RNA/DNA RATIOS IN THE ESTIMATION OF GROWTH STAGES OF OCEANIC ZOOPLANKTON POPULATIONS Dale Eric Baugh DUDLEY KNOX LIBR'iRY NAVAL POSTGRADUATE SCHOOl MONTEREY. CALIFORNIA 93940 NAVAL POSTGRADUATE SCHOOL Monterey, California THESIS ' RNA/DNA RATIOS IN THE ESTIMATION OF GROWTH STAGES OP OCEANIC ZOOPLANKTON POPULATIONS by . Dale Eric Baugh September 197^ Thesis Advisor: E.D. Traganza Approved for public release; distribution unlimited. r 1^%^° UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE ftfhen Data Entered) 4. TITLE (and Subtitle,) RNA/DNA Ratios in the Estimation of Growth Stages of Oceanic Zooplankton Populations REPORT DOCUMENTATION PAGE 1. REPORT NUMBER 2. GOVT ACCESSION NO 7. AUTHORfe) Dale Eric Baugh READ INSTRUCTIONS BEFORE COMPLETT.NC FORM 3. RECIPIENT'S CATALOG NUMBER 5. TYPE OF REPORT ft PERIOD COVEREO Master's Thesis; September 197^ 6. PERFORMING ORG. REPORT NUMBER t. CONTRACT OR GRANT NUMBERfe) 9. PERFORMING ORGANIZATION NAME AND ADDRESS Naval Postgraduate School Monterey, California 939^0 10. PROGRAM ELEMENT. PROJECT, TASK AREA ft WORK UNIT NUMBERS II. CONTROLLING OFFICE NAME AND ADDRESS Naval Postgraduate School Monterey, California 939^0 12. REPORT DATE September 197^ 13. NUMBER OF PAGES 64 14. MONITORING AGENCY NAME ft AODRESSff/ dltiarmt from Controlling Olllct) Naval Postgraduate School Monterey, California 93940 15. SECURITY CLASS, (of thit report) Unclassified 15a. DECLASSIFI CATION/ DOWN GRADING SCHEDULE 16. DISTRIBUTION STATEMENT (ol thit Report; Approved for public release; distribution unlimited. 17. DISTRIBUTION STATEMENT (ol th» eb.tract entered In Block 20, II different from Report; 18. SUPPLEMENTARY NOTES If. KEY WORDS (Contlnum on fvmrt* etde II neceeeary and ld»ntlty by block number) RNA/DNA Oceanic Zooplankton Deoxyribonucleic Acid Ribonucleic Acid 20. ABSTRACT (Continue on reveree aide II neceeeary and Idmntlty by block number) Current thought holds that the rate of protein synthesis is some function of the ribonucleic acid (RNA) concentration in growing animals. It is possible that measurements of the ratio of RNA to deoxyribonucleic acid (DNA) might provide an index of growth stages In gross analysis of mixed zooplankton populations. RNA concentrations are found by measuring the ultra-violet dd ,: (Page 1) FaT,3 1473 EDITION OF 1 NOV 65 IS OBSOLETE S/N 0102-014-6601 I 1 UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGe (Whmn Data tnfr.j UNCLASSIFIED CLtUWITY CLASSIFICATION OF THIS P»GEf*li»n 0«l« Enr.r.d. (20. ABSTRACT Continued) (UV) absorption of Its purine and pyrimidine base groups. Interference from protein in the RNA measurement is accounted for by employing differential UV absorption. DNA concentrations are found by measuring the UV absorption of an indole- deoxyribose adduct. This study indicates that RNA/DNA ratios are related to growth stages of the splash zone copepod Tigriopus californicus . These ratios have the potential to be applied to models which relate zooplankton populations to the food chain and therefore to the sound scattering parameters which are' of great interest to the Navy. DD Form 1473 (BACK) , 1 Jan 73 TTNHT.AS.S-TPTFn S/N 0102-014-G601 „ " SECURITY CLASSIFICATION OF THIS PAGE(T»?».n D.I. EnCrtd) RNA/DNA Ratios in the Estimation of Growth Stages of Oceanic Zooplankton Populations by Dale Eric Baugh Lieutenant Junior Grade, United States Navy B.S., United States Naval Academy, 1972 Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN OCEANOGRAPHY from the NAVAL POSTGRADUATE SCHOOL September 1974 C. r DUDLEY KNOX LIBR',RY NAVAL POSTGRAC'JATE £ MONTEREY. CALIFORNIA 93940 ABSTRACT Current thought holds that the rate of protein synthe- sis is some function of the ribonucleic acid (RNA) concen- tration in growing animals. It is possible that measurements of the ratio of RNA to deoxyribonucleic acid (DNA) might provide an index of growth stages in gross analysis of mixed zooplankton populations. RNA concentrations are found by measuring the ultraviolet (UV) absorption of its purine and pyrimidine base groups. Interference from protein in the RNA measurement is accounted for by employing differential UV absorption. DNA concentra- tions are found by measuring the UV absorption of an indole-deoxyribose adduct. This study indicates that RNA/DNA ratios are related to growth stages of the splash zone copepod Tigriopus calif ornicus. These ratios have the potential to be applied to models which relate zooplankton populations to the food chain and therefore to the sound scattering parameters which are of great interest to the Navy. TABLE OF CONTENTS I. INTRODUCTION 10 II. HISTORICAL 12 III. EXPERIMENTAL PROCEDURE 28 A. COLLECTION AND HANDLING OP T. CALIFORNICUS 28 B. PRE-ANALYSIS 29 C. SAMPLE ANALYSIS 29 D. PREPARATION OF STANDARDS 30 E. PRECIPITATION OF RNA AND DNA 31 F. HYDROLYSIS OF RNA MACRO-MOLECULAR STRUCTURE 31 G. DIGESTION AND MEASUREMENT OF DNA 31 H. MEASUREMENT OF RNA AND THE PROTEIN CHECK 34 IV. RESULTS 40 V. DISCUSSION 48 A. TIGRIOPUS CALIFORNICUS .AS AN EXPERIMENTAL ORGANISM 48 B. CHEMICAL PROCEDURE 49 C. PREVIOUS WORK 51 D. RNA/DNA VERSUS GROWTH STAGE 52 E. APPLICABILITY 57 F. FUTURE METHODS 58 G. A CRITIQUE 59 LIST OF REFERENCES 60 INITIAL DISTRIBUTION LIST ' 63 LIST OF TABLES TABLE I. TABLE II. TABLE III. A description of the composition of the five experimental groups used in the analysis DNA and RNA values in micrograms per milliliter for all the experimental data taken • RNA to DNA ratios in micrograms per milliliter to micrograms per milliliter for both experimental runs, and the value of their mean and mean derivation 41 43 44 TABLE IV. Absorption values for a differential UV absorption analysis of a bovine serum albumin protein standard taken at 260 nm and 280 nm 45 LIST OP FIGURES FIGURE 1, FIGURE 2. The basic three-part structure of a DNA and an RNA nucleotide 14 Hydrogen bonding as it occurs betwen complimentary base pairs in the DNA double helix or between the different forms of DNA and RNA as found in protein synthesis 15 FIGURE 3. The physical size relationship between complimentary base pairs and the DNA molecule is such so that only purines can bond to pyrimidines and vice-versa 16 FIGURE 4. The logical sequence of the formation of a specific amino acid from a specific order of codons on a DNA strand 18 FIGURE 5(a). The beginnings of protein synthesis from the "protein message" carried on the DNA molecule to the formation of a mRNA molecule that corresponds to the same protein 19 FIGURE 5(b). Protein synthesis from the placement of the mRNA molecule on the ribosome to the formation of tRNA molecules that attach to their specific amino acids and then line up along the mRNA molecule causing the proper sequence and positioning of amino acids to occur resulting in the formation of a protein molecule 20 FIGURE 6. Number of individuals versus time for an idealized population growth exhibiting Sigmoid growth characteristics 21 FIGURE 7. Growth (% per day) versus RNA (yg/mg dry weight) for six species (after Sutcliffe, 1969) 24 FIGURE 8. Micrograms RNA per mg dry weights versus age (days) for cultures of Artemia salina and Euchaeta elongata (after Dagg and Littlepage , 1972) 26 FIGURE 9- FIGURE 10, FIGURE 11. FIGURE 12. FIGURE 13. FIGURE 14. FIGURE 15. FIGURE 16, FIGURE 17. Perchloric acid (PCA) precipitation of nucleic acid, phospholipids, and tissue protein from a homogenate of T. californicus 32 The hydrolysis of RNA from the PCA precipitate of the T. californicus homogenate by controlled digestion and the separation of RNA from DNA by centrifugation and extraction — 33 The digestion of DNA from the PCA precipitate after RNA was extracted off, and the spectrophotometric measurement of the deoxyribose- indole adduct formed in the process 35 The UV absorption of the RNA bases and the differential UV absorption of carried-over proteins from the combined supernatant and washings of the PCA precipitation of DNA and RNA hydrolysis step 37 DNA (yg/ml) versus absorbance (units) for a calf thymus DNA standard 33 RNA (yg/ml) versus absorbance (units) for a yeast RNA standard 39 An absorption spectrum of pure liver RNA, pure peptide contaminant, and a RNA sample after 18 hrs. hydrolysis in 1 N KOH at 37°C, to demonstrate the effect that protein carried over with the RNA extract from the PCA precipi- tated homogenate has on RNA absorption readings taken at 260 mn (after Munro, 1969) 46 RNA to DNA ratios in micrograms per milliliter to micrograms per milliliter versus population age of natural occuring populations of T. californicus 47 A superposition of Dagg and Littlepage's (1972) A. Salina plots of growth (#/day) versus age (days) RNA/DNA (yg/mg: yg/mg) versus age (days), and the author's T. californicus RNA/DNA (yg/ml: yg/ml) versus population age — ' 56 ACKNOWLEDGMENT The author wishes to thank Dr. Eugene Traganza for the basic idea from which this research is derived, and for the funding of this work which came from his NPS Foundation research grant. A special appreciation is also extended to Dr. Charles Rowell for his technical and chemical assistance. The technical aid received from Mr. Kenneth Graham, Mr. Peter Savo, and Mr. Thomas Burson added much to the completion of this work. I would also like to thank the Oceanography Office secretaries for their continual help. I. INTRODUCTION From the Navy's point of view sound transmission is a most important physical characteristic of the sea. Detec- tional, navigational, and weapons sensors all rely upon this phenomenon. Studies of sound in the sea reveal the presence of scatterers that can reduce the effectiveness of acoustic sensors [Tucker, 1957]. This sound scattering appears as a volume reverberation term and reduces the recognition differential level associated with a specific sensor and a specific target. This scattering has been associated with zooplankton in the sea [Traganza and Stewart, 1973]. Zooplankton levels are not constant in that there are marked diurnal, seasonal, and regional variations [Cushing, 1959]. While zooplankton biomass variability does not cause fluctuations in low frequency volume reverberation levels directly, it indirectly influences the levels through the food chain. Therefore, a measurement of the rate of biomass change could lead to a prediction of biological volume reverberation fluctuations. Models designed to estimate the growth rate of zooplank- ton populations are based on field measurements of a change in biomass. This type of measurement requires two biomass samples over a period of time. Current techniques are plagued with too many variables to make these measurements 10 anything but a rough estimate [Pease, 1973]. In addition to the variability, the amount of time, equipment, and personnel needed to produce these measurements make this method a very limited procedure; that is, not an in_ situ measurement . In the search for better methods of biomass measurement the field of marine biochemistry holds a great potential [Riley and Chester, 1971]. Chemical measurements may be made quickly, accurately, and with certainty. Early research in this concern approaches the idea of relating a cellular constituent like ribonucleic acid or deoxyribonucleic acid to a population growth rate [Sutcliffe, 19653- A specific chemical parameter has not yet been accurately related to predicting growth rates in marine zooplankton. It is to this end that this research was undertaken. 11 II. HISTORICAL Current thought holds that RNA is a necessary precursor to protein synthesis [Brachet, I960; Roth, 1961; Sutcliffe, 1965; Miller, 1969; Hoagland, 1959; Clark and Marcker, 1968; Yanofsky, 1967; Gale, 1956; Romberg, i960]. To better understand this relationship between RNA and protein synthe- sis, it is necessary to examine the basic chemistry of RNA, DNA, and protein molecules. Early cytochemical observations showed that RNA was abun- dant in rapidly growing cells such as onion root tips. At the same time it was found that cells with a high physiological activity but with a slow growth rate such as heart muscle, contained little RNA. THese observations lead to the conclu- sion that cells that synthesize large amounts of protein contain large amounts of RNA. Qualitative observations have shown that there is a direct correlation between RNA synthesis and the synthesis of proteins in populations of exponentially growing cells [Brachet, 1959]. The role of RNA is inseparably related to the equally important DNA. The Nobel prize winning work of Watson and Crick in discovering the structure of the DNA molecule was the basis for understanding the DNA control and continuance of cell functions [Crick, 1968]. DNA is the carrier of the genetic message from one generation to the next, and thus is the basis for the control and continuance of cellular 12 functions. This means that it is DNA that ensures that progeny carry on similar functions over many generations [Kornberg, i960]. These two DNA functions of control and continuance are executed by the actions of RNA but can be best understood by examining the basic structure of a DNA molecule. DNA and RNA molecules are composed of an ordered se- quence of basic units known as nucleotides. A nucleotide is structured from a sugar, a nitrogenous base, and a phos- phate group as illustrated in Figure 1. Two linear chains of complimentary nucleotides bonded together in the form of a double helix are the basis for DNA molecular structure. The two complimentary chains of nucleotides are joined across the central axis of the double helix by hydrogen bonding between base pairs (see Figure 2). RNA structure is similar except for the following: ribose replaces deoxyribose, uracil replaces thymine, and RNA occurs as a single, coiled chain. DNA's abilities to continue and direct cell functions originate with the nature of the bonding between bases in the complimentary nucleotides in the DNA helix. The physical size of the different bases dictates that purines can only bond to pyrimidines and still fit the DNA helical structure (see Figure 3). Linear groups of 3 nucleotides (a codon) occuring along one strand of a DNA molecule can be. associated to a specific 13 DNA NUCLEOTIDE □ - BASE SUGAR PHOSPHATE 1 DEOXYRIBOSE RNA NUCLEOTIDE _ ® ® SUGAR I' RIBOSE Fiqure 1. The basic three-cart structure of a DNA and an RNA nucleotide. 14 HYDROGEN BONDING IN BASE PAIRS GUANINE O . cxmsiNE _ H— N H C— H ADENINE THYMINE H—N— H- ^h C— H o ii -H N- V c — c — o=c. •N H c— H ^H-h n / -c — c c- I I I H H H Figure 2. Hydroqen bonding as it occurs between comnl i men tary base Dairs in the DfiA double helix or between the different forms of DMA and RNA as found in nrotein synthesis. 15 guanine / J I N — \ H i i / A, *H DNA central axis — — — ^ ■;'L/- DNA molecular diameter o -7 A •N- N o=c ^ C — c — N' _» V-X. -11 % diameter of Durine bases * o 4 A -H — M. ode n i ne r V X. N ^ PURINES diameter of nyrimidine bases O t hym j ne || I N' o=c ,C~ \ / I c— c c ( I I PYRIMIDINES Fiqure 3. The physical size relationship between comnlimen tary base oairs and the DNA molecule is such so that only purines can bond to nyrimidines and vice-versa. 16 amino acid from which proteins are built (see Figure 4) . Thus a section of DNA molecule with the proper number of codons can be associated to any protein molecule [Kornberg, I960]. DNA directs the building of a protein molecule in- directly through mRNA, tRNA, and ribosomal RNA (see Figure 5). The complimentary bonding between base pairs in DNA and RNA molecules is the basis for this method of protein structure . In any given cell the more proteins that are to be synthesized, the more RNA that must be present to perform this function. Thus a measure of the RNA concentration in a cell will give some idea of the rate of protein synthesis. Essentially DNA is entirely located in the nucleus of the cell (except in bacterial and uiral forms) and forms the genes characteristic of that cell species. Any given mature organism will have a characteristic number of genes (diploid) , the same for all mature cells excepting sex cells in that organism (haploid) . With the same number of genes, the same amount of DNA will be found in the cells and a characteristic amount of DNA can be associated to any organism at a given time in its life. This idea provides a basis for measuring the number of cells present in an organism or in a population [Mirsky and Osawa, 1961]. Many populations go through a sigmoid growth curve [Miller, 1969], with four distinct stages of growth (see Figure 6). Each stage is characterized by its particular 17 DNA molecule + composed of linear arrangements of codons a codon describes an amino acid an amino acid is part of a protein molecule + protein molecule formed by linear arrangement of amino acids linear arrangement of codons determines linear arrangement of amino acids * protein molecule Fiaure 4. The loqical sequence of the formation of a snecific amino acid from a specific order of codons on a DNA strand. 18 FUNCTION control and continuation k — codon 1 — J< — codon 2 J A G C T T A I 1 i i i i r i i i i i i i i 1 1 1 1 1 1 i I i G A T C C G nucleotide unit complimentary hydrogen bonding DNA double helix as normally found in the interphase nucleus — sequence of codons determines protein to be synthesized codon 1 CO don 2 ENACTMENT A G C T T A formation of "protein message" i i ! i 1 1 1 1 i • . ■ i i i t l l carrier in nucleus G A T C C G 4- single "unwound" DNA strand mRNA molecule formed in nucleus mRNA migrates to the cytoplasm to the ribosomes , single DNA strand "r-ewinds" with its complimentary strand codon 1 codon 2 ESTABLISHMENT OF CONSTRUCTION SITE protein message carrier in cytoplasm + G A | T C C A I mRNA molecule ribosomal RNA structure base Figure 5(a). The beqinninqs of nrotein synthesis from the ''protein messaae" carried on the DNA molecule to the forma- tion of a mRNA molecule that corresoonds to the same nrotein 19 RAW MATERIALS SUPPLY attachment of amino acids to tRNA molecules ana migration to ribosomes ammo acid 1 tRNA molecule anti-codon 1 amino acid 2 1 1 T T G tRNA molecule anti-codon 2 This is a cytochemical process -peptide bond CONSTRUCTION 4. formation of protein molecule resulting from pairing of codons and anti-codons on the ribosomal RNA structure amino acids 1 II 1 A G c T T G 1 1 1 1 ( 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 1 G A T C C A g V j^ M M H tRNA mRNA ribosomal RNA proper allignrcent of amino acids results from specific allignment of tRNA molecules along the mRNA molecule's axis Fiaure 5(b). Protein synthesis from the placement of the mRNA molecule on the ribosome to the formation of tRNA mole- cules that attach to their specific amino acids and then line up alonq the mRNA molecule causina the proper seauence and positioning of amino acids to occur resultinn in the formation of a protein molecule. 20 CO Q o TIME -> Figure 6. Number of individuals versus time for an idealized population qrowth exhibitina Siqmoid arowth characteristics. 21 type of growth. Stage I is an initial growth stage with a small number of individuals, but with a large progeny potential. Stage II is the exponential growth stage where young are rapidly produced and the old members die off. Stage III Is a maturation period where the young age and where no net progeny are produced. Stage IV growth depends upon the population dynamics of the particular species. If the number of new progeny equals the number of deaths, the population reaches steady state. Likewise, if the death rate or removal rate is higher than the birth rate, the population will decrease in number. It is during this stage that environmental interactions with the population have a large effect upon the population numbers. An individual organism goes through stages very similar to sigmoid population dynamics. A newly born individual goes through an initial hyperplastic stage characterized by a rapid increase in cell numbers. This is followed by a mixed stage in which hyperplastic and hypertrophic (cytoplasmic) growth occurs [Miller, 1969]. It has been shown that protein synthesis is a necessary precursor to cell growth, and that RNA production is directly related to the rate of protein synthesis. Thus one could expect to find the highest relative concentration of RNA in the fastest growing individuals, Sigmoid stage II or mixed hyperplastic and hypertrophic stages; with decreasing relative concentrations of RNA being found in older and 22 slower growing individuals. Furthermore, so basic a meta- bolic process, such as protein synthesis, is not likely to be species specific within broad limits. Thus the RNA- growth relationship from individual organisms should be applicable to populations of organisms, and RNA concentra- tions as normalized with DNA concentrations might permit a prediction of growth rate and growth stage, even when applied to mixed populations, such as zooplankton. In 1965 W. H. Sutcliffe, Jr. was one of the first researchers to obtain significant results indicating RNA- growth relationships [Sutcliffe, 1965]. Sutcliffe used synchronous cultures of Artemia salina and Nassarsisus obsoletus as test organisms. He made RNA-to-dry-weight determinations on the amphipod Orchestia platensis and used this result to produce a growth rate-RNA relationship. He then applied his growth rate model to the mud snail and brine shrimp cultures' actual growth and found that a reasonable correlation existed between these cultures and cultures of 0. platensis. Sutcliffe then concluded that the RNA-growth rate relationship could be useful in predicting growth rates in other psecies or mixed populations. Sut- cliffe extended his early work by comparing a collection of curves of growth rate-RNA relationships that involved 2k species (see Figure 7). Again he suggested that this data showed a positive relationship between growth rate and RNA concentration [Sutcliffe, 19693. 23 lOOOOt 1000 * □ vs. o O 100 10 *_ 1 5 mjc roorgani sms £-Aedes aegypt i o-coc kroach n - bacteria X~ amphipod X~l ru it { t y °o° Xo 1 10 100 1000 RNA ( g/mg dry weight) Fiaure 7. Growth [% ner day) versus RNA ( a/ma dry weinht) for six snecies (after Daaq and Li ttl enane , ]°7?) . 2k In contrast to Sutcliffe's work are the results of the work done by Dagg and Littlepage [Dagg, 1972]. Dagg and Llttlepage collected the copepod Euchaeta elongata and used Artemla sallna to develop synchronous cultures from which to work. They made analysis of RNA versus dry weight, protein, DNA,and percent growth to examine the RNA-growth rate relationship. The results of their work showed that neither Sutcliffe's 1965 RNA-growth rate equation nor their own equation derived from Artemia salina growth, accurately predicted growth rates for Euchaeta elongata. Dagg and Littlepage concluded that even though there was a statisti- cally significant relationship between growth rates and RNA/dry wt. ratios in A. salina and E. elongata, the wide range of growth rates associated with a small range of RNA/dry wt . ratios caused the relationship to lack sufficient acuity to be used in a RNA-growth rate growth prediction (see Figure 8) . Sutcliffe's work was tested by Pease [1968]. Pease found that Sutcliffe's relationship was valid only during the most rapid growth stage of the experimental organism. He concluded that the growth rate to RNA relationships were specific only to organisms in their most rapid growth stages, and that the rest of the time they were related only to the organism from which they were derived. Notwithstanding their difference of opinion on the applicability of the general trend in the RNA-growth rate relationships as seen in the results of Pease, Sutcliffe and 25 30 I 20 DC c 10 Euchaeta elonaata -i— M A AGE (months) -t- M 60 40 i- < ex: 20 Artemi a sal i na 20 60 40 AGE (days) Figure R. "i crograms RNA oer mo dry weinhts versus aoe (days) for cultures of Artemi a s a 1 i n a and Euchaeta el on oat, a (after Daqa and Littlenaoe, 1^72). 26 Dagg and LIttlepage, Is the work of a host of others, using a spectrum of organisms [Leick, 1968; Vickers and Mitlin, 1965-66; Haines, 1973; Lay, 1965; Bulow, 1970]. The direct application of RNA growth rate predictions to marine zooplankton populations is as yet untested. 27 III. EXPERIMENTAL PROCEDURE A. COLLECTION AND HANDLING OF T. CALIFORNICUS Populations of Tlgriopus californicus were collected in splash pools found above the mean high-water mark. The pools were found along the rocks that line the beach around Lover's Point at the southern end of Monterey Bay, California. The animals were scooped from the pools with a number 10 plankton bucket. Presumably natural food found in the splash pools was transported with the copepods. Identification of the species was simplified insofar as T. californicus is the only species of marine copepod found in splash pools above the mean high-water mark along Monterey Bay and that they possess a distinctive reddish-orange color [Egloff, 1966]. Micro- scopic examination confirmed the visual analysis and alos indicated that the catches were homospecific. Once collected, the copepods were kept in plastic containers at room temperature (ca. 23°C) near a source of sunlight (the copepods are algae feeders). Those copepods that were to be kept over a period of time longer than a week were refrigerated at 8°C. A Pasteur pipette with a length of thin tubing for applying suction by mouth was used to separate experimental groups, e.g., gravid females, from the others. Immediately prior to analysis the copepods were filtered on to a millipore 0.45 um HA filter and washed with distilled water. 28 The organisms were "dried" for weighing by being scraped onto a dry Whatman number 3 filter paper. They were then manually separated from the debris into a plastic weighing dish and weighed to the nearest tenth of a milligram (mg) . B. PRE-ANALYSIS A glass tissue grinder, distilled water, 0.6 normal (N) perchloric acid (PCA) , and 1.2 N PCA were placed over ice prior to analysis. Two water baths were set up sufficiently far in advance so that they had stabilized at temperatures of 37°C and 100"C at the time of analysis. A 200 mg "semi-dry weight" sample of copepods, ca. 1000, was taken from the experimental group being analyzed and was placed into an ice packed tissue grinder with ice-cold distilled water. The copepods were ground for 10 minutes or until a fine homogenate was formed. C. SAMPLE ANALYSIS The Schmidt-Thannhauser method [Schmidt, 19^5] as modified by Munro and Fleck [Munro, 1969] was used to separate and measure the RNA fraction of the sample. Interfering protein was checked by using differential ultra- violet (UV) spectrometic techniques [Munro, 1969]. DNA concentration was found by employing Ceriotti's indole method [Cerriotti, 1952] as modified by Munro and Fleck [Munro, 1969]. 29 D. PREPARATION OF STANDARDS DNA stock solution standards were prepared by dissolving 20 mg of calf thymus DNA in 50 milliliters (ml) distilled water. Five ml of 1 N NaOH were added to the DNA to aid the dissolution of the thymus strands by ensuring the separation of DNA molecules in a basic solution. It took 30 minutes for the thymus to dissolve, as heating is prohibited to prevent the breakdown of the DNA molecules. The stock solution was kept refrigerated until needed. Working solutions were prepared by diluting the stock standard 1:25 with distilled water to produce a standard solution of 32 micrograms (yg) DNA/ml from which multiple dilutions were made. A RNA stock solution standard was prepared by dissolving 32 mg of yeast RNA in 1 liter (1) of 0.1 N PCA. The solution was heated in a boiling water bath to dissolve the RNA. The stock solution containing 32 yg of RNA/ml was refrigerated until needed. Working solutions were made by diluting 5 ml portions of the stock solution with 0.1 N PCA. A protein standard was prepared by dissolving 5 mg bovine serum albumin in 50 ml distilled water. This produced a stock solution of 100 yg protein per ml of solution. Repeated analysis of all of the stock standards over a period of three months indicated that the standards did not deteriorate to any detectable extent. 30 E. PRECIPITATION OP RNA AND DNA Two and one-half ml of ice-cold 0.6 N PCA were added to 50 ml of copepod homogenate in a centrifuge tube. The centrifuge tube contents were mixed and allowed to stand for ten minutes. The mixture was centrifuged and the supernatant was discarded. The residue was washed with 5.0 ml 0.2 N PCA. The supernatant was again discarded after centrifuging. The residue wash was repeated with another 5.0 ml 0.2 N PCA to ensure precipitation of all of the nucleic acid molecules. The supernatant was drawn off with a Pasteur pipette and discarded (see Figure 9). F. HYDROLYSIS OF RNA MACRO-MOLECULAR STRUCTURE Four milliliters 0.3 N KOH was added to the residue and this combination was heated for one hour at 37° C. Munro and Fleck, 1969 j showed that the least amount of protein carry- over into the RNA solution will occur at this combination of temperature and time (there is some danger of digesting protein molecules at higher temperatures). Two and one-half milliliters of cold 1.2 N PCA were added and the mixture was stirred. The mixture was cooled for ten minutes in an ice bath and was then centrifuged. The supernatant was held for RNA determination and the residue was used for DNA determination (see Figure 10). G. DIGESTION AND MEASUREMENT OF DNA The precipitate from above was washed twice with 5 ml of 0.2 N PAC and the washings combined with the supernatant 31 I SAMPLE 1 :20 in ice cold K2O HCtfCGENATE T (250 ms. wet wt. ) 5 ml. + 2.5 ml. ice cold 0.6 N PCa and mix LET STAND 10 KIN. CENTRIFUGE 1-- precipitate WASH WITH 9.2 N PCA (super) BISCaRD ->( super) DISCARD DRAIN tflTH Pasteur pipette — ^( super) DISCARD Figure 9. Perchloric' acid (PCA) precipitation of nucleic, acid, phospholipids, arid tissue protein from a homo gen ate of T. cal i form' cus . 32 Precipitate Containing Nucleic Acids, etc. HEAT FOR 1 Hr. AT 37° C. add 2.5 ml. cold 1.2 N. PC-a, stir COOL FOR 10 MIN. OVER ICE ( precipitate )<- To DNA Analysis CENTRIFUGE -^(super) To RNA and Protein Analysis Figure 10. The hydrolysis of RNA from the PCA precipi- tate of the T. cal i form' cus homoqenate by controlled digestion and the separation of RNA from DNA by centrifu- gation and extraction. 33 from above and saved for the RNA analysis. Four milli- liters of 0.3 N KOH was added to the precipitate and the mixture was heated to a high temperature in a Bunsen flame until the precipitate was digested indicating that the DNA molecules had been broken up into basic nucleotide units. Twelve milliliters of 0.3 N KOH was added to. the digestion and the total volume was brought up to 50 ml with distilled water. To 2.0 ml of this solution was added 1.0 ml of concentrated HC1 and 1.0 ml of 0.04$ indole color reagent. The mixture was heated for 15 minutes in a boiling water bath then cooled in running water and extracted three times with CHC1 . The last extraction was centrifuged at 300 revolutions per minute for five minutes before the CHCl^ was removed. The extract was discarded and the absorbance of the aqueous layer was read at 490 nm to find the relative DNA concentration (see Figure 11). H. MEASUREMENT OF RNA AND THE PROTEIN CHECK Ten milliliters 0.6 N PCA was added to the RNA super- natant which was combined with the RNA washings from the DNA digestion step. The solution was made to 100 ml with distilled water. The absorbance of the solution was read at 260 nm in order to measure the relative RNA concentrations The optical absorbance was also read at 280 nm to obtain a differential protein value. Munro and Fleck, 1969, showed that RNA has a major absorption peak at 260 nm, while pure bovine serum protein has its major absorption peak at 280 nm. 34 DNA Containing Precipitate W^SH TwICE WITH — >( super) 5 ml. 0.211 jFCA to the precipitate add 4 ml.0.3N KOH Save for RNA Analysis HEAT TILL DISSOLVES iadd 12 ml. ; 0.3N, KOH BRING VOLUME TO 50 ml. WITH H20 take 2 ml. + 1 ml. concentrated HCL + 1 ml. o.o4% indole, mix 1 ' HEAT FOR 15 mm. IN BOILING H2C B^iTH cool in me running H?0 EXTRACT 3 TINES WITH CHCI3 (extract) -> DISCARD Measure DNA Absorbance of Solution at 490 nm. Figure 11. The digestion of DNA from the PCA precipitate after RNA was extracted off, and the snectrophotometri c measurement of the deoxyri bose-i ndol e adduct formed in the process. 35 They also ran a series of absorption spectra for different RNA, RNA and protein, and protein samples, all of known concentrations. From these values they determined readings at 280 nm that would indicate significant amounts of protein carried over into the RNA extract that would give erroneous relative RNA concentrations. Therefore the reading is made at 280 nm to ensure that a protein free RNA extraction had been performed (see Figure 12). 36 Washings from DNA Precipitate Combined with Supernatant from RNA Hydrolysis step add 10 ml. 0.6N PC A, make to 100 ml. with distilled H20 Measure RNA Absorbance at 260 nm. Measure Protein Absorbance at 280 nm. Figure 12. The UV absorption of the RNA bases and the differential UV absorption of carried-over proteins from the combined supernatant and washings of the PCA precipitation of DNA and RNA hydrolysis step. 37 .4 .8 1.0 ABSORBANCE (UNITS) Figure 13. DNA (yg/ml versus absorbance (units) for a calf thymus DNA standard. 38 60 ._ a. 40 35 30 25 20 15 10 .1 .2 .3 .4 .5 .6 ABSORBMCE (UNITS.) Figure 14. RNA (y/ml ) versus absorbance (units) for a yeast RNA standard. 39 IV. RESULTS Data was obtained on five groups of T. californlcus , each group representing a different stage in the development of a population. RNA and DNA analyses were applied to each group in duplicate.' Each experimental group was composed of approximately 500 individuals (about 200 mg dry weight body mass). Growth stages were established by the relative size of individuals within each group, or by collecting copepods from splash pools predominated by young or old individuals (see Table I). The five experimental groups consisted of the following collections of copepods: (1) The old group was taken from a natural wild population observed to have existed over a period of at least two months. (2) The young group was collected from a new splash pool and the older individuals were removed from the group . (3) The all-gravid- female group was made by collecting gravid females from several splash pools. (lj) An all-but-gravid-female group was then composed of the individuals left after the gravid females were removed from those collections. (5) A group of undetermined mixture was taken from a wild population found in the splash zone. i»0 Table I. A description of the composition of the five experimental groups used in the analysis. GROUP old young all gravid females all but gravid females mixed DESCRIPTION 100 gravid females per 1000 — mostly large in size (2-3 mm.) 10 gravid females per 1000 — many small individ- uals (l mm. or less) many mating pairs all late stage gravid females (black egg sacks) mixed population from which gravid females were removed natural population in late stage of development but not fully documented 41 The results of the duplicate experimental runs are shown in Table II. The all-gravid female group had the highest RNA/DNA ratio of 4.62. This was followed by the "young" group with a ratio of 3.84. The "mixed" group had a ratio of 3.15 and was followed by the all-but-gravid-female group with a ratio of 2.l6. The "old" group had the lowest ratio of 1.082. Run 2 was taken from the same homogenate as the first run, but was performed after the completion of the first run. Table III indicates the precision of the measurements made. Possible protein carryover in the RNA extraction was estimated by taking UV absorbance readings at 260 nm and 280 nm for the range of protein standards. The results are given in Table IV. Munro and Fleck [1969] ran absorption spectra for pure RNA, a RNA fraction contaminated with peptide but digested as in their experimental procedure, and the peptide material after digestion. The results are shown in Figure 15 and will be used in conjunction with the data in Table IV to establish the status of protein carryover in the RNA extraction procedure. A plot of RNA to DNA ratios versus population age is shown in Figure 16. • 42 Table II. DNA and RNA values In micrograms per milliliter for all the experimental data taken. GROUP DNA RATIO RNA RUN I RUN II old 6.82 = 1.084 6.29 6.80 = 1.079 6.30 young 43.28 = 3.840 11.27 43.24 = 3.843 11.15 all gravid females 19.414 = 4.622 4.20 9.707 = 4.627 2.098 all but ' 11.72 = 2.158 gravid females 5.43 5.095 = 2.158 2.361 ; mixed 25.88 = 3.156 8.20 19.56 = 3.155 6.20 1 *Run II was performed separately from Run original homoginate. ■ I on a portion of the 43 ' Table III. DNA and RNA ratios in micrograms per milliliter to micro- grams per milliliter for both experimental runs, and the value of their mean and mean deviation. GROUP old young all gravid females RUN I 1.084 3.840 all but gravid females 4.622 2.158 mixed 3.156 RUN II MEAN MEAN DEVIATION 1.079 3.843 4.627 2.158 3.155 1.0815 3.8415 4.6245 2.158 3.155 + .0025 + .0015 + .002 + .0001 + .0004 M Table TV. Absorption values for a differential UV absorption analysis of a bovine serum albumin protein standard taken at 260 nm and 280 nm. PROTEIN CONCENTRATION Cg/ml) ABSORPTION 260 nm. 280 nm. 250 .409 .428 125 .211 .231 i 1 62.5 .102 .110 f 32.25 * .03 ! *below minimum sensitivity level • i '15 1-2 i o 200 220 240 260 280 300 WAVE LENGTH (nm) Figure 15. An absorption spectrum of Dure liver RMA, pure peptide contaminant, and a RNA sample after IP hrs. hy- drolysis in 1 M KOH at 37°C, to demonstrate the effect that protein carried over with the RNA extract from the PCA oreci pi tated homoqenate has on RMA absorption readinos taken at 260 mn (after Munro, 1969). H6 4' 3 D 3- ex: a 1 young mixed all but old q r a v i d POPULATION ARE all qravi d Fiqure 16. RNA to DMA ratios in micronrams per milliliter to micrograms o&r milliliter versus nonulation aoe of natural occurinq populations of T. cal i forni cus . i»7 V. DISCUSSION A. TIGRIOPUS CALIFORNICUS AS AN EXPERIMENTAL ORGANISM Tlgriopus callfornlcus is a harpacticoid, supra-littoral benthic, copepod that is related to the pelagic, planktonic, calanoid copepods. It was important to use an organism that was convenient, but at the same time was similar to typical zooplanktonic species. The Copepoda comprise over 60% of the pelagic animal families and are, as such, the most common zooplankton in number. Populations of T. californicus were very easy to obtain since they can only live in splash pools, and their species occur exclusive of any other species along thewest coast of California [Egloff, 1966]. Other kinds of organisms generally lack these characteristics, so that field collections made on splash pools contained for the most part Tigriopus californicus . Tigriopus californicus is alos a hardy species. They tolerate large temperature, salinity, and food ranges making them an excellent specimen to keep in the laboratory. T. californicus hatch from eggs and grow through six navpliar and six copepodid stages [Egloff, 1966]. Each stage can be characterized by size and by the development of distinctive features such as setae. Thus growth stages can be readily estimated by microscopic examination of the individuals present in the population. Also the location of the splash pools can give some insight as to the age of the '18 population. Those pools well above the mean high water mark contained more older and more mature individuals than fresh pools. These pools remain undisturbed the longest due to their position, and allow the copepod, population to develop since the copepods are not continually being washed away. The extent of the algae development in a pool can be related to the copepod population age. These characteristics of T. californicus were of additional value in the use of T. californicus as experimental organisms. B. CHEMICAL PROCEDURE The chemistry as modified by Munro is presumed to be the best procedures that are now available. The mean deviation for these procedures (Table III), was ± 0.002 ratio units. The high precision of the methods is apparent In the standard curves (see figures). Both DNA and RNA standards produced a negligible amount of scatter in three separate standard curve determinations (see Figures 13 and 14). At the end of the five-month experimentation period standards were run against the initial stock solutions of RNA, DNA, and protein. The absorbance was found to be the same as that originally recorded. Protein carried over into the RNA extract from the RNA digestion step was of concern to Sutcliffe in his work [Sutcliffe, 1965]. He pointed out this fault in Dagg and Littlepage's work in personal correspondence to Dr. Eugene Traganza, and recommended that a Lowry protein determination ^9 be performed on the RNA extract ir\ order to correct the value of RNA absorption for absorption from protein carryover. In modifying the basic Schmidt-Thannhauser procedure Munro went to great lengths to insure that the concentration of base, and the time and temperature of digestion would be sufficient to hydrolize all the RNA, but not the tissue proteins present. As a result one can expect not to have protein interference in the RNA absorption. However, to insure that negligible protein carryover was occurring, the UV absorption of bovine serum protein standards was made at 260 nm and 2 80 nm (Table IV) to compare with Munro 's results (Figure 15) and the results of the RNA extraction absorption readings. The low level of absorptions obtained at 280 nm from relatively high concentrations of pure protein indicates that the higher absorptions read at 2 80 nm are due to the "tail" of the RNA 260 nm spectrum curve, not to protein interference. Figure 11 from Munro indicates this RNA "tail effect". Thus by ensuring that all the readings at 280 nm were significantly less than the readings at 260 nm and that these 280 nm readings follow the trend in RNA readings at 260 nm, protein interference is not significant to the RNA absorptions. Munro's standard curves shown in Figure' 15 indicate that the RNA hydrolysis step, if performed for 15 min . in 0.3 N KOH at 37°C, will not appreciably hydrolyze any of the phospholipids and tissue proteins present in the extract. There is a degradation factor involved with the analysis of DNA. Nuclear DNA will decompose at normal room temperatures 50 Early DNA measurements indicated this when care was not taken to minimize the time between the filtering of the copepods and the time when homogenization took place. Accordingly the analysis should proceed as rapidly as possible and the homogenation must occur at low temperatures. C. PREVIOUS WORK Sutcliffe [1969] showed a positive trend between RNA concentration and growth rate during some organisms most rapid growth stage. Dagg and Littlepage [1972] pointed out that this trend generally holds, but only during the most rapid period of growth. They maintained that the trend was non-specific at other growth times. Sutcliffe [1969] himself has admitted that his data lacked a sufficient number of points to make the RNA-growth rate trend specific to an entire life-cycle as well as to life cycles of different organisms. Thus the RNA-growth rate relationship can only be considered well defined during the organisms most rapid growth period. Much of the work done along this line has involved the use of synchronous, laboratory cultures. Apparently no one has tried to extend the RNA-growth rate trend to a natural occurring population of mixed organisms or even of mixed growth stages, i.e., some young, some old, some maturing. Prom these facts the author feels that if a meaningful and workable relationship between RNA and growth rate is to be obtained naturally occurring mixed populations should be used in the analysis. 51 Dagg and Littlepage did not check their work for protein carryover in the RNA extraction step. Though it was expected that not much error would be introduced in the RNA absorption, it was necessary to ensure that the chemical technique was not producing an erroneous RNA concentration. In personal correspondence to Dr. Eugene Traganza, Sutcliffe stated that he found up to 50% error in Dagg's first RNA readout method due to protein carryover. This source of error according to Sutcliffe could easily account for the data scatter that Dagg and Littlepage mentioned in their work. Again it becomes obvious that protein interference must be accounted for or must be prevented. D. RNA/DNA VERSUS GROWTH STAGE Figure 16 presents the relationship between RNA/DNA and growth stage as developed in this work. The arrangement of the five points is not fixed to the sequence shown in Figure 16, but can arbitrarily be shifted to any position. The arrangement shown was used since it is consistent with the RNA-growth rate relationship. The "young" group has the highest ratio of any of the mixed groups which indicates that the RNA concentration on a cellular level is the highest of any of the mixed groups. This result is expected in that the individuals in this category are young and immature. They are going through a definite growth sequence which implies that the RNA concentration should be going through a definite high level. 52 The "young" group was not a natural population. Young Individuals were added to over-emphasize the "youngness". The intentions here were to bracket the other mixed popula- tions of various ages. The "old" group was taken from a wild population and was experimentally tested as a naturally occurring group. It had the lowest RNA-DNA ratio of any of the groups. Again the result would be expected from knowledge of the RNA-protein synthesis relationship. The "old" group was mostly mature individuals with a few late stage gravid females. It appeared that most of the females had dropped their egg sacs since there were a large number of large females present without egg sacs. The "mixed" group fits In between the old and young groups. Again this trend follows since the "mixed" group could not be as young as the "young" group, but yet was not as "old" a population as was the "old" group. The lack of documentation for this group was unfortunate, for a good description would tell how close to either extreme, young or old, the group should fit. What little was known about this group indicated that it was closer to a young population than an older one and this evidence is supported by its RNA-DNA ratio. The all-but- gravid-female group was the remaining individuals from a population originally dense in gravid females. Because the population was very dense in gravid females it was presumably in a later stage of development than the young group which would explain its lower RNA/DNA ratio. In fact, this group could be likened to the old group that had few gravid females. 53 The position of this group before the gravid females were removed is indeterminate, but could be estimated to fall between the "young" and "old" group boundaries. The all- gravid- female-group represents the highest RNA/DNA ratio. It is not known whether the high RNA concentration is due to the eggs or to the females carrying the eggs. The work of Vickers and Mitlin [1965-66] showed that boll weevil eggs contained very little RNA so that it might be surmised that the high RNA concentration is related to the gravid females' high protein production rate. Since eggs are basically protein, this idea seems to be reasonable. The positioning of the point representing the all-gravid-female- group is quite debatable. The all-gravid-female-group can be looked at as representing the maximum population potential This view point would require that the all-gravid-female- group be placed after the "old" group on the population age axis since a population must mature before it can reproduce. The all-gravid-female-group can also be considered to represent the group with the greatest protein synthesis rate. This viewpoint would require the placement of the group before the "young" group on the population age axis. The all-gravid- female-group is not a real population and its placement is strictly arbitrary. What is important is that a definite trend of ratio versus growth stage has been established. Younger populations have a higher RNA/DNA ratio than do older populations. 54 Another approach to explaining the shape of Figure 16 can be made by comparing population dynamics to the charac- teristics of an individual's growth. Dagg and Littlepage's Artemla salina culture A exhibited a Sigmoid growth curve (see Figure 17). A rough plot of their RNA/DNA ratios for the same culture is shown in Figure 17 . Superimposing the two curves on similar axes (see Figure 17) shows the relationship of their RNA/DNA ratios to the Sigmoid growth stage that their synchronous culture was in. Their trend was for a decreasing RNA-DNA ratio in stage I with a minimum somewhere in stage II. From that point on through stage III the trend is for an increasing RNA-DNA ratio. When the author's five different growth stage points are placed on the same growth curves in accordance with the reasoning explained earlier in this section, a very similar curve emerges (see Figure 17). This results in the implication that individual growth tendencies can be related to population growth tendencies. There is still a large, important area that is in question, and that occurs at the end of Sigmoid stage III. The trends of the natural populations were not measured in this stage, and the synchronous cultures usually die off after they reach maturity. Data obtained in this phase would greatly help to clarify the actual shape of the growth stage to RNA/DNA curve. Too little was known about the dynamics of Tigriopus populations to predict a trend for this stage, nor was the time available to pursue measurements in this area. 55 200 3 to T3 O C7 100 1 2 Q - qrowth ("-/day) vs. aae (days ) (after D and L , 1972) for A. sal ina - RNA/DNA (ua/mq: ua/mq) vs . aae (days ) ( after D and L, 19 7?) for' A. sal ina RNA/DNA (ua/ml: uq/ml ) vs. popu- lation aqe for T. californicus 20 40 60 AGE (days) young mi xed al 1 but ol d qra vi d POPULATION AGE all qravi d Fiqure 17. A superposition of Oaqq and Little^ane's (1972^ A. sal i na plots of qrowth (D'/day) versus aae (days) RNA/DNA (pq/mq: pq/mq) versus aqe (days), and the author's T. cal i forni cus RNA/DNA (ug/ml: uq/ml ) versus pooulat'ion aqe. 56 E. APPLICABILITY Due to the large amount of time involved in collecting and preparing groups for analysis and the time necessary for the analysis (3 hours per trial), data had to be limited to boundary values. More data points would have given a clearer picture as to how the RNA/DNA-growth stage trend changes. What is important from this work is that there is a definite trend existing between RNA and growth stages of a natural, mixed population of Tigriopus. It is felt that further work along these lines of RNA/DNA-growth stage indications would better elucidate the trend that has been established. The actual value of this trend, though, is not without question. Even if the trend could be established through all growth stages, it would still have to be re-established for actual zooplankton populations. Total RNA concentrations may not be as sensitive a measure as is needed to get a firm grasp on zooplankton growth. Eighty percent of the RNA analyzed was ribosomal RNA which is a fairly constant quantity over the life span of any one species [Pan, 1961], The quantity of ribosomal RNA present in a cell is a function of cell size. Thus mature cells have a constant amount of ribosomal RNA. tRNA and mRNA are generated as needed in a cell so that at any one time ribosomal RNA is the largest contributor to the total amount of RNA present. This is a result of the molecular structure of the various forms of RNA. tRNA has a molecular weight of 10,000 to 30,000; mRNA has a molecular weight of 40,000 to 57 60,000; and ribosomal RNA ranges up to 1,300,000 [Fan,196l]. Thus ribosomal RNA Is the major contributor to RNA quantitative analysis and could act to mask out changes in the amounts of tRNA and mRNA over a cell's life. P. FUTURE METHODS The analysis could have been made more sensitive by measuring chemical, cellular quantities that are more vital to the actual rate of protein synthesis. Pease [1973] suggested that ribonuclease be used. Messenger RNA may be more directly indicative of protein synthesis than is total RNA. An enzyme that triggers protein synthesis, e.g., RNA synthetase or one that is directly involved in constructing all proteins may be a more specific growth indication than total RNA. The problem remains to isolate this indicator and to devise a chemical analysis that will accurately and in real time give a measure of the indicators concentration. Most important is the knowledge that a chemical measurement of some cellular indicator common to all zooplankton, has the potential to provide an accurate, reliable measurement of zooplankton growth rates, biomass or population. More research is necessary to determine the best course to pursue. Once this is done it could become possible to adapt the chemical analysis to an automated method. This adaption would lead to real-time, in situ growth rate measurements that could lead to the continuous, accurate prediction of zooplankton populations in the sea and ultimately the predic- tion of sound scattering through the food chains. 58 G. A CRITIQUE The author's own work indicates the importance of employing control groups in studying population characteris- tics, for his lack of using such leaves his results in some question as to interpretation. Employing controls would lead to more specific results. A control population developed in the laboratory and sampled at periodic inter- vals would give the RNA/DNA ratios more identification, and would provide information to better determine the growth stage of a natural population. All phases of collection and analysis should be well documented to provide statistics that are complete and relevant. The author could have made his statistics more complete and meaningful by fully documenting all the stages cf this work, and especially by paying particular attention to the collection stage. By carefully noting the physical location, the algae development, and the general size and number of copepods in the splash pools, a good idea of the population age can be formulated. 59 LIST OF REFERENCES 1. Brachet, Jean, The Biological Role of Ribonucleic Acids, p. 1-49, Elsevier, New York, I960. " 2. Brachet, Jean, "Nucleocytoplasmic Interactions", from The Cell, v. II, edited by Brachet and Mirsky, p. 819- 835, Academic, New York, 1961. 3. Bulow, Frank J. , "RNA-DNA Ratios as Indicators of Recent Growth Rates of a Fish", Journal of the Fisheries Research Board of Canada, v. 27, no. 12, p. 2343-49 , 1970. 4. Ceriotti, G. , "A Microchemical Determination of Desoxyribonucleic Acid" , Journal of Biological Chemistry, v. 198, p. 297-303, 1952. 5. Clark, Brian F. C, and Kjeld A. Marcker, "How Proteins Start", Scientific American, v. 218, No. 1, 1968, p. 36. 6. Crick, F. H. C. , "Nucleic Acids", Scientific American, v. 197, P. 188-200, September, 1957. 7. Cushing, D. H. , "The Seasonal Variation in Oceanic Production as a Problem in Population Dynamics" , International Council on the Exploration of the Sea, Journal Du Conseil, v. XXIV (3), p. 455-464, 1959. 8. Dagg, M. J., and J. L. Littlepage, "Relationships Between Growth Rate and RNA, DNA, Protein and Dry Weight in Artemia salina and Euchaeta elongata" , Marine Biology, v. 17, p. 162-176, 1972. 9. Egloff, David Allen, "Ecological Aspects of Sex Ratios and Reproduction in Experimental and Field Populations of the Marine Copepod Tigriopus californicus" , a Dissertation submitted to the Department of Biological Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Stanford University, August, 1966. 10. Fan, David P., and Akiko Higa, and Cyrus Levinthal, "Messenger RNA Decay and Protection", Journal of Molecular Biology, v. 8, p. 210, January-June, 1964. 11. Gale, Ernest P., "Experiments in Protein Synthesis", Scientific American, v. 194, p. 42-46, March, 1956. 60 12. Gay, Helen, "Nuclear Control of the Cell", Scientific American, v. 202, p. 126-136, January, i960. 13. Haines, T. A., "An Evaluation of RNA-DNA Ratio as a Measure of Long-Term Growth in Fish Populations", Journal of the Fisheries Research Board of Canada, v. 30, no. 2, p. 195, February, 1973. 14. Hoagland, Mahlon B. , "Nucleic Acids and Proteins", Scientific American, v. 201, p. 55-61, December, 1959. 15. Holm-Hansen, Sutcliffe, Sharp, "Measurement of DNA in the Ocean and its Ecological Significance", Limnology and Oceanography, v. 13, p. 507-514, 1966. 16. Kornberg, Arthur, "Biological Synthesis of Deoxyribo- nucleic Acid", Science, v. 101, p. 1503, I960. 17. Lang, C. A. H. Y. Lau, and D. J. Jefferson, "Protein and Nucleic Acid Changes During Growth and Aging in the Mosquito", Biochemistry Journal, v. 95, p. 372- 377, 1965. 18. Lehninger, Albert L. , "Energy Transformation in the Cell", Scientific American, v. 202, p. 102-114, May, I960. 19. Leick, Vagan, "Ratios Between Content of DNA, RNA and Protein in Different Micro-Organisms as a Function of Maximal Growth Rate", Nature, v. 217, p. 1153-1155, March, 1968. 20. Miller, S. A. "Protein Metabolism During Growth and Development", from Mammalian Protein Metabolism, v. Ill, edited by H. N. Munro, p. 185-227, Academic Press, New York, 1969. 21. Munro, H. N., and Fleck, from Mammalian Protein Meta- bolism, v. Ill, edited by H. N. Munro, p. 439-457, 465-4«3, Academic Press, New York, 1969. 22. Pease, A. K. , "The Use of Estimations of Ribonucleic Acid to Predict the Growth Rates of Zooplanktonic Organisms", MSc thesis, University of British Columbia, p. 107, 1968. 23. Pease, Alan K. , "Uptake of Radioactive Substrates by Cell Suspensions of Artemia salina and Their Subsequent Incorporation into Protein and RNA", submitted in partial fulfillment of the requirements for the degree of PH.D. at Dalhousie University, August, 1973- 61 24. Riley, J. P. and R. Chester, Introduction to Marine Chemistry, p. 271-277, Academic Press, New York, 1971. 25. Roth, Jay S. , "Ribonucleic Acid and Protein Synthesis", Journal of Chemical Education, v. 38 (4), p. 217-220, 1961. 26. Schmidt, Gerhard, and S. J. Thannhauser, "A Method for the Determination of Desoxyribonucleic Acid, RNA, and Phospho-proteins in Animal Tissue", Journal of Biological Chemistry, v. 16, p. 83-89, November-December, 19^5. 27. Sutcliffe, W. H. , Jr., "Growth Estimates from Ribonucleic Acid Content in Some Small Organisms", Limnology and Oceanography (Suppl.) 10, p. 253-358, 1965- 28. Sutcliffe, W. H. , Jr., "Relationship Between Growth Rate and Ribonucleic Acid Concentration in Some Inverte- brates" , Journal of Fisheries Research Board of Canada, v. 27, p. 606-609, 1969. 29. Traganza, E. D., and R. P. Stewart, "A Seasonal Volume Reverberation Model of the North Atlantic and North Pacific Oceans" , Journal of Underwater Acoustics (USN) , July, 1973. 30. Tucker, Gordon H., "Relation of Fishes and Other Organisms to the Scattering of Underwater Sound" , Journal of Marine Research, v. X, p. 215-239, 1951. 31. Vickers, D. H., and N. Mitlin, "Changes in Nucleic Acid Content of the Boll Weevil Anthonomus grandis Bohemian During its Development", Physiological Zoology, v. 38-39, P. 70-76, 1965-1966. 32. Watson, James D. , The Double Helix, Atheneum, New York, 1968. 33. Yanofsky, Charles, "Gene STructure and Protein Structure", Scientific American, v. 216, no. 5, p. 80, 1967. 62 INITIAL DISTRIBUTION LIST No. Copies 1. 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