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Bromophenols in Rhodomela larix : a study in chemical ecology Phillips, David William 1980

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BROMOPHENOLS IN RHODOMELA LARIX: A STUDY IN CHEMICAL ECOLOGY by DAVID WILLIAM PHILLIPS B.S.,  Western C a r o l i n a U n i v e r s i t y , 1976  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE  FACULTY OF GRADUATE STUDIES (Department o f 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  THE  standard  UNIVERSITY OF BRITISH COLUMBIA June 1980  0DAVID WILLIAM  PHILLIPS, 1980  In presenting t h i s thesis in p a r t i a l f u l f i l m e n t o f t h e requirements f o r f  an advanced degree at the U n i v e r s i t y 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 a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives.  It i s understood that copying or p u b l i c a t i o n  of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department nf  BOTANY  The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  30 JUNE 1980  ABSTRACT  In t h i s  study  and  chemistry  are  i n v e s t i g a t e d and  findings  aspects  an  Environmental p o o l s which might b r o m o p h e n o l s by  red  introduced.  i n the p o o l s  t i d e p o o l and  low  l a r i x i n d i c a t e s t h a t the  to  be  previously to  be  thought  rate of  developed  f o r the  are  artifacts  n a t u r a l constituents of A high-performance  was  two  tideof  over  biomass  and  larix  compounds. release  morphologically  sufficiently  same s p e c i e s .  t o be  pro-  or exudation  i n t e r t i d a l populations  R.  the  f o r the  amounts o f t h e s e  implications of this  considered  the  show t h a t R.  Chemotaxonomic c o m p a r i s o n o f t h e different  Agardh  algae.  the p r o d u c t i o n  exudes c o p i o u s  are  o f the reasons  Measurements o f a l g a l  of t o t a l phenols  ecological  C.  a l g a were e x a m i n e d i n summer  a t h r e e month p e r i o d .  The  (Turner)  biology  factors occurring, i n l o c a l  affect  this  ecology,  e f f o r t made t o r e l a t e  to a determination  p r o d u c e s and  the  o f Rhodomela l a r i x  d u c t i o n o f b r o m o p h e n o l s by  levels  of  Several a r e now  of similar  bromophenols considered  algae.  l i q u i d c h r o m a t o g r a p h i c method  separation,  identification  and  iii quantitative determination of red algal  bromophenols.  The m e t h o d i s e v a l u a t e d on t h e b a s i s o f s e v e r a l  chromato-  g r a p h i c p a r a m e t e r s , and s t e p s i n i t s d e v e l o p m e n t  and  improvement a r e d i s c u s s e d . P r e v i o u s s t u d i e s on r e d a l g a l p h e n o l s h a v e ally  overlooked quantitative considerations.  presents data f o r v a r i o u s aspects of the a n a l y s i s of the major bromophenolic 2,3-dibromo-4,5-dihydroxybenzyl Temporal  gener-  This study  quantitative  substance  (lanosol,  a l c o h o l ) i n R.  larix.  d e t e r m i n a t i o n o f l a n o s o l c o n c e n t r a t i o n s demon-  s t r a t e s t h a t the h i g h e s t l e v e l s o c c u r i n w i n t e r months. P o p u l a t i o n a l d i f f e r e n c e s w e r e o b s e r v e d and t h e h i g h e s t l e v e l s w i t h i n a s i n g l e p l a n t w e r e f o u n d t o be i n t h e youngest  regions.  C o n t r o l o f the e x u d a t i o n o f bromophenols R.  l a r i x was  studied using a r t i f i c i a l  by  conditions  which  f e l l w i t h i n the ranges of environmental c o n d i t i o n s p r e v i o u s l y monitored.  E x u d a t i o n o f l a n o s o l i s enhanced i n  light,  a t h i g h e r t e m p e r a t u r e s and a t l o w e r  pH h a s  little  e f f e c t on e x u d a t i o n .  The  salinities.  rates of  exu-  d a t i o n found i n these experiments correspond to those measured,in  the  tidepool.  Examination of the broad spectrum a n t i b i o t i c ity at  of l a n o s o l proves t h i s  compound t o be e f f e c t i v e  low l e v e l s a g a i n s t a wide v a r i e t y o f organisms.  activeven The  iv e f f e c t i s e n h a n c e d a t l o w pH, exudates  and c o m p a r i s o n  w i t h R.  s u g g e s t s l a n o s o l t o be t h e m a j o r a c t i v e  larix  com-  ponent . A d i s c u s s i o n of the e c o l o g i c a l s i g n i f i c a n c e b r o m o p h e n o l p r o d u c t i o n i s p r e s e n t e d and an  argument  made f o r t h e p r o d u c t i o n o f t h e s e compounds a s substances.  antibiotic  Comparison w i t h p r e v i o u s experiments  i n g the temporal a n t i b i o t i c a c t i v i t y of another species supports t h i s conclusion.  of  describRhodomela  V  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  v i i  ACKNOWLEDGEMENTS  ix  PROEM CHAPTER I. CHAPTER I I .  CHAPTER I I I . CHAPTER IV.  CHAPTER V. CHAPTER VI.  1 Aspects o f the ecology o f Rhodomela larix  3  Bromophenols of. Rhodomela l a r i x : Chemotaxonomy o f morphologica1 forms  23  HPLC S e p a r a t i o n o f r e d a l g a l bromophenols . . .  31  Temporal, i n t e r p o p u l a t i o n a l and i n t r a t h a l l i a l measurement o f l a n o s o l l e v e l s i n Rhodomela larix  53  Exudation o f bromophenols by Rhodomela l a r i x  67  Broad spectrum a n t i b i o t i c activity of bromophenols from Rhodomela larix  83  PERSPECTIVES -FOOTNOTES REFERENCES  101 .  106 107  vi  LIST OF TABLES  I.  II. III.  IV.  V.  VI.  VII.  VIII.  IX.  Dominance, frequency and importance v a l u e s f o r the algae i n the two t i d e p o o l s . . . . 2 X Species a s s o c i a t i o n of algae i n the two pools  17  21  R e t e n t i o n times, c a p a c i t y f a c t o r s and r e l a t i v e r e t e n t i o n s of standards of bromophenols separated by r e v e r s e phase HPLC .  41  R e s o l u t i o n as a f u n c t i o n of column e f f i c i e n c y , s e l e c t i v i t y and c a p a c i t y u s i n g d i f f e r e n t s o l v e n t systems i n the separ a t i o n of 3,5-dibromo-4-hydroxybenzyl a l c o h o l and the corresponding a c i d ...  45  A summary of data a v a i l a b l e on q u a n t i t a t i v e aspects of bromophenols i n r e d algae  55  Q u a n t i t a t i v e comparison of l a n o s o l conc e n t r a t i o n s i n three d i s t i n c t populat i o n s of Rhodomela l a r i x  63  Exudation measured as t o t a l phenols over a twenty-four hour p e r i o d by t i d e p o o l and s u b t i d a l forms of Rhodomela l a r i x . .  73  Q u a n t i t a t i v e d e t e r m i n a t i o n of exuded l a n o s o l and other o b s e r v a t i o n s under v a r y i n g experimental c o n d i t i o n s  76  A n t i b i o t i c a c t i v i t y of l a n o s o l a g a i n s t three s p e c i e s of marine f u n g i  97  vii  LIST OF FIGURES  1.  2.  3.  4.  5. 6. 7.  8.  9.  10.  Map  o f Bath I s l a n d , B r i t i s h Columbia, and environs, and d e t a i l showing l o c a t i o n of the t i d e p o o l s s t u d i e d . . . . . . . .  7  Diagram o f pools 1 and 2 showing ranges i n s t r a t i f i c a t i o n o f s a l i n i t y and temperature and l i g h t a t t e n u a t i o n w i t h depth  11  Environmental measurements made i n p o o l 1 and p o o l 2 during three consecutive months  13  T o t a l phenols i n p o o l 1 and p o o l 2 over a twenty-four hour p e r i o d given as p a r t s per m i l l i o n of p h l o r o g l u c i n o l . .  19  Temperature programmed GLC a n a l y s i s o f Rhodomela l a r i x bromophenols  28  Reverse phase HPLC s e p a r a t i o n o f f o u r t e e n bromophenol standards  40  HPLC A n a l y s i s of the petroleum ether and c h l o r o f o r m f r a c t i o n s from the l a r g e s c a l e e x t r a c t i o n of Rhodomela l a r i x . .  46  HPLC A n a l y s i s o f the e t h y l a c e t a t e and methanol f r a c t i o n s from the l a r g e s c a l e e x t r a c t i o n o f Rhodomela l a r i x . .  48  Reverse phase s e p a r a t i o n o f bromophenols e x t r a c t e d from P r i o n i t i s l y a l l i i and Ceramium washingtoniense  51  A t y p i c a l HPLC s e p a r a t i o n o f Rhodomela l a r i x bromophenols used f o r the q u a n t i t a t i v e determination o f l a n o s o l . . . .  60  v i i i 11.  12. 13. 14.  15.  16.  17.  18.  19.  20.  21.  Seasonal v a r i a t i o n o f l a n o s o l content in Rhodomela l a r i x and change i n t h e d r y to wet weight r a t i o over the p e r i o d of one y e a r  62  Lanosol content i n Rhodomela l a r i x  65  HPLC o f t h e subtidal  different thallus  parts  of  t w e n t y - f o u r hour exudate f o r m o f Rhodomela l a r i x  the of the . . . .  75  E f f e c t o f t e m p e r a t u r e and s a l i n i t y on t h e p r o d u c t i o n o f c o l o r e d m a t t e r by Rhodomela l a r i x d u r i n g an e i g h t h o u r e x u d a t i o n e x periment  78  E f f e c t o f pH and l i g h t on t h e p r o d u c t i o n o f c o l o r e d m a t t e r by Rhodomela l a r i x d u r i n g an e i g h t h o u r e x u d a t i o n e x p e r i m e n t . . . .  79  E f f e c t o f l i g h t , s a l i n i t y , t e m p e r a t u r e and pH o n e x u d a t i o n o f l a n o s o l b y R h o d o m e l a larix  81  A n t i b i o t i c a c t i v i t y of lanosol against Candida a l b i c a n s , E s c h e r i c h i a c o l i , Sac c h a r o m y c e s c e r e v i s i a e a n d ~ Staphy1ococcus aureus  91  The e f f e c t o f pH o n t h e a n t i b i o t i c o f l a n o s o l and l a n o s a l t a g a i n s t Escherichia coli  93  activity  Effect of varying concentrations of lanosol i n p u r e f o r m , i n a l g a l e x t r a c t s and i n exudates on the g r o w t h r a t e o f E s c h e r i c h i a coli  96  A t y p i c a l s n a i l repulsion experiment showing t h e e f f e c t s o f l a n o s o l and l a n o s a l t on t h e b e h a v i o r o f L i t t o r i n a scutulata  99  Degree o f r e p u l s i o n o f L i t t o r i n a scutulata by v a r y i n g c o n c e n t r a t i o n s o f l a n o s o l and l a n o s a l t  100  ix  ACKNOWLEDGEMENTS  I wish Robert  t o t h a n k D r s . Raymond A n d e r s e n ,  De Wreede, R o n a l d  Foreman, G i l b e r t  Scagel,  Tony Swain and N e i l  reading  and  criticism  Special generous trips of  assistance  to Bath  t h e use  Dr. helpful  and  t o Dr. Dr.  Raymond A n d e r s e n  De Wreede f o r h i s d u r i n g t h e many  James C r a i g i e k i n d l y  s u p p o r t and My edited  deserves  t o Dr.  Neil  syntheses.  identification  E. Graham w i t h  Towers f o r h i s c o n t i n u e d  tolerated,  typed the manuscript, through t h i s  "thanks" i s i n s u f f i c i e n t  tributions .  f o r t h e many  experiments.  encouragement throughout  t o o k t o g e t us  thanks  Z. Abramowski and Ms.  w i f e , Helen,  and  pro-  Guenther  O l i v e r i a h e l p e d w i t h the  microbiological  Many t h a n k s  field  R o n a l d Foreman f o r u s e  c o n c e r n i n g s p e c t r o s c o p y and  Celestino  initial  careful  o f t h e GLC-MS.  t h e a l g a e and Ms.  say,  Robert  enthusiasm  there.  discussions  Julie  the  it  and  Robert  manuscript.  t h e b r o m o p h e n o l s t a n d a r d s and Dr.  Eigendorf  Ms.  t o Dr.  Island,  the f a c i l i t i e s  vided  of  thanks  Hughes,  Towers f o r t h e i r  of this  B r u c e Bohm,  the p a s t f o u r y e a r s .  supported, and  encouraged,  d i d whatever  experience. gratitude  else  Needless  f o r h e r many  to con-  1  PROEM  The e c o l o g i s t ' s and s u b t l e access small tion  chemical  t o modern  of  Chemical  the  levels.  Whether or  called the  plant  tic,  of  plants  interaction  to microbe, or  of  interactions  defensive  Vuillemin  of  (1889)  the  another,  first  a concept  used the  thousands  w h i c h has  the  complex  deals  biolog-  with  creatures,  (Harborne  plant,  evoluchemical  and  and  1972).  plant  to  as b e i n g  ecologists  animal  antagonis-  to  The  the  plant  grown t o  (herbivory)  kingdom.  organisms,  origin.  the  include  antag-  production  predation  one o r g a n i s m t o  terrestrial  find  term " a n t i b i o s i s "  produced by marine of  called  field  interesting.  response  of  the  to  chemical  described  common t h r o u g h o u t  counteraction  antibiotics  those  is  led  living  phytochemical  in  detect  indifferent.  the most  compounds  and p a r a s i t i s m  scribe  of  to  intricate  chemist's  unravel  both  ecology  can be  mutually  The m a j o r i t y  to  broad  plant  the  him to  science  and o t h e r  is  it  at  this  phytochemical  beneficial  onistic  of  have  of  attempts  organisms  One c o r n e r  interactions  has been  of  products  of  and t h e  enabling  branch  ecology  interrelationships  awareness  in nature  natural  a new and e x c i t i n g  ecology.  ical  balance  instrumentation  quantities of  increasing  life  the not  to  deof  study to  mention  2  T h i s d i s s e r t a t i o n takes a step i n f u r t h e r i n g understanding  of the r o l e o f an unusual  group of  "secondary"  m e t a b o l i t e s which are widespread among r e d algae. of the ecology,  our  Aspects  chemistry and b i o l o g y of a p a r t i c u l a r r e d  a l g a are approached from the standpoint of attempting  to  r e l a t e "form to f u n c t i o n , " form being the p r o d u c t i o n of the compounds and f u n c t i o n t h e i r r a i s o n d ' e t r e .  I sincerely  hope that even i f the reader remains unconvinced  by  final verdict,  the  I w i l l have at l e a s t s t i m u l a t e d i m a g i n a t i v e  s p e c u l a t i o n , i f not the d e s i r e to i n t e n s i f y r e s e a r c h i n t o the chemical ecology of the oceans.  CHAPTER I  ASPECTS OF THE ECOLOGY OF RHODOMELA LARIX  3  4  INTRODUCTION  Tidepools tidal  zone  Canada. found,  and et  generally  prefer  than  of  1974)  do m a n y o f  should  that  of  quite  a variety for  of  physical  parts  of  Pyefinch little coast  British of  the  low  intertidal  periods  of  can  i n pools  to  in  1962,  and o f t e n  physiological to  survive  (Doty  adaptations in  the  Ganning  1924,  1930).  tide-  must  have  monitoring  pools  North  The above  oc-  habitat.  in  other  1928,  and McLachlan  been done on t h e  and Macy  in  indicating  on t h e in  zone  problems  Johnson and Skutch  1971, Edelstein  such work has  littoral  tidepool  conditions  1946)  The  conditions  extreme,  1967),  Johnson  and u n u s u a l  some w o r k h a s b e e n d o n e  (Klugh  the  sub-  Scagel  immersion  high  be  (Rhodophyta).  1945,  (Biebl  The e n v i r o n m e n t a l  (see Humphrey  organisms  algae  (Fritsch  inter-  Columbia,  brown and g r e e n m a c r o p h y t e s .  unstable of  rocky  red  intensities  longer  the  of  p o s e many d i f f e r e n t  the world  o r no  in  and e n v i r o n m e n t a l  1943,  of  a variety  seashore  such algae  Although  throughout  coast  species  red algae  species.  are  curred  the  therefore  these  pools  pools  light  and/or  the  occur  the  lower  occurrence  for  these  i n c l u d i n g many  regions  al.  scattered  a l o n g much o f  Within  Red a l g a e tidal  are  1975),  Pacific  studies  5  considered ature,  factors  salinity  tribution  of  s u c h as  algae  in  the  coastal  s u c h as  (Khailov  and B u r l a k o v a  (Ragan and J e n s e n presence  habitat.  or  release  tidepools. of  1969)  1979)  and o t h e r  of  an a l g a l  factors  between p l a n t s  s u c h as  or  plants  frequently': overlooked.  These  role  distribution  in.determining In  this  performed British  context,  during  the  Columbia.  environmental  the  Physical  conditions  identified  standing  crop)  in  inter-  or  and c h e m i c a l pools flora  in  the  (percentage  of  classes  compounds.  tion  of  were  subjected  the  the  experiments  physiological during  stresses  changing  in  in the  would give  environmental  and addition,  from the  It  to which  In  was some  these  over  pools  cover  exudation  of  experiments  two  the  phase  tidepools.  were m o n i t o r e d  w a s made t o m o n i t o r  this  been  important  changes  an a t t e m p t  that  have  tidepools  determined.  chemical  intra-specific  in  was  of  to  particular  o n some  1978 o n two  abundance  a  an  algae  report  these  substances  and a n i m a l s  of  have  matter  relation  species  dis-  recently  chemical in  temper-  the  organic  species  several  each  in  affect  could play  to  The a l g a l  and t h e of  wish  summer o f  a t h r e e month p e r i o d . were  I  too  light,  Only  dissolved  been c o n s i d e r e d  absence  Biological  competition  oxygen,  a n d p H a n d how t h e s e m i g h t  factors  the  dissolved  algae  hoped indicaalgae  conditions.  6  MATERIALS AND METHODS  The S t u d y  Site  The Island, of  two  British  Georgia.  surrounds level  were  at  (MLLW,  lb)  tively.  at  Canadian Chart 1)  and 3.7  Levels  of  with  the pools  using  Point  the  on  Bath  la),  in  the  a series  of  tidepools  of  about  Datum).  m (pool of  Standard  chosen  tidal  respec-  cycles  and C u r r e n t  a reference  zero  a b o v e MLLW  53 a n d 4 1 cm  and r e l e v a n t  as  m above  The p o o l s  2)  Canadian Tide  Atkinson  4.5  Strait  port  surveying  and  Tables Silva  instruments  employed.  Measurement  of  Light Model L I - 1 8 5 in  located  (Figure  containing  a height  Bay as a s e c o n d a r y p o r t . were  Canada  a n d h a d maximum d e p t h s  determined  (1978),  ledge  island  4.4 m (pool  (Figure  s t u d i e d were  Columbia,  A rock  the  tide  were  tidepools  intensity  and Chemical readings  quantum meter,  a vertical  surface  Physical  position .  except  1  in  the  with  Factors  w e r e made u s i n g the probe  depth vs.  Li-Cor  ( M o d e l UWQ 2 1 9 2 )  R e a d i n g s w e r e made a t  case o f  a  each  light  pool's  intensity  profiles. Temperature salinity, YSI  °/oo)  Salinometer,  and s a l i n i t y  profiles, were  (given  as p a r t s  as m e a s u r e d w i t h  determined  over  a  per  thousand  calibrated  a 24 h o u r  period  7  F i g u r e 1. Map o f B a t h I s l a n d , B r i t i s h C o l u m b i a , and e n v i r o n s ( l A ) and d e t a i l (IB) showing l o c a t i o n o f t h e t i d e pools studied. y  8  in  each of  three  September,  consecutive  months  equipped w i t h  Standard b u f f e r s used f o r  (Strickland  a Beckman M o d e l  (pH 7 . 0 0  o x y g e n was m e a s u r e d b y  done on e a c h w a t e r  sample,  t o mg  portable  electrode. Seattle)  were  The  the  Winkler  Three  replicate  and t h e  average  method  titrations value  was  C^/l.  Sampling  of  39502  a n d 1 0 . 0 0 Amachem,  1968).  converted  cover  a Beckman Expandmate  and Parsons  Quadrat  and  calibration.  Dissolved  were  August  1978).  pH was m e a s u r e d u s i n g pH m e t e r  (July,  Methods  surface  areas  each a l g a l  of  species  the  pools  and t h e  contained within  determined using quadrat frames. 2 i n ( 1 0 cm) s q u a r e s was u s e d f o r  A  ( 1 . 5 m)  pool  1,  percentage them were  o  frame  and a  divided 2 ( 4 m)  2 frame  divided  The number to  of  calculate  algal  each  were  surface (all  Abbott square  Frequency,  (25  squares  species  1974 o r for  into  squares  of  The p e r c e n t a g e  which were  and H o l l e n b u r g and t h e n  for  was u s e d  for  pool  e n c o m p a s s e d b y e a c h p o o l was area.  dominance  determined  cm)  and,  identified  cover  1976)  was  first  calculated  for  the  subsequently,  each species  entire  to  each  Widdowson  estimated  importance  according  used  for  using  2.  Cox  pool. values (1972).  2 X  species  relative  to  association Rhodomela  values larix  were  assigned  (Turner)  C.  to  Agardh  all  species  (Kershaw  9  1964). Standing larix  crop measurements  and P r i o n i t i s  lyallii  were  Harvey  obtained  by  for  scraping  a  R. series  o of  (10  cm)  sections  ious  combinations  were  determined  dry weight Chemical  was  to  the  after  chemical  taken, Total  Langlois  (1975) set  S e a w a t e r was u s e d as centrations  of  calibration  and t h e  phenols  carbohydrate 1951) of  cover.  analysis  laboratory.  Spectrophotometer  total  percentage 5 days  at  apparent  species  Algal  at  90°C  of  tidepool  frozen  (or  in  dry  until  var-  weights  constant  phenols  a blank,  tidepool  (Dubois  et  constituents changes  in  in  al.  in  the  of  the  returned  SP 8 0 0  but  Attempts  the  low  (e.g. over  the  Scanning  (445  nm).  known,  were used  and p r o t e i n  pools  concentration  ml  concentration  water.  to  and  wavelength  seawater  1956)  due  100  were measured u s i n g  and v a r y i n g ,  estimation  the  Ice  on a Unicam Model  phloroglucinol  in  water,  on Dry  a constant  proved unsuccessful  these  two  achieved).  samples were  method o f  these  Measurements  For water  of  containing  to  confor  of  measure  (Lowry  e_t  concentrations there  were  no  time).  RESULTS AND DISCUSSION  The p r o b l e m o f  monitoring  physical  and  chemical  al.  10  conditions by  the  in  systems  s u c h as  stratification  of  these  within  an i n d i v i d u a l  pool.  Figure  2,  salinity  cally of  pool  1 had  and 11-25  °/oo  ocean wash o r  of  water  algae  in  the  able  rock  these ture  predominated are  surface,  ranges  in  as  11-28  the  is  the  pool  over  that  broad.  verti-  on t h e  amount  pool.  Pool  less less  of  two p o o l s  is  dense The  the  avail-  tolerance  The d e g r e e also  2  mixing  surface.  most  their  in  °/oo  but  salinity,  the  occur  shown  depending  a low  indicating  stratification  of  entering  over  complicated  w h i c h may  example,  distributed  salinities  is  stratification,  therefore,  pools  varying  For  horizontally  salinity  ocean w a t e r ;  layer  factors  precipitation  showed a s i m i l a r with  tidepools  to  of  tempera-  shown  in  2 Figure for  2.  the  Swedish  tidepool their  Ganning  biota  lack  of  (1971)  tidepools are  desiccation problems  (1924),  other  of  temperature  parameter The graphs ing  in  depths  ities  for  hand,  and i t s the  Figure  to  results  He b e l i e v e s  that  that  habitat  because  of  tolerance  and n o t  because  of  they might  has  stressed as  encounter. the  Klugh  importance  the main  limiting  organism.  and s a l i n i t i e s averages  and l o c a t i o n s that  similar  studied.  fluctuations  tidepool  3 are  reported  which  temperatures  indicate  he  confined  osmoregulatory on t h e  has  of  readings  throughout  precipitation  which  the  (as  appear taken  pools.  land  at  Low  surface  in  the  varysalin-  runoff)  ( FRESH WATER  STRAIT OF GEORGIA (23 /oo)u  Q - "1700 10 - -1200 2 0 - -1100  21. 2H TEMPERATURE <°C)  3 0 " -1050  POOL 1  43  DEPTH (cm)  J  nooo LIGHT_2 (uE m  F i g u r e 2. D i a g r a m o f p o o l s 1 and 2 showing r a n g e s i n s t r a t i f i c a t i o n o f s a l i n i t y (°/oo) and t e m p e r a t u r e and l i g h t a t t e n u a t i o n w i t h d e p t h .  _ sec ) x  12  is  occurring while  ities the  indicate  pool,  the  the  pool  degree  reducing  its  the  of  o c c u r when t h e  The r e s u l t s Figure  3 clearly  respiration  of  sequently  the  dissolved  the is  level  produced  (1924)  of  the  than  determines, cannot  rence  tidepools.  cation at  of  light  be  depths  out  biota  the  in  but  up,  to  the the  the  high, the  is  top  con-  readings,  as  shown  the  is  day  not  due  to  at  and the  in  night  is  pool's  pH o c c u r s .  If  factor  obvious,  have  is  true  species  then  occur-  measurements  actually  received  by  As m u c h as  pool  CG^  rather  accurate  20 cm o f  when  suggested  a wholly  is  level  Klugh  are not  surface  in-  consumed,  this for  sub-  the  oxygen  (1928)  to  active  evolved  a reduction  present.  in  photosynthesis  determined by,  pools.  pool's  stable  Conversely,  immediately  in  process,  During  goes  of  of  oxygen  limiting  irradiance  striking  filtered  is  intensities  the  varying  pool  tidepools  Although not light  ratio  the pool.  the  pH a l o n e  surface  the  oxygen,  of  the  in  in.  Johnson and Skutch  acidity  in  is  salin-  occurring  pH d e t e r m i n a t i o n s  and a l o w e r i n g  that  in  The more  place;  respiration  and l a t e r  and,  the pool.  the  CC^ i n  of  in  taking  pH o f  creased presence of  the  and h i g h  evaporation  tide  indicate  is  exposed,  present.  of  algae  photosynthesis  of  volume  centrating course,  salts  is  off  on a  indi-  algae  35% o f  reflected water  the  of  the or  calm  is  13  Figure  3-.  E n v i r o n m e n t a l m e a s u r e m e n t s made i n  pool  2 during  were  July  6-7  three  18-19  (  ).  Surface  (  ),  Tide  light  consecutive months.  levels  intensities  eratures  in  salinity  and d i s s o l v e d  scale  is  divided  hours  on t h e  relation  to  horizontal Readings surface  August  °C,  into  Q  zero  tide  were  taken  and i n  the  at  study.  level the of  in  is  above -2  uE m parts  level  indicated  graph marked each  temp-  thousand The  the the the  of  time at  pool  1200 in  fine  "TIDES".  10 cm b e l o w pool.  ,  beginning of  dates  September  sec per  by  and  MLLW. -1  i n mg 1 * .  The  1  sampling •) a n d  intervals  a depth of  center  in  (O^)  two hour  through  are  (°/oo)  oxygen  the  (  i n meters  (I )  salinities  day o f  line  are  14-15  The  pool  pool's  '-'Q_  N3  U l  o  o o "I  LA  r f i r  pH  / oo  TEMPERATURE  I  o  TIDES  15  day  ( F i g u r e 2).  w i t h turbulence  T h i s i r r a d i a n c e would decrease  even f u r t h e r  (e.g. wind or waves) on the p o o l s u r f a c e  or w i t h an i n c r e a s e i n d i s s o l v e d o r g a n i c matter i n the p o o l s . Surface i n s o l a t i o n a l s o c o n t r o l s the temperature of the water i n the pools to a great extent e i t h e r by h e a t i n g the water d i r e c t l y or by h e a t i n g the surrounding  rock.  D i s s o l v e d oxygen i n the pools i s again with photosynthesis  and,  t h e r e f o r e , pH.  correlated  F i g u r e 3 shows  that as pH i n c r e a s e s so does the l e v e l of d i s s o l v e d oxygen. The  oxygen l e v e l s of the pools were h i g h even at the  h i g h e r temperatures  (warmer s o l u t i o n s h o l d l e s s d i s s o l v e d  gas), i n d i c a t i v e of the h i g h p h o t o s y n t h e t i c r a t e and, p r o d u c t i v i t y of the algae i n the p o o l s .  A levelling  hence, effect  i n the oxygen content of the pools would occur when they are reimmersed and the turbulence of waves r e l e a s e s or d i l u t e s the excess gases present.  P y e f i n c h (1943) s t a t e s  that the d i v e r s i t y of the f l o r a i n a rock p o o l i s the f a c t o r i n governing  key  the range of pH and oxygen v a l u e s .  T h i s appears to be the case i n the two pools s t u d i e d here. The  l a r g e r and more densely populated p o o l  (see below)  shows the g r e a t e r r a t e of change i n the f o u r v a r i a b l e s so far discussed.  The  c o n t r i b u t i o n of phytoplankton  to the  p r o d u c t i v i t y of the p o o l has not been c o n s i d e r e d s i n c e i t should be minimal when compared to t h a t of the flora.  attached  16  Tabulation  of  the  quadrat  sampling  data  revealed  2 that  pool  1 had a surface  o 165 ( 1 0 cm) quadrats) 2 10.88 m (encompassing of  percentage  for  the  algae  dominance,  in  two p o o l s  the  was  dominant  Kylin  and R a l f ' s i a than  portant  in  in  the  the  lower  are  were more  pool,  pool  crop  m  frequency  both pools.  crusts  lower  Standing of  in  1.65  (encompassing  and p o o l 2 a s u r f a c e a r e a o f 2 1 3 4 ( 2 5 cm) quadrats). Values  cover,  larix  pool  area of  importance  shown i n  Table  Geramium  washingtoniense  important  a n d P.  than  and  lyallii  in  I.  the  the  higher.  measurements  (as  g dry weight,  gave  averages  of  higher  was m o r e  in  Rhodomela and P r i o n i t i s  R.  1.81  im-  g d.  wt.)  (+ 0 . 5 6 )  g  2 and 7.67 pools.  (+ 2.53) Using  a sphere,  one  Rhodomela  in  attempt ing I  to  and t h a t  in  surface  area  can e s t i m a t e  the  overall  standing  1  (with  to  be  the the  there  was  the  level  of  antibiotic,  ( 1 0 cm)  for  correlate  per  then  The t w o p o o l s high percentage  of  R.  closely  production sole  total  day. they  its  0.39  each g d.wt.  phenols  per  formulae  that  Therefore, total  the  pool  Rhodomela  found  g respectively  total  source)  with  g d.wt./l  of  phenols  and volume crop  spherical  of  this  shape)  and (assum-  volume. alga  0.5  in  mg/1  pool 3 day.  of  Rhodomela exudes  1.41  mg  of  If  we a s s u m e h a l o p h e n o l s  to  be  s h o u l d be e f f e c t i v e were  initially  larix  in  each.  at  such  chosen because It  of  of  phenols  pool  reached  both  was h o p e d  levels. of  the  that  1  Table I. Dominance, frequency and importance values f o r the algae i n the two t i d e p o o l s . * The f i r s t f i g u r e i n each column r e f e r s to p o o l 1 and the second f i g u r e t o p o o l 2. SPECIES  COVER(%)  DOMINANCE  FREQUENCY  IMPORTANCE (7„)  Rhodomela larix  45.9*  45.1*  0.76  4.91  90.3  96.0  39.8  38.6  9.3  3.6  0.15  0.39  63.6  33.3  16.5  7.4  0.8  20.3  0.01  2.21  7.9  79.9  1.9  23.5  5.8  1.6  0.10  0.17  29.7  19.5  8.4  4.1  38.2  29.4  0.63  3.20  77.6  70.7  33.5  26.5  Ceramium washingtoniense Prionitis lyallii Ralfsia crusts Bare rock  18  since  R.  larix  exudation  of  is  very  vidual  phenolic  be  low  sible in  to  the  for  get  time  tration  of in  Figure  stress  the the  4.  pools The  of  that  cellular  metabolites  a stress  response. response  Sieburth Exudates  tidepool believe by  may a c t Jensen  the as  the  pool  others  phenol 18-19  by  the  1975).  the  at  is  shown just  indicating of  extra-  of  and  be  this  McLachlan  1969).  in  Some  a form of  sufficient  pool  algae might  (Craigie  important  substances  the  concen-  examples  and Jensen  that  appear-  of  the  each case,  literature  (Langlois  in  in  are numerous  e x u d a t i o n may b e  once  increased  subsequent  in  pos-  present  t o maximum v a l u e s  phenols)  probably  algae,  in  An  plants  to  was o n l y  increased production  Sieburth  antibiotic  1978)  the the  1969,  organisms that  increase  There  are  on the  indi-  proved  a reflection  The c h a n g e  (e.g.  in  time.  and t h e i r  of  phenols  as m e a s u r e d o n J u l y  levels  reimmersion  of  placed  It  total  was j u d g e d  sampling.  the p o s s i b i l i t y  posal  the  phenols  physiological  1964,  pool water  of  pool water  plant  the  any p a r t i c u l a r  the  from t h i s  purposes.  of  the  concentrations  at  in  content,  the  pool water  ance  kind  in  an i n d i c a t i o n  exudation  before  However,  identification  of  in  phenolic  compounds  components  rate  the  in  characteristic  c o u l d be m o n i t o r e d .  too  high  the  of  researchers  waste  components  (Langlois  ecology  product of  1975,  concentrations  in  dis-  exudates Ragan the  and  POOL 1  POOL 2  F i g u r e 4. T o t a l phenols i n pool 1 and pool 2 over a twenty-four hour p e r i o d given as p a r t s per m i l l i o n o f phloroglucinol. The p e r i o d o f dark i s i n d i c a t e d by stippling.  20  tidepool. exudates is  Although has  yet  to  some i n d i c a t i o n  bacteria  the  antibiotic  be d e m o n s t r a t e d that  exudates  and i n v e r t e b r a t e  and S i e b u r t h  1966,  activity  life  Sieburth  of  algal  conclusively,  have  there  some e f f e c t s  history  and Jensen  stages 1969,  on  (Conover Langlois  1975) . o  The X that the  only  of  pool  in  the  Rhodomela  association. (see  Chapter  and t h a t lower  (personal Since III),  it  is  by  Ceramium i s  a common  show no  37% o f  the  if  epiphyte  hence  the  close  halophenols these  algae,  effect  gross  must  a significant  constitute this  is  in  is  compounds  their  against  the  levels form of  of  matter  fraction or  portion  into  (1969)  produc-  epiphyti-  the  indicates  productivity  organic  active  a large  released  such h i g h  especially  dissolved  release  however,  an a l g a  question  red  and B u r l a k o v a  as  of  in  that  in  Rhodomela  contains  unlikely  function  1977 . ) ,  indicate  associated  with  may b e r e l e a s e d  present;  closely  II)  Ceramium.  as much as  Whether  (Table  association  Ceramium a l s o  Work b y K h a i l o v that  data  observation  by. Rhodomela w o u l d  zation  no  pool.  s h o w some a n t i b i o t i c tion  association  Rhodomela and Ceramium a r e  upper  occurs  species  of  passive of  the  is  complex molecules  alga Phenols  exudates.  unknown  carbon  compound  an  (DOM) .  these  environment.  waste  of  at  budget  One  would  production, such  as  Table I I . X s p e c i e s a s s o c i a t i o n o f a l g a e i n t h e two p o o l s . p r o b a b i l i t i e s given are that the plant i n question i s not a s s o c i a t e d w i t h Rhodomela l a r i x . POOL 1 SPECIES Ceramium  ~  .  washingtoniense Prioriitis  ~  TTT lyallix  Ralfsia  ~~  crusts  -  X  2  ' P(%)  The  POOL 2 X  2  P(%)  8.77  1  0.34  59.0  1.52  23.2  0.50  48.8  2.51  14.1  0.80  78.6  22  halophenols  or polyphenols.  T h e s e compounds must, i t  seems, have some o t h e r f u n c t i o n accumulates biotic  until  t o t h e c o n t r a r y , t h e y c a n be  substances.  reviewed  and  Silva  the l i t e r a t u r e  and  on  Bittner  further  evidence  considered  (1979) have  the a n t i b i o t i c  anti-  recently  activity  of  algal metabolites. In  this  chapter  I have p r e s e n t e d p r e l i m i n a r y e v i -  dence f o r t h e e x u d a t i o n o f p h e n o l i c compounds by R. found  i n tidepools.  a stressful  This habitat  from  one/promoting, a t l e a s t  i n c r e a s e d p r o d u c t i o n o f DOM Whether t h e s e undetermined.  If antibiotic,  p o o l organisms  c o u l d be  a l l indications  substances.  as a n t i b i o t i c s  their  is  i n t h e c a s e o f Rhodomela,  as p h e n o l i c  compounds f u n c t i o n  larix  action  remains  against  tide-  e n h a n c e d u n d e r t h e i n c r e a s e d en-  vironmental p r e s s u r e s i n h e r e n t to t h i s  habitat.  CHAPTER  II  BROMOPHENOLS OF RHODOMELA  LARIX:  CHEMOTAXONOMY OF MORPHOLOGICAL FORMS  23  24  INTRODUCTION  Algae diversity they  the  in  phenols.  the  in  this  1973,  and Amiya  Chevolot-Magueur et  al.  1976,  and C r a i g i e 1979,  et  Combaut 1978,  Kurata  form of  family  et  al.  Lundgren  and Amiya  suggested  debated  (Fenical  1975).  species  are  useful  within  a single  Rhodomela variable Two o f upper  in  in  forms--one  intertidal  intertidal  to  zone,  upper  al.  It  is  et  1976,  1979, use  1964,  differentiating (Fenical  as  other  Augier  subtidal  zone  of  Ragan et  al.  closely  that  and these  related  1975). in  several forms.  in  the  the middle  and  the  Kurata  1965)  agreed  tidepools to  al.  taxonomic  morphological  common t o the  1978,  Pedersen  Columbia occurs  distinct  e_t  1975,  generally  habitat  distributed  e_t a l . al.  in  terpen-  Kurata  Pedersen  Their  (Peguy  British  and sometimes  these  et  1980).  has been  1975,  the  which  rich  are w i d e l y  Saenger  in  compounds  halogenated  Weinstein  1978,  unique  especially  either  1976,  markers  compounds  is  (Fenical  1975,  al.  are  carbon-halogen  The b r o m o p h e n o l s  among g e n e r a Kurata  of  Rhodophyta  The R h o d o m e l a c e a e  compounds or  division  and abundance  contain.  these oids  of  lower  seashore--are  25  grouped w i t h i n  a single  To d a t e , for  all  bromophenols  (Weinstein  et  vestigations essary  to  Since mass  related  the  should  spectrometry  ration  to  in  the  fact  of  m e t h o d was  considered  species  the  this  1967).  the  forms  two  considered  of  examined  in-  became  (tidepool the  same  and  species.  for  the  sepa-  halometabolites,  and i n t e r t i d a l for  their  nec-  chromatography-  commonly u s e d  suitable  Agardh.  During  it  gas-liquid  algal  C.  intertidal  study,  is  tidepool  species  lower  e_t a l .  combined  and i d e n t i f i c a t i o n of  of  (Turner)  present  be  (GLC-MS)  comparison  the  Katsui  whether  technique  larix  come f r o m t h e  1975,  determine  intertidal)  R.  collections  have  al.  taxon,  forms  by  a this  assignment  at  level.  MATERIALS AND METHODS  Collection  of  Samples R.  larix  Island.  Samples of  the  were  collected  After  removal  washed i n  fresh water  Dry  Voucher  Ice.  logical  Extraction  in of  September,  visible  the  to  the  of  TMS  the method o f  of  on  plants  laboratory  have been p l a c e d  University  forms  1978,  epiphytes,  and r e t u r n e d  and P r e p a r a t i o n general  and i n t e r t i d a l  early  specimens  Herbarium at  In  tidepool  in  British  the  (1978)  Bath were on Phyco-  Columbia.  Derivatives  Pedersen  of  was  26  followed.  One g o f  homogenizer 5.0)  with  25 m l  and s o n i c a t e d  filtered ethyl  combined and t h e The w a t e r  layer  reextracted  solvent  with  (C),  as  removed a t to  60°C  layer  moved u n d e r  fractions  nitrogen  of  bromophenols,  were  38°C  into  layers  (fraction  were  A).  2 N NaOH a n d B).  acidified  t a k e n up  and t h e  For to  in  each o f  trimethylchlorosilane added t o  vials.  a  final  pH 2  with ethyl  (TMS)  dried  each v i a l  For  and  containing  Chemical  ml  methanol  over  rephos-  the  derivatives  acetonitrile  (Pierce  0.5  M e t h a n o l was  a vacuum d e s i c c a t o r .  trimethylsilyl 25 u l  in  samples were  trimethylsilyl-trifluoroacetamide  15  was  and e x t r a c t e d w i t h  to m i c r o s i l a t i o n  pentoxide  preparation  for  (pH  before.  and t r a n s f e r r e d  were  Virtis  buffer  acetate  (fraction  (15 m i n )  a  The e x t r a c t  pH 8 w i t h  was  in  and p a r t i t i o n e d  The e t h y l  acetate  the water to  ground  (200 W). 1 paper  adjusted  ethyl  The d r i e d  phorous  15 m i n  50 m l ) .  was  1 N HC1, h e a t e d acetate  for  (3 x  t y p e was  0.1 M sodium phosphate  t h r o u g h Whatman No.  acetate  fraction  each a l g a l  of  the  N,0-bis1%  Co.)  as  catalyst  by h e a t i n g  to  60°C  on a VG-Micromass  mass  followed  min.  Analysis Samples were spectrometer liquid  analyzed  interfaced  chromatograph.  to  a Pye-Unicam  A glass  column o f  Series  104  3% S E - 3 0  gason  27  a c i d washed 8 0 - 1 0 0 mesh Chromosorb-W used at  a He f l o w  from 100-250°C s p e c t r a were m/e  73 w a s  m/e w e r e  of  at  30 m l / m i n .  50°C/min  taken  at  Temperature  gave b e s t  70 e V .  excessive;  (Varian Assoc.)  In  therefore,  programming  results.  every only  case ions  was  Mass  an i o n of  at  greater  recorded.  RESULTS AND D I S C U S S I O N  Five of  bromophenols  two m o r p h o l o g i c a l  tures  of  these  tidal  fraction  contained 4;  The the  five  are  given  1:  m/e  are  2,  only)  (100),  271  (27),  222 345  m/e  75  (82), 74  Figure  extracts The  GLC t r a c e 5.  C,  Fraction  2  struc-  (inter-  f o r m o n l y ) , 2_, 3  fraction  mass  spectral  Relative  A and  (intertidal  77  223 347  (100),  (91),  (18), (91),  75  257 360  (74),  351  (24),  353  (15),  423  (53),  438  (32),  440  (56),  442  (35).  data were  intensities  following  (82),  (18),  the  only),  4.  parentheses  (73),  221  shown i n  and  in  Rhodomela l a r i x .  and a t y p i c a l  4 and 5;  following  in  of  identified  1 (intertidal  compounds.  74  pound 2:  A)  B,  3_ ( t i d e p o o l  forms  compounds  compounds  fraction  were  77  425  obtained  (>10%  each m/e. 147  (27),  (45), (36),  only)  Compound 205  259 362  (18),  (36), (27).  (21),  349  (12),  (100),  427  (59),  Compound 3 :  for  m/e  74  Com-  (25),  2 5x  2.5x  2  30  28  26  24  22  CHO  '  20  18  16  14  R = H=1  12  10  8  6  4  2  0  Time (min) 250  225  200  V75  Temperature  150  125  (°C)  F i g u r e 5. T e m p e r a t u r e programmed GLC a n a l y s i s o f Rhodomela l a r i x b r o m o p h e n o l s . The r e l a t i v e p o s i t i o n o f compound 5 i s shown by an i n s e r t e d d a s h e d l i n e .  100  29  75 331 4:  (32),  147  (14), m/e  (27),  345  75  149  (20),  (80),  147  (100),  257  (84),  259  (93),  (20),  434  (34),  436  (41).  347  (48),  149  (100),  335  339  (32),  411  (12),  423  (16),  425  (24),  435  (32),  512  (20),  514  (44),  516  (24).  74  (28),  75  (30), 148  77  (12),  Compound 5 :  137  (13),  (40),  339  (23),  345  (18),  347  (23),  411  (65),  413  (35),  424  (10),  426  (20),  428  (12).  about  3 m (12  Columbia  shores.  distinct, of  the  it  same  is  ft)  form.  facts  of  Saenger  vertical  Although  in  one o r  cannot  If  al.  these  the  other  259  (12),  409  (32),  occur  with-  British  morphologies  are  they  all  used  contain  into  then  of  the  this  the  extracts  are  to  be  quite but  presence  types  is  of  the  one  inter-  considered  (Weinstein  e_t a l .  of  this  arti-  1975, compound  inconsequential.  supposition  such a r t i f a c t s  but  under  can o n l y  the m i l d  I question  extraction  here.  Most workers halometabolites  in  procedures  against of  only  aldehydes  1976),  the production  lations  that  m/e (17),  (13),  on  (28),  bromophenols.  argue  procedures  distribution  their  interesting  extraction et  207  Rhodomela examined h e r e  C o m p o u n d 1_ o c c u r r e d tidal  (12),  139  337  in  (72), 433  (22),  of  198  (16),  335  forms  (12),  337  (100),  two  149  (20),  427  (11),  Compound  147  The  (22),  105  (32),  267  who h a v e  find  distinct  examined marine  sufficient chemical  evidence types  if  to not  algae  for  separate in  fact  popu-  30 distinct  species  C r e w s et_ a l . larix, tidal  differences ration other  at  it  in  this  and N o r r i s  1979).  appears  the  tidepool  that  classed w i t h i n  level.  to  Fenical  al.  bromophenol  credence  1975,  Caccamese e t  s h o u l d be  heteromorphic  further algae.  1977,  however, forms  (Fenical  Similar  species the  chemistry  concept  a single not  chemical  should, of  For  in  1976,  Rhodomela  and  inter-  species,  warranting  sepa-  investigation the  future,  pleomorphism i n  of  lend marine  CHAPTER I I I  HPLC SEPARATION OF RED ALGAL  31  BROMOPHENOLS  32  INTRODUCTION  Since products ceived  increasing  of  the  tography  et  1978,  1972,  Pedersen  Craigie  Stoffelen  (Chevolot-Magueur et  al.  1978,  Glombitza (GLC)  et  (Saenger  al.  1972,  1972) et  Pedersen  II)  separation  leading  this  of  group  et  and F r i e s  see C h a p t e r  al.  al.  gas  layer  chroma-  al.  1975,  Ragan  et  Glombitza  Chantraine  1976, al.  e_t a l .  Kurata  1976,  1975,  Pedersen  et  al.  1979, spectrometry  1978,  Pedersen  Pedersen  phenols.  subsequent Perhaps  Stoffelen  and  DaSilva  and F r i e s  structural the most  1980,  chromatography  have been used e x t e n s i v e l y  to  al.  Combaut  and Amiya  liquid  Pedersen  1974,  et  and  chromatography  Kurata  1979,  1978,  or-  thin  1967,  1972,  column  1976,  re-  been  several  a n d c o m b i n e d GLC-mass  al.  Pedersen  1964),  Weinstein  1972),  (Ragan and C r a i g i e  (GLC-MS)  also  al.  and S t o f f e l e n  e_t a l .  1973,  1974),  Lundgren  Stoffelen  et  et  compounds h a v e  and G r u e n i g et  have  1975).  (Peguy  1973,  natural  halophenols  representing  (Fenical  al.  e_t a l .  These  algae  chromatography  (Kurata  Stoffelen  of  as m a r i n e  1949),  attention.  Rhodophyta  and C r a i g i e  discovery  and A u g i e r  f r o m a number  Paper  1973,  initial  (Mastagli  isolated ders  their  1977,  for  the  elucidation  useful  of  information  33  regarding far  the  examined has  such complex available. of  array  of  instruments  high-performance  are  recent liquid  chromatographic  and t h e r e f o r e  more w i d e l y  organisms  various  tography  for (LC)  of  packed w i t h  generating  up  A variety  of  available  which,  the  of  most  Because tization  as  in  sensitivity to  the  larix  to  of  several  et  al.  red  the  algae  o r g a n i s m on w h i c h  to  to  development  (HPLC)  the  systems  inexpensive  screening Liquid  the point  that  theoretical  of  chroma-  it  and p r e c i s i o n .  rivals  HPLC  beads  are  capable  plates  per  meter.  detection  systems  is  t o m o d e r n LC e q u i p m e n t , of  micro-  of  to  nanogram  sample p r e p a r a t i o n  necessary) this  and t h e  method,  of  bromophenols  in  British  C. A g a r d h ,  Katsui  the  un-  makes  quan-  possible.  ease  GLC n o t  and g e n e r a l l y  compounds.  and s e n s i t i v e  compounds  previous  1975,  of  for  um m i c r o p o r o u s  25,000  achieved by  (Turner)  of  available  application 5-10  in  so  Unfortunately,  method c o m p a r a t i v e l y  and d e t e c t i o n  of  each a l g a  chromatography  when c o u p l e d  determination  species  advances  groups  accurate  separation  tities  expensive  h a s now a d v a n c e d  p a c k e d c o l u m n GLC i n columns  within  come f r o m GLC-MS a n a l y s e s .  However,  make t h i s  halophenols  having  chemical e_t a l . test  resolution  chose  Columbia. received  was  to  and  apply  from three  it  common  Rhodomela the  investigations  1967) the  I  (deriva-  scrutiny (Weinstein  c h o s e n as  effectiveness  of  a  model  the  HPLC  34  method.  MATERIALS AND METHODS  Chemicals Standards alcohol (3),  (2)  of  2,3-dibromo-4,5-dihydroxybenzyl  (lanosol),  3,5-dibromo-4-hydroxybenzyl  4-hydroxybenzoic methyl  ether  (Atlantic  acid  (14)  Regional  benzaldehyde HPLC g r a d e chemicals the  acetonitrile  of  The  provide  crude  extracts.  procedures  part  produce  mg  a small  ( 3 0 uM) amount  and  Nova  J.  S.  Craigie  Scotia).  3,4-dihydroxy-  from A l d r i c h  Chemical  Scientific.  from various  syntheses  sources  additional  the  small  were performed  standards of  the  Co.  and  All  other  and  were  utilized  are w e l l  a single  product. alcohol  slightly  on a  was  involved.  (1).  The  re-  the  Approximately  slowly  cooled methanol  with  not  known and f o r  was  micro-  for. comparison products  quantities  sodium b o r o h y d r i d e of  3,5-dibromo-  obtainable.  3-Bromo-4,5-dihydroxybenzyl 1.0  by Dr.  Halifax, (TMA)  Purification  due t o  provided  from Fisher were  (5),  Bromophenols  following  to  most  quality  Other  scale  action  chloride  alcohol  3,5-dibromo-4-hydroxybenzyl  Laboratory,  and s o l v e n t s  Synthesis  and  were purchased  highest  attempted  (6)  were k i n d l y  Tetramethylammonium  of  3-bromo-4,5-dihydroxybenzaldehyde  added  to  containing  35  2 . 0 mg  (1.0  uM)  0.1 ml water free  the  of  3_.  When t h e  was a d d e d t o  hydrolyze  the  reaction  hydrochloric hr  mixture  acid  methyl  methyl  ceased,  hydroborane  added.  a mixture  ether  ether  f r o m !L a b o v e ,  (HC1) w e r e  on a steam b a t h ,  benzyl  the  had  was.  of  (4).  two  To 0 . 3  drops  After  unreacted  1_ a n d  of  sulfoxide  dissolved  anhydride  (Albright  in  0.3  ml  dimethyl  of  and Goldman 1965)  2 N HC1 w e r e  methanol.  Heating  2 N for  to  yield  added t o  methyl 1.0  on a steam b a t h  for  a single  mg  ether (0.4  for  15  uM)  Two  2 in  compound has  g i v e n by Lundgren  et  al.  1979.  3.4- dihydroxybenzaldehyde starting  material.  crystals  ethanol  = 0.38  and p r o t o n m a g n e t i c reported  in  the  this  Freshly g,  Concentration  produced white (yield  (1.8  of  which were  g,  of  The  8.  detailed  M) w a s u s e d  the  reaction  recrystallized  spectra  ml  been  sublimed  2 1 % ) , mp 1 7 8 - 1 8 0 ° C .  resonance  above  0.13  0.5  produced  procedure  preparation  ml  min  (8).  one h r  uM)  product.  (9).  the  (0.4  and 0.2  3,6-Dibromo-4,5-dihydroxybenzaldehyde for  one  its  One mg  and h e a t e d on a steam b a t h  2.3- Dibromo-4,5-dihydroxybenzyl drops  ml  found. (7).  2_ w a s  of  heating  2,3-Dibromo-4,5-dihydroxybenzaldehyde  acetic  and  alcohol.  3-Bromo-4,5-dihydroxybenzyl of  reaction  as solvent from  Infrared  agreed w i t h  those  references.  3.5- Dibromo-4-hydroxyberizaldehyde  (10).  One mg  (0.5  uM)  36  of  5_ w a s w o r k e d u p a s  produced to  three  10 t h e  acetal Aqueous  products  other  (12_)  addition  to  the  are  thought of  the  which yielded  sisted  heating  for  the  that  was  the  In  to  hemi-  be  the  starting  aldehyde  15 m i n  dioxide  1.0  in  same a s  in  m e t h a n o l was r e p l a c e d  ml  material. caused  total  oxidation  sole the  addition  product  alcohol  con(5)  methanol.  ethyl  the  by  as  mg o f  0.5  reaction  b y HPLC.  An a l t e r n a t i v e  2,3-Dibromo-4,5-dihydroxybenzyl procedure  this  and c o n t i n u e d h e a t i n g  aldehyde.  a n d 2 mg m a n g a n e s e  However,  as m o n i t o r e d  (13)  procedure of  7_ a b o v e .  products  and a c e t a l  acid  conversion  in  ether  (11).  preparation  of  The  8_ e x c e p t  ethanol.  Chromatography HPLC w a s p e r f o r m e d linked  to  constant sisted  a Variscan wavelength  of  on a V a r i a n Model  of  280 nm.  40% a c e t o n i t r i l e  (in  The e l u t i o n glass  set  at  TMA a n d d i b a s i c  (buffer).  The pH o f  solvent  the  was  solvent  distilled  10 mM e a c h o f  con-  water)  sodium  adjusted  a  phosphate  to  3.2-3.5  c o n c e n t r a t e d HC1. Initially,  and w a t e r optimal dards  LC  634 S S p e c t r o p h o t o m e t e r  containing  with  5000  in  elution  (both  solvent the with  gradient  containing strength  shortest the  elution with additives)  for  time.  best  80%,  acetonitrile  was u s e d t o  resolution  of  Once e s t a b l i s h e d ,  40% a c e t o n i t r i l e  solvent  determine  the  stan-  isocratic  proved  the  most  useful  and  economical.  Octadecylsilane (Varian) mode.  were  All  used  roughly  for  compounds  concentrations  of  Evaluation  of  analyzed  the  1-10  for  selectivity  and e f f i c i e n c y :  (4:6)  B, A p l u s  only;  10 mM TMA;  D,  additives. cation to  chromatographic factor  (k ),  retical in  these  land  1  a whole. parameters  relative  plates  (n)  1974):  used to  in  give vol-  Method and a m i x t u r e  the  evaluation  to  of  solvent  of  column water  C, A  containing  independently  gave  an i n d i v i d u a l  plus both  an  retention  («.) , n u m b e r  as  (R).  follows  four  capacity of  theo-  Formulae  (Snyder  indi-  component  From t h e s e measurements calculated:  of  solvent  and  (pH 3 . 2 ) ;  were  are  the  acetonitrile  and r e s o l u t i o n  calculations  phase  Injection  addition  10 mM b u f f e r  effectiveness  t h e m e t h o d as  were  i n methanol  (in  40% a c e t o n i t r i l e  the  reverse  standards  systems  A,  Each system used  of  the  MCH-10  ul.  solvent  above)  in  responses.  5_ a n d 6_ w e r e u s e d  system described  as  Chromatographic  Three m o d i f i e d compounds  Micropak  1 mg p e r m l  detector  typically  of  separations  about  equivalent  umes w e r e  columns  and  used Kirk-  38  n = 5.554 /  R = s/n  V  4  t  height, ° ' k'-^  = retention t  o  and k*2=  (1 e l u t i n g tion  = retention capacity  before  indicated  represent  by  2). the  in min,  time  of  factors The  \  +  J  1  w,= peak w i d t h  a nonretained for  terms  bracketed  column e f f i c i e n c y ,  2  (c)  (b)  time  k'  / I k'  «  (a)  where  lW  r-oj-  t \  two in  at  solute  compounds  the  (a),  selectivity  and  and  separated  resolution  letters  half  (b)  equa-  and  (c)  capacity  respectively. Collection  and E x t r a c t i o n  Rhodomela l a r i x . a l g a were Visible  in  the  having  fresh water  Following  lyophilization,  (yield  to  pass  subtidal  fresh weight zone  and i m m e d i a t e l y  through  = 447 g d . w t . ) .  2 kg  been removed,  washed i n  Wiley m i l l  Algae  Approximately  collected  epiphytes  of  the  the  Soxhlet  Bath  plants  frozen  algae were  a 2 mm m e s h  off  at  ground  of Island. were  -80°C. in  a  screen  extraction  of  the  dried  39  material ether,  using  a series  chloroform,  fractions. (£40°C)  ethyl  about  50 m l .  (42.9  g)  deposited  tract  was r e m o v e d b y  dipotassium  to  dryness,  b y HPLC f o r  of  boiling bath.  salt  of  each o f  redissolved  volume of  in  four vacuo  solid  the methanol  (Weinstein fractions  ml methanol  as  the  et  al.  was  and  ex-  taken  analyzed  bromophenols.  80% m e t h a n o l The e x t r a c t s 60°C f o r  Kylin ground  were  and t h e  times  ethyl  acetate.  combined and t h e  solvent  were  5.0  in  in  for  acidified  to  one h r  The e t h y l removed i n  on a  pH 2 w i t h  remaining water  ml methanol  lyallii  a Waring blender  15 m i n a n d f i l t e r e d .  vacuo  t a k e n up  and P r i o n i t i s  and r e f l u x e d  removed i n with  of  gave  and i d e n t i f i e d  four  1.0  in  amount  lanosol  the  in  each a l g a were  warmed t o  large  filtration  petroleum  and m e t h a n o l )  was r e d u c e d The  Ceramium w a s h i n g t o n i e n s e Ten g o f  (light  on c o n c e n t r a t i o n  sulfate  One m l  solvents  acetate  Each f r a c t i o n  to  1975).  of  Harvey. in  steam  1 N HC1,  The m e t h a n o l extracted  acetate vacuo.  layers The  and used f o r  was  three were  residues  HPLC  analyses.  RESULTS AND DISCUSSION  Separation  of  Figure  Standard  Compounds  6 illustrates  the  separation  of  14  bromophenol  40  i  0  '  i  2  "  i  4  1  1  6  1  1  1  8  1  10  1  1  12  1  1  r  14  T i m e (min)  F i g u r e 6. R e v e r s e p h a s e HPLC s e p a r a t i o n o f f o u r teen bromophenol s t a n d a r d s . The r e l a t i v e p o s i t i o n o f compound 11 i s shown by a d a s h e d l i n e .  41  T a b l e I I I . R e t e n t i o n t i m e s , c a p a c i t y f a c t o r s and r e t e n t i o n s o f standards o f bromophenols s e p a r a t e d r e v e r s e p h a s e HPLC. t = 1 . 8 min. o  relative by  r  COMPOUND  t  R  k'  a  (min) 1)  3-bromo-4,5-dihydroxybenzy1 alcohol  3. 1  2)  2,3-dibromo-4,5-dihydroxybenzyl alcohol  4 . 15  3)  3-bromo-4,5-dihydroxybenzaldehyde  4 : 55  4)  3-bromo-4,5-dihydroxybenzyl methyl ether  4. 9  5)  3,5-dibromo-4-hydroxybenzyl alcohol  5 .,4  2. 0  6)  3,5-dibromo-4-hydroxybenzoic acid  5 . ,8  2 ., 2 2  7)  2,3-dibromo-4,5-dihydroxybenzaldehyde  6 . ,3  8)  2,3-dibromo-4,5-dihydroxybenzyl methyl ether  7. . 7  9)  2,5-dibromo-3,4-dihydroxybenzaldehyde  8. .6  10)  3,5-dibromo-4-hydroxybenzaldehyde  9. , 2 5  I D  2,3-dibromo-4,5-dihydroxybenzyl ethyl ether  1 0 .. 0  12)  H e m i a c e t a l o f 7 above  1 0 .. 4  13)  Acetal  14)  3,5-dibromo-4-hydroxybenzyl methyl ether  o f 7_ above  0 . 72 1 . 82 1 . 13 1 . 17 1 . 53 1 . 12 1 . 72 1 . 16 1. 11 1 .,13 2 . ,5 1 ., 3 1 3. , 2 8 1 ..15 3. , 7 8 1 ,, 1 0 4 ., 1 4 1 ,, 1 3 4 ,. 6 7 1 ,. 0 2 4 ,. 7 8 1 .. 2 7  12 . 7  6, . 0 6 1 ,. 0 2  1 2 ,. 9  6, . 1 7  42  standards  at  Retention  times,  data  for  a flow  these  reverse  more p o l a r aldehydes polarity Methyl  compounds  of  alcohols  while  their  ether  of  lanosol chain  of  are  is  by r e v e r s e  broad  that  repeated  increased  to  before  first,  of  the p-  (9)  by  increase polarity.  The  show t h e  time.  The  nonpolar  counterparts. to  expected,  followed  quite  included  as  separation.  rendered  Chromatographic at  retained  the  versus  com-  ethyl  effect  Also  of  of  interest  the  o-  give  level the  of  point  elution  little  gave  peaks.  injections  of  of  column, long  retention  all  occurs.  showed times  have  S u m e r e e_t a l . is  column r e a c t i v e Such g r o s s  with  noted  retained  compound r e t a i n e d  that  lanosol  acids  Some a u t h o r s  (van  bromophenols  success:  an i r r e v e r s i b l y  results the  Method  separation  on t h e  and a l d e h y d e s  and t a i l i n g  the  is,  functions  on r e t e n t i o n  attempts  pound e v e n t u a l l y however,  elute  retention  (7).  irreversibly  very  compounds  psi).  I I I .  reduces  p h a s e LC m e t w i t h  no r e t e n t i o n  Table  function  hydroxyl  length  the  Initial  filled  ethers  (11)  in  o-Dihydroxy  decreased p o l a r i t y  Evaluation  the  1120  and r e l a t i v e  given  and a c i d s  free  dibromoaldehyde  was  of  an o - d i b r o m o  to  the  factors  (pressure  a normal phase  and e t h e r s .  pared  is  of  1 ml/min  are  order  that  and e t h y l  extended  of  capacity  The e l u t i o n the  rate  com-  1979) ; probably sites  are  contamination  43  must ens  be  considered  column In  solvent tivity most  life,  undesirable  not  an e f f o r t  modifiers  to mention to  converted method  to  chosen  are  reduce  were  and e f f i c i e n c y  phenols  since its  these  while  acidic  ( k = 10  phenoxide  anions  in  improve  LC o f  (van  S u m e r e e_t a_.  peak  symmetry by  simply agent  and e f f e c t i v e l y to  (Burce, tively  irreversible  the  solvent.  personal bonds  to  packing material. ary  phase  on a r e v e r s e  a completely  nonpolar  I  in  the  believe  occurs  1979)  as  there which  it  generally  is  still  can be  been used This  for  in  the  with then  the  overcome re-  this  modifier  groups  purpose selec-  column  the  station-  exposed  The p o s s i b i l i t y  functions of  first  tailing.  column are  case  the  a phase-altering  environment.  silanol  easily  interacting  phase  to  and a r e  the  silanol  hydrogen bonding  Since  for  adding  Compounds  capacity.  commonly u s e d  communication). unblocked  selec-  ion  however,  TMA h a s  suitable  was  retention, by  efficiency.  capability  suppressing  F o r many c o m p o u n d s , problem of  is  short-  improve  solution,  retention  technique  on  problems,  maintaining  This  improves  effect  quite  to  inevitably  sought which would  suppression. phenols  it  in  lanosol,  the is  to of  packing,  as  therefore .  negated. The c o m b i n a t i o n improves  separation  of  ion  capability  suppression  a n d TMA  considerably.  The  addition results  44  of  the  evaluation  then  in  this  table,  combination  creases  are  a closely  component  a high  column  level.  related  Analysis  of  applied  a crude  test to  of  the  Figures  in  trace  Lanosol  is  and t h e  aldehyde  Methyl  ethers  the  The  amounts  (7) 8,  of  of  still be  from in-  compounds Selectivity  although  in  the  maintained increased  at  by  rate.  extract  the  component in  the  14)  a complex m i x t u r e  Most  in  s y s t e m comes w h e n  separations  Soxhlet  7 and 8.  (4,  is  a chromatographic  the major  and a l s o  and a c i d .  buffer,  flow  separation  of  As n o t e d  separation  can e a s i l y or  IV.  and  Extracts  extract.  fractions  appear  the  the  strength  Rhodomela  plant  shown i n  of  separately  selectivity  alcohol  Selectivity  solvent  The  in  taken  Table  system e f f i c i e n c y  reducing  four  shown i n  TMA t r i p l e s  reduced by a d d i t i o n  dual  is  each component  column e f f i c i e n c y  5 and 6, is  of  of  achieved  of  the  R.  petroleum in  the  ethyl  appear  ether  only  in  for  as  the  are  compounds fraction.  chloroform  acetate  such  larix  standard  it  fraction,  fraction.  the  methanol  extract. In previous of  lanosol  studies  and r e l a t e d  be a r t i f a c t s  of  1975,  Saenger  et  ethyl  and m e t h y l  fall  There into  and m e t h y l  have been  procedures  1976).  ethers  aldehydes  compounds  isolation al.  the  considered  (Weinstein  is this  ethers  little  et  doubt  category;  to  al. that  however,  Table IV. R e s o l u t i o n as a f u n c t i o n o f c o l u m n e f f i c e n c y , selectivi t y and c a p a c i t y u s i n g d i f f e r e n t s o l v e n t systems i n the s e p a r a t i o n o f 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l a l c o h o l and t h e c o r r e s p o n d i n g a c i d . A b b r e v i a t i o n s used are given i n the t e x t . SOLVENT SYSTEM A)  Acetonitrile:water  EFFICIENCY 11 8  SELECTIVITY 0  CAPACITY  R  -  0  B) A p l u s  lOmM b u f f e r  8.72  0.28  0.46  1.12  C) A p l u s  lOmM TMA  9.22  0.88  0.44  3.61  D)  lOmM TMA  8.96  0.26  0.45  1.05  B plus  46  F i g u r e 7. HPLC a n a l y s i s o f t h e p e t r o l e u m e t h e r and c h l o r o f o r m f r a c t i o n s from the l a r g e s c a l e ( S o x h l e t ) e x t r a c t i o n o f Rhodomela l a r i x . The a s s i g n m e n t o f peaks i n p a r e n t h e s i s i s u n c e r t a i n .  47  Figure 7  48  F i g u r e 8. HPLC a n a l y s i s o f t h e e t h y l a c e t a t e and methanol f r a c t i o n s from the l a r g e s c a l e (Soxhlet) e x t r a c t i o n o f Rhodomela l a r i x .  49  7  50  the  presence  of  aldehyde  c a u s e s me t o  believe  stituents  the  of  procedures presence existence fore,  be  that  algae.  (Pedersen  of  these  in  7_ t h r o u g h o u t  s u c h compounds  1978,  see a l s o  species  In  so  far  con-  extraction II)  The p o s s i b i l i t y  this  Analysis study  two  of  Other  other  washingtoniense  and P r i o n i t i s  the  bromophenols.  presence of  of  these  algae  contain  major  compound  species  are n a t u r a l  Chapter  examined  fractions  show  of  the  their  should,  there-  reevaluated.  Chromatographic  tracts  four  Even t h e most m i l d  compounds.  other  all  two  species  bromophenols, (2).  The  was v e r i f i e d  standard  at  different  Prionitis, bromophenols, m-dibromo traction  acid of  by  like  the most (6)  of  algal  Algae species,  lyallii,  were  appear  in  Ceramium  examined  T h e LC t r a c e s Figure  of  lanosol  cochromatography  the  9.  Both  solvent  strengths  Rhodomela, abundant  and a l c o h o l  being (5_).  the  lanosol A more  array  of  authentic rates.  a variety and  of  the  careful  a n d C e r a m i u m may l e a d  an even w i d e r  one  this  and f l o w  contains  ex-  only  in  with  for  for  Ceramium c o n t a i n i n g  identity  both P r i o n i t i s  identification  Red  to  phenolic  exthe  com-  pounds . In for  the  conclusion,  determination  Column s e l e c t i v i t y  HPLC i s of  a rapid  and s e n s i t i v e  bromophenolic  and e f f i c i e n c y  in  compounds  reverse  in  phase  method algae.  51  T  -1  0  i  1  2  1  1  1  1  4  6  Time  1  1  1  8  1  10  1  1  12  1  1—  14  (min)  6  1  0  i — i — i — i — i — i — i — i — i — i — i — i — i — i —  2  4  6  Time  8  10  12  1  14  (min)  F i g u r e 9. Reverse phase s e p a r a t i o n o f bromophenols e x t r a c t e d f r o m P r i o n i t i s l y a l l i i (bottom) and Ceramium w a s h i n g t o n i e n s e ( t o p ) . Compounds i n parenthesis are t e n t a t i v e l y i d e n t i f i e d .  52  separations  c a n be i m p r o v e d b y t h e a d d i t i o n o f m o d i f i e r s  which a l t e r  t h e n a t u r e o f t h e s t a t i o n a r y p h a s e o r sup-  press  i o n i z a t i o n when, f o r example,  acidic metabolites with  solvents  separation  i s necessary.  containing modifiers  Isocratic elution produces  adequate  o f a wide v a r i e t y o f bromophenols  many t y p e s o f f u n c t i o n a l g r o u p s . the need  the separation o f  f o r chromatographs  forming devices  Under  these  equipped with  i s eliminated.  containing conditions  complex  gradient \  CHAPTER IV  TEMPORAL, INTERPOPULATIONAL  AND  INTRATHALLIAL  MEASUREMENT OF LANOSOL LEVELS IN RHODOMELA LARIX  53  54  INTRODUCTION  Fenical  (1975)  metabolites , rather bolic the  pathways,  alga  has  than being  function  containing  The n a t u r e  is  however,  ter  (DOM) b y a l g a e  Wangersky may o n l y also  be  1978,  (Langlois  1975,  The of  study  bromophenols  Our p r e s e n t derived fully  compounds literature bility  is  in  in  the  well  produced  of  of  in  red  algae  consists  extractions  invariably plants.  Table  meta-  providing  if  it  exists,  organic  (Hellebust Such  mat-  1974,  materials  but  they  may  substances  1978).  in  the  temporal  received  only  of  which,  lead  primary  dissolved  antibiotic  has  halo-  environmental  metabolism,  variations  algal  system  1979).  Ragan and J e n s e n of  in  advantage,  documented  as  red  a selective  this  products  from algal must  an e x o c r i n e  excretion  knowledge  done,  of  that  involved  Ragan and J e n s e n  be w a s t e actively  in  them w i t h  advantage. unclear;  suggested  to  abundance  little  attention.  quantitative unless  very  data  care-  underestimation  V summarizes  in  this  area  and i l l u s t r a t e s  the  data  even  for  multiple  the  the  available  great  extracts  of  of  variaa  single  species. As a r e s u l t ,  the  physiological  and  ecological  T a b l e V. A summary o f d a t a a v a i l a b l e o n q u a n t i t a t i v e a s p e c t s o f b r o m o p h e n o l s i n r e d algae. A l l e s t i m a t e s a r e based on t h e abundance o f t h e n a t u r a l l y o c c u r r i n g p h e n o l , n o t o n d e r i v a t i v e s w h i c h may h a v e b e e n p r e p a r e d i n i s o l a t i o n p r o c e d u r e s . The compounds l i s t e d a r e as f o l l o w s : 1) 3 , 5 - d i b r o m o - 4 - h y d r o x y p h e n y l a c e t i c a c i d , 2) 3,5-dibromo-4h y d r o x y p h e n y l p y r u v i c a c i d , 3) p e r d e s m e t h y l c y c l o t r i b r o m o v e r a t r y l e n e , 4) 2 , 3 - d i b r o m o 4 , 5 - d i h y d r o x y b e n z y l m e t h y l e t h e r , 5) 2,3,2',3'-tetrabromo-4,5,4',5 -tetrahydroxydiphenyl methane, 6) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l a l c o h o l , 7) 2,3-dibromo-5-hydroxyb e n z y l - 1 ' , 4 - d i s u l f a t e ( p o t a s s i u m ) , 8) 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l a l c o h o l , 9) 2 , 3 d i b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e , 10) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l e t h y l e t h e r , 11) 3 - b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e , 12) 3,3'-dibromo-4,4',5,5 -tetrahydroxybibenzyl, 13) 3 - b r o m o - 4 , 5 - d i h y d r o x y b e n z y l m e t h y l e t h e r , 14) 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l methyl e t h e r , 15) 2 , 4 - d i b r o m o - l , 3 , 5 - t r i h y d r o x y b e n z e n e , 16) 5,6,3',5'-tetrabromo-3,4,2',4',6'p e n t a h y d r o x y d i p h e n y l m e t h a n e , 17) b i s ( 2 , 3 , 6 - t r i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l ) ether. * = e s t i m a t e d abundance. ! = tentative identification. 1  1  COMPOUND  ALGA  REFERENCE  % d.wt.. (7 w . w t . ) 0  Halopytis H.  incurvus  pinastroides  Odonthalia  corymbifera  1  (0.003)  2  (0.002)  1  (0.003)  2  (0.003)  3  (0.003)  4  (0.01)  5  0.  dentata  Polysiphonia  brodiaei  !  Chantraine  et  Combaut  al.  Kurata  et  et  al.  al.  1973  1978  1973  (0.004)  6  (0.008)  7  (2.0)  6  0.5-2.0*  Craigie  8  0.1,  (0.024)  i b i d . , Glombitza S t o f f e l e n 1972  8  0.5,  0.005  Pedersen Lundgren  et et  al. al.  1974, 1979  9  0.08  Lundgren  et  'al.  1979  and G r u e n i g  1967  and  T a b l e V.  P.  continued.  lanosa  10  0.006  5  0.005  6  1-5,  7  1.0  Glombitza  2-3  H o d g k i n e t a l . 1966 Ragan and C r a i g i e 1978 and S t o f f e l e n  1972  P.  morrowii  11  0.07  Saito  P.  urceolata  11  0.08  Kurata  12  0.005  13  0.02  14  0.03  .6  0.02-0.06-*, (0.003)  C r a i g i e and G r u e n i g 1967, G l o m b i t z a and S t o f f e l e n 1972  8  0.003  Craigie  4  0.005, (0.009)  Katsui et Weinstein  a l . 1967, e_t a l . 1 9 7 5  9  0.03  Katsui  al.  7  (0.04)  Weinstein  4  0.06  Kurata  7  9.2  9  0.02  6  (0.009)  10  (0.009)  15  (0.009)  16  (0.004)  Rhodomela  R.  R.  confervoides  larix  subfusca  Rytiphlea  tinctoria  Symphyo c 1 a d i a  latis'c'u'la  17  a n d Ando 1955 et  al.  1976  and G r u e n i g  et  1967  et a l .  1975  and A m i y a 1975  Chevolot-Maguer  Kurata  1967  et  al.  and Amiya 1980  1976  57  significance  of  Bromophenols  have been  (Silva  bromophenols  and B i t t n e r  on q u a n t i t a t i v e be h e l p f u l  in  1979),  brown algae  red in  For  a l g a Rhodomela the  levels  species  (see  of  Chapter  examined were three  tative  chemical  a clearer phenols  red  shown t h a t of  chose C.  the major  to  over in  examination of  plant. of  polyphenols  in  regarding examine  Agardh  a one y e a r  and w i t h i n  the  the  for  in  content  alga would  raison  the  this  period.  a single  d'etre  of  their  changes  Also  among  plant  an e x t e n s i v e  this  might  a knowledge  to  bromophenol  of  available  bromophenol  was h o p e d t h a t  understanding  in  importance  (Turner)  III),  It  which  I  populations  (intrathallial).  occurrence,  reason  differences  distinct  their  information  lanosol,  antibiotics is  useful  larix  unknown.  be e f f e c t i v e  concentrations  this  is  information  have  can p r o v i d e  significance.  algae  no  their  (1978) in  but  of  assessing  seasonal v a r i a t i o n  red  shown t o  aspects  Ragan and J e n s e n  in  quanti-  lead  to  bromo-  algae.  MATERIALS AND METHODS  Collection  of  Unless collected off  Bath  in  Algal  Samples  otherwise the  Island  lower during  indicated, intertidal the  highest  all to  algal  upper  tide  of  samples  subtidal the  were zone  month.  58  The p l a n t s large  for  area,  in  plastic  or  frozen  pools  bags  one week  at  90°C.  in  particulate  with  a Virtis  matter  vacuo. with  of  dry  residue  phenol  of  taken  from the in  off  water  up  near  samples  of  plant,  to  in  having  each a l g a l in  been  sample  boiling  on a steam  807  bath,  and t h e m e t h a n o l layer 60°C  was for  re-  acidified 15 m i n  continuously  5.0  o  acetate  (to  extracted  was  ml methanol  refor  analysis. other  the  content  the  triplicate;  The e t h y l  chromatographic  parts  branches  lab  tide-  in  and t h e n  (4 h r s ) .  For measurement ferent  from  zone  the  N HC1, h e a t e d  w e r e w o r k e d up  parison  a  immediately  low i n t e r t i d a l  reflux  filtered  sulphates)  Collections above)  came e i t h e r  microhomogenizer  The r e s i d u a l  acetate  moved and t h e subsequent  the  then used  One g o f  one h o u r  was  1.0  ester  ethyl  done  duplicate,  Following  hydrolyze  were  in  ground  pH 2 . 0  from the  taken  methanol.  to  samples  to  over  Columbia.  extractions  were  moved i n  or  and r e t u r n e d  Algae were  Other  selected  Procedures  All  for  randomly  epiphytes  ice.  Island  British  Extraction  was  over  (-20°C).  Bamfield,  dried  sample were  cleaned of  on B a t h  weights  each  two  same w a y  among  w i t h many l a t e r a l s ,  for  (see  relative  com-  populations.  lanosol 1.0  locations  concentration  g each o f  branches  in  growing  with  few  dif-  tips,  laterals  59  and h o l d f a s t s  was e x c i s e d  Chromatographic  scribed length  in of  sorption  the  chromatographic I I I .  In  2 9 2 nm w a s u s e d lanosol  c h r o m a t o g r a p h was Injections potential cedure.  determination  Chapter  of  of  this rate  L a n o s o l was authentic  part was  half  height  by  Calibration  to mg/g  t h e maximum  1975).  ab-  The  calibrated used  de-  a wave-  to  loop.  minimize  quantification  comparison w i t h curves peak  method and c o n v e r t i n g  and s u b s e q u e n t l y  performed  pro-  1 ml/min.  identified  standard.  al.  the  however,  is  always  of  was  system and e l u a n t s  a 10 u l  m e t h a n o l w e r e made b y m e a s u r i n g at  before.  lanosol  this et  with  of  case,  since  volume were  in  flow  this  (Weinstein  fitted  this  error The  as  Methods  Quantitative b y HPLC u s i n g  and e x t r a c t e d  dry weight  for  areas  the  lanosol by  t o mg/ml  the  in  width  of  lanosol  as q u a n t i t i e s  in  the  alga. The  life  were p r o v i d e d  history  by U l l a  data  for  Visscher  algae  in  (personal  these  samples  communication).  RESULTS AND D I S C U S S I O N  Figure  10 s h o w s  bromophenols  f r o m R.  indicator  the  of  a typical  larix.  level  of  HPLC s e p a r a t i o n  L a n o s o l was  total  of  c h o s e n as  bromophenols  in  the  the an plant.  60  T  0.02AU  1  i — i — i — i — i — i — i — i — i — i — r 0  2  4 TIME  6 (min)  8  .10  F i g u r e 10. A t y p i c a l HPLC s e p a r a t i o n o f Rhodomela l a r i x b r o m o p h e n o l s u s e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f l a n o s o l ( i n d i c a t e d by the a r r o w ) . The c h r o m a t o g r a p h i c c o n d i t i o n s a r e l i s t e d i n the text.  61  The p e a k  corresponding  can e a s i l y  1.2  to  levels  range  in winter  are  of  the  well  separated  by  levels  basis  essentially  in  this in  three  times  (1978)  larix  11).  The  those  of  found  brown algae which  method.  R.  (Figure  and  the  similar  they  examined  polyphenols. The r e s u l t s  are  shown i n  among t h e the  months field  of  Table  three  variation  tidepool  the  VI.  populations  observed  for  f o r m had h i g h e r  than  did  either  range  lanosol  examined here this  species  lanosol  the  of  levels  subtidal  comparisons  is  levels  typical  of  (Table  V).  in  summer  Bath  the  Island  The  or  Bam-  populations.  higher  levels  during  the  (spring  of  minimum.  period  of  a n d summer)  For  the  but  in  early  cover  by e a r l y  in  by  during  the  brown algae  the  spring  to mid  Rhodomela e p i f l o r a  the  smaller  and s u b t i d a l  tized  have p o i n t e d  maximum p o t e n t i a l  Rhodomela  tidepool  (1978)  polyphenols  when c o l o n i z a t i o n  Both  interpopulational  The w i d e  Ragan and J e n s e n  of  lanosol  Ragan and J e n s e n  maxima and m i n i m a  is  be q u a n t i f i e d  3.8% on a d r y w e i g h t  summer m o n t h s .  for  lanosol  and a c c u r a t e l y  The y e a r l y is  to  out  occur  fall  increasing  summer. decreases  algae  is  to  months  at  a  observed.  typically  epiphy-  a maximum  Thereafter until  not  and w i n t e r  same p a t t e r n w a s are  the  epiphytization  marine  plants  that  the  in winter  epiphyte  diversity none  is  REPRODUCTIVE  STATUS  50 -veg-  -HI  ® -  veg-  II-  40  40  30  30  r > a o w o 1  rt  /"-\  3  3 ft) rt S3 rt  UP 0Q  20  20 £  rt  10  10  J  M  M  J TIME  1J  A  0  N  (months)  Figure 11. Seasonal v a r i a t i o n o f l a n o s o l c o n t e n t i n Rhodomela l a r i x (solid l i n e ) a n d c h a n g e i n t h e d r y t o w e t w e i g h t r a t i o o v e r t h e p e r i o d o f one y e a r (dashed l i n e ) . T h e d e v i a t i o n f r o m t h e mean f o r a l l s a m p l e s i s i n d i c a t e d b y v e r t i c a l b a r s and t h e r e p r o d u c t i v e s t a t e o f the p l a n t s by h o r i z o n t a l bars (® = t e t r a s p o r i c p l a n t s , o_ = c a r p o s p o r i c p l a n t s , v e g = v e g e t a t i v e p l a n t s ) .  Table VI. Q u a n t i t a t i v e comparison o f l a n o s o l concentrations i n t h r e e d i s t i n c t p o p u l a t i o n s o f Rhodomela l a r i x ( c o l l e c t e d i n A u g u s t 1978) . POPULATION  B a m f i e l d (low intertidal  )  Bath I s . (low i n t e r to h i g h s u b t i d a l ) Tidepool  (Bath I s . )  LANOSOL (mg/g d.wt.)  REPRODUCTIVE CONDITION  -,-,  „ tetrasponc  /. n Q A \  11.8 (+0.30) ±  g  Q  (  o.17) ^-  21.7 (+0.55)  v e g e t a t i v e and tetrasporic vegetative  64 observed. The d a t a  for  intrathallial  (Figure  12)  show m a x i m a l  content in  the  the  youngest  algal  phytized other  and most  thallus. in  the  species  older  of  c o u l d be a r e s u l t in  of  these p o r t i o n s ,  Leaching riod  of  levels  out  or  less of  the  rapid  ceeds.  Higher  drop  temperatures,  or  the  rate  or  the p l a n t s . active vide  growth w i t h  epiphyte  Again, the  as w i t h  in  in of  of  the  to  the  understanding  the  content  of  of  the  lead  as  This lanosol tips. a  to  of  pe-  lower could  summer  pro-  water affect  lanosol  period  levels  many  This  of  the  role  are  might  levels  lanosol  epi-  during  and h i g h e r  factors  the  of  of  plant.  correlating in  as  growing  would  entire  in  compound  1979).  compounds  insolation  changes  thallus  concentrations  lanosol  study  the  invariably  (Ballantine  the  change  has been  and a n t i f u n g a l  ecological  may b e  of  (summer)  for  lanosol  of  should  lanosol  in most  proin  control.  Lanosol bacterial  almost  a combination  Further  a better  of  growth  levels  is  lower  exudation  of  in  growing portions  contrasted  compounds  explain  levels  algae  the  as  active  the  regions  marine  levels  rapidly  Rhodomela  variation  the  shown t o agent  brown a l g a l significance control  of  be an e f f e c t i v e  (Silva  phenols of  and B i t t n e r  1979).  (Ragan and J e n s e n  bromophenols  pathogens  anti-  rather  to  than  red  1978),  algae  epiphytes.  65  LANOSOL (mg/g d.  wt.)  45.0  TIPS  42.1  REGION OF DENSE LATERALS  21.3  REGION OF FEW LATERALS  8.6  HOLDFAST  F i g u r e 12. Lanosol content i n d i f f e r e n t p a r t s o f t h e Rhodomela l a r i x t h a l l u s , ( c o l l e c t e d f r o m t i d e p o o l s on Bath I s . , B.C. i n September, 1 9 7 9 ) .  66 The  control of herbivory  excluded. and,  The a u t h o r h a s shown  a t much h i g h e r  sulfate  salt  snails. ators  (Chapter  effect  o f metabolism Biochemical  that  as r e p e l l a n t s a g a i n s t  tidepool  p o t e n t i a l pred-  these  compounds a r e w a s t e  ( P e d e r s e n e_t a l . 1979) seems h i g h l y evolutionary  lead to the formation  trends  do n o t g e n e r -  o f b y - p r o d u c t s more  p o t e n t i a l l y more t o x i c t h a n t h e s t a r t i n g  from which they should  are synthesized  accumulations occur  when e n v i r o n m e n t a l synthesis  From t h e r e s u l t s conclude  that  storage during  c o n d i t i o n s would f a v o r  and, s u b s e q u e n t l y ,  increased  o f these  materials Neither  products  since  the p e r i o d increased  food  photo-  storage.  e x p e r i m e n t s one c a n  the scope o f p o s s i b l e f u n c t i o n s  p h e n o l s has b e e n c o n s i d e r a b l y n a r r o w e d . careful  complex  (Swain 1 9 7 7 ) .  b r o m o p h e n o l s be c o n s i d e r e d  t h e i r minimum  f o r bromo-  Until  a more  a s s e s s m e n t o f t h e e f f e c t i v e n e s s o f l a n o s o l as  antibiotic,  antiepiphyte  o r a n t i h e r b i v o r e agent  s i g n i f i c a n c e o f bromophenols i n the e c o l o g y  algae w i l l will  lanosol  i t s dipotassium  could extend to other  possibility  unlikely.  the  VI) that  as w e l l .  products  and  compounds i s a l s o n o t  concentrations,  are e f f e c t i v e  This  The  ally  by t h e s e  remain unclear.  be p r e s e n t e d  Work  i n Chapter VI.  done a l o n g  i s made, of red  these  lines  CHAPTER V  EXUDATION OF BROMOPHENOLS BY RHODOMELA LARIX  67  68  INTRODUCTION  The e x u d a t i o n reducing  substances  been r e c o g n i z e d have r e c e n t l y  of  tannins,  polyphenols  ("Gelbstoff")  (Wangersky  examined  the  whose  When s u c h a l g a e their  yellow  antibiosis bacteria  against  to  Less  release  ized,  Ragan and J e n s e n  control  of  (or  of  1969,  carbon-halogen  pounds  (Khailov  that  of  of  of  1975).  (Silva are  Red a l g a e  compounds  exuded,  then  ranging  animals  contain 1975),  1979). their  If  (Conover  red  1975).  algae,  Langlois  organic  or  (Khailov  from  Langlois  1969,  phenols  (Fenical  and B i t t n e r  in  1975)  matter  been c h e m i c a l l y  total  light.  effective  marine  dissolved  have n o t  in  be  1969,  of  in  of  marine  exudates  (1979)  accumulation  organisms,  and B u r l a k o v a  amounts  estimates  the  long  produces  enhanced  and Jensen  the  alga  known t o  a s DOM) h a v e b e e n made  Langlois  antibiotic  is  and s e s s i l e  are  exudates  only  is  exudation  this  tidepools,  Sieburth  studied  copious  both  release  many g r o u p s  1966,  These but  in  planktonic  w h i c h a r e known  (DOM).  occur  of  has  1978).  brown exudates  and S i e b u r t h  to  rate  other  by brown algae  A s c o p h y l l u m nodosum and shown t h a t polyphenols  and  character-  carbohydrates and  high  Burlakova  levels  many o f  of  which  are  it  is  these  com-  presence  in  ocean  waters  69  in  significant  logical  concentrations raises  q u e s t i o n s as  producing  them  to the advantage  (Pedersen  Such compounds may  function  herbivory or f o u l i n g animals  or  e_t aL.  1974,  s u c h as pH,  and  processes and  rate  and  Fenical  salinity  and  physical  i n the p h y s i o l o g y o f macrophytes  S t e n g e l 1974)  and  should also  s u c h as t h e r a t e o f r e l e a s e  chemical  p l a y an (Biebl  affect  o f DOM  as  im1962,  exudation polyphenols  study I examined  the  o f s e l e c t e d e n v i r o n m e n t a l m o d i f i c a t i o n s on  the  quality  In t h i s  o f e x u d a t i o n by Rhodomela l a r i x  Columbia.  During  t h e day  coastal pools containing this  coloration Chapter  1975).  and  an  increasing  is attributable  attempts  components o f t h e e x u d a t e s  of  apparent  Much o f  at the  in  amount  t o p h e n o l i c compounds  I ) ; however, e a r l i e r  of the i n d i v i d u a l  species.  (Turner)  tidepools  y e l l o w t o r e d d i s h brown d i s c o l o r a t i o n becomes in  alga  epiphytization,  temperature  C. A g a r d h , o b t a i n e d f r o m h i g h i n t e r t i d a l British  the  o f m a r i n e a l g a e by o t h e r a l g a e ,  as o t h e r s u b s t a n c e s .  effects  eco-  microorganisms.  portant part Soeder  afforded  in controlling  In the marine environment, factors  interesting  this  (see  identification were  un-  successful . I h a v e now  reexamined  the exudates  controlled  laboratory  experiments  to c o n t r o l  e x u d a t i o n w i t h i n normal  and  am  ranges  o f R.  larix  able not of  in  only  environmental  70  factors,  but  the major  also  to  phenolic  identify  component  and q u a n t i f y of  separately  exudates.  MATERIALS AND METHODS  Collection  and M a i n t e n a n c e  Algae were intertidal Plants water  or  were  in  collected  from the  cleaned  over  filtered  returned  in  rotary  (60 rpm)  (330 uE. this  m~ sec 2  _ 1  ,  "adjustment"  1 "low  light period  for  the  lab  urn),  of  Bath  high  fresh  plants  rechecked  under  = 12:12)  sea wrapped  on  medium  in  gyro-  light  15°C.  algae were used  washed  epiphytes  flasks  at  Island.  were  for  culture  48 h r s  the  the  laboratory  the  form"  cycle  zone  in  washed i n  to  the  (0.45  and m a i n t a i n e d shakers  2.5  In  Specimens  subtidal  epiphytes,  ice.  sea w a t e r  Algal  from tidepools  upper  of  and i m m e d i a t e l y  i n newspaper  of  After exudation  experiments. Experiments  Involving  Ten grams collections) transferred Erlenmeyer up  of  each a l g a l  were washed (in  0.22 to  control to  to  and t h e  light.  temperature  Total  Phenols  (subtidal  urn f i l t e r e d  100 m l  by wrapping  exclude  of  type  An a d d i t i o n a l  aluminum f o i l shaker  in  duplicate)  flasks.  as a d a r k  the  Measurement  of  pair  the of  them i n The  same i n  several  to  tidepool  sea w a t e r  flasks  flasks  raised  and  were 20°C.  and  250  was  ml  set  layers  of  returned Total  71  phenols  were measured at  the method o f  Langlois  o n a Pye U n i c a m M o d e l and c o n v e r t e d  to  0,  4,  8 and 24 h r s  (1975).  Optical  SP-8500 UV-VIS  phenol  according  density  was  to  read  Spectrophotometer  concentrations  a s ppm o f  phloro-  glucinol. Exudation under Three  Controlled  chambers were Light  used at  conditions  0-1000 For  Different  attenuation  layers Light  of  Environments  temperatures  were  uE m~ s e c "  set  to  the  lower  which were  intensity.  flasks  For  25 a n d 35 ° / o o , pHs  (6, 7 ,  with  glass  35 ° / o o ) Into to.be  or  used at  full  100 m l  over LI-  a series  of  or  per  parts  the  each f l a s k tested,  water  pH was (in  10.0  The  20°C  light Vita  for ml  sea w a t e r  increasing  chamber. multiple boxes. meter  source  the  higher  exudation were  set  salinities  sea w a t e r  (or  partially  adj usted w i t h  freshly  for  was  each  set  of  15,  and  either  HC1 o r  up  (5,  diluted  evaporated  dilute  in  lights  thousand of . s a l i n i t y )  duplicate) g of  from  foil-lined  and f i f t y  8 and 9), f i l t e r e d distilled  of  30°C.  185 Q u a n t u m  output  filtered  growth  20 a n d  the  24 D u r o - t e s t  Two h u n d r e d  containing  as b e f o r e .  of  in  E15)  intensities,  a M o d e l UWQ 2 1 9 2 p r o b e .  e a c h chamber was a b a n k  light  light  a Li-Cor  10,  a range  c h e e s e c l o t h were p l a c e d  with  (72T12)  so t h a t  Conditions  (Model  of  c o u l d be a c h i e v e d  was m e a s u r e d w i t h  fitted  Environmental  for NaOH.  conditions  washed and g e n t l y  blotted  tidepool the  R.  growth  Dissolved the  larix  chambers oxygen  end o f  obtained  Extraction  of  chamber,  the  pH 2 w i t h  The w a t e r  was  ethyl  anhydrous vacuo  period Five  1 wk a t  90°C)  averaged  for  experimental  8 hrs  each  algae  filtered  conditions.  initially  (8 h r s )  as dry  above  estimation  and  an  determinations as  of  of  weight were  (38°C),  the  made  and  the  the  initial  Exudates  was  off  removed  from the  and t h e medium  extracted with  acetate  at  indi-  layers  were  After  residue  chromatographic  ethyl  was  acetate  combined the  bath.  (3x100  and d r i e d  solvent  t a k e n up  growth  acidified  1 N HC1 a n d w a r m e d 15 m i n o n a s t e a m then  in  samples.  flask  sodium s u l f a t e .  and used f o r  placed  replicate  the  and Chromatography  After  and t h e  appropriate  g samples washed and b l o t t e d  dry weights  m  for  flasks  were m o n i t o r e d  viability.  drying  to  levels  the  algal  (after  values  under  experimental  of  10.0  added and t h e  the  cation  for  were  in  ml),  over  was  removed  0.5  ml  methanol  analysis.  RESULTS AND DISCUSSION  A definite stances moderate  is  increase  Brentamine  o b s e r v e d when Rhodomela  irradiation  laboratory  in  larix  and t e m p e r a t u r e s  environment  (Table  VII).  in This  reactive is a  sub-  exposed  to  controlled release  occurs  Table V I I . E x u d a t i o n m e a s u r e d as t o t a l p h e n o l s o v e r a t w e n t y f o u r h o u r p e r i o d by t i d e p o o l and s u b t i d a l ( b o t h i n t h e l i g h t and d a r k ) forms o f Rhodomela l a r i x . Q u a n t i t i e s g i v e n a r e as parts per m i l l i o n of phloroglucinol. COLLECTION  TIME  (hours)  4  8  24  5.7  6.4  46.5  Subtidal form (m light)  o i c 21.5  n/ n 24.0  ^ o i 63.1  Subtidal form (in dark)  6.3  6.5  Tidepool  form  /. -i . i  J  \  47.3  74  from algal to  the  "shock"  habitat rather or  thalli  to  the  total  more  intense  form.  chamber.  whether the  it  periods  (Biebl  "stress"  of  sities  1962,  and c o o l e r  water  counterpart  increases  the  which  for  rate  HPLC a n a l y s i s exudate  (Figure  shows  13).  compound i n on Swedish exudation even f o r  of  perature  of  to  the  of  longer  the  many  to  and  than  year,  Increased case  of  light  inten-  high  inter-  its  such  subtidal  macro-  the  lower  and  physio-  1974).  stress  in  be  the major  (1974)  have  it  appears  a widespread  their  natural  conditions  does  phenols  Polysiphonia  is  exuded are so  does  light  releases  to  and i n  individual  Therefore,  still  increases  the  al.  bromophenols  lanosol  of  the  et  The e x p e r i m e n t a l of  exposed  the  is  .  exudation.  Pedersen  algae  response  lanosol  shores.  than  natural  larix  adapted  temperatures  of  sea w a t e r  R.  and S t e n g e l  most  of  form of  be t y p i c a l  is  adjust  release  in  i n marine micro-  Soeder  to  of  are kept  insolation  a greater  form,  The r a t e  algae  to  time  from t h e i r  seems b e t t e r  examined  promotes  subtidal  mg/ml  the  tidepool  phenols,  responses  phytes  24 h r  transferred  This would appear  logical  tidal  being  culture  Since  less  the  of  constant  dark.  w h i c h have been g i v e n  given lanosol  the  component  also  found  brodiaei likely  this  zone  that  phenomenon,  habitats.  and r e s u l t s in  in  Table  given  VIII.  exudation  and  As  as tem-  75  F i g u r e 13. HPLC o f t h e t w e n t y - f o u r h o u r e x u d a t e o f t h e s u b t i d a l f o r m o f Rhodomela l a r i x kept i n t h e l i g h t f o r 24 h r s ( s e e T a b l e V I I and p. 7 0 ) .  76 Table VIII. Q u a n t i t a t i v e d e t e r m i n a t i o n o f exuded l a n o s o l and other observations under v a r y i n g experimental c o n d i t i o n s . Unless s p e c i f i e d , t e m p e r a t u r e = 20°C, ° / o o = 2 5 , l i g h t i n t e n s i t y = 330 uE n T ^ s e c - ' a n d pH = 8 . The i n i t i a l o x y g e n c o n c e n t r a t i o n was t a k e n as 0 ppm a n d t h e mean i n i t i a l d r y w e i g h t was 1 . 8 5 g . ( * ) = r e p l i c a t e a t 1000 uE m ' ^ s e c " to determine r a t i o of l a n o s o l t o l a n o s a l t exuded over t h e e x p e r i m e n t a l p e r i o d . -  CONDITION  T E M P. S A L I N I T Y  L I G H T  FINAL pH  FINAL dry wt.  OXYGEN (ppm)  10°C  8.0  1.78  7.02  0.02  20°C  8.0  1.90  6.51  0.05  30°C  4.5  1.41  1.59  0.56  5  5.4  1.33  5.28  0.61  15°/oo  8.6  1.83  5.98  0.04  25°/oo  8.6  1.80  7.13  0.04  35°/oo  8.5  1.85  6.95  0.03  0  6.7  1.66  1.66  0.04  100  7.8  1.93  6.35  0.03  330  8.0  1.90  6.51  0.05  1000  4.9  1.51  5.25  3.0  1000(-H )  4.6  1.35  5.15  3.3  1000(+H )  -  -  -  0.17  6  7.9  1.79  5.19  0.03  7  7.4  1.77  6.25  0.05  8  8.9  1.78  6.51  0.04  9  8.9  1.98  6.86  0.04  /oo  +  (*)  +  P H  LANOSOL (mg/ml)  discoloration effect  is  of  seen w i t h  salinities  in  from several  effect  yields  algae  release to  for  red  al.  levels  (Figure  algae  of  higher 15)  other  under  without  hand,  leaching  The  initial  these  out  of  This  light  them pigment  pH h a d  upon  little  intensities  apparent  highest  damaging  conditions  (respira-  rates  were  in-  damage  The  The g r e a t e r  lanosol  in  including  data).  were  (1970)  floridorubin  halophenols  photosynthesis).  the  Saenger  Varying  Lower  pigmentation  by p l a c i n g  oxygen e v o l u t i o n  on the  algae  of  14).  opposite  salinity.  red pigment  1976).  while  An  increasing  (Figure the  14)^  extended periods.  et  (see  exceeding  to  a variety  exudation  temperatures,  due  of  on e x u d a t i o n ,  the  tion  for  (Saenger  creased to  species  (Figure  highest  extracted  water  hydrolysis  the  t h e medium  has p r e v i o u s l y  distilled  regard  produced  and l a n o s o l  lanosol  t h e medium  of  probably  f r o m dead o r  dying  cells.  this  factor  there  was  rounding to  was  pH o f  all  media,  altered,  was  8.0.  a large the  release  algae,  an a c c u m u l a t i o n  (k  phenol  = 10 ^ )  the of in  of  except In  lanosol  acidic  where  case  where  every into  pH d r o p p e d w e l l  the  those  the water  below  5.0  sur-  due  phenolics  t h e medium.  However,  had  the  cl  phenols  b e e n e x u d e d as  pH c h a n g e w o u l d n o t  sulfate . esters,  h a v e b e e n as  great.  the  observed  As a m e a n s  of  78  F i g u r e 14. E f f e c t o f t e m p e r a t u r e and s a l i n i t y on t h e p r o d u c t i o n o f c o l o r e d m a t t e r by Rhodomela l a r i x d u r i n g an e i g h t h o u r e x u d a t i o n experiment" TEe a l g a e h a v e b e e n removed f r o m the m e d i a t o improve c l a r i t y o f t h e c o l o r s . (see Table V I I I f o r f u r t h e r d e t a i l s o f c o n d i t i o n s )  79  F i g u r e 15. E f f e c t o f pH and l i g h t on t h e p r o d u c t i o n o f c o l o r e d m a t t e r by Rhodomela l a r i x d u r i n g an e i g h t h o u r e x u d a t i o n experiment. The a l g a e have b e e n removed f r o m the media to improve c l a r i t y o f the c o l o r s , (see T a b l e V I I I f o r f u r t h e r d e t a i l s o f c o n d i t i o n s )  80 determining  the  chemical  nature  of  exuded -2  replicate,  illuminated  and e x t r a c t e d w i t h o u t  at  1 0 0 0 uE m  then reextracted  additional  lanosol  constituted  5.2% o f  extraction  (Table  VIII).  lanosol  e x u d e d as  Another esterified  lanosol  is  broken  to  question,  of  this  lanosol  "dirty"  sea w a t e r  f r o m w h i c h R. 24 h r s  (see  at  detected  20°C u n d e r even at. the  Conversion lanosol rates the  exuded/g  of  physical  this  fall  this  species  in  set  arises  upon  III)  light,  fraction  unacidified  conclude  that  whether  added t o  from the  lanosol  Table  VIII  hour  gives  the  in  Figure  modifications  ranges  summer m o n t h s  normally (see  of  conditions  the  range  from about  1-150  other In  order  sulfate  10  ml  tidepool After  could  be  sensitivities.  in  illustrated  or  collected.  no  sulfate  dipotassium  detector  data  dry weight  The  t h e medium.  was  directly  highest the  I  is  the  originally  high  within  into  1 mg o f  and c h e m i c a l  tidepools  within  of  exudation  experiments the  was  The  phenol.  exudation  obtained  larix  acidified  down b y b a c t e r i a  Chapter  used  acidification.  As a r e s u l t , free  , was  acidified.  obtained  question which  after  salt  that  the  microorganisms answer  sec  removed f r o m the  only  is  after  a  -1  being previously  m e d i u m was  lanosol,  mg  comparative  16.  All  used i n  of these  occurring  Chapter  exudation ug/g  into  I),  in  and  rates  d.wt.hr.  for  o.oooi — 1  1  0 100  1  1  330 LIGHT  1000  1  1  5  1  15  I  1  25  SALINITY  35  1 (  6  )  ' 10  1 (  7  )  20  !  1 (  8  TEMPERATURE  F i g u r e 16. Effect of l i g h t , s a l i n i t y , e r a t u r e a n d pH (" ) on e x u d a t i o n o f l a n o s o l by Rhodomela l a r i x .  temp-  )  1  30 (pH).  (  9  )  82  R.  larix  contains  f r o m 1-4%  basis  (see  Chapter  IV).  here,  this  alga  exuding  content hence,  per  hour,  synthesis  ecological ings  is  are  that  significant IV  over in  I  the  period  when t h e  ducive  to  high  lanosol  in  the  fact  due  to  the  levels  a year lowest  exudation plants  of  of  and,  plant.  of  The  these  find-  e x u d a t i o n may b e  more  in  that  the  In  R.  during  The  larix  concentrations  are most  lower  levels  and n o t  to  this  period  epiphytization  of  the  algae  may b e a r e f l e c t i o n  of  increased  to  attempted  be of  (spring  an  exudation  in bio-  low  1978) indicator  maximum to  con-  slowed  (Ragan and J e n s e n to  pre-  of  may  correspondingly  lanosol  Chap-  summer m o n t h s ,  The  exudation  (assuming  of  been  conditions  rates.  bromophenols.  levels  the  lanosol  has  lanosol  during  both polyphenols  higher  turnover  (1979) , i t  and f o u n d  levels  tial  found  total  f r o m May t h r o u g h A u g u s t  increased  phenols)  its  a n d summer m o n t h s .  of  total  weight  release  implications  rates  synthesis  of  of  an a c t i v e  environmental  and bromophenols  of  compound w i t h i n  spring  of  are  cisely  of  0.3-1.6%  Ragan and J e n s e n  increased  in  plants  be  this  rates  on a d r y  intriguing.  in  examined  the  the  of  lanosol  the  indicating  most  As n o t e d  ter  At  and p h y s i o l o g i c a l  thus  suggested  of  poten-  mid- summer) as  epiphytization.  a  response  CHAPTER V I  BROAD SPECTRUM ANTIBIOTIC A C T I V I T Y OF BROMOPHENOLS FROM RHODOMELA LARIX  83  84  INTRODUCTION  The ecology nized  significance  of  land plant  (Whittaker  effort  to  rine  algae  many o f  which are  and B i t t n e r compasses been 1973,  in  Most antibiosis  has  of  we h a v e by  the  for  broad  little  Baslow  class, of  with  been  the  an  marine  therein).  Ma-  metabolites, (Silva  compounds and t h e y  algae  en-  have  (Scheuer  regarding  crude  isolated  and,  1969,  extracts.  and  identified  (Lewin  Bhakuni  these  algal  algal  subsequently,  subject  few o f  several  1962,  and  Wolters  Silva  compounds h a v e  spectrum a n t i b i o t i c  group  recog-  active  these  information  compounds  knowledge  compound o r  in  the  1977).  appeared on t h e 1964,  of  to  there  "secondary"  divisions  from t e s t i n g  Unfortunately,  examined  of  available  of  has  significance  chemical  substantially  1964,. S i e b u r t h 1974).  every  the major  the  l o n g been  recently  The d i v e r s i t y  t h e number  have  has  be b i o l o g i c a l l y  and F e n i c a l  comes  increased  reviews  known t o  all  production  1973 a n d r e f e r e n c e s  a variety  1979).  Faulkner  However,  a similar  virtually  found  Only  (Burkholder produce  antibiotic  populations  1970).  determine  environment  of  benefit  activity.  of  the  of  compounds w h i c h  Hence,  afforded it  been  an  alga  produces.  85  Antimicrobial used,  often  range  of  tests,  provide  although little  biological  rapid,  simple  information  effects  and  widely  regarding  a particular  the  c o m p o u n d may  possess. With ity  of  rally  this  in mind,  a bromophenolic occurring  salt  I  chose  substance,  (here  them m a r i n e .  stituent C.  of  Agardh  all  along  the marine  (Weinstein the  Hollenburg  1976).  activity  et  1953,  1960, tive  Saito  Weinstein study  of  by McLachlan the  effect  ber  of  effort  of  lanosol  algae.  to  the  antibiosis,  both  from p u r i f i e d  occasions has  The o n l y  (1966),  this  quantitative  from extracts  compounds.  ecological to  this of  Allen  activity  study  I  they  and exudates  of  anti-  Dawson  quantitawas  done  examined on a num-  h a v e made  aspects  or  red  the  and  phenolics  and  (Mautner  truly  in which  other  (Abbott  mention  1955,  con-  abundant  b e e n made  antibiotic  In  the  organisms,  (Turner)  is  bromophenols  and two  planktonic include  of  1975).  bromophenol  which  of  phenolic  larix  known o f  and Sameshima  e_t a l .  the major  natu-  2,3-dibromo-  North America  bromophenols  and C r a i g i e  of  of  is  on numerous  biotic al.  Little  significance  however,  is  activ-  and i t s  on a v a r i e t y  1975),  coast  the  "lanosalt,"  a l g a Rhodomela  §_t a_l.  Pacific  physiological alga;  Lanosalt  examine  lanosol,  called  5-hydroxy-benzyl-l',4-disulfate) many o f  to  an  bromophenol  as w e l l  as  86  MATERIALS AND METHODS  Algal  Extraction R.  larix  collected January,  in  and I s o l a t i o n (approx.  the  1980.  upper  2.5  of  kg,  subtidal  The a l g a was  Compounds 500 g d r y w e i g h t )  zone  ( 2 mm m e s h )  and e x t r a c t e d  ratus  a series  solvents  Evaporation amount with  of  of  of  the methanol  solid  (44.7  g)  and r e c r y s t a l l i z e d  for  this  compound w e r e  the  dipotassium  in  extract  w h i c h was  identical salt  of  et  a  1975) .  large washed  Physical  those  in  appa-  al_. a  collected,  lanosol  in  a Soxhlet  produced  to  Island  ground  (Weinstein  from methanol.  sulfate  Bath  lyophilized,  Wiley m i l l with  off  was  data  reported  (Weinstein  for et  al.  1975). L a n o s o l was p r e p a r e d b y et  a l . (1966).  duced  1 2 3 mg o f  identical isolated for  to  the  the  lanosalt  free  Hodgkin  upon h y d r o l y s i s  alcohol.  authentic  compounds was  This  standard.  compound was  The p u r i t y  c h e c k e d b y HPLC  pro-  (see  of  the  Chapter  III  details). Other  R.  One g o f  the method of  larix  i n vacuo  in  extracts hot  were p r e p a r e d  80% m e t h a n o l .  and t h e w a t e r  by g r i n d i n g  The m e t h a n o l  acidified  to  1 g  was  pH 2 w i t h  of  removed  dilute  87 HC1 a n d h e a t e d ethyl left of  acetate a dark  for  15 m i n  (50%)  The q u a n t i t y  of  lanosol  of  Algal  Ten g o f  to  the  sea w a t e r  bright  were  light  for  in to  the  again  for  agar  by  in  antibiotic  1.0  ml  experiments.  was  ascertained  curve  prepared  as  for  (diameter,  the  of  100 m l  20°C w h i l e _ 1  )  for  extracted  1 g extract  with  of  of  being  8 hrs.  with  lanosol  0.22  um  exposed The  ethyl  already  algae  acetate  described.  in  Standard Test  using  this  exudate  test  organisms  for  be  bacteria Agar  tested  plates.  or  or  coli,  cerevisiae  was  plates  done  were  The d i a m e t e r  either dextrose  seeded filter  loaded w i t h  lanosalt  on  and  Sabouraud  and s m a l l  6mm) p r e v i o u s l y lanosol  Organisms  Escherichia  Saccharomyces  fungi. to  in  HPLC.  (Difco)  concentration  at  determination  aureus,  the microorganism  of  the  screening  albicans  (Difco)  surface  in  Activity  Staphylococcus  discs  t a k e n up  algae were placed  2  as  Initial  agar  solvent  Exudates  and k e p t  achieved  Screening  nutrient  the  extracts  ( 1 0 0 0 uE m ~ s e c  The q u a n t i t a t i v e  Candida  the  of  a calibration  removed and t h e w a t e r  a n d w o r k e d up  was  removal  with  standard.  Preparation  filtered  Extraction  w h i c h was  and used  b y HPLC a n d c o m p a r i s o n the  60°C.  and subsequent  green residue  ethanol  using  at  a  with paper  known  were p l a c e d  on  the  of  of  growth  the  zone  88 inhibition  was  determined  Subsequent  Experiments  L i q u i d media were p r e p a r e d by bactopeptone one  set  either small  of  (Difco)  dilute test  HC1 o r  tubes  to  density  of  Fifty  lanosol  and t h e  or  distilled  To 2 . 0 ml  ul  of  (change  ml of  of  in  to  a 10 h r  sity  w a s r e a d o n a P y e U n i c a m SP 6 - 5 0 0 at  other  and b a c t e r i a lanosol,  experiments  susoptical  containing each  density)  35°C).  in  0.5  mg  tube  monitored  Optical  den-  spectrophotometer  extracts  were  or  Varying  exudates  at  pH 8  concentrations  in  small  added and g r o w t h r a t e s  amounts  monitored  of of  over  a  period.  Experiments  w i t h Marine  Three marine Thompson,  British sp.  an  media were prepared  a d d e d as b e f o r e .  algal  50% e t h a n o l  by T.  at  with  520 nm. In  24 h r  give  g  In  pH 6 - 1 0  added t o  optical  (incubation  water.  each medium  50% e t h a n o l were  and 5.0  a bacterial  phase  lanosalt  experiments  g glucose  over  set  period  1.0  m e d i a w e r e made t o  stationary  5 mg o f  growth rate  of  added 0.5  g r o w n up  35°C.  growth rate  liter  NaOH.  was  0.1.  one  at  coli  bacterial  to  experiments  24 h r s  E.:  addition  pension  of  with  for  the  after  fungal  Department  Columbia,  Crane,  Fungi isolates of  Vancouver,  Dendryphiella  were k i n d l y  Botany, B.  salina  C.  provided  University  These were  (Suth.)  Pugh e t  of  Sigmoidea Nicot  and  89  Z a l a r i o n maritimum with  these  using  (Linder) Anastasiou.  f u n g i were p e r f o r m e d on  sea water  were grown up  (pH  on  7.0).  the  A l l experiments  D i f c o c o r n meal  Stock c u l t u r e s of each  above a g a r and  small  agar  agar species  blocks  2 (0.5  cm  ) were e x c i s e d and  plates. was  placed  cific trol  Around the  placed  lanosol concentrations  o f the  three  no  species,  amined a f t e r  1-2  Dendryphiella  an  ture plates of  (3.0-0.03 mg/cm ) .  the  Growth was  sporulating mycelia p l a t e s to  were i n v e r t e d  seed the  conidia  m y c e l i a l g r o w t h c o u l d be  the  Tests with  Island  and  Tidepool  t h e method o f O h t a (Whatman No. larger  ex-  cul-  and  e n t i r e agar  filter  paper  surdiscs  observed w i t h i n  the two  measured.  i n sea water at  o r g a n i s m was (1979).  1) were p l a c e d  glass p e t r i  two  Germination of  s c u t u l a t a G o u l d , was  this  con-  Snails  maintained  testing with  as b e f o r e .  zone o f i n h i b i t i o n  Littorina  used.  Lanosol-containing surface  cm spe-  slow f o r  Stock  on  days and  A  a l t e r n a t e method was  the  2  zone o f i n h i b i t i o n was  were p l a c e d and  o f about  For  conidia.  test  paper d i s c s c o n t a i n i n g 2  lanosol. and  of  weeks a t room t e m p e r a t u r e .  t a p p e d over c l e a n t e s t face with  center  inoculum at a distance  a s e r i e s of f i l t e r  disc contained  i n the  dishes,  done by  N i n e cm  collected 10°C.  3 ml  a modification  filter  of  Bath  Antibiotic  paper  i n the bottom o f and  on  discs  slightly  s e a w a t e r were  of  90 added t o  each.  containing  A series  (0.1-10.0  over  surface  5.5  the cm;  rings  inner  to  of  snails  in  the  (diameter,  dispersal  of  paper  4.0  center 4-6 the  experiment  with  two r e p l i c a t e s  cm;  to  rings.  This  results  solutions  in water  filter  diameter,  control  positioned  The  mg/cm )  h u n g up and a l l o w e d  applied  test  2 ( 0 . 0 0 1 - 0 . 1 mg/cm ) 2  lanosol  lanosalt  of  area,  11.2  dry.  the  applied  diameter,  cm )  Only the  and  rings  dishes  and  mm) w e r e p l a c e d  inside  each  was n o t e d  per  after  on t h r e e  in  were  were five ring.  10 m i n .  separate  concentration  the  solvents  petri  snails  each)  or  evenly  (outer  Once d r i e d , of  ethanol  were  rings  air  was r e p e a t e d  were v e r y  in  (100 u l  occasions  each case.  The  consistent. RESULTS AND DISCUSSION  Of lanosol  the  and  organisms  lanosalt  used i n  activity,  susceptible  (Figure  17).  was u s e d i n  further  antibiosis  showed no e f f e c t while  the  ranged able  at  lanosol  at  effects  effective 2 0 . 0 1 mg/cm . liquid  E.  culture  initial  coli  o n E. at  screening  proved  As a r e s u l t ,  the  of  most  this  bacterium  experiments.  Lanosalt  concentrations  from very  Fpr  the  as h i g h as  coli  1 mg/cm  experiments  in 2  2  ,  particular to  a  1 mg/cm  barely  lanosol  detect-  91  ZONE OF INHIBITION ( d i a m e t e r i n mm)  F i g u r e 17. A n t i b i o t i c a c t i v i t y of l a n o s o l a g a i n s t Candida a l b i c a n s ( ), Escherichia coli( ), Saccharomyces c e r e v i s i a e (—-——) and S t a p h y l o c o c c u s a u r e u s (- — — -) . -  92  concentration not  to  stop  centration  of  0.2  ppt  completely  was  the  corresponds  f o u n d t o be  g r o w t h o f E.  effective coli.  but  This  con-  t o t h e m i d r a n g e dose r e s p o n s e  of  2 the  i n i t i a l plating  L a n o s a l t was ten  again  times those  C o n o v e r and l i n e pH nins  of  of  lanosol  Sieburth  reducing  substances  toxicity  ineffective,  herbivorous  substances,  this  effect  o f l a n o s o l and  were t e s t e d a t d i f f e r e n t only  illustrate  the  two  the  effect  the  compounds, b u t o f pH  on  the  one  notes  that  are  roughly  the  g r o w t h r a t e o f E.  pHs  18).  activity  animals.  and As  a  lanosalt against  coli  E.  results  effects  (control),  not but  10,  growth  only  slows  i t seems i n  mechanism o f A possible  of phenolate anions  of  antibiosis.  of b a c t e r i a l pH  of  some i n d i c a t i o n  c o n t r o l a t pH  higher  The  The  of lanosol  to the  to d e t o x i f y l a n o s o l .  plants  the  the maximal l e v e l s  i s the p r o d u c t i o n  other  as  u s i n g bromophenols,  chemistry  coli  tan-  increased  on m i c r o o r g a n i s m s .  also give  The  alka-  etc.)  This  d i f f e r e n c e s i n the  l a n o s o l curve  equivalent.  on  the  of a l g a l  polyphenols,  d e t o x i f i c a t i o n p r o c e d u r e i s unknown. tion  concentrations  ( F i g u r e 18).  they  the  Comparing  some way  mg/cm ) .  (1966) have s u g g e s t e d t h a t  a n i m a l s as w e l l as  levels  not  even a t  includes effects  means o f e x a m i n i n g  0.1  (see F i g u r e  to p l a n k t o n i c  probably  toxicity  (e.g.  sea water promotes the  (e.g.  toxic  experiments  from  this explanathe  Figure 18. The e f f e c t o f pH on t h e a n t i b i o t i c a c t i v i t y o f l a n o s o l and l a n o s a l t a g a i n s t E s c h e r i c h i a c o l i (pH 6 , 7— — — , 8 - — • • • , 9 — — — , 10 • — • — • ) .  94  bromophenols. inactivity  These  as  s a l t s would approximate  of the l a n o s a l t  the  as shown i n F i g u r e 18.  I n C h a p t e r V I have e x a m i n e d t h e e x u d a t i o n o f b r o m o p h e n o l s f r o m t i d e p o o l R.  larix  and  p e r i o d s o f most c o p i o u s p h e n o l r e l e a s e nite  and r a p i d  algae. quite  As  change i n pH  a result  acidic,  which  i n contrast  E.  pH  18,  t o t h e above  against,  at  one  least  plants  i s never  amine t h i s exudates  sure i f t h i s  extracts  larix,  are used.  quantifying  a l l three i n another  R.  an  larix.  "action  spectrum"  the h i g h c h l o r o p h y l l  e x t r a c t s and  series  of  (controls)  bacterial was  thought  activity  especially  ex-  i n these,  compound  of b i o l o g i c a l  the p o t e n t i a l  to  producing  type o f experiment  T e c h n i c a l problems,  extracts, minimized example,  This  and  the l a n o s o l  and  t o be  s e e n when w h o l e  I attempted  these q u a n t i t i e s w i t h pure  growth experiments.  i n pure  i s the o n l y source or  duplicating testing  argument  substance  q u e s t i o n by m a k i n g e x t r a c t s  o f R.  be  6).  the major source o f a c t i v i t y  or crude  defi-  f o r example,  When d e a l i n g w i t h an a n t i b i o t i c form,  during  s u r r o u n d i n g the  t h e s u r f a c e o f t h e p l a n t may  activity  (Figure  there i s a  o f the water  promotes a n t i b i o t i c coli  found that  for  i n tests  o f t h e method.  with For  c o n c e n t r a t i o n o f the  i t s p r o g r e s s i v e d e g r a d a t i o n masked t h e  of the b a c t e r i a  as m o n i t o r e d by v i s i b l e  growth  spectrophotometry  95 (Figure  19).  T h e r e f o r e , one  quantitative except  aspects  to r e f e r  can  of lanosol  extracts  of red algae.  however, showed a c l o s e trols  ( F i g u r e 19).  the b a c t e r i a l sion it  that  given has  substance  results  i n Table  IX.  As  a l g a e and  testing  procedure  i n w h i c h D.  in  their  and  that  i n R.  effects  salina  are  marine  on m a r i n e f u n g i .  The  c o n i d i a were p e p p e r e d  t h e most r a p i d  larix.  o t h e r work  d e r i v e d from  shown t o i n h i b i t  and n o t j u s t m y c e l i a l The  snail and  L.  i s o f t e n found  no  evidence t h i s may  growth  scutulata  essentially  It  for  aware, no  antibiotics  of  to the c o n c l u -  as an a n t i b i o t i c  I am  con-  the r e s u l t s  lead  onto  and i n f o r m a t i v e  degradation could occur.  l a n o s o l was  tidepools  lanosol  l o n g e x p e r i m e n t a l p e r i o d s were n e c e s s a r y i n  which l a n o s o l cedure  exudates,  of t h i s k i n d found  f a r as  surface proved  t h a t no  with  o f the assays w i t h marine f u n g i  b e e n done u t i l i z i n g  the agar  given here  which gives  bromophenols  experiments  T h i s i n f o r m a t i o n and  i s produced  i s the primary  literature,  of suspected  The  the  i n extracts  c o r r e l a t i o n w i t h the  experiments  lanosol  The  activity  the r e a d e r to the  numerous examples o f a c t i v i t y in  say n o t h i n g about  that be  on and  Using  spore  this  pro-  germination  alone. i s v e r y common i n  throughout around  R.  the  larix,  local  intertidal but  i t e v e r consumes t h e a l g a .  zone.  there i s The  reason  the h i g h c o n c e n t r a t i o n s o f bromophenols  LANOSOL (and c o n t r o l )  0.6  EXTRACT  EXUDATE  0.5  0.4  0.3  0.2  0.1  J  L  8  24  8  24  8  24  TIME (hours)  F i g u r e 19. E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f l a n o s o l i n p u r e form, i n a l g a l e x t r a c t s a n d i n e x u d a t e s on t h e g r o w t h r a t e o f E s c h e r i c h i a c o l i . The i n i t i a l c o n c e n t r a t i o n was 0.25 p p t ( — — — ) and t h e d i l u t i o n s a r e 0.13 (• — — -), 0.06 (• ), 0.03 ( ) and 0 ( — — ) p p t .  Table IX. A n t i b i o t i c a c t i v i t y of lanosol against three species of m a r i n e f u n g i ( g i v e n as t h e d i a m e t e r o f t h e zone o f g r o w t h i n h i b i t i o n i n mm). * A c t i v i t y a g a i n s t D. s a l i n a c o n i d i a l g e r m i n a t i o n , n o t t h e a d u l t m y c e l i u m as i n t h e o t h e r t h r e e e x a m p l e s . * * SPECIES  LANOSOL (mg/cm )  Dendryphiella salina  Dendryphiella salina*  Sigmoidea sp.  Zalerion maritimum  3.0  10  18  9  20  0.3  7  12  7  16  0.03  0  7  0  10  0  0  0  0  0  2  * * - T h r e e - r e p l i c a t e s were data are averages o b t a i n e d  used f o r each, s p e c i e s t e s t e d and t h e f r o m each set o f e x p e r i m e n t s .  above  98 in  the  alga.  Results  a typical  and t h e  salt  results  of  Lanosol  produced r e p u l s i o n  0.001 at  to  are  of  all  pictured  experiments  0 . 1 mg/cm  concentrations  This  method o f  a better area  by r e s i d u a l the  filter  while 100  of  were  rings  the  those  was of  1979)  before  at  21.  levels  only  the  phenol.  was m o d i f i e d  by  to  of  so  the  snail  thoroughly  surrounding  of  effective  free  applied  lanosol  combined  Figure  problems  overcome  both  20 a n d t h e  snails  lanosalt  Also,  with  shown i n  quantities  obtained.  paper  of  (Ohta  the  solvent  Figure  are  times  testing  control  c o u l d be  in  test  the  that sample  repulsion drying  snails  with  them. Evidence scale  production  an a n t i b i o t i c this red  has been p r e s e n t e d h e r e or  at  substance  compound t o algae would  at  the  this  of  lanosol  enough t o  the  implications  ment.  More  by r e d  b y R.  broad  of  stage  larix. role  the  be m e r e l y  further  s u c h phenomena  algae.  reasons  the  analysis for  the  will  large  lanosol  ecology  in  investigation  work  coupled  invariably  production  of  of  Dem-  activity  the marine  as  of  conjecture.  spectrum a n t i b i o t i c  encourage  chemical  of  the  .The a s s i g n m e n t  in  detailed microecological  quantitative determine  utilization  a particular  onstration is  least  for  of into  environwith  help  to  bromophenols  99  Figure 20. A t y p i c a l s n a i l repulsion experiment showing t h e e f f e c t s o f l a n o s o l and l a n o s a l t on t h e behavior of L i t t o r i n a scutulata. This photograph w a s t a k e n 10 m i n a f t e r f i v e s n a i l s h a d b e e n p l a c e d i n the c e n t e r o f each d i s h .  100  NUMBER OF S N A I L S ESCAPING SAMPLE RING  Figure 21. Degree o f r e p u l s i o n o f L i t t o r i n a scutulata by v a r y i n g c o n c e n t r a t i o n s of l a n o s o l ( ) and" l a n o s a l t (• •). L a n o s a l t c o n c e n t r a t i o n s a r e 100 t i m e s t h o s e l i s t e d on t h e v e r t i c a l a x i s .  101  PERSPECTIVES  This ing  a role  algae.  dissertation to  the  production  Each c h a p t e r  formulation cause  of  ters,  the  of  the  a conclusion  first,  each to  explain  that  in  the  the  increase  in  produced by  impetus  of  level  tidepools the  tidepool  culture  of  is  over  range  environmental  e n a b l e d me t o  because  will  first  chapter  the  the  phenols course  habitat  algae might  of  which,  I  the  chapas  a  therefore significance  within  set  conditions  lay  in  the  noticeably of  for  in-  a day.  two  I  reasons:  system--essentially  in nature--and  organisms  later  of  the  Be-  dissertation  and the  a "closed"  on o t h e r  pools  the  brown c o l o r a t i o n  tidepool  of  role.  matter  for  total  tidepool  flask  yellow  the  effect the  this  subject  clarification.  taken  red  step.  the  examine  the  a large  regarding  of  The i m p o r t a n c e  to  towards  contribution  separate  chose  a step  the  of  creases  by  in  to  assign-  bromophenols  diversity  attempt  discovery  of  represents  w h o l e may n e e d m i n o r  each  has been c o n c e r n e d w i t h  secondly, (phenolic  very  well  the  pools.  factors  the  substances)  have  used f e l l  some  Measurements  occurring  up e x u d a t i o n  rapid  in  the  experiments  within  102 environmental  ranges,  physiological  stresses  For to  their  assignment  occurring ferent  some r e d  in  subtidal forms  The  zone.  as  being still  at  R.  unnatural  confusion  Populations to  dif-  change  in  gross  response larix  growing  as  exposed  often  form of  no  tested.  level.  an a d a p t i v e  from that  in  has  the  their  a  quite  upper  comparison  of  least,  calling  for  to  these  two the  larix. of  and i n f o r m a t i v e  further  place  being  stress,  this  The m e t h o d w h i c h  for  w a s made e a s y  two  habitats,  tidepool  chemistry  and exudates  is  species  The c h e m i c a l  f o r m R.  b y HPLC.  of  the  natural  appearance  The  was  at  algae  there  g a v e me some b a s i s ,  tidepool  rapid  algae  conceivably  environment. different  of  assumed t o  on t h e  different  degrees  morphology,  were  for  species  I the  and a c c u r a t e .  additional  in  species,  further  developed proved screening  bromophenols.  demonstrated  was  of  algal  Quantification  The e f f i c i e n c y the P.  to  analysis  lyallii  and  of  for  examined be extracts  of  lanosol  the  method  bromophenols  C.  washingtoniense. Demonstration content,  both  temporal  first  real  clue  these  compounds.  Polysiphonia  of  into  the  in  and i n t r a t h a l l i a l ,  the  ecological  Sieburth  lanosa,  variation  which  e_t a l . also  bromophenol provided  significance  (1974)  contains  have  of  shown  lanosol  the  in  that  103  concentrations supports  of  up  a diverse  the  yeasts  and i n w i n t e r ,  If  the  levels  as  do t h o s e  dant,  of  the  the  lanosa  of  a l g a was  of  of  the  epiflora  in  t h e numbers  (e.g. the  levels  appeared  to  The covery in  particular)  Brown a l g a e fouling  by  (Conover Ryland  in  species  the  arises.  P.  shown t o  abun-  algal  pounds  threatened  to  that  to  toward of  the  the  this of in  see  of  1964,  a definite  the  present  P.  study  to  thallus  tips. to  exudates control  the  dis-  (lanosol of  R.  in  larix.  surface substances  and Conover  the n o t i o n  ecological  changes or  lanosa  led  1968).  lanosol  abundance  Interestingly,  bromophenols  Sieburth  the  antibiotics  growing  the  by  seasonal  polyphenolic  Sieburth  furthers  in  same s p e c i e s . of  be  (fouled)  Variations  due e i t h e r  production  exudates  have  l a n o s a was  l o n g been known t o  also  temporally  correlation  quantities  and S i e b u r t h  vary  an i n t r i g u i n g  bromophenols--particularly red  this  were p r e s e n t  have  1974,  fungi.  "contamination"  large  In  and f i l a m e n t o u s  algal  second phase  epiflora.  diatoms  tolerance by  V) ,  of  species  of  (Table  composed m a i n l y  present.  be m i n i m a l  that  changing  a particular  bromophenols) of  of  c o u l d be of  dry weight  is  i n no way  organisms  levels  of  larix,  epiflora  number  changing  epiflora  lanosol  R.  its  and s e a s o n a l l y  summer  Although  P.  5°L o f  to  1965,  The p r e s e n c e the  free  that  function.  of  form--in  these  com-  The  rates  104 of  exudation  of  The e f f e c t i v e E.  coli  was  0.1-0.2 seems  surface the  less  than  Thus  fouling,  Testing of  mg/cm  rate  order  of  to the  all  2  is  as  did  of  affected  against  bromophenols to  control  microorganisms E.  they at  show  coli.  activity  that  d.wt.  approximately  necessary  other  ppt/g  compound  , which  antibiotic  were  0.15  exudation  that  proved  as  this  that  lanosol  organisms  such a response:  of  of  assuming  same r e s p o n s e  as h i g h  dose o f  0.3  the  be on t h e  a variety  were  antibiotic  ppt.  to  lanosol  of  lanosol  did  some  in  fact  on show  concentration  9 between was  0.03  greatest  exudates Results the  and 0.3 at  lower  closely of  door  the  to  mg/cm pHs,  array  in  repulsion  potential  lanosol than  was  that  Further  the  the  with of  for  possible  a snail the  with  a variety  full  spectrum of  of  and p o s s i b l y  most  ecological  significance  of  the  Roos  antibiotic  (1957). activity  be  of  R.  lanosol.  snails  opened  level  repellent  which  animals  type  of  bromophenols  of  the  lower  antibiotic.  convincing  extracts  in at  R.  could  effect.  argument comes  He e x a m i n e d t e m p o r a l of  for  was much  be  marine  larix  pure  involved  The  this  coli  functions  compound t o  A final  of  activity  tidepool  herbivores. as  o n E.  seen f o r  t h e y may a l s o  effective  testing  the work in  that  necessary  demonstrate  that  experiments  another  The e f f e c t  and t h e  resembled  bromophenols of  .  for  from  variation  subfusca  hr.  105 on s e v e r a l have  species  shown t h i s  dry weight that the  the  of  to  contain  (or  as  lanosol,  basis  summer m o n t h s  activity  larix  the  same c o n c e n t r a t i o n s ,  for  this  exists  of  antibiotic  an i n d i r e c t  temporal  R.  and t h e to  activity  antibiotic  activity  Roos  as  lanosol  (1975) on  a  observed  s u b f u s c a was twice  lowest  great in  in  the  approximately  s e a s o n a l maxima  R.  of  subfusca.  lanosol  found  for  in  the  s e a s o n a l maxima and  for  correlation  lanosalt  5.1%).  contains  compound c o r r e s p o n d  and Amiya  9.2%> o f  and e s s e n t i a l l y  Rhodomela  minima of  Kurata  alga  antibiotic  winter.  bacteria.  minima and  Hence  content  extracts  there  with  of  red  algae. Most ning  in  applying  understand to for  certainly,  them i n  the vast further  organisms occurs, tailed  the  is  not  h a v e made b u t  concepts  the  sense  of of  and complex marine analyses  of  steadily too  research  ecosystems.  I  far in  the  the  the  chemical their  and I  future,  begin-  ecology,  a s we  terrestrial  interactions  chemical  barest  environment.  increasing  into  the  The  among  hope  ecology  of  such  potential  marine  that  much more  origins,  there  and  de-  complex  106  FOOTNOTES  (1)  This  light  measures region  (2)  This  meter  wavelengths  of  the  figure  in  Figure  in  the  This  figure  phenols single  day  basis,  0.05  per  is  in  (about  content  exclusive of  or  in  rather  it  exists  between  the  each o f  pool  total  of  data  factors  the  in  over  phenols  environmental  of  period  on an  total  of  a  hourly  w o u l d be p r o d u c e d  water  (or  alga,  0.14  as  amount  for  produced  mg/hr).  and  is  mutually  intended.  "correlation"  simply  discussed  pools.  the  Hence,  presented  between medium d i s c o l o r a t i o n  any o f  necessarily  the  changes  some r e l a t i o n s h i p  the word  similarity  not  the  tidepool  No c o r r e l a t i o n  use  in  gram d r y w e i g h t  lanosol  effect  10 h r s ) .  mg o f of  some o f  the  photosynthesis.  b a s e d o n t h e maximum l e v e l  reached  each l i t e r  (4)  occurring  for  a summary o f  which might  effectively  400 and 800 nm,  spectrum u t i l i z e d  represents  text  combination  between  3 and i n c l u d e s  conditions  (3)  and probe  its  implies  two  that the  t w o may be  may e x i s t . implies  many u s e s  events,  been  The  no  in  established.  of  addition,  mathematical  this  a mutual basis  In  thesis;  relationship which  has  107  REFERENCES  Abbott,  I . A . a n d G. J . H o l l e n b u r g . 1976. Marine algae of California. Stanford Univ. Press, Stanford? BT7 p p .  A l b r i g h t , J . 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