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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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E F F E C T S OF NUTRIENT  LIMITATION ON BIOCHEMICAL  THREE MICROALGAE AND  COMPOSITION OF  THEIR FOOD VALUE TO OYSTER LARVAE,  CRASSOSTREA GIGAS By G A I L SIBBALD CALDERWOOD B.Sc,  The U n i v e r s i t y  A T H E S I S SUBMITTED  of Victoria,  1980  I N P A R T I A L FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department o f Oceanography  We  accept t h i s to  thesis  the required  as conforming standard  THE UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t , 1989 © Gail  S i b b a l d C a l d e r w o o d , 1989  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2/88)  6cf  S.  /f33  ABSTRACT Three m i c r o a l g a e commonly used i n b i v a l v e a q u a c u l t u r e were grown i n u n i a l g a l b a t c h c u l t u r e and h a r v e s t e d f o r chemical a n a l y s e s a t r i g o r o u s l y monitored stages o f n i t r a t e , phosphate, o r 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 a n a l y s e d ; t h e f i r s t was l i g h t - l i m i t e d .  Light-saturated  c u l t u r e s o f t h e second s e r i e s were used i n o y s t e r  larvae  growth t r i a l s . C e l l u l a r c a l o r i c v a l u e was reduced by n u t r i e n t s t a r v a t i o n o f T a h i t i a n Isochrysis  galbana  phosphate) and Chaetoceros due t o d e p l e t i o n  (clone T-Iso) ( n i t r a t e o r calcitrans  o f c e l l u l a r p r o t e i n and l i p i d ,  carbohydrate content increased. pseudonana and  ( n i t r a t e or s i l i c a t e )  (clone 3H) i n c r e a s e d  Conversely,  F a t t y a c i d composition  Thalassiosira  c e l l u l a r p r o t e i n and l i p i d  decreased carbohydrate under s i l i c a t e  r e s u l t i n g i n an i n c r e a s e  while  starvation  i n f i n a l c a l o r i c content.  (percent  of the t o t a l f a t t y acids) of  l i g h t - s a t u r a t e d , n u t r i e n t - l i m i t e d diatoms was remarkably s t a b l e through 6 hours of s t a r v a t i o n .  I. galbana  (T-Iso)  polyunsaturated:monounsaturated f a t t y a c i d r a t i o s dropped dramatically s e r i e s 2.  by t h e second day o f n u t r i e n t s t a r v a t i o n i n  Changes observed i n s e r i e s 1 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 long s t a r v a t i o n  periods.  Polyunsaturated:monounsaturated f a t t y a c i d r a t i o s  declined  w i t h time i n both diatoms i n s e r i e s 1.  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 composition among a l l t h r e e a l g a l  iii  species during  occurred  under phosphate l i m i t a t i o n  w h i c h 20:5n3 d e c r e a s e d f r o m 22%  increased  I. galbana  two-fold  (T-Iso)  f r o m 19%  was  to  t o 6%  the  belief  more s i g n i f i c a n t n u t r i t i o n a l is  found i n high  i n the  diatoms.  increased  a c i d 20:5n3 may  i n I. galbana  be  The  (T-Iso)  When c a l o r i c have  I t i s recognized  l e v e l s were s u b - o p t i m a l i n t h e  diet  trials.  a  latter but  l a r v a e grew f a s t e s t when f e d  c a r b o h y d r a t e may  f a s t e r growth.  larvae  of nutrient status,  e n e r g y w h i c h s p a r e d e s s e n t i a l p r o t e i n s and permitting  16:0  f a c t o r t h a n 22:6n3.  concentration C. gigas  fatty  most e n e r g y r i c h d i a t o m t r e a t m e n t s . were s i m i l a r ,  and  a p o o r d i e t f o r C. gigas  that the  pseudonana  37%.  compared t o b o t h d i a t o m d i e t s r e g a r d l e s s supporting  o f T.  not  the  values  provided  lipids, that  thereby  ration  iv  TABLE OF CONTENTS Page Abstract L i s t of Tables L i s t of Figures Acknowledgements  i i v i ix xvii  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 C h e m i c a l C o m p o s i t i o n 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. Nitrogen Starvation 9 ii. Silicon Starvation 11 iii. 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. Series 1 17 ii. Series 2 17 iii. A l g a l Analyses 19 2. L a r v a l C u l t u r e " 20 3. C h e m i c a l A n a l y s i s 21 i. Fatty Acid Analysis 22 ii. Gross Composition Analyses 23 RESULTS I.  .' 26 A l g a l G r o w t h i n S e r i e s 1 a n d S e r i e s 2 C u l t u r e s . 26 1. Series 1 26 2. S e r i e s 2 27 II. Biochemical 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. 3.  B.  Chaetoceros Thalassiosira  41 46  calcitrans pseudonana  Fatty A c i d Composition 1. C o m p a r i s o n o f M i d - L o g P r o f i l e s .... 2. C o m p a r i s o n 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 . . a. T a h i t i a n Isochrysis i. Fatty Acid Class P r o f i l e ii. Essential Fatty Acids iii. F a t t y A c i d Peaks  b.  Chaetoceros i. ii. iii.  c.  Thalassiosira i. ii. iii.  calcitrans  Fatty Acid Class P r o f i l e Essential Fatty Acids F a t t y A c i d Peaks  pseudonana  Fatty Acid Class P r o f i l e Essential Fatty Acids F a t t y A c i d Peaks  46 51 61 61 61 66 66  79 79 84 85  98 99 99 104  V  III.  Growth o f O y s t e r L a r v a e 1. T a h i t i a n Isochrysis  2. 3. IV.  117 117  122 122  Chaetoceros calcitrans Thai assiosira pseudonana  Factors  A f f e c t i n g Crassostrea  gigas  Growth  DISCUSSION 1. 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 G r o s s Composition i. Nitrate Starvation ii. Silicate Starvation 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 i. Nitrate Starvation ii. S i l i c a t e Starvation iii. Phosphate S t a r v a t i o n 3. E f f e c t o f L i g h t upon F a t t y A c i d C o m p o s i t i o n 4. E f f e c t o f A l g a l C h e m i c a l C o m p o s i t i o n on L a r v a l Growth i. Fatty Acids ii. Gross Composition 5. G r a z i n g R a t e s and R a t i o n S i z e  129 132 133 133 135 137 137 139 140 142 146 146 147 148  SUMMARY AND CONCLUSIONS  152  FUTURE WORK  155  REFERENCES  157  APPENDICES A. C a l o r i c v a l u e and c e l l u l a r w e i g h t o f g r o s s b i o c h e m i c a l components o f m i c r o a l g a e grown i n series 2 cultures B. T a b l e s o f c o m p l e t e f a t t y a c i d p r o f i l e s o f m i c r o a l g a e grown i n s e r i e s 1 a n d s e r i e s 2 cultures  168  169  175  vi  List  of  Tables Page  Table I.  Concentrations 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 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  of  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 a d d e d d u r i n g l o g p h a s e growth. ( S i l i c a t e was n o t a d d e d t o T a h i t i a n Isochrysis g r o w t h medium.) Table I I .  F i n a l s u r v i v a l o f Crassostrea gigas larvae i n three unialgal diet t r i a l s l a s t i n g f r o m 12 t o 19 d a y s . Tahitian Isochrysis d i e t s consisted 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 a t m i d - l o g g r o w t h p h a s e , o r a f t e r 2 o r 6 d a y s o f 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 control) 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 s e a w a t e r whrn t h e l a r v a e were f e d nitrate-starved cells. 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 a t 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-control, Mid-log Si-control) 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 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 starved 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. V a l u e s a r e mean percentages ± 1 standard d e v i a t i o n , of l i v e l a r v a e from a sample o f g r e a t e r t h a n 100  Table I I I .  18  a n i m a l s from each r e p l i c a t e  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 g r o w t h 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 o f 20,000 c e l l s mL f o r 19 d a y s , o f 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 m i d - l o g g r o w t h p h a s e , 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 o r 6 d ) . A second mid-log treatment (Mid-log control) 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  118  vii  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 seawater. Least squares means o f s h e l l l e n g t h ± s t a n d a r d e r r o r were c a l c u l a t e d by n e s t e d a n a l y s i s o f v a r i a n c e , a t t h e 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 t h e number o f m e a s u r e m e n t s made. Square b r a c k e t s e n c l o s e l e t t e r s of s i g n i f i c a n c e from Tukey's m u l t i p l e c o m p a r i s o n t e s t , (p<.05). M a t c h i n g l e t t e r s among t r e a t m e n t s w i t h i n t h e day o f measurement s i g n i f y no d i f f e r e n c e among t h e t r e a t m e n t s b e a r i n g t h o s e letters Table IV.  T a b l e V.  Table VI.  . 121  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 g r o w t h 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 , f o r 14 d a y s as i n T a b l e I I , o f 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, 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-control, Mid-log Si-control) 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 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 starved c e l l s in 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  125  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 g r o w t h 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 f o r 12 d a y s as i n T a b l e I I , o f Thalassiosira pseudonana h a r v e s t e d f r o m 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, 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-control, Mid-log Si-control) 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 fed n i t r a t e - s t a r v e d 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 - 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 d a y s 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 o f T a h i t i a n Isochrysis (T-ISO)  128  A summary o f t h e b i o c h e m i c a l c o m p o s i t i o n and p e r f o r m a n c e o f 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  viii  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 t h e v a l u e o f t h e diet i n increasing larval shell length. Two r e p l i c a t e s were a n a l y z e d and averaged i n each treatment  131  ix  List  of Figures Page  Fig.  Fig.  1.  2.  G r o w t h c u r v e s (shown by c e l l numbers) o f 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 m e d i a , showing the c o n c e n t r a t i o n o f t h e l i m i t i n g n u t r i e n t s i n t h e 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 s a m p l e s were t a k e n Growth c u r v e s  vivo  Fig.  Fig.  Fig.  Fig.  3.  4.  5.  6.  (shown by c e l l  28  numbers o r in  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 - , p h o s p h a t e - a n d s i l i c a t e - l i m i t e d media, showing t h e concentration of the l i m i t i n g nutrients 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 o m i t t e d s i n c e i t f o l l o w s same c u r v e a s o t h e r treatments.) A r r o w s i n d i c a t e when s a m p l e s were t a k e n  30  Growth c u r v e s (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 m e d i a , showing t h e c o n c e n t r a t i o n o f t h e l i m i t i n g n u t r i e n t s i n t h e media. Dashed l i n e i n s t a t i o n a r y phase o f growth c u r v e s r e p r e s e n t s e s t i m a t e d c e l l number; a c c u r a t e c o u n t s were n o t p o s s i b l e due t o c e l l c l u m p i n g . Arrows i n d i c a t e when s a m p l e s were t a k e n  32  Growth c u r v e s (shown by c e l l numbers) o f t h e 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 m e d i a . Arrows mark t h e t i m e a t w h i c h t h e l i m i t i n g n u t r i e n t was e x h a u s t e d f r o m t h e 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 t h e 6 h s t a r v a t i o n p o i n t . Tahitian Isochrysis ( T - I s o ) was f e d a t 2 d a n d 6 d starvation  35  C e l l u l a r weight o f gross biochemical components i n T a h i t i a n Isochrysis sampled d u r i n g m i d - l o g 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 ) . B a r s r e p r e s e n t mean ± s t a n d a r d e r r o r o f c e l l weight. See A p p e n d i x A, T a b l e 4 f o r s t a n d a r d 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 a n d b)  38  C a l o r i c v a l u e o f T a h i t i a n Isochrysis d u r i n g m i d - l o g phase (ML), o r a f t e r  j  sampled 2 or 6 d  X  Fig.  Fig.  Fig.  Fig.  Fig.  7.  8.  9.  10.  11.  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 e n e r g y 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 A p p e n d i x A, T a b l e 1 f o r standard deviation of analyses of duplicate c u l t u r e s (a a n d b)  40  C e l l u l a r weight o f gross biochemical components i n Chaetoceros calcitrans sampled d u r i n g m i d - l o g p h a s e (ML) o r 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 ) . B a r s r e p r e s e n t mean ± s t a n d a r d e r r o r o f c e l l weight. See A p p e n d i x A , T a b l e 5 f o r s t a n d a r d 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  C a l o r i c v a l u e o f Chaetoceros calcitrans s a m p l e d d u r i n g m i d - l o g p h a s e (ML) o r 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, -Si); A) c e l l u l a r e n e r g y e q u i v a l e n t s , B) percent 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 A p p e n d i x A , Table 2 f o r standard deviation of analyses of d u p l i c a t e c u l t u r e s (a a n d b)  45  C e l l u l a r weight o f gross biochemical components i n Thalassiosira pseudonana s a m p l e d d u r i n g m i d - l o g p h a s e (ML) o r 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, -Si). B a r s r e p r e s e n t mean ± s t a n d a r d e r r o r of c e l l weight. See A p p e n d i x A , T a b l e 6 f o r standard error of analyses of duplicate c u l t u r e s (a a n d b)  48  C a l o r i c v a l u e o f Thalassiosira pseudonana s a m p l e d d u r i n g m i d - l o g p h a s e (ML) o r 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, -Si); A) c e l l u l a r e n e r g y e q u i v a l e n t s , B) percent 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 A p p e n d i x A , Table 3 f o r standard d e v i a t i o n of analyses of d u p l i c a t e c u l t u r e s (a a n d b)  50  Fatty acid class profiles  of Tahitian  Isochrysis,  pseudonana a n d  Thalassiosira  Chaetoceros calcitrans c e l l s harvested during t h e m i d - l o g growth phase i n b a t c h c u l t u r e : A) S e r i e s 1 B) S e r i e s 2 Fig.  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 composition o f T a h i t i a n Isochrysis harvested during mid-log growth phase. The l o w e r g r a p h e n l a r g e s t h e s c a l e o f t h e s m a l l e r peaks o f t h e upper  53  X I  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  13.  14.  15.  16.  17.  18.  19.  graph. Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e cultures (n=2 i n s e r i e s 1, n=3 i n s e r i e s 2)  55  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 midl o g growth phase. The l o w e r g r a p h e n l a r g e s the s c a l e o f t h e s m a l l e r peaks o f t h e upper graph. Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e cultures (n=2 i n s e r i e s 1, n = l i n s e r i e s 2)  57  Comparison o f s e r i e s 1 and s e r i e s 2 h i s t o g r a m s of 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 m i d - l o g g r o w t h phase. The lower g r a p h e n l a r g e s t h e s c a l e o f t h e s m a l l e r peaks o f t h e upper graph. Error bars represent ± 1 standard deviation 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=l i n s e r i e s 1, n=3 i n s e r i e s 2)  59  Changes o v e r t i m e i n t h e 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 series 1 c u l t u r e (see F i g . l f o r growth c u r v e and sampling time): 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  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (ML) o r a f t e r 2 o r 6 d a y s 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 ) . Error bars represent ± 1 standard deviation 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)  65  Changes o v e r t i m e i n t h e m a j o r 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 c u r v e and s a m p l i n g t i m e s ) : A) n i t r a t e - l i m i t e d medium, a n d B) p h o s p h a t e l i m i t e d medium  68  Changes o v e r t i m e i n t h e m a j o r 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 ( s e e F i g . 4 f o r growth curve) and h a r v e s t e d a t m i d - l o g growth p h a s e (ML) o r a f t e r 2 o r 6 d a y s 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 represent ± 1 standard deviation of analyses o f r e p l i c a t e c u l t u r e s (n=3).  70  Changes o v e r t i m e acids of Tahitian  i n the intermediate f a t t y Isochrysis d u r i n g growth i n  xii  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 s a m p l i n g t i m e ) : A) n i t r a t e - l i m i t e d medium, a n d B) p h o s p h a t e - l i m i t e d medium  72  Fig.  20.  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r growth curve) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (ML) o r a f t e r 2 o r 6 d a y s 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 bars represent ± 1 standard deviation 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) 74  Fig.  21.  Changes o v e r t i m e i n t h e m i n o r f a t t y T a h i t i a n Isochrysis d u r i n g growth i n c u l t u r e (see F i g . 1 f o r growth c u r v e sampling times): A) n i t r a t e - l i m i t e d and B) p h o s p h a t e - l i m i t e d medium  Fig.  Fig.  Fig.  Fig.  22.  23.  24.  25.  acids of series 1 and medium, 76  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r growth curve) and h a r v e s t e d a t m i d - l o g growth p h a s e (ML) o r a f t e r 2 o r 6 d a y s 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 represent ± 1 standard deviation of analyses o f r e p l i c a t e c u l t u r e s (n=3)  78  Changes o v e r t i m e i n t h e 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 series 1 c u l t u r e (see F i g . 2 f o r growth c u r v e and sampling time): 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, a n d C) s i l i c a t e l i m i t e d medium  81  Changes o v e r t i m e i n t h e 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 c u r v e ) i n A) n i t r a t e - l i m i t e d medium, o r B) s i l i c a t e - l i m i t e d medium a n d h a r v e s t e d a t m i d l o g g r o w t h p h a s e (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 o r 6 h , - S i 2 o r 6 h). Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e cultures (n=2 i n 2 h s a m p l e s , n=3 i n 6 h s a m p l e s )  83  Changes o v e r t i m e i n t h e m a j o r f a t t y a c i d s o f Chaetoceros calcitrans grown i n s e r i e s 1 ( S e e F i g . 2 f o r growth c u r v e and s a m p l i n g 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, a n d C) s i l i c a t e - l i m i t e d medium  87  xiii  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  26.  27.  28.  29.  30.  31.  Changes o v e r t i m e i n t h e m a j o r f a t t y a c i d s o f Chaetoceros calcitrans cultured i n series 2 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 ( s e e F i g . 4 f o r growth curve) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 a n d - S i 2 h or 6 h ) . Error bars represent ± l standard deviation of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h samples)  89  Changes o v e r t i m e i n t h e 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 s a m p l i n g 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, a n d C) s i l i c a t e - l i m i t e d medium  91  Changes o v e r t i m e i n t h e 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 cultured 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 c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 a n d S i - l i m 2 h or 6 h ) . Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h samples)  93  Changes o v e r time i n t h e 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 g r o w t h c u r v e a n d sampling times): 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, a n d C) s i l i c a t e l i m i t e d medium  95  Changes o v e r t i m e i n t h e m i n o r f a t t y a c i d s o f Chaetoceros calcitrans cultured i n series 2 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 ( s e e F i g . 4 f o r growth curve) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 a n d - S i 2 h or 6 h ) . Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h samples)  97  Changes o v e r t i m e i n t h e 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 g r o w t h c u r v e a n d sampling times): A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e - l i m i t e d medium  101  xiv  Fig.  Fig.  Fig.  Fig.  Fig.  32.  33.  34.  35.  36.  Changes o v e r t i m e i n t h e 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 c u r v e ) i n A) n i t r a t e - l i m i t e d medium, o r B) s i l i c a t e - l i m i t e d medium a n d h a r v e s t e d a t m i d l o g g r o w t h p h a s e (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 or 6 h ) . Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e cultures (n=2 i n N - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d samples)  103  Changes o v e r t i m e i n t h e 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 g r o w t h c u r v e a n d sampling times): A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e - l i m i t e d medium. T h e S i 66 h s a m p l e was i n c l u d e d i n t h e 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 t h e m i d - l o g (ML) fatty acid levels.  106  Changes o v e r t i m e i n t h e m a j o r 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 ( s e e F i g . 4 f o r growth curve) and h a r v e s t e d a t m i d - l o g 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 ) . Error bars represent ± 1 standard deviation 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 - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s )  108  Changes o v e r t i m e i n t h e 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 g r o w t h c u r v e and s a m p l i n g t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e - l i m i t e d medium. The S i 66 h s a m p l e was i n c l u d e d i n t h e 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 t h e m i d - l o g f a t t y acid levels  110  Changes o v e r t i m e i n t h e 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, o r s i l i c a t e - l i m i t e d medium ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 ) . Error bars represent ± 1 standard deviation 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 - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s )  112  X V  Fig.  Fig.  Fig.  Fig.  37.  38.  39.  40.  Changes o v e r t i m e i n t h e m i n o r 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 c u r v e and sampling times): A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e - l i m i t e d medium. T h e S i 66 h s a m p l e was i n c l u d e d i n t h e 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 t h e m i d - l o g f a t t y acid levels  114  Changes o v e r t i m e i n t h e m i n o r 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 ( s e e F i g . 4 f o r growth c u r v e ) and h a r v e s t e d a t m i d - l o g growth p h a s e (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 represent ± 1 standard deviation 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 - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s )  116  G r o w t h o f 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 m i d - l o g g r o w t h p h a s e ( M L ) , or 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 s e c o n d m i d - l o g t r e a t m e n t (ML-control) c o n t r o l l e d f o r t h e e f f e c t o f holding larvae fed nitrate-starved c e l l s i n n i t r a t e - f r e e seawater. Starved larvae d i d n o t r e c e i v e any a l g a l f o o d d u r i n g t h e experiment. L e a s t s q u a r e s means o f l a r v a l l e n g t h w e r e a n a l y s e d by n e s t e d a n a l y s i s o f variance of 3 r e p l i c a t e s ; standard errors a l l l i e w i t h i n t h e symbols except f o r t h e (-N 6 d) t r e a t m e n t (See T a b l e I I ) . Asterisks 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 t h e 95% confidence level  120  G r o w t h ( s h e l l l e n g t h ) o f Crassostrea gigas l a r v a e f e d Chaetoceros calcitrans harvested f r o m 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 m i d - l o g phase (ML), o r 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 (ML-N-control, ML-Sicontrol) controlled f o r 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 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 n o t r e c e i v e any a l g a l f o o d during the experiment. L e a s t s q u a r e s means o f l a r v a l l e n g t h were a n a l y s e d b y n e s t e d analysis of variance of 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 t h e range o f t h e s y m b o l s (See T a b l e I I I ) . A s t e r i s k s  xv i 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 confidence l e v e l F i g . 41.  95%  Growth ( s h e l l length) of Crassostrea gigas l a r v a e fed 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 a t mid-log phase (ML) 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 (ML-N-control, ML-Sic 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 starved c e l l s i n nutrient stripped, 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 i e v e any a l g a l food d u r i n g the experiment. L e a s t squares means o f l a r v a l l e n g t h were a n a l y s e d by nested a n a l y s i s of v a r i a n c e of 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 T a b l e I V ) . Asterisks 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% confidence l e v e l  124  127  ACKNOWLE DGEMENTS  F u n d i n g p r o v i d e d by t h e Department o f F i s h e r i e s Canada, i s g r a t e f u l l y  acknowledged.  was p e r f o r m e d a t t h e P a c i f i c Neil lab. to  and Oceans,  A l lexperimental  Biological  work  S t a t i o n , where D r .  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 scallop  I am i n d e b t e d t o D r . I a n Whyte who was n e v e r t o o b u s y  e d u c a t e me i n t h e 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 n e v e r s h o r t o f c o n v e r s a t i o n about nutrition.  bivalve  D r . P a u l J . H a r r i s o n o p e n e d up t h e w o r l d 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 b e h i s  student.  T h a n k s a r e owed t o a l l members o f my t h e s i s c o m m i t t e e , a n d to  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  through  My  critical  'pillar'  review  me  o f my w o r k .  a n d my f r i e n d , Mark S a u n d e r s , e n d u r e d 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 a n d c o m p a s s i o n .  1  INTRODUCTION  The  B.C.  dollars  oyster  i n d u s t r y i s p r e s e n t l y worth about 3  annually,  realized.  yet  i t s f u l l p o t e n t i a l i s f a r from  Production  potential  i n 1985  annual production  a p p r o x i m a t e 29,000 t  Three species  of the  nutrition  Ostrea  occurs  e s t u a r i e s and  abundance i n B r i t i s h l i m i t e d due and  Crassostrea introduced  The  but  to i t s small  first  are  significant  the  Strait  has  become t h e  Production size,  External  commercially native  in  in  in  o f O. lurida  coast  on  oyster,  found  slow growth,  from the  importation  gigas  was  is  high  (Quayle  1988).  oyster,  was  e a r l y 1900's t o  favoured  gigas  from Japan o f t h e  i n 1926  o f G e o r g i a were p l a n t e d  S p a w n i n g o f C.  interest  about  left.  o y s t e r Crassostrea  24°C.  i t i s not  native east  t o the west c o a s t  few  The  s p e c i f i c t e c h n i c a l requirements the  reliable  t o lower C a l i f o r n i a  lagoons but  Columbia.  virginica,  industry i s  larvae.  t h e U.S.A.  from A l a s k a  saltwater  should  p r o m o t e d an  of b i v a l v e  o f Canada and  mortality  oyster  o f o y s t e r s have been h a r v e s t e d  lurida,  estimated  l e a s e d grounds The  being  hatchery produced seed f o r  the west c o a s t  1940  3420 t w h e r e a s  o p e r a t i o n s , which i n t u r n has  understanding the  very  was  (Quayle, 1988).  g r o w i n g more d e p e n d e n t on culture  million  species  Pacific  (Quayle 1988).  i n t e n s i v e l y and  Areas C.  gigas  in culture practices.  r e q u i r e s t e m p e r a t u r e s b e t w e e n 19  f e r t i l i z a t i o n marks t h e  beginning  of  and the  of  2  pelagic  life  o f t h e l a r v a e . The l a r v a e q u i c k l y p a s s  the trochophore  stage t o the v e l i g e r  r e t r a c t a b l e v e l u m w i t h i n 24 h o u r s . participates  simultaneously  through  stage, developing The c i l i a t e d  a  velum  i n swimming and f e e d i n g .  D e v e l o p m e n t o f t h e umbone a t t h e s h e l l h i n g e , t h e d i g e s t i v e s y s t e m , and a c i l i a t e d  foot preceeds the p e d i v e l i g e r  S h o r t l y b e f o r e r e a c h i n g t h e maximum s h e l l b l a c k eye spots appear, s i g n a l l i n g for  l e n g t h o f 300  that the larva  u.m,  i s prepared  setting.  During  s e t t l e m e n t , t h e l a r v a e seek a s u i t a b l e  substrate f o r  attachment.  Once f o u n d ,  from a g l a n d  a t t h e b a s e o f t h e f o o t and t h e l e f t  a cementing substance  i s secreted  becomes p e r m a n e n t l y a t t a c h e d t o t h e s u b s t r a t e . then as  stage.  undergo metamorphosis, becoming j u v e n i l e s  valve  The o y s t e r s commonly  known  spat.  Oyster  culture operations require a r e l i a b l e  Seed i s g e n e r a l l y c o l l e c t e d  supply  f r o m n a t u r a l s e t s on  ( p o r t a b l e s u b s t r a t e ) and a l l o w e d  of  seed.  cultch  t o grow i n p r o d u c t i v e  areas.  However, s i n c e t e m p e r a t u r e s r e q u i r e d f o r spawning a r e h i g h relative  t o B.C. w a t e r s , w i l d s e t s c a n be  S e e d o y s t e r s c a n now be o b t a i n e d coast o f North a reliable  f r o m h a t c h e r i e s on t h e w e s t  A m e r i c a , i n c l u d i n g B.C.  source  erratic.  Such p r a c t i c e s  o f s e e d on demand, a l l o w c o n t r o l l e d  provide the potential  f o r genetic  selection.  provide s e t s and  3  The  o p e r a t i o n o f an e f f i c i e n t  d e p e n d s on p r o d u c t i o n o f l i v e most c o s t l y  requirements  A defined a r t i f i c i a l requirements.  support  As  and  e r r o r but  of  studies  limited.  i s r e q u i r e d t o determine d i e t s cannot  specific  completely  for bivalve  d i e t s have been d e v e l o p e d  larvae, which  l i m i t e d g r o w t h i n b i v a l v e a d u l t s ( C a s t e l l and  1984,  (Langdon and P a r k e r and  (Gabbot e t a l . 197 6, Langdon 1983). virginica  artificial  d i e t , but  p o o r and  B o l t o n 1984,  L a n g d o n and Chu  Langdon  S e l i v o n c h i c k 1986) W a l d o c k 1981,  and  larvae  Chu  et a l . raise  f a r a s m e t a m o r p h o s i s on  s u r v i v a l t o the eyed l a r v a l  development lagged  Trider  and  e t a l . (1987) were a b l e t o  l a r v a e as  Crassostrea  In  and  (Urban  the  determined  as a f o o d s o u r c e  some a r t i f i c i a l  Siegfried  larvae  algal  s p e c i e s have been  yet, a r t i f i c i a l  1974), j u v e n i l e s  1982,  foods,  have been  diet  replace phytoplankton although  unicellular  Suitable algal  a process of t r i a l  nutritional  hatchery  area of l a r v a l b i v a l v e production  Langdon 1984). through  oyster or bivalve  f r o m 9 t o 14  stage  days behind  an was  that of  fed algae.  the absence of a s u i t a b l e a r t i f i c i a l  c a n p o t e n t i a l l y be phytoplankton  larval  nutritional  T h i s o b j e c t i v e c a n be met  knowledge o f f i r s t ,  cost  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  that f u l f i l l  optimal growth.  diet,  the e f f e c t s  c o m p o s i t i o n on t h e g r o w t h and  with  requirements  only with  of phytoplankton  survival  efficiency  for  the  chemical  o f 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 of the a l g a l cells.  I n a d d i t i o n , p h y t o p l a n k t o n must meet c e l l digestibility assimilation Mytilus  t o ensure  (Romberger a n d E p i f a n i o veligers  edulis  i n diameter 1984a).  requirements  i n this  and U k e l e s  e t a l . 1984, S p r u n g ,  s i z e range a r e  and d i g e s t i o n  of  i n Bivalve  was  Chlorella  Nutrition  Studies o f the chemical composition of b i v a l v e shown t h e i m p o r t a n c e phytoplankton.  of the quality  Although  larvae  have  and q u a n t i t y o f l i p i d s i n  glycogen reserves are important  s o u r c e s f o r a d u l t o y s t e r s , t h e y have n o t been  c o r r e l a t e d w i t h Ostrea S t a r v e d O. edulis than p r o t e i n  edulis  spat y i e l d  r e l y more upon e n e r g y  and c a r b o h y d r a t e t o g e t h e r  Initial  positively release  10 u,m  1979).  Importance o f L i p i d s  1962).  and  C. virginica less than  and  a s a s s e s s e d 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  autotrophica  energy  filtration  P o o r g r o w t h p e r f o r m a n c e o f C. virginica  attributed to a lack of l y s i s  1.  1981).  s e l e c t phytoplankton  However n o t a l l c e l l s  (Babinchak  efficient  ( R i i s g a r d e t a l . 1980, F r i t z  digestible.  s i z e and  from  1957).  triglycerides  ( M i l l a r and S c o t t  g r o w t h r a t e o f n e w l y r e l e a s e d O. edulis  correlated with l i p i d  from t h e brood  d e p e n d on n e u t r a l  lipid  content of the larvae  (Helm e t a l . 1 9 7 3 ) .  O. edulis  stores b u i l t during the l a r v a l  t o p r o v i d e n e c e s s a r y energy Spencer 1973).  (Collyer  f o r metamorphosis  was upon larvae stages  ( H o l l a n d and  T h i s phenomenon a l s o o c c u r s i n l a r v a e o f f o u r  5  g a s t r o p o d , Littorina Patinopectin  species  yessoensis  (Holland  e t a l . 1975)  scallop larvae  and  (Whyte e t a l . 1 9 8 7 ) .  Fatty acids e s t e r i f i e d with  g l y c e r o l are  blocks  i m p o r t a n t components o f  of  lipids.  membranes, and serve  t h e y a c t as  storage  as m e t a b o l i c p r e c u r s o r s .  contain with  They a r e  the main b u i l d i n g  and  are  known  (Ackman e t a l . 1968,  P o h l and  Zurheide 1979).  specific  and  algae  can  unsaturated  Schwarzenbach  The  n3  the  c a r b o n b o n d s c a n n o t be  position f o r the  n6  f a t t y a c i d s are  (Holland  composition  fatty  1978).  22:6n3  ( J o n e s e t a l . 1979,  Sulkin  1984).  during  larval  is  of the  desaturated  Riley  same s p e c i e s  p r i o r to the  fatty  acids  d e v e l o p m e n t o f C. virginica  related  fatty acids to give  ability  o f j u v e n i l e Crassostrea  (Chu  because  requirement and  Levine  22:6n3  efficiently  20:5n3  and  and  increases Webb 1 9 8 4 ) .  e x t e n d 18:3n3  (Ackman e t a l . 1 9 6 8 ) ,  gigas  larvae to  22:6n3 f r o m s h o r t e r c h a i n e d a c i d s  i n a d e q u a t e f o r optimum g r o w t h  of  n9  20:5n3  Kanazawa e t a l . 1979,  can  1969,  species  Marine organisms have a  feeders  numbered  e s s e n t i a l i n a l l animals  L a r v a l c o n t e n t o f 20:5n3 and  A l t h o u g h some f i l t e r  acids  1978).  long chain polyunsaturated  20:5n3 and  C h u e c a s and  v a r y e v e n among c l o n e s  ( F i s h e r and  and  Lipid  and  generally  e v e n numbered c a r b o n c h a i n s , a l t h o u g h some odd  chains  cell  products f o r energy  Marine microalgae  straight-chained saturated  in  (Waldock and  is  and  the  synthesize apparently  Holland  1984).  An  * F a t t y a c i d n o m e n c l a t u r e u s e d i n t h i s t h e s i s i s a:bnx where a i s t h e number o f c a r b o n s i n t h e c h a i n , b i s t h e number o f d o u b l e ( u n s a t u r a t e d ) b o n d s and x i s t h e p o s i t i o n o f t h e f i r s t d o u b l e bond from t h e methyl end.  6  artificial  d i e t r e p l a c i n g corn o i l w i t h cod  g r o w t h o f C. virginica,  liver  the l a t t e r d i e t having  a  o i l improved higher  content of long chained polyunsaturated f a t t y acids and  Trider  1974).  L a n g d o n and W a l d o c k  (1981)  t h a t e n c a p s u l a t e d 22:6n3 i m p r o v e d C. gigas was  used t o supplement a d i e t d e f i c i e n t  20:5n3.  demonstrated  s p a t g r o w t h when i t  i n both  In a d d i t i o n t o the h i g h l y unsaturated  n e u t r a l hydrocarbons organisms,  may  be  essential  functioning at least  (Castell  lipids  22:6n3 fatty  and  acids,  t o marine  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 . 1 9 8 7 ) .  Nevertheless, larval dietary  lipid  positively  growth i s not n e c e s s a r i l y r e l a t e d  content.  S i z e o f C. gigas  correlated with l a r v a l  neither algal  lipid  various algal  d i e t s c o u l d be  (Waldock and  2.  Nascimento  Variation  Nutritional  content nor  al.  1985  and and  acid composition  Composition  of A l g a l  nature of a l g a l chemical  Dupuy 1980, 1986,  content;  correlated with l a r v a l  (Epifanio  cells  1979,  of  growth  composition. composition  (Ackman e t a l . 1968,  P i o r r e c k and  Whyte 1 9 8 7 ) .  Diets  Pohl  1984,  Cells harvested  have  Conover  Fabregas from  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 o r lower growth than  but  d i e t s must r e c o g n i z e  i n a l g a l chemical  b e e n a t t r i b u t e d t o c u l t u r e age Chu  fatty  studies using phytoplankton  Demonstrated d i f f e r e n c e s  1975,  triacylglycerol  been  1979).  of Chemical  the t r a n s i t o r y  l a r v a e has  to  et  the larval  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  Cary  e t a l . 1981).  U n f o r t u n a t e l y , much o f  the  7  literature  assessing the chemical composition of  has  to i d e n t i f y the p h y s i o l o g i c a l  failed  c u l t u r e conditions a f f e c t i n g the c e l l s .  state of the c e l l Growth  conditions  The  to interpret. i s necessary  Stringent control  s t u d i e s become  of a l l culture  for reproducible results.  relevance of the gross chemical composition of algae to  the n u t r i t i o n  of the g r a z e r i s not c l e a r .  and Mercenaria  virginica  Growth o f  j u v e n i l e s was  mercenaria  of v a r i o u s combinations  of 4 a l g a l  carbohydrate together with a decrease i n j u v e n i l e Crassostrea  c h e m i c a l c o m p o s i t i o n was phosphorus r a t i o s Chu  1979).  increase i n  in protein yielded  the  f e d a l g a e whose  virginica  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  of the phytoplankton  (1982) f o u n d h i g h p r o t e i n  related to better  composed  species (Epifanio  H o w e v e r , W i k f o r s e t a l . (1984) c o n c l u d e d t h a t an  best results  C.  not  c o r r e l a t e d with gross chemical composition of d i e t s  and  or  limiting  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 , c o m p a r a t i v e difficult  phytoplankton  c u l t u r e medium.  i n the a l g a l  food f o r l a r v a e .  Amino a c i d  diet  Webb  was  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), a l t h o u g h p r o p o r t i o n s o f amino a c i d s proportion required (Webb and  Chu  Dietary l i p i d protein  i n the l a r v a l  t i s s u e m i g h t be  to  the  important  1982).  c o m p o s i t i o n m i g h t be more i m p o r t a n t t h a n t h a t o f  o r c a r b o h y d r a t e t o the development of b i v a l v e  Among f i v e u n i a l g a l d i e t s gigas),  i n algae r e l a t i v e  f e d t o queen conch l a r v a e  o n l y t h e p o o r e s t d i e t had  a v e r y low  lipid  larvae.  (Strombus content  and  8  l a c k e d b o t h 20:5n3 and differences  1985).  However not  c o n t e n t n o r t h e p r o p o r t i o n o f 20:5n3  E p i f a n i o e t a l . (1981) n o t e d t h a t j u v e n i l e grew w e l l on a d i e t o f Thalassiosira  virginica  and Phaeodactylum  t h a t a lower r a t i o  2 0 : 5 n 3 , 22:6n3 and  C.  f o r 0. edulis  was  suggested  a better  juveniles.  The  c a r b o h y d r a t e were c r i t i c a l .  et  f a t t y a c i d c o n t e n t was  Additional  a p p a r e n t l y adequate  i n experiments  employing  amino  (Enright  phytoplankton  dependent upon c o n t r o l o f f a c t o r s a f f e c t i n g  metabolism  and  Environmental composition 1983,  1983,  factors  shown t o a f f e c t a l g a l  T e s h i m a e t a l . 1983,  intensity  Gostan  Brzezinski  Effect  and  ( O r c u t t and  concentration  diets  growth,  chemical composition of the algae.  i n c l u d e temperature  (Opute 1974,  3_.  of  a l . 1986b).  Reproducibility is  fatty  levels  c a r b o h y d r a t e i n c r e a s e d growth as l o n g as t h e e s s e n t i a l a c i d and  (3H)  pseudonana  E n r i g h t e t a l . (1986b)  o f s a t u r a t e d : ( n 6 + n3)  composition d i e t  and  b o t h o f w h i c h were l a c k i n g i n  tricornutum  22:6n3, b u t h i g h i n 20:5n3.  acid  (Pillsbury  i n g r o w t h among t h e g o o d d i e t s was  attributable to l i p i d 22:6n3.  22:6n3  1985,  chemical  (Opute 1974,  R e d a l j e and  Seto e t a l . 1984), l i g h t  L e c h u g a - D e v e z e 1986)  P a t t e r s o n 1973,  S c o t t 1980,  Thompson e t a l . ,  (See f o l l o w i n g s e c t i o n s  of Nutrient Limitation  and  1989)  and  Laws  quality  light Terry et a l . nutrient  for references).  on A l g a l  Composition  N i t r o g e n and p h o s p h o r u s a r e i m p o r t a n t m i c r o n u t r i e n t s n e c e s s a r y for  phytoplankton growth.  Silicon  i s a l s o necessary f o r the  9  growth o f diatoms.  Depletion  a f f e c t phytoplankton critical  ratio  chemical composition  o f an a l g a l c e l l ' s  of the c r i t i c a l environmental  requirement  (Terry 1980).  requirements  conditions  (as l o n g  Phytoplankton  i.  used  light  or a nutrient  species  nutrient i s cells  eventually  nutrients are replenished.  Starvation i n nitrogen-deficient conditions  content.  under n i t r o g e n - r e p l e t e  t o continue c e l l  specific  accumulation  reduces  algal  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  c u l t u r e medium i s s p e n t  1974,  until  Many o f t h e s t a r v e d  n i t r a t e which accumulate are  e t a l . 1985).  I n 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  i n t h e medium.  division  protein  of the  to the c e l l s  t h e s t a t i o n a r y p h a s e when t h e l i m i t i n g  Nitrogen  Cell  by  supply of nutrients i s a v a i l a b l e .  grow e x p o n e n t i a l l y  becomes l i m i t i n g .  side  as excess n u t r i e n t s a r e n o t a t t o x i c  In batch c u l t u r e , a f i n i t e  die unless  f o r t h e two  On e i t h e r  a n d by t h e c o n c e n t r a t i o n  (Droop 1974, Rhee 1978, T e r r y  exhausted  The  r a t i o , p h y t o p l a n k t o n growth i s r e g u l a t e d  single nutrient least available, relative  enters  (Droop 1 9 7 4 ) .  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 p h y t o p l a n k t o n growth  levels)  can  o f two n u t r i e n t s i n t h e a l g a l g r o w t h medium, i s  equal t o the r a t i o nutrients.  of these micronutrients  g r o w t h when e x t e r n a l  (Dortch 1982).  (Shifrin  and C h i s h o l m  o f non-nitrogenous  conditions, nitrogen  The r e s u l t , w h i c h i s 1981), c a n be t h e  compounds s u c h a s l i p i d  E l - F o u l y e t a l . 1985, W i k f o r s  i n the  (Opute  1986) o r c a r b o h y d r a t e  10  ( M y k l e s t a d and 1985). in  A  Haug 1972,  shift  nitrogen  synthesis nitrogen  starvation  must f a l l  i s increased  and  also  Chisholm  greater  (Richardson et a l . 1969).  influence l i p i d  storage  (1981) f o u n d t h a t  than the  total  De novo s y n t h e s i s  responsible  for increasing neutral  conversion  significant There can  be  among l i p i d nitrogen fatty 1984,  lipid  stress, neutral  Phospholipid  Silicon  Silicon  lipids  (Pohl  production  and  1975).  unit  volume  active  in  primarily nitrogen-stressed i s not  P a r r i s h and  s u b s i d e s due  a  (Suen e t a l . 1 9 8 7 ) .  and  Z u r h e i d e 1982,  ( P a r r i s h and  lipid  composition  Marimura, 1966).  increase  start  Under  polyunsaturated Piorreck  Wangersky  et a l .  1987).  to a requirement f o r  Wangersky  the  1987).  Starvation  i s supplied  metasilicate  lipids  accumulation  e t a l . 1985,  amino a c i d c y s t i d i n e  ii.  mass p e r  i s considered  f r a c t i o n s ( O t s u k a and  of  the  (Conover  a marked d i f f e r e n c e i n f a t t y a c i d  El-Fouly  form  nitrogen-stressed  of n o n - l i p i d s to l i p i d s  factor in l i p i d  acids decrease  The  o r i g i n a l biomass at the  synthesis.  and  lipid  ammonium o r u r e a ) a v a i l a b l e i n  of n u t r i e n t d e p r i v a t i o n , thus confirming  cells  occur  I n some c a s e s  levels before  p h y t o p l a n k t o n c u l t u r e s p r o d u c e d new w h i c h was  et a l .  s t o r a g e can  (Werner 1 9 7 0 ) .  t o v e r y low  (e.g. n i t r a t e ,  g r o w t h medium c a n  Shifrin  El-Fouly  from c a r b o h y d r a t e t o l i p i d  advanced n i t r o g e n  cell  M y k l e s t a d 1974,  i n the  c u l t u r e medium as  sodium  (Na2Si03•9H20) which h y d r o l y z e s t o  acid, Si(OH)4.  When s i l i c a t e  orthosilicic  i s exhausted from the  medium,  11  the  cells  pass through a p e r i o d d u r i n g which c e l l s  c y t o k i n e s i s and  initiate  c e l l wall  formation  w a l l d e v e l o p m e n t i s a r r e s t e d a s d i a t o m s do sufficient formation  i n t r a c e l l u l a r pools  Not  only  f r u s t u l e , but  the production  of the  ( V o l c a n i 1977,  Silicate  by  is silicate  enzymes DNA  increase  Vaulot  and  increased  g r e a t l y as  b e e n shown t o  and  increased  r a t e due  1988).  lipid  Cyclotella  cryptica,  and  synthesis  closely  a greater than  (Werner 1 9 7 7 ) .  100%  Ratios  Although t o t a l  fatty  of acids  accumulation carbon  of non-1ipid  carbon  o f newly a s s i m i l a t e d c a r b o n (Shifrin  Changes i n S i : C and  f o l d under severe s i l i c a t e diatoms  lipid  i s accumulated at a g r e a t l y  to conversion  partitioning  e t a l . 1987).  increase the  a r e s u l t of t r i a c y l g l y c e r o l  (Roessler  and  thymidylate  a r r e s t e d w i t h i n 4 hours followed  f i x a t i o n decreases, l i p i d increased  synthesis  monounsaturated t o polyunsaturated  i n C. cryptica  formation  e t a l . 1987).  i n fatty acid synthesis  saturated  f o r the  p o l y m e r a s e and  In s i l i c a t e - s t a r v e d  s y n t h e s i s was  change i n d i a t o m  required  a c e s s a t i o n i n pigment p r o d u c t i o n  lipid,  valve  i t i s a l s o n e c e s s a r y i n DNA  d e f i c i e n c y has  content of diatoms. protein  cell  maintain  f o r new  s t a r v a t i o n leads to considerable  metabolism.  kinase  not  further  ( B r z e z i n s k i 1985).  Silicate  of the  of s i l i c o n  but  complete  and  ( H a r r i s o n e t a l . 1977).  a c i d s , p r o t e i n s , carbohydrates,  Net  into  C h i s h o l m 1981,  Si:N  limitation  into  Taguchi  r a t i o s were 2 t o  i n three  species  3  of  synthesis of nucleic  c h l o r o p h y l l s and  fucoxanthins  is  r e d u c e d ; however d i a d i n o x a n t h i n  (Volcani  iii.  synthesis  1977).  Phosphate  Starvation  E n e r g y r i c h p h o s p h a t e b o n d s o f ATP cellular  a r e an i n t e g r a l p a r t o f  energy t r a n s f e r through processes o f photo-,  substrate-, or oxidative phosphorylation. p h o s p h a t e r e d u c e s ATP metabolism necessary al.  continues  1984a and b ) .  Depletion of  l e v e l s , thereby impairing f o r the f i x a t i o n  of carbon  energy (Cembella e t  I n t r a c e l l u l a r polyphosphate pools harbour  e n e r g y and p h o s p h a t e r e s e r v e s w h i c h a l l o w c e l l s  to  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  1964).  Scenedesmus  sp. c e l l s  synthesizing protein  i n p h o s p h a t e - d e f i c i e n t medium  Radioisotope  P) h a s d e t e r m i n e d t h a t e x t e r n a l p h o s p h a t e  phosphate donor The  Chlorella.  s y n t h e s i s o f DNA Phosphate  f o r RNA, latter  metabolites  results  ( M i y a c h i and T a m i y a  and Holm-Hansen 1977,  and t r i a c y l g l y c e r o l s  In yeast, t o t a l  and  to biomass  Lehman 1976, Konopka and  1 9 8 1 , and Wynne and Rhee 1 9 8 6 ) .  Douglas  and p r o t e i n  and b o o s t s c a r b o h y d r a t e p e r u n i t  ( S a k s h a u g and M y k l e s t a d 1973,  variously  1961).  i n decreases i n phosphate-  Holm and A r m s t r o n g 1 9 8 1 ) , RNA/DNA r a t i o s  sterols  direct  a c t as i n d i r e c t phosphate donors f o r  ( P e r r y 1972, S a k s h a u g  carbohydrate r a t i o s  is a  labeling  p h o s p h o l i p i d s and p o l y p h o s p h a t e s i n  and p r o t e i n  limitation  continued  at rates equivalent to 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 d a y s . (  (Kylin  survive  Schnur  lipids,  decrease while phospholipids are  a f f e c t e d when t h e y a r e p h o s p h a t e - s t a r v e d (Ramsay and  1979).  4.  OBJECTIVES  It  i s generally  recognized that  combinations o f a l g a l  produce t h e b e s t l a r v a l growth r a t e s Romberger and E p i f a n i o studies  (Webb a n d Chu 1 9 8 2 ,  1981, E p i f a n i o 1979), however, i n such  i ti sdifficult  t o separate the e f f e c t s o f factors  s u c h a s a l g a l s i z e and d i g e s t i b i l i t y . single  species  effects  d i e t s may b e t t e r  T h u s t h e employment o f  serve t o i l l u m i n a t e the  o f a l g a l c h e m i c a l c o m p o s i t i o n w h i c h c a n b e v a r i e d by  altering has  species  growth c o n d i t i o n s .  been e x p l o r e d u s i n g  Nutrition of juvenile  t h i s technique.  Tissue  j u v e n i l e c l a m s was c o r r e l a t e d t o n i t r o g e n Thalassiosira  pseudonana  bivalves  growth i n  content o f  e x p r e s s e d a s a v a r i a t i o n i n C:N  ratios;  a C:N r a t i o b e t w e e n 8.4 a n d 10.5 p r o d u c e d  results  ( G a l l a g e r a n d Mann 1 9 8 1 ) .  Chaetoceros  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 a n d s i l i c a t e a f f e c t e d g r o w t h o f O. edulis  juveniles  superior calcitrans  limitation  (Enright  e t a l . 1986b).  A h i g h e r c o n t e n t o f 22:6n3 was s u g g e s t e d a s a p o s s i b l e for  improved o y s t e r  saturated  cells  g r o w t h when t h e y were f e d n u t r i e n t -  rather  composition could  reason  than n u t r i e n t - l i m i t e d c e l l s ;  not supply a s a t i s f a c t o r y  gross  explanation.  Growth r e s p o n s e s 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 r e s p e c i e s specific,  (Helm a n d L a i n g  1 9 8 7 , C a r y e t a l . 1981) a n d  r e q u i r e m e n t s change w i t h t h e s t a g e o f t h e l i f e bivalve. for  T h e r e f o r e t h e m e r i t o f a l g a l d i e t s must b e t e s t e d  each b i v a l v e  research  cycle of the  species  at specific  life  cycle stages.  Most  o f b i v a l v e n u t r i t i o n h a s 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 r e q u i r e m e n t s , and knowledge o f t h e n u t r i t i o n a l requirements o f b i v a l v e  larvae  Furthermore, the l a r v a l  s t a g e i s t h e most c r i t i c a l ,  and This  c o s t l y phase o f a s u c c e s s f u l  following  on l a r v a l  oyster  lacking.  bivalve hatchery  study has endeavoured t o c o n t r i b u t e  information  1.  i s clearly  intensive operation.  t o present  n u t r i t i o n by m e e t i n g t h e  objectives:  Grow i n b a t c h c u l t u r e a n d h a r v e s t a t v a r y i n g n i t r a t e , phosphate o r s i l i c a t e microalgal  species  which a r e p r e s e n t l y  favourable foods f o r b i v a l v e d i a t o m s , Isochrysis  calcitrans  starvation,  larvae;  a f f . galbana  a n d Thalassiosira  stages o f  three  r e c o g n i z e d as  a flagellate  (T-Iso),  a n d two  Chaetoceros  pseudonana ( 3 H ) ,  respectively.  2.  3.  Assess the e f f e c t s of nutrient  s t a r v a t i o n on f a t t y  and  gross chemical composition of s i n g l e species  the  three phytoplankton  larvae  fed single species  phytoplankton species has  it  gigas  i n which t h e chemical  was s e l e c t e d  Crassostrea  diets of the three  been m o d i f i e d by n i t r a t e o r s i l i c a t e  Crassostrea  diets of  species.  A s s e s s t h e r e s u l t a n t growth performance o f gigas  acid  composition  limitation.  f o r the bioassays  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  y e a r and i s a r e l a t i v e l y h a r d y c u l t u r e  because  most o f t h e  organism.  15  This  i s the f i r s t  nutrient  limitation  Crassostrea limiting  gigas  of algal  larvae.  on t h e e f f e c t s  biochemical  of well  defined  c u l t u r e s on t h e g r o w t h o f  Strict  f a c t o r s of the algae  information and g r o s s  report of the e f f e c t s  c o n t r o l o f t h e growth  cultures provides  new  o f t h e s e f a c t o r s on t h e f a t t y  composition of the  algae.  acid  16  MATERIALS AND  1.  Algal  Unialgal  METHODS  Cultures  c u l t u r e s o f Isochrysis  (henceforth  r e f e r r e d t o as  T a h i t i a n Isochrysis),  calcitrans  ( P a u l s e n ) T o k a n o and  (clone  ( H u s t . ) H a s l e and  3H)  Northeast P a c i f i c  Culture  Thalassiosira  i n the  300  18  collector.  (1 |im)  a i r and  2 min.  L o f c u l t u r e , and  Irradiance  3.5  f r o m two at the  L of  sides with  vessel  sparged with pure carbon d i o x i d e s t i r r i n g was  a p p l i e d by  r e m a i n e d b e t w e e n 8.0  Culture  medium was  enriched  natural  and  filter  surface  swirling  seawater  was  a  filtered h  the  Culture  pH  8.3.  sterilized  (0.45  |im)  s e a w a t e r f r o m D e p a r t u r e Bay,  n u t r i e n t e n r i c h m e n t was  natural  cool-  e v e r y 0.5  c u l t u r e s when g r o w t h p a r a m e t e r s were m o n i t o r e d .  The  the  culture.  B a t c h c u l t u r e s were b u b b l e d w i t h  Further  phase  L  -s -- m e a s u r e d w i t h a L i - c o r l i g h t m e t e r w i t h  cosine  for  supplied  i n 20  1  |iE-m  B.C.,  exponential  first  second i n 6 L Erlenmeyer f l a s k s c o n t a i n i n g C o n t i n u o u s i l l u m i n a t i o n was  of  ± 1.5°C.  s e r i e s o f c u l t u r e s were grown; t h e  polycarbonate carboys containing  the  Columbia, Vancouver,  A l l c u l t u r e s were m a i n t a i n e d  white fluorescent tubes.  pseudonana  C o l l e c t i o n , Department  o f g r o w t h a t a t e m p e r a t u r e o f 21  Two  Chaetoceros  H e i m d a l were p r o c u r e d f r o m  Oceanography, U n i v e r s i t y of B r i t i s h Canada.  ( c l o n e T-ISO) G r e e n  galbana  b a s e d on  (ESNW) u s e d by  the  Harrison  nutrient Nanaimo,  formula  for  B.C.  enriched  e t a l . (1980),  plus  10~  M sodium s e l e n i t e  9  ( 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 e q u i m o l a r amount o f F e S 0 4 » 7 H 2 0 , s o d i u m g l y c e r o p h o s p h a t e was  replaced with  and  Na2H2P04«H20.  N u t r i e n t r a t i o s were m o d i f i e d a s i n T a b l e I t o e n s u r e limitation  of a specific nutrient.  A l lphytoplankton  were  grown i n n i t r a t e - l i m i t e d o r p h o s p h a t e - l i m i t e d medium. a n d T. pseudonana  calcitrans limited  cultured  in silicate-  medium.  1;  i.  Series  for  fatty acid  The f i r s t  diet t r i a l s .  inoculated cells-mL  - 1  series  Filter-sterilized  acid  (0.45  From e a c h c a r b o y , 3 L o f c u l t u r e analysis  was  t o a d e n s i t y o f 2 0,000 were t a k e n f o r  at i n t e r v a l s , starting at mid-logarithmic  growth phase and c o n t i n u i n g u n t i l Bacteria,  were grown  urn) n u t r i e n t - e n r i c h e d  i n 20 L p o l y c a r b o n a t e c a r b o y s  with stock culture .  of algal cultures  a n a l y s e s o n l y and were n o t f e d t o o y s t e r s i n  seawater contained  fatty  were a l s o  C.  the culture  was  senescent.  w h i c h were m o n i t o r e d u s i n g a p h a s e - c o n t r a s t  m i c r o s c o p e , were n o t a b u n d a n t u n t i l  algal  senescence  was  reached.  ii.  Series  2:  Since series  1 cultures  were t o o l a r g e  to  a v o i d t h 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 , a second s e r i e s grown f o r d i e t t r i a l s Three l i t e r s and of  i n smaller light-saturated  o f medium e n r i c h e d w i t h n u t r i e n t s  T a b l e I were c o n t a i n e d cultures  was  inoculated  was  cultures.  as i n s e r i e s 1  i n 6 L Erlenmeyer f l a s k s . d a i l y t o ensure a uniform  A  series  daily  18 Table I: Concentrations 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 of 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 of 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 brackets) was added d u r i n g log phase growth. ( S i l i c a t e was not added t o T a h i t i a n I s o c h r y s i s growth medium.)  Nutrient Concentration ()1H) Series  Treatment  NaH03  Na2*H2P04>H20  Ha2Si03»9B20  1  P-liiited H-liaited Si-liaited  5490 549 2196  9.9 39 39  528 (+276) 528 (*276) 106  2  H-linited Si-United  275 2196  20 20  528 106  19  supply  of n u t r i e n t - d e f i c i e n t algae  to feed t o the  oyster  larvae.  T a h i t i a n Isochrysis and  was  6 d of n i t r a t e  chemical  t h e y c o u l d n o t be harvesting.  analyses.  a l s o taken  experiments with  a t m i d - l o g p h a s e and  s t a r v a t i o n , t o f e e d t o l a r v a e and  composition  s t a r v a t i o n was  harvested  An  intermediate  f o r chemical  analyses.  i n the  h e l d as  l o n g a s Isochrysis  starvation  after  2 and  f o r chemical  mid-log c e l l s  iii.  i n g e s t and  A l g a l Analyses:  Only the  as o y s t e r l a r v a e  (Strickland  c o u n t s were d i l u t e d w i t h  and  112  In vivo  fluorometer.  and  then  Parsons 1972).  a 3%  d e t e r m i n e d u s i n g a M o d e l ZB yim a p e r t u r e .  c e l l counts of  the  2  silicate 6 h starved,  and  diets.  a sterile  syringe  s i l i c a t e were m e a s u r e d t w i c e  c o n c e n t r a t i o n s became l o w ,  made h o u r l y  large for  S a m p l e s were drawn f r o m c u l t u r e s  n i t r a t e , p h o s p h a t e and  until  cells  Thus, diatoms i n s e r i e s  6 h of n i t r a t e or  analyses.  were a s s e s s e d  too  through a permanently f i x e d g l a s s tube with and  Preliminary  before  making a c c u r a t e  clumps i m p o s s i b l e .  were h a r v e s t e d  for  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  c l u m p e d , c r e a t i n g a f o o d p a r t i c l e t h a t was  cells  d  sample a t 4 d  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  oyster larvae to  at 2  a  m e a s u r e m e n t s were Samples f o r c e l l  sodium c h l o r i d e s o l u t i o n  and  C o u l t e r Counter equipped with  f l u o r e s c e n c e was  day  measured w i t h  a  a  75  Turner  20  2.  Larval  Culture  Oyster larvae  supplied  by B a y n e s Sound O y s t e r C o . , U n i o n B a y ,  B.C., were s h i p p e d a t l o w t e m p e r a t u r e s Biological  Station.  10°C s e a w a t e r sterilizer. the  L a r v a e were r e s u s p e n d e d  mL s a m p l e s  i n a bucket o f  t e m p e r a t u r e was a l l o w e d t o i n c r e a s e  day t o t h e experimental temperature  concentration  (26°C).  p a s s i v e l y drawn i n t o a p i p e t t e  Larval  plunger.  from water  agitated  E q u a l a l i q u o t s o f 5000 t o 6000  l a r v a e were t h e n d i s t r i b u t e d among 4 L g l a s s  bath  over  was e s t i m a t e d i n t h e b u c k e t by c o u n t s o f s i x , 1  by a p e r f o r a t e d  containing  to the Pacific  t o 1 urn a n d p a s s e d t h r o u g h a UV  filtered Bucket  (~10°C)  3.5 L o f s e a w a t e r  beakers  i n a temperature c o n t r o l l e d  water  (26°C).  Seawater used  i n larval  n u t r i e n t s which  Nereocystis  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 t h e  were l i m i t i n g  i n algal diets.  a n d t h e d i a t o m Ditylum  luetkeana  (West) G r u n . were u s e d t o s t r i p silicate  respectively.  seawater  Following  The b u l l  brightwellii  o f n i t r a t e and  1 \im f i l t r a t i o n  t r e a t m e n t , n i t r a t e - s t r i p p e d s e a w a t e r was f i l t e r e d (Jim p o l y c a r b o n a t e N u c l e p o r e membrane f i l t e r s stripped  s e a w a t e r was f i l t e r  Millipore  sterilized  and  charcoal  t h r o u g h 0.5  and s i l i c a t e -  t h r o u g h 0.45 u.m  filters.  A c o n t r o l treatment of starved treated  kelp  identically  (unfed) l a r v a e , o t h e r w i s e  t o t h e remainder o f l a r v a l  t r e a t m e n t s , was i n c l u d e d  i n each experiment.  culture Starved  larvae  and t h o s e f e d m i d - l o g p h a s e p h y t o p l a n k t o n were grown i n  unstripped,  1 |xm f i l t e r e d ,  A d d i t i o n a l mid-log stripped  UV s t e r i l i s e d  fed larval  seawater t o c o n t r o l  stripping nutrients  natural  seawater.  c u l t u r e s were grown i n n u t r i e n t f o rpossible  side effects of  from t h e seawater.  L a r v a e underwent a complete water exchange d a i l y . algal  diets  different  ( i . e . d i f f e r e n t nutrient treatments, harvested at  a l g a l g r o w t h p h a s e s ) were f e d t o t h e l a r v a e  concentration  of 20,000  cells  mL  -1  .  Every  microscope equipped with a micrometer. e s t i m a t e s were made f r o m l i v e sample o f g r e a t e r carried  than  out i n t r i p l i c a t e .  a t each sample  Chemical  a n d d e a d c o u n t s i n a random A l l e x p e r i m e n t s were  L e n g t h d a t a were a n a l y s e d b y forsignificant  differences  among  date.  Analysis  W e i g h t s f o r a l l a n a l y s e s were m e a s u r e d on a n to  larval  Approximate s u r v i v a l  100 i n d i v i d u a l s .  nested analysis o f variance  3.  few d a y s  at a  was m e a s u r e d t o t h e n e a r e s t 5 urn u n d e r a d i s s e c t i n g  length  diets  The v a r i o u s  ± 1 u,g u n l e s s o t h e r w i s e  Phytoplankton harvested  ultramicrobalance  stated.  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 b y c e n t r i f u g a t i o n i n 2 5 0 mL b o t t l e s a t 2 0 0 0 rpm. P e l l e t s were c o m b i n e d a n d c e n t r i f u g e d 4000 and  rpm i n a d e s k - t o p c e n t r i f u g e . t h e p e l l e t was f r o z e n  Freeze-dried sealed  again  i n 1 0 mL t u b e s a t  S u p e r n a t a n t was  a t -80°C b e f o r e b e i n g  freeze-dried.  s a m p l e s were p a c k a g e d u n d e r n i t r o g e n  i n oxygen b a r r i e r b a g s .  discarded  a n d vacuum-  C e l l s were c o l l e c t e d f r o m  22  known v o l u m e s o f a l g a l  samples  onto pre-washed, pre-combusted GF/A  filters  freeze-dried  i.  i n weighed f o i l  and r i n s e d w i t h  sea s a l t s .  packages  Loaded  3.4% were  (± 10 p.g) t o d e t e r m i n e  F a t t y a c i d methyl e s t e r s  f r o m 20 t o 50 mg  of freeze-dried  samples  prepared  capped w i t h M i n i n e r t  i n 5 mL  (Pierce Co.).  Reacti-Vials  (FAME) were employing  (1988).  Methyl esters  were  valves  U n s a p o n i f i a b l e s were removed i n h e x a n e  t r e a t m e n t w i t h 0.5 Boron t r i f l u o r i d e  after  M methanolic potassium hydroxide at i n methanol  transesterification.  bicarbonate.  (12%) was  The  d r y n e s s i n a vacuum o v e n  85°C.  used f o r  FAME were d i s s o l v e d  w i t h s a t u r a t e d aqueous sodium  i n h e x a n e and  c h l o r i d e and  saturated  h e x a n e s o l u t i o n was  septum.  d e t e r m i n e d and d i s s o l v e d  washed  aqueous  evaporated to  (50°C), flushed with nitrogen  capped w i t h a t e f l o n - l i n e d was  mm  filters  t h e method d e v e l o p e d by Whyte  sodium  25  dry weight.  Fatty Acid Analysis:  prepared  concentration  (500°C) and p r e - w e i g h e d  by g e n t l e vacuum f i l t r a t i o n  ammonium f o r m a t e t o remove any  cellular  o f known c e l l  Weight o f o r g a n i c  and material  i n ethyl acetate to a  1  c o n c e n t r a t i o n o f 10 iJ.g--u.L~ .  A H e w l e t t P a c k a r d , m o d e l 5890 g a s - l i q u i d equipped w i t h a flame i o n i z a t i o n I n t e g r a t o r was  \im f i l m )  rise  2°C-min~  d e t e c t o r and HP  3393A  u s e d t o a n a l y z e 2 u.L o f FAME i n j e c t e d  Supelcowax-10 f u s e d s i l i c a 0.25  chromatograph  capillary  column  (30m  x 0.32  h e a t e d t o 190°C and programmed a f t e r 1  to a final  onto mm  32 m i n  t e m p e r a t u r e o f 2 4 0 ° C f o r 16  min.  a ID, to  23  C a r r i e r gas  was  e x t r a dry helium, passed  p u r i f i e r , w i t h a f l o w r a t e o f 20 m L m i n " 100:1.  F a t t y a c i d s were i d e n t i f i e d  PUFA-1 m e t h y l e s t e r s ( S u p e l c o a c c o r d i n g t o Ackman  ii.  1  and  a heated  a split  ratio  against a standard and  analyses  Analyses:  o f Whyte e t a l . ( 1 9 8 7 ) .  were  Analytical  schemes f o l l o w e d  F r e e z e - d r i e d samples  (4  mg)  water  (2:4:1,  5 mL).  i n an  i c e bath  c e l l s were s o n i c a t e d a t 30 W  s p u n i n a d e s k - t o p c e n t r i f u g e a t 4000 rpm supernatant was  was  passed  through  e x t r a c t e d t w i c e more.  a GF/C  Filtered  t w i c e w i t h t h e a d d i t i o n o f 50 mL (v/v)  t o m i m i c B l i g h and  c h l o r o f o r m l a y e r was  Dyer  D r y n e s s was  evaporations  of chloroform.  c h l o r o f o r m and  M a r s h and  was  used t o determine t o t a l Chloroform  was  The  two  lipid  a  and  residue separated  c h l o r o f o r m and  on  v/v,  The  the  water  The rotary  sequential f r a c t i o n was  i n a volumetric flask to  The  standard.  supernatant  to dryness  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 Weinstein  f i l t e r and  o f 1:1  a s s u r e d by  diluted  f o r 2 min.  (1959) e x t r a c t i o n .  evaporated  evaporator.  in  of  of  were e x t r a c t e d i n c h l o r o f o r m , m e t h a n o l and Algal  gas  (1986).  Gross Composition  those  Inc.)  through  analysis  picked  up  an  (5 t o 25  mL).  (1966) s u l p h u r i c a c i d c h a r r i n g method lipid  against a  tripalmitin  s o l u t i o n s were e v a p o r a t e d  t o dryness  in  a vacuum o v e n 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 acid.  The  same r o u n d - b o t t o m f l a s k was  m e t h a n o l l a y e r t o r e t a i n any  used t o evaporate  the  aqueous  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 separatory was  funnel.  diluted  mL  initial of  1 M  pulled 50  mL  The  chloroform layer  5 mL  i n a volumetric  t o t a l m o n o s a c c h a r i d e s and  extraction  sulphuric  r e s i d u e and  acid  t h r o u g h a GF/C  and  Dubois et  the  a l . (1956).  fraction  for  oligosaccharides.  f i l t e r were h y d r o l y s e d  of  the  h,  25  or  polysaccharides.  phenol/sulphuric acid  D - g l u c o s e was  in 2  100°C f o r 2  d i l u t e d with water to  flask for analysis  S u g a r s were measured u s i n g t h e of  flask  i n a sealed tube at  filter  i n a volumetric  from  c o n c e n t r a t e d aqueous methanol  i n water to  determination of The  i n the  reagent  calibration  standard.  A n o t h e r sample o f determination. in  an  2 mL  Cells  ice bath. of  m i x e d on  10%  dry  algae i n 2 mL  Protein  (w/v)  (4-20  was  mg)  precipitated  i n an  i n a d e s k - t o p model a t  r e s i d u e was  dissolved  50°C f o r a t  to  25  mL  least  i n a volumetric  The  a l b u m i n was  Ash on for  u s e d as  c o n t e n t was  h.  protein  of  of  was  f o r 8 min.  The heating  d i l u t e d with for total  water  protein  Bovine  serum  standard.  burning dry  pre-combusted  W  and  1 M NaOH and  analysed  30  addition  solution  (Hartree, 1972).  calibration  a s s e s s e d by  p r e - w a s h e d and 24  the  The  s o l u t i o n was  f l a s k and  u s i n g a m o d i f i e d Lowry method  with the  4000 rpm  a d d i n g 5 mL  1 h.  for  i c e b a t h f o r 4 min  centrifuged  by  used  o f w a t e r were s o n i c a t e d a t  trichloroacetic acid.  a Vortex, held  to  was  algal  (500°C) GF/C  samples weighed filters  at  500°C  25  D u p l i c a t e s a m p l e s o f e a c h a l g a l t r e a t m e n t were a n a l y s e d Mean c a l o r i c v a l u e was c a l c u l a t e d and  4.3 k c a l g  respectively  - 1  for lipid,  using  factors  c a r b o h y d r a t e and  (Beukema and D e B r u i n  1979).  o f 8.42,  protein  twice. 4.1  RESULTS  I.  A l g a l Growth i n S e r i e s  1 and  Series  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 .  was  grown i n l a r g e c o n t a i n e r s  limitation.  The  chemical composition of this  oyster  Series  saturated  1.  conditions  Series  Plots  feeding  trials.  i n smaller  of Series  the  prior  series  light  2 was  not  used  in  grown u n d e r  light  vessels.  1  1 T a h i t i a n Isochrysis  nutrient concentration that  first  s e r i e s 1 i s presented  s e r i e s was  culture  Cultures  The  which r e s u l t e d i n  f o r comparative purposes, but larvae  2  i n the  cell  density  and  n i t r a t e - l i m i t e d c u l t u r e , show  s t a t i o n a r y p h a s e b e g a n a t a b o u t 13 0 h , w h i c h  to n i t r a t e l i m i t a t i o n  primarily  a f f e c t e d the  c u l t u r e was  185  both l i g h t -  phase samples from the  (Fig. 1).  Thus, l i g h t  h s a m p l e ; and  and  a f t e r 185  nitrate-limited.  was  limitation h  the  A l l stationary  phosphate-limited culture  (185  h  a f t e r w a r d s ) were p h o s p h a t e - s t a r v e d ; h o w e v e r , t h e y were light-limited  due  C. calcitrans  cells  light  limitation  t o the  l a r g e volume o f t h e  ensuing s t a t i o n a r y growth.  p h a s e g r o w t h o c c u r r e d a t a b o u t 90 silicate  depletion  i n the  Nitrate  or  119  Although l i g h t  h.  silicate  b e f o r e p h o s p h a t e was  depleted,  cultures  occurred  limitation  onset  i n the  d i d not  i t must be  of  Stationary  h, p r i o r t o both n i t r a t e  respective  depletion  also  culture.  began t o clump t o g e t h e r w i t h t h e  and  and  and  (Fig. 2). sample t a k e n  at  become a p p a r e n t assumed t h a t  in a l l  27  series  1 treatments nutrient depletion coincided with  light  limitation.  T. pseudonana Therefore  c e l l s a l s o clumped a t h i g h e r  C o u l t e r Counter c e l l  without fluorescence data c o u l d n o t be f i t t e d . fatty later)  light limitation  series  1 T. pseudonana  starved  stages  occurred  b y 92 h  curve  discussed  i s assumed t o h a v e a l s o a f f e c t e d a l l cultures.  A l l samples from t h e  c u l t u r e f r o m 108 h a n d l a t e r were p h o s p h a t e -  (Fig. 3).  pale  o f t h e growth  ( t o be  The c u l t u r e c o n t i n u e d  f o r 5.5 d a y s i n t h e  S i l i c a t e was n o t d e p l e t e d  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 .  turned  2.  c o u n t s were u n r e l i a b l e a n d  the f i n a l  phosphate s t a r v a t i o n phase. the  densities.  However, s i n c e t h e l a r g e s t c h a n g e s i n  a c i d composition  phosphate-limited  cell  I t aged  from  rapidly,  a n d s a n k when t h e s a m p l e was t a k e n a t 163 h .  Series 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 v o l u m e were grown i n t h e s e c o n d series  f o r larval oyster  before  cell  S a m p l e s were t a k e n  c l u m p i n g t o o k p l a c e , a t m i d - l o g phase and 2 and 6  h after the limiting l o n g e r be d e t e c t e d curves  feeding t r i a l s .  are depicted  nutrient, nitrate  or s i l i c a t e ,  i n t h e c u l t u r e medium. i n F i g . 4.  c o u l d no  Average growth  28  Figure  1:  G r o w t h c u r v e s (shown b y c e l l numbers) o f T a h i t i a n Isochrysis grown i n s e r i e s 1 p h o s p h a t e - a n d n i t r a t e - l i m i t e d media, showing t h e c o n c e n t r a t i o n of t h e l i m i t i n g n u t r i e n t s i n t h e 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 m e d i u m ) . A r r o w s i n d i c a t e when s a m p l e s were t a k e n .  Tahitian Isochrysis S e r i e s 1 M i 11 i o n c e l l s  /mL  Medium Nutrient  Concentration  (uM)  600  500 0-  tI  400h  25Qh  (  N 0 3  330h 355h only) ( P 0 4 only)  450h  P 0 4 - l i m c e l l no.  400  300  200  N 0 3 - l i m c e l l no,  0.01 0  100  200  300 Time (h)  uM P 0 4  (P04-lim)  uM N 0 3  (N03-lim)  400  100  0 500  30  Figure  2:  G r o w t h c u r v e s (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 t h e c o n c e n t r a t i o n o f t h e l i m i t i n g n u t r i e n t s i n t h e media. (Nitrate-limited f l u o r e s c e n c e was o m i t t e d s i n c e i t f o l l o w s same curve as other treatments.) A r r o w s i n d i c a t e when s a m p l e s were t a k e n .  Chaetoceros 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  calcitrans (rel, u n i t s )  Series 1 Medium nutrient concentration  100  H 500 150h  174h  P 0 4 - l i m flu - x -  0  50  S i 0 4 - l i m flu  a  P 0 4 - l i m c e l l no,  o  N 0 3 - l i m c e l l no,  x  S i 0 4 - l i m c e l l no.  — -B  uM P 0 4  (P04-lim)  -0-  uM N 0 3  (N03-lim)  -X-  uM S i 0 4  100 Time (h)  -  —  -  (Si04-lim)  150  200  32  Figure  3:  Growth c u r v e s (shown by c e l l numbers) o f Thalassiosira pseudonana grown i n s e r i e s 1 p h o s p h a t e - and s i l i c a t e - l i m i t e d m e d i a , s h o w i n g t h e concentration of the l i m i t i n g nutrients i n the media. Dashed l i n e i n s t a t i o n a r y p h a s e o f g r o w t h c u r v e s r e p r e s e n t s e s t i m a t e d c e l l number; a c c u r a t e c o u n t s were n o t p o s s i b l e due t o c e l l c l u m p i n g . A r r o w s i n d i c a t e when s a m p l e s were t a k e n .  Thalassiosira  pseudonana S e r i e s 1  Million cells /mL  0  Medium Nutrient  50  100  Time (h)  150  Concentration  200  (uM)  250 OO OO  34  F i g u r e 4: G r o w t h c u r v e s (shown b y c e l l numbers) o f t h e 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 m e d i a . A r r o w s mark t h e t i m e a t w h i c h t h e l i m i t i n g n u t r i e n t was e x h a u s t e d f r o m t h e medium. C. calcitrans a n d T. pseudonana were f e d t o o y s t e r larvae at the 6 h s t a r v a t i o n point. T a h i t i a n Isochrysis (TI s o ) was f e d a t 2 d and 6 d s t a r v a t i o n .  Series 2 Algal Growth Curves 100  Million cells / m L A.  Nitrate-starved  Nitrate-limited medium  Ni trate-starved  —  T. pseudonana  —  C. Calcitrans  • T -  so  0.01 50  100  150  200  250  T i m e (h)  100  Million cells / m L B.  Silicate-limited medium  Si starvation  0.1 | ; — - . T. pseudonana —  C. calcitrans  0.01 50  •100  150  T i m e (h)  200  250  36  II.  A.  No  Biochemical Composition  Gross  Biochemical Composition  significant differences  c u l t u r e treatments phytoplankton  of  in algal  cell  Algae  of  Algae  mass among t h e  were d e t e c t e d i n e a c h o f t h e  species.  C e l l weights  three  were t h e r e f o r e p o o l e d  g i v e a f i n a l mean w e i g h t common t o a l l t r e a t m e n t s  to  w i t h i n each  species.  R e c o v e r y o f o r g a n i c s by  experimental procedure  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 . and  carbohydrate  standards  i n the a l g a l  of bovine  e q u i v a l e n t s and  estimates  do n o t n e c e s s a r i l y r e f l e c t F i g u r e s 5, 7 and  chemical  recovery of t o t a l p l u s a s h do  C.  They r e v e a l t h a t n i t r a t e  phytoplankton  composition  o r g a n i c s was  n o t add  up  weight  9 include c e l l  starvation  affected  cell  mass  T.  calculated  i.e., total mass.  organics  I t i s not  have been u n d e r e s t i m a t e d  or  unidentified  t o the oyster's n u t r i t i o n .  Tahitian  Isochrysis  Cellular  energy p o t e n t i a l l y  greatest  i n the mid-log  total  glucose  and  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  1.  against  the a c t u a l  such t h a t  t o the t o t a l  and  by  lipid  calcitrans  impaired  known what o r g a n i c components may  fraction  Protein,  tripalmitin,  f o r T a h i t i a n Isochrysis,  pseudonana.  affected  s a m p l e s were c a l c u l a t e d  serum a l b u m i n ,  o f t h e g r o s s components.  was  available to oyster larvae  s a m p l e o f T a h i t i a n Isochrysis  e n e r g e t i c e q u i v a l e n t o f 276  kcal-10  - 1 2  cells  was with  a  ( F i g . 6a).  37  F i g u r e 5:  ^  C e l l u l a r w e i g h t o f 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 m i d - l o g 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 ) . B a r s r e p r e s e n t mean ± s t a n d a r d error of c e l l weight. See A p p e n d i x A, T a b l e 4 f o r standard error of analyses of duplicate cultures (a and b ) .  38  Tahitian Isochrysis  Cellular Weight of C o m p o n e n t s pg  70  /cell  39  Figure  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 m i d - l o g 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 ) ; A) c e l l u l a r e n e r g y 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 g r o s s b i o c h e m i c a l components. See A p p e n d i x A, T a b l e 1 f o r s t a n d a r d 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 a n d b ) .  40  Caloric Value of Tahitian Isochrysis ^  Cellular Energy equivalents 300  K o a l /10  12  cells  250  200-  150'  100  -N 2 d b Culture Age  B  P e r c e n t of Total Energy in G r o s s C o m p o n e n t s 100%  75% -  50%  25%  -N 2 d b Culture Age  41  P r o t e i n and o f 41%  and  lipid 44%  Carbohydrate cellular  The  comprised  of t o t a l  was  approximately equal c a l o r i c  cell  protein.  s t a r v a t i o n , was  P r o t e i n dropped  pg-cell  - 1  14 p g - c e l l  from  (Fig. 5). - 1  a large depletion of 27 p g - c e l l  t o 11 p g c e l l  - 1  Chaetoceros  The  c a l o r i c v a l u e o f Chaetoceros  .  or s i l i c a t e  calcitrans  to  127-151 k c a l - 1 0  pg  starvation  f r o m a mid  further  and  pg-cell  - 1  t o g r e a t e r than slightly  starvation  from  change.  - 1 2  from  starvation  the energy  S i x hours  of  content of - 1 2  cells  cells.  c e l l s was  attributable to a  f r o m a m i d - l o g v a l u e o f 14 pg  9 pg a t m i d - l o g phase t o 5 pg (Fig. 7).  Carbohydrate  increased  ( F i g . 8b).  after  to  6 h  Carbohydrate  o v e r a m i d - l o g v a l u e o f 10 p g - c e l l  t o 43-48%  diminished  (Fig. 8a).  reduced  from n i t r a t e - s t a r v e d  lipid  was  calcitrans  (mono/oligosaccharide + polysaccharide) gained 4  starvation.  two  cellular  l o g v a l u e o f 166-179 k c a l - 1 0  i n cellular protein  of n i t r a t e  energy  total  calcitrans  C.  6-9  - 1  Prolonged  when t h e c e l l s were n u t r i e n t - s t a r v e d  reduction  to 5  content decreased  in little  2.  loss  - 1  from 9 p g c e l l  Lipid  2 days t o 6 days r e s u l t e d  Caloric  of  change i n t h e g r o s s c o m p o s i t i o n , w i t h i n  while carbohydrate increased  nitrate  ( F i g . 6b).  energy.  days o f n i t r a t e  from  respectively  a r e l a t i v e l y m i n o r component a t 15%  most s t r i k i n g  26-31  energy  values  x  during from  pg-cell  - 1  nitrate  23-26% o f t o t a l  cell  C e l l u l a r w e i g h t 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 m i d - l o g p h a s e (ML) o r 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 ) . B a r s r e p r e s e n t mean ± standard error of c e l l weight. See A p p e n d i x A, Table 5 f o r standard e r r o r of analyses of d u p l i c a t e c u l t u r e s (a and b ) .  43  Chaetoceros  calcitrans  Cellular Weight of Components pg  /cell  ML a  ML b  -N -N a b Culture Age  Lipid  Mono/ol igo  Protein  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 m i d - l o g p h a s e (ML) o r 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 e n e r g y 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 g r o s s b i o c h e m i c a l components. See A p p e n d i x A, T a b l e 2 f o r s t a n d a r d 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 a n d b ) .  45  Caloric Value of Chaetoceros calcitrans Cellular Energy equivalents  200  Kcal / 1 0  1 2  cells  150-  100-  -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  I n s i l i c a t e - s t a r v e d C. calcitrans reduced loss  from  in  cellular  increased energy  from  saw  decrease  3.  10 p g c e l l  to 6 p g c e l l  caloric value  9 p g t o 12-14  The  25%  (Fig. 7), creating Ash  from  t o 18%  32%  t o 40%  - 1 2  i n cellular protein  cells,  F i g . 10a).  from  Energy  (Fig. 9).  silicate-starved  to a decline  pgcell  Nitrate pg-cell  - 1  cells  due  6 h of  ,  Fig. 8).  content increased  Other  from  cells  low  in lipid  from  9 pgcell  - 1  content  - 1  .  The  8  to 7  t o t a l l e d the lowest of - 1 2  cells  Fatty Acid  a c i d complement.  proportion of p a r t i c u l a r  fatty  (4-5 ash  resultant the  ( F i g . 10a).  Composition  F a t t y a c i d c o m p o s i t i o n i s r e p o r t e d as a p e r c e n t a g e fatty  from  lost in  o r g a n i c s d i d not change but  t r e a t m e n t s a t 79-85 k c a l - 1 0  total  a  carbohydrate  in protein  8 t o 10 p g c e l l  c a l o r i c value of the c e l l s  B.  by  starvation.  starvation yielded - 1  was  i n b o t h a s h , from  + p o l y s a c c h a r i d e ) which f e l l after  a  pseudonana  cells  C e l l mass was  a t m i d - l o g p h a s e g r o w t h t o 5 p g , and  ,  - 1 2  8 p g t o 11 pg and  4 pg t o 5 p g , r e s p e c t i v e l y  (mono/oligo  and  ( F i g . 8b).  s t a r v a t i o n y i e l d e d t h e most e n e r g y - r i c h T.  - 1  cell  pseudonana  (119-126 k c a l - 1 0  pg-cell  a  content  augmented f r o m m i d - l o g v a l u e s o f 97-113 k c a l • 1 0 rise  was  balance of t o t a l  i n protein  i n c a r b o h y d r a t e from  Silicate  - 1  (Fig. 8a).  pg.  an a m p l i f i c a t i o n  Thalassiosira  cells  - 1  c a r b o h y d r a t e mass  Changes i n t h e  of  the  relative  a c i d s c a n n o t be  interpreted  as  47  /  F i g u r e 9:  C e l l u l a r w e i g h t o f g r o s s b i o c h e m i c a l components i n Thalassiosira pseudonana s a m p l e d d u r i n g m i d - l o g p h a s e (ML) o r 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 ) . B a r s r e p r e s e n t mean ± standard error of c e l l weight. See A p p e n d i x A, Table 6 f o r standard e r r o r of analyses of d u p l i c a t e c u l t u r e s (a and b ) .  48  Thalassiosira  pseudonana  Cellular Weight of C o m p o n e n t s pg  /cell  ML a Lipid Protein  ML b  -N -N a b Culture Age ! •  Mono/oligo Ash  •Si a  -Si b  Polysaccharide  49  Figure  10:  C a l o r i c v a l u e o f Thalassiosira pseudonana sampled d u r i n g m i d - l o g p h a s e (ML) o r 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 e n e r g y 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 g r o s s b i o c h e m i c a l components. See A p p e n d i x A, T a b l e 3 f o r s t a n d a r d 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 ) .  Caloric value of A  Thalassiosira pseudonana  Cellular Energy equivalents 140  Kcal / 1 0  1 2  -i  ML a  cells  ML b  B  -N -N a b Culture Age  -Si a  -Si b  P e r c e n t of Total Energy in G r o s s C o m p o n e n t s 100%  I to  iim  ML a  i  mmmml  ML b  mmmm  1  :  -N -N a b Culture Age  -.i  1  '<m  -Si a  -Si b  51  changes i n the c e l l t h e c h a n g e s do or  de  quota of t h a t p a r t i c u l a r  fatty  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  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  acid,  as  but  catabolism  fatty  acid  composition.  Fatty acid  p r o f i l e s were a n a l y z e d  i n two  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 20  L carboys  until  1.  they  used i n s e r i e s  reached  fatty  11 acid  Chaetoceros  response  and  cultures.*  i n the  T h e s e c u l t u r e s were  dense  sampled  summarizes t h e profiles  Profiles  intraspecific  differences  of healthy Tahitian  and Thalassiosira  calcitrans  i n the  Isochrysis,  harvested  pseudonana  d u r i n g t h e m i d - l o g a r i t h m i c growth s t a g e . levels  of  senescence.  Comparison o f Mid-Log  Figure  1.  series  Saturated  fatty  the t o t a l p o l y u n s a t u r a t e t o monounsaturate  w e r e h i g h e s t i n T a h i t i a n Isochrysis  (about  acid  ratio  50:20 i n b o t h  series).  Notable  differences  i n the  levels  important  polyunsaturated  two  algal  classes represented  had  high levels  o f 20:5n3  T a h i t i a n Isochrysis Isochrysis  had  had  fatty  of the  nutritionally  a c i d s a r e a p p a r e n t between  in this  study.  ( 1 5 - 2 2 % , F i g . 13  o n l y 1%  ( F i g . 12).  a h i g h p r o p o r t i o n o f 22:6n3  diatoms possessed  4%  or l e s s .  * Tables of complete f a t t y A p p e n d i x B.  The  and  14),  (16%)  profiles  are  diatoms  while  Instead,  T a h i t i a n Isochrysis  acid  two  the  Tahitian  while  the  also  included i n  52  c o n t a i n e d a moderate l e v e l series  1,  while the  w h i c h had  (18:2n6, 4 t o trace  5%);  linolenic  d i a t o m s had  of n6-polyunsaturated Isochrysis  of  conditions.  culture vessel  T h e r e were no  two  influenced  13).  calcitrans  had  2 than i n s e r i e s  C.  series  to  of  the  in fatty  density  usable  and  acid  the  size  irradiance  0.65)  b e t w e e n s e r i e s 1 and  from m i d - l o g growth phase  h i g h e r n3 to  fatty acid  increased  18:4n3  levels in series 2  r i c h e r i n 16:2n4 t h a n  sensitive to  1 and  of  mid-log phase  2 cultures  s i g n i f i c a n t (Figure  irradiance.  vs.  ( F i g . 12). in series 2  n6  2  (Fig. series  1.21)  and  Series (8%  1  vs  b o t h 18:4n3  Apparent d i f f e r e n c e s  o f T. pseudonanana 14).  levels in  (4.81  Thompson e t a l . (1989) h a v e shown t h a t  16:2n4 a r e  not  1 due  was  calcitrans  2%).  vs  series  of diatom f a t t y acids  Cell  significant differences harvested  (2.07  in  culture.  Isochrysis  22:6n3  acid  therefore, light-deficient  series.  Tahitian C.  two  1 must a c c o u n t f o r d i f f e r e n c e s  the  Tahitian  linoleic  between t h e  Phytoplankton i n the  c o m p o s i t i o n between t h e  penetrating  Fractions  were p r e s e n t o n l y  sensitivity  g r o w t h were n u t r i e n t - r e p l e t e ;  of the  in  in  diatoms.  demonstrates the  growth i n s e r i e s  amounts.  complement o f  Small compositional d i f f e r e n c e s  culture  (18:3n3)  were g r e a t e s t  these f a t t y acids  amounts i n t h e  cultures  only trace  fatty acids a large  acid  f a t t y acids  and in are  53  \  Figure  11:  Fatty 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 harvested during the mid-log growth phase i n b a t c h c u l t u r e : A) S e r i e s 1 B) S e r i e s 2.  Mid-log Fatty Acid Profiles Series 1 100  % of total fatty acids  MM  HH |  n6  x  n3  \  | other  polyunsat'd  /  Monounsaturated Saturated  Isochrysis  T. pseudonana C. calcitrans  Series 2 100  % of total fatty acids  i l l  n6  iHi  n3  liliil  other/  v  \  Polyunsat'd  Monounsaturated Hi  Isochrysis  T. pseudonana C. c a l c i t r a n s  Saturated  55  Figure  12:  C o m p a r i s o n o f s e r i e s 1 and s e r i e s 2 h i s t o g r a m s o f 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 l o w e r graph e n l a r g e s the s c a l e o f t h e s m a l l e r peaks o f the upper graph. Error bars represent ± 1 standard deviation of analyses 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 ) .  M i d - L o g F a t t y A c i d s S e r i e s 1 and 2 Tahitian Isochrysis  57  Figure  13:  C o m p a r i s o n o f s e r i e s 1 and s e r i e s 2 h i s t o g r a m s o f f a t t y a c i d c o m p o s i t i o n o f Chaetoceros calcitrans h a r v e s t e d d u r i n g mid-log growth phase. The l o w e r 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. Error bars represent ± 1 . standard deviation of analyses 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 ) .  M i d - L o g F a t t y A c i d s S e r i e s 1 and 2  Chaetoceros calcitrans  59  F i g u r e 14:  C o m p a r i s o n o f s e r i e s 1 and s e r i e s 2 h i s t o g r a m s o f f a t t y a c i d c o m p o s i t i o n o f Thalassiosira pseudonana h a r v e s t e d d u r i n g m i d - l o g g r o w t h p h a s e . The l o w e r 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. Error bars represent ± 1 standard deviation of analyses 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 ) .  M i d - L o g F a t t y A c i d s S e r i e s 1 and 2  Thalassiosira pseudonana  61  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  a.  Tahitian  The  same f a t t y a c i d t r e n d s were s e e n i n b o t h s e r i e s b u t l e v e l s  attained at  Isochrysis  i n series  1, a f t e r 240 h o f n i t r a t e s t a r v a t i o n  450 h s a m p l i n g t i m e ) , were r e a c h e d i n s e r i e s  n i t r a t e was e x h a u s t e d f r o m t h e medium. the  cultures)  because t h e c u l t u r e s  i.  Fatty  Acid  In  series  Class  1, t o t a l  i n both nitrate-and Total 40%, the  indicates  saturation  were g r o w i n g  saturated  fatty acid  starvation  (series 2  faster.  l e v e l s changed  phosphate-limited treatments fractions declined  little  (Fig. 15).  f r o m 46% t o a b o u t  l a r g e l y due t o a d e c r e a s e i n t h e n 3 - p o l y u n s a t u r a t e s , a s monounsaturated f a t t y a c i d p r o p o r t i o n s  a b o u t 27% b y 400 h i n b o t h m e d i a .  fraction 33:30.  increased  Culturing  l o n g e r t h a n 400 h f u r t h e r  f r o m 18%  in nitrate-  d i m i n i s h e d t h e n3  and t h e p o l y u n s a t u r a t e t o monounsaturate r a t i o t o T h e n6 f a t t y a c i d s  were p r e s e n t a t a b o u t 7%, i n  diminutive proportion t o the n3-polyunsaturated  series  also  that  Profile  polyunsaturated  l i m i t e d medium  In  2, 48 h a f t e r  c h a n g e s i n c h e m i c a l c o m p o s i t i o n due t o n i t r a t e  p r o c e e d e d more r a p i d l y u n d e r l i g h t  to  This  (i.e.  2, t h e t o t a l  fraction.  p o l y u n s a t u r a t e t o monounsaturate r a t i o  dropped s u b s t a n t i a l l y  f r o m 49:19 a t m i d - l o g p h a s e t o  33:28 b y t h e s e c o n d d a y o f n i t r a t e s t a r v a t i o n p r o p o r t i o n o f n6 i n s e r i e s  (Fig. 16).  2 was s i m i l a r t o s e r i e s  1.  The  The  62  Figure  15:  Changes o v e r t i m e i n t h e 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 c u r v e and s a m p l i n g t i m e ) : A) n i t r a t e - l i m i t e d medium, a n d B) p h o s p h a t e - l i m i t e d medium.  1  Tahitian Isochrysis S e r i e s F a t t y A c i d C l a s s Profile Nitrate-limited M e d i u m 100  % of total fatty acids  HI  n6  m I  n3 |  . Polyunsat'd  other• Saturated Monounsaturated  85 h  185 h 250 h 3 3 0 h 4 0 0 h 450 h  Culture age  B 100T  Phosphate-limited Medium  % of total fatty acids 1  85 h  185 h  250 h  355 h  Culture age  400 h  64  Figure  16:  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (ML) or 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 , -N 6 d ) . E r r o r b a r s r e p r e s e n t ± 1 s t a n d a r d 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  fatty acid class profile nitrate  ii.  Tahitian  Fatty  had  Isochrysis  and  13%  as  starved c e l l s  Eicosopentaenoic acid component  a high l e v e l of cultures  22:6n3  (1%)  and  was  reached s t a t i o n a r y nitrate-starved  Fatty  2  1  (Fig.  (185  h,  6 days i n s e r i e s  of T a h i t i a n  22:6n3.  phosphate-starved treatment i n s e r i e s  was  s e e n i n 14:0  nitrate levels acid  The  s i m i l a r responses to largest (19%  starvation r o s e and  ( F i g s . 17  to  12%),  in series  18:ln9 and  decline  and  (Figs.  and  when  20).  1  19),  Discriminate  the age  i n both media. (330  h),  16:0,  major  in  fatty  i n b o t h media 1  Following 1 6 : l n 7 and  most p r o m i n e n t  intermediate  fatty  u n i d e n t i f i e d peak,  fatty acid  phosphate-limited cultures  increases  in relative levels in series  w h i l e the the  16:0,  treatment than  1,  culture  l e v e l s became t h e  2 0 : l n 9 , 2 0 : 3 n 3 , 20:5n3 and decreased.  small  culture  Although  w e r e more p r o n o u n c e d i n the' n i t r a t e - s t a r v e d  ( F i g . 17).  a  F i g . 19)  2  and  18).  were 14:0,  Isochrysis  1 8 : l n 9 , 18:2n6, 18:3n3, 18:4n3 and  a c i d p e a k s had  ( F i g . 17),  p r e s e n t o n l y as  phase i n s e r i e s  which  Peaks  Major f a t t y acids  the  1  r e d u c e d f u r t h e r when t h e  for 2 to  Acid  from s e r i e s  (20:5n3) was  (17%)  aged i n b o t h n i t r a t e -  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  2 d-nitrate  iii.  prolonged  Acids  decreased s l i g h t l y to  in  change a f t e r  starvation.  Essential  limited  d i d not  22:0 fatty  acids  N,  r e s p o n s e s between n i t r a t e -  evident i n series  1 fatty  acids  67  Figure  17:  Changes o v e r t i m e i n t h e m a j o r 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 c u r v e and s a m p l i n g t i m e s ) : A) n i t r a t e - l i m i t e d medium, a n d B) p h o s p h a t e - l i m i t e d medium.  )  A  Tahitian Isochrysis S e r i e s Major F a t t y A c i d s N03-limited Medium  1  69  Figure  18:  Changes o v e r t i m e i n t h e m a j o r 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (ML) or 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 , -N 6 d ) . E r r o r b a r s r e p r e s e n t ± 1 s t a n d a r d 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 Major Fatty Acids 30  % of total f a t t y a c i d s ^  25  ML -N 2 d  X T  -N 4 d  20  i  15 -  10 -  5  -N 6 d  I  I  P ll  0 14:0  16:0  18:1n9  18:2n6  18:3n3  18:4n3  22:6n3 o  71  Figure  19:  Changes o v e r t i m e i n t h e 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 c u r v e and sampling time): A) n i t r a t e - l i m i t e d medium, a n d B) p h o s p h a t e - l i m i t e d medium.  Tahitian Isochrysis S e r i e s Intermediate F a t t y A c i d s N03-limited Medium  A  1  % of total fatty acids 85 h  111  185 h  1  H D 330 h •  400 h 450 h  skM. 16:1n7  I•1 18:1n7  20:1n9  20:3n3  20:5n3  I III 1  22:0  22:5n6  P04-limited Medium  B % of total fatty acids  85 h  if 1 -  i  I 16:1n7  1H  185 h  •  250 h  EES 3 5 5 h 400h  •| ii 18:1n7  20;1n9  I 1 w ll  20:3n3  20:5n3  22:0  22:5n6  73  Figure  20: 1  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (ML) or 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 , -N 6 d ) . E r r o r b a r s r e p r e s e n t ± 1 s t a n d a r d 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 Intermediate Fatty Acids % of total f a t t y a c i d s  N  16:1n7  18:1n7  20:1n9  20:3n3  20:5n3  22:0  22;5n6.  75  Figure  21:  Changes o v e r t i m e i n t h e m i n o r 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 ( s e e F i g . 1 f o r g r o w t h curve- a n d s a m p l i n g times): A) n i t r a t e - l i m i t e d medium, a n d B) p h o s p h a t e - l i m i t e d medium.  76 Tahitian Isochrysis S e r i e s 1 Minor F a t t y A c i d s N03-limited Medium  A  % of total fatty acids  16:2n4  18:1n13  B 1.4  19:?  P04-limited  20:a  21:5n3  Medium  % of total fatty acids 85 h  1.2 1  185 h  mmni  355  h  400 h  mm  0.8  111  0.6 0.4  I  0.2 0  I  I 16:2n4  mm 18:1n13  u .  1  I19:?  20:a  21:5n3  77  F i g u r e 22:  Changes o v e r t i m e i n t h e m i n o r 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) a n d h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (ML) or 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 , -N 6 d ) . E r r o r b a r s r e p r e s e n t ± 1 s t a n d a r d 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  included the  nitrate-limited  onset of in  18:4n3 w h i c h d e c r e a s e d o v e r t i m e  phosphate s t a r v a t i o n .  (Figs.  17  and  series  l e v e l by (Figs.  18  series  2,  as  found i n the  20).  After  day  250  h  and  in  series  among t h e  and  resulted  decreased  intermediate  peaks  final  i n trends  same  sample i n s e r i e s  acid  1  slow  proportions  in series  i n 22:5n6, may  2, be  in  e.g.,  a  attributed  limitation.  Isochrysis  g e n e r a l l y s m a l l enough t o be and  levels  2 many p e a k s showed a  increase  Changes i n m i n o r T a h i t i a n  21  the  D i s c r e p a n c i e s between f a t t y  1 p r i o r to  light  increased s l i g h t l y with  dominant f a t t y a c i d s reached the  d e c r e a s e i n 1 6 : l n 7 and to  in  19).  and  decline.  9%)  22:0  20:5n3 l e v e l s  2 a l l the day  to  Phosphate s t a r v a t i o n  augmented 1 6 : l n 7 , 1 8 : l n 7 , and  2 0 : l n 9 , 20:3n3 and  In  t r e a t m e n t and  ( f r o m 14%  22), 1,  but, was  one  fatty acids  considered  notable difference  were  insignificant among t h e  a d e c r e a s e i n 16:2n4 i n t h e  (Figs.  treatments  nitrate-limited  medium, w h e r e a s p h o s p h a t e l i m i t a t i o n p r o d u c e d t h e  opposite  effect.  b.  Chaetoceros  i.  Fatty Acid Class  Chaetoceros similarly  calcitrans  calcitrans  Profile  fatty acid  i n a l l three culture  class  p r o f i l e s were  media i n s e r i e s  1  R e s p o n s e s w i t h t i m e were more a c u t e i n p h o s p h a t e silicate-limited  culture  than i n n i t r a t e - l i m i t e d  affected  (Fig.  23).  and culture  (Fig.  80  Figure  23:  Changes o v e r t i m e i n t h e 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 c u r v e and s a m p l i n g 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, a n d 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  58 h  97  h  119 h  150 h  174 h  C u l t u r e age  Phosphate-limited Medium % of total fatty acids  106  58 h  97 h  119 h  Culture  150 h  age  Silicate-limited Medium % of total fatty acids  10Q  99 h  119 h  150 h  82  F i g u r e 24:  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) i n A) n i t r a t e - l i m i t e d medium, o r B) s i l i c a t e - l i m i t e d medium a n d h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 represent ± 1 standard deviation of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h s a m p l e s ) .  Series 2 Fatty Acid Class Profile  Chaetoceros  calcitrans  'd  Mid-log  Si-lim 2 h Si-lim 6 h Culture Age  84  23).  In the s i l i c a t e - l i m i t e d  evident u n t i l silicate  s a m p l e a t 150 h , ( i . e . a f t e r 30 h o f  s t a r v a t i o n , when t h e c e l l s were s e n e s c e n t ) .  increases final  the l a s t  t r e a t m e n t , e f f e c t s were n o t  i n unknowns  % of total  such as N  fatty acids  ( F i g . 27c) e x p l a i n  Large  t h e low  i n the s i l i c a t e - l i m i t e d  treatment.  As  w i t h t h e 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  polyunsaturate  fraction declined  decreases occurred within particularly  20:5n3  small portion  In the  ii.  The  t h e n3 f a t t y a c i d s  (Fig. 25).  cultures  2, n u t r i e n t  largest  (Fig. 23),  The n6 f a t t y a c i d s  comprised a  (Fig. 23).  The  Fatty  ( F i g . 25b and c ) .  deprivation  fatty acid class profile  Essential  1, t h e  16:2n6, was d e p l e t e d o v e r t i m e i n p h o s p h a t e -  silicate-limited  series  over time.  of the polyunsaturated p r o f i l e  d o m i n a n t n6 a c i d , and  i n series  h a d no s i g n i f i c a n t e f f e c t on  (Fig. 24).  Acids  W h i l e 22:6n3 c o m p r i s e d o n l y a m i n o r component o f t h e t o t a l fatty acid  complement o f Chaetoceros  contained large all  this  alga  amounts o f 20:5n3 w h i c h d e c r e a s e d w i t h t i m e i n  treatments i n series  were n o t o b s e r v e d u n t i l senescence i n s i l i c a t e 2, 6 h o f s i l i c a t e (Fig. 26).  calcitrans,  1 (Fig. 25). after nutrient  Pronounced depletion  reductions and upon  and p h o s p h a t e - l i m i t e d m e d i a .  starvation  d i d n o t c h a n g e 20:5n3  In series levels  C h a n g e s i n t h e l e v e l o f 20:5n3 f r o m 17% t o 13% was  seen i n t h e s e r i e s  1 nitrate-limited culture  only with the  85  o n s e t o f l i g h t - l i m i t e d g r o w t h a t t h e 97 h s a m p l e . followed during nitrate nitrate  starvation  20:5n3 l e v e l s  The  levels  (Fig. 27).  ( a f t e r 119 h ) .  i n series  1 affected  intermediate  o f 22:6n3 d i f f e r e n t l y i n Chaetoceros  l e a d t o a d e c r e a s e by  Silicate  starvation  i n series  f r o m a m i d - l o g l e v e l o f 2%  calcitrans  (Fig. 28).  growth, n i t r a t e  22:6n3 i n s e r i e s  levels  Fatty Acid  o f 0.5%, down  While t h i s  with time i n  starvation  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 fatty  acid  C. calcitrans  i n both n i t r a t e - s t a r v e d  i n series  2 (Figs.  Only s l i g h t changes o c c u r r e d calcitrans in  (after  119 h ) .  and  i n series  1.  silicatei n the fatty  silicate-starved  i n the fatty acid  p e a k s o f C.  medium i n s e r i e s  f r o m 10% t o 14%  l i g h t - l i m i t e d growth  starvation  was  2 6 , 28 and 3 0 ) .  grown i n n i t r a t e - l i m i t e d  16:0 w h i c h i n c r e a s e d  during  upon  Peaks  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 composition  and  h a d no e f f e c t  E x c e p t f o r a d e c r e a s e i n 22:6n3 when c e l l s were  acid  intermediate  (58 h) t h r o u g h l i g h t -  f o r the increase i n t h i s  and  senescence.  m i n o r component i n c r e a s e d s t e a d i l y  nitrate-limited  i i i .  2 yielded  1, f r o m t h e m i d - l o g p h a s e  responsible  fatty  P h o s p h a t e d e p l e t i o n h a d no s i g n i f i c a n t e f f e c t  starvation  series  2  (Fig. 26).  silicate  fatty acid  Similarly,  c a u s e d no s i g n i f i c a n t c h a n g e i n s e r i e s  three treatments  acid  starvation  Stability  (Figs.  1 except  25 and 27)  (97 t o 119 h) and p r i o r t o n i t r a t e 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  Figure  25:  Changes o v e r t i m e i n t h e m a j o r 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 c u r v e and s a m p l i n g 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, a n d 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  58 h  mm 97 h  m  14:0  0 16:0  16:1n7  H0150 h  J  I  II  16:2n4 16:3n4  Silicate-limited Medium  18:4n3 20:5n3  1 1 99 h 111150 h  Inn  I: 14:0  16:0  16:1n7  16:2n4  16:3n4  18:4n3  20:5n3  88  Figure  26:  Changes o v e r t i m e i n t h e m a j o r f a t t y a c i d s o f Chaetoceros calcitrans cultured i n series 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 and - S i 2 h o r 6 h ) . E r r o r bars represent ± 1 standard deviation of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h samples).  Chaetoceros  calcitrans  Series  2  Major Fatty Acids % of total f a t t y a c i d s ML -N-2 h  -N 6 h -Si 2 h -Si 6 h  1 P  I 11 ii  1  14:0  16:0  16:1n7  16:2n4  16:3n4  18:4n3  20:5n3  90  Figure  27:  Changes o v e r t i m e i n t h e 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 c u r v e and s a m p l i n g times): 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, a n d 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  58 h I  I 97 h  IMP  N  ii 1 J I II 16:2n6  n  1174 h  Li |1  60  16:4n1  17:0  a I  18:1n7  22:5n3 22:6n3  * of total f a t t y a c i d s  Phosphate-limited Medium  Ml  I  N 12  16:2n6  % of total f a t t y  16:4n1  17:0  18:1n7 22:5n3 22:6n3  acids  Silicate-limited Medium  l i i l 99 h  10  H I 150 h  1 16:2n6  16:4n1  17:0  18:1n7 22:5n3  22:6n3  92  Figure  28:  Changes o v e r t i m e i n t h e 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 cultured in series 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 and - S i 2 h o r 6 h ) . E r r o r bars represent ± 1 standard d e v i a t i o n of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h samples).  Series 2 Intermediate Fatty Acids  Chaetoceros  calcitrans  % of total fatty a c i d s  N  .16:2n6  16:4n1  17:0  18:1n7  22:5n3  22:6n3  94  Figure  29:  Changes o v e r t i m e i n t h e m i n o r 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 g r o w t h c u r v e a n d s a m p l i n g 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, a n d 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  * of total fatty acids 58 h  1.4 •  Nitrate-limited Medium  97 h  1.2  m  1.0  1 5 0  n  174 h  0.8 0.6 0.4 0.2 0 1  1.6  16:1n5 16;3n3 18:1n13 18:1n9 18:2n6 18:2n4 18;3n1 20:4n6  * of total fatty acids 58 h  1.4  Phosphate-limited Medium  97 h 119 h  1.2  150 h 1.0 0.8 0.6 0.4 0.2  T II II  •if  0 16:1n5 16:3n3 18:1n13 18:1n9 18:2n6 18:2n4 18:3n1 20:4n6 * of total fatty acids 1  I6;1n5 16:3n3 18:ln13 18:1n9 18:2n6 18:2n4 18:3n1 20:4n6  96  Figure  30:  Changes o v e r t i m e i n t h e m i n o r f a t t y a c i d s o f Chaetoceros calcitrans cultured i n series 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) and h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 and - S i 2 h o r 6 h ) . Error bars represent ± 1 standard deviation of analyses of r e p l i c a t e c u l t u r e s (n=2 i n 2 h s a m p l e s , n=3 i n 6 h samples).  Chaetoceros  Series 2  calcitrans  Minor Fatty Acids 2.5  % of total f a t t y a c i d s ML  2 -  -N 2 h  -N 6 h -Si 2 h  ,5  0,5 -  0  -Si 6 h  1 1 1 1I  Ii  1  1  •  16:1n5  II  1 16:3n3  18:1n13  18:1n9  1 ti  18:2n6  1. p  18:3n4 ~0  98  series  1 n i t r a t e - l i m i t e d medium r e s u l t e d  increase  i n 22:6n3  (see  Essential  Responses t o phosphate d e p l e t i o n intermediate 2.4%.  fatty acid  Except i n 16:ln7  until  the  Fatty  i n an  immediate  A c i d s and  18:ln7 which i n c r e a s e d  f r o m 0.5  ( F i g . 25),  not  l a s t s a m p l e when c e l l  changes d i d  d e a t h was  fatty acids  silicate-limited  culture  senescence  and  The  occurred at  1 8 : l n 7 , 16:3n3 and  the  the to  develop  occurring.  i n c r e a s e s among t h e  16:0,  27).  were s i m i l a r , e x c e p t i n  Most s u b s t a n t i a l  including  Fig.  in series (160  unknown N  1  h),  ( F i g s . 25,  27  29).  minor f a t t y a c i d  series  1 was  medium  ( F i g . 29).  composition of  d e p e n d e n t on After  the  limiting nutrient  calcitrans i n the  in  culture  mid-log phase growth i n n i t r a t e -  l i m i t e d medium, 1 8 : 3 n l i n c r e a s e d dramatically  Chaetoceros  f r o m 0.25%  to  decreased again under prolonged  1%  and  then  nitrate  starvation.  c.  Thalassiosira  T. pseudonana series medium.  1 and  pseudonana fatty acid  2 only  c o m p o s i t i o n can  for cells  S a m p l e s were l o s t and  nitrate-limited  cultures  t h a t no  m i d - l o g s a m p l e was  culture  in series  representative of  grown i n data  in series  be  compared between  silicate-limited  i s unavailable 1.  I t must be  t a k e n from the first  for stressed  phosphate-limited  1.  Instead the  sample i s  the  l a t e - l o g p h a s e when p h o s p h a t e l i m i t a t i o n  99  was  i n f l u e n c i n g the chemical  first the  sample o f t h e s i l i c a t e - l i m i t e d  i n series  proportional values of  f a t t y a c i d s were p r e s e n t  i n T. pseudonana  The p o l y u n s a t u r a t e  both  from a mid-log  phosphate-limited  respectively. was s u b s t a n t i a l  i n both  The  rapidly  o f t h e n3 f r a c t i o n although  c u l t u r e s aged, of polyunsaturates more p r o n o u n c 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 2 0 : 5 n 3 , 18:4n3 a n d 22:6n3 f a t t y a c i d s  n6-polyunsaturated  1( F i g .  v a l u e o f 46:22 t o 20:30 a n d 25:30 a s  treatments,  in the phosphate-limited reduction  t o monounsaturate r a t i o  and s i l i c a t e - l i m i t e d  Reduction  at levels  o f 16:0 a n d 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  decreased  1 is  Profile  which v a r i e d with the content  31).  The  i n the series.  Fatty Acid Class  Saturated  of the algae.  culture  s o l e r e p r e s e n t a t i v e of the mid-log  f a t t y acids content  i.  composition  i n the  (Fig. 33).  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  c o m p a r i s o n t o t h e n3 c o m p o n e n t , b u t was e n h a n c e d when p h o s p h a t e was  limiting  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  acid  (20:4n6, F i g . 3 5 a ) .  S e r i e s 2 T. pseudonana  p r o f i l e s were n o t 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  ii.  Essential  Thalassiosira and  (Fig. 32).  Fatty Acids  pseudonana  contained high l e v e l s  of both  22:6n3 w i t h t h e f o r m e r a l w a y s i n g r e a t e r p r o p o r t i o n  33 a n d 3 4 ) .  In series  1, s i g n i f i c a n t  20:5n3 (Figs.  r e d u c t i o n s o f 20:5n3  100  Figure  31:  Changes o v e r t i m e i n t h e 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 g r o w t h c u r v e a n d s a m p l i n g t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e l i m i t e d medium.  Thalassiosira A  pseudonana  Series 1  F a t t y A c i d C l a s s Profile Phosphate-limited Medium  102  Figure  32:  Changes o v e r t i m e i n t h e 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) i n A) n i t r a t e - l i m i t e d medium, o r B) s i l i c a t e - l i m i t e d medium a n d h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 or 6 h ) . E r r o r bars represent ± 1 standard deviation o f analyses of r e p l i c a t e c u l t u r e s (n=2 i n N - s t a r v e d s a m p l e s , n=3 i n 2 h S i s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s ) .  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 t h e 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 starvation  i n series  only s l i g h t l y . pronounced  2 r e d u c e d t h e amount o f t h e f a t t y  Apparently l i g h t  l i m i t a t i o n h a d a more  e f f e c t on p r o p o r t i o n s o f 20:5n3 t h a n  starvation.  However, phosphate  greater reduction  acid  starvation  silicate  c a u s e d an  even  i n amount o f 2 0 : 5 n 3 , f r o m 22% t o a l o w o f  6%, i n T . pseudonana  i n series  1.  T h i s was t h e m o s t p r o f o u n d  e f f e c t o b s e r v e d among a l l t r e a t m e n t s among a l l 3 p h y t o p l a n k t o n species  iii.  All  employed.  Fatty  Acid  Peaks  effects of nutrient  starvation  on T . pseudonana  fatty  c o m p o s i t i o n were v e r y s l i g h t i n s e r i e s  2 and appear  within  (Figs  the limits  of natural  S i n c e changes i n f a t t y a c i d  variation  levels i n series  l i m i t e d medium t o o k p l a c e p r i o r t o s i l i c a t e t h e y were c a u s e d b y l o w l i g h t that suffered  ( F i g . 33b).  decreases i n f a t t y acid  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 were 16:3n4, 18:4n3 and 20:5n3  fall  3 4 , 36 and 3 8 ) .  1  silicate-  starvation Major  fatty  (92 h ) , acids  l e v e l s i n both culture  (Fig. 33).  i n c r e a s e s were 1 6 : 0 , 1 6 : l n 7 and 1 8 : l n 7  to  acid  conditions,  Those  showing  ( F i g s . 33 a n d 3 5 ) .  P h o s p h a t e - l i m i t e d c e l l s u n d e r w e n t a 100% i n c r e a s e  i n 16:0 and  a reduction  Relatively  large and  i n 20:5n3 t o 25% o f o r i g i n a l l e v e l s .  i n c r e a s e s were a l s o  observed  20:.4n6, and a d e c r e a s e was  l i m i t e d T. pseudonana  seen  (Fig.35).  i n 18:lnl3,  18:ln9,  18:ln7,  i n 16:2n4 i n p h o s p h a t e -  105  Figure  33:  Changes o v e r t i m e i n t h e m a j o r 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 g r o w t h c u r v e a n d s a m p l i n g t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e l i m i t e d medium. The S i 66 h s a m p l e i s i n c l u d e d i n t h e 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 m i d - l o g (ML) fatty acid levels.  Thalassiosira  pseudonana  Series 1  Major Fatty Acids P04-limited Medium % of total fatty acids  14:0  16:0  B  16:1n7  16:3n4  18:4n3  20:5n3  22:6n3  20:5n3  22:6n3  Si04-limited Medium % of total fatty acids  14:0  16:0  16:1n7  16:3n4  18:4n3  107  Figure  34:  Changes o v e r t i m e i n t h e m a j o r 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) a n d h a r v e s t e d a t m i d - l o g 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 ) . Error bars represent ± 1 standard deviation 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 - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s ) .  Thalassiosira  pseudonana  Series  2  Major Fatty Acids % of total f a t t y a c i d s  14:0  16:0  16:1n7  16:3n4  18:4n3  20:5n3  22:6n3 o co  109  Figure  35:  Changes o v e r t i m e i n t h e 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 series 1 c u l t u r e ( S e e . F i g . 3 f o r growth c u r v e and sampling times): A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e - l i m i t e d medium. The S i 66 h s a m p l e i s i n c l u d e d i n t h e 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 . t  Thalassiosira A 4  pseudonana  Series 1  Intermediate F a t t y A c i d s P04-limited Medium % of total fatty acids  -i  N  16:2n6  16:2n4  18:1n13  18:1n9  18:1n7  20:4n6  Ill  F i g u r e 36:  Changes o v e r t i m e i n t h e 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, o r s i l i c a t e l i m i t e d medium ( s e e F i g . 4 f o r g r o w t h c u r v e ) a n d h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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 or 6 h ) . Error bars represent ± 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 - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s ) .  Thalassiosira  Series 2  pseudonana  Intermediate Fatty Acids 5  % of total f a t t y a c i d s ML -N 2 h  4 4  -N 6 h -Si 2 h  3  -Si 6 h  •  X  T  N  16:2n6  16:2n4  18:1n13  • 18:1n9  ^  18:1n7  m m.  20:4n6  t o  113  F i g u r e 37:  Changes o v e r t i m e i n t h e m i n o r 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 g r o w t h c u r v e a n d s a m p l i n g t i m e s ) : A) p h o s p h a t e - l i m i t e d medium, a n d B) s i l i c a t e l i m i t e d medium. T h e S i 66 h s a m p l e i s i n c l u d e d i n t h e 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 m i d - l o g fatty acid levels.  Thalassiosira  pseudonana  A  Minor F a t t y A c i d s P04-limited Medium  B  Si03-limited Medium  Series 1  % of total fatty acids 1,6 i  17:0  16:4n1  18:1n5  18:2n6  18:3n6  20:4n3  115  Figure  38:  Changes o v e r t i m e i n t h e m i n o r 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 ( s e e F i g . 4 f o r g r o w t h c u r v e ) a n d h a r v e s t e d a t m i d - l o g g r o w t h p h a s e (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). Error bars represent ± 1 standard deviation 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 - s t a r v e d s a m p l e s , n=3 i n 2 h S i - s t a r v e d s a m p l e s , n=4 i n 6 h S i - s t a r v e d s a m p l e s ) .  Thalassiosira  Series 2  pseudonana  Minor Fatty Acids 1.2  % of total f a t t y a c i d s  N 2 h  M ML  H i  -N 6 d  -Si 2 h  ^ 1 -Si 6 h  0.8  0.6 -  0.4 -  0.2  0 17:0  16:4n1  18:2n6  18:3n4  20:4n3  117  Changes i n t h e minor peaks o f s e r i e s range  o f l e s s t h a n 1%  ( F i g . 37).  p h o s p h a t e - l i m i t e d c e l l s was component. limitation  1 were o v e r a v e r y s m a l l  The  o n l y n o t a b l e change i n  a reduction  i n the  18:3n6  In the s i l i c a t e - l i m i t e d treatment,  light  c a u s e d a d e c r e a s e i n 18:3n6, w h i l e 1 8 : l n 5  following s i l i c a t e  starvation.  Ill.  Growth o f O y s t e r  Larvae  starved controls  (no a l g a l  In a l l feeding t r i a l s , oyster larvae  showed l i t t l e  o r no g r o w t h .  food)  In mid-log  o f a l l e x p e r i m e n t s , no e f f e c t on l a r v a l  shell  e x h i b i t e d when l a r v a l  stripped of  nitrate  1.  or s i l i c a t e  Tahitian  increased  c u l t u r e w a t e r was  length  for nutrient-starved algal  of  controls was  either  treatments.  Isochrysis  T a h i t i a n Isochrysis  was  a poor d i e t  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  f o r C. gigas level  l a r v a e when  When c e l l s were h a r v e s t e d a t t h e m i d - l o g p h a s e o f g r o w t h fed to larvae larger  f o r f i v e days, s h e l l  (90 p.m,  l e n g t h a t subsequent variations  may  F i g . 39, T a b l e I I I ) . measurements was  in algal diet.  ( T a b l e I I ) and first,  l e n g t h was  poor  and  nitrate-  However,  not a f f e c t e d  S u r v i v a l was  mL .  significantly  (99 |im, p<0.05, T u k e y T e s t ) t h a n t h o s e f e d  starved c e l l s  -1  o f 20,000 c e l l s  larval  by  i n a l l treatments  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  have b i a s e d r e s u l t s as t h e experiment  progressed.  118 Table II: F i n a l survival of Crassostrea gigas larvae in three unialgal diet t r i a l s l a s t i n g from 12 to 19 days. Tahitian Isochrysis diets consisted of a l g a l c e l l s harvested fton n i t r a t e - U n i t e d medium at aid-log growth phase, or after 2 or 6 days of n i t r a t e starvation (-N 2 d or 6 d). I second aid-log treatsent (Hid-log control) controlled for the effect of holding larvae fed nitrate-starved c e l l s in n i t r a t e - f r e e seavater. Diatom diets consisted of c e l l s harvested from n i t r a t e - and s i l i c a t e limited nediuii at aid-log phase, or after 6 h of nutrient starvation (-8 6 h, -Si 6 h). Two additional mid-log treatments (Hid-log N-control, Hid-log Si-control) controlled for the effect 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 in nutrient stripped, nitrate-free or s i l i c a t e - f r e e seavater, respectively. Valaes are mean percentages t 1 standard deviation, 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 .  Tahitian Isochrysis  Chaetoceros c a l c i t r a n s  Thalassiosira pseudonana  19 days  14 days  12 days  Hid-log Mid-log control -H-2 d -H-6 d  29 25 13 6  t • t t  20 4 5 5  Mid-log Hid-log H-control Hid-log Si-control -N 6 h -Si 6 h  59 61 46 41 32  t t • i t  6 17 22 24 20  Hid-log Hid-log H-control Hid-log Si-control -H 6 h -Si 6 h  78 93 86 90 94  t i t • t  15 7 11 9 1  119  Figure  39:  G r o w t h o f 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 f r o m n i t r a t e - l i m i t e d medium a t m i d - l o g 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 s e c o n d mid-log treatment (ML-control) c o n t r o l l e d f o r t h e effect of holding larvae fed nitrate-starved c e l l s i n n i t r a t e - f r e e seawater. Starved larvae d i d not r e c e i v e any a l g a l f o o d d u r i n g t h e e x p e r i m e n t . L e a s t s q u a r e s means o f l a r v a l l e n g t h were a n a l y s e d by n e s t e d 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 n d a r d e r r o r s a l l l i e w i t h i n t h e symbols except f o r t h e (-N 6 d) t r e a t m e n t ( s e e T a b l e I I ) . Asterisks represent s i g n i f i c a n t difference at 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  250  Shell Length  (urn)  250  200  200  150 -  150  100  i  5  10 T i m e (days)  15  100  20  O  121 Table I I I : Shell length !)IB, part a) and Interval growth rates (part b| of l a r v a l Crassostrea gigas fed a unialgal d i e t , at a d a i l y concentration of 20,000 cells-aL-1 for 19 days, of Tahitian Isochrysis harvested fron nitrate-limited aediura at raid-log grovth phase, or a f t e r 2 or 6 days of n i t r a t e starvation (-N 2 d or 6 d). A second mid-log treatment (Hid-log control) controlled for the e f f e c t of holding larvae fed nitrate-starved c e l l s in nitrate-free seavater. Least squares Beans of shell length t standard error vere calculated by nested analysis of variance, at the 95\ confidence l e v e l , for three replicates. 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 multiple coaparison test, (p<.05). Hatching letters among treatments within the day of measurement s i g n i f y no difference among the treatments bearing those l e t t e r s . a) Time (Days) Treatment Replicate ML  1 2 3 LS Mean  0  5  85 t 0.8 (54)  99 t 1.4 (51) 98 t 1.3 (57) 99 ! 1.4 (55)  85 t 0.8  99 i 0.7 [ a l  119 • 124 t 132 t 125 t  97 i 1.7 (57) 98 i 1.9 (55) 100 t 2.4 (29)  HL-control 1 2 3 LS Hean -N 2 d  -S 6 d  85 t 0.8  1 2 3 LS Hean  85 i 0.8  1 2 3 LS Hean  10  85 i 0. 8  19  2.3 (53) 3.1 (56) 3.3 (55)  156 i 5.9 (44) 185 t 7.9 (47) 167 ± 7.1 (44)  181 t 8.3 (36) 198 i 12.5 (30) 238 i 10.3 (51)  1.9 (al  170 • 4.2 [ab]  206 t 5.0 lab]  123 • 3.2 (71) 111 ! 2.4 (50) 114 t 3.3 (56)  168 i 6.3 (52) 160 t 7.7 (50) 152 t 8.9 (53)  216 i 7.8 (36) 152 t 10.0 (45) 151 t 6.9 (30)  99 i 0.8 [a]  116 i 1.8 lb]  160 t 3.9 label 173 i 5.1 lab]  89 t 1.3 (57) 89 t 1.2 (62) 92 i 1.4 (46) 90 • 0.7 [b]  109 t 96 t 108 t 104 i  159 i 161 i 172 t 164 i  90 t 0.6 (60) 92 t 0.8 (60) 92 • 1.0 (55)  111 i 3.1 (56) 108 i 3.8 (48) 114 t 5.0 (48)  156 t 4.7 (48) 195 ± 6.8 (50) 198 i 8.7 (47)  204 i 32.6 (4) 220 t 10.0 (36) 191 i 9.0 (28)  111 i 1.9 I d  181 t 4.1 lac]  205 i 9.9 lac]  91 i 0.7 l b !  3.1 (55) 3.8 (54) 5.0 (64) 1.8 [be]  b) Treatment  0-5 days  5-10 days  ML HL-control -8 2 d -H 6 d  2.8 2.8 2.8 4.0  5.2 3.4 12.0 14.0  1.0 1.2  15  6.6 (54) 7.2 (46) 6.9 (50) 4.0 lac)  188 t 9.7 (30) 169 i 7.6 (49) 190 t 8.7 (37) 182 i 5.0 lac]  Grovth ()lB'day-l) 10-15 days 15-20 days 9.0 8.8 4.5 6.0  9.0 3.3  122  Thus i t i s d i f f i c u l t T a h i t i a n Isochrysis  t o assess the r e l a t i v e value of the diets.  When f e d T a h i t i a n Isochrysis, o n l y 205 |i.m a f t e r  19 d a y s o f f e e d i n g  was c o m p a r e d b e t w e e n m i d - l o g Thalassiosira  pseudonana  T a b l e V) .  i n another  Larval  growth  and  experiment (see  l e n g t h , a t 12 d a y s , was 160 p.m d i e t , w h i l e a d i e t o f T.  y i e l d e d much l a r g e r  pseudonana length  Isochrysis  ( F i g . 39) .  T a h i t i a n Isochrysis  diets  F i n a l mean s h e l l  with the mid-log  l a r v a e grew t o a maximum mean o f  larvae ranging  i n mean  f r o m 215 t o 256 \im d e p e n d i n g on t h e t r e a t m e n t  shell (Table  V). 2.  Chaetoceros  Crassostrea  calcitrans  gigas  l a r v a e grew b e s t on a d i e t o f  when f e d a l g a e h a r v e s t e d d u r i n g t h e m i d - l o g  calcitrans  ( F i g . 4 0 ) . N i t r a t e - s t a r v e d C. calcitrans food v a l u e . Table  Chaetoceros  Shell  ranked second i n  l e n g t h was s i g n i f i c a n t l y  IV) among t h e t h r e e t r e a t m e n t s  phase  after  different Day 3.  (p<0.05,  Final  shell  l e n g t h s o n Day 14 were 265 |i.m, 209 u.m a n d 173 p,m f o r m i d - l o g , nitrate-limited  and s i l i c a t e - l i m i t e d  treatments,  I n t e r v a l growth r a t e s i n a l l treatments l a r v a e reached  lines  were h i g h e s t u n t i l t h e  a l e n g t h o f a b o u t 190 nm, a t w h i c h p o i n t g r o w t h  r a t e s were s l o w e r the  respectively.  again  i n F i g . 40).  ( T a b l e I V , and s e e a l s o t h e s l o p e o f  123  Figure  40:  G r o w t h ( s h e l l l e n g t h ) o f Crassostrea gigas larvae 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 m i d - l o g p h a s e ( M L ) , o r 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 m i d - l o g t r e a t m e n t s (ML-Nc o n t r o l , ML-Si-control) controlled 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 starved 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, r e s p e c t i v e l y . Starved l a r v a e d i d n o t r e c e i v e any a l g a l f o o d d u r i n g t h e experiment. L e a s t s q u a r e s means o f l a r v a l l e n g t h were a n a l y s e d b y n e s t e d 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 errors a l l l i e within the range o f t h e symbols (see T a b l e I I I ) . Asterisks 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 t h e 95% confidence l e v e l .  Crassostrea gigas larval growth on Chaetoceros calcitrans diet  125 Table IV: Shell length [p, pact a) and i n t e i v a l growth rates (part b) of l a r v a l Crassostrea gigas fed a unialgal d i e t , for 14 days as in Table I I , of Chaetoceros c a l c i t r a n s harvested fron 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 after 6 h of nutrient starvation (-N 6 h, - S i 6 h). Two additional aid-log treatments (Hid-log N-control, Hid-log Si-control) controlled for the effect 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 seavater, respectively.  Time (Days) Treatment Replicate Hid-log  1 2 3  114 i 0 .9 (165)  LS Hean Hid-log H-control  0  114 • 0.9  1 2 3 LS Hean  114 t 0.9  Hid-log 1 Si-control 2 3 LS Hean -1  6h  1 2 3 LS Hean  -Si 6 h  114 t 0.9  114 • 0.9  1 2 3 LS Hean  114 i 0.9  3  6  10  14  165 • 2.6(63) 149 i 2.9 (76) 163 t 2.5 (77)  208 197 195  t •  2.5 (69) 2.9 (69) 2.4 (72)  242 i 4.2 (63) 232 • 3.8 (72) 230 i 3.6 (67)  279.i 4.5 (66) 276 i 5.0 (69) 268 i 4.9 (69)  159 t 1.3 [ a l  200  +  1.6 lab]  235 i 2.3 [ a 1  274 t 2.6 (al  147 i 3.3 (68) 159 i 2.5 (67) 163 • 2.5 (71)  205 195 198  4.6 (66) 3.3 (69) 3.4 (66)  233 ± 3.8 (66) 224 • 3.9 (66) 222 i 3.2 (66)  281 t 6.0 (60) 248 i 4.9 (66) 245 i 4.3 (66)  156 i 1.3 [al  199  +  149 i 2.4 (54) 158 i 2.0 (60) 164 i 2.7 (71)  193 176 207  +  3.4 (73) 2.4 (60) 2.8 (80)  238 t 5.8 (66) 221 t 4.4 (66) 235 t 4.4 (64)  268 t 5.1 (60) 258 t 3.8 (66) 258 t 3.4 (69)  157 i 1.4 [ a l  192  +  1.6 Ibcl  232 t 2.3 [a]  262 i 2.7 [bl  124 • 1.6 (71) 136 t 1.9 (65) 137 • 1.7 (63)  159 166 158  +  2.8 (80) 3.2 (80) 2.6 (53)  192 t 4.7 (63) 198 • 5.1 (60) 182 i 3.8 (60)  215 t 5.3 (75) 203 i 4.6 (69)  133 i 1.3 (bl  161  +  1.6 [dl  191 i 2.4 [bl  209 i 3.1 [ c l  130 i 1.8 (72) 121 t 1.5 (57) 128 i 1.5 (57)  144 137 144  +  2.3 (62) 2.1 (45) 2.3 (69)  159 • 3.2 (60) 159 i 3.2 (38) 161 i 3.3 (60)  178 t 5.0 (63) 172 i 5.3 (29) 170 t 3.1 (72)  126 i 1.4 [ c l  142  1.8 [ e l  160 • 2.7 [ c l  173 i 3.2 [dl  b) Treatment  0-3 days  Mid-log N-control Si-control -H 6 h -Si 6 h  14.0 14.3 6.3  •  +  +  1.6 [abcl 226 t 2.3 [ a l  Growth (HiHay-1) 3-6 days 6-10 days 15.0 14.3 11.7 9 .3 12.0  13.7 6.8 10.0 7.5 5.3  258 • 2.7 [bl  10-14 days 8.8 8.0 7.5 4.5 4.5  9.8  3.3  126  Figure  41:  Growth ( s h e l l length) o f Crassostrea gigas l a r v a e f e d 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 a t mid-log phase (ML) o r 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 (ML-N-control, 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 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, nitrate-free 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 t h e experiment. L e a s t squares means o f l a r v a l l e n g t h were a n a l y s 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 t h e range o f t h e symbols (see T a b l e I V ) . 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 t h e 95% c o n f i d e n c e level.  Crassostrea gigas larval growth on Thalassiosira pseudonana diet  128 Table V: Shell 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 for 12 days as in Table IV, of Thalassiosira pseudonana harvested from n i t r a t e - and s i l i c a t e limited mediuQ. Grovth vas also compared against a diet of Tahitian Isochrysis (T-ISO). a) 0  Time (Days) Treatment Replicate Hid-log •  1 2 3 LS Hean  Hid-log N-control  -N 6 h  •  135 i 1.5 [cd]  187 t 3.1 [b]  227 t 4.2 [bed]  143 t 2.8 (42) 144 i 2.9 (55) 141 • 3.1 (53)  205 t 6.0 (53) 192 i 5.9 (54) 198 i 5.9 (51)  232 i 5.9 (55) 237 t 6.8 (50) 235 t 7.0 (49)  143 i 1.5 [be]  198 • 2.9 [b]  235 t 3.7 [be]  133 i 2.9 (49) 143 t 3.0 (54) 145 ± 2.7 (54)  189 i 6.0 (35) 203 t 6.1 (38) 196 ± 4.4 (51)  141 t 1.5 [bcdl 196 t 3.4 [bl 145 t 3.3 (42) 139 t 2.8 (54) 145 t 2.8 (58)  110 i 1.6  1 2 3 LS Hean  T-ISO  110 t 1.6  1 2 3 LS Hean  -Si 6 h  228 • 6.2 (53) 231 t 10.0 (34) 221 • 6.7 (40)  110 t 1.6  110 i 1.6  • 110 i 1.6  1 2 3 LS Hean  b) Treatment Hid-log Hid-log-N-control Hid-log-Si-control -N 6 h -Si 6 h T-ISO  110 i 1.6  12  181 • 5.3 (42) 186 t 5.4 (52) 195 t 5.2 (54)  (136) 138 t 1.7 (50) 131 • 2.5 (47) 137 t 2.5 (58)  Hid-log 1 Si-control 2 3 LS Hean  7  110 t 1.6  1 2 3 LS Hean  3  174 t 4.6 (52) 166 i 4.2 (54) 163 t 4.6 (54)  220 ± 7.2 (45) 210 i 8.4 (48) 249 i 8.7 (36)  142 • 1.9 [bcdl 170 i 3.6 [cl  215 i 4.8 (cdl  162 t 2.8 (39) 152 i 3.1 (47) 137 t 2.7 (51)  208 t 5.5 (47) 227 i 4.2 (47) 163 i 5.5 (52)  243 i 6.5 (47) 270 f 4.5 (41) 232 t 8.6 (44)  157 * 2.0 [al  217 i 3.8 [a]  256  131 i 1.7 (54) 126 i 2.3 (48) 133 • 2.4 (47)  147 t 2.5 (33) 147 i 1.8 (65) 139 i 3.0 (49)  165 i 3.2 (37) 156 t 3.1 (54)  133 • 1.2  144 t 1.4  161 i 2.3  0-3 days 8.3 11.0 10.3 10.7 15.7 7.7  t  4.9 [al  Grovth (um-day-l) 3-7 days 7-12 days 13.0 13.8 13.8 7.0 15.0 2.8  8.0 7.4 9.0 7.8 3.4  129  3..  Thalassiosira  pseudonana  S i l i c a t e - s t a r v e d T. pseudonana pseudonana  d i e t f o r oyster  c e l l s p r o v e d t o b e t h e b e s t T.  larvae.  T h i s was c o n t r a r y  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  to the  w h i c h was t h e  w o r s t a l g a l d i e t compared t o a l l t h e o t h e r t r e a t m e n t s f o r C. calcitrans.  M i d - l o g p h a s e T. pseudonana  second b e s t d i e t  length  o f 110 \im, s i l i c a t e - s t a r v e d T.  y i e l d e d a mean s h e l l  length  o f 157 \im w h i c 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, T u k e y t e s t ) produced by a l l t h e o t h e r treatments the  (Table  f r o m 140 \im V).  By d a y 7,  l a r v a e consuming m i d - l o g and s i l i c a t e - s t a r v e d  g r o w i n g a t 14 ^m-d larvae V).  rated the  ( F i g . 4 1 ) . 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 pseudonana  cells  -1  d i e t s were  1  a n d 15 ^ m d " , r e s p e c t i v e l y w h i l e t h e  fed nitrate-starved cells  grew a t o n l y  A f t e r d a y 7, d a i l y g r o w t h r a t e s  7 um'd"  i n t e r v a l growth r a t e s  f o r larvae  experiment.  between a h i g h pseudonana  (Table  as seen i n  l a r g e r t h a n 190 |xm i n t h e C.  F i n a l mean s h e l l  o f 256 \im f o r l a r v a e  a n d 215 p.m f o r l a r v a e  1  slowed t o from 7 t o 9  n m - 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 t h e same t r e n d  calcitrans  the  lengths  ranged  f e d s i l i c a t e - s t a r v e d T.  f e d n i t r a t e - s t a r v e d T.  pseudonana.  IV.  Factors  T a h i t i a n Isochrysis 20:5n3 diet  A f f e c t i n g Crassostrea was r i c h  gigas  Growth  i n 22:6n3 a n d i m p o v e r i s h e d i n  ( F i g . 1 2 ) . The i n f e r i o r p e r f o r m a n c e o f t h i s  f o r C. gigas  larvae  suggests that  unialgal  22:6n3 may h a v e  little  130  nutritional value f o r oyster larvae. l a r g e amounts o f 20:5n3 a n d l i t t l e  The d i a t o m s , p o s s e s s i n g  22:6n3  were a b l e t o s u p p o r t a c c e p t a b l e l a r v a l  survival  H o w e v e r , i f 22:6n3 i s r e q u i r e d a t l e a s t decrease  (Figs.  i n l a r v a l g r o w t h when t h e y were f e d s i l i c a t e - s t a r v e d may h a v e b e e n due t o a d r o p  calcitrans  levels  2% t o 0.5% ( F i g s . 40 a n d 2 8 ) .  from  Among t h e d i a t o m s , t h e most e n e r g y - r i c h a l g a l the f a s t e s t growing mid-log c e l l s  larvae  (Table V I ) .  i n 22:6n3  cells  produced  Chaetoceros  a n d s i l i c a t e - s t a r v e d Thalassiosira  calcitrans  pseudonana  had t h e 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 t h e s p e c i e s .  balance of l i p i d , percentage  pseudonana  The  c a r b o h y d r a t e and p r o t e i n , e x p r e s s e d a s a  o f a s h f r e e d r y w e i g h t , was s i m i l a r  d i e t s , a t 27:30:43 i n C. calcitrans  Trends  (Table I V ) .  i n low l e v e l s , t h e  Chaetoceros  cells  13 a n d 14)  i n the best  a n d 22:31:47 i n T.  (TableV I ) .  favouring energetic equivalents also apply within the  T. pseudonana  experiment.  The o r g a n i c  ratio  ( 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 t h e m i d - l o g t r e a t m e n t o f 18:42:39. However t h e f o r m e r  c o n t a i n e d t h e l e a s t energy  yielded the smallest larvae  (TableV I ) .  Superiority of nitrate-starved calcitrans  cells  cannot  f o r both treatments  differences  l i e i n the protein  nitrate-starved  over s i l i c a t e - s t a r v e d  be a t t r i b u t e d t o e n e r g y  was s i m i l a r  The  p o t e n t i a l and  (Table V I ) .  content as i t  Gross  and c a r b o h y d r a t e  C.  composition  fractions.  c e l l s had a h i g h e r c a r b o h y d r a t e  content,  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 larvae, i n experiment for each phytoplankton species investigated the a f f e c t of nutrient starvation on the value of the d i e t in increasing l a r v a l s h e l l length. Tvo replicates vere analyzed and averaged in each treatment.  Species  Treatment  Tahitian Isochrysis  Hid-log  C. c a l c i t r a n s  Hid-log 6h-S-lim 6h-Si-lim  T. pseudonana  Hid-log Sh-H-lii 6h-Si-lim  Diet Rating  C a l o r i c Value (kcal/1012 c e l l s )  Lipid : Carbohydrate : Protein (% of ash free dry veight)  276 244 213  28 :: 19 : 53 24 :: 64 :; 12 27 :: 63 ;: 10  1st 2nd 3rd  173 138 143  27 ;: 30 ;: 43 19 :: 54 : 27 27 :: 22 : 51  2nd 3rd 1st  106 82 122  18 :: 42 :; 39 22 :: 49 : 29 22 :: 31 : 47  2d-I-lii  6d-H-lim  a t t h e expense o f p r o t e i n , than t h e s i l i c a t e - s t a r v e d  cells  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 starved c e l l s  a n d 27:22:51 i n t h e s i l i c a t e - s t a r v e d  cells.  133  DISCUSSION  Nutrient starvation  of phytoplankton c e l l s  n u t r i t i o n a l value of While s i l i c a t e  single  starvation  species diets  produced the  t r e a t m e n t s o f T. pseudonana n u t r i t i o n a l v a l u e o f C. starvation  reduced the  investigated.  to  assess  C a r e must be and  within  between s e r i e s  poor with  nitrate starvation  1 and  A  grown i n s e r i e s  comparison of  series  2 cultures  at  similar  2 Tahitian  w h i c h had  supply, with a c e l l  density  s p e c i f i c growth r a t e in series  nitrate  a cell  starvation  of  rates  cell  assumption.  densities.  were  For  j u s t exhausted i t s n i t r a t e of  1.0-10  0.04-h  1 grew a t  density  The  1 were  c u l t u r e s , growth r a t e s  Isochrysis  f a s t e r even a t h i g h e r c e l l  example, a c u l t u r e  the  s p e c i f i c growth  In  at  difficult  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 .  provides evidence supporting t h i s  - 1  were  Tahitian  r e c o g n i z e which of  densities  h  Nitrate  Table I I ) .  probably l i g h t - l i m i t e d .  0.02  the  diatoms  s u r v i v a l were s o  d e n s e , l a r g e volume a l g a l c u l t u r e s  Isochrysis  i t reduced  ( F i g . 40).  phytoplankton species to  considerably  larvae.  gigas  t a k e n when c o m p a r i n g c h e m i c a l c o m p o s i t i o n among  numerous v a r i a b l e s  series  the  b e s t g r o w t h among  food value of both  G r o w t h and  ( F i g . 39,  f o r C.  ( F i g . 41),  calcitrans  that e f f e c t s of  Isochrysis  (3H)  affected  of  -1  .  In  7  cells-mL  - 1  comparison,  , had  Tahitian  a s p e c i f i c growth r a t e 3.6-10  which occurred at  6  c e l l s mL 6 10  6  -1  cells  a  and  of prior  -1  mL .  to  134  S i m i l a r c o m p a r i s o n s may in  s e r i e s 1 and  s e r i e s 2.  were g r o w i n g a t cells-mL  - 1  ,  contrast,  0.10-h"  0. 0 7 - h "  s e r i e s 1 T.  at  at a density  greater  p r i o r to  2.8  10  6  from 0 . 1 0 - h -1  than  3•10  In  silicate-  s t a r v a t i o n , showed  -1  and  6  imminent.  c u l t u r e s growing i n  silicate  c e l l s mL ,  cultures  at  1-10  6  0.004-h"  cells-mL 1  at  a  - 1  to  3.0-10  6  mL" .  Evidence of  5-10  2 T. pseudonana  cultures  1  cells  C.  Series  pseudonana  i n growth r a t e 1  1  made b e t w e e n T. pseudonana  e v e n when n i t r a t e s t a r v a t i o n was  l i m i t e d medium and decline  be  light  cultures.  calcitrans c e l l s mL  density  of  limitation  i s also  Series  seen i n growth r a t e s  2 growth r a t e s  ^ were h i g h e r t h a n g r o w t h r a t e s  3•10  6  c e l l s mL  -1  in series 1  Thus f a t t y a c i d c o m p o s i t i o n o f c e l l s influenced  s i m u l t a n e o u s l y by  t h e s e two cultures  factors.  The  profound  i m p a c t on  analysed  in this  lower  was  probably  light  effects  of  in series 2  were a s s e s s e d  to  s t a r v a t i o n w h i c h had  a  phytoplankton  study.  E f f e c t s of Nutrient  i.  Nitrate  Starvation  on  Gross  Composition  Starvation  reduction  in nitrogen-stressed  (Fogg 1956)  and  i s also  cells  shown by  is  well  data  at  ).  algal cells  gross composition of the  1.  established  and  separate the  composition of  e f f e c t s of n u t r i e n t  -1  from s e r i e s 1  (under h i g h l i g h t c o n d i t i o n s )  determine the  Protein  to  0.15-h  at a  (0.10-h  both nutrient  l i m i t a t i o n , making i t d i f f i c u l t  of  of -1  in  this  135  study. levels of  T a h i t i a n Isochrysis after  nitrate  p r o t e i n d e c l i n e d t o 20%  2 days o f n i t r a t e  starvation  reduced  starvation  In  response  and  c a r b o h y d r a t e and phytoplankton  lipid  classes  compounds, b u t  quotas  direct shifts  and  1970,  E l - F o u l y e t a l . 1985).  pseudonana 9).  metabolism in  El-Fouly et a l .  change w i t h d u r a t i o n o f n i t r o g e n s t r e s s  carbohydrate  No  (Werner  changes o c c u r r e d i n l i p i d  i n n i t r o g e n - s t r e s s e d T. pseudonana  concurs with the r e s u l t s of S h i f r i n  and  ( F i g . 9)  Chisholm  carbohydrate  d e c r e a s e d when t h e s e  levels  r o s e and  lipid  c u l t u r e s were n i t r a t e - s t a r v e d (1984) a l s o  found  1970).  ( F i g s . 5 and  nitrogen deficiency  (1981) n i t r a t e - s t a r v e d  content  most s p e c i e s . lipid  C.  7).  11  increased only s l i g h t l y Algal  c e l l s may  s t o r a g e under prolonged T h i s p o s s i b i l i t y was  shift  and  diatoms,  carbohydrate rose i n  nitrogen starvation  to  (Werner  not explored i n the c u r r e n t  (too l a r g e a food p a r t i c l e ) necessary  Shifrin  from c a r b o h y d r a t e  undesirable  be  When  species of  since older cells  I t may  Thomas e t a l .  increased carbohydrate  experiments  food.  calcitrans  i n mass c u l t u r e s o f T a h i t i a n Isochrysis.  Chisholm  lipid  which  species.  and  and  and  (1981) u s i n g  C o n v e r s e l y , i n T a h i t i a n Isochrysis  yield  hours  v a r y among and w i t h i n a l l  (Ben-Amotz e t a l . 1985,  1985)  T.  mid-log  Six  ( F i g s . 7 and  to nitrogen stress, algal c e l l s  towards the non-nitrogenous  t h e same  (Fig. 5).  C. calcitrans  p r o t e i n t o one-half t h e i r mid-log values  of  c l u m p e d t o g e t h e r and  for cell  f o r use  division  as  became larval  to stop before  136  lipid  s t a r t s accumulating i n phytoplankton  Wangersky  ii.  I n Cyclotella  Starvation  an  cryptica,  e s t a b l i s h e d d i a t o m model f o r e a r l y  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  a f f e c t e d by  a decline  i n s i l c a t e metabolism,  p h o t o s y n t h e s i s i s r e d u c e d when s i l i c a t e w i t h DNA  synthesis  (Volcani  1977,  and  the  absence of c e l l  I n T. pseudonana  division  both l i p i d  Enright  (1984)  Cyclotella  as  I t has  cryptica,  protein  f o r at  and  protein stores  been p r e v i o u s l y  newly a s s i m i l a t e d  C a r b o h y d r a t e , w h i c h was  increased  lipid  b e e n shown t o gracilis al.  may  least 6 h  carbon  under s i l i c a t e  while  in  lipid  synthetic  i n Cyclotella  cryptica  limitation  1986b, V a u l o t e t a l . 1987,  (Roessler  1988).  starvation in for  r a t e s have and  1988).  T a g u c h i e t a l . ( 1 9 8 7 ) , w h i c h a l s o show l i p i d  in  previously  (Werner 1970,  Roessler  by  is  a carbon source  Lipid  in  organics.  increased  and  into lipids  have c o n t r i b u t e d  increase  of  continued  established that  reduced under s i l i c a t e  production.  this  t h o s e o b s e r v e d i n T. pseudonana  carbon i s converted  T. pseudonana  In  However i n c r e a s e s  disproportionately partitioned into lipids assimilated  synthesis  r e s u l t i n g i n increased  (Fig. 9).  l a r g e as  .  starvation interferes  Presumably, carbon f i x a t i o n  carbohydrate l e v e l s f e l l s t o r e s were n o t  but  V a u l o t et a l . 1987).  synthesized  starvation.  directly  consequently a r r e s t s p r o t e i n  Werner 1977,  s t u d y , T. pseudonana silicate  and  1987).  Silicate  silicate  (Parrish  also  Chaetoceros Enright  Data  of  increases  in  et  137  Chaetoceros  gracilis,  Hantzchia  sp. and Cyclotella  s u s p e c t a s t h e c e l l s were i n o c u l a t e d into  low  light  cultures  sp., a r e  from t h e s t a t i o n a r y  phase  i n w h i c h pH v a l u e s became h i g h e r t h a n  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. lipids (Fig.  and  carbohydrates resulted  7).  a decrease i n  calcitrans,  i n a l o s s o f o r g a n i c mass  T h i s s p e c i e s grew v e r y r a p i d l y  (4-5  divisions•day-1  d u r i n g m i d - l o g p h a s e ) u n d e r c o n d i t i o n s d e s c r i b e d and senesced  shortly  a f t e r t h e y were s a m p l e d .  p h o t o s y n t h e s i s was lipid  and  quickly arrested  i n C.  cultures  Apparently, and  calcitrans,  c a r b o h y d r a t e were consumed i n t h e a b s e n c e o f  carbon  fixation.  Silicate  r e q u i r e m e n t s v a r y among s p e c i e s  do n i t r o g e n a n d p h o s p h o r u s r e q u i r e m e n t s Gotham 1980,  Wynne and Rhee 1 9 8 6 ) .  s o u r c e and  light quality.  ( T e r r y 1980,  on g r o w t h  studies  nutritional  studies.  and  ratios  rates,  The  are used  literature  for  abounds w i t h  i n w h i c h p h y t o p l a n k t o n f r o m 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 nutrient  Rhee  as  Growth m e d i a must  accommodate t h e s e r e q u i r e m e n t s when c e l l s comparative  1985)  Optimum n u t r i e n t  can a l s o v a r y w i t h i n s p e c i e s depending nitrogen  (Brezezinski  i s not  requirements  identified.  o f each  food v a l u e , but the  Knowledge o f t h e  elemental  species allows preparation of  media which a r e l i m i t e d  i n a known, s p e c i f i c  limiting  culture  nutrient.  138  2.  E f f e c t of Nutrient  S t a r v a t i o n on  Lipid  P h y t o p l a n k t o n have a g r e a t v a r i e t y o f taxonomic c h a r a c t e r i s t i c s Riley  1969,  P o h l and  triacylglycerols phospholipids the  structural  Z u r h e i d e 1982, al.  1987).  (Chu  and  f a t t y a c i d s w h i c h show  algal  Zurheide 1979).  monounsaturated f a t t y f o r energy i n the  among t h e  a c i d s s u c h as  Composition  classes  The 16:0  saturated and  16:1,  neutral l i p i d s , particularly ( F i s h e r and  components s u c h as  cell  P i o r r e c k e t a l . 1984,  Dupuy 1980)  age  a f f e c t s the  due  and  and are  stored  The  polar  the  Schwarzenbach 1978).  conserve the polyunsaturated  Culture  (Chuecas  fatty  acids  within  membranes  (Pohl  and  A r a o e t a l . 1987, proportions  to growth-limiting  of  Suen  fatty  f a c t o r s such  et  acids as  nutrient deprivation.  i.  Nitrate Starvation  T a h i t i a n Isochrysis s i m i l a r way of 14  C  lipids  to that reported  little  largely  increased  of saturated  the  polyunsaturated  the  polar lipids  1984, levels  conversion  (Suen e t a l . 1 9 8 7 ) .  stimulates  literature.  Accumulation  and  compounds  of t r i g l y c e r i d e s  monounsaturated f a t t y  f a t t y a c i d s and  ( P o h l and  ( p r i m a r i l y 18:ln9)  of other  t o de nova into  Nitrate deficiency generally  synthesis  Suen e t a l . 1 9 8 7 ) .  a l m o s t 50%  i n the  starvation in a  i n 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 i s due  f i x a t i o n with  lipids  responded t o n i t r a t e  while  decrease  with  Piorreck et a l .  study, monounsaturated  increased  w i t h i n 2 d of n i t r a t e  acids,  glycolipids  Z u r h e i d e 1982, In t h i s  composed  i n T a h i t i a n Isochrysis  s t a r v a t i o n , and  a  by  139  p r o p o r t i o n a l drop l a r g e l y due  (17%)  t o reduced  occurred  i n polyunsaturated  levels,  proportions of n3-polyunsaturates  (Fig.  16) .  Predominant f a t t y 16:0,  18:ln9,  10%;  and  12).  18:ln9.  by  18:3n3 w i t h  ( E n r i g h t 1984,  Nitrate  Pillsbury  starvation  1985,  levels  due  to nitrogen starvation.  was  fairly  p r o p o r t i o n o f 16%.  f r o m 13%  t o 15%,  g r o w t h , however i t i s not  except  fatty  and  14:0, study  acid  and  a  acutus 22:6n3  starvation  falling  Similarly,  Ben-Amotz e t a l .  only a small increase i n the i n Isochrysis  o f 16:0  i n c r e a s e i n 18:1  important  r e s i l i e n t under n i t r a t e  (Fig.  Laing  and Scenedesmus  vulgaris The  5%  In comparison, a  E l - F o u l y e t a l . (1985) showed a 250%  than  other  Helm and  acids levels,  p o l y u n s a t u r a t e s ) were r e d u c e d .  (1985) o b s e r v e d  14:0,  and  with that of  enhanced t h e  d e c r e a s e i n 16:0 o f Chlorella  the  l e v e l s b e t w e e n 3%  agrees  A l l o t h e r major f a t t y  from a mid-log  were  22:6n3, a l l w i t h l e v e l s g r e a t e r  general composition  researchers  (i.e.  18:4n3 and  18:2n6 and  The  1987).  a c i d s o f T a h i t i a n Isochrysis  after  10  only  fatty acid  days of  3%  22:6,  stationary  c l e a r what f a c t o r l i m i t e d g r o w t h  of  culture.  Short-term c a u s e d an  nitrate  starvation  insignificant  of the diatoms i n t h i s  change i n f a t t y  m a i n f a t t y a c i d s o f C. calcitrans 16:3n4, 18:4n3 and differed  20:5n3  were 14:0,  ( F i g . 13).  (1979) and  Helm and  Laing  composition. 16:0,  Relative  c o n s i d e r a b l y from t h a t observed  Nascimento  acid  The  16:ln7,  composition  by W a l d o c k  (1987).  study  The  and  main  fatty  140  a c i d s o f T. pseudonana  were 14:0,  16:0,  1 8 : 4 n 3 , 20:5n3 and  22:6n3  (1978) and  (1984) r e p o r t e d  Enright  which d e v i a t e d pseudonana  ( F i g . 14).  16:ln7,  16:2n4,  F i s h e r and  fatty acid  composition  somewhat f r o m t h a t d e s c r i b e d h e r e .  d i f f e r e d mainly i n the  much l o w e r i n t h e  other  not  be  The  only  studies.  c h a n g e i n C. calcitrans  1.  E f f e c t s c o u l d not  during  but  cultures  should  be  saturated  and  attributed to  and  values.  However E n r i g h t  t r e a t m e n t was  cultures  \i.E-m - s e c  ii.  Starvation  Although f a t t y acid first  levels  Chaetoceros of c o n t r o l  L)  i t had  acid composition. and  the  s y n t h e s i s can  In view of  available irradiance analysing  i n c r e a s e more t h a n  6 to 9 h of s i l i c a t e  same  a more s u b d u e d  ) i t i s p o s s i b l e t h a t t h e y were  cells.  Silicate  Enright et a l .  acids i n  T. pseudonana,  (18  light-limited  have been  (1984) a l s o f o u n d t h a t when t h e  e f f e c t upon changes i n f a t t y s i z e of t h e i r  nitrate  composition  22:6n3 l e v e l s were r e d u c e d t o 20%  i m p o s e d on  growth i n  s t a r v a t i o n produced higher  monounsaturated f a t t y  gracilis  composition  light-limited  investigations.  (1986b) f o u n d t h a t n i t r a t e  w i t h i n the  fatty acid  Large changes i n f a t t y a c i d  found i n diatoms i n other  (300  among t h e  a  ignored.  limitation.  of  was  Differences are probably  of genetic v a r i a t i o n  o b s e r v e d i n e i t h e r s e r i e s was series  T.  amount o f 20:5n3 w h i c h  r e s u l t of c u l t u r e c o n d i t i o n s used i n each study, possibilities  Schwarzenbach  100%  s t a r v a t i o n (Werner  the  141  1977), t h e f a t t y was  pseudonana  acid composition  largely unaffected i n this  c u l t u r e was h e l d u n t i l change seen  o f C. calcitrans  senescence  study unless the  was r e a c h e d .  The o n l y  i n h e a l t h y c e l l s was a d r o p i n C.  22:6n3 f r o m a m i d - l o g e t a l . (1986b) f o u n d substantial  level  o f 2% t o 0.5%  t h a t C. gracilis  and T.  calcitrans  (Fig. 28).  cells  showed a  i n c r e a s e i n m o n o u n s a t u r a t e s and a d e c r e a s e i n  20:5n3 a n d 22:6n3 when t h e y were s i l i c a t e - s t a r v e d but undefined time.  T h e i r r e s u l t s are supported  s t u d y o f t h e same d i a t o m (1988).  Total  Effects series  ( C . gracilis)  f o r a longer by a  with  later  by M o r t e n s e n e t a l .  n3 and l o n g - c h a i n e d h i g h l y u n s a t u r a t e d  acids decreased continuous  Enright  increased s i l i c a t e  fatty  limitation i n  culture.  of s i l i c a t e  s t a r v a t i o n were much more p r o n o u n c e d i n  1, b u t o n l y a f t e r c e l l s h a d c l u m p e d and were  from  suspension.  i i i .  Phosphate  sinking  Starvation  Unfortunately, effects  o f p h o s p h a t e s t a r v a t i o n were n o t  investigated  2 and e f f e c t s  i n series  superimposed with p o s s i b l e  light  i n series  limitation  1 are  effects.  N e v e r t h e l e s s when c o m p a r i n g f a t t y a c i d c o m p o s i t i o n o f phosphate-limited cultures with that of cultures  limited  other nutrients  that  i n series  phosphate l i m i t a t i o n acid  composition  1, i t becomes a p p a r e n t  can have a profound  o f some s p e c i e s .  effect  by  on t h e f a t t y  142  Phosphate levels  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  o f Thalassiosira  on t h e f a t t y  acid  The monounsaturate  pseudonana.  f r a c t i o n was e n r i c h e d a t t h e e x p e n s e o f t h e p o l y u n s a t u r a t e s (Fig. 31).  Of t h e l a t t e r f r a c t i o n ,  (20:4n6) i n c r e a s e d , f r o m l e s s nutritionally  t h a n 1% t o 4%  i m p o r t a n t 20:5n3 d r o p p e d  c o n c e n t r a t i o n o f 22% w h i c h nutrient limitation (Fig. 33).  only arachidonic  (Fig. 35).  i n any e x p e r i m e n t  T h e s a t u r a t e d C16 f a t t y  The  t o 25% o f i t s o r i g i n a l  was t h e most s i g n i f i c a n t  observed  acid  acid  effect of  i n this  study  i n c r e a s e d b y 100%  compared t o i t s m i d - l o g 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 did  calcitrans  n o t show a n y s i g n i f i c a n t  cultured  changes i n f a t t y  i n series  acid  composition  r e l a t i v e t o t h e 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 T a h i t i a n Isochrysis by phosphate results  limitation  c o m p o s i t i o n was l e s s  than n i t r a t e  s u g g e s t t h a t Thalassiosira  smaller internal normally r e l y (Kylin  fatty acid  1964).  limitation.  pseudonana  phosphate  Polyphosphates  which  are thermodynamically  1966,  Kulaev  cells  structural  needs d u r i n g  affected  These  phytoplankton deficiencies  c o n t a i n phosphoanhydride  e q u i v a l e n t t o t h o s e o f ATP  1975) a n d c a n t h e r e f o r e requirements  fulfill  f o r phosphate  medium.  maintains a  r e s e r v e o f polyphosphate which  on t o f i l l  1  bonds (Harold  not only the but also the  e n e r g e t i c demands o f m e t a b o l i s m .  It  i s l o g i c a l t h a t phosphate  reduction fatty  d e p l e t i o n should cause a  i n the long chained polyunsaturates since  acids are normally associated with c e l l  these  membranes i n t h e  143  phospholipid  fraction of algal c e l l s .  microalgal culturist  Therefore  the  should ensure t h a t the n u t r i e n t r a t i o s  c u l t u r e s grown f o r m a r i n e a n i m a l  food provide  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 o f growth t o prevent 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 t h e d i e t . n i t r o g e n t o phosphorus r a t i o s  a r e known t o v a r y  53:1  i n the case  o r e v e n a s h i g h a s 200:1 (Rhee and  japonica  Gotham 1980,  of  f r o m 7:1  optimum N:P  3..  Effect  ratio  T e r r y 1980).  and  The  ratio  for that particular  separated  i n t h i s p a r t of the study.  results  light-limited) effects  i t was  of l i g h t  Light limitation  However, by  of l i g h t  limitation  in series  on  1  acid  ( F i g . 17).  14:0  only  1 cultures  composition.  appeared  to  limitation  under l i g h t  Otherwise f a t t y a c i d  plus  qualitative  1 T a h i t i a n Isochrysis to nitrate  from  nutrient-  (nutrient-  a l g a l chemical  decreased  limitation  comparing r e s u l t s  t h e c u l t u r e grew more s l o w l y ) w i t h one  Saturated fatty series  limitation  p o s s i b l e t o d i s c e r n some  h a v e o n l y s l o w e d t h e c h a n g e s due (because  influenced series  of nutrient  from t h o s e  from s e r i e s  the  Composition  2 c u l t u r e s , w h i c h a r e p r e s u m e d t o be  l i m i t e d , with  the  phytoplankter.  nutrient l i m i t a t i o n probably  clearly  can  are  s h o u l d be w e l l b e l o w  simultaneously, effects  c o u l d n o t be  series  ratio  o f L i g h t upon F a t t y A c i d  Since l i g h t 1 cultures  t h e N:P  to  Asterionella  Thus, t o ensure t h a t c u l t u r e s  phosphorus-replete,  loss  Optimum  a l s o v a r y w i t h i n s p e c i e s d e p e n d i n g on g r o w t h r a t e s and nitrogen source.  of  levels  exception. limitation attained  in in  144  series  1, 240 h f o l l o w i n g  were r e a c h e d i n s e r i e s f r o m t h e medium  In  series  ( i . e . a t 450 h ) ,  2, 48 h a f t e r n i t r a t e was  increased  Tahitian  18:2n6 and 18:4n3 l e v e l s i n c o n t r a s t  i n t h e s e two f a t t y a c i d s 17).  exhausted  (Fig. 18).  1, p h o s p h a t e s t a r v a t i o n  Isochrysis  (Fig.  nitrate starvation  t o decreases  when t h i s s p e c i e s was  Otherwise both s t a r v a t i o n  nitrate-starved  treatments  affected  composition s i m i l a r l y .  Light  a p p e a r s t o have had a g r e a t e r e f f e c t t h a n  starvation  on f a t t y a c i d  1 cultures.  nutrient  composition o f the diatoms i n s e r i e s  The p o l y u n s a t u r a t e d t o m o n o u n s a t u r a t e d  d e c r e a s e d m o d e r a t e l y i n l i g h t - l i m i t e d C. calcitrans nitrate  starvation  f r o m 17% t o 13%  (Fig. 25).  observations since nitrate-starved  due t o a r e d u c t i o n Low  ratios prior to  i n t h e amount o f 20:5n3  l i g h t must a c c o u n t f o r t h e s e  no c h a n g e s were o b s e r v e d i n h i g h - l i g h t ,  cultures  i n series  2.  The m o d e r a t e  elevation  o f p r o p o r t i o n s o f 22:6n3 and 16:0 f o u n d u n d e r l i g h t l i m i t a t i o n were s i m p l 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 a n d  27) .  The  p o l y u n s a t u r a t e d t o m o n o u n s a t u r a t e d r a t i o was  dramatically  reduced i n s e r i e s  In the n 3 - f r a c t i o n by  (Fig. 33).  pseudonana. reduced  o r p h o s p h a t e was e x h a u s t e d f r o m t h e  Proportional  16:3n4, 18:4n3 and 22:6n3 w h i l e is  1, l i g h t - l i m i t e d T.  f o r e x a m p l e , l e v e l s o f 20:5n3 were  h a l f before s i l i c a t e  medium  also  decreases also 16:0 and 1 6 : l n 7  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  occurred i n increased.  (1984) saw s i m i l a r  It  fatty  145  acid  increases  light  i n T. pseudonana  conditions  c u l t u r e s grown u n d e r s i m i l a r  t o those o f experiments described  a l t h o u g h no c h a n g e was s e e n i n 16:0 a n d 1 6 : l n 7 . comparisons with t h i s cell  study are not v a l i d  d e n s i t i e s and c u l t u r e  deprivation  age o r l e n g t h  were n o t d e s c r i b e d  These r e s u l t s a r e i n c o n t r a s t al.  of nutrient (1984) w o r k .  t o p r e v i o u s work b y M o r t e n s e n e t u n s a t u r a t e d n3  acids  o f C. gracilis  were shown t o i n c r e a s e  light  i n t e n s i t y f r o m 83 t o 1395 \xE-m~ • s e c  with - 1  2  a t 28°C.  Direct  however, because  i n Enright's  (1988) i n w h i c h l e v e l s o f h i g h l y  cultures  here,  fatty  increased  i n 200  However, c e l l s were h a r v e s t e d  mL  i n the late  l o g p h a s e a n d i t i s unknown what f a c t o r s may h a v e b e e n limiting. algal  Changes i n l i g h t  nutrient  and  Fragilaria  nitrogen 1981). not  may  Therefore  interact.  only affected  factors affecting culture  the subsistence  as i r r a d i a n c e decreased  R e d a l j e a n d Laws 1 4  cell  light  into  protein,  o f Thalassiosira  allenii,  a f f e c t e d t h e range o f v a l u e s over which n u t r i e n t temperature e f f e c t s  intensity  but i t also and  varied.  Interactive effects of nutrient l i m i t a t i o n may  quota f o r  (Rhee a n d Gotham  (1983) showed t h a t  C-incorporated  and l i p i d  ratios  I n n i t r a t e - l i m i t e d Scenedesmus sp.  crotonensis,  increased  polysaccharide  influence  r e q u i r e m e n t s b y a l t e r i n g optimum N:P  (Wynne a n d Rhee 1 9 8 6 ) . conditions  q u a l i t y can s t r o n g l y  l i m i t a t i o n with  e x p l a i n why e f f e c t s o f l i g h t  d i f f e r e d between s i l i c a t e -  light  limitation  and p h o s p h a t e - l i m i t e d  cultures.  146  Combined p h o s p h a t e and yielded  similar  l i g h t l i m i t a t i o n i n C.  results  were more a c u t e and  the  to  nitrate-starved  calcitrans  cells  although  main changes o c c u r r e d o n l y  they  upon  senescence.  Effects and  of  l i g h t i n t e n s i t y may  Patterson  levels  (1973) a l s o  i n Nitzschia  and T.  light/dark pseudonana  raised  (Fisher  c o n c l u s i o n s must be irradiance  was  not  made when t h e  cultures  i m p o s e d on p h a s e and  the  and  amount o f  this  i n the  t h i s manner on  investigation g r o w t h on  of  benefit  of  light/dark  fatty acid  The  in  authors  cycle  to  account  day  by  the  was  culture,  effects  of  only  the  at very l a t e  a control  log  the an  factor.  used i n the  e f f e c t of  effects  of  larval diet  phytoplankton c e l l s i s unknown.  in this thesis, the  irradiance  ( i . e . a v a l i d c o m p a r i s o n can  oyster nutrition  r e s u l t s presented  of  light  however, because  l i g h t r e c e i v e d per  c e l l s were n o t the  Effects  tentative  continuous l i g h t cultures  s t u d y so  (1984)  F u r t h e r m o r e , when a d a r k c y c l e  u n d e f i n e d growth l i m i t i n g  of  al.  Schwarzenbach 1978).  h a v e b e e n c o n f o u n d e d by  Light-limited  20:5n3  have been demonstrated p r e v i o u s l y  adjusted  without the  r e s u l t s may  20:5n3.  r e g a r d e d as  i s equal).  Orcutt  responsed to high  minutissima  f o r the. reduced l i g h t p e r i o d be  l i g h t reduced  w h i l e Seto et  l e v e l s of  cycles  species dependent.  f o u n d low  closterium,  r e p o r t e d t h a t Chlorella with s l i g h t l y  be  a well  In  portion  treated  l i g h t of  the  controlled  l i g h t - l i m i t e d phytoplankton  c o m p o s i t i o n and  n u t r i t i o n a l value  to  in  147  filter is  feeders i s warranted.  i m p e r a t i v e when one  facilities  An  understanding of t h i s  considers  t h a t most c o m m e r c i a l  grow l a r g e v o l u m e s o f a l g a e w h i c h a r e  l i g h t - l i m i t e d because a r t i f i c i a l large  culture  i.  Fatty  chained  L a n g d o n and  W a l d o c k 1981,  Holland  study, the  d i a t o m s C.  20:5n3 and  sustaining  1984).  not  reported  g r o w t h and  Growth  are  This  L a n g d o n and edulis  Sulkin  rich  larvae  suggests that  Waldock  on  successful a d i e t of  20:5n3 may  a l g a was  rich  in  only  be  n u t r i t i o n o f C. gigas  (1981) d e m o n s t r a t e d  The  were  this  i n 22:6n3.  j u v e n i l e s when Dunaliella  s u p p l e m e n t e d w i t h 22:6n3.  1984,  T. pseudonana  was  to  (Jones e t a l .  h e r e , were n o t  to the  considered  phytoplankton used i n  and  s u r v i v a l of  significant contributor  g r o w t h o f O.  the  calcitrans  T a h i t i a n Isochrysis.  22:6n3.  Of  L e v i n e and  T a h i t i a n Isochrysis  Repeated t r i a l s ,  a more  than  improved was  tertiolecta  void  of both  20:5n3  22:6n3.  Evidence f o r d i f f e r i n g the  fatty acids  e s s e n t i a l f o r growth of marine organisms  W a l d o c k and  and  Larval  Acids  Highly unsaturated long  in  generally  volumes.  E f f e c t o f A l g a l C h e m i c a l C o m p o s i t i o n on  1979,  oyster  i l l u m i n a t i o n i s used f o r very  4.  be  problem  literature.  fatty  T a h i t i a n Isochrysis  e x c e l l e n t d i e t f o r Crassodoma larvae  and  a c i d requirements i s present  juveniles  p o o r f o o d f o r C. gigas  i s considered  gigantea  ( C a r y e t a l . 1981) (Helm and  Laing  (Hinnites and  an multirugosus)  O. edulis,  1987).  in  A  rare  but  a  analysis  148  of e s s e n t i a l mussel  Mytilus  amino a c i d s  f o r a b i v a l v e was p e r f o r m e d  I t was assumed t h a t l a c k o f  californianus.  radioisotope label  incorporation  p r o v e d an i n a b i l i t y  i n t o an a r r a y o f amino a c i d s  t o s y n t h e s i z e t h e compounds a n d t h u s  implied a requirement  f o r them  and T. pseudonana  C. calcitrans  on t h e  growth and a c c e p t a b l e l a r v a l  (Harrison  1975).  were a b l e t o s u p p o r t g o o d  survival  e s p e c i a l l y when t h e  f o r m e r was h a r v e s t e d d u r i n g m i d - l o g p h a s e and t h e l a t t e r following s i l i c a t e  starvation  However, t h e diatoms 22:6n3 low  (Figs.  ( F i g s . 40 a n d 4 1 , T a b l e I I ) .  were r i c h  13 and 1 4 ) .  i n 20:5n3 b u t i m p o v e r i s h e d i n  I f 22:6n3 i s r e q u i r e d a t l e a s t i n  l e v e l s , the diet of silicate-starved  h a v e l o s t n u t r i t i o n a l v a l u e due t o a d r o p this  fatty  acid.  ii.  Gross  Composition  Silicate-starved C. calcitrans  T. pseudonana  (Figs.  from  2% t o 0.5% o f  10 and 41) and m i d - l o g  ( F i g s . 8 and 40) p r o d u c e d  c o n t e n t and c o n s e q u e n t l y , t h e y r e s u l t e d g r o w t h o f C. gigas.  may  C. calcitrans  the highest c a l o r i c i n the fastest  larval  O t h e r w i s e , when c a l o r i c v a l u e s were  s i m i l a r , differences apparently l a y i n the carbohydrate content.  Nitrate  calcitrans  cells  starvation  i n c r e a s e d t h e v a l u e o f C.  over the e n e r g e t i c a l l y  equivalent  silicate-  s t a r v e d c e l l s w i t h a c o r r e s p o n d i n g change i n a s h - f r e e compositional ratios  from  lipid:carbohydrate:protein assume t h a t e s s e n t i a l  1:0.8:1.9 t o 1:2.8:1.4 ( F i g . 40, T a b l e V I ) .  One  may  r e q u i r e m e n t s were p r e s e n t i n b o t h  diets  149  and  that carbohydrate  p r o t e i n s and observed diets  lipids.  p r o v i d e d a s p a r i n g e f f e c t on A d d i t i o n a l carbohydrate  has  ( E n r i g h t e t a l . 1986b).  A  final  gracilis  balance  (lipid:carbohydrate:protein)  i m p r o v e d g r o w t h o f O.  j u v e n i l e s whereas a d d i t i o n a l  lipid  The  resulted  the n u t r i t i o n a l  i s t o the requirements  amount o f e n e r g y u t i l i z e d efficiency  o f an  submarina  e f f i c i e n c y was components  i n i t s conversion.  a c q u i r e d w i t h a 1:1:1  i n t a k e was  Ration  ratio  Optimal  gross  Ratios producing study  the  were  Size  ration  factor controlling  the  s i z e must h a v e b e e n  inadequate.  T h i s e x p l a i n s the decrease  i n the  p e r i o d of the t r i a l s  V).  of the  by  food,  ( S c o t t 1980).  employed i n t h i s  the primary  outcome o f t h e d i e t t r i a l s ,  final  regulated  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 ( T a b l e V I ) .  G r a z i n g R a t e s and  caloric  algal  Conversion  of i t s algal  (lipid:carbohydrate:protein).  1:1.1:1.6 and  of the  was  picatilis  var. pulsifera  b e s t growth w i t h phytoplankton  and  growth  organism, the smaller i s the  i n a r o t i f e r Brachionus  Brachiomonas  As  edulis  i n reduced  composition  the q u a l i t y , or biochemical composition  5.  o f 1:1.5:1  juveniles.  more c o m p a t i b l e  food  been  t o e n h a n c e t h e g r o w t h p e r f o r m a n c e i n C.  also  rates of  essential  in larval  ( F i g s . 40  Assessment of the n u t r i t i o n a l  and  growth  41, T a b l e s  requirements  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 o f  rate  of  the  optimal  c a l o r i c v a l u e but d e v i a n t chemical  composition.  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  IV  150  a c c o m p l i s h e d 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 efficiencies  f o r appropriate  Unfortunately, efficiencies study. that and  rations.  measurement o f 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  f o r e a c h a l g a l d i e t was b e y o n d t h e s c o p e o f t h i s  Single  species  d i e t s allow  the researcher  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 o p t i m a l r a t i o n s c a n be e s t a b l i s h e d  (Enright  and a s s i m i l a t i o n  e t a l . 1986b).  t o assume  a r e e q u a l among d i e t s f o r each  treatment  However t h e a s s u m p t i o n t h a t  equal i s not n e c e s s a r i l y t r u e .  Digestibility  could  rates are be  a f f e c t e d by changes i n c h e m i c a l c o m p o s i t i o n , o r c e r t a i n culture conditions example, g r a z i n g the  r a t e s o f O. edulis  m e d i a o f Isochrysis  early  during  s t a t i o n a r y growth phases  (Wilson  intake  late  exponential  by  and  1979).  may n o t b e p o s s i b l e w i t h s p e c i f i c  algae  b i v a l v e s h a v e a maximum r a t e o f • i n g e s t i o n , g o v e r n e d b y  changes i n f i l t r a t i o n concentrations 1984a). algal  rates  activity  (mL-h  -1  •g  -1  ) may d i f f e r  ( E p i f a n i o and E w a r t 1 9 7 7 ) .  since a further increase production  for different particle  (Winter 1973, E p i f a n i o and Ewart 1977, S p r u n g ,  Ingestion  species  rates  g r o s s growth e f f i c i e n c y  The  For  l a r v a e were s t i m u l a t e d  galbana  Optimal c a l o r i c as  might produce p h a g o s t i m u l a n t s .  f o r each  A maximum v a l u e f o r  approaches an o p t i m a l r a t i o n  i n a v a i l a b l e food r e s u l t s i n t h e  o f p s e u d o f e c e s and h i g h l y r e d u c e d  filtration  (Winter and Langton 1976, M a l o u f and B r e e s e  maximum a n d minimum c o n c e n t r a t i o n  a f f e c t e d by t h e s i z e  level  1977).  of algal cells i s  ( c e l l volume) o f a l g a l c e l l s  (Epifanio  151  and  Ewart 1977).  food  Filtration  c o n c e n t r a t i o n due  the volume o f  food  digestibility  of the  filtration  F o r Mytilus  (JLL  efficiencies constant In the  pass through the  food.  up  - 1  In d i l u t e  insufficient  larvae transition  cells  filtration  a daily  f o r C. gigas  10  concentrations  ration.  .  by  determination  of the  maximum and  minimum a l g a l  concentrations  ( E p i f a n i o and  as  different  efficiency. the  composition  Cell  concentrations  on  an  reducing  optimal  species  i n which chemical  controlled  remained  (Sprung  1984b). - 1  cells• M-L  feeding  ration  permitting  The  were  and  up  be the  efficient  most i d e a l  situation  parameters f o r each  diet  c o u l d cause changes i n d i g e s t i o n c o u l d be  feeding  feeding  composition  growth c o n d i t i o n s .  and  feeding rations  optimum d a i l y  maintained  within  intervals.  b i v a l v e d i e t may  combining knowledge o f o p t i m a l  20  1  f e e d i n g regime c o u l d  of these  cell  r e q u i r e d r a n g e by  Information  optimal  Ewart 1977).  would r e q u i r e d e t e r m i n a t i o n  of  of  assessed  An  M.L"  and  growth  Generally accepted  employ a g r a d a t i o n - 1  between 5  Net  f o r mussel l a r v a e  - 1  cells'nL  ingestion  the  ingestion rate  occurs  cells  60  50  on  filtration-limited  (Sprung 1984a).  nL  work, c e l l  from  to  to  and  concentrations,  i n g e s t i o n and  reached a plateau at  as  food  gut  by  concentration.  o f Isochrysis  t o 40  increasing  ingestion capacity, limited  ingestion-limited  present  regimes  t h a t can  food  edulis  ingestion to cells  t o an  r a t e i s u n l i m i t e d by  increases with  10  rate declines with  be  gathered  strategies  for  i s manipulated  Nevertheless,  by algal by  hypotheses  152 regarding the requirements o f s p e c i f i c  compounds r e q u i r e s  artificial  may b e c o n t r o l l e d  d i e t i n which a l l n u t r i e n t s  individually.  U n t i l such t e c h n o l o g y has been  acquired, algal diets w i l l  continue t o play  nutritional  of bivalve  investigations  successfully  a central role i n  larvae.  Work p r e s e n t e d h e r e s e r v e s t o d e m o n s t r a t e t h e e p h e m e r a l o f t h e c h e m i c a l c o m p o s i t i o n o f a l g a e and t h e n e c e s s i t y rigid  control  o f p h y t o p l a n k t o n growth c o n d i t i o n s  performing comparative s t u d i e s . capable o f y i e l d i n g diverse  Single  species  growth r a t e s  dependent upon t h e p h y s i o l o g i c a l  state  t o t h i s information  other l i m i t i n g  nutrients  nature of the  when o f algae are  of oyster  larvae  and c o r r e s p o n d i n g  chemical composition of the a l g a l c e l l s . with attention  an  Continued  research  may a l l o w an e v a l u a t i o n  i n l a r v a l d i e t s s u c h a s amino  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  to fatty  of  acids,  acids.  153  SUMMARY AND  1.  CONCLUSIONS  The g r o s s c h e m i c a l c o m p o s i t i o n o f u n i a l g a l the  f l a g e l l a t e Isochrysis  d i a t o m s , Chaetoceros (clone  pseudonana  (clone  and  calcitrans  T - I s o ) and two  Thalassiosira  limitation.  were a n a l y s e d ; t h e f i r s t  volume, l i g h t - l i m i t e d c u l t u r e s  Two  series of  consisted  that also  of  cultures  t h a t o n l y became  Tahitian  Isochrysis  large  became  s t a r v e d , a n d t h e s e c o n d was grown i n s m a l l e r  2.  of  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 cultures  galbana  cultures  nutrient-  high-light  nutrient-starved.  m i d - l o g phase c e l l s  had g r e a t e r  c a l o r i c value than e i t h e r n i t r a t e - or phosphate-limited cells.  Nitrate  protein  and l i p i d  respectively;  3.  starvation  f o r 2 days d e p l e t e d  t o 2 5% and 75% o f m i d - l o g  c a r b o h y d r a t e i n c r e a s e d by  Nitrate  starvation  further  change.  caloric  c o n t e n t o f C. calcitrans.  starvation, 50%,  cellular protein  a t t h e expense o f a r i s e  Silicate  starvation  content  three-fold.  f o r another 4 days r e s u l t e d  S i x h o u r s o f n i t r a t e and s i l i c a t e  resulted  cellular  starvation  in little  reduced  Under n i t r a t e  and l i p i d  were d e p l e t e d b y  i n carbohydrate  content.  i n a 60% l o s s o f  carbohydrate.  4.  Increases  i n cellular protein  respectively,  and l i p i d  o f 38% and 25%  increased c a l o r i c content of  silicate-  s t a r v e d Thalassiosira by  25%.  Nitrate  pseudonana.  starvation  Carbohydrate  depleted c e l l  decreased  protein  and  added a s h c o n t e n t .  The m a j o r f a t t y a c i d s Tahitian  of the nutrient-replete 14:0, 1 6 : 0 , 1 6 : l n 7 ,  Isochrysis:  a l g a e were: 18:ln9,  18:2n6, 18:3n3, 18:4n3,20:3n3, 22:5n6 a n d 22:6n3, Chaetoceros  14:0, 1 6 : 0 , 1 6 : l n 7 ,  calcitrans:  16:2n4, 16:3n4, 18:4n3 Thalassiosira  20:5n3  and 22:6n3  14:0, 1 6 : 0 , 1 6 : l n 7 ,  pseudonana:  16:2n6, 16:2n4, 16:3n4, 18:4n3, 20:5n3  Changes i n T a h i t i a n  saturation limited  r a t i o s dropped starvation phosphate  p r o c e e d e d more r a p i d l y u n d e r  (series 2 cultures)  i n series  1.  and 22:6n3.  f a t t y a c i d c o m p o s i t i o n due  Isochrysis  to n i t r a t e starvation  16:2n6,  The  dramatically  t h a n when l i g h t  light  was  polyunsaturated:monounsaturated by t h e s e c o n d d a y o f  and d i d n o t c h a n g e t h e r e a f t e r . l i m i t a t i o n (and l i g h t  Responses t o  l i m i t a t i o n ) were  similar  to those of n i t r a t e l i m i t a t i o n .  Nutrient deprivation the  fatty acid  f o r 6 h h a d no s i g n i f i c a n t e f f e c t on  composition o f the diatoms  H o w e v e r , c h a n g e s were o b s e r v e d e i t h e r be a t t r i b u t e d nitrate,  silicate  to light  i n series  1, w h i c h  l i m i t a t i o n , o r , long  o r phosphate  Polyunsaturated:monounsaturated decreased with time  i n series  starvation fatty acid  i n both diatoms  periods. levels  i n series  1.  2.  could  155  8.  T h e most d e c i s i v e e f f e c t all  three algal  on f a t t y  acid  c o m p o s i t i o n among  s p e c i e s o c c u r r e d i n T. pseudonana  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 ,  under  20:5n3  d e c r e a s e d t o 25% o f i t s m i d - l o g v a l u e a n d 16:0 i n c r e a s e d by 100%.  9.  T a h i t i a n Isochrysis, little  rich  i n 22:6n3, a n d p o s s e s s i n g v e r y  2 0 : 5 n 3 , was a p o o r 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  status.  Y e t , t h e d i a t o m s w h i c h h a d h i g h 20:5n3  and were i m p o v e r i s h e d survival.  o f 22:6n3 s u p p o r t e d g o o d g r o w t h and  T h i s s u g g e s t s t h a t 20:5n3 may b e 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 C. gigas 10.  22:6n3 t o t h e n u t r i t i o n o f  larvae.  Among t h e d i a t o m s , produced  levels  t h e most e n e r g y  rich  algal  t h e f a s t e s t growing o y s t e r l a r v a e .  when c a l o r i c v a l u e s were s i m i l a r ,  cells Otherwise,  increased carbohydrate  may h a v e s p a r e d any e s s e n t i a l p r o t e i n s a n d l i p i d s . therefore recognized that ration optimal i n the diet  trials.  l e v e l s were l e s s  I ti s than  FUTURE WORK  An  understanding  of the n u t r i t i o n a l  r e q u i r e s much f u r t h e r w o r k .  of  composition.  r e q u i r e d t o grow a l g a e Any  stringent control of culture  the optimal r a t i o n  levels  presented here  o f e a c h d i e t and  l a r v a e , may  optimal l a r v a l survival  w o u l d a l s o add  f o r each  of both the algae  help to identify c r i t i c a l g r o w t h and  survival.  s h o u l d be m o n i t o r e d  metamorphosis.  i s to  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  analyses of the biochemical composition the  and  conditions.  A n a t u r a l e x t e n s i o n of the experiments  stage of oyster l a r v a e .  of  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 o f a l g a l p h y s i o l o g y  determine  bivalves  Past s t u d i e s have r e p e a t e d l y  ignored the necessary procedures consistent chemical  requirements  through  Determination  nutrients  of a s s i m i l a t i o n  valuable insight  for  I d e a l l y , growth the c r i t i c a l  and  and  stages  of  efficiencies  i n t o t h e most e f f e c t i v e  diet  composition.  Although  fatty  differences study, the potential  acid  i n food value of the microalgae f a t t y acid composition of series  areas  irradiances  and  1 indicated  A range  p h o s p h a t e l i m i t a t i o n may  yield  in fatty  in diet trials.  Live diets w i l l  two  of  diets with  a c i d composition which c o u l d  the be  c o n t i n u e t o pose  p r o b l e m s b e c a u s e i t i s i m p o s s i b l e t o a f f e c t one component w i t h o u t  any  used i n t h i s  f o r continued research.  greatest variation tested  composition c o u l d not e x p l a i n  nutritional  c a u s i n g c o n c u r r e n t changes i n o t h e r  compounds.  Nevertheless, microencapsulation technology  permit hypothesis t e s t i n g o f the importance o f s p e c i f i c compounds i n t h e g r o w t h o f b i v a l v e  larvae.  may  158  REFERENCES Ackman, R.G. 1986. WCOT ( c a p i l l a r y ) g a s - l i q u i d chromatography. In: R . J . H a m i l t o n and J . B . R o s s e l l ( E d s . ) , A n a l y s i s o f O i l s and F a t s , E l s e v i e r A p p l i e d S c i e n c e P u b l i s h e r s L t d . N.Y., p p . 137-206. Ackman, R.G., C.S. T o c h e r and J . M c L a c h l a n . 1968. 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B i o c h e m i c a l c o m p o s i t i o n and e n e r g y content of s i x s p e c i e s of phytoplankton used i n mariculture of bivalves. A q u a c u l t u r e 60: 2 3 1 - 2 4 1 . W h y t e , J.N.C. 1988. F a t t y a c i d p r o f i l e s from d i r e c t methanolysis of l i p i d s i n t i s s u e of cultured species. A q u a c u l t u r e 75: 193-205. W h y t e , J . N . C , N. B o u r n e and C A , H o d g s o n . 1987. Assessment o f b i o c h e m i c a l c o m o s i t i o n and e n e r g y r e s e r v e s i n l a r v a e o f t h e s c a l l o p Patinopecten yessoensis. J . Exp. Mar. B i o l . E c o l . 113: 113-124. W i k f o r s , G.H. 1986. A l t e r i n g g r o w t h and g r o s s c h e m i c a l c o m p o s i t i o n o f two m i c r o a l g a l m o l l u s c a n f o o d s p e c i e s by v a r y i n g n i t r a t e and p h o s p h a t e . A q u a c u l t u r e 59: 1-14. W i k f o r s , G.H., J.W. T w a r o g , J r . , and R. U k e l e s . 1984. Influence of chemical composition of a l g a l food sources on g r o w t h o f j u v e n i l e o y s t e r s , Crassostrea virginica. B i o l . B u l l . 167: 251-263. W i l s o n , J.H. 1979. O b s e r v a t i o n s on t h e g r a z i n g r a t e s and g r o w t h o f Ostrea edulis L . l a r v a e when f e d a l g a l c u l t u r e s of d i f f e r e n t ages. J . E x p . Mar. B i o l . E c o l . 38: 187-199. W i l s o n , J.H. 1980. P a r t i c l e r e t e n t i o n and s e l e c t i o n by l a r v a e and s p a t o f Ostrea edulis i n a l g a l suspensions. M a r . B i o l . 57: 135-145. Winter, J.E. 1973. The f i l t r a t i o n r a t e o f Mytilus edulis i t s d e p e n d e n c e on a l g a l c o n c e n t r a t i o n m e a s u r e d by a continuous automatic r e c o r d i n g apparatus. Mar. B i o l . 317-328.  and 22:  W i n t e r , J . E . , and R.W. Langton. 1976. Feeding experiments w i t h Mytilus edulis L. at small l a b o r a t o r y s c a l e . I. The i n f l u e n c e o f t h e t o t a l amount o f f o o d i n g e s t e d and f o o d c o n c e n t r a t i o n on g r o w t h . In: G. P e r s o o n e and E . J a s p e r s ( E d s . ) , Proceedings of the Tenth European Symposium i n M a r i n e B i o l o g y , V o l . 1, p p . 5 6 5 - 5 8 1 , Universa Press, 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 t h e r e l a t i v e N and P r e q u i r e m e n t ( t h e optimum N:P r a t i o ) o f m a r i n e p l a n k t o n i c a l g e . J. P l a n k t o n R e s . 8: 9 1 - 1 0 3 .  169 APPENDICES  Appendix & Table 1: C a l o r i c value of Tahitian Isochrysis sampled during aid-log phase (ML), 01 a f t e r 2 or 6 d of nitrate starvation (-H 2 d , -H 6 d). Hean and standard deviations of duplicate cultures (a and b) are presented. Brackets enclose percent of t o t a l c a l o r i e s . Energy equivalents - kcal-10-12 c e l l s Treatment  lipid  HL  112.07 132.97  a a Hean STD  -N 2d  a a  b b  100.01 6.69  (44%)  a a  86.13 4.22  (42%)  b b  81.58 4.04  (35%)  96.28 1.50  116.17 4.66  138.39 1.49  (38%)  120.67 1.32  (48%)  95.42 1.55  (411)  24.42 5.44  (56%)  21.88 0.05  (10%)  15.24 2.22  (9%)  16.79 0.50  246.40 217.01 217.99  (7%)  17.29 16.29 (46%)  240.61 • 243.72 249.08  17.46 13.02 (55%)  275.69  237.21 244.02  21.93 21.84  93.87 96.97 (46%)  112.70 4.68  269.15 282.23  18.98 29.87  122.00 119.35  97.78 94.78 Mean STD  (151)  139.88 136.89  77.55 85.62 Mean STD  40.47 0.76  Total  117.37 108.02  111.52 120.83  81.92 90.35 Mean STD  Protein  39.71 41.23  106.71 93.32 Mean STD  -H 6d  122.52 10.45  Carbohydrate  217.50 208.94 208.05  (8%)  208.49  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 after 6 h of nitrate or s i l i c a t e starvation (-N, - S i ) . Hean and standard deviations of duplicate cultures (a and b) are presented. Brackets enclose percent of t o t a l c a l o r i e s . Energy equivalents - kcal-10-12 c e l l s Treatment  Lipid  a a  Carbohydrate  76.40 80.02 Hean STD  b b  78.21 1.81  41.24 42.87 (44*)  65.25 73.68 Hean STD  69.46 4.22  41.10 0.30  (42*)  -Si 6h  a a  48.62 1.14  (32*)  b b  65.88 2.30  (33*)  56.59 0.32  (26*)  60.95 0.07  64.43 2.09  (44*)  26.81 0.39  (48*)  24.52 2.32  53.58 1.24  24.78 1.01  (43*)  36.42 0.19  (32*)  58.41 0.78  (20*)  55.23 2.05  126.82 146.44 152.51  (24*)  149.48  154.57 147.62 (39*)  57.28 53.18 (18*)  166.38  128.20 125.44  59.18 57.63 (18*)  179.24 166.32 166.43  36.61 36.23  22.20 26.84 (42*)  (33*)  25.79 23.77  27.20 26.41  56.27 56.91 Hean STD  43.33 2.92  58.98 1.23  175.39 183.09  54.82 52.34  62.34 66.52  68.18 63.57 Hean STD  (23*)  61.01 60.88  47.48 49.76 Hean STD  42.06 .81  Total  57.75 60.20  46.25 40.41  41.41 40.80 Hean STD  Protein  151.09 135.75 136.93  (40*)  136.34  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 aid-log phase (HL) or after 6 h of nitrate or s i l i c a t e starvation (-N, - S i ) . Hean and standard deviations of duplicate cultures (a and b) are presented. Brackets enclose percent of t o t a l c a l o r i e s . Energy equivalents - k c a l / l e l 2 c e l l s Treatment HL  Lipid  a a  Carbohydrate  34.41 35.03 Hean STD  34.72 0.31  41.07 43.15 (311)  29.73 28.95  b b Hean STD  29.34 0.39  24.88 0.40  (30%)  Silis  a a  34.02 0.04  (32%)  b b  42.68 0.85  (40%)  46.44 0.25  (37%)  36.72 2.55  28.85 0.62  (36%)  30.69 1.44  (47%)  31.99 0.15  32.33 0.03  16.91 0.57  (34%)  21.90 0.21  (33%)  44.66 0.06  (21%)  47.64 1.73  78.52 85.65 83.90  (26%)  84.77  117.50 118.55 (38%)  49.38 45.91 (25%)  98.59  75.80 81.24  44.71 44.60 (26%)  113.35 97.26 99.92  22.12 21.69  31.84 32.14 (37%)  (32%)  16.34 17.48  29.26 32.13  46.19 46.69 Hean STD  36.92 1.69  36.52 0.87  111.13 115.57  32.30 32.36  29.46 28.23  43.53 41.83 Mean STD  (37%)  34.17 39.28  34.07 33.98 Hean STD  42.11 1.04  Total  35.65 37.39  35.23 38.61  25.28 24.48 Hean STD  Protein  118.02 127.40 124.74  (38%)  126.07  173  Appendix A Table 4: C e l l u l a r weight of gross biochemical components in series 2 Tahitian Isochrysis. Cultures vere grovn i n n i t r a t e - l i m i t e d medium and harvested at aid-log phase (ML) and after 2 and G days of nitrate starvation (-N 2 d or 6 d ) . Hean and standard deviations of duplicate cultures (a and b) are presented. T r i p l i c a t e ash determinations vere used to calculate mean ash. Treatment  C e l l u l a r l e i g h t of Components (pg-cell-1) Cell leight pg-cell-1  ML  n  a a Hean STD  55.79 5.52  15  -H a 2d a Hean STD  55.79 5.52  15  b b Hean STD  55.79 5.52  15  -8 a 6 d a Hean STD  55.79 .85  15  b b Hean STD  55.79 5.52  15  Lipid  Carbohydrate Hono/Oligo Poly  Protein  13.31 15.79  .00 .53  9.23 9.06  28.63 26.35  14.55 1.24  .53 .00  9.15 .09  27.49 1.14  12.67 11.08  .77 .75  25.17 27.35  4.63 7.28  11.88 .80  .76 .01  26.26 1.09  5.96 1.33  9.73 10.73  .95 .80  31.58 31.04  5.35 5.33  10.23 .50  .87 .08  31.31 .27  5.34 .01  9.21 10.17  .70 .73  27.67 27.02  4 .26 3.17  9.69 .52  .72 .48  27.35 .02  3.72 .32  11.61 11.26  1.26 1.48  20.57 21.07  4.22 3.97  11.43 .18  1.37 .11  20.82 .25  4.10 .12  Ash  Total Organics 51.17 51.73  6.79 .07  51.72  43.24 46.47 5.35 .01  44.85 47.61 47.89  5.25 .25  47. 75 41.84 41.10  6.63 .54  41.47 .20 37.66 37.78  9.89 .39  37.72  174 ippendix A Table 5: C e l l u l a r weight of gross biocheaical components i n series 2 Chaetoceros c a l c i t r a n s . Cultures 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 at mid-log phase (HL) and after 6 hours of nutrient starvation (-N 6 h, - S i 6 h). Hean and standard deviations of duplicate cultures (a and b) are presented. T r i p l i c a t e ash determinations vere used to .calculate mean ash. Treatment  C e l l u l a r leight of Components (pg-cell-1) Cell leight pg«cell-l  HL  Lipid  n  a a  9jT 9.50 Hean STD  42.12 5.94  13  b b Hean STD  42.12 5.94  13  -H a 6 h a Hean STD  42.12 5.94  13  b b Hean STD  42.12 5.94  13  -Si a 6 h a Hean STD  42.12 5.94  13  b b Hean STD  42.12 5.94  13  Carbohydrate Mono/Oligo Poly Tl 1768 .56 9.41  Protein  Ash  14J9 14.68  9.29 .21  .73 .17  9.05 .36  14.38 .30  7.75 8.75  .99 .48  9.77 8.91  13.37 12.77  8.25 .50  .74 .25  9.34 .43  13.07 .30  4.92 4.85  1.08 1.12  13.11 13.04  6.29 5.80  4.88 .04  1.10 .02  13.07 .03  6.04 .25  5.64 5.91  1.49 1.25  13.00 14.22  8.93 8.84  5.77 .14  1.37 .12  13.61 .61  8.88 .05  8.10 7.55  .88 .99  5.44 5.15  14.44 14.06  7.82 .27  .94 .05  5.30 .15  14.25 .19  6.68 6.76  .62 1.24  4.54 5.00  13.97 12.97  6.72 .04  .93 .31  4.77 .23  13.47 .50  9.07 .25  Total Organics 32.75 34.16 33.45 31.88 30.92  9.92 .72  31.40  25.40 24.80 14.29 .03  25.10 29.07 30.22  8.75 .17  29.64  28.86 27.75 12.16 .15  28.30 25.82 25.97  14.28 .03  25.89  175  Appendix A Table 6: Cellular weight of gross biochemical components i n Thalassiosira pseudonana. Cultures vere grown in nitrate-limited or s i l i c a t e - l i m i t e d medium and harvested at aid-log phase (MLl and after 6 hours of nutrient starvation (-H 6 h, - S i ( h). Hean and standard deviations of duplicate cnltures (a and b) are presented. T r i p l i c a t e ash determinations vere used to calculate 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 pg'cell-1  HL  Lipid  n  a a Hean STD  29.58 4.14  14  b b Hean STD  29.58 4.14  14  -H a 6h a Hean STD  29.58 4.14  14  b b • Hean STD  29.58 4.14  14  -Si a 6 h a Hean STD  29.58 4.14  14  b b Hean STD  29.58 4.14  14  Carbohydrate Hono/Oligo Poly  Protein  4.09 4.16  .58 .35  8.97 9.68  8.70 9 .12  4.12 .04  .47 .12  9.33 .36  8.91 .21  3.53 3.44  .37 .43  7.83 8.55  7.88 7.89  3.49 .05  .40 .03  ' 8.19 .36  7.89 .01  3.00 2.91  .50 .36  7.45 8.77  3.99 4.26  2.96 .05  .43 .07  8.11 .66  4.12 .14  4.05 4.04  .57 .51  6.28 6.06  5.39 5.29  4.04 .01  .54 .03  6.17 .11  5.34 .05  5.17 4.97  .00 .49  6.80 6.99  10.91 10.88  5.07 .10  .49 .00  6.90 .09  10.89 .01  5.49 5.55  .32 .29  7.08 7.19  12.04 11.20  5.52 .03  .31 .02 -  7.13 .05  11.62 .42  Ash  Total Organics 22.33 23.31  7 .85 1.09  22.82 19.60 20.31  8.64 .10  19.96 14.94 16.30  10.45 1.05  15.62 16.29 15.89  10.43 .00  16.09 22.88 23.32  5.41 .05  23.34 24.93 24.22  5.98 .13  24.57  176  Appendix B. Table 1: Changes over tine i n the f a t t y acids expressed as a percentage of the t o t a l , i n series 1 Tahitian Isochrysis 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 acids I and N are unknowns. Fatty Acid  Algal Sample Mid-log  14:0 I 16:0 fl  16 16 16 16 16 16 16 18 18 18 18 18 18 18 18 11 19 20 20 20 20 20 20 20 20 22 22 21 22 22 22 22  ln7 2n6 2n4 3n4 3n3 3nl 4nl lnl3 ln9 ln7 2n9 2n6 2n4 3n6 3n3 4n3 ?  In9 2f 2n6 A 3n6 4n6 3n3 5n3 0 ln9 5n3 4n6 5n6 5n3 6n3  Saturated Honounsat'd Polyunsat'd Total n3 n6  19.0i0.1 0.3 12.9i0.3 0.9i0.1 2.6+0.0 0.3 0.6*0.0 0.3*0.0  N-limited 85 h 185 h 250 h 330 h 400 h 450 h  P-limited 85 h 185 h 250 h 355 h 400 h  19.1  18.9 0.3 12.7 1.0 2.6 0.3 0.6 0.2  14.8 0.4 13.6 0.5 1.9 0.1 0.4 0.3 0.1 0.1 0.2 0.3 20.1 2.3 0.1 4.8 0.1 0.2 4.4 10.4 0.5 0.1  12.8 0.4 16.0 2.0 0.1 0.3 0.3 0.2 0.1 0.2 0.4 21.1 1.9 0.2 3.7 0.1 0.1 4.3 9.9 0.6 0.1  0.4 0.3 0.1  0 0. 0.  0.1 0.5 22.1 1.9 0.1 3.3  0 0. 24. 1. 0. 2.  0.1 3.9 10.6 0.5 0.2  0. 3. 8. 0 0  0.1 0.1 0.2 0.3 0.3 0.5 0.7 0.2 0.4 0.5 4.9 0.2 13.9  0.1  0.2  16.9  17.2 0.4 13.4 0.5 2.1 0.1 0.5 0.3 0.1 0.1 0.2 0.2 18.1 1.9 0.1 3.9 0.1 0.1 3.9 11.6 0.4 0.2 0.1 0.1 0.1 0.2 0.2 0.4 0.4 0.5 0.1 0.6 0.2 4.6 0.2 14.5  0.2 0.4 0.2 0.7 1.0 0.3 0.4 0.2 2.4 0.2 15.2  0.2 0.3 0.4 0.7 1.2 0.4 0.7 0.3 2.2 0.2 15.7  0. 0 0. 0. 0. 0. 1. 0. 0. 0. 1. 0. 13. 0  32.1*0.4 17.5*0.9  32.4 18.2  31.2 22.8  29.2 24.8  30.0 25.8  29.7 27.7  46.1*1.6 38.3*0.9 6.6*0.6  46.7 38.5 7.0  42.5 31.8 9.4  42.3 30.1 11.0  39.3 31.1 7.1  39.8 32.5 6.5  0.2 0.1 0.3*0.1 12.4*0.6 1.6*0.1 0.1 4.2*0.4 0.1 0.2 3.9*0.2 14.0*0.3 0.2*0.1 0.6*0.1 0.3*0.0 0.1 0.5*0.0 0.2 0.1 2.0*0.1 1.1*0.2 0.3*0.0 0.1 0.5*0.0 0.3*0.1 2.1*0.1 0.2 16.8*0.2  13.1 0.8 2.7 0.6 0.2  0.4 12.9 1.6 4.5 3.8 14.1 0.3 0.7 0.3 0.5 1.9 1.2 0.3 0.5 0.3 2.2  11.8 0.4 16.7 0.4 2.5  11 0 19 0 2  0.2 0.1 0.3 12.0 1.5 0.1 3.9 0.1 0.2 4.0 13.8 0.2 0.5 0.3  18.1 0.4 12.4 1.4 3.5 0.3 0.9 0.3 0.1 0.3 0.2 0.2 11.5 2.1 0.1 3.4 0.1 0.3 4.9 16.4 0.2 0.3 0.2  16.9 0.5 12.3 1.2 3.6 0.3 1.0 0.3 0.1 0.2 0.2 0.2 12.2 2.3 0.2 3.9 0.1 0.2 5.3 15.3 0.2 0.2 0.2  0.5 0.2 0.1 2.0 1.0 0.2 0.2 0.5 0.2 2.0 0.2 16.7  0.3 0.1 0.2 1.0 0.7 0.2 0.2 0.5 0.1 1.7 0.1 14.2  0.2 0.1 0.3 0.9 0.7 0.3 0.2 0.5 0.1 1.7 0.2 15.2  0.2 0.1 0.3 0.3 0.2 0.3 15.3 18.1 2.4 2.5 0.1 0.1 4.5 4.4 0.2 0.1 0.2 0.2 5.2 5.1 15.2 15.0 0.2 0.3 0.27 0.2 0.1 0.1 0.1 0.1 0.3 0.1 0.5 0.3 0.7 0.7 0.5 0.7 0.3 0.4 0.4 0.3 0.2 0.3 2.0 1.7 0.2 0.1 14.2 12.1  32. 5 30 3  31.8 17.2  30.7 17.9  29.4 18.8  27.6 20.1  28.8 25.6  33 3 26 8 56  46.9 38.3 6.9  46.0 37.8 6.0  46.8 38.1 6.5  45.7 36.3 7.5  42.3 33.6 6.9  13.8 0.5 13.4 1.2 4.0 0.2 0.8 0.4  13.6 0.5 14.5 1.1 4.1 0.2 0.8 0.4  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 series 2 Tahitian Isochrysis cultures and harvested either at nid-log phase or after 2 days of n i t r a t e starvation. T r i p l i c a t e measurements vere taken at each phase and reported as mean • 1 S.D. The fatty acids I and 8 are unknowns. Fatty Acid Mid-log 1 2 14:0 I 16:0 16:ln7 16:2n6 16:2n4 16:3n4 16:3n3 18:lnl3 18:ln9 18:ln7 18:ln5 18:2n9 18:2n6 18:3n6 18:3n3 18:4n3 19:? 20:ln9 20:2f 20:a 20:3n3 20:5n3 22:0 22:ln9 21:5n3 22:4n6 22:5n6 22:5n3 22:6n3 Saturated Honounsat'd Polyunsat'd Total n3 n6  3  xts  Algal Sample 2 day K-starved 1 2 3  12.7 .1 13.3 1.2 1.2 .3 .2 .1 .1 .7 17.4 1.7 0.1 .1 6.5 .6 4.0 14.4 .2 .7 .4 .5 2.1 .5 .4  18.9 .2 11.3 1.0 2.2 .6 .4 .3 .2 .2 11.7 1.5 0.1 .1 3.4 .5 4.2 15.5 .2 .9 .4 .6 2.5 .9 .3  12.6 .1 12.3 1.4 1.2 .3 .3 .1 .1 .5 14.2 1.9 0.1 .1 6.6 .6 4.4 14.8 .2 1.2 .4 .5 2.4 .6 .3  14.7+3.6 0.2*0.1 12.3il.O 1.2+0.2 1.6*0.6 0.4*0.2 0.3+0.1 0.2*0.1 0.2*0.1 0.4+0.3 14.4*2.8 1.7+0.2 0.1+0.0 0.1+0.0 5.5+1.8 0.6+0.1 4.2+0.2 14.9+0.5 0.2+0.0 0.9+0.3 0.4+0.0 0.5+0.1 2.3+0.2 0.7+0.2 0.3+0.1  .4 2.5 .4 15.8  .2 .2 2.6 .3 16.6  .4 2.8 .3 16.6  0.4+0.1 2.6+0.2 0.3+0.1 16.3+0.5  11.2 .2 18.9 .3 2.4 .2 .3 .2 .1 .5 22.8 1.8 0.1 .1 4.00 .1 3.0 10.2 .5 .2 .5 .3 1.2 .4 .4 .3 2.4 .2 12.9  15.1 14.6 .2 .1 21.6 19.6 .4 .4 2.2 2.0 .1 .1 .3 .3 .1 .2 .1 .5 .5 1.8 0.1 .1 5.0 2.7 7.8 .5 .2 .4 .3 1.1 .3 .2 2.3 11.8  xts 13.7+2.1 0.2+0.0 20.0 1.4 0.4+0.0 2.2+0.2 0.1+0.0 0.3+0.0 0.2+0.0 0.1+0.0 0.5+0.1  1.6 0.1 .1 5.3 .1 3.3 8.8 .4 .2  1.8+0.1 0.1+0.0 0.1+0.0 4.7+0.7 0.1+0.1 3.0+0.3 9.0+1.1 0.5+0.0 0.2+0.0  .4 .3 1.0 .2 .4 .2 2.1 .2 11.5  0.4+0.1 0.3+0.0 1.1+0.1 0.3+0.1 0.3+0.1 0.2+0.1 2.3+0.2 0.1+0.1 12.1+0.8  27.4+4.7 19.1+4.1  34.8+3.7 . 24.1+1.2  49.5+4.5 38.9+1.8 9.4+2.3  33.1+3.6 25.2+2.4 7.4+1.1  178 Appendix B. Table 2b: The f a t t y acids expressed as a percentage of the t o t a l , i n series 2 Tahitian Isochrysis cultures 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 acids I and N are unknovns. Patty Acid 4 day N-starved 1 2 3 14:0 I 16:0 N 16:ln7 16:2n6 16:2n4 16:3n4 16:3n3 18:lnl3 18:ln9 18:ln7 18:2n9 18:2n6 18:3n6 . 18:3n3 18:4n3 19:?? 20:ln9 20:4n6 20:3n3 20:5n3 22:0 22:ln9 21:5n3 22:4n6 22:5n6 22:5n3 22:6n3 Saturated Honounsat'd Polyunsat'd Total n3 n6  14.57 .18 17.62 .34 2.90 .19 .38 .23 .05 .28 22.63 1.52 .11 4.18 .13 3.20 10.24 .46 .19 .18 .50 .53 .9  17.17 .20 17.73 .50 2.88 .16 .41 .16 .09 .27 20.97 1.47 .11 3.66  .53 .28 2.17 .19 . 13.27  .40 .20 2.10 .19 12.42  3.10 11.28 .54 .26 .26 .44 .34 1.04  xts  Algal Sample 6 day (l-starved 1 2 3  15.59 15.78il.31 .17 0.18*0.02 20.94 . 18.76il.89 .35 0.40*0.09 2.47 2.75*0.24 .09 0.15*0.05 .31 0.37*0.05 .15 0.18+0.04 .06 0.07*0.02 .45 0.33+0.10 25.50 23.03*2.29 1.52 1.50+0.03 .11 0.11*0.00 4.79 4.21+0.57 .14 0.14*0.01 3.14 3.15+0.05 7.88 9.80*1.75 .45 0.48+0.05 .17 0.21*0.05 .18 0.18*0.00 .32 0.42*0.09 .23 0.37+0.15 1.06 1.00*0.09 .26 .21 1.86 .13 9.88  0.40+0.14 0.21*0.04 2.04+.0.16 0.17*0.03 11.86+1.76  11.31 .14 17.24 .57 2.78 .17 .36 .16 .04 .51 24.88 1.56 .09 4.57 .16 3.04 9.56 .54 .20 .27 .40 .30 1.26 .48 .60 .32 2.42 .22 13.15  13.92 12.84 .23 .21 20.22 16.71 .47 .23 3.38 3.63 .16 .24 .72 .50 .20 .25 .05 .33 .36 21.69 21.39 2.01 1.76 .15 2.91 2.95 .10 2.86 2.60 9.66 11.83 .71 .60 .17 .21 .10 .27 .49 .36 .52 1.43 1.29 .68 .52 .64 .77 .27 .46 2.11 2.11 .21 .23 12.99 14.10  x+s 12.69+1.31 0.19+0.05 18.06+1.89 0.42+.0.17 3.26+0.44 0.19+0.04 0.53+0.18 0.20+0.05 0.03+0.03 0.40+0.10 22.65+1.93 1.78+0.23 0.12+0.04 3.48+0.95 0.13+10.04 2.83+0.22 10.35+1.28 0.62+0.09 0.12+0.11 0.19+0.09 0.39+0.11 0.39+0.11 1.33+0.09 0.56+0.11 0.67+0.09 0.35+0.10 2.21+0.18 0.22+0.01 13.41+0.64  35.54+3.29 27.82+2.71  32.08+3.29 28.86+3.00  33.83+4.91 26.24*3.99 6.93+0.83  35.65*4.20 28.29+2.49 6.51+1.44  179 Appendix B. Table 3a: Changes over tiae i n the fatty acids expressed as a percentage of the t o t a l , i n series 1 Chaetoceros calcitrans 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 acids I and N are unknown. Fatty Acid  Algal Sample Mid-log  N-limited 58 h  97 h  119 h  150 h  174 h 18.66 0.52 15.70 1.19 27.63 0.30 0.60 2.63 5.52 3.68 0.15  14:0 I 16:0 N 16:ln7 16:ln5 17:0 16:2n6 16:2n4 16:3n4 16:3n3 16:3nl 16:4nl 18:lnl3 18:lnll 18:ln9 18:ln7 18:ln5 18:2n6 18:2n4 18:3n3 18:3nl 18:4n3 19:? 20:4n6 20:5n3 22:0 22:5n3 22:6n3  14.45il.48 0.49+0.04 9.43t0.28 1.96+0.54 23.68+2.79 0.57+0.04 0.90+0.16 3.52+0.98 7.86+0.80 7.55+1.24 0.09+0.00 0.14+0.05 0.73+0.47 0.42+0.21  15.52 0.51 9.63 2.34 25.65 0.54 1.01 2.83 7.29 8.43 0.09 0.17 0.39 0.57  17.74 0.55 13.73 1.72 28.43 0.42 0.81 2.40 5.61 5.28 0.12 0.14 0.50 0.47  18.94 0.53 14.79 . 1.17 29.36 0.35 0.54 2.40 5.44 4.52 0.14  0.33+0.27 0.53+0.23 0.10+0.02 0.35+0.15 0.13 0.10+0.05 0.25+0.09 1.21+0.21 0.11+0.02 0.14 16.03+1.68 0.07+0.03 0.65+0.08  0.52 0.69 0.11 0.45 0.13 0.13 0.31 1.06 0.12 0.14 17.22 0.07 0.05 0.71  0.50 0.53 0.13 0.35 0.37 0.13 0.95 0.26 0.20 0.42 13.56 0.10 0.08 0.89  0.58 0.51 0.14 0.30 0.40 0.11 1.01 0.17 0.20 0.45 12.91 0.10 0.08 1.02  18.80 0.53 15.58 2.02 28.09 0.32 0.58 2.39 5.23 3.68 0.14 0.05 0.39 0.57 0.07 0.52 0.62 0.29 0.29 0.44 0.11 1.09 0.12 0.22 0.49 12.05 0.12 0.11 1.30  Saturated Honounsat'd Polyunsat'd Total n3 n6  24.78+1.92 25.63+3.56  26.23 28.08  32.38 30.48  34.38 31.43  35.08 30.48  35.17 30.23  37.43+5.22 18.15+2.05 3.87+1.13  38.53 19.26 3.42  29.47 15.04 3.17  27.94 14.43 3.15  26.35 13.83 3.17  27.38 14.33 3.37  0.46 0.49 .  0.43 0.57 . 0.17 0.43 0.61 0.32 0.24 0.48 0.09 1.14 0.21 0.24 0.50 12.39 0.21 0.11 1.38  180  Appendix 8. Table 3b: Changes ovei time in the fatty acids expressed as a percentage of the t o t a l , i n series 1 Chaetoceros calcitrans grovn in phosphate-limited or s i l i c a t e - l i m i t e d medium. The fatty acids I and N are unknowns. Fatty Acid  Algal Sample P-limited 58 h 97 h  14:0 16:0  I  16:ln7 16:ln5 17:0 16:2n6 16:2n4 16:3n4 16:3n6 16:3n3 16:4nl 18:0 18:lnl3 18:ln9 18:ln7 18:ln5 T 18:2n6 18:2n4 18:3n4 18:3n3 18:3nl 18:4n3 19:? 20:4n6 20:4n3 20:5n3 22:0 22:5n3 22:6n3 Saturated Honounsat'd Polyunsat'd Total n3 n6  13.40 9.23 1.57 21.71 0.59 0.78 4.22 8.42 6.67 0.09 0.10 1.66 0.14 0.28 0.14 0.37 0.08  14.89 7.49 1.80 30.31 0.44 0.78 3.22 8.10 7.57 0.15 0.17 1.06  0.24  0.63 0.31  0.13 0.29 0.60 0.14  119 h  150 h  13.72 10.72 2.46 30.34 0.39 0.45 2.79 7.23 5.52 0.16 0.11 0.64 0.05 0.46 0.45 2.06  18.80 15.58 2.02 28.09 0.32 0.58 1.59 4.57 2.63 0.13 0.20 0.27  0.15 0.45 0.39 0.08 0.18 0.86 0.33 0.18 0.49  Si-limited 119 h 99 h  150 h  0.97 0.11 2.43 0.15 0.12 0.64 0.30 0.12 0.17 0.57 0.28 0.16 0.41 0.07 6.98 0.19 0.09 0.52  13.68 6.69 3.69 28.28 0.38 1.67 3.84 8.90 5.94 0.23 0.15 1.06 0.10 0.17 0.18 0.81  13.86 7.06 2.69 29.07 0.30 1.31 3.93 9.41 5.28 0.21 0.10 0.91 0.18 0.19  0.18 0.40 0.41 0.07 0.18 0.67 0.55 0.22 0.22 0.07 15.64 0.10 0.08 0.67  0.21 0.31 0.56 0.07 0.17 0.80 0.43 0.24 0.30 0.07 15.21 0.75  3.87 0.25 0.24 0.24  1.29  9.86 13.39 10.19 15.58 0.22 1.19 1.08 2.62 1.08 0.92 0.39 0.23 0.16 0.70 0.44 7.27 0.23 0.57 0.17 0.15 0.09 0.29 0.25 0.12 0.09 0.09  0.27 0.06 0.18 1.35 0.09 0.08 0.13 14.84  16.12  0.09 0.59  0.52  12.52 0.11 0.10 0.73  23.59 23.17  23.16 31.91  25.05 33.70  35.15. 32.07  22.24 29.82  18.41 30.85  24.85 32.07  38.99 17.16 4.63  39.87 17.76 4.37  32.58 13.97 3.85  19.54 8.31 2.77  39.07 17.34 4.69  37.45 16.73 4.75  11.83 5.15 2.26  0.27 0.70 0.68 0.17 0.37  181  Appendix B. Table 4a: 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 calcitrans cultures and harvested at either 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 duplicate measurements taken at starved phases are reported as mean t l S.D. The fatty acids I and N are unknowns. Patty Acid  Algal Sample Hid-log 1  2 h N-starved 1 2  6 h N-starved 1 2  Saturated Honounsat'd Polyunsat'd Total n3 n6  24.72 22.47  29.38+1.15 25.63*2.87  30.06*5.02 24.68+1.63  37.29 23.25 1.85  38.82+5.22 25.49*3.88 1.74+0.80  34.06+6.83 22.66+2.85 2.45+1.60  16-00*0.70 0.49*0.04 12.65*0.37 1.75*0.25 23.87*2.35 0.52*0.02 0.53*0.04 1.16+0.64 2.14*0.10 8.70+0.34 0.26*0.04 0.07+0.00 0.20+0.08 0.11+0.03 0.38+0.15 0.46+0.21 0.40+0.14 0.48+0.14 0.48+0.02 0.07+0.04 5.46+0.78 0.10+0.02 0.28+0.08 0.28+0.08 16.70+0.91 0.09*0.01 0.74*0.80 0.23+0.22 1.94*1.23  15.88 .52 16.99 .96 22.20 .78 .43 .81 1.88 5.00 .18  x+s  15.51 .47 11.07 1.74 21.47 .48. .88 1.41 2.15 9.23 .19 .12 .28 .11 .24 .29 .28 .34 .41 .04 4.81 .10 .23 .23 15.53 .05 .38 .17 2.07  .20 .14 .55 .70 .56 .49 .50 .06 4.68 .11 .37 .37 15.84 .09 1.63 .49 3.35  16.33 .48 12.75 1.82 25.66 .52 .48 1.45 2.03 8.67 .29 .07 .13 .10 .31 .38 .33 .62 .46 .11 5.46 .11 .22 .22 17.65 .10 .09 .07 1.08  xts  14:0 I 18:0 N lS:ln7 ' 16:ln5 17:0 16:2n6 16:2n4 16:3n4 16:3n3 16:3nl 16:4nl 18:0 18:lnl3 18:ln9 18:ln7 18:2n6 18:3n4 18:3n3 18:4n3 20:4n6 20:c 20:4n3 20:5n3 22:0 22:5n3 Z 22:6n3  16.47 .53 12.97 1.47 24.75 .54 .54 .43 2.19 9.05 .27 .07 .28 .09 .28 .30 .32 .34 .47 .04 6.24 .08 .24 .24 16.62 .09 .50 .14 1.38  15.19 .45 12.24 1.96 21.21 .51 .56 1.60 2.20 8.37 .22  3  .05 .13 .52 1.14 .58 1.80 1.62 .16 5.22 .53 .48 .48 13.29 .09 .90 .31 1.76  14.66 .45 11.38 1.46 22.79 .45 .41 1.24 1.83 6.98 .24 .08 .17 .07 .24 .34 .29 .43 .40 .07 4.96 .07 .18 .18 14.60 .06 1.30 .40 3.39  15.27+0.86 0.49+0.05 14.19+3.97 1.21+0.35 22.50+0.42 0.62+0.23 0.42+0.01 1.03+0.30 1.86+0.04 5.99+1.40 0.21+0.04 0.08 0.11+0.08 0.10*0.04 0.38+0.20 0.74*0.57 0.44+0.21 1.12*0.97 1.01+0.86 0.12*0.06 5.09+0.18 0.30+0.33 0.33+0.21 0.33*0.21 13.95+0.93 0.08+0.02 1.10+0.28 0.36+0.06 2.58+1.15  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 series 2 Chaetoceros c a l c i t r a n s cultures 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 duplicate measurements taken at starved phases are reported as mean ±1 S.D. The f a t t y acids I and N are unknowns. Fatty Acid 2 h Si-starved 1 2 3 14:0 I 16:0 N 16:ln7 16:ln5 17:0 16:2n6 16:2n4 16:3n4 16:3n3 16:4nl 18:0 18:lnl3 18:ln9 18:ln7 18:2n6 18:3n4 18:4n3 20:c 20:4n3 2 0:5 n 3 22:5n3 Z 22:6n3 Saturated Honounsat'd Polyunsat'd Total n3 n6  14.78 .49 10.08 1.52 22.02 .61 .67 1.71 2.83 9.73 .23 .18 .15 .38 .42 .99 .18 .37 5.01 .32 .32 16.49 .06 .29 .06  15.27 .49 10.06 2.40 23.89 .72 .61 2.37 2.88 10.22 .35 .29 .11 .29 .28 .40 .22 .28 4.34 .20 .20 17.40 1.16 .15 1.16  14.63 .54 11.79 2.32 20.40 .61 .55 1.53 2.21 8.49 .37 .31 .11 .31 .33 1.18 .19 .38 5.54 .24 .24 14.36 .25 0.25  Algal Sample 6 h Si-starved xts 1 2 14.89t0.33 0-51*0.03 10.64t0.99 2.08+0.49 22.10+1.75 0.65+0.06 0.61+0.06 1.87.+0.44 2.64+0.37 9.48+0.89 0.32*0.08 0.26+0.07 0.12+0.02 0.33+0.05 0.34+0.07 0.86+0.41 0.20+0.02 0.34+0.06 4.96+0.60 0.25+0.06 0.25+0.06 16.08tl.56 0.49+0.59 0.22+0.10 0.49+0.59  13.41 .48 12.81 3.60 27.89 .37 .59 1.48 2.61 6.07 .25 .19 .14 .71 .70 2.08 .25 .25 3.62 .31 .31 13.25 .81 .45 .81  15.96 .54 10.73 1.38 21.83 .66 .67 1.93 2.92 10.62 .40 .46 .13 .19 .22 .31 .18 .32 5.80 .21 .21 16.99 .74 .19 .74  x+s 14.69+1.80 0.51+0.04 11.77+1.47 2.49+1.57 24.86+4.29 0.52+0.21 0.63+0.06 1.71t0.32 2.77+0.22 8.35+3.22 0.33+0.11 0.33+0.19 0.14+0.01 0.45+0.37 0.46+0.34 1.20+1.25 0.22+0.05 0.29t0.05 4.71+1.54 0.26t0.07 0.26+0.07 15.12+2.64 0.78t0.05 0.32+0.18 0.78+0.05  . 26.26+1.40 24.28*2.34  27.23+3.34 27.49+6.46  37.38+5.33 22.59*3.48 2.07+0.46  36.57*8.51 22.76*4.46 1.93*0.37  183 Appendix B. Table 5: Changes over tiae i n the fatty acids expressed as a percentage of the t o t a l , i n series 1 Thalassiosira pseudonana cultures 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 fatty acids I and N are unknowns. The aid-log sample i s represented by the Si (6 h sample. Fatty Acid  Algal Sample P-limited 62 h 108 h  14:0 I 16:0 N 16:ln7 16:ln5 17:0 16:2n6 16:2n4 16:3n4 16:3n3 16:4nl 18:lnl3 18:ln9 18:ln7 18:ln5 18:2n9 18:2n6 18:3•6 18:3nl 18:4n3 20:3n6 20:4n6 20:4n3 20:5n3 22:6n3 Saturated Honounsat'd Polyunsat'd Total n3 • n6  158 h  207 h  6.12 0.67 36.42 0.38 28.92 0.11 0.13 0.92 1.02 1.81 0.07 0.04 2.82 2.75 1.96 0.19 0.20 0.71 0.73 0.11 1.65 0.23 2.17 0.11 5.67 1.77  7.83 0.68 36.67 0.52 26.28 0.09  0.14 0.37 0.80 0.21 0.07 0.06 0.53 1.50 0.08 7.66 0.14 1.20 0.17 12.26 1.97  8.43 0.69 39.01 0.92 25.78 0.13 0.08 0.96 0.99 2.94 0.06 0.08 1.29 3.06 0.21 0.13 0.17 0.63 0.96 0.08 2.64 0.10 2.12 0.06 5.87 1.64  39.18 23.90  47.52 30.60  42.67 36.75  35.00 22.06 4.46  19.30 10.27 4.77  17.17 9.27 4.72  8.59 0.65 30.30 0.70 22.34 0.11 0.29 1.09 2.15 6.05  Si-limited 66 h 92 h  163 h  7.41 0.65 . 18.64 2.86 20.69 0.16 0.49 2.06 2.62 7.70 0.11 0.41 0.26 0.34 0.26  7.36 0.72 29.56 0.88 31.96 0.21 0.23 1.33 2.19 3.90 0.10 0.17 0.34 0.26 0.49 0.14  7.04 0.75 22.11 1.51 26.34 0.24 0.36 1.49 2.26 5.98 0.10 0.30 0.47 0.55 3.34 0.80  0.40 0.60 0.29 4.33 0.11 0.61 0.28 21.70 4.44  0.18 0.14 0.27 2.41 0.23 0.12 12.32 2.14  0.55 0.14 0.39 1.88 0.07 0.19 0.12 11.13 2.45  44.50 32.63  26.54 21.71  37.15 33.40  29.51 30.94  20.81 10.28 6.82  45.66 30.86 3.78  25.50 17.09 1.88  27.05 15.68 2.44  0.99 0.88 2.44 0.07 0.06 2.08 3.71 0.34 0.13 0.23 0.94 1.05 0.10 1.95 0.22 3.62 0.09 5.98 2.19  ,  184  appendix B. Table 6a: The i a t t y acids expressed as a percentage of the t o t a l , i n series 2 Thalassiosira pseudonana cultures and harvested at either Hid-log phase or after 2 h or 6 h of n i t r a t e starvation. T r i p l i c a t e or duplicate measurements vere taken at each phase and reported as mean t 1 S.D. The fatty acids I and N are unknowns. Fatty k i d  A l g a l Sample Hid-log 1 2  14:0 I 16:0 N 16:ln7 16:ln5 17:0 16:2n6 16:2n4 16:3n4 16:4nl 18:lnl3 18:ln9 18:ln7 18:2n6 18:3n4 18:4n3 20:4n6 20:4n3 20:5n3 22:6n3 Saturated Honounsat'd Polyunsat'd Total n3 n6  3  8.13 8.24 6.24 .63 .62 .59 24.42 22.93 30.93 .40 .28 .89 26.02 25.28 21.42 .17 .16 .16 .51 .47 .48 1.50 2.07 1.52 1.96 2.25 1.90 6.00 6.16 6.11 .31 .23 .60 .32 .38 .27 .40 .41 .30 .29 .35 .31 .24 .22 .33 .37 4.25 3.32 4.18 .32 .48 .37 .36 .23 19.00 19.41 18.36 3.32 4.16 3.43  x±s  2 h N-starved 1 2  x+s  6 h N -starved 1 2  x+s  7.54+1.12 7.79 8.08 0.61+0.02 .53 .54 26.10.+4.25 29.20 29.29 0.52+0.32 .88 .53 24.24+2.47 25.87 26.07 0.16.+0.01 .15 .17 0.49+0.02 .36 .34 1.70+0.32 1.89 1.49 2.04+0.19 1.61 1.39 6.09+0.08 5.30 4.23 0.38+0.19 .19 .16 0.32+0.06 .54 .53 0.37+0.06 .76 1.02 0.32+0.03 .34 .34 0.23+0.02 .31 .42 0.35+0.03 .33 .57 3.92+0.52 2.56 3.33 0.40+0.11 .31 .66 0.32+0.08 .27 .33 18.92+0.53 16.24 15.77 3.64+0.46 2.58 2.68  7.94+0.21 7.62 6.84 0.54+0.01 .55 .50 29.25+0.06 27.56 33.76 0.71+0.25 .48 .55 25.97+0:14 24.99 25.40 0.16+0.01 .16 .16 0.35+0.01 .29 .36 1.69+0.28 1.54 1.24 1.50+0.16 1.32 1.21 4.77+0.76 4.14 3.56 0.18+0.02 .26 .21 0.54+0.01 .60 .56 0.89+0.18 1.09 .63 0.34+0.00 .39 .38 0.37+0.08 .59 .31 0.45+0.17 .73 .40 2.95+0.54 3.52 3.55 0.49+0.25 1.00 .42 0.30+0.04 .36 .28 16.01+0.33 17.39 14.33 2.63+0.07 3.18 2.74  7.23+0.55 0.53+0.04 30.66+4.38 0.52+0.05 25.20+0.29 0.16+0.00 0.33+0.05 1.39+0.21 1.27+0.08 3.85+0.41 0.24+0.04 0.58+0.03 0.86+0.33 0.39+0.00 0.45+0.20 0.57+0.23 3.54+0.02 0.71+0.41 0.32+0.06 15.86+2.16 2.96+0.31  34.13+5.39 25.41+2.63  37.54+0.28 27.90+0.34  38.22+4.98 27.19+0.65  37.99+2.53 26.80+1.59 2.33+0.45  31.34+2.70 21.89+0.98 2.55+0.61  31.16+4.13 22.38+2.54 2.55+0.82  185 Appendix B. Table 6b: The f a t t y acids expressed as a percentage of the t o t a l , i n series 2 Thalassiosira pseudonana cultures and harvested at 2 h or 6 h of s i l i c a t e starvation. 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 fatty acids I and N are unknowns. Patty Acid 2 h Si-starved 1 2  3  14:0 I 16:0 M 16:ln7 16:ln5 17:0 16:2n6  7.34 .66 26.61 1.14 26.73 .17 .57 1.16  7.82 .64 24.13 .88 25.77 .19 .54 1.57  8.19 .63 27.41 .73 26.10 .14 .52 1.26  16:3n4 16:3n3 16:4nl 18:0 18:lnl3 18:ln9 18:ln7 18:2n6 18:3n4 18:3nl 18:4n3 20:4n6 20:4n3 20:5n3 22:6n3  4.74 .13 .44 .08 .33 .36 .38 .28  5.29 .09 .28 .09 .34 .33 .32 .29 .25 .19 4.28 .22 .24 18.28 3.56  4.77 .12 .30 .08 .51 .40 .31 .31 .22 .15 4.05 .20 .25 17.10 3.15  UiM  Saturated Honounsat'd Polyunsat'd Total n3 n6 1  hU  4.46 .25 17.76 3.41  1,11  xts  Algal Saiple 6 h Si-starved 1 2 7.78i0.43 0.64*0.02 27.05i2.76 0.92*0.21 26.20i0.49 0.17+0.03 0.54*0.03 1.33i0.21 2,05*0,11 4.93+0.31 0.11+0.02 0.34+0.09 0.08+0.01 0.39*0.10 0.36+0.04 0.34*0.04 0.29*0.02 0.24+0.02 0.17+0.03 4.26*0.21 0.21+0.01 0.25*0.01 17.7h0.59 3.37+0.21  8.37 .62 24.95 .94 28.69 .19 .46 1.41 2.18 4.23 .11 .17 .07 .34 .40 .27 .27 .31 .16 3.57 .34 .24 16.60 3.06  3  4  6.84 .71 27.78 .52 27.31 .16 .45 1.03 1.99 4.61 .12 .58 .07 .30 .32 .28 .30 .20 .14 4.96 .16 .21 16.11 3.22  8.59 .61 27.15 .69 27.14 .18 .41 1.20 1.96 3.83 .14 .14 .07 .32 .36 .21 .29 .29 .13 3.82 .32 .22 14.58 2.31  Xts  8.08 .64 26.43 .45 29.05 .17 .48 1.10 2.15 4.11 .08 .25 .07 .32 .37 .23 .31 .28 4.10 .31 .27 16.49 2.95  7.97*0.78 0.65*0.05 26.58*1.22 0.65*0.22 28.05i0.96 0.18*0.01 0.45*0.03 1.19*0.17 2.07*0.11 4.20*0.32 0.11*0.03 0.29*0.20 . 0.07*0.01 0.32+0.02 0.36+0.03 0.25+0.03 0.29*0.02 0.27+0.05 0.14+0.02 4.1h0.61 0.28*0.08 0.24+0.03 15.95i0.93 2.89.+0.40  35.45*3.23 28.46*0.70  35.07i2.04 29.16il.05  31.89*1.91 22.33*1.11 1.83*0.24  32.16t2.99 23.43*2.02 1.76*0.27  

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