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Isolation and ultrastructural study of a lytic virus in the small marine phytoflagellate, Micromonas… Mayer, Jolie Arden 1978

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ISOLATION AND ULTRASTRUCTURAL STUDY OF A LYTIC VIRUS IN THE SMALL MARINE PHYTOFLAGELLATE, MICROMONAS PUSILLA (PRASINOPHYCEAE) by JOLIE ARDEN MAYER B . S c , Stanford Un i ve r s i t y , 1972 M.A., Stanford Un i ve r s i t y , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept th i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1978 @ J o l i e Arden Mayer In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that 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 a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thou t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 i i ABSTRACT The d i scovery, i s o l a t i o n , and u l t r a s t r u c t u r a l inves t i ga t ion of a l y t i c v i rus i n f ec t i on in the naked, marine phy to f l a ge l l a te , Micromonas p u s i l l a , are descr ibed. Infected c e l l s were f i r s t seen in A p r i l , 1975, during a transmission e lec t ron microscope examination of a week old enrichment cu l ture contain ing nanoplankton c o l l e c t e d at the mouth of the Burrard In let in Vancouver, B r i t i s h Columbia. Se lec t i ve l y s i s occurred in th i s c u l t u r e » removing near ly a l l M. p u s i l l a c e l l s without v i s i b l y a f f e c t i n g the other m i c ro f l a ge l l a te populat ions. U l t r a s t ruc tu ra l examination of the cu l ture ind icated the presence of polyhedral inc lus ions with in c e l l s of M. p u s i l l a . Medium from th i s i n fec ted cu l ture was observed to cause the destruct ion of unia lga l s t r a in s of M. p u s i l l a maintained in the Northeast P a c i f i c Culture C o l l e c t i o n . U l t r a s t ruc tu ra l s tudies o f the host organism, M. pusi11a, confirmed the e a r l i e r f ind ings of Manton and Parke (1959) and provided add i t iona l information on the f i ne s t ructure of th i s nanof l age l l a te . Terminal f l a g e l l a r ha i r s , a v e s t i g i a l , second basal body, and a per iphera l system of microtubules are descr ibed. The polyhedral v i rus p a r t i c l e s in s i t u wi th in in fec ted c e l l s , or when i so l a ted and negat ive ly sta ined with uranyl acetate , appear t a i l - l e s s and present p r o f i l e s cons i s tent with t h e i r having icosahedral symmetry. The v i r i on s have an average diameter of 135 nm. Surface substructures analogous to v i r a l capsomeres were observed on p a r t i a l l y d isrupted p a r t i c l e s . V i r a l entry takes place with in 15 minutes fo l lowing introduct ion of inoculum and occurs by l o c a l i z e d fus ion of adjacent c e l l and v i rus sur faces , i i i fol lowed by d i r e c t penetrat ion of v i r a l genome. A f t e r an ec l i p se period of approximately three hours in durat ion progeny v i rus appear in the per inuc-lear cytoplasm. Associated with the v i r a l assembly process are cytoplasmic f i b r i l s and c lu s te r s of membrane-bound v e s i c l e s . V i r a l egress occurs by l o c a l i z e d rupture of the plasmalemma. V i r a l a c t i v i t y i s monitored by an assay system based upon the reduction in the in vivo chlorophyl1 a_ f luorescence of in fected c e l l s . Solut ions of v i rus p a r t i c l e s stored at 4°C in m i l l i p o r e - f i l t e r e d , autoclaved seawater or cu l ture medium re ta in t h e i r i n f e c t i v e capac i ty f o r nine months. The v i rus i s s tab le in a pH range between 4 and 10. Thermal i nac t i v a t i on occurs between 55° and 63°C. Host range studies ind ica te that the l y t i c propert ies of the v i rus are probably s p e c i f i c to M. p u s i l l a . The v i rus has been designated Micrbmbnas p u s i l l a v i r u s , MPV. i v TABLE OF CONTENTS ABSTRACT .. i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES vi i i ACKNOWLEDGEMENTS x v i i INTRODUCTION 1 MATERIALS AND METHODS 15 I. ALGAL MAINTENANCE AND GENERAL TECHNIQUES . . . 15 1.1. Cultures 15 1 .2. Ce l l counts 17 II. VIRUS MAINTENANCE AND GENERAL TECHNIQUES 17 11.1. General maintenance 17 11.2. General experimental techniques 17 11.3. Virus f i l t e r a b i l i t y 18 III. LIGHT MICROSCOPY 18 IV. ELECTRON MICROSCOPY 18 IV.1. Whole mounts - metal-shadowed c e l l s 18 IV.2. Th in-sect ioned material 19 IV.3. Scanning e lect ron microscopy 22 IV.4. Negative s ta in ing of v i r i on s 23 IV.5. Metal-shadowed v i rus 23 V. FLUOROMETRIC METHODS 23 V . l . F luorometric apparatus and general procedures 23 V.2. Determination of p ropo r t i ona l i t y and l i n e a r i t y 24 V.3. Cor re la t ion between in vivo f luorescence and c e l l dens i ty 25 V.4. "N i te -Owl " , 48 hour experiment 26 V Table of Contents (Continued) VI. HOST RANGE STUDIES 29 VII. VIRUS STABILITY - PHYSICAL AND CHEMICAL PROPERTIES 29 LIST OF ABBREVIATIONS USED 32 OBSERVATIONS AND RESULTS 33 I. MICROMONAS PUSILLA: GENERAL CHARACTERISTICS AND ULTRASTRUCTURE , 33 1.1. H i s t o r i c a l survey 33 1.2. General c e l l s t ructure 36 1.3. The ch lo rop la s t 42 1.4. The mitochondrion 45 1.5. The nucleus and nuclear-ER system 48 1.6. The golgi and ER system 52 1.7. The f l a g e l l a r apparatus and associated cytoplasmic microtubules 52 1.8. Miscel laneous inc lus ions 64 II. VIRUS MORPHOLOGY 66 III. ULTRASTRUCTURAL ASPECTS OF THE INFECTION PROCESS 76 111.1. Ear ly host -v i rus i n te rac t i on 76 111.2. I n t r a c e l l u l a r morphological features of the i n f e c t i o n 83 111.3. Observations on v i r a l re lease — . 92 IV. DEVELOPMENT OF A FLUORESCENCE ASSAY FOR VIRAL ACTIVITY 96 IV.1. Culture growth studies 96 IV.2. In vivo f luorescence and cu l ture death 99 vi Table of Contents (Continued) DISCUSSION 105 I. MICROMONAS PUSILLA: ULTRASTRUCTURE 105 II. THE VIRUS 112 III. ULTRASTRUCTURAL ASPECTS OF THE INFECTION PROCESS 118 IV. THE USEFULNESS OF IN VIVO FLUORESCENCE AS AN ASSAY OF VIRAL INFECTIVITY 126 SUMMARY AND CONCLUDING REMARKS 130 APPENDIX I 134 HOST RANGE 134 APPENDIX II_ 137 PHYSICOCHEMICAL PROPERTIES OF THE VIRUS 137 APPENDIX III 141 PROCEDURES FOR COLLECTING AND CONCENTRATING MPV PARTICLES -PRELIMINARY RESULTS 141 LITERATURE CITED 144 v i i LIST OF TABLES Table I. Previous studies of eukaryot ic v iruses and VLPs . . . . . . . 2 Table II. I solates of Micromonas p u s i l l a 16 Table III. F i xa t ion and dehydration schedule I 20 Table IV. F ixa t ion and dehydration schedule II 21 Table V. "Nite-Owl" experiment 28 Table VI. Host s p e c i f i c i t y 135 Table VII. Physicochemical propert ies of v i rus 139 v i i i LIST OF FIGURES F i g . 1. Phase in ter ference l i g h t micrograph of M. p u s i l l a . . 40 F i g . 2. A long i tud ina l sect ion through a c e l l showing r e l a t i v e pos i t i on of major c e l l organel les 40 F i g . 3. Whole c e l l preparat ion: specimen f i xed in osmic varor on copper g r i d then shadowed with evaporated platinum and graphite 40 F i g . 4. Scanning e lec t ron micrograph: specimen was c r i t i c a l point dr ied on nucleopore f i l t e r s of pore diameter 0.4 um. . . 40 F i g . 5. Transverse sect ion through a c e l l — 43 F i g . 6. Longitudinal sect ion through a c e l l o r ientated perpend icu lar ly with respect to sect ion shown in F i g . 5 43 F i g . 7. Oblique sect ion showing c l o se l y packed thylakoids 43 F i g . 8. High magni f icat ion of stacked thylakoids 43 F i g . 9. High magni f icat ion of pyrenoid-starch sheath complex 43 F i g . 10. Sect ion through a c e l l showing an ea r l y d i v i s i o n stage of the ch lorop la s t and pyrenoid 43 F i g . 11. Late d i v i s i o n stage of the ch lorop la s t 43 F i g . 12. Longitudinal sect ion through a c e l l i l l u s t r a t i n g i t s p o l a r i t y 46 F i g . 13. Transverse sect ion i l l u s t r a t i n g pos i t i on of mitochondrion with in the centra l depression of the ch lorop la s t 46 ix L i s t of Figures (Continued) F i g . 14. Two small p r o f i l e s of the mitochondrion are shown demonstrating the contoured form — 46 F i g . 15. High magn i f i cat ion of a mitochondrion 46 F i g . 16. Longitudinal sect ion through a c e l l . 49 F i g . 17. Longitudinal sect ion or ientated at ca. 90° ro ta t ion with respect to F i g . 16 49 F i g . 18. High magni f icat ion showing s t ructure and contents o f per inuc lear channel 49 F i g . 19. Channel of nuclear ER f i l l e d with long f i be r s (cf . F i g . 18). 49 F i g . 20. Sect ion showing swollen nuclear envelope conta in ing unorganized f i b r i l l a r material 49 F i g . 21. Oblique sect ion through golgi body 53 F i g . 22. Longitudinal sect ion through f l age l lum and basal body 53 F i g . 23. Section through the mu l t i ve s i cu l a te body showing i t s contents to be a heterogeneous c o l l e c t i o n of ve s i c l e s and membranous elements 53 F i g . 24. Sect ion showing shape and pos i t i on of mitochondrion, golg i body and f l a g e l l a r apparatus 53 X L i s t of Figures (Continued) F i g . 25. Flagel lum and mitochondrion are moving out of the plane of sect ion simultaneously as nucleus and pyrenoid starch sheath begin to appear 53 F i g . 26. The cytoplasmic extension of the nuclear space, i . e . per inuc lear region (PER, th i s F i g . on l y ) , i s beginning to appear 53 F i g . 27. Pyrenoid begins to appear as golg i body moves out of the plane of sect ion 53 F i g . 28. Shadowed whole mount of f l age l lum showing terminal hairs 56 F i g . 29. Longitudinal sect ion o f junct ion between proximal f l a g e l l a r region and d i s t a l extension 56 F i g . 30. Longitudinal sect ion through f l a g e l l a r t r a n s i t i o n zone, basal p l a t e , and d i s t a l port ion of the basal body 56 F i g . 31. Cross sect ion through f l a g e l l a r axoneme. 56 F i g . 32. Cross sect ion through "tapered c y l i n d e r " reg ion, shown here as a membrane surrounding the centra l two microtubules. 56 F i g . 33. Cross sect ion below basal p la te 56 F i g . 34. Cross sect ion through d i s t a l end of basal body — 56 F i g . 35. Longitudinal view of an i n t r a c e l l u l a r axoneme showing a 9+2 set of microtubules l y i ng ins ide the plasmalemma 56 xi L i s t of Figures (Continued) F i g . 36. Cross sect ion through i n t r a c e l l u l a r axoneme which i s surrounded by lobed nucleus . . . . 56 F i g . 37. Cross sect ion through f l a g e l l a r basal body (bb-1) and second non-funct ional basal body (bb-2) 59 F i g . 38. Longitudinal sect ion through funct iona l and non-funct iona l basal bodies (bb-2). 59 F i g . 39. D i v i s i on of the f l a g e l l a r apparatus: c e l l contain ing three basal bodies shown in transverse and long i tud ina l views 59 F i g . 40. Longitudinal sect ion through c e l l containing four basal bodies. 59 F i g . 41. Sect ion showing two mucocyst-1ike bodies (MCB) located adjacent to c e l l surface and near basal bodies 59 F i g . 42. Sect ion through two MCBs which are interconnected. 59 F i g . 43. Transverse sect ion showing the two systems of per iphera l microtubules (MT) 62 F i g . 44. Higher magni f icat ion showing axoneme and associated s ing le MT 62 F i g . 45. High magni f icat ion of pa ired per ipheral MTs located with in a protruding r idge of cytoplasm external to the ch lo rop la s t 62 F i g . 46. Longitudinal view of f l a g e l l a r apparatus and assoc iated cytoplasmic MTs 62 xi i L i s t of Figures (Continued) F i g . 47. Longitudinal sect ion through f l a g e l l a r t r a n s i t i o n zone and basal body 62 F i g . 48. Cross sect ion through r idge of cytoplasm conta in -ing the s ing le per ipheral microtubule 62 F i g . 49. Longitudinal view of per ipheral microtubule system 62 F i g . 50. Glancing long i tud ina l sect ion showing doublet MTs 62 F i g . 51. Infected c e l l contain ing polyhedral p a r t i c l e s 67 F i g . 52. Thick sect ion through an in fec ted c e l l showing numerous polyhedral v i rus p a r t i c l e s containing densely s ta in ing core s t ruc tures . 67 F i g . 53. V i r i o n , i l l u s t r a t i n g f i v e - f o l d symmetry, has absorbed uranyl acetate to become p o s i t i v e l y contrasted 70 F i g . 54. Hexagonally shaped p a r t i c l e i l l u s t r a t i n g three-f o l d axis of symmetry 70 F i g . 55. Negatively s ta ined v i rus p a r t i c l e showing two-f o l d axis of symmetry 70 F i g . 56. P a r t i c l e demonstrating f i v e - f o l d symmetry 70 F i g . 57. P a r t i c l e demonstrating t h ree - f o l d symmetry 70 F i g . 58. P a r t i c l e demonstrating two-fold symmetry 70 F i g . 59. Metal-shadowed v i r i on s f i xed in uranyl acetate p r i o r to shadowing to diminish d i s t o r t i o n due to drying 73 xi i i L i s t of Figures (Continued) F i g . 60. Higher magni f icat ion of a v i r i o n shown in F i g . 59 73 F i g . 61. Negatively contrasted v i rus p a r t i c l e showing prominence of ap ica l region resembling that seen in F i g . 60 73 F i g . 62. Two negat ive ly contrasted specimens 73 F i g . 63. P o s i t i v e l y contrasted v i r i o n showing d i s t i n c t per iphera l banding 73 F i g . 64. Morphological subunits in terpreted to be capsomeres are shown organized into a t r i angu l a r array 73 F i g . 65. Subunits v i s i b l e here do not demonstrate obvious pattern of organ izat ion 73 F i g . 66. Ce l l with attached v i r i on s p o s i t i v e l y contrasted with uranyl acetate 78 F i g . 67. Higher magni f icat ion of v i rus p a r t i c l e s on f l a g e l l a r surface 78 F i g . 68. Section showing ear l y stage of v i r a l attachment 78 F i g . 69. V i r i o n on c e l l surface 78 F i g . 70. Two v i r i o n s on c e l l sur face, one showing densely s ta in ing core 78 F i g . 71. Sect ion through v i r i o n at c e l l / s u r f a c e 78 F i g . 72. Glancing sect ion through attaching v i r i o n • 81 F i g . 73. Attaching v i r i o n is shown on protrus ion produced at s i t e of contact 81 F i g . 74. V i r i o n attached by " s t a l k " - l i k e p ro jec t ion to c e l l surface 81 xiv L i s t of Figures (Continued) F i g . 75. F i b r i l l a r material v i s i b l e wi th in v i r i o n i s a l so apparent ins ide host, adjacent to attachment s i t e 81 F i g . 76. Apparent t rans fe r of e lec t ron dense material between v i r i o n and host c e l l 81 F i g . 77. Sect ion through attaching v i r i o n showing associated s ta lk or t a i l - l i k e p ro jec t ion 81 F i g . 78. Sect ion through attaching v i r i o n 81 F i g . 79. Section through attaching v i r i o n . 81 F i g . 80. Sect ion through c e l l containing numerous membrane bound ves i c l e s (mbv) and one la rger polyhedral v i r a l p a r t i c l e 84 F i g . 81. High magni f icat ion of mbv's and polyhedral p a r t i c l e located adjacent to surface of ER 84 F i g . 82. Ce l l contain ing abnormally ac t i ve golg i body. 84 F i g . 83. Higher magni f icat ion of golg i body and associated s t ructures 84 F i g . 84. Sect ion through c e l l contain ing polyhedral v i rus p a r t i c l e s , tnbv's and large amounts of cytoplasmic f i b r i l s - 87 F i g . 85. High magni f icat ion of cytoplasmic f i b r i l s 87 F i g . 86. Section showing region of densely sta ined cytoplasm (arrow) pos i t ioned beside nuclear envelope 87 F i g . 87. Ce l l with d iv ided ch lo rop la s t showing evidence of v i r a l i n f ec t i on 87 XV L i s t of Figures (Continued) F i g . 88. Ce l l containing extensive amounts of cytoplasmic f i b r i l s , and l im i ted numbers of mby's and v i rus p a r t i c l e s 90 F i g . 89. Numerous v i r a l p a r t i c l e s located near periphery of c e l l 90 F i g . 90. Ce l l showing extensive cytoplasmic d i sorgan izat ion 90 F i g . 91. F ina l stages of i n f e c t i o n 90 F i g . 92. Late i n f e c t i o n stage showing v i r i on s near periphery of c e l l 93 F i g . 93. mbv's and v i rus p a r t i c l e s shown gathering at periphery of c e l l . 93 F i g . 94. Loca l i zed b l i s t e r i n g of c e l l 93 F i g . 95. Plasmalemma beginning to rupture adjacent to s i t e of v i r i o n attachment 93 F i g . 96. Loca l i zed rupture of c e l l surface is complete, a l lowing re lease of v i r i on s 93 F i g . 97. Loca l i zed rupture of c e l l surface 93 F i g . 98. In vivo ch lorophy l l ^ f l u o r e s c e n c e of f l a sk and tube batch cu l tures of M. p u s i l l a . 97 F i g . 99. In vivo ch lorophy l l a_ f luorescence and c e l l dens i ty of M. p u s i l l a grown in tube batch cu l tures 100 xv i L i s t of Figures (Continued) F i g . TOO. Results of "Nite-Owl" experiment: The e f f e c t of the add i t ion of v i rus on in vivo f luorescence and c e l l dens i ty of M. p u s i l l a grown in tube batch cu l tures 102 F i g . 101. Pre l iminary procedures f o r c o l l e c t i n g and concentrat ing MPV p a r t i c l e s 143 xv i i ACKNOWLEDGEMENTS I wish to express my s incere apprec iat ion to my research superv i sor , Dr. F. J . R. Tay lo r , f o r his pat ience, adv ice, to lerance, and support throughout th i s i n ve s t i g a t i on . His valuable c r i t i c i s m s and ass i s tance during the preparat ion of th i s manuscript are g r a t e f u l l y acknowledged. I would a lso l i k e to thank Drs. T. B i s a l pu t r a , R. I. Hamilton, and P. J . Harrison f o r t h e i r c r i t i c a l reading of the manuscript. Numerous people have provided ass i s tance throughout the various aspects of my s tud ies . I thank Dr. T. B i sa lput ra f o r generously o f f e r i n g the extensive use of his e lec t ron microscope and photographic f a c i l i t i e s , and Drs. P. J . Harrison and R. I. Hamilton f o r sharing t h e i r s c i e n t i f i c exper t i se . I would e s p e c i a l l y l i k e to thank Mr. L. Veto f o r his a s s i s t -ance and exce l len t technica l advice on e lec t ron microscopy and photography, and Mrs. R. Waters, whose knowledge of v i ruses and technica l expert i se with t iny f l a g e l l a t e s i n i t i a t e d th i s i nve s t i g a t i on . I a lso thank Mrs. M. Douglas and my s i s t e r Candace f o r t h e i r ass i s tance with the l i n e drawings, and Ms. L e s l i e Borleske fo r her rap id and extremely accurate typing of the thes i s . I w i l l always remember the i n sp i r a t i on and encouragement I received from my f r i ends . In p a r t i c u l a r , I thank B i l l Thompson fo r his help with data processing and s t a t i s t i c a l analyses, and Ariane Neleman fo r her ass i s tance with the typing of ea r l y dra f t s of th i s manuscript. I. am e s p e c i a l l y gratefu l to Steve Hosford f o r his enthus ia s t i c support and his extensive ass i s tance in preparat ion of the photographic p la tes . His thoughtfulness w i l l always be remembered and appreciated. x v i i i The greatest thanks of a l l must go to my husband, Roy, who persevered through those years when the end seemed out of s i ght . Without his c o n t i n -ued support, encouragement, to lerance, and good s p i r i t s the d i f f i c u l t i e s involved in preparing f o r a Ph.D. degree would have been unbearable. During the tenure of th i s i nves t i ga t ion the author was the r e c i p i e n t of a Ki l lam Predoctoral Fel lowship and a Un iver s i t y of B r i t i s h Columbia Graduate Fel lowship. This research was supported by National Research Council of Canada grant #A"-6137 to Dr. F. J . R. Tay lor . 1 INTRODUCTION From the l i t e r a t u r e on a lga l v iruses i t i s apparent that near ly a l l studies published to date have been examinations of v iruses i n fec t i n g prokaryot ic , blue-green algae (cyanobacter ia) . At present very l i t t l e i s known about v iruses in the c e l l s of eukaryot ic algae. With the exceptions of a suggestion that the l y s i s of C h l o r e l l a pyrenoidosa might be caused by a v i r a l i n f e c t i o n (Zavarzina and Protsenko, 1958) there were no hints of v i rus i n fec t ions in eukaryot ic algae un t i l the present decade. Since 1970, numerous pub l i ca t ions c i t i n g observations of v i r u s - l i k e p a r t i c l e s (VLPs) ins ide c e l l s of eukaryotes have appeared in the l i t e r a t u r e . These reports have been l im i ted l a r ge l y to de sc r i p t i ve accounts of VLPs apparently found i n c i d e n t a l l y in the course of u l t r a s t r u c t u r a l studies of p a r t i c u l a r algae. (For an exce l l en t summary of the ear l y work done with a lgal v i ruses and v iruses of cyanobacteria the reader i s re fer red to a review by Brown, 1972.) General ly these VLPs appear polygonal in u l t r a t h i n sect ion (e i ther 5- or 6-sided) and are found assoc iated with var i ab le degrees of cytoplasmic d i sorgan izat ion or d i s rupt ion of the a lga l host. VLPs have been re fer red to as v i ruses by some authors. However, with only two exceptions (Gibbs et a l . , 1975; O l i v e i r a and B i s a l pu t ra , 1978), proof of the v i r a l nature of the i n f e c t i o n ( i . e . p a r t i c l e transmiss ion into healthy c e l l s ) remains to be demonstrated and the q u a l i f i c a t i o n " v i r u s - l i k e " must s t i l l be app l i ed . A b r i e f h i s t o ry of per t inent e a r l i e r observations fo l l ows , of which a summary i s provided in Table I. TABLE I. Previous Studies of E u k a r y o t i c V i r u s e s REFERENCE SOURCE Zav a r z i n a & Protsenko (1958) Zav a r z i n a (1961, 1964) Tikhonenko & Z a v a r z i n a (1966) Schnepf e t a l . (1970) Mixed protozoan c u l t u r e Lysate Open a i r basins Germany Lee (1971) Mattox e t a l . (1972) C u l t u r e C o l l e c t i o n Indiana Univ. CCIU #1499 CCIU #174, #610, #1252, and #439 Pickett-Heaps (1972) Toth .& Wilce (1972) Baker & Evans (1973) C l i t h e r o e & Evans (1974) Chapman & Lang (1973) Swale & B e l c h e r (1973) Freshwater Lake A u s t r a l i a A t l a n t i c Ocean Hodgkins Cove, Mass. A t l a n t i c Ocean Coast o f B r i t a i n CCIU #161 Wild sample Hoffman & Stanker (1974, 1976) Manton & Leadbeater Danish nanoplankton (1974) Wild sample Moestrup & Thomsen (1974) ' Water samples I s e f j o r d (Tempelkrogen) and VLPs ' HOST C h l o r e l l a pyrsnoidosa Aphelidium i n Scenedesmus armatus S i r o d o t i a tenuissima Uronema gfgag '. Choleochaetae s c u t a t a  Radiofilum t r a n s v e r s a l e  Stiqeocloniurn farctum Oedogonium  Chorda tomentosa  Ectocarpus f a s c i c u l a t u s Porphyridium purpureum Aulacomonas ( c o l o r l e s s f l a g e l l a t e ) C y l i n d r o c a p s a Chrysochromulina mantorn'ac Pyramimonas o r i e n t a l i s HOST TYPE Ch l o r o / ankton phycomycete p a r a s i t e o f C h l o r o / p l a n k t o n Rhodo/FW/benthic Chloro/FW/benthic Chloro/FW/benthic Phaeo/marine/benthic Phaeo/marine/benthic Rhodo/FW/plankton Chloro/FW/plankton Chloro/FW/benthic Hapto/marine/plankton Prasino/marine/plankton DIMENSION 30-40 nm 200 nm 50-60 nm 240 nm ro 170 nm 150-170 nm 40 nm 200 nm 200-230 nm 120 nm a) 60 nm b) 200 nm TABLE I. Cont'd REFERENCE Markey (1974) Pearson & N o r r i s (1974) Dodds e t a l . (1975) Gibbs e t . a l . (1975) S k o t n i k i et a l . (1976) SOURCE Wild sample P a c i f i c Ocean C u l t u r e Santa C a t a l i n a Island CCIU § 174 Freshwater Lake A u s t r a l i a HOST P y l a i e l l a l i t t o r a l i s  Platymonas sp. Uronema gigas Chara c o r a l l i n a Pienaar (1975) Franca (1976) La C l a i r e & West (1977) Hoffman (1978) Soyer (1978) Nanoplankton San Juan Islands, USA Wild sample, enrichment c u l t u r e s Portugal Wild material Wild material a) Hvmenomonas b) Cryptomonas. c) .HicrpmonAs, Gyrodinium  resplendens Streblonema  Hydrurus f o e t i d u s Blastodinium O l i v e i r a & B i s a l p u t r a (1978) CCIU LB #1534 Sorocarpus uvaeformis Shape i s polyhedral unless s t a t e d . 'Algal c l a s s , e.g. Chloro represents Chlorophyceae. 3FW = freshwater. Host growth h a b i t , i . e . planktom'c or bent h i c . HOST TYPE Phaeo/marine/benthic Prasino/marine/plankton Chloro/FW/benthic Charo/FW/benthic a) Hapto ( a l l b) Crypto Marine c) Prasino plankton) Dino/marine/plankton Phaeo/marine/benthic Chryso/FW/plankton D i n o ( p a r a s i t i c ) / m a r i n e / plankton Phaeo/marine/benthic DIMENSION 130-170 nm 60 nm 400 nm (head) 1000 nm ( t a i l ) rod-shaped 532 nm by 18 nm a) 65 nm b) 99 nm & 240 nm c) 129 nm 35 nm ( c r y s t a l ) 20 nm (po l y -gonal ) 135-150 nm 200-230 nm 30 nm 170 nm 4 Previous studies of eukaryot ic a lga l v i ruses Zavarzina and Protsenko (1958) were the f i r s t researchers to suggest the existence of a v i r a l i n f ec t i on in a eukaryot ic a lga. In lysed cu l tures of C h l o r e l l a pyrenoidosa they found large numbers of spher ica l p a r t i c l e s , ca. 30 to 40 nm in diameter. They suggested these p a r t i c l e s resembled e i t he r higher plant v iruses or bacteriophages, but provided no photographic evidence. The presumptive v i rus maintained i n f e c t i v i t y when exposed to temperatures as high as 60°C f o r 10 minutes; longer exposures at t h i s temperature caused i n a c t i v a t i o n . Further studies by Zavarzina (1961, 1964) prompted her to conclude that the presumptive v i rus was morphological ly more s im i l a r to bacteriophages than to higher p lant v i ruses . The VLPs were also seen attached to Caulobacter v i b r i o i d e s , a paras i te of C h l o r e l l a  pyrenoidosa. There was no observed l y s i s of the paras i te and Zavarzina (1964) suggested that v i r a l i n f e c t i o n might occur through the paras i t i sm of C_. v i b r i o i de s on C_. pyrenoidosa. The morphology of th i s l y t i c agent was described in more de ta i l by Tikhonenko and Zavarzina (1966). Using phosphotungstic ac id as a negative s ta in they were able to d i s t ingu i sh the s o - ca l l ed "Chlorel lophage" as a polyhedral p a r t i c l e ca. 41 nm in diameter possessing a t a i l 24 nm in length. In 1970, Schnepf et a l . reported t h e i r observations of l y s i s of the aquatic phycomycete, Aphel idium, which was p a r a s i t i z i n g the green alga Scenedesmus armatus. Only the paras i te showed l y s i s , the a lga l host remained i n t a c t . Associated with th i s l y t i c a c t i v i t y were polyhedral VLPs measuring ca. 200 nm in diameter. No evidence of i n f e c t i v i t y was provided. One year l a t e r these researchers reported rod-shaped VLPs occurr ing in the 5 cytoplasm of a number of Aphel idiurn- infected Scenedesmus c e l l s (Schnepf et a l . , 1971). These p a r t i c l e s measured 35 nm in length and 10 nm in diameter, and looked qu i te d i f f e r e n t than the polyhedral p a r t i c l e s observed in the pa ra s i te . Lee (1971) reported c r y s t a l l i n e arrays of polyhedral VLPs in the freshwater red a l ga , S i rodot i a tenuiss ima. The p a r t i c l e s , which measured 50 to 60 nm, occurred f ree in the cytoplasm and did not appear to cause any c e l l l y s i s . Plants contain ing the putat ive v i r i on s were cu l tured in f l a sk s contain ing uninfected species of S i rodot i a and a re la ted genus, Batrachospermum. No evidence of transmiss ion of the VLPs was detected. VLPs were reported by Pickett-Heaps (1972) in germlings of the freshwater green alga Oedogonium. The polyhedral p a r t i c l e s were larger than any descr ibed prev ious ly , measuring 240 nm in diameter, and appeared to be assoc iated with an abnormal accumulation of cytoplasm in the basal ha l f of the germling c e l l s . Young germlings contained fewer p a r t i c l e s than d id o lder germlings, the l a t t e r correspondingly showed more cytoplasmic d i s rup t i on . The VLPs were observed only in germling stages of the plants and Pickett-Heaps suggested that i n f ec t i on would most l i k e l y occur while the alga ex i s ted as naked, mot i le zoospores. No VLPs were observed in the zoospores however. During a study of f i n e s t ructure in u lo t r i cha lean a lgae, Mattox et a l . (1972) observed v i r u s - l i k e inc lus ions in four genera of algae. They described polygonal p a r t i c l e s in Uronema gigas, Coleochaete scutata and Radiof i lum t ransver sa le . These p a r t i c l e s occurred e i t he r in the nuclear region or scattered throughout the cytoplasm of c e l l s contain ing d isrupted n u c l e i . Also included in this report was a s ing le micrograph of Stigeocloneum 6 fareturn showing c e l l s contain ing f i b r i l l a r inc lus ions ins ide the ch lo rop la s t . No information about any of the p a r t i c l e s izes was provided. More de ta i l was provided however in a report by Dodds et a l . (1975) who a l so observed VLPs in Uronema gigas. They descr ibed p a r t i c l e s s i g n i f i c a n t l y la rger than any reported prev ious ly , measuring ca. 400 nm in sec t i on . P a r t i c l e s negat ive ly sta ined in 2% uranyl acetate revealed a head component (ca. 500 nm in diameter) and a t a i l component (ca. 1000 nm in length, and seen only 20% of the t ime). Toth and Wilce (1972) were the f i r s t to report evidence of a v i r a l - l i k e i n f ec t i on in a marine eukaryot ic a lga. Free-swimming three-day-o ld zoospores of the brown alga Chorda tomentosa were found to contain polyhed-ra l p a r t i c l e s approximately 170 nm in diameter. The in fected spores d isp layed signs of abnormal development and u l t r a s t r u c t u r e . Normal c e l l wall formation appeared to be i n h i b i t e d and no signs of germination were observed. Ce l l organel les showed abnormal morphology: ch lo rop la s t thylakoids were convoluted and separated, and mitochondria appeared swollen and d i s t o r t e d . Toth and Wi lce, l i k e Pickett-Heaps (1975), suggested that i n f e c t i v e p a r t i c l e s might enter during the very e a r l i e s t stages of zoospores development, while the c e l l s are s t i l l naked and mot i le . Chapman and Lang (1973) publ ished a b r i e f report of polyhedral VLPs in the freshwater red alga Porphyridium purpureum. The p a r t i c l e s were 40 nm in diameter, and appeared morpholog ica l ly s im i l a r to those descr ibed by Lee (1971) f o r S i r o d o t i a . In two e a r l i e r pub l i cat ions Chapman et a l . (1971) and Chapman (1972) reported unusual nuclear inc lus ions in th i s alga which were termed "concentrosomes". These bodies which measured between 50 to 100 nm in diameter and which appeared u l t r a s t r u c t u r a l l y as 7 " c o n c e n t r i c a l l y arranged, e lec t ron opaque, c i r c u l a r p r o f i l e s " were a lso observed in the polyhedral p a r t i c l e - i n f e c t e d c e l l s . This simultaneous occurrence led the authors to suggest an ontogenetic re l a t i onsh ip might e x i s t between the two. No u l t r a s t r u c t u r a l evidence was o f fered to support th i s conc lus ion. A presumptive v i r a l i n f e c t i o n was reported by Swale and Belcher (1973) f o r the sma l l , co l o r l e s s vol vocalean f l a g e l l a t e Aulacomonas. Polyhedral p a r t i c l e s were observed which measured 200 to 230 nm in diameter. Ce l l s contain ing these p a r t i c l e s showed u l t r a s t r u c t u r a l d i sorgan izat ion and loss of nuclear membranes. The authors reported what they be l ieved to be associated t a i l s t ruc tures , and therefore suggested that the p a r t i c l e s resembled bacteriophages. As Aulacomonas i s a phagotrophic f l a g e l l a t e , i t i s poss ib le that the i n f e c t i o n could occur during feeding. A study of the benthic brown alga Ectocarpus f a sc i cu l a tu s by Baker and Evans (1973) revealed the occasional occurrence of po lyhedra l , v i r u s - l i k e p a r t i c l e s (ca. 150 nm in diameter) in mot i le zoospores. The p a r t i c l e s occupied the an te r i o r regions of the c e l l s , the area normally occupied by the nucleus. The p a r t i c l e s appeared to d i s rupt normal cy to log i ca l development and seemed to i n h i b i t normal s e t t l i n g and attachment of the c e l l s . In a re l a ted study C l i theroe and Evans (1974) observed s i m i l a r p a r t i c l e s (ca. 170 nm) in the p l u r i l o c u l a r sporangia of a wi ld sample of Ectocarpus. The zoospores ins ide the in fected sporangia showed evidence of p a r t i c u l a t e inc lus ions and disrupted nuclear membranes, while zoospores in uninfected sporangia showed d i s t i n c t and en t i re nuclear membranes. Occas iona l ly locu les in the sporangia showed advanced stages of i n fec t i on 8 accompanied by complete l y s i s of the spores. Genera l ly , however, organel les other than the nucleus appeared to be unaf fected, and as the spores themselves appeared to have matured, the authors suggested that the in fec ted zoospores could be re leased. Markey (1974) reported VLPs ca. 130 to 170 nm in diameter in the marine brown alga P y l a i e l l a l i t t o r a l i s . The p a r t i c l e s , which appeared hexagonal in u l t r a t h i n s ec t i on , were observed in the un i l ocu l a r sporangia of a wi ld specimen. Infected plants exh ib i ted modif ied u l t r a s t ruc tu re inc lud ing dark - s ta in ing regions wi th in the nucleus (not considered to be heterochromatin) and "add i t iona l numerous membranous components". As the sporangia normally are protected with a th ick c e l l w a l l , the author be l ieved that i n f e c t i o n by d i r e c t entry of the p a r t i c l e s was un l i ke l y . He suggested that the p a r t i c l e s may have gained access through wound t i s s u e , or by i n i t i a l l y i n f e c t i n g naked reproduct ive c e l l s , remaining dormant during p lant development, and only l a t e r being t r i ggered into a c t i v i t y . A l l observations came from a s ing le wi ld p lant specimen; no other in fected specimens were found. Pearson and Norn's (1974) descr ibed in t ranuc lear VLPs in the marine prasinophycean alga Platymonas sp. The p a r t i c l e s were polyhedral and smal ler than many of those reported e a r l i e r , measuring only 50 to 60 nm in diameter. In th i s respect they resembled p a r t i c l e s seen by Lee (1971) in S i rodot i a and by Chapman (1973) in Porphyridium. P a r t i c l e d i s t r i b u t i o n was r e s t r i c t e d to ins ide the nucleus, with the nuclear membrane remaining i n t a c t . L y t i c a c t i v i t y was not observed. No information on the mechanism of i n f e c t i o n or re lease of VLPs was provided, but the authors did suggest 9 that u l t imate ly the in fected nucleus could i n h i b i t normal c e l l funct ions , eventua l ly leading to cytoplasmic degeneration, c e l l l y s i s , and re lease of the p a r t i c l e s . Photographic evidence f o r th i s was not inc luded. During an u l t r a s t r u c t u r a l examination of another marine prasinophyte, Pyramimonas o r i en ta l i s , Moestrup and Thomsen (1974) observed one c e l l (from among the hundreds sectioned) contain ing polyhedral p a r t i c l e s . These p a r t i c l e s occurred in the area usua l ly occupied by the nucleus. Two d i s t i n c t c lasses of p a r t i c l e s were observed. Those p a r t i c l e s occurr ing in greatest number were very s i m i l a r in s i ze (ca. 60 nm) to p a r t i c l e s described in Platymonas (ca. 50-60 nm, Pearson and Nor r i s , 1975). A few larger p a r t i c l e s were a l so seen, these measuring approximately 200 nm in diameter. As only a s ing le c e l l was found to contain p a r t i c l e s , the authors were unable to determine whether the two types of inc lus ions represented d i f f e r e n t developmental stages of the presumed i n f e c t i o n . Manton and Leadbeater (1974) presented a b r i e f report of polygonal VLPs in the marine haptophycean alga Chrysochromulina mantoniae prepared from a sample of wi ld Danish nanoplankton. The p a r t i c l e s were extremely sma l l , only 22 nm in diameter. The authors ind icated that the nanoplankton-i c host showed no evidence of being phagotrophic, suggesting that the p a r t i c l e s probably d id not enter during feeding. Polygonal VLPs were observed by Hoffman and Stanker (1975, 1976) in attached, one -ce l l ed germlings of the f i lamentous, freshwater green alga Cyl indrocapsa germine l la . These p a r t i c l e s were seen in 3 to 10% of the germlings produced by heat shock-induced zoosporogenesis. The p a r t i c l e s were 200 to 230 nm in diameter and were found to occupy the basa l , nuclear 10 region of the germling. Intact nucle i were never seen in p a r t i c l e - c o n t a i n -ing germlings, and progress ive degeneration of organel les was observed in in fected c e l l s . As in fected germlings could only be produced a f t e r heat shock treatment, and as no p a r t i c l e s were ever seen in the adul t p l an t s , the authors suggested that the apparently healthy cu l ture might be carry ing a l a t e n t , induc ib le i n f e c t i o n . Not a l l VLPs reported to occur in eukaryot ic algae are polygonal and l y t i c . Gibbs et a l . (1975) i s o l a ted rod shaped p a r t i c l e s (532 nm by 18 nm) from the charophyte Chara coral 1 ina. These p a r t i c l e s were observed to c l o s e l y resemble tobacco mosaic v i rus (TMV). The p a r t i c l e s were p u r i f i e d , cha rac te r i zed , and used to experimental ly i n f e c t v i r u s - f r e e Chara_ c e l l s . Further studies (Skotniki et a l . , 1976) showed these p a r t i c l e s to have a number of propert ies in common with the tobamoviruses of higher plants ( i . e . amino ac id composition of coat p ro te i n , base composition of genome RNA) although d i f f e r i n g in some others (e.g. overa l l p a r t i c l e length, s i ze of coat p ro te in ) . The s i m i l a r i t y of Chara c o r a l ! i n a v i rus (CCV) with that of TMV has led to the suggestion by Skotnik i et a l . (1975) that the d e f i n i t i o n of the tobamovirus group (Harrison et a l . , 1966) be changed to include CCV. This study provided the f i r s t unequivocal proof of v iruses in eukaryot ic algae. During a study of nanoplankton occurr ing in the waters surrounding San Juan I s land, Washington, U. S. A . , Pienaar (1976) observed VLPs in three species of phytoplankton: Hymenomohas carterae (Haptophyceae), Micromonas p u s i l l a (Prasinophyceae), and Cryptombnas sp. (Cryptophyceae). The p a r t i c l e s in Hymenomonas appeared hexagonal in sec t i on , measured ca. 1.1 65 nm in diameter, and were located with in an apparently degenerated nucleus. Infected c e l l s were abnormally large (roughly twice as large as normal c e l l s ) and lacked t h e i r c h a r a c t e r i s t i c external layer of cocco l i t h s . Micromonas c e l l s d isp layed polyhedral p a r t i c l e s in the cytoplasm which measured 129 nm. Unl ike Hymenomonas, the nucleus of these remained i n t a c t . The c e l l s themselves a l so showed no signs of l y s i s . Two d i f f e r e n t c lasses of p a r t i -c les were observed in Cryptomonas. Occurring within the degenerative nuclear region were numerous hexagonally-shaped p a r t i c l e s ca. 99 nm in diameter which appeared s i m i l a r to VLPs descr ibed f o r the other eukaryot ic algae. The p a r t i c l e s located external to the nucleus were markedly d i f f e r -ent in s i z e and form. These were composed of a " s t a l k - l i k e region which terminated in a swollen head". The p a r t i c l e s were 240 nm in length with an average diameter o f ca. 120 nm. The two p a r t i c l e types d id not show any obvious r e l a t i o n s h i p . Infected c e l l s appeared to be senescing. LaC la i re and West (1977) reported the presence of icosahedral VLPs in vegetat ive c e l l s of the brown alga Streblonema. The p a r t i c l e s , which measured ca. 135 to 150 nm were found densely packed in the cytoplasm of c e l l s causing the displacement of c e l l u l a r organe l les . The nuc le i of p a r t i c l e - r i d d e n c e l l s t y p i c a l l y were l y sed. The cu l tures possessing the VLPs, however, appeared to be growing v igorous ly . In general morphology, the p a r t i c l e s c l o se l y resembled those reported in reproduct ive c e l l s of other brown algae (see e.g. Baker and Evans, 1973; C l i theroe and Evans, 1974; Markey, 1974; Toth and Wi lce, 1972). Hoffman (1978) observed VLPs in c e l l s of the co lon ia l chrysophycean alga Hydrurus foet idus which had been c o l l e c t e d and f i xed d i r e c t l y in the 12 f i e l d . Only two VLP- infected c e l l s were found from the many thousands examined suggesting that the rate of i n f e c t i o n was extremely low. The polyhedral p a r t i c l e s measured ca. 50 to 60 nm in diameter. O l i v e i r a and B i sa lput ra (1978) have recent ly reported t h e i r observa-t ions of p a r t i c l e s found in a stock cu l tu re of the brown alga Sorocarpus  uvaeformis. The i s omet r i c a l l y shaped p a r t i c l e s which measured 170 nm were observed in zoospores and less f requent ly in vegetat ive c e l l s of the f i laments of mature plants during a study of zoospore germination. Culture medium contain ing i n fec ted zoospores was f i l t e r e d and tested with healthy plants and zoospores taken from other stock cu l tu res . Zoospores inoculated into th i s " i n f e c t e d medium" were observed to lyse with in three days. Mature plants however were able to grow in " i n f e c t e d medium", with less than 5% developing signs of i n f e c t i o n . Plants i n t e n t i a l l y wounded were more suscept ib le . The authors concluded, on the basis of t h e i r u l t r a s t r u c t u r a l observations and i n f e c t i v i t y experiments, that the p a r t i c l e s are v i r a l in nature. This study represents only the second such report of an es tab l i shed v i r a l i n f e c t i o n in benthic eukaryot ic algae (the f i r s t being that of Gibbs et a l . , 1975). Pecu l i a r inc lus ions other than d i s t i n c t polyhedral (or rod-shaped) p a r t i c l e s have a l so been descr ibed in eukaryot ic algae. As mentioned prev ious ly , Chapman and Lang (1973) descr ibed bodies c a l l e d "concentrosomes" in Porphyridium. Franca (1976) observed c r y s t a l l i n e arrays of p a r t i c l e s in the marine d i n o f l a g e l l a t e Gyrodinium resplendum. The p a r t i c l e s were ca. 35 nm and appeared as "more or less i n d i v i d u a l i z e d c i r c u l a r sub-units form-ing a polygonal matrix of e lectron-dense material around a centra l t r an s luc -ent a rea " . No morphological or u l t r a s t r u c t u r a l changes were evident in 13 c e l l s contain ing these i nc lu s i ons . Some c e l l s examined contained membrane l im i ted regions contain ing " p a r t i c l e s " 20 nm in diameter which appeared more t yp i ca l of polyhedral VLPs descr ibed by other researchers. These smaller p a r t i c l e s were subsequently shown by Soyer (1978) not to be v i r a l re l a ted and instead were c ro s s - sec t iona l p r o f i l e s of " t r i c h o c y s t o i d f i l aments " ( f i laments at the end of t r i c h o c y s t s ) . During the same study, Soyer reported the presence of in t ranuc lear p a r t i c l e s measuring ca. 30 nm in the p a r a s i t i c d i n o f l a g e l l a t e Blastodinium. She suggested that these in t ranuc-l ea r inc lus ions resembled the l a rger p a r t i c l e s seen by Franca (1976) in Gyrodinium. In tumor- l ike t i s sue of G r a c i l a r i a verrucosa (Rhodophyceae) Tr ipod i and Beth (1976) observed unusual c a t e r p i l l a r - l i k e bodies contain ing rows of fus i form c e l l s . These bodies were usua l ly located adjacent to p l a s t i d s , with one observed to occur wi th in the nucleus. The authors proposed that these inc lus ions might be v i r a l l y induced, but they o f fered no evidence favor ing th i s proposal over the p o s s i b i l i t y of bac te r i a l induct ion. No conc lus ive evidence f o r the presence of v iruses in marine planktonic algae has been published to date, only occasional observations of VLPs having been made in some phytoplankters. The present study descr ibes the d i scovery, i s o l a t i o n , and u l t r a s t r u c t u r a l desc r ip t i on of a l y t i c v i rus i n f ec t i on in the marine nanoplanktonic f l a g e l l a t e Micromonas p u s i l l a (Butcher) Manton and Parke. Discovery of the v i rus In A p r i l , 1975, a surface sample of seawater c o l l e c t e d from a s ta t ion s i tuated approximately midway between Point Atkinson and Point Grey in 14 Engl ish Bay at Vancouver, Canada, was processed as part of an inves t i ga t ion of the loca l nanoplankton. Following one week of enrichment c u l t u r i n g , an examination made with a l i g h t microscope ind icated that the dominant phy to f l age l l a te in the cu l tu re was the prasinophyte Micromonas p u s i l l a (which comprised 80 to 90% of the c e l l s in the c u l t u r e ) ; the remaining organisms were various m i c r o f l a g e l l a t e s . A sample from the cu l ture was f i xed f o r transmiss ion e lec t ron microscopy (TEM). E lectron micrographs showed Micromonas c e l l s contain ing hexagonal s t ructures resembling VLPs. Many of the c e l l s appeared to be in a s ta te of l y s i s , with the presumptive v i r a l p a r t i c l e s scattered through the surrounding embedding medium. A re-examination of the o r i g i n a l cu l ture with the l i g h t microscope ind icated an apparent disappearance of the Micromonas populat ion, with no change in the other m i c ro f l a ge l l a t e populat ions. An a l i quot of the " i n fec ted c u l t u r e " was added to a un ia lga l laboratory cu l tu re of another clone of M. p u s i l l a (whose c e l l s as examined in TEM did not exh ib i t p a r t i c l e s ) , and the c e l l s were examined at i n te rva l s fo l lowing incubat ion. Within 24 hr , the op t i ca l density of the cu l ture had dec l ined v i s i b l y , and with in 48 hr, microscopic examination showed few l i v i n g c e l l s . The i n f e c t i v e sample has subsequently been maintained by weekly or biweekly t rans fe r . 15 MATERIALS AMD METHODS I. ALGAL MAINTENANCE AND GENERAL TECHNIQUES 1.1. Cultures Unia lgal cu l tures of Micromonas p u s i l l a (Butcher) Manton and Parke were obtained from four d i f f e r e n t sources. Four geographic i so l a tes were maintained throughout the course of t h i s study. Deta i l s of these i so l a tes are l i s t e d in Table II. A l l glassware used in these experiments was b o r o s i l i c a t e (Pyrex) unless otherwise s tated. Cultures were maintained in 125 ml Erlenmeyer f l a sks contain ing 100 ml of P rovaso l i ' s enriched seawater (PES) (P rovaso l i , 1968). For experimental work cu l tures were grown at 16-18°C in a con t ro l l ed environment chamber equipped with f luorescent tubes (General E l e c t r i c Cool White) which provided l a t e r a l i l l umina t ion on a 12 hr:12 hr l i gh t :dark regime. L ight i n t e n s i t y , as -2 -1 measured with a L i co r quantum-meter, averaged 70 uE'm -s . Throughout the experimental per iod , stock cu l tures were t rans fer red into f resh medium every 10 to 14 days. The Micromonas i so l a te s chosen fo r experimental work were grown in 250 ml or 500 ml Erlenmeyer f l a sks conta in ing 200 ml or 400 ml of PES re spec t i ve l y . A f t e r 7 to 10 days of growth the cu l tures were e i ther prepared f o r transmission e lec t ron microscopy or subdivided into 20 ml volumes and t rans fer red into 40 ml capac i ty , metal-capped cu l ture tubes. Unialgal cu l tures used fo r host s p e c i f i c i t y tests were provided by the Northeast P a c i f i c Culture C o l l e c t i o n (NEPCC), U. B. C , Vancouver, Iv6 TABLE II. I solates of Micrombnas p u s i l l a , Sources Pa r t i cu l a r s Plymouth Culture C o l l e c t i o n #27 Iso lated in 1973 by Dr. Parke from the Engl ish Channel M. Un iver s i t y of Washington Culture Co l l e c t i on #15-8-42 Isolated from seawater co l l e c ted at 2200 m o f f Baja, C a l i f , by Dr. 0. Holm-Hansen Northeast P a c i f i c Culture Co l l e c t i on #P29 Isolated in Jan. 1971 from Engl ish Bay, Vancouver, B r i t i s h Columbia by R. Waters Northeast P a c i f i c Culture Co l l e c t i on #P29a Isolated in March 1971 by R. Waters from Weather Ship Stat ion P in the N. E. P a c i f i c (50°11 'N , 144°51'W) 17 Canada. These cu l tures were grown in 50 ml Erlenmeyer f l a sks contain ing 40 ml of PES. Fol lowing one week of growth at the l i g h t and temperature regime descr ibed above, each cu l ture was d iv ided equal ly and t rans fer red into two, 40 ml metal-capped cu l tu re tubes., I. 2. Ce l l counts Three samples taken from growing cu l tures were preserved by mixing with 0.2 ml of Rodhe's mod i f i ca t ion of Lugo l ' s iodine so lu t ion (Rodhe et a l . , 1958). Preserved c e l l samples were observed with a photomicroscope equipped with phase o p t i c s , and counted with a Palmer-Maloney Counting Chamber. The microscope ocular was f i t t e d with a Whipple d i sk , the area of the Whipple f i e l d was measured, and the preserved samples were counted fo l lowing the method described by G u i l l a r d (1973). II. VIRUS MAINTENANCE AND GENERAL TECHNIQUES 11.1. General maintenance For general maintenance 2 to 3 ml of v i r a l lysate were added to 100 ml o f Micromonas i s o l a t e s . Cultures inoculated in th i s manner demonstrated l y s i s with in 2 to 3 days. A l t e r n a t i v e l y , lysed cu l tures were pooled and centr i fuged in the GSA ro tor o f a Sorval centr i fuge at 10,000xg f o r 30 to 45 min to remove c e l l debr i s . The c l a r i f i e d lysate was then stored e i t he r at 4°C or at growth chamber temperature (16 -18°C ) . 11.2. General experimental techniques For experimental te s t ing of v i r a l a c t i v i t y 0.5 ml of v i rus source was added to 40 ml capac i ty , metal-capped cu l tu re tubes containing 20 ml 18 volumes of Micromonas cu l tures in logar i thmic growth. The in vivo f l u o r -escence of these batch cu l tures was read and recorded d a i l y (see sect ion on Fluorometric Methods). The r e l a t i v e concentrat ion of i n f e c t i v e v i rus p a r t i c l e s was determined by s e r i a l endpoint d i l u t i o n of a given v i rus source in autoclaved seawater. The d i l u t i on s of v i rus stock were tested f o r v i r a l a c t i v i t y as descr ibed above. II. 3. Virus f i l t e r a b i l i t y To determine the f i l t e r a b i l i t y of the v i rus a c l a r i f i e d v i r a l suspen-sion was sequent i a l l y f i l t e r e d a s e p t i c a l l y through M i l l i p o r e f i l t e r s of 0.45 ym and 0.22 ym pore diameter contained with in M i l l i p o r e Swinnex-25 f i l t e r holders. F i l t r a t e s were stored at 4°C and l a t e r tested fo r v i rus a c t i v i t y . III. LIGHT MICROSCOPY L iv ing material was observed using a photomicroscope equipped with phase-contrast or Nomarski op t i c s . Ce l l shape and motion were recorded. Ce l l s were photographed with I I ford FP4 (ASA 125) f i l m using phase op t i c s . IV. ELECTRON MICROSCOPY IV.1. Whole mounts - metal-shadowed c e l l s Ce l l s of Micromonas from exponent ia l ly growing cu l tures were placed on 200 mesh, Formvar-coated (0.35%), copper g r id s . The grids were set in pe t r i dishes which had been prev ious ly saturated with 0 s0 4 vapor (hanging 19 drops of 4% aqueous OsO^ were suspended on the ins ide of the pe t r i dish l i d s ) . A f t e r 5 to 6 sec the gr ids were removed and the c e l l s allowed to s e t t l e f o r 5 min. The drop of cu l ture was then removed and the gr ids washed gently with d i s t i l l e d water. Grids were placed in a Balzers BA 360 M High Vacuum Coating Un i t , shadowed with platinum (8 sec) fol lowed by graphite (8 sec) at a vacuum of 0.6x10 To r r , and examined with a Zeiss EM 9A e lec t ron microscope at 60 KV. IV.2. Th in- sect ioned materia l Ce l l s of Micromonas in exponential growth were harvested by c e n t r i -fugat ion at 580xg f o r 15 min OT l lOOxg f o r 7 min in an Internat ional Model HN cent r i fuge . The f i x a t i o n and dehydration schedule is shown in Table III. A l t e r n a t i v e l y , f i x a t i v e s were added d i r e c t l y to the cu l tures without any pre l iminary cent r i fuga t ion and the f i x a t i o n and dehydration schedule shown in Table IV was fo l lowed. A f t e r dehydration the c e l l s were i n f i l t r a t e d w i th \ increas ing concentrat ions of Spurr low v i s c o s i t y embedding medium in acetone (Spurr, 1969), l e f t overnight in 100% r e s i n , and t rans fer red through three more changes of 100% r e s i n . I n f i l t r a t e d c e l l s were pe l l e ted and t rans fer red to BEEM capsules to which f resh 100% re s in was added. The BEEM capsules were e i t he r l e f t f o r 6 hr to al low materia l to s e t t l e or centr i fuged 2 to 5 min at 1000xg to concentrate the c e l l s . The embedded material was polymerized at 70°C in a vacuum oven at 10 psi f o r 8 to 12 hr. Sections were cut on a Reichert 0MU 3 ultramicrotome using a Dupont diamond kn i f e , c o l l e c t e d on 200 mesh, 0.35% formvar-coated, copper gr ids or on carbon-coated, s l o t g r id s , and post s ta ined f o r 25 to 40 min with 5% uranyl acetate in 50% ethanol fol lowed by 10 min in Reynolds' lead c i t r a t e 20 Table III. F i xa t i on and dehydration schedule I Medium Time 1.5 % g lutara ldehyde, 0.1 M sodium cacodylate buf fer pH 7.3, 0.23 M sucrose (8%), 0.005 M C a C l 2 45 minutes b u f f e r , 0.12 M sucrose 15 minutes bu f fe r 15 minutes b u f f e r 15 minutes 1% 0 s 0 4 , bu f fe r 1 hour bu f fe r 15 minutes bu f fe r 15 minutes d i s t i l l e d water 15 minutes 30% acetone 10 minutes 60% acetone 10 minutes 90% acetone 10 minutes 100% acetone 10 minutes 100% acetone 10 minutes 100% acetone 10 minutes \ 21 Table IV. F ixa t ion and dehydration schedule II Medium Time 25% glutaraldehyde added d i r e c t l y to l i q u i d cu l tu re to give f i n a l concentrat ion of 1.5% 4% OsO, added d i r e c t l y to above mixture to give f i n a l concentrat ion of 0.4% P r o v a s o l i ' s enriched seawater medium (PES) PES medium PES medium 30% acetone 60% acetone 90% acetone 100% acetone 100% acetone 100% acetone 5 minutes 10 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 22 (Reynolds, 1963). Sections were viewed with e i the r a Zeiss 9A e lec t ron microscope at 60 KV or a Zeiss EM 10 operated at 60 or 80 KV. For examination of v i r a l entry and morphological studies of v i r a l r e p l i c a t i o n a l iquots of c l a r i f i e d v i r a l lysate were added to host cu l tures p r i o r to f i x a t i o n . The time between add i t ion of v i r a l lysate and f i x a t i o n var ied from 15 min to 4 hr. Normal f i x a t i o n procedures were fol lowed (described above). IV.3. Scanning e lec t ron microscopy For scanning e lec t ron microscope (SEM) examination, cu l tures were f i xed and dehydrated as descr ibed prev ious ly . Dehydrated material was t rans fer red into amy! acetate v i a a ser ies of subs t i tu t i on steps. The materia l was t rans fe r red into a mixture of 3:1 acetone:amyl acetate (10 min), fol lowed by 1:1 acetone:amy! acetate (10 min), 1:3 acetone:amy! acetate (10 min), and three changes of 100% amyl acetate (15 min each). Ce l l s in 100% amyl acetate were centr i fuged to form a loose p e l l e t and drops of the concentrated suspension were placed onto pieces of 0.4 urn pore diameter Nucleopore f i l t e r which had been cut to approximately the diameter of aluminum SEM stubs. , The amyl acetate was allowed to dry 5 min then the f i l t e r s were placed into s t a in le s s s tee l mesh baskets and c r i t i c a l point d r ied by the C0£ method (Anderson, 1966). The pieces of f i l t e r containing the dr ied c e l l s were attached to aluminum SEM stubs by the use of c o l l o i d a l s i l v e r "dag" and the f ree edges of the f i l t e r were sealed with s i l v e r . The stubs were coated with gold using a Mikros Vacuum Evaporator VE 10 to _5 10 To r r , then covered and stored and examined l a t e r with a Cambridge Mark 2A Stereoscan e lec t ron microscope. 23 IV.4. Negative s ta in ing of v i r i on s Regardless of what previous treatment a suspension of v i rus p a r t i c l e s might have received the procedure f o r negative s ta in ing remained the same. A drop of the v i r a l suspension was placed onto carbon-coated, copper gr ids using a microp ipet te . The drop was allowed to stand 10 min then the g r id was held with forceps and any remaining l i q u i d was washed with 10 to 15 drops of 2% uranyl acetate in d i s t i l l e d water. The grids were allowed to dry and examined d i r e c t l y by e lec t ron microscopy (described above). IV. 5. Metal-shadowed v i rus V i r a l l y sate se lected f o r heavy metal shadowing was centr i fuged in the GSA rotor of a Sorval centr i fuge at 10,000Xg f o r 30 to 45 min to remove c e l l debr i s , or was c o n c e n t r a t e d by u l t r a c e n t r i f u g a t i o n ( s e e A p p e n d i x I I I ) . B e s t re su l t s were obtained i f th i s v i r a l suspension was placed on gr ids and f i xed with 2% uranyl acetate as descr ibed prev ious ly fo r negative s ta in ing of the v i ru s . Only 5 drops of uranyl acetate were app l i ed , however, and gr ids were then thoroughly washed with d i s t i l l e d water to remove a l l traces of uranyl acetate. The gr ids were shadowed with _5 plat inum/pal ladium in a Mikros VE 10 Vacuum Evaporator uni t at 10 Torr and l a t e r examined with the e lec t ron microscope. V. FLU0R0METRIC METHODS V . l . F lubrometric apparatus and general procedures To f a c i l i t a t e the measurement of in vivo f luorescence of batch cu l tures a mod i f i ca t ion of Lorenzen's method fo r continuous measurement of in vivo ch lorophy l l was employed (Lorenzen, 1966). A Turner Model 111 f luorometer 24 equipped with a red sen s i t i ve photomul t ip l ie r (R 136) and a temperature-con t ro l l ed high s e n s i t i v i t y door was used. In order to maintain batch cu l ture tubes at growth chamber temperatures during periods of data c o l l e c t i n g an aluminum water bath was designed. The bath was f i l l e d with tap water at 15-17°C which was c i r c u l a t e d v ia a p e r i s t a l t i c pump through the temperature con t ro l l ed door and returned to the bath. The f luorometer, pump, and water bath were connected and allowed to e q u i l i b r a t e f o r at l eas t 10 min before the f luorescence of samples was determined. During th i s period experimental cu l ture tubes were t rans fer red from the cont ro l l ed environment chamber to the water bath. Culture tubes were removed i n d i v i d u a l l y from the bath and mixed fo r 10 sec by v i b ra t i on on a vortex mixer. A 3 ml sample was poured into a 4 ml cuvette and in vivo f l u o r -escence was recorded a f te r a 15 sec s t a b i l i z a t i o n per iod. Following the reading the sample was returned to i t s source. Culture medium was used to zero the f luorometer before i n i t i a l readings and before each change to a d i f f e r e n t s e n s i t i v i t y s ca le . V.2. Determination of p ropo r t i ona l i t y and l i n e a r i t y Although the Turner f luorometer has four s e n s i t i v i t y scales l abe l l ed XI, X3, X10, and X30, the exac t .p ropor t i ona l i t y var ies from one instrument to the next. The actual r e l a t i on sh ip between scales was determined by s e r i a l d i l u t i o n of disodium f luorosce in in 0.05 M sodium phosphate buf fer (Turner, 1972). The p ropo r t i ona l i t y of the sca les was XI, X3.71, X9.83, and X30.97, f o r scales 1, 3, 10, and 30, re spec t i ve l y . The f luorescence readings obtained from.the dye d i l u t i on s were examined s t a t i s t i c a l l y and regress ion ana lys i s of the data ind icated the 25 p scales were l i n e a r (r >0.98). V.3. Cor re la t i on between in vivo f luorescence and c e l l density In order to determine the re l a t i on sh ip between c e l l number and in  vivo f luorescence in batch cu l tures of M. p u s i l l a , and to ascer ta in whether th i s r e l a t i on sh ip var ied with cu l ture age, growth experiments were undertaken. Growth curve experiment: To obtain a growth curve f o r M. p u s i l l a , c e l l counts and in vivo f luorescence were measured d a i l y f o r a set of cu l tures over a period of approximately one month. The procedures f o r counting c e l l s and measuring f luorescence were those described prev ious ly . The experimental design w i l l be descr ibed here. A one l i t e r Erlenmeyer f l a sk contain ing PES medium was inoculated with a cu l tu re of M. p u s i l l a in l a te exponential or ea r l y s ta t ionary growth providing a f i n a l d i l u t i o n of 1/50. A sample of the inoculum was preserved with Rodhe's mod i f i ca t ion of Lugo l ' s iodine so lut ion and counted l a t e r . The f l a sk was placed on a magnetic s t i r r i n g platform and gently mixed while 20 ml a l iquots of cu l tu re were p ipetted into each of f i v e metal -capped, cu l ture tubes designated f o r f luorescence measurements. A s ix th tube conta in ing 20 ml of PES was set up to provide a d a i l y measurement of cu l tu re temperature. The o r i g i na l f l a sk cu l tu re was included in th i s experiment because i t s cu l tu re volume was s u f f i c i e n t to permit the d a i l y removal of samples (to be preserved f o r c e l l counts).over an extended per iod. The f l a s k , designated as "counting f l a s k " and the s i x te s t tubes were placed in the growth chamber to be observed and sampled d a i l y . A seventh tes t tube was stored at the cu l tu re chamber temperature and used 25 to c o l l e c t a d a i l y sample from the counting f l a sk . Dai ly between 11:30 AM and 2:00 PM the counting f l a sk was mixed and a sample added to the seventh cu l tu re tube. The seven sample tubes were placed into an aluminum water bath kept at growth chamber temperature and the in vivo f luorescence of each sample was measured d a i l y (method descr ibed in V . l ) . In add i t i on , a 3 ml a l i quo t was removed from the seventh cu l tu re tube and preserved with Rodhe's Lugo l ' s so lu t ion fo r c e l l counts l a t e r . The remaining contents of the seventh sample were returned to the counting f l a s k . Water bath and cu l ture temperatures were recorded at the beginning and end of each sampling per iod. By the twel f th day of the experiment the f luorescence of the f i v e tube cu l tures had increased markedly and was o f f the sca le and therefore the fo l lowing add i t i ona l procedures were i n i t i a t e d : Before measuring the f luorescence of a p a r t i c u l a r tube a 0.5 ml a l i quot of the cu l ture was withdrawn and mixed with 4.5 ml of PES, and the f luorescence of th i s 1/10 d i l u t i o n was measured and recorded. The d i l u t i o n tube which reg i s tered the d a i l y median f luorescence value was sampled and preserved f o r c e l l counts l a t e r ( in add i t ion to d a i l y counting f l a sk samples). By day 17 these procedural modi f icat ions were appl ied to the counting f l a sk cu l ture as wel 1. V.4. "N i te -Owl " , 48 hour experiment In order to determine whether in vivo f luorescence could be used to measure in fec ted cu l ture death a 48 hour experiment, designated as " N i t e -Owl", was undertaken. 27 A 500 ml Erlenmeyer f l a sk contain ing PES was inoculated with a cu l tu re of M. p u s i l l a ( in exponential growth) to give a f i n a l d i l u t i o n of 1/20. The f luorescence of the inoculum cu l tu re was measured and a sample preserved f o r c e l l counts. Twenty ml of t h i s cu l ture were p ipetted into each of 14 metal-capped cu l ture tubes. The tubes were incubated in the growth chamber and the in vivo f luorescence measured to determine the i n i t i a t i o n of logar i thmic growth. Twenty-six hours l a t e r , 0.5 ml of autoclaved seawater was added to two of the fourteen tubes (designated as experimental c o n t r o l s ) . Five of the remaining twelve tubes were inoculated with 0.5 ml of c l a r i f i e d v i r a l l y sate and the in v ivo f luorescence of these seven treatments was recorded. One hour l a t e r two more contro l tubes were inoculated with seawater, the remaining f i v e tubes were inoculated with v i r a l l y s a te , and the in vivo f luorescence of these seven tubes was recorded. One hour l a t e r and every two hours f o r 28 consecutive hours the in  vivo f luorescence of a l l 14 tubes was recorded. Staggering the i n i t i a l i nocu la t ion time by one hour allowed hourly f luorescence values to be c o l l e c t e d by sampling only b ihour ly . Because of the l im i ted volume of these batch cu l tures a d i f f e r e n t tube was se lected at each sampling time fo r c e l l counting so as to decrease the e f fec t s of volume d i f fe rences between tubes. S im i l a r l y a d i f f e r e n t contro l tube was sampled fo r c e l l counts every four hours. The sampling scheme i s shown in Table V. The experiment was terminated ear ly (28 hr a f t e r inocu la t ion) as the in vivo f luorescence of the v i r u s - i n f e c t e d cu l tures reg i s tered below the s e n s i t i v i t y of the f luorometer. During the course of the experiment, due to a mechanical f a i l u r e , TABLE V. "Nite-Owl" Experiment. Scheme f o r c o l l e c t i n g c e l l count samples. Cul t u r e I d e n t i f i c a t i o n Hours a f t e r a d d i t i o n o f inoculum 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 C - l 1 C-2 C-3 + + + + C-4 N O T . - S A M P L E D v - i 2 V-2 V-3 V-4 V-5 V-6 V-7 V-8 + + + + + + + + + + + + - + + V-9 V-10 N O T S A M P L E D C = c o n t r o l t e s t s , i . e . no v i r u s added, = v i r u s t e s t s , i . e . v i r u s added. 29 l i gh t s in the growth chamber remained on fo r 24 hr and the cu l tures experienced a d i s rupt ion of t h e i r normal 12:12 l i gh t :dark regime. VI. HOST RANGE STUDIES The host range of the v i rus was examined by means of a ser ies of experiments conducted with unia lga l cu l tures provided by NEPCC. A l i s t of these cu l tures i s presented in Table VI (see Appendix I). As described prev ious l y , 40 ml of cu l ture was grown in 50 ml E r l e n -meyer f l a sks fo r one week then d iv ided equal ly and t rans fer red into two, 40 ml cu l ture tubes. One tube from each pa i r was treated as a cu l ture v i a b i l i t y contro l and inoculated with 0.5 ml autoclaved seawater; the second tube was inoculated with 0.5 ml v i r a l l y sa te . Nine or ten d i f f e r e n t species were studied in each experiment inc lud ing one i s o l a te of M. p u s i l l a which served as a contro l te s t of v i r a l potency. The in vivo f luorescence of a l l tubes was measured d a i l y f o r one week. These experiments were only intended to examine the spectrum of l y t i c behaviour of the v i rus and no attempts were made to examine any of the species u l t r a s t r u c t u r a l l y . VII. VIRUS STABILITY - PHYSICAL AND CHEMICAL PROPERTIES The s t a b i l i t y of the v i rus when exposed to a va r ie ty of environmental condit ions was studied experimental ly using l y t i c capacity as an i nd i ca to r . To determine the s e n s i t i v i t y of the v irus to temperature changes f i v e water baths were set up at the fo l lowing temperatures: 2 3 ° , 3 3 ° , 4 3 ° , 5 5 ° , and 62°C. Each bath held a f l a sk conta in ing 45 ml of autoclaved seawater which had been allowed to e q u i l i b r a t e at bath temperature f o r 45 min. 30 A 5 ml sample of c l a r i f i e d v i r a l l y sate was added to each f l a sk (g iv ing a f i n a l d i l u t i o n of 1/10), the so lu t ion mixed thoroughly, and a sample removed immediately and placed on i c e . Each f l a sk was sampled at 15.min i n te rva l s f o r two hours. A l l samples were cooled on i c e , then stored at 4°C f o r l a t e r i n f e c t i v i t y assay on healthy cu l tu res . The s t a b i l i t y of the v i rus as a funct ion of environmental pH was tested in the fo l lowing manner: Autoclaved seawater was buffered with 0.1 M sodium acetate or 0.1 M T r i s HC1 and the pH adjusted to 3, 4, 5, 6, 7, 8, 9, and 10. A 1/10 d i l u t i o n of v i rus in buffered seawater was made at each pH and the samples stored at 4°C overnight. The e f f e c t of treatment with the organic solvents to luene, ethyl e ther , and chloroform, was a lso s tud ied. Toluene was added to a v i r a l suspension to g ive a f i n a l concentrat ion o f 10% and ethyl ether was added to a second suspension to give a concentrat ion of 33%. The mixtures were poured into separatory funnels and allowed to s e t t l e f o r 15 sec a f t e r which the heavier bottom f rac t i ons containing the v i rus were removed. A t h i r d sample of v i rus was mixed with 10% chloroform in a graduated c y l i n d e r ; the chloroform was allowed to s e t t l e and the treated v irus suspension removed. A l l so lvent treated samples were stored at 4°C. A l l v i rus treatments descr ibed above were tested simultaneously by inocu la t ing tes t tubes contain ing 20 ml of host cu l ture with 0.5 ml v i rus suspension. This included 45 temperature treatments, 8 pH treatments, and 3 solvent treatments. Th i r teen contro l tubes were a l so prepared: Three control tubes were inoculated with autoclaved seawater as checks of cu l ture v i a b i l i t y , two tubes were inoculated with a 1/10 d i l u t i o n of untreated v i rus 31 to check v irus potency, and e ight contro l s were inoculated with seawater of varying pH. In vivo f luorescence values were recorded da i l y f o r a l l tubes. The re su l t s of these tests are presented in Table VII (see Appendix II). 32 LIST OF ABBREVIATIONS USED bb basal body of f l age l lum ch ch lo rop la s t ch^ inner surface of ch lo rop la s t c h Q outer surface of ch lo rop la s t c f cytoplasmic f i b r i l s d f l d i s t a l f l a g e l l a r region er endoplasmic ret iculum f f i b r i l s or f i b r i l l a r materia l f l f l age l lum gb golg i body m mitochondrion mbv membrane bound ves i c l e s mcb mucocyst-1ike body mt microtubule mvb mu l t i ve s i cu l a te body n nucleus ne nuclear envelope ner nuclear endoplasmic ret icu lum p pyrenoid p f l proximal f l a g e l l a r region pi plasmalemma rer rough endoplasmic ret icu lum s starch or s tarch sheath tc tapered cy l i nde r 33 OBSERVATIONS AND RESULTS I. MICROMONAS PUSILLA: GENERAL CHARACTERISTICS AND ULTRASTRUCTURE 1.1. H i s t o r i c a l survey Due to the very small s i ze of Micromonas p u s i l l a (Butcher) Manton and Parke and a number of other s i m i l a r small f l a g e l l a t e s , t h e i r systematic pos i t ion has been uncertain f o r many years . F r i t s ch (1935) assigned f l a g e l l a t e s of th i s general type to the fami ly Polyblepharidaceae in the c lass Chlorophyceae, but described them as an "unnatural assemblage". The advent of e lect ron microscopy enabled Manton, Parke, and t h e i r co-workers (Manton, 1959; Manton and Parke, 1960; Manton et a l . , 1963; Parke and Rayns, 1964; Manton, 1965) to examine the f i ne s t ructure of these t iny u n i c e l l s and led them to conclude that a number of s o - ca l l ed "naked" c e l l s were a c tua l l y covered with a layer of minute organic scales i n v i s i b l e with the l i g h t microscope. As th i s sca ly covering often included the f l a g e l l a r apparatus and, as no c e l l s with sca ly f l a g e l l a had been observed prev ious ly , these researchers suggested that the systematic scheme of the green algae should be re-examined. Christensen (1962, 1966), extending the e a r l i e r ideas of Chadefaud (1941, 1950) proposed two d i s t i n c t c lasses to accept these non-volvocalean f l a g e l l a t e s : the Loxophyceae, which included a l l those "naked" c e l l s which lacked a c e l l wall and any type of f l a g e l l a r decorat ion, and the Pras ino-phyceae, which included the s c a l y - f l a g e l l a t e d representat ives . Parke and Manton (1965) approved of th i s d i s t i n c t i o n from the Chlorophyceae but favored the adoption of only a s ing le new c l a s s , the Prasinophyceae, 34 because u l t r a s t r u c t u r a l s i m i l a r i t i e s between organisms placed in the d i f f -erent c lasses by Christensen made the d i s t i n c t i o n seem "unnatural and awkward". Studies by R icketts (1966, 1967, 1970) demonstrating the existence of a unique pro toch lo rophy l l -1 ike pigment (magnesium 2,4 div inylphaeoporphyrin a^ monomethyl ester) not found in other green f l a g e l l a t e s , provided add i t iona l evidence in favor of th i s new taxonomic grouping. On the basis of these u l t r a s t r u c t u r a l and biochemical features th i s group of green monads i s cu r ren t l y c l a s s i f i e d e i the r as Prasinophyceae, wi th in the Chlorophyta (sensu Pe te r f i and Manton, 1968), or as Prasinophyta (sensu Round, 1971). The f i r s t reports imp l i ca t ing M. p u s i l l a (under i t s o r i g i na l designa-t ion as Chromulina pus i l l a .Bu tcher ) as an important component of the phytoplankton community were published in 1951 by- Knight-Jones (1951) who wrote: "The most genera l ly abundant organism in B r i t i s h coastal waters and the North Sea seems to be the 1.5 ym chrysomonad Chromulina p u s i l l a Butcher . . . Th i s f l a g e l l a t e can only have escaped desc r ip t i on e a r l i e r because of i t s minute s i z e . " Because of i t s t i ny s i z e C_. p u s i l l a was o r i g i n a l l y misc lass -i f i e d as a member of the Chrysophyceae by Butcher (1952) but he was insecure in th i s dec i s i on : "The assignment of th i s organism to i t s true pos i t i on i s a matter of some d i f f i c u l t y as, in so small a spec ies , the c e l l contents are very hard to d e f i n e . " M. p u s i l l a has been found to be cosmopolitan. S i g n i f i c a n t numbers q C ( 1 0 - 1 0 c e l l s / l i t e r ) of th i s f l a g e l l a t e have been reported from A r c t i c waters of the Barents Sea (Throndsen, 1970), temperate waters of the B r i t i s h Channel (Manton and Parke, 1960), and the Norwegian (Throndsen, 1969) and North Seas (Knight-Jones, 1951). It has a l so been observed in 35 t rop i ca l waters of the E. P a c i f i c , the Caribbean, and the Sargasso Sea (cf. Throndsen, 1976). Observations on both wi ld populations and those in cu l tu re have ind icated that th i s species i s euryhal ine (Throndsen, 1976), being capable of growth with in a s a l i n i t y range of 5-35°/oo. Throndsen (1973a) has studied the m o t i l i t y of M. p u s i l l a . Based upon i t s r e l a t i v e swimming rate of more than 50 c e l l lengths per second ( in accordance with Knight-Jones and Walne, 1951) he ca l cu la ted i t s probable migration capacity to be less than f i v e meters/day. M. p u s i l l a has been observed to occur well below the euphotic zone, even though i t can be phototact ic (Manton and Parke, 1960). In open waters o f f the Gulf of Panama, Throndsen (cf . 1976) 5 observed concentrat ions of 10 c e l l s / l i t e r at 100 m. Manton and Parke (1960) reported th i s species occurr ing down to depths of 500 m in the Bay of Biscay. The f i ne s t ructure of M. p u s i l l a was examined as part of a rout ine inves t i ga t ion of " c i l i a t e d plant c e l l s " by Manton (1959). However, i t was not r e c l a s s i f i e d un t i l 1960, at which time Manton and Parke found u l t r a -s t ruc tu ra l evidence supporting Chlorophyte a f f i n i t i e s (Manton and Parke, 1960). Based upon t h e i r observations on th i s and a second prev ious ly undescribed f l a g e l l a t e , they created a new genus, Micromonas, with two spec ies : p u s i l l a (Butcher) Manton and Parke as the type, and adding squamata Manton and Parke. The l a t t e r was separated from M. p u s i l l a on the basis of s i ze (M. squamata being cons iderably l a rger with a length of 3-6.5 ym as opposed to 1-3 ym), f l a g e l l a r s t ruc tu re , and the presence of both body and f l a g e l l a r s ca le s . As noted prev ious ly ,Chr i s tensen suggested grouping these anomolous 36 green f l a g e l l a t e s into two c lasses based on the presence or absence of sca les . If th i s scheme was fol lowed the two species o r i g i n a l l y assigned to the genus Micromonas would have been placed in d i f f e r e n t a lga l c la s ses . The combination of Chr i s tensen ' s Loxophyceae with Prasinophyceae circum-vents th i s d i f f i c u l t y . Desikachary (1977) has recent ly removed M. squamata from the genus Micromonas and renamed i t Mantoniel la squamata (Manton and Parke), th i s separat ion being based s o l e l y on the presence of sca les and the d i f fe rences in the nature of f l a g e l l a r i n s e r t i o n . The only u l t r a s t r u c t u r a l desc r ip t i on of M. pusi11a p r i o r to th i s study i s that by Manton in 1959, and although e a r l i e r , l e s s - r e f i n e d techniques were employed, i t was remarkably accurate. In th i s study improved equip-ment and methodology have provided an updated p i c tu re . In the sect ion which fol lows emphasis i s placed on newly i d e n t i f i e d features . 1.2. General c e l l s t ruc ture M. p u s i l l a has been claimed to be the smal lest and s implest f l a g e l l a t e (Manton, 1965; Throndsen, 1976). The minute s i ze and simple external appearance of th i s monad i s i l l u s t r a t e d in F i g . 4. Ce l l s are 1.0 to 3.0 ym in length and 0.75 to 1.0 ym in width. Re lat ive to t h e i r small s i ze the c e l l s are extremely f a s t moving, and, as a r e su l t of t h e i r t i ny s i ze makes them d i f f i c u l t to study with the l i g h t microscope. Using high-power, phase microscopy i t was poss ib le to study c e l l s i ze and shape (F ig . l ) .and to recognize the s ing le emergent f lage l lum which, in add i t ion to i t s s i z e , i s th i s organism's only outstanding, s u p e r f i c i a l morphological character. The c e l l s are roughly reniform in shape. In 37 some c u l t u r e s , however, spher ica l or pear-shaped ind i v idua l s were predomin-ant, suggesting that c e l l morphology i s va r i ab le . There i s a subt le asymmetry of the c e l l , obvious only a f t e r examining organe l la r shape and d i s t r i b u t i o n in numerous sect iona l views. In reniform c e l l s the c e l l body appears to be d iv ided unequally into two lobes, a c h a r a c t e r i s t i c which was descr ibed by Manton and Parke (1960) as "reminiscent of a comma". A long i tud ina l sect ion through one c e l l i s shown in F i g . 2. The general morphology of Micromonas i s summarized diagrammatical ly in Diagram 1. A s ing le emergent f lage l lum i s inser ted l a t e r a l l y into a depression and i s d i rec ted ob l ique ly across the smaller body lobe. Because the c e l l genera l ly swims with i t s f l age l lum d i rec ted backwards the smaller lobe has been designated as the pos ter io r end of the c e l l . A second basal body, not g iv ing r i s e to a f l age l lum, i s s i tuated adjacent to the basal body of the emergent f lage l lum (F ig . 37). Occas iona l ly a second, stubby f lage l lum i s v i s i b l e protruding from the c e l l surface (F igs . 38 and 40). Much of the i n t e r i o r of the c e l l i s f i l l e d by a large saddle-shaped ch lo rop la s t which occupies the convex s ide of the c e l l opposite the point of f l a g e l l a r i n s e r t i o n . The ch lo rop la s t contains a conspicuous pyrenoid which i s enclosed by a s tarch sheath and protrudes into the centra l region of the cytoplasm. The nucleus, which contains a poorly def ined and weakly s ta in ing nuc leo lus , occupies the,smal ler pos te r io r lobe of the c e l l . In more elongated c e l l s th i s "nuc lear lobe" appears to extend as a remote peninsula of cytoplasm r e l a t i v e to the centra l c e l l body (F ig . 12). A large d i s co id mitochondrion i s pos i t ioned d i r e c t l y over the pyrenoid region of the ch l o rop l a s t , curving to fo l low the contour of the p l a s t i d ' s centra l 38 Diagram 1. Schematic representat ion of the general u l t r a s t ruc tu re of Micromonas p u s i l l a . (For abbreviat ions see p. 32.) 39 0.5 pm A* 40 Figs. 1-4. General s t ruc tura l c h a r a c t e r i s t i c s of M. p u s i l l a . F i g . 1. Phase in ter ference 1ight micrograph of M. p u s i l l a . Note reniform shape and th i cker proximal region of emergent f l age l lum. X 4,500. F i g . 2. A long i tud ina l sect ion through a c e l l showing r e l a t i v e pos i t ion of major c e l l organe l les . Mitochondrion is not obvious in th i s plane of s ec t i on . Bar = 1.0 urn. X 53,000. F i g . 3. Whole c e l l preparat ion: specimen f i xed in osmic vapor on copper g r id then shadowed with evaporated platinum and graphite (deta i l s given in t e x t ) . B i p a r t i t e s t ructure of f lage l lum i s v i s i b l e . Bar = 1.0 ym. X 19,000. F i g . 4. Scanning e lect ron micrograph: specimen was c r i t i c a l point dr ied on nucleopore f i l t e r s of pore diameter 0.4 ym (deta i l s given in t e x t ) . X 15,500. 42 depression (F ig . 5). S i tuated near the base of the f l age l lum, s l i g h t l y an te r i o r to the pyrenoid bulge, i s a s ing le golg i body (dictyosome) which demonstrates some as soc ia t ion with an expanded per inuc lear space and the endoplasmic ret iculum (ER). 1.3. The ch lorop la s t In transverse sect ion the ch lorop la s t appears to be cup-shaped (F igs . 5 and 13). However, long i tud ina l views (Figs. 6 and 16) suggest that th i s "cup" shape i s not symmetrical ly complete. A gap at the pos ter io r and an te r i o r ends of the ch lo rop la s t suggests i t to be a more saddle-shaped s t ructure which cradles most of the c e l l contents in a s l i g h t l y asymmetric fashion as shown in F i g . 6. One s ide of the saddle is s l i g h t l y la rger than the other and houses a proport ionate ly greater share of the pyrenoid-starch complex. This s t ruc tu ra l asymmetry is evident in F igs . 6, 7, and 16. Thylakoids grouped in to stacks of two to e ight extend throughout the organe l le , l y ing p a r a l l e l to the dorsal surface (Figs. 7 and 8). These stacks are f requent ly in terrupted by small pockets of granular ch lo rop la s t stroma which resu l t s in separation and/or stacking of lamellae (F ig . 8) . Thylakoid groups a l so appear to form by fo ld ing and invag inat ion of a s ing le lamel la into mu l t ip le c l o se l y appressed lamellae (Figs. 8 and 9). The centra l port ion of the ch lorop las t houses a prominent pyrenoid measuring approximately 0.65 ym in diameter (F i g . 5) which i s surrounded by a d i s t i n c t starch s h e l l . This pyrenoid-starch complex was a constant and conspicuous feature of every c e l l examined. The pyrenoid matrix was e lec t ron opaque and granular in appearance (F i g . 2) and contained no inc lus ions or thy lako id i n t ru s ions . The starch she l l appeared u l t r a -43 F igs . 5-11. U l t r a s t ruc tu ra l features of the ch lo rop la s t . (A l l bars = 0.5 ym). F i g . 5. Transverse sect ion through a c e l l . Ch loroplast appears :.: cup-shaped in th i s view. Proiminent pyrenoid surrounded by a double- layered starch sheath i s the most obvious feature of the c e l l . Note the r e l a t i v e pos i t ions of the mitochondrion, golg i body, and endoplasmic ret icu lum (ER). Bar = 0.5 ym. X 53,000. F i g . 6. Longitudinal sect ion through a c e l l o r ientated perpendicu-l a r l y with respect to sect ion shown in F i g . 5. ( i . e . o r i e n -tated at ca. 90° ro ta t ion with respect to F i g . 5) . Note asymmetric "saddle" shape of ch lo rop la s t . Bar = 0.5 ym. X 29,000. F i g . 7. Oblique sect ion showing c l o s e l y packed thy lako ids . Mito-chondrion i s pos i t ioned d i r e c t l y adjacent to ch lo rop la s t . Bar = 0.5 ym. X 26,500. F i g . 8. High magni f icat ion of stacked thy lako ids . Note in termi t tent pocket of ch lo rop la s t stroma (arrow). Bar = 0.5 ym. X 52,000. F i g . 9. High magni f icat ion of pyrenoid-starch sheath complex. F i b -r i l s poss ib ly represent ing ch lo rop la s t genome are v i s i b l e near the edge of s tarch sheath. Bar = 0.5 ym. X 52,000. F i g . 10. Sect ion through a c e l l showing an ear l y d i v i s i o n stage of the ch lorop la s t and pyrenoid. The cleavage furrow is be-ginning to form (arrowheads). Bar = 0.5 ym. X 32,500. F i g . 11. Late d i v i s i o n stage of the ch lo rop la s t . Organel le and pyrenoid are completely b i sected. Bar = 0 . 5 ym. X 40,500. 45 s t r u c t u r a l l y to be composed of two d i s t i n c t layers (F igs. 2, 5, 12, and 15). The innermost layer was e lec t ron transparent showing l i t t l e d e t a i l ; the outermost layer was less e lec t ron transparent and f requent ly appeared to be separated from the e lec t ron t rans lucent layer by a d i s t i n c t boundary (F ig . 5). The region between the outermost l ayer and the ch lorop la s t matrix was character ized by the interming l ing of stromal and starch sheath elements (F igs. 5, 8, and 9). E lectron t rans lucent areas, containing networks of f i b r i l l a r mater ia l be l ieved to be p l a s t i d DNA (Bouck, 1965; B i sa lput ra and B i s a l pu t ra , 1967a, b ) , were observed in th i s t r an s i t i ona l region (F ig . 9 ) . The f i n e s t f i b r i l s were estimated to measure 5.5 nm in diameter and the coarser f i b r i l s 8.0 nm. Ear ly and l a te d i v i s i o n stages of the ch lo rop la s t and pyrenoid are shown in F igs . 10 and 11 re spec t i ve l y . Ch lorop last d i v i s i o n appears to begin by an invaginat ion of the ch lo rop la s t envelope which exaggerates the b i lobed appearance of the ch lo rop la s t (F ig . 6) and causes an inpocketing of the pyrenoid and i t s surrounding starch sheath (F ig . 10). This cleavage furrow continues to increase u n t i l the ch lorop la s t lobes and pyrenoid halves are completely b i sected (F i g . 11). 1.4. The mitochondrion The c e l l contains a s ing le mitochondrion approximately 0.70 ym in length and 0.25 ym in width d i sp lay ing c h a r a c t e r i s t i c ch lorophycean- l ike, f l a t tened c r i s t a e (F igs. 5, 12, and 15). The d i s co id organel le i s d i sp laced s l i g h t l y to one s ide of the median axis of the c e l l and i s pos i t ioned d i r e c t l y over the centra l depression in the ch l o rop l a s t , curving to fo l low the contours, of the pyrenoid (see Diagram 1). The extent of th i s curvature 46 F igs . 12-15. Location and u l t r a s t r u c t u r a l features of the mitochond-r i o n . (A l l bars = 0.5 ym). F i g . 12. Longitudinal sect ion through a c e l l i l l u s t r a t i n g i t s p o l a r i t y (a b i t exaggerated in th i s rather elongated c e l l ) . The major i ty of organel les ( i . e . mitochondrion, ch l o rop l a s t , golg i body, ER) are located in the an te r i o r ha l f of the c e l l . Note how the nucleus appears to be i s o l a ted in the pos ter io r lobe of the c e l l . A lso note the d i s t i n c t i v e torous shape of ER. X 52,500. F i g . 13. Transverse sect ion i l l u s t r a t i n g pos i t i on of mitochond-r i on with in the centra l depression of the ch lo rop la s t . An e lect ron transparent region contain ing f i b r i l l a r material be l ieved to be mitochondrial DNA i s shown by arrowhead. Note the loca t ion of the golg i body. X 50,000. F i g . 14. Two small p r o f i l e s of the mitochondrion are shown demonstrating the contoured form of th i s organel le . Note the expanded per inuc lear space (arrowhead). X 51,000. F i g . 15. High magni f icat ion of a mitochondrion. F lattened c r i s t a e t yp i ca l of chlorophycean algae are shown. Note the f i b r i l l a r materia l located with in the e lec t ron transparent region (arrow). X 85,000. 47 48 i s apparent in the glancing sect ion shown in F i g . 14 in which two sma l l , i r r e g u l a r p r o f i l e s of the mitochondrion are separated by a s i g n i f i c a n t expanse of cytoplasm and pyrenoid. The near-centra l l oca t ion and contoured shape of th i s organel le are a l so evident in F i g . 23. E lectron transparent areas were rou t ine l y observed with in the matrix of the mitochondrion. These regions f requent ly contained networks of f i n e f i b r i l s (Figs. 7, 13, and 15) s i m i l a r in appearance to f i b e r s observed and descr ibed at mitochondrial DNA by Nass et a l . (1965) and by B i sa lputra and B i sa lputra (1967b). These f i b r i l s measured approximately 3-5.5 nm in diameter. These dimensions compared favorably with those obtained f o r mitochondrial DNA by Nass and her co-workers (2-5 nm). The s l i g h t l y asymmetric arrangement of c e l l u l a r organel les with respect to each other i s demonstrated by the s e r i a l sect ions shown in F igs . 24 through 27. These micrographs i l l u s t r a t e the proximity of the mitochondrion to the f lage l lum (see a lso F igs . 2 and 22) and reveal that both the mitochondrion and the f l a g e l l a r apparatus are d i sp laced to one s ide of the c e l l (F igs. 24 and 25) while the nucleus and pyrenoid complex are s l i g h t l y o f f s e t towards the opposite s ide (F igs. 26 and 27), the nucleus appearing to be the most c e n t r a l l y pos i t ioned organel le with respect to the long i tud ina l axis of the c e l l . 1.5. The nucleus and nuclear-ER system The nucleus measuring approximately 0.85 ym in diameter occupies the pos ter io r lobe of the c e l l (F igs. 2, 12, and 17); the nucleolus was seldom conspicuous. Darker s ta in ing areas resembling heterochromatin were seen 49 Figs. 16-20. U l t r a s t ruc tu ra l features of the nucleus and per inuc lear ER. F i g . 16. Longitudinal sect ion through a c e l l . Ovoid nucelus is pos i t ioned at pos ter io r end of the c e l l . Expanded nuc lear -assoc iated ER region extends a n t e r i o r l y and contains materia l of a f ibrous nature (arrowhead). Note mu l t i ve s i cu l a te body. Bar = 0.5 ym. X 44,000. F i g . 17. Longitudinal sect ion or ientated at ca. 90° ro ta t ion with respect to F i g . 16. Cont inu i ty of expanded per inuc lear region is i l l u s t r a t e d with arrows. Bar = 0.5 ym. X 47,500. F i g . 18. High magni f icat ion showing s t ructure and contents of per inuc lear channel. Materia l shown here appears to be organized into long f i b e r s . Bar = 0.25 ym. X 73,000. F i g . 19. Channel o f nuclear ER f i l l e d with long f i be r s (c f . F i g . 18). Note ribosomes attached to membrane cons t i tu t ing rough ER (RER). Bar = 0.25 ym. X 75,000. F i g . 20. Sect ion showing swollen nuclear envelope containing unorganized f i b r i l l a r mater i a l . Note the nuclear pore ( fac ing arrowheads) and the as soc ia t ion of ribosomes with the outer nuclear membrane. Bar = 0.25 ym. X 81,000. 51 (Figs. 16 and 20). Nuclear pores were a lso observed rou t i ne l y . In a num-ber of sect ions these pores appeared to be qu i te large (80 nm in diameter, F i g . 20). Regions of the per inuc lear space appeared expanded, the outer membrane of the nuclear envelope often being widely separated from the inner membrane (F igs. 14, 16, 17, and 20). This swe l l ing , which may be a f i x a t i o n a r t i f a c t , i s a common feature of other green f l a g e l l a t e s (Norris and Pearson, 1975; Pickett-Heaps and Ot t , 1974). The distended per inuc lear region often contained f i b r i l l a r material (F igs. 17 and 20) or what appeared to be f i ne "ha i r s ca le s " (F igs. 16 and 18). Structures termed ha i r scales have a lso been seen in the per inuc lear region of the prasinophycean f l a g e l l a t e Mantoi i iel la squamata (Barlow, 1977), a c lose u l t r a s t r u c t u r a l r e l a t i v e of M. p u s i l l a . (See Bouck, 1972, f o r a review of mastigonemes and re la ted s t ruc tures . ) A s ing le channel of the swollen per inuc lear region was f requent ly observed to penetrate the cytoplasm (F igs. 16 and 17). The ult imate course of th i s channel was not obvious from s ing le sect iona l views. Evidence from mul t ip le and s e r i a l sect ions demonstrated cont i gu i ty with in t he . go l g i , endoplasmic ret iculum (ER), and nuclear membrane systems suggestive of a c lose r e l a t i o n s h i p , although d i r e c t con t inu i t y of these membrane systems was not observed. S im i l a r membrane continua have been observed in Heteromastix (Manton et a l . , 1965) and in Mantoniel la squamata (Barlow, 1977). Ribosomes were attached to the outermost nuclear membrane (F ig . 20). 52 1.6. The golg i and ER system A torous sheet of ER l i ne s the ventral s ide of the an te r i o r lobe of the c e l l , l y ing ju s t beneath the plasmalemma. This sheet can be seen in long i tud ina l sect ion in F igs . 12 and 14, in transverse sect ion in F i g . 5, and in obl ique sect ion in F igs . 5 and 21. The cytoplasm adjacent to the s i t e of f l a g e l l a r i n se r t i on f requent ly contained ve s i c l e s and membranous elements which a l so appeared to be part of the ER system (Figs. 15 and 22). Only the nuc lear -assoc iated ER appeared to contain ribosomes and was of the "rough" type. A s ing le golg i body i s located in the an te r i o r lobe of the c e l l , pos i t ioned above the s i t e of f l a g e l l a r i n se r t i on and between the mitochond-r ion and the ER (Figs. 21 and 22). In the major i ty of c e l l s examined the golg i body demonstrated no obvious d i f fe rence between forming and maturing faces (F igs. 21 and 22). 1.7. The f l a g e l l a r apparatus and associated cytoplasmic microtubules The s ing le emergent f lage l lum i s inser ted l a t e r a l l y , c lose to the pyrenoid-starch complex (F ig . 2) . The observations of Manton (1959) regarding the general shape of the f lage l lum have been confirmed by shadowed whole mounts and scanning e lec t ron micrographs of osmium-fixed c e l l s (F igs. 3, 4, and 28). These revealed that the f lage l lum i s b i p a r t i t e , cons i s t ing of a short (1 ym), broad, proximal sha f t , housing the normal 9+2 axonemal set of microtubules, and a conspicuously th inner , d i s t a l port ion cons i s t ing of only the two centra l axonemal microtubules surrounded by the f l a g e l l a r membrane. The d i s t a l sec t ion extended f o r 2 to 3 ym. S im i l a r abbreviated axonemal s t ructures have been observed in zoospores and gametes of some 53 F igs . 21-27. U l t ra s t ruc ture of golg i body and endomembrane system. (A l l bars = 0 . 5 ym, except as noted.) F i g . 21. Oblique sect ion through golgi body. Note i n f l a t e d areas of golgi c i s te rnae . Bar = 0.25 ym. X 70,000. F i g . 22. Longitudinal sect ion through f lage l lum and basal body. Golgi body i s located an te r i o r to f l a g e l l a r base and juxtapos i t ioned between mitochondrion and ER. X 60,500. F i g . 23. Sect ion through the mu l t i ve s i cu l a te body showing i t s contents to be a heterogeneous c o l l e c t i o n of ve s i c l e s and membranous elements. X 49,000. F igs . 24-27. Ventral region of c e l l shown in s e r i a l sect ions . X 38,500. F i g . 24. Section showing shape and pos i t i on of mitochondrion, golg i body and f l a g e l l a r apparatus. Note the second basal body (1 arrow) and the microtubules (arrowhead) which extend p o s t e r i o r l y toward nucleus. F i g . 25. Flagel lum and mitochondrion are moving out of the plane of sect ion simultaneously as nucleus and pyrenoid starch sheath begin to appear. Note that mu l t i ve s i cu -l a te body shows' no cont inu i ty with the c e l l ' s endomem-brane system (cf. F igs . 26 and 27). F i g . 26. The cytoplasmic extension of the nuclear s p a c e , i . e . p e r i n u -c l ea r region(PER, th i s F i g . only) i s beginning to. appear. F i g . 27. Pyrenoid begins to appear as golg i body moves out of the plane of sec t i on . Note the f i b r i l - l i k e material (arrow-head) ins ide the cytoplasmic extension of the per inuc lear space. 5.5 phaeophycean algae and among other r e l a t i v e s of the Chlorophyceae (Manton, 1965). A long i tud ina l view of the extension of the two centra l micro-tubules beyond those of the per ipheral r ing i s shown in the u l t r a t h i n sect ion in F i g . 29. Metal-shadowed specimens a d d i t i o n a l l y revealed the presence of a t u f t of three or four very f i n e ha irs attached to the f l a g e l l a r t i p , not reported prev ious ly by Manton (1959). Only 30 to 40% of the c e l l s examined displayed these ha i r s . Due to t h e i r de l i c a te s t ructure i t was assumed that c e l l s lack ing these hairs had l o s t them during process ing. The " garn i ture " of terminal f l a g e l l a r ha irs seen on Mantoniel l a squamata (Barlow, 1977) were found to f a l l o f f f requent ly during f i x a t i o n procedures. In add i t ion to the extreme shortness of the "9+2" region of the f lage l lum i t i s u l t r a s t r u c t u r a l l y unusual in other respects , two of these invo lv ing the t r a n s i t i o n region. Although two electron-opaque cy l inders and a transverse septum are v i s i b l e in long i tud ina l sect ion (F i g . 30), the d i s t i n c t i v e " s t e l l a t e s t ruc ture " of Lang (Lang, 1963) was not seen in any transverse sect ions . This may have been due to a paucity of cross sect ions through th i s region of the t r a n s i t i o n zone. However i t may be re l a ted instead to a second unusual feature of the f l age l lum: an e lect ron opaque s t ructure pos i t ioned d i s t a l to the f l a g e l l a r t r a n s i t i o n zone which, in long i tud ina l view, has the shape of a tapered cy l i nder and was apparently connected to the per iphera l r ing of microtubule doublets (F igs. 30 and 47). The centra l pa i r of axonemal microtubules extended through th i s tapered s t ructure but appeared to terminate j u s t an te r i o r to the d i s t a l c y l i nder of the " s t e l l a t e region" (F ig . 30). In transverse sect ion th i s s t ructure appeared as a r ing e n c i r c l i n g the two centra l microtubules (F ig . 32) 56. F igs . 28-36. F l a g e l l a r u l t r a s t r u c t u r e : General features . F i g . 28. Shadowed whole mount of f l age l lum showing terminal ha i r s (arrowheads). Bar = 0.5 ym. X 43,500. F i g . 29. Longitudinal sect ion of junct ion between proximal f l a g e l l a r region and d i s t a l extens ion. Bar = 0 . 5 ym. X 49,000. F i g . 30. Longitudinal sect ion through f l a g e l l a r t r a n s i t i o n zone, basal p l a t e , and d i s t a l port ion of the basal body. Note tapered cy l i nde r (TC) s i tuated d i r e c t l y above t r a n s i t i o n reg ion. Numbered arrows r e f e r to various planes of cross sect ion i l l u s t r a t e d in F igs . 31-34. Bar = 0.5 ym. X 99,500. F i g . 31. Cross sect ion through f l a g e l l a r axoneme. Corresponds to leve l of arrow #1 in F i g . 30. Bar = 0.25 ym. X 64,000. F i g . 32. Cross sect ion through "tapered cy l i nde r " reg ion, shown here as a membrane surrounding the centra l two microtub-u les . Corresponds to leve l of arrow #2 in F i g . 30. Bar = 0.25 ym. X 71,000. F i g . 33. Cross sect ion below basal p l a te . Central two microtubules are no longer v i s i b l e . Corresponds to leve l of arrow #3 in F i g . 30. Bar = 0.25 ym. X 74,500. F i g . 34. Cross sect ion through d i s t a l end of basal body. Micro-tubules are grouped into t r i p l e t s . Corresponds to leve l of arrow #4 in F i g . 30. Bar = 0.25 ym. X 74,500. F i g . 35. Longitudinal view of an i n t r a c e l l u l a r axoneme showing a 9+2 set of microtubules l y ing ins ide the plasmalemma. Associated cytoplasmic microtubules are l abe l l ed (arrow-heads). Bar = 0.5 ym. X 52,500. F i g . 36. Cross sect ion through i n t r a c e l l u l a r axoneme (arrow) which i s surrounded by lobed nucleus. Bar = 0.5 ym. X 65,500. 58 g iv ing the same impression as a c ros s - sec t iona l view of t h e . " c o i l e d f i b r e " discussed by Bouck (1971) and by Moestrup and Thompson (1974). The small c i r c u l a r p r o f i l e s of a c o i l i n g " f i b e r " described by Bouck f o r Ochromonas (1971) were not seen. Some c e l l s contained an axonemal set of 9+2 microtubles deep with in the cytoplasm (Figs. 35 and 36). Pickett-Heaps and Ott observed th i s type of phenomenon in the reduced prasinophycean f1agel1 ate Pedinomorias (1974) but d id not comment on i t s s i g n i f i c a n c e . The withdrawal of f l a g e l l a into the c e l l body has been observed in u n i c e l l u l a r stages of members of the Phaeophyceae (Baker and Evans, 1973; D. C. Walker, pers. comm.) and has been thought to be re la ted to s e t t l i n g and germination. In o lder cu l tures of Micromonas,cells were f requent ly observed to s e t t l e to the bottom of growth f l a s k s . However, as only a c t i v e l y swimming c e l l s were used f o r u l t r a s t r u c t u r a l studies i t i s un l i ke l y that the numerous ind iv idua l s observed to contain i n t r a c e l l u l a r axonemes were a l l s e t t l i n g c e l l s . Manton (1960) described a non-moti le, pa lmel lo id phase of M. p u s i l l a , although th i s stage was never observed to occur in any of the cu l tures used f o r t h i s study. Interphase c e l l s normally have one f u n c t i o n a l , emergent f lage l lum (Figs. 2 and 38). Those c e l l s with two f l a g e l l a (F ig . 40) were presumed to have been enter ing c e l l d i v i s i o n . Observations of a second basal body (F igs. 37, 38, and 41) were at f i r s t a lso in terpreted as a sign of f o r t h -coming c e l l d i v i s i o n . However the incidence of c e l l s containing three or four basal bodies (F igs. 39 and 40) led to the conclus ion that M. p u s i l l a does contain a second, non- funct iona l ,basa l body. Manton (1959) and •,.59 F igs . 37-42. Views of extra basal bodies and miscellaneous i nc lu s i ons . (A l l bars =0 .5 ym, except where noted.) F i g . 37. Cross sect ion through f l a g e l l a r basal body (bb-1) and second non-funct ional basal body (bb-2). X 61,500. F i g . 38. Longitudinal sect ion through funct iona l and non-funct ional basal bodies (bb-2). Note that second basal body pro-trudes s l i g h t l y from c e l l body. X. 43,000. F i g . 39. D i v i s i on of the f l a g e l l a r apparatus: c e l l contain ing three basal bodies shown in transverse and long i tud ina l views (arrowheads). Fourth basal body is not v i s i b l e in th i s sec t i on . X 36,000. F i g . 40. Longitudinal sect ion through c e l l contain ing four basal bodies. Note the d i f ferences in the lengths of emergent ( f l ) vs. "stubby" f l a g e l l a (arrowhead). X 38,000. F i g . 41. Section showing two mucocyst- l ike bodies (MCB) located adjacent to c e l l surface and near basal bodies. Note the organizat ion of mcb matrix material into bands (arrowhead). X 52,000. F i g . 42. Section through two MCBs which are interconnected ( s ing le arrow). Note banding of matrix material and protrus ion of MCB through plasmalemma (arrowhead). Bar = 0.25 ym. X 89,500. 61 Pienaar (1975) a l so observed a second basal body in Micromonas but suggest-ed that th i s s t ructure was associated with c e l l d i v i s i o n . The second basal body was occas iona l l y observed to protrude s l i g h t l y from the c e l l body (Figs. 38 and 40) but in general i t appeared to be more reduced than the "stubby f l age l lum" recent ly described fo r Mantbriiella squamata (Barlow, 1977). No s t r i a t e d f i b e r or rh i zop l a s t - 1 i ke s tructures were seen associated with the f l a g e l l a r apparatus. The c e l l s were observed to contain two per ipheral microtubular systems, composed of one and two microtubules re spec t i ve l y (here ina f ter re fer red to as the s i n g l e t and doublet micro-tubu les ) , both of which emanate from the basal body region and extend around the c e l l ' s periphery d i r e c t l y beneath the plasmalemma (Figs. 46-50). The prec i se extent and arrangement of these microtubules remains unclear. In the obl ique transverse sect ion shown in F i g . 43 the two systems of microtubules are seen in cross sec t i on . The doublet seems to o r i g ina te between the basal body and the plasmalemma and runs ob l i que l y , t r a v e l l i n g approximately ha l f way around the c e l l . A higher magni f icat ion of the r idge of cytoplasm enclos ing th i s feature is shown in F i g . 45. The course of the s i n g l e t i s less c l ea r . It appears to o r i g ina te on the inner s ide of the basal body and extends p o s t e r i o r l y f o r an undetermined d istance (Figs. 43, 44, 47, and 49). Figure 46 o f fer s a long i tud ina l view of the f l a g e l l a r apparatus and i t s associated microtubules. Based on these observations i t was concluded that the two per iphera l microtubular systems fo l low d i f f e r e n t obl ique paths around the c e l l . 62 F igs . 43-50. F l a g e l l a r apparatus: associated per iphera l cytoplasmic micro-tubules. F i g . 43. Transverse sect ion showing the two systems of per iphera l micro-tubules (MT). Paired MTs are v i s i b l e d i r e c t l y beneath plasma-lemma external to basal body and ch lorop la s t (double arrowheads). S ing le MT (s ing le arrow)is s i tuated between basal body and nucleus. Bar = 0.5 ym. X 66,500. F i g . 44. Higher magni f icat ion showing axoneme and associated s ing le MT. Bar = 0.25 ym. X 91,500. F i g . 45. High magni f icat ion of paired per ipheral MT located with in a protruding r idge of cytoplasm external to the ch lo rop la s t . Bar = 0.25 ym. X 91,000. F i g . 46. Longitudinal view of f l a g e l l a r apparatus and associated cy to -plasmic MTs Presence of i n t r a c e l l u l a r axoneme makes i d e n t i f -i c a t i on of respect ive MTs d i f f i c u l t . Bar = 0.5 ym. X53,500. F i g . 47. Longitudinal sect ion through f l a g e l l a r t r a n s i t i o n zone and basal body. Note tapered cy l i nder (TC, arrowheads) located above t r a n s i t i o n zone and s ing le r o o t l e t MT. Bar = 0.5 ym. X 37,500. F i g . 48. Cross sect ion through r idge of cytoplasm containing the s ing le per iphera l microtubule. Bar = 0.25 ym. X37,500. F ig . 49. Longitudinal view of per ipheral microtubule system. MTs fo l low the contours of the c e l l surface and move rap id l y out of the plane of sec t i on . Two glancing sect ions through s i ng l e t MT shown here i l l u s t r a t e i t s course around periphery of c e l l . Bar = 0.5 ym. X 53,500. F i g . 50. Glancing long i tud ina l sect ion showing doublet MTs. (See legend, of F i g . 49). Bar = 0.5 ym. X 85,000. 64 1.8. Miscel laneous inc lus ions Located adjacent to the f l a g e l l a r basal bodies and juxtaposed between the per inuc lear ER and the mitochondrion, i s a s i n g l e , vacuo le - l i ke s t ructure (F igs. 16, 23, and 25), approximately 0.4 ym in diameter. In sect ion the body appears round or ovoid and f requent ly contains membranous elements and/or numerous small ve s i c l e s of var iab le dens i ty . Consequently, i t i s re fe r red to here as the mu l t i ve s i cu l a te body. While i t appears to be d i s c r e t e , showing no d i r e c t connection with the golg i or ER membrane systems, small cytoplasmic ve s i c l e s often occur in c lose proximity (Figs. 23 and 35). The general form of th i s vacuo le - l i ke body i s s trongly reminiscent of the Corps de Maupas descr ibed in cryptomonads (c f . Lucas, 1970).. Based upon a h i s t o l o g i c a l examination Lucas (1970) suggested that the Corps de Maupas might be lysosomal in character (see Mat i l e , 1975, fo r a review of l y t i c compartments in p lant c e l l s ) . Histochemical work under r i g i d l y con t ro l l ed condit ions w i l l be necessary before the funct ion of the mu l t i ve s i cu l a te body seen here can be f i rm ly e s tab l i shed. Also located with in the cytoplasm, adjacent to the plasmalemma, are smal l , membrane-bound organel les which present round to i r r e g u l a r l y shaped p r o f i l e s in sect ion (F igs. 41 and 42). The number and frequency of occurrence of these bodies are v a r i a b l e , but when present they genera l ly occur in pa irs and measure approximately 0.25 ym in diameter. The matrix cons i s t s of an amorphous, semi-e lectron dense material which i s ne i ther d i s t i n c t l y granular nor f i b r i l l a r and which appears to be organized into bands or layers on the s ide of the organel le adjacent to the c e l l ' s 65 periphery (Figs. 41 and 42). Extensions of these organel les have been observed to protrude through the plasmalemma suggesting that they may be some form of extrusome (Hovasse and Mignot, 1970). They bear no s t ruc tura l resemblance to t r i chocys t s described prev ious ly f o r the Prasinophyceae (Manton, 1969; Nor r i s , 1975), nor are they s i m i l a r to those seen in other a lga l c lasses (Hovasse and Mignot, 1970). These s tructures may represent a form of mucocyst, but cytochemical inves t i ga t ion is necessary to determine the chemical nature of th i s body. 66 II. VIRUS MORPHOLOGY Studies o f sect ioned and negat ive ly s ta ined specimens ind ica ted v i rus p a r t i c l e s were qui te uniform with regard to general morphology. Deta i led s t ructure var ied in appearance however, according to the observat ional techniques employed, and, in sect ioned ma te r i a l , according to the state of maturity and angle of cut. The i n t r a c e l l u l a r appearance of the Micromonas p u s i l l a v i rus i s presented in F igs . 51 and 52. From these micrographs i t can be c l e a r l y seen that the v i r i on s are polyhedral in form and may d i sp lay an e lect ron opaque centra l region (F i g . 52), the presence of which appeared to depend upon the f i x a t i o n technique employed and the thickness of sec t ion . Based upon sect iona l views, the edge to edge diameter (as seen on a t h ree - f o l d axis of symmetry) was measured to be approximately 130 nm. Examinations of s e r i a l sect ions suggested that poorly def ined and incomplete p a r t i c l e s (F igs. 51 and 52) should genera l ly be in terpreted as g lancing sect ions of f u l l y matured v i r i o n s , rather than as intermediate developmental stages. The smal ler subc i r cu l a r p r o f i l e s seen in F i g . 51 probably represent these e a r l i e r developmental and precursor stages. E lectron microscope examination of c l a r i f i e d v i r a l lysate and u l t r a -centr i fuged v i r i o n s sta ined with 2% uranyl acetate revealed p a r t i c l e s with hexagonal or pentagonal out l ines d i sp lay ing no obvious t a i l - l i k e s t ructures or other external appendages. It must be noted that associated external s t ructures were cons i s ten t l y lack ing in a l l types of specimen preparat ions examined, with the exception of ea r l y i n f ec t i on stages in which forms which might be l ikened to t a i l s t ructures were occas iona l l y Figs. 51-52. I n t r a c e l l u l a r appearance of v i rus p a r t i c l e s . F i g . 51. Infected c e l l containing polyhedral p a r t i c l e s . Note d i s t i n c t hexagonal shape of v i r i o n (s ing le arrow) and weaker p r o f i l e s of t angent i a l l y sectioned v i r i on s (arrowheads). Bar = 0 . 5 ym. X 55,000. F i g . 52. Thick sect ion through an in fected c e l l showing numerous polyhedral v i rus p a r t i c l e s conta in ing densely s ta in ing core s tructures (arrows). Bar = 0.25 ym. X 102,500. 69 observed associated with attaching v i r i ons ( see- Infect ion Process). Sta in ing with 1% phosphotungstic ac id (PTA) (buffered at various pH va lues ) , resu l ted in the o b l i t e r a t i o n of f i n e s t ruc ture . The average diameter of negat ively sta ined p a r t i c l e s was 135 nm, approximately equal to the diameter of p a r t i c l e s examined in th i s sec t i on . This minor d i f fe rence might have been due to the e r ro r associated with accurate measurement of s ta in embedded v i r i o n s , or conversely i t might be re la ted to d i s t o r t i o n of p a r t i c l e s during dehydration and embedding fo r sec t ion ing . The polyhedral p a r t i c l e s present p r o f i l e s cons i s tent with t h e i r having icosahedral symmetry. In the l i n e drawing of Ser ies I, a-c icosahedra are presented on t h e i r f i v e - , t h ree - , and two-fold axes of symmetry. Ser ies II, a-c of the p late i l l u s t r a t e s p r o f i l e s which might re su l t from the models of Ser ies I. The three- and two-fold p r o f i l e s represent median sect ions whi le the f i v e - f o l d p r o f i l e presents a tangentia l view; a median sect ion through the f i v e - f o l d o r ien ta t i on model would appear e i the r near ly c i r c u l a r (at lower reso lut ion) or ten-s ided (at higher r e so lu t i on ) . F igs . 53 to 58 provide u l t r a s t r u c t u r a l examples of each of these p r o f i l e s . The v i rus p a r t i c l e s shown in F igs . 53-55 have been sta ined with aqueous uranyl acetate ; the v i r i o n in F i g . 53 absorbed the chemical and was p o s i t i v e l y contrasted with the s ta in while those p a r t i c l e s in F i g . 54 and 55 were negat ively contrasted. F igs . 56-58 i l l u s t r a t e the three c h a r a c t e r i s t i c p r o f i l e s of icosahedral p a r t i c l e s found in sect ioned mate r i a l . The v i rus p a r t i c l e s appeared to f l a t t e n s i g n i f i c a n t l y upon drying making i t necessary to introduce a f i x a t i v e before shadowing with evaporated metal. The optimum resu l t s were obtained by the app l i ca t i on of a drop of -70 Series I. I l l u s t r a t i o n s of icosahedra on f i v e - (a) , three- (b), and two-fold (c) axes of symmetry. Ser ies II. P r o f i l e s of sect ions through icosahedra. F i g . I la i l l u s t r a t e s a tangent ia l sect ion through f i v e - f o l d or ient -at ion model. F i g s . . l i b and c represent median sec t ion . F igs . 53-55. Views of i s o l a ted v i rus p a r t i c l e s s ta ined with aqueous uranyl acetate. Bars = 100 nm. F i g . 53. V i r i o n , i l l u s t r a t i n g f i v e - f o l d symmetry, has absorbed uranyl acetate to become p o s i t i v e l y contrasted. Note how centra l region i s densely sta ined and per ipheral areas show d i s t i n c t l ayer s . X 230,800. F i g . 54. Hexagonally shaped p a r t i c l e i l l u s t r a t i n g t h r e e - f o l d axis of symmetry. Deposit ion of s t a in in layers (arrowhead) a lso evident in th i s negat ive ly contrasted specimen. X 198,500. F i g . 55. Negatively sta ined v i rus p a r t i c l e showing two-fold axis of symmetry. X 197,500. F igs. 56-58. U l t r a th i n sect ions of i n t r a c e l l u l a r v i r i on s showing char-a c t e r i s t i c icosahedral p r o f i l e s . Bars = 100 nm. F i g . 56. P a r t i c l e demonstrating f i v e - f o l d symmetry. X 196,000. F i g . 57. P a r t i c l e demonstrating th ree - f o l d symmetry. X 196,000. F i g . 58. P a r t i c l e demonstrating two-fold symmetry. X 196,000. 71 72 aqueous uranyl acetate to gr ids contain ing v i r a l suspensions. The uranyl acetate and seawater were removed by r in s ing with d i s t i l l e d water and the gr ids were dr ied and shadowed. These procedures did not e l iminate th i s a l t e r a t i o n of p a r t i c l e shape but did diminish i t s e f f e c t . I n i t i a l l y th i s d i s t o r t i o n due to f l a t t e n i n g was be l ieved to be the r e su l t of the p a r t i a l co l l apse of the v i r a l caps id , re su l t i ng in what appeared to be the exaggerated protrus ion of the polyhedral ap ices. These p a r t i a l l y co l lapsed p a r t i c l e s when viewed in metal-shadowed preparations presented d i s t i n c t l y non-spherical contours with sca l loped-shaped, f i v e - and s i x - s i ded p r o f i l e s , reminiscent of s tars (F igs. 59 and 60). This star-shaped contour was not, however, r e s t r i c t e d to metal-shadowed preparat ions, s i m i l a r p r o f i l e s f requent ly being observed among groups of negat ive ly contrasted p a r t i c l e s (F ig . 61). Protrus ion of ap ica l regions has been observed, but not d iscussed, in other icosahedral v i ruses (e.g. T ipu la i r i de scen t v i r u s , Wil l iams and Smith, 1958) but not to the s i g n i f i c a n t extent as was evidenced here. As sect ioned material f a i l e d to demonstrate th i s morpho-l og i ca l f ea ture , there i s a p o s s i b i l i t y that th i s p e c u l i a r i t y of shape i s a r t i f a c t u a l . An outer she l l and an inner body can be d i s t ingu i shed in v i rus p a r t i c l e s which have been negat ively or p o s i t i v e l y contrasted with uranyl acetate. The outer s h e l l , taken to represent the v i r a l caps id , appears to be a mul t i l ayered s t ructure with no obvious surrounding envelope (Figs. 53, 62, and 63). As presented in F igs . 62 and 63 the mul t i l ayered nature of the v i r a l coat i s manifested as a l t e rna t ing bands of e lec t ron opaque and e lect ron transparent mater i a l . 73 Figs. 59-65. Morphological features of i s o l a ted v i r i o n s . Specimens were sta ined with aqueous uranyl acetate unless otherwise noted. Bars = 100 nm except where noted. F i g . 59. . Metal-shadowed v i r i on s f i xed in uranyl acetate p r i o r to shadowing to diminish d i s t o r t i o n due to dry ing. Note pecu l i a r " s tar " - shape bel ieved to be the r e su l t of p a r t i a l co l l apse of v i r a l caps id . Bar = 200 nm. X 53,000. F i g . 60. Higher magni f icat ion of a v i r i o n shown in F i g . 59. Note exaggerated appearance of the polyhedral apices (arrowhead). Bar = 200 nm. X 86,000. F i g . 61. Negatively contrasted v i rus p a r t i c l e showing prominence of ap ica l region resembling that seen in F i g . 60. X 145,000. F i g . 62. Two negat ive ly contrasted specimens. Note double- layered appearance of p a r t i c l e coats ( fac ing arrowheads). X 147,000. F i g . 63. P o s i t i v e l y contrasted v i r i o n showing d i s t i n c t per ipheral banding (adjacent to arrowhead). X 287,500. F igs. 64-65. P a r t i a l l y d isrupted v i r i o n s . Sta in has penetrated into the v i r a l capsid al lowing reso lu t ion of morphological subunits. F i g . 64. Morphological subunits in terpreted to be capsomeres are shown organized into a t r i angu l a r array. Subunits appear hollow. X 230,000. F i g . 65. Subunits v i s i b l e here do not demonstrate obvious pattern of organ izat ion. X 230,000. 74 75 A number of negat ively contrasted specimens contained an i r r e g u l a r , e lec t ron opaque body of var i ab le s i ze located with in the centra l port ion of the p a r t i c l e (F igs. 54 and 55). Some sect ioned samples a l so demonstrated a small e lec t ron dense, mid-zonal spot, d i s t ingu i shab le by i t s smal ler s i ze from the darkly s ta in ing core s t ructures observed in v i r i on s of F i g . 52. These features were not cons i s ten t l y seen. These e lec t ron opaque areas may be re la ted to the condensation of v i r a l genome, or may be a r t i f a c t u a l . Surface substructures analogous to the morphological subunits or capsomeres of other v i ruses were not regu la r l y detected on i n t ac t v i rus p a r t i c l e s contrasted with negative s t a i n . Addi t iona l capsid e laborat ion was observed, however, on v i r i on s that had been p a r t i a l l y d i s rupted, the amount of de ta i l depending upon the degree to which the s ta in penetrated into the i n t e r s t i c e s of the v i r i on s (Madeley, 1972). In F igs . 64 and 65 specimens in which s ta in penetrat ion was s u f f i c i e n t to permit v i s u a l i z a t i o n of v i r a l capsomeres are shown. The subunits seen in F i g . 64 appear as an equ i l a te ra l t r i angu l a r assemblage not unl ike the trisymmetrons-described by wrigley (1969, 1970) and S t o l t z (1971, 1973) f o r some i r i d o v i r u s e s . The arrangement of capsomeres i s not as obvious in F i g . 65. The subunits appear to rad iate from a centra l locus on the p a r t i c l e suggesting the specimen may have been or iented on i t s f i v e - f o l d axis of r o t a t i o n . In F i g . 64 the capsomeres can be c l e a r l y d i s t ingu i shed as being hollow while in F i g . 65 l i t t l e substructure i s revealed. 76 III. ULTRASTRUCTURAL ASPECTS OF THE INFECTION PROCESS The fo l lowing sect ion i s devoted to the u l t r a s t r u c t u r a l aspects of the passage of v i r a l i n f e c t i o n . More than f o r t y d i f f e r e n t f i xa t i on s of in fected cu l tures were examined. In the major i ty of cases in fected c e l l s did not preserve w e l l . The re su l t s presented here represent observations of a small number of adequately preserved, in fec ted specimens. Three main aspects of the i n f e c t i o n were examined: 1) v i r a l attachment and penetrat ion (ear ly ho s t - v i r i on i n t e r a c t i o n ) , 2) morphological a l t e ra t i on s of in fected c e l l s associated with v i r a l m u l t i p l i c a t i o n , and 3) u l t r a s t r u c -tura l aspects of v i r a l re lease. Observation resu l t s have been grouped accord ing ly. I I I . l . Ear ly host -v i rus i n te rac t i on Healthy cu l tures were exposed to v i r a l inocula and incubated at growth chamber temperature f o r various lengths of time before being harvested and prepared f o r u l t r a s t r u c t u r a l study. Samples were harvested and f i xed every 15 min f o r two hours, then every 30 min f o r three hours. The greatest numbers of v i r a l p a r t i c l e s were seen on the outer surface of c e l l s a f t e r 15 min of exposure. By 30 min the number o f external p a r t i c l e s in the v i c i n i t y of the c e l l s had begun to decrease, and by one hour v i r t u a l l y no e x t r a c e l l u l a r v i rus p a r t i c l e s were observed. The f i r s t i n t r a c e l l u l a r p a r t i c l e s appeared in c e l l s harvested a f t e r three hours o f exposure. C e l l s prepared f o r the e lec t ron microscope a f t e r exposure to inocula f o r less than three hours showed no morphological evidence of i n f e c t i o n . 77 Figures 66 through 71 i l l u s t r a t e adsorption of the v i r i ons to the c e l l surface and what is in terpreted to be ear ly stages of v i r a l attachment leading to v i r a l entry. As seen in F igs . 66 and 67, the v i r i o n s can be d i s t r i bu ted randomly over the en t i re surface of the c e l l inc lud ing the f l a g e l l a r apparatus. The specimens in these f igures were sta ined with uranyl acetate then thoroughly washed before the s ta in was allowed to dry in order to e l iminate the p o s s i b i l i t y that the v i r i on s might adsorb onto the c e l l s during dry ing. The accumulation of s t a in ( in F igs . 66 and 67) prevented re so lu t i on of f i n e r de ta i l s and thus i n h i b i t e d determination of the intimacy of th i s b inding. More information was provided by sect iona l views of c e l l s exposed to an inoculum fo r 15 minutes (F igs. 68-79). Figures 68 through 79 i l l u s t r a t e v i r a l attachment and what i s in terpreted here to be the means by which the v i r a l genome gains entry into the host c e l l . . In the sequence of events which fol lows i t is assumed that a l l v i r i on s seen attached to the surface of c e l l s were in the process of entry rather than e x i t . Only c e l l s harvested a f t e r 15 minute exposure were used to i l l u s t r a t e these processes. I n i t i a l l y , the v i r i on s l i e in proximity to the apparently unaltered c e l l surface (F igs. 68 and 69). In some instances where v i r i ons showed no d i r e c t contact with host c e l l s there appeared to be a weakly s ta in ing bridge of materia l between the v i r a l coat and the plasmalemma (F ig . 69). Small project ions of the c e l l surface appeared to be the f i r s t v i s i b l e sign of response of the host surfaces to the proximity of the v i rus (F igs. 70 and 71). At th i s stage of i n i t i a l i n te rac t i on no d i s i n teg ra t i on o r ' 78 Figs. 66-71. Ear ly i n te r ac t i on of v i r i o n and M. p u s i l l a . Ce l l s shown in F igs . 68-71 were exposed to v i rus f o r 15 min before f i x a t i o n . (Bars = 100 nm except where noted.) F i g . 66. Ce l l with attached v i r i on s p o s i t i v e l y contrasted with uranyl acetate. Note the random d i s t r i b u t i o n of v i rus p a r t i c l e s over a l l surfaces inc lud ing f l age l lum. Bar = 0.5 ym. X 32,500. F i g . 67. Higher magni f icat ion of v i rus p a r t i c l e s on f l a g e l l a r surface (from F i g . 66). Note that no t a i l - l i k e s t ructures or other external appendages are evident. X 104,000. F i g . 68. .Section showing ear l y stage o f . v i r a l attachment. Interact ion between host and v i r i on s appears to be s u p e r f i c i a l (arrow-heads). V i r i o n at bottom of F i g . (arrow) appears to be surrounded by double- layered membrane. X 113,000. F i g . 69. V i r i on on c e l l sur face. Note amorphous, semi-e lectron dense materia l located between p a r t i c l e and plasmalemma (arrow-heads). X 112,000. F i g . 70. Two v i r i on s on c e l l sur face, one showing densely s ta in ing core (arrow). Note pro ject ion of c e l l surface (arrowhead) at s i t e of v i r a l attachment (cf. F igs . 72-79). X 113,000. F i g . 71. Section through v i r i o n at c e l l surface. Virus and host membranes appear to be i n tac t and show no evidence of i n t e r -ac t i on . X 109,000. 80 other morphological changes in the v i r a l coat were observed (Figs. 69-71). The region of attachment always appeared to involve only a small area of the surface of the v i r i o n inc lud ing an apex of the polyhedral p a r t i c l e (F igs. 68-79). The s i z e of th i s region d id not appear to be re la ted to the intimacy of b ind ing , demonstrating only minimal v a r i a t i on between specimens (F igs. 72-79). The next stage of i n te rac t i on appeared to be a change in the conf i gur -at ion of the cytoplasmic membrane in the proximity of v i r a l attachment, character ized by the lack of a d i s t i n c t boundary separating v i rus and c e l l surfaces (F igs. 72-74). This apparent " f u s i on " of the v i r a l coat to the c e l l , with a d i s rupt ion of t h e i r respect ive sur faces , permitted d i r e c t cont inu i ty between the cytoplasm and the in terna l compartment of the v i r i o n (Figs. 72-76). At s i t e s of presumed v i r a l penetrat ion, fi lamentous or f i b r i l l a r ma te r i a l , in terpreted to represent the v i r a l genome, was observed at a stage of t r a n s i t into the c e l l (F i g . 75). In F i g . 76 " f i l aments " are v i s i b l e , extending from the v i r a l p a r t i c l e , through the s i t e of attachment, and into the cytoplasm. V i r i ons were occas iona l l y observed attached to c e l l s v ia a s t a l k - or t a i l - l i k e p ro jec t ion (Figs. 77-79). The o r i g i n of th i s p ro jec t ion was unclear. In. some views the s t ructure appeared to be an e laborat ion of the c e l l surface (F i g . 77), while in other instances a re l a t i onsh ip with the v i r i o n appeared more l i k e l y (Figs. 78 and 79). As ne i ther i s o l a ted not i n t r a c e l l u l a r v i r i on s demonstrated s i m i l a r appendages i t was assumed that these project ions were hos t -der ived, instances where the host membrane appeared to c lose o f f the bottom of th i s p ro jec t ion (F ig . 79) being the poss ib le r e s u l t of the plane of sec t i on . 81 F igs . 72-79. Views of v i r a l attachment and host -v i rus i n t e r a c t i o n . Ce l l s in these f i gures received 15 min exposure to v i rus before f i x a t i o n . (Bars = TOO nm.) F i g . 72. Glancing sect ion through attaching v i r i o n . Note the lack of d i s t i n c t boundary between v i rus and c e l l surface (arrow-head). Note a lso condensed f i b r i l l a r material ins ide v i r i o n (arrow). X 127,000.. F i g . 73. Attaching v i r i o n i s shown on protrus ion produced at s i t e of contact. F i b r i l l a r materia l resembling that seen in F i g . 72 i s v i s i b l e near the base of the v i r i o n (arrow). X 136,500. F i g . 74. V i r i o n attached by " s t a l k " - l i k e p ro jec t ion to c e l l surface. X 136,500. F i g . 75. F i b r i l l a r material v i s i b l e ;within v i r i o n i s a l so apparent ins ide host, adjacent to attachment s i t e (arrow). X 136,500. F i g . 76. Note what appears to be a t rans fe r of e lec t ron dense materia l between v i r i o n and host c e l l (arrows). Also note contents of r e se rvo i r (e lectron transparent area) beneath plasmalemma (arrowheads). X 139,000. F i g . 77. Sect ion through attaching v i r i o n showing associated s ta lk or t a i l - l i k e p ro jec t ion (arrowhead). Pro ject ion appears to be der ived from host surface. X 139,000. F i g . 78. See text to F i g . 77. (Represents a d i f f e r e n t sect ion than that shown in F i g . 77.) X 139,000. F i g . 79. See text to F i g . 77. X 139,000. 83-Stages in the disappearance of parental v i r i on s were not documented. Inoculum v i r i on s were never observed to enter host c e l l s v i a phagocytosis ( v i ropex i s ) . III.2. I n t r a c e l l u l a r morphological features of the i n f ec t i on The e a r l i e s t in terna l u l t r a s t r u c t u r a l evidence of i n f e c t i o n was observed in c e l l s a f t e r three hours of exposure to the inoculum. This may be considered to be the termination of the " e c l i p s e per iod" of the i n f ec t i on ( i . e . p r i o r to the formation of recognizable v i r i on s with in the c e l l ; Fenner et a l . , 1974). The u l t r a s t r u c t u r a l appearance of c e l l s in "3 hour" preparations was qui te v a r i a b l e , some c e l l s showing no signs of i n f e c t i o n . The major i ty of c e l l s examined, however, showed some morphological manifestat ions of the i n f e c t i o n , with varying degrees of cytoplasmic degradation (Figs. 80-91). Figures 80 through 83 i l l u s t r a t e what i s in terpreted here to be ear ly i n t r a c e l l u l a r evidence of i n f e c t i o n . The cytoplasm of these c e l l s contained large numbers of sma l l , membrane-bound ves i c le s (mbv) which were f requent ly seen in assoc ia t ion with the c e l l ' s endomembrane system (Figs. 81 and 83). Most mbv's were d i s t i n c t l y smal ler in diameter (ca. 60-80 nm) and less regu la r l y shaped than the polyhedral v i r a l p a r t i c l e s . No s t ructures s i m i l a r in appearance to mbv's were ever observed in uninfected c e l l s from control cu l tu res . Functional aspects of the spa t i a l a s soc ia t ion of the mbv's, the polyhedral v i r i o n s , and the golgi body shown in F igs . 82 and 83 were not c l e a r . By convention (Morrg et a l . , 1971), when the golg i body demonstrates 84 Figs. 80-83. Ear ly i n t r a c e l l u l a r evidence of v i rus i n f e c t i o n . Ce l l s show no obvious signs of cytoplasmic degradation. F i g . 80. Sect ion through c e l l contain ing numerous membrane bound ve s i c l e s (mbv) and one l a rger polyhedral v i r a l p a r t i c l e . Note that a l l v i r a l re la ted (presumably) s t ructures are located in the cytoplasm external to organe l les . Bar = 0.5 ym. X 54,000. F i g . 81. High magni f icat ion of mbv's and polyhedral p a r t i c l e located adjacent to surface of ER. Membranes around mbv's are d i s t i n c t l y less e lec t ron dense than e i t he r those of endo-membrane system (e.g. ER) or those surrounding the polyhedral p a r t i c l e . Bar = 200 nm. X 92,000. F i g . 82. Ce l l containing abnormally ac t i ve golg i body. Golgi appears to be p r o l i f e r a t i n g mater ia l associated with mbv's. Bar = 0 . 5 ym. X 53,000. F i g . 83. Higher magni f icat ion of golg i body and associated structures shown in F i g . 82. Note greater e lec t ron density of c i s ternae membranes (double arrowhead) as compared with those of mbv's ( s ing le arrowhead). In f lated golg i c i s ternae near periphery of c e l l contain f i b r i l - l i k e materia l (arrow). Bar = 200 nm. X 96,500. 86 s t ruc tu ra l po l a r i t y , the "forming face" i s considered to be the region of f l a t t e n e d , und i f fe rent i a ted c i s t e rnae , whi le the "maturing face" i s associated with curved and enlarged or swollen c i s ternae. According to the s i ze of the c i s te rnae , flow of materia l through the golg i system shown in F igs. 82 and 83 i s toward the c e l l sur face. However, the inner "forming face" a lso appears to be p r o l i f e r a t i n g material in the d i r ec t i on of the mbv's, away from the c e l l ' s sur face, and i s more "curved" than,the pole with more swollen c i s te rnae . General ly golg i a c t i v i t y was more apparent in in fected c e l l s than ' i n non- infected c e l l s . Ce l l s in intermediate stages of i n f e c t i o n are shown in F igs. 84 through 87. In add i t ion to mbv's, these c e l l s genera l ly contained greater numbers of v i r a l p a r t i c l e s than did c e l l s assumed to be in e a r l i e r i n f e c t i o n stages. Further morphological modi f icat ions were ev ident. Large amounts of long, th in f i b r i l s were observed, dispersed throughout the cytoplasm (F igs. 84-88). These cytoplasmic f i b r i l s (cf) measured ca. 5-8 nm in diameter. Present only occas iona l ly , and less obvious, were small regions of densely s ta in ing cytoplasm (Figs. 84 and 86) which were never evident in uninfected c e l l s . These e lec t ron dense patches were occas iona l l y observed to l i e adjacent to the nuclear envelope (Figs. 86). E lectron dense regions with in the nucleus, assumed to represent nuc leo lar material ,were a lso more evident in in fected c e l l s (F i g . 84). Ce l l s in F igs . 88 through 91 show u l t r a s t r u c t u r a l manifestat ions in terpreted to be advanced stages of i n f e c t i o n . These c e l l s contained not iceably greater amounts of cytoplasmic f i b r i l s (F ig . 88) and showed s i g n i f i c a n t signs of cytoplasmic degeneration (Figs. 88, 90, and 91). 87 Figs. 84-87. Ce l l s showing morphological manifestat ions of v i r a l i n f e c t i o n , representing intermediate stages of i n f e c t i o n . Bars = 0.5 ym. F i g . 84. Sect ion through c e l l containing polyhedral v i rus p a r t i c l e s , mbv's and large amounts o f cytoplasmic f i b r i l s . Note densely sta ined areas of nucleus and cytoplasm (arrows). X 49,000. F i g . 85. High magni f icat ion of cytoplasmic f i b r i l s (arrowheads). No associated mbv's are v i s i b l e . X 56,500. F i g . 86. Sect ion showing region of densely sta ined cytoplasm (arrow) pos i t ioned beside nuclear envelope. X 48,000. F i g . 87. Ce l l with d iv ided ch lo rop la s t showing evidence of v i r a l i n f e c t i o n . Note the presence of polyhedral p a r t i c l e s , mbv's and cytoplasmic f i b r i l s . X 40,500. 8 8 89 Organelles appeared less d i s t i n c t and f requent ly were d i f f i c u l t to i d e n t i f y . V i r a l p a r t i c l e s f requent ly were concentrated near the c e l l surface (F igs. 89 and 90), which showed evidence of a loss of s t ruc tura l i n t e g r i t y in the v i c i n i t y of the p a r t i c l e s . Small masses of mbv's were s t i l l v i s i b l e , f r e q u e n t l y located near the periphery of the c e l l in a s soc ia t ion with numbers of v i r a l p a r t i c l e s (F igs. 90 and 91). The s i t e s of ea r l y formation of v i r a l s t ruc tu ra l components were not apparent in sect ioned mate r i a l . Large masses of v i rogen ic materia l f requent ly assoc iated with the morphogenesis of other cytoplasmic v i ruses (Devauchelle, 1977) were not observed. Morphogenesis of v i r a l p a r t i c l e s appeared to be e n t i r e l y cytoplasmic. V i r i on s were never seen with in the nucleus, nor were they ever encountered with in c i s ternae of the ER or golg i complex. No phys ica l a s soc ia t ion with the mitochondrion or ch lo rop la s t was evident. The in terna l s t ructure and surface features of progeny v i r i o n s were genera l ly i n d i s t i n c t . The contents of the p a r t i c l e s could at times be v i s u a l i z e d as a densely s ta in ing core region (Figs. 52,.89, 91, and 95). These "cores " appeared to be very l a b i l e s tructures and extremely var i ab le in appearance (Figs. 84 and 89). Production of progeny v i r a l p a r t i c l e s did not appear to be synchron-ous. The appearance of v i r a l p a r t i c l e s d id not vary much between c e l l s in " e a r l y " i n f e c t i o n stages and those in terpreted to be in advanced stages. The number of p a r t i c l e s increased, and t h e i r d i s t r i b u t i o n with in the c e l l ( c loser to the c e l l periphery in more advanced stages) changed during the course of i n f e c t i o n . 90 F igs . 88-91. U l t r a s t ruc tu ra l mani festat ion of advanced stages of i n f e c t i o n . F i g . 88. Ce l l containing extensive amounts of cytoplasmic f i b r i l s , and l im i ted numbers of mbv's and v i rus p a r t i c l e s . C e l l u l a r organel les appear to be degenerating, showing loss of s t ruc tu ra l i n t e g r i t y . Bar =0 .5 ym. X 45,500. F i g . 89. Numerous ' v i r a l . p a r t i c l e s located .near-periphery of c e l l . Cytoplasm surrounding v i r i o n s contains f i b r i l s and d i s -t i n c t l y fewer ribosomes than seem in healthy c e l l s . Bar = 200 nm. X 57,000. F i g . 90. Ce l l showing extensive cytoplasmic d i sorgan iza t ion . V i r ions appear to be concentrated at ruptur ing c e l l sur face. Bar = 200 nm. X 55,000. F i g . 91. F ina l stages of i n f e c t i o n . Ce l l shows extensive loss of cytoplasm. Note v i r i o n adjacent to ruptur ing plasmalemma. (arrowhead). Bar = 0 . 5 ym. X 32,000. 91 92 III.3. Observations on v i r a l re lease Figures 92 through 97 i l l u s t r a t e the proposed sequence of events. As mentioned above, during l a t e r stages of i n f e c t i o n (a f ter three hours) virions were often observed to be congregated near the periphery of the c e l l . V i r ions d i r e c t l y opposed to the plasmalemma f requent ly appeared to have one apex pos i t ioned t angen t i a l l y to the c e l l surface (Figs. 92, 94, and 95; an exception being shown in F i g . 93), and often appeared to be attached to the plasmalemma by means of an extremely short strand of e lect ron opaque material (F igs. 92 and 93). S im i l a r short " s trands" were observed associated with some more c e n t r a l l y located v i r i on s (Figs. 93 and 94). Adjacent to (but not d i r e c t l y at) the point of contact between v i r i o n and host membrane, the plasmalemma was observed to swell and b l i s t e r (Figs. 92-95). This b l i s t e r i n g response appeared to be very l o c a l i z e d and was d i s t ingu i shab le from the normal undulate appearance of the plasmalemma (Figs. 94-97). V i r a l p a r t i c l e s were never seen with in the " b l i s t e r reg ion" nor were they ever observed to bud through the c e l l membrane. U l t imate ly the surface of these c e l l s ruptured, re leas ing v i r i on s and associated cytoplasm into the surrounding medium. This method of v i r a l re lease appeared to l i b e r a t e only a small number of v i r i on s at one time. The progeny v i rus p a r t i c l e s may be released over an extended period of time through seve ra l , separate l o c a l i z e d ruptures in the plasmalemma, but th i s was impossible to v e r i f y using sect ioned mate r i a l . The c e l l shown in F i g . 91 appears to have already ruptured in two regions (both located near the bottom of the micrograph) and i s beginning to rupture at a t h i r d s i t e . This postulated Figs . 92-97. Successive stages in v i r a l re lease. Bars = 200 nm. F i g . 92. Late i n f e c t i o n stage showing v i r i on s near periphery of c e l l . Note that c e l l surface shows evidence of b l i s t e r i n g adjacent to (arrowhead), but not at point of contact (arrow). X 98,000. F i g . 93. mbv's and v irus p a r t i c l e s shown gathering at periphery of c e l l . Note b l i s t e r i n g of plasmalemma (arrowhead). X 101 ,500. F i g . 94. B l i s t e r i n g of c e l l membrane appears to be l o c a l i z e d , i . e . note lack of d i s t o r t i o n fu r ther along membrane (arrows). X 99,000. F i g . 95. Plasmalemma beginning to rupture adjacent to s i t e of v i r i o n attachment. X 99,000. F i g . 96. Loca l i zed rupture of c e l l surface is complete, al lowing re lease of v i r i o n s . X 102,500. F i g . 97. See text to F i g . 96. X 102,500. 95 sequential system of v i r a l re lease w i l l eventua l ly cause c e l l mor ta l i t y ( f u l l l y s i s ) . 96 IV. DEVELOPMENT OF A FLUORESCENCE ASSAY FOR VIRAL ACTIVITY IV.1. Culture growth studies The in vivo f luorescence growth curves of f l a sk and tube batch cu l tures of M. pusi11a (grown under the condit ions noted in the sect ion on methods) are shown in F i g . 98. It is evident from the graph that the growth rates in the f l a sk and tubes were s i m i l a r fo r the f i r s t ten days of the experiment a f t e r which the two patterns of growth rate diverged. A f te r day 10, cu l tu re tube f luorescence continued to increase at a f a i r l y steady rate un t i l day 17, a f t e r which a steady dec l ine occur-red. A smooth curve drawn through a l l the data points ( i . e . days 0 through 24) ind icated some general trends: 1) a lag period of two to three days, fol lowed by 2) a per iod o f logar i thmic growth cont inuing un t i l days 17 to 19, fol lowed by 3) a period during which f luorescence s t ead i l y dec l ined un t i l the termination of the experiment. "Counting f l a sk " f luorescence demonstrated a s i m i l a r lag period and ear l y logar i thmic phase, then plateaued between days 10 and 15. This plateau was fol lowed by a second per iod of increas ing f luorescence. This data could a lso be in terpreted as a s ing le more gradual and prolonged logar i thmic growth phase which continued throughout the duration of the experiment. As the purpose of th i s i nves t i ga t ion was to determine the r e l a t i o n -ship between in v ivo f luorescence and c e l l number fo r eventual use as a b i o l o g i c a l assay of batch cu l ture growth (and death), and as most succeed-ing experiments were to be conducted in cu l ture tubes, the data obtained 97 F i g . 98. In vivo ch lorophy l l f luorescence of f l a sk and tube batch cu l tures of M. p u s i l l a . I N V I V O F L U O R E S C E N C E - T U B E S A N D F L A S K 900 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r D a y s 99 from the tes t tubes was se lected p r e f e r e n t i a l l y f o r the determination of the c o r r e l a t i o n between in vivo f luorescence and c e l l number. Although tube f luorescence was observed to increase un t i l day 17, c e l l counts ind icated that c e l l number began to leve l o f f at day 12 (see F i g . 99). Pre l iminary i nves t i ga t i on of the to ta l experimental data suggested the d i v i s i o n of the data into two blocks f o r s t a t i s t i c a l ana ly s i s . In vivo f luorescence values and c e l l counts from days 0 through 11 were grouped and treated as representing lag and logar i thmic growth, and the data obtained a f t e r day 11 was in terpreted to represent s ta t ionary growth and culture-senescence. Regression ana lys i s of each block of data ind icated a strong l i n e a r re l a t i on sh ip ex i s ted between f luorescence and c e l l number in each case (r >0.98). However, the slopes of the two regressions were s i g n i f i c a n t l y d i f f e r e n t ( i . e . days 0-11, Fluorescence = 2.1 X Ce l l Density (X 10 ); days 12-24, Fluorescence = 3.0 X Ce l l Density (X 1 0 " 5 ) ; F-, 2 1 = 9.1, p<.005). This suggested that : 1) a strong l i n e a r re l a t i on sh ip did ex i s t between in vivo f luorescence and c e l l number in batch cu l tures of . M. p u s i l l a , and that 2) th i s r e l a t i on sh ip appeared to be dependent upon age and/or the phys ica l cond i t ion of the cu l ture being studied (see F i g . 99). IV.2. In vivo f luorescence and cu l tu re death The growth experiments descr ibed above demonstrated that in vivo f luorescence could be used as a s i g n i f i c a n t l y prec i se i nd i ca to r of cu l ture growth. The usefulness of in vivo f luorescence as an i nd i ca to r of in fected cu l ture death was inves t i ga ted. The resu l t s of the "Nite-Owl" experiment are presented in F i g . 100. 100 F i g . 99. In vivo ch lorophyl l a_ f luorescence and c e l l density of M. p u s i l l a grown in tube batch cu l tures . 102' F i g . 100. Results of "Nite-Owl" experiment: The e f f e c t of the addi t ion of v i rus on in v ivo f luorescence and c e l l dens i ty of M. pusi11a grown in tube batch cu l tu res . Arrow ind icates the time of addi t ion of the inoculum. 104 P r i o r to the add i t ion of the v i rus inoculum, and f o r the f i r s t two hours t he rea f te r , the f luorescence of a l l experimental c e l l cu l tures showed l i t t l e v a r i a t i o n . Three hours a f t e r the addi t ion of the inoculum, in vivo f luorescence of the " t rea ted " cu l tures began to decrease and continued to dec l ine s tead i l y un t i l the experiment was terminated. As in the previous experiment c e l l number and cu l ture f luorescence demonstrated a strong c o r r e l a t i o n . Pre l iminary attempts to use the in vivo f luorescence assay to determine the r e l a t i v e concentrat ion of i n f e c t i v e v i rus p a r t i c l e s in a v i rus source were not e n t i r e l y succes s fu l . When very d i l u t e v i rus samples were added to tube batch cu l tures of M. p u s i l l a ( i . e . low m u l t i p l i c i t y of i n f e c t i o n ) , the f luorescence response was delayed. Frequently th i s delay exceeded the l i f e span of the batch cu l tu res . 105 DISCUSSION I. MICROMONAS PUSILLA: ULTRASTRUCTURE The inves t i ga t ion of the cytopatholog ica l e f f ec t s of the v irus i n -fec t ion on Micromonas p u s i l l a has provided add i t iona l information on the s t ruc ture of th i s nano f l age l l a te . The general features of th i s minute c e l l inc lud ing i t s s i z e , external morphology, and arrangement of major organe l le s , prev ious ly descr ibed by Manton (1959), have been confirmed by th i s study. The only new feature of i n te re s t in the ch lorop la s t was the e lect ron transparent f i b r i l - c o n t a i n i n g regions observed adjacent to the pyrenoid complex. F i b r i l l a r material observed in the p l a s t i d s of other green algae (e.g. Chlamydomohas, Ris and P laut , 1962) contained component f i b r i l s measuring ca. 2.5 nm in diameter. The greater diameter of the f i b r i l s observed in Micromonas (ca. 5.5-8 nm) suggest that they may represent a more condensed form of p l a s t i d genophore or they may be a r t i f a c t u a l . With the exception of the well def ined ring-shaped arrange-ment of ch l o rop l a s t DNA observed in members of brown and golden-brown algal c lasses (B i sa lputra and B i s a lpu t ra , 1969) the arrangement of a lga l p l a s t i d genophores i s not h ighly s p e c i f i c (B i sa lput ra , 1974). An apparently constant feature of the c e l l s was the i n f l a t e d channel of nuc lear -assoc iated ER (NER) which was f requent ly observed to be conf luent with the per inuc lear space. This cont inu i ty was not su rp r i s i ng as s i m i l a r connections between ER and nuclear envelope are commonly seen, not only in other green f l a g e l l a t e s (e.g. Pyramimohas, Norr is and Pearson, 1 0 6 1975; Mantonie l la , Barlow, 1977), but in representat ives of other a lga l c lasses (e.g. the ch lorop las t -ER-nuc lear membrane system of chrysomonads, Gibbs, 1962, 1970). This type of in terna l membrane cont inu i ty along with observation of the s t ruc tu ra l a s soc ia t ion of golg i and ER c i s ternae membranes led Morre" et a l . (1971) to descr ibe a continuous endomembrane system as a bas ic feature of eukaryot ic c e l l s . One d i s t i n c t i v e feature of the NER requires fu r ther comment. Within th i s expanded region of ER s i g n i f i c a n t amounts of f i b r i l - l i k e materia l were f requent ly observed. The o r i g i n of these f i b r i l s was not known. However, Bouck (1971) has observed that the f i b r i l l a r material observed with in the boundaries of the nuclear envelope and i t s contiguous membrane-l im i ted regions of f l a g e l l a t e d c e l l s cons i s t s of s t ruc tura l components of f l a g e l l a r hairs in many cases. The transport of f l a g e l l a r hairs to the base of the f lage l lum v ia golg i c i s ternae i s well documented fo r Ochromonas (Bouck, 1971). No such golgi -mediated t rans fe r of the presumed "ha i r s " was ever observed in Micromonas, and as only terminal f l a g e l l a r hairs were observed on Micromonas, i t i s unclear how these i n t r a c e l l u l a r , " h a i r s t ruc tures " reach t h e i r u lt imate l o ca t i on . The s a c - l i k e i nc lu s i on bodies observed in Micromonas appear to be unique to th i s organism. Because of t h e i r proximity to the c e l l surface and t h e i r tendency to protrude and occas iona l l y expel material through the plasmalemma, these bodies have been in terpreted here as representing some form of extrusome. Tr ichocysts seen in other green f l a g e l l a t e s (Manton, 1969; Norr is and Pearson, 1975) bear no resemblance to these bodies. Neither are these inc lus ions s i m i l a r in appearance to the "muciferous 107 bodies" described in Pyramimonas r e t i c u l a t a by Belcher (1968). The term "mucocyst" has been used to descr ibe a wide va r ie ty of small organel les located d i r e c t l y beneath the c e l l surface in p ro t i s t s (Tokuyasu and Scherbaum, 1965). The i nc lu s i on bodies described here are qui te s i m i l a r in appearance to mucocysts descr ibed in Tetrahymena pyr i formis (Tokuyasu and Scherbaum, 1965). Based upon the ava i l ab le s t ruc tura l evidence, the regular inc lus ions observed in Micromonas have been t e n t a t i v e l y c a l l e d mucocyst-1ike bodies. A number of i n te re s t i ng observations re l a ted to the f l a g e l l a r appar-atus require add i t iona l comment. The discovery of a c l u s t e r of terminal hairs on the emergent f lage l lum of Micromonas i s not su rpr i s ing in view of the presence of an analogous c l u s t e r of terminal f l a g e l l a r ha irs on the re la ted nanof lage l l a te Mantoniel la squamata. Despite i t s extremely c lose resemblance to Manton ie l la , which has been considered a c l a s s i c a l and c h a r a c t e r i s t i c representat ive of the Prasinophyceae (Peter f i and Manton, 1968), Micromonas, p r imar i l y because of i t s "naked" c e l l e x t e r i o r , has been considered an anomolous member of th i s c lass (Manton, 1967). The u l t r a -s t ruc tura l observations presented here provide add i t iona l evidence of the c lose l i nk between Micromonas p u s i l l a and Mantoniel la squamata and suggest that the former now can be considered to represent a prasinophycean f l a g e l l a t e in which the f l a g e l l a r scales or ha i r s , normally present in the c lass ( Pe te r f i ' and Manton, 1968), have been l o s t (also see below). The presence of a second, apparently nonfunct iona l , basal body i s a lso not surpr i s ing in view of the number of p r o t i s t s o r i g i n a l l y i d e n t i f i e d as u n i f l a g e l l a t e which l a t e r were shown to contain a vest ige of a second f lage l lum (Manton, 1967). Chromulina (Rou i l l e r and Faure-Fremiet, 1958), 10'8 Monomastix (Manton, 1967), Pedinbmbnas (Pickett-Heaps and Ot t , 1974) and Mantoniel la (Barlow, 1977) are examples. Among the reduced b i f l age l . l a te representat ives in the Prasinophyceae (Peter f i and Manton, 1968), only in Mantbii iel la squamata (Barlow, 1977) has the v e s t i g i a l basal body been observed to protrude from the c e l l body in a s i m i l a r manner to what was observed here f o r Micromonas.' Based upon the s t r i k i n g u l t r a s t r u c t u r a l s i m i l a r i t y of these two genera th i s resemblance might be expected. A commonly observed and c h a r a c t e r i s t i c u l t r a s t r u c t u r a l feature of the t r a n s i t i o n a l region of the f l a g e l l a fo r a number of chromophyte a lgal c lasses i s a s t ructure c a l l e d the " t r a n s i t i o n a l he l i x " (TH; Hibberd, 1976, 1978). S im i l a r s t ructures have been reported in co lo r l e s s f l a g e l l a t e s (Brugerol le and Joyon, 1975), and in one instance an apparently analogous s t ructure descr ibed as a " c o i l e d f i b e r " (Moestrup and Thomsen, 1974) was observed in a green f l a g e l l a t e . In these organisms the feature described could be resolved as a h e l i c a l s t ructure located with in the t r a n s i t i o n region of the f lage l lum. The e lect ron opaque form described here as a "tapered c y l i n d e r " (TC), which was observed in long i tud ina l views of the f lage l lum of Micromonas cannot be considered homologous to the TH of Hibberd (1978). The tapered cy l i nde r of Micromonas is located d i s t a l to the t r a n s i t i o n a l region of the f lage l lum and therefore apparently i s a supplementary feature present in add i t ion to the t r a n s i t i o n zone. In transverse sect ion the TC cannot be resolved into t iny c i r c u l a r p r o f i l e s suggestive of a h e l i x , and unl ike e i t he r the TH of the chromophytes or the c o i l e d f i b e r o f Pyramimonas th i s s t ruc ture appears to taper l i k e a funnel at i t s proximal end. 10'9 During a d i scuss ion of the phylogenetic s i gn i f i c ance of the TH in representat ives of the Chromophyta, Hibberd (1978) suggested that the analogous s t ructure described in an i so l a ted report f o r Pyramimonas (Moestrup and Thomsen, 1974) probably had a separate phylogenetic o r i g i n . As s tructures s i m i l a r to the "court c y l i n d r e " seen in Karotomorpha (Brugerol le and Joyon, 1975), the " c o i l e d f i b e r " observed in Pyramimonas, and the "tapered c y l i n d e r " reported here in Micromonas, are not outstanding u l t r a s t r u c t u r a l f ea tures , i t i s probable that s i m i l a r s t ruc tura l mod i f i ca -t ions of f l a g e l l a r s t ructure are not uncommon, but f requent ly over looked. At present the funct iona l s i gn i f i c ance of such s tructures must remain a matter of conjecture. I n t r a c e l l u l a r axonemal s t ructures r e s u l t i n g from withdrawn f l a g e l l a have been observed in zoospores of Ulva during the process of s e t t l i n g and germination (Braten, 1971). Although such s t ructures are not commonly seen in f l a g e l l a t e d p ro t i s t s (Pickett-Heaps and Ot t , 1974), a s im i l a r s i t u a t i o n has been reported in Pedinomonas (Pickett-Heaps and Ott , 1974), a sca ly green f l a g e l l a t e tha t , based upon features of i t s m i to t i c process (presence of per s i s ten t interzonal sp ind le and absence of a phycoplast ) , recent ly has been postulated as an extant ancestor at the base of an evolut ionary l i n e terminating with the ulvacean algae (Stewart and Mattox, 1978). The i n t r a c e l l u l a r axonemal s t ructures and v e s t i g i a l basal bodies observed in both Pedinomonas (Pickett-Heaps and Ot t , 1974) and Micromonas are features which may be found in a number of seemingly unrelated algae and are not by themselves r e l i a b l e ind ica tor s of a c lose re l a t i on sh ip no between these two organisms. Information on the process of c e l l d i v i s i o n in Micromonas might be useful in determining i t s phy le t i c a f f i n i t i e s . However, despite examination of c e l l s representing more than f i f t y d i f f e r e n t e lec t ron microscope preparat ions, no sp indle s t ructures a s s o c i -ated with nuclear d i v i s i o n , or stages of cy tok ines i s were even observed. A l l f i xa t i on s f o r these u l t r a s t r u c t u r a l studies were done during the day. The doubling time of c e l l s in logar i thmic growth phase was determined to be approximately one c e l l d i v i s ion/day (see F i g . 99). These facts suggested that c e l l d i v i s i o n might be occurr ing during the "dark" period of the c e l l c y c l e . Although information on c e l l d i v i s i o n i s not a va i l ab l e f o r Micromonas, a recent study of d i v i s i o n in Mantoniel la squamata has ind icated that i t , l i k e Pedinomonas, a lso possesses a per s i s tent interzonal sp indle and lacks a phycoplast. Based on the c lose s i m i l a r i t y observed and discussed prev ious ly f o r Micromonas and Manton ie l l a , i t i s probable that u l t r a s t r u c -tura l examination of c e l l d i v i s i o n in Micromonas w i l l reveal a s i t ua t i on s i m i l a r to that observed in Mantoniel 1 a and Pedinomonas. A common feature of the f l a g e l l a r apparatus of many p r o t i s t s i s the presence of groups of cytoplasmic microtubules which appear to o r i g ina te in the v i c i n i t y of f l a g e l l a r basal bodies and extend f o r some s p e c i f i e d distance around the c e l l , l y ing beneath the plasmalemma. In green a lgal f l a g e l l a t e s these f l a g e l l a r " r o o t l e t s " (Manton et a l . , 1955) f requent ly occur as four ind iv idua l sets and c o l l e c t i v e l y demonstrate a " c ruc i a te arrangement" (Moestrup, 1972) which, because of i t s apparent conservat ive-ness, has been used as a s i g n i f i c a n t c h a r a c t e r i s t i c in phenetic and I l l phylogenetic schemes,(Moestrup, 1978; Mattox and Stewart, 1978). Micromonas was observed to contain only two such sets of r o o t l e t m ic ro -tubules associated with the basal body of the emergent f l age l lum. The v e s t i g i a l basal body was never seen to be associated with cytoplasmic microtubules. By comparison, Pedinomonas, a u n i f l a g e l l a t e with a second basal body, contains a complete c ruc ia te set of roo t le t s (Pickett-Heaps and Ot t , 1974) in add i t ion to a more elaborate " s t r i a t e d r o o t l e t " . Mantonie l la , which has one funct iona l emergent f lage l lum and one stubby aberrant f lage l lum (Barlow, 1977), contains (1 ike Micromonas) only "ha l f " the c ruc ia te s t ructure with two roo t le t s cons i s t ing of 4 microtubules and 2 microtubules, r e spec t i ve l y . If the number of composite microtubules in these r o o t l e t s i s s i gn i f i c an t , a s suggested by Moestrup (1978), then i t i s i n te re s t i n g to note that the " h a l f c ruc i a te " roo t le t s of Micromonas contain exact ly ha l f the number of composite microtubules observed in roo t l e t s of Manton ie l la . On the basis of the s t ruc tu ra l evidence presented i t can be sa id that Micromonas p u s i l l a i s s i m i l a r in many respects to a small number of presumably reduced green f l a g e l l a t e s . In p a r t i c u l a r , i t s resemblance to Mantoniel la squamata i s s t r i k i n g . In accordance with the widely held view that b i f l a g e l l a t e is a p r im i t i ve c h a r a c t e r i s t i c and u n i f l a g e l l a t e represents a reduced state (Tay lor , 1976), Micromonas p u s i l l a can be thought of as a h ighly reduced and s p e c i a l i z e d prot i s tan form. 112 II. THE VIRUS Observation of v i r u s - l i k e p a r t i c l e s (VLPs) i s now commonplace in a wide range of c e l l types. In the major i ty of cases these observations represent inc identa l f ind ings reported during a general u l t r a s t r u c t u r a l i nves t i ga t ion of some organism. Frequently in such instances i t i s impossible to e s tab l i sh the v i r a l i d e n t i t y of the observed p a r t i c l e s because the sample material containing the inc lus ions i s no longer ava i l ab le and the data provided i s inadequate. This problem i s e s p e c i a l l y prevalent in studies of natural populations in which processing of mul t ip le samples and large volumes makes cursory cataloguing and temporary storage the most p r a c t i c a l procedures. A d d i t i o n a l l y , the i s o l a t i o n of i nd iv idua l s from mixed, wi ld populations f o r the purpose of e s tab l i sh ing clones genera l ly makes i t un l i ke l y that a na tu ra l l y occurr ing v i r a l pathogen w i l l be found because any in fec ted ind iv idua l s probably would not c u l t u r e . . In the present i nves t i ga t i on the i d e n t i f i c a t i o n of. i n t r a c e l l u l a r agents respons ib le fo r the l y s i s of a wi ld population of Micromonas  p u s i l l a i s reported. These pathogenic agents have been introduced i n to , and observed to cause l y s i s of four d i f f e r e n t geographical i so l a tes of th i s species. They can be s e r i a l l y passaged through "heal thy" c e l l s , pass through a 0.22 ym f i l t e r , and possess morphological c h a r a c t e r i s t i c s of known v i ruses . The evidence descr ibed above has been taken as s u f f i c i e n t to e s tab l i sh the v i r a l nature of these agents. The v irus has been named Micromonas p u s i l l a v i rus (MPV). 113 The most outstanding morphological features of the v i rus p a r t i c l e s are t h e i r large s i ze (ca. 130 nm), t h e i r uniform hexagonal (and occas ion-a l l y pentagonal) p r o f i l e s in sect ioned and negat ive ly sta ined ma te r i a l , and t h e i r lack of d i s t i n c t i v e t a i l - l i k e or other external s t ructures . Based on the features descr ibed above, the MPV c l o se l y resembles the i r i de scen t v i ruses observed in insects ( B e l l e t , 1968) and repor ted, for a marine worm (Devauchelle and Durchon, 1973; Devauchelle, 1977). These v i ruses have been so named because c ry s t a l s or p e l l e t s of the p a r t i c l e s appear b l u e - v i o l e t and green when viewed with obl ique i l l umina t i on (Will iams and Smith, 1957, 1958; B e l l e t , 1968). In th i s respect they d i f f e r from MPV which has not been observed to demonstrate such i r idescence. S u p e r f i c i a l l y MPV a lso resembles the large polyhedral v iruses of some amphibians (Granhoff, 1969) and the A f r i can swine "fever v i rus (Hess, 1971). These v i ruses have a lso been grouped with the i r i de s cen t v iruses (Fenner, 1976), although they do not form i r i de scen t c r y s t a l s (Ke l ly and Robertson, 1973). Observations of p a r t i c l e s showing two-, t h ree - , and f i v e - f o l d axes of ro ta t iona l symmetry have been in terpreted here as evidence that MPV has icosahedral symmetry (Klug and Caspar, 1960). As more than one polyhedral form can have icosahedral symmetry (cf . Klug and Caspar, 1960) demonstration of regular icosahedral shape can only be shown by means of b i d i r e c t i o n a l metal shadowing (Williams and Smith, 1958). This remains to be done. Its simple polyhedral p r o f i l e and lack of external appendages d i s -t inguishes MPV from the major i ty of bacteriophages and cyanophages which genera l ly have some assoc iated t a i l s t ructures to be used f o r attachment 114 to and/or penetrat ion of host surfaces (Tikhonenko, 1970). T a i l « l e s s polyhedral phages have been reported (e.g. 0X174, Tromans and Home, 1961) but these are s i g n i f i c a n t l y smal ler in diameter than MPV. In being " t a i l - l e s s " MPV resembles v iruses i n f e c t i n g animal c e l l s . Unl ike the r e l a t i v e l y r i g i d c e l l wall of most prokaryotes, the animal c e l l plasmalemma i s a l a b i l e s t ructure which v i r i on s can penetrate by means of , v i ropex i s ( i . e . phagocytosis) or simple membrane fus ion (Dales, 1973). Although i t can be recognized as a "p lant c e l l " because of the presence of ch lorop las t s (Manton and Parke, 1959, 1960), Micromonas i s surrounded only by i t s plasmalemma l i k e most "animal" c e l l s . This f ac t makes i t f e a s i b l e that the mechanism of entry employed by MPV might resemble that of animal v i ruses . Therefore the c l a s s i c a l d i s t i n c t i o n of "p lant " and "animal" v i ruses may not be useful when deal ing with v iruses in p r o t i s t s . The mul t i l ayered external appearance of negat ive ly and p o s i t i v e l y contrasted specimens requires fur ther d i scus s ion . Structures morphologic-a l l y s im i l a r to uni t membranes of c e l l s have been observed and in terpreted to be "common and c h a r a c t e r i s t i c features " of mature, i n tac t p a r t i c l e s o f mosquito i r i de s cen t v i rus (MIV) by S t o l t z (1971, 1973) who concluded that such s t ructures were a l so a common feature of a number of cytoplasmic-r e p l i c a t i n g DNA v i ruses . These "un i t membranes" which were observed f requent ly on both negat ive ly sta ined and sect ioned specimens of MIV were a lso reported to be present on pox v iruses ( S to l t z and Summers, 1972). In the present i nves t i ga t i on no such obvious membranes were observed in sect ioned mate r i a l . This makes i t d i f f i c u l t to determine at th i s . t ime whether the mul t i l ayered surface appearance of sta ined v i r i o n s a c tua l l y represents a c h a r a c t e r i s t i c s t ruc tu ra l feature of MPV. 115 Deta i led surface substructures revealed during the negative s ta in ing of ce r t a in specimens were reminiscent of those.seen on other large icosahedral v i ruses . Wrigley (1969, 1970) described arrays of morphologic-al subunits on Se r i ce s th i s i r i de scen t v i rus (SIV) and T ipu la i r i de scen t v i rus (TIV) that had been treated with the nasal decongestant " A f r i n " p r i o r to negative s t a in ing . A d d i t i o n a l l y he observed that v i r i o n s which were stored in d i s t i l l e d water f o r prolonged periods tended to f a l l apart in to t r i angu l a r fragments o f subunits which he re fer red to as "trisymmet-rons" . Morphological subunits resembling those of SIV and TIV could be resolved on MPV without the add i t ion of A f r i n . Intact p a r t i c l e s of MPV f a i l e d to reveal surface subunits. A s i m i l a r s i t ua t i on was noted by Wrigley (1969, 1970) fo r SIV and TIV and by S t o l t z (1971, 1973) f o r Chironomus icosahedral cytoplasmic deoxyribovirus (ICDV) and MIV. Those v i r i on s on which substructure could be resolved appeared i r r e g u l a r in shape, and although d i s rupt ion into fragments was never observed, i t was assumed that the s t ruc tura l i n t e g r i t y of these p a r t i c l e s had been damaged. I solated "trisymmetrons" were never observed. However, the s t r i k i n g s i m i l a r i t y o f the surface features observed here f o r MPV with those described f o r i r i de scen t v i ruses must be noted. Observations on the morphogenesis of MPV presented here suggest that assembly takes place in the per inuc lear cytoplasm. A wide va r ie ty of RNA v iruses are known to assemble in the cytoplasm (Fenner et a l . , 1974). However, these RNA v iruses s u p e r f i c i a l l y do not resemble MPV. They are e i t he r s i g n i f i c a n t l y smal ler or s t r u c t u r a l l y more complex than the p a r t i c l e s descr ibed here. 116 On the basis of i t s s i z e , general morphology, and th i s apparent cytoplasmic s i t e of assembly MPV a lso resembles a large number of v iruses which have been re fer red to c o l l e c t i v e l y as " icosahedral cytoplasmic deoxyr iboviruses" (ICDV) ( S t o l t z , 1971; Ke l l y and Robertson, 1973). These DNA-containing v i ruses whose r e p l i c a t i o n cyc les are confined to the c e l l cytoplasm have been i s o l a ted from a wide range of animal and insect hosts. This group i n c l u d e s the lymphocystis v i ru s from marine and freshwater f i s h (Weissenberg, 1965; Zwil lenberg and Wolf, 1968), the A f r i can swine fever v i rus (Hess, 1971), the polyhedral v i ruses of frogs and newts (Granhoff, 1961, and, in p a r t i c u l a r , the i r i de scen t v iruses of insects ( B e l l e t t , 1968) as noted above. This assemblage of v i ruses (the ICDV's) has been the subject of a number of reviews ( S t o l t z , 1971, 1973; Ke l ly and Robertson, 1973; McAuslan and Armentrout, 1974). The v a l i d i t y of th i s grouping of v iruses i s a matter of content ion. From a purely s t ruc tura l standpoint S t o l t z (1973) suggests that a l l ICDV's are re l a ted and that " . . . t h e term i cosa -hedral cytoplasmic deoxyribovirus remains a u s e f u l . . . d e s c r i p t i v e phrase emphasizing those obvious features which can at present be considered d iagnost ic fo r th i s group of v i r u s e s " . Ke l l y and Robertson (1973) present an opposing view,arguing that i t i s premature to create, a "super group of ICDV's" s ince the ava i l ab le evidence ind icates that there are s i g n i f i c a n t d i f fe rences between these v i ruses which they be l ieve have been grouped on an a r b i t r a r y bas i s . Viruses demonstrate a very l im i ted range of morphologies (Caspar and Klug, 1962; Subak-Sharpe, 1971) and without add i t iona l information morphology i s an i n s u f f i c i e n t c r i t e r i o n on which to def ine re l a t i on sh ip (Ke l ly and Robertson, 1973). 117 Consequently, in view of the d iscuss ion presented above, i t i s obvious that e s tab l i sh ing the d e f i n i t i v e r e l a t i on sh ip of th i s newly i d e n t i f i e d v i r a l pathogen with known v i rus groups w i l l be poss ib le only a f te r information on i t s biochemical and b iophys ica l propert ies i s a v a i l a b l e , although i t i s suspected that the resemblance to i r i de scen t v iruses w i l l be strengthened. 118 III. ULTRASTRUCTURAL ASPECTS OF THE INFECTION PROCESS The major u l t r a s t r u c t u r a l events associated with the i n f ec t i on of Micromonas by MPV have been descr ibed. The fo l lowing d i scuss ion w i l l focus on the i n te rp re ta t i on and a comparison of these observations with those of s i m i l a r studies of v i r a l morphogenesis. An important cons iderat ion associated with the i n te rp re ta t i on of e lec t ron microscopy i s the f a c t that the images presented are s t a t i c representat ions of dynamic processes. Problems associated with techno log i -cal l im i t a t i on s of the e lec t ron microscope have been dea l t with by Dales (1973). For example, in studying v i r a l ent ry , inadequate membrane preservat ion and the lack of uni formity in the angle and thickness of sect ions may r e s u l t in the mi s in terpre ta t ion of the geometric re l a t i on sh ip between the v i rus and the c e l l sur face. In view of such problems, port ions of the fo l lowing d i scuss ion must be considered specu la t i ve . Based upon the u l t r a s t r u c t u r a l observations the fo l lowing scenario can be proposed f o r MPV i n f e c t i o n : 1) Within 15 min fo l lowing in t roduc-t ion of the inoculum v i r i on s adsorb onto, and penetrate, host c e l l s ; 2) "Penetrat ion" i s fol lowed by an e c l i p s e - l i k e period during which v i r a l synthet ic processes are occurr ing at the molecular l e v e l ; 3) I n t r a c e l l u l a r v i r i o n development becomes v i s i b l e wi th in three hours as v i r a l assembly takes p lace ; and 4) A v i r a l egress phase occurs during which v i r i on s move adjacent to the plasmalemma where they induce mul t ip le l o c a l i z e d ruptures of the c e l l membrane. Virus receptors have been defined as natural components which bind v i rus under in vivo condit ions (Lonberg-Holm and Ph i l i p son , 1974) and 119 may occur on regions of membranes covering c i l i a and f l a g e l l a as well as on the more genera l ized port ions of the plasmalemma and i n t r a c e l l u l a r membrane system (Dales, 1973). Based on th i s d e f i n i t i o n i t i s poss ib le that the apparent random arrangement of v i r i on s observed on host c e l l s during ea r l y i n f e c t i o n stages may r e f l e c t a d i s t r i b u t i o n of receptor s i t e s which encompass the en t i re plasmalemma (cf . Dales, 1973). A number of v i ruses are known to attach n o n s p e c i f i c a l l y to c e l l and other surfaces (e.g. Vacc in ia v i rus adsorbs to aluminum, g lass , and c e l l s , equal ly w e l l ; Lonberg-Holm and P h i l i p s o n , 1974). The p o s s i b i l i t y that MPV adsorbs n o n s p e c i f i c a l l y to nonbio log ica l surfaces was not inves t i ga ted. However, negat ive ly s ta ined preparations of MPV added to enrichment cu l tures contain ing mixed plankton populat ions, showed that the v i r i on s could adsorb onto other sma l l , u n i d e n t i f i e d , naked f l a g e l l a t e s , but were never observed on the surfaces of bac te r i a l c e l l s . These pre l iminary f ind ings suggest that MPV demonstrates a l im i ted degree of attachment s p e c i f i c i t y , adhering only to some eukaryot ic c e l l membranes. The intimacy of i n i t i a l i n terac t ions between host c e l l s and v i rus could not be ascerta ined by u l t r a s t r u c t u r a l observat ion. The "penetra t ion " step of ea r l y c e l l - v i r i o n i n te rac t i on requires that e i t he r the whole v i rus or the v i r a l genome must penetrate into the host c e l l (Lonberg-Holm and P h i l i p s o n , 1974). Detection of penetrat ion by means of e lec t ron microscopy i s l a r ge ly a matter of conjectura l i n te rp re ta t i on (see d iscuss ion above). In the present instance "penetra-t i o n " of host c e l l s u l t r a s t r u c t u r a l l y appeared to invo lve: 1) the pro-duction of a l o c a l i z e d swel l ing of the host surface in response to the 120 presence of attached v i r i o n s , 2) the apparent fus ion of "shared" a t tach -ment s i t e ...surfaces, permitt ing cont inu i t y of the inner compartment of the v i r i o n with the c e l l cytoplasm, and 3) the transmittance of f i b r i l l a r material ( in terpreted to represent v i r a l genome) into the host c e l l . The ser ies of events described here s u p e r f i c i a l l y resembles t yp i ca l bac te r i o - and cyanophage i n f e c t i o n , but d i f f e r s fundamentally in that in the present system the attachment i s d i r e c t l y to the c e l l membrane and no h ighly s pec i a l i zed t a i l s t ructures appear to be involved in penetrat ion of the host sur face. However, the sma l l , s p h e r i c a l , DNA-phage 0X174 has been shown to attach and uncoat ( i . e . re lease and transmit i t s nuc le i c acid) at the c e l l surface without the use of the typ i ca l phage t a i l appendage (Newbold and Sinsheimer, 1970), although " sp ikes " present on the v i r i o n surface at capsid apices have been impl icated to act as t a i l analogues (Tromans and Home, 1961). The u l t r a s t r u c t u r a l l y unmodified apices of MPV may s i m i l a r l y be assoc iated with the process of v i r a l attachment and/or penetrat ion (F igs. 68-79). MPV i n f ec t i on bears no resemblance to that of v i ruses i n f e c t i n g p lant p ro top la s t s , the process there being l ikened to endocytosis (Takebe, 1975). It i s genera l ly accepted that most animal v iruses enter t h e i r host c e l l s by means of v i ropex i s (a process equivalent to phagocytos is) . Penetrat ion by means of membrane fu s i on , although less common, has been observed (Lonberg-Holm and P h i l i p s o n , 1974) and is genera l ly associated with highly d i f f e r e n t i a t e d , nondeformable c e l l surfaces (Dales, 1973). The fus ion of Sendai v i rus with membranes of host c e l l s , f o r example, has been c l e a r l y demonstrated (Dourmashkin and T y r r e l l , 1970; Morgan and Howe, 1968). This method of entry by means of fus ion appears to be 121 r e s t r i c t e d to enveloped animal v i ruses . Although th i s process i s super-f i c i a l l y s i m i l a r to that observed here f o r MPV there i s no substant ia l evidence f o r the presence of an envelope in the l a t t e r and therefore the mechanism does not appear to be the same. If a d i r e c t analogy i s suggested with animal v i r a l i n f e c t i o n (non-enveloped v i r u s e s ) , then i n t e r n a l i z a t i o n of MPV through the apparently "unspec i a l i zed " plasmalemma of Micromonas might be expected to occur by v i r opex i s . This was never observed. The o r i g i n of the e lec t ron opaque processes (Figs. 70, 77-79) occas iona l l y observed to connect e x t r a c e l l u l a r v i r i on s with the host c e l l surface remains a matter o f conjecture. No such s t ructures have been reported f o r other spher ica l v i rus i n f e c t i o n s . However, a s i tua t i on s u p e r f i c i a l l y resembling that observed here was noted by Morgan and Rose (1968) and Morgan et a l . (1968) during u l t r a s t r u c t u r a l studies of the entry of in f luenza and herpes simplex v iruses into animal c e l l s . They suggested that project ions might r e f l e c t an except iona l l y int imate s tate of v i r a l attachment poss ib ly causing the adjacent surfaces to become d i s to r ted during f i x a t i o n manipulations. The s tructures observed here have been in terpreted to represent a c e l l surface e laborat ion in response to v i r a l attachment. The temporal sequence of events associated with new v i r a l development in th i s system i s conjectura l as the i n f e c t i o n process appeared to be asynchronous, c e l l s observed three hours post inocu la t ion exh ib i t i ng extremely va r i ab le degrees of i n f e c t i o n . In genera l , ea r l y events appeared to take place in the centra l cytoplasmic region of the c e l l which, at th i s stage, demonstrated minimal evidence of u l t r a s t r u c t u r a l 122 a l t e r a t i o n . Two types of unique s t ructures were encountered un i ve r s a l l y wi th in the cytoplasm of these in fec ted c e l l s . These were 1) c lu s te r s of sma l l , membrane bound ves i c l e s (mbv) and 2) s ca t te red , va r i ab le amounts of cytoplasmic f i b r i l s ( c f ) . Ves ic les assoc iated with the process of v i r a l development have been observed in a v a r i e t y of c e l l types. Co l l e c t i on s of ve s i c l e s ca. 50 to 150 nm in diameter were observed wi th in i n f l uenza - i n fec ted chick embryo f i b r o b l a s t c e l l s , s i tuated d i r e c t l y below the plasmalemma,by Compans and Dimmock (1968). Labe l l i ng experiments led them to suggest that the ves i c l e s might r e f l e c t increased a c t i v i t y o f the c e l l membrane which d id not appear to be re l a ted to synthesis of v i r a l envelope prote ins . The mbv's observed in i n fec ted Micromonas c e l l s were of s i m i l a r s i ze (ca. 60 to 80 nm) but of less var i ab le dimension than those observed in the i n f l uenza - i n fec ted c e l l s . Also un l ike those which appeared to be plasma-membrane-associated, the mbv's showed no c h a r a c t e r i s t i c pos i t i on in in fec ted c e l l s . Ves i cu l a t i on appears to be a common c h a r a c t e r i s t i c of p lant v i rus i n f e c t i o n (Hamilton, 1974). However, un l ike the mbv's of Micromonas the ves i c le s o f higher plants are genera l l y found assoc iated with some c e l l u l a r organe l le . Ves i c le s have been observed with in the ch lorop las t s of c e l l s i n fec ted with turn ip yel low mosaic v i rus (TYMV) and barley s t r i p e mosaic v i rus (Cha lcrof t and Matthews, 1966; C a r r o l , 1970). Ves i c le s have a lso been reported with in the per inuc lear space of c e l l s in fected with pea enation mosaic v i rus (de Zoeten et a l . , 1972). Although the funct ion of these ves i cu la ted membrane systems cu r ren t l y i s not well 123 understood (Hamilton, 1974), the ch lorop la s t ve s i c l e s of TYMV-infected c e l l s have been impl icated in v i r a l RNA synthesis (Matthews, 1973). Cytoplasmic ve s i c l e s have been observed in parenchyma c e l l s of sugarbeet in fec ted with beet western yel low v i rus (BWYV; Esau and Hoefert, 1972). Unl ike the ves i c l e s described here, the s tructures seen in sugarbeet appear to be derived from ER c i s te rnae , contain f i b r i l l a r material resembling nuc le i c ac id f i b r i l s described f o r ch lorop las t s and mitochondria, and appear to be involved in the transport of th i s f i b r i l l a r mater ia l into the nucleus. No such sequence of events was seen here. C lusters of mbv's were however predominantly c e n t r a l l y located during ear ly stages of i n f ec t i on and f requent ly per ipheral in l a t e r stages. F i b r i l l a r materia l resembling the "cytoplasmic f i b r i l s " described here has been reported in ve s i c l e s of v i r u s - i n f e c t e d plants (mentioned above) and plant protoplasts (Burgess et a l . , 1974a), but has not been reported to occur f r e e l y dispersed in cytoplasm of in fected c e l l s . The resemblance of the f i b r i l s seen here with the network of f i b r i l s observed in ch lorop las t s and mitochondria (B i sa lputra and B i s a lpu t ra , 1976b) suggest that they may contain nuc le ic a c id . The presence of ves i c le s and cytoplasmic f i b r i l s , plus the appearance of the i n t ac t v i rus p a r t i c l e s with in the cytoplasm, were the only cons i s tent in terna l signs of i n f e c t i o n . The u l t r a s t r u c t u r a l appearance of the ch lorop la s t and the mitochondrion remained unaltered throughout the course of the i n f e c t i o n . With the exception of the occasional appearance of a s l i g h t l y more densely s ta in ing nuc leo lar region the 124 nucleus s i m i l a r l y showed no i nd i ca t i on of t h e ' i n f e c t i o n process. These observations suggest that the i n f ec t i on process of MPV d i f f e r s s i g n i f i -cant ly from that of many plant v iruses which are known to cause s i g n i f i c a n t d i s rupt ion of o rgane l l a r s t ructure (cf . Esau, 1968). In i t s apparent "cytoplasmic s i t e " of assembly and/or maturation MPV resembles a number of l a rge , icosahedral cy top l a sm ic - rep l i ca t ing v iruses which have been grouped under the heading of i r i dov i ru se s (Fenner, 1976). Several authors have proposed models to expla in i n t r a c e l l u l a r maturation of i r i dov i ru se s (Xeros, 1964; Younghusband and Lee, 1969; Yule and Lee, 1973). One opinion is that the assembly of the v i r a l she l l occurs synchronously with the incorporat ion of a v i rop lasmic matrix (Younghusband and Lee, 1969). Yule and Lee (1973) and Devauchelle (1977) suggest,however, that p a r t i c l e s are i n i t i a l l y formed as hollow she l l s which are f i l l e d during subsequent morphogenetic steps. A l l models maintain that v i r a l assembly and maturation are in t imate ly associated with v i r a l - i n d u c e d , cytoplasmic inc lu s ion bodies c o l l e c t i v e l y re fer red to as viroplasm. General ly no such d i s t i n c t i v e e lec t ron dense v i rogenic areas were evident in MPV-infected c e l l s and although the present observations suggest that d i r e c t e laborat ion of v i rus p a r t i c l e s may be occurr ing in the cytoplasm, none of the current models adequately expla in the sequence of events seen here. U l t r a s t ruc tu ra l evidence suggests that the l y s i s of MPV-infected c e l l s may be co r re l a ted with the c lose appos i t ion (or attachment) of progeny v i r i on s with the in terna l s ide of the plasmalemma. It was a lso observed that v i r a l re lease genera l ly appeared to occur at mu l t ip le s i t e s along the c e l l surface in the form of l o c a l i z e d ruptures rather than by 125 overa l l c e l l l y s i s . An enzyme-extractable " l y t i c p r i n c i p l e " (cf. Dales, 1973) has been i so l a ted from some animal v iruses (Barbanti-Brodans et a l . , 1971; Hosaka and Shimizu, 1972) which in s p e c i f i c cases appears to be c l o se l y coupled with a c e l l fus ing f ac to r (Howe and Morgan, 1969). This fus ing f ac to r apparently i s re leased at the s i t e of i n te rac t i on between v i r i on s and plasmalemma but may e l i c i t i t s e f f e c t at some d i s tant point from the s i t e of attachment (cf . Dales, 1973). The observed l o c a l i z e d ruptures of MPV-infected Micromonas c e l l s may represent the response of the c e l l surface to a s i m i l a r " l y t i c p r i n c i p l e " released by plasmalemma-associated progeny v i r i o n s . V i r a l egress by budding through the plasma-lemma as seen f o r a number of i r i dov i ru se s ( T r i p i e r et a l . , 1974; Webb et a l . , 1976), was not observed here. 126 IV. THE USEFULNESS OF IN VIVO FLUORESCENCE AS AN ASSAY OF VIRAL INFECTIVITY By d e f i n i t i o n a v i rus i s an i n fec t i ous en t i t y . Consequently any study.that attempts to deal with the fundamental nature of a v i rus should be concerned, at l ea s t in par t , with methods of assaying i n f e c t i v i t y . The conventional assay method f o r l y t i c v i r a l i n fec t ions involves enumeration of plaques ( i . e . l o c a l i z e d zones of i n fec t i on ) which form on monolayers of cu l ture c e l l s (Adams, 1959; c f . Safferman, 1973). Several attempts to use plaque assay methods with the present system were not succes s fu l . Micromonas d id not grow on s o l i d or semi - so l id medium (Nichols , 1973), and bacter ia present in the nonaxenic, unia lga l cu l tures contaminated a l l such e f f o r t s . Furthermore numerous attempts to cu l ture Micromonas axen i ca l l y in th i s laboratory (and elsewhere) have f a i l e d . Personal communications'with'M. R. Droop arid R. Lev/ in 'have ' indicated that to date th i s phy to f l a ge l l a te grows, at best , "monaxenical ly" (s ic) (M.R. Droop). D i rec t c e l l counts of Micromonas populations a lso were found to be impract ica l f o r measuring v i r a l a c t i v i t y because 1) the c e l l s are so sma l l , and 2) there is a d i f f i c u l t y in d i sc r im inat ing between healthy and in fected c e l l s using l i g h t microscope techniques. Ear ly in th i s i n v e s t i -gat ion, the obvious deco lorat ion of in fected cu l tures suggested that in vivo ch lorophy l l a^  f luorescence might be used to assay i n f e c t i v i t y . The usefulness of photosynthetic pigments fo r est imating plant, biomass has been recognized f o r many years (c f . Vol lenweider, 1969). Since the work of Lorenzen (1966), the measurement of in Vivo f luorescence-o f 127 ch lorophy l l has become a rout ine method of i n d i r e c t l y est imating biomass (e.g. Blasco, 1973). The re su l t s of growth experiments with Micromonas have demonstrated that a s i g n i f i c a n t l i n e a r re l a t i on sh ip ex i s t s between c e l l number and in v ivo f luorescence f o r a c t i v e l y growing batch cu l tures (r >0.98). These re su l t s agree with those of Loftus and Se l i ge r (1975) who observed that the amount of ch lorophy l l a^  per c e l l , and the r a t i o (R) of in vivo f luorescence to that of extractab le ch lorophy l l a_ remained e s s e n t i a l l y constant f o r l o ga r i thmica l l y growing cu l tu re s . A s i m i l a r r e l a t i on sh ip between in vivo f luorescence and c e l l number was observed during experiments with v i r u s - i n f e c t e d cu l tures (e.g. the "Nite-Owl" experiment). The r a p i d i t y of the f luorometr ic response in v i r u s - i n f e c t e d c e l l s i s noteworthy, fo r i t suggests that th i s assay has an unexpectedly high degree of s e n s i t i v i t y . The timing of the in vivo f luorescence decrease was found to be co inc identa l with the occurrence of i n t r a c e l l u l a r polyhedral v i rus p a r t i c l e s in in fected cu l tu res . The rap id decrease in f luorescence of in fec ted cu l tures , however, could not be simply explained by pos tu la t ing ch lorop la s t de s t ruc t i on , f o r the p l a s t i d s maintained a normal u l t r a s t r u c t u r a l appearance throughout the i n f ec t i on process (with destruct ion presumed to fo l low c e l l l y s i s ) . The observed dec l ine might have been the r e su l t of e i t he r of the fo l lowing p o s s i b i l i t i e s : 1) a decrease in the amount of c h l o r o p h y l l / c e l l , occurr ing without a l t e r a t i o n of d i s rupt ion of ch lo rop la s t s t ruc tu re , or 2) a loss o f , or decrease i n , the overa l l f luorescent capacity of the apparently i n t ac t ch lorop las t s of i n fec ted c e l l s . 128 The f i r s t p o s s i b i l i t y requires that ch lorophy l l ins ide the ch lorop la s t of in fected c e l l s i s degraded rap id l y and i s not being replenished. Chlorophyl l b iosynthes is has been shown to be sens i t i ve to i n h i b i t i o n of both cytoplasmic (cyclohexamide) and organe l le (chloramphenicol) ribosomes (cf . Meeks, 1974). It has been demonstrated fo r a number of eukaryot ic algae (e.g. Chlamydomonas, Ohad et a l . , 1972) that ch lorophy l l b iosynthes is occurs ins ide the p l a s t i d but is e s s e n t i a l l y under nuclear and cytoplasmic c o n t r o l . This suggests that v i r a l in ter ference with normal cytoplasmic prote in synthesis could have a profound negative e f f e c t on ch lorophy l l synthes i s . The turnover rate of ch lorophy l l f o r some eukarot ic algae (e.g. diatoms) has been found to be as f a s t as f i v e to twenty hours (Falkowski et a l . , 1978). In view of the apparent cytoplasmic s i t e of MPV assembly, these facts suggest that the rap id decrease in in vivo f luorescence might be a manifestat ion of the blockage of ch lorophy l l synthesis at the cytoplasmic leve l occurr ing simultaneously with a rapid turnover of c h l o r o p h y l l . The f luorescent spectra of ch lorop las t suspensions ( i . e . in v i t r o f luorescence) are genera l ly s i m i l a r to those of i n t ac t c e l l s ( i . e . in vivo f luorescence, Goedheer, 1972). Exceptions to th i s have been observed in ch lorop la s t preparations from some eukaryot ic algae (e.g. Euglena g r a c i l i s , Brown, 1966) in which the breakage of c e l l s re su l t s in a loss o f , or decrease in f luorescence occurr ing without apparent damage in ch lorop la s t s t ruc ture . The observed dec l ine of in vivo f luorescence in MPV-infected c e l l s might s i m i l a r l y be due to the i n s t a b i l i t y of i s o l a ted ch lorop las t s in l y s ing Micromonas c e l l s . 129 The in vivo f luorescence assay fo r MPV i n f e c t i v i t y does appear to be u se fu l . However, there are two s i g n i f i c a n t drawbacks associated with the assay: 1) i t measures the number of host c e l l s destroyed in a manner which i s q u a l i t a t i v e rather than quant i ta t i ve in terms of information abso lute ly re l a ted to the numbers of v i rus p a r t i c l e s present in i nocu la , and 2) at low " m u l t i p l i c i t i e s of i n f e c t i o n " (moi, i . e . very few v i r i on s per c e l l ) the delay in the f luorescence response can exceed the l i f e span of batch cu l tures of l im i ted volumes (e.g. Micromonas cu l tures grown in tes t tubes). Measurement of adenosine tr iphosphate (ATP), an essent ia l const i tuent of a l l l i v i n g organisms, has been shown to be a h ighly sens i t i ve assay f o r measuring l i v i n g biomass of organisms (Holm-Hansen and Booth, 1966; Bottomley and Stewart, 1976). Such an assay might be sens i t i ve enough to monitor changes in cu l tures exposed to v i rus in extremely low concentra-t ions such as might be encountered during v i r a l endpoint d i l u t i o n te s t s . However, the presence of contaminating bacter ia precludes app l i ca t i on of an ATP assay at present. 130 SUMMARY AND CONCLUDING REMARKS This i nves t i ga t ion has confirmed, and i l l u s t r a t e d by more recent , improved techniques, the u l t r a s t r u c t u r a l observations of the minute, naked phytof1 age!1 ate Micromonas p u s i l l a (Butcher) Manton and Parke which were f i r s t reported by Manton and Parke in 1959. The discovery of terminal f l a g e l l a r hairs i s cons i s tent with the c l a s s i f i c a t i o n of M. p u s i l l a as a member of the c lass Prasinophyceae although f l a g e l l a r sca les present in other members of the group are d e f i n i t e l y absent. A number of new observations on c e l l morphology are presented. 1) A t u f t of four f l a g e l l a r ha irs i s located at the t i p of the s ing le emergent f l age l lum. The o r i g i n of these hairs may be associated with the expanded nuclear-ER system. However, t h e i r course from th i s proposed o r i g i n to t h e i r eventual locus on the f l a g e l l a r t i p is unknown. 2) A second basal body has been observed as part of the basic morphology of the c e l l . No r o o t l e t systems or mot i le capac i t i e s are associated with th i s basal body. 3) Located pos ter io r to the t r a n s i t i o n zone with in the emergent f lage l lum i s a unique s t ructure re fer red to here as a "tapered c y l i n d e r " . Its funct iona l s i gn i f i c ance is not known. 4) The c e l l contains a per ipheral microtubular system associated with the basal body of the emergent f lage l lum which is composed of two microtubular roo t le t s of 1 and 2 microtubules, re spec t i ve l y . 5) Two prev ious ly unknown cytoplasmic s t ructures are descr ibed: a vacuo le - ! i ke , mu l t i ves i cu la ted body, c e n t r a l l y located between the base 131 of the f lage l lum and the major organe l les , and spher ica l mucocyst-1ike bodies, s i tuated d i r e c t l y beneath the plasmalemma and adjacent to the f l age l lum. Regular ly shaped, po lyhedra l , i n t r a c e l l u l a r s t ruc tures , f i r s t observed in a na tu ra l l y occurr ing populat ion of Micromonas p u s i l l a , have been i so l a ted and descr ibed. The fo l lowing evidence i s taken as s u f f i c i e n t to e s tab l i sh the v i r a l nature of these e n t i t i e s : 1) The s t ructures can be introduced i n t o , and cause l y s i s o f , four geographical i s o l a te s of M. p u s i l l a . Furthermore they can be s e r i a l l y passaged through cu l tures of these i s o l a t e s . 2) The s t ructures pass through a 0.22 ym f i l t e r . This f i l t r a t e causes l y s i s as descr ibed in 1). 3) The morphology of these s t ructures i s s im i l a r to that of known v i ruses . 4) The loca t ion of the s t ructures r e l a t i v e to the time from i n t r o -duction un t i l c e l l l y s i s i s cons i s tent with that of a v i r a l i n f ec t i on (see below). The v i rus has been designated Micromonas p u s i l l a v i rus (MPV). It appears to be species s p e c i f i c in terms of i t s l y t i c behavior and occurs under natural condit ions in the loca l waters of the southern S t r a i t of Georgia, near Vancouver, Canada. MPV represents the f i r s t v i rus i d e n t i f i e d and i so l a ted from a marine or freshwater phytoplankter. On the basis of i t s large s i z e , i t s icosahedral symmetry, the arrangement of morphological surface subunits, and i t s apparent cytoplasmic s i t e of r e p l i c a t i o n , MPV resembles v iruses of the i r i d o v i r u s group. U l t r a s t ruc tu ra l examination of the i n f ec t i on of M. p u s i l l a by MPV has 132 suggested the fo l lowing summary of events: 1) V i r a l entry takes place with in 15 min fo l lowing in t roduct ion o f inoculum and occurs by a combination of l o c a l i z e d fus ion of adjacent c e l l and v i rus sur faces , fol lowed by d i r e c t penetrat ion of v i r a l genome. V iropexis does not occur. 2) V i r a l entry is fol lowed by an ec l i p se period of approximately three hours in durat ion. 3) Progeny v i rus assembly occurs asynchronously and appears to take place in the per inuc lear cytoplasm. It i s associated with large amounts of scattered cytoplasmic f i b r i l s and c lu s te r s of smal l , membrane-bound v e s i c l e s , never present in uninfected c e l l s . No v i r i on s can be seen with in the nucleus OT d i r e c t l y associated with s p e c i f i c c e l l organe l les . 4) Progeny v i r i on s are released at mu l t ip le s i t e s along the plasma-lemma by l o c a l i z e d rupture of the c e l l membrane. V i r a l egress by budding does not occur. None of the current models f o r assembly of i r i dov i ru se s adequately expla in the events observed here. An assay has been designed which monitors v i r a l a c t i v i t y based upon the reduction in the in vivo f luorescence of in fected c e l l s . The assay, however, does not permit d i r e c t q u a n t i f i c a t i o n of the v i rus and i s not sens i t i ve enough to be used at extremely low " m u l t i p l i c i t i e s of i n f e c t i o n " . Much work remains to be done with th i s v i ru s / a l g a l p r o t i s t system. Suggestions f o r future work inc lude: 1) The p u r i f i c a t i o n and biochemical charac te r i za t i on of Micromonas  p u s i l l a v i ru s . 2) Development of a; more re f ined assay of v i r a l i n f e c t i v i t y provid ing 133 fo r quan t i f i c a t i on of the pathogens, probably dependent on the axenic growth of Micromonas. 3) A de ta i l ed examination of the mechanism of the e f f e c t of MPV i n fec t i on on the photosynthetic apparatus of th i s simple eukaryot ic auto-troph (as a counterpart to ex i s t i n g studies of v i r a l e f f ec t s on photosyn-thes i s of higher p l an t s , p lant p ro top la s t s , and cyanobacter ia) . 4) An examination of the general d i s t r i b u t i o n of th i s v i rus in the marine environment and i t s e f f e c t on the population dynamics of th i s spec ies . 134 APPENDIX I HOST RANGE Virus samples were tested in 46 un ia lga l i s o l a te s representing 35 d i f f e r e n t species from f i v e a lgal c l a s ses . The re su l t s are presented in Table VI. Lys i s was observed to occur only in the four i s o l a te s o f Micromonas p u s i l l a . Due to the large number of specimens inves t i ga ted , in vivo f luorescence of c u l t u r e s , rather than u l t r a s t r u c t u r a l evidence was used to check fo r v i r a l a c t i v i t y . 135 TABLE VI. Host s p e c i f i c i t y . The fo l lowing marine phytoplankters have been tested with MPV: ., Lys i s Observed Algal Class Species (+ or -) Prasinophyceae Micromonas pusiTLa (4 i s n l a t p Q * + Cymbomonas sp. NEPCC #P130 Halosphaera sp. NEPCC #P232 Heteromastix l o n g i f i l i s NEPCC #P195 Mantoniel la squamata NEPCC #P208 Nephroselmis g i l v a NEPCC #P129 Nephroselmis sp. NEPCC #P193 Platymonas spp. (4 i so l a te s ) NEPCC #P86, #P236, #P46, #P84 Pyramimonas g r o s s j NEPCC #P235 Pryamimonas obovata NEPCC #P234 Pyramimonas o r ien ta l i s NEPCC #P193 Pyramimonas sp. NEPCC #P36 Scour fe ld ia sp. NEPCC #PSc Dinophyceae Katodinium rotundatum NEPCC #D44 Gymnodinium piacidum NEPCC #D75 Gymnodiniurn simplex NEPCC #D119a Gymnodinium spp. (2 i s o l a te s ) NEPCC #D176, #D217 Gymnodini urn v i t i 1 i go NEPCC #D27 Northeast P a c i f i c Culture Co l l e c t i on #D166 Cryptophyceae Chroomonas s a l i na (3 i so l a te s ) NEPCC #CrBB2. #Cr76, #CrLl Chroomonas sp. NEPCC #Cr48 Cryptomonas profunda NEPCC #Cr65 136 TABLE VI (Continued) Lys i s Observed Algal Class Species (+ or -) Cryptophyceae (Cont.) Cryptombhas spp. (2 i so l a te s ) NEPCC #Cr98, #Crl60 Rhodomonas lens NEPCC #Cr22 Northeast P a c i f i c Culture Co l l e c t i on #Crl61 Chrysophyceae Apedine l la s p i h i f e r a NEPCC #Chl08 Chromulina sp. NEPCC #Ch239 Ochromonas sp. NEPCC #Ch83 Qlisthodiscus 1 uteus NEPCC #Ch221 Haptophyceae Chrysochromulina a l i f e r a NEPCC #H82 (- Prymnesiophyceae) corymbellus aureus NEPCC #H222 Chry s ida l i s sp. NEPCC #H184 C r y s t a l l o l i t h u s hyal inus NEPCC #H240 Emiliana huxleyi NEPCC #H55 Pavlova l u the r i NEPCC #H5 A l l cu l tures provided by the Northeast P a c i f i c Culture Co l l e c t i on (NEPCC) unless otherwise s p e c i f i e d See Table II f o r sources 137 APPENDIX II PHYSICOCHEMICAL PROPERTIES OF THE VIRUS: In order to determine the storage c a p a b i l i t i e s of i s o l a ted v i r i on s and to begin understanding t h e i r physicochemical p roper t ie s , the e f fec t s of a va r ie ty of environmental f ac tor s and chemical agents on v i r a l a c t i v i t y were examined. The re su l t s of these studies are summarized in Table VII. a) Thermal i n s t a b i l i t y . Thermal i n ac t i v a t i on studies revealed that i n f e c t i v i t y i s destroyed completely at 63°C a f t e r 15 min and by 30 min at 55°C, but i s maintained at 55°C f o r 15 min. For general storage the leas t de leter ious method was r e f r i g e r a t i o n at 4°C in M i l l i p o r e - f i l t e r e d , autoclaved seawater or PES medium. Such samples reta ined t h e i r i n f e c t i v e capacity f o r at l eas t nine months. b) pH s e n s i t i v i t y . The e f f e c t o f environmental pH on v i r a l s t a b i l i t y was a lso examined. Under normal storage condit ions ( i . e . at 4°C) the f l u i d of v i rus stock cu l tures had a pH value of 8.12 ± .02. Virus samples were tested and found to be s tab le over a broad range of pH va lues, maintaining t h e i r i n f e c t i v i t y a f t e r an incubation period of 24 to 48 hours at 4°C in a pH range between 4 and 10. I n f e c t i v i t y was l o s t at pH 3. c) S e n s i t i v i t y to organic so lvents . The e f f e c t of treatment with organic solvents was a l so examined. The v i rus demonstrated no s e n s i t i v i t y to toluene (10% by volume, in seawater), 138 moderate s e n s i t i v i t y to chloroform (10% by volume, in seawater) and extreme s e n s i t i v i t y to ethyl ether (33% by volume, in seawater). d) F i l t e r a b i l i t y . F i l t e r e d v i rus which had been s e r i a l l y passaged through 0.45 ym and 0.22 ym Nucleopore f i l t e r s was found to be capable of i n f ec t i n g host cu l t u re s , but at a s i g n i f i c a n t l y slower rate than that of c l a r i f i e d , but u n f i l t e r e d v i r a l suspensions. This diminished i n f e c t i v e capacity was bel ieved to be due to the tendency of v i rus p a r t i c l e s to assoc iate with c e l l debris and consequently be adsorbed onto the f i l t e r surface. Evidence f o r th i s a s soc ia t ion of v i rus with c e l l u l a r debris was a lso provided by the f i nd ing that the p e l l e t produced during rout ine low-speed c l a r i f i c a t i o n of v i r a l lysate could be used to i n f e c t and cause l y s i s of host cu l tu res . 139 T a b l e V I I . Physicochemical propert ies of v i r u s . E f f e c t of var ied environmental f ac tor s and phys ica l treatments on v i r a l i n f e c t i v i t y . Parameter Tested Treatment Time Lys i s (+ or -) I. Thermal i n s t a b i l i t y 4°C 6-8 months + 15°C 1 year + 23°C 2 hr + 33°C 1-3 min + 30 min + 1 hr + • 1.5 hr + 20 hr + 43°C 1-3 min + 30 min + 1 hr + 1.5 hr + 20 hr + 55°C 1-3 min + 15 min + 30 min -1 hr -1.5 hr -20 hr -63°C 1-3 min + 15 min -30 min -1 hr -1.5 hr -20 hr -II. pH S e n s i t i v i t y pH 3 48 hr pH 4 48 hr + pH 5 48 hr + pH 6 48 hr + pH 7 48 hr + pH 8 48 hr + pH 9 48 hr + pH 10 48 hr + -140 TABLE VII. Cont. Parameter tested Treatment Time Lys i s (+ or -) III. S e n s i t i v i t y to Organic so lvents 10% Toluene 15 min + 10% Chloroform 15 min 33% Ethyl ether 15 min -IV. F i l t e r a b i l i t y 0-.45 ym nucleopore f i l t e r - - - + 0.22 ym nucleopore f i l t e r - - - + 1 Culture l y s i s observed but occurred at a s i g n i f i c a n t l y slower rate than that of cu l tures inocu lated with untreated v i ru s . 141 APPENDIX III PROCEDURES FOR COLLECTING AND CONCENTRATING MPV PARTICLES -PRELIMINARY RESULTS The scheme shown in F i g . 101 summarizes the procedures fol lowed during pre l iminary attempts to c o l l e c t and concentrate MPV from v i r a l l y sa te . I n i t i a l procedures inc luded: se t t i ng up 850 ml batch cu l tures of M. p u s i l l a in 1 l i t e r Erlenmeyer f l a s k s , inocu la t ing these cu l tures with 25 ml of v i r a l stock s o l u t i o n , and incubating the cu l tures un t i l s i g n i f i c a n t l y s i s was evident (3 to 5 days). Lysed cu l tures were centr i fuged at low speed (10,000 Xg) in the GSA Rotor of a Sorval centr i fuge to p r e c i p i t a t e c e l l u l a r debr i s . For polyethylene g lyco l (PEG, 6000-7500) p r e c i p i t a t i o n (Yamamoto et a l . , 1970) concentrat ions of PEG between 6% and 8%, and NaCl between 0.1 M and 0.5 M were tes ted. Samples being processed at high speeds (70,000 Xg) were sedimented by cent r i fuga t ion in a Type 30 Rotor of a Spinco u l t r acen t -r i f u ge . The re su l t s of concentrat ing MPV by PEG p r e c i p i t a t i o n , u l t r a c e n t r i -fugat ion, and low-speed cent r i fuga t ion were compared by means of d i r e c t examination of p a r t i c l e density using e lec t ron microscopy (negative s ta in ing technique), and by in vivo f luorescence assay. In fect ive v i rus p a r t i c l e s were found in a l l three preparat ions. Prel iminary f ind ings ind icated that the u l t r a cen t r i f u ga t i on preparat ion contained s i g n i f i c a n t l y more i n f e c t i v e p a r t i c l e s than did the other two preparat ions. 142 Attempts to concentrate MPV for fu r ther p u r i f i c a t i o n studies were hampered by three main f a c to r s : 1) the apparently low numbers of v i rus p a r t i c l e s in large volumes of .: " cu l tu re medium; 2) the tendency f o r s i g n i f i c a n t amounts of v i r u s to be removed with the c e l l u l a r debris during low-speed c l a r i f i c a t i o n of v i r a l l y s a te ; and 3) the general i n e f f i c i e n c y of repeated u l t r a cen t r i f u ga t i on of r e l a t i v e l y small volumes. 143 M. p u s i l l a (850 m l ) + V i r a l L y s a t e (25 m l ) 3-5 d a y s I n c u b a t i o n 0 1 8 ° C C e n t r i f u g e 0 1 0 , 0 0 0 Xg 30 m in P e l l e t / P - 1 S u p e r n a t a n t / S - 1 (500 ml ) 0 . 5 M N a C l ; 10% PEG S u p e r n a t a n t / S - 1 (250 m l ) 1 S u p e r n a t a n t / S - 1 (100 m l ) C e n t r i f u g e _@ 1 0 , 0 0 0 X g . 30 m in C e n t r i f u g e . p 7 0 , 0 0 0 X g , 2 h r S u p e r n a t a n t / S - 2 P e l l e t / P - 2 + S t e r i l e SW (30 m l ; 2 8 . 5 ° / o o ) j S t o r e 0 4 ° C 72 h r S u p e r n a t a n t / S - 3 P e l l e t / P - 3 + S t e r i l e SW ( 4 . 5 m l ; 2 8 . 5 ° / o o ) S t o r e 0 4 ° C 72 h r C e n t r i f u g e @ 7 0 , 0 0 0 Xg 2 h r C e n t r i f u g e 0 7 0 , 0 0 0 Xg 2 h r S u p e r n a t a n t P e l l e t / P - 2 A + S t e r i l e SW (5 m l ; 2 8 . 5 ° / o o ] C e n t r i f u g e . 0 10 ,000 Xg 30 min S u p e r n a t a n t P e 1 1 e t / P - 3 A + S t e r i l e SW ( 2 . 5 m l ; 2 8 . 5 ° / o o l S u p e r n a t a n t / S - I A (100 mU P e l l e t / P - I A + S t e r i l e SW (9 m l ; 2 8 . 5 ° / q o ) V i r a l Q u a n t i f i c a t i o n : I. 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