Environmental DNA最新文献

Short amplicon sequencing is a commonly used method to study the diversity of organisms in various habitats. The hypervariable regions of the small subunit rRNA gene (18S rDNA) are the most general barcodes for eukaryotes, which can provide detailed taxonomic information across a wide range of eukaryotic diversity. However, some organisms are often missed by universal primers, which have difficulty amplifying their barcodes. In this study, specific primers were designed for the amplification of the highly diverse 18S-V9 region of the Euglenozoa and Metamonada groups. The performance of the newly designed primers—V9Eug and V9Meta—was compared with the universal V9 primer on cultured communities derived from a range of freshwater environments of the Soos Natural Reserve and the Slavkov Forest in the Czech Republic. The V9Eug primer was more specific with Euglenozoa representing 91.8% of reads and 57.0% of OTUs, while the V9Meta primer showed lower specificity with only 48.4% of reads and 19.7% of OTUs assigned to Metamonada. Both the Euglenozoa and Metamonada primer pairs significantly improved recovery of their target groups compared to the universal V9 primer pair, detecting 2.7 and 1.8 times more OTUs, respectively. These results provide a more sensitive protocol for studying the diversity of these eukaryotic taxa.

Environmental DNA (eDNA) sampling from rivers has emerged as a promising new method for monitoring freshwater organisms of management concern. However, eDNA sampling cannot yet offer reliable estimates of a target species' abundance/biomass or confident determinations of a species' absence from a river segment. To unlock these abilities—and thereby greatly improve eDNA as a tool for management decision-making—the influence of local environmental factors on eDNA fate must be better understood. At nine river sites across the central California coast, we added a known quantity of novel eDNA (Brook Trout, Salvelinus fontinalis) and collected eDNA at sequential downstream distances for qPCR analysis. We then used random forest modeling to identify the most important environmental factors to reach-scale (≤ 200 m) sampling outcomes and characterize salmonid eDNA fate. Our final model identified six factors important to sampling outcomes, including five environmental factors (discharge, local catchment calcium oxide content, average depth of the sampling cross-section, presence of pools, and impervious cover of the watershed) and one factor regarding our experimental design (the number of qPCR technical replicates). Our results highlight the notable effects of cross-sectional area, turbulence, and catchment geology on eDNA fate, and we suggest the discharge and presence of pools as useful proxies for evaluating a site's favorability for eDNA recovery.

Parasite transmission occurs in complex environments comprising multiple matrices. Trematode parasites of ruminant livestock such as the liver fluke, Fasciola hepatica and the rumen fluke, Calicophoron daubneyi, show affinity with freshwater environments shared with their amphibious snail intermediate host, Galba truncatula. Isolation of environmental DNA (eDNA) from these parasites and their snail hosts in water draining from grazing land provides opportunities for improved molecular diagnostic detection and can help identify infection risks at farm level. The detection and quantification of eDNA from other environmental matrices has received less attention but would improve the understanding of parasite dynamics on pasture. Our study has considerably extended eDNA sampling methods for the detection of parasitic trematodes of ruminant livestock and their snail intermediate host by including for the first time the analysis of soil and herbage environmental samples alongside water collections. A droplet digital PCR (ddPCR) workflow was developed to detect parasite and snail eDNA from soil, herbage, and water collected from livestock farms. For the first time, C. daubneyi eDNA was isolated from agricultural soil alongside water samples and G. truncatula eDNA was detected in water, soil, and herbage samples. No environmental samples were positive for F. hepatica eDNA. Assessing multiple environmental matrices increased the number of positive sites. Future implementation of eDNA detection methods alongside traditional parasite diagnostics can underpin more holistic evaluations of the environmental components of parasite epidemiology and facilitate adaptation to changing disease patterns.

Many benthic deep-sea animals rely on carcasses from the overlying water column that sink to the seafloor and form local organic enrichments known as food falls. This flux of organic carbon from the shallow pelagic to the deep sea is part of the biological carbon pump (BCP) and as such contributes to carbon sequestration. For a complete understanding of local carbon budgets, it is crucial to identify the diversity and distribution of sinking carcasses which are difficult to detect by observational methods. Here, we analyzed the diet of the abundant amphipod scavenger, Eurythenes gryllus, by DNA metabarcoding to assess their potential to identify food falls in the Fram Strait, a gateway to the Arctic. E. gryllus scavenges on nekton but so far it was not certain whether this represents their main diet. We detected dietary taxa (26 in total) in 20 out of 101 analyzed amphipods. We found that amphipods primarily fed on larger nekton including fish, cephalopods, and mammals, with bony fish being the most targeted food source in terms of diversity and abundance. Only one amphipod had fed on a gelatinous organism. These results support the hypothesis that E. gryllus targets mostly nekton food falls. The diversity of dietary taxa differed between the Eastern and Western Fram Strait, which suggests regional variability in food falls availability. We also detected, for the first time in E. gryllus, infections with the parasitic dinoflagellate Hematodinium. This detection demonstrates the potential of metabarcoding for revealing both food web dynamics and host–parasite interactions in the deep sea. E. gryllus seems a promising “natural sampler” to monitor the diversity of deep-sea food falls which will help to investigate the importance of medium-sized food falls in local vertical carbon export in a rapidly changing Arctic Ocean.

Environmental DNA (eDNA) consists of genetic material shed by living organisms, including those that are deceased, offering a unique opportunity to detect and identify terrestrial insect pests without requiring visual identification. The sweetpotato whitefly, Bemisia argentifolii, and the twospotted spider mite, Tetranychus urticae, are notorious for causing crop losses through virus transmission and direct feeding. Our study aimed to: (1) assess the effectiveness of B. argentifolii literature-based PCR primers compared to newly developed primers for eDNA amplification, (2) evaluate the sensitivity of conventional PCR (cPCR) and real-time quantitative PCR (qPCR) for detecting eDNA of B. argentifolii and T. urticae, (3) establish a rapid eDNA processing methodology using the LGC Biosearch Technologies QuickExtract DNA extraction kit and the Qiagen DNeasy Blood and Tissue kit, and (4) test the specificity of the developed primers against non-target species. B. argentifolii and T. urticae were confined to tomato leaves (Solanum lycopersicum) using clip cages for 24 h, after which eDNA was collected from leaf surfaces using a water spray method, filtered, and processed for DNA amplification. While literature-based primers showed sufficient sensitivity, their specificity for eDNA applications was inadequate, prompting the design of novel PCR primers for both pest species. Positive eDNA detection was achieved with both amplification methods, with qPCR proving more reliable than cPCR due to the latter's inconsistent performance with positive control samples. We also introduced a rapid eDNA processing approach using the QuickExtract DNA extraction kit, contrasting it with the more conventional Qiagen DNeasy Blood and Tissue kit. We believe that our findings are the first step toward the practical use of eDNA as a highly sensitive, early detection technique.

National standardization of ecological sampling protocols between different agencies in Aotearoa New Zealand has historically been difficult to attain, creating challenges for combining datasets for national scale analyses. The introduction of new methods for biological monitoring, such as environmental DNA (eDNA), presents an opportunity to standardize aquatic sampling protocols prior to widespread adoption. The objective of this study was to optimize eDNA sample replication for the consistent characterization of freshwater fish and macroinvertebrate communities in flowing waters, and ultimately, to inform the development of robust national monitoring standards. A comparison of field replication and extraction methods (pooling of preservation buffer) was also trialed as part of this high replication (n = 16) eDNA study to assess any potential benefits in measuring species richness and reducing processing costs alongside replication optimisation. This involved two ‘syringe’ sampling methods (‘standard’ and ‘boosted’, eight each) conducted across 54 riverine sites throughout the country. No significant difference was found for species richness between the standardized (eight replicates) or boosted (16 replicates composited to eight) eDNA methods for fish and macroinvertebrates. Results indicated that six replicates were needed to consistently detect 89.5% of fish species likely to be present using field-based syringe eDNA sampling and preservation. However, an altitudinal species richness effect was observed for fish. For macroinvertebrates, six replicates were required to identify 86% of taxa identified to the NEMS (National Environmental Monitoring Standards) level used for the Macroinvertebrate Community Index (MCI: usually genera) while eight replicates were required to detect 89% of NEMS taxa. For fish and macroinvertebrate biodiversity, this study suggests that six replicates are a reasonable trade-off between effective community characterization and cost in New Zealand lotic systems.

The use of environmental DNA to detect species is now widespread in freshwater ecology. However, the detectability of species depends on many factors, such as the quantity of eDNA particles available in the environment and their state (e.g., free DNA fragments, organellar, or aggregated DNA particles). To date, the most advanced knowledge of the production and state of DNA particles concerns teleosts. Most often, these studies target mitochondrial genes, since they are present in multiple copies in a cell. However, it is likely that the characteristics of eDNA molecules vary greatly among taxa and genetic compartments, with direct consequences for species detection. Using an indoor mesocosm experiment, we compared the rate of mitochondrial and nuclear eDNA production and particle size distribution (PSD) of four distinct and common aquatic taxa (zebrafish, tadpole, isopod and mollusk). The tank water was filtered through a series of filters with decreasing porosity and mitochondrial and nuclear eDNA at each size fraction were quantified by qPCR. We found that the production and the size of eDNA particles varied greatly among taxa and genetic compartments. For most taxa, the number of nuclear eDNA particles released in water was higher than that of mitochondrial origin. The PSD of mt-eDNA showed a pattern common to all taxa: the relative number of particles increased from the smallest size fractions (0.2 μm and less) to the largest (over 1.2 μm), while the distribution of nu-eDNA was very different from one taxon to another. We also observed a high temporal variability in the quantity of eDNA particles and in PSD, although the latter was more complex to model. These results call for caution in how to sample and analyze eDNA in aquatic environments, particularly for organisms that emit small particles in small quantities such as isopods.

Swinhoe's (or Yangtze) giant softshell turtle (Rafetus swinhoei) is a large, critically endangered freshwater turtle and considered among the rarest species in the world. As of 2024, only two individuals have been confirmed to remain alive, one at the Suzhou Zoo in China and one in Xuan Khanh Lake, Viet Nam. The only hope for the long-term survival of R. swinhoei is finding additional, as yet undiscovered, animals that have thus far eluded detection by traditional survey methods. In recent years, numerous studies have been published on the use of environmental DNA (eDNA) for species detection and monitoring. This method takes advantage of the persistence of DNA in the environment, such as in water, soil, and air. An organism's DNA is shed into the environment through urine, feces, and the sloughing of skin. Species-specific quantitative polymerase chain reaction (qPCR) testing can be used to detect eDNA in samples collected from the environment. eDNA trials to detect R. swinhoei were initiated by the Asian Turtle Program and Washington State University in 2013. To expand the use of eDNA for species detection by conservationists, we developed and validated a first-of-its-kind innovative point-of-detection (POD) qPCR platform for the rapid, onsite detection of R. swinhoei from water samples. Here we show that the portable eDNA test kit can be used for the successful detection of R. swinhoei in a large body of water and that pooling filters may be a useful strategy to reduce test costs and improve detection efficiency. Use of this test can expand the search for R. swinhoei in unexplored and understudied lakes, reservoirs, and other bodies of water where this species may be present and could inform field surveys utilizing eDNA for other threatened species that are rare in nature.

Salmonid aquaculture, a major component of the Northern European, North American, and Chilean coastal economies, is under threat from challenges to gill health, many of which originate from plankton communities. A first step toward mitigating losses is to characterize the biological drivers of poor gill health. Numerous planktonic taxa have been implicated, including toxic and siliceous microalgae, hydrozoans, and scyphozoans; however, rigorous longitudinal surveys of plankton diversity and gill health have been lacking. In the current study, we present and assess an exhaustive identification approach combining both morphological and molecular methods together with robust statistical models to identify the planktonic drivers of proliferative gill disease (PGD) and fish mortality. We undertook longitudinal evaluation at two marine aquaculture facilities on the west coast of Scotland using daily data collected during the 2021 growing season (March–October). Examining these two different sites, one sheltered and one exposed to the open sea, we identified potentially new, important, and unexpected planktonic drivers of PGD and mortality (e.g., doliolids and appendicularians) and confirmed the significance of some established threats (e.g., hydrozoans and diatoms). We also explored delayed or “lagged” effects of plankton abundances on gill health and undertook a comparison of environmental DNA (eDNA) metabarcoding and microscopy in their ability to identify and quantify planktonic species. Our data highlight the diversity of planktonic threats to salmonid aquaculture as well as the importance of using both molecular and morphological approaches to detect these. There is now an urgent need to expand systematic longitudinal molecular and morphological approaches across multiple sites and over multiple years. The resultant catalogue of main biological drivers will enable early warning systems, new treatments, and, ultimately, a sustainable platform for future salmonid aquaculture in the marine environment.

There are numerous downsides and risks associated with electrofishing; hence, environmental DNA (eDNA) metabarcoding is becoming increasingly common in aquatic ecological studies. Generally, researchers agree that eDNA metabarcoding is more sensitive than electrofishing, and that eDNA metabarcoding is better at detecting rare species. As predatory species tend to be rarer than prey species, eDNA metabarcoding should hypothetically detect more predator species than electrofishing. Instead of supporting the notion that eDNA must replace electrofishing, or that eDNA and electrofishing must display the same results, the current study aims to establish the strengths and weaknesses of eDNA metabarcoding when compared to electrofishing. eDNA metabarcoding and electrofishing data were collected on three sampling dates at four experimental sites. A RV coefficient analysis confirmed that the eDNA metabarcoding data (RV = 0.395, p = 0.057) are statistically different from the electrofishing data. A paired Wilcoxon signed rank test revealed that eDNA data collection techniques detect more predatory species than electrofishing (p = 0.041). When the analysis was conducted for prey species a statistically significant difference did not occur (p = 0.661). Overall, the results of the study suggest that eDNA metabarcoding does not display the same results as electrofishing due to eDNA metabarcoding detecting predatory species at higher rates. The combined use of eDNA alongside electrofishing can help mitigate electrofishing's bias against predatory species, while electrofishing can address reliability concerns associated with eDNA. This collaborative approach ultimately enhances the accuracy of fish community assessments.