3

PHYSIOLOGICAL, BIOCHEMICAL, AND GENETIC STATUS OF MARINE ORGANISMS

Scientific Questions

There are profound consequences of the strong relationships among the physical, chemical, and biological processes in the ocean. The ocean varies over time and space due to natural factors and human activities. The variability is evident in the latitudinal and vertical changes in the spectral intensity of light; in the gradients of temperature, pressure, and salinity; in the distribution of suspended particulate matter and of a multitude of chemicals such as biologically important nutrients, carbon dioxide, oxygen, and trace gases; in the distribution and abundance of organisms; and in the effects of ocean hydrodynamic factors such as currents, eddies, fronts, storms, and turbidity currents.

Some environmental variability is periodic and is the pacemaker for daily and seasonal events such as animal migrations, phytoplankton blooms, and sexual reproduction. Long-term change, detected by the study of sedimentary fossils, can be episodic, as in the case of the Permian-Triassic extinctions, or periodic, as in the sequence of major mass extinctions during the Phanerozoic. The triggering mechanisms for mass extinctions are not fully known, but primary productivity under the stress of global change may be an important factor. The response of marine organisms that inhabit the upper ocean to an increased flux of ultraviolet radiation due to depletion of stratospheric ozone is only now beginning to be studied (e.g., Cullen et al., 1992).

Marine organisms respond to environmental cues by behavioral, physiological, immunological, humoral, and genetic mechanisms. Ultimately, such responses at the level of individual organisms aggregate to produce



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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS 3 PHYSIOLOGICAL, BIOCHEMICAL, AND GENETIC STATUS OF MARINE ORGANISMS Scientific Questions There are profound consequences of the strong relationships among the physical, chemical, and biological processes in the ocean. The ocean varies over time and space due to natural factors and human activities. The variability is evident in the latitudinal and vertical changes in the spectral intensity of light; in the gradients of temperature, pressure, and salinity; in the distribution of suspended particulate matter and of a multitude of chemicals such as biologically important nutrients, carbon dioxide, oxygen, and trace gases; in the distribution and abundance of organisms; and in the effects of ocean hydrodynamic factors such as currents, eddies, fronts, storms, and turbidity currents. Some environmental variability is periodic and is the pacemaker for daily and seasonal events such as animal migrations, phytoplankton blooms, and sexual reproduction. Long-term change, detected by the study of sedimentary fossils, can be episodic, as in the case of the Permian-Triassic extinctions, or periodic, as in the sequence of major mass extinctions during the Phanerozoic. The triggering mechanisms for mass extinctions are not fully known, but primary productivity under the stress of global change may be an important factor. The response of marine organisms that inhabit the upper ocean to an increased flux of ultraviolet radiation due to depletion of stratospheric ozone is only now beginning to be studied (e.g., Cullen et al., 1992). Marine organisms respond to environmental cues by behavioral, physiological, immunological, humoral, and genetic mechanisms. Ultimately, such responses at the level of individual organisms aggregate to produce

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS population-level effects, the focus of Chapter 2. Study of responses by individual organisms to environmental conditions, both biotic and abiotic, is a fundamental aspect of marine biology and is necessary for understanding how both natural variability and human activities (e.g., global climate change and pollution) affect marine ecosystems. Knowledge about the effect of ocean processes on the cycling of biologically important elements, such as carbon and nitrogen, is important for estimating the potential impact of global climate change on marine systems. For example, the ocean is both a sink and a source in the global carbon cycle, with processes such as burial of organic carbon (Lyle, 1988) and dissolution of carbonates in marine sediments (Archer and Maier-Reimer, 1994) affecting atmospheric carbon dioxide on time scales of months to thousands of years. To understand the carbon cycle, we must understand the biochemical and molecular mechanisms that regulate metabolism and food webs. Research on organismal responses to environmental conditions is focused on three questions: What is the physiological condition (e.g., nutritional, immunological, developmental, reproductive) of marine organisms? How do marine organisms respond to deviations in environmental factors (e.g., food, temperature, oxygen, dissolved nutrients, chemical contaminants, light, pressure, current velocity)? How do environmental factors regulate ocean processes (e.g., organic carbon burial, development of anoxic zones, nutrient regeneration, sea-air exchange of volatile compounds, primary productivity)? Responses of organisms to environmental parameters (including other organisms) are believed to account for much of the variability in population dynamics and community structure in marine food webs. Organisms can respond to deviations in environmental conditions through changes in gene expression and metabolism that yield changes in physiological state and performance. These responses may result from changes in the expression of relevant genes. The responsiveness of gene expression to environmental factors is well documented for many terrestrial organisms but for only a few marine organisms (Davidson, 1986, 1989; Koban et al., 1991; Sanders et al., 1992). For most marine organisms, including those of major ecological and economic importance, the influence of environmental factors on biochemistry, physiology, and gene expression is virtually unknown. Understanding the molecular details of these regulatory processes could allow the development of genetic and immunological probes and biochemical tests to quantify the physiological condition and health of marine organisms and their responses to environmental stress. It is important to characterize sublethal effects (even though the subject organism is alive and appears healthy by other measures) because even small organismal effects can sum to large effects on the population

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS or global levels. This type of information is important for global biogeochemical models that include information about the biosphere and its interactions with the physical environment. Patterns and mechanisms of gene expression may differ among taxa, so that studies of a broad range of marine organisms could demonstrate a variety of patterns of gene expression. For example, glycolytic pathways differ among marine invertebrates and between vertebrates and marine invertebrates. Although today's molecular methods can identify genetic variation (Chapter 2), assessment of physiological status and the diversity of physiological processes of individual organisms within these communities is not easily accomplished. It is desirable to be capable of both identifying the species present and predicting the response of each species to a number of environmental variables. Development of appropriate diagnostic tests will require considerable fundamental knowledge about the basic biology of the target organisms, such as a description of their life histories and knowledge of sensitive life stages, determination of whether their reproduction and development depends on natural environmental cues, and identification of stress manifestations in the biochemistry and genes of these organisms. In the future, molecular techniques could help resolve questions related to environments that place unusual stresses on the physiology of marine organisms, such as high temperature, high salinity, and high pressure. Also of interest are how organisms change metabolically as they proceed through different environments in different life history stages--for example, salmon living as juveniles in fresh water, maturing in the ocean, and returning to fresh water to spawn. Techniques to Address the Scientific Questions Several molecular techniques can be used to study the physiological condition of marine organisms in relation to environmental parameters. Many of the key ecological and physiological attributes of natural populations, such as rates of reproduction, recruitment, growth, energy flow, and the level of environmental stress to which organisms are exposed, are difficult to assess directly, especially when large numbers of individuals must be analyzed. Because the physiology of organisms reflects underlying biochemical and molecular activities, indices of such activities should, in principle, provide useful means to assess physiological state and permit rapid analysis of large numbers of samples. The development of biochemical and molecular indices of physiological state requires precise laboratory-based calibration, in which the environmental factor of

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS interest is varied and changes in biochemical and molecular properties are assessed quantitatively. When significant correlations are found between environmental variables and biochemical or molecular processes, useful indices can be developed. The use of nucleic acid concentrations and immunological techniques is described below; other methods (delineated in Chapter 2) are applicable for determining physiological status when applied to the appropriate biological function. Nucleic Acid Concentration: Some ecological studies, including growth rate measurements, have relied on measurement of RNA and DNA concentrations and their ratios. Fisheries biologists have shown that under certain circumstances the ratio of RNA (over 95 percent of which is ribosomal RNA) to DNA can provide a rough estimate of growth rate (Bulow, 1987) and is a useful indicator of nutritional status (Clemmesen, 1990; Ueberschaer and Clemmesen, 1990). However, this technique is useful only if proper controls and standards are employed, including restricting comparisons to single species, size classes, life history stages, environmental temperature, and the physical activity of the individuals being tested. Because the majority of the RNA pool is ribosomal RNA, ratios of RNA to DNA cannot be used to quantify the synthesis of specific proteins. Development of nucleic acid probes to quantify the synthesis of individual proteins could yield more specific indicators of physiological or biochemical status, such as growth rate, reproductive status, stress level, and nutritional state. Since the first step in protein synthesis is the production of messenger RNA (mRNA), mRNAs for any particular protein are represented by a tiny fraction of the total RNA pool. In order to quantify a specific mRNA species, it is necessary to clone and characterize a cDNA probe that will exclusively hybridize to the specific mRNA in question (e.g., Crawford and Powers, 1989). These cDNA probes can be used to quantify the relative concentration of a locus-specific mRNA (e.g., Crawford and Powers, 1989) and/or they can be used to determine transcriptional rates (e.g., Crawford and Powers, 1992) (Figure 7). As other locus-specific cDNAs are cloned and characterized, they can be used to determine the effects of short-term environmental changes on the physiological status of marine organisms. The major barriers to the expanded use of this methodology are (1) the paucity of fundamental knowledge about the protein-specific mRNAs that reflect physiological, biochemical, or genetic status, (2) attaining the levels of amplification (or signal detection) needed so that the signal can be quantified, and (3) the cloning and characterization of appropriate cDNA probes that can be used to quantify the specific mRNAs.

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS FIGURE 3-1. Relationship between transcription rate from the Ldh-B locus and the amount of LDH-B enzyme (Crawford and Powers, 1992).

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS Immunological Techniques: In addition to being useful for detecting species and population differences (Chapter 2), antibodies are effective tools for determining how environmental factors, such as temperature, salinity, organic toxins, and metals, affect the concentrations of specific proteins (heat shock proteins, cytochrome P450, metallothioneins, metabolic enzymes, growth and reproductive hormones, and others) (e.g., Chen, 1983; Gedamu et al., 1983; Koban et al., 1991; Sanders et al., 1992). Because some of these specific proteins occur with similar amino acid sequences in a range of organisms, antibodies can sometimes be designed to react and bind selectively with sequences common to several species. In other cases, it is necessary to develop species-specific antibodies. These immunological probes can then be used to monitor and quantify the impact of environmental variables on the physiological status of marine organisms. The main barriers to the use of immunological methods to assess the physiological, biochemical, or genetic status of marine organisms are (1) the identification of appropriate indicator proteins that reflect important physiological or biochemical processes and (2) the purification of these indicator proteins and the subsequent generation of antibodies directed against them. Practical Implications Variations and changes in the global environment from natural events and human-induced changes can produce effects on marine ecosystems with great biological and economic impacts. For example, El Niño events severely disrupt marine ecosystems and marine fisheries in the Pacific Ocean. Global changes could likewise affect marine organisms, yet specific physiological information required for predictive capabilities is lacking. Molecular techniques offer opportunities to identify environmental impacts on marine species by detecting and characterizing changes in the synthesis of important macromolecules such as proteins at transcriptional, translational, and product levels. Physiological Condition of Marine Organisms Knowledge of the physiological condition of organisms and how organisms respond to optimal and suboptimal environmental conditions could contribute to improved management of commercially important and endangered species, enhance monitoring for water quality and environmental change, and provide a means to monitor other species. Resource managers need information describing the physiological conditions of populations, how these conditions vary over time,

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS and how they are affected by the environment. These data, which can be obtained by using molecular techniques, will allow better marine resource management; potentially stronger, more productive industries; and responsible protection of marine resources. Study of stress responses of marine organisms to environmental factors could lead to the development of methods to produce specialty chemicals (pharmaceuticals, enzymes, hormones), by “optimizing” stressful conditions. Many marine algae produce chemicals of commercial value in response to stressful external environmental conditions. For example, when certain species of marine algae are grown under salinity stress or nitrogen limitation, they produce large quantities of β-carotene, alcohols, glycerol, and hydrocarbons (e.g., Ben-Amotz and Avron, 1990; Bubrick, 1991; Behrens and Delente, 1991). Mariculture Even with improved fisheries management strategies, the catch of fish from the sea could decline. The culture of marine organisms (mariculture) could partially compensate for declines in wild populations (National Research Council, 1992). An understanding of how environmental stress affects the biochemistry and physiology of cultured organisms is essential for the successful expansion of mariculture. Such knowledge would allow mariculturists to monitor the health, nutritional state, and reproductive status of their crop to provide cultured organisms with an optimal environment. An understanding of the genetics and biochemistry of growth hormones and environmental impacts on hormone production and gene expression could enhance mariculture production and provide the foundational knowledge for eventual genetic engineering of organisms to increase the production of hormones and other biochemicals that increase growth or other desirable characteristics of cultured organisms (Fischetti, 1991). Mariculture poses risks to adjacent environments, including release of waste products and excess food and the possible release of exotic (nonnative) species into coastal ecosystems. Molecular techniques can be exploited to reduce these problems. For example, molecular methods can be used to monitor waste effluents and then to treat mariculture waste in a manner similar to that used to treat urban waste. Molecular methods also can be used to monitor effluents from mariculture systems to prevent escape of exotic species to neighboring waters. Genetic engineering techniques can be used to ensure that cultured exotic species are reproductively sterile, so that they cannot reproduce if they inadvertently escape. Genetic manipulation of eggs can produce sterile triploid organisms that

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS also have the advantage in some organisms that growth is enhanced (Allen and Downing, 1986). Environmental Quality One of the consequences of the world's growing population is the degradation of highly productive coastal ocean ecosystems as they receive increasing discharges of plant nutrients such as nitrate and phosphate, metals and other toxicants, and particulate matter. Many ecologically and economically important species spend all or a portion of their lives in these threatened habitats. Bioremediation can be an effective means to restore the natural condition of coastal ecosystems. Bioremediation could benefit greatly from identification and characterization of relevant genes encoding key proteins and enzymes involved in biodegradation. An improved understanding of the physiology and biochemistry of marine organisms capable of transforming contaminants could enhance our ability to restore marine habitats. The impact of the introduction or stimulation of selected species in natural populations could also be predicted, in part, if the selected species are understood more completely. The ocean has been and will continue to be used for disposal of human wastes. Waste treatment and disposal must be carried out in ways that preserve the food, recreational potential, and resources of the seas. Many species are sensitive indicators of changes in water quality or eutrophication on local, regional, and even global scales. In addition, molecular probes and enzymatic tests may provide a rational basis to regulate the ocean disposal of industrial sludge, sewage, or radioactive substances. The expression of genes that respond to environmental stresses, such as changes in temperature, oxygen, and pollution (e.g., genes encoding heat shock proteins, cytochrome P450, metallothionein, and proteins), can potentially provide excellent sublethal indicators that could be used to monitor the impact of oceanic dump sites and other pollution problems. As these sublethal indicators are refined for monitoring the impact of environmental pollution, methods can be automated to allow rapid and continuous analysis of water quality. This can be accomplished by the development of in situ sampling devices and reporting systems that either record the data directly or relay the information to a collecting station. The detecting system can be developed in the form of a biosensor or a microanalyzer system, and the information can be transferred via an acoustic, microwave, or optical system (National Research Council, 1993).

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS Food Safety Food safety is of great importance to producers, processors, and consumers of seafood products. Improper disposal of human and industrial wastes and nonpoint source pollution have led to deteriorating coastal water quality, forcing the closing of shellfish and other fisheries, costing millions of dollars, and devastating local economies and businesses. Marine animals can harbor disease-causing organisms and contaminants (Ahmed, 1991). The present method of determining the safety of shellfish is based on antiquated techniques that require detection of coliform bacteria that often are not specific to human sewage. Techniques that rapidly and specifically detect contaminants and pathogens are available but have not been implemented. These capabilities need to be developed more fully and implemented for rapid and inexpensive monitoring of the food quality of marine resources. Bioremediation Contaminated waters, sediments, and living resources in areas from which seafood is harvested are an economic liability, a detriment to public health, and a social and recreational liability. Emerging technologies that include new instrumentation and biochemical, physiological, and molecular methods can be used to identify and exploit marine organisms to transform harmful contaminants to less harmful forms, improve water and sediment quality, and mitigate damage to coastal habitats. The Federal Coordinating Council for Science, Engineering, and Technology (FCCSET) budget document on biotechnology (FCCSET, 1992) identified bioremediation—the transformation of pollutants, toxic substances, and metals into less harmful forms by living organisms, primarily microorganisms—as a potential direction for marine biotechnology research. Essentially every country has urban waste treatment plants and industrial effluents that discharge billions of tons of polluted water into the lakes, rivers, estuaries, and coastal waters of the world. Aquatic microorganisms and plants have been used to treat human and agricultural wastes for hundreds of years. The use of natural microbes and aquatic plants continues to be an efficient and cost-effective method to treat urban waste. Molecular techniques may be useful for identifying and even producing species that are more effective. A number of studies suggest that mixed anaerobic bacterial communities are preferable to single species for the treatment of urban waste (Ahmed et al., 1984; Wetegrove, 1984). Industrial effluents often contain man-made chemicals, toxins, and unique derivatives of naturally occurring compounds that may not be commonly degraded by aquatic microbes. In addition, massive spills of oil, toxins, and other

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS anthropogenic pollutants sometimes overwhelm the ocean's ability to cope efficiently with the huge excessive concentration of substrates, with disastrous consequences. Oil spills in Alaska and elsewhere are typical examples of the tremendous cost and ecological impact associated with our inability to cope with such spills. During the Exxon Valdez oil spill, nitrogen fertilization of small sections of selected intertidal areas resulted in the enhanced growth of a naturally occurring bacterium that could use the oil as a carbon source. Although this experiment was confined to small test areas, the results strongly suggest that supplying critical, rate-limiting nutrients to such bacteria may be one of the more efficient and cost-effective methods for dealing with the final cleanup phases of these disasters. In fact, over a decade ago, one of the first patented genetically engineered organisms was a multiplasmid bacterium for the rapid degradation of crude oil (Friello et al., 1976). This approach is being applied to the degradation of other man-made toxins and pollutants. The U.S. Environmental Protection Agency has identified hundreds of chemical pollutants that are major problems in the aquatic environment because they are not readily degraded by naturally occurring microbes. The halogenated hydrocarbons, which make up the majority of these toxins, are particularly persistent in the aquatic environment and are toxic to a variety of organisms. A number of researchers have been using recombinant DNA techniques to create microorganisms that will degrade these toxins. Some success has been achieved in the degradation of halogenated hydrocarbons, phenols, and aromatic amines, using engineered microbes and mixed-species bioreactors containing a marine polysaccharide matrix (Portier and Fujisaki, 1986; Portier et al., 1987a; Sayler, 1990). For example, immobilization of microorganisms on a solid matrix has been shown to maximize their ability to degrade hazardous chemicals, such as chlorinated phenols and polychlorinated biphenyls. Properly engineered microbial bioreactors can remove such toxins continuously for periods of several months, with residual effluent levels of less than 100 parts per billion (see Attaway, 1989 and references therein).