Environmental DNA (eDNA) is the genetic material from a plant or animal that is found in the air, water or soil. Fish, such as invasive carp, release DNA into the environment in the form of skin cells, secretions, and feces. This DNA can be collected from water samples in the field and be used to indicate the potential presence of an individual species.
A positive eDNA hit does not necessarily indicate the presence of a live carp. eDNA evidence cannot verify whether live invasive carp are present, whether the DNA may have come from a dead fish, or whether water containing Asian carp DNA may have been transported from other sources, such as bilge water.
eDNA testing is useful as a potential early indicator of invasive carp presence and was developed to improve monitoring of such aquatic invasive species. All fish release DNA into the environment and scientific sampling can detect and identify species-specific organisms (bighead and silver carp). However, as there remain many uncertainties about what a positive eDNA sample indicates, it is used in conjunction with other detection tools. This comprehensive approach includes electro-fishing, netting, the operation of electric barriers, and the construction of fencing to prevent carp from crossing between waterways, among dozens of other measures.
The Great Lakes Region of the U.S. Fish and Wildlife Service has been conducting eDNA testing since 2013 within the Chicago Area Waterway System and other areas of concern within the Upper Mississippi River, Ohio River and Great Lakes. To ensure reliable results the Service maintains the Quality Assurance Project Plan (QAPP) for eDNA monitoring of bighead and silver carps which details the eDNA monitoring process, including methods and quality control.
The U.S. Fish and Wildlife Service processes eDNA samples at the Whitney Genetics Lab in Onalaska, Wisconsin.
eDNA Calibration Study
In late 2011, the Invasive Carp Regional Coordinating Committee funded a study to better understand environmental DNA or eDNA. This study was referred to as the eDNA Calibration Study which became known as ECALS.
The main purpose of ECALS was to improve the application of eDNA methodology to assess and mange uncertainty. ECALS investigated alternate sources of invasive carp DNA, improved existing genetic markers and investigated the relationship between the number and distribution of positive eDNA samples with the density of invasive carp populations. The results of this study have allowed project managers to better interpret eDNA results, as well as investigate ways to make the eDNA process more efficient by decreasing processing time and cost.
Study objectives:
Objective 1. eDNA vector assessment
Identify potential vectors, or sources, for invasive carp DNA to enter the Chicago Area Waterways (CAWS) without originating from a live, free-swimming bighead or silver carp (or hybrid). Potential vectors include the introduction of tissue and other cell matter containing invasive carp DNA from:
- Movement of carp and carp material via fish-eating birds, particularly eDNA deposits entering the water via bird excrement
- Combined sewer outfalls (specifically in regions of Chicago where invasive carp are sold in markets on the street) as a vector of eDNA
- Fish carcasses that are transported via barges that pass through the electric dispersal barrier
Objective 2. Develop eDNA markers for improved population inference
Develop high-fidelity, sensitive genetic markers for detecting the presence of invasive carp DNA and for making broad estimates of invasive carp abundance. Using multiple markers may aid researchers in their ability to estimate invasive carp populations and movement.
Objective 3.1 Increase the efficiency of eDNA processing
Develop a way to process eDNA samples more efficiently by decreasing processing time and refining the process so it is more sensitive to lower concentrations of eDNA. As a result, eDNA processing time may be reduced from 10 working days to five working days, allowing for much quicker turnaround time for results and more rapid response to new eDNA hits. Also, assess whether different water sampling protocols might improve invasive carp detection probabilities (depth- integrated sampling, new filtering techniques, appropriate volume of water to sample).
Objective 3.2 Determine eDNA assay sensitivity under no flow conditions
Determine the relationship between invasive carp size, number and behavior on eDNA loading rates (DNA shedding, sloughing), minimum amounts of eDNA required for detection and time to detection, and the rate at which detectable amounts of eDNA fills a volume of water during non-flowing conditions.
Objective 3.3 Determine eDNA assay sensitivity under flowing water conditions
Determine the rate at which detectable amounts of eDNA fills a volume of water during flowing conditions.
Objective 3.4 Quantify relationships between major environmental factors and eDNA degradation under no flow conditions
Determine the relationship between environmental factors -- water temperature, light exposure, zooplankton and microbial biomass, pH -- on eDNA degradation rates in no flow systems.
Objective 3.5 Develop guidance on using calibration data to broadly estimate invasive carp abundance under no flow conditions
Evaluate likely spread of eDNA from source points in no flow systems so that sampling can be planned such that sample points are reasonably expected to represent independent samples (not from same eDNA plume).
Objective 3.6 Develop a hydrodynamic eDNA transport predictive model to characterize fish occurrence
Models based on fish behavior and CAWS water-flow dynamics will be developed to better understand eDNA behavior in the system. Models will also help identify likely carp population scenarios.
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify a species.
Calibration refers to investigating the suite of parameters that may influence the detection, degradation or persistence of DNA, and defining how those parameters affect these variables. For example, what effect does temperature have on DNA? What effect does flowing or non-flowing water have on the movement of DNA in a system?
When vectors are described, the source of DNA has to also be considered.
A source is a point of entry into the environment where the fate and transport of eDNA goes from being controlled to being uncontrolled. Think of eDNA as a pollutant. A source is defined by a location and a mode of release.
A vector is a mode of transport in the environment. It may be a bird, rainfall runoff, storm or sewer flows, or a barge, etc. For example, barges may transport dead fish or fish slime on the hull. Runoff may carry fertilizer, fishy ice, etc.
For example: Live fish below the electric dispersal barriers are an obvious potential source of eDNA. They release eDNA into the environment. Once released into the environment, the eDNA may move upstream by one of several vectors - bird, barge, recreational boat or fisherman.
The purpose of ECALS was to investigate alternative sources and pathways for eDNA detections beyond a live fish. The study also examined how environmental variables such as light, temperature and water velocity impact eDNA detections, explored the correlation between the number of positive samples and the strength of the DNA source, developed more efficient eDNA markers to cut the sampling processing time in half and modeled eDNA transport specific to the Chicago Area Waterway System. The intent was that all entities using eDNA as a monitoring tool would be able to use this information to refine their own monitoring and management programs.
ECALS is an interagency study, with U.S. Army Corps of Engineers, U.S. Geological Survey and U.S. Fish and Wildlife Service participating and was fully funded by the Great Lakes Restoration Initiative.
The ECALS Team conducted a storm sewer experiment in late 2011 to investigate our ability to detect DNA being transmitted to the river from a storm sewer after being deposited into a storm drain. This was in response to a report from an Illinois Department of Natural Resources biologist who observed ice holding dead invasive carp being dumped directly into a storm sewer in Chicago's Chinatown- an area where we have detected eDNA in the river nearby. The team simulated this event and demonstrated that, indeed, DNA can be deposited to the river by this mechanism. This study was repeated summer 2012.
Lab filtering of DNA is a time-consuming step of the eDNA sampling process. The ECALS team investigated alternative sampling protocols to make eDNA sampling more efficient. One way is to minimize, or eliminate, the lab component by conducting field filtering. Using sieve cloth, river water was filtered in the field, enabling the samples to be processed directly. It was assumed that eDNA is attracted to particulate material. Extracting the particulate material in the field, the team was able to process those samples for the presence of invasive carp DNA.