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Effects of nutrient limitation on biochemical composition of three microalgae and their food value to… Calderwood, Gail Sibbald 1989

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EFFECTS OF NUTRIENT LIMITATION ON BIOCHEMICAL COMPOSITION OF THREE MICROALGAE AND THEIR FOOD VALUE TO OYSTER LARVAE, CRASSOSTREA GIGAS By GAIL SIBBALD CALDERWOOD B . S c , The U n i v e r s i t y o f V i c t o r i a , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f Oceanography We a c c e p t t h i s t h e s i s as conforming t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August, 1989 © G a i l S i b b a l d Calderwood, 1989 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 The University of British Columbia Vancouver, Canada Date 6cf S. /f33 DE-6 (2/88) ABSTRACT Three microalgae commonly used i n bivalve aquaculture were grown i n u n i a l g a l batch culture and harvested f o r chemical analyses at rigorously monitored stages of n i t r a t e , phosphate, or s i l i c a t e l i m i t a t i o n . Two serie s of cultures were analysed; the f i r s t was l i g h t - l i m i t e d . Light-saturated cultures of the second series were used i n oyster larvae growth t r i a l s . C e l l u l a r c a l o r i c value was reduced by nutrient starvation of Tahitian Isochrysis galbana (clone T-Iso) (nitr a t e or phosphate) and Chaetoceros calcitrans ( n i t r a t e or s i l i c a t e ) due to depletion of c e l l u l a r protein and l i p i d , while carbohydrate content increased. Conversely, Thalassiosira pseudonana (clone 3H) increased c e l l u l a r protein and l i p i d and decreased carbohydrate under s i l i c a t e starvation r e s u l t i n g i n an increase i n f i n a l c a l o r i c content. Fatty acid composition (percent of the t o t a l f a t t y acids) of light-saturated, n u t r i e n t - l i m i t e d diatoms was remarkably stable through 6 hours of starvation. I. galbana (T-Iso) polyunsaturated:monounsaturated f a t t y acid r a t i o s dropped dramatically by the second day of nutrient starvation i n ser i e s 2. Changes observed i n series 1 could e i t h e r be at t r i b u t e d to l i g h t l i m i t a t i o n or long starvation periods. Polyunsaturated:monounsaturated f a t t y acid r a t i o s declined with time i n both diatoms i n series 1. The most decisive e f f e c t on f a t t y acid composition among a l l three a l g a l i i i s p e c i e s o c c u r r e d under phosphate l i m i t a t i o n o f T. pseudonana d u r i n g which 20:5n3 decreased from 22% t o 6% and 16:0 i n c r e a s e d t w o - f o l d from 19% t o 37%. I. galbana (T-Iso) was a poor d i e t f o r C. gigas l a r v a e compared t o both diatom d i e t s r e g a r d l e s s o f n u t r i e n t s t a t u s , s u p p o r t i n g the b e l i e f t h a t the f a t t y a c i d 20:5n3 may be a more s i g n i f i c a n t n u t r i t i o n a l f a c t o r than 22:6n3. The l a t t e r i s found i n h i g h c o n c e n t r a t i o n i n I. galbana (T-Iso) but not i n the diatoms. C. gigas l a r v a e grew f a s t e s t when fe d the most energy r i c h diatom t r e a t m e n t s . When c a l o r i c v a l u e s were s i m i l a r , i n c r e a s e d carbohydrate may have p r o v i d e d energy which spared e s s e n t i a l p r o t e i n s and l i p i d s , thereby p e r m i t t i n g f a s t e r growth. I t i s r e c o g n i z e d t h a t r a t i o n l e v e l s were sub-optimal i n the d i e t t r i a l s . i v TABLE OF CONTENTS Page A b s t r a c t i i L i s t o f T a b l e s v i L i s t o f F i g u r e s i x Acknowledgements x v i i INTRODUCTION 1 1. Importance o f L i p i d s i n B i v a l v e N u t r i t i o n 4 2. V a r i a t i o n o f Chemical Composition o f A l g a l D i e t s .. 6 3. E f f e c t o f N u t r i e n t L i m i t a t i o n on A l g a l Composition 9 i . N i t r o g e n S t a r v a t i o n 9 i i . S i l i c o n S t a r v a t i o n 11 i i i . Phosphate S t a r v a t i o n 12 4. O b j e c t i v e s . 13 MATERIALS AND METHODS 16 1. A l g a l C u l t u r e s 16 i . S e r i e s 1 17 i i . S e r i e s 2 17 i i i . A l g a l Analyses 19 2. L a r v a l C u l t u r e " 20 3. Chemical A n a l y s i s 21 i . F a t t y A c i d A n a l y s i s 22 i i . Gross Composition Analyses 23 RESULTS .' 26 I . A l g a l Growth i n S e r i e s 1 and S e r i e s 2 C u l t u r e s . 26 1. S e r i e s 1 26 2. S e r i e s 2 27 I I . B i o c h e m i c a l Composition o f Algae 36 A. Gross Biochemical Composition o f Algae 36 1. T a h i t i a n Isochrysis 36 2. Chaetoceros calcitrans 41 3. Thalassiosira pseudonana 46 B. F a t t y A c i d Composition 46 1. Comparison o f Mid-Log P r o f i l e s . . . . 51 2. Comparison o f N u t r i e n t - S t a r v e d P r o f i l e s .. 61 a. T a h i t i a n Isochrysis 61 i . F a t t y A c i d C l a s s P r o f i l e 61 i i . E s s e n t i a l F a t t y A c i d s 66 i i i . F a t t y A c i d Peaks 66 b. Chaetoceros calcitrans 79 i . F a t t y A c i d C l a s s P r o f i l e 79 i i . E s s e n t i a l F a t t y A c i d s 84 i i i . F a t t y A c i d Peaks 85 c. Thalassiosira pseudonana 98 i . F a t t y A c i d C l a s s P r o f i l e 99 i i . E s s e n t i a l F a t t y A c i d s 99 i i i . F a t t y A c i d Peaks 104 V I I I . Growth of Oyster Larvae 117 1. T a h i t i a n Isochrysis 117 2. Chaetoceros calcitrans 122 3. Thai assiosira pseudonana 122 IV. F a c t o r s A f f e c t i n g Crassostrea gigas Growth 129 DISCUSSION 132 1. E f f e c t s of N u t r i e n t S t a r v a t i o n on Gross Composition 133 i . N i t r a t e S t a r v a t i o n 133 i i . S i l i c a t e S t a r v a t i o n 135 2. E f f e c t o f N u t r i e n t S t a r v a t i o n on L i p i d Composition 137 i . N i t r a t e S t a r v a t i o n 137 i i . S i l i c a t e S t a r v a t i o n 139 i i i . Phosphate S t a r v a t i o n 140 3. E f f e c t o f L i g h t upon F a t t y A c i d Composition 142 4. E f f e c t o f A l g a l Chemical Composition on L a r v a l Growth 146 i . F a t t y A c i d s 146 i i . Gross Composition 147 5. G r a z i n g Rates and R a t i o n S i z e 148 SUMMARY AND CONCLUSIONS 152 FUTURE WORK 155 REFERENCES 157 APPENDICES 168 A. C a l o r i c v a l u e and c e l l u l a r weight o f gros s b i o c h e m i c a l components o f microalgae grown i n s e r i e s 2 c u l t u r e s 169 B. T a b l e s o f complete f a t t y a c i d p r o f i l e s o f mic r o a l g a e grown i n s e r i e s 1 and s e r i e s 2 c u l t u r e s 175 v i L i s t of T a b l e s Page T a b l e I . C o n c e n t r a t i o n s of l i m i t i n g n u t r i e n t s used i n n u t r i e n t enrichment s o l u t i o n s o f v a r i o u s a l g a l growth media used i n s e r i e s 1 and s e r i e s 2 c u l t u r e s o f T a h i t i a n Isochrysis, Chaetoceros calcitrans and Thalassiosira pseudonana. A second a d d i t i o n of s i l i c a t e ( i n b r a c k e t s ) was added d u r i n g l o g phase growth. ( S i l i c a t e was not added t o T a h i t i a n Isochrysis growth medium.) 18 T a b l e I I . F i n a l s u r v i v a l of Crassostrea gigas l a r v a e i n t h r e e u n i a l g a l d i e t t r i a l s l a s t i n g from 12 t o 19 days. T a h i t i a n Isochrysis d i e t s c o n s i s t e d of a l g a l c e l l s h a r v e s t e d from n i t r a t e - l i m i t e d medium at mid-log growth phase, o r a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d or 6 d ) . A second mid-log treatment (Mid-log c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of growing l a r v a e i n n i t r a t e - f r e e seawater whrn the l a r v a e were f e d n i t r a t e - s t a r v e d c e l l s . Diatom d i e t s c o n s i s t e d of c e l l s h a r v e s t e d from n i t r a t e - and s i l i c a t e - l i m i t e d medium at mid-log phase, or a f t e r 6 h of n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h ) . Two a d d i t i o n a l mid-log treatments (Mid-log N - c o n t r o l , M i d - l o g S i - c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d or s i l i c a t e -s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e - f r e e or s i l i c a t e - f r e e seawater, r e s p e c t i v e l y . Values are mean percentages ± 1 standard d e v i a t i o n , o f l i v e l a r v a e from a sample of g r e a t e r than 100 animals from each r e p l i c a t e 118 T a b l e I I I . S h e l l l e n g t h (p,m, p a r t a) and i n t e r v a l growth r a t e s ( p a r t b) o f l a r v a l Crassostrea gigas f e d a u n i a l g a l d i e t , a t a d a i l y c o n c e n t r a t i o n of 20,000 c e l l s mL f o r 19 days, of T a h i t i a n Isochrysis h a r v e s t e d from n i t r a t e -l i m i t e d medium at mid-log growth phase, or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d or 6 d ) . A second mid-log treatment (Mid-log c o n t r o l ) c o n t r o l l e d f o r the e f f e c t o f h o l d i n g v i i l a r v a e f e d n i t r a t e - s t a r v e d c e l l s i n n i t r a t e - f r e e seawater. L e a s t squares means of s h e l l l e n g t h ± standard e r r o r were c a l c u l a t e d by nested a n a l y s i s of v a r i a n c e , a t the 95% c o n f i d e n c e l e v e l , f o r t h r e e r e p l i c a t e s . Round b r a c k e t s e n c l o s e the number of measurements made. Square b r a c k e t s e n c l o s e l e t t e r s o f s i g n i f i c a n c e from Tukey's m u l t i p l e comparison t e s t , (p<.05). Matching l e t t e r s among treatments w i t h i n the day o f measurement s i g n i f y no d i f f e r e n c e among the treatments b e a r i n g those l e t t e r s . 121 T a b l e IV. S h e l l l e n g t h (u,m, p a r t a) and i n t e r v a l growth r a t e s (part b) of l a r v a l Crassostrea gigas f e d a u n i a l g a l d i e t , f o r 14 days as i n T a b l e I I , of Chaetoceros calcitrans h a r v e s t e d from n i t r a t e - and s i l i c a t e - l i m i t e d medium at mid-log phase, or a f t e r 6 h o f n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h ) . Two a d d i t i o n a l mid-log treatments (Mid-log N - c o n t r o l , M i d - l o g S i - c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d or s i l i c a t e -s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e - f r e e or s i l i c a t e - f r e e seawater, r e s p e c t i v e l y 125 T a b l e V. S h e l l l e n g t h (|xm, p a r t a) and i n t e r v a l growth r a t e s ( p a r t b) of l a r v a l Crassostrea gigas f e d a u n i a l g a l d i e t f o r 12 days as i n T a b l e I I , of Thalassiosira pseudonana h a r v e s t e d from n i t r a t e - and s i l i c a t e - l i m i t e d medium at mid-log phase, or a f t e r 6 h of n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h ) . Two a d d i t i o n a l mid-log treatments (Mid-log N - c o n t r o l , M i d - l o g S i - c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d or s i l i c a t e -s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e - f r e e or s i l i c a t e - f r e e seawater, r e s p e c t i v e l y . o f Thalassiosira pseudonana f o r 12 days as i n T a b l e 2. Growth was a l s o compared a g a i n s t a d i e t of T a h i t i a n Isochrysis (T-ISO) 128 T a b l e V I . A summary of the b i o c h e m i c a l composition and performance of t h r e e u n i a l g a l d i e t s f e d t o Crassostrea gigas l a r v a e . An experiment f o r each phytoplankton v i i i s p e c i e s i n v e s t i g a t e d the a f f e c t o f n u t r i e n t s t a r v a t i o n on the v a l u e o f the d i e t i n i n c r e a s i n g l a r v a l s h e l l l e n g t h . Two r e p l i c a t e s were analyzed and averaged i n each treatment 131 i x L i s t o f F i g u r e s Page F i g . 1. Growth curves (shown by c e l l numbers) of T a h i t i a n Isochrysis grown i n s e r i e s 1 phosphate- and n i t r a t e - l i m i t e d media, showing the c o n c e n t r a t i o n o f the l i m i t i n g n u t r i e n t s i n t he media (e.g. phosphate c o n c e n t r a t i o n i n the p h o s p h a t e - l i m i t e d medium). Arrows i n d i c a t e when samples were taken 28 F i g . 2. Growth curves (shown by c e l l numbers o r in vivo f l u o r e s c e n c e ) o f Chaetoceros calcitrans grown i n s e r i e s 1 n i t r a t e - , phosphate- and s i l i c a t e - l i m i t e d media, showing the c o n c e n t r a t i o n o f the l i m i t i n g n u t r i e n t s i n the media. ( N i t r a t e - l i m i t e d f l u o r e s c e n c e was omitted s i n c e i t f o l l o w s same curve as o t h e r treatments.) Arrows i n d i c a t e when samples were taken 3 0 F i g . 3. Growth curves (shown by c e l l numbers) o f Thalassiosira pseudonana grown i n s e r i e s 1 phosphate- and s i l i c a t e - l i m i t e d media, showing the c o n c e n t r a t i o n o f the l i m i t i n g n u t r i e n t s i n the media. Dashed l i n e i n s t a t i o n a r y phase o f growth curves r e p r e s e n t s estimated c e l l number; a c c u r a t e counts were not p o s s i b l e due t o c e l l clumping. Arrows i n d i c a t e when samples were taken 32 F i g . 4. Growth curves (shown by c e l l numbers) o f the t h r e e a l g a l s p e c i e s grown i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d media. Arrows mark the time a t which the l i m i t i n g n u t r i e n t was exhausted from the medium. C. calcitrans and T. pseudonana were f e d t o o y s t e r l a r v a e at the 6 h s t a r v a t i o n p o i n t . T a h i t i a n Isochrysis (T-Iso) was f e d a t 2 d and 6 d s t a r v a t i o n 35 F i g . 5. C e l l u l a r weight o f gross b i o c h e m i c a l components i n T a h i t i a n Isochrysis sampled d u r i n g mid-log phase (ML), or a f t e r 2 or 6 d of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . Bars r e p r e s e n t mean ± standard e r r o r o f c e l l weight. See Appendix A, Tabl e 4 f o r standard e r r o r o f an a l y s e s o f d u p l i c a t e c u l t u r e s (a and b) 38 F i g . 6. C a l o r i c v a l u e o f T a h i t i a n Isochrysis sampled d u r i n g mid-log phase (ML), or a f t e r 2 or 6 d j X o f n i t r a t e s t a r v a t i o n (-N 2 d,-N 6 d ) ; A) c e l l u l a r energy e q u i v a l e n t s , B) pe r c e n t o f t o t a l c e l l energy i n gros s b i o c h e m i c a l components. See Appendix A, Tabl e 1 f o r standard d e v i a t i o n o f an a l y s e s o f d u p l i c a t e c u l t u r e s (a and b) 40 F i g . 7. C e l l u l a r weight o f gros s b i o c h e m i c a l components i n Chaetoceros calcitrans sampled d u r i n g mid-log phase (ML) or a f t e r 6 h o f n i t r a t e o r s i l i c a t e s t a r v a t i o n (-N, - S i ) . Bars r e p r e s e n t mean ± standard e r r o r o f c e l l w eight. See Appendix A, T a b l e 5 f o r standard e r r o r o f a n a l y s e s o f d u p l i c a t e c u l t u r e s (a and b) 43 F i g . 8. C a l o r i c v a l u e o f Chaetoceros calcitrans sampled d u r i n g mid-log phase (ML) or a f t e r 6 h o f n i t r a t e o r s i l i c a t e s t a r v a t i o n (-N, - S i ) ; A) c e l l u l a r energy e q u i v a l e n t s , B) perc e n t o f t o t a l c e l l energy i n gros s b i o c h e m i c a l components. See Appendix A, Ta b l e 2 f o r standard d e v i a t i o n o f a n a l y s e s o f d u p l i c a t e c u l t u r e s (a and b) 45 F i g . 9. C e l l u l a r weight o f gros s b i o c h e m i c a l components i n Thalassiosira pseudonana sampled d u r i n g mid-log phase (ML) or a f t e r 6 h o f n i t r a t e o r s i l i c a t e s t a r v a t i o n (-N, - S i ) . Bars r e p r e s e n t mean ± standard e r r o r of c e l l weight. See Appendix A, T a b l e 6 f o r standard e r r o r of an a l y s e s o f d u p l i c a t e c u l t u r e s (a and b) 48 F i g . 10. C a l o r i c v a l u e o f Thalassiosira pseudonana sampled d u r i n g mid-log phase (ML) or a f t e r 6 h o f n i t r a t e o r s i l i c a t e s t a r v a t i o n (-N, - S i ) ; A) c e l l u l a r energy e q u i v a l e n t s , B) pe r c e n t o f t o t a l c e l l energy i n gros s b i o c h e m i c a l components. See Appendix A, Ta b l e 3 f o r standard d e v i a t i o n o f a n a l y s e s o f d u p l i c a t e c u l t u r e s (a and b) 50 F i g . 11. F a t t y a c i d c l a s s p r o f i l e s o f T a h i t i a n Isochrysis, Thalassiosira pseudonana and Chaetoceros calcitrans c e l l s h a r v e s t e d d u r i n g the mid-log growth phase i n batch c u l t u r e : A) S e r i e s 1 B) S e r i e s 2 53 F i g . 12. Comparison o f s e r i e s 1 and s e r i e s 2 histograms o f f a t t y a c i d c o mposition o f T a h i t i a n Isochrysis h a r v e s t e d d u r i n g mid-log growth phase. The lower graph e n l a r g e s the s c a l e o f the s m a l l e r peaks o f the upper X I graph. E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n s e r i e s 1, n=3 i n s e r i e s 2) 55 F i g . 13. Comparison o f s e r i e s 1 and s e r i e s 2 histograms o f f a t t y a c i d composition o f Chaetoceros calcitrans h a r v e s t e d d u r i n g mid-l o g growth phase. The lower graph e n l a r g e s the s c a l e o f the s m a l l e r peaks o f the upper graph. E r r o r b a rs r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n s e r i e s 1, n=l i n s e r i e s 2) 57 F i g . 14. Comparison of s e r i e s 1 and s e r i e s 2 histograms o f f a t t y a c i d composition o f Thalassiosira pseudonana h a r v e s t e d d u r i n g mid-log growth phase. The lower graph e n l a r g e s the s c a l e o f the s m a l l e r peaks of the upper graph. E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=l i n s e r i e s 1, n=3 i n s e r i e s 2) 59 F i g . 15. Changes over time i n the f a t t y a c i d c l a s s p r o f i l e f o r T a h i t i a n Isochrysis i n s e r i e s 1 c u l t u r e (see F i g . l f o r growth curve and sampling t i m e ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium 63 F i g . 16. Changes over time i n the f a t t y a c i d c l a s s p r o f i l e f o r T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=3) 65 F i g . 17. Changes over time i n the major f a t t y a c i d s o f T a h i t i a n Isochrysis i n s e r i e s 1 c u l t u r e (see F i g . 1 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, and B) phosphate-l i m i t e d medium 68 F i g . 18. Changes over time i n the major f a t t y a c i d s o f T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and ha r v e s t e d a t mid-log growth phase (ML) or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r b a rs r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=3). 70 F i g . 19. Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f T a h i t i a n Isochrysis d u r i n g growth i n x i i s e r i e s 1 c u l t u r e (see F i g . 1 f o r growth curve and sampling t i m e ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium 72 F i g . 20. Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) o r a f t e r 2 or 6 days o f n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r b a r s r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=3) 74 F i g . 21. Changes over time i n the minor f a t t y a c i d s o f T a h i t i a n Isochrysis d u r i n g growth i n s e r i e s 1 c u l t u r e (see F i g . 1 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium 76 F i g . 22. Changes over time i n the minor f a t t y a c i d s o f T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r b a r s r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s of r e p l i c a t e c u l t u r e s (n=3) 78 F i g . 23. Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Chaetoceros calcitrans i n s e r i e s 1 c u l t u r e (see F i g . 2 f o r growth curve and sampling t i m e ) : A) n i t r a t e - l i m i t e d medium, B) p h o s p h a t e - l i m i t e d medium, and C) s i l i c a t e -l i m i t e d medium 81 F i g . 24. Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Chaetoceros calcitrans grown i n s e r i e s 2 c u l t u r e (see F i g . 4 f o r growth curve) i n A) n i t r a t e - l i m i t e d medium, or B) s i l i c a t e - l i m i t e d medium and h a r v e s t e d a t mid-l o g growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 o r 6 h, - S i 2 or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f analyses o f r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples) 83 F i g . 25. Changes over time i n the major f a t t y a c i d s o f Chaetoceros calcitrans grown i n s e r i e s 1 (See F i g . 2 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, B) phosphate-l i m i t e d medium, and C) s i l i c a t e - l i m i t e d medium 87 x i i i F i g . 26. Changes over time i n the major f a t t y a c i d s o f Chaetoceros calcitrans c u l t u r e d i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) o r a t 2 or 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h and - S i 2 h or 6 h ) . E r r o r b a r s r e p r e s e n t ± l standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples) 89 F i g . 27. Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f Chaetoceros calcitrans grown i n s e r i e s 1 c u l t u r e (see F i g . 2 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, B) p h o s p h a t e - l i m i t e d medium, and C) s i l i c a t e - l i m i t e d medium 91 F i g . 28. Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f Chaetoceros calcitrans c u l t u r e d i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h and S i - l i m 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples) 93 F i g . 29. Changes over time i n the minor f a t t y a c i d s o f Chaetoceros calcitrans grown i n s e r i e s 1 c u l t u r e (See F i g . 2 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, B) p h o s p h a t e - l i m i t e d medium, and C) s i l i c a t e -l i m i t e d medium 95 F i g . 30. Changes over time i n the minor f a t t y a c i d s o f Chaetoceros calcitrans c u l t u r e d i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 o r 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h and - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples) 97 F i g . 31. Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Thalassiosira pseudonana i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e - l i m i t e d medium 101 x i v F i g . 32. Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e (see F i g . 4 f o r growth curve) i n A) n i t r a t e - l i m i t e d medium, or B) s i l i c a t e - l i m i t e d medium and h a r v e s t e d a t mid-l o g growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples) 103 F i g . 33. Changes over time i n the major f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e - l i m i t e d medium. The S i 66 h sample was i n c l u d e d i n the phosphate-l i m i t e d p r o f i l e t o show the mid-log (ML) f a t t y a c i d l e v e l s . 106 F i g . 34. Changes over time i n the major f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium or s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML), or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h o r 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples) 108 F i g . 35. Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e - l i m i t e d medium. The S i 66 h sample was i n c l u d e d i n the phosphate-l i m i t e d p r o f i l e t o show the mid-log f a t t y a c i d l e v e l s 110 F i g . 36. Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium, or s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples) 112 X V F i g . 37. Changes over time i n the minor f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e - l i m i t e d medium. The S i 66 h sample was i n c l u d e d i n the phosphate-l i m i t e d p r o f i l e t o show the mid-log f a t t y a c i d l e v e l s 114 F i g . 38. Changes over time i n the minor f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium, o r s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and ha r v e s t e d a t mid-log growth phase (ML) o r a t 2 o r 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h o r 6 h, - S i 2 h o r 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples) 116 F i g . 39. Growth of Crassostrea gigas l a r v a e f e d T a h i t i a n Isochrysis h a r v e s t e d from n i t r a t e -l i m i t e d medium a t mid-log growth phase (ML), or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d or 6 d ) . A second mid-log treatment (ML-control) c o n t r o l l e d f o r the e f f e c t o f h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d c e l l s i n n i t r a t e - f r e e seawater. Starved l a r v a e d i d not r e c e i v e any a l g a l food d u r i n g the experiment. Least squares means o f l a r v a l l e n g t h were analysed by nested a n a l y s i s o f v a r i a n c e o f 3 r e p l i c a t e s ; standard e r r o r s a l l l i e w i t h i n the symbols except f o r the (-N 6 d) treatment (See Tabl e I I ) . A s t e r i s k s r e p r e s e n t s i g n i f i c a n t d i f f e r e n c e a t the 95% co n f i d e n c e l e v e l 120 F i g . 40. Growth ( s h e l l length) o f Crassostrea gigas l a r v a e f e d Chaetoceros calcitrans h a r v e s t e d from n i t r a t e - a n d s i l i c a t e - l i m i t e d medium a t mid-log phase (ML), or a f t e r 6 h of n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h ) . Two a d d i t i o n a l mid-log treatments (ML-N-control, ML-Si-c o n t r o l ) c o n t r o l l e d f o r the e f f e c t o f h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d or s i l i c a t e -s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e -f r e e o r s i l i c a t e - f r e e seawater, r e s p e c t i v e l y . S t a r v e d l a r v a e d i d not r e c e i v e any a l g a l food d u r i n g the experiment. L e a s t squares means of l a r v a l l e n g t h were analysed by nested a n a l y s i s o f v a r i a n c e of 3 r e p l i c a t e s ; s t andard e r r o r s a l l l i e w i t h i n the range o f the symbols (See Table I I I ) . A s t e r i s k s xv i represent s i g n i f i c a n t difference at the 95% confidence l e v e l 124 F i g . 41. Growth ( s h e l l length) of Crassostrea gigas larvae fed Thalassiosira pseudonana harvested from n i t r a t e - and s i l i c a t e - l i m i t e d medium at mid-log phase (ML) or a f t e r 6 h of nutrient starvation (-N 6 h, - S i 6 h). Two additional mid-log treatments (ML-N-control, ML-Si-control) controlled for the e f f e c t of holding larvae fed nitrate-starved or s i l i c a t e -starved c e l l s i n nutrient stripped, n i t r a t e -free or s i l i c a t e - f r e e seawater, respectively. Starved larvae did not recieve any a l g a l food during the experiment. Least squares means of l a r v a l length were analysed by nested analysis of variance of 3 r e p l i c a t e s ; standard errors a l l l i e within the range of the symbols (See Table IV). Asterisks represent s i g n i f i c a n t difference at the 95% confidence l e v e l 127 ACKNOWLE DGEMENTS Funding p r o v i d e d by the Department o f F i s h e r i e s and Oceans, Canada, i s g r a t e f u l l y acknowledged. A l l experimental work was performed a t the P a c i f i c B i o l o g i c a l S t a t i o n , where Dr. N e i l Bourne g r a c i o u s l y p r o v i d e d f a c i l i t i e s i n the s c a l l o p l a b . I am indebted t o Dr. Ian Whyte who was never too busy t o educate me i n the p r e c i s e t e c h n i q u e s o f chemical a n a l y s e s , and never s h o r t o f c o n v e r s a t i o n about b i v a l v e n u t r i t i o n . Dr. Paul J . H a r r i s o n opened up the world o f a l g a l p h y s i o l o g y f o r me. I t was a p r i v i l e g e t o be h i s s t u d e n t . Thanks are owed t o a l l members of my t h e s i s committee, and t o P e t e r Thompson, f o r s h a r i n g t h e i r e x p e r t i s e w i t h me through c r i t i c a l review o f my work. My ' p i l l a r ' and my f r i e n d , Mark Saunders, endured i t a l l w i t h g r e a t r e s o u r c e s o f humour and compassion. 1 INTRODUCTION The B.C. o y s t e r i n d u s t r y i s p r e s e n t l y worth about 3 m i l l i o n d o l l a r s a n n u a l l y , y e t i t s f u l l p o t e n t i a l i s f a r from b e i n g r e a l i z e d . P r o d u c t i o n i n 1985 was 3420 t whereas estimated p o t e n t i a l annual p r o d u c t i o n of the l e a s e d grounds should approximate 29,000 t (Quayle, 1988). The o y s t e r i n d u s t r y i s growing more dependent on hatchery produced seed f o r r e l i a b l e c u l t u r e o p e r a t i o n s , which i n t u r n has promoted an i n t e r e s t i n u n d e r s t a n d i n g the n u t r i t i o n of b i v a l v e l a r v a e . Three s p e c i e s of o y s t e r s have been h a r v e s t e d commercially on the west c o a s t of Canada and the U.S.A. The n a t i v e o y s t e r , Ostrea lurida, occurs from A l a s k a t o lower C a l i f o r n i a i n e s t u a r i e s and s a l t w a t e r lagoons but i t i s not found i n abundance i n B r i t i s h Columbia. P r o d u c t i o n of O. lurida i s v e r y l i m i t e d due t o i t s s m a l l s i z e , slow growth, h i g h m o r t a l i t y and s p e c i f i c t e c h n i c a l requirements (Quayle 1988). Crassostrea virginica, the n a t i v e e a s t c o a s t o y s t e r , was i n t r o d u c e d t o the west co a s t from the e a r l y 1900's t o about 1940 but few are l e f t . The f i r s t s i g n i f i c a n t i m p o r t a t i o n from Japan of the P a c i f i c o y s t e r Crassostrea gigas was i n 1926 (Quayle 1988). Areas of the S t r a i t of Georgia were p l a n t e d i n t e n s i v e l y and C. gigas has become the favoured s p e c i e s i n c u l t u r e p r a c t i c e s . Spawning o f C. gigas r e q u i r e s temperatures between 19 and 24°C. E x t e r n a l f e r t i l i z a t i o n marks the b e g i n n i n g o f the 2 p e l a g i c l i f e o f the l a r v a e . The l a r v a e q u i c k l y pass through t h e trochophore stage t o the v e l i g e r s t a g e , d e v e l o p i n g a r e t r a c t a b l e velum w i t h i n 24 hours. The c i l i a t e d velum p a r t i c i p a t e s s i m u l t a n e o u s l y i n swimming and f e e d i n g . Development o f t h e umbone a t the s h e l l h i n g e , t h e d i g e s t i v e system, and a c i l i a t e d f o o t preceeds the p e d i v e l i g e r s t a g e . S h o r t l y b e f o r e r e a c h i n g the maximum s h e l l l e n g t h o f 300 u.m, b l a c k eye spo t s appear, s i g n a l l i n g t h a t the l a r v a i s prepared f o r s e t t i n g . During s e t t l e m e n t , the l a r v a e seek a s u i t a b l e s u b s t r a t e f o r attachment. Once found, a cementing substance i s s e c r e t e d from a g l a n d a t the base of the f o o t and the l e f t v a l v e becomes permanently a t t a c h e d t o the s u b s t r a t e . The o y s t e r s then undergo metamorphosis, becoming j u v e n i l e s commonly known as s p a t . O y s t e r c u l t u r e o p e r a t i o n s r e q u i r e a r e l i a b l e supply o f seed. Seed i s g e n e r a l l y c o l l e c t e d from n a t u r a l s e t s on c u l t c h ( p o r t a b l e s u b s t r a t e ) and allowed t o grow i n p r o d u c t i v e a r e a s . However, s i n c e temperatures r e q u i r e d f o r spawning are h i g h r e l a t i v e t o B.C. waters, w i l d s e t s can be e r r a t i c . Seed o y s t e r s can now be obtained from h a t c h e r i e s on the west c o a s t o f North America, i n c l u d i n g B.C. Such p r a c t i c e s p r o v i d e a r e l i a b l e source o f seed on demand, a l l o w c o n t r o l l e d s e t s and p r o v i d e the p o t e n t i a l f o r g e n e t i c s e l e c t i o n . 3 The o p e r a t i o n o f an e f f i c i e n t o y s t e r or b i v a l v e h a tchery depends on p r o d u c t i o n of l i v e u n i c e l l u l a r a l g a l f o o d s , the most c o s t l y area of l a r v a l b i v a l v e p r o d u c t i o n (Urban and Langdon 1984). S u i t a b l e a l g a l s p e c i e s have been determined through a p r o c e s s of t r i a l and e r r o r but s t u d i e s o f n u t r i t i o n a l requirements have been l i m i t e d . A d e f i n e d a r t i f i c i a l d i e t i s r e q u i r e d t o determine s p e c i f i c r e q u i r e m e n t s . As y e t , a r t i f i c i a l d i e t s cannot comp l e t e l y r e p l a c e phytoplankton as a food source f o r b i v a l v e l a r v a e , a lthough some a r t i f i c i a l d i e t s have been developed which support l i m i t e d growth i n b i v a l v e a d u l t s ( C a s t e l l and T r i d e r 1974), j u v e n i l e s (Langdon and B o l t o n 1984, Langdon and S i e g f r i e d 1984, Parker and S e l i v o n c h i c k 1986) and l a r v a e (Gabbot e t a l . 197 6, Langdon and Waldock 1981, Chu e t a l . 1982, Langdon 1983). Chu e t a l . (1987) were a b l e t o r a i s e Crassostrea virginica l a r v a e as f a r as metamorphosis on an a r t i f i c i a l d i e t , but s u r v i v a l t o the eyed l a r v a l stage was poor and development lagged from 9 t o 14 days behind t h a t of l a r v a e f e d a l g a e . In the absence of a s u i t a b l e a r t i f i c i a l d i e t , c o s t e f f i c i e n c y can p o t e n t i a l l y be i n c r e a s e d by p r o v i d i n g l a r v a e w i t h p h y t o p l a n k t o n t h a t f u l f i l l l a r v a l n u t r i t i o n a l requirements f o r o p t i m a l growth. T h i s o b j e c t i v e can be met o n l y w i t h the knowledge of f i r s t , the e f f e c t s of phytoplankton chemical c o m p o s i t i o n on the growth and s u r v i v a l of l a r v a e , and secondly 4 the f a c t o r s a f f e c t i n g the chemical composition o f the a l g a l c e l l s . In a d d i t i o n , phytoplankton must meet c e l l s i z e and d i g e s t i b i l i t y requirements t o ensure e f f i c i e n t f i l t r a t i o n and a s s i m i l a t i o n (Romberger and E p i f a n i o 1981). C. virginica and Mytilus edulis v e l i g e r s s e l e c t phytoplankton l e s s than 10 u,m i n diameter ( R i i s g a r d e t a l . 1980, F r i t z e t a l . 1984, Sprung, 1984a). However not a l l c e l l s i n t h i s s i z e range are d i g e s t i b l e . Poor growth performance o f C. virginica was a t t r i b u t e d t o a l a c k o f l y s i s and d i g e s t i o n o f Chlorella autotrophica as assessed by a u t o f l u o r e s c e n c e t e c h n i q u e s (Babinchak and Ukeles 1979). 1. Importance o f L i p i d s i n B i v a l v e N u t r i t i o n S t u d i e s o f the chemical composition o f b i v a l v e l a r v a e have shown the importance o f the q u a l i t y and q u a n t i t y o f l i p i d s i n ph y t o p l a n k t o n . Although glycogen r e s e r v e s are important energy sources f o r a d u l t o y s t e r s , they have not been c o r r e l a t e d w i t h Ostrea edulis spat y i e l d ( C o l l y e r 1957). S t a r v e d O. edulis r e l y more upon energy from t r i g l y c e r i d e s than p r o t e i n and carbohydrate t o g e t h e r ( M i l l a r and S c o t t 1962). I n i t i a l growth r a t e o f newly r e l e a s e d O. edulis was p o s i t i v e l y c o r r e l a t e d w i t h l i p i d c ontent o f t h e l a r v a e upon r e l e a s e from the brood (Helm e t a l . 1973). O. edulis l a r v a e depend on n e u t r a l l i p i d s t o r e s b u i l t d u r i n g the l a r v a l stages t o p r o v i d e necessary energy f o r metamorphosis (Holland and Spencer 1973). T h i s phenomenon a l s o occurs i n l a r v a e of f o u r 5 g a s t r o p o d , Littorina s p e c i e s (Holland e t a l . 1975) and i n Patinopectin yessoensis s c a l l o p l a r v a e (Whyte e t a l . 1987). F a t t y a c i d s e s t e r i f i e d w i t h g l y c e r o l are the main b u i l d i n g b l o c k s o f l i p i d s . They are important components o f c e l l membranes, and they a c t as storage products f o r energy and s e r v e as m e t a b o l i c p r e c u r s o r s . Marine m i c r o a l g a e g e n e r a l l y c o n t a i n s t r a i g h t - c h a i n e d s a t u r a t e d and u n s a t u r a t e d f a t t y a c i d s w i t h even numbered carbon c h a i n s , although some odd numbered c h a i n s are known (Ackman e t a l . 1968, Chuecas and R i l e y 1969, Pohl and Zurheide 1979). L i p i d composition i s s p e c i e s s p e c i f i c and can v a r y even among c l o n e s of the same s p e c i e s of a l g a e ( F i s h e r and Schwarzenbach 1978). The n3 and n6 f a t t y a c i d s are e s s e n t i a l i n a l l animals because the carbon bonds cannot be d e s a t u r a t e d p r i o r t o the n9 p o s i t i o n ( H o l l a n d 1978). Marine organisms have a requirement f o r the l o n g c h a i n p o l y u n s a t u r a t e d f a t t y a c i d s 20:5n3 and 22:6n3 (Jones e t a l . 1979, Kanazawa e t a l . 1979, Levine and S u l k i n 1984). L a r v a l content of 20:5n3 and 22:6n3 i n c r e a s e s d u r i n g l a r v a l development of C. virginica (Chu and Webb 1984). Although some f i l t e r f e e d e r s can e f f i c i e n t l y extend 18:3n3 and r e l a t e d f a t t y a c i d s t o g i v e 20:5n3 (Ackman e t a l . 1968), the a b i l i t y o f j u v e n i l e Crassostrea gigas l a r v a e t o s y n t h e s i z e 20:5n3 and 22:6n3 from s h o r t e r chained a c i d s i s a p p a r e n t l y inadequate f o r optimum growth (Waldock and H o l l a n d 1984). An * F a t t y a c i d nomenclature used i n t h i s t h e s i s i s a:bnx where a i s the number o f carbons i n the c h a i n , b i s the number of double (unsaturated) bonds and x i s the p o s i t i o n of the f i r s t double bond from the methyl end. 6 a r t i f i c i a l d i e t r e p l a c i n g c orn o i l w i t h cod l i v e r o i l improved growth of C. virginica, t h e l a t t e r d i e t having a h i g h e r content o f l o n g chained p o l y u n s a t u r a t e d f a t t y a c i d s ( C a s t e l l and T r i d e r 1974). Langdon and Waldock (1981) demonstrated t h a t e n c a p s u l a t e d 22:6n3 improved C. gigas spat growth when i t was used t o supplement a d i e t d e f i c i e n t i n both 22:6n3 and 20:5n3. In a d d i t i o n t o the h i g h l y u n s a t u r a t e d f a t t y a c i d s , n e u t r a l hydrocarbons may be e s s e n t i a l l i p i d s t o marine organisms, f u n c t i o n i n g a t l e a s t i n p a r t as a n t i o x i d a n t s . (Ben-Amotz e t a l . 1987). N e v e r t h e l e s s , l a r v a l growth i s not n e c e s s a r i l y r e l a t e d t o d i e t a r y l i p i d c o n t e n t . S i z e o f C. gigas l a r v a e has been p o s i t i v e l y c o r r e l a t e d w i t h l a r v a l t r i a c y l g l y c e r o l c o n t e n t ; but n e i t h e r a l g a l l i p i d content nor f a t t y a c i d c o m p o s i t i o n of v a r i o u s a l g a l d i e t s c o u l d be c o r r e l a t e d w i t h l a r v a l growth (Waldock and Nascimento 1979). 2. V a r i a t i o n of Chemical Composition of A l g a l D i e t s N u t r i t i o n a l s t u d i e s u s i n g phytoplankton d i e t s must r e c o g n i z e the t r a n s i t o r y nature o f a l g a l chemical c o m p o s i t i o n . Demonstrated d i f f e r e n c e s i n a l g a l chemical composition have been a t t r i b u t e d t o c u l t u r e age (Ackman e t a l . 1968, Conover 1975, Chu and Dupuy 1980, P i o r r e c k and Pohl 1984, Fabregas e t a l . 1985 and 1986, Whyte 1987). C e l l s h a r v e s t e d from the s t a t i o n a r y growth phase y i e l d e d e i t h e r h i g h e r or lower l a r v a l growth than c e l l s h a r v e s t e d d u r i n g the e x p o n e n t i a l phase ( E p i f a n i o 1979, Cary e t a l . 1981). U n f o r t u n a t e l y , much of the 7 l i t e r a t u r e a s s e s s i n g the chemical composition of phytoplankton has f a i l e d t o i d e n t i f y the p h y s i o l o g i c a l s t a t e o f the c e l l or c u l t u r e c o n d i t i o n s a f f e c t i n g the c e l l s . Growth l i m i t i n g f a c t o r s must be d e f i n e d , o t h e r w i s e , comparative s t u d i e s become d i f f i c u l t t o i n t e r p r e t . S t r i n g e n t c o n t r o l of a l l c u l t u r e c o n d i t i o n s i s necessary f o r r e p r o d u c i b l e r e s u l t s . The r e l e v a n c e o f the g r o s s chemical composition o f a l g a e t o the n u t r i t i o n o f the g r a z e r i s not c l e a r . Growth of C. virginica and Mercenaria mercenaria j u v e n i l e s was not c o r r e l a t e d w i t h g r o s s chemical composition of d i e t s composed o f v a r i o u s combinations of 4 a l g a l s p e c i e s ( E p i f a n i o 1979). However, Wi k f o r s e t a l . (1984) concluded t h a t an i n c r e a s e i n carbohydrate t o g e t h e r w i t h a decrease i n p r o t e i n y i e l d e d the b e s t r e s u l t s i n j u v e n i l e Crassostrea virginica f e d a l g a e whose chemical c o m p o s i t i o n was m o d i f i e d by v a r y i n g n i t r o g e n t o phosphorus r a t i o s o f the phytoplankton c u l t u r e medium. Webb and Chu (1982) found h i g h p r o t e i n i n the a l g a l d i e t was r e l a t e d t o b e t t e r food f o r l a r v a e . Amino a c i d composition i s g e n e r a l l y c o n s i d e r e d i n c o n s e q u e n t i a l ( E n r i g h t e t a l . 1986a), alth o u g h p r o p o r t i o n s o f amino a c i d s i n a l g a e r e l a t i v e t o the p r o p o r t i o n r e q u i r e d i n the l a r v a l t i s s u e might be important (Webb and Chu 1982). D i e t a r y l i p i d c omposition might be more important than t h a t of p r o t e i n or carbohydrate t o the development of b i v a l v e l a r v a e . Among f i v e u n i a l g a l d i e t s f e d t o queen conch l a r v a e (Strombus gigas), o n l y the p o o r e s t d i e t had a v e r y low l i p i d content and 8 l a c k e d both 20:5n3 and 22:6n3 ( P i l l s b u r y 1985). However d i f f e r e n c e s i n growth among the good d i e t s was not a t t r i b u t a b l e t o l i p i d content nor the p r o p o r t i o n of 20:5n3 and 22:6n3. E p i f a n i o e t a l . (1981) noted t h a t j u v e n i l e C. virginica grew w e l l on a d i e t of Thalassiosira pseudonana (3H) and Phaeodactylum tricornutum both of which were l a c k i n g i n 22:6n3, but h i g h i n 20:5n3. E n r i g h t e t a l . (1986b) suggested t h a t a lower r a t i o o f s a t u r a t e d : ( n 6 + n3) was a b e t t e r f a t t y a c i d c o m p o s i t i o n d i e t f o r 0. edulis j u v e n i l e s . The l e v e l s o f 20:5n3, 22:6n3 and carbohydrate were c r i t i c a l . A d d i t i o n a l carbohydrate i n c r e a s e d growth as l o n g as the e s s e n t i a l amino a c i d and f a t t y a c i d content was a p p a r e n t l y adequate ( E n r i g h t e t a l . 1986b). R e p r o d u c i b i l i t y i n experiments employing phytoplankton d i e t s i s dependent upon c o n t r o l of f a c t o r s a f f e c t i n g growth, metabolism and chemical composition o f the a l g a e . Environmental f a c t o r s shown t o a f f e c t a l g a l chemical c o m p o s i t i o n i n c l u d e temperature (Opute 1974, R e d a l j e and Laws 1983, Teshima e t a l . 1983, Seto e t a l . 1984), l i g h t q u a l i t y (Opute 1974, Gostan and Lechuga-Deveze 1986) and l i g h t i n t e n s i t y (Orcutt and P a t t e r s o n 1973, S c o t t 1980, T e r r y e t a l . 1983, B r z e z i n s k i 1985, Thompson e t a l . , 1989) and n u t r i e n t c o n c e n t r a t i o n (See f o l l o w i n g s e c t i o n s f o r r e f e r e n c e s ) . 3_. E f f e c t o f N u t r i e n t L i m i t a t i o n on A l g a l Composition N i t r o g e n and phosphorus are important m i c r o n u t r i e n t s necessary f o r p hytoplankton growth. S i l i c o n i s a l s o necessary f o r the 9 growth o f diatoms. D e p l e t i o n o f these m i c r o n u t r i e n t s can a f f e c t phytoplankton chemical composition (Droop 1974). The c r i t i c a l r a t i o o f two n u t r i e n t s i n the a l g a l growth medium, i s equal t o the r a t i o o f an a l g a l c e l l ' s requirement f o r the two n u t r i e n t s . At t h i s r a t i o , both n u t r i e n t s are p o t e n t i a l l y l i m i t i n g t o phytoplankton growth (T e r r y 1980). On e i t h e r s i d e o f the c r i t i c a l r a t i o , phytoplankton growth i s r e g u l a t e d by environmental c o n d i t i o n s and by the c o n c e n t r a t i o n o f the s i n g l e n u t r i e n t l e a s t a v a i l a b l e , r e l a t i v e t o the c e l l s requirements (as lo n g as excess n u t r i e n t s are not a t t o x i c l e v e l s ) (Droop 1974, Rhee 1978, T e r r y e t a l . 1985). In b a t c h c u l t u r e , a f i n i t e supply o f n u t r i e n t s i s a v a i l a b l e . Phytoplankton grow e x p o n e n t i a l l y u n t i l l i g h t o r a n u t r i e n t becomes l i m i t i n g . In l i g h t - s a t u r a t e d c o n d i t i o n s , growth e n t e r s the s t a t i o n a r y phase when the l i m i t i n g n u t r i e n t i s exhausted i n the medium. Many of the s t a r v e d c e l l s e v e n t u a l l y d i e u n l e s s n u t r i e n t s are r e p l e n i s h e d . i . N i t r o g e n S t a r v a t i o n C e l l d i v i s i o n i n n i t r o g e n - d e f i c i e n t c o n d i t i o n s reduces a l g a l p r o t e i n c o n t e n t . I n t r a c e l l u l a r p o o l s o f amino a c i d s and n i t r a t e which accumulate under n i t r o g e n - r e p l e t e c o n d i t i o n s , are used t o c o n t i n u e c e l l growth when e x t e r n a l n i t r o g e n i n the c u l t u r e medium i s spent (Dortch 1982). The r e s u l t , which i s s p e c i e s s p e c i f i c ( S h i f r i n and Chisholm 1981), can be the accumulation o f non-nitrogenous compounds such as l i p i d (Opute 1974, E l - F o u l y e t a l . 1985, Wikfors 1986) or carbohydrate 10 (Myklestad and Haug 1972, Myklestad 1974, E l - F o u l y e t a l . 1985). A s h i f t from carbohydrate t o l i p i d s t o r a g e can occur i n advanced n i t r o g e n s t a r v a t i o n (Werner 1970). In some cases c e l l n i t r o g e n must f a l l t o v e r y low l e v e l s b e f o r e l i p i d s y n t h e s i s i s i n c r e a s e d (Richardson e t a l . 1969). The form of n i t r o g e n (e.g. n i t r a t e , ammonium or urea) a v a i l a b l e i n the growth medium can a l s o i n f l u e n c e l i p i d s t o r a g e (Conover 1975). S h i f r i n and Chisholm (1981) found t h a t n i t r o g e n - s t r e s s e d p h y t o p l a n k t o n c u l t u r e s produced new l i p i d mass per u n i t volume which was g r e a t e r than the t o t a l o r i g i n a l biomass a t the s t a r t o f n u t r i e n t d e p r i v a t i o n , thus c o n f i r m i n g a c t i v e l i p i d s y n t h e s i s . De novo s y n t h e s i s i s c o n s i d e r e d p r i m a r i l y r e s p o n s i b l e f o r i n c r e a s i n g n e u t r a l l i p i d s i n n i t r o g e n - s t r e s s e d c e l l s and c o n v e r s i o n of n o n - l i p i d s t o l i p i d s i s not a s i g n i f i c a n t f a c t o r i n l i p i d accumulation (Suen e t a l . 1987). There can be a marked d i f f e r e n c e i n f a t t y a c i d c omposition among l i p i d f r a c t i o n s (Otsuka and Marimura, 1966). Under n i t r o g e n s t r e s s , n e u t r a l l i p i d s i n c r e a s e and p o l y u n s a t u r a t e d f a t t y a c i d s decrease (Pohl and Zurheide 1982, P i o r r e c k e t a l . 1984, E l - F o u l y e t a l . 1985, P a r r i s h and Wangersky 1987). P h o s p h o l i p i d p r o d u c t i o n s u bsides due t o a requirement f o r the amino a c i d c y s t i d i n e ( P a r r i s h and Wangersky 1987). i i . S i l i c o n S t a r v a t i o n S i l i c o n i s s u p p l i e d i n the c u l t u r e medium as sodium m e t a s i l i c a t e (Na2Si03•9H20) which h y d r o l y z e s t o o r t h o s i l i c i c a c i d , S i ( O H )4. When s i l i c a t e i s exhausted from the medium, 11 the c e l l s pass through a p e r i o d d u r i n g which c e l l s complete c y t o k i n e s i s and i n i t i a t e c e l l w a l l f o r m a t i o n but f u r t h e r c e l l w a l l development i s a r r e s t e d as diatoms do not m a i n t a i n s u f f i c i e n t i n t r a c e l l u l a r p o o l s of s i l i c o n f o r new v a l v e f o r m a t i o n ( B r z e z i n s k i 1985). S i l i c a t e s t a r v a t i o n l e a d s t o c o n s i d e r a b l e change i n diatom metabolism. Not o n l y i s s i l i c a t e r e q u i r e d f o r the f o r m a t i o n o f the f r u s t u l e , but i t i s a l s o necessary i n DNA s y n t h e s i s and the p r o d u c t i o n o f the enzymes DNA polymerase and t h y m i d y l a t e k i n a s e ( V o l c a n i 1977, V a u l o t e t a l . 1987). S i l i c a t e d e f i c i e n c y has been shown t o i n c r e a s e the l i p i d c o n t ent of diatoms. In s i l i c a t e - s t a r v e d Cyclotella cryptica, p r o t e i n s y n t h e s i s was a r r e s t e d w i t h i n 4 hours f o l l o w e d c l o s e l y by a c e s s a t i o n i n pigment p r o d u c t i o n and a g r e a t e r than 100% i n c r e a s e i n f a t t y a c i d s y n t h e s i s (Werner 1977). R a t i o s of s a t u r a t e d and monounsaturated t o p o l y u n s a t u r a t e d f a t t y a c i d s i n c r e a s e d g r e a t l y as a r e s u l t of t r i a c y l g l y c e r o l accumulation i n C. cryptica ( R o e s s l e r 1988). Although t o t a l carbon f i x a t i o n d e c r e a s e s , l i p i d i s accumulated a t a g r e a t l y i n c r e a s e d r a t e due t o c o n v e r s i o n of n o n - 1 i p i d carbon i n t o l i p i d , and p a r t i t i o n i n g of newly a s s i m i l a t e d carbon i n t o i n c r e a s e d l i p i d s y n t h e s i s ( S h i f r i n and Chisholm 1981, Taguchi e t a l . 1987). Changes i n S i : C and Si:N r a t i o s were 2 t o 3 f o l d under severe s i l i c a t e l i m i t a t i o n i n t h r e e s p e c i e s of diatoms ( H a r r i s o n e t a l . 1977). Net s y n t h e s i s o f n u c l e i c a c i d s , p r o t e i n s , c a r b o h y d r a t e s , c h l o r o p h y l l s and f u c o x a n t h i n s i s reduced; however d i a d i n o x a n t h i n s y n t h e s i s c o n t i n u e s ( V o l c a n i 1977). i i i . Phosphate S t a r v a t i o n Energy r i c h phosphate bonds o f ATP are an i n t e g r a l p a r t o f c e l l u l a r energy t r a n s f e r through pr o c e s s e s o f photo-, s u b s t r a t e - , o r o x i d a t i v e p h o s p h o r y l a t i o n . D e p l e t i o n of phosphate reduces ATP l e v e l s , thereby i m p a i r i n g energy metabolism necessary f o r the f i x a t i o n o f carbon (Cembella e t a l . 1984a and b ) . I n t r a c e l l u l a r polyphosphate p o o l s harbour energy and phosphate r e s e r v e s which a l l o w c e l l s t o s u r v i v e s h o r t term e x t e r n a l phosphate d e f i c i e n c y ( K y l i n 1964). Scenedesmus sp. c e l l s i n p h o s p h a t e - d e f i c i e n t medium conti n u e d s y n t h e s i z i n g p r o t e i n a t r a t e s e q u i v a l e n t t o c e l l s i n p h o s p h a t e - s u f f i c i e n t medium f o r 2 days. R a d i o i s o t o p e l a b e l i n g ( P) has determined t h a t e x t e r n a l phosphate i s a d i r e c t phosphate donor f o r RNA, p h o s p h o l i p i d s and polyphosphates i n Chlorella. The l a t t e r a c t as i n d i r e c t phosphate donors f o r s y n t h e s i s of DNA and p r o t e i n (Miyachi and Tamiya 1961). Phosphate l i m i t a t i o n r e s u l t s i n decreases i n phosphate-m e t a b o l i t e s (Perry 1972, Sakshaug and Holm-Hansen 1977, and Holm and Armstrong 1981), RNA/DNA r a t i o s and p r o t e i n t o carbohydrate r a t i o s and boosts carbohydrate per u n i t biomass (Sakshaug and Myklestad 1973, Lehman 1976, Konopka and Schnur 1981, and Wynne and Rhee 1986). In y e a s t , t o t a l l i p i d s , s t e r o l s and t r i a c y l g l y c e r o l s decrease w h i l e p h o s p h o l i p i d s are v a r i o u s l y a f f e c t e d when they are phosphate-starved (Ramsay and Douglas 1979). 4. OBJECTIVES I t i s g e n e r a l l y r e c o g n i z e d t h a t combinations o f a l g a l s p e c i e s produce the b e s t l a r v a l growth r a t e s (Webb and Chu 1982, Romberger and E p i f a n i o 1981, E p i f a n i o 1979), however, i n such s t u d i e s i t i s d i f f i c u l t t o separate the e f f e c t s o f f a c t o r s such as a l g a l s i z e and d i g e s t i b i l i t y . Thus the employment of s i n g l e s p e c i e s d i e t s may b e t t e r serve t o i l l u m i n a t e the e f f e c t s o f a l g a l chemical composition which can be v a r i e d by a l t e r i n g growth c o n d i t i o n s . N u t r i t i o n o f j u v e n i l e b i v a l v e s has been e x p l o r e d u s i n g t h i s t e c h n i q u e . T i s s u e growth i n j u v e n i l e clams was c o r r e l a t e d t o n i t r o g e n content o f Thalassiosira pseudonana expressed as a v a r i a t i o n i n C:N r a t i o s ; a C:N r a t i o between 8.4 and 10.5 produced s u p e r i o r r e s u l t s ( G a l l a g e r and Mann 1981). Chaetoceros calcitrans c o m p o s i t i o n m o d i f i e d by n i t r a t e and s i l i c a t e l i m i t a t i o n a f f e c t e d growth o f O. edulis j u v e n i l e s ( E n r i g h t e t a l . 1986b). A h i g h e r content o f 22:6n3 was suggested as a p o s s i b l e reason f o r improved o y s t e r growth when they were f e d n u t r i e n t -s a t u r a t e d c e l l s r a t h e r than n u t r i e n t - l i m i t e d c e l l s ; g r o s s c o m p o s i t i o n c o u l d not supply a s a t i s f a c t o r y e x p l a n a t i o n . Growth responses o f b i v a l v e l a r v a e t o a l g a l d i e t s a re s p e c i e s s p e c i f i c , (Helm and L a i n g 1987, Cary e t a l . 1981) and requirements change wi t h the stage o f the l i f e c y c l e o f the b i v a l v e . T h e r e f o r e the m e r i t o f a l g a l d i e t s must be t e s t e d f o r each b i v a l v e s p e c i e s a t s p e c i f i c l i f e c y c l e s t a g e s . Most r e s e a r c h o f b i v a l v e n u t r i t i o n has c o n c e n t r a t e d on a d u l t and 14 j u v e n i l e requirements, and knowledge o f the n u t r i t i o n a l requirements o f b i v a l v e l a r v a e i s c l e a r l y l a c k i n g . Furthermore, the l a r v a l stage i s the most c r i t i c a l , i n t e n s i v e and c o s t l y phase of a s u c c e s s f u l b i v a l v e h a tchery o p e r a t i o n . T h i s study has endeavoured t o c o n t r i b u t e t o p r e s e n t i n f o r m a t i o n on l a r v a l o y s t e r n u t r i t i o n by meeting the f o l l o w i n g o b j e c t i v e s : 1. Grow i n ba t c h c u l t u r e and h a r v e s t a t v a r y i n g stages o f n i t r a t e , phosphate or s i l i c a t e s t a r v a t i o n , t h r e e m i c r o a l g a l s p e c i e s which are p r e s e n t l y r e c o g n i z e d as f a v o u r a b l e foods f o r b i v a l v e l a r v a e ; a f l a g e l l a t e and two diatoms, Isochrysis a f f . galbana ( T - I s o ) , Chaetoceros calcitrans and Thalassiosira pseudonana (3H), r e s p e c t i v e l y . 2. Assess the e f f e c t s o f n u t r i e n t s t a r v a t i o n on f a t t y a c i d and g r o s s chemical composition o f s i n g l e s p e c i e s d i e t s o f the t h r e e phytoplankton s p e c i e s . 3. Assess the r e s u l t a n t growth performance o f Crassostrea gigas l a r v a e f e d s i n g l e s p e c i e s d i e t s o f the t h r e e phytoplankton s p e c i e s i n which the chemical composition has been m o d i f i e d by n i t r a t e or s i l i c a t e l i m i t a t i o n . Crassostrea gigas was s e l e c t e d f o r the b i o a s s a y s because i t i s r e a d i l y a v a i l a b l e from l o c a l h a t c h e r i e s most of the year and i s a r e l a t i v e l y hardy c u l t u r e organism. 15 T h i s i s the f i r s t r e p o r t o f the e f f e c t s o f w e l l d e f i n e d n u t r i e n t l i m i t a t i o n o f a l g a l c u l t u r e s on the growth o f Crassostrea gigas l a r v a e . S t r i c t c o n t r o l o f the growth l i m i t i n g f a c t o r s o f the algae c u l t u r e s p r o v i d e s new i n f o r m a t i o n on the e f f e c t s o f these f a c t o r s on the f a t t y a c i d and g r o s s b i o c h e m i c a l composition of the a l g a e . 16 MATERIALS AND METHODS 1. A l g a l C u l t u r e s U n i a l g a l c u l t u r e s o f Isochrysis galbana (clone T-ISO) Green ( h e n c e f o r t h r e f e r r e d t o as T a h i t i a n Isochrysis), Chaetoceros calcitrans (Paulsen) Tokano and Thalassiosira pseudonana (clone 3H) (Hust.) Hasle and Heimdal were pro c u r e d from the N o rtheast P a c i f i c C u l t u r e C o l l e c t i o n , Department o f Oceanography, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C., Canada. A l l c u l t u r e s were maintained i n the e x p o n e n t i a l phase o f growth a t a temperature of 21 ± 1.5°C. Two s e r i e s of c u l t u r e s were grown; the f i r s t i n 20 L p o l y c a r b o n a t e carboys c o n t a i n i n g 18 L of c u l t u r e , and the second i n 6 L Erlenmeyer f l a s k s c o n t a i n i n g 3.5 L o f c u l t u r e . Continuous i l l u m i n a t i o n was s u p p l i e d from two s i d e s w i t h c o o l -white f l u o r e s c e n t t u b e s . I r r a d i a n c e a t the v e s s e l s u r f a c e was 300 |iE-m -s -1- measured wi t h a L i - c o r l i g h t meter w i t h a c o s i n e c o l l e c t o r . Batch c u l t u r e s were bubbled w i t h f i l t e r e d (1 |im) a i r and sparged w i t h pure carbon d i o x i d e every 0.5 h f o r 2 min. F u r t h e r s t i r r i n g was a p p l i e d by s w i r l i n g the c u l t u r e s when growth parameters were monitored. C u l t u r e pH remained between 8.0 and 8.3. C u l t u r e medium was f i l t e r s t e r i l i z e d (0.45 |im) n u t r i e n t e n r i c h e d n a t u r a l seawater from Departure Bay, Nanaimo, B.C. The n u t r i e n t enrichment was based on the formula f o r e n r i c h e d n a t u r a l seawater (ESNW) used by H a r r i s o n e t a l . (1980), p l u s 10~ 9 M sodium s e l e n i t e ( P r i c e e t a l . , 1987) . FeNH4(S04)2•6H20 was r e p l a c e d w i t h an equimolar amount of F e S 04» 7 H20 , and sodium glycerophosphate was r e p l a c e d w i t h N a2H2P 04« H20 . N u t r i e n t r a t i o s were m o d i f i e d as i n Tab l e I t o ensure l i m i t a t i o n o f a s p e c i f i c n u t r i e n t . A l l phytoplankton were grown i n n i t r a t e - l i m i t e d or p h o s p h a t e - l i m i t e d medium. C. calcitrans and T. pseudonana were a l s o c u l t u r e d i n s i l i c a t e -l i m i t e d medium. i . S e r i e s 1; The f i r s t s e r i e s o f a l g a l c u l t u r e s were grown f o r f a t t y a c i d a n a l y s e s o n l y and were not f e d t o o y s t e r s i n d i e t t r i a l s . F i l t e r - s t e r i l i z e d (0 .45 urn) n u t r i e n t - e n r i c h e d seawater c o n t a i n e d i n 20 L pol y c a r b o n a t e carboys was i n o c u l a t e d w i t h s t o c k c u l t u r e t o a d e n s i t y o f 2 0,000 c e l l s - m L- 1. From each carboy, 3 L of c u l t u r e were taken f o r f a t t y a c i d a n a l y s i s a t i n t e r v a l s , s t a r t i n g a t m i d - l o g a r i t h m i c growth phase and c o n t i n u i n g u n t i l the c u l t u r e was senescent. B a c t e r i a , which were monitored u s i n g a p h a s e - c o n t r a s t microscope, were not abundant u n t i l a l g a l senescence was reached. i i . S e r i e s 2: S i n c e s e r i e s 1 c u l t u r e s were too l a r g e t o a v o i d the e f f e c t s o f l i g h t l i m i t a t i o n , a second s e r i e s was grown f o r d i e t t r i a l s i n s m a l l e r l i g h t - s a t u r a t e d c u l t u r e s . Three l i t e r s o f medium e n r i c h e d w i t h n u t r i e n t s as i n s e r i e s 1 and T a b l e I were c o n t a i n e d i n 6 L Erlenmeyer f l a s k s . A s e r i e s o f c u l t u r e s was i n o c u l a t e d d a i l y t o ensure a uniform d a i l y 18 Table I: Concentrations of l i m i t i n g nutrients used in nutrient enrichment solutions of various a l g a l growth media used in series 1 and series 2 cultures of Tahitian Isochrysis, Chaetoceros calcitrans and Thalassiosira pseudonana. A second addition of s i l i c a t e (in brackets) was added during log phase growth. ( S i l i c a t e was not added to Tahitian Isochrysis growth medium.) N u t r i e n t Concentration ()1H) Series Treatment NaH03 Na2*H2P04>H20 Ha2Si03»9B20 1 P - l i i i t e d 5490 9.9 528 (+276) H - l i a i t e d 549 39 528 (*276) S i - l i a i t e d 2196 39 106 2 H - l i n i t e d 275 20 528 S i - U n i t e d 2196 20 106 19 supply o f n u t r i e n t - d e f i c i e n t algae t o feed t o the o y s t e r l a r v a e . T a h i t i a n Isochrysis was h a r v e s t e d a t mid-log phase and a t 2 d and 6 d o f n i t r a t e s t a r v a t i o n , t o feed t o l a r v a e and f o r chemical composition a n a l y s e s . An i n t e r m e d i a t e sample a t 4 d s t a r v a t i o n was a l s o taken f o r chemical a n a l y s e s . P r e l i m i n a r y experiments w i t h n u t r i e n t - s t a r v e d diatoms e s t a b l i s h e d t h a t they c o u l d not be h e l d as long as Isochrysis b e f o r e h a r v e s t i n g . A f t e r 6 h of n u t r i e n t s t a r v a t i o n the c e l l s clumped, c r e a t i n g a food p a r t i c l e t h a t was too l a r g e f o r o y s t e r l a r v a e t o i n g e s t and making a c c u r a t e c e l l counts of the c e l l s i n the clumps i m p o s s i b l e . Thus, diatoms i n s e r i e s 2 were h a r v e s t e d a f t e r 2 and 6 h of n i t r a t e or s i l i c a t e s t a r v a t i o n f o r chemical a n a l y s e s . Only the 6 h s t a r v e d , and mid-log c e l l s were assessed as o y s t e r l a r v a e d i e t s . i i i . A l g a l A n a l y s e s : Samples were drawn from c u l t u r e s through a permanently f i x e d g l a s s tube w i t h a s t e r i l e s y r i n g e and n i t r a t e , phosphate and s i l i c a t e were measured twice a day u n t i l c o n c e n t r a t i o n s became low, and then measurements were made h o u r l y ( S t r i c k l a n d and Parsons 1972). Samples f o r c e l l counts were d i l u t e d w i t h a 3% sodium c h l o r i d e s o l u t i o n and determined u s i n g a Model ZB C o u l t e r Counter equipped w i t h a 75 yim a p e r t u r e . In vivo f l u o r e s c e n c e was measured w i t h a Turner 112 f l u o r o m e t e r . 20 2. L a r v a l C u l t u r e O y s t e r l a r v a e s u p p l i e d by Baynes Sound Oy s t e r Co., Union Bay, B.C., were shipped a t low temperatures (~10°C) t o the P a c i f i c B i o l o g i c a l S t a t i o n . Larvae were resuspended i n a bucket o f 10°C seawater f i l t e r e d t o 1 urn and passed through a UV s t e r i l i z e r . Bucket temperature was allowed t o i n c r e a s e over the day t o the experimental temperature ( 2 6°C) . L a r v a l c o n c e n t r a t i o n was estimated i n the bucket by counts o f s i x , 1 mL samples p a s s i v e l y drawn i n t o a p i p e t t e from water a g i t a t e d by a p e r f o r a t e d p l u n g e r . Equal a l i q u o t s o f 5000 t o 6000 l a r v a e were then d i s t r i b u t e d among 4 L g l a s s beakers c o n t a i n i n g 3.5 L of seawater i n a temperature c o n t r o l l e d water bath ( 2 6°C) . Seawater used i n l a r v a l c u l t u r e s was t r e a t e d t o d e p l e t e the n u t r i e n t s which were l i m i t i n g i n a l g a l d i e t s . The b u l l k e l p Nereocystis luetkeana and the diatom Ditylum brightwellii (West) Grun. were used t o s t r i p seawater o f n i t r a t e and s i l i c a t e r e s p e c t i v e l y . F o l l o w i n g 1 \im f i l t r a t i o n and c h a r c o a l t r e a t m e n t , n i t r a t e - s t r i p p e d seawater was f i l t e r e d through 0.5 (Jim p o l y c a r b o n a t e Nuclepore membrane f i l t e r s and s i l i c a t e -s t r i p p e d seawater was f i l t e r s t e r i l i z e d through 0.45 u.m M i l l i p o r e f i l t e r s . A c o n t r o l treatment o f s t a r v e d (unfed) l a r v a e , otherwise t r e a t e d i d e n t i c a l l y t o the remainder o f l a r v a l c u l t u r e t r e a t m e n t s , was i n c l u d e d i n each experiment. S t a r v e d l a r v a e and those f e d mid-log phase phytoplankton were grown i n u n s t r i p p e d , 1 |xm f i l t e r e d , UV s t e r i l i s e d n a t u r a l seawater. A d d i t i o n a l mid-log f e d l a r v a l c u l t u r e s were grown i n n u t r i e n t -s t r i p p e d seawater t o c o n t r o l f o r p o s s i b l e s i d e e f f e c t s o f s t r i p p i n g n u t r i e n t s from the seawater. Larvae underwent a complete water exchange d a i l y . The v a r i o u s a l g a l d i e t s ( i . e . d i f f e r e n t n u t r i e n t t r e a t m e n t s , h a r v e s t e d at d i f f e r e n t a l g a l growth phases) were f e d t o the l a r v a e a t a c o n c e n t r a t i o n o f 2 0 , 0 0 0 c e l l s mL- 1. Every few days l a r v a l l e n g t h was measured t o the ne a r e s t 5 urn under a d i s s e c t i n g microscope equipped w i t h a micrometer. Approximate s u r v i v a l e s t i m a t e s were made from l i v e and dead counts i n a random sample o f g r e a t e r than 100 i n d i v i d u a l s . A l l experiments were c a r r i e d out i n t r i p l i c a t e . Length data were analys e d by nested a n a l y s i s o f v a r i a n c e f o r s i g n i f i c a n t d i f f e r e n c e s among d i e t s a t each sample d a t e . 3. Chemical A n a l y s i s Weights f o r a l l a n a l y s e s were measured on an u l t r a m i c r o b a l a n c e t o ± 1 u,g u n l e s s otherwise s t a t e d . Phytoplankton h a r v e s t e d f o r chemical a n a l y s i s were c o n c e n t r a t e d by c e n t r i f u g a t i o n i n 250 mL b o t t l e s a t 2000 rpm. P e l l e t s were combined and c e n t r i f u g e d a g a i n i n 10 mL tubes at 4000 rpm i n a desk-top c e n t r i f u g e . Supernatant was d i s c a r d e d and the p e l l e t was f r o z e n a t -80°C b e f o r e b e i n g f r e e z e - d r i e d . F r e e z e - d r i e d samples were packaged under n i t r o g e n and vacuum-s e a l e d i n oxygen b a r r i e r bags. C e l l s were c o l l e c t e d from 22 known volumes o f a l g a l samples of known c e l l c o n c e n t r a t i o n onto pre-washed, pre-combusted (500°C) and pre-weighed 25 mm GF/A f i l t e r s by g e n t l e vacuum f i l t r a t i o n and r i n s e d w i t h 3.4% ammonium formate t o remove any sea s a l t s . Loaded f i l t e r s were f r e e z e - d r i e d i n weighed f o i l packages (± 10 p.g) t o determine c e l l u l a r d r y weight. i . F a t t y A c i d A n a l y s i s : F a t t y a c i d methyl e s t e r s (FAME) were prepared from 20 t o 50 mg o f f r e e z e - d r i e d samples employing the method developed by Whyte (1988). Methyl e s t e r s were prepared i n 5 mL R e a c t i - V i a l s capped w i t h M i n i n e r t v a l v e s ( P i e r c e C o . ) . U n s a p o n i f i a b l e s were removed i n hexane a f t e r treatment w i t h 0.5 M methanolic potassium h y d r o x i d e a t 85°C. Boron t r i f l u o r i d e i n methanol (12%) was used f o r t r a n s e s t e r i f i c a t i o n . FAME were d i s s o l v e d i n hexane and washed w i t h s a t u r a t e d aqueous sodium c h l o r i d e and s a t u r a t e d aqueous sodium b i c a r b o n a t e . The hexane s o l u t i o n was evaporated t o dryness i n a vacuum oven ( 5 0°C), f l u s h e d w i t h n i t r o g e n and capped w i t h a t e f l o n - l i n e d septum. Weight o f o r g a n i c m a t e r i a l was determined and d i s s o l v e d i n e t h y l a c e t a t e t o a c o n c e n t r a t i o n o f 10 iJ.g--u.L~1. A Hewlett Packard, model 5890 g a s - l i q u i d chromatograph equipped w i t h a flame i o n i z a t i o n d e t e c t o r and HP 3393A I n t e g r a t o r was used t o analyze 2 u.L of FAME i n j e c t e d onto a Supelcowax-10 fused s i l i c a c a p i l l a r y column (30m x 0.32 mm ID, 0.25 \im f i l m ) heated t o 190°C and programmed a f t e r 32 min t o r i s e 2° C - m i n ~1 t o a f i n a l temperature of 240°C f o r 16 min. 23 C a r r i e r gas was e x t r a dry h e l i u m , passed through a heated gas p u r i f i e r , w i t h a flow r a t e of 20 m L m i n "1 and a s p l i t r a t i o o f 100:1. F a t t y a c i d s were i d e n t i f i e d a g a i n s t a standard of PUFA-1 methyl e s t e r s (Supelco Inc.) and a n a l y s e s were a c c o r d i n g t o Ackman (1986). i i . Gross Composition Analyses: A n a l y t i c a l schemes f o l l o w e d those o f Whyte e t a l . (1987). F r e e z e - d r i e d samples (4 mg) were e x t r a c t e d i n c h l o r o f o r m , methanol and water (2:4:1, v/v, 5 mL). A l g a l c e l l s were s o n i c a t e d a t 30 W i n an i c e bath and spun i n a desk-top c e n t r i f u g e a t 4000 rpm f o r 2 min. The supernatant was passed through a GF/C f i l t e r and the r e s i d u e was e x t r a c t e d t w i c e more. F i l t e r e d supernatant was separated t w i c e w i t h the a d d i t i o n of 50 mL o f 1:1 c h l o r o f o r m and water (v/v) t o mimic B l i g h and Dyer (1959) e x t r a c t i o n . The c h l o r o f o r m l a y e r was evaporated t o dryness on a r o t a r y e v a p o r a t o r . Dryness was assured by two s e q u e n t i a l e v a p o r a t i o n s o f c h l o r o f o r m . The l i p i d f r a c t i o n was p i c k e d up i n c h l o r o f o r m and d i l u t e d i n a v o l u m e t r i c f l a s k t o an a p p r o p r i a t e volume f o r q u a n t i t a t i v e a n a l y s i s (5 t o 25 mL). The Marsh and W e i n s t e i n (1966) s u l p h u r i c a c i d c h a r r i n g method was used t o determine t o t a l l i p i d a g a i n s t a t r i p a l m i t i n s t a n d a r d . Chloroform s o l u t i o n s were evaporated t o dryness i n a vacuum oven a t 50°C b e f o r e b e i n g c h a r r e d w i t h s u l p h u r i c a c i d . The same round-bottom f l a s k was used t o evaporate the aqueous methanol l a y e r t o r e t a i n any h y d r o p h i l i c components t h a t may have been t r a n s f e r r e d i n the c h l o r o f o r m l a y e r from the s e p a r a t o r y f u n n e l . The c o n c e n t r a t e d aqueous methanol f r a c t i o n was d i l u t e d i n water t o 5 mL i n a v o l u m e t r i c f l a s k f o r d e t e r m i n a t i o n o f t o t a l monosaccharides and o l i g o s a c c h a r i d e s . The i n i t i a l e x t r a c t i o n r e s i d u e and f i l t e r were h y d r o l y s e d i n 2 mL o f 1 M s u l p h u r i c a c i d i n a s e a l e d tube a t 100°C f o r 2 h, p u l l e d through a GF/C f i l t e r and d i l u t e d w i t h water t o 25 or 50 mL i n a v o l u m e t r i c f l a s k f o r a n a l y s i s of p o l y s a c c h a r i d e s . Sugars were measured u s i n g the p h e n o l / s u l p h u r i c a c i d reagent of Dubois e t a l . (1956). D-glucose was the c a l i b r a t i o n s t a n d a r d . Another sample o f dry a l g a e (4-20 mg) was used f o r p r o t e i n d e t e r m i n a t i o n . C e l l s i n 2 mL of water were s o n i c a t e d a t 30 W i n an i c e b a t h . P r o t e i n was p r e c i p i t a t e d w i t h the a d d i t i o n of 2 mL o f 10% (w/v) t r i c h l o r o a c e t i c a c i d . The s o l u t i o n was mixed on a V o r t e x , h e l d i n an i c e bath f o r 4 min and c e n t r i f u g e d i n a desk-top model at 4000 rpm f o r 8 min. The r e s i d u e was d i s s o l v e d by adding 5 mL of 1 M NaOH and h e a t i n g t o 50°C f o r a t l e a s t 1 h. The s o l u t i o n was d i l u t e d w i t h water t o 25 mL i n a v o l u m e t r i c f l a s k and a n a l y s e d f o r t o t a l p r o t e i n u s i n g a m o d i f i e d Lowry method ( H a r t r e e , 1972). Bovine serum albumin was used as the c a l i b r a t i o n s t a n d a r d . Ash c o ntent was assessed by b u r n i n g dry a l g a l samples weighed on pre-washed and pre-combusted (500°C) GF/C f i l t e r s a t 500°C f o r 24 h. 25 D u p l i c a t e samples o f each a l g a l treatment were a n a l y s e d t w i c e . Mean c a l o r i c v a l u e was c a l c u l a t e d u s i n g f a c t o r s o f 8.42, 4.1 and 4.3 k c a l g- 1 f o r l i p i d , carbohydrate and p r o t e i n r e s p e c t i v e l y (Beukema and DeBruin 1979). RESULTS I . A l g a l Growth i n S e r i e s 1 and S e r i e s 2 C u l t u r e s Two s e r i e s o f a l g a l c u l t u r e s were a n a l y s e d . The f i r s t s e r i e s was grown i n l a r g e c o n t a i n e r s which r e s u l t e d i n l i g h t l i m i t a t i o n . The chemical composition o f s e r i e s 1 i s presented f o r comparative purposes, but t h i s s e r i e s was not used i n o y s t e r l a r v a e f e e d i n g t r i a l s . S e r i e s 2 was grown under l i g h t s a t u r a t e d c o n d i t i o n s i n s m a l l e r c u l t u r e v e s s e l s . 1. S e r i e s 1 P l o t s o f S e r i e s 1 T a h i t i a n Isochrysis c e l l d e n s i t y and n u t r i e n t c o n c e n t r a t i o n i n the n i t r a t e - l i m i t e d c u l t u r e , show t h a t the s t a t i o n a r y phase began a t about 13 0 h, which was p r i o r t o n i t r a t e l i m i t a t i o n ( F i g . 1 ) . Thus, l i g h t l i m i t a t i o n p r i m a r i l y a f f e c t e d the 185 h sample; and a f t e r 185 h the c u l t u r e was both l i g h t - and n i t r a t e - l i m i t e d . A l l s t a t i o n a r y phase samples from the p h o s p h a t e - l i m i t e d c u l t u r e (185 h and afterwards) were phosphate-starved; however, they were a l s o l i g h t - l i m i t e d due t o the l a r g e volume of the c u l t u r e . C. calcitrans c e l l s began t o clump t o g e t h e r w i t h the onset of l i g h t l i m i t a t i o n and ensuing s t a t i o n a r y growth. S t a t i o n a r y phase growth o c c u r r e d a t about 90 h, p r i o r t o both n i t r a t e and s i l i c a t e d e p l e t i o n i n the r e s p e c t i v e c u l t u r e s ( F i g . 2 ) . N i t r a t e or s i l i c a t e d e p l e t i o n o c c u r r e d i n the sample taken a t 119 h. Although l i g h t l i m i t a t i o n d i d not become apparent b e f o r e phosphate was d e p l e t e d , i t must be assumed t h a t i n a l l 27 s e r i e s 1 treatments n u t r i e n t d e p l e t i o n c o i n c i d e d w i t h l i g h t l i m i t a t i o n . T. pseudonana c e l l s a l s o clumped a t h i g h e r c e l l d e n s i t i e s . T h e r e f o r e C o u l t e r Counter c e l l counts were u n r e l i a b l e and without f l u o r e s c e n c e data the f i n a l stages o f the growth curve c o u l d not be f i t t e d . However, s i n c e the l a r g e s t changes i n f a t t y a c i d c omposition o c c u r r e d by 92 h (to be d i s c u s s e d l a t e r ) l i g h t l i m i t a t i o n i s assumed t o have a l s o a f f e c t e d a l l s e r i e s 1 T. pseudonana c u l t u r e s . A l l samples from the p h o s p h a t e - l i m i t e d c u l t u r e from 108 h and l a t e r were phosphate-s t a r v e d ( F i g . 3 ) . The c u l t u r e c o n t i n u e d f o r 5.5 days i n the phosphate s t a r v a t i o n phase. S i l i c a t e was not d e p l e t e d from the s i l i c a t e - l i m i t e d c u l t u r e u n t i l 92 h. I t aged r a p i d l y , t u r n e d p a l e and sank when the sample was taken a t 163 h. 2. S e r i e s 2 H i g h - l i g h t c u l t u r e s o f s m a l l e r volume were grown i n the second s e r i e s f o r l a r v a l o y s t e r f e e d i n g t r i a l s . Samples were taken b e f o r e c e l l clumping took p l a c e , a t mid-log phase and 2 and 6 h a f t e r the l i m i t i n g n u t r i e n t , n i t r a t e o r s i l i c a t e , c o u l d no l o n g e r be d e t e c t e d i n the c u l t u r e medium. Average growth curves are d e p i c t e d i n F i g . 4. 28 F i g u r e 1: Growth curves (shown by c e l l numbers) of T a h i t i a n Isochrysis grown i n s e r i e s 1 phosphate- and n i t r a t e - l i m i t e d media, showing the c o n c e n t r a t i o n of the l i m i t i n g n u t r i e n t s i n the media (e.g. phosphate c o n c e n t r a t i o n i n the p h o s p h a t e - l i m i t e d medium). Arrows i n d i c a t e when samples were ta k e n . Tahitian Isochrysis Ser ies 1 Mi 11 ion c e l l s / m L M e d i u m N u t r i e n t C o n c e n t r a t i o n (uM) 0.01 0 0-t I 3 3 0 h 3 5 5 h 25Qh ( N 0 3 o n l y ) ( P 0 4 o n l y ) 4 0 0 h 4 5 0 h P 0 4 - l i m c e l l no. N 0 3 - l i m c e l l no, uM P 0 4 ( P 0 4 - l i m ) uM N 0 3 ( N 0 3 - l i m ) 600 500 400 300 200 100 0 100 200 300 Time (h) 400 500 30 F i g u r e 2: Growth curves (shown by c e l l numbers or in vivo f l u o r e s c e n c e ) o f Chaetoceros calcitrans grown i n s e r i e s 1 n i t r a t e - , phosphate- and s i l i c a t e - l i m i t e d media, showing the c o n c e n t r a t i o n o f the l i m i t i n g n u t r i e n t s i n the media. ( N i t r a t e - l i m i t e d f l u o r e s c e n c e was omitted s i n c e i t f o l l o w s same curve as othe r treatments.) Arrows i n d i c a t e when samples were ta k e n . Chaetoceros calcitrans Ser ies 1 100 M i l l i o n c e l l s /mL or f l u o r e s c e n c e (re l , u n i t s ) M e d i u m n u t r i e n t c o n c e n t r a t i o n 150h 174h 0 50 100 Time (h) 150 H 500 -P 0 4 - l i m f lu - x - S i 0 4 - l i m f lu a P 0 4 - l i m ce l l no, o N 0 3 - l i m ce l l no, x S i 0 4 - l i m ce l l no. — — -B uM P 0 4 ( P 0 4 - l i m ) - 0 - uM N 0 3 ( N 0 3 - l i m ) -- X - uM S i 0 4 ( S i 0 4 - l i m ) 200 32 F i g u r e 3: Growth curves (shown by c e l l numbers) of Thalassiosira pseudonana grown i n s e r i e s 1 phosphate- and s i l i c a t e - l i m i t e d media, showing the c o n c e n t r a t i o n o f the l i m i t i n g n u t r i e n t s i n the media. Dashed l i n e i n s t a t i o n a r y phase o f growth curves r e p r e s e n t s estimated c e l l number; a c c u r a t e counts were not p o s s i b l e due t o c e l l clumping. Arrows i n d i c a t e when samples were t a k e n . Thalassiosira pseudonana Ser ies 1 M i l l i o n c e l l s / m L M e d i u m N u t r i e n t C o n c e n t r a t i o n (uM) 0 50 100 150 200 250 Time (h) OO OO 34 F i g u r e 4: Growth curves (shown by c e l l numbers) o f the t h r e e a l g a l s p e c i e s grown i n s e r i e s 2 n i t r a t e - and s i l i c a t e -l i m i t e d media. Arrows mark the time a t which the l i m i t i n g n u t r i e n t was exhausted from the medium. C. calcitrans and T. pseudonana were f e d t o o y s t e r l a r v a e a t the 6 h s t a r v a t i o n p o i n t . T a h i t i a n Isochrysis (T-Iso) was f e d a t 2 d and 6 d s t a r v a t i o n . 100 Series 2 Algal Growth Curves Mill ion cel ls /mL 0.01 Nitrate-starved A. Nitrate-l imited medium Ni trate-starved 50 — T. pseudonana — C. Calc i trans • T - so 100 150 T i m e (h) 200 250 100 Mill ion cel ls /mL 0.1 | ; 0.01 Si starvation B. Si l icate- l imited medium — - . T. pseudonana — C. calc i trans 50 •100 150 T i m e (h) 200 250 36 I I . B iochemical Composition of Algae A. Gross Biochemical Composition o f Algae No s i g n i f i c a n t d i f f e r e n c e s i n a l g a l c e l l mass among the c u l t u r e treatments were d e t e c t e d i n each of the t h r e e p h y t o p l a n k t o n s p e c i e s . C e l l weights were t h e r e f o r e pooled t o g i v e a f i n a l mean weight common t o a l l treatments w i t h i n each s p e c i e s . Recovery of o r g a n i c s by experimental procedure was a f f e c t e d by the c o l o u r i m e t r i c t e s t s chosen f o r a n a l y s i s . P r o t e i n , l i p i d and carbohydrate i n the a l g a l samples were c a l c u l a t e d a g a i n s t standards o f bovine serum albumin, t r i p a l m i t i n , and g l u c o s e e q u i v a l e n t s and do not n e c e s s a r i l y r e f l e c t the a c t u a l weight of the g r o s s components. F i g u r e s 5, 7 and 9 i n c l u d e c e l l mass es t i m a t e s f o r T a h i t i a n Isochrysis, C. calcitrans and T. pseudonana. They r e v e a l t h a t n i t r a t e s t a r v a t i o n a f f e c t e d p h ytoplankton chemical composition such t h a t c a l c u l a t e d r e c o v e r y o f t o t a l o r g a n i c s was impaired i . e . , t o t a l o r g a n i c s p l u s ash do not add up t o the t o t a l c e l l mass. I t i s not known what o r g a n i c components may have been underestimated or the p o s s i b l e e n e r g e t i c c o n t r i b u t i o n of the u n i d e n t i f i e d f r a c t i o n t o the o y s t e r ' s n u t r i t i o n . 1. T a h i t i a n Isochrysis C e l l u l a r energy p o t e n t i a l l y a v a i l a b l e t o o y s t e r l a r v a e was g r e a t e s t i n the mid-log sample of T a h i t i a n Isochrysis w i t h a t o t a l e n e r g e t i c e q u i v a l e n t of 276 k c a l - 1 0- 1 2 c e l l s ( F i g . 6 a ) . 37 F i g u r e 5: ^ C e l l u l a r weight of g r o s s b i o c h e m i c a l components i n T a h i t i a n Isochrysis sampled d u r i n g mid-log phase (ML), o r a f t e r 2 o r 6 d o f n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . Bars r e p r e s e n t mean ± standard e r r o r of c e l l weight. See Appendix A, T a b l e 4 f o r standard e r r o r of a n a l y s e s of d u p l i c a t e c u l t u r e s (a and b ) . 38 Tahitian Isochrysis Cellular Weight of Components pg / c e l l 70 39 F i g u r e 6: C a l o r i c v a l u e of T a h i t i a n Isochrysis sampled d u r i n g mid-log phase (ML), o r a f t e r 2 or 6 d o f n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) ; A) c e l l u l a r energy e q u i v a l e n t s , B) p e r c e n t o f t o t a l c e l l energy i n gross b i o c h e m i c a l components. See Appendix A, Tab l e 1 f o r standard d e v i a t i o n of a n a l y s e s of d u p l i c a t e c u l t u r e s (a and b ) . 40 Caloric Value of Tahitian Isochrysis ^ Cellular Energy equivalents 300 Koal /1012 c e l l s 250 200 -1 5 0 ' 100 -N 2 d b Culture Age B Percent of Total Energy in G r o s s Components 100% 75% -50% 25% -N 2 d b Culture Age 41 P r o t e i n and l i p i d comprised approximately equal c a l o r i c v a l u e s o f 41% and 44% of t o t a l c e l l energy r e s p e c t i v e l y ( F i g . 6b). Carbohydrate was a r e l a t i v e l y minor component a t 15% o f t o t a l c e l l u l a r energy. The most s t r i k i n g change i n the g r o s s c o m p o s i t i o n , w i t h i n two days o f n i t r a t e s t a r v a t i o n , was a l a r g e d e p l e t i o n of c e l l u l a r p r o t e i n . P r o t e i n dropped from 27 p g - c e l l- 1 t o 5 p g - c e l l- 1 w h i l e carbohydrate i n c r e a s e d from 9 p g c e l l- 1 t o g r e a t e r than 26-31 p g - c e l l- 1 ( F i g . 5 ) . L i p i d content decreased s l i g h t l y from 14 p g - c e l l- 1 t o 11 p g c e l l- 1. Prolonged s t a r v a t i o n from 2 days t o 6 days r e s u l t e d i n l i t t l e f u r t h e r change. 2. Chaetoceros calcitrans The c a l o r i c v a l u e o f Chaetoceros calcitrans was d i m i n i s h e d when the c e l l s were n u t r i e n t - s t a r v e d ( F i g . 8 a ) . S i x hours of n i t r a t e o r s i l i c a t e s t a r v a t i o n reduced the energy content of C. calcitrans from a mid l o g v a l u e of 166-179 k c a l - 1 0- 1 2 c e l l s t o 127-151 k c a l - 1 0- 1 2 c e l l s . C a l o r i c l o s s from n i t r a t e - s t a r v e d c e l l s was a t t r i b u t a b l e t o a r e d u c t i o n i n c e l l u l a r p r o t e i n from a mid-log v a l u e o f 14 pg t o 6-9 pg and l i p i d from 9 pg a t mid-log phase t o 5 pg a f t e r 6 h o f n i t r a t e s t a r v a t i o n ( F i g . 7 ) . Carbohydrate (mono/oligosaccharide + p o l y s a c c h a r i d e ) gained 4 p g - c e l l- 1 over a mid-log v a l u e o f 10 p g - c e l l x d u r i n g n i t r a t e s t a r v a t i o n . Carbohydrate i n c r e a s e d from 23-26% o f t o t a l c e l l energy t o 43-48% ( F i g . 8b). C e l l u l a r weight o f g r o s s b i o c h e m i c a l components Chaetoceros calcitrans sampled d u r i n g mid-log phase (ML) or a f t e r 6 h of n i t r a t e o r s i l i c a t e s t a r v a t i o n (-N, - S i ) . Bars r e p r e s e n t mean ± standard e r r o r of c e l l weight. See Appendix A, T a b l e 5 f o r standard e r r o r o f a n a l y s e s of d u p l i c a t e c u l t u r e s (a and b ) . Chaetoceros calcitrans Cellular Weight of Components 43 pg / c e l l ML a ML b Lipid Protein -N -N a b Cu l tu re Age Mono/ol igo Ash -Si a -Si b Polysaccharide 44 F i g u r e 8: C a l o r i c v a l u e o f Chaetoceros calcitrans sampled d u r i n g mid-log phase (ML) or a f t e r 6 h o f n i t r a t e o r s i l i c a t e s t a r v a t i o n (-N, - S i ) ; A) c e l l u l a r energy e q u i v a l e n t s , B) p e r c e n t o f t o t a l c e l l energy i n gross b i o c h e m i c a l components. See ' Appendix A, Tab l e 2 f o r standard d e v i a t i o n o f a n a l y s e s of d u p l i c a t e c u l t u r e s (a and b ) . 4 5 Caloric Value of Chaetoceros calcitrans Cellular Energy equivalents 200 Kcal / 1 0 1 2 cel ls 1 5 0 -1 0 0 --N -N a b Culture Age B Percent of Total Energy in Gross Components 100% 75% -50% -25% -N -N a b Culture Age 46 In s i l i c a t e - s t a r v e d C. calcitrans carbohydrate mass was reduced from 10 p g c e l l- 1 t o 6 p g c e l l- 1 ( F i g . 7 ) , c r e a t i n g a l o s s i n c e l l u l a r c a l o r i c v a l u e ( F i g . 8 a ) . Ash content i n c r e a s e d from 9 pg t o 12-14 pg. The balance of t o t a l c e l l energy saw an a m p l i f i c a t i o n i n p r o t e i n from 32% t o 40% and a decrease i n carbohydrate from 25% t o 18% ( F i g . 8 b ) . 3. Thalassiosira pseudonana S i l i c a t e s t a r v a t i o n y i e l d e d the most e n e r g y - r i c h T. pseudonana c e l l s (119-126 k c a l - 1 0- 1 2 c e l l s , F i g . 10a). Energy was augmented from mid-log v a l u e s of 97-113 k c a l • 1 0- 1 2 c e l l s by a r i s e i n c e l l u l a r p r o t e i n from 8 pg t o 11 pg and i n l i p i d from 4 pg t o 5 pg, r e s p e c t i v e l y ( F i g . 9 ) . C e l l mass was l o s t i n s i l i c a t e - s t a r v e d c e l l s due t o a d e c l i n e i n both ash, from 8 p g - c e l l- 1 a t mid-log phase growth t o 5 pg, and carbohydrate (mono/oligo + p o l y s a c c h a r i d e ) which f e l l from 9 p g c e l l- 1 t o 7 p g c e l l- 1, a f t e r 6 h of s t a r v a t i o n . N i t r a t e s t a r v a t i o n y i e l d e d c e l l s low i n p r o t e i n content (4-5 p g - c e l l- 1, F i g . 8 ) . Other o r g a n i c s d i d not change but ash content i n c r e a s e d from 8 t o 10 p g c e l l- 1. The r e s u l t a n t c a l o r i c v a l u e o f the c e l l s t o t a l l e d the lowest of the treatments a t 79-85 k c a l - 1 0- 1 2 c e l l s ( F i g . 10a). B. F a t t y A c i d Composition F a t t y a c i d c o mposition i s r e p o r t e d as a percentage o f the t o t a l f a t t y a c i d complement. Changes i n the r e l a t i v e p r o p o r t i o n of p a r t i c u l a r f a t t y a c i d s cannot be i n t e r p r e t e d as 4 7 / F i g u r e 9: C e l l u l a r weight of g r o s s b i o c h e m i c a l components i n Thalassiosira pseudonana sampled d u r i n g mid-log phase (ML) o r a f t e r 6 h o f n i t r a t e or s i l i c a t e s t a r v a t i o n (-N, - S i ) . Bars r e p r e s e n t mean ± standard e r r o r of c e l l weight. See Appendix A, T a b l e 6 f o r standard e r r o r o f a n a l y s e s o f d u p l i c a t e c u l t u r e s (a and b ) . Thalassiosira pseudonana Cellular Weight of Components 48 pg / c e l l M L a Lipid Protein M L - N -N b a b Cu l t u re Age ! • Mono/oligo Ash •Si a -Si b Polysaccharide 49 F i g u r e 10: C a l o r i c v a l u e of Thalassiosira pseudonana sampled d u r i n g mid-log phase (ML) o r a f t e r 6 h o f n i t r a t e or s i l i c a t e s t a r v a t i o n (-N, - S i ) ; A) c e l l u l a r energy e q u i v a l e n t s , B) p e r c e n t of t o t a l c e l l energy i n g r o s s b i o c h e m i c a l components. See Appendix A, T a b l e 3 f o r standard d e v i a t i o n of a n a l y s e s of d u p l i c a t e c u l t u r e s (a and b ) . Caloric value of Thalassiosira pseudonana A Cellular Energy equivalents Kcal / 1 0 1 2 ce l ls 140 -i ML ML -N -N -Si -Si a b a b a b Culture Age B Percent of Total Energy in Gross Componen ts 100% I to iim i mmmml mmmm 1 : -.i 1 '<m ML ML -N -N -Si -Si a b a b a b Culture Age 51 changes i n the c e l l quota o f t h a t p a r t i c u l a r f a t t y a c i d , but t h e changes do r e f l e c t b i o c h e m i c a l a c t i v i t y such as c a t a b o l i s m o r de novo s y n t h e s i s which a f f e c t s the o v e r a l l f a t t y a c i d c o m p o s i t i o n . F a t t y a c i d p r o f i l e s were analyzed i n two s e r i e s o f c u l t u r e s . * S e l f - s h a d i n g promoted a l i g h t - l i m i t e d response i n the dense 20 L carboys used i n s e r i e s 1. These c u l t u r e s were sampled u n t i l they reached senescence. 1. Comparison of Mid-Log P r o f i l e s F i g u r e 11 summarizes the i n t r a s p e c i f i c d i f f e r e n c e s i n the f a t t y a c i d p r o f i l e s o f h e a l t h y T a h i t i a n Isochrysis, Chaetoceros calcitrans and Thalassiosira pseudonana h a r v e s t e d d u r i n g the m i d - l o g a r i t h m i c growth s t a g e . S a t u r a t e d f a t t y a c i d l e v e l s and the t o t a l p o l y u n s a t u r a t e t o monounsaturate r a t i o were h i g h e s t i n T a h i t i a n Isochrysis (about 50:20 i n both s e r i e s ) . N otable d i f f e r e n c e s i n the l e v e l s o f the n u t r i t i o n a l l y important p o l y u n s a t u r a t e d f a t t y a c i d s are apparent between the two a l g a l c l a s s e s r e p r e s e n t e d i n t h i s s t u d y . The two diatoms had h i g h l e v e l s o f 20:5n3 (15-22%, F i g . 13 and 14), w h i l e T a h i t i a n Isochrysis had o n l y 1% ( F i g . 12). I n s t e a d , T a h i t i a n Isochrysis had a h i g h p r o p o r t i o n of 22:6n3 (16%) w h i l e the diatoms possessed 4% or l e s s . T a h i t i a n Isochrysis a l s o * T a b l e s of complete f a t t y a c i d p r o f i l e s are i n c l u d e d i n Appendix B. 52 c o n t a i n e d a moderate l e v e l of l i n o l e n i c a c i d (18:3n3) i n s e r i e s 1, w h i l e the diatoms had o n l y t r a c e amounts. F r a c t i o n s o f n 6-polyunsaturated f a t t y a c i d s were g r e a t e s t i n T a h i t i a n Isochrysis which had a l a r g e complement o f l i n o l e i c a c i d (18:2n6, 4 t o 5%); these f a t t y a c i d s were p r e s e n t o n l y i n t r a c e amounts i n the diatoms. Small c o m p o s i t i o n a l d i f f e r e n c e s between the two s e r i e s of c u l t u r e s demonstrates the s e n s i t i v i t y o f diatom f a t t y a c i d s t o c u l t u r e c o n d i t i o n s . Phytoplankton i n the mid-log phase of growth were n u t r i e n t - r e p l e t e ; t h e r e f o r e , l i g h t - d e f i c i e n t growth i n s e r i e s 1 must account f o r d i f f e r e n c e s i n f a t t y a c i d c o m p o s i t i o n between the two s e r i e s . C e l l d e n s i t y and the s i z e of the c u l t u r e v e s s e l i n f l u e n c e d the u s a b l e i r r a d i a n c e p e n e t r a t i n g the c u l t u r e . There were no s i g n i f i c a n t d i f f e r e n c e s between s e r i e s 1 and 2 T a h i t i a n Isochrysis h a r v e s t e d from mid-log growth phase ( F i g . 13). C. calcitrans had h i g h e r n3 f a t t y a c i d l e v e l s i n s e r i e s 2 than i n s e r i e s 1 due t o i n c r e a s e d 18:4n3 (4.81 v s . 1.21) and 22:6n3 (2.07 vs 0.65) l e v e l s i n s e r i e s 2 ( F i g . 12). S e r i e s 1 C. calcitrans was r i c h e r i n 16:2n4 than i n s e r i e s 2 (8% vs 2 % ) . Thompson e t a l . (1989) have shown t h a t both 18:4n3 and 16:2n4 are s e n s i t i v e t o i r r a d i a n c e . Apparent d i f f e r e n c e s i n s e r i e s 1 and 2 c u l t u r e s of T. pseudonanana n6 f a t t y a c i d s are not s i g n i f i c a n t ( F i g u r e 14). \ 53 F i g u r e 11: F a t t y a c i d c l a s s p r o f i l e s of T a h i t i a n Isochrysis, Thalassiosira pseudonana and Chaetoceros calcitrans c e l l s h a r v e s t e d d u r i n g the mid-log growth phase i n batch c u l t u r e : A) S e r i e s 1 B) S e r i e s 2. 100 Mid-log Fatty Acid Profiles Series 1 % of total fatty acids MM n6 x H H n3 \ polyunsat'd | | other / Monounsaturated Saturated Isochrysis T. pseudonana C. calc i t rans Series 2 100 % of total fatty acids i l l n6 v iHi n3 \ Polyunsat'd l i l i i l o t h e r / Monounsaturated H i Saturated Isochrysis T. pseudonana C. calc i t rans 5 5 F i g u r e 12: Comparison of s e r i e s 1 and s e r i e s 2 histograms of f a t t y a c i d composition of T a h i t i a n Isochrysis h a r v e s t e d d u r i n g mid-log growth phase. The lower graph e n l a r g e s the s c a l e of the s m a l l e r peaks of the upper graph. E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s of r e p l i c a t e c u l t u r e s (n=2 i n s e r i e s 1, n=3 i n s e r i e s 2 ) . Mid-Log Fatty Ac ids Se r i es 1 and 2 Tahit ian Isochrysis 57 F i g u r e 13: Comparison of s e r i e s 1 and s e r i e s 2 histograms of f a t t y a c i d composition of Chaetoceros calcitrans h a r v e s t e d d u r i n g mid-log growth phase. The lower graph e n l a r g e s the s c a l e of the s m a l l e r peaks of the upper graph. E r r o r bars r e p r e s e n t ± 1 . standard d e v i a t i o n of a n a l y s e s of r e p l i c a t e c u l t u r e s (n=2 i n s e r i e s 1, n=l i n s e r i e s 2 ) . Mid-Log Fatty Ac ids Se r i es 1 and 2 Chaetoceros calcitrans 59 F i g u r e 14: Comparison of s e r i e s 1 and s e r i e s 2 histograms of f a t t y a c i d composition of Thalassiosira pseudonana h a r v e s t e d d u r i n g mid-log growth phase. The lower graph e n l a r g e s the s c a l e of the s m a l l e r peaks o f the upper graph. E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s of r e p l i c a t e c u l t u r e s (n=l i n s e r i e s l , n=3 i n s e r i e s 2 ) . Mid-Log Fatty Ac ids Se r i es 1 and 2 Thalassiosira pseudonana 61 2. Comparison of N u t r i e n t - S t a r v e d P r o f i l e s a. T a h i t i a n Isochrysis The same f a t t y a c i d t r e n d s were seen i n both s e r i e s but l e v e l s a t t a i n e d i n s e r i e s 1, a f t e r 240 h of n i t r a t e s t a r v a t i o n ( i . e . a t 450 h sampling t i m e ) , were reached i n s e r i e s 2, 48 h a f t e r n i t r a t e was exhausted from the medium. T h i s i n d i c a t e s t h a t the changes i n chemical composition due t o n i t r a t e s t a r v a t i o n proceeded more r a p i d l y under l i g h t s a t u r a t i o n ( s e r i e s 2 c u l t u r e s ) because the c u l t u r e s were growing f a s t e r . i . F a t t y A c i d C l a s s P r o f i l e In s e r i e s 1, t o t a l s a t u r a t e d f a t t y a c i d l e v e l s changed l i t t l e i n both n i t r a t e - a n d p h o s p h a t e - l i m i t e d treatments ( F i g . 15). T o t a l p o l y u n s a t u r a t e d f r a c t i o n s d e c l i n e d from 46% t o about 40%, l a r g e l y due t o a decrease i n the n 3 - p o l y u n s a t u r a t e s , as the monounsaturated f a t t y a c i d p r o p o r t i o n s i n c r e a s e d from 18% t o about 27% by 400 h i n both media. C u l t u r i n g i n n i t r a t e -l i m i t e d medium l o n g e r than 400 h f u r t h e r d i m i n i s h e d the n3 f r a c t i o n and the p o l y u n s a t u r a t e t o monounsaturate r a t i o t o 33:30. The n6 f a t t y a c i d s were p r e s e n t a t about 7%, i n d i m i n u t i v e p r o p o r t i o n t o the n3-polyunsaturated f r a c t i o n . In s e r i e s 2, t h e t o t a l p o l y u n s a t u r a t e t o monounsaturate r a t i o a l s o dropped s u b s t a n t i a l l y from 49:19 a t mid-log phase t o 33:28 by the second day of n i t r a t e s t a r v a t i o n ( F i g . 16). The p r o p o r t i o n o f n6 i n s e r i e s 2 was s i m i l a r t o s e r i e s 1. The 62 F i g u r e 15: Changes over time i n the f a t t y a c i d c l a s s p r o f i l e f o r T a h i t i a n Isochrysis i n s e r i e s 1 c u l t u r e (see F i g . l f o r growth curve and sampling t i m e ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium. Tahitian Isochrysis Series 1 Fatty Ac id C lass Prof i le Nitrate- l imited Medium 100 % of total fatty acids 85 h 185 h 250 h 330 h 400 h 450 h Culture age H I n6 m n3 I | other• . Polyunsat 'd Saturated Monounsaturated B Phosphate- l imi ted Medium % of total fatty acids 1 0 0 T 1 85 h 185 h 250 h 355 h 400 h Culture age 64 F i g u r e 16: Changes over time i n the f a t t y a c i d c l a s s p r o f i l e f o r T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) o r a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r b a rs r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=3). Tahitian Isochrysis Series 2 Fatty Acid Class Profile 66 f a t t y a c i d c l a s s p r o f i l e d i d not change a f t e r prolonged n i t r a t e s t a r v a t i o n . i i . E s s e n t i a l F a t t y A c i d s T a h i t i a n Isochrysis had a h i g h l e v e l of 22:6n3 (17%) which decreased s l i g h t l y t o 13% as c u l t u r e s aged i n both n i t r a t e -l i m i t e d and p h o s p h a t e - l i m i t e d media i n s e r i e s 1 ( F i g . 17), and i n 2 d - n i t r a t e s t a r v e d c e l l s from s e r i e s 2 ( F i g . 18). E i c o s o p e n t a e n o i c a c i d (20:5n3) was p r e s e n t o n l y as a s m a l l component (1%) and was reduced f u r t h e r when the c u l t u r e reached s t a t i o n a r y phase i n s e r i e s 1 (185 h, F i g . 19) and when n i t r a t e - s t a r v e d f o r 2 t o 6 days i n s e r i e s 2 ( F i g s . 2 0 ) . i i i . F a t t y A c i d Peaks Major f a t t y a c i d s of T a h i t i a n Isochrysis were 14:0, 16:0, 18:ln9, 18:2n6, 18:3n3, 18:4n3 and 22:6n3. Although i n c r e a s e s were more pronounced i n the' n i t r a t e - s t a r v e d treatment than i n the phosphate-starved treatment i n s e r i e s 1, the major f a t t y a c i d peaks had s i m i l a r responses t o c u l t u r e age i n both media ( F i g . 1 7 ) . The l a r g e s t d e c l i n e i n r e l a t i v e l e v e l s i n s e r i e s 1 was seen i n 14:0 (19% t o 12%), i n both media. F o l l o w i n g n i t r a t e s t a r v a t i o n i n s e r i e s 1 (330 h ) , 16:0, 16:ln7 and 22:0 l e v e l s r o s e and 18:ln9 l e v e l s became the most prominent f a t t y a c i d ( F i g s . 17 and 19), w h i l e the i n t e r m e d i a t e f a t t y a c i d s 2 0:ln9, 20:3n3, 20:5n3 and the u n i d e n t i f i e d peak, N, d e c r e a s e d . D i s c r i m i n a t e f a t t y a c i d responses between n i t r a t e -and p h o s p h a t e - l i m i t e d c u l t u r e s e v i d e n t i n s e r i e s 1 f a t t y a c i d s 67 F i g u r e 17: Changes over time i n the major f a t t y a c i d s o f T a h i t i a n Isochrysis i n s e r i e s 1 c u l t u r e (see F i g . 1 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium. ) Tahitian Isochrysis Series 1 Major Fatty Ac ids A N03- l im i ted Medium 69 F i g u r e 18: Changes over time i n the major f a t t y a c i d s o f T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s of r e p l i c a t e c u l t u r e s (n=3). Tahitian Isochrysis Series 2 Major Fatty Acids 30 25 20 15 -10 -5 0 % of total fat ty ac ids X T i I P l l ^ ML -N 2 d -N 4 d -N 6 d I 14:0 16:0 18:1n9 18:2n6 18:3n3 18:4n3 22:6n3 o 71 F i g u r e 19: Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s o f T a h i t i a n Isochrysis d u r i n g growth i n s e r i e s 1 c u l t u r e (see F i g . 1 f o r growth curve and sampling t i m e ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium. A Tahitian Isochrysis Series 1 Intermediate Fat ty Ac ids N03- l im i ted Medium % of total fatty acids 85 h 111 185 h H D 330 h • 400 h 450 h skM. I • 1 III 1 I 1 16:1n7 18:1n7 20:1n9 20:3n3 20:5n3 22:0 22:5n6 B P04- l im i ted Medium % of total fatty acids 1 -i i f I ii •| I w 1 ll 85 h 1H 185 h • 250 h EES 355 h 400h 16:1n7 18:1n7 20;1n9 20:3n3 20:5n3 22:0 22:5n6 73 F i g u r e 20: Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s of T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e 1 i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s of r e p l i c a t e c u l t u r e s (n=3). Tahitian Isochrysis Series 2 Intermediate Fatty Acids % of total fat ty ac ids N 16:1n7 18:1n7 20:1n9 20:3n3 20:5n3 22:0 22;5n6. 75 F i g u r e 21: Changes over time i n the minor f a t t y a c i d s o f T a h i t i a n Isochrysis d u r i n g growth i n s e r i e s 1 c u l t u r e (see F i g . 1 f o r growth curve- and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, and B) p h o s p h a t e - l i m i t e d medium. 76 Tahitian Isochrysis Series 1 Minor Fat ty Ac ids A N03- l im i ted Medium % of total fatty acids 16:2n4 18:1n13 19:? 20:a 21:5n3 B P04- l im i ted Medium % of total fatty acids 1.4 1.2 1 0.8 0.6 0.4 0.2 0 I mm I I mm 1 85 h 111 185 h mmni 3 5 5 h 400 h u . I-16:2n4 18:1n13 19:? 20:a 21:5n3 77 F i g u r e 22: Changes over time i n the minor f a t t y a c i d s o f T a h i t i a n Isochrysis grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d, -N 6 d ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=3). Tahitian Isochrysis Series 2 Minor Fatty Acids 79 i n c l u d e d 18:4n3 which decreased over time (from 14% t o 9%) i n the n i t r a t e - l i m i t e d treatment and i n c r e a s e d s l i g h t l y w i t h the onset o f phosphate s t a r v a t i o n . Phosphate s t a r v a t i o n r e s u l t e d i n augmented 16:ln7, 18:ln7, and 22:0 l e v e l s and decreased 20:ln9, 20:3n3 and 20:5n3 l e v e l s among the i n t e r m e d i a t e peaks ( F i g s . 17 and 19). In s e r i e s 2 a l l the dominant f a t t y a c i d s reached the same l e v e l by day 2, as found i n the f i n a l sample i n s e r i e s 1 ( F i g s . 18 and 2 0 ) . A f t e r day 2 many peaks showed a slow d e c l i n e . D i s c r e p a n c i e s between f a t t y a c i d p r o p o r t i o n s i n s e r i e s 1 p r i o r t o 250 h and i n t r e n d s i n s e r i e s 2, e.g., a decrease i n 16:ln7 and i n c r e a s e i n 22:5n6, may be a t t r i b u t e d t o l i g h t l i m i t a t i o n . Changes i n minor T a h i t i a n Isochrysis f a t t y a c i d s were g e n e r a l l y s m a l l enough t o be c o n s i d e r e d i n s i g n i f i c a n t ( F i g s . 21 and 22), b u t , one n o t a b l e d i f f e r e n c e among the treatments i n s e r i e s 1, was a decrease i n 16:2n4 i n the n i t r a t e - l i m i t e d medium, whereas phosphate l i m i t a t i o n produced the o p p o s i t e e f f e c t . b. Chaetoceros calcitrans i . F a t t y A c i d C l a s s P r o f i l e Chaetoceros calcitrans f a t t y a c i d c l a s s p r o f i l e s were a f f e c t e d s i m i l a r l y i n a l l t h r e e c u l t u r e media i n s e r i e s 1 ( F i g . 23). Responses w i t h time were more acute i n phosphate- and s i l i c a t e - l i m i t e d c u l t u r e than i n n i t r a t e - l i m i t e d c u l t u r e ( F i g . 80 F i g u r e 23: Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Chaetoceros calcitrans i n s e r i e s 1 c u l t u r e (see F i g . 2 f o r growth curve and sampling t i m e ) : A) n i t r a t e - l i m i t e d medium, B) p h o s p h a t e - l i m i t e d medium, and C) s i l i c a t e - l i m i t e d medium. Chaetoceros calcitrans Series 1 Fatty Acid Class Profile Nitrate-limited Medium % of total fatty acids 106 58 h 9 7 h 119 h 150 h C u l t u r e age Phosphate-limited Medium % of total fatty acids 10Q 58 h 97 h 119 h C u l t u r e age Silicate-limited Medium % of total fatty acids 174 h 150 h 99 h 119 h 150 h 82 F i g u r e 24: Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Chaetoceros calcitrans grown i n s e r i e s 2 c u l t u r e (see F i g . 4 f o r growth curve) i n A) n i t r a t e - l i m i t e d medium, or B) s i l i c a t e - l i m i t e d medium and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h or 6 h ) . E r r o r b a rs r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples). Chaetoceros calcitrans Series 2 Fatty Acid Class Profile 'd Mid-log Si- l im 2 h Si- l im 6 h C u l t u r e A g e 84 23 ) . In the s i l i c a t e - l i m i t e d treatment, e f f e c t s were not e v i d e n t u n t i l the l a s t sample a t 150 h, ( i . e . a f t e r 30 h of s i l i c a t e s t a r v a t i o n , when the c e l l s were s e n e s c e n t ) . Large i n c r e a s e s i n unknowns such as N ( F i g . 27c) e x p l a i n the low f i n a l % o f t o t a l f a t t y a c i d s i n the s i l i c a t e - l i m i t e d t r e a t m e n t . As w i t h the o t h e r two s p e c i e s i n v e s t i g a t e d i n s e r i e s 1, the p o l y u n s a t u r a t e f r a c t i o n d e c l i n e d over t i m e . The l a r g e s t decreases o c c u r r e d w i t h i n the n3 f a t t y a c i d s ( F i g . 2 3 ), p a r t i c u l a r l y 20:5n3 ( F i g . 25). The n6 f a t t y a c i d s comprised a s m a l l p o r t i o n o f the p o l y u n s a t u r a t e d p r o f i l e ( F i g . 2 3 ) . The dominant n6 a c i d , 16:2n6, was d e p l e t e d over time i n phosphate-and s i l i c a t e - l i m i t e d c u l t u r e s ( F i g . 25b and c ) . In s e r i e s 2, n u t r i e n t d e p r i v a t i o n had no s i g n i f i c a n t e f f e c t on the f a t t y a c i d c l a s s p r o f i l e ( F i g . 2 4 ) . i i . E s s e n t i a l F a t t y A c i d s While 22:6n3 comprised o n l y a minor component o f the t o t a l f a t t y a c i d complement o f Chaetoceros calcitrans, t h i s a l g a c o n t a i n e d l a r g e amounts of 20:5n3 which decreased w i t h time i n a l l treatments i n s e r i e s 1 ( F i g . 2 5 ) . Pronounced r e d u c t i o n s were not observed u n t i l a f t e r n u t r i e n t d e p l e t i o n and upon senescence i n s i l i c a t e and p h o s p h a t e - l i m i t e d media. In s e r i e s 2, 6 h of s i l i c a t e s t a r v a t i o n d i d not change 20:5n3 l e v e l s ( F i g . 2 6 ) . Changes i n the l e v e l of 20:5n3 from 17% t o 13% was seen i n the s e r i e s 1 n i t r a t e - l i m i t e d c u l t u r e o n l y w i t h the 85 onset of l i g h t - l i m i t e d growth a t the 97 h sample. S t a b i l i t y f o l l o w e d d u r i n g n i t r a t e s t a r v a t i o n ( a f t e r 119 h ) . S i m i l a r l y , n i t r a t e s t a r v a t i o n caused no s i g n i f i c a n t change i n s e r i e s 2 20:5n3 l e v e l s ( F i g . 2 6 ) . The t h r e e treatments i n s e r i e s 1 a f f e c t e d i n t e r m e d i a t e f a t t y a c i d l e v e l s o f 22:6n3 d i f f e r e n t l y i n Chaetoceros calcitrans ( F i g . 2 7 ) . Phosphate d e p l e t i o n had no s i g n i f i c a n t e f f e c t and s i l i c a t e s t a r v a t i o n l e a d t o a decrease by senescence. S i l i c a t e s t a r v a t i o n i n s e r i e s 2 y i e l d e d l e v e l s o f 0.5%, down from a mid-log l e v e l o f 2% ( F i g . 2 8 ) . While t h i s i n t e r m e d i a t e f a t t y a c i d minor component i n c r e a s e d s t e a d i l y w i t h time i n s e r i e s 1, from the mid-log phase (58 h) through l i g h t - and n i t r a t e - l i m i t e d growth, n i t r a t e s t a r v a t i o n had no e f f e c t upon 22:6n3 i n s e r i e s 2; t h e r e f o r e , l i g h t l i m i t a t i o n p r o b a b l y was r e s p o n s i b l e f o r t h e i n c r e a s e i n t h i s f a t t y a c i d i n s e r i e s 1. i i i . F a t t y A c i d Peaks Except f o r a decrease i n 22:6n3 when c e l l s were s i l i c a t e -s t a r v e d , t h e r e were no s i g n i f i c a n t a l t e r a t i o n s i n the f a t t y a c i d c o m p o s i t i o n i n both n i t r a t e - s t a r v e d and s i l i c a t e - s t a r v e d C. calcitrans i n s e r i e s 2 ( F i g s . 26, 28 and 30). Only s l i g h t changes o c c u r r e d i n the f a t t y a c i d peaks of C. calcitrans grown i n n i t r a t e - l i m i t e d medium i n s e r i e s 1 except i n 16:0 which i n c r e a s e d from 10% t o 14% ( F i g s . 25 and 27) d u r i n g l i g h t - l i m i t e d growth (97 t o 119 h) and p r i o r t o n i t r a t e s t a r v a t i o n ( a f t e r 119 h ) . L i g h t l i m i t a t i o n d u r i n g growth i n 86 F i g u r e 25: Changes over time i n the major f a t t y a c i d s o f Chaetoceros calcitrans grown i n s e r i e s 1 (See F i g . 2 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, B) p h o s p h a t e - l i m i t e d medium, and C) s i l i c a t e - l i m i t e d medium. Chaetoceros calcitrans Series 1 Major Fatty Acids % of total fatty acids 14:0 16:0 16:1n7 16:2n4 16:3n4 18:4n3 20:5n3 Phosphate-limited Medium 0 m 58 h mm 97 h H 0 1 5 0 h J I II 14:0 16:0 16:1n7 16:2n4 16:3n4 18:4n3 20:5n3 Silicate-limited Medium 11 99 h 111150 h I: Inn 14:0 16:0 16:1n7 16:2n4 16:3n4 18:4n3 20:5n3 88 F i g u r e 26: Changes over time i n the major f a t t y a c i d s of Chaetoceros calcitrans c u l t u r e d i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2^ h or 6 h and - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s of r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples). Chaetoceros calcitrans Ser ies 2 Major Fatty Acids % of total fat ty ac ids 1 ML -N-2 h -N 6 h -Si 2 h -Si 6 h i i 1 P I 1 1 14:0 16:0 16:1n7 16:2n4 16:3n4 18:4n3 20:5n3 9 0 F i g u r e 27: Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s of Chaetoceros calcitrans grown i n s e r i e s 1 c u l t u r e (see F i g . 2 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, B) phosphate-l i m i t e d medium, and C) s i l i c a t e - l i m i t e d medium. Chaetoceros calcitrans Series 1 Intermediate Fatty Acid Peaks % of total fatty acids Nitrogen-limited Medium |1 L i ii I II 58 h I I 97 h IMP 6 0 n 1174 h 1 J a I N 16:2n6 16:4n1 17:0 18:1n7 22:5n3 22:6n3 * of total fa t ty a c i d s Phosphate-limited Medium I Ml 12 10 N 16:2n6 16:4n1 17:0 18:1n7 22:5n3 22:6n3 % of total fa t ty a c i d s Silicate-limited Medium l i i l 99 h H I 150 h 1 16:2n6 16:4n1 17:0 18:1n7 22:5n3 22:6n3 92 F i g u r e 28: Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s of Chaetoceros calcitrans c u l t u r e d i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h and - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s of r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples). Chaetoceros calcitrans Series 2 Intermediate Fatty Acids % of total fatty ac ids N .16:2n6 16:4n1 17:0 18:1n7 22:5n3 22:6n3 94 F i g u r e 29: Changes over time i n the minor f a t t y a c i d s o f Chaetoceros calcitrans grown i n s e r i e s 1 c u l t u r e (See F i g . 2 f o r growth curve and sampling t i m e s ) : A) n i t r a t e - l i m i t e d medium, B) p h o s p h a t e - l i m i t e d medium, and C) s i l i c a t e - l i m i t e d medium. Chaetoceros calcitrans Series 1 Minor Fatty Acids 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 * of total fatty acids 58 h • 97 h m 1 5 0 n 174 h Nitrate-limited Medium 1 16:1n5 16;3n3 18:1n13 18:1n9 18:2n6 18:2n4 18;3n1 20:4n6 * of total fatty acids 58 h 97 h 119 h 150 h Phosphate-limited Medium T II II •if 1 16:1n5 16:3n3 18:1n13 18:1n9 18:2n6 18:2n4 18:3n1 20:4n6 * of total fatty acids I6;1n5 16:3n3 18:ln13 18:1n9 18:2n6 18:2n4 18:3n1 20:4n6 96 F i g u r e 30: Changes over time i n the minor f a t t y a c i d s of Chaetoceros calcitrans c u l t u r e d i n s e r i e s 2 n i t r a t e - and s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 o r 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h and - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s of r e p l i c a t e c u l t u r e s (n=2 i n 2 h samples, n=3 i n 6 h samples). Chaetoceros calcitrans Series 2 Minor Fatty Acids 2.5 % of total fat ty ac ids 2 -,5 0,5 -0 ML - N 2 h -N 6 h -Si 2 h -Si 6 h 1 1 1 I 1 1 • 1 16:1n5 1 Ii II 1 ti p 1. 16:3n3 18:1n13 18:1n9 18:2n6 18:3n4 ~0 98 s e r i e s 1 n i t r a t e - l i m i t e d medium r e s u l t e d i n an immediate i n c r e a s e i n 22:6n3 (see E s s e n t i a l F a t t y A c i d s and F i g . 2 7 ) . Responses t o phosphate d e p l e t i o n were s i m i l a r , except i n the i n t e r m e d i a t e f a t t y a c i d 18:ln7 which i n c r e a s e d from 0.5 t o 2.4%. Except i n 16:ln7 ( F i g . 25), changes d i d not develop u n t i l the l a s t sample when c e l l death was o c c u r r i n g . Most s u b s t a n t i a l i n c r e a s e s among the f a t t y a c i d s i n s e r i e s 1 s i l i c a t e - l i m i t e d c u l t u r e o c c u r r e d a t senescence (160 h ) , i n c l u d i n g 16:0, 18:ln7, 16:3n3 and the unknown N ( F i g s . 25, 27 and 2 9 ) . The minor f a t t y a c i d composition of Chaetoceros calcitrans i n s e r i e s 1 was dependent on the l i m i t i n g n u t r i e n t i n the c u l t u r e medium ( F i g . 2 9 ) . A f t e r mid-log phase growth i n n i t r a t e -l i m i t e d medium, 18:3nl i n c r e a s e d from 0.25% t o 1% and then d r a m a t i c a l l y decreased agai n under prolonged n i t r a t e s t a r v a t i o n . c. Thalassiosira pseudonana T. pseudonana f a t t y a c i d composition can be compared between s e r i e s 1 and 2 o n l y f o r c e l l s grown i n s i l i c a t e - l i m i t e d medium. Samples were l o s t and data i s u n a v a i l a b l e f o r n i t r a t e - l i m i t e d c u l t u r e s i n s e r i e s 1. I t must be s t r e s s e d t h a t no mid-log sample was taken from the p h o s p h a t e - l i m i t e d c u l t u r e i n s e r i e s 1. Instead the f i r s t sample i s r e p r e s e n t a t i v e o f the l a t e - l o g phase when phosphate l i m i t a t i o n 9 9 was i n f l u e n c i n g the chemical composition o f the a l g a e . The f i r s t sample of the s i l i c a t e - l i m i t e d c u l t u r e i n s e r i e s 1 i s the s o l e r e p r e s e n t a t i v e o f the mid-log p r o p o r t i o n a l v a l u e s of f a t t y a c i d s content i n the s e r i e s . i . F a t t y A c i d C l a s s P r o f i l e S a t u r a t e d f a t t y a c i d s were p r e s e n t i n T. pseudonana a t l e v e l s which v a r i e d w i t h the content o f 16:0 and was h i g h e s t a t e a r l y s t a t i o n a r y phase r e g a r d l e s s o f treatment i n s e r i e s 1 ( F i g . 31). The p o l y u n s a t u r a t e t o monounsaturate r a t i o r a p i d l y decreased from a mid-log v a l u e o f 46:22 t o 20:30 and 25:30 as both p h o s p h a t e - l i m i t e d and s i l i c a t e - l i m i t e d c u l t u r e s aged, r e s p e c t i v e l y . Reduction o f the n3 f r a c t i o n o f p o l y u n s a t u r a t e s was s u b s t a n t i a l i n both t r e a t m e n t s , although more pronounced i n the p h o s p h a t e - l i m i t e d c u l t u r e , and r e s t e d l a r g e l y i n the r e d u c t i o n i n 20:5n3, 18:4n3 and 22:6n3 f a t t y a c i d s ( F i g . 33). The n 6 - p o l y u n s a t u r a t e d f r a c t i o n o f f a t t y a c i d s was s m a l l i n comparison t o the n3 component, but was enhanced when phosphate was l i m i t i n g due t o i n c r e a s e s i n a r a c h i d o n i c a c i d (20:4n6, F i g . 35a). S e r i e s 2 T. pseudonana p r o f i l e s were not s i g n i f i c a n t l y a f f e c t e d by n u t r i e n t s t a r v a t i o n ( F i g . 32). i i . E s s e n t i a l F a t t y A c i d s Thalassiosira pseudonana c o n t a i n e d h i g h l e v e l s o f both 20:5n3 and 22:6n3 w i t h the former always i n g r e a t e r p r o p o r t i o n ( F i g s . 33 and 3 4 ) . In s e r i e s 1, s i g n i f i c a n t r e d u c t i o n s o f 20:5n3 100 F i g u r e 31: Changes over time i n the f a t t y a c i d c l a s s p r o f i l e of Thalassiosira pseudonana i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e -l i m i t e d medium. Thalassiosira pseudonana Series 1 Fatty Ac id C lass Prof i le A Phosphate- l imi ted Medium 102 F i g u r e 32: Changes over time i n the f a t t y a c i d c l a s s p r o f i l e o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e (see F i g . 4 f o r growth curve) i n A) n i t r a t e - l i m i t e d medium, or B) s i l i c a t e - l i m i t e d medium and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h or 6 h ) . E r r o r b a rs r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i -s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples). 103 Thalassiosira pseudonana Series 2 Fatty Acid Class Profile % of total fatty acids 100 i  B .'d Mid-iog -Si 2 h -Si 6 h 104 (92 h) i n the s i l i c a t e - l i m i t e d medium, w h i l e 6 h o f s i l i c a t e s t a r v a t i o n i n s e r i e s 2 reduced the amount of the f a t t y a c i d o n l y s l i g h t l y . A p p a r e n t l y l i g h t l i m i t a t i o n had a more pronounced e f f e c t on p r o p o r t i o n s o f 20:5n3 than s i l i c a t e s t a r v a t i o n . However, phosphate s t a r v a t i o n caused an even g r e a t e r r e d u c t i o n i n amount of 20:5n3, from 22% t o a low of 6%, i n T. pseudonana i n s e r i e s 1. T h i s was the most profound e f f e c t observed among a l l treatments among a l l 3 phytoplankton s p e c i e s employed. i i i . F a t t y A c i d Peaks A l l e f f e c t s o f n u t r i e n t s t a r v a t i o n on T. pseudonana f a t t y a c i d c o m p o s i t i o n were ve r y s l i g h t i n s e r i e s 2 and appear t o f a l l w i t h i n the l i m i t s o f n a t u r a l v a r i a t i o n ( F i g s 34, 36 and 38). S i n c e changes i n f a t t y a c i d l e v e l s i n s e r i e s 1 s i l i c a t e -l i m i t e d medium took p l a c e p r i o r t o s i l i c a t e s t a r v a t i o n (92 h ) , they were caused by low l i g h t ( F i g . 33b). Major f a t t y a c i d s t h a t s u f f e r e d decreases i n f a t t y a c i d l e v e l s i n both p h o s p h a t e - l i m i t e d and s i l i c a t e - l i m i t e d c u l t u r e c o n d i t i o n s , were 16:3n4, 18:4n3 and 20:5n3 ( F i g . 33). Those showing i n c r e a s e s were 16:0, 16:ln7 and 18:ln7 ( F i g s . 33 and 35). P h o s p h a t e - l i m i t e d c e l l s underwent a 100% i n c r e a s e i n 16:0 and a r e d u c t i o n i n 20:5n3 t o 25% of o r i g i n a l l e v e l s . R e l a t i v e l y l a r g e i n c r e a s e s were a l s o observed i n 1 8 : l n l 3 , 18:ln9, 18:ln7, and 20:.4n6, and a decrease was seen i n 16:2n4 i n phosphate-l i m i t e d T. pseudonana ( F i g . 35). 105 F i g u r e 33: Changes over time i n the major f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e -l i m i t e d medium. The S i 66 h sample i s i n c l u d e d i n the p h o s p h a t e - l i m i t e d p r o f i l e t o show mid-log (ML) f a t t y a c i d l e v e l s . Thalassiosira pseudonana Series 1 Major Fat ty Ac ids P04 - l im i ted Medium % of total fatty acids 14:0 16:0 16:1n7 16:3n4 18:4n3 20:5n3 22:6n3 B S i04- l im i ted Medium % of total fatty acids 14:0 16:0 16:1n7 16:3n4 18:4n3 20:5n3 22:6n3 107 F i g u r e 34: Changes over time i n the major f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium or s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML), o r a t 2 or 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n of a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples). Thalassiosira pseudonana Ser ies 2 Major Fatty Acids % of total fa t ty ac ids 14:0 16:0 16:1n7 16:3n4 18:4n3 20:5n3 22:6n3 o co 109 F i g u r e 35: Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s of Thalassiosira pseudonanatgrown i n s e r i e s 1 c u l t u r e ( S e e . F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e - l i m i t e d medium. The S i 66 h sample i s i n c l u d e d i n the p h o s p h a t e - l i m i t e d p r o f i l e t o show mid-log f a t t y a c i d l e v e l s . Thalassiosira pseudonana Series 1 Intermediate Fatty Ac ids A P04- l im i ted Medium % of total fatty acids 4 - i N 16:2n6 16:2n4 18:1n13 18:1n9 18:1n7 20:4n6 I l l F i g u r e 36: Changes over time i n the i n t e r m e d i a t e f a t t y a c i d s of Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium, o r s i l i c a t e -l i m i t e d medium (see F i g . 4 f o r growth curve) and ha r v e s t e d a t mid-log growth phase (ML) o r a t 2 or 6 h o f n u t r i e n t s t a r v a t i o n (-N 2 h or 6 h, - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f an a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples). Thalassiosira pseudonana Series 2 Intermediate Fatty Acids 5 % of total fat ty ac ids 4 4 3 ML - N 2 h - N 6 h - S i 2 h • - S i 6 h T 18:1n13 X • 18:1n9 ^ m m. 20:4n6 N 16:2n6 16:2n4 18:1n7 t o 113 F i g u r e 37: Changes over time i n the minor f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 1 c u l t u r e (See F i g . 3 f o r growth curve and sampling t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, and B) s i l i c a t e -l i m i t e d medium. The S i 66 h sample i s i n c l u d e d i n the p h o s p h a t e - l i m i t e d p r o f i l e t o show mid-log f a t t y a c i d l e v e l s . Thalassiosira pseudonana Series 1 Minor Fat ty Ac ids A P04- l im i ted Medium B S i 03 - l im i ted Medium % of total fatty acids 1,6 i 17:0 16:4n1 18:1n5 18:2n6 18:3n6 20:4n3 115 F i g u r e 38: Changes over time i n the minor f a t t y a c i d s o f Thalassiosira pseudonana grown i n s e r i e s 2 c u l t u r e i n n i t r a t e - l i m i t e d medium, o r s i l i c a t e - l i m i t e d medium (see F i g . 4 f o r growth curve) and h a r v e s t e d a t mid-log growth phase (ML) or a t 2 or 6 h of n u t r i e n t s t a r v a t i o n (-N 2 h o r 6 h, - S i 2 h or 6 h ) . E r r o r bars r e p r e s e n t ± 1 standard d e v i a t i o n o f a n a l y s e s o f r e p l i c a t e c u l t u r e s (n=2 i n N-starved samples, n=3 i n 2 h S i - s t a r v e d samples, n=4 i n 6 h S i - s t a r v e d samples). Thalassiosira pseudonana Ser ies 2 Minor Fatty Acids 1.2 % of total fa t ty ac ids 0.8 0.6 -0.4 -0.2 0 M ML N 2 h H i -N 6 d -Si 2 h ^ 1 -Si 6 h 17:0 16:4n1 18:2n6 18:3n4 20:4n3 117 Changes i n the minor peaks of s e r i e s 1 were over a v e r y s m a l l range o f l e s s than 1% ( F i g . 37). The o n l y n o t a b l e change i n p h o s p h a t e - l i m i t e d c e l l s was a r e d u c t i o n i n the 18:3n6 component. In the s i l i c a t e - l i m i t e d t r e a t m e n t , l i g h t l i m i t a t i o n caused a decrease i n 18:3n6, w h i l e 18:ln5 i n c r e a s e d f o l l o w i n g s i l i c a t e s t a r v a t i o n . I l l . Growth of Oyster Larvae In a l l f e e d i n g t r i a l s , s t a r v e d c o n t r o l s (no a l g a l food) of o y s t e r l a r v a e showed l i t t l e o r no growth. In mid-log c o n t r o l s of a l l experiments, no e f f e c t on l a r v a l s h e l l l e n g t h was e x h i b i t e d when l a r v a l c u l t u r e water was s t r i p p e d o f e i t h e r n i t r a t e o r s i l i c a t e f o r n u t r i e n t - s t a r v e d a l g a l t r e a t m e n t s . 1. T a h i t i a n Isochrysis T a h i t i a n Isochrysis was a poor d i e t f o r C. gigas l a r v a e when f e d as a u n i a l g a l d i e t a t a r a t i o n l e v e l o f 20,000 c e l l s mL- 1. When c e l l s were h a r v e s t e d a t the mid-log phase o f growth and f e d t o l a r v a e f o r f i v e days, s h e l l l e n g t h was s i g n i f i c a n t l y l a r g e r (99 |im, p<0.05, Tukey Test) than those f e d n i t r a t e -s t a r v e d c e l l s (90 p.m, F i g . 39, Table I I I ) . However, l a r v a l l e n g t h a t subsequent measurements was not a f f e c t e d by v a r i a t i o n s i n a l g a l d i e t . S u r v i v a l was poor i n a l l treatments (Table II) and a p r o p e n s i t y f o r s m a l l e r l a r v a e t o succumb f i r s t , may have b i a s e d r e s u l t s as the experiment p r o g r e s s e d . 118 Table I I : F i n a l s u r v i v a l of Crassostrea gigas larvae in three u n i a l g a l d i e t t r i a l s l a s t i n g from 12 to 19 days. T a h i t i a n I s o c h r y s i s d i e t s c o n s i s t e d of a l g a l c e l l s harvested f t o n n i t r a t e - U n i t e d medium at a i d - l o g growth phase, or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d or 6 d ) . I second a i d - l o g t r e a t s e n t (Hid-log c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of holding larvae fed n i t r a t e - s t a r v e d c e l l s i n n i t r a t e - f r e e seavater. Diatom d i e t s c o n s i s t e d of c e l l s harvested from n i t r a t e - and s i l i c a t e -l i m i t e d nediuii at a i d - l o g phase, or a f t e r 6 h of n u t r i e n t s t a r v a t i o n (-8 6 h, - S i 6 h). Two a d d i t i o n a l mid-log treatments (Hid-log N-control, Hid-log S i - c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of holding larvae fed n i t r a t e - s t a r v e d or s i l i c a t e - s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e - f r e e or s i l i c a t e - f r e e seavater, r e s p e c t i v e l y . Valaes are mean percentages t 1 standard d e v i a t i o n , of l i v e larvae from a sample of greater than 100 animals from each r e p l i c a t e . T a h i t i a n I s o c h r y s i s Chaetoceros c a l c i t r a n s T h a l a s s i o s i r a pseudonana 19 days 14 days 12 days Hid-log 29 t 20 Mid-log 59 t 6 Hid-log 78 t 15 Mid-log c o n t r o l 25 • 4 Hid-log H-control 61 t 17 Hid-log H-control 93 i 7 -H-2 d 13 t 5 Hid-log S i - c o n t r o l 46 • 22 Hid-log S i - c o n t r o l 86 t 11 -H-6 d 6 t 5 -N 6 h 41 i 24 -H 6 h 90 • 9 - S i 6 h 32 t 20 - S i 6 h 94 t 1 119 F i g u r e 39: Growth of Crassostrea gigas l a r v a e f e d T a h i t i a n Isochrysis h a r v e s t e d from n i t r a t e - l i m i t e d medium a t mid-log growth phase (ML), o r a f t e r 2 o r 6 days o f n i t r a t e s t a r v a t i o n (-N 2 d o r 6 d ) . A second mid-log treatment (ML-control) c o n t r o l l e d f o r the e f f e c t o f h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d c e l l s i n n i t r a t e - f r e e seawater. S t a r v e d l a r v a e d i d not r e c e i v e any a l g a l food d u r i n g the experiment. L e a s t squares means of l a r v a l l e n g t h were analysed by nested a n a l y s i s o f v a r i a n c e o f 3 r e p l i c a t e s ; s t a ndard e r r o r s a l l l i e w i t h i n the symbols except f o r the (-N 6 d) treatment (see Tabl e I I ) . A s t e r i s k s r e p r e s e n t s i g n i f i c a n t d i f f e r e n c e a t the 95% c o n f i d e n c e l e v e l . Crassostrea gigas larval growth on Tahitian Isochrysis diet 2 5 0 S h e l l L e n g t h (urn) 200 150 -100 5 10 Time (days) 15 2 5 0 2 0 0 150 i 100 20 O 121 Table I I I : S h e l l length !)IB, part a) and I n t e r v a l growth rates (part b| of l a r v a l Crassostrea gigas fed a u n i a l g a l d i e t , at a d a i l y concentration of 20,000 c e l l s - a L - 1 for 19 days, of T a h i t i a n I s o c h r y s i s harvested fron n i t r a t e - l i m i t e d aediura at raid-log grovth phase, or a f t e r 2 or 6 days of n i t r a t e s t a r v a t i o n (-N 2 d or 6 d). A second mid-log treatment (Hid-log c o n t r o l ) c o n t r o l l e d for the e f f e c t of holding larvae fed n i t r a t e - s t a r v e d c e l l s in n i t r a t e - f r e e seavater. Least squares Beans of s h e l l length t standard e r r o r vere c a l c u l a t e d by nested a n a l y s i s of v a r i a n c e , at the 95\ confidence l e v e l , f o r three r e p l i c a t e s . Round brackets enclose the namber of measurements made. Square brackets enclose l e t t e r s of s i g n i f i c a n c e i r o n Tukey's m u l t i p l e coaparison t e s t , (p<.05). Hatching l e t t e r s among treatments within the day of measurement s i g n i f y no d i f f e r e n c e among the treatments bearing those l e t t e r s . a) Time (Days) 0 5 10 15 19 Treatment R e p l i c a t e ML 1 85 t 0.8 (54) 99 t 1.4 (51) 119 • 2.3 (53) 156 i 5.9 (44) 181 t 8.3 (36) 2 98 t 1.3 (57) 124 t 3.1 (56) 185 t 7.9 (47) 198 i 12.5 (30) 3 99 ! 1.4 (55) 132 t 3.3 (55) 167 ± 7.1 (44) 238 i 10.3 (51) LS Mean 85 t 0.8 99 i 0.7 [ a l 125 t 1.9 (al 170 • 4.2 [ab] 206 t 5.0 lab] HL-control 1 97 i 1.7 (57) 123 • 3.2 (71) 168 i 6.3 (52) 216 i 7.8 (36) 2 98 i 1.9 (55) 111 ! 2.4 (50) 160 t 7.7 (50) 152 t 10.0 (45) 3 100 t 2.4 (29) 114 t 3.3 (56) 152 t 8.9 (53) 151 t 6.9 (30) LS Hean 85 t 0.8 99 i 0.8 [a] 116 i 1.8 lb] 160 t 3.9 l a b e l 173 i 5.1 lab] -N 2 d 1 89 t 1.3 (57) 109 t 3.1 (55) 159 i 6.6 (54) 188 t 9.7 (30) 2 89 t 1.2 (62) 96 t 3.8 (54) 161 i 7.2 (46) 169 i 7.6 (49) 3 92 i 1.4 (46) 108 t 5.0 (64) 172 t 6.9 (50) 190 t 8.7 (37) LS Hean 85 i 0.8 90 • 0.7 [b] 104 i 1.8 [be] 164 i 4.0 lac) 182 i 5.0 lac] -S 6 d 1 90 t 0.6 (60) 111 i 3.1 (56) 156 t 4.7 (48) 204 i 32.6 (4) 2 92 t 0.8 (60) 108 i 3.8 (48) 195 ± 6.8 (50) 220 t 10.0 (36) 3 92 • 1.0 (55) 114 t 5.0 (48) 198 i 8.7 (47) 191 i 9.0 (28) LS Hean 85 i 0. 8 91 i 0.7 l b ! 111 i 1.9 I d 181 t 4.1 lac] 205 i 9.9 lac] b) Grovth ()lB'day-l) Treatment 0-5 days 5-10 days 10-15 days 15-20 days ML 2.8 5.2 9.0 9.0 HL-control 2.8 3.4 8.8 3.3 -8 2 d 1.0 2.8 12.0 4.5 -H 6 d 1.2 4.0 14.0 6.0 122 Thus i t i s d i f f i c u l t t o assess the r e l a t i v e v a l u e o f the T a h i t i a n Isochrysis d i e t s . When f e d T a h i t i a n Isochrysis, l a r v a e grew t o a maximum mean of o n l y 205 |i.m a f t e r 19 days o f f e e d i n g ( F i g . 39) . L a r v a l growth was compared between mid-log T a h i t i a n Isochrysis and Thalassiosira pseudonana d i e t s i n another experiment (see Ta b l e V) . F i n a l mean s h e l l l e n g t h , a t 12 days, was 160 p.m w i t h the mid-log Isochrysis d i e t , w h i l e a d i e t o f T. pseudonana y i e l d e d much l a r g e r l a r v a e r a n g i n g i n mean s h e l l l e n g t h from 215 t o 256 \im depending on the treatment (Table V ) . 2. Chaetoceros calcitrans Crassostrea gigas l a r v a e grew b e s t on a d i e t o f Chaetoceros calcitrans when f e d algae h a r v e s t e d d u r i n g the mid-log phase ( F i g . 4 0 ) . N i t r a t e - s t a r v e d C. calcitrans ranked second i n food v a l u e . S h e l l l e n g t h was s i g n i f i c a n t l y d i f f e r e n t (p<0.05, Ta b l e IV) among the t h r e e treatments a f t e r Day 3. F i n a l s h e l l l e n g t h s on Day 14 were 265 |i.m, 209 u.m and 173 p,m f o r m i d - l o g , n i t r a t e - l i m i t e d and s i l i c a t e - l i m i t e d t r e a t m e n t s , r e s p e c t i v e l y . I n t e r v a l growth r a t e s i n a l l treatments were h i g h e s t u n t i l the l a r v a e reached a l e n g t h of about 190 nm, a t which p o i n t growth r a t e s were slower a g a i n (Table IV, and see a l s o the s l o p e o f the l i n e s i n F i g . 4 0 ) . 123 F i g u r e 40: Growth ( s h e l l length) o f Crassostrea gigas l a r v a e f e d Chaetoceros calcitrans h a r v e s t e d from n i t r a t e -and s i l i c a t e - l i m i t e d medium a t mid-log phase (ML), or a f t e r 6 h of n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h ) . Two a d d i t i o n a l mid-log treatments (ML-N-c o n t r o l , M L - S i - c o n t r o l ) c o n t r o l l e d f o r the e f f e c t o f h o l d i n g l a r v a e f e d n i t r a t e - s t a r v e d o r s i l i c a t e -s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e - f r e e or s i l i c a t e - f r e e seawater, r e s p e c t i v e l y . S t a r v e d l a r v a e d i d not r e c e i v e any a l g a l food d u r i n g the experiment. L e a s t squares means of l a r v a l l e n g t h were analys e d by nested a n a l y s i s o f v a r i a n c e o f 3 r e p l i c a t e s ; standard e r r o r s a l l l i e w i t h i n the range o f the symbols (see Tabl e I I I ) . A s t e r i s k s r e p r e s e n t s i g n i f i c a n t d i f f e r e n c e a t the 95% co n f i d e n c e l e v e l . Crassostrea gigas larval growth on Chaetoceros calcitrans diet 125 Table IV: S h e l l length [p, pact a) and i n t e i v a l growth r a t e s (part b) of l a r v a l Crassostrea gigas fed a u n i a l g a l d i e t , for 14 days as i n Table I I , of Chaetoceros c a l c i t r a n s harvested f r o n n i t r a t e - and s i l i c a t e - l i m i t e d medium at mid-log phase, or a f t e r 6 h of n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h). Two a d d i t i o n a l a i d - l o g treatments (Hid-log N-control, Hid-log S i - c o n t r o l ) c o n t r o l l e d f o r the e f f e c t of holding larvae fed n i t r a t e - s t a r v e d or s i l i c a t e - s t a r v e d c e l l s i n n u t r i e n t s t r i p p e d , n i t r a t e - f r e e or s i l i c a t e - f r e e seavater, r e s p e c t i v e l y . Time (Days) 0 3 6 10 14 Treatment R e p l i c a t e Hid-log 1 114 i 0 .9 (165) 165 • 2.6(63) 208 t 2.5 (69) 242 i 4.2 (63) 279.i 4.5 (66) 2 149 i 2.9 (76) 197 • 2.9 (69) 232 • 3.8 (72) 276 i 5.0 (69) 3 163 t 2.5 (77) 195 2.4 (72) 230 i 3.6 (67) 268 i 4.9 (69) LS Hean 114 • 0.9 159 t 1.3 [ a l 200 + 1.6 lab] 235 i 2.3 [ a 1 274 t 2.6 (al Hid-log 1 147 i 3.3 (68) 205 4.6 (66) 233 ± 3.8 (66) 281 t 6.0 (60) H-control 2 159 i 2.5 (67) 195 3.3 (69) 224 • 3.9 (66) 248 i 4.9 (66) 3 163 • 2.5 (71) 198 • 3.4 (66) 222 i 3.2 (66) 245 i 4.3 (66) LS Hean 114 t 0.9 156 i 1.3 [al 199 + 1.6 [abcl 226 t 2.3 [ a l 258 • 2.7 [bl Hid-log 1 149 i 2.4 (54) 193 + 3.4 (73) 238 t 5.8 (66) 268 t 5.1 (60) S i - c o n t r o l 2 158 i 2.0 (60) 176 2.4 (60) 221 t 4.4 (66) 258 t 3.8 (66) 3 164 i 2.7 (71) 207 2.8 (80) 235 t 4.4 (64) 258 t 3.4 (69) LS Hean 114 t 0.9 157 i 1.4 [ a l 192 + 1.6 Ibcl 232 t 2.3 [a] 262 i 2.7 [bl -1 6 h 1 124 • 1.6 (71) 159 2.8 (80) 192 t 4.7 (63) 2 136 t 1.9 (65) 166 + 3.2 (80) 198 • 5.1 (60) 215 t 5.3 (75) 3 137 • 1.7 (63) 158 + 2.6 (53) 182 i 3.8 (60) 203 i 4.6 (69) LS Hean 114 • 0.9 133 i 1.3 (bl 161 + 1.6 [dl 191 i 2.4 [bl 209 i 3.1 [ c l - S i 6 h 1 130 i 1.8 (72) 144 + 2.3 (62) 159 • 3.2 (60) 178 t 5.0 (63) 2 121 t 1.5 (57) 137 2.1 (45) 159 i 3.2 (38) 172 i 5.3 (29) 3 128 i 1.5 (57) 144 + 2.3 (69) 161 i 3.3 (60) 170 t 3.1 (72) LS Hean 114 i 0.9 126 i 1.4 [ c l 142 1.8 [ e l 160 • 2.7 [ c l 173 i 3.2 [dl b) Growth (HiHay-1) Treatment 0-3 days 3-6 days 6-10 days 10-14 days Mid-log 15.0 13.7 8.8 9.8 N-control 14.0 14.3 6.8 8.0 S i - c o n t r o l 14.3 11.7 10.0 7.5 -H 6 h 6.3 9 .3 7.5 4.5 - S i 6 h 12.0 5.3 4.5 3.3 126 Figure 41: Growth (s h e l l length) of Crassostrea gigas larvae fed Thalassiosira pseudonana harvested from n i t r a t e - and s i l i c a t e - l i m i t e d medium at mid-log phase (ML) or a f t e r 6 h of nutrient starvation (-N 6 h, - S i 6 h). Two additional mid-log treatments (ML-N-control, ML-Si-control) controlled for the e f f e c t of holding larvae fed nitrate-starved or s i l i c a t e - s t a r v e d c e l l s i n nutrient-stripped, n i t r a t e - f r e e or s i l i c a t e - f r e e seawater, respectively. Starved larvae did not receive any a l g a l food during the experiment. Least squares means of l a r v a l length were analysed by nested analysis of variance of 3 r e p l i c a t e s ; standard errors a l l l i e within the range of the symbols (see Table IV). Asterisks represent s i g n i f i c a n t difference at the 95% confidence l e v e l . Crassostrea gigas larval growth on Thalassiosira pseudonana diet 128 Table V: S h e l l length ( p i , part a) and i n t e r v a l grovth rates (part b) of l a r v a l Crassostrea gigas fed f o r 12 days as i n Table IV, of T h a l a s s i o s i r a pseudonana harvested from n i t r a t e - and s i l i c a t e -l i m i t e d mediuQ. Grovth vas a l s o compared against a d i e t of T a h i t i a n I s o c h r y s i s (T-ISO). a) Time (Days) 0 3 7 12 Treatment R e p l i c a t e Hid-log 1 110 t 1.6 (136) 138 t 1.7 (50) 181 • 5.3 (42) 228 • 6.2 (53) 2 131 • 2.5 (47) 186 t 5.4 (52) 231 t 10.0 (34) • 3 137 t 2.5 (58) 195 t 5.2 (54) 221 • 6.7 (40) LS Hean 110 t 1.6 135 i 1.5 [cd] 187 t 3.1 [b] 227 t 4.2 [bed] Hid-log 1 143 t 2.8 (42) 205 t 6.0 (53) 232 i 5.9 (55) N-control 2 144 i 2.9 (55) 192 i 5.9 (54) 237 t 6.8 (50) 3 141 • 3.1 (53) 198 i 5.9 (51) 235 t 7.0 (49) LS Hean 110 i 1.6 143 i 1.5 [be] 198 • 2.9 [b] 235 t 3.7 [be] Hid-log 1 133 i 2.9 (49) 189 i 6.0 (35) S i - c o n t r o l 2 143 t 3.0 (54) 203 t 6.1 (38) 3 145 ± 2.7 (54) 196 ± 4.4 (51) LS Hean 110 t 1.6 141 t 1.5 [bcdl 196 t 3.4 [bl -N 6 h 1 145 t 3.3 (42) 174 t 4.6 (52) 220 ± 7.2 (45) 2 139 t 2.8 (54) 166 i 4.2 (54) 210 i 8.4 (48) 3 145 t 2.8 (58) 163 t 4.6 (54) 249 i 8.7 (36) LS Hean 110 i 1.6 142 • 1.9 [bcdl 170 i 3.6 [ c l 215 i 4.8 (cdl - S i 6 h 1 162 t 2.8 (39) 208 t 5.5 (47) 243 i 6.5 (47) 2 152 i 3.1 (47) 227 i 4.2 (47) 270 f 4.5 (41) • 3 137 t 2.7 (51) 163 i 5.5 (52) 232 t 8.6 (44) LS Hean • 110 i 1.6 157 * 2.0 [ a l 217 i 3.8 [a] 256 t 4.9 [al T-ISO 1 131 i 1.7 (54) 147 t 2.5 (33) 2 126 i 2.3 (48) 147 i 1.8 (65) 165 i 3.2 (37) 3 133 • 2.4 (47) 139 i 3.0 (49) 156 t 3.1 (54) LS Hean 110 i 1.6 133 • 1.2 144 t 1.4 161 i 2.3 b) Grovth (um-day-l) Treatment 0-3 days 3-7 days 7-12 days Hid-log 8.3 13.0 8.0 Hid-log-N-control 11.0 13.8 7.4 H i d - l o g - S i - c o n t r o l 10.3 13.8 -N 6 h 10.7 7.0 9.0 - S i 6 h 15.7 15.0 7.8 T-ISO 7.7 2.8 3.4 129 3.. Thalassiosira pseudonana S i l i c a t e - s t a r v e d T. pseudonana c e l l s proved t o be the bes t T. pseudonana d i e t f o r o y s t e r l a r v a e . T h i s was c o n t r a r y t o the r e s u l t s w i t h s i l i c a t e - s t a r v e d C. calcitrans which was the worst a l g a l d i e t compared t o a l l the ot h e r treatments f o r C. calcitrans. M i d - l o g phase T. pseudonana c e l l s r a t e d the second b e s t d i e t ( F i g . 41). A f t e r j u s t 3 days o f f e e d i n g l a r v a e w i t h an i n i t i a l l e n g t h o f 110 \im, s i l i c a t e - s t a r v e d T. pseudonana y i e l d e d a mean s h e l l l e n g t h o f 157 \im which was s i g n i f i c a n t l y d i f f e r e n t (p>0.05, Tukey t e s t ) from 140 \im produced by a l l the ot h e r treatments (Table V ) . By day 7, the the l a r v a e consuming mid-log and s i l i c a t e - s t a r v e d d i e t s were growing a t 14 ^m-d-1 and 15 ^ md"1, r e s p e c t i v e l y w h i l e the l a r v a e f e d n i t r a t e - s t a r v e d c e l l s grew a t o n l y 7 um'd"1 (Table V ) . A f t e r day 7, d a i l y growth r a t e s slowed t o from 7 t o 9 nm-d-l i n a l l t r e a t m e n t s , f o l l o w i n g the same t r e n d as seen i n i n t e r v a l growth r a t e s f o r l a r v a e l a r g e r than 190 |xm i n the C. calcitrans experiment. F i n a l mean s h e l l l e n g t h s ranged between a h i g h o f 256 \im f o r l a r v a e f e d s i l i c a t e - s t a r v e d T. pseudonana and 215 p.m f o r l a r v a e f e d n i t r a t e - s t a r v e d T. pseudonana. IV. F a c t o r s A f f e c t i n g Crassostrea gigas Growth T a h i t i a n Isochrysis was r i c h i n 22:6n3 and impoverished i n 20:5n3 ( F i g . 12). The i n f e r i o r performance o f t h i s u n i a l g a l d i e t f o r C. gigas l a r v a e suggests t h a t 22:6n3 may have l i t t l e 130 n u t r i t i o n a l v a l u e f o r o y s t e r l a r v a e . The diatoms, p o s s e s s i n g l a r g e amounts of 20:5n3 and l i t t l e 22:6n3 ( F i g s . 13 and 14) were a b l e t o support a c c e p t a b l e l a r v a l s u r v i v a l (Table I V ) . However, i f 22:6n3 i s r e q u i r e d a t l e a s t i n low l e v e l s , the decrease i n l a r v a l growth when they were f e d s i l i c a t e - s t a r v e d Chaetoceros calcitrans may have been due t o a drop i n 22:6n3 l e v e l s from 2% t o 0.5% ( F i g s . 40 and 2 8 ) . Among the diatoms, the most e n e r g y - r i c h a l g a l c e l l s produced the f a s t e s t growing l a r v a e (Table V I ) . Chaetoceros calcitrans mid-log c e l l s and s i l i c a t e - s t a r v e d Thalassiosira pseudonana c e l l s had the h i g h e s t c a l o r i c v a l u e s w i t h i n the s p e c i e s . The bal a n c e o f l i p i d , carbohydrate and p r o t e i n , expressed as a percentage o f ash f r e e dry weight, was s i m i l a r i n the be s t d i e t s , a t 27:30:43 i n C. calcitrans and 22:31:47 i n T. pseudonana (Table V I ) . Trends f a v o u r i n g e n e r g e t i c e q u i v a l e n t s a l s o a p p l y w i t h i n the T. pseudonana experiment. The o r g a n i c r a t i o ( l i p i d : c a r b o h y d r a t e : p r o t e i n ) , 22:49:29, i n n i t r a t e - s t a r v e d c e l l s was s i m i l a r t o the mid-log treatment o f 18:42:39. However the former c o n t a i n e d the l e a s t energy p o t e n t i a l and y i e l d e d the s m a l l e s t l a r v a e (Table V I ) . S u p e r i o r i t y o f n i t r a t e - s t a r v e d over s i l i c a t e - s t a r v e d C. calcitrans c e l l s cannot be a t t r i b u t e d t o energy content as i t was s i m i l a r f o r both treatments (Table V I ) . Gross composition d i f f e r e n c e s l i e i n the p r o t e i n and carbohydrate f r a c t i o n s . The n i t r a t e - s t a r v e d c e l l s had a h i g h e r carbohydrate c o n t e n t , Table VI: A summary of the biochemical composition and peiformance of three u n i a l g a l d i e t s fed to Crassostrea gigas l a r v a e , i n experiment for each phytoplankton species i n v e s t i g a t e d the a f f e c t of n u t r i e n t s t a r v a t i o n on the value of the d i e t i n i n c r e a s i n g l a r v a l s h e l l length. Tvo r e p l i c a t e s vere analyzed and averaged i n each treatment. Species Treatment Diet C a l o r i c Value L i p i d : Carbohydrate : P r o t e i n Rating (kcal/1012 c e l l s ) (% of ash free dry veight) T a h i t i a n Hid-log 276 28 : : 19 : 53 I s o c h r y s i s 2 d - I - l i i 244 24 : : 64 : ; 12 6d-H-lim 213 27 : : 63 ; : 10 C. c a l c i t r a n s Hid-log 1st 173 27 ; : 30 ; : 43 6h-S-lim 2nd 138 19 : : 54 : 27 6h-Si-lim 3rd 143 27 : : 22 : 51 T. pseudonana Hid-log 2nd 106 18 : : 42 : ; 39 S h - H - l i i 3rd 82 22 : : 49 : 29 6h-Si-lim 1st 122 22 : : 31 : 47 a t the expense o f p r o t e i n , than the s i l i c a t e - s t a r v e d c e l l s L i p i d : c a r b o h y d r a t e : p r o t e i n r a t i o s were 19:54:27 i n n i t r a t e s t a r v e d c e l l s and 27:22:51 i n the s i l i c a t e - s t a r v e d c e l l s . 133 DISCUSSION N u t r i e n t s t a r v a t i o n of phytoplankton c e l l s a f f e c t e d the n u t r i t i o n a l v a l u e of s i n g l e s p e c i e s d i e t s f o r C. gigas l a r v a e . While s i l i c a t e s t a r v a t i o n produced the b e s t growth among treatments o f T. pseudonana (3H) ( F i g . 41), i t reduced the n u t r i t i o n a l v a l u e o f C. calcitrans ( F i g . 4 0 ) . N i t r a t e s t a r v a t i o n reduced the food v a l u e of both diatoms i n v e s t i g a t e d . Growth and s u r v i v a l were so poor w i t h T a h i t i a n Isochrysis t h a t e f f e c t s of n i t r a t e s t a r v a t i o n were d i f f i c u l t t o a s s e s s ( F i g . 39, T a b l e I I ) . Care must be taken when comparing chemical c o m p o s i t i o n among and w i t h i n phytoplankton s p e c i e s t o r e c o g n i z e which of the numerous v a r i a b l e s might have m o d i f i e d t h a t c o m p o s i t i o n . The dense, l a r g e volume a l g a l c u l t u r e s grown i n s e r i e s 1 were p r o b a b l y l i g h t - l i m i t e d . A comparison o f s p e c i f i c growth r a t e s between s e r i e s 1 and s e r i e s 2 c u l t u r e s a t s i m i l a r c e l l d e n s i t i e s p r o v i d e s evidence s u p p o r t i n g t h i s assumption. In s e r i e s 2 T a h i t i a n Isochrysis c u l t u r e s , growth r a t e s were c o n s i d e r a b l y f a s t e r even a t h i g h e r c e l l d e n s i t i e s . For example, a c u l t u r e which had j u s t exhausted i t s n i t r a t e s u p p l y , w i t h a c e l l d e n s i t y of 1.0-107 c e l l s - m L- 1, had a s p e c i f i c growth r a t e of 0.04-h- 1. In comparison, T a h i t i a n Isochrysis i n s e r i e s 1 grew at a s p e c i f i c growth r a t e o f 0.02 h- 1 a t a c e l l d e n s i t y o f 3.6-106 c e l l s mL- 1 and p r i o r t o n i t r a t e s t a r v a t i o n which o c c u r r e d a t 6 106 c e l l s mL- 1. 134 S i m i l a r comparisons may be made between T. pseudonana c u l t u r e s i n s e r i e s 1 and s e r i e s 2. S e r i e s 2 T. pseudonana c u l t u r e s were growing a t 0.10-h"1 a t a d e n s i t y g r e a t e r than 3•106 c e l l s - m L- 1, even when n i t r a t e s t a r v a t i o n was imminent. In c o n t r a s t , s e r i e s 1 T. pseudonana c u l t u r e s growing i n s i l i c a t e -l i m i t e d medium and p r i o r t o s i l i c a t e s t a r v a t i o n , showed a d e c l i n e i n growth r a t e from 0.10-h- 1 a t 1-106 c e l l s - m L- 1 t o 0. 07-h"1 a t 2.8 106 c e l l s mL- 1, and 0.004-h"1 a t 3.0-106 c e l l s mL"1. E vidence o f l i g h t l i m i t a t i o n i s a l s o seen i n growth r a t e s of C. calcitrans c u l t u r e s . S e r i e s 2 growth r a t e s of 0.15-h- 1 at 5-10 c e l l s mL ^ were h i g h e r than growth r a t e s a t a lower d e n s i t y o f 3•106 c e l l s mL- 1 i n s e r i e s 1 ( 0 . 1 0 - h- 1) . Thus f a t t y a c i d composition of c e l l s from s e r i e s 1 was i n f l u e n c e d s i m u l t a n e o u s l y by both n u t r i e n t and p r o b a b l y l i g h t l i m i t a t i o n , making i t d i f f i c u l t t o separate the e f f e c t s of t h e s e two f a c t o r s . The c o mposition of a l g a l c e l l s i n s e r i e s 2 c u l t u r e s (under h i g h l i g h t c o n d i t i o n s ) were assessed t o determine the e f f e c t s of n u t r i e n t s t a r v a t i o n which had a profound impact on gross composition of the phytoplankton a n a l y s e d i n t h i s s t u d y . 1. E f f e c t s of N u t r i e n t S t a r v a t i o n on Gross Composition i . N i t r a t e S t a r v a t i o n P r o t e i n r e d u c t i o n i n n i t r o g e n - s t r e s s e d c e l l s i s w e l l e s t a b l i s h e d (Fogg 1956) and i s a l s o shown by data i n t h i s 135 s t u d y . T a h i t i a n Isochrysis p r o t e i n d e c l i n e d t o 20% o f mid-log l e v e l s a f t e r 2 days o f n i t r a t e s t a r v a t i o n ( F i g . 5 ) . S i x hours of n i t r a t e s t a r v a t i o n reduced C. calcitrans and T. pseudonana p r o t e i n t o o n e - h a l f t h e i r mid-log v a l u e s ( F i g s . 7 and 9 ) . In response t o n i t r o g e n s t r e s s , a l g a l c e l l s d i r e c t metabolism towards the non-nitrogenous compounds, but s h i f t s i n carbohydrate and l i p i d quotas v a r y among and w i t h i n a l l phytoplankton c l a s s e s (Ben-Amotz e t a l . 1985, E l - F o u l y e t a l . 1985) and change w i t h d u r a t i o n of n i t r o g e n s t r e s s (Werner 1970, E l - F o u l y e t a l . 1985). No changes o c c u r r e d i n l i p i d and carbohydrate i n n i t r o g e n - s t r e s s e d T. pseudonana ( F i g . 9) which concurs w i t h the r e s u l t s o f S h i f r i n and Chisholm (1981) u s i n g the same s p e c i e s . C o n v e r s e l y , i n T a h i t i a n Isochrysis and C. calcitrans carbohydrate l e v e l s rose and l i p i d decreased when these c u l t u r e s were n i t r a t e - s t a r v e d ( F i g s . 5 and 7 ) . Thomas e t a l . (1984) a l s o found n i t r o g e n d e f i c i e n c y i n c r e a s e d carbohydrate y i e l d i n mass c u l t u r e s of T a h i t i a n Isochrysis. When S h i f r i n and Chisholm (1981) n i t r a t e - s t a r v e d 11 s p e c i e s of diatoms, l i p i d c ontent i n c r e a s e d o n l y s l i g h t l y and carbohydrate rose i n most s p e c i e s . A l g a l c e l l s may s h i f t from carbohydrate t o l i p i d s t o r a g e under prolonged n i t r o g e n s t a r v a t i o n (Werner 1970). T h i s p o s s i b i l i t y was not e x p l o r e d i n the c u r r e n t experiments s i n c e o l d e r c e l l s clumped t o g e t h e r and became u n d e s i r a b l e (too l a r g e a food p a r t i c l e ) f o r use as l a r v a l f o o d . I t may be necessary f o r c e l l d i v i s i o n t o stop b e f o r e 136 l i p i d s t a r t s accumulating i n phytoplankton ( P a r r i s h and Wangersky 1987). i i . S i l i c a t e S t a r v a t i o n In Cyclotella cryptica, an e s t a b l i s h e d diatom model f o r e a r l y s i l i c a t e s t a r v a t i o n work, p h o t o s y n t h e s i s i s not d i r e c t l y a f f e c t e d by a d e c l i n e i n s i l c a t e metabolism, but p h o t o s y n t h e s i s i s reduced when s i l i c a t e s t a r v a t i o n i n t e r f e r e s w i t h DNA s y n t h e s i s and consequently a r r e s t s p r o t e i n s y n t h e s i s ( V o l c a n i 1977, Werner 1977, V a u l o t e t a l . 1987). In t h i s s t u d y , T. pseudonana s y n t h e s i z e d p r o t e i n f o r a t l e a s t 6 h of s i l i c a t e s t a r v a t i o n . Presumably, carbon f i x a t i o n c o n t i n u e d i n the absence o f c e l l d i v i s i o n r e s u l t i n g i n i n c r e a s e d o r g a n i c s . In T. pseudonana both l i p i d and p r o t e i n s t o r e s i n c r e a s e d w h i l e carbohydrate l e v e l s f e l l ( F i g . 9 ) . However i n c r e a s e s i n l i p i d s t o r e s were not as l a r g e as those observed i n T. pseudonana by E n r i g h t (1984) . I t has been p r e v i o u s l y e s t a b l i s h e d t h a t i n Cyclotella cryptica, newly a s s i m i l a t e d carbon i s d i s p r o p o r t i o n a t e l y p a r t i t i o n e d i n t o l i p i d s and p r e v i o u s l y a s s i m i l a t e d carbon i s converted i n t o l i p i d s ( R o e s s l e r 1988). Carbohydrate, which was reduced under s i l i c a t e s t a r v a t i o n i n T. pseudonana may have c o n t r i b u t e d a carbon source f o r i n c r e a s e d l i p i d p r o d u c t i o n . L i p i d s y n t h e t i c r a t e s have a l s o been shown t o i n c r e a s e i n Cyclotella cryptica and Chaetoceros gracilis under s i l i c a t e l i m i t a t i o n (Werner 1970, E n r i g h t e t a l . 1986b, V a u l o t e t a l . 1987, R o e s s l e r 1988). Data of Taguchi e t a l . (1987), which a l s o show l i p i d i n c r e a s e s i n 137 Chaetoceros gracilis, Hantzchia sp. and Cyclotella sp., are suspect as the c e l l s were i n o c u l a t e d from the s t a t i o n a r y phase i n t o low l i g h t c u l t u r e s i n which pH v a l u e s became h i g h e r than 9.5. C o n v e r s e l y , i n s i l i c a t e - s t a r v e d C. calcitrans, a decrease i n l i p i d s and carbohydrates r e s u l t e d i n a l o s s o f o r g a n i c mass ( F i g . 7 ) . T h i s s p e c i e s grew ve r y r a p i d l y (4-5 d i v i s i o n s • d a y - 1 d u r i n g mid-log phase) under c o n d i t i o n s d e s c r i b e d and c u l t u r e s senesced s h o r t l y a f t e r they were sampled. A p p a r e n t l y , p h o t o s y n t h e s i s was q u i c k l y a r r e s t e d i n C. calcitrans, and l i p i d and carbohydrate were consumed i n the absence of carbon f i x a t i o n . S i l i c a t e requirements v a r y among s p e c i e s ( B r e z e z i n s k i 1985) as do n i t r o g e n and phosphorus requirements ( T e r r y 1980, Rhee and Gotham 1980, Wynne and Rhee 1986). Optimum n u t r i e n t r a t i o s can a l s o v a r y w i t h i n s p e c i e s depending on growth r a t e s , n i t r o g e n source and l i g h t q u a l i t y . Growth media must accommodate these requirements when c e l l s are used f o r comparative n u t r i t i o n a l s t u d i e s . The l i t e r a t u r e abounds wi t h s t u d i e s i n which phytoplankton from s t a t i o n a r y and e x p o n e n t i a l phases are assessed f o r r e l a t i v e food v a l u e , but the l i m i t i n g n u t r i e n t i s not i d e n t i f i e d . Knowledge of the elemental requirements of each s p e c i e s a l l o w s p r e p a r a t i o n of c u l t u r e media which are l i m i t e d i n a known, s p e c i f i c n u t r i e n t . 138 2. E f f e c t o f N u t r i e n t S t a r v a t i o n on L i p i d Composition Phytoplankton have a g r e a t v a r i e t y of f a t t y a c i d s which show taxonomic c h a r a c t e r i s t i c s among the a l g a l c l a s s e s (Chuecas and R i l e y 1969, Pohl and Zurheide 1979). The s a t u r a t e d and monounsaturated f a t t y a c i d s such as 16:0 and 16:1, are s t o r e d f o r energy i n the n e u t r a l l i p i d s , p a r t i c u l a r l y the t r i a c y l g l y c e r o l s ( F i s h e r and Schwarzenbach 1978). The p o l a r p h o s p h o l i p i d s conserve the p o l y u n s a t u r a t e d f a t t y a c i d s w i t h i n the s t r u c t u r a l components such as c e l l membranes (Pohl and Zurheide 1982, P i o r r e c k e t a l . 1984, Arao e t a l . 1987, Suen e t a l . 1987). C u l t u r e age a f f e c t s the p r o p o r t i o n s o f f a t t y a c i d s (Chu and Dupuy 1980) due t o g r o w t h - l i m i t i n g f a c t o r s such as n u t r i e n t d e p r i v a t i o n . i . N i t r a t e S t a r v a t i o n T a h i t i a n Isochrysis responded t o n i t r a t e s t a r v a t i o n i n a s i m i l a r way t o t h a t r e p o r t e d i n the l i t e r a t u r e . Accumulation o f l i p i d s i n n i t r o g e n - s t r e s s e d phytoplankton i s due t o de nova 1 4C f i x a t i o n w i t h l i t t l e c o n v e r s i o n of o t h e r compounds i n t o l i p i d s (Suen e t a l . 1987). N i t r a t e d e f i c i e n c y g e n e r a l l y s t i m u l a t e s i n c r e a s e d s y n t h e s i s of t r i g l y c e r i d e s composed l a r g e l y o f s a t u r a t e d and monounsaturated f a t t y a c i d s , w h i l e the p o l y u n s a t u r a t e d f a t t y a c i d s and g l y c o l i p i d s decrease wi t h the p o l a r l i p i d s (Pohl and Zurheide 1982, P i o r r e c k e t a l . 1984, Suen e t a l . 1987). In t h i s s t u d y , monounsaturated l e v e l s ( p r i m a r i l y 18:ln9) i n c r e a s e d i n T a h i t i a n Isochrysis by almost 50% w i t h i n 2 d of n i t r a t e s t a r v a t i o n , and a 139 p r o p o r t i o n a l drop (17%) o c c u r r e d i n p o l y u n s a t u r a t e d l e v e l s , l a r g e l y due t o reduced p r o p o r t i o n s of n 3 - p o l y u n s a t u r a t e s ( F i g . 16) . Predominant f a t t y a c i d s of T a h i t i a n Isochrysis were 14:0, 16:0, 18:ln9, 18:4n3 and 22:6n3, a l l w i t h l e v e l s g r e a t e r than 10%; and 18:2n6 and 18:3n3 w i t h l e v e l s between 3% and 5% ( F i g . 1 2 ) . The g e n e r a l composition agrees w i t h t h a t o f o t h e r r e s e a r c h e r s ( E n r i g h t 1984, P i l l s b u r y 1985, Helm and L a i n g 1987). N i t r a t e s t a r v a t i o n enhanced the l e v e l s of 16:0 and 18:ln9. A l l o t h e r major f a t t y a c i d s l e v e l s , except 14:0, ( i . e . p o l y u n s a t u r a t e s ) were reduced. In comparison, a study by E l - F o u l y e t a l . (1985) showed a 250% i n c r e a s e i n 18:1 and a decrease i n 16:0 o f Chlorella vulgaris and Scenedesmus acutus due t o n i t r o g e n s t a r v a t i o n . The important f a t t y a c i d 22:6n3 was f a i r l y r e s i l i e n t under n i t r a t e s t a r v a t i o n f a l l i n g o n l y 3% from a mid-log p r o p o r t i o n of 16%. S i m i l a r l y , Ben-Amotz e t a l . (1985) observed o n l y a s m a l l i n c r e a s e i n the f a t t y a c i d 22:6, from 13% t o 15%, i n Isochrysis a f t e r 10 days of s t a t i o n a r y growth, however i t i s not c l e a r what f a c t o r l i m i t e d growth of the c u l t u r e . Short-term n i t r a t e s t a r v a t i o n of the diatoms i n t h i s study caused an i n s i g n i f i c a n t change i n f a t t y a c i d c o m p o s i t i o n . The main f a t t y a c i d s of C. calcitrans were 14:0, 16:0, 16:ln7, 16:3n4, 18:4n3 and 20:5n3 ( F i g . 13). R e l a t i v e c omposition d i f f e r e d c o n s i d e r a b l y from t h a t observed by Waldock and Nascimento (1979) and Helm and L a i n g (1987). The main f a t t y 140 a c i d s o f T. pseudonana were 14:0, 16:0, 16:ln7, 16:2n4, 18:4n3, 20:5n3 and 22:6n3 ( F i g . 14). F i s h e r and Schwarzenbach (1978) and E n r i g h t (1984) r e p o r t e d f a t t y a c i d c o m p o s i t i o n which d e v i a t e d somewhat from t h a t d e s c r i b e d h e r e . T. pseudonana d i f f e r e d mainly i n the amount of 20:5n3 which was much lower i n the o t h e r s t u d i e s . D i f f e r e n c e s are p r o b a b l y a r e s u l t o f c u l t u r e c o n d i t i o n s used i n each s t u d y , but p o s s i b i l i t i e s o f g e n e t i c v a r i a t i o n among the c u l t u r e s should not be i g n o r e d . The o n l y change i n C. calcitrans f a t t y a c i d c o m p o s i t i o n observed i n e i t h e r s e r i e s was d u r i n g l i g h t - l i m i t e d growth i n s e r i e s 1. E f f e c t s c o u l d not be a t t r i b u t e d t o n i t r a t e l i m i t a t i o n . Large changes i n f a t t y a c i d c o m p o s i t i o n have been found i n diatoms i n o t h e r i n v e s t i g a t i o n s . E n r i g h t e t a l . (1986b) found t h a t n i t r a t e s t a r v a t i o n produced h i g h e r l e v e l s of s a t u r a t e d and monounsaturated f a t t y a c i d s i n Chaetoceros gracilis and 22:6n3 l e v e l s were reduced t o 20% of c o n t r o l v a l u e s . However E n r i g h t (1984) a l s o found t h a t when the same treatment was imposed on T. pseudonana, i t had a more subdued e f f e c t upon changes i n f a t t y a c i d c o m p o s i t i o n . In view of the s i z e of t h e i r c u l t u r e s (18 L) and the a v a i l a b l e i r r a d i a n c e (300 \i.E-m -sec ) i t i s p o s s i b l e t h a t they were a n a l y s i n g l i g h t - l i m i t e d c e l l s . i i . S i l i c a t e S t a r v a t i o n Although f a t t y a c i d s y n t h e s i s can i n c r e a s e more than 100% w i t h i n the f i r s t 6 t o 9 h of s i l i c a t e s t a r v a t i o n (Werner 141 1977), the f a t t y a c i d composition o f C. calcitrans and T. pseudonana was l a r g e l y u n a f f e c t e d i n t h i s study u n l e s s the c u l t u r e was h e l d u n t i l senescence was reached. The o n l y change seen i n h e a l t h y c e l l s was a drop i n C. calcitrans 22:6n3 from a mid-log l e v e l o f 2% t o 0.5% ( F i g . 2 8 ) . E n r i g h t e t a l . (1986b) found t h a t C. gracilis c e l l s showed a s u b s t a n t i a l i n c r e a s e i n monounsaturates and a decrease i n 20:5n3 and 22:6n3 when they were s i l i c a t e - s t a r v e d f o r a l o n g e r but u n d e f i n e d t i m e . T h e i r r e s u l t s are supported by a l a t e r study o f the same diatom (C. gracilis) by Mortensen e t a l . (1988). T o t a l n3 and long-chained h i g h l y u n s a t u r a t e d f a t t y a c i d s decreased w i t h i n c r e a s e d s i l i c a t e l i m i t a t i o n i n continuous c u l t u r e . E f f e c t s o f s i l i c a t e s t a r v a t i o n were much more pronounced i n s e r i e s 1, but o n l y a f t e r c e l l s had clumped and were s i n k i n g from s u s p e n s i o n . i i i . Phosphate S t a r v a t i o n U n f o r t u n a t e l y , e f f e c t s o f phosphate s t a r v a t i o n were not i n v e s t i g a t e d i n s e r i e s 2 and e f f e c t s i n s e r i e s 1 are superimposed w i t h p o s s i b l e l i g h t l i m i t a t i o n e f f e c t s . N e v e r t h e l e s s when comparing f a t t y a c i d c o mposition o f p h o s p h a t e - l i m i t e d c u l t u r e s w i t h t h a t o f c u l t u r e s l i m i t e d by o t h e r n u t r i e n t s i n s e r i e s 1, i t becomes apparent t h a t phosphate l i m i t a t i o n can have a profound e f f e c t on the f a t t y a c i d c o mposition of some s p e c i e s . 142 Phosphate l i m i t a t i o n had a s t r i k i n g e f f e c t on the f a t t y a c i d l e v e l s o f Thalassiosira pseudonana. The monounsaturate f r a c t i o n was e n r i c h e d a t the expense of the p o l y u n s a t u r a t e s ( F i g . 3 1 ) . Of the l a t t e r f r a c t i o n , o n l y a r a c h i d o n i c a c i d (20:4n6) i n c r e a s e d , from l e s s than 1% t o 4% ( F i g . 35). The n u t r i t i o n a l l y important 20:5n3 dropped t o 25% of i t s o r i g i n a l c o n c e n t r a t i o n o f 22% which was the most s i g n i f i c a n t e f f e c t o f n u t r i e n t l i m i t a t i o n observed i n any experiment i n t h i s study ( F i g . 3 3 ) . The s a t u r a t e d C16 f a t t y a c i d i n c r e a s e d by 100% compared t o i t s mid-log v a l u e o f 18%. P h o s p h a t e - l i m i t e d Chaetoceros calcitrans c u l t u r e d i n s e r i e s 1 d i d not show any s i g n i f i c a n t changes i n f a t t y a c i d composition r e l a t i v e t o the same s p e c i e s grown i n n i t r a t e - l i m i t e d medium. T a h i t i a n Isochrysis f a t t y a c i d c omposition was l e s s a f f e c t e d by phosphate l i m i t a t i o n than n i t r a t e l i m i t a t i o n . These r e s u l t s suggest t h a t Thalassiosira pseudonana m a i n t a i n s a s m a l l e r i n t e r n a l r e s e r v e o f polyphosphate which phytoplankton n o r m a l l y r e l y on t o f i l l phosphate needs d u r i n g d e f i c i e n c i e s ( K y l i n 1964). Polyphosphates c o n t a i n phosphoanhydride bonds which are thermodynamically e q u i v a l e n t t o those o f ATP (Harold 1966, Kulaev 1975) and can t h e r e f o r e f u l f i l l not o n l y the c e l l s s t r u c t u r a l requirements f o r phosphate but a l s o the e n e r g e t i c demands o f metabolism. I t i s l o g i c a l t h a t phosphate d e p l e t i o n should cause a r e d u c t i o n i n t h e lo n g chained p o l y u n s a t u r a t e s s i n c e these f a t t y a c i d s are normally a s s o c i a t e d w i t h c e l l membranes i n the 143 p h o s p h o l i p i d f r a c t i o n o f a l g a l c e l l s . T h e r e f o r e the m i c r o a l g a l c u l t u r i s t should ensure t h a t the n u t r i e n t r a t i o s of c u l t u r e s grown f o r marine animal food p r o v i d e phosphate-r e p l e t e c o n d i t i o n s d u r i n g a l l phases of growth t o prevent l o s s o f v a l u a b l e p o l y u n s a t u r a t e d compounds i n the d i e t . Optimum n i t r o g e n t o phosphorus r a t i o s are known t o v a r y from 7:1 t o 53:1 o r even as h i g h as 200:1 i n the case o f Asterionella japonica (Rhee and Gotham 1980, T e r r y 1980). The r a t i o can a l s o v a r y w i t h i n s p e c i e s depending on growth r a t e s and the n i t r o g e n s o u r c e . Thus, t o ensure t h a t c u l t u r e s are p h o s p h o r u s - r e p l e t e , the N:P r a t i o should be w e l l below the optimum N:P r a t i o f o r t h a t p a r t i c u l a r p h y t o p l a n k t e r . 3.. E f f e c t of L i g h t upon F a t t y A c i d Composition S i n c e l i g h t and n u t r i e n t l i m i t a t i o n probably i n f l u e n c e d s e r i e s 1 c u l t u r e s s i m u l t a n e o u s l y , e f f e c t s of n u t r i e n t l i m i t a t i o n c o u l d not be c l e a r l y separated from those of l i g h t l i m i t a t i o n i n t h i s p a r t of the s t u d y . However, by comparing r e s u l t s from s e r i e s 2 c u l t u r e s , which are presumed t o be o n l y n u t r i e n t -l i m i t e d , w i t h r e s u l t s from s e r i e s 1 c u l t u r e s ( n u t r i e n t - p l u s l i g h t - l i m i t e d ) i t was p o s s i b l e t o d i s c e r n some q u a l i t a t i v e e f f e c t s o f l i g h t l i m i t a t i o n on a l g a l chemical c o m p o s i t i o n . L i g h t l i m i t a t i o n i n s e r i e s 1 T a h i t i a n Isochrysis appeared t o have o n l y slowed the changes due t o n i t r a t e l i m i t a t i o n (because the c u l t u r e grew more slowly) w i t h one e x c e p t i o n . S a t u r a t e d f a t t y a c i d 14:0 decreased under l i g h t l i m i t a t i o n i n s e r i e s 1 ( F i g . 17). Otherwise f a t t y a c i d l e v e l s a t t a i n e d i n 144 s e r i e s 1, 240 h f o l l o w i n g n i t r a t e s t a r v a t i o n ( i . e . a t 450 h ) , were reached i n s e r i e s 2, 48 h a f t e r n i t r a t e was exhausted from the medium ( F i g . 18). In s e r i e s 1, phosphate s t a r v a t i o n i n c r e a s e d T a h i t i a n Isochrysis 18:2n6 and 18:4n3 l e v e l s i n c o n t r a s t t o decreases i n t h e s e two f a t t y a c i d s when t h i s s p e c i e s was n i t r a t e - s t a r v e d ( F i g . 17). Otherwise both s t a r v a t i o n treatments a f f e c t e d c o m p o s i t i o n s i m i l a r l y . L i g h t appears t o have had a g r e a t e r e f f e c t than n u t r i e n t s t a r v a t i o n on f a t t y a c i d composition o f the diatoms i n s e r i e s 1 c u l t u r e s . The p o l y u n s a t u r a t e d t o monounsaturated r a t i o s decreased moderately i n l i g h t - l i m i t e d C. calcitrans p r i o r t o n i t r a t e s t a r v a t i o n due t o a r e d u c t i o n i n the amount of 20:5n3 from 17% t o 13% ( F i g . 2 5 ) . Low l i g h t must account f o r these o b s e r v a t i o n s s i n c e no changes were observed i n h i g h - l i g h t , n i t r a t e - s t a r v e d c u l t u r e s i n s e r i e s 2. The moderate e l e v a t i o n of p r o p o r t i o n s o f 22:6n3 and 16:0 found under l i g h t l i m i t a t i o n were simpl y enhanced under n i t r a t e s t a r v a t i o n ( F i g s . 25 and 27) . The p o l y u n s a t u r a t e d t o monounsaturated r a t i o was a l s o d r a m a t i c a l l y reduced i n s e r i e s 1, l i g h t - l i m i t e d T. pseudonana. In the n 3 - f r a c t i o n f o r example, l e v e l s o f 20:5n3 were reduced by h a l f b e f o r e s i l i c a t e o r phosphate was exhausted from the medium ( F i g . 33). P r o p o r t i o n a l decreases a l s o o c c u r r e d i n 16:3n4, 18:4n3 and 22:6n3 w h i l e 16:0 and 16:ln7 i n c r e a s e d . I t i s i n t e r e s t i n g t o note t h a t E n r i g h t (1984) saw s i m i l a r f a t t y 145 a c i d i n c r e a s e s i n T. pseudonana c u l t u r e s grown under s i m i l a r l i g h t c o n d i t i o n s t o those o f experiments d e s c r i b e d h e r e , a l t h o u g h no change was seen i n 16:0 and 16:ln7. D i r e c t comparisons w i t h t h i s study are not v a l i d however, because c e l l d e n s i t i e s and c u l t u r e age o r l e n g t h o f n u t r i e n t d e p r i v a t i o n were not d e s c r i b e d i n E n r i g h t ' s (1984) work. These r e s u l t s are i n c o n t r a s t t o p r e v i o u s work by Mortensen e t a l . (1988) i n which l e v e l s o f h i g h l y u n s a t u r a t e d n3 f a t t y a c i d s o f C. gracilis were shown t o i n c r e a s e w i t h i n c r e a s e d l i g h t i n t e n s i t y from 83 t o 1395 \xE-m~2 • s e c- 1 i n 200 mL c u l t u r e s a t 28°C. However, c e l l s were h a r v e s t e d i n the l a t e l o g phase and i t i s unknown what f a c t o r s may have been l i m i t i n g . Changes i n l i g h t q u a l i t y can s t r o n g l y i n f l u e n c e a l g a l n u t r i e n t requirements by a l t e r i n g optimum N:P r a t i o s (Wynne and Rhee 1986). T h e r e f o r e f a c t o r s a f f e c t i n g c u l t u r e c o n d i t i o n s may i n t e r a c t . In n i t r a t e - l i m i t e d Scenedesmus sp. and Fragilaria crotonensis, the s u b s i s t e n c e c e l l quota f o r n i t r o g e n i n c r e a s e d as i r r a d i a n c e decreased (Rhee and Gotham 1981). R e d a l j e and Laws (1983) showed t h a t l i g h t i n t e n s i t y not o n l y a f f e c t e d 1 4C - i n c o r p o r a t e d i n t o p r o t e i n , p o l y s a c c h a r i d e and l i p i d o f Thalassiosira allenii, but i t a l s o a f f e c t e d the range of v a l u e s over which n u t r i e n t and temperature e f f e c t s v a r i e d . I n t e r a c t i v e e f f e c t s o f n u t r i e n t l i m i t a t i o n w i t h l i g h t l i m i t a t i o n may e x p l a i n why e f f e c t s o f l i g h t l i m i t a t i o n d i f f e r e d between s i l i c a t e - and p h o s p h a t e - l i m i t e d c u l t u r e s . 146 Combined phosphate and l i g h t l i m i t a t i o n i n C. calcitrans y i e l d e d s i m i l a r r e s u l t s t o n i t r a t e - s t a r v e d c e l l s a lthough they were more acute and the main changes o c c u r r e d o n l y upon senescence. E f f e c t s o f l i g h t i n t e n s i t y may be s p e c i e s dependent. O r c u t t and P a t t e r s o n (1973) a l s o found low l i g h t reduced 20:5n3 l e v e l s i n Nitzschia closterium, w h i l e Seto e t a l . (1984) r e p o r t e d t h a t Chlorella minutissima responsed t o h i g h l i g h t w i t h s l i g h t l y r a i s e d l e v e l s of 20:5n3. E f f e c t s of i r r a d i a n c e and l i g h t / d a r k c y c l e s have been demonstrated p r e v i o u s l y i n T. pseudonana ( F i s h e r and Schwarzenbach 1978). The authors c o n c l u s i o n s must be regarded as t e n t a t i v e however, because i r r a d i a n c e was not a d j u s t e d i n the l i g h t / d a r k c y c l e t o account f o r the. reduced l i g h t p e r i o d ( i . e . a v a l i d comparison can o n l y be made when the amount of l i g h t r e c e i v e d per day by the c u l t u r e s i s e q u a l ) . Furthermore, when a dark c y c l e was imposed on the continuous l i g h t c u l t u r e s a t v e r y l a t e l o g phase and without the b e n e f i t of a c o n t r o l c u l t u r e , the r e s u l t s may have been confounded by the e f f e c t s o f an u n d e f i n e d growth l i m i t i n g f a c t o r . L i g h t - l i m i t e d c e l l s were not used i n the l a r v a l d i e t p o r t i o n of t h i s study so the e f f e c t of phytoplankton c e l l s t r e a t e d i n t h i s manner on o y s t e r n u t r i t i o n i s unknown. In l i g h t o f the r e s u l t s p r e s e n t e d i n t h i s t h e s i s , a w e l l c o n t r o l l e d i n v e s t i g a t i o n o f the e f f e c t s of l i g h t - l i m i t e d phytoplankton growth on f a t t y a c i d composition and n u t r i t i o n a l v a l u e t o 147 f i l t e r f e e d e r s i s warranted. An understanding of t h i s problem i s i m p e r a t i v e when one c o n s i d e r s t h a t most commercial o y s t e r f a c i l i t i e s grow l a r g e volumes of a l g a e which are g e n e r a l l y l i g h t - l i m i t e d because a r t i f i c i a l i l l u m i n a t i o n i s used f o r v e r y l a r g e c u l t u r e volumes. 4. E f f e c t o f A l g a l Chemical Composition on L a r v a l Growth i . F a t t y A c i d s H i g h l y u n s a t u r a t e d l o n g chained f a t t y a c i d s are c o n s i d e r e d t o be e s s e n t i a l f o r growth of marine organisms (Jones e t a l . 1979, Langdon and Waldock 1981, Levine and S u l k i n 1984, Waldock and H o l l a n d 1984). Of the phytoplankton used i n t h i s s t u d y , the diatoms C. calcitrans and T. pseudonana were r i c h i n 20:5n3 and T a h i t i a n Isochrysis was r i c h i n 22:6n3. Repeated t r i a l s , not r e p o r t e d h e r e , were not s u c c e s s f u l i n s u s t a i n i n g growth and s u r v i v a l of l a r v a e on a d i e t of o n l y T a h i t i a n Isochrysis. T h i s suggests t h a t 20:5n3 may be a more s i g n i f i c a n t c o n t r i b u t o r t o the n u t r i t i o n of C. gigas than 22:6n3. Langdon and Waldock (1981) demonstrated improved growth of O. edulis j u v e n i l e s when Dunaliella tertiolecta was supplemented w i t h 22:6n3. The a l g a was v o i d o f both 20:5n3 and 22:6n3. Evidence f o r d i f f e r i n g f a t t y a c i d requirements i s p r e s e n t i n t h e l i t e r a t u r e . T a h i t i a n Isochrysis i s c o n s i d e r e d an e x c e l l e n t d i e t f o r Crassodoma gigantea (Hinnites multirugosus) l a r v a e and j u v e n i l e s (Cary et a l . 1981) and O. edulis, but a poor food f o r C. gigas (Helm and L a i n g 1987). A r a r e a n a l y s i s 148 o f e s s e n t i a l amino a c i d s f o r a b i v a l v e was performed on the mussel Mytilus californianus. I t was assumed t h a t l a c k o f r a d i o i s o t o p e l a b e l i n c o r p o r a t i o n i n t o an a r r a y o f amino a c i d s proved an i n a b i l i t y t o s y n t h e s i z e the compounds and thus i m p l i e d a requirement f o r them ( H a r r i s o n 1975). C. calcitrans and T. pseudonana were a b l e t o support good growth and a c c e p t a b l e l a r v a l s u r v i v a l e s p e c i a l l y when the former was h a r v e s t e d d u r i n g mid-log phase and t h e l a t t e r f o l l o w i n g s i l i c a t e s t a r v a t i o n ( F i g s . 40 and 41, T a b l e I I ) . However, the diatoms were r i c h i n 20:5n3 but impoverished i n 22:6n3 ( F i g s . 13 and 14). I f 22:6n3 i s r e q u i r e d a t l e a s t i n low l e v e l s , the d i e t o f s i l i c a t e - s t a r v e d C. calcitrans may have l o s t n u t r i t i o n a l v a l u e due t o a drop from 2% t o 0.5% of t h i s f a t t y a c i d . i i . Gross Composition S i l i c a t e - s t a r v e d T. pseudonana ( F i g s . 10 and 41) and mid-log C. calcitrans ( F i g s . 8 and 40) produced the h i g h e s t c a l o r i c c o n t ent and consequently, they r e s u l t e d i n the f a s t e s t l a r v a l growth o f C. gigas. Otherwise, when c a l o r i c v a l u e s were s i m i l a r , d i f f e r e n c e s a p p a r e n t l y l a y i n the carbohydrate c o n t e n t . N i t r a t e s t a r v a t i o n i n c r e a s e d the v a l u e o f C. calcitrans c e l l s over the e n e r g e t i c a l l y e q u i v a l e n t s i l i c a t e -s t a r v e d c e l l s w i t h a co r r e s p o n d i n g change i n a s h - f r e e c o m p o s i t i o n a l r a t i o s from 1:0.8:1.9 t o 1:2.8:1.4 l i p i d : c a r b o h y d r a t e : p r o t e i n ( F i g . 40, Tab l e V I ) . One may assume t h a t e s s e n t i a l requirements were p r e s e n t i n both d i e t s 149 and t h a t carbohydrate p r o v i d e d a s p a r i n g e f f e c t on e s s e n t i a l p r o t e i n s and l i p i d s . A d d i t i o n a l carbohydrate has been observed t o enhance the growth performance i n C. gracilis d i e t s a l s o ( E n r i g h t e t a l . 1986b). A f i n a l balance o f 1:1.5:1 ( l i p i d : c a r b o h y d r a t e : p r o t e i n ) improved growth o f O. edulis j u v e n i l e s whereas a d d i t i o n a l l i p i d r e s u l t e d i n reduced growth r a t e s o f j u v e n i l e s . The more compatible the n u t r i t i o n a l c o mposition of t h e a l g a l food i s t o the requirements of an organism, the s m a l l e r i s the amount o f energy u t i l i z e d i n i t s c o n v e r s i o n . Conversion e f f i c i e n c y i n a r o t i f e r Brachionus picatilis was r e g u l a t e d by t h e q u a l i t y , or b i o c h e m i c a l composition o f i t s a l g a l f o o d , Brachiomonas submarina var. pulsifera ( S c o t t 1980). Optimal e f f i c i e n c y was a c q u i r e d w i t h a 1:1:1 r a t i o o f the g r o s s components ( l i p i d : c a r b o h y d r a t e : p r o t e i n ) . R a t i o s producing the b e s t growth w i t h phytoplankton employed i n t h i s study were 1:1.1:1.6 and 1:1.4:2.1 l i p i d : c a r b o h y d r a t e : p r o t e i n (Table V I ) . 5. G r a z i n g Rates and R a t i o n S i z e As c a l o r i c i n t a k e was the primary f a c t o r c o n t r o l l i n g the outcome o f the d i e t t r i a l s , r a t i o n s i z e must have been inadequate. T h i s e x p l a i n s the decrease i n l a r v a l growth r a t e i n the f i n a l p e r i o d of the t r i a l s ( F i g s . 40 and 41, T a b l e s IV and V ) . Assessment of the n u t r i t i o n a l requirements o f the l a r v a e would n e c e s s i t a t e comparison of d i e t s of o p t i m a l c a l o r i c v a l u e but d e v i a n t chemical c o m p o s i t i o n . When employing m u l t i s p e c i e s d i e t s , t h i s c o u l d o n l y be a c c u r a t e l y 150 accomplished by e s t a b l i s h i n g i n g e s t i o n r a t e s and a s s i m i l a t i o n e f f i c i e n c i e s f o r a p p r o p r i a t e r a t i o n s . U n f o r t u n a t e l y , measurement of i n g e s t i o n r a t e s and a s s i m i l a t i o n e f f i c i e n c i e s f o r each a l g a l d i e t was beyond the scope o f t h i s s t u d y . S i n g l e s p e c i e s d i e t s a l l o w the r e s e a r c h e r t o assume t h a t i n g e s t i o n r a t e and d i g e s t i b i l i t y are equal among d i e t s and o p t i m a l r a t i o n s can be e s t a b l i s h e d f o r each treatment ( E n r i g h t e t a l . 1986b). However the assumption t h a t r a t e s are equal i s not n e c e s s a r i l y t r u e . D i g e s t i b i l i t y c o u l d be a f f e c t e d by changes i n chemical c o m p o s i t i o n , o r c e r t a i n c u l t u r e c o n d i t i o n s might produce p h a g o s t i m u l a n t s . For example, g r a z i n g r a t e s o f O. edulis l a r v a e were s t i m u l a t e d by the media o f Isochrysis galbana d u r i n g l a t e e x p o n e n t i a l and e a r l y s t a t i o n a r y growth phases (Wilson 1979). Optimal c a l o r i c i n t a k e may not be p o s s i b l e w i t h s p e c i f i c algae as b i v a l v e s have a maximum r a t e o f • i n g e s t i o n , governed by changes i n f i l t r a t i o n r a t e s f o r d i f f e r e n t p a r t i c l e c o n c e n t r a t i o n s (Winter 1973, E p i f a n i o and Ewart 1977, Sprung, 1984a). I n g e s t i o n r a t e s (mL-h- 1•g- 1) may d i f f e r f o r each a l g a l s p e c i e s ( E p i f a n i o and Ewart 1977). A maximum v a l u e f o r gr o s s growth e f f i c i e n c y approaches an optimal r a t i o n l e v e l s i n c e a f u r t h e r i n c r e a s e i n a v a i l a b l e food r e s u l t s i n the p r o d u c t i o n o f pseudofeces and h i g h l y reduced f i l t r a t i o n a c t i v i t y (Winter and Langton 1976, Malouf and Breese 1977). The maximum and minimum c o n c e n t r a t i o n o f a l g a l c e l l s i s a f f e c t e d by the s i z e ( c e l l volume) o f a l g a l c e l l s ( E p i f a n i o 151 and Ewart 1977). F i l t r a t i o n r a t e d e c l i n e s w i t h i n c r e a s i n g food c o n c e n t r a t i o n due t o an i n g e s t i o n c a p a c i t y , l i m i t e d by the volume o f food t h a t can pass through the gut and on the d i g e s t i b i l i t y o f the f o o d . In d i l u t e food c o n c e n t r a t i o n s , f i l t r a t i o n r a t e i s u n l i m i t e d by i n g e s t i o n and i n g e s t i o n r a t e i n c r e a s e s w i t h food c o n c e n t r a t i o n . For Mytilus edulis l a r v a e t r a n s i t i o n from f i l t r a t i o n - l i m i t e d i n g e s t i o n t o i n g e s t i o n - l i m i t e d f i l t r a t i o n o c c u r s between 5 and 10 c e l l s ( J L L- 1 of Isochrysis (Sprung 1984a). Net growth e f f i c i e n c i e s reached a p l a t e a u a t 10 c e l l s M.L"1 and remained c o n s t a n t up t o 40 c e l l s n L - 1 f o r mussel l a r v a e (Sprung 1984b). In the p r e s e n t work, c e l l c o n c e n t r a t i o n s of 20 c e l l s • M - L- 1 were i n s u f f i c i e n t as a d a i l y r a t i o n . G e n e r a l l y accepted f e e d i n g regimes f o r C. gigas employ a g r a d a t i o n o f f e e d i n g r a t i o n s up t o 50 t o 60 c e l l s ' n L - 1 . An o p t i m a l f e e d i n g regime c o u l d be a s s e s s e d by d e t e r m i n a t i o n of the optimum d a i l y r a t i o n and the maximum and minimum a l g a l c o n c e n t r a t i o n s p e r m i t t i n g e f f i c i e n t i n g e s t i o n ( E p i f a n i o and Ewart 1977). The most i d e a l s i t u a t i o n would r e q u i r e d e t e r m i n a t i o n of these parameters f o r each d i e t as d i f f e r e n t c e l l c omposition c o u l d cause changes i n d i g e s t i o n e f f i c i e n c y . C e l l c o n c e n t r a t i o n s c o u l d be maintained w i t h i n the r e q u i r e d range by r e d u c i n g f e e d i n g i n t e r v a l s . I n f o r m a t i o n on an optimal b i v a l v e d i e t may be gathered by combining knowledge of optimal f e e d i n g s t r a t e g i e s f o r a l g a l s p e c i e s i n which chemical composition i s manipulated by c o n t r o l l e d growth c o n d i t i o n s . N e v e r t h e l e s s , hypotheses 152 r e g a r d i n g the requirements o f s p e c i f i c compounds r e q u i r e s an a r t i f i c i a l d i e t i n which a l l n u t r i e n t s may be c o n t r o l l e d i n d i v i d u a l l y . U n t i l such technology has been s u c c e s s f u l l y a c q u i r e d , a l g a l d i e t s w i l l c o n t i n u e t o p l a y a c e n t r a l r o l e i n n u t r i t i o n a l i n v e s t i g a t i o n s o f b i v a l v e l a r v a e . Work pr e s e n t e d here serves t o demonstrate the ephemeral nature o f the chemical composition o f algae and the n e c e s s i t y o f the r i g i d c o n t r o l o f phytoplankton growth c o n d i t i o n s when pe r f o r m i n g comparative s t u d i e s . S i n g l e s p e c i e s o f algae are capable o f y i e l d i n g d i v e r s e growth r a t e s o f o y s t e r l a r v a e dependent upon the p h y s i o l o g i c a l s t a t e and c o r r e s p o n d i n g chemical c o m p o s i t i o n o f the a l g a l c e l l s . Continued r e s e a r c h w i t h a t t e n t i o n t o t h i s i n f o r m a t i o n may a l l o w an e v a l u a t i o n o f o t h e r l i m i t i n g n u t r i e n t s i n l a r v a l d i e t s such as amino a c i d s , s t e r o l s , v i t a m i n s and m i n e r a l s , i n a d d i t i o n t o f a t t y a c i d s . 153 SUMMARY AND CONCLUSIONS 1. The gro s s chemical composition o f u n i a l g a l c u l t u r e s o f the f l a g e l l a t e Isochrysis galbana (clone T-Iso) and two diatoms, Chaetoceros calcitrans and Thalassiosira pseudonana (clone 3H) were m o d i f i e d by n i t r a t e , phosphate, and/or s i l i c a t e l i m i t a t i o n . Two s e r i e s o f c u l t u r e s were ana l y s e d ; the f i r s t c o n s i s t e d o f l a r g e volume, l i g h t - l i m i t e d c u l t u r e s t h a t a l s o became n u t r i e n t -s t a r v e d , and the second was grown i n s m a l l e r h i g h - l i g h t c u l t u r e s t h a t o n l y became n u t r i e n t - s t a r v e d . 2. T a h i t i a n Isochrysis mid-log phase c e l l s had g r e a t e r c a l o r i c v a l u e than e i t h e r n i t r a t e - or p h o s p h a t e - l i m i t e d c e l l s . N i t r a t e s t a r v a t i o n f o r 2 days d e p l e t e d c e l l u l a r p r o t e i n and l i p i d t o 2 5% and 75% of mid-log content r e s p e c t i v e l y ; carbohydrate i n c r e a s e d by t h r e e - f o l d . N i t r a t e s t a r v a t i o n f o r another 4 days r e s u l t e d i n l i t t l e f u r t h e r change. 3. S i x hours o f n i t r a t e and s i l i c a t e s t a r v a t i o n reduced c a l o r i c content o f C. calcitrans. Under n i t r a t e s t a r v a t i o n , c e l l u l a r p r o t e i n and l i p i d were d e p l e t e d by 50%, a t the expense of a r i s e i n carbohydrate c o n t e n t . S i l i c a t e s t a r v a t i o n r e s u l t e d i n a 60% l o s s o f c a r b o h y d r a t e . 4. In c r e a s e s i n c e l l u l a r p r o t e i n and l i p i d o f 38% and 25% r e s p e c t i v e l y , i n c r e a s e d c a l o r i c content o f s i l i c a t e -s t a r v e d Thalassiosira pseudonana. Carbohydrate decreased by 25%. N i t r a t e s t a r v a t i o n d e p l e t e d c e l l p r o t e i n and added ash c o n t e n t . The major f a t t y a c i d s o f the n u t r i e n t - r e p l e t e a l g a e were: T a h i t i a n Isochrysis: 14:0, 16:0, 16:ln7, 18:ln9, 18:2n6, 18:3n3, 18:4n3,20:3n3, 22:5n6 and 22:6n3, Chaetoceros calcitrans: 14:0, 16:0, 16:ln7, 16:2n6, 16:2n4, 16:3n4, 18:4n3 20:5n3 and 22:6n3 Thalassiosira pseudonana: 14:0, 16:0, 16:ln7, 16:2n6, 16:2n4, 16:3n4, 18:4n3, 20:5n3 and 22:6n3. Changes i n T a h i t i a n Isochrysis f a t t y a c i d c o m p o s i t i o n due t o n i t r a t e s t a r v a t i o n proceeded more r a p i d l y under l i g h t s a t u r a t i o n ( s e r i e s 2 c u l t u r e s ) than when l i g h t was l i m i t e d i n s e r i e s 1. The polyunsaturated:monounsaturated r a t i o s dropped d r a m a t i c a l l y by the second day of s t a r v a t i o n and d i d not change t h e r e a f t e r . Responses t o phosphate l i m i t a t i o n (and l i g h t l i m i t a t i o n ) were s i m i l a r t o those o f n i t r a t e l i m i t a t i o n . N u t r i e n t d e p r i v a t i o n f o r 6 h had no s i g n i f i c a n t e f f e c t on the f a t t y a c i d composition of the diatoms i n s e r i e s 2. However, changes were observed i n s e r i e s 1, which c o u l d e i t h e r be a t t r i b u t e d t o l i g h t l i m i t a t i o n , o r , l o n g n i t r a t e , s i l i c a t e o r phosphate s t a r v a t i o n p e r i o d s . Polyunsaturated:monounsaturated f a t t y a c i d l e v e l s decreased w i t h time i n both diatoms i n s e r i e s 1. 155 8. The most d e c i s i v e e f f e c t on f a t t y a c i d c o mposition among a l l t h r e e a l g a l s p e c i e s o c c u r r e d i n T. pseudonana under phosphate l i m i t a t i o n . Under t h i s c o n d i t i o n , 20:5n3 decreased t o 25% o f i t s mid-log v a l u e and 16:0 i n c r e a s e d by 100%. 9. T a h i t i a n Isochrysis, r i c h i n 22:6n3, and p o s s e s s i n g v e r y l i t t l e 20:5n3, was a poor d i e t r e g a r d l e s s o f i t s n u t r i e n t s t a t u s . Y e t , the diatoms which had h i g h 20:5n3 l e v e l s and were impoverished o f 22:6n3 supported good growth and s u r v i v a l . T h i s suggests t h a t 20:5n3 may be a more s i g n i f i c a n t c o n t r i b u t o r than 22:6n3 t o the n u t r i t i o n o f C. gigas l a r v a e . 10. Among the diatoms, the most energy r i c h a l g a l c e l l s produced the f a s t e s t growing o y s t e r l a r v a e . Otherwise, when c a l o r i c v a l u e s were s i m i l a r , i n c r e a s e d carbohydrate may have spared any e s s e n t i a l p r o t e i n s and l i p i d s . I t i s t h e r e f o r e r e c o g n i z e d t h a t r a t i o n l e v e l s were l e s s than o p t i m a l i n the d i e t t r i a l s . FUTURE WORK An und e r s t a n d i n g o f the n u t r i t i o n a l requirements o f b i v a l v e s r e q u i r e s much f u r t h e r work. Past s t u d i e s have r e p e a t e d l y i g n o r e d the necessary procedures r e q u i r e d t o grow a l g a e of c o n s i s t e n t chemical c o m p o s i t i o n . Any f u t u r e work must i n c o r p o r a t e a b a s i c knowledge of a l g a l p h y s i o l o g y and s t r i n g e n t c o n t r o l o f c u l t u r e c o n d i t i o n s . A n a t u r a l e x t e n s i o n of the experiments p r e s e n t e d here i s t o determine the o p t i m a l r a t i o n l e v e l s o f each d i e t and f o r each stage o f o y s t e r l a r v a e . T h i s , t o g e t h e r w i t h more e x t e n s i v e a n a l y s e s o f the b i o c h e m i c a l composition of both the a l g a e and the l a r v a e , may h e l p t o i d e n t i f y c r i t i c a l n u t r i e n t s f o r o p t i m a l l a r v a l growth and s u r v i v a l . I d e a l l y , growth and s u r v i v a l s h o u l d be monitored through the c r i t i c a l stages of metamorphosis. Determination of a s s i m i l a t i o n e f f i c i e n c i e s would a l s o add v a l u a b l e i n s i g h t i n t o the most e f f e c t i v e d i e t c o m p o s i t i o n . Although f a t t y a c i d composition c o u l d not e x p l a i n any d i f f e r e n c e s i n food v a l u e of the microalgae used i n t h i s s t u d y , the f a t t y a c i d composition o f s e r i e s 1 i n d i c a t e d two p o t e n t i a l areas f o r continued r e s e a r c h . A range of i r r a d i a n c e s and phosphate l i m i t a t i o n may y i e l d d i e t s w i t h the g r e a t e s t v a r i a t i o n i n f a t t y a c i d c o mposition which c o u l d be t e s t e d i n d i e t t r i a l s . 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J a s p e r s ( E d s . ) , Proceedings of the Tenth European Symposium i n Marine B i o l o g y , V o l . 1, pp. 565-581, U n i v e r s a P r e s s , Wetteren. Wynne, D. and G.-Y. Rhee. 1986. E f f e c t s of l i g h t i n t e n s i t y and q u a l i t y on the r e l a t i v e N and P requirement (the optimum N:P r a t i o ) of marine p l a n k t o n i c a l g e . J . P l a n k t o n Res. 8: 91-103. 169 APPENDICES Appendix & Table 1: C a l o r i c value of T a h i t i a n I s o c h r y s i s sampled during a i d - l o g phase (ML), 01 a f t e r 2 or 6 d of n i t r a t e s t a r v a t i o n (-H 2 d , -H 6 d). Hean and standard d e v i a t i o n s of d u p l i c a t e c u l t u r e s (a and b) are presented. Brackets enclose percent of t o t a l c a l o r i e s . Energy e q u i v a l e n t s - kcal-10-12 c e l l s Treatment l i p i d Carbohydrate P r o t e i n T o t a l HL a 112.07 39.71 117.37 269.15 a 132.97 41.23 108.02 282.23 Hean 122.52 (44%) 40.47 (151) 112.70 (411) 275.69 STD 10.45 0.76 4.68 -N a 106.71 111.52 18.98 237.21 2 d a 93.32 120.83 29.87 244.02 Mean 100.01 (42%) 116.17 (48%) 24.42 (10%) 240.61 STD 6.69 4.66 5.44 b 81.92 139.88 21.93 • 243.72 b 90.35 136.89 21.84 249.08 Mean 86.13 (35%) 138.39 (56%) 21.88 (9%) 246.40 STD 4.22 1.49 0.05 -H a 77.55 122.00 17.46 217.01 6 d a 85.62 119.35 13.02 217.99 Mean 81.58 (38%) 120.67 (55%) 15.24 (7%) 217.50 STD 4.04 1.32 2.22 b 97.78 93.87 17.29 208.94 b 94.78 96.97 16.29 208.05 Mean 96.28 (46%) 95.42 (46%) 16.79 (8%) 208.49 STD 1.50 1.55 0.50 Appendix A Table 2: C a l o r i c value of Chaetoceros c a l c i t r a n s sampled during mid-log phase (ML) or a f t e r 6 h of n i t r a t e or s i l i c a t e s t a r v a t i o n (-N, - S i ) . Hean and standard d e v i a t i o n s of d u p l i c a t e c u l t u r e s (a and b) are presented. Brackets enclose percent of t o t a l c a l o r i e s . Energy e q u i v a l e n t s - kcal-10-12 c e l l s Treatment L i p i d Carbohydrate P r o t e i n T o t a l a 76.40 41.24 57.75 175.39 a 80.02 42.87 60.20 183.09 Hean 78.21 (44*) 42.06 (23*) 58.98 (33*) 179.24 STD 1.81 .81 1.23 b 65.25 46.25 54.82 166.32 b 73.68 40.41 52.34 166.43 Hean 69.46 (42*) 43.33 (26*) 53.58 (32*) 166.38 STD 4.22 2.92 1.24 41.41 40.80 61.01 60.88 25.79 23.77 128.20 125.44 Hean STD 41.10 0.30 (32*) 60.95 0.07 (48*) 24.78 1.01 (20*) 126.82 47.48 49.76 62.34 66.52 36.61 36.23 146.44 152.51 Hean STD 48.62 1.14 (33*) 64.43 2.09 (43*) 36.42 0.19 (24*) 149.48 - S i a 68.18 27.20 59.18 154.57 6 h a 63.57 26.41 57.63 147.62 Hean 65.88 (44*) 26.81 (18*) 58.41 (39*) 151.09 STD 2.30 0.39 0.78 b 56.27 22.20 57.28 135.75 b 56.91 26.84 53.18 136.93 Hean 56.59 (42*) 24.52 (18*) 55.23 (40*) 136.34 STD 0.32 2.32 2.05 172 fippendix & Table 3: C a l o r i c value of Chaetoceros c a l c i t r a n s sampled during a i d - l o g phase (HL) or a f t e r 6 h of n i t r a t e or s i l i c a t e s t a r v a t i o n (-N, - S i ) . Hean and standard d e v i a t i o n s of d u p l i c a t e c u l t u r e s (a and b) are presented. Brackets enclose percent of t o t a l c a l o r i e s . Energy e q u i v a l e n t s - k c a l / l e l 2 c e l l s Treatment L i p i d Carbohydrate P r o t e i n T o t a l HL a 34.41 41.07 35.65 111.13 a 35.03 43.15 37.39 115.57 Hean 34.72 (311) 42.11 (37%) 36.52 (32%) 113.35 STD 0.31 1.04 0.87 b 29.73 35.23 32.30 97.26 b 28.95 38.61 32.36 99.92 Hean 29.34 (30%) 36.92 (37%) 32.33 (33%) 98.59 STD 0.39 1.69 0.03 25.28 24.48 34.17 39.28 16.34 17.48 75.80 81.24 Hean STD 24.88 0.40 (32%) 36.72 2.55 (47%) 16.91 0.57 (21%) 78.52 34.07 33.98 29.46 28.23 22.12 21.69 85.65 83.90 Hean STD 34.02 0.04 (40%) 28.85 0.62 (34%) 21.90 0.21 (26%) 84.77 S i - a 43.53 29.26 44.71 117.50 l i s a 41.83 32.13 44.60 118.55 Mean 42.68 (36%) 30.69 (26%) 44.66 (38%) 118.02 STD 0.85 1.44 0.06 b 46.19 31.84 49.38 127.40 b 46.69 32.14 45.91 124.74 Hean 46.44 (37%) 31.99 (25%) 47.64 (38%) 126.07 STD 0.25 0.15 1.73 173 Appendix A Table 4: C e l l u l a r weight of gross biochemical components i n s e r i e s 2 T a h i t i a n I s o c h r y s i s . Cultures vere grovn i n n i t r a t e - l i m i t e d medium and harvested at a i d - l o g phase (ML) and a f t e r 2 and G days of n i t r a t e s t a r v a t i o n (-N 2 d or 6 d ) . Hean and standard d e v i a t i o n s of d u p l i c a t e c u l t u r e s (a and b) are presented. T r i p l i c a t e ash determinations vere used to c a l c u l a t e mean ash. Treatment C e l l u l a r l e i g h t of Components ( p g - c e l l - 1 ) C e l l l e i g h t L i p i d Carbohydrate P r o t e i n Ash T o t a l p g - c e l l - 1 n Hono/Oligo Poly Organics ML a 13.31 .00 9.23 28.63 51.17 a 15.79 .53 9.06 26.35 51.73 Hean 55.79 15 14.55 .53 9.15 27.49 6.79 51.72 STD 5.52 1.24 .00 .09 1.14 .07 -H a 12.67 .77 25.17 4.63 43.24 2 d a 11.08 .75 27.35 7.28 46.47 Hean 55.79 15 11.88 .76 26.26 5.96 5.35 44.85 STD 5.52 .80 .01 1.09 1.33 .01 b 9.73 .95 31.58 5.35 47.61 b 10.73 .80 31.04 5.33 47.89 Hean 55.79 15 10.23 .87 31.31 5.34 5.25 47. 75 STD 5.52 .50 .08 .27 .01 .25 -8 a 9.21 .70 27.67 4 .26 41.84 6 d a 10.17 .73 27.02 3.17 41.10 Hean 55.79 15 9.69 .72 27.35 3.72 6.63 41.47 STD .85 .52 .48 .02 .32 .54 .20 b 11.61 1.26 20.57 4.22 37.66 b 11.26 1.48 21.07 3.97 37.78 Hean 55.79 15 11.43 1.37 20.82 4.10 9.89 37.72 STD 5.52 .18 .11 .25 .12 .39 174 ippendix A Table 5: C e l l u l a r weight of gross b i o c h e a i c a l components i n s e r i e s 2 Chaetoceros c a l c i t r a n s . C u l t u r e s were grovn i n n i t r a t e - l i m i t e d or s i l i c a t e - l i m i t e d media and harvested a t mid-log phase (HL) and a f t e r 6 hours of n u t r i e n t s t a r v a t i o n (-N 6 h, - S i 6 h ) . Hean and standard d e v i a t i o n s of d u p l i c a t e c u l t u r e s (a and b) are presented. T r i p l i c a t e ash determinations vere used to .ca l c u l a t e mean ash. Treatment C e l l u l a r l e i g h t of Components (pg-cell-1) C e l l l e i g h t L i p i d Carbohydrate P r o t e i n Ash T o t a l pg«cell-l n Mono/Oligo Poly Organics HL a 9 j T Tl 1768 1 4 J 9 32.75 a 9.50 .56 9.41 14.68 34.16 Hean 42.12 13 9.29 .73 9.05 14.38 9.07 33.45 STD 5.94 .21 .17 .36 .30 .25 b 7.75 .99 9.77 13.37 31.88 b 8.75 .48 8.91 12.77 30.92 Hean 42.12 13 8.25 .74 9.34 13.07 9.92 31.40 STD 5.94 .50 .25 .43 .30 .72 -H a 4.92 1.08 13.11 6.29 25.40 6 h a 4.85 1.12 13.04 5.80 24.80 Hean 42.12 13 4.88 1.10 13.07 6.04 14.29 25.10 STD 5.94 .04 .02 .03 .25 .03 b 5.64 1.49 13.00 8.93 29.07 b 5.91 1.25 14.22 8.84 30.22 Hean 42.12 13 5.77 1.37 13.61 8.88 8.75 29.64 STD 5.94 .14 .12 .61 .05 .17 - S i a 8.10 .88 5.44 14.44 28.86 6 h a 7.55 .99 5.15 14.06 27.75 Hean 42.12 13 7.82 .94 5.30 14.25 12.16 28.30 STD 5.94 .27 .05 .15 .19 .15 b 6.68 .62 4.54 13.97 25.82 b 6.76 1.24 5.00 12.97 25.97 Hean 42.12 13 6.72 .93 4.77 13.47 14.28 25.89 STD 5.94 .04 .31 .23 .50 .03 175 Appendix A Table 6: C e l l u l a r weight of gross biochemical components i n T h a l a s s i o s i r a pseudonana. Cultures vere grown in n i t r a t e - l i m i t e d or s i l i c a t e - l i m i t e d medium and harvested at a i d - l o g phase (MLl and a f t e r 6 hours of n u t r i e n t s t a r v a t i o n (-H 6 h, - S i ( h). Hean and standard d e v i a t i o n s of d u p l i c a t e c n l t u r e s (a and b) are presented. T r i p l i c a t e ash determinations vere used to c a l c u l a t e mean ash. Treatment C e l l u l a r Weight of Components ( p g * c e l l - l ) C e l l Weight L i p i d Carbohydrate P r o t e i n Ash T o t a l p g ' c e l l - 1 n Hono/Oligo Poly Organics HL a 4.09 .58 8.97 8.70 22.33 a 4.16 .35 9.68 9 .12 23.31 Hean 29.58 14 4.12 .47 9.33 8.91 7 .85 22.82 STD 4.14 .04 .12 .36 .21 1.09 b 3.53 .37 7.83 7.88 19.60 b 3.44 .43 8.55 7.89 20.31 Hean 29.58 14 3.49 .40 ' 8.19 7.89 8.64 19.96 STD 4.14 .05 .03 .36 .01 .10 -H a 3.00 .50 7.45 3.99 14.94 6 h a 2.91 .36 8.77 4.26 16.30 Hean 29.58 14 2.96 .43 8.11 4.12 10.45 15.62 STD 4.14 .05 .07 .66 .14 1.05 b 4.05 .57 6.28 5.39 16.29 b 4.04 .51 6.06 5.29 15.89 • Hean 29.58 14 4.04 .54 6.17 5.34 10.43 16.09 STD 4.14 .01 .03 .11 .05 .00 - S i a 5.17 .00 6.80 10.91 22.88 6 h a 4.97 .49 6.99 10.88 23.32 Hean 29.58 14 5.07 .49 6.90 10.89 5.41 23.34 STD 4.14 .10 .00 .09 .01 .05 b 5.49 .32 7.08 12.04 24.93 b 5.55 .29 7.19 11.20 24.22 Hean 29.58 14 5.52 .31 7.13 11.62 5.98 24.57 STD 4.14 .03 .02 - .05 .42 .13 176 Appendix B. Table 1: Changes over t i n e i n the f a t t y a c i d s expressed as a percentage of the t o t a l , i n s e r i e s 1 T a h i t i a n I s o c h r y s i s grown i n n i t r a t e - l i m i t e d or phosphate-limited median. Mid-log values are mean + 1 S.D. |n=2). The f a t t y a c i d s I and N are unknowns. F a t t y A c i d A l g a l Sample Mid-log N - l i m i t e d P - l i m i t e d 85 h 185 h 250 h 330 h 400 h 450 h 85 h 185 h 250 h 355 h 400 h 14:0 19.0i0.1 19.1 17.2 14.8 12.8 11.8 11 18.9 18.1 16.9 13.8 13.6 I 0.3 0.4 0.4 0.4 0.4 0 0.3 0.4 0.5 0.5 0.5 16:0 12.9i0.3 13.1 13.4 13.6 16.0 16.7 19 12.7 12.4 12.3 13.4 14.5 fl 0.9i0.1 0.8 0.5 0.5 0.4 0 1.0 1.4 1.2 1.2 1.1 16 ln7 2.6+0.0 2.7 2.1 1.9 2.0 2.5 2 2.6 3.5 3.6 4.0 4.1 16 2n6 0.3 0.1 0.1 0.1 0.3 0.3 0.3 0.2 0.2 16 2n4 0.6*0.0 0.6 0.5 0.4 0.3 0.4 0 0.6 0.9 1.0 0.8 0.8 16 3n4 0.3*0.0 0.2 0.3 0.3 0.3 0.3 0. 0.2 0.3 0.3 0.4 0.4 16 3n3 0.1 0.1 0.2 0.1 0. 0.1 0.1 16 3nl 0.2 0.1 0.1 0.1 0.2 0.3 0.2 0.2 0.1 16 4nl 0.1 0.2 0.2 0.2 0.1 0 0.1 0.2 0.2 0.3 0.3 18 l n l 3 0.3*0.1 0.4 0.2 0.3 0.4 0.5 0. 0.3 0.2 0.2 0.2 0.3 18 ln9 12.4*0.6 12.9 18.1 20.1 21.1 22.1 24. 12.0 11.5 12.2 15.3 18.1 18 ln7 1.6*0.1 1.6 1.9 2.3 1.9 1.9 1. 1.5 2.1 2.3 2.4 2.5 18 2n9 0.1 0.1 0.1 0.2 0.1 0. 0.1 0.1 0.2 0.1 0.1 18 2n6 4.2*0.4 4.5 3.9 4.8 3.7 3.3 2. 3.9 3.4 3.9 4.5 4.4 18 2n4 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 18 3n6 0.2 0.1 0.2 0.1 0.1 0. 0.2 0.3 0.2 0.2 0.2 18 3n3 3.9*0.2 3.8 3.9 4.4 4.3 3.9 3. 4.0 4.9 5.3 5.2 5.1 11 4n3 14.0*0.3 14.1 11.6 10.4 9.9 10.6 8. 13.8 16.4 15.3 15.2 15.0 19 ? 0.2*0.1 0.3 0.4 0.5 0.6 0.5 0 0.2 0.2 0.2 0.2 0.3 20 In9 0.6*0.1 0.7 0.2 0.1 0.1 0.2 0 0.5 0.3 0.2 0.27 0.2 20 2f 0.3*0.0 0.3 0.1 0.3 0.2 0.2 0.1 20 2n6 0.1 0.1 0.1 0.1 0.2 0. 0.1 20 A 0.5*0.0 0.5 0.1 0.1 0 0.5 0.3 0.2 0.1 20 3n6 0.2 0.2 0.2 0.2 0.2 0. 0.2 0.1 0.1 0.1 20 4n6 0.1 0.2 0.3 0.4 0.3 0. 0.1 0.2 0.3 0.3 0.1 20 3n3 2.0*0.1 1.9 0.4 0.3 0.2 0.4 0. 2.0 1.0 0.9 0.5 0.3 20 5n3 1.1*0.2 1.2 0.4 0.5 0.7 0.7 0. 1.0 0.7 0.7 0.7 0.7 22 0 0.3*0.0 0.3 0.5 0.7 1.0 1.2 1. 0.2 0.2 0.3 0.5 0.7 22 ln9 0.1 0.1 0.2 0.3 0.4 0. 0.2 0.2 0.2 0.3 0.4 21 5n3 0.5*0.0 0.5 0.6 0.4 0.4 0.7 0. 0.5 0.5 0.5 0.4 0.3 22 4n6 0.3*0.1 0.3 0.2 0.5 0.2 0.3 0. 0.2 0.1 0.1 0.2 0.3 22 5n6 2.1*0.1 2.2 4.6 4.9 2.4 2.2 1. 2.0 1.7 1.7 2.0 1.7 22 5n3 0.2 0.2 0.2 0.2 0.2 0. 0.2 0.1 0.2 0.2 0.1 22 6n3 16.8*0.2 16.9 14.5 13.9 15.2 15.7 13. 0 16.7 14.2 15.2 14.2 12.1 Saturated 32.1*0.4 32.4 31.2 29.2 30.0 29.7 32. 5 31.8 30.7 29.4 27.6 28.8 Honounsat'd 17.5*0.9 18.2 22.8 24.8 25.8 27.7 30 3 17.2 17.9 18.8 20.1 25.6 Polyunsat'd T o t a l 46.1*1.6 46.7 42.5 42.3 39.3 39.8 33 3 46.9 46.0 46.8 45.7 42.3 n3 38.3*0.9 38.5 31.8 30.1 31.1 32.5 26 8 38.3 37.8 38.1 36.3 33.6 n6 6.6*0.6 7.0 9.4 11.0 7.1 6.5 5 6 6.9 6.0 6.5 7.5 6.9 177 Appendix B. Table 2a: The f a t t y acids expressed as a percentage of the t o t a l , i n s e r i e s 2 T a h i t i a n I s o c h r y s i s c u l t u r e s and harvested e i t h e r at n i d - l o g phase or a f t e r 2 days of n i t r a t e s t a r v a t i o n . T r i p l i c a t e measurements vere taken at each phase and reported as mean • 1 S.D. The f a t t y a c i d s I and 8 are unknowns. F a t t y Acid A l g a l Sample Mid-log 2 day K -starved 1 2 3 xts 1 2 3 xts 14:0 12.7 18.9 12.6 14.7+3.6 11.2 15.1 14.6 13.7+2.1 I .1 .2 .1 0.2*0.1 .2 .2 .1 0.2+0.0 16:0 13.3 11.3 12.3 12.3il.O 18.9 21.6 19.6 20.0 1.4 1.2 1.0 1.4 1.2+0.2 .3 .4 .4 0.4+0.0 16:ln7 1.2 2.2 1.2 1.6*0.6 2.4 2.2 2.0 2.2+0.2 16:2n6 .3 .6 .3 0.4*0.2 .2 .1 .1 0.1+0.0 16:2n4 .2 .4 .3 0.3+0.1 .3 .3 .3 0.3+0.0 16:3n4 .1 .3 .1 0.2*0.1 .2 .2 .1 0.2+0.0 16:3n3 .1 .2 .1 0.2*0.1 .1 .1 0.1+0.0 18:lnl3 .7 .2 .5 0.4+0.3 .5 .5 .5 0.5+0.1 18:ln9 17.4 11.7 14.2 14.4*2.8 22.8 18:ln7 1.7 1.5 1.9 1.7+0.2 1.8 1.8 1.6 1.8+0.1 18:ln5 0.1 0.1 0.1 0.1+0.0 0.1 0.1 0.1 0.1+0.0 18:2n9 .1 .1 .1 0.1+0.0 .1 .1 .1 0.1+0.0 18:2n6 6.5 3.4 6.6 5.5+1.8 4.00 5.0 5.3 4.7+0.7 18:3n6 .6 .5 .6 0.6+0.1 .1 .1 0.1+0.1 18:3n3 4.0 4.2 4.4 4.2+0.2 3.0 2.7 3.3 3.0+0.3 18:4n3 14.4 15.5 14.8 14.9+0.5 10.2 7.8 8.8 9.0+1.1 19:? .2 .2 .2 0.2+0.0 .5 .5 .4 0.5+0.0 20:ln9 .7 .9 1.2 0.9+0.3 .2 .2 .2 0.2+0.0 20:2f .4 .4 .4 0.4+0.0 20:a .5 .6 .5 0.5+0.1 20:3n3 2.1 2.5 2.4 2.3+0.2 .5 .4 .4 0.4+0.1 20:5n3 .5 .9 .6 0.7+0.2 .3 .3 .3 0.3+0.0 22:0 .4 .3 .3 0.3+0.1 1.2 1.1 1.0 1.1+0.1 22:ln9 .4 .3 .2 0.3+0.1 21:5n3 .2 .4 .2 .4 0.3+0.1 22:4n6 .4 .2 .4 0.4+0.1 .3 .2 0.2+0.1 22:5n6 2.5 2.6 2.8 2.6+0.2 2.4 2.3 2.1 2.3+0.2 22:5n3 .4 .3 .3 0.3+0.1 .2 .2 0.1+0.1 22:6n3 15.8 16.6 16.6 16.3+0.5 12.9 11.8 11.5 12.1+0.8 Saturated 27.4+4.7 34.8+3.7 Honounsat'd 19.1+4.1 . 24.1+1.2 Polyunsat'd T o t a l 49.5+4.5 33.1+3.6 n3 38.9+1.8 25.2+2.4 n6 9.4+2.3 7.4+1.1 178 Appendix B. Table 2b: The f a t t y a c i d s expressed as a percentage of the t o t a l , i n s e r i e s 2 T a h i t i a n I s o c h r y s i s c u l t u r e s and starved of n i t r a t e for 4 or 6 days. T r i p l i c a t e measurements vere taken at each phase and reported as mean t 1 S.D. The f a t t y a c i d s I and N are unknovns. Pat t y Acid A l g a l Sample 4 day N-starved 6 day ( l-starved 1 2 3 xts 1 2 3 x+s 14:0 14.57 17.17 15.59 15.78il.31 11.31 13.92 12.84 12.69+1.31 I .18 .20 .17 0.18*0.02 .14 .23 .21 0.19+0.05 16:0 17.62 17.73 20.94 . 18.76il.89 17.24 20.22 16.71 18.06+1.89 N .34 .50 .35 0.40*0.09 .57 .23 .47 0.42+.0.17 16:ln7 2.90 2.88 2.47 2.75*0.24 2.78 3.38 3.63 3.26+0.44 16:2n6 .19 .16 .09 0.15*0.05 .17 .16 .24 0.19+0.04 16:2n4 .38 .41 .31 0.37*0.05 .36 .72 .50 0.53+0.18 16:3n4 .23 .16 .15 0.18+0.04 .16 .20 .25 0.20+0.05 16:3n3 .05 .09 .06 0.07*0.02 .04 .05 0.03+0.03 18:l n l 3 .28 .27 .45 0.33+0.10 .51 .33 .36 0.40+0.10 18:ln9 22.63 20.97 25.50 23.03*2.29 24.88 21.69 21.39 22.65+1.93 18:ln7 1.52 1.47 1.52 1.50+0.03 1.56 2.01 1.76 1.78+0.23 18:2n9 .11 .11 .11 0.11*0.00 .09 .15 0.12+0.04 18:2n6 4.18 3.66 4.79 4.21+0.57 4.57 2.91 2.95 3.48+0.95 18:3n6 . .13 .14 0.14*0.01 .16 .10 0.13+10.04 18:3n3 3.20 3.10 3.14 3.15+0.05 3.04 2.86 2.60 2.83+0.22 18:4n3 10.24 11.28 7.88 9.80*1.75 9.56 9.66 11.83 10.35+1.28 19:?? .46 .54 .45 0.48+0.05 .54 .71 .60 0.62+0.09 20:ln9 .19 .26 .17 0.21*0.05 .20 .17 0.12+0.11 20:4n6 .18 .26 .18 0.18*0.00 .27 .21 .10 0.19+0.09 20:3n3 .50 .44 .32 0.42*0.09 .40 .27 .49 0.39+0.11 20:5n3 .53 .34 .23 0.37+0.15 .30 .36 .52 0.39+0.11 22:0 .9 1.04 1.06 1.00*0.09 1.26 1.43 1.29 1.33+0.09 22:ln9 .48 .68 .52 0.56+0.11 21:5n3 .53 .40 .26 0.40+0.14 .60 .64 .77 0.67+0.09 22:4n6 .28 .20 .21 0.21*0.04 .32 .27 .46 0.35+0.10 22:5n6 2.17 2.10 1.86 2.04+.0.16 2.42 2.11 2.11 2.21+0.18 22:5n3 .19 .19 .13 0.17*0.03 .22 .21 .23 0.22+0.01 22:6n3 . 13.27 12.42 9.88 11.86+1.76 13.15 12.99 14.10 13.41+0.64 Saturated 35.54+3.29 32.08+3.29 Honounsat'd 27.82+2.71 28.86+3.00 Polyunsat'd T o t a l 33.83+4.91 35.65*4.20 n3 26.24*3.99 28.29+2.49 n6 6.93+0.83 6.51+1.44 179 Appendix B. Table 3a: Changes over t i a e i n the f a t t y acids expressed as a percentage of the t o t a l , i n s e r i e s 1 Chaetoceros c a l c i t r a n s grovn i n n i t r a t e - l i m i t e d medium. Nid-log values are mean • 1 S.D. (n=2). The f a t t y a c i d s I and N are unknown. F a t t y Acid A l g a l Sample Mid-log N - l i m i t e d 58 h 97 h 119 h 150 h 174 h 14:0 14.45il.48 15.52 17.74 18.94 18.80 18.66 I 0.49+0.04 0.51 0.55 0.53 0.53 0.52 16:0 9.43t0.28 9.63 13.73 14.79 . 15.58 15.70 N 1.96+0.54 2.34 1.72 1.17 2.02 1.19 16:ln7 23.68+2.79 25.65 28.43 29.36 28.09 27.63 16:ln5 0.57+0.04 0.54 0.42 0.35 0.32 0.30 17:0 0.90+0.16 1.01 0.81 0.54 0.58 0.60 16:2n6 3.52+0.98 2.83 2.40 2.40 2.39 2.63 16:2n4 7.86+0.80 7.29 5.61 5.44 5.23 5.52 16:3n4 7.55+1.24 8.43 5.28 4.52 3.68 3.68 16:3n3 0.09+0.00 0.09 0.12 0.14 0.14 0.15 16:3nl 0.14+0.05 0.17 0.14 0.05 16:4nl 0.73+0.47 0.39 0.50 0.46 0.39 0.43 18:lnl3 0.42+0.21 0.57 0.47 0.49 . 0.57 0.57 . 1 8 : l n l l 0.07 0.17 18:ln9 0.33+0.27 0.52 0.50 0.58 0.52 0.43 18:ln7 0.53+0.23 0.69 0.53 0.51 0.62 0.61 18:ln5 0.10+0.02 0.11 0.13 0.14 0.29 0.32 18:2n6 0.35+0.15 0.45 0.35 0.30 0.29 0.24 18:2n4 0.13 0.13 0.37 0.40 0.44 0.48 18:3n3 0.10+0.05 0.13 0.13 0.11 0.11 0.09 18:3nl 0.25+0.09 0.31 0.95 1.01 1.09 1.14 18:4n3 1.21+0.21 1.06 0.26 0.17 0.12 0.21 19:? 0.11+0.02 0.12 0.20 0.20 0.22 0.24 20:4n6 0.14 0.14 0.42 0.45 0.49 0.50 20:5n3 16.03+1.68 17.22 13.56 12.91 12.05 12.39 22:0 0.07 0.10 0.10 0.12 0.21 22:5n3 0.07+0.03 0.05 0.08 0.08 0.11 0.11 22:6n3 0.65+0.08 0.71 0.89 1.02 1.30 1.38 Saturated 24.78+1.92 26.23 32.38 34.38 35.08 35.17 Honounsat'd 25.63+3.56 28.08 30.48 31.43 30.48 30.23 Polyunsat'd T o t a l 37.43+5.22 38.53 29.47 27.94 26.35 27.38 n3 18.15+2.05 19.26 15.04 14.43 13.83 14.33 n6 3.87+1.13 3.42 3.17 3.15 3.17 3.37 180 Appendix 8. Table 3b: Changes ovei time i n the f a t t y a c i d s expressed as a percentage of the t o t a l , i n s e r i e s 1 Chaetoceros c a l c i t r a n s grovn i n phosphate-limited or s i l i c a t e - l i m i t e d medium. The f a t t y acids I and N are unknowns. F a t t y A c i d A l g a l Sample P - l i m i t e d S i - l i m i t e d 58 h 97 h 119 h 150 h 99 h 119 h 150 h 14:0 13.40 14.89 13.72 18.80 13.68 13.86 9.86 16:0 9.23 7.49 10.72 15.58 6.69 7.06 13.39 I 1.57 1.80 2.46 2.02 3.69 2.69 10.19 16:ln7 21.71 30.31 30.34 28.09 28.28 29.07 15.58 16:ln5 0.59 0.44 0.39 0.32 0.38 0.30 0.22 17:0 0.78 0.78 0.45 0.58 1.67 1.31 1.19 16:2n6 4.22 3.22 2.79 1.59 3.84 3.93 1.08 16:2n4 8.42 8.10 7.23 4.57 8.90 9.41 2.62 16:3n4 6.67 7.57 5.52 2.63 5.94 5.28 1.08 16:3n6 0.09 0.15 0.16 0.13 0.23 0.21 0.92 16:3n3 0.10 0.17 0.11 0.20 0.15 0.10 0.39 16:4nl 1.66 1.06 0.64 0.27 1.06 0.91 0.23 18:0 0.14 0.05 0.10 0.18 0.16 18: l n l 3 0.28 0.13 0.46 0.97 0.17 0.19 0.70 18:ln9 0.14 0.29 0.45 0.11 0.18 0.44 18:ln7 0.37 0.60 2.06 2.43 0.81 1.29 7.27 18:ln5 0.08 0.14 0.15 0.23 T 0.15 0.12 0.18 0.21 0.57 18:2n6 0.24 0.63 0.45 0.64 0.40 0.31 0.17 18:2n4 0.31 0.39 0.30 0.41 0.56 0.15 18:3n4 0.27 0.08 0.12 0.07 0.07 0.09 18:3n3 0.06 0.27 0.18 0.17 0.18 0.17 0.29 18:3nl 0.18 0.70 0.86 0.57 0.67 0.80 0.25 18:4n3 1.35 0.68 0.33 0.28 0.55 0.43 0.12 19:? 0.09 0.17 0.18 0.16 0.22 0.24 0.09 20:4n6 0.08 0.37 0.49 0.41 0.22 0.30 0.09 20:4n3 0.13 0.07 0.07 0.07 20:5n3 14.84 16.12 12.52 6.98 15.64 15.21 3.87 22:0 0.11 0.19 0.10 0.25 22:5n3 0.09 0.10 0.09 0.08 0.24 22:6n3 0.59 0.52 0.73 0.52 0.67 0.75 0.24 Saturated 23.59 23.16 25.05 35.15. 22.24 18.41 24.85 Honounsat'd 23.17 31.91 33.70 32.07 29.82 30.85 32.07 Polyunsat'd T o t a l 38.99 39.87 32.58 19.54 39.07 37.45 11.83 n3 17.16 17.76 13.97 8.31 17.34 16.73 5.15 n6 4.63 4.37 3.85 2.77 4.69 4.75 2.26 181 Appendix B. Table 4a: The f a t t y a c i d s "expressed as a percentage of the t o t a l , i n s e r i e s 2 Chaetoceros c a l c i t r a n s c u l t u r e s and harvested at e i t h e r mid-log phase or a f t e r 2 h or 6 h of n i t r a t e s t a r v a t i o n . T r i p l i c a t e or d u p l i c a t e measurements taken at starved phases are reported as mean t l S.D. The f a t t y a c i d s I and N are unknowns. Patty A c i d A l g a l Sample Hid-log 2 h N-starved 6 h N-starved 1 1 2 3 xts 1 2 x+s 14:0 15.51 16.47 15.19 16.33 16-00*0.70 15.88 14.66 15.27+0.86 I .47 .53 .45 .48 0.49*0.04 .52 .45 0.49+0.05 18:0 11.07 12.97 12.24 12.75 12.65*0.37 16.99 11.38 14.19+3.97 N 1.74 1.47 1.96 1.82 1.75*0.25 .96 1.46 1.21+0.35 l S : l n 7 ' 21.47 24.75 21.21 25.66 23.87*2.35 22.20 22.79 22.50+0.42 16:ln5 .48. .54 .51 .52 0.52*0.02 .78 .45 0.62+0.23 17:0 .88 .54 .56 .48 0.53*0.04 .43 .41 0.42+0.01 16:2n6 1.41 .43 1.60 1.45 1.16+0.64 .81 1.24 1.03+0.30 16:2n4 2.15 2.19 2.20 2.03 2.14*0.10 1.88 1.83 1.86+0.04 16:3n4 9.23 9.05 8.37 8.67 8.70+0.34 5.00 6.98 5.99+1.40 16:3n3 .19 .27 .22 .29 0.26*0.04 .18 .24 0.21+0.04 16:3nl .12 .07 .07 0.07+0.00 .08 0.08 16:4nl .28 .28 .20 .13 0.20+0.08 .05 .17 0.11+0.08 18:0 .11 .09 .14 .10 0.11+0.03 .13 .07 0.10*0.04 18:lnl3 .24 .28 .55 .31 0.38+0.15 .52 .24 0.38+0.20 18:ln9 .29 .30 .70 .38 0.46+0.21 1.14 .34 0.74*0.57 18:ln7 .28 .32 .56 .33 0.40+0.14 .58 .29 0.44+0.21 18:2n6 .34 .34 .49 .62 0.48+0.14 1.80 .43 1.12*0.97 18:3n4 .41 .47 .50 .46 0.48+0.02 1.62 .40 1.01+0.86 18:3n3 .04 .04 .06 .11 0.07+0.04 .16 .07 0.12*0.06 18:4n3 4.81 6.24 4.68 5.46 5.46+0.78 5.22 4.96 5.09+0.18 20:4n6 .10 .08 .11 .11 0.10+0.02 .53 .07 0.30+0.33 20:c .23 .24 .37 .22 0.28+0.08 .48 .18 0.33+0.21 20:4n3 .23 .24 .37 .22 0.28+0.08 .48 .18 0.33*0.21 20:5n3 15.53 16.62 15.84 17.65 16.70+0.91 13.29 14.60 13.95+0.93 22:0 .05 .09 .09 .10 0.09*0.01 .09 .06 0.08+0.02 22:5n3 .38 .50 1.63 .09 0.74*0.80 .90 1.30 1.10+0.28 Z .17 .14 .49 .07 0.23+0.22 .31 .40 0.36+0.06 22:6n3 2.07 1.38 3.35 1.08 1.94*1.23 1.76 3.39 2.58+1.15 Saturated 24.72 29.38+1.15 30.06*5.02 Honounsat'd 22.47 25.63*2.87 24.68+1.63 Polyunsat'd T o t a l 37.29 38.82+5.22 34.06+6.83 n3 23.25 25.49*3.88 22.66+2.85 n6 1.85 1.74+0.80 2.45+1.60 182 Appendix B. Table 4b: The f a t t y acids expressed as a percentage of the t o t a l , i n s e r i e s 2 Chaetoceros c a l c i t r a n s c u l t u r e s grovn i n s i l i c a t e - U n i t e d medium. T r i p l i c a t e or d u p l i c a t e measurements taken at starved phases are reported as mean ±1 S.D. The f a t t y acids I and N are unknowns. F a t t y Acid A l g a l Sample 2 h S i - s t a r v e d 6 h Si - s t a r v e d 1 2 3 xts 1 2 x+s 14:0 14.78 15.27 14.63 14.89t0.33 13.41 15.96 14.69+1.80 I .49 .49 .54 0-51*0.03 .48 .54 0.51+0.04 16:0 10.08 10.06 11.79 10.64t0.99 12.81 10.73 11.77+1.47 N 1.52 2.40 2.32 2.08+0.49 3.60 1.38 2.49+1.57 16:ln7 22.02 23.89 20.40 22.10+1.75 27.89 21.83 24.86+4.29 16:ln5 .61 .72 .61 0.65+0.06 .37 .66 0.52+0.21 17:0 .67 .61 .55 0.61+0.06 .59 .67 0.63+0.06 16:2n6 1.71 2.37 1.53 1.87.+0.44 1.48 1.93 1.71t0.32 16:2n4 2.83 2.88 2.21 2.64+0.37 2.61 2.92 2.77+0.22 16:3n4 9.73 10.22 8.49 9.48+0.89 6.07 10.62 8.35+3.22 16:3n3 .23 .35 .37 0.32*0.08 .25 .40 0.33+0.11 16:4nl .18 .29 .31 0.26+0.07 .19 .46 0.33+0.19 18:0 .15 .11 .11 0.12+0.02 .14 .13 0.14+0.01 18:lnl3 .38 .29 .31 0.33+0.05 .71 .19 0.45+0.37 18:ln9 .42 .28 .33 0.34+0.07 .70 .22 0.46+0.34 18:ln7 .99 .40 1.18 0.86+0.41 2.08 .31 1.20+1.25 18:2n6 .18 .22 .19 0.20+0.02 .25 .18 0.22+0.05 18:3n4 .37 .28 .38 0.34+0.06 .25 .32 0.29t0.05 18:4n3 5.01 4.34 5.54 4.96+0.60 3.62 5.80 4.71+1.54 20:c .32 .20 .24 0.25+0.06 .31 .21 0.26t0.07 20:4n3 .32 .20 .24 0.25+0.06 .31 .21 0.26+0.07 2 0:5 n 3 16.49 17.40 14.36 16.08tl.56 13.25 16.99 15.12+2.64 22:5n3 .06 1.16 .25 0.49+0.59 .81 .74 0.78t0.05 Z .29 .15 0.22+0.10 .45 .19 0.32+0.18 22:6n3 .06 1.16 0.25 0.49+0.59 .81 .74 0.78+0.05 Saturated . 26.26+1.40 27.23+3.34 Honounsat'd 24.28*2.34 27.49+6.46 Polyunsat'd T o t a l 37.38+5.33 36.57*8.51 n3 22.59*3.48 22.76*4.46 n6 2.07+0.46 1.93*0.37 183 Appendix B. Table 5: Changes over t i a e i n the f a t t y a c i d s expressed as a percentage of the t o t a l , i n s e r i e s 1 T h a l a s s i o s i r a pseudonana c u l t u r e s grown i n s i l i c a t e - l i m i t e d or n i t r a t e - l i m i t e d medium. The f a t t y a c i d s I and N are unknowns. The a i d - l o g sample i s represented by the Si (6 h sample. F a t t y A c i d A l g a l Sample P - l i m i t e d S i - l i m i t e d 62 h 108 h 158 h 207 h 66 h 92 h 163 h 14:0 8.59 8.43 6.12 7.83 7.41 7.36 7.04 I 0.65 0.69 0.67 0.68 0.65 0.72 0.75 16:0 30.30 39.01 36.42 36.67 . 18.64 29.56 22.11 N 0.70 0.92 0.38 0.52 2.86 0.88 1.51 16:ln7 22.34 25.78 28.92 26.28 20.69 31.96 26.34 16:ln5 0.11 0.13 0.11 0.09 0.16 0.21 0.24 17:0 0.29 0.08 0.13 0.49 0.23 0.36 16:2n6 1.09 0.96 0.92 0.99 2.06 1.33 1.49 16:2n4 2.15 0.99 1.02 0.88 2.62 2.19 2.26 16:3n4 6.05 2.94 1.81 2.44 7.70 3.90 5.98 16:3n3 0.06 0.07 0.07 0.11 0.10 0.10 16:4nl 0.14 0.08 0.04 0.06 0.41 0.17 0.30 18:lnl3 0.37 1.29 2.82 2.08 0.26 0.34 0.47 18:ln9 0.80 3.06 2.75 3.71 0.34 0.26 0.55 18:ln7 0.21 0.21 1.96 0.34 0.26 0.49 3.34 18:ln5 0.07 0.13 0.19 0.13 0.14 0.80 18:2n9 0.06 0.17 0.20 0.23 18:2n6 0.53 0.63 0.71 0.94 0.40 0.18 0.55 18:3•6 1.50 0.96 0.73 1.05 0.60 0.14 0.14 18:3nl 0.08 0.08 0.11 0.10 0.29 0.27 0.39 18:4n3 7.66 2.64 1.65 1.95 4.33 2.41 1.88 20:3n6 0.14 0.10 0.23 0.22 0.11 0.07 20:4n6 1.20 2.12 2.17 3.62 0.61 0.23 0.19 20:4n3 0.17 0.06 0.11 0.09 0.28 0.12 0.12 20:5n3 12.26 5.87 5.67 5.98 21.70 12.32 11.13 22:6n3 1.97 1.64 1.77 2.19 4.44 2.14 2.45 Saturated 39.18 47.52 42.67 44.50 26.54 37.15 29.51 Honounsat'd 23.90 30.60 36.75 32.63 21.71 33.40 30.94 Polyunsat'd , T o t a l 35.00 19.30 17.17 20.81 45.66 25.50 27.05 n3 • 22.06 10.27 9.27 10.28 30.86 17.09 15.68 n6 4.46 4.77 4.72 6.82 3.78 1.88 2.44 184 appendix B. Table 6a: The i a t t y a c i d s expressed as a percentage of the t o t a l , i n s e r i e s 2 T h a l a s s i o s i r a pseudonana c u l t u r e s and harvested at e i t h e r Hid-log phase or a f t e r 2 h or 6 h of n i t r a t e s t a r v a t i o n . T r i p l i c a t e or d u p l i c a t e measurements vere taken at each phase and reported as mean t 1 S.D. The f a t t y a c i d s I and N are unknowns. F a t t y k i d A l g a l Sample Hid-log 1 2 3 x±s 2 h N-starved 1 2 x+s 6 h N 1 -starved 2 x+s 14:0 8.13 8.24 6.24 7.54+1.12 7.79 8.08 7.94+0.21 7.62 6.84 7.23+0.55 I .63 .62 .59 0.61+0.02 .53 .54 0.54+0.01 .55 .50 0.53+0.04 16:0 24.42 22.93 30.93 26.10.+4.25 29.20 29.29 29.25+0.06 27.56 33.76 30.66+4.38 N .40 .28 .89 0.52+0.32 .88 .53 0.71+0.25 .48 .55 0.52+0.05 16:ln7 26.02 25.28 21.42 24.24+2.47 25.87 26.07 25.97+0:14 24.99 25.40 25.20+0.29 16:ln5 .17 .16 .16 0.16.+0.01 .15 .17 0.16+0.01 .16 .16 0.16+0.00 17:0 .51 .47 .48 0.49+0.02 .36 .34 0.35+0.01 .29 .36 0.33+0.05 16:2n6 1.50 2.07 1.52 1.70+0.32 1.89 1.49 1.69+0.28 1.54 1.24 1.39+0.21 16:2n4 1.96 2.25 1.90 2.04+0.19 1.61 1.39 1.50+0.16 1.32 1.21 1.27+0.08 16:3n4 6.00 6.16 6.11 6.09+0.08 5.30 4.23 4.77+0.76 4.14 3.56 3.85+0.41 16:4nl .31 .23 .60 0.38+0.19 .19 .16 0.18+0.02 .26 .21 0.24+0.04 18:lnl3 .32 .38 .27 0.32+0.06 .54 .53 0.54+0.01 .60 .56 0.58+0.03 18:ln9 .40 .41 .30 0.37+0.06 .76 1.02 0.89+0.18 1.09 .63 0.86+0.33 18:ln7 .29 .35 .31 0.32+0.03 .34 .34 0.34+0.00 .39 .38 0.39+0.00 18:2n6 .24 .22 0.23+0.02 .31 .42 0.37+0.08 .59 .31 0.45+0.20 18:3n4 .33 .37 0.35+0.03 .33 .57 0.45+0.17 .73 .40 0.57+0.23 18:4n3 4.25 3.32 4.18 3.92+0.52 2.56 3.33 2.95+0.54 3.52 3.55 3.54+0.02 20:4n6 .32 .48 0.40+0.11 .31 .66 0.49+0.25 1.00 .42 0.71+0.41 20:4n3 .37 .36 .23 0.32+0.08 .27 .33 0.30+0.04 .36 .28 0.32+0.06 20:5n3 19.00 19.41 18.36 18.92+0.53 16.24 15.77 16.01+0.33 17.39 14.33 15.86+2.16 22:6n3 3.32 4.16 3.43 3.64+0.46 2.58 2.68 2.63+0.07 3.18 2.74 2.96+0.31 Saturated Honounsat'd Polyunsat'd T o t a l n3 n6 34.13+5.39 25.41+2.63 37.99+2.53 26.80+1.59 2.33+0.45 37.54+0.28 27.90+0.34 31.34+2.70 21.89+0.98 2.55+0.61 38.22+4.98 27.19+0.65 31.16+4.13 22.38+2.54 2.55+0.82 185 Appendix B. Table 6b: The f a t t y a c i d s expressed as a percentage of the t o t a l , i n s e r i e s 2 T h a l a s s i o s i r a pseudonana c u l t u r e s and harvested at 2 h or 6 h of s i l i c a t e s t a r v a t i o n . T r i p l i c a t e or quadruplicate measurements were taken at each phase and reported as mean • 1 S.D. The f a t t y acids I and N are unknowns. Patty Acid A l g a l S a i p l e 2 h S i - s t a r v e d 6 h S i - s t a r v e d 1 2 3 x t s 1 2 3 4 Xts 14:0 7.34 7.82 8.19 7.78i0.43 8.37 6.84 8.59 8.08 7.97*0.78 I .66 .64 .63 0.64*0.02 .62 .71 .61 .64 0.65*0.05 16:0 26.61 24.13 27.41 27.05i2.76 24.95 27.78 27.15 26.43 26.58*1.22 M 1.14 .88 .73 0.92*0.21 .94 .52 .69 .45 0.65*0.22 16:ln7 26.73 25.77 26.10 26.20i0.49 28.69 27.31 27.14 29.05 28.05i0.96 16:ln5 .17 .19 .14 0.17+0.03 .19 .16 .18 .17 0.18*0.01 17:0 .57 .54 .52 0.54*0.03 .46 .45 .41 .48 0.45*0.03 16:2n6 1.16 1.57 1.26 1.33i0.21 1.41 1.03 1.20 1.10 1.19*0.17 UiM hU 1,11 2,05*0,11 2.18 1.99 1.96 2.15 2.07*0.11 16:3n4 4.74 5.29 4.77 4.93+0.31 4.23 4.61 3.83 4.11 4.20*0.32 16:3n3 .13 .09 .12 0.11+0.02 .11 .12 .14 .08 0.11*0.03 16:4nl .44 .28 .30 0.34+0.09 .17 .58 .14 .25 0.29*0.20 . 18:0 .08 .09 .08 0.08+0.01 .07 .07 .07 .07 0.07*0.01 18:lnl3 .33 .34 .51 0.39*0.10 .34 .30 .32 .32 0.32+0.02 18:ln9 .36 .33 .40 0.36+0.04 .40 .32 .36 .37 0.36+0.03 18:ln7 .38 .32 .31 0.34*0.04 .27 .28 .21 .23 0.25+0.03 18:2n6 .28 .29 .31 0.29*0.02 .27 .30 .29 .31 0.29*0.02 18:3n4 .25 .22 0.24+0.02 .31 .20 .29 .28 0.27+0.05 18:3nl .19 .15 0.17+0.03 .16 .14 .13 0.14+0.02 18:4n3 4.46 4.28 4.05 4.26*0.21 3.57 4.96 3.82 4.10 4.1h0.61 20:4n6 .22 .20 0.21+0.01 .34 .16 .32 .31 0.28*0.08 20:4n3 .25 .24 .25 0.25*0.01 .24 .21 .22 .27 0.24+0.03 20:5n3 17.76 18.28 17.10 17.7h0.59 16.60 16.11 14.58 16.49 15.95i0.93 22:6n3 3.41 3.56 3.15 3.37+0.21 3.06 3.22 2.31 2.95 2.89.+0.40 Saturated 35.45*3.23 35.07i2.04 Honounsat'd 28.46*0.70 29.16il.05 Polyunsat'd T o t a l 31.89*1.91 32.16t2.99 n3 22.33*1.11 23.43*2.02 n6 1 1.83*0.24 1.76*0.27 

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