Environmental DNA最新文献

The marine aquaculture industry and regulators are in the process of implementing environmental DNA (eDNA) metabarcoding of microbial communities for compliance monitoring. This requires standardization of sampling, laboratory, and data analysis protocols. Towards this goal, we in this study completed two further milestones using samples collected from two Scottish salmon farms: (i) We tested the effect of using two different PCR protocols (i.e., different DNA polymerases, master mixes, and annealing temperatures), which are frequently being used in eDNA biomonitoring of aquaculture installations, for the amplification of the taxonomic marker gene (V3-V4 hypervariable region of the bacterial 16S rRNA gene). (ii) We quantified sampling background noise obtained from eDNA samples and statistically compared results with the sampling bias observed in macrofaunal samples from the same source sediments. We detected differences in bacterial community structures resulting from the performance of different PCR protocols, profoundly influencing the interpretation of biomonitoring results. Furthermore, we found that sampling-induced errors for eDNA samples were similar to errors for macrofaunal samples collected according to compliance monitoring protocol (~25% variability in both cases). Finally, we showed that within-grab variances of microbial community structures were in the same order of magnitude (less than 10× difference in all cases) as the one obtained from replicate grabs collected from the same locale (impact category). Based on our findings, we suggest using a consistent PCR protocol for biomonitoring efforts to improve the comparability of results, especially when different service providers are conducting the biomonitoring. We propose a sampling scheme to be considered in eDNA biomonitoring that includes taking three replicate grabs at each locale, with one replicate sample from each grab. This minimizes sampling-induced errors and makes upcoming eDNA-based monitoring results comparable with previous compliance monitoring results obtained from macrofaunal data.

Environmental nucleic acid-based assessments are powerful tools for understanding microbial ecology, and environmental degradation in aquatic environments. This approach is particularly useful in guiding restoration in estuaries, some of the most degraded ecosystems in the world. The recent popularity of this approach has been accompanied by a parallel increase in the diversity of applied methods. A range of best practice methods exist across the field that can be employed and are selected based on environmental considerations such as physicochemical gradients, maximizing yield, and quality of nucleic acids sampled across sites within a study area. A consistent approach to intra-study nucleic acid sampling also ensures accurate comparison between those sites. This study evaluates environmental nucleic acid (eNA) sampling methods across salinity gradients in aquatic ecosystems, focusing on the impact of preservation techniques on environmental DNA (eDNA) yield and environmental RNA (eRNA) yield and quality. Fieldwork was conducted at three sites within the Coorong estuary system in South Australia, representing low salinity, marine, and hypersaline conditions. Snap freezing and LifeGuard preservation solution treatments were applied in situ to compare their effects on nucleic acid yields and eRNA integrity. Snap freezing enhanced eDNA yield in low salinity sediments but negatively impacted eRNA integrity in marine and hypersaline conditions. Conversely, treatment with preservation solution consistently improved both eDNA and eRNA recovery across all salinity levels, which makes this approach a good candidate for preserving eNA molecules across environmental gradients. The study underscores the necessity of tailoring sample preservation methods to specific environmental conditions for accurate eNA-based microbial community assessments in coastal ecosystems. These findings contribute to the development of robust eNA sampling protocols for benthic communities under varying salinity conditions.

Ecologists, biologists, and conservation scientists are increasingly interested in the use of environmental DNA (eDNA) data for research and potentially decision-making. While commercial DNA extraction kits are typically user-friendly and accessible, they may fail to deliver the desired results with inherently complex eDNA samples, necessitating protocol optimization or educated selection of alternative approaches. To this end, knowledge of the basic steps and principles of DNA extractions is essential, but traditional education tracks in ecology, conservation, and environmental management typically do not include in-depth training in molecular methods. The primary objective of this paper is to enable scientists with an ecological background and limited molecular training to understand the four key steps of eDNA isolations, and to use this expertise to their advantage. We describe the purpose of commonly used reagents and chemicals, point out alternatives for each key step, explain the impact of certain choices regarding isolation approaches on DNA integrity and purity, and highlight the possibility of a tailor-made “mix and match” approach. We anticipate that this paper will enable field ecologists to develop a deeper understanding of the mechanisms and chemistry underlying eDNA extractions, thus allowing them to make informed decisions regarding the best eDNA extraction method for their research goals. Our intention is not to provide comprehensive, step-by-step protocols, but to offer guiding principles while highlighting alternative solutions. Finally, we hope that this paper will act as a useful resource to support knowledge transfer and teaching.

The environmental DNA (eDNA) approach is an emerging tool for monitoring marine biodiversity. However, the sampling effort needs optimization according to the site characteristics and target taxonomic groups. In this study, we optimized the eDNA sampling effort in terms of sample volume and number of replicates to monitor the diversity of marine vertebrates (mainly fish) in Hong Kong's subtropical waters that show a gradient of estuarine to oceanic waters. To maximize detection, we used three pairs of metabarcoding primers (12S-v5, MiFish-U, and MiFish-E). We compared vertebrate diversity in 78 water samples, ranging from 1 to 10 L, collected from oceanic and estuarine sites. Metabarcoding yielded a total of 140 vertebrate species, of which 18 were unique to the estuarine site, 66 unique to the oceanic site, and 56 shared between both sites. The detected species were predominantly ray-finned fish (136 species), and the three primer pairs exhibited differential sensitivity toward different taxa, especially cartilaginous fish and cetaceans. Increasing sampling volume per replicate generally increased the total detected species, average species per replicate, and species coverage, and sampling 3 or 4 × 4 L represented the most efficient sampling effort for the estuarine and oceanic sites, respectively. The diversity analysis revealed that sampling >2 L per replicate reduced variability and improved diversity analysis. The results also showed that a larger sampling volume per replicate increased the probability of detecting endangered, indicator, invasive, and elusive species, with 4 L representing the most efficient volume. This study recommended sampling 4 L per replicate and 3 replicates for estuarine and 4 for oceanic sites, respectively for effectively monitoring marine fish in subtropical waters using the eDNA approach.

Management and conservation of species-rich tropical freshwater systems require reliable information on the diversity and distribution of species present. Here, we used environmental DNA metabarcoding to reveal the diversity of the fishes in the Rufiji River catchment of central Tanzania. Across 174 samples from 49 sites, and using a newly developed reference library, we mapped the presence of 66 fish species from an estimated 91 that we are confident are present in the system. We found clear evidence of community structuring of the assemblage linked to key environmental gradients—elevation, temperature, and turbidity. We also identified core distributions of rare or threatened taxa, including migratory species such as the anguillid eels. With a focused analysis of 50 samples collected over a small spatial scale (<2 km) from the Kilombero River, we showed that each single sample can capture an average of 23.1 species, while three samples can capture 39.4 species, from a total of 56 species encountered in the 50 samples. Collectively the results help to identify species vulnerable to ongoing change in the catchment, including dam construction and agricultural intensification. The results clearly demonstrate how eDNA-based metabarcoding can reliably describe the diversity and distributions of riverine fish species across a catchment, providing standardized information that will be valuable for environmental management.

Monitoring alien species is critical to their management. However, early detection of invading alien freshwater fish can be challenging due to the difficulty of observing fish in low abundance. Environmental DNA (eDNA) has emerged as a new and potentially more sensitive method for sampling invasive species as compared to conventional methods, but the comparative financial cost is not often assessed. Adoption of eDNA by managers requires studies that showcase its cost-effectiveness relative to conventional approaches. Here we use eDNA to assist in the management of an aggressive alien fish, the pearl cichlid (Geophagus brasiliensis), that is invading an urban river in south-western Australia. We applied an occupancy model to survey data collected 6 years apart (2015, 2021) to assess how the species' distribution had changed and to evaluate whether an instream barrier had the potential to limit upstream invasion. To understand the effectiveness of eDNA, we used our model to quantify the relative efficiency (capture probability) of two eDNA sampling methods (active eDNA and passive eDNA) and fyke netting, as well as the number of replicate samples required per site to deliver >95% detection. We coupled the number of replicates needed with the cost per replicate to determine the cost-efficiency of each method. We found that G. brasiliensis abundance was higher in downstream reaches in both survey years, and there was no evidence that its distribution had changed through time. However, G. brasiliensis was present above the instream barrier. Active eDNA sampling was considerably better at detecting G. brasiliensis than the other methods, making it the most cost-effective method. Fyke nets came in a close second, and passive eDNA was a very distant third. Our results directly inform management in the study river and broadly highlight the cost-effectiveness of active eDNA as a freshwater biosecurity tool.

Nature-like bypasses refer to fishways that simulate natural streams. Apart from facilitating fish migrations, bypasses possess the capacity to enhance biodiversity in dammed rivers. Feasibility of environmental DNA (eDNA) as a tool for bypass assessments is unknown. This study investigated fish eDNA in 10 bypasses and their main channels. Initially, the relative DNA flow-through was estimated in bypasses. Subsequently, the impact of environmental factors and bypasses on fish assemblages was evaluated, and the robustness of the eDNA and electrofishing methods was assessed pertaining to bypass monitoring. The eDNA flow-through was computed using an equation to estimate the residual DNA at specified distances downstream of the source site. The relative DNA flow-through was lowest in the longest bypass with low flow rate and highest in the shortest bypass with higher flow rate and was dependent on the DNA decay rate coefficient used. The redundancy analysis revealed significant effects of spatial location, agriculture, catchment area, and bypass length on the species composition. The within-river analyses indicated significant and nonsignificant bypass effects on species composition and total species richness, respectively. Higher richness and DNA abundance of migratory and threatened species were observed in the bypasses than in the main channels. The eDNA samples displayed higher species richness compared to electrofishing. The species composition of the bypass eDNA samples was intermediate between that of the main channel eDNA and bypass electrofishing samples, which further corroborated performance of eDNA flow-through in bypasses. Therefore, bypass eDNA samples represented variable mixtures of local and main channel assemblages, indicating relatively low robustness of eDNA for quantitative and spatially accurate bypass assessments. Nevertheless, these results demonstrate practical applicability of eDNA in surveying the presence of desired species and evidence of the benefits of bypasses in supporting biodiversity and species threatened by damming.

Understanding the distribution and habitat use of endangered species is essential for conservation efforts. Environmental DNA (eDNA) analysis has become a more common approach to defining species habitat occupancy through identification of residual DNA in water samples and has potential to detect populations that are in low abundance or use habitats over a large geographical range. Here, we optimized an eDNA protocol to detect the presence of the endangered white sturgeon (Acipenser transmontanus). We implemented lab-based experiments to understand the sensitivity and persistence of white sturgeon eDNA and then applied these methods to habitats with known white sturgeon abundances categorized as high, low, or not present. Using quantitative PCR (qPCR) and a modified StrAci1N-flap primer set, white sturgeon eDNA was detected in water collected from tanks holding white sturgeon down to a dilution of 10,000× (estimated eDNA concentration of 0.00035 μg/L—0.00176 μg/L). Following the removal of white sturgeon from the tanks, the eDNA signal decreased with time but could be detected for up to 7 days. In the field, all sites with high abundances of white sturgeon returned positive eDNA detections. We did not detect white sturgeon eDNA at sites with low abundance or in areas where they were not expected to be present. Results from this work further advance our interpretation of eDNA from wild populations and provide a noninvasive method to advance recovery efforts by identifying species presence in areas of suspected use or to guide additional inventory efforts.

Freshwater ecosystems, home to a remarkable diversity of species, are facing severe threats from human activities such as climate change, habitat degradation, over-extraction of water for irrigation, and pollution. The platypus, an iconic species in freshwater ecosystems around Australia, is threatened by all these activities, both singly and in combination. The scale and complexity of these intersecting and reinforcing threats makes cost-effective monitoring tools essential to better understand how platypus populations are responding. In this study, we optimized a loop-mediated isothermal amplification (LAMP) assay for the rapid and cost-effective detection of platypus DNA in environmental water samples, offering an attractive alternative to quantitative polymerase chain reaction (qPCR). We improved a water filtration protocol for in-field use, employing suitable filter membranes for processing large volumes of water, thereby maximizing DNA recovery from dilute samples. The limit of detection for the platypus LAMP (Plat-LAMP) assay was determined to be 12.4 copies/μL using a standard plasmid positive reference and 7 × 10⁻⁶ ng/μL when applied to DNA extracted from platypus tissue, with improved sensitivity achieved through the incorporation of locked nucleic acid primers. In comparative testing against qPCR, the Plat-LAMP assay exhibited greater sensitivity in detecting platypus DNA in known positive water samples collected from platypus habitat at Healesville Sanctuary. Furthermore, the Plat-LAMP assay demonstrated 100% specificity when performed on water samples collected from non-platypus habitats. In field testing across waterways in Victoria and New South Wales, the Plat-LAMP assay detected platypus in 36.96% of samples, compared to 54.35% of samples using qPCR. These findings underscore the Plat-LAMP assay's potential as a faster and more cost-effective complementary method to qPCR, rendering it suitable for point-of-application water testing. The ability to conduct eDNA surveys without the need for cold-chain logistics would significantly assist conservation organizations and water managers map platypus distributions and facilitate conservation efforts around Australia.

Environmental DNA (eDNA) is increasingly used in biodiversity assessments, but there remain uncertainties regarding its congruence with data based on traditional approaches involving habitat sampling and morphological-based taxonomy. Using eDNA for biomonitoring has several advantages, including improved processing efficiencies and precision of taxonomic identification. In contrast, traditional biomonitoring is time-consuming and expensive, often limiting the number of sites monitored. Establishing that eDNA-derived metrics are congruent with their traditional equivalents on a national scale would support its wider use in biomonitoring. Our study compared ecosystem health assessments made by traditional biomonitoring techniques to those using eDNA from 53 sites throughout Aotearoa New Zealand. Because eDNA sampling was not done concurrently with benthic sampling at most sites, we used the average community composition at each site based on previous sampling occasions. We also allocated species identified by eDNA to the traditional level of identification to allow comparisons with eDNA data identified to broader taxonomic groups. We assessed similarities between the three datasets and found a high degree of correlation and convergence between biotic indices calculated from the different methods. eDNA did, however, appear to under-represent some taxa, reflecting challenges in matching barcodes with an often-incomplete sequence library. eDNA data did not always perform better in terms of showing the effects of land use on invertebrate community composition, but all datasets produced similar patterns. Multivariate analyses (redundancy analysis and variation partitioning) identified congruent relationships between environmental and spatial variables with the invertebrate community structure described by the three methods. eDNA data replicated the environmental responses and showed the same overall patterns in community composition as the traditionally collected data. We suggest that eDNA biomonitoring can complement traditional methods, and will perform at least as well as traditional data at detecting patterns in invertebrate community composition and ecosystem health at a national scale.