Researchers use biotechnology to identify foods made from endangered species
The burgeoning global meat trade has taken its toll on the Kouilou region of the Congo. A stretch of unprotected rainforest supplies a clandestine gorilla meat market, and last fall, investigators revealed that Kouilou gorillas are poached at a rate of two per week.
“The population of gorilla that we located is in sharp decline, and will probably become extinct in a few years if we do not stop that trend,” says Pierre Fidenci, president of the San Francisco-based Endangered Species International, which conducted the investigation.
This is easier said than done—even if intervention procedures can be organized, there is still the problem of busting poachers. Even when they are caught red-handed, it’s often hard to prove the meat they are trafficking was harvested from an endangered gorilla. Finding a gorilla hand on a market table is a dead give away, but when gorilla flesh is sold for human consumption, law officers are just as likely to find slabs of meat in coolers. Identification starts to get tricky, especially if the meat is partially processed.
This is why George Amato, director of the American Museum of Natural History’s Sackler Institute for Comparative Genomics, turned to DNA sequencing. It turns out there is a short fragment of DNA found in all animals that contains a species-specific identifier. Called the barcode region, it’s comparable to the black stripes found on food labels at grocery stores—at least in concept.
“It tells you what species you are dealing with. If we extract DNA from a piece of meat, and the barcode sequence matches up with the western lowland gorillas of Kouilou, we know the meat came from this species,” says Amato. This is because the DNA barcode region accumulated mutations at a high rate during evolution. Individuals within the same species have the same sequence, but when comparing two different types of an animal, the barcodes differ enough for researchers to tell them apart.
Yet extracting DNA barcodes from real-world meat and leather samples might not give a clean read—at least this is what critics said of Amato’s work. In the real world endangered species are trafficked in shipments of crocodile purses, for example. Even if extracting DNA is possible, researchers might not get clean sequences for the barcode region.
Last fall, Amato put these fears to rest. Using partially processed flesh collected from a prior study in the Congo, and museum samples of dried animal skin, Amato and his colleagues extracted DNA, and isolated the barcode sequence—officially named Cytochrome Oxidase I, or COX-1. He compared samples from gorilla, crocodile and African antelope.
It turns out the gorilla samples were clearly distinguished from crocodile and antelope species. Most importantly, even when nothing was available but a piece of crocodile or antelope skin, Amato was able to name the species correctly—he could even distinguish between different types of gorillas, crocodiles and antelopes. “It seems trivial now that we have done it, but people originally wondered whether you could get DNA from market meat,” says Amato. “Samples are not refrigerated, and have often been handled, and this can impact quality.”
Last winter high school students started sending Amato products to barcode. They gave him butter and goat cheese, not to mention dog biscuits that purportedly contain bison meat. “We wondered about the dog biscuits, but we were able to get a clear barcode and verify the species—even despite the processing,” says Amato.
He admits croc skin purses were a challenge, but these, too, provided clean barcode sequences. “We just had to dig for the best pieces,” he says. By peeling away layers of scales under a microscope, he found pieces of embedded bone that could be used. DNA was extracted, and clear sequences were obtained.
Mike Sutton, vice president of the Monterey Bay Aquarium, says the findings could help stop the illegal trade of species. Fishermen from Baja Mexico bring fillets of endangered Totoaba (Totoaba macdonaldi) across the boarder by labeling imports as rockfish or red snapper. “Fillets are hard to identify, and if a custom’s official can’t identify the meat, a consumer certainly can’t,” he says. “DNA barcoding could help fix this.”
Critics are now beginning to come round to the idea. “In several cases the smell alone would be an easier, faster and cheaper method of identification, but barcoding could be very useful when meats have been partially processed,” says Karina Lucas of the Piracicaba, Brazil-based Escola Superior de Agricultura. Lucas has written about problems with barcoding, but says most of these address other uses—like whether the technique can help classify newly discovered species. These criticisms don’t apply to bush meat applications, she says.
The Real McCoy?
With these successes behind him, Amato decided to tackle a more difficult problem—tuna sushi. “Threatened gorillas and tigers invoke images from National Geographic specials, but we don’t think much about the endangered fish market because we think of fish as dinner,” he says.
When it comes to land-based animals, humans usually eat from the middle of the food chain. A hundred years ago western civilization stopped hunting wildlife for market distribution, and our meat now mainly comes from herbivores—plant-eating animals like cattle and bison. But when it comes to the fish we take from the sea, primary carnivores are still fair game.
“When we talk about selling bluefin tuna, it’s comparable to grizzly bear stakes—at least as far as the ecosystem is concerned,” says Mike Sutton, vice president of the Monterey Bay Aquarium. “We don’t eat carnivores on land, but these are our favorite sea foods.”
Tuna are considered apex predators—they eat squid and other fish, and are at the top of the fish food chain. This means for every tuna killed, it takes a lot of ocean resources to replace the animal. As the most prized cuts of fish come from 350-pound tuna—it takes a lot of squid and smaller animals to support large-scale fishing, and the fish can’t reproduce faster than they are caught. This is one reason many tuna stocks are collapsing, says Sutton.
Yet selling rare tuna remains entirely legal. In fact the Scotts Valley-based Yamamori Sushi Boat Grill regularly carries bluefin. Even while north atlantic and southern stocks are scarce, owner Sunwoo Kimn says he rarely knows which species he is selling. “We don’t differentiate what species of bluefin we get,” he says.
The tuna bring in a lot of money. While a slice or two of less endangered yellowfin tuna might only be $4.00, bluefin sells for $7.00. Fresh wild caught is available through most of the year, and farm raised comes in during the late spring and summer.
In an ideal world, Sutton says these options would be left off the menu. “Bluefin is on the redlist of our seafood watch cards,” says Sutton. “Both wild and farmed are an unacceptable choice from an environmental perspective.”
This is because “farmed” tuna are not grown on typical fish farms. They are captured in the wild, held in captivity—either on land, or in nets under the ocean—and fattened up. Sardines from the Monterey Bay are imported to feed the fish, representing a net loss to the ocean.
“When tuna feed in open waters they play a role in the ecosystem—they eat a variety of foods, and are sometimes preyed on by other species. If you put them in captivity it’s very wasteful—it’s a whole other thing,” says Sutton.
Despite these harms, restaurants have no reason to keep the fish off the menu, and Sutton says it’s up to consumers to boycott. Yet avoiding tuna in the marketplace is a lot harder than it sounds. Lets say you go to the restaurant and get a tuna role – it could be yellowfin or big eye—both are over fished. It could also be a species of bluefin.
Big eye and bluefin are especially similar in texture and taste. Yellowfin also mimic’s the soft, buttery flavor of its rare cousin—especially when the meat has been a little beaten up. Without taste-testing two kinds of tuna side-by-side, you might not be able to tell which fish species you are eating.
This makes it hard to tell whether a critically overfished species is being sold under the guise of albacore or yellowfin. “There is no restaurant that has any way to verify the kind of meat they are getting,” says Chris Mumma, sushi chef at the Santa Cruz local favorite, Aqua Blue, as he prepares a tempura roll with shrimp. “Most chefs can tell the difference between albacore tuna and yellow-tailed amberjack, which is not related to tuna at all, but has the same color and a similar flavor. But when comparing tuna to tuna, we just have to take the word of the distributor.”
Yet by the time tuna sushi makes it to your plate, the meat has changed hands several times, and the distributor might not be sure what they are selling.
First, tuna is pulled from the water by a fisherman. It dies on the boat, where it might be gutted, skinned and single cut. Fish are usually flash frozen on site, and then shipped to a market. Here they are reduced to smaller pieces. Distributors then buy the slices and sell to local sushi bars.
“There is no regulation that requires a piece of the fish [like skin or bone] be left for identification,” says Sutton. “By the time you see it, it’s just a piece of meat.”
Sometimes mix-ups even happen on the boat. “We always ask for yellowfin, but there is no way to know for sure what we get—at least not without DNA testing,” says Mumma.
Yet as Amato discovered, tuna DNA is elusive. Evolutionarily speaking, it turns out tuna species are very closely related. “Most species are clearly different, but genetically speaking, tuna look a lot a like,” he says.
Identifying Mystery Meat
Research-like databases, and the scientists who study barcoding are no exception. Over the years barcodes for beetles, butterflies and other animals have been filed away, and their COX-1 sequences are now available in databases that everyone can access.
When researchers have a piece of mystery meat, they extract its DNA and sequence the region where the barcode is found. Then computers compare the barcode region to all the other animals in the database. The mystery meat barcode is lined up next to pig, bird and primate, and eventually a match is found.
If the meat is a piece of Nile crocodile—or Crocodylus niloticus—its barcode will match up with the DNA archived for other members of the species. It will differ no more than 2 percent from other Nile crocodiles. When compared to a turkey or a frog—or even another species of crocodile—the sequence will be more than 4-10 percent different.
Yet when comparing two different species of tuna, things are a little more complicated. Their barcode sequences are 98 percent similar—this means two different types of tuna will match up, appearing to look like members of the same species. This is why previous researchers were unsuccessful when trying to apply barcodes to tuna.
To get around this problem, Amato and his graduate student tried to think outside of the box. They decided not to compare the entire barcode region, and opted against calculating similarities. Instead, they looked for areas within each species’ barcode that could be used for identification.
They started with known samples of Northern Bluefin, for example, and looked for sub-regions within the barcode sequence that were unique to this species alone. After doing this with many different tuna, they were surprised to find unique, distinguishing regions for each tuna species. “We were able to tell different species apart very easily by comparing these sub-regions,” says Amato.
For the next step of the project, Amato sent his graduate student, Jake Lowenstein, out for dinner. The goal was to collect pieces of tuna to bring back to the lab, and see if barcoding could be used to identify the species.
After stopping in for sushi at 31 restaurants in New York City and Denver, Colo., Lowenstein discreetly slipped 68 pieces of meat into test tubes. “We tried to capture the gamut of sushi bars, from one of the most expensive restaurants in New York right through to neighborhood sushi bars,” says Amato, who claims he resisted the temptation to taste-test back at the lab. “I ate some of the caviar when we did a caviar study a while ago, but I didn’t eat any of the sushi,” he says.
Back at the lab, the researchers extracted DNA from the sushi slices, sequenced the samples, and identified each species. What came next was a big surprise.
Nineteen of the samples sold as albacore turned out to be northern and southern bluefin (T. thynnus, and T. Maccoyii). Both of these species are severely depleted—northern bluefin have declined by more than 90 percent since the 1970s, and conservationists warn that southern bluefin may soon face extinction.
Sutton says he is saddened by the findings, but not entirely surprised. “We can’t afford to make any mistakes with bluefin because it is so severely overfished,” he says, “but mislabeling is more common in the fish market than anyone realizes. You don’t know what you are getting most of the time.”
The United States imports 80 percent of its seafood, and most of this comes in as fillets. Even when the fish species is correctly named on the label, other information might be wrong. For example, farm-raised salmon are sometimes listed as wild—or vice versa. Farmed salmon pick up pesticides and heavy metals when farmed, and contaminate wild stocks with parasites. Wild caught salmon are generally considered a better choice, as the Alaskan industry is well regulated. Yet as Sutton says, “mislabeling defeats the purpose of the sustainable seafood movement.”
Busting Bad Menus and Bush Meat Traffickers
Overall, more than two-thirds of the tuna listed on menus was misidentified. Five out of nine samples sold as “white tuna” were not even tuna at all, but rather escolar (Lepidocybium flavorunneum), a species banned for sale in Italy and Japan because it causes stomach upset, Other menus sold yellowfin or albacore under the auspice of bluefin.
“We were surprised we found so many misidentified items on the menu,” says Amato. “We were initially just looking for a way to distinguish between tuna that have very similar DNA sequences.”
Sutton says the findings are concerning. “It’s critical that fish are properly labeled, if they aren’t, consumer awareness programs fall apart pretty rapidly,” he says.
Making the barcoding process faster and more portable might help customs officials bust meat traffickers and those who mislabel fish. “If a simple barcoding test can be developed, it will help,” says Sutton. “But it has to be feasible for large bureaucracies.”
The Convention on International Trade in Endangered Species (CITES) agrees with this point, and says barcoding—if developed into a simple tool—might help enforce the trade restrictions around the world.
“The ability to be able to easily identify specimens of the species in international trade with an existing tool would be helpful,” says David Morgan, of the Chief, Scientific Support Unit at the CITES Secretariat. While total tuna sales are hard to track, international trade in wildlife products has a global market of about $15 billion. As much as $8 billion of this is illegal, and involves species protected by national laws and international wildlife conventions.
Most stages of the barcoding process can be done on site—at least with the right technology. “You can do a quick DNA extraction at the market,” says Amato. Copying the DNA for analysis can also be done on-site, at least with the right equipment.
Instead of sequencing the barcodes, researchers are investigating tiny chips called microarrays, which can be designed to hold matching-strands of barcode DNA from any number of endangered species. After extracting and copying DNA samples from a shipment, the barcodes could be washed over the microarray onsite.
“You could have a chip that tells you whether the meat comes from an endangered animal,” says Amato.
Even if onsite analysis isn’t yet possible, he says government labs can incorporate these methods into inspections. Amato trained customs scientists on the technique in April, and hopes to work with U.S. Fish and Wildlife, the U.S. Department of Agriculture, and the Food and Drug Administration as well. “Any standard lab will have everything you need to barcode meat samples,” he says. It’s possible even local agencies could use barcoding—they might just have to outsource the sequencing stage.
There is no question that the technology has already come a long way. Twenty years ago researchers could not have completed a barcoding study without a lot of hassle. Before barcoding, if scientists wanted to find out if monkey meat came from an endangered species, they would have to find a good region of DNA to use as a basis for comparison, and sequence all the monkey species out there. Other scientists in the field were working with different regions of the genome, so their findings couldn’t be compared.
This is why the COX-1 barcode region is so important. In 2003 the New York City-based Sloan Foundation organized a meeting at the Cold Spring Harbor Lab in Long Island. The goal was to select a region to use for species identification, and decide whether there was utility in having researchers all work with the same fragment. COX-1 was later selected as the DNA region of choice.
“I have been trying for 20 years to do this – to take something being smuggled, and identify the species,” says Amato. “But I had to start from scratch with each animal. We have come a long way since then, and my hope now is that this technology be put to use by people who can make a difference.”
Location, Location, Location
Knowing where your fish comes from is just as important as identifying the species, researchers say
Northern bluefin tuna are no different than other fish—they like to eat. Yet unlike other sea creatures, who like to stay close to home, Tuna are globetrotters. Once a year stocks around the world travel thousands of miles to feast on the rich squid and smaller fish near Nova Scotia, Canada. The feeding area extends down along the coast toward North and South Carolina.
According to Randy Kochevar, a researcher at Stanford’s Hopkins Marine Station, in Monterey, this makes the tuna populations particularly vulnerable to hunting.
“If you go up in the north Atlantic when the tuna are feeding, and you catch 10 fish, five might have originated from the Mediterranean and four might be from the Gulf of Mexico,” he says. Even if fishing is regulated near home, fragile stocks of tuna can be diminished by actions that happen miles away.
To make matters more complicated, the fish breed back home in the local scene. So removing members of a population during feeding season could directly impact population breeding trends months down the road.
“Even though the populations mix for feeding, they don’t actually breed together,” says Kochevar. “Breeding season is right now—in the Gulf of Mexico, among other places, as the oil is spilling out all over the place.”
Kochevar’s colleagues used electronic tags to follow the tuna, and found the fish were eating and breeding separately. They published the findings in 2005 in the academic journal Nature.
A follow up study traced the fish found in feeding grounds back to their birthplace. As the water in the Mediterranean has a different chemical signature than the Gulf of Mexico’s H20, the researchers were able to tell each fish’s ocean of origin. They pulled samples from the ear bones of the fish, and analyzed isotopes.
“In a fish the ear bone grows gradually over time—it lays down new material, like the growth rings on a tree,” says Kochevar. “If you sample the innermost rings you can determine where the tuna came from.” This study was published in the academic journal Science in 2008. The findings bolstered the idea that tuna eat together, but breed apart. as the fish found in feeding grounds originated miles away from separate stock populations. The fish come from their neighborhood to eat, and return home for breeding after, the findings show.
The findings indicate that better fishing quotas should be established. Overfishing near feeding grounds removes bluefin from two very different geographic populations, and has a global impact.
These two studies were among those reviewed by the Convention on International Trade in Endangered Species (CITES) at their March meeting in Doha Quatar. CITES bans the international trade of vulnerable species, but to qualify for protection, an animal must be endangered and also traded on a major market. This year Northern bluefin were nominated for protection, and a vote was held to determine their status.
Things didn’t go well for the fish. When delegates met for the vote, Japan, Canada and other nations opposed the proposal. Many countries argued management groups were better equipped to tackle the decline of bluefin tuna stocks. In the end there were 20 votes in favor of listing the Tuna, and 68 against.
If the fish had received protection, their meat could not be legally sold on the international market. The United States would have cracked down on domestic sales.
“I was not entirely surprised, but still very disappointed,” says George Amato, of the New York, NY-based American Museum of Natural History. “We know Northern bluefin is endangered, this is unambiguous. Whether we choose to protect it or not, the fish are endangered. It’s too bad the fish won’t have a legal designation to represent this reality.”
This means it’s up to consumers to protect the fish, says Mike Sutton, vice president of the Monterey Bay Aquarium. Consumers should avoid purchasing Northern bluefin all together.
Avoiding other kinds of tuna is also recommended, as bluefin are often mistakenly marketed under the guise of less vulnerable relatives.
When shopping for seafood Sutton recommends looking for the blue and white Marine Stewardship Council eco-label. “They trace the source of the seafood and ensure the species being sold are not overfished,” he says. | AC