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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., University of C a l i f o r n i a Santa Barbara, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of BOTANY We accept t h i s thesis as conforming to the 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the 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 able f o r reference 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 extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of 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 not be allowed without my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Department of i i ABSTRACT Spores of 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 to determine s t r u c t u r a l changes during germination. L i g h t microscopic observations i n d i c a t e that few changes occur p r i o r to 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 microscopic observations show that a number of changes occur w i t h i n the p r o t o p l a s t before emergence. An ovoid nucleus becomes i r r e g u l a r and lobed; smooth, c i s t e r n a l endo-plasmic r e t i c u l u m develops; and concurrent development of dictyosomes and c e n t r i o l e occurs. The dictyosomes and c e n t r i o l e are 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 the proximal c y l i n d e r of the c e n t r i o l e d i f -f e r e n t i a t e s i n t o a b a s a l body of a f u t u r e f l a g e l l u m . When the spore case r u p t u r e s , the inner l a y e r of the w a l l disappears and the nucleus r e v e r t s to i t s o r i g i n a l ovoid form. The p r o t o p l a s t emerges through a wedge-shaped s p l i t i n the w a l l and g r a d u a l l y develops i n t o a f l a g e l -l a t e d c e l l o r , sometimes, a myxamoeba. Simultaneously, c o n t r a c t i l e and food vacuoles develop, the c i s t e r n a l endoplasmic r e t i c u l u m becomes ribosome-coated, and a f l a g e l l u m develops from the b a s a l body. i i i i TABLE OF CONTENTS In t r o d u c t i o n 1 M a t e r i a l s and Methods 4 Res u l t s L i g h t M i c r o s c o p i c R e s u l t s 6 E l e c t r o n M i c r o s c o p i c Results Stage One - Re s t i n g Spore 7 Stage Two - S i x Hour Stage i n Germination . . . . . . . . 10 Stage Three - Twelve Hour Stage i n Germination 12 Stage Four - Seventeen Hour Stage i n Germination 13 A. Spores P r i o r to 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 Di s c u s s i o n 19 Summary 30 Bi b l i o g r a p h y 32 Appendix i v TABLE OF PLATES L i g h t micrographs of F u l i g o s e p t i c a spores at 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 spores. 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 the 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 carbon-f i l t e r e d , d i s t i l l e d water or 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 of F u l i g o s e p t i c a spores twelve hours subsequent to w e t t i n g spores. 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 p r i o r to 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 of F u l i g o s e p t i c a spore at time of emergence from spore case. 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 p r o t o p l a s t s a f t e r emergence from spore cases. V ACKNOWLEDGEMENT The author g r e a t l y appreciates and wishes to thank the 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 advice during the p r e p a r a t i o n of t h i s t h e s i s . Dr. R. J . Bandoni, 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 advice and techniques f o r the e l e c t r o n microscopy p o r t i o n of 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 suggestions which proved h e l p f u l . Alice-Ann B i s a l p u t r a and Grace Wood, who helped i n the t y p i n g of t h i s t h e s i s . And, to other members of the Department of Botany, who have helped a s s i s t the author during her graduate st u d i e s at the U n i v e r s i t y of B r i t i s h Columbia. 1. INTRODUCTION Germination of Myxomycete spores was described f i r s t by de Bary i n 1854. He observed 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 from spores of T r i c h i a r u b i f o r m i s (now c a l l e d H e m i t r i c h i a vesparium). Sub-sequent st u d i e s on spore germination i n d i c a t e d that the r a t e of germina-t i o n , percentage of germination, and method of germination vary from species to 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 of e x t e r n a l f a c t o r s on spore germination i n Myxomycetes. Smart (1937) i n v e s t i g a t e d the e f f e c t of medium on germination. He found that the r a t e and percentage of spore germination of many Myxomycete species were increased i f spores were sown i n weak decoctions of 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 decayed leaves. L i s t e r (1901) and Durand (1894) i n d i c a t e d that repeated w e t t i n g and d r y i n g of spores increased the r a t e and percentage of ger-mination. E l l i o t t (1949) noted that repeated w e t t i n g and d r y i n g had no e f f e c t on germination i n some of the Myxomycetes. He emphasized that the germination r a t e increased when spores were wetted w i t h a one percent b i l e s a l t s o l u t i o n . Other w e t t i n g agents used i n attempting to i n c r e a s e the r a t e and percentage of germination were a l c o h o l , detergents, mer-c u r i c c h l o r i d e , and t r i s o d i u m phosphate (Cayley, 1929"; E l l i o t t , 1949). C o n f l i c t i n g r e s u l t s were reported. Temperature was shown to be an important e x t e r n a l f a c t o r a f f e c t -i n g both r a t e and percentage of germination as w e l l as method of germination. Smart (1937) i n d i c a t e d that spore germination was best at 2. approximately 25°C to 30°C (see a l s o Constantineanu, 1906), and that the r a t e and percentage of germination were reduced 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 of l i g h t , hydrogen i o n c o n c e n t r a t i o n , spore c o n c e n t r a t i o n , and spore age are other f a c t o r s which have been i n v e s t i -gated. L i g h t appears to have no i n f l u e n c e on germination (Smart, 1937). The optimum pH f o r spore germination i n most species i s between pH 4.5 and pH 7.0 (Smart, 1937). Concentration of sown spores i n f l u e n c e s r a t e and percentage of germination (Scholes, 1962; Smart, 1937; Wilson and Cadman, 1928). Scholes (1962) has s t a t e d that spores germinate more r a p i d l y and the percentage of germination i s g r e a t e r w i t h d i l u t e suspensions of spores; whereas, Smart (1937) and Wilson and Cadman (1928) have report e d the opposite e f f e c t of spore c o n c e n t r a t i o n on germination. Spore age i n f l u e n c e s the r a t e and percentage of germination i n some species (Alexopoulos, 1963). Several types of spore germination have been described. In some Myxomycetes the p r o t o p l a s t escapes through a wedge-shaped crack i n the spore w a l l ( G i l b e r t , 1928; Howard, 1931; McManus, 1961; Smart, 1937). In other species the p r o t o p l a s t e x i t s the spore case through an i r -r e g u l a r pore ( G i l b e r t , 1928; Smart, 1937). The p r o t o p l a s t , at the time of emergence, has been described as a myxamoeba or f l a g e l l a t e d swarm c e l l , depending on the species and on environmental c o n d i t i o n s ( G i l b e r t , 1928; Smart, 1937). In some Myxomycetes the myxamoeba remains quiescent 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 c e l l (Howard, 1931; Smart, 1937). The swarm c e l l has been report e d to 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 cytoplasmic o r g a n e l l e changes during germination of fungal spores have been published. I n v e s t i g a t o r s have noted the development and enlargement of a vacuole p r i o r to germination ( G i l b e r t , 1928; Smart, 1937). Concurrent w i t h vacuolar formation i s movement of cytoplasmic granules. Gradually the spore w a l l becomes s t r e t c h e d and a pore develops through which the p r o t o p l a s t emerges ( G i l b e r t , 1928; Smart, 1937). F l a g e l l a r formation i s reported to occur subsequent to pore or crack development i n the spore 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 during germination have not been r e p o r t e d , and few papers have been pu b l i s h e d on the f i n e s t r u c t u r e of Myxomycete spores (Loquin, 1959; Schuster, 1964; Wohlfarth-Bottermann, 1959). The f i n e s t r u c t u r e of myxamoebae has been observed i n Didymium n i g r i p e s (Schuster, 1964), and Cohen (1959) has s t u d i e d 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 that occur from the time of spore formation to the p e r i o d i n which swarm c e l l s or myxamoebae are r e l e a s e d . Thus, the purpose of t h i s study i s to i n v e s t i g a t e the s e q u e n t i a l changes o c c u r r i n g i n Myxomycete spores during germination. F u l i g o s e p t i c a (L.) Weber, spores have been s e l e c t e d f o r t h i s study because they germinate r a p i d l y and i n high percentages i n the l a b o r a t o r y . MATERIALS AND METHODS Spores of F u l i g o s e p t i c a were obtained from a e t h a l i a which had been c o l l e c t e d on the U n i v e r s i t y of B r i t i s h Columbia Endowment Lands and i d e n t i f i e d by R. J . Bandoni, J u l y , 1963. Approximately 0.1 g of spores was wetted i n a one percent 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 minute and r i n s e d twice w i t h s t e r i l e carbon-f i l t e r e d , d i s t i l l e d water. The spores were sown i n 60 x 20 mm s t e r i l e p l a s t i c disposable p e t r i dishes which contained e i t h e r 25 ml of 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 of s t e r i l e l e a f - e x t r a c t decoction. The leaves used i n making the l e a f - e x t r a c t decoction were from deciduous trees and had been picked at random on the U n i v e r s i t y of B r i t i s h Columbia Endowment Lands. The percentage of germination and r a t e of germination were determined by averaging the r e s u l t s of d u p l i c a t e spore suspensions subjected to the same c o n d i t i o n s . To determine the percentage of germi n a t i o n , one drop of spore suspension was p i p e t t e d onto a glass s l i d e , s t a i n e d w i t h i o d i n e potassium i o d i d e ( I K I ) , and examined by l i g h t microscopy. The f i r s t 300 spores examined were used to determine the germination percentage. Approximately 90 to 95 % of the spores germi-nated 17 to 20 hours a f t e r they had been wetted i n the b i l e s a l t 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 decoction. The optimum temperature f o r germination was determined by i n -c u b a t i n g the spore suspension i n temperature c o n t r o l l e d chambers set at approximately 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C. The temperature at which the r a t e of germination and percentage of germi-5. n a t i o n was grea t e s t was approximately 25°C. This temperature was designated as the optimum temperature f o r spore germination and f u r t h e r germination studies were performed at t h i s e s t a b l i s h e d optimum temperature. A l i g h t microscopic study of the germination process was performed 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 . Spores were examined w i t h a L i e t z D i a l u x microscope using b r i g h t f i e l d i l l u m i n a t i o n , dark f i e l d i l l u m i n a t i o n , and phase c o n t r a s t . Micrographs were taken w i t h a L i e t z Orthomat microscope. The m a t e r i a l f or e l e c t r o n microscopy was f i x e d at four d i f f e r e n t times during the germination p e r i o d . The times a r b i t r a r i l y were decided on and designated as stages one, two, t h r e e , and four. Stage one represented r e s t i n g spores which were not subjected to germination treatment. Spores of stage two were wetted and sown i n the germination medium f o r s i x hours, and those of stage three f o r twelve hours. Stage four represented the spores i n which the p r o t o p l a s t s were about to emerge from the spore cases or which had emerged from the spore cases. F i x a t i o n of m a t e r i a l f o r e l e c t r o n microscopy was w i t h unbuffered 1.5 % KMnO^ f o r 20 minutes at 0°C or 1 % 0 s 0 4 b u f f e r e d w i t h .15 M phosphate at pH 7.2 f o r one hour at 0°C. 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 water, they were embedded i n a drop of 4 % water agar. The agar drop was cut i n t o s m a ll pieces and the pieces were dehydrated i n a graded s e r i e s of 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 Weier, 1963) was used to embed the dehydrated m a t e r i a l and the blocks were poly-merized 18 to 24 hours i n a vacuum oven at 65°C. The m a t e r i a l was sec t i o n e d w i t h glass knives w i t h e i t h e r a Porter-Blum MI 1 or MI 2 microtome. A f t e r mounting the sections on c o l l o d i a n - c o a t e d copper g r i d s , the specimens were p o s t - s t a i n e d w i t h u r a n y l acetate and l e a d c i t r a t e (Reynolds, 1963). A l l sec t i o n s were examined w i t h a H i t a c h i HU-11A microscope. 6. RESULTS Approximately 90 to 95 % of the spores of F u l i g o s e p t i c a germi-nated a f t e r 17 to 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 decoction 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 decoction tended to 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 water. L i g h t Microscopy Few changes w i t h i n the spore case during germination can be observed w i t h the r e s o l u t i o n of the l i g h t microscope. The spores appear s p h e r i c a l ( F i g . 1 ) , 6 to 9 y. i n diameter, and the spore w a l l i s spinu-l o s e . The p r o t o p l a s t , at f i r s t quiescent, 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 nucleus seems to maintain i t s form during germination. There appears to be a s l i g h t increase i n the number of vacuoles 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. Approximately 17 hours a f t e r w e t t i n g the spores, wedge-shaped s p l i t s develop i n the spore cases. 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 ength equals at l e a s t t h r ee-fourths the diameters of the spores. Simultaneously, there i s considerable movement of the protoplasm and the p r o t o p l a s t s begin to emerge from the spore cases ( F i g s . 2 - 5 ) . The p e r i o d of time f o r the emergence of the 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 spore. When r e l e a s e d , the 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 the mouths of the rupture f o r 10 to 15 minutes ( F i g s . 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 are seen u n t i l a f t e r the p r o t o p l a s t s become amoeboid. Most of the swarm c e l l s are u n i f l a g e l l a t e ( F i g s . 9 - 10); however, b i f l a g e l l a t e d c e l l s are not uncommon ( F i g . 8). The f l a g e l l a of 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 are about the same length as the bodies of the swarm c e l l s ( F i g s . 8 - 10). 7. E l e c t r o n Microscopy Stage One - Rest i n g Spore The t y p i c a l s t r u c t u r e of most r e s t i n g spores observed 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 : W a l l : 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 the spore p r o t o p l a s t (W, F i g . 11). I t appears s t r u c t u r a l l y s i m i l a r to the spore w a l l s of D. n i g r i p e s (Schuster, 1964). The outer l a y e r of the w a l l (OW, F i g . 12) i s e l e c t r o n dense, gr a n u l a r , and i s approximately 50 mu wide. E l e c t r o n dense spines a r i s e at i r r e g u l a r i n t e r v a l s on the surface of the outer l a y e r ( F i g . 11). The spine m a t e r i a l a l s o appears g r a n u l a r , and each spine i s about 150 mu long. 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 -parent inner l a y e r (IW, F i g . 12) l i e s between the e l e c t r o n dense outer l a y e r and the plasma membrane. This inner l a y e r i s approximately the same width as the outer l a y e r , and i t i s sub-divided i n t o two se c t i o n s by a 100 A° e l e c t r o n dense l i n e . The e l e c t r o n l i g h t s e c t i o n outside the e l e c t r o n dense l i n e i s approximately 40 mu wide. The s e c t i o n adjacent to the plasma membrane v a r i e s i n wi d t h , ranging from 2 mu to 4 mu. The e l e c t r o n dense l i n e appears s i m i l a r to the e l e c t r o n dense outer l a y e r of the w a l l ( F i g . 12) . Plasma Membrane: A d i s t i n c t plasma membrane envelops the protoplasm of the spore (PM, F i g . 11). 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 symmetrically 3 l a y e r e d (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, are not inf r e q u e n t . 8. Vacuoles: There are s e v e r a l vacuoles i n the cytoplasm of the spore (V, F i g . 11). These vacuoles are 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 transparent or granular vacuolar sap; membrane-like fragments; and some e l e c t r o n dense, granular matter ( F i g . 13). The vacuoles range i n s i z e from 1 to 2 p. Transparent V e s i c l e s : Two types of e l e c t r o n transparent v e s i c l e s are dispersed randomly throughout the cytoplasm of the spore. One type of v e s i c l e i s membrane-bound and i s about .1 to .3 fi i n diameter ( F i g . 11). Some of the membranes about these v e s i c l e s appear broken. The other type of e l e c t r o n transparent v e s i c l e (TVe, F i g . 14) i s not bound by a membrane. These v e s i c l e s are i r r e g u l a r l y shaped and vary from .2 to .4 ju i n diameter. Mitochondria: Mitochondria w i t h t u b u l a r - t y p e c r i s t a e are d i s t r i b u t e d randomly w i t h i n the p r o t o p l a s t (M, F i g . 11). The s i z e , shape, and number of mitochondria per p r o t o p l a s t v a r i e s from spore to spore. Most mito-chondria are somewhat s p h e r i c a l to ovoid and are about 1 to 1.5 p. i n diameter (M, F i g . 15). Each mitochondrion i s surrounded by an inner u n i t membrane and an outer u n i t membrane. The inner membrane, approximately 80 A° t h i c k , invaginates forming tubular c r i s t a e which are about 25 mu i n diameter. Each c r i s t a : may branch s e v e r a l times forming s i d e t u b ules. Most of the c r i s t a e extend g e n e r a l l y towards the c e n t r a l r e gion of the mitochondrion; however, a few of the c r i s t a e appear to be continuous w i t h c r i s t a e a r i s i n g from other regions of the mitochondrion ( F i g . 15). The i n t e r n a l space o f the tu 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 the 9 . cytoplasm (Fig. 15). An electron dense, coarsely granular matter occupies the c e n t r a l region of most of the mitochondria (Figs. 15 - 16). This dense core i s about 100 mu i n diameter and i t appears to lack any organized structure ( F i g . 16). The outer membrane surrounds the mitochondrion. This membrane does not invaginate and i t i s approxi-mately as thick as the inner membrane. The mitochondrial membranes of 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 the plasma membranes and the vacuolar membranes (Fig. 16). Cytoplasm and Other Inclusions: C h a r a c t e r i s t i c of the r e s t i n g spore i s a granular cytoplasm which i s intermediate i n density between the electron dense outer layer of the wall and the less electron dense vacuolar sap (Fig. 11). Some of the 100 to 130 A° granules i n the cytoplasm resemble ribosomes i n s i z e and density (Fig. 14). Nucleus: A s i n g l e , ovoid nucleus i s present i n the protoplast of each spore of F. se p t i c a (N, F i g . 11). This nucleus, about 3 to 3.5 p long and 2 p wide, contains a sin g l e nucleolus (Nu, Fi g . 17). The nucleolus i s approximately 1 p. i n diameter and i s electron dense and granular. At lea s t one nucleolar vacuole i s present i n each nucleolus, and neither the nucleolar vacuole nor the nucleolus i s surrounded by a membrane. The chromatin material i n the nucleus i s almost as electron dense as the nucleolar granules, and i t appears granular and randomly dispersed throughout the less electron dense nucleoplasm (Fig. 17). The nuclear material i s encompassed by an envelope which i s composed of two membranes, each about 80 A 0 thick. A perinuclear space, about 110 A° wide, i s seen between the two membranes. Both the outer and the inner membranes of 10. the envelope are smooth, no ribosomes are present 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 the nuclear envelope form "pores" which are 200 to 400 A° i n diameter. These pores are simple and occur at 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 are seen at the circumference of the pores and no diaphrams cross the diameter of the pores to separate the nuclear m a t e r i a l from the cytoplasm. Bleb formation commonly i s observed i n the nuclear envelope (NB, F i g s . 18 - 19). One type of bleb appears to be formed by the evagination of the outer membrane of the nuclear envelope and the formation of another membrane w i t h i n t h i s e vagination (NB, F i g . 18). Thus, t h i s type of bleb i s encompassed by two membranes. A second type of bleb (NB, F i g . 19), surrounded by only one membrane, a l s o i s seen i n a number of the nuclear envelopes. I t appears to be formed by the evagination of the outer membrane and the i n v a g i n a t i o n of the inner membrane of the envelope. Stage Two - S i x Hour Stage i n Germination 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 occurred. These spores appear s i m i l a r to the one shown i n f i g u r e 22; however, vacuoles c o n t a i n i n g membrane-like fragments, not present i n f i g u r e 22, are present i n most spores at t h i s stage. Many spore o r g a n e l l e s do not 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 only when s t r u c t u r a l changes occur or when new orga n e l l e s are encountered. Nucleus: The n u c l e i of spores at t h i s stage of germination assume a v a r i e t y of forms (N, F i g s . 22 - 23). The nuclear envelope becomes more 11. d i s t i n c t and no bleb formation i s d i s t i n g u i s h a b l e ( F i g . 23). The chro-matin m a t e r i a l , nucleoplasm, and nucleolus do not appear to be a l t e r e d ( F i g . 23). Mitochondria: Both the inner and the outer membranes of the mitochondria f i x more d i s t i n c t l y than they do i n the r e s t i n g spore stage ( F i g s . 22 and 24), and the dark cores w i t h i n the m i t o c h o n d r i a l m a t r i x become more prominent. Each core, approximately 100 to 150 mu i n diameter, l i e s p a r a l l e l to the l o n g i t u d i n a l a x i s of the mitochondrion, and each i s com-posed of 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 diameter. Some f i b r i l s are seen i n c r o s s - s e c t i o n near the edges of the core, w h i l e other f i b r i l s run p a r a l l e l to the l o n g i t u d i n a l a x i s of the mitochondrion ( F i g . 24). Transparent V e s i c l e s : There i s an increase i n the number of s m a l l , membrane-bounded v e s i c l e s i n the cytoplasm (Ve, F i g . 25). The membranes encompassing the v e s i c l e s are approximately 80 to 100 A° i n t h i c k n e s s , w h i l e the diameter of the v e s i c l e s ranges from 50 to 100 mu. These v e s i c l e s are randomly d i s t r i b u t e d w i t h i n the p r o t o p l a s t . Endoplasmic Reticulum: C i s t e r n a l endoplasmic r e t i c u l u m (ER, F i g . 25) i s present i n some of the spores. 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 of about 100 to 120 A° i n width. The bounding membrane i s about 80 A° t h i c k , and no ribosomes are present on the outer surface of t h i s membrane (ER, F i g . 25). 12. Stage Three - Twelve Hour Stage i n Germination A number of changes occur w i t h i n the spore p r o t o p l a s t w i t h i n twelve hours a f t e r w e t t i n g the spores. Organelles not "detected" i n the e a r l i e r stages of germination are observed, and some of the organ-e l l e s described p r e v i o u s l y continue to change. The s p i n u l o s e spore w a l l , plasma membrane, vacuoles c o n t a i n i n g membrane-like fragments, transparent v e s i c l e s , and membrane-bound v e s i c l e s remain unchanged ( F i g . 26). The nucleus continues to change, c e n t r i o l e s and dictyosomes appear, and the c i s t e r n a l endoplasmic r e t i c u l u m becomes more extensive (ER, F i g . 27). Nucleus: Most n u c l e i have become lobed (N, F i g . 27); however, the nuclear m a t e r i a l appears to have changed l i t t l e . A s i n g l e n ucleolus i s present i n each nucleus (Nu, F i g . 27). C e n t r i o l e : In some spores, 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 to the nucleus and i n the c e n t r a l r e g ion of the proto-p l a s t . Each c e n t r i o l e i s composed of a p a i r of c y l i n d e r s , 400 to 430 mju long and 180 mu i n diameter. The c y l i n d e r s are open at both ends and l i e at r i g h t angle to one another. Each c y l i n d e r i s made up of nine l o n g i t u d i n a l , evenly spaced, tubular f i b r i l s (Ce, F i g . 29), and each f i b r i l i s about 350 A° i n diameter and i s composed of two sub-u n i t s . At the end of the c y l i n d e r c l o s e s t to the adjacent c y l i n d e r , the f i b r i l s bend inward f o r about 300 A° at a 90° angle ( F i g s . 28 - 29). Two l o n g i t u d i n a l , tubular f i b r i l s l i e i n the c e n t r a l r e g i o n of the c y l i n d e r s , and each f i b r i l i s about 300 A° i n diameter. In a l l 13. centrioleSj, there i s cytoplasm of 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 the c y l i n d e r . Dictyosomes: Most spores c o n t a i n at l e a s t one or two dictyosomes, and each dictyosome i s composed of a stack of three to four f l a t t e n e d c i s t e r n a e w i t h small v e s i c l e s at each end of the c i s t e r n a e (D, F i g s . 30 - 31). The c i s t e r n a e resemble smooth-surfaced, c i s t e r n a l - t y p e endo-plasmic r e t i c u l u m . The membranes forming each c i s t e r n a e are u n i t membranes, about 80 A° i n t h i c k n e s s , and are separated 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 approximately 150 A° wide (D, F i g . 31). The dictyosomal c i s t e r n a e l i e p a r a l l e l to each other and are separated by a space of about 120 A°. The small membrane-bound v e s i c l e s concentrated on each s i d e of the stacks of f l a t t e n e d c i s t e r n a e ( F i g . 31) are about 200 to 400 A° i n diameter. Stage Four - Seventeen Hour Stage i n Germination There are four main developmental forms of F. s e p t i c a spore proto-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 are con-t a i n e d w i t h i n the spore case ( F i g . 32), others are emerging through s p l i t s i n spore cases ( F i g . 38), and many of the p r o t o p l a s t s have developed i n t o swarm c e l l s ( F i g . 39) or myxamoebae ( F i g . 40). A. Spores P r i o r to 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 not occurred, both the inner and outer l a y e r s of the 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 of d i s s o l u t i o n or c r a c k i n g of any part of the w a l l (W, F i g . 32). Many n u c l e i s t i l l are lobed and s e c t i o n s through these lobes may give the impression 14. that more than one nucleus i s present i n a s i n g l e spore (N, F i g . 32). However, i n c e r t a i n spores, the n u c l e i are 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). C e n t r i o l e s : Changes i n c e n t r i o l e s t r u c t u r e are seen p r i o r to p r o t o p l a s t emergence. The c e n t r i o l e s assume the form of b a s a l bodies w i t h s e v e r a l types of r o o t l e t s extending from them (BB, F i g s . 33 - 35). The nine o u t e r f i b r i l s are r o t a t e d evenly i n the same d i r e c t i o n and to a s l i g h t degree about a c e n t r a l f i b r i l . Each outer f i b r i l i s connected to the ad-jacent f i b r i l s and to the 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 extensions, thereby, forming a "pin-wheel" p a t t e r n (BB, F i g s . 33-B - 35). The outer f i b r i l s are about 350 A° i n diameter, the c e n t r a l f i b r i l 450 A° i n diameter, and the f i b e r - l i k e extensions are about 40 A° wide. One form of r o o t l e t tapers from the outer f i b r i l l a r r e g i o n of the proximal c y l i n d e r and i n t o the cytoplasm f o r about 100 to 150 nyu ( F i g . 33-B). The r o o t l e t s appear to be made of s e v e r a l tubules which are about 150 A° i n width. Another form of r o o t l e t i s seen i n f i g u r e s 34 and 35. A sheath of l o n g i -t u d i n a l f i b r i l s p a r t i a l l y surrounds the proximal c y l i n d e r of the c e n t r i o l e . These sheathing r o o t l e t s are about 300 mu long and 30 mu i n diameter, and they appear to be connected to part of the w a l l of the proximal c y l i n d e r by f i n e , r a d i a t i n g f i b e r s about 30 to 40 A° i n width ( F i g . 35). The s h e a t h - l i k e r o o t l e t s do not l i e p a r a l l e l to the proximal c y l i n d e r but seem to extend i n t o the cytoplasm at an obtuse angle (R, F i g . 34). 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 c y l i n d e r ( F i g . 35). 15. Mitochondria: An e l e c t r o n dense, s t r u c t u r e d core i s no longer 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 mitochondria; however, the area formerly occupied by the core now appears l e s s e l e c t r o n dense than the surrounding m a t r i x (M, F i g . 36). Vacuoles: Many vacuoles c o n t a i n i n g membrane remnants are surrounded by c i s t e r n a l endoplasmic r e t i c u l u m . This endoplasmic r e t i c u l u m i s not coated w i t h ribosomes ( F i g . 37). B. P r o t o p l a s t Emergence In the second form, the p r o t o p l a s t s are seen emerging through s p l i t s i n the spore w a l l s ( F i g . 38). Only the s p i n u l o s e , e l e c t r o n dense, outer l a y e r of the spore w a l l remains ( F i g s . 41 - 42). The inner l a y e r , composed of two e l e c t r o n transparent s e c t i o n s separated by a dense l i n e , i s no longer v i s i b l e . The nucleus r e v e r t s to; an ovoid form and i s i n the cytoplasm which f i r s t e x i t s the spore case. Small vacuoles are s c a t t e r e d throughout cytoplasm and the other o r g a n e l l e s are not v i s i b l y a l t e r e d . This f r e 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 ( F i g . 39) or a myxamoeba ( F i g . 40). 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 the myxamoeba i s of i n -d e f i n i t e form. The myxamoeba and the swarm c e l l are s t r u c t u r a l l y s i m i l a r and both are bound by a s i n g l e u n i t membrane, the plasma membrane (PM, F i g s . 39 - 40). Both possess a s i n g l e ovoid nucleus (N), a c o n t r a c t i l e vacuole (CV), mitochondria w i t h tubular-type c r i s t a e (M) , rough c i s t e r n a l endoplasmic r e t i c u l u m (RER), food vacuoles (V), dictyosomes (D), and 16. membrane-bound v e s i c l e s (Ve) ( F i g s . 39 - 51). In swarm c e l l s , micro-tubules (MT, F i g . 39) pass about the nucleus i n the cytoplasm and extend towards the b a s a l body of the f l a g e l l u m . Endoplasmic 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 surface i s present i n a l l p r o t o p l a s t s that have emerged from spore cases. Each membrane i s about 80 A° t h i c k , and each i n t r a -c i s t e r n a l space i s approximately 150 A° i n width. Ribosomes are seen on the outer surfaces of the c i s t e r n a e and are about 100 A° i n diameter (RER, F i g . 43). Food Vacuoles: Several vacuoles c o n t a i n i n g food p a r t i c l e s are dispersed randomly thoughout the cytoplasm of the myxamoebae and swarm c e l l s . Each vacuole i s bound by a s i n g l e u n i t membrane, about 75 to 80 A° t h i c k . The vacuoles range from .7 to 2 p i n s i z e and c o n t a i n a dense, g r a n u l a r , membrane-bound m a t e r i a l . These gr a n u l a r , membrane-encompassed p a r t i c l e s are suspended i n an e l e c t r o n transparent vacuolar sap (V, F i g . 44). They vary i n s i z e , from .2 to .5^u i n diameter, and i n shape. C o n t r a c t i l e Vacuole: U s u a l l y one c o n t r a c t i l e vacuole i s present in.each c e l l (CV, F i g s . 39 - 40). In swarm c e l l s , the c o n t r a c t i l e vacuole g e n e r a l l y i s s i t u a t e d i n the p o s t e r i o r of the c e l l , or near dictyosomes; i n myxamoebae, i t does not appear to 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 vacuoles vary i n s i z e and shape, depending on whether they are i n an expanded s t a t e (CV, F i g . 45) or i n a cont r a c t e d s t a t e (CV, F i g . 46). Bordering contracted vacuoles are many small v e s i c l e s ranging from 40 to 70 mu i n diameter. The membranes surrounding 17. both c o n t r a c t i l e vacuoles and bordering v e s i c l e s are about 80 A° t h i c k . Dictyosomes: Dictyosomes are found i n most myxamoebae and f l a g e l l a t e d c e l l s . They are 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 to the c o n t r a c t i l e vacuoles. In swarm c e l l s , the dictyosomes are i n the a n t e r i o r of the c e l l s between the ba s a l bodies of f l a g e l l a and the n u c l e i . Many dictyosome-like v e s i c l e s are v i s i b l e i n the a n t e r i o r of the c e l l s ( F i g s . 38 and 46). S t r u c t u r a l l y , the dictyosomes i n swarm c e l l s and myxamoebae appear more organized and defined than they do i n p r o t o p l a s t s of stage three. F l a g e l l a : Most swarm c e l l s are u n i f l a g e l l a t e d ; however, b i f l a g e l l a t e d c e l l s are not uncommon. Each f l a g e l l u m i s 6 to 9 long, about 230 mu i n diameter, and each i s l o c a t e d i n the a n t e r i o r of the c e l l ( F i g . 39). In c r o s s - s e c t i o n , the f l a g e l l a r s t r u c t u r e i s seen to be made of nine p e r i p h e r a l f i b r i l s evenly spaced about two c e n t r a l f i b r i l s ( F i g . 50). Secondary elements are present between the two c e n t r a l f i b r i l s and the 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 are composed of two sub-u n i t s , each about 140 A° i n diameter. On' some of the p e r i p h e r a l f i b r i l s , two arms appear to r a d i a t e out from one of the two sub-units. Each arm serens to be about 50 A° t h i c k and approximately 70 A° long. The c e n t r a l f i b r i l s l a c k sub-unit s t r u c t u r e and are about 170 A° i n diameter. The distance between the two c e n t r a l f i b r i l s and the nine p e r i p h e r a l f i b r i l s i s about 350 A°, and the distance between the p e r i p h e r a l f i b r i l s and the u n i t membrane which surrounds the a x i a l f i l a m e n t i s about 200 A°. The p e r i -p h e ral f i b r i l s are about 120 A° apart and there appears to be a space of approximately 50 A° between the two c e n t r a l f i b r i l s . The membrane about the a x i a l f ilament i s approximately 90 A° t h i c k . 18. The b a s a l bodies of f l a g e l l a are sub-dermal and s t r u c t u r a l l y s i m i -l a r to the b a s a l bodies p r e v i o u s l y described i n t h i s study. In c r o s s -s e c t i o n , each b a s a l body i s composed of nine 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 . Two sub-units comprise each of the outer f i b r i l s . The dimensions of the sub-structure of the b a s a l bodies i s s i m i l a r to t h a t of the f l a g e l l u m . R o o t l e t s , which appear to be composed of l i n e a r aggregates of f i b r i l s , extend from the periphery of the b a s a l body i n t o the cytoplasm (R, F i g . 47). Each f i b r i l i n the r o o t l e t i s about 120 A° 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 other r o o t l e t s (R, F i g . 48). These r o o t l e t s , 110 mu wide near the base of the f l a g e l l u m , g r a d u a l l y taper and extend p o s t e r i o r l y i n the c e l l . Microtubules (MT, F i g . 47) a l s o are dispersed p e r i p h e r a l l y i n the a n t e r i o r of the swarm c e l l . They appear to terminate near the b a s a l body of the f l a g e l l u m . In f i g u r e 47, there are i n d i c a t i o n s that some of the microtubules 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 a s i n g l e , ovoid nucleus (N, F i g s . 39 - 40). The 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 the swarm c e l l near the f l a g e l l a r b a s a l body. No d i r e c t connection to the b a s a l body from the nucleus has been observed. In the myxamoeba, the nucleus 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 gion of the c e l l . Each ovoid nucleus contains a s i n g l e , e l e c t r o n dense, granular n u c l e o l u s which i s not bound by a membrane and which has one to two n u c l e o l a r vacuoles (Nu, F i g s . 52 - 53). The n u c l e o l a r vacuoles are e l e c t r o n transparent and are not encompassed by a membrane. 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 transparent nucleoplasm and i t appears as c l u s t e r s of e l e c t r o n dense granules ( F i g s . 51 and 54). A nuclear envelope surrounds the nuclear m a t e r i a l . No bleb formation of t h i s 19. envelope i s seen and few pores are v i s i b l e i n the membranes of the envelope. The membranes forming the envelope are about 80 A° t h i c k and the p e r i n u c l e a r space between the two membranes i s about 130 A° wide (NE, F i g . 54). DISCUSSION F u l i g o s e p t i c a spores germinate 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 decoction subsequent to being wetted i n a low percentage b i l e s a l t s o l u t i o n . Other i n v e s t i g a t o r s (Constantineanu, 1906, G i l b e r t , 1927; Scholes, 1962; Smart, 1937) report 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 spores; 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 that a low percentage and a low r a t e of germination i s obtained w i t h spores of F. s e p t i c a under s i m i l a r c o n d i t i o n s . Germination 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 of the same s p e c i e s , as has been observed i n t h i s study. Smart (1937) and others (Constantineanu, 1906; Wilson and Cadman, 1928; Scholes, 1962) have demonstrated that temperature, pH of the medium, age of spores, c o n c e n t r a t i o n of spores, and/or t o x i c sub-stances i n the medium i n h i b i t or r e t a r d the r a t e of germination i n many Myxomycete species. One or more of these f a c t o r s may account f o r the poor r e s u l t s obtained by Cook and H o l t (1928) and f o r the d i f f e r e n t r a t e s of germination f o r two p o r t i o n s of the same c o l l e c t i o n . The p r o t o p l a s t s of F. s e p t i c a are freed from the spore cases i n a manner s i m i l a r to that described by G i l b e r t (1928) and others (Howard, 1931; McManus, 1961). Only the time r e q u i r e d p r i o r to the 20. s p l i t t i n g of the spore case appear to v a r y , w i t h the way i n which the spore case s p l i t s and the swarm c e l l s develop being s i m i l a r . A f t e r the development of 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 outside the mouth of the rupture. I t remains there s e v e r a l minutes before 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 to 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 species ( E l l i o t t , 1949; G i l b e r t , 1928; Howard, 1931), and Kerr (1960) suggests that f l a g e l l a r formation occurs at t h i s time. Contrary to these observations, Smart (1937) s t a t e s that f o r F. s e p t i c a , "In those experiments of the w r i t e r i n which the temperatures between 25°C and 31°C were used, the protoplasm of the spores always began to escape from the spore membrane immediately upon the formation of the aperature and g r a d u a l l y emerged. The s p l i t i n the w a l l progressed as the p r o t o -p l a s t escaped u n t i l the empty spore case showed a rupture equal to one-h a l f or more the diameter of the spore. The p r o t o p l a s t then moved away from the empty spore membrane through amoeboid a l t e r a t i o n s of 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 to be mostly u n i f l a g e l l a t e , but isokont b i f l a g e l l a t e c e l l s are not uncommon. Contrary to these f i n d i n g s , E l l i o t t (1949) and G i l b e r t (1928) have report e d t h a t , i n t h e i r germination s t u d i e s , n e a r l y a l l the swarm c e l l s are heterokont b i -f l a g e l l a t e s . Information on other Myxomycete species provides a p a r t i a l e x p l a n a tion f o r these seeming c o n t r a d i c t i o n s . Cohen (1960 - see Alexopoulos, 1963) has demonstrated that the age of swarm c e l l s , 21. c o n d i t i o n of swarm c e l l s , and species i n f l u e n c e s the percentage of b i -f l a g e l l a t e c e l l s which form. Cohen (1959) and Koevenig (1961) (see Alexopoulos, 1963) both have shown that one or more p s e u d o f l a g e l l a may be mistaken f o r t r u e f l a g e l l a . Kerr (1960) has observed i n Didymium  n i g r i p e s that 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 that some of these c e l l s may 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 hours. Thus, discrepancies i n r e s u l t s could be due to 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 presence of 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 during 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 which remain i n t a c t u n t i l the spores have ruptured to r e l e a s e the p r o t o p l a s t s . At t h i s time, no e l e c t r o n transparent inner l a y e r i s v i s i b l e and only the e l e c t r o n opaque, spin u l o s e outer l a y e r remains. The spore w a l l s s t r u c t u r a l l y resemble the spore w a l l s of D. n i g r i p e s (Schuster, 1964; Wohlfarth-Bottermann, 1959). And, the spines of the w a l l appear to have been formed i n a manner s i m i l a r to that which Schuster (1964) describes f o r D. n i g r i p e s r a t h e r than as Cadman (1931-32) suggests. The l a t t e r author i n d i c a t e s that the protoplasm sh r i n k s and causes the w a l l s to buckle and form ridges on which spines develop. However, there i s no space between the w a l l and the p r o t o p l a s t i n F. s e p t i c a spores which would a l l o w f o r the c o l l a p s e of the spore w a l l . The l i g h t area between the w a l l and 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 space, probably i s an e l e c t r o n transparent inner l a y e r of the w a l l s i m i l a r to the one i n spores of F. s e p t i c a . How the inner l a y e r breaks down 22. a f t e r the spore ruptures i s not known, but s e v e r a l p o s s i b l e explana-t i o n s can be suggested. The inner l a y e r might r a p i d l y d i s s o l v e i n the medium once the spore r u p t u r e s , or the inner l a y e r of the w a l l might be subject to enzymatic degradation p r i o r to the s p l i t t i n g of the w a l l . R e s u lts of McManus (1961) tend to support the l a t t e r p o s s i b i l i t y . She has observed that i n some germinating spores of Clastoderma debaryanum, one s i d e of the spore case g r a d u a l l y becomes l e s s dense and appears to d i s s o l v e . However, the inner l a y e r of the w a l l s remain v i s i b l e i n unruptured spores of F. s e p t i c a . The transparent v e s i c l e s which are not bound by membranes appear to be s i m i l a r to those which have been described i n the plasmodial and spore stages of D. n i g r i p e s , i n plasmodia of D. c l a v u s , Stemonitis f u s c a ,  H e m i t r i c h i a vesparium, Clastoderma debaryanum (Mc Manus, 1965; Schuster, 1964; Wohlfarth-Bottermann, 1959) , and i n many Eumycota (Blondel and T u r i a n , 1960; Hawker and Abbott, 1963a, 1963b; Thyagarajan, C o n t i , and Naylor, 1961, 1962). McManus (1965) s t a t e s that the transparent v e s i c l e s resemble s e c r e t o r y granules as found i n animal gland c e l l s , and Schuster (1964) b e l i e v e s that they have some type of c o n t r a c t i l e vacuolar a c t i v i t y . However, most of these v e s i c l e s c l o s e l y resemble saturated and unsaturated l i p i d d r o p l e t s which are dispersed throughout the cytoplasm. The l i p i d d r o p l e t s then could: "serve as a l o c a l s t o r e of energy and a p o t e n t i a l source of short carbon chains that can be used by the c e l l i n the syn t h e s i s of 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 the 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 products" (Fawcett, 1966). 23. Vacuoles c o n t a i n i n g membrane-like fragments and e l e c t r o n dense granular matter i n the c e l l sap change l i t t l e p r i o r to swarm c e l l and myxamoeba development. However, once swarm c e l l s or myxamoebae are formed, these vacuoles disappear. S i m i l a r appearing o r g a n e l l e s have been described i n other Myxomycetes as food vacuoles c o n t a i n i n g b a c t e r i a l carcasses i n v a r y i n g degrees of d i g e s t i o n (Schuster, 1964; Wohlfarth-Botterman, 1959). These vacuoles a l s o resemble food vacuoles i n some Protozoa ( E l l i o t t and Clemmons, 1966; Mercer, 1959; Schuster, 1963). Lindegren (1962) s t a t e s that yeast c e l l s a l s o possess vacuoles which c o n t a i n mem-branes. However, he b e l i e v e s that these vacuoles are not r e s i d u a l food vacuoles but membrane s y n t h e s i z i n g vacuoles. The synthesized membranes then a r e extruded i n t o the cytoplasm forming endoplasmic r e t i c u l u m of the c e l l . Concurrent w i t h the disappearance of the membrane-containing vacuoles i s the appearance of d e f i n i t e food vacuoles ( F i g . 44). The membrane-bound granular matter i n these vacuoles s t r u c t u r a l l y resembles b a c t e r i a l c e l l protoplasm (Leene and van I t e r s o n , 1965; Schuster, 1963). Thus, i t seems that once the p r o t o p l a s t e x i t s the spore case, i t begins to ingest b a c t e r i a as a food source. C o n t r a c t i l e vacuoles develop at about the same time as food vacuoles, and u s u a l l y one c o n t r a c t i l e vacuole i s present i n each swarm c e l l or myxamoeba. These vacuoles s t r u c t u r a l l y are s i m i l a r to c o n t r a c t i l e vacuoles of some Protozoa (Trager, 1964), and they probably f u n c t i o n to e l i m i n a t e excess water i n a comparable manner. The small v e s i c l e s about the s y s t o l i c vacuole suggest that r u p t u r i n g of the tonoplast has occurred. 24. When the tonoplast fragments, many small v e s i c l e s are formed and the excess water and s o l u b l e wastes are e l i m i n a t e d from the c e l l . Soon a f t e r the vacuole discharges i t s contents, the small v e s i c l e s fuse and excess water and waste products d i f f u s e i n t o the vacuole u n t i l the pressure becomes too great and i t again c o n t r a c t s . No n e p h r i d i a l tubules or endoplasmic r e t i c u l u m appear to tr a n s p o r t s o l u b l e wastes to the c o n t r a c t i l e vacuole as E l l i o t t and Bak (1964) s t a t e occurs i n the 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 the endoplasmic r e t i c u l u m during germination seem to be c o r r e l a t e d c l o s e l y w i t h the changes i n the p h y s i o l o g i c a l a c t i v i t y of the c e l l s : the more a c t i v e the c e l l , the more developed the endoplasmic r e t i c u l u m . No c i s t e r n a l or tubular endoplasmic r e t i c u l u m i s present i n the r e s t i n g spore; only smooth-surfaced v e s i c l e s are v i s i b l e i n the ribosome-dispersed cytoplasm of these c e l l s . Whether the smooth sur-faced v e s i c l e s a c t u a l l y are 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 the m o t i l e c e l l s of some Phycomycetes which form no endoplasmic r e t i c u l u m (Cantino, e t . a l . , 1963), the p r o t o p l a s t s of F. s e p t i c a develop smooth surfaced, 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, the endoplasmic r e t i c u l u m becomes ribosome-coated and appears s i m i l a r to the endoplasmic r e t i c u l u m which Blondel and Turian (1960) describe i n Allomyces macrogynus c e l l s . Thus, i t seems that i n F. s e p t i c a the formation 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 the more a c t i v e s t a t e of the p r o t o p l a s t . The development of ribosome-coated endoplasmic r e t i c u l u m i n swarm c e l l s of F. s e p t i c a does not appear to be the 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 nuclear a s s o c i a t e d v acuoles, and no rough endoplasmic r e t i c u l u m i s seen being extruded i n t o the cytoplasm from these vacuoles, as Lindegren (1962) describes o c c u r r i n g i n yeast c e l l s . The ribosomes i n the cytoplasm p r i o r to swarm c e l l development appear to become attached to the c i s t e r n a l endoplasmic r e t i c u l u m to form ribosome-coated endoplasmic r e t i c u l u m . Dictyosomes, which are not v i s i b l e i n the r e s t i n g spores of F. s e p t i c a , appear to develop c o n c u r r e n t l y w i t h the endoplasmic r e t i c u l u m . Thus, the most organized form of the dictyosomes are seen i n the swarm c e l l s and myxamoebae. From these observations, and from observations of others ( F u l l e r and R e i c h l e , 1965; McManus, 1965), i t appears that both the dictyosomes and the endoplasmic r e t i c u l u m can be broken down and r e -synthesized during the l i f e - c y c l e of the organism. The more me t a b o l i -c a l l y a c t i v e the p r o t o p l a s t , the more s t r u c t u r a l l y organized i s the o r g a n e l l e . The dictyosomes and endoplasmic r e t i c u l u m probably are most a c t i v e during the swarm c e l l and myxamoeba stage because they appear best developed at t h i s time. The dictyosomes always maintain a juxtanuclear 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 McAlear, 1963), and i t suggests that some type of c o n t r o l might be exerted by the nuc-l e u s . However, McManus (1965) reports that i n plasmodia of s e v e r a l Myxomycetes, the dictyosomes are not l o c a l i z e d i n juxtanuclear s i t e s but are s c a t t e r e d throughout the cytoplasm. In swarm c e l l s the d i c t y o -somes probably f u n c t i o n i n the se c r e t o r y processes of the c e l l as they do 26. i n other organisms (Dalton, 1961; Fawcett, 1966). In some Protozoa, the dictyosomes and c o n t r a c t i l e vacuoles are i n c l o s e p r o x i m i t y to each other and share water removal a c t i v i t i e s i n the c e l l (Cohn, 1964). 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 the dictyosomes and c o n t r a c t i l e vacuoles e x i s t s i n swarm c e l l s of F. s e p t i c a . These or g a n e l l e s might f u n c t i o n i n a manner comparable to the dictyosomes i n Protozoa. 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 are s i m i l a r to those i n other Myxomycetes (Dugas and Bath, 1962; McManus, 1965; Schuster, 1965; Wohlfarth-Bottermann, 1959). However, they are u n l i k e those of most other f u n g i . The mitochondria i n Hemiascomycetes (Bandoni, B i s a l p u t r a , B i s a l p u t r a , i n press; Lindegren, 1962; Thyagarajan, et. a l . , 1961), Euascomycetes (Moore and McAlear, 1962, 1963b; Shatkin and Tatum, 1959), some Phycomycetes (Hawker and Abbott, 1963a; Moore and McAlear, 1963b; Cantino, et. a l . , 1963), Basidiomycetes (Bandoni, personal communication; Moore and McAlear, 1963b), and Fungi I m p e r f e c t i (Hawker and Hendy 1963; Thyagarajan, et. a l . , 1962, 1963; W e l l s , 1964) a l l have l a m e l l a r - t y p e c r i s t a e mitochondria. Only a few f u n g i other than Myxomycetes possess mitochondria w i t h tubular-type c r i s t a e (Hawker and Abbott, 1963b; F u l l e r and R e i c h l e , 1965), and i n these forms, the tubules do not branch as they do i n the mitochondria of F. s e p t i c a p r o t o p l a s t s and other Myxomycete plasmodia. A l s o , the m o t i l e c e l l s of F. s e p t i c a possess numerous mitochondria, whereas, the m o t i l e c e l l s of some f u n g i possess only a s i n g l e mitochondrion (Cantino, et. a l . , 1963). The e l e c t r o n dense cores which are v i s i b l e i n the e a r l y stages of germination are probably n u c l e i c a c i d cores which have been shown to be present i n some 27. Myxomycetes (Schuster, 1965). The d i f f e r e n t appearance of these cores during germination may be a r e s u l t of p h y s i o l o g i c a l a l t e r a t i o n s o c c u r r i n g w i t h i n the c e l l . Such changes might i n f l u e n c e , d i r e c t l y or i n d i r e c t l y , the r e a c t i o n of the cores to the f i x a t i v e . 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 studies have been made. Ross (1957) and others (Howard, 1931; McManus, 1961; Smart, 1937) show that 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 p r o t o p l a s t s have been freed from spore cases. Kerr (1960) and Ross (1957) a l s o have demonstrated that each f l a g e l l u m i s anchored i n the c e l l by a b a s a l body. E l e c t r o n micrographs of F. s e p t i c a swarm c e l l s show that the f l a g e l l a have the t y p i c a l 9 + 2 f i b r i l l a r arrangement which c h a r a c t e r i z e s m o t i l e processes i n other f u n g i ( F u l l e r and R e i c h l e , 1965; Kock, 1956, Cantino, et. a l . , 1963; Renaud and S w i f t , 1964) and other organisms (Fawcett, 1966). These f l a g e l l a a r i s e from b a s a l bodies which may or may not be s i m i l a r to basal bodies i n other f u n g i . Of the few f l a g e l l a t e d fungal c e l l s which have been examined by e l e c t r o n microscopy ( F u l l e r and R e i c h l e , 1965; Cantino, et. a l . , 1963; Renaud and S w i f t , 1964), the s t r u c t u r e of the basal body i s s i m i l a r but not i d e n t i c a l . However, u n l i k e some fungal ( B e r l i n and Bowen, 1964) and animal (Fawcett, 1966) c e n t r i o l e s and b a s a l bodies, i n which each of the nine p e r i p h e r a l f i b r i l s i s composed of three s u b - u n i t s , the nine p e r i p h e r a l f i b r i l s of the b a s a l bodies and c e n t r i o l e s i n F. s e p t i c a are made up of two sub-units. As i n other f u n g i (Renaud and S w i f t , 1964), the b a s a l body develops from the proximal c y l i n d e r of the 28. c e n t r i o l e which i s i n a juxtanuclear p o s i t i o n . However, u n l i k e other fungi (Renaud and S w i f t , 1964; 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 bodies of F. s e p t i c a swarm c e l l s i s not maintained during 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 connection 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 , only dietyosome-like v e s i c l e s appear between the b a s a l body and the nucleus. The f u n c t i o n of these dictyosome-like 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 that the v e s i c l e s fuse and form the f l a g e l l a r sheath (Sorokin, 1962). The f u n c t i o n of the r o o t l e t s extending from the b a s a l bodies i s unknown, but i t i s probably that they f u n c t i o n i n support and anchorage of the f l a g e l l a . A s i m i l a r f u n c t i o n may e x i s t f o r the microtubules which r a d i a t e about the base of the f l a g e l l u m . The nucleus undergoes a s e r i e s of changes during germination and a few of these changes have been reporte d i n other Myxomycetes. The ovoid nucleus, which i s c h a r a c t e r i s t i c of the r e s t i n g spore, resembles^ the n u c l e i Dugas and Bath (1962) describe i n Physarum polycephalum Plasmodia. However, w h i l e n u c l e i of P. polycephalum u s u a l l y c o n t a i n as many as four n u c l e o l i , those of F. s e p t i c a possess only a s i n g l e n u c l e o l u s . B l ebs, which may or may not c o n t a i n nuclear materiaL, are seen i n the nuclear envelope of only the r e s t i n g spores. These nucleo-cytoplasmic blebs s t r u c t u r a l l y are s i m i l a r to those i n other organisms (Schuster, 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 able to d i r e c t c e r t a i n cytoplasmic r e a c t i o n s and syntheses i f the blebs should pass 29. i n t o the cytoplasm. The pores i n the nuclear envelope a l s o a l l o w f o r a c e r t a i n amount of nucleo-cytoplasmic exchange. Schuster (1963) sta t e s that Porter (1960) has suggested that such p a r t i c l e s as r i b o s e n u c l e i c a c i d (RNA) might pass through these pores i n t o the cytoplasm. The extreme p l a s t i c i t y of the nucleus, as seen i n F. s e p t i c a spores during germination and i n p r o t o p l a s t s of other Myxomycetes (Dugas and Bath, 1962; McManus, 1965; Locquin, 1949), i n d i c a t e s that there i s i n t e r a c t i o n between the nucleus and the cytoplasm. The more i r r e g u l a r and lobed the nucleus, the greater i s the surface f o r these i n t e r -a c t i o n s to occur. Thus, i t appears that the greatest amount of i n t e r a c t i o n might occur during germination and not i n the r e s t i n g spore and swarm c e l l or myxamoeba stages. The r e s u l t s of t h i s study i n d i c a t e that Myxomycetes, such as F. s e p t i c a , are not c l o s e l y r e l a t e d to true f u n g i . They s t r u c t u r a l l y appear more c l o s e l y r e l a t e d to Protozoa. S i m i l a r i t i e s i n o r g a n e l l e s t r u c t u r e tend to support t h i s i d e a . Myxomycetes and most Protozoa 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 feeding h a b i t s of the two groups are s i m i l a r , and n e i t h e r group possesses a w a l l during most of t h e i r l i f e - c y c l e . However, the presence of a w a l l during the spore stage and the c e n t r i o l e and b a s a l body sub-structure 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 Approximately 90 to 95 % of the spores of F. s e p t i c a germinate a f t e r 17 to 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 decoction 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 s a l t s o l u t i o n . Spores which have been sown i n l e a f - e x t r a c t decoction germinate s l i g h t l y sooner than spores which have been sown i n carbon-f i l t e r e d , d i s t i l l e d water. An ovoid nucleus 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 , mitochondria w i t h t u b u l a r - t y p e c r i s t a e , vacuoles 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 tting the spores, the nucleus i s very i r -r e g u l a r i n shape and many small v e s i c l e s are found i n the cytoplasm. Small amounts of 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 cytoplasm. Before the spore ruptures to r e l e a s e t h e i r p r o t o p l a s t s , 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 nucleus becomes lobed; and more smooth, c i s t e r n a l endo-plasmic r e t i c u l u m develops. The proximal c y l i n d e r s of the c e n t r i o l e s develop i n t o b a s a l bodies s h o r t l y before the s p l i t t i n g of the spore cases. With the r u p t u r i n g of the spore case, the inner l a y e r of the w a l l disappears. Simultaneously, the p r o t o p l a s t s escape. They r e s t 31. a short time near the mouth of the rupture and then develop i n t o f l a g e l l a t e d swarm c e l l s or myxamoebae. 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Edinburgh 55: part 3. number 24. Wohlfarth-Bottermann, K. E. 1959. G e s t a t t e t das elektronenmikroskopische b i l d aussagen zur dynamik i n der z e l l e ? . Z e i t . f ur Z e l l f o r s c h . 50: 1 - 2 7 . Yuasa, A. 1935. On the s c l e r o t i u m of 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. F i g s . 2 - 5 . Emergence of 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. Released p r o t o p l a s t assumes s p h e r i c a l shape outside mouth of rupture. X 1,900. PLATE 2 F i g . 7. S p h e r i c a l p r o t o p l a s t i n o s c i l l a t o r y s t a t e outside mouth of rupture. X 1,900. F i g . 8. B i f l a g e l l a t e d swarm c e l l . X 1,600. F i g . 9. U n i f l a g e l l a t e d swarm c e l l . X 1,600. F i g . 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 1,150. • ® 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 small membrane-bound v e s i c l e s . W = w a l l , PM = plasma membrane, M = mitochondrion, 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. Spinulose spore w a l l . OW = e l e c t r o n dense outer l a y e r , IW = e l e c t r o n transparent inner 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. Vacuoles c o n t a i n i n g membrane-like fragments and e l e c t r o n dense granular matter i n c e l l sap. Phos-phate b u f f e r e d OsO^ f i x a t i o n . X 37,150. F i g . 14. Transparent v e s i c l e s . Note absence of 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 tubular-type 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 branch forming other c r i s t a e w i t h i n the mitochondrion. M = mitochondrion, TVe = transparent v e s i c l e , V = vacuole. Phosphate bu f f e r e d OsO^ f i x a t i o n . X 43,900. F i g . 16. Mitochondrion w i t h e l e c t r o n dense, granular core i n the matrix. M = mitochondrion, PM = plasma membrane, IW = inner l a y e r of w a l l , OW = outer 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 127,700. PLATE 6 F i g . 17. Nucleus. NE = nuclear envelope, 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 36,650. F i g . 18. Nuclear envelope showing one type of bleb commonly observed i n the r e s t i n g spore stage. Note simple pore i n envelope. NB = nuclear b l e b , N = nuclear 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. F i g . 19. Second type of nuclear bleb observed i n the r e s t i n g spore stage nucleus. Note simple pores i n envelope. NB = nuclear b l e b , P = pore. Phosphate b u f f e r e d OsO, f i x a t i o n . X 39,650. PLATE 7 F i g . 20. Nucleus. Note number of simple pores i n envelope and membrane s t r u c t u r e of envelope. NE = nuclear envelope, P = pore, N = nuclear m a t e r i a l , Nu = nuc l e o l u s . KMnO. f i x a t i o n . X 37,500. 4 F i g . 21. Section of the nuclear envelope showing the two u n i t membranes of the envelope. Note the simple pores. NE = nuclear envelope, P = pores, N = nuclear 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. Cr o s s - s e c t i o n of a spore s i x hours a f t e r wetting. Note nuclear shape and number of small v e s i c l e s i n the cytoplasm. W = w a l l , M = mitochondrion, TVe = transparent v e s i c l e , N = nuclear m a t e r i a l . Phosphate buff 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 spores. 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 the nucleus. 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 56,000. PLATE 10 F i g . 24. Mitochondrion of spore i n stage two. Note f i b r i l l a r appearance of core. 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 of spore s i x hours a f t e r the beginning of germination. Note the number of s m a l l , membrane-bound v e s i c l e s and the small amount of c i s t e r n a l endoplasmic r e t i c u l u m . TVe = transparent v e s i c l e , M = mitochondrion. 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. Section through a spore twelve hours a f t e r wetting. The nucleus i s not shown i n t h i s section. OW = outer layer of w a l l , IW = inner layer of w a l l , PM = plasma membrane, V = vacuole, M = mitochondrion. Phosphate buffered OsO. f i x a t i o n . X 26,600. PLATE 12 F i g . 27. Lobed nucleus c h a r a c t e r i s t i c of spores twelve hours a f t e r the beginning of germination. Note c i s t e r n a l endoplasmic r e t i c u l u m about the nucleus. ER = endo-plasmic r e t i c u l u m , NE = nuclear envelope, N = nuclear m a t e r i a l , Nu = n u c l e o l u s , M = mitochondrion. Phos-phate 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. Centrio l e adjacent to nucleus. Cylinders of the c e n t r i o l e are open at both ends. N = nuclear m a t e r i a l , Ce = c e n t r i o l e . Phosphate buffered OsO^ f i x a t i o n . X 72,850. Fig . 29. Centriole at a greater magnification. Ce = c e n t r i o l e . Phosphate buffered OsO, f i x a t i o n . X 133,575. PLATE 14 Fig . 30. Two dictyosomes. i n close proximity to nucleus. D = dictyosome, N = nuclear m a t e r i a l , Nu = nucleolus. Phosphate buffered OsO, f i x a t i o n . X 111,100. Fig . 31. Dictyosome at higher magnification. Each dictyo-some i s composed of three to four cisternae l y i n g p a r a l l e l to one another. D = dictyosome, N = nuclear material. Phosphate buffered OsO, f i x a -t i o n . X 156,950. PLATE 15 Fig . 32. Spore p r i o r to protoplast emergence. W = w a l l , PM = plasma membrane, TVe = transparent v e s i c l e , M = mito-chondrion, ER = endoplasmic reticulum, V = vacuole, N = nuclear material. Phosphate buffered OsO. f i x a -t i o n . X 41,400. PLATE 16 F i g . 33-A. Section through a spore p r i o r to protoplast emergence. Note that the basal body i s present i n the c e n t r a l region of the spore and i n a juxtanuclear p o s i t i o n . BB = basal body, N = nuclear m a t e r i a l , TVe = trans-parent v e s i c l e , M = mitochondrion, W = w a l l . Phos-phate buffered OsO^ f i x a t i o n . X 22,000. Fi g . 33-B. Cross-section of a basal body at high magnification. Note that r o o t l e t s extend from periphery of basal body. BB = basal body, R = r o o t l e t s . Phosphate buf-fered OsO^ f i x a t i o n . X 85,550. Fi g . 34. Longitudinal section through proximal c y l i n d e r of a basal body. Note that a d i f f e r e n t type of r o o t l e t than seen i n figure 33-B extends into the cytoplasm from the basal body periphery. BB = basal body, R = r o o t l e t s . Phosphate buffered OsO^ f i x a t i o n . X 49,950. F i g . 35. Cross-section through proximal c y l i n d e r of the basal body. Note that the r o o t l e t s radiate from the basal 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 = basal body, R = r o o t l e t s . Phosphate buffered OsO^ f i x a t i o n . X 59,250. PLATE 17 F i g . 36. Mitochondrion t y p i c a l of spores p r i o r to p r o t o p l a s t emergence. Membranes of mitochondrion appear d i s t i n c t and the "core" r e g i o n l e s s e l e c t r o n dense than the 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 core r e g i o n . M = mitochondrion, V = vacuole, Phosphate buff e r e d OsO. f i x a t i o n . X 83,100. F i g . 37. 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 to vacuoles 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 = vacuole. Phosphate b u f f e r e d OsO. f i x a t i o n . X 79,200. PLATE 18 Sect i o n through emerging p r o t o p l a s t . Note absence of inner l a y e r of spore w a l l . The nucleus has r e v e r t e d to ovoid shape and i s i n the.cytoplasm which f i r s t e x i t s the spore case. OW = outer l a y e r of w a l l , M = mito-chondrion, N = nuclear 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 F i g , 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 bas a l body of the f l a g e l l u m i s present i n the an-t e r i o r of the c e l l . Microtubules 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 vacuole i s seen i n the p o s t e r i o r of the 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 the surface of the endoplasmic r e t i -culum. PM = plasma membrane, BB = b a s a l body, MT = microtubules, N = nuclear m a t e r i a l , M = mito-chondrion, CV = c o n t r a c t i l e vacuole, RER = r i b o -some coated endoplasmic r e t i c u l u m . Phosphate buf-fered OsO. f i x a t i o n . X 33,750. PLATE 20 F i g . 40. Myxamoeba. Note the ovoid shape of the nucleus and the presence of a c o n t r a c t i l e vacuole. PM = plasma membrane, CV = c o n t r a c t i l e vacuole, M = mitochondrion, RER = ribosome-coated endoplasmic r e t i c u l u m , N = nuclear 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 . In upper part 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 to be completely i n t a c t . In the lower part of the micrograph, the spore case has ruptured r e -l e a s i n g the p r o t o p l a s t and only the outer l a y e r of the w a l l i s v i s i b l e . OW = outer l a y e r of w a l l , IW = inner 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 that only the spinulose outer l a y e r of the w a l l remains. OW = outer l a y e r of w a l l . Phosphate buf-f e r e d OsO^ f i x a t i o n . X 88,900. F i g . 43. Ribosomes present on outer surface of c i s t e r n a l endo-plasmic r e t i c u l u m . RER = ribosome-coated endoplasmic r e t i c u l u m , 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 F i g . 44. Vacuoles c o n t a i n i n g e l e c t r o n dense granular matter. The granular matter 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. F i g . 45. C o n t r a c t i l e vacuole i n expanded s t a t e . CV = c o n t r a c t i l e vacuole, RER = ribosome-coated 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 i n c c o n t r a c t e d s t a t e . A l s o , 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 nucleus. N = nuclear m a t e r i a l , D = dictyosome, Ve = v e s i c l e , CV = c o n t r a c t i l e vacuole. Phosphate b u f f e r e d OsO, f i x a t i o n . X 108,250. PLATE 24 F i g . 47. C r o s s - s e c t i o n through the b a s a l body of the f l a g e l -lum. The b a s a l body i s composed of nine 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 sub-units. Extending from the p e r i p h e r a l area of the b a s a l body are r o o t l e t s which are 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 poster-i o r l y i n the c e l l . PM = plasma membrane, MT = micro-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. F i g . 48. L o n g i t u d i n a l s e c t i o n through the a n t e r i o r of a swarm c e l l . R o o t l e t s and dictyosome-like v e s i c l e s are present i n t h i s area. 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. F i g . 49. C r o s s - s e c t i o n through 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 = nuclear mater-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 37,500. F i g . 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 which are present between the nine 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 nucleus of swarm c e l l s and myxamoebae. The nuclear m a t e r i a l i s surrounded by a d i s t i n c t nuclear envelope. A s i n g l e nucleolus i s present i n each nucleus. NE = nuclear envelope, N = nuclear mater-i 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 vacuoles. No membrane encompasses the n u c l e a l a r vacuoles. Nu = nuc 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 62,475. * F i g . 54. Nuclear envelope at higher m a g n i f i c a t i o n . Note the two u n i t membranes forming the envelope. N = nuclear m a t e r i a l , NE = nuclear envelope, RER = ribosome-coated 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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