TRA Grant Detail

The funding provided by this grant will fund the laboratory expenses required to complete the study. Materials requested for funding include two DNEasy Power Water DNA Extraction kits ($1,072 each).

This project serves a diverse group of "cavers" and "non-cavers," including the scientific community, the public, landowners, and many more. Because the field work required of this project occurs in Merritt's Mill Pond, accessible through the public Jackson County State Park area, community members and cave divers in the area have expressed interest in the nature of the project, how it effects them, and what they can do to assist. This provides an unique opportunity for myself and my fellow researchers to share our work with the public and educate the community on the purpose of our work. In addition, contributing non-invasive monitoring techniques to the scientific community and natural resource agencies alike is a main priority of this project.

Optimization of an eDNA detection assay will aid natural resource managers with a methodology to allow for continued, long-term monitoring of imperiled species in dangerous habitats, such as the Georgia Blind Cave Salamander. In addition, raising awareness to the diversity and presence of such an interesting creature in these cave environments is crucial in advocating for the protection of this vulnerable species.

As previously stated, the main outcome of this project is to provide an effective methodology for natural resource managers and scientists to detect and monitor the Georgia Blind Cave Salamander, as well as assess the ecological relationships of present animals in an environment that requires extensive training and monetary support to work in. In addition, the outcomes of this project will be made publicly available to community members, the caving community and government agencies. Several components of the project will be published in multiple scientific journals, including Conservation Biology, The Journal of the American Society of Ichthyologists and Herpetologists, and others.

The objective of this study is to target the Georgia Blind Cave Salamander (Eurycea wallacei) in several historical sites in northwestern Florida to determine the feasibility of using environmental DNA (eDNA) assays to detect a species of low abundance using both passive and active sampling techniques, as well as conduct an ecological survey of other co-occurring metazoan species within the system.

This project began in August of 2023 and will continue into late summer of 2025 as the applicant's Master's Thesis at Southeastern Louisiana University under the advisement of Dr. Christopher Beachy and Dr. Kyle Piller. The project is broken down into these phases:
1. Preliminary method testing and species-specific primer and protocol development.
2. Cave-Diving certification
3. Field collection
4. Laboratory analysis
5. Next-Generation Sequencing
6. Data Analysis
7. Thesis defense, publication of findings

According to the project timeline phases, the following goals will be completed:
1. Preliminary method testing and species-specific primer and protocol development.
a. Species-specific methodology will be developed in the laboratory to ensure that
we can effectively detect the salamander in a captive colony of the species
before utilizing the techniques with field samples.
2. Cave-Diving certification
a. Due to the certifications required for the PI to enter the underwater cave
systems to collect water samples, Gretchen will complete her TDI Cave
certification in Summer 2025 prior to collecting any cave samples.
3. Field collection
a. 1500ml of water will be collected at three locations within each cave locality -
surface, cavern entrance, and within the cave. These three locations were
chosen to gauge the distance in which researchers must enter the system in
order to detect the salamander.
4. Laboratory analysis
a. Water samples will be filtered, followed by DNA extraction, PCR amplification,
visualization of successful DNA detection with gel electrophoresis, and
prepared to be sent to a third-party organization for next-generation
sequencing.
5. Next-Generation Sequencing
a. Amplified DNA samples will be sent to a third-party organization for
next-generation sequencing. This will sequence every strand of unique DNA
present in the sample.
6. Data Analysis
a. Sequence data will be statistically analyzed by the applicant. Bioinformatics
techniques will be used by the applicant to determine presence or absence of
Eurycea wallacei, as well as characterize the metazoan DNA present in each
sample.
7. Thesis defense, publication of findings
a. The culmination of this project will be presented and defended during the
applicant's public thesis defense. Any and all findings of the project will be
published through applicable scientific journals, as well as shared with the
community, government agencies, etc.

The determination of the validity of using environmental DNA to detect Eurycea wallacei could give us two answers - it worked, or it didn't work. Both answers are just as effective in determining next steps of monitoring the elusive species. If the assay was successful, this could be applied to many different rare species from a variety of different biomes. If the assay was ineffective in detecting the salamander, then we can pursue alternative monitoring techniques to ensure the protection of this salamander.

The culmination of this project will be presented and defended during the applicant's public thesis defense. Any and all findings of the project will be published through applicable scientific journals, as well as shared with the community, local government agencies, etc. Scientific journals we hope to publish the outcomes of this project in include NSS, Conservation Biology, The Journal of the American Society of Ichthyologists and Herpetologists, and others.

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After thinking each day how to make cavers happy I will refresh myself at Starbucks

NSS

Cavers everywhere will rejoice

A bunch of rejoicing cavers

To make cavers happy

6 months

Each day I will think of something to make cavers happy and then hit Starbucks for a refreshment

I will ask cavers if they are happy

Will shout from the rooftops that cavers are rejoicing