Jessica Beyer
Marion College
Fond du Lac, WI
Mentor: Dr. James Maki

Yellowstone National Park is one of the most tectonically active regions on the planet. Within the park lies the Yellowstone Caldera; a 600,000 year-old volcanic feature that marks off a geographic region with some of the highest measured geothermal heat fluxes in the world. The caldera itself overlies magma chambers that are the sources of heat that create the park’s unique geothermal features: geysers, hot springs, fumaroles, and mud pots.

One of the most prominent features in the park is Yellowstone Lake. It is the largest (352km2) high altitude (>2000m) lake in North America. The lake is glacial in origin and is located almost entirely within the caldera. The portion of the lake within the caldera has an abundance of thermal features including fumaroles, hot springs, and geysers. Associated with many of these thermal areas are unique biological communities comprised of a variety of microbial populations.

Previously, investigators have observed the presence of worms, primarily leeches (Hirudinea), in the vicinity of hydrothermal vents. The goal of my project was to examine leeches collected from Yellowstone Lake to determine the presence and diversity of any microorganisms associated with them. 

Three different leeches (A, B, and C) were examined. All three leeches were identified as Nephelopsis obscura. The first leech (A, ~6 cm long) was collected from the surface of a remote operating vehicle (ROV) after it completed a dive. Leech B (~4.5 cm long) was found on an anchor after it was hauled to the surface. Leech C (~4 cm long) was found on a vent cap that had been retrieved from a hydrothermal vent. Each leech was cut into four sections and the tissues were homogenized in buffer. DNA was extracted from the homogenized samples. 

The DNA from the different sections was amplified using the polymerase chain reaction (PCR) and Bacterial and Archael 16S small subunit rRNA gene-specific primers. Leech A had amplification product from three of the four sections using the Bacterial 16S primers. No amplification product was observed using Archael primers. No product was observed from any sections of leeches B and C after amplification using either Archael or Bacterial primers over a variety of template concentrations. It is currently unclear (1), why only one worm apparently had bacterial populations associated with it, and (2), why these were not distributed in all sections of the worm. 

The PCR products from leech A were cloned and subsequent restriction fragment length polymorphism (RFLP) analysis was performed. RFLP data was obtained from a total of 180 clones. Thirteen different RFLP patterns were discovered, and some patterns were unique to certain sections of the worm. Section I had two unique patterns, section III had one, and section IV had four unique patterns. Two patterns were found in all three sections examined, and six patterns were found in two out of the three sections. These data suggest that the leech contains distinct microbial habitats that support these unique populations of bacteria. Sequencing of clones representing the different RFLP patterns will be performed. This will allow the phylogenetic analysis of the bacterial populations associated with this leech.