Anfíbios anuros representam um grupo de vertebrados cujo plano corpóreo apresenta uma série de modificações associadas ao desempenho locomotor através de saltos, sendo estas especializações muito antigas e conservadas filogeneticamente. Embora estudos comparativos venham demonstrando associações entre desempenho de salto e diversidade de habitat e história de vida para esse grupo filogenético, as relações entre o desempenho locomotor e o comportamento antipredatório dentro do contexto da diversificação do uso do micro-habitat permanecem inexploradas. Primeiramente, nós testamos modelos adaptativos de evolução morfológica associados com a diversidade do uso do micro-habitat (aquático, arborícola, fossorial, reofílico e terrestre) em espécies de anuros e foi examinada a relação da distância máxima do salto como uma função dos componentes das variáveis morfológicas e do uso do micro-habitat. Nós também investigamos, tanto em nível intra quanto interespecífico a influência da complexidade do microambiente (arena vazia, arena com folhiços ou arena com folhiço e arbustos) e do tipo de estímulo (aproximação versus toque) na manifestação do comportamento antipredatório em anuros. Adicionalmente, nós investigamos o efeito do uso de diferentes tipos de refúgio pelos anuros quando sujeitos aos testes de simulação predatória no laboratório. Nossos resultados demostram a existência de múltiplos ótimos adaptativos para os comprimentos dos membros associados aos diferentes usos do micro-habitat, com uma tendência de aumento dos membros posteriores em espécies reofílicas, arborícolas e aquáticas quando comparadas com espécies terrestres e fossoriais, as quais evoluíram em direção ao ótimo adaptativo com membros posteriores mais curtos. Além disso, espécies reofílicas, arborícolas e aquáticas apresentaram maior desempenho para o salto e membros posteriores mais longos quando comparadas com espécies terrestres e fossoriais. Em seguida foi abordada a influencia da complexidade do ambiente no comportamento antipredatório e nossas análises intraspecíficas mostraram que o número de respostas ativas (salto) e passivas é fortemente dependente do tipo de estímulo e varia de acordo com a complexidade estrutural do ambiente. Simultaneamente, nossas análises comparativas interespecíficas mostraram que anuros modulam a distância saltada em resposta ao toque do predador de acordo com a complexidade ambiental, e nós ressaltamos uma variação interespecífica associada ao uso de micro-habitat. Por fim, foi investigado o uso de refúgios pelos anuros expostos a testes de simulação predatória. Nossos resultados mostram que os anuros usam a vegetação arbustiva, o folhiço e a água como areas de refúgio quando sujeitadas a simulação predatória. Além disso, ocorreu variação interespecífica na escolha de refúgios potencialmente associados à diversificação do uso de microhábitat / Anuran amphibians represent a group of vertebrates whose body plan presents a series of changes associated with jumping locomotor performance; these specializations are very old and phylogenetically conserved. Although comparative studies have shown associations among jumping performance, habitat diversity and life history for this phylogenetic group, the relationship between locomotor performance and antipredator behavior remains unexplored within the context of diversification of microhabitat use and habitat. First, we tested adaptive models of morphological evolution associated with the diversity of microhabitat use (aquatic, arboreal, fossorial, torrent and terrestrial) in species of anurans and examined the relation of the maximum distance jumped as a function of components of morphological variables and microhabitat use. We also investigated, both at intra and interspecific levels, the influence of the complexity of microenvironment (empty arena, with leaf litter or bushes) and stimulus type (approach versus touch) on the manifestation of antipredator behavior in anurans. In addition, we investigated the effect of the use of different types of refuges by anurans when subjected to simulated predator tests in the laboratory. Our results demonstrate the existence of multiple optima of limb lengths associated to different microhabitats, with a trend of increasing hindlimbs in torrent, arboreal, aquatic species whereas fossorial and terrestrial species evolve toward optima with shorter hindlimbs. Moreover, arboreal, aquatic and torrent anurans have higher jumping performance and longer hindlimbs, when compared to terrestrial and fossorial species. Then, was addressed the influence of the complexity of environment on antipredator behavior and our intraspecific analyzes showed that the number of active responses (jump) and passive responses is strongly dependent on stimulus type and varies according to the structural complexity of the environment. At the same time, our interspecific comparative analyzes showed that anurans modulate the distance jumped in response to the predator\'s touch according to environmental complexity, and we highlight an interspecific variation associated with the use of microhabitat. Lastly, was investigated the use of refuges by anurans exposed to simulated predation events. Our results show that anurans use bushes, leaf litter and water as refuge areas when subjected to simulated predator. Moreover, there is interspecific variation in the choice of refuges potentially associated to diversification of microhabitat use

No description available.

The western toad (Anaxyrus boreas, formerly Bufo boreas) is one of many amphibian species considered to be at risk of extinction (COSEWIC status is Special Concern). I examined habitat use patterns of the western toad using several methods to gain a better understanding of its habitat requirements. I examined the relationship between relative abundance of the western toad and two sympatric amphibian species (wood frog, Lithobates sylvaticus; and boreal chorus frog, Pseudacris maculata) and habitat features at eight scales of spatial extent at 24 wetlands in the Lake Utikuma region of Alberta, Canada. I radio-tracked adult western toads in three study areas in the Aspen Parkland and Boreal regions of north-central Alberta to examine 1) whether patterns of habitat selection change with different scales of spatial extent, spatial resolution, habitat composition, temporal period, and between males and females during the active period, 2) habitat used for hibernation, and 3) factors influencing the timing and nature of movements to hibernation sites. I found that the abundance of the three amphibian species was best described at different spatial extents and was related to the biology of each species. Resource Selection Function (RSF) models, created using radio-telemetry data, indicated that habitat selection was scale-dependent for western toads; differences in selection were observed among study designs, study areas, time periods, and sexes. Predictive ability did not differ significantly among study designs. However, models that were created using a fine-grained map and home-range spatial extent generally produced models with greater predictive ability than models using a coarse-grained map or population-range extent. During the active season toads selected open habitat types such as wet shrub, disturbed grass, and crop/hay fields. Western toads hibernated terrestrially in pre-existing tunnels and the majority of toads hibernated in forest stands dominated by spruce. Toads used hibernation sites 1461936 m from breeding ponds and 68% of hibernacula were communal. Arrival at and entry into hibernation sites was influenced by temperature and/or day length; larger toads moved to hibernation sites later in the year. My research results can be used to identify and protect habitat for western toads in Canada. / Environmental Biology and Ecology

The amphibian decline phenomenon now involves in excess of a third of the roughly 6000 species of amphibians on the planet. The problems that drive the declines are diverse with no end in sight. The Amphibian Conservation Action Plan (ACAP) aims to stem amphibian decline through four recommended actions by researchers and conservation biologists: (1) Expand scientific understanding of amphibian declines and extinctions; (2) continue to document amphibian diversity and ecology and how they are changing; (3) develop and implement long-term conservation programs; (4) prepare emergency response actions for eminent crises. This Dissertation focused on two of those recommendations: expanding scientific understanding of amphibian declines and extinctions and continuing to document amphibian diversity and ecology and how they are changing. The first chapter is a review of the amphibian decline phenomenon. The second, third, and fourth chapters focus on expanding scientific understanding of amphibian diversity and ecology with the description of a formerly unknown species (chapter 2), and ecological papers on two poorly known species (chapters 3 and 4). Chapter five focuses on the first ACAP recommendation in improving scientific understanding of the causes behind amphibian decline. The chapter is an experimental examination of two related species and their developmental reactions to common heavy metal contaminants. The goal of this Dissertation is to contribute toward the general amphibian knowledge base relative to the recommendations of ACAP.

The aim of this thesis was to elucidate the role of carbonic anhydrase (CA) in the modulation of central pH/CO2-sensitive fictive breathing (measured using in vitro brainstem-spinal cord preparations) in leopard frogs (Rana pipiens) following exposure to chronic hypercapnia (CHC) and chronic hypoxia (CH). CHC caused an augmentation in fictive breathing compared to the controls (normoxic normocapnic). Addition of acetazolamide (ACTZ), a cell-permeant CA inhibitor, to the superfusate reduced fictive breathing in the controls and abolished the CHC-induced augmentation of fictive breathing. ACTZ had no effect on preparations taken from frogs exposed to CH. Addition of bovine CA to the superfusate did not alter fictive breathing in any group, suggesting that the effects of ACTZ were due to inhibition of intracellular CA. Taken together, these results indicate that CA is involved in central pH/CO2 chemoreception and the CHC-induced increase in fictive breathing in the leopard frog.

The aim of this thesis was to elucidate the role of carbonic anhydrase (CA) in the modulation of central pH/CO2-sensitive fictive breathing (measured using in vitro brainstem-spinal cord preparations) in leopard frogs (Rana pipiens) following exposure to chronic hypercapnia (CHC) and chronic hypoxia (CH). CHC caused an augmentation in fictive breathing compared to the controls (normoxic normocapnic). Addition of acetazolamide (ACTZ), a cell-permeant CA inhibitor, to the superfusate reduced fictive breathing in the controls and abolished the CHC-induced augmentation of fictive breathing. ACTZ had no effect on preparations taken from frogs exposed to CH. Addition of bovine CA to the superfusate did not alter fictive breathing in any group, suggesting that the effects of ACTZ were due to inhibition of intracellular CA. Taken together, these results indicate that CA is involved in central pH/CO2 chemoreception and the CHC-induced increase in fictive breathing in the leopard frog.

Currently, a variety of methods are available to monitor anurans, and little standardization of methods exists. New methods to monitor anurans have become available over the past twenty years, including PVC pipe arrays used for tree frog capture and Automated Digital Recording Systems (ADRS) used to remotely monitor calling activity. In addition to ADRS, machine-learning computer software, automated vocalization recognition software (AVRS), has been developed to automatically detect vocalizations within digital sound recordings. The use of a combination of ADRS and AVRS shows the promise to reduce the number of people, time, and resources needed for an effective call survey program. However, little research exists that uses the described tools for wildlife monitoring, especially for anuran monitoring. In the study, there were two problems addressed relating to AVRS. The first was the poorly understood relationship between auditory survey methods and physical survey methods. I tested this problem by using current auditory monitoring methods, ADRS and the AVRS Song Scope© (v.3.1), alongside more traditional physical monitoring methods that included drift fences, a PVC pipe array, and visual encounter transects. No significant relationship between physical and auditory community population measures was found. Auditory methods were also effective in the detection of call characteristic differences between urban and rural locations, further suggesting an influence of noise pollution. The second problem addressed was the call identification errors found in auditory survey methods. I examined the influence of treatments including the ADRS location, listener group, species, and season on the error rates of the AVRS Song Scope© (v.3.1) and groups of human listeners. Computer error rates were higher than human listeners, yet less affected by the treatments. Both studies suggested that AVRS was a viable method to monitor anuran populations, but the choice of methods should be dependent upon the species of interest and the objectives of the study.

Frogs, toads, and salamanders are well known for harboring an array of distasteful (and poisonous) secondary metabolites, presumably as antipredator defenses; yet few experiments have rigorously demonstrated the efficacy of amphibian chemical defenses against ecologically relevant consumers. For example, despite an absence of rigorous statistical evidence showing their distastefulness to predators, eastern newts (Notophthalmus viridescens (Rafinesque))--a common salamander in lentic North American habitats--are assumed to tolerate diverse predator assemblages because newts secrete tetrodotoxin (TTX), a neurotoxin. Here we combine laboratory and field-based ecology with bioassay-guided separation of chemical extracts to show that eastern newts--although chemically protected against ecologically important consumers in lentic systems--nonetheless suffer substantial predation when tethered in the field. When offered newts with alternative prey (paedomorphic Ambystoma talpoideum), red swamp crayfish (Procambarus clarkii) and largemouth bass (Micropterus salmoides) were 9-10x as likely to feed on A. talpoideum as newts. Additionally, juvenile bluegill (Lepomis machrochirus) were 70% less likely to consume newt eggs compared to control food pellets. We also show that different newt tissues were differentially palatable to predatory fish. All bluegill tested consumed a palatable control food, but only 20% consumed dorsal skin, only 35% ate ventral skin, but 75% fed on newt viscera, suggesting that deterrent metabolites are concentrated in the skin. Bioassay-guided fractionation revealed that crude and water-soluble newt chemical extracts inhibited bluegill feeding, definitively establishing the chemical nature of newt antipredator defenses, although we were unsuccessful at isolating the chemical compounds responsible for unpalatability. Yet, deterrent activity in the polar but not the lipophilic chemical fraction and bioassay results demonstrating that naıve predators rapidly learn to avoid natural concentrations of TTX support the possible role of TTX in suppressing predation on newts. However, when tethered in the field, newt mortality was 55% higher in ponds with predatory fishes than in ponds lacking fishes (62% vs. 40% respectively), indicating the possible existence of other predators that are resistant to (or tolerant of) newt chemical defenses. Together, these results stress the importance of rigorous, ecologically relevant, and hypothesis-driven experimentation to better understand the complexity of chemically- mediated predator-prey interactions, even for well-studied species like N. viridescens.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

Cannibalism is a seemingly aberrant interaction, appearing counter to the fitness of individuals. Yet cannibalism is not overly uncommon, and naturally occurs among aquatic organisms, including larval amphibians. In temporary wetlands larval amphibians are in a race to complete metamorphosis before their aquatic habitat disappears. When intraspecific competition intensifies, eating conspecifics may represent a beneficial if not necessary strategy. The research presented within this thesis aims to characterize factors that influence cannibalism within populations of larval amphibians. Wood frog tadpoles (Lithobates sylvaticus) were used to test potential benefits of cannibalism as a diet, determine if dietary quality and nutritional stress influence cannibalism, and investigate the roles of competition and chemical cues in influencing cannibalism. Larval long-toed salamanders (Ambystoma macrodactylum), and ringed salamanders (A. annulatum) were used to investigate a functional link between trophic polymorphism and cannibalism in natural populations. Results suggest that perceived increases in competition may stimulate some individuals to become less risk averse, and more aggressive, which may in turn facilitate cannibalistic behaviour. Cannibalism itself provided only conditional benefits to larval wood frogs, rather than the optimal growth that would be expected from an ideal diet. However, this may have been the result of individual variation in response to the diet and/or conspecific cues as opposed to a nutritional deficit. In conditions where tadpoles could perceive increased competition they altered their behaviour and morphology in ways that may improve their foraging success and potentially promote cannibalism. Finally, a functional link appears to exist between head morphology and cannibalism in natural wetlands. However, the appearance of this morphology appears related to conditions that may facilitate increased population densities through rapid pond drying.