Finding the Invisible: A new way to look for invasive species using eDNA

By 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 pikeminnow swim carefree through the gaps of 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, one of the largest freshwater lakes in the west. It lies just south of Glacier National Park and remains a particularly clean water source, despite human pressures of runoff from agriculture and cities.

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

Next to the lakeshore, a sign warns of invasive aquatic plant species found in the water.

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 on 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 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 in the lab 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, urine, or pollen—it enters the “environment”.

To find out how scientists are chasing down DNA, 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 I have seen.

Standing at the shore, Gordon Luikart watches the mountains to track if rain or lightning will interrupt his experiment. He is as 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’s 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, soft-spoken researcher, who works with eDNA in his lab, rinses the remaining debris with ethanol into small vials.

A student holds up a vial filled with lake water.

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’ll look for DNA from invasive species that aren’t supposed to be in the lake as well as others 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’ll never find. Zebra and quagga mussels. They will also look for the mussels with eDNA in the environment.

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

To that end, Luikart 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 and beam the results back to shore. He also just received funding to start design on another sampler that will sit in place at heavily used sites to take samples over time to monitor for invasive species.

AN AGE OF INVADERS

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 for sport fishing in 1905.

But now, new invasive species are coming on their own. No one introduced them on purpose. They have a will of their own.

“Flathead lake is by far the most likely location of invasion,” says Luikart.

There are many aquatic invasive species already in Montana, including Eurasian watermilfoil, curlyleaf pondweed, flowering rush and New Zealand mudsnails. In addition, there are two mussels that aren’t here yet but must be kept out forever:  Zebra and quagga mussels.

To look at an invasion that has already happened, I drive south from the 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 rushes fill in the southern shore of Flathead Lake.

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 will drift to a new location and take root. With a reproductive strategy like this, it’s easy to see why a plant or animal could be considered invasive.

It’s a hot sunny day and Virgil Dupuis wears a Hawaiian-printed shirt as he stands by the water.

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

Dupuis is Extension Director at the Salish Kootenai College and a member of the Confederated Salish and Kootenai Tribes.  He has studied 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, around the lake and down into the Flathead, Clark Fork and Columbia Rivers.  It’s now creeping toward Glacier Park.

He 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 aren’t 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 says.

Vigil Dupuis examines the stalk of a flowering rush.

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.

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 Coho Salmon in Washington, at a fraction of the cost of traditional field sampling methods that require capturing multiple individuals.

 However, one drawback is that you can’t pinpoint the exact location of the species since the eDNA it shed is constantly drifting in the water.

 There is a potential for false positives with eDNA, as in detection of Asian carp in the Great Lakes in 2010 that caused a panic, briefly, when they weren’t known to be there.

 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 isn’t expected to be.

THE NEW FOOD CHAIN

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 don’t 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. 

A zebra mussel is compared to a quagga mussel.

 “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 water and at the shorelines can lead to algal blooms that feed on nutrients and cause die-offs of fish.

It’s no surprise how these mussels come to be at the top of each ecosystem where they’ve found 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 by clinging to a hull.

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’ll be stopped is if someone actually sees them.  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 hasn’t found a mussel in a Montana lake or river, except the many that are stopped in trailered boats on their way to the state.

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

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.

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

INVADERS IN DISGUISE

On an early morning, I make my way up a twisting a 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’s only one other boat on the lake as a pair of loons call and an osprey circles over overhead.

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

A close-up view of Eurasian Watermilfoil

On a paddle of the lake, I encounter a 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, you have to count each strand on a branch or perform a genetic test in the lab. The invasive version has a feathery with a reddish stalk, but it’s nearly impossible to tell apart from the native, apart from its rapacious growth.

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

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.

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.”

eDNA can help to do just that.

The Daily Inter Lake published Ken Rand's story on January 23rd, 2016. The Solutions Journal also ran this story in their March 2016 issue. You can check out an extended version of "New Invaders, New Solutions," with additional graphics, on Ken Rand's Atavist page.