SPECIES DETECTION: MOLLUSCS
BIVALVES
Currier C. A., Morris T. J., Wilson C. C., Freeland J. R. (2018): Validation of environmental DNA (eDNA) as a detection tool for at‐risk freshwater pearly mussel species (Bivalvia: Unionidae). Aquatic Conservation: Marine and Freshwater Ecosystems 28: 545-558.
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Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms. Previous eDNA studies have yet to develop species‐specific markers that target taxa with many potentially sympatric confamilials. Forty‐one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging owing to morphological crypsis and species scarcity. Species‐specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling. The paired sampling design showed that species‐specific markers can be designed even within speciose families, and that eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.
Dysthe J. C., Rodgers T., Franklin T. W., Carim K. J., Young M. K., McKelvey K. S., Mock K. E., Schwartz M. K. (2018): Repurposing environmental DNA samples—detecting the western pearlshell (Margaritifera falcata) as a proof of concept. Ecology and Evolution 8: 2659-2670.
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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.
Gasparini L., Crookes S., Prosser R. S., Hanner R. (2020): Detection of freshwater mussels (Unionidae) using environmental DNA in riverine systems. Environmental DNA 2: 321-329.
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Environmental DNA (eDNA) methods are being developed for use in conservation biology to improve upon conventional species survey techniques. Validation of eDNA methods in different environmental contexts is required if they are to be widely adopted. One potential application of eDNA methods is for the detection of freshwater mussels (Bivalvia: Unionidae), which are among the most imperiled species in North America. Conventional unionid survey methods are highly invasive and can be difficult to conduct due to issues with morphological identification and their cryptic use of habitat. eDNA methods can potentially provide a non-invasive, extremely specific, and highly sensitive alternative. Here, we examine the effectiveness of eDNA methods at detecting an imperiled unionid, the wavy-rayed lampmussel (Lampsilis fasciola), in lotic systems with moderate discharge. We developed a novel qPCR assay for the detection of L. fasciola eDNA, which included a custom internal positive control to check for PCR inhibition. We used different experimental densities of caged L. fasciola specimens as a point source of eDNA within two rivers of the Grand River watershed in Southern Ontario. Sampling occurred at set distances downstream of the cage using purpose-built sampling equipment. Detection was obtained at the cage (i.e., point of eDNA shedding) but not downstream at distances ≥10 m during stream discharges of approximately 1,632–2,332 L/s. The results indicate that eDNA is diluted rapidly in rivers with moderate discharge and that high-resolution spatial sampling efforts may be necessary to obtain meaningful eDNA-based distribution data of unionids, and other sessile organisms, present at low density in lotic systems.
Prié V., Valentini A., Lopes-Lima M., Froufe E., Rocle M., Poulet N., Taberlet P., Dejean T. (2020): Environmental DNA metabarcoding for freshwater bivalves biodiversity assessment: methods and results for the Western Palearctic (European sub-region). Hydrobiologia 848: 2931-2950.
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Freshwater ecosystems are the most vulnerable worldwide and freshwater bivalves rank amongst the most threatened animals in the world. Surveying and monitoring freshwater bivalves are difficult tasks: they are difficult to find, hard to identify (taxonomic expertise is needed), and working underwater is technically challenging. It is therefore crucial to find more efficient methods to survey and monitor these species. Here, we present the first metabarcoding approach for freshwater bivalves and compare environmental DNA (eDNA) and traditional surveys. We describe two sets of primers (for Unionida and Venerida) developed for freshwater bivalves eDNA metabarcoding. These primers have been tested in the field, with about 300 studied sites. Results were compared to freshwater bivalves’ surveys using traditional methods, with eDNA always detecting more species than traditional surveys, especially when Sphaerids were taken into account. While our study initially focused on Western Palearctic freshwater bivalve species, our primers were confronted in silico with available sequences and have proven to be effective at a global scale. The results show that eDNA metabarcoding, with our developed primers, is a remarkable tool allowing for non-invasive surveys, detection of rare and inconspicuous species, absence data and overall freshwater bivalves routine monitoring.
Coghlan S. A., Currier C. A., Freeland J., Morris T. J., Wilson C. C. (2021): Community eDNA metabarcoding as a detection tool for documenting freshwater mussel (Unionidae) species assemblages. Environmental DNA 3: 1172-1191.
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Documenting species occurrences and habitat occupancy of unionid mussels can often be challenging. Environmental DNA (eDNA) has been shown to be a reliable tool for detecting unionids with comparable or greater sensitivity than conventional sampling and has the added advantages of not disturbing individuals or occupied habitats. However, single-species eDNA assays are limited to targeting individual species of interest and are functionally blind to the presence of other species. Community eDNA assays have the potential to characterize local species assemblages simultaneously but are currently less extensively developed and implemented than single-species eDNA testing. We tested the effectiveness of community eDNA markers to identify unionid species assemblages, using two overlapping conserved primers that target the maternal mitochondrial 16S rDNA region. Both primer sets were optimized using three mock communities and successfully amplified 62.5%–81.6% of species with largely consistent results between the primer sets. Following optimization, eDNA from water samples from 24 reference sites with known mussel communities was amplified and sequenced to quantify species richness and diversity within and among sites. Metabarcoding results from the monitoring sites largely mirrored those from the mock communities, with >80% of species detections identified by both assays. The results were broadly consistent with species data from quadrat-based manual field surveys, although both community eDNA and conventional sampling detected some species that the other method did not. These results demonstrate that community eDNA assays using conserved primers and next-generation sequencing have the potential to simultaneously target eDNA from multiple unionid species and provide a powerful tool for complementing or augmenting conventional field surveys to characterize and monitor unionid species assemblages.
Klymus K. E., Richter C. A., Thompson N., Hinck J. E., Jones J. W. (2021): Metabarcoding assays for the detection of freshwater mussels (Unionida) with environmental DNA. Environmental DNA 3: 231-247.
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Freshwater mussels of the order Unionida are a widely distributed taxon that are important in maintaining freshwater ecosystems and are also highly imperiled throughout the world. Monitoring of mussel populations with environmental DNA (eDNA) is an attractive alternative to traditional methods because it is noninvasive and requires less labor and taxonomic knowledge from field personnel. We developed eDNA metabarcoding assays specific to freshwater mussels and tested them at six sites in the Clinch River, located in the southeastern United States. Our objective was to determine the utility of eDNA metabarcoding for future monitoring of mussel populations and restoration efforts in this watershed. Two metabarcoding assays that target the mitochondrial DNA regions of the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit (ND1) genes were developed and tested. Our assays appear to be order specific, amplifying members from the two families found in North America, Unionidae and Margaritiferidae, while not amplifying nontarget fish or other bivalve species. From the field collected samples, our assays together detected 19 species, eight of which are listed as federally endangered. The assays also detected 42%, 58%, and 54% of the species identified by recent quantitative visual mussel surveys at three sampling sites. Increased sampling effort by processing a greater water volume or number of samples will likely increase species detections. These eDNA metabarcoding assays may enable enhanced monitoring of freshwater mussel assemblages and subsequently inform conservation efforts.
Preece E. P., Bryan M., Mapes S. M., Wademan C., Dorazio R. (2021): Monitoring for freshwater mussel presence in rivers using environmental DNA. Environmental DNA 3: 591-604.
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In 2013, the U.S. Environmental Protection Agency (USEPA) updated its national recommended water quality criteria for the protection of aquatic life from the toxic effects of ammonia in freshwaters. From its updated national dataset, USEPA determined freshwater mussels in the family Unionidae were the most sensitive taxa to ammonia. Thus, it has become necessary to determine whether freshwater mussels exist in waterbodies near discharge locations from Publically Owned Treatment Works (POTWs), to ensure that POTWs are regulated appropriately with regard to ammonia levels in their treated effluent. However, because mussels are notoriously difficult to detect using traditional survey methodologies, most POTWs have not determined whether freshwater mussels exist in their receiving waters. Environmental DNA (eDNA) presents a promising methodology to survey for freshwater mussels in POTW receiving waters. We developed and validated a quantitative polymerase chain reaction (qPCR) assay for the three freshwater mussel taxa that are currently known to exist in California’s Central Valley. We then placed a cage with 20 Gonidea angulata into a riverine environment and collected water samples at evenly spaced downstream intervals to determine how habitat type and distance from the caged mussels affected eDNA detection. Using two-stage occupancy modeling, we determined higher detection probabilities occur in riffle habitats compared to pool or run habitats. We also found surface samples work well for collecting freshwater mussel eDNA in riffle habitats, but are less likely to capture mussel eDNA in pools or runs. The maximum downstream distance at which the eDNA from the 20 placed G. angulata mussels was detected was 8 km. Our study demonstrates that eDNA is a reliable tool for determining freshwater mussel presence in riverine systems, but that sampling should consider habitat type, distance between sampling locations, and time of year for the most effective results.
Coutts A., O’Brien A., Weeks A. R., Swearer S. E., van Rooyen A., Branigan S., Morris R. L. (2022): An environmental DNA approach to informing restoration of the functionally extinct oyster, Ostrea angasi. Aquatic Conservation: Marine and Freshwater Ecosystems 32: 1732-1744.
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The success of oyster reef restoration can be enhanced by data on the distribution of remnant populations to inform the selection of suitable restoration locations. A quantitative polymerase chain reaction-based environmental DNA (eDNA) assay was designed to provide distribution data for the oyster, Ostrea angasi, whose reefs are functionally extinct in Port Phillip Bay, Australia. Ostrea angasi eDNA accumulation and decay was measured in aquaria containing oysters in low and high densities, prior to testing the efficacy of the eDNA approach for detection of oysters at 15 field sites. Ostrea angasi eDNA accumulated significantly faster in aquaria where more individuals were present, while eDNA became undetectable 2–6 days after oysters were removed in low-density treatments. The eDNA samples were successful at detecting O. angasi in the field when taken in close proximity of an oyster population. Increasing the sample number and volume could maximize oyster detection, demonstrating the potential of eDNA to identify suitable sites for the restoration of functionally extinct marine ecosystems.
Egeter B., Veríssimo J., Lopes‐Lima M., Chaves C., Pinto J., Riccardi N., Beja P., Fonseca N. A. (2022): Speeding up the detection of invasive bivalve species using environmental DNA: A Nanopore and Illumina sequencing comparison. Molecular Ecology Resources 22: 2232-2247.
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Traditional detection of aquatic invasive species via morphological identification is often time-consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina-based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and substantially decrease sample turnaround times. However, nanopore-sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools (msi package) to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves while comparing it with Illumina-based sequencing. We sampled water from sites with pre-existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. Samples were extracted, amplified, and sequenced by the two platforms. The mean agreement between sequencing methods was 69% and the difference between methods was nonsignificant. The lack of detections of some species at some sites could be explained by their known low abundances. This is the first reported use of MinION to detect aquatic invasive species from eDNA samples.
Steiner K., Dyer N., Lee C. K., Vandergoes M. J., Wood S. A. (2022): Development of a triplex droplet digital polymerase chain reaction assay for the detection of three New Zealand native freshwater mussels (Echyridella) in environmental samples. Environmental DNA 4: 1065-1077.
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The freshwater mussel Echyridella (also known as kākahi) is the only freshwater mussel found in New Zealand’s lakes and rivers and is endemic to the country. They play a crucial role in freshwater ecosystems and have high cultural significance. The three putative species of Echyridella are all classified as at risk. Monitoring is required to enhance knowledge on their distribution and to provide management, but conventional approaches (diving, wading, or swimming and visually looking for mussels) are time-consuming, costly, and require good visibility. The use of environmental DNA (eDNA) in combination with PCR techniques offer advantages over conventional methods. In this study, we developed a triplex droplet digital PCR assay, targeting a region of the mitochondrial cytochrome oxidase subunit 1 gene, that differentiates all three kākahi species. The limits of quantification using gblocks were 0.6 × 10−03–6 × 10−03 fg/µl, yielding 2–8 copies/reaction, and for tissue DNA 10,000–100,000 fg/µl, yielding 9–15 copies/reaction. The method was applied to water samples from 48 lakes, lagoons, or ponds and two samples from streams. A total of 58% of samples from lakes, lagoons, or ponds and all samples from the streams were positive for kākahi. Sediment samples (n = 15) were also tested. Only those collected in close proximity (<1 m) to living kākahi were positive highlighting the patchy distribution of eDNA in sediment. This new assay will assist in determining the current distribution of kākahi at large temporal and spatial scales and has the potential to be used for the assessment of historic distributions as well.
Sugawara K., Sasaki Y., Okano K., Watanabe M., Miyata N. (2022): Application of eDNA for monitoring freshwater bivalve Nodularia nipponensis and its glochidium larvae. Environmental DNA 4: 908-919.
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Habitat loss, the introduction of invasive species, and climate change due to human activity have threatened many freshwater unionid bivalves worldwide. Unionid bivalves represent important members of freshwater ecosystems, providing bitterling fish with spawning grounds and contributing to water clarification via the filtration of suspended solids. This study examined an environmental DNA (eDNA) approach for monitoring the unionid bivalve Nodularia nipponensis in a lake environment. We developed oligonucleotide primers for the specific detection of mitochondrial DNA from N. nipponensis and evaluated the decay and release rates of eDNA in a laboratory tank experiment under two bivalve densities (49 and 164 individuals m−2) selected to ensure sufficient eDNA yields. The eDNA release rate was weakly and positively correlated with bivalve density but strongly and positively correlated with inorganic nitrogen and phosphorus excretion from the bivalve. In a eutrophic lake, Lake Hachiro, 67% of the samples (16 out of 24) collected from June to July yielded detectable amounts of eDNA and were not detected at all in May and August (0 out of 20). This difference was probably due to the release of N. nipponensis glochidium larvae. During these months, unionid glochidia adhered to bundles of polyvinylidene chloride fibers placed on the lake bottom for 8 h, confirmed by microscopic inspection. We successfully obtained N. nipponensis eDNA from all bundles examined over the months, demonstrating the efficacy of bundle capture in quantifying unionid bivalve eDNA. Our eDNA-based technique with specific oligonucleotide primers and fiber bundles may be a promising tool for monitoring N. nipponensis in freshwater environments. Fiber bundles are useful for tracing eDNA derived from glochidium larvae, even at low unionid bivalve densities, which may be conducive to conservation efforts.
CEPHALOPODS
Mauvisseau Q., Parrondo M., Fernández M. P., García L., Martínez J. L., García-Vázquez E., Borrell Y. J. (2017): On the way for detecting and quantifying elusive species in the sea: The Octopus vulgaris case study. Fisheries Research 191: 41-48.
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Environmental DNA (eDNA) can be a powerful method for assessing the presence and the distribution of aquatic species. We used this tool in order to detect and quantify eDNA from the elusive species Octopus vulgaris, using qPCRs (SybrGreen protocol). We designed species-specific primers, and set up an experimental aquarium approach to validate the new molecular tool in different controlled conditions. Field validation was conducted from sea water samples taken from 8 locations within an octopus fishery area in the Cantabrian Sea during February–March 2016. A significant positive correlation between the total biomass (g of O. vulgaris within thanks) and the amount of O. vulgaris eDNA detected (p-value = 0.01261) was found in aquarium experiments. The species was also detected by PCR in 7 of the 8 water samples taken at sea, and successfully quantified by qPCR in 5 samples. This preliminary study and innovative method opens very promising perspectives for developing quick and cheap tools for the assessment of O. vulgaris distribution and abundance in the sea. The method could help in a close future for quantifying unseen and elusive marine species, thus contributing to establish sustainable fisheries.
Wada T., Doi H., Togaki D., Kaida R., Nagano M., Katano I., Suzuki M., Ohtani T., Mitsuhashi H. (2020): Exploring a legendary giant squid: An environmental DNA approach. Marine Biology 167: 160.
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The giant squid, Architeuthis dux, has been reported for centuries, but its distribution and ecology have remained famously mysterious. We investigated the A. dux distribution in the Sea of Japan using the environmental DNA (eDNA) method, which has recently been considered as a useful technique for evaluating the distributions of rare species. To develop the eDNA method to detect A. dux, we first created a species-specific polymerase chain reaction (PCR) primer–probe and tried to detect the eDNA in the western region of the Sea of Japan where A. dux has recently been observed. We successfully collected and amplified the A. dux eDNA in our study area using field water sampling and real-time PCR measurements. A. dux eDNA was detected in winter but not in summer, reflecting the historical record of A. dux observations in the region. The use of eDNA techniques could be a potential method for monitoring “invisible” and rare organisms, even in open ocean habitats.
GASTROPODS
Goldberg C. S., Sepulveda A., Ray A., Baumgardt J., Waits L. P. (2013): Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshwater Science 32: 792-800.
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Early detection of aquatic invasive species is a critical task for management of aquatic ecosystems. This task is hindered by the difficulty and cost of surveying aquatic systems thoroughly. The New Zealand mudsnail (Potamopyrgus antipodarum) is a small, invasive parthenogenic mollusk that can reach very high population densities and severely affects ecosystem functioning. To assist in the early detection of this invasive species, we developed and validated a highly sensitive environmental deoxyribonucleic acid (eDNA) assay. We used a dose–response laboratory experiment to investigate the relationship between New Zealand mudsnail density and eDNA detected through time. We documented that as few as 1 individual in 1.5 L of water for 2 d could be detected with this method, and that eDNA from this species may remain detectable for 21 to 44 d after mudsnail removal. We used the eDNA method to confirm the presence of New Zealand mudsnail eDNA at densities as low as 11 to 144 snails/m2 in a eutrophic 5th-order river. Combined, these results demonstrate the high potential for eDNA surveys to assist with early detection of a widely distributed invasive aquatic invertebrate.
Clusa L., Ardura A., Gower F., Miralles L., Tsartsianidou V., Zaiko A., Garcia-Vazquez E. (2016): An easy phylogenetically informative method to trace the globally invasive Potamopyrgus mud snail from river’s eDNA. Plos One 11: e0162899.
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Potamopyrgus antipodarum (New Zealand mud snail) is a prosobranch mollusk native to New Zealand with a wide invasive distribution range. Its non-indigenous populations are reported from Australia, Asia, Europe and North America. Being an extremely tolerant species, Potamopyrgus is capable to survive in a great range of salinity and temperature conditions, which explains its high invasiveness and successful spread outside the native range. Here we report the first finding of Potamopyrgus antipodarum in a basin of the Cantabrian corridor in North Iberia (Bay of Biscay, Spain). Two haplotypes already described in Europe were found in different sectors of River Nora (Nalon basin), suggesting the secondary introductions from earlier established invasive populations. To enhance the surveillance of the species and tracking its further spread in the region, we developed a specific set of primers for the genus Potamopyrgus that amplify a fragment of 16S rDNA. The sequences obtained from PCR on DNA extracted from tissue and water samples (environmental DNA, eDNA) were identical in each location, suggesting clonal reproduction of the introduced individuals. Multiple introduction events from different source populations were inferred from our sequence data. The eDNA tool developed here can serve for tracing New Zealand mud snail populations outside its native range, and for inventorying mud snail population assemblages in the native settings if high throughput sequencing methodologies are employed.
Mulero S., Boissier J., Allienne J. F., Quilichini Y., Foata J., Pointier J. P., Rey O. (2020): Environmental DNA for detecting Bulinus truncatus: A new environmental surveillance tool for schistosomiasis emergence risk assessment. Environmental DNA 2: 161-174.
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Under ongoing climate changes, the development of large-scale monitoring tools for assessing the risk of disease emergence constitutes an urging challenge. This is particularly the case for snail-borne diseases such as the urogenital bilharziasis that emerged in Corsica and threat European countries. The expansion of this tropical disease mainly relies on the local presence of competent snail hosts such as Bulinus truncatus. Unfortunately, very little is known about the actual repartition of freshwater snails worldwide which makes new emergences difficult to predict. In this study, we developed two ready-to-use environmental DNA-based methods for assessing the distribution of B. truncatus from water samples collected in the field. We used two approaches, a quantitative PCR (qPCR) and a droplet digital PCR (ddPCR) approach. We successfully detected B. truncatus in natural environments where the snail was previously visually reported. Our environmental DNA diagnostic methods showed a high sensitivity (≈60 DNA copy per mL of filtered water) and a high specificity to B. truncatus. Results obtained in qPCR and ddPCR were very similar. This study demonstrates that environmental DNA diagnostics tools enable a sensitive large-scale monitoring of snail-borne diseases hence allowing the delimitation of areas potentially threatened by urogenital schistosomiasis.
Ponce J. J., Arismendi I., Thomas A. (2021): Using in-situ environmental DNA sampling to detect the invasive New Zealand Mud Snail (Potamopyrgus antipodarum) in freshwaters. PeerJ 9: e11835.
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Environmental DNA (eDNA) detection of aquatic invasive species is currently at the forefront of aquatic conservation efforts because the methodology provides a cost effective and sensitive means to detect animals at low densities. Developments in eDNA technologies have improved detection probabilities for rare, indicator, and invasive species over the past decade. However, standard lab analysis can take days or weeks before results are available and is prohibitive when rapid management decisions are required for mitigation. Here, we investigated the performance of a real-time quantitative PCR system for on-site eDNA detection of New Zealand mud snails (Potamopyrgus antipodarum). Six sites in western Washington, USA were sampled using the rapid eDNA technique and traditional methods, with five samples per site. On-site eDNA detection of mud snails resulted in a 10% increase in positive sites (16/30 = 53% positive) relative to visual surveys (13/30 = 43% positive). In addition, positive associations were observed between mud snail eDNA concentration (eDNA copies per reaction) and the number of mud snail individuals at each site (R2 = 0.78). We show that the rapid on-site eDNA technology can be effective for detection and quantification of New Zealand mud snails in freshwaters. This on-site eDNA detection approach could possibly be used to initiate management protocols that allow for more rapid responses during the onset of biological invasions.
Woodell J. D., Neiman M., Levri E. P. (2021): Matching a snail’s pace: successful use of environmental DNA techniques to detect early stages of invasion by the destructive New Zealand mud snail. Biological Invasions 23: 3263-3274.
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Early detection of invasive species allows for a more rapid and effective response. Restoration of the native ecosystem after an invasive population has established is expensive and difficult but more likely to succeed when invasions are detected early in the invasion process. Containment efforts to prevent the spread of known invasions also benefit from earlier knowledge of invaded sites. Environmental DNA (eDNA) techniques have emerged as a tool that can identify invasive species at a distinctly earlier time point than traditional methods of detection. Here, we focus on whether eDNA techniques can be successfully applied to detect new invasions by the destructive New Zealand Mud Snail Potamopyrgus antipodarum (NZMS). It is an opportune time to apply eDNA-based detection in P. antipodarum, which is currently expanding its invasive range across eastern North America. We collected water samples from eight sites in central Pennsylvania that prior evidence indicated were not yet invaded by the NZMS but were part of the same watershed as other previously documented invaded sites. We found evidence for NZMS invasion at five of the eight sites, with subsequent physical confirmation of mud snails at one of these sites. This study is the first example of successful application of eDNA to detect a previously unidentified invasive population of NZMS, setting the stage for further monitoring of at-risk sites to detect and control new invasions of this destructive snail. This study also shows potential opportunities for invasion monitoring offered by using low-cost efforts and methods that are adaptable for citizen science.
Dimond J. L., Gathright B. R., Bouma J. V., Carson H. S., Sowul K. (2022): Detecting endangered pinto abalone (Haliotis kamtschatkana) using environmental DNA: Comparison of ddPCR, qPCR, and conventional diver surveys. Environmental DNA 4: 1397-1406.
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Abalone populations along the Pacific Coast of North America are threatened. In the Salish Sea (Washington, USA), pinto abalone (Haliotis kamtschatkana) have failed to recover from intensive harvest after over 25 years of fishery closure, prompting a growing restoration effort. As these efforts expand, a persistent challenge is simply locating this rare and highly cryptic species in the field, limiting the ability to identify critical habitat and locate wild adults to serve as restoration broodstock. Here, we tested the use of environmental DNA (eDNA) to detect pinto abalone. Using a quantitative PCR (qPCR) assay previously developed for larval pinto abalone, we first evaluated its sensitivity to abalone eDNA in aquaria settings, finding a positive relationship between abalone biomass and the concentration of abalone DNA. We then tested abalone eDNA detection in the field by collecting replicate water samples from abalone restoration sites, using an occupancy model to estimate detection probability in relation to abalone biomass estimated via diver surveys. Both eDNA concentration and detection probability were positively associated with diver-estimated abalone biomass. By modifying the assay for droplet digital PCR (ddPCR), detection probability increased by 32%–89% over qPCR. eDNA surveys using ddPCR had higher error (CV = 96.9%) than diver surveys (CV = 29.4%) but were more efficient, taking approximately 1/10th of the person-hours per site of a diver survey. For the final phase of the study, we collected water samples at 80 sites throughout the region, obtaining positive abalone eDNA detections at 11 sites with qPCR and 19 additional sites with ddPCR. Our results provide novel survey data on abalone populations within the Salish Sea and show that eDNA is a viable tool for cost-effective, efficient, and non-invasive abalone detection.