Detection of the endangered European weather loach (Misgurnus fossilis) via water and sediment samples: Testing multiple eDNA workflows

The European weather loach (Misgurnus fossilis) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle (C_t) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis. We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis, total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis.     

Improved environmental DNA sampling scheme for Alabama sturgeon provides new insight into a species once presumed extinct

Detection of rare species can be challenging and time-consuming using conventional methods, but environmental DNA (eDNA) is becoming a commonly used tool for detection in conservation and management of species. This study demonstrates the utility of the precipitation method (precipitated and preserved in 3 M sodium acetate and 95% ethanol) for collection of eDNA to detect the seasonal distribution of the critically endangered Alabama sturgeon (Scaphirhynchus suttkusi). Surface and benthic water samples were collected across a wider geographic area than previously published for Alabama sturgeon eDNA. Surface and benthic samples both yielded detections and resulted in a similar proportion of positive detections to previous work. However, by sampling a greater portion of the distribution of the Alabama sturgeon, further insight was provided on potential sturgeon movement. The results of the precipitation method show that Alabama sturgeon detections increase during spawning months, and that the fish may be overwintering in the Tombigbee River. High detections from winter benthic samples suggest that habitat choice may play a role in detectability and highlight the need to consider natural history when designing environmental DNA studies. When designing environmental DNA collection for rare species, sampling design should factor in species ecology, habitat use, site characteristics, and specific questions driving the research.     

Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.     

Space invaders: Searching for invasive Smallmouth Bass (Micropterus dolomieu) in a renowned Atlantic Salmon (Salmo salar) river

Humans have the ability to permanently alter aquatic ecosystems and the introduction of species is often the most serious alteration. Non‐native Smallmouth Bass (Micropterus dolomieu) were identified in Miramichi Lake c. 2008, which is a headwater tributary to the Southwest Miramichi River, a renowned Atlantic Salmon (Salmo salar) river whose salmon population is dwindling. A containment programme managed by the Department of Fisheries and Oceans, Canada (DFO) was implemented in 2009 to confine Smallmouth Bass (SMB) to the lake. We utilized environmental DNA (eDNA) as a detection tool to establish the potential escape of SMB into the Southwest Miramichi River. We sampled at 26 unique sites within Miramichi Lake, the outlet of Miramichi Lake (Lake Brook), which flows into the main stem Southwest Miramichi River, and the main stem Southwest Miramichi River between August and October 2017. We observed n = 6 positive detections located in the lake, Lake Brook, and the main stem Southwest Miramichi downstream of the lake. No detections were observed upstream of the confluence of Lake Brook and the main stem Southwest Miramichi. The spatial pattern of positive eDNA detections downstream of the lake suggests the presence of individual fish versus lake‐sourced DNA in the outlet stream discharging to the main river. Smallmouth Bass were later confirmed by visual observation during a snorkeling campaign, and angling. Our results, both eDNA and visual confirmation, definitively show Smallmouth Bass now occupy the main stem of the Southwest Miramichi.     

Environmental DNA detects a rare large river crayfish but with little relation to local abundance

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Environmental DNA Marker Development with Sparse Biological Information: A Case Study on Opossum Shrimp (Mysis diluviana)

The spread of Mysis diluviana, a small glacial relict crustacean, outside its native range has led to unintended shifts in the composition of native fish communities throughout western North America. As a result, biologists seek accurate methods of determining the presence of M. diluviana, especially at low densities or during the initial stages of an invasion. Environmental DNA (eDNA) provides one solution for detecting M. diluviana, but building eDNA markers that are both sensitive and species-specific is challenging when the distribution and taxonomy of closely related non-target taxa are poorly understood, published genetic data are sparse, and tissue samples are difficult to obtain. To address these issues, we developed a pair of independent eDNA markers to increase the likelihood of a positive detection of M. diluviana when present and reduce the probability of false positive detections from closely related non-target species. Because tissue samples of closely-related and possibly sympatric, non-target taxa could not be obtained, we used synthetic DNA sequences of closely related non-target species to test the specificity of eDNA markers. Both eDNA markers yielded positive detections from five waterbodies where M. diluviana was known to be present, and no detections in five others where this species was thought to be absent. Daytime samples from varying depths in one waterbody occupied by M. diluviana demonstrated that samples near the lake bottom produced 5 to more than 300 times as many eDNA copies as samples taken at other depths, but all samples tested positive regardless of depth.     

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

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Riverine distribution of mussel environmental DNA reflects a balance among density,transport, and removal processes

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A sensitive environmental DNA (eDNA) assay leads to new insights on Ruffe (<Emphasis Type="Italic">Gymnocephalus cernua</Emphasis>) 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.     

Abundance estimation of riverine macrophyte Egeria densa using environmental DNA: effects of sampling season and location

Invasive macrophytes can have a variety of effects on aquatic ecosystems. The early detection and abundance estimation of an invasive species is important to effectively control it and minimize the ecosystem impacts. It is imperative to develop more efficient sampling methods for the abundance quantification of aquatic plants in large riverine systems. We examined (1) relationships between the environmental DNA (eDNA) concentrations of the invasive macrophyte, Egeria densa, and the upstream coverage area on the multiple life-history stages (dormant, growing, and senescence seasons) in a large riverine system in Japan and (2) if the relationships between the eDNA concentrations and coverage area could vary with the lateral sampling locations (left bank, middle, and right bank). The eDNA concentrations had significant positive relationships with the upstream coverage area of E. densa at multiple spatial scales for the dormant and senescence seasons. These results suggest that the eDNA analysis could be useful to quantify the relative abundance of this aquatic macrophyte in the riverine system; however, the selection of the eDNA sampling season could be important to accurately estimate abundance. Our results also showed that the eDNA concentrations of E. densa did not significantly differ from the lateral sampling location, suggesting that the eDNA samples could reflect the relative abundance of E. densa upstream of the study sites regardless of the lateral sampling location.

     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6–65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.     

Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi‐aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water‐borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus‐specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.     

Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries

The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next‐generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom‐designed group‐specific primer set and next‐generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture‐based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co‐occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture‐based methods, for improved mapping of species distribution patterns.     

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, ‘early detection and rapid response’; (ii) for conserving imperilled native species, ‘protection of biodiversity hotspots’; and (iii) for assessing biosecurity risk, ‘an ounce of prevention equals a pound of cure.’ However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism’s DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next‐generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.     

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2–0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies.     

Occupancy in dynamic systems: accounting for multiple scales and false positives using environmental DNA to inform monitoring

Occupancy is an important metric to understand current and future trends in populations that have declined globally. In addition, occupancy can be an efficient tool for conducting landscape-scale and long-term monitoring. A challenge for occupancy monitoring programs is to determine the appropriate spatial scale of analysis and to obtain precise occupancy estimates for elusive species. We used a multi-scale occupancy model to assess occupancy of Columbia spotted frogs in the Great Basin, USA, based on environmental DNA (eDNA) detections. We collected three replicate eDNA samples at 220 sites across the Great Basin. We estimated and modeled ecological factors that described watershed and site occupancy at multiple spatial scales simultaneously while accounting for imperfect detection. Additionally, we conducted visual and dipnet surveys at all sites and used our paired detections to estimate the probability of a false positive detection for our eDNA sampling. We applied the estimated false positive rate to our multi-scale occupancy dataset and assessed changes in model selection. We had higher naïve occupancy estimates for eDNA (0.37) than for traditional survey methods (0.20). We estimated our false positive detection rate per qPCR replicate at 0.023 (95% CI: 0.016–0.033). When the false positive rate was applied to the multi-scale dataset, we did not observe substantial changes in model selection or parameter estimates. Conservation and resource managers have an increasing need to understand species occupancy in highly variable landscapes where the spatial distribution of habitat changes significantly over time due to climate change and human impact. A multi-scale occupancy approach can be used to obtain regional occupancy estimates that can account for spatially dynamic differences in availability over time, especially when assessing potential declines. Additionally, this study demonstrates how eDNA can be used as an effective tool for improved occupancy estimates across broad geographic scales for long-term monitoring.     

基于环境DNA宏条形码技术的辽东湾典型围海养殖池塘内水母多样性研究

研究使用环境DNA宏条形码技术(eDNA metabarcoding)检测辽东湾东北部河口区围海养殖池塘水母种类多样性,探索适用于水母种类物种鉴定和监测的新方法。利用环境DNA宏条形码技术,分别基于18S rDNA和COI宏条形码检测了辽东湾东北部河口区围海养殖池塘水母种类多样性,通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从围海养殖池塘7个采样点中获得可检测的采样点数据。结果显示,基于18S rDNA宏条形码检测出8种水母种类,其中钵水母纲大型水母2种、水螅水母总纲小型水母6种;基于COI宏条形码技术共检测出19种水母种类,其中钵水母纲大型水母5种、水螅水母总纲小型水母14种;两种DNA条形码标记都显示养殖种类海蜇(Rhopilema esculentum)为优势种。研究结果表明,环境DNA宏条形码技术作为一种新兴的生物多样性监测手段可用于快速检测水母种类多样性,在水母类物种鉴定、监测及早期预警中有较大的应用潜能。     

Mitochondrial Genome Sequencing and Development of Genetic Markers for the Detection of DNA of Invasive Bighead and Silver Carp (Hypophthalmichthys nobilis and H. molitrix) in Environmental Water Samples from the United States

Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American aquatic ecosystems. Recently, environmental DNA (eDNA), genetic material shed by organisms into their environment that can be detected by non-invasive sampling strategies and genetic assays, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American surface waters. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with those from other common and closely related fish species from the Illinois River watershed to identify and design new species-specific markers for the detection of bighead and silver carp DNA in environmental water samples. We then tested these genetic markers in the laboratory for species-specificity and sensitivity. Newly developed markers performed well in field trials, did not have any false positive detections, and many markers had much higher detection rates and sensitivity compared to the markers currently used in eDNA surveillance programs. We also explored the use of multiple genetic markers to determine whether it would improve detection rates, results of which showed that using multiple highly sensitive markers should maximize detection rates in environmental samples. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp and will improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.     

Assessing Environmental DNA Detection in Controlled Lentic Systems

Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m³). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m³ (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3–5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42–73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m³ or 1.75 g/m³).