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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 L i c e n c i a t u r a , U n i v e r s i t y of P o r t o - P o r t u g a l ,  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 t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1983 (c)MARIA EMILIA DE ANDRADE ALVES DE SA KLUT  DE-6  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the head of  department or by h i s or her  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department o f The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  (3/81)  Columbia  written  ii  ABSTRACT  The  effects  growth  of  enriched The  of 50-200  five  axenic  chlorophyte  growth  rate  diatom  seawater  Dunalieila  weissfloqii and  (F) a d d i t i o n s  species  (14-15%o  )  tertiolecta  were v i r t u a l l y  maximum  Chaetoceros  fluoride  phytoplankter  half-salinity  Thalassiosira  mg/L  /  growth  gracilis  were  and  appeared  growth  the prymnesiophyte  Pavlova  inhibited,  a t 150-200  concentration.  inhibition  was  at  these  fluoride  carterae  was  by more  with  than  exposed  to  allowed  t o adapt  period the  of  this  normal  changes  strain  of  was  f o r growth. was  which  were  arrested  ii  over  resumed.  However,  a t 150  mg/L  species  was  an  or  was  extended  Interestingly,  did  not  during  by  require  adaptation  undergoing  accompanied  major  biochemical,  alterations.  biosynthesis  a t the time  fluoride  concentration,  apparently  chlorophyll  this  level  this  Amphidinium  20-25%  Amphidinium  and u l t r a s t r u c t u r a l  Although t o be  growth  F  %  transfers  the highest by  The  35-50  However,  repeated  F . When  in F  to the highest  fluoride  physiological  seemed  increases  dinoflagellate  metabolic  inhibited  9 0 % a t 2 0 0 mg/L  F-resistant  additional  to  growth-  was  dinoflagellate  sensitive  growth  gradual  time,  The  another  tested.  lutheri  upon  diatom i n both  be  concentrations  overcome  levels.  t h e most  concentration, and  partially  F  the  to  by  mg/L  examined.  whereas  stimulated of  nutrient-  unaffected  density,  a l l the fluoride  in  on t h e  of  Amphidinium  of F - i n h i b i t i o n ,  normal  biosynthetic the  total  rates  carotenoid  development dark the  were  of  content  restored  during  recovery  was  efficiency  the  to  by  in  the  formation  was  appearance pyrenoid like  a  matrix.  may  in  be  also  a  with  suggesting  an  by  observed  the  adaptation which  in  appears  characteristic  and by  The  the  was  that  this  photosynthetic  F-adapted  Amphidinium  chloroplast  (especially  the  nucleus.  of  appearance cells  the  and  entailed permanent  some and  mutant.  iii  form  the the  prolamellar-  found  that  the These  in  in  the close  chloroplast, dependence  cells  showed  nucleolar  activity, It  of  assembly.  were  cells.  to in  inclusions  F-adapted  mitotic  leading  suggests  thylakoid  in  Thylakoid  configuration  metabolic  rings  phenotypic  in  in  Although  i t appears  mitochondria  F-inhibited  be  the  dinoflagellate  osmiophilic  nuclei  of  with  enhancement  fluoride,  microbodies the  have to  the  for  large  signs  may  of  F-adapted  center  concentric  accompanied  in  increased  photorespiration. light  studies  unexpected  Large  association  and  period,  affected  the  showed  mitochondria.  this  prolamellar-like This  structures  pyrenoid cells  of  of  mitochondria,  greatly  an  while  photorespiration.  features  pyrenoid),  Concurrent  was  reduction  increased  abnormal  F-adaptation,  photorespiration.  rate  a  Ultrastructural revealed  in  adaptation  impaired  due  there  possibly  photosynthetic  after  increased.  F-resistance,  r e s p i r a t i o n and normal  resumed  is of  expressed  which inferred genetic as  a  on dark  region, were that  not F-  change,  iv  TABLE OF CONTENTS  ABSTRACT  i i  TABLE OF CONTENTS  iv  L I S T OF FIGURES  vi  L I S T OF TABLES  ix  ACKNOWLEDGEMENTS  X  INTRODUCTION  1  MATERIAL AND METHODS  5  PHYTOPLANKTON  5  SPECIES  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 PART 2. PIGMENT  CONTENT,  10  PHOTOSYNTHETIC AND  RESPIRATORY RATES  13  CHLOROPLAST PIGMENTS  13  PHOTOSYNTHETIC AND RESPIRATORY RATES  14  PART 3. ELECTRON MICROSCOPY  14  TABLE I I  20  TABLE I I I  21  ^ABLE IV  22  TABLE V  23  iv  V  DISCUSSION  24  PART 1. FLUORIDE EFFECT ON PHYTOPLANKTER GROWTH PART 2. PIGMENT CONTENT, RESPIRATION  PHOTOSYNTHESIS  OF THE WILD-TYPE  ..  24  AND  AND  F-RESISTANT STRAINS OF A. CARTERAE  28  CHLOROPLAST PIGMENTS  28  PHOTOSYNTHESIS  29  RESPIRATION  30  PART 3. ULTRASTRUCTURE OF F-TREATED A. CARTERAE  ..  33  CONCLUSION  42  KEY FOR FIGURES  43  FIGURES  .  LITERATURE CITED  44 56  V  vi  L I S T OF  FIGURES  FIGURE  1  Growth c u r v e s o f D u n a l i e l l a exposure  2  to d i f f e r e n t  Growth c u r v e s Chaetoceros different  3  4  first 44  on  first  weissflogii exposure  and  to 44  F-concentrations of Pavlova  lutheri  from  single 45  repeated F-treatments  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 single  5  on  F-concentrations  of T h a l a s s i o s i r a  gracilis  Growth c u r v e s and  tertiolecta  and  repeated  Growth c u r v e s  o f A.  45  F-treatments c a r t e r a e from  F-adaptation 46  series 6  E l e c t r o n micrograph  o f F - u n e x p o s e d A.  7  E l e c t r o n micrograph  of a d i v i d i n g  8  P y r e n o i d and  9  Lipid-like  c a r t e r a e . . 47  cell.  47  p o l y s a c c h a r i d e cap  inclusion  and  47  the p o l y s a c c h a r i d e cap.. 47  10  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  11  C h l o r o p l a s t morphology  48  12  C l o r o p l a s t w i t h dense o s m i o p h i l i c m a t e r i a l  48  13  Cloroplast  with vesiculated  48  14  Autophagic  vacuoles  15  P y r e n o i d showing d i s j o i n t e d  structures  49 thylakoid  bands  49  vii  16  Mitochondria  containig  rudimentary  electron 49  dense i n c l u s i o n s . 17  Microbody - endoplasmic  18  Electron  19  Axoneme o f a f l a g e l l u m  20  P y r e n o i d morphology  21  Pyrenoid with d i s j o i n t e d thylakoids  micrograph  reticulum  association.  o f F - a d a p t e d A. c a r t e r a e .  Pyrenoid with  L 50 extending t o 51  a prolamellar-like  membrane 51  system 23  I n t e r c o n n e c t e d membranous t u b u l e s i n t h e 51  pyrenoid 24  ... 50 50  the c h l o r o p l a s t . 22  .. 49  Crystalline lattice  of t h e p r o l a m e l l a r - l i k e 51  body 25  Mitochondria  52  26  M i t o c h o n d r i a w i t h dense i n c l u s i o n  52  27  M i t o c h o n d r i a w i t h 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 w i t h dense i n c l u s i o n  52  29  Microbody morphology  53  30  Microbody - mitochondria  morphology  -  chloroplast  association  53  31  Nucleolus  53  32  Autophagic  vacuoles  and c h l o r o p l a s t 53  degeneration 33  Electron  micrograph  of F - r e s i s t a n t  vii  A. c a r t e r a e .  . 54  viii  34  P r o l a m e l l a r - l i k e bodies i n the pyrenoid m a t r i x . . 54  35  Nucleus with a t y p i c a l n u c l e o l u s  36  Microbody - endoplasmic r e t i c u l u m a s s o c i a t i o n .  37  Schematic drawing of F-adapted A. c a r t e r a e  viii  54 .. 54 55  ix  LIST  TABLE  I.  Algal  TABLE  II.  Summary on  IV.  exposure  in this  growth  t o each  growth  F  suffering  Photosynthetic type  and  carterae by  an  and  oxygen  F  respiratory  from  electrode  ix  inhibition  of wild-type  strains 0  2  to  levels  of Amphidinium  F-resistant measured  two  partial  to high  pigments  strains  of  20  repeated exposure  exposure  Chloroplast  from  level  response  fluoride  first  effects  parameters  on  after  9  study  concentration  phytoplankters  resistant  TABLE V.  TABLES  fluoride  I I I . Adaptative  from  TABLE  of  used  phytoplankter  first  TABLE  strains  OF  and  changes  F-  carterae  rates  of  21  of  ...  22  wild-  Amphidinium registered 23  X  ACKNOWLEDGEMENTS  I  wish  to  express  my  sincere  Bisalputra  f o r h i s encouragement  this  a very  to  study  Dr.  N.  criticism  Garbary many the  and  t o Dr.  Botany  from  Investigacao gratefully  preparation  Cole  role  of  D r . P.  i n this  thesis.  from  Cientifica,  the Lisboa  and c o n s t r u c t i v e thesis.  help  acknowledged.  x  An  I  am  D r . D.  in clarifying  I am a l s o  grateful  to  Columbia f o r  extended  University  Instituto -  made  extended  J . Harrison,  endeavours.  Biomedicas,  have  i s also  a t University of B r i t i s h i n my  t o D r . T.  which  this  and f o r t h e i r  t h e I . C.  scholarship  one. G r a t i t u d e  Oliveira,  Department  absence a  L.  expressed  supportive  and advice  f o r h i s suggestions  the  a n d D r . K.  ideas  their of  J. Antia during  indebted  rewarding  gratitude  of  Nacional  Portugal,  are  leave Porto de also  INTRODUCTION  The a  element f l u o r i n e , i n the form of f l u o r i d e i o n , i s  normal  minor  constituent  salinity-dependent F  of  seawater,  ocurring  c o n c e n t r a t i o n of approximately 1-2  regimes, c o n c e n t r a t i o n s as high as 1,627  have  reported  been  Miller  and  (Kilham and  Kester  (1976),  Hecky the  1973).  ionic  endogenous f l u o r i d e  i n seawater  c o n s i s t s of 50%  F~,  47% MgF+, 2.1% C a F  biological  of seawater  apart  role  from  the  stimulatory  phytoplankton  or  (Antia  effluents  and  (Leblanc et a l . 1971, 1974,  Hocking 1981, areas,  Roberts  widespread urban  additional use  of  particularly  a l . 1979,  Fluoride  growth species  i t s natural  pollution been  Thompson  Pankhurst et a l . 1980,  domestic  from  documented  et  al.  1979,  Barbaro et a l .  supply i n c e r t a i n  pollution  from the c u r r e n t  t o o t h p a s t e i s a l s o expected  intensity  high i n e s t u a r i n e  urban communities  a  Marier and Rose 1971, Harbo et  fluoridized  sewage. The  as  from  has  Pandey 1981). F l u o r i n a t i o n of water and  NaF°. The  for certain  Aside  emissions  35%« , a t 25°C,  serving  fluoride  1972,  et  et a l . 1980,  it  1980).  of  i s v i r t u a l l y unknown,  a micronutrient  anthropogenic  industrial  al.  of  F  According t o  and 1.1%  +  mg/L  speciation  of s a l i n i t y  fluoride  possibility  factor  occurrence,  via  mg/L  (Warner 1971, Sen Gupta et a l . 1978). However, i n c e r t a i n  freshwater  bf  at  of  such  pollution  may  areas where i n d u s t r i a l  be and  tend t o congregate (Moore 1971). at elevated  concentration  1  i s known t o  be  highly  toxic  to  microorganisms and  VanDemark  1980,  terrestrial  animals,  (Eagers 1969, D i o u r i s  plants  and  and Penot 1977, Yost  1978, Roberts e t a l . 1979, Pankhurst e t a l .  Conover  organic  many  and  Poole  compounds such  1982).  In a d d i t i o n ,  as f l u o r o a c e t a t e  fluorinated  and f l u o r o c i t r a t e  are known t o accumulate i n s e v e r a l p l a n t s n a t u r a l l y exposed t o i n o r g a n i c f l u o r i d e (Peters and Shorthouse 1972). Although itself, toxic food  fluoroacetate  i t can be  converted  (Marier  fluoroacetate Vickery  i s synthesized  1975)  and  reported  (Marier  interferes  these  strains  (Vickery and  with  the Krebs  and Rose 1971). f l u o r i d e - r e s i s t a n t s t r a i n s have cells  (Wiggert  and S u t t i e 1967, W i l l i a m s  Hamilton 1969, Q u i s s e l that  the c e l l  f o r b a c t e r i a l and mammalian  Werkman 1939, C a r l s o n  shown  within  apparently  A number of s e l e c t e d  1968,  f l u o r o c i t r a t e which i s  and Rose 1971). In F - r e s i s t a n t p l a n t s ,  c y c l e intermediates  and  into  t o the p l a n t  and may c o n s t i t u t e a potent hazard t o members of the chain  been  i s not t o x i c  1967,  and S u t t i e 1972). I t has been  have  the a b i l i t y  t o adapt  to  apparently  i n h i b i t o r y f l u o r i d e concentrations.  resistance  ( b e l i e v e d t o be phenotypic) disappeared when the  cells  were  cultured On  in  (Williams  1967).  permanent  or genotypic  medium  t h e other  without hand,  resistance  However t h i s  added  fluoride  the development of  has a l s o  been  reported  (Hamilton 1969, Q u i s s e l and S u t t i e 1972, Bunick and Kashket 1981). Apparently t h i s gene.  Furthermore,  resistance  spontaneous  2  i s regulated  by a s i n g l e  f l u o r i d e - r e s i s t a n t mutants  are  known  cells)  to  occur  and  content  differ  (Kelly In  most  1972,  Hamilton  view  seawater  showing  minor  of  that  the  complexes  (Oliveira  .et  salinity the  a l .  reduction  potential  One  of  the  the  effects  euryhaline  been  1968,  million  enzymatic  of  fluoride  postulated of  with  changes  Quissel  with  of  in  and  Suttie  of  marine  found  inhibition  at  lack  may  some If  this would  and  coastal  l o c a t i o n s than objectives  of  elevated  species  of  the  in  and  from  such  is  then  diminish toxic  Furthermore,  such  areas  from  river-water,  and  comparable to  present  study  fluoride phytoplankton  situations. was  to  concentrations in  seawater  true, to  of  true  coastal  phytoplankton  F,  of  estuarine  with  species  formation  the  other  fluoride  3  the  expected  detrimental  in  mg/L  to  seawater.  more  100  expose  seawater  be  three  hypothesis be  nutrient-  only  component(s)  implication,  could  to  toxicity  due  thereby  in  added  of  be  salt  species  F  salinity,  a c t u a l l y occurs  by  of  dozen  1 0 - 1 0 0 mg/L  salinity  reduction  estuarine  their  induction  a  surprising  fluoride  concentrations in  26%  formation  therefore,  the  with  with  1978).  of  of  mixing  per  mechanism  have  on  concentrations  complex  the  (Williams  growth  innocuous  such  in  2  1977).  enriched  the  based  tested  fluoride  (ca.  wild-type  hypothesis  investigation  suggested  frequency  explain  permeability  phytoplankton,  high  the  to  several  An  low  1968).  common  membrane  very  from  order  resistance, the  at  examine on  five  nutrient-enriched  seawater  of  was  examine  to  reduced  salinity  the  (14-15%o ) . A n o t h e r  underlying  causes  i n h i b i t i o n t h a t m i g h t be o b s e r v e d . The adaptation to  after i n i t i a l  successive  concentration  exposure levels  as  inhibition, of  any  likelihood  alga by  to  Stockner  growth  of growth  f o l l o w e d by  the  suggested  of  objective  adaptation increasing and  Antia  (1976) was  a l s o examined.  considered  important t o i n v e s t i g a t e both the physiology  cytology  o f a t l e a s t one a l g a l  inhibition added  As p a r t o f t h i s o b j e c t i v e , i t was  and  subsequent  species  development  fluoride.  4  t h a t showed  and  growth-  of r e s i s t a n c e t o the  MATERIALS  PHYTOPLANKTON  I were  CULTURE  for  the  the  grow  the  by  medium  for  150,  200 250  F  2)  was  was  used  a  this  mg/L on  four  studies,  F.  long as  the  solubility  1978)  observed  0.53  a  the  to  in  is  in  NaF  The  of  used.  It  twice  seawater  of  the  of  of  NaF  0,  this was  50,  100,  calculated (of  mg/L  F  saturation  is  final  same m e d i u m  200  solubility  and  component the  precipitate of  to  Antia  content  additions  level  the  of  water; F  inocula  adapted  seawater  mg/L.  gradual  salinity  limit  medium  the  concentration  standing,  at  of  as  previously  endogenous  with  produced  equivalent  were  seawater  The  tests  listed  algae.  glass-distilled  about  a  of  a l l strains, serving  salinity  (+0.5)%. .  Since  strains  MEASUREMENTS  tests,  with  at  euryhaline  classes  GROWTH  the  growth  F,  seawater  The the  15  mg/L  previously al.  with  estimated the  regarded in  from  five  nutrient-enriched  dilution  salinity  C  growth  (1975)  halved  be  chosen  of  fluoride-effect  on  Cheng  cultures  C O N D I T I O N S AND  For  METHODS  SPECIES  Axenic Table  AND  pointed  can  2  + be  of  NaF  out  that  of  that  magnitude  26%» s a l i n i t y  MgF  to  (Oliveira  .gt  aliquots  of  . growth  0 - 2 0 0 mg/L  tests  were  F-containing  conducted medium  5  in  with  4  culture  ml  tubes,  inoculated stock  with  0.2  culture,  ml  and  incubated  continuous  cool-white  The  was  growth  measurement after were  of  collected after  batch  Millipore  of  phaeophytin  disc  was  overnight  at PYE  to  the  SP8-100 a  and  spectrophotometric  ml  at  600  nm  techniques  a  test  After  of  time,  pigment  2  i  n  the extracts  6  MgCO^  TA  to  onto  culture cell  I I . Each 5  were  extracts  filter of  90%  stored  were  then  f o r pigment  the light light  ml  of  prevent  of  collected  (1 cm  days  i n the presence  extracts  was  were  (13  appropriate  These  the  filtered  t o which  measuring  i n cuvets  growth  measurements  tube  of  fluoride,  were  size)  spectrophotometer, c  of  Counter  supernatant  aliquots  or without  for  a t 0-4°C.  wavelengths  chlorophylls  3  suspension  The  determination.  Unicam  1  density  and  A t t h e same  added.  and  specific  with  the Coulter  i n darkness  centrifuged  grown  withdrawn  using  were  in  c a . 65 u E irT^s""- -  and a s e p t i c  conditions,  aqueous  transferred  acetone  content  an  C)  studies.  (HA, 0.45 jum p o r e  were  concentration  cultures  algal  (at 16-20°  by  optical  initiation)  formation.  aliquots  monitored  t h e l o g a r i t h m i c phase  culture  discs  drops  light  culture,  during  appropriate  DETERMINATIONS  dim  dinoflagellate  an  stationary  culture's  these  of  (of irradiance  Axenic  PIGMENT  Under  two  each  throughout  CHLOROPLAST  light  periodically  vortex-mixing. used  aliquots  absorbance  path)  with  a  the concentrations of were  calculated  from  the improved equations formulated aqueous  acetone  chlorophyll c  2  o  n  extracts i  and  by Humphrey  dinoflagellates  g 6 4  _ .40 E  [ C h l o r o p h y l l c ] = 24.88 E  6 3 0  - 3.80 E  2  chlorophyll  acetone  extract  wavelengths carotenoid  and E  content  0  concentrations  shown.  At  = 10 E  where  carotenoid  6 6 4  a r e expressed  i n jag/ml  the absorbance  the  time,  of each  [Carotenoid]  6 3 0  represents  P a r s o n s - S t r i c k l a n d equation  the  containing  ( J e f f r e y e t a l . 1975),  y  [ C h l o r o p h y l l a] = 11.43 E  where  (1979) f o r 90%  same  the  a t the  approximate  e x t r a c t was c a l c u l a t e d from t h e ( S t r i c k l a n d and Parsons 1972),  4 8 Q  concentration  is  expressed  in  a r b i t r a r i l y s p e c i f i c pigment u n i t s (;i SPU/ml e x t r a c t ) . All  calculated  correlated density  to  pigment  concentrations  the c u l t u r e  i n order  t o obtain  volumes pigment  i n extracts  used  and  their  concentrations  were cell  per 10^  cells.  PHOTOSYNTHETIC AND RESPIRATORY MEASUREMENTS  Photosynthetic  and r e s p i r a t o r y r a t e s of d i n o f l a g e l l a t e  c u l t u r e s of known c e l l  density  measured  methods  by  standard  7  ( c a . 6-8 x 10^ c e l l s / m l were (Jassby  1978a,b)  using  an  oxygen-electrode  (Hansatech,  D.  W.)  at  18°C  and  the  p h o t o s y n t h e t i c i r r a d i a n c e a r b i t r a r i l y f i x e d a t 350 pE m"~2s~l The  source  of i l l u m i n a t i o n was  lamp of a s l i d e  provided by the incandescent  projector.  ELECTRON MICROSCOPY  For samples  of  ultrastructural  the  studies,  dinoflagellate,  appropriate  i n v a r i o u s phases of growth  and f l u o r i d e treatments, were concentrated by the technique for in was  1.5  d e s c r i b e d by  h  saline  phosphate  then p o s t - f i x e d  buffer.  Thorough  fixation,  using  dehydrating  in  (0.17  on  Zeiss  M)  rinsing  with 2% buffer  the a  same graded  was  9S  Electron  (v/v) g l u t a r a l d e h y d e  (v/v) OsO^  carried  buffer.  fixed  (pH 7.4). The m a t e r i a l  out,  This  methanol  was  series  i  n  the same  after followed and  each by  finally  (Luft 1961). U l t r a - t h i n s e c t i o n s were  a R e i c h e r t OMU-3 Ultramicrotome EM  filtration  e t a l . (1973), and  f o r 1 h with 1%  embedding i n Epon 812 cut  Bisalputra  at room temperature  culture  microscope  a c e t a t e and lead, c i t r a t e  after  and  s t a i n i n g with u r a n y l  (Reynolds 1963).  8  examined with a  TABLE I . A l g a l  Alga  s t r a i n s used i n t h i s s t u d y  Culture  Strain/clone  collection*  designation  Isolator  Local of isolation  CHLOROPHYCEAE Dunaliella  tertiolecta  Woods Hole  Dun  Millport  60  Butcher  PRYMNESIOPHYCEAE Pavlova  (Monochrysis)  lutheri  (Droop)  Green  M.Droop  Cumbrae, Scotland  BACILLARIOPHYCEAE Chaetoceros  gracilis  U.W.D.O.S.  TO-5 8-2  W.Thomas  Schutt T/halassjosira weissflogii  Gulf of Tehuantepec  (fluviatilis)  (Grunow) P r y x e l l  U.W.D.O.S.  Actin  R.Guillard  & Hasle  Long  Island  Sound, N . Y .  DINOPHYCEAE AmphidiniuTn  carterae  Woods Hole  Arnphi 1  Hulburt  R.Guillard  Falmouth G r e a t Pond, Mass  * O b t a i n e d by Dr.N.J. A n t i a from: Woods H o l e O c e a n o g r a p h i c I n s t i t u t i o n a t Woods H o l e , Mass, MjJlport, Marine Station a t M i l l p o r t , S c o t l a n d ; U.U.D.Q.S. U n i v e r s i t y o f Washington, Department o f Oceanography a t S e a t t l e , c o u r t e s y o f J o y c e Lewin. r  9  RESULTS  PART 1. GROWTH MEASUREMENTS  Phytoplankter fluoride II  growth response t o t h e f i r s t  at various concentrations,  and i l l u s t r a t e d Dunaliella  were  barely  affected  parameters  while  Chaetoceros  considerably  (with  gracilis  (Table  stimulation  Cj. g r a c i l i s  flocculation  growth,  which  i n Table  weissflogii  3-11% i n h i b i t i o n )  appeared  II,  to  of t h e c e l l s  was not evident  be  tested,  stimulated  r a t e by a l l the F  F i g . 1, was  in their  F concentration  45%) i n growth  concentrations  marked  and T h a l a s s i o s i r a  by the h i g h e s t  (averaging  of  i s summarized  i n F i g s . 1-4.  tertiolecta  growth  exposure of  2).  visibly after  The  growth  accompanied  by  e a r l y log-phase  (to the naked  eye) i n the  c o n t r o l c u l t u r e without added F. The prymnesiophyte Pavlova l u t h e r i inhibited  ( c a . 20%) i n growth  density,  by f i r s t  3) .  higher  The  reduced growth  rate,  was s i g n i f i c a n t l y  but not i n maximum  exposure t o 100 mg/L F F  concentrations  (150-200  (Table I I , mg/L)  Fig.  further  the growth r a t e t o about 50% and a l s o the maximum density  t o about 65% of the c o n t r o l without  added  f l u o r i d e . When the c u l t u r e grown t o s t a t i o n a r y phase on 200 mg/L F was used as inoculum f o r a s e r i e s of t e s t s over a range  of F c o n c e n t r a t i o n s  (Table  I I I , F i g . 3: a d a p t a t i o n  s e r i e s ) , the growth-rate i n h i b i t i o n obtained a f t e r the  1 0  first  exposure  successive partial F  growth  not  maximum  at  growth  in  in  Tables  on  200  This  F  of  was  of  less  (compare  similar  at  150  mg/L  cases,  20%  by  the  repeated  corresponding the  by  during  A  both  a  data  repeated late  adaptation (Fig.  to 3:  growth  second  log-  this  200-200  that  was  incubated  than  days the  inoculum  was  was  by  suggesting  concentrations  increased to  showed of  to  90-95%  presence  medium  the  200  the  this  a  at  the  than  after  20-25%  of  exposure  from  fresh  that  not  F  f l u o r i d e had  taken  resumed  from  15%  rather  in  was  d i n o f l a g e l l a t e Amphidinium  examination  36  an  release  in about  progress  intensified  division  division  F  the  in  the  F-inhibition  When  0F) ,  improved  characterized  inhibition  after  growth  was  still  Microscopic  cell  However,  that  was  severely  indicated  observed  III). Interestingly,  and  inhibited  cells  was  II  growth  greatest  concentration.  rate  F.  overcome  F  F  of  series).  The  4).  mg/L  suggesting  and  growth  F  was  containing  adaptation  F,  same  F  medium  concentration  mg/L.  the  density  mg/L  phase,  mg/L  in  200  growth  100  at  improvement  but  was  to  short  and  up  at  to  of  200  to  150  mg/L  culture  mg/L  F  (Fig.  with some  mg/L  100  the  motile  F.  This  inhibited  a  certain  aspect  acting  a  lethal  toxin.  as  severely  inhibited  without  F,  lag  could  be  culture  nearly  period  fluoride-induced  permanent  carterae  (Fig. damage  normal 4:  200-  to  cell  alleviated  upon  fluoride stress.  Further  experiments  were  11  undertaken  to  examine  the  possibility highest  of growth  adaptation  F concentration  of A., c a r t e r a e  tested after  s u c c e s s f u l growth on  lower c o n c e n t r a t i o n s which produced p a r t i a l this 150  purpose, mg/L  100*200 Under  an inoculum  F was used mg/L  F  these  (Table  was markedly  obtained  on  a new s e r i e s of t e s t s on  I I I , F i g . 4: the  adaptation  repeated  superior  first  pre-exponential  However,  even  exposure  experiment,  lag period  after  this  63  latter  growth  to  from  growth  as the  and  30 days  ( F i g . 4) .  initiation  amounted  of the  t o only  h a l f of  (Table I I I ) .  the c u l t u r e t h a t was grown i n 200 mg/L F f o r 63 days  inoculum, same  a second  leading  to  exponential The carterae  This  growth  without  of c e l l  l a g (see F i g . 4: 150+200 (I) -*200 (II) adaptation  days  (involving  time  experiment  the h i g h e s t  F  was  repeated  concentration This  a f t e r a pre-exponential  ( F i g . 5) .  maintained a four-week  Furthermore,  for  seven  period  12  the  division  significant  to sub-inhibitory F l e v e l s .  carterae  was attempted a t  showed a complete recovery  s u c c e s s f u l l y achieved 51  experiment  concentration.  normal  to  exposure  growth  fluoride  dinoflagellate  ca.  a l lF  fluoride,  t h a t obtained i n the absence of added f l u o r i d e Using  at  on 200 mg/L F a f t e r  of about  days  series).  t o the corresponding  c o n s i d e r a b l e c e l l d i v i s i o n was observed a  i n h i b i t i o n . For  from the c u l t u r e grown on  to i n i t i a t e  conditions,  concentrations growth  taken  on the  F) . exposing  without  A.  prior  adaptation  was  l a g p e r i o d of  the growth  consecutive  of growth  pre-  of A..  transfers  per t r a n s f e r t o  fresh  medium)  medium  i n 200  without  added  F-medium F.  Such  was  tested  tests  on the normal  showed  that  the F-  r e s i s t a n t s t r a i n d i d n e i t h e r 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 a d d i t i o n , i t was i n t e r e s t i n g t o n o t i c e that  when  again  this  i n 200  growth  rate  period  new  strain  F-medium, and  a  on 0 F-medium was  i t showed  markedly  little  reduced  subcultured  change  i n the  pre-exponential  lag  (see F i g . 5: 200 (VII) -f /*0 (III) *200 F) .  PART 2. PIGMENT CONTENT, PHOTOSYNTHETIC AND RESPIRATORY RATES  CHLOROPLAST  PIGMENTS  The acetone-extracted from  the F - r e s i s t a n t  difference the  in i t s visible  wild-type  content  carterae  dinoflagellate.  However,  e r  i n both forms of A., c a r t e r a e the t o t a l  appeared  carotenoid  t o be  i n d i v i d u a l carotenoids  content  significantly  detectable  q u a n t i t a t i v e l y , the c  e  ± i  w  e  c  e  virtually  (Table I V ) . of the F - r e s i s t a n t greater,  were not analysed.  13  no  spectrum from t h a t of  Even p  pigment mixture  showed  absorption  of c h l o r o p h y l l s a and  identical  strain  A.  photosynthetic  though the  PHOTOSYNTHETIC AND  The  RESPIRATORY RATES  net  photosynthesis  of  the  two  strains  Amphidinium c a r t e r a e  (wild-type and  by oxygen e v o l u t i o n ,  showed i n s i g n i f i c a n t d i f f e r e n c e s ,  when  measurements  darkness certain  or  were  after  period  performed  leaving  of  time  the  respiration  showed  upon  whether  measurements  exposure  to  darkness.  light In  the  dinoflagellate the  cells  after in  darkness  a  to  a  long  condition,  the  respiratory  while  in  the  rate  90%  latter  rate of the F - r e s i s t a n t  for  a  depending continuous  exposure  to  F-resistant greater  condition  s t r a i n was  to  endogenous  were made a f t e r  subsequent  even  exposure  s i g n i f i c a n t differences  former  showed  wild-type,  respiratory  or  just  quantified  (Table V) . However, the  dark  the  F-resistant)  of  30%  than the  greater.  PART 3. ELECTRON MICROSCOPY  In  presenting  the  reference  will  following Amphidinium  u l t r a s t r u c t u r a l observations, be  made  to  different  types  the of  cells:  "CONTROL"  (F-unexposed), w i l d - t y p e  stock c u l t u r e ,  never exposed to any  over the l e v e l  (1-2  mg/L)  cells  in  normal  fluoride concentration  normally present i n seawater  (see  growth curve f o r 0 F i n F i g . 4); "F-INHIBITED", c e l l s 37  days  of  exposure  to  severely 200  mg/L  14  i n h i b i t e d i n growth upon F added to  seawater  (see  curve  for  200  F  in  "F-ADAPTED", mg/L  F  curve  and  Fig. cells  examined  for  cells  culture  of  after  showed  this  maintained  features,  and  in  variant  Particular  chloroplast  (Fig.  an  the  6,  of  9).  6)  microtubules commonly  majority  10%  the  cells  and  of  they  in  differed  ultrastructural  this from  1973,  appeared from  loss  culture the  aspects.  1971,  which  show  The  to of  15  certain  pronounced lobed by  inclusions  (Fig.  Ris  7)  remained cells of  the  was for  rounder  the in  Approximately  motile, in  of  1969).  microscope,  hyaline,  a  contained  described  flagella.  stroma  1973).  tunnelling  and  of  Dodge  traversed  the  light  a  being  highly  movement  be  F,  is  to  lipid-like  the  F-  cytological  cytoplasm  Kubai  control The  mg/L  Dodge  6-9  cells,  the  1962a,b,  Figs.  The  and  Under  growth  descriptions  pyrenoid  8).  200  carterae.  normal  previously  (Dodge  cells  immobilized  in  chromosome  CELLS".  and  Taylor  a  to  (see  200  Gibbs  cells,  as  shape,  1957,  dividing  on  Since,  A.  earlier  contained  mitosis  these  showed  with  grains  grow  culture  F-resistant  (Fig.  to  dinoflagellate  cells.  observed  of  of  F-treated  the  "F-INHIBITED  to  cells  these  starch  related  dinoflagellate  resistance  drawn  bands  In  4).  1971,  is  of  thylakoid  abundance  (Figs.  Dodge  features in  Fig.  (Hulburt  1968,  alterations  in  agreement  attention  important  in  F  as  These  carterae  Crawford  to  adapted  days  strain  referred  general  Amphidinium  of  42  lasting  "CONTROL C E L L S " .  number  successfully  150-*200 ( I ) +200 ( I I )  adapted  and  4);  the  pigmented following chloroplast  appeared  more  accumulation vesicles,  of  was  (Figs.  12,  degree  of  the  stacking the  intact  markedly  in  feature well  developed, was  0.16-0.26  /am  endoplasmic endoplasmic scarcely  cells  autophagic  small  16) .  ( F i g . 10) ,  greater  11-13).  In  appeared  to cap  In  vesicles  general, a  became  rudimentary  i n size  observed  reticulum  and  by  Golgi  the  through  cell  the  (Fig.  reticulum  tunnels  in  from  vicinity  prominent  17).  dense  0.21-0.47  of  x  rough  Furthermore,  the  systems  the nucleus  appeared  the  appeared  F-inhibition  sections,  a  electron  ranging  to  containing  mitochondria  microbodies were  decreased  relative  vacuoles  (Fig. 16).  to  ( F i g . 10) .  were seen  suggesting  be No  i n any of  the absence  of  divisions.  and  CELLS".  F-inhibited  ultrastructural similar  a  inclusions  noticed  "F-ADAPTED normal  (Figs.  occasionally  F-inhibited  mitotic  while  although  affected  cytoplasmic  without periphery  with  bands  and  but the polysaccharide  and l i p i d - l i k e  dense,  14,  Ellipsoid  the  starch  of  (Figs.  inclusion  seen  thylakoid  disjointed,  or  chloroplast  frequently  individual  cells,  amounts  with  the thylakoids  the F-inhibited  control  large  of  the  10-13),  ( F i g . 15) .  Cytoplasmic  the  was  (Figs.  regions,  near  13) . D i l a t i o n  increasingly  remained  dense  osmiophilic  observed  pyrenoid,  become  electron  electron  features. density  These  cells  cells  in  The as  several  chloroplast  that  16  differed  stroma  of c o n t r o l  cells,  from  the  important displayed but the  thylakoids obtained  showed from  dilation  was  even  greater  F-inhibition.  prominent  plastoglobuli  d i s o r g a n i z a t i o n than  seemed  near  to  In  the  particular,  thylakoid  c h l o r o p l a s t envelope,  increase  in size  and  number  the t r a n s i t i o n from F - i n h i b i t i o n t o F-adaptation While pyrenoid after  the p o l y s a c c h a r i d e  showed  remarkable  F-adaptation.  already  seen  pyrenoid,  but  appeared  from  this  perforated  23,  part  of  a  from  etioplasts  1978).  of  22  prolamellar-like It  should  bands as  be  continuous  depicts  the  connection the  pointed  out  adjacent  the  adapted  through  that  the  pyrenoid forming  those  known  Tilney-Basset, between  perforated  unperforated  c h l o r o p l a s t lobes  detail  membranes  (Kirk and  with  the  resembling  and  the  thylakoids  F-adapted  t h y l a k o i d s were r e s t r i c t e d to the pyrenoid to  of  Sections  plants  be  present,  in  higher  structure  with  so i n t e n s i f i e d t h a t i t  the  p r o l a m e l l a r - l i k e system  Fig.  bands.  these  of  and  ( F i g . 18).  internal  bands  20-24) .  portion  of  persisted  d i s a r r a y was  revealed  still  alteration  disjointed  (Figs.  central  24)  cap was  F-inhibition  now  cap-adjoining (Figs.  The  that  the  thylakoid  the  disjointed  and y e t appeared  thylakoids  ( F i g . 20) . F i g u r e 23  of  the  represents  a s e c t i o n through the p r o l a m e l l a r - l i k e s t r u s t u r e showing a mosaic  of  interconnected  Figure  24  shows  prolamellar-like bands  of  two  to  a  network  central  structure, three  of  membranous  tubules.  crystalline-type lattice r a d i a t i n g from  apposing  the  thylakoids  in  perforated  that  project  f u r t h e r i n t o the c h l o r o p l a s t lobe i l l u s t r a t e d i n F i g . 20.  17  a  The the  axonemes  F-treatments  However, an  many  abnormal  changed  of  1.08  pm.  A  of  i n F i g , 30.  F-inhibition,  cytoplasmic A  starch  prominent  presence dark  growth  spot  recovery  of  dark  at  the  of  successful  from  was and  feature and  light  center  nuclei are  of dividing  significant  accumulation degeneration and  of of  cytoplasmic  the  recovery  adaptation,  after  reappearance  mitotic by  microtubular and  nucleus  rings  nucleolus  confirmed  These  F-adapted  other  was  encircling  ( F i g . 31) . division the  the  F-induced  The from  sighting  tunnels  a  in  of the  changes  i n F i g . 37.  illustrated A  cells.  the  normal  dinoflagellate-characteristic  between  of  the  x  inclusions.  concentric  was  although  1.33-1.67  expected,  by  was  microbodies  normal;  the F-adapted  of  apparently  F-adaptation  As  and  It  association  accompanied  of  tubular  the order  successful  lipid  inclusions  F-adapted  chloroplasts  .  showed  appearance.  of  and  dense  than  by  ( F i g . 19)  F-adaptation  remained  larger was  affected  normal  large  close  mitochondria,  i s illustrated  initial  size  remarkably  photosynthetic  of  the  fold  their  from  normal  that  hardly  remain  cristae  their  4-5  approximately elliptical,  of  the  observe  s t i l l  cells  were  to  i n the form  from  to  microbodies,  appeared  while  l i t t l e  became  flagella  the mitochondria  feature  interesting  the  and  25-28),  (Figs.  of  proportion autophagic  plastids membrane  of the F-adapted vacuoles  cells  and  ( F i g . 32) , m i t o c h o n d r i a , systems.  18  These  autolytic  showed  extensive nuclear symptoms  suggested growth  premature  under  fluoride  Further permanence observed as  a  of  the  growth  further  of  These  (ii)  the  nucleolus  growth  of  A.,  on  (cf.  31),  confirmed  different  the wild-type  major  resistance  cytological was  ultrastructural phenotypic  after  followed  f  by  added  tested  showed  t h e same  major  that  from  matrix  (cf.  modifications,  by  such  as  30)  i n the  was  carterae  acquisition  are  of  . These  strain  o f A.  apparently  24),  presence  29,  strain  22,  noted  the  Figs.  that  include:  prolamellar-like  F-resistant  and  F-adapted  33-36,  pattern  (normal)  accompanied  the  (cf. Figs.  ( i i i )  the  features,  of  i n Figs.  and  19  and  without  ring  mutants.  culture  medium  microbodies  observations,  in  culture  radiating  concentric  Fig.  mg/L  the  features  During  200  on  illustrated  peculiar  from  the  t o determine  carterae.  i n the pyrenoid  large  from  maintained  characteristic  thylakoids  extraordinarily  in  now  strain  samples  features  located  cells  ultrastructural  cells,  features  disjointed  bodies,  this  a l l culture  ultrastructural  some  performed  abnormal  strain  repeated  fluoride,  (i)  were  i n the F-adapted  repeated  of  stress.  experiments  F-resistant  cells.  senescence  o f F-  irreversible known  for  TABLE I I . Summary of f l u o r i d e phytoplankter  c o n c e n t r a t i o n e f f e c t s on  growth parameters* from  first  exposure t o each F l e v e l  Species tested  % E x p o n e n t i a l growth r a t e ( a ) , and % maximum growth d e n s i t y  ( b ) , from  fluoride  (mg/L) of  concentration  0 a  50 b  a  100  b  a  150  b  added  200  a  b  a  b  97  95  CHLOROPHYCEAE  p.. t e r t i o l e c t a  100 100  -  -  100 100  91 100  82  £. 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 DINOPHYCEAE  100 100  96  95  90  109  90  89  A.  100 100  100 86  80  74  5  9  to  the  PRYMNESIOPHYCEAE  P_. l u t h e r i  95  52  65  53 63  BACILLARIOPHYCEAE  *  carterae  Each  parameter  shown  corresponding  value  obtained  without added  fluoride.  111  is  100 88  expressed  from  20  108  controls  relative (0 mg/L  F) taken  TABLE I I I . Adaptative growth response of two phytoplankters on repeated exposure to f l u o r i d e a f t e r suffering  p a r t i a l i n h i b i t i o n from f i r s t exposure to high  F levels.  Percent exponential growth rate (a) , and maximum growth density concentration involved  (mg/L) used  at each  treatment.  growth on the concentration at l e f t  Each  (b) , obtained from F  step in the treatment sequence  of arrow and using inoculum from  this  growth to i n i t i a t e new growth on the concentration shown at r i g h t of arrow.  Pavlova l u t h e r i * 200-MJF  200-*50F  200+100F  a  a  a  b  100(121) 100(106)  b  97(118) 97(103)  200*150F  b  200*200F  a  101(122) 96(103)  b  a  60(73) 81(87)  b  44(54) 78(83)  flffiPhjcJiniUffi carterae ** 150*100F  150-150F b  a  100(167) 100(126)  *  200-*0F  taken  150*200(I)+200(II)F  a  b  a  b  a  b  39(64)  73(91)  24(39)  40(50)  86(143)  82(103)  A l l growth parameters  culture  150+200F  as  shown without control,  while  parentheses those  shown  are expressed  relative  in parentheses  to the  are expressed  r e l a t i v e to 0 mg/L F (of Table II) taken as c o n t r o l . **  A l l growth parameters  shown without  c u l t u r e 150-»100 F taken as control,  parentheses  are expressed  relative  to the  while those shown i n parentheses are expressed  r e l a t i v e to 0 mg/L F (of Table II) taken as c o n t r o l .  21  TABLE IV. C h l o r o p l a s t pigments of wild-type s t r a i n s of  Pigment  Amphidinium  units/10  and F - r e s i s t a n t  carterae  c e l l s of  6  Wild-type  C h l o r o p h y l l a*  c*  Chlorophyll  0  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  Chlorophyll c /chlorophyll a 2  Total  carotenoids**  T. c a r o t e n o i d s / c h l o r o p h y l l a  *  Calculated  equations  of  according Humphrey  . to  the  (1979)  F-resistant  new  f o r pigment  spectrophotometric solutions  i n 90%  acetone and assuming presence of both c h l o r o p h y l l s a and **  Calculated  (Strickland  according  t o the P a r s o n s - S t r i c k l a n d  and Parsons 1972)  specified for dinoflagellates,  where the pigment u n i t  (SPU)  s a i d t o be c o n s i d e r a b l y  smaller  p r i n c i p a l carotenoid,  used i s a r b i t r a r y  and u-SPU i s  than ug when p e r i d i n i n i s the  representing  i n A. c a r t e r a e according  equation  65-68% of t o t a l  carotenoid  t o J e f f r e y , S i e l i c k i and Haxo  22  (1975)  TABLE V. Photosynthesis  and r e s p i r a t i o n of 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 of Amphidinium c a r t e r a e measured from  changes r e g i s t e r e d  by oxygen  A l l measurements were made a t  Process  Previous  measured  darkness exposure *  Rates of  electrode.  18°C.  [0 ] changes 2  Wild-type A11c~ 1t-u a l 1  a  (ug/min/cell) F-resistant  i  %control I  1*  *  n  Actual „j_  o  j  - 1 * *  %control'  (min)  Net  Photo-  synthesis  Respiration  Nil  1.04x10-7  100  0.97x10-7  93  150  1.41xl0  100  1.52x10-7  108  2  0.83x10-7  100  1.58x10-7  190  150  1.82x10-7  100  2.36x10-7  130  Nil  1.87x10-7  100  2.55x10-7  136  150  3.23x10-7  100  3.88x10-7  120  Nil  0.80  100  1.63  204  150  1.29  100  1.55  120  _ 7  Gross Photosynthesis***  Respiration/ Photosynthesis  * A l l the c u l t u r e samples used had been maintained i n continuous l i g h t ( i r r a d i a n c e ca. 65 pE m"2s-l) p r i o r to t h i s darkness exposure. ** Values obtained f o r the w i l d - t y p e c e l l s were taken t o r e p r e s e n t t y p i c a l " c o n t r o l " p r o p e r t i e s 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 E F F E C T  Under  nutrient  euryhaline additions may  up  t o near  of  resulting  the  in  In  considering  level,  by S t o c k n e r salinity  of  from  level.  comparison  in  unexpected  by  1978).  some  salt  then,  three  this  displayed  tolerance  Similar  cases  of  at  i s due  an  o f t h e same  concentration  Oliveira  level  showed  to  little  salinity  content of double  of  while  immediate two  o f F~ et a l .  estuarine  the highest  et al.(1978),  showed  that  phytoplankton  (Oliveira  potential f o r adaptation.  24  estuarine  the hypothesis  seawater  salinity  species  toxicity,  t o the complexation  of  of  toxicants  coastal  toxicity  the examination by  the  gradual  (1976).  on f l u o r i d e  not support  the lower  tested  adequate  at  fluoride  and  environmental  inhibition  component(s)  has enabled  concentration  does  elevated  develop  and A n t i a  effected  seawater  Furthermore,  seawater  to  lack  full-strength  ion  F  those  to  species  can  f o r t h e same F c o n c e n t r a t i o n ,  difference  fluoride  Other  exposure  of  time  effects  growth  tolerate  growth.  other  species  enough  they  normal to  some  i n seawater.  but, given  adaptation  observations  the  readily  saturation  virtually  discussed  This  can  concentration,  been  salinity  conditions,  a f f e c t e d on t h e f i r s t  F  phytoplankton have  sufficient  concentration,  build-up  PHYTOPLANKTERS  phytoplankton  be a d v e r s e l y  fluoride  ON  other  and, even tolerance strains  In a c t i n g as a metabolic poison i n animals, p l a n t s and microorganisms, glycolysis  fluoride  i s commonly considered  and r e s p i r a t i o n by a t t a c k i n g  t o damage  the enzyme enolase  (Ross e t a l . 1962, Kanapka and Hamilton 1971, Sargent and Taylor Singh  1972, Wang and Himoe 1974, Yost and VanDemark 1978, and Setlow  direct algal  1979). The present  correlation sensitivity  inhibitable extracts  between  the occurrence  to f l u o r i d e .  enolase a c t i v i t y  of D u n a l i e l l a  results  the same  enolase,  the  in. v i v o  of enolase and of f l u o r i d e -  was demonstrated  in cell-free  and Pavlova l u t h e r i by  A n t i a e t a i . (1966), who could not detect using  techniques.  i t i n Amphidinium  Despite  sensitivity  of  this  Suttie  1967)  and  nucleic  acid  metabolism  corn-seedling  investigators several  Miller  growth  have pointed  respiratory  mitochondrial and  root  governing  1974).  reported  (Chang 1968). T h i s and other  out t h a t  enolase i s only one of  enzymes a f f e c t e d  membrane  to  (Carlson  p r o c e s s e s of c e l l d i v i s i o n ; such e f f e c t s have been for  l a c k of  Amphidinium  exogenous f l u o r i d e suggests e f f e c t s on n u c l e o t i d e and  no  The presence  tertiolecta  carterae  suggested  by f l u o r i d e and t h a t  systems may a l s o be damaged ( M i l l e r  Interestingly,  Dunaliella  i s known t o  possess a l l these r e s p i r a t o r y enzymes (Kwon and Grant 1971) as yet  well  as well-developed  mitochondria  i t appears t o be t o t a l l y  (Eyden 1975), and  i n s e n s i t i v e t o f l u o r i d e . Some  s t u d i e s of f l u o r i d e - t o l e r a n t p l a n t s have shown t h a t and  other  vitro,  respiratory  are apparently  enzymes, known  t o be  not a f f e c t e d i n v i v o  25  enolase  inhibited in  (Peters  et a l .  1965) . The  o b s e r v a t i o n of Chaetoceros  growth-stimulation  f l u o r i d e a d d i t i o n o f f e r s another  example of s i m i l a r  noted  by  for  other  However,  the  promoted  by  this  stimulatory the  study,  coastal  lower  since  salinity  flocculation, diatom,  and  i t was  not  by  the  excessive  known  Myklestad  (Hoyt  £.  e_t a l . (1978) . may  be  used  in  at  the  investigators.  The  manifested  latter growth  from  s t i m u l a t i o n of  production  several  1976) , and  of  1970) , or  extracellular  of  material  (>50,000) with  this  polysaccharide  species  this  effects  gracilis  Extracellular  have h i g h molecular weight properties  on  clearly  the  material.  is  Oliveira  (estuarine-level) s a l i n i t y  tested  suggests  production  effect  accompanying  mucilage-like  (Haug  phytoplankters  from  Chaetoceros i s said  friction-reducing  to cause s p e c i e s f l o a t a t i o n  forming h i g h l y buoyant f l o e s  (Lewin  to  by  1973).  D e s p i t e phytoplankter t o l e r a t i o n of e l e v a t e d f l u o r i d e concentration accumulation injury  of  trophic  in must  seawater, be  accumulation  of  probability  considered,  planktonivorous levels  the  the  since this  animals  or  those  marine  food  of  F-bio-  c o u l d l e a d to i n the  chains.  upper  High  F  has been found i n amphipods which are known t o  c o n s t i t u t e one of the major components of feeding h a b i t s of juvenile that  salmon  fish  and  et  a l . 1980) . I t i s a l s o known  can accumulate high l e v e l s  osseous t i s s u e s uptake  (Hocking  of f l u o r i d e mainly i n  (Marier and Rose 1971). S t u d i e s of  assimilation  by  an  26  estuarine  crab  fluoride indicated  hazardously  high  concentration Certain  and  i n the  zooplankters  fluoride found  several  in  normal  contain  F  in  form  1979)  .  as  It  of  of  are  concentrations  growth  when  apparently these were strain  i t  the  to  was  designed this  not  that  neither  without to  to  for  medium  overcome  the  and  animal of  However,  i t  was  strain  months  new  from  possibility  in  upon  without  F  a l .  fluoride  depend  this  et  ingestion  several nor  to  weight)  of  F-resistant  of high  F  for  added  F.  strain  fluoride, stress.  cytological  characterize  27  dry  levels  additional  dinoflagellate.  found  (Gregson  study,  that  easily  was of  examined.  physiological order  10%  by  require  in  live  in  Hempel  high  the  maintained  noticed  prepared  1979,  sponge  present  bioacumulate  Braekkan  concentrating  was  observations,  of  In  1971).  levels  (about  such  F  than  fluorosilicate  subcultured  Furthermore, habituated  did  marine  of  observe  carterae  and  to  when  higher  facilitated  capable  to  Amphidinium  a  that  accumulation  interesting  F  (Soevik  tissue (Moore  appear  magnitude  constituent  possible  seawater.  fluoride  of  muscle  20 mg/L  crustaceans  potassium  phytoplankters  in  exceeded  while  major  bioaccumulation  unpolluted  and  seawater  a  is  water  orders  1981),  Manthey  the  bioaccumulation  the  Based  now was on  studies  F-resistant  PART 2. PIGMENT CONTENT, PHOTOSYNTHESIS AND RESPIRATION OF THE WILD-TYPE AND F-RESISTANT STRAINS OF AMPHIDINIUM CARTERAE  CHLOROPLAST PIGMENTS  Pigment  and  distinctive  features  frequently  associated  with  a  symptom  phyto-F-toxicity  a r e two of  injury  (Wander  and  and Newman 1961, LeBlanc et a l . 1971,  and Rose 1971, Comeau and LeBlanc 1972, Hocking e t  al.  1980, Pandey 1981, Conover  the  extent  species, their  degradation  of c h l o r o s i s ,  McBride 1956, McNulty Marier  chloroplast  of  this  phenomenon can vary  i t i s not d i r e c t l y  affected  and Poole  related  areas and may  1982). from  Although  species to  t o the F content of  d i m i n i s h with prolonged  time  of exposure. In the case of A. c a r t e r a e , c h l o r o s i s was o n l y apparent  at  possibility needed  to  showed  that  not  the  time  of  of "hidden be  F  injuries"  examined.  however,  i n the F - r e s i s t a n t  In t h i s  connection,  the  strain  this  study  the c h l o r o p h y l l content of the new s t r a i n d i d  change  Furthermore,  significantly i t was  from  observed  c h l o r o p h y l l a was predominant  that that,  of  the w i l d - t y p e .  i n both  strains,  over  the c h l o r o p h y l l c , and  chlorophyll  t o c h l o r o p h y l l a, ( v i z .  the r a t i o  of secondary  0.3)  of comparable  was  inhibition;  order  2  to t h a t  (0.4) observed  by  J e f f r e y e t a l . (1975) f o r two i s o l a t e s of the same s p e c i e s . Of  particular  interest,  the c a r o t e n o i d s known t o p l a y an  28  important  role  pigments,  have  destruction present  i n the p h o t o - p r o t e c t i o n of the been  from  study,  F  suggested  pollution  to  offer  (LeBlanc  chloroplast  resistance  et  a l . 1971).  to The  showed t h a t i n the F - r e s i s t a n t s t r a i n ,  both  the t o t a l c a r o t e n o i d content and the c a r o t e n o i d - c h l o r o p h y l l a r a t i o , were s i g n i f i c a n t l y g r e a t e r than those of the w i l d type, by  which  i n t u r n were s i m i l a r  McAllister  Although,  the  peridinin  is  et  known  among  possesing  only  be  to  the  be  the  major  with  c  2  (Jeffrey  minor  (Johansen  same  species.  were  not  analysed,  xanthophyll  of  dinoflagellates  1976)  and  carotenoids dinoxanthin,  et  calculated  the  peridinin-containing  pyrrhoxanthin,  peridininol  for  carotenoids  chlorophyll  associated  diatoxanthin, and  (1964)  individual  carterae  to  al.  to the v a l u e s  (  i s known -carotene,  diadinoxanthin  a l . 1974,  Jeffrey  et a l .  1975) .  PHOTOSYNTHESIS  In conformity with other p h o t o s y n t h e t i c organisms, A. carterae  is  able  to  synthesize  i n o r g a n i c m a t e r i a l using trapped carbon be  organic light  compounds  energy. The path of  flow i n d i n o f l a g e l l a t e photosynthesis  intermediate  between C  3  and  C  4  from  plants  i s b e l i e v e d to  ( B e a r d a l l e_t a l .  1976) . Previous inhibits  i n v e s t i g a t i o n s have suggested  photosynthesis  of  29  higher  plants  that  fluoride  from  first  exposure  without  However,  after  adaptation adaptation,  Amphidinium d i d not photosynthetic wild-type  r a t e per  strain,  cell  the  (PEPCase and  RuDPCase)  1976) .  It  that  two  McBride  ( S l a t y e r and be  1956) .  strain  of  d i f f e r e n c e i n the net c h l o r o p h y l l a from fluoride  key  should  and  F-resistant  or per  suggesting  with  al.  the  show s i g n i f i c a n t  interferred  et  (Wander  may  not  the have  photosynthetic  enzymes  T o l b e r t 1971,  Beardall  pointed  out  that  these  experiments with both s t r a i n s of Amphidinium were conducted at  constant  (ca.  350  temperature  pE  intensities  m~ s~ ). 2  are  Amphidinium  that  with  Amphidinium  1978).  In  carterae  light that  any) al.  light  species  inhibition 1964,  particular, can  irradiance high  t o l e r a t e d by  (if et  same  known  easily  little  Chan  the  is  (McAllister  1968,  photosynthetic  It  generally  photosynthesis Richardson  1  using  of of  Brown  and  i t i s known  maintain  normal  r a t e s at l i g h t i r r a d i a n c e s as high as 800 liE  m""2 -l which i s s a i d to be s l i g h t l y g r e a t e r than s a t u r a t i n g s  (Humphrey 1979).  RESPIRATION  Fluoride respiration specific 1974,  i s g e n e r a l l y b e l i e v e d to reduce or  apparently  enzymes  Sarkar  stimulation  et  (Yang al.  by  interfering  and 1982  of t h i s process  Miller for  with  1963,  recent  inhibit  some  Wang  review).  of  and  its  Himoe  However,  i n p l a n t s has been shown to be  30  caused In  by  high  agreement  F concentrations with  dinoflagellate  used  increase  in  increase  after  dark  such  the  this  endogenous placement  endogenous  respiration  the  90%  observed  suggests that  in  such  measurements  could  be  dark  resistant  to  strain  known  high  be  (Humphrey  1978b)  than the  case  of  F-resistant  even h i g h e r ,  having  appropriate It  appears  in  restoring  to  that  and  although  the  further  new  of  strain  i t is  expected  gross photosynthesis  i n terms  event  greater  but  20-100%  been  made  rate  from  adaptation of  be  other  in  the  offset Even are  algae present  appears  the  F-  ratios  to  ratio  for  the  excretion.  1978),  this  in  t h i s may  glycolate  Chan  31  two  continuation  the  compared  the  the  methodology  photosynthetic  photosynthesis  a  that  that  carterae,  has  between  addition  by  increased  allowance  But  in  1977,  A.  photosynthesis.  in  wild-type,  loss  Burris  of  photorespiration  dinoflagellates  1975,  augmentation  deprivation  significantly  respiratory  among  F-  include  occur  r e s p i r a t i o n . In  photorespiratory  though  light  30%  development of  rates  after  the  of  respiration  exaggerated  may  considerable  measure  of  known  F-resistant  true  simultaneous  from t h e s e c a l c u l a t i o n s , t h a t C-fixation  a  independent  (Jassby  showing  enhanced  by  as the  t h i s measurement c o u l d  photorespiration  the  showed  darkness  the  shortly  Newman 1 9 5 7 ) .  r e s p i r a t i o n . Regarding  in  entailed  difference  strains  of  study  r e s p i r a t i o n , i t i s inferred that has  to  observations,  in  resistance  of  ( M c N u l t y and  to  be  wild-type i f  photorespiration. may  F-resistant  have  succeeded  Amphidinium  to  nearly  normal  reduction probable  in  levels, the  (involving understood.  In  studies  be  will  was  of  At  acid  respect,  required  32  on  by  account point,  a of the  photorespiration  pathway)  is  appropriate  specifically  group.  limited  this  dinoflagellate  glycolic this  still  efficiency  photorespiration.  role the  success  photosynthetic  enhanced  physiological  this  for  this  not  well  biochemical taxonomic  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 i n the  F-adapted  affects  mostly  nucleus. and  the  i t appears chloroplast,  The a l t e r a t i o n s  taking  that  fluoride-treatment  mitochondria, place  and the  i n the c h l o r o p l a s t  e s p e c i a l l y i n the p y r e n o i d a r e unique. At  that  cells,  the time  thylakoid  of c r i t i c a l  organization  F - i n h i b i t i o n , i t was c l e a r  was  subjected  to  extensive  a l t e r a t i o n both i n the c h l o r o p l a s t and the p y r e n o i d . Within the c h l o r o p l a s t , the changes appeared t o be d i r e c t e d from  the envelope  resulting  in dilation  and s e p a r a t i o n  the t h y l a k o i d bands away from the c h l o r o p l a s t Such  effects  chloroplast not it  reversed is  suggested  membrane  systems.  not  necessarily  an  and growth  the c h l o r o p l a s t  change  However, t h i s  within  situation i s  to  successful  cells.  In higher  and  1981). C h l o r o p l a s t  disorganization,  and Adams 1956, McNulty  LeBlanc  1972) ,  disorganization  but  were  site  (Chang and Thompson 1966a, L a i and in  conjunction  w i t h c h l o r o s i s , from exposure t o f l u o r i d e has been (Solberg  the  i n d i c a t i n g that  impediment  of Amphidinium  of  envelope.  has been shown t o be the main  of f l u o r i d e accumulation Ambasht  osmotic  f o r the F-adapted c h l o r o p l a s t ,  photosynthesis plants,  an  inward  and Newman 1961, Comeau  the s t r u c t u r a l not  reported  described.  details In  of  this  another  i n v e s t i g a t i o n , 1 mM f l u o r i d e was found t o r e t a r d or i n h i b i t the  development  of  thylakoids  33  i n the p l a s t i d s  of a bog  moss,  with  simultaneous  promotion showed  of  plastoglobuli,  signs  symptoms  not  strategy  achieved  might  growth  formation  chloroplast 1977).  and  membranes  These  toxicity  retarding,  revival  like  complex  for of  the  but  from  the  adaptation  adjoining  These pyrenoid  of  thylakoid phenomenon  prolamellar-  the  led  thus  to  pyrenoid,  at  Pyrenoids  composed  been  for  reported  However,  uniformly  represents  fact,  the  i t with  the  granular,  and  appears the  other of  the  bulk  of  packing that  of this  t h y l a k o i d s of  the  lobes.  Amphidinium  observed  a  thylakoid  packing  remains  observations  membrane.  F-inhibition)  pyrenoid of  extreme  representing the  i s continuous  chloroplast  of  in  survival  of  complete  role  1980).  In  face  critical  F-adapted  complex  material. network  the  to  material  successful  development  have  matrix,  prolamellar-like  membranous  a  in  led  lattice  (Griffiths  pyrenoid  membranous  be  at  possible  material  dinoflagellates  to  membranous  dinoflagellates.  crystalline  proteinaceous  F-adapted  i n the  certain  a  have  surprising  r e - e v a l u a t i o n of  least  the  Amphidinium  otherwise  membrane  of  (already apparent  The  the  starch  growth  appears  by  degeneration.  mainly  be  appearance  pyrenoid  disorganization  a  the  (Simola  to  successful  lattice  F-adapted  that  disruption stated  of  while  of  explored.  The the  of  were  possibility was  inhibition  may  This in  led be  to a  concept  the  the  conclusion  center is  for  the  based  on  development  34  of  the  that  assembly a  the of  parallel  prolamellar  bodies in  and t h e p r o t h y l a k o i d s ,  higher  plants.  pigments,  lipids,  diferentiation resumption leaves  of  these  This  inhibit/retard  analogy  some  differentiation  i n  has  on  the  chloroplasts.  Since  membrane  i s  linked  synthesis  i n the higher  that  fluoride  affecting  inhibits  fluoride  by  F-induced  chlorosis  Newman of  1961),  a large  time  a  While  with  detailed  retaining  1976)  plant  that plant  to granal  chlorophyll  i ti s p o s s i b l e  biosynthesis  leaves  was  (McNulty  and  by t h e o b s e r v a t i o n  Amphidinium  before  supportive of  o f t h e mechanism o f  study  apparently  changes  i n the proteinaceous  of  effect  of  may  cells  at the  t h e s u c c e s s f u l growth  adaptation.  mitochondria  nature  1978,  t h y l a k o i d development by  of chlorotic  F-inhibition  etiolated  higher  steps  investigation  i n higher  upon  thylakoid  of prolamellae  chlorophyll  adapted  osmiophilic  the of  (Bogorad  the  fluoride  b i o s y n t h e s i s . Evidence  proportion  from  to  final  and i n t h e present  of critical  recovery  plant  interference  suggested  way  Amphidinium  chlorophyll  that  development  the  the  TAmphidinium  the conversion to  for  and T i l n e y - B a s s e t  of  similar  contain  thylakoid  when  suggests  step(s) a  into  synthesis (Kirk  etioplasts  required  membranes  to light  of  components  proteins,  chlorophyll  a r e exposed  darkness  membrane  or  of  1981).  Lutz  These  i n t h e stroma  inclusion t h e changes  seemed  was  to  normal  have  undergone  matrix,  observed  since  after  i s not entirely  35  cristae,  an  t h e F-  important intensely  adaptation.  clarified  by  The this  study  and  may  l i e in  dinoflagellate  the  underlying  r e s p i r a t i o n which  biochemistry  i s yet poorly  of  understood  ( L o e b l i c h 1966). Apart from a l t e r i n g the r e s p i r a t o r y r a t e s , fluoride  is  mitochondrial  known  population  tissues  ( Miller  inferred  a decrease  to  a l t e r a t i o n of  reason  cells  Krebs C y c l e  the  and  biosynthesis  (Miller  mitochondrial  DNA  1974).  Teal  and  a  their  possible  of F-  t h a t the  own  (Avers on both DNA f o r  mutation  any of the 10% p r o t e i n s  i n vivo  in  known  ribosomes.  has been  the f l u o r o a c e t a t e  reported  matrix  are known t o depend  1979),  can a l t e r  enzymes  that  respiration and  membranes. The  but i t i s p o s s i b l e  the c e l l  also  e f f i c i e n c y due  matrix enzymes a r e a f f e c t e d  of  the plant  authors  p r o b a b i l i t y of f l u o r i d e i n h i b i t i o n  respiratory possible  These  i n the m i t o c h o n d r i a l  t o be produced by m i t o c h o n d r i a l The  in  i n higher  mitochondrial  mitochondria  system  increase  i n the p h o s p h o r y l a t i o n  i s not c l e a r  Although  an  and p r o t e i n l e v e l  the inner  and other  genetic  induce  Miller  f o r the change  adapted  1976).  to  of the a l g a l  discussed.  inhibition  It is  of endogenous  f o r another d i n o f l a g e l l a t e (Hochachka  1964) may be r e l a t e d t o a f l u o r i d e e f f e c t ,  if it  i s considered  t h a t exogenously s u p p l i e d F i s metabolized t o  fluoroacetate  within  the  the c e l l ,  case of F - r e s i s t a n t p l a n t s  Interestingly, often  seen  as i t i s known t o occur i n (Vickery  and V i c k e r y  1975).  the F-adapted Amphidinium mitochondria were  i n close  association  with  chloroplasts  and  m i c r o b o d i e s , i n d i c a t i n g an a c t i v e r o l e i n p h o t o r e s p i r a t i o n .  36  The  juxtaposing  functional  of these  advantage  o r g a n e l l e s i s known  f o r metabolic  exchanges  p h o t o r e s p i r a t i o n of C - p i a n t l e a f c e l l s (MB) occur  a  required i n  (Avers 1976).  3  Microbodies  to offer  i n a wide range  of e u k a r y o t i c  cells  ( F r e d e r i c k et a l . 1968, Bibby and Dodge 1973, Heywood  1974,  Herzog  1975,  Silverberg  1980, a  and Fahimi 1974, White and Brody 1974, Mu'ller 1975,  Spector  and  Carr  1979,  Pueschel  P a i s 1981). In the F - a d a p t e d / r e s i s t a n t c e l l s t h e r e i s  significant  microbodies. suggest  This  that  circumvent  increase  a  a  (ca. f i v e  enlargement major  basic  times)  i n the s i z e  of microbodies  metabolic  shift  (key) b i o c h e m i c a l  was  of  appears  to  required  to  lesion  caused  by  i n t r a c e l l u l a r F-accumulation. According t o the most c u r r e n t and  tenable  opinion  endoplasmic  reticulum  microbodies (ER)  are  (Silverberg  derived  from  1975, Spector  Carr 1979, Pueschel 1980). In Amphidinium, a c l o s e  the and  spatial  a s s o c i a t i o n found between the ER and MB and the o b s e r v a t i o n (Figs.  29,  organelles, in  30)  of  suggest  the ER. L i t t l e  microbodies are  well  direct  connections  the t r a n s f e r  i s known about  between  of p r o t e i n s  for their  roles  in glycolate  r o l e of  Microbodies metabolism  (peroxisomes)  and i n the c o n v e r s i o n of s t o r e d l i p i d s  carbohydrates  (glyoxysomes),  Spector the  into  (Muller 1975, S i l v e r b e r g 1975,  and Carr 1979, T o l b e r g and Essner 1981). Based on  standard  Bibby  two  manufactured  the p h y s i o l o g i c a l  i n photosynthetic d i n o f l a g e l l a t e s .  known  these  3,3-diaminobenzidine  and Dodge  (DAB) cytochemical  test  (1973) r e p o r t e d t h a t c a t a l a s e i s absent i n  37  the  wild-type  Amphidinium  dinoflagellates.  However,  negative  does  enzyme  finding,  activity  fixation  since  process  content  cells.  The  microbodies  aethiopica  spathe  and  other  some  Beezley  results  that  through  that  be  occur  the  size  obtained  However  coupled  possibility  microbodies  glyoxylate  formed  microbodies  may  may  in  as  within  euglenoids, Collins  i t could  also  Amphidinium of  s t i l l  further  enzyme electron  exists  that  metabolize  of  an  be  might  the  i n which  glyoxysomes  and 1975,  et a l .  mitochondrial  i n the mitochondria, function  Paul  these  1981).  diatoms,  activity to  of  an  Zantedeschia  (Pais  e t a l . 1979)  catalytic  the  of  1975,  photorespiration  as  reported i n  e t aJL. 1973,  (Frederick  Bullock  the  dinoflagellate  with  the  i n t h e F-  activity  processes  of  enzyme  interpreted  enzyme  this  during  photorespiration  of  of  the absence  Whatever  fructification  dehydrogenase  transport.  case  the the  these  unsual  or  type. A  ability been  may  enhanced  a n d V o l c a n i 1974,  e t a l . 1976,  glycolate  novel  during  Paul  hypothesized occur  in  known  imply  .  their  greening  chlorophytes  1975,  Merret  1975)  genera  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 with  on  increase  other  also  could  stimulation  connection  Based  i s  the inactivation  strain  an  13  in  not necessarily  microbodies,  adapted/resistant for  i t  (Silverberg  of these  evidence  and  unique  aspect  to regain  completely  of  mitotic  the F-adapted capacity,  suppressed  at  38  the  Amphidinium  which time  appeared of  was i t s t o have  critical  F-  inhibition.  This  biochemical  recovery  changes  must  within  the  changes accounted  rings  seen i n the n u c l e o l u s , which in  eukaryotic  been reported Rose 1971,  cells  In  another  investigation  fluoride  was  also  reduce  to  inhibition  corn  found  not  the  of DNA  number  1976).  has  (Marier  and  cause changes i n the  RNA  the  of  of  concentric  RNase a c t i v i t y  to  some  Fluoride  seedling roots  of  only  light  major  i s the s i t e of ribosome  (Avers  et al.1982) and  s t r u c t u r e of germinating  Perhaps  the dark and  to i n t e r f e r e with  Sarkar  entailed  nucleus.  these  assembly  for  have  same  (Chang 1968) .  plant  reduce RNA  mitotic  (deoxyribonucleic  material,  synthesis  figures  acid)  but  suggesting  synthesis  during  i n t e r p h a s e of the m i t o t i c c y c l e (Chang and Thompson 1966b). In  addition, fluoride  has  been reported  the enzyme r e s p o n s i b l e f o r DNA 1971)  having  organisms  replication  adverse e f f e c t s on (Hocking  et  Amphidinium. there was  aJL.  to i n t e r f e r e  the  with  (Marier and Rose  r e p r o d u c t i o n of marine  1980) .  In  the  case  no doubt t h a t m i t o s i s was  completely  a r r e s t e d a t the time of F - i n h i b i t i o n , which i n f e r s t h a t s y n t h e s i s was question recover One  likewise arrested. This  about  the  mechanism  both m i t o s i s  and  l i k e l y answer may  repair  or  (Sager  known  and  1977) . Another p o s s i b i l i t y to  fluoride  through  which  Amphidinium  synthesis after  a  in  conjunction  K i t c h i n 1975,  DNA  intriguing could  F-adapation.  l i e i n some c r y p t i c mechanism f o r  modification  microorganisms  DNA  by  r a i s e s an  of  with  Samson and  DNA  other Cairns  i s the development of r e s i s t a n c e  genetic  39  change mediated by  plasmid-  like  extrachromosomal  confer Olson  resistance  to Hg  cells  developed  during  either  seem  temporary,  1967)  o r permanent.  resistance, without Suttie  Bunick of  and  chromosome young  (Mohamed  material,  n o t c o n f i r m e d (Temple In  t h e case  added  fluoride  the r a t i o s  1969,  still  tomato  in  and  However,  the  NaF  i n onion root agent  was tips.  inducing  microsporocytes  effect  and  1968, Mohamed  investigation  t h e mutagenic  medium  Quissel  (1966a)  a mutagenic  i n another  (Williams  controversial.  e t a l . 1966b, Mohamed  Nevertheless,  plant  in  resistance,  stress  1981).  aberrations  a c t as  abnormalities  leaves  1969).  could  F  c a n be  known a s " g e n o t y p i c "  (Hamilton  is  1976,  resistance  adaptation  h i s co-workers  t o i n d u c e chromosomal HF  the  reculturing  Kashket  to  w i t h b a c t e r i a and  that  i s also  fluoride  t o Mohamed  addition,  with  and  known  1976, Reanney  the  after  additions  effect  According  remains  those  "phenotypic"  removing  The l a t t e r  which  1972,  indicate  so-called  after  fluoride  mutagenic  the  i o n (Cohen  2 +  to  the  disappears  same  as  the process of f l u o r i d e  which  In  such  e t aj,. 1979) . F u r t h e r m o r e , s t u d i e s  mammalian  able  elements  using  the  of f l u o r i d e  was  and W e i n s t e i n 1 9 7 8 ) .  o f Amphidinium, may  have  i t i s also possible  c a u s e d m u t a t i o n by  of the various  ions present  that  interfering  i n t h e seawater  used f o r p r e p a r i n g  t h e n o r m a l g r o w t h medium. I n p a r t i c u l a r ,  it  the a v a i l a b i l i t y  appeared  growth seawater  might  that be  affected,  of s a l i n i t y  since  of M g  endogenous  35% i s known t o e x i s t  40  and  2 +  Ca  + 2  for  fluoride  largely  in  as M g F  +  and  to  a  smaller  extent  seawater of a s a l i n i t y Mg +  and  2  Ca  approximately added ratios  of  ionic  presumably  mM  Mg/F  and  (=0.4) of  Mg  In  +  these  experiments  of approximately 14% was used where  4.2  resulting  would  be Ca  in  correspond  respectively.  of 200 mg/L,  and  2 +  would  mM,  concentration  shifts  CaF ,  concentrations  2+  22  fluoride  as  2 +  to  towards  MgF  limitation  With  the  mM,  the  or 10.5  expected  or  to  cause  large  and  CaF ,  +  +  depletion  of  b i o l o g i c a l l y a v a i l a b l e s p e c i e s of Mg and Ca i o n s . In normal c u l t u r e s , occur  in  very  population  low  shift  spontaneous mutants  number from  (ca.  the  5%),  normal  are known t o  however wild-type  a  major  to  the  spontaneous mutant which had an " o p p o r t u n i t y " t o s u r v i v e by becoming  tolerant  occur. An to  the  adverse f l u o r i d e  effects,  "opportunistic"  bryozoan s p e c i e s  has been shown  withstand NaF  species  declined  Fluoroacetate in  certain  organisms)  to  toxicity  and become dominant  significantly  spontaneous  bacteria,  mutants  a t very  ( K e l l y 1968).  41  (Pankhurst are a l s o  et  might  while other a l . 1980).  known to occur  low frequency (2 per  million  CONCLUSION  Under  nutrient  phytoplankters  are  concentrations saturation spectrum  sufficient  (200  of  effects  better of  of  shown  that  in  order  to  in  cellular  of  the  mainly  solubility  in  strain  metabolic  conducted  to  the  some  studies  wide  the  three the  F-tolerant represents  of  required  the  should  within  The  combined w i t h the  a to  "cryptic"  address  the  cells;  the  the  microbodies,  enzymatic  clarify  the  These  of  fluoride  salinity.  by  this  F-accumulation  complemented w i t h  underlying  changes,  understanding  fluoride.  cytochemistry  be  of  known t o c o n t a i n DNA,  suggest  high  m u t a n t . However, f u r t h e r s t u d i e s a r e  possibility  also  of  euryhaline  approaches  irrespective  differences  dinoflagellate,  a  which  ultrastructural  physiological  phenotypic  tolerant  mg/L  i n seawater),  o r g a n e l l e s that are  gain  fairly  conditions,  which  could  be  assays i n s u b c e l l u l a r f r a c t i o n s  role,  i f any,  pathways; examine  development  microalga.  42  of  these  cytogenetic  possible of  organelles  studies  should  genetic a l t e r a t i o n s  F-resistance  in  this  KEY FOR FIGURES  ax  axoneme  b  basal  C  chloroplast  c  crystalline  lattice  ER  endoplasmic  reticulum  F  flagellum  G  golgi  apparatus  L  lipid  inclusion  M  mitochondria  MB  microbody  Mt  microtubules  mt  membranous  N  nucleus  Nu  body  tubules  nucleolus  P  prolamellar-like  Pc  polysaccharide  Pg  plastoglobuli  Py  structure  cap  pyrenoid  S  starch  grain  s  chloroplast  T  trichocyst  V  autophagic  stroma  vacuole  43  Fig.  1.  Growth first  curves exposure  (mg/L)  tertiolecta  to the fluoride  on  concentrations  shown.  Growth  curves  Chaetoceros fluoride case  of m m a l i e l l a  of Thalassiosira  gracilis  on  concentrations  of £. g r a c i l i s ,  fluoride-promoted arrows.  44  first (mg/L)  the f i r s t  flocculation  weissflogii exposure shown.  and  to the In t h e  observation i s indicated  of by  1.2 |  1.0  o o  Dunaliella tertiolecta (first  exposure)  F  >V 200 F| T  Jr  CD 0.8 +•>  Co  £0.6 (0  c 0) Q  0.4  (0  o  O  6 Growth  12 P e r i o d (days}  44a  18  Fig.  3.  Growth (see  curves  first  of Pavlova  exposure)  adaptation  series)  concentrations adaptation on  2 0 0 mg/L  provide shown  Fig.  4.  F  treatments  first  each  Growth  curves  single  see f i r s t  adaptation  In the case  exposure)  o f Amphidinium exposure)  series)  was  of the  used  to  growth-curves  (mg/L)  indicate  t h e sequence  for  each  preceding  for  t h e next  sequence with  arrows  with  the  (see fluoride  In the case a r e used  of  to  of F concentrations  treatment  providing  sequence.  indicate  used  inocula  T h e Roman  an F c o n c e n t r a t i o n i n a  t h e same  45  from  and repeated  shown.  treatment  following  carterae  treatments  treatments,  treatments  fluoride  to the F concentrations  repeated  treatment  the  arrowhead.  concentrations  numerals  (see  with  f o r the repeat  corresponding  facing  shown.  single  t h e s t a t i o n a r y - p h a s e growth  (from  inoculum  from  and r e p e a t e d  (mg/L)  series,  lutheri  consecutive  F concentration.  t*5 a  0.41-  Amphidinium carterae  (first exposure)  f- Amphidinium 2£2<£^°° carterae 2oo-*-o r (adaptation series)/ i50-*>200 (I)-*-200(H) F F  ,  150-*-150 F  150-*»200F  6  12 18 24 30 36 Growth Period (days)  6  45a  12  18  •r-r • i _l 24 30 36 42 48 Growth Period (days)  I  L  54  60  66  Fig.  5.  Growth  curves  of Amphidinium  carterae  "repeat"  a d a p t a t i o n o n 2 0 0 mg/L  followed  by  without  repeated  added  recultured  fluoride  t h e sequence  for  each  preceding  for  t h e next  sequence the  on c u l t u r e  (see curve  treatment  D,E,G) a n d H ) . The  providing  T h e Roman  a F concentration i n a  arrows used  inocula  numerals treatment  consecutive treatments  same F c o n c e n t r a t i o n .  46  medium  of F concentrations  treatment.  indicate  (A,B,C,D),  (see curves  i n 2 0 0 mg/L  indicate  following  growth  F  from  with  46 a  Amphidinium carterae (Repeat adaptation series)  A: o —• 200 F B: 200(1)—•200(1)F C: 2 0 0 ( 1 ) 2 0 0 (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)  46a  Figs.  6-9.  E l e c t r o n micrographs of v a r i o u s s e c t i o n s of F-unexposed  Fig.  6.  (normal) Amphidinium  P o r t i o n of a c e l l with n u c l e o l u s the nucleus  (N), and  carterae.  (Nu)  within  lobed c h l o r o p l a s t  (C)  c o n t a i n i n g p l a s t o g l o b u l i (Pg). Note s t a r c h grains  Fig.  7.  (S) i n the cytoplasm, x 19,700.  A d i v i d i n g c e l l c h a r a c t e r i z e d by t u n n e l i n g of microtubules nucleus,  Fig.  8.  (Mt)  the  through  x 18,400.  S e c t i o n through the pyrenoid  (Py)  traversed  by numerous t h y l a k o i d bands (arrows) c h a r a c t e r i s t i c polyssacharide  cap  9.  P o r t i o n of a c e l l showing inclusions  (L) and  polyssacharide  cap  47  the  lipid-like  pyrenoidal  (Pc). x 43,200.  and  (Pc).  x 18,900.  Fig.  the  k7cL  47a  E l e c t r o n micrographs details  of F - i n h i b i t e d  Longitudinal a flagellum (b).  showing  section  cells.  of a " m o t i l e "  of c y t o p l a s m i c  inclusions  (L) i s g r e a t l e y  endoplasmic apparatus stroma  cell  (F) and t h e a s s o c i a t e d  The p r e s e n c e  lipid  various  reticulum  (G) a p p e a r  basal  body  starch  (S) and  reduced.  The  (ER) and t h e g o l g i t o be n o r m a l .  (C) has a c q u i r e d  t h a t was n o t o b s e r v e d  showing  Chloroplast  an e l e c t r o n  density  i n the control  cells,  x 14,200.  Transverse section  of the c h l o r o p l a s t  displaying  thylakoids  dilated  (C)  (arrows).  x 34,800.  Accumulation of o s m i o p h i l i c to the chloroplast of  the thylakoids  Osmiophilic structures  envelop  (arrow) and  (arrow) w i t h  the c h l o r o p l a s t  x 40,900.  48  adjacent stacking  ( s t ) . x 32,200.  material near  material  vesiculated  (C)  periphery,  48a  F i g s . 14-17.  U l t r a s t r u c t u r a l d e t a i l s of F - i n h i b i t e d cells.  F i g . 14.  Large autophagic vacuoles (V) c o n t a i n i n g dense g r a n u l a r and membranous v e s i c l e s , x 11,100.  F i g . 15.  P y r e n o i d a l t h y l a k o i d bands of d i s j o i n t e d appearance  (arrows) and a p p a r e n t l y normal  p o l y s s a c h a r i d e cap  F i g . 16.  ( P c ) . x 46,100.  M i t o c h o n d r i a (M) c o n t a i n i n g a e l e c t r o n dense i n c l u s i o n  rudimentary  (arrow) and  large  autophagic v a c u o l e s (V). x 18,700.  F i g . 17.  Microbodies  (MB)  of s m a l l s i z e are l o c a t e d i n  the v i c i n i t y of endoplasmic x 57,000.  49  reticulum  (ER).  4 9 a  49a  F i g s . 18-20.  F i g . 18.  Transverse s e c t i o n s  Portion  of F-adapted  cells.  of a c e l l showing c h l o r o p l a s t  (C) e x h i b i t i n g v a r i o u s  lobes  degrees of s t r u c t u r a l  changes, i n p a r t i c u l a r the d i l a t i o n of thylakoids  near the c h l o r o p l a s t  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.  F i g . 19.  Section  through the axoneme (ax) o f a  flagellum.  F i g . 20.  x 57,000.  The arrows i n d i c a t e the d i s j o i n t e d thylakoid  pyrenoidal  bands which extend i n t o the  c h l o r o p l a s t lobes  50  (C). x 18,800.  50 a  5oa  Figs.  Different  21-24.  pyrenoid  Fig.  21.  Section system  22.  thylakoids  Tangential  lobe  Fig.  23.  44,000.  s e c t i o n of another membrane  network,  x  aspect  system  showing  membranous  tubules  (mt) s i m i l a r  prolamellar  Section pyrenoid and  x  bodies  through  the  of higher  interconnected t o those  of  plant  the central portion  showing  the polyssacharide  the crystalline  side  (P) a n d t h e  46,100.  prolamellar-like and  of the  36,300.  section  etioplasts.  24.  projecting into the  Tangential  the  Fig.  cells.  indicate the pyrenoidal  (C).x  prolamellar-like connected  of the  t h e p r o l a m e l l a r - l i k e membrane  ( P ) .The arrows  chloroplast  aspects  (Py) o f F - a d a p t e d  through  disjointed  Fig.  structural  view),  lattice  structure x  51  47,500.  of the c a p (Pc)  (c) o f t h e (P)  (both  surface  Sla  51a  F i g s . 25-28.  D i f f e r e n t morphological  aspects of  mitochondria of F-adapted c e l l s . The arrows i n d i c a t e e l e c t r o n dense  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  inclusions.  52 a  52a  F i g s . 29-32.  U l t r a s t r u c t u r a l d e t a i l s of F-adapted cells.  F i g . 29.  Section  showing a l a r g e microbody (MB).  x 47 300. f  F i g . 30.  S e c t i o n of a c e l l  i l l u s t r a t i n g the c l o s e  s p a t i a l a s s o c i a t i o n between the microbody (MB),  the m i t o c h o n d r i a  (C). G o l g i  F i g . 31.  (M) and t h e c h l o r o p l a s t  (G). x 47,000.  S e c t i o n through  the t y p i c a l n u c l e o l u s  d i s p l a y i n g i n t e r n a l dark and l i g h t  (Nu)  concentric  r i n g s , x 39,400.  F i g . 32.  P o r t i o n of a c e l l showing the accumulation of autophagic v a c u o l e s degeneration  (V) and c h l o r o p l a s t  (C). Pyrenoid  53  ( P y ) . x 17,600.  53 a  53a  F i g s . 33-36.  E l e c t r o n micrographs of v a r i o u s  sections  of F - r e s i s t a n t s t r a i n .  F i g . 33.  P o r t i o n of a c e l l showing the pyrenoid  (Py)  c o n t a i n i n g p r o l a m e l l a r - l i k e bodies. Chloroplast  (C), g o l g i apparatus (G).  x 17,500.  F i g . 34.  Section i l l u s t r a t i n g  the 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 from the p r o l a m e l l a r - l i k e bodies (P) of the pyrenoid  F i g . 35.  (Py). x 46,600.  S e c t i o n through the nucleus peculiar nucleolus  (N) c o n t a i n i n g the  (Nu) with a dark and l i g h t  r i n g p a t t e r n , x 19,600.  Fig.36.  Large microbodies connections  with  (ER). x 41,400.  54  (MB) showing d i r e c t the endoplasmic  reticulum  54 a  54  a  Fig.  37.  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