Real-Time Monitoring on the Chinese Giant Salamander Using RPA-LFD.

The Chinese giant salamander (Andrias davidianus), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the A. davidianus, presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of A. davidianus and its close relatives. Our results indicated that four of these primer/probe sets accurately identified A. davidianus, distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of A. davidianus genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where A. davidianus is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for A. davidianus.

Using low volume eDNA methods to sample pelagic marine animal assemblages.

Environmental DNA (eDNA) is an increasingly useful method for detecting pelagic animals in the ocean but typically requires large water volumes to sample diverse assemblages. Ship-based pelagic sampling programs that could implement eDNA methods generally have restrictive water budgets. Studies that quantify how eDNA methods perform on low water volumes in the ocean are limited, especially in deep-sea habitats with low animal biomass and poorly described species assemblages. Using 12S rRNA and COI gene primers, we quantified assemblages comprised of micronekton, coastal forage fishes, and zooplankton from low volume eDNA seawater samples (n = 436, 380-1800 mL) collected at depths of 0-2200 m in the southern California Current. We compared diversity in eDNA samples to concurrently collected pelagic trawl samples (n = 27), detecting a higher diversity of vertebrate and invertebrate groups in the eDNA samples. Differences in assemblage composition could be explained by variability in size-selectivity among methods and DNA primer suitability across taxonomic groups. The number of reads and amplicon sequences variants (ASVs) did not vary substantially among shallow (<200 m) and deep samples (>600 m), but the proportion of invertebrate ASVs that could be assigned a species-level identification decreased with sampling depth. Using hierarchical clustering, we resolved horizontal and vertical variability in marine animal assemblages from samples characterized by a relatively low diversity of ecologically important species. Low volume eDNA samples will quantify greater taxonomic diversity as reference libraries, especially for deep-dwelling invertebrate species, continue to expand.

Passive eDNA sampling facilitates biodiversity monitoring and rare species detection.

Environmental DNA (eDNA) technology has revolutionized biomonitoring, but challenges remain regarding water sample processing. The passive eDNA sampler (PEDS) represents a viable alternative to active, water filtration-based eDNA enrichment methods, but the effectiveness of PEDS for surveying biodiverse and complex natural water bodies is unknown. Here, we collected eDNA using filtration and glass fiber filter-based PEDS (submerged in water for 1 d) from 27 sites along the final reach of the Yangtze River and the coast of the Yellow Sea, followed by eDNA metabarcoding analysis of fish biodiversity and quantitative PCR (qPCR) for a critically endangered aquatic mammal, the Yangtze finless porpoise. We ultimately detected 98 fish species via eDNA metabarcoding. Both eDNA sampling methods captured comparable local species richness and revealed largely similar spatial variation in fish assemblages and community partitions between the river and sea sites. Notably, the Yangtze finless porpoise was detected only in the metabarcoding of eDNA collected by PEDS at five sites. Also, species-specific qPCR revealed that the PEDS captured porpoise eDNA at more sites (7 vs. 2), in greater quantities, and with a higher detection probability (0.803 vs. 0.407) than did filtration. Our results demonstrate the capacity of PEDS for surveying fish biodiversity, and support that continuous eDNA collection by PEDS can be more effective than instantaneous water sampling at capturing low abundance and ephemeral species in natural waters. Thus, the PEDS approach can facilitate more efficient and convenient eDNA-based biodiversity surveillance and rare species detection.

Species-specific eDNA assay development for enhanced box jellyfish risk management in coastal environments.

Human interaction with marine creatures holds both positive and negative dimensions. Coastal communities benefit from marine environments, relying on them for sustenance and livelihoods. Fishing activities support economies, and marine biodiversity contributes to overall ecosystem health. However, challenges like overfishing, habitat destruction, and pollution pose threats to both marine life and human communities. Recently, there has been widespread concern regarding the potential increase in jellyfish populations across global marine ecosystems, attributed mainly to environmental factors such as climate drivers and anthropogenic forces, or their complex interactions. Encounters with hazardous marine species, such as box jellyfish, exemplify the dangers associated with coastal activities. Unintended interactions may lead to stings, injuries, and even fatalities, necessitating proactive measures and advanced technologies. This study addresses the inadequacies of existing measures in preventing box jellyfish incidents by introducing environmental DNA (eDNA) assays for detecting the deadly Chiropsoides buitendijki and focuses on developing qPCR and dPCR-based eDNA assays. Emphasising prevention over treatment, the study establishes a proactive system to assess C. buitendijki distribution across 63 tourist beaches in the Gulf of Thailand. Comparative analysis highlights the superior performance of dPCR over qPCR and traditional surveys. The dPCR experiment yielded positive results for all eDNA samples collected at sites where C. buitendijki had previously been identified. Remarkably, the eDNA testing also detected positive results in 16 additional sample locations where no physical specimens were collected, despite reported jellyfish stings at some of these sites. These findings underscore the precision and efficacy of the proposed eDNA detection technology in the early detection and assessment of box jellyfish distribution. This advancement therefore not only aids ecological research but also serves as a valuable tool for safeguarding public health, providing an early warning system for potential jellyfish encounters. Balancing positive human-marine interactions with effective risk mitigation strategies is crucial for sustainable coexistence, the preservation of marine ecosystems, and human well-being.

Combining environmental DNA and remote sensing for efficient, fine-scale mapping of arthropod biodiversity.

Arthropods contribute importantly to ecosystem functioning but remain understudied. This undermines the validity of conservation decisions. Modern methods are now making arthropods easier to study, since arthropods can be mass-trapped, mass-identified, and semi-mass-quantified into 'many-row (observation), many-column (species)' datasets, with homogeneous error, high resolution, and copious environmental-covariate information. These 'novel community datasets' let us efficiently generate information on arthropod species distributions, conservation values, uncertainty, and the magnitude and direction of human impacts. We use a DNA-based method (barcode mapping) to produce an arthropod-community dataset from 121 Malaise-trap samples, and combine it with 29 remote-imagery layers using a deep neural net in a joint species distribution model. With this approach, we generate distribution maps for 76 arthropod species across a 225 km2 temperate-zone forested landscape. We combine the maps to visualize the fine-scale spatial distributions of species richness, community composition, and site irreplaceability. Old-growth forests show distinct community composition and higher species richness, and stream courses have the highest site-irreplaceability values. With this 'sideways biodiversity modelling' method, we demonstrate the feasibility of biodiversity mapping at sufficient spatial resolution to inform local management choices, while also being efficient enough to scale up to thousands of square kilometres. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Measuring the state of aquatic environments using eDNA-upscaling spatial resolution of biotic indices.

Aquatic macroinvertebrates, including many aquatic insect orders, are a diverse and ecologically relevant organismal group yet they are strongly affected by anthropogenic activities. As many of these taxa are highly sensitive to environmental change, they offer a particularly good early warning system for human-induced change, thus leading to their intense monitoring. In aquatic ecosystems there is a plethora of biotic monitoring or biomonitoring approaches, with more than 300 assessment methods reported for freshwater taxa alone. Ultimately, monitoring of aquatic macroinvertebrates is used to calculate ecological indices describing the state of aquatic systems. Many of the methods and indices used are not only hard to compare, but especially difficult to scale in time and space. Novel DNA-based approaches to measure the state and change of aquatic environments now offer unprecedented opportunities, also for possible integration towards commonly applicable indices. Here, we first give a perspective on DNA-based approaches in the monitoring of aquatic organisms, with a focus on aquatic insects, and how to move beyond traditional point-based biotic indices. Second, we demonstrate a proof-of-concept for spatially upscaling ecological indices based on environmental DNA, demonstrating how integration of these novel molecular approaches with hydrological models allows an accurate evaluation at the catchment scale. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Harnessing eDNA metabarcoding to investigate fish community composition and its seasonal changes in the Oslo fjord.

In the face of global ecosystem changes driven by anthropogenic activities, effective biomonitoring strategies are crucial for mitigating impacts on vulnerable aquatic habitats. Time series analysis underscores a great significance in understanding the dynamic nature of marine ecosystems, especially amidst climate change disrupting established seasonal patterns. Focusing on Norway's Oslo fjord, our research utilises eDNA-based monitoring for temporal analysis of aquatic biodiversity during a one year period, with bi-monthly sampling along a transect. To increase the robustness of the study, a taxonomic assignment comparing BLAST+ and SINTAX approaches was done. Utilising MiFish and Elas02 primer sets, our study detected 63 unique fish species, including several commercially important species. Our findings reveal a substantial increase in read abundance during specific migratory cycles, highlighting the efficacy of eDNA metabarcoding for fish composition characterization. Seasonal dynamics for certain species exhibit clear patterns, emphasising the method's utility in unravelling ecological complexities. eDNA metabarcoding emerges as a cost-effective tool with considerable potential for fish community monitoring for conservation purposes in dynamic marine environments like the Oslo fjord, contributing valuable insights for informed management strategies.

Cost-effort analysis of Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) in monitoring marine ecological communities.

Monitoring the diversity and distribution of species in an ecosystem is essential to assess the success of restoration strategies. Implementing biomonitoring methods, which provide a comprehensive assessment of species diversity and mitigate biases in data collection, holds significant importance in biodiversity research. Additionally, ensuring that these methods are cost-efficient and require minimal effort is crucial for effective environmental monitoring. In this study we compare the efficiency of species detection, the cost and the effort of two non-destructive sampling techniques: Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) metabarcoding to survey marine vertebrate species. Comparisons were conducted along the Sussex coast upon the introduction of the Nearshore Trawling Byelaw. This Byelaw aims to boost the recovery of the dense kelp beds and the associated biodiversity that existed in the 1980s. We show that overall BRUV surveys are more affordable than eDNA, however, eDNA detects almost three times as many species as BRUV. eDNA and BRUV surveys are comparable in terms of effort required for each method, unless eDNA analysis is carried out externally, in which case eDNA requires less effort for the lead researchers. Furthermore, we show that increased eDNA replication yields more informative results on community structure. We found that using both methods in conjunction provides a more complete view of biodiversity, with BRUV data supplementing eDNA monitoring by recording species missed by eDNA and by providing additional environmental and life history metrics. The results from this study will serve as a baseline of the marine vertebrate community in Sussex Bay allowing future biodiversity monitoring research projects to understand community structure as the ecosystem recovers following the removal of trawling fishing pressure. Although this study was regional, the findings presented herein have relevance to marine biodiversity and conservation monitoring programs around the globe.

Environmental DNA as a tool to reconstruct catch composition for longline fisheries vessels.

Global wild-capture fisheries are a large and diverse sector requiring various tools for fisheries-dependant data collection and effective Monitoring, Control and Surveillance (MCS). Here we present a novel protocol to collect eDNA from brine tanks onboard commercial longline vessels to reconstruct catch composition. We collected samples from nine vessels operating out of the Eastern Tuna Billfish Fishery, Australia, validating eDNA results with reliable catch data consisting of seven target and bycatch species. Environmental DNA was highly effective for detecting species retained on vessels without contamination or false positives. For four vessels, logbook data and eDNA were consistent with detections of all species. The remaining vessels detected all species except for rare catches of short-billed spearfish (Tetrapturus angustirostris). Similarities between rank abundance distributions of catch and eDNA reads were observed with logbook data mirrored when eDNA sequences were organised into rank order abundance. The method was effective at identifying highly abundant taxa retained in brine tanks- tuna (Thunnus spp.), swordfish (Xiphias gladius), marlin (Kajijia audax), and Atlantic Pomfret (Brama brama). Further research is required to validate how eDNA and other molecular monitoring tools can be scaled and applied to provide solutions for monitoring challenges in the fisheries sector.

An overlooked source of false positives in eDNA-based biodiversity assessment and management.

Biodiversity conservation and management in urban aquatic ecosystems is crucial to human welfare, and environmental DNA (eDNA)-based methods have become popular in biodiversity assessment. Here we report a highly overlooked source of significant false positives for eDNA-based biodiversity assessment in urban aquatic ecosystems supplied with treated wastewater - eDNA pollution originating from treated wastewater represents a noteworthy source of false positives. To investigate whether eDNA pollution is specific to a certain treatment or prevalent across methods employed by wastewater treatment plants, we conducted tests on effluent treated using three different secondary processes, both before and after upgrades to tertiary treatment. We metabarcoded eDNA collected from effluent immediately after full treatment and detected diverse native and non-native, commercial and ornamental fishes (48 taxa) across all treatment processes before and after upgrades. Thus, eDNA pollution occurred irrespective of the treatment processes applied. Release of eDNA pollution into natural aquatic ecosystems could translate into false positives for eDNA-based analysis. We discuss and propose technical solutions to minimize these false positives in environmental nucleic acid-based biodiversity assessments and conservation programs.

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

Revolutionizing aquatic eco-environmental monitoring: Utilizing the RPA-Cas-FQ detection platform for zooplankton.

The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas technology has revolutionized molecular diagnostics and pathogen detection due to its unparalleled sensitivity and trans-cleavage ability. However, its potential in the ecological and environmental monitoring scenarios for aquatic ecosystems remains largely unexplored, particularly in accurate qualitative/quantitative detection, and its actual performance in handling complex real environmental samples. Using zooplankton as a model, we have successfully optimized the RPA-CRISPR/Cas12a fluorescence detection platform (RPA-Cas-FQ), providing several crucial "technical tips". Our findings indicate the sensitivity of CRISPR/Cas12a alone is 5 × 109 copies/reaction, which can be dramatically increased to 5 copies/reaction when combined with RPA. The optimized RPA-Cas-FQ enables reliable qualitative and semi-quantitative detection within 50 min, and exhibits a good linear relationship between fluorescence intensity and DNA concentration (R2 = 0.956-0.974***). Additionally, we developed a rapid and straightforward identification procedure for single zooplankton by incorporating heat-lysis and DNA-barcode techniques. We evaluated the platform's effectiveness using real environmental DNA (eDNA) samples from the Three Gorges Reservoir, confirming its practicality. The eDNA-RPA-Cas-FQ demonstrated strong consistency (Kappa = 0.43***) with eDNA-Metabarcoding in detecting species presence/absence in the reservoir. Furthermore, the two semi-quantitative eDNA technologies showed a strong positive correlation (R2 = 0.58-0.87***). This platform also has the potential to monitor environmental pollutants by selecting appropriate indicator species. The novel insights and methodologies presented in this study represent a significant advancement in meeting the complex needs of aquatic ecosystem protection and monitoring.

Development and Testing of Species-Specific Primers for Detecting the Presence of the Northern Pacific Sea Star (Asterias amurensis) from Environmental DNA.

The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North China such as offshore Qingdao. Effective monitoring and prevention measures are urged to minimize its negative impacts. Compared with traditional biomonitoring methods, environmental DNA technology has emerged as a powerful and cost-efficient tool for inferring species' presence and abundance. In this study, we developed a pair of species-specific primers (i.e., Ast-F and Ast-R) for the A. amurensis mitochondrial COI gene and tested its utility in amplifying and quantifying the DNA fragments from environmental samples under both laboratory and field conditions. The results of controlled water tank experiments demonstrated that the amount of eDNA released by A. amurensis was positively related to its biomass; after the removal of the starfish, the eDNA degraded significantly in 24 h and remained detectable for 8 days. The number of eDNA copies enriched tended to increase with smaller pore size of filter membrane and larger volume of filtered water. For field tests, we confirmed the validation of our approach in six locations in Qingdao by filtering 1000 ml water per sample with a 0.45-µm pore size filtration. All the amplification products generated a single and bright band via gel electrophoresis, and the quantitative PCR results unveiled significant differences in eDNA copies. This study provided an eDNA-based approach for investigating the distribution and biomass of A. amurensis, which may help to formulate early warning and management strategies in coastal Qingdao and other regions.

Emerging use of air eDNA and its application to forensic investigations - A review.

Biological material is routinely collected at crime scenes and from exhibits and is a key type of evidence during criminal investigations. Improvements in DNA technologies allow collection and profiling of trace samples, comprised of few cells, significantly expanding the types of exhibits targeted for DNA analysis to include touched surfaces. However, success rates from trace and touch DNA samples tend to be poorer compared to other biological materials such as blood. Simultaneously, there have been recent advances in the utility of environmental DNA collection (eDNA) in identification and tracking of different biological organisms and species from bacteria to naked mole rats in different environments, including, soil, ice, snow, air and aquatic. This paper examines the emerging methods and research into eDNA collection, with a special emphasis on the potential forensic applications of human DNA collection from air including challenges and further studies required to progress implementation.

Linkage between spatiotemporal distribution of environmental DNA and phenological activity in an amphidromous fish, ayu Plecoglossus altivelis altivelis, in a river located in its northernmost distributional area.

Environmental DNA (eDNA) is a promising tool for the continuous monitoring of fish ecology and diversity. However, its potential for describing the phenological activity of fish has rarely been examined. This study aimed to elucidate a linkage between the spatiotemporal distribution of eDNA and the phenology of an amphidromous fish, ayu Plecoglossus altivelis altivelis, in a river in Hokkaido, Japan, which is its northernmost distributional area. A significant positive correlation between eDNA concentration and catch per unit effort of P. a. altivelis in the river confirmed the use of eDNA as a surrogate for the abundance of P. a. altivelis. eDNA of P. a. altivelis was first detected in late April on a sandy beach adjacent to the river mouth. Subsequent to its first detection at the lowest site in the river in early May, eDNA spread throughout the river, indicating the upstream migration of P. a. altivelis. Spawning activity was also represented by a rapid increase in eDNA concentration and its surge at night in the lowest reaches of the river during September and October. These results suggest that upstream migration and spawning primarily commenced when the water temperature reached 10°C and decreased below 20°C, respectively. This observation is consistent with the behavioral responses observed in P. a. altivelis populations from other regions of Japan. Consequently, this study demonstrated that eDNA distribution was closely linked to the phenological activity of P. a. altivelis and that eDNA is a powerful tool for studying the phenology of migratory fishes.

A qPCR assay for the rapid and specific detection of Shining ram's-horn snail (Segmentina nitida) eDNA from Stodmarsh National Nature Reserve, UK.

Segmentina nitida Müller 1774 is a freshwater snail which was formerly widespread throughout England and south Wales. Since the 1840s it has seen a rapid decline in its range which has been attributed to deteriorating water quality due to nutrient enrichment, lowering of water tables and over-management of the ditches in which it resides. S. nitida has therefore been identified as a UK Biodiversity Action Plan (UKBAP) priority species which recommends further research for its conservation. Here we have developed a Taqman based qPCR eDNA assay for the detection of S. nitida at the Stodmarsh National Nature Reserve and compared the results with a manual survey of the ditches at this location. 32 ditches were surveyed in November 2020 (22 at Stodmarsh) and February 2021 (10 outside the known range of S.nitida). Our eDNA analysis exhibited an observed percentage agreement of 84% with a kappa coefficient of agreement between manual and eDNA surveys of 0.56 (95% CI 0.22 to 0.92). Three ditches determined to be negative for S. nitida by eDNA analysis were manual survey positive, and a further two ditches that were negative by manual survey were positive by eDNA analysis revealing the potential for improved overall detection rates using a combination of manual and eDNA methodologies. eDNA analysis could therefore augment manual survey techniques for S. nitida as a relatively quick and inexpensive tool for collecting presence and distribution data that could be used to inform manual surveys and management of ditches.

Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure.

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, and is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC molecules changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data support a model in which transport of environmental DNA occurs through the entire cell surface by a dynamic pilus, mediating efficient uptake through the cell wall into the periplasm, where DNA diffuses to a cell pole containing the localized transport machinery mediating passage into the cytosol.

Quantitative PCR (qPCR) assay for the specific detection of the Chinese mystery snail (Cipangopaludina chinensis) in the UK.

Cipangopaludina chinensis Gray 1833 is an East Asian freshwater snail and invasive species in many parts of the world (Global Invasive Species Database, 2022). Within the UK, it was first found at the Pevensey Levels, Sussex, and has since been reported at a second site at Southampton Common, Hampshire. Both sites are designated as Sites of Special Scientific Interest (SSSI) for their wildlife importance. Although the impacts of this species within the UK have not yet been investigated several exotic parasites of the snail have been reported and research suggests that its presence can negatively impact native snail species. This is especially important at the Pevensey Levels due to the presence of the rare freshwater mollusc Anisus vorticulus (Little Whirlpool Rams's-horn snail). Here, we have developed a qPCR-based eDNA assay for the detection of C. chinensis and compared water samples tested for eDNA with results from manual survey of the ditches at the Pevensey Levels. Our eDNA analysis exhibited an overall observed percentage agreement of 80% with a kappa coefficient of agreement between manual and eDNA surveys of 0.59 (95% CI 0.31 to 0.88). Some samples which were qPCR negative for C. chinensis were positive by manual survey, and vice versa revealing the potential for improved overall detection rates when using a combination of manual and eDNA methodologies. eDNA analysis can therefore augment manual survey techniques for C. chinensis as a relatively quick and inexpensive tool for collecting presence and distribution data that could be used to inform further manual surveys and control measures within the ditches.

Development and validation of an environmental DNA assay to detect federally threatened groundwater salamanders in central Texas.

The molecular detection of DNA fragments that are shed into the environment (eDNA) has become an increasingly applied tool used to inventory biological communities and to perform targeted species surveys. This method is particularly useful in habitats where it is difficult or not practical to visually detect or trap the target organisms. Central Texas Eurycea salamanders inhabit both surface and subterranean aquatic environments. Subterranean surveys are challenging or infeasible, and the detection of salamander eDNA in water samples is an appealing survey technique for these situations. Here, we develop and validate an eDNA assay using quantitative PCR for E. chisholmensis, E. naufragia, and E. tonkawae. These three species are federally threatened and constitute the Septentriomolge clade that occurs in the northern segment of the Edwards Aquifer. First, we validated the specificity of the assay in silico and with DNA extracted from tissue samples of both target Septentriomolge and non-target amphibians that overlap in distribution. Then, we evaluated the sensitivity of the assay in two controls, one with salamander-positive water and one at field sites known to be occupied by Septentriomolge. For the salamander-positive control, the estimated probability of eDNA occurrence (ψ) was 0.981 (SE = 0.019), and the estimated probability of detecting eDNA in a qPCR replicate (p) was 0.981 (SE = 0.011). For the field control, the estimated probability of eDNA occurring at a site (ψ) was 0.938 (95% CRI: 0.714-0.998). The estimated probability of collecting eDNA in a water sample (θ) was positively correlated with salamander relative density and ranged from 0.371 (95% CRI: 0.201-0.561) to 0.999 (95% CRI: 0.850- > 0.999) among sampled sites. Therefore, sites with low salamander density require more water samples for eDNA evaluation, and we determined that our site with the lowest estimated θ would require seven water samples for the cumulative collection probability to exceed 0.95. The estimated probability of detecting eDNA in a qPCR replicate (p) was 0.882 (95% CRI: 0.807-0.936), and our assay required two qPCR replicates for the cumulative detection probability to exceed 0.95. In complementary visual encounter surveys, the estimated probability of salamanders occurring at a known-occupied site was 0.905 (SE = 0.096), and the estimated probability of detecting salamanders in a visual encounter survey was 0.925 (SE = 0.052). We additionally discuss future research needed to refine this method and understand its limitations before practical application and incorporation into formal survey protocols for these taxa.