Environmental DNA最新文献

Natural matrices affect environmental DNA (eDNA) detections. Effects of matrix-eluted compounds on the polymerase chain reaction (PCR) step have been the focus of most inhibition studies. Factors affecting eDNA detections in a typical laboratory workflow, i.e., DNA extraction and PCR steps, are mostly unknown. Here, we assessed the effect of four metal ions (Ca²⁺, Fe³⁺, Mn²⁺, Cu²⁺) present in marine sediments on DNA detectability for both the extraction and the PCR detection steps. A single metal ion and exogenous DNA were added to marine sediments treated chemically to remove inhibitors. Our results showed that natural concentrations of calcium, iron, and manganese ions in surface marine sediments can impede completely DNA detections. Alternatively, copper ions added to the matrix increased DNA detectability by 7.7%. We also observed bimodal inhibitory effects of calcium and iron ions on DNA detectability, suggesting that the extraction and the PCR steps are both affected. Our findings highlight new limitations of eDNA detections. Avenues to optimize eDNA detection protocols applicable to multiple matrices are discussed.

Detecting environmental DNA (eDNA) of numerous organisms from the same samples has been revolutionized by metabarcoding. However, utilizing the vast amounts of data generated from metabarcoding to predict occupancy probabilities for co-occurring species is currently rare. Here, we demonstrate how metabarcoding data can be used to advance community ecology research through a case study using replicate stream water samples and Bayesian occupancy models to test hypotheses of eDNA occurrence for a native fish (brook trout, Salvelinus fontinalis), its major ectoparasite (gill lice, Salmincola edwardsii), and an introduced potential competitor (brown trout, Salmo trutta). Gill lice DNA occupancy was positively associated with brook trout biomass determined via electrofishing conducted near eDNA sampling sites, suggesting gill lice occupancy is dependent on host density. Leveraging site-specific molecular operational taxonomic units identified from metabarcoding, DNA occupancy of trout and gill lice was often positively predicted by species richness of aquatic insect orders trout commonly feed on, which are also environmental quality indicators. Thus, high-quality habitats that environmentally sensitive salmonids and their primary prey rely on may promote higher fish occupancy rates, further facilitating the spread of fish parasites. An increasing amount of community-level data is being generated from global metabarcoding efforts, and we suggest our framework could be broadly implemented to enhance understanding of factors impacting distributions of co-occurring species, reveal new ecological phenomena, and support management and conservation efforts.

Targeted environmental DNA (eDNA) studies mainly rely on quantitative real-time polymerase chain reaction (qPCR) to amplify extremely low concentrations of DNA present in environmental samples. Understanding factors that influence targeted eDNA assay performance and detection in field samples, such as Taq DNA polymerase enzyme type and thermocycle protocol, is critical for the interpretation of qPCR results. We completed a systematic performance evaluation of five distinct targeted eDNA assays (eANFI6, eFISH1, eANBO5, eGLIN1, and eLICA3 targeting sablefish, general fish, Boreal toad, wood turtle, and the American bullfrog) by subjecting the same samples to analysis by five different Taq enzyme reagent mixes (Immolase, Environmental Master Mix (EMM), Amplitaq, QIAcuity, and QuantiNova) and the commonly used 2-step (95°C, 60°C) and 3-step (95°C, 64°C, 72°C) thermocycle protocols. We evaluated assay performance using a standardized dilution series of synthetic dsDNA target sequences and calculated limits of detection (LOD) and quantification (LOQ) and 95% confidence intervals for each combination of Taq enzyme and thermocycle protocol. All assays performed within acceptable performance criteria as defined by the Canadian national standard for targeted eDNA assays. Based on data generated by synthetic dsDNA fragments, the eDNA assays performed comparably regardless of the enzyme reagent mix and protocol used, except for eANFI6 and eGLIN1 using EMM and the 3-step protocol, where no amplification was observed. On freshwater field samples, EMM and Immolase performed best. On marine field samples, Immolase, EMM, Qiacuity, and QuantiNova performed equally well, although EMM failed to amplify some samples. The work reveals that an enzyme reaction mix or thermocycle protocol can affect the result of an eDNA assay, but the appropriate choice also depends on the nature of the field sample. It is therefore imperative that these are considered when selecting appropriate reaction conditions and that they are clearly reported.

Wetlands are ecologically and culturally significant ecosystems that are experiencing biodiversity declines globally. Biomonitoring techniques that use environmental DNA (eDNA) to detect and monitor biodiversity are well established in lake, riverine, and marine ecosystems. However, their use in wetlands requires further development due to the presence of sediments that block eDNA filters to limit water filtration, alongside a lack of standardized methodology. In this study, we examined eDNA dynamics to understand spatiotemporal biodiversity patterns in an Aotearoa New Zealand wetland and to optimize their application to wetland-specific challenges. We sampled four sites across Opuatia Wetland at three time points during an austral spring. We conducted conventional taxonomic surveys, tested three different filter sizes (1.2 μm, 5 μm, and semi-quantitative dacron filters), and assessed our ability to detect foreign DNA (from kea; Nestor notabilis) at different time points and distances post-release. We found significant differences in DNA sequence composition across time and space, and when using different sized filters. eDNA data generally complemented (versus replaced) conventional survey and identification methods, with certain species only detected by one method or the other. Taxonomic resolution of conventional sampling and identification methods often exceeded that of eDNA. Foreign DNA was detectable 10 m from its release point for up to 1 week post-release. Our results provide new considerations for future eDNA research in wetland environments, where rapid biomonitoring techniques are needed to support conservation and preservation.

The introduction of the signal crayfish Pacifastacus leniusculus to British rivers has led to ecological degradation and the decline of the native white-clawed crayfish Austropotamobius pallipes. To manage and mitigate the impact of the signal crayfish, conservation agencies and government bodies employ multiple conservation strategies. These take the form of proactive native crayfish breeding and stocking programs and reactive invasive crayfish control programs. Here, we used eDNA to assess the populations of native and invasive crayfish species across 50 sites in 10 river catchments in Norfolk, United Kingdom (UK). The sites were chosen to enable assessment of the potential of eDNA to inform proactive and reactive crayfish conservation strategies. Three of the catchments sampled were selected to assess the success of recent A. pallipes reintroduction, whereas the remaining seven were selected to better understand the distribution of each species at the landscape scale. Combining results of eDNA-based methods with net searches within an occupancy model enabled us to confidently determine the presence of P. leniusculus at eight sites, and A. pallipes at three sites, which was more than visual searches alone (five and two study sites, respectively). Neither eDNA nor net searches detected A. pallipes at sites where A. pallipes had been reintroduced. We recommend that practitioners using eDNA-based surveys for management and conservation of crayfish should consider: (1) designing eDNA surveys with an emphasis on large spatial scales to comprehensively describe the distributions of native and invasive crayfish in a region of interest; (2) work with local conservation organizations and/or government bodies to inform the selection of study sites to generate results that are meaningful to real-world conservation actions; and (3) use results from eDNA-based crayfish surveys to target limited conservation resources to appropriate proactive and/or reactive conservation actions.

Environmental DNA consists of multiple states including but not limited to membrane-bound, adsorbed, and dissolved states. Because of their chemical and physical properties, each of these states may have different degradation and transport potential. Essential to the study of eDNA states is being able to isolate them from an environmental sample. Here, we focus on improving the DNA recovery of the dissolved state of eDNA from water. We compared three recovery methods, namely isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis. We evaluated the effectiveness of these methods based on the measured DNA recovery of two different species' DNA of different sizes. DNA recovery was assessed with qPCR. We also checked for the effect of inhibitor removal, and lastly, we estimated the cost of reagents and consumables (not labor). The DNA recovery varied among extraction methods, with isopropanol precipitation yielding the highest recovery (52.47 ± 19.69%), followed by centrifugal dialysis (12.58 ± 7.15%) and magnetic bead extraction (9.92 ± 3.89%). However, isopropanol precipitation's efficacy was influenced by humic acid concentration in the water matrix. The presence of humic acids significantly affected isopropanol precipitation, with higher humic acid concentrations leading to increased DNA recovery. This suggests that humic acids act as co-precipitators during isopropanol precipitation. We observed that longer DNA fragments (chicken) had lower recovery compared to shorter fragments (salmon) across all extraction methods. Magnetic bead extraction effectively removed inhibitors, while centrifugal dialysis and isopropanol extraction required an additional inhibitor removal step. Isopropanol, magnetic bead extraction, and centrifugal dialysis methods have estimated costs of 7.74, 8.53, and 23.62 USD in 2023 respectively. Overall, isopropanol precipitation was the least expensive and most effective with the highest measured recoveries, but it was dependent on humic acid concentration in the environmental sample.

Fram Strait, the gateway between the Arctic and Atlantic Oceans, is undergoing major climate change-induced physical and biological transformations. In particular, rapid warming and ongoing “Atlantification” are driving species range shifts and altering food web structures in the Arctic. Understanding and predicting the consequences of these processes on future ecosystems requires detailed assessments of local and pelagic biodiversity. Gelatinous zooplankton (GZP) is an important component of pelagic communities, and recent evidence indicates that such communities are undergoing major changes in the Fram Strait. However, as sampling GZP is challenging, they are regularly underestimated in biodiversity, distribution, and abundance. To overcome this and address existing ecological knowledge gaps, we investigated patterns of pelagic metazoan diversity in Fram Strait using environmental DNA (eDNA) metabarcoding of the cytochrome c oxidase I (COI) gene. We successfully detected a broad range of taxa from the marine metazoan and GZP communities across sampling locations and ocean depth zones. We demonstrate the vertical structuring of diversity and elucidate relationships between taxa and water mass indicators, such as salinity and temperature. Furthermore, when comparing eDNA data with net and video transect data for GZP at the same period and location, we found that eDNA uncovered a higher number of taxa, including several that were not detected by the other methods. This study is a contribution to the formation of baseline Arctic GZP biodiversity datasets, as well as future research on changing marine metazoan biodiversity and community composition.

The loss of tidal wetlands in the San Francisco Bay estuary have led to declines in native fish presence. Restoration of tidal wetlands in this area has intensified, with a primary goal of increasing the number of native fishes. We compared the presence of longfin smelt in naturally accreted and beneficial dredge reuse wetlands as a measure of successful restoration. We used environmental DNA (eDNA) analyses as our metric for fish presence and fish community composition, employing two different water sampling methods for comparison (standard and high-volume). Longfin smelt were present in multiple sites, but at numbers too low for accurate comparisons across sites. Community composition varied based on the water sampling method, but the presence/absence of longfin smelt was consistent across sampling methods. As this represents a pilot study, further refinement of methodology is necessary, but the use of high-volume water sampling methods is promising.

Gelatinous zooplankton (GZP), i.e., ctenophores, cnidarian medusae, chaetognaths, appendicularians and salps, are considered climate change winners. This becomes particularly obvious in the Southern Ocean, which has undergone a significant shift from a krill-based to a salp-based ecosystem over the last decades. A better knowledge on the role of gelatinous invertebrates as prey is needed to predict the impact of such a gelatinous shift. Until recently, GZP was considered as a “trophic dead end”. However, their true importance in diets has remained unresolved due to the rapid digestion of their watery and soft tissues in predators' stomachs. In this study, we want to validate the paradigm shift from GZP being considered as “survival food” to be considered a “regular” prey item for two demersal fish species (Notothenia rossii and N. coriiceps) of Potter Cove, South Shetland Islands, using a multimarker (COI and 18S) metabarcoding approach. We found that GZP taxa commonly occurred in the diets of both species, represented by pelagic tunicates (appendicularians, salps), cnidarians, chaetognaths and ctenophores. Salps were the most abundant prey group, preyed upon by each individual of both species, reaching 98.7% relative read abundance for 18S. We recovered a wide range of different taxa in their diets, from primary producers to highly abundant invertebrates, thus the two nototheniid species can be regarded as “natural samplers” of the ecosystem in study. Finally, we want to point out the importance of multimarker metabarcoding approaches for broad ecological assessments, given the differential amplification and sequencing success of different markers for specific groups and the unequal taxonomic coverage of the reference databases. The output of each marker was highly complementary, since an important prey item such as salps, was only detected with 18S, while other taxa (e.g., Arthropoda) were represented with a higher taxonomic resolution with COI.

The Northern muriqui (Brachyteles hypoxanthus) is one of the world's most critically endangered primates, with only ~1000 mature individuals remaining in the wild. Habitat loss and hunting have led to its sharp decline, making conservation efforts crucial. Analyses of gut microbiomes in wild populations can provide valuable information on host health and vulnerability, and ultimately, contribute to baseline knowledge toward improving conservation programs and reintroduction efforts. In this study, we analyzed the microbiome (16S rRNA metabarcoding) of fecal samples belonging to 53 uniquely genotyped individuals from three social groups from the Caparaó National Park, aiming to provide the first assessment of the microbiome diversity and composition for this species. Our results showed the muriqui gut microbiome was predominantly composed of the phyla Bacteroidetes and Firmicutes, with the dominant classes represented by Bacteroidia and Clostridia. High similarity in bacterial diversity and composition was found for individuals from distinct groups, suggesting a negligible geographical effect at the fine spatial scale analyzed. No significant effect of host genotype heterozygosity levels on microbiota diversity was recovered, but a significant influence of genetic distance on microbiota community structure and composition was demonstrated. Our findings stress the importance of considering associations between host genetics and the microbiome and suggest that the analyzed populations host a similar microbiome composition. This detailed microbiome assessment can aid conservation actions, including future anthropogenic impact assessments and animal reintroductions.