As part of an effort to develop environmental DNA into a forensic tool, diver Evan Kovacs photographs the wreck of a World War II plane off the coast of Saipan.

Can sophisticated research tools, such as plucking environmental DNA from water and the sea floor, speed the recovery of long-missing service members?

A version of this story appeared in Science, Vol 377, Issue 6610.Download PDF

For 3 weeks in June 1944, bombs rained down on the tiny, lush island of Saipan, then an important Japanese stronghold in the western Pacific Ocean. As part of its World War II campaign, the United States had mounted an assault by sea and air. Amid flaming palm trees and churning seas, a sturdy U.S. Hellcat fighter plane and its lone pilot went down in a harbor west of the island. His body was never found.

Nearly 80 years later, on a calm morning in early March, maritime archaeologist Calvin Mires and underwater imaging expert Evan Kovacs dove off a boat anchored in the harbor and swam 10 meters down through the warm turquoise water toward the wreck of the Hellcat.

Another team of archaeologists would later thoroughly excavate the half-buried wreck, seeking the pilot’s remains. Mires and Kovacs had a different job: They circled the wreck, collecting plugs of sediment and 6-liter bottles of seawater. They were exploring whether they could detect human DNA seeping out of the crash site and into the environment.

The work of Mires, of the Woods Hole Oceanographic Institution, and Kovacs, of Marine Imaging Technologies, is part of a new collaboration between the U.S. military and outside scientists to apply the tools of research science to help bring missing service members home. More than 81,500 U.S. service members are considered missing in action (MIA); their remains lie buried in untold locations worldwide or are interred as unknowns in military cemeteries (see map, below). About 41,000 of them were lost over oceans and require underwater recovery.

Carrier-based bombers took part in the 1944 invasion of Saipan. Many planes were lost and some of their crews remain unaccounted for.

Mires, Kovacs, and others are working to develop environmental DNA (eDNA) into a forensic tool to hasten these slow, daunting underwater missions. After surveying wrecks in the area for nearly a decade, researchers located the Hellcat in 2018 and are still exploring whether it holds remains. An eDNA tool, if it existed, might have sped the search in this and thousands of similar MIA cases.

“Rather than taking out a full team of divers, scouring the wreck, and excavating the site, we could potentially have a much faster way to tell” whether a service member’s remains are nearby, says Kirstin Meyer-Kaiser, a benthic ecologist at Woods Hole and member of the team.

“We are a force multiplier,” says Charles Konsitzke, leader of the University of Wisconsin (UW), Madison’s MIA Recovery and Identification Project, which is part of the eDNA project. He and his team are among dozens of scientists and specialists from outside the U.S. government who have teamed up with an arm of the Department of Defense (DOD) called the Defense POW/MIA Accounting Agency (DPAA) to find service members still missing from past wars. In other projects, scientists are surveying the sea floor with instruments normally used for oceanographic research and developing artificial intelligence systems to efficiently pick out the shape of a downed plane from a morass of sonar data. Although methods vary, all the efforts share a common goal, Konsitzke says: “to hasten this process and bring more people home.”

A few days before Christmas 1944, Patricia Krueger received a telegram from the U.S. Army. She hoped it would contain a belated birthday greeting from her husband, Army flight engineer 1st Lt. Charles Krueger, whom she had not heard from in 2 weeks. Instead, the message said he wasn’t coming home: His B-29 had been lost over Mukden, Manchuria, and he was later declared MIA. Their son, John Krueger of Middleton, Wisconsin, now 78, still tears up when he recounts this story.

Charles and Patricia Krueger in 1943 on his family’s farm in Monroe, Wisconsin.

Decades later, the military continues to work to bring back the remains of service members like Charles Krueger. The job of finding them falls to DPAA, created in 2015 after critics charged that the previous MIA search process was slow, burdened by bureaucracy, and behind on innovations in science and technology. Between 1973 and 2014, the remains of only 1849 missing service members were returned to their families, according to DPAA figures, although the pace has ticked up in recent years; in 2021, the agency accounted for the remains of 141 MIAs.

To accelerate the work, Congress gave DPAA the authority to develop public-private partnerships with scientists and groups outside government. “They tasked us with going out there and leveraging the great expertise and interest in the outside world,” says military historian Michael Dolski. He oversees about 200 partnerships at DPAA, which has an annual budget of nearly $130 million.

Teaming up with academic scientists introduces new ways of thinking, Dolski says. Academics are more plugged into scientific advances and often have more leeway than the government to be flexible and creative, he explains. “Working with partners allows us to tap into their technologies and capabilities in ways that we just can’t maintain.”

UW researchers, who work on Wisconsin-based cases independently in addition to partnering with DPAA, took up Charles Krueger’s case in 2020. His family welcomed the input: Before then, they had only his 1946 casualty file, which the UW team later found conflicted with other historical documents. “There’s a lot of uncertainty with DPAA, and often you get radio silence,” John Krueger says. (An agency representative says, “DPAA and our partners work at the speed of science and accuracy.”)

Each case involves historical research, finding remains, and then identifying them with DNA and skeletal and dental traits. “It’s like detective work,” Konsitzke says.

Recovering the fallen

More than 81,500 service members from past conflicts around the world are considered missing in action (MIA), most from World War II. To date, the remains of more than 3000 MIAs have been identified by the U.S. government.* Scientists are developing new methods to locate and retrieve others.

In the case of Charles Krueger, the UW team, which includes historians, archaeologists, geneticists, and others, searched military archives and pored over WWII-era photos and video. They discovered contemporaneous Japanese newsreel footage of Krueger’s downed B-29. And, wading through documents, they found evidence that remains matching Krueger’s were recovered from Manchuria and buried in an unknowns tomb in Honolulu’s National Memorial Cemetery of the Pacific. “We wouldn’t have gotten any of this without UW,” says John Krueger, who has been in touch with the team for every step.

The next move is to exhume and analyze the remains in hopes of making a genetic match to the Krueger family, who have submitted DNA samples. But the unknowns tomb may contain other remains, too, and before disinterment the military requires a means of identifying at least half of all possible people buried in the grave, as well as approval from a web of federal offices and DOD. UW made the request in late 2021, but the researchers—and the family—are still waiting.

The UW team has had previous successes, for example recovering two WWII service members whose aircraft crash sites weren’t initially found. Archaeologists walked the French countryside, interviewed eyewitnesses to the crash, and used ground-penetrating radar to find the long-overgrown crash locations. Then they excavated and found the remains.

We all feel the gravity of the importance of our science and the value of what we’re doing.

• Kirstin Meyer-Kaiser
• Woods Hole Oceanographic Institution

For the nearly 41,000 U.S. MIAs presumed lost at sea, the historical research is similar. But finding remains underwater requires a specialized suite of scientific tools. For example, in February 2020, scientists from Project Recover—a nonprofit research collaboration between the University of Delaware (UD) and the Scripps Institution of Oceanography—deployed a fleet of torpedo-shaped autonomous underwater vehicles (AUVs), each about as long as a bathtub, off the coast of Vietnam. They were searching for missing Air Force Maj. Paul Avolese, who had gone down in a B-52 bomber in the region in 1967, according to historical research. The military had searched for his remains since 1993.

The AUVs deployed side-scan sonar, which shines a “flashlight” of acoustic pulses across the seabed, and magnetometers, which pick up metallic signals. They detected a B-52 wreck and what looked like human remains on the sea floor, and the team’s divers and archaeologists brought them up. A DPAA lab later used DNA analysis to confirm they were Avolese’s remains.

Project Recover has found more than 50 other aircraft associated with at least 185 MIAs with these methods, according to the organization’s own tallies. “Our ability to locate these is pretty unique,” says Mark Moline, an oceanographer at UD Lewes and Project Recover.

A 2017 ceremony honored World War II Aviation Radioman Second Class Albert P. “Bud” Rybarczyk, whose crash site in the waters off Palau was located by Project Recover, a collaboration of academic scientists.

Still, challenges remain. Such underwater recoveries are logistically challenging and expensive, Dolski says. (A DPAA representative declined to provide an average cost for each mission “as each has unique variables.”) Now, Dolski and others want to use DNA—already vital to identifying remains once found—to more quickly home in on the right spot underwater.

Organisms constantly shed DNA into their surroundings, and a liter of water can yield telltale genetic material from everything from bacteria to blue whales, depending on where the sample was taken. Today, scientists use eDNA extensively to survey biodiversity in rivers, lakes, and oceans, and to detect early clues to invasive species.

“This method is really sensitive and powerful,” says Annette Govindarajan, a biological oceanographer at Woods Hole who uses eDNA to study how organisms migrate up and down the ocean’s water column. With eDNA, “we don’t even need the animals themselves. It’s like ocean forensics.”

In fact, research suggests eDNA could be a useful tool in criminal forensics on dry land. Fungal DNA in dust or plant, insect, and bacterial DNA in soil might reveal where dirt in a suspect’s car came from, for example. In 2017, researchers at the University of Tennessee found human mitochondrial DNA, which is much more plentiful than nuclear DNA, in the soil beneath four decomposing cadavers, suggesting the method could link a site to a death even if remains have been removed.

But so far, eDNA hasn’t been applied extensively to forensic cases, says Kelly Meiklejohn, a forensic scientist at North Carolina State University. “As far as I’m aware, there are no law enforcement labs that do eDNA for casework,” she says. And despite eDNA’s ubiquity in marine biology, Meyer-Kaiser and Konsitzke say they don’t know of any studies using it to detect remains in the ocean.

The team plans to change that. Mires was at an archaeology conference in January 2020 when he ran into his former student Kara Davis, then working at DPAA. They got to talking about the potential of eDNA, with Davis suggesting it could serve as a clue to underwater human remains.

For example, once searchers find the first glimmers of a wreck with sonar, magnetometer, or imaging data from an AUV, scientists could sample nearby water and sediments seeking human DNA. If they get a DNA signal, searchers would have some assurance that remains are present before sending down an expensive and disruptive excavation team. And because wrecks are often partially buried and scattered across the ocean floor, eDNA could help marine archaeologists know where to look.

With eDNA, searchers could impact the environment less. “We also save time and energy [spent] looking in the wrong place,” says Hannah Fleming, a maritime archaeologist with DPAA who is coordinating the project.

I’m the thing I’m looking for, in a way, and so I don’t want to get any of myself in the sample.

• Bridget Ladell
• University of Wisconsin, Madison

“It’s a really interesting idea,” agrees Elena Zavala, a paleogeneticist at the University of California, Berkeley, who has partnered with DPAA on a different challenge: extracting DNA for identification from WWII and Korean War remains coated in DNA-degrading mortuary chemicals. “There’s a lot of overlap between ancient DNA and forensic science, and we’re just starting to build that bridge,” she says.

In her academic research, Zavala and colleagues have detected eDNA from extinct human relatives in cave sediments thousands of years old. By comparison, the decades-old DNA from WWII crash sites seems practically fresh.

But Zavala wonders whether contamination, either from the samplers themselves or a passing scuba diver, could mask the signal of a fallen soldier. She’d also like data on whether human remains in the ocean continue to shed DNA for decades, and whether that DNA is still detectable in water samples.

UW geneticist Joshua Hyman notes that heat and light break down DNA. He says the best bet for preserving it is a cold, dark environment unperturbed by currents—as close to a –80°C freezer as you can get. DNA might degrade beyond recognition in sediment or water from a sunny tropical place such as Saipan. “It would be helpful to do some feasibility testing,” Zavala says.

Calvin Mires and Evan Kovacs dive to collect plugs of sediment and bottles of water around an aircraft wreck near Saipan.

As part of a project to test human environmental DNA detection, a researcher samples the sea floor near a wreck near Saipan.

Molecular biologist Bridget Ladell works to extract DNA from samples in a “clean room” at the University of Wisconsin, Madison.

A bomber flies over Tanapag Harbor during the Battle of Saipan on 15 June 1944.

U.S. Marines land on the beach in Saipan on 24 June 1944 as part of an operation to capture the island.

That’s why the team visited Saipan’s 30°C waters in March. To test detection across environments, they also traveled to the cold, fresh water of Lake Huron in August and will visit milder waters near Palermo, Italy, later this month. At each location they’ll sample an area containing confirmed remains, one with possible remains, and one that’s empty; together these combinations will help them figure out whether and how eDNA detection works, Fleming says.

In Saipan, UW molecular biologist Bridget Ladell transformed a hotel room into a makeshift lab where the group filtered and packaged the samples on dry ice to ship to Wisconsin, where she extracted the DNA. She’ll do the same with sediment and water from Lake Huron and Italy. As of press time, the samples were queued for sequencing on machines nicknamed Bessie, Louise, Valentina, and Willa, in honor of pioneering female aviators. Once the sequence data are available, bioinformaticians will comb through them, looking for human signatures and trying to match them to samples from any remains. The ultimate goal would be to match eDNA data to families and make an immediate identification, but DPAA’s Fleming stresses that the research is still exploratory.

Scientists on the team are also tacking their own research questions onto the work. Meyer-Kaiser, for example, wants to use eDNA to quantify biodiversity at the sampling sites and study how anemones, fish, and other organisms colonize wrecks.

The eDNA work is one part of a “complex toolkit” of underwater innovations DPAA and scientists are working on, Fleming says. Project Recover researchers are pushing the abilities of their AUVs, tinkering with the vehicles and their sensors to survey previously unreachable sites as deep as 600 meters. “Those areas are incredibly difficult to get to, and have not been studied well,” Fleming says.

The group is also creating and training a machine learning algorithm they hope will be able to sift through the “firehose” of sonar data collected by AUVs to not only identify an aircraft wreck deep on the sea floor, but also discern its make and model. “Within a year we’re hoping to have an automated system [that] spits out 20 highlighted areas from an entire mission that we can then examine,” Moline says.

For scientists, the work is more than technically satisfying. It’s “the most rewarding aspect of my career,” Mires says. When diving in Saipan, despite the lagoon’s warm water and beautiful scenery, the gravity of the work was top of mind, he says. “In other archaeology sites I’ve worked on, the history is remote,” he says. “Here, you’re doing something not for a thing or artifact, but for a person, and all the people they touched.”