### SUMMARY OF DISCUSSION, WORKING GROUP I:
BIOCHEMICAL AND MOLECULAR METHODS FOR MEASURING PHYSIOLOGICAL RATES AND CONDITION

Discussion Leaders: George Somero and Joseph Torres

#### Overview

Numerous biochemical and molecular "indicator" techniques are available for determining physiological capacities (rates) of organisms and their physiological status (condition). Indicator methodologies may be used to generate a "calibration curve" which relates the physiological trait of interest, e.g., metabolic rate, to a readily measured biochemical or molecular characteristic, e.g., activity of a respiratory enzyme. The physiological rates (e.g., metabolism or growth) or status (e.g., nutritional or reproductive status) of large numbers of individuals from field populations can then be determined simply by making a biochemical (or molecular) measurement, without need for difficult and time-consuming experimentation with living specimens.

Several biochemical or molecular indicator methodologies are well established for use in species of terrestrial and freshwater organisms, but they have not been applied widely to marine species. Below we briefly discuss several methodologies which are appropriate for addressing questions of central importance to GLOBEC, specifically, questions concerning rate processes and physiological states of zooplanktonic organisms. This information is intended to serve as background material for the draft "Request for Proposals" (RFP) ("GLOBEC Call for Proposals for Research Concerning the Exploitation of Biochemical and Molecular Techniques for Gauging Physiological State of Marine Zooplankton", Appendix II of this report).

#### General Performance Capacity

Metabolism and Locomotory Ability. Two critical rate processes of interest to physiological ecologists are rates of metabolism and locomotory capacities of organisms. Metabolic rates may be used to estimate energy turnover and flux in an ecosystem. Locomotory capacity is critical in determining predator-prey relationships in motile species.

Enzymatic analyses can provide accurate estimates of the metabolic potential--both "basal" and "active" rates--of organisms. The enzymes of energy metabolism, e.g., those of ATP generation, are common to most marine species. Assay of these enzymes under physiologically realistic conditions, e.g., of temperature and pH, can provide an estimate of the maximal ATP- generating potential of an organism or a specific tissue. Likewise, in the locomotory muscles, activities of ATP-generating enzymes in both aerobically- and anaerobically-poised pathways can provide an estimate of capacities for both low-speed (aerobically-powered) and high-speed (anaerobically-powered) swimming.

To exploit enzymatic methods successfully, one must have an understanding of the pathways of ATP generation in different species. For fishes this information is available; for many invertebrates data are lacking. It is imperative that initial efforts to adapt enzymatic analyses to marine systems more completely characterize the metabolic capabilities of ecologically important invertebrate species such as copepods. A general theme of this document is that much additional background information on the physiology, biochemistry, and molecular biology of marine species must be determined before appropriate biochemical indices for evaluating ecologically important characteristics can be fully developed.

Several factors must be considered in designing effective methods for using enzyme activities as estimators of metabolic rate and locomotory capacity. Among these are the following. First, enzymatic activities tend to be size-dependent. Thus, size must be incorporated into experimental design and data analysis. Second, enzymatic activities are highly dependent on diet (a fact which makes these methods very useful for estimating physiological state). In laboratory calibration studies, e.g., studies in which oxygen consumption rates and metabolic enzymes are measured, dietary effects should be considered as an experimental treatment. Third, the stability of the enzymes of interest during storage should be determined. Although many enzymes are generally stable during long-term storage at -80°C (or even in conventional freezers), it is appropriate to test for stability whenever a new enzyme, or new type of organism, is to be analyzed.

Specific choices of indicator enzymes for gauging metabolic and locomotory capacities must be based on knowledge of the primary pathways used by the species or tissue of interest and of the stabilities of these enzymes during storage. The aerobic metabolic potential of an organism or tissue can be estimated by determining citrate synthase (CS) activity. CS is a good indicator of the Krebs citric acid cycle activity of an organism or tissue, and CS is generally stable during long- term storage of frozen specimens. Cytochrome c oxidase is another appropriate enzyme, but its stability is typically not good when subjected to freeze-thaw cycles. Lactate dehydrogenase (LDH) is a very good indicator of anaerobic locomotory power in fishes, and is stable during long-term frozen storage. However, in copepods and other planktonic invertebrates, other enzymes might be better indices of locomotory capacity, especially if the planktonic invertebrates rely more on aerobic than on anaerobic schemes for supplying the ATP required for locomotory processes.

Nutritional Status. Enzymes of digestive processes may provide an important source of information about physiological state. Individuals living under conditions of food shortage may contain reduced levels of substrate-inducible digestive enzymes. To use digestive enzymes in analyses of nutritional status, it is necessary to determine the appropriate enzymes for analyses. For example, protease activity may be appropriate for carnivorous species, whereas enzymes like laminarinase are more suitable for herbivores. Possible diel variations in digestive enzyme activities should be considered in designing experiments. Nutritional status also could be indexed by measuring the amounts of stored nutrients, e.g., glycogen or triacylglycerides, deposited in storage sites such as muscle and liver.

Molecular Biological Approaches. Although enzymatic analysis has the benefits of being well- developed, rapid, usually inexpensive, and relatively simple, there may be circumstances under which molecular methods offer increased sensitivity and resolving power. Using specific primers to quantify the amounts of specific nucleic acids in natural samples is one example of a situation where molecular approaches may be especially powerful. Methods of this type will depend on the generation of specific primers of use to workers studying zooplankton.

Other Considerations. Depending on the question being asked, sorting of organisms may or may not be necessary. if the total metabolic potential of all organisms in a given volume of water is of interest, then measurement of, for example, CS activity in an entire community (made via separating the organisms by centrifugation, settling, or screening, and, then, homogenizing the entire community to obtain a source of soluble enzyme) is possible. This type of measurement obviously is crude, but it seems likely to give information about the integrated capacity for ATP generation in the entire community of organisms in the water sample. Refining the sampling and sorting regimes could involve separation and analysis of individual sizes or species, and the analysis of single tissues, e.g., muscle tissues or digestive organs, to obtain more specific information about swimming ability or digestive capacity, respectively. Analysis of diagnostic tissues can provide especially clear biochemical signals about the organism's physiological state.

#### Morbidity

In many contexts it is helpful to have an index of the degree to which an organism has been stressed, and the extent to which an organism is living near the limits of its tolerance, e.g., to anoxia or temperature. At present there are no clear cut methods for quantifying stress level or "morbidity." However, several biochemical attributes would seem to offer promise in this context

Stress Proteins. A diverse family of proteins known as stress proteins ("heat shock proteins" was the expression formerly used to describe these molecules) can be induced in response to a variety of environmental stresses, including high temperature, anoxia, heavy metal pollution, and exposure to ethanol. The appearance of stress proteins typically occurs within an hour of exposure to the environmental stress, and a high level of stress proteins may be maintained in the cell for extended periods. The appearance of stress proteins is an indication of two things: the occurrence of an environmental stress, and the ability of the organism to respond adaptively to this stress. Thus, the synthesis of stress proteins does not indicate morbidity per se; it may reflect just the opposite condition, i.e., an organism which is coping well with an altered environment. However, stress protein appearance could serve, at least in theory, as a useful biochemical indicator of exposure to stress, even though other physiological and biochemical indicators would be needed to gauge accurately whether that stress leads to some level of morbidity.

Choice of Tissue. Tissues and organs differ in their environmental sensitivities, and the choice of appropriate tissue for examining morbidity is important. In fishes, brain tissue seems to be the most highly "defended" tissue, showing physiological decline only after more "expendable" tissues, e.g., locomotory muscle, have begun to show physiological deterioration (e.g., in protein synthetic capacity and ATP generating potential). Thus, physiological studies under controlled laboratory conditions should be conducted to determine which tissue(s) are most appropriate for use as indicator tissues of morbidity.

#### Diapause

Diapause, whether it occurs during early development of copepods (diapause eggs) or during later stages (CII-adult) is a very important stage in the life history of many important calanids. From an ecological standpoint, the absence of important metazoan grazers in the trophic pyramid changes the character of the ecosystem. From a physiological standpoint, it is an important (and perhaps flexible) response to times of environmental stress, a metabolic refugium that may increase the odds of species survival during global warming periods.

Our current lack of knowledge about the physiology and biochemistry of marine invertebrates is perhaps best illustrated by the minimal understanding we possess about factors regulating diapause in copepods. Regulatory mechanisms that affect the reversible shut-down of active metabolism are totally unknown. Metabolic costs/benefits of the diapause state likewise need much additional study. Biochemical methods should be used in background studies to elucidate these regulatory mechanisms, and to provide insights into the metabolic benefits (and costs) of these states of reduced activity. Perhaps the large data base on brine shrimp ( Artemia ) diapause and quiescence could provide insights into the most appropriate models for copepod diapause. Changes in tolerance of anoxia might also be examined for species that settle into low-oxygen water (e.g., oxygen minimum layers) during periods of diapause.

#### Egg Production Rates

Biochemical and molecular approaches for quantifying egg production rates are currently not in use. Sorting and counting methods are time-consuming, but are accurate and effective. Several interesting biochemical alternatives were suggested as worthy of further exploration.

First, immunological tests for the occurrence and/or abundance of eggs of a particular species in theory could provide a rapid and accurate means for surveying egg production. For example, if highly specific antibodies could be generated to a species-specific protein such as a sperm-binding protein, then sensitive antibody tests could be developed for characterizing unsorted populations of eggs. Such antibody tests might be restricted to species that spawn unfertilized eggs. Species with internal fertilization might not be amenable to this type of analysis.

Second, chemicals (e.g., pheromones) that induce spawning by stimulating reproductively competent individuals to release eggs over a short period of time might provide an index of the reproductive activity that is occurring. Sensitive analytical methods, e.g., HPLC techniques, will be needed to detect, identify, and quantify these highly dilute chemicals.

#### Growth Rates

Biochemical and molecular indicators of growth rate potentials have been used for over a decade with considerable success. Further refinement of these methods will enhance their accuracy, speed, and utility for use in generating "real time" data on board ships.

RNA:DNA Ratios. The ethidium bromide method for quantifying RNA and DNA in small (<0.5 mg) samples is now commonly used for determining RNA:DNA ratios. Dozens of samples can be analyzed per day by an individual worker, so this method is well suited for sampling large numbers of individuals. Microtitre plates and readers could speed-up the analysis of numerous samples. The method needs more testing and development with invertebrates, but its general applicability to animals seems likely.

There are several precautions that must be taken in using this method. Among these are: (i) DNA concentration may be size-dependent, e.g., in fish locomotory muscle DNA concentration may decrease with size, and (ii) DNA content per nucleus varies widely among species. Thus, there is no single term for the denominator of the RNA:DNA expression.

DNA Polymerase. One enzyme involved in DNA replication, DNA polymerase, could in theory serve as an excellent indicator of growth rate, i.e., of DNA synthetic rate. With the advent of non- radioactive fluorescent substrates for quantifying DNA synthesis, the measurement of DNA polymerase activity at sea seems a very reasonable and attractive strategy for measuring growth rates. This method requires further development for marine species for which growth involves increases in cell number (DNA synthesis). For species in which growth involves increases in cell size, but no increase in the amount of DNA per cell, e.g., later stages of copepods, the DNA polymerase method is clearly not applicable.

Chitin Polymerization Enzymes and Molting Hormones. For species in which chitin synthesis correlates strongly with growth, measurement of the activities of one or more enzymes of chitin synthesis could be a useful biochemical method for estimating growth potential. Assay of molting hormone concentrations in seawater could also be useful for estimating growth. These potentially useful methods require development, including detailed laboratory calibration.

#### Developmental Stage

For fishes, identifying developmental stage appears to provide few major problems, and biochemical or molecular approaches appear unnecessary. For invertebrates, screening large populations for developmental stage is more problematical. Here, 2-dimensional (2-D) gel electrophoresis methods could be a real boon, assuming that each life stage has a unique "protein signature" that could be detected by highly sensitive 2-D gel methods.

#### Age

There is no known biochemical or molecular method for estimating age. No chemical species appears to build up merely as a consequence of age. Lipofuscin methods appear unreliable. Size-related changes in biochemical properties should not be confused with age-related changes.

#### Applicability of Biochemical and Molecular Methods to Shipboard Study

Several factors must be taken into account in evaluating the applicability of biochemical and molecular methods for use on board ships, e.g., for gathering "real time" data. Some of these considerations are briefly discussed below.

Stability of Samples. Many--probably most--enzymes and RNA and DNA are generally stable during prolonged storage at freezing temperatures. As a general rule, the colder the temperature of initial freezing and storage, the more likely is the biochemical or molecular system to be stable. Freezing in liquid nitrogen is optimal, but often not necessary to ensure adequate stability. The presence of a -80°C freezer on board ship is almost certain to benefit biochemical and molecular analyses.

The decision as to whether samples should be worked-up on board ship must be based in part on considerations of stability. Labile samples should, if possible, be worked-up immediately, without freezing. More stable samples can be collected in large numbers (i.e., in larger numbers than can be conveniently assayed on board ship during an expedition) and returned to the home laboratory for analysis.

Weighing and Storage of Reagents. Because most biochemical and molecular methods employ several labile reagents, often in minute quantities, it is advisable to pre-weigh as many reagents as possible before going to sea. if a motion-compensated shipboard balance is not available, then pre- weighing of reagents is a must. On board ship the reagents must be stored under appropriate conditions to ensure stability.

Centrifugation. Table-top centrifuges ("microfuges") work well at sea, and even larger centrifuges work well if placed on gimbals. Thus, preparation of high-speed supernatants for enzymatic analysis should pose few problems.

#### Other Considerations

1. Normalization of Enzymatic Activities. No one normalization format may be suitable for all purposes. Very small organisms, e. g., individuals with fresh (=wet) weights of less than 0.5 mg, may be difficult to weigh accurately when wet. Thus, normalization of enzymatic activity to dry weight or to total body protein may be more suitable than normalization to fresh weight. For larger individuals, tissue samples can be run and normalization to wet weight may be the best means of expressing enzymatic activity per mass of organisms. Whatever the normalization procedure adopted, procedures must be followed that will allow comparisons to be made between different organisms and, when possible, between data from different laboratories. Often the lack of consistent normalization procedures between laboratories makes data sets difficult, if not impossible, to cross-calibrate.
2. Standardization of Analytical Procedures. The caveats raised above apply equally strongly in the context of designing precise analytical protocols for biochemical and molecular work. Assay temperature, pH, ionic strength, substrate concentration, etc. will affect the results of in vitro experiments. Ideally, different workers should agree on common in vitro conditions, thereby making their data sets as amenable to cross-calibration as possible. A particularly strong caveat applies to assay temperatures for enzymatic analyses: a 1°C change in assay temperature typically leads to a 12% change in enzymatic activity. Thus, it is imperative to use thermostatted cuvette holders for running spectrophotometric enzyme assays. Running assays at "room temperature," i.e., without precise temperature control, will lead to enormous variation in the data, and a reduced ability to compare samples from different days or laboratories.
3. Minimal Data Base on Marine Invertebrate Species, Especially Zooplanktonic Species and Life Stages. A major theme running through our discussions of biochemical and molecular indicators of physiological state is the dearth of information on the biochemistry and molecular biology of marine invertebrates. Emphasis should be given to enlarging this data base, to enable informed decisions to be reached concerning the best biochemical indicators to study, and the best technical approaches to be developed for specific applications to invertebrates.
4. Field versus Laboratory Populations. In several contexts it was pointed out that laboratory- reared individuals may differ in physiological or biochemical status from field-caught individuals of similar size, age, or life stage. This difference, which may reflect differences in diet, exercise level, predation, or other factors, should be taken into account when laboratory studies are conducted to generate "calibration curves," e.g., of metabolic rate versus enzymatic activity. Fortunately, however, it seems likely from what is currently known that extrapolation of such calibration curves to higher or lower values of the variable(s) of interest is apt to be valid.