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Physiological and cytological effects of sodium fluoride additions to cultures of euryhaline phytoplankters… Klut, Maria Emilia de Andrade Alves de Sá 1983

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PHYSIOLOGICAL AND CYTOLOGICAL EFFECTS OF SODIUM FLUORIDE ADDITIONS TO CULTURES OF EURYHALINE PHYTOPLANKTERS WITH EMPHASIS ON A SENSITIVE DINOFLAGELLATE by MARIA EMILIA DE ANDRADE ALVES DE SA KLUT Licenciatura, University of Porto-Portugal, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE DEPARTMENT OF BOTANY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1983 (c)MARIA EMILIA DE ANDRADE ALVES DE SA KLUT In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT The e f f e c t s o f / 5 0 - 2 0 0 mg/L f l u o r i d e (F) a d d i t i o n s on t h e g r o w t h o f f i v e a x e n i c p h y t o p l a n k t e r s p e c i e s i n n u t r i e n t -e n r i c h e d h a l f - s a l i n i t y s e a w a t e r (14-15%o ) were examined. The c h l o r o p h y t e D u n a l i e i l a t e r t i o l e c t a and t h e d i a t o m T h a l a s s i o s i r a w e i s s f l o q i i were v i r t u a l l y u n a f f e c t e d i n b o t h g r o w t h r a t e and maximum g r o w t h d e n s i t y , whereas a n o t h e r d i a t o m C h a e t o c e r o s g r a c i l i s a p p e a r e d t o be g r o w t h -s t i m u l a t e d by a l l t h e f l u o r i d e c o n c e n t r a t i o n s t e s t e d . The g r o w t h o f t h e p r y m n e s i o p h y t e P a v l o v a l u t h e r i was 35-50 % i n h i b i t e d , a t 150-200 mg/L F c o n c e n t r a t i o n . However, t h i s i n h i b i t i o n was p a r t i a l l y overcome upon r e p e a t e d t r a n s f e r s a t t h e s e f l u o r i d e l e v e l s . T he d i n o f l a g e l l a t e A m p h i d i n i u m  c a r t e r a e was t h e most s e n s i t i v e t o t h e h i g h e s t f l u o r i d e c o n c e n t r a t i o n , w i t h g r o w t h i n h i b i t e d by 20-25% a t 150 mg/L and by more t h a n 90% a t 200 mg/L F. When t h i s s p e c i e s was e x p o s e d t o g r a d u a l i n c r e a s e s i n F c o n c e n t r a t i o n , o r was a l l o w e d t o a d a p t t o t h e h i g h e s t F l e v e l o v e r an e x t e n d e d p e r i o d o f t i m e , n o r m a l g r o w t h was resumed. I n t e r e s t i n g l y , t h e F - r e s i s t a n t s t r a i n o f A m p h i d i n i u m d i d n o t r e q u i r e a d d i t i o n a l f l u o r i d e f o r g r o w t h . However, d u r i n g a d a p t a t i o n t h i s d i n o f l a g e l l a t e was a p p a r e n t l y u n d e r g o i n g major m e t a b o l i c c h a n g e s w h i c h were a c c o m p a n i e d by b i o c h e m i c a l , p h y s i o l o g i c a l and u l t r a s t r u c t u r a l a l t e r a t i o n s . A l t h o u g h c h l o r o p h y l l b i o s y n t h e s i s o f A m p h i d i n i u m seemed t o be a r r e s t e d a t t h e t i m e o f F - i n h i b i t i o n , n o r m a l ii b i o s y n t h e t i c r a t e s were resumed a f t e r F - a d a p t a t i o n , w h i l e t h e t o t a l c a r o t e n o i d c o n t e n t i n c r e a s e d . C o n c u r r e n t w i t h t h e d e v e l o p m e n t o f F - r e s i s t a n c e , t h e r e was an enhancement i n d a r k r e s p i r a t i o n and p o s s i b l y i n p h o t o r e s p i r a t i o n . A l t h o u g h t h e n o r m a l p h o t o s y n t h e t i c r a t e o f t h i s d i n o f l a g e l l a t e was r e s t o r e d d u r i n g t h e a d a p t a t i o n p e r i o d , i t a p p e a r s t h a t t h i s r e c o v e r y was i m p a i r e d by a r e d u c t i o n i n t h e p h o t o s y n t h e t i c e f f i c i e n c y due t o i n c r e a s e d p h o t o r e s p i r a t i o n . U l t r a s t r u c t u r a l s t u d i e s o f F - a d a p t e d A m p h i d i n i u m r e v e a l e d a b n o r m a l f e a t u r e s i n t h e c h l o r o p l a s t ( e s p e c i a l l y i n t h e p y r e n o i d ) , m i t o c h o n d r i a , and t h e n u c l e u s . T h y l a k o i d f o r m a t i o n was g r e a t l y a f f e c t e d by f l u o r i d e , l e a d i n g t o t h e a p p e a r a n c e o f a p r o l a m e l l a r - l i k e c o n f i g u r a t i o n i n t h e p y r e n o i d m a t r i x . T h i s u n e x p e c t e d a p p e a r a n c e o f p r o l a m e l l a r -l i k e s t r u c t u r e s i n t h e F - a d a p t e d c e l l s s u g g e s t s t h a t t h e p y r e n o i d may be a c e n t e r f o r t h y l a k o i d a s s e m b l y . T h e s e c e l l s a l s o showed l a r g e o s m i o p h i l i c i n c l u s i o n s i n t h e m i t o c h o n d r i a . L a r g e m i c r o b o d i e s were f o u n d i n c l o s e a s s o c i a t i o n w i t h t h e m i t o c h o n d r i a and c h l o r o p l a s t , s u g g e s t i n g an i n c r e a s e d m e t a b o l i c dependence on p h o t o r e s p i r a t i o n . The n u c l e i o f F - a d a p t e d c e l l s showed d a r k and l i g h t c o n c e n t r i c r i n g s i n t h e n u c l e o l a r r e g i o n , a c c o m p a n i e d by s i g n s o f m i t o t i c a c t i v i t y , w h i c h were n o t o b s e r v e d i n t h e F - i n h i b i t e d c e l l s . I t i s i n f e r r e d t h a t F-a d a p t a t i o n may have e n t a i l e d some f o r m o f g e n e t i c change, w h i c h a p p e a r s t o be permanent and e x p r e s s e d as a c h a r a c t e r i s t i c p h e n o t y p i c m utant. i i i i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v i LIST OF TABLES i x ACKNOWLEDGEMENTS X INTRODUCTION 1 MATERIAL AND METHODS 5 PHYTOPLANKTON SPECIES 5 CULTURE CONDITIONS AND GROWTH MEASUREMENTS 5 CHLOROPLAST PIGMENT DETERMINATIONS 6 PHOTOSYNTHETIC AND RESPIRATORY MEASUREMENTS 7 ELECTRON MICROSCOPY 8 TABLE I 9 RESULTS 10 PART 1. GROWTH MEASUREMENTS 10 PART 2. PIGMENT CONTENT, PHOTOSYNTHETIC AND RESPIRATORY RATES 13 CHLOROPLAST PIGMENTS 13 PHOTOSYNTHETIC AND RESPIRATORY RATES 14 PART 3. ELECTRON MICROSCOPY 14 TABLE II 20 TABLE I I I 21 ^ABLE IV 22 TABLE V 23 i v V DISCUSSION 24 PART 1. FLUORIDE EFFECT ON PHYTOPLANKTER GROWTH .. 24 PART 2. PIGMENT CONTENT, PHOTOSYNTHESIS AND RESPIRATION OF THE WILD-TYPE AND F-RESISTANT STRAINS OF A. CARTERAE 28 CHLOROPLAST PIGMENTS 28 PHOTOSYNTHESIS 29 RESPIRATION 3 0 PART 3. ULTRASTRUCTURE OF F-TREATED A. CARTERAE .. 33 CONCLUSION 42 KEY FOR FIGURES 43 FIGURES . 44 LITERATURE CITED 56 V v i LIST OF FIGURES FIGURE 1 Growth curves of D u n a l i e l l a t e r t i o l e c t a on f i r s t exposure to d i f f e r e n t F - c o n c e n t r a t i o n s 4 4 2 Growth curves of T h a l a s s i o s i r a w e i s s f l o g i i and Chaetoceros g r a c i l i s on f i r s t exposure to d i f f e r e n t F - c o n c e n t r a t i o n s 4 4 3 Growth curves of Pavlova l u t h e r i from s i n g l e and repeated F-treatments 4 5 4 Growth curves of Amphidinium c a r t e r a e from s i n g l e and repeated F-treatments 4 5 5 Growth curves of A. c a r t e r a e from F - a d a p t a t i o n s e r i e s 46 6 E l e c t r o n micrograph of F-unexposed A. c a r t e r a e . . 47 7 E l e c t r o n micrograph of a d i v i d i n g c e l l . 47 8 Pyrenoid and p o l y s a c c h a r i d e cap 47 9 L i p i d - l i k e i n c l u s i o n and the p o l y s a c c h a r i d e cap.. 47 1 0 E l e c t r o n micrograph of F - i n h i b i t e d A . c a r t e r a e . . 4 8 1 1 C h l o r o p l a s t morphology 4 8 1 2 C l o r o p l a s t with dense o s m i o p h i l i c m a t e r i a l 4 8 1 3 C l o r o p l a s t with v e s i c u l a t e d s t r u c t u r e s 4 8 1 4 Autophagic v a c u o l e s 4 9 1 5 Pyrenoid showing d i s j o i n t e d t h y l a k o i d bands 4 9 v i i 16 M i t o c h o n d r i a c o n t a i n i g rudimentary e l e c t r o n dense i n c l u s i o n s . 49 17 Microbody - endoplasmic r e t i c u l u m a s s o c i a t i o n . .. 49 18 E l e c t r o n micrograph of F-adapted A. c a r t e r a e . ... 50 19 Axoneme of a f l a g e l l u m 50 20 Pyrenoid morphology L 50 21 Pyrenoid w i t h d i s j o i n t e d t h y l a k o i d s extending t o the c h l o r o p l a s t . 51 22 Pyrenoid with a p r o l a m e l l a r - l i k e membrane system 51 23 Interconnected membranous t u b u l e s i n the pyren o i d 51 24 C r y s t a l l i n e l a t t i c e of the p r o l a m e l l a r - l i k e body 51 25 M i t o c h o n d r i a morphology 52 26 M i t o c h o n d r i a with dense i n c l u s i o n 52 27 M i t o c h o n d r i a with dense i n c l u s i o n s 52 28 M i t o c h o n d r i a with dense i n c l u s i o n 52 2 9 Microbody morphology 53 30 Microbody - mitochondria - c h l o r o p l a s t a s s o c i a t i o n 53 31 Nucleolus 53 32 Autophagic v a c u o l e s and c h l o r o p l a s t degeneration 53 33 E l e c t r o n micrograph of F - r e s i s t a n t A. c a r t e r a e . . 54 v i i v i i i 34 Prolamellar-like bodies in the pyrenoid matrix. . 54 35 Nucleus with a t y p i c a l nucleolus 54 36 Microbody - endoplasmic reticulum association. .. 54 37 Schematic drawing of F-adapted A. carterae 55 viii i x L I S T OF TABLES TABLE I . A l g a l s t r a i n s u s e d i n t h i s s t u d y 9 TABLE I I . Summary o f f l u o r i d e c o n c e n t r a t i o n e f f e c t s on p h y t o p l a n k t e r g r o w t h p a r a m e t e r s f r o m f i r s t e x p o s u r e t o e a c h F l e v e l 20 TABLE I I I . A d a p t a t i v e g r o w t h r e s p o n s e o f two p h y t o p l a n k t e r s on r e p e a t e d e x p o s u r e t o f l u o r i d e a f t e r s u f f e r i n g p a r t i a l i n h i b i t i o n TABLE IV. C h l o r o p l a s t p i g m e n t s o f w i l d - t y p e and F-r e s i s t a n t s t r a i n s o f A m p h i d i n i u m c a r t e r a e ... 22 TABLE V. P h o t o s y n t h e t i c and r e s p i r a t o r y r a t e s o f w i l d -t y p e and F - r e s i s t a n t s t r a i n s o f A m p h i d i n i u m c a r t e r a e m easured f r o m 0 2 c h a n g e s r e g i s t e r e d f r o m f i r s t e x p o s u r e t o h i g h F l e v e l s 21 by an oxygen e l e c t r o d e 23 ix X ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my s i n c e r e g r a t i t u d e t o Dr. T. B i s a l p u t r a f o r h i s encoura g e m e n t and a d v i c e w h i c h have made t h i s s t u d y a v e r y r e w a r d i n g one. G r a t i t u d e i s a l s o e x t e n d e d t o Dr. N. J . A n t i a f o r h i s s u g g e s t i o n s and c o n s t r u c t i v e c r i t i c i s m d u r i n g t h e p r e p a r a t i o n o f t h i s t h e s i s . I am i n d e b t e d t o D r . L. O l i v e i r a , D r. P. J . H a r r i s o n , D r . D. G a r b a r y and Dr. K. C o l e and f o r t h e i r h e l p i n c l a r i f y i n g many i d e a s e x p r e s s e d i n t h i s t h e s i s . I am a l s o g r a t e f u l t o t h e B o t a n y D e p a r t m e n t a t U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r t h e i r s u p p o r t i v e r o l e i n my e n d e a v o u r s . An e x t e n d e d l e a v e o f a b s e n c e from t h e I . C. B i o m e d i c a s , U n i v e r s i t y o f P o r t o and a s c h o l a r s h i p f r o m t h e I n s t i t u t o N a c i o n a l de I n v e s t i g a c a o C i e n t i f i c a , L i s b o a - P o r t u g a l , a r e a l s o g r a t e f u l l y a c k n o w l e d g e d . x INTRODUCTION The element f l u o r i n e , in the form of fluoride ion, i s a normal minor constituent of seawater, ocurring at salinity-dependent concentration of approximately 1-2 mg/L F (Warner 1971, Sen Gupta et a l . 1978). However, in certain freshwater regimes, concentrations as high as 1,627 mg/L F have been reported (Kilham and Hecky 1973). According to M i l l e r and Kester (1976), the ionic speciation of endogenous fluoride in seawater of s a l i n i t y 35%« , at 25°C, consists of 50% F~, 47% MgF+, 2.1% CaF + and 1.1% NaF°. The b i o l o g i c a l role of seawater fluoride i s v i r t u a l l y unknown, apart from the p o s s i b i l i t y of i t serving as a growth stimulatory factor or a micronutrient for certain species bf phytoplankton (Antia 1980). Aside from i t s natural occurrence, anthropogenic fluoride p o l l u t i o n from i n d u s t r i a l effluents and emissions has been documented (Leblanc et a l . 1971, 1972, Marier and Rose 1971, Harbo et a l . 1974, Roberts et a l . 1979, Thompson et a l . 1979, Hocking et a l . 1980, Pankhurst et a l . 1980, Barbaro et a l . 1981, Pandey 1981). Fluorination of water supply in certain areas, and additional domestic p o l l u t i o n from the current widespread use of f l u o r i d i z e d toothpaste i s also expected v i a urban sewage. The intensity of such p o l l u t i o n may be p a r t i c u l a r l y high in estuarine areas where i n d u s t r i a l and urban communities tend to congregate (Moore 1971). Fluoride at elevated concentration i s known to be 1 highly toxic to many t e r r e s t r i a l animals, plants and microorganisms (Eagers 1969, Diouris and Penot 1977, Yost and VanDemark 1978, Roberts et a l . 1979, Pankhurst et a l . 1980, Conover and Poole 1982). In addition, fluorinated organic compounds such as fluoroacetate and f l u o r o c i t r a t e are known to accumulate in several plants naturally exposed to inorganic f l u o r i d e (Peters and Shorthouse 1972). Although fluoroacetate i s not toxic to the plant i t s e l f , i t can be converted into f l u o r o c i t r a t e which i s toxic and may constitute a potent hazard to members of the food chain (Marier and Rose 1971). In F-resistant plants, fluoroacetate i s synthesized within the c e l l (Vickery and Vickery 1975) and apparently interferes with the Krebs cycle intermediates (Marier and Rose 1971). A number of selected f l u o r i d e - r e s i s t a n t strains have been reported for b a c t e r i a l and mammalian c e l l s (Wiggert and Werkman 1939, Carlson and Suttie 1967, Williams 1967, 1968, Hamilton 1969, Quissel and Suttie 1972). It has been shown that these strains have the a b i l i t y to adapt to apparently i n h i b i t o r y f l u o r i d e concentrations. However t h i s resistance (believed to be phenotypic) disappeared when the c e l l s were cultured in medium without added flu o r i d e (Williams 1967). On the other hand, the development of permanent or genotypic resistance has also been reported (Hamilton 1969, Quissel and Suttie 1972, Bunick and Kashket 1981). Apparently t h i s resistance i s regulated by a single gene. Furthermore, spontaneous f l u o r i d e - r e s i s t a n t mutants 2 a r e known t o o c c u r a t v e r y low f r e q u e n c y ( c a . 2 p e r m i l l i o n c e l l s ) and d i f f e r f r o m t h e w i l d - t y p e i n t h e i r e n z y m a t i c c o n t e n t ( K e l l y 1 9 6 8 ) . I n o r d e r t o e x p l a i n t h e mechanism o f f l u o r i d e r e s i s t a n c e , s e v e r a l h y p o t h e s i s have been p o s t u l a t e d w i t h t h e most common v i e w b a s e d on t h e i n d u c t i o n o f c h a n g e s i n membrane p e r m e a b i l i t y ( W i l l i a m s 1968, Q u i s s e l and S u t t i e 1972, H a m i l t o n 1 9 7 7 ) . An i n v e s t i g a t i o n w i t h a d o z e n s p e c i e s o f m a r i n e p h y t o p l a n k t o n , t e s t e d w i t h 10-100 mg/L F added t o n u t r i e n t -e n r i c h e d s e a w a t e r o f 26% s a l i n i t y , f o u n d o n l y t h r e e s p e c i e s s howing m i n o r growth i n h i b i t i o n a t 100 mg/L F, and s u g g e s t e d t h a t t h e s u r p r i s i n g l a c k o f t o x i c i t y f r o m s u c h h i g h f l u o r i d e c o n c e n t r a t i o n s may be due t o t h e f o r m a t i o n o f i n n o c u o u s c o m p l e x e s w i t h some s a l t c omponent(s) o f s e a w a t e r ( O l i v e i r a .et a l . 1 9 7 8 ) . I f t h i s h y p o t h e s i s i s t r u e , t h e n t h e r e d u c t i o n o f s a l i n i t y would be e x p e c t e d t o d i m i n i s h s u c h complex f o r m a t i o n and t h e r e b y e x p o s e t h e t r u e t o x i c p o t e n t i a l o f f l u o r i d e i n s e a w a t e r . F u r t h e r m o r e , s u c h s a l i n i t y r e d u c t i o n a c t u a l l y o c c u r s i n e s t u a r i n e a r e a s f r o m t h e m i x i n g o f c o a s t a l s e a w a t e r w i t h r i v e r - w a t e r , and t h e r e f o r e , by i m p l i c a t i o n , c o m p a r a b l e f l u o r i d e c o n c e n t r a t i o n s c o u l d be more d e t r i m e n t a l t o p h y t o p l a n k t o n i n e s t u a r i n e l o c a t i o n s t h a n i n o t h e r c o a s t a l s i t u a t i o n s . One o f t h e o b j e c t i v e s o f t h e p r e s e n t s t u d y was t o examine t h e e f f e c t s o f e l e v a t e d f l u o r i d e c o n c e n t r a t i o n s on f i v e e u r y h a l i n e s p e c i e s o f p h y t o p l a n k t o n i n n u t r i e n t - e n r i c h e d 3 seawater of reduced s a l i n i t y (14-15%o ) . Another o b j e c t i v e was t o examine t h e u n d e r l y i n g causes of any growth i n h i b i t i o n t h a t might be o b s e r v e d . The l i k e l i h o o d of growth a d a p t a t i o n a f t e r i n i t i a l i n h i b i t i o n , f o l l o w e d by a d a p t a t i o n t o s u c c e s s i v e exposure of t h e a l g a t o i n c r e a s i n g c o n c e n t r a t i o n l e v e l s as s uggested by S t o c k n e r and A n t i a (1976) was a l s o examined. As p a r t of t h i s o b j e c t i v e , i t was c o n s i d e r e d i m p o r t a n t t o i n v e s t i g a t e b o t h t h e p h y s i o l o g y and c y t o l o g y o f a t l e a s t one a l g a l s p e c i e s t h a t showed growth-i n h i b i t i o n and subsequent development of r e s i s t a n c e t o t h e added f l u o r i d e . 4 MATERIALS AND METHODS PHYTOPLANKTON SPECIES A x e n i c c u l t u r e s o f f i v e e u r y h a l i n e s t r a i n s l i s t e d i n T a b l e I were c h o s e n from f o u r c l a s s e s o f a l g a e . CULTURE CONDITIONS AND GROWTH MEASUREMENTS F o r t h e g r o w t h s t u d i e s , a l l s t r a i n s , s e r v i n g as i n o c u l a f o r t h e f l u o r i d e - e f f e c t t e s t s , were p r e v i o u s l y a d a p t e d t o grow on t h e n u t r i e n t - e n r i c h e d s e a w a t e r medium o f A n t i a and Cheng (1975) w i t h t h e s a l i n i t y o f t h e s e a w a t e r component h a l v e d by d i l u t i o n w i t h g l a s s - d i s t i l l e d w a t e r ; t h e f i n a l s a l i n i t y was 15 ( +0.5)%. . The endogenous F c o n t e n t o f t h i s medium was e s t i m a t e d a t a b o u t 0.53 mg/L. The same medium was u s e d f o r t h e g r o w t h t e s t s w i t h NaF a d d i t i o n s o f 0, 50, 100, 150, 200 mg/L F. S i n c e a c o n c e n t r a t i o n o f NaF c a l c u l a t e d t o be 250 mg/L F, p r o d u c e d a g r a d u a l p r e c i p i t a t e ( o f MgF 2 + C a F 2 ) on l o n g s t a n d i n g , t h e l e v e l o f 200 mg/L F c a n be r e g a r d e d as e q u i v a l e n t t o t h e s o l u b i l i t y s a t u r a t i o n o f NaF i n s e a w a t e r a t t h e s a l i n i t y u s e d . I t i s p o i n t e d o u t t h a t t h i s s o l u b i l i t y l i m i t i s t w i c e t h e m a g n i t u d e o f t h a t p r e v i o u s l y o b s e r v e d i n s e a w a t e r o f 26%» s a l i n i t y ( O l i v e i r a .gt a l . 1978) . The g r o w t h t e s t s were c o n d u c t e d w i t h 4 ml a l i q u o t s o f t h e 0-200 mg/L F - c o n t a i n i n g medium i n c u l t u r e t u b e s , 5 i n o c u l a t e d w i t h 0.2 ml a l i q u o t s o f an a p p r o p r i a t e a l g a l s t o c k c u l t u r e , and i n c u b a t e d s t a t i o n a r y ( a t 1 6 - 2 0 ° C) i n c o n t i n u o u s c o o l - w h i t e l i g h t ( o f i r r a d i a n c e c a . 65 uE irT^s""-1-The g r o w t h was p e r i o d i c a l l y m o n i t o r e d by s p e c t r o p h o t o m e t r i c measurement o f e a c h c u l t u r e ' s o p t i c a l d e n s i t y a t 600 nm a f t e r v o r t e x - m i x i n g . A x e n i c c u l t u r e s and a s e p t i c t e c h n i q u e s were us e d t h r o u g h o u t t h e s e s t u d i e s . CHLOROPLAST PIGMENT DETERMINATIONS Under dim l i g h t c o n d i t i o n s , 3 ml a l i q u o t s o f t h e d i n o f l a g e l l a t e c u l t u r e , grown w i t h o r w i t h o u t f l u o r i d e , were c o l l e c t e d d u r i n g t h e l o g a r i t h m i c p h a s e o f g r o w t h (13 d a y s a f t e r b a t c h c u l t u r e i n i t i a t i o n ) a nd were f i l t e r e d o n t o M i l l i p o r e d i s c s (HA, 0.45 jum p o r e s i z e ) i n t h e p r e s e n c e o f two d r o p s o f an aqueous s u s p e n s i o n o f MgCO^ t o p r e v e n t p h a e o p h y t i n f o r m a t i o n . A t t h e same t i m e , a p p r o p r i a t e c u l t u r e a l i q u o t s were w i t h d r a w n f o r measurements o f c e l l c o n c e n t r a t i o n u s i n g t h e C o u l t e r C o u n t e r TA I I . Each f i l t e r d i s c was t r a n s f e r r e d t o a t e s t t u b e t o w h i c h 5 ml o f 90% a c e t o n e were added. The p i g m e n t e x t r a c t s were s t o r e d o v e r n i g h t i n d a r k n e s s a t 0-4°C. T h e s e e x t r a c t s were t h e n c e n t r i f u g e d and t h e s u p e r n a t a n t was c o l l e c t e d f o r p i g m e n t c o n t e n t d e t e r m i n a t i o n . A f t e r m e a s u r i n g t h e l i g h t a b s o r b a n c e a t s p e c i f i c w a v e l e n g t h s i n c u v e t s (1 cm l i g h t p ath) w i t h a PYE Unicam SP8-100 s p e c t r o p h o t o m e t e r , t h e c o n c e n t r a t i o n s o f c h l o r o p h y l l s a and c 2 i n t h e e x t r a c t s were c a l c u l a t e d f r o m 6 the improved equations formulated by Humphrey (1979) for 90% aqueous acetone extracts and din o f l a g e l l a t e s containing chlorophyll c 2 o n i y (Jeffrey et a l . 1975), [Chlorophyll a] = 11.43 E g 6 4 _ 0.40 E 6 3 0 [Chlorophyll c 2]= 24.88 E 6 3 0 - 3.80 E 6 6 4 where chlorophyll concentrations are expressed in jag/ml acetone extract and E represents the absorbance at the wavelengths shown. At the same time, the approximate carotenoid content of each extract was calculated from the Parsons-Strickland equation (Strickland and Parsons 1972), [Carotenoid] = 10 E 4 8 Q where the carotenoid concentration i s expressed in a r b i t r a r i l y s p e c i f i c pigment units (;i SPU/ml extract) . A l l calculated pigment concentrations in extracts were correlated to the culture volumes used and th e i r c e l l density in order to obtain pigment concentrations per 10^ c e l l s . PHOTOSYNTHETIC AND RESPIRATORY MEASUREMENTS Photosynthetic and respiratory rates of di n o f l a g e l l a t e cultures of known c e l l density (ca. 6-8 x 10^ cells / m l were measured by standard methods (Jassby 1978a,b) using an 7 oxygen-electrode (Hansatech, D. W.) at 18°C and the photosynthetic irradiance a r b i t r a r i l y fixed at 350 pE m"~2s~l The source of illumination was provided by the incandescent lamp of a s l i d e projector. ELECTRON MICROSCOPY For u l t r a s t r u c t u r a l studies, appropriate culture samples of the d i n o f l a g e l l a t e , in various phases of growth and flu o r i d e treatments, were concentrated by the f i l t r a t i o n technique described by Bisalputra et a l . (1973), and fixed for 1.5 h at room temperature with 2% (v/v) glutaraldehyde in saline phosphate (0.17 M) buffer (pH 7.4). The material was then post-fixed for 1 h with 1% (v/v) OsO^ i n the same buffer. Thorough rinsing was carried out, after each f i x a t i o n , using the same buffer. This was followed by dehydrating in a graded methanol series and f i n a l l y embedding in Epon 812 (Luft 1961). Ultra-thin sections were cut on a Reichert OMU-3 Ultramicrotome and examined with a Zeiss EM 9S Electron microscope after staining with uranyl acetate and lead, c i t r a t e (Reynolds 1963). 8 TABLE I. A l g a l s t r a i n s used i n t h i s study Alga Cu l t u r e S t r a i n / c l o n e I s o l a t o r Local of c o l l e c t i o n * d e s i g n a t i o n i s o l a t i o n CHLOROPHYCEAE D u n a l i e l l a t e r t i o l e c t a Butcher Woods Hole Dun PRYMNESIOPHYCEAE Pavlova (Monochrysis) l u t h e r i (Droop) Green BACILLARIOPHYCEAE Chaetoceros g r a c i l i s Schutt T/halassjosira ( f l u v i a t i l i s ) w e i s s f l o g i i (Grunow) P r y x e l l & Hasle M i l l p o r t U.W.D.O.S. 60 TO-5 8-2 U.W.D.O.S. A c t i n M.Droop Cumbrae, Scotland W.Thomas Gulf of Tehuantepec R . G u i l l a r d Long I s l a n d Sound, N .Y . DINOPHYCEAE AmphidiniuTn c a r t e r a e Hulburt Woods Hole Arnphi 1 R . G u i l l a r d Falmouth Great Pond, Mass * Obtained by Dr.N.J. A n t i a from: Woods Hole r Oceanographic I n s t i t u t i o n at Woods Hole, Mass, M j J l p o r t , Marine S t a t i o n at M i l l p o r t , Scotland; U.U.D.Q.S. U n i v e r s i t y of Washington, Department of Oceanography at S e a t t l e , courtesy of Joyce Lewin. 9 RESULTS PART 1. GROWTH MEASUREMENTS Phytoplankter growth response to the f i r s t exposure of fluor i d e at various concentrations, i s summarized in Table II and i l l u s t r a t e d in Figs. 1-4. Dunaliella t e r t i o l e c t a and Thalassiosira w e i s s f l o g i i were barely affected (with 3-11% inhibition) in th e i r growth parameters by the highest F concentration tested, while Chaetoceros g r a c i l i s appeared to be stimulated considerably (averaging 45%) in growth rate by a l l the F concentrations (Table II, F i g . 1, 2). The growth stimulation of Cj. g r a c i l i s was v i s i b l y accompanied by marked f l o c c u l a t i o n of the c e l l s after early log-phase growth, which was not evident (to the naked eye) in the control culture without added F. The prymnesiophyte Pavlova l u t h e r i was s i g n i f i c a n t l y i n h i b i t e d (ca. 20%) in growth rate, but not in maximum density, by f i r s t exposure to 100 mg/L F (Table II, F i g . 3) . The higher F concentrations (150-200 mg/L) further reduced the growth rate to about 50% and also the maximum growth density to about 65% of the control without added f l u o r i d e . When the culture grown to stationary phase on 200 mg/L F was used as inoculum for a series of tests over a range of F concentrations (Table I I I , F i g . 3: adaptation s e r i e s ) , the growth-rate i n h i b i t i o n obtained after the 1 0 f i r s t e x p o s u r e t o 100 mg/L F was overcome d u r i n g s u c c e s s i v e g r o w t h a t t h e same F c o n c e n t r a t i o n . A s i m i l a r p a r t i a l improvement i n g r o w t h r a t e was o b s e r v e d a t 150 mg/L F b u t n o t a t 200 mg/L F. However, i n b o t h c a s e s , t h e maximum g r o w t h d e n s i t y was i m p r o v e d a b o u t 20% by r e p e a t e d g r o w t h i n medium c o n t a i n i n g F (compare c o r r e s p o n d i n g d a t a i n T a b l e s I I and I I I ) . I n t e r e s t i n g l y , t h e r e p e a t e d g r o w t h on 200 mg/L F was c h a r a c t e r i z e d by a l a t e s e c o n d l o g -p h a s e , s u g g e s t i n g t h a t t h e a d a p t a t i o n t o t h i s F c o n c e n t r a t i o n was s t i l l i n p r o g r e s s ( F i g . 3: 200-200 F o f a d a p t a t i o n s e r i e s ) . The g r o w t h o f t h e d i n o f l a g e l l a t e A m p h i d i n i u m c a r t e r a e was i n h i b i t e d l e s s t h a n 15% by F c o n c e n t r a t i o n s up t o 100 mg/L. T h i s i n h i b i t i o n was i n c r e a s e d t o 20-25% a t 150 mg/L F, and s e v e r e l y i n t e n s i f i e d t o 90-95% a t 200 mg/L F ( F i g . 4 ) . M i c r o s c o p i c e x a m i n a t i o n o f t h e c u l t u r e w i t h t h e g r e a t e s t F - i n h i b i t i o n showed t h e p r e s e n c e o f some m o t i l e c e l l s a f t e r 36 d a y s o f e x p o s u r e t o 200 mg/L F. T h i s i n d i c a t e d t h a t t h e f l u o r i d e had i n h i b i t e d a c e r t a i n a s p e c t o f c e l l d i v i s i o n r a t h e r t h a n a c t i n g as a l e t h a l t o x i n . When an i n o c u l u m t a k e n f r o m t h i s s e v e r e l y i n h i b i t e d c u l t u r e was i n c u b a t e d i n f r e s h medium w i t h o u t F, n e a r l y n o r m a l g r o w t h was resumed a f t e r a s h o r t l a g p e r i o d ( F i g . 4: 200-0F) , s u g g e s t i n g t h a t t h e f l u o r i d e - i n d u c e d damage t o c e l l d i v i s i o n was n o t permanent and c o u l d be a l l e v i a t e d upon r e l e a s e f r o m f l u o r i d e s t r e s s . F u r t h e r e x p e r i m e n t s were u n d e r t a k e n t o examine t h e 11 p o s s i b i l i t y of growth adaptation of A., carterae on the highest F concentration tested after successful growth on lower concentrations which produced p a r t i a l i n h i b i t i o n . For this purpose, an inoculum taken from the culture grown on 150 mg/L F was used to i n i t i a t e a new series of tests on 100*200 mg/L F (Table I I I , F i g . 4: adaptation s e r i e s ) . Under these conditions, the repeated growth at a l l F concentrations was markedly superior to the corresponding growth obtained on f i r s t exposure to f l u o r i d e , and considerable c e l l d i v i s i o n was observed on 200 mg/L F after a pre-exponential lag period of about 30 days (Fig. 4) . However, even after 63 days from i n i t i a t i o n of the experiment, t h i s l a t t e r growth amounted to only half of that obtained in the absence of added f l u o r i d e (Table I I I ) . Using the culture that was grown in 200 mg/L F for 63 days as inoculum, a second growth experiment was attempted at the same fluoride concentration. This time the di n o f l a g e l l a t e showed a complete recovery of c e l l d i v i s i o n leading to normal growth without s i g n i f i c a n t pre-exponential lag (see Fig . 4: 150+200 (I) -*200 (II) F) . The adaptation experiment was repeated exposing A. carterae to the highest F concentration without prior exposure to sub-inhibitory F l e v e l s . This adaptation was successfully achieved after a pre-exponential lag period of ca. 51 days (Fig. 5) . Furthermore, the growth of A.. carterae maintained for seven consecutive transfers (involving a four-week period of growth per transfer to 12 fresh medium) in 200 F-medium was tested on the normal medium without added F. Such tests showed that the F-resistant s t r a i n did neither need nor depend upon the added F for v i a b i l i t y and growth (see F i g . 5: 200 (VII) -*0 (I) •* 0(II)••O(III) F) . In addition, i t was interesting to notice that when thi s new s t r a i n on 0 F-medium was subcultured again in 200 F-medium, i t showed l i t t l e change in the growth rate and a markedly reduced pre-exponential lag period (see Fi g . 5: 200 (VII) -f /*0 (III) *200 F) . PART 2. PIGMENT CONTENT, PHOTOSYNTHETIC AND RESPIRATORY RATES CHLOROPLAST PIGMENTS The acetone-extracted photosynthetic pigment mixture from the F-resistant A. carterae showed no detectable difference in i t s v i s i b l e absorption spectrum from that of the wild-type d i n o f l a g e l l a t e . Even quantitatively, the content of chlorophylls a and p e r c e ± i w e c e v i r t u a l l y i d e n t i c a l in both forms of A., carterae (Table IV). However, the t o t a l carotenoid content of the F-resistant s t r a i n appeared to be s i g n i f i c a n t l y greater, though the indi v i d u a l carotenoids were not analysed. 13 PHOTOSYNTHETIC AND RESPIRATORY RATES The net photosynthesis of the two strains of Amphidinium carterae (wild-type and F-resistant) quantified by oxygen evolution, showed i n s i g n i f i c a n t differences, even when measurements were performed just after exposure to darkness or after leaving the c e l l s in darkness for a certain period of time (Table V) . However, the endogenous dark re s p i r a t i o n showed s i g n i f i c a n t differences depending upon whether the measurements were made after continuous exposure to l i g h t or subsequent to a long exposure to darkness. In the former condition, the F-resistant d i n o f l a g e l l a t e showed a respiratory rate 90% greater than the wild-type, while in the l a t t e r condition the respiratory rate of the F-resistant s t r a i n was 30% greater. PART 3. ELECTRON MICROSCOPY In presenting the u l t r a s t r u c t u r a l observations, the following reference w i l l be made to d i f f e r e n t types of Amphidinium c e l l s : "CONTROL" (F-unexposed), wild-type c e l l s in normal stock culture, never exposed to any fluoride concentration over the l e v e l (1-2 mg/L) normally present in seawater (see growth curve for 0 F in F i g . 4); "F-INHIBITED", c e l l s severely inhibited in growth upon 37 days of exposure to 200 mg/L F added to seawater (see 14 c u r v e f o r 200 F i n F i g . 4 ) ; "F-ADAPTED", c e l l s s u c c e s s f u l l y a d a p t e d t o grow on 200 mg/L F and examined a f t e r 42 d a y s i n c u l t u r e ( s e e g r o w t h c u r v e f o r 150-*200 (I) +200 ( I I ) F i n F i g . 4 ) . S i n c e , t h e F-a d a p t e d c e l l s showed l a s t i n g r e s i s t a n c e t o 200 mg/L F, a c u l t u r e o f t h i s v a r i a n t s t r a i n o f d i n o f l a g e l l a t e i s b e i n g m a i n t a i n e d and r e f e r r e d t o as F - r e s i s t a n t A. c a r t e r a e . "CONTROL CELLS". T h e s e c e l l s showed n o r m a l c y t o l o g i c a l f e a t u r e s , i n g e n e r a l agreement w i t h e a r l i e r d e s c r i p t i o n s o f A m p h i d i n i u m c a r t e r a e ( H u l b u r t 1957, G i b b s 1962a,b, Dodge and C r a w f o r d 1968, Dodge 1971, T a y l o r 1971, Dodge 1 9 7 3 ) . P a r t i c u l a r a t t e n t i o n i s drawn i n F i g s . 6-9 t o c e r t a i n i m p o r t a n t f e a t u r e s o f t h e s e c e l l s , w h i c h show p r o n o u n c e d a l t e r a t i o n s i n t h e F - t r e a t e d c e l l s . The h i g h l y l o b e d c h l o r o p l a s t ( F i g . 6) c o n t a i n e d a p y r e n o i d t r a v e r s e d by a number o f t h y l a k o i d bands ( F i g . 8 ) . The c y t o p l a s m c o n t a i n e d a n abundance o f s t a r c h g r a i n s and l i p i d - l i k e i n c l u s i o n s ( F i g s . 6, 9 ) . I n t h e d i v i d i n g c e l l s , t h e t u n n e l l i n g o f m i c r o t u b u l e s r e l a t e d t o chromosome movement ( F i g . 7) was commonly o b s e r v e d as p r e v i o u s l y d e s c r i b e d f o r d i n o f l a g e l l a t e m i t o s i s (Dodge 1973, K u b a i and R i s 1 9 6 9 ) . "F-INHIBITED CELLS". Under t h e l i g h t m i c r o s c o p e , t h e m a j o r i t y o f t h e s e c e l l s a p p e a r e d t o be h y a l i n e , r o u n d e r i n s h a p e , and i m m o b i l i z e d f r o m l o s s o f f l a g e l l a . A p p r o x i m a t e l y 10% o f t h e c e l l s i n t h i s c u l t u r e r e m a i n e d m o t i l e , p i g m e n t e d and t h e y d i f f e r e d f r o m t h e c o n t r o l c e l l s i n t h e f o l l o w i n g u l t r a s t r u c t u r a l a s p e c t s . The s t r o m a o f t h e c h l o r o p l a s t 15 a p p e a r e d more e l e c t r o n dense ( F i g s . 1 0 - 1 3 ) , and a c c u m u l a t i o n o f o s m i o p h i l i c r e g i o n s , w i t h or w i t h o u t v e s i c l e s , was o b s e r v e d n e a r t h e c h l o r o p l a s t p e r i p h e r y ( F i g s . 12, 13) . D i l a t i o n o f t h e t h y l a k o i d s w i t h a g r e a t e r d e g r e e o f s t a c k i n g was f r e q u e n t l y s e e n ( F i g s . 1 1 - 1 3 ) . I n t h e p y r e n o i d , t h e i n d i v i d u a l t h y l a k o i d bands a p p e a r e d t o become i n c r e a s i n g l y d i s j o i n t e d , b u t t h e p o l y s a c c h a r i d e cap r e m a i n e d i n t a c t ( F i g . 15) . C y t o p l a s m i c s t a r c h and l i p i d - l i k e i n c l u s i o n s d e c r e a s e d m a r k e d l y i n t h e F - i n h i b i t e d c e l l s ( F i g . 10) , r e l a t i v e t o t h e c o n t r o l c e l l s , w h i l e a u t o p h a g i c v a c u o l e s c o n t a i n i n g l a r g e amounts o f d e n s e , s m a l l v e s i c l e s became a p r o m i n e n t f e a t u r e ( F i g s . 14, 16) . I n g e n e r a l , m i t o c h o n d r i a a p p e a r e d w e l l d e v e l o p e d , a l t h o u g h a r u d i m e n t a r y e l e c t r o n d ense i n c l u s i o n was o c c a s i o n a l l y n o t i c e d ( F i g . 1 6 ) . E l l i p s o i d m i c r o b o d i e s r a n g i n g i n s i z e f r o m 0.21-0.47 x 0.16-0.26 /am were o b s e r v e d i n t h e v i c i n i t y o f t h e ro u g h e n d o p l a s m i c r e t i c u l u m ( F i g . 1 7 ) . F u r t h e r m o r e , t h e e n d o p l a s m i c r e t i c u l u m and G o l g i s y s t e m s a p p e a r e d t o be s c a r c e l y a f f e c t e d by t h e F - i n h i b i t i o n ( F i g . 10) . No c y t o p l a s m i c t u n n e l s t h r o u g h t h e n u c l e u s were s e e n i n any o f t h e F - i n h i b i t e d c e l l s e c t i o n s , s u g g e s t i n g t h e a b s e n c e o f m i t o t i c d i v i s i o n s . "F-ADAPTED CELLS". T h e s e c e l l s d i f f e r e d f r o m t h e n o r m a l and F - i n h i b i t e d c e l l s i n s e v e r a l i m p o r t a n t u l t r a s t r u c t u r a l f e a t u r e s . The c h l o r o p l a s t s t r o m a d i s p l a y e d s i m i l a r e l e c t r o n d e n s i t y as t h a t o f c o n t r o l c e l l s , b u t t h e 16 thylakoids showed even greater disorganization than that obtained from F - i n h i b i t i o n . In p a r t i c u l a r , thylakoid d i l a t i o n was prominent near the chloroplast envelope, and p l a s t o g l o b u l i seemed to increase in size and number with the t r a n s i t i o n from F - i n h i b i t i o n to F-adaptation (Fig. 18). While the polysaccharide cap was s t i l l present, the pyrenoid showed remarkable a l t e r a t i o n of internal d e t a i l after F-adaptation. The disjointed bands of thylakoids already seen from F - i n h i b i t i o n persisted in the adapted pyrenoid, but now t h i s disarray was so i n t e n s i f i e d that i t appeared perforated (Figs. 20-24) . Sections through the cap-adjoining central portion of the F-adapted pyrenoid (Figs. 23, 24) revealed these bands as membranes forming part of a prolamellar-like system resembling those known from e t i o p l a s t s of higher plants (Kirk and Tilney-Basset, 1978). Fig. 22 depicts the connection between the prolamellar-like structure and the perforated thylakoid bands. I t should be pointed out that the d i s j o i n t e d thylakoids were r e s t r i c t e d to the pyrenoid and yet appeared to be continuous with the unperforated thylakoids of the adjacent chloroplast lobes (Fig. 20) . Figure 23 represents a section through the prolamellar-like strusture showing a mosaic of interconnected network of membranous tubules. Figure 24 shows a central c r y s t a l l i n e - t y p e l a t t i c e in a prolamellar-like structure, radiating from the perforated bands of two to three apposing thylakoids that project further into the chloroplast lobe i l l u s t r a t e d in F i g . 20. 17 The axonemes o f t h e f l a g e l l a were h a r d l y a f f e c t e d by t h e F - t r e a t m e n t s and a p p e a r e d t o r e m a i n n o r m a l ( F i g . 19) . However, many o f t h e m i t o c h o n d r i a f r o m F - a d a p t a t i o n showed an a b n o r m a l f e a t u r e i n t h e f o r m o f l a r g e dense i n c l u s i o n s ( F i g s . 25-28), w h i l e t h e c r i s t a e r e m a i n e d t u b u l a r and c h a n g e d l i t t l e f r o m t h e i r n o r m a l a p p e a r a n c e . I t was i n t e r e s t i n g t o o b s e r v e t h a t t h e F - a d a p t e d m i c r o b o d i e s became a p p r o x i m a t e l y 4-5 f o l d l a r g e r t h a n n o r m a l ; a l t h o u g h s t i l l e l l i p t i c a l , t h e i r s i z e was o f t h e o r d e r 1.33-1.67 x 1.08 pm. A r e m a r k a b l y c l o s e a s s o c i a t i o n between m i c r o b o d i e s , m i t o c h o n d r i a , and c h l o r o p l a s t s o f F - a d a p t e d c e l l s i s i l l u s t r a t e d i n F i g , 30. As e x p e c t e d , t h e r e c o v e r y o f p h o t o s y n t h e t i c g r o w t h f r o m s u c c e s s f u l a d a p t a t i o n , a f t e r i n i t i a l F - i n h i b i t i o n , was a c c o m p a n i e d by t h e r e a p p e a r a n c e o f c y t o p l a s m i c s t a r c h and l i p i d i n c l u s i o n s . A p r o m i n e n t f e a t u r e o f t h e F - a d a p t e d n u c l e u s was t h e p r e s e n c e o f d a r k and l i g h t c o n c e n t r i c r i n g s e n c i r c l i n g a d a r k s p o t a t t h e c e n t e r o f t h e n u c l e o l u s ( F i g . 31) . The r e c o v e r y o f a p p a r e n t l y n o r m a l m i t o t i c d i v i s i o n f r o m s u c c e s s f u l F - a d a p t a t i o n was c o n f i r m e d by t h e s i g h t i n g o f d i n o f l a g e l l a t e - c h a r a c t e r i s t i c m i c r o t u b u l a r t u n n e l s i n t h e n u c l e i o f d i v i d i n g c e l l s . T h e s e and o t h e r F - i n d u c e d c h a n g e s a r e i l l u s t r a t e d i n F i g . 37. A s i g n i f i c a n t p r o p o r t i o n o f t h e F - a d a p t e d c e l l s showed a c c u m u l a t i o n o f a u t o p h a g i c v a c u o l e s and e x t e n s i v e d e g e n e r a t i o n o f p l a s t i d s ( F i g . 32) , m i t o c h o n d r i a , n u c l e a r and c y t o p l a s m i c membrane s y s t e m s . T h e s e a u t o l y t i c symptoms 18 s u g g e s t e d p r e m a t u r e s e n e s c e n c e o f some c e l l s f r o m t h e g r o w t h under f l u o r i d e s t r e s s . F u r t h e r e x p e r i m e n t s were p e r f o r m e d t o d e t e r m i n e t h e permanence o f t h e ab n o r m a l u l t r a s t r u c t u r a l f e a t u r e s o b s e r v e d i n t h e F - a d a p t e d c e l l s , now m a i n t a i n e d i n c u l t u r e as a F - r e s i s t a n t s t r a i n o f A., c a r t e r a e . D u r i n g and a f t e r r e p e a t e d g r o w t h o f t h i s s t r a i n on 200 mg/L f f o l l o w e d by f u r t h e r r e p e a t e d g r o w t h on c u l t u r e medium w i t h o u t added f l u o r i d e , a l l c u l t u r e s a m p l e s t e s t e d showed t h e same major u l t r a s t r u c t u r a l f e a t u r e s c h a r a c t e r i s t i c o f t h e F - a d a p t e d c e l l s . T h e s e f e a t u r e s i l l u s t r a t e d i n F i g s . 33-36, i n c l u d e : ( i ) d i s j o i n t e d t h y l a k o i d s r a d i a t i n g f r o m p r o l a m e l l a r - l i k e b o d i e s , l o c a t e d i n t h e p y r e n o i d m a t r i x ( c f . F i g s . 22, 24), ( i i ) t h e p e c u l i a r c o n c e n t r i c r i n g p a t t e r n n o t e d i n t h e n u c l e o l u s ( c f . F i g . 31), and ( i i i ) t h e p r e s e n c e o f e x t r a o r d i n a r i l y l a r g e m i c r o b o d i e s ( c f . F i g s . 29, 30) . T h e s e o b s e r v a t i o n s , c o n f i r m e d t h a t t h e F - r e s i s t a n t s t r a i n was d i f f e r e n t f r o m t h e w i l d - t y p e (normal) s t r a i n o f A. c a r t e r a e i n m a j o r c y t o l o g i c a l f e a t u r e s , and t h a t a c q u i s i t i o n o f F-r e s i s t a n c e was a c c o m p a n i e d by a p p a r e n t l y i r r e v e r s i b l e u l t r a s t r u c t u r a l m o d i f i c a t i o n s , s u c h as a r e known f o r p h e n o t y p i c m u t a n t s . 19 TABLE I I . Summary of fluo r i d e concentration e f f e c t s on phytoplankter growth parameters* from f i r s t exposure to each F l e v e l Species tested % Exponential growth rate (a), and % maximum growth density (b), from added fl u o r i d e concentration (mg/L) of 0 50 100 150 200 a b a b a b a b a b CHLOROPHYCEAE p.. t e r t i o l e c t a 100 100 - - 97 95 PRYMNESIOPHYCEAE P_. l u t h e r i 100 100 91 100 82 95 52 65 53 63 BACILLARIOPHYCEAE £. g r a c i l i s 100 100 148 138 162 122 144 136 126 126 T_. w e i s s f l o a i i 100 100 96 111 95 108 90 109 90 89 DINOPHYCEAE A . carterae 100 100 100 86 100 88 80 74 5 9 * Each parameter shown i s expressed r e l a t i v e to the corresponding value obtained from controls (0 mg/L F) taken without added f l u o r i d e . 20 TABLE III. Adaptative growth response of two phytoplankters on repeated exposure to fluoride after suffering partial inhibition from f i r s t exposure to high F levels. Percent exponential growth rate (a) , and maximum growth density (b) , obtained from F concentration (mg/L) used at each treatment. Each step in the treatment sequence involved growth on the concentration at l e f t of arrow and using inoculum from this growth to i n i t i a t e new growth on the concentration shown at right of arrow. Pavlova lutheri* 200-MJF 200-*50F 200+100F 200*150F 200*200F a b a b a b a b a b 100(121) 100(106) 97(118) 97(103) 101(122) 96(103) 60(73) 81(87) 44(54) 78(83) flffiPhjcJiniUffi carterae ** 150*100F 150-150F 150+200F a b a b a b 150*200(I)+200(II)F a b 100(167) 100(126) 39(64) 73(91) 24(39) 40(50) 86(143) 82(103) * A l l growth parameters shown without parentheses are expressed relative to the culture 200-*0F taken as control, while those shown in parentheses are expressed relative to 0 mg/L F (of Table II) taken as control. ** A l l growth parameters shown without parentheses are expressed relative to the culture 150-»100 F taken as control, while those shown in parentheses are expressed relative to 0 mg/L F (of Table II) taken as control. 21 TABLE IV. Chloroplast pigments of wild-type and F-resistant st r a i n s of Amphidinium carterae units/10 6 c e l l s of Wild-type F-resistant Pigment Chlorophyll a* Chlorophyll c0* Chlorophyll c 2 / c h l o r o p h y l l a Total carotenoids** T. carotenoids/chlorophyll a ug 2.16 2.17 ug 0.69 0.62 0.31 0.29 ju-SPU 3.8 4.7 /j-SPU/ug 1.75 2.16 * Calculated according . to the new spectrophotometric equations of Humphrey (1979) for pigment solutions in 90% acetone and assuming presence of both chlorophylls a and ** Calculated according to the Parsons-Strickland equation (Strickland and Parsons 1972) specified for d i n o f l a g e l l a t e s , where the pigment unit (SPU) used i s a r b i t r a r y and u-SPU i s said to be considerably smaller than ug when peridinin i s the p r i n c i p a l carotenoid, representing 65-68% of t o t a l carotenoid in A. carterae according to J e f f r e y , S i e l i c k i and Haxo (1975) 22 TABLE V. Photosynthesis and r e s p i r a t i o n of wild-type and F-resistant s t r a i n s of Amphidinium carterae measured from changes registered by oxygen electrode. A l l measurements were made at 18°C. Process measured Previous darkness exposure (min) Rates of [0 2] changes (ug/min/cell) Wild-type F-resistant * 11 ~ 1 - 1 a i I * * n „j_ o j - 1 * * Actual %control 1 Actual %control' Net Photo-synthesis N i l 1.04x10-7 100 150 1.41xl0 _ 7 100 0.97x10-7 93 1.52x10-7 108 Respiration Gross Photo-synthesis*** 2 150 N i l 150 Respiration/ Photosynthesis N i l 150 0.83x10-7 100 1.82x10-7 100 1.87x10-7 100 3.23x10-7 100 0.80 100 1.29 100 1.58x10-7 190 2.36x10-7 130 2.55x10-7 136 3.88x10-7 120 1.63 204 1.55 120 * A l l the culture samples used had been maintained in con-tinuous l i g h t (irradiance ca. 65 pE m"2s-l) pr i o r to this darkness exposure. ** Values obtained for the wild-type c e l l s were taken to represent t y p i c a l "control" properties of the d i n o f l a g e l l a t e . The illumination irradiance for the photosynthetic measurements was 350 juE m"2s"l. *** Values obtained by c a l c u l a t i o n . 23 DISCUSSION PART 1. FLUORIDE EFFECT ON PHYTOPLANKTERS Under n u t r i e n t s u f f i c i e n t c o n d i t i o n s , some s p e c i e s o f e u r y h a l i n e p h y t o p l a n k t o n c a n r e a d i l y t o l e r a t e f l u o r i d e a d d i t i o n s up t o n e a r s a t u r a t i o n i n s e a w a t e r . O t h e r s p e c i e s may be a d v e r s e l y a f f e c t e d on t h e f i r s t e x p o s u r e t o e l e v a t e d f l u o r i d e c o n c e n t r a t i o n , b u t , g i v e n enough t i m e and g r a d u a l b u i l d - u p o f F c o n c e n t r a t i o n , t h e y c a n d e v e l o p t o l e r a n c e r e s u l t i n g i n v i r t u a l l y n o r m a l g r o w t h . S i m i l a r c a s e s o f p h y t o p l a n k t o n a d a p t a t i o n t o o t h e r e n v i r o n m e n t a l t o x i c a n t s have been d i s c u s s e d by S t o c k n e r and A n t i a ( 1 9 7 6 ) . In c o n s i d e r i n g s a l i n i t y e f f e c t s on f l u o r i d e t o x i c i t y , t h e o b s e r v a t i o n s o f g r o w t h i n h i b i t i o n a t an e s t u a r i n e s a l i n i t y l e v e l , f o r t h e same F c o n c e n t r a t i o n , showed l i t t l e d i f f e r e n c e f r o m t h o s e e f f e c t e d a t t h e c o a s t a l s a l i n i t y l e v e l . T h i s c o m p a r i s o n does n o t s u p p o r t t h e h y p o t h e s i s t h a t t h e u n e x p e c t e d l a c k o f f l u o r i d e t o x i c i t y t o p h y t o p l a n k t o n i n f u l l - s t r e n g t h s e a w a t e r i s due t o t h e c o m p l e x a t i o n o f F~ i o n by some s a l t c o mponent(s) o f s e a w a t e r ( O l i v e i r a e t a l . 1 9 7 8 ) . F u r t h e r m o r e , t h e l o w e r s a l i n i t y c o n t e n t o f e s t u a r i n e s e a w a t e r has e n a b l e d t h e e x a m i n a t i o n o f d o u b l e t h e h i g h e s t F c o n c e n t r a t i o n t e s t e d by O l i v e i r a e t a l . ( 1 9 7 8 ) , and, ev e n t h e n , t h r e e o f t h e same s p e c i e s showed i m m e d i a t e t o l e r a n c e t o t h i s c o n c e n t r a t i o n l e v e l w h i l e two o t h e r s t r a i n s d i s p l a y e d a d e q u a t e p o t e n t i a l f o r a d a p t a t i o n . 24 In acting as a metabolic poison in animals, plants and microorganisms, flu o r i d e i s commonly considered to damage gl y c o l y s i s and respiration by attacking the enzyme enolase (Ross et a l . 1962, Kanapka and Hamilton 1971, Sargent and Taylor 1972, Wang and Himoe 1974, Yost and VanDemark 1978, Singh and Setlow 1979). The present results suggested no di r e c t c o r r e l a t i o n between the occurrence of enolase and algal s e n s i t i v i t y to f l u o r i d e . The presence of f l u o r i d e -i n h i b i t a b l e enolase a c t i v i t y was demonstrated in c e l l - f r e e extracts of Dunaliella t e r t i o l e c t a and Pavlova l u t h e r i by Antia et a i . (1966), who could not detect i t in Amphidinium  carterae using the same techniques. Despite t h i s lack of enolase, the in. vivo s e n s i t i v i t y of Amphidinium to exogenous flu o r i d e suggests e f f e c t s on nucleotide (Carlson and Suttie 1967) and nucleic acid metabolism governing processes of c e l l d i v i s i o n ; such e f f e c t s have been reported for corn-seedling root growth (Chang 1968). This and other investigators have pointed out that enolase i s only one of several respiratory enzymes affected by fluoride and that mitochondrial membrane systems may also be damaged (Miller and M i l l e r 1974). Interestingly, D u n a l i e l l a i s known to possess a l l these respiratory enzymes (Kwon and Grant 1971) as well as well-developed mitochondria (Eyden 1975), and yet i t appears to be t o t a l l y i n s e n s i t i v e to f l u o r i d e . Some studies of fluo r i d e - t o l e r a n t plants have shown that enolase and other respiratory enzymes, known to be inhi b i t e d in  v i t r o , are apparently not affected i n vivo (Peters et a l . 25 1965) . The observation of Chaetoceros growth-stimulation from fl u o r i d e addition offers another example of similar e ffects noted for other phytoplankters by O l i v e i r a e_t a l . (1978) . However, the stimulatory effect on £. g r a c i l i s may be promoted by the lower (estuarine-level) s a l i n i t y used in t h i s study, since i t was not c l e a r l y manifested at the coastal s a l i n i t y tested by the l a t t e r investigators. The f l o c c u l a t i o n , accompanying the growth stimulation of t h i s diatom, suggests excessive production of e x t r a c e l l u l a r mucilage-like material. E x t r a c e l l u l a r polysaccharide production i s known from several species of Chaetoceros (Haug and Myklestad 1976) , and t h i s material i s said to have high molecular weight (>50,000) with fri c t i o n - r e d u c i n g properties (Hoyt 1970) , or to cause species f l o a t a t i o n by forming highly buoyant floes (Lewin 1973). Despite phytoplankter t o l e r a t i o n of elevated f l u o r i d e concentration in seawater, the pr o b a b i l i t y of F-bio-accumulation must be considered, since this could lead to injury of planktonivorous animals or those in the upper trophic le v e l s of the marine food chains. High F accumulation has been found in amphipods which are known to constitute one of the major components of feeding habits of juvenile salmon (Hocking et a l . 1980) . It i s also known that f i s h can accumulate high lev e l s of fluoride mainly in osseous tissues (Marier and Rose 1971). Studies of fluoride uptake and assimilation by an estuarine crab indicated 26 h a z a r d o u s l y h i g h b i o a c c u m u l a t i o n i n m u s c l e t i s s u e when F c o n c e n t r a t i o n i n t h e w a t e r e x c e e d e d 20 mg/L (Moore 1971). C e r t a i n z o o p l a n k t e r s and c r u s t a c e a n s a p p e a r t o b i o a c u m u l a t e f l u o r i d e s e v e r a l o r d e r s o f m a g n i t u d e h i g h e r t h a n l e v e l s f o u n d i n n o r m a l s e a w a t e r ( S o e v i k and B r a e k k a n 1979, Hempel and Manthey 1981), w h i l e a m a r i n e sponge was f o u n d t o c o n t a i n F as a m a j o r c o n s t i t u e n t ( a b o u t 10% o f d r y w e i g h t ) i n t h e f o r m o f p o t a s s i u m f l u o r o s i l i c a t e ( G r e g s o n e t a l . 1979) . I t i s p o s s i b l e t h a t s u c h h i g h l e v e l s o f a n i m a l b i o a c c u m u l a t i o n a r e f a c i l i t a t e d by i n g e s t i o n o f p h y t o p l a n k t e r s c a p a b l e o f c o n c e n t r a t i n g f l u o r i d e f r o m u n p o l l u t e d s e a w a t e r . I n t h e p r e s e n t s t u d y , t h e p o s s i b i l i t y o f f l u o r i d e a c c u m u l a t i o n was n o t e xamined. However, i t was i n t e r e s t i n g t o o b s e r v e t h a t t h e F - r e s i s t a n t s t r a i n o f A m p h i d i n i u m c a r t e r a e m a i n t a i n e d f o r s e v e r a l months i n h i g h F c o n c e n t r a t i o n s d i d n e i t h e r r e q u i r e n o r depend upon F f o r g r o w t h when s u b c u l t u r e d i n medium w i t h o u t added F. F u r t h e r m o r e , i t was n o t i c e d t h a t t h i s new s t r a i n now h a b i t u a t e d t o l i v e w i t h o u t a d d i t i o n a l f l u o r i d e , was a p p a r e n t l y p r e p a r e d t o e a s i l y overcome F s t r e s s . B a s e d on t h e s e o b s e r v a t i o n s , p h y s i o l o g i c a l and c y t o l o g i c a l s t u d i e s were d e s i g n e d i n o r d e r t o c h a r a c t e r i z e t h e F - r e s i s t a n t s t r a i n o f t h i s d i n o f l a g e l l a t e . 27 PART 2. PIGMENT CONTENT, PHOTOSYNTHESIS AND RESPIRATION OF THE WILD-TYPE AND F-RESISTANT STRAINS OF AMPHIDINIUM CARTERAE CHLOROPLAST PIGMENTS Pigment and chloroplast degradation are two d i s t i n c t i v e features of chlorosis, a symptom of injury frequently associated with phyto-F-toxicity (Wander and McBride 1956, McNulty and Newman 1961, LeBlanc et a l . 1971, Marier and Rose 1971, Comeau and LeBlanc 1972, Hocking et a l . 1980, Pandey 1981, Conover and Poole 1982). Although the extent of this phenomenon can vary from species to species, i t i s not d i r e c t l y related to the F content of the i r affected areas and may diminish with prolonged time of exposure. In the case of A. carterae, chlorosis was only apparent at the time of F i n h i b i t i o n ; however, the p o s s i b i l i t y of "hidden i n j u r i e s " in the F-resistant s t r a i n needed to be examined. In thi s connection, t h i s study showed that the chlorophyll content of the new s t r a i n did not change s i g n i f i c a n t l y from that of the wild-type. Furthermore, i t was observed that, in both stra i n s , chlorophyll a was predominant over the chlorophyll c 2 , and the r a t i o of secondary chlorophyll to chlorophyll a, (viz. 0.3) was of comparable order to that (0.4) observed by Jeffrey et a l . (1975) for two iso l a t e s of the same species. Of p a r t i c u l a r i n t e r e s t , the carotenoids known to play an 28 important role in the photo-protection of the chloroplast pigments, have been suggested to offer resistance to destruction from F p o l l u t i o n (LeBlanc et a l . 1971). The present study, showed that in the F-resistant s t r a i n , both the t o t a l carotenoid content and the carotenoid-chlorophyll a r a t i o , were s i g n i f i c a n t l y greater than those of the wild-type, which in turn were similar to the values calculated by McAllister et a l . (1964) for the same species. Although, the i n d i v i d u a l carotenoids were not analysed, p e r i d i n i n i s known to be the major xanthophyll of carterae among the peridinin-containing d i n o f l a g e l l a t e s possesing only chlorophyll c 2 (Jeffrey 1976) and i s known to be associated with minor carotenoids ( -carotene, diatoxanthin, pyrrhoxanthin, dinoxanthin, diadinoxanthin and p e r i d i n i n o l (Johansen et a l . 1974, Jeffrey et a l . 1975) . PHOTOSYNTHESIS In conformity with other photosynthetic organisms, A. carterae i s able to synthesize organic compounds from inorganic material using trapped l i g h t energy. The path of carbon flow in d i n o f l a g e l l a t e photosynthesis i s believed to be intermediate between C 3 and C 4 plants (Beardall e_t a l . 1976) . Previous investigations have suggested that fluoride i n h i b i t s photosynthesis of higher plants from f i r s t 29 exposure without adaptation (Wander and McBride 1956) . However, after adaptation, the F-resistant s t r a i n of Amphidinium did not show s i g n i f i c a n t difference in the net photosynthetic rate per c e l l or per chlorophyll a from the wild-type s t r a i n , suggesting that fluoride may not have in t e r f e r r e d with the two key photosynthetic enzymes (PEPCase and RuDPCase) (Slatyer and Tolbert 1971, Beardall et a l . 1976) . It should be pointed out that these experiments with both strains of Amphidinium were conducted at constant temperature using the same l i g h t irradiance (ca. 350 pE m~ 2s~ 1). It i s known that high l i g h t i n t e n s i t i e s are generally e a s i l y tolerated by species of Amphidinium with l i t t l e ( i f any) i n h i b i t i o n of photosynthesis (McAllister et a l . 1964, Brown and Richardson 1968, Chan 1978). In p a r t i c u l a r , i t i s known that Amphidinium carterae can maintain normal photosynthetic rates at l i g h t irradiances as high as 800 liE m""2s-l which i s said to be s l i g h t l y greater than saturating (Humphrey 1979). RESPIRATION Fluoride i s generally believed to reduce or i n h i b i t r e s p i r a t i o n apparently by i n t e r f e r i n g with some of i t s s p e c i f i c enzymes (Yang and M i l l e r 1963, Wang and Himoe 1974, Sarkar et a l . 1982 for recent review). However, stimulation of t h i s process in plants has been shown to be 30 caused by high F c o n c e n t r a t i o n s (McNulty and Newman 1957). In agreement with such o b s e r v a t i o n s , the F - r e s i s t a n t d i n o f l a g e l l a t e used i n t h i s study showed c o n s i d e r a b l e i n c r e a s e i n the endogenous r e s p i r a t i o n . Regarding the 30% i n c r e a s e a f t e r placement i n darkness as a true measure of dark r e s p i r a t i o n , i t i s i n f e r r e d t h a t the development of F-r e s i s t a n c e has e n t a i l e d the simultaneous augmentation of endogenous r e s p i r a t i o n independent of p h o t o s y n t h e s i s . But the 90% d i f f e r e n c e i n r e s p i r a t i o n r a t e s between the two s t r a i n s observed s h o r t l y a f t e r l i g h t d e p r i v a t i o n f u r t h e r suggests t h a t t h i s measurement co u l d i n c l u d e a c o n t i n u a t i o n of p h o t o r e s p i r a t i o n known to occur i n the methodology of such measurements (Jassby 1978b) and t h a t the new s t r a i n c o u l d be showing exaggerated p h o t o r e s p i r a t i o n i n a d d i t i o n t o enhanced dark r e s p i r a t i o n . In t h a t event i t i s expected from these c a l c u l a t i o n s , t h a t gross photosynthesis i n terms of C - f i x a t i o n may be s i g n i f i c a n t l y g r e a t e r i n the F-r e s i s t a n t s t r a i n than the w i l d - t y p e , but t h i s may be o f f s e t by p h o t o r e s p i r a t o r y l o s s by g l y c o l a t e e x c r e t i o n . Even though high r e s p i r a t o r y to p h o t o s y n t h e t i c r a t e r a t i o s are known among d i n o f l a g e l l a t e s compared to other algae (Humphrey 1975, B u r r i s 1977, Chan 1978), i n the present case of F - r e s i s t a n t A. c a r t e r a e , t h i s r a t i o appears to be even h i g h e r , having i n c r e a s e d 20-100% from the w i l d - t y p e i f a p p r o p r i a t e allowance has been made f o r p h o t o r e s p i r a t i o n . I t appears t h a t although the a d a p t a t i o n may have succeeded i n r e s t o r i n g p h o t o s y n t h e s i s of F - r e s i s t a n t Amphidinium to 31 n e a r l y n o r m a l l e v e l s , t h i s s u c c e s s was s t i l l l i m i t e d by a r e d u c t i o n i n t h e p h o t o s y n t h e t i c e f f i c i e n c y on a c c o u n t o f p r o b a b l e enhanced p h o t o r e s p i r a t i o n . A t t h i s p o i n t , t h e p h y s i o l o g i c a l r o l e o f d i n o f l a g e l l a t e p h o t o r e s p i r a t i o n ( i n v o l v i n g t h e g l y c o l i c a c i d pathway) i s n o t w e l l u n d e r s t o o d . In t h i s r e s p e c t , a p p r o p r i a t e b i o c h e m i c a l s t u d i e s w i l l be r e q u i r e d s p e c i f i c a l l y f o r t h i s t a x o n o m i c g r o u p . 3 2 PART 3. ULTRASTRUCTURE OF F-TREATED AMPHIDINIUM CARTERAE Judging from the u l t r a s t r u c t u r a l changes observed in the F-adapted c e l l s , i t appears that fluoride-treatment a f f e c t s mostly the chloroplast, mitochondria, and the nucleus. The alte r a t i o n s taking place in the chloroplast and e s p e c i a l l y in the pyrenoid are unique. At the time of c r i t i c a l F - i n h i b i t i o n , i t was clear that thylakoid organization was subjected to extensive a l t e r a t i o n both in the chloroplast and the pyrenoid. Within the chloroplast, the changes appeared to be directed inward from the envelope resulting in d i l a t i o n and separation of the thylakoid bands away from the chloroplast envelope. Such ef f e c t s suggested an osmotic change within the chloroplast membrane systems. However, t h i s s i t u a t i o n i s not reversed for the F-adapted chloroplast, indicating that i t i s not necessarily an impediment to successful photosynthesis and growth of Amphidinium c e l l s . In higher plants, the chloroplast has been shown to be the main s i t e of fluoride accumulation (Chang and Thompson 1966a, Lai and Ambasht 1981). Chloroplast disorganization, in conjunction with chl o r o s i s , from exposure to fluo r i d e has been reported (Solberg and Adams 1956, McNulty and Newman 1961, Comeau and LeBlanc 1972) , but the structural d e t a i l s of th i s disorganization were not described. In another investigation, 1 mM f l u o r i d e was found to retard or i n h i b i t the development of thylakoids in the pl a s t i d s of a bog 33 moss, w i t h s i m u l t a n e o u s i n h i b i t i o n o f s t a r c h f o r m a t i o n and p r o m o t i o n o f p l a s t o g l o b u l i , w h i l e t h e c h l o r o p l a s t membranes showed s i g n s o f d i s r u p t i o n ( S i m o l a 1 9 7 7 ) . T h e s e t o x i c i t y symptoms were s t a t e d t o be g r o w t h r e t a r d i n g , b u t t h e p o s s i b i l i t y o f s u c c e s s f u l g r o w t h r e v i v a l from a d a p t a t i o n was n o t e x p l o r e d . The l a t t i c e a p p e a r a n c e o f t h e membranous m a t e r i a l i n t h e F - a d a p t e d p y r e n o i d a p p e a r s t o be a s u c c e s s f u l s u r v i v a l s t r a t e g y a c h i e v e d by A m p h i d i n i u m i n t h e f a c e o f e x t r e m e d i s o r g a n i z a t i o n ( a l r e a d y a p p a r e n t a t c r i t i c a l F - i n h i b i t i o n ) t h a t m i g h t o t h e r w i s e have l e d t o c o m p l e t e t h y l a k o i d d e g e n e r a t i o n . The s u r p r i s i n g d e v e l o p m e n t o f a p r o l a m e l l a r -l i k e membrane complex i n t h e F - a d a p t e d p y r e n o i d l e d t h u s t o a r e - e v a l u a t i o n o f t h e p o s s i b l e r o l e o f t h e p y r e n o i d , a t l e a s t f o r c e r t a i n d i n o f l a g e l l a t e s . P y r e n o i d s composed m a i n l y o f a c r y s t a l l i n e l a t t i c e r e p r e s e n t i n g t h e p a c k i n g o f p r o t e i n a c e o u s m a t e r i a l have been r e p o r t e d f o r o t h e r d i n o f l a g e l l a t e s ( G r i f f i t h s 1 9 8 0 ) . However, t h e b u l k o f t h e F - a d a p t e d p y r e n o i d m a t r i x , r e m a i n s u n i f o r m l y g r a n u l a r , and t h e p r o l a m e l l a r - l i k e complex r e p r e s e n t s t h e p a c k i n g o f membranous m a t e r i a l . In f a c t , i t a p p e a r s t h a t t h i s membranous network i s c o n t i n u o u s w i t h t h e t h y l a k o i d s o f t h e a d j o i n i n g c h l o r o p l a s t l o b e s . T h e s e o b s e r v a t i o n s l e d t o t h e c o n c l u s i o n t h a t t h e p y r e n o i d o f A m p h i d i n i u m may be a c e n t e r f o r t h e a s s e m b l y o f t h y l a k o i d membrane. T h i s c o n c e p t i s b a s e d on a p a r a l l e l phenomenon o b s e r v e d i n t h e d e v e l o p m e n t o f t h e p r o l a m e l l a r 34 b o d i e s and t h e p r o t h y l a k o i d s , i n t h e s t r o m a o f e t i o p l a s t s i n h i g h e r p l a n t s . T h e s e membrane components c o n t a i n p i g m e n t s , l i p i d s , o r p r o t e i n s , r e q u i r e d f o r t h e d i f e r e n t i a t i o n o f t h e s e membranes i n t o t h y l a k o i d upon r e s u m p t i o n o f c h l o r o p h y l l s y n t h e s i s when t h e e t i o l a t e d l e a v e s a r e e x p o s e d t o l i g h t ( K i r k and T i l n e y - B a s s e t 1978, L u t z 1981). T h i s a n a l o g y s u g g e s t s t h a t f l u o r i d e may i n h i b i t / r e t a r d some s t e p ( s ) o f TAmphidinium t h y l a k o i d d i f f e r e n t i a t i o n i n a s i m i l a r way t o t h e e f f e c t t h a t d a r k n e s s has on t h e d e v e l o p m e n t o f h i g h e r p l a n t c h l o r o p l a s t s . S i n c e t h e c o n v e r s i o n o f p r o l a m e l l a e t o g r a n a l membrane i s l i n k e d t o t h e f i n a l s t e p s o f c h l o r o p h y l l s y n t h e s i s i n t h e h i g h e r p l a n t ( B o g o r a d 1976) i t i s p o s s i b l e t h a t f l u o r i d e i n h i b i t s A m p h i d i n i u m t h y l a k o i d d e v e l o p m e n t by a f f e c t i n g c h l o r o p h y l l b i o s y n t h e s i s . E v i d e n c e s u p p o r t i v e o f f l u o r i d e i n t e r f e r e n c e w i t h c h l o r o p h y l l b i o s y n t h e s i s was s u g g e s t e d by a d e t a i l e d i n v e s t i g a t i o n o f t h e mechanism o f F - i n d u c e d c h l o r o s i s i n h i g h e r p l a n t l e a v e s ( M c N u l t y and Newman 1961), and i n t h e p r e s e n t s t u d y by t h e o b s e r v a t i o n o f a l a r g e p r o p o r t i o n o f c h l o r o t i c A m p h i d i n i u m c e l l s a t t h e t i m e o f c r i t i c a l F - i n h i b i t i o n b e f o r e t h e s u c c e s s f u l g r o w t h r e c o v e r y f r o m a d a p t a t i o n . W h i l e r e t a i n i n g a p p a r e n t l y n o r m a l c r i s t a e , t h e F-a d a p t e d m i t o c h o n d r i a seemed t o have u n d e r g o n e i m p o r t a n t c h a n g e s i n t h e p r o t e i n a c e o u s m a t r i x , s i n c e an i n t e n s e l y o s m i o p h i l i c i n c l u s i o n was o b s e r v e d a f t e r a d a p t a t i o n . The n a t u r e o f t h e c h a n g e s i s n o t e n t i r e l y c l a r i f i e d by t h i s 35 study and may l i e in the underlying biochemistry of di n o f l a g e l l a t e r e s p i r a t i o n which i s yet poorly understood (Loeblich 1966). Apart from a l t e r i n g the respiratory rates, fluoride i s known to induce an increase in the mitochondrial population and protein l e v e l in higher plant tissues ( M i l l e r and M i l l e r 1974). These authors also inferred a decrease in the phosphorylation e f f i c i e n c y due to a l t e r a t i o n of the inner mitochondrial membranes. The reason for the change in the mitochondrial matrix of F-adapted c e l l s i s not clear but i t i s possible that the Krebs Cycle and other matrix enzymes are affected (Avers 1976). Although mitochondria are known to depend on both the genetic system of the c e l l and t h e i r own DNA for biosynthesis (Miller 1979), a possible mutation in mitochondrial DNA can a l t e r any of the 10% proteins known to be produced by mitochondrial ribosomes. The p r o b a b i l i t y of fluoride i n h i b i t i o n of the algal respiratory enzymes i n vivo has been discussed. I t i s possible that the fluoroacetate i n h i b i t i o n of endogenous res p i r a t i o n reported for another d i n o f l a g e l l a t e (Hochachka and Teal 1964) may be related to a fluoride e f f e c t , i f i t i s considered that exogenously supplied F i s metabolized to fluoroacetate within the c e l l , as i t i s known to occur in the case of F-resistant plants (Vickery and Vickery 1975). Interestingly, the F-adapted Amphidinium mitochondria were often seen in close association with chloroplasts and microbodies, indicating an active role in photorespiration. 36 The juxtaposing of these organelles i s known to offer a functional advantage for metabolic exchanges required in photorespiration of C 3-piant leaf c e l l s (Avers 1976). Microbodies (MB) occur in a wide range of eukaryotic c e l l s (Frederick et a l . 1968, Bibby and Dodge 1973, Heywood 1974, Herzog and Fahimi 1974, White and Brody 1974, Mu'ller 1975, Silverberg 1975, Spector and Carr 1979, Pueschel 1980, Pais 1981). In the F-adapted/resistant c e l l s there i s a s i g n i f i c a n t increase (ca. fi v e times) in the size of microbodies. This enlargement of microbodies appears to suggest that a major metabolic s h i f t was required to circumvent a basic (key) biochemical lesion caused by i n t r a c e l l u l a r F-accumulation. According to the most current and tenable opinion microbodies are derived from the endoplasmic reticulum (ER) (Silverberg 1975, Spector and Carr 1979, Pueschel 1980). In Amphidinium, a close s p a t i a l association found between the ER and MB and the observation (Figs. 29, 30) of dire c t connections between these two organelles, suggest the transfer of proteins manufactured in the ER. L i t t l e i s known about the physiological role of microbodies in photosynthetic d i n o f l a g e l l a t e s . Microbodies are well known for the i r roles in glycolate metabolism (peroxisomes) and in the conversion of stored l i p i d s into carbohydrates (glyoxysomes), (Muller 1975, Silverberg 1975, Spector and Carr 1979, Tolberg and Essner 1981). Based on the standard 3,3-diaminobenzidine (DAB) cytochemical test Bibby and Dodge (1973) reported that catalase i s absent in 37 t h e w i l d - t y p e A m p h i d i n i u m and i n 13 o t h e r g e n e r a o f d i n o f l a g e l l a t e s . However, i t i s a l s o known t h a t t h i s n e g a t i v e f i n d i n g , does n o t n e c e s s a r i l y i m p l y t h e a b s e n c e o f enzyme a c t i v i t y s i n c e t h e i n a c t i v a t i o n may o c c u r d u r i n g t h e f i x a t i o n p r o c e s s ( S i l v e r b e r g 1975) . Whatever t h e enzyme c o n t e n t o f t h e s e m i c r o b o d i e s , t h e i r e nhanced s i z e i n t h e F-a d a p t e d / r e s i s t a n t s t r a i n c o u l d be i n t e r p r e t e d a s a n e v i d e n c e f o r an i n c r e a s e i n p h o t o r e s p i r a t i o n o f t h e s e c e l l s . The s t i m u l a t i o n o f enzyme a c t i v i t y w i t h i n m i c r o b o d i e s and p h o t o r e s p i r a t i o n i n c r e a s e , were r e p o r t e d i n c o n n e c t i o n w i t h g r e e n i n g p r o c e s s e s o f Z a n t e d e s c h i a  a e t h i o p i c a s p a t h e d u r i n g f r u c t i f i c a t i o n ( P a i s 1981). B a s e d on o t h e r r e s u l t s o b t a i n e d w i t h d i a t o m s , e u g l e n o i d s , and some c h l o r o p h y t e s ( F r e d e r i c k e t aJL. 1973, C o l l i n s and M e r r e t 1975, P a u l and V o l c a n i 1974, 1975, P a u l e t a l . 1975, B e e z l e y e t a l . 1976, B u l l o c k e t a l . 1979) i t c o u l d a l s o be h y p o t h e s i z e d t h a t p h o t o r e s p i r a t i o n i n A m p h i d i n i u m m i g h t o c c u r t h r o u g h t h e c a t a l y t i c a c t i v i t y o f t h e enzyme g l y c o l a t e d e h y d r o g e n a s e c o u p l e d t o m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t . However t h e p o s s i b i l i t y s t i l l e x i s t s t h a t t h e d i n o f l a g e l l a t e m i c r o b o d i e s may f u r t h e r m e t a b o l i z e t h e g l y o x y l a t e formed i n t h e m i t o c h o n d r i a , i n w h i c h c a s e t h e s e m i c r o b o d i e s may f u n c t i o n as g l y o x y s o m e s o f an u n s u a l o r n o v e l t y p e . A u n i q u e a s p e c t o f t h e F - a d a p t e d A m p h i d i n i u m was i t s a b i l i t y t o r e g a i n m i t o t i c c a p a c i t y , w h i c h a p p e a r e d t o have been c o m p l e t e l y s u p p r e s s e d a t t h e t i m e o f c r i t i c a l F-38 i n h i b i t i o n . This recovery must have entailed major biochemical changes within the nucleus. Perhaps some of these changes accounted for the dark and l i g h t concentric rings seen in the nucleolus, which i s the s i t e of ribosome assembly in eukaryotic c e l l s (Avers 1976). Fluoride has been reported to i n t e r f e r e with RNase a c t i v i t y (Marier and Rose 1971, Sarkar et al.1982) and cause changes in the RNA structure of germinating corn seedling roots (Chang 1968) . In another investigation of the same plant material, flu o r i d e was found not only to reduce RNA synthesis but also to reduce the number of mitotic figures suggesting i n h i b i t i o n of DNA (deoxyribonucleic acid) synthesis during interphase of the mitotic cycle (Chang and Thompson 1966b). In addition, fl u o r i d e has been reported to i n t e r f e r e with the enzyme responsible for DNA r e p l i c a t i o n (Marier and Rose 1971) having adverse e f f e c t s on the reproduction of marine organisms (Hocking et aJL. 1980) . In the case of Amphidinium. there was no doubt that mitosis was completely arrested at the time of F - i n h i b i t i o n , which inf e r s that DNA synthesis was likewise arrested. This raises an i n t r i g u i n g question about the mechanism by which Amphidinium could recover both mitosis and DNA synthesis after F-adapation. One l i k e l y answer may l i e in some crypti c mechanism for DNA repair or modification known in conjunction with other microorganisms (Sager and Kitchin 1975, Samson and Cairns 1977) . Another p o s s i b i l i t y i s the development of resistance to fl u o r i d e through a genetic change mediated by plasmid-39 l i k e extrachromosomal elements such as those known to confer r e s i s t a n c e t o H g 2 + i o n (Cohen 1976, Reanney 1976, Olson et aj,. 1979) . Furthermore, s t u d i e s with b a c t e r i a and mammalian c e l l s seem to i n d i c a t e t h a t the r e s i s t a n c e developed during the process of f l u o r i d e a d a p t a t i o n can be e i t h e r temporary, the s o - c a l l e d "phenotypic" r e s i s t a n c e , which disappears a f t e r removing the F s t r e s s (Williams 1967) or permanent. The l a t t e r i s a l s o known as "genotypic" r e s i s t a n c e , which remains a f t e r r e c u l t u r i n g i n medium without f l u o r i d e a d d i t i o n s (Hamilton 1969, Q u i s s e l and S u t t i e 1972, Bunick and Kashket 1981). However, the mutagenic e f f e c t of f l u o r i d e i s s t i l l c o n t r o v e r s i a l . A ccording t o Mohamed and h i s co-workers (1966a) NaF was abl e t o induce chromosomal a b e r r a t i o n s i n onion ro o t t i p s . In a d d i t i o n , HF c o u l d a ct as a mutagenic agent i n d u c i n g chromosome a b n o r m a l i t i e s i n tomato microsporocytes and young l e a v e s (Mohamed et a l . 1966b, Mohamed 1968, Mohamed 1969). N e v e r t h e l e s s , i n another i n v e s t i g a t i o n using the same p l a n t m a t e r i a l , the mutagenic e f f e c t of f l u o r i d e was not confirmed (Temple and Weinstein 1978). In the case of Amphidinium, i t i s a l s o p o s s i b l e t h a t the added f l u o r i d e may have caused mutation by i n t e r f e r i n g w ith the r a t i o s of the v a r i o u s ions present i n the seawater used f o r pr e p a r i n g the normal growth medium. In p a r t i c u l a r , i t appeared t h a t the a v a i l a b i l i t y of Mg 2 + and C a 2 + f o r growth might be a f f e c t e d , s i n c e endogenous f l u o r i d e i n seawater of s a l i n i t y 35% i s known to e x i s t l a r g e l y as MgF + 40 and to a smaller extent as CaF +, In these experiments seawater of a s a l i n i t y of approximately 14% was used where Mg2+ and Ca 2 + concentrations would correspond to approximately 22 mM and 4.2 mM, respectively. With the added f l u o r i d e concentration of 200 mg/L, or 10.5 mM, the r a t i o s of Mg/F (=0.4) would be expected to cause large ionic s h i f t s of Mg 2 + and C a 2 + towards MgF+ and CaF +, presumably resulting in l i m i t a t i o n or depletion of b i o l o g i c a l l y available species of Mg and Ca ions. In normal cultures, spontaneous mutants are known to occur in very low number (ca. 5%), however a major population s h i f t from the normal wild-type to the spontaneous mutant which had an "opportunity" to survive by becoming tolerant to the adverse f l u o r i d e e f f e c t s , might occur. An "opportunistic" bryozoan species has been shown to withstand NaF t o x i c i t y and become dominant while other species declined s i g n i f i c a n t l y (Pankhurst et a l . 1980). Fluoroacetate spontaneous mutants are also known to occur in certain bacteria, at very low frequency (2 per m i l l i o n organisms) (Kelly 1968). 41 CONCLUSION Under n u t r i e n t s u f f i c i e n t c o n d i t i o n s , e u r y h a l i n e p h y t o p l a n k t e r s are f a i r l y t o l e r a n t of high f l u o r i d e c o n c e n t r a t i o n s (200 mg/L which approaches s o l u b i l i t y s a t u r a t i o n i n seawater), i r r e s p e c t i v e of s a l i n i t y . The wide spectrum of u l t r a s t r u c t u r a l changes, mainly i n the three o r g a n e l l e s t h a t are known to c o n t a i n DNA, combined with the p h y s i o l o g i c a l d i f f e r e n c e s shown by the F - t o l e r a n t d i n o f l a g e l l a t e , suggest t h a t t h i s s t r a i n r e p r e s e n t s a phenotypic mutant. However, f u r t h e r s t u d i e s are r e q u i r e d to g a i n a b e t t e r understanding of some of the " c r y p t i c " e f f e c t s of f l u o r i d e . These s t u d i e s should address the p o s s i b i l i t y of F-accumulation w i t h i n the c e l l s ; the c y t o c h e m i s t r y of the microbodies, which c o u l d be complemented with enzymatic assays i n s u b c e l l u l a r f r a c t i o n s i n order to c l a r i f y the r o l e , i f any, of these o r g a n e l l e s i n c e l l u l a r metabolic pathways; c y t o g e n e t i c s t u d i e s should a l s o be conducted to examine p o s s i b l e g e n e t i c a l t e r a t i o n s u n d e r l y i n g the development of F - r e s i s t a n c e i n t h i s m i c r o a l g a . 42 KEY FOR FIGURES ax axoneme b b a s a l body C c h l o r o p l a s t c c r y s t a l l i n e l a t t i c e ER endoplasmic r e t i c u l u m F f l a g e l l u m G g o l g i apparatus L l i p i d i n c l u s i o n M mitochondria MB microbody Mt m i c r o t u b u l e s mt membranous t u b u l e s N nucleus Nu n u c l e o l u s P p r o l a m e l l a r - l i k e s t r u c t u r e Pc p o l y s a c c h a r i d e cap Pg p l a s t o g l o b u l i Py pyrenoid S s t a r c h g r a i n s c h l o r o p l a s t stroma T t r i c h o c y s t V autophagic vacuole 43 F i g . 1. Growth c u r v e s o f m m a l i e l l a t e r t i o l e c t a on f i r s t e x p o s u r e t o t h e f l u o r i d e c o n c e n t r a t i o n s (mg/L) shown. Growth c u r v e s o f T h a l a s s i o s i r a w e i s s f l o g i i and C h a e t o c e r o s g r a c i l i s on f i r s t e x p o s u r e t o t h e f l u o r i d e c o n c e n t r a t i o n s (mg/L) shown. In t h e c a s e o f £ . g r a c i l i s , t h e f i r s t o b s e r v a t i o n o f f l u o r i d e - p r o m o t e d f l o c c u l a t i o n i s i n d i c a t e d by a r r o w s . 44 1.2 | 1.0 Dunaliella tertiolecta (first exposure ) o o CD +•> Co 0.8 £ 0 . 6 (0 c 0) Q (0 o 0.4 O F >VT 200 F| Jr 6 12 18 G r o w t h P e r i o d (days } 4 4 a F i g . 3 . Growth c u r v e s o f P a v l o v a l u t h e r i f r o m s i n g l e ( s e e f i r s t e x p o s u r e ) and r e p e a t e d ( s e e a d a p t a t i o n s e r i e s ) t r e a t m e n t s w i t h t h e f l u o r i d e c o n c e n t r a t i o n s (mg/L) shown. I n t h e c a s e o f t h e a d a p t a t i o n s e r i e s , t h e s t a t i o n a r y - p h a s e g r o w t h on 200 mg/L F ( f r o m f i r s t e x p o s u r e ) was u s e d t o p r o v i d e i n o c u l u m f o r t h e r e p e a t g r o w t h - c u r v e s shown c o r r e s p o n d i n g t o t h e F c o n c e n t r a t i o n s f a c i n g e a c h a r r o w h e a d . F i g . 4. Growth c u r v e s o f A m p h i d i n i u m c a r t e r a e f r o m s i n g l e s e e f i r s t e x p o s u r e ) and r e p e a t e d ( s e e a d a p t a t i o n s e r i e s ) t r e a t m e n t s w i t h t h e f l u o r i d e c o n c e n t r a t i o n s (mg/L) shown. I n t h e c a s e o f r e p e a t e d t r e a t m e n t s , a r r o w s a r e us e d t o i n d i c a t e t h e s e q u e n c e o f F c o n c e n t r a t i o n s u s e d f o r e a ch p r e c e d i n g t r e a t m e n t p r o v i d i n g i n o c u l a f o r t h e n e x t t r e a t m e n t s e q u e n c e . The Roman n u m e r a l s f o l l o w i n g an F c o n c e n t r a t i o n i n a t r e a t m e n t s e q u e n c e i n d i c a t e c o n s e c u t i v e t r e a t m e n t s w i t h t h e same F c o n c e n t r a t i o n . 45 t*5 a 0.41- Amphidinium carterae (first exposure) 2oo-*-o r 6 12 18 24 30 36 Growth Period (days) f- Amphidinium 2£2<£^°° F carterae (adaptation series)/,i50-*>200 (I)-*-200(H) F 150-*-150 F 150-*»200F • r - r • i 6 12 18 24 30 36 42 48 Growth Period (days) _l I L 54 60 66 4 5 a F i g . 5. Growth c u r v e s o f A m p h i d i n i u m c a r t e r a e from " r e p e a t " a d a p t a t i o n on 200 mg/L F (A,B,C,D), f o l l o w e d by r e p e a t e d g r o w t h on c u l t u r e medium w i t h o u t added f l u o r i d e ( s e e c u r v e s D,E,G) and r e c u l t u r e d i n 200 mg/L ( s e e c u r v e H ) . The a r r o w s i n d i c a t e t h e s e q u e n c e o f F c o n c e n t r a t i o n s u s e d f o r e ach p r e c e d i n g t r e a t m e n t p r o v i d i n g i n o c u l a f o r t h e n e x t t r e a t m e n t . The Roman n u m e r a l s f o l l o w i n g a F c o n c e n t r a t i o n i n a t r e a t m e n t s e q u e n c e i n d i c a t e c o n s e c u t i v e t r e a t m e n t s w i t h t h e same F c o n c e n t r a t i o n . 46 46 a Amphidinium carterae A: o —• 200 F (Repeat adaptation series) B: 200(1)—•200(1)F C: 200(1)200 (M)F D: 200(2H ) - »0F E : 200(71) -> 0(1) 0(I)F G : 200(211) 0(1) -* 0(1) 0(m)F H : 200(211)-^ 0(m) 200 F Growth Period (days) 4 6 a Figs. 6-9. Electron micrographs of various sections of F-unexposed (normal) Amphidinium carterae. Fi g . 6. Portion of a c e l l with nucleolus (Nu) within the nucleus (N), and lobed chloroplast (C) containing p l a s t o g l o b u l i (Pg). Note starch grains (S) in the cytoplasm, x 19,700. Fig . 7. A dividing c e l l characterized by the tunneling of microtubules (Mt) through the nucleus, x 18,400. Fig. 8. Section through the pyrenoid (Py) traversed by numerous thylakoid bands (arrows) and c h a r a c t e r i s t i c polyssacharide cap (Pc). x 18,900. Fig. 9. Portion of a c e l l showing l i p i d - l i k e inclusions (L) and the pyrenoidal polyssacharide cap (Pc). x 43,200. 47 k7cL 47a E l e c t r o n micrographs showing v a r i o u s d e t a i l s of F - i n h i b i t e d c e l l s . L o n g i t u d i n a l s e c t i o n of a " m o t i l e " c e l l showing a f l a g e l l u m (F) and the a s s o c i a t e d b a s a l body (b ) . The presence of c y t o p l a s m i c s t a r c h (S) and l i p i d i n c l u s i o n s (L) i s g r e a t l e y reduced. The endoplasmic r e t i c u l u m (ER) and the g o l g i apparatus (G) appear t o be normal. C h l o r o p l a s t stroma (C) has a c q u i r e d an e l e c t r o n d e n s i t y t h a t was not observed i n the c o n t r o l c e l l s , x 14,200. Transverse s e c t i o n of the c h l o r o p l a s t (C) d i s p l a y i n g d i l a t e d t h y l a k o i d s (arrows). x 34,800. Accumulation of o s m i o p h i l i c m a t e r i a l adjacent t o the c h l o r o p l a s t envelop (arrow) and s t a c k i n g of the t h y l a k o i d s ( s t ) . x 32,200. O s m i o p h i l i c m a t e r i a l (arrow) with v e s i c u l a t e d s t r u c t u r e s near the c h l o r o p l a s t (C) p e r i p h e r y , x 40,900. 48 48a Figs. 14-17. Ul t r a s t r u c t u r a l d e t a i l s of F-inhibited c e l l s . F i g . 14. Large autophagic vacuoles (V) containing dense granular and membranous v e s i c l e s , x 11,100. Fig. 15. Pyrenoidal thylakoid bands of di s j o i n t e d appearance (arrows) and apparently normal polyssacharide cap (Pc). x 46,100. Fi g . 16. Mitochondria (M) containing a rudimentary electron dense inclusion (arrow) and large autophagic vacuoles (V). x 18,700. Fi g . 17. Microbodies (MB) of small size are located in the v i c i n i t y of endoplasmic reticulum (ER). x 57,000. 49 4 9 a 49a Figs. 18-20. Transverse sections of F-adapted c e l l s . F i g . 18. Portion of a c e l l showing chloroplast lobes (C) exhibiting various degrees of structural changes, in p a r t i c u l a r the d i l a t i o n of thylakoids near the chloroplast envelop (arrows), the breakdown of the envelop membranes (double arrow) and the reappearance of p l a s t o g l o b u l i (Pg). x 17,100. Fi g . 19. Section through the axoneme (ax) of a flagellum. x 57,000. Fi g . 20. The arrows indicate the disjointed pyrenoidal thylakoid bands which extend into the chloroplast lobes (C). x 18,800. 50 50 a 5oa F i g s . 21-24. D i f f e r e n t s t r u c t u r a l a s p e c t s o f t h e p y r e n o i d (Py) o f F - a d a p t e d c e l l s . F i g . 21. S e c t i o n t h r o u g h t h e p r o l a m e l l a r - l i k e membrane s y s t e m ( P ) . The a r r o w s i n d i c a t e t h e p y r e n o i d a l d i s j o i n t e d t h y l a k o i d s p r o j e c t i n g i n t o t h e c h l o r o p l a s t l o b e ( C ) . x 44,000. F i g . 22. T a n g e n t i a l s e c t i o n o f a n o t h e r a s p e c t o f t h e p r o l a m e l l a r - l i k e membrane s y s t e m (P) and t h e c o n n e c t e d n e t w o r k , x 36,300. F i g . 23. T a n g e n t i a l s e c t i o n showing t h e i n t e r c o n n e c t e d membranous t u b u l e s (mt) s i m i l a r t o t h o s e o f t h e p r o l a m e l l a r b o d i e s o f h i g h e r p l a n t e t i o p l a s t s . x 46,100. F i g . 24. S e c t i o n t h r o u g h t h e c e n t r a l p o r t i o n o f t h e p y r e n o i d showing t h e p o l y s s a c h a r i d e cap (Pc) and t h e c r y s t a l l i n e l a t t i c e (c) o f t h e p r o l a m e l l a r - l i k e s t r u c t u r e (P) ( b o t h s u r f a c e and s i d e v i e w ) , x 47,500. 51 S l a 51a Figs. 25-28. Different morphological aspects of mitochondria of F-adapted c e l l s . The arrows indicate electron dense inclusions. F i g . 25. x 59,100 F i g . 26. x 57,000 F i g . 27. x 51,100 F i g . 28. x 44,300 52 52 a 52a Figs. 29-32. Ultra s t r u c t u r a l d e t a i l s of F-adapted c e l l s . F i g . 29. Section showing a large microbody (MB). x 47 f300. Fig. 30. Section of a c e l l i l l u s t r a t i n g the close s p a t i a l association between the microbody (MB), the mitochondria (M) and the chloroplast (C). Golgi (G). x 47,000. Fig. 31. Section through the t y p i c a l nucleolus (Nu) displaying i n t e r n a l dark and l i g h t concentric rings, x 39,400. Fi g . 32. Portion of a c e l l showing the accumulation of autophagic vacuoles (V) and chloroplast degeneration (C). Pyrenoid (Py). x 17,600. 53 53 a 53a Figs. 33-36. Electron micrographs of various sections of F-resistant s t r a i n . F i g . 33. Portion of a c e l l showing the pyrenoid (Py) containing prolamellar-like bodies. Chloroplast (C), golgi apparatus (G). x 17,500. Fi g . 34. Section i l l u s t r a t i n g the disjointed thylakoids radiating from the prolamellar-like bodies (P) of the pyrenoid (Py). x 46,600. Fi g . 35. Section through the nucleus (N) containing the peculiar nucleolus (Nu) with a dark and l i g h t ring pattern, x 19,600. Fig.36. Large microbodies (MB) showing dire c t connections with the endoplasmic reticulum (ER). x 41,400. 54 54 a 54 a F i g . 37. A s c h e m a t i c d i a g r a m i l l u s t r a t i n g t h e p r o m i n e n t f e a t u r e s o f F - a d a p t e d c e l l s : The p r o l a m e l l a r -l i k e b o d i e s (P) l o c a t e d i n t h e p y r e n o i d ( P y ) , t h e m i t o c h o n d r i a (M) c o n t a i n i n g e l e c t r o n d e n s e i n c l u s i o n s , t h e t y p i c a l n u c l e o l u s (Nu) showing c o n c e n t r i c d a r k and l i g h t c o n c e n t r i c r i n g s , and l a r g e m i c r o b o d i e s (MB) i n c l o s e s p a t i a l a s s o c i a t i o n w i t h t h e c l o r o p l a s t ( C ) , m i t o c h o n d r i a (M), and t h e e n d o p l a s m i c r e t i c u l u m ( E R ) . N u c l e u s (N), g o l g i a p p a r a t u s ( G ) , a u t o p h a g i c v a c u o l e s ( V ) , t r i c h o c y s t ( T ) , p o l y s s a c h a r i d e c a p ( P c ) , and f l a g e l l u m ( F ) . 55 LITERATURE CITED A n t i a , N. J . 1980. 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