Canned tuna is high in protein, low in fat and by far the most popular shelf-stable seafood in the United States.

It can also be mysterious, questionable and scandalous. As The Washington Post reported in late January, Subway — the world’s largest sandwich chain — is currently facing a class-action lawsuit in the state of California that claims its tuna sandwiches “are completely bereft of tuna as an ingredient.”

After the news broke, the jokes swiftly followed. Jessica Simpson (who famously didn’t know whether Chicken of the Sea was chicken or tuna back in 2003) tweeted: “It’s OK @SUBWAY. It IS confusing.” Jimmy John’s, a competitor, started sending email blasts with subjects like: “Tuna 👏 Sandwiches 👏 Should 👏 Use 👏 Real 👏 Tuna 👏.”

Subway, for its part, has categorically denied the allegations. “There simply is no truth to the allegations in the complaint that was filed in California,” a spokeswoman wrote in an email to The New York Times. “Subway delivers 100 percent cooked tuna to its restaurants, which is mixed with mayonnaise and used in freshly made sandwiches, wraps and salads that are served to and enjoyed by our guests.”

From a reporter’s perspective, however, the case bore further investigation — a deep dive, if you will.

So, I procured more than 60 inches worth of Subway tuna sandwiches. I removed and froze the tuna meat, then shipped it across the country to a commercial food testing lab. I spent weeks chatting with tuna experts. I waited, and waited, until the lab results came back.

Here’s what I found.

Tuna, in the Abstract

Canned tuna has its die-hards and naysayers, but it certainly sells. According to Nielsen Holdings, a global data and analytics company, about 700 million cans of tuna were sold in the U.S. over the last year.

“I think part of it is just nostalgia,” said Ryan Sutton, the chief food critic for Eater NY, when asked to explain the ascendancy of the canned fish. “It’s what a lot of people grew up eating.”

Tuna sandwiches rose to prominence in the early 1900s, when people realized canned fish could translate into a quick and cheap meal that involved no cooking. By the 1950s, tuna had surpassed salmon in popularity, and during the 1980s, an estimated 85 percent of Americans had canned tuna in their pantries despite growing concerns about high levels of mercury in the fish.

After a decades-long decline in tuna consumption, a 2018 Wall Street Journal story suggested that millennials were to blame (“many can’t be bothered to open and drain the cans”), though newer brands offering more sustainable options were seeing their market share grow.

Then, in 2020, canned goods took on great urgency as shoppers scrambled to buy shelf-stable food, not knowing how long the pandemic might last. That year, the global canned tuna market was valued at $8.57 billion, according to Grand View Research.

Subway has nearly 40,000 locations worldwide, about half of them in the U.S. (As Grub Street once calculated, the average distance between Subways in Manhattan is 1,154 feet, or about four and a half blocks.) The total number of locations has been in decline since 2015, a trend that a New York Times investigation attributed, in part, to targeted and manipulated inspections.

Still, Subway’s storefronts are ubiquitous, and according to the company, its tuna sandwiches are some of the best selling. “Subway’s tuna sandwich ranks among our guests’ favorite sandwiches,” the chain’s spokeswoman wrote in an email.

But it’s safe to say that Karen Dhanowa and Nilima Amin — the plaintiffs in the lawsuit — do not love Subway’s tuna, which they believe is “anything but tuna,” according to their filing from January. (Ms. Dhanowa and Ms. Amin’s legal team declined to comment on the case for this article.)

What exactly the plaintiffs believe the sandwiches contain, they wouldn’t say. But they are alleging that Subway has deliberately misled customers by selling products “falsely advertised as ‘tuna’” in order to charge a “premium price.”

Subway’s spokeswoman, when asked about the progress of the case, reiterated the statement shared when the original complaint was filed.

“The taste and quality of our tuna make it one of Subway’s most popular products and these baseless accusations threaten to damage our franchisees,” she wrote in an email.

“Given the facts, the lawsuit constitutes a reckless and improper attack on Subway’s brand and goodwill,” she added.

With all of that in mind, I began searching for a commercial lab that could test a sample of Subway’s product. A handful of them politely declined my inquiries, citing technical limitations and company policies that made my tuna ineligible for analysis. Eventually, I found myself on the phone with a spokesman for a lab that specializes in fish testing. He agreed to test the tuna but asked that the lab not be named in this article, as he did not want to jeopardize any opportunities to work directly with America’s largest sandwich chain.

For about $500, his lab could conduct a PCR test — which rapidly makes millions or billions of copies of a specific DNA sample — and try to tell me whether this substance included one of five different tuna species.

From the Sea to Your Submarine

According to the Seafood List, which is compiled by the U.S. Food and Drug Administration, there are 15 species of nomadic saltwater fish that can be labeled “tuna.”

Subway’s tuna and seafood sourcing statement says the chain only sells skipjack and yellowfin tuna — species that a lab would recognize as Katsuwonus pelamis and T. albacares.

Before it lands on a Subway sandwich, that tuna, like the majority of commercially sold tuna, is caught by fishermen working in exclusive economic zones. (E.E.Z.s are areas that extend roughly 200 nautical miles from each country’s coast; the U.S., with over 13,000 miles of coastline, controls the largest E.E.Z. in the world, containing 3.4 million square nautical miles of ocean.)

There are five organizations that manage regional fisheries within those economic zones. Their job is to enforce regulations and “make sure the predators of ocean ecosystems remain in the ocean and not on our plates,” according to Barbara Block, a professor of marine sciences at Stanford University who co-directs Stanford’s Tuna Research and Conservation Center.

“Removal at sustainable levels is a priority of many,” Dr. Block added in an email, “but practiced by few.” Bluefin tuna, for example, is now an endangered species following decades of overfishing.

There are three methods these fisheries use to catch tuna: purse seining, longlining and pole-and-line fishing.

Pole-and-line fishing is the kind hobbyists take part in: sitting on a boat and bringing in one catch at a time. Larger fishing operations tend to rely on the other two methods.

Purse seiners drop a large, round wall of netting around a school of fish and then “purse” the bottom of the net shut to prevent fish from escaping. Longliners drop one 30- or 40-mile-long line into the water, then wait for the fish to catch on hundreds (or thousands) of hooks.

Then the fish is cleaned, sorted and, eventually, canned. Dave Rudie, the president of Catalina Offshore Products in San Diego (the former tuna capital of the world), works with a cannery that sells about a million cans of tuna each year, 10,000 of which contain bigeye sourced from him.

“The really perfect colored tuna — the brightest red — goes to the sushi bars,” Mr. Rudie said. “Tuna that’s a little bit paler in color will go more for the cooking, where you sear the outside and throw out the middle.”

“We also get some more off-color tuna and that off-color tuna, we cut it up and freeze it,” he continued. “And we send it up to a cannery in Oregon.”

Canneries tend to follow the same general process. “Most canned tuna is caught by purse seiners and it’s frozen on the boat,” Mr. Rudie said. “They’re going to take it to a cannery, where they’re most likely going to cook it once, and then they’re going to pull the meat off the bone, and they’re going to put it in a can, and then it’s going to get retorted — cooked a little bit — to sterilize it the last time before they seal the can.”

Then there’s the issue of labeling. When Oceana, an organization focused on ocean advocacy, conducted one of the largest “fish fraud” investigations in the early 2010s, it discovered that “seafood may be mislabeled as often as 26 to 87 percent of the time for commonly swapped fish such as grouper, cod and snapper, disguising fish that are less desirable, cheaper or more readily available.”

Such distinctions might not be noticeable, or all that troubling, to every consumer. But when you’re trying to figure out what’s in a Subway tuna sandwich, they matter.

I was told that if I packed a Ziploc of Subway tuna into a Styrofoam shipping cooler with a few ice packs and mailed it across the country, the lab could test it.

To procure the sandwich specimens, I visited three different Subway locations around Los Angeles. It seemed logical to order only tuna on the sandwiches — no extra vegetables, cheese or dressing — as the lab was already wary about the challenges of identifying a fish that’s been cooked at least once, mixed with mayo, frozen and shipped across the country.

My first frozen tuna shipment, which cost upward of $150, was lost in transit. But on second try, the sample arrived intact. In two to three weeks, the lab would tell me whether it contained any tuna.

Sandwich Artists Weigh In

Though Subway declined to disclose its tuna suppliers, Sage, who has been a Subway manager in California for three years, shared some details about how the product arrives at her location. (Sage asked not to use her full name out of fear of reprisal from her employer.)

“The tuna comes in a case and inside the case, there are six aluminum pouches and it’s just like a pressed, vacuum sealed slab of tuna,” Sage said. “It’s flaky and it’s clearly soaked in water — it’s like a brine, so it’s just soaked in salt water — and it’s just flaky tuna. We just spread it apart with our hands” — gloved, of course — “and mix it with mayo.”

Sage said that each store follows corporate guidelines, which instruct that certain meats can stay out in the store’s refrigerated sandwich bar for up to 24, 48 or 72 hours.

Tuna, she said, has a 72-hour counter life (the time frame was also confirmed by Subway’s spokeswoman), though Sage said her store often replaces it before it hits three days. “We all agree — all of us that work there — it gets kind of gross,” she said.

Jen, a former Subway “sandwich artist” who worked at a location in Iowa for a year, said she couldn’t imagine what incentive Subway would have to replace the tuna with anything else. (Jen also asked not to use her full name out of fear of reprisal from her employer).

“I dealt with the tuna all the time,” Jen said. “The ingredients are right on the package and tuna is a relatively cheap meat. There would be no point to making replacement tuna to make it cheaper.”

And as an occasional consumer of Subway’s tuna, Jen said she’s confident it’s fish.

“I personally have a really weak stomach to fish, which is how we know the tuna is real,” she said. “Last time I ate it, I puked my guts out.”

But Sage said that beyond meeting these food safety standards, she’s not very concerned about whether this tuna is real or not.

“We don’t really care at all,” she said of herself and her fellow sandwich artists. “Which may sound kind of weird, I guess, but customers will bring it up and we just go, ‘I don’t know. What kind of cheese do you want?’”

The Lab Results

Finally, after more than a month of waiting, the lab results arrived.

“No amplifiable tuna DNA was present in the sample and so we obtained no amplification products from the DNA,” the email read. “Therefore, we cannot identify the species.”

The spokesman from the lab offered a bit of analysis. “There’s two conclusions,” he said. “One, it’s so heavily processed that whatever we could pull out, we couldn’t make an identification. Or we got some and there’s just nothing there that’s tuna.” (Subway declined to comment on the lab results.)

To be fair, when Inside Edition sent samples from three Subway locations in Queens out for testing earlier this year, the lab found that the specimens were, indeed, tuna.

With all testing, there are major caveats to consider. Once tuna has been cooked, its protein becomes denatured — meaning that the fish’s characteristic properties have likely been destroyed, making it difficult, if not impossible, to identify.

All of the people I spoke with also questioned why Subway would swap out its tuna.

“I don’t think a sandwich place would intentionally mislabel,” Mr. Rudie from Catalina Offshore Products said. “They’re buying a can of tuna that says ‘tuna.’ If there’s any fraud in this case, it happened at the cannery.”

Peter Horn, the director of the Ending Illegal Fishing Project at the Pew Charitable Trusts, agreed that it would be difficult to place blame on Subway if this were the case.

“In the defense of Subway, or quite a lot of these fishmongers, the further you get the fish from the bone, the harder it is to recognize what that fish is,” he said.

“Most of us see the fish on the bone, skin intact, and we can recognize what sort of fish that is,” he continued. “You drop the head and the tail off, it becomes more difficult, but you can still probably recognize it. You take the skin off it, you take it off the bone and you cut it into slices then you’re only sort of saying, ‘Right, what’s the color and texture?’”

Mr. Sutton, the food critic, suggested that this incident could encourage consumers to take more interest in where their food comes from.

“I would hope that an issue like this would cause more people across the country and all across the world to spend more time thinking about every step of the environmental, labor and economic supply chains that supply their food,” he said.

And even as Subway’s prices have risen beyond the days of $5 footlongs, Mr. Horn said the company’s notably cheap sandwiches raise more important questions than the integrity of their tuna.

“We can’t just continue to have a downward pressure on the price,” Mr. Horn said, “because if we all want everything at rock bottom prices, that means something, somewhere is going to be exploited, whether that’s people or the ocean — probably both.”