eDNA Calibration Study

In late 2011, the ICRCC funded a study to better understand eDNA. This study was referred to as the eDNA Calibration Study, or 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 (decrease processing time and cost).

The study had a number of specific 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.

Publications

ECALS 2014 Major Milestones

May 2014

Probabilistic model fully parameterized. Draft report on parameterization of the graphical model. Begin analysis of historical monitoring results.

December 2014

Third Interim Report

Download Report (PDF)

Suggested citation: “Asian Carp Regional Coordinating Committee. 2014. Environmental DNA Calibration Study, Interim Technical Review Report. Kelly Baerwaldt, editor. 234 pages. Available at http://www.asiancarp.us/”
Final report describing the probabilistic model and summarizing the analysis of historical monitoring results

This report summarizes efforts to resolve some of the ambiguity that surrounds interpretation of eDNA monitoring results in the Chicago Area Waterway System (CAWS). This has been accomplished by developing a model that supports probabilistic statements about the source(s) of eDNA detected in water samples and the presence of live bighead carp and silver carp upstream of the electric fish barrier, which is located at Romeoville, Illinois. Numerous other methods and models developed in the course of this effort are described in the report. These include methods to estimate the probability of detecting bighead carp and silver carp eDNA in the water column, methods to make inferences about target marker concentrations from eDNA monitoring results, methods to assess the probability of target species presence using data on conventional fishing effort, and hydrodynamic fate and transport models to simulate target marker concentrations. The major take-away messages with respect to using eDNA for early detection of invasive species is that, at lower concentrations, invasive carp target markers are difficult to detect in the water column and the polymerase chain reaction (PCR) assay has a high false negative rate. This is attributed to processing and division of water samples prior to the assay. With respect to secondary sources of invasive carp eDNA in the CAWS, we found that CSOs and navigation were likely to be the largest contributors of second-hand eDNA and the most likely sources of second-hand eDNA detected in monitoring samples. The probabilistic model has been parameterized and implemented for silver carp. With respect to the presence of silver carp in the CAWS, we found that detection of silver carp eDNA can either increase or decrease a prior degree of belief in target species presence depending on location within the CAWS and the extent of prior fishing effort. While eDNA monitoring results strongly suggest that larger quantities of invasive carp are not present in the CAWS, inferences based on eDNA evidence are more difficult for smaller quantities. Overall, ECALS studies suggest that secondary sources are an important contributor of eDNA to the CAWS.

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ECALS 2013 Major Milestones

February 2013: Second interim ECALS Report

July 2013: Markers

Part 1 of a report describing the development of a marker

The eDNA Calibration Study continues its work to develop important information on the genetics of invasive bighead and silver carp, and to develop enhanced DNA-based tools for detection and monitoring of these species. To date, the ECALS team has produced an highly-representative DNA library of reference mitochondrial haplotypes for both species, is nearing completion of efforts to characterize genetic patterns in North American populations of both species, and is nearing completion of tests on several potential new conventional eDNA markers and quantitative real-time PCR (qPCR) eDNA markers for both species. The close phylogenetic relationship between silver and bighead carp and the high level of similarity in their DNA sequences, in addition to DNA from other related species that may be found in eDNA samples, makes new marker development a particular challenge. The new markers being tested by the ECALS team, along with those already established, will have different strengths and limitations, but should, either singly or in some combination, result in a more robust eDNA monitoring tool set that optimizes assay cost and throughput efficiency.

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July 2013: Calibration

Summary of Loading/Shedding Report:

Water samples can be quantified for carp eDNA using qPCR. The sensitivity of the assay will depend on time-consuming but necessary optimization of the analysis (temperature, reagent amounts).
The eDNA signal can be highly variable, likely reflecting clumped eDNA distribution.
We found no correlation between water temperature and eDNA shedding rates.
We observed a correlation between eDNA loading and fish density. We expect to see a stronger correlation when larger treatment differences are used in upcoming pond studies.
Inhibition of the polymerase chain reaction by compounds in the sample that are co-extracted with the DNA will lead to false negatives. Positive internal controls can be used to identify inhibited samples, and thus avoid false negatives.

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September 2013

Calibration - Part 1 of a report describing the relationship between environmental factors on eDNA degradation rates

ECALS researchers have completed a series of trials focused on the degradation of invasive carp eDNA. The majority of the DNA in these trials degraded either rapidly or very rapidly over a few days, but in all cases a small portion of DNA persisted beyond 2 to 4 weeks. Both temperature and pH affected the rate of eDNA degradation in these trials, whereas microbial loads and turbulence did not.

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Draft problem formulation report-including a proposed graphical model and preliminary plans for parameterization of network nodes.

November 2013

Draft problem formulation report revised in response to comments. Summary of comments and ECALS responses to comments on the problem formulation report.

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December 2013

Calibration - Part 2 of a report describing shedding studies

Here we follow up the loading studies interim report from July 2013 and include results from laboratory studies assessing the effects of diet on eDNA shedding rates by bigheaded carps (silver and bighead carp). In order to understand how eDNA behaves in the environment, we must understand how it enters the system. In our July interim report, we addressed three of our four hypotheses that could influence the shedding rate of eDNA by these fish (fish density, warmer water, and spawning). We now provide results from studies that tested the fourth hypothesis: cellular debris from the gut-lining shed via excrement is a major source of shed eDNA. Our conclusion from this study found that fish do shed DNA at higher rates when fed, likely due to cells sloughed off in the excrement. Non-fed fish still shed detectable amounts of DNA but at approximately 10-fold lower rates compared to the fed fish (especially those fed algae).

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ECALS 2012 Major Milestones

March: First Interim Report

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April: Begin Bird Studies (field and lab)

A telemetry study on cormorants in the Illinois Waterway down to Peoria to document migratory and foraging behavior, as well as testing the feces for invasive carp DNA. Lab studies will also be conducted to determine the detectable amounts of invasive carp DNA in cormorant and bald eagle feces when they are fed these fish, and also feathers will be sampled to determine if DNA can be carried on the bodies of these birds.

May: Hi-throughput technique Report

Report documenting the results from studies conducted to decrease the processing time and cost of eDNA samples. We expect a follow up to this report in late October. Download Report (PDF)

August: Storm Sewers Final Report

The storm sewer experiment in Chinatown where invasive carp contaminated-ice is deposited in a storm sewer then tested for viable eDNA detections in the sewer and river was conducted in June. This experiment was also conducted in November of last year.
Download the report (PDF)

December: Optimized Sampling Protocol Report

Experiments to test different methods of sampling and extracting DNA from the river were conducted in June, this is a follow up to an experiment also conducted in 2011. Testing the limits of detection for three different methods including the standard 2L surface sample with filtering, in-field filtering with 10L of water, and finally centrifuging 50mL Falcon tubes. The goal is to determine the most efficient processes that can be used to sample for eDNA, perhaps by giving resource managers options that are standardized to meet their needs.

ECALS Frequently Asked Questions

How is ECALS funded?
ECALS is an interagency study, with USACE, USGS and USFWS participating and is fully funded by the Great Lakes Restoration Initiative.

What is the timeframe of this study?
ECALS is a two year study, with completion expected at the end of 2013. However, the team will be providing interim products frequently.

What is the purpose of ECALS?
The purpose of ECALS is to investigate alternative sources and pathways for eDNA detections beyond a live fish. The study will also examine how environmental variables such as light, temperature and water velocity impact eDNA detections, explore the correlation between the number of positive samples and the strength of the DNA source, develop more efficient eDNA markers to cut the sampling processing time in half and model eDNA transport specific to the Chicago Area Waterway System. The intent is that all entities using eDNA as a monitoring tool, not just the ICRCC, will be able to use this information to refine their own monitoring and management programs.

What will you do with the results?
Results will be provided frequently, and will be shared with the ICRCC, specifically the Monitoring and Rapid Response Work Group. The results will inform current eDNA sampling strategies and will make the sampling more efficient and allow for more informed interpretation by managers for rapid response decisions in the Chicago Area Waterway System.

What is a vector?
When vectors are described vectors, 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.

What is calibration?
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?

Why would invasive carp DNA be found in fertilizers?
Invasive carp are used as ingredients in some fertilizer products, which may then be used in the Chicago region and (via runoff as a vector) end up in the water.

What's a storm sewer experiment?
The ECALS Team conducted a storm sewer experiment in late 2011 to investigate the viability of DNA (or, 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 DNR 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 will be repeated summer 2012.

What are alternative sampling trials?
Lab filtering of DNA is a time-consuming step of the eDNA sampling process. The teams is investigating 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 is assumed that eDNA is attracted to particulate material, and, therefore, extracting the particulate material in the field, the team was able to process those samples for the presence of invasive carp DNA.

What is an eDNA marker?
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify a species. The team is testing water samples for the markers for bighead and silver carp and intends to develop improved markers to decrease processing time.

What is modeling eDNA transport?
The ECALS Team will be investigating the influence of environmental variables, including water velocity and degradation rates, to model how eDNA can be transported, or how it moves, through the Chicago Area Waterway System.