Molecular Approaches

## Molecular Approaches to Identify a Species and Assess its Physiological Status In Oceanic Plankton

### by Douglas L. Crawford

Molecular techniques are useful for identifying species and defining distinct populations and for quantifying cellular or physiological status. A variety of molecular methods, sequence analysis, and Restriction Fragment Length Polymorphism's (RFLPs) have been successfully applied to marine problems. Carlton and Geller (1993) demonstrated that many bays and estuaries are constantly invaded by plankton carried in ship ballast water. Finnerty and Block (1992) demonstrated that although marlin are found world wide, they are not panmictic--i.e., different ocean basins consist of genetically distinct populations.

These studies are only a few examples of how molecular techniques can be used to address important biological and conservational questions in the ocean. Quantitative molecular analysis is similarly useful. A few researchers have used gross measures of RNA and DNA concentrations to ascertain an index of growth or physiological status (see article by Buckley and McNamara). More specific measurements, e.g., determination of specific gene products, are almost exclusively performed on culture organisms where there is little or no problem with identification. These endeavors have been applied most successfully to the identification of developmentally specific gene expression, growth-specific gene products or genes characteristic of disease states. For cultured oceanic species, Clarke et al (1992) were able to demonstrate that the amount of certain enzymes is indicative of the recent feeding regimes and, therefore, likely linked to growth or other important physiological indices.

What is needed is a way to combine these two seemingly disparate technical goals, that is, to be able to both (1) identify specific species or populations and (2) quantify specific genes or their products as an index of physiological status. My laboratory is currently working toward this goal by utilizing quantitative PCR to both identify the copepod Calanus finmarchicus, and to quantify its expression of specific genes. The level of enzyme expression from a single species is determined by isolating nucleic acids from these copepods, converting its mRNA into cDNA, and quantitatively amplifying the cDNAs that code for key metabolic enzymes. The PCR products for each enzyme's mRNA are of different size and are electrophoretically separable. As a first step, we have cloned and characterized genes that code for several key metabolic enzymes and used them to design species specific primers. This research makes the assumption that mRNA concentration is highly correlated to the concentration of the enzyme they encode. Although this has to be verified experimentally, it has been demonstrated in a number of laboratory studies. For example, diet-related changes in enzyme concentrations in rabbits can be accurately determined by measuring mRNAs (Granner and Pilkis, 1990). Additionally, enzyme concentration in the small minnow Fundulus heteroclitus is a function of its mRNA concentration, and both of these measures of enzyme expression are sensitive to environmental temperature experienced by these fish (Crawford and Powers, 1989). Thus, by designing the proper molecular probes, it will be possible to identify a specific species and determine its physiological status by quantifying mRNA expression.

This approach has several advantages over other methods. First it does not depend upon identifying and sorting adults; the level of enzyme expression can be measured in any stage of the life cycle, even the most difficult to identify. Second, many gene products can be measured simultaneously, each separated by size electrophoretically. And, finally, many individuals may be assessed simultaneously without any sorting at all. In a mixed species sample, the number of Calanus finmarchicus could be determined by measuring the concentration of a nuclear gene (this is really a measure of the number of cells), and the average physiological condition could be assessed by measuring the concentration of an enzyme's mRNA. Both nuclear DNA and mRNA can be assayed in the same reaction because their PCR products are of different size. The ratio of these two measures may vary due to changes in recent feeding conditions, temperature, developmental state or other physiological conditions. By grouping of many individuals, one loses information concerning inter-individual variability in physiological condition, but gains the ability for rapid survey of many populations. Where populations (samples) differ, individual organisms would need to be analyzed using the same methods outlined for mixed populations to determine the source of the differences.

This goal of simultaneously identifying a species and quantifying gene expression faces many hurdles. There are obvious molecular and biochemical assumptions to be tested (e.g., the relationship between enzyme concentration and its mRNA, the correlation between dry weight and nuclear DNA concentration, etc.). Just as important are problems arising when taking a basic laboratory technique into a field setting. To design probes that are species-specific yet inclusive of all individuals within a species, one has to know the sequence variation both within and between species. Thus, it is not adequate to sequence a single clone from an individual. One must measure the sequence variation within the species and compare this variation to the phylogenetically most similar species. If a good phylogeny is not available, one must be determined. Fortunately, for C. finmarchicus the most closely related species have been identified, and the relationship between these sister groups is reasonably well established. Theoretical considerations suggest that much of the genetic variability will be observed if approximately ten individuals are sequenced for each gene. Aligning these sequences will identify species-specific oligo-nucleotide that will be used to design primers. Another challenge is to make sure the molecular probes do not exclude genetic variants that may exist in different populations. Primers could be too specific and may not work on all the allelic variants at a locus. If the molecular probes did not assess all alleles, one might find a variation in gene expression due to a change in allelic frequency. This can be addressed by examining the sequence variation between widely dispersed populations and by judiciously choosing molecular probes that are unlikely to vary between populations (sites).

By melding two fields of molecular biology--molecular population genetics and molecular physiology--we hope to streamline the assessment of zooplankton physiological status. There is a vast potential in such an approach, but caution must be taken to verify all the assumptions. Applying known molecular techniques to field problems shows promise, but one must understand the underlying premises inherent in these techniques before applying them to questions in the field. (Douglas Crawford is an Assistant Professor in the Department of Organismal Biology and Anatomy at the University of Chicago)

### References

Carlton, J. T. and J. B. Geller. 1993. Ecological roulette - the global transport of nonindigenous marine organisms. Science, 261, 78-82.

Clarke, M. E., C. Calvi, M. Domeier, M. Edmonds, and P. J. Walsh. 1992. Effects of nutrition and temperature on metabolic enzyme activities in larval and juvenile red drum, Sciaenops ocellatus, and lane snapper, Lutjanus synagris. Mar. Biol., 112, 31-36.

Crawford, D. L. and D. A. Powers. 1989. Molecular basis of evolutionary adaptation at the lactate dehydrogenase-B locus in the fish Fundulus heteroclitus. Proc. Natl. Acad. Sci., 86, 9365-9369.

Finnerty, J. R. and B. A. Block. 1992. Direct sequencing of mitochondrial DNA detects highly divergent haplotypes in blue marlin (Makaira nigricans). Mar. Mol. Biol. Biotech., 1, 206-214.

Granner, D. and S. Pilkis. 1990. The genes of hepatic glucose metabolism. J. Biol. Chem., 265, 10173-10176.