertilised eggs and early embryos. Using this line, I showed that fertilisation triggers a dramatic increase in intracellular ROS levels, and this increase in ROS levels is sustained throughout early embryogenesis. Lowering ROS levels using antioxidants (e.g. NAC) impairs mesoderm formation and results in severe developmental defects, which are associated with a down-regulation of several signalling pathways, including PI3K/Akt, TGF-β/Nodal and Wnt/β-catenin signalling. I also showed that the dysregulation of these signalling pathways could be partially rescued by addition of the oxidant H2O2 or the ROS inducer menadione, suggesting that the fertilisation induced ROS helps activate several signalling pathways required for mesoderm formation. Further investigations through loss and gain of function analyses revealed that fertilisation induced calcium wave, which is dependent on IP3, activates ROS production from mitochondria, via the mitochondrial complexes II, III and IV. In addition, treatment of early embryos with mitochondrial inhibitors results in defects in cell division during early embryogenesis. Together, my findings suggest that a sustained high level of ROS is essential for cell cycle progression and the activation of early signalling events, therefore ensuring proper early embryonic development in X. laevis.

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No description available.

No description available.

Global biodiversity is being lost at an unprecedented rate, such that we have entered the sixth mass extinction in the history of the earth with emerging infectious diseases (EID) recognised as an important contributor to this loss. Amphibian chytridiomycosis is an EID that has driven very rapid declines in, or even extinctions of, hundreds of amphibian species. Infectious diseases such as chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), often persist in biological and non-biological reservoirs making them difficult to eradicate. In turn, this makes reintroductions of target species challenging due to the risk of infection. This thesis investigates the critically endangered mountain chicken (Leptodactylus fallax) as a case study of the population impacts of a chytridiomycosis epidemic and to test the effectiveness of strategies to mitigate the effects of the disease. Specifically, this research (1) charts the decline of the mountain chicken on the only two islands on which it exists, and determines the impact on genetic diversity; (2) tests whether anti-fungal treatment can improve the survival of mountain chickens with Bd infection in the wild; (3) examines the role of Bd reservoir species in causing Bd infections of reintroduced mountain chickens; and (4) determines habitat features that are predictors of infection at release sites. Chytridiomycosis drove the mountain chicken to near extinction on Dominica in 2002 and Montserrat in 2009, in one of the fastest recorded vertebrate species declines, leading to a significant loss of genetic diversity. On Montserrat, treating mountain chickens with an anti-fungal drug (itraconazole) during the chytridiomycosis epidemic improved survival rates and reduced Bd infection rates in the short term, but did not provide long-term protection. Although mountain chickens have been driven to near-extinction by Bd infection on Montserrat, the pathogen persists in two sympatric reservoir species which are not impacted by Bd infection, the most prolific of which (Eleutherodactylus johnstonei) displays strong seasonality in Bd infection prevalence and load. Timing mountain chicken reintroduction to occur during the period when tree frog Bd infection was at its lowest was tested to determine the impact on reintroduction success. Multi-state mark-recapture modelling applied post-release showed that optimising the timing of release reduced Bd infection rates and increased survival. Radio-tracking was utilised with geographic profiling to determine that release site water bodies were likely sources of Bd infection in reintroduced mountain chickens. This could inform targeted mitigation of the pathogen and improve future reintroduction success. Where species have been extirpated in the wild, and an irreversible threat such as an EID persists, novel reintroduction strategies are required. These include optimising the timing and conditions of release in order to minimise the impact of the threat along with targeted mitigation measures such as individual level treatments.

Tree frogs adhere to surfaces using their toe pads. These pads secrete a fluid which enables them to adhere using a temporary adhesive mechanism known as wet adhesion. In keeping with the principles that govern this type of mechanism, the toe pads should be capable of adhering sufficiently and detaching effortlessly. However, the vast majority of research in this area has centred on static experiments, thus giving no real indication of the dynamic activity of the animal. Therefore, one aspect of this PhD project was to investigate the biomechanics of tree frog locomotion, focussing particularly on the kinematics and forces involved during toe pad detachment. Tree frog toe pads do indeed possess a rapid, efficient detachment mechanism, the toe pads simply peeling off of the surface from the rear, requiring very little force. The impact of this at higher scales was reflected in the force profiles of individual limbs, and in the global kinematics of the frog during locomotion. In addition to this, the wet adhesive mechanism itself was investigated by assessing the performance of tree frogs in a range of challenging conditions, including on overhangs and flooded surfaces. The latter condition compromises a key component of the wet adhesive mechanism; however, torrent frogs perpetually clamber across wet rocks in the vicinity of waterfalls, and yet are able to maintain their attachment. Therefore, the second aspect of this project was to compare the adhesive capabilities of tree- and torrent frogs, and propose explanations for the differences in their performance. Whilst comparable on dry, smooth surfaces, the adhesion of torrent frogs on rough, flooded substrates was far superior to that of tree frogs. Several explanations for this are offered, including the utilisation of a greater proportion of their ventral contact area, and a toe pad morphology that is optimised for such conditions. The results of this PhD project may provide a functional and morphological ‘blueprint’ for the development of toe pad mimics that can adhere in wet conditions.

Ranavirus infection has caused severe disease and mass mortality in UK common frogs for more than twenty years resulting in serious declines in some populations. The pathogen has been studied since 1992. These studies generated two valuable resources exploited in this thesis: an archive of tissues and virus isolates and a database of reports from citizen scientists on ranavirus-consistent mortality. The previous studies yielded modest evidence suggesting that introductions from North America initiated ranavirus emergence in the UK, though little else was known about the pattern of introduction or spread. This thesis conducts a more detailed investigation, extending existing knowledge of ranavirus diversity and spread through molecular epidemiology and phylogenetics, an in vivo infection experiment, and in silico models. Non-lethal sampling protocols for ranavirus screening were assessed in a controlled setting and shown to be as effective as traditional protocols. The database of citizen science reports was utilised in spatio-temporal models of the spread of ranavirus disease, finding that ranavirus infection is spreading by transmission between ponds but that new outbreaks are also correlated with both human population density and regional temperatures. The first whole genome sequence from a UK ranavirus is presented. Analysis of the genome shows that it is an isolate of the ranavirus type species, FV3, on the basis of its near identical genome arrangement and a ‘supergene’ phylogenetic analysis. An unexpected finding was evidence for recent lateral transfer of host DNA into the FV3 genome. A candidate gene survey of European ranaviruses revealed considerable diversity that may explain the variation in virulence and host range in Spain. Two proposed new species of Ranavirus are described there - one highly virulent, the other seemingly asymptomatic – and the previously described CMTV is shown to be a likely cause of catastrophic decline across multiple hosts. A lack of monophyly among Spanish ranaviruses and the spatial pattern of incidence suggest recent introduction(s). Together, the evidence presented in this thesis underlines the key role that humans have played in the spread of this group of virulent wildlife pathogens in two European countries.