Using eDNA in the fight against Asian Carp

If you live North America, you may be concerned
about Asian carp, specifically the species of Asian carp known as silver and bighead.
Silver and bighead carps were originally imported into the southern United States for use in
aquaculture ponds in the 1970s. Through flooding they escaped and entered the Mississippi River
and its tributaries, where they have been invading northward ever since.
These two species of Asian carp have caused immense damage to the country’s aquatic
habitats, out-competing desirable fishes for food. That competition results in fewer and
slower growing native fishes. The U.S. Fish and Wildlife Service is part
of an international, federal and state collaborative that is working hard to prevent the spread
of self-sustaining populations of bighead and silver carps into the Great Lakes.
One of our newest tools in the detection of Asian carp comes from a scientific method
developed by the University of Notre Dame, and utilized extensively by the U.S. Army
Corps of Engineers for monitoring, that analyzes water samples for traces of Asian carp DNA
left behind in the environment, known as environmental DNA, or “eDNA.”
A fish’s genetic material can be left behind in the form of scales, cells, feces or mucus. Research has shown that such sources for eDNA
are more commonly found on the surface of the water. To find Asian carp eDNA, we therefore
take water samples from the top two inches of the water column.
The location of every water sample collected for eDNA analysis is recorded, along with
depth and water temperature. To ensure that water samples from different
locations are not accidently mixed, the fish biologist collecting water samples puts on
a new pair of gloves before each water sample is collected.
Once collected, the water samples are placed in a cooler on ice and taken back to the lab
for filtering. At the lab the water samples are carefully
handled to prevent contamination. Prior to filtering a new sample, a work station
is disinfected with a 10 percent bleach solution. The work station is then set up with sterilized
equipment. To ensure that the work station is not contaminated
with eDNA from earlier water samples, the lab technician runs an equipment control test.
A clean filter paper is placed on the vacuum manifold and 500 milliliters of deionized
water is run through the filter. Once the water has completely filtered, the filter
paper is removed and placed into a small vial. The filter paper will later be tested to ensure
that the equipment was completely clean before the actual water sample was filtered.
A new piece of filter paper is placed on the vacuum manifold and the process is repeated,
this time with the water taken from the bay, until the entire sample is filtered.
The green on the filter paper is caused by algae in the water. Mixed in with the algae
may or may not be the traces of Asian carp eDNA that scientists are looking for. To find out, the filter papers from each sample
are placed into a freezer and are eventually sent to a lab for DNA analysis. In order to determine if Asian carp DNA is
in a water sample, all DNA has to be extracted and tested. Several rinses with detergents break apart
cells to release DNA, and wash away fats, proteins and other organic matter. Ethanol
is used to precipitate DNA out of the solution and become a solid. We can’t see DNA with
the naked eye at this point, but sometimes a white substance can be observed at the bottom
of the test tube. At this stage in the process the origins of
the DNA in the sample are unknown. They may be from Asian Carp, or possibly from other
animals in the water. In order to find only the Asian carp DNA, we use a test called polymerase
chain reaction, or “PCR” for short. PCR uses species specific strands of DNA called
primers. We test the water samples for two species of Asian carp, bighead and silver.
Therefore we use specific primers to match bighead carp DNA and specific primers to match
silver carp DNA. These primers, along with the extracted DNA
from the water sample, are put into a mixture and undergo many cycles of heating and cooling
that create thousands and thousands of copies of any Asian carp DNA that was in the water
sample. These copies of Asian carp DNA are a specific
size, or length, of DNA that can be separated and then seen with the human eye on a starch
gel with a process called electrophoresis. In the gel there are bands, which in this
instance are the pieces of silver carp DNA that were replicated in the PCR. In one lane lab technicians ran a positive
control. This positive control was DNA extracted from a known source of silver carp DNA, in
this instance silver carp tissue. The control is used to determine which water samples have
silver carp DNA in them, and are considered positive, and which samples did not have carp
DNA in them. The PCR band, or product, is sequenced so
that we can confirm that the band we see on the gel matches silver carp DNA exactly. If
the sequences match, then the positive result is confirmed.
The absence of a band in the gel means that there was no Asian carp DNA in the sample. A positive eDNA sample does not always mean
that there was or is a live fish in the water. eDNA evidence cannot verify whether the DNA
may have come from a live or dead fish, or from other sources such as contaminated bilge
water, storm sewers or fish-eating birds. Though one positive eDNA finding for silver
or bighead carp may be alarming at first, what researchers are looking for are patterns
in where and when positive eDNA samples are found.
As a result, researchers will take multiple water samples from the same site over time.
When one or more areas routinely supply positive eDNA samples, it is of great interest to researchers
because it means that there is a sustained source of Asian carp eDNA in the vicinity. The science of eDNA holds great potential
for other invasive species detection and prevention. Researchers are currently developing the technology
to detect other carp species, as well as northern snakehead, round goby and goldfish.
eDNA may also one day be useful in the detection of threatened and endangered species, making
it a valuable tool in species recovery. The U.S. Fish and Wildlife Service, U.S. Army
Corps of Engineers and U.S. Geological Survey are currently conducting a study to improve
the understanding and interpretation of eDNA results. Together, we will work with our international,
federal and state partners to continue to push the limits of eDNA science to refine
its use.

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