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UBC Theses and Dissertations

Some aspects of conjugation in Stentor coeruleus Webb, Terry Lavern 1968

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SOME ASPECTS OF CONJUGATION IN STENTOR COERULEUS by TERRY LAVERN WEBB. B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1968  In presenting this thesis in p a r t i a l  f u l f i l m e n t of the requirements  for an advanced degree at the 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 for extensive copying of  this  thesis for s c h o l a r l y purposes may be granted by the Head of my Department or by h.iJs representatives.  It  is understood that copying  or p u b l i c a t i o n of this thesis for 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 of Z o o l o g y The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date August; 2 7 ,  196$  ii  ABSTRACT  Conjugation i n S t e n t o r coeruleua was i n v e s t i g a t e d u s i n g two  experimental methods which r e g u l a r l y y i e l d e d  l a r g e numbers of mating p a i r s .  One method i n v o l v e s a  p a r t i c u l a r c u l t u r e technique, the other r e q u i r e s a mixture of c e l l s from d i f f e r e n t s t o c k s . peared  pairs  ap-  i n the form of b u r s t s of conjugation, e i t h e r  by mixing  c e r t a i n stocks or o c c u r r i n g spontaneously  some stock c u l t u r e s . of  Mating  induced in  Spontaneous b u r s t s , i n the m a j o r i t y  cases, occurred during a d e f i n i t e i n t e r v a l i n the  development of a c u l t u r e .  Morphologically d i s t i n c t  conjugator c e l l s appear immediately  before as w e l l as d u r i n g  the i n i t i a l stages of a burst of conjugation. were formed by the union of two  pre-  Mating  pre-conjugators.  Mixing eight stocks i n a l l p o s s i b l e combinations twos and observing t h e i r subsequent response they were separable i n t o two  pairs  of  revealed  complementary mating types.  The m a j o r i t y of mating p a i r s formed i n mixtures  of stocks  c o n s i s t e d of i n d i v i d u a l s of d i f f e r e n t mating types. Evidence  i s presented which i s compatible with the hypo-  t h e s i s that c e l l to c e l l contacts between i n d i v i d u a l s of d i f f e r i n g mating type are necessary a mating r e a c t i o n .  f o r the i n i t i a t i o n of  iii TABLE OF CONTENTS Page INTRODUCTION METHODS AND  1 MATERIALS  4  1.  Stocks of Stentor  4  2.  P e t r i Dish C u l t u r e Technique  4  3.  Photography  6  4.  I n d u c t i o n of Conjugation  6  RESULTS  7  1. 2.  The Appearance of Conjugation i n Stock Cultures  7  Pre-conjugators and the Formation of Mating P a i r s  14  3.  P r o d u c t i o n of S e l f i n g Clones  20  4.  Induced  21  5.  S p e c i f i c Mating Between Stocks  24  6.  Demonstration  26  7. 8.  Role of C e l l to C e l l Contacts i n the I n d u c t i o n of Conjugation S u r v i v a l of Exconjugants  31 36  9.  Abnormal Mating  38  Conjugation  of Cross-mating  DISCUSSION  40  SUMMARY  51  BIBLIOGRAPHY  52  APPENDIX  56  iv LIST OF TABLES  Table I  Page The appearance o f mating p a i r s i n stock c u l t u r e s .  II  9  Number o f days from the i n i t i a t i o n of c u l t u r e s  to the f i r s t  appearance  of mating p a i r s i n stocks CH, H, S, T, and W III  13  I n t r a c l o n a l conjugation ( s e l f i n g ) i n cultures  s t a r t e d from s i n g l e  cells. IV  S p e c i f i c mating i n mixtures of stocks.  V  22  25  Observed c l a s s e s o f mating p a i r s scored on the b a s i s o f r e l a t i v e pigmentation, presence of carmine, and r e l a t i v e s i z e .  30  LIST OF FIGURES Figure 1.  Page Estimated change i n the percentage of  the p o p u l a t i o n i n v o l v e d i n con-  j u g a t i o n through a spontaneous conjugation b u r s t . 2.  11  Representatives o f normal v e g e t a t i v e i n d i v i d u a l s and pre-conjugators.  3.  a)  V e n t r a l view of a normal c e l l .  15  b)  A n t e r i o r view of a normal c e l l .  15  c)  V e n t r a l view of a pre-conjugator.  15  d)  A n t e r i o r view of a pre-conjugator.  15  A n t e r i o r view o f a pre-conjugator showing the bulge w i t h i n the f o l d e d down p o r t i o n o f the f r o n t a l f i e l d .  4.  17  Two pre-conjugators with the f o l d e d down p o r t i o n o f t h e i r f r o n t a l  fields  i n c l o s e a p p o s i t i o n ; bulges not y e t joined. 5.  Two pre-conjugators with bulges  17 just  joined. 6.  Contracted c e l l s j u s t before they  18 pull  apart. 7.  18  A sequence showing the development o f a mating p a i r from i n i t i a l adhesion  till  j u s t before s e p a r a t i o n , a t o t a l o f 28  hours. a)  Bulges j u s t u n i t e d .  b)  4 hours.  c)  9 hours.  d)  21 hours.  e)  28 hours, s e p a r a t i o n came d u r i n g the f o l l o w i n g  hour.  Mating p a i r from a mixture of stocks C and  S.  Squashed p a i r from a mixture of l a b e l l e d stock S c e l l s and u n l a b e l l e d  stock C  cells. D i f f u s i o n chamber (a) before wrapping w i t h b o l t i n g c l o t h and wrapping  (b) a f t e r  i s complete.  P e t r i dish culture with d i f f u s i o n chambers i n p l a c e . An example o f a mating t r i p l e t found i n a mixture o f stocks C and  S.  Mating p a i r with one p a r t n e r at about stage 7 o f d i v i s i o n .  vii ACKNOWLEDGEMENTS  I would l i k e to thank my s u p e r v i s o r Dr. D. W. F r a n c i s f o r h i s p a t i e n t guidance and encouragement d u r i n g t h i s study. I am indebted to Mr. Charles M. Harden f o r a l l o w i n g me to read h i s paper while i t was i n manuscript  form.  I am g r a t e f u l to Dr. Vance T a r t a r and Dr. T. M. Sonneborn f o r t h e i r u s e f u l comments and c r i t i c i s m d u r i n g the study. A p p r e c i a t i o n i s expressed t o Dr. A. B. Acton, Dr. C. V. Finnegan, and Dr. D. Suzuki f o r t h e i r c r i t i c a l reading o f the manuscript.  1 INTRODUCTION  An e s s e n t i a l c h a r a c t e r i s t i c of a d i p l o i d organism i s its ability  to e f f e c t , i n one way  or another,  a t i o n of i t s genes i n i t s o f f s p r i n g . protozoans  the recombin-  Among the  ciliated  t h i s i s brought about by the sexual process  conjugation which i n v o l v e s the temporary union of two  of indiv-  i d u a l s , d u r i n g which they exchange p a r t s of the n u c l e a r apparatus. The phenomenon of conjugation has been observed  and  studied i n a l a r g e number of c i l i a t e d protozoans. i n f o r m a t i o n concerning lacking...  Conjugation  However,  sexual r e p r o d u c t i o n i n Stentor i s i n Stentor coeruleus has been  observed  by Moxon (1869), B a l b i a n i (1891), Hamburger (1908), Mulsow (1913), and more r e c e n t l y by T a r t a r (1961), B u r c h i l l and Harden and Holland  (1968).  The  (1967),  e a r l y workers found  that  exconjugants d i d not s u r v i v e long a f t e r engaging i n sexual reproduction.  Mulsow,, however, was  able to keep them a l i v e  long enough to examine the c y t o l o g i c a l events of conjugation. His s t u d i e s showed that the events of conjugation the recombination found  accomplish  of chromosomes i n a manner s i m i l a r to that  i n other c i l i a t e s ; . namely, the f u s i o n of h a p l o i d pro-  n u c l e i and  subsequent renewal of the macronucleus from  nuclear d e r i v a t i v e s .  T a r t a r i n reviewing the subject  microsuggests  that the proper c o n d i t i o n s f o r conjugation seldom occur. He draws a t t e n t i o n to s t u d i e s i n which Stentor c u l t u r e s have  2 laeen c a r r i e d on continuously f o r s e v e r a l years with very few or no mating p a i r s detected. Recently,  two  separate s t u d i e s ( B u r c h i l l , 1967;  and Holland, 1968)  reported the occurrence  of abundant con-  j u g a t i o n i n mass c u l t u r e s of Stentor coeruleus. found  Harden  Burchill  that conjugation occurred most o f t e n i n c u l t u r e s  which had been f e d r e c e n t l y , observing up to f i f t y  percent  of p o p u l a t i o n i n v o l v e d i n conjugation at one time.  Harden  and Holland  (1968) d i s c u s s the e f f e c t s of conjugation on a  l a r g e number of conjugating p a i r s , s t a t i n g that S. seems to be able to f o l l o w one another a f t e r a short i n t e r v a l .  coeruleus  episode of conjugation with They a l s o observed  a high  m o r t a l i t y r a t e among exconjugants but were unable to demons t r a t e any  i n c r e a s e i n m o r t a l i t y r a t e due  to i n b r e e d i n g .  To date, a l l s t u d i e s of sexual processes been confined to the occurrence c o l l e c t i o n s or c u l t u r e s .  i n Stentor have  of conjugation i n mass  Such s t u d i e s , although p r o v i d i n g a  c o n s i d e r a b l e amount of knowledge, are l i m i t e d because the occurrence  of conjugation i s r a t h e r u n p r e d i c t a b l e .  attempts to induce conjugation ( T a r t a r , 1961;  Recent  Burchill,  1967)  i n t h i s genus have been u n s u c c e s s f u l . In s e v e r a l c i l i a t e genera conjugation has been brought under s t r i n g e n t experimental  c o n t r o l by v i r t u e of the f a c t  that mating type d i f f e r e n t i a t i o n s account f o r the union cells.  of  When c u l t u r e s of complementary mating type are mixed  a mating r e a c t i o n occurs because of cross-mating  between  3 the two types.  U n t i l the p a r t i c u l a r mating system of a  species i s known the c o n t r o l l e d matings r e q u i r e d f o r g e n e t i c s t u d i e s are i m p r a c t i c a l . The purpose o f t h i s study was to i n v e s t i g a t e sexual r e p r o d u c t i o n i n Stentor coeruleus w i t h the aim of developi n g an experimental  system w i t h which to c o n t r o l conjugation,  and to add to the g e n e r a l knowledge of conjugation i n t h i s genus.  The .study was occasioned by the unexpected d i s c o v e r y  of p e r s i s t e n t conjugation i n stock c u l t u r e s o f t h i s  ciliate.  4. METHODS AND  1.  MATERIALS  Stocks of Stentor The  study was  conducted  u s i n g nine d i f f e r e n t stocks of  Stentor coeruleus which were obtained from v a r i o u s sources (see Appendix). S, SC,  They were designated as f o l l o w s :  ST, T, CH,  each stock was  H and W.  To provide g e n e t i c u n i f o r m i t y ,  (thus r e p r e s e n t i n g a clone) and  t h e r e a f t e r v/as maintained F i v e of the stocks ( C , DF, because each one was The  DF,  e s t a b l i s h e d i n c u l t u r e from the v e g e t a t i v e  progeny of a s i n g l e c e l l  ulation.  C,  s e p a r a t e l y i n continuous  culture.  S, SC, T) were g e n e t i c a l l y  distinct  d e r i v e d from a d i f f e r e n t n a t u r a l pop-  c o l l e c t i o n s i t e s range from the west coast to  the east coast of North America; t h e r e f o r e these  stocks  should represent a wide range of g e n e t i c d i v e r s i t y .  Stocks  CH and H were o r i g i n a l l y d e r i v e d from stock T and had been maintained  i n separate l a b o r a t o r i e s f o r a p e r i o d of at l e a s t  two years before being e s t a b l i s h e d i n c u l t u r e f o r the present study.  Stock ST was  o r i g i n a l l y d e r i v e d from organisms belong-  i n g to stock SC but was  a l s o maintained  i n a separate l a b o r -  atory before being e s t a b l i s h e d i n c u l t u r e f o r the present study.  2.  The o r i g i n of stock W i s not known.  P e t r i Dish C u l t u r e Technique A f t e r being e s t a b l i s h e d i n c u l t u r e each stock was  t a i n e d i n continuous  main-  c u l t u r e by the f o l l o w i n g subculture  5 method. All dishes  c u l t u r e s were maintained i n s t e r i l e p l a s t i c (100  x 25 mm).  Each p e t r i d i s h was  bottom with a t h i n l a y e r of one 1966).  The  petri  coated on  per cent agar (De  the  Terra,  agar l a y e r appeared to f a c i l i t a t e the attachment  of s t e n t o r s to the bottom of the p e t r i d i s h ; i n i t s presence c o n s i s t e n t l y good c u l t u r e s were produced.  The  culture  dishes  were prepared by a d d i t i o n of approximately 75 ml of m i l l i p o r e ' f i l t e r e d pond water along with two  b o i l e d wheat g r a i n s .  To  i n i t i a t e a c u l t u r e an inoculum of 0.5-1.0 ml o f water, cont a i n i n g a species of c o l o r l e s s f l a g e l l a t e s ( c u l t u r e d separatel y i n the same medium), was  added to the c u l t u r e d i s h  24 hours l a t e r by the i n t r o d u c t i o n of 25-50 s t e n t o r s . colorless flagellates,  The  c l o s e l y resembling Rhabdomonas c o s t a t a ,  acted as food organisms. the l a b o r a t o r y  followed  A l l c u l t u r e s were maintained i n  at room temperature .(21°-25°C).  Stocks  c u l t u r e d i n t h i s manner c o n s i s t e n t l y y i e l d e d a vigorous popu l a t i o n of r a p i d l y d i v i d i n g s t e n t o r s which approached maximum  d e n s i t y i n 12-16  the p o p u l a t i o n which few,  A f t e r reaching  maximum d e n s i t y  entered a s t a t i o n a r y phase of growth  i f any,  c u l t u r i n g was  days.  d i v i d i n g s t e n t o r s were present.  during Sub-  c a r r i e d out when the stentors were w e l l i n t o  the s t a t i o n a r y growth phase but before the c u l t u r e began to decline  (the c u l t u r e was  then 20-25 days o l d ) .  were maintained f o r 30. days r e g a r d l e s s culture.  A l l cultures  o f t h e i r time of sub-  Cultures were observed d a i l y or every second  day  6 to f o l l o w t h e i r progress from the time o f i n i t i a t i o n to the 30  day i n c u l t u r e .  Observations were made by t r a n s -  mitted l i g h t u s i n g a Wild M-5 stereomicroscope stage  3.  with sub-  illumination.  Photography Organisms were photographed, as they were, i n the c u l t u r e  dishes i n order to record the events o f conjugation i n an undisturbed s t a t e .  Photomicrographs were taken with Kodak  high c o n t r a s t copy f i l m u s i n g a Wild Mka 1 camera which was mounted d i r e c t l y on a Wild Mo stereomicroscope.  The l i g h t  source f o r photography was provided by a L e i t z m i c r o - f l e s h device with a f l a s h d u r a t i o n o f 1/1000 second..  4.  Induction o f Conjugation A method was developed  j u g a t i o n i n Stentor .  which s u c c e s s f u l l y induced  con-  The e s s e n t i a l f e a t u r e which i s neces-  sary f o r the i n d u c t i o n o f conjugation i s the mixing o f sexu a l l y r e a c t i v e c e l l s from two d i f f e r e n t s t o c k s .  This feature  was i n c o r p o r a t e d i n t o s e v e r a l experiments designed  to demon-  s t r a t e the extent o f mating between a l l stocks, the extent of cross-mating between c e l l s o f two.stocks,  and the a c t u a l  stimulus which leads to the formation o f mating p a i r s .  Dis-  cussion o f each o f these experiments w i l l be reserved f o r l a t e r s e c t i o n s to avoid any confusion which might a r i s e from an awkward s e p a r a t i o n o f a d i s c u s s i o n of the methods used and the r e s u l t s  obtained.  7 RESULTS  The  i n i t i a l observations i n t h i s i n v e s t i g a t i o n were made  w i t h the unexpected d i s c o v e r y o f conjugating p a i r s i n s e v e r a l c u l t u r e s o f stock S s t e n t o r s .  At that time i t was considered  p o s s i b l e that the c u l t u r e technique was p r o v i d i n g c o n d i t i o n s which favored conjugation.  With t h i s i n mind a number o f  subcultures were made u s i n g e x a c t l y the same technique as before; again, numerous conjugating p a i r s appeared.  These  p r e l i m i n a r y observations l e a d to the s t a r t o f an i n v e s t i g a t i o n which ran from November 1966. to May 1968, the r e s u l t s of which are presented  1.  i n this  thesis.  The Appearance o f Conjugation Conjugation,  observed  i n Stock  Cultures  o f t e n i n v o l v i n g l a r g e numbers o f c e l l s ,  was  f r e q u e n t l y throughout the p e r i o d o f study i n stock  c u l t u r e s maintained  i n the manner p r e v i o u s l y d e s c r i b e d .  C u l t u r e s o f each o f nine stocks maintained subculture were observed  i n continuous  d a i l y or every second day to f o l l o w  "t h. t h e i r progress from the time o f i n i t i a t i o n to the 30 in culture.  day  A f t e r 30 days the c u l t u r e s were d e c l i n i n g and  date were no longer  recorded.  A t o t a l of 602 stock c u l t u r e s were made and  observed;  of these, 292 (49%) contained mating p a i r s a t some time during t h e i r development.  The number o f p a i r s present at one time  i n a c u l t u r e d i s h v a r i e d from l e s s than 1% to g r e a t e r 30% o f the t o t a l p o p u l a t i o n .  than  These f i g u r e s are derived from  8 the accumulated data f o r a l l nine stocks and, as such, are somewhat m i s l e a d i n g .  They are i n c l u d e d here merely to  i n d i c a t e that the c u l t u r e method does appear to provide c o n d i t i o n s which are f a v o r a b l e f o r sexual r e p r o d u c t i o n . A more d e t a i l e d i n s p e c t i o n o f the data obtained from the c o n s i d e r a t i o n o f each stock i n d i v i d u a l l y , r e v e a l e d a d e f i n i t e v a r i a t i o n between stocks i n their'tendency to produce c u l t u r e s which contained mating p a i r s (Table I ) .  These date  show a well, defined tendency f o r some stocks (CH, DF, H, S, T and W) to produce subcultures which r e g u l a r l y c o n t a i n mating p a i r s , but a t the same time show other stocks (C, SC and ST) to have l i t t l e o r no tendency to produce c u l t u r e s which c o n t a i n mating p a i r s .  On t h i s b a s i s , the stocks can  be d i v i d e d i n t o two groups:  those which r e g u l a r l y conjugate  w i t h i n stock c u l t u r e s , and those which do n o t . T h i s d i v i s i o n i s f u r t h e r s u b s t a n t i a t e d by the o b s e r v a t i o n that conjugation i n c u l t u r e s o f stocks C, SC and ST never i n v o l v e d the format i o n o f more than f i v e mating p a i r s i n any c u l t u r e , while, i n c o n t r a s t , conjugation i n c u l t u r e s o f stocks CH, DF, H, S, T and W u s u a l l y i n v o l v e d the formation of l a r g e numbers of p a i r s , o f t e n numbering i n the hundreds. P e t r i d i s h c u l t u r e s i n i t i a t e d with food organisms and s t e n t o r s u s u a l l y e x h i b i t e d a d e f i n i t e sequence o f events with respect to t h e i r growth p a t t e r n and the appearance o f mating p a i r s through the 30 day o b s e r v a t i o n a l p e r i o d . During the f i r s t few days the food organisms m u l t i p l i e d r a p i d l y followed c l o s e l y by an i n c r e a s e i n the number o f  9  Table I .  stocks  The a p p e a r a n c e o f m a t i n g p a i r s i n s t o c k  t o t a l number o f s u b c u l t u r e s made  cultures.  number o f s u b c u l t u r e s containing mating p a i r s  C  51  6(12%)  CH  40  19(48%)  DF  11  8(73%)  H  123  85(64%)  S  242  104(43%)  SC  19  1(5%)  ST  19  0  T  47  44(94%)  W  50  25 (,50%)  10 stentors. density  The p o p u l a t i o n o f s t e n t o r s approached maximum  C e s t i m a t e d from the decreasing number of d i v i d i n g  i n d i v i d u a l s ) a f t e r about 14-16 days.  During  the f o l l o w i n g  f i v e or s i x days the p o p u l a t i o n entered a s t a t i o n a r y phase d u r i n g which few d i v i d i n g s t e n t o r s appeared.  As mentioned  p r e v i o u s l y , by the time a c u l t u r e was 30 days o l d i t u s u a l l y had begun to d e c l i n e .  When mating p a i r s appeared i n a c u l -  ture d i s h , they most f r e q u e n t l y d i d so i n the form of a burst of  conjugation l a s t i n g s e v e r a l days.  In any conjugation  burst a l a r g e number o f mating p a i r s appeared on the f i r s t day,, however, p a i r s i n decreasing numbers were formed on succeeding  days.  The graph i n f i g u r e 1 f o l l o w s the change  i n the percentage o f the p o p u l a t i o n i n v o l v e d i n conjugation through a t y p i c a l b u r s t .  The data f o r the graph were obtained  from d i r e c t counts taken at i n t e r v a l s d u r i n g the course o f the b u r s t .  The counts ranged from 730 to 1,273 c e l l s ,  each  c e l l being scored as to whether or not i t was conjugating. From these f i g u r e s an estimate was made o f the percentage of the t o t a l p o p u l a t i o n i n v o l v e d i n conjugation at the time of the count.  A s i g n i f i c a n t o b s e r v a t i o n from t h i s curve i s that  during the f o u r hour i n t e r v a l between the f i r s t counts there was approximately percentage of" mating c e l l s .  and second  a 2.5 f o l d i n c r e a s e i n the  E q u a l l y ss s t r i k i n g i s the f u r t h e r  o b s e r v a t i o n that 24 hours e a r l i e r there were no p a i r e d c e l l s present i n the c u l t u r e . When a burst o f conjugation appeared i n a c u l t u r e , i t did  so only once and at a d e f i n i t e stage during the 30 day  F i g u r e 1.  Estimated change i n the percentage of the p o p u l a t i o n i n v o l v e d i n conjugation through a spontaneous conjugation b u r s t . count was  taken as time zero.)  (The  first  ESTIMATED P E R C E N T OF P O P U L A T I O N I N V O L V E D IN CONJUGATION o  cn  o  cn  o  cn  12 l i f e of the c u l t u r e . conjugation  To demonstrate the stage at which  appeared, a number of c u l t u r e s o f stocks  S, T and W were set up and  CH,  H,  observed d a i l y to record the  number of days elapsed  from i n i t i a t i o n to the f i r s t  ance of mating p a i r s .  R e c a l l that these f i v e stocks  to the group which r e g u l a r l y produced c u l t u r e s l a r g e numbers of mating p a i r s .  The  exact  appearbelong  containing  r e s u l t s from the above  experiment (shown i n Table I I ) i n d i c a t e the burst of conj u g a t i o n begins i n the i n t e r v a l 7-24  days a f t e r the  t i o n of the c u l t u r e , w i t h an o v e r a l l mean of 12.2 A c l o s e r look at the data r e v e a l s that 98% of the c u l t u r e dishes had  (123  the beginning of the  burst f a l l i n the i n t e r v a l 7-18  days.  initia-  days.  o f 126  conjugation  This adjusted  figure  probably represents  a more r e a l i s t i c i n t e r v a l than that  previously  Under the c u l t u r e c o n d i t i o n s  stated.  i n v e s t i g a t i o n a prevalent  observation  emerges::  of t h i s mating  p a i r s r a r e l y , i f ever, appeared i n young c u l t u r e s (1-6 or i n o l d e r c u l t u r e s  total)  days)  (19-30 days), but p a i r s d i d appear with  some r e g u l a r i t y i n c u l t u r e s which were 7-18  days o l d .  The  reasons f o r the appearance of mating w i t h i n t h i s r e l a t i v e l y d i s t i n c t i n t e r v a l may  r e s i d e at l e a s t i n part with  n u t r i t i o n a l s t a t e o f c u l t u r e and  the  w i l l , be d e a l t with i n the  discussion.  The three numbers excluded are marked with an a s t e r i s k i n the raw data which appear i n Figure I I .  13 Table I I .  Number o f d a y s f r o m t h e i n i t i a t i o n o f t h e c u l t u r e to t h e f i r s t appearance o f mating p a i r s i n s t o c k s CH, H, S, T and W.  CH  H  16 11 11 9 16 16 13 14 16 14 14 11 11  10 7 7 7 10 12 10 15 10 24* 18 14 18 17 14 18 13 10 12 12 12 113 12 12 11 10 11 12 14 14 11 10 12 10 10 10  13.2  12.3  S  T  W  18 14 14 11 10 15 14 18 11 12 12 11 11 10 12 9 9 10 16 12 10 12 8. 8 8 8 9 9 •  7 11 11 12 11 11 9 10 12 12 14 16 16 10 19* 10 10 11 14 14 11 19 20* 16 16 13 14 14 14 14 11 10 14 10 14  10 7 7 11 7 10 10 10 10 14 11 11 14 14  11.5  12.9  10.4  OVERALL. MEAN:- 12.2 * These f i g u r e s w e r e e x c l u d e d t o g i v e t h e a d j u s t e d , i n t e r v a l (see text f o r explanation).  14 2.  Pre-conjugators  and the Formation o f Mating P a i r s  The accumulated observations through a great many b u r s t s of conjugation, both i n stock c u l t u r e s and i n induced  bursts  (induced b u r s t s w i l l , be discussed i n a l a t e r s e c t i o n ) , make i t p o s s i b l e to d e s c r i b e some o f the f e a t u r e s which c h a r a c t e r i z e the mating behavior o f S t e n t o r .  As p r e v i o u s l y mentioned,  the beginning o f a burst o f conjugation was c h a r a c t e r i z e d by the appearance o f a great many mating p a i r s .  At the same time,  the e a r l y stages of a burst were marked by the appearance o f numerous m o r p h o l o g i c a l l y d i s t i n c t c e l l s which are designated as "pre-conjugator  cells"..  These unique c e l l s are d i s t i n c t  from normal v e g e t a t i v e s t e n t o r s i n having a p o r t i o n o f t h e i r l e f t f r o n t a l f i e l d and a s s o c i a t e d membranellar band f o l d e d down (.compare Figure 2a and b with c and d ) .  Within the  folded-down p o r t i o n there appears a prominent bulge (see Figure 3) which e v e n t u a l l y becomes the l o c u s o f the i n i t i a l mating union when two c e l l s j o i n to form a mating p a i r . Preconjugators  were most evident during the f i r s t day o f a  conjugation burst, becoming l e s s numerous on succeeding days.  The a c t u a l mating behavior which r e s u l t s i n the j o i n -  i n g o f two preconjugators  to form a mating p a i r i n v o l v e s an  a c t i v e s e r i e s of events d i r e c t e d toward the formation o f a mating p a i r . Preconjugators  u s u a l l y remained attached to the bottom of  the c u l t u r e d i s h by t h e i r h o l d f a s t s thus l e a v i n g t h e i r ends f r e e to wave about. first  "head"  Two c e l l s , which were about to j o i n ,  o r i e n t e d themselves i n such a way that the folded-down  F i g u r e 2.  Representatives o f normal v e g e t a t i v e i n d i v i d u a l s and pre-conjugators.  (For the preconjugators  the folded-down p o r t i o n of the f r o n t a l f i e l d i s i n d i c a t e d by a bracket.)  Magnification llOx.  a)  V e n t r a l view of a normal  cell.  b)  A n t e r i o r view of a normal  c)  V e n t r a l view of a pre-conjugator.  d)  A n t e r i o r view of a pre-conjugator.  cell.  16 p o r t i o n s o f t h e i r r e s p e c t i v e f r o n t a l f i e l d s were i n c l o s e a p p o s i t i o n (.Figure 4 ) . for  Often they remained  i n this position  up to f i f t e e n minutes with the c i l i a of t h e i r membranel-  l a r bands touching and c o n t i n u a l l y b e a t i n g .  E v e n t u a l l y the  bulges on each c e l l touched and stuck, thus u n i t i n g them (Figure 5 ) ,  At t h i s p o i n t the union between the c e l l s  was  not.very strong f o r i f they were s t i m u l a t e d to c o n t r a c t (Figure 6) they o f t e n p u l l e d a p a r t . of  the f r o n t a l f i e l d was  The folded-down p o r t i o n  never seen to s t i c k to any other  area of any other c e l l , although having ample opportunity to do so as i t made c o n t a c t s w i t h neighbouring c e l l s ,  ^here-  f o r e , the complementary s t i c k i n e s s appears to r e s i d e only w i t h i n the folded-down p o r t i o n of the f r o n t a l f i e l d  and  a s s o c i a t e d membranellar band, p o s s i b l y only on the bulge itself.  The'formation of a mating p a i r was  s u l t of two pre-conjugators adhering.  always the r e -  On no o c c a s i o n were  two v e g e t a t i v e c e l l s or a. v e g e t a t i v e and a- pre-conjugator seen to j o i n together. A f t e r two  c e l l s became f i r m l y u n i t e d they u s u a l l y r e -  mained r e l a t i v e l y motionless as the union between them broadened.  They remained  j o i n e d f o r v a r y i n g lengths of time,  u s u a l l y longer than 24 hours.  F i g u r e 7 (a, b, c, d, e) shows  a sequence of photomicrographs  of a mating p a i r , t r a c i n g i t s  development from i n i t i a l adhesion t i l l i n t o two  exconjugant  j u s t before s e p a r a t i o n  c e l l s , a t o t a l of 28  hours.  F i g u r e 3.  A n t e r i o r view of a pre-conjugator showing the bulge ( i n d i c a t e d by the arrow) w i t h i n the folded-down p o r t i o n of the f r o n t a l  field.  Magnification llOx.  F i g u r e 4.  Two  pre-conjugators with the folded-down  p o r t i o n of t h e i r f r o n t a l f i e l d s i n c l o s e a p p o s i t i o n ; bulges not y e t j o i n e d . Magnification llOx.  Figure 5 .  Two  pre-conjugators with bulges j u s t  joined.  Magnification llOx.  F i g u r e 6.  Contracted c e l l s j u s t before they p u l l a p a r t . Magnification  llOx.  F i g u r e 7.  A sequence  showing  the development  p a i r from i n i t i a l adhesion t i l l s e p a r a t i o n , a t o t a l of 28 hours.  of a mating  just  before  M a g n i f i c a t i o n 74x.  a)  Bulges j u s t u n i t e d .  b)  4 hours.  c)  9 hours.  d)  21 hours.  e)  28 hours, s e p a r a t i o n came during the f o l l o w i n g hour.  19  20 3.  P r o d u c t i o n of S e l f i n g The  Clones  term s e l f i n g , as used by students of c i l i a t e g e n e t i c s ,  r e f e r s i n i t s broadest  sense, to the occurrence  t i o n among i n d i v i d u a l s derived, by asexual from a s i n g l e parent  cell  of conjuga-  reproduction,  (.i.e. i n t r a c l o n a l  conjugation).  In t h i s respect Stentor appears to have a h i g h tendency f o r s e l f i n g s i n c e s i x of the nine stocks r e g u l a r l y produced subc u l t u r e s i n which i n t r a c l o n a l conjugation occurred.  To f u r t h e r  i n v e s t i g a t e the s e l f i n g a b i l i t y o f Stentor, s i n g l e c e l l s were i s o l a t e d , placed i n separate p e t r i d i s h c u l t u r e s and allowed to reproduce a s e x u a l l y . were observed  In each case, the r e s u l t i n g progeny  f o r the presence o f i n t r a c l o n a l  S i n g l e c e l l s from two sources were s e l e c t e d :  conjugation. normal vege-  t a t i v e s t e n t o r s from each o f the nine stocks and s t e n t o r s from stock S which had j u s t become p a i r e d a t conjugation. For the former group a s i n g l e v e g e t a t i v e c e l l was i s o l a t e d from each stock and placed s e p a r a t e l y i n t o a p e t r i d i s h c u l ture.  The source o f c e l l s f o r the l a t t e r group r e q u i r e s  e x p l a n a t i o n i n some d e t a i l .  In order to o b t a i n stock S c e l l s  which had j u s t become p a i r e d at conjugation i t was to  necessary  c l o s e l y observe a c u l t u r e i n the e a r l y stages of a con-  j u g a t i o n burst i n which there were a l a r g e number of p r e conjugators  evident.  When two pre-conjugators  were seen to  come together and s t i c k f i r m l y they were q u i c k l y removed and t r a n s f e r r e d along with approximately i n t o a small, p e t r i d i s h (35 x 10 mm)..  2 ml c u l t u r e f l u i d  Before any n u c l e a r  exchange could take p l a c e , the connection between the c e l l s  21 was  severed with a f i n e g l a s s needle.  separated c e l l s was ture.  Each of the  now  placed i n t o a separate p e t r i d i s h c u l -  A t o t a l of 12 c e l l s  (6 s p l i t p a i r s ) was  and grown i n c u l t u r e by t h i s  prepared  procedure.  The progress of a l l . 21 c u l t u r e s produced by the two sources was  followed f o r 30 days.  tures, no conjugation was subcultured and observed  observed,  I f , i n any  of the  cul-  then those dishes were  f o r an a d d i t i o n a l 30 days.  process o f s u b c u l t u r i n g was  above  This  repeated with each c u l t u r e  d i s h u n t i l conjugation appeared, or u n t i l f i v e subcultures had been made.  The r e s u l t s (.Table I I I ) show that s i n g l e  c e l l s f r o m stocks CH,  DF, H, S, T and W possess the  to produce clones which w i l l e x h i b i t i n t r a c l o n a l  ability  conjugation.  Further, the r e s u l t s show that c e l l s i s o l a t e d just, a f t e r they have become p a i r e d at conjugation a l s o have the a b i l i t y to self.  4.  Induced  Conjugation  In some cases, conjugation was  found to be induced  the i n t r o d u c t i o n of c e l l s belonging to one  by  stock i n t o a c u l -  ture d i s h c o n t a i n i n g c e l l s of d i f f e r e n t stock.  The  essential  f e a t u r e , necessary f o r the i n d u c t i o n , i s to mix s e x u a l l y r e a c t i v e c e l l s from two d i f f e r e n t stocks. complished  by the f o l l o w i n g technique.  Two  Mixtures were ac12 day o l d p e t r i  d i s h c u l t u r e s , not yet s e l f i n g , belonging to d i f f e r e n t were s e l e c t e d . was  One  c u l t u r e , which was  stocks  to act as the donor,  placed on the stage of the stereomicroscope  and the other  Table I I I .  I n t r a c l o n a l conjugation ( s e l f i n g ) i n s t a r t e d from s i n g l e c e l l s  Source of s i n g l e cells*  Presence of mating p a i r s i n the f i n a l subculture  Stock C.  5  no  Stock DF  0.  yes  Stock S  1  yes  Stock SC  5  no  Stock ST  5  no  Stock T  3  yes  Stock CH  1  yes  Stock H  0  yes  Stock W  3  yes  cell a  1.  yes  cell b  died  cell a  2  yes  celL b  0  yes  Split pair:  Split pair:  Split pair:  Split pair:  Split pair:  Split pair:  *  Number of s u b c u l t u r e s made a f t e r the i n i t i a l culture  cultures  cell a  died  cell b  2  yes  cell a  1  yes  cell b  1  yes  cell a  3  yes  cell b  1  yes  cell a  1  yes  cell b  1  yes  A l l . s p l i t p a i r s were from stock S..  23 beside the microscope.,  With the a i d o f the microscope and  u s i n g a Pasteur p i p e t , approximately  300 s t e n t o r s were  c a r e f u l l y removed from the agar surface o f the donor c u l ture d i s h and introduced i n t o the other c u l t u r e d i s h . Subsequent observation o f the r e c i p i e n t c u l t u r e d i s h revealed a s t r i k i n g sequence o f events which w i l l be r e f e r r e d to as the mating r e a c t i o n .  A f t e r a r e f r a c t o r y p e r i o d l a s t i n g about  5^-7 hours there was the gradual appearance o f numerous p r e conjugator c e l l s .  T h i s was followed immediately  a t i o n o f mating p a i r s .  by the form-  The r e c i p r o c a l experiment, i n which  the donor stock c u l t u r e of the previous experiment became the r e c i p i e n t o f c e l l s from the extraneous stock, showed a s i m i l a r mating r e a c t i o n , again l e a d i n g to the formation of mating p a i r s .  The induced  conjugation b u r s t s were d i f f e r e n t ,  i n some r e s p e c t s from the b u r s t s o f conjugation which occurred spontaneously First,  i n p e t r i d i s h c u l t u r e s o f the nine s t o c k s .  there were f a r more pre-conjugators  duced conjugation b u r s t s .  Pre-conjugators  found  i n the i n -  numbering i n  excess o f 100 were f r e q u e n t l y seen at one time during  induced  conjugation b u r s t s , whereas, i n spontaneous b u r s t s u s u a l l y l e s s than f o r t y pre-conjugators Secondly,  were evident at any one time.  i t was p o s s i b l e , i n the induced b u r s t s , to observe  many mating p a i r s being formed as t h e i r bulges adhered, while i n spontaneous bursts i t was d i f f i c u l t j u s t as they were being formed.  to f i n d mating p a i r s  T h i s d i f f e r e n c e i s probably  due to the g r e a t e r number o f pre-conjugators i n the induced  conjugation b u r s t .  being  present  24 5.  S p e c i f i c Mating Between Stocks E a r l y i n the i n v e s t i g a t i o n i t was found t h a t not a l l  combinations  o f c e l l s from the v a r i o u s stocks gave the induced  mating r e a c t i o n .  I n order to determine which  would r e s u l t i n an induced  combinations  conjugation burst, the stocks were 2  mixed together i n a l l p o s s i b l e combinations  o f twos .  To ac-  complish t h i s , each stock was t e s t e d i n d i v i d u a l l y by adding c e l l s from the remaining  seven d i f f e r e n t stocks (donors) to  a s e r i e s o f p e t r i d i s h c u l t u r e s ( r e c i p i e n t s ) o f the stock being tested«  The c o n t r o l i n each case was made by adding  c e l l s which belonged  to the stock being t e s t e d , to a c u l t u r e  of that same stock.  The mixtures were conducted  i n the man-  ner p r e v i o u s l y d e s c r i b e d f o r the i n d u c t i o n of conjugation. Each mixture was subsequently  observed  of an induced conjugation b u r s t .  f o r the appearance  A mixture was assumed to  be l a c k i n g i n any c a p a c i t y to g i v e a mating r e a c t i o n i f a f t e r 24 hours no pre-conjugators or mating p a i r s were formed. The r e s u l t s (Table IV) show the stocks to be d i v i s i b l e  into  two groups with r e s p e c t to t h e i r mating r e a c t i v i t y i n mixtures:  group I c o n s i s t s o f stocks C, SC and ST,, while group  II c o n s i s t s o f stocks S, T, CH, H and W. was observed  No, mating r e a c t i o n  i n mixtures o f c e l l s o f two stocks belonging to  the same group.  But, a mating r e a c t i o n d i d occur i n a l l mix-  tures where c e l l s from any stock i n group I were combined  Stock DF was not i n c l u d e d i n t h i s experiment because i t was not obtained u n t i l the l a t t e r part o f the i n v e s t i g a t i o n .  25  Table IV.  S p e c i f i c mating i n mixtures of stocks.  Stocks t e s t e d ( r e c i p i e n t s ) * S  T  CH  H  W  SC  +  +  +•  +  +  ST  +  +  +  +  +  C  SC  ST  Donor stocks C  S  +  T  +  CH  +  H  +  +  + •f-  +  +  W  *  Each v e r t i c a l column represents a s e r i e s of c u l t u r e s of a s i n g l e stock which were t e s t e d f o r t h e i r a b i l i t y to g i v e a mating r e a c t i o n w i t h c e l l s from the donor stocks. A p l u s (+) s i g n i n d i c a t e s a mating r e a c t i o n occurred. A minus (-) s i g n i n d i c a t e s no mating r e a c t i o n occurred.  with c e l l s from any  stock i n group I I .  These r e s u l t s s t r o n g l y suggest p a i r s i n appropriate mixtures sence o f two reasonable  that the formation of mating  i s the d i r e c t r e s u l t of the pre-  complementary mating types.  I t , t h e r e f o r e , seems  to t e n t a t i v e l y a s s i g n mating types to group I and II..  Hence,, the stocks i n group I become mating type I and those i n group I I become mating type I I .  6.  Demonstration o f I t was  Cross-mating  d e s i r e d to determine whether the mating  d u r i n g induced  conjugation b u r s t s was  mating, with one  observed  i n the form of c r o s s -  c e l l of a p a i r belonging to one  stock  and  the other c e l l belonging to the other stock. Several p r e l i m i n a r y observations l e d to the e a r l y b e l i e f that cross-mating  d i d occur during induced  conjugation b u r s t s .  These came from the s c r u t i n y of i n d i v i d u a l mating p a i r s formed i n mixtures;  i n the m a j o r i t y o f p a i r s , the two  cells  were r e c o g n i z a b l y d i f f e r e n t with respect to s i z e and i n t e n s i t y of pigmentation,  r e l a t i v e to each other.  Figure 8  shows a c h a r a c t e r i s t i c mating p a i r formed i n a mixture stock C and stock S c e l l s .  of  The l a r g e r more darkly p i g -  mented c e l l almost c e r t a i n l y belongs to stock S, whereas the smaller, more l i g h t l y pigmented c e l l almost belongs to stock C.  T h i s judgement was  certainly  r e i n f o r c e d by  measuring the diameter of contracted c e l l s of both stocks; measurement revealed a d i f f e r e n c e i n s i z e such that c e l l s of stock S averaged 272 microns while c e l l s of stock C were  27 smaller, averaging only 220 microns.  Further, when c e l l s  from stocks S and C were placed together i n a drop of water on a s l i d e and observed with the stereomicroscope, the d i f ference i n pigmentation i n t e n s i t y , although not great,  was  apparent. L a b e l l i n g w i t h carmine was used to i n v e s t i g a t e the occurrence of cross mating more f u l l y and to o b t a i n quant i f i a b l e results.  L a b e l l e d c e l l s of a group I stock were  mixed with u n l a b e l l e d c e l l s o f a group I I stock and both c e l l s o f the r e s u l t i n g mating p a i r s were i n d i v i d u a l l y on the b a s i s of three d i s t i n g u i s h i n g  scored  characteristics:  presence or abs.ence o f l a b e l , l i g h t or dark pigmentation with r e s p e c t to each other, and l a r g e or small s i z e with respect to each other.  For the experiment, stocks C and  S were chosen because the d i f f e r e n c e between them, w i t h respect to s i z e and pigmentation,was most e a s i l y seen. L a b e l l i n g was accomplished by the i n t r o d u c t i o n o f a carmine p a r t i c l e suspension (prepared by suspending 0.1 g powdered carmine i n 1 ml c u l t u r e f l u i d ) i n t o a c u l t u r e cont a i n i n g stock G.  Immediately, the c e l l s began to ingest  the carmine and a f t e r 15 minutes t h e i r cytoplasms contained l a r g e q u a n t i t i e s o f the l a b e l .  Following t h i s treatment,  approximately 300 newly l a b e l l e d c e l l s were removed, washed by c e n t r i f u g a t i o n i n three changes of f r e s h medium to remove any f r e e carmine p a r t i c l e s , and then added to a p e t r i c u l t u r e c o n t a i n i n g stock S.  dish  Twelve hours l a t e r the r e s u l t -  i n g mating p a i r s were removed, placed one at a time on a  g l a s s s l i d e i n a drop o f c u l t u r e f l u i d , and separated w i t h a f i n e g l a s s needle.  Each c e l l o f the p a i r was then p l a c e d  i n a separate drop o f c u l t u r e f l u i d on the s l i d e ,  squashed  l i g h t l y w i t h a cover g l a s s (Figure 9 ) , and examined f o r the presence o f carmine p a r t i c l e s u s i n g the steromicroscope. A l t o g e t h e r , three separate experiments were performed stocks C and S u s i n g the above l a b e l l i n g technique. results  with The  (Table V) f o r each mating p a i r were scored on the  b a s i s o f pigmentation, s i z e and presence o f carmine, r e s u l t i n g i n the emergence of s i x c l a s s e s o f mating  pairs  from the combined r e s u l t s o f a l l three experiments.  How-  ever, a c l o s e examination of the data i n Table V revealed that some c l a s s e s may be amalgamated to g i v e a t o t a l o f three d i s t i n c t c l a s s e s :  first,  a cross-mating c l a s s (com-  b i n i n g c l a s s e s 1 , 2 and 3) i n which mating p a i r s c o n s i s t o f one stock C c e l l and one stock S c e l l ;  secondly, a s e l f i n g  c l a s s (combining c l a s s e s 4 and 5) i n which mating  pairs  c o n s i s t o f two stock G c e l l s ; and t h i r d l y , an a d d i t i o n a l s e l f i n g c l a s s ( c l a s s 6 only) i n which mating p a i r s c o n s i s t of two stock S c e l l s .  T h i s r e d i s t r i b u t i o n i s based on the  p r o b a b i l i t y that some o f the l a b e l l e d stock C c e l l s had extruded the f o r e i g n carmine p a r t i c l e s thus l e a v i n g them unlabelled.  T h i s assumption  i s supported by the presence,  a f t e r 12 hours, o f carmine p a r t i c l e s on the bottom o f the c u l t u r e d i s h where at the beginning o f the experiment had been none.  there  Figure 8 .  Mating p a i r from a mixture of stocks C and S. M a g n i f i c a t i o n 104x.  F i g u r e 9.  Squashed  p a i r from a mixture of l a b e l l e d  stock S c e l l s and u n l a b e l l e d stock C c e l l s . (The arrows i n d i c a t e the carmine p a r t i c l e s . ) Magnification  104x.  Table V.  Observed C l a s s e s of Mating P a i r s Scored on the B a s i s o f R e l a t i v e Pigmentation, Presence o f Carmine, and R e l a t i v e S i z e .  Split pair ° Cell a class of pair  pigment  carmine  1  lgt  +  2  Igt  3  lgt  4  lgt  5  lgt  6  drk  Cell b size  Number of mating p a i r s  pigment  carmine  sml  drk  -  -*  sml  drk  +  sml  drk  sml  lgt  +  sml  lgt  -  lrg  drk  '  +  -  Expt 1  Expt 2  Expt 3  Total each class  lrg  33  29  41  103  lrg  4  2  4  10  lrg  0  1  0  '1  sml  1  1  1  3  sml  0  0  1  1  lrg  _1  _0  _1  2  Totals:  39  33  48  size  T o t a l p a i r s sampled:  120  Symbols and a b b r e v i a t i o n s : + carmine p a r t i c l e s p r e s e n t ; - no carmine p a r t i c l e s p r e s e n t ; l g t = l i g h t l y pigmented c e l l ; drk = d a r k l y pigmented c e l l ; sml = s m a l l e r c e l l ; l r g = l a r g e r * The absence o f carmine p a r t i c l e s i n these c e l l s i s probably due t o the c e l l s having extruded the f o r e i g n p a r t i c l e s (see t e x t f o r a f u r t h e r e x p l a n a t i o n ) . * T h i s c e l l contained a s i n g l e very small carmine p a r t i c l e which was probably picked up from the bottom of the c u l t u r e dish'.  cell  31 The r e d i s t r i b u t e d r e s u l t s s t r o n g l y suggest  that 95% of  the mating p a i r s formed i n the mixture were of the c r o s s mating type, while the remaining 5% were of the s e l f i n g  type.  The above l a b e l l i n g experiments were performed only with mixtures  of c e l l s from stocks G and S.  cant that i n mixtures induced  However, i t i s s i g n i f i -  of other stocks, which r e s u l t e d i n an  conjugation b u r s t , the m a j o r i t y of m&ting p a i r s formed  appeared to be o f the cross-mating type, judged  on the  d i f f e r e n c e s i n s i z e and pigmentation  c e l l s making  of the two  observed  up the p a i r s . The high i n c i d e n c e of cross-mating  overwhelmingly  supports  the c o n c l u s i o n that the mating r e a c t i o n , which occurred only i n c e r t a i n mixtures,was due mating types  7.  to the presence  of complementary  ( i . e . mating types I and I I ) .  The Role of C e l l to C e l l Contacts i n the I n d u c t i o n of Conjugation The a c t u a l stimulus which caused  when c e l l s from two  the mating r e a c t i o n  d i f f e r e n t stocks were mixed was  obvious, but, two d i s t i n c t  p o s s i b i l i t i e s were considered.  F i r s t l y , the c e l l s o f e i t h e r or both stocks may some f a c t o r i n t o the medium which caused  have put  other c e l l s to  become pre-conjugators thus l e a d i n g to the observed reaction.  not  The second p o s s i b i l i t y  mating  (suggested by Sonneborn  i n a personal communication) i s that the mating r e a c t i o n may  have been a consequence of a "contact i n t e r a c t i o n "  between s e x u a l l y r e a c t i v e c e l l s .  Such contact phenomena  32 have been found to e x i s t i n Euplotes (Heckman and S i e g e l , 1964) where i t has been shown that the c e l l s i n a mixture of two mating types a c t u a l l y have to make contacts, not contacts which r e s u l t i n s t i c k i n g together, but simply make and break contacts which s t i m u l a t e the c e l l s to become ready f o r conjugation some hours l a t e r . In order to determine the cause o f the mating r e a c t i o n i n Stentor, an experiment was performed i n which a s e r i e s of three d i f f u s i o n chambers was used to i s o l a t e groups of cells.  The c e n t r a l i d e a was to prevent the i s o l a t e d  cells  from c o n t a c t i n g c e l l s of another mating type, while, at the same time, s u b j e c t i n g them to the i n f l u e n c e of any d i f f u s i b l e f a c t o r s which might cause a mating r e a c t i o n . The d i f f u s i o n chambers (Figure 10a, b) were made from 2 cm lengths o f s l o t t e d p l e x i g l a s tubing (one i n c h i n diameter) wrapped w i t h nylon b o l t i n g c l o t h having a pore s i z e o f 62 microns (obtained from John S t a n i a r & Co., S t r e e t , Manchester, England).  Sherborn  The three chambers  (lettered  A, B and C i n F i g u r e 11) were placed i n t o a 12 day p e t r i d i s h c u l t u r e o f stock C, each chamber i n an area which had p r e v i o u s l y been c l e a r e d o f c e l l s .  The chambers were  pressed f i r m l y i n t o the agar l a y e r thus e f f e c t i v e l y i n g the bottom of the chamber.  seal-  D i f f u s i o n chambers p r e -  pared i n t h i s manner prevented the passage o f s t e n t o r s e i t h e r i n t o or out o f the chamber, but d i d not prevent the passage of the food organisms which were about 30 microns i n l e n g t h .  Figure 10.  D i f f u s i o n chamber (a) before wrapping w i t h b o l t i n g c l o t h and (b) a f t e r wrapping i s complete.  33  Figure 1 1 .  P e t r i d i s h c u l t u r e w i t h d i f f u s i o n chambers in  place.  34  To i n i t i a t e an experiment, c e l l s belonging to stocks C and S were a l l o t t e d to the three chambers i n the f o l l o w i n g way:  chamber A r e c e i v e d 100 stock C c e l l s ,  chamber B r e c e i v e d 100 stock S c e l l s , and chamber C r e c e i v e d 50 stock C c e l l s p l u s 50 stock S c e l l s .  Addition-  a l c e l l s o f stock S were added to the p e t r i d i s h i n the area o u t s i d e o f the d i f f u s i o n chambers.  The  procedure  d e s c r i b e d so f a r represented one h a l f of a two part experiment  with both p a r t s being executed  concurrently.  The  other h a l f . o f the experiment was the r e c i p r o c a l of the p a r t j u s t d e s c r i b e d and was performed i n the same manner  except  that the d i f f u s i o n chambers were placed i n t o a p e t r i d i s h c u l t u r e o f stock S i n s t e a d of stock C.  Both c u l t u r e dishes  were subsequently  observed  f o r the presence  o f pre-conjugators  or mating p a i r s .  A f t e r seven hours there were, i n both  dishes, pre-conjugators and mating p a i r s present i n chamber C and i n the area o u t s i d e the d i f f u s i o n chambers; both o f these areas contained c e l l s from stocks C and S mixed together. At the same time, chamber A ( c o n t a i n i n g only stock C c e l l s ) and chamber B ( c o n t a i n i n g only stock S c e l l s ) showed no evidence  of a mating r e a c t i o n w i t h no pre-conjugators or  mating p a i r s being formed even a f t e r 24 hours.  This e x p e r i -  ment was repeated three times with the same r e s u l t s . The r e s u l t s of t h i s s e r i e s o f experiments i n d i c a t e a l a c k of any d i f f u s i b l e f a c t o r a f f e c t i n g the mating a b i l i t y of c e l l s .  I t , t h e r e f o r e , seems evident that the only d i f -  ference between c e l l s i n chambers A or B and those i n the  3 6  other areas, l i e s i n the i n a b i l i t y o f the c e l l s  contained  i n chambers A and B to make contacts with c e l l s o f a d i f f e r e n t mating type.  F u r t h e r evidence  suggesting the  absence o f any d i f f u s i b l e f a c t o r a c t i n g i n the i n d u c t i o n o f conjugation was found  i n the i n a b i l i t y of conditioned medium  taken from a c t i v e l y conjugating c u l t u r e s to induce a t i o n when added to a non-conjugating of the f l u i d had been drained.  conjug-  c u l t u r e from which most  T h i s was repeated  several  times u s i n g d i f f e r e n t c u l t u r e s with no apparent e f f e c t o f c u l t u r e f l u i d upon c e l l s . These r e s u l t s s t r o n g l y suggest  that contacts between  c e l l s belonging to d i f f e r e n t mating types play an e s s e n t i a l r o l e i n the i n d u c t i o n o f conjugation.  8.  S u r v i v a l o f Exconjugants Although  an extensive study of exconjugants was not made,  the dominant impression acquired from the f o l l o w up o f exconjugants  was t h e i r apparent high m o r t a l i t y r a t e .  The high  m o r t a l i t y i s evident a f t e r a conjugation burst when l a r g e numbers of s l u g g i s h , poorly pigmented c e l l s appear which l i v e only a few days. ;  In s e v e r a l attempts to o b t a i n l i v i n g exconjugants, t o t a l o f 8 0 mating p a i r s was i s o l a t e d from d i f f e r e n t  a cul-  tures at v a r i o u s times during the course o f the i n v e s t i g a tion.  I n each case the mating p a i r s were placed separately  i n t o small p e t r i dishes c o n t a i n i n g new medium and food organisms.  A f t e r a p a i r separated,  one c e l l was t r a n s f e r r e d to  37 another  small p e t r i d i s h c o n t a i n i n g new medium and food  organisms.  Of the t o t a l o f 160 exconjugants produced by  the s e p a r a t i o n o f the 80 p a i r s , only two c e l l s , d e r i v e d from d i f f e r e n t p a i r s i s o l a t e d from a conjugation burst i n stock W,  survived long enough to begin d i v i s i o n .  These  two s u r v i v i n g exconjugants produced clones which were subcultured continuously f o r s i x months, however, no mating p a i r s were observed  i n e i t h e r l i n e of descent.  The remain-  i n g 158 exconjugants d i e d w i t h i n a few days a f t e r  isolation.  The most s u c c e s s f u l attempts to keep exconjugants a l i v e were with l a r g e groups of mating p a i r s which were i s o l a t e d and placed i n t o a p e t r i d i s h c u l t u r e .  Four groups of mating  p a i r s , ranging i n s i z e from 10 p a i r s to 60 p a i r s , were i s o l a t e d and placed i n t o new p e t r i d i s h c u l t u r e s .  One group  came from a conjugation burst i n a stock T c u l t u r e and the other three groups came from induced mixtures  o f stocks C and S.  Although  conjugation bursts i n a l a r g e number of the  exconjugants i n these groups d i e d , a few d i d survive i n each d i s h , e v e n t u a l l y g i v i n g r i s e to a p o p u l a t i o n .  A f t e r 30 days  i n c u l t u r e , during which no conjugation was observed, a l l f o u r dishes were subcultured.  The p o p u l a t i o n which descended  from the stock T exconjugants showed conjugation w i t h i n the first  subculture.  T h i s was a l s o true i n two o f the three  l i n e s of descent which o r i g i n a t e d from the mixtures C and S.  The remaining  o f stocks  l i n e d i d not show any conjugation  a f t e r four subcultures. To b r i e f l y summarize, i n the presence o f a high m o r t a l i t y  38 r a t e some exconjugants d i d s u r v i v e and d i v i d e ; i n some cases they gave r i s e to a p o p u l a t i o n o f c e l l s which conjugated i n the f i r s t  subculture, l e s s than 60 days a f t e r  ;  t h e i r p r e v i o u s mating.  9.  Abnormal  Mating  During the course of the i n v e s t i g a t i o n , two types o f abnormal mating were observed.  F i r s t , a small number  (approximately 15) of mating c e l l s showing  "multiconjugation"  were observed i n which three c e l l s were j o i n e d together i n stead o f the u s u a l two.  Figure 12 shows an example d i s c o v -  ered i n a conjugation burst induced by a mixture of c e l l s from stocks C and S.  A second type of abnormal mating,  found i n a stock S c u l t u r e , was  seen i n a s i n g l e mating  p a i r i n which one o f the c e l l s was at a l a t e stage o f d i v i s i o n (Figure 13).  The d i v i d i n g c e l l appeared to be at stage  7 ( T a r t a r , 1961) and remained at that stage throughout the f o u r hour p e r i o d d u r i n g which i t was observed. Abnormal mating, o f the two types d e s c r i b e d , appeared i n f r e q u e n t l y throughout the i n v e s t i g a t i o n .  F i g u r e 12..  An example of a mating t r i p l e t mixture  of stocks C and S.  found  (The two  in a larger  c e l l s appear to be stock S; the smaller probably belongs to stock  cell  C.)  Magnification llOx.  Figure 13.  Mating p a i r with one partner at about stage 7 of  division.  Magnification llOx.  39  40 DISCUSSION  In many c i l i a t e s , conjugation i s most r e a d i l y by mixing c u l t u r e s of d i f f e r e n t o r i g i n .  induced  From the r e s u l t s  of these mixtures, the c u l t u r e s can be c l a s s i f i e d i n t o or more mating types, being c l a s s e d as complementary types i f conjugation i s induced by the mixture 1957).  two  mating  (Sonneborn,  By t h i s d e f i n i t i o n i n d i v i d u a l s belonging to the  same mating type do not conjugate of s e l f i n g ) .  (except i n c e r t a i n cases  But when i n d i v i d u a l s of two d i f f e r e n t  types are mixed they w i l l u n i t e .  mating  Mating types have been  found i n seven genera of c i l i a t e s ; namely, Colpidium, Euplotes, Oxytricha, Paramecium, Tetrahymena, Sty1onychia, and Tokophyra (Sonneborn,  1957;  A l l e n , 1967).  A f t e r the i n i t i a l r e c u r r e n t observations of conjugation i n stock c u l t u r e s i t seemed reasonable to begin a search f o r mating was  types i n Stentor coeruleus.  The success of the search  enhanced by the d i s c o v e r y of a method to induce conjugation,  i n which the e s s e n t i a l f e a t u r e was two d i v e r s e s t o c k s .  the mixture of c e l l s  I t became apparent from mixture  ments that the eight stocks t e s t e d represented two  from  experi-  comple-  mentary mating types on the b a s i s of t h e i r response when mixed, two Table I V ) .  at a time, i n a l l p o s s i b l e combinations I t was,  (see  t h e r e f o r e , p o s s i b l e to a s s i g n stocks  to e i t h e r mating type I or mating type I I . of stocks i n t o mating  types was  This d i v i s i o n  d i s t i n c t i n that mixtures  c o n s i s t i n g of c e l l s of the same mating type d i d not r e s u l t  41 i n a mating r e a c t i o n .  But, when c e l l s o f d i v e r s e mating  type were mixed, a mating r e a c t i o n occurred.  By t h i s  d e f i n i t i o n , stocks C, SC, and ST belong to mating type I and r e a c t i n a p a r a l l e l manner when mixed w i t h stocks belonging to mating type I I .  I n a s i m i l a r way, stocks  CH, H, S, T and W belong to mating  type I I and r e a c t i n a  p a r a l l e l manner when mixed with mating type I s t o c k s . The occurrence o f two mating types i n the present study should not be taken to imply that only two mating types e x i s t i n this species.  In s e v e r a l c i l i a t e s p e c i e s , which have  been studied e x t e n s i v e l y , r a t h e r complex systems of i n t e r breeding mating types have been recognized.  I n these, a  p a r t i c u l a r species may have e i t h e r a s i n g l e v a r i e t y with two or more i n t e r b r e e d i n g mating  types (e.g. Euplotes  crassus Heckman, 1964) or s e v e r a l v a r i e t i e s , each with two or more i n t e r b r e e d i n g mating a u r e l i a , Sonneborn, 1957).  types (e.g. Paramecium  I n t e r b r e e d i n g between i n d i v i d u a l s  of d i f f e r e n t v a r i e t i e s does not occur.  By t h i s  interpreta-  t i o n , the eight stocks o f the present study belong to the same v a r i e t y s i n c e they c o n s t i t u t e two i n t e r b r e e d i n g mating types.  However, the small number of g e n e t i c a l l y  stocks (probably f i v e ; see Appendix) used i n t h i s t i o n emphasizes i t s p r e l i m i n a r y nature.  distinct investiga-  I t seems reasonable,  t h e r e f o r e , to suppose that f u t u r e s t u d i e s , u s i n g l a r g e r numbers o f stocks c o l l e c t e d from widely d i s t r i b u t e d areas, w i l l r e v e a l a d d i t i o n a l mating types and p o s s i b l y varieties.  different  42 In most c i l i a t e s , mating p a i r s formed i n mixtures o f two  complementary mating types always c o n s i s t o f i n d i v i d u a l s  of d i f f e r e n t mating type.  There have, however, been a few  cases reported where a small f r a c t i o n o f the p a i r s formed i n mixtures was apparently due to the union o f c e l l s o f the same mating type 1961;  (Hiwatashi, 1951; Metz, 1954; Katashima,  L a r i s o n and S i e g e l , 1961).  P a i r s formed f o l l o w i n g  mixtures o f d i v e r s e c u l t u r e s o f Stentor r e g u l a r l y of c e l l s o f d i v e r s e types.  consist  T h i s c o n c l u s i o n i s based on the  r e s u l t s o f the l a b e l l i n g experiments i n which the m a j o r i t y of p a i r s c o n s i s t e d o f one l a b e l l e d end one u n l a b e l l e d c e l l (Table V ) . The explanation f o r the occurrence o f the few p a i r s which appeared to be i n the form o f s e l f i n g i s not clear.  Metz (1954) showed that i n Paramecium c e l l s  belonging  to a s i n g l e clone can mate amongst themselves a f t e r some o f the i n d i v i d u a l s - have made t r a n s i e n t contacts with c e l l s o f a complementary mating type.  The explanation Metz o f f e r e d  was that c e l l s which have been temporarily u n i t e d with  individ-  u a l s of a complementary mating type may i n some cases a c q u i r e that mating type s p e c i f i c i t y i n the course of the contact. Thus a t r a n s i t o r y s h i f t o f mating type may occur i n these cases, r e s u l t i n g i n the formation o f s e l l i n g Hiwatashi  pairs.  (1951) found that s e l f i n g occurred i n f i v e  cent o f mating p a i r s formed i n mixtures  per-  o f Paramecium  caudaturn. In Stentor, s e l f i n g i s a l s o evident i n the occurrence of conjugation i n some stock c u l t u r e s and c l o n a l  cultures.  43 This type o f s e l f i n g probably  has a d i f f e r e n t b a s i s i n i t s  spontaneous appearance than that which appeared i n mixtures. The appearance o f the majority o f spontaneous conjugation bursts d u r i n g the i n t e r v a l 7-18 days a f t e r the i n i t i a t i o n of the c u l t u r e s (see Table I I ) seems best explained as being due to the n u t r i t i o n a l s t a t e o f the c u l t u r e . The n u t r i t i v e s t a t e o f the protozoan important  seems to be an  f a c t o r i n f l u e n c i n g conjugation i n a l l c i l i a t e s .  Sonneborn (1939) observed  that i n Paramecium a u r e l i a the  mating r e a c t i o n does not take p l a c e i n c u l t u r e s that are e i t h e r over-fed or completely  starved.  e x i s t s i n P. caudatum (Gilman, P. c a l k i n s i  A similar  situation  1939) and P. b u r s a r i a and  (Wichterman, 1953).  appeared to be the most important  Geise  (1939) found that food  s i n g l e f a c t o r i n regard to  conjugation i n P. multimicronucleatum  and that a d e c l i n e i n  a v a i l a b l e food supply a f t e r a p e r i o d o f p l e n t y was r e q u i r e d . In p r a c t i c e , c i l i a t e s are brought i n t o mating c o n d i t i o n f o r conjugation s t u d i e s by s u b j e c t i n g them to a d e c l i n i n g t i o n a l regime (Sonneborn, 1957; A l l e n , 1967).  nutri-  Stentors  c u l t u r e d i n the manner described are i n i t i a l l y provided  with  a period o f n u t r i t i o n a l abundance during which they m u l t i p l y rapidly.  But, as they approach maximum d e n s i t y the food  organism p o p u l a t i o n dwindles and the s t e n t o r s enter a p e r i o d of famine.  I t i s during the t r a n s i t i o n a l p e r i o d , when the  food p o p u l a t i o n i s d e c l i n i n g that the burst o f conjugation appears.  Therefore, Stentor appears to respond to a d e c l i n e  44 i n food supply a f t e r a period o f plenty by  selfing.  The f a c t that Stentor s e l f s b r i n g s up a problem i n regard to i t s mating type system.  The problem l i e s i n the  explanation of the b a s i s o f p a i r formation i n a p o p u l a t i o n which has not been mixed with another mating type.  Selfing  i n pure c u l t u r e s i s not uncommon i n c i l i a t e s i n which mating types have been found to e x i s t .  The s e l f i n g  conjugations  p r e v i o u s l y reported f o r Paramecium b u r s a r i a (Jennings, P. a u r e l i a  1941)  (Kimball, 1939a; Sonneborn, 1947), P. m u l t i m i c r o -  nucleatum (Sonneborn, 1957), Tetrahymena  p y r i f o r a l i s , ('Nanneyl  and Chaughey, 1955) and Euplotes crassus (Heckman,  1964)  always r e s u l t e d from the d i f f e r e n t i a t i o n o f i n d i v i d u a l s o f complementary  mating type w i t h i n the s e l f i n g c u l t u r e .  In  these cases, the s e l f i n g p a i r s were formed as e r e s u l t o f the union of c e l l s of complementary  mating,  type.  In the few  cases i n which such i n t r a c l o n a l mating has been s t u d i e d i t was explained as e i t h e r due to macronuclear heterogeneity and assortment o f macronuclear subunits during c l o n a l  expan-  s i o n ( A l l e n and Nanney, 1958), or as a consequence o f v a r i a b l e gene .expression (Butzel, 1955).  Recent s t u d i e s  have shown the l a t t e r to be the most l i k e l y  interpretation.  Heckman (1964, 1967) found i n Euplotes crassus that  selfing  i n o l d heterozygotes was due to a normally r e c e s s i v e being expressed.  In Paramecium a u r e l i a . s e l f i n g ,  allele  interpret-  able as due to changes i n gene a c t i v i t y , has been e x t e n s i v e l y studied  (Sonneborn, 1966; Taub, 1966; and Bleyman,  Returning, now,  1967a).  to the i n f l u e n c e of n u t r i t i o n on conjugation,  45 Hiwatashi (1958) found that s e l f i n g i n Paramecium caudatum was  s u s c e p t i b l e to environmental ( n u t r i t i o n a l ) c o n t r o l .  Mating type changes were i n f l u e n c e d by v a r y i n g growth c o n d i t i o n s , high f i s s i o n r a t e favored (Hiwatashi,  1960).  one  type,  low  another  B l eyman (1967b) found that i n Paramecium  a u r e l i a a period of r a p i d growth at 27° C followed  by a  d e p l e t i o n of food at 19° C would b r i n g about maximal r e a c t i v i t y , both f o r s e l f i n g and mating with standard  testers.  Both- Hiwatashi and Bleyman observed that t h e i r  respective  s e l f i n g stocks tended to produce c u l t u r e s which c o n s i s t e d predominantly of one mating type, ranged from those which d i d not  but that the  s e l f to those which r e g u l a r l y  s e l f e d , depending upon t h e i r o r i g i n and This observation  i s reminiscent  found i n the s e l f i n g stocks of  cultures  culture conditions.  of the c o n d i t i o n which i s Stentor.  I t i s i n t e r e s t i n g that a l l the stocks which s e l f r e g u l a r l y belong to mating type I I , while those which only r a r e l y s e l f belong to mating type I.  Gilman (1941) s i m i l a r l y  reported  that i n Paramecium caudatum s e l f i n g i s common i n some v a r i e t i e s and  i t may  occur more o f t e n i n c u l t u r e s of one p r e v a i l i n g  mating type than i n c u l t u r e s of the In view of the f i n d i n g s reported i n g i n Stentor may  be due  mating type expression  other. f o r other  ciliates,  to a p e r s i s t e n t i n s t a b i l i t y  self-  of  such that t r a n s i t o r y changes i n mating  type would lead to the s i t u a t i o n where two mating types were present  i n a culture.  complementary  Further,  the  cultures  46 may  be predominantly  of one mating type, but a p e r i o d o f  r a p i d growth followed by a d e p l e t i o n of food may  cause some  i n d i v i d u a l s to temporarily change mating type, thus p e r m i t t i n g the formation of mating p a i r s . ance of s e l f i n g a f t e r 7-18  T h i s would e x p l a i n the appear-  days i n c u l t u r e .  Depending upon  the o r i g i n o f the c u l t u r e the mating type changes may  involve  type I expressing type I I or a l t e r n a t i v e l y , type II expressing type I. t i o n s may ing  However, the d i s t i n c t p o s s i b i l i t y that f u t u r e i n v e s t i g a r e v e a l a d d i t i o n a l mating types capable of i n t e r b r e e d -  with the present types makes i t necessary  to consider the  p o s s i b i l i t y that i n d i v i d u a l s of type I or type I I may to  be able  express a mating type not yet discovered, a s i t u a t i o n  which would r e s u l t i n s e l f i n g .  I t was  p o s s i b l e to determine  the mating type of the s e l f i n g stocks by choosing c u l t u r e s which had not begun to s e l f , presumably c o n t a i n i n g c e l l s  ex-  p r e s s i n g only one mating type. When mating types I and II of Stentor were brought t o gether under c o n d i t i o n s which were f a v o r a b l e f o r conjugation, the onset of mating behavior occurred only a f t e r a w a i t i n g period of 5-7  hours.  The w a i t i n g period ( o f t e n c e l l e d  r e f r a c t o r y period) i s apparently widespread among the for  i t has been reported f o r Euplotes  the ciliates  (Kimball, 1939b; Katashima  1959;  Heckman 1963,  1952)  O x y t r i c h a ( S i e g e l , 1956), and Tetrahymena ( E l l i o t  Gruchy, 1952;  1964), S t y l o n y c h i a ( G r e l l , 1951;  Nanney and Caughey, 1953).  In mixtures  Downs, and  of  complementary mating types i n Euplotes. i t has been shown that during the w a i t i n g p e r i o d repeated c e l l contacts are  47 made between the two mating types, l e a d i n g subsequently to formation of mating p a i r s (Heckman and S i e g e l , 1964).  The  hypothesis here was that the contacts b r i n g about an exchange of i n f o r m a t i o n which causes the c e l l s to become ready f o r conjugation some hours l a t e r .  Heckman and S i e g e l provide  support f o r t h e i r hypothesis by demonstrating  that the w a i t -  ing p e r i o d can be eliminated i f c e l l s o f d i f f e r e n t mating types are each permitted to make previous contact w i t h of a t h i r d mating type.  cells  Thus, by p r e - t r e a t i n g the c e l l s  they found that when the mixture was made, mating occurred immediately  between the f i r s t two types.  In Stentor, c e l l to c e l l contacts appear necessary f o r the i n i t i a t i o n o f the mating r e a c t i o n . appears to be a l a c k o f any d i f f u s i b l e a f f e c t the mating a b i l i t y of c e l l s .  In a d d i t i o n , there f a c t o r which might  When c e l l s o f e i t h e r  mating type I or type I I were placed i n t o a d i f f u s i o n chamber i n such a way that they could not contact c e l l s of the other mating type but a t the same time were subjected to the e n v i r o n mental i n f l u e n c e s o f the other mating type, they d i d not form pre-conjugators or mating p a i r s .  In sharp c o n t r a s t , when  c e l l s o f mating type I and I I were placed together i n t o a d i f f u s i o n chamber the normal w a i t i n g p e r i o d was followed by the appearance o f pre-conjugators and, subsequently, pairs.  The r e s u l t s o f these experiments provide  mating  powerful  evidence i n support o f the hypothesis that contact between c e l l s o f d i f f e r e n t mating type provides a stimulus which causes the c e l l s to become ready f o r conjugation some hours  48 later. The nature of the i n f o r m a t i o n (stimulus) which appears to be exchanged i s unknown.  Metz (1954) presented an hypo-  t h e s i s which holds that c e l l unions l e a d i n g to conjugation are brought about by the i n t e r a c t i o n of complementary  mating  type substances which he s a i d were p r o t e i n i n nature. Evidence i n d i c a t i n g that p r o t e i n s y n t h e s i s i s necessary f o r the appearance and maintenance of mating a b i l i t y i s provided by Bleyman (1964) f o r Paramecium a u r e l i a and by Cohen (1965) f o r P. b u r s a r i a . 1964)  I t has been suggested  (Heckman and  Siegel,  that upon i n i t i a l contact c e l l s of complementary  meting  type cannot u n i t e but molecular amounts of mating type substances could be exchanged.  Then, the " f o r e i g n " p r o t e i n  could s t i m u l a t e s y n t h e s i s of a d d i t i o n a l mating type substances so that i n due course the c e l l u l a r c o n c e n t r a t i o n s would be s u f f i c i e n t l y high to allow c e l l unions.  The  wait-  ing p e r i o d would, of course, be the time d u r i n g which the new  mating type substances were being s y n t h e s i z e d . Stentor seems to respond to contact with c e l l s of comple-  mentary mating type i n a manner which i s at l e a s t  partially  s i m i l a r to that suggested f o r Euplotes by Heckman and  Siegel.  But, Stentor shows an a d d i t i o n a l response i n the f o l d i n g down of a p o r t i o n of i t s l e f t f r o n t a l f i e l d the form of a pre-conjugator.  as i t takes on  I f contacts w i t h c e l l s of  another mating type s t i m u l a t e s s y n t h e s i s of new  mating  type  substances i n t h i s genus, then such s y n t h e s i s apparently  49 r e s u l t s i n a h i g h l y l o c a l i z e d b u i l d up o f such  substances.  The i n i t i a l union at conjugation has i t s l o c u s s o l e l y  with-  in' the folded-down p o r t i o n o f the f r o n t a l f i e l d , p o s s i b l y only on the bulge produced w i t h i n i t .  A l s o , synthesis i s  e v i d e n t l y not completed u n t i l the c e l l has taken on the form o f a pre-conjugator, time do not r e s u l t i n c e l l F i n a l l y , the observed  s i n c e contacts previous to that unions. high m o r t a l i t y o f exconjugants  produced i n s e l f i n g c u l t u r e s may be due to inbreeding degene r a t i o n r e s u l t i n g from the accumulation  of d e l e t e r i o u s  r e c e s s i v e genes i n homozygous c o n d i t i o n . found  Nanney (1956, 1957)  that during the course o f inbreeding i n s t r a i n s o f  Tetrahymena v a r i o u s signs o f inbreeding degeneration one o f which was death at conjugation. evidence  appeared,  A l s o , there i s good  to i n d i c a t e that the use o f o l d stocks  (clones)  may r e s u l t i n a general n o n - v i a b i l i t y among exconjugants CSiegel, 1967).  The stocks employed i n the present  study  have i n some cases been c u l t u r e d f o r periods i n excess o f ten y e a r s .  A l a r g e p o r t i o n o f the a g e - c o r r e l a t e d m o r t a l i t y  i n c i l i a t e s seems to be due to p r o g r e s s i v e d e t e r i o r a t i o n o f micronuclei.  Several instances o f chromosomal i r r e g u l a r i -  t i e s i n o l d stocks have been reported f o r Paramecium .(Bippell, 1955; Sonneborn and S c h n e l l e r , ,1960) and T e t r a hymena (Nanney and Nagel,  1964; Wells, 1965).  A high m o r t a l i t y among exconjugants o f Stentor has a l s o been encountered by B u r c h i l l Holland  (1968).  (1967) and by Harden and  However, both o f these s t u d i e s r e p o r t the  50  s u r v i v a l o f some exconjugant study.  c e l l s , as does the present  These v i a b l e exconjugants appeared  q u i t e normal,  and were able to p r o l i f e r a t e , e v e n t u a l l y producing c l o n e s . The f a c t that some exconjugant ing  c e l l s do s u r v i v e i s encourag-  and should serve as a stimulus to seek the c o n d i t i o n s  necessary to i n c r e a s e v i a b i l i t y . In r e t r o s p e c t , the reported occurrence o f mating  types  i n Stentor should provide an experimental system with which to i n v e s t i g a t e i n h e r i t a n c e i n t h i s genus.  In addition,  i f conjugation can be brought under p r e c i s e and s t r i n g e n t experimental c o n t r o l then b i o l o g y would be provided with a most powerful t o o l toward analyses o f very fundamental tions.  ques-  Cross breeding could be combined with microsurgery  so that one could ask, f o r example, whether the macronucleus from a mating type I c e l l , when t r a n s p l a n t e d to a mating type I I c e l l , w i l l cause the host c e l l now to become mating type I - again, w i l l cytoplasm from a mating  type I c e l l  plus macronucleus from a mating type II. c e l l g i v e , i n combina t i o n , a mating type I I c e l l .  The outcome o f such  experiments  would provide v a l u a b l e i n f o r m a t i o n on the r e l a t i o n s h i p s which e x i s t between the nucleus and the cytoplasm.  51 SUMMARY  1.  Two experimental  methods, which r e g u l a r l y y i e l d l a r g e  numbers of mating p a i r s were used to i n v e s t i g a t e conjugation i n Stentor coeruleus. a p a r t i c u l a r c u l t u r e technique, mixture 2.  of d i f f e r e n t  One method i n v o l v e s  the other r e q u i r e s a  stocks.  Mating p a i r s appeared i n the form of conjugation b u r s t s , e i t h e r induced by mixing spontaneously  c e r t a i n stocks, or o c c u r r i n g  i n some stock c u l t u r e s and i n clones  produced by s i n g l e c e l l s i s o l a t e d from them. 3.  Spontaneous conjugation b u r s t s , i n the m a j o r i t y of cases occurred d u r i n g a d e f i n i t e i n t e r v a l i n the development of a c u l t u r e , p o s s i b l y due to the n u t r i t i o n a l c o n d i t i o n s w i t h i n the c u l t u r e .  4.  M o r p h o l o g i c a l l y d i s t i n c t pre-conjugator immediately  .cells appear  before as w e l l as during the i n i t i a l  of a conjugation b u r s t .  stages  Mating p a i r s are formed by the  union o f pre-conjugators. 5.  Mixing eight stocks i n a l l p o s s i b l e combinations of twos and observing t h e i r subsequent response revealed  they  were separable i n t o two complementary mating types. 6.  The m a j o r i t y o f mating p a i r s formed i n mixtures were o f the cross-mating  7.  type.  Contact between c e l l s of d i f f e r i n g mating type appears to be necessary  f o r the i n i t i a t i o n of a mating r e a c t i o n .  52 BIBLIOGRAPHY  A l l e n , S.L., 1967. Chemical Genetics of Protozoa. I n "Chemical Zoology." V o l . I . Academic P r e s s . New York pp. 617-694. A l l e n , S.L., and D.L. Nanney, 1958. A n a l y s i s o f n u c l e a r d i f f e r e n t i a t i o n , i n s e l f e r s o f Tetrahymena. Amer. 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Wells, C , 1965. Tetrahymena.  Age a s s o c i a t e d nuclear anomalies i n J . Proto. 12: 561-563.  Wichterman, R., 1953. "The B i o l o g y o f Paramecium". B l a k i s t o n Co., New York.  The  56 APPENDIX  O r i g i n and H i s t o r y o f Stocks  Stock C T h i s stock was obtained i n August  1967 from C a r o l i n a  B i o l o g i c a l Supply Company ( B u r l i n g t o n , North C a r o l i n a ) . I t was c o l l e c t e d i n the v i c i n i t y o f B u r l i n g t o n and had been maintained i n c u l t u r e f o r approximately 35 y e a r s . Stock DF T h i s stock was i s o l a t e d from a water sample taken from Ross Lake, B r i t i s h Columbia  i n March, 1968.  Stock S T h i s stock was obtained i n June 1966 from Dr. Vance T a r t a r . I t was c o l l e c t e d from S t e l l a Lake, Washington and had been maintained i n c u l t u r e since 1954. Stock SC T h i s stock was obtained i n February 1968 from S c h e t t l e B i o l o g i c a l s (P.O. Box 184, S t i l l w a t e r , Minnesota). c o l l e c t e d i n the v i c i n i t y o f Greensboro,  I t was  North C a r o l i n a and  had been maintained i n c u l t u r e f o r three y e a r s . Stock ST T h i s stock was obtained i n February 1968.from E.G. S t e i n h i l b e r + Co. (P.O. Box 888, Oshkosh, Wisconsin). originally  s t a r t e d from organisms  I t was  belonging to Stock SC.  57 Stock T This stock was  obtained i n August 1967  from the  General  B i o l o g i c a l Supply House (8200 South Hoyne Ave.., Chicago, Illinois).  I t was  had been maintained  c o l l e c t e d i n the v i c i n i t y of Chicago i n continuous  and  c u l t u r e f o r approximately  15 y e a r s . Stock  CH  T h i s stock was  obtained i n October 1967  from Charles  Harden (Science + Engineering Inc., 140 Fourth Waltham, Massachusetts).  I t was  organisms belonging to Stock  originally  Ave.,  s t a r t e d from  T.  Stock H T h i s stock was  obtained i n November 1966  De T e r r a (The I n s t i t u t e f o r Cancer Research, Ave.,  P h i l a d e l p h i a , Pennsylvania).  I t was  s t a r t e d from organisms belonging to Stock  from Noel 7701  Burlholme  originally T.  Stock W T h i s stock was Establishment  (P.O. Box  had been maintained o r i g i n was  obtained from Wards Natural Science 1712,  Rochester,  New  York).  It  i n c u l t u r e f o r three years, but i t s  not known.  

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