5

CHEMICAL ECOLOGY

Scientific Questions

As in the terrestrial environment, the complex interactions of many marine plants and animals are mediated by chemical interactions. These chemical signals are important for maintaining stable community structures by regulating predator-prey interactions, establishing territories, and assisting in reproductive strategies. For example, predators find their prey, and potential prey avoid their predators, in part by detecting water-borne chemical cues; organisms avoid predation by generating distasteful or even toxic compounds; individuals find mates of their species by following trace odors through the sea; and swimming larvae of bottom-living animals are recruited to adult populations or find habitats where they can survive as adults by responding to specific chemical substances. In addition, many marine plants and animals have evolved solutions to unique problems in the sea, such as production of special skeletal materials and glues. For each of the phenomena cited here, interesting and unique questions arise: What is the chemical nature of the products involved? How are they produced and disseminated? How are they detected? Such compounds are potentially useful to humans.

In the ocean, predator-prey dynamics have been shown to be mediated by chemical effects such as attraction and deterrence (Rittschof and Bonaventura, 1986; Faulkner et al., 1990; Rittschof, 1990; Paul, 1992). Chemicals appear to be important, for example, in limiting food selection by zooplankters, placing in question the concept that measurements of chlorophyll alone adequately assess food availability and total productivity. Chlorophyll is an excellent measure of total primary producers; however, because all phytoplankton are not grazed upon equally, this measure may not be as useful as previously assumed until the relative food distribution of the species to zooplankton has been determined. Copepods



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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS 5 CHEMICAL ECOLOGY Scientific Questions As in the terrestrial environment, the complex interactions of many marine plants and animals are mediated by chemical interactions. These chemical signals are important for maintaining stable community structures by regulating predator-prey interactions, establishing territories, and assisting in reproductive strategies. For example, predators find their prey, and potential prey avoid their predators, in part by detecting water-borne chemical cues; organisms avoid predation by generating distasteful or even toxic compounds; individuals find mates of their species by following trace odors through the sea; and swimming larvae of bottom-living animals are recruited to adult populations or find habitats where they can survive as adults by responding to specific chemical substances. In addition, many marine plants and animals have evolved solutions to unique problems in the sea, such as production of special skeletal materials and glues. For each of the phenomena cited here, interesting and unique questions arise: What is the chemical nature of the products involved? How are they produced and disseminated? How are they detected? Such compounds are potentially useful to humans. In the ocean, predator-prey dynamics have been shown to be mediated by chemical effects such as attraction and deterrence (Rittschof and Bonaventura, 1986; Faulkner et al., 1990; Rittschof, 1990; Paul, 1992). Chemicals appear to be important, for example, in limiting food selection by zooplankters, placing in question the concept that measurements of chlorophyll alone adequately assess food availability and total productivity. Chlorophyll is an excellent measure of total primary producers; however, because all phytoplankton are not grazed upon equally, this measure may not be as useful as previously assumed until the relative food distribution of the species to zooplankton has been determined. Copepods

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS and bivalve mollusks select phytoplankton on the basis of its quality and often reject phytoplankters that produce toxins and other deterrent chemicals (Ward and Targett, 1989). Despite their apparent inability to keep from releasing chemical clues to their whereabouts, many marine organisms use chemicals to avoid predation. For example, accumulating evidence indicates that the ink clouds of squids, octopuses, and sea hares probably do much more than confuse potential predators visually. In schooling squid they may chemically signal other members of the school of the need for flight (Gilly and Lucero, 1992), and in sea hares there are probably components that dull the senses of potential predators (Carefoot, 1987). Sea slugs, under attack by a predator, secrete specific compounds into their mucous trails that, when encountered by another slug, cause it to move rapidly away; such substances have been dubbed “alarm pheromones.” Terrestrial examples of such compounds exist. Foul-tasting or toxic substances are known to be harmlessly ingested from plant sources by caterpillars of many butterfly species; their presence in the tissue of the caterpillars and butterflies serves as a deterrent to predatory birds. Similar events occur in the sea; sea hares stockpile toxic products from their algal foods and remain relatively predator-free as an apparent result (reviewed by Carefoot, 1987). On coral reefs, plants and animals that produce chemical deterrents and toxins are often less preyed upon. Soft-bodied invertebrates like sponges, ascidians, and soft corals produce large quantities of organic substances that provide elaborate chemical defenses (Paul, 1992). Recent studies demonstrate that even the minute larvae of a number of invertebrates are provided with chemical defenses from the mother's eggs. Many sessile colonial invertebrates (sponges, ascidians, bryozoans, corals, soft corals) typically form colonies that enlarge by outward growth around the edges. As space is nearly always limiting in the habitats occupied by such organisms, they are in almost constant competition to gain new space and simultaneously keep from being overgrown by other colonies of their own or other species. Most encounters are carried out slowly by chemical means; in many such interactions, an unoccupied band occurs between interacting colonies where release of a chemical by one species leads to the retreat of another. In corals and ascidians it has been established that a colony can distinguish among other colonies those that were derived from the same sexually produced larva as itself and those that were not (reviewed by Rinkevich and Weissman, 1987; Grosberg, 1988). That is, they recognize “self ” from “nonself.” Founding and maintaining populations of most bottom-living marine invertebrates depend on chemical cues released by one member of a species and detected by another. Many, if not most, mobile marine animals find their mates by following water-borne pheromones to their sources (e.g., Karplus, 1981; Hadfield

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS and Switzer-Dunlop, 1984; Zimmer-Faust et al., 1985). Spawning is often stimulated across populations by chemical means (Giese and Pearse, 1974-1987). Sperm find, recognize, and fuse with eggs as a function of chemical recognition (Miller, 1985). Free-swimming larvae use chemical cues from adults of their species to choose sites for settlement and metamorphosis (Crisp, 1974; Castro, 1978; Morse and Morse, 1984). It has been discovered for many marine invertebrates that continued recruitment of new settling larvae to an area depends on the presence of adults (Tegner and Dayton, 1977; Prince et al., 1988); recruitment potential is thus not just a function of larval availability, with important consequences for harvesting practices. Larvae of other species metamorphose in response to chemical cues from the habitat itself, including the specific plant or animal upon which it must feed after metamorphosis, or metabolites from microorganisms associated with the habitat (Morse and Morse, 1984; Hadfield, 1986; Hadfield and Pennington, 1990; Jensen and Morse, 1990; Pawlik, 1992). Many marine organisms exist only in complex relationships with other organisms in various forms of symbiosis. Tropical corals and giant clams must harbor specific single-celled algae to exist; shipworms, actually a kind of clam, can feed on wood only when their digestive systems harbor specific protozoa containing specific cellulose-degrading bacterial symbionts; anemone fish live only among the tentacles of anemones; and parasites can survive only on or in specific host species. Many of these symbiotic relationships rely on the establishment of chemical cues in each new generation. While there is evidence that specific chemicals are involved in the ecological interactions cited above, for only a small fraction are the chemicals themselves actually known, much less the mechanisms of their biological synthesis and detection. Chemical disruption of large areas of the intertidal or sea bottom by pollutants, oil spills, or even detergents used to clean up chemical spills can thus have grave and far-reaching consequences if they destroy, alter, or overwhelm the chemical signals upon which communities rely. Because very little is currently understood about such disturbances, an effort should be made to understand the impact of such disturbances on individuals, populations, species, and communities. Unique conditions of aquatic realms have led to the development of unique solutions to such problems as maintenance of form and adhesion. Certain marine algae produce vast quantities of complex molecules with elastic properties to serve structural functions. Barnacles, mussels, and other marine organisms have evolved special glues that rapidly set in water. Interesting questions concern the molecular and cellular bases for the production of such chemical forms, although their usefulness in many applications has already been recognized (e.g., Rzepecki et al., 1991) (Table 5-1).

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS Table 5-1. Purification of Polyphenolic Protein from Mytilus edulis from Rzepecki et al. (1991). STEP TOTAL PROTEIN, mg TOTAL DOPA/ µmol DOPA/ PROTEIN, µmol/mg YIELD, % FOLD PURIFICATION From 32 g of feet: S-1, perchloric acid 210.0 71.3 0.34 100 1 S-3, acetone precipitate 66.7 41.6 0.62 58 1.8 Sephadex G-200(a) 29.4 30.5 1.04 43 3.1 HPLC 13.1 10.2 0.79 15 2.3(b) (a) Gel filtration was omitted for most mussel proteins. (b) The specific activity of DOPA proteins decreases with time and manipulation (such as lyophilization) as a result of oxidation, but for M. edulis both gel filtration and HPLC chromatography were necessary for electrophoretic homogeneity. The deceptively low numbers for low purification result from the high relative proportion of polyphenolic protein in the initial extract. Techniques to Address the Scientific Questions The major questions in chemical ecology will yield answers more rapidly by the application of most of the methods of molecular biology described in Chapter 2. Specifically, isozyme techniques, as well as DNA sequence analysis, will be important in clarifying species identity questions, especially those involving specific interactions, be they predator-prey, symbiotic, or intraspecific. In some of the complex interactions involved in symbiosis, DNA sequence data will be necessary to determine which of a symbiotic pair is responsible for the production of compounds of interest. This can be accomplished by cloning and sequencing one or more of the genes involved in the synthesis of the compound or using other molecular probes when the symbiont can be cultured separate from its host. In situ hybridization methods will also greatly aid in such clarification, especially where microorganisms residing within larger multicellular organisms are suspected of being the source of compounds of interest. Because many pheromones are peptides, cloning and sequencing will be important tools in locating the genes responsible for the synthesis of potentially useful pheromones. Peptides can be readily employed to generate specific

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS antibodies, and they, in turn, can be used to locate both a source organism and a specific site in the organism where the peptide is generated. This method will thus also be useful in determining whether a host or an endosymbiont is the source of a peptide. Eventually, recombinant DNA methods may be useful in splicing desired genes into microorganisms that can be readily mass generated. Practical Implications “Drugs from the Sea” and Agrichemicals Over the past 30 years, a strong commitment has been made to utilize nature and its vast content of natural resources in the development of new drugs and agrichemicals. While terrestrial plants and microorganisms have been the traditional sources for well over half of today's medications, the potential of marine species is only now being recognized. Because most marine plants and animals are taxonomically and genetically distinct from those on land (Wesselds and Hopson, 1988), they often produce unique chemical compounds that provide the foundation for the next several decades of biomedical and agrichemical research. Recent studies of marine invertebrates have shown that they contain substances, many of which are defensive compounds, with significant potential for treatment of many human diseases. Marine invertebrate animals have already yielded substances that appear promising for the treatment of cancer (de Silva and Scheuer, 1980; Rinehart et al., 1981; Look et al., 1986). Sponges and soft corals also produce biomedically important compounds, some of them showing significant antiinflammatory capabilities (Powers, 1990). Extracts of a Pacific sponge contain manoalide, a compound that reduces inflammation in both arthritis and asthma by specifically inhibiting the major enzyme involved in initiation of the inflammatory process. Pseudopterosins, compounds from a Caribbean soft coral, are also potent antiinflammatory agents. Although it is expected that herbicidal and insecticidal activity will be discovered among the many marine defensive compounds, few studies have been initiated to explore the possibilities. In one case, however, the chemically defended soft coral (Briareum polyanthes) has been found to contain compounds that deter feeding by grasshoppers. Compounds used in chemical communication by marine organisms form a potentially rich source of repellents to insects and other invertebrate pests (e.g., nematodes and slugs) of commercially important plants. The production of toxic compounds by marine organisms can have detrimental effects both on commercially important species and on humans. The

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS frequency of occurrence of “red tides” and other catastrophic toxic events in the sea appears to be increasing, causing growing concern in both environmental and public health contexts (Ahmed, 1991, and references therein). Human illness and death associated with toxic algal blooms and the consumption of contaminated shellfish include paralytic, neurotoxic, amnesic, and diarrhetic shellfish poisoning. These illnesses are caused by biotoxins produced by marine phytoplankton. In addition to humans, whales, dolphins, and seabirds that eat contaminated fish can become victims of the toxins, which are concentrated as they are passed up the food chain from phytoplankton to zooplankton to fish and ultimately to the final consumer. Consumption of contaminated fish can result in paralysis and even death. The causes and source organisms of toxic outbreaks are poorly known and almost totally unpredictable. Present monitoring techniques rely on measurements of toxins in the food product. It would be beneficial to predict future higher-level outbreaks by monitoring the abundance and distribution of the microorganisms responsible. Recent evidence reveals that many marine toxins are produced by microorganisms, including microalgae and bacteria, the distribution and population dynamics of which are almost totally unknown. Given the increasing frequency of poisoning from seafood and the growing dependence on seafood in the United States (Ahmed, 1991; Powers, 1990), it is imperative that we attain a full comprehension of the ecological and biochemical roles of marine microbial toxins and their sources. Recently, molecular methods have been developed to discriminate between toxic and nontoxic Pseudonitzachia species (Scholin et al., in press) that cause domoic acid accumulation. Such methods will allow reliable identification of microorganisms in environmental samples, thus potentially allowing problems to be recognized before they reach catastrophic levels. Marine microorganisms also represent a vast and rich biomedical resource that remains virtually untapped (Fenical and Jensen, 1992). Minute free-living algae, bacteria, and fungi can be found in all parts of the ocean, and many more exist as symbionts on the surface and in the tissues of marine plants and animals. They represent a resource at least as diverse and prolific as the microorganisms from soil that form the basis of the production of most antibiotics. A few recent studies have already demonstrated the potential that exists: an unidentified deep-sea bacterium was shown to produce a new chemical class of compounds that inhibited tumor cell proliferation and replication of human immunodeficiency virus, the causative agent in AIDS (Gustafson et al., 1989). Several marine fungi have been studied and found to produce antibiotics and neuroactive substances. These few fundamental discoveries demonstrate the pharmacological resource potential of the microbial marine environment. Several impediments to progress in the area arise from our rudimentary understanding of marine microbial identities and ecology. In molecular biology lies the major hope for solving these problems and reaping the rewards to be gained from studying the biology and biochemistry of marine microorganisms.

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS Across the entire taxonomic spectrum, marine organisms produce compounds that are useful in a variety of human applications. They are sources of amino acids, vitamins, lipids, fats, waxes, sugars, polysaccharides, and pigments, as illustrated by the following examples. Chitin Chitin, a complex nitrogenous polysaccharide, is one of the most abundant biopolymers in the world, and in the sea it makes up a large portion of the exoskeleton of many marine invertebrates, especially crustaceans. Chitin has an untapped commercial potential because it can be used for a large variety of industrial applications, including adhesives, chelating agents, paper and textile additives, and structural matrices, and its potential for promoting wound healing and other biomedical applications is enormous (Muzzarelli et al., 1986). Complex Polysaccharides Complex polysaccharides from marine algae, especially agar, carrageenans, and alginates, already form the basis of a $300 million per year industry for their uses as emulsifiers, stabilizers, and thickeners and for their biomedical applications. As the demand for these products grows and the natural supply declines, the necessity for genetically engineered, high-quality, fast-growing algae will become apparent, and the current U.S. dependence on foreign sources will become less tolerable. Already the most up-to-date methods of molecular and cellular biology are being utilized to produce new, culturable strains of high-quality, agar-producing algae. There are also examples of ecologically and physiologically important plant pigments (phycobilins, carotenoids) of current and future commercial use in the pharmaceutical and food industries. Vitamins and Other Essential Nutrients A number of commonly cultured marine algae are excellent sources of a broad spectrum of vitamins and other essential nutrients, contributing to an industry whose sales now exceed $2 billion per year for human and animal uses. Vitamin B12 and pantothenic acid are commonly excreted by several species of blue-green algae, opening the possibility for continuous culture methods for producing these vitamins. Algae are already a commercial source of food additives, including β-carotene, glycerol, amino acids, alcohols, and other hydrocarbons (Chapter 3; Ben-Amotz and Arvon, 1990; Behrens and Delente, 1991; Bubrick, 1991). However, only about 60 of the estimated 22,000 to

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS 26,000 living algal species have been surveyed for their content of vitamins or other useful chemicals or bioactive metabolites. They clearly provide an untapped resource of biological diversity that merits extensive research. Adhesives Because they evolved in and still inhabit hostile environments where wave action requires a truly tight grip, many marine mussels, barnacles, and other invertebrates have incredibly strong glues that serve this purpose. In addition, these glues are secreted and “set” in a watery medium, a property lacking in most commercially available adhesives. One of these glues has been isolated and structurally characterized; it is a protein with unique structural modifications that allow it to harden underwater. Attempts are now being made to produce the protein with recombinant methods. This type of glue will be in high demand for a large spectrum of aqueous cement applications, including repair of broken bones and dental repairs. Microbial Products and Biochemical Processes The existence of bacteria living in very high temperature water at deep-sea hydrothermal vents opens the possibility of manipulating them for commercial purposes, especially isolating—and probably genetically engineering—their heat-resistant enzymes. The ability of these enzymes to withstand very high temperatures could greatly enhance chemical reaction rates in commercial applications, and promising results have already been obtained. In fact, a new form of the enzyme tac polymerase that makes it possible to do the PCR at high temperatures (discussed in Chapter 2) was cloned from a thermophilic bacterium isolated from a hydrothermal vent and is now commercially produced by Stratagene (La Jolla, Calif.). A number of hyperthermophilic bacteria have been isolated and cultured at temperatures between 90° and 110°C, and enzymes of several types have been isolated from them and found to be active and stable at elevated temperatures (Brown et al., 1990). Some of these enzymes have been patented for future commercial applications. The fact that these enzymes are stable at high temperatures makes them particularly useful for chemical processes that require reactions at or near 100°C. Some of the bacteria of hydrothermal vent communities have another potentially very useful attribute: the ability to oxidize hydrogen sulfide and use the energy to fix carbon dioxide into complex organic compounds. Virtually all other ecosystems on earth derive such energy from the sun via photosynthesis. Hydrothermal vent bacteria should be useful for converting industrial hydrogen

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS sulfide into less toxic products and for desulfurizing coal. Other bacteria in these hydrothermal vent communities reproduce extremely rapidly and produce methane (Brock, 1985), offering the potential for more efficient biomass conversion into methane than is obtained with low-temperature bacteria. Clearly, tremendous possibilities exist for the use of hydrothermal vent microorganisms in the chemical, energy, biomedical, and pollution treatment industries. This report highlights many important sources and types of chemical/biochemical substances to be found in studies of “chemical ecology.” Textbooks, reviews, and classical research papers abound with examples of chemical interactions in marine organisms that provide promise as opportunities for economic utilization (e.g., Attaway, 1988, 1989; Ahmed and Attaway, 1987; Ahmed, 1991; Attaway and Zaborsky, 1992; Colwell, 1983; Colwell and Zilinskas, 1993; Powers 1990; Zaborsky, 1993).

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