Embracing environmental DNA? How values influence the integration of a new technology into an oceanographic expedition.

During the past decade, environmental DNA (eDNA) methodology has become an important non-invasive tool to monitor aquatic micro- and macro-organisms, including freshwater crayfish. In Europe, noble crayfish Astacus astacus is the most widespread native freshwater crayfish. However, the species is threatened in its entire distribution range. It is therefore included on the International Union for Conservation Nature (IUCN) red list, and on several national red lists. Reliable monitoring is essential for implementation of conservation measures. For crayfish, traditional population trends have been obtained from catch per unit effort (CPUE) data. In order to successfully apply and use eDNA monitoring for noble crayfish, or any species, it is a prerequisite to know the strengths and weaknesses of the applied methods and how they perform compared to traditional methodology. Sampling strategy and analysis methodology also depends on choice of species to be monitored, and which questions to be answered. Further, refinement of the employed methods may improve the detection probability for eDNA monitoring. Here we report the results from 1) a recently published study on noble crayfish eDNA monitoring (Johnsen et al. 2020) and 2) an ongoing study comparing and optimising the methods used for monitoring noble crayfish. 1) We compared eDNA monitoring (transects with ten 5L samples) with traditional trapping (transects with 50 traps) for noble crayfish in lentic habitats, in order to evaluate detection probability and if eDNA concentration correlates with relative density of crayfish. We also compared two commonly used analytical methods [quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR)] for eDNA monitoring. We found that qPCR outperformed ddPCR in detection frequency (Fig. 1), most likely due to some inhibition in the ddPCR analysis. eDNA monitoring provided reliable presence/absence data for noble crayfish, even in lakes with very low crayfish densities. Detection frequency increased with increasing CPUE (Fig. 1). However, we did not observe any correlation between relative crayfish densities and eDNA concentrations of crayfish. eDNA concentrations were consistently very low, even in lakes with very high crayfish densities. For lakes with very low crayfish densities, we estimated that ~5 samples (5L samples) are needed for 95 % detection likelihood, while for lakes with high densities 2 samples were needed. 2) We compared two eDNA sampling strategies (sampling from bottom or the surface), commonly used for crayfish or fish in Norway to investigate how both strategies perform. The sampled filters were divided and two DNA extraction protocols were evaluated (CTAB based vs Column based). We found that the DNA yield was higher from the column based DNA extraction protocol, and that eDNA concentrations from fish (brown trout Salmon trutta, northern pike Esox lucius and European perch Perca fluviatilis) were significantly higher than for crayfish. For crayfish and brown trout, there was little difference between detection probability for bottom and surface samples, while for northern pike and European perch the detection probability was higher for the bottom samples. Currently, we are analysing eDNA samples collected with glass fibre filters and NatureMetrix filters for noble crayfish in both lentic and lotic habitats and the preliminary results will be presented. We conclude that eDNA monitoring cannot substitute CPUE monitoring for freshwater crayfish, but it offers reliable presence-absence data, provided sufficient sampling efforts. Thus, it is suitable for large scale monitoring of threatened crayfish and combined with eDNA analysis of alien crayfish and diseases such as crayfish plague, this is a cost-efficient supplement offering a more holistic approach for aquatic environments and native crayfish conservation. Furthermore, the synergy effect of using collected eDNA samples from different projects to monitor additional species is substantial.

Environmental DNA (eDNA) extracted from water samples has recently shown potential as a valuable source of population genetic information for aquatic macroorganisms. This approach offers several potential advantages compared with conventional tissue‐based methods, including the fact that eDNA sampling is noninvasive and generally more cost‐efficient. Currently, eDNA approaches have been limited to single‐marker studies of mitochondrial DNA (mtDNA), and the relationship between eDNA haplotype composition and true haplotype composition still needs to be thoroughly verified. This will require testing of bioinformatic and statistical software to correct for erroneous sequences, as well as biases and random variation in relative sequence abundances. However, eDNA‐based population genetic methods have far‐reaching potential for both basic and applied research. In this paper, we present a brief overview of the achievements of eDNA‐based population genetics to date, and outline the prospects for future developments in the field, including the estimation of nuclear DNA (nuDNA) variation and epigenetic information. We discuss the challenges associated with eDNA samples as opposed to those of individual tissue samples and assess whether eDNA might offer additional types of information unobtainable with tissue samples. Lastly, we provide recommendations for determining whether an eDNA approach would be a useful and suitable choice in different research settings. We limit our discussion largely to contemporary aquatic systems, but the advantages, challenges, and perspectives can to a large degree be generalized to eDNA studies with a different spatial and temporal focus.

There is increasing interest in the health of waterways in Aotearoa New Zealand. The National Policy Statement for Freshwater Management 2020 (NPS-FM) places a greater onus on resource managers to report on the state of freshwater ecosystem health, including fish. Routine fish monitoring is time intensive and is principally conducted in ‘wadeable’ stream reaches. In comparison, environmental DNA (eDNA) sampling is a relatively recent and rapid technique that likely detects fish and other vertebrate and invertebrate taxa over a broader spatial scale. In this study, we present data for five wadeable streams comparing diversity derived from multi-year standardised electrofishing with eDNA samples collected from the same reaches. Further, we explore whether the number of eDNA monitoring ‘reads’ for species at a site may provide an approximate (semi-quantitative) indication of their ‘near field’ relative abundance based on one-pass electrofishing captures. Results indicated that while some aspects of the methodology require fine-tuning, eDNA sampling shows substantial promise for complimenting state of the environment (SOE) reporting for describing fish diversity in wadeable streams. The use of aquatic eDNA monitoring to provide a cost-effective indication of broader catchment scale biodiversity (e.g. birds and exotic pests) in wadeable and non-wadeable streams is also discussed.

Fungal diversity drives key processes in terrestrial ecosystems, but remains challenging to measure in the field and to monitor over time. In particular, we lack methods capable of describing both the regional biotas and fungal related conservation values, such as habitats for rare and threatened fungi. Environmental DNA (eDNA) could serve this purpose, once its validity to capture fungal assemblage properties is established. Using three approaches (full‐season eDNA sampling from air, snapshot visual surveys, and eDNA sampling from dead wood), we present the first comparative assessment of fungal diversity at forest stand scale. The focus was on two well‐known fungal groups (lichens and wood inhabiting basidiomycetes) in old hemiboreal forests. Aerial eDNA revealed regional‐scale cryptic diversity not detected in visual surveys, particularly among lichens and including species of conservation concern. However, at the local population scale, both aerial eDNA and eDNA sampled from dead wood often missed visually detected species. In particular, eDNA sampled from dead wood performed only as a supplementary source of species data. The methods differed in detecting the spatial covariance of wood fungal and lichen diversity, with the combined (most complete) dataset indicating the strongest congruence. Synthesis and applications. Neither eDNA approaches nor visual surveys were able to detect full diversity of local fungi and all species of specific conservation value. However, there is clear merit in combined use of these methods, particularly in pairing aerial eDNA with visual observation‐based surveys on selected taxon groups. This approach can be especially valuable in fungal groups that have challenging taxonomy, inhabit partly inaccessible microhabitats, or have overall high diversity (such as lichens).

Advances in environmental DNA (eDNA) methodologies have led to improvements in the ability to detect species and communities in aquatic environments, yet the majority of studies emphasize biological diversity at the species level by targeting variable sites within the mitochondrial genome. Here, we demonstrate that eDNA approaches also have the capacity to detect intraspecific diversity in the nuclear genome, allowing for assessments of population‐level allele frequencies and estimates of the number of genetic contributors in an eDNA sample. Using a panel of microsatellite loci developed for the round goby (Neogobius melanostomus), we tested the similarity between eDNA‐based and individual tissue‐based estimates of allele frequencies from experimental mesocosms and in a field‐based trial. Subsequently, we used a likelihood‐based DNA mixture framework to estimate the number of unique genetic contributors in eDNA samples and in simulated mixtures of alleles. In both mesocosm and field samples, allele frequencies from eDNA were highly correlated with allele frequencies from genotyped round goby tissue samples, indicating nuclear markers can be reliably amplified from water samples. DNA mixture analyses were able to estimate the number of genetic contributors from mesocosm eDNA samples and simulated mixtures of DNA from up to 58 individuals, with the degree of positive or negative bias dependent on the filtering scheme of low‐frequency alleles. With this study we document the application of eDNA and multiple amplicon‐based methods to obtain intraspecific nuclear genetic information and estimate the absolute abundance of a species in eDNA samples. With proper validation, this approach has the potential to advance noninvasive survey methods to characterize populations and detect population‐level genetic diversity.

BackgroundSchistosomiasis is a neglected tropical parasitic disease associated with severe pathology, mortality and economic loss worldwide. Programs for disease control may benefit from specific and sensitive diagnostic methods to detect Schistosoma trematodes in aquatic environments. Here we report the development of novel environmental DNA (eDNA) qPCR assays for the presence of the human-infecting species Schistosoma mansoni, S. haematobium and S. japonicum.Methodology/Principal findingsWe first tested the specificity of the assays across the three species using genomic DNA preparations which showed successful amplification of target sequences with no cross amplification between the three focal species. In addition, we evaluated the specificity of the assays using synthetic DNA of multiple Schistosoma species, and demonstrated a high overall specificity; however, S. japonicum and S. haematobium assays showed cross-species amplification with very closely-related species. We next tested the effectiveness of the S. mansoni assay using eDNA samples from aquaria containing infected host gastropods, with the target species revealed as present in all infected aquaria. Finally, we evaluated the effectiveness of the S. mansoni and S. haematobium assays using eDNA samples from eight discrete natural freshwater sites in Tanzania, and demonstrated strong correspondence between infection status established using eDNA and conventional assays of parasite prevalence in host snails.Conclusions/SignificanceCollectively, our results suggest that eDNA monitoring is able to detect schistosomes in freshwater bodies, but refinement of the field sampling, storage and assay methods are likely to optimise its performance. We anticipate that environmental DNA-based approaches will help to inform epidemiological studies and contribute to efforts to control and eliminate schistosomiasis in endemic areas.

Objective Environmental DNA (eDNA) metabarcoding has become an important method for inventorying and monitoring biota in aquatic systems. The Texas Parks and Wildlife Department conducts regular fishery-independent sampling of biotic communities using traditional sampling gears, such as gill nets and bag seines, in all of the major estuaries of adjacent to the Gulf of Mexico in Texas. Previous studies have shown that eDNA approaches can complement traditional sampling methods. Methods We compared fish community structure data in the Cedar Lakes estuary system obtained with traditional sampling gears with data obtained using eDNA sampling using a small sequence of mitochondrial 12S ribosomal RNA gene and a validated taxonomic reference file. Result For spring and fall of 2022, eDNA metabarcoding detected a larger number of species than either bag seines or gill nets. Species richness detected via eDNA in two seasons in a single year was comparable with the species richness of agency’s historical record based on traditional gears for Cedar Lakes. Conclusion Seasonal and spatial variation in species richness was similar between traditional and eDNA sampling; however, eDNA metabarcoding allowed detection of several species that would be difficult or impossible to capture with either bag seines or gill nets. We observed two limitations of eDNA metabarcoding. Read depth was not a good index of relative abundance, which limits our ability to infer relative biomass using single samples. Secondly, we observed detection bias in our eDNA results. Specifically, eDNA failed to detect two species of elasmobranchs present when water sampling was performed and eDNA also performed poorly compared to traditional sampling gears for some species of bony fishes. Despite these limitations, eDNA metabarcoding proved to be an efficient and cost-effective alternative and compliment to traditional fisheries sampling gears for fishery-independent monitoring of community structure and composition in estuaries of the Gulf of Mexico.

One can extract DNA from any environmental sample irrespective of the organism i.e., Soil, Water, Air. This DNA is identified as environmental DNA or eDNA. The application of the novel eDNA approaches, particularly NGS techniques, has evolved biodiversity surveys taking into account both the budget and the time. eDNA has revolutionized our thinking about biogeography. Results obtained from eDNA approaches have given some crucial insights into the study of ancient environments that are useful in the sustainable management of contemporary biodiversity in aquatic and terrestrial ecosystems. Advancements in eDNA technologies also enhance the knowledge of molecular ecology and make it possible to answer different ecological questions by using genetic methods.

The creation of Marine Protected Areas (MPAs) is central to the European Union’s strategy for protecting and restoring marine environments. However, current research indicates that monitoring of MPAs is insufficient to ensure their protective role. In response, the European Commission encourages the development and adoption of more effective monitoring tools for MPAs. Environmental DNA (eDNA) methods may serve this function, but their use in MPA monitoring and management remains relatively unexplored. The current study addresses this gap by examining the potential for eDNA to enhance monitoring within Mediterranean MPAs, where monitoring challenges are especially severe. We addressed the gap by synthesizing the results of two analyses. First, a systematic literature review examined existing applications of eDNA metabarcoding to monitor marine habitats. From the experiences documented in the reviewed literature, we applied a SWOT analysis (a framework used to identify and analyze strengths, weaknesses, opportunities and threats) to assess the tool’s appropriateness for EU monitoring requirements. Second, we probed the tool’s suitability for Mediterranean MPAs with a questionnaire on monitoring and other challenges completed by the managers of 29 MPAs in the region. The findings of our SWOT analysis demonstrated that when compared to conventional monitoring methods, eDNA frequently outperformed them or acted as a crucial supplement. We also found that eDNA is suitable for most criteria set forth in EU requirements. Our questionnaire revealed monitoring strategy limitations that eDNA approaches may alleviate. Still, eDNA approaches do have drawbacks which may jeopardize its accuracy and sufficiency. Incorporating eDNA monitoring methods has great potential to enhance protection efforts in MPAs in the Mediterranean and elsewhere. Their successful adoption will require improved coordination among stakeholders and current, comprehensive, and customized recommendations for MPA managers. This will improve the overall functioning of MPA networks and thus support the role of MPAs in safeguarding vulnerable marine environments.

[This corrects the article DOI: 10.1371/journal.pone.0162277.].

BackgroundOver the past decade, environmental DNA (eDNA) has become a resourceful tool in conservation and biomonitoring. Environmental DNA has been applied in a variety of environments, but the application to studies of marine fish, particularly at tropical latitudes, are limited. Since many commercially important Caribbean fishes are overexploited, these species are optimal candidates to explore the use of this method as a biomonitoring tool. Specifically, for many of these species, the formation of fish spawning aggregations (FSAs) marks a critical life history event where fishes will gather in large numbers for reproduction. These FSAs are ephemeral in nature, lasting only a few days, but are predictable in time and space which makes them susceptible to overfishing.MethodsIn this study, we test the feasibility of using an eDNA sampling approach (water and sediment collection) to detect the presence of known FSAs off the west coast of Puerto Rico, with cytochrome c oxidase subunit 1 (CO1) and 12S rRNA (12S) primers designed to target specific species. A total of 290 eDNA samples were collected and, of those, 206 eDNA samples were processed. All eDNA samples varied in DNA concentration, both between replicates and collection methods. A total of 12 primer sets were developed and tested using traditional PCR and qPCR.ResultsDespite validation of primer accuracy and sample collection during known peak spawning times, the use of traditional PCR and qPCR with both molecular markers failed to produce species-specific amplification. Thus, a trial test was conducted using the CO1 primers in which target fish DNA was ‘spiked’ at various concentrations into the respective eDNA samples to determine the target species DNA concentration limit of detection. Upon successful amplification of the trial, results indicated that eDNA samples were below the detection threshold of our methods, suggesting that the number of fish present at the spawning aggregations was inadequate for single-species detection methods. In addition, elements such as the unavoidable presence of non-target DNA, oceanic environmental conditions, shedding rates of target fish, among other biotic and abiotic factors could have affected DNA persistence and degradation rates at the sites.ConclusionWe provide recommendations for species-specific fish detection in lower latitudes, and suggestions for studies aiming to monitor or detect fish spawning aggregations using eDNA sampling.

Analysis of environmental DNA (eDNA) has gained widespread usage for taxonomically based biodiversity assessment. While interest in applying noninvasive eDNA monitoring for population genetic assessments has grown, its usage in this sphere remains limited. One barrier to uptake is that the effectiveness of eDNA detection below the species level remains to be determined for multiple species and environments. Here, we test the utility of this emergent technology to obtain within‐species haplotypic variation of New Zealand (NZ) blackfoot pāua ( Haliotis iris) . We compare mitochondrial haplotype diversity recovered from marine eDNA samples against traditional tissue samples of blackfoot pāua collected at the same NZ coastal site. Targeting the ATP8‐ATP6 region, we recovered four mitochondrial haplotypes from eDNA versus six haplotypes from tissue samples. Three common haplotypes were recovered with both eDNA and tissue samples, while only one out of three rare haplotypes – represented in tissue samples by one individual each – was recovered with our eDNA methods. We demonstrate that eDNA monitoring is an effective tool for recovering common genetic diversity from pāua, although rare (<5%) haplotypes are seldom recovered. Our results show the potential of eDNA to identify population‐level haplotypic diversity for gastropods in the marine environment below the species level. This work demonstrates that eDNA methods can be an effective, noninvasive tool for genetic monitoring. Noninvasive eDNA sampling could minimize target organism stress and human interaction enabling population genetic research for hard‐to‐sample, delicate, or sensitive species.

eDNA, also known as environmental DNA, has garnered significant attention due to its potential applications in various fields such as biodiversity assessment, species distribution monitoring, ecological interaction analysis, and quantitative analysis. However, the presence of non-selective DNA signals in eDNA samples poses challenges in accurately detecting species, assessing biodiversity, and conducting quantitative analysis. To address these limitations, this study developed a novel method for selectively detecting iDNA from specific species in eDNA samples. The method involved the application of PMA treatment to Alexandrium spp. effectively preventing the detection of non-selective exDNA signals. Additionally, by optimizing the filter size used in the sampling process, the researchers were able to selectively collect and analyze iDNA from species of interest, particularly Alexandrium spp. Furthermore, the study successfully demonstrated the selective collection and analysis of iDNA from Alexandrium spp. cysts present in the sediment layer, further strengthening the findings. The results indicated that the combined use of PMA treatment and filter size optimization significantly enhanced the selective detection capability of iDNA. The successful selective detection of iDNA from eDNA in the sediment layer highlights the practical applicability of the developed method. This study holds promise for advancing eDNA monitoring technology by providing a selective iDNA detection method utilizing PMA. Moreover, these findings lay the foundation for effectively utilizing iDNA in environmental conservation, monitoring, and ecological research.

Conservation and management of marine biodiversity depends on biomonitoring of marine habitats, but current approaches are resource‐intensive and require different approaches for different organisms. Environmental DNA (eDNA) extracted from water samples is an efficient and versatile approach to detecting aquatic animals. In the ocean, eDNA composition reflects local fauna at fine spatial scales, but little is known about the effectiveness of eDNA‐based monitoring of marine communities at larger scales. We investigated the potential of eDNA to characterize and distinguish marine communities at large spatial scales by comparing vertebrate species composition among marine habitats in Qatar, the Arabian Gulf (also known as the Persian Gulf), based on eDNA metabarcoding of seawater samples. We conducted species accumulation analyses to estimate how much of the vertebrate diversity we detected. We obtained eDNA sequences from a diverse assemblage of marine vertebrates, spanning 191 taxa in 73 families. These included rare and endangered species and covered 36% of the bony fish genera previously recorded in the Gulf. Sites of similar habitat type were also similar in eDNA composition. The species accumulation analyses showed that the number of sample replicates was insufficient for some sampling sites but suggested that a few hundred eDNA samples could potentially capture >90% of the marine vertebrate diversity in the study area. Our results confirm that seawater samples contain habitat‐characteristic molecular signatures and that eDNA monitoring can efficiently cover vertebrate diversity at scales relevant to national and regional conservation and management.

Species monitoring using environmental DNA (eDNA) is a powerful new technique for natural resource scientists and the number of research groups employing eDNA detection is growing rapidly. However, current eDNA sampling technologies consist mainly of do‐it‐yourself solutions, and the lack of purpose‐built sampling equipment is limiting the efficiency and standardization of eDNA studies. Here, we describe the first fully integrated sampling system (Smith‐Root eDNA Sampler) designed by a team of molecular ecologists and engineers for high‐throughput eDNA sample collection. It consists of a backpack portable pump that integrates sensor feedback, a pole extension with remote pump controller, custom‐made filter housings in single‐use packets for each sampling site and onboard sample storage. The system is optimized for sampling speed and replicability, while minimizing risk of contamination. We present an example pilot study designed to identify optimal eDNA Sampler system parameter values (i.e. pump pressure, flow rate, filter pore size, sample volume) in a new sampling environment. We identified a peak in filtration efficiency at a flow rate threshold of 1.0 L/m, and found that 5 μm filters captured significantly more target eDNA than 1 μm filters. Results also suggest that high filtration pressures may reduce eDNA retention, which implies that pressure should be standardized to avoid biasing detection data. Similar to the technological evolution of backpack electrofishers, eDNA sampling technology is in the process of transitioning from a nascent phase to professionally engineered research tools. Such innovations will be essential as eDNA monitoring becomes one of the industry standard methods used for species detection and management.