DNA & eDNA

What is eDNA?

eDNA refers to the genetic material (nuclear, mitochondrial and chloroplast DNA) that is released by an organism to its environment as dead skin/plant cells, hair, feces, urine, saliva, mucous, gametes, etc.  This material can be collected from the environment through water, sediment, soil, or air sampling, subjected to DNA isolation and tested to determine presence of the target species without observing the organism directly.

In essence, organisms leave a trail of DNA in the environments they occupy, which persists for a limited period of time.  The environment can be surveyed through eDNA testing of collected samples to determine if the target organism was/is present.  This principle and application of DNA technology is much like how forensic biologists analyze DNA found on samples collected at crime scenes to determine the culprit.

eDNA is particularly advantageous for aquatic and semi-aquatic species as shed DNA is transported through the aquatic environment away from the source, improving the ability to detect the target species.

It’s been just over a decade now since Ficetola et al.1 published the first eDNA study in 2008, having tested freshwater samples collected from wetlands in France for presence of the invasive American bullfrog.  Since this first report of applied eDNA testing, there has been an explosion of scientific studies with hundreds of peer-reviewed articles published each year.  The number of species that eDNA testing has been applied to continues to grow and we have learned more about the persistence and dispersion of eDNA in the environment, best practices for eDNA sampling and preservation, requirements for robust laboratory assay design and the impact that the number of replicates tested on a sample has on the statistical confidence of eDNA testing results.

Why test eDNA?

Ecological biodiversity and species-specific surveys are used to support environmental assessments for a number of purposes including resource management and extraction, commercial, industrial, and residential development projects, conservation efforts, determining successful uptake of restored habitat by desired species and monitoring for early presence of invasive species.

When either the abundance of the target species, or the ability to detect the target species within an environment is high, conventional methods of visual and audible observation, trapping, electrofishing, netting, etc. often offer the best and most cost effective approach.  However, when species are cryptic or abundance of a target species is low, as is the case with endangered species and at the early stages of an invasive species, there is considerable time and effort required to detect the species using traditional survey methods.  It is also possible that conventional surveys will miss the target species at low numbers given that the environmental area to survey may be large.

eDNA has several advantages over conventional methods, particularly when the sampling medium is mobile. Some of these advantages include:

  • A greater opportunity for the target eDNA to be sampled at a distance from the individual organism in aquatic systems.
  • Improved sensitivity to detect cryptic and rare species compared with conventional methods, providing a higher success2 rate at a lower overall cost and time investment.
  • Ability to discriminate the target species with accuracy and precision, reducing reliance upon qualified experts to identify these species in the field.
  • No requirement to obtain a permit or license, since only environmental samples are being taken as opposed to conventional methods that may involve trapping and handling of species at risk.
  • Allowance of a broader window of time during periods that are safer for staff to operate in the field since the species itself does not need to be observed directly. For example, sampling during daylight hours as opposed to conducting surveys at night for species that are more active after dark.
  • A positive impact on the habitat and individual organisms that occupy it by eliminating the need for intrusive investigations (trapping, electrofishing, netting, extensive habitat searches). Thus, also reducing the risk of pathogen transfer.

How does eDNA laboratory testing work?

Several methodologies for eDNA analysis exist.  Most methods are designed to detect a single target species using quantitative polymerase chain reaction (qPCR), also known as real-time PCR.  qPCR is a highly sensitive and specific DNA analysis that allows for detection of very low quantities of DNA through repeated (up to 50) cycles of PCR that exponentially generate many copies of the target DNA; typically, a short sequence of DNA that is unique to the study organism.  The generated copies of target DNA are visualized by lab instrumentation that detect a fluorescent reporter dye, which is released from a probe that specifically binds the target DNA sequence, thus confirming detection of the study species.

qPCR technology has been around for 25 years.  It is a modification of standard PCR, which was discovered in 1983 by Kary Mullis, who was awarded the 1993 Nobel Prize for chemistry for the discovery.  qPCR technology is a reliable and rapid method used for a number of important tests we rely upon in our daily lives. It detects infectious diseases, inherited genetic abnormalities, harmful pathogenic bacteria in food products, genetically modified crops and water quality.

Limitations of eDNA Testing

Contrary to the name, “quantitative” PCR, qPCR as applied to eDNA samples can only reliably report if the target species was detected or not detected in a tested sample.  We can understand why this is the case by examining a scenario in which water samples are collected from the environment, filtered and analyzed for eDNA presence of a rare amphibian.  If we discover a large quantity of DNA in one of those samples, it could be the result of a large number of individuals of the target species having been present, or it could equally be the case that the sample was collected in close proximity to a single individual of the species, resulting in more eDNA capture due to proximity alone, not due to there being a large number of individuals from the species present.

Complicating the situation further is the fact that the species will shed DNA to the environment at different rates during different lifecycle stages.  This eDNA will persist in the environment for varying lengths of time depending upon the specific environmental conditions including temperature, microbial activity and exposure to ultraviolet light, all of which work to breakdown DNA to the point where it is no longer of sufficient quality for detection by the qPCR assay.  For aquatic environments, eDNA starts to degrade within hours of release from the organism and can be reliably detected by an eDNA assay on average 7 to 21 days3 post release.  eDNA thus provides a recent picture of a species’ presence in an environment.

eDNA is shed from the organism, however it does not indicate whether the individual source organism is alive, or dead.  Furthermore, eDNA provides no information on the age, size, gender, or reproductive status of the organism.

Despite these limitations, one must not be discouraged and dismiss eDNA, but rather be reminded of the benefits that eDNA presents as a scientific tool and examine the utility when properly executed for completing species surveys that are more sensitive, more accurate and with reduced cost and overall investment of time.

References

1 Ficetola, G.F.; Miaud, C.; Pompanon, F.; Taberlet, P. 2008. Species detection using environmental DNA from water samples. Biology Letters 4 (4):423–425.

2 Jerde, C.L.; Mahon, A.R.; Chadderton, W.L.; Lodge, D.M. 2011. “Sight-unseen” detection of rare aquatic species using environmental DNA. Conservation Letters 00:1–8.

3 Dejean, T.; Valentini, A.; Duparc, A.; Pellier-Cuit, S.; Pompanon, F.; Taberlet, P.; Miaud, C. 2011. Persistence of Environmental DNA in Freshwater Ecosystems. PLoS ONE 6 (8):1-4.

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