Search

Abstract A model was developed to forecast and compare changes in species presence assessed with either eDNA or traditional observations. We use it to explore how ecosystem conditions could affect the suitability of eDNA for Environmental Impact Assessment. First, a deterministic model simulated the dynamics of the impacted population (called “receptor” in EIA) and their shed DNA fragment concentrations. Second, random distributions of receptor organisms and eDNA fragment quantities at steady state were simulated within the impacted spatial domain (called “project area”). Then, simple random samplings were performed for both the receptor and eDNA. Third, post‐sampling processes (eDNA extraction, amplification, and analysis) were simulated to estimate the taxon detection probability. Fourth, we simulated an impact by modifying the growth, mortality, and mobility (null, passive, and active) parameters of the receptor taxon. eDNA detection probability curves were then estimated for a range of environmental sample volumes by fitting a Weibull cumulative distribution function. A F‐like statistic compared detection curves before and after impact. Statistically significant differences were detected with eDNA in impact scenarios where receptor taxon growth rate decreased and receptor mobility was null or passive. In scenarios where the project area accumulates DNA shed from multiple categories of the same taxon (e.g., from dead organisms if mortality increased or when individuals can cross project area boundaries), it is difficult to assess impact. Our study shows that results obtained from eDNA sampling will not always agree with an impact classically assessed on a receptor population. One reason is that sources of the total eDNA pool are not identified. The modeling highlights the need: to do preliminary testing of sample sizes, to develop new approaches that will identify sources from the pool of extracted DNA, and to improve descriptions of the ecogeochemical processes required to forecast shed DNA reactivity.

AbstractAquatic biodiversity monitoring to inform conservation and management efforts in‐stream systems has increasingly begun to incorporate environmental DNA (eDNA)‐based sampling methods. We conducted a comparison of eDNA metabarcoding to a traditional protocol of combined seining and electrofishing methods to assess fish biodiversity of wadeable stream sites in six separate drainages in the Ozark Highlands of Missouri (USA). The study further focused on the headwaters of the Meramec River, which included 11 sites and seasonal sampling (summer and winter). We compared estimates of diversity across sampling methods, assessed the influences of water flow (depth, velocity, and discharge) and season, and tested the effects of sampling method and site locality on fish assemblage composition. eDNA sampling detected approximately double the number of species compared to traditional methods, providing higher diversity estimates while maintaining the relative ranking of sites. eDNA detection probabilities were positively associated with stream depth and velocity and were generally higher in summer than in winter but not for all species. Estimated species richness was positively associated with discharge for both methods but the relationship was stronger with eDNA sampling. Assemblage differences between tributary and mainstem sites were attributable to a small number of species that were found predominantly in one stream size category or the other, indicating that eDNA was sensitive to within‐drainage assemblage structure relationships. We highlight improved species detection, a more comprehensive understanding of assemblage structural dynamics, and the potential ability to integrate data across sampling methods as important benefits that encourage the use of eDNA metabarcoding as a primary collection method in future stream biodiversity assessment and monitoring programs.

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Course-based undergraduate research experiences (CUREs) increase student access to high impact research experiences. CUREs engage students in the scientific process by learning how to pose scientific questions, develop hypotheses, and generate data to test them. Environmental DNA (eDNA) is a growing field of research that is gaining accessibility through decreasing laboratory costs, which can make a foundation for multiple, engaging CUREs. This manuscript describes three case studies that used eDNA in an upper year undergraduate course. The first focusses on a systematic literature review of eDNA metadata reporting. The second describes the biomonitoring of brook trout in southern Ontario using eDNA. The third involves eDNA metabarcoding for freshwater fish detection in southern Ontario. Undergraduates were involved in the development and execution of experiments, scientific communication, the peer review process, and fundraising. Through this manuscript, we show the novel application of eDNA CUREs and provide a roadmap for other instructors interested in implementing similar projects. Interviews with seven students from these courses indicate the benefits experienced from taking these courses. We argue that the use of eDNA in CUREs should be expanded in undergraduate biology programs due to the benefit to students and the increasing accessibility of this technology., Competing Interests: The authors declare no competing interests.

Determining biological status of freshwater ecosystems is critical for ensuring ecosystem health and maintaining associated services to such ecosystems. Freshwater macroinvertebrates respond predictably to environmental disturbances and are widely used in biomonitoring programs. However, many freshwater species are difficult to capture and sort from debris or substrate and morphological identification is challenging, especially larval stages, damaged specimens, or hyperdiverse groups such as Diptera. The advent of high throughput sequencing technologies has enhanced DNA barcoding tools to automatise species identification for whole communities, as metabarcoding is increasingly used to monitor biodiversity. However, recent comparisons have revealed little congruence between morphological and molecular-based identifications. Using broad range universal primers for DNA barcode marker cox1, we compare community composition captured between morphological and molecular-based approaches from different sources - tissue-based (bulk benthic and bulk drift samples) and environmental DNA (eDNA, filtered water) metabarcoding - for samples collected along a gradient of anthropogenic disturbances. For comparability, metabarcoding taxonomic assignments were filtered by taxa included in the standardised national biological metric IBMWP. At the family level, bulk benthic metabarcoding showed the highest congruence with morphology, and the most abundant taxa were captured by all techniques. Richness captured by morphology and bulk benthic metabarcoding decreased along the gradient, whereas richness recorded by eDNA remained constant and increased downstream when sequencing bulk drift. Estimates of biological metrics were higher using molecular than morphological identification. At species level, diversity captured by bulk benthic samples were higher than the other techniques. Importantly, bulk benthic and eDNA metabarcoding captured different and complementary portions of the community - benthic versus water column, respectively - and their combined use is recommended. While bulk benthic metabarcoding can likely replace morphology using similar benthic biological indices, water eDNA will require new metrics because this technique sequences a different portion of the community., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Environmental DNA (eDNA) is widely used in biodiversity, conservation, and ecological studies but despite its successes, similar approaches have not yet been regularly applied to assist in wildlife crime investigations. The purpose of this paper is to review current eDNA methods and assess their potential forensic application in freshwater environments considering collection, transport and persistence, analysis, and interpretation, while identifying additional research required to present eDNA evidence in court. An extensive review of the literature suggests that commonly used collection methods can be easily adapted for forensic frameworks providing they address the appropriate investigative questions and take into consideration the uniqueness of the target species, its habitat, and the requirements of the end user. The use of eDNA methods to inform conservationists, monitor biodiversity and impacts of climate change, and detect invasive species and pathogens shows confidence within the scientific community, making the acceptance of these methods by the criminal justice system highly possible. To contextualise the potential application of eDNA on forensic investigations, two test cases are explored involving i) species detection and ii) species localisation. Recommendations for future work within the forensic eDNA discipline include development of suitable standardised collection methods, considered collection strategies, forensically validated assays and publication of procedures and empirical research studies to support implementation within the legal system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The marine dinophyte Alexandrium tamiyavanichii is a toxigenic species that produces a group of neurotoxins that is responsible for paralytic shellfish poisoning in humans. Early detection of the species is essential for efficient monitoring. Harmful microalgal monitoring systems have evolved over the years with the advent of environmental DNA (eDNA)-based species detection techniques. In this study, eDNA samples were collected from a large-scale sampling covering the southern South China Sea. The sensitivity and specificity of metabarcoding of the V4 and V9 18S ribosomal DNA barcodes by high-throughput sequencing (HTS) were compared to the species-specific real-time qPCR targeting the A. tamiyavanichii ITS2 region. Environmental samples were screened for A. tamiyavanichii by qPCR (n = 43) and analyzed with metabarcoding (n = 30). Our results revealed a high occupancy profile across samples for both methods; 88% by qPCR, and 80-83% by HTS. When comparing the consistency between the two approaches, only two samples out of 30 were discordant. The V4 and V9 molecular units detected in each sample were positively correlated with the qPCR ITS2 gene copies (V4, r s  = 0.67, p < 0.0001; V9, r s  = 0.65, p < 0.0001), indicating that metabarcoding could be used as a useful tool for early detection of the species. Our results also revealed that the estimation of A. tamiyavanichii cell abundances based on the HTS read abundances was comparable to that of the qPCR quantification. For long-term monitoring, metabarcoding could serve as a cost-effective screening of detecting not only single HAB species but also simultaneously detecting a multitude of potentially harmful species, which is valuable in informing the subsequent implementation of species-specific monitoring strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The comprehensive analysis of multiple biological communities is essential for assessing diversities within mangrove ecosystems, yet such studies are infrequent. Environmental DNA (eDNA) facilitates the simultaneous exploration of organisms across various levels within a single ecosystem. In this investigation, 16S rRNA, cytochrome C oxidase I (COI), and Mito-fish primers were employed to characterize the microbiome, eukaryotic plankton, and fish communities, along with their intricate interactions, across 24 samples from three Chinese mangrove reservoirs. The resulting dataset encompasses 3779 taxonomic groups (genus level), spanning from the microbiome to vertebrates. Diversity analysis unveiled a higher level of stability in the microbiome community compared to plankton, underscoring the superior site-specificity of plankton. The association analysis revealed that biodiversity was primarily affected by temperature, turbidity, and fluorescent dissolved organic matter (fDOM). Notably, the physicochemical factors, turbidity, and fDOM had a more pronounced impact on the microbiome than on plankton, explaining their distinct sensitivities to site-specific conditions. Network analysis constructed 15 biological interaction subnetworks representing various community connections. The most connected genera in each subnetwork, highly responsive to different environmental factors, could serve as potential indicators of distinct ecosystem states. In summary, our findings represent the first comparison of the response sensitivities of different communities and the construction of their interaction networks in mangrove environments. These results contribute valuable insights into marine ecosystem dynamics and the role of environmental factors in shaping biodiversity., Competing Interests: Declaration of competing interest There is no conflict of interest in the manuscript, which is approved by all authors for publication. We also declare that the work described is original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Population outbreaks of the crown-of-thorns starfish (COTS) seriously threaten the sustainability of coral reef ecosystems. However, traditional ecological monitoring techniques cannot provide early warning before the outbreaks, thus preventing timely intervention. Therefore, there is an urgent need for a more accurate and faster technology to predict the outbreaks of COTS. In this work, we developed an electrochemical biosensor based on a programmed catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cyclic amplification strategy for sensitive and selective detection of COTS environmental DNA (eDNA) in water bodies. This biosensor exhibited excellent electrochemical characteristics, including a low limit of detection (LOD = 18.4 fM), low limit of quantification (LOQ = 41.1 fM), and wide linear range (50 fM - 10 nM). The biosensing technology successfully allowed the detection of COTS eDNA in the aquarium environment, and the results also demonstrated a significant correlation between eDNA concentration and COTS number (r = 0.990; P < 0.001). The reliability and accuracy of the biosensor results have been further validated through comparison with digital droplet PCR (ddPCR). Moreover, the applicability and accuracy of the biosensor were reconfirmed in field tests at the COTS outbreak site in the South China Sea, which has shown potential application in dynamically monitoring the larvae before the COTS outbreak. Therefore, this efficient electrochemical biosensing technology offers a new solution for on-site monitoring and early warning of the COTS outbreak., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Infectious diseases stem from disrupted interactions among hosts, parasites, and the environment. Both abiotic and biotic factors can influence infection outcomes by shaping the abundance of a parasite's infective stages, as well as the host's ability to fight infection. However, disentangling these mechanisms within natural ecosystems remains challenging. Here, combining environmental DNA analysis and niche modelling at a regional scale, we uncovered the biotic and abiotic drivers of an infectious disease of salmonid fish, triggered by the parasite Tetracapsuloides bryosalmonae. We found that the occurrence and abundance of the parasite in the water-i.e., the propagule pressure- were mainly correlated to the abundances of its two primary hosts, the bryozoan Fredericella sultana and the fish Salmo trutta, but poorly to local abiotic environmental stressors. In contrast, the occurrence and abundance of parasites within fish hosts-i.e., proxies for disease emergence-were closely linked to environmental stressors (water temperature, agricultural activities, dams), and to a lesser extent to parasite propagule pressure. These results suggest that pathogen distribution alone cannot predict the risk of disease in wildlife, and that local anthropogenic stressors may play a pivotal role in disease emergence among wild host populations, likely by modulating the hosts' immune response. Our study sheds light on the intricate interplay between biotic and abiotic factors in shaping pathogen distribution and raises concerns about the effects of global change on pathogen emergence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Simon Blanchet reports financial support was provided by French Biodiversity Office. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study thoroughly examines three cetacean monitoring methods and assessing their advantages and limitations, establishing a foundational basis for comprehensive information on composition, distribution, and behavior. While real-time and non-invasive, visual surveys favor surface-active cetaceans and are weather-dependent. Local ecological knowledge supplements insights into group behavior. Environmental DNA (eDNA) analysis efficiently detects species like the narrow-ridged finless porpoise (Neophocaena asiaeorientalis) and common bottlenose dolphin (Tursiops truncatus), offering non-invasive, and spatially adept monitoring. Furthermore, eDNA provides prey species information, revealing the narrow-ridged finless porpoise's winter migration to deeper waters due to prey distribution. The study identifies prevalent prey species, like the Japanese Anchovy (Engraulis japonicus) and Osbeck's grenadier anchovy (Coilia mystus), offering insights into the porpoise's feeding ecology and adaptation to changing prey availability in winter. This study systematically compares diverse methodologies employed in cetacean surveys, thereby yielding a comprehensive understanding of cetacean distribution, behavior, and feeding ecology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Analysis of environmental DNA (eDNA) enables indirect detection of species without the need to directly observe and sample them. For biosecurity and invasion biology, eDNA-based methods are useful to address biological invasions at all phases, from detecting arrivals to confirming eradication of past invasions. We conducted a systematic review of the literature and found that in biosecurity and invasion biology, eDNA has primarily been used to detect new incursions and monitor spread in marine and freshwater ecosystems, with much slower uptake in terrestrial ecosystems, reflecting a broader trend common to the usage of eDNA tools. In terrestrial ecosystems, eDNA research has mostly focussed on the use of eDNA metabarcoding to characterise biodiversity, rather than targeting biosecurity threats or non-native populations. We discuss how eDNA-based methods are being applied to terrestrial ecosystems for biosecurity and managing non-native populations at each phase of the invasion continuum: transport, introduction, establishment, and spread; across different management options: containment, control, and eradication; and for detecting the impact of non-native organisms. Finally, we address some of the current technical issues and caveats of eDNA-based methods, particularly for terrestrial ecosystems, and how these might be solved. As eDNA-based methods improve, they will play an increasingly important role in the early detection and adaptive management of biological invasions, and the implementation of effective biosecurity controls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Agricultural run-off in Australia's Mackay-Whitsunday region is a major source of nutrient and pesticide pollution to coastal and inshore ecosystems of the Great Barrier Reef. While the effects of run-off are well documented for the region's coral and seagrass habitats, the ecological impacts on estuaries, the direct recipients of run-off, are less known. This is particularly true for fish communities, which are shaped by the physico-chemical properties of coastal waterways that vary greatly in tropical regions. To address this knowledge gap, we used environmental DNA (eDNA) metabarcoding to examine fish assemblages at four locations (three estuaries and a harbour) subjected to varying levels of agricultural run-off during a wet and dry season. Pesticide and nutrient concentrations were markedly elevated during the sampled wet season with the influx of freshwater and agricultural run-off. Fish taxa richness significantly decreased in all three estuaries (F = 164.73, P = <0.001), along with pronounced changes in community composition (F = 46.68, P = 0.001) associated with environmental variables (largely salinity: 27.48% contribution to total variance). In contrast, the nearby Mackay Harbour exhibited a far more stable community structure, with no marked changes in fish assemblages observed between the sampled seasons. Among the four sampled locations, variation in fish community composition was more pronounced within the wet season (F = 2.5, P = 0.001). Notably, variation in the wet season was significantly correlated with agricultural contaminants (phosphorus: 6.25%, pesticides: 5.22%) alongside environmental variables (salinity: 5.61%, DOC: 5.57%). Historically contaminated and relatively unimpacted estuaries each demonstrated distinct fish communities, reflecting their associated catchment use. Our findings emphasise that while seasonal effects play a key role in shaping the community structure of fish in this region, agricultural contaminants are also important contributors in estuarine systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Individuals leave behind traces of their DNA wherever they go. DNA can be transferred to surfaces and items upon touch, can be released into the air, and may be deposited in indoor dust. The mere presence of individuals in a location is sufficient to facilitate either direct or indirect DNA transfer into the surrounding environment. In this study, we analyzed samples recovered from commonly touched surfaces such as light switches and door handles in an office environment. We evaluated two different methods to isolate DNA and co-extract DNA and RNA from the samples. DNA profiles were compared to the references of the inhabitants of the different locations and were analyzed taking into consideration the type of sampled surface, sampling location and information about the activities in a room during the sampling day. Results from DNA samples collected from surfaces were also compared to those from air and dust samples collected in parallel from the same areas. We characterized the amount and composition of DNA found on various surfaces and showed that surface DNA sampling can be used to detect occupants of a location. The results also indicate that combining information from environmental samples collected from different DNA sources can improve our understanding of DNA transfer events in an indoor setting. This study further demonstrates the potential of human environmental DNA as an investigative tool in forensic genetics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Applications of environmental DNA (eDNA) analysis methods for biomonitoring have grown exponentially over the last decade and provide a wealth of new information on the distribution of species. However, eDNA methods have limited application for estimating population-level metrics. Environmental RNA (eRNA) has the potential to address ecological questions by gathering population demographic information from environmental media but may be challenging to detect and analyze. We developed gene-specific eRNA assays targeting keratin-associated genes in two focal species, American bullfrogs (Lithobates catesbeianus) and tiger salamanders (Ambystoma mavortium) to answer an important question in amphibian management: whether species detections represent breeding populations versus transitory adults. We performed an extensive laboratory validation with amphibians housed across development stages, where we collected 95 and 127 environmental samples for bullfrogs and salamanders, respectively. Both assays were highly specific to the larval stage and amplified with high sensitivity (90% in bullfrog and 88.4% in tiger salamander samples). We then applied our validated assays to multiple natural systems. When larvae were present, we found 74.1% overall detection in bullfrog field samples and 70.8% and 48.5% overall detection in field samples from ponds with A. macrodactylum and A. californiense larvae, correlating with eDNA detection rates. When only adults were present, we did not detect larvae-specific eRNA in A. macrodactylum ponds, despite high eDNA detection rates. Although much work is ahead for optimizing assay design, sampling and filtering methods, we demonstrate that eRNA can successfully be used to discern life stages with direct application for ecology and conservation management., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

The approach of combining cost-effective nanopore sequencing and emerging environmental DNA (eDNA) metabarcoding could prove to be a promising tool for biodiversity documentation, especially in Malaysia. Given the substantial funding constraints in recent years, especially in relation to the country's biodiversity, many researchers have been limited to conduct restricted research without extended monitoring periods, potentially hindering comprehensive surveys and could compromise the conservation efforts. Therefore, the present study aimed to evaluate the application of eDNA metabarcoding on freshwater fish using short reads generated through nanopore sequencing. This assessment focused on species detection in three selected rivers within the Endau Rompin Landscape in Malaysia. Additionally, the study compared levels of species detection between eDNA metabarcoding and conventional sampling methods, examined the effectiveness of primer choice, and applied both metabarcoding and shotgun sequencing to the eDNA approach. We successfully identified a total of 22 and 71 species with an identification threshold of >97% and >90%, respectively, through the MinION platform. The eDNA metabarcoding approach detected over 13% more freshwater fish species than when the conventional method was used. Notably, the distinction in freshwater fish detection between eDNA primers for 12S rRNA and cytochrome oxidase I was insignificant. The cost for eDNA metabarcoding proved to be more effective compared to conventional sampling with cost reduction at 33.4%. With favourable cost-effectiveness and increased species detection, eDNA metabarcoding could complement existing methods, enhance holistic diversity documentation for targeted habitats and facilitate effective conservation planning., (© 2024 John Wiley & Sons Ltd.)

Stream ecosystems are under pressure due to multiple stressors. Restoration measures can halt further degradation and improve their ecological status. However, assessment of the effectiveness of the implemented measures is often insufficient because of logistic and financial constraints. DNA-metabarcoding has been proposed to scale up sample processing, although its application as a diagnostic tool has received less attention. The aim of our study was to evaluate if DNA-metabarcoding of stream macroinvertebrates can be used to compute a stressor-specific index to assess the effectiveness of a stream restoration project. For this purpose, we sampled the upstream, restored, and downstream section of a recently restored lowland stream in the Netherlands. At each site, we applied three different methods of macroinvertebrate identification: morphological identification of bulk samples (morphology), DNA-metabarcoding of the same bulk samples (DNA) and metabarcoding of eDNA extracted from the water (eDNA). First, we compared the community composition identified by each method. The communities identified by morphology and DNA were highly similar, whereas the communities generated by the eDNA differed. Second, we analysed whether the identification methods could be used to assess the effectiveness of the restoration project, focussing on a stressor-specific index for flow as the restoration measures aimed at improving flow conditions. Both the morphology and bulk DNA samples indicated improved flow conditions in the restored section of the stream (i.e., less stress from the reduction or absence of flow than in the unrestored sections). Contrary, the eDNA-water samples did not differentiate the amount of stress throughout the catchment, although applying recent developments in eDNA sampling could lead to more robust results. In conclusion, this study forms proof of concept that DNA from bulk samples can be utilized to assess the effectiveness of restoration measures, showing the added value of this approach for water managers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gea H. van der Lee reports financial support was provided by Dutch Ministry of Agriculture, Nature and Food Quality. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography., (© 2024 John Wiley & Sons Ltd.)

As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi-marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior-supported primers for immediate applications. The ITS2 was the best-performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications., (© 2024 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and non-invasiveness. This approach emerges as a game-changer for the elusive realm of endangered and rare species-think nocturnal, environmentally elusive amphibians. Here, we have established a framework for constructing a reliable metabarcoding pipeline for amphibians, covering primer design, performance evaluation, laboratory validation, and field validation processes. The Am250 primer, located on the mitochondrial 16S gene, was optimal for the eDNA monitoring of amphibians, which demonstrated higher taxonomic resolution, smaller species amplification bias, and more extraordinary detection ability compared to the other primers tested. Am250 primer exhibit an 83.8% species amplification rate and 75.4% accurate species identification rate for Chinese amphibians in the in silico PCR and successfully amplified all tested species of the standard samples in the in vitro assay. Furthermore, the field-based mesocosm experiment showed that DNA can still be detected by metabarcoding even days to weeks after organisms have been removed from the mesocosm. Moreover, field mesocosm findings indicate that eDNA metabarcoding primers exhibit different read abundances, which can affect the relative biomass of species. Thus, appropriate primers should be screened and evaluated by three experimental approaches: in silico PCR simulation, target DNA amplification, and mesocosm eDNA validation. The selection of a single primer set or multiple primers' combination should be based on the monitoring groups to improve the species detection rate and the credibility of results., (© 2024 John Wiley & Sons Ltd.)

Taxon-specific quantitative PCR (qPCR) assays are commonly used for environmental DNA sampling-based inference of animal presence. These assays require thorough validation to ensure that amplification truly indicates detection of the target taxon, but a thorough validation is difficult when there are potentially many non-target taxa, some of which may have incomplete taxonomies. Here, we use a previously published, quantitative model of cross-amplification risk to describe a framework for assessing qPCR assay specificity when there is missing information and it is not possible to assess assay specificity for each individual non-target confamilial. In this framework, we predict assay specificity against unsampled taxa (non-target taxa without sequence data available) using the sequence information that is available for other confamilials. We demonstrate this framework using four case study assays for: (1) An endemic, freshwater arthropod (meltwater stonefly; Lednia tumana), (2) a globally distributed, marine ascidian (Didemnum perlucidum), (3) a continentally distributed freshwater crustacean (virile crayfish; Faxonius virilis, deanae and nais species complex) and (4) a globally distributed freshwater teleost (common carp; Cyprinus carpio and its close relative C. rubrofuscus). We tested the robustness of our approach to missing information by simulating application of our framework for all possible subsamples of 20-all non-target taxa. Our results suggest that the modelling framework results in estimates which are largely concordant with observed levels of cross-amplification risk using all available sequence data, even when there are high levels of data missingness. We explore potential limitations and extensions of this approach for assessing assay specificity and provide users with an R Markdown template for generating reproducible reports to support their own assay validation efforts., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

The analyses of environmental DNA (eDNA) and environmental RNA (eRNA) released by organisms into their surrounding environment (water, soil and air) have emerged as powerful tools for monitoring biodiversity. While eDNA has been widely adopted for the non-invasive detection of species and characterization of community composition, the utilization of eRNA is still in its infancy. Due to its functional nature, eRNA holds intriguing potential for biodiversity monitoring offering new avenues of research beyond species detection. For example, conspecifics that are almost genetically identical can exhibit distinct transcriptomic differences depending on their life stage. In this issue of Molecular Ecology Resources, Parsley and Goldberg (2024) demonstrate, through a lab-validated field study, that eRNA can be used to detect distinct life stages of amphibians. This study elegantly demonstrates that eRNA can be used not only to detect invasive or endangered species but also to reveal population demographic information important for guiding effective conservation strategies., (© 2024 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Fungi play a vital role in ecosystem functioning, yet significant knowledge gaps persist in understanding their diversity and distribution leading to uncertainties about their threat status and extinction risk. This is partly owed to the difficulty of monitoring fungi using traditional fruiting body surveys. The present study evaluates airborne environmental DNA (eDNA) sampling as a monitoring tool with a focus on grassland macrofungi. We applied active and passive air sampling methods, complemented by extensive field surveys of waxcap and clavarioid fungi-species groups of high relevance for conservation. Twenty-nine species were recorded during the field surveys, 19 of which were also detectable by ITS2 metabarcoding of the collected samples. An additional 12 species from the studied genera were identified exclusively in air eDNA. We found that the patterns of species detection and read abundance in air samples reflected the abundance and occurrence of fruiting bodies on the field. Dispersal kernels fitted for the three dominant species predicted rapidly decreasing spore concentrations with increasing distance from fruitbodies. Airborne assemblages were dominated by a high diversity of common species, while rare and threatened red-listed species were under-represented, which underscores the difficulty in detecting rare species, not only in conventional surveys. Considering the benefits and drawbacks of air sampling and fruitbody surveys, we conclude that air sampling serves as a cost- and time-efficient tool to characterize local macrofungal communities, providing the potential to facilitate and improve future fungal monitoring efforts., (© 2024 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Adsorption of biomacromolecules onto polymer surfaces, including microplastics (MPs), occurs in multiple environmental compartments, forming an ecocorona. Environmental DNA (eDNA), genetic material shed from organisms, can adsorb onto MPs which can potentially either (1) promote long-range transport of antibiotic resistant genes or (2) serve to gain insights into the transport pathways and origins of MPs by analyzing DNA sequences on MPs. However, little is known about the capacity of MPs to adsorb eDNA or the factors that influence sorption, such as polymer and water chemistries. Here we investigated the adsorption of extracellular linear DNA onto a variety of model MP fragments composed of three of the most environmentally prevalent polymers (polyethylene, polyethylene terephthalate, and polystyrene) in their pristine and photochemically weathered states. Batch adsorption experiments in a variety of water chemistries were complemented with nonlinear modeling to quantify the rate and extent of eDNA sorption. Ionic strength was shown to strongly impact DNA adsorption by reducing or inhibiting electrostatic repulsion. Polyethylene terephthalate exhibited the highest adsorption capacity when normalizing for MP specific surface area, likely due to the presence of ester groups. Kinetics experiments showed fast adsorption (majority adsorbed under 30 min) before eventually reaching equilibrium after 1-2 h. Overall, we demonstrated that DNA quickly binds to MPs, with pseudo-first- and -second-order models describing adsorption kinetics and the Freundlich model describing adsorption isotherms most accurately. These insights into DNA sorption onto MPs show that there is potential for MPs to act as vectors for genetic material of interest, especially considering that particle-bound DNA typically persists longer in the environment than dissolved DNA.

Background: Grasses populate most biogeographical zones, and their diversity influences allergic sensitisation to pollen. Previously, the contribution of different Poaceae subfamilies to airborne pollen has mostly been inferred from historical herbarium records. We recently applied environmental (e)DNA metabarcoding at one subtropical site revealing that successive airborne grass pollen peaks were derived from repeated flowering of Chloridoid and Panicoid grasses over a season. This study aimed to compare spatiotemporal patterns in grass pollen exposure across seasons and climate zones., Methods: Airborne pollen concentrations across two austral pollen seasons spanning 2017-2019 at subtropical (Mutdapilly and Rocklea, Queensland) and temperate (Macquarie Park and Richmond, New South Wales) sites, were determined with a routine volumetric impaction sampler and counting by light microscopy. Poaceae rbcL metabarcode sequences amplified from daily pollen samples collected once per week were assigned to subfamily and genus using a ribosomal classifier and compared with Atlas of Living Australia sighting records., Results: eDNA analysis revealed distinct dominance patterns of grass pollen at various sites: Panicoid grasses prevailed in both subtropical Mutdapilly and temperate Macquarie Park, whilst Chloridoid grasses dominated the subtropical Rocklea site. Overall, subtropical sites showed significantly higher proportion of pollen from Chloridoid grasses than temperate sites, whereas the temperate sites showed a significantly higher proportion of pollen from Pooideae grasses than subtropical sites. Timing of airborne Pooid (spring), Panicoid and Chloridoid (late spring to autumn), and Arundinoid (autumn) pollen were significantly related to number of days from mid-winter. Proportions of eDNA for subfamilies correlated with distributions grass sighting records between climate zones., Conclusions: eDNA analysis enabled finer taxonomic discernment of Poaceae pollen records across seasons and climate zones with implications for understanding adaptation of grasslands to climate change, and the complexity of pollen exposure for patients with allergic respiratory diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:JMD, AH and PJB report grants from Australian Research Council (DP210100347; DP170101630) related to this research. JMD, AH and PJB report the grant from the Australian National Health and Medical Research Council GNT1116107, with financial co-contribution from Asthma Australia and Stallergenes Greer Australia Pty Ltd, as well as in kind support from Australasian Society of Clinical Immunology and Allergy, Asthma Australia, Bureau of Meteorology, Commonwealth Scientific and Industrial Research Organisation, Stallergenes Greer Australia Pty Ltd, and Federal Office of Meteorology and Climatology (MeteoSwiss), for the conduct of part of this project. JMD reports grants from the Australian Research Council (DP190100376; LP190100216), National Foundation of Medical Research Innovation, Abionic SA, The Emergency Medicine Foundation, and Queensland Chief Scientist Citizen Science Grant, outside the scope of submitted work. PJB and JMD report grants from Bureau of Meteorology outside the submitted work. JMD reports QUT has patents broadly relevant US PTO 14/311,944 and AU 2008/316,301 issued. SvH, BC, AM, BAS, JAK declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

China has become one of the most serious countries suffering from biological invasions in the world. In the context of global climate change, invasive alien species (IAS) are likely to invade a wider area, posing greater ecological and economic threats in China. Western mosquitofish (Gambusia affinis), which is known as one of the 100 most invasive alien species, has distributed widely in southern China and is gradually spreading to the north, causing serious ecological damage and economic losses. However, its distribution in China is still unclear. Hence, there is an urgent need for a more convenient way to detect and monitor the distribution of G. affinis to put forward specific management. Therefore, we detected the distribution of G. affinis in China under current and future climate change by combing Maxent modeling prediction and eDNA verification, which is a more time-saving and reliable method to estimate the distribution of species. The Maxent modeling showed that G. affinis has a broad habitat suitability in China (especially in southern China) and would continue to spread in the future with ongoing climate change. However, eDNA monitoring showed that occurrences can already be detected in regions that Maxent still categorized as unsuitable. Besides temperature, precipitation and human influence were the most important environmental factors affecting the distribution of G. affinis in China. In addition, by environmental DNA analysis, we verified the presence of G. affinis predicted by Maxent in the Qinling Mountains where the presence of G. affinis had not been previously recorded., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Dams and weirs impede the continuity of rivers and transit of migratory fish. To overcome this obstacle, fishways are installed worldwide; however, management after installation is important. The Miyanaka Intake Dam has three fish ladders with different flow velocities and discharges and has been under adaptive management since 2012. Fish catch surveys, conducted as an adaptive management strategy, place a heavy burden on fish. Furthermore, a large number of investigators must be mobilized during the 30-day investigation period. Thus, a monitoring method using environmental DNA that exerts no burden on fish and requires only a few surveyors (to obtain water samples) and an in-house analyst was devised; however, its implementation in a fishway away from the point of analysis and with limited flow space and its effective water sampling frequency have not been reported. Therefore, in 2019, we started a trial aiming to evaluate the methods and application conditions of environmental DNA surveys for the continuous and long-term monitoring of various fish fauna upstream and downstream of the Miyanaka Intake Dam. To evaluate the fish fauna, the results of an environmental DNA survey (metabarcoding method) for 2019 to 2022 were compared to those of a catch survey in the fishway from 2012 to 2022. The results confirmed the use of environmental DNA surveys in evaluating the contribution of fishways to biodiversity under certain conditions and introduced a novel method for sample collection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nakai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Sedimentary ancient DNA (sedaDNA) has rarely been used to obtain population-level data due to either a lack of taxonomic resolution for the molecular method used, limitations in the reference material or inefficient methods. Here, we present the potential of multiplexing different PCR primers to retrieve population-level genetic data from sedaDNA samples. Vaccinium uliginosum (Ericaceae) is a widespread species with a circumpolar distribution and three lineages in present-day populations. We searched 18 plastid genomes for intraspecific variable regions and developed 61 primer sets to target these. Initial multiplex PCR testing resulted in a final set of 38 primer sets. These primer sets were used to analyse 20 lake sedaDNA samples (11,200 cal. yr BP to present) from five different localities in northern Norway, the Alps and the Polar Urals. All known V. uliginosum lineages in these regions and all primer sets could be recovered from the sedaDNA data. For each sample on average 28.1 primer sets, representing 34.15 sequence variants, were recovered. All sediment samples were dominated by a single lineage, except three Alpine samples which had co-occurrence of two different lineages. Furthermore, lineage turnover was observed in the Alps and northern Norway, suggesting that present-day phylogeographical studies may overlook past genetic patterns. Multiplexing primer is a promising tool for generating population-level genetic information from sedaDNA. The relatively simple method, combined with high sensitivity, provides a scalable method which will allow researchers to track populations through time and space using environmental DNA., (© 2024 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Environmental DNA (eDNA) techniques are emerging as promising tools for monitoring marine communities. However, they have not been applied to study the integrated effects of anthropogenic pressures on marine biodiversity. This study examined the relationships between demersal community species composition, key environmental features, and anthropogenic impacts such as fishing effort and seafloor litter using eDNA data in the central Tyrrhenian Sea. The results indicated that both fishing effort and seafloor litter influenced species composition and diversity. The adaptive traits of marine species played a critical role in their response to debris accumulation and fishing. Mobile species appeared to use relocation strategies, while sessile species showed flexibility in the face of disturbance. Epibiotic species relied on passive transport. The use of eDNA-based methods is a valuable resource for monitoring anthropogenic impacts during scientific surveys, enhancing our ability to monitor marine ecosystems and more effectively assess the effects of pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA -operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para -cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCE Clostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile -specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing., Competing Interests: The authors declare no conflict of interest.

As one of the powerful tools of species biomonitoring, the utilization of environmental DNA (eDNA) technology is progressively expanding in both scope and frequency within the field of ecology. Nonetheless, the growing dissemination of this technology has brought to light a multitude of intricate issues. The complex effects of environmental factors on the persistence of eDNA in water have brought many challenges to the interpretation of eDNA information. In this study, the primary objective was to examine how variations in the presence and development of biofilms impact the persistence of grass carp eDNA under different sediment types and flow conditions. This investigation encompassed the processes of eDNA removal and resuspension in water, shedding light on the complex interactions involved. The findings reveal that with an elevated biofilm development level, the total removal rate of eDNA gradually rose, resulting in a corresponding decrease in its residence time within the mesocosms. The influence of biofilms on the persistence of grass carp eDNA is more pronounced under flowing water conditions. However, changes in bottom sediment types did not significantly interact with biofilms. Lastly, in treatments involving alternating flow conditions between flowing and still water, significant resuspension of grass carp eDNA was not observed due to interference from multiple factors, including the effect of biofilms. Our study offers preliminary insights into the biofilm-mediated mechanisms of aquatic eDNA removal, emphasizing the need for careful consideration of environmental factors in the practical application of eDNA technology for biomonitoring in natural aquatic environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (β) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Environmental DNA and RNA (eDNA and eRNA; collectively eNA) analyses have the potential for non-invasive and cost-efficient biomonitoring compared with traditional capture-based surveys. Although various types of eNA particles, including not only mitochondrial eDNA but also nuclear eDNA and their transcripts, are present in the water, performances of eNA detection and quantification have not yet been evaluated sufficiently across multiple mitochondrial and nuclear genes. We conducted a tank experiment with ayu (Plecoglossus altivelis) to compare the detection sensitivity, yields per water sample, and quantification variability between replicates of each type of eNAs. The assay targeting the multi-copy nuclear gene exhibited a higher sensitivity than the assay targeting the mitochondrial gene, and both the target eDNA and eRNA concentrations per water sample were higher for the nuclear gene. On the contrary, variation in eRNA quantifications per sample does not necessarily correspond to that in eDNA, and the intra-sample quantification variability (represented as the CVs between PCR replicates) tended to be larger for eRNA than eDNA. Our results suggested that, even if suitable to the sensitive detection of species occurrence, the use of eRNA particularly derived from multi-copy nuclear gene may not be necessarily appropriate for the reliable assessment of species abundance. The findings in this study would help optimize eNA analyses for making biomonitoring and stock assessment in aquatic environments more efficient and reliable., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Batrachochytrium dendrobatidis (Bd) is a lethal fungal species that parasitizes vertebrates and is associated with the worldwide decline of amphibian populations. The development of sensitive, rapid detection methods, particularly DNA-based techniques, is critical for effective management strategies. This study evaluates the efficacy of DNA extraction and a portable PCR device in a mountable field laboratory setup for detecting Bd near the habitats of three critically endangered Atelopus toad species in Ecuador. We collected skin swabs from Atelopus balios, A. nanay, and A. bomolochos, and environmental DNA (eDNA) samples from streams in Andean and coastal regions of Ecuador. For eDNA, a comparison was made with duplicates of the samples that were processed in the field and in a standard university laboratory. Our findings revealed Bd detection in eDNA and swabs from 6 of 12 water samples and 10 of 12 amphibian swab samples. The eDNA results obtained in the field laboratory were concordant with those obtained under campus laboratory conditions. These findings highlight the potential of field DNA-based monitoring techniques for detecting Bd in amphibian populations and their aquatic habitats, particularly in remote areas. Furthermore, this research aligns with the National Action Plan for the Conservation of Ecuadorian Amphibians and contributes to the global effort to control this invasive and deadly fungus., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riascos-Flores et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Environmental DNA (eDNA) carrying antibiotic resistance gene (ARG) has attracted a great deal of attention because of its threat to the ecology and human health. Traditional porous adsorbents, such as microporous biochar and natural mineral, are low-effective in removing eDNA from sewage. This study used cuttlefish-bone (CB), a fishery waste, as an anticipated material to adsorb a model compound of eDNA from herring sperm (hsDNA). An interesting result was firstly observed that extremely high DNA adsorption on cuttlefish-bone pyrolysis derivative (CCB) was up to 88.7 mg/g, 3-10 folds higher than that of most other adsorbents in the existing literatures, which was attributed to the carbon film and large pores. To achieve an adsorption rate of 75 %, hsDNA adsorption took 96 h on CB but only 24 h on CCB, which was attributed to the fluent channel of CCB. The ligand exchange, Ca 2+ bridge and π-π interaction were identified as dominated adsorption mechanisms, based on FTIR and phosphate competition experiments. This study exploited a high-efficient, environmentally friendly, and low-cost adsorbent for treating ARG-contaminated soil and water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Environmental DNA metabarcoding reveals a vast genetic diversity of marine eukaryotes. Yet, most of the metabarcoding data remain unassigned due to the paucity of reference databases. This is particularly true for the deep-sea meiofauna and eukaryotic microbiota, whose hidden diversity is largely unexplored. Here, we tackle this issue by using unique DNA signatures to classify unknown metabarcodes assigned to deep-sea foraminifera. We analyzed metabarcoding data obtained from 311 deep-sea sediment samples collected in the Clarion-Clipperton Fracture Zone, an area of potential polymetallic nodule exploitation in the Eastern Pacific Ocean. Using the signatures designed in the 37F hypervariable region of the 18S rRNA gene, we were able to classify 802 unassigned metabarcodes into 61 novel lineages, which have been placed in 27 phylogenetic clades. The comparison of new lineages with other foraminiferal datasets shows that most novel lineages are widely distributed in the deep sea. Five lineages are also present in the shallow-water datasets; however, phylogenetic analysis of these lineages separates deep-sea and shallow-water metabarcodes except in one case. While the signature-based classification does not solve the problem of gaps in reference databases, this taxonomy-free approach provides insight into the distribution and ecology of deep-sea species represented by unassigned metabarcodes, which could be useful in future applications of metabarcoding for environmental monitoring., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barrenechea Angeles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Many studies have shown that environmental DNA (eDNA) sampling can be more sensitive than traditional sampling. For instance, past studies found a specific qPCR probe of a water sample is better than a seine for detecting the endangered northern tidewater goby, Eucyclogobius newberryi . Furthermore, a metabarcoding sample often detects more fish species than a seine detects. Less consideration has been given to sampling costs. To help managers choose the best sampling method for their budget, I estimated detectability and costs per sample to compare the cost effectiveness of seining, qPCR and metabarcoding for detecting endangered tidewater gobies as well as the associated estuarine fish community in California. Five samples were enough for eDNA methods to confidently detect tidewater gobies, whereas seining took twice as many samples. Fixed program costs can be high for qPCR and seining, whereas metabarcoding had high per-sample costs, which led to changes in relative cost-effectiveness with the number of locations sampled. Under some circumstances (multiple locations visited or an already validated assay), qPCR was a bit more cost effective than metabarcoding for detecting tidewater gobies. Under all assumptions, seining was the least cost-effective method for detecting tidewater gobies or other fishes. Metabarcoding was the most cost-effective sampling method for multiple species detection. Despite its advantages, metabarcoding has gaps in sequence databases, can yield vague results for some species, and can lead novices to serious errors. Seining remains the only way to rapidly assess densities, size distributions, and fine-scale spatial distributions., Competing Interests: The authors declare there are no competing interests., (©2024 Lafferty.)

Green tides, characterized by excessive Ulva prolifera blooms, pose significant ecological and economic challenges, especially in the South Yellow Sea. We successfully employed 18S environmental DNA (eDNA) metabarcoding to detect Ulva prolifera micropropagules, confirming the technique's reliability and introducing a rapid green tide monitoring method. Our investigation revealed notable disparities in the eukaryotic microbial community composition within Ulva prolifera habitats across different regions. Particularly, during the early stages of the South Yellow Sea green tide outbreak, potential interactions emerged between Ulva prolifera micropropagules and certain previously undocumented microorganisms from neighboring waters. These findings enhance our comprehension of early-stage green tide ecosystem dynamics, underscoring the value of merging advanced molecular techniques with conventional ecological methods to gain a comprehensive understanding of the impact of green tide on the local ecosystem. Overall, our study advances our understanding of green tide dynamics, offering novel avenues for control, ecological restoration, and essential scientific support for sustainable marine conservation and management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Environmental DNA (eDNA) is becoming an established tool across the biological and medical sciences. Despite the evident successes and wide adoption of eDNA approaches, some fundamental questions remain. For instance, there is almost a dogma in the field around the superiority of mitochondrial DNA for use in eDNA studies, however robust comparison with nuclear eDNA is widely lacking. The dominance of mitochondrial-based eDNA for animal and plant studies appears to be largely settled, despite a widespread lack of rigorous nuclear eDNA testing. Outside of the source organism the protections conferred on eDNA by the cell, mitochondrial and nuclear membranes are poorly understood, including the contribution of each to eDNA persistence and degradation. Utilizing shotgun sequencing to unbiasedly assess the level of nuclear and mitochondrial eDNA across samples, we reveal stark differences in nuclear versus mitochondrial eDNA persistence and abundance. By focusing too heavily on mitochondrial DNA alone the field is underutilizing eDNA's full potential., Competing Interests: Declaration of competing interest The authors declare having no financial interests/personal relationships which may be considered as potential competing interests. Funding agencies played no role in the research, interpretation or wiriting and review of the paper., (Published by Elsevier B.V.)

The Dongjiang River, a major tributary of the Pearl River system that supplies water to more than 40 million people in Guangdong Province and neighboring regions of China, harbors rich biodiversity, including many endemic and endangered species. However, human activities such as urbanization, agriculture, and industrialization have posed serious threats to its water quality and biodiversity. To assess the status and drivers of phytoplankton diversity, which is a key indicator of aquatic ecosystem health, this study used Environmental DNA (eDNA) metabarcoding combined with machine learning methods to explore spatial variations in the composition and structure of phytoplankton communities along the Dongjiang River, including its estuary. The results showed that phytoplankton diversity exhibited spatial distribution patterns, with higher community structure similarity and lower network complexity in the upstream than in the downstream regions. Environmental selection was the main mechanism shaping phytoplankton community composition, with natural factors driving the dominance of Pyrrophyta, Ochrophyta, and Cryptophyta in the upstream regions and estuaries. In contrast, the downstream regions was influenced by high concentrations of pollutants, resulting in increased abundance of Cryptophyta. The random forest model identified temperature, dissolved oxygen, chlorophyll a, NO 2 - , and NH 4 + as the main factors influencing the primary phytoplankton communities and could be used to predict changes during wet periods. This study provides valuable insights into the factors influencing phytoplankton diversity and community composition in the Dongjiang River, and demonstrates the application value of eDNA metabarcoding technique in large-scale, long-distance river biodiversity monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Urbanization and the persistent environmental changes present a major challenge for urban freshwaters and availability of water for humans and wildlife. In order to increase understanding of urban ecohydrology, we investigated the variability of planktonic bacteria and benthic diatoms - as two key biological indicators - coupled with insights from hydrochemistry and stable water isotopes across four urban streams characterized by different dominant water sources in Berlin, the German capital, over a period of one year (2021-2022). DNA metabarcoding results show that substantial spatio-temporal variability exists across urban streams in terms of microbial diversity and richness, with clear links to abiotic factors and nutrient concentrations. Bacterial communities showed clear distinction between effluent-impacted and non-effluent impacted streams as well as clear seasonal turnover. In-stream benthic diatom assemblages also showed robust seasonal variation as well as high species diversity. Our multiple-tracer approach is relevant for emerging questions regarding the increased use of treated effluent to supplement declining baseflows, the assessment of stream restoration projects and the impact of storm drainage and surface pollution on aquatic ecosystem health. eDNA analysis allows analysis of spatial and temporal patterns not feasibly studied with traditional analyses of macroinvertebrates. This can ultimately be leveraged for future water resource management and restoration planning and monitoring of urban freshwater systems across metropolitan areas., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Doerthe Tetzlaff reports financial support was provided by German Research Foundation. Doerthe Tetzlaff reports financial support was provided by Einstein Foundation Berlin. Doerte Tetzlaff reports financial support was provided by Federal Ministry of Education and Research Berlin Office. Michael Monaghan reports financial support was provided by Federal Ministry of Education and Research Berlin Office., (Copyright © 2023. Published by Elsevier Ltd.)

Monitoring is essential to ensure that environmental goals are being achieved, including those of sustainable agriculture. Growing interest in environmental monitoring provides an opportunity to improve monitoring practices. Approaches that directly monitor land cover change and biodiversity annually by coupling the wall-to-wall coverage from remote sensing and the site-specific community composition from environmental DNA (eDNA) can provide timely, relevant results for parties interested in the success of sustainable agricultural practices. To ensure that the measured impacts are due to the environmental projects and not exogenous factors, sites where projects have been implemented should be benchmarked against counterfactuals (no project) and control (natural habitat) sites. Results can then be used to calculate diverse sets of indicators customized to monitor different projects. Here, we report on our experience developing and applying one such approach to assess the impact of shaded cocoa projects implemented by the Instituto de Manejo e Certificação Florestal e Agrícola (IMAFLORA) near São Félix do Xingu, in Pará, Brazil. We used the Continuous Degradation Detection (CODED) and LandTrendr algorithms to create a remote sensing-based assessment of forest disturbance and regeneration, estimate carbon sequestration, and changes in essential habitats. We coupled these remote sensing methods with eDNA analyses using arthropod-targeted primers by collecting soil samples from intervention and counterfactual pasture field sites and a control secondary forest. We used a custom set of indicators from the pilot application of a coupled monitoring framework called TerraBio. Our results suggest that, due to IMAFLORA's shaded cocoa projects, over 400 acres were restored in the intervention area and the community composition of arthropods in shaded cocoa is closer to second-growth forests than that of pastures. In reviewing the coupled approach, we found multiple aspects worked well, and we conclude by presenting multiple lessons learned., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The quantity and quality of DNA isolated from environmental samples are crucial for getting robust high-throughput sequencing data commonly used for microbial community analysis. The differences in the nature and physicochemical properties of environmental samples impact DNA yields, and therefore, an optimisation of the protocols is always recommended. For instance, samples collected from corroded areas contain high concentrations of metals, salts, and hydrocarbons that can interfere with several steps of the DNA extraction protocols, thereby reducing yield and quality. In this study, we compared the efficiency of commercially available DNA extraction kits and laboratory-adopted methods for microbial community analysis of iron incrustations and oilfield-produced water samples. Modifications to the kits manufacturers' protocols were included to maximise the yield and quality. For iron incrustations, the modified protocol for FastDNA Spin Kit for Soil yielded higher DNA and resulted in higher diversity, including the recovery of low-abundant and rare taxa in the samples, compared to DNeasy PowerSoil Pro Kit. The DNA extracted with modified phenol-chloroform methods yielded higher DNA but failed to pass quality control PCR for 16S sequencing with and without purification. The protocols mentioned here can be used to maximise DNA recovery from iron incrustations and oilfield-produced water samples., (© 2024. The Author(s).)

The richness of plankton communities determines the fish productivity in the ocean, including important resources that rely on extractive fisheries, such as hakes (genus Merluccius) and tunas (genus Thunnus). Their preys forage on zooplankton, and the latter feed on phytoplankton. Inventories of plankton communities for scientific advice to sustainable fishing are essential in this moment of climate change. Plankton is generally inventoried using conventional methodologies based on large water volumes and visual morphological analyses of samples. In this study, we have employed metabarcoding on environmental DNA (eDNA) samples extracted from small water volumes for plankton inventory from twelve distant sampling stations in the East Atlantic Ocean. Zones rich in hake and tuna prey were detected from eDNA, and multivariate multiple regression analysis was able to predict those zones from diatom-based indices and planktonic diversity based on functional groups. Salinity was negatively correlated with the proportion of diatoms in phytoplankton, highlighting expected impacts of current global change on marine plankton communities. The results emphasise the importance of the plankton richness for fish productivity and support the utility of environmental DNA as a tool to monitor plankton composition changes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In recent years, environmental DNA (eDNA) has received attention from biologists due to its sensitivity, convenience, labor and material efficiency, and lack of damage to organisms. The extensive application of eDNA has opened avenues for the monitoring and biodiversity assessment of amphibians, which are frequently small and difficult to observe in the field, in areas such as biodiversity survey assessment and detection of specific, rare and threatened, or alien invasive species. However, the accuracy of eDNA can be influenced by factors such as ambient temperature, pH, and false positives or false negatives, which makes eDNA an adjunctive tool rather than a replacement for traditional surveys. This review provides a concise overview of the eDNA method and its workflow, summarizes the differences between applying eDNA for detecting amphibians and other organisms, reviews the research progress in eDNA technology for amphibian monitoring, identifies factors influencing detection efficiency, and discusses the challenges and prospects of eDNA. It aims to serve as a reference for future research on the application of eDNA in amphibian detection., (© 2023 Wiley Periodicals LLC.)

Our limited knowledge about the ecological drivers of global arthropod decline highlights the urgent need for more effective biodiversity monitoring approaches. Monitoring of arthropods is commonly performed using passive trapping devices, which reliably recover diverse communities, but provide little ecological information on the sampled taxa. Especially the manifold interactions of arthropods with plants are barely understood. A promising strategy to overcome this shortfall is environmental DNA (eDNA) metabarcoding from plant material on which arthropods leave DNA traces through direct or indirect interactions. However, the accuracy of this approach has not been sufficiently tested. In four experiments, we exhaustively test the comparative performance of plant-derived eDNA from surface washes of plants and homogenized plant material against traditional monitoring approaches. We show that the recovered communities of plant-derived eDNA and traditional approaches only partly overlap, with eDNA recovering various additional taxa. This suggests eDNA as a useful complementary tool to traditional monitoring. Despite the differences in recovered taxa, estimates of community α- and β-diversity between both approaches are well correlated, highlighting the utility of eDNA as a broad scale tool for community monitoring. Last, eDNA outperforms traditional approaches in the recovery of plant-specific arthropod communities. Unlike traditional monitoring, eDNA revealed fine-scale community differentiation between individual plants and even within plant compartments. Especially specialized herbivores are better recovered with eDNA. Our results highlight the value of plant-derived eDNA analysis for large-scale biodiversity assessments that include information about community-level interactions., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Monitoring environmental DNA can track the presence of organisms, from viruses to animals, but requires continuous sampling of transient sequences from a complex milieu. Here we designed living sentinels using Bacillus subtilis to report the uptake of a DNA sequence after matching it to a preencoded target. Overexpression of ComK increased DNA uptake 3,000-fold, allowing for femtomolar detection in samples dominated by background DNA. This capability was demonstrated using human sequences containing single-nucleotide polymorphisms (SNPs) associated with facial features. Sequences were recorded with high efficiency and were protected from nucleases for weeks. The SNP could be determined by sequencing or in vivo using CRISPR interference to turn on reporter expression in response to a specific base. Multiple SNPs were recorded by one cell or through a consortium in which each member recorded a different sequence. Sentinel cells could surveil for specific sequences over long periods of time for applications spanning forensics, ecology and epidemiology., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Our ability to assess biodiversity at relevant spatial and temporal scales for informing management is of increasing importance given this is foundational to identify and mitigate the impacts of global change. Collecting baseline information and tracking ecological changes are particularly important for areas experiencing rapid changes and representing data gaps such as Arctic marine ecosystems. Environmental DNA has the potential to provide such data. We extracted environmental DNA from 90 surface sediment samples to assess eukaryote diversity around Greenland and Svalbard using two separate primer pairs amplifying different sections of the 18S rRNA gene. We detected 27 different phyla and 99 different orders and found that temperature and the change in temperature explained the most variation in the community in a single linear model, while latitude, sea ice cover and change in temperature explained the most variation in the community when assessed by individual non-linear models. We identified potential indicator taxa for Arctic climate change, including a terebellid annelid worm. In conclusion, our study demonstrates that environmental DNA offers a feasible method to assess biodiversity and identifies warming as a key driver of differences in biodiversity across these remote ecosystems.

Deforestation is considered a major threat to biodiversity across many parts of the globe, but the biological impacts of this dramatic ecosystem disturbance often remain incompletely understood. In New Zealand - the world's last major landmass to be colonised by humans - widespread deforestation over recent centuries has left a highly fragmented suite of relict forest stands, ideal for assessing anthropogenic biological change. We hypothesise that this widespread environmental disturbance has underpinned repeated and predictable ecological shifts across distinct rivers and regions. Here we use freshwater environmental DNA (eDNA) data (113 samples across 38 locations; 89 insect taxa) to test for concordant biological shifts linked to this deforestation. eDNA analyses highlight consistent compositional and functional differentiation between forested versus deforested assemblages, including turnover of 'cryptic' congeneric taxa that are morphologically similar yet ecologically and genetically distinct. These dramatic biological shifts are evident even over fine spatial scales within streams, emphasising the widespread emergence of a novel 'deforested' assemblage. Our results illustrate that environmental change can drive predictable biological shifts across broad geographic regions, and highlight the power of eDNA for assessing anthropogenic ecosystem change over large geographic scales., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jonathan M Waters reports financial support was provided by Royal Society of New Zealand Marsden Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)