Development and application of an antibody-based protein microarray to assess stress in grizzly bears (Ursus arctos)

Abstract

There is an inherent conflict over land use between humans and wildlife. Human activities can alter habitat, creating pressure on North American large carnivore populations. Traditional wildlife techniques can be slow to show population declines, especially in long lived species with slow reproduction rates and high mortality of young, such as grizzly bears (Ursus arctos), which leads to delayed information for land managers trying to find the balance between human use of land and preservation of wildlife. Concern about population health of grizzlies in Western Alberta, Canada has lead to investigation of the impacts of current land use within grizzly bear habitat. The objective of this work was to develop a protein microarray that could detect patterns of physiological stress in a rapid manner with small samples of grizzly bear tissue. Sampling from four regions in the foothills of the Rocky Mountains in Alberta resulted in the capture of 133 bears. During the developmental phase, proteins involved with mitochondrial function were found, using two dimensional gel electrophoresis, to be altered in situations of increased stress. Limited cross-reactivity was found when evaluating grizzly bear stress protein expression using commercially available protein microarrays. The protein microarray developed in this thesis consists of 31commercial antibodies validated for grizzly bears. These antibodies recognize proteins associated with different aspects of the stress response, including the hypothalamic-pituitary-adrenal axis, apoptosis/cell cycle, cellular stress, and oxidative stress and inflammation. Skin was selected as the tissue for evaluation of protein expression. Strong correlations were found between many of the proteins within functional categories. Model selection for the protein categories revealed variation that corresponded with region, serum markers of stress (total cortisol and hsp60), growth, the density of roads in the habitat and the amount of anthropogenic change in the bear’s home range. Regional trends of expression found bears in Swan Hills and bears from North highway 16 having elevated expression of the proteins measured by the microarray. The protein microarray was thus able to detect expression patterns reflecting physiological and environmental markers. The array shows great promise for future use in detection of potential distress in wildlife populations due to alterations of their habitat.