The skin is the largest organ of the body and provides many functions. Among tetrapod vertebrates, amphibian skin is semi-permeable and responsible for a greater proportion of water absorption and gas exchange. Myriad factors affect the physiological performance of amphibian skin. Morphological traits linked with amphibian skin physiology or ecology have remained difficult to discern because of a lack of quantitative comparative research and the discovery of sources of intraspecific variation that are mostly ignored in study designs. This thesis aims to address the effects of these sources of variation using a trait that is known to vary between sexes, among seasons, and among body regions and thought to be linked with physiology or ecology, skin thickness. The first source of variation addressed is sexual dimorphism. Specimens of the white-lipped treefrog, Litoria infrafrenata, that display sexual dimorphism in body size and skin thickness were used to test if body size was the main determinate of sexually dimorphic skin thickness. Size corrected values did not significantly differ between males and females, although the sample size was small. Seasonal variation in skin thickness has also been documented in some species, so the American bullfrog (Lithobates catesbeianus), the Northern leopard frog (L. pipiens), and the spring peeper (Pseudacris crucifer) from multiple months of the year were sampled to determine if skin thickness increased in the autumn or winter months. Seasonal skin thickening was only detected in L. catesbeianus, and skin from autumn and winter was significantly thicker than from earlier in the year. This pattern was also detectable in museum specimens collected 80 years ago, although the signal was damped, possibly due to preservation. Using a dataset of 10 species and published data, a general pattern was uncovered whereby the dorsal skin is the thickest region and the ventral thigh region is the thinnest. However, this pattern is not always true for every individual of every species (L. pipiens and P. crucifer) and in some species the dorsal skin is thinnest (Bokermannohyla alvarengai and Litoria infrafrenata). The same dataset found that skin thickness is significantly related to body size, as was found in the chapter on Litoria infrafrenata. Summer specimens of Lithobates catesbeianus were outliers below the interspecific regression line and winter specimens fell within the range of variation of other species, hinting that seasonal skin thickening could be renamed seasonal skin thinning in this species. Finally, a link between ecology and skin thickness was tested using the 10 species from previous analyses and data from the literature. At a phylogenetially broad scale, body size explained a greater amount of the variation in environmental parameters than skin thickness. At smaller taxonomic scales, skin thickness appears more closely linked with ecology. It is concluded that amphibians generally follow an allometric trend for skin thickness and when faced with suboptimal conditions over long periods of time, evolve integumentary structures like iridiophores to compensate for any physiological disadvantage of an ‘ideal’ skin thickness. In the interim, however, skin thickness may change, thus sacrificing e.g. mechanical support.

In 1897 Davenport measured the wet and dry weights of amphibian embryos from the stage of hatching onwards. He observed that there was a continuous increase in the wet weight but that the dry weight remained constant until the embryo began feeding. From this he concluded that "growth is due chiefly to imbibed water". Schaper (1902) noted a similar constancy of dry weight from the early tail bud stages until the time of feeding in embryos of Rana fusca. These early observations have been confirmed by Dempster (1933) who, working with Amblystoma punctatwn, extended his experiments to include the earliest developmental stages. The increase in volume, and hence the growth, of amphibian embryos is therefore due to the uptake of water from the environment. Many embryologists have attempted to correlate this water uptake with the osmotic pressure of the embryos. The early work in this field has been extensively reviewed by Needham (1931).

Tree frogs have evolved specialised toe pads which allow them to efficiently climb vertical surfaces. The toe pad stick by using ‘wet adhesion’ – a combination of forces produced by a thin layer of fluid between the pad and the surface which provide temporary adhesion to allow quick attachment and detachment for climbing. Most studies on tree frogs have been based on their adhesive capabilities on surfaces which are flat, clean and dry (usually glass). However, climbing tree frogs in the wild will come across a variety of surfaces which could affect their adhesive abilities. This PhD investigated whether tree frog adhesion is affected by various ‘challenging’ surfaces, which reflect conditions that tree frogs may encounter whilst climbing. These include rough surfaces, wet conditions, surfaces with loose particulate and hydrophobic surfaces. Experiments were predominantly conducted using a force transducer to measure adhesive and frictional forces of single toe pads, as well as whole animal attachment experiments involving a rotating tilting board. The toe pads of tree frogs were shown to possess a self-cleaning mechanism, whereby the pads will remove contaminants (and subsequently recover adhesive forces) through repeated use, thanks to shear movements of the pad and the presence of pad fluid which aids contaminant deposition. To investigate how torrent frogs (frogs which inhabit waterfalls) can adhere to rough and flooded surfaces, the performance of torrent frogs species Staurois guttatus was compared to a tree frog species (Rhacophorus pardalis). Torrent frogs could produce higher adhesive forces than tree frogs with their toe pads, and possess a specialised toe pad morphology (directional fluid channels on the pad periphery) which may contribute to better performance in flooded conditions. Torrent frogs utilise large areas of ventral skin to stay attached on overhanging surfaces, while tree frogs display a reduction in contact area resulting in a failure to stay attached. This combination of ability and behaviour will help torrent frogs to stay attached on the rough and flooded surfaces that make up their waterfall habitat. On rough surfaces, tree frogs showed improved (compared to smooth surface performance) performance on smaller scale roughness (asperity size <10 µm), and poorer performance on the larger scale roughnesses tested (30 – 425 µm). Interference reflection microscopy (IRM) revealed that larger asperities result in pad fluid being unable to fill the larger gaps of such surfaces, which was confirmed by adding water to rough surfaces to improve attachment performance. The soft pad does however aid in conforming to some rough surfaces, which could account for the better performance on the smaller scale roughness. Many plant surfaces exhibit hydrophobic properties, and so the adhesive performance of tree frogs on hydrophobic surfaces was compared to that on hydrophilic surfaces. It was found that the toe pads could produce similar adhesive and frictional forces on both surfaces. The pad fluids contact angles were then measured on hydrophobic surfaces using IRM, where droplets of pad fluid formed lower contact angles (and are therefore exhibiting higher wettability) than water. Though the exact composition of pad fluid is unknown, some form of surfactant must be present which aids wetting of surfaces (either a surface modification or detergent present in the fluid) to allow wet adhesion to occur - goniometer experiments of water on dried footprints on hydrophobic surfaces confirmed this. The ability to stick in a variety of conditions could provide inspiration for ‘smart’ adhesives, which mimic the adaptable adhesion of tree frog toe pads.

Amphibians are facing an extinction crisis, and in many cases it has become necessary to conserve species in captivity. Unfortunately, our understanding of the nutritional requirements of amphibians in captivity is currently limited. There have been anecdotal reports that captive bred amphibians have duller skin colouration than wild members of the same species. Many amphibians use carotenoid pigments in skin colouration. Carotenoids are yellow-red pigments synthesised by photosynthetic tissues, however vertebrates cannot synthesise them de novo and rely entirely on dietary sources. Reduced colour in captive bred amphibians could therefore result from limited carotenoid availability in the diet. Limited access to carotenoids could have further negative consequences on health and reproductive success since carotenoids are known to function in the immune, antioxidant and reproductive systems of other vertebrate taxa. The role of carotenoids in amphibian fitness is currently unknown, and the aim of this PhD was to assess the importance of carotenoids to anuran amphibians. Skin colour may be influenced by carotenoid availability, and it was therefore important to establish a suitable method for quantifying skin colour in amphibians. In Chapter 1 I assessed two methods for colour quantification, including digital photography and spectrometry. Neither method was significantly more accurate than the other, however I chose to use photography for my studies for reasons of practicality. In Chapters 2 and 3 I assessed the role of carotenoids in the reproductive systems of wild frogs. I found that carotenoid-based skin colouration was involved in the breeding behaviour of wild Agalychnis moreletii frogs. I found positive assortative mating by colour in that frogs were found mating with individuals with similar carotenoid-based colouration. This assortative mating by colour may be driven by mutual mate choice, or may have evolved to prevent mismatched pairing with a sympatric and phenotypically similar species (A. callidryas). I then analysed the carotenoid composition of eggs produced by wild A. moreletii and Phyllomedusa trinitatis, which are tree frogs with similar reproductive ecology but different egg deposition strategies: Agalychnis moreletii deposits green eggs on the surface of leaves while P. trinitatis wraps pale cream eggs in leaves. The concentration and diversity of carotenoids was significantly higher in A. moreletii than P. trinitatis and potential ecological explanations for this are discussed. In Chapter 4 I investigated carotenoid availability in the diet of captive amphibians by analysing the carotenoid composition of commonly used feeder-invertebrates. I assessed interspecific variation in carotenoid accumulation in three feeder-cricket species (Gryllus bimaculatus, Gryllodes sigillatus and Acheta domesticus), which were all fed one of three diets (wheat-bran, fish-food based diet, fresh fruit and vegetables). All three cricket species were a poor source of carotenoids unless they were gut-loaded on a carotenoid-rich diet. Nutrient retention over a two-day period was poor across species. Finally, there were significant interspecific differences in gut-loading capacity, with G. bimaculatus having the greatest capacity. This study shows that carotenoid availability to captive amphibians will be limited unless they are fed specific feeder invertebrate species, recently gut-loaded on carotenoid-rich foods. In Chapters 5, 6 and 7 I assessed the influence of carotenoids on fitness related traits in captive anurans. I firstly assessed colour degradation in captive-reared A. moreletii frogs and looked at whether skin colour could be improved through increased carotenoid availability in the diet. I found that carotenoid-based skin colour degraded quickly in captivity but could be improved through dietary supplementation with carotenoids, however there was a critical period during post-metamorphic growth for deposition of carotenoids in the skin. Next, I investigated the effect of carotenoids on larval growth and development, and post-metamorphic growth, development, skin colour and reproductive success in captive A. callidryas frogs. I found no direct effect of carotenoids during larval stages, however there were carry-over effects on post-metamorphic growth and skin colour. Increased post-metamorphic carotenoid availability positively influenced growth, skin colour and reproductive success. Similarly to A. moreletii, there was a critical period during post-metamorphic growth for deposition of carotenoids in the skin. Finally, I examined the protective potential of carotenoids in Silurana (Xenopus) tropicalis tadpoles that were exposed to ultraviolet (UV) light. Exposure to UV light significantly reduced tadpole survival and there was no evidence that carotenoids afforded any protection. Nevertheless, in tadpoles that were not exposed to enhanced UV light, carotenoids increased survival. Furthermore, carotenoids had a significantly positive effect on the developmental rate of tadpoles in both UV limited and UV enhanced environments. The studies in this PhD show that carotenoid availability has important consequences on fitness related traits in anuran amphibians, which include effects on growth, skin colour, reproductive success and survival. Carotenoid availability to captive amphibians will, however, be low unless they are provided with feeder invertebrates that have been recently gut-loaded on carotenoid-rich foods. These findings should be considered when recommendations are made on the nutritional requirements of amphibians in captivity as they could significantly improve the fitness of captive-bred individuals, and thus increase the success of ex situ conservation programmes.

At the outset of my PhD, my aim was to investigate the mechanisms responsible for the directed migration of primitive myeloid cells (PMCs) to wounds in Xenopus embryos. PMCs are the first blood cells to differentiate and become functional in Xenopus embryos, and have a notable migratory ability to be recruited by embryonic wounds before a functional vasculature is established. To find the mechanism underlying PMCs migration toward embryonic wounds, I first performed a screen to identify candidate cytoskeleton related genes, which might be responsible for facilitating the inflammatory response to injury in embryos. In situ hybridization and RT-PCR showed that coronin1a and l-plastin were specifically expressed in PMCs. I carried out loss-of-function experiments for coronin 1a and l-plastin in Xenopus embryos. Unfortunately neither knockdown affected the ability for PMCs to migrate during embryonic development or during the wound healing process. Loss-of-function experiments on coronin 1a and l-plastin also did not affect epidermal wound closure speed. Thus, although coronin 1a and l- plastin are expressed specifically in PMCs, they do not appear to be necessary for the migration of PMCs during development and during wound healing in Xenopuos embryos. Since my initial aim failed to provide insight into the mechanisms that mediate 9the inflammatory response to embryonic wounds, I decided to investigate the function of a previously identified monomeric actin protein during embryonic wound healing and appendage regeneration: namely Thymosin beta4 (Tβ4). In situ hybridization experiments showed that Tβ4 is expressed exclusively in the epidermis of developing frog embryos. Tβ4 knockdown embryos resulted in a significantly delay in the speed of wound closure during the early phase of wound healing. This delay correlated with a decrease in the actin contractile ring at the wound margin. Furthermore I found that the cell shapes of epidermal cells in the Tβ4 knockdown embryos were different from epidermal cells in control embryos. I hypothesize that this reduction caused the actin filaments changes in the epidermal cells, and were responsible for the failure of the cells to form an actin contractile ring, thus delaying the initial speed of wound closure. I tried to confirm that most of these defects specific to Tβ4, by performing rescue experiments with Tβ4 mRNA injections. Furthermore, I discovered that Tβ4 knockdown embryos displayed defects in tail development, including the absence of blood vessel branching within the fin of the tail. Finally, I found that the tails in Tβ4 knocked-down tadpoles failed to regenerate, while tails in control embryos regenerated completely following amputation. Both in situ hybridization and real-time PCR showed that Tβ4 was up regulated in the regenerated part of the tail in Xenopus tadpoles. Together with the tail amputation results, Tβ4 might be important for tail development and regeneration. These findings suggest that Tβ4 might play an important roles in the modulation of the actin cytoskeleton, which are essential for the proper behavior of epidermal cells during wound healing and appendage regeneration.

No description available.

Concern over unexplained population declines in the common toad (Bufo bufo) has led to it being recognised as a priority species for conservation research. The general consensus among herpetologists is that the most important cause of the declines seen as part of a global amphibian extinction crisis is habitat loss and degradation. The aim of thesis is to investigate the effect of habitat availability and quality on common toad populations in the United Kingdom, with a particular emphasis on the effect of land under agricultural use. I use occupancy modelling, resource selection function modelling and genetic techniques to determine the effects of both terrestrial and aquatic habitat on common toads. Based on my findings, I consider the impacts of habitat at local, landscape and national scales. My research revealed the importance of pond density and presence of woodland in increasing relative probability of toad occurrence in the terrestrial habitat (Chapters 2 and 3) and the importance of water quality, woodland coverage and available terrestrial habitat coverage in determining common toad presence in ponds. The isolating effect of urban areas is demonstrated in Chapter 4 where Isolation By Barriers (IBB) explains genetic distance between common toad breeding populations better than Isolation By Distance (IBD). In Chapter 5 I again use the resource selection function for terrestrial habitat use developed in Chapter 3, to investigate the importance of terrestrial habitat availability in determining pond use by common toads at a national scale. In my concluding chapter, I make recommendations for management and further research including consideration of potential interactions between the effect of habitat loss, fragmentation and degradation with other potential causes of common toad decline.

My thesis has accomplished 3 significant contributions to neuroscience. Firstly, I have discovered a novel example of vertebrate deep-brain photoreception. Spontaneously generated fictive locomotion from the isolated nervous system of pro-metamorphic Xenopus tadpoles is sensitive to the ambient light conditions, despite input from the classical photoreceptive tissues of the retina and pineal complex being absent. The photosensitivity is found to be tuned to short wavelength UV light and is localised to a small region of the caudal diencephalon. Within this region, I have discovered a population of neurons immuno-positive for a UV-specific opsin protein, suggesting they are the means of phototransduction. This may be a hitherto overlooked mechanism linking environmental luminance to motor behaviour. Secondly, I have advanced the collective knowledge of how both nitric oxide and dopamine contribute to neuromodulation within motor control systems. Nitric oxide is shown to have an excitatory effect on the occurrence of spontaneous locomotor activity, representing a switch in its role from earlier in Xenopus development. Moreover, this excitatory effect is found to be mediated in the brainstem despite nitric oxide being shown to depolarise spinal neurons. Thirdly, I have developed a new preparation for patch-clamp recording in pro-metamorphic Xenopus tadpoles. My data suggest there are several changes to the cellular properties of neurons in the older animals compared with the embryonic tadpole; there appears to be an addition of Ih and K[sub](Ca) channels and the presence of tonically active and intrinsically rhythmogenic neurons. In addition, I have shown that at low doses dopamine acts via D2-like to hyperpolarise the membrane potential of spinal neurons, while at higher doses dopamine depolarises spinal neurons. These initial data corroborate previously reported evidence that dopamine has opposing effects on motor output via differential activation of dopamine receptor subtypes in Xenopus tadpoles.

Mutation within the human ALS2 gene, which encodes the protein Alsin, causes a number of recessive motor neuron diseases. The ALS2 gene encodes a 180kDa protein, which has been shown to localize to early endosomes. The Alsin protein comprises three predicted guanine exchange factor (GEF) domains, the best characterised of which is the VPS9 domain for Rab5 GTPase, which is involved in the endocytosis membrane trafficking pathway, particularly in the docking and fusion of early endosomes. Furthermore, Alsin contains a Rho-GEF domain which specifically interacts with Rac-1 GTPase in the PI3K/AKT signal transduction pathway. This pathway has been implicated in numerous biological processes, including control of protein translation, via the mTOR branch of the pathway. To date, most work on the human ALS2 disease phenotypes has focused on the role of alsin in membrane trafficking, and neglected alsin’s potential role in signalling via its Rho-GEF domain. The focus of this project was to study the role of alsin in signalling during early Xenopus development, a period rich in well-characterised cell-cell signalling. I have shown that alsin is maternally loaded and zygotically expressed in the early Xenopus embryo. In cell culture, alsin is localised to early endosomes. Knockdown of alsin protein through the use of mopholinos (MO), resulted in a gastrulation defect, in particular, failure to close the blastopore caused by disrupted mesoderm induction and convergent extension movements. An animal cap assay was used to study mesoderm induction in the presence of als2-MO and activin protein, a potent mesoderm inducer. These animal caps extended normally, indicating proper mesoderm induction. By contrast, als2-MO animal caps failed to extend when co-injected with activin mRNA suggesting that alsin is important for the production and/or secretion of the activin ligand in the source cell. Subsequently it was determined that knockdown of alsin reduced the precursor protein levels of TGF-β family members activin and Xnr-2. These results suggest a novel role for alsin in mRNA stability, translational regulation or post-translational control of specific mesoderm-inducer mRNAs.