Characterization of anthropogenic Influence on the gut microbiota of captive and wild specimens of Ursus americanus

Ursus americanus, the American Black bear, is native to North America and has an expansive range that covers most of Canada, parts of the Pacific Northwest, and the Atlantic Northeast including New York, Pennsylvania, and the Virginias. Within California, black bears inhabit most of the mountainous regions, which span from the Klamath Mountains in Northern California to the San Bernardino Mountains in Southern California. Black bears are extremely adaptable to different environments but typically utilize forested areas that provide both food resources and a suitable barrier from human encroachment. As landscapes change due to human growth and expansion, bears have adapted. Bears living at the wildland-urban interfaces have altered their behavior in response to human encroachment into their territory. A novel anthropogenic food source in the form of trash has been adopted by the bears because it is easily accessible and calorie dense. This food source is temporally and spatially predictable and is regularly replenished. Another important factor potentially influencing bear health and disease is the species composition and relative abundance of bacteria comprising the gut microbiota. Collectively, the gut microbiota includes all bacteria, archaea, viruses, and eukaryotic microbe inhabitants. Black bears compensate for the lack of an anatomically specialized digestive tract and cellulose-digesting enzymes by housing cellulolytic microorganisms in their gastrointestinal (GI) system. Utilizing fecal samples provides a non-invasive method to characterize the bacterial species that are present within the GI system. Many factors can influence the results of microbiota studies such as collection, storage, and extraction methods, experimental design, and data analysis making it critical to maintain methodological consistency throughout a study. Ensuring efficient DNA extraction from every sample resulting in high DNA yield and quality is necessary to accurately analyze microbial communities. One of the current popular techniques for studying complex microbial communities is through targeted 16S rRNA amplicon sequencing. Accuracy of genus and species level identification can vary depending on the region of the 16S rRNA gene that is sequenced. Research has shown that the V4 region is reliable for representation of the full-length 16S rRNA sequence for phylogenetic analysis. Another factor that affects the results of microbial community studies is how the resulting data is analyzed. The field of microbiota characterization, specifically in Ursus americanus, has left several unanswered questions including: How does anthropogenic influence affect the species distribution and relative abundance of bacteria within the gastrointestinal system of Ursus americanus? Additionally, are there notable differences in observed bacterial species between the three study groups of captive and wild Ursus americanus specimens? Finally, how does bacterial DNA extraction method and data analysis affect the outcome of the characterization of bacterial species? To answer the above questions, I have designed an experiment that targets three different groups of captive and wild Ursus americanus specimens. Fecal samples from these specimens will be utilized to investigate my questions using the following specific aims: Specific Aims I. To compare and assess extraction of DNA from fecal samples of Ursus americanus from three different study groups; captive, wildland, and urban-wildland bears. a. Two different extraction methods will be compared using the QIAamp Fast DNA Stool Mini Kit. Each method follows the methods outlined by Qiagen with specific added steps and modifications to ensure efficient DNA extraction. The two extraction methods will be analyzed by next generation sequencing. b. Extraction efficiency will be assessed by the total DNA yield from each sample. DNA yield will be measured using the NanoDrop spectrometer and Quantus fluorometer. II. To characterize the relative abundance and species distribution of the gut microbiota of the three Ursus americanus study groups through next generation sequencing (NGS). a. Amplification, library preparation, and sequencing will be performed in-house using the Illumina Miseq platform. The V3 and V4 hypervariable regions of the 16s rRNA gene will be sequenced in both the forward and reverse directions using the Miseq v3 reagent kit to produce a full length read spanning both hypervariable regions. b. A bioinformatic analysis of the 16s rRNA NGS data will be performed using the QIIME2 pipeline. Different quality control filters will be applied to the data at different points during the analysis. Hypotheses The above specific aims will allow me to address the following hypotheses: I. Due to the ability of lysozyme to hydrolyze the 1,4-beta linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine in the peptidoglycan of Gram-positive bacteria, treated samples will result in a higher yield of DNA as well as a higher abundance of Gram-positive bacteria in comparison to untreated samples. II. Due to differences in diet, specimens of Ursus americanus from the captive study group will harbor a less diverse gut microbiota in comparison to the wildland and urban-wildland study groups. Statistical methods Using the software R, comparison of DNA yield between treatment groups will be analyzed with a paired t-test. Differences in relative abundances of predominant phyla will be analyzed with a one-way ANOVA. Comparison of the relative abundance and species distribution of the gut microbiota of the three Ursus americanus study groups will be analyzed with a PERMANOVA test.