eDNA Testing for Great Crested Newts

This week I answered a plea for help and stepped in to carry out some e-DNA testing of five ponds at Tintwistle near Glossop in the Peak District, as part of the PondNet project.

Environmental DNA (e-DNA) testing is a relatively new procedure and the technology is still being trialled to a certain extent. It aims to offer another way of surveying for Great Crested Newts. Traditional surveys have to be carried out at night, and at least four visits should be completed between April and June to demonstrate whether newts are present or absent. This is time-consuming and has it's issues, such as the health and safety of wandering in dark woodlands near water at night, the cost of paying for consultants, working unsociable hours, cleaning bottle traps, etc.

One advantage of e-DNA is that only one visit is needed to collect the water samples, and this can be done in the daytime, making it ideal for remote sites. DNA is 'lost' by newts through skin, faeces etc and can persist in the pond's water for up to three weeks. The e-DNA samples therefore need to be taken within the normal window for newt surveying ... My survey was therefore a little late in the season, due to another volunteer being unable to do the sampling, but it could still pick up some DNA.

It was an easy hour's drive down the M1 and across the Pennine moors via the Woodhead Pass.  Parking up at the side of an angling club, it was then an pleasant twenty minute walk around the reservoir and into the nature reserve at Swallow's Wood. Armed with a map, I managed to find the ponds without too much trouble, and after ignoring a 'closed footpath' sign and battling my way through the woodland undergrowth, I reached the site.

The first three ponds I surveyed were really pools in a stream separated by weirs. As well as recording environmental data and noting any amphibians seen, I collected e-DNA samples following a strict protocol.  This took around 30 minutes per pond and the process is described below.

One kit is provided for each pond and contains everything you need to carry out the water sampling. This is all disposable. The first stage is to put on a pair of nitrile gloves. This is to minimise contamination from other ponds/water that have been touched. 

Twenty water samples are taken from around the pond, trying to space these out as evenly as possible and make sure that some are from open water and other around vegetation.

Without standing in the pond (again to avoid cross-contamination), reach into the pond with the test-tube ladle and fill it with water. You should swirl the water around a little to mix the water column, but avoid disturbing the sediment on the bottom of the pond as DNA can persist here for a long time and may give a false result.

Pour the ladle full of water into the 'whirl-pak' bag provided. This should stand up on its own, but take care not to knock it over. When all twenty samples are placed in the bag, close the bag and shake it for ten seconds to mix the water.

For each pond, six test tubes are provided. Each contains an alcohol-based fluid that will preserve any DNA until it can be tested.

Putting on a new pair of gloves, decant some of the collected water from the whirl-pak bag into test tubes using a plastic pipette. I found the pipettes, being disposable, were not very robust and I had to take several pipettes worth to make up the sample. Each test tube is filled up to the 50 ml mark. Replace the screw cap firmly, and shake the bottle for ten seconds. This is to mix up the water sample and the preserving fluid.

The water in the whirl-pak bag should then be shaken up again, as the DNA constantly sinks to the bottom, and the next sample taken. In total, six test tubes are filled, forming replicates of the test.

Each of the six test tubes are labelled with a bar code which is repeated on the box that they come in. It's really important to make sure that the test tubes all go back into the same box they came out of, and that this is clearly labelled.  As well as writing the pond name/code on the box, the DNA test code needs to be recorded on the paperwork so that the results and the pond can be married up together afterwards.

It was a really useful experience to carry out this testing and see what was involved. The results should be available in September, and it will be interesting to see if Great Crested Newts are present on the site.

Whilst carrying out the tests, I also spotted some good examples of woodland and pond plants (Sweet Flag, Betony, Wood Vetch), several species of butterfly (Speckled Wood, Red Admiral) and added Large Red damselfly to my list. It is a lovely site to have a wander around if you are ever in the area.