Carp Hills area resident Mike Nash wanted to put his new molecular biology knowledge to practical use. He reached out to Friends of the Carp Hills in the summer about conducting a DNA study. After researching the options, we decided to experiment with a relatively new technology called Environmental DNA (eDNA).
In October 2021, we conducted a pilot eDNA project on three ponds in the Carp Barrens and sent the samples to a lab for sequencing. We received the results about a month later.
The DNA analysis was self-funded by the participants.
$300 raised so far.
You can support the project with a small donation. Use PayPal or e-transfer.
Friends of the Carp Hills is a non-profit organization, but we are not a registered charity.
What is eDNA?
Environmental DNA is an emerging and important technique to help measure biodiversity. It usually involves taking a water sample although soil and even air can be sampled. All living organisms shed DNA (e.g. skin, hair, feces, urine), which can be found in water for aquatic species, and even from terrestrial wildlife near the sampled water body.
Rise in the use of eDNA is driven by reduced costs in sequencing, advances in Bioinformatics, and efforts by industry and academia to catalog the DNA of all life on the planet.
How is sequencing done? A combination of chemicals (polymerase+primers) are added to the DNA in the sample and amplified many times (by heat cycling). The end result is sufficient DNA so that a sequencer can read a statistically significant number of small regions that can identify a species. In our testing, the sequencer read about 80,000 DNA molecules from some very small water samples we took. The sequencing approach is referred to as short-read amplicon sequencing.
The lab we used – Jonah Ventures – performed NGS (Next Generation sequencing) and not qPCR. NGS “casts a net” – what lives in this pond? qPCR sequencing is done when you want to focus on a single species; e.g. “I think that there are Blanding’s turtles in this pond, let me prove that this is true.”
Description of Carp Hills eDNA Pilot Project
Part 1
We took four water samples at three ponds in the Carp Barrens in October 2021 (Step 1). Water was added to a syringe and forced through a filter to concentrate the suspended solids. The filter was removed and sent to Jonah Ventures for analysis (Steps 2 -8).
We paid for four assays:
- Vertebrates (12S) – mammals
- Phytoplankton (23S)
- Macroinvertebrates (COI) – insects and microscopic
- Fish (12S)
What did we find?
We collected the samples two days after a major rainfall so the water levels were high and the ponds would have experienced recent terrestrial runoff. The species found are those that have interacted with a pond within the last 21 days. Over 90 species were identified with >97% confidence.
Vertebrates
- Canus lupus familiaris – Domestic dog DNA accounted for 50% of vertebrate species recorded. All dog DNA was found in one pond where the trail runs very close to the shoreline. DNA specific to ten breeds was identifiable (e.g. West Highland Terrier, Toy Poodle, Gordon Setter). Dogs are prohibited from the Carp Barren Trail so clearly there are people not following this conservation measure. Dog DNA likely entered the pond from feces and urine flushing into it from runoff and dogs drinking the water.
- Muskrat, Beaver, and Coyote were also found.
Phytoplankton
- Phytoplankton form the base of marine and freshwater food webs. They are key players in the global carbon cycle, photosynthesizing carbon dioxide and sunlight into oxygen and carbohydrates.
- Each pond had a different phytoplankton profile.
- The dominant species were algae.
- When there are too many nutrients (e.g. fertilizer and feces runoff) in the water, algal growth can get out of hand and cause “blooms” where the pond turns green. The blooms reduce oxygen levels in the water and have other deleterious effects on pond dwellers.
- Cyanobacteria are present in two of the ponds. These are a group of photosynthetic bacteria evolutionarily optimized for environmental conditions of low oxygen. When they die, they become toxic. If ingested in sufficient quantity, the toxins can damage major organs for vertebrates like dogs and humans and even cause death.
Macroinvertebrates
- Macroinvertebrates are small aquatic animals and the aquatic larval stages of insects that form an important part of the food web in a pond. They include dragonfly and stonefly larvae, snails, worms, and beetles.
- Aquatic macroinvertebrates are good indicators of water quality because they are affected by the physical, chemical, and biological conditions. They can’t escape pollution and show the effects of short- and long term events.
- Rotifer species were the main macroinvertebrates. They are the pond’s garbage colllectors, eating particulate organic detritus, dead bacteria, algae, and protozoans.
Fish
- The top four species found were Central Mudminnow, Brook Stickleback, Finescale Dace, and Pumpkinseed. These are widely found throughout the Ottawa area. They are warm water tolerant, and can tolerate low oxygen conditions.
- There were some anomalous species (out-of-range for this area), which may indicate a DNA library or sample problem.
- Each pond had it own mix of species.
Spreadsheets with the species list details can be downloaded using the link below.
What Happens Next?
Part 2 – Finish the pilot project.
We would like to run sequences for: reptiles and amphibians, bacteria, plants, and possibly mollusks.
- We’re looking for:
- species like Chorus Frogs, turtles, and snakes that have been heard or seen in the ponds to confirm that the eDNA technology captures their presence,
- species that we are not aware of, and
- information on the bacteriological community including E. coli.
The cost for this is approximately $800.
Part 3 – Future possibilities.
Our eDNA project was an informal pilot to understand how to use the technology and what information it might provide.
We are considering larger, more formal projects in the future.
- Create a comprehensive species inventory of the Carp Barrens ponds.
- Monitor the ponds for changes over time to determine any impacts from increased human presence due to the trail.
- Compare Carp Hills ponds near the Carp Barrens Trail and Crazy Horse Trail with those that are isolated from significant human presence.
- Other ideas as may be offered by the scientific community.
We need support from biologists to carry on.
How Can You Help?
Biologists. Join our eDNA working group – contact us to start the conversation.
Support the Project. Donate to help us finish sequencing the three Carp Barrens ponds as part of our pilot project. Use PayPal or e-transfer.
- This will cost approximately $800.
- Any addition funds will be used for further studies.
$300 raised so far!
Friends of the Carp Hills is a non-profit organization, but we are not a registered charity.
Learn more
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Carp Hills eDNA Project Information
YouTube: Casting a Net for DNA Webinar Video from 25 January 2022.
Presentation: Casting a Net for DNA Webinar (PDF) slides from 25 January 2022.
List of References by slide number (PDF).
Link to eDNA wordcloud.
Jonah Ventures – the lab that did our analysis.
Link to eDNA species spreadsheets. Eight files – two for each of: 12S Vertebrates, Fish, 23S Phytoplankton, Macroinvertebrates (labeled ArthCOI).
Sample test methods (PDF). Four files – 12S Vertebrates, Fish, 23S Phytoplankton, Macroinvertebrates (labeled ArthCOI).
References