Environmental DNA (eDNA) Applications—Recent Advances and Future Prospects

  • Publications on eDNA Are Increasing Exponentially
  • qPCR and Sequencing are Main Analytical Methods
  • Metabarcoding Is Powerful for Communal eDNA Analysis

Introduction

Sparked by the Human Genome Project completed in 2003, the genomic revolution has fundamentally changed how we survey the biodiversity of the Earth, from complex mammalian species, to the simplest life form. High-throughput sequencing (aka next-generation sequencing (NGS)) platforms now enable the rapid sequencing of DNA or RNA from diverse environmental samples, termed “environmental DNA” (eDNA). According to Taberlet et al., this term was first used in a 1987 publication by Ogram on extracting DNA from sediments, but it did not really emerge until the early 2000s, when it was first used by microbiologists. In its simplest sense, eDNA refers to DNA extracted from any type of environmental sample (e.g. soil, water, air, etc.), without isolation of a particular organism. 

The field of molecular ecology uses NGS and other genetic methods to address ecological questions, and it has recently seen an explosion of scientific activity on the development and use of eDNA. This is evidenced in the chart shown here, by the annual number of articles published in PubMed since 2000 on the topic. Even greater numbers on eDNA can be found in Google Scholar by searching all publications and patents, including citations.

PubMed search and chart by Jerry Zon

Main eDNA sources: soil, water, and air

In view of this voluminous literature, and the Zone’s focus on “what’s trending in nucleic acid research,” this blog will provide a brief overview of eDNA methodologies, as well as a synopsis of recent advances in molecular ecology based on eDNA derived from the Earth’s main environmental reservoirs: soil, water, and air. 

Readers should keep in mind that the analytical methods employed for these sources of eDNA also apply to the diverse microbial communities found in and on humans, animals, and fish. Increasing interest in these types of “microbiomes” is driven mainly by health-related attention, as already discussed in a four-part series of Zone blogs.  

Overview of eDNA Methodologies

Unlike traditional biodiversity assessment methods, where captured or recorded individuals are used to determine presence or abundance, eDNA-based biodiversity assessment relies on our ability to capture the “genetic signature” left behind by organisms through shedding, excreting, decaying, etc., according to a 2019 review by Seymour. Environmental DNA-based research is therefore dependent on the ability to accurately match the left-behind genetic signature to the correct species. Seymour adds that this matching is challenged by various factors, including life-time of eDNA in water (short), soil or ice (long), and transport dynamics, as indicated by this graph.

Sample types of eDNA have different spatial and temporal scopes of inference from different habitats. Consider each sample type as a single sample from that environment. Placement of a sample type in a quadrant is not quantitative, but represents a common scale at which it has been used. Dashed arrows indicate the potential for a sample type to confer information at multiple scales of inference, but additional research to quantify these possibilities is needed. Taken from Deiner et al. Molecular Ecology, 26(21):5872-5895 and freely available under Creative Commons Attribution 4.0 International (CC BY 4.0).

qPCR traces

At this time, eDNA studies can be categorized into two basic approaches: targeted (species-specific), and semi-targeted (community). Both categories are often discussed simultaneously, says Seymour, but differ drastically in their methodology, interpretations, and accuracy. Species-specific studies use assays tailored to particular species to target specific DNA fragments in an environmental sample. Presence or absence of the targeted DNA, as in the classical eDNA studies, is still conducted using standard end-point PCR. However, the standard for quantification is becoming quantitative PCR (qPCR) vs standard or absolute quantification by single-molecule counting via digital PCR (dPCR) in plates or digital droplet PCR (ddPCR).

ddPCR. Taken from commons.wikimedia.org and free to use.

Asian carp

Targeted eDNA, which is often attributed to the rise in eDNA research, was first used for detection of the invasive American bullfrog in France, followed by extensive Asian carp (pictured here) monitoring efforts in the United States. These examples have been expanded to government-supported eDNA monitoring programs and assay developments for a wide range of invasive and conservation-status species, according to Seymour. However, he adds, far greater scientific potential is being unveiled within the realm of community eDNA studies, and this is beginning to open the door to multi-disciplinary research and a new wave of discovery science.

Illustration of eDNA metabarcoding

The eDNA community-based methods encompass a wide range of NGS techniques, including metabarcoding (illustrated here), long-read shot-gun mitogenomics, genome skimming, and more. Of these, metabarcoding, which is receiving the most attention and is reviewed elsewhere, generates a large number of individual barcode sequences in a single NGS run, enabling the simultaneous identification of individuals in large mixed communities, such as a trap sample containing many different insect species. Santos et al. very recently reported metabarcoding for single-molecule nanopore sequencing of 16S ribosomal RNA (16S rRNA) genes, as they are widely used in environmental microbiology. Nanopore sequencing has been previously featured in a number of Zone blogs as enabling portability into the field. This type of sequencing-on-site can greatly expand the use of eDNA metabarcoding.

Soil Samples for eDNA

Illustration of soil bacteria

According to a 2020 report by Pathan et al., extracellular eDNA can represent up to 40% of the total soil DNA pool. The interest from the scientific community on eDNA in soil is quite recent; however, according to the researchers, studies have already found that eDNA in soil can act as a constituent of biofilms, as well as a source of nutrients and signaling- or chemoattractant molecules, the latter of which is amazing to the Zone. In addition to soil bacteria (illustrated here), fungi, and molds, “dirt” has a remarkably complex eDNA composition derived from animals that live above and under the surface, as detailed in a lengthy publication by Andersen et al. (2012) titled Metabarcoding of ‘dirt’ DNA from soil reflects vertebrate biodiversity.

Millipede

As for the composition of communal eDNA in and on soil, “size matters,” as recently reported in a 2020 publication by Lucie et al. titled Body size determines soil community assembly in a tropical forest. These investigators note that tropical forests shelter unparalleled biological diversity. The relative influence of environmental selection (i.e., abiotic conditions, biotic interactions) and stochastic-distance-dependent neutral processes (i.e., demography, dispersal) in shaping communities has been extensively studied for various organisms, but has rarely been explored across a large range of body sizes. Consequently, researchers built a detailed census of the whole-soil biota in a 120,000 square meter tropical forest plot in French Guiana, using soil DNA metabarcoding. The distribution of 19 taxonomic groups of various sizes was found to be primarily stochastic. This discovery suggests that neutral processes are prominent drivers of the assembly of these communities at this scale, which ranged from tiny microbes to mesofauna (0.1–2 mm) such as nematodes, mites, and pauropods (millipede-like arthropods, pictured here).

Degradation dynamics of eDNA in soils were investigated by Sirois et al., who examined the impacts of soil moisture, temperature, agricultural management, and habitat type. Synthetic eDNA was added to soil microcosms, and its disappearance over time was measured using both NGS and qPCR. The synthetic eDNA was degraded rapidly, but a small fraction remained detectable throughout the experiments (39–80 days). The eDNA degradation rate was positively correlated with moisture and temperature, but negatively correlated with soil organic carbon content. End‐point stabilization of eDNA was highest at low moisture and temperature, but exhibited no relationship with soil organic carbon. Tilled soils had higher rates of degradation and less stabilization than no‐till soils. Among different habitats, researchers observed that forest soils had the slowest degradation rate, and meadow soils had the greatest stabilization of eDNA. Among the conclusions, “small amounts of eDNA may persist in soils indefinitely.”

Water Samples for eDNA

J. Craig Venter—uber-famous for successfully pioneering “shotgun sequencing” of the human genome—grabbed scientific headlines in the mid-2000s for embarking on metagenomic sequencing of global ocean samples. He did so in the Northwest Atlantic through the Eastern Tropical Pacific, as shown here, from his yacht The Sorcerer II. 

Numbered ocean sample locations. Taken from Rusch et al. PLoS Biol 5(3): e77 with permission under the Creative Commons Attribution 2.5 Generic license via commons.wikimedia.org

Ocean plankton

The initial findings of this first of its kind eDNA study were published by the Venter team in the prestigious Nature journal in 2010, in an article titled Genomic and functional adaptation in surface ocean planktonic prokaryotes, which reported the sequencing of 137 diverse marine isolates collected from around the world (see map). Metagenomic data analysis aimed to gain insights into the ecology of the surface ocean prokaryotic picoplankton (0.1–3.0 μm size range), pictured here. 

The results suggest that the sequenced genomes define two microbial groups: one composed of only a few taxa that are nearly always abundant in picoplanktonic communities, and the other consisting of many microbial taxa that are rarely abundant. The genomic content of the second group suggests that these microbes are capable of slow growth and survival in energy-limited environments, and rapid growth in energy-rich environments. By contrast, the abundant and cosmopolitan picoplanktonic prokaryotes, for which there is genomic representation, have smaller genomes, are probably capable of only slow growth, and seem relatively unable to sense or rapidly acclimate to energy-rich conditions. Their genomic features also lead to the proposal that one method used to avoid predation by viruses and/or bacterivores is slow growth and maintenance of low biomass.

A decade later, we have moved forward from this metagenomic sequencing using Sanger-based methodology with ABI 3730XL DNA sequencers (Applied Biosystems Inc.), to sequencing-by-synthesis on Illumina platforms, to sequencing-by-scanning with the Oxford Nanopore Technologies (ONT) systems mentioned above.  

In searching Google Scholar for recent metagenomic nanopore-based sequencing of plankton, the Zone found an item titled Zooplankton Communities, the M.S. Thesis of Stijn Willemse (Ghent University, Belgium), downloadable at this link. Briefly, this work investigated whether it is possible to use an ONT MinION sequencer for metabarcoding of marine zooplankton communities in samples taken during a biomonitoring campaign on the North Sea. Different DNA extraction techniques were tested, after which PCR amplification and purification were optimized by selection of an optimal primer set, PCR master mix, inhibition prevention measures, and purification protocols. The amplicons were then sequenced and subdivided into species using a reference database. Finally, a comparison was made using ZooScan hardware and software analysis, in which species are determined based on their morphology, as described in detail elsewhere. The results were largely conformed to ZooScan determinations and showed no discrepancies from what was found in the literature. It was concluded that “metabarcoding with MinION sequencing is a valuable innovation in the field of biomonitoring.”

Great white shark

In a conceptually and technically related investigation, Truelove et al. used a portable ONT MinION sequencer to study eDNA from the great white shark (Carcharodon Carcharias, pictured here), contained in seawater samples collected in the high seas, where the sharks have historically been identified with biologging datasets. Onboard an oceanographic vessel, a total of 10 sequencing runs were performed on the MinION, with a turnaround time from water collection to sequence results and annotation of ~48 hr. It was concluded that “identifying vertebrates by amplifying eDNA from seawater provides a novel approach for sampling and detecting the presence of elusive species of conservation importance in remote locations.”

Northern pike

Sepulveda et al. tested the sensitivity of a portable, battery operated, field-based eDNA PCR platform (Biomeme Inc.), comparing it to widely used lab-based eDNA approaches for detecting invasive northern pike (Esox lucius), pictured here, in eight lakes on Alaska’s Kenai Peninsula. The field-based platform takes ~1 hr from sample collection to final results, and uses a field-based shelf-stable DNA extraction kit. It was found that the field-based approach was less sensitive than lab-based approaches and was more prone to inhibition, thus increasing potential for false-negatives. Until sensitivity and inhibition issues can be resolved, this portable, field-based approach is best viewed as a complement to, rather than a replacement for, standard eDNA lab-based approaches.

For additional exemplary publications about eDNA metabarcoding in freshwater systems, readers can consult Fujii et al. (2019) Environmental DNA metabarcoding for fish community analysis in backwater lakes: A comparison of capture methods and Tessler et al. (2016) A Global eDNA Comparison of Freshwater Bacterioplankton Assemblages Focusing on Large-River Floodplain Lakes of Brazil.

Mimivirus

Mimiviridae (depicted here) is a group of viruses with large genomes and virions that are thought to influence the population dynamics of eukaryotic microorganisms, such as amoeba, in marine environments by preying on the host microorganisms. To better study and understand the ecological relevance of Mimiviridae, Prodinger et al. (2020) developed an optimized metabarcoding method for these viruses using a large set of primers and a combination of qPCR and sequencing.

Finally, it is worth noting that Turner et al. have found that fish eDNA is more concentrated in aquatic sediments than surface water, as exemplified in the case of carp eDNA. They found that carp was 8–1,800 times more concentrated per gram of sediment than per milliliter of water, and was detected in sediments up to 132 days after carp removal. This was explained by retarded degradation of sediment-adsorbed DNA molecules. Compared to aqueous eDNA, sedimentary eDNA could provide a more abundant and longer-lasting source of genetic material for inferring current or past site occupancy by aquatic macrofauna, particularly benthic species.

Air Samples for eDNA

Blue grama

According to a publication by Johnson et al., the studies that have examined airborne eDNA have focused solely on pollen, potentially overlooking other eDNA sources and elements of the ecology of airborne eDNA. Therefore, in a subsequent study, Johnson et al. collected airborne eDNA using commercially available dust traps and quantified the amount of eDNA present for a flowering wind-pollinated genus, blue grama (Bouteloua), and insect-pollinated honey mesquite (Prosopis glandulosa), both of which are pictured here. Importantly, the honey mesquite was not flowering during  the study at Texas Tech University Native Rangeland in Northern Texas. 

Honey mesquite

The researchers were able to use primer-specific qPCR to detect airborne eDNA from both species. As honey mesquite is insect-pollinated and was not flowering during the time of this study, the results confirm that airborne eDNA consists of, and can detect species through, more than just pollen. Additionally, Johnson et al. were able to detect temporal patterns reflecting Bouteloua reproductive ecology, and suggested that airborne honey mesquite eDNA responded to weather conditions during their study. These findings suggest that further study of the ecology of airborne eDNA is needed in order to uncover its potential for single-species and whole-community research, as well as for management in terrestrial ecosystems. 

Future Prospects

From a technology perspective, the major changes in eDNA research and applications have involved evolution of traditional PCR/qPCR and Sanger-based sequencing to higher throughput/absolute quantitation methods by, for example, digital PCR, and much higher throughput NGS. Among the latter technologies, the advances in ONT nanopore sequencing are particularly impressive, and have proven to be applicable in the field, which greatly expands the locations where eDNA sampling and sequencing “on the spot” can be carried out. 

To quote from eDNA expert Seymour, “eDNA has provided a catalyst for an amazing wave of research. Combined with advances in sequencing technologies, computer-assisted learning, and chemical analyses, we are potentially facing a renaissance in biological science, most assuredly within the realm of molecular ecology.”

He adds that “the impact that eDNA has already had on all aspects of relevant research, despite its recent development, is astounding and will undoubtedly continue to influence careers and policies for years to come, with all manner of mistakes and updates along the way. It will be paramount to ensure collaboration is maintained within and among investigation bodies and to put effort into conveying the importance of such findings to the public and managers.”

The Zone fully agrees with these views, and looks forward to future advances based on eDNA.

Your comments are welcomed, as usual.

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