The past decade has seen an explosion of accomplishments in biotechnology, because researchers have made rapid strides in understanding the structure and function of molecules and cells. Sea Grant’s innovative biotechnology research is providing new products and processes from the sea.

Biotechnology simply means using living organisms to accomplish some task, and humans have been doing just that for thousands of years. For example, baking bread with yeast, fermenting wine or beer, making cheese, yogurt, or stonewashed jeans are all processes that utilize biological organisms. And humans have been genetically modifying organisms through classical selective breeding ever since agriculture began. Currently, Sea Grant–sponsored scientists are exploring areas of biotechnology such as molecular genetics, biochemistry and pharmacology, immunology and pathology, bioprocessing, and bioremediation.

The difference between the new technology and the old is that today, thanks to the breaking of the odd 4-letter code that spells out the structure of DNA, the blueprint of our genetic information, we can now directly insert genetic information into organisms and accomplish desired change much more quickly and efficiently, with great specificity. It’s a powerful new biological toolbox.

Our bodies contain thousands of genes, each made of sequences of DNA, that determine the characteristics that make us who we are and make a human different from, say, an acorn or a jellyfish. Because they contain "instructions" for the manufacture of specific proteins, genes also determine the structure of the hormones and enzymes that initiate, inhibit, and regulate our bodies’ physiological processes.

Historically, the practice of agriculture involved generations of classical selective breeding of plants and animals to enhance some desirable characteristic by increasing the expression of a particular gene in the population over subsequent generations. Because we can manipulate cells and molecules, genes in individuals can now be modified directly to include a specific characteristic, with rapid and predictable results.

Besides correcting genetic disorders, genetic engineering is often used in agriculture and aquaculture to increase resistance of farmed species to pests and diseases or to help crops grow larger and faster.

"It’s like editing and revising a manuscript; you can take out mistakes and insert pieces to improve it," says Hans Laufer, University of Connecticut (UCONN) molecular and cell biology professor. Laufer has examined the endocrine systems of economically important crustaceans for more than a decade, discovering key hormones that regulate processes of molting, development, and reproduction. Understanding these hormones will allow aquaculturists to manipulate the life cycle of crabs and shrimp, he says. By identifying and cloning sequences of DNA that code for enzymes that activate key reproductive hormones, Laufer has doubled egg production in shrimp.

Feeding animals hormones can make them grow faster and reproduce more prolifically, but if a growth hormone gene is successfully transferred to an animal, the animal then produces the hormone itself, and the trait may be carried on to offspring. Tom Chen, UCONN Biotechnology Center director, has successfully transplanted a foreign gene into a crustacean, for the first time in history!

The gene Chen inserted was a "reporter" gene—one that is easy to detect and confirm in the animal’s cells—to prove that the procedure was successful. This demonstration opens the way to exciting possibilities. The technique could be applied one day to shrimp and other crustaceans such as crabs and lobster to improve characteristics such as color, taste, growth rate, size, and disease resistance, for aquaculture.

Having received a new gene from a bacterium, this female bears eggs containing 10-day old F₁ transgenic crayfish embryos.  Photo by T. Chen, UCONN Biotechnology Center.

Chen explains that transplanting genes in crustaceans is difficult because it requires access to unfertilized eggs, and the eggs laid by wild females are usually fertilized by the time they are collected.

"We are now one step ahead in our adventure," says Chen. "We are able to insert the gene directly into the immature gonads." Using a small, sharp knife, Chen makes a small hole in the exoskeleton of an immature crayfish near its thorax, where the testes and ovaries are located. Then, using Krazy Glue®, he delivers a new gene, in the form of a "pantropic retroviral vector"—a small segment of the desired DNA encased in the shell of a virus. The new gene then appears in the eggs or sperm, making injection unnecessary. Chen’s past efforts have included transferring genes in finfish and mollusks. For example, he introduced growth-enhancing hormones into young scallops, hypothesizing that faster-growing scallops might better survive winters in Long Island Sound and reach market size sooner. In an earlier project sponsored by Maryland Sea Grant, Chen and Yonathan Zohar, University of Maryland Center for Marine Biotechnology director, cloned genes for striped bass gonadotropin releasing hormone, a hormone that regulates reproduction in this fish.

Genetic engineering is by no means limited to animals. Don Cheney, Northeastern University biology associate professor, and Subash Minocha, University of New Hampshire plant biology professor, have both been working on genetic manipulation in macroalgae. Both are among a team of investigators involved in a regional Sea Grant effort to develop and enhance the aquaculture of various strains of Porphyra, a red alga (known as nori to sushi eaters) commercially farmed for the food and pharmaceutical industries. Minocha says there is much yet to be accomplished in genetic engineering for the algae, as the practice is in its infancy. His lab has succeeded in using an electric shock technique to transplant a bacterial gene into two species of algae, Porphyra and Ulva spp., both consumed as food.

Cheney has used bioengineering techniques such as protoplast fusion to induce genetic variation in several seaweeds, including nori, thereby developing new strains. His lab was successful in producing a fast-growing polyploid strain of Porphyra, which is excellent for growing nori on nets. The new strain is being used by Coastal Plantations, Inc. in Eastport, Maine, a nori aquaculture facility. In another project, sponsored by MIT Sea Grant, Cheney’s lab produced a new strain of Chondrus crispus. This species, commonly called Irish moss, is harvested for its carrageenan, a smoothing gel that thickens products like ice cream, pet food, and toothpaste. Cheney’s new strain has different gelling characteristics from those of the native species, and while the applications are uncertain, it proves that carrageenan composition can be altered with the new techniques, encouraging new product development.

Disease Detection

Immunology and disease detection are another important application of marine biotechnology. Monoclonal antibody technology is one technique that researchers are using to develop new tests for diagnosing plant, animal, and human diseases. This technique uses immune system cells that make proteins called antibodies. The cells making these antibodies can be cloned in large amounts in the laboratory. Because of their molecular structure, the antibodies bind with specific antigens (foreign material invading the body), much as a key fits into a lock. When the antibodies bind chemically to antigens of interest, such as tumor cells or specific enzymes, scientists can use a fluorescent tag or colorimetric signal to help detect, and sometimes eliminate, pathogens or contaminants.

DNA polymerase is a key enzyme that makes the base pair sequence of a strand of DNA and also "proofreads" the sequence of DNA it creates. Using a technique called the polymerase chain reaction (PCR), scientists can copy a fragment of DNA in a test tube millions of times, making it much easier to detect the DNA segment of interest.

Thomas Kocher, zoology professor and director of the DNA Sequencing Facility at the University of New Hampshire (UNH), is using PCR and microsatellite DNA markers (genetic tags) to determine genetic differences in flounder used for breeding in hatcheries and stock enhancement programs. This research, sponsored by Maine/New Hampshire Sea Grant, will help breeders select a genetically superior stock and establish a paradigm for the genetic domestication of other marine finfish. In previous work, Kocher compared barely detectable genetic variations in cod and harbor porpoises. Such research aids in tracking the movements and evolutionary history of populations, and can help in monitoring and managing stocks of depleted species (see Nor’easter Spring/Summer 1995).

This new technology can also diagnose and control infectious diseases in commercially grown finfish. In research funded by Maine/New Hampshire Sea Grant, John T. Singer and Bruce L. Nicholson, University of Maine, Orono, biochemistry, microbiology, and molecular biology professors, have developed PCR assays to identify several fish pathogens, including infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia (VHSV), and other diseases that attack farmed fish such as salmon.

Because viral diseases can spread rapidly through aquaculture facilities, cause high mortalities, and be difficult to eradicate, they can cause huge economic loss. Working with industry collaborators, Singer and Nicholson developed one of the first PCR assays to detect a fish pathogen, and the first one effective on birnaviruses, a group of aquatic viruses that share the unusual characteristic of double-stranded RNA. The team has been able to use IPNV viral proteins in vaccines, introducing them by means of Vibrio anguillarum, a marine fish pathogen. The genetically engineered bacterium then serves double duty as a vaccine for both Vibrio and birnaviruses (see Fall/Winter 1992 Nor’easter).

Dinner Table to Drug Counter

We need look no further than the dinner table to see how biotechnology can improve our lives. Ensuring the safety and quality of seafood is the goal of Garth Rand, University of Rhode Island (URI) food sciences professor. In a recent Rhode Island Sea Grant project, Rand developed two biosensors that use very different methods to detect viral fish pathogens. The first is a piezoelectric sensor, which uses a vibrating quartz crystal, similar to those in wristwatches. The crystal is coated with antibody molecules that bind with a specific pathogen, Vibrio parahaemolyticus. When an extract of shellfish tissue is put in contact with the sensor’s probe, any Vibrio pathogen cells present will bind chemically, increasing the weight on the crystal and reducing the vibration, and alerting the tester that the fish is contaminated. This test can be used on several seafood pathogens and greatly speeds up the testing process before fish can reach the market. With a hand-held instrument and a computer hookup, tests that once took 24 to 48 hours can now be completed in 10 to 15 minutes.

The second biosensor, developed by Rand with Steve Letcher, URI physics professor, operates by means of laser beams travelling in a fiber optic cable coated with antibodies. This biosensor can quickly detect Salmonella, a food-poisoning organism, and other pathogens at low concentrations in 30 to 60 minutes.

In the Woods Hole Oceanographic Institution (WHOI) biology department, Senior Scientist John Stegeman and Associate Scientist Mark Hahn have been using "biomarkers" in several Sea Grant research pro-jects. Biomarkers, such as certain enzymes or metabolic products, can be used to determine how susceptible marine mammals and seabirds are to pollutants such as dioxins, furans, and aromated hydrocarbons. Such contaminants accumulate in the fatty tissue of animals and have severe health impacts. Animals near the top of the food chain, such as seals, whales, and predatory seabirds, have the highest accumulations of toxic substances in their tissues.

Biomarkers can also be used to monitor contaminated environments, either with animals captured at the sites or in cages in situ. Certain genes can be used as biomarkers to indicate the level of exposure of animals to contaminants. Keeping records of the biomarker responses from animals in various locations can map contaminant distribution, on the small scale of a pollution site, or on a larger, even global scale. Biomarkers can also be used to monitor remedial efforts at contaminated sites (see Nor’easter Spring/Summer 1994).

In the past, Hahn says, determining the susceptibility of animals to toxic substances involved direct testing of the affected animals, a practice that raises ethical concerns and endangered species issues. However, now scientists can take a small tissue sample, for example, from a stranded animal, clone the gene for the hydrocarbon receptor, make the protein in a test tube, and study its characteristics.

In other WHOI Sea Grant projects, Hahn and colleagues are establishing a molecular basis for explaining and monitoring the effects of chemicals on the endocrine systems and development of fish. Some chemicals, called endocrine disruptors, have been found to alter reproduction and reduce immune system function in wildlife populations. The researchers are looking at a Superfund site in southeastern Massachusetts, where decades of industrial polychlorinated biphenyls (PCBs) contaminated New Bedford Harbor sediments. These studies will help determine the relationship between such pollutants and the health of sensitive marine birds, fish, and mammals. The scientists use biomarkers and DNA amplification of small tissue samples to perform nondestructive bioassays to assess the problem. Terns, beluga and pilot whales, and salt marsh minnows are among the species these researchers are studying by cloning and sequencing genes that metabolize and activate or inactivate chemical compounds.

Chemical biology and pharmacology represent another key frontier in biotechnology, and one that is making humans healthier. Jon Clardy, Cornell University’s Horice White chemistry and chemical biology professor, and a team of organic chemists and X-ray crystallographers have been investigating potential pharmaceutical agents from the sea, with support from New York Sea Grant. The goals of the research are to characterize new compounds that may be useful therapeutic agents, especially as antitumor or immunosuppressive agents; identify the physiological protein target of these active compounds; and determine the three-dimensional structure of the compound bound to its target. Analysis of the three-dimensional structure provides clues about how the compound works on the molecular level. This information can facilitate the design of improved drugs with increased potency and fewer side effects.

Among the compounds under study in the Clardy lab is Didemnin B, a peptide isolated from a Caribbean tunicate. It was the first natural marine product to enter clinical trials as an antitumor agent, investigated for the treatment of breast, ovarian, cervical, myeloma, and lung cancers. It may also prove useful for treating autoimmune diseases or for preventing organ transplant rejection.

But it’s not easy to apply the new discoveries. "While thousands of compounds from marine organisms have been shown to have biochemical properties that may be useful for pharmaceutical products or processes, very few have ever made it to the marketplace because large-scale culture methods still need to be developed," says Yuzuru Shimizu, URI biomedical studies and pharmacognosy professor. Shimizu and colleagues carried out a Rhode Island Sea Grant project to culture vast quantities of a strain of a photosynthetic marine dinoflagellate, Amphidium operculatum. They designed a bioreactor to mass-propagate this planktonic organism, which produces a metabolic compound that has been shown to be highly effective in treating colon and eye cancers. In fact, the compound, Q10, was 20 times more effective in tests on a liver tumor than the medical treatments currently available.

As Clardy, Shimizu, and others continue to discover new pharmaceutical compounds and make it more practical to use marine organisms with antitumor and antibacterial properties, the biologists’ toolbox will rapidly expand and affect every aspect of our lives. Biotechnology offers the opportunity to better conserve our treasure trove of ocean resources, and with the growth of applications, we can look forward to improvements in food supply and safety, health and medicine, aquaculture methodology, bioremediation, and wildlife management as the next millennium begins.