Because of growing concern of habitat fragmentation and its adverse effects on salamander communities in Appalachian region, sympatric populations of ambystomatid salamanders A. opacum and A. maculatum were studied in Northeast Tennessee to address a number of questions: i) the extent of sexual size dimorphism (SSD) in both species, ii) what traits influence the dorsal skin pigmentation and how, iii) whether gender differences in developmental stability occur, and iv) the extent of phenotypic variation within each species. The findings of this study revealed SSD in both species of salamanders. The most parsimonious statistical model was developed that explained the influence of body mass, dorsal body area, and sex on development of dorsal white pigmentation in marbled salamanders. Data on asymmetry indicate that females are under more stress than males in marbled salamanders, while for spotted salamanders nonsignificant asymmetry is indicative of similar level of stress in both sexes. Data on coefficient of variation (CV) suggest stabilizing selection on optimal body size and mass in female marbled salamanders compared to males; however, for spotted salamanders CV indicates relatively similar selection pressure for body size and mass for both sexes.

<p>Per- and polyfluoroalkyl substances (PFAS) are a group of emerging global contaminants used in a variety of industrial processes and consumer products, such as personal care products and fast-food wrappers. However, due to their carbon-fluorine bonds, these chemicals resist degradation and persist in the environment. PFAS toxicity is driven by a compound’s functional group and chain length with perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide dimer acid (GenX) being of focal concern due to their toxicity to wildlife and presence in the environment. Despite growing concern regarding these contaminants, inadequate attention has been given to evaluating what organismal characteristics influence uptake and depuration of these chemicals, such as life stage and sex. <em>Xenopus laevis</em> tadpoles are a useful model to assess the influence of sex on PFAS kinetics since they have a life history that includes a gill to lung transition. Previous studies have shown that air-breathing organisms depurate PFAS more slowly than water-breathing organisms, but this relationship has never been directly tested. Sex has been shown to be an important factor in the depuration of PFOA for rats, with female rats depurating PFOA in four hours while males depurate in four days. The early portion of bioaccumulation curves are also understudied even though tadpoles accumulate PFAS rapidly, reaching steady state within 48 hours of exposure. <em>Xenopus laevis</em> are used to study multiple endpoints for endocrine disrupting chemicals including PFAS. Despite this, toxicity reference values (TRVs) have not been described for the uptake and elimination of PFAS using <em>X. laevis</em>. To address these gaps in knowledge, I first developed TRVs for <em>X. laevis</em> tadpoles exposed to PFOA throughout metamorphosis and evaluated the influence of sex on phenotypic endpoints. Results showed a no observed effect concentration (NOEC) of 11.1 ppm for body mass at day 14 and no effect of sex on apical endpoints. Next, I described the early bioaccumulation of four PFAS with differing structure (chain lengths and functional groups). PFOS was the only chemical to bioaccumulate with a log bioconcentration factor (BCF) at 10 and 1,000 ppm of 1.33 and 1.18, respectively. PFHxS, PFOA, and GenX had BCFs less than 0. Finally, I examined the impact of life-stage and sex on <em>X. laevis</em> tadpole and juvenile depuration rates. Larval tadpoles depurated four times faster than juveniles, indicating a significant effect of life stage on elimination rates. Sex had no influence on elimination rates. These are the first studies conducted evaluating the significance of life stage and sex in toxicokinetics of PFAS in amphibians.</p>

Thyroid hormones (THs) are essential signaling molecules for the postembryonic development of all vertebrates. THs are capable of initiating a diverse set of developmental programs across multiple tissues. The role of TH in regulating gene expression is well-known, but the initiation of TH signaling is still not fully understood. In amphibians, THs are the sole hormones required for the metamorphosis from tadpole to juvenile froglet. Amphibians are a useful model for studying TH signaling, as they undergo extensive, tissue-specific response programs in response to exogenous TH. The metamorphosis of the American bullfrog, Rana [Lithobates] catesbeiana is temperature sensitive. R. catesbeiana tadpoles do not undergo metamorphosis at cold temperatures (4-5 °C) even in the presence of THs that should otherwise prompt it. However, tadpoles undergo metamorphosis at an accelerated rate when returned to warm temperatures (24-25 °C) forty days after their initial TH exposure. R. catesbeiana tadpoles possess a “molecular memory” of TH exposure which establishes the TH signal at cold temperatures and prompts accelerated metamorphosis after a return to warmer temperatures. The mechanisms of the molecular memory which allow it to uncouple the initiation of TH signaling from the execution of the TH response program are not fully understood. Previous research has established that transcripts encoding transcription factors are a substantial component of the TH-dependent transcriptomic response of cultured tailfin (C-Fin) at cold temperatures. However, not all of these putative transcripts encoding transcription factors required active transcription and translation for their induction, which suggests that the initiation of a TH signal involves mechanisms other than regulating gene expression. Herein, we used quantitative polymerase chain reaction (qPCR) and RNA-Sequencing (RNA-Seq) to investigate the TH-dependent transcriptomic response of the back skin, a tissue that undergoes extensive remodeling during metamorphosis. Cultured back skin (C-Skin) was TH-responsive in warm, cold and temperature shift conditions. Forty-four transcripts underwent significant changes in abundance in response to TH in cold temperatures under which the molecular memory is established. Seven of these transcripts encoded putative transcription factors. Surprisingly, the only TH-responsive transcript significantly changed at 4 °C in both the C-Skin and the previously studied C-Fin was thyroid hormone-induced basic leucine zipper-containing protein (thibz). Thibz has been found to be TH-responsive at cold temperatures in the liver, lung, liver, brain, tailfin and back skin of whole animals, which suggests it may be an important regulator of initiating TH signaling. The lack of overlap in the transcriptomic responses of C-Skin and C-Fin may suggest that even the early initiation of TH signaling has tissue-specificity. Alternately, the molecular memory may include mechanisms that do not require active transcription and translation. Transcripts associated with epigenetic modifications and post-transcriptional changes to mRNA stability were also significantly expressed at 4 °C within the C-Skin. Previous investigation of the putative transcription factors in C-Fin revealed that active transcription and translation was not always required for changes in transcript abundance. Multiple mechanisms may be at play in the TH response at different temperatures. In cold temperatures, TH may modulate mRNA stability to influence transcript abundance as a part of initiating TH signaling without executing metamorphosis. Further research is needed to explore potential alternative mechanisms of establishing the molecular memory and the accelerated metamorphic response. The temperature sensitivity of R. catesbeiana’s TH response is incredibly valuable in investigating mechanisms of early TH signaling during postembryonic vertebrate development. / Graduate

Salamanders are one of the most at-risk taxa in the world due to habitat destruction, pollution, climate change, invasive diseases, and more. This has led to a need for conservation breeding programs that are often associated with zoos and aquaria. Salamanders can be difficult to breed in captivity, though, due to their dependence on specific environmental cues and other unknown factors that stimulate sperm and egg production. To overcome these challenges, assisted reproductive technologies (ART) such as exogenous hormone administration, sperm cryopreservation, and in-vitro fertilization have been developed to increase offspring propagation and maintain genetic diversity. If genetically robust populations of salamander species can be sustainably managed ex situ into the future, then their species can be protected in situ through practices such as reintroductions into native habitats. Given the importance of salamanders to healthy, functioning ecosystems, my doctoral research focused on methods to advance caudate conservation efforts through ART. This dissertation addresses four key areas targeting salamander reproduction for conservation: 1) Novel, non-invasive hormone administration routes; 2) Sperm extender toxicity; 3) Novel sperm cryoprotectants; and 4) Application of ART to target salamander species. The objectives were to: 1) Compare nasal, oral, and intramuscular delivery routes of gonadotropin-releasing hormone on spermic response; 2) Compare sperm extenders at varying osmolalities for maintaining sperm quality over time; 3) Test dimethyl sulfoxide versus dimethylformamide as cryoprotectants to increase frozen-thawed sperm viability, motility, and fertilization capability in the eastern tiger salamander (Ambystoma tigrinum); and 4) Transfer ART protocols developed from the eastern tiger salamander to the eastern hellbender (Cryptobranchus alleganiensis) and spotted salamander (Ambystoma maculatum). This work answers critical questions that should help advance salamander ART research into the future and lead to more sustainably managed caudate populations.

This project evaluated the influence of habitat parameters and distance from low-order streams on herpetofauna communities, in addition to evaluating the efficacy of 2 sampling methods. Amphibians were associated with mature hardwood forests with high density of large, highly decayed woody debris. Reptiles were associated with mixed pine-hardwood forests, woody debris, and vertical canopy structure. Overall, woody debris was an important habitat feature for amphibians and reptiles. Amphibians were detected in greater abundance and species richness near the stream. Reptiles were similar in abundance and species richness as distance increased from low-order streams. I recommend a SMZ width ¡Ý50 m on each side of the low-order streams in east-central Mississippi. Area-constrained and funnel-pitfall traps each detected species that the other method was unable to sample due to inherent biases. I recommend the use of both survey methods when investigating the entire terrestrial herpetofauna community in mature, riparian and upland forests.

I conducted surveys of amphibian, reptile, and small mammal communities surrounding 4 isolated, upland and 6 stream-connected temporary wetlands on Tombigbee National Forest and Noxubee National Wildlife Refuge in north Mississippi from May 2001 - March 2006. Trap captures yielded 17 amphibian species (n = 11,142), 21 reptile species (n = 541), and 10 small mammal species (n = 472). Upland pools supported greater diversity of Ambystomatid salamanders, anurans, lizards, and mice (Peromyscus spp.), than floodplain pools. Factors including landscape position of pools, proximity to alternate water sources, and barriers to dispersal potentially influenced faunal communities of temporary wetlands. Infrared-triggered cameras were used to monitor mammalian activity surrounding ephemeral wetlands to determine potential depredation of pitfall traps. Raccoons (Procyon lotor) accounted for most images (35% total images). I assessed relative effectiveness of sampling techniques to capture and retain herpetiles in presence of depredation. I tested pitfall trap, funnel trap, and pitfall trap combined with exclusion cover along driftence arrays. Overall, pitfall traps of both designs produced more captures than funnels, and excluded-pitfalls yielded greater captures than un-excluded pitfalls for most amphibians. Overall mortality rates were <2% of total captures with anurans accounting for most (63.30%) mortality. I submit that in long-term studies, pitfall traps with exclusion are prudent to limit sampling bias and mortality occurring with depredation of captured herpetofauna. During trapping, pit-traps of both designs yielded incidental captures of small mammals. Capture rates for small mammals were similar in un-excluded pitfall traps and excluded pitfall traps. Southern short-tailed shrews (Blarina carolinensis) and mice accounted for 93% of total captures and suffered 76% and 52% mortality, respectively, potentially due to exposure, starvation, flooding, and/or depredation. Additionally, one species listed as rare in Mississippi was captured during herpetofaunal surveys, oldfield mouse (Peromyscus polionotus). Due to potential impact of pitfall trapping on small mammals, I recommend that researchers either alter trapping methods to address non-target hazards (frequency of checking traps, providing shelter) or work cooperatively using an integrated survey approach for herpetiles and small mammals to limit trap mortality.

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