Guess Who Came for Dinner?
Researchers use DNA clues to study the diets of
Chesapeake Bay fish

THE BLUE CATFISH HAS A HUGE APPETITE, and it is not a picky eater. Its dinner menu includes plants, insects, crustaceans, worms, and other fish, like menhaden, shad, and river herring.

Recreational fishers have a big appetite of their own for hooking blue catfish as trophies because of their size. The largest landed in Maryland waters, caught in 2012 in the Potomac River, weighed 84 pounds. Blue catfish (Ictalurus furcatus) are among the Chesapeake Bay's largest predators, and a supersize fish needs a lot to eat.

To resource managers, the blue catfish has fast become a big nuisance. The species is not native to the Bay; introduced to Virginia rivers in the 1970s as a game fish, blue catfish rapidly spread to all of the Chesapeake's major tributaries. Like a boorish dinner guest who won't leave, these fish have proceeded to chow down on a variety of native species like menhaden and blue crab that are important to maintaining the estuary's ecosystem and fishing industries.

To curb these effects, federal and state managers drew up plans in 2014 for reducing the abundance and range of this invasive species in the Chesapeake. The plans call in part for finding out more about exactly what blue catfish are eating in the estuary and where. But getting those answers is not easy. Fisheries scientists can remove and examine the stomach contents of a blue catfish. But if it ate its last meal more than 12 hours or so before it was caught, the contents may be partially digested goop, difficult to identify.

Now scientists studying Chesapeake Bay fisheries are beginning to apply new scientific tools that promise to help them learn more about what blue catfish and other predators are eating. They are using DNA sequencing, a technology used by police on TV shows like "CSI: Miami" to identify samples taken from crime scenes based on their unique genetic signatures. This technique can also yield clues about a different kind of remains — decomposed fish taken from blue catfish stomachs.

A Genetic Library

One of the scientists doing this work is Matt Ogburn of the Smithsonian Environmental Research Center (SERC), in Edgewater, Maryland. The marine ecologist is interested in how communities of fish interact with their environment and how their dining habits change as they grow older and larger and move around. Because blue catfish are voracious eaters and their populations are rising in the Chesapeake, they are a potentially useful species to study.

Ogburn and his colleagues suspected that DNA sequencing was a more reliable way of cataloging catfish stomach contents than the traditional method, which relies on appearance. A trained biologist has to recognize the prey species by characteristics like body shape or, for crustaceans, pieces of shells left behind in the stomach.

Using DNA sequencing can offer a more precise method. Biologists use a particular technique called genetic barcoding because it's something like scanning the unique code printed on a label on a grocery-story product. This approach is possible because of a discrete, single stretch of DNA, a gene called COI, for "cytochrome c oxidase subunit 1." COI was selected as a useful gene for DNA barcoding animal species because it is very nearly unique for many species of animals, including the smaller fish and invertebrates (animals without backbones, such as worms) that predator fish in the Chesapeake like to eat. Biologists can take the remains of an animal — even one partially digested in a catfish stomach — determine the sequence of its COI gene, and compare the sequence to a library of known COI sequences to discover which species the flesh came from.

Ogburn has used a library called the Barcode of Life Database (BOLD) developed by biologists worldwide for this purpose. But the SERC scientists knew it needed some updating before they could use it to study Chesapeake Bay predators and prey. The database contains COI sequences for nearly 12,000 species of fish, including many native to the estuary. In many cases, though, the individual fish from which those gene sequences were originally derived were caught in another region of the world. Ogburn and his colleagues knew that might complicate the job of identifying stomach contents of Bay predators. That is because within a single species, genetic sequences can vary slightly across regions. A COI sequence of a small fish appearing in the BOLD database might differ from the sequence of the same species found inside the stomach of a catfish in the Chesapeake.

The researchers, including SERC biologist Robert Aguilar, set about to plug that information gap by starting a new project called the Chesapeake Bay Barcode Initiative. In 2011, they began obtaining specimens of fish and invertebrates caught in the estuary, determined the COI sequence for each, and contributed the information to the BOLD database. So far, they've found COI sequences for more than 220 of the Bay's 315 fish species. The work has created a resource that can be used for other kinds of fisheries research in the future.

CSI for Fish Stomachs
Gene map of some fish species. Graphic, Chesapeake Bay Barcoding Initiative, Smithsonian Environmental Research Center

These portions of a DNA segment (above) show unique signatures of eight species of fish and invertebrates. Scientists can use this segment, named COI, to identify prey species in the stomach contents of larger predator fish, such as blue catfish, living in the Chesapeake Bay. Each color represents a separate "letter" in the genetic code contained within the DNA (except for gray, which represents a gap in the sequence). Species that are closely related tend to have sequences that are more similar than do species that are less closely related. Graphic, Chesapeake Bay Barcoding Initiative, Smithsonian Environmental Research Center

Dining Choices of Blue Catfish
Robert Aguilar. Photograph, Nicky Lehming
Robert Aguilar, a biologist at the Smithsonian Environmental Research Center, helped develop a library of these DNA segments. Photograph, Nicky Lehming

With that improved tool in hand, Ogburn's team wanted to know which method was more reliable for identifying stomach contents, genetic barcoding or the traditional technique of examining appearance. The team compared both methods to analyze contents from 319 blue catfish caught in four tidal freshwater areas in Maryland — the Patuxent River, Marshyhope Creek, the Sassafras River, and Swan Creek.

It wasn't much of a contest: the SERC researchers identified the species of only nine percent of tissue samples by observing morphology but 90 percent using genetic barcoding. The latter represented 23 different fish species, a sign of the blue catfish's wide-ranging palate. The researchers even found an unexpected piece of tissue — from a black cormorant. Ogburn speculates that the blue catfish scavenged upon the bird's carcass after it wound up in the water.

Not everything in a fish's stomach is easily identifiable using genetic techniques, especially the remains of invertebrates. These animals, such as mysids (small crustaceans) and polychaetes (worms), can make up a large portion of the diet of some predator fish. But there are gaps in the DNA library for these species. So far Ogburn and his colleagues have determined DNA sequences for only 250 species of the larger Bay invertebrates while the number in the estuary is estimated to total more than 1,000. The species of each invertebrate has to be correctly identified through other techniques before researchers can label it with its DNA code, and identifying invertebrates, which are small, can be time-consuming and tricky. That is why the SERC study of blue catfish, published in 2017 in the journal Environmental Biology of Fishes, covered only the fish species they ate and not invertebrates. George Mason University researchers are conducting a pilot study using DNA barcoding to determine what kinds of invertebrates are eaten by predator fish in the Potomac River.

Maryland Sea Grant has also funded research to develop this kind of technology. Rose Jagus, a molecular biologist, and graduate student Ammar Hanif used DNA barcoding to analyze the stomach contents of menhaden, an important prey species for striped bass. The researchers used a genetic sequence other than COI to identify what kinds of phytoplankton menhaden eat.

A Next-Generation Genetic Tool

Given the limitations of DNA barcoding, it is unlikely to replace visual observation of stomach contents any time soon. Examiners can often see enough detail about partially digested fish and the bones and shell left in the stomach to assign particular samples to at least a broad taxonomic grouping, such as a genus or family, if not a particular species. Those details have given resource managers important information about what predator fish are eating in the Chesapeake Bay.

But emerging genetic techniques offer scientists a complementary, powerful, and efficient way to study the estuary's fisheries. In contrast to DNA barcoding of a single tissue sample at a time, an approach called "next-generation sequencing" can quickly identify all of the species in a fish stomach in a single laboratory test run. This is a quicker method than analyzing individual items one by one. The next-generation method should also indicate the relative abundances of prey species within a stomach, such as whether the predator recently ate more menhaden or more bay anchovies.

The same approach can also be used to examine other important questions in fisheries biology, like whether a water sample from a particular river contains DNA of an endangered, rare fish like river herring or sturgeon or an invasive species like blue catfish. That information could help inform managers about where to focus their efforts to protect the endangered fish and reduce the populations of unwelcome, invasive fish.

Next-generation sequencing has not yet been used to study Chesapeake Bay fisheries, but progress to date suggests that it and more traditional DNA barcoding have a lot of promise, Ogburn says. "It's been an exciting area to try to push into. It's producing new opportunities for new kinds of research. We're always looking for ways to get better data, to get it more efficiently, and to answer important questions for management or conservation."

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