eDNA Sampling Surprises TIC Students

by John Davenport

Denver Trout Unlimited has a TU EmbraceA-Stream grant entitled “eDNA Sampling by Denver South Platte Youth.” Forty samples have been taken starting in the Fall of 2021. It was expected that the sampling would shed light on the biodiversity of the sampled water (mostly the South Platte Watershed) and over time highlight some successes, or focus attention on some problems as species decline.

The grant funds sample kits from JonahDNA in

Boulder, consisting of a 60cc syringe to suck up the water sample, a filter that screws on the end of the syringe to filter out molecules of DNA, rubber gloves, a DNA preservative and a mailing envelope to send the sample back to JonahDNA. Upon receiving the mailed in sample, JonahDNA sends the PCR amplified and fish targeted DNA to Texas A&M for sequencing. After a month results are returned as a data file with a count of the number of copies of specific strains of fish found in the sample. It costs approximately $50/ sample.

Traditionally, fish biodiversity in the Denver South Platte Watershed has been measured by Paul Winkle, Aquatic Biologist for Colorado Parks and Wildlife (CPW), and other CPW staff and volunteers, by dropping electroshocking probes into the water and capturing, counting, measuring, and assessing the stunned fish. This process costs more than $50/sample; however, electroshock sampling tells us a lot about the health, size, and abundance of the various species.

In 2008, 18 species were found between Chatfield and Confluence Park by electroshock surveying. In the fall of 2020, Jordan Parman, Water Quality Scientist from Metro Wastewater Reclamation, found 19 species by taking an eDNA sample at 120th Avenue in Denver. In the Spring of 2022, Trout in the Classroom students found 17 species with eDNA samples at Ruby Hill and Pasquinells landing, and Paul Winkle found similar results electroshocking on the opposite riverbank.

We have been pleased to see that eDNA and electroshock sampling methods were producing similar results. However, surprises came when the students took a pooled eDNA sample of their own Trout in the Classroom (TIC) tank and water from the site where they will be releasing their trout in the Spring.

Surprise # 1

WHOA! A ton of Atlantic Menhaden, California Anchovy, Pacific Pilchard, and Atlantic Mackerel DNA came up in our TIC tank eDNA sample!

Complex discussions ensued and students, educators, and volunteers concluded that these results were likely coming from the food they were feeding our TIC trout! We are feeding our fish…fish. Our TIC trout food is likely made up of Atlantic Menhaden, California Anchovy, Pacific Pilchard, and Atlantic Mackerel.

Surprise # 2

The TIC rainbow trout the students are raising, of course, were not all the same 170 base pairs but there were two distinct strains of DNA from our TIC tanks.

Picture #1 shows a GAC on that triplet (positions #19-21), and Picture #2 shows GGC (positions #1921). The "usual" sequence in that region is CAC and CGC respectively, but both strains show a G instead of C at position #19.

These two strains varied by a single Guanine (G) instead of a Cytosine (C) (which are two of four nitrogen-containing bases in DNA sequencing also known as nucleotides) at position 19 along the rainbow trout DNA strain.

Students concluded that our trout are therefore related to one another and siblings. Which CPW has also confirmed!

Surprise # 3

One of these distinct strains was also found in the river in a sample taken by another school. Could this be used to track how our released fish did over the following year?

It appears these fish had moved from their TIC release site in July of 2021 at Overland Park to eDNA sampling site at Ruby Hill in Dec. 2022.

More sampling will help confirm this theory. If it is true, it opens an interesting way to track the movement of fish without the cost of tagging or expensive electronics.

Surprise # 4

When American Indi- an Academy of Denver students compared the unique 170 base pair strains of rainbow trout with samples of rainbows from the Denver South Platte, Clear Creek, the Fraser River, and the Snake River in Dillon, they found five distinct strains.

One of the strains in their tank was also found in the Snake River.

Are these fish related? Are these fish former TIC trout? Were they stocked by CPW previously?

The students are eager to learn more, and we hope to continue our research.

Eighteen more eDNA samples will be taken by students this Spring to complete the grant study. The 170 base pair strain analysis will be continued next year as part of the TIC program. It will be expanded to sample the water near their TIC classrooms