Thesis (M.S.) -- Central Connecticut State University, 2006. / Thesis advisor: Tiffany M. Doan. "... in partial fulfillment of the requirements for the degree of Master of Science in Biology." Includes bibliographical references (leaves 44-49). Also available via the World Wide Web.

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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

Currently published data on the role of the lymphatic system in amphibians are inadequate and contradictory. Estimates of the rate of formation of lymph and the role of the lymph hearts in returning this fluid to the circulation are not based on actual volume determinations but rather estimates derived from changes in hematocrit using published values of plasma and blood volume. The lymph hearts are known to be vital to the maintenance of normal fluid compartment physiology and to increase their rate of activity during episodes of hypovolemic stress. Yet, significant redistribution of body fluids following hemorrage appears to occur in animals without lymph hearts. In this study, plasma and blood volumes were determined by the dye dilution technique using injected Evan's blue dye to label the plasma. Eight intact and 6 animals with their lymph hearts destroyed were hemorrhaged to 78% and 75% of their initial blood volumes, respectively. Changes in blood volume were measured following the hemorrhage by analysis of Evan's blue washout and hemodilution.

The present study documents that baroreceptors located in the pulmocutaneous artery (PCA) are key components in control of lymph heart rate in amphibians. A negative feedback control loop exists between arterial pressure and lymphatic heart rate. The recurrent laryngeal nerve (rLN), which innervates the PCA baroreceptors, transmits information on arterial pressure to integration centers in the central nervous system. Lymphatic heart rate (LHR) is reduced as a result of increases in arterial pressure. This loop was determined using three experimental protocols. First, the correlation between LHR reduction and hormonally induced vasoconstriction was determined. Increases in arterial pressure due to pressor actions of angiotensin II and arginine vasotocin at high concentrations was negatively correlated to LHR. Second, lymphatic heart rate changes due to natural increases in arterial pressure were compared to rate changes due to increase in arterial pressure after bilateral denervation of the rLN. Post-denervation LHR was not affected by natural increase in arterial pressure prior to the establishment of a new resting arterial pressure. Increase in arterial pressure due to administration of vasoconstricting hormones was negatively correlated with LHR following denervation. Third, the effect on LHR due to direct stimulation of the rLN was studied. Stimulation of the rLN caused LHR to stop without increases in arterial pressure. Presumably, this negative feedback loop is present to limit fluid return to the cardiovascular system from the lymphatic system during periods of acute hypertension. Reduction in the return of lymph volume to the cardiovascular system could eliminate potential damage to pulmonary tissues due to high arterial pressures.

Many plants and animals are defended by toxic compounds, and circumvention of those defenses often has involved the evolution of elaborate mechanisms for tolerance or resistance of the toxins. Toads synthesize potent cardiotonic steroids known as bufadienolides (BDs) from cholesterol and store those toxins in high concentrations in their cutaneous glands. Those toxins protect toads from the majority of predators, including most snakes that readily consume other species of frogs. BDs exert their effect by inhibiting ion transport by the Na+/K+-ATPase (NKA). This ubiquitous transmembrane enzyme consists of a catalytic alpha-subunit, which carries out the enzyme's functions, and a glycoprotein beta-subunit, which provides structural stability. Inhibition of the NKA causes highly elevated intracellular Ca2+ levels and results in often lethal increased cardiac contraction strength. Molecular resistance to bufadienolides in snakes is conferred by mutations in the alpha-subunit of the Na+/K+-ATPase. I have found that these mutations are more prevalent in snakes than previously suggested, and that many genetically resistant species do not feed on toads. This suggests that possession of the mutations alone does not carry substantial negative consequences, and that feeding on toads may have been an ancestral habit in some groups of snakes. I have further found evidence of tissue-specific variation in resistance to bufadienolides, and gene expression investigations revealed that the bufadienolide resistance-conferring mutations are not expressed equally among different organs. Variation in resistance among different tissues indicates that possession of the mutations does not protect all cells equally. Finally, by testing the physiological responses of resistant snakes to exposure to cardiotonic steroid, I have found that feeding on toads incurs negative consequences and that toad-specialized resistant snakes respond differently from nontoad-specialized resistant snakes. The presence of physiological consequences of toxin exposure may explain why feeding on toads has been lost in some lineages of snakes that retain resistance-conferring mutations. In summary, these findings indicate that genetic resistance of the Na+/K+-ATPase is necessary in order for snakes to survive acute toxicity of bufadienolides, but it is not sufficient to explain fully the physiological mechanisms involved in dealing with chronic exposure to the toxins.

Habitat requirements and habitat use for Pseudophryne australis and Heleioporus australiacus were investigated to aid management of these threatened frogs around Sydney, Australia. Much of the work focussed on roads, commonly encountered features in the habitat of both species. The habitat requirements based on locality records of both frogs in the Sydney Basin were investigated at four spatial scales. Both species are habitat specialists. They showed a strong geological association with Hawkesbury Sandstone and occupy upper topographic areas with ephemeral watercourses of gentle gradients. Both frogs occur predominantly in areas of higher precipitation and milder temperature regimes compared to averages representative of the region. Leaf litter is an important feature of P. australis breeding sites, whereas H australiacus generally associate with crayfish burrows. Both species are dependent on natural vegetation with a complex structure. H australiacus have a relatively long larval period (3 - 12 months) and breed in ephemeral pools, exposing their tadpoles to the risk of dying due to early pond drying. In the laboratory, tadpoles responded to decreasing water levels by shortening their larval periods and metamorphosing earlier than siblings held at constant water level. Despite this plastic response, a number of pools in the field failed to produce metamorphs due to early drying, an observation also made on P. australis. Regular monitoring of breeding sites revealed increased reproductive success away from roads for both species probably because of relatively longer hydroperiods. Spatial distributions and associations with habitat features, and movement patterns of both frogs were further investigated using mark-recapture methods. Both species showed strong site fidelity. P. australis formed small aggregations and predominantly selected leaf litter piles despite their relatively low availability. Leaf litter piles in creeks moved over time and the animals moved with these piles. In contrast, H australiacus individuals formed no aggregations and showed no preference for any available structural vegetation type. Locations of individuals were independent of relative distances to creeks and artificial drains, but males appeared to be more common near culverts. However, individuals were randomly distributed in space and nearest-neighbour distances were high relative to individual movement distances, suggesting minimal overlap between relatively large home ranges. Radio-telemetry demonstrated that some H australiacus individuals burrow in the road environment. There they would be at risk of being dug up and possibly injured during road works. The results are discussed in relation to the spatial requirements of both species and the protection of utilised habitat features. Management options are suggested to mitigate the impacts of road works. Differences in spatial dynamics of both frogs with overlapping habitats highlighted in this study require species-specific management approaches.

Habitat requirements and habitat use for Pseudophryne australis and Heleioporus australiacus were investigated to aid management of these threatened frogs around Sydney, Australia. Much of the work focussed on roads, commonly encountered features in the habitat of both species. The habitat requirements based on locality records of both frogs in the Sydney Basin were investigated at four spatial scales. Both species are habitat specialists. They showed a strong geological association with Hawkesbury Sandstone and occupy upper topographic areas with ephemeral watercourses of gentle gradients. Both frogs occur predominantly in areas of higher precipitation and milder temperature regimes compared to averages representative of the region. Leaf litter is an important feature of P. australis breeding sites, whereas H australiacus generally associate with crayfish burrows. Both species are dependent on natural vegetation with a complex structure. H australiacus have a relatively long larval period (3 - 12 months) and breed in ephemeral pools, exposing their tadpoles to the risk of dying due to early pond drying. In the laboratory, tadpoles responded to decreasing water levels by shortening their larval periods and metamorphosing earlier than siblings held at constant water level. Despite this plastic response, a number of pools in the field failed to produce metamorphs due to early drying, an observation also made on P. australis. Regular monitoring of breeding sites revealed increased reproductive success away from roads for both species probably because of relatively longer hydroperiods. Spatial distributions and associations with habitat features, and movement patterns of both frogs were further investigated using mark-recapture methods. Both species showed strong site fidelity. P. australis formed small aggregations and predominantly selected leaf litter piles despite their relatively low availability. Leaf litter piles in creeks moved over time and the animals moved with these piles. In contrast, H australiacus individuals formed no aggregations and showed no preference for any available structural vegetation type. Locations of individuals were independent of relative distances to creeks and artificial drains, but males appeared to be more common near culverts. However, individuals were randomly distributed in space and nearest-neighbour distances were high relative to individual movement distances, suggesting minimal overlap between relatively large home ranges. Radio-telemetry demonstrated that some H australiacus individuals burrow in the road environment. There they would be at risk of being dug up and possibly injured during road works. The results are discussed in relation to the spatial requirements of both species and the protection of utilised habitat features. Management options are suggested to mitigate the impacts of road works. Differences in spatial dynamics of both frogs with overlapping habitats highlighted in this study require species-specific management approaches.

Bill Brooks is worried about the future of his organization, perhaps even more than he is about the future of horned lizards. Brooks is the president and a founding member of the Horned Lizard Conservation Society, a nonprofit with the goal of protecting all species of horned lizard. The HLCS was founded in Austin in 1991, after its members realized that populations of Texas horned lizards, Phrynosoma cornutum, had crashed at some point in the preceding decades; they wanted to both figure out what had happened to the lizards and how to bring them back. Cornutum is an iconic species and the state reptile, and almost all Texans over the age of 40 can remember seeing them in the hundreds as children. Flash-forward a quarter of a century, and a lot of these founding members are either old and grey or deceased. HLCS membership is down, and Brooks thinks that if there aren’t any of his kind left (Texans that grew up with “horny toads”), there’ll be no one left to care. Herpetologists are working on returning healthy horned lizard populations to the wild, and both reintroduction and relocation programs are showing signs of success. But can they work, and will it be too late for Brooks’ organization and a Texas identity dependent upon horned lizards? / text