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Physiological, ultrastructural and growth studies of Prymnesiophyceae coccolithophorid species subjected… Price, Laurel Lynne 1992

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PHYSIOLOGICAL, ULTRASTRUCTURAL AND GROWTH STUDIES OF PRYMNESIOPHYCEAE INCLUDING COCCOLITHOPHORID SPECIES SUBJECTED TO CONTINUOUS LIGHT AND D:N CYCLE REGIMES, AND VARIOUS PHOSPHATE CONCENTRATIONS By LAUREL LYNNE PRICE R. N., George Brown College, 1973 B . S c , The University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September  1992  ® Laurel Lynne Price,  1992  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  study.  scholarly  or for  her  I further  purposes  gain  (Signature)  Department  of  Botany  The University of British Vancouver, Canada  Date  DE-6 (2/88)  Oct.  15/92  Columbia  It  shall not  permission.  requirements that  agree  may  representatives.  financial  the  be  that  the  by  understood be  allowed  an  advanced  Library shall  permission  granted  is  for  for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ii ABSTRACT  Thirteen species of marine phytoplankton belonging to the taxonomic division Prymnesiophyceae, eleven of which were coccolithophorids, were tested for their growth, physiological, and morphological responses to continuous light and D:N cycle regimes as well as three phosphate concentrations. The Prymnesiophyceae, Isochrysis qalbana and Chrysochromulina sp. and the non-coccolith forming strain of Coccolithus pelagicus were unable to grow under continuous light. The remaining species showed a trend for lower growth rates under continuous light compared to the D:N cycle. These results suggest that for some species, diel periodicity may be beneficial. The non-coccolith forming coccolithophorids were found to be more sensitive to continuous light than the coccolith-forming strains. No significant differences in pigments, POC, PON, or C:N ratios were observed for species grown under the two light regimes. The coccolith-forming coccolithophorids contained 3-5 times more chl a and chl c than the non-coccolith forming coccolithophorids. These results suggest that the coccosphere reflects light and shades the cell. The cell compensates by producing more chl a. No differences in cell and coccolith dimensions or morphology were observed under either light regime. Four of the five coccolithophorids examined showed no significant differences in growth rates when they were grown  iii  under three different phosphate concentrations (13.0, 10.0, and 3.0 or 0.9 uM). The maximum yields were significantly lower at 3.0 and 0.9 uM compared to 13.0 and 10.0 uM phosphate concentrations for all of the coccolithophorids. Phosphate concentration did not affect coccolith formation or morphology. Efforts to induce coccolith formation in noncoccolith forming strains of E. huxleyi under phosphate and nitrate limitation were unsuccessful. Morphological changes were observed in Coccolithus neohelis under the three phosphate concentrations. At 3.0 uM phosphate, 90-100% of cells had flagella and 10-60% had one or more coccoliths, whereas at 13.0 uM phosphate 0-10% of cells had flagella and 100% had coccoliths.  iv TABLE OP CONTENTS ABSTRACT  ii  LIST OF TABLES  vi  LIST OF FIGURES  viii  ACKNOWLEDGEMENTS  X  INTRODUCTION  1  Coccolithophorid Ecology  1  Coccoliths and Their Formation  4  Objectives  8  CHAPTER 1.  CONTINUOUS LIGHT VS. 14:10 D:N CYCLES OF  ELEVEN PRYMNESIOPHYCEAE SPECIES: EFFECTS OF LIGHT REGIMES ON GROWTH RATES, PHYSIOLOGICAL PARAMETERS, AND CELL AND COCCOLITH ULTRASTRUCTURE AND MORPHOLOGY  9  Background  9  Materials and Methods  12  General Culture Maintenance  12  Growth Tests  13  Analytical Methods  15  Results  19  Growth Studies  19  Maximum Yield  25  Effects of Culture Volume  27  Cell Volume Changes  28  Pigment Analyses  32  POC, PON Analyses  36  Cellular and Coccolith Ultrastructure  42  V  Calcium Content Studies Discussion CHAPTER 2.  47 68  EFFECTS OF PHOSPHATE AND NITRATE  CONCENTRATION ON GROWTH, MAXIMUM YIELD, CELL AND COCCOLITH MORPHOLOGY, AND COCCOLITH INDUCTION  78  Background  78  Materials and Methods  81  General Culture Maintenance  81  Growth Experiments  81  Analytical Methods  82  Results  85  Growth Studies  85  Maximum Yield  85  Coccolith Formation and Induction  94  Ultrastructure  94  Discussion  101  General Conclusions  106  Future Research  108  References  109  Appendix  117  vi LIST OF TABLES  TABLE 1  Species of Prymnesiophyceae used in this study  20  2  Growth under continuous light and D:N cycle  24  3  Growth of 2 coccolithophorids under continuous light and D:N cycle  30  4  Cell and coccolith sizes of 4 coccolithophorids  46  5  Calcium counts using SEM X-ray microanalysis  58  6  Comparison of growth parameters between coccolith-forming and non-coccolith forming strains of E. huxleyi under continuous light and D:N cycle regimes  7  73  Comparison of growth parameters between coccolith-forming and non-coccolith forming strains of C. pelagicus under continuous light and D:N cycle regimes  8  Effects of phosphate concentration on growth parameters  9  93  Effects of phosphate concentration on the % of cells with coccoliths  11  89  Effects of phosphate concentration on E. huxlevi 88E growth parameters  10  74  95  Coccolith induction experiments on strains of E. huxleyi during nitrogen and phosphorus limitation  12  Effects of phosphate concentration on growth  96  vii  parameters as a % of maximum growth rate and yield using 13.0 /iM PC>4~3 as a control 13  Formulae used for pigment concentration analyses  14  117  Growth rates and pigment analyses for E. huxleyi as reported in the literature  15  102  124  Growth rates and pigment analyses for coccolithophorids and Prymnesiophyceae as reported in the literature  125  viii LIST OF FIGURES  FIGURE 1  Semi-log plots of growth curves over time for 11 species of Prymnesiophyceae/Haptophyceae  2  21  Semi-log plots of growth curves as a function of cell density over time  29  3  Plots showing changes in cell volume over time  31  4  Changes in pigment concentration over time  33  5  Changes in pigment ratios over time  35  6  Changes in F Cell - 1 and F Chi a - 1 over time  37  7  Changes in POC and PON over time  40  8  Changes in C:N ratios over time  41  9  SEM micrographs of 4 coccolithophorids grown under continuous light or a 14:10 D:N cycle  10  43  SEM X-ray micrographs for calcium detection over a 2 minute acquisition period  49  11  SEM X-ray microanalysis micrographs  59  12  Semi-log plots of growth curves as a function of vivo  in  fluorescence over time for 5 species of f  t  — T  coccolithophorids grown under 3 PO4 concentrations 13  86  Growth of E. huxleyi 88E as a function of cell density, in  vivo  fluorescence, and % cells with  coccoliths 14  90  Effects of phosphate concentration on the morphology of Coccolithus neohelis  97  ix  15  SEM micrographs of C. neohelis cell types under 3 phosphate concentrations  16  Growth curves over time as a function of in  98 vivo  fluorescence for E. huxleyi (646 and 88E) and C. pelaqicus (241 and COPEL)  118  17  Changes in pigment cell volume - 1 over time  119  18  Plots of C. pelaqicus COPEL subjected to continuous light after reaching stationary growth under D:N cycle  19  120  SEM X-ray microanalysis micrograph of a cyst-like formation observed in cultures of C. pelaqicus 241  122  X  ACKNOWLEDGEMENTS  I wish to express my sincere thanks and appreciation to my research supervisor, Dr. PAUL J. HARRISON, who has supported my work in the last few years and made this thesis possible, to Dr. LUIS OLIVEIRA for letting me use the facilities in his research laboratory, to Dr. L. SAMUELS and Mr. M. WEIS for their technical assistance, to all those in the labs who helped and encouraged me during my stay in the laboratory, to Mr. PAT HARRISON and the people in the office who have helped me in the last few years. Last but not least, to LAURIE TORMBOM and Dr. BRIAN NICHOL who always encouraged me to stick with it and helped me to solve my research problems.  1 INTRODUCTION Phytoplankton belonging to the class Prymnesiophyceae commonly called coccolithophorids are the only known group of intracellularly calcifying algae. They are predominantly oceanic but a few species are neritic (Gaarder, 1971; Okada and Honjo, 1973, 1975; Schei, 1975; Okada and Mclntyre, 1977; Heimdal and Gaarder, 1980, 1981; Reid, 1980; Hallegraeff, 1984). Since coccolithophorids are the most important calcifying organisms in the oceans and the third largest group of phytoplankton, biomineralization by them occupies a major role in the global carbon cycle. Calcification results in the transfer of carbon from the atmosphere to the lithosphere where limestone sediments constitute the largest single reservoir of carbon. Thus the formation, functions, and factors affecting coccolith synthesis and morphology become important for us to understand. Coccolithophorid Ecology and Life Cycles Coccolithophorids are unicellular autotrophic organisms. Most species are less than 20 jum in diameter and are thus members of the nanoplankton. Some species undergo what appears to be an alternation of generations between a motile and a non-motile stage with one or both of the stages forming coccoliths (species specific calcareous plates in an organic matrix). Cell division occurs mostly by longitudinal fission of the mother cell with sharing of the coccoliths and further regeneration of the coccosphere, or, rarely by  2  repeated divisions of the mother cell within the coccosphere and release of motile or non-motile daughter cells. These daughter cells may or may not form coccoliths, and the coccoliths may or may not be the same as the mother cell. Parke and Adams (1960) found this to be the case for Coccolithus pelaaicus which may alternate between a motile (Crystallolithus hyalinus) and a non-motile coccolith bearing (Coccolithus pelaaicus) phase in their life cycle. Cricosphaera carterae has also been observed to alternate between a motile diploid phase and a non-motile haploid phase (Rayns, 1962). It can be appreciated that this led to a great deal of taxonomic confusion as more information on the life cycles made it apparent that motile and non-motile phases of some species had been classified as separate species. Further confusion occurs because most of the taxonomy is based on coccolith morphology. Coccolith malformations have been found to occur in Emiliania huxleyi and are apparently related to temperature (Watabe and Wilbur, 1966) or possibly a nutrient deficiency (Okada and Honjo, 1973; Wilbur and Watabe, 1963). Watabe and Wilbur (1966) found that above and below the maximum growth temperature of 18°C, coccolith malformations increased. However, malformed coccolithophores are rarely observed in tropical to temperate pelagic areas (Okada and Honjo, 1975), but may often occur in subarctic blooms (Okada and Honjo, 1973) and neritic and marginal waters of the western Pacific. Very  3  little work has been done on this group's physiology especially with regard to nutrient and light responses which may elucidate the causes of coccolith malformations. Since most coccolithophorids are photosynthetic organisms, they are restricted to the photic zone, with maximum population densities found between 50 and 150 m in most oceanic areas (Okada and Honjo, 1973). This group of organisms also has a fairly ubiquitous geographic distribution in both oceanic and neritic environments (Okada and Honjo, 1973), with Emiliania huxleyi having the widest distributions and Cricosphaera spp. being restricted to inshore waters (Smayda, 1958). Even though coccolithophorids have only about 200 species, quantitatively (cells 1 ) , they come third after the diatoms and dinoflagellates, respectively. Very often in oligotrophic oceanic areas, they may be more abundant than dinoflagellates, and may outnumber diatoms in some inshore areas (Gaarder, 1971). Based on coccolith formation being light dependent and the motile and non-motile (with or without coccoliths) life cycle phases existing for many species, it has been postulated that the formation of coccoliths may be a means of taking advantage of the nutricline (Munk and Riley, 1952; Powys unpubl. results, 1986). Munk and Riley (1952) pointed out the possible advantages of sinking as a way of reaching regions of greater nutrient concentrations, and increasing the rate of nutrient supply to the cell membrane by disturbing the unstirred layers around the cell. Powys  4  (1986) speculated that the nutrient replete motile cell migrates upward toward higher light intensities to undergo greater photosynthesis and growth. The higher light intensities and subsequent lower nutrient concentrations stimulate coccolith formation. The coccosphere increases the cell's weight, allowing it to sink out of the low nutrient environment into more nutrient rich water where cell division takes place resulting in the motile stage again. Raven (1976, 1980) speculated that flagella are also important in both decreasing the thickness of unstirred layers around the cells and in moving the cells to areas with higher nutrient concentrations.  Coccoliths and Their Formation The delicately shaped CaC03 crystallites (1.5-50 /JTCI in diameter) are formed within specialized cisternae of the Golgi apparatus (Manton and Leedale, 1963; Wilbur and Watabe, 1963) and are mostly found in the calcite form and rarely in the aragonite or vaterite forms (Wilbur and Watabe, 1963). These crystallites are called coccoliths, crystalloliths, placoliths, etc. depending upon their morphology (Klaveness, 1972a; Klaveness and Paasche, 1979). Formation of the coccoliths is a complex process involving the synthesis of both organic and inorganic components and their assembly in an ordered manner. The formation of these coccoliths is thought to be regulated by a complicated acidic polysaccharide, containing Ca2+-binding carboxyl and  5  ester-sulphate groups (De Jong et al., 1976; van der Wal et al., 1983; van Emburg et al., 1986; Kok et al., 1986; Borman et al., 1987; Westbroek et al., 1989) which bind Ca 2 + preferentially from a medium containing Na + and Mg 2 + . All coccolithophorids examined to date, except Emiliania huxleyi, have an organic baseplate as a constituent of the coccolith. It has been postulated that the coccolith polysaccharide acts as a nucleating agent and functions as a matrix in the calcification process. ATPase activity has been found at the sites of the coccolith vesicles (Klaveness, 1976) and a Ca2+-ATPase has been isolated from the coccolithophorid Cricosphaera roscoffensis by Okazaki et al. (1984). Alkaline phosphatase has also been detected at the sites of calcification by McComb et al. (1979) who also showed that alkaline phosphatase may be the same as a Ca 2 + ATPase. Both of these enzymes have been connected with calcium transport. The formation of coccoliths requires the uptake of carbon and calcium ions, the synthesis of organic compounds, the transport of these elements into the coccolith vesicle, and their ordered assembly. Calcification is light dependent. The ratio of carbon taken up for photosynthesis and calcification is close to one (Wilbur and Watabe, 1963; Paasche, 1964, 1965). Paasche (1966) observed coccolith production to be greatest in red and blue wavelengths, and during very low production rates in the dark. This is understandable when the  6  coccolithophorid maximum is usually found between 50-150 m in the photic zone (Okada and Honjo, 1973) under natural conditions. Emiliania huxleyi was shown to use both CO2 and HC0 3 ~ in photosynthesis (Paasche, 1964). Sikes et al. (1980) revealed that CO2 was the substrate for photosynthesis and HCC>3~ was the form of carbon for calcification. Nimer and Merrett (1992) found that a high-calcifying strain of E. huxleyi (strain 88E) utilized HC03~ as the preferred carbon source for photosysthesis. Coccoliths are made of the calcite form of CaCC>3, but it has been found that under nitrogen limitation, coccoliths of Emiliania huxleyi are composed of the aragonite and vaterite forms of CaC03 (Wilbur and Watabe, 1963). This implies that these cells are unable to sufficiently produce the polysaccharide matrix necessary for coccolith formation, but can still transport Ca 2 + ions into the coccolith vesicle. The most extensively studied of the coccolithophorids has been Emiliania huxleyi even though it is not a typical example of its class for at least two reasons; it is easy to maintain in culture and its coccoliths do not have an organic baseplate. Also, under laboratory culture conditions E. huxleyi gradually loses the ability to produce coccoliths (Mjaaland, 1956; Paasche, 1963; Klaveness and Paasche, 1979). A culture of 100% coccolith-forming cells (C-cells) gradually becomes a culture of all non-coccolith forming or naked cells (N-cells).  7  Another cell type that appears under laboratory culture conditions are motile, scale-forming cells (S-cells) (Klaveness and Paasche, 1971). When viewed under a light microscope, both N-cells and S-cells appear naked. S-cells may be distinguished from N-cells under the light microscope only if the X-body and/or flagella are apparent. N-cells may not produce coccoliths, but they still have a coccolith vesicle and are identical ultrastructurally to C-cells when observed under the electron microscope (Klaveness and Paasche, 1971; Klaveness, 1972b). S-cells and N-cells may only be positively identified under TEM (transmission electron microscopy). C-cells and S-cells were thought to be part of the life cycle of E. huxleyi (Klaveness and Paasche, 1971; Klaveness, 1972b), whereas the change from C-cells to N-cells was thought to be a permanent effect of culture conditions (Klaveness and Paasche, 1971; Klaveness, 1972a). Since N-cells have never been reported under natural oceanic conditions, but deformed coccoliths have been reported (Okada and Honjo, 1973, 1975; Wilbur and Watabe, 1966) there have been several attempts to induce recalcification in E. huxleyi. Recalcification of S-cells has been reported when the cells were subjected to nitrate limitation (Wilbur and Watabe, 1963; Paasche and Klaveness, 1970). It was not stated whether the recalcified cells could be isolated and maintained in a calcified condition by Wilbur and Watabe (1963), but Paasche and Klaveness (1970) reported that only a few cells produced coccoliths and they  8  could reproduce the results of the experiment. Subjecting the clone E. huxleyi 45IB to trace metal, phosphate, and nitrate limitation did not induce coccolith formation (Sikes and Wilbur, 1980). Andersen (unpubl. results) was able to induce coccolith formation in clones of N-cells but not Scells under phosphate limitation and not under nitrate limitation. The function of coccoliths in coccolithophorids has long been investigated with inconclusive results even though some factors affecting coccolith formation and mechanisms of calcification have been elucidated.  OBJECTIVES This thesis addresses the following three objectives: 1. To increase our knowledge of coccolithophorid physiological and morphological responses under two light regimes (continuous light and a 14:10 D:N cycle). 2. To provide a better understanding of coccolithophorid growth and morphology under various phosphate concentrations. 3. To attempt coccolith induction in clones of noncoccolith forming strains of Emiliania huxleyi under nitrate and phosphate limitation.  9 Chapter 1. CONTINUOUS LIGHT VS. 14:10 D:N CYCLES OF ELEVEN PRYMNESIOPHYCEAE SPECIES: EFFECTS OF LIGHT REGIMES ON GROWTH RATES, PHYSIOLOGICAL PARAMETERS, AND CELL AND COCCOLITH ULTRASTRUCTURE AND MORPHOMETRY  BACKGROUND The preceding remarks on coccolithophorid ecology and coccolith formation in the introduction outlined the importance of these organisms to the marine environment and the global carbon cycle (global warming). While the literature is extensive with studies on coccolith formation, comparatively little is known about the physiological and morphological responses of coccolithophorids to environmental factors such as light and nutrient variations. Clearly this is important since light regimes and nutrient (phosphate and nitrate) concentrations probably affect coccolith formation, thereby influencing global warming by —o  reducing or increasing the rate of CO3 * (from atmospheric CO2 flux across the air-sea interface) fixed into CaC03 (coccoliths) by coccolithophorids. The daily day:night cycle is one of the most predictable environmental factors to which phytoplankton are exposed. One would thus expect phytoplankton to have adapted various ecological and cellular responses based upon daily light fluctuations. It may even be probable that some cellular functions may require a day:night cycle (Brand and Guillard, 1981). This may be more likely for oceanic temperate and tropical phytoplankton which do not as a rule  10  experience the water turbidity changes and vertical mixing that coastal phytoplankton encounter. The effects of photoperiods on several unicellular algal species have been examined by a number of researchers (Castenholz, 1964; Paasche, 1967, 1968b; Durbin, 1974; Hobson, 1974; Nelson, 1979; Brand and Guillard, 1981). These studies found that in general most coastal phytoplankton species grow faster in longer photoperiods and continuous light, whereas oceanic species were generally sensitive to continuous light. Brand and Guillard (1981) found this oceanic-coastal trend to be present in diatoms and coccolithophorids, but much less so in the dinoflagellates examined. They observed that one of the four coccolithophorids studied, Cyclococcolithus leptoporus, was unable to reproduce in continuous light at any light intensity, Hymenomonas carterae (Cricosphaera carterae) had a higher growth rate under continuous light than in a day:night cycle, and Emiliania huxleyi was only slightly inhibited by continuous light at greater than 320 ;uE m~2 s"1 (Brand and Guillard, 1981). Paasche (1967) found that the coccolithophorid Emiliania huxleyi grew faster in longer photoperiods, but that the maximum growth rates were reached at a photoperiod of 15:9 D:N cycle. Photoperiods from 15 hours of light to continuous light showed no significant difference in growth rates. In general, other phytoplankton species exhibited photoinhibition under long photoperiods and/or continuous light. The diatom Synedra tabulata reached  11  its maximum growth rate under 15 hours of light with no significant increase in longer photoperiods to continuous light (Castenholz, 1964). Paasche (1968) found that the coastal diatom Ditylum bricrhtwellii was inhibited by continuous light at a number of light intensities, which makes it an exception compared to most other coastal species. Hobson (1974) found that the prymnesiomonad Isochrysis cralbana grew faster in a 12:12 D:N cycle than in a 16:8 D:N cycle at various light intensities but only at 25°C and not at lower temperatures. Division patterns in phytoplankton reveal a preference for nighttime division. The timing of cell division in Emiliania huxleyi, has been shown to be determined by the onset of the previous photoperiod, and to occur during the dark period (Paasche, 1967; Chisholm and Brand, 1981). Depending on the photoperiod, Chisholm and Brand (1981) observed that the cell cycle phasing was determined by the D:N cycle and persisted for at least three days under continuous light for two strains of E. huxleyi and Hymenomonas carterae. Cell division phasing which persisted under continuous light was observed in species whose growth rates approached one doubling/day in continuous light (Chisholm and Brand, 1981). Since most of the phytoplankton species examined have been diatoms and dinoflagellates, of which most had higher growth rates in long photoperiods or continuous light, the purpose of the present study was to increase our knowledge  12  of coccolithophorid physiology and cell and coccolith morphology by examining a number of species of the class Prymnesiophyceae (of which 9 species are coccolithophorids) under continuous light and a 14:10 D:N cycle regime. This was done by comparing the growth rates (divisions day -1 ) as a function of in vivo  chl a fluorescence over time. Of these  11 species, a coccolith-forming and a non-coccolith forming strain of the coccolithophorids Emiliania huxleyi and Coccolithus pelacricus were examined in more detail (growth rates, physiological parameters, and cell and coccolith ultrastructure) under both light regimes.  13 MATERIALS AND METHODS General Culture Maintenance Stock cultures of the algae listed in Table 1 were maintained in filter sterilized (0.22 /zm Millipore) ESenriched natural seawater (HESNW) (Harrison et al. 1980) with several modifications to the original medium. The natural seawater was obtained from a depth of 10 m beyond the dock at the Dept. Fisheries and Oceans, West Vancouver. Silicon was omitted while Na2glyceroP04 and FeNH4(SO4)2'6H2O were replaced by equimolar concentrations of Na2HP04 and FeCl3*6H20, respectively. Na2HP04 and NaN03 were added to a final concentration of 13 ^M and 300 fxK respectively. Tris buffer was added to a final concentration of 1.0 mM. Selenium in the form of Na2Se03 was added to a final concentration of 10 nM. Deionized distilled water (DDW) and reagent grade chemicals were used in preparing nutrient enrichment solutions. Culture vessels employed throughout this research were soaked in 10% HCl(v/v) for at least 24 h and then rinsed thoroughly with DDW prior to sterilization in an autoclave. All cultures (i.e. stock and experimental) were grown at 17±1°C (except Coccolithus neohelis which was maintained at 21°C) without stirring, because most of these species were found to be sensitive to stirring (Jeffrey and Allen, 1964; 0. K. Andersen, unpubl. results, 1986). Stock cultures were maintained on a 14:10 lightrdark (L:D) cycle with illumination from cool-white fluorescent tubes at an  14  irradiance of 100 /zE m ^ s~ . Growth tests Experimental cultures were maintained either on a 14:10 L:D cycle or under continuous illumination both at an irradiance of 100 /xE m~2 s"1 and at 17±1°C using cool-white fluorescent tubes. All initial growth studies were carried out in culture tubes containing 1.25 ml of exponentially growing stock culture diluted with 25.0 ml of HESNW. Growth was monitored directly in the culture tubes by reading in vivo  chlorophyll a fluorescence at the same time every day  using a Turner Designs fluorometer after brief hand mixing. This mixing did not appear to affect the growth or morphology of the cells. Plots of the log of fluorescence against incubation time were used to calculate the following three parameters: (1) adaptation period = number of days from inoculation to the first significant increase in fluorescence, (2) exponential growth rate = maximum increase in fluorescence per day, and (3) maximum yield = maximum fluorescence of the growth curve. Microscopic examination of the cultures was carried out at the same time every day to observe the health and morphology of the cells. Extensive growth studies were carried out on noncoccolith forming and coccolith-forming strains of Coccolithus pelacficus (motile Crystallolithus hyalinus stage) and Emiliania huxleyi. They were grown in 6 L flasks containing 250 ml of exponentially growing stock cultures in  15  5 L of modified HESNW. Cultures were bubbled continuously with filter-sterilized air (sterilized 0.22 jum Millipore and A/E glass fibre filters) containing 2-4% CO2 to control changes in pH for the Coccolithus pelagicus strains. Using this method, C limitation was prevented and the pH was maintained between 7.95-8.90 during the course of the experiments. Continuous air bubbling was not used on the Emiliania huxleyi cultures until late exponential growth when the pH reached 8.2. The pH for these experimental cultures was maintained between 7.95-8.80. It was found that air bubbling in the early growth stages reduced the pH below normal seawater pH values (<7.95). Temperature was maintained at 17±1°C with illumination at 100 /xE m~2 s - 1 using cool-white fluorescent tubes either under continuous illumination or a 14:10 L:D cycle. Sampling was done through a siphon, and the first part of the sample was discarded. The cultures were gently swirled by hand to resuspend them prior to sampling.  This did not appear to affect the growth  of the cultures whereas continuous gentle stirring with a teflon stir bar at 60 rpm resulted in massive clumping of cells, especially for the Coccolithus pelagicus cultures, and appeared to increase the number of loose coccoliths in the coccolith-forming Emiliania huxleyi cultures. These observations were based upon visually comparing continuously stirred and hand swirled cultures under light microscopy, and data reported by 0. K. Andersen (unpubl. results, 1986) and Jeffrey and Allen (1964).  16  Several variables were measured, including cell density (CD), average cell volume (CV), chlorophyll a (chl a ) , chlorophyll c (chl c), phaeopigments (phaeo), carotenoids (caro), in vivo  fluorescence (F-chl a), and particulate  organic carbon and nitrogen (POC/PON). Morphological changes in coccoliths and cell ultrastructure were monitored during the course of an experiment using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. Analytical Methods i) Fluorometry Growth was monitored using a Turner Designs Model 10 fluorometer to measure in vivo  fluorescence of 5 ml  subsamples of 2 clones each of Coccolithus pelagicus (COPEL and 241) and Emiliania huxleyi (88E and 646). Samples were taken at the same time every day to minimize the effect of the L:D cycle on fluorescence, ii) Cell Counts Cell densities and cell volumes were measured using a particle counter (Coulter). Samples of experimental cultures were diluted by a factor of 15 in 3% filter-sterilized NaCl for cell densities > 10  cells ml" 1 but no sample dilutions  were made at cell densities <10 4 cells ml - 1 . Microscopic counts were made to compare with cell densities obtained using the particle counter. The observation of the presence or absence of coccoliths on the  17  cells was carried out using a Zeiss phase contrast microscope at 250-400X magnification, because it was found that stirring in the particle counter tended to knock off some of the coccoliths of Emiliania huxleyi but not Coccolithus pelagicus. Samples were examined at the same time every day following fluorescence measurements. iii) Particulate Organic Carbon/Nitrogen Duplicate samples of 10 to 100 ml were filtered (/JL 1/4 atm) every 2 days onto a 13 mm GF/F precombusted glass fibre filter during the course of an experiment. The filtered samples were stored flat, in petri dishes over desiccant. Samples were kept frozen until analysis, where upon the samples were dried in an oven at 60°C for 24 h and analyzed with a CHN analyser (Carlo Erba). For a blank, a sample of DDW was filtered and treated as above for particulate organic carbon and nitrogen. iv) Spectrophotometric Chl a Analysis Extractions for spectrophotometric determinations of chl a, chl c, phaeopigment, and carotenoid concentrations were carried out according to Strickland and Parsons (1972) . Calculations of chl a, chl c, phaeopigments, and carotenoid concentrations were determined using the equations given in Appendix 1. v) Scanning Electron Microscopy (SEM) Fixations for SEM observations of coccoliths and cells  18  were always carried out at the same time of day and the same stage of the growth curve to minimize the effect of the culture conditions on cellular and coccolith ultrastructure. Samples were concentrated by gentle filtration onto 0.45 or 1.0 lira. Nuclepore filters and fixed for 1 h at room temperature with 2% glutaraldehyde (v/v) and 1% OSO4 (v/v) in saline phosphate (1.7 M) buffer (PBS pH 8.0). The cells were rinsed thoroughly with PBS buffer prior to dehydration in a graded ethanol series. Next, cells were subjected to critical point drying in a Balzer's Union CPD 02 0 Critical Point Dryer and gold coated before insertion into a scanning electron microscope.  vi) Energy-Dispersive X-ray Microanalysis Fixations for X-ray microanalysis were the same as for SEM fixations above. Calcium presence and concentrations were determined using an AN10000 X-ray Analyser. Over an Xray spectrum acquisition period of 2 min at the Ca peak at 3.700 keV, the integral windows and apparent concentrations of Ca were determined by integrating the areas under the Ca peak and subtracting background Ca counts. As well, a visual X-ray map of Ca, Os, K, and background distributions of a single cell viewed under SEM were acquired via computer over an acqusition period of six minutes.  19  RESULTS I. Growth Studies Eleven species belonging to the Prymnesiophyceae, of which nine species were coccolithophorids (Table 1), were used in this study. Isochrysis galbana and the Chrysochromulina sp. do not form coccoliths, but they do form organic scales. Coccolithus pelaqicus 241, Emiliania huxleyi 55a and 646 are non-coccolith forming strains. Coccolithus pelaqicus COPEL, Coccolithus neohelis. Cricosphaera carterae. Cyclococcolithus leptoporus. Emiliania huxleyi 88E, and Syracosphaera elonqata are coccolith-forming coccolithophorids. These eleven species were tested for their ability to grow under two different light regimes; continuous light and a 14:10 D:N cycle (Table 2 and Figures 1A-K). Three patterns; no change, an increase, or a decrease in growth rate were observed under a continuous light regime compared to a D:N cycle. Cultures were grown under a D:N cycle (control) and then shifted to continuous light. I. galbana (Fig. 1A), Chrysochromulina sp. (Fig. IB), C. pelaqicus 241 (Fig. IE), C. pelaqicus COPEL (Fig. IF), C. neohelis (Fig. IH), and C. carterae (Fig. II) all exhibited significant decreases in maximum growth rate under continuous light compared to the 14:10 D:N cycle ranging from a 90% decrease for the Chysochromulina sp. to a 25% decrease for Coccolithus neohelis. For C. pelaqicus 241  20 Table 1  Species of Prymnesiophyceae used in this study.  ALGA  Oceanic/ Coastal  N 1 ,S3 Chrvsochromulina sp. (Lackey) N,S  NEPCC strain number  245  Isochrvsis qalbana (Parke)  Tahiti  (isolator/source)  (unknown/49A36'N 140*37'W)  (K. Haines/unknown)  COCCOLITHOPHORIDS C  Coccolithus neohelis (Mclntyre and Be)  448  (J. West/Hawaii)  N  Coccolithus pelaqicus (Gaarder et Markali)  241  (R. Waters/Stn. 8 in N. Pacific Ocean)  C  Coccolithus pelaqicus (Schwarz)  COPEL  (unknown/unknown)  C  Cricosphaera carterae C (Braarud et Fagerl.) Braarud  453  (J. Lewin/unknown)  C  Cyclococcolithus leptoporus O (Murray et Blackman) Kamptner  244  (R. Waters/Stn. 5 in N. Pacific Ocean)  N  Emiliania huxlevi (Lohmann) Hay et Mohler  0,C  646  (E. Paasche/Oslo Fjord, Norway)  C  Emiliania huxlevi (Lohmann) Hay et Mohler  o,c  88E  (R. Selvin/unknown)  N  Emiliania huxlevi (Lohmann) Hay et Mohler  o,c  55a  (Waters/50A11'N 144 A 57'W)  N  Emiliania huxleyi (Lohmann) Hay et Mohler  o,c  556  (J. Acreman/Stn. P 49*5'N 144M0'W)  S  Emiliania huxleyi (Lohmann) Hay et Mohler  o,c  451B  (R.R.L. Guillard/Sargasso Sea)  C  Syracosphaera elonqata (Lohmann)  635  (J. Leftley/unknown)  *Northeast Pacific Culture Collection, Dept. of Oceanography, U. B. C. Non-coccolith forming (N) Coccolith forming (C) Scale forming (S)  21 Figures 1A-K. Semi-log plots showing growth curves as a function of in vivo fluorescence over time for 11 species of the class Prymnesiophyceae/Haptophyceae of which 9 species are coccolithophorids (Table 1). All curves represent triplicate experiments each done in triplicate. Vertical error bars represent ± 2 S.D. (n = 9) and are smaller than the symbol where not apparent. At the arrow, three of the non-cocoolith forming species growing under continuous light were shifted to a 14:10 D:N cycle.  22  N0N-COCCOI.ITH FORMING S P E C I E S 100  Isochrysis golbone  „•-•-•-.  O—OContinuous Light • • 14:10 D:N Cycle 100 5  10  15  Time (Doys)  £milionio huxleyi 55e (Non—coceolith # — • forming stroin)  10  2, I 0.10 100  |  •  Chrysoehromulinc so.  E  O—OContinuous Light • — 0 1 4 : 1 0 D:N Cycle  —•  0.01 4 5 Time(Ooys)  / u ii  10  !>--;:«  10  •-o-o  "  O •  OContinuous Light • 14:10 D:N Cycle 100 15  10 Time (Doys)  20  Coccolithus pelcgic.'S 241 (Non—coceolith forming motile strain) .  ,6? -*trr  10  0 •4 0.100  A .  100  4  i  i  O OContinuous light • — • 14:10 D:N Cycle Emitionie huxleyi 6*6 (Non—coceolith forming stroin) 10 Time (Doys)  s«-«-«.# O.,  O-O-O 10 Time (Doys)  u  /•  i'j  •o-»—o-m O-O—O-O  0.001  -•-•  -•-• •-•/o-o-o-o-°  "i '1  O OContinuous Light O — 3 14:10 D:N Cycle  1  0-0-0—«_»'  0.010  £ * I  15  20  15  20  23  COCCOLITH FORMING IOOO  SPECIES  1000  r CoceoBthus pelogicus COPEL ( C o c c o l i t h - f o r m i n g motile strain)  Crtcosphoero ccrteroe  S S J2=^ ^' ° mi  100  w  10 •  1.0  r.,0  Kf  »  | j  i/rv°  -S-8 .•/ •o  10  O — O Continuous Light • • 1 4 : 1 0 D:N Cycle  1  O — O Continuous Light • • 14:10 D:N Cycle  ?*'°  0.1  5  100  10 Time (Ooys)  10 15 Time (Doys)  20  20  25  100 Cyclocoeeoli'.hus leptoporus  Emilicnis huxleyi 88E ( C o c c o l i t h - f o r m i n g stroin)  o—?—4—& 1  T u.  15  .§-?^?=r=8=8 6"Vl '  10  10  .o ^«  8*c-.^°'  O O  s=©  O Continuous Light © 14:10 D:N Cycle  ^  S^1  4  10  4 6. Time (Doys)  O e  OCor.tinuous Light O 14:10 D:N Cycle  10  6  Time (Doys)  100  1000 Coccoliihus neohelis  ^  Syrocosphoero elongoto  -•=5=5=8=9  '  _ c o I O — O Continuous Light • — - . • 1 4 : 1 0 D-JN Cycle  100  10  .•' o-°' ,•0-"° O •  ft 5  10  15  5  »  10  Time (Doys) Time (Doys)-  OContinoous Bght • 14:10 D-.N Cycle  15  20  Table 2  — 2 —1 Growth of mlcroalgae under contlnuoua light and 14:10 L:D cycle (100 /JE m a Irradlance, 17±1°C temperature), (a) Maximum growth period (days)a; (b) maximum growth rate (divisions day - 1 2 S.D.); (c) maximum yield (in vivo fluorescence In relative units 1 2 S.D.); (d) final pH of cultures. For all experiments n = 6 to 9.  ALGA  Continuous Light  14:10 L:D Cycle  NOM-COCCOLITH FORMING ChrvBochromullna op. Cont. 0.1110.03 1-9 L:D (10-12)1 (0.7410.07)'  6.011.0 (28.512.2)1  8.5 8.8  1-5  1.0810.03  43111  9.0  CpccpUtJlMg pelaqjcuB 2  0.0510.01 (0.0510.01):  8.2 8.3  2-8  0.5310.08  2817  8.9  3011  8.3  1-4  2.7210.01  3810  8.4  4116  8.7  2-4  0.9810.05  8319  8.6  2911 (5015)1  9.2 9.1  1-3  0.8410.04  5616  8.9  2-4 (6-9)1  0.010.08 (0.2010.13)1  Emlliania huxleyi 55a" 3-6  2.6610.04  1-3  0.8910.06  1-4 (8-11)1  0.5910.10 (0.3910.05)1  Emlllanla huxlevi 6465 iBochrvBia oalbana COCCOLITH-rORMINO CoccolithuB neohella  1-4  1.9510.16  53110  9.2  1-3  2.6010.16  5718  9.2  Coccolithus pelaqicuB  2-6  0.7010.27  35121  8.9  1-6  1.0810.03  43111  8.9  Crjcosphaera, carterae  2-9  0.5110.10  182110  9.4  1-3  0.7110.02  185148  9.5  CvclococcolithuB leptoporuB  1-4  1.1210.17  3212  8.7  1-5  0.6610.17  2716  8.7  EmiltaptA huxleyi 8BE4  1-4  1.4510.44  3112  8.4  1-6  1.1110.07  3612  8.3  SvracoBPhaera elonoata8  1-3  1.2710.04  6719  8.9  1-3  1.2410.05  85117  8.8  This refers to the time period used to calculate (b), the maximal growth rate. Species subjected to a 14il0 LiD cycle after reaching stationary growth under continuous light. Non-coccolith forming motile atraln 241. Coccolith forming motile strain COPEL. Coccollth-forming strain.  25  (Fig. IE) cultures grown under continuous light, in  vivo  fluorescence dropped by two orders of magnitude over a 48 hour period and then remained at a stationary low level while under a 14:10 D:N cycle the decrease was much reduced. Two of the nine species of coccolithophorids tested (Table 1), Emiliania huxleyi 646 (non-coccolith forming naked strain) (Table 2, Fig. 1C) and Syracosphaera elonqata (Table 2, Fig. IK) showed no significant difference in growth rates under either continuous light or a 14:10 D:N cycle. Three species, E. huxlevi 55a (non-coccolith forming naked strain) (Fig. ID), E. huxleyi 88E (coccolith-forming strain) (Fig. IG), and Cyclococcolithus leptoporus (Fig. IJ) exhibited significantly higher growth rates (from 5 to 41%) under continuous light compared to a D:N cycle (Table 2). It should be pointed out that Cyclococcolithus leptoporus did not grow under continuous light when summer natural seawater was used in the modified HESNW medium, but exhibited growth in continuous light only when winter  natural seawater was  used in the culture medium. Light microscopy did not show any obvious morphological differences between summer and winter cultures of C. leptoporus. II. Maximum Yield Maximum yields were determined by in vivo  fluorescence  and cell density was not measured. From Table 2 and Figs. 1A-1K, all of the coccolith-forming species of coccolithophorid, Coccolithus pelaqicus COPEL, Emiliania  26 huxleyi 88E, Coccolithus neohelis. Cricosphaera carterae. Cvclococcolithus leptoporus. and Syracosphaera elongata, showed no significant (p < 0.05) differences in maximum yields (in vivo  fluorescence) under either a continuous  light or a 14:10 D:N cycle regime. Both of the non-coccolithophorids, Isochrvsis aalbana and Chvsochromulina sp., and all of the non-coccolith forming species, Emiliania huxlevi 55a and 646, and Coccolithus pelagicus 241 exhibited significantly lower maximum yields (ranging from 20 to 560%) under a continuous light regime (Fig. 1A-E) compared to a D:N cycle. When continuous light cultures of I. galbana and Chrysochromulina sp. were subjected to a 14:10 D:N cycle, maximum yields increased significantly (p < 0.05) (Fig. 1A, B ) . None of the 11 species achieved a higher maximum yield under the continuous light regime, even though the maximum growth rates for E. huxleyi 88E and Cyclococcolithus leptoporus were higher. The final pH of the cultures was recorded. Of the two non-coccolithophorids, I. qalbana showed a higher final pH and Chrysochromulina sp. exhibited a lower final pH under continuous light compared to the pH reached under the D:N cycle. Of the three non-coccolith forming species, C. pelagicus 241 had a lower final pH under continuous light compared to the D:N cycle, whereas both E. huxleyi strains showed no pH differences under either light regime. There was no significant difference in final pH recorded for the  27  coccolith-forming species under either light regime. III. Effects of Culture Volume Greater in-depth studies were conducted on two coccolithophorids that had naked and coccolith-forming strains; these were Emiliania huxleyi 88E and Coccolithus pelaaicus COPEL (coccolith-forming strains) and Emiliania huxleyi 646 and Coccolithus pelaaicus 241 (non-coccolith forming strains). These experiments were done using 5.25 L batch cultures under the same conditions as the 25 ml batch cultures already discussed, except that C. pelaaicus cultures were air-bubbled to maintain the pH between 8.08.8. It was found that the E. huxleyi cultures were able to maintain their pH between 7.9-8.3 without air-bubbling (Table 3). Based on in vivo  fluorescence (Table 3, Appendix 2 and  4A) and cell densities (Table 3, Figs. 2A-D, Appendix 4B) the same growth rate trends occurred in the 5.25 L cultures as in the 25 ml batch cultures. However, there were some significant differences. The non-coccolith forming strain E. huxleyi 646 revealed a significantly higher growth rate under continuous light (28% higher) when grown in 5.25 L cultures (Table 3, Fig. 2D and Appendix 2D) compared to those cultures grown in 25 ml cultures (Table 2, Fig. 1C), whereas C. pelaaicus 241 had 2 times the growth rate under the D:N cycle regime in the 5.25 L cultures (Table 3, Fig. 2B, and Appendix 2B) than in the 25 ml cultures (Table 2, Fig. 1H) based on in  vivo  28 fluorescence. Examining Table 3, it becomes apparent that growth rates based on cell densities showed either no significant difference or were lower by 23-44% than the growth rates determined using in vivo  fluorescence for all four strains  of coccolithophorids. When comparing Tables 2 and 3, it was observed that under continuous light C. pelaqicus 241, E. huxleyi 646 (non-coccolith forming strains), and E. huxleyi 88E (coccolith-forming strain) had higher maximum yields calculated from in vivo  fluorescence in the 5.25 L cultures  compared to the 25 ml cultures (55 times, 30%, and 2 times higher respectively), but the opposite was found under D:N conditions for C. pelaqicus 241 and E. huxleyi 646 (10 and 3 times lower, respectively). E. huxleyi 88E showed no significant differences in maximum yields under the D:N cycle in the 5.25 L and 25 ml cultures. The coccolith forming strain, C. pelaqicus COPEL, showed the opposite results In maximum yield compared to E. huxleyi 88E. IV. Cell Volume Changes Cell volumes tended to be variable (Figs. 3A-D, Appendix 4C) but three trends were noticed. One was that cell volumes increased as the cultures entered stationary growth. Another trend was that there were no significant differences in cell volumes under either light regime. Lastly, C. pelaqicus COPEL exhibited a significant increase in cell volume within 24-48 h of being subjected to  29  100  Coccolithus pelagicus COPEL {Coccolith forming motile strain)  Emiliania huxleyi 88E (Coccolith forming strain)  1000  »100 o u A.  o -  10  X in  § a  i  A A A14:10 D:N Cycle A— AContinuous Light  o U 0.10 5 10 Time (Days)  5 10 Time (Oays)  Coccolithus pdagicus 241 (Non—coccolith forming motile strain) 100  100.  15  Emiliania huxleyi 646 (Non—coccolith forming strain)  B  A  S  i  / / A  A'  10  i  m o  f  fc i u  A A 14:10 D:N Cycle A— AContinuous Light  l*\  A  a  A 14:10 D:N Cyde  A— AContinuous Light 0.10  4 6 Time (Days)  10  2  4 6 Time {Oays)  8  10  Figures 2A-D. Semi-log plots showing growth curves as a function of cell density over time for 2 coccolithophorids each consisiting of a coccolith-forming (Coccolithus pelaqicus COPEL and Emiliania huxlevi 88E) and a noncoccolith forming strain (Coccolithus pelaqicus 241 and Emiliania huxlevi 646) . The growth curves are the result of 4, 5.25 L batch cultures started in a 14:10 D:N cycle and at the arrow 2 cultures were switched to a continuous light regime. All curves represent duplicate experiments each done in quadruplicate. Vertical error bars represent ± 2 S.D. (n = 4 to 8) and are smaller than the symbol where not apparent.  Table 3  Growth of two species of coccolith and non-coccolith forming coccollthophorids under continuous light and a 14»10 DsN cycle (100 fiE m~2 B~1, 1711BC). (a) maximum growth period (days)1; (b) maximum \t (divisions day" 4 2 S.D.) calculated from in vivo I luorencence (top line) and cell density (bottom line); (c) maximum yield (in vivo fiuoieecence 1 2 S.O.); (d) maximum yield (xlO cells ml" 4 2 S.D.); (e) final pi! of cultures i 2 S.D. All cultures were grown in 5.25 L batch cultures, n = <1-B.  ALGA  14:10 LiO Cycle  Continuous Light  NON-COCCOLITH FORMING Coccollthus pelaqicuB 241  2.7510.28  8.310.1 0.1310.03  Emiliania huxlevl 646  2-5  1.23*0.02  2-6  0.8310.13  57.7546.72  8.410.3 23.3315.54  2-4  1.1610  3.3010il0  —  2-4  0.7010.0  —-  0.1540  2-5  0.9340.06  24.5042.12  —  2-6  0.8710.11  —  6-9  0.6810.06  62.7545.30  6-9  0.5340.11  3-7  0.8540.13  3-6  0.4840.03  8.010.1  8.310.2  20.4046.59  COCCOLITH FORMING Coccollthua pelaalcuB COPEL  6-9 0.7340.02 (3-6)2 (1.82)2 6-9 0.7910.11 (3-5)2 (1.26)2  Emiliania huxlevl 88E  3-7  1.0840.07  3-6  1.1340.59  8.810.1  57.0412.7 (29.0)2 2.0940.01 (1.52)2  69.2548.84  fl.010.3 18.0043.96  |This refers to the time period (days) used to calculate (b and c), the maximal growth rate. '14il0 LiD cycle culture transferred Into 5.25 L of new modified HESNW at 1/21 dilution and subjected to continuous light (Appendix 4A, 4B).  8.610.2  1.6440.08  7.910.3  44.5045.66 6.4011.17  u o  20  Coccolithus pelogicus COPEL (Coccolith forming motile strain)  •  100  #14:10 D:N Cycle  O— OContinuous Light 90  15  10  Emiliania huxleyi 88E (Coccolith forming strain) • • 14:10D:N Cycle O— OCont Light  to  E ^^ a> E  H  r vw^  80  "5 O  * * * ! !  T oi\ 1  J O-Q o  50 5 10 Time (Days)  15  5 10, Time (Days)  Coccolithus pelogicus 241 (Non—coccolith forming motile strain) 16 • • 14:10 D:N CycleT B C— OCont. Light  55  E  ^^  15  Emiliania huxleyi 646 (Non—coccolith forming strain)  • #14:10 D:N Cycle O— OContinuous Light  45  V  E _3 O  Is>  35  O  25  2  4 6 Time (Days)  8  10  4 6 Time (Days)  Figures 3A-D. Changes in cell volume over time for 2 coccolithophorids under the same conditions as in Figure 2.  10  32 continuous light but then it dropped to a volume comparable to the D:N cycle regime (Fig. 3A and Appendix 4C). V. Pigment Analyses Pigment determinations (chl a, chl c, and carotenoids) over time was one of the parameters used to assess the physiological responses of the coccolithophorids to the two light regimes. When comparing pigment concentrations between the coccolith-forming and the non-coccolith forming strains, it was observed that during the exponential growth phase the coccolith-forming strains had 1-7 times more pigment cell -1 than the non-coccolith forming strains (Figs. 4A-D). Compared to the 14:10 D:N cycle cultures, the noncoccolith forming strains E. huxleyi 646 (Fig. 4D) and C. pelaqicus 241 (Fig. 4B) showed a significant decrease in chl a (x 10~ 6 /xg Cell"1) after 24 h of continuous light but no significant difference by 48 h. The chl c and carotenoid concentrations showed no significant differences under either light regime. Of the coccolith-forming strains, E. huxleyi 88E (Fig. 4C) exhibited no significant difference in pigment concentrations per cell under either light regime. The other coccolith-forming strain, C. pelaqicus COPEL (Fig. 4A) demonstrated a repeated dramatic significant increase in both chl a and c after 24 h of continuous light with a subsequent decrease to D:N cycle levels by 48 h. This was again demonstrated to occur when a D:N cycle culture sample was added to new culture medium and subjected to continuous light (Appendix 4D). There was no observed significant  33  Emiliania huxleyi 88E (Coccolith f o r m i n g strain)  Coccolithus pelagicus COPEl (Coccolith forming motile strain)  5 10 Time (Days)  5 10 Time (Days)  Coccolithus pelagicus 241 (Non—coccolith forming motile strain) 20 A AChl a • •Chi c •—•Carotenoids  1.5  Emiliania huxleyi 646 (Non—coccolith f o r m i n g strain) -AChl a -•Chi c -•Carotenoids  o % 1.0  I o  c o  0.5  £ b. 0.0 2  4 6 Time (Days)  8  10  4 6 Time (Days)  Figures 4A-D. Changes in pigment concentration (x 10 cell""1) over time for 2 coccolithophorids under the same conditons as in Figure 2.  10  /xg  34 difference in carotenoid concentrations under either light regime. Pigment ratios (Figs. 5A-D, and Appendix 4E) revealed no significant differences between the coccolith-forming and non-coccolith forming strains of E. huxlevi under either light regime. However, the coccolith-forming motile strain of C. pelaqicus (COPEL) had significantly higher chl a and chl c:carotenoid ratios (ranging from 3 to 8 times) under both light regimes compared to the non-coccolith forming motile strain (241). The chl arc ratios for C. pelaqicus revealed no significant differences under the D:N cycle between the coccolith-forming motile strain and noncoccolith forming motile strain, but under continuous light the chl arc ratios were significantly higher (1.6 times) in the coccolith-forming motile strain. Examining the pigment ratios (Figs. 5A-D, and Appendix 4E), it was observed that there were no significant differences between 14rl0 DrN cycle and continuous light regimes except for C. pelaqicus 241 (Fig. 5B) which showed a significant decrease in the chl crcarotenoids ratios cell -1 after 24 h of continuous light. In the non-coccolith forming strains, E. huxleyi 646 and C. pelaqicus 241, no significant differences were observed in F Cell -1 (Figs. 6B and D) under either light regime. The coccolith forming strains (E. huxleyi 88E and C. pelaqicus COPEL) showed a significant difference in F Cell"1 (Figs. 6A andC) in the first 24-48 h of continuous light  Coccoiith us pelagicus CO PEL {Coccoiith forming motile stroin) arc axarotenoid cxorotenoidy.  40  30  20  Emiliania huxleyi 88E (Coccoiith forming stroin) • Barcarotenoids •—•crcarotenoids • 9a:c  SI  jo a cc c  20  V  E 10 •  ,t=st 15  5 10 Time (Days)  5 10 Time (Days)  Coccolithus pelagicus 241 (Non—coccoiith forming motile strain)  20  30  9—t)a:c  15  Emiliania huxleyi 646 (Non—coccoiith forming strain) •—tax •—Barcarotenoids • •crcarotenoids  •—•a:carotenoids • — • crcarotenoids  15 n _o  20 -  o tr c V  E  10  * 2  4 6 Time (Days)  B  10  4 6 Time (Days)  10  Figures 5A-D. Changes in pigment ratios over time for 2 coccolithophorids under the same conditions as in Figure 2 and a = chl a and c = chl c.  36  compared to a 14:10 D:N cycle but no significant difference by 48-72 h. It was observed that E. huxlevi 88E had approximately 3x's the F Cell -1 of E. huxlevi 646 under both light regimes whereas the two strains of C. pealgicus showed no such difference (Figs. 6A-D). Another parameter examined was F Chi a - 1 (Figs. 6E-H). The coccolith-forming (Fig. 6G) and non-coccolith forming (Fig. 6H) strains of E. huxleyi showed significant increases in F Chi a - 1 under continuous light compared to a 14:10 D:N cycle. The coccolith-forming C. pelaqicus COPEL (Fig. 6E) showed a significant decrease in F Chi a  under continuous  light whereas the non-coccolith forming C. pelaaicus 241 (Fig. 6F) showed no significant difference. Under both light regimes, the coccolith-forming E. huxleyi 88E was observed to have lOx's less the F Chi a - 1 than the non-coccolith forming E. huxleyi 646, and the coccolith-forming C. pelaqicus COPEL was observed to have 2x's more the F Chi a"*1 than the non-coccolith forming C. pelaqicus 241 (Figs. 6EH). As seen in Appendices 3A-D and 4F, the pigment cell volume -1 (x 10~ 9 /xg Mm -3 ) curves revealed the same patterns as seen in the pigment concentrations cell  —i  .  V. POC. PON Analyses Changes in particulate organic carbon (POC) and particulate organic nitrogen (PON) over time for the coccolith forming and the non-coccolith forming strains of Coccolithus pelaqicus (Figs. 7A and 7B), and Emiliania  37  Coccolithus pelagicus COREL ICoccolith forming motile strain)  30  T o  u  u .-? 20  c 3  I o o  o  \  *o  ,o o 1 o  u  5 10 Time (Days)  ;  12  B  §  e  I  (O  I  o x  —o 1 \  ©•  i YJ\ W'V 1  i  >14:10D:N Cycle O— OContinuous Light  Lu  2 Time (Days)  10  Emiliania huxleyi 646 (Non-coccolith forming strain)  a o  ^--•*-&3-*  /  4 6 8 Time (Days)  a U  u  ft!/  15  Coccolithus pelagicus 24-1 (Non—coccolith forming motile strain)  9  / *  w10  114: 10D:N Cycle T l O— OCont. Light  U:10D:N Cycle OContinuous Light  /  m I o  \  xJ  Emiliania huxleyi B8E (Coccolith forming strain) I ©14:10 D:N Cycle : O— OContinuous Light  4 6 Time (Days)  8  Figure 6A-H. Changes in F Cell-1 (Figs. 6A-D) and F Chi a - 1 (Figs. 6E-H) over time for 2 coccolithophorids under the same conditions as in Figure 2.  10  38  Coccolithus pelagicus COPEL (Coccolith forming motile strain)  12  inn  • 14:10 D:N Cycle O— OCont Ught y  10  Emilia nia huxleyi 88E (Coccolith forming strain) | • — • 14:10 D:N Cycle G O— OContinuous Ught T  80 -O  8 60  a  O  S 6  u "^  40  /  20  *f  4 2 0 4 6 8 Time (Days)  0  Coccolithus pelagicus 241 (Non—coccolith forming motile strain)  400  • 14:10 D:N Cycle O— OContinuous Light  2  *  I  4 6 8 Time (Days)  10  Emiliania huxleyi 646 (Non—coccolith fonninq strain) (14:10 D:N Cycle T H O— OContinuous Light  300 o Z *o  § 2  ./K 1  2  i  •  *  4 Time (Days)  •  T  Or  —  ti  200  / J 100  '  6  12  2  4 Time (Days)  6  O  39  huxlevi (7C and 7D) showed no significant differences in PON under either light regime. There were significant differences in POC for the coccolith forming strains only. After 24 h of continuous light, C. pelacricus COPEL (Fig. 7A) exhibited a significant 3-fold increase in POC compared to the D:N cycle levels. By 72 h of continuous light, the POC concentrations were not significantly different from the D:N cycle concentrations; therefore this may have been only a transient response. E. huxleyi 88E (Fig. 7C) showed no significant difference in POC under either light regime except for a significant decrease after 48 h of continuous light only. The coccolith forming strain of E. huxleyi had 3-5 times more POC cell -1 , and 2-4 times more PON cell -1 than the non-coccolith forming strain during the exponential growth phase. C. pelagicus, unlike E. huxleyi, showed no significant difference in POC and PON concentrations between the coccolith forming and the non-coccolith forming strains during the exponential growth phase. When C:N ratios are examined (Figs. 8A-D), no significant differences under either light regime were observed, except for the non-coccolith forming strain of E. huxleyi (Fig. 8D) which exhibited a significant increase after 24 h of continuous light, but no significant differences after three days of continuous light. The two strains of C. pelagicus had the same C:N ratios (Figs. 8A and 8B), but E. huxleyi 88E (coccolith forming strain) revealed a 40-75% higher C:N ratio than the non-coccolith  40 Coccolithus pelagicus COPEL (Coccolith—forming motile strain)  Emiliania huxleyi 88E (Coccolith—forming strain)  u o> 3  Z  o a. o* o a.  V  u  4 6 8 Time (Days) Coccolithus pelagicus 241 (Non—coccolith forming motile strain) 20 •—•POC B • — A PON 15  10  12  Emiliania huxleyi 646 (Non—coccolith forming strain)  o C7> 3  (O  •«•  I  4 6 8 Time(Days)  V  Ol 3  o  2  I  10  o  zo a.  o a. o* oa.  o" o Q.  Time (Days)  4 6 Time (Days)  Figures 7A-D.Changes in particulate organic carbonfPOC) and particulate organic nitrogen (PON) (x 1( Mg cell -1 ) over time for 2 coccolithophorids under the same conditions as in Figure 2.  4 Coccolithus pelagicus COPEL 10 {Coccolith forming motile strain) • — • 1 4 : 1 0 D:N Cycle O— OContinuous Light  Erniliania huxleyi 88E {Coccolith forming strain) • 14:10 D:N Cycle OContinuous Light  12  10 •  / '  o  T '1  \ J'  X^  z 6  -I-:: ii i i i i i i  6  8 10 Time (Days)  0  12  Coccolithus pelagicus 241 {Non-coccolith forming motile strain)  2  4 6 8 Time (Days)  10  12  Erniliania huxleyi 646 (Non—coccolith forming strain) • #14:10 D:N Cycle C— OContinuous Light  B  o  »—»14:10D:N Cycle O— OContinuous Light  -6 2L 2  4 Time (Days)  6  2  4 Time (Days)  6  Figures 8A-D. Changes in carbon:nitrogen (C:N) ratios over time for 2 coccolithophorids under the same conditions as in Figure 2.  42 forming strain, E. huxleyi 646 (Fig. 8D).  VI. Cellular and coccolith ultrastructure Scanning electron microscopy (SEM) was conducted on four coccolith-forming coccolithophorid species grown under continuous light and a 14:10 D:N cycle; Emiliania huxlevi 88E (Figs. 9A and B), Coccolithus neohelis (Figs. 9C and D ) , Cricosphaera carterae (Figs. 9E and F), and Coccolithus pelaaicus COPEL (coccolith-forming motile strain) (Figs. 9G and H ) . In the coccolith-forming strains, SEM revealed no significant differences in either cell or coccolith diameters (Table 4) under either light regime during the exponential growth phase. E. huxleyi 88E had the smallest cell diameter and the largest coccoliths, and C. carterae had the largest cell diameter and the smallest coccoliths. Emiliania huxleyi 88E appeared to be the only species that shed its coccoliths. There were no observable differences in coccolith ultrastructure among any of the coccolithophorids under either light regime. The two non-coccolith forming strains of coccolithophorid, C. pelagicus 241 and E. huxleyi 646, (Table 4) exhibited no significant differences in cell dimensions under continuous light or the D:N cycle. The smaller size for the non-coccolith forming strains compared to the coccolith-forming strains of the same species is due to the lack of a coccolith shell which was included in the  43 Figures 9A-H. Scanning electron micrographs of 4 coccolithophorids grown under continuous light or a 14:10 D:N cycle regime. Emiliania huxleyi 88E grown under a 14:10 D:N cycle (Fig. 9A)and continuous light(Fig. 9B). Scale = 2 /xm. Coccolithus neohelis grown under a 14:10 D:N cycle (Fig. 9C) and continuous light (Fig. 9D). Scale = 2 fim. Cricosphaera carterae grown under a 14:10 D:N cycle (Fig. 9E) and continuous light (Fig. 9F) . Scale = 4 /urn. Coccolithus pelaaicus COPEL grown under a 14:10 D:N cycle (Fig. 9G) and continuous light (Fig. 9H). Scale = 4 pm. Coccolith (C), Flagellum (F), Haptonema (H) shown in figures.  is  Mggr: " t ^  Table 4  Cell and coccolith sizes of 4 species of coccollthophorids grown under continuoui light and a 14il0 DiN cycle regime at 100 ^E m s and 17il°C (except Coccolithua neohellB which was grown at 21'C). Horphometrlc analysis was conducted on 10 cells and 20 coccollths. All values Include 12 S.D.  Coccollthophorid  Cell Dimensions 0'm) 14:10 D:N cycle  Continuous light  Emlllanla huxlevi 88E  7.710.2* (3.9±0.7)2  7.010.5* (3.810.2)2  Emillanla huxleyl 646  4.310.2  4.010.1  Coccollthus neohells  8.911.2 x 7.6i0.73  7.711.4 x 7.010.23  Coccolithua pelaqicuB COPEL  13.111.8 x 11.211.9  10.710.9 x 10.110.9  Coccolithua pelaqlcua 241  12.210.3  12.6±0.1  Cricosphaera carterae  11.110.9 x 9.710.2  13.511.1 x 10.810.8  Coccollth Dimensions (fim) 14:10 DtN cycle  Continuous light  EmUJLaiUa huxleyt 88E  3.510.5 x 2.5±0.6J  3.110.5 x 2.110.6-  Coccolithua neohells  3.110.3 x 2.110.2  2.910.3 x 1.810.3  Coccolithua pelaqicuB COPEL  2.210.1 x 1.410.2  2.110.2 x 1.310.2  Cricosphaera carterae  2.110.2 x 1.310.1  1.710.2 x 1.010.1  Assume spherical shape. Therefore value «= cell diameter. Cell diameter without coccolith shell. Length x width (assuming an elliptical shape). Non-coccollth forming strains.  as  47 cell diameters and volumes of the coccolith-forming species. What was initially thought to be the presence of an occasional coccolith shell in the D:N cycle cultures of the non-coccolith forming motile strain of C. pelaqicus 241, turned out to lack calcium upon SEM X-ray microanalysis (Appendix 5). These rare observations may, therefore, be the presence of the remains of organic cyst formations. Transmission electron microscopy of the coccolithforming (C. pelaqicus COPEL and £. huxleyi 646) and noncoccolith forming (C. pelaqicus 241 and E. huxlevi 88E) strains of coccolithophorids used in this study revealed the presence of intracellular and extracellular coccoliths and the typical intracellular structure that confirmed the species used are coccolithophorids (Appendix 6A-D).  VII. Calcium content studies Calcium atom localization and content of individual cells of the coccolith-forming and the non-coccolith forming strains of Emiliania huxleyi and Coccolithus pelaqicus subjected to both light regimes was determined during the exponential growth phase utilizing SEM X-ray microanalysis. Figures 10A-H are representative SEM X-ray micrographs which constitute the raw data for Table 5. Table 5 shows that only the non-coccolith forming strain of E. huxleyi had a significant decrease in atomic calcium under continuous light compared to the values obtained under the D:N cycle regime. The continuous light  48  regime resulted in cells with approximately 3 times less cellular calcium than that observed in cells under the D:N cycle. The coccolith forming strains of both species exhibited no significant differences in calcium content (cellular and coccolith) under either light regime. It should be pointed out that the calcium detected in the noncoccolith forming strains is cellular calcium and accounts for 1-4% of the total calcium detected in the coccolith forming strains which includes both cellular and coccolith calcium. Thus, it is probable that 96-99% of calcium detected in coccolith forming cells is present in the coccoliths. This is apparent from the calcium window (Ca) of the X-ray micrograph prints (Figs. 11A-H) where the coccoliths are outlined in the coccolith forming strains but no calcium is seen, even though it is detected above background levels in the non-coccolith forming strains. It should be pointed out that the brightness of the calcium, background, and osmium X-ray windows would increase at higher cycles per second than that used in this experiment (> 1000 cps), but would also possibly result in equipment and sample damage.  49 Figures 10A-H. SEM X-ray micrographs for calcium detection at 3.700 keV over a 2 minute acquisition period (Live: 120 s) for individual cells of coccolith forming and noncoccolith forming strains of 2 coccolithophorids (Coccolithus pelagicus and Emiliania huxleyi). Labelled peaks detected are osmium (Os), gold (Au), potassium (K), and calcium (Ca)._C. pelagicus COPEL (coccolith forming motile strain) grown under a 14:10 D:N cycle (Fig. 10A) and continuous light (Fig. 10B). C. pelagicus 241 (non-coccolith forming motile strain) grown under a 14:10 D:N cycle (Fig. 10C) and continuous light (Fig. 10D). E. huxleyi 88E (coccolith forming strain) grown under a 14:10 D:N cycle (Fig. 10E) and continuous light (Fig. 10F). E. huxleyi 646 (non-coccolith forming strain) grown under a 14:10 D:N cycle (Fig. 10G) and continuous light (Fig. 10H). The Real (time the computer clock was running in seconds) and %Dead (all cells are dead) in the X-ray legends are not pertinent.  V -  RflV: Live: Real:  0 - 20 keU 120 s P r e se t : 120 s Rema i ni ng: 141 s 1 5 * Dead  Of.  0 fi  Mrf£  A.  ^S  < 1.1 FS= HK OS = - 2 4 MEM1:  3.700 eh  keU 195=  2603  6 . -J /  cts  X-RRV: Live: R ea 1 :  0 - 20 keU 120s P r e s e t : 120s Remaining! 141 s 15* Dead  sa toe  X-RflV: Live: R ea 1 :  141s  Preset: 120 s Rema i ni nq: 15* Dead  0s  JJ-&<^-  < 1.1 FS= 4K OS= MEM1:  3.700 24  keU ch 195=  6.3 > 272 ets  X-RflV: Live: Real:  0 - 20 keU 120s P r e s e t : 120: Rema i ni ng: 141s 155s Dead  < 1,1 FS= 4K OS= MEM1 :  3.700 24  eh  keU 195=  0s  6.3 > 313 • c t s  &t /0£  X-RflVs Live: Real:  - 20 keU 120s Rema i n i ngs 12n y P r e s e t : ; 1 4 * Dead 139:  ^m < 1.1 FS= 4K OS= MEMis  0s  -fyTT-HUm...., >* . T V  3.700  keU ch 195=  6.3 > 1985 c t s  I  X-RflV: 0 - 2 0 keU Live: 120 s Preset: 120 s Rema i ni ng: Real: 140 s 14>. Dead  ^-wyr^r-*..!,!, (%K\J  < 1.1 FS= 4K OS= MEN 1 :  3.700 1<Z  keU ch 195=  0s  * .. ^  6 . 3 ?^ 'Z'CffZ Q  i^ + •-  .•  I . tj. ^ .  I__I  r^i  I_I  s*  X-RflV: Live: Real:  0 - 20 keU 120 s P r e s e t : 120s R e m a i n i n g : 141s 155s Dead  Osl  fm  ^J\Aj.,.,.A»,§^,  < 1.1 FS= 4K GS= MEN 1 :  Q  3.700  keU ch 195=  W——hMfci  6.3 > 362  CtS  S7  X-RRV: Live: Real:  0 - 20 keU 120s P r e s e t : 120s R e m a i n i n g : 141s 15^. Dead  K  C  0s  p  -u^_-  < 1.1 FS= 4K 0S= MEM:  3.700 «_•  keU ch 195=  t.,  404  ts  Table 5  X-ray m i c r o a n a l y s i s . Ca counts at 3.700 keV over a 2 m i n acquisition time interval ± 2 S.D. ( n = 1 0 ) . Coccolith forming and non-coccolith forming strains of two species of coccolithophorids subjected to a 14:10 D:N cycle and continuous light r e g i m e s .  Species  Coccolith forming  D:N Cycle  Continuous  Non-coccolith forming  D:N Cycle  Continuous  Coccolithus pelaaicus  1993±260  2718±419  28±2  37±8  Emiliania huxleyi  2507±424  2267±637  113±28  31±19  tn oo  59 Figures 11A-H. SEM X-ray microanalysis micrographs for coccolith forming and non-coccolith forming strains of Coccolithus pelaqicus and Emiliania huxleyi. The micrographs were obtained over a 5.5-6.5 min acquisition period (Live: 322-399 s) at about 1000 cps. The windows shown are the scanning electron micrograph of the cell being examined (SEM), the calcium detection window (Ca), the background detection window (Bkg), and the osmium detection window (Os). The Real (time the computer clock was running in seconds) and the %Dead (all cells are dead) in the X-ray legends are not pertinent. C. pelagicus COPEL (coccolith forming motile strain) grown under a 14:10 D:N cycle (Fig. 11A) and continuous light (Fig. 11B). C. pelaqicus 241 (non-coccolith forming motile strain) grown under a 14:10 D:N cycle (Fig. 11C) and continuous light (Fig. 11D). E. huxleyi 88E (coccolith forming strain) grown under a 14:10 D:N cycle (Fig. H E ) and continuous light (Fig. 11F). E. huxleyi 646 (non-coccolith forming strain) grown under a 14:10 D:N cycle (Fig. 11G) and continuous light (Fig. 11H).  60 Fig.11 A  X-RflV: 0 - 20 keU Live: 393s Preset: 3200s Remaining: 2807s Real: 443 s 11 >: Dead  < 1.1 FS= SK OS= -19 MEN1:  3.700  keU ch 195=  6.3 ? 8336  ct s  * /  Fig. 11 B  X-RflVs 0 - 20 keU Live: 371s P r e s e t : 3200s R e m a i n i n g : 2829 Real: 414s 10": Dead  a u m u n i m •*.  < 1.1 FS= 8K 0S=-147 MEM 1 :  3.700 ch  keU 195=  7714  6.3 > cts  F/q. IIC  K-RflV: Lives Real:  0 30 U 0 Dead  401  a i n i n g: 267 u s  pltJJllr^h^Wiu*—liivMfH.-Ul.N  "^^^^t^rli K  1.1  !FS= 4K OS=-440 iMEMi:  III 3.700  keU ch 195=  "•/V-WLT,  t.  341  c t. s  63 Fiq- 1ID  X-RflV: 0 - 20 keU Live: 322s P r e s e t s 3000s R e m a i n i n g : 2678: r. t d i : 402 s 20* Dead  c  A_/ alaH  < 1.1 FS= 16K OS MEN1:  isil  gfegr~'"",~"  3.700 teQ  r keU ch 195=  6.3 > 1215 cts  X-RAY: 0 - 20 keU L i v e: 393 s Preset: 320 0 s Remaining: 2307s Real: 468 s 165i Dead  iMEM1 :  Rg.11 F  X-RflV: 0 - 20 keU L i ve: 393 s Pre se t : 320 0s R ema i ni n9: 280? Real: 433s 10* Dead  u*^"*  < 1.1 FS= 4K OS= MEMls  3 . 70 0 19  ch  keU 195=  2564  cts  u Fig.11 6  X-RftV: 0 - £0 keU Live: 399s Preset: 3200s Remaining: 2801s Real: 462s 14": Dead  < 1.1 FS= 8K 0S=-14~ MEM 1 :  i.700  keU ch 195=  o  6 . -• y .-. + -  LL =;  Fig.11 H  X-RflV: 0 - 20 ke'J Live: 399s Preset: 3200s Remaining: 2801 Real: 462s 14"-: Dead  1 . 1 |FS= 8K OS= -147 MEM1!  'fin keU ch 195  1044  ctsl  68  DISCUSSION Growth of several species of Prymnesiophyceae, many of which were coccolithophorids, have been examined previously under various conditions including continuous light and/or D:N cycle regimes (Mjaaland, 1956; Jeffrey and Allen, 1964; Paasche, 1967; Paasche and Klaveness, 1970; Hobson, 1974; Brand and Guillard, 1981; Chisholm and Brand, 1981; Brand, 1982a, b, 1984). The current work is the first to examine a number of growth, physiological and morphological parameters of several species and strains of coccolith-forming and noncoccolith forming Prymnesiophyceae under continuous light and 14:10 D:N cycle regimes. I. Growth Studies My results clearly demonstrate the variability within this taxonomic group of organisms. The Prymnesiophyceae, I. qalbana and Chrvsochromulina sp. exhibited significant sensitivity to continuous light (Figs. 1A and B, Table 2) compared to a D:N cycle as shown by a significant decrease in growth rate. Of the 9 coccolithophorids examined, Coccolithus neohelis, Cricosphaera carterae, Coccolithus pelaqicus 241, and C. pelaqicus COPEL (Table 2, Figs. IE, F, H, and I) exhibited a significant decrease in growth rate under continuous light. All of these species are oceanic species except Cricosphaera carterae which is a coastal species. The remaining coccolithophorids, the E. huxleyi strains 88E, 55a, and 64 6, Syracosphaera elongata, and Cyclococcolithus leptoporus (Table 2, Figs. IC, D, G, J, and  69  K) were either not affected by continuous light or exhibited an increase in growth rate when compared to a D:N cycle. S. elongata and C. leptoporus are oceanic species while E. huxleyi is a more ubiquitous species found in both coastal and oceanic regions. Also, C. leptoporus had a significantly higher growth rate under continuous light when cultured in enriched winter  natural seawater which is contrary to what  Brand and Guillard (1981) found. However, C. leptoporus did not show a detectable growth rate under continuous light when cultured in summer enriched natural seawater which agrees with Brand and Guillard's (1981) results. It is possible this species may have been in a different life cycle phase that resulted in a sensitivity to continuous light, even though there were no visible morphological differences when observed under a light microscope. In addition, the C. leptoporus strain used in this study was originally isolated from Stn. 5 in the North Pacific Ocean whereas the species used by Brand and Guillard (1981) was an isolate from the Sargasso Sea which may indicate possible genetically distinct strains of this species as a result of environmental adaptation (Mclntyre et al., 1970). The differences in growth rates under both light regimes among the coccolith-forming and non-coccolith forming strains of E. huxlevi and C. pelaqicus may also indicate possible genetic variability. Paasche and Klaveness (1970) found differences in growth rates between a coccolith-forming and a naked strain of E. huxleyi as well as growth rate  70  differences between batches of seawater used. Brand (1982a) also found different growth rates in clones of E. huxleyi which was attributed to genetic variability. My results demonstrated that growth rate sensitivity to continuous light is present for some species of coccolithophorids and for both the Prymnesiophyceae species, but it cannot be stated that oceanic species are less tolerant of continuous light than coastal species. Brand and Guillard (1981) demonstrated that oceanic coccolithophorids as well as other oceanic phytoplankton species are sensitive to continuous light. Diel periodicity has been demonstrated for a number of phytoplankton species. Emiliania huxleyi, Hvmenomonas carterae, and Isochrysis galbana have been found to divide primarily during the night (Paasche, 1967; Nelson and Brand, 1979; Chisholm and Brand, 1981). In this study, only 2 species' growth rates (E. huxlevi and §.. elonqata) were not significantly affected by continuous light (Table 2). It may be speculated that these species are influenced more by a biological clock than by a D:N cycle or continuous light. The remaining 7 species, which includes I. galbana and Cricosphaera (Hvmenomonas) carterae, exhibited a significant change in growth rate under continuous light, indicating they may also be influenced by a biological clock as well as a D:N cycle. When comparing growth rates between coccolith-forming and non-coccolith forming strains of E. huxleyi and C.  71  pelaqicus. it was found that based on growth rates calculated from cell densities, the non-coccolith forming strains were more sensitive to continuous light than the coccolith-forming strains (Tables 6 and 7 ) . Growth rates calculated in this study were comparable to growth rates reported by other researchers (Tables 8 and 9 in the Appendix). II. Maximum Yield The observation of a significant decrease in the maximum yields of the non-coccolith forming coccolithophorids and the two species of Prymnesiophyceae under continuous light compared to the 14:10 D:N cycle regime, further supports the sensitivity of these species to continuous light compared to the coccolith-forming species (Table 2). Even though cultures were grown at an irradiance of 100 /xE m  s~ , which is at the threshold of light  saturation for growth for many phytoplankton species, including coccolithophorids (Castenholz, 1964; Durbin, 1974; Brand and Guillard, 1981; and Glover et al., 1987), this continuous light irradiance level may be damaging over time to the non-coccolith forming strains due to a lack of a coccosphere. It has been hypothesized that coccoliths may reflect light, thus protecting the cell from cellular and photosystem damage at high irradiances (Braarud et al., 1952). There are no data to support this hypothesis, and there is no reported research comparing a number of coccolith-forming and non-coccolith forming  72  coccolithophorids' responses to continuous light and D:N cycle regimes. The diel periodicity of I. qalbana (Nelson and Brand, 1979) would account for the decreased maximum yield observed under continuous light since a dark cycle is needed for cell division. This may also be the case for Chrysochromulina sp. and C. pelagicus 241. Diel periodicity would not account for the significant decrease in maximum yield in the noncoccolith forming strains of E. huxleyi (55a and 646) since their maximum growth rates were not significantly different under either light regime. III. Physiological Responses Pigment determinations (chl a, chl c, and caroteniods) over time (Figs. 4A-D) was one parameter used to assess the physiological responses of the coccolithophorids to continuous light and the 14:10 D:N cycle regimes. No significant changes in pigment concentrations, ratios and fluorescence cell -1 were observed under either light regime. The coccolith-forming strains, however, had significantly higher concentrations of chl a and chl c (3 to 5 times) than the non-coccolith forming strains (Figs. 4A-D, Tables 6 and 7). Paasche and Klaveness (1970) reported that a coccolithforming strain of E. huxleyi had a higher concentration of chl a than a non-coccolith forming strain. One hypothesis to explain this observation, is that the coccosphere may be reflecting light and thus shading the cells. This would result in the chloroplasts being exposed  Table 6  Growth rates, chlorophyll a content, carbon and nitrogen content, and cell volumes of a coccolith-forming (C) and non-coccolith forming (N) strain of Emiliania huxlevi. Cultures were grown at 100 /JE m~ s~ irradiance under a 14:10 L:D cycle or continuous light regime, and 17 ± 1°C. All values are calculated with * 2 S.D.  Emiliania huxlevi Continuous Light  Cell Parameter  Growth rate (div. day - 1 ) Chi a (pg cell - 1 )  C  1.13*0.59 ,1 1.08±0.07 1 0.7*0.3  14:10 L:D Cycle  N  N/C (%)  0.83±0.131 1.23±0.022  86*38 114*5  0.2±0.0  0.48*0.03^ 0.85*0.13'  24*4 0.7*0.3  Carbon (pg cell" 1 )  50±8  Nitrogen (pg cell" 1 )  4.5*1.4  C:N ratio  8.0±1.1  13±2  25*0 51*15  2.1±0.4  48*7 5.3*2.0  4.8*0.5  60*2  8.2*0.7  N  N/C (%)  0.87*0.11A 0.93±0.062  181*12 110*10  0.2*0.0  29*7  13*3  25*2  2.1*0.2  5.3*0.9  43*13  68*3  Cell Volume (A/m3)  64±9  35*3  55*2  66*4  47*5  71*3  C/Chl a  78*24  63*11  84*13  76*11  65*13  84*5  N/Chl a  6.1±0.2  7.7*0.5  6.1*0.2  80*3  10.5*2.0  171*28  T =T Maximum \i (divisions day * 2 S. D.) calculated from cell density. Maximum fi (divisions day" * 2 S. D.) calculated from in vivo fluorescence.  -J CO  Table 7 Growth rates, chlorophyll a content, carbon and nitrogen content, and cell volumes of a coccolith-forming (C) and a non-coccolith forming (N) strain of Coccolithus pelaqicus. Cultures were grown at 100 /uE m s irradiance and 17 ± 1°C under a 14:10 L:D or continuous light regime. All values are calculated with ± 2 S.D.  Coccolithus pelaqicus Continuous Light  Cell Property  Growth rate (div. day" 1 )  N  14:ilO L:D Cycle  N/C (%)  0.7910.11-10.73±0.022  N/C (%)  C  N  0.53±0..ll1 0.68±0.,062  O^OtO.O 1 1.16±02  136±29 171±15  Chi a (pg cell" 1 )  9.0±1.2  1.8±0.2  20±1  6.9±1.,5  2.5±0.3  37±3  Carbon (pg cell" 1 )  452±202  366±83  88±23  349±127  3891129  112±4  Nitrogen (pg cell" 1 )  46±12  60±16  130±12  51±12  64±8  125±15  C:N ratio  5.0±0.9  4.7±0.3  95±11  4.9±0.7  4.7±0.5  Cell Volume (fjm3)  815±141  1053±86  130±12  996135  755±120  C/Chl a  49±16  201±24  431±97  42±1  156±6  372±5  N/Chl a  5.1±0.7  32.9±5.3  644±16  7.4±0.1  25.6*0.0  346±5  1  ;  =-T '' Maximum /u (divisions day ± 2 S. D.) calculated from cell density. Maximum /i (divisions day" ± 2 S. D.) calculated from in vivo fluorescence.  96±3 76±9  75  to lower irradiances than the chloroplasts in the noncoccolith forming strains. To maintain maximum photosynthesis the coccolith-forming cells might possibly compensate by increasing the density and/or size of the photosynthetic units (Prezelin, 1980). This would be reflected by observing coccolith-forming cells containing higher concentrations of chl a and c per cell than noncoccolith forming cells. POC, PON determinations over time was another physiological parameter used to assess coccolithophorids' responses to continuous light and D:N cycle regimes. No significant differences were found in POC, PON, and C:N ratios during exponential growth under continuous light compared to the D:N cycle. This suggests that cellular processes involved in the fixation of carbon and nitrogen are not affected by continuous light. The C:N ratios for the coccolith-forming strain, E. huxleyi 88E, was found to be 89. This is in good agreement with data from Sakshaug et al. (1983) and 0. K. Andersen (unpubl., 1986a and b) who found C:N ratios for E. huxleyi to be 7.1 and 7.4-11, respectively. The C:N ratios for the naked species E. huxlevi 646, C. pelagicus 241, and C. pelaqicus COPEL were found to be 4-6, 4-5, and 4-6, respectively (Figs. 8A-D). The 0.7±0.3 pg carbon cell -1 found in the coccolithforming strain E. huxlevi 88E, was within the range of that found by 0. K. Andersen (unpubl., 1986a) which was approximately 0.65 pg carbon cell"1. The coccolith-forming  76  strain, E. huxlevi 88E had 4 times the carbon cell~x and 2 times the nitrogen cell-  compared to the non-coccolith  forming strain, E. huxlevi 646 (Table 6 ) . The differences in carbon cell"  may be attributed to the presence of multiple  layers of coccoliths in E. huxleyi 88E which are composed of CaCC>3 in an organic matrix. It is suggested that 75% of the carbon in the coccolith-forming strain is contained in the coccoliths since E. huxleyi 88E has the same cell dimensions without coccoliths as the non-coccolith forming strain, E. huxleyi 646 (Table 4 ) . No significant differences in the carbon cell  x  and nitrogen cell  x  in the coccolith-forming  and non-coccolith forming strains of C. pelagicus (Table 7) may be attributed to the large standard deviations and the variations in cell size that were observed (Tables 4 and 7 ) . IV. Coccolith Ultrastructure and Calcium Content No differences in coccolith dimensions or morphology were observed among any of the species examined under either light regime (Table 4, Figs. 9A-H). Differences in calcium cell"  were found between the non-coccolith forming and  coccolith forming strains of coccolithophorids (Table 5, Figs. 11A-H). The coccolith-forming strains had 22-70 times more calcium than the non-coccolith forming strains under both light regimes. This is to be expected since the majority of the calcium is of the inorganic type found in the coccoliths. Since the calcium in the non-coccolith forming strains is intracellular, 96-99% of the calcium detected in the coccolith-forming strains is present in the  77  coccoliths. Linschooten et al. (1991) estimated that a coccolith-forming strain of E. huxlevi contained 0.21-0.26 pg calcium coccolith" . From SEM micrographs, it is estimated that E. huxlevi 88E contained 10-14 coccoliths cell -1 . As a result, the total coccolith calcium cell -1 may be estimated to be 2.10-2.64 pg calcium coccosphere"1. It is known that cells of E. huxlevi do not form coccoliths in the dark and only produce enough coccoliths to complete the coccosphere (van der Wal et al., 1987; Linschooten, 1991). This would account for the lack of difference in the calcium counts under continuous light and the D:N cycle regimes. There are also low-calcifying and high-calcifying strains of E. huxlevi (Nimer and Merrett, 1992). This would imply that calcium uptake rates and coccolith formation are variable between and among species of cocclolithophorids.  78 CHAPTER 2 EFFECTS OF PHOSPHATE AND NITRATE CONCENTRATION ON GROWTH, MAXIMUM YIELD, CELL AND COCCOLITH MORPHOLOGY, AND COCCOLITH INDUCTION  BACKGROUND Since nutrient concentrations have been demonstrated to affect algal yield, but not maximal growth rate for other phytoplankton groups, it is likely to be similar for coccolithophorids. Since Ca  transport is achieved by an  active transport system (most likely across the coccolith vesicle membrane in coccolithophorids) requiring a Ca + 2 ATPase (Klaveness, 1976; Okasaki et al. , 1984), it is possible that phosphate concentrations may affect cellular and coccolith formation and/or morphology. A large amount of research pertaining to coccolithophorids has been based on the mechanisms of coccolith formation, almost all with inconclusive results. The possibility of inducing coccolith formation under nitrate and phosphate limitation was proposed (Wilbur and Watabe, 1963; Paasche and Klaveness, 1970; Klaveness and Paasche, 1971; Sikes and Wilbur, 1980; O. K. Andersen, unpubl. 1986a and b), but no in-depth definitive or reproducible research on this aspect has been conducted. If nutrient limitation could induce coccolith formation or alter cell morphology, this may have implications for the global carbon cycle. If oceanic nutrient concentrations of phosphates and nitrates should rise due to anthropogenic causes, would  79  coccolithophorids reduce their coccolith formation rate? If so, the reduction in coccolith formation would have an affect on the conversion of atmospheric and aqueous forms of carbon (CO2 and HCO3"") into coccolith CaC03. If nutrient enrichment of the oceans were to occur, this may affect the concentration of atmospheric CO2 and thus global warming. Emiliania huxleyi has been observed to vary the size and number of coccoliths under laboratory culture conditions (personal observation). Size variation has also been observed in the natural oceanic environment (Mclntyre and Be, 1967). Variations in coccolith morphology within the same species have been attributed to coccolith malformations. Coccolithophorids with malformed coccoliths have been observed from the warm Australian waters (Hallegraeff, 1984), the marginal seas of the western Pacific Ocean (Okada and Honjo, 1975) and the subarctic waters of the north Pacific Ocean (Okada and Honjo, 1973). Okada and Honjo (1975) were unable to find any correlation between the malformations and physical or chemical factors. Okada and Honjo (1975) hypothesized that because the malformations involved structural differences in the coccoliths, the malformations were a result of an alteration in the coccolith organic matrix or the shape of the coccolith vesicle during coccolith formation. To date no research has examined this particular aspect of coccolithogensis. This chapter characterizes quantitative and qualitative  80  responses of several coccolithophorids (coccolith-forming and non-coccolith forming) to various phosphate concentrations and examines the induction of coccolith formation under phosphate and nitrate limitation.  81 MATERIALS AND METHODS General Culture Maintenance Eight stock cultures listed in Table 1 consisted of four coccolith-forming species (Coccolithus neohelis. Coccolithus pelaqicus, Emiliania huxleyi 88E, and Svracosphaera elongata) and four strains of non-coccolith forming Emiliania huxleyi (646, 55a, 451B, and 556). These strains were used were maintained as in Chapter 1 except that the ESAW-enriched (HESNW) natural seawater (Harrison et al. 1980) was modified. Na 2 HP0 4 was added to final concentrations of 13.0 juM, 8.0 jzM, while 3.0 /xM PC>4~3 was the concentration of the natural seawater used in this experiment, except for Emiliania huxleyi 88E which was maintained at 13.0, 10.0, and 0.9 /zM P0 4 ~ 3 (the P04""3 concentration of the natural seawater used). NaN03 was added to a final concentration of 3 00 /xM. Culture vessels and stock and experimental cultures employed throughout this research were maintained as mentioned in Chapter 1. Growth Experiments Experimental cultures were maintained at PC>4~3 concentrations of 13.0, 8.0, and 3.0 /zM (except for E. huxleyi 88E which was maintained at 13.0, 10.0, and 0.9 /xM PO4""3 concentrations) all on a 14:10 D:N cycle (except E. huxleyi 88E cultures at 10.0 /zM P0 4 ~ 3 which were also maintained under a continuous light regime) at an irradiance of 100 /xE m~2 s - 1 and 17±1°C using cool-white fluorescent  82  tubes. Coccolithus neohelis was maintained at 21°C. All growth studies were carried out in culture tubes containing 1.25 ml of exponentially growing stock culture diluted with 25.0 ml ESAW-enriched (HESNW) natural seawater. Growth was monitored directly in the culture tubes by reading in  vivo  fluorescence at the same time every day using a Turner Designs fluorometer after brief hand mixing. This mixing did not appear to affect the growth or morphology of the cells. Plots of the log of fluorescence against incubation time were used to calculate the growth parameters described in Chapter 1. Visual examination of the cultures was carried out at the same time every day to observe the health and morphology of the cells. Analytical Methods i) Fluorometry Growth was monitored using a Turner Designs Model 10 fluorometer to measure in vivo  flourescence of the  experimental cultures. Fluorometry was done at the same time every day to minimize the effect of the D:N cycle on fluorescence. ii) Coccolith Formation The percentage of cells with coccoliths was calculated by visually counting the cell density and the number of cells with one or more coccoliths in 0.1 - 1.0 ml of the experimental cultures. This was done in triplicate at the same time every day following fluorometry measurements. Cultures of Emiliania huxleyi 88E were allowed to enter  83  senescence. At this point, cells did not form coccoliths and gentle agitation by hand removed the coccoliths from most of the cells in culture. Cultures of Coccolithus neohelis, maintained at the three phosphate concentrations (3.0, 8.0, and 13.0 juM) , were allowed to reach senescence before transfer to new medium at the same phosphate concentrations. At this point, cells in the 3.0 juM phosphate medium lost their coccoliths easily such that only about 10% of cells had coccoliths when transferred to new medium. The cells in 8.0 and 13.0 /zM phosphate medium did not lose their coccoliths when transferred to new medium. A 1.25 ml sample of these cells were then added to 25.0 ml of ESAW-enriched (HESNW) natural seawater at 0.9 (the P0 4 ~ 3 of the natural seawater used), 10.0, and 13.0 ixM P0 4  concentrations. Each  experiment was done in triplicate (n = 9). iii) Scanning Electron Microscopy (SEM) Fixations for SEM observations of coccoliths and cells were always carried out at the same time of day and the same stages of the growth curve (early, mid, and late exponential growth) for Coccolithus neohelis to minimize the effect of the culture conditions on cellular and coccolith ultrastructure. Samples were concentrated by gentle filtration onto 0.45 /zm Nuclepore filters and fixed for 1 h at room temperature with 2% glutaraldehyde (v/v) and 1% 0s0 4 (v/v) sodium cacodylate (0.2 M) buffer (NaCac pH 7.3). The cells were rinsed thoroughly with NaCac buffer prior to dehydration in a graded ethanol series. Next, cells were  84  subjected to critical point drying in a Balzer's Union CPD 020 Critical Point Dryer and gold coated before insertion into a scanning electron microscope, iv) Coccolith Induction Nitrate (NO3"") and phosphate (P04~3) limited experimental and stock cultures were maintained at 300 juM N0 3 " and 0.9 JUM PO4-3 or 30 /xM N0 3 ~ and 13.0 /xM P0 4 " 3 concentrations respectively. All cultures were maintained at 17 ± 1°C on a 14:10 D:N cycle at 100 juE m~2 s - 1 irradiance using cool-white fluorescent tubes. The natural seawater used in the modified ESAW-enriched (HESNW) natural seawater had initial nitrate and phosphate concentrations of 3 0 juM and 0.9 fj.M respectively. No phosphate was added to the phosphate-limited cultures of Emiliania huxleyi and no nitrate was added to the nitrate-limited cultures of E. huxleyi. All coccolith induction studies were carried out in culture tubes containing 1.25 ml of exponentially growing stock culture diluted with 25.0 ml ESAW-enriched (HESNW) natural seawater. The percentage of cells with coccoliths was calculated by determining with the light microscope, the cell density and the number of cells with one or more coccoliths in 0.1 - 1.0 ml of the experimental cultures. This was done in triplicate at the same time every day.  85 RESULTS I. Growth Studies Six species of coccolithophorids were tested for their ability to grow under different P0 4 ~ 3 concentrations (Tables 2.2 - 2.4 and Figs 2.1A-E and 2.2A-C). Under the D:N cycle regime significant differences in growth rates were observed in the non-coccolith forming strain, Emiliania huxleyi 55a and the coccolith-forming strain, Coccolithus neohelis. E. huxleyi 55a and C. neohelis had significantly lower growth rates (60 and 20%, respectively) at 3.0 /iM P04~ 3 compared to 13.0 and 8.0 JUM P0 4 ~ 3 (Fig. 2.1A and D, and Table 2.2). The other four species (E. huxlevi 88E and 64 6, C. pelagicus COPEL, and S. elongata) showed no significant differences in growth rates under the three phosphate concentrations (Table 2.2 and 2.3, and Figs. 2.1B-D and 2.2A and C) . II. Maximum Yield The maximum yields were not significantly different at 13.0 and 8.0 juM P04-3 for the species E. huxleyi 646, C. neohelis, and C. pelagicus COPEL, but at 3.0 /iM PO4""3 the maximum yields were significantly lower than yields at the two higher PC>4~3 concentrations for all of the species studied (Table 2.2 and Figs. 2.1A and 2.ID). E. huxlevi 88E showed the same trend in maximum yields based on both cell density and in vivo B).  fluorescence (Table 2.3 and Figs. 2.2A-  86  Figures 2.1A-E. Semi-log plots showing growth curves as a function of in vivo fluorescence over time for 5 species of coccolithophorids subjected to 3 P(>4~3 concentrations (13.0, 10.0, and 3.0 MM). All curves represent triplicate experiments each done in triplicate. Vertical error bars represent ± 2 S. •D. (n = 9) and are smaller than the symbol where not apparent.  Emiliania huxleyi 55a (Non—coccolith forming stroln)  100 0  Syracosphaera elongata  100.0  =!••• Z io.o -C  o  1o  ,> c  1.0  10.0 JC  f  u.  /  o  O [p04^ O — O 3 . 0 uM • — • 8 . 0 uM A — A 13.0 uM  c  /  0.1  0.1 5  10 Time (Days)  15  I.«, ,.»„•„ mlm,*,.»-A .t-l»*..A~*..fc. !•,>••  0  20  Emiliania huxleyi 646 (Non-coccolith forming strain)  100  [PO4 3 ] O — O 3 . 0 uM • — # 8 . 0 uM A—A13.0uM  1.0 t  5  10 15 20 Time (Days)  25  30  Coccollthus neohelis  1000.0 B 100.0  -O -A  u  •i  10.0  o %  ^-3i [P04J] O — O 3 . 0 uM • — # 8 . 0 uM A—A13.0uM M.  ._ . . . . . . .  I  .  .  .  .  I  5 10 Time (Days)  15  1.0  r  Q  '•*  1  0  [PO4 3 ] O — O 3 . 0 uM • — 9 8 . 0 uM A — A 13.0 uM * l ^ 1 ' I*  5  J  l ' * ' * * * * ' t * * * * . . . . I  10 15 20 Time (Days)  25  30  Coccolithus pelagicus COPEL (Coccolith forming motile strain)  100.00  10.00  [PO43] O — O 3 . 0 uM • — • S . O uM A A 1 3 . 0 uM  0.01 10  0 Time (Days)  00 09  Table 2.2  Effects of phosphate concentration at 17±1°C and a 14:10 D:N cycle at (b) maximum growth rate (divisions fluorescence in relative units l 2  on coccolithophorid growth parameters. Batch cultures grown 100 uE m s irradiance. (a) maximum growth period (days) ; day" ± 2 S.D. where n = 6 to 9); (c) maximum yield {in vivo S.D.)  13.0 /iM  ALGA  b  8.0 /iM  3.0 ^M  b  b  NON-COCCOLITH FORMING Emlllania huxlevi 55a  2-6  1.11±0.06  57.3±11.0  2-6  1.16±0.11  30.8±10.0  3-6  0.45±0.11  5.4±2.0  Emllianla huxlevi 646  1-4  0.9110.06  54.0126.9  1-4  0.9910.27  66.017.1  1-4  0.9510.18  40.3±1.8  Coccolithus pelaqlcus COPEL  2-6  1.72±0.14  30.014.8  2-6  1.8510.14  25.013.2  2-5  1.84±0.17  12.911.2  Coccolithus neohelisJ  1-7  1.34±0.04  99.7±17.0  1-7  1.3510.09  89.0±26.9  1-7  1.07±0.15  38.3±4.6  Svracosphaera elonqata  0-16  0.46±0.03  28.6±4.3  0-16  0.43±0.01  18.711.0  0-16  0.4110.04  15.211.0  COCCOLXTH-FORMING  This refers to the time period used to calculate (b), the maximal growth rate. Coccolith-forming motile strain. Q. neohelig maintained at 21°c. oo  90  Figures 2.2A-C. Plots showing growth curves as a function of cell density (Fig. 2.2A), the % of cells with coccoliths (Fig. 2.2B), and in vivo fluorescence (Fig. 2.2C) over time for Emiliania huxlevi 88E.  Fig. 2.2A 1000.00  Emilianio huxleyi 8BE 13.0|iM[P0 4 ~ 3 ] 14:10 D:N Cycle  14:10 D:N Cycle  1100.00 « u  A A"  A  10.00  -^-A-A ^A-A-A-4  T  IA. . A — A | \ AA Al 1 N ./i  A  \ 1.00  5  o3  ^ 0.10 t  V  u  -3  10.0 uM [ P 0 4 ] A — A 14:10 D:N Cycle A— A Cont Light  y?#—•0.9 MM  A ^ s^i'T /# A  A10.0uM  0.01 _  •  » |i '  *  -  ' •  » » » ,  2  10  »  ». I  .  «  »  »  I  .  .  4 6 Timo (Days)  . .  I .  8  .  .  ..  I  .  .  .  .  I  .  .  .  . .1  .  10  .  .  .  I  *  15  *.. . . ! _ * .  J__A_.  20  25  Hg. 2.2B 100  Emilianio huxleyi 88E 14:10 D:N Cycle TA  91  80  A — - A + 2 mM Ca2+ 14:10 D:N Cycle 13.0|JM[P04-J]  £  o o o o o  60  *  40  *  20  i  [PC*3]• — © 0 . 9 uM * 10.0 uM [P0 ~ 3 ] I 4 A—A10.0JJM k A D:N Cycle i *— A Cont Light t  10  p  •  -  r  f i « i . i . .  1  I  . i . « . . i  4 6 Time (Days)  T 25 to  Fig. 2.2C 100  Emiliania huxleyi 88E  10.0 JJM [ P 0 4-" 3" Ji ]  13.0uM[P0 4 ~ 3 ]  0 ^0;f-8^8"  l'*T  IT  "7  10  /  t  •  •  - • 14:10 D:N Cycle O - O Cont Light •  '  ILL  *•  /,  .  &  ML  1  -/  o/  S  •  i  t  *  *  * i •  i  r  Bi i *  '  •  '  '  '  *  '  •  K i  i  8 0 5 Time (Days)  i  i  «  10  • • •  i  i  15  i  i  i  i  20  » i  25  Table 2.3  P0 4 -3  13.0  IJH  10.0 fjH  Effects of phosphate concentration on Emiliania huxlevi 88E growth parameters. Batch cultures grown at 17±1°C and a 14:10 D:N cycle or continuous light at 100 (JE m~ s~ irradiance. (a) maximum growth period (days) ; (b) maximum growth rate (divisions day" 1 2 S.D. where n = 6 to 9); (c) maximum yield (in vivo fluorescence (F) in relative units 1 2 S.D. and/or cell density x 10 cells m l - 1 (CD) ± 2 S.D.)  D:N Cycle/ Continuous  Fluorescence/ Cell Density  F  5-10  0.7710.10  41.3126.1  CD  5-9  0.66±0.06a  47.5133.5  F  2-5  0.87±0.06  36.013.3  CD  6-11 2-5  1.1110.27b 0.9710.10°  39.615.0  F  1-4  1.2610.01  31.312.0  CD  1-6  1.0510.05°  107131  CD  6-10  1.1710.35b  7.910.4  D:N  D:N  Cont  0.9 ^M  D:N  Coccolith-forming strain. This refers to the time period used to calculate (b), the maximal growth rate. a Growth rate based on growth curve in Fig. 2.2A right panel. Growth rate based on growth curve in Fig. 2.2A left panel. c Growth rate based on growth curve in Fig. 2.2A middle panel.  94 III. Coccolith Formation and Induction The increase in the percentage of cells with coccoliths over time for E. huxleyi 88E was not significantly different at any of the three PO4  concentrations when examined under  a 14:10 D:N cycle (Fig. 2.2B). Under continuous light at 10.0 /zM P04~ 3 , there was a significant rise in the percentage increase in cells with coccoliths day  x  (Table  2.4) compared to that observed under a 14:10 D:N cycle (24 ± 2% under continuous light compared to 13±4% under a 14:10 D:N cycle). The percentage of cells with coccoliths over time for Coccolithus neohelis was significantly different at the three phosphate concentrations. At 3.0 /JLM. phosphate, the cells lost their coccoliths easily when trasferred to new medium, and then the percentage of cells with coccoliths rose from 10% at the time of transfer (day 0) to 70% by the eighth day of exponential growth (Fig. 2.3 based on cells observed in cultures from Fig. 2.ID). At 8.0 and 13.0 //M phosphate, the cells did not lose their coccoliths upon transfer to new medium which resulted in 100% of cells having coccoliths at time 0 (Fig. 2.ID and 2.3). Efforts to induce coccolith formation in strains of non-coccolith forming E. huxleyi under nitrate and phosphate limitation were unsuccessful (Table 2.5). IV. Ultrastructure The only species which showed any morphological (cellular and coccolith) differences at the three PO4 concentrations was Coccolithus neohelis. The SEM micrographs  Table 2.4  Effects of phosphate concentration on the percentage (%) of cells with coccoliths over time in Emiliania huxlevi 88E . Batch cultures grown at 17±1°C under a 14:10 D:N cycle and/or continuous light at 100 /JE m s~ irradiance. (a) time period of maximum increase in cells with coccoliths (days); (b) range of % of cells with coccoliths over time period in (a) ± 2 S.D.; (c) % increase in the number of cells with coccoliths day" (n = 90 cells).  [P04~3]  D:N/ Cont  a (Days)  b (Range)  (% rise day" )  13.0 fjH  D:N  0-7  10.0 fiH  D:N  0-3 4-10  45±0 1±1  Cont  0-2  47±2 - 93±2  24±2  D:N  4-10  1±1 - 97±2  16±0  0.9 /uM  2±0 - 72±20  79±12 98±2  10±3  11±4 13±4  Coccolith-forming strain.  to oi  Table 2.5  Coccolith induction experiments on different strains of Emiliania huxleyi during nitrogen and phosphorus limitation. Values are the % of cells with one or more coccoliths visible in the light microscope  Strain  - P0>  - N0 3 '  <1  <1  55a  NJ  556  N  0  0  646  N  0  0  0  0  451B 88E a  Cell Type  97±2  90±3  3 00 fifl NC>3~ and 0.9 /iM P04~ J concentrations used. No P 0 4 ~ J was added to the natural seawater used as medium. b 30 juM N 0 3 ~ and 13.0 juM PO4- 3 concentrations used. No N 0 3 ~ was added to the natural seawater used as medium. *N = non-coccolith forming (naked) strain. 2 S = scale-forming strain. 3 C = coccolith-forming strain.  100  13.0 uM 8.0 uM 3.0 uM  10 Time (Days)  Figure  2.3  Time (Days)  E f f e c t s of p h o s p h a t e c o n c e n t r a t i o n on t h e m o r p h o l o g y of C o c c o l i t h u s n e o h e l i s o v e r t i m e d u r i n g e x p o n e n t i a l g r o w t h (Fig. 2 . I D ) . S e m i - c o n t i n u o u s b a t c h c u l t u r e s w e r e g r o w n u n d e r a 1 4 : 1 0 D:N c y c l e at 2 1 ° C and 100 nE m - 2 s - 1 i r r a d i a n c e . T h r e e s e p a r a t e a r e a s / S E M grid w i t h a p p r o x i m a t e l y 30 c e l l s / a r e a / e x p e r i m e n t w e r e e x a m i n e d u n d e r SEM (n = 90 c e l l s / e x p e r i m e n t ) . A t t r a n s f e r t o n e w m e d i u m , c e l l s at 3.0 /iM p h o s p h a t e lost t h e i r c o c c o l i t h s e a s i l y w h e r e a s c e l l s at 8.0 and 13.0 /iM p h o s p h a t e retained t h e i r c o c c o l i t h s . T h i s is r e f l e c t e d in t h e % c e l l s w i t h c o c c o l i t h s at  time 0.  vo  98  Plate l. SEM micrographs of the various morphological changes that occurred over time in Coccolithus neohelis when cultured in 13.0, 10.0, and 3.0 fM P04~ J medium. A naked non-flagellated cell (1A); A coccolith-forming nonflagellated cell (IB); Type 1A and IB cells in the same culture (IC); flagellated coccolith bearing cells (ID); and a flagellated non-coccolith bearing cell (IE).  PLATC A  99  100  in Plate 1 are typical of the types of cells observed. The percentages of cells with and without coccoliths and with and without flagella were observed to vary dramatically and significantly at the three PO4  concentrations (Fig. 2.3)  and over time. At 3.0 juM P 0 4 - 3 90-100% of the cells of C. neohelis had flagella throughout the exponential growth phase (2, 4, and 8 days on Fig. 2.ID). This is significantly different from the 0-10% of cells observed to have flagella at 8.0 and 13.0 lM PO4  . The 3.0 /xM PO4  cultures showed an increase in  the number of cells with coccoliths over time from 10% at 2 days to 70% by 8 days. This is significantly different from the 8.0 and 13.0 /xM P0 4 ~ 3 cultures which maintained 100% of cells with coccoliths (Fig. 2.3). As mentioned above, the cells in 3.0 /J,K phosphate lost their coccoliths easily upon trasfer to new medium whereas the cells at the two higher phosphate concentrations did not (Fig. 2.3). There were no significant differences in the morphology of the coccoliths at any of the three P0 4 ~ 3 concentrations.  101  DISCUSSION I. Growth Studies and Maximum Yield Phosphate limitation may be described in terms of cell yield or growth rate. For this study, growth rates were calculated based on the exponential growth phase. When nutrients are saturating, phosphate limitation was indicated by a significant reduction in maximum yield compared to that obtained when phosphate was saturating for growth. The culture medium contained 300 /zM nitrate. This would result in medium N:P ratios of 23-37:1 at 8.0-13.0 £iM phosphate concentrations. Sakshaug et al. (1983) found E. huxleyi to have an N:P balance point of 17. Even at these higher phosphate concentrations (8.0-13.0 MM), the E. huxleyi strains may have been under phosphate limitation, but this was only apparent at 0.9 and 3.0 fxM. phosphate concentrations as evidenced by a significant decrease in maximum yield (Table 2.6) for all species. II. Coccolith Formation and Induction Phosphate concentrations did not appear to significantly affect the increase in the percentage of cells with coccoliths over time for the coccolith-forming strain E. huxleyi 88E (Fig. 2.2B and Table 2.4). This would tend to suggest that the active transport of Ca + 2 by a Ca+2-ATPase was not affected by the phosphate concentrations used in this study. Attempts to induce coccolith formation in naked strains of E. huxleyi under phosphate or nitrate limitation  102  was unsuccessful (Table 2.5). O. K. Andersen (unpubl. paper, 1986a and b) was able to induce coccolith formation in all the cells of a culture of a naked strain of E. huxleyi under phosphate limitation (300 ;uM nitrate and 0.77 juM phosphate) in medium containing a N:P ratio of 389:1, but not in scaleforming strains. Earlier reports of coccolith induction involved only a few cells in a culture of a naked strain of E. huxleyi (Wilbur and Watabe, 1963; Paasche and Klaveness, 1970; Klaveness and Paasche, 1971; Sikes and Wilbur, 1980). Even though flagella were not observed, it is possible the E. huxleyi strains used in this study were scale-forming and not naked. Also, not using medium with phosphate concentrations lower than 0.9 jiiM, might account for the lack of coccolith formation in the E. huxleyi strains used in this study. III. Ultrastructure Two of the physiological responses of phytoplankton to nutrient limitation involve changes in cell morphology and a change in life cycle stage. Under phosphate limitation, morphological changes may involve colony formation in Phaeocystis pouchetii (Veldhuis and Admiraal, 1987), or an increase in  cell size in Pediastrum duplex (Lehman, 1976).  These two morphological changes may result in an increase in sinking rates which would allow cells to reach more nutrient-rich waters. Another morphological change that may occur is flagellation. Flagella are important in both  Table 2.6  Effects of Phosphate concentration on growth parameters as a % of maximum growth rate, and maximum yield with 13.0 ^H. P0 4 used as a control (100 % ) . Batch cultures grown at 17 1 1"C under a 14tl0 DiN cycle at 100 JJE m a irradlance. Values based on results from Table 2.2 and 2.3. means values are significantly different from the 13.0 fjH P0 4 ~ values used as control. Arrows represent the increase or decrease from the 100% maximum values observed at 13.0 ;JM P0 4 .  B.O pM  Species  J  PO4  % of maximum  3.0  IJH P O 4  J  % of maximum  growth rate  yield  »5  I4S  tl6  191  le  143  U  {10  li  112  (20  t61  »7  lie  t7  •si  le  ill  HI  in  growth rate  yield  NON-COCCOLITH FORMING Emlllanla huxlevi 55a Emlllania huxlevi 646 COCCOLITH-FORMING Coccolithua neohells Coccolithue pelaqlcuB COPEL Svracosphaera elonaata  Emiliania huxlevi 88E  10.0 //M P0 4 " J  0.9  % of maximum  * of maximum  growth rate  yield  F1  il2  f20  CD 2  •26-44  *29  /JM  P04"J  growth rate  yield  r 77  {73  DiN 3  { 64  t6  • 52  • 210  Cont^  CD  Values based on in vivo fluorescence. Values based on cell density, •jBatch cultures grown under a 14tl0 DiN cycle. Batch cultures grown under a continuous light regime.  104  decreasing the nutrient-deplete unstirred layers around the cell and in moving the cell to areas of higher nutrient concentrations (Raven, 1976 and 1980). This is evidenced by the vertical, diurnal migration of some dinoflagellate species. Coccolithophorids are known to have different life cycle stages (an alternation of generations) in which one or both involve a flagellated stage (Parke and Adams, 1960). One or both of these stages produce coccoliths. Coccolithus pelagicus may alternate between a motile and non-motile phase in which both produce coccoliths, and Cricosphaera carterae includes these two phases as well as a benthic filamentous and naked motile stages (Tappan, 1980). The triggering factors for these life cycle changes are unknown, but one hypothesis involves nutrient depletion. The morphological changes observed in Coccolithus neohelis in this study may be attributed to phosphate concentration (Fig. 2.3 and Plate 1). Under high phosphate concentrations (8.0 and 13.0 /xM) , cells of C. neohelis were non-flagellated with coccoliths, whereas under low phosphate conditions (3.0 juM) 100% of cells were flagellated with the percentage of coccolith-forming cells increasing over time from 10% at time 0 days to 70% by day 8 of exponential growth (Figs. 2.ID and 2.3). It may be suggested that by day 8 of exponential growth, the cells in 3.0 /xM phosphate medium lost their coccoliths easily whereas cells in the higher phosphate concentrations did not lose their  105  coccoliths. This may indicate a decrease in adhesion of the coccoliths to the cell surface and to each other at 3.0 MM phosphate concentrations Under low phosphate conditions, flagellated cells would have an advantage by being able to move to a region of nutrient-rich water. The same is true for the increase in sinking rate of a coccolith-forming cell compared to a noncoccolith forming cell. Phosphate limitation does not fully explain the observed morphological cell types in C. neohelis.  106 GENERAL CONCLUSIONS Various aspects of coccolithophorid and prymnesiophyte responses to continuous light and D:N cycle regimes, and phosphate concentrations were examined. The major conclusions and contributions are summarized below.  1.  Despite the variability demonstrated within this  taxonomic group, a decrease in growth rate under continuous light compared to a D:N cycle regime suggests a sensitivity to continuous light. 2.  The non-coccolith forming coccolithophorids and the  two Prymnesiophyceae species were more sensitive to continuous light than the coccolith-forming coccolithophorids of the same species. This may suggest the presence of genetic variability and diel periodicity. 3.  No significant changes in pigment parameters were  observed under either continuous light or a D:N cycle. 4.  Coccolith-forming strains of E. huxleyi and C.  pelaqicus had 3-5 times higher concentrations of chl a and chl c than the non-coccolith forming strains of the same species. This suggests that the coccosphere may be shading the cell requiring an increase in the density and/or size of the photosynthetic units to maintain maximum photosynthesis. 5.  No significant differences in POC, PON, or C:N  ratios were observed under either light regime. This suggests that cellular processes involved in the fixation of carbon and nitrogen are not affected by continuous light.  107  6.  The coccolith-forming strain of E. huxleyi had 4  times the carbon cell -1 and 2 times the nitrogen cell -1 compared to the non-coccolith forming strain under both light regimes. 7.  The coccolith-forming strains had 22-70 times more  calcium cell"1 than the non-coccolith forming strains under both light regimes, and it is suggested that 96-99% of the calcium is in the coccoliths. 8.  Phosphate concentrations (0.9, 10.0, and 13.0 nK)  did not affect the increase in the percentage of cells with coccoliths over time for the coccolith-forming strain of E. huxleyi. 9.  Coccolithus neohelis underwent morphological  changes under various phosphate concentrations from flagellated non-coccolith forming cells at low phosphate concentrations to non-flagellated coccolith-forming cells at higher phosphate concentrations.  108  FUTURE RESEARCH This thesis suggests a number of areas amenable to future research that would add to the knowledge about this ecologically important group of organisms. 1.  Examine coccolith induction in naked strains of E.  huxleyi under phosphate limitation at concentrations lower than 0.9 /xM, and other factors such as nitrate, trace metals, vitamins, and other organics should be explored. 2.  Examine the possibility of isolating and  maintaining the different morphological cell types (flagellated and non-flagellated; non-coccolith forming and coccolith-forming) observed in C. neohelis in order to conduct physiological comparisons under various environmental conditions (nutrient uptake, pigments, photosynthesis, responses to light quantity and quality, etc.). 3.  DNA analysis and morphological examination (TEM and  SEM) on the C. neohelis cell types. 4.  Examine the responses of these and other  coccolithophorids to phosphate limitation with emphasis on cell quotas of phosphorus, alkaline phosphatase activities, the amount of calcium as calcium carbonate in coccoliths (CjJ and in the cell (C0) and their ratios, and calcification rates (C^/C 0 ). 5.  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Smayda, T. J. 1958. Biogeographical studies of marine phytoplankton. Oikos, 9:158-191. Strickland, J. D. H. and T. R. Parsons. 1972. A Practical Handbook of Seawater Analysis. Bulletin 167, Bull. Fish. Res. Board Canada, Ottawa, p. 185192. Tappan, H. 1980. The Paleobiology of Plant Protists. W. H. Freeman and Co., 1028 pp. van der Wal, P., E. W. de Jong, and P. Westbroek. 1983. Ultrastructural polysaccharide localization in calcifying and naked cells of the coccolithophorid Emiliania huxleyi. Protoplasma. 118:157-168.  116 van der Wal, P., J. P. M. de Vrind, E. W. de Vrind-de Jong, and A. H. Borman. 1987. Incompleteness of the coccosphere as a possible stimulus for coccolith formation in Pleurochrysis carterae (Prymnesiophyceae). J. Phycol.. 23:218-221. van Emburg, P. R., E. W. deJong, and W. Th. Daems. 1986. Immunochemical localization of a polysaccharide from biomineral structures (coccoliths) of Emiliania huxleyi. J. Ultrastr. and Mol. Struc. Res.. 94:246-259. Veldhuis, M. J. W. and W. Admiraal. 1987. Influence of phosphate depletion on the growth and colony formation of Phaeocystis pouchetii (Hariot) Lagerheim. Mar. Biol.. 95:47-54. Westbroek, P., J. R. Yuong, and K. Linschooten. 1989. Coccolith production (biomineralization) in the marine alga Emiliania huxleyi. J. Protozool., 36:368-373. Wilbur, K. M., and N. Watabe. 1963. Experimental studies on calcification in molluscs and the alga Coccolithus huxleyi. Annals N. Y. Acad. Sci., 109:82-112. Wilbur, K. M., and N. Watabe. 1966.Effects of temperature on growth, calcification and coccolith form in Coccolithus huxleyi. Limnol.Oceanocrr. . 11:567-575. Yentsch, C. S. and D. W. Menzel. 1963. A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep Sea Res.. 10:221-223.  Appendix 1  Formulae used for pigment concentration analyses, measured on a spectrophotometer (spec), or a fluorometer (fluor).  Pigment  Formulae (/jg L-l) X  Method  Reference  Spec  (Jeffrey and Humphrey, 1975)  Chi a  S(11.47E664-0.40E630)V-11  Chi a 1  1.40(Fo-Fa)(1/0.586763)(v/V)  Fluor  (Yentsch and Menzel, 1963)  Chi c  S(24.36E 630 -3.73E 664 )V" 1 1 _1  Spec  (Jeffrey and Humphrey, 1975)  Phaeopigments  26.7[1.7(E664a)-E664o]SV-ll-l  Spec  (Lorenzen, 1967)  Phaeopigments  1.40(1.8Fa-Fo)(1/0.586763)  Fluor  (Parsons and Strickland, 1963)  Spec  (Yentsch and Menzel, 1963)  Carotenoids  •S(E480)xlO  S = vol. of acetone (ml) V = vol. of sample filtered (L) v = vol. of acetone (L) E 664 = a n d other extinction measurements must be corrected for turbidity by subtraction of measurement at E750. E 480 = corrected for turbidity by subtraction of measurement at 3E75Q. 1 = path length of cuvette (1 cm cuvettes used in this study) F = in vitro fluorescence of chl a Subscript ' a' = with acid addition (2 drops 10% HC1) Subscript 'o'= without acid addition  1.40(1/0.586763)= calibration factor for Turner Designs Fluorometer (factor=Door value x q/q-1)  118  Coccolithus pelogicus COREL (Coccolith formino motile strain^  100  Emiliania huxleyi 88E (Coccolith forming stroinV  10C  vO-o<K>  -***££  f y  •-0  10  flO JZ u  o l  o  T / *  I  1  ii-  A  u. o  >  >  0.10  l  0.01  10  c 1  • • 14:10 D:N Cycle O— OContinuous Light 5 10 Time (Doys)  0.10  15  Coccolithus pelogicus 241 (Non—coccolith forming motile strain)  1II  • • 1 4 : 1 0 0:N Cycle O— OContinuous Light  5 10 ' Time (Days)  15  Emiliania huxleyi 64-6 (Non—coccolith formino stroin^  100  ^o-o  -C  f  o  u  o'S  I  ^- 1 o  / '  o  >  >  c 1  /  X  *'t • — • 14:10 D:N Cycle O— OContinuous Light  — • 14:10 D:N Cycle O— OContinuous Light  x,oLL  0.10 4 6 Time (Days)  10  4 6 Time (Days)  10  Appendix 2A-D. Semi-log plots showing growth curves as a function of in vivo fluorescence over time for 2 coccolithophorids each consisiting of a coccolith-forming (Coccolithus pelaaicus COPEL and Emiliania huxleyi 88E) and a non-coccolith forming strain (Coccolithus pelaaicus 241 and Emiliania huxleyi 646). The growth curves are the result of 4, 5.25 L batch cultures started in a 14:10 D:N cycle and at the arrow, 2 cultures were switched to a continuous light regime. All curves represent duplicate experiments each done in quadruplicate. Vertical error bars represent ± 2 S.D.(n = 4 to 8) and are smaller than the symbol where not apparent.  119  Coccolithus pelagicus COPEL ICoccolrth forming motile strain) 60 t — A C h l a T • •Chi c | • •Carot. 4  r*) I  E 3 o» 3  I o  /  I  o  E  /  20  /  25  E 3 o> 3  o> I o  A  40  _3  I  Emiliania huxleyi 58E (Coccolith forming strain) • • Ca roten oids •—AChl a A—AChl c  \  \  a  E  \ \  "5 O •*•> c o  "5 O o  E  o»  5 10 Time (Days)  K  15  Coccolithus pelagicus 241 -coccolith forming motile strain)  I  E  5 10 Time (Days)  I  E  E  3  3  15  Emiliania huxleyi 646 (Non—coccolith forming strain)  en 3 Ci  I  o o  E _3  "5 U •4-1  c a>  E  a»  ~  2  4  6  Time (Days)  2  4 6 Time (Days)  8  -x ,-1 Appendix 3A-D. Changes in pigment cell volume (x 10-9 "<3 /xm~ ) over time for 2 coccolithophorids under the same conditions as in Appendix 2.  10  120 Appendix 4A-F. Plots of Coccolithus pelaaicus COPEL (coccolith forming motile strain) subjected to a continuous light regime after reaching stationary growth under a 14:10 D:N cycle regime and then being diluted (1/21) into 5.25 L of fresh modified HESNW. Figs. 4A and B. Semi-log plots showing growth curves as a function of in vivo fluorescence over time and cell density over time. Figs. 4C-F. Changes in cell volume (4C)/changes in pigments (chl a, chl c, and carotenoids x 10" 6 ng cell"-1) , (4D); pigment ratios (chl a:c, chl crcarotenoids, and chl a:carotenoids) (4E); changes in pigment cell volume -1 (chl a, chl c, and carotenoids x 10~ 9 Mg Mm" 3 ), (4F).  CoccoMhim pafaglcua OOPQ. (CooooBh forming cnotHo (train)  CooooRthui polagloui OOPQ. (Coeeolth forming motfl* (train)  50.0 L 40.0 o  A- -AChla O- •OChle Q- - O CaroUnold*  A  ri* 30.0 o £ 20.0  £  0.0' S  1'  \  V 10.0* O—  &*.*-  ^  10 Tim* (Day*)  100.00  i,M0 —  1.00  10  19  11rn((Dayt)  A- — A Continuous Ught  '  -^  -9-  9  B  A-A—A-*  ^ A  /  , /  r\ A-  A-~*  0.01 8  10  IS  9  10 Ttm*(Doy«)  71m«(Day()  9.00  • £ 60.0  »—*CMa •—•CWo •—•Carolonoldt  ?  t -  40.0  20.0  v. c  0  10 T1m«(Doyt)  £  0.0  0  5  10 T1rm(Dayt)  10  122 Appendix 5. SEM X-ray microanalysis micrograph of the rare appearance of non-calcium containing cyst-like formations in D:N cycle cultures of the non-coccolith forming motile strain of C. pelaqicus 241.  /J?3  App^ngei^ IT.  X-RflVs L i ve: Real:  0 - 20 kcU 353s P r c s e t : 3000s Remai ni ng! 2647 403s \2's. Dead  < 1.1 FS= 8K OS= MEM1S  1  3.700  eh  keU 195  b • —• 7  635  ct s  Table 8  Growth rates and pigment analyses for Emiliania huxlevi as reported in the literature.  Species  Author  fi  Pigments (x 10  pg cell  )  Pigment Ratios  (div/day) Chi a  Chi c  Carotenoids  a:c  Growth Conditions  chltCarotenoids Cont l i g h t (100 /iE m~2 s - 1 ) 18.5°C  Emiliania huxlevi (coccoliths)  Mjaalund, 1956  1.0  Emilianla huxlevi, (coccoliths)  Paasche, 1967  1.4 1.4  Emilianla h u x l e v i (coccoliths)  Paatsche & Klaveness, 1970  1.4-1.7 0.1-0.2 0.64  Cont light (260 /iE m~2 s"1) Cont light (22 /iE m"2 s"1) 21°C  1.2-1.4 0.40  Cont light (260 /iE m~2 B - 1 ) Cont light (22 /iE m"2 s"1) 21DC  EmiUnnia, huxlevi (naked)  Emilianla huxleyi (coccoliths)  Brand, 1982  Emiliania huxlevi (coccoliths)  Brand & 1.8-2.1 Guillard, 1981  Emiliania huxlevi (naked)  Haxo, 1985  EmlUftnJa. hWt;?Yi (coccoliths)  Jeffrey & Allen, 1964  Cont l i g h t (100 /iE m"2 s" 1 ) 16:8 DtN (100 /iE m~2 a" 1 ) 21-C  0.5 0.7  0.1-0.2  14il0 D«N (64 /iE m"2 B " 1 ) 16°C  0.6-1.0  Cont & 1 4 i l 0 DtN (100 /iE m-2 s - 1 ) , 24°C 0.28  0.15  0.27  1.9  1.6  cont l i g h t (12 //E m~2 s" 1 )  1.55  2.39  Cont light (500 ft.c.) 14-16°C  Table 9 Growth rates and pigment analyses results for coccollthophorids and Prymneslophyceae as reported in the literature.  Species  fi  Author  Pigments (x 10~ pg cell  )  Pigment Ratios  (div/day) Chi a CvclococcolithuB leptoporus  Chi c  Carotenoids  a:c  Growth Conditions  Chl:Carotenoids 14:10 DiN (100 fiE m~2 a"1) Cont light (100 j/E m~2 s"1) 24"C  Brand fi 0.9-1.0 Guillard, 0 1981  Gephvrocapsa oceanlca  1.5 2.0  Cont light (100 //E m~2 a"1) 14:10 D:N (100 fiE m"2 a"1) 24°C  Hvmenomonas carterae Brand, 1982  0.8  14:10 D:N (64 jiE m~2 a"1) 16°C  Hvmenomonas carterae Brand & Guillard, 1981  2.0 1.8  14:10 D:N (100 /uE m"2 a"1) Cont light (100 pE m~2 a"1) 24°C  Hvmenomonaa sp.  Jeffrey fi Allen, 1964  5.78  1.50  Cont light (500 ft.c.) 14-16-C  lapchryala qalbana  Jeffrey fi Allen, 1964  1.84  1.28  Cont light (500 ft.c.) 14-16»C  jBochrysJB qalbana  Brand,  1 4 : 1 0 DtN (100 /iE m"2 a" 1 ) 24°C  0.8  1982  Syracoaphaeyft  Jeffrey & Allen, 1964  4.3  2.67  Cont light (500 ft.c.) 14-16°C  

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