• 14Jan

    eDNA testing for great crested newts

    Image: Great crested newt

    Environmental DNA (eDNA) testing is a new method of surveying for species that is being hailed as cheaper and less resource intensive. But what is it, and is it the panacea it’s claimed to be?

    eDNA is nuclear or mitochondrial DNA that is released from an organism into the environment. It can be secreted via faeces, mucous, and gametes; shed as skin and hair; and is also found in carcasses. In aquatic environments, eDNA is diluted and distributed in the water. In suitable conditions, it can persist in the water for up to 21 days before being broken down by microbial activity and UV light. The DNA from a range of aquatic organisms can be detected in water samples at very low concentrations using qPCR (quantitative polymerase chain reaction) methods.

    Surveying for protected species such as great crested newts (Triturus cristatus) plays a large part in the role of a consultant ecologist. As part of the planning process, developers need to evidence that they have considered any protected species that may be on the site to ensure they are not harmed during construction works. Spending most of their time hidden underground and in nooks and crannies, the easiest time to survey for newts is in the Spring when they return to ponds to breed. Aside from commercial developments, understanding the distribution of newts is also important to conservation biologists and land managers when planning habitat management and translocation programmes.

    Historical Natural England guidelines state that a minimum of four surveys have to be carried out between April and June, using three different survey methods on each occasion, to demonstrate the likely presence or absence of newts on site1. The survey effort is intensive and during unsociable hours: torching is carried out after sunset, with bottle traps being set before dusk and retrieved early the next morning.

    eDNA testing is advertised as a quick and accurate alternative to traditional survey methods. A single sample from each pond, sent off for laboratory analysis, can quickly establish the presence/absence of newts without the need for further visits. In 2014, supported by evidence from field experiments, Nature England approved this as a method of demonstrating the likely presence/absence of GCN in ponds2.

    The eDNA procedures for great crested newts were developed during a DEFRA research project3. Controlled experiments compared the results of eDNA analysis with traditional methods at ponds where newts were known to be present, and allowed a robust procedure to be developed.

    Sampling procedures have been developed to maximise the chance of collecting eDNA if newts are present. Surveyors take 20 x 30 ml samples from around the perimeter of the pond, which are then mixed together. From this, 6 x 15 ml sub-samples are decanted into sterile tubes and sent to the laboratory for analysis. There, a fragment of the mitochrondrial cytochrome oxidase I gene (cyt-b) is amplified. After extraction, qPCR analysis is run. In early trials4, the initial success rate of 63% detection was made more effective by increasing the volume of DNA in the PCR. Tests were also done to ensure that the DNA of closely related species did not produce a false positive.

    Degradation of DNA was an issue, caused by both temperatures above 20°C and the interval between collection and analysis. To prevent this, ethanol was added to collection tubes as a preservative. An artificial DNA marker has also added to check for unexpected decay of DNA between sampling and sample analysis. This will reduce the number of false negatives, as laboratories will be alerted to the fact that material has decayed.

    Correctly identifying the presence of newts in ponds using eDNA has been cited as between 84% to 91%, based on different field experiments4,5. Newts are a cryptic species: using traditional methods, four negative samples of the same site are needed to give reasonably high confidence that they are truly absent. In comparison5, eDNA can give the same result in just one visit.

    This new survey method has many more advantages. As well as a reducing the minimum number of visits from four to one, surveys can be done at any time. From a health and safety perspective, not working at night has obvious benefits to surveyors, and commercially it reduces the manpower required.

    Traditional methods require surveyors to have suitable knowledge/experience and hold a European Protection Species (EPS) licence through the relevant home nation’s statutory body (eg. Natural England). A benefit of using the eDNA technique is that, when not in connection to mitigation work, a licence is not needed to collect water samples2. This is of particular benefit to organisations such as the Freshwater Habitat’s Trust that gather information about the status of water bodies and their species using volunteers. In 2014 they assessed the use of eDNA for their citizen science based monitoring programme. 91.3% of sites were correctly identified as supporting newts. The remainder were false negatives, either due to small numbers of newts or difficulties in collecting water samples.

    eDNA sampling is non-invasive – removing direct contact with newts, the risk of spreading amphibian diseases such as chytridiomycosis is reduced. A single-use sterile pack is used to take water samples for eDNA testing. By reducing the amount of equipment used, biosecurity is further improved. In turn, this also reduces environmental impact, as chemical disinfectants are only needed to clean boots rather than traps and nets too.

    As eDNA persists for up to 21 days, it can detect newts even if they are not physically in the pond, an obvious benefit over surveying which requires first-hand observations. Using eDNA, a negative result is believed to be a much stronger indication of true absence. GCN are more likely to be detected using eDNA in conditions not suited to traditional sampling methods, such as murky water when torching, or shallow water preventing bottle traps from being set.

    Despite all of these benefits, some ecologists are sceptical about eDNA testing and have been slow to embrace it. So what are the perceived and actual drawbacks?

    The cost of an eDNA kit, couriered to the laboratory and it’s analysis (per pond) is typically £250+. Whilst eDNA testing may free up manpower, these additional costs mean the cost saving is only estimated to be around 20%, depending on the number of ponds on each site. As there are few laboratories offering the service the analysis has to be booked in well in advance, meaning it is virtually impossible to carry out ad hoc surveys. There is a lot of demand for the service in a survey season that is still very restrictive: limited by the rate of eDNA degradation, it can be extended only by a few weeks.

    Although eDNA can assess the presence of newts, it is less useful in assessing the size of a population. Whilst more precision in eDNA testing might improve this, other factors mean the amount of DNA collected still cannot accurately be correlated to newt numbers. These include environmental factors such as temperature, pH and UV (that affect the rate of DNA degradation) and water sampling methods.

    If newts are present and the pond is in an area of development, a population size class needs to be calculated. For this, Natural England requires six surveys using traditional methods, carried out between mid-March and mid-June. And this is where the eDNA process falls down. A major drawback occurs in the timescales for receiving results back, which is 2-3 months. Even if eDNA testing were done at the very start of the season, there would not be sufficient time to do the additional surveys required to establish a population size. eDNA testing is therefore most suited to projects with a long lead in time.

    In summary, the 2015 survey season was the first in which eDNA testing was widely used for great crested newt surveys. It offers a faster, non-invasive method of detecting the presence of newts in ponds, however the survey window is still limited and cost benefits are small. Due to the timescales for receiving results back, it is most suited to long-term planning proposals or conservation projects with no pressing schedule. It will not replace traditional surveys methods, which are still required to establish population sizes for mitigation. However, it is an additional valuable item in the surveyor’s toolbox, and will give benefits when used appropriately.

     

    by Kate Wright MSc Biodiversity & Conservation (Year 1)

     

    References

    1. Natural England & Department for Environment, Food & Rural Affairs (2015) Great crested newts: surveys and mitigation for development projects [Online] Available at: https://www.gov.uk/guidance/great-crested-newts-surveys-and-mitigation-for-development-projects
    2. Natural England (2015) “EPS Mitigation Licensing: Latest Developments”, Newsletter, February 2015 [Online] Available at: https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/407710/eps-newsletter-feb-2015.pdf
    3. Biggs J, Ewald N, Valentini A, Gaboriaud C, Griffiths RA, Foster J, Wilkinson J, Arnett A, Williams P and Dunn F (2014) “Analytical and methodological development for improved surveillance of the Great Crested Newt” Appendix Technical advice note for field and laboratory sampling of great crested newt (Triturus cristatus) environmental DNA. Freshwater Habitats Trust, Oxford.
    4. Rees HC, Bishop K, Middleditch DJ, Patmore JRM, Maddison BC, Gough KC (2014) “The application of eDNA for monitoring of the Great Crested Newt in the UK”, Ecology and evolution. 4(21):4023-32
    5. Biggs J, Ewald N, Valentini A, Gaboriaud C, Dejean T, Griffiths RA, et al (2015) “Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus)”, Biological Conservation. 183:19-28

Discussion One Response

  1. February 21, 2016 at 11:18 pm

    Hi Kate,

    Nice article, however I’m happy to report that turnaround times are now much less than the 2-3 months experienced in 2014.

    Most labs (including ours) now get results back within 2 weeks as standard and can often turn them around in 2-3 days if there is real urgency. That means that if you get the test done at the beginning of the season, it’s possible to get results back in time to do the population surveys where necessary. It does mean the labs get very busy at the beginning of the season though, so if you’re flexible and not under time pressure then it’s best to plan to do your eDNA surveys in the second half of May.

    Kat

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