“Oi, the water is very high today,” says David Ekkers cheerfully. The molecular ecologist from Waardenburg Ecology is standing in the middle of the Gasterense Diep, one of the upper reaches of the Drentsche Aa. The water almost reaches the edge of his armpit-high waders. Ekkers holds his arms high to keep them out of the cold water for a while. But now he has to face it: he has to bend over to get under the bridge. And then he has to place a filter capsule in a device there, two decimeters below the water surface.
The device is an autosampler: a cassette that can hold 24 of these filter capsules, and that you can program to allow a certain amount of water to flow through one of those filters at regular intervals, thanks to cleverly linked motors, tubes and valves. The capsule then closes automatically again – until the researcher comes to pick it up again. The goal: filter DNA from the water.
This is the cutting edge of the development of e-DNA technology, according to Ekkers. E-dna, or environmental DNA is hereditary material that is present loosely in soil, water and even air, and that reveals which organisms are present or have recently visited. “Well, not really just DNA,” the researcher immediately qualifies, after he has smoothly climbed back out of the water via the steep bank. “In this case, it concerns loose cells or cell parts that float in the water. Really loose DNA is so small that it passes directly through the filter.”
The cell material comes from the plants, animals and microorganisms that live in this stream. Ekkers’ project focuses on three types of fish: eel, river lamprey and roach. The first two are rare, and move from fresh to salt water or vice versa over the course of their lives. The latter is common and lives in this stream system all year round. This serves as a reference. “The e-DNA that these fish leave behind mainly comes from their poop,” explains the biologist. “In this filter we capture the cells that have been released from the intestinal lining.”
E-DNA has a number of advantages over other methods of monitoring, Ekkers emphasizes. “It is relatively cheap, you do not have to catch animals, and the technique is sensitive enough to detect rare species. With other methods, such as trapping, you have a good chance of missing rare species.” In recent years, rare fish species have been found in new places using e-DNA, including brook bullhead in the Brabantse Dommel and the big mud crawler in Flanders.
But there are also limitations to the technology, Ekkers notes. “Due to currents, it is sometimes difficult to trace the location of the fish. And you do not get any information about the age structure of fish populations.” There is an additional complication with the river lamprey: the young fish live deeply buried in the mud – and within a few kilometers of this stream valley at most. Ekkers: “This is the only remaining spawning location for river lampreys in the Northern Netherlands.”
Investigating dynamics
Waardenburg Ecology works here together with ecologist Peter Paul Schollema of the Hunze en Aa’s Water Board. They are not actually concerned with the fish, Ekkers emphasizes. “Yes, in the long term of course. But this is mainly a pilot project for the next step in e-DNA technology: that you can automatically take a series of samples in one place, over longer periods of time. Or at the same time in different places along a river course.” Then you can investigate the dynamics of these migratory fish species, he explains. When exactly are they in a certain area? How fast do they swim through it? And ultimately: in what numbers? “Then it gets really interesting.”
Before that happens, there are still many hurdles to overcome. Ekkers talks about it while he takes out some things from his car and sets them up. He will soon use this to filter a liter of stream water. This serves as a check for the measurement that he also has carried out at the same time by the autosampler under the bridge.
“One of those hurdles is that we do not always know how long DNA remains detectable in the water,” he says. “Sometimes it dilutes very quickly, or it is broken down, depending on the circumstances. And exactly how much DNA the fish release also depends on the species and their activity. At the moment, for example, there are no river lampreys swimming through the stream, but they are all deeply buried in the mud. Yes, they also release DNA. We are now trying to see how much we can pick up.”
The European eel (left) and the river lamprey.
Photos Getty Images, ANP/KINA
The Hunze en Aa’s Water Board also ‘physically’ monitors the young river lampreys at other times, with a special grabber that you use to bring up mud. This is done in collaboration with Wageningen Marine Research, as part of the mandatory monitoring for this Natura 2000 area.
Another hurdle with e-DNA research is the detection threshold: how much DNA you need to prove the species. That threshold depends on various factors: the species, the time of year, the temperature and substances in the water that make DNA analysis difficult. Ekkers: “But also the liquids you use to process and store your sample. And the DNA technology used. We are now investigating the effect of all these factors. And we try to concentrate the DNA of the target species in all kinds of ways in order to lower the detection threshold.”
And in this sub-experiment, with the autosamplers, Ekkers wants to find out whether there is a difference between what those filters yield over the course of a season, and what you ‘catch’ if you take a sample on site and immediately analyze it in the lab. “Suppose I leave such an autosampler somewhere for a few months,” says Ekkers, “then such a filter cassette hangs in that stream all that time. How does that change your measurement results, compared to a sample that I immediately analyze? We are going to find out now, so that we can properly interpret the data from such an autosampler later.”
Not a goal in itself
Suddenly the sun breaks through. The old oaks of the Drentsche Aa show their most vibrant autumn colours. The stream meanders through it like in a painting. “One of the best preserved stream valleys in the Netherlands,” notes Ekkers.
He now filters a liter of stream water using a stick with a suction hose and a pump – a construction that he put together himself. The suction hose has the same filter capsule attached to it as the one he previously placed in the autosampler. The capsules were designed by Sylphium, a start-up from the University of Groningen. Ekkers invented the autosampler himself and built it together with two friends who are handy in programming and electrical engineering. “I really like that technical part,” he says. “Think about what exactly you want to know and measure, and then achieve that with the simplest possible means.”
But it should not become an end in itself, he emphasizes. “I am and will remain a biologist. What matters to me is that we continue to refine these techniques in order to monitor nature better and better. And that is what you need to better protect these species and their habitats.”
So, the liter of stream water has been filtered. Ekkers unscrews the filter capsule from the hose. It goes with me to the lab. The other items also go back in the car. On to the next location: a kilometer upstream he will soon repeat the entire exercise. He is curious about what concentrations of e-DNA he will find there, closer to the ‘source population’ of the river lampreys.
“What we ultimately want is to develop a reliable and scalable technology,” he concludes. He means a technology that can be used relatively easily and affordably on a larger scale. “This will allow researchers, nature organizations and water boards to find out for themselves where the bottlenecks are in fish migration, and then solve them as best as possible.”
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