Reptile populations are suffering substantial global losses and roads are identified as one of the leading threats to their persistence. Currently, efforts to mitigate this threat are being implemented with various levels of success. I studied the effectiveness of exclusion structures (i.e., fencing) at preventing reptiles from gaining access to the road, and reducing road mortality. I also examined if population connectivity structures (i.e., ecopassages) were effective at reducing habitat and population fragmentation and allowing individuals to access habitats, resources, and mates on both sides of a major road (4 lane highway). I found that the fence was ineffective at preventing reptiles from gaining access to the road; however, reptiles were observed using the ecopassages to cross the road. Behavioural trials testing painted turtles’ (Chrysemys picta) willingness to use an ecopassage demonstrated that refusal was twice more likely than use of an ecopassage. I also examined the potential for roads to pose a physiological threat to roadside populations of reptiles by examining corticosterone (CORT), a stress hormone linked to negative health effects in cases of elevated levels over the long-term. To assess if individuals living near a major road had higher CORT levels than individuals from a less impacted population, I developed a novel means of measuring CORT from painted turtle claws in partnership with Dr. Gabriela Mastromonaco (Toronto Zoo). With long-term CORT levels considered as a proxy for chronic physiological stress, I did not find evidence that populations near roads had altered stress levels. However, this seminal study will provide the framework for further examination of more species, including species-at-risk, and a better understanding of effects of anthropogenic environments on wildlife health. As road ecologists strive to expand our understanding of the threats roads pose to reptiles, it is important that this field spans multiple disciplines, so that we can both understand the direct and indirect threats that roads cause and develop effective mitigation that preserves biodiversity within our anthropogenic landscape.

abstract: There is considerable recent interest in the dynamic nature of immune function in the context of an animal’s internal and external environment. An important focus within this field of ecoimmunology is on how availability of resources such as energy can alter immune function. Water is an additional resource that drives animal development, physiology, and behavior, yet the influence hydration has on immunity has received limited attention. In particular, hydration state may have the greatest potential to drive fluctuations in immunity and other physiological functions in species that live in water-limited environments where they may experience periods of dehydration. To shed light on the sensitivity of immune function to hydration state, I first tested the effect of hydration states (hydrated, dehydrated, and rehydrated) and digestive states on innate immunity in the Gila monster, a desert-dwelling lizard. Though dehydration is often thought to be stressful and, if experienced chronically, likely to decrease immune function, dehydration elicited an increase in immune response in this species, while digestive state had no effect. Next, I tested whether dehydration was indeed stressful, and tested a broader range of immune measures. My findings validated the enhanced innate immunity across additional measures and revealed that Gila monsters lacked a significant stress hormone response during dehydration (though results were suggestive). I next sought to test if life history (in terms of environmental stability) drives these differences in dehydration responses using a comparative approach. I compared four confamilial pairs of squamate species that varied in habitat type within each pair—four species that are adapted to xeric environments and four that are adapted to more mesic environments. No effect of life history was detected between groups, but hydration was a driver of some measures of innate immunity and of stress hormone concentrations in multiple species. Additionally, species that exhibited a stress response to dehydration did not have decreased innate immunity, suggesting these physiological responses may often be decoupled. My dissertation work provides new insight into the relationship between hydration, stress, and immunity, and it may inform future work exploring disease transmission or organismal responses to climate change. / Dissertation/Thesis / Doctoral Dissertation Biology 2016

Uluru is the more famous of the two namesake inselbergs found in Uluru Kata-Tjuta National Park (UKTNP). Uluru is roughly 10 km around its base, stands nearly 350 m tall, and can cast a shadow more than a kilometre long during the early morning and late afternoon hours. Additionally, there are many areas near the base of Uluru that receive nearly continuous shade throughout the day, which may also mean that these places could be buffered against extreme temperatures. This study investigated Uluru's influence on the climate at various distances from its base, and simultaneously assessed if the structure of the reptile community was related to the observed climatic variation. We used iButton data loggers to record the temperature and relative humidity at 26 sample locations surrounding Uluru. We used ArcGIS to map the path of Uluru's shadow and to determine when and how long data loggers recorded in and out of Uluru's shading influence. We found that the temperature and relative humidity were strongly influenced by Uluru's shadow, and that reptile assemblages were strongly correlated with the amount of time a sampled site was influenced by Uluru's shadow.

An organism’s spatial ecology allows for access to essential resources such as food, mates, and escape from predators. Home range size, or the total area an organism inhabits, varies in relation to numerous factors including seasonality. During the breeding season, home range size increases in males across taxa. In addition, males usually also have larger home range sizes than females. This implicates testosterone (T) as a possible mediator of this relationship. Indeed, T causes an increase in home range size of males in numerous species of lizards. In addition to T causing an increase in home range size, it also causes an increase in coloration, which is used as a signal to deter or elicit aggressive behaviors in lizards. Potentially, contests are less common in natural settings than in the lab due to this signaling despite increased frequency overlap of home ranges in males. The larger the home range size of males, mediated through an increase in T, the more overlap with conspecifics. With this increase in spatial demand, or home range size, there is often a corresponding increase in spatially related brain regions. In reptiles, these brain regions are the medial and dorsal cortices (MC and DC respectively). The increase in cortical brain region size due to an increase in spatial demand may be mediated by an increase in neurogenesis. Proliferation of neurons occurs along the ventricles and radiate to numerous regions in the brain including the MC. With respect to the MC, immature neurons, which express the protein doublecortin (DCX), migrate from the ventricles, through the inner plexiform layer and are integrated into the cell layer. Because DCX is only expressed in recently born, migrating neurons, it can be used to measure neurogenesis. In mammals and birds, neurogenesis and growth of certain brain regions is affected by steroid hormones, including T. Here we tested two hypotheses: (1) T affects the home range size of Sceloporus occidentalis and (2) cortical brain region volumes are related to home range size and/or T which is mediated through changes in rates of neurogenesis. We surgically castrated individuals and implanted subjects with either a T-filled implant or blank implant and then released them at their initial capture sites. In addition to these castrated individuals, subjects not subjected to castration served as unmanipulated controls. Home range size of individuals in the field was quantified using a global positioning system (GPS) unit and later delineating those GPS points using minimum convex polygons (MCPs). We predicted that (1) castrated, T-treated lizards and unmanipulated control lizards would have larger home range sizes than castrated, control lizards c and (2) MC and DC cortices would be larger in volume and contain more DCX-immunoreactive cells in the lizards with the highest circulating T levels and with the largest home range sizes. We found that increased T caused an increase in the number of blue abdominal scales. We found no differences in home range size relating to T. Likewise, T did not affect MC volumes. However, we did observe a decrease in DC volume with increasing plasma levels of T. Because T did not affect home range size, it follows that we did not find an effect of T on MC volume. However, the significant result of T causing a decrease in DC volume implies a possible trade off with regards to energetics and the maintenance of brain region volumes as prior research indicates that T in increases energy expenditure and decreases foraging efforts.

Reptiles exhibit neurogenesis throughout the brain during adulthood. However, very few studies have quantified telencephalon-wide neurogenesis in adulthood, and no studies have performed these investigations in snakes. Quantifying neurogenesis in the adult snake is essential to understanding class-wide adult neurogenesis and providing insight into the evolution of this trait. The thymidine analog 5-bromo-2’-deoxyuridine (BrdU) was used to quantify cell proliferation, migration, and survival in the ball python (Python regius). First, to determine the proper dose of BrdU for injection we subcutaneously injected 50mg/kg, 100mg/kg, and 250mg/kg into 15 adult male P. regius. We found the 250mg/kg dose marked significantly more cells than the 50mg/kg dose, but not the 100mg/kg dose. Then we subcutaneously injected 100mg/kg BrdU into 15 juvenile male P. regius at 3 different time points (2 days, 2 weeks, 2 months) prior to sacrifice to quantify proliferation, migration, and survival of cells in several telencephalic subregions. After sectioning and immunohistochemical staining, we found proliferation to be highest in the accessory olfactory bulb (AoB), retrobulbar regions (AD, AV), dorsal ventricular ridge (DVR), and dorsolateral amygdala/lateral amygdala (DLA/LA). Of the proliferating cells, the proportions of cells that migrated after 2 weeks were highest in the ventral lateral region (VL), anterior medial and lateral cortices (aMC, aLC), and anterior NS (aNS). After 2 months, the highest proportional survival was in the AoB, aLC, aMC, aNS, DVR, and ventral medial regions (VM). Regions involved in long-term functions like spatial memory may require less proliferation and longer survival, while regions involved in short-term functions undergo more proliferation with higher relative attrition.

The influence of feeding juvenile female leopard tortoises (Geochelone pardalis, n=18) a commercially available, complete, extruded feed three (3) or seven days (7) per week on dry matter and digestible energy intake, apparent digestibility of dry matter, organic matter, gross energy and fiber fractions, animal body weight and measurements, digesta transit time, rate of passage, and indigestible fill was evaluated. Both feeding frequencies are commonly practiced with captive tortoises. When fed 7 compared to 3 days per week, dry matter and digestible energy intake was greater. Tortoises gained more g BW, but not when adjusted per kg initial BW. When fed 7 compared to 3 days per week, tortoises grew more in plastron width (PW) and carapace height (CH), but not midline straight carapace length (MSCL), and grew more in calculated shell volume (i.e., a calculated estimate of shell volume using MSCL, PW, and CH), with a higher calculated body condition index (BCI). Providing short fasts (i.e., feeding 3 compared to 7 days per week) may be useful in slowing tortoise growth when animals are provided food ad libitum. In general, ad libitum feeding, especially of a highly digestible extruded feed, is not recommended for captive juvenile G. pardalis, especially when offered food daily. With two data points (detected as outliers) removed due to low fecal output (and resulting unrealistically high apparent digestibility of all nutrients analyzed) of two animals when fed 3 days per week, apparent digestibility of cellulose in tortoises fed 7 (n=18) compared to 3 (n=16) days per week was lower, but no differences were detected in DM, OM, GE, or any other fiber fractions analyzed. Transit time (TT1) was shorter and indigestible fill was higher in tortoises (n=18) fed 7 compared to 3 days per week, regardless of percent Cr marker recovered. With four animals removed due to <50% Cr marker recovery, tortoises fed 7 compared to 3 days per week exhibited shorter mean retention time (RGIT), with no differences in digesta transit or indigestible fill. Longer digesta retention when food availability included short periods of fasting may have allowed tortoises to extract more energy from cellulose.

Doctor of Philosophy / Division of Biology / Walter K. Dodds / Tallgrass prairie evolved with fire-grazer interactions. Fire and grazing are vital processes for maintaining grasslands and cattle production, and therefore will be continued as land management schemes. The effects of fire and grazers on prairie streams are understudied, but may significantly influence stream ecology. This dissertation examined how prescribed burning, bison grazing, and patch-burn grazing (by cattle) influence water quality, stream biota, and riparian amphibians and reptiles at Konza Prairie, Kansas, or Osage Prairie, Missouri. Using Global Positioning System, we monitored bison and cattle distribution throughout watersheds. The immediate effects of prescribed burning were examined at both Konza and Osage Prairies. The impacts of bison on water quality were determined by using a long-term dataset from Konza Prairie and compared watersheds with and without bison. Amphibian and reptile assemblages were monitored for two years at Osage, and assemblage data were analyzed using redundancy analysis, permuted analysis of variance, and occupancy modeling. A patch-burn grazing experiment occurred for 5 years at Osage (2 years pretreatment data and 3 years of treatments) and was analyzed using a before-after, control-impact design. Prescribed burning had minimal effects on water chemistry. At Konza Prairie, bison did not alter water quality likely because they spent negligible time (<5%) in streams. Contrarily, cattle at Osage Prairie significantly increased stream concentrations of total suspended solids, nutrients, Escherichia coli bacteria, algal biomass, and primary production. Unlike bison, cattle spent significant time (~21%) in streams if allowed access to riparian zones. In watersheds with cattle excluded from streams by riparian fencing, water quality contaminant concentrations increased significantly, but not to the magnitude of unfenced streams. Amphibian abundance and richness were not different among patch types; instead, they were restricted to specific basins. However, reptiles displayed preference for certain patch-types, and had the highest abundance and richness in watersheds with fire and grazing. These results have implications for natural resource management. Riparian fencing of cattle may be a useful practice in areas where water resource protection is the priority. However, overland flow may alter water quality in watersheds with grazers despite fencing. Land managers will need to define management objectives and accept trade-offs in water quality, amphibian and reptile habitat, and cattle production.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / Knowledge of global reptile ecology is limited and there remains much to understand in terms of detailed reptile species information, including that of their distributions. In South Africa, despite being one of SANParks best-studied reserves, surprisingly little is known about the distributions and spatial ecology of reptiles within Kruger National Park (KNP). Management within KNP follows a strategic adaptive management strategy which monitors the statuses of animals using species or group specific indicators. Indicators are given predetermined upper and lower ranges of acceptable fluctuation before actions are taken. These ranges are referred to as thresholds of potential concern (TPCs), and for reptiles these are based on changes to their distributions across the landscape of KNP. An apparent lack of high-quality reptile distribution data inhibits the effective monitoring of the statuses of these animals within KNP, which in turn limits management and conservation options. In this study, I use several methods to quantify available reptile occurrence data which formed the foundations for predicting the distributions of these species across KNP by means of species distribution modelling, with a view to gaining novel insight into reptile assemblage structure across the landscape of KNP.

Na cascavel Crotalus durissus terrificus ocorre o armazenamento de longo prazo do esperma (LTSS), no trato genital da fêmea, durante o intervalo entre a cópula (outono) e a ovulação (primavera). Peptídeos e peptidases estão entre os principais componentes que influenciam a atividade espermática em mamíferos. O presente estudo objetivou caracterizar a presença de peptidases em C. d. terrificus, com esta função reconhecida em mamíferos e/ou que clivam peptídeos atuantes nesta função, bem como avaliar a hipótese do envolvimento destas peptidases na preservação dos espermatozóides desta serpente. O aspecto morfo-funcional dos espermatozóides foi comparado na presença dos peptídeos angiotensina II (AngII), arginina vasotocina (AVT), bradicinina (BK), peptídeo promotor da fecundação (FPP) e hormônio liberador de tireotrofina (TRH). Caracterizamos os efeitos de agentes quelante, tiol-redutor, cofator e inibidores, bem como dos peptídeos supracitados, sobre as atividades enzimáticas relacionadas, quais sejam: aminopeptidases ácida (APA), básica (APB), alanil sensível (APN-PS) e insensível à puromicina (APN-PI), cistil (CAP), dipeptidil-peptidase IV (DPPIV), piroglutamil tipo 1 (PAP-I) e prolil-imino- (PIP), bem como da prolil endopeptidase (POP), em frações solúvel (FS) e/ou de membrana solubilizada (FM) do sêmen proveniente do ducto deferente, assim como deste próprio tecido e dos tecidos uterino e vaginal de C. d. terrificus, ou seja, tecidos por onde passam ou armazenam-se os espermatozóides. Verificamos a variação sazonal destas mesmas atividades, nestes tecidos, incluindo o sêmen armazenado no útero durante o LTSS. O sêmen coletado do ducto deferente foi fracionado para avaliação da distribuição destas atividades peptidásicas. Nossos dados mostraram características de liquefação do sêmen e movimento dos espermatozóides da cascavel que os diferenciam do padrão humano. FPP com cálcio e BK melhoraram a preservação da viabilidade espermática, similarmente ao que ocorre em mamíferos. Em todos os tecidos e no sêmen as atividades APB, PIP e POP foram detectadas apenas da FS, enquanto as demais estão presentes em FS e FM, seguindo um padrão de distribuição observado na maioria dos tecidos de mamíferos. Amastatina e bestatina inibiram APB e APN, enquanto a diprotina A foi o inibidor mais eficiente para a DPPIV em FM. PAP-I e PIP foram inibidas, respectivamente, por bestatina e puromicina. Este perfil de inibição também é similar ao encontrado para as aminopeptidases em tecidos de mamíferos. Todas as atividades peptidásicas foram influenciadas por algum dos peptídeos estudados, sugerindo que tais peptídeos são substratos potenciais e/ou moduladores destas atividades na cascavel. As atividades APB e APN foram caracterizadas como metalopeptidases. APB, CAP e DPPIV foram inibidas por MnCl2. CAP e PAP-I foram caracterizadas como enzimas sulfidril-dependentes. As atividades APB, APN-PI e APN-PS predominaram, em relação às demais peptidases, em todas as estações e na maioria dos tecidos e no sêmen, sugerindo sua maior relevância na fisiologia reprodutiva da C. d. terrificus. Os níveis de todas as atividades peptidásicas estudadas variaram sazonalmente, sugerindo que sua ação moduladora sobre peptídeos susceptíveis está integrada ao ciclo reprodutivo desta serpente. O fracionamento do sêmen do ducto deferente revelou a presença de fluido seminal e espermatozóides, bem como de uma estrutura prostassoma-símile, até então somente identificada em mamíferos. Em todas estas frações há atividade peptidásica, predominando a APN-PI no prostassoma-símile e no fluido seminal, e APN-PS e APN-PI na FS e FM dos espermatozóides, caracterizando um envolvimento com a redução da motilidade espermática, tal qual ocorre em mamíferos. Concluimos que as atividades peptidásicas estudadas apresentam características sazonais e tecido-específicas que sugerem uma atuação relevante na preservação dos espermatozóides de C.d. terrificus. / In the rattlesnake Crotalus durissus terrificus occurs the long-term sperm storage (LTSS), in the female tract, during the interval between mating (autumn) and ovulation (spring). Peptides and peptidases are among the main components that influence the spermatic activity in mammals. The present study aimed to characterize the presence of peptidases in C. d. terrificus, that are well-recognized to exert this function and/or that have the ability to hydrolyze peptides that exert this function in mammals, as well to evaluate whether these peptidases are related to the preservation of spermatozoids in this snake. The morphological and functional characteristics of spermatozoids were compared in the presence of angiotensin II (AngII), arginine-vasotocin (AVT), bradykinin (BK), fertilization promoting peptide (FPP) and thyrotropin-releasing hormone (TRH). We have checked the effect of chelating and thiol-reducing agents, cofactor and inhibitors, as well the effect of aforementionated peptides on related enzyme activities such as acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive alanyl (APN-PI), cystyl (CAP), pyroglutamyl type 1 (PAP-I) and prolyl-imino (PIP) aminopeptidases, and dipeptidyl-peptidase IV (DPPIV), as well as on prolyl endopeptidase (POP), in soluble (FS) and/or solubilized membrane-bound (FM) fractions of semen from vas deferens, of this own tissue, and vagina and uterus tissues of C. d. terrificus, i.e. tissues where spermatozoids pass through or where they are stored. The seasonal variation of these peptidase activities, in all tissues, including the semen stored in uterus during the LTSS, were evaluated. The semen from vas deferens was fractioned in order to know the distribution of these peptidase activities. The features of seminal liquefaction and movement of spermatozoids were different between the rattlesnake and human. Similar to mammals, FPP plus calcium and BK improved the preservation of the viability of spermatozoids from C. d. terrificus. In all tissues and semen, the APB, PIP and POP activities were detected only in FS, while others peptidases were present in FS and FM, following a similar pattern of distribution usually observed in mammalian tissues. Amastatin and bestatin inhibited APB and APN activities, while diprotin A was the most efficient inhibitor of DPPIV in FM. PAP-I and PIP activities were inhibited by bestatin and puromycin, respectively. This inhibition profile was similar to that of mammalian tissues. All peptidase activities were influenced at least by one of the peptides under study, suggesting these peptides as potential substrates and/or modulators for these peptidases of the rattlesnake. The APB and APN activities were characterized as metallopeptidases. APB, CAP and DPPIV were inhibited by MnCl2. CAP and PAP-I were characterized as sulfhydryl-dependent enzymes. The APB, APN-PI and APN-PS activities were predominant, in relation to the other examined peptidases, in all seasons and in most tissues and semen, suggesting their great relevance in the reproductive physiology of the C. d. terrificus. The levels of all studied peptidase activities were seasonally variable, suggesting that their modulator actions on susceptible peptides are integrated to the reproductive cycle of this snake. The fractionation of C. d. terrificus semen revealed the presence of seminal fluid and spermatozoids, as well a prostasome-like structure, until then identified only in mammals. In all of these fractions, there are peptidase activities, predominating the APN-PI in prostasome and seminal fluid, and the APN-PS and APN-PI in FS and FM of spermatozoids, suggesting their involvement in the reduction of the spermatic mobility, such as in mammals. In conclusion, the studied peptidase activities present seasonal and tissue-specific characteristics, which suggest a relevant role in the preservation of the spermatozoids of C. d. terrificus.

As tartarugas na Amazônia são particularmente sensíveis à pressão humana, já que os impactos nas áreas de nidificação podem afetar negativamente a taxa de reprodução das populações. A seleção do local de nidificação é um componente fundamental para o sucesso de desova, no entanto, a associação entre a escolha do local e a aptidão do local escolhido ainda é pouco estudada. Neste trabalho testamos um conjunto de variáveis para explicar os padrões de nidificação em Tracajás (Podocnemis unifilis), com base em dados coletados em 73 locais de nidificação ao longo de 118 km de rio na Amazônia Oriental Brasileira. Modelos lineares generalizados (GLMs) foram usados para avaliar as influências antropogênicas e ambientais nos padrões de seleção do local de nidificação (número de ninhos, densidade de ninhos, distância do ninho à água e variação na distância à água) e adequabilidade do local de nidificação (remoção de ninhos por humanos). Como resultado, encontramos que o número e densidade de ninhos foram fortemente explicados por variáveis ambientais, e a distancia do ninho á água diminuiu com a proximidade as casas. A remoção humana foi o principal responsável pela perda de ninhos (47% dos ninhos foram removidos) principalmente nos trechos do rio com maior atividade humana. Nossos dados sugerem que mudanças antropogênicas estão levando a diferenças entre a escolha de local de nidificação e a sua adequabilidade, assim, nos rios onde humanos tem maior acesso, sinais usados pelas fêmeas para selecionar áreas de nidificação não estão permitindo desovas bem-sucedidas. Ao contrário das previsões nas hipóteses, os achados demonstram que as fêmeas não parecem evitar desovar em locais perigosos e inadequados. Portanto, ações diretas, como a proteção de áreas de nidificação, são vitais para a conservação das tartarugas na Amazônia. / Amazonian freshwater turtles are particurlarly sensitive to human pressure, since impacts on their nesting areas can negatively affect reproductive rate of populations. Nest-site selection is a fundamental component of freshwater turtle nesting success, however, linking oviposition choices to overall suitability of nest-site selection remains poorly tested. We tested a set of variables to explain nesting patterns in the yellow-spotted river turtle (Podocnemis unifilis), based on data collected from 73 nesting sites along 118 km of river in the eastern Brazilian Amazon. General Linear Models (GLMs) were used to evaluate anthropogenic and environmental influences on patterns in nest-site selection (four responses: number of nests, nest density, distance from nest to water and variation in distance to water) and nest-site suitability (removal of nests by humans). Number and density of nest were largely explained by environmental variables, and distance from nest to water decreased with the proximity to human habitations. Human removal of nests was the primary driver of nest failure (47% of the nests were removed) and removal was higher in sections of river with higher human activity. We show that anthropogenic changes are driving differences between nest-site selection and suitability, whereby the signals used by females to select nesting areas no longer enable successful nesting along rivers accessible to humans. Contrary to predictions from the hypotheses, our findings demonstrate that females do not appear to avoid nesting in dangerous and unfit sites. Therefore, direct actions including the protection of nesting areas are vital for the conservation of Amazonian freshwater turtles.