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USGS2023

A nitrifier-enriched microbial community contributes to the degradation of environmental DNA

Environmental DNA (eDNA) surveys are a promising alternative to traditional monitoring of invasive species, rare species, and biodiversity. Detecting organism-specific eDNA reduces the need to collect physical specimens for population estimates, and the high sensitivity of eDNA assays may improve detection of rare or cryptic species. However, correlating estimated concentrations of eDNA with species abundance can be difficult due to the many abiotic and biotic factors that influence eDNA persistence and degradation. Here, we assessed the impact of a nitrifier-enriched microbial (NEM) community on the persistence and degradation of Hypophthalmichthys molitrix (silver carp) milt eDNA using experimental aquatic mesocosms and a quantitative PCR approach. The NEM community was cultured from combined sediment and water samples collected from a golf course pond in Columbia, Missouri (USA), and experiments were conducted in the dark at 22°C. We found that the NEM community transformed organic nitrogen from silver carp milt to measurable amounts of nitrate, both in the presence and absence of ammonia nitrogen. Additionally, regardless of ammonia availability, milt eDNA followed a one-phase exponential decay pattern after an initial 24-h plateau in the presence of the NEM community. However, milt eDNA had a shorter half-life (12.5 h) in the absence of exogenous ammonia compared to when ammonia was present (15 h). In sterile mesocosms, eDNA was stable during the 72-h experiment. Together, these results suggest that the presence of microorganisms is necessary for short-term degradation of eDNA. Furthermore, nitrifying microbial communities, which are ubiquitous in most soil and water environments, could limit eDNA persistence in the environment. Understanding the contributions of environmental microbial communities will allow more confidence in sampling design and eDNA result interpretations for biodiversity management applications.

Environmental DNA↗

USGS2018

An analytical framework for estimating aquatic species density from environmental DNA

Environmental DNA (eDNA) analysis of water samples is on the brink of becoming a standard monitoring method for aquatic species. This method has improved detection rates over conventional survey methods and thus has demonstrated effectiveness for estimation of site occupancy and species distribution. The frontier of eDNA applications, however, is to infer species density. Building upon previous studies, we present and assess a modeling approach that aims at inferring animal density from eDNA. The modeling combines eDNA and animal count data from a subset of sites to estimate species density (and associated uncertainties) at other sites where only eDNA data are available. As a proof of concept, we first perform a cross-validation study using experimental data on carp in mesocosms. In these data, fish densities are known without error, which allows us to test the performance of the method with known data. We then evaluate the model using field data from a study on a stream salamander species to assess the potential of this method to work in natural settings, where density can never be known with absolute certainty. Two alternative distributions (Normal and Negative Binomial) to model variability in eDNA concentration data are assessed. Assessment based on the proof of concept data (carp) revealed that the Negative Binomial model provided much more accurate estimates than the model based on a Normal distribution, likely because eDNA data tend to be overdispersed. Greater imprecision was found when we applied the method to the field data, but the Negative Binomial model still provided useful density estimates. We call for further model development in this direction, as well as further research targeted at sampling design optimization. It will be important to assess these approaches on a broad range of study systems.

Ecology and Evolution↗

USGS2018

Ethanol and sodium acetate as a preservation method to delay degradation of environmental DNA

Environmental DNA (eDNA) samples that are collected from remote locations depend on rapid stabilization of the DNA. The degradation of eDNA in water samples is minimized when samples are stored at ≤ 4 °C. Developing a preservation technique to maintain eDNA integrity at room temperature would allow a wider range of locations to be sampled. We evaluated an ethanol and sodium acetate solution to maintain the integrity of the DNA samples for the time between collection and lab testing. For this evaluation, replicate water samples taken from a tank housing Asian carp were placed on ice or held at room temperature. At both temperatures, water samples were left untreated or were preserved with an ethanol and sodium acetate solution (EtOH–NaAc). Every day for 6 days following collection, a subset of the samples was removed from each preservation method and DNA was extracted and nuclear and mitochondrial markers were assayed with qPCR. Results showed comparable persistence of DNA between iced samples without the EtOH–NaAc treatment and samples that received EtOH–NaAc treatment that were kept at room temperature. We found that DNA can be amplified from preserved samples using an EtOH–NaAc solution after up to 7 days at room temperature.

Conservation Genetics Resources↗

USGS2020

Influence of sediment and stream transport on detecting a source of environmental DNA

Environmental DNA (eDNA) can be used for early detection, population estimations, and assessment of potential spread of invasive species, but questions remain about factors that influence eDNA detection results. Efforts are being made to understand how physical, chemical, and biological factors—settling, resuspension, dispersion, eDNA stability/decay—influence eDNA estimations and potentially population abundance. In a series of field and controlled mesocosm experiments, we examined the detection and accumulation of eDNA in sediment and water and the transport of eDNA in a small stream in the Lake Michigan watershed, using the invasive round goby fish ( Neogobius melanostomus ) as a DNA source. Experiment 1: caged fish (average n = 44) were placed in a stream devoid of round goby; water was collected over 24 hours along 120-m of stream, including a simultaneous sampling event at 7 distances from DNA source; stream monitoring continued for 24 hours after fish were removed. Experiment 2: round goby were placed in laboratory tanks; water and sediment were collected over 14 days and for another 150 days post-fish removal to calculate eDNA shedding and decay rates for water and sediment. For samples from both experiments, DNA was extracted, and qPCR targeted a cytochrome oxidase I gene (COI) fragment specific to round goby. Results indicated that eDNA accumulated and decayed more slowly in sediment than water. In the stream, DNA shedding was markedly lower than calculated in the laboratory, but models indicate eDNA could potentially travel long distances (up to 50 km) under certain circumstances. Collectively, these findings show that the interactive effects of ambient conditions (e.g., eDNA stability and decay, hydrology, settling-resuspension) are important to consider when developing comprehensive models. Results of this study can help resource managers target representative sites downstream of potential invasion sites, thereby maximizing resource use.

PLoS ONE↗

USGS2022

Using structured decision making to evaluate potential management responses to detection of dreissenid mussel (Dreissena spp.) environmental DNA

Environmental (e)DNA tools are sensitive and cost-effective for early detection of invasive species. However, the uncertainty associated with the interpretation of positive eDNA detections makes it challenging to determine appropriate natural resource management responses. Multiple sources of error can give rise to positive detections of eDNA in a sample when individuals of that species are not present at the site or a widespread infestation is not imminent. Acting on an erroneous eDNA inference could result in needless costs or reductions in desirable resources. Alternatively, failure to rapidly act on eDNA results that truly indicate invader presence could compound negative impacts and lead to high, long-term costs to manage infestations. We used a structured decision making (SDM) process, which incorporates tradeoffs and uncertainties, to evaluate appropriate response actions following hypothetical eDNA detections of invasive dreissenid mussel ( Dreissena spp.) eDNA in Jordanelle Reservoir, Utah (USA). We worked with decision-makers and stakeholders to identify objectives and discrete management action alternatives to assess consequences and tradeoffs. Alternatives ranged from no action to intensive and expensive control efforts. The best performing alternative was delayed containment described by immediate attempts to confirm the eDNA detections using non-molecular sampling techniques followed by mandatory watercraft exit inspections to prevent dreissenid mussel spread to regional water bodies. Non-molecular sampling increased public support for management by demonstrating a commitment to monitor the invasion state before action, whereas containment decreased likelihood of regional spread to other waters. Delayed containment had the lowest downside risk, and the highest upside gains relative to other alternative actions. Sensitivity analyses showed our results to be robust to parameter and outcome uncertainty.

Management of Biological Invasions↗

USGS2021

Time to get real with qPCR controls: The frequency of sample contamination and the informative power of negative controls in environmental DNA studies

Environmental (e)DNA methods have enabled rapid, sensitive and specific inferences of taxa presence throughout diverse fields of ecological study. However, use of eDNA results for decision-making has been impeded by uncertainties associated with false positive tests putatively caused by sporadic or systemic contamination. Sporadic contamination is a process that is inconsistent across samples and systemic contamination occurs consistently over a group of samples. Here, we used empirical data and laboratory experiments to (i) estimate the sporadic contamination rate for each stage of a common, targeted eDNA workflow employing best practice quality control measures under simulated conditions of rare and common target DNA presence, (ii) determine the rate at which negative controls (i.e., “blanks”) detect varying concentrations of systemic contamination, and (iii) estimate the effort that would be required to consistently detect sporadic and systemic contamination. Sporadic contamination rates were very low across all eDNA workflow steps, and, therefore, an intractably high number of negative controls (>100) would be required to determine occurrence of sporadic contamination with any certainty. Contrarily, detection of intentionally introduced systemic contamination was more consistent; therefore, very few negative controls (<5) would be needed to consistently alert to systemic contamination. These results have considerable implications to eDNA study design when resources for sample analyses are constrained.

Molecular Ecology Resources↗

USGS2019

Reporting the limits of detection and quantification for environmental DNA assays

Background Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ). Aims Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. Materials & Methods/ Results We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method. Discussion/Conclusions Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays.

Environmental DNA↗

USGS2018

Inferring presence of the western toad (Anaxyrus boreas) species complex using environmental DNA

Western toads (species complex comprised of Anaxyrus boreas , A. canorus , A. exsul , and A. nelsoni ) are widely distributed in the western United States but are declining, particularly in the southeastern extent of their range. The subspecies A. b. boreas is listed as a Species of Greatest Conservation Need in New Mexico, Colorado, Utah, and Wyoming. Reliable and sensitive methods for delineating distributions of western toads are critical for monitoring the status of the species and prioritizing conservation efforts. We developed two qPCR assays for detecting western toad DNA in environmental DNA samples. Both markers efficiently and reliably detect low concentrations of western toad DNA across their range in the conterminous U. S. without detecting non-target, sympatric species. To determine the optimal annual sampling period, we then tested these markers using repeated sampling in ponds where western toads were known to be present. Quantities of collected eDNA varied widely across samples, but sample-level detections across sites exceeded 80% for June sampling. In the later summer, detection dropped off sharply with only a single detection in the ten samples collected throughout August.

Global Ecology and Conservation↗

USGS2015

The effect of dilution and the use of a post-extraction nucleic acid purification column on the accuracy, precision, and inhibition of environmental DNA samples

Isolation of environmental DNA (eDNA) is an increasingly common method for detecting presence and assessing relative abundance of rare or elusive species in aquatic systems via the isolation of DNA from environmental samples and the amplification of species-specific sequences using quantitative PCR (qPCR). Co-extracted substances that inhibit qPCR can lead to inaccurate results and subsequent misinterpretation about a species’ status in the tested system. We tested three treatments (5-fold and 10-fold dilutions, and spin-column purification) for reducing qPCR inhibition from 21 partially and fully inhibited eDNA samples collected from coastal plain wetlands and mountain headwater streams in the southeastern USA. All treatments reduced the concentration of DNA in the samples. However, column purified samples retained the greatest sensitivity. For stream samples, all three treatments effectively reduced qPCR inhibition. However, for wetland samples, the 5-fold dilution was less effective than other treatments. Quantitative PCR results for column purified samples were more precise than the 5-fold and 10-fold dilutions by 2.2× and 3.7×, respectively. Column purified samples consistently underestimated qPCR-based DNA concentrations by approximately 25%, whereas the directional bias in qPCR-based DNA concentration estimates differed between stream and wetland samples for both dilution treatments. While the directional bias of qPCR-based DNA concentration estimates differed among treatments and locations, the magnitude of inaccuracy did not. Our results suggest that 10-fold dilution and column purification effectively reduce qPCR inhibition in mountain headwater stream and coastal plain wetland eDNA samples, and if applied to all samples in a study, column purification may provide the most accurate relative qPCR-based DNA concentrations estimates while retaining the greatest assay sensitivity.

Biological Conservation↗

USGS2018

An experimental comparison of composite and grab sampling of stream water for metagenetic analysis of environmental DNA

Use of environmental DNA (eDNA) to assess distributions of aquatic and semi-aquatic macroorganisms is promising, but sampling schemes may need to be tailored to specific objectives. Given the potentially high variance in aquatic eDNA among replicate grab samples, compositing smaller water volumes collected over a period of time may be more effective for some applications. In this study, we compared eDNA profiles from composite water samples aggregated over three hours with grab water samples. Both sampling patterns were performed with identical autosamplers paired at two different sites in a headwater stream environment, augmented with exogenous fish eDNA from an upstream rearing facility. Samples were filtered through 0.8 μm cellulose nitrate filters and DNA was extracted with a cetyl trimethylammonium bromide procedure. Eukaryotic and bacterial community profiles were derived by amplicon sequencing of 12S ribosomal, 16S ribosomal, and cytochrome oxidase I loci. Operational taxa were assigned to genus with a lowest common ancestor approach for eukaryotes and to family with the RDP Classifier software for prokaryotes. Eukaryotic community profiles were more consistent with composite sampling than grab sampling. Downstream, rarefaction curves suggested faster taxon accumulation for composite samples, and estimated richness was higher for composite samples as a set than for grab samples. Upstream, composite sampling produced lower estimated richness than grab samples, but with overlapping standard errors. Furthermore, a bimodal pattern of richness as a function of sequence counts suggested the impact of clumped particles on upstream samples. Bacterial profiles were insensitive to sample method, consistent with the more even dispersion expected for bacteria compared with eukaryotic eDNA. Overall, samples composited over 3 h performed equal to or better than triplicate grab sampling for quantitative community metrics, despite the higher total sequencing effort provided to grab replicates. On the other hand, taxon-specific detection rates did not differ appreciably and the two methods gave similar estimates of the ratio of the common fish genera Salmo and Coregonus at each site. Unexpectedly, Salmo eDNA dropped out substantially faster than Coregonus eDNA between the two sites regardless of sampling method, suggesting that differential settling affects the estimation of relative abundance. We identified bacterial patterns that were associated with eukaryotic diversity, suggesting potential roles as biomarkers of sample representativeness.

PeerJ↗

USGS2024

Environmental DNA

The widespread adoption of environmental DNA (eDNA) detection tools for biodiversity monitoring has led to the need for universal data standards to inform principled eDNA data applications. Improvements in understanding the meaning and possible uncertainty of eDNA data can minimize erroneous conclusions, increase confidence in eDNA data, and maximize conservation outcomes. -Environmental DNA (eDNA) is the genetic material left by organisms in the environment. -eDNA is increasingly being used to detect the presence of species and assess biodiversity, but broad-scale best practices are still being developed. -This affects the quality, accessibility, and usefulness of data. -Non-invasive eDNA sampling can complement or enhance conventional approaches, as it can be a highly sensitive, rapid, and cost-effective tool for biodiversity monitoring. -Creating unified eDNA data best practices and developing DNA reference libraries will ensure eDNA detection methods are applied consistently to accurately inform conservation and wildlife management.

Report↗

USGS2021

Editorial: Environmental DNA innovations for conservation

Environmental DNA (eDNA) analysis refers to the collection of bulk environmental samples such as water, sediment, or air, and studying the genetic remnants that organisms have shed into their environment to gain information about species presence.

Frontiers in Ecology and Evolution↗

USGS2015

Environmental DNA sampling protocol - filtering water to capture DNA from aquatic organisms

Environmental DNA (eDNA) analysis is an effective method of determining the presence of aquatic organisms such as fish, amphibians, and other taxa. This publication is meant to guide researchers and managers in the collection, concentration, and preservation of eDNA samples from lentic and lotic systems. A sampling workflow diagram and three sampling protocols are included as well as a list of suggested supplies. Protocols include filter and pump assembly using: (1) a hand-driven vacuum pump, ideal for sample collection in remote sampling locations where no electricity is available and when equipment weight is a primary concern; (2) a peristaltic pump powered by a rechargeable battery-operated driver/drill, suitable for remote sampling locations when weight consideration is less of a concern; (3) a 120-volt alternating current (AC) powered peristaltic pump suitable for any location where 120-volt AC power is accessible, or for roadside sampling locations. Images and detailed descriptions are provided for each step in the sampling and preservation process.

Techniques and Methods↗

USGS2016

Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA

A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp ( Ctenopharyngodon idella ) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty – indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis, and forensic and clinical diagnostics.

Molecular Ecology Resources↗

USGS2025

Evaluation of rapid DNA extraction methods to better enable point-of-use environmental DNA detection

Recent developments in molecular testing have created the opportunity for biologists and managers to detect environmental DNA (eDNA) of target species rapidly and without the requirement of a laboratory. These point-of-use protocols may be especially useful for early detection and rapid response for invasive species or surveillance for at-risk native species, where timely management decisions are critical. Point-of-use eDNA protocols also facilitate wider and less expensive implementation of eDNA methods. One of the key components to an effective point-of-use protocol is a rapid DNA extraction method. Several rapid extraction protocols are suitable for implementation in the field, but information regarding their relative effectiveness is lacking. We evaluated extraction efficiency of four DNA rapid extraction protocols using filters spiked with primary cultured grass carp ( Ctenopharyngodon idella ) gill cells. The extraction methods included two syringe-based column extractions, a lysis and extraction solution, and a divalent cation chelation resin (Chelex) extraction protocol alongside a laboratory-based control kit. We estimated DNA yield using a newly designed quantitative polymerase chain reaction (qPCR) assay targeting the grass carp nuclear genome. We evaluated two additional factors, filter type (mixed cellulose ester [MCE] and polyethersulfone [PES]) and background eDNA source (aquaculture or river). The lysis and extraction solution and Chelex extraction both had the highest overall yield, with MCE filters further increasing Chelex yield while the enzyme extraction yield was dependent on interaction with both filter and eDNA source. Our results indicate that rapid extraction protocols, such as solutions with short heating steps, are effective for DNA isolation and help to increase the overall accessibility of eDNA analyses.

Environmental DNA↗

USGS2017

Comparing efficiency of American Fisheries Society standard snorkeling techniques to environmental DNA sampling techniques

Analysis of environmental DNA (eDNA) is an emerging technique used to detect aquatic species through water sampling and the extraction of biological material for amplification. Our study compared the efficacy of eDNA methodology to American Fisheries Society (AFS) standard snorkeling surveys with regard to detecting the presence of rare fish species. Knowing which method is more efficient at detecting target species will help managers to determine the best way to sample when both traditional sampling methods and eDNA sampling are available. Our study site included three Navajo Nation streams that contained Navajo Nation Genetic Subunit Bluehead Suckers Catostomus discobolus and Zuni Bluehead Suckers C. discobolus yarrowi . We first divided the entire wetted area of streams into consecutive 100-m reaches and then systematically selected 10 reaches/stream for snorkel and eDNA surveys. Surface water samples were taken in 10-m sections within each 100-m reach, while fish presence was noted via snorkeling in each 10-m section. Quantitative PCR was run on each individual water sample in quadruplicate to test for the presence or absence of the target species. With eDNA sampling techniques, we were able to positively detect both species in two out of the three streams. Snorkeling resulted in positive detection of both species in all three streams. In streams where the target species were detected with eDNA sampling, snorkeling detected fish at 11–29 sites/stream, whereas eDNA detected fish at 3–12 sites/stream. Our results suggest that AFS standard snorkeling is more effective than eDNA for detecting target fish species. To improve our eDNA procedures, the amount of water collected and tested should be increased. Additionally, filtering water on-site may improve eDNA techniques for detecting fish. Future research should focus on standardization of eDNA sampling to provide a widely operational sampling tool.

Arizona, New Mexico↗

USGS2013

Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples

Environmental DNA (eDNA) methods for detecting aquatic species are advancing rapidly, but with little evaluation of field protocols or precision of resulting estimates. We compared sampling results from traditional field methods with eDNA methods for two amphibians in 13 streams in central Idaho, USA. We also evaluated three water collection protocols and the influence of sampling location, time of day, and distance from animals on eDNA concentration in the water. We found no difference in detection or amount of eDNA among water collection protocols. eDNA methods had slightly higher detection rates than traditional field methods, particularly when species occurred at low densities. eDNA concentration was positively related to field-measured density, biomass, and proportion of transects occupied. Precision of eDNA-based abundance estimates increased with the amount of eDNA in the water and the number of replicate subsamples collected. eDNA concentration did not vary significantly with sample location in the stream, time of day, or distance downstream from animals. Our results further advance the implementation of eDNA methods for monitoring aquatic vertebrates in stream habitats.

Canadian Journal of Fisheries and Aquatic Sciences↗

USGS2015

Environmental DNA calibration study interim technical review report December 2014

The Environmental DNA Calibration Study (ECALS) is a multi-year study to improve the understanding and interpretation of the detection of Asian carp DNA in environmental samples (eDNA) used in early detection monitoring. eDNA surveillance programs seek to detect the presence of genetic material (DNA in cells sloughed off in slime, feces, urine, etc.) extracted from water samples; the detection of genetic material is linked to the possible presence of Asian carp. The study involves collaboration between the U.S. Army Corps of Engineers, the U.S. Geological Survey, and the U.S. Fish and Wildlife Service. ECALS addresses three major Action Items from the Asian Carp Regional Coordinating Committee (ACRCC) Asian Carp Control Strategy Framework, of which results to date are presented below. Initial ECALS efforts focused on eDNA vectors whereas marker development and calibration experiments received greater attention in 2013.

Report↗