Originally published by The Scientist. Original article available here.

For a little more than a decade, scientists have been filtering water samples from aquariums, rivers, lakes, and even the ocean to obtain DNA that was shed by fish and other aquatic life. The goal: to use this environmental DNA (eDNA) to monitor aquatic species. Now, a trio of papers—two on animals, and one on plants—suggest it’s also possible to detect and identify terrestrial organisms using eDNA floating in the air.

Although the research (along with the entire field of eDNA) is in early stages, experts tell The Scientist that the technology could make it more logistically and financially feasible to find and monitor rare, endangered, invasive, or shy species. Such studies will likely complement rather than replace existing monitoring methods such as camera traps, say scientists working with eDNA, but the ability to fill in the blind spots left by current methods could be immensely beneficial to ecologists.

Genetic analyses including eDNA are “a way of democratizing and enhancing our ability to know what’s going on in the natural world, and also what we’re doing to it,” Mark Stoeckle, an environmental genomicist at the Rockefeller University who uses eDNA to monitor fish populations and was not involved in any of the new studies, tells The Scientist.

Two of the three studies, both published today (January 6) in Current Biology, demonstrated the successful collection and analysis of airborne eDNA shed by animals. Those experiments, one conducted at and around the Hamerton Zoo in the UK and the other at the Copenhagen Zoo in Denmark, relied on the assumption that animals in pens, enclosures, and indoor exhibits would give off strong, consistent signals. The authors of both papers were able to detect and identify the DNA of dozens of different animal species.

By sheer coincidence, the two experiments were conducted in parallel without either team knowing about the other until one team led by York University molecular ecologist Elizabeth Clare, then at Queen Mary University of London, posted its work as a preprint on bioRxiv just days before the other group, led by evolutionary genomicist Kristine Bohmann from the University of Copenhagen’s GLOBE Institute planned to submit its own. After Bohmann’s and first author Christina Lynggaard’s panic over being “scooped” subsided, they tell The Scientist, the two teams got in touch—it’s a small community and they already knew each other—and decided to submit their papers to journals as a package deal. Having “two independent confirmations of the same thing,” Clare tells The Scientist, makes her “feel way more confident that what we’ve done is really replicable.”

York University molecular ecologist Elizabeth Clare collecting airborne eDNA at the Hamerton ZooELIZABETH CLARE

The studies differ in important ways, but their similarities are more prominent. Both captured eDNA using vacuums to pass air through a filter at various sites at their respective zoos. Both used PCR amplification with primers for known species in the area to identify and verify zoo animals, a process called eDNA metabarcoding. And in both cases, their results blew their authors’ expectations out of the water, especially for proof-of-concept research. Based on their findings, the researchers conclude that animal DNA can travel much farther through the air than they expected—both teams were able to detect zoo animals as well as those living outside the zoo, even from hundreds of meters away.

“We were seriously worried it wouldn’t work,” Clare tells The Scientist. Lynggaard echoes that sentiment. “When I was planning this, I thought of the worst-case scenario. . . Most likely we’re not going to get anything,” she says of her initial expectations. But the results were unexpectedly robust, with each sample yielding detectable DNA from between 6 and 21 species.

Altogether, Clare’s team was able to identify DNA from 25 different mammal and bird species that live in or near the zoo, as well as DNA from the food being fed to those animals. Sometimes a sensor located outside of a building would pick up identifiable quantities of DNA from a species housed inside, or from an enclosure located all the way across the zoo. Meanwhile, Bohmann’s team detected 49 vertebrate species: 30 mammals, 13 birds, a handful of fishes, one amphibian, and one reptile—a taxonomic range that left Bohmann and her colleagues “sitting in awe,” she tells The Scientist.

There’s something in the air

The studies follow up on earlier work in which Clare’s team detected airborne eDNA from a colony of naked mole rats maintained in a laboratory setting—an environment with far fewer variables than the zoo.

“The perfect thing about zoos is you have all these nonnative species you cannot mix up with anything else,” Clare tells The Scientist. “And you also know precisely where they are. That became really important for both of us because we were picking up the animals we were near [the sensors], but a lot of other animals as well.”

The zoo research is still considered proof-of-concept for terrestrial eDNA monitoring, though taking the eDNA sensors outdoors represents a notable step forward for the field. In this case, the two teams took a variety of approaches to collection, which the study authors say should be informative as airborne eDNA monitoring makes its way into applied ecological research. Bohmann’s team developed three different types of sensors that sucked in air through both conventional and water vacuums and positioned them in and around the zoo, where they filtered air for hours at a time. By contrast, Clare’s team only used one kind of sensor and ran collections for, at most, half-hour bursts. Having both approaches published side by side, experts tell The Scientist, will serve as a valuable reference when determining which approaches are better for various settings.

“We had forensically-tiny amounts of DNA,” Clare tells The Scientist. “They had tons of DNA,” she adds, but because the other team ran fewer collections for longer periods of time, “they didn’t have as much detail on where it [came from].”

For now, the process is far from perfect. Some animals living in the zoos, such as a tiger that Clare’s team attempted to detect, were missing from the eDNA samples altogether. That might be due to experimental error or the animal shedding less DNA than other creatures, or a combination of myriad other factors. For now, any attempt to explain why some animals were more readily detected than others, Clare says, would be pure speculation.

Two of the air collection devices used by the research team in CopenhagenCHRISTIAN BENDIX

The tiger question also confused Stoeckle, who didn’t work on either of the new papers. He tells The Scientist he would have liked to see more discussion of possible reasons that some animals went undetected, but is overall very complimentary of both zoo studies.

“When you’re starting out, the positive results are the most important ones,” he says. “The negative ones are less important, and the positive results in these papers are great.”

Passively detecting plants

Meanwhile, research on airborne plant eDNA is a few steps ahead, giving animal researchers hints as to what they might attempt next. Last month, Texas Tech University doctoral candidate Mark Johnson, his advisor, ecologist Matthew Barnes’, and their colleagues reported in BMC Ecology and Evolution the results of a study in which they sequenced eDNA from dust traps, which passively collect pollen as well as any other airborne molecules, in a field owned by the university. The team found several species of grass, fungi, and even an invasive species called tree of heaven (Ailanthus altissima) that had all been overlooked by more conventional surveys.

“Airborne eDNA continues to surprise us with how much material is in the environment,” Johnson tells The Scientist.

Johnson and Barnes have conducted similar experiments before, but this paper looked at a year’s worth of collection data, offering new insight into how seasonal changes, weather, and other factors impact the species detected by eDNA, offering new insight into the ecosystem’s dynamics. 

Other researchers are also trying to do the same with insect eDNA. Preprint research presented at last month’s Ecology Across Borders conference reportedly identified 85 insect species—and some vertebrates—from airborne eDNA.

The scientists behind the trio of recent papers all agree that there’s lots of work to be done in order to make eDNA an established and useful tool for ecological research. “We’ve shown that it works, now we need to try to understand some of the nuances of it,” Johnson tells The Scientist. “How does wind, how does weather, how does height affect our collection?”

Leaving the lab

As the field forges ahead, airborne eDNA scientists do have one major source of guidance: the field of aquatic eDNA research, in which researchers have several years’ worth of a head start. Scientists working with aquatic eDNA have already thoroughly demonstrated that the technology works and are now making strides toward using it as a standard ecological tool. Airborne eDNA research is a few years behind, but it’s “following a similar trajectory,” Johnsonexplains.

For both animal and plant studies, the next stage of research involves taking collections out of artificial environments and into natural settings. In some cases, this work is already underway: Johnson is now working on follow-up research in natural environments that takes a closer look at specific variables such as distance, weather, and altitude, and a paper in which he uses his passive dust traps to collect animal eDNA is making its way through the peer review process.

Bohmann, Lynggaard, and Clare note that many basic questions remain unanswered. For example, they won’t be able to glean any sort of temporal resolution—how long ago an animal can pass through the area and still get detected—until they bring their work out of a zoo and into a forest or jungle, where animals roam free rather than being confined to one area. Unfortunately, that kind of research brings new challenges.

“We can’t plug a water vacuum in in the rainforest in Madagascar,” Bohmann tells The Scientist. “And also we can’t make too much noise,” which would disturb the wildlife. That’s why her team tested a few different types of sensors, and why Johnson’s passive collection research will likely prove valuable. “We wanted something that would be transferrable to a natural environment,” Bohmann adds.

University of Guelph biologist Robert Hanner, who didn’t work on any of the studies but helped shape the field of eDNA research, says that the aquatic eDNA research community still has plenty of challenges of its own; although it has progressed further than the airborne eDNA field, scientists don’t yet have all the answers they need to make eDNA surveys practical. For example, ecologists are often interested in measuring the abundance of a given species in an area, and so far, aquatic eDNA surveys only detect their presence.

Airborne eDNA continues to surprise us with how much material is in the environment.—Mark Johnson, Texas Tech University

“There are so many caveats,” Hanner tells The Scientist, adding that the two zoo studies serve as valuable proof-of-concept papers, but that he’s skeptical of their practical utility. Their success “warrants a bit of cautious optimism rather than irrational exuberance,” he says.

Much like Clare’s issue with the zoo’s tiger, Hanner recalls a researcher working in his lab who failed to gather eDNA from a crustacean from water in its immediate vicinity. The challenge, he explains, is that the field doesn’t yet know why that would happen. The conventional explanation would be that the PCR amplification somehow went wrong. But it’s also possible, Hanner says, that certain organisms shed less eDNA than others, or that the primers used to identify them are faulty or can’t handle the degree to which eDNA tends to be fragmented. For all he knows, certain sediments in the water might bind to eDNA or the particles ferrying it, preventing collection of that DNA from happening in the first place.

And that’s just to name a few; Hanner notes that factors such as air or water flow, seasonal changes, time of day, temperature, and, as Johnson examined, altitude, may all play a role in determining how much eDNA is obtained or what species are detected. Yet these details often go unreported in the literature, which has primarily been saturated with proof-of-concept studies focused on showing that eDNA analysis works at all. That, Hanner says, is currently holding the field back.

Still, many researchers are hopeful that eDNA holds the key to understanding what happens in natural environments when scientists are not around to see or hear it.

“It’s surprising how much we don’t know about the natural world, even for familiar animals,” Stoeckle tells The Scientist. “These new technologies are going to help us understand that better, and hopefully be better stewards of the environment. That’s ultimately the goal, and in that way, I’m optimistic.”