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An ultrastructural study of Peridinium trochoideum with special reference to the theca and its formation Kalley, John Peter 1971

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AN ULTRASTRUCTURAL STUDY OF PERIDINIUM TROCHOIDEUM WITH SPECIAL REFERENCE TO THE THECA AND ITS FORMATION by John Peter K a l l e y B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY, 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of • g x o L Q 6V  The University of British Columbia Vancouver 8, Canada Date H vfouy .t'HI i i ABSTRACT Freeze-etching and t h i n s e c t i o n i n g were used to examine the f i n e s t r u c t u r e of the marine d i n o f l a g e l l a t e Peridinium trochoideum (Stein) Lemm. Among the cytoplasmic i n c l u s i o n s described were three t y p i c a l d i n o f l a g e l l a t e o r g a n e l l e s : a di n o c a r y o t i c nucleus with condensed interphase chromosomes, c h l o r o p l a s t s with thylakoids associated i n groups of three, and t r i c h o c y s t s contained i n membranous sacs. In ad d i t i o n to the above, dictyosomes, cytoplasmic membrane systems, f i b r o u s bodies and 'segregated bodies' were observed and described. Upon examining the gross morphology of the theca, i t was found that t h e c a l age could be determined by the extensiveness of sutures, p i t s , and b l i s t e r s which became predominant on the external t h e c a l membranes with age. C h a r a c t e r i s t i c a l l y , older c e l l s sometimes had continuous, deeply scored sutures with adjacent 'marginal suture bands' and i n t e r -c a l a r y bands. I n d i v i d u a l t h e c a l p l a t e s of mature c e l l s were not com-p l e t e l y enclosed w i t h i n membrane sacs as commonly assumed and some adjacent p l a t e s were found to be continuous. Four membrane systems were found to be associated with the t h e c a l p l a t e s : the t h e c a l membrane and outer p l a t e membrane systems l a y above the p l a t e s , the inner p l a t e membrane and plasmalemma l a y below. Sutures were formed by i n f o l d i n g of the outer p l a t e membrane between adjacent p l a t e s . With the exception of the plasmalemma, the membranes associated with the p l a t e s displayed membrane asymmetry. In the mature, thecate,non-dividing c e l l s , densely s t a i n i n g i n c l u s i o n s termed 'pro-thecal bodies' were found to be d i s t r i b u t e d through-out the cytoplasm. Before ecdysis, each amorphous prothecal body t r a n s -formed i n t o many v e s i c l e s each of which contained f i b r o u s m a t e r i a l i n an e l e c t r o n transparent matrix. I t appeared that the vast number of v e s i c l e s i i i so formed may have increased the c e l l ' s osmotic pressure enough to i n i t i a t e ecdysis. At ecdysis, the t h e c a l p l a t e s and o v e r l y i n g mem-branes were l o s t and the new w a l l was formed by deposition of material from prothecal bodies at the p r o t o p l a s t surface. The newly formed w a l l was continuous over the p r o t o p l a s t and no p l a t e s e x i s t e d as such. Pores, however, were present. The sutures, when f i r s t formed were shallow and discontinuous. TABLE OF CONTENTS page LIST OF PLATES AND FIGURES V ACKNOWLEDGEMENTS V±±± / INTRODUCTION 1 METHODS AND MATERIALS i+ OBSERVATIONS ULTRASTRUCTURE OF THE CYTOPLASM AND ITS INCLUSIONS 6 ULTRASTRUCTURE OF THE THECA 9 ULTRASTRUCTURE OF THE THECAL PLATES AND ASSOCIATED MEMBRANES ... I 2 ULTRASTRUCTURE OF WALL FORMATION 15 DISCUSSION CYTOPLASM AND ORGANELLES 19 THECAL MORPHOLOGY 2 2 PLATES AND ASSOCIATED MEMBRANES • • • • 2 3 FORMATION OF THE WALL 2 ^ PLATES AND EXPLANATIONS • • »• • ° • 30 V LIST OF PLATES AND FIGURES EXPLANATION OF FIGURES page PLATE I f i g u r e 1 f i g u r e 2 f i g u r e 3 Chloroplast . c h l o r o p l a s t s sparse stroma thylakoids 31 PLATE I I f i g u r e 4 f i g u r e 5 Nucleus interphase nucleus chromosomes 32 PLATE I I I f i g u r e 6 f i g u r e 7 Nucleus nucleolus nuclear membrane 33 PLATE IV f i g u r e 8 f i g u r e 9 f i g u r e 10 f i g u r e 11 Fibrous Bodies p e r i n u c l e a r extension p e r i n u c l e a r f i b r o u s body pe r i n u c l e a r f i b r o u s body cytoplasmic f i b r o u s body 3^ PLATE V f i g u r e 12 f i g u r e 13 f i g u r e 14 f i g u r e 15 Dictyosomes and Segregated Bodies ... dictyosome, o r i e n t a t i o n and l o c a t i o n dictyosome segregated body, l o c a t i o n segregated body 35 PLATE VI f i g u r e 16 f i g u r e 17 f i g u r e 18 f i g u r e 19 f i g u r e 20 f i g u r e 21 PLATE VII f i g u r e 22 f i g u r e 23 f i g u r e 24 f igure 25 f igure 26 f i g u r e 27 Tri c h o c y s t s t r i c h o c y s t s , cross s e c t i o n t r i c h o c y s t s , l o n g i t u d i n a l section t r i c h o c y s t , freeze-etching pore plug, freeze-etching pore plug, t h i n s e c t i o n i n g pores, freeze-etching 36 L i f e Cycle , mature thecate c e l l ecdysis naked f l a g e l l a t e stage r e s t i n g stage c y t o k i n e s i s immature thecate daughter c e l l 37 EXPLANATION OF FIGURES v i page PLATE VIII Morphology of the Theca ... • 38 f i g u r e 28 young theca f i g u r e 29 theca, Nomarski.- i n t e r f e r e n c e f i g u r e 30 theca, phase contrast PLATE IX Morphology of the Theca 39 f i g u r e 31 olde r theca, i n t e r c a l a r y band PLATE X Morphology of the Theca ^0 f i g u r e 32 b l i s t e r s and p i t t i n g f i g u r e 33 suture and marginal suture band PLATE XI Thecal Plates and Membranes • • ^1 f i g u r e 34 plaques and sutures f i g u r e 35 marginal suture band f i g u r e 36 p l a t e - membrane ass o c i a t i o n s f i g u r e 37 p l a t e - membrane ass o c i a t i o n s PLATE XII Thecal Plates and Membranes ^2 f i g u r e 38 p l a t e - membrane ass o c i a t i o n s f i g u r e 39 suture c o n t i n u i t y f i g u r e 40 suture d i s c o n t i n u i t y PLATE XIII Thecal Plates and Membranes ^3 f i g u r e 41 membrane ass o c i a t i o n s and asymmetry f i g u r e 42 the four membrane systems f i g u r e 43 c o n t i n u i t y of the inner p l a t e membrane PLATE XIV Thecal Plates and Membranes ^+ f i g u r e 44 inner p l a t e membrane f i g u r e 45 scars on the inner p l a t e membrane PLATE XV Prothecal Body ^5 f i g u r e 46 young, mature c e l l f i g u r e 47 pre-ecdysis f i g u r e 48 pre-ecdysis f i g u r e 49 pre-ecdysis/ecdysis v i i EXPLANATION OF FIGURES page PLATE XVI Prothecal V e s i c l e s ^6 f i g u r e 50 pre-ecdysis f i g u r e 51 pre-ecdysis f i g u r e 52 prothecal v e s i c l e s at w a l l f i g u r e 53 prothecal v e s i c l e s i n w a l l PLATE XVII Cytokinesis • ^7 f i g u r e 54 e a r l y c y t o k i n e s i s , Nomarski i n t e r f e r e n c e f i g u r e 55 e a r l y c y t o k i n e s i s , phase contrast f i g u r e 56 e a r l y c y t o k i n e s i s , e l e c t r o n microscopy PLATE XVIII Cytokinesis ^ 8 f i g u r e 57 pr o t h e c a l v e s i c l e formation at c e l l isthmus f i g u r e 58 v e s i c u l a r separation of daughter c e l l s PLATE XIX Cytokinesis ^9 f i g u r e 59 new w a l l at isthmus f i g u r e 60 new inner p l a t e membrane PLATE XX Immature Daughter C e l l s 50 f i g u r e 61 daughter c e l l s , u n d i f f e r e n t i a t e d thecae PLATE XXI Immature Daughter C e l l s 51 f i g u r e 62 pore i n u n d i f f e r e n t i a t e d theca f i g u r e 63 appearance of sutures ACKNOWLEDGEMENTS I am indebted to Dr. T. B i s a l p u t r a f o r h i s i n s t r u c t i o n and guidance during the duration of t h i s study and f o r h i s assistance i n preparing t h i s t h e s i s . I would a l s o l i k e t o thank Dr. F.J.R. Taylor f o r h i s h e l p f u l comments and suggestions throughout the p r o j e c t . INTRODUCTION For the most part, c l a s s i f i c a t i o n of thecate d i n o f l a g e l l a t e s i s based on the p a r t i c u l a r shape and arrangement of c e l l w a l l p l a t e s which form the theca*of a given genus or species. Since non-thecate forms cannot be c l a s s i f i e d on the basis of t h e c a l morphology, they are u s u a l l y c l a s s i f i e d according to c e l l shape. Recently, Dodge and Crawford (19) "have est a b l i s h e d a p o t e n t i a l l y valuable system f o r the u l t r a s t r u c t u r a l c a t e g o r i z a t i o n of d i n o f l a g e l l a t e c e l l surface l a y e r s . Thus non-thecate and thecate forms can be c l a s s i -f i e d u l t r a s t r u c t u r a l l y using the same comparative system. The simplest degree of organization e x i s t s i n c e l l s which possess a s i n g l e l a y e r of f l a t t e n e d v e s i c l e s at the c e l l surface. Progressive degrees of organiza-t i o n are based p r i m a r i l y on the a c q u i s i t i o n of rudimentary pla t e s within the v e s i c l e s and subsequent thickening and elaboration of such p l a t e s i n h e a v i l y thecate forms. Their survey shows that when p l a t e s occur, each p l a t e i s f u l l y enclosed by a membrane sac. I f one assumes that thecate forms arose from non-thecate forms, the most obvious sequence would appear to be v i a the establishment of w a l l m a t e r i a l w i t h i n surface v e s i c l e s s i m i l a r to those found i n Amphidinium (17) or Oxyrrhis (19). In other words, a phylogenetic sequence of p l a t e development may be r e f l e c t e d by the phenetic sequence of t h e c a l structure beginning from the simple non-thecate forms to the elaborate thecate forms. This premise i s based on the assumption that p l a t e s of the thecate forms are enclosed by membrane sacs or v e s i c l e s . Although one might expect to * The term theca i s \ised to describe both the thecal plates and the associated membranes collectively. 2. be able to f o l l o w the development of such p l a t e s within these v e s i c l e s , to date there i s no e l e c t r o n microscopic evidence of such t h e c a l onto-geny. On the contrary, there are reports which describe young thecate d i n o f l a g e l l a t e s as having continuous, u n d i f f e r e n t i a t e d walls (7.25). To complicate the matter, the sequence of events leading to daughter c e l l formation i n d i n o f l a g e l l a t e s can be q u i t e v a r i a b l e . In a few the-cate d i n o f l a g e l l a t e s an e c d y s i a l stage may occur whereby the theca i s l o s t e i t h e r p r i o r to or a f t e r c y t o k i n e s i s . For instance, i n Peridinium w e s t i i (35) cy t o k i n e s i s takes place i n s i d e the parent theca p r i o r to ecdysis, whereas i n Gyrodinium c o h n i i (Crypthecodinium cohnii) (25) and Gonyaulax polyedra (22) cy t o k i n e s i s occurs w i t h i n a c y s t a f t e r ecdysis. Forms such as Pyrodinium bahamense (7) and Peridinium triquetrum (6) do not undergo ecdysis. Instead, each of the daughter c e l l s r e t a i n s one-half of the parent theca and subsequently reforms the missing h a l f . The l a t t e r i s most common w i t h i n the group. As Braarud (6) has shown, the theca of Peridinium trochoideum (Stein) Lemm. i s l o s t completely p r i o r to cy t o k i n e s i s and each of the daughter c e l l s must form an e n t i r e l y new theca. When l i v i n g m a t e r i a l of P.  trochoideum i s observed, i t becomes apparent that c e l l w a l l formation takes place r a p i d l y . This would suggest that a great deal of w a l l m a t e r i a l or i t s precursors are probably synthesized and stored p r i o r to cy t o k i n e s i s . With l i g h t microscopy, i t i s d i f f i c u l t to follow the development of the theca. The cl o s e a s s o c i a t i o n of the newly formed w a l l m a t e r i a l on the plasma membrane with the underlying cytoplasmic matrix and i n -c l u s i o n s obscures the d e l i c a t e d e t a i l s of p l a t e d i f f e r e n t i a t i o n . Examina-t i o n of thecae of f u l l y developed c e l l s i n s i t u i s a l s o d i f f i c u l t f o r the 3. same reason and therefore, gross d e t a i l s of the p l a t e s , t h e i r sutures, and pores have had to be determined from d i s s o c i a t e d thecae, often stained. Thecal s t r u c t u r e has been used as an important c r i t e r i o n i n the recog-n i t i o n of species i n the Dinophyceae. Consequently, the added d e t a i l provided by the e l e c t r o n microscope should r e s u l t i n a more complete understanding of t h i s important d i n o f l a g e l l a t e s t r u c t u r e . E l e c t r o n microscopic studies on d i n o f l a g e l l a t e c e l l walls have i n the past been accomplished by shadow-casting (14), negative s t a i n i n g (31), or t h i n s e c t i o n i n g techniques (26). Adequate assessment of the i n vivo topography of the t h e c a l p l a t e s i s d i f f i c u l t to obtain using these techniques. To obtain a three-dimensional representation of the c e l l w a l l from e l e c t r o n micrographs of t h i n sections, a large number of s e r i a l sections must be taken from both t a n g e n t i a l and c r o s s - s e c t i o n a l planes through the theca. Shadow-casting and negative s t a i n i n g tech-niques require chemical cleansing and drying of the w a l l which may destroy or d i s t o r t some of the three-dimensional features and d e t a i l s . In t h i s study, freeze-etching and t h i n sectioning techniques were used to e l u c i d a t e the f i n e s t r u c t u r e and formation of the theca of the marine d i n o f l a g e l l a t e P. trochoideum (Stein) Lemm. Freeze-etching enables three-dimensional r e l i e f s to be obtained from l i v i n g or f i x e d m a t e r i a l using p h y s i c a l rather than chemical methods (32). 4. Mater i a l s and Methods Peridinium trochoideum (Stein) Lemm. was obtained from the Culture C o l l e c t i o n of Algae, Indiana U n i v e r s i t y ( C o l l e c t i o n Number LB 1017) and was grown i n Chihara medium (10) supplemented with soil-water extract. Cultures were maintained at 20-25°C. and were i l l u m i n a t e d with f l u o r e s c e n t l i g h t f o r 16 hrs. per 24 hr. period. The majority of m a t e r i a l f o r freeze-etching was frozen d i r e c t l y i n the c u l t u r e medium. However, some mat e r i a l was f i x e d f o r 1 hr. i n 2.5% glutaraldehyde buffered with sodium cacodylate (pH. 6.8) p r i o r to f r e e z -in g . The freeze-etching technique was s i m i l a r to that described by Moor (32) and was performed on a Balzers BA 360M Freeze-Etching u n i t . Freezing was accomplished by p l a c i n g a suspension of ma t e r i a l i n a 3 mm. gold support cup, immersing the cup i n l i q u i d Freon 22 (cooled i n l i q u i d n i t -rogen) and then q u i c k l y t r a n s f e r r i n g i t to l i q u i d nitrogen. Deep f r a c t u r -i n g was achieved by taking low speed cuts across the m a t e r i a l . Cutting and sublimation were performed at -100°C. and sublimation time was 1 min. at 3 x 10 -^ Torr. pressure. The etched material was shadowed with platinum/carbon and subsequently strengthened by carbon evaporation. The r e p l i c a was released d i r e c t l y i n 70% sulphuric a c i d and a f t e r 1 hr., i t was t r a n s f e r r e d to d i s t i l l e d water. The r e p l i c a was then placed i n sodium hypochlorite f o r a f u r t h e r 1 hr. to remove remaining debris. A f t e r thorough washing i n d i s t i l l e d water, the r e p l i c a was picked up and mounted on a Formvar coated g r i d . M a t e r i a l f o r t h i n sectioning was f i x e d i n one of two ways: (a) f o r 1 hr. i n a combination of 2.5% glutaraldehyde and 2.5% formaldehyde (from paraformaldehyde) i n 0.1M sodium cacodylate b u f f e r , pH 6.8. 2% sucrose (w/v) was added to adjust the osmolarity of the f i x a t i v e t o that of the growth medium; (b) f o r 1 hr. at 4°C. with 2.5% g l u t a r a l d e -hyde i n 0.1M sodium cacodylate b u f f e r , pH 6.8. 10% DCMU (v/v) was added to the f i x a t i v e to i n h i b i t oxygen evolution during f i x a t i o n . A f t e r p o s t - f i x a t i o n with 1% 0s04 i n cacodylate b u f f e r f o r 1 hr., the c e l l s were concentrated by gentle c e n t r i f u g a t i o n and embedded i n 1% agar (w/v). The agar-embedded c e l l s were dehydrated using a graded s e r i e s of ethanol and i n f i l t r a t e d with i n c r e a s i n g concentrations of Spurr's embedding medium (40) i n ethanol or Maraglas (4) i n propylene oxide. F i n a l l y , the agar-embedded c e l l s were cured i n 100% r e s i n . 'Sections were cut on a P o r t e r -Blum MT-1 Ultramicrotome using a du-Pont diamond k n i f e and were post stained with saturated uranyl acetate i n 70% methanol and lead c i t r a t e (36). Both sections and r e p l i c a s were observed with a Zeiss EM 9A e l e c t r o n microscope. Wall m a t e r i a l f o r l i g h t microscopy was obtained by c o l l e c t i n g thecae shed from c e l l s which had been l e f t i n hypotonic d i s t i l l e d water over-night. Micrographs of thecae and l i v i n g m a t e r i a l were obtained using a Z e i s s Photomicroscope with phase contrast or Nomarski i n t e r f e r e n c e i l l u m i n a t i o n systems*-6. OBSERVATIONS ULTRASTRUCTURE OF THE CYTOPLASM AND ITS INCLUSIONS Among the most common cytoplasmic i n c l u s i o n s observed with the el e c t r o n microscope i n c e l l s of P. trochoideum are c h l o r o p l a s t s (C, f i g . 1), starc h grains (SG, f i g . 4), n u c l e i (N, f i g . 4), pe r i n u c l e a r f i b r o u s bodies (PFB, f i g . 9), cytoplasmic f i b r o u s bodies (CFB, f i g . 11), dictyosomes (D, f i g . 12), 1 segregated bodies' (SB, f i g . 14), mitochondria (M, f i g . 14), and t r i c h o c y s t s (T, f i g . 16). Within the c h l o r o p l a s t s of P. trochoideum i t i s p o s s i b l e t o d i s t i n -guish the associated thylakoids (TL) and two d i s t i n c t stromal regions -the dense stroma (DS) and sparse stroma (SS) ( f i g . 1). The dense stroma appears to occupy the regions between t h y l a k o i d lamellae ( f i g . 1) while the sparse stroma occupies the c e n t r a l region of the c h l o r o p l a s t ( f i g . 2). P a r t i c u l a t e components of both stromal types occur i n equal density. I t appears that the sparse^stroma i s l e s s densely stained because i t lacks a moderately dense amorphous component which i s prevalent i n the dense stroma. F i b r i l s (F, f i g . 2) s i m i l a r to DNA f i b r i l s of ch l o r o p l a s t s (2, 38) and mitochondria (3) al s o e x i s t i n the sparse stromal region. In preliminary f l u o r e s c e n t studies, c h l o r o p l a s t s of P. trochoideum showed a p o s i t i v e fluorescence f o r DNA. As expected, the thylakoids of P. trochoideum c h l o r o p l a s t s are associated i n c l o s e l y appressed groups of three ( f i g . 3). This i s s i m i l a r t o other d i n o f l a g e l l a t e p l a s t i d s (16). The extent of the a s s o c i a t i o n , however, i s not constant and i t i s not uncommon to see pseudo-grana of various s i z e s w i t h i n a given length of th y l a k o i d surface. D i s c o n t i n u i t i e s of s i n g l e t h y l a k o i d lamellae are marked i n f i g u r e 3 by arrows. Dodge has made s i m i l a r observations i n Aureodinium pigmentosum (15). The nucleus (N, f i g . 4) i s a t y p i c a l d i n ocaryotic type with nucleolus (NU), double membrane envelope (NE), and interphase chromo-somes which remain condensed and c o i l e d . Within the chromosomes ( f i g . 5) i t i s p o s s i b l e to detect two d i r e c t i o n s of c o i l i n g , ( c o i l s p a r a l l e l to the chromosomal l o n g i t u d i n a l axis are marked by black arrows, c o i l s normal t o the l o n g i t u d i n a l axis are marked by white arrows). The chromatin of the chromosomes r e a d i l y resembles the sparse stroma of c h l o r o p l a s t s (cf. f i g s . 2 and 6). The nucleolus (NU, f i g . 6) appears to contain equal amounts of f i b r o u s and granular m a t e r i a l . Except f o r small n u c l e o l a r 'vacuoles' (arrows, f i g . 6) the s t a i n i n g density i s r e l a t i v e l y uniform. Although the nuclear envelope has never been observed to t o t a l l y break down, there are stages when the envelope has many large gaps ( f i g . 7). At such times, the nucleus i s surrounded by many membranous v e s i c l e s (NV, f i g . 7) which might have a r i s e n from the nuclear envelope, and/or may contribute to i t s completion. An unusual f e a t u r e of the nucleus i s the large d i l a t i o n s of the p e r i n u c l e a r cisternum (PE, f i g . 8) wi t h i n which i s sometimes found a p e r i n u c l e a r f i b r o u s body (PFB, f i g s . 9 and 10). At a po i n t where the nuclear envelope i s expanded (arrow, f i g . 9), the inner nuclear membrane (INM) remains around the nucleoplasm while the outer nuclear membrane (ONM) expands to accommodate the f i b r o u s body and i t s granular matrix. 8. In other instances f i b r o u s bodies occur w i t h i n membrane bound i n c l u s i o n s i n the cytoplasm (CFB, f i g s . 11, 13, and 14). Both the nuclear and cytoplasmic f i b r o u s bodies appear i d e n t i c a l to f i b r o u s bodies found i n Peridinium w e s t i i (31), Woloszynskia micra (26), and Symbiodinium microadriaticum (23). Their o r i g i n , and f u n c t i o n are un-known. In c l o s e proximity to the nucleus l i e two other organelles. Dictyosomes are u s u a l l y found i n the juxtanuclear cytoplasm o r i e n t a t e d with the d i s t a l f a c e (secreting face) towards the nucleus (D, f i g s . 12 and 13). Neither the dictyosome nor the extensive membrane system often located with i t appear to be d i r e c t l y involved i n any r e a d i l y recognizable or detectable f u n c t i o n during w a l l formation. The second s t r u c t u r e associated with the nucleus has been termed a 'segregated body' (SB, f i g . 14). I t i s always lo c a t e d immediately below the nucleus at the a n t a p i c a l pole of the c e l l . There i s no apparent de-l i m i t i n g membrane surrounding i t , yet i t i s d i s t i n c t l y segregated from the cytoplasm and not an extension of i t ( f i g . 15). I t contains f i b r o u s m a t e r i a l (FB) s i m i l a r to that observed i n nuclear and c y t o -plasmic f i b r o u s bodies and s p h e r i c a l globules (GL), the periphery of which st a i n s denser than the center. Both f i b r o u s bodies and globules are contained within an amorphous matrix which appears to have a some-what dense center and l e s s dense border, (arrows, f i g . 15). The f u n c t i o n of t h i s i n c l u s i o n i s not known. The l a s t organelle to be discussed i s the t r i c h o c y s t . These organelles have the capacity to e j e c t from the c e l l . E j e c t i o n , however, has never been observed i n c u l t u r e s of P. trochoideum. In cross section the organelles appear as c r y s t a l l i n e square cores, 0.20^ i n diameter, bounded by a s i n g l e membrane (TS) which forms a sac around the core ( f i g . 16). 9. In l o n g i t u d i n a l view, f i b e r s can be seen extending from the pointed a p i c a l t i p of the core to the t r i c h o c y s t sac (TF, f i g s . 17 and 18). Tr i c h o c y s t sacs do not appear to be within t h e c a l pores (P, as seen i n f i g u r e 17). In most cases t r i c h o c y s t s are s p a t i a l l y r e l a t e d to the pores but the sacs and pores are independent. . Examination of the pore shows that i t i s an apparently i s o l a t e d s t r u c t u r e c o n s i s t i n g of a canal through the p l a t e m a t e r i a l (W) and a semi-spherical, membranous, pore plug occupying the canal ( f i g s . 19 and 20). The aperture of the canal i s ridged on the outer w a l l surface (R, f i g . 20) and when the p l a t e (W) i s observed i n surface view both the ridged aperture and pore plug can be i d e n t i f i e d ( f i g . 21). The average pore diameter i n P. trochoideum i s 0.12yi. ULTRASTRUCTURE OF THE THECA Excluding encystment, Peridinium trochoideum has a r e l a t i v e l y simple l i f e c y c l e (6). Mature c e l l s are u s u a l l y pear shaped and have short a p i c a l spines ( f i g . 22). In l i v i n g c e l l s , the g i r d l e i s e a s i l y observed but i n d i v i d u a l p l a t e s are rather d i f f i c u l t to discern. During ecdysis, the theca i s broken along the g i r d l e and the p r o t o p l a s t becomes s l i g h t l y enlarged and somewhat s p h e r i c a l ( f i g . 23). I t i s i n t e r e s t i n g to note that the p r o t o p l a s t appears to have already acquired an appreciably t h i c k surface l a y e r . Although Braarud (6) observed a hyaline excretion between the p r o t o p l a s t and theca during e a r l i e r studies on P. trochoideum, such an event was never observed i n the present clone obtained from Indiana. A f t e r f r e e i n g i t s e l f from the theca, the naked p r o t o p l a s t swims about f o r some time and i n doing so acquires an elongate shape ( f i g . 24). Later, the f l a g e l l a are shed and the p r o t o p l a s t assumes a r e s t i n g stage ( f i g . 25). 10. A s l i g h t c o n s t r i c t i o n i n the midregion i s the f i r s t i n d i c a t i o n of cyto-k i n e s i s ; subsequently a d i s t i n c t cleavage furrow i s formed ( f i g . 26). A f t e r separation, the young daughter c e l l s appear non-motile, s p h e r i c a l , t h i c k walled, and u n d i f f e r e n t i a t e d (with respect to thecal p l a t e s ) ( f i g . 27). The epitheca of mature c e l l s of P. trochoideum i s composed of 1, 2 three s e r i e s of p l a t e s two of which are v i s i b l e i n v e n t r a l view ( f i g . 28). The a p i c a l p l a t e s 1', 2', and 4' (four p l a t e s comprise t h i s set) are located around the apex while the precingular p l a t e s 1", 2", and 7" (seven p l a t e s comprise t h i s set) are adjacent to the g i r d l e . The small, narrow a p i c a l c l o s i n g p l a t e (CP) l i e s a n t e r i o r to the f i r s t a p i c a l p l a t e (1') between p l a t e s 2' and 4'. What has been p r e v i o u s l y described as a horn (27) appears to be two spines (H) which a r i s e from the a n t e r i o r end of the second and fo u r t h a p i c a l p l a t e s and extend upwards on both sides of the a p i c a l c l o s i n g p l a t e p a r a l l e l with the c e l l ' s c e n t r a l a x i s . A c t u a l l y , the spines are not elaborately tapered ( f i g . 29), rather they appear acute due to the perspective rendered by the f r a c t u r e plane. Normally, an a p i c a l pore e x i s t s i n the a p i c a l c l o s i n g p l a t e (12), but i n some c e l l s i t i s not observed ( f i g . 30). The two f l a g e l l a o r i g i n a t e on the v e n t r a l side from a s i n g l e f l a g e l l a r pore (FP) s i t u a t e d i n the deeply excavated g i r d l e (G, f i g . 28). The g i r d l e i s left-handed, that i s , the g i r d l e forms a descending s p i r a l with the r i g h t end of the g i r d l e l y i n g below the l e f t ( f i g . 29). The transverse f l a g e l l u m l i e s i n the e n c i r c l i n g g i r d l e whereas the l o n g i -t u d i n a l f l a g e l l u m l i e s i n the sulcus (SL, f i g . 29), a groove s i t u a t e d d i r e c t l y beneath the f i r s t a p i c a l p l a t e , p a r a l l e l to the axis of l o c o -motion. The sulcus tends to broaden towards the c e l l antapex. There are no a n t a p i c a l spines or horns. •^Notation according to Kofoid, r e f . 24. Opiate formula same as given by Lebour, r e f . 27. 11. Plates from both the epitheca and hypotheca can be i d e n t i f i e d from the d o r s o l a t e r a l view of the c e l l shown i n f i g u r e 31. An i n t e r -c a l a r y p l a t e of the epitheca, 2a (three p l a t e s comprise t h i s s e t ) , l i e s above the t h i r d and f o u r t h precingular p l a t e s . The t h i r d a p i c a l p l a t e which l i e s above the i n t e r c a l a r y p l a t e cannot be seen since the a n t e r i o r end of the epitheca i s embedded i n the matrix. The p o s i t i o n of the c e l l , however, allows the f i r s t a n t a p i c a l p l a t e , 1' 1'' (two p l a t e s comprise t h i s set) t o be seen l y i n g beside the f i r s t p o s t c i n g u l a r p l a t e , l 1 ' ' , and below the second and t h i r d p o s t c i n g u l a r p l a t e s , 2' 1', and 3 1 , 1 ( f i v e p l a t e s comprise t h i s s e t ) . As a r u l e , the g i r d l e i s subdivided i n t o three p l a t e s demarcated by sutures. The sutures are d i s t i n c t from the ridges since they are depressed i n the w a l l rather than protruding from i t (see S, f i g . 32). The f i r s t suture of the g i r d l e (Si) l i e s beneath the f i r s t p r e c i n g u l a r p l a t e , 1'1 ( f i g s . 28 and 29), and the second (S 2) i s found adjacent to the junction of the second and t h i r d p recingular p l a t e s , 2"' and 3'1 ( f i g . 31). 'The t h i r d (S^Jis found beneath the seventh pr e c i n g u l a r p l a t e , 7'' ( f i g . 29).: The g i r d l e does not have extensive l i s t s , but the a n t e r i o r edge i s more deeply ridged than the p o s t e r i o r ( f i g s . 1 and 4). Although the s i z e v a r i a t i o n of c e l l s i n a c u l t u r e i s due to un-equal f i s s i o n during c e l l d i v i s i o n , aging a l s o accounts f o r d i f f e r e n c e s i n c e l l s i z e and c e r t a i n changes i n c e l l morphology. In general, young c e l l s which acquire a theca soon a f t e r maturity are pear-shaped ( f i g . 28), but growth leads to the expansion of the c e l l i n t o an almost e l l i p -s o i d shape ( f i g . 31) p r i o r to d i v i s i o n . Hence, the shape of the c e l l can be used to independently assess t h e c a l age. The texture of membranes 12. covering the t h e c a l p l a t e s a l s o i n d i c a t e s the developmental stage of the theca. A young but r e l a t i v e l y well-developed theca has surface membranes which have a r e l a t i v e l y smooth e x t e r i o r surface ( f i g . 28), whereas an older theca shows a s t r i k i n g a l t e r a t i o n of the surface. The outer surface membranes of the p l a t e s become h i g h l y undulated, p i t t e d , and b l i s t e r e d (B, f i g s . 31 and 32). There are, however, c e r t a i n regions on the surface of older thecae that are f r e e of p i t t i n g . These regions appear as small 'plaque-like' areas scattered over the t h e c a l surface (PL, f i g s . 33 and 34). In o l d c e l l s , sutures are sometimes bordered by a d i s t i n c t i v e 'marginal suture band" (MB, f i g . 31, and 33). The band width i s f a i r l y constant f o r any one c e l l but the width varies among c e l l s with thecae of d i f f e r e n t ages. Older c e l l s u s u a l l y have wider bands, 0.38 - 0.5 JA. wide. The band represents a s l i g h t l y elevated region of the theca adjacent to the recessed suture (MB, f i g . 32 and 33). I n t e r -c a l a r y bands are also present i n some older thecae and appear on the opposite side of the suture as the marginal suture band (IB, f i g . 31). Since they are not s t r i a t e or pronounced they cannot be r e a d i l y observed with the l i g h t microscope. ULTRASTRUCTURE OF THE THECAL PLATES AND ASSOCIATED MEMBRANES One s t r i k i n g external feature of both mature and o l d c e l l s i s the r e l a t i v e d i s c o n t i n u i t y of p l a t e demarcation (sutures) at the t h e c a l surface (arrows, f i g s . 34 and 35). This i s due to the o v e r l y i n g p l a t e membranes. The t h e c a l membrane i s the outermost membrane and i t covers 13. the e n t i r e c e l l (TM, f i g s . 36, 37, and 40). I t can be seen that the th e c a l membrane ( f i g . 36) must be s l i g h t l y invaginated at the surface immediately above p l a t e junctions to form what i s recognized as a suture (S, f i g . 35). I n t e r c a l a r y bands have not been detected i n t h i n s e c t i o n i n g ; how-ever, marginal suture bands appear as thickened regions at the edges of two overlapping p l a t e s ( f i g . 36). In some cases the p l a t e s may bear a r i d g e (R, f i g . 36). The p l a t e m a t e r i a l i s e l e c t r o n transparent and hence appears nondescript. C l o s e l y appressed to and below the t h e c a l membrane i s the outer p l a t e membrane (OPM, f i g s . 36 and 40). The outer p l a t e membrane w i l l be defined as the membrane which l i e s immediately above the p l a t e . I n -f o l d i n g of such a membrane between adjacent pla t e s occurs wi t h i n each suture (S, f i g s . 36 - 41). I t i s quite c l e a r from f i g u r e 37 that when sutures between p l a t e s are incomplete, the outer p l a t e membrane r e f l e c t s on i t s e l f and thus does not extend completely around the p l a t e . For the most part, the outer p l a t e membrane does not surround p l a t e s even when the p l a t e s are separate (arrow, f i g . 38). Possib l e attachment or c o n t i n u i t y of the suture with a membrane beneath the p l a t e has been ob-served only once (arrow, f i g . 39). I t can be proposed therefore, that such a membrane system does not completely enclose each p l a t e i n a sac-l i k e manner. Instead, the pl a t e s generally appear to be flanked by two separate membrane systems, the outer p l a t e membrane immediately above and the inner p l a t e membrane immediately below (IPM, f i g s . 38 - 40). The inner p l a t e membrane cannot be regarded as the plasmalemma. The plasmalemma, defined herein, (PM, f i g s . 38 and 40) l i e s below the inner p l a t e membrane and d e l i m i t s a narrow band of cytoplasm at the pr o t o p l a s t periphery. This cytoplasmic band i s the r e s u l t of extensive vacuolation (V) of the p a r i e t a l cytoplasm and i s a common c h a r a c t e r i s t i c 14. i n mature c e l l s of P. trochoideum. Furthermore, the plasmalemma i s r e l a t i v e l y t h i n , whereas the inner and outer p l a t e membranes (and the th e c a l membrane) are not only t h i c k e r but are al s o asymmetric ( f i g s . 40 and 41) ; the l a t t e r are about 150A* t h i c k with p a r t i t i o n s of 75/50/258 o whereas the plasmalemma i s a symmetrically 100A t h i c k . In c e l l s with mature thecae, the thicker, p a r t i t i o n of both the inner and outer p l a t e membranes i s always found c l o s e s t to the p l a t e ( f i g s . 40 and 41). Where sutures e x i s t , the outer p l a t e membrane i n f o l d s and p a i r s . The two thinner p a r t i t i o n s become appressed and form a c e n t r a l p a i r i n g l i n e thus g i v i n g the suture an apparent p e n t a p a r t i t e appearance (S, f i g . 41). The f r a c t u r e d face shown i n f i g u r e 42 reveals a l l four membrane systems. Both the t h e c a l membrane and outer p l a t e membrane possess regions of p i t t i n g . The p l a t e m a t e r i a l has a r e l a t i v e l y smooth sur-face. The underlying inner p l a t e membrane has a r e t i c u l a t e surface whereas the plasmalemma appears s l i g h t l y r e t i c u l a t e and undulated. Since the four membrane systems can be recognized from t h e i r l o c a t i o n and morphology i n freeze-etched preparations, i t i s now p o s s i b l e to f u r t h e r demonstrate the r e l a t i o n s h i p between the suture and the underlying p l a t e membrane. In f i g u r e 43, both the plasmalemma and inner p l a t e membrane appear to be continuous over the cytoplasm even at p l a t e junctions. This supports the observations from t h i n sectioned m a t e r i a l ( f i g s . 37, 38, and 40) that there i s no connection between the outer and inner p l a t e membrane systems. Figures 44 and 45 r e v e a l the p l a t e - s i d e view of the inner p l a t e membrane. I f each p l a t e were surrounded by a p l a t e membrane one would 15. expect a f r a c t u r e below the p l a t e and above the inner p l a t e membrane to re v e a l sutures. Instead, no sutures, t e a r s , or membrane pr o j e c t i o n s occur on the membrane immediately below the p l a t e . The membrane, how-ever, does e x h i b i t d i s t i n c t scars (SC). These scars may represent places where the membranes of the inner and outer membrane systems may once have been c l o s e l y associated or even p o s s i b l y attached. ULTRASTRUCTURE OF WALL FORMATION In a d d i t i o n to the i n c l u s i o n s described p r e v i o u s l y one f i n d s l a r g e o s m i o p h i l i c bodies d i s t r i b u t e d c i r c u m f e r e n t i a l l y around the nucleus (PB, f i g . 46). These i n c l u s i o n s p l a y an important r o l e i n the formation of the w a l l , and w i l l be r e f e r r e d to as 'prothecal bodies.' In mature c e l l s , these structures a r e / f o r the most part, f a i r l y amor-phous, although i t i s common to f i n d the structure permeated by vary-i n g amounts of membrane (MC, f i g . 47). P r i o r to ecdysis, the pro t h e c a l bodies of the mature c e l l undergo transformation, at which time the almost wholly amorphous i n c l u s i o n becomes traversed with i n c r e a s i n g amounts of membrane ( f i g . 48). Subsequently, the prothecal body becomes almost t o t a l l y composed of l o o s e l y packed f l a t t e n e d v e s i c l e s ( f i g . 49). Further transformation r e s u l t s i n the formation of v e s i c l e s whose l i m i t i n g membranes and con-tents are assumed to be derived from the prothecal body (see PV, f i g . 57). Since the v e s i c l e s o r i g i n a t e from p r o t h e c a l bodies they w i l l be c a l l e d p rothecal v e s i c l e s . As prothecal v e s i c l e s migrate to the surface of the pr o t o p l a s t , an amorphous substance can be seen to accumulate between the plasma-lemma and the inner p l a t e membrane (arrow, f i g . 50). A f t e r migration of the prothecal v e s i c l e s to the plasmalemma, the deposition of ma t e r i a l 16. across the plasmalemma becomes extensive r e s u l t i n g i n the formation of a cushion of new w a l l m a t e r i a l (WM) between the plasmalemma and the inner p l a t e membrane ( f i g . 51). The c e l l u s u a l l y undergoes ecdy-s i s at t h i s stage and the t h e c a l membrane, outer p l a t e membrane, and p l a t e s are l o s t . The prothecal v e s i c l e s c o n s t i t u t e a s u b s t a n t i a l p a r t of the p e r i p h e r a l cytoplasm of c e l l s at t h i s stage ( f i g . 52). Prothecal v e s i c l e s contain l i t t l e , i f any, of the amorphous component found i n the prothecal body; instead, they contain l o o s e l y intertwined f i b r o u s m a t e r i a l ( f i g s . 51 and 52). I t i s probably the i n c o r p o r a t i o n of t h i s m a t e r i a l together with the prothecal v e s i c l e membrane that gives the new c e l l w a l l a moderately dense s t a i n i n g c h a r a c t e r i s t i c at t h i s stage of development ( f i g . 53). During c y t o k i n e s i s , l i g h t microscope observations i n d i c a t e that new c e l l w a l l m a t e r i a l i s r e a d i l y d i s c e r n i b l e , e s p e c i a l l y at the isthmus of the d i v i d i n g . c e l l (WM, f i g . 54). I f plasmolysis i s induced at t h i s stage, the p r o t o p l a s t , with a d e f i n i t e new w a l l l a y e r (W), becomes separated from the o v e r l y i n g inner p l a t e membrane of the parent c e l l (IPMp, f i g . 55). The new w a l l material appears to be reasonably f l e x i b l e at t h i s time and remains c l o s e l y associated with the under-l y i n g p r o t o p l a s t . E l e c t r o n microscope observations of the same stage i n d i c a t e that an appreciable amount of w a l l material has been l a i d down at the proto-p l a s t surface (W, f i g . 56), thus confirming the l i g h t microscope observations. The prothecal bodies have decreased i n number by t h i s stage but p r o t h e c a l v e s i c l e s are s t i l l abundant i n the p e r i p h e r a l cyto-plasm adjacent to the new w a l l (PV, f i g . 56). Wall formation takes place 17. very r a p i d l y at the isthmus where, during l a t e c y t o k i n e s i s , the development of prothecal v e s i c l e s from prothecal bodies i s very evident ( f i g . 57). Eventually, the pro t h e c a l v e s i c l e s become d i s -t r i b u t e d i n two bands across the isthmus (arrows, f i g . 58). Their subsequent f u s i o n and maturation r e s u l t s i n the separation of the parent p r o t o p l a s t i n t o daughter halves and the eventual formation of t h e i r new walls. The w a l l m a t e r i a l at the separation p o i n t appears as a very d e f i n i t e band (W, f i g . 59) and i s not nearly as f i b r o u s as that observed i n e a r l i e r stages (cf. f i g . 53). There i s no under-l y i n g inner p l a t e membrane at t h i s stage. A f t e r cytokineses and/or during e a r l y maturation, each of the daughter c e l l s acquires an inner p l a t e membrane (IPM, f i g . 60). This membrane i s asymmetrical and has i t s t h i c k e r p a r t i t i o n adjacent to the wa l l s i m i l a r to the inner p l a t e membrane of a mature c e l l . The membrane appears to o r i g i n a t e i n a zone between the plasmalemma and the w a l l and although i t i s d i f f i c u l t to e s t a b l i s h , i t appears that the new inner p l a t e membrane i s derived from moderately e l e c t r o n opaque ma t e r i a l associated with the w a l l (arrows, f i g s . 59 and 60). This m a t e r i a l probably represents aggregates of membrane components derived from the prothecal v e s i c l e s . Although the new inner p l a t e membrane i s now established, the outer p l a t e membrane and t h e c a l membrane are s t i l l absent. Some of the membrane covering the w a l l at t h i s time i s new and some rep-resents inner p l a t e membrane of the parent c e l l (IPMp, f i g . 60). The 18. l a t t e r can be recognized because i t has i t s thinner p a r t i t i o n adja-cent to the w a l l - the r e s u l t of w a l l deposition between i t and the plasmalemma. I t should be made c l e a r that both the outer p l a t e membrane and t h e c a l membrane of mature c e l l s have t h e i r t h i c k e r p a r t i t i o n s adjacent to the w a l l as described p r e v i o u s l y . Each young daughter c e l l of P. trochoideum thus possesses a c e l l w a l l which i s continuous but u n d i f f e r e n t i a t e d . That i s , there are no i n d i v i d u a l p l a t e s or sutures ( f i g . 61). Prothecal bodies observed i n young daughter c e l l s appear s i m i l a r to those of young, mature c e l l s . There are no signs of the ,amorphous component being traversed with membrane although membrane components can be seen adjacent to some amorphous i n c l u s i o n s (MC, f i g . 61). Examinations of a young c e l l with a developing theca shows that both sutures and p i t t e d regions are la c k i n g , but a f u l l y developed pore (P), i d e n t i c a l to that on the mature theca, (P. f i g . 28), i s present on the smooth surface ( f i g . 62). Figure 63 i l l u s t r a t e s an intermediate stage of p l a t e morphogenesis as defined by the f o r -mation of the suture. At t h i s stage, the sutures (S) are shallow, and very discontinuous (arrows). Again pores are present and a l -though p i t t e d regions can be observed on the t h e c a l surface, they are not as d i s t i n c t as i n the mature thecae shown i n f i g u r e s 32 and 42. 19. DISCUSSION CYTOPLASM AND ORGANELLES With the p o s s i b l e exception of the segregated body, none of the organelles described i n the observations are unique to Peridinium  trochoideum. The c h l o r o p l a s t s with t h e i r thylakoids associated i n groups of three are t y p i c a l f o r the group (16). What i s not common how-ever, i s the pseudogranal as s o c i a t i o n s between thylakoids of vary-ing s i z e . The stromal d i f f e r e n t i a t i o n i s another elaboration of c h l o r o p l a s t morphology. With two types of stroma, dense and sparse, i t seems l i k e l y that each type may p o s s i b l y have d i f f e r e n t yet probably l i n k e d f u n c t i o n s . On the basis of preliminary f l u o r e s c e n t studies and c h l o r o p l a s t u l t r a s t r u c t u r e of p l a s t i d genophores (2, 38) i t i s suggested that the sparse stroma may contain DNA.• The nucleus of P. trochoideum can be described as a di n o c a r y o t i c nucleus (46) on the basis of having c o i l e d and condensed interphase chromosomes. I t would appear from the u l t r a s t r u c t u r e of the chromo-somes that the chromatin f i b e r s lack the histone and r e s i d u a l p r o t e i n complex common to l a r g e r eucaryotic chromatin f i b e r s (11, 13, 30, 37). Unlike the nucleolus of eucaryotic c e l l s (animal c e l l s s p e c i f i c a l l y ) , the nucleolus of P. trochoideum i s r e l a t i v e l y simple and lacks the three detectable regions of the nucleolus r e f e r r e d to as the pars amorpha, pars f i b r o s a , and pars chromosoma (21). Although the nucleoplasm i s t y p i c a l f o r a dinocaryotic nucleus, the envelope displays c h a r a c t e r i s t i c s which tend to suggest a po s s i b l e nucleo-cytoplasmic r e l a t i o n s h i p common to a n o c t i c a r y o t i c 20. envelope. The n o c t i c a r y o t i c nucleus, as defined by Zingmark (46), has been described by A f z e l i u s (1) as having no interphase chromosomes and having nucleo-cytoplasmic communication through blebbing of the nuclear envelope. The v e s i c l e - a s s o c i a t e d stage observed i n P. t r o c h o i -deum could p o s s i b l y be an analogous type of nucleo-cytoplasmic communication since not only are there large gaps i n the nuclear envelope but the v e s i c l e s themselves may be agents of transport. The presence of nucleo-cytoplasmic communication by the envelope of a d i n o c a r y o t i c nucleus has been reported by Dodge and Crawford f o r Gymnodinium fuscum (18). In P. trochoideum there are.also large d i l a t e d areas between the inner and outer nuclear membranes where f i b r o u s bodies are observed. Nuclear associated f i b r o u s bodies have a l s o been described by Dodge f o r Aureodinium (15) and by Taylor f o r a symbiotic marine d i n o f l a g e l l a t e (42). These f i b r o u s bodies have a l s o been observed as membrane bound i n c l u s i o n s i n the cytoplasm of P. trochoideum and are very s i m i l a r , i f not the same as, structures observed i n Peridinium w e s t i i (31), Wolosynskia micra (26) and Symbiodinium microadriaticum (23). There i s a p o s s i b i l i t y , there-f o r e , that cytoplasmic f i b r o u s bodies might a r i s e from the p e r i -nuclear f i b r o u s bodies which are released from the nuclear envelope at some stage. Other nuclear envelope elaborations have been des-c r i b e d by Kofoid and Swezy C}440 i n Gyrodinium corallinum and G. virgatum where the outer part of the nucleus appears as a t h i n a l v e o l a r l a y e r of e l l i p s o i d a l vacuoles and by Taylor (45) who described p e r i -nuclear s t r u c t u r a l elements i n Gonyaulax p a c i f i c a . 21. The t r i c h o c y s t s observed i n P. trochoideum are morphologically s i m i l a r to those described i n other species (5, 18, 25, 31) but are smaller i n diameter, whereas reports from the above sources describe t r i c h o c y s t diameters of 0.2 to 0.4 p., the l a r g e s t found . i n P. trochoideum were 0.2 y.Trichocyst f i b r i l s s i m i l a r to those described by Bouck and Sweeney (5) and Messer and Ben-Shaul (31) were seen attached to the t r i c h o c y s t core. The a n t e r i o r end of the t r i c h o c y s t sac containing the core and f i b r i l s was not continuous with the pore plug but appeared to be terminal beneath the w a l l at the plasma membrane as i n P. w e s t i i (31). This s i t u a t i o n i s somewhat d i f f e r e n t to that described f o r Gonyaulax and Prorocentrum where the t r i c h o c y s t sac i s s i t u a t e d i n the w a l l adjacent to the p l a t e mem-brane which o v e r l i e s the pore (5). In P. trochoideum. the t r i c h o c y s t sac i s s i t u a t e d below the pore i n the w a l l which i s covered by the p l a t e membrane and the t h e c a l membrane. These membranes must be punctured during the release of the shaft. Although i t was not ob-served, the t r i c h o c y s t sac may p r o j e c t i n t o the canal p r i o r to d i s -charge. The t h e c a l pores through which the shafts are ejected have an average diameter of 0.12 u which i s much smaller than pores (0.2 -0.3 |a) described i n P. w e s t i i (31). However, t h i s can be r e l a t e d to the corresponding t r i c h o c y s t diameter of the two species. Assuming the discharged t r i c h o c y s t shafts are of smaller diameter than the r e s t i n g or charged form (31), no problem should a r i s e during e x i t of s t r i a t e rods of P. trochoideum through a 0.12 u pore. 22. THECAL MORPHOLOGY From the observations, i t i s c l e a r that surface morphology of the t h e c a l membranes of P. trochoideum r e f l e c t s i t s stage of development. Smooth surfaces are c h a r a c t e r i s t i c of young c e l l s ; with aging, p i t s , b l i s t e r s , sutures, marginal suture bands, and i n t e r - c a l a r y bands become p r o g r e s s i v e l y d i f f e r e n t i a t e d . Pores, on the other hand, are formed very e a r l y i n the ontogeny of the thecae. B l i s t e r i n g at the c e l l surface has been demonstrated i n Crypthe-3 codjnium c o h n i i (Seligo) Javornicky . where i t i s evident that the outer t h e c a l membrane and inner p l a t e membrane become separated from the w a l l (25). I t i s uncertain, however, whether t h i s i s a n a t u r a l occurrence analogous to the b l i s t e r s on the surface of P.  trochoideum. Both marginal suture bands and i n t e r c a l a r y bands occur on thecae of P. trochoideum. Each i s d i s c r e t e i n morphology; the marginal suture band i s a d i s t i n c t i v e elevated region at the p l a t e margin adjacent to the suture, whereas the i n t e r c a l a r y band i s not elevated and borders the suture on the opposite s i d e to the marginal suture band. The i n t e r c a l a r y band i s g e n e r a l l y twice the width of the marginal suture band^ however both surface features appear as a f u n c t i o n of aging. Some i n v e s t i g a t o r s (29, 34) b e l i e v e that the i n t e r c a l a r y band represents a region of growth. The marginal suture band may a l s o f u l f i l l the same fu n c t i o n . 3 This taxon was r e f e r r e d to as "Gyrodinium c o h n i i ( S c h i l l e r ) " ( s i c ) . 23. PLATES AND ASSOCIATED MEMBRANES Compared to other species i n the genus, the c e l l w a l l m a t e r i a l of Peridinium trochoideum takes up l i t t l e , i f any, s t a i n and there-f o r e appears quite nondescript. In both P. w e s t i i (31) and P. cinctum (19, 22) the w a l l m a t e r i a l s t a i n s appreciably and one can e a s i l y detect i t s f i b r i l l a r nature. The m u l t i l a y e r e d cyst w a l l of P y r o c y s t i s spp. (M) has been examined using freeze-etching technique and the f i b r i l l a r nature of the w a l l i s q u i t e evident. As shown i n the observations, the mature w a l l of P. trochoideum shows no f i b r i l s e i t h e r i n t h i n sectioned or freeze-etched m a t e r i a l . Dodge (19) has shown that p l a t e s of Heterocapsa t r i g u e t r a possess two or more sides which bear r i d g e s ; the remaining sides have tapered flanges. This p l a t e c h a r a c t e r i s t i c was a l s o suggested as common i n P. trochoideum. However, from the current i n v e s t i g a t i o n , i t appears that i n P. trochoideum such a c h a r a c t e r i s t i c may occur only when a marginal suture band i s present. In most cases, p l a t e overlap occurs without any elaboration. In t h i s work, a d i s t i n c t i o n has been made between the two separate membrane systems which are i n contact with the plates. The outer p l a t e membrane was defined as that membrane which l i e s between the t h e c a l membrane and the p l a t e and the inner p l a t e membrane as l y i n g between the p l a t e and the plasmalemma. The reason f o r d e f i n i n g two separate p l a t e membranes i s .based on the f o l l o w i n g observations. F i r s t l y , each p l a t e was not . . completely enclosed nor t o t a l l y separated by one continuous membrane. Secondly, the membrane immediately under the p l a t e s was shown by t h i n s e c t i o n i n g and freeze-etching to be 24. continuous over the pro t o p l a s t . F i n a l l y , a c y t o l o g i c a l l y w e l l defined membrane surrounding each p l a t e i n a manner s i m i l a r to that observed by Dodge (19) i n Wolozynskia coronata, Ceratium  h i r u n d i n e l l a , and Peridinium cineturn could not be shown. Only once was the a s s o c i a t i o n of the outer p l a t e membrane and the inner p l a t e membrane observed t o form a p l a t e enclosure. Be-cause of t h i s , i t i s beli e v e d that the outer p l a t e membrane may have been continuous with the inner p l a t e membrane at one time^only to become d i s s o c i a t e d from i t during maturation. The v e s t i g i a l p o i n t of a s s o c i a t i o n i s probably represented by the scars observed on the inner p l a t e membrane. Whether the outer p l a t e membrane i s continuous with the inner p l a t e membrane or whether i t i n f o l d s i n a s s o c i a t i o n with a suture, apparently only the t h i c k e r p a r t i t i o n of the membrane l i e s adjacent to the p l a t e s . The organization of the theca c l o s e l y resembles that of Crypthe- codinium c o h n i i (25) i n which the outer p l a t e membrane was reported to form sutures by i n f o l d i n g . The fo l l o w i n g diagram i l l u s t r a t e s the r e l a t i o n s h i p between the various membrane systems and t h e c a l p l a t e s of P. trochoideum. Diagram 1. 25. The asymmetric t h e c a l membrane (TM) i s the outermost membrane and i s continuous over the e n t i r e c e l l . The outer p l a t e membrane (OPM) may at the e a r l i e s t stages of t h e c a l ontogeny surround each p l a t e (region 1). However, lack of a s s o c i a t i o n between the outer p l a t e membrane (OPM) and inner p l a t e membrane (IPM) i s d e f i n i t e l y predominant i n most c e l l s (region 3). I f one assumes that the outer p l a t e membrane and inner p l a t e membrane are one and the same during e a r l y development, then an intermediate stage of separation must e x i s t (region 2). In e i t h e r case, the t h i c k e r p a r t i t i o n of the membrane(s) always l i e s adjacent to the p l a t e . Sutures (S) have a t y p i c a l p e n t a p a r t i t e appearance as a r e s u l t of apposition of p a i r i n g membranes. The plasmalemma (PM) i s a r e l a t i v e l y t h i n symmetrical membrane containing the cytoplasm. In most c e l l s a p a r i e t a l cytoplasmic band i s a r e s u l t of vacuolation (V) at the c e l l periphery. Most of the r e s u l t s i n d i c a t e that c o n t i n u i t y between outer and inner p l a t e membranes i s l a c k i n g , i n mature c e l l s . I t i s not l i k e l y that d i s r u p t i o n at f i x a t i o n causes the breakage of a mem-brane which would otherwise enclose a p l a t e . I t would be d i f f i c u l t to j u s t i f y the f a c t that such a membrane would be subject to break-age at exactly the same poin t i n every c e l l observed. 26. The l a s t membrane to be discussed i s the plasmalemma. The question of determining and d e f i n i n g the plasmalemma may involve a semantic problem. Dodge contends that the outermost membrane of the d i n o f l a g e l l a t e i s the plasmalemma (19). I f t h i s i s the case, then at ecdysis the plasmalemma of P. trochoideum w i l l be l o s t and \ . . . an inner membrane must therefore be designated the plasmalemma. This problem of r e d e f i n i n g a plasmalemma i s superfluous i n P. trochoideum i f the plasmalemma i s defined as that membrane which i s i n d i r e c t contact with the cytoplasm. I t i s , u n l i k e the other membranes associated with the p l a t e s , symmetrical. FORMATION OF THE WALL The pro t h e c a l body i n non-dividing c e l l s was shown to be an e l e c t r o n dense, amorphous body. P r i o r to ecdysis, membrane com-ponents appear and traverse throughout the amorphous component of the body and there i s a marked decrease i n s t a i n a b i l i t y . At t h i s stage, the pro t h e c a l bodies appear i d e n t i c a l to the " a t y p i c a l p l a s -t i d s " described i n Crypthecodinium c o h n i i (25). At ecdysis the amor-phous component of the pro t h e c a l body i s almost gone, being replaced by membranes which l a t e r form p r o t h e c a l v e s i c l e s . The essence of t h i s transformation appears to be the probable production of some soluble, e l e c t r o n transparent component from the amorphous material of the prot h e c a l body. Such a conversion r e s u l t s i n the formation of pro-t h e c a l v e s i c l e s , the consequence of which p o s s i b l y increases the osmotic pressure within the c e l l . Thus, at ecdysis such an increase 27. i n pressure i n s i d e the p r o t o p l a s t may be the a c t i v e f o r c e e f f e c t i n g the rupture of the theca. Although Braarud (6) believes that ecdysis i n P. trochoideum occurs as the r e s u l t of an excretion of hy a l i n e substance between the p r o t o p l a s t and theca, no such event occurred i n the clone of P. trochoideum from Indiana. A f t e r escape from the theca, the pr o t o p l a s t proceeds through z c y t o k i n e s i s . Having l o s t the p l a t e s and o v e r l y i n g membranes, i t i s surrounded by the inner p l a t e membrane and plasmalemma of the parent c e l l . The plasmalemma (as .defined herein) i s the only s t a b l e , w a l l -associated membrane retai n e d during the c e l l c y c l e . I t i s assumed that the w a l l m a t e r i a l i s derived from prothecal bodies, packaged i n pro t h e c a l v e s i c l e s / a n d deposited by the l a t t e r at the s i t e of the new w a l l . I t i s most i n t e r e s t i n g that Bursa (8, 9) has observed discharges of ectoplasmic c o l l o i d from Woloszynskia, Gyrodinium, and Peridinium which are capable of d i f f e r e n t i a t i n g i n t o membrane and p l a t e s t r u c t u r e s . Indeed, i f the ectoplasmic m a t e r i a l were prothecal v e s i c l e s or t h e i r counterparts one might expect such a phenomenon to occur. ' When the w a l l i s formed, m a t e r i a l i s deposited between the plasmalemma and the inner p l a t e membrane of the parent c e l l . There are no sutures and hence no d i s c e r n i b l e p l a t e s at t h i s stage and the w a l l e x i s t s as a continuous sphere over the p r o t o p l a s t . This s i t u a t i o n confirms the f a c t that the inner p l a t e membrane i s continuous. S i m i l a r studies have shown that Pyrodinium bahamense (7), Crypthecodinium  c o h n i i (25), and Gonyaulax polygramma (44) a l s o e x h i b i t a stage i n which w a l l m a t e r i a l i s present but apparently not d i f f e r e n t i a t e d i n t o p l a t e s . 28. In P. trochoideum the f i r s t plate-membrane system to become es t a b l i s h e d a f t e r w a l l formation i s the inner p l a t e membrane system. The t h i c k e r p a r t i t i o n of t h i s membrane l i e s , as expected, adjacent to the w a l l . Formation of i n d i v i d u a l t h e c a l p l a t e s i s presumed to, take pl a c e concurrently with the formation of outer p l a t e membrane and t h e c a l membrane systems. I t seems quite probable that the mem-brane components f o r these systems e x i s t i n the u n d i f f e r e n t i a t e d w a l l as a consequence of the t o t a l incorporation of prothecal v e s i c l e s i n t o the w a l l . Indeed i f a prothecal v e s i c l e were to remain i n the w a l l as such, i t could w e l l form a pore plug. This could e x p l a i n the presence of pores i n very young thecae and e l i m i -nates the ne c e s s i t y of invoking a d i s s o l u t i o n of w a l l m a t e r i a l i n the formation of a pore a f t e r w a l l deposition has taken place. In Prorocentrum, i t has been suggested that the f i r s t w a l l covering i s complete (14) and i t was assumed that d i s s o l u t i o n of w a l l m a t e r i a l r e s u l t e d i n pore formation. Sutures are not present on young thecae but apparently develop some time a f t e r w a l l m a t e r i a l has been l a i d down. Taylor (43) has found from l i g h t microscopy that young thecae of Gonyaulax tamarensis with l i t t l e or no surface markings do not break along expected suture l i n e s but fragment i n t o odd shaped pieces of w a l l m a t e r i a l . When sutures i n P. trochoideum f i r s t appear they are shallow and quite discontinuous. To f u l l y understand the process of suture formation i t w i l l be necessary to f o l l o w the development of the two ov e r l y i n g membranes of the w a l l (the t h e c a l and outer p l a t e membrane) and t h e i r subsequent c o n t r i b u t i o n i n su b d i v i s i o n of the w a l l i n t o d i s t i n c t l y shaped p l a t e s . Presumably the mechanism f o r c o n t r o l over p l a t e shape 29. must l i e within the c e l l and hence i t would appear l o g i c a l to expect a c t i v e s i t e s at the plasmalemma and/or inner p l a t e membrane that determine the format f o r a l l p l a t e s types. However, i t must be recognized that wherever incomplete sutures have been observed, d i s s o l u t i o n of w a l l m a t e r i a l appears to have been i n i t i a t e d on the e x t e r n a l face of the w a l l . Undoubtedly p l a t e formation i s an extremely complicated process, e s p e c i a l l y i f one were to attempt to explain the process from the b a s i c g e n e t i c a l c o n t r o l systems to the a c t u a l p h y s i c a l outcome of determining p l a t e demarcation. 30. PLATES AND EXPLANATIONS LEGEND B = b l i s t e r C = c h l o r o p l a s t CFB = cytoplasmic f i b r o u s body CH = chromosome CP = a p i c a l c l o s i n g p l a t e D = dictyosome DL = dense l a y e r DS = dense stroma F = f i b r i l s FB = f i b r o u s body FL = f o l d s FP = f l a g e l l a r pore G = g i r d l e GL = globules H - spine IB = i n t e r c a l a r y band INM = inner nuclear membrane IPM = inner p l a t e membrane • IPMp = parent c e l l inner p l a t e : M = mitochondria MC = membrane component N = nucleus NE = nuclear envelope NU = nucleolus NV = nuclear v e s i c l e s ONM = outer nuclear membrane OPM = outer p l a t e membrane P = pore PB = prothecal body PE = pe r i n u c l e a r extension PFB = p e r i n u c l e a r f i b r o u s bod PL = plaque PM plasmalemma PP pore plug R = ridge S = suture SB = segregated body SC scar SG s t a r c h g r a i n SL = sulcus SS = sparse stroma T = t r i c h o c y s t TF = t r i c h o c y s t f i b e r s TL = thylakoids TM - t h e c a l membrane TS = t r i c h o c y s t sac W = w a l l WM = w a l l m a t e r i a l PLATE I 31. F i g u r e I. Chloroplasts (C) showing thylakoid lamellae (TL) i n ass o c i a t i o n s of three and dense (DS) and sparse (SS) stromal regions. x 21,600 (inset^ x 36,600 Figure 2. Sparse stroma showing f i b e r s (F) beli e v e d to be DNA. x 73,200 Figure 3. Thylakoids (TL) i n associations of three. The margins of some of the small thylakoids are marked by arrows. x154,000 PLATE I I 3 2 . Figure 4. T y p i c a l mesocaryotic nucleus (N) with c o i l e d and condensed interphase chromosomes (CH), nucleolus (NU), and double membrane envelope (NE). x 9,700 Figure 5. Interphase chromosomes (CH) showing two d i r e c t i o n s of c o i l i n g : c o i l s p a r a l l e l t o the chromosome l o n g i t u d i n a l axis (black v e r t i c a l arrows) and c o i l s normal to the chromo-some l o n g i t u d i n a l axis (white h o r i z o n t a l arrows). x 59,000 PLATE I I I 33. Figure 6. Fibre—granular nucleolus (NU) with small c l e a r n u c l e o l a r vacuoles (arrows). x 41,800 Figure 7. P a r t i a l breakdown of nuclear envelope (NE) and a s s o c i a t i o n with nearby v e s i c l e s (NV). x 57,000 PLATE IV Figure 8. Extension of outer nuclear membrane around f i b r o -granular matrix of the pe r i n u c l e a r extension (PE). x 17,000 Figure 9. Perinuclear f i b r o u s body (PFB) wit h i n p e r i n u c l e a r extension. Note separation of inner (INM) and outer (ONM) nuclear membranes (arrow.) x 57,000 Figure 10. Per i n u c l e a r f i b r o u s body (PFB) apparently i n close a s s o c i a t i o n with the nuclear m a t e r i a l (CH). x 57,000 Figure 11. Cytoplasmic f i b r o u s body (CFB). The f i b e r s do not occupy the e n t i r e i n c l u s i o n . x 12,100 PLATE V 35. Figure 12. Dictyosome (D) with d i s t a l (secreting) face towards nucleus. (N) x 28,500 Figure 13. Dictyosome (D), cytoplasmic f i b r o u s body (CFB), and complex membrane system adjacent to nucleus. x 25,500 Figure 14. Location of segregated body (SB) ju s t below nucleus (N) at a n t a p i c a l end of c e l l . x 18,000 Fig u r e 15. The segregated body contains f i b e r s (FB) s i m i l a r to those i n nuclear and cytoplasmic f i b r o u s bodies and globules (GL) with dark s t a i n i n g p e r i p h e r i e s . These are embedded i n an amorphous matrix which c o n s i s t e n t l y has a large c e n t r a l dense region and a l e s s dense periphery (arrows). x 21,600 PIATE VI 36. F i g u r e 16. Trichoc y s t s (T) i n cross s e c t i o n appear as square, c r y s t a l l i n e structures bounded by a s i n g l e membrane (TS). The c r y s t a l l i n e l a t t i c e of the t r i c h o c y s t core i s e a s i l y seen. x 59,000 Figure 17. A t r i c h o c y s t (T) can be seen d i s l o c a t e d from a pore (P) and l y i n g adjacent to the c e l l membrane beneath the w a l l (W). Tri c h o c y s t f i b e r s (TF) can be seen at the core t i p . The over-lapping w a l l p l a t e s are separated by a suture (S). x 35,000 Figure 18. As s o c i a t i o n of t r i c h o c y s t s (T) with the t r i c h o c y s t sac. The t r i c h o c y s t (T) has f i b e r s (TF) extending from i t s core to the top of i t s sac. 37,800 Figures 19 and 20. A continuous pore plug (PP) i s present i n the w a l l i n d i c a t i n g that i t e x i s t s as an independent st r u c t u r e from the t r i c h o c y s t s . x 60,000 x 132,000 PLATE VII 37. Figure 22. Thecate c e l l of P. trochoideum. x 940 Figure 23. C e l l undergoing ecdysis. Note r e l a t i v e thickness of the p r o t o p l a s t surface l a y e r . x 940 Figure 24. Free-swimming, elongate, naked p r o t o p l a s t . ( F l a g e l l a r s t r u c t u r e s enhanced photog_phically). x 940 Figure 25. Resting p r o t o p l a s t a f t e r l o s s of f l a g e l l a . x 940 Fi g u r e 26. C e l l undergoing c y t o k i n e s i s . Note the presence of w a l l m a t e r i a l across the isthmus. x 940 Figure 27. A newly formed daughter c e l l . x 940 PLATE VIII 38. F igure 28. V e n t r a l view of the epitheca and g i r d l e (G) of P. trochoideum showing a p i c a l (1 1, 2', 4'), p r e c i n g u l a r (1'', 2 1'/ 7''), and a g i r d l e p l a t e (G) demarcated by a suture ( S ^ ) . The a p i c a l spines (H) a r i s e from the second and f o r t h a p i c a l p l a t e s (2 1 and 4') which border the a p i c a l c l o s i n g p l a t e (CP). The s i n g l e f l a g e l l a r pore (FP) l i e s i n the g i r d l e (G) immediately below the f i r s t a p i c a l p l a t e (1'). x 12,000 Fig u r e 29. Light micrograph (Nomarski interference) showing the v e n t r a l view of an empty theca. To the l e f t of the f l a g e l l a r pore (FP) i s the f i r s t g i r d l e suture (S^) and below the pore and f i r s t a p i c a l p l a t e ( l 1 ) i s the sulcus (SL). x 1,200 F i g u r e 30. Phase contrast micrograph of an empty theca showing the absence of the a p i c a l pore i n the a p i c a l c l o s i n g p l a t e (CP). The t h e c a l p l a t e s correspond to those seen i n the -freeze-etched m a t e r i a l i n f i g u r e 28. x 1,200 PLATE IX 39. Figure 31. View of the l e f t side of an older theca showing p l a t e s of the epitheca and hypotheca: i n t e r c a l a r y (2a), p r e c i n g u l a r ( 2 " , 3 1 1 ) , postcin g u l a r ( 1 ' " , 2"} 3'"), and a n t a p i c a l ( l " " ) . Pores (P), b l i s t e r s (B), and f o l d s (FL) can be seen on the t h e c a l surface. Only sutures (S) of the epitheca and hypotheca have an accessory marginal band (MB). The f o l d s are a r t i f a c t s probably derived by c e n t r i f u g a t i o n of c e l l s . The i n t e r c a l a r y band (IB) i s f a i n t e r and wider than the marginal suture band. I t l i e s on the opposite s i d e of the suture than the l a t t e r . x 9,500 PLATE X Figure 32. The rough t h e c a l membrane surface i s scored by a suture (S) which i s associated with a marginal suture band (MB). Note that the suture (S) i s depressed i n t o the w a l l and the marginal band (MB) represents a s l i g h t l y elevated part of the theca. x 30,000 Figure 33. Arrows i n d i c a t e points at which the sutures (S) are discontinuous. Note tha t the marginal band (MB), however, s t i l l maintains a continuous b e l t 0.36 ji i n width regardless of suture inconsistency. P l a q u e - l i k e areas (PL) f r e e of p i t s are scattered randomly on the surface. x 82,800 PLATE XI 41. Figure 34. Porti o n of a mature t h e c a l surface showing sutures (S), pores (P), c i r c u l a r plaques (PL), and p i t t i n g . Where the t h e c a l membrane i s continuous over the p l a t e s the sutures cannot be detected (arrows). x 21,000 Figure 35. Porti o n of an o l d t h e c a l surface showing the marginal suture band (MB) adjacent to the suture (S). B l i s t e r i n g (B) i s common on olde r thecae. x 14,000 Figure 36. Cross s e c t i o n through the marginal suture band showing the thickening of both p l a t e s along the length of the suture (S). The p l a t e on the r i g h t bears a ridge (R). The nondescript w a l l (W) i s covered by the outer p l a t e membrane (OPM) and t h e c a l membrane (TM). The inner p l a t e membrane (IPM) l i e s below the w a l l . Note the absence of a connection between the inner p l a t e membrane and the suture. x 68,000 Figure 37. Cross s e c t i o n through the marginal suture band at a poin t where adjacent t h e c a l p l a t e s are continuous. x 68,000 PLATE XII 42. F i g u r e 38. The outer p l a t e membrane appears to terminate at the inner s i d e of the overlapping p l a t e s . Although there i s no connection between the suture (OPM) and the inner p l a t e membrane (IPM), dense m a t e r i a l i s l o c a t e d between the two systems (arrow). The plasmalemma (PM) contains a narrow p a r i e t a l cytoplasmic band which r e s u l t s from vacuolation (V) at or near the c e l l periphery. x 66,000 Figure 39. The suture (S) appears to be attached to the inner p l a t e membrane (IPM) by means of a f o o t (arrow). Note that only the terminal p o r t i o n of the f o o t appears to be attached. x 40,200 Fig u r e 40. The t h i c k e r p a r t i t i o n of the continuous asymmetric inner p l a t e membrane (IPM) l i e s adjacent to the t h e c a l p l a t e s . Note that there i s no d i s r u p t i o n of the inner p l a t e membrane along i t s length. One would expect such a membrane t o be disrupted at the p o i n t where a suture might be attached. x 110,000 PLATE XIII 43. F i g u r e 41. Thin s e c t i o n through a complex junction of four t h e c a l p l a t e s . The asymmetric t h e c a l (TM) and p l a t e membranes (OPM) are about 15OS o t h i c k having p a r t i t i o n s of 75/50/25A. The sutures (S) appear penta-p a r t i t e as a r e s u l t of the apposition of two outer p l a t e membranes. The t h i c k e r p a r t i t i o n of the outer p l a t e membrane always l i e s adjacent to the p l a t e (W). x 144,000 Figure 42. Fractured surface showing the t h e c a l p l a t e s (W) and the four associated membranes. The t h e c a l membrane (TM) and outer p l a t e membrane (OPM) are p i t t e d ; the inner p l a t e membrane (IPM) s l i g h t l y r e t i c u l a t e , and the plasmalemma (PM) appears undulated. x 14,200 Figu r e 43. The plasmalemma (PM) and inner p l a t e membrane (IPM) appear t o be continuous over the pr o t o p l a s t and independent of the o v e r l y i n g suture (S). x 23,200 PLATE XIV 44. Figures 44 and 45. Continuity of the inner p l a t e membrane (IPM) over the p r o t o p l a s t . The outer surfaces of the inner p l a t e mem-branes bear scars (SC) which may represent s i t e s of previous a s s o c i a t i o n with sutures = outer p l a t e membrane. x 15,360 x 18,600 PLATE XV 45. F i g u r e 46. Densely s t a i n i n g prothecal bodies (PB) as they appear i n non-dividing mature c e l l s . x 12,000 Fig u r e 47. An older c e l l of P. trochoideum. The p r o t h e c a l body (PB) i s composed of a membranous component (MC) and an amorphous com-ponent (AC). Prothecal v e s i c l e s (PV) can be seen forming at the l e f t . x 22,200 Fi g u r e 48. Moderately s t a i n i n g prothecal bodies (PB) as they appear p r i o r to ecdysis. The p r o t h e c a l body at the top has progressed to a f u r t h e r s t a t e of d i f f e r e n t i a t i o n than the one below. Note the d i f f e r e n c e i n r e l a t i v e amounts of amorphous (AC) and membrane com-ponents (MC). x 13,500 Fig u r e 49. F l a t t e n e d prothecal v e s i c l e s (PV) from a prothecal body i n which the amorphous component (AC) has s u b s t a n t i a l l y diminished. x 15,000 PLATE XVI 4 6 . Figures 50 and 51. Progressive stages showing the accumulation of w a l l m a t e r i a l between the plasmalemma (PM) and the inner p l a t e membrane (IPM). x 47,500 x 25,000 Figure 52. Incorporation of prothecal v e s i c l e s (PV) i n t o the w a l l . The f i b r i l l a r m a t e r i a l i n the v e s i c l e s and the membrane of the v e s i c l e s account f o r the moderate s t a i n i n g c h a r a c t e r i s t i c s of the w a l l . x 15,600 Fig u r e 53. Fibrous nature of the newly formed c e l l w a l l (W). Pro-t h e c a l v e s i c l e s (PV) can be seen entrapped within the w a l l . x 25,000 PLATE XVII 47. Figure 54. Nomarski i n t e r f e r e n c e micrograph of a d i v i d i n g c e l l . Note the accumulation of w a l l m a t e r i a l (WM) across the cleavage furrow (arrow). x 1,500 Figure 55. Phase contrast micrograph of a plasmolysed d i v i d i n g c e l l . Note the separation of the w a l l (W) from the o v e r l y i n g inner p l a t e membrane of the parent c e l l (IPMp). x 1,100 Figure 56. E l e c t r o n micrograph of a d i v i d i n g c e l l s i m i l a r to that stage shown i n f i g u r e 54. The c e l l has a r e l a t i v e l y t h i c k w a l l (W). Note the prothecal v e s i c l e s (PV) l i n i n g the p r o t o p l a s t p e r i -phery. x 5,600 PLATE XVIII 48. Figure 57. Formation of prothecal v e s i c l e s (PV) from a p r o t h e c a l body (PB) at the isthmus of a d i v i d i n g c e l l . One of the v e s i c l e s i s being released (arrow). x 39,600 Figure 58. Isthmus of a c e l l i n l a t e c e l l d i v i s i o n . The p r o t h e c a l v e s i c l e s have aligned i n two rows across the isthmus (arrows). These rows represent the s i t e s where the two new c e l l walls of the daughter c e l l s w i l l be formed p r i o r to complete separation. x 25,800 PLATE XIX 49. F i g u r e 59. Small link, between daughter c e l l s . Note the d e f i n i t e l a y e r of w a l l m a t e r i a l present at t h i s s i t e (W). Below the l i g h t e r w a l l l a y e r (W) l i e s a denser layer (DL) which appears to be sandwiched between the w a l l and cytoplasm. x 57,000 Figure 60. Presence of a new inner p l a t e membrane (IPM). The membrane appears to have a r i s e n within the dense l a y e r (DL) beneath the w a l l . Note membrane fragments (arrow) within the w a l l which may be involved i n membrane formation. The parent c e l l inner p l a t e membrane (IPMp) i s s t i l l present. x 104,500 PLATE XX ' 50' F i g u r e 61. Two daughter c e l l s nearly separated. The w a l l m a t e r i a l appears to cover the e n t i r e p r o t o p l a s t without i n t e r r u p t i o n - that i s , there are no p l a t e s or sutures present at t h i s time. x 6,190 PLATE XXI 51. Figure 62. An u n d i f f e r e n t i a t e d theca which lacks f u l l y delineated p l a t e s . The c e l l surface i s r e l a t i v e l y f r e e of p i t s . The f o l d s probably arose during c e n t r i f u g a t i o n of the c e l l s . Note the presence of the f u l l y developed pore (P) with i t s r i d g e . x 25,000 Figure 63. I n i t i a l stages i n the d i f f e r e n t i a t i o n of the sutures. The sutures (S) are s t i l l not deeply scored i n t o the w a l l and are not continuous (arrows). x 15,000 REFERENCES 52. 1. A f z e l i u s , B.A. 1962. The nucleus of Noctiluca s c i n t i l l a n s . Aspects of nucleo-cytoplasmic exchanges and the formation of nuclear membrane. J. C e l l B i o l . 19:229-238. 2. B i s a l p u t r a , T, and B i s a l p u t r a , A.A, 1967a, The occurrence of DNA f i b r i l s i n chlorop l a s t s of Lawrencia s p e c t a b i l i s . J . U l t r a s t r u c t . Res. 17:14-22. 3. B i s a l p u t r a , T. and B i s a l p u t r a , A.A. 1967b. Chloroplast and mitochondrial DNA i n a brown alga Egregia m e n z i e s i i . J . C e l l B i o l . 33:511-520. 4. B i s u l p u t r a , T. and Weier, T.E. 19^3• The c e l l w a l l of Scenedesmus quadricauda. Amer. J . Bot, 50:1011-1019. 5. Bouck, G.B. and Sweeney, B.M. 1966. The f i n e structure and ontogeny of t r i c h o c y s t s i n marine d i n o f l a g e l l a t e s . Protoplasma 61:205-223. 6. Braarud, T. 1957. Observations on Peridinium trochoideum (Stein) Lemm. i n c u l t u r e . Nytt. Mag. Bot. 6:39-42. 7. Buchanan, R.J. 1968, Studies at Oyster Bay i n Jamaica, West Indies. IV. Observations on the morphology and sexual cycle of Pyrodinium  bahamense P l a t e . J.. Phycbl. 4:272-277. 8. Bursa, A, 1958. The freshwater d i n o f l a g e l l a t e Woloszynskia l i m n e t i c a n.sp. Membrane and protoplasmic s t r u c t u r e s , J, Protozool. 5:299-304. 9. Bursa, A, 1966. Ectoplasm as a morphogenetic f a c t o r i n the dino-f l a g e l l a t e Woloszynskia l i m n e t i c a . Verh. i n t . Ver Limnol, 16:1589-1594. 10. Chihara, M. 1968. F i e l d , c u l t u r i n g , and taxonomic studies of ' Ulva f e n e s t r a t a P. and R, and Ulva s c a g e H i sp, nov. (Chlorophyceae) i n B r i t i s h Columbia and Northern Washington. Syesis 1:87-103. 11. Chunosoff, L. and H i r s h f i e l d , H.I. 1967. Nuclear structure and mitosis i n the d i n o f l a g e l l a t e Gonyaulax monilata. J . Pro t o z o o l . 14:157-163. 12. Dickensheets, R.E. 1970. Preliminary studies of the u l t r a s t r u c t u r e of the theca of Peridinium trochoideum (Stein) Lemm, J:. Phycol-C ( s u p p . ) : 1 0 . 13. Dodge,. J , D . 1964. Chromosome structure i n the Dinophyceae. I I Cytochemical s t u d i e s . Arch, M i k r o b i o l . 48:66-80. 14. Dodge, J.D. 1965. Thecal f i n e structure i n the d i n o f l a g e l l a t e genera Prorocentrum and Exuviae11a. J, Mar. B i o l . Ass. U.K. 45:607-614. 53. 15. Dodge, J.D. 1967. Fine structure of d i n o f l a g e l l a t e Aureodinium pigmentosum gen. e t sp. nov. B r i t . Phycol. B u l l . 3 : 327 - 3 3°. 1 6 . Dodge, J.D. 1968. Fine structure of chlorop l a s t s and pyrenoids i n some marine d i n o f l a g e l l a t e s (Aureodinium pigmentosum and Glenodinium sp.) J . C e l l S c i . 3:41-48. 17. Dodge, J.D. and Crawford, R.M. 1968. The f i n e structure of the d i n o f l a g e l l a t e Amphidinium c a r t e r i Hulburt. P r o t i s t o l o g i c a 4 :231-242. 18. Dodge, J.D. and Crawford, R.M. 1969. The f i n e structure of Gymnodinium fuscum (Dinophyceae) New. Phytol. 68:613-618, 19. Dodge, J.D. and Crawford, R.M. 1970. A survey of t h e c a l f i n e structure i n the Dinophyceae. Bot, J . Linn. Soc. London, 63:53-6?. 20. F r i t s c h , F.E. I965. Structure and Reproduction i n the Algae, Vol. I, Cambridge U n i v e r s i t y Press, Cambridge.p, 694. 21. Hay, E.D. 1968, Structure and f u n c t i o n of the nucleolus i n develop-in g c e l l s , i n The Nucleus, edited by A.J. Dalton and F. Haguenau, Academic Press, New York. 22. Kalley, J.P., personal observation, 2 3 . Kevin, M.J., H a l l , M.T., McLaughlin, J.J.A. and Zahl, P.A. 1969. Symbiodinium microadraaticum Freudenthal. A revised taxonomic d e s c r i p t i o n , u l t r a s t u r c t u r e . J . Phycol 5:341-350. 24. Kofoid, C A . 1909. On Peridinium s t e i n i jorgensen, with a note on the nomenclature of the skeleton of the P e r i d i n i d a e 0 Arch. F. Protistenk 1 6 : 2 5-47. 24a Kofoid, C A . and Swenzy, 0. 1921. The fr e e l i v i n g unarmoured D i n o f l a g e l l a t a . Mem. Univ. C a l i f . 5:1-562. 2 5 . Kubai, D.F. and Ri s , H. 1968. D i v i s i o n i n the d i n o f l a g e l l a t e Gyrodiniura c o h n i i ( S c h i l l e r ) . J . C e l l B i o l . 4 0 : 5 0 8 - 5 2 8 . 26. Leadbeater, B. and Dodge, J.D. 1966. The f i n e structure of Woloszynskia miera sp. nov., a new marine D i n o f l a g e l l a t e , B r i t . Phycol, B u l l . 3:1-17. 2 7 . Lebour, M.V. I 9 2 5 . The D i n o f l a g e l l a t e s of the Northern Seas. Mar. B i o l . Ass. U.K., Plymouth. 28. Loeblich A.R, I I I 1969. Thecal u l t r a s t r u c t u r e and composition of modern d i n o f l a g e l l a t e s , J, Paleontology 43:892. 2 9 . Mangiri, L. 1911. M o d i f i c a t i o n s de l a cuirasse des P e r i d i n i e n s . Internat. Rev. Hydrobiol. 4:44-45. 54. 30. Matthys, E. and Puiseux-Dao, S. 1968. Les effets du bromure d'ethidium sur les ultrastructures des mitochondries et des plastes chez l'Amphidinium cart e r i . C.R. Acad. Sci., Paris. 267:2123-2125. 31. Messer, G. and Ben-Shaul, Y. 1969. Fine structure of Peridinium westii Lemm., a freshwater Dinoflagellate. J. Protozool. 16:272-280, 32. Moor, D.H. 1965. Freeze-etching. Balzers High Vacuum. Rep. 2. 33. Mornin, L. and Francis, D. 1967. The fine structure of rtematodinium armatum a naked dinoflagellate. J, Microsc. Paris. 6:759-772. 34. Peters, N. 1927. Das Wachstum dis Peridinium - Panzers. Zool. Anzeig. 73:14-3-148. 35. Rahat, M. 1968. Observations on the l i f e cycle of Peridinium westii i n a mixed culture. Isr, J, Bot. 17:200-206. 36. Reynolds, S. 1963. The use of lead citrate at high pH as an electron opaque stain i n electron microscopy. J. Cell B i o l . 17:208-211. 37. Ris, H. 1962, Interpretation of ultrastructure i n the c e l l nucleus, i n The Interpretation of Ultrastructure, edited by R.CJ. Harris, Academic Press, New York pp. 69-88. 38. Ris, H, and Plant, W. 1962. Ultrastructure of DNA-containing areas i n the chloroplast of Chlamydomonas. J. Cell B i o l . 13:383-391. 39. Soyer, M-0. 1969. L'enveloppe nucleaire chez Noctiluca miliaris Suriray (Dinoflagellata). I. Quelques donnees sur son ultrastructure et son,evolution au cours de l a sporogenese. J, Microsc. Paris, 8:569-580. 40. Spurr, A.R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy, J„ Ultrastruct. Res. 26:31-43. 41. Swift, E. and Remsen, C.C. 1970. The c e l l wall of Pyrocystis spp. (Dinococcales). J. Phycol, 6:79-86. 42. Taylor, D.L. 1968. In s i t u studies on cytochemistry and ultra-strucutre of a symbiotic marine dinoflagellate. J. Mar, Bio l . Ass. U.K. 48:349-366. 43. Taylor, F.J.R., personal cmmunication. 44. Taylor, F.J.R., 1962. Gonyaulax polygramma Stein i n Cape Waters: A taxonomic problem related to developmental morphology, J.S.Afr. Bot. 38:237-242. 45. Taylor, F.J.R, 1969. Peri-nuclear structural elements formed i n the dinoflagellate Gonyaulaux pacifica Kofoid. Protistologica 2:165-167. 46, Zingmark, R.G. 1970. Ultrastructural studies on two kinds of mesocrayotic dinoflagellate nuclei. Amer, J. Bot. 57 !586-592. APPENDIX CHIHARA MARINE MEDIUM To 1000 cc. f i l t e r e d * sea water add: materials quantity minor elements (see below) NaNOo NaHgPfy.lZHgO 2 cc. 0.200 g. 0.025 g. Minor Elements Solution To 1000 cc. d i s t i l l e d water add: materials quantity EDTA - Mag 3.0000 g. FeCl3 . 61^0 0.0800 g. MnCLg . 4H 20 0.1200 g. ZnCl 2 0.0150 g. CoCl 2 . 211^ 0 0.0030 g. CuCl 2 . 21-IgO 0.0012 g. NagMoO^  . 2H 20 0.0500 g. H 3B0 3 0.6000 g. * millipore f i l t e r ; pore diameter 0.22^. 

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