Global climate change is already contributing to the extirpation of numerous species worldwide, and sensitive species will continue to face challenges associated with rising temperatures throughout this century and beyond. It is especially important to evaluate the thermal ecology of endangered ectotherm species now so that mitigation measures can be taken as early as possible. A recent study of the thermal ecology of the federally endangered Blunt-Nosed Leopard Lizard (Gambelia sila) suggested that they face major activity restrictions due to thermal constraints in their desert habitat, but that large shade-providing shrubs act as thermal buffers to allow them to maintain surface activity without overheating. We replicated this study but added a population of G. sila with no access to large shrubs to facilitate comparison of the thermal ecology of G. sila in shrubless and shrubbed populations. We found that G. sila without access to shrubs spent more time sheltering inside rodent burrows than lizards with access to shrubs, especially during the hot summer months. Lizards from a shrubbed population had higher midday body temperatures and therefore poorer thermoregulatory accuracy than G. sila from a shrubless population, suggesting that greater surface activity may represent a thermoregulatory tradeoff for G. sila. Lizards at both sites are currently constrained from using open, sunny microhabitats for much of the day during their short active seasons, and our projections suggest that climate change will exacerbate these restrictions and force G. sila to use rodent burrows for shelter even more than they do now, especially at sites without access to shrubs. The continued management of shrubs and of burrowing rodents at G. sila sites is therefore essential to the survival of this endangered species.

Chapter 1: Animals can respond to extreme climate by behaviorally avoiding it, or by physiologically coping with it. We understand behavioral thermoregulation and physiological thermal tolerances, but water balance has largely been neglected. Climate change includes both global warming and changes in precipitation regimes, so improving our understanding of organismal water balance is increasingly urgent. We assessed the hydric physiology of endangered Blunt-nosed Leopard Lizards (Gambelia sila) by measuring cutaneous evaporative water loss (CEWL), plasma osmolality, body mass, and body condition throughout their active season. On average, G. sila had low CEWL that is likely desert-adaptive, and high plasma osmolality that is indicative of dehydration. Given that our study was in a drought year, it is reasonable to believe that every lizard measured was dehydrated to a degree. We hypothesized that throughout the G. sila active season, as their habitat got hotter and drier, G. sila would become increasingly dehydrated and watertight. Instead, CEWL and plasma osmolality showed minimal change for females and nonlinear change for males, which we hypothesize is connected to sex-specific reproductive behaviors and changes in food availability. We also measured thermoregulation and microhabitat use, expecting that more hydrated lizards would have higher body temperature, better thermoregulatory accuracy, and spend more time aboveground. However, we found no effect of CEWL, plasma osmolality, body mass, or body condition on these thermal and behavioral metrics. We posit either that G. sila tolerate dehydration to maintain activity during their brief active season, or that because every lizard was dehydrated due to the drought, they all experienced equally constrained thermoregulation and microhabitat use. Finally, G. sila spend considerable time underground in burrows, and we believe burrows serve as essential hydric, not only thermal, refugia. Our findings suggest that these lizards might benefit from artificial humid refugia and supplemental hydration, especially during drought. Chapter 2: Testing acclimation plasticity informs our understanding of functional biodiversity and applies to conservation management amidst our rapidly changing climate. While there is a wealth of research on the plasticity of thermal and hydric physiology in response to temperature acclimation, there is a comparative gap for research on acclimation to different hydric regimes, as well as the interaction between water and temperature. We sought to fill this gap by acclimating Western Fence Lizards (Sceloporus occidentalis) to experimental climate conditions (crossed design of Hot or Cool, Dry or Humid) for eight days, and measuring cutaneous evaporative water loss (CEWL), plasma osmolality, hematocrit, and body condition before and after acclimation under common conditions. CEWL changed plastically in response to the different climates, with lizards acclimated to Hot Humid conditions experiencing the greatest increase in CEWL. Change in CEWL among individuals was negatively related to treatment vapor pressure deficit. Plasma osmolality, hematocrit, and body condition all showed greater changes in response to temperature than to humidity or vapor pressure deficit. CEWL and plasma osmolality were positively related across treatment groups before acclimation and within treatment groups after acclimation, but the two variables showed different responses to acclimation, suggesting that they are interrelated but governed by different mechanisms. This study is among just a small number of studies that assess more than one metric of hydric physiology and that test the interactive effects of temperature and humidity. Such measurements will be essential for predictive models of activity and survival for animals under climate change.

Bat populations in the eastern United States are currently declining at unprecedented rates as a result of habitat loss, commercial wind energy development, and white-nose syndrome. Effective conservation of these declining populations requires knowledge of several aspects of summer and winter ecology, including daytime habitat use (day-roost selection and social behaviors), nocturnal habitat use (foraging habitat selection, prey selection, and prey abundance), and winter hibernation (torpor) patterns. This dissertation addresses these questions for Rafinesque’s big-eared bat (Corynorhinus rafinesquii), a species of conservation concern in the southeastern United States. Kentucky represents the northern edge of the range of Rafinesque’s big-eared bat, and summer and winter behaviors in Kentucky are likely to differ from what has been observed in southern portion of the range, where available habitats and climate are different. My research occurred in two study areas in Kentucky, Mammoth Cave National Park in central Kentucky, and the Ballard Wildlife Management areas in western Kentucky. This dissertation includes all of the work done in western Kentucky, where I radio-tagged 48 adult big-eared bats and documented daytime and nighttime habitat use. Also included is a portion of the work done in central Kentucky, focusing on hibernation patterns of 14 adult big-eared bats radio-tagged during the winter at Mammoth Cave. Data disseminated in this dissertation provide insights into the summer and winter ecology of Rafinesque’s big-eared bat in Kentucky, and can be used to manage populations threatened by habitat loss and white-nose syndrome.

One hypothesis explaining the numerous independent evolutionary transitions from oviparity to viviparity among squamates (snakes and lizards) proposed that squamate embryonic development is independent of eggshell calcium. Recent research showed at least 25% of the calcium in hatchling oviparous squamates is extracted from the shell. Though not a direct test, these results are inconsistent with the hypothesis. To directly test the hypothesis, we removed eggshell calcium (through peeling) early in development of Pantherophis guttatus (corn snake) eggs. Survivorship to hatching did not differ between peeled and intact eggs. Yet hatchlings from peeled eggs were shorter (273.6 ± 3.4 vs. 261.0 ± 3.7 mm, p=0.0028, n=16), lighter (6.36 ±0.22 vs. 5.75 ± 0.23 g, p=0.0158, n=16), and had reduced calcium (40.8 ± 1.7 vs. 30.5 ± 1.8 mg, p

This study addresses the nature of physiological and phenological evolutionary strategies of root growth dynamics and energy allocation followed by Atriplex confertifolia, Ceratoides lanata, and Artemisia tridentata growing in their natural cool desert environment. Root observation chambers with inclined Plexiglass windows were installed in monospecific desert communities of Atriplex confertifolia, Ceratoides lanata and Artemisia tridentata. Soil temperature and water potential measurements taken immediately adjacent to the observation windows indicated a minimal disturbance was caused by the presence of these chambers. For the three species initiation of root growth was observed before initiation of shoot activity, furthermore, active root growth extended over much longer periods during the year than shoot growth. Initial growth was observed for the three species in the upper soil layers in the spring. Later in the season most of the growth activity was measured at progressively greater depths in the soil. Measurable root growth was observed for Atriplexin August when the soil water potentials were in the range of -70 bars for 1972, and at water potentials of -60 bars for all three species during 1973. Detectable growth for these three species was recorded as late as January in 1974. Except for the main extension roots, individual apical meristems were seldom active for more than 2 weeks. Atriplex confertifolia and Ceratoides lanata plots were labeled during the growth season with 14Co2 in polyethylene enclosures to study both the partitioning of photosynthates to plant parts and their total allocation of carbon at the community level. A definite seasonal pattern of partitioning of recent photoassimilates corresponding to phenological events emerged. In the spring, photoassimilates were principally directed to shoot growth, especially expanding new leaves and vegetative buds. In terms of relative energy allocated to plant parts per unit dry weight basis, it appears that Ceratoides lanata expends less energy for reproductive organs. For both species, carbon used for new stems and previous years shoot growth appears to constitute a significant sink for energy use and storage. Relative translocation of carbon to roots was minimal during the spring for both species. It increased with the progression of the season reaching a maximum in July for Atriplex and at the end of the season for Ceratoides. Energy allocation at the community level for these species showed that approximately 60 and 40 percent of the recently photoassimilated 14C for the Atriplex-dominated community in July and September, respectively, appeared localized in the new shoot growth, the remaining was distributed in nearly equal amounts between previous year's shoot growth and the root system. The scheme of energy allocation in Ceratoides showed similar patterns of carbon utilization in July and September; approximately 80 percent of the fixed carbon was al located in approximately equal amounts to roots and new shoot growth with the remainder to the previous year's shoot growth. In the Ceratoides-dominated community 65 percent and 36 percent of 14C photoassimilated in April and July, respectively, and still remaining in the plant by September, was localized in the underground structures. Similarly, in the Atriplex community, 35 percent and 29 percent of the 14C incorporated in April and July appeared in the root system. From the total 14C photoassimilated in July for both communities, approximately 60 percent and 50 percent was retained in the plants by September in the Atriplex- and Ceratoides-dominated communities, respectively.

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the effects of GH on cellular proliferation; TUNEL-labeling was used to assess the effects of GH on apoptosis in the zebrafish inner ear following acoustic trauma. The time-course of hair cell bundle density, cell proliferation, and apoptosis was established by combining data for baseline fishes and sound-exposed fishes at post-sound exposure day 1 (psed1), psed2, and psed3. GH-injected fish exhibited greater densities of hair cells than bufferinjected controls. In addition, GH-injected fish had higher levels of cell proliferation and lower levels of apoptosis than buffer-injected controls. This suggests that GH may play an important role in zebrafish inner ear hair cell regeneration by stimulating cellular proliferation and inhibiting cellular apoptosis.

Ecdysis triggering hormone (ETH) is a neuropeptide known for its role in the orchestration of ecdysis. However, its role in the regulation of Juvenile Hormone (JH) synthesis is unknown. In Aedes aegypti, JH is synthesized by the corpora allata (CA) and titers are tightly regulated by allatoregulatory factors. In this study I describe the effect of ETH on JH synthesis during the late pupal stage and in the adult female after blood feeding. Analysis of ETH receptor (ETHRs) expression showed that ETHRs are present in both the CA and the corpora cardiaca (CC), a neurohemal organ. The data suggest that ETH regulates JH synthesis directly through its receptors in CA. Our results show that in pupa, ETH has a stimulatory effect on JH synthesis while in adult blood fed females, ETH is inhibitory. These findings constitute the first evidence of ETH as a regulatory peptide in mosquito JH synthesis.

This research examined the effects of acclimation to lowered salinity, elevated temperature, and hypoxia on aerobic metabolism of the Gulf killifish, Fundulus grandis, a common estuarine resident of the Gulf of Mexico. Standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were each influenced by one or more acclimation treatments. Assessing the consistency of these traits measured in the same individuals over time, all were found to be significantly repeatable with no indication that the repeatability of any traits was affected by acclimation conditions. Significant correlations were found between SMR and Pcrit (positively correlated), between SMR and AAS (negatively correlated), between MMR and AAS (positive), and between AAS and Pcrit (negative). This study, therefore, documents the effects of acclimation on these traits, their repeatability, and correlations among them. It further suggests that repeatability of these traits is not context dependent.

Despite the threat of climate change, the physiological mechanisms by which temperature drives the distribution of species are unclear. Here we used chronic temperature exposures to determine that the upper limit for positive growth in the eastern brook trout (Salvelinus fontinalis) is 23.4 °C. Additionally, brook trout exposed to daily temperature oscillations of 8 °C, around a mean of 21 °C, exhibited growth rates that were 43 and 35% lower by length and weight respectively, than in constant 21 °C controls. Limitations in growth were associated with increases in indicators of the physiological stress response. Individuals exposed to 22 or 24 °C for 24 days exhibited plasma cortisol levels that were 12 and 18 fold greater than at 16 °C. Similarly, gill heat shock protein (Hsp)-70 levels were 10.7 and 56 fold higher at 22 and 24 °C than at 16 °C. Brook trout exposed to daily temperature oscillation of 4 or 8 °C had gill Hsp-70 levels that were 40 and 700 fold greater than controls. Acute (6 h) temperature exposures were used to demonstrate a threshold for induction of the Hsp-70 and plasma glucose responses of 20.7 °C and 21.2 °C respectively. Finally, we conducted field surveys that demonstrated increased plasma cortisol, plasma glucose, and gill Hsp-70 at temperatures above 21 °C. Induction of the cellular and endocrine stress responses is associated with decreased growth in brook trout. Thermal limitations on growth may provide a mechanism by which temperature drives the distributions of this cold-water species.

<p>Ammonia is the primary nitrogenous waste product in teleost fish, which is produced primarily through protein metabolism. Fish experience natural elevations in internal ammonia loads, including during digestion where luminal ammonia concentrations in the intestine rise substantially. Furthermore, the intestine may absorb a portion of this ammonia, despite it being toxic to the fish. Based on this, <em>in vitro </em>techniques were employed in order to develop a model for teleost intestinal ammonia handling.</p> <p>Ammonia absorption and endogenous ammonia production occur along the entire length of the intestine. However, section-specific differences exist in terms of both endogenously produced ammonia and ammonia flux rates, with the highest rates in the anterior and mid intestine. Feeding stimulated an increase in production rates in all intestinal sections. Overall, ammonia originating from the gut may account for up to 42% of post-prandial whole-fish ammonia excretion. This could partly be attributed to the increased activity of the ammonia-producing enzyme glutamate dehydrogenase, and decreased activity of the ammonia-fixing glutamine synthetase. Furthermore, gut tissue ammonia concentrations surpassed typical chyme concentrations and were well regulated independent of high luminal ammonia, suggesting active transport across the intestinal epithelium.</p> <p>Seawater (60%) acclimation caused no substantial changes in the ammonia handling properties of the intestine. Ammonia transport in the intestine of both freshwater and seawater trout appears to occur via active means, coupled to Na⁺/K⁺ ATPase activity. Specifically, this involves Na⁺ linked transport through substitution of NH₄⁺ for K⁺on the apical Na⁺/K⁺/2Cl^- co-transporter occurring predominantly in the anterior and mid intestine, and solvent drag through fluid transport (osmotically driven by active NaCl absorption) in all sections. Additionally, Rhesus glycoprotein mediated ammonia transport likely occurs through basolateral Rhbg1, supporting previous molecular evidence. Overall this thesis illuminates the quantitative importance and mechanisms of gut ammonia transport in fish, and highlights future research avenues.</p> / Master of Science (MSc)