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The influence of irradiance, nitrogen limitation, and temperature on the biochemical composition of marine… Thompson, Peter Allan 1991

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T H E I N F L U E N C E O F I R R A D L A N C E , N I T R O G E N L I M I T A T I O N , A N D T E M P E R A T U R E O N T H E B I O C H E M I C A L C O M P O S I T I O N O F M A R I N E P H Y T O P L A N K T O N A N D T H E I R N U T R I T I O N A L V A L U E T O L A R V A L CRASS OS TREA GIGAS B y P E T E R A L L A N T H O M P S O N B . S c , Univers i ty of B r i t i s h Columbia, 1977 M . M . S t . , Univers i ty of Toronto, 1983 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Oceanography) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A October 1991 © Peter A l l a n Thompson, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The major objective of this study was to determine how marine planktonic autotrophs responded biochemically and physiologically to physical variation in their environment. The results were interpreted from ecological and evolutionary perspectives. Phytoplankton are the basis for the world's largest food web. If naturally occurring variations in temperature, light, and nutrient availability influence the biochemical, and perhaps nutritional value of the phytoplankton, then these variations may be significant in determining the efficiency of biomass transfer between trophic levels. For 10 species of marine phytoplankton, the cellular changes in carbon quota, cell volume, and nitrogen quota which occurred under different levels of light (energy) limitation were determined. Generally, cell volume, carbon, and nitrogen quotas all decreased with the decreasing availability of light energy. Fatty acid composition also responded to variation in light energy. Frequently the lowest percentage of the fatty acid 16:0 and the greatest percentage of the essential fatty acid 20:5co3 occurred at the lowest irradiance. A n increase in the percentage of the other essential fatty acid, 22:6co3, occasionally occurred at the highest irradiance, particularity for Pavlova lutheri (Droop). Variation in temperature also influenced fatty acid composition. Most species showed an increase in the fatty acid 16:4col, and a decrease in 16:3co4 at low temperatures. Only 1 of 8 species (Thalassiosira ii pseudonana, (Hustedt)), showed a significant relationship between temperature and the degree of unsaturation of the fatty acids. Thalassiosira pseudonana cells varied in their biochemical composition in response to variation in irradiance and nitrogen availability. At saturating irradiances, cells were relatively high in carbon, primarily short chain saturated fatty acids and carbohydrate. T. pseudonana cells grown at a saturating irradiance were a better food item for larval osyters (Crassostrea gigas, (Thunberg)) than cells grown at low levels of irradiance. When fed saturating rations of T. pseudonana cells grown at a high irradiance, C. gigas larvae had significantly higher growth rates and significantly lower mortalities. iii T A B L E O F C O N T E N T S A B S T R A C T i i L IST O F T A B L E S vii L I S T O F F I G U R E S x A C K N O W L E D G E M E N T S xii G E N E R A L I N T R O D U C T I O N 1 Irradiance and cell physiology .2 Irradiance and fatty acids 3 Temperature and fatty acids 3 Nutritional value 4 C H A P T E R 1. T H E I N F L U E N C E O F I R R A D I A N C E O N C E L L V O L U M E A N D C A R B O N Q U O T A F O R T E N S P E C I E S O F M A R I N E P H Y T O P L A N K T O N 7 Introduction 7 Material and Methods 10 semi-continuous culture 10 continuous culture 11 Biomass and cell volume 12 Transients 13 P O C and P O N 14 Results 16 Growth rates versus irradiance 16 Cell rowth rates versus irradiance 16 Cell volume versus irradiance 16 Carbon quota versus irradiance 22 Interrelationships 29 Transients in irradiance 29 Comparison of chemostat and turbidostat cultures 35 Discussion Cell volume versus irradiance 41 Carbon quota versus irradiance 46 Transients 47 Comparison of chemostat and turbidostat cultures 48 Ecological significance 49 Summary 52 C H A P T E R 2. T H E E F F E C T S O F V A R I A T I O N IN I R R A D I A N C E O N T H E F A T T T Y ACID C O M P O S I T I O N O F M A R I N E P H Y T O P L A N K T O N . . . 53 Introduction 53 Materials and Methods 56 Semi-continuous 56 Biomass and biochemistry 57 iv Fatty acids 57 Results 59 Growth rates 59 Biochemistry 59 Fatty acids 59 The essential fatty acids 69 Variation in fatty acids . . . 69 Discussion 73 Variation in fatty acids 73 Fatty acids as storage products influenced by irradiance 74 Fatty acids associated with photosynthesis 76 Fatty acids associated with pigments 77 Theoretical consisderations: PFD versus F A composition 80 Summary 83 C H A P T E R 3. T H E E F F E C T S OF VARIATION IN T E M P E R A T U R E O N T H E F A T T Y ACID COMPOSITION OF MARINE P H Y T O P L A N K T O N 84 Introduction 84 Materials and Methods .88 Semi-continuous cultures 88 Biomass and biochemistry 89 Fatty acids 90 Results 91 Growth 91 General biochemical parameters 91 Fatty acids. . . 98 Temperature 105 Growth rate 115 Chlorophyll a 115 Nutrition 116 Unusual responses 116 Discussion 120 Temperature versus growth rate 120 Carbon, nitrogen and chlorophyll a 121 Temperature and growth rate versus fatty acid composition . . . . 122 General responses 122 Specific fatty acids 127 Summary 131 C H A P T E R 4. E F F E C T S OF MONOSPECIFIC A L G A L DIETS OF VARYING BIOCHEMICAL COMPOSITION O N T H E GROWTH AND SURVIVAL O F GRASSOSTREA GIGAS L A R V A E 132 Introduction 132 Materials and Methods 135 Algal culture and medium 136 General 136 Algal and oyster biochemical composition 139 Oyster culture 140 v Results 143 Phytoplankton 143 Oysters 143 Fatty acids 147 In algae 147 In algae versus oysters 147 In oysters 150 Models 150 Discussion 158 General 158 Fatty acids 160 Summary 163 G E N E R A L CONCLUSIONS 165 R E F E R E N C E S 171 vi LIST OF T A B L E S Table 1.1 Data for maximum growth rates, 1/2 Ik, maximum and minimum cell volumes, and maximum and minimum carbon quotas 21 Table 1.2 A survey of the recent literature values on cell volume and carbon quotas .43 Table 2.1 Growth rates, pigments, lipid, carbon, and nitrogen quotas for eight species of marine phytoplankton 60 Table 2.2A Fatty acid composition for 2 species of marine phytoplankton at 4 different light intensities 63 Table 2.2B Fatty acid composition for 2 species of marine phytoplankton at 4 different light intensities 64 Table 2.2C Fatty acid composition for 2 species of marine phytoplankton at 4 different light intensities 65 Table 2.2D Fatty acid composition for 2 species of marine phytoplankton at 4 different light intensities 66 Table 2.3 Variation in recent literature values for the fatty acid content of the marine phytoplankter, Thalassiosira pseudonana .72 Table 3.1A The biochemical and summary fatty acid data for 2 species of marine phytoplankton at 5 different temperatures 94 Table 3. IB The biochemical and summary fatty acid data for 2 species of marine phytoplankton at 5 different temperatures 95 Table 3.1C The biochemical and summary fatty acid data for 2 species of marine phytoplankton at 5 different temperatures 96 vii Table 3. ID The biochemical and summary fatty acid data for 2 species of marine phytoplankton at 5 different temperatures 97 Table 3.2A The fatty acid data for Chaetoceros calcitrans at 5 different temperatures 107 Table 3.2B The fatty acid data for Thalassiosira pseudonana at 5 different temperatures 108 Table 3.2C The fatty acid data for Chaetoceros gracilis at 5 different temperatures 109 Table 3.2D The fatty acid data for Chaetoceros simplex at 5 different temperatures . 110 Table 3.2E The fatty acid data for Phaeodactylum tricornutum at 5 different temperatures I l l Table 3.2F The fatty acid data for Dunaliella tertiolecta at 5 different temperatures 112 Table 3.2G The fatty acid data for Pavlova lutheri at 5 different temperatures 113 Table 3.2H The fatty acid data for Isochrysis galbana (T-iso) at 5 different temperatures . 114 Table 4.1 A summary of the culture conditions for T. pseudonana in Chapter 4 137 Table 4.2 A summary of the culture conditions for the oysters in Chapter 4 138 Table 4.3 The oyster growth rates, mortalities and phytoplankton biochemistry from Chapter 4 144 Table 4.4 The fatty acid composition of the algal diets fed to larval oysters 148 Table 4.5 The fatty acid composition of the larval oysters 149 Table 4.6 Relationships of the fatty acid composition in the algal diets and the larval oysters 152 viii Table 4.7 Details of the mutiple regression models of oyster growth rates as a function of the biochemistry of their diets 153 ix LIST OF FIGURES Figure 1.1 Growth rates, cell volumes, and carbon quotas for 10 species of marine phytoplankton as a function of irradiance 18,19, 20 Figure 1.2A The relationship between relative growth rate and relative cell volume for 10 species of marine phytoplankton 24 Figure 1.2B The relationship between relative growth rate and relative carbon quota for 10 species of marine phytoplankton 26 Figure 1.2C The relationship between relative growth rate and relative nitrogen quota for 10 species of marine phytoplankton 28 Figure 1.3A Changes in cell volume for Thalassiosira pseudonana during a transient in irradiance 32 Figure 1.3B Changes in carbon quota for Thalassiosira pseudonana during a transient in irradiance 34 Figure 1.4A Cell volumes for Heterosigma akashiwo grown in either light-limited or nutrient-limited cultures 38 Figure 1.4B Carbon quotas for Heterosigma akashiwo grown in either light-limited or nutrient-limited cultures 40 Figure 2.1A The percentage of carbon found as lipid versus irradiance for 8 species of marine phytoplankton 62 Figure 2. IB The percentage of carbon found as lipid versus growth rate for 8 species of marine phytoplankton 62 Figure 2.2 The cummulative amount of essential fatty acids (20:5co3 + 22:6oo3) as a percentage of total fatty acids found in 8 species of marine phytoplankton at 4 different irradiances 71 X Figure 3.1 The growth rates and Qios for 8 species of marine phytoplankton grown at temperatures ranging from 10 to 25 °C 93 Figure 3.2 The carbon to nitrogen ratio for 8 species of marine phytoplankton grown at temperatures ranging from 10 to 25 °C 100 Figure 3.3 The chlorophyll a quotas for 8 species of marine phytoplankton grown at temperatures ranging from 10 to 25 °C 102 Figure 3.4 The carbon to chlorophyll a ratio for 8 species of marine phytoplankton grown at temperatures ranging from 10 to 25 °C 104 Figure 3.5 The cummulative amount of essential fatty acids (20:5co3 + 22:6co3) as a percentage of total fatty acids found in 8 species of marine Ehytoplankton grown at temperatures ranging •om 10 to 25 °C 119 Figure 3.6 The ratio of unsaturated to saturated fatty acids versus growth rate for three species of marine phytoplankton 126 Figure 4.1 Increase in the mean size of larval oysters fed saturating rations of T. pseudonana cells 146 Figure 4.2 The relationship between oyster growth rates and the fatty acid composition of their algal food 155 Figure 4.3 The relationship between the fatty acid composition of the larval oysters and their growth rates 157 xi Acknowledgements I would like to thank my family, especially J . & S. Thompson, M . & J . Chepesuik, Elizabeth Anne, Michael and Andrew, for their support and encouragement which made it possible to undertake this degree. Interactions with a large number of colleagues and friends including, J . Parslow, M . Levasseur, N. Price, A. Waite, W. Cochlan, J. Berges, C. Suttle, G. Doucette, and D. Montagnes, helped in formulating my viewpoints on algal physiology and biological oceanography. My thanks to Drs. J .N.C. Whyte, T.R. Parsons, N . Bourne and R.A. Anderson for their support of this project, and to the external examiner, R. Mann, for his thorough review of the thesis. Financial support was provided by Chevron Canada Ltd., The B.C. Science Council, NSERC operating and strategic grants. Most of the work herein would not have been completed without the technical assistance of Ming-xin Guo, D. Jones, K. Jeffries, H . Heckel, N. Ginther, and P. Clifford. I would like to thank Dr. Paul J . Harrison for his enthusiasm and commitment to my research, but mostly for providing a superlative role model for a developing scientist. Peter Thompson xii I N T R O D U C T I O N The research presented in this thesis documents the biochemical and physiological responses of marine planktonic autotrophs to variation in the availability of some of the resources considered to restrict their growth in the marine environment. The ecological and evolutionary success of phytoplankton is, in part, a function of their biochemical and physiological capabilities for physiological adaptation (acclimatization) to environmental variation (Turpin and Harrison 1979, Goldman and Ryther 1976). Since phytoplankton have evolved to exploit the available ecological niches (Harris 1986) it is also possible to learn something about their ecology by examining their capability for acclimatization. Resource limitation of phytoplankton reproduction (by either light or nutrients) is complicated by the multitude of scales, both in time and space, over which the availability of a resource can fluctuate. There are predictable cycles in the light field on seasonal, daily and wave-cycle time scales, as well as unpredictable fluctuations due to weather systems providing cloud cover and physical mixing processes associated with wind induced turbulence and langmuir circulation. Furthermore in many marine ecosystems there is a seasonal progression from light to nutrient limitation. In this study research was primarily focused upon elucidating the physiological responses of marine phytoplankton to temporally stable but different levels of the resources which can limit their growth. In general terms, the major hypothesis was: " H o w do mar ine p l ank ton i c autotrophs r e spond to phys i ca l va r i a t ion i n the i r env i ronment ? ' and the corollaries: l what type of acclimatizations occur? can these be interpreted from a biochemical, physiological, ecological and evolutionary perspective? Research on the biochemical and physiological responses of marine phytoplankton to variation in irradiance is presented in Chapters 1 and 2. There is also research on the physiological and biochemical responses of marine phytoplankton to variation in temperature (Chapter 3) and nutrient availability (Chapter 1). The ecological significance of variation in the biochemical composition of phytoplankton was investigated in Chapter 4. If naturally occurring variation in temperature, light, and nutrient availability influence the biochemical, and perhaps nutritional value, of the phytoplankton, then these variations may be significant in determining the efficiency of biomass transfer between trophic levels. Using the information gained from the first portion of the research, a further question was posed: can the environmental conditions be manipulated to increase the efficiency of biomass transfer up the food chain? Chapter 4 is an investigation of how irradiance, nitrogen limitation and temperature can be used to modify the biochemical composition of marine phytoplankton and subsequently improve the growth and reduce the mortality of larval bivalves. Further details of the material contained in each chapter are given below. 2 Chapter 1 examines biochemical and physiological responses of phytoplankton to variation in irradiance, including some of the energy saving mechanisms used by phytoplankton under conditions of low irradiance. Different patterns have been reported for the relationship between cell size and irradiance, however, the majority of species studied have shown some increase in cell volume with increasing irradiance (Brown and Richardson 1968). Any strategy that reduces the energetic costs of growth should result in an improvement in individual fitness within a light-limited environment. Since carbon fixation represents the major energetic cost of photoautotrophic growth, Chapter 1 emphasizes the cellular changes in carbon quota, cell volume, and nitrogen quota which occurred in ten phytoplankton species grown under different levels of light (energy) limitation. The principal objective of Chapter 2 was to assess the effect of variation in light intensity on the fatty acid (FA) profiles of some phytoplankton species commonly used in aquaculture. The analysis of FAs in phytoplankton has assumed new importance following the discovery that a dietary deficiency of 20:5co3 and 22:6co3 (the essential fatty acids = EFAs) limits growth in several species of bivalves (de Moreno et al. 1976, Trider and Castell 1980, Langdon and Waldock 1981). Continued interest in phytoplankton FAs is due to their importance in cell physiology, classification and taxonomy, and because of the significance of essential fatty acids (EFAs) in herbivore diets. At present, aquaculturalists are primarily interested in the transfer of EFAs from phytoplankton diets to cultured herbivorous species (e.g. Watanabe et al. 3 1983) and ecologists in using FAs as biomarkers in the marine food chain (Sargent and Whittle 1981). Lower temperatures may increase the degree of F A unsaturation (Ackman et al. 1968, Lynch and Thompson 1982, Mortensen et al. 1988), perhaps leading to increased EFAs but there is a paucity of data. More highly unsaturated membranes are thought to be generally required at lower temperatures in order that membranes may remain reasonably homeoviscous (Sinensky 1974, Quinn 1981). There are a number of well documented physiological responses that are associated with changes in temperature and are propounded to confer some measure of homeoviscosity to cellular membranes (Hadley 1985). The data from Chapter 3 provide some information regarding which of these mechanisms may be functioning within marine phytoplankton and may at least partially determine how the principle of homeoviscosity might be extended to marine phytoplankton. Variations in the percent composition of specific fatty acids were compared with variation in other biochemical/physiological parameters for associations that may give some insights into the role of fatty acids in algal physiology. The usefulness of temperature to manipulate fatty acid composition in order to maximize the percentage EFAs and maintain membranes in a homeoviscous state was assessed. The study of marine invertebrate nutrition is a subject of broad commercial and ecological significance. Factors influencing the transfer of biomass up the food chain may be similar in both the natural and hatchery environments, particularly if the natural system is not limited by food quantity (at least on short time scales). If food quantity does not 4 limit growth then food quality may be of increased importance in determining the efficiency of biomass transfer between trophic levels. It is possible in the laboratory or hatchery to test, qualitatively, the relative nutritional value (food quality) of different phytoplankton species. Such work has been conducted at the hatchery in Conwy for the past 60 years (Walne 1974). In early work, the nutritional value of phytoplankton was determined, but variability within one species was often high, and differences between species frequently could only be ascribed to qualitative factors such as digestibility or toxicity. Since the seminal work of Parsons et al. (1961), an increasing number of researchers have examined the biochemical composition of phytoplankton for their nutritionally important components (primarily protein, lipid and carbohydrate). Unfortunately relationships between phytoplankton biochemical composition and animal growth rates have been difficult to elucidate (Epifanio 1979, Webb and Chu 1982, Gallager and Mann 1981, Enright et al. 1986a, b), resulting in different conclusions about the significance of phytoplankton gross biochemical composition as a factor influencing bivalve growth. Since the discovery that certain long chain polyunsaturated fatty acids (PUFAs) are essential (EFAs) for many marine organisms, particularly during larval stages, considerable research has focused on the availability of EFAs in both the hatchery (e.g. Watanabe et al. 1983), and in the natural environment (Sargent and Whittle 1981). Phytoplankton which are deficient in the EFAs 20:5co3 and 22:6co3 have been shown to be poor food items for the oyster larvae Crassostrea gigas (Langdon and Waldock 1981). The nutritional suitability of phytoplankton for bivalves 5 has been assessed, at least partially, by the phytoplankton's F A profile (Webb and Chu 1982), however, within the large group of phytoplankton with adequate EFAs there remains considerable variability in their nutritional value to oysters (Enright et al. 1986a). Attempts to determine why one species of phytoplankton is more nutritious than another have been largely unsuccessful. Furthermore, studies utilizing more than one species of phytoplankton are difficult to interpret because it is impossible to control for interspecific differences in digestibility, toxicity, cell size, and the possible sparing effects facilitated by a mixed species diet (Webb and Chu 1982). Chapter 4 examines the growth rates and survival of the larval oyster C. gigas, when fed one species of phytoplankton grown under a range of conditions. The marine diatom, Thalassiosira pseudonana, was grown under different conditions of light quality, quantity, temperature and nutrient limitation to provide diets that varied in carbon, nitrogen, protein, lipid, carbohydrate and percent fatty acid composition. It was hypothesized that C. gigas larvae consuming biochemically distinct, monospecific algal diets maintained at cell densities which saturate the animals' feeding capabilities would show differences in growth rates which might be ascribed solely to variation in the nutritional value of the food. It was anticipated that this approach should minimize differences caused by interspecific variation in digestibility, cell size and palatability which may have confounded previous studies using multispecies diets. It is recognized that continuous (i.e. no 24 h cycle) but different levels of irradiance are "unnatural". It is the author's intention that these studies will form the basis for more complex experimental designs involving variable light and temperature regimes. 6 CHAPTER 1 T H E I N F L U E N C E OF IRRADIANCE O N C E L L V O L U M E A N D CARBON Q U O T A FOR T E N SPECIES OF MARINE P H Y T O P L A N K T O N I N T R O D U C T I O N Since light derived energy is the major abiotic factor limiting photoautotrophic growth in the aquatic environment it seems reasonable to predict that photoautotrophic organisms should be capable of physiologically adapting to variation in this resource. The physiological responses of phytoplankton to light limitation can be categorized into: 1) those responses that improve the cell's light-energy harvesting abilities, and 2) those that reduce the energy required for growth. Many researchers have documented physiological responses of the first category. Typically, these include an increase in pigment content and some adjustments to the photosynthetic apparatus that accompany a decrease in the total daily irradiance received by the phytoplankton (Falkowski 1980, Pr6zelin 1981). The kinetics of pigment synthesis/degradation have also been determined for cells that have been suddenly switched from one extreme irradiance level to another (Rivkin et al. 1982a, Falkowski 1984). High rates of physiological adaptation (=k, notation from Falkowski 1984) could be expected to be associated with r-selected (high reproductive output, (Pianka 1970)) species. An r-selected species should acclimatize 7 quickly either to develop high growth rates in response to an improvement in environmental conditions or to maintain high growth rates within a fluctuating environmental regime. Claustre and Gostan (1987) suggested that the rate of adaptation (k) should be proportional to cell size. Different patterns have been reported for the relationship between cell size and irradiance, however, the majority of species studied have shown some increase in cell volume with increasing irradiance. In diatoms the relationships between growth rate and cell size are variable, possibly due their unique silica frustule. Increased growth rates in diatoms (grown under stable conditions) have been associated with increases in cell size (Davis et al. 1973, Harrison et al. 1976, Costello and Chisholm 1981), decreases in cell size (Findlay 1972, Paasche 1973, Durbin 1977, Furnas 1978), or shown to have no change with variation in cell size (Werner 1971a,b,c). It appears to be unknown whether the variation in cell volume associated with variation in irradiance is due to increases in cell width (valve width) or cell length. This chapter examines the second category of responses to a reduction in irradiance, that is, some of the energy saving mechanisms used by phytoplankton under conditions of low photon flux densities (PFDs). Any strategy that reduces the energetic cost of growth should result in an improvement in individual fitness within a light-limited environment. Since carbon fixation represents the major energetic cost of photoautotrophic growth, this study emphasizes the cellular changes in carbon quota. Additionally, evidence of the changes in cell volume, and nitrogen quota which occurred in ten phytoplankton species grown under different levels of 8 light (energy) limitation is presented. This study rigorously examines the relationship between variation PFD and the resulting variation in cell volume and carbon quota for ten species of marine phytoplankton. For one species, Thalassiosira pseudonana, the transient responses of cell volume and carbon quota are documented. The kinetic parameters describing these changes in cell volume in response to the sudden variation in irradiance are also presented. A summary discussion of the significance of these results to the overall thesis is found in the general conclusions following Chapter 4. 9 M A T E R I A L S A N D M E T H O D S Phytoplankton cultures were obtained from the Northeast Pacific Culture Collection (NEPCC), Department of Oceanography, University of British Columbia. All culture work was conducted in enriched artificial seawater (ESAW) based on the formulations of Harrison et al. (1980). The medium was modified by replacing sodium glycerophosphate with an equimolar concentration of sodium phosphate, ferrous ammonium sulphate with an equimolar concentration of ferric chloride; batch cultures also had the addition of selenite and molybdate to achieve 1 nM final concentrations. Chemostat and turbidostat cultures had 25 and 50 uM ammonium respectively, as the sole nitrogen source. Semi-continuous cultures: Seven marine diatoms, Chaetoceros calcitrans (Paulsen) Tokano (NEPCC #590), Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (NEPCC #58), Chaetoceros simplex (Ostenfeld) (NEPCC #591), Chaetoceros gracilis Schutt (NEPCC #645), Phaeodactylum tricornutvm Bohlin (NEPCC #640), and Ditylum brightwellii (West) Grunow (NEPCC #649), and three flagellates, Dunaliella tertiolecta Butcher (NEPCC #1), Pavlova lutheri (Droop) (NEPCC #5) and Isochrysis aff. galbana (Green, clone T-iso, termed Tahitian IsochrysisXNEPCC #601) were grown at 17.5 ± 0.5 °C. A wide range of continuous photon flux densities were used, ranging from a minimum of 4.5 to a maximum of 225 umol photons m-2s-l (measured with a Li-CorR model LI 185 light meter, using a 10 2K collector). Light was provided by Vita-lite^ fluorescent tubes and attenuated by distance and/or neutral density screening. At least eight generations (1 transfer) occurred prior to the collection of any presented data, and a minimum of five additional generations (i.e. second transfer, grown to mid-log phase) occurred under the defined experimental conditions prior to the determination of cell volume or carbon quota. Culture vessels varied from 50 mL borosilicate glass test tubes with teflon-lined caps to 12 L round, flat-bottomed borosilicate flasks stirred at 60 rpm and bubbled with a mixture of sterile air and ca. <2% C O 2 . Continuous cultures: Chemostat and turbidostat cultures of Heterosigma akashiwo (Handa) Handa (NEPCC #630) and turbidostat cultures of Micromonas pusilla (Butch.) Manton et Parke (NEPCC #29) were grown in sealed 2 L round, flat-bottomed borosilicate flasks in constant temperature tanks at 18 °C. Cultures were diluted with medium at fixed rates using piston pumps (Fluid Metering Inc., model RP). The overflow was collected and the volume used to estimate the dilution rate. Cultures were monitored daily using 10 mL syringes to remove samples for measurements of fluorescence and cell numbers. Turbidostats were operated manually with small daily adjustments in flow rate to ensure high ambient levels of all nutrients (full ESAW concentrations except for nitrogen), and an ammonium surplus (inflow=50 uM, outflow ca. 25 uM) was confirmed by colorimetric analysis (Slawyk and Maclsaac 1972). Continuous light of 20, 40,80,160 and 10, 20, 40, 80,160 ^imol photons n r V 1 (H. akashiwo and M. pusilla, respectively) was provided by Vita-liteR fluorescent tubes and 11 attenuated by distance and/or neutral density nylon screening. Blue Plexiglas (3 mm thick, Rohm and Hass #2069) was used to simulate an underwater light spectrum (Jitts et al. 1964). Chemostat cultures of H. akashiwo were limited by inorganic nitrogen supplied as 25 uM ammonium, and were diluted so that u was 80, 60, 40, and 20% of Umax at each of the four following irradiances (I) = 20,40, 80,160 umol photons m " 2 s"l (total = 16 chemostats). The specific dilution rates were: 0.15, 0.22, 0.34, and 0.47 d-1 at 20 umol photons n r V 1 ; 0.56, 0.47,0.37, and 0.18 d" 1 at 40 umol photons m-2 s - l ; 1.04, 0.73, 0.55, and 0.23 d-1 at 80 umol photons n r V 1 ; and 0.22, 0.5, 0.78, and 0.99 d-1 160 umol photons n r 2 s " l . Samples were collected for analyses after a minimum of five days at steady state (variation in biomass <> ± 5% per day). Duplicate turbidostat cultures of H. akashiwo were run at 0.50, 0.96, 0.96,1.25, and 1.34 d-1 at 20, 30, 40, 80, and 160 umol photons m" 2 s"l . Biomass and cell volume: Determination of biomass (in vivo fluorescence, frequently determined from insertion of the 50 mL culture tube into a Turner Designs^ Model 10 fluorometer and/or cell counts utilizing a Coulter Counter^ model TAII equipped with a population accessory) were made once or twice per day. Growth rates were calculated as: u = ln(F 1/F 0)/(t 1-t 0) where F i = biomass at time 1 (ti) and Fo = biomass at time 0 (to). Estimates of growth rates made via in vivo fluorescence from cells adapted to a specific photon flux density (PFD) for a minimum of eight generations and those 12 made from cell counts were not significantly different (Thompson, unpubl. data). All of the species studied were normally single-celled. Dilution prior to counting (with GF/F filtered 3% NaCl) depended on culture density and cell size, but ranged from 1:100 (M. pusilla) to zero (H. akashiwo). Background counts were made regularly and subtracted from sample counts. Coulter Counter apertures of 70 and 200 nm were used depending on the size of the phytoplankton cell. The 70 nm aperture was calibrated with 5.07 um polystyrene beads, and cross calibrated with 2.02 and 19.42 beads. The majority of cells (often 90%) were distributed into two channels. Cell volumes were calculated from the distribution of cells into a variable number of the 16 channels available on the Coulter Counter, (e.g. for T. pseudonana): 6-12 (Ca/Ct)MVa where Ca = (count in channel a - blank for channel a), Ct = (count in channels 6-12 - total blank), MVa = mean volume for channel a. The calculated volumes are based on equivalent spherical diameters and therefore are expected to be intraspecifically consistent, but may be in error with regard to absolute volume for cell shapes that were significantly different from spheres, such as D. brightwellii, and P. tricornutum. Transients: Two experiments on the effects of transients in irradiance were conducted. High and low light T. pseudonana cultures were grown at 110 nmol photons m'^s ' l and 14 (imol photons m'^s'l , respectively. High and low light D. brightwellii cultures received 275 and 11.5 |imol photons m'^s'l , 13 respectively. Transients were achieved instantaneously, merely by switching the neutral density screens from the low light to the high light cultures (T=0). Experiments were conducted with cultures between early- and mid-log phase. For the transient experiments involving T. pseudonana, visual examination of the cells was made using a Zeiss inverted microscope to determine the number of cells occurring as singlets, doublets, and triplets, or clumps prior to the transient. For the T. pseudonana transient, measurements of cell volume were made at t = 0.5, 1, 2, 3, 6,12, 18, 24, 48, and 72 h. D. brightwellii was measured (length and width) using a calibrated eyepiece micrometer fitted to a Zeiss inverted microscope at t = 0 and 78 h. A minimum of 50 cells were measured. Coulter counter measurements of cell volume ofD. brightwellii were made at t = 0, 0.5, 1, 3, 7, 11, 20, 26, 46, 54, 69,77, and 141 h. POC and PON: Particulate organic carbon and nitrogen (POC and PON) subsamples were collected on precombusted 13 mm Gelman A/E filters and were analyzed on a Carlo Erba C H N analyzer. No data were omitted from the figures, but the number of measurements of u was greater than the number of measurements of cell volume and there were fewer measurements of carbon and nitrogen quotas. A few datum points at high PFDs were omitted from some statistical analyses. These are clearly marked in the figures and may have been affected by photoinhibition. In the few cases where parameters were measured at identical PFDs, mean coefficients of variation (CV) were: for 14 growth rate CV=1.9%, n=18; for carbon quota CV=5.7%, n=24; for nitrogen quota CV=7.5%, n=23; and for cell volume CV=2.8%, n=25. 15 R E S U L T S Growth rate versus irradiance For each species, measurements of u were modeled as a function photon flux density (PFD or irradiance (I)) using the relationship u = a + b * log PFD (Falkowski and Owens 1980, Terry et al. 1983). Other equations, such as the Michaelis-Menten curve n^maxMIk+llrnax) and the hyperbolic tangent (Piatt and Jassby 1976) were also tested on the data, but they did not provide a consistently better fit. Over the range of PFDs studied, all species had a significant linear regression of n on log PFD (Fig. 1.1, A panels). To reduce the problem associated with the interspecific variability in the PFD at which photoinhibition may occur, \i at 200 nmol photons m"2 s ' l was estimated and reported here as Umax' (Table 1.1). Both |imax' and 1/21 '^ (the PFD where n=l/2nmax') showed considerable interspecific variability (Table 1.1). Umax' ranged from 0.89 d"l for Af. pusilla to 2.1 d"l for T. pseudonana. 1/2 Ik' ranged from a low of 7.9 n m o l photons m"2 s"l for P. tricornutum to 29 nmol photons m"2 s"* for /. galbana. Cell volume versus irradiance When cells were grown in nutrient replete semi-continuous or turbidostat cultures, all species except P. lutheri (Fig 1.1, panel 5B) showed a significant linear response in cell volume (CV) as a function of log PFD 16 Fig. 1.1 Ten marine phytoplankton species were grown under a range of photon flux densities at saturating nutrient concentrations in turbidostats or semi-continuous cultures: Chaetoceros calcitrans (1), Thalassiosira pseudonana (2), Chaetoceros gracilis (3), Chaetoceros simplex (4), Phaeodactylum tricornutum (5), Dunaliella tertiolecta (6), Pavlova lutheri (7), Isochrysis aff. galbana (T-iso) (8), Heterosigma akashiwo (9), and Micromonas pusilla (10). Panels A are growth rate (\i), panels B are cell volume, and panels C are carbon quotas. Open circles are variables vs log PFD (bottom horizontal axis) with a least squares linear regression (solid line) and 95% confidence intervals (dotted lines) shown fitted to the data. Closed circles are variables plotted against PFD (top horizontal axis). Curves are calculated from parameters a, b derived from variables = b * log PFD + a. Open squares are data not included in the regression analysis (suspected of photoinhibition). 17 -1 , Growth rale (d ) Growth rate (d ) .-1 - 1 , Growth rate (d ) Growth rate (d ) Cell volume (fim ) Cell volume (fim ) Cell volume (firry ) Cell volume (fim ) K) U » U l Ol v l D o o o o o o o o o o o o o o o o o U l Ol s i o o o -» -• Nj to CJ Ol m o m o tn o cn o o o o o H » r - i 1 l l _ i i v Carbon quota (pg cell ') Ol CO O to O) 03 o o o 1 '4V \ ^ o \ \ * " - o A i i Carbon quota (pg cell ') O 00 O W A Carbon quota (pg cell ) - • M O l A O i O l v l O -1 1 1 1 ° O o i i i o o so o o -1, Growth rate (d ) O O O O O -* -» Growth rate (d ) p p o o p b to bi bo b -1, Growth rate (d ) O p p o p r - » : - » r * ; - » b ( o * b i a ) b f o * b i Growth rate (d ') p O O p .-* 7* .-* ."* t o 4 » c n b o b r o - * > b > 1 — i — c r m o Cell volume (tjm ) Cell volume (urn ) - " W O i r U i o i s i a i O O O O O O O O o , Cell volume (jLtm ) —» —* M ho K) cn o to cn o cn o cn o Cell volume (/xm ) to ut cn oi OD O O O O O O O i 1 CT> ro —4 o -tots!_ ro 1 • \ \ 1 1 \ N o o Carbon quota (pg cell ) 03 to Carbon quota (pg cell ) to *• O) - 1 . Carbon quota (pg cell ) 04 o o cn o Carbon quota (pg cell ) OJ os o to > cn oo o o o 1 ' 0 0 i -v2 • o O o o o - 0 _ o o o a o o o o o \ 1 8 0 0 " ^ 1 6 0 0 . 3 - 1 4 0 0 -akashiwo^ — <o 6 0 0 1 1 0 1 0 0 1 0 0 0 0 1 0 0 2 0 0 1 0 1 0 0 1 0 0 0 1 0 0 2 0 0 10E .' / 1 m/ // / 1 / 1 0 0 1 0 0 0 1 0 1 0 0 1 0 0 0 1 0 1 0 0 1 0 0 0 - 2 -1 P h o t o n f lux d e n s i t y (fu,m photons m s ) 20 Table 1.1. Ten species of marine phytoplankton were grown under a range of irradiances (4.5 to 225 umol photons m -2 s - l ) to determine the effects of variation in irradiance on growth, cell volume, and carbon quota. Parameters were calculated from data fit to a significant linear regression against log PFD (photon flux density=irradiance). Umax' is M- at 200 umol photons m" 2 s*1, and 1/21 '^ is the PFD where \L=V2 Umax'- Minimum values of cell volume (CVmin) and carbon quota (Qcmin) were calculated at PFD (I) = 10 umol photons m"2 s"l, and maximums at 200 umol photons m-2 s-1. Species M-max' ld-1 l/2I k ' (umol E nr 2 i s-1) (Jim3) CVmin % difference Qcmax (pgcell-1) Qcmin % difference C. calcitrans 2.07 20 30.8 17.7 45% T. pseudonana 2.11 19 56.4 35.5 37% 12.6 9.21 26% C. gracilis 1.80 11 53.0 38.6 27% 16.7 9.20 45% C. simplex 2.05 20 74.6 34.8 53% P. tricornutum 1.36 8 65.7 24.4 63% 15.0 4.95 67% D. tertiolecta 1.45 28 227 174 23% P. lutheri 1.12 24 I. galbana (T-iso) 1.39 29 50.2 33.7 33% H. akashiwo 1.51 26 1763 491 72% 429 119 72% M. pusilla 0.89 16 5.8 2.5 56% (Fig. 1.1, panels B). To minimize the errors associated with extrapolation, the maximum cell volume (CVmax') was defined as CV at 200 umol photons m-2 s-1 and CVmin' as CV at 10 umol photons m"2 s"*. Over this range of PFDs, there was considerable interspecific variation in the magnitude of the decreases in cell volume which ranged from a large difference (72% for H. akashiwo) to not significantly different from zero (for P. lutheri) (Table 1.1). Carbon quota versus irradiance Carbon quota showed a similar dependence on irradiance as did CV (Fig. 1.1, panels C). This relationship between carbon quota and log PFD was significant for all four species where n>8 (i.e. T. pseudonana, C. gracilis, P. tricornutum and H. akashiwo). For the other 6 species where n<5, / . galbana was the only species which showed no indication of a trend towards increasing carbon quota with increasing PFD (Fig. 1.1, panel 8C). If the maximum carbon quota (Qcmax') was defined as Qc at 200 umol photons m-2 s-1 and Qcmin' as Qc at 10 umol photons m"2 s ' l then for those species which showed a significant regression of Qc on log PFD, decreases in carbon quota ranged from 26% for T. pseudonana to 72% for H. akashiwo (Table 1.1). 22 Fig. 1.2. (A) Relative growth rate (u/umax) versus relative cell volume (cell volume/maximum cell volume) for 10 marine phytoplankters grown at a range of photon flux densities at saturating nutrient concentrations in turbidostats or semi-continuous cultures: Chaetoceros calcitrans (O), Thalassiosira pseudonana (•), Chaetoceros gracilis (A), Chaetoceros simplex (A), Phaeodactylum tricornutum (•), Dunaliella tertiolecta (•), Pavlova lutheri (V), Isochrysis aff. galbana (T-iso) (T), Heterosigma akashiwo (0), and Micromonas pusilla (•). 23 1.0 A 0.8 CD E O > = 0.6 CD O CD > CD CC 0.4 O • O A A • • • • A • A • • • A A • raotJP V O A 0 V A • A o •& o o o 0.2 0.00 0.25 0.50 0.75 1 . 0 0 Relative growth rate 24 Fig. 1.2. (B) ([i/Hmax) versus relative carbon quota (carbon quota/maximum carbon quota) for 10 species of marine phytoplankton grown at a range of photon flux densities at saturating nutrient concentrations in turbidostats or semi-continuous cultures: Chaetoceros calcitrans (O), Thalassiosira pseudonana (•), Chaetoceros gracilis (A), Chaetoceros simplex (A), Phaeodactylum tricornutum (•), Dunaliella tertiolecta (•), Pavlova lutheri (V), Isochrysis aff. galbana (T-iso) (T), Heterosigma akashiwo (0), and Micromonas pusilla (•). 25 CO o CT O JD t_ CO o CD > CD CC 1.00 B 0.75 0.50 • 0.25 O O o A o o o o v # o o • A 0. A 0.00 0.00 0.25 0.50 0.75 Relative growth rate 1.00 26 Fig. 1.2. (C) (u/umax) versus relative nitrogen quota (nitrogen quota/maximum nitrogen quota) for 10 species of marine phytoplankton grown at a range of photon flux densities at saturating nutrient concentrations in turbidostats or semi-continuous cultures: Chaetoceros calcitrans (O), Thalassiosira pseudonana (•), Chaetoceros gracilis (A), Chaetoceros simplex (A), Phaeodactylum tricomutum (•), Dunaliella tertiolecta (•), Pavlova lutheri (V), Isochrysis aff. galbana (T-iso) (T), Heterosigma akashiwo (0), and Micromonas pusilla (•). 27 JS o cr c CD D) O 1.00 0.75 0.50 O o • CD JS CD DC 0.25 O O 0.00 0.00 0.25 0.50 0.75 1.00 Relative growth rate 28 Interrelationships The similarity between u. versus log PFD and CV versus log PFD curves suggested that a relationship between u. and CV should also exist. For the ten species examined here, relative CV (relative CV= measured CV/maximum measured CV) was correlated with the light dependent variation in relative growth rate, u/u.max (Fig. 1.2A) (r=0.591, n = l l l , p^O.Ol). Similarly, a significant correlation (r=0.664, n=55, p<0.01) between u. and Qc was shown by expressing carbon quota (Qc) and |i as relative measurements (relative Qc = Qc/Qcmax and relative u. = |i/umax» respectively) (Fig. 1.2B). There was a significant but weaker correlation between relative nitrogen quota and relative growth rate (r = 0.40, n = 55, p<0.05)(Fig. 1.2C). Transients in irradiance Two cultures of T. pseudonana were allowed to adapt to 110 umol photons m-2 s-1 (HL), and 2 additional cultures were adapted to 14 umol photons m-2 s-1 (LL) for 160 h (ca. 19 generations at H L , and 8 generations at LL) prior to the transient in light. Just prior to the transition in irradiance, microscopic examination revealed high light cells were 91% singlets, 8% doublet cells, and ca. 1% triplets or clumps. Low light cells were 90% singlets, 7% doublet cells, and ca. 3% triplets or clumps. To minimize errors associated the difference between the number of triplets in H L and L L cultures, particles equal to, or greater than three times the mean cell 29 volumes, were not used to calculate the mean cell volumes (calculated from Coulter Counter data). L L cells were growing at 0.83 ± 0.10 d ' l and H L cells were growing at 2.00 ± 0.15 d" 1 (means ± 1 S.D., n = 2). The volume of the H L cells was 60.8 ± 0.05 u m 3 and L L cells were 48.7 ± 0.58 u m 3 (means ± 1 S.D., n = 2) at 160 h (Fig. 1.3A). The transient was effected by transposing light regimes (i.e. 110<=>14 umol photons m'^s'l). Subsequent changes in cell volume were fit to the model: At = Af+(Ai-Af)*e-kt (Falkowski 1984) or -k = ln[(At-Af)/(Ai-Af) (Harding 1988) where At is the mean value of the cellular parameter at time t, Ai is the mean initial value, Af is the minimum or maximum value obtained, k is the rate of adaptation (h-1) and t is time. Higher k values indicate more rapid adaptation, k (mean ± 1 S.D.) was 0.74 ± 0.11 h"l for the L L - » H L culture and 0.53 ± 0.08 h ' l for the HL-»LL culture. Adaptation in cell volume reached a maximum at 5 h (LL—»HL) and a minimum at 12 h (HL—>LL) (Fig. 1.3A). The overshoot in the minimum and maximum cell volumes achieved was corrected within 24 h (Fig. 1.3A). 30 Fig. 1.3 (A) Changes in cell volume for Thalassiosira pseudonana in duplicate cultures grown at 14 umol photons m " 2 s"l (O), or 110 umol photons m-2 s-1 (•); transposition to higher or lower irradiance occurred at arrow. Bars represent ± 1 S.D. (where not shown, error bars were smaller than symbols). * indicates a significant difference between treatments (t-test) ** p<0.01, *** p<0.001. 31 Fig. 1.3.(B) Changes in carbon quota for Thalassiosira pseudonana grown in duplicate cultures at 14 umol photons m"2 s"l (•), or 110 umol photons m"2 s ' l (•); transposition to higher or lower irradiance occurred at arrow. Bars represent ± 1 S.D. (where not shown, error bars were smaller than symbols). * indicates a significant difference (t-test) ** p<0.01, *** p^O.001. 33 34 Carbon quotas also changed in response to the light transient (Fig. 1.3B). Although initial estimates of Qc for H L and L L cells are not significantly different, H L - » L L cells showed an immediate decrease in Qc similar to their decrease in cell volume. Similarly L L - » H L cells showed a sudden increase in Qc with increasing PFD and cell volume. At the peak of the transient (after 12 h) the ratio of carbon quotas for (HL-»LL)/(LL-»HL) cells reached 0.41 (Fig. 1.3B). The kinetic parameters (k values) determined from changes in growth rates (growth rates calculated from changes in cell numbers) during the light transient were 0.63 ± 0.3 h"l (LL-»HL) and 0.10 ± 0.01 h ' 1 (HL-»LL). One D. brightwellii culture was grown in semi-continuous culture at 11.5 umol photons m-2 s-1 for 430 h, split into 2 fractions and both moved to 275 umol photons m-2 s-1. Coulter Counter measurements revealed that cell volume increased for 80 h from 3287 unv* to 6511 um^ (data not shown). Microscopic examination revealed mean cell width remained at 17.5 ± 1.8 um and mean cell length increased from 44.1 ± 9.0 to 62.8 ± 21.2 um (means ± 1 S.D., n>50 cells). Comparison of turbidostat and chemostat cultures: The cell volumes (Fig. 1.4A) for H. akashiwo grown in light-limited turbidostat cultures had a significant linear relationship with log PFD (slope=978±141, n=10). Chemostat cultures, limited by nitrogen and diluted so that u. was 80, 60,40, and 20% of Umax at each of the 4 following irradiances (PFD) = 20, 40, 80,160 umol photons m ' 2 s"1 (total = 16 35 chemostats) showed no significant trend (slope=-94±99, n=14) in cell volume with log PFD (Fig. 1.4A). The relationships between cell volume and log PFD were significantly different for chemostats versus turbidostats (p<0.01). Carbon quotas were also a significant linear function of log PFD (slope=195±25, n=10) for H. akashiwo grown in light-limited turbidostat cultures (Fig. 1.4B). Over all chemostats, however, Qc showed no significant trend (slope=0.07±0.15, n=14) with log PFD (Fig. 1.4B). The relationships between carbon quota and log PFD were significantly different for chemostats versus turbidostats (p<0.001). 36 Fig. 1.4 (A). The cell volume versus log PFD (I) for Heterosigma akashiwo grown under nitrogen saturation in light-limited turbiodstats at umax (O), in NH^-limited chemostats, 80% umax (•), 60% umax (A), 40% umax (A), 20% umax (•). Turbidostat data are fitted to the model where cell volume or carbon quota = b * log PFD + a. Least" squares linear regressions (solid line) plus 95% C.I. (dotted lines) are shown fitted to either turbidostat or chemostat data. 37 250 L • 1 ' 1 10 100 1000 Photon flux density (^ mol photons rrr2s1) 38 Fig. 1.4 (B) Same as Fig. 1.4A except carbon quota versus log PFD (I). 39 10 100 1000 Photon flux density (/umol photons rrvV) 4 0 DISCUSSION Cell volume versus irradiance Different patterns have been reported for the relationship between cell size and irradiance (Table 1.2). The majority of species studied have shown some increase in cell volume with increasing irradiance. In this study, all species except P. lutheri showed an significant increase in cell volume with the log of increasing irradiance over the range from 4.5 to 225 umol photons m-2 s-1. Even P. lutheri showed a strong, but statistically insignificant, trend to increased cell volume with increasing PFD, similar to the other species. At high PFDs some researchers (cf. Table 1.2) have observed a decrease in cell volume with increasing PFD. Above the PFDs where u saturates, the responses of CV to PFD may be affected by interspecific variability in the onset of photoinhibition. The mechanism(s) responsible for the changes in cell volume with irradiance is not well understood. It has been demonstrated that microalgal cell volume may be under the control of variation in internal ion concentration (Riisgard et al. 1980), or variation in the low molecular weight metabolites of photosynthetic origin (Ahmad and Hellebust 1985, Dor 1985, Reed and Stewart 1985). For diatom cells encased in silica frustules, it seems highly unlikely that variation in the amount of internal metabolites should influence cell volume. Recent evidence that the cell volume is under the control of carbon metabolism (Claustre and Gostan 1987), is substantiated by the data presented here. At least when a cell's ability to grow is constrained 41 by light limitation, cell volume and carbon quota are frequently a function of irradiance. In diatoms, various relationships between growth rates and cell size have been reported. Increased growth rates in diatoms (grown under stable conditions) have been associated with increases in cell size (Davis et al. 1973, Harrison et al. 1976, Costello and Chisholm 1981), decreases in cell size (Findlay 1972, Paasche 1973, Durbin 1977, Furnas 1978), or shown to have no change with variation in cell size (Werner 1971a,b,c). For diatoms grown under constant conditions, variation in u may be more closely related to sexual reproduction associated with changes in valve width (Davis et al. 1973, Costello and Chisholm 1981). Diatoms have two methods of increasing cell volume, increased valve width via sexual reproduction or increased pervalvar length via additional girdle (intercallary) bands. Based on our results for D. brightwellii it is suggested that for diatoms undergoing a transient in light, the pervalvar length is likely to be altered by the addition of girdle bands. Our results could explain the results of Joint et al. (1987) who found a 50% increase in the length of the pervalvar axis of Skeletonema costatum during the initial stages of a bloom (assuming the bloom was initiated by a transient to greater light per cell per day). i 42 Table 1.2. Relative changes in cell volume and carbon quota with increasing irradiance in marine and freshwater phytoplankters from 11 algal classes. Values derived from literature (many from graphs, 100%=maximum size): Nc=no change; PI=peak at intermediate irradiance; up = an increase in cell volume or carbon quota with increasing irradiance; down = a decrease in cell volume or carbon quota with increasing irradiance. Class/Species Cell Volume Carbon quota Notes Reference CO Bacillariophyceae Chaetoceros protuberans Chaetoceros furcellatus Cyclotella meneghiniana Cyclotella meneghiniana Nitzschia closterium Nitzschia delicatissima Phaeodactylum tricornutum Phaeodactylum tricornutum Phaeodactylum tricornutum (clone TFX) (clone BB) Skeletonema costatum Skeletonema costatum Skeletonema costatum (6NY17) (Skel) (UP45) Skeletonema costatum Skeletonema costatum Skeletonema costatum Thalassiosira antarctica Thalassiosira weissflogii Up Nc Nc Down 55% Nc Nc Up (small) Up Up 15% Up 55% Up (small) Up 33% Nc Up 60% Down 25% Up 72% Up 22% Up 66% Down 30% Down Up 63% Up 47% Down 60% Clone specific 2 clones Clone specific Daylength dependent n=24, Morel et al. (1987) Hegseth (1989) Jorgensen (1964) Rosen and Lowe (1984) Brown and Richardson (1968) Hegseth (1989) Fawley(1984) Beardall and Morris (1976) Terry et al. (1983) Gallagher and Alberte (1985) Falkowski and Owens (1980) Gallagher and Alberte (1985) Sakshaug and Andresen (1986) Yoder(1979) 5 temperatures Langdon (1986) Hegseth (1989) PI Sosik et al. (1989) Table 1.2 Continued. Class/Species Cell Volume Carbon quota Notes Reference Cryptophyceae Cryptomonas erosa @ 1°C Up 30% @ 4 ° C Up 58% @15°C Up 60% @23.5°C Up 49% Rhaphidophyceae Heterosigma akashiwo Up 51% Prymnesiophyceae Isochrysis galbana Nc Hymenomonas elongata Up 25% Hymenomonas carterae Up 20% Chrysophcyceae Ochromonas danica Down 32% Dinophyceae Amphidinium sp. Down 23% Amphidinium carteri Up 23% Ceratium furca Up 15% Dissodinium lunula Up (small) Gonyaulax polyedra Up 62% Gonyaulax polyedra @20°C Down 12% @ 15°C Down 44% Gonyaulax tamarensis Up 24% Pyrocystis fusiformis Up 87% Pyrocystis noctiluca Up 67% Pyrocytis noctiluca Up 42% Morgan and Kalff(1979) PI Carbon quota correlated very well with cell volume Up 23% (Protein quota up 22%) Up 40% PI PI PI Temperature dependent Up Langdon(1986) Claustre and Gostan (1987) Claustre and Gostan (1987) Sosik et al.(1989) Brown and Richardson (1968) Brown and Richardson (1968) Sosik et al. (1989) Meeson and Sweeney (1982) Swift and Meunier (1976) Prezelin and Sweeney (1978) Meeson and Sweeney (1982) Langdon (1986) Swift and Meunier (1976) Swift and Meunier (1976) Rivkin et al. (1982a) Table 2.1 Continued. Class/Species Cell Volume Carbon quota Notes Reference Bangiophyceae Porphyridium cruentum Porphyridium aerugineum Euglenophyceae Euglena sp. Euglena gracilis Class Chlorophyceae Astasia longa Chlamydomonas reinhardii Chlamydomonas reinhardii Chlorella pyrenoidosa Chlorella pyrenoidosa Chlorella vulgaris Chlorella 8 strains (averaged) Chlorococcum wimmeri Dunaliella tertiolecta Dunaliella tertiolecta Scenedesmus obliquus Cyanophyceae Anacystis nidulans Cyanidium caldarum Synechococcus Chroococcaceae Gloecocapsa alpicola Up 45% Up 68% Down 20% Up 30% Up 43% Down 21% Up 17% Up 44% Up 95% Up 60% Down PI Small change in I PI Up 52+10% Up 50+20% Up 24% PI Up 72% Down 27% Up 10% Small change in I Up 50% Down 62% Up 53% Down Nc Up 60% Brown and Richardson (1968) Brown and Richardson (1968) Cook(1963) Brown and Richardson (1968) Brown and Richardson (1968) Osborne and Raven (1986) Brown and Richardson (1968) Brown and Richardson (1968) Myers and Graham (1971) Jorgensen (1964) Winokur(1948) Brown and Richardson (1968) (1968) Falkowski and Owens 1980 Osborne and Raven (1986) Senger and Fleischhacker (1978) Brown and Richardson (1968) Brown and Richardson (1968) Kana and Glibert (1987) Brown and Richardson (1968) Carbon quota versus irradiance The relationship between cell carbon quota and irradiance is variable (Table 1.2). Considerable data exist to show that cell protein (Chan 1978, Osborne and Raven 1986), pigments (Falkowski 1980, Richardson et al. 1983), chloroplast volume (Brown and Richardson 1968) and thylakoid membranes (Forde and Steer 1976) all increase at low PFD. Sakshaug and Andresen (1986) showed that carbon quota increased substantially with the combination of low irradiance and short daylength, but much less with low irradiance and long daylengths for Skeletonema costatum. For autotrophic phytoplankton the relationship between irradiance and carbon quota may be complex, especially under variable conditions of daylength and PFD. For those species that did show a significant relationship between log PFD and carbon quota, it is suggested that outside the range of PFDs used in this study and in studies using L D cycles, the relationship may not exist. In spite of this, the data presented here indicate that with reductions in PFD, there was often a reduction in Qc. For the ten species examined here, the relationship between Qcmin/Qcmax averaged 0.44, not 1 as predicted by Goldman and McCarthy (1978). Those species where carbon quota increased at low PFD (e.g. Sakshaug and Andresen 1986), apparently manifest a markedly different strategy in their approach to growth and survival in a low light environment compared to species reducing Q c at low PFD (this study). Since many cellular components require considerably more energy for synthesis than simple carbohydrates (Myers 1980, Morris 1981), the determination of carbon 46 quota alone may not adequately reflect the energetic costs of growth in an Hght-limited environment. Transients A n organism's rate of response is a manifestation of an ecological strategy (Post et al. 1984). High rates of physiological adaptation could be expected to be associated with r-selected species. A n r-selected species should adapt quickly either to develop high growth rates in response to an improvement in environmental conditions or to maintain high growth rates within a fluctuating environmental regime. The response of cell size in T. pseudonana to a light transient was maximized in 5 h (LL-»HL) or 12 h (HL-»LL), and the rates of adaptation (k) for cell volume were 0.74 h ' l (LL-»HL) and 0.53 h"l (HL-»LL). These rates are considerably faster than those determined for Hymenomonas elongata by Claustre and Gostan (1987), who also suggest that k should be proportional to cell size. It is proposed that k is more likely a function of both cell size and Umax- Unfortunately since cell size is frequently proportional to Umax (Fenchel 1974), this difference may be difficult to distinguish and requires careful testing over similar differences in initial and final conditions relative to the species' physiological tolerances (Hegseth 1989). That is, the transients must be made within a range which is similar relative to a species' ability to grow, otherwise interspecific comparisons cannot be meaningful. This study provides insufficient additional data to resolve the issue of whether k is proportional to cell size or growth rate. 47 Pell division continued in both the HL-»LL and the L L - » H L T. pseudonana cultures following their transfer to different light regimes. Oxygen evolution vs irradiance data (not presented) indicated that the carbon metabolism of the H L culture would have been dominated by respiration immediately after transfer to L L . Respiration may have provided a large portion of the energy for cell division during the light transition (HL-»LL). Carbon quota fell 42% and cell volume fell 22% in less than 12 h following the change in light regime (HL-»LL). The catabolism of carbohydrate during the transition from H L - » L L has been documented by Post et al. (1985). In contrast, a 10 min exposure of the L L culture to H L indicated a high rate of photosynthesis occurred immediately, probably resulting in the production of excess photosynthate (Post et al. 1985). Rates of adaptation'in growth rate were slower in the HL—»LL culture. Comparison of chemostat and turbidostat cultures At high irradiances there were large differences in CV and Qc between chemostats and turbidostats. High light chemostats were not energy-limited and would not have an energy restraint on carbon metabolism. Nutrient-limited cells growing at 80% of u m a x and PFD = 160 umol photons m - 2 s ' l were smaller than light-limited cells at every growth rate above 37% of umax> PFD = 20 umol photons m ' 2 s ' l , and higher. Therefore the effects of PFD on carbon quota and on cell volume may be different when factors other than PFD control u. These data suggest strongly that, at least for H. akashiwo, nutrient limitation has an overriding effect on cell volume compared with irradiance. 48 Ecological significance A general reduction in phytoplankton cell volume and carbon quota with decreasing PFD may have significant implications for cells sinking out of the euphotic zone. Since carbon quota is reduced by more than ca. 60% as PFD approaches zero and respiration dominates the cell's energy metabolism during light transients (Post et al. 1985), the possibility exists that phytoplankton may respire a considerable portion of their carbon quota while sinking out of the euphotic zone, or at night. If phytoplankton respire 60% of their carbon quotas while sinking out of the euphotic zone, there are important implications for determinations of global C02 budgets. Conventional measures of primary production may seriously overestimate actual primary production if they fail to adequately assess respiratory carbon losses either at night or during sinking (a transition to lower PFDs). Alternatively, upon sinking, one large cell may simply become smaller by dividing into two smaller ones. Regardless, cells existing below the 1% light depth (assuming full sunlight gives 1500 umol photons mf^s'l) would divide at ca. 30% of their maximal rate and should require only 58% of their maximum carbon quota, thus providing a significant energy saving in the capital costs of growth (Richardson et al. 1983). Cells sinking out of the photic zone could be expected to experience decreasing irradiance over a time frame adequate for a reduction in cell size. In this situation the associated reduction in carbon quota could reduce the energy required for reproduction, thereby improving fitness at low PFD (Swift and Meunier 1976). Also, at the level of the individual phytoplankter, the ecological advantages of a smaller 49 size and a smaller carbon quota may include a reduction in sinking rate (Smayda 1970). The reduction in cell volume which occurs in association with low growth irradiances (this study) may partly explain the lower sinking rates of T. weissfloglii grown in light-limited culture compared to the light-saturated culture observed by Bienfang et al. (1983). Although recent research demonstrates that the relationship between sinking rate and cell size is not controlled simply by physical-size relationships and requires increased consideration of the cells' physiological condition (Waite et al. in press). Since many filter feeding organisms have narrow filtration ranges (Gauld 1966) and few calanoid copepods can filter particles smaller than 5 um in diameter (Boyd 1976) a significant reduction in cell volume may effectively alter the grazing pressure upon phytoplankton. Given the large changes in size of some species as PFD varied (this study) it seems likely that the filtration efficiency of some herbivores would not be constant across such a gradient of particle sizes. Assuming an approximately spherical shape, at least four species, T. pseudonana, C. gracilis, C. simplex, and P. tricornutum, were at or near the 5 um diameter when growing at high PFDs and significantly below this diameter at low PFDs. Eight of the ten phytoplankton species in this study are used extensively for invertebrate aquaculture, and significant changes in cell size and carbon quota may influence the invertebrate's feeding efficiency, ingestion rate, and nutritional status. 50 Finally, a reduction in cell size increases the cell's surface area/volume ratio which may serve to improve a cell's ability to sequester nutrient resources from the surrounding environment (Raven 1986). If small size is an ecological benefit, then there must be some compelling improvement in fitness forcing the evolution of the ability for a rapid increase in cell volume when cells move from limiting to saturating light. Light absorption, given equal pigment quotas, may be improved in larger cells due to a reduction in the intracellular self-shading package effect (Raven 1986). Also, an overall reduction in the package effect may be enhanced by the concommitment decrease in pigment quota usually associated with the larger cells found at higher irradiances. Larger cells are predicted to have an ecological advantage over smaller cells in reduced leakage of solutes (including CO2 in C02-concentrating species) and resource storage (Raven 1986). If light-saturated growth produces a surplus of carbohydrates and lipids, storage of these compounds may result in an increase in cell size or quota. The resulting larger high-light grown cells would contain enough cellular material to supply considerable energy or cellular building materials to these same cells if they temporarily experienced a low energy environment (Rivkin et al. 1982b). Given the low maintenance costs of low growth at very low irradiances (Geider et al. 1985) the 66% extra carbon quota of high-light cells may provide enough energy/material to survive many weeks. Thus growth, cell volume and carbon quota all appear to be linked to the photoautotroph's ability to sequester and allocate energy. 51 Finally, a reduction in cell size increases the cell's surface area/volume ratio which may serve to improve a cell's ability to sequester nutrient resources from the surrounding environment (Raven 1986). If small size is an ecological benefit, then there must be some compelling improvement in fitness forcing the evolution of the ability for a rapid increase in cell volume when cells move from limiting to saturating light. Light absorption, given equal pigment quotas, may be improved in larger cells due to a reduction in the intracellular self-shading package effect (Raven 1986). Also, an overall reduction in the package effect may be enhanced by the concommitment decrease in pigment quota usually associated with the larger cells found at higher irradiances. Larger cells are predicted to have an ecological advantage over smaller cells in reduced leakage of solutes (including C02 in C02-concentrating species) and resource storage (Raven 1986). If light-saturated growth produces a surplus of carbohydrates and lipids, storage of these compounds may result in an increase in cell size or quota. The resulting larger high-light grown cells would contain enough cellular material to supply considerable energy or cellular building materials to these same cells if they temporarily experienced a low energy environment (Rivkin et al. 1982b). Given the low maintenance costs of low growth at very low irradiances (Geider et al. 1985) the 66% extra carbon quota of high-light cells may provide enough energy/material to survive many weeks. Thus growth, cell volume and carbon quota all appear to be linked to the photoautotroph's ability to sequester and allocate energy. 5 1 SUMMARY Ten species of marine phytoplankton were grown under a range of photosynthetic photon flux densities (PFDs) and examined for variation in cell volume, and carbon quota. Phytoplankton generally reduced their cell volume and frequently reduce their carbon quota in response to low PFDs. A significant linear relationship between the log of PFD (I) and cell volume (in nine of ten species) and log PFD and carbon quota (four of ten species) was demonstrated. When Thalassiosira pseudonana was exposed to a transient in light intensity it underwent a rapid adaptation in cell volume and carbon quota. Cells going from low light to high light reached maximum mean cell volume within 5 h, and cells going from high light to low light reached a minimum mean cell volume within 1 2 h. The resulting kinetic constant (k; a measure of the rate of adaptation) was considerably larger than previously reported k values. Ditylum brightwellii was observed to undergo an increase in length but no increase in width during a transient to increased irradiance. Nutrient Hmitation was shown to override PFD in determining cell volume and carbon quota for Heterosigma akashiwo. Cells grown at equivalent irradiances but N-limited were smaller than light-limited and nutrient-saturated cells. Therefore cell volume and carbon quota do not have the same relationship with PFD when factors other than PFD control u. The ecological implications of reduced cell volumes and carbon quotas with decreasing PFD include: possible impacts on C O 2 budgets, an influence on sinking rates, potential changes in predation rates and surface area/cell volume benefits. 52 CHAPTER 2 T H E E F F E C T S OF VARIATION IN IRRADIANCE O N T H E F A T T Y ACID COMPOSITION OF MARINE PHYTOPLANKTON. I N T R O D U C T I O N The total fatty acid complement of a large number of marine organisms has been determined using gas chromatographic techniques (Morris and Culkin 1976). Continued interest in phytoplankton fatty acids (FAs) stems from their importance in studies of cell physiology, classification and taxonomy, and because of the importance of essential fatty acids (EFAs) in herbivore diets. At present aquaculturalists are interested in the transfer of EFAs from phytoplankton diets to cultured herbivorous species (e.g. Watanabe et al. 1983). The ecological significance of FAs in the marine food chain has been recognized (Sargent and Whittle 1981). The analysis of FAs in phytoplankton has assumed new importance following the discovery that a dietary deficiency of 20:5co3 and 22:6co3 limits growth in several species of bivalves (de Moreno et al. 1976, Trider and Castell 1980, Langdon and Waldock 1981). The nutritional suitability of phytoplankton for bivalves has been assessed, at least partially, by the phytoplankton's F A profile (Webb and Chu 1982). Phytoplankton species identified as poor food for bivalves have had their nutritional inferiority attributed to inadequate amounts of EFAs (Landgon and Waldock 1981). 53 With the advent of commercial bivalve hatcheries, the culture of phytoplankton with EFAs adequate for larval growth and development has gained new importance. Methods of optimizing the production in phytoplankton of essential dietary components, including EFAs, by manipulating the conditions under which the phytoplankton are grown, have focused primarily on nutrient stress (e.g. Enright et al. 1986b). Extended nutrient starvation of batch cultures can, however, lead to the formation of multi-cellular aggregates (clumping), abrupt sedimentation (crashes) and higher bacterial contamination. These problems may be reduced by using various levels of irradiance or photon flux density (PFD) to manipulate F A composition. Researchers have shown an increase in the percentage of polyunsaturated fatty acids (PUFAs) under low light (Orcutt and Patterson 1974), whereas others have observed a decrease (Constanopoulos and Bloch 1967, Opute 1974, Mortensen et al. 1988). Lower temperatures can also increase the degree of F A unsaturation (Ackman et al. 1968) but at the cost of low phytoplankton growth rates. The literature contains many inconsistencies in fatty acid profiles of phytoplankton species and indeed specific clones. Use of F A profiles for taxonomy or as biochemical tracers requires that they be reproducible and reasonably robust to variation in environmental conditions. Few studies have substantiated the temporal stability of FA profiles (Napolitano et al. 1988, Volkman et al. 1989) which have been widely applied in classification (Chuecas and Riley 1969, Nichols et al. 1983, Nichols et al. 1987). 54 The principal objective of the present study was to assess the effect of variation in light intensity on the F A profiles of some phytoplankton species commonly used in aquaculture. In this study, 8 species of marine phytoplankton grown at 4 different PFDs were assayed for growth rate, cellular carbon, nitrogen, and chlorophyll a per cell. Detailed fatty acid compositions determined using capillary column gas chromatography are reported. The changing proportions of individual fatty acids were tested for correlations with other physiological parameters. Defining the irradiance conditions under which different phytoplankton species produce the largest quantities of certain FAs may be useful to aquaculturalists, and may yield insights into the F A physiology of phytoplankton. 55 M A T E R I A L S A N D M E T H O D S Five marine diatoms, Chaetoceros calcitrans (Paulsen) Tokano (NEPCC #590), Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (NEPPC #58), Chaetoceros simplex Ostenfeld (NEPCC #591), Chaetoceros gracilis Schutt (NEPCC #645), and Phaeodactylum tricornutum Bohlin (NEPCC #640), and three flagellates, Dunaliella tertiolecta Butcher (NEPCC #1), Pavlova lutheri Droop (NEPCC #5) and Isochrysis aff. galbana (Green, clone T-iso, termed Tahitian Isochrysis, N E P C C #601) were obtained from the Northeast Pacific Culture Collection (NEPCC), Department of Oceanography, University of British Columbia. All culture work was conducted in enriched artificial seawater (ESAW) based on the recipe by Harrison et al. (1980). The medium was modified as described in Chapter 1. Cultures were kept in exponential phase and preconditioned for approximately 8 generations to the specific PFDs in 30 mL of medium in 50 mT, borosilicate glass test tubes with teflon-lined caps. All cultures were grown at 17.5 ± 0 . 5 °C. The following PFDs were used: 6 or 14, 24 or 44, 80 or 125, and 200 or 225 umol photons m " 2 s ' l (Li-Cor R model LI 185 meter, 2* collector). Duplicate cultures of T. pseudonana and triplicate cultures of D. tertiolecta at 44 umol photons m " 2 s*l, plus duplicate cultures of C. gracilis at 125 umol photons m* 2 s"* were grown. Continuous light was provided by Vita-liteR fluorescent tubes and attenuated by distance and/or neutral density screening. The 30 mL cultures were used to inoculate 6 L cultures (in 12 L round flat bottom flasks) providing a dilution of 1:200 so that at least five additional generations occurred under the defined experimental 56 conditions prior to harvest. The 6 L cultures were stirred at 60 rpm with a 7.6 cm teflon-coated magnetic bar and bubbled with a mixture of approximately 2% C02 and air. The pH of most cultures was maintained at 8.2 although some increases to ca. 8.5 occurred during culture growth in the case of the fastest growing diatoms. No lag phase was observed following dilution, and all cultures were harvested at mid-exponential phase to ensure nutrient limitation did not occur. Determination of biomass (in vivo fluorescence via a Turner Designs^ Model 10 fluorometer and cell counts utilizing a Coulter Counter^ model TAII equipped with a population accessory) were made once or twice per day. Growth rates were calculated as: u = lnCFx/FoVCti-tQ) Where F i = biomass at time 1 (tl) and Fo = biomass at time 0 (to). Chlorophyll a was measured on 25 mL subsamples filtered through Whatman GF/F filters. Filters were stored frozen and desiccated at -20 °C prior to extraction in 90% acetone, sonication for 5 min and held for 24 h in the dark at 4 °C. Chlorophyll o concentrations were calculated from in vitro fluorescence (Parsons et al. 1984). Particulate organic carbon and nitrogen (POC and PON) subsamples were collected on 13 mm Gelman A/E filters and were analyzed on a Carlo Erba C H N analyzer. Samples for total lipid were analyzed by the lipid charring technique of Marsh and Weinstein (1966) using tripalmitin as a standard. Total lipid also contains chl a. Samples for fatty acid determinations were collected on precombusted GF/F filters, placed inside a petri dish and sealed in plastic bags filled with 57 nitrogen. Prior to analysis they were frozen at -20 °C, for periods of less than 3 weeks, or for longer periods at -80 °C. Samples were saponified and methylated as in Whyte (1988). Intra- and inter-sample duplicates were prepared and analyzed. FAs were analyzed on a Hewlett-Packard 5890A gas liquid chromatograph fitted with a Supelcowax 10 fused silica capillary column (30 m x 0.32 mm ID, 0.25 um film) and identified by comparison with saturated and PUFA-1 methyl ester standards (obtained from Supelco Inc.) in accord with Ackman (1986). The shorthand notation used in fatty acid identification is LtBcoX where L is the chain length, B is the number of double bonds, and coX is the position of the double bond closest to the terminal methyl group. 58 RESULTS Batch cultures were grown under PFDs designed to provide very suboptimal (6-14 umol photons m " 2 s'l), moderately subsaturating (ca. 24-44 umol photons m " 2 s"1), saturating (80-125 umol photons m " 2 s"1), and suprasaturating (200-225 umol photons m " 2 s'l) continuous light conditions (u versus PFD curves are shown in Chapter 1, Fig. 1.1, A panels). These cultures were grown through a minimum of 13 generations (i.e. two transfers) and harvested for biochemical and fatty acid analyses at mid-log phase. In this study, variation in growth rate is considered to be controlled by variation in PFD. Data for PFD, growth rate, chl a cell-1, total lipid, cellular carbon, and nitrogen show that when u declined due to a reduction in PFD, chl a cell'l increased (Table 2.1). The lipid cell-1 was relatively constant with decreasing u, but since the carbon cell'l generally declined with u, the percentage of carbon stored as lipid increased significantly as PFD and u declined (Fig. 2.1). On average 44.5% of the total cellular carbon was lipid. Correlation analysis was used to examine the data for trends and possible relationships of u, PFD and chl a cell"* with the relative proportions of specific fatty acids. The relative amounts of many FAs were correlated with u. For example, results for C. gracilis (Table 2.2) showed an increase in the relative proportions of 15:0 0-2=0.845), 16:0 (r2=0.851), 16:4col (r2=0.869), and 22:6co3 (r2=0.804) which were positively correlated with u. Similarly the relative proportions of 16:lco9 (r2=0.846), 16:lco5 (r2=0.993), and 20:5co3 59 Table 2.1. Biochemical analyses for 8 marine phytoplankters growing exponentially in semi-continuous culture at 4 PFDs ranging from light-limited (<, 44 umol photons m"2 s* *•) to light-saturated (> 80 umol photons m " 2 s-1). Irradiance Growth Pigments chl a Lipid Carbon Nitrogen (umol photons) rate m-2 s-1) (d-l) (fgcelM) (pg cell-l) (pg cell-l) (pg cell-l) C. calcitrans 225 2.00 59.5 2.71 5.94 1.18 80 1.39 78.6 N/A 7.66 0.808 24 1.22 52.9 1.45 3.64 0.311 6 0.48 64.3 1.80 3.04 0.639 T. pseudonana 225 1.84 62.4 3.87 12.5 1.16 125 1.87 79.1 3.11 12.2 1.67 44 1.59 132 3.79 9.99 1.70 6 0.39 170 3.76 9.55 1.58 C. gracilis 225 1.54 49.3 3.31 7.00 1.29 125 1.61 73.8 3.98 21.0 1.95 44 1.40 133 3.70 9.68 1.98 6 0.55 164 4.73 6.88 2.13 C. simplex 225 1.95 84.8 3.45 15.1 2.28 125 1.75 148 3.64 16.5 2.10 44 1.33 133 3.66 9.35 1.68 6 0.27 150 3.51 8.24 1.59 P. tricornutum 225 1.22 31.5 2.93 10.9 2.13 125 1.34 79.6 5.36 12.9 2.10 44 1.11 77.1 3.84 9.07 1.57 14 0.61 74.6 2.96 6.65 1.10 D. tertiolecta 225 1.41 527 14.4 50.0 8.06 125 1.32 713 18.4 48.7 10.1 44 0.91 781 16.3 40.2 7.88 14 0.44 801 17.4 37.1 7.42 P. lutheri 225 0.56 34.3 7.25 12.3 1.45 125 0.63 57.5 4.62 12.7 1.49 44 0.69 117 7.52 15.1 1.96 14 0.48 211 5.98 13.2 1.75 /. aff. galbana (T-iso) 225 0.43 32.8 2.63 9.95 1.18 125 0.56 46.4 4.40 9.36 1.23 44 0.60 75.0 3.48 10.0 1.52 14 0.71 121 4.97 9.89 1.47 60 Fig. 2.1. The percentage of cellular carbon found as lipid [equal to {(ug lipid cell-l)/(|ig carbon cell"l)}xlOO] in 2 graphs: A) versus photon flux density in umol photons m" 2 s"l and, B) versus growth rate (u in d"l) for C. calcitrans (O), T. pseudonana (•), C. gracilis (A), C. simplex (A), P. tricornutum (•), D. tertiolecta (M), P. lutheri (V), 7. aff. galbana (T-iso) (T). Linear regressions are shown fitted to the data. 61 62 TABLE 2.2A. Fatty acid composition of 2 phytoplankton species grown under 4 different PFDs (umol photons n r 2 s"l). Values are reported as percentage of total. In 1 case duplicate cultures were grown and 1 standard deviation (S.D.) is given. Species Chaetoceros calcitrans Thalassiosira pseudonana PFD Fatty Acid 225 80 24 6 225 125 44 n=2 S.D. 6 12:0 0.2 0.1 0.2 0.1 0.1 14:0 16.8 20.4 17.3 16.2 4.9 5.5 7.0 .9 6.4 14:la>7 0.2 14:10)5 0.4 0.2 0.5 0.6 0.2 0.3 0.3 .0 0.9 15:0 0.8 0.8 0.5 0.5 0.8 1.0 1.1 .0 0.8 Prist 0.3 0.3 0.5 0.7 0.3 0.4 0.6 .1 0.5 16:0 9.6 11.0 4.1 4.3 30.7 37.7 21.9 .4 10.2* 16:10)9 1.0 0.8 1.6 2.4 0.8 1.3 1.6 .3 1.7 16:10)7 20.1 25.9 23.1 28.2 20.5 25.1 26.1 .5 38.5** 16:10)5 0.7 0.5 0.3 0.3 0.5 0.2 .0 0.2 17:0 iso 1.5 1.1 1.3 0.9 0.7 0.9 1.1 .0 0.4 16:20)7 1.8 2.9 3.7 4.2U 1.0 1.2 3.2 .1 6.5*A 16:20)4 2.3 2.6 3.9 7.7 1.7 1.7 3.6 .1 2.6 17:0 0.4 0.2 0.1 0.1 16:3©4 6.1 9.6 11.5 8.4 4.6 4.8 9.2 .2 4.2 ? 0.8 0.2 0.3 0.2 0.8 0.2 0.2 .0 0.4 16:40)3 16:4tol 4.6 4.0 3.6 2.4** 2.9 2.0 1.0 .0 0.9IIA 18:0 0.4 0.5 0.0 0.3 0.4 0.3 .0 0.2 18:10)9 0.7 0.7 0.2 0.4 0.2 0.4 0.4 .1 0.5 18.10)7 3.6 0.8 0.5 0.8 0.4 0.5 1.0 .2 0.5 18:2co6 0.3 0.4 0.5 0.3 0.2 0.2 .0 0.2 18:3o)6 0.6 18:30)1 0.3 0.6 0.2 .0 0.6 18:40)3 4.8 1.8 1.1 0.711 8.2 5.3 4.6 .2 1.2BA 20:0 0.3 20:2(06 0.3 1.6 20:30)6 20:4o)6 0.3 0.3 0.4 20:40)3 0.8 0.4 0.2 0.2 .0 20:50)3 15.7 12.5 15.4 14.1 13.8 7.7 12.0 .6 16.5 22:0 0.5 0.2 0.2 0.2 22:40)6 1.2 1.5 22:5co6 0.3 22:40)3 0.6 0.3 22:6(03 1.5 0.6 0.7 0.7 3.2 1.4 1.9 .2 2.1 correlation with growth rate; *p<0.05 **p<0.01 correlation with chlacell-1; Ap<0.05 AAp<0.01 correlation with PFD; • p£ 0.05 ••pSO.Ol 63 TABLE 2.2B. Fatty acid composition of 2 phytoplankton species grown under 4 different PFDs (umol photons m~% s-*). Values are reported as percentage of total. In 1 case duplicate cultures were grown and 1 standard deviation (S.D.) is given. Species Chaetoceros gracilis Chaetoceros simplex PFD Fatty Acid 225 125 n=2 S.D. 44 6 225 125 44 6 12:0 0.1 .1 0.1 0.2 14:0 16.0 16.8 .1 20.0 26.6**A 31.1 26.2 27.6 16.9+ 14:1(09 0.3 14:loo7 0.5 14:lco5 0.1 0.1 .0 0.6 0.5 0.4 0.6 0.5 2.8 15:0 ante 0.5 15:0 0.4 0.5 .0 0.4 0.3* 0.6 0.6 0.5 0.4* Prist 0.1 .1 0.2 0.4 0.4 0.5 0.6 1.1* 16:0 18.4 21.4 .0 13.4 7.6*A 9.5 7.8 6.8 3.8* 16:lco9 0.2 0.4 .2 0.8 1.4*A 1.0 1.9 1.7 3.0 16:la)7 30.3 30.8 .5 22.3 20.0AA 24.9 19.9 16.0 8.9*B 16:lo)5 0.3 0.3 .0 0.3 0.7** 1.2 1.2 1.0 0.7 17:0 iso 0.4 1.5 1.5 1.5 1.5 16:2(07 0.9 0.9 .2 1.5 0.8 3.5 3.3 3.3 3.2 16:2(04 2.8 2.6 .2 3.0 12.3 3.0 3.1 3.7 3.1 16:3o>4 4.1 4.1 .2 10.0 6.2 7.2 8.6 14.3 17.4+ ? 0.2 0.2 0.1 0.7 0.4 0.3 0.5 16:4(03 16:4(01 3.6 3.3 .0 2.2 l . l * B A J t . 3.5 3.3 2.6 4.1 18:0 0.4 0.5 .1 0.2 0.2 0.2 0.3 0.2 0.2 18:loo9 0.5 0.7 .2 0.4 0.6 0.3 0.5 0.3 0.9 18:1(07 0.7 0.7 .1 0.6 0.9 0.6 0.6 0.8 5.4 18:1(09 0.3 0.3 0.1 0.3 18:2(06 0.8 0.8 .1 0.7 1.2 0.2 0.2 0.3 0.3 18:3(06 0.1 .1 0.2 0.2 0.1 18:3(03 0.2 0.2 18:30)1 0.3 0.2 .1 0.2 0.2 0.5 18:4(o3 1.4 1.6 .2 1.6 0.8 0.6 1.2 1.2 0.5 20:0 0.2 20:2(06 0.5 0.5 0.7 0.3 0.7 20:4(06 0.2 0.3 20:4co3 0.3 0.2 .0 0.2 0.1 0.2 0.4 0.2 20:5(03 11.6 11.3 .5 12.3 14.9* 6.1 5.4 9.90 15.5* 22:0 0.3 22:4o6 0.4 22:4(03 0.3 0.2 22:5(03 0.2 0.1 .1 0.3 0.3 22:6(03 1.5 1.2 .0 1.1 0.5* 1.1 1.0 1.1 1.2 correlation with growth rate; *pS0.05 **p<0.01 correlation with chl acell-1; Ap<0.05 AAp<0.01 correlation with P F D ; • p< 0.05 ••p<0.01 + strong trend 64 TABLE 2.2C. Fatty acid composition of 2 phytoplankton species grown under 4 different PFDs (umol photons m"2 s"*). Values are reported as percentage of total. In 1 case triplicate cultures were grown and 1 standard deviation (S.D.) is given. Species Phaeodactylum tricornutum Dunaliella tertiolecta PFD Fatty Acid 225 125 44 14 225 125 44 n=3 S.D. 14 10:0 0.3 0.1 0.4 .1 0.1 12:0 0.1 0.0 .0 13:0 0.1 .1 14:0 4.8 6.5 6.4 7.5 0.2 0.9 0.1 .1 0.8 14:1(07 0.1 .1 15:0 ante 0.3 1.0 .5 Prist 0.1 0.3 0.4 0.5 0.5 0.4 0.7 .5 0.8 16:0 19.4 16.8 13.6 13. I l l 17.3 18.1 15.6 1.6 15.2 16:1©9 0.4 0.9 1.4 1.611 1.2 1.5 2.6 .6 2.911 16:1(07 33.7 23.4 19.2 19.5B 0.2 16:1(05 0.2 0.3 0.1 17:0 iso 0.5 1.0 1.4 2.2 2.9 3.1 3.0 .4 2.8 16:2(07 1.4 1.4 1.4 1.1 0.8 1.2 1.2 .2 1.1 16:2(04 2.5 4.4 6.4 5.2 phytanic 0.7 1.0 1.4 .4 l.Q 17:0 0.1 16:3(04 1.9 1.7 4.0 5.5+ 0.4 0.8 0.9 0.4 3.2 3.2 3.1 .5 2.6 16:4(03 11.2 11.6 14.2 1.8 14.7 16:4(01 2.4 6.1 7.5 4.8 18:0 0.4 0.4 0.3 0.2 0.4 0.2 0.1 .2 18:1(09 1.7 0.3 0.4 0.8 8.1 6.5 4.6 1.0 3 . 3 H 18:10)7 1.3 0.7 0.7 1.8 0.9 1.2 1.1 .2 1.3 18:10)9 0.2 1.4 1.3 1.2 .1 0.9* 18:2(06 0.9 0.8 1.1 1.7** 9.3 8.7 7.2 1.1 4.5** 18:30)6 0.4 4.4 3.6 3.8 .1 3.5 18:3(03 30.7 30.7 35.9 2.2 38.2* 18:30)1 0.1 0.3 0.7 .1 18:4(03 0.5 0.9 0.6 0.6 0.5 0.9 20:0 0.2 20:4(06 0.4 20:4(03 0.3 0.3 0.4 0.2 20:5(03 15.6 23.9 24.9 20.5 0.1 0.1 .1 22:0 0.3 0.2 0.4 0.2 0.2 .3 0.1 22:4(06 0.2 0.3 22:5(06 0.2 0.2 0.1 22:4(03 0.1 0.2 0.3 0.4B 0.1 22:5(03 0.2 0.3 0.3 0.2 22:6(03 2.8 3.9 3.0 1.1* .1 0.1 correlation with growth rate; *p<0.05 **p<0.01 correlation with chl acell-1; Ap<0.05 ••p<0.01 correlation with PFD; • p< 0.05 ••p<0.01 + strong trend 65 T A B L E 2.2D. Fatty acid composition of 2 phytoplankton species grown under 4 different PFDs (umol photons m"2 s'l). Values are reported as percentage of total FAs. Species Pavlova lutheri Isochrysis galbana (T-iso) PFD Fatty Acid 225 125 44 14 225 125 44 14 12:0 0.1 0.2 0.1 0.1 0.1 14:0 12.3 11.4 11.7 13.5 17.6 18.7 22.3 31.2A 14:lu9 0.2 14:10)7 0.2 0.7 14:10)5 0.2 0.1 0.2 0.3 0.4 0.4 15:0 0.2 0.1 0.4 0.2 0.2 0.3 0.6A Prist 0.1 0.2 0.8 0.7 0.2 0.8 16:0 17.3 19.3 18.5 19.1 10.3 10.0 12.8 15.4 16:lo)9 0.5 0.7 2.1 1.8 0.4 0.6 0.6 2.3 16:lo)7 23.7 23.3 19.4 22.4 0.8 1.1 1.9 3.6AA 16:10)5 0.2 0.2 16:2co7 0.1 0.2 0.4 1.0 0.3 0.2 16:20)4 0.7 0.7 0.4 1.1 0.4 0.6 0.9 0.5 16:30)4 0.3 0.3 0.4 0.5 0.0 0.0 0.1 0.2 0.3 0.4 18:0 0.3 0.3 0.3 0.3 0.9 0.3 0.3 0.4 18:10)9 0.9 1.1 1.1 0.9 22.1 16.3 14.9 15.0 18:lto7 1.0 1.3 1.7 4.0AA 1.7 1.2 2.2 3.3 18:20)7 0.3 0.3 0.3 0.5A 18:20)6 2.9 3.2 1.4 0.5* 6.3 4.1 2.9 2.8a 18:30)6 0.2 0.3 0.2 18:30)3 0.4 0.4 0.5 1.1 3.7 4.5 5.5 4.4 18:4co3 6.4 6.7 9.5 8.4 8.5 11.5 10.7 4.3 18:5to3 3.8 5.2 3.1 0.5 20:2(06 0.3 0.2 0.2 0.2 20:3(06 0.3 0.3 20:4o)6 0.2 0.1 20:3(03 0.2 0.2 20:5(03 16.9 17.0 19.4 16.2 0.6 0.6 0.6 0.3 22:0 0.3 0.2 0.3 21:50)3 0.6 0.4 0.5 0.6 22:4(06 0.5 0.5 0.3 0.2 0.0** A ABB 22:50)6 0.7 0.5 0.5 0.4 2.2 2.3 2.1 0.6 22:5(03 0.56 0.6 0.4 0.3 0.0A 22:6(03 9.7 8.2 6.9 3.6AA 14.4 16.7 10.9 2.1 correlation with growth rate; *p^0.05 **p<0.01 8 correlation with chlacell-1; Ap£0.05 AApSO.Ol correlation with PFD; B p< 0.05 BBp<0.01 + strong trend 66 (r2=0.948) were all negatively correlated with u. The relative proportion of 16:4col (r2=0.806) and 22:6co3 (r2=.868) were also both positively correlated with PFD. The percentage of two FAs, 14:0 (r2=0.884) and 16:lco9 (r2=0.885), were positively correlated with chl a cell-1. Negative correlations between chl a celM and the percentage of 16:0 (r2=0.814), 16:lco7 (r2=0.926) and 16:4col (r2=0.963) also occurred. No consistent correlation between the relative proportion of any single F A and u or chl a cell'l w a s apparent over all 8 species examined. The diatom species all had large amounts of 14:0,16:0, 16:lco7, 20:5co3, and usually 16:3co4 fatty acids (Table 2.2), as was previously reported for diatoms (Kates and Volcani 1966, Chuecas and Riley 1969, Volkman et al. 1989). The diatoms showed their largest consistent changes in the relative proportions of 16:0 or 16:lco7 as u was varied by changes in PFD. The relative proportion of 16:0 always declined with u, reaching from 30-60% of its maximum at the lowest growth rates. The large response in the percentage of 16:lo)7 to increases in u was interspecifically variable. Among the diatoms, positive correlations of the percentage of specific FAs with chl a cell-1 were limited to 14 and 16 carbon FAs. Chuecas and Riley (1969) reported that FAs of the 18 carbon class (except 18:1) were less than 1% of the total FAs in diatoms. In this study, however, the percentage of 18:4co3 ranged from 5-8% of the total FAs for C. calcitrans and T. pseudonana grown at high PFD (Table 2.2). As also noted by Chuecas and Riley (1969) the virtual absence of 67 18:2 and 18:3 could be useful in mstinguishing diatoms from the two other classes of phytoplankton examined here. D. tertiolecta showed relatively little variation in F A composition at any PFD (Table 2.2), although the % 16:lco9 was negatively correlated with PFD (r2=0.931) and the % 18:lto9 was positively correlated with PFD (r2=0.975). The proportion of 18:3co3 ranged from 30 to 38% of the total FAs. The relative proportions of several FAs were correlated with |i, including 18:3co3 (negative correlation; r2=0.963),and 18:2co6, 18:2co9 (positively correlated; r2=0.999 and r2=0.937, respectively). In sharp contrast to the other species, D. tertiolecta had almost no 14:0, 16:lco7 or PUFA greater than 18:4co3. P. lutheri contained large amounts of 14:0,16:0, 16:la>7, 20:5co3, and 22:6u)3 (Table 2.2). Correlations were found between the relative proportions of 18:lco7,18:2u)7 (positive correlations; r2=0.981 and r2=0.938, respectively) and 22:6co3 (negative correlation; r2=0.983) and the amount of chl a cell'l. This was the only species examined that showed a significant positive correlation between an 18 carbon F A and chl a cell-l. I. &S.galbana (T-iso) contained large proportions of 14:0 and 16:0. Of all the species examined, it was unique due to the presence of a large percentage of 18:lco9.1, aff. galbana (T-iso) also contained the largest proportion of 22:6co3 which, however, declined sharply at low light with substantial redistribution to 14:0. The percentage of 14:0 (r2=0.979), 15:0 (r2=0.959) and 16:lco7 (r2=0.990) were positively correlated with chl a cell"*, a pattern reminiscent of the diatoms. The percentage of 18:2to6 and 22:4co6 68 were both positively correlated with PFD (r2=0.959 and r2=0.949, respectively). Furthermore u. was also correlated with 22:4to6 (r2=0.989) and 22:5co3 (r2=0.955). Of the EFAs considered to be important for bivalves, the relative percentages of both 20:5co3 and 22:6co3 responded to changes in u.. All diatoms, except C. calcitrans, exhibited the highest proportions of 20:5co3 at the lowest u.. For two species, C. gracilis and C. simplex, the percentage of 20:5co3 decreased significantly with log increasing PFD. The percentage of 20:5Q)3 was negatively correlated with, and changed substantially in response to u., for C. gracilis (Table 2.2). The relative proportion of 22:6co3 was variable in its response to u., but in two of five diatoms the percentage was positively correlated with u. The percentage of 22:6co3 was positively correlated with log PFD for C. gracilis and P. lutheri. Thus where these two EFAs responded to PFD they varied inversely thereby making it impossible to maximize both EFAs simultaneously by varying PFD. The average percentage of 20:5u)3 plus 22:6co3 calculated over the 4 PFDs was highest in P. lutheri (24.4% of total FAs), followed by P. tricornutum (23.9%), with the other species ranging from 10-15%, except D. tertiolecta which contained only trace amounts (Fig. 2.2). The total variability for technique and replicate cultures was assessed for three species by growing, extracting and analyzing duplicates. The average coefficient of variation (CV.) for T. pseudonana in this study was 7.7.% (Table 2.3). The variability of values reported in the literature for the F A composition of T. pseudonana was very large, with the average C V . of 134% (Table 2.3). 69 Fig. 2.2. The percentage of total FAs found as 22:6co3 plus 20:5co3 (two of the nutritionally important FAs for bivalves) calculated as an average over all 4 PFDs for C. calcitrans, T. pseudonana, C. gracilis, C. simplex, P. tricornutum, D. tertiolecta, P. lutheri, I. aHi.galbana (T-iso). The half bar represents half the range obtained over the four different PFDs. 70 20:5w3 + 22:6w3 (% of total FA) _•. _ l fO o cn o f\3 cn o cn o C. gracilis C. simplex C. calcitrans T. pseudonana P. tricornutum H D. tertiolecta P. lutheri I. galbana (T-iso) Table 2.3. Comparison of different fatty acid analyses (literature values compared with this study) for T. pseudonana (all studies are on clone 3H, with the possible exception of Orcutt and Patterson 1974). Fatty acids are given as a percentage of total. C V . = coefficient of variation. Source 1* 2* 3* 4* 5* C V . 6* C V . mean mean Fatty Acid l to5 % n=2 % 14:0 5.0 15.5 20.0 6.1 7.5 10.8 54 7.0 13 14:lco5 0.6 0.0 0.0 0.0 0.0 0.1 200 0.3 8 15:0 0.6 1.0 1.8 0.0 0.0 0.7 101 1.1 3 Pristanic 0.0 0.0 0.0 0.0 0.0 0.0 na 0.6 12 16:0** 10.0 9.7 21.5 11.5 26.1 15.8 43 21.9. 2 16:10)9^  16:lco7 0.1 0.0 0.0 0.0 0.0 0.0 na 1.6 18 23.7 29.5 22.5 26.0 24.2 25.2 10 26.1 2 17:0 iso M 16:2o)7 0.0 0.0 0.0 0.0 0.5 0.1 200 1.1 1 2.9 0.0 0.0 0.0 0.0 0.6 200 3.2 2 16:2o>4 4.0 0.0 0.0 0.0 2.0 1.2 133 3.6 2 17:1 0.0 0.0 0.0 8.0 0.0 1.6 200 0.0 na 16:30)4^  16:4col 6.7 6.3 12.5 0.0 6.1 6.3 64 9.2 2 0.6 0.0 3.5 0.0 0.4 0.9 148 1.0 0 18:0 0.0 0.0 0.0 5.4 0.0 1.1 200 0.3 12 18:lco9 0.5 0.0 0.0 2.0 0.4 0.6 128 0.4 14 18:lo)7 0.5 0.0 0.0 0.0 0.3 0.2 128 1.0 19 18:2o)9 0.0 1.0 0.0 2.8 0.0 0.8 143 0.0 na 18:2co6 0.3 0.0 0.0 0.0 0.2 0.1 126 0.2 14 18:3co6^ 18:4o)3 0.3 0.0 0.0 0.0 0.0 0.1 200 0.0 na 5.7 0.0 9.0 0.0 3.9 3.7 93 4.6 4 20:0 0.0 0.0 0.0 22.5 0.0 4.5 200 0.0 na 20:2co6 0.0 0.0 0.0 0.8 0.0 0.2 200 0.0 na 20:4o)6 0.0 14.2 0.0 0.0 0.0 2.8 200 0.0 na 20:4co3 0.0 0.0 0.0 0.0 0.3 0.1 200 0.2 0 20:5o)3 23.9 15.0 10.0 12.3 18.9 16.0 31 12.0 5 22:1 0.0 0.0 0.0 1.2 0.0 0.2 200 0.0 na 22:6co3 4.6 0.0 1.3 0.0 3.6 1.9 100 1.9 12 24:0 0.0 0.0 0.0 1.9 0.0 0.4 200 0.0 na average C V . 134 8 Sources: *1 Whyte 1988: PFD=16 umol photons n r 2 g-l,T=20°C *2 Orcutt and Patterson 1974: PFD=60 jimol photons m" 2 s"1, T=17-18°C *3 Fisher and Schwarzenbach 1978: (from graph) PFD=93 umol photons n r 2 s'1, T=18°C, L:D cycle *4 Epifanio et al. 1981: PFD=29 umol photons n r 2 a*1, T=18°C *5 Calderwood 1989: PFD=300 umol photons n r 2 s'1, T=21°C. *§this study: PFD=44 umol photons n r 2 s"1, T=17.5°C percentage shown to be significantly correlated with PFD or u in present study. 72 D I S C U S S I O N Variation in fatty acid analyses Fatty acid profiles are used in taxonomic classification and in the assessment of the food value of a phytoplankton species, yet the very large intraspecific variation in previously published profiles suggests the need for continued systematic investigation of the causes of variation in F A composition. Some of the variation seen in literature values are undoubtedly associated with differences in culturing conditions, including differences in light and temperature. In general, the data published here, agreed very well with similar work conducted under different, although well controlled, conditions by Whyte (1988), Volkman et al. (1989), and Calderwood (1989), suggesting that improvements in culturing techniques and technology may reduce the magnitude of this problem. Published values of 14:0 in T. pseudonana range from ca. 5% (Whyte 1988, Epifanio et al. 1981, this study) to near 15-20% (Orcutt and Patterson 1975, Fisher and Schwarzenbach 1978). The percentage of 20:4 was reported as 14.2% by Orcutt and Patterson (1975) and near zero by Whyte (1988), Fisher and Schwarzenbach (1978), Volkman et al. (1989), and this study. Finally, the percentage of 20:0 was reported as 22.5% by Epifanio et al. (1981) but this F A was rare or absent in other analyses of this species. In some previous studies adequate documentation of culture conditions was not given and merely to report the culture age or growth phase without including which nutrient/factor was responsible for the cessation of exponential growth 73 is insufficient. Assessments of the effects of culture conditions on the F A composition of phytoplankton requires rigorous control of light, temperature, pH, salinity, macro and micronutrient availability which all influence cell physiology and probably F A composition (Ackman et al. 1968, Cohen et al. 1988, Chapter 2, Chapter 3). In batch culture some aspects of the physiology and biochemistry of cells start to change prior to an observable reduction in u.. Apart from culture conditions, variabiUty in F A profiles can result from the many methods and procedures used for FA analysis. The plethora of techniques for storage, extraction, fractionation, derivatization, and finally chromatographic separation, all affect reliability and reproducibility of the F A profiles (Pocklington, 1986). The advent of wall-coated open tubular columns and improved bonded stationary phases in flexible fused silica columns, has substantially improved the separation of FAs (Ackman 1986) and eliminated co-chromatographing of many acids which were misidentified in earlier packed column separations. Whyte (1988) attempted to minimize variance in F A analysis by direct derivatization of acids in the tissue, without prior lipid extraction, and has provided a reliable procedure for convenient monitoring of total F A profiles in cultured species. Fatty acids as storage products influenced by PFD Fatty acids produced in large quantities which increased with [i or PFD were considered to be probable storage products. Few studies have examined the relationships between light intensity and F A profiles for phytoplankton (Nichols 1965, Constanopoulos and Bloch 1967, Opute 1974, Orcutt and Patterson 1974, Mortensen et al. 1988). Given the limited number 74 of species examined, the only generalizations that seem possible are that diatoms adjust their levels of 16:0 and 16:1 (in this study, 16:lco7) in response to PFD (Opute 1974, Orcutt and Patterson 1974, this study). With the exception of Nitzschia palea (Opute 1974), 16:0 always declines with decreasing PFD. Similar results were obtained by Fisher and Schwarzenbach (1978) and Sicko-Goad et al. (1988) in experiments with T. pseudonana and Cyclotella meneghiniana, respectively. These diatoms were shown to have short term responses in their F A composition to darkness. In T. pseudonana the percentage of 16:0 and 16:1 declined sharply during a dark period; in C. meneghiniana, the % 16:0 decreased, and the % 16:1 increased during the light portion of a L:D cycle. A substantial portion of 16:0 and 16:1 is located in triacylglycerides (Opute 1974, Fisher and Schwarzenbach 1978). Therefore, it seems reasonable to consider that the FA 16:0 is largely a storage product for excess energy in diatoms. Further corroboration of this interpretation can be seen in the work of Roessler (1988) and Calderwood (1989). For two diatom species, they found that the percentage of 16:0 increased sharply when cells suddenly became Si- or P- limited (respectively) when still receiving a photon flux. Also comparing the results obtained by Whyte (1988) and Calderwood (1989) indicates an increase in 16:0 from 10 to 26.1% in T. pseudonana as irradiance increased from 10 to 300 umol photons m-2 s-1. In the present study, diatoms also showed substantial changes in the proportion of the total 16:1 isomers as a response to variation in PFD. These responses were not as consistent in direction as were those of 16:0; sometimes 75 the proportions of 16:1 isomers increased with PFD (two species), and sometimes they decreased (three species). In the diatoms, other fatty acids which showed a positive correlation in their % abundance with jx included: 16:4u)l (two times) and 22:6co3 (two times). Both these FAs were usually present in relatively small amounts (ca. 4%) and therefore were unlikely to be a significant storage product for energy. In C. simplex the % composition of 14:0 fitted loosely the criterion for a storage product, although the correlation with u or PFD was not significant. Fatty acids stored by species other than diatoms included 18:2 isomers which have also been associated mainly with the neutral lipids (Cohen et al. 1988). In the present study, the % 18:2o>6 increased considerably as PFD approached Imax for the three non-diatoms (Table 2.2). Cohen et al. (1988) also observed an increase in 18:2 under conditions where u was reduced (due to salinity, or cell density) but PFD was saturating in Porphyridium cruetum. Both D. tertiolecta and /. aff. galbana (T-iso) contained large percentages of the F A 18:lco9 which also increased as u approached Umax, thereby suggesting that in these species, 18 carbon FAs may act as energy reserves. Fatty acids associated with photosynthesis Under steady-state conditions, where growth rate is controlled by PFD, photosynthesis is directly proportional to u. Intraspecific positive correlations between the % of a specific F A and u may indicate its association with photosynthesis. The membranes of photosynthetic organelles may represent 75% of the total cellular membrane (Forde and Steer 1976) and are highly unsaturated, containing as high as 90% PUFA (Fuller and Nes 1987). The 7 6 monogalactyl diglycerides of the chloroplast are mainly polyunsaturated 16 and 18 carbon FAs (Opute 1974). Any associations, therefore, between u and saturated or mono-unsaturated FAs were not considered here (see above section on storage products). The relative proportions of the following PUFAs were positively correlated with u,: 22:6co3 (two times), 16:4col (two times), 22:4co6 (once) and 22:5co3 (once). Since Bacillariophyceae was the only class found to contain 16:4col and I. a££.galbana (T-iso) was the only species with a correlation between 22:4co6 or 22:5co3 and u,, these FAs are unlikely to be generally associated with photosynthesis. Nevertheless, the % 22:6co3 was positively correlated with u twice and was often greatest at high PFD (five out of seven times). The association of this F A with another molecule that performs a rate step in photosynthesis or in some anti photo-oxidation procedure in specific species is suggested. The proportions of PUFAs positively correlated with PFD included 16:4tol (two times), 18:4co3 (two times) and 22:4a>6 (once). The reason for a relationship between the percentage of these PUFAs and PFD in only a few species suggests that the abundance of these PUFAs is not associated with the photosynthetic process itself but perhaps with a specific pigment component that increases with PFD (e.g. Ben-Amotz and Avron 1983). Alternatively it is suggested that highly unsaturated FAs may be required in the highly active membranes found in cells growing more rapidly (see Chapter 3). Fatty acids associated with pigments A n association between FAs and pigment molecules is suggested by a positive correlation between chl a cell-l and the percentage of a specific FA. 77 Over all 8 species the relationship between chl a cell" 1 and polyunsaturates (Fig. 3b) supports the argument that chlorophyll molecules are associated with PUFAs in marine phytoplankton (Kates and Volcani 1966). These authors and Cohen et al. (1988) have suggested that diatoms use pairs of 16:1 and 20:5 to accommodate the phytol side chain of chl a. This suggestion was not substantiated by my results as no consistent correlations were evident between 20:5co3 and 16:1 or between these 2 FAs and chl a cell"l. Nevertheless, for the diatoms, 20:5co3 is the only major FA to remain consistently high (two species) or increase with decreasing PFD (three species). The FA, 20:5o)3, was previously shown to increase during the dark portion of a L:D cycle in C. meneghiniana (Sicko-Goad et al. 1988). This conservation or increase in the relative proportion of 20:5o)3 with decreasing PFD supports the contention that this F A represents ail important component of the chloroplast's lipids (Kates and Volcani 1966, Cohen et al. 1988) for some marine phytoplankton. The results of Cohen et al. (1988) indicated an increase in 20:5co3 as PFD increased from 30 to 170 umol photons m ' 2 s"l. This result would seem to contradict the possibility of an association between chl a and this F A since chl a could be predicted to decrease over a change in PFD from 30 to 170 umol photons m " 2 s"l. Whether the % of 20:5co3 increased soley in response to increased PFD (Cohen et al. 1988) is difficult to assess given the confounding effect of their very high cell densities (reported only as mg chl a L-l). The increase in 20:5co3 observed by Cohen et al. (1988) over a change in PFD from 30 to 170 umol photons m " 2 s ' l was not analogous to the increases 78 in 18:3co3 reported by Rosenberg and Pecker (1964) during the transfer of their etoliated heterotrophically grown cells to photoautotrophic conditions. Generally chl a cell-l is zero in etoliated cells under heterotrophic conditions (Rosenberg and Pecker 1964), reaches a maximum at low PFD and decreases slowly as PFD increases (Falkowski 1980, Thompson et al. 1989). Lipids associated with chl a could be expected to behave similarly. The high % of 18:3co3 inD. tertiolecta is typical of green algae. Its relative proportion increased with decreasing PFD (negatively correlated with (i). The near absence of any PUFAs greater than 18:4co3 in D. tertiolecta is consistent with most recent analyses of this species (Ackman et al. 1968, Langdon and Waldock 1981, Volkman et al. 1989) although Chuecas and Riley (1969) reported 10.2% 20:5 and 6.2% 22:5. The lack of PUFAs greater than 18:4co3 in D. tertiolecta suggests that the phytol side chain of chl a can be accommodated in lipid membranes without the 20 carbon PUFAs found in diatoms and prymnesiophytes. Therefore any association between the 20 carbon PUFAs and pigments found in diatoms and prymnesiophytes may involve pigments other than chlorophyll a. Both diatoms and prymnesiophytes contain high levels of chlorophyll c and abundant carotenoids (Jeffrey and Vesk 1981), mostly fucoxanthin (Wright and Jeffrey 1987), not normally found in the Chlorophyceae. Certain carotenoids have also been shown to increase with increasing PFD (Ben-Amotz and Avron 1983) making the possible associations of pigments with specific FAs complex. Although differences in F A composition may represent genetic differences in synthesis and desaturation pathways, more sophisticated 79 analysis of F A composition and pigment composition may help resolve any functional associations between specific pigments and specific FAs. In P. lutheri the relative proportions of both 18:lco7 and 18:2o)7 were positively correlated with chl a cell-1. This species was unique because the % composition of the 18 carbon FAs was correlated with chl a cell-1. p. lutheri also demonstrated the conservation of relatively large amounts of 20:5co3 with decreasing PFD. Theoretical considerations: PFD versus F A composition This study showed that the relative proportions of many individual FAs were significantly correlated with growth rates controlled by variation in PFD. Greater effects on the F A composition of a species may be achieved by reducing PFD further, but some limited evidence suggests that relatively little change in F A composition can be expected at higher PFDs. The recent work by Mortensen et al. (1988) on C. gracilis indicated only small changes in F A composition over PFD ranging from 83-1395 umol photons m " 2 s ' l . The present results showed large changes in specific FAs of C. gracilis over the PFD range of 6-225 umol photons m"2 s ' l . We suggest that F A profiles will change most rapidly within the PFD (or temperature, pH, nutrient, salinity) range where adaptation will enhance the growth rate. The range from 83-1395 umol photons m " 2 s ' l is fully saturating for growth of C. gracilis (Chapter 1, Table 1.1), and if the relative proportion of a FA is correlated with u, the relative proportion of that F A could be expected to plateau once PFD exceeds Ik. 80 The general positive correlation between u, and the ratio of unsaturated/saturated FAs can be interpreted as a need for greater unsaturation at higher rates of photosynthesis and growth (see also Chapter 3) or that energy-limited cells synthesize the metabolically cheapest FAs that will suffice. Some evidence is available to suggest that both mechanisms may operate in adjusting F A profiles, including some trends in F A composition which were not previously discussed. A large increase in 14:0, which represented ca. 15-30% of the total FAs in /. aff.galbana (T-iso) and C. gracilis, occurred with decreasing u,. As anabolic processes involving chain elongation of FAs are energy demanding, the increased percentage of 14:0 with dechning u. implied that a restriction on chain elongation occurred in these energy-limited cells. It is unlikely that 14:0 was incorporated, in any significant amount, into the increasing chloroplast membranes, but rather formed part of the reduced energy storage capacity of the cells grown at low PFDs. Alternatively the covariation of specific PUFAs with u rather than PFD suggests that, in general, unsaturation is not only a function of energy availability but perhaps associated with the rate of metabolic processes (Chapter 3). Considering the data presented, interspecific variability in the FA composition of chloroplast membranes must be high, perhaps associated with the various accessory pigments. F A composition appears to be influenced by photosynthetic rate and pigment concentration. Research on whether variation in PFD can be used to improve the net nutritional value of phytoplankton used as food by the herbivore C. gigas is 81 presented in Chapter 4. The response of EFAs to PFD appears to be species specific. In some species the PFD controlled growth rates are well correlated with the percentage of EFAs produced (Table 2.2). Results suggest that where larger amounts of EFAs are required, the aquaculturalists growing C. simplex or C. gracilis should consider low levels of PFD and those using P. lutheri or 7. aff. galbana (T-iso) should avoid low light conditions. Species specific production rates of the EFAs can be maximized by selecting the correct PFD. Continued standardization of techniques and strict attention to culture conditions need to be emphasized in future studies of F A composition if meaningful interpretations are to be made. Furthermore, more estimates of statistical variation should be included so that normal variation is not confused with significant change. 82 S U M M A R Y Eight species of marine phytoplankton commonly used in aquaculture were grown under a range of photon flux densities (PFDs) and analyzed for their fatty acid (FA) composition. Fatty acid composition was shown to change considerably at different PFDs although no consistent correlation between the relative proportion of a single F A and u or chl a cell"! w a s apparent in the 8 species examined. Within an individual species the percentage of certain fatty acids varied with PFD, growth rate and/or chl a cell-1. The light conditions which produced the greatest proportion of the essential fatty acids was species specific. Eicosapentaenoic acid, 20:5a>3 increased from 6.1% to 15.5% of the total fatty acids of Chaetoceros simplex grown at PFDs which decreased from 225 umol photons m " 2 s"l to 6 umol photons m-2 s"l, respectively. Most species had their greatest proportion of 20:5oo3 at low levels of irradiance. Conversely docosahexaenoic acid, 22:6co3, decreased from 9.7 to 3.6% of the total fatty acids in Pavlova lutheri as PFD decreased. The percentage of 22:6co3 generally decreased with decreasing irradiances. In all diatoms the percentage of 16:0 was significantly correlated with PFD, and in three of five diatoms, with growth rate (u). Results suggest that fatty acid composition is a highly dynamic component of cellular physiology, which responds significantly to variation in PFD. 83 CHAPTER 3 T H E E F F E C T S OF VARIATION IN T E M P E R A T U R E O N T H E F A T T Y ACID COMPOSITION OF P H Y T O P L A N K T O N I N T R O D U C T I O N If natural environmental fluctuations of temperature, light, salinity and nutrient availability influence the nutritional value of phytoplankton, then these fluctuations may be significant in determining the efficiency of biomass transfer between trophic levels. Relatively small changes in the ecological efficiency of biomass transfer between primary producers and herbivores have been calculated to result in order of magnitude changes in the biomass produced at higher trophic levels (Parsons and Takahashi 1973). Relatively little is known about the nutritional requirements of planktonic marine herbivores and how the biochemical composition of marine phytoplankton may influence the efficiency of biomass transfer. There are increasing number of herbivorous species with a demonstrated requirement for certain long chain polyunsaturated fatty acids (essential fatty acids=EFAs)(Watanabe et al. 1983). The effects of environmental conditions on the fatty acid composition of marine phytoplankton has come under increasing scrutiny in recent years (Ackman et al. 1968, Cohen et al. 1988, Chapter 2). Phytoplankton grown in aquacultural operations can have their food value varied by mixing several species (Epifanio 1979) or by adjusting 84 the conditions under which they are grown (Gallager and Mann 1982, Chapter 4). In phytoplankton, methods of optimizing the production of essential dietary components, including EFAs, by manipulating the conditions under which the phytoplankton are grown, have focused primarily on nutrient stress (e.g. Enright et al. 1986b). Extended nutrient starvation of batch cultures can, however, lead to muti-cellular aggregates (clumping), abrupt sedimentation (crashes) and higher bacterial contamination.' These problems may be reduced by using various levels of irradiance or photon flux density (PFD) to manipulate phytoplankton F A composition (Orcutt and Patterson 1974, Chapter 2). Responses of fatty acid compositions to light intensity have been shown to be interspecifically variable (Orcutt and Patterson 1974, Chapter 2). Lower temperatures may also increase the degree of F A unsaturation (Ackman et al. 1968, Lynch and Thompson 1982, Mortensen et al. 1988), but there is a paucity of data as these studies utilized very few temperatures and/or species. Since the essential fatty acids are poly-unsaturated there is some reason to presume cells may increase these PUFAs at lower temperatures to maintain membrane homeoviscosity (Hochachka and Somero 1984). The fatty acids of thermophilic cyanobacteria adapt to decreases in temperature by increasing unsaturation (Holton et al. 1964, Sato and Murata 1980), and the review of White and Somero (1982) suggests that increased unsaturation is a normal response to lower temperatures for most organisms. The underlying principle for this response is believed to be the greater membrane fluidity (homeoviscosity at decreasing temperatures) of 85 membranes composed of more highly unsaturated FAs, although there exists considerable debate about the validity of this hypothesis (Quinn 1988). Such membranes are thought to be generally required at lower temperatures in order that membranes may remain reasonably homeoviscous (Sinensky 1974, Quinn 1981). There are a number of other well documented physiological responses that are associated with changes in temperature and are propounded to confer some measure of homeoviscosity to cellular membranes. These include: variation in the protein:lipid ratio or cholesterol:lipid ratio (Chapman et al. 1983, Oldfield and Chapman 1972, respectively), variation in fatty acid chain length (Hadley 1985), and variation in the position of the cis double bonds within the fatty acid (Hochachka and Somero 1984). The data from this study provide some information regarding which of these mechanisms may be functioning within marine phytoplankton and may at least partially determine how the principle of homeoviscosity might be extended to marine phytoplankton. There are at least 2 lines of reasoning, however, to suggest that the principle of homeoviscous membranes may not generally apply to marine phytoplankton. First, the fatty acids of marine phytoplankton frequently are considerably more unsaturated than terrestrial plants living in far colder environments. Second, although the rate of membrane adaptation is reasonably rapid (Kasai et al. 1976, Lynch and Thompson 1982) it is anticipated that many marine phytoplankters could be broadly eurythermal in "average" physiological conditions. Any physiological adaptation(s) that would render a cell incapable of functioning over a broad 86 range of temperatures might be potentially detrimental in many marine environments due to episodic mixing. In this study, the range of temperatures and the number of species examined was expanded over previous studies in an effort to determine whether these observations of a change in the ratio of unsaturated to saturated F A s are representative of a general pattern for marine phytoplankton. Eight species of marine phytoplankters commonly used in aquaculture and representing 3 taxonomic classes were grown at saturating photon flux densities and varied temperatures from 10 to 25 °C. Variation in the percent composition of specific fatty acids were compared with variation in other biochemical/physiological parameters for associations that may give some insights into the role of fatty acids in algal physiology. The usefulness of temperature to manipulate fatty acid composition in order to maximize the percentage E F A s and maintain membranes in a homeoviscous state was assessed. 87 MATERIALS AND METHODS Five marine diatoms, Chaetoceros calcitrans (Paulsen) Tokano (NEPCC# 590, Cc), Thalassiosira pseudonana (Hustedt) Hasle and Heimdal, clone 3H (NEPCC# 58, Tp), Chaetoceros simplex Ostenfeld (NEPCC# 591, Cs), Chaetoceros gracilis Schutt (NEPCC# 645, Cg), and Phaeodactylum tricornutum Bohlin (NEPCC# 640, Pt), and 3 flagellates, Dunaliella tertiolectar Butcher (NEPCC# 1, Dt), Pavlova lutheri Droop (NEPCC# 5, PI) and Isochrysis aff. galbana (Green, clone T-iso, termed Tahitian Isochrysis) (NEPCC# 601, Ig) were obtained from the Northeast Pacific Culture Collection, Department of Oceanography, University of British Columbia. All cultures were grown in enriched artificial seawater (ESAW) based on the recipe by Harrison et al. (1980). The medium was modified as in Chapter 1. Cultures were kept in exponential phase and preconditioned for a minimum of 8 generations to the specific temperatures in 30 mL of medium in 50 mL borosilicate glass test tubes with teflon-lined caps. All cultures were grown at =220 umol photons m-2 s-1 (Li-CorR model LI 185 meter, 2* collector). Cultures were grown at 10, 15, 17.5 20, and 25 ± 0.5 °C. Duplicate cultures of C. calcitrans, and T. pseudonana were grown at 10 °C. Continuous light was provided by Vita-lite^ fluorescent tubes and attenuated by distance and/or neutral density screening. The 30 mL cultures were used to inoculate 3 L cultures (in 6 L round flat-bottom flasks) providing at least 4 additional generations under the defined experimental conditions prior to harvest. The 3 L cultures were stirred at 88 60 rpm with a 7.6 cm teflon-coated magnetic bar and bubbled with a mixture of approximately 2% C02 and air. The pH of most cultures was maintained at 8.2. No lag phase was observed following dilution, and all cultures were harvested at mid-exponential phase to ensure nutrient limitation did not occur. For growth rate calculations, biomass was measured as in vivo fluorescence using a Turner Designs^ Model 10 fluorometer and/or cell counts utilizing a Coulter CounterR model TAII equipped with a population accessory. Measurements were made once or twice per day. Growth rates were calculated as: [i = ln(Fi/Fo)/(ti-to) Where F l = biomass at time 1 (tl) and Fo = biomass at time 0 (to). Chlorophyll a was measured on 25 mL subsamples filtered through Whatman GF/F filters. Filters were stored frozen and desiccated at -20°C. The filters were immersed in 90% acetone, sonicated for 5 min and extracted for 24 h in the dark at 4° C. Chlorophyll a concentrations were calculated from in vitro fluorescence (Parsons et al. 1984). Particulate organic carbon and nitrogen (POC and PON) subsamples were collected on 13 mm Gelman A/E filters and were analyzed on a Carlo Erba CNS analyzer. Subsamples for total lipid were analyzed by the potassium dichromate technique of Parsons et al. (1984) using tripalmitin as a standard. The total lipid fraction also contained most chl a. 89 Subsamples for fatty acid determinations were collected on precombusted GF/F filters, placed inside a petri dish and sealed in plastic bags filled with nitrogen. Prior to analysis they were frozen at -20 °C for periods of less than 3 weeks, or for longer periods at -80 °C. Samples were saponified and methylated as in Whyte (1988). Intra- and inter-sample duplicates were prepared and analyzed. FAs were analyzed on a Hewlett-Packard 5890A gas liquid chromatograph fitted with a Supelcowax 10 fused silica capillary column (30 m x 0.32 mm E D , 0.25 um film) and identified by comparison with saturated and PUFA-1 methyl ester standards (obtained from Supelco Inc.) in accord with Ackman (1986). The shorthand notation used in fatty acid identification is L:Bo)X where L is the chain length, B is the number of double bonds, and coX is the position of the double bond closest to the terminal methyl group. Unknowns were deleted if they did not occur more than once. Fatty acids that were < 0.3% of total fatty acids were also deleted. The data presented in Chapter 3 include the 225 umol photons m'2 s'l , 17.5 °C data from Chapter 2. Inclusion of these data here allows the comparison of 5 distinct temperature regimes. Most biochemical procedures were identical in both chapters, except the determination of total lipid. In Chapter 3, total lipid was determined by the potassium dichromate technique (Parsons et al. 1984), whereas in Chapter 2 the lipid charring technique (Marsh and Weinstein 1966) was used. The potassium dichromate method was used in latter studies because it proved to be more precise (unpubl. data). A large number of correlation and regression analyses were made. The Bonferroni-adjusted probabilities were not calculated. 90 R E S U L T S Growth Semi-continuous cultures were grown under a range of temperatures from 10 to 25 °C. Although the growth rates of individual cultures were occasionally variable, over this range of temperatures, growth rates (u.) for all species were well described as a linear function of temperature (Fig. 3.1). Growth rates increased from a minimum of 0.29 d-1 for D. tertiolecta at 10 °C to 3.4 d"l for C. calcitrans at 25 °C. Qios for growth rates ranged from 2.1 for C. gracilis to 4.7 forD. tertiolecta. The mean ± 1 S.D. of Qios averaged over all 8 species was 3.1 ± 1.0. General biochemical parameters Due to the change in techniques for determining the total lipid, the 17.5 °C data (i.e. from Chapter 2) were eliminated from the comparisons of lipid quota with temperature that follow. The lipid per cell showed no significant linear relationships with temperature or growth rate (Table 3.1) although some species exhibited weak trends towards less lipid per cell with increasing temperature (statistically insignificant). The percentage of carbon stored as lipid also varied with temperature, generally decreasing with increasing temperature but again showed no significant correlations (Fig. 3.1). The average percentage of carbon stored as lipid (=[pg lipid cell-l/pg carbon cell-l]*100) over all species was 30%. 91 Fig. 3.1. Growth rates as a function of temperature for 8 species of marine phytoplankton. Least squares multiple regressions (solid lines) and 95% confidence intervals (dotted lines) are shown fitted to the data. Q i o s were calculated from the least squares linear regression of growth rate as a function of temperature. 92 93 Table 3.1 A. Biochemical and summary fatty acid compositional data for two marine phytoplankters grown at various temperatures and saturating light (=220 umol photons m"2 s"1). Correlations with: growth rate, *P<0.05, **P<0.01; temperature, •P<005, ••P<0.01; chl a, AP<0.05, AAP<0.01, na=not available. Chaetoceros calcitrans Thalassiosira pseudonana Temperature (°C) 10.0 10.0 15.0 17.5 20.0 25.0 10.0 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.94 0.97 1.87 2.00 1.52 3.39 0.69 0.69 1.25 1.84 1.71 2.35 108 cells L-l 11.3 11.4 11 3.1 5.9 4.1.0 6.9 6.8 7.1 6.2 5.7 3.6 carbon cell-l (pg) 6.8 6.6 7.1 5.9 7.1 7.8 11.9 11.5 9.8 12.5 11.2 16.8 nitrogen cell-l (pg) 0.84 0.82 0.86 1.18 0.87 1.11 2.06 1.87 1.52 1.16 1.43 2.03 protein cell-l (pg) 3.8 3.9 3.5 7.7 4.4 2.6 9.4 9.4 6.1 8.4 5.4 10.6 lipid cell-l (pg) 1.2 1.2 1.0 2.7 2.6 1.8 3.1 3.2 2.0 3.9 2.4 3.4 carbohydrate cell-l(pg) 1.1 1.0 3.5 na na 1.7 2.2 2.0 2.2 na 2.3 5.3 chl a cell-l (fg) 96 100 113 60 221 331 152 150 110 62 174 418 cell size (um^ ) 37.3 36.3 40.6 29.5 47.0 42.0 49.7 49.9 41.5 48.6 46.2 70.4 Fatty acid summary saturated (%) 33.0 29.8 34.0 30.3 38.0 31.4 25.6 23.7 33.8 37.4 44.0 39.6*4 mono-unsaturated (%) 26.6 25.7 26.1 26.8 31.0 26.8 26.8 25.5 27.7 22.0 33.5 26.1 duo-unsaturated (%) 6.6 6.7 5.9 4.7 5.9 6.8 5.3 5.4 3.4 2.9 3.1 3.8 poly-unsaturated (%) 31.4 34.5 32.9 35.5 23.1 31.9 37.3 40.3 32.5 33.9 15.7 27.3 unsatVsaturated 2.0 2.3 1.9 2.2 1.6 2.1 2.7 3.0 1.9 1.6 1.2 1.4*4 weighted unsat. ratio 2.8 2.8 2.8. 2.9 2.4 2.7 3.0 3.1 2.8 2.7 2.0 2.50 mean chain length 16.3 15.8 16.2 16.1 15.7 15.8 15.9 15.9 16.4 16.1 15.3 15.8 HUFAco3(%) 18.8 18.1 21.0 23.4 14.4 17.4 24.0 25.7 23.5 25.5 9.9 19.5 to3 (%) 18.8 18.1 21.0 23.4 14.4 17.4 24.0 25.7 23.5 25.5 9.9 19.5 0)6 (%) 0.7 2.9 0.2 2.1 2.1 0.0 1.2 1.3 0.0 1.2 2.0 1.3 (o3/oo6 27 6.3 138 11.0 6.9 oo 20 21 oo 21 4.9 15 Table 3. IB. Biochemical and summary fatty acid compositional data for two marine phytoplankters grown at various temperatures and saturating light (=220 umol photons m" 2 s"l). Correlations with: growth rate, *P20.05, **P<0.01; temperature, •P<0.05, ••P<0.01; chl a, AP£0.05, AAP<0.01, na=not available. Chaetoceros gracilis Chaetoceros simplex Temperature (°C) 10.0 15.0 17.5 20.0 25.0 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.74 1.10 1.54 1.67 1.01 0.65 1.19 1.95 1.33 2.23 108 cells L-l 6.0 10.5 4.4 4.1 7.7 5.7 6.4 2.5 6.3 4.9 carbon cell-1 (pg) 4.80 8.79 7.00 7.66 6.77 10.5 7.79 15.1 9.72 8.17 nitrogen cell-1 (pg) 0.70 1.20 1.29 1.26 0.96 1.76 1.18 2.28 1.46 1.31 protein cell-1 (pg) 3.8 3.6 5.0 2.7 4.1 7.6 5.3 11.0 4.5 3.5 lipid cell-1 (pg) 3.2 2.6 3.3 1.8 1.8 4.6 2.5 3.5 1.9 1.7 carbohydrate cell-1 (pg) 1.8 0.9 na 1.1 0.8 1.1 0.7 na 1.0 0.8 chl a celM (fg) 111 86 50 194 173 106 163 85 201 238 cell size (um3) 44.0 52.0 49.5 44.8 46.9 38.9 83.7 67.4 53.8 76.0 Fatty acid summary saturated 40.2 49.2 35.6 43.5 44.4 35.8 40.3 43.0 48.7 45.0 mono-unsaturated (%) 37.4 36.7 32.0 38.2 37.9 29.8 24.8 28.7 29.6 25.3 duo-unsaturated (%) 3.4 4.4 5.5 4.3 3.8 3.8 10.2 4.7 8.2 7.1 poly-unsaturated (%) 13.2 7.5 24.3 12.1 12.0 27.3 20.9 14.5 9.3 17.8 unsatd./8aturated 1.3 1.0 1.7 1.3 1.2 1.7. 1.4 1.1 1.0 1.1 weighted unsat. ratio 1.8 1.7 2.4 1.9 1.9 2.5 1.9 1.9 1.8 2.1 mean chain length 14.5 15.2 15.6 15.4 15.4 16.0 14.0 14.3 14.7 14.8 HUFA 0)3 (%) 7.9 3.8 14.8 6.9 6.9 18.4 9.9 8.0 2.7 9.2 co3 (%) 7.9 3.8 14.8 6.9 6.9 18.4 10.2. 8.0 2.7 9.2 0)6 (%) 3.8 0.9 2.8 0.9 1.6 0.0 7.3 0.9 1.0 1.1 o)3/o)6 2.1 4.4 5.3 7.4 4.4* oo 1.4 8.7 2.8 8.1 Table 3.1C. Biochemical and summary fatty acid compositional data for two marine phytoplankters grown at various temperatures and saturating light (=220 umol photons m" 2 s'l). Correlations with: growth rate, *PS0.05, **P<0.01; temperature, 4P<0.05, ••P<0.01; chl a, AP<0.05, AAP<0.01, na=not available. Phaeodactylum tricornutum Dunaliella tertiolecta Temperature (°C) 10.0 15.0 17.5 20.0 25.0 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.67 1.37 1.22 1.47 1.56 0.29 0.77 1.41 1.14 1.41 108 cells L ' 1 4.9 5.3 2.0 2.9 2.5 1.0 1.4 2.1 1.7 0.81 carbon cell-1 (pg) 13.3 10.8 10.9 11.1 15.5 43.3 49.2 50.0 35.7 53.1 nitrogen cell-1 (pg) 2.14 1.73 2.13 1.99 2.64 8.62 8.48 8.06 5.69 9.72 protein cell-1 (pg) 10.2 7.2 9.3 5.1 9.1 30.7 22.2 51.7 43.4 66.3 lipid cell-1 (pg) 2.7 2.1 2.9 2.6 4.3 11.2 11.5 14.4 9.99 19.8 carbohydrate cell-1 (pg) 2.9 2.9 na 1.4 2.5 17.4 35.5 na 23.2 46.4 chl a cell-1 (fg) 132 92.1 31.5 152 232 941 1081 527 918 2408 cell size (urn3) 30.2 37.6 51.3 56.0 73.0 224 179 216 209 195 Fatty acid summary saturated 27.6 28.5 27.7 29.6 35.0 21.6 25.8 22.3 22.7 18.5**4 mono-unsaturated (%) 27.6 28.1 38.8 30.3 40.0 15.7 13.8 10.2 10.9 8.4 duo-unsaturated (%) 7.4 6.1 5.1 6.2 5.9 5.1 10.5 10.6 10.2 11.7 poly-unsaturated (%) 34.6 35.1 24.9 32.0 17.6 36.9 29.6 31.3 30.6 28.5 unsatVsaturated 2.5 2.4 2.5 2.3 1.84 2.7 2.1 2.3 2.3 2.6 weighted unsat. ratio 3.1 3.1 2.7 3.0 2.3 2.9 2.6 2.7 2.7 2.6 mean chain length 15.8 16.0 15.9 16.0 16.1 15.9 15.8 15.5 15.3 14.1 HUFAo)3(%) 27.4 28.6 20.1 25.8 12.5 14.5 9.5 11.9 11.5 13.3 0)3 (%) 0.6 1.2 1.0 0.7 0.5 0.2 0.0 0.0 0.0 0.0 0)6 (%) 0.9 1.3 1.3 0.9 0.6* 5.8 12 14 13 15* co3/o)6 0.6 0.9 0.8 0.8 0.9* 0.0 0.0 0.0 0.0 0.0 Table 3. ID. Biochemical and summary fatty acid compositional data for two marine phytoplankters grown at various temperatures and saturating light (=220 umol photons m" 2 s"1). Correlations with: growth rate, *P£0.05, **P£0.01; temperature, •P<0.05, ••PfiO.Ol; chl a, AP<0.05, AAP<0.01, na=not available. Pavlova lutheri Isochrysis galbana (T-iso) Temperature (°C) 10.0 15.0 17.5 20.0 25.0 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.62 1.09 0.56 1.79 2.36 0.63 1.33 0.43 0.84 1.38 108 cells L-l 6.5 7.2 5.2 7.5 7.1 7.1 8.0 5.6 6.8 7.0 carbon cell-l (pg) 10.9 10.3 12.3 11.6 16.9 10.0 6.8 10.0 11.2 10.1 nitrogen cell-l (pg) 1.80 1.52 1.45 1.51 1.87 1.66 0.89 1.18 1.40 1.30 protein cell-l (pg) 8.6 6.7 9.8 7.5 9.7 6.0 1.9 7.4 6.4 7.0 lipid cell-l (pg) 2.3 2.0 7.3 3.3 3.2 18.9 1.1 2.6 4.3 2.6 carbohydrate cell-l (pg) 0.9 4.8 na 2.7 4.6 1.1 1.2 na 4.5 3.0 chl a celM (fe) cell size ([im'j 136 124 34 176 347 80 119 33 150 231 33.9 38.6 64.8 47.8 44.5 38.8 42.5 50.0 47.4 40.9 Fatty acid summary saturated (%) 28.3 33.7 30.0 44.0 41.2* 32.0 49.2 29.3 31.4 35.7 mono-unsaturated (%) 23.6 28.3 26.2 28.7 25.9 27.0 36.7 29.2 24.8 26.2 duo-unsaturated (%) 3.9 3.1 4.4 3.4 4.1 3.8 4.4 6.9 7.4 7.0 poly-unsaturated (%) 34.0 32.4 34.7 19.8 24.3* 19.5 7.5 31.5 30.4 25.7 unsat./saturated 2.2 1.9 2.2 1.2 1.3* 1.6 1.0 2.3 2.0 1.7 weighted unsat. ratio 2.8 2.8 3.1 2.2 2.4 2.7 1.7 3.0 2.8 2.5 mean chain length 14.4 15.4 14.5 15.3 15.3 15.1 15.3 14.2 14.3 14.5 HUFA co3 (%) 22.4 23.5 33.4 14.2 17.4 20.0 3.8 24.6 22.8 19.2 o>3(%) 2.7 0.2 0.7 0.0 0.0 20.0 3.8 28.3 27.4 22.8 0)6 (%) 1.7 0.0 4.1 0.6 0.5 2.7 0.9 9.7 10.2 7.2 0)3/0)6 1.6 oo 0.2 0.0 0.0 7.3 4.4 2.9 2.7 3.2 Protein and carbohydrate cell" 1 showed no significant trends with temperature or growth rate. Carbon cell-l showed some tendency to occasionally increase at higher temperatures (and/or higher growth rates), particularily in T. pseudonana, P. tricornutum and P. lutheri (Table 3.1). Trends in nitrogen cell-l were similar (Table 3.1). For two species, T. pseudonana and P. lutheri, the C:N ratio increased significantly with increasing temperatures (Fig. 3.2). The chlorophyD a quotas from some samples in Chapter 2 appeared low in relation to the data collected more recently for this study. For this reason, all of the 17.5 °C data were eliminated from the comparisons of chlorophyll a quota with carbon quota and chlorophyll a quota with temperature that follow. In all species, chl a cell-l was greatest at the highest temperature (Fig. 3.3). Similarily the carbon:chl a ratio was lowest at the highest temperature (Fig. 3.4). Fatty acids Correlation and regression analyses were used to examine the data for trends and possible relationships of fatty acids with temperature, u, and chl a-cell-1. General trends in fatty acid composition were examined by partitioning the FAs into the following categories: % saturated, % mono-unsaturated, % di-unsaturated, % poly-unsaturated, and the ratio of unsaturated:saturated FAs (Table 3.1). 98 Fig. 3.2. The carbon/nitrogen ratio (by atoms) versus temperature for C. calcitrans (O), T. pseudonana (•), C. gracilis (V), C. simplex (Y), P. tricornutum (•), D. tertiolecta (•), P. lutheri (A), / . aff. galbana (T-iso) (A). 99 - [ - . , I 1 C. calcitrans ' 1 T. pseudonana i i 1 i • i C. gracilis • 1 —• 1 1 1 ' 1 C. simplex \ / • • \ , / . . • P i . i i - V 1 , 1 . 1 7 • I i . i . i i 10 15 20 25 10 15 20 25 10 15 20 25 10 15 20 25 Temperature (°C) I — ' — I — ' - I—'• p -P. tricornutum i" • r • • i • i D. tertiolecta , - , i i i i i P. lutheri I . ] . | . . 1. galbana (T-iso) -" / A / " / I I I • I I I i / . I . I . I i . i . i . i 10 15 20 25 10 15 20 25 10 15 20 25 10 15 20 25 Temperature (°C) 100 Fig. 3.3. Chlorophyll a quota (fg cell"1) versus temperature for C. calcitrans (O), T. pseudonana (•), C. gracilis (V), C. simplex (T), P. tricornutum (•), D. tertiolecta (•), P. lutheri (A), /. aff. galbana (T-iso) (A). 101 2000 1000. 500 CD U O) 3 o Z3 cr o . p 400 300 200 o 6 100 10 15 20 25 Temperature (°C) 30 102 Fig. 3.4. The carbonrchlorophyll a ratio versus temperature for C. calcitrans (O), T. pseudonana (•), C. gracilis (V), C. simplex (T), P. tricornutum (•), Z>. tertiolecta (•), P. lutheri (A), /. aff. galbana (T-iso) (JO. 103 160 r-140 -5 10 15 20 25 30 Temperature (°C) 104 i) Temperature T. pseudonana was the only species to show significant linear variation in the percentage of saturated FAs as a function of temperature. In T. pseudonana, there were significant increases in 14:0 and a trend towards more 16:0 with increasing temperature. D. tertiolecta was the only species to have a significant linear relationship between the percentage of all mono-unsaturated FAs and temperature (Table 3.1). In this species 18:lco7 was shown to decrease significantly with increasing temperature, and 18:3co3 showed a strong, similar trend. None of the species studied had a significant relationship between their relative content of polyunsaturated FAs and temperature. The ratio of unsaturated/saturated FAs versus temperature was again a significant linear regression for only T. pseudonana. With only 1 of 8 species showing this trend, there was no evidence of a general relationship between this measure of unsaturation and temperature. Other methods of increasing membrane fluidity include adding more double bonds or decreasing mean chain length (Hochachka and Somero 1984). A weighted calculation of the degree of unsaturation (=WUnSat) was made, where the fraction of each F A was multiplied by 1+the number of double bonds (#DB), and summed over the entire FA profile: WUnSat=Z{%FA/100*(l+#DB)} This approach yielded no significant correlations of WUnsat with temperature or growth rate for any species or over all species (Table 3.1), although trends were similar to those for the simpler unsat./saturated ratio. 105 An index of mean chain length (MCL) was calculated as the fraction of each F A multiplied by the number of carbon atoms. This approach showed only D. tertiolecta to significantly vary mean chain length in response to temperature and the result for this species was contrary to expectations. The mean chain length decreased from 15.9 to 14.1 with temperature increasing from 10 to 25 °C (Table 3.1C). No other species had a significant trend in M C L with either temperature or growth rate. The mean chain length was remarkably consistent, over all species and temperatures; the mean ± 1 S.E. was 15.33 ± 0.50. Variation in the percent composition of specific fatty acids as a function of temperature include: 10:0 (Dt), 14:0 (Tp,Pt), 15:0 (Cc.Tp), 16:lco9 (Cc,Pl), 16:40)1 (Cc,Tp,Cs), 18:lco7 (Dt), 18:2o9 (Dt), 18:2co6 (Dt), 18:4o)3 (Dt) and 22:6u)3 (Tp) (Table 3.2). Of these FAs, 16:4col consistently declined with increasing temperature. For each species, the percentage of 16:4col was less at 25°C than at 10°C. This trend was significantly different from random (chi squared, P<0.05). The percentages of 16:4o)l ranged from 0 to 8.4%. D. tertiolecta was the only species to show a significant linear response in the percent composition of any 18 carbon FA as a function of temperature. 106 Table 3.2A. The fatty acid composition of Chaetoceros calcitrans grown at five different temperatures at saturating PFDs (=220 umol photons m"l s*l). Duplicate cultures were grown at 10 °C, mean and 1 S.D. for percent fatty acid composition are given. Regressions of percent composition with: growth rate, *P£0.05, **P^0.01; temperature, •P<0.05, ••P^O.01; correlations with chl a cell-l, AP<0.05, AAP<0.01 (excluding 17.5 °C data). See text for details. Temperature (°C) 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.96±0.03 1.87 2.00 1.52 3.39 Fatty acid mean SD 14:0 16.2 0.52 20.9 16.8 23.3 19.0 14:1(05 0.3 0.04 0.1 0.4 0.4 0.3 15:0 1.0 0.06 0.9 0.8 0.8 0.6**4J Pristanic 0.0 0.00 0.0 0.3 0.4 0.44 16:0 10.4 1.01 10.1 9.6 12.0 9.9 16:lco9 0.5 0.06 0.2 1.0 1.4 1.44 16:10)7 23.8 0.68 24.3 20.1 26.6 23.7 16:lco5 0.7 0.05 0.8 0.7 0.9 0.8 tl6:lcol3 0.0 0.00 0.0 0.0 0.0 0.0 17:0 iso 1.5 0.02 1.9 1.5 1.7 1.5 16:2co7 2.9 0.01 2.9 1.8 2.2 3.3 16:2co4 3.4 0.05 2.9 2.3 2.5 3.5 16:3u)4 4.9 0.18 5.9 6.1 5.4 13.4**A unknown#l 0.7 0.02 0.1 0.8 0.3 0.6 unknown#2 0.0 0.0 0.0 0.0 0.0 0.0 16:4col 7.8 0.52 5.8 4.6 2.1 1.14A 18:0 0.1 0.10 0.2 0.4 0.2 0.2 18:lco9 0.5 0.07 0.2 0.7 0.4 0.2 18:lco7 0.0 0.00 0.5 3.6 0.7 0.5 18:1(05 0.0 0.00 0.0 0.0 0.0 0.0 18:2co6 0.0 0.00 0.2 0.3 0.3 0.0 18:40)3 2.7 0.17 3.0 4.8 3.0 1.9 18:4col 0.0 0.00 0.0 0.0 0.0 0.0 20:2co6 0.2 0.20 0.0 0.3 0.8 0.0 20:40)3 0.3 0.03 0.3 0.8 0.2 0.0 20:5co3 14.1 0.63 16.5 15.7 10.5 14.6 unknown#3 0.0 0.00 0.0 0.0 0.0 0.0 22:4co6 1.2 0.76 0.0 1.2 1.0 0.0 22:50)6 0.2 0.19 0.0 0.0 0.0 0.0 22:4o)3 0.2 0.20 0.0 0.6 0.0 0.0 22:5co3 0.0 0.00 0.0 0.0 0.0 0.0 unknown#4 0.1 0.11 0.0 0.0 0.0 0.2 22:6co3 1.2 0.10 1.3 1.5 0.7 0.9 unknown#5 0.2 0.23 0.0 0.0 0.3 0.0 107 Table 3.2B. The fatty acid composition of Thalassiosira pseudonana grown at five different temperatures and saturating PFDs (=220 umol photons m " 2 s-1). Duplicate cultures were grown at 10 °C, mean and 1 S.D. for percent fatty acid composition are given. Regressions of percent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P£0 .05 , ••P<0.01; correlations with chl a celM, AP£0.05, AAP<0.01 (excluding 17.5 °C data). See text for details. Temperature (°C) Growth rate (d-1) 10.0 0.69±0.00 15.0 1.25 17.5 1.84 20.0 1.71 25.0 2.35 Fatty acid mean SD 14:0 3.7 0.06 5.5 4.9 9.1 11.3*4 14:lco5 0.3 0.12 0.0 0.2 0.2 0.3 15:0 1.0 0.01 0.9 0.8 0.9 0.7**4A Pristanic 0.2 0.01 0.0 0.3 0.0 0.2 16:0 19.7 0.87 27.1 30.7 33.3 27.3 16:lo)9 0.7 0.09 0.3 0.8 0.5 1.0 16:lco7 23.8 0.60 25.7 20.5 30.7 23.2 16:10)5 0.0 0.00 0.0 0.0 0.0 0.0 tl6:lo)13 1.0 0.01 0.7 0.0 0.8 0.9 17:0 iso 0.0 0.00 0.0 0.7 0.0 0.0 16:2co7 1.9 0.05 1.2 1.0 0.8 1.1* 16:2co4 3.0 0.09 2.1 1.7 1.8 2.4 16:3o)4 4.5 0.11 5.1 4.6 3.5 6.5 unknown#l 1.3 0.27 0.0 0.8 0.0 0.3 unknown#2 0.0 0.00 0.0 0.0 0.0 0.0 16:4col 7.9 0.31 3.9 2.9 0.8 0 3**4+ 18:0 0.0 0.00 0.4 0.3 0.4 0.3 18:lco9 0.3 0.29 0.4 0.2 0.4 0.3 18:lto7 0.0 0.00 0.6 0.4 0.5 0.4 18:10)5 0.0 0.00 0.0 0.0 0.0 0.0 18:2o)6 0.1 0.10 0.0 0.3 0.2 0.3 18:4o)3 3.8 0.17 5.0 8.2 3.6 5.0 18:40)1 0.5 0.04 0.0 0.0 0.0 0.0*4 20:2w6 0.0 0.00 0.0 0.0 0.2 0.0 20:4co3 0.0 0.00 0.2 0.3 0.0 0.3 20:5co3 15.4 0.70 13.6 13.7 5.1 11.9 unknown#3 1.1 0.14 0.0 0.9 1.2 1.0 22:4co6 0.0 0.00 0.0 0.0 0.0 0.0 22:5co6 0.0 0.00 0.0 0.0 0.4 0.0 22:4co3 0.0 0.00 0.0 0.0 0.0 0.0 22:5co3 0.1 0.14 0.0 0.0 0.0 0.0 unknown#4 0.3 0.13 0.0 0.0 0.3 0.0 22:60)3 5.5 0.14 4.7 3.2 1.1 2.3*4 unknown#5 0.0 0.00 0.0 0.0 0.4 0.0 108 Table 3.2C. The fatty acid composition of Chaetoceros gracilis grown at five different temperatures and saturating PFDs (=220 umol photons m " 2 s"1). Regressions of precent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P^0.05, ••P<0.01; correlations with chl a cell" 1, AP<0.05, AAP^O.01 (excluding 17.5 °C). See text for details. Temperature (°C) Growth rate (d-1) 10.0 0.74 15.0 1.10 17.5 1.54 20.0 1.67 25.0 1.01 Fatty acid 14:0 16.3 29.4 16.0 21.3 17.8 14:10)5 0.4 0.2 0.1 0.3 0.6 15:0 0.6 1.0 0.4 0.5 0.4 Pristanic 0.0 0.0 0.0 0.0 0.0 16:0 22.1 15.4 18.4 21.2 25.4 16:lo)9 0.4 0.5 0.2 1.3 0.4 16:lco7 31.3 32.0 30.3 34.2 35.1 16:lo)5 0.3 1.2 0.3 0.4 0.3 tl6:lo)13 0.0 0.0 0.0 0.0 0.0 17:0 iso 0.8 2.7 0.4 0.4 0.0 16:2co7 0.4 1.4 0.9 0.7 1.0 16:2co4 1.9 2.6 2.8 2.9 2.2* 16:3co4 1.0 1.9 4.1 3.6 3.7 unknown* 1 0.0 0.0 0.1 0.0 0.0 unknown#2 0.7 0.0 0.0 0.0 0.0 16:4col 1.8 1.3 3.6 1.3 0.4 18:0 0.4 0.6 0.4 0.0 0.5 18:lco9 0.6 0.4 0.5 0.6 0.5 18:lco7 2.8 1.7 0.7 0.8 1.0 18:10)5 0.3 0.7 0.0 0.2 0.0 18:2co6 0.7 0.3 0.8 0.7 0.6 18:40)3 0.6 0.9 1.4 0.7 0.6 18:4o)l 0.0 0.0 0.0 0.0 0.0 20:2co6 0.4 0.0 0.5 0.0 0.0 20:4u)3 0.0 0.0 0.3 0.0 0.0 20:5co3 6.3 2.8 11.6 5.8 5.3 unknown#3 2.3 0.6 1.1 0.2 1.0 22:4co6 0.0 0.0 0.0 0.0 0.0 22:50)6 0.0 0.0 0.2 0.0 0.0 22:40)3 0.0 0.0 0.0 0.0 0.0 22:5to3 0.0 0.0 0.0 0.0 0.0 unknown#4 0.0 0.0 0.0 0.2 0.2 22:6o)3 0.0 0.0 1.5 0.5 0.6 unknown#5 0.7 0.0 0.0 0.0 0.0 109 Table 3.2D. The fatty acid composition of Chaetoceros simplex grown at five different temperatures and saturating PFDs (=220 umol photons n r 2 s"1). Regressions of percent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P<0.05, ••P<0.01; correlations with chl a cell - 1 , AP<0.05, AAP^O.01 (excluding 17.5 °C). See text for details. Temperature (°C) 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.65 1.19 1.95 1.33 2.23 Fatty acid 14:0 4.5 27.7 31.1 35.4 33.0 14:lco5 0.0 0.3 0.4 0.3 0.2 15:0 1.0 0.6 0.6 1.0 0.5 Pristanic 0.0 0.0 0.4 0.0 0.0 16:0 29.2 10.2 9.5 10.6 8.4 16:lco9 0.0 0.3 1.0 1.4 0.5 16:lco7 28.5 22.2 24.9 23.3 20.4 16:lco5 0.0 0.8 1.2 1.4 0.7 tl6:lcol3 0.0 0.0 0.0 0.0 0.0 17:0 iso 0.6 1.1 1.5 1.5 1.1 16:2co7 1.6 3.0 3.5 3.8 3.1 16:2co4 2.2 2.4 3.0 3.4 3.2 16:3CD4 3.5 4.7 7.2 5.5 7.7**A unknown#l 0.0 0.0 0.0 0.0 0.0 unknown#2 0.3 0.7 0.0 0.5 0.2 16:4col 5.1 3.6 3.5 1.1 0.64A 18:0 0.2 0.0 0.2 0.3 0.3 18:lco9 0.2 0.2 0.3 0.3 0.3* 18:lco7 0.8 0.7 0.6 1.9 1.6 18:lco5 0.0 0.2 0.2 0.5 0.54 18:2o)6 0.0 0.2 0.2 0.3 0.3 18:40)3 3.7 0.4 0.6 0.2 0.3 18:4col 0.0 0.0 0.0 0.0 0.0 20:2co6 0.0 4.6 0.7 0.7 0.6 20:4o)3 0.0 0.2 0.2 0.0 0.0 20:5co3 10.4 3.7 6.1 2.0 6.8 unknown#3 0.3 4.4 0.0 0.0 0.9 22:4co6 0.0 0.0 0.0 0.0 0.0 22:5co6 0.0 0.0 0.0 0.0 0.0 22:40)3 0.0 0.0 0.0 0.0 1.7 22:5co3 0.0 0.4 0.0 0.0 0.0 unknown#4 0.2 0.5 0.0 0.0 0.0 22:6co3 4.0 0.7 1.1 0.5 1.2 unknown#5 0.3 0.0 0.0 0.4 0.8 110 Table 3.2E. The fatty acid composition of Phaeodactylum tricornutum grown at five different temperatures and saturating PFDs (=220 umol photons m"2 s-1). Regressions of percent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P^O.05, ••P^O.01; correlations with chl a cell-1, AP<0.05, AAP^O.01 (excluding 17.5 °C data). See text for details. Temperature (°C) 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.67 1.37 1.22 1.47 1.56 Fatty acid 10:0 0.0 0.0 0.0 0.0 0.0 12:0 0.0 0.0 0.0 0.0 0.0 14:0 8.4 7.2 4.8 6.0 4.3^ 14:lco5 0.0 0.0 0.0 0.0 0.0 15:0 0.5 0.4 0.3 0.4 0.4 Pristanic 0.0 0.0 0.2 0.0 0.0 16:0 15.5 17.2 19.7 19.9 25.94 16:lco9 0.0 0.0 0.6 0.7 0.4 16:lco7 26.0 26.4 34.2 27.2 37.1 16:10)5 0.2 0.3 0.1 0.4 0.5 17:0 iso 0.9 0.9 0.5 0.9 0.4 16:2o)7 1.5 1.3 1.4 1.7 1.8A 17:0 anteiso 0.0 0.0 0.0 0.0 0.0 16:2o)4 5.2 4.0 2.5 3.7 3.7 phytanic 0.0 0.0 0.0 0.0 0.0 16:3co4 1.0 0.9 1.9 1.6 2.9A unknown#l 0.0 0.0 0.0 0.0 0.0 unknown#2 0.2 0.4 0.5 0.7 0.3 16:4o)3 0.0 0.0 0.0 0.0 0.0 16:4col 5.9 5.2 2.5 4.7 2.0 18:0 0.4 0.5 0.4 0.8 0.9 18:10)9 0.3 0.5 1.8 0.7 0.4AA 18:10)7 0.6 0.4 1.3 0.8 1.3 18:lco5 0.0 0.0 0.3 0.0 0.2 18:2co9 0.0 0.0 0.2 0.0 0.0 18:20)6 0.7 0.8 0.9 0.9 0.3 18:3co6 0.0 0.0 0.0 0.0 0.0 18:3co3 0.0 0.0 0.0 0.0 0.0 18:4o)3 0.6 0.6 0.5 0.7 0.4 18:5co3 0.0 0.0 0.0 0.0 0.0 20:4co6 0.2 0.3 0.3 0.3 0.0 20:50)3 22.1 23.3 16.2 20.9 10.1 A 22:0 0.0 0.0 0.4 0.0 0.3 21:5u)3 0.0 0.0 0.0 0.0 0.0 unknown#3 0.0 0.4 0.2 0.0 0.3 22:4co6 0.0 0.0 0.0 0.0 0.0 22:5co6 0.0 0.0 0.0 0.0 0.0 22:50)3 0.4 0.4 0.2 0.4 0.3 unknown#4 2.0 2.3 1.7 1.8 1.8 22:6co3 4.1 3.9 2.9 3.5 1.8A unknown#5 0.4 0.6 0.0 0.6 0.0 111 Table 3.2F. The fatty acid composition of Dunaliella tertiolecta grown at five different temperatures and saturating PFDs (=220 umol photons m " 2 s"1). Regressions of percent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P<0.05, ••P<0.01; correlations with chl a cell"1, AP<0.05, AAP^O.01 (excluding 17.5 °C data). See text for details. Temperature (°C) 10.0 15.0 17.5 20.0 25.0 Growth rate (d-1) 0.29 0.77 1.41 1.14 1.41 Fatty acid 10:0 0.0 0.3 0.3 0.5 0.54 12:0 0.0 0.0 0.0 0.0 0.0 14:0 0.6 0.2 0.2 0.0 0.3 14:lco5 0.4 0.3 0.0 0.6 0.7 15:0 0.0 0.0 0.0 0.0 0.0 Pristanic 0.0 0.0 0.5 0.4 0.4 16:0 16.4 21.1 17.3 16.4 12.4 16:1©9 0.3 0.3 1.2 1.7 1.6 16:lco7 2.1 0.5 0.0 0.9 0.3 16:lo)5 0.0 0.0 0.0 0.0 0.0 17:0 iso 2.6 2.9 2.8 2.9 1.8A 16:20)7 0.0 0.0 0.0 0.0 0.0 17:0 anteiso 0.3 0.7 0.8 1.3 2.2 16:2co4 0.0 0.0 0.0 0.0 0.0 phytanic 0.2 0.5 0.7 1.2 1.8 16:3co4 0.2 0.0 0.0 0.0 0.0 unkno wn# 1 3.3 2.8 3.3 2.9 2.4 unknown#2 0.0 0.0 0.0 0.0 0.0 16:4u)3 12.5 8.5 11.2 10.5 11.7 16:4col 0.3 0.0 0.0 0.0 0.0 18:0 0.5 0.6 0.4 0.3 0.2 18:lo)9 7.3 10.4 8.1 6.4 4.9 18:10)7 3.4 2.1 0.9 1.2 0.9**4 18:lco5 0.6 0.2 0.0 0.0 0.0 18:2o)9 1.5 1.4 1.4 1.2 0.84 18:2u)6 3.7 9.1 9.3 9.0 11.0*4 18:3co6 2.1 3.1 4.3 3.9 3.5* 18:3o)3 34.6 28.6 30.7 29.2 26.7+ 18:40)3 1.3 0.6 0.6 0.3 0.44 18:50)3 0.0 0.0 0.0 0.0 0.0 20:4co6 0.0 0.0 0.0 0.0 0.0 20:5o)3 0.6 0.0 0.0 0.0 0.0 22:0 0.0 0.0 0.0 0.0 0.4 21:5o)3 0.0 0.3 0.0 0.6 0.6 unknown#3 0.0 0.0 0.0 0.0 0.0 22:4o)6 0.0 0.0 0.0 0.0 0.0 22:5o)6 0.0 0.0 0.0 0.5 0.2 22:50)3 0.0 0.0 0.0 0.0 0.0 unknown#4 0.4 0.0 0.0 0.6 0.0 22:6co3 0.7 0.0 0.0 0.0 0.6 unknown#5 1.6 0.0 0.0 0.0 0.0 112 Table 3.2G. The fatty acid composition of Pavlova lutheri grown at five different temperatures and saturating PFDs (=220 umol photons m " 2 s'l). Regressions of percent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P£0 .05 , ••P<0.01; correlations with chl a cell' 1 , AP<0.05, AAP<0.01 (excluding 17.5 °C data). See text for details. Temperature (°C) Growth rate (d-1) 10.0 0.62 15.0 1.09 17.5 0.56 20.0 1.79 25.0 2.36 Fatty acid 0.0 0.0 0.0 10:0 0.0 0.0 12:0 2.0 0.0 0.0 0.0 0.0 14:0 3.7 5.0 12.3 9.0 9.8+ 14:10)5 0.0 0.0 0.2 0.0 0.3 15:0 0.9 0.7 0.2 0.9 0.7 Pristanic 0.0 0.0 0.1 0.0 0.0 16:0 18.2 27.0 17.3 32.9 29.8+ 16:lco9 0.0 0.4 0.5 0.3 0.7^ 16:lco7 22.7 27.1 23.6 27.5 24.7 16:lco5 0.0 0.0 0.0 0.0 0.0 17:0 iso 0.8 0.7 0.0 0.7 0.6 16:2o)7 1.4 1.1 0.1 0.9 1.4 17:0 anteiso 0.0 0.0 0.0 0.0 0.0 16:2co4 2.1 2.0 0.7 1.9 2.2 phytanic 0.0 0.0 0.0 0.0 0.0 16:30)4 3.3 5.2 0.3 4.7 6.6 unknown#l 0.0 0.0 0.7 0.0 0.0 unknown#2 0.0 0.4 0.0 0.0 0.4 16:40)3 0.0 0.0 0.0 0.0 0.0 16:40)1 6.4 3.7 0.0 1.0 0.3 18:0 0.0 0.3 0.2 0.4 0.2 18:lco9 0.0 0.3 0.9 0.3 0.0 18:lo)7 0.6 0.5 1.0 0.6 0.3 18:lco5 0.0 0.0 0.0 0.0 0.0 18:2o)9 0.0 0.0 0.0 0.0 0.0 18:2co6 0.0 0.0 2.9 0.2 0.2 18:3o)6 0.0 0.0 0.2 0.0 0.0 18:3o)3 0.0 0.0 0.3 0.0 0.0 18:4co3 3.7 4.7 6.4 4.3 4.4 18:5o)3 0.0 0.0 0.0 0.0 0.0 20:4o)6 0.5 0.0 0.0 0.0 0.0 20:5o)3 11.7 14.0 16.9 7.9 10.8 22:0 0.0 0.0 0.0 0.0 0.0 21:5to3 0.0 0.0 0.0 0.0 0.0 unknown#3 0.0 0.0 0.0 0.0 0.0 22:4©6 0.0 0.0 0.0 0.0 0.0 22:5u)6 1.0 0.0 0.7 0.0 0.0 22:5o)3 1.3 0.0 0.5 0.0 0.0 unknown#4 2.8 0.0 0.0 0.0 0.0 22:6co3 5.2 4.9 9.7 1.9 2.3+ unknown#5 0.0 0.0 0.0 0.0 0.0 113 Table 3.2H. The fatty acid composition of Isochrysis galbana (T-iso) grown at five different temperatures and saturating PFDs (=220 umol photons n r 2 s-1). Regression of percent composition with: growth rate, *P<0.05, **P<0.01; temperature, •P<0.05, ••P<0.01; correlations with chl o cell - 1 , AP<0.05, AAP^O.01 (excluding 17.5 °C). See text for details. Temperature (°C) Growth rate (d_l) 10.0 0.63 15.0 1.33 17.5 0.43 20.0 0.84 25.0 1.38 Fatty acid 10:0 0.0 0.0 0.0 0.0 0.0 12:0 0.0 0.0 0.0 0.0 0.0 14:0 4.3 29.4 17.6 19.4 21.0+ 14:10)5 0.0 0.2 0.0 0.2 0.0 15:0 1.0 1.0 0.2 0.0 0.2 Pristanic 0.0 0.0 0.0 0.0 0.0 16:0 25.2 15.4 10.3 11.5 13.9+ 16:lco9 0.0 0.5 0.4 0.4 0.6 16:lco7 26.2 32.0 0.8 1.2 4.4+ 16:10)5 0.0 1.2 0.0 0.0 0.0 17:0 iso 0.8 2.7 0.0 0.0 0.0 16:2co7 1.4 1.4 0.0 0.0 0.5 17 anteiso 0.0 0.0 0.0 0.0 0.0 16:2co4 2.1 2.6 0.4 0.5 1.0 phytanic 0.0 0.0 0.0 0.0 0.0 16:3co4 3.1 1.9 0.0 0.0 1.5 unknown#l 0.0 0.0 0.0 0.0 0.0 unknown#2 0.8 0.0 0.0 0.2 0.5 16:4co3 0.0 0.0 0.0 0.0 0.0 16:4col 5.8 1.3 0.0 0.0 0.0 18:0 0.2 0.6 0.9 0.5 0.6 18:lco9 0.3 0.4 22.1 18.1 16.9+ 18:lco7 0.5 1.7 1.7 1.3 1.6 18:lo)5 0.0 0.7 0.0 0.0 0.2 18:2o)9 0.0 0.0 0.0 0.0 0.0 18:2co6 0.0 0.3 6.3 5.8 5.2+ 18:3o)6 0.0 0.0 0.0 0.2 0.2 18:3co3 0.0 0.0 3.7 4.6 3.6 18:4o)3 4.4 0.9 8.5 9.8 8.5 18:5co3 0.0 0.0 3.8 3.6 2.4 20:4co6 0.0 0.0 0.0 0.2 0.0 20:50)3 11.6 2.8 0.5 0.6 1.9+ 22:0 0.0 0.0 0.3 0.0 0.0 21:5o)3 0.0 0.0 0.6 0.6 0.3 unknown#3 2.2 0.6 0.0 0.0 0.0 22:4o)6 0.0 0.0 0.5 0.5 0.2 22:5o)6 0.0 0.0 2.2 1.7 1.3 22:5o)3 0.0 0.0 0.6 0.5 0.2 unknown#4 0.0 0.0 0.0 0.0 0.0 22:6o)3 4.0 0.0 14.4 11.1 8.2+ unknown#5 0.0 0.0 0.4 0.0 0.0 114 ii) Growth rate There were 2 species, T. pseudonana and P. lutheri, that showed significant increases in the percentage of total saturated FAs as a function of increasing growth rate (Table 3.1). The degree of unsaturation, measured as the ratio of unsaturated fatty acids/saturated FAs, also showed in a linear relationship with growth rate for these 2 species. In both cases, with increasing growth rates, the percentage decrease of saturated FAs was mostly shifted to increased poly-unsaturates rather than to mono or di-unsaturated FAs (Table 3.1). D. tertiolecta was the only species to show a change in the percent composition of total mono-unsaturated FAs as a function of growth rate. The following individual FAs showed a significant regression of percent composition as a function of growth rate: 14:0 (Tp), 15:0 (Cc.Pt), 16:2o>4 (Cg), 16:3o>4 (Cc.Cs), 16:2co7 (Tp), 16:4oil (Tp),18:lco9 (Cs),18:lco7 (Dt),18:2co6 (Dt), 18:3co6 (Dt), 18:4col (Tp), and 22:6co3 (Tp) (Table 3.2). Several times 16:4ool and 16:3o>4 appeared to covary strongly in opposite directions as growth rates and temperature varied. There was, however, no individual FA with a completely consistent, statistically significant positive or negative regression in percent composition with growth rate across all species. iii) Chlorophyll a For the reasons given above, the associations of chl a with various fatty acids are analyzed without the 17.5 °C data. None of the fatty acid categories were correlated with an individual species' chl a concentration, 115 except P. tricornutum. In the case of P. tricornutum, the chl a concentrations were correlated with the percentages of to6 FAs. Correlations of specific fatty acids with chl a include: 15:0 (Cc, Tp), 16:2co7 (Pt), 16:3co4 (Cc, Cs, Pt), 16:4col (Cc, Cs), 17 iso (Dt), 18:lco7 (Pt), 20:5co3 (Pt), 22:6co3 (Pt), (Table 3.2). iv) Nutrition Of the FAs considered to be essential for bivalves, only T. pseudonana showed a response in either E F A as a linear function of temperature. The E F A 22:6co3 decreased significantly with increasing temperature for this species. For T. pseudonana, both the proportion of 20:5co3 and 22:6co3 showed an indication of a reversal in this trend as the temperature exceeded 20 °C (Table 3.2B), perhaps as a response to supraoptimal temperatures. Amongst the diatoms the percentage of 22:6co3 was frequently (in 4 of 5 species) lowest at the highest temperature (Table 3.2). P. tricornutum contained the highest average percentage of 20:5co3 plus 22:6oo3, (21.7% of total FAs), followed by P. lutheri (17.1%), with the other species averaging from 5-15%, except D. tertiolecta which contained only trace amounts (Fig. 3.5). v) Unusual Responses 7. galbana showed very pronounced and unsual responses in its' FA composition in response to temperature. This species apparently underwent a major elongation of 16:lo)7 to 18:lo)9 at temperatures of 17.5 °C and above. The percent composition of 16:lco7 at 10 and 15 °C was significantly greater than the percent composition of this F A at 17.5, 20 and 25 °C (t-test, P<0.05). 116 Similarity the percent composition of 18:lo>7 at 10 and 15 °C was significantly less than the percent composition of this F A at 17.5, 20 and 25 °C (t-test, P^O.05). This strong, nonlinear response in fatty acid composition highlights one of the major problems of relying on linear regressions and correlations for interpretative analysis, that is, nonlinear responses are not well characterized. 117 Fig. 3.5. Mean percentage (relative to total FA) of 22:6co3 plus 20:5co3 (the nutritionally essential FAs for bivalves) from C. calcitrans, T. pseudonana, C. gracilis, C. simplex, P. tricornutum, D. tertiolecta, P. lutheri, I. aff. galbana (T-iso). The bar represents 1/2 the range of values obtained over a temperature range of 10 to 25 °C. 118 "O CD O CD' CO Sum 20:5w3 + 22:6CJ3 (% of total FA) ro o Ol o C. gracilis C. simplex C. calcitrans T.pseudonana P. tricornutum f D. tertiolecta P. lutheri I. galbana (T-lso) ro cn co o co tn o D I S C U S S I O N Temperature versus growth rate Temperature is known to influence the growth rates of most organisms (Hochachka and Somero 1984) and marine phytoplankton are no exception (Eppley 1972). For many species and a relatively large range of temperatures, the relationship of temperature and growth rate has been determined to be exponential (Eppley 1972), logarithmic (Li and Morris 1982), or linear (Rhee and Gotham 1981). Within 1 species and over the range from 10 to 25°C, growth rates appeared to be a linear function of temperature (this study). The average r2 value for the linear regressions of growth rate as a function of temperature was 0.777 ±0.097 and for log growth rate as a function of absolute T - l (Li and Morris 1982) was 0.757 ±0.126. Thus slightly more variance was described by the former (linear) model over the 15 °C temperature range used in this study. The majority of Qio values determined in this study were above most literature values (Eppley 1972 and references therein), but since they were determined from a large number of points (a minimum of 19 independent cultures per species) there can be some confidence in their validity. The variable response of growth rate to temperature in I. galbana might be partially explained by this species' nonlinear pattern of variation in FA composition with temperature. Such a sharp transition in F A composition might suggest a change in slope for biochemical rate reactions as described 120 by Arrhenius formulations, perhaps suggesting a sharp change in growth rate with variation in temperature. Carbon, nitrogen and chlorophyll a Both carbon (Qc) and nitrogen (Qn) quotas showed only modest variation over the temperature range of 10 to 25 °C used in this study. A sustantial portion of the previous research on the influence of temperature on cellular quotas was conducted in continuous culture (Goldman 1977, Goldman 1979, Rhee and Gotham 1981) where nutrient, light and temperature interactions are not easily separated (Goldman and Mann 1980). One previous study (Goldman and Mann 1980) reporting the effects of temperature on carbon and nitrogen quotas for P. tricornutum in nutrient replete, and possibly light-limited, cultures demonstrated a U shaped relationship, with a broad band of minimum quotas generally occuring between 10 and 20 °C. Goldman and Mann (1980) found the major increase in cellular quotas as temperatures reached 5 °C, a temperature well outside the range of this study. Thus for P. tricornutum both the absolute magnitude of Qc. Qn and the pattern of their variation with temperature was consistent with previous research. P. tricornutum has been reported to maintain a stable C:N ratio over the temperature range of 5 to 25 °C (Goldman and Mann 1980). The results presented here confirm this observation. In this study, 2 other species, T. pseudonana and P. lutheri, both showed significant linear increases in C:N as temperature increased, indicating that at least occasionally the growth rate in terms of carbon may increase faster than the growth rate in terms of 121 nitrogen. Therefore there are species specific reponses of growth in nitrogen and carbon with variation in temperature which should be considered in ecosystem models. In this study there was a general trend for chl a cell-l to increase and carbon-.chl a to decrease with increasing temperature as previously shown by Eppley and Sloan (1966), Eppley (1972) and Morris and Glover (1974). These trends suggest a decrease in energy saturation with increasing temperature, or at least a shift in cell physiology from excess photonsxarbon fixation towards a closer balance. This is interpreted to mean that cells might require more light energy to saturate growth as temperature increases, and further implies that the PFD where growth saturates (Ik) may be temperature dependent. Chlorophyll a cell-l showed only a few significant correlations with specific fatty acids in this data set. Lack of consistency between species suggests no universal associations between chlorophyll molecules and specific fatty acids (see later). Temperature and growth rate versus fatty acid composition i) General responses Most temperate phytoplankters live in environments that are susceptible to periodic mixing events (Harris 1980). Phytoplankton advected or sinking through the seasonal thermocline may find themselves in a low temperature and low light environment. Such cells may have little energy available for membrane modifications. Since desaturation reactions require 122 energy, broadly eurythermal membranes may be evolutionarily "favored" at least in some species of marine phytoplankton. Phytoplankton with membranes adapted to a limited temperature range may encounter physiological difficulties if advected rapidly from one temperature regime to another substantially different one. The physiological advantages of temperature induced variations in the fluidity of membranes seem more likely to occur in homotherms or those organisms either capable of controlling their position and therefore thermal environment, or existing within a stable thermal environment. Many species of phytoplankton do not fulfill either of these latter qualifications. This line of argument is concluded by suggesting that there should be a wide range of species specific capabilities (i.e. evolutionary "strategies") for adapting fatty acid composition and the resulting membrane viscosity in response to temperature variation. There is very little research on the question of whether decreasing temperature causes a general increase in the degree of unsaturation of fatty acids in marine phytoplankton. Three previous studies (Ackman et al. 1968, Lynch and Thompson 1982, Mortensen et al. 1988) have suggested this is indeed the situation, but the small number of species and limited number of temperatures that were utilized limits the generality of their conclusions. In this study, and in Mortensen et al. (1988), experiments were conducted with C. gracilis. They concluded that FAs did indeed become more unsaturated at lower temperatures, although there is no evidence that their apparent change in unsaturation was statistically significant. The results presented here are very similar to their results, except that the temperature range was 123 expanded below 18 °C (Mortensen et al.s' minimum temperature) to include 10 and 15 °C. In this study there was no significant trend in the ratio of unsaturated/saturated FAs over the temperature range of 10 to 25 °C for this species. Mortensen et al. (1988) noted that the ratio of co3/o)6 fatty acids covaried with temperature, while in this study it covaried with growth rate for only C. gracilis. Thus it would appear that the ratio of 0)3/o)6 fatty acids does not generally covary with temperature or growth rate in most marine phytoplankton. The data presented here demonstrated that between 10 and 25 °C, for 8 species of marine phytoplankton, there was no consistent relationship between temperature and the degree of saturation. In this study, neither the weighted ratio of unsaturated/saturated FAs nor the mean chain length varied consistently with temperature in such a way as to maintain membrane homeoviscosity. Where the ratio of unsaturated/saturated FAs varied with temperature or growth rate (e.g. T. pseudonana and P. lutheri) it increased at low temperatures. In a new analysis of previously published data on the influence of photon flux density (PFD) on fatty acid composition (from Chapter 2), 2 species (T. pseudonana and C. simplex) were found that demonstrated a significant negative linear relationship between the ratio of unsaturated/saturated FAs and log PFD. The effects of log PFD and temperature on the physiology of phytoplankton showed an obvious similarity in their linear effect on growth rate. For the 8 species studied here, both increasing temperature (this study) and increasing log PFD (Chapter 1) had significant linear relationships with increased growth rates. 124 Fig. 3.6. The ratio of unsaturated/saturated fatty acids as a function of growth rate for 3 species of marine phytoplankton. Cultures where growth rate was controlled by PFD (•); cultures where growth rate was controlled by variation in temperature (O). Least squares linear regressions are shown fitted to the data. 125 W o.O 0.5 1.0 1.5 2.0 2.5 0.5 I 1 1 1 1 1 0.0 0.5 1.0 1.5 2.0 2.5 Growth rates (d1) 126 If the ratio of unsaturated/saturated FAs are plotted versus growth rate for both temperature and PFD data sets for these 3 species (Fig. 3.6) it is clear that the degree of unsaturation is responding to variation in u. Since the degree of unsaturation is a function of u resulting from either variation in PFD (constant temperature) or variation in temperature (constant PFD), it is not likely that the degree of unsaturation is associated only with the need to maintain membrane fluidity at low temperatures. The results of this study strongly suggest that, at least for these 3 species, the degree of F A unsaturation was influenced by the overall rate of cellular metabolism. Therefore it is suggested that, at least in marine phytoplankton, more unsaturated membranes may facilitate higher rates of metabolic activity. Thus, in general, marine phytoplankton do not appear to use a change in the degree of unsaturation as a method of maintaining homeoviscous membranes. ii) Specific fatty acids The major fatty acids, i.e. those FAs which were, at least occasionally, above 10% of total FAs, were 14:0, 16:0,16:lco7, 18:3co3 (Dt), occasionally either 20:5o)3 or 22:6co3(Ig),. There was no clear pattern of variation in these major fatty acids with temperature. This is in sharp contrast to the response of these same FAs in the same species to variation in photon flux density (Chapter 2). The FAs 16:0 and 16:lco7 frequently varied with PFD (Chapter 2). 127 The membranes of photosynthetic organelles may represent 75% of the total cellular membrane (Forde and Steer 1976) and are highly unsaturated, containing as high as 90% P U F A (Fuller and Nes 1987). The chloroplast lipids are mainly monogalactosyl diglycerides containing mostly poly-unsaturated FAs (Opute 1974). Considering only poly-unsaturated fatty acids that showed some variation with temperature or growth rate, the most consistently variable FAs were 16:4o)l and 16:3co4. Since these FAs seemed to co-vary inversely, it is suggested that the incorporation of 16:3co4 instead of 16:4col may function to increase the fluidity of cellular membranes. The (ol FAs were relatively unique and the positioning of the terminal double bond at the methyl end of the fatty acid chain may increase the distance between 16:4col and other co3 membrane FAs by reducing the van der Waals interactions (Hochachka and Somero 1984). This may result in a homeoviscous membrane with constant fluidity characteristics as temperature decreases. The fatty acid 16:4col reached more than 20% of the total poly-unsaturated fatty acids and may thus impart a substantial change in homeoviscosity. In Chapter 2 it was demonstrated that percentage of the F A 16:4col was frequently highest at the greatest growth rates and highest PFD. In this study, the percentage of 16:4col was highest at the lowest growth rates. This sharply contrasting pattern proves that the percentage of 16:4col does not vary in response to growth rate. In both cases this pattern represents a negative association with chlorophyll a quota. It is suggested that in the situation where PFD was varied (Chapter 2) high 16:46)1 results in more 128 fluid membranes, perhaps required at higher rates of cellular metabolism. In this study greater relative amounts of 16:4col are also considered to increase membrane fluidity but in response to lower temperatures. It may be noteworthy that the percentage of the other major PUFAs (20:5co3 and 22:6co3) although quite variable in response to temperature, either increased or showed no significant decreases with decreasing temperature. In D. tertiolecta the percentage of 18:lco7, 18:2o)9 increased and 18:2co6 decreased significantly as temperature decreased. There was also a nearly 10% increase in 18:3co3 over a 15 °C decrease in temperature from 25 to 10 °C. These responses were nearly identical to those in the fatty acids from the total polar lipids of the chloroplasts and microsomes of its congener, Dunaliella salina (Table 4 in Lynch and Thompson 1982) during temperature transition studies. These changes in fatty acid composition took more than 60 h to complete during the temperature transition from 30 to 12 °C (Lynch and Thompson 1982). Sixty hours is a long time relative to the growth rate of this organism (approximately 1 generation for D. tertiolecta at 12 °C). The relatively long time required for adaptation of membrane lipids suggests that these lipids cannot fully adapt to temperature transitions occurring on shorter time scales. In the marine environment significant variations of light and temperature can occur on time scales of less than 60 h (Harris 1980), and thus marine phytoplankton might be expected to adapted to an average condition. 129 The response of EFAs to temperature appears to be species specific. Only for T. pseudonana was the percentage of either E F A a linear function of temperature over the range from 10 to 25 °C. For most species, variation in the percentage of EFAs was not a linear function over this range of temperatures. Over all species, the range (max-min), in percent of EFAs was greater with variation in temperature from 10 to 25 °C (this study) than it was for PFD varying from 6 to 225 umol photons m " 2 s"* (Chapter 2)(paired t-test, P<0.05). This suggests that selection of the correct temperature may be more important than the correct PFD in maximizing the percentage of EFAs, although in some species the PFD-controlled growth rates were well correlated with the percentage of EFAs produced (Chapter 2) so that both factors should probably be considered. Interactions and possible synergistic effects of light and temperature have not been assessed. In general, lower temperature seems to favor the maximum percentage composition of EFAs, but with a reduction in production per unit of time (Ahern et al. 1983, this study), 130 S U M M A R Y Most species of marine phytoplankton studied here showed significant variation in fatty acid composition in response to variation in temperature. With the data collected in this study it was demonstrated that the ratio of unsaturated/saturated fatty acids was a linear function of growth rate, whether growth rate was varied by temperature or PFD. To the extent of my knowledge this is the first study to demonstrate that the ratio of unsaturated/saturated fatty acids varies in response to the overall rate of cellular metabolism and not the growth temperature per se. Furthermore evidence is provided that some marine phytoplankton may utilize an unusual method of variation in fatty acid composition to maintain membrane homeoviscosity. It was found that a consistent increase in the percent composition of the fatty acid 16:4col and a general decrease in the percentage of 16:3o>4 occurred as temperature declined. The positioning of the double bond at the terminal end of the acyl chain may increase the distance between the col and other to3 fatty acids resulting in a lower melting point for such a membrane (Hochachka and Somero 1984). This mechanism of maintaining membrane homeoviscosity may have been largely overlooked because many temperature adaptation studies have not determined the positions of the double bonds in poly-unsaturated fatty acids. The choice of an appropriate temperature can be useful in maximizing the percentage of EFAs. For T. pseudonana the percent composition of the E F A 22:6co3 increased linearly with decreasing temperature over the range from 25 to 10 °C. 131 CHAPTER 4 E F F E C T S OF MONOSPECIFIC A L G A L DIETS OF VARYING BIOCHEMICAL COMPOSITION O N T H E GROWTH A N D SURVIVAL OF THE PACIFIC OYSTER (CRASSOSTREA GIGAS Thunberg) L A R V A E I N T R O D U C T I O N The study of marine invertebrate nutrition is a subject of broad commercial and ecological significance. Factors influencing the transfer of energy or biomass up the food chain may be similar in both the natural and hatchery environments, particularly if the natural system is not limited by food quantity (at least on short time scales). If food quantity does not limit growth then food quality may be of increased importance in determining the efficiency of energy transfer. It is possible in the laboratory or hatchery to test, qualitatively, the relative nutritional value (food quality) of different phytoplankton species. Such work has been conducted at the hatchery in Conwy for the past 60 years (Walne 1974). In early work, the nutritional value of phytoplankton was determined, but variability within one species was often high, and differences between species frequently could only be ascribed to qualitative factors such as digestibility or toxicity. Since the seminal work of Parsons et al. (1961), an increasing number of researchers have examined the biochemical composition of phytoplankton for their nutritionally important components (primarily protein, lipid and carbohydrate). Unfortunately relationships between 132 phytoplankton biochemical composition and animal growth rates have been difficult to elucidate (Epifanio 1979, Webb and Chu 1982, Gallager and Mann 1982, Enright et al. 1986a, b), resulting in different conclusions about the significance of phytoplankton gross biochemical composition as a factor influencing bivalve growth. Since the discovery that certain long chain polyunsaturated fatty acids (PUFAs) are essential (EFAs) for many marine organisms, particularly during larval stages, considerable research has focused on the availability of EFAs in both the hatchery (e.g. Watanabe et al. 1983), and in the natural environment (Sargent and Whittle 1981). Phytoplankton which are deficient in the EFAs 20:5o)3 and 22:6co3 have been shown to be poor food items for the oyster larvae Crassostrea gigas (Langdon and Waldock 1981). The nutritional suitability of phytoplankton for bivalves has been assessed, at least partially, by the phytoplankton's FA profile (Webb and Chu 1982). However within the large group of phytoplankton with adequate EFAs there remains considerable variability in their nutritional value to at least some species of oyster (Enright et al. 1986a). Attempts to determine why one species of phytoplankton is more nutritious than another have been largely unsuccessful. Furthermore, studies utilizing more than one species of phytoplankton are difficult to interpret because it is impossible to control for interspecific differences in digestibility, toxicity, and cell size (Webb and Chu 1982). This study examines the growth rates and survival of the larval oyster C. gigas, fed one species of phytoplankton grown under a range of conditions. 133 The marine diatom, Thalassiosira pseudonana, was grown under different conditions of light quality, quantity, temperature and nutrient limitation to provide diets that varied in carbon, nitrogen, protein, lipid, carbohydrate and percent fatty acid composition. It has already been demonstrated that variation in light intensity influences phytoplankton fatty acid composition (Orcutt and Patterson 1974, Chapter 2). Blue light can be used to increase protein content and red light to increase cellular carbohydrate (Kowallik 1978). Similarly, nitrogen limitation also can be used to increase the cellular carbohydrate of phytoplankton cells (Shrifrin and Chisholm 1981, Harrison et al. 1990) thereby modifying their nutritional value to bivalves (Flaak and Epifanio 1978, Gallager and Mann 1981). Finally, temperature can influence the fatty arid composition of marine phytoplankton (Ackman et al. 1968, Chapter 3). It was hypothesized that C. gigas larvae fed saturating diets of cells grown under these different conditions would show differences in growth rates which might be ascribed solely to variation in the nutritional value of the food. It was anticipated that this approach should minimize differences caused by interspecific variation in digestibility, cell size and palatability which may have confounded previous studies using multispecies diets. 1 3 4 M A T E R I A L S A N D M E T H O D S Algal culture and medium The marine diatom, Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (NEPCC #58, clone 3H), was obtained from the Northeast Pacific Culture Collection, Department of Oceanography, University of British Columbia. Cultures were grown in enriched natural seawater using the enrichment solutions (ES) of Harrison etal. (1980). The medium was modified as in Chapter 1. Phytoplankton cultures for experiments A, B, and C, were grown at 25, 26, and 29°C, respectively (Table 4.1). The biochemical composition of T. pseudonana was also manipulated by varying the quality and quantity of irradiance. All of the 20 L phytoplankton cultures were continuously illuminated from one side by 4 Vita-liteR fluorescent tubes. Irradiance was attenuated by distance, Roscolux filters and/or neutral density screening. The following irradiances were measured (averages of 4 measurements, top and bottom at both back and front of carboy at normal operating cell densities): saturating white light culture (HIGH), 110 umols photons m " 2 s"l; the low blue light culture (BLUE), 22 umols photons m " 2 s"l after passage through Roscolux # 69; the low white light culture (LOW), 13 umols photons m " 2 s ' l (irradiance reduced by neutral density screening); and the chemostat culture (N-LIMITED), 34 umols photons m " 2 s"l after passage through Roscolux # 19 (moderate red light) (Table 4.1). Three algal cultures (LOW, HIGH, BLUE) were grown in turbidostats adjusted such that cell densities were maintained near 9 x 10$ cells L - l . In the turbidostats, inflow nutrient concentrations (ES enrichment; 135 Harrison et al. 1980) were high and therefore never limiting and these cells grew at the maximum rates for the prevailing conditions of light and temperature. The turbidostat growth rates (Table 4.1) were determined from independent batch cultures grown under identical environmental conditions, but in 50 mL tubes (n=3). The N-LIMITED cultures were grown in N O 3 -limited chemostats. For the chemostats, the inflow N O 3 concentration was approximately 40 uM and the dilution rate was adjusted to be approximately 30% of U-max (ca. 0.33 d"*). The cultures were stirred at 60 rpm with a 7.6 cm teflon-coated magnetic bar and bubbled with a mixture of approximately 0-4% C O 2 and air. The pH of most cultures was maintained at 8.2 although some increases to ca. 8.7 occurred during culture growth in the HIGH culture. Determination of phytoplankton biomass in each culture was made once per day by measuring in vivo fluorescence using a Turner Designs^ Model 10 fluorometer and cell counts utilizing a Coulter Counter^ model TAII equipped with a population accessory. General Three sequential experiments (A,B,C) were conducted using separate batches of oyster larvae ranging in mean size from 107 to 144 um, obtained from Coast Oyster Co. Ltd. (Seattle). Initial conditions for the oyster cultures are summarized in Table 4.2. In experiments A and B, five different treatments (i.e. different algal diets, referred to as: HIGH, LOW, B L U E , N -LIMITED, starved (=no algae)) (Table 4.1) were used (no replication). In experiment C, three algal different diets, HIGH (n=2), LOW (n=4), and food-starved (n=l) were used. 136 Table 4.1. A summary of the conditions under which T. pseudonana was grown to produce variation in biochemical composition for three sequential experiments (A,B»C) on the growth and survival of larval C. gigas. Plus or minus 1 S.D. is given for the algal growth rates of independent batch cultures grown under identical conditions to turbidostat cultures. L A B E L Irradiance Nitrogen quantity quality status (umol photons m - 2 s-i) u. Experiment @ 2 5 ° C ~ A B C (n=3) Temperature (d-1) (°C) HIGH 110 white in excess 1.71±.01 25 26 29 LOW 13 white in excess 0.29±.02 25 26 29 B L U E 22 blue in excess 1.48±.03 25 26 N-LIMITED 34 red limiting £0.33 25 26 137 Table 4.2. A summary of the conditions for larval C. gigas in three sequential experiments (A,B>C) on their growth and survival when fed unicellular diets of T. pseudonana of varying biochemical composition. Experiment A B C Initial age of animals (days) 5 9 7 Initial size of animals (um)* 107±26 140±26 144±19 Duration of experiment (days) 14 10 12 or 14** Mean temperature 22 22 26 Initial density (animals ml-1) 2.5 2.5 5.0 Antibiotics (50 ppm sulfamethazine) no yes yes * mean ± 1 S.D. ** the 2 fastest treatments (HIGH diets) were terminated at 12 rather than 14 days due to large numbers of animals undergoing metamorphosis. 138 Equal phytoplankton cell densities were maintained in each fed treatment. Statistical analysis was mainly by paired t-tests, that is, between treatment comparisons, paired by experiment, across all three experiments. T-tests were used to test the statistical significance of some oyster responses to different treatments within experiment C. Algal and oyster biochemical composition Samples for phytoplankton biochemical composition were collected approximately every 2 or 3 days. The data from each phytoplankton culture over the length of the experiment were pooled to provide a mean for each biochemical parameter for each treatment (=algal diet). Particulate organic carbon and nitrogen (POC and PON) subsamples (25 mL) were collected on precombusted 13 mm Gelman A/E filters and were analyzed on a Carlo Erba C H N analyzer. Subsamples (100 mL) for total lipid were extracted in chloroform:methanol:water (Bligh and Dyer 1959) and analyzed by the lipid charring technique of Marsh and Weinstein (1966) using tripalmitin as a standard. Total lipid also contained chl a. Samples for determination of total polymeric carbohydrates were collected from the methanol.water fraction of the total lipid extraction, hydrolyzed in sealed borosilicate glass tubes containing 3 mL of 1 N H2SO4 for 20 h at 100°C and analyzed by the phenol-sulphuric acid technique of Dubios et al. (1956). Subsamples (50 mL) for total protein were collected on precombusted 25 mm GF/F filters, extracted immediately in 3% TCA, separated into protein and free amino acid fractions and stored at -20°C for later protein analysis by the modified Lowry technique (Lowry et al. 1951, Clayton et al. 1988). 139 Phytoplankton subsamples (1L) for fatty add determinations were collected on precombusted 47 mm GF/F filters, placed inside a petri dish and sealed in plastic bags filled with nitrogen gas. At the end of experiments B and C, oysters were collected on Nitex netting, rinsed briefly with distilled deionized water, freeze-dried and stored frozen under nitrogen gas. Prior to analysis, samples were frozen at -20° C for periods of less than 3 weeks, or for longer periods at -80° C. Samples were saponified and methylated as in Whyte (1988). FAs were analyzed on a Hewlett-Packard 5890A gas liquid chromatograph fitted with a Supelcowax 10 fused silica capillary column (30 m x 0.32 mm ID, 0.25 um film) and identified by comparison with saturated and PUFA-1 methyl ester standards (obtained from Supelco Inc.) in accord with Ackman (1986). The shorthand notation used in fatty acid identification is L:BcoX where L is the chain length, B is the number of double bonds, and G)X is the position of the double bond closest to the terminal methyl group. Oyster culture Unenriched natural seawater (collected from a minimum depth of 7 m, salinity 28 ppt) for the oyster experiments was prefiltered, first through 25 um, then through 1 um cartridge filters and finally through a 142 mm Gelman glass fiber filter. Starved animals were not fed, and their water was not bubbled or changed. Starved animals were sampled only at the beginning and end of each experiment. Subsequent descriptions apply only to fed animals. Water was changed every 2, or occasionally, 3 days. To change the water, larvae were gently siphoned onto Nitex netting, concentrated to 1 L, mixed, a 10 mL subsample removed for growth and 1 4 0 survival rate measurements and the remainder carefully added back to 20 or 40 L of filtered natural seawater water at the correct temperature (Table 1). After each water change all fed treatments (diets) received equal numbers of phytoplankton cells (initial number). The initial density of phytoplankton cells was increased with increasing larval age. The initial cell density was approx. 50,000 cells mL-1 for young larvae (7 days old, ca. 75-150 um in size), increasing to approx. 110,000 cells mL-1 for older larvae (20 days old, ca. 250-300 um in size). Over a 24 h period, cell density decreased to as low as 20,000 cells mL-1 due to grazing. At 24 h the cell densities were "topped up" to their initial numbers. At 48 h (or occasionally 64 h) the water was changed again and the oysters provided with fresh phytoplankton cells. Antibiotics (50 ppm sulfamethazine) were also added at each water change for experiments B and C. Changes in the phytoplankton's biochemical composition over a 24 h period was tested in a simulated experiment. Phytoplankton cells were diluted with filtered natural seawater and allowed to stand 24 h under the same conditions as the oyster carboys. These cells were then collected and any changes in carbon, nitrogen and protein cell-l that occurred over 24 h were determined. Cells from the B L U E , LOW and N-LIMITED cultures showed no change in these parameters over 24 h. Cells from the HIGH culture initially had greater carbon and nitrogen quotas than cells from the other cultures and over 24 h showed a decline in carbon and nitrogen quotas to values closer to those for the other cultures (Thompson unpub. data). 141 Starting when the larvae were 7 days old, carboys were bubbled vigorously with air injected through air stones placed near the bottom of the carboys. Oyster cultures received < 1 umol photons m " 2 s ' l . Animals were measured from umbo (edge at hinge) to opposite edge using an eyepiece micrometer fitted to an inverted microscope. After concentration, resuspension into IL, and vigorous mixing, a constant volume of water was removed from each treatment for enumeration. Due to mortality, fewer animals were measured in later samples. A maximum of 250 to a minimum of 25 live animals were measured per sample to estimate growth rates. Growth rates were calculated as the slope of the least squares linear regression of ln animal size versus time, that is: [In {(size@time tl)/(size ©time to)}]/(tl-to) Similarly, to estimate mortality, an initial sample containing a maximum of approximately 3,000 to 5,000 animals was examined and towards the end of the experiments a minimum of 250 animals was examined. The slope of a least squares linear regression of the percentage of dead animals versus time was used to estimate the mortality rate. 142 R E S U L T S Phytoplankton Variation in the biochemical composition of T. pseudonana was such that high light grown (HIGH) cells provided a diet significantly (paired t-test, p<0.05) higher in carbon cell"* and significantly (paired t-test, p<0.05) lower in protein cell-l than low light grown (LOW) cells (Table 4.3). Generally HIGH cells were also higher in carbohydrate, and lower in lipid than LOW cells (Table 4.3). The nitrogen-limited (N-LIMITED) red light grown cells were significantly lower in protein than the low blue light grown (BLUE) cells (paired t-test, p<0.05). The changes in fatty acid composition of T. pseudonana grown under the different conditions are presented below. Oysters C. gigas larvae fed LOW T. pseudonana cells were consistently smaller at the end of each experiment than those fed HIGH T. pseudonana cells (paired t-test, p<0.05) (Fig. 4.1). Over all experiments, growth rates (Table 4.3) of animals fed LOW cells were significantly lower than those fed HIGH cells (paired t-test, p<0.05) and also within experiment C (t-test p<0.05). Generally, mortality was higher (Table 4.3) in the oysters fed LOW cells (Experiment C, t-test, p£0.05). 143 Table 4.3. Growth and mortality responses of larval Crassostrea gigas in three sequential experiments (A,B,C) fed Thalassiosira pseudonana of varying food quality (3 to 5 diets, including control (starved=no food)). T. pseudonana was grown under one of four conditions {high white light (HIGH), low white light (LOW), low blue light (BLUE) N-limited chemostat under moderate red light (N-LIMITED)} to produce variation in biochemical composition. Phytoplankton cultures were subsampled every 2 or 3 days throughout each experiment. Values reported here are means. See text for details. Experiment Oyster results Biochemical Composition of Algal Diets Growth Mortality Carbon Nitrogen Protein Lipid Carbohydrate % of total fatty acids DIET rate 20:50)3 22:60)3 (d-1) (%day-l) (pg cell-l) A LOW 0.063 5.6 9.2 1.5 10.0 10.2 0.14 6.5 0.9 BLUE 0.071 6.2 11.0 1.7 9.2 7.0 0.20 7.0 1.2 HIGH 0.070 4.5 10.7 1.4 8.8 4.4 0.54 5.8 1.1 N-LIMITED 0.072 6.8 9.9 1.4 7.8 12.3 0.16 4.6 1.3 starved 0.006 >6.4 B LOW 0.033 2.7 7.0 0.82 8.7 6.9 0.13 11.9 1.6 BLUE 0.050 4.0 8.0 1.2 8.6 7.1 0.26 7.8 1.3 HIGH 0.051 2.8 9.9 1.3 8.0 6.7 0.50 8.8 1.8 N-LIMITED 0.052 5.3 8.6 1.2 7.1 4.9 0.40 6.6 1.8 starved 0.011 >6.0 C LOW (n=4) 0.040+005 6.7±1.0 12.2 1.9 7.2 7.7 0.21 18.0 2.4 HIGH (n=2) 0.067+.003 2.6±0.8 14.6 1.9 7.1 6.1 0.26 15.7 3.1 starved 0.000 >6.4 Fig. 4.1. Increases in the mean size of larval Crassostrea gigas over time for three sequential experiments (A,B,C). Animals were fed one of five diets, T. pseudonana grown under high white light (HIGHXO), low white light (LOW)U), low blue light (BLUE)(Q), moderate red light and N O 3 limitation (N-LIMlTED)(Y) or starved (no algae=Q). Vertical axis increases logarithmically (ln), and hence oyster growth rates are the slope of the least squares linear regression through data. Linear regressions of HIGH (short dash line), and LOW (solid line) diets extend to graph edges. Other regressions extend through data only. 1 4 5 0 5 10 15 Time (days) 146 Fatty adds 1) In T. pseudonana Variation in the fatty add composition was assodated with variation in irradiance (Table 4.4). Increased irradiance increased the FAs 14:0,16:0, 16:3co4, and decreased 16:lco7 (paired t-test p<0.05). Although not statistically significant, increases in temperature were associated with increases in the percentage of 20:5co3, 22:6co3 and decreases in 16:0 and 16:lo)7 (Table 4.4). Overall, the experimental diets gave a range in the percent composition of 20:5co3 from 4.6% (Experiment A, N-LIMITED) to 18% (Experiment C, LOW) and of 22:6co3 from 0.9% (Experiment A, LOW) to 3.1% (Experiment C, HIGH) (Table 4.4). The three most common fatty adds in T. pseudonana were 14:0,16:0 and 16:lco7 (Table 4.4). ii) F A in T. pseudonana versus C. gigas growth and survival Regression analyses (n=14) indicated that the growth rates of larval C. gigas were a positive function of the percent composition of the 14:0 (r2=0.30) and 16:0 (r2=0.45) FAs in their diets of T. pseudonana cells (Fig. 2). Similarly the animals' growth rates were a negative function of the percent composition of 16:2co4 (r2=0.42), 16:2co7 (r2=0.50), and 20:5co3 (r2=0.52) (Fig. 2) in their algal diets (Table 4.4). iii) FAs in T. pseudonana vs FAs in C. gigas The oysters contained a number of fatty acids not found or found in low concentrations in their algal diets (Table 4.5). On average the percent composition of 16:4co3, 18:lo)7, 20:lco7 and 22:2j was at least one order of magnitude more in C. gigas than was found in their algal diets (Table 4.5). 147 Table 4.4. The major (£1%) fatty adds as a percent of total fatty acids found in T. pseudonana grown under one of four different conditions of irradiance: {high white light (HIGH), low white light (LOW), low blue light (BLUE), moderate red light (N-LIMITED)}, with variation in temperature and nitrogen limitation (N-LIMITED) and used as food for larval C. gigas in three experiments (A,B,C). Values are averages of samples taken throughout each experiment. See text for details. Die t H I G H Irradiance 110 u E n r 2 s - l Color white Temp. (°C) 25 26 29 Exper iment A B C L O W 15 u E m-2 s-l white 25 26 29 A B C N - L I M I T E D B L U E 3 4 u E m - V l 2 2 u E m -2 s - i red blue 25 26 25 26 A B A B Fat ty acid 14:0 12.5 15:0 1.0 16:0 27.1 16:lco9 2.2 16:1(07 27.2 16:2o)7 1.3 16:2o4 2.9 16:30)4 3 . 7 18:10)9 0.6 18:10)7 0.7 18:30)6 0.4 18:40)3 5 .2 20:40)6 0.2 20:50)3 5 . 8 22:50)3 0.4 22:60)3 1.1 10.6 10.6 10.4 0.9 0.7 1.0 20.5 20.9 17.7 1.9 1.9 3.1 25.0 20.4 39.6 1.7 1.7 3.4 3.7 4.0 3.5 5.7 6.8 1.6 0.4 0.3 0.9 0.8 0.7 1.2 0.2 0.2 0.2 5.4 3.6 1.8 0.2 0.3 0.2 11.7 15.5 6.5 0.4 0.1 0.5 2.4 3.1 0.9 9.0 9.4 9.2 0.8 0.6 0.8 13.5 11.8 30.8 1.1 3.1 1.7 33.0 26.1 24.2 3.4 3.3 1.7 3.8 8.0 0.0 3.4 2.9 1.7 0.6 0.5 2.4 0.7 0.6 2.0 0.3 0.4 1.7 2.9 3.5 3.3 0.3 0.5 2.8 17.2 18.0 4.6 0.3 0.0 0.4 2.4 2.5 1.3 7.1 11.8 11.3 0.7 1.0 0.9 31.7 20.2 17.6 0.8 2.0 3.1 27.1 34.3 31.6 3.2 2.6 2.5 1.0 3.3 4.3 4.3 1.7 3.4 1.3 0.8 0.5 1.3 1.5 0.7 0.3 0.3 0.2 4.7 1.2 2.7 0.2 0.2 0.3 8.6 7.0 8.6 0.3 0.5 0.5 2.2 1.2 1.3 148 Table 4.5. The major (2:1%) fatty acids as a percent of total fatty acids and growth rates (d-1) of C. gigas larvae fed T. pseudonana grown under different conditions of irradiance (HIGH=high white light, LOW=low white light, BLUE=low blue light, N-LIMITED=moderate red light with nitrogen limitation) in two sequential experiments (B,C). Means ±1 S.D. are given for Experiment C. The R values for the significant correlations between the oysters' percent fatty acid composition and oyster growth rates are also given. See text, Table 4.1, and Table 4.2 for details. Experiment C Experiment B Diet-» HIGH (n=2) Htd"1)-* .067±.003 LOW (n=4) Starved LOW BLUE HIGH CHEMO Starved .040±.005 .01 .033 .049 .051 .052 .01 R Fatty acid 16:0 4.4 ±.0 3.6 ±.5 0.0 2.3 4.4 4.6 4.1 0.8 ++0.87 16:0 14.0 ±.7 11.3 ±1.1 8.2 10.9 15.6 21.8 22.3 8.1 +0.63 16:1(09 0.6 ±.0 0.7 ±.1 1.3 0.3 0.2 0.1 0.2 0.9 -0.62 16:1(07 8.6 ±.4 8.4 ±1.3 1.8 8.9 11.8 8.9 11.8 2.1 -0.61 17:0(iso) 1.0 ±.1 1.0 ±.3 <1 0.3 0.2 0.3 0.2 0.9 16:2(07 1.2 ±.0 1.3 ±.4 0.0 1.6 1.3 0.3 1.2 0.0 16:2o>4 1.6 ±.0 1.6 ±.4 <1 0.5 0.4 0.6 0.5 1.1 17:0 0.4 ±.0 0.5 ±.0 0.0 0.5 0.7 0.5 0.5 1.6 16:3(04 2.0 ±.1 0.9 ±.3 0.0 0.0 0.0 0.0 0.0 0.0 16:4(03 7.2 ±.5 7.6 ±1.7 5.5 8.9 9.1 10.4 10.6 12.8 18:0 3.2 ±.0 2.7 ±.1 5.2 2.4 3.1 4.5 4.7 4.8 18:10)9 0.7 ±.0 0.9 ±.2 4.2 0.6 0.7 0.7 1.1 1.8 --0.75 18:lo)7 9.3 ±.4 10.5 ±1.3 3.7 12.2 15.3 12.6 11.9 4.3 +0.63 18:20)4 0.8 ±.1 1.0 ±.3 0.0 0.0 0.0 0.0 0.0 0.0 18:4(03 2.0 ±.6 1.2 ±.2 0.0 0.8 0.9 0.6 0.9 0.0 ++0.72 19:30)6 0.6 ±.0 0.7 ±.9 0.0 2.3 2.1 1.6 1.4 2.2 20:10)11 1.8 ±.0 1.8 ±.1 5.0 1.7 1.7 2.4 2.2 3.0 -0.72 20:10)7 3.9 ±.2 5.1 ±.5 4.6 5.5 6.2 6.2 5.2 5.0 20:2f 0.7 ±.1 0.9 ±.2 1.6 0.9 1.1 1.3 1.0 1.9 +0.69 20:2h 0.1 ±.1 0.3 ±.2 0.0 0.6 0.6 0.9 0.7 1.1 20:40)6 1.0 ±.2 1.5 ±.2 4.4 0.0 0.0 0.0 0.0 0.0 20:5(03 15.7 ±.2 18.0 ±1.5 7.2 17.9 9.4 4.8 5.7 7.8 22:2j 4.5 ±.3 5.9 ±1.2 7.2 7.8 7.1 6.5 6.0 12.4 -0.64 21:5(03 0.5 ±.0 0.5 ±.1 2.9 0.5 0.5 0.2 0.2 0.8 -0.69 22:50)6 0.1 ±.1 0.6 ±.5 2.8 0.1 0.0 0.0 0.0 1.1 -0.78 22:5co3 0.7 ±.1 1.1 ±.2 <1 1.1 0.0 0.9 0.4 4.4 24:0 0.5 ±.5 0.5 ±.5 0.0 0.0 0.0 0.2 0.0 0.0 22:6(03 6.4 ±.1 6.5 ±1.0 2.7 7.1 3.2 3.0 3.2 11.4 + positive correlation p<0.05 ++ positive correlation p<0.01 - negative correlation p<0.05 - negative correlation p<0.01 149 Correlation analysis of the oyster's main 2% of the total F A composition) FAs with the percent FA composition of their dietary sources indicates a significant relationship between 18:lco7, 20:lco7 and 22:2j in the oysters and 16:lo>7 in their algal diets, also between 16:4co3 and 20:5co3 (diets) (Table 4.6). iv) Oyster F A composition and oyster growth The oysters contained £ 5% of the following FAs: 16:0,16:lco7,16:4co3, 18:lco7, 20:lco7, 20:5co3, 22:2j, and 22:6co3 (Table 4.5). Within the oysters, the percent composition of the following major (>2%) FAs were positively correlated with oyster growth rates: 14:0,16:0, 16:lco7,18:lo)7, and 18:4o)3; and major FAs negatively correlated with the oysters' growth rates included: 16:10)9, 18:10)9, 20:lcoll, 20:2f, 22:2j, and 22:5co6 (Table 4.5). Correlations which were not strongly influenced (R>0.3, starved animals removed from correlation) by the outliers associated with the starved animals include 14:0, 16:0, 18:4w3 (Fig. 4.3A) and 22:2j (Fig. 4.3B). Alternatively, the correlations between oyster growth rates and oyster fatty acids could be derived from a relationship between oyster FA composition and body size since growth rate was proportional to mean body size at the time of sampling for F A analysis. v) Models Oyster growth rate as a function of the biochemical composition of their algal diets was modeled using a stepwise multiple regression (model 1, Table 4.7). Initial input data included the mean values of carbon, nitrogen, protein, lipid, carbohydrate, %20:5u)3 and %22:6o)3 for each diet in each experiment versus the resulting mean larval oysters' growth rate (i.e. data as 150 in Table 4.3). The model indicated that 85% of the observed variation in oyster growth rates was explained as a positive function of carbon and a negative function of the percent composition of the E F A 20:5co3 per phytoplankton cell. In order to elucidate which biochemical component of the phytoplankton carbon was positively associated with oyster growth rates, the model was run again with only phytoplankton biochemical data (protein, lipid, carbohydrate and major fatty acids) as input. The growth rates of the oysters were shown to be a positive function of the percent composition of 14:0 and 22:6co3 and a negative function of 20:5co3 in their diets (model 2, Table 4.7). Finally, the oyster growth rates were adjusted for the differences in temperature (assuming Ql0=2), and model 1 and 2 run again. This resulted in a substantial improvement in the fit for model 2 only (multiple r2=0.93) with no change in the selected variables (model 2A, Table 4.7). Using a stepwise multiple regression (input data as above), mortality was shown to be negatively associated with carbohydrate, protein and the % composition of 22:6co3, and positively with nitrogen per phytoplankton cell (model 3, Table 4.7). 151 Table 4.6. Pearson correlations (R) between the percent composition of the major (^ 2%) fatty acids in C. gigas larvae and the major (£2%) fatty acids in their algal diets of T. pseudonana (R values in brackets). Bold fatty acids indicate possible dietary sources. Oyster correlated with phytoplankton fatty acids (R values) fatty acids 14:0 none 16:0 16:0 (0.85) 18:4co3 (0.90) 20:50)3 (-0.75) 16:lo)7 20:50)3 (-0.71) 22:6co3 (0.82) 17:0(iso) 16:10)9 (0.79) 16:2co4 (0.74) 20:4co6 (0.94) 20:5o)3 (0.73) 16:2o)4 16:lco9 (0.92) 16:2co4 (0.86) 20:40)6 (0.92) 20:5o)3 (0.73) 16:3co4 16:2o)7 (-0.77) 16:4co3 16:0 (0.78) 16:lco9 (-0.84) 16:2co4 (-0.82) 18:lco7 (0.76) 20:4co6 (-0.83) 20:5co3 (0.83) 18:0 16:0 (0.81) 16:2co4 (0.75) 18:10)7 (0.72) 18:40)3 (0.86) 18:lto7 16:lu)7 (0.72) 18:4co3 none 19:3co6 20:4w6 (0.90) 20:lcoll 18:4co3 (0.89) 20:lo)7 16:lco7 (0.73) 22:6co3 (-0.72) 20:4co6 16:10)9 (0.82) 16:2co4 (0.92) 20:4o)6 (0.92) 20:5co3 18:4o)3 (-0.81) 20:4co6 (0.74) 20:5co3 (0.80) 22:2j 16:lco7 (0.80) 22:5co3 20:5co3 (0.85) 22:6co3 18:4co3 (-0.74) 20:5co3 (0.77) 152 Table 4.7. Results of stepwise multiple regression models for oyster growth and survival as a function of food chemistry (see text for descriptions of the input data). ANOVA Model Variables t-value probability multiple r2 F P 1 oyster growth rate carbon cell-1 % 20:50)3 6.2 -7.6 0.001 0.001 0.85 30.3 <0.001 2 oyster growth rate % 14:0 % 20:50)3 % 22:60)3 3.2 -4.8 2.9 0.009 0.001 0.015 0.81 14.0 <0.001 2A temperature corrected oyster growth rates 0.93 44.3 <0.001 3 oyster mortality carbohydrate % 22:60)3 nitrogen protein -2.5 -3.6 3.3 -2.6 0.030 0.006 0.009 0.030 0.68 4.8 <0.02 153 Pig. 4.2. Specific growth rates (d"*) of larval Crassostrea gigas as a function of the mean value (averaged over the duration of the experiment) of the percent composition of a fatty acid in their algal diet (Thalassiosira pseudonana) in three sequential experiments A(O), B(#) and C(V). Least squares linear regressions are shown fitted to the data. 154 .1 Oyster growth rates (d ) Oyster growth rates (d*1) Oyster growth rates (d*1) o o o o o o o o o o o a o o to OJ cn cn -vi oo / / o -c / • / / •/ / / < < • i / i i I Fig. 4.3. Correlations between the fatty acid composition (specific fatty acids as a percent of total fatty acids) of larval Crassostrea gigas and their growth rate (A) positive correlations, (B) negative correlation. A least squares linear Une is shown fitted to the data. 156 25 20 15 10 h 1 ; r O 14:0 • 16:0 V 18:4u;3 O rfflb'& O - - cf v 0.00 0.02 0.04 0.06 0.08 Oyster growth rates (cf1) 14 12 10 8 6 4 O B O 22 :2 j 0 o O OCT 0.00 0.02 0.04 0.06 0.08 Oyster growth rates (d*) 157 DISCUSSION The fact that environmental conditions influence the biochemical composition of phytoplankton is well established. Unfortunately most of the research in this area has examined only elemental composition with relatively little work on gross chemical composition (proximate analysis) and even less research on the detailed biochemistry. Similarly, most of the research on light adaptation has focused on pigment and photosynthetic apparatus adaptations. The more important nutritional adaptations variations in protein, lipid and carbohydrate, have received relatively little attention despite the fact that phytoplankton form the basis of the marine food web. Evidence presented in this paper demonstrates that variations in environmental conditions, which can occur in either hatcheries or the natural environment, influenced phytoplankton biochemical composition and the growth rates of animals grazing on them. General Highest growth rates of larval C. gigas were obtained with diets of phytoplankton grown at higher irradiances. Higher irradiances produced diets that were significantly higher in carbon cell-l, the percent composition of the fatty acids 14:0,16:0,16:3co4 and lower in protein cell'l. A negative relationship between growth of C. gigas larvae and increases in the protein content of their food has been previously documented (Utting 1986). Positive associations between increased growth of larval bivalves and increased amounts of specific biochemical constituents in their diets are rare. 158 Researchers have suggested that the amount of carbohydrate per phytoplankton cell is important in determining phytoplankton food quality for various juvenile bivalves (Flaak and Epifanio 1978, Gallager and Mann 1982, Enright et al. 1986a,b) although data for larval bivalves (Whyte et al. 1989) are scarce. Carbohydrate is rapidly mobilized by marine bivalves, preferentially used as a respiratory substrate in juvenile and adult bivalves, normally high in healthy animals and depleted in starved ones (Bayne 1973, Gabbott and Bayne 1973, Mann 1979). In proximate analysis of phytoplankton, carbohydrate can be the lowest percentage of total biomass (Ricketts 1966) perhaps depending upon whether the species stores lipids or carbohydrates as a reserve energy source (Morris 1982, Lewin and Guillard 1963, Fogg 1965). In this study it was demonstrated that higher growth rates of larval C. gigas were associated with algal diets that were high in carbon per cell. Further analysis revealed that cells high in carbon were also high in carbohydrate, and the fatty acids 14:0, 16:0, 16:3co4. Multiple regression models suggests that the growth rates of larval C. gigas were responding more significantly to the increased percentage of 14:0 in their diets than to the carbohydrate. In the case of T. pseudonana, such cells were produced under conditions of saturating light, and there are indications that saturating light often produces cells high in carbon (Chapter 1), short chain saturated fatty acids (Orcutt and Patterson 1974, Chapter 2) and often high in carbohydrate (Morris 1981). 159 Fatty Acids Since 20:5co3 and 22:6co3 PUFAs were shown to be E F A for larval oysters (Langdon and Waldock 1981) researchers have concentrated on methods of increasing lipids and EFAs in their phytoplankton (e.g. Enright et al. 1986b, Calderwood 1989, Chapter 2, Chapter 3). However, the research presented here shows that increasing the amount of the E F A 20:5co3 in the experimental diets had a negative impact on oyster growth rates, and this is interpreted to mean that T. pseudonana has more than adequate 20:5o)3 to support the growth of larval C. gigas. T. pseudonana is typical in lipid and E F A content of many phytoplankton species fed to larval bivalves (Chuecas and Riley 1969, Volkman et al. 1989, Chapter 2), suggesting that, in general, these phytoplankters contain adequate 20:5o>3 for larval bivalves to achieve their optimum growth. It was determined by multiple regression analysis that increases in the E F A 22:6co3 had a small but significant role in increasing the growth and survival of the larvae suggesting that increasing the content of 22:6GJ3 from 1 to 3% of total fatty acids was beneficial. It has been demonstrated for three species of oyster larvae that the amount of their neutral lipid (triacylglycerides) was correlated with larval vigor, growth and survival (Holland and Spencer 1973, Creekman 1977, Waldock and Nascimento 1979), thus emphasizing the importance of lipid energy reserves for larval marine invertebrates (Holland 1978). In this study the significant positive correlations between the animals' percent complement of specific short chain saturated FAs (typically found in the triacylglycerides) and their growth rates supports this conclusion. A 160 relationship between the percent composition of saturated FAs in the total lipid of larval oysters (C. virginica) and the age of the animals was not detected by Chu and Webb (1984). Therefore it is suggested that these results which demonstrate a relationship between the growth rates of larval oysters and percent composition of the FAs 14:0 and 16:0 may provide a useful method of assessing the physiological status of the larvae. Furthermore the positive associations between dietary carbon, carbohydrate and short chain saturated FAs and the larval growth rates suggests strongly that their diets were energy-deficient even in environments containing a surplus of food items. This interpretation allows the observations that the growth of larvae was improved by diets high in carbohydrate (larval nutritional "condition" in the case of Whyte et al. 1989) and short chain saturated FAs (this study) to be combined into one consistent framework. Both carbohydrate and short chain saturated FAs are good dietary sources of rapidly metabolizable energy. Positive associations between the energy content of the diet and larval oysters' growth rates have been observed previously (Calderwood 1989). Several F A occurred in the oyster tissue that were not present in significant amounts in their diets. These included: 16:4co3, 18:lco7, 19:3co6, 20:lco7, 22:2j. From correlation analysis the production of 18:lco7 and 20:lco7 by elongation of T. pseudonana's large amounts of 16:lco7 would appear likely. Whether 18:lco7 and 20:lco7 have a structural or storage function (Ackman 1982, Sargent and Whittle 1981) in these animals is not evident from the data presented here. Correlation analysis also suggested that 161 16:4co3 could arise via the shortening of 20:5co3. The large amounts of 22:2j were not seen by Langdon and Waldock (1981) but were identified by comparison with the chromatograms shown by Ackman and Hooper (1973) as a non-methylene interrupted (NMID) 22 carbon FA. Similarly 20:2f, 20:2g and 22:2i are identified as NMID FAs as seen by Klingensmith (1982) and Whyte (1988) in C. gigas. The ecological significance of these NMID fatty acids is unknown. The fact that 22:2j increased significantly in the starved animals and was negatively correlated with oyster growth rates suggests a structural rather than a storage function (Ackman and Hooper 1973) for this F A in larval bivalves. The exact biosynthetic pathway through which 20 and 22 NMID FAs originate is unknown but hypothesized to be from a monoenoic fatty acid, such as 16:lco7 via elongation to 20:lco7, desaturation between carbons 5 and 6 (now a 20 carbon NMID FA), and then further elongation to 22:2co7 (22:A7A15)(Ackman 1982, Whyte 1988). This interpretation is supported by the significant positive correlation between the dietary supply of 16:lo)7 and the oysters' compliment of 22:2j. 162 SUMMARY By growing the marine diatom, Thalassiosira pseudonana under different conditions of light, nutrient status and temperature, it was possible to produce cells which were different in their biochemical composition. Variation in the phytoplankter's carbon, nitrogen, protein, lipid, carbohydrate and fatty acid composition was examined for its influence on the growth and survival of larval Crassostrea gigas. It is concluded that in T. pseudonana, and perhaps in other phytoplankton species of a similar composition, higher amounts of cellular carbon, primarily short chain saturated FAs and carbohydrate, improve the food quality of this algae. Greater carbohydrate (Morris 1981), a higher percent composition of saturated fatty acids (Orcutt and Patterson 1974, Chapter 2) and greater cell volume and carbon per cell (Chapter 1) can frequently be achieved by growing cells under saturating light. Therefore it seems plausible to speculate that the nutritional value of phytoplankton in the natural environment will vary depending on their position in the water column. In the hatchery situation, growing phytoplankton under high light also has the advantage of producing the greatest food per unit time. Unfortunately a constant high level of irradiance can be difficult to obtain in large scale, batch cultures which can rapidly increase in density and turbidity. As the cell densities increase the mean PFD incident upon the phytoplankon must decline, possibly resulting in a substantial drop in food quality. Futhermore it is concluded that the maximum growth rates and 163 r n a Y i T T H i m survival of larval oysters can be achieved with the correct balance of major biochemical constituents which can be provided either by a mixture of phytoplankton species (Epifanio 1979, Webb and Chu 1982) and/or by adjusting the environmental conditions under which the phytoplankton are grown (this study). Growth rates of larval C. gigas were significantly higher when fed T. pseudonana cells high in carbon, carbohydrate and the saturated fatty acids 14:0 and 16:0. Lower growth rates of larval C. gigas were associated with diets high in protein and the fatty acid 20:5co3. Variation in the fatty acid composition of the larval oysters' diet accounted for up to 93% of the variation in the oyster's growth rates. It is concluded that high energy T. pseudonana cells grown under high light and high nutrients are a superior diet for C. gigas larvae. C. gigas larvae contained ten times the percent composition of the FAs 16:4cu3, 18:1CO7, 20:lo>7 and 22:2j compared with their diet. Correlation analysis suggests that the dietary source of 16:4u)3 was 20:5o)3 and the other FAs were derived from 16:lco7. Faster growing larvae contained higher percentages of 14:0,16:0,18:4co3, and lower percentages of 22:2j. 164 G E N E R A L C O N C L U S I O N S Overall, this research on the influence of environmental conditions upon the biochemistry and physiology of marine phytoplankton, provides new information on a variety of adaptive responses. Starting with such basic physiogical data as the light intensities at which growth saturates (Ik) and the extent with which growth rate increases in response to temperature (QlO) are both presented. More detailed data on the responses of cellular carbon, nitrogen, chlorophyll a, protein, lipid, carbohydrate and fatty acid composition to light, temperature, and ocassionally nutrient limitation are provided. Efforts were made to interpret the phytoplankters' responses in 2 broad categories, those responses which were general, and those that were species specific. For example, general responses included increases in growth rates with increasing irradiance and temperature, increasing cell volume with increasing irradiance, and increasing chlorophyll a quotas with increasing temperature. Species specific responses included variation in certain fatty acids in response to variation in temperature, irradiance or growth rate. When possible, these results were interpreted in a framework of increasing scale from biochemical, to physiological, and ecological perspectives. This study is not viewed as being generally conclusive, but as a beginning. A n improved understanding of a phytoplankters' biochemical and physiological capacity to respond to environmental variation was the major objective. Efforts were made to determine whether artificial (and by logical 165 extension, natural) variation in the phytoplankton's environment, influence the efficiency of biomass transfer up the food chain. A brief summary of the highlights from each chapter is provided below. Ten species of marine phytoplankton were grown under a range of photosynthetic photon flux densities (PFDs) and examined for variation in cell volume, and carbon quota. Results suggest that in response to low PFDs phytoplankton generally reduce their cell volume and frequently reduce their carbon quota. A significant linear relationship between the log of PFD (I) and cell volume (in nine of ten species) and log PFD and carbon quota (four of ten species) was demonstrated. Responses to transient light regimes can occur on time frames of less than 1 day. When Thalassiosira pseudonana was exposed to a transient in light intensity it underwent a rapid adaptation in cell volume and carbon quota. Cells going from low light to high light reached maximum mean cell volume within 5 h, and cells going from high light to low light reached a minimum mean cell volume within 12 h. The resulting kinetic constant (k; a measure of the rate of adaptation) was considerably larger than previously reported k values. Ditylum brightwellii was observed to undergo an increase in length but no increase in width during a transient to increased irradiance. Nutrient limitation was shown to override PFD in determining cell volume and carbon quota for Heterosigma akashiwo. Cells grown at equivalent irradiances but N-limited were smaller than light-limited and nutrient-saturated cells. Therefore cell volume and carbon quota do not have 166 the same relationship with PFD when factors other than PFD control growth rate. Eight species of marine phytoplankton commonly used in aquaculture were grown under a range of photon flux densities (PFDs) and analyzed for their fatty acid (FA) composition. Fatty acid composition was shown to change considerably at different PFDs although no consistent correlation between the relative proportion of a single F A and growth rate or chl o cell-1 was apparent in the 8 species examined. Within an individual species the percentage of certain fatty acids covaried with PFD, growth rate and/or chl a cell-1. The light conditions which produced the greatest proportion of the essential fatty acids was species specific. Eicosapentaenoic acid, 20:5co3 increased from 6.1% to 15.5% of the total fatty acids oi Chaetoceros simplex (Ostenfeld) grown at PFDs which decreased from 225 mmol photons m-2 s-1 to 6 mmol photons m-2 s-1, respectively. Most species had their greatest proportion of 20:5co3 at low levels of irradiance. Conversely docosahexaenoic acid, 22:6co3, decreased from 9.7 to 3.6% of the total fatty acids in Pavlova lutheri (Droop) as PFD decreased. The percentage of 22:6co3 generally decreased with decreasing irradiances. In all diatoms the percentage of 16:0 was significantly correlated with PFD, and in three of five diatoms, with growth rate. Results suggest that fatty acid composition is a highly dynamic component of cellular physiology, which responds significantly to variation in PFD. 167 Most species of marine phytoplankton studied here showed significant variation in fatty acid composition in response to variation in temperature. With the data collected in this study it was demonstrated that the ratio of unsaturated/saturated fatty acids was a linear function of growth rate, whether growth rate was varied by temperature or PFD. This may be the first study to demonstrate that the ratio of unsaturated/saturated fatty acids varies in response to the overall rate of cellular metabolism and not the growth temperature per se. Furthermore evidence is provided that some marine phytoplankton may utilize an unusual method of variation in fatty acid composition to maintain membrane homeoviscosity. It was found that a consistent increase in the percent composition of the fatty acid 16:4u)l and a general decrease in the percentage of 16:4co3 occurred as temperature declined. The positioning of the double bond at the terminal end of the acyl chain may increase the distance between the 0)1 and other 0)3 fatty acids resulting in a lower melting point for such a membrane (Hochachka and Somero 1984). This mechanism of maintaining membrane homeoviscosity may have been largely overlooked because many studies on temperature adaptation have not determined the positions of the double bonds in poly-unsaturated fatty acids. The choice of an appropriate temperature can be useful in maximizing the percentage of essential fatty acids. For T. pseudonana the percent composition of the E F A 22:6co3 increased linearly with decreasing temperature over the range from 25 to 10 C. By growing the marine diatom, Thalassiosira pseudonana under different conditions of light, nutrient status and temperature, it was possible 168 to produce cells which were different in their biochemical composition. Variation in the phytoplankter's carbon, nitrogen, protein, lipid, carbohydrate and fatty acid composition was examined for its' influence on the growth and survival of larval Crassostrea gigas. C. gigas larva contained ten times the percent composition of the FAs 16:4co3,18:lo)7, 20:lco7 and 22:2j compared with their diet. Correlation analysis suggests that the dietary source of 16:4to3 was 20:5o)3 and the other FAs were derived from 16:lo)7. Faster growing larvae contained higher percentages of 14:0,16:0,18:4oa3, and lower percentages of 22:2j. Growth rates of larval C. gigas were significantly higher when fed T. pseudonana cells high in carbon, carbohydrate and the saturated fatty acids 14:0 and 16:0. Lower growth rates of larval C. gigas were associated with diets high in protein and the fatty acid 20:5o)3. Variation in the fatty acid composition of the larval oysters' diet accounted for up to 93% of the variation in the oyster's growth rates. We conclude that high energy T. pseudonana cells grown under high light and high nutrients are a superior diet for C. gigas larvae. Greater carbohydrate (Morris 1981), a higher percent composition of saturated fatty acids (Orcutt and Patterson 1974, Chapter 2) and greater carbon per cell (Chapter 1) can frequently be achieved by growing cells under saturating light. 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