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Are environmental DNA methods ready for aquatic invasive species management?

Multiple studies have demonstrated environmental (e)DNA detections of rare, invasive species. However, invasive species managers struggle with using eDNA results because detections might not indicate species presence. We evaluated if eDNA methods have matured to a point where they can be widely applied to aquatic invasive species management. We found that eDNA methods meet legal standards for being admissible as evidence in most courts, suggesting that eDNA method reliability is not the problem. Rather, we suggest that the interface between results and management needs attention since there are few tools for integrating uncertainty into decision-making. Solutions include decision support trees based on molecular best practices that integrate the temporal and spatial trends in eDNA positives relative to human risk tolerance.

Trends in Ecology and Evolution

Integrating environmental DNA results with diverse data sets to improve biosurveillance of river health

Autonomous, robotic environmental (e)DNA samplers now make it possible for biological observations to match the scale and quality of abiotic measurements collected by automated sensor networks. Merging these automated data streams may allow for improved insight into biotic responses to environmental change and stressors. Here, we merged eDNA data collected by robotic samplers installed at three U.S. Geological Survey (USGS) streamgages with gridded daily weather data, and daily water quality and quantity data into a cloud-hosted database. The eDNA targets were a rare fish parasite and a more common salmonid fish. We then used computationally expedient Bayesian hierarchical occupancy models to evaluate associations between abiotic conditions and eDNA detections and to simulate how uncertainty in result interpretation changes with the frequency of autonomous robotic eDNA sample collection. We developed scripts to automate data merging, cleaning and analysis steps into a chained-step, workflow. We found that inclusion of abiotic covariates only provided improved insight for the more common salmonid fish since its DNA was more frequently detected. Rare fish parasite DNA was infrequently detected, which caused occupancy parameter estimates and covariate associations to have high uncertainty. Our simulations found that collecting samples at least once per day resulted in more detections and less parameter uncertainty than less frequent sampling. Our occupancy and simulation results together demonstrate the advantages of robotic eDNA samplers and how these samples can be combined with easy to acquire, publicly available data to foster real-time biosurveillance and forecasting.

Idaho, Wyoming, Montana

Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data

The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis ) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

PLoS ONE

Optimization of wetland environmental DNA metabarcoding protocols for Great Lakes region herpetofauna

Many species of reptiles and amphibians (herpetofauna) rely on wetlands that are being degraded and lost at a high rate. Characterization of herpetofauna diversity in different wetland types may help guide conservation strategies. However, traditional survey methods often involve sampling within small temporal windows, and the gear deployed may be taxonomically biased, thus, they may fail to accurately characterize species presence/absence and diversity. In contrast, environmental (e)DNA metabarcoding has been shown to effectively survey entire aquatic communities and can provide a useful complement to traditional surveys. The objective of this study was to design and optimize eDNA sampling and laboratory protocols for wetland herpetofauna. Protocols evaluated included different water sampling approaches (point versus transect sampling), seasonality of sampling, and choice of metabarcoding marker (mitochondrial 12S versus 16S rDNA). Samples collected from 10 sites across southern Michigan detected 17 amphibian and five reptile species, including four species of conservation concern ( Ambystoma texanum , Clemmys guttata , Rana palustris , and Sternotherus odoratus ). We observed no difference in the number of species detected between point and transect samples ( p = 0.70), but point sampling required less time ( p = 0.03) and allowed significantly larger volumes of water to be filtered ( p = 1.13e-5). No difference in species richness was observed between the 12S and 16S mitochondrial DNA markers ( p = 0.96). However, a greater number of taxa were identifiable at the species level when using the 16S locus. There was also a significant difference in the number of species detected between early and late summer sampling periods (more species detected in the earlier period; p = 6.31e-6), and some species were only found in the early or late sampling period. Sampling during multiple periods to fully characterize species composition, the use of point sampling, and the 16S mtDNA marker for herpetofauna eDNA metabarcoding studies may increase efficiency and reliability of results.

Environmental DNA

It’s complicated…environmental DNA as a predictor of trout and char abundance in streams

The potential to provide inferences about fish abundance from environmental (e)DNA samples has generated great interest. However, the accuracy of these abundance estimates is often low and variable across species and space. A plausible refinement is the use of common aquatic habitat monitoring data to account for attributes that influence eDNA dynamics. We therefore evaluated the relationships between eDNA concentration and abundance of bull trout ( Salvelinus confluentus ), westslope cutthroat trout ( Oncorhynchus clarkii lewisi ) and rainbow trout ( Oncorhynchus mykiss ) at 42 stream sites in the Intermountain West (USA and Canada) and tested whether accounting for site-specific habitat attributes improved the accuracy of fish abundance estimates. eDNA concentrations were positively associated with fish abundance, but these relationships varied by species and site, and there was still considerable variation unaccounted for. Random site-level differences explained much of this variation, but specific habitat attributes of those sites explained relatively small amounts of this variation. Our results underscore that either eDNA sampling or environmental characterization will require further refinement before eDNA can be used reliably to estimate fish abundance in streams.

Idaho, Montana, Oregon, Washington

Robotic environmental DNA bio-surveillance of freshwater health

Autonomous water sampling technologies may help to overcome the human resource challenges of monitoring biological threats to rivers over long time periods and large geographic areas. The Monterey Bay Aquarium Research Institute has pioneered a robotic Environmental Sample Processor (ESP) that overcomes some of the constraints associated with traditional sampling since it can automate water sample filtration and preservation of the captured material or homogenize it for immediate analyses in situ. The ESP was originally developed for marine environment applications. Here we evaluated whether the ESP can provide reliable, timely information on environmental (e)DNA detections of human and fish pathogens and introduced fishes at U.S. Geological Survey streamgage sites in freshwater rivers. We compared eDNA collected via ESP at high frequency (e.g., every 3 h) with manual eDNA collections collected at lower frequency (e.g., weekly). We found that water samples filtered and preserved by ESPs successfully detected the DNA of human pathogens, fish pathogens and introduced fishes. Both ESP and manually collected samples provided similar information about target DNA presence. We suggest that the greatest current benefit of the ESP is the cost savings of high frequency, bio-surveillance at remote or hard to access sites. The full potential of robotic technologies like the ESP will be realized when they can more easily execute in situ analyses of water samples and rapidly transmit results to decision-makers.

Idaho, Montana, Wyoming

A workshop to advance invasive species early detection capacity of The Rapid Environmental DNA Assessment and Deployment Initiative & Network (READI-Net)

Early detection and rapid response (EDRR) can minimize the impacts of invasive species, which cost billions of dollars globally. To bolster EDRR across the United States, the U.S. Department of the Interior is working with the U.S. Geological Survey and other partners to advance a National EDRR Framework that strengthens tools, actions, and processes to find and eradicate invasive species before they establish and cause negative impacts. An important component of this framework is to strengthen molecular tools for detecting new invasions. The Rapid Environmental (e)DNA Assessment and Deployment Initiative & Network (READI-Net) project is developing automating eDNA sampling tools, processes to ensure that eDNA results are reliable for management decision-making, and information structures to deliver eDNA results to end-users. To improve the potential uptake of this molecular EDRR toolbox, READI-Net investigators met with a group of end-users, partners, developers and subject-matter experts from federal agencies, tribes, universities, and an NGO representing state agencies from February 28 to March 1, 2023, in Moss Landing, CA. Here, we summarize the READI-Net project and the corresponding participant feedback.

Management of Biological Invasions

Detection of environmental DNA of bigheaded carps in samples collected from selected locations in the St. Croix River and in the Mississippi River

The use of molecular methods, such as the detection of environmental deoxyribonucleic acid (eDNA), have become an increasingly popular tool in surveillance programs that monitor for the presence of invasive species in aquatic systems. One early application of these methods in aquatic systems was surveillance for DNA of Asian carps (specifically bighead carp Hypophthalmichthys nobilis and silver carp H. molitrix) in water samples taken from the Chicago Area Waterway System. The ability to identify DNA of a species in an environmental sample presents a potentially powerful tool because these sensitive analyses can presumably detect the presence of DNA in water even when the species is not abundant or are difficult to catch or monitor with traditional gear. Prior to research presented in this report, an initial eDNA surveillance effort was completed in selected locations in the Upper Mississippi and St. Croix Rivers in 2011 after the capture of a bighead carp in the St. Croix River near Prescott, WI. Data presented in this report were developed to duplicate the 2011 monitoring results from the Upper Mississippi and St. Croix Rivers and to provide critical insight into the technique to inform future work in these locations. We specifically sought to understand the potential confounding effects of other pathways of eDNA movement (e.g., fish-eating birds, watercraft) on the variation in background DNA by collecting water samples from (1) sites within the St. Croix River and the upper Mississippi River where the DNA of silver carp was previously detected, (2) sites considered to be free of Asian carp, and (3) a site known to have a large population of Asian carp. We also sought to establish a baseline Asian carp eDNA signature to which future eDNA sampling efforts could be compared. All samples taken as part of this effort were processed using conventional polymerase chain reaction (PCR) according to procedures outlined in the U.S. Army Corps of Engineers Quality Assurance Project Plan with minor deviations designed to enhance the rigor of our data. Presence of DNA in PCR-positive samples was confirmed by Sanger sequencing (forward and reverse) and sequences were considered positive only if sequences (forward and reverse) of ≥150 base pairs had a match of ≥95% to those of published sequences for bighead carp or silver carp. The DNA of bighead carp and silver carp was not detected in environmental samples collected above and below St. Croix Falls Dam on the St. Croix River, above and below the Coon Rapids Dam and below Lock and Dam 1 on the Upper Mississippi River, and from two negative control lakes, Square Lake and Lake Riley. The DNA of silver carp was detected in environmental samples collected below Lock and Dam 19 at Keokuk, Iowa, a reach of the river with high silver carp abundance. The portion (68%) of environmental samples taken below Lock and Dam 19 that were determined to contain the DNA of silver carp was similar to that reported in the scientific literature for other abundant species. The DNA of bighead carp, however, was not detected in environmental samples collected below Lock and Dam 19, a reach of the river known to have bighead carp. Previous reported detections of the DNA of silver carp in samples collected in 2011 were not replicated in this study. Additional analyses are planned for the DNA extracted from the samples collected in 2012. Those analyses may provide additional information regarding the lack of amplification of bighead carp DNA and the lengths of the sequences of silver carp DNA present in samples taken below Lock and Dam 19. These additional analyses may help inform the use of eDNA monitoring in large, complex systems like the Mississippi River.

Minnesota, Wisconsin

Prospects of pollinator community surveillance using terrestrial environmental DNA metagenetics

Current pollinator survey methods exhibit bias, require highly-trained practitioners, and are difficult to scale to large sample sizes. High-throughput sequencing of terrestrial eDNA could provide a complementary tool for studying pollinator communities, but eDNA methods have not been extensively evaluated. We conducted metagenetic analysis of whole arthropod community eDNA from 20 flower and seven honey bee-collected pollen samples and compared eDNA-derived data with traditional netting-based surveys of the pollinator communities present during sampling. We focused our analysis on Anthophila (bees) and detected eight bee genera belonging to four families across COI, 16S, and 28S markers. Results varied considerably by marker and eDNA substrate. Detected bee genera were plausible for the study system and about 43 percent of total bee genera were detected with both eDNA and net-based surveys, though netting resulted in more detections across a wider diversity of genera. Data from sequenced controls suggest that eDNA identifications were unlikely to have resulted from cross-contamination. Our results demonstrate that bee communities can be documented with eDNA techniques and that the choice of marker and substrate substantially influences detection. Future improvements to our methods are required, but eDNA surveys appear well-suited to characterize diverse pollinator communities and provide novel sampling perspectives within plant-pollinator networks. Future efforts should focus on improving the selection of markers available for pollinator eDNA metagenetics, addressing taxonomic gaps within reference sequence databases and optimizing sampling and eDNA isolation protocols. We anticipate that such improvements are highly feasible and that eDNA will be a useful tool to those who study pollinators and plant-pollinator interactions.

Environmental DNA

Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum)

Early detection of aquatic invasive species is a critical task for management of aquatic ecosystems. This task is hindered by the difficulty and cost of surveying aquatic systems thoroughly. The New Zealand mudsnail (Potamopyrgus antipodarum) is a small, invasive parthenogenic mollusk that can reach very high population densities and severely affects ecosystem functioning. To assist in the early detection of this invasive species, we developed and validated a highly sensitive environmental deoxyribonucleic acid (eDNA) assay. We used a dose–response laboratory experiment to investigate the relationship between New Zealand mudsnail density and eDNA detected through time. We documented that as few as 1 individual in 1.5 L of water for 2 d could be detected with this method, and that eDNA from this species may remain detectable for 21 to 44 d after mudsnail removal. We used the eDNA method to confirm the presence of New Zealand mudsnail eDNA at densities as low as 11 to 144 snails/m 2 in a eutrophic 5 th -order river. Combined, these results demonstrate the high potential for eDNA surveys to assist with early detection of a widely distributed invasive aquatic invertebrate.

Freshwater Science

Potential water-quality and hydrology stressors on freshwater mussels with development of environmental DNA assays for selected mussels and macroinvertebrates in Big Darby Creek Basin, Ohio, 2020–22

The richness and abundance of freshwater mussels in the Big Darby Creek Basin has declined in recent decades, according to survey results published by the Ohio Biological Survey. In October 2016, a major mussel die-off of undetermined cause reportedly affected over 50 miles of Big Darby Creek; however, fishes and other wildlife were not noticeably impacted. Pollution, habitat destruction, climate change, and hydrologic modification have all been theorized as potential reasons for the widespread declines in freshwater mussel populations in North America. To better understand potential stressors to mussels and other aquatic organisms in the Big Darby Creek Basin, the U.S. Geological Survey, in cooperation with the Ohio Water Development Authority, evaluated water quality and temporal changes in hydrology at selected locations. In addition, environmental deoxyribonucleic acid (eDNA) quantitative polymerase chain reaction (qPCR) assays were developed to detect the presence of selected mussels and macroinvertebrates using stream water. Time-weighted average concentrations of pesticides, organic wastewater compounds (OWCs), and polycyclic aromatic hydrocarbons (PAHs) were determined for selected locations within the Big Darby Creek Basin. Passive samplers designed to mimic the respiratory exposure of aquatic organisms and the bioconcentration of organic contaminants into their fatty tissues were deployed three times annually at three sites within the Big Darby Creek Basin in 2020 and 2021. Analyses were done for 204 pesticide compounds, 38 OWCs, and 33 PAHs. Of the 204 pesticide compounds, 70 were detected in at least one sample; 30 were detected in all samples. Herbicides and herbicide degradates were the pesticides most frequently detected and also had some of the highest concentrations of the pesticides detected in this study. Three herbicides (atrazine, ametryn, and metribuzin) were detected in at least 88 percent of samples and two fungicides (azoxystrobin and propiconazole) were detected in all samples. Of the 38 OWCs, 24 were detected in at least one sample; however, only one ( N , N -diethyltoluamide [DEET]) was detected in all samples. Of the 33 PAHs, 29 were detected in at least one sample; 12 were detected in all samples. A continuous water-quality monitor was operated seasonally on Big Darby Creek above Georgesville, Ohio, from 2020 to 2022. Dissolved oxygen concentrations generally followed a daily cycle, peaking in early evening and troughing around sunrise. There were occasional 24-hour swings in dissolved oxygen concentration that had a range exceeding 10 milligrams per liter. However, dissolved oxygen concentrations never fell below Ohio’s aquatic life criteria for warmwater habitats (outside of mixing zones) of 4.0 milligrams per liter as an instantaneous minimum and 5.0 milligrams per liter as a minimum 24-hour average. The Ohio water-quality criteria for temperatures are 29.4 degrees Celsius as an instantaneous maximum and 27.8 degrees Celsius as a 24-hour average maximum. In 2020, there were 10 days when the maximum instantaneous value for temperature was exceeded and 3 consecutive days when the maximum 24-hour average temperature was exceeded. Streamflow time-series data from three gaging stations within the Big Darby Creek Basin were evaluated for trends in annual flow statistics and daily nonexceedance probabilities over time. In general, the evaluation of streamflow conditions at the Big Darby Creek gage (with 97 years of record) indicated that streamflow changed between water years 1922 and 2021. During that time span, flows in general increased, the number of high-flow pulses became more frequent, and low-flow pulses and extreme low-flow periods became less frequent. The only strong indication of trends over time in annual flow statistics for the relatively short records for the other two gages (on Little Darby Creek, with 25 years of record, and Hellbranch Run, with 29 years of record) was that as time went on, reversals between rising and falling periods became more frequent. The U.S. Geological Survey Ohio Water Microbiology Laboratory developed eDNA qPCR assays to detect Epioblasma rangiana (northern riffleshell mussels), Chimarra obscura (a species of caddisfly), Maccaffertium pulchellum (a species of mayfly), and optimized a preexisting eDNA qPCR assay to detect for Ptychobranchus fasciolaris (kidneyshell mussels). The assays were validated by using environmental sampling methods. Assay sensitivity was established by determining the limits of detection and quantification. Water samples were collected at 12 sites in the Big Darby Creek Basin between 2020 and 2022 and analyzed for eDNA with the qPCR assays developed for this study.

Ohio

Occurrence of a suite of stream-obligate amphibians in timberlands of Mendocino County, California, examined using environmental DNA

Stream-obligate amphibians are important indicators of ecosystem health in the Pacific Northwest, but distributional information to improve forest management is lacking in many regions. We analyzed archived DNA extracted from water samples in 60 pools in streams on private timberlands in Mendocino County, California, for 3 California Species of Special Concern—Coastal Tailed Frogs ( Ascaphus truei ), Foothill Yellow-legged Frogs ( Rana boylii ), and Southern Torrent Salamanders ( Rhyacotriton variegatus )—to better understand their distributions in the region. Detection probabilities for eDNA of Foothill Yellow-legged Frogs and Coastal Tailed Frogs were positively influenced by water temperature. eDNA occurrence for both frogs was affected by whether silt or organic matter was a dominant substrate in the sampled pool, and Foothill Yellow-legged Frog eDNA occurrence was also affected by water temperature. Foothill Yellow-legged Frog eDNA occurrence had a strong, positive association with water temperature, with occurrence unlikely below 14°C and very likely above 16°C, and a positive association with silt or organic substrates in pools, which was likely an indicator of higher-order stream reaches. In contrast, Coastal Tailed Frogs had a negative association with silt or organic substrates. Historical visual detections were generally congruent with findings using eDNA, but differences highlight important areas for further study. We did not detect Southern Torrent Salamanders using eDNA at any sites. Our study reinforces that ecological relationships of these species are varied, and shows the importance of maintaining the integrity of streams with diverse characteristics for conserving stream amphibians.

California

An evaluation of the efficacy of using environmental DNA (eDNA) to detect giant gartersnakes (Thamnophis gigas)

Detecting populations of rare or cryptic species is essential for their conservation. For species like giant gartersnakes ( Thamnophis gigas ), conventional survey methods can be expensive and inefficient. These sampling difficulties might be overcome by modern techniques that detect deoxyribonucleic acid (DNA) shed by organisms into the environment (eDNA). We evaluated the efficacy of detecting giant gartersnake eDNA in water samples from the laboratory and at locations with known giant gartersnake populations in the Sacramento Valley of California, and failed to detect giant gartersnake DNA in most laboratory and all field samples. Aspects of giant gartersnake biology—such as highly keratinized skin and spending extensive time in the terrestrial environment, as well as hot, sunny, and turbid conditions in wetlands and canals of the Sacramento Valley—likely contributed to low detection probabilities. Although detection of eDNA shows promise under many conditions, further development is needed before sampling for eDNA is a viable option for detecting giant gartersnake populations.

California

Environmental DNA calibration study. Interim technical review report

Invasive aquatic nuisance species pose a major threat to aquatic ecosystems worldwide. Invasive Asian carps, including bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) have been steadily dispersing upstream through the Mississippi, Illinois, and Des Plaines Rivers since the 1990s. To prevent further movement up the Illinois River into the Chicago Area Waterway System (CAWS, see Figures 1.1.1 to 1.1.3) and possibly Lake Michigan and the Great Lakes ecosystem, an electrical barrier has been operating near Lockport to deter the advance of Asian carp. Although one adult individual has been detected in Lockport pool of the Illinois Waterway, the leading edge of the invasion of bighead and silver carp is considered to be at RM 281.5 in Dresden Island Pool, 15 miles downstream from the barrier and 55 miles from Lake Michigan, and that front has not progressed upstream since 2006. Although spawning activity has been observed in Marseilles pool in 2012, verified capture of eggs and larvae remain downstream in Peoria pool, over 140 miles from Lake Michigan (Figure 1.1.1). Should a sustainable Asian carp population become established in the Great Lakes, native fish populations, as well as many threatened or endangered plant/animal species populations, could be impacted. In response to this threat, the Asian Carp Regional Control Committee (ACRCC) was formed in part to coordinate efforts to understand and organize against the Asian carp threat. The Asian Carp Control Strategy Framework (2012a) outlined major tasks to be completed for a better understanding of factors related to the advance of Asian carp populations towards the Great Lakes. In addition, the ACRCC formed the Monitoring and Rapid Response Workgroup to address Asian carp monitoring and removal (ACRCC 2012b).

Midwest waterways

National Aquatic Environmental DNA Strategy

Aquatic life is the engine of ecosystems and economies. In environments ranging from freshwater through marine, this biodiversity underpins the health, culture, opportunities, and economic wellbeing of the Nation -- from local communities to the entire country. The ability to evaluate the status, trends, and future projections of nature is key to maintaining national prosperity, and this requires timely and trusted information about the condition of aquatic biodiversity on a vast scale. With one of the largest Exclusive Economic Zones in the world and extensive estuaries, lakes, rivers and streams, it is a grand challenge for the United States to explore, monitor, and understand aquatic life.

Report

Best practice guidelines for targeted environmental DNA-based proficiency testing in non-regulatory contexts

The effective use of environmental DNA (eDNA) tools is contingent on strict adherence to established and validated methods. Differences in eDNA methods and quality assurance protocols may contribute to variability in results. However, quality assurance measures such as proficiency testing can provide independent evaluation of laboratory performance against pre-established test criteria. With this commentary, we discuss how broad implementation of recurring proficiency testing in eDNA laboratories can build decision-maker confidence in eDNA results. It can also create a culture of continuous evaluation and improvement that minimizes error and meets performance requirements to inform the sustainable use or monitoring of natural resources. We provide an overview of proficiency testing across molecular disciplines, review the state of proficiency testing in eDNA applications, and draft a roadmap for the expanded application of proficiency testing informed by best practices for targeted eDNA detection. We suggest that best practice proficiency testing can be conducted by an independent, third-party sample provider. By demonstrating that laboratories are competent and capable of producing reliable results, implementation of proficiency testing best practices should foster confidence in eDNA measurements and its use in decision-making processes. Increased confidence in eDNA methods and a clear expectation of what is considered satisfactory performance are also likely to create more favorable conditions for investments in eDNA-based monitoring.

Environmental DNA