This article was originally featured on Undark.
On a sunny fall morning, biologist Andy Hubbard set up a temporary lab next to small pools at a national park’s outcropping of ancient granite rocks. Fully engrossed in the quietness of a cactus forest, he and his team filled small bags with cloudy water and meticulously filtered it through tiny filters.
Those filters would later be sent to a laboratory to test for genetic material shed by animals in the water. By collecting environmental DNA, or eDNA, Hubbard and three other members of a National Park Service team hoped to detect signs of native animals and the invasive bullfrogs that have proven devastating for their survival.
“This technology will end up being critical because it’s a more efficient way to detect invasive species, as well as rare species,” said Hubbard, program manager for the National Park Service Sonoran Desert Network in Tucson, Arizona.
Around the world, scientists like Hubbard are increasingly turning to eDNA to detect species from discarded bits of skin, scales, and mucus in water, soil, and air. In the field of conservation research, the emerging technology is opening new frontiers to monitor endangered species, track invasive ones, and sample general biodiversity. It’s also cheaper. And while the field still faces limitations around accuracy and precision, scientists say eDNA is quickly becoming a game-changer for wildlife conservation efforts.
“Environmental DNA has become more and more important as we are putting increased emphasis on understanding biodiversity and importantly, biodiversity loss,” said Adam Sepulveda, a research scientist with the U.S. Geological Survey’s Northern Rocky Mountain Science Center in Bozeman, Montana.
In such a rapidly growing field, technological innovation has been key to advancing eDNA methodologies, especially as scientists attempt to move past constraints in the field: While some researchers continue to use traditional tools, like visual surveys, traps, and nets, others are counting on portable robots to collect more frequent samples, or advanced lab analysis methods to take stock of biodiversity. Meanwhile, Sepulveda and other scientists have said there should be a national strategy for eDNA application, which, they say, would avoid inconsistent guidelines across agencies, and make the eDNA analysis more efficient.
The scientific community now recognizes that eDNA is a useful perspective through which much can be learned about species that are difficult to identify using only traditional tools, said Sepulveda: “eDNA gives us some power to understand what could be happening with these harder-to-find species.”
The exploration of environmental DNA started in the 1980s with researchers studying microbes or microorganisms, such as bacteria, fungi, and algae. But it wasn’t until the early 2000s that the use of eDNA started gaining momentum as methods and technologies evolved, and more scientists began adopting the approach. Research into aquatic ecosystems exploded after the 2008 publication of a study Researchers in France used eDNA testing on samples collected from wetlands to find the presence of invasive American bullfrogs.
Detecting and managing invasive species early on has been an area where eDNA has shown great potential. Instead of conducting time-consuming and intrusive surveys, researchers can now use a small sample of eDNA to identify species before they cause serious harm to ecosystems, as well as organisms too small to see with the naked eye.
This technology is very useful for tracking invasive species because it is highly sensitive. It can detect DNA evidence from just a few individuals at the beginning of an invasion, according to Sepulveda.
Time is crucial because once invasive species spread and establish themselves in a new environment, they pose a major threat to native animals and their habitats. They can also cause significant damage to infrastructure. For instance, the zebra mussel, a shellfish discovered in the Great Lakes in the 1980s, can clog water intake for power and water plants and damage boats and equipment. It is believed that zebra mussels first arrived with ballast water discharged from European ships, and they now live in the Mississippi River Basin, Great Lakes, and many other bodies of water throughout the nation.
“This technology will end up being critical because it’s a more efficient way to detect invasive species, as well as rare species.”
The annual cost of managing invasive species in the United States has risen from $2 billion per year in the 1960s to $21 billion per year since 2010, according to a publication in 2021. Globally, the economic cost related to invasive species over nearly 50 years is estimated at around $1.3 trillion. study In southern Arizona, the American bullfrog, which is native to the eastern U.S. and Canada, is causing major issues. These voracious creatures eat everything in their path, destroying native species like the federally threatened Chiricahua leopard frog.
The amphibians not only compete with smaller native frog species for food and space but can also spread disease-causing pathogens like chytrid fungus and ranaviruses, contributing to the decline of native populations. According to Hubbard, the American bullfrog is probably the most important and impactful non-native invasive animal in the Southwest.
In the hopes of tracking their spread, Hubbard and his springs-monitoring crew have collected eDNA in several national parks within Arizona and neighboring New Mexico.
During the eDNA collection last fall, Hubbard’s team spent a morning at Tucson’s Saguaro National Park, filtering 1,000 milliliters—roughly four cups—of water from the pool. They used filters the size of a dollar coin to trap loose cells and DNA in water samples, and then carefully folded them with tweezers into tiny triangles and placed them in sterilized test tubes. Hubbard expects to have lab results from the DNA analysis by the early part of the year.
Like other researchers, Hubbard has faced some of the limitations of eDNA. For example, DNA can degrade quickly in water, especially in warm weather. “As the water temperature increases, the remaining DNA starts to break down,” he said.
And eDNA cannot reveal the number of species, such as native frog species that are decreasing. In Saguaro National Park, a spring called 'The Grotto' has lowland leopard frogs based on my results, but I cannot tell you how many, and if that number has changed over time. The technology can only indicate if certain eDNA is present or not.
He has also discovered that eDNA may not work well for detecting certain species, like those that release small amounts of genetic material. This includes the northern Mexican garter snake, which visual surveys have seen in marshy areas, but eDNA has not found.
We are attempting to improve our sampling for multiple species, Hubbard said. There's always a trade-off when you do that, and there will be different approaches that will be more or less successful in detecting species, depending on their habitat or behaviors. So, this may not be the best way to detect northern Mexican garter snakes.
Hubbard mentioned the chance of false negatives, when eDNA might miss species that could be present, or false positives, when the technology detects species that are not there, which could also be challenging. Nonetheless, in the case of the garter snake, he said potential errors may be overcome with intensified water sampling combined with traditional tracking methods, such as placing wooden boards for snakes to hide under in wetland habitats. This allows researchers to attract snakes into areas they can easily access during surveys.
“Environmental DNA has become more and more important as we are putting increased emphasis on understanding biodiversity and importantly, biodiversity loss.”
Hubbard considers the most valuable use of the eDNA parks project for monitoring frog populations to plan conservation actions for native species, which includes removing invasive bullfrogs that have been detected. That's our highest priority and biggest concern at the moment, he said.
Another limitation of the technology is related to sampling: While Hubbard’s team spent a few hours filtering water, they couldn’t stay all day—or overnight. Sepulveda, the USGS research scientist, says that robots roughly the size of carry-on luggage could help.
At the Northern Rocky Mountain Science Center, he and his colleagues are working on how best to incorporate eDNA and robot technology to address shortcomings in freshwater bodies battling the infestation of zebra mussels, quagga mussels—originally from Eastern Europe—and other aquatic invasive species. By using robots that can collect multiple eDNA samples over longer periods, fewer researchers will have to do the task.
Sepulveda’s work mainly focuses on invasive species and supporting natural resource managers throughout the country in dealing with them, including those in the Columbia River Basin. That is—or was until a few months ago—the last major water basin in the lower 48 to not yet be invaded by zebra or quagga mussels,” he said.
In September, quagga mussels were found in Idaho’s Snake River, the largest tributary of the Columbia River. The Columbia flows through seven states and a Canadian province. Natural resources agencies continue to monitor the area after moving quickly to contain the invasive species with a chemical treatment that also killed thousands of fish. Trying to control mussel infestation is an uphill battle, Sepulveda said. Female zebra and quagga mussels can produce up to 1 million eggs a year. Besides destroying native aquatic life, mussels attach themselves to hard surfaces like boat hulls that can carry them over long distances.
The scientists have tried out robots that can gather eDNA water samples to lower the cost of monitoring for invasive species and address some of the issues with the technology, such as the incorrect negative results of the northern Mexican garter snake found in the wild.
The robots, also known as autonomous samplers, are created to filter and preserve eDNA not just from invasive species, but also any aquatic and semi-aquatic organisms that leave genetic traces behind. Sepulveda leads a project called the “Rapid environmental DNA Assessment and Deployment Initiative and Network” in which researchers are developing a slimmer version of a robot that was built some years ago for ocean exploration at the Monterey Bay Aquarium Research Institute in Moss Landing, California.
When prepared for use in freshwater, the robots will be positioned on shores, on fixed floating docks, or on boats. “They’re not meant to be submerged,” Sepulveda stated. “They have tubes that go into the water to pull the water in, but the actual robotics are meant to be on land.” READI-NetUnlike humans, the freshwater robots will be capable of collecting samples much more frequently, day or night, increasing the likelihood of species detection. Collecting eDNA can be difficult, Sepulveda explained, because the currents that form in lakes, rivers, and oceans can carry it away from the organisms that discard it. Additionally, given its continued decay, the more time that passes, the harder it is to capture it.
“If you get your scoop of water, there is a chance, especially when something is very rare, that you were there 10 minutes too early or potentially 10 minutes too late—or a day too early, or a day too late,” he said. “And so, one of the ways that we can increase our ability to detect a new invader, just like our ability to detect the Covid virus, is by collecting more samples.”
The robots will be capable of gathering up to 144 samples before a scientist or technician would need to collect them for analysis, he said. “It can be left to collect samples for weeks to months.”
Jim Birch, director of the SURF Center—Sensors: Underwater Research of the Future—at the Monterey Bay Aquarium Research Institute, described the robots as a simplified version of the environmental sample processor, an older underwater robot originally built to study harmful algal blooms, also known as red tides, that can kill animals.
As eDNA technology became more popular, Birch said, two postdoctoral researchers at the institute tested if the robot could successfully gather eDNA samples from the aquarium. The experiment worked. Filtered water samples not only picked DNA traces of fish, but also identified genetic bits of the turkey and chicken feed that fish had consumed.
“If you get your scoop of water, there is a chance, especially when something is very rare, that you were there 10 minutes too early or potentially 10 minutes too late—or a day too early, or a day too late.”
After some field testing, the robot is now being reimagined for optimal use in freshwater, Birch mentioned: “It will have a big impact on invasive species, but its use is much broader.”
Sepulveda mentioned that field deployments of a prototype are just commencing this year. “We’re hoping by 2025 to have 10 that are ready to go and that we can get out to our partners,” he mentioned.
The method of collecting eDNA with robots will be particularly helpful in programs with limited manpower, he said. “If we can find these invasive species early on and identify what and where they are, then we are likely to reduce the economic and ecological costs of the invasion.”
The process of gathering eDNA is just the initial stage in attempting to identify species from the remnants they leave behind as they move through different environments. The dead skin, saliva, waste, and other cellular material that organisms shed must then be examined in a laboratory using molecular techniques.
At Washington State University’s School of the Environment, associate professor Caren Goldberg isolates the DNA caught in the filters sent from southern Arizona by Hubbard. “We handle one species at a time, and sometimes we need to do extra cleaning on the samples, and then we analyze all the data, and double-check it to ensure everything looks good before sending it back,” she said.
Environmental technology is a valuable tool for finding elusive species like frogs, Goldberg said. She knows how hard it can be to find the creatures because, as a University of Arizona student, she completed her master’s thesis after chasing barking frogs in the mountains and surveying Chiricahua leopard frogs that can be difficult to see in murky, and often deep, water holes.
“The reason why I could see how exciting the whole field would be is because I spent so much time out in Arizona looking at these ponds and not being able to find the frogs,” she said.
Goldberg now collaborates with various federal and state agencies to use eDNA techniques in their conservation programs. For the southern Arizona project, she searches for genetic signs of species such as local leopard frogs and the large bullfrogs that consume the small, native amphibians, contributing to their decline. The federal government considers the Chiricahua leopard frog, which now emits its snore-like mating call in a shrinking habitat, a threatened species.
“If we can detect these invasive species and figure out what they are and where they are very early in the invasion process, then we’re likely to limit the economic and ecological costs of that invasion.”
In previous samples from Hubbard, Goldberg has detected the invasive frog. She has also checked for the presence of endangered jaguars that trail cameras have captured in the borderlands. The sample was from ponds where Hubbard had hoped a stealthy, thirsty feline might have left its saliva, but no DNA traces were found.
Since Goldberg aims to identify single species at a time, she uses a laboratory technique called quantitative polymerase chain reaction, or qPCR. It’s an analytical technique similar to the one used to test for Covid-19 infection. And while the methodology is widely used in single-species detection, it’s not suitable to detect multiple species from a mixed sample. Instead, scientists are increasingly using eDNA metabarcoding, which is a relatively new approach that can detect a large number of DNA sequences.
The emergence of eDNA metabarcoding and next generation sequencing has been a breakthrough, said Jan Gogarten, an evolutionary community ecologist at the Helmholtz Institute for One Health in Germany. “We can sequence the diversity in a single sample, and that unlocks the opportunity to look at different sources of eDNA that animals shed in the environment,” he said. “You don’t have to now have a pure sample, like a biopsy of the animal.”
At Kibale National Park in Uganda, Gogarten and his colleagues worked on an eDNA project and were surprised by the results. They collected samples from low-hanging plant leaves in the park’s tropical forest and later found various species in the lab analysis, including African elephants and grey crowned cranes.
Gogarten, also associated with the Department of Applied Zoology and Nature Conservation at the University of Greifswald, expressed astonishment at the diversity they discovered with just one swab.
One unexpected finding was the detection of a fish, specifically a catfish genus, on the plant leaves in the park. This was surprising because catfish usually live in rivers, so their presence on the leaves was unexpected.
The presence of the catfish on the leaves perplexed Gogarten and his colleagues. They speculate that a bird species may have consumed the catfish and then defecated on the leaves while perched on a tree.
Contamination in the lab remains a challenge for eDNA researchers due to the sensitive methods used in analysis. Gogarten noted that other molecules and traces of animal DNA from previous experiments can cause issues.
To minimize the risk of contamination, researchers can establish lab controls and separate workspaces for eDNA analysis. Gogarten emphasized the need for meticulous care in eDNA work to maintain confidence in the results.
While eDNA lab methods are promising, they have limitations. Identifying specimens relies on comparing barcodes with those in DNA reference libraries, so if a species is not in the database, eDNA cannot identify it.
EDNA technology is beneficial for assessing biodiversity in specific areas, providing a detailed mapping of the variety of species. This could allow for high-resolution biodiversity mapping along park edges like Kibale.
Gogarten mentioned that eDNA could assist in determining biodiversity distribution and developing effective conservation strategies on a large scale. He expressed excitement about the increasing accessibility of the technology.
Current eDNA research has focused on identifying species in various water bodies, and the ease and low cost of collecting eDNA samples means the technology is likely to remain in use. Hubbard and his team have been collecting eDNA since spring 2022 and plan to continue in the coming year.
He anticipates that the technology will continue to get better as it provides information that can assist natural resource managers in making decisions. Several companies are developing portable lab units to perform real-time detections in the field. These devices may take several years to be ready to use, and some level of lab work may always be necessary, he says, but it’s one of the exciting advancements for this technology.
Currently, he and his team will use eDNA results to create a plan for the best way forward. “Our ultimate goal is to have these be healthy wetlands that are supporting native species again,” he said. “So we’re trying to take a brief pause and understand the bigger picture and use the data that we have to guide that.”
Sepulveda anticipates that the national strategy, which he is currently working on with the Biden administration, could enhance eDNA application and data-management tools in natural resource management, among other potential results. That strategy is set to be made public in June 2024.
“It really blew our minds at how much diversity we were detecting just by one swab.”
“This eDNA strategy is aiming to establish a framework, or at least a plan, for how to be more coordinated and more effective and how to advance eDNA science, how to keep improving it for the next 10 to 20 years,” he said.
Meanwhile, the technology is increasingly being used in global conservation efforts. In South Africa, scientists rediscovered a species that had not been seen since 1937 by tracking its eDNA in sand. In Brazil, eDNA helped scientists rediscover a species they thought had become extinct since it had not been seen since 1968. And in the Mediterranean Sea, scientists gathered genetic material shed by the endangered and elusive species.
However, the vast oceans are still largely unexplored and eDNA is a crucial tool to uncover the unknown, said Kelly Goodwin, a marine molecular microbiologist with the National Oceanic and Atmospheric Administration. The technology is enhancing the agency’s numerous tools that enable scientists to explore ocean life and protect it, she said.
“There are as many microbes in the ocean as there are stars in the sky,” she said. “There’s a great deal that we do not know about the biology on our planet, including the invisible majority, which is the microbial Earth.” golden mole Goodwin said eDNA technology advances will help address questions not only about unseen organisms in the deep ocean, but also inform the variety of species—rare, endangered, or invasive—throughout the ecosystem. frog “There is a lot of life on this planet that we know very little about,” she said. “And the only lens we have to view it is through their DNA.” angel shark.
To survey the planet’s vast biodiversity, some scientists are relying on environmental DNA, robots, and more.
“There are as many microbes in the ocean as there are stars in the sky,” she said. “There’s a great deal that we do not know about the biology on our planet, including the invisible majority, which is the microbial Earth.”
Goodwin said eDNA technology advances will help answer questions not only about invisible organisms in the deep ocean, but also inform the composition of species—rare, endangered, or invasive—throughout the ecosystem.
“There is a lot of life on this planet that we know very little about,” she said. “And the only lens we have to view it is through their DNA.”
