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This fact sheet was created to help biologists and resource managers understand emerging methods for detecting environmental DNA and their potential application for inventorying and monitoring aquatic species. It is a synthesis of published information.
New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket ( Actinonaias ligamentina ), a freshwater mussel species found in the Clinch River, USA.
This study presents a quantitative PCR (qPCR) assay for the detection of the endangered diamond darter Crystallaria cincotta from environmental DNA (eDNA) in water samples. The assay design is based on an alignment of mitochondrial cytochrome b DNA sequences from 58 individuals representing 25 percid species. Leveraging genetic differences, a species-specific qPCR assay was designed, incorporating alocked nucleic acid (LNA)-enriched probe and a secondary blocking probe to enhance specificity. The assay targets a 93-base pair fragment that includes a diagnostic single nucleotide polymorphism in the probe region; combined with multiple primer mismatches, this provides specificity for distinguishing C. cincotta from other sympatric percid species. Specificity was validated by testing genomic DNA from 16 percid species and synthetic templates, confirming no cross-reactivity. Performance metrics, including the standard curve, qPCR efficiency, limit of detection, and limit of quantification, are reported. The qPCR assay exhibited sufficient sensitivity to detect C. cincotta eDNA in environmental water samples collected from occupied riverine habitats. This study illustrates the effectiveness of LNA-enriched and blocking probes in developing species-specific qPCR assays for eDNA applications, demonstrating their utility in accurately distinguishing closely related species within diverse fish communities.
Silver carp ( Hypophthalmichthys nobilis ), bighead carp ( H. molitrix ) and their hybrids, collectively known as bigheaded carps, have been introduced to Lake Balaton, Hungary. The current stock sizes are difficult to assess. We investigated environmental DNA (eDNA) techniques targeted for bigheaded carps, assessed the spatial distribution of eDNA in Lake Balaton, compared eDNA concentrations to environmental variables to assess potential habitat selection based on those variables, and provided an estimate of biomass of bigheaded carps relative to eDNA shedding rates per unit biomass observed in controlled experiments. Water samples were collected from 70 sites in an array across the lake. Biomass estimation was calculated using mean eDNA concentration obtained by quantitative PCR of the samples and previously determined eDNA shedding rates of bigheaded carps under controlled conditions in a laboratory. Concentration of eDNA was highly variable between sites, resulting in wide confidence intervals. Basins did not significantly differ in eDNA concentration, and there were no strong relationships between environmental variables and eDNA concentration, indications that bigheaded carps use the entire lake. The model provided an estimate of 4,830 metric tonnes (2,750–8,030 tonnes) of bigheaded carps in Lake Balaton, or 81.0 kg/ha. The eDNA method produced a value close to previous estimates by traditional means of total biomass of bigheaded carps in the lake, and like traditional methods, there was a broad confidence interval on the estimate of the mean. The results of the present study support the utility of aquatic eDNA analysis, and the need for further comparisons with fisheries methods and supporting data from laboratory studies.
The development and application of approaches to detect and quantify environmental DNA (eDNA) have potential to improve our understanding of the distribution, abundance, and health of Atlantic Salmon Salmo salar and Pacific salmon Oncorhynchus spp. Here, we review 61 articles focusing on eDNA applications pertaining to salmon occupying natural habitat and aquaculture facilities in the context of advances, opportunities, and challenges. Given recent advances, eDNA now serves as a useful tool for detecting Atlantic Salmon and Pacific salmon and understanding threats to the health of fish and their habitats. Opportunities exist to apply sensitive and minimally invasive eDNA approaches to detect fish and assess fish habitat, assess range expansions of salmon and salmon pathogens, and detect invasive species that may threaten salmon health and abundance. Near real-time eDNA detection and quantification approaches to inform fisheries management may be on the horizon. Challenges limiting the widespread application of eDNA approaches for informing salmon management include accounting for the many factors affecting detection and quantification of eDNA, limits of data for deriving inference, and expense. Through continued development and refinement, eDNA approaches are anticipated to become increasingly available to, and utilized by, managers of Atlantic Salmon and Pacific salmon fisheries.
A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae , which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.
Brazilian elodea ( Egeria densa ) is an invasive freshwater plant that demonstrates widespread ecological impacts in freshwater ecosystems and causes substantial economic damage. Here, we developed an environmental DNA assay for detection of E. densa to provide resource managers with a tool for early detection, identification, and monitoring of invasive populations.
Determining species distributions accurately is crucial to developing conservation and management strategies for imperiled species, but a challenging task for small populations. We evaluated the efficacy of environmental DNA (eDNA) analysis for improving detection and thus potentially refining the known distribution of Chinook salmon ( Oncorhynchus tshawytscha ) in the Methow and Okanogan Subbasins of the Upper Columbia River, which span the border between Washington, USA and British Columbia, Canada. We developed an assay to target a 90 base pair sequence of Chinook DNA and used quantitative polymerase chain reaction (qPCR) to quantify the amount of Chinook eDNA in triplicate 1-L water samples collected at 48 stream locations in June and again in August 2012. The overall probability of detecting Chinook with our eDNA method in areas within the known distribution was 0.77 (±0.05 SE). Detection probability was lower in June (0.62, ±0.08 SE) during high flows and at the beginning of spring Chinook migration than during base flows in August (0.93, ±0.04 SE). In the Methow subbasin, mean eDNA concentration was higher in August compared to June, especially in smaller tributaries, probably resulting from the arrival of spring Chinook adults, reduced discharge, or both. Chinook eDNA concentrations did not appear to change in the Okanogan subbasin from June to August. Contrary to our expectations about downstream eDNA accumulation, Chinook eDNA did not decrease in concentration in upstream reaches (0–120 km). Further examination of factors influencing spatial distribution of eDNA in lotic systems may allow for greater inference of local population densities along stream networks or watersheds. These results demonstrate the potential effectiveness of eDNA detection methods for determining landscape-level distribution of anadromous salmonids in large river systems.
Developing fast, cost-effective assessments of wild animal abundance is an important goal for many researchers, and environmental DNA (eDNA) holds much promise for this purpose. However, the quantitative relationship between species abundance and the amount of DNA present in the environment is likely to vary substantially among taxa and with ecological context. Here, we report a strong quantitative relationship between eDNA concentration and the abundance of spawning sockeye salmon in a small stream in Alaska, USA, where we took temporally- and spatially-replicated samples during the spawning period. This high-resolution dataset suggests that (1) eDNA concentrations vary significantly day-to-day, and likely within hours, in the context of the dynamic biological event of a salmon spawning season; (2) eDNA, as detected by species-specific quantitative PCR probes, seems to be conserved over short distances (tens of meters) in running water, but degrade quickly over larger scales (ca. 1.5 km); and (3) factors other than the mere presence of live, individual fish — such as location within the stream, live/dead ratio, and water temperature — can affect the eDNA-biomass correlation in space or time. A multivariate model incorporating both biotic and abiotic variables accounted for over 75% of the eDNA variance observed, suggesting that where a system is well-characterized, it may be possible to predict species' abundance from eDNA surveys, although we underscore that species- and system-specific variables are likely to limit the generality of any given quantitative model. Nevertheless, these findings provide an important step toward quantitative applications of eDNA in conservation and management.
Tools that bolster early detection of invasive species are needed to prevent their spread. In this study, we assessed if environmental DNA (eDNA) can extend the seasonal window for dreissenid mussel early detection beyond that of plankton tows, which are limited to warmer seasons when mussel larvae are present. We focused eDNA sampling efforts at multiple sites in Tiber Reservoir (Montana) where dreissenid mussel abundance is hypothesized to be low. Samples were collected in June and October 2017, when water temperatures were cooler than thermal optima for dreissenid reproduction, and in July 2017 when water temperatures were warmer and conducive for reproduction. We detected dreissenid mussel DNA in June, July and October even though no dreissenid mussels were observed using non-molecular tools in 2017. A subset of positive and negative eDNA samples were analyzed by an independent lab and results were corroborated. We then estimated the effort needed for 95% probability detection of dreissenid DNA at each site within Tiber Reservoir and found that as many as 27, 14, and 34 samples needed to be collected in June, July and October, respectively. To further validate the utility of eDNA, we also present ancillary eDNA results from other waters in the Flathead Reservation (Montana) where dreissenid mussels have never been detected and from waters with established zebra mussel populations in the upper Mississippi River, which were sampled in the spring when water temperatures were cooler than thermal optima for dreissenid reproduction. All Flathead Reservation samples were negative for dreissenid mussel DNA, while all upper Mississippi River samples were positive. This study adds to a growing body of research that demonstrates eDNA is a highly sensitive tool for dreissenid mussel surveillance in newly invaded waters, including colder seasons when non-molecular tools are likely to be less effective or more challenging to employ.
Many cavefishes and cave crayfishes are considered of conservation concern; however, sampling these species is inherently difficult given their occupied environments. The goal of our project was to verify the presence of select karst organisms while developing the foundation for sampling approaches that might be useful to conservation and management agencies. Our project objectives were to develop assays to amplify deoxyribonucleic acid (DNA) from several species of Ozark cavefishes and cave crayfishes and complete an initial surveillance of locations across the Ozark Highlands using environmental DNA (eDNA). Using DNA either provided by agency cooperators or that we extracted from tissue samples, we PCR amplified and then sequenced the Cytochrome Oxidase 1 (CO1) gene for cave crayfishes and the NADH Dehydrogenase Subunit 2 (ND2) gene for cavefishes. We developed species-specific primers and probes for five cave crayfishes and two cavefishes. From February 2017 to May 2017, we sampled 1–5 sampling units from 42 caves, wells, and springs (i.e., sites) using eDNA and traditional visual surveys. We measured physicochemical parameters at each sampling unit to estimate detection probability associated with both techniques. We also calculated two occupancy covariates for each site using geospatial data. We successfully amplified Troglichthys rosae DNA from the environment and detected DNA representing this species at 24 of 40 sites. At 16 of the sites where we detected T. rosae DNA, we did not visually observe the species. Although our assay for Typlichthys eigenmanni successfully amplified the target DNA from the environment, it also resulted in false absences where the species was visually confirmed. Using eDNA to detect cave crayfishes was much more difficult. The assay for Cambarus subterraneus did not work for eDNA samples and we were unable to pick up DNA from the environment, even at locations where it was visually confirmed. Alternatively, the eDNA surveys worked well for C. tartarus and we were able to amplify DNA at every site where it was visually observed. Our assay for C. aculabrum was based on a single sample obtained from GenBank, and did not amplify eDNA from field samples. Lastly, our eDNA results from samples in the known range of Orconectes stygocaneyi suggested the species may be found at an additional cave. Detection using eDNA based on our O. stygocaneyi assay was likely low because it was designed from a pseudogene; however, positive eDNA samples were sequenced to confirm species-specific DNA. Detection probability of both cavefishes and cave crayfishes varied by survey technique and was influenced by water volume, water clarity, water velocity, and substrate. Detection of cavefishes and cave crayfishes via visual surveys decreased when water volume increased, whereas detection using eDNA increased with greater water volume. Detection between taxa using either sample method was highest in habitats classified by fine substrates, except for eDNA detection of crayfishes which was greatest in coarse substrates. Detection of cavefishes increased with water clarity, but detection of cave crayfishes increased with turbidity. Detection probability of both cavefishes and crayfishes using eDNA increased slightly with water velocity, but decreased with visual surveys as water velocity increased. Occupancy by both taxa was positively related to particular geologic series. Crayfish occupancy was negatively related to fine-scale anthropogenic disturbance (i.e., 500-m buffer around the site), whereas crayfish showed no relationship with disturbance. Our results suggest possible range extensions, provide insights to factors driving detection using both sample techniques, and suggest areas where recharge zones may be shared among caves. Future efforts focused on a comprehensive evaluation of genetic diversity among cave crayfishes to improve assay design could improve detection and the applicability of eDNA as a supplemental and non-invasive sampling approach.
Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike ( Esox lucius ) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water.
Bullseye Snakehead Channa marulius (Hamilton 1822) was first detected in the southern Florida town of Tamarac in 2000 and has been expanding its geographic range since. Environmental DNA (eDNA) analysis is a newly-developed technique used to noninvasively detect cryptic or low-density species or those that are logistically difficult-to-study. Genetic material shed into the environment through tissue and body fluids is concentrated from water samples and analyzed for the presence of target species eDNA. To help delineate Bullseye Snakehead’s geographic range, we developed and validated a species-specific eDNA assay for both quantitative and droplet digital PCR (ddPCR). We then used ddPCR to assess 16 locations in southeast Florida using 222 water samples collected from 2015 to 2018. Positive eDNA detections were obtained at all six locations that were within the known geographic range of Bullseye Snakehead. Furthermore, eDNA was detected in six of 10 locations that were previously thought to be outside the periphery of the range but hydrologically connected through the extensive canal system. Over the four years of sampling, estimated occurrence rates (ψ) remained stable and relatively high (ψ = 0.67 [95% credible interval (CI) 0.33–0.95]) near Tamarac, Florida, as compared to the most southern sampling locations (ψ = 0.0–0.37). Bulls- eye Snakehead eDNA estimated occurrence rates in the middle region increased between 2016 (0.28 [95% CI 0.03–0.94]) and 2017 (0.66 [95% CI 0.24–0.98]), potentially reflecting eDNA detections related to a growing or expanding population. Bullseye Snakehead eDNA was detected at low concentrations on the northern and eastern borders of Everglades National Park, which is an important conservation area and UNESCO World Heritage Site. Despite extensive sampling via electrofishing, no Bullseye Snakehead were visually detected in several locations that yielded positive eDNA samples. It is unclear whether eDNA was transported through flowing water or another vector. To date, collection records for this species are confined to urban canals; however, Bullseye Snakehead may use the interconnected system of canals to disperse to natural conservation areas such as Everglades National Park, Big Cypress National Preserve, and Water Conservation Areas, where it may impact native species via predation and competition.
Seattle Public Utilities (SPU) has a history of conducting traditional fish surveys in urban streams of Seattle, Washington. Limited staff resources have reduced SPU's capacity to monitor fish, and environmental DNA (eDNA) was recognized as an alternative survey method that could potentially improve the efficiency and capacity of SPU-sponsored fish surveys. We performed spatiotemporal surveys of eDNA to assess occupancy and distribution of Chinook Salmon ( Oncorhynchus tshawytscha ), Coho Salmon ( O . kisutch ), and Coastal Cutthroat Trout ( O . clarkii clarkii ) in Thornton Creek, Seattle, between October 2018 and December 2020. Peak Chinook and Coho eDNA detections occurred in October and October–November, respectively, coinciding with expected adult return time. Chinook and Coho eDNA was detected in May at the time when juveniles outmigrate through the Lake Washington basin. Coastal Cutthroat Trout eDNA was widespread and detected at high rates across seasons, reflecting their ubiquitous distribution. Results from multiscale occupancy modeling suggested that distance upstream affected site-level occupancy probabilities for adult Chinook, but not Coho. Model results also suggested that the probability of Coho and Chinook eDNA occurring in water samples was affected by survey year. Finally, model results suggested that the probability of detecting Chinook eDNA in PCR technical replicates was affected by survey year and collection day but detection of Coho eDNA was only affected by collection day. This study indicates eDNA surveys are effective for assessing distribution and occupancy of salmonids in Seattle's urban streams. Integrating eDNA surveys into urban stream monitoring programs can help alleviate the burden of limited assets facing many resource managers.
Invasive species management frameworks, such as the early detection of and rapid response to invasive species, use monitoring programs to detect new species occurrences. Resource managers use environmental DNA (eDNA) as one tool for these monitoring programs. An eDNA detection in a new location may lack perspective for resource managers and researchers because of the rarity of potential invaders and the randomness in their dispersal and detection. An example monitoring program is the eDNA-based sampling approach used by the U.S. Fish and Wildlife Service for bigheaded carps Hypophthalmichthys spp. in the upper Mississippi River and Great Lakes Basins that collects hundreds of water samples per event. The U.S. Fish and Wildlife Service detected a single positive sample for Bighead Carp Hypophthalmichthys nobilis during the spring 2021 sampling event in the Kinnickinnic River within the Milwaukee River Basin, and detected a second single positive sample for bigheaded carps during the fall 2021 sampling event in the Milwaukee River. The U.S. Fish and Wildlife Service did not detect any bigheaded carps in previous years (2015 to 2020) or in either the spring or fall 2022 sampling events. These detections lacked perspective, such as detection numbers for other species. We reanalyzed the 2021 and 2022 samples for four existing species of fish: two fairly common species (Common Carp Cyprinus carpio and Gizzard Shad Dorosoma cepedianum ) and two fairly rare species (Burbot Lota lota and Grass Carp Ctenopharyngodon idella ). We detected Common Carp during all four sampling events, Gizzard Shad during three of four sampling events, and Burbot and Grass Carp during two of four sampling events. These results demonstrated that current sampling efforts could detect other species, and bigheaded carp eDNA was not common in the Milwaukee River compared to these species. More specifically, this finding indicates bigheaded carp eDNA detections are as rare as, or rarer than, Grass Carp eDNA detections, a recent invader to the basin. Our findings also demonstrated how reanalyzing eDNA samples after positive detections for targeted species can help managers understand the context of the detections and provide perspective for the relative abundance of the targeted species. Additionally, our results highlight the importance of completing long-term eDNA-based monitoring rather than a single sampling or inventory event. These detections may have been missed in a single year or sampling event, whereas a multiyear monitoring program provides an opportunity to observe trends through time.
The Black Carp, Mylopharyngodon piceus, is an increasingly widespread invasive species in North America that threatens freshwater mussel populations. We developed four qPCR assays for detecting environmental DNA (eDNA) from these Black Carp populations. Assays were designed to target four mitochondrial DNA loci and were based on 34 complete mitochondrial genome sequences, including 29 generated in this study from samples obtained in three countries. Assays were validated for taxon specificity with in silico comparisons against archived DNA sequences and with in vitro tests of 41 DNA samples from Black Carp, as well as DNA samples from 30 non‐target fish species, all from the Mississippi River Basin. All four assays were able to detect the DNA of all Black Carp samples and did not exhibit any positive results with DNA from other tested species. Tests conducted in round‐robin fashion among three different laboratories found that all four assays were able to detect DNA at very low template concentrations (limits of detection = 3 copies/qPCR, limits of quantification = 16‐64 copies/qPCR) and, as part of in situ validation, were successful in detecting eDNA from Black Carp in aquaculture ponds. Despite some challenges with other attempts at in situ validation, the assays were also effective in detecting Black Carp eDNA in water samples from a drainage ditch in the upper reaches of the species’ range that was known to contain juvenile Black Carp, as well as in water samples from the Missisippi River and a connected oxbow lake in the lower reaches of the species range.
We appreciate the conversation put forward by Iversen et al. (2015) in their response to our article “Quantification of eDNA shedding rates from invasive bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix ” in the 2015 environmental DNA special issue of Biological Conservation. We agree with Iversen et al.'s concern about overly optimistic conclusions that could be drawn from the current eDNA literature. One hope for eDNA technology is that it can be used in estimating abundance or population density. Evidence suggests that eDNA measurements correlate with total biomass (Takahara et al., 2012) rather than abundance. We demonstrate a similar relationship between biomass and eDNA shedding rates. Nevertheless, without field testing of these methods and specific survey protocols, we cannot make strong conclusions regarding the technique's field applicability. In our manuscript, we attempted to point out areas in which more research is needed.
Annual redd counts are used to monitor the status and trends of salmonid populations, but methods to easily and reliably determine which of sympatric species made specific redds are lacking. We explored whether environmental DNA (eDNA) analysis might prove useful for determining the species of salmon redds. We collected eDNA samples from the interstitial spaces of redds of Chinook Salmon Oncorhynchus tshawytscha, redds of Coho Salmon O. kisutch, and areas of undisturbed gravel (n = 10, each), as well as from the water column adjacent to each of those sites in the Sandy River basin, Oregon, USA during the fall of 2013. The concentrations of Chinook and Coho eDNA were quantified within each sample using real-time PCR. The water in the interstitial spaces of redds contained significantly higher eDNA concentrations of the species that made the redd than (1) the other species and (2) the adjacent water column. In contrast, neither Chinook nor Coho eDNA was significantly more concentrated than the other in the water from the interstitial spaces of undisturbed gravel. The interstitial water of undisturbed gravel contained significantly higher eDNA concentrations of Coho than the adjacent water column. In contrast, Chinook eDNA concentration was similar in the interstitial water of undisturbed gravel and the adjacent water column. Both species’ redds had significantly higher concentrations of their respective species’ eDNA than did undisturbed gravel, but conclusions were confounded by differences in the timing and locations of sampling. This initial investigation highlights the potential value and some of the complexity of using eDNA analysis to indicate redd species.