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Aspects of iron and nitrogen nutrition in two red tide dinoflagellates, Gymnodinium sanguineum Hirasaka… Doucette, Gregory John 1988

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ASPECTS OF IRON AND NITROGEN NUTRITION IN TWO RED TIDE DINOFLAGELLATES, GYMNODINIUM SANGUINEUM HIRASAKA AND PROTOGONYAULAX TAMARENSIS (LEBOUR) TAYLOR By GREGORY JOHN DOUCETTE B.Sc, Bowling Green State University, 1979 M.Sc, Texas A & M University, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1988 © Gregory John Doucette, 1988 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. Department of BOTANY The University of British Columbia Vancouver, Canada Date (o £><SC€lK6&i 1*188 DE-6 (2/88) ABSTRACT Iron stress-mediated effects on growth, biochemical composition, iron and nitrogen uptake, and ultrastructure have been examined in the red tide dinoflagellates Gymnodinium sanguineum Hirasaka and Protogonyaulax tamarensis (Lebour) Taylor. The influence of nitrogen source (i.e. NO3 or NH4) on certain iron stress-mediated effects was studied, and some comparisons were made with nitrogen stress-mediated changes in biochemical composition The half-saturation constant for iron-limited growth (K^ = 2 0 1.7*10 M) of G. sanguineum was estimated to be 10-1000 times greater than for other neritic species investigated previously. Also, the iron requirement of this dinoflagellate, in terms of Fe/C ratios, exceeded those of certain coastal diatoms by one to two orders of magnitude. Fe/N ratios demonstrated a larger (1.5-fold) minimum iron requirement for NO3- than NH^-grown c e l l s , l i k e l y reflectin the iron content of NO3 assimilatory enzymes. Acquisition of nitrogen by Fe-deplete, NC^-grown cells was sufficiently inhibited to yield symptoms of N deficiency, revealed by decreased (ca. 1.4-fold) N quotas and free amino acid/protein ratios compared to Fe-deplete, NH^-grown cells Reductions in chlorophyll a (chl a) quotas (Qcn].) a n c* photosynthetic electron transport (PET) efficiency (as measured by in vivo fluorescence indices) occurred under Fe depletion, and are consistent with the essential role of iron in chl a and PET component (i.e. cytochromes and Fe-S proteins) biosynthesis. Nitrogen depletion affected Qcni similarly, but altered P E T efficiency to a markedly lesser extent than did Fe depletion. Iron-deplete G. sanguineum exhibited an enhanced iron transport capacity, which failed to be manifested following a transition from NH^  to NO3 nutrition. This suppression may result from concurrent iron and nitrogen stress, due to the ina b i l i t y of Fe-deplete, NH^-grown cells to rapidly assimilate NO3. The complete i n i t i a l inhibition of NO3 uptake when Fe-deplete, NH^-grown cell s were given saturating iron additions supports this idea. Iron stress caused reductions in chloroplast number and some degeneration of lamellar organization in this species. For P. tamarensis, iron limitation induced the formation of temporary (= pellic l e ) and resting (= hypnozygotes) cysts. Degenerative changes in organelle (i.e. chloroplasts, mitochondria and chromosomes) ultrastructure were largely restricted to pellicular cysts, consistent with their hypothesized role of maintaining v i a b i l i t y over brief, rather than extended (cf. hypnozygotes) exposure to adverse conditions. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES i x LIST OF FIGURES x i ACKNOWLEDGEMENTS x x i v INTRODUCTION 1 Overview 1 I r o n and n i t r o g e n i n marine systems 1 Ecology of r e d t i d e s : i r o n and n i t r o g e n c o n s i d e r a t i o n s 4 M e t a b o l i c r e l a t i o n s h i p s between i r o n and n i t r o g e n 7 O b j e c t i v e s 8 Experimental Organisms 11 CHAPTER 1. THE RED TIDE DINOFLAGELLATE GYMNODINIUM SANGUINEUM HIRASAKA: EFFECTS OF IRON STRESS ON GROWTH, CHLOROPHYLL A, IN VIVO FLUORESCENCE AND ULTRASTRUCTURE, INCLUDING SOME COMPARISONS WITH NITROGEN STRESS 15 Background 15 M a t e r i a l s and Methods 18 General c u l t u r e maintenance 18 I r o n and n i t r o g e n d e p l e t i o n 18 I r o n - l i m i t e d growth 20 A n a l y t i c a l methods 23 U l t r a s t r u c t u r e 24 R e s u l t s 24 V Iron-limited growth 24 Iron and nitrogen depletion 26 Ultrastructure 33 Discussion 39 Iron-limited growth kinetics 39 Cell volume, chl a quota and in vivo fluorescence 44 In vivo fluorescence indices 50 Ultrastructure 52 Ecological considerations . . . . 54 Summary 55 CHAPTER 2. ASPECTS OF IRON AND NITROGEN NUTRITION IN THE RED TIDE DINOFLAGELLATE GYMNODINIUM SANGUINEUM HIRASAKA: EFFECTS OF IRON DEPLETION AND NITROGEN SOURCE ON BIOCHEMICAL COMPOSITION ... 57 Background 57 Materials and Methods 60 General culture maintenance 60 Iron-replete and iron-deplete cultures . 60 Cell density and volume, F, F D, and chl a 63 C, N, protein and free amino acids 63 Iron quotas 64 Data analysis 68 Results .68 Elemental constituents and ratios 69 Biochemical compounds and ratios 7 2 v i In vivo fluorescence ratios and indices 75 Discussion 79 Effects of iron depletion 79 Effects of nitrogen source 85 Qualitative nitrogen composition 88 Ecological considerations 89 Summary . 91 CHAPTER 3. ASPECTS OF IRON AND NITROGEN NUTRITION IN THE RED TIDE DINOFLAGELLATE GYMNODINIUM SANGUINEUM HIRASAKA: EFFECTS OF IRON DEPLETION AND NITROGEN SOURCE ON IRON AND NITROGEN UPTAKE .. 93 Background 93 Materials and Methods 95 General culture maintenance 95 Iron-replete and iron-deplete cultures . 96 Cell pretreatment for uptake experiments 96 Iron and nitrogen uptake, n i t r i t e excretion and internal nitrate pools 99 Results 103 Iron uptake 103 Nitrate uptake 107 Ammonium uptake 110 Nitrite excretion and internal nitrate pools 110 v i i Discussion 113 Iron uptake 113 Nitrogen uptake 120 Conclusions 123 CHAPTER 4. THE ULTRASTRUCTURE OF IRON STRESS-MEDIATED CYSTS IN THE TOXIC RED TIDE DINOFLAGELLATE PROTOGONYAULAX TAMARENSIS (LEBOUR) TAYLOR 125 Background 125 Materials and Methods 127 General cultures maintenance 127 Iron depletion 128 Ultrastructure 129 Results 130 Iron bioassays . • 130 Life history dynamics 130 Vegetative c e l l s 133 Pellicular cysts 136 Hypnozygotes 142 Discussion 145 Life history dynamics 145 Hypnozygotes 148 Pellicular cysts 149 Vegetative c e l l s 152 Summary 153 GENERAL CONCLUSIONS 154 LITERATURE CITED 158 v i i i APPENDIX 1. G r o w t h - i r r a d i a n c e curve of G. sanguineum, w i t h a s s o c i a t e d changes i n c e l l volume 174 APPENDIX 2. Iron d e p l e t i o n of G. sanguineum: pH r e g u l a t i o n and bi o a s s a y s 176 APPENDIX 3. E f f e c t of ammonium c o n c e n t r a t i o n on G. sanguineum growth r a t e 180 APPENDIX 4. Removal of n o n - b i o l o g i c a l i r o n from G. sanguineum: a t e s t of wash volume 182 ix LIST OF TABLES Table 1 . 1 . Molar free ion activities of metals added to iron-limited growth media. Calculations were performed with MINEQL (Westall et a l . 1976) and are based on the sta b i l i t y constants of Ringbom (1963) . 10~ 4 M EDTA i s present in a l l media 21 Table 1 . 2 . Estimates of ^ m a x (d~^) a n d K i j ( M) f ° r iron-limited growth of ten neritic phytoplankton species using a Hanes-Woolf linear transformation (S//J vs. S) 42 Table 2 .1. Average c e l l volume and biochemical composition of iron-replete and iron-deplete cultures grown on nitrate or ammonium. See text for explanation of culture designations 70 Table 2 . 2 . Probability levels of significance for differences between experiments compared (as indicated) using an analysis of variance (nested design). Asterisks (*) identify comparisons precluded by heterogeneous sample variances as determined with an F-test. N.S. = not significantly different at the 95% confidence level 71 Table 2 . 3 . Individual and total percent of c e l l nitrogen quota accounted for by various nitrogen X containing biochemical compounds. See text for explanation of culture designations 7 6 Table 3.1. Size-fractioned rates of 5 5 F e C l 3 incorporation (5 pM additions) as collected on 5.0 pm (CULT/5.0 pm, n = 2) or 0.2 pm (CULT/0.2 pm, n = 2) f i l t e r s , and formation of colloidal iron in 0.2 pm f i l t r a t e (same cultures) retained by 0.2 um f i l t e r s (FILT/0.2 pm). See text for functional definition of size fractions 102 Table 3.2. Calculated values of Kp/K^ for iron-limited growth of G. sanguineum grown on either nitrate or ammonium. The equation employed i s Kp/K^ = P"Femax/P+Femax * ^ max/Qmin' t h e inverse form of equation (9) given by Morel (1987). See text for further details 119 LIST OF FIGURES Figure 1.1. Linear (A) and semi-log (B) plots of growth rate as a function of iron free ion activity for G. sanguineum. Molar iron free ion activ i t i e s in (A) may be determined by multiplyxng the plotted value by 6.3*10 ^ . Error bars = ± 1 S.D. (n = 6 to 12), and are smaller than symbol where not apparent Figure 1.2. Average c e l l volume of G. sanguineum as a function of iron free ion activity, and iron or nitrogen depletion. In Figs. 1.2-1.4: iron- (-Fe) and nitrogen- (-N) deplete cultures are plotted after the scale break; n = 2 for Fe-limited data, n = 3 for Fe-deplete point, n = 1 for N-deplete point; error bars = ± 1 S.D. and are smaller than symbol where not apparent for n 2 2 Figure 1.3. Chlorophyll a quota of G. sanguineum as a function of iron free ion activity, and iron or nitrogen depletion. Symbol labels, sample size and error bars as in Fig. 1.2 Figure 1 .4. In vivo fluorescence (F) and DCMU-enhanced fluorescence (F D) expressed per unit chl a (A), and 1-F/Fjj (B) of G. sanguineum as a function of iron free ion activity, and iron or nitrogen x i i (0,O,o) depletion. Symbol labels, sample size and error bars as in Fig. 1.2 29 Figure 1.5. Changes in c e l l density over time for G. sanguineum batch cultures grown into iron (-Fe) or nitrogen (-N) depletion. Concurrent changes in ambient NO3 concentration are shown for the -N culture 31 Figure 1.6. Plots of in vivo fluorescence (F)/chl a (A), DCMU-enhanced F (F D)/chl a (B), and 1-F/F D (C) over time for G. sanguineum batch cultures (shown in Fig. 1.5) grown into iron (-Fe) or nitrogen (-N) depletion. Values plotted for each culture are mean ± 1 S.D. for duplicate determinations of each variable; error bars are smaller than symbol where not apparent 32 Figures 1.7-1.13. Iron-replete Gymnodinium sanguineum. NEPCC culture #D354. Figure 1.7. Longitudinal section of protoplast showing the nucleus (N), an accumulation body (A), and cortical starch inclusions (S). Chloroplasts appear throughout the c e l l . Note vacuolate nature of cytoplasm. Scale = 10 um 35 Figure 1.8. Section exhibiting reticulate vacuole of pusule system (P) and peripheral regions of both flagellar openings (arrowheads). Note vesicle activity associated with flagellar canal (arrows). Scale = 2 um Figure 1.9. Site of flagellar insertion with basal regions of both flagella (arrowheads) and one flagellar root (arrow) extending into adjacent cytoplasm. Note pusular vesicle (V) bordering on longitudinal flagellar canal (F). Scale = 1 um . . . Figure 1.10. Cross-section of flagellar canal (F) and associated pusular vesicles (V). Flagellar axoneme (arrowhead) and microtubule network (arrows) surrounding the canal are apparent. Scale = 0.5 um Figure 1.11. Area containing extensive rough ER (ER), with several chloroplasts (C) and mitochondria (M) comprising characteristic close association of organelles. Note tubular cristae of mitochondria and similarity of matrix electron density to that of cytoplasm. Scale = 1 pm Figure 1.12. Portion of accumulation body with tightly packed contents showing "fingerprint"-like pattern. Scale = 1 pm Figure 1.13. High magnification of chloroplast lamellae (L) consisting of two or three closely appressed thylakoids. Lamellae are highly organized with no xiv evidence of thylakoid degeneration (cf. Figs. 1.15,1.16). Scale = 0.2 urn 35 Figures 1.14-1.17. Iron-deplete Gymnodinium sanguineum. NEPCC culture #D354. Figure 1.14. Longitudinal section of protoplast containing nucleus (N) and highly vacuolate cytoplasm. Cytoplasmic area and general organelle abundance (e.g. chloroplasts) are sharply reduced from the iron-replete condition (cf. Fig. 1.7). Vacuolar regions appear divided by the tonoplast (arrows). Scale = 5 pm 38 Figure 1.15. Association of organelles showing chloroplasts with "normal" (arrows; note thylakoids occurring only in pairs) and structurally disrupted (arrowheads) lamellae (cf. Fig. 1.13). Mitochondria (M) exhibit a reduction in matrix electron density relative to cytoplasm (cf. Fig. 1.11). Scale = 1 pm 38 Figure 1.16. Enlargement of chloroplast containing few, dilated thylakoids. (arrowheads). Scale = 0.2 pm . 38 Figure 1.17. Accumulation body with loosely arranged contents exhibiting no apparent order (cf. Fig. 1.12) . Scale =1 pm 38 X V Figure 2.1A. Formation of filterable iron ("FeC^ label) by culture f i l t r a t e (0.2 pm f i l t e r : o; 5.0 pm f i l t e r : •) and suspensions of viable (•) or glutaraldehyde-killed (•) cells following 5 pK Fe addition. B. Acquisition of iron {^FeC^, 5 pM addition) by unwashed (o) or washed (5 ml chelexed ESAW salt solution) (•) viable c e l l s and unwashed (•) or washed (•) glutaraldehyde-killed c e l l s . DPM = disintegrations*min~* 66 Figure 2.2. Elemental ratios by atoms of carbon:nitrogen (A), iron:nitrogen (B) and iron:carbon (C) for iron-replete and iron-deplete cultures grown on nitrate or ammonium. Values are mean (n =2) ± 1 S.D. Duplicate samples were analyzed for a l l individual ratio components in each of n = 2 cultures per experiment. Note different scales for Fe-replete and Fe-deplete cultures in (B) and (C) 73 Figure 2.3. Free amino acid (AA):protein (Pr) ratios, expressed as mole percent, of iron-replete and iron-deplete cultures grown on nitrate or ammonium. Average values and error terms presented are as described in Fig. 2.2 74 Figure 2 .4 . In vivo fluorescence normalized per unit chl a (A) and per unit iron (B) of nitrate- or ammonium-grown, iron-replete and iron-deplete x v i c u l t u r e s . Average v a l u e s and e r r o r terms p r e s e n t e d a r e as d e s c r i b e d i n F i g . 2.2. Note d i f f e r e n t s c a l e s f o r F e - r e p l e t e and F e - d e p l e t e c u l t u r e s i n (B) 7 7 F i g u r e 2.5. In vivo f l u o r e s c e n c e (F) and DCMU-enhanced f l u o r e s c e n c e ( F D ) i n d i c e s o f F D - F (A, r e l a t i v e u n i t s n o r m a l i z e d t o c h l a, and r e p r e s e n t a t i v e o f P S I I c a p a c i t y ) and 1-F/F D (B, u n i t l e s s , and r e p r e s e n t a t i v e o f P S I I e f f i c i e n c y ) f o r i r o n -r e p l e t e and i r o n - d e p l e t e c u l t u r e s grown on n i t r a t e o r ammonium. Average v a l u e s and e r r o r terms p r e s e n t e d a r e as d e s c r i b e d i n F i g . 2.2 .... 7 8 F i g u r e 3.1A. Uptake o f c a r b o n (0.1 p C i ' i r t l - 1 N a H 1 4 C 0 3 ) by unwashed (o, n = 3) and washed (•, n = 3) c u l t u r e s . C e l l s were washed by f i l t r a t i o n and r e s u s p e n s i o n u s i n g s t a n d a r d f i l t r a t i o n a p p a r a t u s . B. Uptake o f c a r b o n (0 .4 p C i - m l - 1 N a H 1 4 C 0 3 ) by unwashed ( o f n = 3) and washed (•, n = 3) c u l t u r e s . C e l l s were washed by r e v e r s e - f l o w f i l t r a t i o n (RFF) ( d e t a i l s i n t e x t ) . C. Uptake o f i r o n (0.08 p C i - m l - 1 5 5 F e C l 3 , 5 pM a d d i t i o n ) by unwashed (o, n = 1) and RFF-washed (•, n = 1) c u l t u r e s . E r r o r b a r s f o r (A-C) r e p r e s e n t ± 1 S.D. ( o r o n l y 1 S.D. t o p r e v e n t o v e r l a p ) and a r e s m a l l e r t h a n symbol where not a p p a r e n t f o r x v i i experiments in which n > 1. DPM = disintegrations-min -^ . 98 Figure 3.2. Time course of iron uptake (A,B) and specific iron uptake (C), following resuspension in N O 3 , for iron-replete (A) and iron-deplete (B,C) cultures grown on nitrate (o) or ammonium (•). Rates represent midpoint determinations for two adjacent measurements and are given as the mean ± 1 S.D. for two incubations, one from each of n = 2 cultures per experiment. In certain cases only 1 S.D. (either + or -) i s shown to avoid overlapping and/or to allow use of more expanded scales. Note differences in time scale between Fe-replete and Fe-deplete experiments .... 1 0 4 Figure 3.3. Time course of iron uptake (A,B) specific iron uptake rates (C), following resuspension in NH^ , for iron-replete (A) and iron-deplete (B,C) cultures grown on nitrate (o) or ammonium (•). Values at 12 h in (B) are offset for c l a r i t y . Values at 60 h in (B),(C) were s 0 and are not shown (see text for further explanation). Data points and error terms plotted are as described in Fig. 3.2. Note differences in time scale between Fe-replete and Fe-deplete experiments .... 1 0 5 Figure 3 .4. Time course of nitrate uptake rates for nitrate- (o) or ammonium-grown (•), iron-replete x v i i i (A) and iron-deplete (B) cultures resuspended in 50 pM NO3. Iron-deplete cultures also received saturating iron additions (6.56 pM Fe/14.86 pM EDTA). Data points and error terms plotted are as described in Fig. 3.2. Note differences in time scale between Fe-replete and Fe-deplete experiments 108 Figure 3.5. Time course of ammonium uptake rates for nitrate- (o) or ammonium-grown (•), iron-replete (A) and iron-deplete (B) cultures resuspended in 50 pM NH4. Iron-deplete cultures also received saturating iron additions (6.56 pM Fe/14.86 pM EDTA). Data points and error terms plotted are as described in Fig. 3.2. Note differences in time scale between Fe-replete and Fe-deplete experiments 109 Figure 3.6. Rates of n i t r i t e excretion expressed as a percent of nitrate uptake rate following resuspension in 50 pM NO3 for iron-replete (A) and iron-deplete (B) cultures grown on NO3 (o) or NH4 (•)• Negative values result from NO2- uptake, rather than excretion. Experiments are those presented in Fig. 3.4. Data points and error terms plotted are as described in Fig. 3.2. Note differences in time scale between Fe-replete and Fe-deplete experiments •••• m x i x Figure 3.7. Time course of changes i n i n t e r n a l n i t r a t e pools during NO3 uptake experiments involving iron-replete (A) and iron-deplete (B) cultures grown on NO3 (o) or NH4 (•)• Measurements were made during the experiment shown i n F i g . 3.4. Data points and error terms plotted are as described i n F i g . 3.2, except that values correspond to actual sample times rather than to midpoint determinations. Note differences i n time scale between Fe-replete arid Fe-deplete experiments .... 112 Figure 4.1. Iron bioassays performed on Day 17 (a) and Day 24 (b) showing changes i n c e l l density of -Fe (o) and +Fe (•) cultures 131 Figure 4.2. Changes i n l i f e history stage over a time course of increasing i r o n stress. Values for vegetative c e l l s (±), p e l l i c u l a r (o), and zygotic (•) cysts are expressed as percent of t o t a l c e l l s - m l " 1 132 Figures 4.3-4.8. Protogonyaulax tamarensis vegetative c e l l s . NEPCC culture #D255. Figure 4.3. Amphiesma comprised of outer membrane (0), outer plate membrane (P), thecal plates ( P l ) , p e l l i c l e (Pe), and cytoplasmic membrane (C). Scale = 0.2 ym 135 XX Figure 4.4. Longitudinal section of protoplast. C-shaped nucleus (N) contains nucleolus (Nu) around inner curvature. Crystalline material (arrowhead) is present in hypothecal vacuolar region. Scale = 5 pm 135 Figure 4.5. Chloroplast with lamellae exhibiting closely paired thylakoids (arrows). Scale = 0.5 pm 135 Figure 4.6. Grana-like stacks of tightly appressed thylakoid membranes. Scale = 0.5 pm 135 Figure 4.7. Mitochondrion with tubular cristae and characteristic matrix density. Scale = 0.2 pm .... 135 Figure 4.8. Aggregate of crystalline material showing details of crystal morphology. Vesicle (V) containing concentric membrane swirls i s apparent. Scale = 0.2 pm 135 Figures 4.9-4.12. Protogonyaulax tamarensis pellicular cysts. NEPCC culture #D255. N = nucleus. Figure 4.9. Amphiesma consisting of p e l l i c l e (Pe) subtended by thick layer of granular material (G) possibly originating from vesicles (arrowhead) derived from the cytoplasmic membrane (C). Scale = 0.1 pm 138 x x i Figure 4.10. Cyst with protoplast resembling vegetative c e l l . Note lack of storage products and presence of crystalline material (arrowheads). Scale = 5 pm 138 Figure 4.11. Cyst showing densely aggregated cytoplasm with minimal vacuolar space. Several starch granules (S) and accumulation bodies (A) are present. Scale = 5 um 138 Figure 4.12. Cyst containing numerous starch (S) and l i p i d (L) storage inclusions. Crystalline material (arrowhead) i s apparent. Scale = 5 pm 138 Figures 4.13-4.17. Protogonyaulax tamarensis pellicular cysts. NEPCC culture #D255. Figure 4.13. Chromosomes similar to those of vegetative c e l l s exhibiting highly organized transverse banding of chromatin (arrows). Scale = 0.5 pm .... 141 Figure 4.14. Chromosomes with disrupted chromatin structure. Chromatin i s clumped into electron-dense aggregates (arrows). Nucleolus (Nu) i s apparent. Scale = 1 um 141 Figure 4.15. Chloroplast showing single thylakoids and grana-like stacks containing slightly swollen membranes (arrows). Scale = 0.5 pm 141 x x i i Figure 4.16. Chloroplast exhibiting extensive tubular dilation of thylakoid membranes seen in cross-(arrowheads) and longitudinal- (arrows) section. Scale = 0.2 pm 141 Figure 4.17. Mitochondrion with matrix of reduced density and possibly fewer cristae relative to vegetative c e l l s . Note ' f i l l e d - i n ' appearance of cristae (arrows) Scale = 0.5 pm 141 Figures 4.18-4.23. Protogonyaulax tamarensis hypnozygotes. NEPCC culture #D255. Figure 4.18. Stage 1 cyst with irregular outline and nucleus containing densely-packed, somewhat i l l -defined chromosomes. Crystalline material (arrowhead) and accumulation bodies (A) are apparent. Scale = 10 pm 144 Figure 4.19. Smooth-walled, elongate Stage 2 cyst. Nucleus (N) shows discrete, electron-dense chromosomes. Note presence of crystalline material (arrowhead), cortical l i p i d inclusions (L), and an accumulation body (A) . Scale = 10 pm 144 Figure 4.20. S2 nucleolus (Nu) and associated filaments (arrowhead). Note cross-section of filament penetrating nucleolus (arrow) and presence of large nucleolar organizing chromosome (No). Scale = 1 pm 14 4 x x i i i F i g u r e 4.21. Amphiesma e x h i b i t i n g p e l l i c l e (Pe), g r a n u l a r l a y e r (G), and amorphous, e l e c t r o n - d e n s e m a t e r i a l (A) immediately adjacent t o c y t o p l a s m i c membrane (C). Membrane fragments d i s t a l t o c y t o p l a s m i c membrane are l i k e l y remains of v e s i c l e s d e p o s i t i n g amorphous m a t e r i a l . S c a l e = 0.1 pm 144 F i g u r e A.22. Annulate n u c l e a r v e s i c l e s formed as i n v a g i n a t i o n s of n u c l e a r envelope (Ne). Nu c l e a r pores (arrows) are apparent. S c a l e = 0.5 jjirt 144 F i g u r e 4.23. Aggregation of mito c h o n d r i a showing reduced m a t r i x d e n s i t y . Note d i v i d i n g m itochondrion (arrowhead). S c a l e = 1 pm 144 xxiv ACKNOWLEDGEMENTS I g r a t e f u l l y acknowledge the guidance and support of my s u p e r v i s o r , Dr. P.J. H a r r i s o n . His encouragement throughout a l l phases of my tenure at U.B.C. have p r o v i d e d a s t i m u l a t i n g and rewarding experience. I wish a l s o t o thank the other members of my s u p e r v i s o r y committee, Drs. L. O l i v e i r a , T.R. Parsons and F.J.R. T a y l o r f o r t h e i r c o n t r i b u t i o n s t o t h i s work. Many aspects of my t h i n k i n g and r e s e a r c h , as w e l l as my a c t i v i t i e s a p a r t from academia, have been e n r i c h e d by a s s o c i a t i o n s with my c o l l e a g u e s Drs. W.P. Cochlan, C P . K e l l e r , M.E. Lavesseur, J.A. Parslow, N.M. P r i c e , C.A. S u t t l e , and P.A. Thompson. T h i s work has a l s o b e n e f i t e d from i n t e r a c t i o n s w i t h many oth e r f a c u l t y and graduate students i n the Departments of Botany and Oceanography. Dr. N.M. P r i c e performed MINEQL c a l c u l a t i o n s and Dr. P. Van Der Heyden p r o v i d e d c l e a n H N O 3 f o r i r o n quota d e t e r m i n a t i o n s . The t e c h n i c a l e x p e r t i s e of M. Weis and D. Jones, and the s k i l l f u l work of the Botany Workshop s t a f f are s i n c e r e l y a p p r e c i a t e d . The c o n t i n u e d encouragement of f a m i l y and my f r i e n d s , , e s p e c i a l l y L. Pearce and P. and A. T a n n a h i l l have been i n v a l u a b l e . My t r u l y g r e a t e s t debt of thanks i s owed t o my w i f e and f r i e n d T e r r i Lynn. Your support, p a t i e n c e and l o v e have been co n s t a n t and e s s e n t i a l t o my l i f e and work. F i n a n c i a l support was p r o v i d e d by N a t u r a l S c i e n c e s and E n g i n e e r i n g and Research C o u n c i l (NSERC) Postgraduate S c h o l a r s h i p s , U.B.C. and MacMillan Family F e l l o w s h i p s , and t e a c h i n g a s s i s t a n t s h i p s . An NSERC s t r a t e g i c g r ant awarded to Dr. P.J. H a r r i s o n funded t h i s r e s e a r c h . 1 INTRODUCTION Overview Iron and nitrogen in marine systems. Iron i s the fourth most abundant element in the earth's crust (Taylor 1964) and, of the trace elements essential for phytoplankton growth, i t is quantitatively the most important (Huntsman and Sunda 1980). In natural waters, iron occurs in two oxidation states, Fe (II) and Fe (III). In oxic natural waters, the reduced and most soluble Fe (II) forms are converted to highly insoluble Fe (III) species, which predominate in oxygenated seawater (Byrne and Kester 1976, Sung and Morgan 1980). The distribution of these forms may be influenced by, among other factors, redox potential, pH, the presence of organic matter and photochemical reactions (Hong and Kester 1986). Iron has been hypothesized to be biologically available to phytoplankton as free ferrous or fer r i c ions (Anderson and Morel 1982), dissolved organic complexes (Trick et a l . 1982, Allnutt and Bonner 1987) and recently-formed (i.e. thermodynamically unstable) colloids (Wells et a l . 1983). The concentration and distribution of iron in the ocean have been examined recently by several investigators (Gordon et a l . 1982, Symes and Kester 1985, Landing and Bruland 1987, Martin and Gordon 1988). These studies, employing "ultra-clean" techniques, have minimized previous contamination problems, and yielded much lower values, and more defined and systematic profiles. Surface concentrations of dissolved 2 (i.e. < 0.4 um) iron measured at inshore stations (6000 nM, San Francisco Bay) by Gordon et a l . (1982) exceeded open ocean values (0.13 nM, N.E. Pacific) by over four orders of magnitude. However, in a surface transect across the continental shelf into the apex of the New York Bight, total iron ranged from ca. 3 to 540 nM (Symes and Kester 1985), a^ variation of slightly more than 100-fold. Further, dissolved iron concentrations as low as 0.05 nM have been measured in surface waters of the highly productive upwelling region off California (Martin and Gordon 1988). Vertical profiles of iron distributions generally show minimum surface concentrations which increase with depth and frequently show a maximum associated with the dissolved oxygen minimum (Symes and Kester 1985, Landing and Bruland 1987, Martin and Gordon 1988). The percentage of total iron in surface waters attributable to particulate (i.e. > 0.4 um) forms appears to be highly variable, with values as disparate as ca. 10% (N. Atlantic slope water, Symes and Kester 1985) and 80-90% (nearshore waters off Peru, Hong and Kester 1986) having been reported. Riverine input can be an important source of iron for nearshore environments, with average concentrations of dissolved iron in river water estimated at ca. 700 nM (Martin and Whitfield 1983). The fate of river-borne iron upon mixing with seawater i s , however, quite complex. While certain data indicate that 95% of dissolved iron i s converted to particulate form during estuarine mixing (Sholkovitz 1978), 3 other reports have demonstrated an 8-fold increase in dissolved iron in waters of intermediate salinity, relative to adjacent fresh or more saline water (Fletcher et a l . 1983). Although l i t t l e information i s available on the temporal variation of iron concentrations, factors such as snowmelt and heavy r a i n f a l l would be expected to influence river-borne contributions in coastal regions (Ingle and Martin 1971, Fletcher et a l . 1983). Apart from carbon, hydrogen and oxygen, nitrogen i s quantitatively the most important nutrient required by phytoplankton (Syrett 1981). Nitrogen i s also generally considered to be the nutrient most often limiting marine phytoplankton reproductive rates (Parsons and Harrison 1983). While nitrogen exists in five principle oxidation states in seawater (-3(NH4+, NH3), 0(N 2), +2(N20), +3(N02"), +5(N03~); .Sharp 1983), two species, NH^"*" and N03~, are the predominant sources of inorganic nitrogen utilizable by eukaryotic algae. In the surface waters of central ocean gyres, nitrogen i s consistently near analytical detection limits, with no apparent seasonal pattern (McCarthy 1980, Sharp 1983). Concentrations are invariably higher at depth. Nitrate, rather than ammonium, i s the more abundant form of nitrogen in coastal regions (Sharp 1983), and exhibits distinct seasonal trends. Surface nitrate concentrations are elevated during the winter (> 10 pM) and, following density st r a t i f i c a t i o n during the spring and subsequent u t i l i z a t i o n by phytoplankton, are eventually depleted or reduced to near zero. Ammonium 4 levels show more variability, and although generally not exceeding 3 uM (Sharp 1983), are frequently below 0.5 uM (Paasche 1988). Exceptions occur in areas subject to considerable terrigenous input (e.g. sewage outfalls), where concentrations may range from 10 to 30 pM (Thomas et a l . 1980) . Ecology of red tides: iron and nitrogen considerations. The detrimental aspects of both toxic and non-toxic red tides have been well documented (Steidinger and Haddad 1981, Ragelis 1984, Steidinger and Baden 1984, Taylor 1987). Others have recognized the importance of these blooms in their contribution to local primary production (Vargo et a l . 1987). Irrespective of their perceived effects on the immediate or surrounding environment, red tides are of considerable ecological importance. Accordingly, the identification of factors contributing to or associated with the i n i t i a t i o n and decline of these blooms has been of particular interest. In this regard, environmental cues and l i f e history stage dynamics of the causative organisms have received perhaps the most attention. It i s frequently not possible to discern an obvious relationship between the timing of bloom formation and a particular environmental parameter(s) such as nutrient levels, salin i t y , light or photoperiod (e.g. Anderson and Morel 1979). Some work suggests that favorable temperatures and oxygen concentrations are c r i t i c a l to the successful development of 5 red tides (Anderson and Keafer 1985). Other reports have cited the potential importance of components associated with t e r r e s t r i a l runoff, including trace metals, humates and sali n i t y / s t r a t i f i c a t i o n (Prakash 1975, Anderson and Wall 1975, Provasoli 1979, Anderson et a l . 1983). Among these factors, elevated levels of soluble iron, derived from river discharge, land runoff and also from sediment resuspension, have been frequently correlated with the development of red tides (Ingle and Martin 1971, Kim and Martin 1974, Glover 1978, see Iwasaki 1979, Yamochi 1984). While the stimulatory effects of other trace metals, humates or salinity reductions cannot be discounted without further investigation, this relationship may imply the alleviation of iron-limiting conditions by an increase in iron supply rate. Yet, there have been few studies on the iron nutrition of red tide dinoflagellates (or of dinoflagellates in general). If the growth of these organisms may be at least i n i t i a l l y iron-limited, as some evidence would suggest, the decline of a bloom could result from localized depletion of biologically available iron. A report by Anderson and Morel (1979), noting a sharp decrease in total iron concentration associated with the decline of a Gonyaulax tamarensis bloom, is consistent with this idea. Alternatively, nitrogen limitation has also been implicated in terminating blooms of the same species (Glibert et a l . 1988). Although reduced ambient nutrient levels may contribute to the decline of red tide populations, other explanations, such as reproductive 6 rhythms or depletion of an internally-stored product can be inferred from certain data (Steidinger and Haddad 1981, Anderson et a l . 1983). The dynamics of l i f e history stages, specifically cyst germination and formation, are closely linked with the i n i t i a t i o n and decline, respectively, of red tides (Steidinger 1983, Anderson 1984, Taylor 1987). The induction of encystment and, to a lesser extent, cyst germination, have been investigated previously. Available data indicate that both nitrogen and phosphorus depletion can effect cyst formation in several species (see review by Pfiester and Anderson 1987). Anderson (1980) has demonstrated that excystment can be induced by temperature shifts. However, f i e l d observations suggest that the optimization of other factors, apart from temperature, is required for maximal cyst germination (Anderson et a l . 1983). Given the potential importance of iron in regulating red tide population dynamics, i t i s of interest that no studies to date have examined the relationship between iron nutrition and l i f e history stage. Red tide populations may be controlled by one or a suite of environmental cues, which, further, are l i k e l y to vary with species, location and perhaps stage of bloom development. The essential nutrients iron and nitrogen would appear to be among those factors potentially important in regulating bloom dynamics. 7 Metabolic relationships between iron and nitrogen. Both i r o n and n i t r o g e n are elements e s s e n t i a l t o a l g a l metabolism. Iro n f u n c t i o n s as a coenzyme i n c e r t a i n r e a c t i o n s of the t e t r a p y r r o l e b i o s y n t h e t i c pathway r e s p o n s i b l e f o r c h l o r o p h y l l and cytochrome p r o d u c t i o n (Marschner 1986). T h i s m i c r o n u t r i e n t i s an i n t e g r a l component of cytochromes and i r o n - s u l f u r p r o t e i n s (Hipkins 1983), and of the r e d u c t i v e n i t r a t e a s s i m i l a t o r y enzymes, n i t r a t e and n i t r i t e reductase (NR and NiR, r e s p e c t i v e l y ) (Hewitt 1983). Some or a l l forms of s e v e r a l enzymes such as glutamate synthase, superoxide dismutase, c a t a l a s e , p e roxidase, hydrogenase and a c o n i t a s e r e q u i r e i r o n t o perform t h e i r c a t a l y t i c f u n c t i o n s (Hewitt 1983, R o e s s l e r and L i e n 1984, Marschner 1986, Raven 1988). The p r i n c i p l e and u b i q u i t o u s r o l e of n i t r o g e n as a c o n s t i t u e n t of c e l l u l a r p r o t e i n s and n u c l e i c a c i d s i s w e l l understood. C h l o r o p h y l l pigment molecules of p h o t o s y n t h e t i c organisms c o n t a i n c a . 6% n i t r o g e n (by atoms). A l s o , i t has been c a l c u l a t e d t h a t p h o t o l i t h o t r o p h i c ( i . e . l i g h t - d r i v e n O2 e v o l u t i o n w i t h an i n o r g a n i c carbon source) r e p r o d u c t i o n on n i t r a t e - n i t r o g e n r e q u i r e s 60% more i r o n than f o r growth on ammonium-nitrogen (Raven 1988). C l e a r l y , many aspects of i r o n and n i t r o g e n n u t r i t i o n are c l o s e l y l i n k e d . Of p a r t i c u l a r i n t e r e s t t o the c u r r e n t r e s e a r c h are the m e t a b o l i c i n t e r r e l a t i o n s h i p s of these two n u t r i e n t s as r e l a t e d t o the m e t a l l o p o r p h y r i n s c h l o r o p h y l l a and cytochromes, the n i t r a t e r e d u c i n g enzymes NR and NiR, and a l s o the non-heme i r o n s u l f u r p r o t e i n s (e.g. f e r r e d o x i n ) . 8 S u c c e s s f u l maintenance of the a s s o c i a t i o n s between i r o n and n i t r o g e n are c r i t i c a l t o the processes of p h o t o s y n t h e s i s , r e s p i r a t i o n and n i t r o g e n (predominantly n i t r a t e ) a s s i m i l a t i o n , as w e l l as the s t r u c t u r a l i n t e g r i t y of c e r t a i n o r g a n e l l e s . Because of the i r o n requirement a s s o c i a t e d w i t h NR and NiR, growth on NO3 should impart a g r e a t e r degree of i r o n s t r e s s f o r a g i v e n s u b s a t u r a t i n g ( f o r growth) i r o n c o n c e n t r a t i o n , r e l a t i v e t o growth on NH4. Apart from the work of Reuter and Ades (1987), and a l s o the t h e o r e t i c a l treatment by Raven (1988), no s t u d i e s have examined t h i s obvious i n t e r a c t i o n between i r o n and n i t r o g e n n u t r i t i o n i n phytoplankton. O b j e c t i v e s T h i s t h e s i s addresses the f o l l o w i n g t h r e e o b j e c t i v e s , which are c o n s i d e r e d i n d e t a i l below: 1. To i n c r e a s e our knowledge of d i n o f l a g e l l a t e i r o n n u t r i t i o n , w i t h emphasis on those s p e c i e s producing red t i d e s . 2. To p r o v i d e a b e t t e r understanding of the i n t e r a c t i o n between a l g a l i r o n and n i t r o g e n n u t r i t i o n . 3. To assess the s u s c e p t i b i l i t y t o i r o n - l i m i t e d growth of r e d t i d e d i n o f l a g e l l a t e s , r e l a t i v e t o o t h e r c o a s t a l p hytoplankton. L i t e r a t u r e reviews i n r e l a t i o n t o these o b j e c t i v e s are p r o v i d e d as background i n f o r m a t i o n p r e f a c i n g each chapter. 9 The preceding remarks on iron and nitrogen have attempted to outline the importance of these nutrients at levels ranging from the marine environment to cellular nutrition, as well as some of their metabolic relationships. While the literature is replete with studies of nitrogen-based uptake, metabolic and growth processes of phytoplankton, comparatively l i t t l e is known about phytoplankton iron nutrition. The latter situation clearly applies to dinoflagellates, frequently an important component of the phytoplankton community. The principle objective of the current research was to increase our knowledge of dinoflagellate iron nutrition, with emphasis on those species producing red tides. During this study, responses to iron stress as manifested through growth (Chapter 1), biochemical composition (Chapters 1 and 2), nutrient uptake (Chapter 3) and l i f e history stage (Chapter 4) were examined in red tide-forming dinoflagellates. Several attempts were also made to verify the iron stress-mediated specificity of these observations by conducting analogous experiments u t i l i z i n g nitrogen stress as a stimulus (Chapter 1). The other main goal of this work was to provide a better understanding of the interaction between algal iron and nitrogen nutrition. As the algal requirement for iron varies depending on whether nitrogen i s supplied as nitrate or ammonium, nitrogen source i s lik e l y to influence the nature of iron stress-mediated effects on these organisms. This possibility i s addressed in two chapters herein. Measurements 10 obtained in Chapter 2 characterized the quantitative and qualitative response of several biochemical variables to iron depletion (relative to iron-replete conditions) for nitrate-and ammonium-grown cultures. Nutrient uptake experiments of Chapter 3 determined rates of iron, nitrate and ammonium uptake by iron-replete and iron-deplete ce l l s grown on either nitrate or ammonium. Comparisons were further extended to include data collected during transitions between these two nitrogen sources. The latter results were useful in assessing the effect of nitrogen source and N source transitions on a cell ' s a b i l i t y to successfully adapt to changes in iron a v a i l a b i l i t y . In a more ecological sense, perhaps the most important objective of this thesis was to assess the susceptibility to iron-limited growth of red tide dinoflagellates, relative to other coastal phytoplankton. To this end, comparisons of iron nutritional characteristics were made throughout this work, between red tide and other neritic species for which similar data were available. Information pertaining to the question of growth limitation by iron has implications for the suggested role of this trace element in regulating red tide population dynamics. However, the actual balance between iron supply and phytoplankton demand (or acquisition capabilities) in coastal waters i s the most c r i t i c a l , and as yet the most uncertain factor. Nevertheless, iron stress-mediated properties of growth, composition and uptake determined herein should indicate whether this coupling between iron supply and 11 demand i s p o t e n t i a l l y t i g h t e r f o r r e d t i d e d i n o f l a g e l l a t e s t h a n might be e x p e c t e d f o r o t h e r p h y t o p l a n k t o n g r o u p s . E x p e r i m e n t a l Organisms Two p h o t o s y n t h e t i c d i n o f l a g e l l a t e s , Gymnodinium sanguineum H i r a s a k a and Protogonyaulax tamarensis (Lebour) T a y l o r , were t h e s u b j e c t s o f t h i s i n v e s t i g a t i o n o f r e d t i d e d i n o f l a g e l l a t e i r o n and n i t r o g e n n u t r i t i o n . G. sanguineum, a n o n - t o x i c , a t h e c a t e s p e c i e s , i s t a x o n o m i c a l l y synonymous w i t h G. splendens Lebour and G. nelsoni M a r t i n (Tangen 1979). The c u l t u r e used h e r e i n (#D354, N o r t h E a s t P a c i f i c C u l t u r e C o l l e c t i o n (NEPCC), Dept. o f Oceanography, 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 , Vancouver, Canada) was i s o l a t e d from E s q u i m a l t Lagoon (maximum depth 3.5 m, Watanabe and Robinson 1979), Vancouver I s . , B.C. by A. Chan i n September 1980. S t r a i n #D354 i s c a . 4.6-4.7-10 4 urn^ i n volume and measures c a . 40-50 pm a c r o s s t h e l o n g e s t d i m e n s i o n ( i . e . a n t e r o - p o s t e r o ) . G. sanguineum was employed i n C h a p t e r s 1-3 t o i n v e s t i g a t e v a r i o u s a s p e c t s o f , and r e l a t i o n s h i p s between, i r o n and n i t r o g e n n u t r i t i o n . The d e c i s i o n t o u t i l i z e t h i s s p e c i e s as a t e s t o r g a n i s m was based p r i m a r i l y on two i m p o r t a n t f a c t o r s : i t s a b i l i t y t o form r e d t i d e s and i t s w i d e s p r e a d o c c u r r e n c e t h r o u g h o u t t h e w o r l d ' s c o a s t a l w a t e r s . G. sanguineum was c i t e d by Rob i n s o n and Brown (1983) as t h e c a u s a t i v e o r g a n i s m o f a r e c u r r e n t r e d t i d e i n E s q u i m a l t Lagoon, B.C. f o r a l l but 12 one of six consecutive years documented, achieving peak c e l l densities as high as 1.14-10* c e l l s ' m l - 1 . This species has also been identified as the numerical dominant in red tides off California (Kiefer and Lasker 1975), Virginia (Zubkoff 1979), Norway (Tangen 1979), Peru (Rojas de Mendiola 1979) and West Africa (Dandonneau 1970). Additional reports extend i t s distribution to include Japan and New Zealand (references cited by Watanabe and Robinson 1979). While clearly of interest from an ecological point of view, G. sanguineum i s somewhat problematic with regard to laboratory experimentation. The major hindrance l i e s in i t s extreme sensitivity to mechanical perturbation. Physical l a b i l i t y i s a very important consideration when f i l t e r i n g or washing c e l l s , procedures essential to studies of phytoplankton physiology and biochemical composition. Several such problems were encountered herein and acceptable alternative protocols have been developed where required. Despite these l o g i s t i c a l drawbacks, the ubiquitous nature and numerical importance of G. sanguineum enhance the potential for ecologically relevant extrapolation of laboratory data to coastal dinoflagellate f i e l d populations. The other organism used in this study, P. tamarensis (= excavata), i s a toxic, thecate dinoflagellate responsible for numerous paralytic shellfish poisoning (PSP) outbreaks in many of the world's temperate coastal waters (Taylor 1984). The culture employed was NEPCC #D255, which was isolated by R. 13 Waters off Lummi Is., Washington, U.S.A. in August 1976. The taxonomy of P. tamarensis at the generic level i s the subject of an ongoing discussion in the literature. Although this species (and a group of closely related taxa - the "tamarensis group"), have been removed, by concensus, from the genus Gonyaulax, agreement on an alternative generic designation is yet to be reached. Several authors have assigned members of this group to one (or a combination) of three genera, Protogonyaulax, Alexandrium or Gessnerium, based on various c r i t e r i a (Loeblich and Loeblich 1979, Taylor 1979, Balech 1985). It was of preeminent importance that the identity of the taxonomic entity employed herein, be unambiguously discernable to the reader. Also, while slight emendations to the generic diagnosis of Protogonyaulax may be necessary (Taylor 1985), i t s members are easily distinguished. Furthermore, this designation appears to be gaining relatively widespread acceptance. Based on these c r i t e r i a , and in the absence of a unifying alternative, Protogonyaulax tamarensis was selected to identify the taxon used in this study. P. tamarensis was uti l i z e d in Chapter 4 to examine the effects of iron stress on the l i f e history stage of a red tide dinoflagellate. Of specific interest, was the possibility of iron stress-mediated cyst formation and the ultrastructure of any cysts thus produced. Apart from i t s reputation as an important red tide organism, cyst formation has been extensively documented for P. tamarensis both in laboratory cultures (Turpin et a l . 1978, Anderson et a l . 1984, Anderson 14 and Lindquist 1985) and in f i e l d populations (Anderson 1980, Anderson et a l . 1983). This species was, therefore, considered an appropriate choice to accommodate the goals of the current research. 15 CHAPTER 1. THE RED TIDE DINOFLAGELLATE GYMNODINIUM SANGUINEUM HIRASAKA: EFFECTS OF IRON STRESS ON GROWTH, CHLOROPHYLL A, IN VIVO FLUORESCENCE AND ULTRASTRUCTURE, INCLUDING SOME COMPARISONS WITH NITROGEN STRESS BACKGROUND Marine phytoplankton require a variety of trace metals (e.g. Fe, Mn, Zn, Co, Cu) for c e l l maintenance and growth. Depending on the element, concentrations in coastal regions can exceed those of oceanic waters by as much as four orders of magnitude (Brand et a l . 1983). Recent work has established a distinction in trace metal-limited growth rates between neritic and oceanic phytoplankton species (Brand et a l . 1983, Murphy et a l . 1984). The clearest pattern for those metals compared (i.e. Fe, Mn, Zn) was observed under iron limitation. A l l neritic species exhibited significant reductions in growth rate below the same substrate concentration, whereas the reproduction of oceanic species was generally not (or only slightly) limited at any Fe level tested. Unfortunately, no dinoflagellates were among those coastal species considered. Dinoflagellates account for about 30% of total productivity in the world's oceans (Yentsch et a l . 1980). Included in this ecologically diverse group are species capable of producing red tides, vir t u a l l y monospecific blooms in which c e l l densities often surpass 10^ c e l l s • l - * " (Taylor 1987). Red tide dinoflagellates can be either toxic or non-toxic. Because blooms of either type can result in the death 16 of marine organisms (Steidinger and Haddad 1981, Taylor 1987), these phenomena are of particular ecological significance. Although research continues into the nutritional factors which regulate the population dynamics of these organisms, some evidence suggests that iron bioavailability may be important in this regard (e.g. Ingle and Martin 1971, Glover 1978, Yamochi 1984). There is clearly a need for more information on the iron nutrition of dinoflagellates in general and, specifically, red tide species. Thus, a principle objective of this work was to investigate the growth of a red tide dinoflagellate, Gymnodinium sanguineum Hirasaka, under iron-limiting conditions. G. sanguineum i s a non-toxic species and blooms frequently in a British Columbian lagoon, achieving concentrations greater than 10^ c e l l s ' l - ^ (Robinson and Brown 1983). Other effects of iron limitation on which few data exist for dinoflagellates are alterations to cellular compounds in which iron i s either a constituent or required for biosynthesis. The role of iron in photosynthesis is essential. It i s a component of photosynthetic electron transfer (PET) reactions in the form of cytochromes and iron-sulfur proteins (e.g. ferredoxin), and i s necessary for the biosynthesis of chlorophyll pigments (see Reuter and Peterson 1987). Previous investigations of iron-mediated changes in algal pigments and characteristics associated with PET have employed diatoms and chrysophytes (Glover 1977, Sakshaug and Holm-Hansen 1977), cyanobacteria (Guikema and Sherman 1983, 17 Sandmann and Malkin 1983), and a chlorophyte (Verstreate et a l . 1980). The present study examines the effect of iron limitation and depletion on chlorophyll a quota (Q c nl) a n c* P E T processes (as determined by in vivo fluorescence properties) in semi-continuous and batch cultures of a dinoflagellate. Further, to provide additional support for the specificity of iron stress-mediated changes in these variables, the same measurements were made on batch cultures grown into nitrogen depletion. As nitrogen i s a constituent of both chlorophyll pigments and PET components, variations similar to those occurring under iron stress might be expected. Alterations in the photosynthetic apparatus and general c e l l ultrastructure as a result of iron deficiency have been reported for several algal species (e.g. Meisch et a l . 1980, Douglas et a l . 1986, Hilt et a l . 1987) including one dinoflagellate (Chapter 4) . Such information allows a more direct structural interpretation of an organism's biochemical or physiological response to a given stimulus. As a fin a l part of this study the ultrastructure of iron-replete and iron-deplete G. sanguineum cells was examined. Observations are compared with results from the literature, including those presented in Chapter 4 for another red tide dinoflagellate [Protogonyaulax tamarensis), and also considered with regard to chl a and in vivo fluorescence data. 18 MATERIALS AND METHODS General culture maintenance. Stock cultures of Gymnodinium sanguineum (culture #D354, North East Pacific Culture Collection, Dept. of Oceanography, University of British Columbia) were maintained on f i l t e r - s t e r i l i z e d (Millipore 0.45 pm), ESAW-enriched a r t i f i c i a l seawater (Harrison et a l . 1980) with several adjustments to the original medium. Silicon was omitted while Na2glyceroP0^ and FeNH^(SO4)2•6H2O were replaced by equimolar concentrations of Na2HP04 and FeCl3«6H20, respectively. Na2Mo04'2H20 was added at a concentration of 0.52 pM. Deionized d i s t i l l e d water (DDW) and reagent grade chemicals were used in preparing salt and nutrient enrichment solutions. Culture vessels employed throughout this research were soaked in freshly-made 10% HCl (v/v) for at least 2-3 days and rinsed thoroughly with DDW prior to use. A l l cultures (i.e. stock and experimental) were grown at 17°C without st i r r i n g , due to this species' sensitivity to physical perturbation. Continuous illumination was supplied by eight Vita-Lite UHO fluorescent tubes (four on either side of culture vessels) f i l t e r e d through 3 mm thick blue Plexiglas R (No. 2069, Rohm and Haas) at an irradiance of 145 pE-irT^'s"* (saturating for growth of G. sanguineum, Appendix 1). Iron and nitrogen depletion. Media used to achieve iron (-Fe ESAW) or nitrogen (-N ESAW) depletion were modifications of stock maintenance ESAW with residual trace metal 19 contamination minimized by treatment with Chelex 100 ion exchange resin (Morel et a l . 1979). Iron was omitted from -Fe ESAW. EDTA was combined with the remaining trace metals and i t s concentration reduced to provide an EDTA:trace metal ratio of 1.6. -N ESAW contained no added nitrogen. Media were ster i l i z e d by autoclaving after adjusting the pH to ca. 5.5 using Suprapur R HC1 (Merck). After s t e r i l i z a t i o n , pH was re-equilibrated to ca. 8.0-8.1 by bubbling with st e r i l e (Millipore 0.22 pm) ai r . Chelexed, f i l t e r - s t e r i l i z e d (Millipore 0.22 pm) NaHCC^ was added at a fin a l concentration of 2 mM after autoclaving to avoid possible carbon limitation at high batch culture c e l l densities. Iron (triplicate cultures) and nitrogen (single culture) depletion experiments were carried out in 2.8 1 polycarbonate (PC) Fernbach flasks and initiated by inoculating with early stationary phase stock cultures to ca. 100 cells«ml~^. Continuous bubbling with st e r i l e (Millipore 0.22 pm) air or 1-2% CC>2 was required to control changes in pH (Appendix 2). Several variables, including c e l l density (CD), average c e l l volume (CV), chlorophyll a (chl a), and in vivo fluorescence (F) and DCMU-enhanced F (F D), were monitored during the course of an experiment. Values for nutrient-deplete (-Fe or -N) cultures correspond to exhaustion of the growth-limiting nutrient as determined by no change or a decrease in CD on successive days. Depletion of the desired nutrient was confirmed for both -Fe (Appendix 2) and -N cultures by 20 bioassay and, in the case of the latter, by monitoring ambient NO3 concentrations. Iron-limited growth. Preparation of media was the same as described for iron depletion experiments except that following st e r i l i z a t i o n pH was allowed to re-equilibrate (ca. 8.0-8.1) during dark storage at 4°C, and ESAW NaHC03 concentrations were not supplemented. Also, the following changes in EDTA and trace metal enrichments were made. Six types of ESAW media, with EDTA and trace metals based on the formulation of Brand et a l . (1983), were designed to achieve a range of iron-limited (as well as iron-replete) growth conditions by varying iron free ion a c t i v i t i e s . EDTA and iron (FeCl3*6H20) were combined in single solutions with iron adjusted to provide f i n a l total concentrations ( F e ^ ^ ^ ) of 10~9, 10"8, 10*7, 10~6, 10"5 and IO"4 (= iron-replete) M. The remaining trace metals were prepared as one stock solution without EDTA. Iron and other trace metal free ion act i v i t i e s were buffered by maintaining the f i n a l EDTA concentration at 10~4 M. Transition metal free ion a c t i v i t i e s for each of the six media types (Table 1.1) were calculated using the chemical equilibrium program MINEQL (Westall et a l . 1976) and the sta b i l i t y constants of Ringbom (1963) for an ionic strength of 0.7 at pH 8.1. Computations take into account a l l changes in original ESAW enrichments and thus provide reasonable estimates of pFe (i.e. negative log Fe free ion activity) for this system based on the conditions and st a b i l i t y constants invoked. Changes in pFe due to adsorption of free ions (Fe) 21 Table 1.1. Molar free ion activities of metals added to iron-limited growth media. Calculations were performed with MINEQL (Westall et a l . 1976) and are based on the stability constants of Ringbom ( 1 9 6 3 ) . 1 0 ~ 4 M EDTA i s present in a l l media. P F e t o t a l * _ ** pFe pMn pZn pCu pCo 4.0 , 5.0-9.0+ 16.0 , 18.2-22.2+ 7.6 8.4 9.6 10.9 12.9 14.2 10.4 11.7 * Negative log of total iron concentration. ** Negative log of iron free ion activity. -j- Range includes individual values of 5.0, 6.0, 7.0, 8.0, 9.0. =f Range includes individual values of 18.2, 19.2, 20.2, 2 1 . 2 , 2 2 . 2 . 22 onto polycarbonate culture vessels was assumed to be negligible. It should be recognized that the maximum error in pFe values w i l l be associated with the lowest and highest iron additions due to the assumption of no iron contamination and to the addition of equimolar iron and EDTA, respectively. The latter, more poorly buffered system, exhibits a greater tendency for iron precipitation and higher free ion activities of other transition metals (see Table 1.1). Experiments were run in duplicate as semi-continuous cultures (85 ml polycarbonate Oak Ridge tubes, Nalgene) established by transferring iron-deplete ce l l s into each of the six iron concentrations. Cultures were maintained in early to mid-exponential phase (ca. 100-600 cells'ml -*) by dilution with fresh medium, and specific growth rates calculated based on changes in c e l l density. Growth rates for each pFe represent an average of 6-12 sequential growth curves among which doubling times varied by less than ca. 15%. Kinetic parameters of maximum growth rate (Pmax) a n d half-saturation constant for growth (K^) were estimated from a Hanes-Woolf linear transformation given in Equation (1): S/u = K^/umax + (l/umax)'S, (1) where S = molar substrate concentration, u = specific growth rate, K^ = S at half-maximal growth rate, and umax = maximal growth rate. Approximate steady-state determinations of chl 23 a, F, and F D were made twice on dupl ica te c u l t u r e s of each pFe f o l l o w i n g an accl imation period of at leas t 6-7 generations. Analytical methods. C e l l counts were performed on a Coul ter Counter R model TAII (200 pm aperture, 44.2 pm c a l i b r a t i o n spheres) , with data for average c e l l volume obtained simultaneously from a p a r t i c l e s i z e d i s t r i b u t i o n based on equivalent s p h e r i c a l diameter. Samples were homogenized ( i . e . mixed) p r i o r to counting by gentle i n v e r s i o n . Chl a concentration of f i l t e r e d samples (gravity f i l t r a t i o n , Whatman 934-AH) was determined f l u o r o m e t r i c a l l y (Holm-Hansen et a l . 1965) i n 90% acetone extracts (20 h, 4°C) using a Turner Designs model 10 fluorometer f i t t e d with the f o l l o w i n g set of Corning f i l t e r s : 3-66, reference ; 5-60, e x c i t a t i o n ; 2-64, emission. I t should be noted that gr inding f i l t e r s p r i o r to ex t rac t ion was unnecessary as ground extracts produced s i m i l a r or s l i g h t l y lower y i e l d s . In vivo F and Fp measurements of d a r k - e q u i l i b r a t e d (20 min, see Loftus and S e l i g e r 1975) c u l t u r e a l i q u o t s employed the fluorometer and f i l t e r s used f o r c h l a determinations. Samples f o r FQ were t reated with 10"^ M ( f i n a l concentration) 3-(3,4 d i c h l o r o p h e n y l ) - l , 1 - d i m e t h y l u r e a (DCMU) p r i o r to dark e q u i l i b r a t i o n . Readings were taken f o l l o w i n g 30 s exposure to the fluorometer l i g h t source. Ambient NO3 concentrations (NO3 + NO2) i n - N batch cul ture were measured with a Technicon Autoanalyzer R II according to the procedure of Wood et a l . (1967) . 24 Ultrastructure. Iron-replete and iron-deplete cultures were sampled for transmission electron microscopy (TEM) by collecting cells on 2 um Millipore f i l t e r s (type BS) and processing as follows: primary fixation with 1.5% glutaraldehyde in 0.1 M sodium cacodylate and 0.4 M sucrose (2 h, room temp.), and post-fixation with 1% osmium tetroxide in 0.1 M sodium cacodylate (1 h, room temp.). Samples were en bloc stained using 1% aqueous uranyl acetate, dehydrated in a graded ethanol/propylene oxide series, and embedded in Epon 812. A diamond knife was used to cut random and serial sections which were picked up on formvar-coated 50-mesh copper grids, stained with saturated uranyl acetate (in 50% methanol) and lead citrate, and examined in a Zeiss EM10C transmission electron microscope. RESULTS Iron-limited growth. Specific growth rates (u) of G. sanguineum as a function of iron free ion activity are presented in linear (Fig. 1.1A) and semi-log (Fig. 1.1B) plots. The more conventional linear graph, including a l l iron concentrations examined (note scale break between two highest concentrations), demonstrates the hyperbolic relationship between p and substrate concentration (S). A semi-log plot of these data shows more clearly that the most c r i t i c a l S interval occurs between pFe 20.2 (^ t o t a l = 10""^ ) and 21.2 ( F e t o t a l = 10~ 8), over which u declined from 0.27 to 0.13 d" 1. This difference in p i s at least twice that observed between F i g u r e 1.1. L i n e a r (A) and semi-log (B) p l o t s of growth r a t as a f u n c t i o n of i r o n f r e e i o n a c t i v i t y f o r G. sanguineum. Molar i r o n f r e e i o n a c t i v i t i e s i n (A) may be determined by m u l t i p l y i n g the p l o t t e d v a l u e by 6.3-10 . E r r o r bars = ± S.D. (n = 6 t o 12), and are s m a l l e r than symbol where not app a r e n t . 0.50 < cr o Q: O 0.50 0.10-20 40 60 80 1600 [Fe] (M-6.3-10- 2 0 ) 22.2 21.2 20.2 19.2 18.2 17.2 16.2 pFe (M) 26 any other pair of adjacent S values. The kinetic parameters for iron-limited growth were derived from a Hanes-Woolf linear transformation which plots S/u against S (r* = 0.999). The maximum growth rate O M A X ) i s 0.38 d~*, while the estimated -2 0 half-saturation constant (K^) i s 1.7*10 M. With the exception of the fluorescence index 1-F/FD, a l l variables and their ratios remained essentially constant above pFe 20.2 but changed rapidly as iron free ion activity decreased from pFe 20.2 to 21.2. While average c e l l volume (Fig. 1.2) declined by 35% over this interval, Q c n i (Fig* 1.3) was reduced by half. Conversely, both F and F D normalized per unit chl a (Fig. 1.4A) were ca. two-fold greater at pFe 21.2 than at 20.2. 1-F/FD (Fig. 1.4B) showed a generally decreasing trend with increasing iron stress. Ratios for severely Fe-limited cells (pFe 21.2 and 22.2) were 20-30% below those exhibited under iron-replete conditions (pFe 16.0). Iron and nitrogen depletion. Iron depletion (i.e. terminal point of a batch culture) caused a l l cellular characteristics monitored (Figs. 1.2-1.4) to change in the same relative direction as with increasingly Fe-limited growth (i.e. semi-continuous cultures) below pFe 20.2. Iron-deplete and the most Fe-limited cells (pFe 22.2) differed to the largest extent (ca. two-fold) in Qcni (1.6 and 2.9*10~* g* l i t e r c e l l vol"*, respectively; Fig. 1.3) and F/chl a (320 and 150, respectively; Fig. 1 .4A). 27 Figure 1.2. Average c e l l volume of G. sanguineum as a function of iron free ion activity, and iron or nitrogen depletion. In Figs. 1.2-1.4: iron- (-Fe) and nitrogen- (-N) deplete cultures are plotted after the scale break; n = 2 for Fe-limited data, n = 3 for Fe-deplete point, n = 1 for N-deplete point; error bars = ±1 S.D. and are smaller than symbol where not apparent for n £ 2. 6.0 o g 1.0-< 0 i 1 — / / — i 1 — 1 1 1 1 1 — DEPL 22.2 21.2 20.2 19.2 18.2 17.2 16.2 pFe (M) 28 Figure 1.3. Chlorophyll a quota of G. sanguineum as a f u n c t i o n of iron free ion activity, and iron or nitrogen d e p l e t i o n . Symbol labels, sample size and error bars as in Fig. 1.2. 1.00 0 . 8 0 -< I r— o o £ 0 . 6 0 0) O — 0 . 4 0 -0 . 2 0 -DEPL 22 .2 21 . 2 20 .2 19.2 18.2 17.2 16.2 pFe (M) 29 Figure 1 . 4 . In vivo fluorescence (F) and DCMU-enhanced fluorescence (F D) expressed per unit chl a (A), and 1-F/FD (B) of G. sanguineum as a function of iron free ion activity, and iron or nitrogen (0,0,o) depletion. Symbol labels, sample size and error bars as in Fig. 1.2. 600 DEPL 22.2 21.2 20.2 19.2 18.2 17.2 16.2 pFe (M) 30 Comparison of iron- and nitrogen-deplete c e l l s showed similar measurements of CV (Fig. 1.2) and Qcnx (Fig. 1.3). The most notable distinction between these two nutrient-stressed conditions occurred in ratios of which F and, to a much lesser degree, F D were components. F/chl a (Fig. 1.4A) and F D/chl a (Fig. 1.4A) exceeded N-deplete values by 2.5-fold (ca. 160%) and 1.6-fold (ca. 60%), respectively, while 1-F/FD (Fig. 1.4B) was 40% lower for Fe-deplete c e l l s . Furthermore, the F/chl a ratio of N-deplete ce l l s (125, Fig. 1.4A) was ca. 20% less than for the most Fe-limited condition (pFe 22.2), but s t i l l over 2.5-fold greater than for nutrient sufficiency (pFe 16.0). Nitrogen depletion resulted in a less than 10% decrease of 1-F/FD below the nutrient-replete index (i.e. from 0.68 to 0.62) as compared to 25% (0.51) and 43% (0.39) for severe Fe limitation (avg. for pFe 21.2 and 22.2) and Fe depletion, respectively (Fig. 1.4B). In the absence of available chl a and in vivo fluorescence data for N-limited c e l l s , Figs. 1.5 and 1.6 show changes in ratios of these variables during batch culture growth into nitrogen depletion (-N). Comparable data for iron depletion (-Fe) were also obtained (Figs. 1.5,1.6). Ambient NO3 was undetectable in the -N culture by Day 9 (Fig. 1.5). Thus, chl a and in vivo fluorescence measurements on Day 12 are representative of moderately N-deficient conditions (i.e. continuing growth in the absence of ambient NO3) while those of Day 14 are associated with either severe N deficiency or depletion (i.e. cessation of c e l l division). Under conditions 31 Figure 1.5. Changes in c e l l density over time for G. sanguineum batch cultures grown into iron (-Fe) or n i t r o g e n (-N) depletion. Concurrent changes in ambient NO3 concentration are shown for the -N culture. TIME (d) 32 Figure 1.6. Plots of in vivo fluorescence (F)/chl a ( A ) , DCMU-enhanced F (F D)/chl a (B), and 1-F/Fp (C) over time for G. sanguineum batch cultures (shown xn Fig. 1.5) grown into iron (-Fe) or nitrogen (-N) depletion. Values plotted for each culture are mean ± 1 S.D. for duplicate determinations of each variable; error bars are smaller than symbol where not apparent. 400 ol X o ol X u o u. o 300-200 100 600 400-200 0.80-0.60-0.40 0.20 i 1 i - r • • -Fe O O - N •o-—" • o* -1 . • I - • • I • i i..- i . ! 0 2 4 6 i • i • i 1 i • i • i • i • 8 10 12 14 16 18 20 22 TIME (d) 33 of moderate N deficiency, F/chl a (Fig. 1.6A) and F D/chl a (Fig. 1.6B) were only ca. 25% greater (Day 12: F/chl a, 65; F D/chl a, 165) than for logarithmically growing c e l l s (Day 4; lag phase s t i l l apparent on Day 2, Fig. 1.5). By Day 14 more notable increases in both ratios, to within ca. 15% of N-deplete values (Day 16: F/chl a, 125; F D/chl a, 325), had occurred. Of particular interest were the minimal changes in 1-F/FD (Fig. 1.6C), which remained between 0.58 and 0.69 irrespective of culture nitrogen status. In contrast to N stress, F/chl a (Fig. 1.6A) and F D/chl a (Fig. 1.6B) of the -Fe culture began to rise steadily during mid-exponential phase (Day 9), with both ratios exceeding N-deplete measurements prior to stationary phase (Day 15: F/chl a , 155; F D/chl a, 355). 1-F/FD (Fig. 1.6C) also exhibited a similar (but directionally opposite, i.e. decreasing) trend (Day 15, 0.56) although i t s response lagged those of F/chl a and F D/chl a by one sampling period (i.e. 3 days). Ambient iron concentrations, and thus estimates of iron status, were not determined during -Fe culture growth. Ultrastructure. Iron-replete protoplasts (Fig. 1.7) were characterized by extensive vacuolar space interspersed with regions of cytoplasm. A predominant feature was the nucleus, which contained numerous permanently-condensed chromosomes and exhibited a granular layer associated with i t s double-membrane envelope (enlargement not shown herein; for morphological details see Stone and Vesk 1982). Another distinctive structure was the pusule system (Fig. 1.8). This reticulate 34 Figures 1 . 7 - 1 . 1 3 . Iron-replete Gymnodinium sanguineum. NEPCC culture #D354. Fig. 1 . 7 . Longitudinal section of protoplast showing the nucleus (N), an accumulation body (A), and cortical starch inclusions (S). Chloroplasts appear throughout the c e l l . Note vacuolate nature of cytoplasm. Scale = 10 pm. Fig. 1 . 8 . Section exhibiting reticulate vacuole of pusule system (P) and peripheral regions of both flagellar openings (arrowheads). Note vesicle activity associated with flagellar canal (arrows). Scale = 2 pm. Fig. 1 . 9 . Site of flagellar insertion with basal regions of both flagella (arrowheads) and one flagellar root (arrow) extending into adjacent cytoplasm. Note pusular vesicle (V) bordering on longitudinal flagellar canal (F). Scale = 1 pm. Fig. 1 .10 . Cross-section of flagellar canal (F) and associated pusular vesicles (V). Flagellar axoneme (arrowhead) and microtubule network (arrows) surrounding the canal are apparent. Scale = 0.5 pm. Fig. 1 . 1 1 . Area containing extensive rough ER (ER), with several chloroplasts (C) and mitochondria (M) comprising characteristic close association of organelles. Note tubular cristae of mitochondria and similarity of matrix electron density to that of cytoplasm. Scale = 1 pm. Fig. 1 .12 . Portion of accumulation body with tightly packed contents showing "fingerprint"-like pattern. Scale = 1 pm. Fig. 1 .13 . High magnification of chloroplast lamellae (L) consisting of two or three closely appressed thylakoids. Lamellae are highly organized with no evidence of thylakoid degeneration (cf. Figs. 1 . 1 5 , 1 . 1 6 ) . Scale = 0.2 pm. 35 36 vacuolar network surrounded the area of flagellar insertion (Fig. 1.9). Whether the pusule system comprised one or two pusules (see Taylor in press), could not be distinguished. The presence of vesicles was observed throughout the pusule system, but was concentrated more toward the c e l l surface (Figs. 1.8,1.10) and around the longitudinal flagellar canal (Fig. 1.9). Numerous chloroplasts and mitochondria, as well as dense aggregates of endoplasmic reticulum (most notably rough ER), occurred throughout the c e l l , while starch and l i p i d reserves were restricted primarily to cortical regions (Figs. 1.7,1.11). One or two accumulation bodies, often comprising tightly-packed material in a "fingerprint"-like pattern, were present in some cells (Figs. 1.7,1.12). Organelles were generally grouped closely within small areas of cytoplasm due to the vacuolate nature of the protoplast (Figs. 1.7,1.11). Chloroplasts exhibited lamellae consisting of two or three appressed thylakoids (Fig. 1.13). Orientation of lamellae was variable, ranging from closely parallel to severely undulating. Mitochondria contained tubular cristae within a granular matrix, similar in electron density to the surrounding cytoplasm (Fig. 1.11). Iron-deplete protoplasts (Fig. 1.14) were considerably different from those growing under iron-replete conditions (Fig. 1.7), as demonstrated by comparing similar whole-cell longitudinal sections. Most obvious was a decline in the cytoplasm to vacuole ratio. Vacuolar regions occupied much of the c e l l periphery and appeared to be compartmentalized by the 37 Figures 1.14-1.17. Iron-deplete Gymnodinium sanguineum. N E P C C culture #D354. Fig. 1.14. Longitudinal section of protoplast containing nucleus (N) and highly vacuolate cytoplasm. Cytoplasmic area and general organelle abundance (e.g. chloroplasts) are sharply reduced from the iron-replete condition (cf. Fig. 1.7). Vacuolar regions appear divided by the tonoplast (arrows). Scale =5 um. Fig. 1.15. Association of organelles showing chloroplasts with "normal" (arrows; note thylakoids occurring only in pairs) and structurally disrupted (arrowheads) lamellae (cf. Fig. 1.13). Mitochondria (M) exhibit a reduction in matrix electron density relative to cytoplasm (cf. Fig. 1.11). Scale = 1 um. Fig. 1.16. Enlargement of chloroplast containing few, dilated thylakoids. (arrowheads). Scale = 0.2 pm. Fig. 1.17. Accumulation body with loosely arranged contents exhibiting no apparent order (cf. Fig. 1.12). Scale = 1 pm. I 3 8 39 tonoplast (Fig. 1.14). Organelles remained in close association within the available cytoplasm; however, the abundance of ER was much reduced (Fig. 1.15). Also characteristic of Fe-deplete ce l l s was a notable (albeit unquantified) decrease in chloroplast number (Fig. 1.14). Thylakoids occurred in pairs or singly (cf. Figs. 1.11,1.13). Degenerative structural changes were evident in the separation of adjacent thylakoids (Fig. 1.15) and the dilation of individual thylakoids (Fig. 1.16). Many chloroplast lamellae (and their constituent thylakoids) did, however, retain a "normal" appearance (Fig. 1.15), and no clear difference in the number of lamellae per chloroplast was discernable. Alterations in mitochondrial morphology associated with iron depletion appeared predominantly as reductions in electron density of the matrix (Fig. 1.15, cf. Fig. 1.11). Accumulation body contents of Fe-deplete ce l l s (Fig. 1.17) were loosely arranged and showed no pattern or structural organization (cf. Fig. 1.12). DISCUSSION Iron-limited growth kinetics. Growth rates of several coastal diatom species and a neritic coccolithophorid, as a function of iron bioavailability, have been examined previously (Brand et a l . 1983, Harrison and Morel 1986). The current work i s the f i r s t to examine iron-limited growth in a coastal red tide dinoflagellate, G. sanguineum. Although meaningful comparisons are possible among these data, i t is 40 f i r s t necessary to account for differences between pFe values given in Brand et a l . (1983), and those provided herein and also by Harrison and Morel (1986). Briefly, equilibrium models in seawater employ a series of interdependent calculations to estimate concentrations of chemical species. These thermodynamic models are formulated sequentially according to the major ionic composition of the medium, interactions among these components, and chemical reactions involving trace constituents (Kester 1986). Metal free ion activi t i e s are determined largely by how a system i s defined (e.g. ionic strength, pH, etc.) and the sta b i l i t y constants of chemical species present, and can vary considerably depending on the data input to satisfy these c r i t e r i a . Media formulations used in the present study and by Brand et a l . (1983) are very similar; however, estimates of iron free ion act i v i t i e s based on the st a b i l i t y constants of Ringbom (1963) and S i l l e n and Martell (1964), respectively, d i f f e r by an order of magnitude (higher in the former case). For purposes of this discussion pFe calculations of Brand et a l . (1983) w i l l be considered as ten-fold underestimates to allow direct comparison of kinetic constants for iron-limited growth. In other words, while erroneous values are not inferred for either study, pFe 20.2 of Brand et a l . (1983), for example, is taken as equivalent to pFe 19.2 herein. Values of pFe given by Harrison and Morel (1986) are roughly equivalent to those determined herein, as free ion activities of both media were 41 calculated using MINEQL (Westall et a l . 1976) with similarly defined systems and the st a b i l i t y constants of Ringbom (1963). Iron-limited growth of G. sanguineum i s similar to that reported for other neritic phytoplankton (Brand et a l . 1983, Harrison and Morel 1986) in that a l l growth rates begin to decline rapidly below pFe 20.2. However, a more c r i t i c a l comparison of these data i s possible based on the kinetic parameters of p m a x and K^. These parameters were estimated herein and by Harrison and Morel for Thalassiosira weissflogii using a Hanes-Woolf linear transformation. Although Brand et a l . (1983) did not calculate these growth constants for the nine coastal species examined, I determined values of Umax and KJJ from S/u vs. S (i.e. Hanes-Woolf) plots of the numerical data provided by these authors. It was assumed that the maximum growth rate had been achieved for these species as u varied by 10% or less between the two highest substrate concentrations in a l l but one case, which was not considered. Estimated values of u m a x and for the iron-limited growth of ten neritic phytoplankton species are given in Table 1.2. Maximum growth rates range four-fold from 0.38 (G. sanguineum) to 1.54 (T. weissflogii) d~*. However, the molar free ion concentration which limits growth by 50% (i.e. K^) of the dinoflagellate i s 10-1000 times greater than calculated for the other predominantly diatom species. This large disparity in K^, although i n i t i a l l y unexpected, may be better understood by examining minimum iron requirements of 42 Table 1 . 2 . Estimates of Pmax (d~*) and (M) for iron-limited growth of ten neritic phytoplankton species using a Hanes-Woolf linear transformation (S/(i vs. S). Species 'max K, reference Dinophyceae Gymnodinium sanguineum 0 . 3 8 1 . 7 - 1 0 - 2 0 Bacillariophyceae Asterionella glacialis Bacteriastrum hyalinum Ditylum brightwellii Lithodesmium undulatum Skeletonema costatum Streptotheca tamesis Thalassiosira pseudonana Thalassiosira weissflogii Haptophyceae Hymenomonas carterae 1 . 2 0 1 . 7 •10 0 . 9 0 1 . 8 •10 0 . 9 6 1 . 0 •10 1 . 0 2 6 . 2 •10 1 . 3 7 1 . 2 •10 1 . 0 0 1 . 7 •10 1 . 1 6 3 . 7 •10 1 . 5 4 1 . 1 •10 0 . 6 8 9 . 0 •10 , - 2 2 , - 2 2 R 2 2 , - 2 2 , - 2 2 , - 2 2 , - 2 3 >-21 2 2 2 2 2 2 1+ • * - 2 3 *1 - this study **2 - Brand et a l . ( 1 9 8 3 ) : pFe values given were recalculated using MINEQL (Westall et a l . 1976) and the st a b i l i t y constants of Ringbom ( 1 9 6 3 ) prior to linear transformation (see text for explanation) +3 - Harrison and Morel ( 1 9 8 6 ) 43 the two species for which such data are available, G. sanguineum (Chapter 2) and T. weissflogii (Harrison and Morel 1986). The carbon content per unit c e l l volume i s inherently different between dinoflagellates and diatoms (Chan 1978, 1980). Thus, minimum iron quotas (QFemin) expressed per unit c e l l carbon are l i k e l y the most valid means of comparing iron requirements. Because carbon quotas (Q^) associated with Q F emin were not provided by Harrison and Morel (1986), Qc of log phase cel l s for the same T. weissflogii clone (Blasco et a l . 1982) was used in the calculation. Further, i f the iron stress resulting in Q F emin reduces Qc of T. weissflogii, as noted for G. sanguineum (16%, NC^-grown c e l l s ; Chapter 2), Fe:C ratios may be slightly underestimated in the former species. Fe:C (by atoms) values of 1.1-10-4 and 7.6-10"^ obtained for G. sanguineum and T. weissflogii, respectively, indicate that the minimum cellular iron requirement of this red tide dinoflagellate may exceed that of a coastal diatom by over 100-fold. It should be noted that in an earlier study of T. weissflogii iron nutrition, Anderson and Morel (1982) obtained a Q F emin 4-9 times greater than reported by Harrison and Morel (1986). Nevertheless, i t would appear that the order of magnitude difference in for Fe-limited growth between G. sanguineum and T. weissflogii may be related to the correspondingly large cellular iron requirement of the former species. 44 Cell volume, chl a quota, and in vivo fluorescence. CV, Qjchl' and F and F D/chl a determinations are consistent with those of growth rate in identifying pFe 20.2-21.2 as the most c r i t i c a l range of substrate concentrations. Changes in these variables were clearly associated with iron limitation. However, comparable trends (i.e. increases or decreases) have been observed with other forms of environmental stress and, in certain cases, the possibility of a more generalized response to lower growth rates must also be considered. For example, while iron depletion reduced CV by ca. 60%, nitrogen depletion or low irradiance levels (Appendix 1) can cause CV to decline by over 50% in G. sanguineum. While the response of c e l l characteristics derived from chl a and in vivo fluorescence measurements can also be similar under various types of nutrient deficiency (e.g. Sakshaug and Holm-Hansen 1977), physiological mechanisms specific to the limiting nutrient are lik e l y responsible. By comparing iron and nitrogen stress-mediated variation in these characteristics for G. sanguineum and other species in the literature, the specific effects of Fe stress can be discerned with more confidence. The current research shows that depletion of either iron or nitrogen in G. sanguineum lowers Q c n]. to 20-25% of nutrient-sufficient levels. Chl a i s a magnesium-containing metalloporphyrin derived from the tetrapyrrole biosynthetic pathway (Granick and Beale 1978). Iron and nitrogen participate in porphyrin biosynthesis as cofactors of certain enzymes (Fe only) and constituents of products (N only) 45 occurring in the pathway up to and including chl a (see Pushnik et a l . 1984). Thus, i t i s not surprising that available data (e.g. Glover 1977, Guikema and Sherman 1983, Reuter and Ades 1987), including studies of Fe and N stress in a single species (Sakshaug and Holm-Hansen 1977, this study), demonstrate reduced Qcn]_ under either Fe or N deficiency for representatives of five algal groups. The active heme prosthetic group of photosynthetic electron transfer (PET) cytochromes i s an iron metalloporphyrin and also a product of the tetrapyrrole biosynthetic pathway responsible for chl a synthesis. In addition, PET components include non-heme forms of Fe comprising iron-sulfur proteins such as ferredoxin (Jensen 1986). Given the involvement of Fe and N in the biosynthesis of PET cytochromes and Fe-S proteins, and the functional relationship between PET reactions and in vivo fluorescence (Lawlor 1987), depletion of either nutrient might be expected to effect similar changes in F/chl a. Indeed, previous laboratory results generally demonstrate a two- to four-fold increase in this ratio above nutrient-sufficient values (e.g. Kiefer 1973, Sakshaug and Holm-Hansen 1977, Guikema and Sherman 1983), irrespective of limiting nutrient (i.e. Fe or N). However, considering the extreme cases of nutrient depletion in the present work, F/chl a of Fe-deplete G. sanguineum increased seven times compared to less than three-fold for N-deplete c e l l s . Even under conditions of severe nutrient limitation (as opposed to depletion, cf. Fig. 46 1.6) F/chl a was ca. two times greater for Fe than N stress. Sakshaug and Holm-Hansen (1977) also observed larger Fe stress-mediated increases in this ratio compared to those associated with N depletion for the diatom Skeletonema costatum. Differences in F/chl a, coupled with similar chl a quotas for Fe- and N-stressed G. sanguineum, indicate a greater specific Fe stress-mediated response of in vivo fluorescence and thus PET activity. Among the factors which may contribute to increases in F/chl a are enhanced light energy capture per unit pigment and/or reduced efficiency of light energy transfer to photochemical reaction centers and of electron transport within the PET system. It is well documented that Fe-limited growth reduces both quantities of PET components (i.e. cytochromes and Fe-S proteins) and activity of the PET system in algae (Glover 1977, Sandmann and Malkin 1983, Sandmann 1985) and higher plants (Spiller and Terry 1980, Terry 1983). These specific detrimental effects on PET processes may be sufficient to account for the difference in F/chl a between Fe- and N-deplete G. sanguineum. Fluorescence may be further augmented by more efficient capture of light energy (i.e. increased specific absorption coefficient) by chl a through iron stress-mediated reductions in Qcn\ a n d thus self-shading (cf. N-limited Chaetoceros gracilis, Cleveland and Perry 1987) . 47 Another potentially important consideration i s evidence indicating that iron deficiency preferentially reduces the electron and light energy transfer components and capacity of Photosystem I (PSI) relative to Photosystem II (PSII) (Oquist 1974, Nishio et a l . 1985, Sandmann 1985). When PSII is reduced, fluorescence i s emitted from chlorophyll in 10"^ s (F is slower for oxidized PSII). By comparison, electron 1 o transport from the reaction centers of PSI occurs in 10 s (Lawlor 1987). Back reactions of photochemical events associated with PSI thus account for less than 10% of (room temperature) in vivo fluorescence measurements (Prezelin 1981). The enhanced effect of iron stress on PSI would likely lower both i t s energy and electron transfer efficiency, potentially increasing the contribution of PSI to the fluorescence signal. In this regard, work by Ley (1980) on the red alga Porphyridium omentum, demonstrating that PSI normally receives about twice as much light energy as PSII (irrespective of wavelength), may also be of consequence. However, the ratio of photon flux into PSI and PSII is not constant and i s thought to be optimized by organizational changes in the photochemical apparatus. Such alterations can occur on time scales ranging from seconds or minutes (e.g. State I-State II transitions) to days (e.g. changes in thylakoid morphology, see Ultrastructure below) (Barber 1985 ). While the possibility and significance of iron stress-mediated elevated PSI fluorescence remain unknown, i t s occurrence would represent an additional in vivo F source comprising the larg Fe-deficient F/chl a ratios of G. sanguineum. In a recent study of the diatom Ch. gracilis, Cleveland and Perry (1987) suggested that increases in F/chl a associated with reduced nitrogen quotas resulted from both a rise in the specific absorption coefficient of chl a (due to reduced self-shading as chl a per c e l l decreased) and an uncoupling of photosynthesis (as reflected by lower quantum yields). Interestingly, the authors found no changes in the efficiency of energy transfer between the accessory pigment fucoxanthin and chl a, although fucoxanthin:chl a ratios increased. While similar mechanisms may help to explain the F/chl a increase of N-deficient G. sanguineum, changes associated with iron stress are clearly more effective mediators of elevated in vivo fluorescence in this species. Measurements of F D/chl a, to the extent that F D approximates the potential maximum in vivo fluorescence ( F m a x ) , provide further evidence that fluorescence propertie of G. sanguineum are more sensitive to iron than nitrogen deficiency. Application of the herbicide DCMU blocks PET on the acceptor side of PSII, prohibiting further re-oxidation PSII and thus electron flow between PSII and PSI. As a result, in vivo fluorescence increases to a larger (ca. maximum, Falkowski and Kiefer 1985) steady-state value (F D) considered to represent the energy otherwise available for PSII photochemistry (Prezelin 1981). If F under Fe and N 49 stress were similar, l i t t l e difference in F D/chl a would be expected. Slovacek and Hannan (1977) have demonstrated previously that fluctuations in F/chl a induced by various forms of nutrient limitation can be eliminated by DCMU addition. However, exceptions to a constant fluorescence yield per unit chl a following DCMU application are not uncommon (e.g. Roy and Legendre 1979). In the case of G. sanguineum, v a r i a b i l i t y between F D/chl a ratios was less than exhibited by those of F/chl a. Nevertheless, F D/chl a o f Fe-deplete ce l l s exceeded the N-deplete value by more than 1.5-fold, suggesting a greater F m a x under iron stress. Explanations similar to those proposed for large Fe-mediated F/chl a ratios would also apply here. Results of this study suggest that, while Fe and N are required for the production of both chl a and PET components, G. sanguineum can maintain biosynthesis of cytochromes and Fe-S proteins active in PET more successfully under nitrogen than iron stress. The biosynthetic pathways leading to chl a and heme are identical prior to metal insertion (chl a: Mg, heme: Fe) at which point they diverge (Castelfranco and Beale 1983). While data presented herein provide no biochemical evidence, i t i s interesting to speculate on the possibility o f some N stress-mediated regulation operating at the point o f divergence. Lower nitrogen quotas would favor the pathway t o heme synthesis over that to chl a (under saturating irradiance), thereby reducing the relative effect of N stress on cytochrome production and thus PET activity. This branch 50 point in the tetrapyrrole biosynthetic pathway has been suggested as a potential regulatory site for the production of chlorophyll (see Granick and Beale 1978). In addition, Castelfranco and Jones (1975) have proposed that chlorophyll and heme biosynthesis share a common pool of precursor compounds. It is possible that iron stress more severely limits the supply of these intermediates (e.g..Marsh et a l . 1963). This would explain why, that even i f heme synthesis was also favored under Fe limitation, production of heme by N-stressed c e l l s apparently remains greater. Decreased heme levels have also been induced by addition of Fe chelators (presumably reducing Fe availability) (Duggan and Gassman 1974), an effect thought to be mediated by rapid heme turnover rates (Castelfranco and Jones 1975). In vivo fluorescence indices. In vivo fluorescence data (i.e. F and Fp/chl a) can be expressed as various indices which yield information largely about the operation of PSII. The following are three of these indices and the properties they are suggested to describe: 1) F/F D, the proportion of captured light energy lost as fluorescence; 2) 1-F/FD, the proportion of absorbed light energy ut i l i z e d by PSII (i.e. the efficiency of PSII); and 3) F D-F, the relative output of PSII (i.e. the capacity of PSII) (Prezelin 1981, Droop 1985). It is assumed that PSI fluorescence i s negligible. If this assumption were invalid (see above discussion), F would exhibit a proportionally greater increase than the larger F D signal. These indices would thus shift accordingly and their 51 applicability to s t r i c t l y PSII processes would be questionable. The preceding discussion has demonstrated that F/chl a and F D/chl a (upon which these indices are based) are quite variable depending on species, growth conditions, and physiological state. The above three fluorescence indices are therefore sensitive to similar effects, but would presumably be most useful in comparisons of different growth-limiting factors for one species under otherwise similar conditions (e.g. this study). Data from the present work show increases in both F/chl a and F D/chl a regardless of whether Fe or N limits growth. However, under conditions of Fe depletion, F increases to 60% of F D (F/F D = 0.61), while remaining below 40% of F D for N depletion (F/F D = 0.38). Conversely, the 1-F/FD indices are 0.39 and 0.62 for Fe and N depletion, respectively. The F D-F index (i.e. F D/chl a-F/chl a) i s equivalent for both Fe- and N-deplete c e l l s (F D-F = 200). Information provided by these in vivo fluorescence indices suggest that a greater proportion of harvested light energy is reemitted as fluorescence by iron-deplete G. sanguineum. Further, while the capacity of PSII i s affected similarly by either Fe or N depletion, the former reduces the photochemical efficiency of PSII to ca. 60% of that maintained under the latter. Although such interpretations must be considered in light of the assumptions and limitations associated with these indices, they are indeed consistent with a more pronounced effect of iron over nitrogen stress on PET processes for this species. 52 Ultrastructure. The general ultrastructural features of iron-replete G. sanguineum are consistent with those described for other dinoflagellates (e.g. Dodge 1971). Of particular interest herein are changes in these characteristics associated with iron depletion and their relationship to the variation in chl a and in vivo fluorescence measurements discussed above. Perhaps the most obvious association i s between the decline in Qcn± and decreased chloroplast number. This observation eliminates the possibility that lower chl a quotas are s t r i c t l y a result of smaller chloroplasts. The size of those remaining chloroplasts may also have been reduced, but quantitative comparisons of Fe-replete and -deplete chloroplast dimensions were not made. Fewer chloroplasts present in Fe-deplete cells supports the argument for an increase in the chl a specific absorption coefficient due to reduced self-shading. Degeneration of the normally well-organized chloroplast lamellae and thylakoids did occur under iron stress in G. sanguineum, and similar effects have been reported for other algae (Meisch et a l . 1980, Hardie et a l . 1983, Chapter 4) and higher plants (Platt-Aloia et a l . 1983). While functional interpretation of structural defects i s generally lacking, the integrity of thylakoid membranes and their components clearly influences the efficiency of light harvesting as well as energy and electron transfer processes (Barber 1985). Thus, large in vivo fluorescence measurements of Fe-deplete ce l l s may be at least partly explained by 53 changes observed in photosynthetic membranes. A characteristic of Fe-deplete chloroplasts, which may be an adaptation aimed at reducing the amount of harvested light energy lost as fluorescence, i s a lesser degree of thylakoid appression as evidenced by fewer thylakoids (1-2) per lamella. Several investigators (see Baker and Webber 1987) have shown that decreased thylakoid stacking increases the efficiency of energy transfer between PSII and PSI (several mechanisms are currently postulated) thereby reducing fluorescence; however, other Fe-mediated changes effecting higher fluorescence yields apparently predominate in G. sanguineum. The fact that many lamellae appear "normal" (i.e. no structural disorders) suggests that alterations in photosynthetic components and processes are not always manifested as obvious ultrastructural deviations. Other changes noted in Fe-stressed protoplasts, such as increased vacuolar area, decreased mitochondrial matrix density and ER abundance, and variation in accumulation body morphology, may be non-specific responses to reduced growth rates. Unfortunately, no N-deplete ce l l s were examined for comparison, as in the case of chl a and in vivo fluorescence. Thus, even the iron stress-mediated specificity of chloroplast degeneration i s not certain, although a study of Fe- and N-starved Agmenellum quadruplicatum (Cyanophyceae) (Hardie et a l . 1983) demonstrated that altered thylakoid structure was restricted to the former. Apart from chloroplasts, mitochondria might also be expected to exhibit specific Fe-54 mediated effects related to lower cytochrome production. Decreases in electron density of the matrix were encountered herein and for Fe-stressed Protogonyaulax tamarensis (Chapter 4). Respiratory electron transport (RET) i s associated with cristae membranes, which did not appear to change in either species. It i s possible that reductions in RET components and activity, which remain to be demonstrated in Fe-deplete G. sanguineum, do not alter cristae structure. A considerable portion (over 70%) of RET subunit components extends beyond the plane of the inner mitochondrial membrane (Hackenbrock 1981), yet i t seems unlikely that fewer RET cytochromes would affect the density of the entire matrix, as was observed. A more plausible explanation of lower matrix density in Fe-deplete mitochondria would be loss of TCA cycle constituents due to minimal energetic demands as growth irate declines. Ecological considerations. Elucidation of nutritional factors affecting red tide population dynamics i s essential to understanding the ecology of these natural phytoplankton blooms. To this end, indicators of nutrient limitation are of interest, as certain problems associated with their use may be minimized by the predominance of one or two species. Certain of these indicators exhibit considerable variation according to the nutrient and species in question. Indeed, a rigorous study of N and P limitation in five algal species (Healey and Hendzel 1979) revealed only two of fifteen compositional and metabolic variables to be generally useful indicators of limitation by either nutrient. 55 In addition to the implication of increased iron bioavailability in promoting bloom formation (see Background), a comparatively (cf. Brand et a l . 1983, Harrison and Morel 1986) large iron requirement for two red tide dinoflagellates (Mueller 1985, this study, Chapter 4) further suggests the potential importance of this trace metal in red tide ecology. A l l characteristics of severely iron-limited c e l l s monitored herein (pFe 21.2, 22.2) were easily distinguishable from those of nutrient-sufficient or N-limited G. sanguineum. Comparison of several variables for both Fe- and N-limited growth indicates that F/chl a i s the most specific and sensitive indicator of iron limitation for this species. Perhaps employed in conjunction with other methods of assessing nutritional status (e.g. short-term nutrient enrichment, Healey 1979), the F/chl a ratio would be useful in detecting iron-stressed red tide populations. It must be recognized, however, that the present data reflect only Fe-limited cells of G. sanguineum and comparisons with those growing under N limitation. Thus, the generality of these findings in terms of both species and nutrient specificity requires further investigation. Summary. Examination of iron-limited growth kinetics indicates that reproductive rates of G. sanguineum may be limited by Fe concentrations at least ten-fold greater than for other neritic (predominantly diatom) species. This disparity may be related to the comparatively large iron requirement per unit c e l l carbon of this red tide 56 dinoflagellate. While these data do not confirm a role of iron in affecting red tide population dynamics, they do suggest that this species may be more susceptible to reduced iron bioavailability than many other coastal phytoplankters. In vivo fluorescence properties clearly demonstrate the detrimental effects of Fe stress on the u t i l i z a t i o n of harvested light energy. The type and magnitude of observed changes, when compared with those for N-stressed cultures, provide evidence for the iron stress-mediated specificity of these effects on G. sanguineum. Because of the extent to which Fe limitation modifies F/chl a and the distinction from N-mediated changes, this ratio, in conjunction with other probes of nutritional status, may be a useful indicator of Fe-stressed red tide populations. Ultrastructural observations of Fe-deplete G. sanguineum, especially those revealing changes in chloroplast number and morphology, are consistent with and provide a structural interpretation of variation noted in chl a quotas and in vivo fluorescence. 57 CHAPTER 2 . ASPECTS OF IRON AND NITROGEN NUTRITION IN THE RED TIDE" DINOFLAGELLATE GYMNODINIUM SANGUINEUM HIRASAKA: EFFECTS OF IRON DEPLETION AND NITROGEN SOURCE ON BIOCHEMICAL COMPOSITION BACKGROUND Phytoplankton growth in marine systems i s thought to be limited most frequently by macronutrients such as nitrogen or occasionally si l i c o n (see Parsons and Harrison 1983). Several studies of offshore communities indicate that iron may also regulate primary production (Menzel and Ryther 1961, Entsch et a l . 1983, Subba Rao and Yeats 1984, Martin and Fitzwater 1988). By comparison, i t has been suggested that iron concentrations of inshore or neritic environments are less l i k e l y to limit the growth of phytoplankton (Brand et a l . 1983, Murphy et a l . 1984, Martin and Gordon 1988). Yet, episodic pulses of iron from land drainage or sediment resuspension have been implicated in the i n i t i a t i o n of red tide dinoflagellate blooms (Glover 1978, see Iwasaki 1979). Although direct iron mediation of red tides remains equivocal, to suggest that iron controls these dinoflagellates' reproductive rate, while adequately supporting growth of other coastal species, implies a disparity in requirements for and/or a b i l i t i e s to acquire this trace metal. Evaluation of iron supply versus demand by phytoplankton relies largely on extrapolation of laboratory results to natural species assemblages. However, the majority of work useful in this regard pertains almost exclusively to diatoms 58 (e.g. Anderson and Morel 1982, Brand et a l . 1983, Murphy et a l . 1984, Harrison and Morel 1986). A half-saturation constant for iron-limited growth of a red tide dinoflagellate 10-1000 times greater than calculated for several coastal, predominantly diatom, species was reported in Chapter 1. This finding provides some of the f i r s t evidence suggesting that these dinoflagellates may be more susceptible to reduced iron bioavailability than certain other neritic phytoplankton. The principle goal of the present research was a better understanding of red tide dinoflagellate iron nutrition, as well as that of coastal dinoflagellates in general. Herein, the biochemical composition of iron-replete (i.e. nutrient-sufficient) and iron-deplete c e l l s are compared, while the following chapter (Chapter 3) examines nutrient uptake rates under these same conditions. Biochemical constituents monitored for the present report included cellular quotas of carbon, nitrogen, iron, protein, free amino acids and chlorophyll a. Although not a compositional variable per se, in vivo fluorescence was also determined. Fluorescence properties provide an indirect measure of photosynthetic electron transport, a process involving (iron-containing) cytochromes and iron-sulfur proteins (see Chapter 1). The organism employed in this study was Gymnodinium sanguineum Hirasaka. Iron bioavailability i s largely a function of the relatively low solubility of hydrous ferric oxides, this 59 element's predominant form in oxygenated seawater (Byrne and Kester 1976). However, both biological and non-biological interactions of iron with other trace metals (Harrison and Morel 1986, and references therein) and/or macronutrients (see Huntsman and Sunda 1980) may also influence (and be influenced by) phytoplankton iron nutrition. Many aspects of cellular nitrogen metabolism, including certain assimilatory and biosynthetic pathways, exhibit an iron requirement (see Reuter and Peterson 1987). Of the two major sources of inorganic nitrogen available to eukaryotic algae, namely nitrate and ammonium, only NO3 must be reduced prior to i t s incorporation into amino acids (Syrett 1981). Nitrate and n i t r i t e reductase, both iron-containing metalloenzymes (see Galvan et a l . 1986), are necessary for the reduction of NO3 to NH4. Nitrogen source would thus appear to be an important factor in determining phytoplankton iron requirements. Further, the nitrogen status (e.g. N quota) of iron-deficient c e l l s might also be expected to vary depending on whether N i s supplied as NO3 or NH4. An additional objective of the present work was to investigate the possibility that N source mediates the quantitative and/or qualitative effects of iron depletion. This was accomplished by extending determinations of compositional variables (this study) and nutrient uptake rates (following chapter) for iron-replete and -deplete c e l l s to include both NO3- and NH^-grown cultures. 60 MATERIALS AND METHODS General culture maintenance. Nitrate-grown stock cultures of Gymnodinium sanguineum (culture #D354, North East P a c i f i c Culture C o l l e c t i o n , Dept. of Oceanography, University of B r i t i s h Columbia) were maintained on f i l t e r - s t e r i l i z e d ( M i l l i p o r e 0.45 pm), ESAW-enriched a r t i f i c i a l seawater (Harrison et a l . 1980) prepared and modified as outlined i n Chapter 1. Medium for ammonium-grown stock cultures was si m i l a r to t h i s formulation, except that 80 pM NH4C1 replaced 550 pM NaN03 as the sole source of nitrogen. It should be noted here that G. sanguineum i s extremely s e n s i t i v e to elevated NH4 concentrations, with toxic growth l i m i t a t i o n occurring at 2150 pM NH4 (Appendix 3). Maximum growth rates ( i . e . equivalent to those of N03~grown cultures) are supported by 80 pM NH4. Containers used i n culture maintenance and experiments were pre-treated with freshly-prepared 10% HC1 (v/v) for 2-3-days, followed by several deionized d i s t i l l e d water r i n s e s . A l l cultures employed herein ( i . e . stock and experimental, both unstirred) were held i n a c i r c u l a t i n g water bath at 17°C, and continuously illuminated with 145 p E « m - 2 ' S - 1 of blue fluorescent l i g h t (further d e t a i l s i n Chapter 1). Iron-replete and iron-deplete cultures. The basal E S A W s a l t s o l u t i o n and i n d i v i d u a l enrichment stocks (except trace metals and vitamins) were passed over Chelex 100 ion exchange r e s i n to reduce ambient trace metal contamination (Morel et a l . 1979). For a l l experiments, 2.5 1 of a given medium was 61 dispensed into a 2.8 1 polycarbonate Fernbach flask, the pH lowered to ca. 5.5 with SuprapurR HCl (Merck), and autoclaved. Sterile medium was bubbled with 0.22 pm (Millipore)-filtered air to re-equilibrate the pH at ca. 8.0-8.1 prior to in i t i a t i n g an experiment. Due to high c e l l densities achieved during iron depletion experiments, these media were supplemented with chelexed, f i l t e r - s t e r i l i z e d (Millipore 0.22 pm) NaHCG"3 to a fi n a l concentration of 2 mM after autoclaving to prevent carbon limitation. Iron-replete culture medium contained either 80 pM NaNC^ (designated as +Fe/NC>3 hereafter) or 80 pM NH4C1 (designated as +Fe/NH4 hereafter); otherwise, these two media were identical to general maintenance ESAW medium. The medium employed to deplete cultures of iron (-Fe) received no iron addition. EDTA and a l l other trace metals (TM) were added as one solution, with [EDTA] reduced to maintain an EDTA:TM ratio of 1.6. -Fe medium was supplied with either 550 pM NaNC^ (designated as -Fe/NC^ hereafter) or 80 pM NH4C1 (designated as -Fe/NH4 hereafter). As noted, an NH4 concentration of ca. 80 pM i s required to avoid growth limitation due to ammonium toxicity. However, at this concentration, NH4-nitrogen was exhausted from cultures prior to achieving iron depletion. Thus, NH4 levels were monitored on fi l t e r e d (Whatman 934-AH) samples using a Technicon AutoAnalyzer R (method of Slawyk and Maclsaac 1972) and maintained between ca. 5-80 pM, with additions made as needed to permit depletion of available iron. Iron depletion was verified for both -Fe/NC>3 and 62 -Fe/NH4 cultures by bioassay (Appendix 2). Changes in the pH of these cultures with increasing c e l l density were controlled by bubbling with sterile (Millipore 0.22 um) air or 1-2% C0 2 (Appendix 2). Late logarithmic phase stock cultures acclimated to the appropriate nitrogen source were used to inoculate experiments (i.e. NO3- and NH^-grown stocks used as inocula for +Fe/NC>3 and -Fe/NC^, and +Fe/NH^ and -Fe/NH^ experiments, respectively) at a starting c e l l density of ca. 100 cells-ml -*. A l l experiments were run in duplicate, with each flask initiated from a different stock culture to enhance the potential for variation between flasks. Iron-replete cultures (+Fe/NC>3 and +Fe/NH4) were harvested in early to mid exponential phase for determination (duplicate measurements of a l l variables for each culture) of c e l l density (CD), average c e l l volume (CV), in vivo fluorescence (F) and DCMU-enhanced F (F D), cellular quotas of carbon, nitrogen, iron, protein (Pr), free amino acids (AA) and chlorophyll a (chl a), and also several ratios comprising these variables. Iron-deplete cultures (-Fe/NG*3 and -Fe/NH^) were sampled for these same variables upon depletion of biologically available iron, as indicated by no change or a decline in c e l l density on successive days. Measurements of iron and nitrogen (NO3 and NH^) uptake rates were also performed on a l l experimental cultures concurrent with sampling for biochemical constituents and are described in Chapter 3. 63 Cell density and volume, F, FD and chl a. C u l t u r e c e l l d e n s i t y was monitored u s i n g a C o u l t e r Counter 1* model TAII (200 pm a p e r t u r e , 44.2 pm c a l i b r a t i o n s p h e r e s ) , w i t h the average volume of c e l l s c a l c u l a t e d based on e q u i v a l e n t s p h e r i c a l diameter of the p a r t i c l e s i z e d i s t r i b u t i o n . D e t a i l s of methods f o r F, F D and c h l a d e t e r m i n a t i o n s are g i v e n i n Chapter 1. B r i e f l y , in vivo f l u o r e s c e n c e was measured on d a r k - e q u i l i b r a t e d (20 min) samples a f t e r 30 s exposure t o the f l u o r o m e t e r l i g h t source. The F D tube r e c e i v e d 10 -^ M DCMU ( f i n a l c o n c e n t r a t i o n ) p r i o r t o dark treatment. C h l a was determined a c c o r d i n g t o the f l u o r o m e t r i c method of Holm-Hansen et a l . (1965). As G. sanguineum i s s e n s i t i v e t o p h y s i c a l p e r t u r b a t i o n , c e l l s c o l l e c t e d f o r a l l a n a l y s e s were f i l t e r e d by g r a v i t y whenever p o s s i b l e or u s i n g vacuum p r e s s u r e s of 2 5-50 mm Hg f o r l a r g e r volumes and/or h i g h e r c e l l d e n s i t i e s (care was taken t o a v o i d f i l t e r i n g t o d r y n e s s ) . Cf N, protein and free amino acids. P a r t i c u l a t e o r g a n i c carbon and n i t r o g e n c o n c e n t r a t i o n s were asse s s e d on f i l t e r e d samples (Whatman 934-AH, precombusted a t 460°C f o r 4 h) u s i n g a C a r l o Erba Elemental A n a l y z e r (model 1106) and s t a n d a r d i z e d t o a c e t a n i l i d e (Eastman Kodak Co.). F i l t e r s were s t o r e d at -15°C between sampling and a n a l y s i s times (ca. 1 month). C e l l u l a r p r o t e i n and f r e e amino a c i d measurements f o l l o w e d a s l i g h t m o d i f i c a t i o n (Q. Dortch p e r s . comm.) of the method o u t l i n e d by Dortch e t a l . (1984). A technique v e r y s i m i l a r to t h a t of Dortch e t a l . (1984) has been recommended r e c e n t l y , over s e v e r a l o t h e r phytoplankton e x t r a c t i o n / a n a l y s i s p r o t o c o l s 64 ( C l a y t o n e t a l . 1988). C e l l s h a r vested on precombusted f i l t e r s (Whatman 934-AH) were ground i n 3% TCA ( r a t h e r than 10% used by Dortch e t a l . 1984) and separated by c e n t r i f u g a t i o n i n t o p e l l e t (Pr) and supernatant (AA) f r a c t i o n s , which were f r o z e n i n d i v i d u a l l y and s t o r e d (-15°C) u n t i l a n a l y s i s (ca. 2 months). P r o t e i n - N c o n t a i n e d i n the p e l l e t was determined a g a i n s t a bovine serum albumin standard ( u s i n g %N of BSA, Sigma Chemical Co.) a c c o r d i n g t o Packard et a l . ' s (1972) m o d i f i c a t i o n of the Lowry method (Lowry e t a l . 1951). The supernatant was assayed f l u o r o m e t r i c a l l y (employing fluorescamine) f o r f r e e amino a c i d s u t i l i z i n g glutamate standards (Undenfriend e t a l . 1972, Packard and Dortch 1975). J r o n quotas. Measurement of i r o n quotas r e q u i r e s t h a t i r o n adsorbed t o the c e l l through n o n - p h y s i o l o g i c a l mechanisms (e.g. f e r r i c hydroxy c o l l o i d s ) be e l i m i n a t e d p r i o r t o a n a l y s i s . Anderson and Morel (1982) demonstrated t h a t f o r the diatom Thalassiosira weissflogii, 10"^ M a s c o r b i c a c i d d i s s o l v e s surface-bound i r o n (by r e d u c t i o n of f e r r i c t o more s o l u b l e f e r r o u s i r o n ) , thereby f a c i l i t a t i n g i t s removal by f i l t r a t i o n . T h i s technique proved u n s u c c e s s f u l i n the present study due t o c e l l l y s i s upon ascorbate a d d i t i o n . However the f o l l o w i n g experiments, employing ^ F e C l 3 (5 pM a d d i t i o n s , New England Nuclear) as a l a b e l , suggest a fundamental d i f f e r e n c e i n i r o n a d s o r p t i o n p r o p e r t i e s between G. sanguineum and T. weissflogii. R e s u l t s i n d i c a t e t h a t n o n - b i o l o g i c a l , 65 f i l t e r a b l e i r o n i s a sm a l l percentage of t o t a l f i l t e r a b l e i r o n a s s o c i a t e d w i t h c e l l s of G. sanguineum. The f o r m a t i o n of f i l t e r a b l e i r o n r e t a i n e d by e i t h e r 0.2 or 5.0 pm f i l t e r s (polycarbonate, Nuclepore) f o r c u l t u r e f i l t r a t e and suspensions of v i a b l e or g l u t a r a l d e h y d e (GTA, 1 . 5 % ) - k i l l e d c e l l s (4000 c e l l s • m l " ^ ) was f o l l o w e d over a time-course (note: GTA d i d not cause c e l l l y s i s ) . Experiments were conducted i n 85 ml Oak Ridge tubes (polycarbonate, Nalgene) under c u l t u r e c o n d i t i o n s d e s c r i b e d above. As observed by Anderson and Morel (1982) i n a s i m i l a r experiment wi t h T. weissflogii ( f o r m a l i n - k i l l e d c e l l s , 0.1 and 3.0 pm f i l t e r s ) , i r o n c o l l o i d s appeared r a p i d l y and were c o l l e c t e d o n l y on the 0.2 (or 0.1) pm f i l t e r ( F i g . 2.1A). L a b e l r e t a i n e d by dead G. sanguineum a f t e r 4 h r e p r e s e n t e d a smal l f r a c t i o n ( c a . 20%) of t h a t a s s o c i a t e d w i t h v i a b l e c e l l s ( F i g . 2.1A), which i s i n sharp c o n t r a s t t o the e q u i v a l e n t l a b e l l i n g of T. weissflogii c e l l s , i r r e s p e c t i v e o f t h e i r v i a b i l i t y ( t h e i r F i g . 2.1). The predominantly p h y s i o l o g i c a l nature of i r o n a s s o c i a t e d w i t h G. sanguineum c e l l s and the n e g l i g i b l e t r a p p i n g of c o l l o i d a l i r o n by 5.0 pm f i l t e r s i n d i c a t e t h a t reasonable measurements of i r o n quotas f o r t h i s s p e c i e s can be ob t a i n e d without ascorbate treatment. In a d d i t i o n , removal of f i l t e r a b l e (5.0 pm pore diam.) i r o n from v i a b l e and G T A - k i l l e d c e l l s ( c a . 2-10 4 c e l l s ) by chelexed ESAW s a l t s o l u t i o n (CSS, 5 ml) was examined (5-40 ml CSS e f f e c t s c a . e q u i v a l e n t removal of Fe from t h i s number of c e l l s , Appendix 4 ) . F o l l o w i n g 2-8 h i n c u b a t i o n s ( F i g . 2.IB) dead c e l l s r e t a i n e d o n l y 15-20% of 66 F i g u r e 2.1A. Formation of f i l t e r a b l e i r o n ( 5 5 F e C l 3 l a b e l ) by c u l t u r e f i l t r a t e (0.2 um f i l t e r : o; 5.0 um f i l t e r : •) and su s p e n s i o n s of v i a b l e (•) or g l u t a r a l d e h y d e - k i l l e d (pi c e l l s f o l l o w i n g 5 pM Fe a d d i t i o n . B. A c q u i s i t i o n of i r o n ( 5 5 F e C l 3 , 5 /JM a d d i t i o n ) by unwashed (o) or washed (5 ml c h e l e x e d ESAW s a l t s o l u t i o n ) (•) v i a b l e c e l l s and unwashed (•) or washed (•) g l u t a r a l d e h y d e - k i l l e d c e l l s . DPM = d i s i n t e g r a t i o n s ' m i n - 1 . TIME (h) 67 i r o n a s s o c i a t e d w i t h v i a b l e G. sanguineum ( r e g a r d l e s s of CSS washing), w h i l e a s i m i l a r percentage of v i a b l e T. weissflogii i r o n i n c o r p o r a t i o n was accounted f o r by f o r m a l i n - k i l l e d c e l l s a f t e r a s c o r b a t e treatment of each ( i . e . v i a b l e and f o r m a l i n -k i l l e d c e l l s ; Anderson and Morel 1982, F i g . 2.3A). CSS washing e l i m i n a t e d c a . 10% of t o t a l f i l t e r a b l e i r o n ( v i a b l e c e l l s ) h e r e i n ( F i g . 2.IB), whereas c a . 70% of t h i s i r o n f r a c t i o n was removed by ascorbate from T. weissflogii ( t h e i r F i g . 2.3A). Based on the above r e s u l t s , use of 5.0 pm f i l t e r s and CSS washing appear t o p r o v i d e r e l i a b l e d e t e r m i n a t i o n s of G. sanguineum i r o n quota. The c o n c e n t r a t i o n of c e l l u l a r i r o n was measured s p e c t r o p h o t o m e t r i c a l l y based on methods d e s c r i b e d by Stookey (1970) and M u e l l e r (1985), which d e t e c t the s t a b l e c o l o r complex of f e r r o z i n e (Sigma Chem. Co.) and f e r r o u s i r o n . C e l l s c o l l e c t e d on 5.0 pm f i l t e r s ( p olycarbonate, Nuclepore) were washed wit h CSS and s t o r e d i n a d e s i c c a t o r a t room temperature u n t i l a n a l y s i s . Iron d e t e r m i n a t i o n s were performed by d i g e s t i n g c e l l s w i t h c l e a n ( s i n g l e b o i l i n g , double s u b - b o i l i n g d i s t i l l a t i o n ) c a . 3 N HNO3 (20 h at room temp, f o l l o w e d by 1 h a t 50°C), f o l l o w e d by q u a n t i t a t i v e s e p a r a t i o n of d i g e s t a t e from the i n t a c t f i l t e r , and a d d i t i o n of reagents (see Stookey 1970, note: HC1 omitted from f e r r o z i n e - h y d r o x y l a m i n e h y d r o c h l o r i d e reagent) t o d i g e s t a t e . Absorbance along a 10 cm path l e n g t h was read a t 562 nm a g a i n s t a d i s t i l l e d water blank. 68 Data analysis. The e f f e c t s of i r o n d e p l e t i o n and of n i t r o g e n source on v a r i a b l e s measured h e r e i n were determined from s e v e r a l comparisons. F i r s t , f o r c u l t u r e s grown on e i t h e r n i t r a t e or ammonium, i r o n - r e p l e t e and i r o n - d e p l e t e c e l l s were compared w i t h i n a g i v e n n i t r o g e n source (e.g. +Fe/NC"3 v s . -Fe/NG^). Second, f o r e i t h e r F e - r e p l e t e or F e - d e p l e t e c u l t u r e s , NO3- and NH^-grown c e l l s were compared w i t h i n a g i v e n i r o n s t a t u s (e.g. -Fe/NC>3 v s . -Fe/NH^). Sampling design allowed comparison of v a r i a b l e s by a n a l y s i s of v a r i a n c e (ANOVA) employing a nested d e s i g n , as each experiment i n c l u d e d d u p l i c a t e f l a s k s w i t h two measurements of a v a r i a b l e o b t a i n e d and averaged per f l a s k . The assumption of homogeneity of v a r i a n c e s was v e r i f i e d p r i o r t o an ANOVA u s i n g an F - t e s t . R a t i o s c o m p r i s i n g v a r i a b l e s (e.g. C/N, F / c h l a, e t c ) are based on an n = 2 sample s i z e ( d u p l i c a t e f l a s k s ) f o r each component of the q u o t i e n t . E r r o r terms f o r r a t i o s take i n t o c o n s i d e r a t i o n the p r o p a g a t i o n of e r r o r a s s o c i a t e d w i t h combining two components and t h e i r r e s p e c t i v e v a r i a n c e s i n t o a s i n g l e term. The a p p r o p r i a t e formulae are g i v e n by Yates (1981). Comparisons between r a t i o s were made by eye from bar graphs of t h e i r means ± 1 S.D. RESULTS N o r m a l i z a t i o n of measured v a r i a b l e s i s an important c o n s i d e r a t i o n when d e s c r i b i n g chemical c o m p o s i t i o n . The c e l l volume of G. sanguineum d i f f e r e d both as a r e s u l t of i r o n d e p l e t i o n and as a f u n c t i o n of n i t r o g e n source (Table 2 . 1 ) . 69 NH 4-grown c e l l s were c o n s i s t e n t l y l a r g e r than those grown on NO3, w i t h the d i s p a r i t y most e v i d e n t under Fe d e p l e t i o n (-Fe/NH4 c a . 35% > -Fe/NC^). To e l i m i n a t e the predominating e f f e c t of c e l l volume, a l l i n d i v i d u a l v a r i a b l e s were expressed as c o n c e n t r a t i o n s (by atoms or weight) per l i t e r of c e l l volume. C e l l quotas of elements (C, N and Fe) and b i o c h e m i c a l compounds (Pr, AA and c h l a) are g i v e n i n T a b l e 2.1 and ANOVA p r o b a b i l i t y l e v e l s of s i g n i f i c a n c e f o r comparisons are p r o v i d e d i n T a b l e 2.2. Elemental constituents and ratios. I r o n d e p l e t i o n s i g n i f i c a n t l y reduced the carbon quotas (Q c) of both NO3-(16%) and NH 4- (28%) grown c e l l s , w h i l e Q c was r o u g h l y e q u i v a l e n t between N sources f o r a g i v e n Fe s t a t u s . The most n o t a b l e d i f f e r e n c e i n n i t r o g e n quota was a f u n c t i o n of N source under Fe d e p l e t i o n , w i t h NH 4-grown c e l l s c o n t a i n i n g almost 50% more N than those growing on NO3. -Fe/NH 4 n i t r o g e n quotas were even l a r g e r than f o r +Fe/NH 4 c u l t u r e s (ca. 20%), although not s t a t i s t i c a l l y d i s t i n g u i s h a b l e . In terms of the more c o n v e n t i o n a l C/N r a t i o ( F i g . 2.2A), -Fe/NH 4 c u l t u r e s averaged 3.8, w h i l e C/N v a l u e s f o r a l l o t h e r c o n d i t i o n s were between 6.2 and 7.0. V a r i a b i l i t y among Qp e d e t e r m i n a t i o n s , p a r t i c u l a r l y f o r N03~grown c u l t u r e s , obscured any p o t e n t i a l d i f f e r e n c e s due to n i t r o g e n source and f u r t h e r , p r o h i b i t e d c e r t a i n s t a t i s t i c a l comparisons by ANOVA (Tables 2.1,2.2). Regardless of t h i s f a c t , i r o n d e p l e t i o n d i m i n i s h e d i r o n quotas by c a . 1.5 orders T a b l e 2.1. Average c e l l volume and b i o c h e m i c a l c o m p o s i t i o n of i r o n -r e p l e t e and i r o n - d e p l e t e c u l t u r e s grown on n i t r a t e o r ammonium. See t e x t f o r e x p l a n a t i o n of c u l t u r e d e s i g n a t i o n s . CULTURE 1 CV 2 C 3 N 3 F e 4 P r 3 AA 3 CHL a 5 +Fe/N0 3 4.63 10500 1510 56200 1300 207 634 (0.20) (400) (360) (18200) (50) (34) (102) +Fe/NH 4 4.84 11000 1780 50500 1360 153 585 (0.08) (630) (190) (8500) (60) (8) (44) -Fe/N0 3 2.24 8810 1400 938 1160 106 165 (0.10) (80) (40) (261) (40) (8) (7) -Fe/NH 4 3.05 7920 2070 923 1330 159 154 (0.23) (500) (250) (56) (130) (15) (63) 1. Values f o r a l l experiments are means (n = 2) w i t h one S.D. giv e n i n parentheses; d u p l i c a t e samples were analyzed f o r a l l v a r i a b l e s i n each of n = 2 c u l t u r e s per experiment. 2. pm J-10 4 3. m g - a t * l i t e r c e l l volume"} 4. p g - a t * l i t e r c e l l v o l u m e - 1 5. m g * l i t e r c e l l v o l u m e - 1 T a b l e 2.2. P r o b a b i l i t y l e v e l s o f s i g n i f i c a n c e f o r d i f f e r e n c e s between e x p e r i m e n t s compared (as i n d i c a t e d ) u s i n g an a n a l y s i s o f v a r i a n c e ( n e s t e d d e s i g n ) . A s t e r i s k s (*) i d e n t i f y c o m p a r i s o n s p r e c l u d e d by heterogeneous sample v a r i a n c e s as d e t e r m i n e d w i t h an F - t e s t . N.S. = not s i g n i f i c a n t l y d i f f e r e n t a t the 95% c o n f i d e n c e l e v e l . COMPARISON CV C N Fe P r AA CHL a NO3-GRWN, +Fe v s . -Fe <0.001 <0.01 N.S. * <0.02 <0.01 <0.001 NH4-GRWN, +Fe v s . -Fe <0.001 <0.01 N.S. * N.S. N.S. <0.001 Fe-RPLT, NO3- v s . NH4-GRWN <0.01 N.S. N.S. N.S. N.S. <0.01 N.S. Fe-DPLT, NO3- v s . NH4-GRWN <0.001 N.S. <0.05 N.S. N.S. <0.005 N.S. 72 of magnitude under NO3 or NH 4 growth, y i e l d i n g a minimum Qp e (Q F emin) of 0.9 m g - a t • l i t e r CV" 1. When norm a l i z e d t o N ( F i g . 2.2B) or C ( F i g . 2.2C), the c e l l u l a r i r o n c ontent i n i r o n -r e p l e t e c u l t u r e s was s i m i l a r f o r e i t h e r N source, although the mean Fe/N r a t i o under NO3 supply was 31% g r e a t e r than f o r growth on NH 4. T h i s d i s c r e p a n c y i n Fe/N i n c r e a s e s f u r t h e r t o 50% i n the case of i r o n d e p l e t i o n and i s l i k e l y a r e a l d i f f e r e n c e . Fe/C v a l u e s are the same f o r -Fe/NC^ and -Fe/NH 4 c u l t u r e s (avg. c a . 1 . 1 « 1 0 ~ 4 ) . Biochemical compounds and ratios. The p r o t e i n and f r e e amino a c i d content of NC^-grown c e l l s d e c l i n e d s i g n i f i c a n t l y (11% and 49%, r e s p e c t i v e l y ) w i t h i r o n - d e p l e t i o n , w h i l e i n c u l t u r e s grown on NH 4, these c o n s t i t u e n t s appeared u n a f f e c t e d by i r o n s t r e s s (Table 2.1,2.2). Compared t o c e l l u l a r p r o t e i n ( Q p r ) , f r e e amino a c i d quotas ( Q ^ ) were i n f l u e n c e d more by N source. Q^A. °^ +Fe/NC>3 c u l t u r e s exceeded those of F e - r e p l e t e , NH 4-grown c e l l s by 35%. Conversely, under F e - d e p l e t e c o n d i t i o n s c e l l u l a r AA c o n c e n t r a t i o n s were 50% g r e a t e r f o r NH 4- than NC^-grown c u l t u r e s . R a t i o s of AA/Pr ( F i g . 2.3) predominantly r e f l e c t e d v a r i a t i o n i n Qj^, as i r o n s t r e s s -mediated changes i n t h i s index o c c u r r e d o n l y i n c e l l s grown on NO3 (42% d e c r e a s e ) . As a r e s u l t , -Fe/NG-3 v a l u e s (avg. = 9.2) were s i g n i f i c a n t l y (23%) l e s s than f o r the -Fe/NH 4 experiment (avg. = 12.0). S i m i l a r t o Q F e above, v a r i a b i l i t y i n c h l a quota measurements f o r c e r t a i n c u l t u r e s (e.g. -Fe/NH 4, c o e f f i c i e n t 73 F i g u r e 2 . 2 . E l e m e n t a l r a t i o s by atoms of c a r b o n : n i t r o g e n (A), i r o n : n i t r o g e n (B) and i r o n : c a r b o n (C) f o r i r o n - r e p l e t e and i r o n - d e p l e t e c u l t u r e s grown on n i t r a t e o r ammonium. Values are mean (n = 2) ± 1 S.D. D u p l i c a t e samples were a n a l y z e d f o r a l l i n d i v i d u a l r a t i o components i n each of n = 2 c u l t u r e s per experiment. Note d i f f e r e n t s c a l e s f o r F e - r e p l e t e and Fe-d e p l e t e c u l t u r e s i n (B) and ( C ) . F i g u r e 2.3. Free amino a c i d ( A A ) : p r o t e i n (Pr) r a t i o s , e x p r e s s e d as mole p e r c e n t , of i r o n - r e p l e t e and i r o n - d e p l e t e c u l t u r e s grown on n i t r a t e o r ammonium. Average v a l u e s and e r r o r terms p r e s e n t e d are as d e s c r i b e d i n F i g . 2.2. 75 of v a r i a t i o n - 41%) p r e c l u d e d any d i s t i n c t i o n between N O 3 - and NH^-grown c u l t u r e s (Tables 2.1,2.2). Although an e f f e c t of N source was not d i s c e r n a b l e , i r o n - d e p l e t e c e l l s (-Fe/NC^ and -Fe/NH 4) c o n t a i n e d o n l y 26% of c h l a p r e s e n t under n u t r i e n t s u f f i c i e n c y . To f u r t h e r c h a r a c t e r i z e c e l l n i t r o g e n s t a t u s , the percent of QJJ accounted f o r by these N - r e q u i r i n g compounds (Pr, AA and c h l a; i n t e r n a l NO3 pools from Chapter 3) was e s t i m a t e d (Table 2.3). C a l c u l a t i o n s i n d i c a t e t h a t i n both NO3- and NH^-grown c u l t u r e s , percentages of Q N accounted f o r , were reduced by ca. 10% under i r o n d e p l e t i o n . However, those N - c o n t a i n i n g c o n s t i t u e n t s monitored, i n v a r i a b l y c o n t r i b u t e d t o c a . 15% l e s s of the t o t a l n i t r o g e n i n c e l l s grown on NH 4, i r r e s p e c t i v e of i r o n s t a t u s . In v i v o fluorescence ratios and indices. A t r e n d of i n c r e a s i n g in vivo f l u o r e s c e n c e normalized e i t h e r per u n i t c h l a ( F i g . 2.4A) or per u n i t i r o n ( F i g . 2.4B) o c c u r r e d i n response t o i r o n d e p l e t i o n , but changes i n F/Fe were over an order of magnitude l a r g e r than f o r F / c h l a. Although +Fe/NC>3 and +Fe/NH 4 c u l t u r e s e x h i b i t e d i d e n t i c a l F / c h l a and F/Fe measurements, i r o n - d e p l e t e v a l u e s of both r a t i o s f o r c u l t u r e s grown on NO3 were c a . t w o - f o l d g r e a t e r than f o r those grown on NH^. The in vivo f l u o r e s c e n c e index of F^-F remained u n a l t e r e d f o r a l l experimental treatments (avg. c a . 150, F i g . 2.5A). By comparison, 1-F/F D ( F i g . 2.5B), w h i l e e q u i v a l e n t f o r both i r o n - r e p l e t e experiments (+Fe/N03 and +Fe/NH 4), Table 2.3. I n d i v i d u a l and t o t a l percent of c e l l n i t r o g e n quota accounted f o r by v a r i o u s n i t r o g e n - c o n t a i n i n g b i o c h e m i c a l compounds. See t e x t f o r e x p l a n a t i o n of c u l t u r e d e s i g n a t i o n s . CULTURE P r 1 ' 3 AA 1' 3 N 0 3 2 ' 3 CHL a 1 ' 3 % TOTAL N 4 +Fe/N0 3 86.0 13.7 0.9 0.19 100.8 +Fe/NH 4 76.3 8.6 0.8 0.15 85.9 -Fe/N0 3 82.7 7.6 1.0 0.05 91.4 -Fe/NH 4 64.2 7.7 0.5 0.03 72.4 1. Values f o r p r o t e i n , amino a c i d s and c h l a from Ta b l e 2.1. 2. Data from Chapter 3 f o r i n t r a c e l l u l a r n i t r a t e p o o l s immediately f o l l o w i n g 50 /JM NOO a d d i t i o n t o c u l t u r e s f o r uptake experiments ( i . e . o b t ained a t time z e r o ) ; v a l u e s are per c e n t of t o t a l c e l l n i t r o g e n g i v e n i n Table 2.1. 3. Percent of t o t a l c e l l n i t r o g e n (Table 2 . 1 ) . 4. Percent of t o t a l c e l l n i t r o g e n accounted f o r by a l l compounds c o n s i d e r e d . 77 F i g u r e 2 . 4 . In vivo f l u o r e s c e n c e n o r m a l i z e d p e r u n i t c h l a (A) and per u n i t i r o n (B) of n i t r a t e - o r ammonium-grown, i r o n -r e p l e t e and i r o n - d e p l e t e c u l t u r e s . Average v a l u e s and e r r o r terms p r e s e n t e d a r e as d e s c r i b e d i n F i g . 2.2. Note d i f f e r e n t s c a l e s f o r F e - r e p l e t e and Fe- d e p l e t e c u l t u r e s i n (B). F e - R E P L E T E F e - D E P L E T E 78 F i g u r e 2.5. In vivo f l u o r e s c e n c e (F) and DCMU-enhanced f l u o r e s c e n c e (FQ) i n d i c e s of F n - F (A, r e l a t i v e u n i t s n o r m a l i z e d t o c h l a, and r e p r e s e n t a t i v e of PSII c a p a c i t y ) and 1-F/F D (B, u n i t l e s s , and r e p r e s e n t a t i v e of PSII e f f i c i e n c y ) f o r i r o n - r e p l e t e and i r o n - d e p l e t e c u l t u r e s grown on n i t r a t e or ammonium. Average v a l u e s and e r r o r terms p r e s e n t e d are as d e s c r i b e d i n F i g . 2.2. F e - R E P L E T E F e - D E P L E T E 79 d e c l i n e d s i g n i f i c a n t l y under i r o n s t r e s s ; however, the r e d u c t i o n i n -Fe/NG-3 c u l t u r e s (56%) was more than 1.5 times t h a t of NH^-grown c u l t u r e s (34%). DISCUSSION The c u r r e n t r e s e a r c h p r o v i d e s some of the f i r s t q u a n t i t a t i v e i n f o r m a t i o n on d i n o f l a g e l l a t e i r o n n u t r i t i o n , w i t h p a r t i c u l a r r e f e r e n c e t o r e d t i d e s p e c i e s . R e s u l t s demonstrate t h a t not onl y does i r o n s t r e s s cause s i g n i f i c a n t changes i n the b i o c h e m i c a l composition of G. sanguineum, but the e x t e n t and nature of t h i s v a r i a t i o n i s o f t e n a f u n c t i o n of n i t r o g e n source. E f f e c t s of i r o n d e p l e t i o n common t o both NO3- and NH 4-grown c u l t u r e s are d i s c u s s e d f i r s t . Subsequently, a l t e r a t i o n s unique t o e i t h e r NO3- or NH 4-grown c u l t u r e s w i l l be c o n s i d e r e d . Effects of iron depletion. The c o m p o s i t i o n a l v a r i a b l e most d i r e c t l y r e l e v a n t t o the i r o n n u t r i t i o n of G. sanguineum (and comparisons w i t h o t h e r phytoplankton s p e c i e s ) i s c e l l u l a r i r o n quota, and a l s o those r a t i o s i t comprises. Minimum i r o n quotas (Qp emin) of t h r e e c o a s t a l p h y t o p l a n k t e r s examined p r e v i o u s l y ( i . e . Dunaliella tertiolecta (Chlorophyceae) Davies 1970, Pavlova lutheri (Chrysophyceae) Droop 1973, Thalassiosira weissflogii ( B a c i l l a r i o p h y c e a e ) Anderson and Morel 1982, H a r r i s o n and Morel 1986) have been summarized by H a r r i s o n and Morel (1986, see t h e i r Table 2.2). Values range from a minimum of 2.1*10"^ mol F e * l i t e r c e l l v o l - 1 f o r 80 T. weissflogii (data of H a r r i s o n and Morel 1986) t o a maximum of 3.3-10 - 4 mol Fe«liter c e l l v o l " * f o r D. tertiolecta (Davies 1970). Of the a v a i l a b l e Q F emin data, perhaps the most a c c u r a t e e stimate i s t h a t of H a r r i s o n and Morel (1986) as c o n c u r r e n t measurements of i r o n quota (with removal of surface-bound Fe) and c e l l volume were employed. The Q F emin of G. sanguineum ( t h i s study) was c a l c u l a t e d t o be 9.3 ± O . l ' l O " 4 mol F e * l i t e r c e l l v o l " * (avg. f o r N O 3 - and NH^-grown c e l l s ) . Thus, on a per u n i t c e l l volume b a s i s , the minimum i r o n requirement of t h i s d i n o f l a g e l l a t e i s c a . 3 and 45 f o l d g r e a t e r than those of a c h l o r o p h y t e and a diatom, r e s p e c t i v e l y . Such comparisons of Q F e normalized t o c e l l volume may be b i a s e d by v a r i a t i o n i n the amount of a c t u a l biomass per u n i t c e l l volume among s p e c i e s and groups of phytoplankton (see H i t c h c o c k 1982). C o n v e n t i o n a l l y , t h i s problem i s minimized by e x p r e s s i n g b i o c h e m i c a l c o n s t i t u e n t s based on c e l l carbon. The T. weissflogii Q F e m i n of H a r r i s o n and Morel (1986) has been normalized p r e v i o u s l y t o QQ (Chapter 1), employing carbon quotas of the same c l o n e r e p o r t e d by B l a s c o e t a l . (1982). Fe/C r a t i o s meeting minimum and o p t i m a l growth requirements of phytoplankton have a l s o been estimated by Anderson and Morel (1982) and Morel and Hudson (1985), based on e a r l i e r s t u d i e s of T. weissflogii. C a l c u l a t e d minimum Fe/C v a l u e s range from 7.6-10" 7 t o 7.5*10"^ depending on the data s e t u t i l i z e d , while t h a t a l l o w i n g maximal growth i s c a . 9.4*10"^. E m p i r i c a l l y determined minimum and maximum Fe/C r a t i o s of G. sanguineum 81 (avg. f o r NO3- and NH 4-grown c e l l s ) are 1.1 ± 0.1*10~ 4 and 5.0 ± 0.5*10 , r e s p e c t i v e l y . These r e s u l t s suggest t h a t per u n i t c e l l carbon, the i r o n quota of t h i s d i n o f l a g e l l a t e exceeds t h a t of the diatom T. weissflogii by one t o two o r d e r s of magnitude. T h i s d i s p a r i t y i n c e l l u l a r i r o n requirement was c o n s i d e r e d i n Chapter 1 as a p r i n c i p l e f a c t o r c o n t r i b u t i n g t o the 1 0 - f o l d g r e a t e r h a l f - s a t u r a t i o n c o n s t a n t f o r i r o n - l i m i t e d growth of G. sanguineum over T. weissflogii. While i r o n quotas of these two a l g a l t a x a may be c o n s i d e r a b l y d i f f e r e n t , t h e i r molar i r o n requirements f o r p h o t o s y n t h e t i c and r e s p i r a t o r y e l e c t r o n t r a n s p o r t components (PET and RET, r e s p e c t i v e l y ) , as w e l l as f o r o t h e r c o n s t i t u e n t s e i t h e r c o n t a i n i n g or f u n c t i o n a l l y dependent on i r o n (e.g. n i t r a t e and n i t r a t e r e d u c t a s e , glutamate s y n t h a s e ) , are assumed t o be e q u i v a l e n t . Raven (1988) has proposed a minimum mol Fe:mol C r a t i o of 2.33*10"^ f o r a p l a n t growing p h o t o l i t h o t r o p h i c a l l y ( i . e . l i g h t - d r i v e n 0 2 e v o l u t i o n w i t h an i n o r g a n i c carbon source) at a maximum s p e c i f i c r a t e of 3*10~ 5 s " 1 a t 20°C. His c a l c u l a t i o n s take i n t o c o n s i d e r a t i o n those c a t a l y t i c F e - c o n t a i n i n g components of both PET and RET systems ( i . e . cytochromes and i r o n - s u l f u r p r o t e i n s ) . As the i r o n requirements of n i t r a t e and n i t r i t e r e d u c t a s e are not i n c l u d e d , t h i s Fe/C r a t i o i s more d i r e c t l y a p p l i c a b l e t o NH 4-grown p l a n t s . One can thus determine the p r o p o r t i o n of a c e l l ' s c r i t i c a l i r o n quota ( i . e . t h a t r e q u i r e d t o j u s t s u s t a i n maximal growth, ^ m a x ) accounted f o r by t h i s c a t a l y t i c i r o n w h i l e growing at p m a x (20°C). Raven has performed such 82 c a l c u l a t i o n s u s i n g two of the data s e t s a v a i l a b l e f o r T. weissflogii, and determined t h a t from 16% (data of Morel and Hudson 1985) t o 78% (data of H a r r i s o n and Morel 1986) of i t s c r i t i c a l Q F e i s comprised by c a t a l y t i c i r o n . These percentages may be underestimates, as the c a t a l y t i c Fe requirement was based on NH 4 growth, w h i l e data employed were f o r NG^-grown T. weissflogii. Although a t r u e " c r i t i c a l " Q F e was not o b t a i n e d h e r e i n , ' s i m i l a r c a l c u l a t i o n s can be made u s i n g the Q F emax f o r G. sanguineum (4.58«10~ 3 mol Fe:mol C, +Fe/NH 4, F i g . 2.2A). In marked c o n t r a s t t o the va l u e s of T. weissflogii, o n l y about 0.1% of Q F emax i s accounted f o r by c a t a l y t i c i r o n i n NH 4-grown G. sanguineum. For c e l l s grown on NG^, the percentage i s c l o s e r t o 0.2%. The c r i t i c a l Q F e of T. weissflogii i s 45% of the maximum i r o n quota determined by H a r r i s o n and Morel (1986). I f the same were t r u e f o r G. sanguineum ( p u r e l y s p e c u l a t i v e ) , the Fe accounted f o r would remain below 0.4%. While these v a l u e s are sm a l l r e l a t i v e t o those of the diatom, percentages of c a . 1% have been suggested p r e v i o u s l y f o r hi g h e r p l a n t s (Smith 1984). E l e v a t e d l e v e l s of d i v a l e n t c a t i o n s , i n c l u d i n g i r o n , a s s o c i a t e d w i t h d i n o f l a g e l l a t e chromatin (ca. 6 atoms Fe/ca. 100 n u c l e o t i d e s , Sigee 1983,1984) have not been demonstrated f o r oth e r a l g a e , and would r e p r e s e n t a t l e a s t one n o n - c a t a l y t i c i r o n requirement unique t o d i n o f l a g e l l a t e s . N e v e r t h e l e s s , more i n f o r m a t i o n on the nature and met a b o l i c r o l e of a c o n s i d e r a b l e p o r t i o n of the 83 i r o n quota of G. sanguineum and other d i n o f l a g e l l a t e s i s c l e a r l y needed. R a t i o s of Fe/N d e c l i n e d s i g n i f i c a n t l y under i r o n s t r e s s i r r e s p e c t i v e of n i t r o g e n source, i n f l u e n c e d predominantly by l a r g e r e d u c t i o n s i n Q F e (ca. 1.5 orde r s of magnitude, -Fe/NC^ and -Fe/NH^ c u l t u r e s ) . However, i n c o n t r a s t t o Fe/C r a t i o s , -Fe/NC-3 v a l u e s of Fe/N were 1.5 times g r e a t e r than f o r Fe-d e p l e t e , NH^-grown c e l l s ( F i g . 2.2B). T h i s d i s p a r i t y i s l i k e l y a t t r i b u t a b l e t o the i r o n requirement of NO3 a s s i m i l a t o r y enzymes. By c o n s i d e r i n g both i r o n and n i t r o g e n as a s i n g l e q u o t i e n t , Fe/N q u i t e a c c u r a t e l y r e f l e c t s the 1.6-f o l d l a r g e r Fe requirement c a l c u l a t e d by Raven (1988) f o r c e l l s growing on NO3 over those s u p p l i e d w i t h NH4. Thus, w h i l e measurements of Q F e alone are i n s u f f i c i e n t t o d i s t i n g u i s h t h i s i n h e r e n t d i f f e r e n c e i n i r o n demand, n o r m a l i z i n g t o QJJ a p p a r e n t l y p r o v i d e s the r e q u i r e d r e s o l u t i o n . Reductions i n c h l a a s s o c i a t e d w i t h i r o n d e p l e t i o n , and the r e s u l t i n g c h l a quotas, were s i m i l a r f o r both NO3- and NH^-grown c u l t u r e s (Tables 2.1,2.2). The c h l o r o t i c nature of i r o n - d e p l e t e c e l l s observed i n t h i s and othe r r e p o r t s on i r o n -s t r e s s e d phytoplankton (e.g. Sakshaug and Holm-Hansen 1977, Reuter and Ades 1987) r e f l e c t s i r o n ' s e s s e n t i a l r o l e i n c h l o r o p h y l l b i o s y n t h e s i s (Pushnik e t a l . 1984). G. sanguineum Qchl a a n c * ^ t s r e s P o n s e t o i r o n d e p l e t i o n , i n c l u d i n g u l t r a s t r u c t u r a l m a n i f e s t a t i o n s , were c o n s i d e r e d p r e v i o u s l y (Chapter 1). 84 Cytochromes of both PET and RET c h a i n s not o n l y share s i m i l a r b i o s y n t h e t i c Fe requirements w i t h c h l o r o p h y l l , but they a l s o c o n t a i n a heme p r o s t h e t i c group (Marschner 1986). Data r e p o r t e d h e r e i n and by other i n v e s t i g a t o r s (e.g. Glover 1977, T e r r y 1983, Sandmann 1985) show t h a t a d e c l i n e i n Fe a v a i l a b i l i t y t r a n s l a t e s i n t o fewer PET components and lower PET c a p a c i t y , as i n d i c a t e d i n t h i s study by marked i n c r e a s e s i n F / c h l a ( F i g . 2.4A). The i r o n s t r e s s - m e d i a t e d s p e c i f i c i t y o f , and a d d i t i o n a l f a c t o r s c o n t r i b u t i n g t o , e l e v a t e d F / c h l a r a t i o s i n G. sanguineum are d i s c u s s e d i n Chapter 1. Of p a r t i c u l a r i n t e r e s t t o the p r e s e n t r e s e a r c h i s the v a r i a t i o n i n in vivo f l u o r e s c e n c e r a t i o s and i n d i c e s between -Fe/N03 and -Fe/NH 4 c u l t u r e s . R a t i o s of F / c h l a ( F i g . 2.4A) and F/Fe ( F i g . 2.4B) i n c r e a s e d d r a m a t i c a l l y under i r o n d e p l e t i o n . However, v a l u e s f o r both r a t i o s were 40-50% l e s s i n -Fe/NH 4 than -Fe/N0 3 c u l t u r e s . Perhaps the most i n t u i t i v e e x p l a n a t i o n of these r e s u l t s concerns the d i f f e r e n c e i n Fe requirement f o r growth on NO3 v e r s u s NH 4. The a d d i t i o n a l i r o n needed by N0 3-grown c e l l s f o r N a s s i m i l a t i o n may be s u p p l i e d p a r t l y a t the expense of PET components, thereby l o w e r i n g PET e f f i c i e n c y . As Q F e was s i m i l a r f o r -Fe/N03 and -Fe/NH 4 c u l t u r e s (Table 2.1), t h i s d i s p a r i t y i n apportionment of c e l l u l a r Fe (more d i r e c t l y i n t e r p r e t e d as F/Fe) i m p l i e s t h a t f o r an e q u i v a l e n t r e d u c t i o n i n a v a i l a b l e Fe, PET a c t i v i t y would d e c l i n e most i n N03~grown c e l l s (accompanied by a h i g h e r F y i e l d ) . A d d i t i o n a l support f o r the enhanced e f f e c t of i r o n s t r e s s on PET i n c e l l s grown 85 on N 0 3 was p r o v i d e d by F D - F ( F i g . 2.5A) and 1-F/F D ( F i g . 2.5B). These i n d i c e s are c o n s i d e r e d t o be i n d i c a t i v e of Photosystem I I (PSII) c a p a c i t y and e f f i c i e n c y , r e s p e c t i v e l y (see Chapter 1). Values o b t a i n e d h e r e i n suggest t h a t w h i l e PSII c a p a c i t y i s s i m i l a r among a l l experiments, the e f f i c i e n c y of PSII under Fe d e p l e t i o n i s s i g n i f i c a n t l y l e s s (ca. 35%) i n NO3- than NH^-grown c e l l s . Measurements of F e - c o n t a i n i n g components i n v o l v e d i n PET and NO3/NO2 r e d u c t i o n are r e q u i r e d t o e m p i r i c a l l y v e r i f y t h i s o r an a l t e r n a t i v e i n t e r p r e t a t i o n . Effects of nitrogen source. C e r t a i n r a t i o s were a l t e r e d by i r o n d e p l e t i o n o n l y i n e i t h e r NO3- or NH 4-grown c u l t u r e s (but not bo t h ) , i n d i c a t i n g an o v e r r i d i n g i n f l u e n c e of n i t r o g e n source on the response of some v a r i a b l e s t o i r o n s t r e s s . A depa r t u r e of C/N from the average i r o n - r e p l e t e v a l u e of 6.6 ± 0.5 o c c u r r e d o n l y i n -Fe/NH 4 c u l t u r e s (C/N = 3.8 ± 0.5, F i g . 2.2A). Few comparisons of C/N r a t i o s between NO3- and N H 4 -grpwn phytoplankton are a v a i l a b l e , w h i l e s t i l l fewer e x i s t f o r F e - d e f i c i e n t a l g a e . Reports by s e v e r a l i n v e s t i g a t o r s (Conover 1975, L a r s s o n e t a l . 1985, Thompson e t a l . i n press) demonstrate a n e g l i g i b l e e f f e c t of N source on the C/N r a t i o s of n u t r i e n t - r e p l e t e c u l t u r e s . R e s u l t s o b t a i n e d h e r e i n f o r +Fe / N 0 3 and +Fe/NH 4 G. sanguineum are c o n s i s t e n t w i t h these d a t a . In the o n l y p r e v i o u s study t o examine C/N as a f u n c t i o n of Fe d e p l e t i o n , Sakshaug and Holm-Hansen (1977) were unable t o d e t e c t a c l e a r change i n t h i s r a t i o (range: 7-11, wit h c o n s i d e r a b l e v a r i a t i o n about the mean) f o r e i t h e r a diatom (Skeletonema costatum) or a chrysophyte (Pavlova lutheri) , 86 each grown on NO3-N. S i m i l a r l y , the p r e s e n t experiments r e v e a l e d no d i f f e r e n c e i n C/N between +Fe/NG"3 and -Fe/NC>3 c u l t u r e s (avg. = 6.6 ± 0.5), y e t t h i s r a t i o d e c l i n e d by c a . 40% i n NH^-grown c u l t u r e s under Fe d e p l e t i o n . The C/N r a t i o of n u t r i e n t - s u f f i c i e n t phytoplankton i s g e n e r a l l y c o n s i d e r e d t o approximate the R e d f i e l d r a t i o of 106 C:16 N ( i . e . 6.6), w h i l e a l l o w i n g f o r d i f f e r e n c e s between taxonomic groups. For example, Parsons e t a l . (1961) r e p o r t an average C/N of 6.2 ± 1.8 f o r 11 phytoplankton s p e c i e s d i s t r i b u t e d among f i v e a l g a l c l a s s e s . C/N v a l u e s i n v a r i a b l y r i s e under n i t r o g e n l i m i t a t i o n o r d e p l e t i o n (e.g. Laws and B a n n i s t e r 1980, Dortch e t a l . 1984), and are thus a r e l i a b l e i n d i c a t o r of N d e f i c i e n c y . Conversely, i t f o l l o w s t h a t a decrease i n C/N should r e p r e s e n t a s u r p l u s of n i t r o g e n r e l a t i v e t o carbon, and presumably t o the growth requirement. Iron, d e p l e t i o n s i g n i f i c a n t l y reduced carbon quotas of both NO3- and NH^-grown c u l t u r e s , but o n l y the l a t t e r e x h i b i t e d a lower C/N r a t i o . These data suggest a concomitant drop i n carbon and n i t r o g e n a c q u i s i t i o n by -Fe/NG^ c e l l s , whereas N a s s i m i l a t i o n i s maintained r e l a t i v e t o d e c l i n i n g C f i x a t i o n i n Fe - d e p l e t e c e l l s grown on NH4. T h i s d i s c r e p a n c y l i k e l y r e f l e c t s a reduced c a p a c i t y of Fe-deplete G. sanguineum t o pr o v i d e i r o n needed f o r n i t r a t e a s s i m i l a t i o n , w h i l e ammonium-N i s i n c o r p o r a t e d i n excess of the growth requirement. A comparative i n a b i l i t y of NG-3-grown c e l l s t o a s s i m i l a t e n i t r o g e n under i r o n s t r e s s i s f u r t h e r supported by AA/Pr 87 r a t i o s , which decreased markedly (ca. 40%) and e x c l u s i v e l y i n c u l t u r e s r e c e i v i n g NO3-N ( F i g . 2.3). T h i s r e d u c t i o n was l a r g e l y due t o a 50% d e c l i n e i n f r e e amino a c i d s , overshadowing a s m a l l (10%) l o s s of c e l l u l a r p r o t e i n . In c o n t r a s t , both and Qp r of NH 4-grown c e l l s were u n a f f e c t e d by Fe d e p l e t i o n . The i n c o r p o r a t i o n of N i n t o f r e e amino a c i d s i s a r e q u i r e d s t e p i n the a s s i m i l a t i o n of i n o r g a n i c N i n p r o t e i n and o t h e r nitrogenous c e l l u l a r compounds (Wheeler 1983). I t has been suggested t h a t the f r e e AA p o o l f u n c t i o n s as a n i t r o g e n b u f f e r , and i s u t i l i z e d when c e l l s are d e p r i v e d of N (Admiraal e t a l . 1986). Although - F e / N 0 3 c u l t u r e s c o n t a i n e d s a t u r a t i n g ambient NO3 c o n c e n t r a t i o n s , the lower NO3 a s s i m i l a t o r y c a p a c i t y (a r e s u l t of Fe s t r e s s ) caused c e l l s t o become e f f e c t i v e l y N - l i m i t e d i n r e l a t i o n t o t h e i r growth requirement. Thus, upon c e s s a t i o n of c e l l d i v i s i o n ( i . e . time of sampling h e r e i n ) , f r e e amino a c i d p o o l s were g r e a t l y reduced from p r e v i o u s i r o n - r e p l e t e l e v e l s . Dortch e t a l . (1984) proposed t h a t AA/Pr r a t i o s ( h i g h e s t f o r n u t r i e n t s u f f i c i e n c y ) are a more u n i v e r s a l i n d i c a t o r of N d e f i c i e n c y than C/N, AA/N or Pr/N, with a g r e a t e r range of v a l u e s over s i m i l a r N - r e p l e t e and - d e p l e t e c o n d i t i o n s . R e c a l l t h a t C/N r a t i o s f o r - F e / N 0 3 and -Fe/NH 4 c u l t u r e s d i d not i n d i c a t e a n i t r o g e n shortage i n the former, but r a t h e r an N s u r p l u s i n the l a t t e r . By comparison AA/Pr r a t i o s , w h i l e e q u i v a l e n t f o r both +Fe/NH 4 and -Fe/NH 4 c u l t u r e s , r e v e a l symptoms of N d e f i c i e n c y i n -Fe/NC>3 c e l l s . A c c o r d i n g t o AA/Pr and C/N r a t i o s , the c u r r e n t r e s e a r c h suggests t h a t r e l a t i v e to 88 t h e i r r e s p e c t i v e c e l l u l a r requirements, -Fe/NC^ G. sanguineum c e l l s are N d e f i c i e n t , w h i l e F e - d e p l e t e , NH^-grown c e l l s c o n t a i n an excess of n i t r o g e n . Qualitative nitrogen composition. Phytoplankton n i t r o g e n quotas comprise a v a r i e t y of N - c o n t a i n i n g compounds such as those c o n s i d e r e d h e r e i n ( i . e . Pr, AA, c h l a, i n t e r n a l NO3) and o t h e r s , i n c l u d i n g NH4 p o o l s , RNA and DNA. C a l c u l a t i o n s presented i n Table 2.3 i n d i c a t e t h a t the ni t r o g e n o u s c o n s t i t u e n t s measured account f o r 15-20% l e s s of Q N i n Na-than NO^-grown c e l l s . For the most extreme case ( i . e . -Fe/NH^) c a . 30% of Q N was not i n c l u d e d i n a summation of Pr, AA, c h l a and i n t e r n a l N03. Values exceeding 60% Q N unaccounted f o r , have been r e p o r t e d p r e v i o u s l y i n more d e t a i l e d a n a l y s e s of nitrogenous compounds (Conover 1975, Dortch e t a l . 1984). F a i l u r e t o measure a l l N components i n the AA f r a c t i o n and l o s s of N w i t h a l i p i d f r a c t i o n were suggested as p o s s i b l e e x p l a n a t i o n s . In the prese n t study, however, t h e r e appears t o be a q u a l i t a t i v e d i f f e r e n c e i n N composition unique t o NH^-grown G. sanguineum and a t t r i b u t a b l e t o a c o n s t i t u e n t ( s ) not determined or underestimated. V a r i a t i o n s i n i n t e r n a l NH4, RNA and DNA percentages (of Q N) between NO3- and Nl^-grown c u l t u r e s of Amphidinium carterae (Dinophyceae) and a l s o of Thalassiosira nordenskioldii ( B a c i l l a r i o p h y c e a e ) were observed by Dortch et a l . (1984), but d i d not exceed 2% f o r any s i n g l e component. P l a n t s s u p p l i e d w i t h ammonium can e x h i b i t d i s t i n c t l y d i f f e r e n t 89 f r e e amino a c i d p r o f i l e s compared t o those r e c e i v i n g n i t r a t e (Harada e t a l . 1968). P e r t i n e n t t o these o b s e r v a t i o n s , C l a y t o n (1985) has demonstrated t h a t fluorescamine-based f r e e AA a n a l y s e s s t a n d a r d i z e d t o glutamate (as h e r e i n ) , c o n s i d e r a b l y (by a f a c t o r of 2-3) underestimate c o n c e n t r a t i o n s of c e r t a i n amino a c i d s (e.g. a s p a r t a t e ) . The AA compo s i t i o n of phytoplankton i s r e p o r t e d t o vary w i d e l y among s p e c i e s and as a f u n c t i o n of n u t r i t i o n a l s t a t u s (e.g. Admiraal e t a l . 1986). N e v e r t h e l e s s , the d i s p a r i t y between NH^- and NC^-grown G. sanguineum may r e p r e s e n t amino a c i d s such as a s p a r t a t e , l i k e l y augmented by a d d i t i o n a l f a c t o r s i n c l u d i n g those mentioned above. A l e s s e q u i v o c a l e x p l a n a t i o n awaits a comprehensive i n v e s t i g a t i o n of nitrogenous c e l l c o n s t i t u e n t s . Ecological considerations. S e v e r a l r e p o r t s noted at the o u t s e t of t h i s paper have i d e n t i f i e d i r o n as a p o t e n t i a l l y important f a c t o r i n the i n i t i a t i o n of r e d t i d e s . However, the b e l i e f t h a t c o a s t a l i r o n c o n c e n t r a t i o n s are u n l i k e l y t o l i m i t r e p r o d u c t i v e r a t e s o f phytoplankton r e s u l t s i n an apparent paradox. I t was a l s o noted above t h a t most evidence s u p p o r t i n g the l a t t e r c o n t e n t i o n r e l i e s on s t u d i e s of diatom i r o n n u t r i t i o n . For example, c a l c u l a t i o n s used by M a r t i n and Gordon (1988) t o suggest an adequate i r o n supply f o r n e r i t i c p hytoplankton growth, employ optimum Fe/C r a t i o s of the diatom Thalassiosira weissflogii (Anderson and Morel 1982, Morel and Hudson 1985). Data o b t a i n e d h e r e i n p r o v i d e v a l u e s between one and two ord e r s of magnitude g r e a t e r f o r the d i n o f l a g e l l a t e G. sanguineum. Use of a c r i t i c a l Fe/C ( d i s c u s s e d above) r a t i o 90 may reduce t h i s d i s c r e p a n c y , but d i f f e r e n c e s would s t i l l approach a f a c t o r of t e n . I t i s d i f f i c u l t t o determine the exten t t o which Fe supply exceeds i t s demand by phytoplankton. However, est i m a t e s u t i l i z i n g G. sanguineum Fe/C r a t i o s t i g h t e n the r e l a t i o n s h i p c o n s i d e r a b l y and i n d i c a t e t h a t r e d t i d e d i n o f l a g e l l a t e s may be more s u s c e p t i b l e t o i r o n - l i m i t e d growth than c o a s t a l diatoms. A s i m i l a r c o n c l u s i o n was reached i n Chapter 1, based on the com p a r a t i v e l y l a r g e h a l f - s a t u r a t i o n c o n s t a n t f o r i r o n - l i m i t e d growth of G. sanguineum. Raven (1988) has estimated t h a t a s s i m i l a t i o n of n i t r a t e i n c r e a s e s a p l a n t ' s i r o n requirement by 60% r e l a t i v e t o growth on ammonium. Determinations of b i o c h e m i c a l c o m p o s i t i o n r e p o r t e d i n t h i s study (e.g. Fe/N) p r o v i d e e m p i r i c a l , a l b e i t i n d i r e c t , evidence c o n s i s t e n t w i t h these c a l c u l a t i o n s . Thus, the q u a n t i t y of c e l l u l a r i r o n y i e l d i n g maximum growth r a t e s ( i . e . c r i t i c a l Q F e ) would u l t i m a t e l y be a f u n c t i o n of n i t r o g e n source. By determining the c r i t i c a l Q F e f o r a s p e c i e s , n i t r o g e n source a l s o r e g u l a t e s the degree of growth l i m i t a t i o n f o r a g i v e n s u b s a t u r a t i n g ( f o r growth) i r o n c o n c e n t r a t i o n . The e c o l o g i c a l i m p l i c a t i o n s of these e f f e c t s are u n c e r t a i n , as both predominating N source and blooms of c o a s t a l d i n o f l a g e l l a t e s e x h i b i t temporal v a r i a t i o n (e.g. Anderson et a l . 1983, Robinson and Brown 1983). However, i n the event t h a t i r o n supply and demand are c l o s e l y balanced, the i n f l u e n c e of n i t r o g e n source on phytoplankton i r o n requirements may be important i n deter m i n i n g growth r a t e . 91 A f i n a l comment should be made con c e r n i n g the use of i n d i c e s based on b i o c h e m i c a l c o n s t i t u e n t s t o i d e n t i f y a l i m i t i n g n u t r i e n t . As demonstrated h e r e i n and i n Chapter 1, F / c h l a r a t i o s of NC^-grown G. sanguineum i n c r e a s e c o n s i d e r a b l y (5-7 f o l d ) i n response t o i r o n d e p l e t i o n . Yet, f o r c e l l s grown on NH^, the magnitude of these changes i s only a f a c t o r of t h r e e , approaching t h a t observed under n i t r o g e n d e p l e t i o n (see Chapter 1 ) . I t i s a l s o apparent t h a t i r o n s t r e s s e f f e c t s symptoms of N d e f i c i e n c y (e.g. reduced AA/Pr) i n -Fe/NG"3 c u l t u r e s . In the present case of i r o n d e p l e t i o n , the p o t e n t i a l f o r c o n f l i c t i n g i d e n t i f i c a t i o n of the l i m i t i n g n u t r i e n t as a f u n c t i o n of N source and the index employed i s c l e a r . Such c o m p l i c a t i o n s r e i n f o r c e the need t o c o n s i d e r m u l t i p l e i n d i c a t o r s of n u t r i e n t d e f i c i e n c y i n the f i e l d , and f o r c a r e f u l i n t e r p r e t a t i o n of r e s u l t s . Summary. The f i r s t measurements of i r o n quota f o r G. sanguineum, a red t i d e d i n o f l a g e l l a t e , have been o b t a i n e d . I t s i r o n requirement exceeds those of c e r t a i n n e r i t i c diatoms by one t o two o r d e r s of magnitude. G. sanguineum i r o n quotas, and a comparably l a r g e h a l f - s a t u r a t i o n c o n s t a n t f o r F e - l i m i t e d growth (Chapter 1 ) , support the c o n t e n t i o n t h a t i r o n may r e g u l a t e the growth of c e r t a i n d i n o f l a g e l l a t e s without s i m i l a r l y a f f e c t i n g diatom r e p r o d u c t i v e r a t e s . R a t i o s of b i o c h e m i c a l c o n s t i t u e n t s (e.g. Fe/N) r e v e a l a g r e a t e r Fe requirement f o r NO3- than NH^-grown c e l l s , l i k e l y a t t r i b u t a b l e t o the e s s e n t i a l r o l e of i r o n i n the r e d u c t i v e NO3 a s s i m i l a t o r y enzymes n i t r a t e and n i t r i t e r e d u c t a s e . The magnitude of changes i n F / c h l a and 1-F/F D e x h i b i t e d by -Fe/NO-3 c u l t u r e s suggests t h a t under i r o n s t r e s s , supply of f o r NO3 a s s i m i l a t i o n i s p a r t l y a t the expense of PET components. N e v e r t h e l e s s , a c q u i s i t i o n of n i t r o g e n by c e l l s grown on NO3 i s s u f f i c i e n t l y i n h i b i t e d by i r o n d e p l e t i o n t o y i e l d symptoms of N d e f i c i e n c y . Perhaps the most important e f f e c t mediated by N source would be the d e t e r m i n a t i o n of c r i t i c a l Q p ej thereby r e g u l a t i n g the degree of growth l i m i t a t i o n f o r a g i v e n s u b s a t u r a t i n g i r o n c o n c e n t r a t i o n . 93 CHAPTER 3. ASPECTS OF IRON AND NITROGEN NUTRITION IN THE RED TIDE DINOFLAGELLATE GYMNODINIUM SANGUINEUM HIRASAKA: EFFECTS OF IRON DEPLETION AND NITROGEN SOURCE ON IRON AND NITROGEN UPTAKE BACKGROUND Iron i s q u a n t i t a t i v e l y the most important of a l l t r a c e elements known t o be e s s e n t i a l f o r phytoplankton growth (Huntsman and Sunda 1980). N i t r o g e n , the m a c r o n u t r i e n t r e q u i r e d i n g r e a t e s t abundance a f t e r carbon, hydrogen and oxygen, can account f o r as much as 10% of a l g a l c e l l dry weight ( S y r e t t 1981). Among the b i o c h e m i c a l r o l e s of i r o n i n algae (and p l a n t s i n g e n e r a l ) , s e v e r a l are c l o s e l y l i n k e d w i t h v a r i o u s a s p e c t s of i n o r g a n i c (e.g. a s s i m i l a t o r y ) and o r g a n i c (e.g. b i o s y n t h e t i c ) n i t r o g e n metabolism. Ir o n i s a c o n s t i t u e n t of the r e d u c t i v e n i t r a t e a s s i m i l a t o r y enzymes n i t r a t e and n i t r i t e r eductase (Guerrero e t a l . 1981), and a l s o of cytochromes and i r o n - s u l f u r p r o t e i n s (Marschner 1986). The b i o s y n t h e s i s of c h l o r o p h y l l (ca. 6% N, by atoms) and the c a t a l y t i c a c t i v i t y of some or a l l forms of s e v e r a l enzymes, i n c l u d i n g glutamate synthase, superoxide dismutase, c a t a l a s e , p e r o x i d a s e and hydrogenase ( R o e s s l e r and L i e n 1984, Raven 1988) a l s o r e q u i r e i r o n . The i r o n n u t r i t i o n of a l g a l c e l l s may thus i n f l u e n c e not onl y the i n c o r p o r a t i o n of c e r t a i n i n o r g a n i c N s p e c i e s i n t o amino a c i d s , but a l s o some b i o s y n t h e t i c pathways u t i l i z i n g these compounds and u l t i m a t e l y , the m e t a b o l i c processes dependent upon t h e i r end products (e.g. p h o t o s y n t h e s i s and r e s p i r a t i o n ) . 94 In a q u a t i c environments i n o r g a n i c n i t r o g e n i s a c q u i r e d by e u k a r y o t i c algae p r i m a r i l y as e i t h e r n i t r a t e or ammonium. A s s i m i l a t i o n of the former r e q u i r e s two iron-dependent r e d u c t i o n s t e p s , w h i l e the l a t t e r may be i n c o r p o r a t e d d i r e c t l y i n t o amino a c i d s (Wheeler 1983). Raven (1988) c a l c u l a t e d t h a t f o r p h o t o l i t h o t r o p h i c ( i . e . l i g h t - d r i v e n 0 2 e v o l u t i o n w i t h an i n o r g a n i c carbon source) r e p r o d u c t i o n on n i t r a t e , p l a n t s r e q u i r e 60% more i r o n than when growing a t the same r a t e on ammonium. Thus, the p h y s i o l o g i c a l s t a t u s of phytoplankton under i r o n - l i m i t i n g c o n d i t i o n s would l i k e l y be a f u n c t i o n of n i t r o g e n source (e.g. NO3 or NH4) as w e l l as i r o n b i o a v a i l a b i l i t y . I r o n has been i m p l i c a t e d i n the i n i t i a t i o n of r ed t i d e s (see Chapters 1,2). C o n s i s t e n t w i t h t h i s s u g g e s t i o n are data (Chapter 1) showing a h a l f - s a t u r a t i o n c o n s t a n t f o r F e - l i m i t e d growth of a r e d t i d e s p e c i e s a t l e a s t t e n - f o l d g r e a t e r than r e p o r t e d f o r some n e r i t i c diatoms (e.g. H a r r i s o n and Morel 1986). These d i n o f l a g e l l a t e blooms are of e c o l o g i c a l importance due not o n l y t o t h e i r apparent d e t r i m e n t a l a s p e c t s , i n c l u d i n g t o x i n p r o d u c t i o n and high BOD r e s u l t i n g i n anoxia (Yentsch e t a l . 1980, T a y l o r 1987), but a l s o t h e i r p o t e n t i a l c o n t r i b u t i o n t o l o c a l primary p r o d u c t i o n (Vargo e t a l . 1987). The predominant s p e c i e s of i n o r g a n i c n i t r o g e n i n c o a s t a l waters, v a r i e s both t e m p o r a l l y and s p a t i a l l y a c c o r d i n g t o l o c a l c o n d i t i o n s (Sharp 1983), as do red t i d e s themselves. There are few data p r e s e n t l y a v a i l a b l e on the i r o n n u t r i t i o n of r e d t i d e d i n o f l a g e l l a t e s and c o a s t a l d i n o f l a g e l l a t e s i n 95 g e n e r a l . Furthermore, i t i s a l s o of i n t e r e s t t o a t l e a s t address the p o s s i b i l i t y t h a t n i t r o g e n source may i n f l u e n c e the e f f e c t s of i r o n d e f i c i e n c y on these organisms. From the pr e c e d i n g c hapter (2), i t i s apparent t h a t i r o n d e p l e t i o n a l t e r s the b i o c h e m i c a l composition of Gymnodinium sanguineum, a r e d t i d e s p e c i e s . However, both the exte n t and nature of these changes are, i n f a c t , s t r o n g l y i n f l u e n c e d by n i t r o g e n source ( i . e . NO3 or NH4). In a d d i t i o n , Reuter and Ades (1987) have observed a g r e a t e r e f f e c t ( i . e . r e d u c t i o n ) of i r o n l i m i t a t i o n on the uptake of NO3 than NH4 (enhanced under l i g h t l i m i t a t i o n ) by Scenedesmus quadricauda (Chlorophyceae). I t was the aim of t h i s r e p o r t t o f u r t h e r d e f i n e both the consequences of i r o n s t r e s s and the r e l a t i o n s h i p between i r o n and n i t r o g e n n u t r i t i o n f o r G. sanguineum. T h i s assessment was based on Fe, NO3 and NH 4 uptake r a t e s of i r o n - r e p l e t e (= n u t r i e n t - s u f f i c i e n t ) and i r o n - d e p l e t e c u l t u r e s growing on e i t h e r NO3 or-NH^. I n t e r n a l n i t r a t e p o o l s and r a t e s of n i t r i t e e x c r e t i o n d u r i n g NO3 uptake experiments were measured t o p r o v i d e a d d i t i o n a l i n f o r m a t i o n on n i t r a t e a s s i m i l a t i o n under these c o n d i t i o n s . n MATERIALS AND METHODS General culture maintenance. N i t r a t e - and ammonium-grown s t o c k c u l t u r e s of Gymnodinium sanguineum ( c u l t u r e #D354, North E a s t P a c i f i c C u l t u r e C o l l e c t i o n , Dept. of Oceanography, U n i v e r s i t y of B r i t i s h Columbia) were maintained on m o d i f i e d 96 ESAW-enriched a r t i f i c i a l seawater ( H a r r i s o n e t a l . 1980). Media p r e p a r a t i o n and c u l t u r e c o n d i t i o n s have been d e s c r i b e d i n Chapter 2. Iron-replete and iron-deplete cultures. A l l experimental data a c q u i r e d h e r e i n were ob t a i n e d from a l i q u o t s of those c u l t u r e s u t i l i z e d i n Chapter 2 f o r assessment of b i o c h e m i c a l c o m p o s i t i o n . Samples employed were ha r v e s t e d from c u l t u r e s immediately a f t e r those used t o examine c o m p o s i t i o n a l v a r i a b l e s . Measurements made i n the presen t study (and d e s c r i b e d i n d e t a i l i n the f o l l o w i n g s e c t i o n s ) i n c l u d e i r o n and n i t r o g e n (NO3 and NH 4) uptake r a t e s , n i t r i t e e x c r e t i o n r a t e s and i n t e r n a l NO3 p o o l formation f o r n i t r a t e - or ammonium-grown, i r o n - r e p l e t e ( i . e . +Fe/NC>3 and +Fe/NH 4) and i r o n - d e p l e t e ( i . e . -Fe/NC>3 and -Fe/NH 4) c u l t u r e s . Cell pretreatment for uptake experiments. Determination of i r o n uptake by ^ F e C ^ r e q u i r e s t h a t c e l l s be p l a c e d i n i r o n - f r e e medium b e f o r e l a b e l l i n g t o a l l o w c a l c u l a t i o n of t r a c e r s p e c i f i c a c t i v i t y ( i . e . pCi*pmol ambient F e - 1 ) , and thus t r a n s p o r t and s p e c i f i c uptake r a t e s . C o n v e n t i o n a l washing/resuspension methods f o r phytoplankton employ a standard f i l t r a t i o n apparatus (e.g. Anderson and Morel 1982). As G. sanguineum i s s e n s i t i v e t o p h y s i c a l s t r e s s (see Chapter 2), t h i s technique was f i r s t t e s t e d by comparing carbon uptake (NaH 1 4C03, New England Nuclear) of unwashed (UW) and washed (W) samples ( a l l samples from one c u l t u r e ; n = 3 f o r UW,W). UW experiments i n v o l v e d simply the a d d i t i o n of 1 4 C (0.1 p C i - m l - 1 ) t o an a l i q u o t of u n t r e a t e d c u l t u r e . For W i n c u b a t i o n s , c e l l s were removed from t h e i r o r i g i n a l medium by f i l t r a t i o n u n t i l the meniscus was j u s t above the f i l t e r (5.0 pm f i l t e r , Nuclepore; 25-50 mm Hg), washed by g e n t l y adding c u l t u r e f i l t r a t e (Whatman GF/C f i l t e r e d ) and r e f i l t e r i n g (as b e f o r e ) , and resuspended i n c u l t u r e f i l t r a t e p r i o r t o 1 4 C a d d i t i o n . C e l l d e n s i t i e s of UW and W i n c u b a t i o n s d i f f e r e d by < 5%. Uptake experiments were conducted i n 85 ml Oak Ridge tubes (polycarbonate, Nalgene) and i n c u b a t e d (unbubbled, u n s t i r r e d ) f o r 6 h under c u l t u r e c o n d i t i o n s d e s c r i b e d e a r l i e r (Chapter 2 ) . R e s u l t s c l e a r l y demonstrate an adverse e f f e c t of the c o n v e n t i o n a l washing technique, w i t h W c e l l s showing n e g l i g i b l e 1 4 C uptake even a f t e r 6 h ( F i g . 3.1A). As a p h y s i c a l l y more g e n t l e a l t e r n a t i v e , washing by r e v e r s e - f l o w f i l t r a t i o n (RFF, see Holm-Hansen e t a l . 1970) was t e s t e d . B r i e f l y , RFF i n v o l v e d a l l o w i n g a tube ( T l , 3 cm I.D.), w i t h i t s bottom occluded by s c r e e n i n g (10 pm N i t e x R ) , t o s e t t l e by g r a v i t y through a s l i g h t l y l a r g e r diameter tube (T2, 4.5 cm I.D.) c o n t a i n i n g the c u l t u r e . C u l t u r e medium moves up through the s c r e e n i n g i n t o T l , where the f i l t r a t e i s c o l l e c t e d and c o n t i n u o u s l y removed, l e a v i n g a c o n c e n t r a t e d c e l l suspension (ca. 3-5 ml) i n T2. F i l t r a t i o n p r e s s u r e i s determined by the d i f f e r e n c e between the h e i g h t of the water i n T l and T2. C e l l s were washed by resuspension i n c u l t u r e f i l t r a t e and r e p e a t i n g t h i s procedure. The uptake of 1 4 C (0.4 p C i - m l - 1 ) ( a l l samples from one c u l t u r e ; n = 3 f o r UW,W) by RFF-washed c e l l s (two r e s u s p e n s i o n s ; c a . 10-15 min) was e q u i v a l e n t t o 98 F i g u r e 3.1A. Uptake of carbon (0.1•u C i-ml" 1 N a H i 4 C 0 3 ) by unwashed (o, n = 3) and washed (•, n = 3) c u l t u r e s . C e l l s were washed by f i l t r a t i o n and r e s u s p e n s i o n u s i n g s t a n d a r d f i l t r a t i o n a p p a r a t u s . B. Uptake of carbon ( 0.4• p C i'ml - 1 NaH CO-j) by unwashed (o, n = 3) and washed (•, n = 3) c u l t u r e s . C e l l s were washed by r e v e r s e - f l o w f i l t r a t i o n ( R F F ) ( d e t a i l s i n t e x t ) . C. Uptake of i r o n (0.08 yCi'ml"" 1 5 5 F e C l 3 , 5 a d d i t i o n ) by unwashed (o, n = 1) and RFF-washed (•, n = 1) c u l t u r e s . E r r o r b a r s f o r (A-C) r e p r e s e n t ± 1 S.D. (or on l y 1 S.D. t o p r e v e n t o v e r l a p ) and are s m a l l e r than symbol where not apparent f o r experiments i n which n > 1. DPM = d i s i n t e g r a t i o n s - m i n - 1 . TIME (h) 99 t h a t of UW c e l l s ( F i g . 3.IB), i n d i c a t i n g the s u i t a b i l i t y of t h i s technique f o r washing G. sanguineum. The e f f e c t of RFF on i r o n uptake was a l s o examined by f o l l o w i n g i n c o r p o r a t i o n of 5 5 F e C l 3 (5 pM a d d i t i o n s , 0.08 p C i - m l " 1 , New England Nuclear) i n t o UW and W (RFF procedure) c e l l s over a 6 h p e r i o d ( a l l samples from one c u l t u r e ; n = 1 f o r UW,W). Samples c o l l e c t e d f o r Fe counts were f i l t e r e d and washed of f i l t e r - and surface-bound i r o n as o u t l i n e d below (see Iron uptake). Accumulation of l a b e l was expressed as D P M ' c e l l - 1 due t o the u n c e r t a i n t y of ambient i r o n c o n c e n t r a t i o n s i n the medium, and hence the i n a b i l i t y t o c a l c u l a t e s p e c i f i c a c t i v i t y . I r o n s p e c i f i c a c t i v i t y would be lowest i n UW c u l t u r e s . Both UW and W c u l t u r e s e x h i b i t e d s i m i l a r r a t e s of s a t u r a t e d i r o n uptake ( F i g . 3.1C). Thus, c e l l s employed i n i r o n uptake experiments were washed by the RFF procedure p r i o r t o r e s u s p e n s i o n i n uptake, medium. To ma i n t a i n c o n s i s t e n c y i n treatment of samples, the RFF procedure was a l s o f o l l o w e d f o r a l l n i t r o g e n uptake i n c u b a t i o n s . Iron and nitrogen uptake, nitrite excretion and internal nitrate pools. Iron and n i t r o g e n uptake experiments were conducted i n e i t h e r 85 ml Oak Ridge tubes o r 250 ml Erlenmeyer f l a s k s (both c o n t a i n e r s p o l y c a r b o n a t e , Nalgene) and incubated (unbubbled, u n s t i r r e d ) under 145 pE'm~^*s~ x of continuous i l l u m i n a t i o n a t 17°C. S a t u r a t e d r a t e s of Fe, NO3 and NH 4 uptake by +Fe/N0 3, -Fe/N0 3, +Fe/NH 4 and -Fe/NH 4 c u l t u r e s were measured f o l l o w i n g RFF washing of c e l l s w i t h unenriched ( i . e . no n u t r i e n t a d d i t i o n s ) , chelexed ESAW s a l t s o l u t i o n (CSS, see 100 Chapter 2 ) , and resuspension i n the r e s p e c t i v e uptake media at o i a f i n a l c e l l d e n s i t y of c a . 3 - 5 •IO'' c e l l s •ml"'-L ( a l l i n c u b a t i o n s ) . The c h e l e x i n g p r o t o c o l (Morel e t a l . 1979) minimized r e s i d u a l i r o n contamination, thereby f a c i l i t a t i n g more complete removal of i r o n by washing. Ambient i r o n i s thus assumed t o be n e g l i g i b l e f o l l o w i n g RFF washing, which al l o w s i r o n added o n l y subsequently ( i . e . a f t e r washing) t o be 5 5 c o n s i d e r e d i n c a l c u l a t i o n s of i r o n s p e c i f i c a c t i v i t y f o r J J F e uptake experiments. Iron uptake r a t e s f o r a l l c u l t u r e s i n c l u d e d d e t e r m i n a t i o n s made i n the presence of e i t h e r added NO3 or NH 4 ( i . e . c u l t u r e s grown on NO3 or NH 4 and resuspended i n s a t u r a t i n g Fe p l u s e i t h e r NO3 or NH 4). These uptake experiments employed chelexed ESAW medium ( f u l l enrichment except 50 uU NO3 or 50 uR NH 4) s p i k e d t o a f i n a l ( f u l l ESAW) c o n c e n t r a t i o n of 6 . 5 6 uM ^ F e C l 3 (48 *jCi*mol F e - * , New England N u c l e a r ) / 1 4 . 8 6 /JM EDTA, 20 h p r i o r t o t h e i r i n i t i a t i o n by adding RFF-washed c e l l s . ESAW t r a c e metal enrichment was added immediately f o l l o w i n g Fe/EDTA. T h i s 20 h d e l a y ensured p r e - e q u i l i b r a t i o n of EDTA-trace metal complexes. Incubations of F e - r e p l e t e c e l l s were conducted over 2 4 - 3 3 h, w h i l e those u t i l i z i n g F e - d e p l e t e c u l t u r e s were extended t o 7 2 - 8 2 h, en s u r i n g f u l l r e c o v e r y from i r o n d e p l e t i o n and achievement of n u t r i e n t s a t i e t y . L a b e l l e d c e l l s were c o l l e c t e d on 5 . 0 um f i l t e r s (Nuclepore, 2 5 - 5 0 mm Hg), washed w i t h CSS t o remove c e l l s u r f a c e - and f i l t e r - bound ( i . e . n o n - b i o l o g i c a l ) i r o n (see Chapter 2 ) , and counted by l i q u i d s c i n t i l l a t i o n employing techniques s i m i l a r t o those used f o r 1 4 C ( 5 5 F e i s a low energy 101 /3-emitter, 0.232 MeV). Counts f o r a l l samples were > 3* 10 3 CPM. As n e i t h e r c u l t u r e nor i n c u b a t i o n c o n d i t i o n s were axenic, i t was d e s i r a b l e t o estimate the b a c t e r i a l c o n t r i b u t i o n t o i r o n uptake r a t e s . G. sanguineum c u l t u r e s (CULT, n = 2) and 0.2 p m - f i l t e r e d f i l t r a t e from these c u l t u r e s (FILT, n = 2) were s p i k e d w i t h ^ F e C l 3 (5 pM a d d i t i o n s , New England Nuclear) and i n c u b a t e d f o r 4 h. Samples from CULT i n c u b a t i o n s were s i z e - f r a c t i o n e d u s i n g e i t h e r 5.0 or 0.2 pm f i l t e r s ( Nuclepore), which r e t a i n e d p r i m a r i l y c e l l s (CULT/5.0 pm) or c e l l s , b a c t e r i a and c o l l o i d a l i r o n (CULT/0.2 pm), r e s p e c t i v e l y . FILT samples c o l l e c t e d on 0.2 pm f i l t e r s c o n t a i n e d predominantly i r o n c o l l o i d s (FILT/0.2 pm). A l l samples were washed with CSS (see above) a f t e r f i l t r a t i o n . R e s u l t s (Table 3.1) i n d i c a t e the r a t e of c o l l o i d a l i r o n l a b e l i n c o r p o r a t i o n (FILT/0.2 pm) i s e q u i v a l e n t t o t h a t of b a c t e r i a p l u s c o l l o i d a l i r o n (approximated by s u b t r a c t i n g the c e l l u l a r c o n t r i b u t i o n t o uptake r a t e : = CULT/0.2 pm - CULT/5.0 pm). Thus, the p r o p o r t i o n of G. sanguineum i r o n uptake r a t e s a t t r i b u t a b l e t o b a c t e r i a appears minimal. N i t r o g e n uptake media were i d e n t i c a l t o t h a t u t i l i z e d i n i r o n uptake experiments except f o r the a d d i t i o n of u n l a b e l e d i r o n . A l s o , i n c u b a t i o n times f o r F e - r e p l e t e and - d e p l e t e c u l t u r e s were as f o r Fe uptake experiments ( i . e . 24-33 h and 72-82 h, r e s p e c t i v e l y ) . Disappearance of NO3 and NH4 was monitored on g r a v i t y - f i l t e r e d (Whatman 934-AH) samples with a Table 3.1. S i z e - f r a c t i o n e d r a t e s of •'FeClo i n c o r p o r a t i o n (5 pM a d d i t i o n s ) as c o l l e c t e d on 5.0 um (CULT/5.0 um, n = 2) or 0.2 um (CULT/0.2 pm, n = 2) f i l t e r s , and formation of c o l l o i d a l i r o n i n 0.2 pm f i l t r a t e (same c u l t u r e s ) r e t a i n e d by 0.2 pm f i l t e r s (FILT/0.2 pm). See t e x t f o r f u n c t i o n a l d e f i n i t i o n of s i z e f r a c t i o n s . CULT/5.0 pm1 CULT/0.2 pm1 CULT/0.2 CULT/5.0 um -pm FILT/0.2 pm1 2.8 ± 0.1 9.4 ± 0.4 6.6 6.4 + 1.5 1. Values are mean (n = 2) D P M « h - i (•10*) ± 1 S.D. over 4 h i n c u b a t i o n s . 103 Technicon A u t o A n a l y z e r R I I (methods of Wood e t a l . 1967 and of Slawyk and Maclsaac 1972, r e s p e c t i v e l y ) and used t o c a l c u l a t e uptake r a t e s . Fe, NO3 and NH 4 uptake are expressed as t r a n s p o r t r a t e s per l i t e r of c e l l volume ( i . e . mol Fe or mol N * l i t e r c e l l v o l ~ x « h ~ A ) and/or s p e c i f i c uptake r a t e s ( d - 1 , Fe uptake o n l y ) , the l a t t e r based on p a r t i c u l a t e i r o n c o n c e n t r a t i o n s determined i n Chapter 2 f o r c u l t u r e s employed both i n the p r e c e d i n g paper and h e r e i n . During NO3 uptake experiments, r a t e s of n i t r i t e e x c r e t i o n ( i . e . NC"2~ appearance r a t e i n the c u l t u r e medium d e t e c t e d by A u t o A n a l y z e r R , Wood e t a l . 1967) and i n t e r n a l NO3 p o o l f o r m a t i o n were a l s o f o l l o w e d . NO3 p o o l s were e x t r a c t e d i n t o 20 ml b o i l i n g d i s t i l l e d d e i o n i z e d water a c c o r d i n g t o the C-2 p r o t o c o l of Thoresen e t a l . (1982), and normalized per l i t e r of c e l l volume. The average c e l l volume of a c u l t u r e was d e r i v e d from p a r t i c l e s i z e d i s t r i b u t i o n s ( C o u l t e r Counter model TAII, 200 pm a p e r t u r e , 44.2 pm c a l i b r a t i o n spheres) based on e q u i v a l e n t s p h e r i c a l diameter. RESULTS Data f o r i r o n and n i t r o g e n uptake are pre s e n t e d i n F i g s . 3.2-3.5. A l l v a l u e s p l o t t e d are midpoint d e t e r m i n a t i o n s , approximating the average r a t e between two a d j a c e n t measurements. Iron uptake. Iron uptake r a t e s are pr e s e n t e d f o r +Fe/N03, +Fe/NH 4, -Fe/N0 3 and -Fe/NH 4 c e l l s resuspended i n 104 F i g u r e 3.2. Time course of i r o n uptake (A,B) and s p e c i f i c i r o n uptake (C), f o l l o w i n g resuspension i n NO3, f o r i r o n - r e p l e t e (A) and i r o n - d e p l e t e (B,C) c u l t u r e s grown on n i t r a t e (o) or ammonium (•). Rates r e p r e s e n t midpoint d e t e r m i n a t i o n s f o r two adjacent measurements and are a giv e n as the mean ± 1 S.D. f o r two i n c u b a t i o n s , one from each of n = 2 c u l t u r e s per experiment. In c e r t a i n cases o n l y 1 S.D. ( e i t h e r + or -) i s shown t o a v o i d o v e r l a p p i n g and/or t o a l l o w use of more expanded s c a l e s . Note d i f f e r e n c e s i n time s c a l e between Fe-r e p l e t e and Fe- d e p l e t e experiments. RESUSPENSION IN NO 1.5 1 — TIME (h) 10 F i g u r e 3.3. Time course of i r o n uptake (A,B) s p e c i f i c i r o n uptake r a t e s (C), f o l l o w i n g r e s u s p e n s i o n i n NH4, f o r i r o n -r e p l e t e (A) and i r o n - d e p l e t e (B,C) c u l t u r e s grown on n i t r a t e (o) o r ammonium (•). Values a t 12 h i n (B) are o f f s e t f o r c l a r i t y . V a l u e s a t 60 h i n (B),(C) were s. 0 and are not shown (see t e x t f o r f u r t h e r e x p l a n a t i o n ) . Data p o i n t s and, e r r o r terms p l o t t e d are as d e s c r i b e d i n F i g . 3.2. Note d i f f e r e n c e s i n time s c a l e between F e - r e p l e t e and F e - d e p l e t e experiments. RESUSPENSION IN N H , ~ 1 2 > 1.5-1.2 • 0 .9 -0 .6 -0 . 3 -O-• -- O N O T - C R O W N A ( F . - R E P L E T E ) 12 16 20 t o < X 2 > 1.5-1.2-0.9 • 0 .6 -0.3 • 6 (Fe-DEPLCTE) 8.0-_ ~ 6.0 -0. => 4 .0 -2 . 0 -C (Fe-DEPLETE) 12 24 36 48 60 72 TIME (h) 106 e i t h e r NO3 ( F i g . 3.2) or NH4 ( F i g . 3.3). Maximum s a t u r a t e d i r o n uptake ( i . e . t r a n s p o r t ) r a t e s by -Fe/NC^ c u l t u r e s exceeded those measured f o r +Fe/NC>3 c u l t u r e s by c a . 1.5 - f o l d , i r r e s p e c t i v e of n i t r o g e n s p e c i e s a v a i l a b l e d u r i n g i n c u b a t i o n s ( i . e . whether c e l l s were resuspended i n 50 pM NO3 or NH4) ( F i g s . 3.2A,B and 3.3A,B). Maximum r a t e s of Fe uptake by -Fe /NH4 c e l l s were g r e a t e r than f o r +Fe/NH4 c e l l s (by a f a c t o r of 2.5), o n l y when resuspended i n NH4 ( F i g s . 3.3A,B). Conv e r s e l y , f o l l o w i n g r e s u s p e n s i o n i n NC^-nitrogen, +Fe/NH4 c e l l s e x h i b i t e d the h i g h e s t Fe uptake r a t e s , w i t h v a l u e s twice those determined f o r -Fe /NH4 c e l l s ( F i g s . 3.2A,B). I n i t i a l s p e c i f i c r a t e s of Fe uptake ( i n v a r i a b l y the maximum r a t e s ) f o r a l l i r o n - d e p l e t e c u l t u r e s were roughly e q u i v a l e n t (avg. = 0.075 ± 0.003-h" 1), and exceeded the maximum growth r a t e of 0.0158 h " 1 by a f a c t o r of c a . f i v e ( F i g s . 3.2C,3.3C). The most no t a b l e d i s t i n c t i o n i n i r o n uptake r a t e s between +Fe/NC-3 and +Fe/NH4 c u l t u r e s o c c u r r e d w i t h n i t r o g e n a v a i l a b l e o n l y as NO3 ( F i g . 3.2A). I n i t i a l l y (4 h p o i n t ) , +Fe/NH 4 c e l l s a c q u i r e d i r o n a t 3.5 times the r a t e of +Fe/N03 c e l l s , f o l l o w i n g r e s u s p e n s i o n i n NO3. The e r r o r a s s o c i a t e d w i t h the +Fe/NH4 c u l t u r e s ' average 4 h r a t e i s l a r g e ( F i g . 3.2A), y e t t h i s p o i n t remains d i s t i n c t l y above the comparable d e t e r m i n a t i o n s f o r +Fe/N03 c u l t u r e s . I t should be noted t h a t a t 60 h the i r o n uptake r a t e s of -Fe/NO-3 and -Fe/NH^ c u l t u r e s p r o v i d e d w i t h NH4 ( F i g . 3.3B,C) were £ 0, and thus not i n c l u d e d on the graph. A l l F e - d e p l e t e c u l t u r e s , r e g a r d l e s s of N source accompanying i r o n a d d i t i o n s , e x h i b i t e d no f u r t h e r 107 accumulation of l a b e l a t 72 h. Uptake r a t e s £ 0 l i k e l y r e p r e s e n t l a b e l e q u i l i b r a t i o n due t o extended i n c u b a t i o n p e r i o d s , coupled w i t h enhanced i n i t i a l uptake of i r o n . Nitrate uptake. N i t r a t e uptake r a t e s are p r e s e n t e d f o r +Fe / N 0 3 , +Fe/NH 4, -Fe / N 0 3 and -Fe/NH 4 c e l l s resuspended i n 50 uM N 0 3 and 6.56 pH Fe (plus EDTA) ( F i g . 3.4). The uptake of N 0 3 was markedly a f f e c t e d by i r o n d e p l e t i o n f o r both N0 3- and NH 4-grown c e l l s ( F i g . 3.4). No change i n ambient [N0 3] o c c u r r e d over the f i r s t 24 h of a l l - F e / N 0 3 and -Fe/NH 4 i n c u b a t i o n s a f t e r i r o n r e s u p p l y ( F i g . 3.4B), w h i l e maximal uptake r a t e s were a t t a i n e d i n a l l +Fe / N 0 3 and +Fe/NH 4 experiments w i t h i n £ 18 h ( F i g . 3.4A). The i n i t i a l l a g i n N0 3 uptake under Fe d e p l e t i o n was f u r t h e r exacerbated f o r NH 4-grown c u l t u r e s . These c e l l s showed 36 h N 0 3 uptake r a t e s only 30% of those measured i n - F e / N 0 3 c u l t u r e s . An a c c l i m a t i o n p e r i o d ( c a . 18 h) was a l s o e v i d e n t f o r +Fe/NH 4 c e l l s ( F i g . 3.4A); however, some N 0 3 was taken up immediately (1.5 h r e s o l u t i o n ) w i t h r a t e s i n c r e a s i n g s t e a d i l y t o a s u s t a i n e d maximum a t 18 h. Maximal r a t e s of N 0 3 uptake were expected f o r +Fe / N 0 3 c e l l s , y e t an e a r l y , a l b e i t s h o r t , d e l a y (1.5 h i n t e r v a l ) was observed ( F i g . 3.4A). T h i s l a g may have r e s u l t e d from b r i e f (ca. 0.5 h) exposure t o unenriched ESAW s a l t s o l u t i o n d u r i n g RFF washing and p r i o r t o N 0 3 a d d i t i o n . Upon t e r m i n a t i o n of a l l experiments ( i . e . n u t r i e n t s u f f i c i e n c y ) , N 0 3 uptake r a t e s were e s s e n t i a l l y i n d i s t i n g u i s h a b l e between NO3- and NH 4-grown c e l l s , i r r e s p e c t i v e of p r e v i o u s i r o n s t a t u s ( F i g s . 3.4A,B). 108 F i g u r e 3.4. Time course of n i t r a t e uptake r a t e s f o r n i t r a t e -to) o r ammonium-grown (•), i r o n - r e p l e t e (A) and i r o n - d e p l e t e (B) c u l t u r e s resuspended i n 50 pM NO3. I r o n - d e p l e t e c u l t u r e s a l s o r e c e i v e d s a t u r a t i n g i r o n a d d i t i o n s (6.56 pM Fe/14.86 pM EDTA). Data p o i n t s and e r r o r terms p l o t t e d a r e as d e s c r i b e d i n F i g . 3.2. Note d i f f e r e n c e s i n time s c a l e between F e - r e p l e t e and F e - d e p l e t e experiments. TIME (h) 109 F i g u r e 3.5. Time course of ammonium uptake r a t e s f o r n i t r a t e -Jo) o r ammonium-grown (•)/ i r o n - r e p l e t e (A) and i r o n - d e p l e t e (B) c u l t u r e s resuspended i n 50 pM NH^. I r o n - d e p l e t e c u l t u r e s a l s o r e c e i v e d s a t u r a t i n g i r o n a d d i t i o n s (6.56 pM Fe/14.86 pM EDTA). Data p o i n t s and e r r o r terms p l o t t e d a r e as d e s c r i b e d i n F i g . 3.2. Note d i f f e r e n c e s i n time s c a l e between F e - r e p l e t e and F e - d e p l e t e experiments. 6.0 < DC LJ < I— CL Z> X z A (Fe -REPLETE ) O O N O 3 - G R O W N • • N H 4 - G R 0 W N 30 36 6.0 CN I O £ L S 1 2 » O E 110 Ammonium uptake. Ammonium uptake r a t e s are presented f o r +Fe/N0 3, +Fe/NH 4, -Fe/N0 3 and -Fe/NH 4 c e l l s resuspended i n 50 pM NH 4 and 6.56 pM Fe (plus EDTA) ( F i g . 3.5). I r o n d e p l e t i o n i n h i b i t e d the i n i t i a l uptake of NH 4 ( F i g . 3.5), although not t o the same degree noted f o r N0 3 uptake. A l a g i n r a t e s of NH 4 uptake f o l l o w i n g s a t u r a t i n g i r o n a d d i t i o n s o c c u r r e d i n N0 3- and NH 4-grown c e l l s , w ith the h i g h e s t v a l u e s f o r both o b t a i n e d a t 60 h ( F i g . 3.5B). S i m i l a r l y , maximum N0 3 uptake r a t e s f o r -Fe/N0 3 and -Fe/NH 4 c u l t u r e s were not ac h i e v e d u n t i l c a . 60 h ( F i g . 3.4B). Rates of NH 4 uptake by -Fe/N0 3 and -Fe/NH 4 c u l t u r e s converged toward the end of i n c u b a t i o n s , y e t f o r i n i t i a l samples, N0 3-grown c e l l s showed c a . t h r e e - f o l d h i g h e r r a t e s ( F i g . 3.5B). Nitrite excretion and internal nitrate pools. The i n i t i a l e x c r e t i o n of n i t r i t e , as a percentage of n i t r a t e being taken up, was n e g l i g i b l e ( F i g . 3.6, note: as percentages comprise r a t e measurements, NC>2~ uptake, r a t h e r than e x c r e t i o n , y i e l d s n e g a t i v e v a l u e s ) due t o p a r t i a l or complete s u p p r e s s i o n of N0 3 uptake i n F e - r e p l e t e ( F i g . 3.4A) and Fe-d e p l e t e ( F i g . 3.4B) c e l l s , r e s p e c t i v e l y . As i n c u b a t i o n s p r o g r e s s e d , +Fe/N0 3 and -Fe/N0 3 c u l t u r e s e x h i b i t e d e i t h e r minimal NC<2~ e x c r e t i o n or a removal of ambient NC^" ( F i g . 3.6). In c o n t r a s t , as NH 4-grown c e l l s a d j u s t e d t o the presence of N0 3, p r o g r e s s i v e l y h i g h e r N0 3 uptake r a t e s ( F i g . 3.4) were accompanied by e l e v a t e d r a t e s of NG^ - e x c r e t i o n , e s p e c i a l l y i n -Fe c u l t u r e s (e.g. 36 h, F i g . 3.6B). NC>2~ 1 F i g u r e 3.6. Rates of n i t r i t e e x c r e t i o n e x p r e s s e d as a percent of n i t r a t e uptake r a t e f o l l o w i n g r e s u s p e n s i o n i n 50 JJM NO3 f o i r o n - r e p l e t e (A) and i r o n - d e p l e t e (B) c u l t u r e s grown on NO3 (o) or N H 4 (•)• Negative v a l u e s r e s u l t from NC"2~ uptake, r a t h e r t han e x c r e t i o n . Experiments are those p r e s e n t e d i n F i g 3.4. Data p o i n t s and e r r o r terms p l o t t e d a re as d e s c r i b e d i n F i g . 3.2. Note d i f f e r e n c e s i n time s c a l e between F e - r e p l e t e and F e - d e p l e t e experiments. 1 o < 1— r> o 2 3.0 & 2.0 1.0 0.0 •1.0 1 O NO3-GROWN — • NH4-GROWN c r o x U l I CNI O 2 -2.0 -3.0 4-» = -9.2+14.2 12 18 A (Fe-REPLETE) 24 30 36 l o U l < I— C L K> O OH C_> X U l I C M o 112 F i g u r e 3.7. Time course of changes i n i n t e r n a l n i t r a t e pools d u r i n g NO3 uptake experiments i n v o l v i n g i r o n - r e p l e t e (A) and i r o n - d e p l e t e (B) c u l t u r e s grown on NO3 (o) or NH4 (•). Measurements were made d u r i n g the experiment shown i n F i g . 3 . 4 . Data p o i n t s and e r r o r terms p l o t t e d are as d e s c r i b e d i n F i g . 3.2, except t h a t v a l u e s correspond t o a c t u a l sample times r a t h e r than t o midpoint d e t e r m i n a t i o n s . Note d i f f e r e n c e s i n time s c a l e between F e - r e p l e t e and F e - d e p l e t e experiments. TIME (h) 113 e x c r e t i o n became n e g l i g i b l e (or NC>2~ was taken up) when NO3 uptake r a t e s reached a s t a b l e maximum past 60 h. I n t e r n a l n i t r a t e was d e t e c t e d i n a l l c u l t u r e s immediately f o l l o w i n g NO3 a d d i t i o n s ( F i g . 3.7), the s m a l l e s t i n i t i a l (0 h) po o l s o c c u r r i n g i n -Fe/NH^ experiments ( F i g . 3.7B). A f t e r a sharp i n i t i a l decrease i n both +Fe/N03 and +Fe/NH 4 c u l t u r e s , NH^-grown c e l l s began t o accumulate n i t r a t e over a 9 h p e r i o d ( i . e . from 3-12 h), w h i l e pools of those c e l l s p r e v i o u s l y grown on NO3 c o n t i n u e d t o d e c l i n e s t e a d i l y ( F i g . 3.7A). A t r e n d s i m i l a r t o the l a t t e r was a l s o e v i d e n t i n -F e / N 0 3 c u l t u r e s ( F i g . 3.7B). I n t e r n a l NO3 c o n c e n t r a t i o n s of - F e / N H ^ c e l l s changed l i t t l e d u r i n g the 82 h i n c u b a t i o n s . DISCUSSION Iron uptake. -Fe/N03 and -Fe/NH 4 G. sanguineum c e l l s e x h i b i t e d e l e v a t e d ( c f . +Fe/N0 3 and +Fe/NH 4 c e l l s ) i r o n uptake r a t e s (p = mol F e ' l i t e r c e l l v o l ~ A * h - l ) when n i t r o g e n was a v a i l a b l e as ammonium. However, i n the presence of NO3-n i t r o g e n , a c a p a c i t y f o r e l e v a t e d Fe uptake was r e s t r i c t e d t o those F e - d e p l e t e c e l l s p r e v i o u s l y adapted t o t h i s N source ( i . e . -Fe/N03 c e l l s ) . These r e s u l t s are the f i r s t t o demonstrate an enhancement of Fe uptake f o r an i r o n - s t r e s s e d d i n o f l a g e l l a t e . S p e c i f i c r a t e s of i r o n uptake ( V = h - ± ) by -Fe/N03 and -Fe/NH^ c e l l s , i n i t i a l l y exceeded maximum growth r a t e s ( p m a x ) of t h i s s p e c i e s by a f a c t o r of f i v e . However, u n l i k e measurements of p above, no change i n a c t u a l uptake 114 ( i . e . t r a n s p o r t ) r a t e can be i n f e r r e d based on V. In other words, p can remain eq u a l , w h i l e s p e c i f i c uptake (V) i n c r e a s e s as a r e s u l t of n o r m a l i z i n g r a t e s t o the lower, F e - d e p l e t e c e l l quotas. Enhancement of i r o n uptake has been r e p o r t e d f o r Fe-l i m i t e d c e l l s of the diatom T. weissflogii ( H a r r i s o n and Morel 1986). T h i s response i s a l s o c h a r a c t e r i s t i c of manganese, another t r a c e element e s s e n t i a l f o r phytoplankton growth (Sunda and Huntsman 1986). The 12 h ( c f . £ 1.5 h f o r +Fe c u l t u r e s ) r e s o l u t i o n f o r i r o n - d e p l e t e c u l t u r e s was chosen based on the slow r e c o v e r y of these c u l t u r e s t o t h e i r maximum growth r a t e (ca. 48-72 h, data not shown). While r e c o g n i z i n g t h a t such r a t e s l i k e l y r e p r e s e n t net r a t h e r than gross uptake, a l l v a l u e s w i l l be co n s i d e r e d as s h o r t term measurements i n the ensuing d i s c u s s i o n . As the p o t e n t i a l f o r r e d u c t i o n s i n maximum uptake r a t e s ( p m a x = the maximum t r a n s p o r t r a t e measured d u r i n g an experiment) due t o back r e a c t i o n s and feedback i n h i b i t i o n i n c r e a s e s w i t h d e c r e a s i n g i n i t i a l r e s o l u t i o n ( i . e . i n c r e a s i n g F P i n c u b a t i o n t i m e ) , p m a x of Fe - d e p l e t e c u l t u r e s ( i . e . p~ m a x ) may be underestimated. As a r e s u l t , d i f f e r e n c e s between p ~ F e m a x and p m a x f o r F e - r e p l e t e c u l t u r e s ( i . e . P + F e m a x ) may, s i m i l a r l y , be c o n s e r v a t i v e e s t i m a t e s . For purposes of d i s c u s s i o n , the q u a n t i t a t i v e d i f f e r e n c e between P ~ F e m a x a n d p + F e i n a x f o r an experiment w i l l be r e p r e s e n t e d h e r e a f t e r as the q u o t i e n t p " F e m a x / P + F e i n a x - T h e extreme P ~ F e m a x / p + F e m a x v a l u e s (both low and high) were observed i n NH^-grown c u l t u r e s . A minimum of 0.5 was noted f o r NH^-grown c u l t u r e s resuspended i n 115 NO3 ( F i g s . 3.2A,B), w h i l e the maximum v a l u e of 2.5 o c c u r r e d i n these same c u l t u r e s ( i . e . NH^-grown), but resuspended, i n s t e a d , i n NH 4 d u r i n g Fe uptake experiments ( F i g s . 3.3A,B). T h i s o b s e r v a t i o n i s important i n t h a t P~^ e m ax /'P + F emax °^ G. sanguineum can a p p a r e n t l y vary by a f a c t o r of f i v e , depending on both p r e - c o n d i t i o n i n g N source and the N source s u p p l i e d w h i l e i r o n i s being taken up. An e x p l a n a t i o n w i l l f i r s t be proposed t o account f o r the e f f e c t s of N source, f o l l o w e d by p o s s i b l e i m p l i c a t i o n s f o r the a b i l i t y of c e l l s t o a c c l i m a t e over a range of i r o n c o n c e n t r a t i o n s , as i n f l u e n c e d by N source. The d i f f e r e n c e i n i r o n requirements between NO3- and N H 4 -grown c e l l s i s a t t r i b u t a b l e t o the i r o n c o n t e n t of n i t r a t e and n i t r i t e r e d uctase enzymes (NR and NiR, r e s p e c t i v e l y ) , both e s s e n t i a l f o r NO3 a s s i m i l a t i o n . Once reduced t o NH4 v i a NR-and N i R - c a t a l y z e d r e a c t i o n s , i n c o r p o r a t i o n of NO3-N i n t o amino a c i d s l i k e l y proceeds by the same major pathway as does n i t r o g e n o r i g i n a l l y a c q u i r e d as NH4 ( i . e . the GS-GOGAT pathway i n most phytoplankton, Wheeler 1983). Thus, a p a r t from the demand a s s o c i a t e d w i t h NO3 a s s i m i l a t i o n , a d d i t i o n a l i r o n requirements (e.g. c h l o r o p h y l l and cytochrome b i o s y n t h e s i s , i r o n - s u l f u r p r o t e i n s , glutamate synthase, e t c . ) are presumably common t o both NO3- and NH^-grown c e l l s of the same s p e c i e s . C a l c u l a t i o n s performed by Raven (1988) i n d i c a t e t h a t a p l a n t r e p r o d u c i n g p h o t o l i t h o t r o p h i c a l l y w h i l e s u p p l i e d w i t h NO3, r e q u i r e s 1.6 times as much i r o n t o s u s t a i n the same growth r a t e on NH 4. C o n s i s t e n t w i t h t h i s s u g g e s t i o n are the 1.5 - f o l d 116 g r e a t e r minimum Fe/N r a t i o s of NO3- versus NH^-grown G. sanguineum r e p o r t e d i n Chapter 2. Consi d e r now the two extreme v a l u e s of P~ F e x nax /'P + F emax noted above. Both estimates were ob t a i n e d from c u l t u r e s p r e v i o u s l y grown on NH 4. Thus, the f i v e - f o l d d i f f e r e n c e i n these r a t i o s i s s o l e l y a f u n c t i o n of N source a s s i m i l a t e d ( i . e . NO3 or NH4) d u r i n g i r o n uptake i n c u b a t i o n s . P r i o r t o Fe uptake experiments, NH^-grown c u l t u r e s r e q u i r e d the l e a s t amount of i r o n f o r growth ( i . e . 40% l e s s than NC^-grown c u l t u r e s , Raven 1988). As N source i s u n a l t e r e d by resuspending +Fe/NH 4 c e l l s i n NH 4, the Fe demand of these c e l l s , and consequently p m a x r remains the lowest 1 1 "3 encountered (0.47 mol F e * l i t e r c e l l vol~- L«h • L«10~ , F i g . 3.3A). In c o n t r a s t , P ~ F e m a x of -Fe/NH 4 c e l l s resuspended i n NH 4 (1.2 mol F e - l i t e r c e l l v o l " 1 • h " 1 • 1 0 ~ 3 , F i g . 3.3B) i s > r a t e s determined f o r any oth e r experiment. Because -Fe/NH 4 c u l t u r e s e x h i b i t an excess of c e l l u l a r N (C/N = 3.8, Chapter 2), and w i t h Fe not r e q u i r e d f o r N ( i . e . NH 4) a s s i m i l a t i o n nor s t i l l l i m i t i n g growth, r e c o v e r y toward n u t r i e n t s a t i e t y (e.g. maximum Fe quota) i n the presence of s a t u r a t i n g Fe l e v e l s proceeds unimpeded. The r e s u l t i n g l a r g e P ~ F e m a x (1*2 mol F e * l i t e r c e l l v o l " 1 ' h - 1 • 10~ 3 ), coupled w i t h a minimum P + F e i n a x (0.47 mol F e - l i t e r c e l l v o l - 1 - h " 1 • 1 0 ~ 3 ) , y i e l d e d the h i g h e s t f)~FemsiX/P+Femax r a t i o observed ( i . e . 2.5). P r e c i s e l y the o p p o s i t e t r e n d s i n p m a x and p m a x are apparent when NH 4-grown c e l l s are resuspended i n NO3-N. This 117 change i n n i t r o g e n source r e q u i r e s the maximum p o s s i b l e i n c r e a s e i n i r o n needed t o support growth and, under Fe-r e p l e t e c o n d i t i o n s , t r a n s l a t e s i n t o the l a r g e s t (by 2.5-fold) p + F e m a x measured d u r i n g t h i s study (1.1 mol F e * l i t e r c e l l v o l ' ^ - ' h ' ^ ' l O " 3 , F i g . 3.2A). However, f o r F e - d e p l e t e c e l l s , such a compensatory enhancement of Fe uptake does not occur. The r e s u l t i n g low P ~ F e m a x (5.2 mol F e * l i t e r c e l l v o l " 1 * h _ 1 • 1 0 ~ 3 , F i g . 3.2B) l i k e l y r e f l e c t s c o n t i n u e d p h y s i o l o g i c a l s t r e s s due, however, t o N r a t h e r than Fe d e f i c i e n c y , i n the absence of a f u n c t i o n a l a s s i m i l a t o r y NO3 r e d u c i n g system (see Nitrogen uptake below). The p ~ F e n i a x / p + F e n i a x v a l u e f o r NH^-grown c e l l s resuspended i n NO3 d u r i n g i r o n uptake i n c u b a t i o n s thus r e p r e s e n t s the minimum f o r t h i s r a t i o ( i . e . 0.5). These f i n d i n g s suggest t h a t the q u a l i t a t i v e nature of a t r a n s i t i o n between N sources (e.g. NH4 t o NO3) may cause adjustment of uptake r a t e s , as r e q u i r e d t o f a c i l i t a t e any i n h e r e n t changes i n the c e l l ' s demand f o r i r o n . Such t r a n s i t i o n s might a l s o be expected t o i n f l u e n c e the range of i r o n c o n c e n t r a t i o n s over which a c e l l , through s h o r t term a c c l i m a t i o n , can c o n t i n u e growing a t maximum r a t e s . The p r e c e d i n g statement may be understood a t a more q u a n t i t a t i v e l e v e l w i t h i n the c o n c e p t u a l framework o u t l i n e d r e c e n t l y by Morel (1987). His i n t e r p r e t a t i o n of experimental data, i n the c o n t e x t of proposed h y p e r b o l i c r e l a t i o n s h i p s f o r growth and uptake r a t e s , i d e n t i f i e s the range of s u b s t r a t e c o n c e n t r a t i o n s (S) over which algae can m a i n t a i n growth r a t e s near p m a x as being bounded by K u ( i . e . the h a l f - s a t u r a t i o n 118 c o n s t a n t f o r steady s t a t e growth) and Kp ( i . e . the h a l f -s a t u r a t i o n c o n s t a n t f o r s h o r t term uptake), where K ^ « Kp. The d e r i v a t i o n of p versus S curves f o r T. weissflogii 1 q p r o v i d e d an average Kp of 3.6 ± 1.3*10~ x M between i r o n -l i m i t e d and n o n - l i m i t e d c e l l s (Table 2, H a r r i s o n and Morel 1986). Thus, from the q u o t i e n t K p / K ^ (K^ = 1.1-10" 2 1 M, Table 2, H a r r i s o n and Morel 1986) a g r e a t e r than 3 0 0 - f o l d a c c l i m a t i o n range of i r o n f r e e i o n a c t i v i t i e s can be estimated f o r t h i s diatom. Values of Kp c o u l d not be determined from the s a t u r a t i n g i r o n a d d i t i o n s employed e x c l u s i v e l y h e r e i n . However, through a l g e b r a i c m a n i p u l a t i o n of fundamental equations d e s c r i b i n g growth and uptake, Morel (1987) p r o v i d e s a means by which Kp/K^ can be estimated based on P + F e m a x / p " F e m a x , minimum c e l l u l a r quota ( Q m i n ) and Q m a x : tf Iv — n ~ F e / n + F e • n /Ci • p' u v max'v max wmax / vmin ( i n v e r s e of h i s equation ( 9 ) ) . I t should be noted t h a t i n Morel's t e r m i n o l o g y , P + F e m a x i s gi v e n as P ' L ° i n a x ( i . e . maximum sh o r t term uptake r a t e achieved under n u t r i e n t s a t i e t y ) and p ~ F e i n a x as P ^ m ^ ( i . e . maximum sh o r t term uptake r a t e achieved under severe n u t r i e n t s t r e s s ) . Minimum and maximum i r o n quotas (per c e l l volume) of NO3- and NH^-grown G. sanguineum were o b t a i n e d p r e v i o u s l y i n Chapter 2, and v a r i e d l i t t l e as a f u n c t i o n of N source (Table 2.1). Values f or i n t e r m e d i a t e q u o t i e n t s ( i . e . p F%n a x/p"*" F emax' Qmax/Qmin) a n d K p ^ K p ^ o r a l i experiments h e r e i n a re presented i n T a b l e 3.2. These c a l c u l a t i o n s suggest t h a t the pFe ( i . e . n e g a t i v e l o g i r o n f r e e i o n a c t i v i t y ) a c c l i m a t i o n range of T a b l e 3 . 2 . C a l c u l a t e d v a l u e s of K . / K ^ f o r i r o n - l i m i t e d growth of G. sanguineum grown on e i t h e r n i t r a t e or ammonium. The eq u a t i o n employed i s K /K - p " F e m a x / p ^ m a x • Qmax/Qm\n' the i n v e r s e form of equation (9) g i v e n by Morel ( 1 9 8 7 ) . See t e x t f o r f u r t h e r d e t a i l s . NO3-GROWN/ N 0 3 R E S U S P . 1 NOo N H 4 -GROWN/ R E S U S P . 1 NH 4 -GROWN/ N 0 3 R E S U S P . 1 N H 4 N H 4 -GROWN/ R E S U S P . 1 -Fe / +Fe 2 max' max 1 0 0 . 1 5 1 0 ° .15 1 0 - 0 . 3 0 1 0 ° .40 ^max^Qmin 1 0 1 . 7 6 1 0 1 .76 1 0 1 . 7 6 1 0 1 .76 Kp/Ku 1 01 . 9 1 1 0 1 .91 1 0 1 . 4 6 1 0 2 . 16 1. N i t r o g e n source c u l t u r e grown on ( i . e . N 0 3 or N H 4 ) / n i t r o g e n source c e l l s resuspended i n f o r i r o n uptake experiment ( i . e . N 0 3 or NH 4). 2. Data from F i g s . 3.2 and 3.3 ( t h i s s t u d y ) . 3. Average v a l u e from Chapter 2; Qp e (per c e l l volume) d i d not vary as a f u n c t i o n of n i t r o g e n source (Tables 2.1 and 2 . 2 ) . 120 G. sanguineum may be lower by as much as f i v e - f o l d i f reduced Fe b i o a v a i l a b i l i t y i s accompanied by a t r a n s i t i o n from NH4 t o NO3 n i t r o g e n n u t r i t i o n ( c f . continuous supply of NH4) . V a r i a t i o n s i n p m a x and Q F e made approximately e q u i v a l e n t c o n t r i b u t i o n s t o d e f i n i n g the pFe a c c l i m a t i o n range f o r T. weissflogii (Morel 1987), w h i l e r e d u c t i o n s i n Q F e account f o r 80-100% of t h i s range h e r e i n (Table 3.2). The c o n t r i b u t i o n of P ~ F e m a x / p + F e m a x ^ n t ^ i e c u r r e n t study may, however, be underestimated, as d i s c u s s e d above. In g e n e r a l terms, the magnitude of pFe ranges over which G. sanguineum can m a i n t a i n near maximal growth r a t e s (a f a c t o r of c a . 30-150) i s 2-10 times l e s s than f o r T. weissflogii (a f a c t o r of c a . 300, Morel 1987). F u r t h e r , the a b s o l u t e pFe bounds as d e l i m i t e d by and Kp may be t e n - f o l d h i g h e r f o r the d i n o f l a g e l l a t e based on i t s K^ f o r i r o n - l i m i t e d growth (Chapter 1). As i n Chapters 1 and 2, r e s u l t s o b t a i n e d h e r e i n appear t o support the c o n t e n t i o n t h a t t h i s , and perhaps o t h e r , c o a s t a l d i n o f l a g e l l a t e s p e c i e s may be more s u s c e p t i b l e t o i r o n - l i m i t e d growth than are diatoms from n e r i t i c waters. Nitrogen uptake. The most extreme consequences of i r o n d e p l e t i o n were apparent i n n i t r a t e uptake experiments. F o l l o w i n g s a t u r a t i n g Fe r e s u p p l y , immediate a c q u i s i t i o n of n i t r a t e was completely i n h i b i t e d f o r both NO3- and NH^-grown G. sanguineum, e x h i b i t i n g a 24 h d e l a y r e l a t i v e t o Fe uptake by the same F e - d e p l e t e c u l t u r e s . Maximum NO3 uptake r a t e s were achieved by both -Fe/NO^ and -Fe/NH 4 c u l t u r e s a t 60 h. However, 36 h r a t e s of NO3 uptake i n -Fe/NH^ c u l t u r e s were 3-121 f o l d l e s s than f o r -Fe/NC^ c u l t u r e s and f u r t h e r , were accompanied by the g r e a t e s t r a t e s of n i t r i t e e x c r e t i o n (as a % of c o n c u r r e n t NO3 uptake) measured d u r i n g t h i s study. These data suggest a somewhat more r a p i d development of maximum NO3 uptake and a s s i m i l a t o r y c a p a c i t y f o l l o w i n g i r o n d e p l e t i o n i n NO3- than NH^-grown c e l l s . Iron i s not r e q u i r e d f o r NO3 uptake, and the Fe-dependent form of glutamate synthase (GOGAT), an NH4 a s s i m i l a t o r y enzyme, would be u n a f f e c t e d by N source. Thus, the s u p p r e s s i o n of NO3 uptake and the temporal d i f f e r e n c e between NO3- and NH^-grown c u l t u r e s i s l i k e l y r e l a t e d t o enzymes e s s e n t i a l f o r the a s s i m i l a t i o n of NO3. N i t r a t e a s s i m i l a t i o n i s f a c i l i t a t e d by two i r o n -c o n t a i n i n g enzymes, NR and NiR. Although t h e r e i s c l e a r l y a shortage of c e l l u l a r i r o n f o r c o n t i n u e d enzyme s y n t h e s i s , the p r e c i s e mechanism(s) by which NO3 a s s i m i l a t i o n i s a f f e c t e d are u n c e r t a i n . P o s s i b i l i t i e s i n c l u d e complete breakdown of NR and/or NiR, or perhaps p a r t i a l d e g r a d a t i o n of these enzymes, a l l o w i n g r e t e n t i o n of p r e c u r s o r p r o t e i n molecules. E x t e n s i v e e f f o r t s t o measure NR a c t i v i t y i n n u t r i e n t - s u f f i c i e n t G. sanguineum c e l l s have proved u n s u c c e s s f u l (W. Cochlan unpubl. r e s u l t s ) . While the i m p l i c a t i o n s w i t h r e g a r d t o the b i o c h e m i c a l nature (or the e x i s t e n c e ) of NR remain t o be e l u c i d a t e d , e l e v a t e d Fe/N r a t i o s f o r -Fe/NO-3 over -Fe/NH 4 c e l l s ( 1 . 5 - f o l d , Chapter 2) are c o n s i s t e n t w i t h a g r e a t e r i r o n requirement a s s o c i a t e d with NO3 a s s i m i l a t i o n . F u r t h e r , symptoms of N d e f i c i e n c y observed i n -Fe/NC^ c u l t u r e s (Chapter 2) i n d i c a t e an i r o n s t r e s s - m e d i a t e d f u n c t i o n a l impairment of 122 the a s s i m i l a t o r y NO3 r e d u c i n g system. An i n a c t i v e demolybdo form of n i t r a t e reductase has been r e p o r t e d i n the green a l g a Chlorella (Funkhouser e t a l . 1983); however, t h e r e i s no evidence t o suggest the e x i s t e n c e of an i n a c t i v e NR s p e c i f i c a l l y d e v o i d of a heme s u b u n i t ( s ) . R e v e r s i b l e i n a c t i v a t i o n of NR, such as t h a t o f t e n e f f e c t e d by ammonium uptake and a s s i m i l a t i o n , occurs over s h o r t time s c a l e s and by mechanism u n l i k e l y ( i . e . s p e c i f i c t o NH 4 or a product of i t s a s s i m i l a t i o n ) h e r e i n (see Losada and Guerrero 1979). Based on the more r a p i d e l e v a t i o n of NO3 uptake (and presumably a s s i m i l a t i o n ) upon Fe a d d i t i o n f o r -Fe/NC^ G. sanguineum, i t would appear t h a t de novo s y n t h e s i s of NR and NiR p r o t e i n s , and r e g e n e r a t i o n from i n a c t i v e p r e c u r s o r p r o t e i n molecules (a product of p a r t i a l enzyme d e g r a d a t i o n ) , are l i k e l y of g r e a t e r importance t o NH4- and N0 3-grown c e l l s , r e s p e c t i v e l y . T h i s i s supported by r e p o r t s of NH4, or more c o r r e c t l y a product of i t s metabolism (e.g. g l u t a m i n e ) , r e p r e s s i o n of NR s y n t h e s i s (see Guerrero e t a l . 1981). A de l a y i n a c h i e v i n g maximum NO3 uptake and a s s i m i l a t i o n r a t e s , e x h i b i t e d by F e - r e p l e t e , NH^-grown G. sanguineum, has been demonstrated f o r oth e r a l g a l s p e c i e s (see C o l l o s 1983). However, o p i n i o n s as t o the r e l a t i v e c o n t r i b u t i o n s of de novo p r o t e i n s y n t h e s i s and enzyme a c t i v a t i o n t o maximizing NO3 a s s i m i l a t o r y c a p a c i t y (and thus uptake r a t e s ) i n NH^-grown c e l l s ( i . e . f o l l o w i n g t r a n s i t i o n t o NO3), even under n u t r i e n t s u f f i c i e n c y , remain d i v i d e d (see reviews by Guerrero e t a l . 1981, McCarthy 1981, S y r e t t 1981). 123 The i r o n r e q u i r e d t o a s s i m i l a t e NH4 i s e q u i v a l e n t f o r NO3- and NH^-grown c e l l s , as was t h e i r r a t e of NH^ uptake under F e - r e p l e t e c o n d i t i o n s . However, NH4 uptake f o r -Fe/N0 3 c u l t u r e s exceeded t h a t of -Fe/NH^ c u l t u r e s ( a f t e r Fe resupply) by a f a c t o r of c a . t h r e e . In t h i s case, the n i t r o g e n r a t h e r than i r o n s t a t u s of c e l l s p r i o r t o experiments i s bes t invoked as an e x p l a n a t i o n . -Fe/NG-3 c u l t u r e s e x h i b i t symptoms of N d e f i c i e n c y , w h i l e -Fe/NH^ c u l t u r e s are c h a r a c t e r i z e d by an excess of i n t r a c e l l u l a r N (Chapter 2 ) . I t i s w e l l documented t h a t s a t u r a t e d N ( p a r t i c u l a r l y NH4) uptake r a t e s of N - l i m i t e d phytoplankton are g r e a t e r than i f organisms are N r e p l e t e (see Goldman and G l i b e r t 1983). Thus, as immediate a c q u i s i t i o n and a s s i m i l a t i o n of NH4 i s not pr e c l u d e d by the need t o s y n t h e s i z e / r e g e n e r a t e F e - c o n t a i n i n g enzymes ( c f . NO3 a s s i m i l a t i o n ) , NH^ uptake proceeds a t an i n i t i a l l y h i g h e r r a t e i n NC^-grown c e l l s than f o r " n i t r o g e n - s u f f i c i e n t " -Fe/NH^ c e l l s . Conclusions. The c u r r e n t r e s e a r c h i n d i c a t e s t h a t Fe-s t r e s s e d G. sanguineum develops an enhanced c a p a c i t y f o r the uptake ( i . e . t r a n s p o r t ) of i r o n . However, i n the event Fe-s t r e s s e d c e l l s are r e q u i r e d t o switch from ammonium t o n i t r a t e n u t r i t i o n , e l e v a t e d i r o n uptake r a t e s f a i l t o be manifested. Presumably, the i n a b i l i t y t o immediately a s s i m i l a t e t h i s o x i d i z e d N source ( i . e . NO3) maintains c e l l s under a c e r t a i n degree of n u t r i t i o n a l ( s p e c i f i c a l l y N) s t r e s s . The i n i t i a l d e l a y i n NO3 uptake observed i n these experiments would be c o n s i s t e n t w i t h t h i s i d e a . I n t u i t i v e l y , i t f o l l o w s t h a t a 124 continuous NH^ supply t o NH^-grown c e l l s should c o n f e r an a d a p t i v e advantage w i t h regard t o s h o r t term adjustments t o i r o n s t r e s s . Data r e p o r t e d h e r e i n support t h i s argument, i n s o f a r as the magnitude of the pFe range over which a c e l l can s u c c e s s f u l l y a c c l i m a t e on s h o r t time s c a l e s i s approximated by Kp/K^ (as e l a b o r a t e d by Morel 1987). The importance of these f i n d i n g s as they r e l a t e t o the ecology of r e d t i d e s p e c i e s , and c o a s t a l d i n o f l a g e l l a t e s i n g e n e r a l , i s u n c e r t a i n . Based on the f o r F e - l i m i t e d growth (Chapter 1), the optimum Fe/C r a t i o (Chapter 2) and the magnitude of the pFe a c c l i m a t i o n range ( t h i s chapter) of G. sanguineum as compared t o the n e r i t i c diatom T. weissflogii, the former s p e c i e s i s a p p a r e n t l y more s u s c e p t i b l e t o growth l i m i t a t i o n by i r o n . N i t r o g e n source and N source t r a n s i t i o n s may f u r t h e r a f f e c t a c e l l ' s a b i l i t y t o r a p i d l y a c c l i m a t e t o i r o n s t r e s s . However, i t i s d i f f i c u l t t o q u a n t i f y the balance between Fe supply and phytoplankton demand or a c q u i s i t i o n c a p a b i l i t i e s , and thus the a c t u a l p o t e n t i a l f o r F e - l i m i t e d growth i n c o a s t a l waters. 125 CHAPTER 4. THE ULTRASTRUCTURE OF IRON STRESS-MEDIATED CYSTS IN THE TOXIC RED TIDE DINOFLAGELLATE PROTOGONYAULAX TAMARENSIS (LEBOUR) TAYLOR BACKGROUND Attempts t o e x p l a i n the occurrence of red t i d e d i n o f l a g e l l a t e blooms have c o n s i d e r e d among o t h e r p o s s i b i l i t i e s the p o t e n t i a l importance of f a c t o r s a s s o c i a t e d w i t h t e r r e s t r i a l r u n o f f such as humates, t r a c e metals and s a l i n i t y / s t r a t i f i c a t i o n (Prakash 1975, Anderson and Wall 1978, P r o v a s o l i 1979, Anderson e t a l . 1983). S e v e r a l authors (e.g. Kim and M a r t i n 1974, Glo v e r 1978) have noted an apparent c o r r e l a t i o n between e l e v a t e d l e v e l s of s o l u b l e i r o n d e r i v e d from r i v e r d i s c h a r g e and l a n d r u n o f f , and the development of re d t i d e s . Such a r e l a t i o n s h i p may imply the a l l e v i a t i o n of i r o n - l i m i t i n g c o n d i t i o n s by an i n c r e a s e i n i r o n supply r a t e . Thus, i f growth i s a t l e a s t i n i t i a l l y i r o n - l i m i t e d , the d e c l i n e of a -bloom may r e s u l t from l o c a l i z e d d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n . An important f a c t o r known t o be a s s o c i a t e d w i t h bloom d e c l i n e i s the i n i t i a t i o n of sexual r e p r o d u c t i o n l e a d i n g t o the f o r m a t i o n of dormant r e s t i n g c y s t s (Anderson 1984). As n a t u r a l c y s t p r o d u c t i o n i s t r i g g e r e d by an as y e t undefined s e t of environmental cues, and i r o n b i o a v a i l a b i l i t y may i n f l u e n c e bloom p o p u l a t i o n dynamics, the e f f e c t s of i r o n s t r e s s on r e d t i d e d i n o f l a g e l l a t e l i f e h i s t o r i e s are of i n t e r e s t . F u r t h e r , i f we are t o c o n s i d e r the p o s s i b i l i t y of 126 i r o n s t r e s s - m e d i a t e d p o p u l a t i o n d e c l i n e , the q u e s t i o n of whether i r o n s t r e s s can induce c y s t f o r m a t i o n i n a bloom s p e c i e s must a l s o be addressed. The pr e s e n t study thus i n c l u d e s a d e s c r i p t i o n of changes i n l i f e h i s t o r y stage of a re d t i d e d i n o f l a g e l l a t e s u b j e c t e d t o i n c r e a s i n g i r o n s t r e s s . Protogonyaulax (= Gonyaulax) tamarensis (Lebour) T a y l o r i s a r e d t i d e d i n o f l a g e l l a t e r e s p o n s i b l e f o r numerous p a r a l y t i c s h e l l f i s h p o i s o n i n g (PSP) outbreaks i n many of the world's temperate c o a s t a l waters ( T a y l o r 1984). Cyst f o r m a t i o n has been w e l l documented f o r t h i s s p e c i e s both i n l a b o r a t o r y c u l t u r e s ( T u r p i n e t a l . 1978, Anderson e t a l . 1984 , Anderson and L i n d q u i s t 1985) and i n f i e l d p o p u l a t i o n s (Anderson 1980, Anderson e t a l . 1983). Evidence f o r the importance of c y s t s i n the p o p u l a t i o n dynamics of P. tamarensis blooms has a l s o been r e p o r t e d (Anderson and Morel 1979, Anderson 1984, Anderson and Keafer 1985). Owing t o i t s e c o l o g i c a l importance and the a v a i l a b i l i t y of data r e g a r d i n g c y s t f o r m a t i o n by t h i s s p e c i e s P. tamarensis was s e l e c t e d as a t e s t organism. S e v e r a l d i s t i n c t types of d i n o f l a g e l l a t e c y s t s are p r e s e n t l y r e c o g n i z e d ( T a y l o r i n p r e s s ) . Of thes e , o n l y temporary (= p e l l i c l e ) and r e s t i n g (= hypnozygote) c y s t s are known t o form under c o n d i t i o n s of n u t r i e n t s t r e s s . In P. tamarensis , the former r e s u l t from an as e x u a l process ( i . e . e c d y s i s ) , w h i l e the l a t t e r are products o f sexual r e p r o d u c t i o n . Both c y s t types have been d e s c r i b e d p r e v i o u s l y \ 127 f o r t h i s s p e c i e s (Anderson and Wall 1978, F r i t z 1986). There i s a growing body of l i t e r a t u r e d e s c r i b i n g many as p e c t s of d i n o f l a g e l l a t e c y s t s , i n c l u d i n g c y s t morphology (see reviews by Dale 1983, L o e b l i c h and L o e b l i c h 1984), the pro c e s s of sexu a l r e p r o d u c t i o n (see review by P f i e s t e r 1984) and the environmental c o n t r o l of sexual c y c l e s (see review by P f i e s t e r and Anderson 1987). While r e c e n t work by F r i t z (1986) has produced a d e t a i l e d u l t r a s t r u c t u r a l account of the P. tamarensis l i f e c y c l e , i n c l u d i n g encysted stages from l a b o r a t o r y (induced by n i t r o g e n d e f i c i e n c y ) and f i e l d p o p u l a t i o n s , few data e x i s t on the u l t r a s t r u c t u r e of c y s t p r o t o p l a s t s (Bibby and Dodge 1972, Diirr 1979, see a l s o P f i e s t e r 1984). T h e r e f o r e , i n a d d i t i o n t o some i n s i g h t s c o n c e r n i n g l i f e h i s t o r y response t o i r o n s t r e s s , t h i s study p r o v i d e s an u l t r a s t r u c t u r a l d e s c r i p t i o n of i r o n s t r e s s -mediated P. tamarensis c y s t s and a comparison w i t h n i t r o g e n s t r e s s - m e d i a t e d c y s t s examined by F r i t z (1986). MATERIALS AND METHODS General culture maintenance. Protogonyaulax tamarensis ( c u l t u r e #D255, North E a s t P a c i f i c C u l t u r e C o l l e c t i o n , Dept. of Oceanography, U n i v e r s i t y of B r i t i s h Columbia) was maintained on m o d i f i e d ESNW-enriched n a t u r a l seawater medium ( H a r r i s o n e t a l . 1980). The o r i g i n a l f o r m u l a t i o n was ad j u s t e d t o c o n t a i n 1 pM i r o n (as FeCl3*6H20) and 0.5 pM molybdenum (as Na2Mo04), and de s i g n a t e d +Fe ESNW. Medium used t o grow c e l l s i n t o i r o n d e p l e t i o n (-Fe ESNW) was prepared w i t h n a t u r a l 128 seawater c l e a n e d of o r g a n i c s by treatment w i t h a c t i v a t e d c h a r c o a l , and of r e s i d u a l i r o n by passage over Chelex 100 i o n exchange r e s i n (Morel e t a l . 1979). T h i s c h e l e x e d seawater was e n r i c h e d u s i n g m o d i f i e d ESNW n u t r i e n t s (as d e s c r i b e d above) wi t h the omission of i r o n and EDTA. A l l batch c u l t u r e s ( u n s t i r r e d ) were grown a t 16°C w i t h an i r r a d i a n c e of 120 pE ' i r T 2 * s ~ * s u p p l i e d on a 14:10 L/D c y c l e by S y l v a n i a R VHO D a y l i g h t f l u o r e s c e n t tubes. Iron depletion. P. tamarensis was s u b j e c t e d t o c o n d i t i o n s of i n c r e a s i n g i r o n s t r e s s by i n o c u l a t i n g 10 1 of -Fe ESNW w i t h 1 1 of l a t e e x p o n e n t i a l phase c e l l s maintained on +Fe ESNW (Day 0), and a l l o w i n g the c u l t u r e t o grow u n t i l d e p l e t e d of i r o n (Day 24). On each of t h i r t e e n sampling d a t e s , 1 1 of c u l t u r e was removed f o r c e l l counts, d e t e r m i n a t i o n of l i f e h i s t o r y stage ( i . e . v e g e t a t i v e c e l l , p e l l i c u l a r o r z y g o t i c c y s t ) and d e t e c t i o n of d i s s o l v e d Fe-b i n d i n g s i d e r o p h o r e s (G. Boyer unpubl. d a t a ) . For the f i r s t s i x samplings, 1 1 of f r e s h -Fe ESNW was added back t o the c u l t u r e i n or d e r t o reduce sampling l i m i t a t i o n s imposed by c u l t u r e volume r e s t r i c t i o n s , and t o d i l u t e r e s i d u a l i r o n i n the medium. Data on l i f e h i s t o r y stages are r e p o r t e d as perce n t of t o t a l c e l l s • m l - 1 . A q u a l i t a t i v e assessment of c u l t u r e i r o n s t a t u s was performed twice d u r i n g the course of the experiment (Day 17, Day 24) as f o l l o w s : one p a r t c u l t u r e was combined w i t h two p a r t s -Fe ESNW and s p l i t i n t o two equal volumes; 1 pK F e C l 3 * 6 H 2 0 ( f r e s h l y prepared, without EDTA) was added t o one h a l f , w h i l e n o t h i n g was added t o the other. The 129 growth response of these batch c u l t u r e s , based on l i g h t microscope c e l l counts, was monitored f o r 25 days. Ultrastructure. Samples f o r t r a n s m i s s i o n e l e c t r o n microscopy (TEM) were taken j u s t p r i o r t o ( c o n t r o l , Day 0) and f o l l o w i n g i n o c u l a t i o n i n t o -Fe ESNW (Days 1, 7, 13 and 20). C e l l s were ha r v e s t e d on 0.45 pm M i l l i p o r e f i l t e r s (type HA). Samples prepared f o r TEM were f i x e d on f i l t e r s w i t h 1.5% g l u t a r a l d e h y d e i n 0.1 M sodium c a c o d y l a t e and 0.4 M sucrose (2 h, room temp.), p o s t - f i x e d w i t h 1% osmium t e t r o x i d e i n 0.1 M sodium c a c o d y l a t e (1 h, room temp.), en b l o c s t a i n e d w i t h 1% aqueous u r a n y l a c e t a t e , and dehydrated i n a graded e t h a n o l / p r o p y l e n e oxide s e r i e s . Samples were embedded i n a 1-2 mm t h i c k d i s k of Epon 812 t o f a c i l i t a t e m i c r o s c o p i c i d e n t i f i c a t i o n and s e l e c t i o n of i n d i v i d u a l specimens f o r s e c t i o n i n g . Random and s e r i a l s e c t i o n s were c u t u s i n g a diamond k n i f e , p i c k e d up on uncoated 200- or formvar-coated 50-mesh copper g r i d s , s t a i n e d w i t h s a t u r a t e d u r a n y l a c e t a t e ( i n 50% methanol) and l e a d c i t r a t e , and examined i n a Z e i s s EM10 t r a n s m i s s i o n e l e c t r o n microscope. L i g h t microscopy (LM) of l i v i n g and 2% f o r m a l i n - p r e s e r v e d m a t e r i a l was performed w i t h a Z e i s s Standard microscope. U l t r a s t r u c t u r a l d e s c r i p t i o n s of v e g e t a t i v e c e l l s are based e x c l u s i v e l y on c o n t r o l samples (Day 0), w h i l e treatment of c y s t s i n c l u d e s m a t e r i a l from Days 1, 7, 13, and 20. Terminology used t o d e s c r i b e the amphiesma i s t h a t of M o r r i l l and L o e b l i c h (1983). 130 RESULTS Iron bioassays. I n o c u l a t i o n of i r o n - r e p l e t e P. tamarensis c e l l s i n t o -Fe ESNW r e s u l t e d i n i r o n d e p l e t i o n by Day 24. In the presen t c o n t e x t , i r o n d e p l e t i o n i m p l i e s c e s s a t i o n of growth due t o the absence of b i o l o g i c a l l y a v a i l a b l e i r o n , a t which time i t i s assumed t h a t the i r o n c e l l quota reaches a minimum v a l u e . B i o a s s a y s (Day 24) confirmed t h a t c e l l s were, i n f a c t , d e p l e t e d of i r o n and not of another n u t r i e n t ( F i g s . 4.1A,B). C e l l s h a r vested on Day 17 were not i r o n - d e p l e t e , as a growth response was observed i r r e s p e c t i v e of i r o n a d d i t i o n . Conversely, f o r the Day 24 b i o a s s a y , growth o c c u r r e d o n l y w i t h the a d d i t i o n of i r o n , i n d i c a t i n g t h a t c e l l u l a r i r o n r e s e r v e s were exhausted between Day 17 and 24. The f i r s t b i o a s s a y (Day 17) began t o show a decrease i n c e l l d e n s i t y f o r the -Fe c u l t u r e a f t e r seven days. Assuming minimal i r o n c o ntamination of -Fe ESNW added i n b i o a s s a y s , b i o l o g i c a l l y a v a i l a b l e i r o n was l i k e l y d e p l e t e d i n the primary c u l t u r e v e s s e l ( i . e . source of b i o a s s a y inoculum) on or j u s t p r i o r t o Day 24. Life history dynamics. C u l t u r e composition based on l i f e h i s t o r y stage ( i . e . , v e g e t a t i v e c e l l s , p e l l i c u l a r and z y g o t i c c y s t s ) i s shown i n F i g . 4.2. P e l l i c u l a r c y s t numbers were below d e t e c t i o n l i m i t s of LM counts f o r the c o n t r o l sample (Day 0 ) . Although t h i s c y s t type was encountered on Day 1 i n TEM m a t e r i a l , they were f i r s t observed i n LM counts on Day 5. Values were i n i t i a l l y below 1% and d i d not exceed 5% u n t i l Day 131 F i g u r e 4.1. I r o n b i o a s s a y s performed on Day 17 (A) and Day 24 (B) showing changes i n c e l l d e n s i t y of -Fe (o) and +Fe (•) c u l t u r e s . o E in o >-I— to z L J Q UJ CJ 10 8 6 4 • 2 -0 DAY 17 + Fe O O - F e \ / O o-o- - o -o -o o £ > 8 6 2 0 DAY 24 B / / - o - o - o — o - o - o 10 15 2 0 ' 2 5 TIME (d) 132 F i g u r e 4.2. Changes i n l i f e h i s t o r y stage over a time course of i n c r e a s i n g i r o n s t r e s s . Values f o r v e g e t a t i v e c e l l s p e l l i c u l a r ( o ) , and z y g o t i c (•) c y s t s are e x p r e s s e d as percent of t o t a l c e l l s - m l . 1 0 0 - . A — o-— A -o VEG PEL rYPE 8 0 - \ -— • ZYG _J _J i • i 6 0 -o (— 2 4 0 -LtJ o 1,1 2 0 - o / D_ 0 - — • C ) 5 1 0 1 5 2 0 2 5 3 0 TIME (d) 133 24, when p e l l i c u l a r c y s t s accounted f o r over 25% of c e l l s i n the c u l t u r e . Large (ca. 50-60 um), h e a v i l y pigmented c e l l s comprised about 1% of the c o n t r o l c u l t u r e , and were i d e n t i f i e d as products of sexual f u s i o n ( i . e . z y g o t e s ) . Zygote percentages f l u c t u a t e d near c o n t r o l l e v e l s f o r almost two weeks, i n c r e a s e d t o 11% (8% hypnozygotes, 3% planozygotes) by Day 13, and averaged about 5% f o r the remainder of the experiment. Q u a d r i f l a g e l l a t e planozygotes ( p r e c u r s o r s t o hypnozygotes), f i r s t noted on Day 13, were always l e s s than 3% of the t o t a l c e l l number. V e g e t a t i v e c e l l s exceeded 85% u n t i l Day 24 when i n c r e a s i n g p e l l i c u l a r c y s t percentages reduced them t o 72%. A decrease i n v e g e t a t i v e c e l l s i z e (from c a . 35 jjm t o c a . 25 um, diam.) and reduced pigmentation were a s s o c i a t e d w i t h i n c r e a s i n g i r o n s t r e s s . Vegetative cells. V e g e t a t i v e c e l l s were surrounded by an amphiesma c o n s i s t i n g of f i v e components ( F i g . 4.3). The t h r e e most d i s t a l elements i n c l u d e d the ou t e r membrane (OM), the ou t e r p l a t e membrane (OPM) and a system of t h e c a l p l a t e s . I t d i d not appear t h a t each p l a t e was completely e n c l o s e d w i t h i n an i n d i v i d u a l v e s i c l e . D i r e c t l y below the p l a t e s was a continuous p e l l i c l e . The p e l l i c u l a r l a y e r showed a t r i l a m i n a r s t r u c t u r e and was a s s o c i a t e d w i t h a t h i n m a t r i x of 'fuzzy' m a t e r i a l on both proximal and d i s t a l s i d e s . The innermost component of the amphiesma was the c y t o p l a s m i c membrane (CM). A network of m i c r o t u b u l e s o c c u r r e d immediately beneath the CM. 134 F i g u r e s 4.3-4.8. Protogonyaulax tamarensis v e g e t a t i v e c e l l s . NEPCC c u l t u r e #D255. F i g . 4.3. Amphiesma comprised of outer membrane (0), outer p l a t e membrane (P), t h e c a l p l a t e s ( P I ) , p e l l i c l e (Pe), and c y t o p l a s m i c membrane (C). S c a l e = 0.2 pm. F i g . 4.4. L o n g i t u d i n a l s e c t i o n of p r o t o p l a s t . C-shaped nucleus (N) c o n t a i n s n u c l e o l u s (Nu) around i n n e r c u r v a t u r e . C r y s t a l l i n e m a t e r i a l (arrowhead) i s present i n hypothecal v a c u o l a r r e g i o n . Scale, = 5 pm. F i g . 4.5. C h l o r o p l a s t w i t h l a m e l l a e e x h i b i t i n g c l o s e l y p a i r e d t h y l a k o i d s (arrows). Scale = 0.5 pm. F i g . 4.6. G r a n a - l i k e s t a c k s of t i g h t l y appressed t h y l a k o i d membranes. S c a l e = 0.5 pm. F i g . 4.7. Mitochondrion w i t h t u b u l a r c r i s t a e and c h a r a c t e r i s t i c m a t r i x d e n s i t y . S c a l e = 0.2 pm. F i g . 4.8. Aggregate of c r y s t a l l i n e m a t e r i a l showing d e t a i l s of c r y s t a l morphology. V e s i c l e (V) c o n t a i n i n g c o n c e n t r i c membrane s w i r l s i s apparent. S c a l e = 0.2 pm. 135 136 The v e g e t a t i v e p r o t o p l a s t ( F i g . 4.4) was dominated by a l a r g e C-shaped nucleus w i t h a s i n g l e n u c l e o l u s c o n t a i n e d around i t s i n n e r c u r v a t u r e . Chromosomes were den s e l y packed and e x h i b i t e d a p e r i o d i c t r a n s v e r s e banding p a t t e r n i n l o n g i t u d i n a l s e c t i o n . Dictyosomes and c o n c e n t r a t i o n s of endoplasmic r e t i c u l u m were p a r t i a l l y e n c i r c l e d by the C-shape of the nuc l e u s . C h l o r o p l a s t l a m e l l a e appeared r o u g h l y p a r a l l e l t o each ot h e r and g e n e r a l l y c o n s i s t e d of two c l o s e l y appressed t h y l a k o i d s ( F i g . 4.5). G r a n a - l i k e s t a c k s of as many as e i g h t t h y l a k o i d s were common ( F i g . 4.6). M i t o c h o n d r i a possessed t u b u l a r c r i s t a e embedded i n a r e l a t i v e l y dense mat r i x m a t e r i a l ( F i g . 4.7). Va c u o l a r areas were e x t e n s i v e and e x h i b i t e d a v a r i e t y of membranous i n c l u s i o n s (e.g. F i g . 4.8) and prominent aggregates of a c r y s t a l l i n e m a t e r i a l ( F i g s . 4.4,4.8). C r y s t a l s showed an el e c t r o n - d e n s e o u t l i n e and were approximately 30 nm wide and v a r i a b l e i n l e n g t h . The occurrence of s t a r c h and l i p i d s t o rage r e s e r v e s was n e g l i g i b l e . A s m a l l accumulation body was o c c a s i o n a l l y p r e s e n t . Pellicular cysts. The most c o n s i s t e n t f e a t u r e c h a r a c t e r i z i n g p e l l i c u l a r c y s t s , i r r e s p e c t i v e of c u l t u r e i r o n s t a t u s , was the amphiesma. Of the f i v e v e g e t a t i v e w a l l components, these c y s t s r e t a i n e d o n l y the p e l l i c l e and CM ( F i g . 4.9), y i e l d i n g a rounded c e l l of i r r e g u l a r o u t l i n e ( F i g . 4.10). The t r i l a m i n a r s t r u c t u r e of the c y s t p e l l i c l e was subtended by a l a y e r of g r a n u l a r m a t e r i a l not pr e s e n t i n 137 F i g u r e s 4.9-4.12. Protogonyaulax tamarensis p e l l i c u l a r c y s t s . NEPCC c u l t u r e #D255. N = nucleus. F i g . 4.9. Amphiesma c o n s i s t i n g of p e l l i c l e (Pe) subtended by t h i c k l a y e r of g r a n u l a r m a t e r i a l (G) p o s s i b l y o r i g i n a t i n g from v e s i c l e s (arrowhead) d e r i v e d from the c y t o p l a s m i c membrane (C). S c a l e = 0.1 pm. F i g . 4.10. Cyst w i t h p r o t o p l a s t resembling v e g e t a t i v e c e l l . Note l a c k of storage products and presence of c r y s t a l l i n e m a t e r i a l (arrowheads). S c a l e = 5 pm. F i g . 4.11. Cyst showing densely aggregated cytoplasm w i t h minimal v a c u o l a r space. S e v e r a l s t a r c h granules (S) and accumulation bodies (A) are pr e s e n t . S c a l e = 5 pm. F i g . 4.12. Cyst c o n t a i n i n g numerous s t a r c h (S) and l i p i d (L) sto r a g e i n c l u s i o n s . C r y s t a l l i n e m a t e r i a l (arrowhead) i s apparent. S c a l e = 5 pm. 139 v e g e t a t i v e c e l l s . T h i s l a y e r v a r i e d i n t h i c k n e s s but was g e n e r a l l y about 100 nm wide. M a t e r i a l appeared t o be d e l i v e r e d by v e s i c l e s d e r i v e d from the CM ( F i g . 4.9). P e l l i c u l a r c y s t s were s i m i l a r i n s i z e t o v e g e t a t i v e c e l l s and were a l s o s m a l l e r under i n c r e a s e d i r o n s t r e s s . I n t r a c e l l u l a r o r g a n i z a t i o n v a r i e d c o n s i d e r a b l y among samples c o l l e c t e d on Day 1. U l t r a s t r u c t u r e of these c y s t s ranged from t h a t c l o s e l y resembling v e g e t a t i v e c e l l s ( F i g . 4.10) t o a dense aggregate of c y t o p l a s m i c c o n s t i t u e n t s , i n c l u d i n g accumulation bodies and s t a r c h i n c l u s i o n s ( F i g . 4.11). However, throughout the experiment p e l l i c u l a r c y s t s g e n e r a l l y c o n t a i n e d a moderate amount of cytoplasm and a t l e a s t s e v e r a l s t a r c h and l i p i d r e s e r v e s ( F i g . 4.12). Aggregates of the c r y s t a l l i n e m a t e r i a l o c c u r r i n g i n v e g e t a t i v e c e l l s were e v i d e n t i n most c y s t s ( F i g s . 4.10,4.12). The c y s t nucleus and chromosomes were i n d i s t i n g u i s h a b l e from those of v e g e t a t i v e c e l l s u n t i l Day 20, when changes i n chromatin s t r u c t u r e became e v i d e n t . Regular banding ( F i g . 4.13) was d i s r u p t e d t o v a r y i n g degrees, r e s u l t i n g i n clumping of chromatin i n t o e l e c t r o n - d e n s e aggregates ( F i g . 4.14). D i f f e r e n c e s between c y s t and v e g e t a t i v e c h l o r o p l a s t s were apparent on Day 1, but were c o n s i d e r a b l y more p r e v a l e n t l a t e r i n the time course. A l t e r a t i o n s ranged from s l i g h t s w e l l i n g of t h y l a k o i d s ( F i g . 4.15) t o e x t e n s i v e t u b u l a r d i l a t i o n of membranes ( F i g . 4.16). While l a m e l l a e of Day 20 c y s t s f r e q u e n t l y e x h i b i t e d s i n g l e r a t h e r than p a i r e d t h y l a k o i d s , 140 F i g u r e s 4.13-4.17. Protogonyaulax tamarensis p e l l i c u l a r c y s t s . NEPCC c u l t u r e #D255. F i g . 4.13. Chromosomes s i m i l a r t o those of v e g e t a t i v e c e l l s e x h i b i t i n g h i g h l y o r g a n i z e d t r a n s v e r s e banding of chromatin (arrows). S c a l e = 0.5 pm. F i g . 4.14. Chromosomes with d i s r u p t e d chromatin s t r u c t u r e . Chromatin i s clumped i n t o e l e c t r o n - d e n s e aggregates (arrows). N u c l e o l u s (Nu) i s apparent. S c a l e = 1 um. F i g . 4.15. C h l o r o p l a s t showing s i n g l e t h y l a k o i d s and g r a n a - l i k e s t a c k s c o n t a i n i n g s l i g h t l y s w o l l e n membranes (arrows). S c a l e = 0.5 um. F i g . 4.16. C h l o r o p l a s t e x h i b i t i n g e x t e n s i v e t u b u l a r d i l a t i o n of t h y l a k o i d membranes seen i n c r o s s - (arrowheads) and l o n g i t u d i n a l -farrows) s e c t i o n . S c a l e = 0.2 pm. F i g . 4.17. Mit o c h o n d r i o n w i t h matrix of reduced d e n s i t y and p o s s i b l y fewer c r i s t a e r e l a t i v e t o v e g e t a t i v e c e l l s . Note ' f i l l e d - i n ' appearance of c r i s t a e (arrows) S c a l e = 0.5 pm. 142 g r a n a - l i k e s t a c k s c o n t a i n i n g p a r t i a l l y d i l a t e d membranes a l s o occured i n many c h l o r o p l a s t s ( F i g . 4.15; c f . F i g . 4.6). Due t o the i n h e r e n t m o r p h o l o g i c a l v a r i a b i l i t y of m i t o c h o n d r i a , d e v i a t i o n s from the v e g e t a t i v e s t a t e were d i f f i c u l t t o assess. The most obvious a l t e r a t i o n s were r e d u c t i o n s i n the d e n s i t y of the m a t r i x , and p o s s i b l y i n the number of c r i s t a e p r o f i l e s ( F i g . 4.17). A l s o , c r i s t a e appeared t o be f i l l e d w i t h e l e c t r o n - d e n s e m a t e r i a l . T h i s c o n d i t i o n was p r i m a r i l y a s s o c i a t e d w i t h c y s t s e x h i b i t i n g a b e r r a n t chromosome u l t r a s t r u c t u r e . Hypnozygotes. Hypnozygotes averaged 50-60 pm i n the l o n g e s t dimension and were g e n e r a l l y h e a v i l y pigmented. U n l i k e p e l l i c u l a r c y s t s , d e g e n e r a t i v e u l t r a s t r u c t u r a l a l t e r a t i o n s i n response t o i n c r e a s i n g i r o n s t r e s s were minimal. Among hypnozygotes h a r v e s t e d a t each sampling time, two morphologies (Stages 1 (SI) and 2 (S2)) c o u l d be d i s t i n g u i s h e d based on shape, s u r f a c e o u t l i n e , and n u c l e a r f i n e s t r u c t u r e . SI c y s t s ( F i g . 4.18) were rounded or s l i g h t l y e l o n g a t e w i t h an i r r e g u l a r o u t l i n e w h i l e S2 c y s t s ( F i g . 4.19) were more elongate and g e n e r a l l y smoother. Chromosome p r o f i l e s i n SI n u c l e i were densely-packed, and o f t e n i l l -d e f i n e d and s i m i l a r t o the nucleoplasm i n e l e c t r o n d e n s i t y ( F i g . 4.18). N u c l e i of most S2 c y s t s showed very d i s c r e t e chromosomes of near electron-opaque d e n s i t y ( F i g . 4.19). Many chromosomes c o n t a i n e d one or two s m a l l e l e c t r o n - l u c e n t areas. C l u s t e r s of c a b l e s or f i l a m e n t s (15-25 nm diam.) were 143 F i g u r e s 4.18-4.23. Protogonyaulax tamarensis hypnozygotes. NEPCC c u l t u r e #D255. F i g . 4.18. Stage 1 c y s t w i t h i r r e g u l a r o u t l i n e and nucleus c o n t a i n i n g densely-packed, somewhat i l l -d e f i n e d chromosomes. C r y s t a l l i n e m a t e r i a l (arrowhead) and accumulation bodies (A) are apparent. S c a l e = 10 pm. F i g . 4.19. Smooth-walled, elongate Stage 2 c y s t . Nucleus (N) shows d i s c r e t e , e l e c t r o n - d e n s e chromosomes. Note presence of c r y s t a l l i n e m a t e r i a l (arrowhead), c o r t i c a l l i p i d i n c l u s i o n s ( L ) , and an accumulation body (A). S c a l e = 10 pm. F i g . 4.20. S2 n u c l e o l u s (Nu) and a s s o c i a t e d f i l a m e n t s (arrowhead). Note c r o s s - s e c t i o n of f i l a m e n t p e n e t r a t i n g n u c l e o l u s (arrow) and presence of l a r g e n u c l e o l a r o r g a n i z i n g chromosome (No). S c a l e = 1 pm. F i g . 4.21. Amphiesma e x h i b i t i n g p e l l i c l e (Pe), g r a n u l a r l a y e r (G), and amorphous, e l e c t r o n - d e n s e m a t e r i a l (A) immediately adjacent t o c y t o p l a s m i c membrane (C). Membrane fragments d i s t a l t o c y t o p l a s m i c membrane are l i k e l y remains of v e s i c l e s d e p o s i t i n g amorphous m a t e r i a l . S c a l e = 0.1 um. F i g . 4.22. Annulate n u c l e a r v e s i c l e s formed as i n v a g i n a t i o n s of n u c l e a r envelope (Ne). Nuclear pores (arrows) are apparent. S c a l e = 0.5 pm. F i g . 4.23. Aggregation of m i t o c h o n d r i a showing reduced m a t r i x d e n s i t y . Note d i v i d i n g mitochondrion (arrowhead). S c a l e = 1 pm. Ikk a s s o c i a t e d w i t h and pen e t r a t e d the n u c l e o l u s of c e r t a i n S2 n u c l e i ( F i g . 4.20). SI and S2 c y s t s had s e v e r a l c h a r a c t e r i s t i c s i n common. Although hypnozygote and p e l l i c u l a r c y s t amphiesmae were s i m i l a r , the former had an a d d i t i o n a l l a y e r of amorphous e l e c t r o n - d e n s e m a t e r i a l between the t h i c k e r g r a n u l a r r e g i o n and CM ( F i g . 4.21). Hypnozygote n u c l e i e x h i b i t e d i n v a g i n a t i o n s of the n u c l e a r envelope which appeared as annulate v e s i c l e s i n the nucleoplasm ( F i g . 4.22). I n v a g i n a t i o n s e n c l o s e d a c e n t r a l core of e l e c t r o n - d e n s e m a t e r i a l surrounded by a l e s s e l e c t r o n - d e n s e f i b r o u s r e g i o n Storage pr o d u c t s , i n c l u d i n g l i p i d r e s e r v e s i n the c o r t i c a l cytoplasm and accumulation bodies (as many as t e n per c e l l seen), were pr e s e n t i n a l l z y g o t i c c y s t s examined ( F i g s . 4.18,4.19). S t a r c h bodies were n o t a b l y absent from most of these c y s t s . C h l o r o p l a s t s were g e n e r a l l y s i m i l a r t o those v e g e t a t i v e c e l l s , w h i l e hypnozygote m i t o c h o n d r i a e x h i b i t e d r e d u c t i o n i n the d e n s i t y of the mat r i x and were f r e q u e n t l y grouped i n l a r g e c l u s t e r s ( F i g . 4.23). The c r y s t a l l i n e m a t e r i a l noted i n both v e g e t a t i v e c e l l s and p e l l i c u l a r c y s t was e v i d e n t i n most z y g o t i c c y s t s . DISCUSSION Life history dynamics. L i f e h i s t o r y stages of P. tamarensis were monitored under c o n d i t i o n s of i n c r e a s i n g i r o n s t r e s s . R e s u l t s p r o v i d e the f i r s t evidence of i r o n 146 s t r e s s - m e d i a t e d s e x u a l i t y i n a red t i d e d i n o f l a g e l l a t e . The response of P. tamarensis t o i r o n s t r e s s , as determined by zygote p r o d u c t i o n (ca. 10%, planozygotes + hypnozygotes, t h i s s t u d y ) , appears weaker than f o r d e p l e t i o n of n i t r o g e n (ca. 20%, hypnozygotes on l y , Anderson e t a l . 1984) or phosphorus (ca. 25%, planozygotes + hypnozygotes, Anderson and L i n d q u i s t 1985). However, zygote percentages near 20% have been measured f o r P. tamarensis d u r i n g i r o n d e p l e t i o n experiments not r e p o r t e d here (A. Cembella unpubl. d a t a ) . A d d i t i o n a l work i s r e q u i r e d t o q u a n t i f y zygote p r o d u c t i o n of t h i s s p e c i e s under i r o n s t r e s s . N u t r i e n t d e p l e t i o n has f r e q u e n t l y been employed t o induce d i n o f l a g e l l a t e s e x u a l i t y (see review by P f i e s t e r and Anderson 1987). N e v e r t h e l e s s , a p a r t from s e v e r a l e x c e p t i o n s (e.g. Anderson e t a l . 1985, Anderson and L i n d q u i s t 1985), the temporal r e l a t i o n s h i p between s e x u a l i t y and ambient or i n t e r n a l n u t r i e n t l e v e l s has r e c e i v e d l i t t l e a t t e n t i o n . Although i r o n c o n c e n t r a t i o n s (ambient o r i n t e r n a l ) were not measured, b i o a s s a y s confirmed d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n by t e r m i n a t i o n of the pr e s e n t experiment. O b s e r v a t i o n of maximum zygote percentages more than one week p r i o r t o t h i s i n d i c a t e s t h a t s e x u a l i t y i s a s s o c i a t e d w i t h c o n d i t i o n s of i r o n l i m i t a t i o n r a t h e r than d e p l e t i o n . Anderson and L i n d q u i s t (1985) have suggested t h a t phosphorus s t r e s s -mediated s e x u a l i t y i n P. tamarensis may occur i n response t o n u t r i e n t l i m i t a t i o n a t d e t e c t a b l e phosphorus c o n c e n t r a t i o n s . 147 T h e i r r e s u l t s c o u l d i n d i c a t e a high c e l l u l a r requirement and a low uptake a f f i n i t y f o r t h i s element. Although no k i n e t i c s of i r o n uptake are a v a i l a b l e f o r P. tamarensis, M u e l l e r (1985) has determined c e l l u l a r i r o n c o n c e n t r a t i o n s ( Q F e ) i n i r o n - d e f i c i e n t c e l l s of the same c l o n e — 18 3 used i n the present study. His va l u e of 1.3*10 mol Fe*pm r e p r e s e n t s e s s e n t i a l l y an over estimate of minimum i r o n c e l l quota. I t i s l i k e l y t h a t M u e l l e r ' s c u l t u r e s were not t r u l y d e p l e t e d of i r o n . F u r t h e r , no attempt was made t o remove surface-bound ( i . e . n o n - b i o l o g i c a l ) i r o n . T h i s f r a c t i o n may be a c o n s i d e r a b l e p o r t i o n of i r o n a s s o c i a t e d w i t h diatom c e l l s (Anderson and Morel 1982), but i t appears t o be of l e s s s i g n i f i c a n c e f o r c e r t a i n d i n o f l a g e l l a t e s (see Chapter 2 ) . Ov e r l o o k i n g these u n c e r t a i n t i e s , the "minimum" Q F e of t h i s s p e c i e s appears t o exceed (£ 10-fold) Q F e m i n measured f o r s e v e r a l o t h e r phytoplankton s p e c i e s , i n c l u d i n g Thalassiosira weissflogiiDunaliella tertiolecta and Pavlova lutheri (see H a r r i s o n and Morel 1986, Tab l e 2 ) . These data suggest a high i r o n requirement f o r P. tamarensis and may thus e x p l a i n the occurrence of i r o n s t r e s s e f f e c t s (e.g. sexual i n d u c t i o n ) p r i o r t o the d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n . A l s o c o n s i s t e n t w i t h t h i s i d e a i s a study by Anderson and Morel (1979) of r e d t i d e s caused by P. tamarensis, where a sharp decrease i n t o t a l i r o n c o n c e n t r a t i o n was a s s o c i a t e d w i t h the e a r l y stages of p o p u l a t i o n d e c l i n e . Although t o t a l i r o n was never l e s s than 1 pM, i t i s now accepted t h a t f r e e i o n a c t i v i t y r a t h e r than t o t a l c o n c e n t r a t i o n l a r g e l y r e g u l a t e s the 148 b i o a v a i l a b i l i t y of a metal i n seawater (Morel and M o r e l -Laurens 1983). Iron s t r e s s - m e d i a t e d growth l i m i t a t i o n thus remains a p o s s i b l e e x p l a n a t i o n of d e c l i n i n g c e l l d e n s i t i e s observed by Anderson and Morel (1979); however, the c o n t r i b u t i o n of s e x u a l i t y was not q u a n t i f i e d . Hypnozygotes. S e x u a l i t y i n P. tamarensis i n v o l v e s the f u s i o n of two gametes ( m o r p h o l o g i c a l l y s i m i l a r t o v e g e t a t i v e c e l l s ) t o produce a t h e c a t e , q u a d r i f l a g e l l a t e planozygote which, f o l l o w i n g l o s s of p l a t e s and f l a g e l l a , y i e l d s a hypnozygote. The hypnozygote c e l l w a l l i s i n i t i a l l y i n d i s t i n g u i s h a b l e from the temporary c y s t amphiesma but develops s m a l l (ca. 0.5 pm) o u t f o l d i n g s of the p e l l i c l e (= p a p i l l a e ) , and upon maturation i s smooth and c o n s i s t s of t h r e e l a y e r s ( F r i t z 1986). T h i s process i s accompanied by an e l o n g a t i o n of the hypnozygote. Two c l a s s e s of hypnozygote (SI and S2) were encountered i n t h i s study and appear t o be e a r l y phases i n the development of sexual c y s t s . Although no p a p i l l a e were observed, SI c y s t s are l i k e l y i n t e r m e d i a t e between planozygotes and S2 c y s t s , based on t h e i r i r r e g u l a r o u t l i n e and absence of p l a t e s . S2 c y s t s p r o b a b l y r e p r e s e n t the next stage i n hypnozygote development c h a r a c t e r i z e d by e l o n g a t e shape, smooth s u r f a c e and a l s o the presence of f i l a m e n t s of unknown f u n c t i o n a s s o c i a t e d w i t h the n u c l e o l u s ( F r i t z 1986). Hypnozygotes from c u l t u r e and f i e l d p o p u l a t i o n s examined by F r i t z e x h i b i t e d s e v e r a l n o t a b l e d i f f e r e n c e s from S2 c y s t s . 149 C e l l w a l l t h i c k n e s s was c a . f i v e times g r e a t e r than f o r S2 c y s t s . In a d d i t i o n , S2 c y s t s c o n t a i n s e v e r a l , r a t h e r than one, accumulation bodies and r a r e l y show s t a r c h r e s e r v e s . These d i s c r e p a n c i e s are probably r e l a t e d t o m a t u r a t i o n p e r i o d , and p o s s i b l y d i f f e r e n c e s i n i n d u c t i o n mechanism ( i . e . i r o n v s. n i t r o g e n stress-mediated) and other c u l t u r e c o n d i t i o n s . I t should be noted t h a t the i m p l i c a t i o n s of enhanced chromosome condensation i n S2 over SI z y g o t i c c y s t s are u n c e r t a i n , but may be a s s o c i a t e d w i t h dormancy and reduced n u c l e a r a c t i v i t y . Annulate n u c l e a r v e s i c l e s encountered i n hypnozygotes d u r i n g the p r e s e n t study, have been observed p r e v i o u s l y i n the n u c l e i of f u s i n g P. tamarensis gametes (L. F r i t z p e r s . comm.). Although the v e s i c l e s ' f u n c t i o n may be a s s o c i a t e d w i t h s e x u a l i t y , u n e q u i v o c a l c o n f i r m a t i o n of t h e i r absence from v e g e t a t i v e c e l l s i s s t i l l r e q u i r e d . The occurrence of i n t r a n u c l e a r b a c t e r i a and t h e i r a s s o c i a t i o n w i t h t o x i n p r o d u c t i o n i n P. tamarensis has been suggested (Kodama e t a l . 1988). The P. tamarensis s t r a i n employed i n the p r e s e n t study i s among the more t o x i c i s o l a t e s of t h i s s p e c i e s (Boyer et a l . 1986, Cembella e t a l . 1987). A s i d e from the v e s i c l e s mentioned above (whose f u n c t i o n remains t o be e l u c i d a t e d ) , n u c l e i c o n t a i n e d no other unusual i n c l u s i o n s . Pellicular cysts. In the p r e s e n t study, the percentage of p e l l i c u l a r c y s t s ( l e s s than 5%) was lower than expected, as s i m i l a r experiments w i t h t h i s s p e c i e s have y i e l d e d numbers exceeding 30% upon t r a n s f e r i n t o -Fe ESNW (A. Cembella unpubl. 150 d a t a ) . The sharp i n c r e a s e t o over 25% on Day 24 appears r e l a t e d t o exhaustion of b i o l o g i c a l l y a v a i l a b l e i r o n . Temporary c y s t s r e p r e s e n t a very dynamic stage i n the l i f e h i s t o r y of P. tamarensis. Formed under a v a r i e t y of environmental s t i m u l i (see F r i t z and Triemer 1985 and t h i s s t u d y ) , these c y s t s can germinate t o r e l e a s e v e g e t a t i v e c e l l s w i t h i n 8-24 h of r e t u r n t o f a v o r a b l e growth c o n d i t i o n s ( F r i t z and Triemer 1985). In the presen t study, however, a r e l a t i v e l y c o n s t a n t percentage of p e l l i c u l a r c y s t s ( e x c l u d i n g Day 24) and d e c r e a s i n g i r o n supply suggest t h a t e x t e n s i v e g e r m i n a t i o n was u n l i k e l y . A v a i l a b l e evidence i n d i c a t e s t h a t p e l l i c u l a r c y s t s and hypnozygotes f u n c t i o n i n the maintenance of v i a b i l i t y over b r i e f (days-weeks) and extended (months-years) exposure t o adverse environmental f a c t o r s , r e s p e c t i v e l y . Upon t e r m i n a t i o n of t h i s experiment, d e g e n e r a t i v e changes i n o r g a n e l l e morphology were l a r g e l y r e s t r i c t e d t o p e l l i c u l a r c y s t s . These data are c o n s i s t e n t w i t h the above e c o l o g i c a l r o l e s and support the c o n t e n t i o n t h a t temporary c y s t s are more s u s c e p t i b l e t o sho r t - t e r m s t r e s s than are hypnozygotes. Anderson and Wall (1978) demonstrated t h a t the d u r a t i o n of p e l l i c u l a r c y s t v i a b i l i t y i s r e g u l a t e d , i n p a r t , by the i n d u c t i o n mechanism. Cys t s induced by undef i n e d n u t r i t i o n a l s t r e s s (medium d e f i c i e n t i n N, P, S i and v i t a m i n s ) e x h i b i t e d v i a b i l i t y (50%) s u p e r i o r t o those formed under copper t o x i c i t y (15%) or low temperature (0%) f o l l o w i n g 25 days of encystment. 151 The p r e s e n t r e s u l t s suggest f u r t h e r t h a t v i a b i l i t y might a l s o be a f f e c t e d by p h y s i o l o g i c a l l y - m e d i a t e d changes i n u l t r a s t r u c t u r e s p e c i f i c t o the type of n u t r i e n t d e f i c i e n c y . I r o n i s i n v o l v e d i n c h l o r o p h y l l b i o s y n t h e s i s and i s a component of p h o t o s y n t h e t i c and r e s p i r a t o r y e l e c t r o n t r a n s p o r t cytochromes (Marschner 1986). I r r e g u l a r i t i e s i n c h l o r o p l a s t s and m i t o c h o n d r i a c o u l d t h e r e f o r e r e s u l t from i n s u f f i c i e n t i r o n supply. These e f f e c t s , as w e l l as accumulation of storage p r o d u c t s , are a l s o a s s o c i a t e d w i t h other forms of n u t r i e n t s t r e s s (e.g. S h i f r i n and Chisholm 1981, Doucette e t a l . 1987) and may i n s t e a d r e p r e s e n t a g e n e r a l response t o poor growth c o n d i t i o n s . As t h e r e are no p r e v i o u s r e p o r t s of n u t r i e n t s t r e s s - m e d i a t e d a l t e r a t i o n s i n chromatin s t r u c t u r e (to the exte n t encountered d u r i n g t h i s work), a s i m i l a r e x p l a n a t i o n of ab e r r a n t c y s t chromosomes seems l e s s l i k e l y . D i n o f l a g e l l a t e chromosomes c o n t a i n e l e v a t e d ( r e l a t i v e t o the nucleoplasm and cytoplasm) l e v e l s of t r a n s i t i o n metal c a t i o n s , i n c l u d i n g i r o n (see Sigee 1984,1986), thought t o a i d i n the s t a b i l i z a t i o n of chromatin s t r u c t u r e . Sigee and Kearns (1981) have shown t h a t reduced ambient i o n c o n c e n t r a t i o n s (10% of o r i g i n a l medium) lowered the r e l a t i v e p r o p o r t i o n of d i v a l e n t metal c a t i o n s and i n c r e a s e d the degree of chromatin condensation i n chromosomes of Glenodinium foliacium. D e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n c o u l d thus a l t e r the s p a t i a l o r g a n i z a t i o n of DNA and r e s u l t i n clumping of chromatin f i b e r s e x h i b i t e d by c y s t s l a t e i n the experiment. 152 The s p e c i f i c i t y and i n f l u e n c e on v i a b i l i t y of v a r i o u s i r o n s t r e s s - m e d i a t e d u l t r a s t r u c t u r a l m o d i f i c a t i o n s i s d i f f i c u l t t o a s s e s s ; however, d i s r u p t i o n of chromatin arrangement may be d i r e c t l y r e l a t e d t o i r o n d e p l e t i o n and i s l i k e l y t o reduce the p r o b a b i l i t y of g e r m i n a t i o n . Vegetative cells. The u l t r a s t r u c t u r e of the P. tamarensis v e g e t a t i v e amphiesma and p r o t o p l a s t i s g e n e r a l l y c o n s i s t e n t w i t h t h a t of o t h e r t h e c a t e d i n o f l a g e l l a t e s (see reviews by N e t z e l and Diirr 1984, Dodge and Greuet 1987). A f e a t u r e a l s o observed i n p e l l i c u l a r and z y g o t i c c y s t s i s the c r y s t a l l i n e m a t e r i a l p r e s e n t i n c e r t a i n v a c u o l a r r e g i o n s ( F i g s . 4.4,4.10,4.12,4.18,4.19). A v a r i e t y of c r y s t a l l i n e i n c l u s i o n s have been r e p o r t e d from other d i n o f l a g e l l a t e s (e.g. Bibby and Dodge 1972, Pokorny and Gold 1973, Wedemeyer and Wilcox 1984). In comparison t o those d e s c r i b e d p r e v i o u s l y , c r y s t a l s of P. tamarensis are narrower, not c l o s e l y a s s o c i a t e d w i t h a l i m i t i n g membrane, and are l a r g e l y d i f f e r e n t i n t h e i r l o c a t i o n , g e n e r a l morphology and tendency t o form dense, d i s c r e t e aggregates. S p e c u l a t i o n as t o c r y s t a l c omposition has i n c l u d e d guanine (Hastings e t a l . 1966) and c a l c i u m o x a l a t e ( T a y l o r 1968). Energy d i s p e r s i v e x-ray a n a l y s i s of P. tamarensis c r y s t a l s f a i l e d t o d i s c e r n the primary c o n s t i t u t i v e e l e m e n t ( s ) ; however, sample p r e p a r a t i o n was not designed t o o p t i m i z e t h i s t e c h nique. Some evidence i n d i c a t e s t h a t c r y s t a l s o c c u r r i n g i n p e r i p h e r a l v a c u o l e s may be i n v o l v e d i n c e l l w a l l f o r m a t i o n ( J . Chesnick p e r s . comm.). Although t h i s i s u n l i k e l y i n P. tamarensis due t o the c r y s t a l s ' 153 l o c a t i o n , the p o s s i b i l i t y of a storage ( c f . Gold and Pokorny 1973) or e x c r e t o r y r o l e ( c f . S c h m i t t e r 1971) warrants f u r t h e r i n v e s t i g a t i o n . Summary. I r o n s t r e s s can induce both p e l l i c u l a r and z y g o t i c c y s t f ormation i n the t o x i c r e d t i d e d i n o f l a g e l l a t e P. tamarensis. U l t r a s t r u c t u r a l data i n d i c a t e t h a t temporary c y s t s are more s u s c e p t i b l e than hypnozygotes t o short- t e r m s t r e s s e f f e c t s and are thus c o n s i s t e n t w i t h t h e i r hypothesized e c o l o g i c a l r o l e s . I t i s c l e a r t h a t a c a u s a l r e l a t i o n s h i p between i r o n b i o a v a i l a b i l i t y and red t i d e s cannot be e s t a b l i s h e d i n the l a b o r a t o r y . N e v e r t h e l e s s , these r e s u l t s demonstrate t h a t i r o n d e f i c i e n c y has the p o t e n t i a l t o e f f e c t changes i n l i f e h i s t o r y stage and thus p o p u l a t i o n dynamics of t h i s r e d t i d e s p e c i e s . 154 GENERAL CONCLUSIONS Va r i o u s aspects of red t i d e d i n o f l a g e l l a t e i r o n and n i t r o g e n n u t r i t i o n have been c o n s i d e r e d i n t h i s t h e s i s . C e r t a i n f i n d i n g s r e p r e s e n t new, and much needed, i n f o r m a t i o n on the i r o n n u t r i t i o n of these e c o l o g i c a l l y important organisms. The major c o n c l u s i o n s and c o n t r i b u t i o n s of t h i s work are summarized below. 1. The f i r s t i r o n - l i m i t e d growth k i n e t i c s f o r a c o a s t a l d i n o f l a g e l l a t e (G. sanguineum) have been determined. The h a l f - s a t u r a t i o n c o n s t a n t (K^) was estimated t o be 10-1000 times g r e a t e r than f o r other n e r i t i c s p e c i e s examined p r e v i o u s l y . T h i s d i s p a r i t y i n was e x p l a i n e d , i n p a r t , by t h i s s p e c i e s ' c o m p a r a t i v e l y l a r g e i r o n requirement (as measured by Fe/C r a t i o s ) , which exceeded those of c e r t a i n c o a s t a l diatoms by one t o two orders of magnitude. 2. Reductions i n G. sanguineum c h l o r o p h y l l a ( c h l a) quotas ( Q c n i ) a n d p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t (PET) e f f i c i e n c y (as measured by the index 1-F/F D, where F = in vivo f l u o r e s c e n c e and F D = DCMU-enhanced F) o c c u r r e d under i r o n l i m i t a t i o n and d e p l e t i o n . These a l t e r a t i o n s t r a n s l a t e d i n t o a 7 - f o l d i n c r e a s e i n F / c h l a r a t i o s f o r i r o n - d e p l e t e c e l l s . These o b s e r v a t i o n are c o n s i s t e n t w i t h , and were d i s c u s s e d i n r e l a t i o n t o , the e s s e n t i a l r o l e of i r o n i n c h l a and PET component ( i . e . cytochromes and Fe-S p r o t e i n s ) b i o s y n t h e s i s . 155 3. N i t r o g e n d e p l e t i o n e f f e c t e d a d e c l i n e i n G. sanguineum Q c n]_ °f a magnitude s i m i l a r t o t h a t caused by i r o n d e p l e t i o n . Conversely, N s t r e s s d i d not reduce PET e f f i c i e n c y nor enhance F / c h l a r a t i o s t o the same extent as Fe s t r e s s . These r e s u l t s p r o v i d e d evidence f o r the s p e c i f i c i t y of i r o n s t r e s s - m e d i a t e d e f f e c t s on c e r t a i n PET components. 4. F / c h l a, i n c o n j u n c t i o n w i t h o t h e r probes of n u t r i t i o n a l s t a t u s , was suggested as a p o t e n t i a l l y u s e f u l i n d i c a t o r of i r o n s t r e s s i n f i e l d p o p u l a t i o n s , owing t o the magnitude and apparent s p e c i f i c i t y of changes i n t h i s r a t i o . However, as F / c h l a was e l e v a t e d t o a l e s s e r degree i n i r o n -d e p l e t e , NH^j-grown than NC^-grown c e l l s , and the l a t t e r c e l l s e x h i b i t e d symptoms of N d e f i c i e n c y , c o n s i d e r a t i o n of m u l t i p l e i n d i c e s of n u t r i e n t d e f i c i e n c y and c a r e f u l i n t e r p r e t a t i o n of r e s u l t s were a l s o s t r e s s e d . 5. Fe/N r a t i o s demonstrated a l a r g e r ( 1 . 5 - f o l d ) minimum i r o n requirement f o r NO3- than NH^-grown G. sanguineum c e l l s . T h i s d i f f e r e n c e was c o n s i d e r e d t o be a r e f l e c t i o n of the i r o n requirement of the r e d u c t i v e NO3 a s s i m i l a t o r y enzymes, n i t r a t e and n i t r i t e r e d u c t a s e . 6. A c q u i s i t i o n of n i t r o g e n by i r o n - d e p l e t e , NO-3-grown G. sanguineum c e l l s was s u f f i c i e n t l y i n h i b i t e d t o y i e l d symptoms of n i t r o g e n d e f i c i e n c y . Supporting evidence was p r o v i d e d by decreased (ca. 1.4-fold) n i t r o g e n quotas and f r e e amino a c i d / p r o t e i n r a t i o s . 156 7. The f i r s t demonstration of enhanced i r o n t r a n s p o r t r a t e s (p) i n an i r o n - s t r e s s e d d i n o f l a g e l l a t e (G. sanguineum), has been presented. I t was noted, however, t h a t an e l e v a t e d p f a i l e d t o be manifested i n i r o n - d e p l e t e c e l l s f o l l o w i n g a t r a n s i t i o n from NH4 t o NO3 n u t r i t i o n . T h i s s u p p r e s s i o n was b e l i e v e d t o r e s u l t from co n c u r r e n t i r o n and n i t r o g e n s t r e s s , due t o the i n a b i l i t y of NH^-grown c e l l s t o r a p i d l y a s s i m i l a t e NO3. The complete i n i t i a l i n h i b i t i o n of NO3 uptake when Fe-d e p l e t e , NH^-grown c e l l s were giv e n s a t u r a t i n g i r o n a d d i t i o n s was c o n s i s t e n t w i t h t h i s i d e a . In c o n t r a s t , NH4 was taken up immediately by these, and i r o n - d e p l e t e , NC^-grown c e l l s . 8. Based on the f o r i r o n - l i m i t e d growth, the com p a r a t i v e l y l a r g e Fe/C r a t i o and the r e l a t i v e l y narrow range of i r o n c o n c e n t r a t i o n s over which maximum or near maximum growth r a t e s can be maintained by s h o r t term a d a p t a t i o n (as est i m a t e d t h e o r e t i c a l l y by K p/K^), i t was suggested t h a t G. sanguineum i s more s u s c e p t i b l e t o i r o n - l i m i t e d growth than the n e r i t i c diatom T. weissflogii. 9. I r o n s t r e s s caused r e d u c t i o n s i n c h l o r o p l a s t number and some deg e n e r a t i o n of l a m e l l a r o r g a n i z a t i o n i n G. sanguineum and p r o v i d e d a s t r u c t u r a l b a s i s f o r changes noted i n Qcri\ and f l u o r e s c e n c e p r o p e r t i e s . For P. tamarensis, i r o n l i m i t a t i o n induced the formation of temporary (= p e l l i c l e ) and r e s t i n g (= hypnozygotes) c y s t s . 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E l s e v i e r North H o l l a n d , Inc., N.Y. pp. 279-86. 174 APPENDIX 1 Gro w t h - i r r a d i a n c e curve of G. sanguineum. w i t h a s s o c i a t e d  changes i n c e l l volume Objective: T h i s experiment was performed t o o b t a i n a growth ( u ) - i r r a d i a n c e (I) curve f o r G. sanguineum and t o observe any co n c u r r e n t changes i n c e l l volume as a f u n c t i o n of I. Methods: C u l t u r e s of G. sanguineum were grown a c c o r d i n g t o the c o n d i t i o n s and procedures o u t l i n e d i n the General culture maintenance s e c t i o n of Chapter 1, except t h a t n e u t r a l d e n s i t y s c r e e n i n g (without blue P l e x i g l a s ) was used t o ach i e v e i r r a d i a n c e l e v e l s of 208, 92, 52, 30 and 12 p E«m *s (one c u l t u r e f o r each i r r a d i a n c e ) . F o l l o w i n g s t a b i l i z a t i o n of u, c e l l d i v i s i o n r a t e s and volumes were o b t a i n e d from t h r e e growth curves a t each i r r a d i a n c e . C e l l counts and average c e l l volumes based on e q u i v a l e n t s p h e r i c a l diameter were determined w i t h an e l e c t r o n i c p a r t i c l e counter ( C o u l t e r E l e c t r o n i c s ; see Chapter 1, M a t e r i a l s and Methods f o r a d d i t i o n a l d e t a i l s ) . Values p l o t t e d are mean ± 1 S.D. f o r data d e r i v e d from the t h r e e growth c u r v e s . Results & Conclusions: R e s u l t s presented i n Graph A i n d i c a t e t h a t i r r a d i a n c e s s a t u r a t i n g f o r growth are ca. £ 90 pE«m~ 2'S~*, w i t h no p h o t o i n h i b i t i o n apparent a t the hig h e s t o 1 i r r a d i a n c e examined (208 uEmm~ *s~ ). I t was concluded t h a t 9 1 an i r r a d i a n c e of 145 jjE*m *s , which c o u l d be achieved w i t h 175 e i g h t V i t a - L i t e R UHO f l u o r e s c e n t tubes f i l t e r e d through bl u e P l e x i g l a s R (No. 2069, Rohm and Haas), was w e l l w i t h i n the s a t u r a t e d p o r t i o n o f the p v s . I c u r v e . As t h i s l i g h t i n g c o n f i g u r a t i o n does not a l t e r the maximum growth r a t e from t h a t o b t a i n e d w i t h u n f i l t e r e d l i g h t , and more c l o s e l y approximates an underwater l i g h t environment than white l i g h t , i t was employed e x c l u s i v e l y i n the c u r r e n t r e s e a r c h . Average c e l l volume (Graph B) decreased w i t h i r r a d i a n c e t o a minimum of 2.2-10 pm ( c a . 50% of the c e l l volume a t Pmax) a t the lowest i r r a d i a n c e (12 pE-m""2 • s ~ A ) . Graph A. Growth r a t e s of G. sanguineum a t v a r i o u s i r r a d i a n c e l e v e l s . Graph B. Average c e l l volumes of G. sanguineum a t v a r i o u s i r r a d i a n c e l e v e l s . Values f o r Graphs A and B r e p r e s e n t means ± 1 S.D. f o r n = 3 growth c u r v e s per i r r a d i a n c e l e v e l . E r r o r b a r s a re s m a l l e r than symbols where not apparent. U l u o 1.0 0.0-1 , . , , . 1 . . 1 0 25 50 75 100 125 150 175 200 225 IRRADIANCE 0 i E m ~ 2 - s - 1 ) 176 APPENDIX 2 Iron d e p l e t i o n of G. sanguineum; pH r e g u l a t i o n and b i o a s s a y s Objective: T h i s s e r i e s of experiments was performed t o demonstrate pH r e g u l a t i o n of G. sanguineum batch c u l t u r e c e l l y i e l d and thus, of the d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n from the c u l t u r e medium. Methods: C u l t u r e s of G. sanguineum were grown a c c o r d i n g t o the c o n d i t i o n s and procedures o u t l i n e d f o r i r o n d e p l e t i o n i n the Jr o n and nitrogen depletion s e c t i o n of Chapter 1 ( M a t e r i a l s and Methods), except t h a t one c u l t u r e was not bubbled and the ot h e r was bubbled w i t h a i r or 1-2% C0 2 (50-100 ml-min - 1) as r e q u i r e d t o minimize changes i n pH. Upon c e s s a t i o n of growth, a l i q u o t s of c u l t u r e were t r a n s f e r r e d t o 125 ml Erlenmeyer f l a s k s (polycarbonate, Nalgene) f o r b i o a s s a y experiments (same c u l t u r e c o n d i t i o n s , no b u b b l i n g ) . Three b i o a s s a y treatments were employed t o c o n f i r m the pH ( r a t h e r than i r o n ) c o n t r o l of c e l l y i e l d i n the unbubbled c u l t u r e : 1. a d d i t i o n of ESAW c o n c e n t r a t i o n s of a l l enrichments except i r o n (other t r a c e metals were c h e l a t e d w i t h EDTA a t an EDTA:trace metal r a t i o of 1.6); 2. a d d i t i o n of iron/EDTA a t ESAW c o n c e n t r a t i o n s ; 3. a d d i t i o n of S u p r a p u r R HC1 (Merck) t o lower pH t o 8.2. On Day 6 the pH of treatment f l a s k s 1 and 2 was lowered t o 8.2 w i t h S u p r a p u r R HC1. In the case of the bubbled c u l t u r e , t h r e e b i o a s s a y treatments were run t o demonstrate the d e p l e t i o n of 177 b i o l o g i c a l l y a v a i l a b l e i r o n from the c u l t u r e medium: 1. c o n t r o l ( i . e . no a d d i t i o n s ) ; 2. a d d i t i o n of iron/EDTA at ESAW c o n c e n t r a t i o n s ; 3. a d d i t i o n of ESAW c o n c e n t r a t i o n s of a l l enrichments except i r o n (other t r a c e metals were c h e l a t e d w i t h EDTA at an EDTA:trace metal r a t i o of 1.6). C e l l counts were performed w i t h an e l e c t r o n i c p a r t i c l e c o unter ( C o u l t e r E l e c t r o n i c s ; see Chapter 1, M a t e r i a l s and Methods f o r a d d i t i o n a l d e t a i l s ) . Results & Conclusions: The c e l l y i e l d of the bubbled c u l t u r e (BC) exceeded t h a t of the unbubbled c u l t u r e (UC) by t h r e e - f o l d (Graph A ) . Upon c e s s a t i o n of growth, the pH of UC and BC was 9.3 and 8.3, r e s p e c t i v e l y (Graph B ) . During the middle t o l a t e e x p o n e n t i a l growth phase of BC, pH d i d r i s e t o a maximum of 8.8, but remained a t t h i s l e v e l o n l y f o r a b r i e f p e r i o d (3 d a y s ) . B u b b l i n g with 1-2% C0 2 was i n s u f f i c i e n t t o prevent any e l e v a t i o n i n pH. Although i n c r e a s i n g the percentage of CG*2 may have avoided pH changes, t h i s a c t i o n was c o n s i d e r e d u n d e s i r a b l e due t o a l t e r a t i o n s i n carbon a s s i m i l a t o r y enzymes (e.g. c a r b o n i c anhydrase) induced by h i g h C0 2 c o n c e n t r a t i o n s (Lawlor 1987). Furthermore, the temporary r i s e i n pH d i d not p r o h i b i t the d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n from BC (see b i o a s s a y data below). R e s u l t s of the pH b i o a s s a y f o r UC (Graph C) c l e a r l y demonstrate pH c o n t r o l of batch c u l t u r e y i e l d , as c e l l d i v i s i o n commenced immediately upon the r e t u r n of pH t o 8.2 178 (treatment 3, HCl a d d i t i o n ) . The r e g u l a t o r y r o l e of pH was supported f u r t h e r by the f a c t t h a t no growth response was noted f o r treatments 1 (-Fe ESAW) and 2 (Fe/EDTA) u n t i l a f t e r t h e i r pH was lowered t o 8.2 on Day 6. The i r o n b i o a s s a y conducted on BC (Graph D) confirmed the d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n , as onl y the treatment r e c e i v i n g i r o n showed a resumption of growth (treatment 2, Fe/EDTA). While these i r o n b i o a s s a y data apply t o NO-j-grown c u l t u r e s , s i m i l a r r e s u l t s were ob t a i n e d from i d e n t i c a l treatments w i t h NH^-grown c u l t u r e s . I t was concluded t h a t b u b b l i n g was necessary f o r i r o n d e p l e t i o n experiments and t h a t t e r m i n a t i o n of c e l l d i v i s i o n i n -Fe ESAW d i d r e s u l t from the d e p l e t i o n of b i o l o g i c a l l y a v a i l a b l e i r o n from the c u l t u r e medium. 179 Graph A. Growth c u r v e s of unbubbled (•) and bubbled (o) G. sanguineum b a t c h c u l t u r e s . B. pH changes i n unbubbled (•) and bubbled (o) ba t c h c u l t u r e s shown i n Graph A. C. Bi o a s s a y d e m o n s t r a t i n g pH c o n t r o l of unbubbled b a t c h c u l t u r e (Graph A) c e l l y i e l d . B i o a s s a y treatments i n c l u d e d a d d i t i o n s of ESAW enrichments without Fe ( o ) , ESAW Fe/EDTA enrichments (•), o r S u p r a p u r R HCl t o a pH of 8.2 (•). On Day 6 the pH of the f i r s t two treatments was lowered t o 8.2 u s i n g HCl. D. B i o a s s a y demonstrating i r o n d e p l e t i o n of bubbled c u l t u r e (Graph A ) . B i o a s s a y treatments i n c l u d e d a c o n t r o l ( i . e . no a d d i t i o n ) ( o ) , a d d i t i o n s o f ESAW Fe/EDTA enrichments (•), or ESAW enrichments without Fe (•). 5.0-1 , 1 . 1 1 . • , 1 1 -1 0 1 2 3 4 5 6 7 8 8 TIME (d) 180 APPENDIX 3 E f f e c t 'of ammonium c o n c e n t r a t i o n on G. sanguineum growth r a t e O b j e c t i v e : T h i s e x p e r i m e n t was per f o r m e d t o de t e r m i n e i f e l e v a t e d ammonium c o n c e n t r a t i o n s e f f e c t growth r a t e r e d u c t i o n s i n G. sanguineum. Methods: C u l t u r e s o f G. sanguineum were grown a c c o r d i n g t o t h e c o n d i t i o n s and p r o c e d u r e s o u t l i n e d i n t h e General culture maintenance s e c t i o n o f Ch a p t e r 1, e x c e p t f o r t h e a d d i t i o n o f 50 pM r a t h e r t h a n 550 pM n i t r a t e . C e l l s were a l l o w e d t o exh a u s t t h e ambient n i t r o g e n , a f t e r w h i c h t h e y were t r a n s f e r r e d i n t o one o f s i x t y p e s o f media c o n t a i n i n g f u l l ESAW n u t r i e n t s and t h e f o l l o w i n g s o u r c e s / c o n c e n t r a t i o n s of n i t r o g e n . Two c o n t r o l c u l t u r e s were p r o v i d e d w i t h e i t h e r ESAW n i t r a t e c o n c e n t r a t i o n s ( i . e . 550 pM) o r 80 pM NO3. Four e x p e r i m e n t a l c u l t u r e s c o n t a i n e d e i t h e r 80, 150, 350 o r 450 pM ammonium. D u p l i c a t e t r i a l s were c o n d u c t e d f o r each n i t r o g e n s o u r c e / c o n c e n t r a t i o n . Growth r a t e s ( d ~ x ) were d e t e r m i n e d by c e l l c o u n t s w i t h an e l e c t r o n i c p a r t i c l e c o u n t e r ( C o u l t e r E l e c t r o n i c s ) and a r e g i v e n as t h e mean (n = 2) ± 1 S . D . ( i n p a r e n t h e s e s ) . 181 Results & Conclusions: GROWTH RATES ( d - 1 ) N0 3 550 uM NO3 80 pM NH 4 80 uM NH 4 150 uM NH 4 350 uM NH 4 450 pM 0.35 ( 0 . 0 1 ) 0.35 ( 0 . 0 3 ) 0.34 (0.02) 0.30 (0.01 ) 0.24 (0.06) 0.24 (0.06) R e s u l t s i n d i c a t e a r e d u c t i o n i n growth r a t e w i t h £ 150 JJM NH 4. S i m i l a r f i n d i n g s have been r e p o r t e d p r e v i o u s l y by Thomas et a l . (1980) f o r t h i s s p e c i e s . Growth r a t e s of 80 pM NH 4 and both NO3 c o n t r o l c u l t u r e s appear e q u i v a l e n t . I t was concluded t h a t f o r a l l cases i n which G. sanguineum would be s u p p l i e d w i t h ammonium n i t r o g e n ( i . e . growth or uptake experiments), c o n c e n t r a t i o n s should not exceed 80 JJM. 182 APPENDIX 4 Removal of n o n - b i o l o g i c a l i r o n from G. sanguineum: a t e s t of  wash volume Objective: T h i s experiment was performed t o assess the e f f e c t of i n c r e a s i n g wash volume on the removal of s u r f a c e -bound i r o n from G. sanguineum. Methods: C u l t u r e s of G. sanguineum were grown a c c o r d i n g t o the c o n d i t i o n s and procedures o u t l i n e d i n the General culture maintenance s e c t i o n of Chapter 1, except t h a t u n l a b e l e d ESAW F e C l j was r e p l a c e d by an equimolar c o n c e n t r a t i o n of F e C ^ . At a c e l l d e n s i t y of c a . 4*10 c e l l s •ml""1, 5 ml a l i q u o t s were c o l l e c t e d on 5 pm (pore diameter) f i l t e r s (polycarbonate, N u c l e p o r e ) , washed wit h 5, 10, 20 or 40 ml of chelexed ESAW s a l t s o l u t i o n (CSS, see Chapter 2, M a t e r i a l s and Methods), and counted by l i q u i d s c i n t i l l a t i o n . The experiment was conducted i n d u p l i c a t e (n = 2). Values (mean ± 1 S.D.) are expressed as a perc e n t of the 5 5 F e l a b e l ( i . e . d i s i n t e g r a t i o n s ' m i n " 1 , DPM) removed by the s m a l l e s t (5 ml) wash volume. Results & Conclusions: 5 ml 10 ml 20ml 40 ml 100 94 ± 2 102 ± 18 92 ± 16 183 R e s u l t s demonstrate t h a t the q u a n t i t y of n o n - b i o l o g i c a l i r o n removed from c a . 2*10 4 c e l l s by 5, 10, 20 or 40 ml i s e q u i v a l e n t . As no advantage i s p r o v i d e d by use of g r e a t e r volumes and i t i s d e s i r a b l e t o minimize t o t a l f i l t r a t i o n times, 5 ml wash volumes were employed i n the Fe a d s o r p t i o n experiments i n Chapter 2. PUBLICATIONS (continued from previous page): Fryxell, G.A. , Doucette, G.J. & Hubbard, G.F. 1981. The genus Thalassiosira: the bipolar diatom T. antarctica Comber. Botanica Marina 24:321-35. Fryxell, G.A., V i l l a r e a l , T.A. & Doucette, G.J. 198l. Antarctic phytoplankton: diatom resting spores and Agulhas collections. Antarctic Journal U.S. 1981 Annual Review, pp. 128-30, Fryxell, G.A., Johansen, J.R. & Doucette, G.J. 1982. Phytoplankton cultures and collections around South Georgia. Antarctic Journal U.S. 17:l60-2. Doucette, G.J. 1982. Variability in patterns of resting spore formation in the diatom Thalassiosira antarctica. Eos 63:47 (abstract). Doucette, G.J. & Fryxell, G.A. 1983. Thalassiosira antarctica: vegetative and resting stage chemical composition of an ice-related marine diatom. Marine Biology 78:1-6. Doucette, G.J., Burghardt, R.C. & Fryxell, G.A. 1984. The genus Thalassiosira: protoplast ultrastructure of the bipolar diatom Thalassiosira antarctica Comber. Canadian Journal of Botany 62:1513-22. Doucette, G.J. & Fryxell, G.A. 1985. The genus Thalassiosira •-. (Bacillariophyceae): vegetative and resting stage ultrastructure of an ice-related .marine diatom. Polar Biology 4:107-12. Johansen, J.R., Doucette, G.J. & Fryxell, G.A. 1985. The genus Thalassiosira (Bacillariophyceae): morphology of heterovalvate resting spores of T_. scotia. American Journal of Botany 72:l86l-70". Parslow, J.S., Doucette. G.J., Taylor, F.J.R. & Harrison, P.J. 1986. Feeding by the zooflagellate Pseudobodo sp. on the picoplanktonic prasihomonad Micromonas pu s i l l a . Marine Ecology Progress Series 29:237-46. Doucette, G.J., Price, N.M. & Harrison, P.J. 1987. Effects of selenium deficiency on the morphology and ultrastructure of the coastal marine diatom Thalassiosira pseuddnana (Bacillariophyceae), Journal of Phycology 23:9-17. Johansen, J.R., Doucette, G.J., Barclay, W.R. & Bull, D. 1988. The morphology and physiology of Pleurochrysis carterae var. dentata var. nov. (Prymnesiophyceae), a new coccolithophorid from an inland saline pond in New Mexico, U.S.A. Phycologia 27:78-88. AWARDS (continued from previous page): B r i t i s h Columbia Post Secondary Scholarship, 1987-1988 NSERC Postdoctoral Fellowship, I988-I989 Isaak Walton Killam Postdoctoral Fellowship, 1988-1989 

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