Estimating Growth Rate

## Estimation of Zooplankton and Ichthyoplankton Growth and Condition Using Nucleic Acid Probe Techniques

### by Lawrence Buckley and Peter McNamara

One of the key biological processes controlling population dynamics of marine organisms is zooplankton growth and production rate. Estimation of growth and production in situ is a major goal of biological oceanography, and a better understanding of these parameters as they may be affected by global climate change is a primary objective of the U.S. GLOBEC Program. Presently, a variety of approaches are being used to evaluate copepod growth and condition including cohort analysis, methods based on estimation of egg production, molting frequency, feeding incidence, and metabolic rates. None of these approaches are entirely satisfactory due to drawbacks associated with the required handling and incubation of organisms following shipboard collection. The development of improved biochemical or molecular techniques for estimation of growth and production rates of zooplankton would represent a significant improvement over existing methods.

At least three main biochemical approaches have been applied to the study of zooplankton growth and production: (1) incorporation or metabolic uptake of a labelled precursor, (2) measurement of the rate of a metabolic pathway or enzyme, and (3) estimation of RNA content. The first approach has been used to evaluate both primary production and secondary production. The incorporation of 14C-labeled carbonate by phytoplankton has served as a functional definition of primary production and uptake of 14C-labeled algae by zooplankton has also been investigated. Currently efforts are underway to determine the relation between incorporation of bromodeoxyuridine (BrdU) into DNA (an index of cell proliferation) and growth of zooplankton, including larval fish (see Moore and Stegman, 1992).

The activity of the respiratory electron transport system in marine zooplankton has been related to feeding condition and other environmental variables. Currently, studies relating zooplankton growth with the activities of specific enzymes comprise an active area of research. Enzyme activities are known to be sensitive to nutritional status in adult fish, and to scale with body length in both adult and larval fish. Activities of the key metabolic enzymes lactate dehydrogenase and citrate synthase (representative of anaerobic and aerobic metabolism, respectively) have recently been measured in larval fish and appear to be useful in assessing condition (Clarke et. al., 1992). Citrate synthase activity has also been estimated in individual zooplankton and was found to correlate with feeding (Clarke and Walsh, 1993). Additionally, DNA polymerase activity is known to be responsive to changes in growth rate of Artemia salina nauplii, and may provide another useful marker of cell proliferation and growth in copepods and larval fish.

The third biochemical approach to assessing growth and production rates--estimation of RNA content--has been under active investigation in a number of laboratories and is the primary topic of this article. The RNA content of any tissue or whole organism consists primarily of ribosomal RNA (rRNA), the nucleic acid component of ribosomes. Ribosomes are the protein synthesizing complexes of the cell. Ribosome number, and, therefore, the concentration of rRNA, at any given time is directly related to the protein synthesizing activity of a cell. Since the DNA content of a somatic cell is constant, RNA levels may be normalized to a per cell value after dividing by DNA concentration. The resultant RNA-to-DNA ratio value has proven to be a useful indicator of both nutritional status and growth in larval fish. The relationship between RNA content and growth in copepods, however, appears to be more complex, due to the nature of the molt cycle. (Ota and Landry, 1984).

The successful application of the RNA-to-DNA ratio method to evaluate condition and growth in larval fish can be attributed to several factors. First, there is a direct link between RNA concentration and growth which, in larval fish, involves primarily the generation of muscle tissue (Houlihan et al., 1988). A relationship between the ratio of total RNA to DNA and the growth of a wide variety of temperate marine fish larvae reared in the laboratory has been established (e.g. Buckley 1979). A general model relating RNA-to-DNA ratio and water temperature to larval growth rate was developed from data on 8 laboratory-reared fish species (Buckley, 1984). Second, the larval period in fish is a single, well-defined life stage characterized by rapid growth. Analysis of growth and nutritional condition in fish larvae, therefore, has been relatively free of complicating factors such as age, gender and reproductive status. Finally, extensive laboratory experiments have been conducted to calibrate the method. Consequently, the RNA-to-DNA ratio method (with refinements and variations) has become widely accepted and utilized for the estimation of short-term (days) growth in larval fish. In a recent study, for example, a variation of the approach employed flow cytometry to estimate the RNA and DNA content of individual brain cells from fish larvae (Theilacker and Shen, In press). Two distinct fractions of cells were identified in which RNA levels were sensitive to either feeding or growth.

Growth, as gauged by the weight of fish larvae, is exponential through the larval period, with the majority of protein synthesis directed toward the accretion of muscle tissue. Unlike other life history stages in fish, little metabolic energy is spent in building energy reserves or in reproduction during the larval stage. A consideration of zooplankton growth and physiology as a whole, however, presents a much more complicated picture. Even when considering a single species such as the copepod Calanus finmarchicus, one net haul taken in the field might contain multiple life stages of the organism. Complicating the problem are discontinuous growth and the molt cycle that accompany each developmental stage. These factors can have a profound effect on the biochemical composition of the organism.

### The Potential for Nucleic Acid Probe Techniques

Production and growth are a function of gene expression. Techniques are currently available to estimate the concentration of individual RNA molecules, including rRNA and specific mRNAs. The use of nucleic acid probes can provide information on the identity, growth rate, physiological status and reproductive condition of individual organisms. In addition, information on the genetic relationships among individuals, cohorts and populations is available using this approach (Powers, 1993). Doug Crawford and Lew Incze, for example, are currently using the polymerase chain reaction (PCR) to quantitatively amplify mRNAs from the copepod Calanus finmarchicus which encode enzymes that are indices of metabolic flux (see Crawford article, this newsletter). It is anticipated that PCR primers specific for this copepod will be identified, allowing the indirect quantitation of enzyme concentration (through mRNA concentration) from only C. finmarchicus, even in a mixed sample.

We are using quantitative RNA slot blots to estimate the concentration of rRNA and selected mRNAs in larval fish and copepods. Nucleic acid probes have been developed which are complementary to selected RNA target sequences. These have either been synthesized directly (oligonucleotide probe) or produced using the PCR reaction and labeled with a nonradioactive tag. Extracts containing the RNA from individual copepods or larval fish are blotted and fixed onto a nylon membrane and the labeled probe is allowed to hybridize to the immobilized target RNA molecules. The amount of bound probe is quantified using a chemiluminescent detection procedure followed by film exposure and densitometry.

In a preliminary analysis we have examined changes in the levels of a small subunit rRNA and two abundant mRNAs, coding for the muscle proteins actin and myosin in Atlantic cod larvae. Since growth in larval fish is primarily accomplished by protein synthesis and accumulation as muscle tissue, we reasoned that the levels of these RNA molecules should change in response to changes in feeding conditions and growth rate. Results with first-feeding cod larvae reared in the laboratory indicate that levels of 18S rRNA follow a pattern similar to that of total RNA--peaking around day 4 and then remaining relatively constant from day 6 to day 10 (Figure 2). After day 8, the total RNA content of fed larvae is significantly greater than that of larvae which have been starved since hatching. No appreciable difference, however, is evident in the levels of 18S rRNA from fed and starved fish until day 10. Messenger RNAs from the actin and myosin genes each reach a minimum at day 6, after which levels increase relatively rapidly compared to the levels of 18S rRNA and total RNA over the same time period. Actin and myosin mRNA levels also increase more rapidly in fed than in starved larvae from day 6 to day 10. It should be noted that these experiments are preliminary and it is therefore premature to assess the significance of these apparent trends. In general, however, it does not appear that the patterns of 18S rRNA and actin and myosin mRNA abundance differ greatly from that of total RNA over the time course studied.

An interesting observation made during the course of this work is that the probe for myosin mRNA produced from cod muscle RNA does not hybridize with haddock RNA. This specificity for cod may be useful in distinguishing between cod and haddock eggs that co-occur on Georges Bank (and other spawning grounds) and cannot be distinguished by microscopic examination until just before hatching. This finding highlights one of the major advantages of the nucleic acid hybridization approach to estimation of biological rates (growth, egg production, etc.) and physiological status (nutritional, reproductive, developmental, etc.) of marine organisms. With the appropriate probes available, and the optimal hybridization conditions known, varying degrees of specificity can be obtained from nucleic acid probes.

One of the challenges of using nucleic acid probes lies in identifying a target mRNA that is responsive to feeding conditions and growth rate. We are presently in the process of preparing a subtracted DNA library (a collection of DNA fragments copied from mRNAs) that should be enriched for genes from which transcription is induced or enhanced during starvation in cod. We will use this and other strategies to identify additional mRNAs that are responsive to changes in food availability and growth rate.

Questions concerning zooplankton growth and condition are currently being addressed through the application of both biological and biochemical methods. The use of biochemical and molecular biological techniques offer the advantages of unparalleled sensitivity and specificity at the level of a single gene or gene product. (Larry Buckley is a marine researcher at the National Marine Fisheries Service Laboratory and University of Rhode Island and Peter McNamara is a post-doctoral fellow at the University of Rhode Island)

### References

Buckley, L.J. 1979. Relationships between RNA-DNA ratio, prey density, and growth rate in Atlantic cod (Gadus morhua) larvae. J. Fish. Res. Bd. Can., 36, 1497-1502.

Buckley, L.J. 1984. RNA-DNA ratio: an index of larval fish growth in the sea. Mar. Biol., 80, 291-298.

Clarke, E.M., Calvi, C., Domeier, M., Edmonds, M. and Walsh, P.J. 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.

Clarke, E.M. and Walsh, P.J. 1993. Effect of nutritional status on citrate synthase activity in Acartia tonsa and Temora longicornis. Limnol. Oceanogr., 38, 414-418.

Houlihan, D.F., Hall, S.J., Gray, C. and Noble, B.S. 1988. Growth rates and protein turnover in atlantic cod, Gadus morhua. Can. J. Fish. Aquat. Sci., 45, 951-964.

Moore, M.J. and Stegeman, J.J. 1992. Bromodeoxyuridine uptake in hydropic vacuolation and neoplasms in winter flounder liver. Mar. Environ. Res., 34, 13-18.

Ota, A.Y. and Landry, M.R. 1984. Nucleic acids as growth rate indicators for early developmental stages of Calanus pacificus Brodsky. J. Exp. Mar. Biol. Ecol., 80, 147-160.

Powers, D.A. 1993. Application of Molecular Techniques to Large Marine Ecosystems, pp. 320-352 in Large Marine Ecosystems -- Stress, mitigation, and sustainability., K. Sherman, L. M. Alexander, and B. D. Gold (eds.), AAAS Press.

Theilacker, G.H. and Shen, W. In press. Calibrating starvation-induced stress in larval fish using flow cytometry. Am. Fish. Soc. Symp. Series.