The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

Evolution can occur rapidly, along timescales that are traditionally regarded as 'ecological'. Despite growing acceptance among biologists of rapid evolution, a strong paradigm of contemporary evolution is still absent in many sub-disciplines. Here I apply a contemporary evolution viewpoint to conservation biology. Specifically, I examine the impact of cane toads (Bufo marinus) on Australian snakes. Toads were introduced into Australia in 1935, have spread rapidly and represent a novel, extremely toxic prey item to na�ve Australian predators (including snakes). Based on dietary preferences and geographic distributions I find that 49 species of Australian snake are potentially at risk from the invasion of the toad. Furthermore, examination of physiological resistance to toad toxin in 10 of these �at risk� species strongly suggests that most species of Australian snake are poorly equipped to deal with a likely dose of toad toxin. Even species that are highly resistant to toad toxin (such as the keelback, Tropidonophis mairii) face indirect fitness costs associated with consuming toads. Within a population of snakes however, the impact of toads is unlikely to be random. For example, the examination of several component allometries describing the interaction between snakes and toads revealed that, within a species, smaller snakes are more likely to ingest a fatal dose of toad toxin than are larger snakes. Further consideration of the interaction between snakes and toads suggests that toads will not only be exerting differential impact on snakes based upon morphology, but also exert non-random selection on prey preference and resistance to toad toxin in snake populations. To examine the possibility of a morphological response by snakes to toads, I examined changes in the body size and relative head size of four species of snake as a consequence of time since exposure to toads. Two of the species (green treesnakes and red-bellied blacksnakes) are predicted to face strong impacts from toads. These two species showed an increase in mean body size and a decrease in relative head size as a consequence of time since exposure to toads; both changes in an adaptive direction. In contrast, the other two species (keelbacks and swampsnakes) are predicted to face much lower impact from toads, and these two species showed little or no evidence of morphological change associated with time since exposure to toads. These results indicate an adaptive change in morphology at a rate that is proportional to the predicted level of impact for each species, strongly suggesting an evolved response. Red-bellied blacksnakes (a toad-vulnerable species) were further assessed for evolved responses in prey preference and toxin resistance. Comparisons between toad-exposed and toad-na�ve populations of blacksnakes revealed that snakes from toad-exposed populations exhibited slightly higher resistance to toad toxin and a much-reduced tendency to eat toads, when compared with toad-na�ve snakes. Na�ve snakes exhibited no tendency to learn avoidance of toxic prey, nor were they able to acquire resistance to toxin as a result of several sub-lethal doses, suggesting that the observed differences between populations is evolved rather than acquired. Together, these results strongly suggest that blacksnakes are exhibiting an evolved shift in prey preference and toxin resistance as a consequence of exposure to toads. Thus, it appears that snakes are exhibiting adaptation at multiple traits in response to exposure to toads. Given the high likelihood that these adaptive shifts have an evolved basis, it appears that the impact of toads will decrease with time in many snake populations. But what about toads? Because the outcome of the interaction between a toad and a snake is also mediated by the body size and relative toxicity of toads, it is important to understand how these traits vary in space and time. Exploratory analysis revealed that toads exhibit a decrease in body size and a decrease in relative toxicity as a consequence of time since colonisation, indicating that their impact on native predators decreases with time. Additionally, there appears to be meaningful spatial variation in toad relative toxicity, indicating that some populations of native predators are facing higher impact from toads than others. Overall, these results clearly indicate the importance of assessing the potential for rapid evolutionary response in impacted systems. Doing so may provide evidence that some species are in less trouble than originally thought. Additionally, and as more data accumulate, it may be possible to characterise certain categories of environmental impact by their potential for eliciting adaptive response from �impacted� species. This approach has strong implications for the way conservation priorities are set and the way in which conservation dependent populations are managed.

Amphibians are declining dramatically around the globe, due primarily to two major threats: habitat destruction and emerging infectious diseases. The western toad, Bufo boreas, is an IUCN red-listed species thought to be affected by both these factors. The objectives of this thesis were to (1) determine how forest fragmentation affects western toad movement behaviour; and (2) determine if there is any evidence of the infectious disease, the chytrid fungus (Batrachochytrium dendrobatidis), in this species in British Columbia. I used radio-telemetry to follow daily movement patterns of 23 adult male toads in a fragmented landscape near Vancouver, BC, composed of forest patches and small, recent clear-cuts. Movement parameters were analyzed using a Geographic Information System (GIS) and mixed effects models. Results showed that toads in forests were able to perceive clear-cut edges from as far as 150 m. Greater than 60% of toads released in forest patches actively chose to enter the clear-cuts from adjacent forests, indicating high boundary permeability. In addition, toad movement parameters were not significantly reduced in these disturbed environments, suggesting that clear-cuts do not restrict movement. Although toads appear to favour these disturbed environments, further research is required in larger, more realistically sized clear-cuts. To investigate the prevalence of the chytrid fungus in southwestern British Columbia, I tested a breeding population of western toads in a protected area. Results showed a 28% infection rate, although no toads showed any visual signs of the disease. These results provide the first evidence of the chytrid fungus in western toads in British Columbia and suggest that the disease is potentially more widespread that currently documented. The results of my research suggest that the western toad may not be affected by small scale forest harvesting. In addition, my results support the hypothesis that western toads are reservoirs for the chytrid fungus. Although most populations in Canada appear to be faring well, long-term population monitoring and further testing for chytrid prevalence are required. To minimize the likelihood of western toads experiencing the dramatic declines that are presently taking place in the United States, protecting vital habitats and preventing the spread of chytrid through public awareness are required.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2014. Title from PDF t.p. Includes bibliographical references.

Amphibians are declining dramatically around the globe, due primarily to two major threats: habitat destruction and emerging infectious diseases. The western toad, Bufo boreas, is an IUCN red-listed species thought to be affected by both these factors. The objectives of this thesis were to (1) determine how forest fragmentation affects western toad movement behaviour; and (2) determine if there is any evidence of the infectious disease, the chytrid fungus (Batrachochytrium dendrobatidis), in this species in British Columbia. I used radio-telemetry to follow daily movement patterns of 23 adult male toads in a fragmented landscape near Vancouver, BC, composed of forest patches and small, recent clear-cuts. Movement parameters were analyzed using a Geographic Information System (GIS) and mixed effects models. Results showed that toads in forests were able to perceive clear-cut edges from as far as 150 m. Greater than 60% of toads released in forest patches actively chose to enter the clear-cuts from adjacent forests, indicating high boundary permeability. In addition, toad movement parameters were not significantly reduced in these disturbed environments, suggesting that clear-cuts do not restrict movement. Although toads appear to favour these disturbed environments, further research is required in larger, more realistically sized clear-cuts. To investigate the prevalence of the chytrid fungus in southwestern British Columbia, I tested a breeding population of western toads in a protected area. Results showed a 28% infection rate, although no toads showed any visual signs of the disease. These results provide the first evidence of the chytrid fungus in western toads in British Columbia and suggest that the disease is potentially more widespread that currently documented. The results of my research suggest that the western toad may not be affected by small scale forest harvesting. In addition, my results support the hypothesis that western toads are reservoirs for the chytrid fungus. Although most populations in Canada appear to be faring well, long-term population monitoring and further testing for chytrid prevalence are required. To minimize the likelihood of western toads experiencing the dramatic declines that are presently taking place in the United States, protecting vital habitats and preventing the spread of chytrid through public awareness are required.

Evolution can occur rapidly, along timescales that are traditionally regarded as 'ecological'. Despite growing acceptance among biologists of rapid evolution, a strong paradigm of contemporary evolution is still absent in many sub-disciplines. Here I apply a contemporary evolution viewpoint to conservation biology. Specifically, I examine the impact of cane toads (Bufo marinus) on Australian snakes. Toads were introduced into Australia in 1935, have spread rapidly and represent a novel, extremely toxic prey item to na�ve Australian predators (including snakes). Based on dietary preferences and geographic distributions I find that 49 species of Australian snake are potentially at risk from the invasion of the toad. Furthermore, examination of physiological resistance to toad toxin in 10 of these �at risk� species strongly suggests that most species of Australian snake are poorly equipped to deal with a likely dose of toad toxin. Even species that are highly resistant to toad toxin (such as the keelback, Tropidonophis mairii) face indirect fitness costs associated with consuming toads. Within a population of snakes however, the impact of toads is unlikely to be random. For example, the examination of several component allometries describing the interaction between snakes and toads revealed that, within a species, smaller snakes are more likely to ingest a fatal dose of toad toxin than are larger snakes. Further consideration of the interaction between snakes and toads suggests that toads will not only be exerting differential impact on snakes based upon morphology, but also exert non-random selection on prey preference and resistance to toad toxin in snake populations. To examine the possibility of a morphological response by snakes to toads, I examined changes in the body size and relative head size of four species of snake as a consequence of time since exposure to toads. Two of the species (green treesnakes and red-bellied blacksnakes) are predicted to face strong impacts from toads. These two species showed an increase in mean body size and a decrease in relative head size as a consequence of time since exposure to toads; both changes in an adaptive direction. In contrast, the other two species (keelbacks and swampsnakes) are predicted to face much lower impact from toads, and these two species showed little or no evidence of morphological change associated with time since exposure to toads. These results indicate an adaptive change in morphology at a rate that is proportional to the predicted level of impact for each species, strongly suggesting an evolved response. Red-bellied blacksnakes (a toad-vulnerable species) were further assessed for evolved responses in prey preference and toxin resistance. Comparisons between toad-exposed and toad-na�ve populations of blacksnakes revealed that snakes from toad-exposed populations exhibited slightly higher resistance to toad toxin and a much-reduced tendency to eat toads, when compared with toad-na�ve snakes. Na�ve snakes exhibited no tendency to learn avoidance of toxic prey, nor were they able to acquire resistance to toxin as a result of several sub-lethal doses, suggesting that the observed differences between populations is evolved rather than acquired. Together, these results strongly suggest that blacksnakes are exhibiting an evolved shift in prey preference and toxin resistance as a consequence of exposure to toads. Thus, it appears that snakes are exhibiting adaptation at multiple traits in response to exposure to toads. Given the high likelihood that these adaptive shifts have an evolved basis, it appears that the impact of toads will decrease with time in many snake populations. But what about toads? Because the outcome of the interaction between a toad and a snake is also mediated by the body size and relative toxicity of toads, it is important to understand how these traits vary in space and time. Exploratory analysis revealed that toads exhibit a decrease in body size and a decrease in relative toxicity as a consequence of time since colonisation, indicating that their impact on native predators decreases with time. Additionally, there appears to be meaningful spatial variation in toad relative toxicity, indicating that some populations of native predators are facing higher impact from toads than others. Overall, these results clearly indicate the importance of assessing the potential for rapid evolutionary response in impacted systems. Doing so may provide evidence that some species are in less trouble than originally thought. Additionally, and as more data accumulate, it may be possible to characterise certain categories of environmental impact by their potential for eliciting adaptive response from �impacted� species. This approach has strong implications for the way conservation priorities are set and the way in which conservation dependent populations are managed.

Thesis (Ph.D.)--University of Western Australia, 2003.

Amphibians are declining dramatically around the globe, due primarily to two major threats: habitat destruction and emerging infectious diseases. The western toad, Bufo boreas, is an IUCN red-listed species thought to be affected by both these factors. The objectives of this thesis were to (1) determine how forest fragmentation affects western toad movement behaviour; and (2) determine if there is any evidence of the infectious disease, the chytrid fungus (Batrachochytrium dendrobatidis), in this species in British Columbia. I used radio-telemetry to follow daily movement patterns of 23 adult male toads in a fragmented landscape near Vancouver, BC, composed of forest patches and small, recent clear-cuts. Movement parameters were analyzed using a Geographic Information System (GIS) and mixed effects models. Results showed that toads in forests were able to perceive clear-cut edges from as far as 150 m. Greater than 60% of toads released in forest patches actively chose to enter the clear-cuts from adjacent forests, indicating high boundary permeability. In addition, toad movement parameters were not significantly reduced in these disturbed environments, suggesting that clear-cuts do not restrict movement. Although toads appear to favour these disturbed environments, further research is required in larger, more realistically sized clear-cuts. To investigate the prevalence of the chytrid fungus in southwestern British Columbia, I tested a breeding population of western toads in a protected area. Results showed a 28% infection rate, although no toads showed any visual signs of the disease. These results provide the first evidence of the chytrid fungus in western toads in British Columbia and suggest that the disease is potentially more widespread that currently documented. The results of my research suggest that the western toad may not be affected by small scale forest harvesting. In addition, my results support the hypothesis that western toads are reservoirs for the chytrid fungus. Although most populations in Canada appear to be faring well, long-term population monitoring and further testing for chytrid prevalence are required. To minimize the likelihood of western toads experiencing the dramatic declines that are presently taking place in the United States, protecting vital habitats and preventing the spread of chytrid through public awareness are required. / Forestry, Faculty of / Graduate

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.

O principal objetivo deste trabalho foi estudar a variação morfológica do grupo Bufo granulosus, procurando determinar suas unidades evolutivas. Esse grupo, como concebido até o presente, é comporto por 6 espécies (Bufo granulosus, Bufo bergi, Bufo dorbignyi, Bufo fernandezae, Bufo pygmaeus e Bufo beebei), e 10 subespécies de Bufo granulosus (merianae, nattereri, mini, major, barbouri, lutzi, mirandaribeiroi, azarai, humboldti, goeldii). A distribuição geográfica do grupo é bastante ampla e associada a formações abertas, compreendendo a América Central (Panamá) e quase todos os países da América do Sul, com exceção do Chile e do Equador. Foram analisados cerca de 8700 exemplares depositados em diversas coleções nacionais e estrangeiras de cerca de 880 localidades distintas. Caracteres apontados nas descrições das subespécies do grupo e outros, levantados no decorrer no trabalho, foram conferidos com base no estudo de séries de exemplares, comparados por localidade. Foram tomadas medidas de 23 caracteres morfométricos em 2267 indivíduos, utilizados em uma análise estatística multivariada, juntamente com 9 proporções corporais escolhidas como forma de caracterizar os táxons do grupo granulosus. Caracteres osteológicos e miológicos também foram estudados, e foi realizada uma análise das vocalizações (canto de anúncio) disponíveis para o grupo. São reconhecidas 12 espécies para o grupo granulosus: Bufo granulosus (Sudeste e Nordeste do Brasil); Bufo pygmaeus (Rio de Janeiro); Bufo azarai (Argentina, Paraguai e Mato Grosso do Sul); Bufo mirandaribeiroi (Brasil Central); Bufo major (Argentina, Bolívia, Paraguai e Amazônia, Brasil); Bufo fernandezae (Sul do Brasil, Argentina, Uruguai); Bufo dorbignyi (Sul do Brasil, Argentina, Uruguai); Bufo bergi (Argentina, Paraguai, Mato Grosso do Sul); Bufo nattereri (Roraima); Bufo humboldti (Colômbia, Venezuela, Guiana, Suriname); Bufo merianae (Amazônia, Venezuela, Guiana, Suriname); Bufo sp.n. (Panamá). / The Bufo species of the granulosus group are taxonomically reviewed in order to determine their evolutionary units. The granulosus group is composed by 6 species (Bufo granulosus, Bufo bergi, Bufo dorbignyi, Bufo fernandezae, Bufo pygmaeus and B. beebei), and 10 subspecies of Bufo granulosus (merianae, nattereri, mini, major, barbouri, lutzi, mirandaribeiroi, azarai, humboldti, goeldii). The group presents a wide geographical distribution, occurring in Central America (Panama) and in almost all the countries of South America, except for Chile and Ecuador. A total of 8700 specimens deposited in several national and foreign collections were studied representing about 880 different localities. External morphological caracters currently cited in the literature, and others, chosen at the beginning of the work, were compared within the sample series for each locality. Measurements of 23 morphometric characters were taken on 2267 specimens, and were used in an multivariate statistical analysis along with 9 body ratios chosen as a form of characterizing the taxa of the group. Osteological and miological caracters were also analyzed within the taxa, and an analysis of the vocalization (advertisement call) was performed with the available recordings for the group. Twelve species are recognized for the granulosus group, and one is new: Bufo granulosus (Southeast and Northeast Brazil); Bufo pygmaeus (Rio de Janeiro State, Brazil); Bufo azarai (Argentina, Paraguay and Mato Grosso do Sul, Brazil); Bufo mirandaribeiroi (Central Brazillian Savannas); Bufo major (Argentina, Bolivia, Paraguay and Amazonia, Brazil); Bufo fernandezae (South Brazil, Argentina, Uruguay); Bufo dorbignyi (South Brazil, Argentina, Uruguay); Bufo bergi (Argentina, Paraguay and Mato Grosso do Sul); Bufo nattereri (Roraima State, Brazil); Bufo humboldti (Colombia, Venezuela, Guyana, Surinam); Bufo merianae (Amazonia, Venezuela, Guyana, Surinam); Bufo sp. n. (Panama).