8 Marine Biotechnology

1. 8 Marine Biotechnology Brief Chapter Outline I. Aquaculture A. Gastropod, Bivalve, and Crustacean Production II. Marine Animal Health III. Algal Products IV. Fuels from Algae V. Algal Cell Culture VI. Medical Applications A. Marine Natural Products and Their Medical Potential B. Anticancer and Antiviral Compounds C. Antibacterial Agents D. Marine Toxins VII. Probing the Marine Environment VIII. Conservation IX. Terrestrial Agriculture X. Transgenic Fish A. Biotech Revolution: Fluorescent Fish Pets B. Cause for Concern? The Commercialization and Release of Transgenic Fish

2. Introduction. A. Marine biotechnology is defined as any technique that uses living marine organisms (or parts of these organisms) to make or modify products, to improve plants or animals, or to develop microorganisms for specific uses. B. The marine environment covers almost 71% of the Earth’s surface and contains 97% of the Earth’s available water. C. Oceans are where the first forms of life originated over four billion years ago, and they play important roles in maintaining the Earth’s ecosystems. They are also a large source of food for many other organisms, including humans. D. There is still much that is not known about marine environments and marine organisms, because many marine environments are very difficult to explore.

3. E. Biotechnology has focused on five major areas in marine research: 1. Identifying bioactive compounds produced by marine organisms and elucidation of their function and mode of action. 2. Increasing our understanding of the environmental factors, nutritional requirements, and genetic factors that control the production of primary and secondary metabolites in marine organisms. 3. Understanding the genetics, biochemistry, physiology, and ecology of marine organisms. 4. Developing tools and diagnostics for the improvement of health, reproduction, growth, and cultivation of marine and freshwater organisms. 5. Developing bioremediation tools for better waste processing and disposal, the clean-up of coastal areas, and the remediation of oil spills.

4. II. Aquaculture. • Introduction. 1. Many regions of the world have reduced fish and shellfish stocks, with some areas closed to commercial fishing. People are looking to aquaculture to maintain populations of endangered marine organisms (called “mariculture”) in fresh and salt water. 2. Many countries practice aquaculture, using low input technologies to propagate, harvest, and market fish, algae, crustaceans, and mollusks. 3. Because aquaculture can be harmful to the environment by pollution, biotechnology may play a role in solving some potential environmental problems. 4. Primary product of aquaculture is food, but it also produces food supplements, natural products, medicinal compounds, jewelry, and ornamental fish. 5. By the end of the 21st century, it is believed that aquaculture products will contribute for 20–25% of the world’s fisheries production by weight. 6. Improved methods can lead to, not only increased productivity, but also by-products of aquaculture and agriculture can be used as feed or energy sources.

5. Gastropod, Bivalve, and Crustacean Production. 1. Organisms such as crabs, oysters, mussels, shrimp, and lobsters. 2. Many methods exist for raising these organisms, ranging from using tanks to floating platforms (Figure 8.1). 3. For example, faster growth, maturation, disease resistance, and triploidy can be created by genetic manipulation of oysters: a) Diploid chromosome number is normal in oysters. • Triploid oysters are generated by treating eggs with • cytochalasin B. • c) Cytochalasin B doubles the number of chromosomes so that fertilization causes three sets of chromosomes inside the egg. • d) These oysters are sterile and do not form reproductive organs; they grow larger and faster than normal oysters, and are also more flavorful and meatier. e) An alternative procedure could be mating tetraploid oysters with diploid oysters to generate the triploids, due to safety concerns about cytochalasin B. • 4. Bivalves, such as oysters and gastropods, are cultured by manipulating the reproductive cycle by using chemicals such as hydrogen peroxide and aminobutyric acid (GABA). • 5. Genes are also being identified and cloned for the production of more controlled culturing of abalone and other shellfish.

7. Marine Animal Health. A. Marine animals are susceptible to protozoans, bacteria, viruses, and fungi. Pollution also promotes pathogen growth and hampers marine organism growth. B. Biotechnology may provide early disease detection, development and application of new antibiotics and vaccines, and the development of pathogen-free animals. C. Bacterial Disease: 1. Using antibacterial agents such as disinfectants and antibiotics in ponds or pens. 2. Can lead to antibiotic resistance or humans consuming antibiotics in animals.

8. Viral Disease: 1. Infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) are two of the most deadly viral diseases in salmon. 2. Infected animals become carriers and must be destroyed, and sometimes the whole habitat must be decontaminated. 3. Fish vaccines will greatly aid the fish industry, but very few are available. 4. Fish are not easily vaccinated; it is usually done by injection or by immersing them in water with the vaccine. Using ultrasound can facilitate vaccine entry into fish. 5. A recombinant vaccine against IHN is currently in testing. The vaccine is produced by isolating and expressing genes encoding viral proteins. 6. Isolating and characterizing marine metabolites may also aid in treatments.

9. Algal Products. A. Algae are a very diverse group of photosynthetic eukaryotic organisms that fall into three major groups: 1. Chlorophyta (Green). 2. Rhodophyta (Red). 3. Phaeophyta (Brown). B. In brown seaweeds such as kelp, alginates are the main structural component of the cell wall • and intercellular matrix, which is the space between cells. They are used as food thickeners and stabilizers (Table 8.2).

11. C. Culturing algae: 1. The red algae Porphyra (nori) (Figure 8.3) has been a food source in Japan for over 400 years. It is cultured by collecting reproductive spores and allowing them to grow on horizontal nets. The algae is harvested, dried, and processed. 2. The algae Undaria(wakame) (Figure 8.4) and Laminaria(kombu) are grown in Japan and China by seeding ropes with spores, placing the ropes in tanks, and allowing the algae to grow, before they are harvested and dried. They are used in noodles, soups, salads, and meats.

14. Alginic acids (alginates) and phycocolloid polysaccharides (agar, carrageenans): • Commercially produced in the early twentieth century. • 2. Used in food, industrial products, fertilizer, and energy production. 3. Carrageenan is used as an extender in foods such as evaporated milk and ice cream. 4. Agar is used mostly in foods, but is also used in pharmaceuticals, in capsules, and in the laboratory, to make gels for techniques such as gel electrophoresis. 5. The demand for these products is much more than the amount of algae available, so genetic engineering might improve strains and decrease the cost of products.

15. 6. Algal cell culture is being examined as a way to increase the yields of products such as agar: a) Macroalgae (seaweed): (1) Can be cultured by producing protoplast and callus tissue from which algae can be regenerated. (2) Cell and tissue culture can be used to select for new genes or traits. (3) Protoplast fusion allows for traits from two organisms to be mixed. b) Microalgae (green algae and cyanobacteria): (1) Mostly as food, but also used as pigment sources such as β-carotene. (2) Algae such as Spirulina and Chlorella are of much nutritional value (Tables 8.4 and 8.5), and have been consumed for nearly 500 years in places such as Mexico and Africa. Spirulinais marketed today as dried flakes that are used in fish food and Japanese food. (3) Mass culturing, such as the spiral ponds that Spirulinais grown in, allows large quantities to be grown and harvested in outdoor ponds, possibly lowering production costs of products (Figure 8.6). (4) Phycobiliproteins are pigments involved in algal photosynthesis, and can be used as phycofluors, which can label biological molecules. (5) Algae may be an inexpensive way to harvest proteins in developing countries where farmland is scarce.

19. 7. Modern algal biotechnology involves genetic manipulation to produce algae that grow faster, are resistant to disease, make more of an important metabolite, and produce new products. 8. Algae have potential uses in pharmaceuticals, as fertilizers, as energy sources, and as cell cultures that can produce new chemicals. 9. Many chemicals have been isolated from algae, but the cost of research on these chemicals is too costly because of the costs of screening efforts, difficulties in collecting products, and the small amounts that products are produced in.

20. V. Fuels From Algae. A. Continued use and extraction of fossil fuels such as petroleum and coal pose environmental hazards such as the increase of gases, such as sulfur dioxide, in the atmosphere. Fossils fuels are also a finite energy source. B. Alternatives to fossil fuels may be photosynthetically generated biomass: 1. Renewable and no damage to the environment. Unfortunately, biomass is not economically competitive with current sources of energy. 2. Biomass can be converted by bacteria to fuels such as methane. The bacteria can be obtained from agricultural wastes, estuaries, and high salt environments. 3. Biological gas production is not competitive with other natural gas sources. 4. Dunaliellais an alga that can produce glycerol, which can be converted by bacteria to chemicals such as ethanol and butanol, which can be used as fuels. 5. Algae can produce large amounts of hydrocarbons from fatty acids that can be converted to or used directly as liquid fuels. 6. Algae may also be genetically modified to make gasoline-type fuels, since brown algae and cyanobacteria already make small amounts from fatty acids. 7. Biotechnology may make biomass more viable by enhancing photosynthesis to produce more of a fuel, or modifying biomass to favor fuel production.

21. VI. Algal Cell Culture. A. Used to generate biomass from which cells and metabolites can be obtained. B. Occurs in large ponds or raceways (Figure 8.7), although cultured cells can be used in bioreactors or fermenters, allowing for the controlled culturing of algae. C. Microalgae are usually used in culture, but macroalgae also produce agar and agarose (a polysaccharide used in electrophoresis gels). D. Cell culture technology may contribute to increased agar production. E. Specialty chemicals such as proline can be produced by algae such as Chlorella, along with hydrocarbons, polysaccharides, and rare amino acids like octopamine. F. Cell culture is a high-cost operation, but the products can be very profitable. For example, amino acids yield from $5 to $100 per kilogram produced.

23. VII. Medical Applications. A. Marine Natural Products and Their Medical Potential 1. Marine organisms produce many metabolites that have medical potential because of their unique and sometimes unusual chemical structures. 2. Secondary metabolites are more attractive because the chemicals provide selective advantages to the organism currently or at some point in evolution. 3. Chemicals are isolated, catalogued, and studied for pharmaceutical potential or potential to control insect pests. 4. New chemicals can be isolated and used to generate new chemicals that have potential value. 5. About 20,000 chemicals have been isolated from the world’s organisms, such as bacteria, sponges, corals, and jellyfish. 6. Products from marine organisms such as sponges, sea stars, and starfish may show asimilar significant and untapped promise to plant products, which have only been studied in recent years.

24. Anticancer and Antiviral Compounds. 1. Many marine compounds are toxic to humans, but diluting them sometimes exposes their therapeutic value. 2. Many compounds extracted show anticancer properties: a) Didemnin B is effective against leukemia and melanoma in mice, and is also an effective immunosuppressive agent that could be used in organ transplants to prevent organ rejection. b) Dolastatins are effective against leukemia and melanoma because they inhibit cell division, and may be similar to the anticancer drug vinblastine, which is isolated from the Madagascar periwinkle.

25. 3. Sponges are also a source of natural products: a) Alkaloids, terpenoids, and sterols are examples. b) An example is manoalide, which is derived from the marine sponge Luffariella variablis, which shows anti-inflammatory, analgesic, and antifungal activities. c) Nudibranchs (mollusks that are like snails without a shell) extract compounds from prey and use them for protection. One chemical called “the ulapualides,” protects eggs against eating, and it is also effective against leukemia and fungi. d) Compounds may have antiviral activity, such as eudistomins derived from tunicates, and bryostatins, which inhibit RNA synthesis and stimulate immune reactions such as activating leukocytes and interleukin-2 production. 4. The amount of any natural product is limited by the number of organisms that produce it. Harvesting organisms might not only provide for chemical production but alsomay be useful for maintaining populations of marine organisms.

26. C. Antibacterial Agents. 1. Many marine organisms produce secondary metabolites with antibiotic properties. However, the costs of isolation and screening chemicals do not allow for the extensive characterization of potential antibiotic compounds. 2. For example, squalamine has been isolated from the dogfish and {has activity} against a wide variety of fungi, bacteria, and protozoans. Is this correct?

27. D. Marine Toxins. 1. Toxins are believed to be responsible in predation, defense from predators and pathogenic organisms, and signal transduction in their nervous systems. 2. Toxins such as saxitoxin are produced by cyanobacteria and can be incorporated into shellfish, which can cause paralytic shellfish poisoning. 3. Studied for antitumor, anticancer, and antiviral properties when their concentrations are controlled. They are also useful in neurophysiology and neuropharmacology research, and could be used as models for new compounds. 4. Neurotoxins such as tetrodotoxin (produced by the puffer fish) can be studied because they have specific targets such as receptors or ion channels, and can be useful in studying neuron function. • 5. Other examples of toxins in research include conotoxins from the predatory cone snail, lophotoxinfrom the gorgonian Lophogorgia, and dysidenin from sponges.

28. Probing the Marine Environment. A. Biotechnology has contributed to marine research in the following ways: 1. DNA probes and PCR can be used to identify new organisms without having to capture the organisms. 2. Identifying the country of origin of important fish populations. 3. Tracking larval-stage organisms and microbes in the environment. 4. Identifying currently unknown intermediate life stages of marine organisms. 5. Microplankton (the basis of the marine food chain) can de identified and tagged so that ecological processes such as primary production can be studied. 6. Microbes that cause disease in polluted areas can be identified.

29. Conservation. A. Scientists can use the DNA of animals to determine if protected animals are being used for food, jewelry, or traditional medicines and potions. For example, DNA methods identified unlabeled meat from humpback and fin whales. B. PCR is a powerful technique that can be used wherever electricity is available, and can test for the use of illegal meat in markets and restaurants.

30. Terrestrial Agriculture. • A. Marine organisms can benefit terrestrial (land-based) agriculture in several ways: 1. Cyanobacteria in the form of dried flakes can be used as fertilizer. 2. Genes encoding tolerance to low temperatures could be transferred to plants to possibly allow plants to express these traits and survive at lower temperatures. 3. Genes from salt-tolerant organisms could be transferred to plants to allow them to grow in high- salt environments.

31. Transgenic Fish. A. Transgenic techniques are used to introduce desirable traits into fish, primarily to allow them to increase the growth and weight of cultured finfish. B. Fish have large and transparent eggs, allowing it to be easy for gene transfer. C. The methods available to create transgenic fish include electroporation, microinjection into the egg, and injection through the micropyle, which is the space in the egg where sperm enter. D. Most fish eggs are injected within an hour after fertilization, because they are released from the female and the first division of the egg occurs one hour after fertilization. E. Salmonoid eggs are more difficult to inject because they have a hard outer covering called the “chorion” (Figure 8.10). F. Survival rates for microinjected fish embryos are much higher (35%–80%) than mammals, with 10%–70% of the fish being transgenic.

34. G. Transgenic fish may be able to resist freezing temperatures by producing antifreeze proteins isolated from winter flounder, which survive in subzero temperatures. H. Most transgenic fish studies have focused on the effects of growth hormone genes: 1. Aims to modify fish to allow them to grow faster, and larger. 2. Studies have been done on catfish and salmon. 3. Male fish have been studied because they grow quicker and larger than females. I. Transgenic fish may some day be engineered to be disease-resistant. J. Environmental concerns over transgenic fish include: 1. Fish may not remain isolated in commercial facilities. 2. Fish may alter trophic food webs, disrupting native fish populations. 3. May displace wild counterparts if placed in new environments. 4. Biological and physical controls, like isolation and the elimination of reproductive capabilities, are needed to prevent release of transgenic fish into the environment.

35. K. Biotech Revolution: Fluorescent Fish Pets. 1. In 2001, genetically engineered fish were created at the National Taiwan University as a research tool. 2. The fish contain a gene that encodes for a fluorescent protein that allows the fish to glow bright green when a black light shines on them. 3. Other colors have been developed, such as fluorescent red and a half-green, half-red fish. 4. The fish were first marketed in Taiwan by the Taikong Corporation, and then they were marketed in the Untied States, although Europe has been resistant to them.

36. L. Cause for Concern? The Commercialization and Release of Transgenic Fish. 1. Programs and policies must be in place to help educate the public and ensure the safety of transgenic fish products. 2. Environmental impact studies must be done to help reduce fears, assess risks, and ensure the safety of fish that may be released into the environment. 3. Nonengineered fish must also remain isolated in aquaculture facilities to keep them from release into new habitats where they may threaten current ecosystems.