Environmental DNA Detection Research Articles

Environmental DNA detection of endangered and invasive species in Kejimkujik National Park and Historic Site.

The use of environmental DNA (eDNA) allows the early detection of aquatic species at low densities (e.g., elusive and invasive species), which otherwise could be challenging to monitor using conventional techniques. Here, we assess the ability of eDNA sampling to detect the presence or absence of one species at risk (Blanding's turtle) and two invasive species (chain pickerel and smallmouth bass) in Kejimkujik National Park and National Historic Site, Nova Scotia, where the aquatic system is highly acidic and rich in organic compounds. Five replicates of 1L water samples were taken per sampling site. Water filtration and eDNA extractions were performed on-site, while qPCR reactions were performed in the laboratory using species-specific assays. Samples were treated with an inhibition removal kit and analyzed pre- and post-inhibition removal. Despite the low pH and PCR inhibitors in water samples, our results showed positive eDNA detections in almost all expected positive sites (except in one site for Blanding's turtle). Detections of the target species were also observed at sites where their presence was previously unknown. Our study supports the advantage of eDNA to monitor species at low densities, revealing new distributions or recently invaded areas. We also demonstrate how eDNA can directly instruct management strategies in Kejimkujik.

An Environmental DNA Survey on Distribution of an Endangered Salmonid Species, Parahucho perryi, in Hokkaido, Japan

For protecting endangered species, precise understanding of their distribution is crucial. However, it is often very difficult to estimate at a large scale with conventional methods (e.g., casting nets or electrofishing for aquatic species) because of their low densities in the wild. Sakhalin taimen (Parahucho perryi) is one of the largest and most critically endangered freshwater salmonid fishes in the world. In this study, we applied an environmental DNA (eDNA) detection system for this species to 120 rivers in Hokkaido, the second largest main island of Japan. We successfully detected eDNA from Sakhalin taimen in seven rivers (5.8%). Although these rivers were widely distributed across the island, more than 95% of the total amounts of eDNA were detected from region-A and -I, indicating that local populations in the other regions of Hokkaido are very small and on the brink of extinction. In addition, principal component analyses based on the eDNA-based estimation of Sakhalin taimen distribution and GIS revealed their distribution determinants including limited topographic relief of watershed as well as presence of wetlands and lagoons. Our results suggest that eDNA-based detection systems are an efficient means of monitoring the population status of endangered freshwater species at large scales.

Broad-scale detection of environmental DNA for an invasive macrophyte and the relationship between DNA concentration and coverage in rivers

Environmental DNA (eDNA) techniques have the potential to become useful tools for investigating the distribution and biomass of aquatic organisms. Additionally, Unmanned Aerial Vehicle (UAV) photos have recently been applied to evaluate vegetation cover. We tested the ability of eDNA techniques to evaluate the distribution and biomass of an invasive macrophyte, Egeria densa (Brazilian waterweed). We conducted field surveys in two Japanese rivers to determine the abundance of eDNA from E. densa and compared it with macrophyte coverage by evaluating UAV photographs taken in the summer and winter. We found eDNA of E. densa in all sampled sites, including various sites with no macrophyte coverage, indicating that there was a broad-scale distribution of E. densa eDNA. In summer, we detected a slightly positive relationship between E. densa eDNA and macrophyte coverage. We did not find a clear relationship between E. densa eDNA and macrophyte coverage, probably because of their widespread and dense distribution and the flow of their materials in the rivers. The seasonal differences in the regression may have been caused by the seasonal growing period of E. densa in the summer, with a decline in their biomass in late autumn and winter. We confirmed the ability of eDNA techniques to survey this invasive macrophyte in rivers by comparing results with coverage measurements from UAV photographs.

Sensitive environmental DNA detection via lateral flow assay (dipstick)—A case study on corallivorous crown‐of‐thorns sea star (Acanthaster cf. solaris) detection

AbstractEnvironmental DNA (eDNA) represents an emerging opportunity for species monitoring in the marine environment. One aspect that poses challenges is the ability to detect target DNA without the complexity of specialized laboratory equipment. Lateral flow is an analytical technique that has been adopted in point‐of‐care diagnostics for human, veterinary, and agricultural health. Here, we aim to use lateral flow assay as a detection method for eDNA monitoring using a commercially available nucleic acid lateral flow device (PCRD™) in combination with previously developed species‐specific mtDNA primers. Episodic population explosions of coral‐eating crown‐of‐thorns sea star (CoTS) contribute significantly to the coral reef crisis on tropical Pacific coral reefs. Laboratory testing revealed our lateral flow assay developed for CoTS was as sensitive as digital droplet PCR and able to detect < 10 copies of target DNA, per PCR. Furthermore, the lateral flow assay was completed in less than half the time compared with digital droplet PCR and cost less than half that of digital droplet PCR. We applied this method to eDNA water samples collected in field locations where CoTS were present in low (=nonoutbreak) population densities and found lateral flow assay to be sufficiently sensitive to detect these populations. Detection of low‐density CoTS populations is critical for early warning of outbreaks and leads to early management interventions (e.g., culling). Importantly, we demonstrate that with species‐specific primers, the development and application of lateral flow assay methods for eDNA detection are feasible, for example, for invasive or threatened species, or those with high conservation value.

Evaluating environmental DNA as a tool for detecting an amphibian pathogen using an optimized extraction method.

Environmental DNA (eDNA) detection is a valuable conservation tool that can be used to identify and monitor imperiled or invasive species and wildlife pathogens. Batrachochytrium pathogens are of global conservation concern because they are a leading cause of amphibian decline. While eDNA techniques have been used to detect Batrachochytrium DNA in the environment, a systematic comparison of extraction methods across environmental samples is lacking. In this study, we first compared eDNA extraction methods and found that a soil extraction kit (Qiagen PowerSoil) was the most effective for detecting Batrachochytrium dendrobatidis in water samples. The PowerSoil extraction had a minimum detection level of 100 zoospores and had a two- to four-fold higher detection probability than other commonly used extraction methods (e.g., QIAamp extraction, DNeasy+Qiashredder extraction method, respectively). Next, we used this extraction method on field-collected water and sediment samples and were able to detect pathogen DNA in both. While field-collected water filters were equivalent to amphibian skin swab samples in detecting the presence of pathogen DNA, the seasonal patterns in pathogen quantity were different between skin swabs and water samples. Detection rate was lowest in sediment samples. We also found that detection probability increases with the volume of water filtered. Our results indicate that water filter eDNA samples can be accurate in detecting pathogen presence at the habitat scale but their utility for quantifying pathogen loads in the environment appears limited. We suggest that eDNA techniques be used for early warning detection to guide animal sampling efforts.

Field storage of water samples affects measured environmental DNA concentration and detection

Environmental DNA (eDNA) is an emerging approach for detecting species, yet numerous methodological questions remain unanswered. Here we examined how time to filtration (0–48 h after collection) and sample storage (open vs. chilled in the dark) influenced detection and measured eDNA concentration. Water samples kept in the dark and chilled had no significant decrease in detection or eDNA concentration relative to those filtered immediately upon return from the field. Water samples exposed to light and ambient air temperature had non-detections beginning at 1 h, the majority of these samples were below the limit of detection (LOD) by 6 h, and eDNA was undetectable in these samples by 24 h. These results have important implications for eDNA research where immediate access to refrigeration is not available, or for fieldwork that requires extended sampling time (e.g., canoeing a river). Further, we report faster eDNA degradation times under ambient conditions than some previous aquaria or mesocosm studies, suggesting an ongoing need to study mechanisms related to eDNA persistence and sample storage.

Environmental DNA detection tracks established seasonal occurrence of blacktip sharks (Carcharhinus limbatus) in a semi-enclosed subtropical bay

The integration of eDNA analysis into the population assessment and monitoring of sharks could greatly improve temporal and spatial data used for management purposes. This study aimed to compare eDNA detection against well-established seasonal changes in blacktip shark (Carcharhinus limbatus) abundance in Terra Ceia Bay (FL, USA). We used a species-specific real-time PCR approach to detect C. limbatus eDNA in the bay on a near monthly basis from spring through mid-fall in 2018 and 2019. Previous studies have shown that C. limbatus give birth in the bay in early summer and immature sharks occur there until late fall, when decreasing water temperatures cause them to move offshore and southwards. Water samples (2 L) were collected (4–6 per month) and filtered in the field, with each then being subjected to real-time PCR. Carcharhinus limbatus ‘positive’ filters were significantly more commonly collected during the April-July sampling period than during the August-October sampling period. While following the predicted pattern, eDNA concentration was generally too low for accurate quantification. Our results show that C. limbatus eDNA detection follows known seasonal residency patterns consistently over 2 years of monitoring. Species-specific eDNA analysis using real-time PCR could therefore represent a cost-effective, scalable sampling tool to facilitate improved shark population monitoring in semi-enclosed marine habitats.

Environmental DNA: A promising factor for tuberculosis risk assessment in multi-host settings

Attaining and maintaining the Official Tuberculosis Free status continues to be a challenge when several domestic and wild hosts contribute to the maintenance of the Mycobacterium tuberculosis complex (MTC). Local tuberculosis hotspots are sometimes identified in cattle in low-prevalence regions. We have, therefore, studied one such hotspot in depth in order to produce an epidemiological diagnosis. Host population size and MTC prevalence were estimated in selected wildlife and in livestock, while on-cattle environmental DNA detection was additionally used as a proxy for risk of exposure at the farm (herd) level. Positive skin test reactors were found on16 of the 24 cattle farms studied in the period 2012-2016. Although all goats tested negative to the skin test during this period, MTC was confirmed in four sheep at slaughter, thus indicating an unknown prevalence of infection in this host species. With regard to wildlife, the prevalence of MTC infection based on culture was 8.8% in the case of wild boar (Sus scrofa), and the only road-killed badger (Meles meles) submitted for culture tested positive. Two criteria were employed to divide the cattle farms into higher or lower risk: tuberculosis testing results and environmental DNA detection. Environmental MTC DNA detection yielded significant differences regarding "use of regional pastures" and "proximity to woodland". This study suggests that on-animal environmental DNA sampling may help when assessing contact risk as regards MTC in livestock at the herd level. This tool opens up new avenues of epidemiological research in complex multi-host settings.

Environmental DNA reveals cryptic diversity within the subterranean amphipod genus Pseudocrangonyx Akatsuka & Komai, 1922 (Amphipoda: Crangonyctoidea: Pseudocrangonyctidae) from central Japan

Abstract An environmental DNA (eDNA) detection method confirms the presence of the subterranean amphipod genus PseudocrangonyxAkatsuka & Komai, 1922 in the city of Kyoto, Honshu, Japan for the first time in more than 90 years. Phylogenetic trees using partial sequences of the 16S rRNA gene of eDNA samples from Kyoto and specimens collected from various locations in Japan and Korea highlight that two genetically distinct taxonomic units inhabit Kyoto’s subterranean environments. Results reveal that the ranges of distinct phylogroups of Pseudocrangonyx widely overlap with each other around Lake Biwa in central western Honshu. The eDNA results increase our understanding of the species richness and complicated evolutionary history of these stygobitic amphipods.

Monitoring the silver carp invasion in Africa: a case study using environmental DNA (eDNA) in dangerous watersheds

Biodiverse habitats are increasingly subject to an intensification of anthropogenic stressors that may severely diminish species richness. Invasive species pose a dominant threat to biodiversity and biosecurity, particularly in biodiversity hotspots like Kruger National Park, South Africa. The invasive silver carp, Hypophthalmichthys molitrix, was introduced into the Olifants River and may experience range spread owing to favorable environmental conditions. Intensive monitoring protocols are necessary to effectively manage invasions of species like silver carp. Unfortunately, tropical and sub-tropical aquatic systems are difficult to monitor using conventional methods (e.g., netting, electrofishing and snorkeling) owing to a range of factors including the presence of dangerous megafauna. Conservation of such systems may be advanced by the adoption of novel methods, including environmental DNA (eDNA) detection. Here, we explore the utility of environmental DNA (eDNA) to conduct safe, reliable and repeatable surveys in dangerous watersheds using silver carp as a case study. We conducted eDNA surveys at 12 sites in two neighbouring watersheds, and determined that the species has expanded its range within the Olifants River and to the south in the Sabie River. Expansion in the former is consistent with the presence of suitable spawning conditions. We discuss the implications of this survey for biodiversity monitoring in similar aquatic systems in the tropics and advocate an integrative approach to biomonitoring in these ecosystems.

Spatial and temporal patterns of environmental DNA detection to inform sampling protocols in lentic and lotic systems.

The development of efficient sampling protocols for the capture of environmental DNA (eDNA) could greatly help improve accuracy of occupancy monitoring for species that are difficult to detect. However, the process of developing a protocol in situ is complicated for rare species by the fact that animal locations are often unknown. We tested sampling designs in lake and stream systems to determine the most effective eDNA sampling protocols for two rare species: the Sierra Nevada yellow‐legged frog (Rana sierrae) and the foothill yellow‐legged frog (Rana boylii). We varied water volume, spatial sampling, and seasonal timing in lakes and streams; in lakes we also tested multiple filter types. We found that filtering 2 L versus 1 L increased the odds of detection in streams 5.42X (95% CI: 3.2–9.19X) in our protocol, from a probability of 0.51–0.85 per technical replicate. Lake sample volumes were limited by filter clogging, and we found no effect of volume or filter type. Sampling later in the season increased the odds of detection in streams by 1.96X for every 30 days (95% CI: 1.3–2.97X) but there was no effect for lakes. Spatial autocorrelation of the quantity of yellow‐legged frog eDNA captured in streams ceased between 100 and 200 m, indicating that sampling at close intervals is important.

Analytical validation and field testing of a specific qPCR assay for environmental DNA detection of invasive European green crab (Carcinus maenas)

AbstractEnvironmental DNA (eDNA) methods are providing tools for detecting invasive species in aquatic environments. Targeted qPCR assays applied to eDNA samples promise to overcome limitations of traditional methods, especially for early detection. The European green crab (Carcinus maenas) is considered one of the most successful invasive species globally due to the large range it has invaded and negative impacts on native species, marine habitats, and shellfish industries. We developed, laboratory‐validated, and field‐tested a specific qPCR assay for the detection of green crab from eDNA samples. We also show that the assay can detect green crab in bulk DNA extracted from plankton samples. Assay design, optimization, sensitivity, and specificity testing generally followed the validation pathway recommended by the World Organization for Animal Health for assays used to manage global aquatic animal health and infectious disease. Assay specificity was verified in silico and in vitro by laboratory testing 26 nontarget species, none of which showed potential for amplification. Assay sensitivity was appropriately high, with the limit of detection approaching two gene copies/μl. The assay was field‐tested on eDNA samples collected from filtered seawater at five sites on the Pacific coast of Canada known to harbor green crab based on historical monitoring data; green crab DNA was amplified from all sites. We also present early pilot field testing of the assay done on bulk DNA extracted from plankton samples from four sites from Australia, two sites with and two sites without reported records of green crab presence. Green crab was detected at both sites with known green crab records. Significant inhibition was recorded for some plankton samples but not for eDNA samples. This is the first qPCR assay for detection of European green crab, providing researchers and managers with a valuable new tool to aid early detection and ongoing monitoring.

Environmental DNA detection of Arctic char (Salvelinus alpinus) in Irish lakes: Development and application of a species‐specific molecular assay

AbstractPopulations of Arctic char (Salvelinus alpinus) inhabiting Irish lakes are undergoing a rapid decline. In order to aid monitoring of this species in Irish lakes, a species‐specific qPCR assay was designed and subsequently tested for the detection of Arctic char's environmental DNA (eDNA) in an indoor mesocosm and in natural habitats (six lakes). Specifically, the mesocosm experiment revealed that sample volume can have significant effects on the application of eDNA approaches, whereby probability of detection depends from proximity to the source organism and volume of water filtered. When tested in the wild, positive amplification of Arctic char eDNA was observed in four of the six lakes sampled, proving that this approach can be successfully implemented in natural conditions. However, findings from the present study also showed that suboptimal sampling strategies can lead to false‐negative results (e.g., failure to detect the species when it is present). This study corroborates findings from other research on eDNA detection of fish species, showing that despite its current limitations, this approach can be a fast, noninvasive, and effective tool in aid to the conservation and management of freshwater fish species.

Environmental DNA (eDNA) detection of marine aquatic invasive species (AIS) in Eastern Canada using a targeted species-specific qPCR approach

Environmental DNA (eDNA) detection of marine aquatic invasive species (AIS) in Eastern Canada using a targeted species-specific qPCR approach

Environmental DNA detection and quantification of invasive red-eared sliders, Trachemy scripta elegans, in ponds and the influence of water quality.

Environmental DNA (eDNA) is a powerful tool for monitoring the distribution of aquatic macro-organisms. However, environmental factors, including the water temperature and water quality, can affect the inhibition and/or degradation of eDNA, which complicates accurate estimations of eDNA concentrations and the detection of the presence/absence of species in natural habitats. Further very few eDNA studies have been conducted for reptiles, especially with respect to estimating their biomass and/or abundances. Here we examined the relationship between the visually-observed number of red-eared sliders (Trachemys scripta elegans) and eDNA concentrations across 100 ponds. Additionally, we evaluated the effect of water quality on red-eared slider eDNA concentration in these ponds. We found that there was a significant positive correlation between the observed number of red-eared sliders and the eDNA concentration in the ponds. On comparing various water quality indicators, including dissolved nitrogen, dissolved phosphorous, organic matter, and chlorophyll a (Chl. a), we found that only Chl. a had a negative correlation with the red-eared slider eDNA concentration, while we did not find any inhibition in the quantitative PCR. We conclude that concentrations of eDNA can potentially be used for estimating the abundance of the red-eared slider. Additionally, Chl. a might indirectly influence the degradation of eDNA through the microorganisms bonded to the phytoplankton in the ponds, as microbial activity is thought to decrease eDNA persistence.

Detection of spatiotemporal variation in ranavirus distribution using eDNA

AbstractAmphibian population declines have been associated with emerging diseases including ranaviruses, which can cause mass die‐offs across entire amphibian communities. Understanding and mitigating disease spread requires knowledge of spatial and temporal patterns of pathogen distribution, but also how environmental factors influence pathogen occurrence. We applied environmental DNA (eDNA) detection tools to survey spatial and temporal distributions of ranaviruses by sampling 103 waterbodies in southeastern Ontario, Canada and assessed the role of abiotic factors as predictors of pathogen occurrence. Ten waterbodies sampled during June–August (>30 km between sites) revealed that ranavirus was marginally more prevalent (p = .055) during the latter part of the summer. Ninety‐three sites sampled at a finer scale (<10 km between sites) exhibited seasonal variability in ranavirus detection (site prevalence: 56% May; 66% July). Occupancy modeling revealed that wetland size and elevation influenced ranavirus occurrence while sampling date and water temperature influenced probability of detection. These findings indicate that biotic factors, such as host density and alternative hosts, should be investigated further as likely determinants of ranavirus prevalence across the landscape. Further, these results highlight the sensitivity of eDNA for detecting widespread presence of ranavirus and that abiotic factors may have a limited role in determining its prevalence and infectivity.

Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii).

Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle’s wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.

Radiotelemetry reveals effects of upstream biomass and UV exposure on environmental DNA occupancy and detection for a large freshwater turtle

AbstractBackgroundImperfect detection of hard‐to‐sample organisms has motivated the development of novel monitoring techniques. Environmental DNA (eDNA) can provide a sensitive, relatively low‐cost sampling method in aquatic systems, but biotic and abiotic factors can affect its reliability.AimsWe used a reintroduced population of radio‐tracked alligator snapping turtles (Macrochelys temminckii) to test the efficacy and ecology of eDNA in a lotic system.Materials & MethodsWe collected samples at turtle locations as well as random up‐ and downstream sites. Using a novel eDNA assay, we modeled occupancy and detection of eDNA using turtle biomass and remotely sensed UV exposure and also tested the relationship between these parameters and eDNA concentration.ResultsWe found eDNA occupancy was best explained by upstream biomass and detection probability decreased with greater upstream UV exposure. The concentration of eDNA in a sample was not significantly affected by biomass measures, but decreased with higher upstream UV exposure, and the effect was significant.DiscussionOur results show that UV exposure can affect aquatic eDNA sampling in situ with free‐ranging animals. The use of radiotelemetry allowed for higher control when modeling eDNA persistence and transport in a natural setting relative to laboratory, enclosure, or mesocosm studies.ConclusionWe propose using wildlife tracking techniques to provide added realism to studies of eDNA dynamics.

Comparison of inhibition resistance among PCR reagents for detection and quantification of environmental DNA

AbstractBackgroundReal‐time PCR has been widely employed in environmental DNA (eDNA) surveys to detect and quantify target species. The biggest obstacle in real‐time PCR based eDNA assays is the inhibition of PCR amplification that results in false‐negative detection and uncertainty of quantification.AimsHere, we aimed to evaluate inhibition resistance of PCR reagents for detection and quantification of environmental DNA.Materials & MethodsWe compared six commercial PCR reagents, including four inhibitor‐resistant reagents, for resistance to major PCR inhibitory substances, i.e., humic, fulvic, and tannic acids. Then, we selected three inhibitorresistant reagents to test for their resistance to multiple natural inhibitors using field eDNA samples collected from various habitats.ResultsWe found that each inhibitor‐resistant reagent had its advantage and disadvantage depending on which inhibitory substance was used. Similarly, the inhibitory effects caused by field samples differed among the three inhibitor‐resistant reagents, where none of the reagents consistently showed strong resistance to all field samples.ConclusionOur results indicate that the selection of appropriate PCR reagent would greatly ease PCR inhibition, and consequently improve the estimation of species distribution by real‐time PCR‐based eDNA assays.

An application of PCR-RFLP species identification assay for environmental DNA detection.

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.