TESTING DIFFERENT SAMPLING METHODS TO STUDY ACTINOBACTERIA IN PRISTINE VS HUMAN-IMPACTED KARST SITES

Caves and karst landscapes are important environments for watershed health and biotic diversity. As these sites become increasingly popular ecotourism attractions, balancing the preservation of these environments with the important roles they are beginning to play in local economies is important. There are many metrics by which cave “health” might be measured. Actinobacteria mats are often found along cave surfaces, and studying and comparing strains found within these communities could serve as indicators of natural cave biomes, which may provide a basis for ecotourism management and conservation practices.

In this study, we begin the characterization of microbiological facets of karst environments in The Cedars Natural Area Preserve (Lee County, VA) and other caves in Southwest Virginia. All of the caves included in this study were formed in the Cambro-Ordovician carbonates of the Appalachian Valley and Ridge Province. We are investigating whether human activity impacts the strains of Actinobacteria we see in frequently-visited vs pristine caves in close proximity to each other within the region. We assess the magnitude of human impact on caves according to factors such as how long and how frequently cavers are allowed inside a cave, how many people are allowed at a time, the structure of visitation, gear and hygiene regulations, and the presence of other metazoans. We also test multiple methods for sampling the bacterial mats, including using gas-tight syringes to collect water droplets pooling on the bacterial mats and using gloved hands to gently rub mats and then scraping the sample collected on the glove into a sterile tube. Samples were collected from cave walls in the light, twilight, and dark zones of the caves, suspended in DI water, then plated onto B-4 medium with spectinomycin. We have found that greater than 1:10,000 plating dilution is required. Preliminary PCR results indicate that Actinobacteria is present in all caves and that it can be detected with each of our sampling methods. We are continuing to investigate if there are differences in the strains of Actinobacteria present at the different sites. By studying how human ecotourism may impact these bacterial communities, we can learn how cave tourism management can be improved for optimal preservation, sustainability, and low-impact accessibility.