By filtering and sequencing DNA from river water in Pennsylvania, researchers have uncovered the presence of the invasive New Zealand mud snail, a species that, despite its negative effects on the ecosystem, is difficult to track due to its minute size.

The study, published Tuesday in Biological Invasions, demonstrates how environmental DNA, or genetic material that is sloughed off from cells into an organism's surroundings, can be used to identify the presence of invasive species earlier and with greater precision than older methods, such as visual surveillance or traps.

"All of us moving through our environments shed pieces of ourselves, and most obviously we shed our cells. But in those cells is a cell nucleus, and in that cell nucleus is our DNA," said study co-author Maurine Neiman, an associate professor of biology at the University of Iowa. "Eventually, as those cells degrade in the environment, they release our DNA, sort of like a footprint."

There are over 6,000 invasive species in the United States, and through management expenses and damages from unmanaged invasions, these species cost the U.S. over $120 billion per year. Identifying and tracking smaller invasive species can be a major challenge for researchers because these plants and animals are much harder to see.

"By the time [we're] doing surveys and finding new populations, people have probably already spread them from that population unknowingly, to potentially create new populations," said first author James Woodell, a research support technician at the University of Hawai'i at Mānoa, who worked on the invasive snail project as part of his master's research at the University of Iowa. "So, you're already way behind."

The methods of collecting and sequencing environmental DNA, also known as eDNA, emerged in the late 1980s to help scientists get a clearer picture of microbial communities, as the lab-based culture techniques of the time weren't very accurate. Since then, eDNA has proven to be an important technique for identifying invasive species, because it can pick up species that are otherwise hard to find.

Environmental DNA has been collected from air, soil and even animal products, including honey. But the most frequently used method of isolating eDNA is by collecting and filtering water, the approach the researchers here took.

They focused on the New Zealand mud snail, Potamopyrgus antipodarum, which is thought to have spread through ballast water from ships, beginning as early as the 19th century. Nieman had already studied this species as part of her other work on sexual biology. However, the snail also made a great research subject for an eDNA study, because it's very hard to find with visual methods.

"They're on every single continent except Antarctica," Woodell said. "They're really excellent invaders because they're teeny tiny, only a few millimeters long when they're full grown, and they can become very, very dense. They're able to physically take over an environment so that native species aren't able to establish there." He also explained that the invasive variety of snail reproduces asexually, so unlike other species, you only need one misplaced snail to cause a potential invasion.

Woodell and senior author Edward Levri, a professor of biology at Penn State Altoona, traveled to eight study sites throughout central Pennsylvania, collecting river water from these sites in ordinary plastic storage containers that had been sanitized to prevent contamination.

The samples were then put through an extremely fine mesh to filter out DNA. The researchers then returned to the lab, where they worked with Nieman to isolate and identify mud snail DNA in the samples. While the protocol for the DNA analysis had been developed in previous research from the Goldberg lab at Washington State University, it had yet to be implemented in an area where the snails had never been seen.

Five of the eight sites had evidence of snail DNA. The researchers were also able to find the snails physically at one of the sites to validate the results, though this took much longer than collecting water.

Because each site was selected in part because prior evidence had shown a lack of snails in the area, the findings show that eDNA can catch invasions earlier and more accurately than visual surveillance.

And while some eDNA studies have required sophisticated filtration systems aboard boats to collect samples, one of the benefits of the sampling protocol these researchers used is that it's extremely low-tech, which gives them hope for engaging the community in their research through a citizen science initiative.

"There are people already using these waterways, and the reason they're using them is that they're beautiful and maintained," Woodell said. "If we can communicate that they don't need fancy tools to go out there and contribute to that, that's a very viable future for preservation."

Now, Nieman continues to work on studying the invasion of these snails with her current graduate students. She is also confident that these methods can be applied to other invasive species.

"You should be able, in principle, to do this for any organism where there's been enough groundwork to characterize the genetic variation of the species," she said.

And while Woodell has moved onto other projects, working on soil science in Hawaii and moonlighting as a bartender, he remains passionate about invasive species management.

"If you go fly fishing, if you go boating, clean and bleach your gear. That's not a joke," he said. "It's the biggest thing that you can do to help prevent the spread of invasive species."

The study, "Matching a snail's pace: successful use of environmental DNA techniques to detect early stages of invasion by the destructive New Zealand mud snail," published June 1 in Biological Invasions, was authored by James D. Woodell and Maurine Neiman, University of Iowa; and Edward P. Levri, Penn State Altoona.