Nicrophorus americanus is a federally endangered species whose range has decreased dramatically since the 1920s. It is a nocturnal species that is only active from May to September when temperatures reach at least 15.5°C for three nights in a row. It once ranged throughout North America, from Maine and the southern parts of Canada, west to South Dakota and south to Texas. The historic Texas population consists of four Texas specimens from the 1880s residing at the Philadelphia Academy of Science Invertebrate Collection. Since then, there were no confirmed specimens in Texas, until 2003 when a single individual was found in Lamar County, Texas. The population discovered in Lamar County has been on a steady decrease from 2005, 223 individuals captured, until 2008 only 8 individuals captured. Since 2008, no individuals have been captured in Texas, despite intense surveys. It is possible that the Texas population is a sink population with the Oklahoma population to its north, acting as the source. Genetic analysis of the Texas and Oklahoma populations would help to answer this question and analysis of the entire population may answer questions to why the species declined leaving only the peripheral populations. The specific habitat variables that caused the population to re-colonize and go extinct in Texas are unclear, but when determined could play a critical role in managing the population. / text

I studied a small, enigmatic, and imperiled black bear population in south-central Florida from 2004 - 2006. Annual home ranges of males (96.0 km2) were larger than those of females (32.2 km2). Female home ranges were smaller in winter than in summer or fall. At the landscape scale, bears selected forests, scrub, and citrus, but avoided urban areas. At the home range scale, bears selected bay swamp and hardwood hammock, but avoided urban areas and grassland. Bears selected bay swamp in winter, forests and scrub in summer, and forests, scrub, and marsh in fall. The bear’s diverse diet included citrus fruit. Important foods were acorn, saw palmetto fruit, and Florida carpenter ant. The local landscape is dominated by agriculture on private lands, as opposed to large contiguous forests on public land elsewhere in Florida black bear range. Mean patch size of forests was smaller, while edge density, diversity, and evenness were higher in south-central Florida than elsewhere in the state. Diversity of forest habitat may partially account for the persistence of the black bear in this fragmented landscape. Managers should encourage private landowners to adopt practices that promote bear habitat, and focus on habitat diversity, road crossings, and statewide metapopulation structure.

The study of marine antifreeze proteins has provided new findings. The blue mussel (Mytilus edulis) was known to have antifreeze activity; however, the antifreeze protein or other molecule responsible has never been characterized. Activity was evident in mussels from each of the Maritime provinces, Canada. The antifreeze molecule was shown to alter ice crystal morphology. It functioned over a wide range of pH values and it showed protease resistance. Nonetheless, its purification was not achieved. A winter flounder (Pseudopleuronectes americanus) ?-helical antifreeze protein, wflAFP6, has been shown to form amyloid-like fibrils during freezing. Separation of different aspects of the freezing process demonstrated that equilibrium freezing with an ice template is necessary for conversion of the wflAFP6 to the amyloid-like conformation. Amyloid-like conformation was determined by dye binding and electron microscopy. The effects of wflAFP6 concentration and solution properties were determined in order to better understand the process of conversion.

Wildlife managers use models to aid in predicting high risk areas for human and black bear (Ursus americanus) interactions (HBI). These tools help managers implement management strategies to minimize HBI. Over 3,000 incidents of HBI were compiled from management reports at Great Smoky Mountains National Park (GSMNP) during 1998-2011, a park with 9-10.2 million visitors per year and a black bear population of about 1,600 bears. We used data from bear management reports along with annual visitor use, mast and bear abundance data to develop a series of generalized linear models to assess the spatial and temporal factors affecting HBI. Although HBI occurred throughout the GSMNP, 50% of all HBI occurred in five areas. The best predictor variables of HBI across four subsets of models included interaction between mast production and number of park visitors, month, vegetation cover, visitor activity, and bear abundance. Although there was not a clear relationship between visitor use and mast abundance, the number of park visitors was always relatively high and HBI increased substantially in poor mast years. HBI was more frequent during summer months when park visitation rates and more people and food were present overnight in frontcountry and backcountry camping areas. Over 43% of HBI in hemlock forests were serious. Bear abundance data were not a strong predictor of HBI, and bear bait stations may not provide a sensitive index to bear abundance. GSMNP uses different strategies for managing HBI to protect visitors and bears. In 1991, bear proof waste disposal containers and food storage devices were placed in camping and picnic areas. In combination with aversive conditioning, HBI decreased in some areas of the park. We recommend that proactive bear management programs including education, enforcement of park regulations, and aggressive aversion conditioning of bears be implemented at the identified HBI high risk areas to provide a safer environment for both people and bears in GSMNP.

Understanding species’ distribution, density, and sources of bias in population estimates is critical for reliable conservation strategies. I assessed American black bear distribution, density, and abundance in southern Missouri. Using anecdotal occurrence data, I demonstrated support for a northward trend in extent of occurrences over time and a positive correlation between bear distribution and human–bear incidents. I also used GPS telemetry and camera traps to investigate detection biases in DNA hair snare methods and tested efficacy of two sampling designs for estimating density using spatial capture-recapture models. Results demonstrated that detection probability decreased following a negative asymptotic relationship with decreasing bear proximity to snares and that hair deposition rates decreased over time. Precision of estimates for low density populations with non-uniform distribution increased when using multiple arrays with intensive snare spacing. Optimizing the tradeoff among snare spacing, coverage, and sample size is important for estimating parameters with high precision.

Dens are a critical component of black bear (Ursus americanus) habitat, yet scale-dependent den-site selection has received limited attention. Natural and anthropogenic factors (e.g., vegetation, roads) may also influence bear den-site selection. I quantified black bear denning chronology and den use and evaluated multi-scale den-site selection in Mississippi, USA during 2005–2011. Females entered dens earlier than males and emerged later; multiple den use by both sexes in a single winter was common. I recorded equal numbers of tree and ground dens, with ground dens at higher elevations surrounded by dense vegetation. Chronology and other denning characteristics of bears in Mississippi were similar to other black bear populations in the southeastern United States. Bears exhibited scale-dependent den-site selection selecting sites with greater percentage horizontal cover and farther from roads. Greater percentage horizontal cover may provide security and increase energetic efficiency. Denning farther from roads likely decreases risk of human disturbance.

The population trajectory of woodland caribou (Rangifer tarandus caribou) in Newfoundland is currently determined by low calf survival due to high predation rates during the first 6-8 weeks after parturition. Most caribou in Newfoundland congregate and give birth in open calving grounds; consequently, in order to investigate predator-prey interactions, design research, and develop mitigation strategies, the geographic extent of the caribou calving grounds must be properly identified. We used VHF telemetry locations of caribou calves, collected from 2003-2010, to determine the spatial and temporal extent of caribou calving grounds in three study areas in Newfoundland. We put GPS collars on 47 black bears (Ursus americanus) in 3 caribou ranges where bears are having a significant impact on caribou recruitment by preying on calves during the calving season. Bear movements were greatest during the calving season, potentially increasing encounters with calves. Some bears migrated to the calving grounds just prior to caribou parturition, indicating deliberate broad-scale selection of areas of high calf density. Bears displayed interannual fidelity to calving ground usage patterns during the calving season, with some bears using the calving grounds every year, while others did not. We estimated the probability of a bear spending time in the calving grounds during the calving season as a function of the bear’s sex and mean distance to the calving grounds with logistic regression. We found that as distance increased, the odds of a bear spending time in the calving grounds decreased, and that at any given distance the odds were greater for male bears than for female bears. Our results indicate that some bears in Newfoundland are likely caribou calf predators, while others are not, and that the sex and broad-scale distribution of bears influenced the probability of a bear participating in calf predation during the calving season. The probability distribution of calf-visiting bears could be used to develop management practices to mitigate the impact of bear predation on declining caribou herds in Newfoundland.

Whether active or passive, dispersal accompanied by gene flow shapes the population genetics and evolutionary divergence of species. Indirect methods which use genetic markers have the ability to assess effective dispersal—that which resulted in gene flow. My objective was to see if an aquatic insect and an obligate aquatic invertebrate show similar phylogeographic patterns and genetic uniqueness. Hyalella azteca and Callibaetis americanus were collected from 4-5 springs in each of six basins in the Great Basin of western North America. No dispersal or genetic studies of C. americanus have been conducted to date. However, several studies focusing on mtDNA diversity of H. azteca have revealed a tremendous degree of cryptic diversity in the desert springs of the Great Basin. Nested clade phylogeographical analysis (NCPA), FST values, AMOVA, and Mantel tests were used to examine geographical associations. I also used traditional phylogenetic approaches including maximum parsimony (MP) and likelihood (ML) analyses using cytochrome c oxidase subunit I (COI), 28S, and 16S as genetic markers. The mitochondrial COI sequence divergences in C. americanus were higher than H. azteca COI divergences within springs but lower among springs. FST values were very high in H. azteca reaching near fixation for certain alleles. C. americanus FST values were lower suggesting greater gene flow and, consequently, greater dispersal rates. Even though Mantel tests did not detect significant isolation by distance when evaluating all haplotypes together, nested clade analysis was able to examine smaller networks of related haplotypes and detect significant isolation by distance. Whereas the genetic structure in C. americanus was dominated by restricted gene flow with isolation by distance, H. azteca was characterized more by gradual range expansion followed by fragmentation. Mayflies likely showed more gene flow than amphipods because of their flight capabilities, but movement was still restricted by long distances between isolated springs.

<p> Four stucturally distinct types of macromolecular antifreezes have been previously isolated from the sera of polar marine fish. When the water temperature surrounding these organisms drops below -0.7°C, the freezing point of their bodily fluids, any contact with surrounding ice will nucleate internal ice crystal growth. The antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) synthesized by the fish act to inhibit the growth of existing ice crystals in their sera through direct adsorption to the ice lattice. The α-helical structure of type I AFP from winter flounder has been solved to atomic resolution and its mechanism of ice binding has been proposed. The NMR solution structure of a type III AFP from ocean pout has identified proteins in this class to exist in a β-sandwich conformation, however their mechanism of action remains uncertain.</p> <p> To facilitate the pursuit of an x-ray crystal structure solution, we subcloned the gene for a type III AFP (HPLC6) into pET15b and expressed recombinant His-rHPLC6 AFP in E. coli. Purified rmHPLC6 product has been successfully crystallized, and heavy atom soaks were performed in order to attempt a structure solution by multiple isomorphous replacement. The lone tyrosine in this recombinant AFP has been successfully derivatized in solution with iodine, and the modified protein was crystallized. In order to optimize the measurement of anomalous scattering information, modifications to our data collection system were required. Cryocrystallography techniques were employed to improve the quality of collected data.</p> <p> The expression, purification, crystallization and optimized data collection on an iodine-derivatized type III AFP from ocean pout will be presented here. This work has been instrumental in providing the high quality x-ray data required to solve the crystal structure to atomic resolution. Future examination of the solved structure will promote an increased understanding of the ice-binding mechanism exhibited by this class of proteins.</p> / Thesis / Master of Science (MSc)

The United States Fish and Wildlife Service (USFWS) expressed the need to develop appropriate management strategies for apparently high-density, growing black bear populations in the Roanoke-Neuse-Tar-Cape Fear ecosystem in coastal North Carolina and Virginia. In order to provide the scientific information necessary to develop these strategies, I investigated population densities and genetic structure of black bears at 3 national wildlife refuges [Great Dismal Swamp (GDSNWR), Pocosin Lakes (PLNWR), and Alligator River (ARNWR)]. Density estimates were derived from DNA samples collected noninvasively at each of the 3 refuges for 2 consecutive summers. Hair samples were analyzed for individual identification using 6-7 microsatellite markers. Estimated densities were some of the highest reported in the literature and ranged from 0.56-0.63 bears/km2 at GDSNWR to 0.65-1.12 bears/km2 at ARNWR to 1.23-1.66 bears/km2 at PLNWR. Sex ratios were male-biased in all areas of all refuges. Genetic variability and structure of bears at these refuges was assessed using 16 microsatellite markers for 40 bears from each refuge. Genetic variability of the 3 refuge populations was substantially high compared to other bear populations in North America, with observed heterozygosities ranging from 0.6729 at GDSNWR to 0.7219 at ARNWR. FST and DS values were relatively low (0.0257-0.0895 and 0.0971-0.3640, respectively), indicating movement of bears and gene flow across the landscape is adequate to prevent high levels of genetic differentiation and structure among the refuge bears. Genetic statistics at GDSNWR indicate that this population is isolated to some degree by geography (i.e., the Albemarle Sound) and encroaching urban development (i.e., the towns of Suffolk and Chesapeake). ARNWR has the potential to become isolated in the future if movement corridors to the south of the refuge are not maintained. Harvest of bears is likely warranted at PLNWR and ARNWR, though extreme caution must be taken the first few seasons as hunter success will be extremely high. Further research is needed to determine population growth rates, reproductive parameters, and survival rates at all 3 refuges, particularly if a hunting season will be established and maintained in these areas. Methods for regularly monitoring bear populations at these refuges also should be incorporated into biological programs, as bears comprise a significant component of the ecosystem at these refuges and cannot be ignored when outlining management goals. / Master of Science