Finding the Invisible: A New Way to Look for Invasive Species Using eDNA

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As students look on, members of Gordon Luikart's lab, Steve Amish and Jenna Schabacker, collect filtered water sediment samples with a tow net for eDNA testing at the boat launch on Lake McDonald in Glacier National Park in June 2015. Credit: Ken Rand

On a dock that leads from the pine-bordered shoreline of Flathead Lake, I lean over, and look into the clear green water as hundreds of native Northern pike minnow swim through the gaps in the timber structure of the dock. In the warming water of spring, the fish are spawning. I have watched them since my childhood, carrying out the same cycle year to year, by playfully swirling around each other.

 

Flathead Lake is one of the largest freshwater lakes in the American West. It lies just south of Glacier National Park in Montana and remains a particularly clean water source, despite human pressures of excess nutrients, pollutant runoff, and erosion from agriculture and cities. For now, talk of mining has all but dried up since the upper watershed received protection in 2014.1

 

But, all is not as it seems. Aquatic invasive species are now making slow and steady progress toward not just Flathead Lake, but other rivers and lakes in Montana.

 

The invaders include aquatic plants, fish, amphibians, pathogens, and invertebrates like mollusks and snails. They arrive in new places every day. They can alter ecosystems by taking over large areas, disrupting nutrient cycles, and causing negative economic effects for hydroelectric dams, water, and places that rely on recreation and tourism.

 

Scientists are hoping that a new technology will help protect these waters by providing early detection and monitoring of invasive species without having to spot them visually.

 

In fact, all that is needed is one biological cell.

 

Environmental DNA, or eDNA, is a way of collecting genetic material without having to find or capture a rare or hard-to-find species. Scientists are now using DNA tests that capture water samples and look for unique markers to each species.

 

DNA contains the genetic instructions to make a living thing. Each species of organism has a unique set of genes, and each cell from that organism carries a complete set of those genes. When a plant or animal sheds some of those cells—from a fish’s scale, a leaf or plant tendril, or from blood, sperm, scat, urine, or pollen—it enters the “environment.”

 

To find out how scientists are chasing down eDNA, I spent a summer afternoon at Glacier National Park. Snow-capped Stanton Mountain waits in a raincloud at the opposite end of glacier-fed Lake McDonald, as I stand and watch the first sampling of eDNA that I have seen.

At Lake McDonald in Glacier National Park, students from Gordon Luikart’s summer conservation ecology course hold a tube of sediment collected by a tow net that could potentially hold the DNA of many species that have touched or live in the lake. Credit: Ken Rand

Standing at the shore, Gordon Luikart watches the mountains to track if rain or lightning will interrupt his experiment. Luikart is a conservation geneticist at the Flathead Lake Biological Station, a part of the University of Montana. A transplant from Iowa, he traces his first interests in biology and conservation to his love of fishing and being outdoors.

 

Today, he is speaking to his summer class of conservation biology students while holding a fine-meshed net called a plankton net, usually used to collect small aquatic creatures. “So, you can detect a single cell of anything that has touched the water, defecated from above, or swam through the water,” says Luikart.

A bright flash of lightning strikes at the other end of the lake and a brief echo of thunder rolls across the lake. “Whoa, lightning. Who wants to hold the net?” says Luikart jokingly. No one takes it.

 

On the floating dock, Steve Amish, a genetic researcher at the University of Montana, drags a long, fine mesh net through the water along the dock to capture drifting detritus.

 

On the first try, the net catches mostly cottonwood tree fluff, but on a second pass in deeper water, the sample shows up cleaner. Amish raises the net up in the air to drain most of the water. Jenna Schabacker, a young researcher who works with eDNA in his lab, rinses the remaining debris with ethanol and places it into small vials.

 

The samples they collect don’t look like much, with tiny bits of material sometimes visible in the bottom, but in each of the samples hide the signatures of potentially every species that has touched the lake.

 

Suddenly the students shout in excitement as they look into the vial, holding it up to the clouds. A small pair of eyes peer out. It’s a fish larva.

 

“So, we will probably detect that one with an eDNA test. But what species is it?” Luikart says, “You can’t tell without a DNA test.”

 

Amish and Schabacker take these samples back to the Montana Conservation Genetics Lab in Missoula, where they work. They will look for DNA from invasive species that are not supposed to be in the lake, as well as others that they already know are there, like the non-native lake and rainbow trout, as proof of principle. Their real enemies are the animals and plants they hope they will never find, like zebra and quagga mussels. They will also look for the mussels with eDNA in the environment.

 

To do this, Luikart and his team have to improve the methods of sampling waterborne genetic material. Using eDNA on a large scale in the open environment remains largely unproven. In Flathead Lake, one of the gems of Montana, no one else has undertaken such a task.

 

To that end, the lab will start testing with a small, unmanned submarine that will take water quality and eDNA samples and process them in real-time inside the vehicle while beaming the results back to shore. Luikart also just received funding to start design on another sampler that will sit in place at heavily used sites to take samples over time and monitor for invasive species.

 

Flathead Lake is hardly pristine, despite its visual beauty. Humans have changed and manipulated the lake to fit their needs, starting with the introduction of lake trout in 1905, now the most visible invasive species in the lake today, and other fish to aid sport fishing. This was followed by the introduction of mysis shrimp that fed the lake trout expansion.2

Zebra (top) and quagga (bottom) mussels pose an immediate threat to the waters they are introduced into, often causing widespread ecosystem changes by degrading habitats of native species without viable solutions for eradicating them. Credit: Ken Rand

But now, new invasive species are coming on their own.

 

There are many aquatic invasive species already in Montana, including Eurasian watermilfoil, curlyleaf pondweed, flowering rush, and New Zealand mudsnails. And there are two mussels that are not here yet, but must be kept out:  zebra and quagga mussels.

 

To look at an invasion that has already happened, I drive south from Glacier National Park back to Flathead Lake. At the Ducharme Fishing Access, a few miles east of Polson, a dirt road ends at a small constructed strip of land flanked by cottonwood trees. The shallow water is choked with plants forming dark brown patches in a thick mat. Some are native, but one plant in particular is most dominant; flowering rush, a native of Eurasia, has closed in on the boat ramp in dense drifting swaths.

 

Parts of the flowering rush break loose and float away. Each floating piece, called a rhizome, is a potential rooting plant that may drift to a new location and take root. With a reproductive strategy like this, it is easy to see why a plant or animal might be considered invasive.

 

Virgil Dupuis pulls a stalk with its identifying triangular-sectioned stem, and then tosses it back into the water. “I hate this stuff,” he says with a laugh.

 

Dupuis is Extension Director at the Salish Kootenai College and a member of the Confederated Salish and Kootenai Tribes who has worked on the problem of flowering rush for much of his career.

 

He spent his childhood swimming in the lake. Since then, flowering rush has moved from where it was originally found near Lakeside, Montana, around the lake and down into the Flathead, Clark Fork, and Columbia Rivers. It is now creeping toward Glacier National Park.

 

Dupuis notes how the presence of the invasive plant has a cascading effect on other species. “This kind of a habitat favors invasive fish like bass and perch,” says Dupuis. These fish are not native to the lake, but they thrive by breeding in the flowering rush. They out-compete native fish like endangered bull trout and declining westslope cutthroat trout, Dupuis explains.

Virgil Dupuis, Extension Director at Salish Kootenai College, looks at an invasive flowering rush plant that forms dense mats in parts of Flathead Lake. Credit: Ken Rand

With Peter Rice at the University of Montana, Dupuis is now trying to develop a method to eradicate the plant in small plots by applying herbicide during lower water in the spring.

 

“Aquatics are a weird area, because whose responsibility is it? You know, the landowner doesn’t own it,” he says.

 

“This place here is, to be honest, kind of a lost cause,” says Dupuis, but he still holds out hope that the plant can be controlled by yearly chemical treatments, or at least that such treatments will keep the plant from thriving.

 

How, then, to keep flowering rush from taking over other parts of Montana? And, what about the other rapacious plants and mussels that have fouled other parts of the nation’s waterways?

 

Boat check stations dot the major entryways into Montana’s lakes and streams. There are gaps in coverage, though. eDNA might be the tool that can overcome these limitations.

 

Scientists have used eDNA successfully to detect rare species such as salamanders in Idaho and fish populations, like the Chinook Salmon in Washington,3,4 at a fraction of the cost of traditional field sampling methods that require capturing multiple individuals.

 

However, one drawback to this process is the inability to pinpoint the exact location of the species since the eDNA it sheds is constantly drifting in the water. There is also the potential for false positives with eDNA, as in the detection of Asian carp in the Great Lakes in 2010 that caused a brief panic.5

 

Luikart is frank about eDNA’s uphill climb as an environmental sleuth. “We developed our own test from DNA sequences on zebra and quagga [mussels] and just started applying it,” says Luikart, “so far, the test is sensitive enough to detect invasive species in controlled environments. Outside the laboratory there are many barriers that make accurate tests much more difficult.”

 

DNA can move in many ways and find its way into a place where it is not expected to be. People move plants and animals around all the time; in fact, we surround ourselves with plants and animals that could be considered invasive species. Most do not pose a great danger. But, some invasive species have a real potential to change the places we think of as pristine. One of those is the mussel.

By late summer, flowering rush has entirely overtaken the boat launch on the south shore of Flathead Lake near Polson, Montana. Credit: Ken Rand

“Zebra [and quagga] mussels will take food away from other organisms like young fish, and they will also excrete nutrients and will likely cause fisheries to collapse,” says Luikart. After introduction, the high concentration of nutrients at the surface of the water and at the shorelines can lead to algal blooms that feed on nutrients and cause die-offs of fish.

 

It is no surprise that these mussels come to be at the top of each ecosystem wherever they find a new home.

 

Each female mussel has the ability to produce up to a million offspring in a lifetime of several years. The tiny larvae are no larger than a fingernail. No place is safe as mussels slowly creep their way to new waterways in the bilge of a boat and clinging to hulls.

 

Every year, boats from the Southwest and Great Lakes, with mussels attached to them, come through Montana boat check stations. Currently, the only way they will be stopped is if someone actually sees them or if a trained dog sniffs them out.6 State employees and other agencies collect water samples and send them to the Montana Fish, Wildlife and Parks Aquatic Invasive Species Lab in Helena. At the lab, workers visually examine the samples for evidence of invasive mussels, plants, and snails under a microscope. DNA tests are used only to verify visual findings.

 

“We certainly see a possibility for implementing eDNA, but for now the money’s better spent elsewhere,” says Stacy Schmidt, the manager of the Aquatic Invasive Species Lab, “We do support the research being done.”

 

So far, the lab has not found any mussels in a Montana lake or river, except the many that are stopped in trailered boats on their way in to the state.

 

Luikart argues that it is too easy to miss an invasive species using just visual inspections.

 

A detection process like eDNA can be seen as too sensitive, but that may also be one of its strengths. You need a large piece of an adult or a larva to see under a microscope. With eDNA, a single cell or even molecule could be picked up in extracted DNA.

 

With his collaborators, Luikart would like to start regular eDNA monitoring on Flathead Lake. The Flathead Lake Biological Station estimates that monitoring would cost them USD$40,000 each year, with testing done multiple times every summer in strategic points around high-risk areas such as boat launches and marinas.

Beaver Lake, an isolated lake northwest of Whitefish, Montana. In 2007, invasive Eurasian watermilfoil was discovered in the lake. Since then, it has been treated yearly to eliminate infested areas, and the unwanted plant is thought to be nearly eradicated. Credit: Ken Rand

“Flathead Lake is by far the most likely location of invasion,” says Luikart, who wants to see the prevention and monitoring work done by the state and other agencies to be enhanced by these new quick detection tools.

 

Invasive aquatic plants do not get as much attention as some invasive species, but their potential for damage to rivers and lakes is great, although subtle when compared to mussels, pathogens, or fish, like the lake trout.

 

Another aquatic plant that found its way to Montana is Eurasian watermilfoil, which can turn a lake or river into a dense, light-blocked waterway.

 

On an early morning, I make my way up a twisting, pine-forested back road above Whitefish Lake to the only known site of Eurasian watermilfoil in the Flathead River Basin-Beaver Lake. On this day there is only one other boat on the lake, as a pair of loons call and an osprey circles over head.

 

By chance in 2007, a state employee training at the lake recognized patches of dense Eurasian milfoil not far from the boat launch.

 

Paddling on the lake, I encounter a native Northern milfoil in a small underwater patch at the edge of a marsh. The small lake teems in spots with this native version of the invasive Eurasian species. The wispier Eurasian watermilfoil is scarce on the lake, at least to my novice eyes peering into the water, but still here. To differentiate the two kinds of milfoil, one must count each strand on a branch, or perform a genetic test in the lab. The invasive version has feathery leaves with a reddish stalk, but it is nearly impossible to tell apart from the native, apart from its rapacious growth. To make matters worse, the native milfoil hybridizes with the invasive, making it harder to discern between the two.

 

In my hand the delicate strands of a milfoil break apart, each with the potential to become another plant. I watch the strands drift away and start to realize just how easy it is to move a little strand of life to another place.

The author inspecting Eurasian watermilfoil near Thompson Falls, Montana in the summer of 2015. Credit: Ken Rand

The non-native milfoil can potentially choke an entire waterway if left unchecked, and can stretch up to 30 feet from the floor of a lake to the surface.

 

Further down the Flathead River and on to the Clark Fork and Columbia Rivers, Eurasian Watermilfoil has taken over large swaths of the shoreline and shallow backwaters. At Noxon Reservoir where the river runs wide behind a dam, the waterway is unnavigable by boat, with dense chains of milfoil creating light-blocking mats that change the ecosystem. Flowering rush is there too, as it moved down the river from Flathead Lake.

 

Luikart quotes his colleague, Adam Sepulveda at the United States Geological Survey in Bozeman, who has been working to set up a similar eDNA invasive species detection system around Yellowstone National Park: “If you detect a cancer early,” in this case an invasive species, “like an invasion or tumor, you can excise it early from the body or ecosystem.”7

 

eDNA can help to do just that.

 

Acknowledgments:

This article was produced as part of the Crown Reporting Fellowship at the University of Montana, School of Journalism, with the guidance of mentor Christopher Joyce at NPR to generate environmental stories about the Crown of the Continent.

 

References

  1. Scott, T. Bipartisan Collaboration Preserves the North Fork. Flathead Beacon [online] (2015) http://flatheadbeacon.com/2015/08/26/bipartisan-collaboration-preserves-....
  2. Ellis, B et al. Long-term effects of a trophic cascade in a large lake ecosystem. PNAS 108 (3) 1070-1075 (2011).
  3. Goldberg, C., D. Pilliod, R. Arkle, and L. Waits. Molecular Detection of Vertebrates in Stream Water: A Demonstration Using Rocky Mountain Tailed Frogs and Idaho Giant Salamanders. PLoS ONE 6(7) (2011).
  4. Laramie, M.,D. Pilliod, and C. Goldberg. Characterizing the distribution of an endangered salmonid using environmental DNA analysis. Biological Conservation 183, 29–37 (2015).
  5. Wines, M. Bracing for Carp in Great Lakes, but Debating Their Presence. New York Times [online] (January 2014) http://www.nytimes.com/2014/01/05/us/bracing-for-carp-in-great-lakes-but....
  6. Robbins, J. A Western Showdown with Mussels. New York Times [online] (September 2015) http://www.nytimes.com/2015/09/08/science/a-western-showdown-with-mussel....
  7. Sepulveda, A. et al. Aquatic Invasive Species: Lessons from Cancer Research. American Scientist 100, 234 (2012).