eDNA Assay Research Articles

Monitoring the eradication of the highly invasive topmouth gudgeon (Pseudorasbora parva) using a novel eDNA assay

AbstractAquatic invasive species (AIS) represent an important threat for Biodiversity and are one of the factors determining the ecological integrity of water bodies under the Water Framework Directive. Eradication is one of the most effective tools for the management of invasive species but has important economic and ecological trade‐offs and its success needs to be carefully monitored. We assessed the eradication success of the topmouth gudgeon (Pseudorasbora parva), an invasive fish that poses significant risks to endemic aquatic fauna, in four ponds previously treated with the piscicide Rotenone using a novel qPCR‐based environmental DNA (eDNA) assay. We validated the assay through successfully detecting DNA from topmouth gudgeon in two reservoirs with physically confirmed topmouth gudgeon populations. Topmouth gudgeon were detected in all four treated ponds using 750 ml water samples and in three of the ponds using 15 ml samples, despite the eradication treatment and lack of successful detection using conventional trapping methods. Our results highlight the difficulties of eradicating invasive fish and the need to incorporate reliable monitoring methods as part of a risk management strategy under the water framework directive (WFD).

Efficacy of eDNA as an early detection indicator for Burmese pythons in the ARM Loxahatchee National Wildlife Refuge in the greater Everglades ecosystem

Environmental DNA (eDNA) detection of invasive species can be used to inform management decisions by delimiting occupied ranges and estimating detection probabilities. Environmental DNA is shed into the environment through skin cells and bodily fluids and can be detected in water samples collected from lakes, rivers, and swamps. In south Florida, USA, invasive Burmese pythons (Python bivittatus) occupy much of the Greater Everglades in mostly inaccessible habitat and are credited with causing severe declines in populations of native species. Detection of Burmese pythons by traditional methods, including trapping and visual searching, has been largely ineffective, making eDNA a superior method to identify invaded areas. We adapted a quantitative PCR eDNA assay for use in droplet digital PCR, a state-of-the-art method that improves the precision and accuracy of eDNA detection and quantification. From August 2014 to October 2016, locations in and around Arthur R. Marshall Loxahatchee National Wildlife Refuge in southeast Florida were surveyed for Burmese python eDNA. Positive eDNA detections were made in each of the five sampling events, assessing a total of 399 samples, with moderate-to-high occurrence (ψ = 58–91%) and moderate detection (p = 38–70%) probabilities, potentially reduced by a high presence of PCR-inhibiting compounds in the water. The high occurrence rates and geographic distribution of the positive samples within Loxahatchee suggests a steady release of python eDNA from resident Burmese pythons and reduces support for transport of eDNA by boats or flowing water from the north. The first confirmed sighting of a Burmese python in the Refuge occurred in September 2016 after eDNA testing had indicated the presence of pythons. While the established population boundary is thought to be south of Loxahatchee, the eDNA detections indicate a northern range limit at, or north of, the refuge on the eastern side of the Florida peninsula. Our study demonstrates the utility of eDNA for determining more accurate range limits and expansion information for Burmese pythons, and provides a means for assessing control efforts.

Environmental DNA as a non‐invasive sampling tool to detect the spawning distribution of European anadromous shads (Alosa spp.)

Abstract Populations of the European shads Alosa alosa (Linnaeus, 1758) and Alosa fallax Lacépède, 1800 (Alosa spp.) are protected under legislation because of their vulnerability to human disturbances. In particular, river impoundments block their upstream migration, preventing access to spawning areas. Knowledge on the spatial extent of their spawning is important for informing conservation and river management plans. Determining the spatial extent of Alosa spp. spawning is challenging. They enter rivers over a 2‐3‐month period and the species potentially migrate different distances upstream. Capture and handling can be problematic, spawning events generally occur at night, and kick‐sampling for eggs is limited to shallow water. Assessing their spatial extent of spawning could, however, incorporate non‐invasive sampling tools, such as environmental DNA (eDNA). An eDNA assay for Alosa spp. was successfully developed, based on the cytochrome c oxidase subunit I gene segment and quantitative polymerase chain reaction (qPCR). Application in spring 2017 to the River Teme (River Severn catchment, western England) revealed high sensitivity in both laboratory and field trials. Field data indicated Alosa spp. spawning between May and June, with migrants mainly restricted to areas downstream of the final impoundment. eDNA can thus be used as a non‐invasive sampling tool to determine the freshwater distribution of these fishes in Europe, enhancing their conservation at local and regional scales.

An improved environmental DNA assay for bull trout (Salvelinus confluentus) based on the ribosomal internal transcribed spacer I.

The majority of environmental DNA (eDNA) assays for vertebrate species are based on commonly analyzed regions of the mitochondrial genome. However, the high degree of mitochondrial similarity between two species of charr (Salvelinus spp.), southern Dolly Varden (S. malma lordii) and bull trout (Salvelinus confluentus), precludes the development of a mitochondrial eDNA assay to distinguish them. Presented here is an eDNA assay to detect bull trout based on the first ribosomal internal transcribed spacer (ITSI), a nuclear marker. This assay successfully detects bull trout and avoids detecting Dolly Varden as well as brook trout (S. fontinalis), Arctic char (S. alpinus), and lake trout (S. namaycush). In addition, this assay was compared with an extensively used mitochondrial bull trout assay and it was found that the ITSI-based assay produced higher detectability. Our results suggest this assay should out-perform the published mtDNA assay across the range of bull trout, while the added specificity allows reliable bull trout detection in areas where bull trout co-occur with other charr such as Dolly Varden. While clearly a superior assay in this instance, basing assays on ITSI is not without problems. For vertebrates, there are fewer ITSI sequences available than commonly sequenced regions of the mitochondrial genome. Thus, the initial in silico screening of candidate assays must be preceded by much more extensive sampling and sequencing of sympatric or closely related taxa. Further, all copies of the internal transcribed spacers within an individual may not be identical, which can lead to complications. Lastly, the copy number for ITSI varies widely across taxa; the greater detectability associated with this assay cannot be assumed for other species.

Detecting Southern California’s White Sharks With Environmental DNA

To improve ability to detect white sharks without the need for tags, or visual census, we developed a species-specific environmental DNA (eDNA) assay that targets a 163 bp fragment of the white shark (Carcharodon carcharias) mitochondrial cytochrome B gene on a digital droplet PCR (ddPCR) platform. We used this marker to detect white shark DNA in 250 ml water samples taken from across two sites in Santa Barbara, California (USA) frequented by juvenile white sharks. We did not detect white shark DNA in samples from two neighboring sites where sharks are presumably absent, suggesting that eDNA can indicate nearby white sharks. This marker development, testing, and opportunistic application in a region with known distributions of white sharks indicates that environmental DNA could be developed further to monitor white sharks, thereby informing conservation planning and public safety. With the potential increase in white shark populations due to decades of protection, there is a need for fishery independent methods for assessing white shark distributions, and eDNA may provide be an ideal, non-intrusive tool for coastal assessments.

Environmental DNA in subterranean biology update: from “Where?” to “How many?”

Recent records of Proteus anguinus outside its historically known range (Gorički et al. 2017), discovered through detection of its DNA dissolved in groundwater (environmental DNA or eDNA), mark the beginning of a new era in the study and conservation of cryptic subterranean biodiversity. An upgraded technology, droplet digital PCR (ddPCR), initially developed for studies of gene expression, detection of genetically modified organisms and in medical diagnostics, is being tested for improved detection of the much smaller and rare stygobiont, the cave clam Congeria jalzici. In parallel to eDNA assay development for various stygobiotic species of the Dinaric Karst, a groundwater-sample library is being created. The samples will be available for future analysis of their species composition and will also serve as a source of information on any changes in species distribution over time. In another line of eDNA research, the utility of ddPCR for direct quantification of eDNA molecules in groundwater is being explored by using the large, accessible and well-characterized (Zakšek and Trontelj 2017) natural Proteus population in the Planina Cave (Slovenia) as a model. The eDNA methodology may in the future be applied in estimation and monitoring of Proteus population sizes without having to see, mark or otherwise disturb the animals themselves.

Environmental DNA sampling as a surveillance tool for cane toad Rhinella marina introductions on offshore islands

Containing the spread of established invasive species is critical for minimizing their ecological impact. Effective containment requires sensitive sampling methods capable of detecting new introductions when invaders are at low density. Here we explore whether environmental DNA (eDNA) sampling could be used as a surveillance tool to detect new incursions of aquatic invasive species on offshore islands. We develop an eDNA molecular assay for invasive cane toads (Rhinella marina) in Australia, validate our assay on the mainland, and apply it to an offshore island (Moreton Island) that is a target of ongoing cane toad surveillance. Our eDNA assay correctly identified four mainland sites at which cane toads were observed, as well as a fifth site within 1 km of known populations. Five additional sites outside the cane toad’s current distribution tested negative for cane toad eDNA. Site occupancy detection models indicated that two water samples and three qPCR replicates were sufficient to achieve a cumulate detection probability > 0.95. Applying our eDNA assay to samples from 19 sites on an offshore island over a 2-year period revealed the absence of cane toad eDNA, in line with our current understanding of cane toad distribution. Our results suggest that eDNA sampling could be strategically applied to meet the Australian Commonwealth’s objective of maintaining cane toad-free offshore islands.

Repurposing environmental DNA samples-detecting the western pearlshell (Margaritifera falcata) as a proof of concept.

Information on the distribution of multiple species in a common landscape is fundamental to effective conservation and management. However, distribution data are expensive to obtain and often limited to high‐profile species in a system. A recently developed technique, environmental DNA (eDNA) sampling, has been shown to be more sensitive than traditional detection methods for many aquatic species. A second and perhaps underappreciated benefit of eDNA sampling is that a sample originally collected to determine the presence of one species can be re‐analyzed to detect additional taxa without additional field effort. We developed an eDNA assay for the western pearlshell mussel (Margaritifera falcata) and evaluated its effectiveness by analyzing previously collected eDNA samples that were annotated with information including sample location and deposited in a central repository. The eDNA samples were initially collected to determine habitat occupancy by nonbenthic fish species at sites that were in the vicinity of locations recently occupied by western pearlshell. These repurposed eDNA samples produced results congruent with historical western pearlshell surveys and permitted a more precise delineation of the extent of local populations. That a sampling protocol designed to detect fish was also successful for detecting a freshwater mussel suggests that rapidly accumulating collections of eDNA samples can be repurposed to enhance the efficiency and cost‐effectiveness of aquatic biodiversity monitoring.

Monitoring post-release survival of the northern corroboree frog, Pseudophryne pengilleyi, using environmental DNA

Context Translocations are becoming an increasingly important conservation tool to combat rising levels of species extinction. Unfortunately, many translocation efforts fail; yet, the timing and cause of failure often remain unknown. Monitoring individuals in the days and weeks following release can provide valuable information on their capacity to survive this initial hurdle. In Australia, breeding programs have been established for the endangered northern corroboree frog, Pseudophryne pengilleyi, to enable reintroduction to the wild via captive-reared individuals, typically, early life stages such as eggs or juvenile frogs that cannot be monitored via traditional survey methods that target adult frogs (e.g. shout–response). Environmental DNA (eDNA) detects trace amounts of DNA that organisms release into their environment and could provide a means to infer population persistence for wildlife releases and translocations. Aims In the present study, we aim to develop an eDNA assay capable of detecting both sexes of P. pengilleyi across multiple life stages, and use it to monitor their survival. Methods An eDNA assay was developed to target the two corroboree frog species (P. pengilleyi and P. corroboree, the southern corroboree frog) and was tested for its sensitivity and specificity in silico and in vitro. Pseudophryne pengilleyi eggs were released into three naturally occurring ponds and water samples were, subsequently, collected from each pond on several occasions over a period of 78 days. Quantitative polymerase chain reaction was used to detect P. pengilleyi eDNA from water samples. Key Results The developed assay was shown to be sensitive and specific to corroboree frogs. eDNA monitoring of reintroduced P. pengilleyi detected the species’ DNA at three of three release ponds and DNA remained detectable until at least 78 days post-release at two of three ponds. Conclusions We show how the development of a corroboree frog-specific assay allowed us to monitor the post-release survival of P. pengilleyi in naturally occurring pools. Implications eDNA surveys may provide a useful tool to monitor post-release survival of translocated populations in a non-invasive manner, with the potential to identify the timing and causes of failure. Such knowledge can be used to inform the management of translocated populations and future release strategies.

Development and field validation of an environmental DNA (eDNA) assay for invasive clams of the genus Corbicula

Development and field validation of an environmental DNA (eDNA) assay for invasive clams of the genus Corbicula

A qPCR MGB probe based eDNA assay for European freshwater pearl mussel (Margaritifera margaritifera L.)

Abstract1. Environmental (e)DNA assays are becoming increasingly used to detect rare or invasive aquatic species.2. The Critically Endangered freshwater pearl mussel Margaritifera margaritifera is undergoing range‐wide reduction in population numbers and distribution.3. An eDNA assay to detect the presence of M. margaritifera was developed, based on the mitochondrial cytochrome oxidase I gene, utilizing species‐specific primers, a minor groove binding (MGB) probe and quantitative (q)PCR approaches.4. The results from this pilot study demonstrated high sensitivity both in laboratory and field trials, and provide a valuable non‐invasive tool for detecting M. margaritifera.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

Development of a sensitive detection method to survey pelagic biodiversity using eDNA and quantitative PCR: a case study of devil ray at seamounts

Environmental DNA (eDNA) is increasingly being used in aquatic environments for monitoring species, particularly those that are of conservation concern and/or are difficult to visually observe. Quantitative PCR (qPCR) has been employed to detect low abundance species occurring in environmental water samples. However, the qPCR technique has principally been applied to freshwater habitats, with less application to pelagic marine environments. We developed a species-specific eDNA assay for the Chilean devil ray, Mobula tarapacana, to assess the capability of using eDNA to detect transient pelagic marine animals. For this pilot study, seawater samples taken at seamounts around the Azores (NE Atlantic) were tested to determine the suitability of this approach for detecting the target species. Samples were taken at sites where M. tarapacana has been previously observed, in addition to sites where its presence is not known. eDNA detection was compared to observations carried out on the same day as water sampling. The qPCR assay successfully detected M. tarapacana at four of five seamount sampling opportunities where the species was observed, and there is a statistically significant relationship between genetic and visual detection. Target DNA was found at one location between seamounts in the absence of visual observation. Our results highlight the importance of physical environmental factors in relation to sampling eDNA in the ocean, such as currents and eDNA dispersal ability. This method has been shown to be sensitive for detection of M. tarapacana DNA in seawater and, therefore, in the identification of important seamounts requiring conservation.

Correction: Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study

[This corrects the article DOI: 10.1371/journal.pone.0162277.].

A Noninvasive Tool to Assess the Distribution of Pacific Lamprey (Entosphenus tridentatus) in the Columbia River Basin.

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish once abundant throughout coastal basins of western North America that has suffered dramatic declines in the last century due primarily to human activities. Here, we describe the development of an environmental DNA (eDNA) assay to detect Pacific lamprey in the Columbia River basin. The eDNA assay successfully amplified tissue derived DNA of Pacific lamprey collected from 12 locations throughout the Columbia River basin. The assay amplifies DNA from other Entosphenus species found outside of the Columbia River basin, but is species-specific within this basin. As a result, the assay presented here may be useful for detecting Entosphenus spp. in geographic range beyond the Columbia River Basin. The assay did not amplify tissue or synthetically derived DNA of 14 commonly sympatric non-target species, including lampreys of the genus Lampetra, which are morphologically similar to Pacific lamprey in the freshwater larval stage.

Implementation of Novel Design Features for qPCR-Based eDNA Assessment.

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

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.

A sensitive environmental DNA (eDNA) assay leads to new insights on Ruffe (Gymnocephalus cernua) spread in North America

Detection of invasive species before or soon after they establish in novel environments is critical to prevent widespread ecological and economic impacts. Environmental DNA (eDNA) surveillance and monitoring is an approach to improve early detection efforts. Here we describe a large-scale conservation application of a quantitative polymerase chain reaction assay with a case study for surveillance of a federally listed nuisance species (Ruffe, Gymnocephalus cernua) in the Laurentian Great Lakes. Using current Ruffe distribution data and predictions of future Ruffe spread derived from a recently developed model of ballast-mediated dispersal in US waters of the Great Lakes, we designed an eDNA surveillance study to target Ruffe at the putative leading edge of the invasion. We report a much more advanced invasion front for Ruffe than has been indicated by conventional surveillance methods and we quantify rates of false negative detections (i.e. failure to detect DNA when it is present in a sample). Our results highlight the important role of eDNA surveillance as a sensitive tool to improve early detection efforts for aquatic invasive species and draw attention to the need for an improved understanding of detection errors. Based on axes that reflect the weight of eDNA evidence of species presence and the likelihood of secondary spread, we suggest a two-dimensional conceptual model that management agencies might find useful in considering responses to eDNA detections.

Detection of an Enigmatic Plethodontid Salamander Using Environmental DNA

The isolation and identification of environmental DNA (eDNA) offers a non-invasive and efficient method for the detection of rare and secretive aquatic wildlife, and it is being widely integrated into inventory and monitoring efforts. The Patch-Nosed Salamander (Urspelerpes brucei) is a tiny, recently discovered species of plethodontid salamander known only from headwater streams in a small region of Georgia and South Carolina. Here, we present results of a quantitative PCR-based eDNA assay capable of detecting Urspelerpes in more than 75% of 33 samples from five confirmed streams. We deployed the method at 31 additional streams and located three previously undocumented populations of Urspelerpes. We compare the results of our eDNA assay with our attempt to use aquatic leaf litterbags for the rapid detection of Urspelerpes and demonstrate the relative efficacy of the eDNA assay. We suggest that eDNA offers great potential for use in detecting other aquatic and semi-aquatic plethodontid salamanders.

An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s’ reports and fish community monitorings.