Detection of a diverse marine fish fauna using environmental DNA from seawater samples

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days.Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.     

Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds

Knowledge of the presence of an invasive species is critical to monitoring the sustainability of communities and ecosystems. Environmental DNA (eDNA), DNA fragments that are likely to be bound to organic matters in the water or in shed cells, has been used to monitor the presence of aquatic animals. Using an eDNA-based method, we estimated the presence of the invasive bluegill sunfish, Lepomis macrochirus, in 70 ponds located in seven locales on the Japanese mainland and on surrounding islands. We quantified the concentration of DNA copies in a 1 L water sample using quantitative real-time polymerase chain reaction (qPCR) with a primer/probe set. In addition, we visually observed the bluegill presence in the ponds from the shoreline. We detected bluegill eDNA in all the ponds where bluegills were observed visually and some where bluegills were not observed. Bluegills were also less prevalent on the islands than the mainland, likely owing to limited dispersal and introduction by humans. Our eDNA method simply and rapidly detects the presence of this invasive fish species with less disturbance to the environment during field surveys than traditional methods.     

Using Environmental DNA to Census Marine Fishes in a Large Mesocosm

The ocean is a soup of its resident species'' genetic material, cast off in the forms of metabolic waste, shed skin cells, or damaged tissue. Sampling this environmental DNA (eDNA) is a potentially powerful means of assessing whole biological communities, a significant advance over the manual methods of environmental sampling that have historically dominated marine ecology and related fields. Here, we estimate the vertebrate fauna in a 4.5-million-liter mesocosm aquarium tank at the Monterey Bay Aquarium of known species composition by sequencing the eDNA from its constituent seawater. We find that it is generally possible to detect mitochondrial DNA of bony fishes sufficient to identify organisms to taxonomic family- or genus-level using a 106 bp fragment of the 12S ribosomal gene. Within bony fishes, we observe a low false-negative detection rate, although we did not detect the cartilaginous fishes or sea turtles present with this fragment. We find that the rank abundance of recovered eDNA sequences correlates with the abundance of corresponding species'' biomass in the mesocosm, but the data in hand do not allow us to develop a quantitative relationship between biomass and eDNA abundance. Finally, we find a low false-positive rate for detection of exogenous eDNA, and we were able to diagnose non-native species'' tissue in the food used to maintain the mesocosm, underscoring the sensitivity of eDNA as a technique for community-level ecological surveys. We conclude that eDNA has substantial potential to become a core tool for environmental monitoring, but that a variety of challenges remain before reliable quantitative assessments of ecological communities in the field become possible.     

Establishing an environmental DNA method to detect and estimate the biomass of Sakhalin taimen,a critically endangered Asian salmonid

For field ecologists, detecting a target species in the wild is a severe bottleneck to understanding its distribution and population status. Recently, environmental DNA (eDNA) techniques have been developed as a noninvasive monitoring tool for aquatic organisms. While applications of eDNA techniques for biomass estimation have been proposed, little is known about an applicable size range of the organisms, which might affect relationships between biomass and eDNA concentration. Here, we investigated eDNA from Sakhalin taimen (Parahucho perryi), a giant salmonid species of northern Japan. This species is critically endangered and difficult to detect in the wild by conventional sampling methods. Using quantitative real-time PCR, we tested correlations between eDNA concentration and fish density using fish with a wide range of ages and body sizes in aquarium experiments. We found that our new primers and probe were truly species-specific, and that the eDNA concentration was significantly correlated with fish density and body size (p < 0.001). Furthermore, based on our calculation, the eDNA concentrations were rather constant among aquaria with fish in different age and size groups when their total weight was adjusted. These results suggest that eDNA concentrations can be an indicator of biomass of Sakhalin taimen, although further research is needed for its application in natural environments.     

The application of CRISPR‐Cas for single species identification from environmental DNA

We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.     

Enhanced ecological indication based on combined planktic and benthic functional approaches in large river phytoplankton ecology

Environmental DNA (eDNA) is a powerful method for assessing the presence and distribution of invasive aquatic species. We used this tool to detect and monitor several invasive crayfishes Procambarus clarkii, Orconectes limosus and Pacifastacus leniusculus present in, or likely to invade, the ponds of the Brenne Regional Natural Park. A previous study showed that the eDNA method was not very efficient in detecting P. clarkii. In the present study, we explored new improvements in the detection of invasive crayfish. We designed specific primers for each crayfish species, and set up an experimental mesocosm approach to confirm the specificity of the primers and the sampling protocol. We analysed samples taken from ponds in 2014 and 2015. We compared two qPCR protocols involving either SybrGreen or TaqMan assays. Using these same primers, we were able to detect crayfish eDNA with both assays during the mesocosm experiment. However, crayfish from field samples could only be detected by performing qPCR with a SybrGreen assay. We successfully monitored the presence of three invasive species of crayfish using eDNA. This method is a powerful tool for establishing the presence or absence of invasive species in various freshwater environments.     

基于环境DNA宏条形码的洱海鱼类多样性研究

研究使用环境DNA宏条形码(eDNA metabarcoding)检测洱海鱼类多样性,探索适用于洱海鱼类多样性监测和保护的新方法。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,从洱海16个采样点中获得可检测的9个采样点数据,共检测出17种鱼类,其中土著种5种、外来种12种;鲫(Carassius auratus)、鳙(Hypophthalmichthys nobilis)、麦穗鱼(Pseudorasbora parva)、泥鳅(Misgurnus anguillicaudatus)和食蚊鱼(Gambusia affinis)为优势种。研究结果表明虽然环境DNA宏条形码无法完全替代传统的鱼类监测方法,但作为一种新兴的生物多样性监测手段,其可用于快速检测洱海鱼类多样性及其空间分布。     

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

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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。     

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

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

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.     

Environmental DNA detection of aquatic invasive plants in lab mesocosm and natural field conditions

Aquatic invasive plant species cause negative impacts to economies and ecosystems worldwide. Traditional survey methods, while necessary, often do not result in timely detections of aquatic invaders, which can be cryptic, difficult to identify, and exhibit very rapid growth and reproduction rates. Environmental DNA (eDNA) is a relatively new method that has been used to detect multiple types of animals in freshwater and marine ecosystems through tissues naturally shed from the organism into the water column or sediment. While eDNA detection has proven highly effective in the detection of aquatic animals, we know less about the efficacy of eDNA as an effective surveillance tool for aquatic plants. To address this disparity, we designed mesocosm experiments with Elodea species to determine the ability to detect accumulation and degradation of the DNA signal for aquatic plants, followed by field surveillance of the highly invasive Hydrilla verticillata in freshwaters across several U.S. geographic regions. In both lab and field experiments, we designed a high sensitivity quantitative PCR assay to detect the aquatic plant species. In both experiments, plant eDNA detection was successful; we saw accumulation of DNA when plants were introduced to tanks and a decrease in DNA over time after plants were removed. We detected eDNA in the field in areas of known Hydrilla distribution. Employing eDNA detection for aquatic plants will strengthen efforts for early detection and rapid response of invaders in global freshwater ecosystems.     

Methodological considerations for detection of terrestrial small‐body salamander eDNA and implications for biodiversity conservation

Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine‐scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild‐caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small‐bodied wild terrestrial salamander populations.     

The use of environmental DNA of fishes as an efficient method of determining habitat connectivity

This study demonstrated the use of environmental DNA (eDNA) to determine habitat connectivity for migration of fishes between the sea and river. Environmental DNA is DNA fragments released by fishes in water, which can be used as a species-specific marker of the presence/absence of the target species. A year-round water sampling regime at 15 sites on the Yodo River, Japan, was conducted to determine whether three major man-made barriers on the river inhibited the migration of fishes using species-specific detection of DNA fragments from three target migrant species, temperate seabass, Lateolabrax japonicus, flathead grey mullet, Mugil cephalus, and ayu, Plecoglossus altivelis altivelis. The presence/absence of eDNA from target species was consistent with known patterns of species’ seasonal migration. The detection of the DNA of temperate seabass and flathead grey mullet at sites upstream of the dam closest to the river mouth indicated successful upstream migration of these species via a fish ladder bypassing the dam. On the other hand, DNA of these two species was not detected from the upstream side of the two remaining dams, which are not equipped with fish ladders. Ayu is the only species among the three target species with a land-locked population in Lake Biwa located at the headwater of Yodo River. Ayu DNA was detected at most of the sites in the freshwater area during the warm months; however, in the coldest month of February, eDNA was only detected in the uppermost site of Yodo River at the southern tip of Lake Biwa. The eDNA we detected at this site suggests that it was derived from juvenile ayu spending their winter months in the lake. These results suggest that the eDNA analysis presented here can accurately track the seasonal migration of fishes in a river, demonstrating its application as an indicator of habitat connectivity for fishes in association with man-made barriers in a river. The sampling of eDNA involves merely scooping a tank full of water; therefore, it is a simple, rapid, and cost-effective method for long-term monitoring of habitat connectivity associated with the construction of barriers in a river.     

Identifying spawning sites and other critical habitat in lotic systems using eDNA “snapshots”: A case study using the sea lamprey Petromyzon marinus L.

Many aquatic species of conservation concern exist at low densities and are inherently difficult to detect or monitor using conventional methods. However, the introduction of environmental (e)DNA has recently transformed our ability to detect these species and enables effective deployment of limited conservation resources. Identifying areas for breeding, as well as the ecological distribution of species, is vital to the survival or recovery of a conservation species (i.e., areas of critical habitat). In many species, spawning events are associated with a higher relative abundance of DNA released within an aquatic system (i.e., gametes, skin cells etc.), making this the ideal time to monitor these species using eDNA techniques. This study aims to examine whether a “snapshot” eDNA sampling approach (i.e., samples taken at fixed points in chronological time) could reveal areas of critical habitat including spawning sites for our target species Petromyzon marinus. We utilized a species‐specific qPCR assay to monitor spatial and temporal patterns in eDNA concentration within two river catchments in Ireland over three consecutive years. We found that eDNA concentration increased at the onset of observed spawning activity and patterns of concentration increased from downstream to upstream over time, suggesting dispersal into the higher reaches as the spawning season progressed. We found P. marinus to be present upstream of several potential barriers to migration, sometimes in significant numbers. Our results also show that the addition of a lamprey‐specific fish pass at an “impassable” weir, although assisting in ascent, did not have any significant impact on eDNA concentration upstream after the pass had been installed. eDNA concentration was also found to be significantly correlated with both the number of fish and the number of nests encountered. The application of snapshot sampling techniques for species monitoring therefore has substantial potential for the management of low‐density species in fast‐moving aquatic systems.     

Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.     

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.     

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.     

Detection of Adult Green Sturgeon Using Environmental DNA Analysis

Environmental DNA (eDNA) is an emerging sampling method that has been used successfully for detection of rare aquatic species. The Identification of sampling tools that are less stressful for target organisms has become increasingly important for rare and endangered species. A decline in abundance of the Southern Distinct Population Segment (DPS) of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning Green Sturgeon in the Central Valley are effective at monitoring fish densities in concentrated pool habitats, results do not scale well to the watershed level, providing limited spatial and temporal context. Unlike most traditional survey methods, environmental DNA analysis provides a relatively quick, inexpensive tool that could efficiently monitor the presence and distribution of aquatic species. We positively identified Green Sturgeon DNA at two locations of known presence in the Sacramento River, proving that eDNA can be effective for monitoring the presence of adult sturgeon. While further study is needed to understand uncertainties of the sampling method, our study represents the first documented detection of Green Sturgeon eDNA, indicating that eDNA analysis could provide a new tool for monitoring Green Sturgeon distribution in the Central Valley, complimenting traditional on-going survey methods.     

Investigating the Potential Use of Environmental DNA (eDNA) for Genetic Monitoring of Marine Mammals

The exploitation of non-invasive samples has been widely used in genetic monitoring of terrestrial species. In aquatic ecosystems, non-invasive samples such as feces, shed hair or skin, are less accessible. However, the use of environmental DNA (eDNA) has recently been shown to be an effective tool for genetic monitoring of species presence in freshwater ecosystems. Detecting species in the marine environment using eDNA potentially offers a greater challenge due to the greater dilution, amount of mixing and salinity compared with most freshwater ecosystems. To determine the potential use of eDNA for genetic monitoring we used specific primers that amplify short mitochondrial DNA sequences to detect the presence of a marine mammal, the harbor porpoise, Phocoena phocoena, in a controlled environment and in natural marine locations. The reliability of the genetic detections was investigated by comparing with detections of harbor porpoise echolocation clicks by static acoustic monitoring devices. While we were able to consistently genetically detect the target species under controlled conditions, the results from natural locations were less consistent and detection by eDNA was less successful than acoustic detections. However, at one site we detected long-finned pilot whale, Globicephala melas, a species rarely sighted in the Baltic. Therefore, with optimization aimed towards processing larger volumes of seawater this method has the potential to compliment current visual and acoustic methods of species detection of marine mammals.