Source Of eDNA Research Articles

Using grass inflorescence as source material for biomonitoring through environmental DNA metabarcoding

BackgroundOver the last decade, increasing attention has been directed to using different substrates as sources of environmental DNA (eDNA) in ecological research. Reports on the use of environmental DNA located on the surface of plant leaves and flowers have highlighted the utility of this DNA source in studies including, but not limited to, biodiversity, invasive species, and pollination ecology. The current study assesses grass inflorescence as a source of eDNA for detecting invertebrate taxa.Methods and resultsInflorescences from four common grass species in a central South African grassland were collected for high-throughput sequencing analysis. Universal COI primers were utilised to detect Metazoan diversity. The sequencing results allowed for the detection of three Arthropoda orders, with most OTUs assigned to fungal taxa (Ascomycota and Basidiomycota). Some biases were detected while observing the relative read abundance (RRA) results.DiscussionThe observed biases could be explained by the accidental inclusion of invertebrate specimens during sample collection and DNA extraction. Primer biases towards the amplified taxa could be another reason for the observed RRA results. This study provided insight into the invertebrate community associated with the four sampled grass species. It should be noted that with the lack of negative field controls, it is impossible to rule out the influence of airborne eDNA on the observed diversity associated with each grass species. The lack of the inclusion of PCR and extraction blanks in the sequencing step, as well as the inclusion of negative field controls, including other areas for refinement were highlighted, and suggestions were provided to improve the outcomes of future studies.

EDITH: An R‐package to spatially project eDNA‐based biodiversity across river networks with minimal prior information

Abstract Ecological and ecosystem monitoring is rapidly shifting towards using environmental DNA (eDNA) data, particularly in aquatic systems. This approach enables a combined coverage of biodiversity across all major organismal groups and the assessment of ecological indices. Yet, most current approaches are not exploiting the full potential of eDNA data, largely interpreting results in a localized perspective. In riverine networks, by explicitly modelling hydrological transport and associated DNA decay, hydrology‐based models enable upscaling eDNA‐based diversity information, providing spatially integrated inference. To capitalize on these unprecedented biodiversity data and translate it into space‐filling biodiversity projections, a streamlined implementation is needed. Here, we introduce the eDITH R‐package, implementing the eDITH model to project biodiversity across riverine networks with minimal prior information. eDITH couples a species distribution model relating a local taxon's eDNA shedding rate in streamwater to environmental covariates, a mass balance expressing the eDNA concentration at a river's cross‐section as a weighted sum of upstream contributions, and an observational model accounting for uncertainties in eDNA measurements. By leveraging on spatially replicated eDNA measurements and minimal hydro‐morphological data, eDITH enables disentangling the various upstream eDNA sources, and produces space‐filling maps of a taxon's spatial distribution at any chosen resolution. eDITH is applicable to both eDNA concentration and metabarcoding data, and to any taxon whose DNA can be retrieved in streamwater. The eDITH package provides user‐friendly functions for single‐run execution and fitting of eDITH to eDNA data with both Bayesian methods (via the BayesianTools package) and non‐linear optimization. An interface to the DHARMa package allows model validation via posterior predictive checks. Necessary preliminary steps such as watershed delineation and hydrological characterization are implemented via the rivnet package. We illustrate eDITH's workflow and functionalities with two case studies from published fish eDNA data. The eDITH package provides a user‐friendly implementation of eDITH, specifically intended for ecologists and conservation biologists. It can be used without previous modelling knowledge but also allows customization for experienced users. Ultimately, eDITH allows upscaling eDNA biodiversity data for any river globally, transforming how state and change in biodiversity in riverine systems can be tracked at high resolution in a highly versatile manner.

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring.

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.

Environmental DNA detects Spawning Habitat of an ephemeral migrant fish (Anadromous Rainbow Smelt: Osmerus mordax)

BackgroundAnadromous rainbow smelt (Osmerus mordax) have experienced a large range reduction in recent decades and the status of remnant spawning populations is poorly known in Maine, where these fish have significant ecological, cultural, and commercial relevance. Defining the remnant range of anadromous smelt is more difficult than for many declining fish species because adults are only ephemerally present while spawning in small coastal streams at night during spring runoff periods when traditional assessments can be unreliable or even hazardous. We hypothesized that eDNA might facilitate improved survey efforts to define smelt spawning habitat, but that detection could also face challenges from adult eDNA quickly flushing out of these small stream systems. We combined daytime eDNA sampling with nighttime fyke netting to ascertain a potential window of eDNA detection before conducting eDNA surveys in four streams of varying abundance. Hierarchical occupancy modeling was in turn employed to estimate eDNA encounter probabilities relative to numbers of sampling events (date), samples within events, and qPCR replicates within samples.ResultsResults from the combined eDNA and fyke net study indicated eDNA was detectable over an extended period, culminating approximately 8–13 days following peak spawning, suggesting developing smelt larvae might be the primary source of eDNA. Subsequently, smelt eDNA was readily detected in eDNA surveys of four streams, particularly following remediation of PCR inhibitors. Hierarchical occupancy modeling confirmed our surveys had high empirical detection for most sites, and that future surveys employing at least three sampling events, three samples per event, and six qPCR replicates can afford greater than 90% combined detection capability in low abundance systems.ConclusionsThese results demonstrate that relatively modest eDNA sampling effort has high capacity to detect this ephemerally present species of concern at low to moderate abundances. As such, smelt eDNA detection could improve range mapping by providing longer survey windows, safer sampling conditions, and lower field effort in low density systems, than afforded by existing visual and netting approaches.

Comprehensive insights into profiles and bacterial sources of intracellular and extracellular antibiotic resistance genes in groundwater

Antibiotic resistance genes (ARGs), especially last-resort ARGs (LARGs), are receiving extensive attention as emerging environmental contaminants in groundwater. However, their prevalent intracellular and extracellular patterns and bacterial sources in groundwater remain unclear. Herein, groundwater samples were collected in Tianjin, and characterized based on the profiles of intracellular ARGs (iARGs) and extracellular ARGs (eARGs), as well as the resident bacterial communities and extracellular DNA (eDNA)-releasing bacterial communities. The quantitative real-time PCR assays showed that eARGs presented fewer subtypes than iARGs and generally displayed lower detection frequencies than the corresponding iARGs. Similarly, LARGs exhibited lower detection frequencies than common ARGs, but the total abundance showed no significant differences between them. Genes vanA and blaVIM were the observed dominant LARGs, and aadA was the observed common ARG independent of location inside or outside the bacteria. Furthermore, the top 10 phyla showed much difference between the main eDNA-releasing bacteria and the dominant resident bacteria. Proteobacteria was the predominant resident bacterial phyla while dominating the source of eDNA in groundwater. Despite representing a minor portion of the abundance in the resident bacteria, Actinobacteriota, Acidobacteriota, and Chloroflex surprisingly accounted for a large majority of eDNA release. Co-occurrence patterns among persistent ARGs, the resident bacteria, and eDNA-releasing bacteria revealed that the dominant common iARG aadA and intracellular LARGs blaVIM and vanA had significant positive correlations with Methylobacterium_Methylorubrum and Shewanella. Meanwhile, the dominant extracellular LARG blaVIM may be released by bacteria belonging to at least five genera, including Ellin6067, Bifidobacterium, Blautia, Veillonella, and Dechloromonas. Collectively, the findings of this study extend our understanding regarding the distribution of ARGs and their bacterial sources in groundwater, and indicate the serious pollution of LARGs in groundwater, which poses potential risks to public health.

Spider webs as eDNA samplers: Biodiversity assessment across the tree of life.

The concept of environmental DNA (eDNA) utilizes nucleic acids of organisms directly from the environment. Recent breakthrough studies have successfully detected a wide spectrum of prokaryotic and eukaryotic eDNA from a variety of environments, ranging from ancient to modern, and from terrestrial to aquatic. With their diversity and ubiquity in nature, spider webs might act as powerful biofilters and could thus represent a promising new source of eDNA, but their utility under natural field conditions is severely understudied. Here, we bridge this knowledge gap to establish spider webs as a source of eDNA with far reaching implications. First, we conducted a field study to track specific arthropod targets from different spider webs. We then used high-throughput amplicon sequencing of taxonomic barcodes to investigate the utility of spider web eDNA for biodiversity monitoring of animals, fungi and bacteria. Our results show that genetic remains on spider webs allow the detection of even the smallest target organisms. We also demonstrate that eDNA from spider webs is useful in research of community compositions across the different domains of life, with potentially highly detailed temporal and spatial information.

Reconstruction of 100-year dynamics in Daphnia spawning activity revealed by sedimentary DNA

Environmental DNA (eDNA) is currently developing as a powerful tool for assessing aquatic species dynamics. However, its utility as an assessment tool for quantification remain under debate as the sources of eDNA for different species is not always known. Therefore, accumulating information about eDNA sources from different species is urgently required. The objective of our study was to evaluate whether sedimentary DNA targeting two Daphnia species, D. galeata and D. pulicaria, could track Daphnia population dynamics and resting egg production. Applying a quantitative PCR targeting the mitochondrial 12S rRNA gene on sediment cores collected in Lake Biwa, Japan, we compared sedimentary DNA concentration of Daphnia with the abundance of remains and ephippia, reflecting their abundance and resting egg production, respectively. We found that the sedimentary DNA concentrations of Daphnia for the past century were inconsistent with their population abundance. However, the concentration was highly correlated with the resting egg production. Our results provide evidence that ephippia with resting eggs, released during spawning activities, was a significant source of Daphnia DNA archived in sediments. Our work provides critical insights for using sedimentary DNA as a monitoring tool for egg production dating back 100 years.

Analysis of honey environmental DNA indicates that the honey bee (Apis mellifera L.) trypanosome parasite Lotmaria passim is widespread in the apiaries of the North of Italy

Lotmaria passim is a trypanosomatid that infects honey bees. In this study, we established an axenic culture of L. passim from Italian isolates and then used its DNA as a control in subsequent analyses that investigated environmental DNA (eDNA) to detect this trypasonosomatid. The source of eDNA was honey, which has been already demonstrated to be useful to detect honey bee parasites. DNA from a total of 164 honey samples collected in the North of Italy was amplified with three L. passim specific PCR primers and 78% of the analysed samples gave positive results. These results indicated a high prevalence rate of this trypanosomatid in the North of Italy, where it might be considered another threat to honey bee health.

Degradation modeling of water environmental DNA: Experiments on multiple DNA sources in pond and seawater

AbstractEnvironmental DNA (eDNA) analysis methods have been developed to detect organism distribution and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation. However, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, for example, cells and fragmental DNA. We experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (internal PCR control [IPC]), free cells (from Oncorhynchus kisutch), and resident species. We conducted the experiment with pond and seawater to evaluate the differences between freshwater and marine habitats. We quantified the eDNA copies of free cells, fragmental DNA, and resident species (Cyprinus carpio in the pond and Trachurus japonicus in the sea). We found that eDNA derived from both cells and fragmental DNA decreased exponentially in both the sea and pond samples. The degradation of eDNA from resident species showed similar behavior to the cell‐derived eDNA. We evaluated three degradation models with different assumptions and degradation steps and found that a simple exponential model was effective in most cases. Our findings on cell‐ and fragmental DNA‐derived eDNA provide fundamental information about the eDNA degradation process and can be applied to quantify eDNA behavior in natural environments.

Degradation factors of environmental DNA evaluated by experiments and meta-analysis

Environmental DNA (eDNA) methods have been developed to detect organisms' distributions and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation, but, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, e.g., cell-derived and fragmental DNA. In this study, we conducted the degradation experiments (Saito and Doi 2020a) and a meta-analysis (Saito and Doi 2020b). Firstly, we experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (the DNA of internal positive control, IPC), free cells from Oncorhynchus kisutch, and the resident species (Saito and Doi 2020a). We conducted the experiments with pond and seawater to evaluate the differences between freshwater and marine habitats. Our results showed that eDNA derived from the both cells and fragmental DNA decreased exponentially in the both sea and pond samples. The degradation of eDNA from the resident species showed similar behavior to the cell-derived eDNA. As a meta-analysis, we complied the degradation rates of eDNA in laboratory experiment and field studies from 28 studies (Saito and Doi 2020b). We also collected the related factors, including water sources, water temperature, DNA regions, and PCR amplicon lengths of the measured DNA. Our results suggested that water temperature and amplicon length were significantly related to the degradation rate of eDNA. From the simulation based on the 95% quantile model, we predicted the maximum degradation rate of eDNA in various combinations of water temperature and PCR amplicon length.

Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring.

The last decade brought a spectacular development of so-called environmental (e)DNA studies. In general, "environmental DNA" is defined as DNA isolated from environmental samples, in contrast to genomic DNA that is extracted directly from specimens. However, the variety of different sources of eDNA and the range of taxonomic groups that are targeted by eDNA studies is large, which has led to some discussion about the breadth of the eDNA concept. In particular, there is a recent trend to restrict the use of the term "eDNA" to the DNA of macro-organisms, which are not physically present in environmental samples. In this paper, we argue that such a distinction may not be ideal, because the eDNA signal can come from organisms across the whole tree of life. Consequently, we advocate that the term "eDNA" should be used in its generic sense, as originally defined, encompassing the DNA of all organisms present in environmental samples, including microbial, meiofaunal and macrobial taxa. We first suggest specifying the environmental origin of the DNA sample, such as water eDNA, sediment eDNA or soil eDNA. A second specification would then define the taxonomic group targeted through polymerase chain reaction amplification, such as fish eDNA, invertebrate eDNA and bacterial eDNA. This terminology does also not require assumptions about the specific state of the DNA sampled (intracellular or extracellular). We hope that such terminology will help better define the scope of eDNA studies, especially for environmental managers, who use them as reference in routine biomonitoring and bioassessment.

Aquatic suspended particulate matter as source of eDNA for fish metabarcoding

The use of environmental DNA (eDNA) for monitoring aquatic macrofauna allows the non-invasive species determination and measurement of their DNA abundance and typically involves the analysis of eDNA captured from water samples. In this proof-of-concept study, we focused on the novel use of eDNA extracted from archived suspended particulate matter (SPM) for identifying fish species using metabarcoding, which benefits from the prospect of retrospective monitoring and also analysis of fish communities through time. We used archived SPM samples of the German Environmental Specimen Bank (ESB), which were collected using sedimentation traps from different riverine points in Germany. Environmental DNA was extracted from nine SPM samples differing in location, organic content, and porosity (among other factors) using four different methods for the isolation of high-quality DNA. Application of the PowerSoil DNA Isolation Kit with an overnight incubation in lysis buffer, resulted in DNA extraction with the highest purity and eDNA metabarcoding of these eDNA fragments was used to detect a total of 29 fish taxa among the analyzed samples. Here we demonstrated for the first time that SPM is a promising source of eDNA for metabarcoding analysis, which could provide valuable retrospective information (when using archived SPM) for fish monitoring, complementing the currently used approaches.

Honey as a Source of Environmental DNA for the Detection and Monitoring of Honey Bee Pathogens and Parasites.

Environmental DNA (eDNA) has been proposed as a powerful tool to detect and monitor cryptic, elusive, or invasive organisms. We recently demonstrated that honey constitutes an easily accessible source of eDNA. In this study, we extracted DNA from 102 honey samples (74 from Italy and 28 from 17 other countries of all continents) and tested the presence of DNA of nine honey bee pathogens and parasites (Paenibacillus larvae, Melissococcus plutonius, Nosema apis, Nosema ceranae, Ascosphaera apis, Lotmaria passim, Acarapis woodi, Varroa destructor, and Tropilaelaps spp.) using qualitative PCR assays. All honey samples contained DNA from V. destructor, confirming the widespread diffusion of this mite. None of the samples gave positive amplifications for N. apis, A. woodi, and Tropilaelaps spp. M. plutonius was detected in 87% of the samples, whereas the other pathogens were detected in 43% to 57% of all samples. The frequency of Italian samples positive for P. larvae was significantly lower (49%) than in all other countries (79%). The co-occurrence of positive samples for L. passim and A. apis with N. ceranae was significant. This study demonstrated that honey eDNA can be useful to establish monitoring tools to evaluate the sanitary status of honey bee populations.

EDNA from culture-independent Hirudinaria bpling Phillips 2012 (Annelida: Hirundinidae) as a tool for biodiversity assessment

Recent interest in the use of leeches as a source of eDNA by obtaining and analyzing traces of its last blood meal has proven noteworthy as a tool in screening biodiversity. As the method is fairly new and has relatively unexplored benefits, its assessment as a tool in such fields as ecology and biotechnology prompts further studies. This study was conducted to provide an alternative to collecting samples in poorly-explored areas or in areas where collection is very difficult, if not impossible. Using leeches collected from Angadanan, Isabela, DNA was extracted and cytochrome oxidase I (COI) gene was amplified using conventional PCR. Gene sequences from resulting amplicons were matched with similar sequences using BLASTn, where prey sequences were narrowed down to organisms with the highest match of no less than 85%. Highest score of similarities were obtained and species identified included Cyprinus carpio L. 1758, Homo sapiens L., 1758, Bos taurus L., 1758, Bubalus bubalis carabanensis L.S. Castillo, 1998, Equus caballus L., 1758, and Canis familiaris L., 1758. These results demonstrate that using leech-extracted blood meal may be a successful tool in screening vertebrate biodiversity.

Detection of freshwater mussels (Unionidae) using environmental DNA in riverine systems

AbstractEnvironmental DNA (eDNA) methods are being developed for use in conservation biology to improve upon conventional species survey techniques. Validation of eDNA methods in different environmental contexts is required if they are to be widely adopted. One potential application of eDNA methods is for the detection of freshwater mussels (Bivalvia: Unionidae), which are among the most imperiled species in North America. Conventional unionid survey methods are highly invasive and can be difficult to conduct due to issues with morphological identification and their cryptic use of habitat. eDNA methods can potentially provide a non‐invasive, extremely specific, and highly sensitive alternative. Here, we examine the effectiveness of eDNA methods at detecting an imperiled unionid, the wavy‐rayed lampmussel (Lampsilis fasciola), in lotic systems with moderate discharge. We developed a novel qPCR assay for the detection of L. fasciola eDNA, which included a custom internal positive control to check for PCR inhibition. We used different experimental densities of caged L. fasciola specimens as a point source of eDNA within two rivers of the Grand River watershed in Southern Ontario. Sampling occurred at set distances downstream of the cage using purpose‐built sampling equipment. Detection was obtained at the cage (i.e., point of eDNA shedding) but not downstream at distances ≥10 m during stream discharges of approximately 1,632–2,332 L/s. The results indicate that eDNA is diluted rapidly in rivers with moderate discharge and that high‐resolution spatial sampling efforts may be necessary to obtain meaningful eDNA‐based distribution data of unionids, and other sessile organisms, present at low density in lotic systems.

Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity.

Microbial access to host nutrients is a key factor of the host-pathogen interplay. With their nearly minimal genome, wall-less bacteria of the class Mollicutes have limited metabolic capacities and largely depend on host nutrients for their survival. Despite these limitations, host-restricted mycoplasmas are widely distributed in nature and many species are pathogenic for humans and animals. Yet, only partial information is available regarding the mechanisms evolved by these minimal pathogens to meet their nutrients and the contribution of these mechanisms to virulence. By using the ruminant pathogen Mycoplasma bovis as a model system, extracellular DNA (eDNA) was identified as a limiting nutrient for mycoplasma proliferation under cell culture conditions. Remarkably, the growth-promoting effect induced by supplementation with eDNA was associated with important cytotoxicity for actively dividing host cells, but not confluent monolayers. To identify biological functions mediating M. bovis cytotoxicity, we produced a library of transposon knockout mutants and identified three critical genomic regions whose disruption was associated with a non-cytopathic phenotype. The coding sequences (CDS) disrupted in these regions pointed towards pyruvate metabolism as contributing to M. bovis cytotoxicity. Hydrogen peroxide was found responsible for eDNA-mediated M. bovis cytotoxicity, and non-cytopathic mutants were unable to produce this toxic metabolic compound. In our experimental conditions, no contact between M. bovis and host cells was required for cytotoxicity. Further analyses revealed important intra-species differences in eDNA-mediated cytotoxicity and H2O2 production, with some strains displaying a cytopathic phenotype despite no H2O2 production. Interestingly, the genome of strains PG45 and HB0801 were characterized by the occurrence of insertion sequences (IS) at close proximity to several CDSs found disrupted in non-cytopathic mutants. Since PG45 and HB0801 produced no or limited amount of H2O2, IS-elements might influence H2O2 production in M. bovis. These results confirm the multifaceted role of eDNA in microbial communities and further identify this ubiquitous material as a nutritional trigger of M. bovis cytotoxicity. M. bovis may thus take advantage of the multiple sources of eDNA in vivo to modulate its interaction with host cells, a way for this minimal pathogen to overcome its limited coding capacity.

The effect of bivalve filtration on eDNA-based detection of aquatic organisms.

As the use of environmental-DNA (eDNA) expands as a method to detect the presence and quantity of aquatic taxa, factors potentially impacting the efficacy of this technique must be investigated. Many studies have examined the effects of abiotic parameters on the degradation of environmental-DNA (e.g. UV radiation, pH, temperature, etc.), however, few have focused on biotic effectors. Through high-filtering rates coupled with dense colonization, Asian clams (Corbicula fluminea) are able to drastically alter the quantity of particulate matter through translocation into the sediment, potentially including sources of eDNA in lotic and lentic systems. Using a longitudinal, laboratory experiment, we tested the effect of varying densities of Asian clams on the translocation rate of common goldfish (Carassius auratus) DNA. Target DNA in testing tanks was quantified through quantitative PCR (qPCR) at regular intervals and compared. Tanks housing the highest density of Asian clams produced significantly lower DNA concentrations over time compared to tanks of lower densities. These results show, for the first time, a density-dependent reduction of local eDNA sources by bivalve filtration that may lead to the obstructed detection of target species through the sampling of eDNA. Based on these findings, we recommend highly concentrated bivalve populations be taken into consideration when choosing the time and locality of eDNA sampling efforts.

Spatiotemporal distribution of juvenile chum salmon in Otsuchi Bay, Iwate, Japan, inferred from environmental DNA.

To understand the ecology of juvenile chum salmon during early marine life after their downstream migration, we developed a quantitative PCR-based environmental DNA (eDNA) method specific for chum salmon and investigated the spatiotemporal distribution of eDNA in Otsuchi Bay, Iwate, Japan. Indoor aquarium experiments demonstrated the following characteristics of chum salmon eDNA: (1) the eDNA shedding and degradation were time- and water temperature-dependent and the bacterial abundance could contribute to the eDNA decay, (2) fecal discharge may not be the main source of eDNA, and (3) a strong positive Pearson correlation was found between the number of juveniles and the eDNA amounts. As we discovered strong PCR inhibition from the seawater samples of the bay, we optimized the eDNA assay protocol for natural seawater samples by adding a further purification step and modification of PCR mixture. The intensive eDNA analysis in the spring of 2017 and 2018 indicated that juvenile chum salmon initially inhabited in shallow waters in the shorefront area and then spread over the bay from January to June. The eDNA data also pointed out that outmigration of juvenile chum salmon to open ocean temporarily suspended in April, possibly being associated with the dynamics of the Oyashio Current as suggested by a previous observation. The eDNA method thus enables us large-scale and comprehensive surveys without affecting populations to understand the spatiotemporal dynamics of juvenile chum salmon.

Analyzing airborne environmental DNA: A comparison of extraction methods, primer type, and trap type on the ability to detect airborne eDNA from terrestrial plant communities

AbstractAirborne environmental DNA (eDNA) research has typically focused on the detection of pollen from anemophilous terrestrial plant species; however, recent findings have expanded the definition of airborne eDNA to include a variety of eDNA sources, such as leaf and flower fragments. While methods for capturing pollen are well studied, there is less known about how to best analyze and capture more broadly defined bulk airborne eDNA samples. Therefore, this study aimed to identify efficient techniques for the extraction and amplification of airborne eDNA and how best to capture these samples. First, we collected samples using Big Spring Number Eight dust traps and quantified the influence of three different extraction methods (cetrimonium bromide‐chloroform, DNeasy Plant Maxi Kit, and DNeasy PowerPlant Pro DNA Isolation Kit) and two global plant primer sets (targeting the trnL and ITS2 genes) on quantitative polymerase chain reaction (qPCR)‐based terrestrial plant detection. Additionally, we examined how best to capture airborne eDNA by comparing the performance of three difference passive dust collectors (Big Spring Number Eight, Modified Wilson and Cooke, and Marble dust collectors). We found that the detection of airborne eDNA was significantly influenced by both the extraction methods employed and choice of qPCR primer. Additionally, we found that all three traps preformed similarly but the Big Spring Number Eight dust traps trended as consistently one of the most effective, shedding light on the nonhomogeneous nature of airborne eDNA. This study represents the first methodology paper focusing on airborne eDNA as a bulk environmental sample containing more than just pollen. In the future, additional research should examine more about the ecology of airborne eDNA (origin, state, transport, fate), explore active dust collectors compared to the passive traps used here, and develop the application of metabarcoding for airborne eDNA analysis.