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Spore germination in a myxomycete, Fuligo septica (L.) Weber Corfman, Nancy Anne 1966

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SPORE GERMINATION IN A MYXOMYCETE. FULIGO SEPTICA (L.) WEBER  by NANCY ANNE CORFMAN B. A., U n i v e r s i t y  of C a l i f o r n i a  Santa B a r b a r a , 1963  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of BOTANY  We a c c e p t t h i s t h e s i s as conforming t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1966  In. p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e for  requirements  an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree  t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study,  I f u r t h e r agree t h a t p e r m i s s i o n . f o r e x t e n s i v e c o p y i n g o f t h i s  t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood  that  copying  o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a Vancouver 8, Canada  ii  ABSTRACT Spores o f F u l i g o s e p t i c a (L.) Weber were s t u d i e d by l i g h t and e l e c t r o n microscopy  t o determine  s t r u c t u r a l changes d u r i n g g e r m i n a t i o n .  L i g h t m i c r o s c o p i c o b s e r v a t i o n s i n d i c a t e t h a t few changes occur p r i o r t o p r o t o p l a s t r e l e a s e ; however, e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s show t h a t a number o f changes occur w i t h i n t h e p r o t o p l a s t b e f o r e emergence. An o v o i d n u c l e u s becomes i r r e g u l a r and l o b e d ; smooth, c i s t e r n a l endop l a s m i c r e t i c u l u m d e v e l o p s ; and c o n c u r r e n t development o f and c e n t r i o l e o c c u r s .  The dictyosomes  dictyosomes  and c e n t r i o l e a r e l o c a l i z e d i n  j u x t a n u c l e a r s i t e s , and t h e p r o x i m a l c y l i n d e r o f t h e c e n t r i o l e  dif-  f e r e n t i a t e s i n t o a b a s a l body o f a f u t u r e f l a g e l l u m . When t h e spore case r u p t u r e s , the i n n e r l a y e r o f t h e w a l l d i s a p p e a r s and t h e n u c l e u s r e v e r t s t o i t s o r i g i n a l o v o i d form.  The p r o t o p l a s t emerges  a wedge-shaped s p l i t i n t h e w a l l and g r a d u a l l y develops l a t e d c e l l o r , sometimes, a myxamoeba.  into a flagel-  S i m u l t a n e o u s l y , c o n t r a c t i l e and  f o o d v a c u o l e s d e v e l o p , the c i s t e r n a l endoplasmic ribosome-coated,  through  and a f l a g e l l u m develops  r e t i c u l u m becomes  from t h e b a s a l body.  i  iii TABLE OF CONTENTS  Introduction  1  M a t e r i a l s and Methods  4  Results Light Microscopic Results Electron Microscopic  6  Results  Stage One - R e s t i n g Spore Stage Two - S i x Hour Stage i n G e r m i n a t i o n  7 . . . . . . . .  10  Stage Three - Twelve Hour Stage i n G e r m i n a t i o n  12  Stage Four - Seventeen Hour Stage i n G e r m i n a t i o n  13  A.  Spores P r i o r t o P r o t o p l a s t Emergence  13  B.  P r o t o p l a s t Emergence  15  C.  Swarm C e l l s and Myxamoebae  15  Discussion  19  Summary  30  Bibliography  32  Appendix  iv  TABLE OF PLATES L i g h t m i c r o g r a p h s o f F u l i g o s e p t i c a spores a t Various stages i n germination. U l t r a s t r u c t u r e of F u l i g o s e p t i c a r e s t i n g s p o r e s . U l t r a s t r u c t u r e of F u l i g o s e p t i c a spores s i x hours a f t e r w e t t i n g t h e spores i n a b i l e s a l t s o l u t i o n and i n c u b a t i n g them i n e i t h e r s t e r i l e c a r b o n f i l t e r e d , d i s t i l l e d water o r i n s t e r i l e l e a f - e x t r a c t decoction. U l t r a s t r u c t u r e o f F u l i g o s e p t i c a spores t w e l v e hours subsequent t o w e t t i n g s p o r e s . U l t r a s t r u c t u r e o f F u l i g o s e p t i c a spores p r i o r t o p r o t o p l a s t emergence. U l t r a s t r u c t u r e o f F u l i g o s e p t i c a spore a t time o f emergence from spore c a s e . Ultrastructure of Fuligo septica protoplasts a f t e r emergence from s p o r e c a s e s .  V  ACKNOWLEDGEMENT  The  author g r e a t l y appreciates  and w i s h e s t o thank t h e f o l l o w i n g  persons f o r t h e i r a s s i s t a n c e and a d v i c e d u r i n g t h e p r e p a r a t i o n  of this  thesis. Dr. R. J . B a n d o n i , A s s o c i a t e  P r o f e s s o r , under whose s u p e r v i s i o n  t h i s t h e s i s was c a r r i e d out. Dr. T. B i s a l p u t r a , A s s i s t a n t P r o f e s s o r , who o f f e r e d h i s a d v i c e and  techniques f o r the e l e c t r o n microscopy p o r t i o n o f t h i s study. Dr.  G. C. Hughes, A s s i s t a n t P r o f e s s o r , who o f f e r e d s u g g e s t i o n s  which p r o v e d h e l p f u l . Alice-Ann  B i s a l p u t r a and Grace Wood, who h e l p e d i n t h e t y p i n g o f  this thesis. And,  t o o t h e r members o f the Department o f Botany, who have  h e l p e d a s s i s t t h e author d u r i n g h e r g r a d u a t e s t u d i e s a t t h e U n i v e r s i t y of B r i t i s h Columbia.  1. INTRODUCTION  G e r m i n a t i o n o f Myxomycete spores was i n 1854.  f i r s t by de Bary  He o b s e r v e d the r e l e a s e of f l a g e l l a t e d swarm c e l l s  spores of T r i c h i a r u b i f o r m i s  (now  t i o n , percentage of germination, to  from  c a l l e d H e m i t r i c h i a vesparium).  sequent s t u d i e s on s p o r e g e r m i n a t i o n  species  described  Sub-  i n d i c a t e d t h a t the r a t e of germina-  and method of g e r m i n a t i o n  vary  from  species.  Most i n v e s t i g a t o r s s t u d i e d the i n f l u e n c e o f e x t e r n a l f a c t o r s spore germination  i n Myxomycetes.  of medium on g e r m i n a t i o n . germination  Smart (1937) i n v e s t i g a t e d the e f f e c t  found t h a t the r a t e and p e r c e n t a g e of s p o r e  of many Myxomycete s p e c i e s were i n c r e a s e d i f spores were sown  i n weak d e c o c t i o n s decayed l e a v e s . w e t t i n g and mination.  He  on  o f n a t u r a l s u b s t r a t a such as humus, r o t t e d wood, and  Lister  (1901) and Durand (1894) i n d i c a t e d t h a t r e p e a t e d  d r y i n g of s p o r e s i n c r e a s e d the r a t e and p e r c e n t a g e o f g e r Elliott  (1949) n o t e d t h a t r e p e a t e d w e t t i n g and  e f f e c t on g e r m i n a t i o n the g e r m i n a t i o n  i n some of the Myxomycetes.  He  d r y i n g had  emphasized t h a t  r a t e i n c r e a s e d when spores were w e t t e d w i t h a one  bile salt solution.  Other w e t t i n g agents used i n a t t e m p t i n g  the r a t e and p e r c e n t a g e of g e r m i n a t i o n c u r i c c h l o r i d e , and  no  to  were a l c o h o l , d e t e r g e n t s ,  t r i s o d i u m phosphate ( C a y l e y , 1929"; E l l i o t t ,  percent increase mer1949).  C o n f l i c t i n g r e s u l t s were r e p o r t e d . Temperature was ing  shown t o be an i m p o r t a n t e x t e r n a l f a c t o r a f f e c t -  b o t h r a t e and p e r c e n t a g e of g e r m i n a t i o n  germination.  as w e l l as method o f  Smart (1937) i n d i c a t e d t h a t s p o r e g e r m i n a t i o n  was  b e s t at  2. a p p r o x i m a t e l y 25°C t o 30°C (see a l s o C o n s t a n t i n e a n u , 1906), and t h a t the r a t e and p e r c e n t a g e o f g e r m i n a t i o n were r e d u c e d g r e a t l y i f the temperature was  below 10°C  or above 30°C.  The i n f l u e n c e o f l i g h t , hydrogen i o n c o n c e n t r a t i o n , s p o r e c o n c e n t r a t i o n , and spore age a r e o t h e r f a c t o r s which have been i n v e s t i gated.  L i g h t appears t o have no i n f l u e n c e on g e r m i n a t i o n (Smart, 1937).  The optimum pH f o r s p o r e g e r m i n a t i o n i n most s p e c i e s i s between pH and pH 7.0  (Smart, 1937).  4.5  C o n c e n t r a t i o n o f sown spores i n f l u e n c e s  r a t e and p e r c e n t a g e of g e r m i n a t i o n ( S c h o l e s , 1962; Smart, 1937; W i l s o n and Cadman, 1928).  S c h o l e s (1962) has s t a t e d t h a t spores germinate  more r a p i d l y and the p e r c e n t a g e of g e r m i n a t i o n i s g r e a t e r w i t h  dilute  s u s p e n s i o n s o f s p o r e s ; whereas, Smart (1937) and W i l s o n and Cadman  (1928)  have r e p o r t e d the o p p o s i t e e f f e c t o f s p o r e c o n c e n t r a t i o n on g e r m i n a t i o n . Spore age i n f l u e n c e s t h e r a t e and p e r c e n t a g e of g e r m i n a t i o n i n some s p e c i e s ( A l e x o p o u l o s , 1963). S e v e r a l t y p e s o f s p o r e g e r m i n a t i o n have been d e s c r i b e d .  I n some  Myxomycetes the p r o t o p l a s t escapes through a wedge-shaped c r a c k i n t h e s p o r e w a l l ( G i l b e r t , 1928; Howard, 1931; McManus, 1961; Smart,  1937).  I n o t h e r s p e c i e s t h e p r o t o p l a s t e x i t s t h e s p o r e c a s e t h r o u g h an i r r e g u l a r p o r e ( G i l b e r t , 1928; Smart, 1937).  The p r o t o p l a s t , a t the t i m e  of emergence, has been d e s c r i b e d as a myxamoeba or f l a g e l l a t e d swarm c e l l , depending on the s p e c i e s and on e n v i r o n m e n t a l c o n d i t i o n s 1928; Smart, 1937).  (Gilbert,  I n some Myxomycetes the myxamoeba remains  q u i e s c e n t f o r a few minutes and then develops i n t o a f l a g e l l a t e d swarm cell  (Howard, 1931; Smart, 1937).  The swarm c e l l has been r e p o r t e d t o  3. be u n i f l a g e l l a t e d or b i f l a g e l l a t e d , and i n a few s p e c i e s , t r i f l a g e l l a t e d swarm c e l l s have been observed (Yuasa, 1935, not seen see E l l i o t t ,  1949).  Few s t u d i e s on c y t o p l a s m i c o r g a n e l l e changes d u r i n g g e r m i n a t i o n of  f u n g a l spores have been p u b l i s h e d .  development and enlargement 1928;  Smart, 1937).  I n v e s t i g a t o r s have n o t e d the  of a vacuole p r i o r to germination ( G i l b e r t ,  C o n c u r r e n t w i t h v a c u o l a r f o r m a t i o n i s movement o f  cytoplasmic granules.  G r a d u a l l y the spore w a l l becomes s t r e t c h e d and a  pore develops through which t h e p r o t o p l a s t emerges ( G i l b e r t , 1928; 1937).  F l a g e l l a r f o r m a t i o n i s r e p o r t e d t o o c c u r subsequent  Smart,  t o pore or  c r a c k development i n t h e s p o r e w a l l ( G i l b e r t , 1928; Howard, 1931; McManus, 1961; Smart, 1937). U l t r a s t r u c t u r a l changes i n Myxomycete spores d u r i n g g e r m i n a t i o n have not been r e p o r t e d , and few papers have been p u b l i s h e d on t h e f i n e s t r u c t u r e of Myxomycete spores ( L o q u i n , 1959; S c h u s t e r , 1964; W o h l f a r t h Bottermann,  1959).  The f i n e s t r u c t u r e of myxamoebae has been o b s e r v e d  i n Didymium n i g r i p e s ( S c h u s t e r , 1964), and Cohen (1959) has  studied  f l a g e l l a t i o n i n some Myxomycete swarm c e l l s . L i t t l e i s known about changes t h a t occur from t h e time of spore f o r m a t i o n to the p e r i o d i n which swarm c e l l s or myxamoebae a r e r e l e a s e d . Thus, the purpose of t h i s s t u d y i s to i n v e s t i g a t e t h e s e q u e n t i a l changes o c c u r r i n g i n Myxomycete spores d u r i n g g e r m i n a t i o n . (L.)  Fuligo  Weber, spores have been s e l e c t e d f o r t h i s s t u d y because they  germinate r a p i d l y and i n h i g h p e r c e n t a g e s i n the l a b o r a t o r y .  septica  MATERIALS AND METHODS Spores of F u l i g o s e p t i c a were o b t a i n e d from a e t h a l i a w h i c h had been c o l l e c t e d on t h e U n i v e r s i t y o f B r i t i s h Columbia Endowment Lands and i d e n t i f i e d by R. J . B a n d o n i , J u l y , A p p r o x i m a t e l y 0.1  1963.  g o f spores was w e t t e d i n a one p e r c e n t D i f c o  b i l e s a l t s o l u t i o n f o r one m i n u t e and r i n s e d t w i c e w i t h s t e r i l e c a r b o n f i l t e r e d , d i s t i l l e d water. plastic  The spores were sown i n 60 x 20 mm  sterile  d i s p o s a b l e p e t r i d i s h e s w h i c h c o n t a i n e d e i t h e r 25 ml o f s t e r i l  c a r b o n - f i l t e r e d , d i s t i l l e d water or 25 ml o f s t e r i l e l e a f - e x t r a c t decoction.  The l e a v e s used i n making t h e l e a f - e x t r a c t d e c o c t i o n were  from deciduous t r e e s and had been p i c k e d a t random on t h e U n i v e r s i t y of B r i t i s h Columbia Endowment Lands. The p e r c e n t a g e of g e r m i n a t i o n and r a t e of g e r m i n a t i o n were determined by a v e r a g i n g t h e r e s u l t s o f d u p l i c a t e s p o r e s u s p e n s i o n s s u b j e c t e d t o t h e same c o n d i t i o n s .  To determine t h e p e r c e n t a g e of germi  n a t i o n , one drop of s p o r e s u s p e n s i o n was p i p e t t e d onto a g l a s s s t a i n e d w i t h i o d i n e p o t a s s i u m i o d i d e ( I K I ) , and examined by microscopy.  slide,  light  The f i r s t 300 spores examined were used t o determine t h e  germination percentage.  A p p r o x i m a t e l y 90 t o 95 % o f t h e spores germi-  n a t e d 17 t o 20 hours a f t e r they had been w e t t e d i n t h e b i l e  salt  s o l u t i o n and sown i n e i t h e r s t e r i l e c a r b o n - f i l t e r e d , d i s t i l l e d water or i n l e a f - e x t r a c t d e c o c t i o n . The optimum temperature f o r g e r m i n a t i o n was  determined by i n -  c u b a t i n g the s p o r e s u s p e n s i o n i n temperature c o n t r o l l e d chambers s e t a t a p p r o x i m a t e l y 5°C, 10°C,  15°C,  20°C, 25°C, 30°C, and 35°C.  The  temperature a t which the r a t e o f g e r m i n a t i o n and p e r c e n t a g e of g e r m i -  5. n a t i o n was g r e a t e s t was a p p r o x i m a t e l y 25°C.  T h i s temperature was d e s i g n a t e d  as t h e optimum temperature f o r spore g e r m i n a t i o n and f u r t h e r g e r m i n a t i o n s t u d i e s were performed a t t h i s e s t a b l i s h e d optimum temperature. A l i g h t m i c r o s c o p i c s t u d y o f t h e g e r m i n a t i o n p r o c e s s was w i t h l i v i n g m a t e r i a l and m a t e r i a l s t a i n e d w i t h I K I .  performed  Spores were examined  w i t h a L i e t z D i a l u x m i c r o s c o p e u s i n g b r i g h t f i e l d i l l u m i n a t i o n , dark i l l u m i n a t i o n , and phase c o n t r a s t .  field  M i c r o g r a p h s were t a k e n w i t h a L i e t z  Orthomat m i c r o s c o p e . The m a t e r i a l f o r e l e c t r o n m i c r o s c o p y was f i x e d a t f o u r d i f f e r e n t times d u r i n g t h e g e r m i n a t i o n p e r i o d .  The times a r b i t r a r i l y were d e c i d e d on  and d e s i g n a t e d as s t a g e s one, two, t h r e e , and f o u r .  Stage one r e p r e s e n t e d  r e s t i n g spores which were not s u b j e c t e d t o g e r m i n a t i o n t r e a t m e n t .  Spores o f  s t a g e two were w e t t e d and sown i n t h e g e r m i n a t i o n medium f o r s i x h o u r s , and those of stage three f o r twelve hours.  Stage f o u r r e p r e s e n t e d t h e spores  i n which t h e p r o t o p l a s t s were about t o emerge from t h e s p o r e cases or which had emerged from t h e spore c a s e s . F i x a t i o n o f m a t e r i a l f o r e l e c t r o n m i c r o s c o p y was w i t h u n b u f f e r e d 1.5 % KMnO^ f o r 20 minutes a t 0°C o r 1 % 0 s 0 7.2 f o r one hour a t 0°C.  4  b u f f e r e d w i t h .15 M phosphate a t pH  A f t e r the spores were washed w e l l w i t h d i s t i l l e d  w a t e r , they were embedded i n a drop o f 4 % water agar.  The agar drop was  c u t i n t o s m a l l p i e c e s and t h e p i e c e s were dehydrated i n a graded s e r i e s o f ethanol-propylene oxide s o l u t i o n s .  M o d i f i e d Maraglas  ( B i s a l p u t r a and W e i e r ,  1963) was used t o embed t h e d e h y d r a t e d m a t e r i a l and t h e b l o c k s were p o l y m e r i z e d 18 t o 24 hours i n a vacuum oven a t 65°C.  The m a t e r i a l was s e c t i o n e d  w i t h g l a s s k n i v e s w i t h e i t h e r a P o r t e r - B l u m MI 1 o r MI 2 microtome. mounting  t h e s e c t i o n s on c o l l o d i a n - c o a t e d copper g r i d s , t h e specimens  After were  p o s t - s t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e ( R e y n o l d s , 1963). A l l s e c t i o n s were examined w i t h a H i t a c h i HU-11A m i c r o s c o p e .  6. RESULTS A p p r o x i m a t e l y 90 t o 95 % o f t h e spores o f F u l i g o s e p t i c a germin a t e d a f t e r 17 t o 20 hours i n e i t h e r s t e r i l e l e a f - e x t r a c t d e c o c t i o n or i n s t e r i l e c a r b o n - f i l t e r e d , d i s t i l l e d water.  Spores sown i n s t e r i l e  l e a f - e x t r a c t d e c o c t i o n tended t o germinate s l i g h t l y sooner than spores sown i n s t e r i l e c a r b o n - f i l t e r e d , d i s t i l l e d w a t e r . Light Microscopy Few changes w i t h i n t h e s p o r e case d u r i n g g e r m i n a t i o n can be observed w i t h t h e r e s o l u t i o n o f t h e l i g h t m i c r o s c o p e .  The spores appear  s p h e r i c a l ( F i g . 1 ) , 6 t o 9 y. i n d i a m e t e r , and t h e spore w a l l i s s p i n u lose.  The p r o t o p l a s t , a t f i r s t q u i e s c e n t , becomes a g i t a t e d ; and a  d i s t i n c t nucleus i s observed i n i o d i n e potassium i o d i d e s t a i n e d m a t e r i a l . The s p h e r i c a l n u c l e u s seems t o m a i n t a i n i t s form d u r i n g g e r m i n a t i o n . There appears t o be a s l i g h t i n c r e a s e i n t h e number o f v a c u o l e s w i t h i n the p r o t o p l a s t p r i o r to the s p l i t t i n g of the spore case. A p p r o x i m a t e l y 17 hours a f t e r w e t t i n g t h e s p o r e s , wedge-shaped s p l i t s develop i n t h e s p o r e c a s e s .  The s p l i t s g r a d u a l l y become more  pronounced u n t i l t h e i r l e n g t h equals a t l e a s t t h r e e - f o u r t h s t h e diameters of the spores.  S i m u l t a n e o u s l y , t h e r e i s c o n s i d e r a b l e movement o f t h e  p r o t o p l a s m and t h e p r o t o p l a s t s b e g i n t o emerge from t h e spore cases (Figs. 2 - 5 ) .  The p e r i o d o f time f o r t h e emergence o f t h e p r o t o p l a s t s  i s 3 to 5 minutes.  Only one p r o t o p l a s t emerges from each s p o r e .  When r e l e a s e d , t h e p r o t o p l a s t s assume a s p h e r i c a l shape and remain i n an o s c i l l a t o r y s t a t e near t h e mouths o f t h e r u p t u r e f o r 10 t o 15 minutes  (Figs. 6 - 7 ) .  The p r o t o p l a s t s then become amoeboid and r a p i d l y  develop i n t o f l a g e l l a t e d swarm c e l l s .  No f l a g e l l a a r e seen u n t i l  after  the p r o t o p l a s t s become amoeboid. Most o f t h e swarm c e l l s a r e u n i f l a g e l l a t e ( F i g s . 9 - 1 0 ) ; however, b i f l a g e l l a t e d c e l l s a r e not uncommon ( F i g . 8 ) . The f l a g e l l a o f both u n i f l a g e l l a t e d and b i f l a g e l l a t e d c e l l s a r e about t h e same l e n g t h as t h e b o d i e s o f t h e swarm c e l l s ( F i g s . 8 - 1 0 ) .  7.  E l e c t r o n Microscopy Stage One - R e s t i n g The  Spore  t y p i c a l s t r u c t u r e o f most r e s t i n g spores o b s e r v e d i n t h i s  study i s seen i n f i g u r e 11.  A d e s c r i p t i o n of the u l t r a s t r u c t u r e of the  r e s t i n g spore f o l l o w s : Wall:  A m u l t i - l a y e r e d , s p i n u l o s e w a l l surrounds t h e spore p r o t o p l a s t  (W, F i g . 1 1 ) . I t appears s t r u c t u r a l l y s i m i l a r t o t h e s p o r e w a l l s o f D. nigripes  (Schuster,  1964).  i s e l e c t r o n dense, g r a n u l a r , dense s p i n e s  The o u t e r l a y e r o f t h e w a l l (OW, F i g . 12) and i s a p p r o x i m a t e l y 50 mu wide.  Electron  a r i s e a t i r r e g u l a r i n t e r v a l s on t h e s u r f a c e o f t h e o u t e r  l a y e r ( F i g . 1 1 ) . The s p i n e m a t e r i a l a l s o appears g r a n u l a r , and each s p i n e i s about 150 mu l o n g .  A f i b r i l l a r , and almost e l e c t r o n t r a n s -  p a r e n t i n n e r l a y e r (IW, F i g . 12) l i e s between t h e e l e c t r o n dense o u t e r l a y e r and t h e plasma membrane.  This inner layer i s approximately the  same w i d t h as t h e o u t e r l a y e r , and i t i s s u b - d i v i d e d by a 100 A° e l e c t r o n dense l i n e .  i n t o two s e c t i o n s  The e l e c t r o n l i g h t s e c t i o n o u t s i d e t h e  e l e c t r o n dense l i n e i s a p p r o x i m a t e l y 40 mu wide.  The s e c t i o n a d j a c e n t t o  the plasma membrane v a r i e s i n w i d t h , r a n g i n g from 2 mu t o 4 mu.  The  e l e c t r o n dense l i n e appears s i m i l a r t o t h e e l e c t r o n dense o u t e r l a y e r o f the w a l l ( F i g . 12) . Plasma Membrane:  A d i s t i n c t plasma membrane envelops t h e p r o t o p l a s m  of t h e s p o r e (PM, F i g . 1 1 ) . I t i s a 120 A° t h i c k , s i n g l e u n i t membrane which i s s y m m e t r i c a l l y  3 layered  (45 A° - 30 A° - 45 A ° ) . The plasma  membrane g e n e r a l l y appears smooth; however, i r r e g u l a r i t i e s , as seen i n f i g u r e s 16 and 22, a r e n o t i n f r e q u e n t .  8. Vacuoles:  There a r e s e v e r a l v a c u o l e s i n t h e c y t o p l a s m o f t h e spore (V,  F i g . 1 1 ) . These v a c u o l e s a r e bound by s i n g l e u n i t membranes and c o n t a i n e l e c t r o n t r a n s p a r e n t o r g r a n u l a r v a c u o l a r s a p ; membrane-like fragments; and some e l e c t r o n dense, g r a n u l a r m a t t e r  ( F i g . 1 3 ) . The v a c u o l e s  range  i n s i z e from 1 t o 2 p . Transparent V e s i c l e s :  Two types o f e l e c t r o n t r a n s p a r e n t v e s i c l e s a r e  d i s p e r s e d randomly throughout t h e c y t o p l a s m o f t h e s p o r e .  One type o f  v e s i c l e i s membrane-bound and i s about .1 t o .3 fi i n diameter Some o f t h e membranes about t h e s e v e s i c l e s appear broken.  ( F i g . 11).  The o t h e r t y p e  o f e l e c t r o n t r a n s p a r e n t v e s i c l e (TVe, F i g . 14) i s n o t bound by a membrane. These v e s i c l e s a r e i r r e g u l a r l y shaped and v a r y from .2 t o .4 ju i n d i a m e t e r .  Mitochondria:  Mitochondria with tubular-type c r i s t a e are distributed  randomly w i t h i n t h e p r o t o p l a s t (M, F i g . 1 1 ) . The s i z e , shape, and number o f m i t o c h o n d r i a per p r o t o p l a s t v a r i e s from spore t o s p o r e .  Most m i t o -  c h o n d r i a a r e somewhat s p h e r i c a l t o o v o i d and a r e about 1 t o 1.5 p. i n diameter  (M, F i g . 1 5 ) . Each m i t o c h o n d r i o n i s surrounded by an i n n e r u n i t  membrane and an o u t e r u n i t membrane.  The i n n e r membrane, a p p r o x i m a t e l y  80 A° t h i c k , i n v a g i n a t e s f o r m i n g t u b u l a r c r i s t a e which a r e about 25 mu i n diameter. Each c r i s t a : may branch s e v e r a l times f o r m i n g s i d e t u b u l e s . Most o f t h e c r i s t a e extend g e n e r a l l y towards m i t o c h o n d r i o n ; however,  the c e n t r a l region of the  a few o f t h e c r i s t a e appear t o be c o n t i n u o u s  w i t h c r i s t a e a r i s i n g from o t h e r r e g i o n s of t h e m i t o c h o n d r i o n ( F i g . 1 5 ) . The i n t e r n a l space o f t h e t u b u l a r c r i s t a e i s e l e c t r o n t r a n s p a r e n t , and the m i t o c h o n d r i a l m a t r i x i s s l i g h t l y more e l e c t r o n dense than t h e  9. cytoplasm ( F i g . 15). An e l e c t r o n dense, c o a r s e l y g r a n u l a r occupies  t h e c e n t r a l r e g i o n of most o f t h e m i t o c h o n d r i a  matter  ( F i g s . 15 - 16).  T h i s dense c o r e i s about 100 mu i n diameter and i t appears t o l a c k any organized  s t r u c t u r e ( F i g . 16). The outer membrane surrounds t h e  mitochondrion.  T h i s membrane does not i n v a g i n a t e and i t i s a p p r o x i -  mately as t h i c k as t h e i n n e r membrane.  The m i t o c h o n d r i a l membranes o f  the r e s t i n g spores do not f i x as d i s t i n c t l y as do t h e plasma membranes and  t h e v a c u o l a r membranes ( F i g . 16).  Cytoplasm and Other I n c l u s i o n s : a granular  C h a r a c t e r i s t i c o f t h e r e s t i n g spore i s  c y t o p l a s m which i s i n t e r m e d i a t e  i n d e n s i t y between the e l e c t r o n  dense outer  l a y e r o f t h e w a l l and the l e s s e l e c t r o n dense v a c u o l a r sap  ( F i g . 11).  Some o f the 100 t o 130 A ° granules  ribosomes i n s i z e and d e n s i t y Nucleus:  ( F i g . 14).  A s i n g l e , ovoid nucleus i s present  i n t h e p r o t o p l a s t o f each  spore of F. s e p t i c a (N, F i g . 11). T h i s n u c l e u s , 2 p wide, c o n t a i n s  i n t h e c y t o p l a s m resemble  a s i n g l e nucleolus  about 3 t o 3.5 p  l o n g and  (Nu, F i g . 17). The n u c l e o l u s i s  a p p r o x i m a t e l y 1 p. i n diameter and i s e l e c t r o n dense and g r a n u l a r . l e a s t one n u c l e o l a r v a c u o l e the n u c l e o l a r v a c u o l e  i s present  nor the n u c l e o l u s  At  i n each n u c l e o l u s , and n e i t h e r i s surrounded by a membrane.  The  chromatin m a t e r i a l i n t h e n u c l e u s i s almost as e l e c t r o n dense as t h e nucleolar granules,  and i t appears g r a n u l a r  and randomly  dispersed  throughout t h e l e s s e l e c t r o n dense n u c l e o p l a s m ( F i g . 1 7 ) . The n u c l e a r m a t e r i a l i s encompassed by an envelope which i s composed o f two membranes, each about 80 A  0  thick.  A p e r i n u c l e a r space, about 110 A ° wide, i s seen  between the two membranes.  Both the outer  and the i n n e r membranes o f  10. the envelope a r e smooth, no ribosomes  a r e p r e s e n t on t h e i r s u r f a c e s .  D i s c o n t i n u i t i e s i n t h e n u c l e a r envelope form " p o r e s " which a r e 200 to 400 A° i n d i a m e t e r .  These pores a r e s i m p l e and occur a t i r r e g u l a r  i n t e r v a l s ( P , F i g s . 17 and 20).  No a n n u l i a r e seen a t the c i r c u m f e r e n c e  of t h e pores and no diaphrams c r o s s t h e diameter o f t h e pores t o s e p a r a t e the n u c l e a r m a t e r i a l from t h e c y t o p l a s m . observed i n t h e n u c l e a r envelope appears  B l e b f o r m a t i o n commonly i s  (NB, F i g s . 18 - 1 9 ) . One t y p e o f b l e b  t o be formed by t h e e v a g i n a t i o n o f t h e o u t e r membrane o f t h e  n u c l e a r envelope and t h e f o r m a t i o n o f another membrane w i t h i n t h i s e v a g i n a t i o n (NB, F i g . 1 8 ) . Thus, t h i s t y p e o f b l e b i s encompassed by two membranes.  A second type o f b l e b (NB, F i g . 1 9 ) , surrounded by o n l y  one membrane, a l s o i s seen i n a number o f t h e n u c l e a r e n v e l o p e s . appears  It  t o be formed by t h e e v a g i n a t i o n o f t h e o u t e r membrane and t h e  i n v a g i n a t i o n o f t h e i n n e r membrane of t h e envelope. Stage Two - S i x Hour Stage i n G e r m i n a t i o n Spores examined s i x hours subsequent  to i n i t i a t i o n of germination  i n d i c a t e few u l t r a s t r u c t u r a l changes have o c c u r r e d .  These spores appear  s i m i l a r t o t h e one shown i n f i g u r e 22; however, v a c u o l e s c o n t a i n i n g membrane-like fragments, n o t p r e s e n t i n f i g u r e 22, a r e p r e s e n t i n most spores a t t h i s s t a g e . Many spore o r g a n e l l e s do n o t change i n s t r u c t u r e and, t h e r e f o r e , a d e s c r i p t i o n i s made o n l y when s t r u c t u r a l changes occur o r when new o r g a n e l l e s a r e encountered. Nucleus:  The n u c l e i o f spores a t t h i s s t a g e o f g e r m i n a t i o n assume a  v a r i e t y o f forms (N, F i g s . 22 - 2 3 ) . The n u c l e a r envelope becomes more  11. d i s t i n c t and no b l e b f o r m a t i o n i s d i s t i n g u i s h a b l e ( F i g . 2 3 ) . The c h r o m a t i n m a t e r i a l , n u c l e o p l a s m , and n u c l e o l u s do n o t appear t o be a l t e r e d (Fig. 23). Mitochondria:  Both t h e i n n e r and t h e o u t e r membranes o f t h e m i t o c h o n d r i a  f i x more d i s t i n c t l y than they do i n t h e r e s t i n g spore s t a g e ( F i g s . 22 and 2 4 ) , and t h e dark c o r e s w i t h i n t h e m i t o c h o n d r i a l m a t r i x become more prominent.  Each c o r e , a p p r o x i m a t e l y 100 t o 150 mu i n d i a m e t e r ,  lies  p a r a l l e l t o t h e l o n g i t u d i n a l a x i s o f t h e m i t o c h o n d r i o n , and each i s composed o f numerous f i n e f i b r i l s .  Each f i b r i l i s about 30 A° i n d i a m e t e r .  Some f i b r i l s a r e seen i n c r o s s - s e c t i o n near t h e edges o f t h e c o r e , w h i l e other f i b r i l s run p a r a l l e l t o the l o n g i t u d i n a l a x i s of the mitochondrion (Fig. 24). Transparent V e s i c l e s :  There i s an i n c r e a s e i n t h e number o f s m a l l ,  membrane-bounded v e s i c l e s i n t h e c y t o p l a s m (Ve, F i g . 2 5 ) . The membranes encompassing t h e v e s i c l e s a r e a p p r o x i m a t e l y 80 t o 100 A° i n t h i c k n e s s , w h i l e t h e diameter of t h e v e s i c l e s ranges from 50 t o 100 mu.  These  v e s i c l e s a r e randomly d i s t r i b u t e d w i t h i n t h e p r o t o p l a s t . Endoplasmic  Reticulum:  C i s t e r n a l endoplasmic  i s p r e s e n t i n some o f t h e s p o r e s .  r e t i c u l u m (ER, F i g . 25)  Each c i s t e r n a i s a l o n g , f l a t t e n e d  s a c - l i k e s t r u c t u r e which i s bound by a u n i t membrane w i t h an i n t r a c i s t e r n a l space o f about 100 t o 120 A° i n w i d t h . i s about 80 A° t h i c k , and no ribosomes of  t h i s membrane (ER, F i g . 2 5 ) .  The bounding  membrane  a r e p r e s e n t on t h e o u t e r s u r f a c e  12. Stage Three - Twelve Hour Stage i n G e r m i n a t i o n A number of changes o c c u r w i t h i n t h e spore p r o t o p l a s t t w e l v e hours a f t e r w e t t i n g the s p o r e s .  within  O r g a n e l l e s not " d e t e c t e d " i n  the e a r l i e r s t a g e s of g e r m i n a t i o n a r e o b s e r v e d , and some of the organe l l e s d e s c r i b e d p r e v i o u s l y c o n t i n u e t o change.  The s p i n u l o s e spore  w a l l , plasma membrane, v a c u o l e s c o n t a i n i n g membrane-like f r a g m e n t s , t r a n s p a r e n t v e s i c l e s , and membrane-bound v e s i c l e s remain unchanged (Fig.  26).  The n u c l e u s c o n t i n u e s t o change, c e n t r i o l e s and  dictyosomes  appear, and t h e c i s t e r n a l endoplasmic r e t i c u l u m becomes more e x t e n s i v e (ER, F i g . 2 7 ) . Nucleus:  Most n u c l e i have become l o b e d (N, F i g . 2 7 ) ; however, t h e  n u c l e a r m a t e r i a l appears t o have changed l i t t l e .  A single nucleolus i s  p r e s e n t i n each n u c l e u s (Nu, F i g . 2 7 ) . Centriole:  I n some s p o r e s , a s i n g l e c e n t r i o l e (Ce, F i g . 28) i s seen i h  c l o s e p r o x i m i t y t o the n u c l e u s and i n the c e n t r a l r e g i o n o f t h e p r o t o plast.  Each c e n t r i o l e i s composed o f a p a i r of c y l i n d e r s , 400 t o 430  mju l o n g and 180 mu  i n diameter.  The c y l i n d e r s  and l i e a t r i g h t a n g l e t o one a n o t h e r .  a r e open a t b o t h ends  Each c y l i n d e r  n i n e l o n g i t u d i n a l , e v e n l y spaced, t u b u l a r f i b r i l s each f i b r i l units.  i s made up o f  (Ce, F i g . 2 9 ) , and  i s about 350 A° i n diameter and i s composed o f two  At the end o f the c y l i n d e r  sub-  c l o s e s t t o the a d j a c e n t c y l i n d e r ,  the f i b r i l s bend i n w a r d f o r about 300 A° a t a 90° a n g l e ( F i g s . 28 - 2 9 ) . Two  l o n g i t u d i n a l , tubular f i b r i l s  c y l i n d e r s , and each f i b r i l  l i e i n the c e n t r a l r e g i o n of t h e  i s about 300 A° i n diameter.  In a l l  13. c e n t r i o l e S j , t h e r e i s c y t o p l a s m o f r e l a t i v e l y low e l e c t r o n d e n s i t y i n the c e n t r a l p o r t i o n of t h e c y l i n d e r . Dictyosomes:  Most spores c o n t a i n a t l e a s t one or two d i c t y o s o m e s , and  each dictyosome i s composed of a s t a c k o f t h r e e t o f o u r  flattened  c i s t e r n a e w i t h s m a l l v e s i c l e s a t each end o f t h e c i s t e r n a e (D, F i g s . - 31).  30  The c i s t e r n a e resemble s m o o t h - s u r f a c e d , c i s t e r n a l - t y p e endo-  plasmic reticulum.  The membranes f o r m i n g each c i s t e r n a e a r e u n i t membranes,  about 80 A° i n t h i c k n e s s , and a r e s e p a r a t e d by a low e l e c t r o n dense i n t r a c i s t e r n a l space a p p r o x i m a t e l y 150 A° wide (D, F i g . 3 1 ) .  The d i c t y o s o m a l  c i s t e r n a e l i e p a r a l l e l t o each o t h e r and a r e s e p a r a t e d by a space o f about 120 A°.  The s m a l l membrane-bound v e s i c l e s c o n c e n t r a t e d on each  s i d e of the s t a c k s of f l a t t e n e d c i s t e r n a e ( F i g . 31) a r e about 200 t o 400 A° i n d i a m e t e r . Stage Four - Seventeen Hour Stage i n G e r m i n a t i o n There a r e f o u r main d e v e l o p m e n t a l forms of F. s e p t i c a s p o r e p r o t o p l a s t s seventeen hours a f t e r w e t t i n g .  Some p r o t o p l a s t s s t i l l a r e con-  t a i n e d w i t h i n t h e s p o r e case ( F i g . 3 2 ) , o t h e r s a r e emerging t h r o u g h s p l i t s i n spore cases ( F i g . 3 8 ) , and many of t h e p r o t o p l a s t s have developed i n t o swarm c e l l s A.  ( F i g . 39) or myxamoebae ( F i g . 4 0 ) . Spores P r i o r t o P r o t o p l a s t Emergence  When emergence of the p r o t o p l a s t has n o t o c c u r r e d , b o t h the i n n e r and o u t e r l a y e r s of t h e w a l l remain d i s t i n c t .  There i s no i n d i c a t i o n o f  d i s s o l u t i o n or c r a c k i n g o f any p a r t o f t h e w a l l (W, F i g . 32). s t i l l a r e l o b e d and s e c t i o n s through t h e s e l o b e s may  Many n u c l e i  g i v e the impression  14. t h a t more than one n u c l e u s i s p r e s e n t i n a s i n g l e spore ( N , F i g . 3 2 ) . However, i n c e r t a i n s p o r e s , t h e n u c l e i a r e l e s s i r r e g u l a r and they assume a more o v a l p a t t e r n ( N , F i g . 33-A). Centrioles: emergence.  Changes i n c e n t r i o l e s t r u c t u r e  a r e seen p r i o r t o p r o t o p l a s t  The c e n t r i o l e s assume t h e form o f b a s a l b o d i e s w i t h  several  types of r o o t l e t s e x t e n d i n g from them (BB, F i g s . 33 - 3 5 ) . The n i n e outer f i b r i l s are rotated  e v e n l y i n t h e same d i r e c t i o n and t o a s l i g h t  d e g r e e about a c e n t r a l f i b r i l .  Each o u t e r f i b r i l  i s connected  t o t h e ad-  j a c e n t f i b r i l s and t o t h e c e n t r a l f i b r i l by f i n e f i b e r - l i k e e x t e n s i o n s , t h e r e b y , f o r m i n g a " p i n - w h e e l " p a t t e r n (BB, F i g s . 33-B - 3 5 ) . The o u t e r f i b r i l s a r e about 350 A° i n d i a m e t e r , t h e c e n t r a l f i b r i l 450 A° i n d i a m e t e r , and t h e f i b e r - l i k e e x t e n s i o n s a r e about 40 A° wide.  One form of  r o o t l e t t a p e r s from t h e o u t e r f i b r i l l a r r e g i o n o f t h e p r o x i m a l  cylinder  and i n t o t h e c y t o p l a s m f o r about 100 t o 150 nyu ( F i g . 33-B). appear t o be made o f s e v e r a l Another  The r o o t l e t s  t u b u l e s which a r e about 150 A° i n w i d t h .  form o f r o o t l e t i s seen i n f i g u r e s 34 and 35.  t u d i n a l f i b r i l s p a r t i a l l y surrounds  A sheath o f l o n g i -  t h e p r o x i m a l c y l i n d e r of t h e c e n t r i o l e .  These s h e a t h i n g r o o t l e t s a r e about 300 mu l o n g and 30 mu i n d i a m e t e r , and they appear t o be connected t o p a r t o f t h e w a l l o f t h e p r o x i m a l by f i n e , r a d i a t i n g f i b e r s about 30 t o 40 A° i n w i d t h ( F i g . 3 5 ) . sheath-like  cylinder The  r o o t l e t s do n o t l i e p a r a l l e l t o t h e p r o x i m a l c y l i n d e r b u t seem  t o extend i n t o t h e c y t o p l a s m a t an obtuse a n g l e (R, F i g . 3 4 ) . The d i s t a l c y l i n d e r of the c e n t r i o l e l i e s perpendicular to the proximal (Fig.  35).  cylinder  15. Mitochondria:  An e l e c t r o n dense, s t r u c t u r e d c o r e i s no l o n g e r v i s i b l e  i n the c e n t r a l r e g i o n of the m i t o c h o n d r i a ;  however, t h e a r e a f o r m e r l y  o c c u p i e d by t h e c o r e now appears l e s s e l e c t r o n dense t h a n t h e s u r r o u n d i n g m a t r i x (M, F i g . 3 6 ) . Vacuoles:  Many v a c u o l e s c o n t a i n i n g membrane remnants a r e surrounded by  c i s t e r n a l endoplasmic r e t i c u l u m . c o a t e d w i t h ribosomes B.  T h i s endoplasmic r e t i c u l u m i s n o t  ( F i g . 37).  P r o t o p l a s t Emergence  In t h e second form, t h e p r o t o p l a s t s a r e seen emerging through  splits  i n t h e s p o r e w a l l s ( F i g . 3 8 ) . Only t h e s p i n u l o s e , e l e c t r o n dense, o u t e r l a y e r o f t h e s p o r e w a l l remains of  ( F i g s . 41 - 4 2 ) .  The i n n e r l a y e r , composed  two e l e c t r o n t r a n s p a r e n t s e c t i o n s s e p a r a t e d by a dense l i n e , i s no l o n g e r  visible.  The n u c l e u s r e v e r t s to; an o v o i d form and i s i n the c y t o p l a s m  which f i r s t e x i t s t h e s p o r e case.  S m a l l v a c u o l e s a r e s c a t t e r e d throughout  c y t o p l a s m and t h e o t h e r o r g a n e l l e s a r e n o t v i s i b l y a l t e r e d . p r o t o p l a s t then develops i n t o e i t h e r a f l a g e l l a t e d c e l l  This freed  ( F i g . 39) or a  myxamoeba ( F i g . 4 0 ) . C.  Swarm C e l l s and Myxamoebae  The swarm c e l l i s p y r i f o r m i n shape w h i l e t h e myxamoeba i s o f i n d e f i n i t e form.  The myxamoeba and t h e swarm c e l l a r e s t r u c t u r a l l y  similar  and both a r e bound by a s i n g l e u n i t membrane, t h e plasma membrane (PM, F i g s . 39 - 4 0 ) .  Both possess a s i n g l e o v o i d n u c l e u s ( N ) , a c o n t r a c t i l e  v a c u o l e ( C V ) , m i t o c h o n d r i a w i t h t u b u l a r - t y p e c r i s t a e (M) , rough  cisternal  endoplasmic r e t i c u l u m (RER), f o o d v a c u o l e s ( V ) , dictyosomes ( D ) , and  16. membrane-bound v e s i c l e s  (Ve) ( F i g s . 39 - 5 1 ) .  I n swarm c e l l s ,  micro-  t u b u l e s (MT, F i g . 39) pass about t h e n u c l e u s i n t h e c y t o p l a s m and extend towards Endoplasmic  t h e b a s a l body o f t h e f l a g e l l u m .  Reticulum:  C i s t e r n a l endoplasmic r e t i c u l u m w i t h  ribosomes  on i t s s u r f a c e i s p r e s e n t i n a l l p r o t o p l a s t s t h a t have emerged from spore c a s e s .  Each membrane i s about 80 A° t h i c k , and each  c i s t e r n a l space i s a p p r o x i m a t e l y 150 A° i n w i d t h .  intra-  Ribosomes a r e seen  on t h e o u t e r s u r f a c e s o f t h e c i s t e r n a e and a r e about 100 A° i n diameter (RER, F i g . 4 3 ) . Food V a c u o l e s :  S e v e r a l vacuoles c o n t a i n i n g food p a r t i c l e s are d i s p e r s e d  randomly thoughout  t h e c y t o p l a s m of t h e myxamoebae and swarm c e l l s .  Each v a c u o l e i s bound by a s i n g l e u n i t membrane, about 75 t o 80 A° t h i c k . The v a c u o l e s range from .7 t o 2 p membrane-bound m a t e r i a l .  i n s i z e and c o n t a i n a dense, g r a n u l a r ,  These g r a n u l a r , membrane-encompassed p a r t i c l e s  a r e suspended i n an e l e c t r o n t r a n s p a r e n t v a c u o l a r sap (V, F i g . 4 4 ) .  They  v a r y i n s i z e , from .2 t o .5^u i n d i a m e t e r , and i n shape. C o n t r a c t i l e Vacuole: cell  U s u a l l y one c o n t r a c t i l e v a c u o l e i s p r e s e n t i n . e a c h  (CV, F i g s . 39 - 4 0 ) . I n swarm c e l l s , t h e c o n t r a c t i l e v a c u o l e  g e n e r a l l y i s s i t u a t e d i n t h e p o s t e r i o r o f t h e c e l l , o r near  dictyosomes;  i n myxamoebae, i t does n o t appear t o be l o c a t e d i n any s p e c i f i c r e g i o n of  the c e l l .  C o n t r a c t i l e v a c u o l e s v a r y i n s i z e and shape, depending  on  whether they a r e i n an expanded s t a t e (CV, F i g . 45) o r i n a c o n t r a c t e d s t a t e (CV, F i g . 4 6 ) . B o r d e r i n g c o n t r a c t e d v a c u o l e s a r e many s m a l l v e s i c l e s r a n g i n g from 40 t o 70 mu i n d i a m e t e r .  The membranes s u r r o u n d i n g  17. b o t h c o n t r a c t i l e v a c u o l e s and b o r d e r i n g v e s i c l e s a r e about 80 A° t h i c k . Dictyosomes: cells.  Dictyosomes a r e found i n most myxamoebae and  flagellated  They a r e l o c a t e d u s u a l l y i n c l o s e p r o x i m i t y t o t h e c o n t r a c t i l e  vacuoles.  I n swarm c e l l s , t h e dictyosomes a r e i n t h e a n t e r i o r of t h e  c e l l s between t h e b a s a l b o d i e s o f f l a g e l l a and t h e n u c l e i .  Many  d i c t y o s o m e - l i k e v e s i c l e s a r e v i s i b l e i n the a n t e r i o r o f t h e c e l l s 38 and 4 6 ) .  (Figs.  S t r u c t u r a l l y , t h e dictyosomes i n swarm c e l l s and myxamoebae  appear more o r g a n i z e d and d e f i n e d t h a n t h e y do i n p r o t o p l a s t s o f s t a g e three. Flagella:  Most swarm c e l l s a r e u n i f l a g e l l a t e d ; however,  c e l l s a r e n o t uncommon.  Each f l a g e l l u m i s 6 t o 9  biflagellated  l o n g , about 230  i n d i a m e t e r , and each i s l o c a t e d i n the a n t e r i o r o f t h e c e l l  mu  ( F i g . 39).  In c r o s s - s e c t i o n , t h e f l a g e l l a r s t r u c t u r e i s seen t o be made o f n i n e peripheral f i b r i l s  e v e n l y spaced about two c e n t r a l f i b r i l s  ( F i g . 50).  Secondary elements a r e p r e s e n t between the two c e n t r a l f i b r i l s and t h e nine p e r i p h e r a l f i b r i l s .  The p e r i p h e r a l f i b r i l s a r e composed of two sub-  u n i t s , each about 140 A° i n d i a m e t e r .  On' some of t h e p e r i p h e r a l  two arms appear t o r a d i a t e out from one of t h e two s u b - u n i t s . serens t o be about 50 A° t h i c k and a p p r o x i m a t e l y 70 A° l o n g .  fibrils,  Each arm The  f i b r i l s l a c k s u b - u n i t s t r u c t u r e and a r e about 170 A° i n d i a m e t e r .  central The  d i s t a n c e between the two c e n t r a l f i b r i l s and t h e n i n e p e r i p h e r a l f i b r i l s i s about 350 A°, and the d i s t a n c e between t h e p e r i p h e r a l f i b r i l s and the u n i t membrane w h i c h surrounds t h e a x i a l f i l a m e n t i s about 200 A°.  The  peri-  p h e r a l f i b r i l s a r e about 120 A° a p a r t and t h e r e appears t o be a space o f a p p r o x i m a t e l y 50 A° between the two c e n t r a l f i b r i l s . the  a x i a l f i l a m e n t i s a p p r o x i m a t e l y 90 A° t h i c k .  The membrane about  18. The b a s a l bodies o f f l a g e l l a a r e sub-dermal and s t r u c t u r a l l y lar  t o the b a s a l b o d i e s p r e v i o u s l y d e s c r i b e d i n t h i s s t u d y .  In c r o s s -  s e c t i o n , each b a s a l body i s composed of n i n e p e r i p h e r a l f i b r i l s two c e n t r a l f i b r i l s . The  Two  simi-  s u b - u n i t s c o m p r i s e each o f the o u t e r  about fibrils.  dimensions o f the s u b - s t r u c t u r e o f the b a s a l b o d i e s i s s i m i l a r t o  t h a t o f the f l a g e l l u m .  R o o t l e t s , which appear t o be composed o f l i n e a r  aggregates o f f i b r i l s , extend  from the p e r i p h e r y o f the b a s a l body i n t o  the c y t o p l a s m  (R, F i g . 4 7 ) .  i n diameter.  No f i b r i l l a r s t r u c t u r e i s v i s i b l e i n o t h e r r o o t l e t s (R, F i g .  48).  Each f i b r i l i n the r o o t l e t i s about 120  These r o o t l e t s , 110 mu wide near the base o f the f l a g e l l u m ,  g r a d u a l l y t a p e r and extend p o s t e r i o r l y i n the c e l l . Fig.  A°  Microtubules  (MT,  47) a l s o a r e d i s p e r s e d p e r i p h e r a l l y i n the a n t e r i o r o f the swarm  cell.  They appear t o t e r m i n a t e near the b a s a l body of the f l a g e l l u m .  f i g u r e 47, t h e r e a r e i n d i c a t i o n s t h a t some of the m i c r o t u b u l e s  In  terminate i n  l i n e a r aggregates near the f l a g e l l a r base. Nucleus:  Each swarm c e l l and myxamoeba possesses  (N, F i g s . 39 - 4 0 ) .  The n u c l e u s  nucleus  i s l o c a t e d i n the a n t e r i o r t h i r d of  swarm c e l l near the f l a g e l l a r b a s a l body.  the  No d i r e c t c o n n e c t i o n t o t h e  b a s a l body from the n u c l e u s has been observed. nucleus  a s i n g l e , ovoid  I n the myxamoeba, the  i s not a s s o c i a t e d w i t h a s p e c i f i c r e g i o n of the c e l l .  Each o v o i d  n u c l e u s c o n t a i n s a s i n g l e , e l e c t r o n dense, g r a n u l a r n u c l e o l u s which i s not bound by a membrane and w h i c h has one F i g s . 52 - 5 3 ) .  t o two n u c l e o l a r v a c u o l e s  The n u c l e o l a r v a c u o l e s a r e e l e c t r o n t r a n s p a r e n t and  n o t encompassed by a membrane.  (Nu, are  Chromatin m a t e r i a l i s s c a t t e r e d randomly  throughout an e l e c t r o n t r a n s p a r e n t n u c l e o p l a s m  and i t appears as c l u s t e r s  o f e l e c t r o n dense g r a n u l e s  A n u c l e a r envelope  surrounds  ( F i g s . 51 and 54).  the n u c l e a r m a t e r i a l .  No b l e b f o r m a t i o n o f t h i s  19. envelope i s seen and few pores a r e v i s i b l e i n t h e membranes o f t h e envelope.  The membranes f o r m i n g t h e envelope a r e about 80 A° t h i c k  and t h e p e r i n u c l e a r space between t h e two membranes i s about 130 A° wide (NE, F i g . 5 4 ) .  DISCUSSION F u l i g o s e p t i c a spores g e r m i n a t e r e a d i l y i n c a r b o n - f i l t e r e d , d i s t i l l e d water and i n l e a f - e x t r a c t d e c o c t i o n w e t t e d i n a low p e r c e n t a g e b i l e s a l t s o l u t i o n . ( C o n s t a n t i n e a n u , 1906, G i l b e r t , 1927; S c h o l e s ,  subsequent t o b e i n g Other i n v e s t i g a t o r s 1962; Smart, 1937)  r e p o r t s i m i l a r f i n d i n g f o r F u l i g o s e p t i c a s p o r e s ; however, a few i n v e s t i g a t o r s (Cook and H o l t , 1928) i n d i c a t e t h a t a low p e r c e n t a g e and a low r a t e o f g e r m i n a t i o n similar conditions.  i s o b t a i n e d w i t h spores o f F. s e p t i c a under  G e r m i n a t i o n r a t e s may d i f f e r even w i t h two p o r t i o n s  of a s i n g l e c o l l e c t i o n o f t h e same s p e c i e s , as has been o b s e r v e d i n t h i s study.  Smart (1937) and o t h e r s  Cadman, 1928; S c h o l e s ,  ( C o n s t a n t i n e a n u , 1906; W i l s o n and  1962) have demonstrated t h a t t e m p e r a t u r e , pH o f  the medium, age o f s p o r e s ,  concentration  o f s p o r e s , and/or t o x i c sub-  s t a n c e s i n t h e medium i n h i b i t or r e t a r d t h e r a t e of g e r m i n a t i o n i n many Myxomycete s p e c i e s .  One o r more o f t h e s e f a c t o r s may account f o r  the poor r e s u l t s o b t a i n e d by Cook and H o l t (1928) and f o r t h e d i f f e r e n t r a t e s of g e r m i n a t i o n The  f o r two p o r t i o n s o f t h e same c o l l e c t i o n .  p r o t o p l a s t s of F. s e p t i c a a r e f r e e d from t h e spore cases i n  a manner s i m i l a r t o t h a t d e s c r i b e d by G i l b e r t (1928) and o t h e r s (Howard, 1931; McManus, 1961).  Only t h e t i m e r e q u i r e d p r i o r t o t h e  20. s p l i t t i n g of the spore case appear  t o v a r y , w i t h t h e way  i n which  the spore case s p l i t s and t h e swarm c e l l s develop b e i n g s i m i l a r . A f t e r t h e development o f a wedge-shaped s p l i t i n the spore case does the p r o t o p l a s t emerge and assume a s p h e r i c a l form o u t s i d e t h e mouth o f the r u p t u r e .  I t remains t h e r e s e v e r a l minutes b e f o r e i t becomes  amoeboid and develops i n t o a swarm c e l l or myxamoeba.  This r e s t i n g  p e r i o d p r i o r t o swarm c e l l development i s t y p i c a l i n s e v e r a l Myxomycete s p e c i e s ( E l l i o t t , 1949; G i l b e r t , 1928; Howard, 1931), and K e r r (1960) suggests t h a t f l a g e l l a r f o r m a t i o n o c c u r s a t t h i s t i m e .  Contrary to these  o b s e r v a t i o n s , Smart (1937) s t a t e s t h a t f o r F. s e p t i c a , " I n those experiments of the w r i t e r i n which the temperatures between 25°C and 31°C were used, t h e p r o t o p l a s m o f the spores always began to escape from the spore membrane i m m e d i a t e l y upon t h e f o r m a t i o n o f t h e a p e r a t u r e and g r a d u a l l y emerged.  The s p l i t i n t h e w a l l p r o g r e s s e d as the p r o t o -  p l a s t escaped u n t i l t h e empty spore case showed a r u p t u r e e q u a l t o oneh a l f or more t h e diameter o f t h e spore.  The p r o t o p l a s t then moved away  from t h e empty spore membrane through amoeboid a l t e r a t i o n s o f i t s form and w i t h i n a few minutes become a f l a g e l l a t e swarm c e l l . " The swarm c e l l s i n t h i s study seem t o be m o s t l y u n i f l a g e l l a t e , but i s o k o n t b i f l a g e l l a t e c e l l s a r e not uncommon. findings, E l l i o t t  (1949) and G i l b e r t  Contrary to these  (1928) have r e p o r t e d t h a t , i n t h e i r  g e r m i n a t i o n s t u d i e s , n e a r l y a l l the swarm c e l l s a r e h e t e r o k o n t b i flagellates.  I n f o r m a t i o n on o t h e r Myxomycete s p e c i e s p r o v i d e s a p a r t i a l  e x p l a n a t i o n f o r t h e s e seeming c o n t r a d i c t i o n s .  Cohen (1960 - see  A l e x o p o u l o s , 1963) has demonstrated t h a t t h e age o f swarm c e l l s ,  21. c o n d i t i o n of swarm c e l l s , and  s p e c i e s i n f l u e n c e s the p e r c e n t a g e of b i -  f l a g e l l a t e c e l l s w h i c h form.  Cohen (1959) and K o e v e n i g (1961)  Alexopoulos,  1963)  b o t h have shown t h a t one  be m i s t a k e n f o r t r u e f l a g e l l a .  Kerr  (see  or more p s e u d o f l a g e l l a  (1960) has  may  o b s e r v e d i n Didymium  n i g r i p e s t h a t u n i f l a g e l l a t e d c e l l s develop f i r s t and  t h a t some o f t h e s e  c e l l s may  Thus,  become b i f l a g e l l a t e d a f t e r s e v e r a l h o u r s .  i n r e s u l t s c o u l d be due  discrepancies  t o d i f f e r e n c e s i n F. s e p t i c a s t r a i n s , and/or  the p r e s e n c e o f p s e u d o f l a g e l l a on u n i f l a g e l l a t e d c e l l s . A number of u l t r a s t r u c t u r a l changes occur i n F. s e p t i c a germination.  The  spores have m u l t i - l a y e r e d , s p i n u l o s e w a l l s w h i c h r e m a i n  i n t a c t u n t i l the spores have r u p t u r e d t h i s t i m e , no e l e c t r o n t r a n s p a r e n t  t o r e l e a s e the p r o t o p l a s t s .  i n n e r l a y e r i s v i s i b l e and  the e l e c t r o n opaque, s p i n u l o s e o u t e r  l a y e r remains.  The  W o h l f a r t h - B o t t e r m a n n , 1959).  And,  At  only  spore w a l l s  s t r u c t u r a l l y resemble the s p o r e w a l l s o f D. n i g r i p e s ( S c h u s t e r ,  1964;  the s p i n e s of the w a l l appear t o have  been formed i n a manner s i m i l a r t o t h a t w h i c h S c h u s t e r (1964) f o r D.  during  n i g r i p e s r a t h e r than as Cadman (1931-32) s u g g e s t s .  The  describes latter  author i n d i c a t e s t h a t the p r o t o p l a s m s h r i n k s and causes the w a l l s t o b u c k l e and  form r i d g e s on which s p i n e s  space between the w a l l and  develop.  the p r o t o p l a s t i n F. s e p t i c a s p o r e s w h i c h  would a l l o w f o r the c o l l a p s e of the s p o r e w a l l . the w a l l and  The  l i g h t a r e a between  the p r o t o p l a s t , which Cadman (1931-32) b e l i e v e s i s a s p a c e ,  p r o b a b l y i s an e l e c t r o n t r a n s p a r e n t t o the one  However, t h e r e i s no  i n spores of F. s e p t i c a .  i n n e r l a y e r o f the w a l l s i m i l a r How  the i n n e r l a y e r breaks down  22. a f t e r the s p o r e r u p t u r e s i s not known, but s e v e r a l p o s s i b l e e x p l a n a t i o n s can be suggested.  The i n n e r l a y e r might r a p i d l y d i s s o l v e i n t h e  medium once t h e s p o r e r u p t u r e s , or the i n n e r l a y e r o f the w a l l might  be  s u b j e c t t o enzymatic d e g r a d a t i o n p r i o r t o t h e s p l i t t i n g of t h e w a l l . R e s u l t s of McManus (1961) t e n d t o s u p p o r t the l a t t e r p o s s i b i l i t y .  She  has o b s e r v e d t h a t i n some g e r m i n a t i n g spores o f C l a s t o d e r m a debaryanum, one s i d e of the s p o r e case g r a d u a l l y becomes l e s s dense and appears t o dissolve.  However, the i n n e r l a y e r o f the w a l l s remain v i s i b l e i n  u n r u p t u r e d spores of F.  septica.  The t r a n s p a r e n t v e s i c l e s w h i c h a r e not bound by membranes appear to be s i m i l a r t o t h o s e w h i c h have been d e s c r i b e d i n the p l a s m o d i a l and spore s t a g e s of D. n i g r i p e s , i n p l a s m o d i a o f D. c l a v u s , S t e m o n i t i s f u s c a , H e m i t r i c h i a v e s p a r i u m , C l a s t o d e r m a debaryanum  (Mc Manus, 1965; S c h u s t e r ,  1964; W o h l f a r t h - B o t t e r m a n n , 1959) , and i n many Eumycota ( B l o n d e l and T u r i a n , 1960; Hawker and A b b o t t , 1963a, 1963b; T h y a g a r a j a n , C o n t i , and N a y l o r , 1961, 1962).  McManus (1965) s t a t e s t h a t t h e t r a n s p a r e n t v e s i c l e s  resemble s e c r e t o r y g r a n u l e s as f o u n d i n a n i m a l g l a n d c e l l s , and S c h u s t e r (1964) b e l i e v e s t h a t they have some type of c o n t r a c t i l e v a c u o l a r a c t i v i t y . However, most of t h e s e v e s i c l e s c l o s e l y resemble s a t u r a t e d and u n s a t u r a t e d l i p i d d r o p l e t s w h i c h a r e d i s p e r s e d throughout t h e c y t o p l a s m .  The  lipid  d r o p l e t s then c o u l d : " s e r v e as a l o c a l s t o r e o f energy and a p o t e n t i a l s o u r c e o f s h o r t c a r b o n c h a i n s t h a t can be used by the c e l l of the  i n the s y n t h e s i s  i t s l i p i d - c o n t a i n i n g s t r u c t u r a l components, such as membranes, or i n e l a b o r a t i o n of s p e c i f i c s e c r e t o r y p r o d u c t s " ( F a w c e t t , 1966).  23. V a c u o l e s c o n t a i n i n g membrane-like fragments and e l e c t r o n dense g r a n u l a r m a t t e r i n t h e c e l l sap change l i t t l e p r i o r t o swarm c e l l myxamoeba development.  and  However, once swarm c e l l s or myxamoebae a r e  formed, t h e s e v a c u o l e s d i s a p p e a r .  S i m i l a r a p p e a r i n g o r g a n e l l e s have  been d e s c r i b e d i n o t h e r Myxomycetes as f o o d v a c u o l e s c o n t a i n i n g b a c t e r i a l c a r c a s s e s i n v a r y i n g degrees of d i g e s t i o n ( S c h u s t e r , 1964; 1959).  These v a c u o l e s a l s o resemble  Wohlfarth-Botterman,  food v a c u o l e s i n some P r o t o z o a  ( E l l i o t t and Clemmons, 1966; M e r c e r , 1959; S c h u s t e r , 1963).  Lindegren  (1962) s t a t e s t h a t y e a s t c e l l s a l s o possess v a c u o l e s w h i c h c o n t a i n membranes.  However, he b e l i e v e s t h a t t h e s e v a c u o l e s a r e not r e s i d u a l f o o d  v a c u o l e s but membrane s y n t h e s i z i n g v a c u o l e s .  The s y n t h e s i z e d membranes  t h e n a r e e x t r u d e d i n t o t h e c y t o p l a s m f o r m i n g endoplasmic  r e t i c u l u m o f the  cell. C o n c u r r e n t w i t h t h e d i s a p p e a r a n c e of the membrane-containing v a c u o l e s i s t h e appearance of d e f i n i t e f o o d v a c u o l e s ( F i g . 4 4 ) . membrane-bound g r a n u l a r m a t t e r i n t h e s e v a c u o l e s s t r u c t u r a l l y b a c t e r i a l c e l l p r o t o p l a s m (Leene and van I t e r s o n , 1965;  The  resembles  S c h u s t e r , 1963).  Thus, i t seems t h a t once t h e p r o t o p l a s t e x i t s t h e spore c a s e , i t begins t o i n g e s t b a c t e r i a as a f o o d s o u r c e . C o n t r a c t i l e v a c u o l e s develop a t about the same time as f o o d v a c u o l e s , and u s u a l l y one c o n t r a c t i l e v a c u o l e i s p r e s e n t i n each swarm c e l l or myxamoeba. of  These v a c u o l e s s t r u c t u r a l l y a r e s i m i l a r t o c o n t r a c t i l e v a c u o l e s  some P r o t o z o a ( T r a g e r , 1964), and they p r o b a b l y f u n c t i o n t o e l i m i n a t e  excess water i n a comparable manner.  The s m a l l v e s i c l e s about t h e  s y s t o l i c v a c u o l e suggest t h a t r u p t u r i n g o f t h e t o n o p l a s t has o c c u r r e d .  24. When t h e t o n o p l a s t  f r a g m e n t s , many s m a l l v e s i c l e s a r e formed and t h e  excess water and s o l u b l e wastes a r e e l i m i n a t e d from t h e c e l l . a f t e r the vacuole discharges  i t s contents,  Soon  the small v e s i c l e s fuse  and excess water and waste p r o d u c t s d i f f u s e i n t o t h e v a c u o l e u n t i l t h e pressure  becomes t o o g r e a t and i t a g a i n c o n t r a c t s .  No n e p h r i d i a l  t u b u l e s o r endoplasmic r e t i c u l u m appear t o t r a n s p o r t s o l u b l e wastes t o the c o n t r a c t i l e v a c u o l e as E l l i o t t and Bak (1964) s t a t e o c c u r s i n t h e c i l i a t e , Tetrahymena p y r i f o r m i s . A l t e r a t i o n s i n t h e endoplasmic r e t i c u l u m d u r i n g g e r m i n a t i o n  seem  t o be c o r r e l a t e d c l o s e l y w i t h t h e changes i n t h e p h y s i o l o g i c a l a c t i v i t y o f t h e c e l l s : t h e more a c t i v e t h e c e l l , t h e more developed t h e endoplasmic reticulum.  No c i s t e r n a l o r t u b u l a r endoplasmic r e t i c u l u m i s p r e s e n t i n  t h e r e s t i n g s p o r e ; o n l y smooth-surfaced v e s i c l e s a r e v i s i b l e i n t h e r i b o s o m e - d i s p e r s e d c y t o p l a s m of these c e l l s .  Whether t h e smooth s u r -  f a c e d v e s i c l e s a c t u a l l y a r e v e s i c u l a r endoplasmic r e t i c u l u m i s not known. U n l i k e t h e m o t i l e c e l l s o f some Phycomycetes w h i c h form no endoplasmic r e t i c u l u m ( C a n t i n o , e t . a l . , 1963), t h e p r o t o p l a s t s o f F. s e p t i c a develop smooth s u r f a c e d , c i s t e r n a l endoplasmic r e t i c u l u m .  L a t e r , when  the swarm c e l l s and myxamoebae form, t h e endoplasmic r e t i c u l u m becomes r i b o s o m e - c o a t e d and appears s i m i l a r t o t h e endoplasmic r e t i c u l u m w h i c h B l o n d e l and T u r i a n  (1960) d e s c r i b e i n A l l o m y c e s macrogynus c e l l s .  Thus, i t seems t h a t i n F. s e p t i c a t h e f o r m a t i o n  of ribosome-coated  endoplasmic r e t i c u l u m i s a s s o c i a t e d w i t h t h e more a c t i v e s t a t e o f t h e protoplast.  The development  of r i b o s o m e - c o a t e d endoplasmic  reticulum  i n swarm c e l l s o f F. s e p t i c a does n o t appear t o be t h e same as i t i s  25. i n yeasts.  No rough endoplasmic r e t i c u l u m i s v i s i b l e i n n u c l e a r  a s s o c i a t e d v a c u o l e s , and no rough endoplasmic r e t i c u l u m i s seen b e i n g e x t r u d e d i n t o t h e c y t o p l a s m from t h e s e v a c u o l e s , as L i n d e g r e n (1962) describes occurring i n yeast c e l l s .  The ribosomes i n t h e c y t o p l a s m  p r i o r t o swarm c e l l development appear t o become a t t a c h e d t o t h e c i s t e r n a l endoplasmic r e t i c u l u m t o form r i b o s o m e - c o a t e d endoplasmic reticulum. D i c t y o s o m e s , w h i c h a r e n o t v i s i b l e i n t h e r e s t i n g s p o r e s of F. s e p t i c a , appear t o develop c o n c u r r e n t l y w i t h t h e endoplasmic r e t i c u l u m . Thus, t h e most o r g a n i z e d form of t h e dictyosomes a r e seen i n t h e swarm c e l l s and myxamoebae.  From t h e s e o b s e r v a t i o n s , and from o b s e r v a t i o n s  of o t h e r s ( F u l l e r and R e i c h l e , 1965; McManus, 1965), i t appears t h a t b o t h the  dictyosomes and t h e endoplasmic r e t i c u l u m can be broken down and r e -  s y n t h e s i z e d d u r i n g t h e l i f e - c y c l e o f t h e organism.  The more m e t a b o l i -  c a l l y a c t i v e t h e p r o t o p l a s t , t h e more s t r u c t u r a l l y o r g a n i z e d i s t h e organelle.  The dictyosomes and endoplasmic r e t i c u l u m p r o b a b l y a r e most  a c t i v e d u r i n g t h e swarm c e l l and myxamoeba s t a g e because they appear b e s t developed at t h i s time.  The d i c t y o s o m e s always m a i n t a i n a j u x t a n u c l e a r  p o s i t i o n during germination.  This r e l a t i o n s h i p a l s o i s observed i n the  Eumycota ( F u l l e r and R e i c h l e , 1965; Hawker, 1963; Moore and M c A l e a r , 1963), and i t s u g g e s t s t h a t some t y p e o f c o n t r o l might be e x e r t e d by t h e nucleus.  However, McManus  (1965) r e p o r t s t h a t i n p l a s m o d i a o f s e v e r a l  Myxomycetes, t h e dictyosomes a r e n o t l o c a l i z e d i n j u x t a n u c l e a r but a r e s c a t t e r e d throughout t h e c y t o p l a s m .  sites  I n swarm c e l l s t h e d i c t y o -  somes p r o b a b l y f u n c t i o n i n t h e s e c r e t o r y p r o c e s s e s o f t h e c e l l as they do  26. i n o t h e r organisms  ( D a l t o n , 1961; F a w c e t t , 1966).  I n some P r o t o z o a ,  the dictyosomes and c o n t r a c t i l e v a c u o l e s a r e i n c l o s e p r o x i m i t y t o each o t h e r and s h a r e water removal a c t i v i t i e s i n the c e l l A s i m i l a r s p a t i a l r e l a t i o n s h i p between t h e dictyosomes and v a c u o l e s e x i s t s i n swarm c e l l s o f F. s e p t i c a . f u n c t i o n i n a manner comparable  (Cohn,  1964).  contractile  These o r g a n e l l e s might  t o t h e dictyosomes i n P r o t o z o a .  The m i t o c h r o n d r i a s t r u c t u r a l l y a r e s i m i l a r t o t h o s e i n o t h e r Myxomycetes (Dugas and B a t h , 1962; McManus, 1965; S c h u s t e r , 1965; Wohlfarth-Bottermann, other fungi.  1959).  However, they a r e u n l i k e t h o s e of most  The m i t o c h o n d r i a i n Hemiascomycetes ( B a n d o n i , B i s a l p u t r a ,  B i s a l p u t r a , i n p r e s s ; L i n d e g r e n , 1962; T h y a g a r a j a n , e t . a l . ,  1961),  Euascomycetes (Moore and M c A l e a r , 1962, 1963b; S h a t k i n and Tatum, 1959), some Phycomycetes (Hawker and A b b o t t , 1963a; Moore and M c A l e a r , 1963b; C a n t i n o , e t . a l . , 1963), B a s i d i o m y c e t e s ( B a n d o n i , p e r s o n a l  communication;  Moore and M c A l e a r , 1963b), and F u n g i I m p e r f e c t i (Hawker and Hendy T h y a g a r a j a n , e t . a l . , 1962, 1963; W e l l s , 1964) c r i s t a e mitochondria.  1963;  a l l have l a m e l l a r - t y p e  Only a few f u n g i o t h e r than Myxomycetes possess  m i t o c h o n d r i a w i t h t u b u l a r - t y p e c r i s t a e (Hawker and A b b o t t , 1963b; F u l l e r and R e i c h l e , 1965), and i n t h e s e forms, t h e t u b u l e s do not b r a n c h as they do i n the m i t o c h o n d r i a of F. s e p t i c a p r o t o p l a s t s and o t h e r Myxomycete p l a s m o d i a .  A l s o , t h e m o t i l e c e l l s o f F. s e p t i c a possess  numerous m i t o c h o n d r i a , whereas, the m o t i l e c e l l s of some f u n g i possess o n l y a s i n g l e m i t o c h o n d r i o n ( C a n t i n o , e t . a l . , 1963).  The  electron  dense c o r e s which a r e v i s i b l e i n t h e e a r l y s t a g e s o f g e r m i n a t i o n a r e p r o b a b l y n u c l e i c a c i d c o r e s which have been shown t o be p r e s e n t i n some  27. Myxomycetes ( S c h u s t e r ,  1965).  cores during germination  may  o c c u r r i n g w i t h i n the c e l l .  The  d i f f e r e n t appearance o f these  be a r e s u l t o f p h y s i o l o g i c a l a l t e r a t i o n s Such changes might i n f l u e n c e , d i r e c t l y  i n d i r e c t l y , the r e a c t i o n of the c o r e s to the  fixative.  L i t t l e i s known about f l a g e l l a r s t r u c t u r e and  development i n  Myxomycetes as few c y t o l o g i c a l s t u d i e s have been made. and o t h e r s  (Howard, 1931;  McManus, 1961;  Smart, 1937)  Ross (1957) show t h a t e i t h e r  u n i f l a g e l l a t e d or b i f l a g e l l a t e d c e l l s develop a f t e r the have been f r e e d from s p o r e c a s e s .  Kerr  protoplasts  (1960) and Ross (1957) a l s o  have demonstrated t h a t each f l a g e l l u m i s anchored i n the c e l l by b a s a l body.  f i b r i l l a r arrangement w h i c h  c h a r a c t e r i z e s m o t i l e p r o c e s s e s i n o t h e r f u n g i ( F u l l e r and Kock, 1956,  Cantino,  e t . a l . , 1963;  o t h e r organisms ( F a w c e t t , 1966). b o d i e s w h i c h may Of the few  a  E l e c t r o n m i c r o g r a p h s of F. s e p t i c a swarm c e l l s show t h a t  the f l a g e l l a have the t y p i c a l 9 + 2  1965;  or  or may  Reichle,  Renaud and S w i f t , 1964)  and  These f l a g e l l a a r i s e from b a s a l  not be s i m i l a r t o b a s a l b o d i e s i n o t h e r  f l a g e l l a t e d f u n g a l c e l l s w h i c h have been examined by  m i c r o s c o p y ( F u l l e r and R e i c h l e , 1965;  Cantino,  e t . a l . , 1963;  fungi. electron  Renaud  and S w i f t , 1964), the s t r u c t u r e of the b a s a l body i s s i m i l a r but  not  identical.  and  However, u n l i k e some f u n g a l  a n i m a l ( F a w c e t t , 1966)  ( B e r l i n and Bowen, 1964)  c e n t r i o l e s and b a s a l bodies, i n w h i c h each of  the n i n e p e r i p h e r a l f i b r i l s i s composed o f t h r e e s u b - u n i t s , the p e r i p h e r a l f i b r i l s of the b a s a l b o d i e s and c e n t r i o l e s i n F. are made up of two  sub-units.  As i n o t h e r  f u n g i (Renaud and  1964), the b a s a l body d e v e l o p s from the p r o x i m a l  c y l i n d e r of  nine  septica Swift, the  28. c e n t r i o l e which i s i n a j u x t a n u c l e a r p o s i t i o n . f u n g i (Renaud and S w i f t , 1964;  However, u n l i k e o t h e r  F u l l e r and R e i c h l e , 1965), a c l o s e  a s s o c i a t i o n of the n u c l e i and b a s a l b o d i e s of F. s e p t i c a swarm c e l l s i s not m a i n t a i n e d  d u r i n g or a f t e r f l a g e l l a r development.  There i s no  i n d i c a t i o n of a d i r e c t c o n n e c t i o n by means of a r h i z o p l a s t between the two o r g a n e l l e s , o n l y d i e t y o s o m e - l i k e v e s i c l e s appear between the b a s a l body and the n u c l e u s .  The  f u n c t i o n of these d i c t y o s o m e - l i k e v e s i c l e s  i s unknown; however, some i n v e s t i g a t o r s suggest t h a t the v e s i c l e s and form the f l a g e l l a r sheath  ( S o r o k i n , 1962).  The  f u n c t i o n of  fuse  the  r o o t l e t s e x t e n d i n g from the b a s a l b o d i e s i s unknown, but i t i s p r o b a b l y t h a t they f u n c t i o n i n support and anchorage o f the f l a g e l l a . f u n c t i o n may  A similar  e x i s t f o r the m i c r o t u b u l e s w h i c h r a d i a t e about the base o f  the f l a g e l l u m . The n u c l e u s undergoes a s e r i e s o f changes d u r i n g g e r m i n a t i o n a few o f these changes have been r e p o r t e d i n o t h e r Myxomycetes. o v o i d n u c l e u s , which i s c h a r a c t e r i s t i c o f the r e s t i n g s p o r e ,  and  The  resembles^  the n u c l e i Dugas and B a t h (1962) d e s c r i b e i n Physarum polycephalum Plasmodia.  However, w h i l e n u c l e i o f P. p o l y c e p h a l u m u s u a l l y c o n t a i n as  many as f o u r n u c l e o l i , those o f F. s e p t i c a possess o n l y a s i n g l e nucleolus.  B l e b s , which may  or may  not c o n t a i n n u c l e a r m a t e r i a L , a r e  seen i n the n u c l e a r envelope o f o n l y the r e s t i n g s p o r e s .  These n u c l e o -  c y t o p l a s m i c b l e b s s t r u c t u r a l l y a r e s i m i l a r t o t h o s e i n o t h e r organisms ( S c h u s t e r , 1963;  Gay,  1956).  I f these blebs c o n t a i n nuclear m a t e r i a l ,  as has been suggested  by Gay  (1956), then they might be a b l e t o d i r e c t  c e r t a i n c y t o p l a s m i c r e a c t i o n s and syntheses  i f the b l e b s s h o u l d pass  29. i n t o the c y t o p l a s m .  The pores i n t h e n u c l e a r envelope a l s o a l l o w  f o r a c e r t a i n amount o f n u c l e o - c y t o p l a s m i c exchange. s t a t e s t h a t P o r t e r (1960) has suggested n u c l e i c a c i d (RNA) The  Schuster  (1963)  t h a t such p a r t i c l e s as r i b o s e  might pass through t h e s e pores  i n t o the  cytoplasm.  extreme p l a s t i c i t y o f t h e n u c l e u s , as seen i n F. s e p t i c a spores  d u r i n g g e r m i n a t i o n and i n p r o t o p l a s t s o f o t h e r Myxomycetes (Dugas and B a t h , 1962; McManus, 1965;  L o c q u i n , 1949), i n d i c a t e s t h a t t h e r e i s  i n t e r a c t i o n between the n u c l e u s and t h e c y t o p l a s m .  The more i r r e g u l a r  and l o b e d t h e n u c l e u s , the g r e a t e r i s the s u r f a c e f o r these a c t i o n s to occur.  inter-  Thus, i t appears t h a t the g r e a t e s t amount o f i n t e r a c t i o n  might occur d u r i n g g e r m i n a t i o n and not i n the r e s t i n g spore and swarm c e l l or myxamoeba s t a g e s . The r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t Myxomycetes, such as s e p t i c a , a r e not c l o s e l y r e l a t e d t o t r u e f u n g i . more c l o s e l y r e l a t e d t o P r o t o z o a . tend to support t h i s i d e a .  F.  They s t r u c t u r a l l y appear  Similarities i n organelle structure  Myxomycetes and most P r o t o z o a possess  mito-  chondria w i t h t u b u l a r - t y p e c r i s t a e , c o n t r a c t i l e vacuoles, food vacuoles, and s i m i l a r dictyosome o r g a n i z a t i o n .  The  f e e d i n g h a b i t s of the  groups a r e s i m i l a r , and n e i t h e r group possesses their life-cycle.  However, t h e presence  two  a w a l l d u r i n g most o f  o f a w a l l d u r i n g the  spore  s t a g e and t h e c e n t r i o l e and b a s a l body s u b - s t r u c t u r e more c l o s e l y resemble c h a r a c t e r i s t i c s of the lower p l a n t s and many f u n g i .  30. SUMMARY A p p r o x i m a t e l y 90 t o 95 % o f t h e spores o f F. s e p t i c a  germinate  a f t e r 17 t o 20 hours i n e i t h e r s t e r i l e l e a f - e x t r a c t d e c o c t i o n or i n s t e r i l e c a r b o n - f i l t e r e d , d i s t i l l e d water f o l l o w i n g w e t t i n g i n a b i l e salt solution.  Spores which have been sown i n l e a f - e x t r a c t d e c o c t i o n  germinate s l i g h t l y sooner than spores which have been sown i n c a r b o n f i l t e r e d , d i s t i l l e d water. An o v o i d n u c l e u s c o n t a i n i n g a s i n g l e n u c l e o l u s , m i t o c h o n d r i a w i t h t u b u l a r - t y p e c r i s t a e , v a c u o l e s w i t h membrane-like fragments  and  e l e c t r o n dense m a t e r i a l , l i p i d d r o p l e t s , and membrane-bound v e s i c l e s c h a r a c t e r i z e the r e s t i n g spore p r o t o p l a s t .  Each p r o t o p l a s t i s  surrounded by a m u l t i - l a y e r e d , s p i n u l o s e spore w a l l . S i x hours a f t e r w e t t i n g the s p o r e s , the n u c l e u s i s v e r y  ir-  r e g u l a r i n shape and many s m a l l v e s i c l e s a r e found i n t h e c y t o p l a s m . S m a l l amounts  o f smooth c i s t e r n a l endoplasmic r e t i c u l u m a l s o  appear  i n the c y t o p l a s m . B e f o r e the spore r u p t u r e s t o r e l e a s e t h e i r  protoplasts,  c e n t r i o l e s and dictyosomes develop c o n c u r r e n t l y i n j u x t a n u c l e a r s i t e s . A l s o , the n u c l e u s becomes l o b e d ; and more smooth, c i s t e r n a l endoplasmic r e t i c u l u m develops.  The p r o x i m a l c y l i n d e r s of t h e c e n t r i o l e s  develop i n t o b a s a l b o d i e s s h o r t l y b e f o r e t h e s p l i t t i n g of the spore cases. W i t h the r u p t u r i n g o f t h e spore c a s e , the i n n e r l a y e r of the w a l l disappears.  S i m u l t a n e o u s l y , the p r o t o p l a s t s escape.  They r e s t  31.  a s h o r t time near t h e mouth o f t h e r u p t u r e and then develop  into  f l a g e l l a t e d swarm c e l l s o r myxamoebae. In b o t h swarm c e l l s and myxamoebae, t h e endoplasmic  reticulum  becomes r i b o s o m e - c o a t e d , c o n t r a c t i l e - v a c u o l e s and f o o d v a c u o l e s d e v e l o p , and t h e n u c l e u s i s r e v e r t e d t o i t s o v o i d form.  Flagella  a t t a c h e d t o b a s a l b o d i e s a l s o develop i n t h e swarm c e l l .  Micro-  t u b u l e s a r e seen t o r a d i a t e p o s t e r i o r l y from t h e b a s a l body r e g i o n of t h e f l a g e l l a and p r o b a b l y g i v e more r i g i d i t y t o t h e m o t i l e c e l l s .  32. BIBLIOGRAPHY A l e x o p o u l o s , C. J . 1963. The Myxomycetes I I .  Bot. Rev. 29: 1 - 78.  Bandoni, R. J . , A. A. B i s a l p u t r a , and T. B i s a l p u t r a . In press. Ascospore development i n Hansenula anomala. Can. J . M i c r o b i o l . B a r y , A. de. 1854. E u g l e n a a r t i g e g e b i l d e aus sporen v o n T r i c h i a r u b i f o r m i s . F l o r a 12: 648. B e r l i n , J . and C. Bowen. 1964. C e n t r i o l e s i n t h e fungus Albugo Am. J . Bot. 51: 650 - 652.  Candida.  B i s a l p u t r a , T. and T. E. Weier. 1963. The c e l l w a l l o f Scenedesmus q u a d r i c a u d a . Am. J . Bot. 50: 1011 - 1019. B l o n d e l , B. and G. T u r i a n . , 1960. 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Problems i n t h e s t u d y o f n u c l e a r f i n e s t r u c t u r e . Proc. I n t . Conf. E l e c t r o n M i c r o s c o p . 4 t h . Conf. 2: 186 - 199. (Not Seen) Renaud, F. and H. S e i f t . 1964. The development o f b a s a l b o d i e s and f l a g e l l a i n A l l o m y c e s a r b u s c u l u s . J . C e l l . B i o l . 23: 339 -354. R e y n o l d s , E. S. 1963. The use o f l e a d c i t r a t e a t h i g h pH as an e l e c t r o n opaque s t a i n i n m i c r o s c o p y . J . C e l l B i o l . 17: 208 - 213.  35. Ross, I . K. 1957. Syngamy and Plasmodium f o r m a t i o n i n t h e Am. J . Bot. 44: 843 - 850.  Myxogastres.  S c h o l e s , P. 1962. Some o b s e r v a t i o n s on t h e c u l t i v a t i o n , f r u i t i n g , and g e r m i n a t i o n of F u l i g o s e p t i c a . J . Gen. M i c r o b i o l . 29: 137 - 148. S c h u s t e r , F . 1963. An e l e c t r o n microscope study o f the a m o e b o - f l a g e l l a t e , N a e g l e r i a g r u b e r i ( S c h a r d i n g e r ) . I . The amoeboid and f l a g e l l a t e s t a g e s . J . P r o t o z o o l . 10: 297 - 313. S c h u s t e r , F. 1964. E l e c t r o n m i c r o s c o p e o b s e r v a t i o n s on spore f o r m a t i o n i n the t r u e s l i m e mold Didymium n i g r i p e s . J . P r o t o z o o l . 11: 207 216. S h a t k i n , A. J . and E. L. Tatum. 1959. E l e c t r o n m i c r o s c o p y o f Neurospora c r a s s a m y c e l i a . J . B i o p h y s . Biochem. C y t o l . 6: 423 - 426. Smart, R. F. 1937. I n f l u e n c e on c e r t a i n e x t e r n a l f a c t o r s on spore g e r m i n a t i o n i n the Mycomycetes. Am. J . Bot. 24: 145 - 157. S o r o k i n , S. 1962. C e n t r i o l e and t h e f o r m a t i o n of r u d i m e n t a r y c i l i a by f i b r o b l a s t s and smooth muscle c e l l s . J . C e l l B i o l . 15: 363 - 377. T h y a g a r a j a n , T. R., S. F. C o n t i , and H. B. N a y l o r . 1961. E l e c t r o n microscopy o f yeastt m i t o c h o n d r i a . Exp. C e l l Res. 25: 216. T h y a g a r a j a n , T. R., S. F. C o n t i , and H. B. N a y l o r . 1962. Electron m i c r o s c o p y of R h o d o t o r u l a g l u t i n i s . J . B a c t e r i o l . 83: 381 - 394. T h y a g a r a j a n , T. R., S. F. C o n t i , and H. B. N a y l o r . 1963. Intranuclear and i n t r a c y t o p l a s m i c s t r u c t u r e s o f R h o d o t o r u l a g l u t i n i s as r e v e a l e d by e l e c t r o n micrographs o f s e r i a l s e c t i o n s . Exp. C e l l Res. 29: 235 - 241. T r a g e r , W. 1964. The c y t o p l a s m of P r o t o z o a , p. 81 - 137. I n The c e l l , v o l . 6. B i o c h e m i s t r y , p h y s i o l o g y , morphology. J . B r a c h e t and A. M i r s k y ( e d ) . . Academic P r e s s , New York. W e l l s , K. 1964. The b a s i d i a o f E x i d i a n u c l e a t a . I . M y c o l o g i a 56: 327 - 341.  Ultrastructure.  W i l s o n , M. and E. Cadman. 1928. The l i f e h i s t o r y and c y t o l o g y o f R e t i c u l a r i a l y e o p e r d o n . B u l l . Trans. Roy. Soc. E d i n b u r g h 55: p a r t 3. number 24. W o h l f a r t h - B o t t e r m a n n , K. E. 1959. G e s t a t t e t das e l e k t r o n e n m i k r o s k o p i s c h e b i l d aussagen zur dynamik i n der z e l l e ? . Z e i t . f u r Z e l l f o r s c h . 50: 1-27. Y u a s a , A. 1935. On t h e s c l e r o t i u m o f H e m i t r i c h i a s e r p u l a . ( I : Japanese) Bot. Mag. (Tokyo) 53:.511 - 513. (Not Seen)  APPENDIX  PLATE 1 F i g . 1.  F u l i g o s e p t i c a r e s t i n g spore.  X 1,800.  Figs. 2 - 5 .  Emergence o f spore p r o t o p l a s t through wedge-shaped s p l i t i n w a l l . X 1,900.  F i g . 6.  R e l e a s e d p r o t o p l a s t assumes s p h e r i c a l shape o u t s i d e mouth o f r u p t u r e . X 1,900.  PLATE 2 Fig.  7.  Spherical protoplast i n oscillatory state mouth o f r u p t u r e . X 1,900.  outside  Fig.  8.  B i f l a g e l l a t e d swarm c e l l .  Fig.  9.  U n i f l a g e l l a t e d swarm c e l l .  X 1,600.  Fig.  10.  U n i f l a g e l l a t e d swarm c e l l .  Phase c o n t r a s t .  X 1,600.  X 1,150.  •  ®  W  ®  PLATE 3 F i g . 11.  T y p i c a l r e s t i n g spore. Arrows w i t h i n p r o t o p l a s t i n d i c a t e s m a l l membrane-bound v e s i c l e s . W = w a l l , PM = plasma membrane, M = m i t o c h o n d r i o n , TVe = transparent v e s i c l e , V = vacuole, N = nuclear m a t e r i a l , Nu = n u c l e o l u s . Phosphate b u f f e r e d OsO. f i x a t i o n . X 27,500.  PLATE 4 F i g . 12.  S p i n u l o s e s p o r e w a l l . OW = e l e c t r o n dense o u t e r l a y e r , IW = e l e c t r o n t r a n s p a r e n t i n n e r l a y e r , PM = plasma membrane. Phosphate b u f f e r e d OsO, f i x a t i o n . X 127,000.  F i g . 13.  V a c u o l e s c o n t a i n i n g membrane-like fragments and e l e c t r o n dense g r a n u l a r m a t t e r i n c e l l sap. Phosphate b u f f e r e d OsO^ f i x a t i o n . X 37,150.  F i g . 14.  T r a n s p a r e n t v e s i c l e s . Note absence o f membranes about each v e s i c l e . Phosphate b u f f e r e d OsO, f i x a t i o n . X 42,000.  PLATE 5 F i g . 15.  Mitochondria w i t h t u b u l a r - t y p e c r i s t a e i n r e s t i n g spore p r o t o p l a s t . Arrows i n d i c a t e r e g i o n where c r i s t a e b r a n c h f o r m i n g o t h e r c r i s t a e w i t h i n the m i t o c h o n d r i o n . M = m i t o c h o n d r i o n , TVe = t r a n s p a r e n t v e s i c l e , V = v a c u o l e . Phosphate b u f f e r e d OsO^ f i x a t i o n . X 43,900.  F i g . 16.  M i t o c h o n d r i o n w i t h e l e c t r o n dense, g r a n u l a r c o r e i n the m a t r i x . M = m i t o c h o n d r i o n , PM = plasma membrane, IW = i n n e r l a y e r o f w a l l , OW = o u t e r l a y e r o f w a l l . Phosphate b u f f e r e d OsO. f i x a t i o n . X 127,700.  PLATE 6 Fig.  17.  N u c l e u s . NE = n u c l e a r e n v e l o p e , N = n u c l e a r m a t e r i a l , Nu = n u c l e o l u s . Phosphate b u f f e r e d OsO, f i x a t i o n . X 36,650.  Fig.  18.  N u c l e a r envelope showing one type o f b l e b commonly observed i n t h e r e s t i n g s p o r e s t a g e . Note s i m p l e pore i n envelope. NB = n u c l e a r b l e b , N = n u c l e a r m a t e r i a l , P = pore. Phosphate b u f f e r e d OsO, f i x a t i o n . X 40,000.  Fig.  19.  Second type o f n u c l e a r b l e b observed i n t h e r e s t i n g spore s t a g e n u c l e u s . Note s i m p l e pores i n envelope. NB = n u c l e a r b l e b , P = p o r e . Phosphate b u f f e r e d OsO, fixation. X 39,650.  PLATE 7 Fig.  20.  N u c l e u s . Note number o f s i m p l e pores i n envelope and membrane s t r u c t u r e o f e n v e l o p e . NE = n u c l e a r e n v e l o p e , P = p o r e , N = n u c l e a r m a t e r i a l , Nu = n u c l e o l u s . KMnO. f i x a t i o n . X 37,500. 4  Fig.  21.  S e c t i o n o f t h e n u c l e a r envelope showing t h e two u n i t membranes o f t h e envelope. Note t h e s i m p l e p o r e s . NE = n u c l e a r e n v e l o p e , P = p o r e s , N = n u c l e a r m a t e r i a l . KMnO. f i x a t i o n . X 65,000.  PLATE 8  F i g . 22.  C r o s s - s e c t i o n o f a s p o r e s i x hours a f t e r w e t t i n g . Note n u c l e a r shape and number o f s m a l l v e s i c l e s i n the c y t o p l a s m . W = w a l l , M = m i t o c h o n d r i o n , TVe = t r a n s p a r e n t v e s i c l e , N = n u c l e a r m a t e r i a l . Phosphate b u f f e r e d OsO. f i x a t i o n . X 27,750.  PLATE 9  F i g . 23.  Nucleus s i x hours a f t e r w e t t i n g of s p o r e s . Note the i r r e g u l a r c o n f i g u r a t i o n of t h e n u c l e u s . N = n u c l e a r m a t e r i a l , Nu = n u c l e o l u s . Phosphate b u f f e r e d OsO, f i x a t i o n . X 56,000.  PLATE 10 F i g . 24.  M i t o c h o n d r i o n o f spore i n stage two. Note f i b r i l l a r appearance o f c o r e . Phosphate b u f f e r e d OsO, f i x a t i o n . X 30,600.  F i g . 25.  Cytoplasm o f spore s i x hours a f t e r t h e b e g i n n i n g o f g e r m i n a t i o n . Note t h e number o f s m a l l , membrane-bound v e s i c l e s and t h e s m a l l amount o f c i s t e r n a l endoplasmic r e t i c u l u m . TVe = t r a n s p a r e n t v e s i c l e , M = m i t o c h o n d r i o n . Phosphate b u f f e r e d OsO. f i x a t i o n . X 49,375.  PLATE 11  F i g . 26.  S e c t i o n through a spore twelve hours a f t e r w e t t i n g . The n u c l e u s i s not shown i n t h i s s e c t i o n . OW = outer l a y e r o f w a l l , IW = i n n e r l a y e r o f w a l l , PM = plasma membrane, V = v a c u o l e , M = m i t o c h o n d r i o n . Phosphate b u f f e r e d OsO. f i x a t i o n . X 26,600.  PLATE 12 F i g . 27.  Lobed n u c l e u s c h a r a c t e r i s t i c o f spores t w e l v e hours a f t e r the b e g i n n i n g o f g e r m i n a t i o n . Note c i s t e r n a l endoplasmic r e t i c u l u m about the n u c l e u s . ER = endop l a s m i c r e t i c u l u m , NE = n u c l e a r e n v e l o p e , N = n u c l e a r m a t e r i a l , Nu = n u c l e o l u s , M = m i t o c h o n d r i o n . Phosphate b u f f e r e d OsO. f i x a t i o n . X 41,250.  PLATE 13  F i g . 28.  C e n t r i o l e a d j a c e n t to n u c l e u s . C y l i n d e r s of the c e n t r i o l e a r e open at both ends. N = n u c l e a r m a t e r i a l , Ce = c e n t r i o l e . Phosphate b u f f e r e d OsO^ f i x a t i o n . X 72,850.  F i g . 29.  C e n t r i o l e at a greater magnification. Ce = centriole. Phosphate b u f f e r e d OsO, f i x a t i o n . X 133,575.  PLATE 14  Fig.  30.  Two dictyosomes. i n c l o s e p r o x i m i t y to n u c l e u s . D = dictyosome, N = n u c l e a r m a t e r i a l , Nu = nucleolus. Phosphate b u f f e r e d OsO, f i x a t i o n . X 111,100.  Fig.  31.  Dictyosome a t h i g h e r m a g n i f i c a t i o n . Each d i c t y o some i s composed o f t h r e e t o four c i s t e r n a e l y i n g p a r a l l e l to one another. D = dictyosome, N = nuclear material. Phosphate b u f f e r e d OsO, f i x a tion. X 156,950.  PLATE 15  F i g . 32.  Spore p r i o r t o p r o t o p l a s t emergence. W = w a l l , PM = plasma membrane, TVe = t r a n s p a r e n t v e s i c l e , M = m i t o c h o n d r i o n , ER = endoplasmic r e t i c u l u m , V = v a c u o l e , N = nuclear material. Phosphate b u f f e r e d OsO. f i x a tion. X 41,400.  PLATE 16  F i g . 33-A.  S e c t i o n through a spore p r i o r t o p r o t o p l a s t emergence. Note t h a t the b a s a l body i s p r e s e n t i n the c e n t r a l r e g i o n of the spore and i n a j u x t a n u c l e a r p o s i t i o n . BB = b a s a l body, N = n u c l e a r m a t e r i a l , TVe = t r a n s parent v e s i c l e , M = m i t o c h o n d r i o n , W = w a l l . Phosphate b u f f e r e d OsO^ f i x a t i o n . X 22,000.  F i g . 33-B.  C r o s s - s e c t i o n o f a b a s a l body a t h i g h m a g n i f i c a t i o n . Note t h a t r o o t l e t s extend from p e r i p h e r y o f b a s a l body. BB = b a s a l body, R = r o o t l e t s . Phosphate buff e r e d OsO^ f i x a t i o n . X 85,550.  F i g . 34.  L o n g i t u d i n a l s e c t i o n through p r o x i m a l c y l i n d e r o f a b a s a l body. Note t h a t a d i f f e r e n t type o f r o o t l e t than seen i n f i g u r e 33-B extends i n t o the c y t o p l a s m from the b a s a l body p e r i p h e r y . BB = b a s a l body, R = rootlets. Phosphate b u f f e r e d OsO^ f i x a t i o n . X 49,950.  F i g . 35.  C r o s s - s e c t i o n through p r o x i m a l c y l i n d e r o f the b a s a l body. Note t h a t the r o o t l e t s r a d i a t e from the b a s a l body and appear to be connected to the c y l i n d e r by . f i n e f i b e r - l i k e extensions. BB = b a s a l body, R = rootlets. Phosphate b u f f e r e d OsO^ f i x a t i o n . X 59,250.  PLATE 17 Fig.  36.  F i g . 37.  M i t o c h o n d r i o n t y p i c a l o f spores p r i o r t o p r o t o p l a s t emergence. Membranes o f m i t o c h o n d r i o n appear d i s t i n c t and the " c o r e " r e g i o n l e s s e l e c t r o n dense than t h e matrix. No f i b r i l l a r s t r u c t u r e i s v i s i b l e i n the c o r e r e g i o n . M = m i t o c h o n d r i o n , V = v a c u o l e , Phosphate b u f f e r e d OsO. f i x a t i o n . X 83,100. Smooth, c i s t e r n a l endoplasmic r e t i c u l u m u s u a l l y i s i n c l o s e p r o x i m i t y t o v a c u o l e s c o n t a i n i n g membrane-like fragments. ER = endoplasmic r e t i c u l u m , V = v a c u o l e . Phosphate b u f f e r e d OsO. f i x a t i o n . X 79,200.  PLATE 18 S e c t i o n through emerging p r o t o p l a s t . Note absence o f i n n e r l a y e r o f spore w a l l . The n u c l e u s has r e v e r t e d t o o v o i d shape and i s i n t h e . c y t o p l a s m which f i r s t e x i t s the spore case. OW = o u t e r l a y e r of w a l l , M = m i t o c h o n d r i o n , N = n u c l e a r m a t e r i a l . Phosphate b u f f e r e d OsO, f i x a t i o n . X 31,875.  PLATE 19 Fig,  39.  L o n g i t u d i n a l s e c t i o n through a swarm c e l l . The b a s a l body o f t h e f l a g e l l u m i s p r e s e n t i n t h e ant e r i o r of the c e l l . M i c r o t u b u l e s and r o o t l e t s r a d i a t e i n t o the cytoplasm i n the region of t h i s b a s a l body. A c o n t r a c t i l e v a c u o l e i s seen i n t h e p o s t e r i o r o f t h e f l a g e l l a t e d c e l l , and ribosomes are v i s i b l e on t h e s u r f a c e o f t h e endoplasmic r e t i culum. PM = plasma membrane, BB = b a s a l body, MT = m i c r o t u b u l e s , N = n u c l e a r m a t e r i a l , M = m i t o c h o n d r i o n , CV = c o n t r a c t i l e v a c u o l e , RER = r i b o some c o a t e d endoplasmic r e t i c u l u m . Phosphate b u f f e r e d OsO. f i x a t i o n . X 33,750.  PLATE 20 F i g . 40.  Myxamoeba. Note t h e o v o i d shape o f t h e n u c l e u s and the presence o f a c o n t r a c t i l e v a c u o l e . PM = plasma membrane, CV = c o n t r a c t i l e v a c u o l e , M = m i t o c h o n d r i o n , RER = r i b o s o m e - c o a t e d endoplasmic r e t i c u l u m , N = n u c l e a r m a t e r i a l . Phosphate b u f f e r e d OsO, f i x a t i o n . X 36,800.  PLATE 21 F i g . 41.  C r o s s - s e c t i o n through spore w a l l s . I n upper p a r t of micrograph, the p r o t o p l a s t s t i l l i s r e t a i n e d w i t h i n the spore case and the m u l t i - l a y e r e d w a l l appears t o be c o m p l e t e l y i n t a c t . I n t h e lower p a r t o f the m i c r o g r a p h , t h e spore case has r u p t u r e d r e l e a s i n g the p r o t o p l a s t and o n l y t h e o u t e r l a y e r o f the w a l l i s v i s i b l e . OW = o u t e r l a y e r o f w a l l , IW = i n n e r l a y e r of w a l l . Phosphate b u f f e r e d OsO, f i x a t i o n . X 111,275.  F i g . 42.  Spore w a l l a f t e r the p r o t o p l a s t has been r e l e a s e d . Note t h a t o n l y the s p i n u l o s e o u t e r l a y e r of the w a l l remains. OW = o u t e r l a y e r o f w a l l . Phosphate b u f f e r e d OsO^ f i x a t i o n . X 88,900.  F i g . 43.  Ribosomes p r e s e n t on o u t e r s u r f a c e of c i s t e r n a l endop l a s m i c r e t i c u l u m . RER = r i b o s o m e - c o a t e d endoplasmic reticulum, M = mitochondrion, V = vacuole. Phosphate b u f f e r e d OsO. f i x a t i o n . X 41,150. 4  PLATE 22 Fig.  44.  V a c u o l e s c o n t a i n i n g e l e c t r o n dense g r a n u l a r m a t t e r . The g r a n u l a r m a t t e r i s bound by u n i t membranes. V = vacuole. Phosphate b u f f e r e d OsO^ f i x a t i o n . X 106,350.  Fig.  45.  C o n t r a c t i l e v a c u o l e i n expanded s t a t e . CV = c o n t r a c t i l e v a c u o l e , RER = r i b o s o m e - c o a t e d endoplasmic r e t i c u l u m . Phosphate b u f f e r e d OsO. f i x a t i o n . X 63,475.  PLATE 23 F i g . 46.  C o n t r a c t i l e vacuole inccontracted state. Also, a dictyosome i s seen i n c l o s e p r o x i m i t y w i t h the n u c l e u s . N = n u c l e a r m a t e r i a l , D = d i c t y o s o m e , Ve = v e s i c l e , CV = c o n t r a c t i l e v a c u o l e . Phosphate b u f f e r e d OsO, f i x a t i o n . X 108,250.  PLATE 24 Fig.  47.  C r o s s - s e c t i o n t h r o u g h the b a s a l body o f the f l a g e l lum. The b a s a l body i s composed of n i n e p e r i p h e r a l f i b r i l s about two c e n t r a l f i b r i l s . Each p e r i p h e r a l f i b r i l i s made up of two s u b - u n i t s . E x t e n d i n g from the p e r i p h e r a l a r e a o f the b a s a l body are r o o t l e t s which a r e f i b r i l l a r - l i k e i n s t r u c t u r e . Microtubules are seen r a d i a t i n g from the f l a g e l l a r r e g i o n p o s t e r i o r l y i n the c e l l . PM = plasma membrane, MT = m i c r o t u b u l e s , BB = b a s a l body, R = r o o t l e t s , Ve = v e s i c l e . Phosphate b u f f e r e d OsO^ f i x a t i o n . X 57,500.  Fig.  48.  L o n g i t u d i n a l s e c t i o n t h r o u g h the a n t e r i o r of a swarm cell. R o o t l e t s and d i c t y o s o m e - l i k e v e s i c l e s are p r e s e n t i n t h i s a r e a . R = r o o t l e t s , Ve = v e s i c l e . Phosphate b u f f e r e d OsO^ f i x a t i o n . X 33,700.  Fig.  49.  C r o s s - s e c t i o n t h r o u g h a f l a g e l l u m i n a swarm c e l l . Note the 9 + 2 f i b r i l arrangement. N = n u c l e a r materi a l , Nu = n u c l e o l u s . Phosphate b u f f e r e d OsO fixat i o n . X 37,500.  Fig.  50.  C r o s s - s e c t i o n through a f l a g e l l u m . Note the 9+2 f i b r i l arrangement and the secondary elements w h i c h a r e p r e s e n t between the n i n e p e r i p h e r a l f i b r i l s and the two c e n t r a l f i b r i l s . A u n i t membrane surrounds t h i s a x i a l process. Phosphate b u f f e r e d OsO, f i x a t i o n . X 82,650.  PLATE 25 Fig..51.  T y p i c a l n u c l e u s o f swarm c e l l s and myxamoebae. The n u c l e a r m a t e r i a l i s surrounded by a d i s t i n c t n u c l e a r envelope. A s i n g l e n u c l e o l u s i s p r e s e n t i n each nucleus. NE = n u c l e a r e n v e l o p e , N = n u c l e a r materi a l , Nu = n u c l e a l u s . Phosphate b u f f e r e d OsO, f i x a t i o n . X 44,300.  F i g . 52 - 53. N u c l e o l i w i t h one or two n u c l e o l a r v a c u o l e s . No membrane encompasses t h e n u c l e a l a r v a c u o l e s . Nu = nucleolus. Phosphate b u f f e r e d OsO, f i x a t i o n . X 62,475. * F i g . 54.  N u c l e a r envelope a t h i g h e r m a g n i f i c a t i o n . Note t h e two u n i t membranes forming t h e envelope. N = n u c l e a r m a t e r i a l , NE = n u c l e a r e n v e l o p e , RER = ribosomec o a t e d endoplasmic r e t i c u l u m . Phosphate b u f f e r e d OsO, f i x a t i o n . X 88,000. 4  

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