Using eDNA to sample for great crested newts

Using eDNA to sample for great crested newts

The great crested newt (Triturus cristatus) is the UK’s most heavily protected amphibian species, having suffered huge population declines in the last century. Their habitats cannot be disturbed or destroyed without permission from the relevant Statutory Nature Conservation Organisation.

House builders and other developers, must, survey potential breeding ponds to establish if newts are present on development sites. Ecologists can use eDNA, as the latest addition to a variety of survey methods, to do this.

What is GCN eDNA sampling?

Environmental DNA, generally shortened to eDNA is the genetic material that organisms leave behind in their environment.

Great crested newts (GCN), release eDNA into the ponds in which they live when they deposit; skin cells, faeces, mucus, sperm or eggs into the water. The DNA in this material can persist, and be detected, in the water for several weeks.

Research published in 2013 established a technique for reliably detecting newt eDNA in water bodies, and Natural England subsequently approved a protocol for this to become a survey method.

A number of suppliers including Wildcare, now offer GCN eDNA sampling kits that allow surveyors to collect samples and post them to a lab for testing. The results (positive or negative) for each pond are returned via email, soon after analysis.

When should I use eDNA sampling?

GCN eDNA sampling is approved for use by Natural England during the breeding season only, from 15th April until 30th of June. During this time GCN are using ponds, so if they are present they are likely to leave a genetic signature.

If you suspect newts are present and expect to have to estimate relative abundance, be sure to do your eDNA sampling at the start of the season.

eDNA sampling can save planning applications time and money compared with survey methods such as torch surveys or bottle trapping. These traditional methods require repeat surveys throughout the season, whereas eDNA sampling can establish presence or absence with a single site visit right up to the end of June. However, unlike other methods, eDNA cannot estimate how many newts are present.

Some consultants start bottle trapping while they wait for their eDNA sample results so that if the pond is positive for GCN they have plenty of time for more extensive surveys. If the pond is negative they simply stop all surveying.

How reliable is it?

eDNA sampling is very effective at detecting GCNs, provided Natural England’s protocols are followed and a licenced laboratory is used.

Biggs et al. (2014) estimated that eDNA sampling had a 99.3% detection efficiency, compared with 76% for bottle trapping, 74% for torch surveys and 44% for egg searches.

To ensure high standards among laboratories offering GCN eDNA testing, Natural England run a Proficiency Testing Scheme to check their protocols. Natural England will only accept eDNA results for licence applications where these come from participating labs. Labs can only test samples collected with their own kits as only they can search for their degradation controls.

Wildcare is proud to supply kits from Applied Genomics, who scored 7/7 in their laboratory proficiency testing during 2017. The kits are made to order so the preservative is fresh and contains a control strand of DNA to check for degradation, ensuring your results are reliable.

How do I collect my samples

Most GCN eDNA kits will include instructions, but it is the surveyor’s responsibility to be familiar with, and to follow, the Natural England approved protocol described briefly below.

The protocol steps include:

  1. Take twenty 30ml water samples from around the edge of a pond, taking care not to disturb the sediment (eDNA can be preserved in sediment, leading to a false positive).
  2. Mix the 20 samples.
  3. Use a pipette to add some of the mixture into the six 50ml tubes that will be sent off for analysis.
  4. Ensure the remaining water is returned to the pond.

An eDNA sampling kit will contain everything you need to take the 20 samples and make up the 6 analysis tubes.The tubes contain a preservative that protects any eDNA from degradation while they are sent back to the lab and analyzed.

Tips when using your kit:

  • Avoid collecting from densely vegetated areas.
  • As a rule of thumb, use one kit per 1ha of a pond – most ponds will only need one kit.
  • Kits should be used within 2 weeks of receipt.
  • Keep kits away from high temperatures and direct sunlight.
  • You need to be careful not to transfer eDNA from one pond to another, so never go into the water when taking samples, and never reuse parts from a kit used elsewhere.
  • It is advisable not to survey in muddy conditions where you could come into contact with pond water.
  • If you need to decontaminate boots and equipment, we recommend a chlorine based bleach such as Domestos.
  • If you are using eDNA testing to survey a pond in support of a license application, then the sample must be collected by a licensed great crested newt surveyor.

I’ve collected my samples, what happens next?

Ensure samples are kept out of direct sunlight and away from heat, but don't worry about keeping them in the fridge unless you are storing them for more than a few days. If you do decide to keep the samples longer than a few days they can be stored in the fridge for up to a month before analysis if necessary. Never freeze your samples as this can cause the tubes to crack and leak.

  1. Package and return your samples according to the lab instructions. Wildcare’s service includes a pre-arranged courier collection.
  2. Once the sample is received, the lab extracts the DNA and stores it before PCR at -20°C.
  3. Samples are then tested for inhibition and degradation using real-time PCR assay, before being tested for GCN DNA using real-time PCR assay.
  4. Results will be emailed to you, with a full report.

A positive result means that newts have been present in the water up to 21 days before sampling. A negative result means no GCN eDNA was detected. Very rarely results are inconclusive, meaning no GCN eDNA was found, but the tests show that the PCR reaction was inhibited or the control DNA was degraded - in this case, you should collect a new sample and retest*.

*You will still be charged for an inconclusive result as the lab has had to perform the same amount of work as with a successful result.

Left-over kits and their associated analysis slot are non-refundable. If you wish to donate them to a charity such as, the Amphibian and Reptile Conservation Trust, please let us know and we will be happy to make the necessary arrangements.

Where can I order my equipment from?

To start collecting your samples and order your eDNA kit click here. Alternatively, if you would like to still use more traditional methods instead of or as well as the eDNA kit, click here to view Wildcare's range of pre-cut bottle traps, nets and other trapping equipment.

Where can I find more information?

You can find all the original technical guidance and reports, including the 2014 ‘Biggs report’ (the Final Report) on the DEFRA website.

Click here to find out more about Guidance on using eDNA in Wales:

There are also a number of scientific papers about eDNA, of which below are a couple of recent ones:

Rees et al, 2017 - The detection of great crested newts year-round via environmental DNA analysis.

Buxton et al, 2017 - Seasonal variation in environmental DNA in relation to population size and environmental factors.