Environmental DNA最新文献

Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring aquatic biodiversity in extreme environments like the Qinghai–Tibet Plateau (QTP), where traditional methods face significant challenges. However, key knowledge gaps remain, particularly in selecting optimal sampling substrates for target taxonomic groups and understanding substrate-specific detection differences. Here, using eDNA metabarcoding with five primer sets (targeting mitochondrial, nuclear, and nucleoid sequences), we systematically compared biodiversity detection between sediment and water samples collected from five sites in the Dongjicuona Lake across the tree of life (encompassing archaea, bacteria, algae, fungi, plants, protozoa, invertebrates, fishes, birds, and mammals). The results showed that sediment eDNA exhibited significantly higher amplicon sequence variant (ASV) richness compared to water eDNA for most taxonomic groups. In contrast, the opposite pattern was observed for fishes, while no significant differences were observed for fungi and birds. Community composition differs significantly between sediment and water samples for most taxonomic groups, except for mammals. Across all taxonomic groups, 18.0% of ASVs were shared between sediments and water, with fishes showing the highest overlap (42.6%) and archaea the lowest (4.5%). Additionally, the percentages of ASVs contributing to major community dissimilarity (cumulatively accounting for 80%) ranged from 6.4% (invertebrates) to 17.4% (protozoa), with most taxa exhibiting distinct habitat preferences. These findings support water sampling as an effective method for monitoring fish diversity, while emphasizing the need for a multi-substrate approach to comprehensively monitor other taxonomic groups in plateau lakes.

Fungi play essential roles in key ecosystem functions and processes, yet they often occur in inconspicuous, species-rich, and complex communities that remain difficult to study. Studies of fungal communities based on DNA extracted from environmental samples commonly rely on clustering sequence reads into units of diversity, followed by taxonomic identification and, in some cases, linkage to ecological traits. In this study, we evaluated how two clustering approaches—amplicon sequence variants (ASVs) and operational taxonomic units (OTUs)—affect the characterization of fungal communities. Despite minor differences, both approaches recovered consistent taxonomic patterns and community structure. Although both methods produced a similar total number of sequence clusters, they differed in representation of fungal community composition. All ASV representative sequences matched OTU representative sequences with at least 92.2% similarity, whereas several rare OTUs showed low similarity to ASV reads, suggesting differences in the detection of low-abundance taxa. However, only a small fraction of OTU reads (< 0.1%) lacked a corresponding ASV, indicating that ASVs captured nearly all OTU-defined taxa. In contrast, 14% of ASV reads assigned to species hypotheses (SHs) did not match any OTU reads assigned to SHs, whereas only 1.3% of OTU SH-assigned reads lacked a corresponding ASV match. ASVs generally provided higher resolution than OTUs, as abundant SHs were often represented by multiple ASVs, suggesting that ASVs capture intraspecific diversity. Consequently, ASVs should not be used as direct species proxies but instead require post hoc grouping to reflect species-level diversity. OTUs-based community composition aligned more clearly with soil properties, particularly the N:C ratio. Overall, both approaches provided a similar overview of broad-scale species richness. The choice between two clustering methods depends on the research question and the desired level of taxonomic resolution, and our results provide little support for the claim that ASVs should categorically replace taxonomic units in marker-gene data analysis.

Bennett's wallabies Notamacropus rufogriseus, introduced to New Zealand from Australia in the late 1800s, strongly exemplify the detection challenges posed by invasive terrestrial species that are rare, cryptic, and highly mobile. Across their invasive range, N. rufogriseus occupy large landscapes at low densities, making their surveillance challenging. Recent research has demonstrated that airborne environmental DNA (eDNA) can rapidly identify terrestrial vertebrate diversity in an area. Leveraging these findings, we investigate the utility of airborne eDNA for the targeted monitoring of N. rufogriseus, using a novel, probe-based quantitative PCR assay. The effects of filtration material, collection method (active versus passive), distance from the source, and environmental conditions were examined for their effects on airborne detection probability, using a captive population of wallabies in a controlled park setting. A total of 110 airborne samples were collected, 55 with active (battery-powered fan) samplers and 55 passive (nonpowered) samplers, across six distinct experimental periods at distances of 0, 10, 100, and 1000 m from the closest known source of wallaby DNA. Filters designed to capture coarse particles (> 10 μm) significantly improved detection rates and DNA recovery for actively collected samples, compared to filters targeting finer particles (1–10 μm). Active samplers significantly outperformed passive samplers in overall detection rates, particularly at shorter ranges from the target. Distance from the source had a significant negative effect on detection probability. Detection rates declined sharply beyond 10 m but remained possible up to 1 km from the source for both collection methods. These findings demonstrate that airborne eDNA can detect terrestrial vertebrate species at ecologically relevant distances, supporting its potential for landscape-scale surveillance. Notably, these results underscore the importance of optimizing sampler design when applying airborne eDNA for targeted species monitoring.

The effective use of environmental DNA (eDNA) tools is contingent on strict adherence to established and validated methods. Differences in eDNA methods and quality assurance protocols may contribute to variability in results. However, quality assurance measures such as proficiency testing can provide independent evaluation of laboratory performance against pre-established test criteria. With this commentary, we discuss how broad implementation of recurring proficiency testing in eDNA laboratories can build decision-maker confidence in eDNA results. It can also create a culture of continuous evaluation and improvement that minimizes error and meets performance requirements to inform the sustainable use or monitoring of natural resources. We provide an overview of proficiency testing across molecular disciplines, review the state of proficiency testing in eDNA applications, and draft a roadmap for the expanded application of proficiency testing informed by best practices for targeted eDNA detection. We suggest that best practice proficiency testing can be conducted by an independent, third-party sample provider. By demonstrating that laboratories are competent and capable of producing reliable results, implementation of proficiency testing best practices should foster confidence in eDNA measurements and its use in decision-making processes. Increased confidence in eDNA methods and a clear expectation of what is considered satisfactory performance are also likely to create more favorable conditions for investments in eDNA-based monitoring.

Marine Protected Areas (MPAs) play a crucial role in conserving marine biodiversity while providing ecological, social, and economic benefits. Effective monitoring is essential for assessing changes in biodiversity and ensuring the sustainability of MPAs. In this context, biodiversity monitoring in Karimunjawa National Park (KNP) provides an excellent opportunity to examine effective monitoring practices. Traditionally, biodiversity assessments have been conducted through visual census methods, which have limitations such as challenges in species identification, time constraints, and high survey costs. To complement visual surveys, this study employed environmental DNA (eDNA) metabarcoding with the 12S rRNA gene, utilizing Oxford Nanopore sequencing to assess fish diversity across different zonation systems within KNP. eDNA analysis detected a total of 183 fish species, with 87 species (38% of the 229 species recorded by visual census) and 25 families (71%) shared between the two methods. Alpha diversity (ANOVA, p > 0.05) showed no significant differences between sites and zonation, whereas community structure (PERMANOVA, p < 0.05) revealed significant differences between sites and zonation. Additionally, eDNA offered complementary insights by detecting broader functional traits than the visual census, such as nocturnal behavior, habitat preferences, and migratory variations of fish species, whereas the visual census predominantly only recorded reef-associated and nonmigratory taxa. These findings demonstrate that eDNA, particularly when integrated with Oxford Nanopore sequencing, is a powerful tool for marine biodiversity monitoring. Standardizing bioinformatics workflows is crucial for ensuring data comparability and maximizing the effectiveness of eDNA-based conservation strategies in Indonesia's MPAs.

The ability of environmental DNA (eDNA) to provide rapid assessments of mammal taxa composition at the watershed scale can make it an efficient survey method on large-scale landscapes, complementing camera traps. Due to the rugged and inaccessible terrain of many areas in Bhutan, camera trapping is associated with logistical challenges, increasing the cost of sampling considerably. In this study, conducted in the Upper Punatsangchhu catchment basin of Bhutan, we investigated the ability of eDNA water samples to capture the diversity of terrestrial mammals in comparison with camera trapping, using six watersheds within the basin as a baseline sampling frame. Combined, the two methods detected a total of 72 mammalian species: eDNA metabarcoding identified 60 species, while camera trapping detected 33 species, with an overlap of 21 species between the two methods. In addition, eDNA metabarcoding detected 90% of the IUCN Red List species detected by the camera traps. Small mammals were frequently detected using eDNA metabarcoding, while camera trapping more often detected large mammals. The mean detection probabilities recorded from eDNA were higher for all species grouped by orders and size categories compared with camera trapping. Biodiversity models based on eDNA metabarcoding and camera trapping both retrieved dominant effects of temperature and isolation in structuring the mammal assemblage. We conclude that eDNA sampling based on watersheds accurately represents the spatial distribution of species across each watershed in our study area in Bhutan to provide a rapid assessment of mammals from river water.

Environmental DNA (eDNA) is a non-invasive monitoring approach increasingly used to detect marine organisms; however, misunderstandings of the temporal variability in eDNA detection have limited its integration within management decisions. A clearer understanding of the periodicity (e.g., seasonality) and duration (e.g., weeks, months) of species eDNA detection is essential to optimize sampling design and data interpretation. As such, this study aims to provide a representative assessment of optimal eDNA detection windows across diverse taxonomic groups, primers, and geographic regions using eDNA metabarcoding. Coastal marine presence-absence eDNA data were collected along the Northwest Atlantic coast, in the Bay of Fundy, Scotian Shelf, and Baffin Island. eDNA detection window(s) were defined as unimodal, contiguous months having greater than 75% detection probability and were calculated for each taxon for each primer in each region. Most marine species exhibited short eDNA detection windows (1–2 months). The optimal sampling periods and durations were conserved among closely related species, highlighting the importance of considering biological traits when designing and interpreting eDNA studies. Additionally, primer choice influenced the optimal detection periods, with higher seasonal variation in community composition and detection rates using universal COI and 18S primers compared to fish 16S and 12S primers. These results demonstrate that ignoring seasonal variation may cause false negatives, inefficient sampling, and reduced data comparability across independent studies. Thus, we propose a set of guidelines aimed at the development of optimal sampling designs for coastal ecosystems and the interpretation of trends across datasets.

Infectious hematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) are major pathogens in aquaculture. Detection of both viruses requires lethal sampling, as it is typically performed by testing of internal organs. However, this approach only identifies the pathogens once viral transmission has already occurred and infection is underway. As both viruses can spread through water, an efficient environmental detection method could significantly improve disease control and prevention of transmission, and enhance welfare by reducing lethal sampling. In this study, we assessed the feasibility of detecting IHNV and VHSV environmental RNA (eRNA) in water during in vivo infection trials in Oncorhynchus mykiss, using RT-qPCR. By sampling at multiple time points post-exposure, we could evaluate the efficacy of detection at decreasing viral concentration in water over time. Viral eRNA was recovered using different methods: viral concentration with polyethylene glycol (PEG) precipitation, filter membranes, filtered water, or unprocessed water samples. RT-qPCR values were compared to the quantification of infectious particles using viral titration, with RT-qPCR consistently detecting higher eRNA copies/mL than the cell-based assay to detect infectious particles (TCID₅₀/mL). eRNA detection from the filter membranes significantly outperformed the other tested methods, enhancing eRNA recovery particularly at lower viral concentrations. Notably, eRNA detection in water was still possible after the peak of mortality for both viruses. Additionally, IHNV eRNA was successfully detected in farm water samples, even up to 50 days post initial fish tissue diagnosis, confirming the feasibility under real conditions. This study provides the first quantification of IHNV eRNA from aquaculture water and demonstrates the effectiveness of a filtration-based viral concentration method for environmental surveillance. These findings suggest that eRNA-based RT-qPCR detection of IHNV and VHSV from water could be a valuable addition to current diagnostic tools, potentially enabling earlier detection and improved containment in aquaculture and the surrounding environment.

In the face of ongoing anthropogenic pressures on global biodiversity, effective monitoring strategies are essential for understanding species distributions and guiding conservation. Leptonycteris nivalis, L. yerbabuenae, and Choeronycteris mexicana are nectar-feeding bats of conservation concern that occur in the southwestern United States and Mexico. Leptonycteris nivalis and L. yerbabuenae are morphologically similar and difficult to differentiate in the field, making eDNA a particularly relevant monitoring tool. Since previous studies have shown that eDNA detection of these species is possible when swabs are taken from their dietary flowers, we sought to determine whether they can be detected from artificial feeders, which are commonly used on residential properties to attract hummingbirds and are a known supplemental food source for L. yerbabuenae. Between 2023 and 2024, citizen scientists (n = 12) in Arizona and New Mexico took 306 swabs of hummingbird feeders, which we tested with an existing qPCR assay for L. nivalis and newly-developed qPCR assays for L. yerbabuenae and C. mexicana. Leptonycteris yerbabuenae and C. mexicana showed the highest number of detections (300 and 274 swabs, respectively). Previously only known to occur in the U.S. in Texas and New Mexico, we detected L. nivalis near Portal, Arizona, within 50 km of documented foraging range in western New Mexico. Detections of this endangered species suggest its range extends beyond Hidalgo County, New Mexico and into neighboring Cochise County, Arizona, highlighting a need for increased surveillance of this species. Our work with artificial nectar feeders expands the eDNA detection method for nectar-feeding bats to the human-wildlife interface and shows that citizen science can be successfully used for eDNA surveys. Such methods provide an alternative to mist netting or acoustics, which can help monitor occupancy and clarify range extensions. This work corroborates other studies illustrating that eDNA can effectively detect and monitor terrestrial species.

In recent years, European countries have intensified efforts to control or limit the spread of invasive freshwater crayfish and the crayfish plague pathogen Aphanomyces astaci, while working to conserve native species such as the noble crayfish (Astacus astacus). Although crayfish shed relatively low amounts of DNA into their environment, environmental DNA (eDNA) approaches have proven effective for detecting their presence. A range of protocols and equipment is currently used in eDNA-based monitoring of freshwater crayfish. To evaluate how methodological variation influences detection accuracy, we conducted the first European interlaboratory ring test using eDNA to detect A. astacus, the invasive signal crayfish Pacifastacus leniusculus, a chronic carrier of A. astaci, and the pathogen itself. The aim is to harmonize monitoring methods for crayfish and disease surveillance across laboratories. Eleven teams from thirteen European countries participated, each using its own equipment and protocols to collect and filter water from indoor tanks and outdoor ponds where the presence of A. astacus and P. leniusculus had been experimentally manipulated, as well as from a natural lake containing a P. leniusculus population. The resulting samples were analyzed in each team's laboratory. Despite methodological differences, all teams successfully detected DNA from both crayfish species in indoor tanks (3–10 crayfish/m³). However, detection accuracy declined in outdoor ponds where crayfish density was an order of magnitude lower (0.32 crayfish/m³). Detection was most variable for A. astaci, likely due to its very low prevalence in the host stock. Our study demonstrates the challenges of achieving consistent eDNA results across laboratories and highlights the importance of interlaboratory comparisons. It also underscores the need to identify sources of variability and error, an essential step toward developing robust and standardized protocols. This multinational intercalibration and exchange of knowledge improved methodology and enhanced reliability in crayfish detection.