Environmental DNA最新文献

Molecular detection of environmental DNA (eDNA) and RNA (eRNA) allows highly sensitive qualitative (i.e., presence or absence) and quantitative (i.e., abundance) monitoring of aquatic bacteria. However, bacterial molecular diagnostics are limited by low positive predictive values. Protocols for bacterial eDNA and eRNA molecular monitoring have primarily focused on optimizing specimen collection, and the optimal method to purify bacterial nucleic material from postcollection aquatic specimens to maximize the analytical sensitivity of molecular diagnostics remains poorly defined. Accordingly, strategies to isolate bacterial eDNA and eRNA from fresh and saltwater were investigated. We evaluated two filtration and four nucleic acid purification systems as representative of current generation bacterial eDNA and eRNA isolation strategies for capacity to isolate bacterial eDNA and eRNA from prelysed (i.e., free-nucleic acids) and viable (i.e., colony forming units, CFU) bacterial cells. We also compared the sensitivities of reverse transcription quantitative PCR (RT-qPCR) and metagenomic shotgun microbiome sequencing. The optimal protocol used 0.7 μm borosilicate glass filters (Whatman plc) followed by extraction with the RNeasy PowerWater kit (Qiagen). The protocol had a very high analytical sensitivity (10⁻³–10⁰ ng and 10²–10¹ CFU detected in 500 mL) across multiple species of bacteria, when tested with either RTqPCR or metagenomic sequencing. Importantly, this study highlighted several limitations which are restrictive to both qualitative and quantitative bacterial eDNA and eRNA studies. First, a 12-h time course between sampling and extraction revealed significant species-specific changes in cell number and free-nucleic acid concentrations can occur postspecimen collection. Second, we found Gram-positive bacteria yielded less nucleic material compared to Gram-negative bacteria suggesting bacterial eDNA and eRNA studies could be biased by microorganism genome stability and extraction efficiency. This study highlights the need to define the species-specific diagnostic sensitivity of a protocol when monitoring aquatic bacterial eDNA and eRNA with molecular diagnostics.

Marine scientific trawl surveys are commonly used to assess the distribution and population size of fisheries-related species, yet the method is effort-intensive and can be environmentally destructive. Sequencing environmental DNA (eDNA) from water samples can reveal the presence of organisms in a community without capturing them; however, we expect the detectability of taxa to differ between eDNA and trawl surveys, and understanding how species traits and population variables contribute to detection differences can help calibrate our expectations from each form of sampling. Here, we coupled eDNA metabarcoding and capture trawl surveys in British Columbia, Canada, to examine species traits that explain recurrent differences in detectability between the two methods, including habitat, body size, and biomass. At the regional scale, 17 of 23 fish species (74%) captured by the trawl were detected by eDNA metabarcoding, and 39 additional species were detected by eDNA sampling only. We found that eDNA metabarcoding disproportionately detected trawl-caught species with greater local biomass (i.e., greater biomass in the adjacent trawl). Fish detected only in eDNA had a greater range of body lengths and a broader range of habitat preferences outside the trawls' target size and sampling areas. Our results suggest that with our level of sampling, eDNA metabarcoding can adequately recapitulate detection of fish communities detected by trawl surveys, but with a bias toward fish of high population biomass and greater inclusion of fish from outside the trawled area.

In the face of climate change, the accelerating nature crisis, and other anthropogenic impacts, conserving biodiversity demands effective monitoring. While innovative approaches have emerged to enhance biodiversity assessment, significant data gaps persist, particularly within marine ecosystems. In this study, we assess the utility of combining citizen (community) science with environmental DNA (eDNA) metabarcoding for characterizing and quantifying marine fish biodiversity. Over the summer of 2022, 32 volunteers conducted extensive water sampling in a large Norwegian fjord, yielding 96 samples. Contrasting eDNA findings with conventional observational surveys (such as a national species registration database and beach seine surveys) unveiled a substantial overlap in recorded species inventories. The eDNA citizen science initiative identified previously undocumented species and rediscovered others emphasizing an increase in warm-water species within the study area. Additionally, eDNA data unveiled reduced diversity within the inner fjord relative to the outer fjord. To conclude, our study demonstrates the successful integration of eDNA within a citizen science framework, facilitating comprehensive biodiversity tracking across coastal marine regions. These findings hold promise for advancing marine conservation efforts by providing valuable data to inform critical decision-making processes.

Environmental DNA (eDNA) has recently emerged as an effective tool for invasive species biosecurity. We explored the use of eDNA for the detection of khapra beetle (Trogoderma granarium, Everts 1898), an invasive insect of cereal grains and other food products that has a high global economic impact. We developed a novel method for aggregating khapra beetle eDNA deposited in stored grain that entails washing a sample of rice, filtering the sample, and detecting trace beetle DNA using a standard qPCR workflow. To explore the performance of this method, we raised 500 khapra beetle larvae within 500 g of rice over a 14-day period and then removed them. We then used this “spiked” rice to create a range of simulated densities of khapra beetle larvae. This lab approach mimics conditions that are comparable to field densities of ~1.4 to 180 beetles per 50 kg of rice (1/8 to 16 spiked rice grains per 100 g sample of clean rice), assuming DNA is uniformly distributed within the rice. We detected khapra beetle eDNA from all density levels tested. Logistic models revealed that eDNA amounts equivalent to what is left by ~1 khapra beetle larva in a 50 kg container of rice can be detected with 85% to >97% certainty, depending on the number of qPCR technical replicates run per sample. Based on this model, we estimated that for one 50 kg container of rice where beetle DNA is uniformly distributed, a single sample of 100 g with six technical replicates would be sufficient to be >99% certain that the container was free of khapra beetle eDNA (95% credible intervals: 97.7%–100%). Our results suggest that eDNA surveys may be useful as a cost-effective, first-step detection of khapra beetle in stored grain and provide a means to map the relative magnitude of khapra beetle transport pathways, informing allocation of conventional biosecurity inspection efforts.

Environmental DNA (eDNA) degradation influences the effectiveness of eDNA-based biodiversity monitoring, but the factors that determine the rate of decay of eDNA in terrestrial environments are poorly understood. We assessed the persistence of vertebrate eDNA from a mock vertebrate community created with soil from zoo enclosures holding 10 target species from different taxonomic classes (reptiles, birds, and mammals) and of different biomass (little penguin and giraffe). We examined species detection rates resulting from eDNA metabarcoding, as well as relative eDNA concentrations via qPCR, from soil samples over eight time points (0–12 weeks), during exposure to three ambient temperatures (10, 25, and 40°C) and three levels of ultraviolet B (UV-B) radiation (0%, 50%, and 100% intensity). We recorded considerable variation in detectability between species, independent of temperature, and UV-B effects. Quantitative polymerase chain reaction (PCR) indicated degradation of eDNA over time for all temperature and UV treatments, although it was still possible to detect eDNA from some species after 12 weeks. Degradation rates were lowest for high UV-B treatments, presumably due to UV-B reducing bacterial metabolism. The temperatures investigated did not influence eDNA decay. Our results indicate that eDNA in soil can persist under a range of temperatures and high UV radiation for longer than expected. Sheltered sites with minimal UV-B radiation, which have previously been considered ideal sites for terrestrial eDNA collection, may not be optimal for eDNA persistence in some cases due to microbial decay. A better understanding of eDNA degradation in terrestrial environments is needed to enhance the accuracy of eDNA metabarcoding for surveying terrestrial vertebrate communities.

To reach the renewable energy targets set by the European Commission, a tenfold expansion of the installed offshore wind farms is needed. Since the construction of offshore wind farms may affect local soft-sediment fauna, an efficient monitoring technique is needed to monitor the potential effects on the marine ecosystem. Here, we assess whether eDNA metabarcoding is a suitable alternative to monitor fish and epibenthos biodiversity in these difficult to access marine habitats. Water sampling and trawl surveys were conducted in parallel in 12 coastal and 18 offshore sites, the latter located inside and outside two offshore wind farms in the Belgian part of the North Sea. 12S eDNA metabarcoding retrieved 85.7% of the fish species caught in the beam trawls, whereas the COI eDNA metabarcoding only identified 31.4% of the epibenthic invertebrate species. Furthermore, the 12S marker resulted in an additional detection of 26 unique fish species, whereas the COI marker detected an additional 90 invertebrate species. Spatial patterns in alpha diversity recovered with eDNA metabarcoding were not significantly different from those observed with morphological determination. Significant differences were found in fish and invertebrate community structures between the coastal, transition and offshore zones as well as on the smaller wind farm scales, which agreed with the morphological beam trawl data. Indicator species found with morphological beam trawl monitoring for each of the three zones (coastal, transition, offshore) were also detected with 12S eDNA metabarcoding, and the latter method detected an additional 31 indicator species. Our findings show the need for adequate quality control of the obtained species lists and reveal that 12S eDNA metabarcoding analyses offers a useful survey tool for the monitoring of fish communities in offshore wind farms, but the used COI assay did not adequately capture the epibenthic communities as observed with beam trawl data.

Marine protists are globally distributed and sensitive to environmental conditions, which makes them a focal group when studying the effects of climate change on biodiversity and ocean health. However, they are a highly diverse group with varying evolutionary histories and morphologies and widely variable preservation potential in the fossil record. Thus, their past diversity and composition are poorly known. Paleogenetics, which relies, among other approaches, on DNA metabarcoding of sedimentary ancient DNA (sedaDNA), provides a promising avenue to explore the past history and responses of marine protists to global change. Choosing the right marker for sedaDNA studies is critical, striking a balance between marker length and taxonomic resolution. While marker guides exist for modern environmental DNA surveys, a thorough assessment of existing short markers for sedaDNA studies targeting protists is lacking. In this study, we report on a comparison of in silico PCR for eight short 18S rDNA markers, including one from the Tara Oceans initiative and a longer marker commonly used in modern marine eDNA studies. We analyze their taxonomic coverage and resolution, taxonomic overlap and uniqueness between markers, co-amplification of non-protist taxa, and amplicon size differences across taxonomic groups. Additionally, we provide a detailed analysis of diatoms, dinoflagellates, haptophytes, and chlorophytes. Our study is aimed at supporting project-specific marker choices for characterizing protist composition and diversity. While we focus on marine protists, our results are applicable to other aquatic and terrestrial environments.

The collection of environmental DNA (eDNA) and subsequent metabarcoding are useful tools for assessing marine fish biodiversity noninvasively. It is of particular importance to evaluate biodiversity in regions that are hard to access and thus less well studied. Sampling and preservation methods tailored to the specific circumstances are required. Aquatic eDNA is often captured on filters made of different materials and pore sizes, and subsequently stored under divergent conditions for varying periods of time. Previous studies on multispecies detection in marine systems have primarily focused on capture and extraction effects. Our study, in contrast, examined the effects of filter type, storage method, and storage time on DNA yield, alpha (i.e., ZOTU richness) and beta diversity (i.e., ZOTU composition) recovered from a marine ecosystem in Shark Bay, Western Australia. We compared two different filter types (cellulose-nitrate filters with pore sizes of 0.45 μm; glass-fiber filters with pore sizes of 0.1 μm), two storage methods (preservation in Longmire's solution and drying, respectively), various storage times (30–68 days) on two metabarcoding assays using different fish-specific primers. Our results showed that storage time decreased DNA yield and affected alpha and beta diversity estimates. Cellulose-nitrate filters stored in Longmire's solution proved to be the best combination with the smallest decrease in DNA yield, no effect on alpha diversity and consistent community compositions. Storing glass-fiber filters in Longmire's solution led to a decrease in eDNA yield and alpha diversity estimates with increasing storage time. Furthermore, the largest change in beta diversity for each metabarcode was found for glass-fiber filters regardless of storage method. Our results highlight the importance of considering storage time and interactions between storage method and filter when analyzing eDNA results, especially when storing samples for an extended time period or comparison of samples stored for different durations.

Aquatic fungi drive important ecosystem processes, which may be affected by anthropogenic stressors, including changes in water quality. However, due to the great diversity of aquatic fungi, our understanding of the relationships among water quality variables, fungal community composition, and ecosystem function remains incomplete. Here, we show that aquatic fungal community structure and functional diversity correlate strongly with water quality. We used a multi-marker survey of environmental DNA and long-term water quality data to survey 17 stream, pond, and tidal creek sites within a coastal drainage network in southeastern Virginia, USA, targeting both the ITS2 and LSU barcoding regions. The community composition of Chytridiomycota and Ascomycota (fungal phyla) and aquatic hyphomycetes (a functional group) were strongly correlated with habitat type and water quality variables. Functional diversity of aquatic fungi decreased with warmer water, due mainly to the reduced richness of saprotrophic groups, and increased with total particulate phosphorus, due to the richness of algal parasites. Community dissimilarity analyses of samples using both ITS2 and LSU yielded consistent results, but per-sample alpha diversity differed. This work reveals a surprising degree of local variation in aquatic fungal community composition and functional diversity within a coastal watershed, which was coupled with variation in water quality variables. Furthermore, eDNA sampling is an effective and efficient means to accurately characterize diverse aquatic fungal communities with exciting implications for addressing basic and applied research questions. Our work supports the prediction that community composition and functional diversity of aquatic fungi are impacted by impaired water quality and global change.

Sedimentary ancient DNA (sedaDNA) offers an important opportunity for investigating long-term community dynamics. Nevertheless, sedaDNA is challenging since DNA is degraded and fragmented over time. Of particular interest for such sedaDNA studies are phytoplankton communities, which are sensitive environmental indicators and important producers in aquatic systems. So far, only a few suitable metabarcoding primers for sedaDNA targeting phytoplankton exist. In this study, we introduce new metabarcoding primers targeting cyanobacteria and dinoflagellates. They amplify short, ~200-bp ribosomal 16S and 18S DNA fragments. We compared these primers against published ones, uncovering distinct communities captured by different primer sets. The newly designed dinoflagellate and cyanobacterial primers revealed unique sets of amplicon sequence variants (ASVs) compared to published primers, highlighting the impact of primer choice on describing community composition. We also explored the effect of amplicon length on metabarcoding success over a sample age. Observed trends suggest that amplification success decreases with longer amplicons, probably as a result of DNA degradation in older sediment samples. Lastly, strong DNA preservation challenges emerged in sediment samples older than 7000 BP, corresponding with oxic phases of the Baltic Sea bottom water. This emphasizes the importance of age, sediment type, and preservation conditions when interpreting sedaDNA results. Despite limitations in temporal resolution, the study shows that sedaDNA-based fluctuations in the phytoplankton community are consistent with well-known environmental stages. More research is necessary to understand (1) DNA preservation and its impact on reconstructed communities and (2) impact of abiotic conditions on phytoplankton communities.