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UBC Theses and Dissertations

A developmental, physiological and structural study of the rhodophycean alloparasite Harveyella mirabillis… Goff, Lynda June 1975

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A DEVELOPMENTAL, PHYSIOLOGICAL AND STRUCTURAL STUDY OF THE RHODOPHYCEAN ALLOPARASITE HARVEYELLA MIMBILIS (CHOREOCOLACEAE, CRYPTONEMIALES) by LYNDA JUNE GOFF B. S. (Honours), Oregon College of Education, 1971 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEGREE OF PHILOSOPHY In the Department of BOTANY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1975 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of Brit ish Columbia Vancouver 8, Canada i i Chairman: Professor Kathleen Cole ABSTRACT The nature of the symbiotic as s o c i a t i o n of the red alga HavveyeZZa mi-vabiZis (Reinsch) Schmitz and Reinke (Cryptonemiales) and i t s red a l g a l hosts OdonthaZia and Rhodomela (Ceremiales) was investigated. The d i s t r i b u -t i o n of H. mivabiZis was revised to include a d d i t i o n a l host species as w e l l as a greater range of occurrence i n the northeast P a c i f i c . A study of procarp development confirms that H. mivab-iZis should be retained i n the order Cryptonemiales. Based only upon morphological c r i t e r i a , H, mivabiZis has been defined previously as p a r a s i t i c on i t s red a l g a l hosts. In the present study, a new d e f i n i t i o n of parasitism has been formulated to include p h y s i o l o g i c a l as well as morphological aspects of parasitism. Accordingly, a red a l g a l parasite i s defined as any red alga l i v i n g temporarily or permanently within or on a host, de r i v i n g benefits from i t and causing i t harm. The a s s o c i a t i o n of HavveyeZZa mivabiZis and i t s hosts i s considered with regard to (1) the reproductive and developmental dependence of H. mivabiZis on a s p e c i f i c host, (2) the p o s s i b i l i -ty of metabolite exchange between host and HavveyeZZa, and (3) the e f f e c t s of the presence of HavveyeZZa on i t s host. A f i e l d and laboratory study of the development and reproduction of H. mivabiZis has revealed that the completion of i t s l i f e h i s t o r y i s depend-ent on the presence of a s u i t a b l e host and that reproduction and development are af f e c t e d by seasonal changes i n environmental parameters. I n i t i a l spore germination occurs i n host wounds i n f l i c t e d p r i m a r i l y by grazing isopods and i i i amphipods. Rhizoidal c e l l s penetrate the walls between host c e l l s and esta-b l i s h secondary p i t connections with host c e l l s . Subsequent development i s characterized by rapid p r o l i f e r a t i o n of the r h i z o i d a l c e l l s within the host, a rupturing of the host's outer w a l l region and the f i n a l development of a colourless reproductive pustule. Morphological and c y t o l o g i c a l studies have shown that H. mivabiZis conforms to a t y p i c a l "PoZysiphonia-ty^e-" l i f e h i s t o r y . The e f f e c t s of seasonal f l u c t u a t i o n s i n seawater s a l i n i t y and temperature, and changes i n the hours of bright sunshine (photoperiod) on the reproduction and development of H. mivabilis have been examined over a 20-month period. Gametogenesis occurred i n northeast P a c i f i c populations i n the spring and f a l l between a seawater temperature range of 8.5-11 C whereas tetrasporogenesis occurred i n the l a t e winter as both photoperiod and water temperature increased. H. mivabiZis i s p h y s i o l o g i c a l l y dependent upon the host 0. fZoooosa as a source of n u t r i e n t s . L i q u i d s c i n t i l l a t i o n a n alysis and l i g h t microscopic autoradiography have demonstrated that Hll+C03 i s photosynthetically assimi-la t e d by the host and subsequently transferred to H. mivabiZis. The primary flow occurs from host medullary c e l l s to adjacent r h i z o i d a l c e l l s of H. mivabiZis. A secondary transfer occurs from host c e l l s dispersed i n the pustule to adjacent H. mivabiZis c e l l s . Ion exchange chromatography and chemical extraction techniques were employed to separate labeled f r a c t i o n s of 0. fZoooosa and H. mivabiZis a f t e r various periods of 1 1 + C - t r a n s l o c a t i o n . The change i n r a d i o a c t i v i t y i n the alcohol-soluble neutral f r a c t i o n most c l o s e l y p a r a l l e l e d the t o t a l increase i n r a d i o a c t i v i t y i n H. mivabiZis and the corresponding decrease i n 0. fZoooosa. To i d e n t i f y translocated com-pounds, the labeled n e utral f r a c t i o n s were separated by paper chromatography. An increase i n r a d i o a c t i v i t y was associated with an unknown substance i n i v HarveyeZZa which had an R glucose value s i m i l a r to glucuronic and galacturonic acids i n both a c i d i c and basic solvent systems. A concomitant decrease i n i n 0. fZoooosa neutral sugars separated i n both basic and a c i d i c solvents. C o r t i c a l , medullary, and r h i z o i d a l c e l l s of H. mirabiZis were examined by l i g h t and electron microscopy to determine the s t r u c t u r a l mechanisms involved i n nut r i e n t t r a n s f e r . A membrane system i n the r h i z o i d a l c e l l s , c o n s i s t i n g of the plasmalemma, p i n o c y t o t i c v e s i c l e s , m u l t i v e s i c u l a r and con-c e n t r i c bodies, ER, dictyosomes, microbodies and an extensive vacuolar system may be involved i n the uptake, processing and d i s t r i b u t i o n of nutrients throughout these c e l l s . Histochemical i d e n t i f i c a t i o n was made of proteins, l i p i d s and carbohydrates associated with t h i s v a cuolar/vesicle system. Light autoradiography, supported t h i s proposed membrane uptake mechanism. Plasma-lemmal extensions (plasmalemmavilli) of. H. mirabiZis medullary c e l l s i n the pustule may also be involved i n nutrient uptake. The e f f e c t s of H. mirabiZis on the host 0.^ fZoooosa were also examined by l i g h t and electron microscopy. In host medullary c e l l s adjacent to r h i z o i d a l c e l l s , changes occur i n vacuolation, plasmalemmal v e s i c u l a t i o n , ER, p l a s t i d s , n u c l e i , p i t connections and walls. D i r e c t penetration of host c e l l s by r h i z o i d a l c e l l s of H. mirabiZis o c c a s i o n a l l y occurs r e s u l t i n g i n death of the host c e l l s . Host medullary and c o r t i c a l c e l l s dispersed i n the emergent pustule show few of the degenerative responses noted i n host c e l l s adjacent to H. mirabiZis,rhizoidal c e l l s . On the contrary, host c e l l d i v i s i o n and photosynthetic a s s i m i l a t i o n of HllfC03 a l l increase. S p h e r i c a l . v i r u s - l i k e s o l i t a r y bodies (S-bodies) occur i n a l l c e l l s of H. mirabiZis and i n host c e l l s attached, to H. mirabiZis by secondary p i t connections. The p o s s i b i l i t y r a d i o a c t i v i t y - was associated with b'oth a high R glucose unknown and mannitol V that these structures may induce the infective host response i s discussed In the concluding discussion the possible evolution of H. mivabi'lis considered in relation to proposed theories of the origin of red algal parasites. v i TABLE OF CONTENTS ABSTRACT 1 1 LIST OF TABLES v i i i LIST OF FIGURES i x LIST OF COLOUR PLATES xxv LIST OF APPENDICES x x v i ACKNOWLEDGEMENTS x x v i i I. GENERAL INTRODUCTION 1 I I . HISTORICAL REVIEW OF THE TAXONOMY OF HAHVEYELLA MIRABILIS 4 I I I . METHODS AND MATERIALS 7 A. C o l l e c t i o n and Treatment of Samples for E c o l o g i c a l and D i s t r i b u t i o n a l Studies 7 B. Morphological and Developmental Studies 9 1. Light microscopic techniques 9 2. Electron microscopic techniques 11 C. P h y s i o l o g i c a l Studies 14 1. Histochemical i d e n t i f i c a t i o n of compounds 14 2. Translocation studies: s c i n t i l l a t i o n a n a l y s i s . . 16 3. Translocation studies: autoradiographic analysis 19 4. Separation and analysis of labeled compounds 21 IV. REPRODUCTIVE AND DEVELOPMENTAL DEPENDENCY OF EARVEYELLA MIRABILIS ON A SPECIFIC HOST 25 Introduction 25 Results and Observations 29 A. General Description of Eavveyella mivdbiUs 29 v i i B. D i s t r i b u t i o n 33 C. Reproduction and Development 35 Discussion 76 V. METABOLITE EXCHANGE BETWEEN HARVEYELLA AND ITS HOST 80 Introduction 80 Results and Observations 84 A. S t r u c t u r a l Investigations 84 B. P h y s i o l o g i c a l Investigations. 136 Discussion 156 A. 1 1 + C - f i x a t i o n and Translocation.. 156 B. Proposed Nutrient Uptake System......... 158 VI. EFFECTS OF HARVEYELLA MIRABILIS ON ITS HOST ODONTHALIA. FLOCCOSA 163 Introduction 163 Results and Observations 165 A. Response at Organismal Level 165 B. Response at C e l l u l a r Level 165 1. Formation of secondary p i t connections 168 2. Host c e l l s of i n t e r d i g i t a t i o n zone 170 3. Host c e l l s i n Harveyella pustule 181 Discussion 188 VII. FINAL DISCUSSION AND EVOLUTIONARY CONSIDERATION 194 VIII. LITERATURE CITED 203 IX. APPENDICES 223 v i i i LIST OF TABLES I. Histochemical tests 15 I I . Translocation samples 18 I I I . D i s t r i b u t i o n of Idothea wosnesenskii on some algae 60 IV. Consumption of some algae by Idothea wosnesenskii 60 V. C y t o l o g i c a l data 67 VI. U l t r a s t r u c t u r a l features of EarveyeZZa mirabiZis vegetative c e l l s i n zones I, I I , I I I . . . 85 VII. EarveyeZZa mirabiZis, neutral f r a c t i o n 151 VIII. OdonthaZia fZoooosa, n e u t r a l f r a c t i o n 151 IX. C e l l u l a r responses i n EarveyeZZa i n f e c t e d OdonthaZia fZoooosa 167 i x LIST OF FIGURES FIGURE 1. L i v i n g material. Vegetative and reproductive pustules of H. mivabiZis on 0. fZoooosa 30 2. Isolated host medullary c e l l i n pustule 30 3. Isolated host c o r t i c a l c e l l at pustule surface... 30 4. Drawing. C e l l u l a r zones of H. mirabiZis 31 5. Light micrograph.. Medullary c e l l of H. mirabiZis... 32 6. L i v i n g material. Ultimate c e l l of r h i z o i d a l filament penetrating between host c e l l s . . . . ; 32 7. R h i z o i d a l c e l l s of H. mirabiZis i n i n t e r d i g i t a t i o n zone.... 32 8. Isolated host medullary c e l l s i n i n t e r d i g i t a t i o n . zone 32 9. Secondary p i t connections between H. mirabiZis r h i z o i d a l c e l l s and host medullary c e l l s 32 10. Reported d i s t r i b u t i o n of H. mirabiZis i n the North A t l a n t i c 34 11. D i s t r i b u t i o n of H. mirabiZis on Oregon Coast... 36 12. D i s t r i b u t i o n of H. mirabiZis.on B r i t i s h Columbia and northern Washington coast 37 13. Study area at Simpson's Reef, Cape Arago, Oregon.. 38 14. Study area at Parsons Point, near Sooke, B. C 38 15. Diagram of "PoZysiphonia-type" l i f e h i s t o r y 39 16. Tetraspores of H. mirabiZis stained with t o l u i d i n e blue.... 41 17. Tetrasporangium and tetraspore mother c e l l 41 18. Tetraspores stained with PAS 41 19. Electron micrograph. Section through tetrasporangium 41 20. Tetraspores stained with Sudan black B 41 X FIGURE 21. Section through mature male pustule of H. mivabiZis........ 42 22. Spermatangia and spermatangial mother c e l l s 42 23. Spermatangial v e s i c l e s i n r e l e a s i n g spermatia 42 24. Released spermatia i n outer mucilaginous covering * of pustule 42 25. Releasing, spermatium 42 26. L i v i n g material. Mature carposporophyte-bearing pustule 44 27. Gonimoblast with terminal carpospores... 44 28. Releasing carpospores.. 44 29. Carpospores attached to glass p l a t e s . . . . . . 44 30. Procarp i n i t i a l i n periphery of female gametophyte — . 46 31. F i r s t d i v i s i o n of procarp i n i t i a l . 46 32. Second d i v i s i o n of procarp i n i t i a l 46 33. 4-celled carpogonial branch, a u x i l i a r y c e l l , and y f i r s t s t e r i l e c a l l of procarp 46 34. Mature procarp 46 35. Trichogyne protruding through pustule w a l l 46 36. Trichogyne ramifying under outer pustule w a l l 46 37. Trichogyne "pad" at pustule surface 46 38. Drawings A-D. P o s t - f e r t i l i z a t i o n development i n H. mivabiZis 47 39. Drawing. Mature carposporophyte 48 40. Protruding trichogyne and spermatia 49 41. P o s t - f e r t i l i z a t i o n . Retraction of trichogyne 49 42. Formation of connecting filament from carpogonium to a u x i l i a r y c e l l 49 x i FIGURE 43. D i v i s i o n of a u x i l i a r y c e l l to form c e n t r a l c e l l and foot c e l l 49 44. Fusion of s t e r i l e branch c e l l s and foot c e l l 49 45. D i v i s i o n of c e n t r a l c e l l to- form two gonimoblast i n i t i a l c e l l s 49 46. D i v i s i o n of gonimoblast i n i t i a l to form h o r i z o n t a l gonimoblast c e l l s 49 47. Elongation of h o r i z o n t a l gonimoblast filaments and formation of erect gonimoblast filaments.... 49 48. Erect gonimoblast filaments with terminal carpospores...... 51 49. Terminally borne, elongate carpospores.... 51 50. Remnants of fused foot c e l l and. s t e r i l e branches.. 51 51. Elongate columnar c e l l s i n female gametophyte pustule 51 52.. Open o s t i o l e with elongate carpospore. 51 53. Scanning electron micrograph of o s t i o l e with extruding mucilage 51 54. L i v i n g material. C o r t i c a l wound of host with associated Earveyella pustules 53 55. Spore i n ruptured host medullary c e l l 53 56. Spore i n protoplasm of ruptured host medullary c e l l i n wound area 53 57. Rhiz o i d a l c e l l s of. H. mirabilis penetrating between host c e l l s at wound surface 53 58. H. mirabilis development.in host tissues adjacent to wound surface 53 59. Thick-walled tetraspore 10 hr a f t e r host i n o c u l a t i o n . . 55 60. Germination of tetraspore 24 hr a f t e r host i n o c u l a t i o n 55 61. Penetration of r h i z o i d of attached tetraspore between host medullary c e l l s 55 I x i i FIGURE 62. Germination of unattached tetraspore.. Elongation of r h i z o i d 55 63. Scanning electron micrograph of attached tetraspore of H. mivdbilis 55 64. Primary filament of H. mirabiZis extending along wound surface of host. 5-7 days a f t e r spore germination 56 65. Primary filament entering into host t h a l l u s 56 66. Rhi z o i d a l c e l l s of H. mirabiZis i n i n t e r c e l l u l a r spaces of host t h a l l u s near wound surface. 8 days a f t e r i n o c u l a t i o n . 56 67. Development of H. mirabiZis .in host tissues away from host wound. 3 weeks a f t e r i n o c u l a t i o n . . . . 56 68. Host medullary c e l l s surrounded by H. mirabiZis r h i z o i d a l c e l l s . 5. weeks a f t e r i n o c u l a t i o n . . 58 69. P r o l i f e r a t i o n of.H. mirabiZis near.surface of host t h a l l u s 58 70. Emergence of H. .mirabiZis pustule from inf e c t e d host. 68 days a f t e r i n o c u l a t i o n 58 71. Trichogyne emerging from female pustule. 82 days a f t e r tetraspore i n o c u l a t i o n 58 72. Grazing wounds i n f l i c t e d on 0. fZoooosa by Idothea wosnesenskii 63 73. Localized swelling and d i s c o l o u r a t i o n of grazed host infected with H. mirabiZis. 4 weeks a f t e r i n f e c t i o n 63 74. Emergence of. EarveyeZZa pustule from grazing wound of 0. fZoooosa. 8 weeks a f t e r i n f e c t i o n 63 75. H. mirabiZis spores attached to glass p l a t e s . Degeneration 7-10 days a f t e r release 63 76. Germination of H. mirabiZis spores on medium supplemented with freeze-dried, powdered 0. fZoooosa 63 77. Germination of H. mirabiZis spores on medium supplemented with.freeze-dried, powdered 0. washingtoniensis 65 x i i i FIGURE 78. Meiosis in. tetrasporangium of H. mivabiZis. E a r l y prophase I. 65 79a,b. Meiosis. Pachynema 65 80a,b. Meiosis. Diplonema-diakinesis 65 81a,b. Meiosis. Six chromosomes i n newly-formed tetraspores 65 82a,b.. Late m i t o t i c prophase i n spermatangial mother c e l l 68 83a,b. Six chromosomes i n mature spermatium.... 68 84a,b. Immature spermatangium. with s i x interconnected chromatin, knobs 68 85a,b. Six chromosomes i n prophase vegetative a p i c a l c e l l i n female pustule..... 68 86a,b. Carpogonial nucleus. Six chromosomes.. 68 87a,b. Six chromosomes i n carpogonial branch c e l l 68 88a,b. Gonimoblast filament c e l l . Twelve chromosomes 68 89. Phenology of H. mivabiZis on 0. fZoooosa at Parsons Point, B. C. Seasonal v a r i a t i o n s i n sea water temperature, s a l i n i t y and hours bright sunshine, 1973-1974 69 90. L i f e h i s t o r y of H. mivabiZis. on 0. fZoooosa. 71 91. Comparison of the phenology of H. mivabiZis at Plymouth, U. K., and Parsons Point, B. C 74 Figures 92-108 C o r t i c a l c e l l s of H. mivabiZis. 92. Tangential section through cortex of pustule 86 93. Three-layered wall surrounding c o r t i c a l c e l l 86 94. Primary p i t connection between two c o r t i c a l c e l l s 86 95. Longitudinal section of outer c o r t i c a l c e l l and adjacent w a l l layers 87 x i v FIGURE 96. Outer wall layers of pustule... 87 97. Diagram of 5 c e l l wall layers surrounding outer cortical c e l l 87 98. Formation of primary p i t connection 87 99. Cortical c e l l nucleus 90 100. Tangential section of nuclear envelope with hexagonally arranged nuclear pores 90 101. Peripherally situated ER. 90 102. Saccate mitochondrion associated with dictyosome releasing rough vesicles... 90 103. Double membrane-bound organelle (plastid) 91 104. Dictyosome releasing smooth vesicles... 91 <— 105. Coalescence of dictyosome derived vesicles 91 106. Large coalescent vesicle containing f i b r i l l a r material and concentric body 91 107. Tubular multivesicular bodies associated with concentric body 91 108. Fusion of multivesicular body with plasmalemma 91 Figures 109-130 Medullary, cells of H. mirabilis. 109. Plastic-embedded thick section through pustule. Stained with PAS... 94 110. Wall matrix surrounding outer medullary c e l l 94 111. Inner medullary c e l l embedded in amorphous wall matrix 94 112. Nucleus bounded by pore-fenestrated nuclear envelope 94 113. Double membrane-bound plastid-like structure 95 114. Elongated dumbbell-shaped plastids associated with mitochondrion 95 XV FIGURE 115. Tangential section through c e l l periphery. Convoluted c e l l membrane. ... 95 116. P l a s t i d with a c e n t r i c a l l y positioned e n c i r c l i n g thylakoid 95 117. P l a s t i d with e n c i r c l i n g thylakoid and one traversing thylakoid. 95 118. Dictyosomes associated with nucleus 97 119. Smooth dictyosome v e s i c l e s forming d i r e c t connection with tonoplast of c e n t r a l vacuole... 97 120. Long plasmalemmavilli. extending from plasmalemma of'.outer medullary c e l l 97 121. Shortplasmalemmavilli p r o j e c t i n g from plasmalemma of inner medullary c e l l 99 122. Cross-section of plasmalemmavilli 99 123. Long plasmalemmavilli i n inf o l d e d areas of outer medullary c e l l s 99 124. Primary p i t connections between adjacent medullary c e l l s 99 125. Small v e s i c l e s attached to convoluted membrane surrounding p i t connection 99 126. Smooth ER near p i t connection 99 127. Single tubule of smooth ER 100 128. Central vacuole containing granular material and m u l t i v e s i c u l a r bodies 100 129. M u l t i v e s i c u l a r and concentric bodies i n c e n t r a l vacuole 100 130. Concentric body i n c e n t r a l vacuole 100 x v i FIGURE Figures 131-193 Rhizoidal c e l l s of..ff.. mirdbilis. 131. R h i z o i d a l c e l l connected to medullary c e l l of 0. floooosa by secondary p i t connection. 102 132. Nucleus with abundant nuclear pores 103 133. Nucleus with single microtubule terminating at nuclear envelope 103 134. P a r a l l e l microtubules outside nucleus 103 135. Lobed and elongate p l a s t i d s 105 136. D i v i s i o n of p l a s t i d 105 137. Concentric bodies i n p l a s t i d 105 138. Schematic summary of i n t e r r e l a t i o n s h i p s of s t r u c t u r a l components comprising r h i z o i d a l c e l l membrane system 106 139. Tangential section. Smooth ER i n c e l l perphery 107 140. Cross-section. Smooth ER i n c e l l periphery 107 141. ER-derived v e s i c l e s associated with plasma lemma 107 142. Large primary vacuole associated with mitochondria and p l a s t i d s 108 143. Secondary vacuoles associated with plasmalemma and coalescing to fomr larger vacuoles 108 144. Secondary vacuole associated with plasmalemma and ER 112 145. D i l a t e d t r i p a r t i t e membrane surrounding plasmalemma-associated secondary vacuole 112 146. Invagination of plasmalemma to form d i l a t e d membrane surrounding secondary vacuole 112 147. M u l t i p l e secondary vacuoles forming at l o c a l i z e d membrane l o c i 112 148. I n i t i a t i o n of secondary vacuole invagination and concomitant release of vacuolar contents i n t o cytoplasm.... 114 x v i i FIGURE 149. Completion of secondary vacuole invagination 114 150. Release of secondary. vacuolar contents 114 151. Cross-section of invaginated secondary vacuole 114 152. Remnants of secondary vacuoles surrounding released vacuolar contents 115 153. Contiguous electron transparent cytoplasmic regions with surrounding secondary vacuolar membranes.. 115 154. Concentric bodies forming from remnants of invaginated secondary vacuoles...... 116 155. Concentric body i n cytoplasm 116 156. Concentric body associated with plasmalemma... 116 157. Concentric body within f i b r o u s - f i l l e d vacuole 116 158. Coalescence of plasmalemma-associated secondary vacuole with plasmalemma 117 159. Primary vacuole containing osmiophilic, granular and lamellar debris 117 160. Dictyosome producing electron-dense, granular v e s i c l e s . . . . 119 161. Smooth ER extending throughout cytoplasm 119 162. ER-derived v e s i c l e s coalescing with plasmalemma-associated secondary vacuoles 119 163. D i l a t a t i o n of ER to form ER vacuole. 120 164. D i l a t a t i o n of ER to form ER. vacuole 120 165. Microbody-like organelles associated with ER 120 166. M u l t i v e s i c u l a r body i n cytoplasm 122 167. M u l t i v e s i c u l a r body associated with d i l a t e d ER vacuole.... 122 168. Association of m u l t i v e s i c u l a r body with plasmalemma 122 x v i i i FIGURE 169. M u l t i v e s i c u l a r body adjacent to secondary vacuole.. 122 170. M u l t i v e s i c u l a r body associated with electron transparent cytoplasmic region.. 122 171. Floridean starch i n primary vacuole 124 172. Light micrograph; PAS stained thick section. Floridean starch i n cytoplasm and primary vacuoles 124 173. Spherical osmiophilic granules ( l i p i d s ) i n cytoplasm 124 174. Osmiophilic granule lacking bounding membrane.. 125 175. Hollow osmiophilic. granule. 125 176. Light micrograph; Sudan black B stained thick section. L i p i d s i n cytoplasm and vacuoles... 125 177. Light micrograph; control.treatment for. l i p i d l o c a l i z a t i o n 125 178. Light micrograph; N i l e blue A stained thick section. Fatty acids associated with primary vacuole.... 127 179. Dense osmiophilic i n c l u s i o n with p e r i p h e r a l electron transparent regions (polyphosphate granule) 127 180. Polyphosphate granule associated with d i l a t e d ER 127 181. Polyphosphate granule 127 182. Electron-dense granular material i n cytoplasm 128 183. Electron-dense granular material surrounded by d i l a t e d ER 128 184a,b. Light micrograph; mercuric chloride-bromphenol blue stained thick section. Granular e l e c t r o n -dense substance stains p o s i t i v e for protein 129 185. Non-membrane-bound c r y s t a l l i n e body i n cytoplasm 129 186. Tangential section near plasmalemma. Microtubules present.. 131 187. Numerous so l i t a r y - b o d i e s (S-bodies) associated with ER 131 x i x FIGURE 188. S-body i n mitochondrion 131 189. S-body associated with mitochondrial membrane 131 190. S-bodies associated with secondary vacuoles 133 191. S-body associated with secondary vacuoles 133 192. U l t r a s t r u c t u r e of S-body 133 193. Diagram of S-body 133 194. Negative-stained S-body. " T a i l " adjoining outer membrane 135 195. Negative-stained S-body. Hexagonal shape' and c e n t r a l core evident 135 196. S-body a f t e r 14 hr dig e s t i o n i n RNase at 25 C 135 197. S-body a f t e r 14 hr RNase digestion at 40 C 135 198. S-body a f t e r 14 hr DNase digestion at 25 C 135 199. S-body a f t e r 14 hr DNase digestion at 40 C... 135 200a,b. Autoradiographs. 0 hr t r a n s l o c a t i o n ; host c o r t i c a l c e l l s . . . . 137 201. Translocation of l i +C-labeled compounds between 0. fZoooosa and H. mirabiZis over 36 hr. Dark pretreatment p r i o r to l a b e l i n g 139 202. Translocation of 1 4 C - l a b e l e d compounds between 0. fZoooosa and H. mirabiZis over 36 hr. Light pretreatment p r i o r to l a b e l i n g . . 139 203. R a d i o a c t i v i t y i n seawater during t r a n s l o c a t i o n . . . . . 140 204. Translocation of l l tC-labeled compounds i n controls incubated with lhZ i n l i g h t . . 140 205. Translocation of 1 1 +C-labeled compounds i n controls incubated wi th lkC i n dark 140 XX FIGURE 206a,b. Autoradiography. Density of s i l v e r grains associated with ti s s u e areas of H. mivabilis and 0. floocosa a f t e r various t r a n s l o c a t i o n periods 143 207a,b. Autoradiographs. 0 hr tra n s l o c a t i o n ; illuminated cortex of 0. floocosa 137 208a,b. Autoradiographs. 0 hr tra n s l o c a t i o n ; medulla of 0. floocosa 137 209a,b. Autoradiographs. 4 hr tra n s l o c a t i o n ; illuminated host c o r t i c a l c e l l s 137 210a,b. Autoradiographs. 4 hr tra n s l o c a t i o n ; host medullary c e l l s 144 211a,b. Autoradiographs.., 4 hr tra n s l o c a t i o n ; host medullary c e l l s i n i n t e r d i g i t a t i o n zone 144 212a,b. Autoradiographs. 24 hr tra n s l o c a t i o n ; illuminated host c o r t i c a l c e l l s . . . . . . 144 213a,b. Autoradiographs. 8 hr tr a n s l o c a t i o n . I n t e r d i g i t a t i o n zone 144 214a,b. Autoradiographs.. 24 hr translocation.. R h i z o i d a l c e l l s of H. mivabilis i n i n t e r d i g i t a t i o n zone. 146 215a,b. Autoradiographs. 24 hr tr a n s l o c a t i o n . Medullary c e l l s of H. mivabilis pustule 146 216. Hypothesized flow scheme for 1' tC^-labeled metabolites from 0. floocosa to H. mivabilis 147 217a,b. Autoradiographs. . 0 hr tr a n s l o c a t i o n . Secondary f l u x of 1 I +C. Host medullary c e l l s i n outer pustule of Havveyella , 146 218a,b. Autoradiographs. 4 hr tr a n s l o c a t i o n . Secondary f l u x of 1 4 C . Havveyella c e l l s of pustule adjacent to i s o l a t e d host c e l l s 146 Figures 219-222 D i s t r i b u t i o n of lkC i n separated f r a c t i o n s from 0. floocosa and H. mivabilis a f t e r various periods of t r a n s l o c a t i o n .  219. Change i n t o t a l r a d i o a c t i v i t y over 24 hr tr a n s l o c a t i o n 148 x x i FIGURE 220. Change i n r a d i o a c t i v i t y i n ether extract and insolu b l e residue 148 221. Change i n r a d i o a c t i v i t y i n c a t i o n i c and anionic f r a c t i o n . . . 148 222. Change i n r a d i o a c t i v i t y i n neutral sugars and. starch 148 223. Percentage recovery of t o t a l r a d i o a c t i v i t y from the chromatographically separated n e u t r a l f r a c t i o n of Havveyella. A c i d i c solvent system • 152 224. Percentage recovery of t o t a l r a d i o a c t i v i t y from the chromatographically separated neutral, f r a c t i o n of Havveyella. .- Basic : solvent system 152 225. Percentage recovery of t o t a l r a d i o a c t i v i t y from the chromatographically separated neutral f r a c t i o n of 0. floocosa. A c i d i c solvent system 155 226. Percentage recovery of t o t a l r a d i o a c t i v i t y from the chromatographically separated neutral f r a c t i o n of 0. floocosa. Basic solvent system 155 227. Light micrograph; Sudan black B stained thick section. H. mivabilis and 0. floocosa i n i n t e r d i g i t a t i o n zone 166 228. Cross-section of Havveyella pustule. Host c e l l s dispersed throughout pustule..... 166 229. Secondary p i t connection between host medullary c e l l and Havveyella r h i z o i d a l c e l l i n i n t e r d i g i t a t i o n zone 166 230. T r i p a r t i t e membrane adjacent to p i t plug i n Havveyella r h i z o i d a l c e l l 166 231.. Cytoplasmic protuberance from host c e l l toward adjacent r h i z o i d a l c e l l of H. mivabilis 169 232. Wall adjacent to advancing host c e l l protuberance... 169 233. Secondary p i t connection between two c l o s e l y -situated medullary c e l l s 169 234. S-bodies i n cytoplasm of host medullary c e l l attached to Havveyella r h i z o i d a l c e l l by p i t connection. 169 235. S-body associated with p l a s t i d of. host c e l l connected to Havveyella by secondary p i t connections..- ... 169 x x i i FIGURE 236. Medullary c e l l of 0. fZoooosa from uninfected tis s u e region.. 237. Host medullary c e l l i n HavveyeZZa i n f e c t e d region 238. Plasmalemma v e s i c u l a t i o n i n infe c t e d host c e l l . . . 239. Infected, host medullary c e l l 173 240. Light micrograph; Sudan black B stained, thick section. L i p i d s abundant i n host, medullary c e l l , i n i n f e c t e d t i s s u e region .' 173 241. Large osmiophilic granule i n cytoplasm of infe c t e d host medullary c e l l 173 242. Increase i n ..number, of, osmiophilic granules i n infected host c e l l 173 243. Light micrograph;. Luxol's f a s t blue G stained thick section. Phospholipids i n infe c t e d host medullary c e l l s 174 244. Light micrograph; Luxol's f a s t blue G stained thick section. No phospholipids i n uninfected host medullary c e l l s 174 245. Light micrograph; Luxol's f a s t blue G stained thick section. Phospholipids absent from host c o r t i c a l c e l l s i n HavveyeZZa infected tissues 174 246. Osmiophilic granules associated with secondary p i t connection 174 247. Aggregation of osmiophilic granules i n host medullary c e l l s 174 248. Osmiophilic granule appressed to secondary p i t connection i n host c e l l 174 249. Convoluted membrane surrounding primary p i t connections between adjacent host c e l l s i n infe c t e d t i s s u e region 174 250. Uninfected host medullary c e l l nucleus 176 251. Localized evagination of nuclear envelope i n infec t e d host medullary c e l l 176 171 171 171 x x i i i FIGURE 252. Bleb-nucleus with c e n t r a l l y located chromatin.;. 176 253. I r r e g u l a r l y shaped nucleus i n infec t e d ..host. c e l l . ......... . 176 254. Enlarged nucleus of in f e c t e d host medullary c e l l 176 255. P l a s t i d i n host medullary c e l l showing no u l t r a s t r u c t u r a l changes 177 256. Phycobili'somes on thylakoids of host p l a s t i d . . 177 257. P l a s t i d i n infe c t e d host c e l l 177 258. Degenerating p l a s t i d in,host c e l l of H. mivabilis infected t i s s u e region 177 259. Hypertrophied thylakoids i n infec t e d host c e l l p l a s t i d 179 260. Rupturing of p l a s t i d i n infe c t e d host c e l l 179 261. Plastids. i n rupturing host c e l l 179 262. Bacteria i n degenerating host wall... 179 263. Light micrograph; penetration of host medullary c e l l by H. mivabilis r h i z o i d a l c e l l s 180 264. Light micrograph; penetrating Havveyella c e l l 180 265. Light micrograph; f i n e wall s t r i a t i o n s i n host w a l l near penetrating Havveyella filaments 180 266. Havveyella r h i z o i d a l c e l l adjacent to host c e l l 180 267. Penetration of host medullary c e l l by r h i z o i d a l c e l l of H. mivabilis 182 268. Membranous material i n c e l l w a l l i n t e r f a c e between host and penetrating r h i z o i d a l c e l l s 182 269. Light micrograph; penetration of host medullary c e l l s i n i n t e r d i g i t a t i o n zone by Havveyella r h i z o i d a l c e l l s 182 270. Autoradiograph. Isolated host medullary c e l l i n pustule of Havveyella; uptake of H 1 L tC03~ 184 x x i v FIGURE 271. Interconnected.traversing thylakoids i n p l a s t i d of host medullary c e l l i n pustule of E. mivabilis ... 184 272. D i v i s i o n of p l a s t i d i n host medullary c e l l i n pustule 184 273. P r o l i f e r a t i o n of. rough ER i n host medullary c e l l i n pustule 184 274. Host, c o r t i c a l c e l l i n pustule of Earveyella. 186 275. S-body i n cytoplasm of host c o r t i c a l c e l l connected to Earveyella by secondary p i t connection....... 186 276a,b. Autoradiographs.. C o r t i c a l c e l l s i n pustule forming, c e l l "packets". C e l l s a c t i v e i n photosynthetic a s s i m i l a t i o n of l t f C . 186 277. Osmiophilic.granules associated with p i t connections between host c o r t i c a l c e l l s i n E. mirabilis pustule 187 278. External palisade layer of c o r t i c a l c e l l s i n infec t e d 0. floocosa.. 187 xxv LIST OF COLOURED PLATES COLOUR PLATE 1. PAS stained section i n d i c a t i n g f l o r i d e a n starch 110 2. PAS stained section i n d i c a t i n g carbohydrates i n primary and secondary vacuoles 110 3. N i l e blue A stained section. Fatty acids present i n autophagic v e s i c l e s . . 110 4. N i l e blue A stained section.. Fatty acids i n autophagic v e s i c l e s 110 5. Toluidine blue 0; f l o r i d e a n starch and polyphosphate bodies i n Havveyella r h i z o i d a l c e l l s . . 110 6. Toluidine blue 0; polyphosphate bodies'in Havveyella r h i z o i d a l , c e l l s and i n host medullary c e l l s . . . . . ... 110 7. Sudan black B; host medullary c e l l s surrounded by Havveyella r h i z o i d a l c e l l s i n i n t e r d i g i t a t i o n zone 110 8. Sudan black B; Havveyella c e l l s penetrating host medullary c e l l i n i n t e r d i g i t a t i o n zone.. 110 9. PAS st a i n i n g ; f l o r i d e a n starch i n uninfected host c e l l . . . . I l l 10. Luxol f a s t blue G, ne u t r a l red; phospholipids i n i s o l a t e d host c e l l i n Havveyella pustule I l l 11. Toluidine blue 0; s t a i n i n g differences of host c e l l and Havveyella c e l l s of pustule I l l 12. Toluidine blue 0; f l o r i d e a n starch i n Havveyella c e l l . . . . . I l l 13. A l c i a n blue, a l c i a n yellow; sulfated polysaccharides i n wall layer surrounding outer host medullary c e l l I l l 14. A l c i a n blue, a l c i a n yellow; sulfated polysaccharides i n wall layer surrounding inner host medullary c e l l I l l .15. A l c i a n blue, a l c i a n yellow; sulfated polysaccharides are absent from c e l l w a l l surrounding host c e l l i n pustule I l l 16. A l c i a n blue, a l c i a n yellow; d i f f u s e band of su l f a t e d poly-saccharides surrounding Havveyella c e l l s i n pustule....... I l l x x v i LIST OF APPENDICES APPENDIX I. New d i s t r i b u t i o n a l records f o r Earveyella mirabilis i n the northeast P a c i f i c 223 I I . D i s t r i b u t i o n a l records for Earveyella mirabilis 2 2 6 1. Herbarium annotations 2 2 6 2. L i t e r a t u r e references 2^0 I I I . C o l l e c t i o n data, Sooke, B. C , 1973-1974 2 3 1 IV. Temperature, s a l i n i t y and hours bright sunshine at selected stations.' 2 3 2 V. Modifications of standard techniques 2 3 ^ 1. Modified Karpechenko' s f i x a t i v e 235 2. A n i l i n e blue-HCl s t a i n i n g of reproductive structures.. 2 3 ^ 3. Aceto-iron-Haematoxylin-chloral hydrate s t a i n i n g of chromosomes 2 3 ^ 4. Modified Erdschreiber culture medium 2 3 ^ 5. Hard-cure modification of Spurr's low v i s c o s i t y epoxy r e s i n 2 3 ^ 6. S c i n t i l l a t i o n f l u o r 2 3 8 VI. R values i n two solvent systems... 2 3 ^ x x v i i ACKNOWLEDGEMENTS I wish to s i n c e r e l y thank Dr. Kathleen Cole for her continued d i r e c t i o n , advice and support throughout the course of t h i s study. I would also l i k e to express my appreciation to Dr. G. C. Hughes, Dr. E. B. Tregunna, Dr. R. F. Scagel, Dr. R. E. Foreman and Dr. E. Conway whose guidance helped d i r e c t t h i s research. Thanks are also extended to Mr. L. L. Veto and Mr. M. T. Higham for t h e i r excellent technical assistance i n electron microscopy and photo-graphy, Mr. M. Storm for assistance i n determining s a l i n i t y values, Dr. J . S. C r a i g i e (NRC, A t l a n t i c Regional Laboratory, Halifax, N. S.) for providing f l o r i d o s i d e and i s o f l o r i d o s i d e reference standards, Dr. T. E d e l s t e i n (NRC, A t l a n t i c Regional Laboratory, Halifax, N. S.) and Dr. J . Cabioch (Station Biologique, Roscoff, France) for t h e i r generosity i n sending fresh and pre-served a l g a l material, to the various herbaria for t h e i r promptness i n providing a l g a l specimens, Environment Canada (Inland Water Directorate and Atmospheric Environment Services, V i c t o r i a , B. C.) for providing necessary environmental data, the Department of Botany, U n i v e r s i t y of B r i t i s h Columbia and the P a c i f i c Research Station, Canada A g r i c u l t u r e for use of e l e c t r o n microscopy f a c i l i t i e s , L e s l i e Borleske for typing the f i n a l manuscript, and f i n a l l y , to the many associates who accompanied me and kept me from drowning during the long, cold and wet late-night a l g a l c o l l e c t i n g t r i p s . Special thanks are due to Dr. D. S. Cameron for h i s constant encouragement and help i n proofing the f i n a l text, and to Dr. Janet R. Stein whose continued encouragement and f r i e n d s h i p , as w e l l as her enthusiasm for teaching and learning has contributed greatly to both my p r o f e s s i o n a l and personal development. x x v i i i This research project was supported by National Research Council of Canada grants to Dr. Kathleen Cole and by financial awards to the author provided by the University of British Columbia, National Research Council of Canada, Edith Ashton Memorial Scholarship Fund, and the Western Society of Naturalists. 1 I. GENERAL INTRODUCTION Havveyella mivabilis (Reinsch) Schmitz and Reinke, a small hemispherical colourless member of the Cryptonemiales attached to various species of Khodomela and Odonthalia,has been reported from the north P a c i f i c and north A t l a n t i c oceans (Farlow, 1881; Sturch, 1899, 1924; Holmes and Batters, 1890; Kuckuck, 1894; De Toni, 1897; C o l l i n s , 1900; Chemin, 1927; Newton, 1931; Rosenvinge, 1931; Levring, 1935; SSdnova, 1940, 1954; K y l i n , 1944; Park, 1953; Taylor, 1957; Wilce, 1959; Scagel, 1961, 1963; Ede l s t e i n , Chen and McLachlan, 1970; Norton, 1970). It i s included i n a small group of f l o r i d e a n algae described as p a r a s i t i c on t h e i r red a l g a l hosts ( F r i t s c h , 1945). P a r a s i t i c red algae are delineated from epiphytic and endophytic forms on the basis of the morphological c h a r a c t e r i s t i c s of host penetration, reduction i n t h a l l u s s i z e and loss i n pigmentation ( S e t c h e l l , 1918). A wide range of each of these morphological features i s exhibited within the approxi-mately 40 genera of reported p a r a s i t i c florideophytes. S e t c h e l l (1918) chose the c h a r a c t e r i s t i c s of host penetration and s i z e and pigment reduction i n defining a p a r a s i t i c red alga since he thought they r e f l e c t e d the p h y s i o l o g i -c a l dependence of the p a r a s i t i c alga on i t s host. He assumed that an alga which i s reduced i n stature, possesses reduced amounts of photosynthetic pigments, and s t r u c t u r a l l y penetrates i t s host must be incapable of producing s u f f i c i e n t q u antities of organic carbon to sustain i t s growth requirements and must therefore r e l y upon i t s host as a source of metabolites. A problem has arisen i n using morphological c r i t e r i a to define p a r a s i t -ism i n the red algae. By l i m i t i n g the d e f i n i t i o n to morphological features, p h y s i o l o g i c a l i n t e r a c t i o n s of the host and pa r a s i t e have been generally assumed or ignored. Dixon (1973) has concluded that, although i t i s 2 generally accepted that there i s some degree of " p h y s i o l o g i c a l parasitism" i n the red algae, the evidence i n favour of such an i n t e r p r e t a t i o n i s extremely weak. The emphasis on morphological c h a r a c t e r i s t i c s i n the study of red a l g a l parasitism i s r e f l e c t e d i n the f a c t that most investigations of these forms have focused p r i m a r i l y on morphological and anatomical aspects of the parasite and i t s r e l a t i o n s h i p with i t s host (Oltmanns, 1905; S e t c h e l l , 1905, 1914, 1923; Wilson, 1910; Dawson, 1944; Pocock, 1953; Pocock and Martin, 1953; K y l i n , 1956; Feldmann and Feldmann, 1958, 1963; Fan and Papenfuss, 1959; Tanaka and Nozawa, 1960; Fan, 1961; Adey and Sperapani, 1971; E d e l s t e i n , 1972; Guiry, 1974; Adey, Masaki and Akioka, 1974). Only recently have studies been undertaken to a s c e r t a i n i f the morphologically defined parasites are indeed " p h y s i o l o g i c a l p a r a s i t e s " (Kugrens, 1971; H a r l i n , 1971, 1973a; Evans, Callow and Callow, 1973). Although these in v e s t i g a t i o n s have provided preliminary information on a few s p e c i f i c p arasites, there remains a need for a d d i t i o n a l d e t a i l e d studies of symbiotic red algae encompassing the e n t i r e range of host-parasite dependencies. These inv e s t i g a t i o n s must include studies of developmental and reproductive i n t e r a c t i o n s of host and p a r a s i t e as well as cytochemical, u l t r a s t r u c t u r a l and p h y s i o l o g i c a l i n v e s t i g a t i o n s of nutrient t r a n s l o c a t i o n and the e f f e c t of parasite i n f e c t i o n on the host. In the present i n v e s t i g a t i o n of the r e l a t i o n s h i p of Harveyella mirabilis and i t s hosts, Odonthalia and Rhodomela, i t has been necessary to redefine the previously employed morphological d e f i n i t i o n of red a l g a l parasitism. This new d e f i n i t i o n has been formulated to include p h y s i o l o g i c a l aspects of the host—parasite r e l a t i o n s h i p as s p e c i f i e d i n the c l a s s i c a l concept of parasitism (Hertig, T a l i a f e r r o , and Schwartz, 1937; Henry, 1966; Scott, 1969; <3 Lewis, 1973). A red a l g a l parasite w i l l thus be defined as any red alga l i v i n g temporarily or permanently within or on a host, deriving b e n e f i t s from i t , and causing i t harm. The p u t a t i v e l y p a r a s i t i c red alga Havveyella mivabilis was chosen f o r the present study of a l g a l parasitism for several reasons. F i r s t l y , i t i s considered to be a highly evolved p a r a s i t i c form (Sturch, 1926) possessing a l l the morphological c h a r a c t e r i s t i c s used i n defining a " s t r u c t u r a l para-s i t e " . Secondly, H. mivabilis exhibits a great degree of host s p e c i f i c i t y implying possible p h y s i o l o g i c a l i n t e r a c t i o n s with i t s rhodomelacean hosts. T h i r d l y , s u f f i c i e n t quantities of H. mivabilis could be obtained from l o c a l c oastal waters to enable p h y s i o l o g i c a l studies on the nature of the host-parasite r e l a t i o n s h i p . The r e l a t i v e abundance of t h i s p a r a s i t e also allowed extensive f i e l d and culture studies to a s c e r t a i n the nature of the reproduc-t i v e and developmental dependence of Havveyella on i t s hosts. In determining whether the " s t r u c t u r a l p a r a s i t e " Havveyella i s a c t u a l l y a " p h y s i o l o g i c a l p a r a s i t e " , the following three questions have been investigated: 1) Is the reproduction and development of Havveyella i n any way dependent on the presence of a s p e c i f i c host? 2) Is there an exchange of material between the host and Havveyella7. 3) Does the presence of Havveyella a f f e c t the welfare of the host? 4 I I . HISTORICAL REVIEW OF THE TAXONOMY OF HARVEYELLA MIRABILIS The nature of the p h y s i o l o g i c a l i n t e r a c t i o n between red a l g a l parasites and t h e i r hosts can be correlated with the taxonomic s i m i l a r i t i e s of the two symbionts (Feldmann and Feldmann, 1958). Parasites that are not taxonomi-c a l l y r e l a t e d to t h e i r hosts ( a l l o p a r a s i t e s ) generally i n t e r a c t with them much d i f f e r e n t l y than those that are r e l a t e d (adelphoparasites). Thus i n the case of Harveyella mirabilis on Odonthalia and Rhodomela, i t i s e s s e n t i a l that the o r d i n a l a f f i l i a t i o n s of Harveyella be established before proceeding to an analysis and discussion of the p h y s i o l o g i c a l nature of the host-p a r a s i t e r e l a t i o n s h i p . Although not formally described u n t i l 1875, Harveyella mirabilis was probably f i r s t observed by Lyngbye i n 1819 from c o l l e c t i o n s made i n Norway. Liebman also observed t h i s species i n 1838 at Helsingc^r, Denmark but r e f e r r e d i t to Corynephora marina (Rosenvinge, 1931). Reinsch (1875) f i r s t described H. mirabilis under the name Choreooolax mirabilis Reinsch but noted only the vegetative plant. The f i r s t study of the reproductive structures was included i n a l e t t e r by C. Schmitz to J . Reinke (Reinke, 1889). Schmitz described the antheridia and the mature cystocarp noting the l a t t e r to be s i m i l a r to the cystocarp of Cauloaanthus but d i f f e r e d from Choreoeolax polysiphoniae. He proposed placing the species Choreoeolax mirabilis (and Choreooolax amerioanus Reinsch) i n the new genus Harveyella, named i n honour of W. H. Harvey. Following Schmitz's suggestion, Reinke established the new genus, placing i t i n the Gelidiaceae (Nemalionales). A study of Choreooolax polysiphoniae cystocarps (Richards, 1891) showed that the cystocarps of 5 Choveoaolax and Havveyella d i f f e r , thus j u s t i f y i n g the d i v i s i o n of the genus Choveoaolax by Reinke and Schmitz.. Schmitz (1889) included Havveyella i n the d i s t i n c t subfamily Harveyelleae of the Gelidiaceae. Holmes and Batters (1890) placed i t i n the same p o s i t i o n i n t h e i r l i s t of B r i t i s h marine algae as did De Toni (1897) i n h i s "Sylloge Algavum". Sporangiajwere unknown u n t i l 1894 when Kuckuck published a d e s c r i p t i o n of what he considered to be the tetraspores of Choveoaolax albus, c o l l e c t e d at Helgoland. Sturch (1899) l a t e r reported that Choveoaolax albus was simply a "tetraspore-bearing specimen of H. mivabilis". Antheridia and carpogonial branches were f i r s t described by Schmitz and Hauptfleisch i n 1896 although the development of the carpogonium and the gonimoblast was not observed. Sturch's d e t a i l e d d e s c r i p t i o n of development of H. mivabilis (1899, 1924) provides the f i r s t account of carpogonial development. He noted that an a u x i l i a r y c e l l develops before f e r t i l i z a t i o n and thus he proposed removing H. mivabilis from the Gelidiaceae and placing i t i n the Gigartinaceae ( G i g a r t i n a l e s ) . Eddelbuttel (1910) put Havveyella i n the family Choreocol-aceae which he placed i n the G i g a r t i n a l e s as did Svedelius (1911). The question of the o r d i n a l a f f i l i a t i o n of H. mivabilis became even more confused i n 1924. Upon a d d i t i o n a l examination of the carposporophyte development i n Havveyella, Sturch described that the a u x i l i a r y c e l l "undoubtedly segmented af t e r f e r t i l i z a t i o n " and i n t h i s respect, Havveyella shows taxonomic, a f f i n i -t i e s with the Ceramiales. K y l i n (1923) described the possible rhodomelacean (Ceramiales) a f f i n i t i e s of H. mivabilis by noting that the gonimoblast c e l l i s cut o f f on the outer-side of the a u x i l i a r y c e l l but concluded that i n most other respects, i t d i f f e r s greatly from the Rhodomelaceae and should not be included i n t h i s group. Sturch (1926) concluded that because of the c o n f l i c -6 t i n g evidence,.it i s impossible to decide to which f l o r i d e a n order Earveyella belongs. Subsequently,.Kylin (1937),. ' r e f e r r e d the family Choreocolaceae to the order Cryptonemiales because of c e r t a i n s i m i l a r i t i e s to the Callymeniaceae. Currently, the. monotypic genus Earveyella i s included i n the order Cryptonemiales although as indicated by F r i t s c h (1945), questions remain concerning p o s t - f e r t i l i z a t i o n events which require further i n v e s t i g a t i o n . In the present study, carposporophyte development has been examined i n an e f f o r t to a s c e r t a i n the precise taxonomic, a f f i l i a t i o n of Earveyella mira-bilis. The r e s u l t s of t h i s i n v e s t i g a t i o n are included in. Section I I I (Reproductive and Developmental Dependency of Earveyella- on a S p e c i f i c Host). 7 I I I . METHODS AND MATERIALS A. C o l l e c t i o n and Treatment of Samples for E c o l o g i c a l and D i s t r i b u t i o n a l Studies. The e f f e c t of selected environmental parameters on the development and reproduction of Havveyella mivabilis on Odonthalia floocosa (Esper) Falkenberg was investigated using data obtained from f i e l d and culture studies. Periodic sampling was made of two populations near Sooke, Vancouver Island, B r i t i s h Columbia (48°20'N 123°44'W) from January 1973 to September 1974. Population 1 was located on a r e l a t i v e l y sheltered, sand-inundated area on the south side of Whiffen Spit; whereas population 2 was situated at Parsons Point, a rocky, r e l a t i v e l y exposed area approximately 500 meters southwest of popula-t i o n 1 (Figure 12, i n s e t ) . The v e r t i c a l d i s t r i b u t i o n of Havveyella i n both populations extended from approximately 0 to 2 m above the zero t i d a l datum l e v e l (Canadian hydrographic standard). At le a s t 200 randomly selected, mature Havveyella pustules were c o l l e c t e d biweekly or monthly from population 2 and less frequently from population 1. Twenty-eight a d d i t i o n a l populations of Havveyella located i n Oregon, Washington, and B r i t i s h Columbia were sampled at l e a s t once during the course of t h i s study. C o l l e c t i o n s i t e s , hosts and reproductive stages are summarized i n Appendix I. At the time of each c o l l e c t i o n , surface water temperatures and s a l i n i t i e s were determined at three or more l o c a l i t i e s . Measurements were made at slack low water at a depth of 0.5 m. At le a s t three temperature measurements and two s a l i n i t y samples were taken at each l o c a l i t y . S a l i n i t y was measured using an Auto-lab i n d u c t i v e l y coupled salinometer (Model 601, Mk. I I I ) . Comparison of these data was made with monthly surface water temperatures and s a l i n i t i e s 8 taken from November 1965 to September 1966 at a recording s t a t i o n 700 meters offshore from Parsons Point ( E l l i o t t , 1969a,b) and d a i l y surface temperatures from Neah Bay, Washington (U. S. Coast and Geodetic Survey, 1962). The hours of bright sunlight at Jordan River and V i c t o r i a , B. C. between 1931-1960 and monthly t o t a l s at V i c t o r i a for 1973-1974 were obtained from the Canadian Atmospheric and Environmental Service (Meterol. Branch Canada, Dept. Trans-port, 1962; Environment Canada, Atmospheric Environment Service, 1973; V i c t o r i a Weather O f f i c e s , personal communication, 1974). These data are pre-sented i n Appendix IV. The e f f e c t of variance i n t i d a l exposure on the development of Harveyella was also examined. T h i r t y plants from population 2, covering an approximate 2 m t i d a l range (0-2.0 m), were marked with p l a s t i c tags and observed from February 1973 to January 1974. In order to determine the presence and degree of maturation of the repro-ductive structures of Harveyella, f r e s h l y c o l l e c t e d plants were fi x e d i n a modified Karpechenko's sol u t i o n (Appendix V, 1), rinsed i n water, dehydrated i n a graded ethanol seri e s and stored i n 70% ethanol. Sections 20-25 urn thick were cut on a fre e z i n g microtome, stained i n a n i l i n e blue-hydrochloric acid (Appendix V, 2),,and mounted i n glycerine-water (1:1 v/v). H. mirabilis on 0. flooaosa was maintained for periods up to s i x months i n culture to compare development under co n t r o l l e d conditions to that observed i n the f i e l d . The plants i n various developmental stages were c o l l e c t e d , c a r e f u l l y cleaned by brushing, tagged, and maintained i n 12 1 culture tanks. The plants were cultured i n either aerated m i l l i p o r e — f i l t e r e d (0.22 ym m i l l i p o r e size) seawater or i n aerated modified Erdschreiber's culture medium (Appendix V, 4). This culture mediumr/was used throughout the study. During November to March, 9 the tanks were kept at a constant temperature of 7 C under an 8:16 hour photoperiod at 1000 lux (Sylvania Cool White Fluorescent l i g h t ) (converted from foot-candle measurements). In A p r i l the temperature was elevated to 11°C and the photoperiod lengthened to a 12:12 hour cycle at 1600 lux where i t was maintained u n t i l October. The geographical d i s t r i b u t i o n of H. mivabilis was determined by a c r i t i -c a l review of the l i t e r a t u r e and an examination of herbarium materials from both north A t l a n t i c and north P a c i f i c c o l l e c t i o n s (see Appendix II for c o l l e c t i o n and herbarium l o c a l i t i e s ) . Randomly selected plants from each c o l l e c t i o n were stained i n a n i l i n e blue-hydrochloric a c i d and sectioned. Taxonomic d i s p o s i t i o n and the presence of reproductive structures i n the specimens examined were noted on each herbarium sheet. B. Morphological and Developmental Studies. 1. Light Microscopic Techniques To substantiate the presumed "Polysiphonia-ty-pe" l i f e h i s t o r y of H. mivabilis, chromosomes were counted i n reproductive and vegetative c e l l s . T e trasporic, carposporic, carpogonial and spermatial stages were c o l l e c t e d i n 1972-1973 at Rosario Beach State Park, Washington, and from population 2 at Parsons Point, Vancouver Island, B. C. M a t e r i a l was fi x e d immediately upon c o l l e c t i o n i n a sol u t i o n of 3 parts 95% ethanol to 1 part g l a c i a l a c e t i c a c i d and decolourized i n bright sunlight f o r several days. It was transferred into aceto-iron-haematoxylin-chloral hydrate s t a i n and softener (Appendix V, 3) for 2-3 days. Small pieces of m a t e r i a l were heated f o r 2 minutes i n a drop of fresh s t a i n on a s l i d e and squashed. Chromosomes were counted i n a 10 minimum of 250 c e l l s from each reproductive and vegetative phase. Requirements for germination of H. mirabilis spores were studied i n cultured material. Freshly c o l l e c t e d carposporic and t e t r a s p o r i c pustules were brushed free of contaminants, rinsed f o r 30 seconds i n sodium hypochlor-i t e (1% v/v) and placed i n 500 ml capacity glass culture dishes containing 200 ml culture medium. Carpospores and tetraspores were released onto ground glass d i s c s , microscope s l i d e s and polypropylene s t r i p s (0.004 x 0.90 x 3.0 cm s t r i p s of Olefern obtained from Avisun Corp., Phila d e l p h i a , Pa.; H a r l i n , 1973b). Upon release, some of the spores were pipetted to culture dishes containing 150 ml of culture medium covering s o l i d i f i e d , s t e r i l i z e d agar (2% Difco agar i n culture medium), carageenan (5% V i s c a r i n obtained from Marine C o l l o i d s Inc., S p r i n g f i e l d , N. J.) or agar supplemented with freeze-dried powdered 0. fZoooosa, 0. washingtoniensis K y l i n and Rhodomela larix (Turner) C. Agardh (12 g powdered alga:0.5 g agar:150 ml culture medium). A l l dishes were placed on a shaker and maintained at 10'C at a 12:12 hour photoperiod (1000 l u x ) . Cultures were examined d a i l y with a Wild M 20 micro-scope equipped with a 20X water immersion objective. Cleaned and s u r f a c e - s t e r i l i z e d carposporic and t e t r a s p o r i c pustules were attached with brass insect pins to scored and unscored areas of the main axis of various plants including 0. fZoooosa, Laurencia speotabilis Postels and Ruprecht, Plocamiwn paoifiovm K y l i n , Iridaea oovdata (Turner) Bory, Poly-siphonia paoifioa Hollenberg and Graoilariopsis sjoestedii (Kylin) Dawson. These were placed i n vigorously aerated 12 1 culture tanks containing culture medium. The plants were grown under the April-October culture conditions described on pages 8-9. A l l pustules of sporulating H. mirabilis were removed a f t e r two days and selected host plants were sectioned and observed 11 d a i l y f o r the f i r s t 45 days and weekly during the following three-month period. Attempts were made to determine the agents p r i m a r i l y responsible f o r the natural host wounds i n which successful germination of H, mivabilis occurred. F i e l d observations were made to determine the most common and heavily feeding grazers of 0. floocosa. These grazers were then tested i n the laboratory to determine feeding preference, consumption mass and s p e c i f i c grazing methods. F i f t y isopods of the species Idothea wosnesenskii (Brandt) were placed i n a 12 1 tank containing 0. floocosa. 0. washingtoniensis, R. lavix, Itmvencia spectabilis, Plocamiwn pacificum, Micvocladia bovealis Ruprecht, Delessevia decipiens J . G. Agardh, Ividaea cordata. Viva lactuca L., and Eedophyllum sessile (C. Agardh) S e t c h e l l , a l l commonly found growing near Eavveyella. The type of wounds r e s u l t i n g from grazing was noted as well as the number of grazers on each plant a f t e r two day periods. The f i v e algae most frequented by Idothea were placed i n a separate tank with 30 isopods. The loss i n weight i n each species was calculated f o r a 14 day period. S p e c i f i c procedural d e t a i l s are discussed i n the r e s u l t s and observations (page 59 ) . Analyses were made of f e c a l p e l l e t s and guts of several i n d i v i d u a l isopods c o l l e c t e d i n the f i e l d f o r the laboratory studies. 2. Electron Microscopic Techniques Several electron microscopic f i x a t i o n procedures were employed i n attempts to preserve adequately vegetative and reproductive c e l l s of E. miva-bilis, 0. floocosa, 0. washingtoniensis and B. lavix. The procedures included the use of permanganate, dichrornate, paraformaldehyde, a c r o l e i n , g l u t a r a l d e -hyde and osmium tetroxide. The most s a t i s f a c t o r y f i x a t i o n was obtained using a modifiedclglutaraldehyde-osmium tetroxide procedure. H. mivabilis on 0. floocosa, 0. washingtoniensis and R. lavix was'fixed i n the f i e l d immedi-a t e l y upon c o l l e c t i o n from populations i n Oregon, Washington, and B r i t i s h Columbia. Small pieces of material were placed i n a s o l u t i o n of 50% g l u t a r -aldehyde (v/v), 0.07 M phosphate buffer at pH 7.3 and dimethyl sulfoxide (1:9:0.1) at 0°C f o r 4 hours. They were washed i n pH 7.3 phosphate buffer and transferred on i c e to the laboratory and then f i x e d at 4°C for 2^ hours i n a s o l u t i o n of 2% osmium tetroxide (w/v), phosphate buffer (0.07 M) at pH 7.3 and dimethylsulfoxide (1:1:0.02). A f t e r washing i n buffer, the material was dehydrated i n a graded ethanol seri e s followed by a graded acetone s e r i e s . I t wassslowly i n f i l t r a t e d over a period of three days under vacuum with a hard-cure modification of Spurr's (1969) medium (Appendix V, 5) and subsequent-l y embedded i n f r e s h medium. Thin sections„:were cut on LKB 1 and Reichert OM U3 ultramicrotomes using glass or diamond knives. The sections were post-stained i n a saturated sol u t i o n of uranyl acetate i n 50% methanol (v/v) for 1 hour followed by 30 minutes i n lead c i t r a t e (Reynolds, 1963). The specimens were examined i n H i t a c h i HS 7S, AEI 801 A and P h i l l i p s 200 transmission electron microscopes, a l l operated with an accelerating voltage of 50 KV. Vegetative and reproductive c e l l s of H. mivabilis were subjected to enzyme digestion and negative s t a i n i n g i n an attempt to asc e r t a i n the nature and structure of unusual s p h e r i c a l i n c l u s i o n s present i n a l l of the c e l l s - ( s e e page 132). The enzymes employed i n t h i s procedure included deoxyribonuclease (lx c r y s t a l l i z e d , obtained from N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio), and ribonuclease-A (bovine pancreas, No. R-4875, 5x c r y s t a l l i z e d , type IA, obtained from Sigma Chemical Company, St. Louis, Mo.). Freshly c o l l e c t e d material was f i x e d f o r 2 hours i n 5% (v/v) glutaraldehyde i n 0.07 M phosphate buffer at pH 7.3 at 5 C. I t was rinsed i n buffer and digested at 25 C and 40'C i n either deoxyribonuclease ,[0.4 or 0.2 mg/ml deoxyribonuclease i n 0.07 M phosphate buffer and 0.003 M magnesium s u l f a t e (Ris and Plaut, 1962) at pH 6.8 and 7.4] for 14 hours, or ribonuclease (100 mg/ml ribonuclease i n d i s t i l l e d water at pH 6.8 and 7.4, adjusted with N sodium hydroxide) f o r 2 hours. P r i o r to use, the ribonuclease was heated to 80°C for 10 minutes i n a water bath to in a c t i v a t e any contaminant deoxyribonuclease (Bisalputra and Bisa l p u t r a , 1969). Controls were incubated under p a r a l l e l conditions i n solutions lacking the enzymes. Following enzyme digestion, the material was post-fixed i n 1% buffered osmium tetroxide (v/v) f or 2% hours, washed, dehydrated and embedded i n Spurr's medium as previously described. Negatively-stained dip preparations were made from f r e s h l y cut pieces of a c t i v e l y growing H. mivabilis, mature reproductive pustules and infected and uninfected host t i s s u e . The cut material was dipped i n drops of 1% (w/v) potassium phosphotungstate (pH 6.5) (Ie, 1972) or i n a 2% (w/v) aqueous uranyl acetate s o l u t i o n (Dawes, 1971). A l t e r n a t e l y , materials were ground by mortar and pestle i n 1% (w/v) potassium phosphotungstate (pH 6.5) and dropped onto parlodion and carbon coated g r i d s . Scanning electron microscopy (SEM) of Havveyella was undertaken i n a study of spore release and germination. Carposporic pustules and f i e l d -collected' and cultured 0. floecosa containing germinating Havveyella carpo-spores and tetraspores were prepared f o r SEM by the following procedure: material was f i x e d on i c e for 2 hours i n a so l u t i o n of 5% "(v/v) glutaraldehyde i n 0.07 M phosphate buffer at pH 7.3. The samples were washed i n buffer and post-fixed at 4 C for 30 minutes i n 1% osmium tetroxide (v/v) i n pH 7.3 phos-14 phate buffer (0.07 M) . F i n a l washing i n buffer was followed by three short washes i n d i s t i l l e d water to remove a l l s a l t s . The samples were dehydrated i n a graded ethanol s e r i e s followed by gradual replacement of the 100% ethanol with amyl acetate. Samples were dried by the c r i t i c a l point method using carbon dioxide (Cohen, Marlow and Garner, 1968; Wickham and Worthen, 1973). The specimens were mounted on stubs with c o l l o i d a l s i l v e r , coated with gold i n a rotary evaporator and examined with a Cambridge Stereoscan Electron Microscope (Mark II-A). C. P h y s i o l o g i c a l Studies. 1. Histochemical I d e n t i f i c a t i o n of Compounds Thick sections of H. mirabil-is on. 0. flocoosa and 0. washingtorviensis were stained for proteins,'polysaccharides, l i p i d s and for the metachromasia of various compounds according to the procedures o u t l i n e d , i n Table I. Whenever possible, consecutive thick, and t h i n sections were cut and examined with l i g h t and electron microscopes r e s p e c t i v e l y . In t h i s way, c e l l u l a r components reacting to l i g h t microscopic stains would be i d e n t i f i e d u l t r a s t r u c t u r a l l y ' . M a t e r i a l was f i x e d according to the. transmission electron microscopy f i x a t i o n procedures outlined i n section. B2. In addition, f i x a t i o n procedures excluding the use of osmium tetroxide were used (Feder and O'Brien, 1968). Osmium tetroxide i s known to i n t e r f e r e with the protein s t a i n i n g r e a c t i o n i n v o l v i n g mercuric bromphenol blue (Mazia, Brewer and A l f e r t , 1953). Thus material to be stained by t h i s method was f i x e d according to the following procedure: f r e s h l y c o l l e c t e d specimens were f i x e d i n 10% a c r o l e i n or 5% glutaraldehyde i n unbuffered seawater for 24 hours at 0 C. Half the material from each f i x a t i o n was dehydrated i n ethanol and embedded i n Spurr's medium as previously.describ-ed, while the remaining was dehydrated at 0 C with 2-methoxyethanol and t r a n s f e r r 15 Staining reaction (Source of stain) TABLE I Histochemical tests. Compounds id e n t i f i e d Reaction colour References Fast Green FCF (Al l i e d Chemical) Brom Phenol Blue (All i e d Chemical) Acid Fuchsin (Baker Chemical) Periodic Acid Schiff's (Basic Fuchsin from G. T. Gurr, Ltd.) Alcian Blue 8GN (Mathieson, Coleman & B e l l Co., Inc.) Alcian Yellow (Microme No. 836) (ESBE Lab. Supplies) Sudan Black B (Al l i e d Chemical) Nile Blue A (NA 0686) (Allied Chemical) Luxol's Fast Blue G (Solvent Blue 34) (Mathieson, Coleman & B e l l Co., Inc.) Toluidine Blue 0 (Mathieson, Coleman & B e l l Co., Inc.) proteins proteins proteins proteins sulfated polysaccharides carboxylated polysaccharides l i p i d s , l i p o -proteins fatty acids neutral l i p i d s phospholipids tnetachromasia: high M.W. com-pounds with free carboxyl, sulfate, phosphate groups, i . e . : sulfated polysaccharides, polyuronic acids, polyphosphates bright green blue . red magenta, pink blue-green yellow (orange) l i p i d s : blue lipoproteins: blueblack oxazine component: fatty acids -bright blue. Oxazone component: neutral l i p i d s - red blue-gray (iridescent) 3-metachromasia: (monomeric): blue or blue-purple, y-metachromasia: red or shades of green Ruthmann, 1970. Jensen, 1962. Ruthmann, 1970. Mazia, Brewer and A l f e r t , 1953. Robinow and Marak, 1966. Jensen, 1962. McCully, 1966. Hawker, Abbott and Gooday, 1968. Feder and O'Brien, 1968. Parker and D i b o l l , 1966 Parker and D i b o l l , 1966 Ruthmann, 1970. Ruthmann, 1970 Pearse, 1960. Sorokin, 1967. Tainter, 1971. Pearse, 1960 Feder and O'Brien, 1968. McCully, 1966. Hoefert, 1969. Chamberlain and Evans, 1973. 16 successively to ethanol, n-propanol and n-butanol. Control material used f o r the i d e n t i f i c a t i o n of l i p i d s was a d d i t i o n a l l y subjected to a graded s e r i e s of acetone. The material was embedded i n a g l y c o l methacrylate/polyethylene glycol/"monomer mixture" (Feder and O'Brien, 1968). Sections 1-1.5 ym thick from both Spurr and methacrylate embedded s p e c i -mens were cut with glass knives on a Reichert OM U3 ultramicrotome and mounted on acid-cleaned s l i d e s i n deionized water. Sections to be stained with t o l u i d i n e blue 0 were mounted i n an aqueous so l u t i o n of 0.002 M Ca(H 2P0^) 2.' H 20 to s t a b i l i z e metachromasia (McCully, 1966). The s l i d e s were placed on a s l i d e warmer at 70°C for 24 hours. 15-20 ym control sections of fresh and alcohol or acetone/acetic acid (3:1 v/v) f i x e d material were cut on a freezing microtome and stained f o r comparison with f i x e d and embedded sections. 2. Translocation Studies: S c i n t i l l a t i o n Analysis Approximately 1300 plants of H. mivabilis on 0. floocosa were c o l l e c t e d at Botany Beach near Port Renfrew, Vancouver Island, B. C. (48°33'N 124°26'W). C o l l e c t i o n s were made from mid-February to early A p r i l in- order to obtain plants e s s e n t i a l l y free of epiphytes and endophytes. The plants were trans-ported on i c e to the laboratory where they were cleaned as usual, and excised from the bulk of the host t h a l l u s . The pustules with 4-6 cm of subtending host t i s s u e were placed i n 12 1 tanks containing vigorously aerated m i l l i p o r e -f i l t e r e d seawater. They were kept i n l i g h t (Sylvania Cool White at 1000 lux) or under dark conditions f o r 24 hours at 7'"*C (pretreatment). Following t h i s period, 1 cm segments of host t i s s u e bearing one or more H. mivabilis pustules were excised from the t h a l l u s pieces; 25 of these segments (1 set) were 17 placed i n each, of 41 disposable p e t r i plates (30 ml capacity). In addition, 8 sets co n s i s t i n g of H. mirabilis without subtending host t i s s u e and uninfec t -ed (3. flocoosa were established as controls. A l l plants were covered with 10 ml s t e r i l i z e d seawater containing sodium bicarbonate (0.002 M), sodium chloride (0.034 M) and Naft^CO [20 uCi, s p e c i f i c a c t i v i t y : 59.17 mCi/mmol (694 yCi/mg), obtained from Amersham/Searle Corp., A r l i n g t o n Heights, I l l i n o i s ] . Samples 1-34, 42, 43, 46 and 47 (Table II) were incubated at 7 C under cool white fluorescent l i g h t s (4000 lux) while samples 35-41, 44, 45, 48 and 49 (dark control samples) were placed i n t o t a l darkness at 7 C. Afte r 90 minutes incubation i n the labeled bicarbonate/seawater, each set of plants was quickly washed 3 times i n m i l l i p o r e - f i l t e r e d , unlabeled seawater to remove a l l exogenous l a b e l . A l l 0 hours t r a n s l o c a t i o n sets (1, 2, 35, 36, 42-49; Table II) were removed from the medium, blo t t e d , and placed i n l i q u i d nitrogen. The remaining sets were incubated under continuous l i g h t i n unlabeled sodium bicarbonate (0.004 M) i n seawater for s p e c i f i e d t r a n s l o c a t i o n periods. Sub-sequently, these were immersed i n l i q u i d nitrogen. The frozen plants from sets 1-41 were dissected i n an attempt to separate H. mirabilis and 0. flocoosa from one another. This resulted i n two subsets from each sample ( i . e . from sample set 1, subset 1-H: separated H. mirabilis and subset 1£0: separated 0. fZoooosa). Each subset was further divided to give 2-3 approximate 200 mg f r a c t i o n s . {This l a s t d i v i s i o n was necessary since a maximum of only 250 mg tiss u e could be incorporated into the quantity of s c i n t i l l a t i o n f l u o r employed; t h i s produced m u l t i p l e samples which were summed together upon determination of r a d i o a c t i v i t y (dpm).J A l l subset f r a c t i o n s and samples 42-49 were weighed and 5 ml.70% ethanol was added before capping. A f t e r bleaching i n bright sunlight f o r several weeks, they were 18 Table II Translocation Samples Sample 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 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Translocation period (hours) 0 0 1 2 2 4 4 6 6 8 8 10 10 12 12 14 14 16 16 18 18 22 22 24 24 28 28 30 30 34 34 36 36 0 0 4 8 16 24 36 0 Harveyella only 0 Harveyella only 0 Harveyella only 0 Harveyella only 0 Odonthalia only 0 Odonthalia only 0 Odonthalia only 0 Odonthalia only 0 Odonthalia only Pretreatment Light Dark X X X X X X Incubation condition Light x x x x x x x x x x x X X -X X X X X X X X X X X X X X X Dark Light Light Dark Dark Light Light Dark Dark 19 ground with- a hand homogenizer. The alcohol was evaporated so a l l v i a l s contained approximately 0.5 ml t i s s u e s l u r r y . Protosol Tissue S o l u b i l i z e r (New England Nuclear, Boston, Mass.) was added to each v i a l (2.5 ml/vial) and the material was digested for 72 hours at 55 C. Aft e r the samples were cooled to 25 C, 15 ml toluene-based s c i n t i l l a t i o n fluor.; (Appendix V,6) was added. Each sample (subset f r a c t i o n ) was counted 10 times on a Nuclear Chicago Unilux TM III L i q u i d S c i n t i l l a t i o n System and the mean counts per minute (cpm) were calculated. The e f f e c t s of chemical quench-ing and self-absorption on the counting e f f i c i e n c y were determined by the internal- s t a n d a r d i z a t i o n channels-ratio method (Herberg, 1965; Wang and W i l l i s , 1965). The r e s u l t i n g e f f i c i e n c y value was used to ca l c u l a t e the actual d i s -integrations per minute for each sample (dpm) according to the formula: S - BG d p m = cpm e £2B (1) where, S = sample counts per minute (mean of 10 values) cpm r r \ J^cpm = background r a d i a t i o n (counts per minute) E = counting e f f i c i e n c y (percentage) The 2-3 dpm values from each subset were summed to give a t o t a l dpm f o r each subset. This value was divided by the t o t a l subset wet weight (g)"/> to give a t o t a l dpm/gm subset value. 3. Translocation Studies': Light Microscopic Autoradiographic Analysis Freshly c o l l e c t e d mature and immature H. mirabilis pustules with subtend-ing 0. floocosa were aerated i n the dark for 24 hours as previously described (Part I I I B, page 8-9). These were then incubated under fluorescent l i g h t i n 20 NaH-^COs i - n seawater as described f o r s c i n t i l l a t i o n a n a l y s i s . The plants were washed and placed i n unlabeled bicarbonate i n seawater f or 0, 8, 16 and 24 hour translocation periods. Control plants were incubated i n the dark i n NaH-^COs, washed, and allowed to translocate the l a b e l i n unlabeled medium under dark conditions. A d d i t i o n a l controls consisted of i s o l a t e d H. mirabilis and uninfected 0. ftoooosa. These were incubated separately i n labeled bicarbonate i n seawater under dark and l i g h t conditions. Following transloca-t i o n or immediately a f t e r l a b e l i n g and washing, each Harveyella-Oddnthalia segment was f i x e d , dehydrated and embedded as previously described f o r trans-mission electron microscopy. A l t e r n a t e l y , Harveyella-Odonthalia segments were fi x e d f o r 5 hours i n a c r o l e i n and osmium tetroxide vapours and were prepared by c r i t i c a l point drying as described f o r SEM (page 13). The dried specimens were i n f i l t r a t e d with Spurr's medium over a 5 day period under vacuum. Ethanol and water soluble substances were l o c a l i z e d more e f f e c t i v e l y using t h i s second fixation-embedding technique. Transverse sections, 1 ym thick, were cut through adjoined Harveyella and 0. flocoosa tissues on a Reichert OM U3 ultramicrotome using glass knives. Three specimens from each t r a n s l o c a t i o n period were sectioned. A minimum of s i x s l i d e s (100 sections per s l i d e ) were prepared from every specimen. The sections were a f f i x e d to acid-cleaned glass s l i d e s . Slides were transferred to a 40 C warming table. The warmed s l i d e s were covered with melted (40 C) Kodak NTB-3 l i q u i d nuclear emulsion. A l l procedures inv o l v i n g the l i g h t s e n s i t i v e emulsion were c a r r i e d out under a s a f e l i g h t equipped with a Wratten #2 dark red f i l t e r . The s l i d e s were dried i n the dark f o r s i x hours i n covered paper boxes containing CaCl2 and then transferred to ligh t - p r o o f b a k e l i t e boxes. Exposure proceeded over f i v e days at 4 C. The exposed emulsion was subsequently develop-2 1 ed i n Kodak D-19 developer, f i x e d , and washed according to Jensen (1962). The emulsion was cleared i n 45% ethanol a f t e r washing i n water (Ruthmann, 1970). A f t e r a i r drying, the s l i d e s were mounted with immersion o i l ( r e f r a c -t i v e index 1.515). Photographs were taken on Kodak Panatomic X or High Contrast Copy f i l m using a 40x objective on a Wild M 20 microscope, equipped with phase o p t i c s . A quantitative estimate of the amount of l a b e l incorporated into d i f f e r -ent t i s s u e areas of H. mivabilis and 0. floocosa was obtained by counting s i l v e r grains over randomly selected s l i d e areas. For every t r a n s l o c a t i o n period, 10 f i e l d s were counted from each of four s l i d e s . A L e i t z lOx counting ocular with a 1000 ym2 counting area (when used with a 40x objective) was employed. In each t r a n s l o c a t i o n period, the counts from the 10 f i e l d s were averagedj the background counts subtracted, and an average from the four s l i d e s was calculated. For every value, the mean, variance and standard deviation were calculated using a Fortran SAS1 programme. 4. Separation and Analysis of Labeled Compounds a. Labeling and Translocation Labeling i n NaH^COs i n seawater proceeded under conditions previously outlined i n III B except each sample consisted of 100 H. mivabilis pustules with subtending 0. floocosa i n 40 ml l a b e l i n g medium (80 y C i ^ C ) . A f t e r l a b e l i n g and washing, t r a n s l o c a t i o n proceeded i n the l i g h t f o r 0, 4, 8, and 24 hours and i n the dark f o r 0 and 8 hours (dark c o n t r o l s ) . A d d i t i o n a l controls, c o n s i s t i n g of only H. mivabilis or uninfected 0. floocosa, were incubated under both l i g h t and dark conditions f o r 0 and 8 hours a f t e r l a b e l i n g . A l l plants were k i l l e d following translocation by immersing i n l i q u i d nitrogen. H. miva-22 hiZis was dissected from 0. fZoooosa and each sample, c o n s i s t i n g of 1-1.5 g tis s u e , was weighed i n a previously weighed s c i n t i l l a t i o n v i a l . b. E xtraction and Separation of Major Compounds A l l samples were ground with a hand t i s s u e homogenizer and extracted three times with b o i l i n g 80% ethanol (Craigie, McLachlan and Tocher, 1968). Following each extraction the sample was centrifuged and the combined super-natants evaporated to dryness using a Buchler rotary f l a s h evaporator at 40 C. The residue was suspended i n d i s t i l l e d water and p a r t i t i o n e d between anhydrous eth y l ether and water. The ether layer was c o l l e c t e d and the volume reduced to 1 ml while the aqueous phase was evaporated to a syrup residue: As a r e -suspended aqueous s o l u t i o n , t h i s residue was fractionated and desalted by passing through a column of Rexyh r e s i n 101 (H) (35 ml bed volume, Fisher S c i e n t i f i c Co., F a i r Lawn, N. J.) and a column of Duolite r e s i n A-4 (OH) (42 ml bed volume, Diamond Chemicals, Redwood C i t y , C a l i f o r n i a ) (Whyte and Southcott, 1970). The neutral f r a c t i o n obtained was evaporated to dryness, resuspended i n 1 ml ethanol (50% v/v) and stored at -5 C. E l u t i o n of the Rexyn r e s i n with ammonium hydroxide (2 N) yie l d e d a f r a c -t i o n (cation f r a c t i o n ) containing p r i m a r i l y amino acids. The Duolite r e s i n was eluted with formic acid (0.2 N), y i e l d i n g a f r a c t i o n (anion f r a c t i o n ) containing organic acids. Both f r a c t i o n s were evaporated to dryness, resus-pended and stored as described f o r the neutral sugars. Floridean s t a r c h i n the al c o h o l i n s o l u b l e residue was hydrolyzed with 0.65 mg/ml a.4amyj.ase (hog pancreas, N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio) f o r 5 hours at 25 C (Evans, Callow and Callow, 1973). A f t e r c e n t r i f u g a t i o n , the supernatant (containing the starch hydrolysis products) 23 was stored at -5 C. The remaining residue was hydrolyzed with N s u l f u r i c a c i d f o r 15 hours at 105 C (Majak, C r a i g i e and McLachlan, 1966). The product was n e u t r a l i z e d (Bray, 1960) with barium carbonate and stored at -5 C. c. R a d i o a c t i v i t y Analysis by L i q u i d S c i n t i l l a t i o n Spectrometry The 100 y l aliquots of the cation, anion and n e u t r a l f r a c t i o n s and 500 y l digested f l o r i d e a n starch, hydrolyzed residue, and ether extract were placed i n s c i n t i l l a t i o n v i a l s containing 12 ml Aquasol S c i n t i l l a t i o n f l u o r (New England Nuclear). T r i p l i c a t e samples were prepared from each f r a c t i o n and the a c t i v i t y (cpm) determined using a l i q u i d s c i n t i l l a t i o n spectrometer (Nuclear Chicago Unilux TM III) as previously described (Section I I I B, page The d i s i n t e g r a t i o n s per minute (dpm) were calculated per gram sample by incorporating the d i l u t i o n f a c t o r and sample wet weight (gp)a) i n t o formula 1 (page where d = d i l u t i o n factor W = sample wet.weight (grams) d. Radiochromatography and Autoradiography of Neutral Fractions ^ C - l a b e l e d neutral f r a c t i o n s from a l l samples were separated by descend-ing chromatography on Whatman No. 3 paper i n the following solvent systems (Whyte and Southcott, 1970): A c i d i c solvent system: et h y l acetate: a c e t i c a c i d : formic a c i d : water (18:3:1:4 v/v) Basic solvent system: e t h y l acetate: pyridine: water (10:4:3 v/v) 24 Detection of the compounds on paper chromatograms was accomplished using a l k a l i n e s i l v e r n i t r a t e (Trevelyan, Procter and Harrison, 1950). The R values (R = R . = distance substance t r a v e l s from the o r i g i n divided by g glucose the distance glucose t r a v e l s from the o r i g i n , value m u l t i p l i e d by 100; Smith, 1969) of the separated compounds were calculated and compared to R values of reference standards obtained for each solvent system (Appendix VI). e. Q u a n t i f i c a t i o n of R a d i o a c t i v i t y i n Separated Compounds Autoradiograms were prepared of each radiochromatogram using 14 x 17" sheets of Kodak X-ray f i l m (Blue Brand Medical, BB-14). The f i l m was exposed i n the dark at 25 C for one month. Following development of the radiographs, R values of a l l labeled spots were ca l c u l a t e d . The corresponding areas on each chromatogram were excised, placed i n s c i n t i l l a t i o n v i a l s to which was added 10 ml toluene-based s c i n t i l l a t i o n c o c k t a i l (Appendix V, 6) and counted 10 times on a Nuclear Chicago 720 Series L i q u i d S c i n t i l l a t i o n System (Wang and Jones, 1959; Davidson, 1962). The mean counts per minute for each v i a l was calculated and used to c a l c u l a t e d i s i n t e g r a t i o n s per minute according to formula 1 (page 19). The percentage of l a b e l i n each separated compound was calculated f o r each sample. 25 IV. REPRODUCTIVE AND DEVELOPMENTAL DEPENDENCY OF EARVEYELLA MIRABILIS ON A SPECIFIC HOST Introduction Parasitism i s a complex phenomenon and consequently d i f f i c u l t to define. One problem c l e a r l y apparent i n the red algae i s that there are varying degrees of parasitism between plants. Exactly when a plant stops being merely epiphytic on another and assumes a p a r a s i t i c nature i s often questionable. It i s thus necessary to e s t a b l i s h c h a r a c t e r i s t i c s of parasitism. One such c h a r a c t e r i s t i c i s that parasites are dependent upon the presence of a su i t a b l e host f o r the completion of t h e i r l i f e h i s t o r y (Walker, 1969). Few studies of parasitism i n the red algae have attempted to inve s t i g a t e t h i s aspect. In f a c t , r e l a t i v e l y l i t t l e information i s a v a i l a b l e on the i n f e c t i v e process and early development of p a r a s i t i c red algae. P r e c i s e l y how a red a l g a l p a r a s i t e penetrates i t s host i s an e s s e n t i a l question i n an i n v e s t i g a t i o n of the i n f e c t i o u s process and host s p e c i f i c i t y . The f i r s t observations of host penetration were made by Solms-Laubach i n 1877. He described the epidermal penetration of the host Lauveneia obtusa (Huds.) Lamour by the para s i t e Janozewskia verruoaeformia Solms. Falkenberg (1901) provided a d d i t i o n a l information on t h i s i n f e c t i v e process and applied the term "protonema" to the early developmental stage of the penetrating hyphal system. Feldmann and Feldmann (1958) provided a d e t a i l e d study of the penetration and subsequent development of J. verruoaeformis with i t s host L. obtusa. The adhesion of spores to the host and subsequent r h i z o i d formation and penetra-t i o n through i n t e r c o r t i c a l host c e l l walls were described and i l l u s t r a t e d . In t h e i r study of South A f r i c a n p a r a s i t i c Florideae, Martin and Pocock (1953) 26 described a s i m i l a r method of epidermal penetration of Stveblooladia tenuis-sima Pocock by germlings of Microeolax africana Martin and Pocock. Spores of the melobesioidean parasite Kvaleya epilaeva Adey and Speripani have been recently described to germinate and form a d i f f e r e n t i a t e d p e r i t h a l l i u m and hypothallium before producing u n i c e l l u l a r haustoria (Adey and Sperapani, 1971). These haustoria penetrate the host p e r i t h a l l i u m but undergo no further d i v i s i o n . In an early study of parasitism, Richards (1891) noted that Choveo-aolax polysiphoniae Reinsch was almost always found i n the branch a x i l s of i t s host, Polysiphonia fastigiata Grev. He also noted that there was often an accumulation of organic d e t r i t u s i n the branch a x i l s . Although he did not a c t u a l l y observe the process of spore germination, he reported that spores of Choveoaolax begin germinating i n the organic matter i n the branch a x i l s and subsequently penetrate between the loosely adjoined host c e l l s i n the a x i l s . In h i s study of Choveoaolax, Chemin (1923) also reported that germination occurs external to the host's cortex and that penetration was secondary. Kugrens and West (1973a) observed that the spores of Choveoaolax may e i t h e r germinate outside the host and subsequently penetrate the host cortex or, a l t e r n a t e l y , the spores enter through an i n j u r y , thus germinating within the ti s s u e s of Polysiphonia. Sturch (1899, 1924) reported that the spores of both H. mivabilis and Holmsella paohydevma (Reinsch) Sturch enter t h e i r hosts through an "aperture" before germinating. Few studies have dealt with the a b i l i t y of p a r a s i t i c f l o r i d e a n spores to germinate i n the absence of a s u i t a b l e host. Chemin (1927, 1937a) attempted to germinate carpospores and tetraspores of H. mivabilis on glass p l a t e s . He reported that, immediately upon attachment, spores produced phycoerythrin and divided to form a pigmented c i r c u l a r d i s c of c e l l s which he assumed would 27 subsequently penetrate the host, Rhodomela convervoides (Hudson) S i l v a [sub. R. subfusoa (Woodward) AgardbJ. Both Rosenvinge (1931) and F r i t s c h (1945) commented that t h i s observation was so unusual, that i t required confirmation. Rosenvinge (1931) attempted to repeat the work of Chemin but was unable to obtain sporulating plants. Feldmannaand Feldmann (1958) repeated Chemin's study using material obtained from Roscoff, France, the source of Chemin's plants. They obtained very d i f f e r e n t r e s u l t s and concluded that Chemin a c t u a l l y observed the development of two host encrusting c o r a l l i n e algae. I t was reported that the colourless spores of Harveyella did not germinate i n culture without the host, but simply degenerated upon consumption of th e i r reserve metabolites. Although unable to induce germination of H. mirabilis on glass, Feldmann and Feldmann (1958) were successful i n germinating spores of the p a r a s i t e Janozewskia verruoaeformis. Spores germinated on glass p l a t e s , and produced a m u l t i c e l l u l a r r h i z o i d during the f i r s t 5-6 days of development. In the absence of the host Laurenoia obtusa, the spores degenerated a f t e r 6 days. Similar spore germination r e s u l t s were obtained by Kugrens (1971) i n his study of J. gardneri S e t c h e l l and Guernsey. He reported that tetraspores germinated i n culture but died within two weeks. Kugrens reported successful germination of Erythrocystis sacoata (J. G. Agardh) S i l v a tetraspores i n culture but was unable to germinate spores of the p a r a s i t e Gonimophyllum skottsbergii S e t c h e l l . Sturch's s i g n i f i c a n t studies of H. mirabilis, H. paohyderma and C. poly-siphoniae (1899, 1924, 1926) represented the f i r s t attempt to elucidate the l i f e h i s t o r i e s of any p a r a s i t i c alga. Although s t r i c t l y d e s c r i p t i v e , these inv e s t i g a t i o n s were pioneering studies'; of the developmental dependency of parasites on hosts and on the e f f e c t s of changes i n environmental parameters 28 on the occurrence and reproduction of the p a r a s i t e s . Few other studies have dealt with the e f f e c t of host a v a i l a b i l i t y and environmental factors on the d i s t r i b u t i o n , development and reproduction of red a l g a l p a r a s i t e s . Newroth and Taylor (1968) traced the circumboreal d i s -t r i b u t i o n of Cevatocolax havtzii Rosenvinge i n r e l a t i o n s h i p to the d i s t r i b u -t i o n of i t s host Phylloiphova bvodiaei (Turn.) Endlich but provided l i t t l e a d d i t i o n a l information on the a s s o c i a t i o n . Recently Adey and Sperapani (1971) analyzed the d i s t r i b u t i o n of the parasite Kvaleya epilaeve i n r e l a t i o n to i t s host's d i s t r i b u t i o n and water temperature. In the present study, the reproductive biology of Havveyella mivabilis has been investigated by c o r r e l a t i n g culture studies with a 20-month f i e l d i n v e s t i g a t i o n . How the reproduction and development of Havveyella might be r e l a t e d to the presence of the host Odonthalia floooosa has been the primary question. An analysis has been made of environmental parameters such as water temperature, s a l i n i t y , daylength and invertebrate grazing to determine how these factors might a f f e c t the phenology of both the host and p a r a s i t e . 29 Results and Observations A. General D e s c r i p t i o n of Havveyella mivabilis. The red alga Havveyella mivabilis (Reinsch) Schmitz and Reinke (Choreo-colaceae, Cryptonemiales) has been found i n the northeast P a c i f i c growing on and within the tissues of Odonthalia floocosa, 0. washingtoniensis, and Rhodomela lavix "(Rhodomelaceae, Ceramiales). Although usually white i n colour (Figure 1), the erumpent t h a l l u s (pustule) may at times appear s l i g h t l y pink due to the presence of pigmented host c e l l s i n the p e r i p h e r a l c e l l layers of the pustule (Figures 2, 3). The t h a l l u s of H. mivabilis i s composed of three c e l l u l a r zones designat-ed zones I, I I , and I I I (Figure 4). The outer, p e r i p h e r a l c o r t i c a l layer (zone I ) , consists of uninucleate, densely s t a i n i n g , s p h e r i c a l or s l i g h t l y elongate c e l l s , approximately 10-12 ym i n diameter (Figures 2, 3, arrows). Although v a r i a b l e i n thickness, t h i s layer i s usually composed of 3-8 c e l l l a y e r s . A gradual increase i n c e l l s i z e and vacuolation occurs as zone I merges into zone I I , the medulla. In mature vegetative and reproductive pustules, the uninucleate c e l l s are elongate (30-70 x 15-30 ym) and are highly vacuolate (Figure 5). In zone I I I , extensive c e l l u l a r development i n the i n t e r c e l l u l a r host w a l l regions r e s u l t s i n the formation of r h i z o i d a l -l i k e filaments of uninucleate Havveyella c e l l s , termed r h i z o i d a l c e l l s . The ultimate and subultimate c e l l s of these penetrating filaments are elongate (50-80 x 7-10 ym) (Figure 6), with only a few small vacuoles, whereas the remaining c e l l s of the filament appear to d i f f e r e n t i a t e into vacuolated, starch-containing'cells (Figure 7). The filaments surround and i s o l a t e host c e l l s (Figure 8), and secondary p i t connections are formed between the i n t e r -30 Figures 1 - 3 Vegetative and reproductive pustules of Earveyella mirabilis Figure 1. L i v i n g m a t e r i a l ; mature male ( o* ) and female ( ? ) gameto-phytes on branch of 0. flocoosa; immature vegetative pustule (VP) i n host branch a x i l . Figure 2. X.S. through vegetative pustule; i s o l a t e d host medullary c e l l s (hmc) i n pustule, attached to adjacent E. mirabilis c e l l s , by p i t connection (arrow); c o r t i c a l c e l l s of E. mirabilis at pustule surface. Figure 3. X.S. vegetative pustule; i s o l a t e d host c o r t i c a l c e l l s (hcc) at surface. Note p e r i p h e r a l l y situated p l a s t i d s (P); two a p i c a l c e l l s ( c o r t i c a l ) of E. mirabilis indicated by a s t e r i s k . A l l measurements i n micrometers (urn) unless otherwise i n d i c a t e d . 3 1 *-Figure 4. C e l l u l a r zones i n the t h a l l u s of Earveyelta nrirabi-lis. Zone I, c o r t i c a l zone; Zone I I , medullary zone; Zone I I I , i n t e r d i g i t a t i o n zone. 31 3 2 ^ Figures 5 - 9 Medullary and r h i z o i d a l c e l l s of Havveyella mivabilis i n f e c t i n g 0. floocosa Figure 5. P l a s t i c embedded section; vacuolated (V) medullary c e l l of pustule with c e n t r a l nucleus (N), and a prominent nucleolus (arrow). Figure 6. L i v i n g m a t e r i a l ; ultimate (apical) c e l l of r h i z o i d a l filament penetrating between host medullary c e l l s . A primary p i t con-nection (1° PC) to the subultimate c e l l i s evident. Numerous small vacuoles (V) are present. Figure 7. P l a s t i c embedded section; r h i z o i d a l c e l l s i n the i n t e r d i g i t a -t i o n zone (zone I I I ) . Floridean starch (FS) i s present. Figure 8. Karpechenko-fixed m a t e r i a l ; i s o l a t e d host medullary c e l l s i n i n t e r d i g i t a t i o n zone surrounded by filaments of H. mivabilis. Figure 9. Karpechenko-fixed material; secondary p i t connection (2° PC) from H. mivabilis r h i z o i d a l c e l l to host medullary c e l l . A l l measurements i n micrometers (ym) 33 d i g i t a t i n g c e l l s of Havveyeila and those of the host (Figure 9). Pustules of Harveyella are most often encountered near the base of the host plant, within the branch a x i l s or along the main a x i s . They may o c c a s i o n a l l y be found on subultimate or ultimate vegetative and reproductive branchlets as w e l l as on mature cystocarps of the host. B. D i s t r i b u t i o n . The d i s t r i b u t i o n of H. mirabilis has been determined from herbarium and f i e l d c o l l e c t i o n s and l i t e r a t u r e records. Harveyella was f i r s t reported from Svin^or, Norway (Lyngbye, 1819) and has since been reported from numerous l o c a l i t i e s i n the North A t l a n t i c Ocean. It has been c o l l e c t e d i n t e r t i d a l l y and s u b t i d a l l y on a l i m i t e d number of host species. The A t l a n t i c hosts include Rhodomela aonvervoides (Hudson) S i l v a [Syn: R. subfusca (Woodward) Agardh], R. lycopodioides (L.) C. Agardh and R. virgata Kjellman, a l l members of the family Rhodomelaceae (Ceramiales)]. Carposporophyte development of Choreooolax odonthalia Levring on Odonthalia dentata (L.) Lyngbye has recently been noted to be of the Harveyella-ty-pe. rather than Choreooolax (Edelstein, personal communication; Goff, unpublished). If C. odonthalia i s found to be c o n s p e c i f i c with H. mirabilis, the l i s t of hosts employed by Harveyella i n the North A t l a n t i c could include 0. dentata. C o l l e c t i o n records of H. mira-bilis i n the North A t l a n t i c Ocean are l i s t e d i n Appendix I I , and the d i s t r i b u t i o n indicated i n Figure 10. In the P a c i f i c Ocean, Harveyella was f i r s t reported on Rhodomela lyaopodioides f. tenuissima from the Sea of Japan (Sinova, 1940) and from the Sea of Okhotsk (Sinova, 1954). I t was l a t e r reported on Odonthalia floocosa c o l l e c t e d from the San Juan Ar c h i p e l i g o (Scagel, 1961, 1963). It i s 34^ Figure 10. Reported distribution of Harveyella mi-rabilis in the North Atlantic. Overlays provide mean surface temperatures for December and August. 35 presently known to range i n t e r m i t t e n t l y from Harris Beach, Oregon (42°04'N) to Chatham Sound, B r i t i s h Columbia (54°13'N) on the rhodomelacean hosts 0. floocosa, 0. washingtoniensis and Rhodomela lavix. C o l l e c t i o n ; records for the northeast P a c i f i c are summarized i n Appendix I and the s p e c i f i c c o l l e c t i o n s i t e s noted i n Figures 11 and 12. H. mivabilis most commonly occurs i n the northeast P a c i f i c i n wave-sheltered coastal areas, although s u i t a b l e hosts may be found i n both sheltered and exposed areas. Along the Oregon coast, Havveyella has only been found on hosts situated i n protected coves or behind rock headwalls. One such area i s found at Simpson's reef at North Cove, Cape Arago (43°20'N, 124°23'W) (Figure 13). The rock reef (Figure 13, arrows) provides a n a t u r a l breakwater behind which H. mivabilis i s abundant on 0. washingtoniensis and l e s s commonly on 0. floocosa. The i n t e r t i d a l area west of Sooke Harbour i n l e t (Figures 12, i n s e t , 14) i s s i m i l a r i n wave-exposure to Simpson's reef. Again, Havveyella i s abundant i n t h i s region but i s more commonly found on 0. floo-cosa than on 0. washingtoniensis. C. Reproduction.and Development. I. General L i f e History Havveyella mivabilis has a t y p i c a l "Polysiphonia-type" l i f e h i s t o r y i n which there i s a sequence of gametangial, carposporangial, and tetrasporangial stages (Figure 15). Both the tetrasporangial and gametangial stages are morphologically s i m i l a r while the carposporophyte i s reduced i n s i z e and dependent upon the female gametophyte. C h a r a c t e r i s t i c f l o r i d e a n tetraspores, spermatia (male gametes) and carpogonia (female gametes) are borne i n the peripheral c e l l layers of separate pustules, whereas the carposporophytes are 36 * -Figure 11. C o l l e c t i o n s i t e s of Havveyella mivabilis i n Oregon. B o i l e r Bay 25 D e v i l ' s Punch Bowl 26 Yaquina Head 27 Seal Rock 28 Cape Perpetua. 29 Neptune Beach State Park...... 30 Strawberry H i l l (south beach) 31 Hecata Head 32 Sunset Beach (Cape Arago) 33 North Cove (Cape Arago) 34 South Cove (Cape Arago) 35 P i s t o l River 36 H a r r i s Beach State Park 37 36 37 tt Figure 12. C o l l e c t i o n s i t e s of.. Harveyella mirabilis. i n B r i t i s h Columbia and Washington. Chatham Sound 1 Westerman Bay 2 Mignot Point (Belize I n l e t ) 3 Dalkeith Point 4 Fox Island Channel 5 Burgess Island 6 Brandon Point (Robinson Is.) 7 Duncan Bay, Discovery Passage. 8 Quadra Island 9 Cape Lazo 10 Inside Passage (50°03'N 125°2'W) 11 Point Atkinson. 12 Botany Beach (North) 13 Botany Beach (South).... 14 Gl a c i e r Point 15 Otter Point 16 Parsons Point 17 Waadah Island 18 Deadman Bay (San Juan Is.) 19 Mt. Dal l a s Beach (San Juan I s . ) . . 20 American Camp (San Juan Is.) 21 Ca t t l e Point (San Juan Is.) 22 Goose Island 23 Rosario Beach State Park 24 Map Inset: Study areas near Sooke, Vancouver Island, B. C. 38 et Figures 13 - 14 Study areas Figure 13. Simpson's Reef, North Cove, Cape Arago, Oregon. E. mivabi-'iis-i n f e c t e d 0. washingtoniensis and 0. flocoosa are abundant i n sheltered areas behind rock headwall (arrows). Figure 14. Parsons Point, west of W h i f f i n Spit at the entrance of Sooke Harbour, Vancouver Island, B. C. Locations of study popula-tions 1 and 2 are i n d i c a t e d by arrows. 38 Figure 15. "Polysiphonia-tjpe" l i f e h i s t o r y . 39 'Polysiphonia-Type"Life History TETRASPORE re/wale ga/wetophyte N T CARPOGONIUM (9 gamete) retained zygote Ml <N)<> SPERMATIUM (<fgamete) TETRASPORE a* /wale ga/wetophyte N SPERM A TANG IUM l dependent opljyte \fgametaphyte CARPOSPORE M! tetraspor-ophyte 2N Rl> Meiosit Ml t Mitosit TETRASPORANGIUM Ri 4 TETRASPORES genotypic sex-determination 40 deeply embedded i n the female gametophyte. 2. Reproductive Structures a. Tetraspores As described by Sturch (1899, 1924), tetrasporangia are cut o f f obliquely from terminal or subterminal c e l l s (Figures 16, 17). The tetrasporangium undergoes reductional d i v i s i o n producing four c r u c i a t e l y or t e t r a h e d r a l l y arranged uninucleate tetraspores (Figures 16, 17). The colourless tetraspores range from 13-18 um i n length and are surrounded by a thick tetrasporangial w a l l . The walls of immature sporangial walls contain sulfated polysaccharides as indicated with t o l u i d i n e blue although t h i s compound decreases i n maturing sporangia (Figure 16). The presence of protein or glycoprotein i n the t e t r a -sporangial c e l l w all i s indicated by a p o s i t i v e reaction with mercuric bromphenol blue, f a s t green and ac i d fuchsin. These findings are i n accord-ance with those of Chamberlain and Evans (1973) i n t h e i r study of Cevamium spores. Mature tetraspores contain an abundance of storage carbohydrates which s t a i n with p e r i o d i c a c i d S c h i f f ' s reagent (Figure 18). U l t r a s t r u c t u r a l examination of mature tetraspores reveal these carbohydrates to be f l o r i d e a n starch and dark-cored fibrous v e s i c l e s s i m i l a r to those described i n the tetraspores and carpospores of other f l o r i d e a n algae (Figure 19) (Peyriere, 1972; Kugrens and West, 1972a,1973b;Scott and Dixon, 1973a; Chamberlain and Evans, 1973; Hawkins, 1974). In addition, mature tetraspores contain a r e l a t i v e l y large quantity of l i p i d s i n the cytoplasm and vacuoles as indicated with Sudan black B (Figure 20). b. Spermatangia and Spermatia Spermatangia are borne on spermatangial mother c e l l s that form the e n t i r e peripheral c e l l layer of the smooth hemispherical male gametophyte (Figure 21). 41 \ Figures 16 - 20 Tetraspore production Figures 16-18, 20 - Li g h t microscopy, p l a s t i c embedded thick sections. Figure 16. Tetraspores, p e r i p h e r a l l y situated i n pustule; stained with t o l u i d i n e blue. Sulfated polysaccharides present i n immature tetrasporangial w a l l (itw) but absent from mature tetraspor-angium w a l l (mtw). Figure 17. Tetrasporangium cut o f f terminally and obliquely from t e t r a -spore mother c e l l . Connections between tetraspores i n d i c a t e d by arrows. (Phase contrast) Figure"18. Abundance of small f l o r i d e a n starch granules (FS) (arrows) i n mature tetraspores. Stained with p e r i o d i c a c i d S c h i f f ' s reagent (PAS). Figure 19. El e c t r o n micrograph of mature tetrasporangium i n cross-sec-t i o n . Floridean starch (FS), fibrous v e s i c l e s (FV), and nucleus (N) are i n d i c a t e d . Figure 20. L i p i d s i n mature tetraspores; stained with Sudan black B. A l l measurements i n micrometers (urn) 41 42 * Figures 21 - 25 Spermatangia and spermatia Figure 21. Karpechenko-fixed material; medial X.S. of mature male pustule showing subtending host c e l l s (H), and the i n t e r -d i g i t a t i o n (IZ), medullary (Me) and c o r t i c a l (Co) zones of Harveyella with p e r i p h e r a l l y situated spermatangia and spermatia ( S t ) . Figure 22. Plastic-embedded t h i c k section; spermatangial mother c e l l s (SMC) d i v i d i n g o b l i q u e l y or transversely to form chains of spermatangia ( S t ) . Basal spermatangial v e s i c l e s (StV) prominent. Figure 23. Rupturing of spermatangial v e s i c l e s i n r e l e a s i n g Harveyella spermatia; mitochondria (M) are i n d i c a t e d . Figure 24. Released spermatia (S) (arrows), caught i n mucilaginous covering of male pustule (Mu). Figure 25. Releasing spermatium; nuclear envelope i n t a c t ; f l o r i d e a n starch (FS), fi b r o u s v e s i c l e s (FV) and lamellar bodies (LB) are present. A l l measurements i n micrometers (ym) Spermatangial mother c e l l s undergo s e r i e s of transverse or oblique d i v i s i o n s to give r i s e to either terminally or subterminally borne chains of spermatan-gia (Figure 22). Mature spermatangia are elongate, ranging i n length from 5-6 urn (Figure 22, arrows). Prominent basal spermatangial v e s i c l e s rupture as the spermatia are released from the spermatangial w a l l (Figure 23). A s i m i l a r rupturing of spermatangial v e s i c l e s has been noted i n Levringietta and Evythrooystis (Kugrens and West, 1972b), Ftilota (Scott and Dixon, 1973b) and Janozewskia (Kugrens, 1974). The released spermatia appear to be caught i n the thick mucilaginous covering of the pustule (Figure 24) and are shed as a mass. Electron microscopic f i x a t i o n of spermatia has proved d i f f i c u l t , but enough c e l l u l a r d e t a i l has been preserved to show that the spermatium of HavveyeZZa i s u l t r a s t r u c t u r a l l y s i m i l a r to spermatia described i n other f l o r i d e a n algae (Figure 25) (Peyriere, 1971, 1974; Simon-Bichard-Breaud,1971, 1972a, 1972b; Kugrens and West, 1972b; Scott and Dixon, 1973b). c. Carposporophytes, Carpospores and Procarps Mature carposporophyte-bearing pustules are r e a d i l y recognized macro-s c o p i c a l l y by t h e i r multilobed appearance (Figure 26). A mature gonimoblast and carpospores are contained i n each lobe. Large qu a n t i t i e s of f l o r i d e a n starch are present i n gametophyte medullary c e l l s near the carposporophyte (Figure 27). When contained i n the cystocarp, the carpospores are elongate (Figure 27) but assume a more s p h e r i c a l shape upon release (Figure 28). Carpospores are u l t r a s t r u c t u r a l l y i d e n t i c a l to tetraspores as reported i n the alga Cevamivm (Chamberlain and Evans, 1973). The massive quantity of storage granules i n both carpospores and tetraspores i s responsible for the highly r e f r a c t i v e and granular appearance of the spore cytoplasm (Figure 29). No spore pigmentation could be detected with- either normal bright f i e l d or blue l i g h t fluorescent microscopy. 44 <* Figures 26 - 29 Carposporophytes, carpospores Figure 26. L i v i n g m a t e r i a l ; mature carposporophyte-bearing pustule. Each lobe contains a mature gonimoblast and carpospores. Figure 27. X.S. of pustule lobe. Gonimoblast (G) with terminal elongate carpospores (c) (arrows). Large q u a n t i t i e s of f l o r i d e a n starch (FS) occur i n vegetative c e l l s subtending the gonimo-b l a s t . Figure 28. Releasing carpospores (c) from sectioned carposporophyte-bearing pustule. Carpospores assume s p h e r i c a l dimensions upon release. (Water-immersion objective) Figure 29. Carpospores attached to glass pl a t e s i n c u l t u r e . Granular, r e f r a c t i v e appearance of spore due to large q u a n t i t i e s of storage granules. (Water-immersion objective) A l l measurements i n micrometers (ym) unless otherwise i n d i c a t e d . 45 Carposporophyte development i n Harveyella has been examined i n d e t a i l . The procarp i s formed by d i v i s i o n of a dense peripheral c e l l i n the female gametophytic pustule. Four small c e l l s are cut o f f successively from the base of t h i s dense c e l l (Figures 30-33). The o r i g i n a l c e l l d i f f e r e n t i a t e s into a carpogonium and trichogyne and the four c e l l s form the carpogonial branch and the basal supportive ( a u x i l i a r y ) c e l l . Thus, the mature procarp consists of a basal supportive c e l l (which w i l l act as an a u x i l i a r y c e l l ) , bearing two s t e r i l e branches (sb^, sh^) and a carpogonial branch (cb) (Figures 34, 38a). The carpogonial branch consists of the carpogonium and three subtending chain c e l l s . The trichogyne ( t ) , which extends from the carpogonium, elongates and pierces the external w a l l layer of the pustule (Figure 35). Occasionally, the trichogyne appears unable to break through the " c u t i c l e " and instead, bends and ramifies under the outer wall layer (Figure 36). The trichogyne may also form broad "pads" on the pustule surface (Figure 37). Spermatia can occasionally be observed on or near these "pads" and on the protruding trichogyne (Figure 40). If i t i s not f e r t i l i z e d , the protruding trichogyne continues to elongate and f i n a l l y withers (Figure 34). Following syngamy, the d i p l o i d nucleus passes to the carpogonium and the trichogyne withers or r e t r a c t s , often forming d i s t a l trichogyne knobs (Figure 41). The zygote nucleus within the carpogonium divides m i t o t i c a l l y . One of the n u c l e i i s then transferred to the a u x i l i a r y c e l l (supportive c e l l ) by a 1 short connecting filament which- protrudes from the carpogonial base (cp) (Figures 42, arrow, 38b). The d i p l o i d i z e d a u x i l i a r y c e l l immediately under-goes a transverse d i v i s i o n to form a lower foot c e l l (fc) (with the attached s t e r i l e branches) and an upper c e n t r a l c e l l (cc) (Figures 43, 38c). The foot 46 * Figures 30 - 37 Carposporophyte development Figures 30-52 - Fixed with Karpechenko's f i x a t i v e , stained with a n i l i n e blue. Figure 30. X.S. of female gametophyte; p e r i p h e r a l area of pustule. D i v i s i o n of the densely stained a p i c a l c e l l ( a s t e r i s k 0 ) w i l l give r i s e to the procarp. Figure 31. F i r s t d i v i s i o n of a p i c a l c e l l ( a s t e r i s k 1 ) . Figure 32. Second d i v i s i o n of a p i c a l c e l l ( a s t e r i s k 2 ) . Figure 33. A f t e r the f o u r t h d i v i s i o n of the a p i c a l c e l l and the f i r s t d i v i s i o n of the supporting ( a u x i l i a r y c e l l , au) a four-c e l l e d carpogonial branch (cb) , a u x i l i a r y c e l l and f i r s t s t e r i l e c e l l (sc) (cut o f f from a u x i l i a r y c e l l ) i s recognized. Figure 34. Mature procarp with elongate a u x i l i a r y c e l l , two s t e r i l e branches (sbj, sb2), carpogonial branch with trichogyne (t) extending through pustule surface. Figure 35. Trichogyne p i e r c i n g through outer w a l l (OW) of pustule. Figure 36. Trichogyne ramifying under outer w a l l surface of pustule. Figure 37. Trichogyne forming broad "pad" (tp) at pustule surface. A l l measurements i n micrometers (urn) 4 7 " Figure 38. A-D.. P o s t - f e r t i l i z a t i o n events. Figure 38 A. P r e - f e r t i l i z a t i o n . Mature procarp with carpogonial branch (cp), carpogonium with extended trichogyne ( t ) , a u x i l i a r y c e l l (au) (supporting c e l l ) , and two s t e r i l e branches ( s b i , sb2) o r i g i n a t i n g from the a u x i l i a r y c e l l . Figure 38 B. P o s t - f e r t i l i z a t i o n . Formation of connecting filament (cf) from carpogonium base to a u x i l i a r y c e l l ; formation of knob at end of r e t r a c t i n g trichogyne. Figure 38 C. D i v i s i o n of a u x i l i a r y c e l l to form c e n t r a l c e l l (cc) and foot c e l l (fc) with attached s t e r i l e branch c e l l s . Figure 38 D. D i v i s i o n of c e n t r a l c e l l g i v i n g r i s e to two gonimoblast i n i t i a l s ( g i 1 , g i 2 ) . Foot c e l l f using with s t e r i l e branch c e l l s ; remnants of carpogonial branch adjacent. Elonga-t i o n of haploid vegetative c e l l s to form columnar c e l l s (co). 48 A Figure 3 9 . Mature carposporophyte; each gonimoblast i n i t i a l c e l l has given rise to horizontal gonimoblast filaments (hgf) from which form erect gonimoblast filaments (egf). Carpospores (c) are produced terminally on the erect gonimoblast f i l a -ments. The vegetative columnar cells (col. c) have elongat-ed, pushing the vegetative cortical cells far above the developing carposporophyte. 48 4 9«i Figures 40 - 47 Carposporophyte development Figure 40. Protruding trichogyne (t) and spermatia (S) caught in sur-rounding mucilage. Figure 41. Post-fertilization; retraction of trichogyne (t) and charac-t e r i s t i c formation of a terminal trichogyne - knob indicates f e r t i l i z a t i o n has occurred. Note ruptured segment of the external wall (asterisk). Figure 42. Formation of connecting filament (cf) from carpogonium base (cp) to auxiliary (supporting) c e l l (au). Figure 43. Division of diploidized auxiliary c e l l to form dome-shaped central c e l l (cc) and foot c e l l (fc). Knob - trichogyne and carpogonial branch s t i l l evident. Figure 44. Fusion of st e r i l e branch cells (sb) with foot c e l l (fc) sub-tending central c e l l (cc). Figure 45. Division of central c e l l to form two gonimoblast i n i t i a l c e l ls ( g i 1 , g i 2 ) . Each w i l l give rise to horizontal gonimo-blast filament. Figure 46. Division of each gonimoblast i n i t i a l to form horizontal gonimoblast ce l l s (asterisk). Figure 47. Elongation of horizontal gonimoblast filaments (hgf). Erect gonimoblast filaments (egf) proliferate from the horizontal filaments. A l l measurements in micrometers (ym) 50 c e l l fuses with the s t e r i l e branch c e l l s but undergoes no further development. The c e n t r a l c e l l i s c h a r a c t e r i s t i c a l l y dome-shaped and embedded 4-5 c e l l layers below the pustule surface (Figures 43, 44). Transverse d i v i s i o n of 1 2 the c e n t r a l c e l l forms two gonimoblast i n i t i a l c e l l s (gi , g i ) (Figures 45, 38d) from which the gonimoblast a r i s e s . Each gonimoblast i n i t i a l produces compact chains of small dense c e l l s (Figure 46) which elongate h o r i z o n t a l l y to form h o r i z o n t a l gonimoblast filaments (hgf) (Figures 47, arrows, 38e). Tufts of erect gonimoblast filaments (egf) a r i s e from h o r i z o n t a l gonimoblast filaments (Figures 48, 39). Carpospores are borne terminally on the erect filaments (Figure 49). Occasionally, the remains of the fused foot c e l l and s t e r i l e branches may be observed subtending the mature gonimoblast (Figure 50). Concomitant with gonimoblast development, medullary c e l l s of the female gametophyte near the'gonimoblast i n i t i a l r a p i d l y elongate to form long columnar c e l l s ( c o l . c) which subtend 4-6 c o r t i c a l c e l l l a y e r s . Columnar c e l l s are also formed from medullary c e l l s removed from the gonimoblast i n i t i a l s . Elongation of these c e l l s i s i n i t i a t e d near ramifying h o r i z o n t a l gonimoblast filaments. Thus the e n t i r e gonimoblast i s contained within a loc u l e which i s surrounded by gametophytic c e l l s (Figure 51). Contrary to e a r l i e r reports (Sturch, 1899, 1924), carpospores are r e -leased through small openings i n the pustule surface rather than being released i n mass only upon pustule degeneration. The opening or " o s t i o l e " ranges from 10-12 ym i n diameter, about twice the diameter of mature, elongate carpospores (Figure 52). Large quantities of mucilage are extruded from the o s t i o l e along with the carpospores (Figure 53). 5 1 * Figures 48 - 53 Carposporophyte development Figure 48. Erect gonimoblast filaments (egf) with terminal carpospores (c ) . Figure 49. Cluster of terminally borne, elongate carpospores (c) with c e n t r a l l y located nucleus (N). Figure 50. Remnants of fused foot c e l l and s t e r i l e branches subtending the mature gonimoblast. Figure 51. X.S. of lobe of female gametophyte pustule containing matur-ing carposporophyte. Elongate columnar c e l l s ( c o l . c) have pushed the gametophytic cortex (Co) away from the developing carposporophyte. Figure 52. Open o s t i o l e (asterisk) with elongate carpospore ( c ) . Copious q u a n t i t i e s of mucilage (Mu) f i l l the i n t e r c e l l u l a r spaces and are extruded with the r e l e a s i n g carpospores through the o s t i o l e . Figure 53. Scanning e l e c t r o n micrograph of o s t i o l e (Os) with extruding mucilage (Mu). A l l measurements i n micrometers (ym) 3. Development of Havveyella mivabilis a. F i e l d Observations of the Infection Process In a study of the development of H. mivabilis on 0. floocosa, two popula-tions from Parsons Point (Figure 12,inset) were observed over a 20-month period. In f i e l d c o l l e c t e d materials, developing pustules of Havveyella often occurred near c o r t i c a l l e s i o n s i n Odonthalia. These wounds (Figure 54) were most common near the base or i n branch a x i l s of the host. Spores, s i m i l a r i n s i z e to tetraspores and carpospores of Havveyella^ were observed i n ruptured c e l l s of the hostwwound (Figure 55). These spores lacked pigmentation and possessed large quantities of starch and other storage carbohydrate (Figure 56). Filaments of elongate c e l l s penetrated between loosely adjoined host medullary c e l l s at the wound surface (Figure 57), extending into the c e n t r a l medulla of the host (Figure 58). Pustules developed i n the immediate wound region or, more commonly, at considerable distance from the i n i t i a l i n f e c t i o n s i t e . Vegetative filaments of Havveyella may extend i n t e r c e l l u l a r l y through-out the host, several centimeters from the o r i g i n a l wound before i n i t i a t i n g a reproductive pustule. It i s possible that t h i s i n t e r n a l vegetative growth i s responsible f o r the pustules observed i n the early spring i n new host t i s s u e adjacent to the overwintering basal stubs of 0. floocosa (see page 64). b. Laboratory Studies of the Infection Process Similar development patterns were observed i n Havveyella grown on 0. flocoosa i n the laboratory. Tetraspores and carpospores were released d i r e c t -l y onto a r t i f i c i a l l y wounded, uninfected and uninjured host plants. A l t e r -nately, spores released onto glass plates were transferred by micropipette to a host wound i n f l i c t e d with a razor blade. Ten hours a f t e r i n i t i a l inocu-l a t i o n , numerous thick-walled spores of Havveyella were observed i n the 53 K Figures 54 - 58 I n f e c t i v e process. Development of Harveyella mivabilis on Odonthalia floocosa; f i e l d c o l l e c t e d materials. Figure 54. L i v i n g m a t e r i a l ; host with erumpent Harveyella pustules associated with c o r t i c a l wound of host. Figure 55. Sporexin ruptured host medullary c e l l (hmc). (Phase c o n t r a s t ) . Figure 56. Spore i n protoplasm of ruptured host medullary c e l l i n wound area. Granular appearance of spore due to presence of storage products ( f l o r i d e a n s t a r c h ) . (Phase c o n t r a s t ) . Figure 57. Rh i z o i d a l c e l l s of Harveyella (Hrc) penetrating between lo o s e l y adjoined host medullary c e l l s at wound surface (WS). Figure 58. Development of H. mivabilis between (arrows) and below host medullary c e l l s (hmc) at wound surface. A l l measurements i n micrometers (ym) 53 54 ruptured host c e l l s (Figure 59). Germination and subsequent development of both tetraspores and carpospores were i d e n t i c a l and w i l l be discussed together. Havveyella spores did not germinate or penetrate through the uninjured cortex of Odonthalia. It appears that successful germination and growth of Havveyella i s dependent upon the entry of a spore into a l e s i o n of a tested host plant. Germination of Havveyella spores normally occurred within 24 hours of host i n o c u l a t i o n . A protuberance of the spore w a l l marked the s i t e of sub-sequent r h i z o i d emergence (Figure 60). Some spores attached to the host c e l l w a l l p r i o r to germination (Figure 61). A l t e r n a t e l y , other spores germinated i n the degenerating cytoplasm of the ruptured host c e l l without attaching to the w a l l . A long r h i z o i d developed from t h i s spore and continued to elongate u n t i l contact was made with the host c e l l w all (Figure 62). From scanning electron microscopy, i t appears that the thick outer spore covering acts to attach the spore f i r m l y to the subtending host by forming long wall-extensions (Figure 63). Scott and Dixon (1973a) have noted that the w a l l of spores and gametes may be important i n attachment. Within 5-7 days a f t e r germination, a primary filament, c o n s i s t i n g of several elongate c e l l s , arose from the attached spore. This filament either penetrated d i r e c t l y into the host between loosely adjoined medullary c e l l s or, more commonly, i t extended along the surface of the wound before penetrating into the host (Figure 64, arrow). Af t e r entering the host t h a l l u s , dichoto-mously branching c e l l s were cut o f f (Figure 65). Each dichotomous c e l l gave r i s e to two separate penetrating filaments. Approximately one week a f t e r i n o c u l a t i o n , Havveyella filaments occupied ho'st i n t e r c e l l u l a r spaces immedi-ate l y subtending the wound (Figure 66). Subsequent p r o l i f i c growth extended 55 * Figures 59 - 63 I n f e c t i v e process. Development of Earveyella mirabilis on Odonthalia flocoosa; laboratory studies. Figure 59. 10 hours a f t e r host i n o c u l a t i o n . Thick-walled tetraspore from surface of wounded host. Figure 60. 24 hours a f t e r host i n o c u l a t i o n . Germination of Earveyella tetraspore indicated by spore protuberance which marks the s i t e of subsequent r h i z o i d a l emergence (R). Figure 61. Attachment of Earveyella tetraspore (Sp) to wound surface; the r h i z o i d (R) penetrates between loos e l y adjoined host medullary c e l l s . Figure 62. Germination of tetraspore (Sp) not attached to host w a l l . Rhizoid emerges and elongates u n t i l contact i s made with host c e l l surface. Figure 63. Scanning electron micrograph of tetraspore attached to a host wound surface (WS) by extensions formed from the outer c e l l w a l l (OW). A l l measurements i n micrometers (ym) Figures 6 4 - 6 7 Inf e c t i v e process. Development of Harveyella mirabilis on Odonthalia floocosa; laboratory studies. Figure 64. 5-7 days a f t e r spore germination ( i n o c u l a t i o n ) . Several elongate c e l l s of the primary filament (1°F) extend along the wound surface (WS). This filament a r i s e s from d i v i s i o n of the attached tetraspore. Nucleus (N) i s v i s i b l e i n one c e l l . Figure 65. Primary filament entering into host t h a l l u s . C h a r a c t e r i s t i c dichotomously branching c e l l (asterisk) i s cut o f f . This c e l l w i l l give r i s e to dichotomously branching, penetrating r h i z o i d a l filaments. Figure 66. 8 days a f t e r i n o c u l a t i o n . Penetrating r h i z o i d a l c e l l s are common between host medullary c e l l s (hmc) near wound surface ( a s t e r i s k s ) . Figure 67. Approximately 3 weeks a f t e r i n o c u l a t i o n . X.S. of infected host; much of the development of Harveyella r h i z o i d a l c e l l s (asterisk) occurs away from the o r i g i n a l s i t e of entry (at the wound surface) . A l l measurements i n micrometers (ym) 56 5 7 throughout the host t h a l l u s near the wound. As noted i n the f i e l d c o l l e c t e d m aterial, the greatest amount of development of Havveyella often occurred some distance from the i n i t i a l i n f e c t i o n region (Figure 67). Five weeks a f t e r i n o c u l a t i o n , host medullary c e l l s were t o t a l l y surrounded by filaments of H. mivabilis (Figure 68). Secondary p i t connections were common between Odonthalia and Havveyella. Continued p r o l i f e r a t i o n of i n t e r n a l Havveyella filaments and a concomitant increase i n c o r t i c a l c e l l d i v i s i o n of Odonthalia resulted i n a swelling and d i s c o l o u r i n g of the host t h a l l u s i n the infected region. As the i n t e r n a l filaments of Havveyella reached the c o r t i c a l zone of Odonthalia, the filaments became much branched and c l o s e l y packed (Figure 69). Rapid l o c a l i z e d d i v i s i o n of both Havveyella and Odonthalia resulted i n an erumpent white pustule composed of l a r g e r , more vacuolate Havveyella c e l l s and some host c e l l s (Figure 70). The rupturing of the host cortex and emergence of the Havveyella pustule occurred i n the laboratory material 65-75 days a f t e r i n o c u l a t i o n . Pustules bearing carpogonial branches have been observed 82 days a f t e r i n o c u l a t i o n with tetraspores (Figure 71). c. Host Wounding Agents Observations made at Parsons Point, B. C. have revealed that woundsoof 0. floooosa are produced i n three ways: (1) seasonal host degeneration, (2) abrasion by moving sand and stones, and (3) grazing a c t i v i t i e s of inverte-brates. Degeneration and abrasion account for massive i n j u r i e s sustained by Odonthalia during the f a l l and winter. However, these wounds are of secondary importance i n the i n f e c t i o n process since few Havveyella i n f e c t i o n s are i n i t i a t e d during t h i s period (see page 63). During the spring and summer, when carpospores and tetraspores of Havveyella i n i t i a t e the majority of new i n f e c t i o n s , invertebrate grazing produces numerous c o r t i c a l lesions which 58 a Figures 68 - 71 I n f e c t i v e process. Development of Harveyella mirabilis on Odonthalia flocoosa', laboratory studies. Figure 68. 5 weeks a f t e r i n o c u l a t i o n . Host medullary c e l l s (hmc) sur-- rounded by Harveyella r h i z o i d a l c e l l s (Hrc) i n tissues near host wound. Figure 69. Dichotomously branched, c l o s e l y packed r h i z o i d a l c e l l s (Hrc) p r o l i f e r a t i n g i n l o c a l i z e d host area and pushing host c e l l s outward. Outer wall (OW) of host s t i l l i n t a c t . Figure 70. 68 days a f t e r i n o c u l a t i o n . Rupturing of outer wall (OW) of host and emergence of Harveyella pustule. Figure 71. 82 days a f t e r tetraspore i n o c u l a t i o n . Trichogyne (t) emerg-ing from erumpent female pustule. A l l measurements i n micrometers (ym) 58 59 occur most commonly near branch a x i l s and the t h a l l u s base. The grazing invertebrates include members of the genera Idothea (isopods) and AmphithoS (amphipods). The most commonly encountered grazer i s the large isopod Idothea wosnesenskii (Brandt). Analysis of f e c a l p e l l e t s and guts of several isopods and amphipods c o l l e c t e d on 0. floocosa have been made. Remains of c o r t i c a l c e l l s t y p i c a l of higher red algae comprise approximately 80% of the contents. The remainder includes the diatoms Navioula (Schizonema), Coscinodiscus and Stephanodiscus and the bluegreen alga Devmocarpa, a l l common epiphytes of 0. floocosa. The grazing a c t i v i t i e s of I. wosnesenskii c o l l e c t e d from 0. floocosa at Parsons Point were investigated i n the laboratory as b r i e f l y described on page 11- The data from these grazing experiments are summarized i n Tables III and IV. The d i s t r i b u t i o n of 50 isopods on some a l g a l species a f t e r two-day i n t e r v a l s i s presented i n Table I I I . A f t e r each two-day period, the number of i n d i v i d u a l s on each a l g a l species was noted. The isopods were then removed from the plants and were again randomly released back into the culture tanks. A f t e r each period, the greatest number of i n d i v i d u a l s occurred on 0. floocosa, R. lavix and Laurencia spectabilis r e s p e c t i v e l y . To determine the actual amount of each alga consumed, wet-weight losses of the a l g a l species most frequented by Idothea were measured over a 14-day period (Table IV). Consideration of weight increases due to growth of each alga was made using a c o n t r o l . The control tank containing the f i v e a l g a l species and no isopods was placed under conditions i d e n t i c a l to the tank containing algae and isopods. For each alga, a percentage weight increase was calculated f o r the 14 days (Table IV, Column C). This value was used to compute the expected weight of each t e s t species a f t e r 14 days i f no grazing 60 Table I I I . D i s t r i b u t i o n of Idothea wosnesenskii on some algae i n culture. No. of i n d i v i d u a l s on each species a f t e r 2 d. periods Species 2 4 6 8 10 Odonthalia floooosa 24 26 30 32 19 0. washingtoniensis 4 3 1 2 4 Laxccencia spectabilis 8 9 7 5 9 Plocamium pacificum 0 0 1 0 2 Micvocladia bovealis 0 0 0 0 0 Belessevia deoipiens 0 0 1 0 0 Ividaea covdata 0 1 0 3 5 Viva laotuoa 2 0 2 0 0 Bedophyllum sessile 2 0 2 0 1 Rhodomela lavix 10 11 6 8 10 Table IV. Consumption of some algae by Idothea wosnesenskii. A B C D E F G H i n i t i a l exp. exp. actu a l weight weight % t o t a l a l g a l weight weight weight loss l o s s 5 consump-weight 1 in*-:.;.-. a f t e r a f t e r t i o n 6 crease 2 14 d. 3 14 d.1* Species gms % gms." gms. g t n s - ' % % Odonthalia floocosa . 63. 13 4.15 65 .75 42. 86 22. 89 34. 81 71.24 0. washingtoniensis 15. 00 1.36 15 .20 13. 79 1. 21 7. 96 3.75; Rhodomela lavix 48. 13 2.69 49 .42 45. 53 3. 89 7. 87 12.12 Lauvencia specta-bilis 30. 94 10.39 34 .15 31. 45 2. 70 7. 90 8.40 Ividaea covdata 20. 87 1.82 21 .45 19. 81 . 1. 44 6. 78 4.48 32.13=EF -^wet-weight 2from c o n t r o l (see text):growth increase 3B x A 4D - E 5F/D 6F/ZF 61 was allowed to occur (Column D). The a c t u a l weight a f t e r 14 days grazing by 30 isopods (Column E) was . .subtracted from t h i s "expected weight value" (Column D) to derive the t o t a l amount (maximum) of alga consumed by the isopods (Column F)1. This i s represented as a percentage (percent weight loss or percent consumed) for each a l g a l species (Column G). The percent weight loss of 0. floaaosa i s s u b s t a n t i a l l y greater than the other algae tested. The sum of values i n Column F (EF) gives the t o t a l a l g a l weight con-sumed (maximum) by 30 isopods i n 14 days. Of the t o t a l 32.13 grams consumed, 0. flocoosa accounted for 71.24% (Column H). Not only axe. Idothea r e s t r i c t e d i n a l g a l preference, they have also been observed to be s p e c i f i c i n the type of a l g a l tissue they consume. In both f i e l d and laboratory studies, the isopods grazed the surface c o r t i c a l c e l l s of 0. flocoosa, thus exposing the underlying medullary c e l l s (Figure 72). Shacklock (1975, personal communication) has reported s i m i l a r patterns of c o r t i c a l grazing by the isopods Idothea balthica and Laucna vi-ncta and the amphipod Gammarus Oceanians on Chondrus crispus. Uninfected 0. flocoosa, grazed by I. wosnesenskii,. was placed i n c u l t u r e tanks with 0. flocoosa and sporulating Harveyella. A f t e r four weeks, swelling and d i s c o l o u r i z a t i o n of the grazed areas were noted (Figure 73). Vegetative pustules emerged from the wound and from adjacent areas within eight weeks (Figure 74). d. Spore Germination on Alternate Substrates Spore germination and subsequent development, as previously discussed (page 54) are dependent upon a Harveyella spore contacting a s u i t a b l e host. It i s l i k e l y that the biomass production of the grazed plants was less than the ungrazed plants, thus a l l consumption values are maximal and do not repre-sent absolute values. 62 Several alternate substrates proved incapable of stimulating spore germina-t i o n . Although the spores r e a d i l y attached to glass, agar, carageenan, Olefern (polypropylene s t r i p s ) and other red algae (see page 10) submerged i n culture medium, spore germination did not occur. As described by Feldmann. arid Feldmann (1958), the spores simply degenerated a f t e r 7-10 days (Figure 75). Havveyella spores released into shallow depressions i n agar supplemented with freeze-dried 0. floeeosa germinated within 48 hours. The r h i z o i d elongated but no subsequent development occurred (Figure 76); the germling degenerated within 14 days. Spores from t e t r a s p o r i c Havveyella pustules on 0. floeeosa germinated on agar supplemented with 0. washingtoniensis but no development beyond i n i t i a l r h i z o i d protuberance was observed (Figure 77). A greater per-centage of spores were noted to germinate i n heavily ba c t e r i a l - contaminated cultures of Havveyella spores on wounded hosts and freeze-dried host-supple-mented agar than i n near axenic cultures (treated with p e n i c i l l i n G, strepto-mycin-sulfate and chloramphenicol; G u i l l a r d , 1973). e. L i f e History of Havveyella mivabilis on Odonthalia floocosa Developmental, c y t o l o g i c a l and phenological data obtained during the 20-month study of Havveyella at Parsons Point, B. C. i n d i c a t e that H. mivabilis passes through a complete f l o r i d e a n cycle annually. Carpospores released from Havveyella i n the l a t e summer and f a l l i n i t i a t e overwintering i n f e c t i o n s i n the host, 0. floeeosa. The host p e r s i s t s i n t e r t i d a l l y throughout the winter (November-January) as hollow basal branches i n which much of the development of Havveyella occurs. Remnants of the precedingr-summer's cystocarpic pustules are found oc c a s i o n a l l y on the basal host branches but more commonly, tetraspor-ophytic pustules occur. These pustules may protrude into the c e n t r a l hollow of the host or may emerge exter n a l l y through the host cortex. Pustules c o l l e c t -63^ Figures 72 - 77 Development of Earveyella mirabilis on grazed host and alternate substrates. Figure 72. Grazing wounds i n f l i c t e d on 0. flocoosa by Idothea wosnesenskii (arrows). Figure 73. 4 weeks after infection. Localized swelling and discolour-ation (arrows) of grazed 0. flocoosa infected with Earvey-ella. Figure 74. Approximately 8 weeks after infection. Emergence of vege-tative Earveyella pustule (VP) from grazing wound of 0. flocoosa (arrow). Figure 75. 7-10 days after release onto glass plates. Bacteria abund-ant around degenerating Earveyella spores. Only a few viable spores remain (arrow). No spore germination was noted. (Water-immersion objective). Figure 76. Germination of Earveyella spores on medium supplemented with freeze-dried, powdered 0. flocoosa. Elongation of rhizoid (R) occurs but no further development was observed. (Water-immersion objective). Figure 77. Germination of Earveyella spores on medium supplemented with freeze-dried, powdered 0. washingtoniensis. No development beyond i n i t i a l rhizoid (R) emergence was noted. A l l measurements in micrometers (ym) unless otherwise indicated. 63 ed during the winter contain a small number of tetraspores and few new i n f e c t i o n s occur during t h i s period. A great increase i n the number of t e t r a s p o r i c pustules and tetraspores coincides with an increase i n growth of Odonthalia occurring i n the l a t e winter and early spring. Much of the new growth of Odonthalia occurs from the d i s t a l end of the overwintering basal branches (see page 52). Internal filaments of Earveyella r a p i d l y extend i n t o the new host t i s s u e and p r o l i f e r -ate t e t r a s p o r i c pustules. Meiosis occurs i n the p e r i p h e r a l l y situated t e t r a -sporangia i n the formation of tetraspores. Early prophase I i s signaled by an increased nuclear s i z e and the appearance of a r e t i c u l a t e d mass of chromatin surrounding a heavily stained, c e n t r a l l y located nucleus (Figure 78). It i s d i f f i c u l t to count chromosomes at t h i s stage but there appear to be approxi-mately 12. During the early stages of prophase I (leptonema, zygonema), the reticulum continues to condense, the nucleus decreases i n s i z e (Table V) and the nucleolus begins to disperse." Pachynema i s characterized by the appearance of s i x d i s t i n c t b i v a l e n t s , each apparently composed of two homologous synapsed chromosomes (Figure 79). The nucleolus i s d i f f i c u l t to i d e n t i f y . During diplonema-diakinesis, the nucleus increases s l i g h t l y i n diameter (Table V) and the chromosomes appear more d i f f u s e and bead-like than the uniformly condensed pachytene chromosomes (Figure 80). Bytthe l a t e stages of d i a k i n e s i s , the nucleolus has completely dispersed. The-most d i s t i n c t i v e feature of diplonema-d i a k i n e s i s i s the presence of V and 0 shaped chromosomes (Figure 80) r e s u l t i n g from the p a r t i a l separation of homologous chromosomes. In the newly-formed tetraspores, s i x d i s t i n c t chromosomes and a nucleolus are v i s i b l e (Figure 81). Meiosis and the r e s u l t i n g formation of tetraspores occurs i n the l a t e winter and early spring. 65« Figures 78 - 81 Meiosis i n the tetrasporangium of Earveyella mirabilis Figure 78. Ea r l y prophase I (leptonema, zygonema) i n tetrasporangium; nucleolus d i s t i n c t and chromosomes appear d i f f u s e . Figures 79-81 same magnification. A l l drawings are composite drawings made from- photographs taken at 4-8 d i f f e r e n t f o c a l planes. Figure 79a,b. Pachynema i n tetrasporangium; s i x bivalents are present. Figure 80a,b. Diplonema-diakinesis i n tetrasporangium; two ring-shaped chromosomes are v i s i b l e . Figure 81a,b. Six chromosomes i n newly-formed tetraspores. 65 80a 80b 66 Gametophytic pustules r e s u l t from tetraspore i n f e c t i o n . A haploid chromosome number i s found i n both the male and female reproductive stages. Six d i s t i n c t chromosomes, ranging i n length from 0.5 ym to 1.75 ym (Table V) have been seen i n both the spermatangial mother c e l l (Figure 82) and i n mature and immature spermatia (Figures 83, 84). Six chromosomes have also been observed i n l a t e prophase stages of female vegetative a p i c a l c e l l s (Figure 85), u n f e r t i l i z e d carpogonial c e l l s (Figure 86) and carpogonial branch c e l l s (Figure 87). Spermatial and carpogonial pustules (primary gametophytes) occur from l a t e February to l a t e June, reaching a maximum number i n May (Figure 89). F e r t i l i z a t i o n of the carpogonium by the spermatium r e s u l t s i n the forma-t i o n of the carposporophyte. Twelve chromosomes are evident i n gonimoblast filament c e l l s (Figure 88). Mature carposporophytes occur from July to October and comprise approximately 60% of the t o t a l population i n early August (Figure 89). Heavy grazing by isopods and amphipods throughout the summer r e s u l t s i n an abundance of c o r t i c a l wounds i n Odonthalia i n which released Harveyella carpospores develop. A small f r a c t i o n of the carposporic i n f e c t i o n s may develop into t e t r a s p o r i c pustules i n September and October, but these pustules contain few tetraspores. Most of the carpospores give r i s e to the i n t e r n a l i n f e c t i o n s of Harveyella which overwinter i n the basal host branches. Secondary male and female gametophytes may be produced during September through November (Figure 89). These pustules occur at the margins of the degenerating primary male and female gametophytes. Although many secondary pustules are formed, the number of gametes produced i s s u b s t a n t i a l l y l e s s than i n primary gametophytes and thus the number of carposporophytes formed i s small (12% of sampled population). 67 Table V. Cy t o l o g i c a l data. Stage Aver. d i a . Chrom. Range chrom. . . nucleus ym1 number length ym Harveyella mirab-Llis SMC2 (prophase) 4.62 6 0.50-•1.75 SMC (metaphase) 3.78 * Mature spermatium (prophase) 2.60 6 0.65-•1.00 Female a p i c a l c e l l (prophase) 3.80 6 0.50-•1.75 Carpogonial branch c e l l (prophase) 4.40 6 0.50--1.75 Carpogonium (prophase) 4.40 6 0.50--1.70 GFC 3 (prophase) 3.90 12 0.37--0.87 GFC (interphase) 3.60 * Tetrasporangium (leptonema) 6.60 12? Tetrasporangium (pachynema) Tetrasporangium (dipl.-diak.) 3.50 4.45 6 b i v a l . -ents A 0.40--1.50 Tetraspore (prophase) 3.90 6 0.34--1.75 *Chromosomes could not be counted. •"•Minimum number of c e l l s measured i n each category was 250. Spermatangial mother c e l l s . Gonimoblast filament c e l l s . 68 1 Figures 82 - 88 Chromosomes i n c e l l s of male and female gametophytes and carposporophyte of Eavveyella rrd-vabilis Figures 82-84 same magnification. Figure 82a,b. Late prophase i n spermatangial mother c e l l ; s i x chromo-somes and nucleolus are present. Figure 83a,b. Six chromosomes i n mature spermatium; arrows indic a t e c o n s t r i c t i o n s i n two chromosomes. Figure 84a,b. Immature spermatangium, chromatin appears as s i x i n t e r -connected knobs; a large vacuole i s present at t h i s stage. Figures 85-87 same magnification. Figure 85a,b. Six chromosomes i n prophase vegetative a p i c a l c e l l i n female pustule. Figure 86a,b. Carpogonial nucleus, s i x chromosomes. Figure 87a,b. Six chromosomes i n carpogonial branch c e l l ; nucleolus present. Figure 88a,b. Gonimoblast filament c e l l ( d i p l o i d ) ; twelve chromosomes evident. 68 69 1 Figure 89. Phenology of Harveyella .mirabilis. on Odonthalia, floocosa at Parsons P o i n t , B. C ; 1973-1974. 70 Similar patterns of reproductive cycling to that found in the Parsons Point population have been observed from additional populations studied in Oregon, Washington and Bri t i s h Columbia. The data obtained in these studies are summarized in Appendix I. A diagrammatic summary of the l i f e history of Earveyella mirabilis i s presented in Figure 90 . f. Phenology of Earveyella mirabilis Reproduction of Earveyella can be correlated to changes in water tempera-ture, sunlight, s a l i n i t y , grazing activities and the availability of a suitable host. As shown in Figure 89 , the number of tetrasporic pustules greatly increas-es in early February, reaching a peak in March. During this time, water temperature, salinity and the hours of bright sunshine (photoperiod) increase (Figure 8 9 ) , i n addition to the increase in host biomass. The number and feeding activity of isopods and amphipods on the newgrowth Odonthalia also increases during this time. Released tetraspores infect additional host branches through grazing wounds as previously described. A rise in the percentage of spermatial and carpogonial pustules (primary gametophytes) occurs in the spring (Figure 89) as water temperature, salinity and hours of bright sunshine increase. It i s interesting to note that primary gametophytes develop in the spring as water temperature increases from 9-11 C and secondary gametophytes (page 66) form in the f a l l as the temperature decreases from 11. to 8 .5 C. The number of mature carposporophyte pustules reaches a peak in the summer and early autumn. During this time, water temperatures may reach a maximum of 1 1 . 5 - 1 2 . 0 C. Due to a decrease in the SookeRiver discharge, the salinity reaches a maximum of 3 1 ° / o o (Figure 8 9 ) . Sunlight also increases, 71c Figure 90. L i f e h i s t o r y of HavveyeZZa mivabiZis on Odonthalia fZoooosa. 71 Life History of Harveyella mirabilis on Odonthalia floeeosa 72 a t t a i n i n g a yearly maximum. However, due to the p r o l i f i c growth of such large laminarians as Hedophyllum, Alaria and Maeroaystis, the quantity of sun-l i g h t ' t o which Harveyella i s a c t u a l l y exposed decreases. To test the e f f e c t of decreased incident r a d i a t i o n on gametophyte development, three 6 m square i n t e r t i d a l areas, a l l located at the approximate 0.5 m t i d e l e v e l (Canadian) at Parsons Point were kept stripped of a l l laminarians from February through August. No di f f e r e n c e i n reproduction could be noted between the denuded and the natural areas. The e f f e c t of v e r t i c a l i n t e r t i d a l p o s i t i o n i n g on the reproductive p e r i o d i -c i t y of Harveyella at Parsons Point was investigated as described on page 8. The abundance and reproduction of Harveyella from high i n t e r t i d a l areas (above 1.0 m) has been found to d i f f e r considerably from that of plants i n the middle and low i n t e r t i d a l regions. Very few gametophytes are found i n the high i n t e r -t i d a l zone during the summer, whereas these are commonly found i n the lower i n t e r t i d a l areas. The high i n t e r t i d a l plants are frequently t e t r a s p o r i c i n the summer, producing few tetraspores. A major diffe r e n c e between the high and low i n t e r t i d a l zones i s the temperature reached during summer low t i d e periods. High i n t e r t i d a l areas are exposed to several hours of bright sunlight during summer days, r e s u l t i n g i n tidepool temperatures of 18-25 C. In the lower i n t e r t i d a l areas, exposure occurs over a much shorter period and thus the high water temperatures are not reached. The two areas also d i f f e r i n the amount of sunlight received per day. Either or both of these parameters may act i n regulating the reproduction of Harveyella. Separation of the e f f e c t s of temperature and l i g h t cannot e a s i l y be made i n in situ studies of t h i s type. As pointed out by Gessner (1970), temperature f l u c t u a t i o n s are mainly an expression of diff e r e n c e s i n the 73 i n t e n s i t y of incident sun r a d i a t i o n and are intimately r e l a t e d to f l u c t u a t i o n s i n l i g h t conditions. Major s a l i n i t y differences noted during r i v e r discharge periods at Parsons Point (Figure 78) may be interpreted as i n f l u e n c i n g the reproduction of Harveyella but t h i s could not account for s i m i l a r i t i e s i n reproductive p e r i o d i c i t y noted i n other coastal areas not affected by such large-scale s a l i n i t y f l u c t u a t i o n s . The reproduction of Harveyella mirabilis i n the North A t l a n t i c generally d i f f e r s from that found i n the northeast P a c i f i c i n the timing of gametophyte and carposporophyte production. A l i s t of reproductive stages noted from North A t l a n t i c herbarium c o l l e c t i o n s and from l i t e r a t u r e references i s present-ed i n Appendix I I . Sturch's study (1899) of the reproductive p e r i o d i c i t y of H. mirabilis on Rhodomela convervoides at Plymouth, U. K. exemplifies the generalized reproduc-t i v e cycle of Harveyella i n the North A t l a n t i c . A comparison of the phenology of Harveyella at Plymouth and Parsons Point i s presented i n Figure 91. Gametes and carpospores are found at Plymouth during the winter months of November through February while they occur from A p r i l through August at Parsons Point. The timing of tetraspore production coincides i n the two populations. In both, meiosis and the subsequent formation of tetraspores occurs during February through A p r i l . Tetraspore (and carpospore) release occurs s h o r t l y a f t e r spore formation. This was demonstrated by actual spore release i n the laboratory and by noting the number of "empty" sporangia i n materials c o l l e c t e d from Parsons Point. The phenology of H. mirabilis c o l l e c t e d from European waters and from the southern areas'(Rhode Island to Maine) of the northeast coast of North America 7 4* Figure 91. Comparison of the phenology,of Harveyella mirabilis at Plymouth, U. K. and Parsons Point, B. C. as r e l a t e d to changes i n seawater temperature. months 75 i s s i m i l a r to that noted i n the Plymouth population. I t d i f f e r s from that reported, along the east coast of Greenland (Rosenvinge, 1931) (Appendix II) and from some northern areas i n the northwest A t l a n t i c 1 . Mean sea surface temperatures of the North A t l a n t i c regions i n which Earveyella occurs are given f o r the months of August and December i n Figure 10 (overlays). High August temperatures (13-18 C) occur i n both the northeast and northwest A t l a n t i c (with the exception, of Greenland). Gametes and carpospores are not produced i n these areas u n t i l l a t e f a l l and winter when the water temperature f a l l s to 12 C and below (Figure 10, December overlay). Carpospores are produced: i n December i n both the northeast and northwest A t l a n t i c (except Greenland) although the December water temperatures may d i f f e r considerably between and within these two l o c a l i t i e s (Figure 10). Gametes and carpospores have been found i n the spring and summer i n Greenland (Appendix II) but, as shown i n Figure 10 (August overlay), the summer water temperature i n t h i s region i s much lower than, i n other North A t l a n t i c areas. The reproductive c y c l i n g of Earveyella i n Greenland appears s i m i l a r to that noted i n the north-east P a c i f i c . In a l l North A t l a n t i c populations including those of Greenland, tetraspores are produced i n l a t e winter and ea r l y spring, s i m i l a r to northeast P a c i f i c populations. E d e l s t e i n , Chen and McLachlan (1970) have reported t e t r a s p o r i c Earveyella mirabil'is occurs i n A p r i l and carposporic plants i n June at Digby Neck Peninsula Bay, Bay of Fundy, Nova Scotia. The annual v a r i a t i o n i n water temperature i n t h i s area i s s i m i l a r to the northeast P a c i f i c , ranging from a low of 0.9 C i n January to lOs-l C i n August. 76 Discussion A. Host S p e c i f i c i t y . From t h i s s e r i e s of experiments i t has been demonstrated that Havveyella i s incapable of germinating and completing i t s l i f e h i s t o r y without a sui t a b l e host. The i n a b i l i t y of Havveyella spores to germinate without ruptured and degenerating c e l l s of Odonthalia and Rhodomela indicates that the host must provide the stimulus f o r spore germination. It i s possible that the stimulus may be produced by b a c t e r i a l degeneration of host t i s s u e (page 62). B. E f f e c t of Environmental Parameters on the D i s t r i b u t i o n and Development of Havveyella mivabilis. In addition to the occurrence of a sui t a b l e host plant, various environ-mental parameters may a f f e c t the d i s t r i b u t i o n as well as the development and reproduction of Havveyella mivabilis i n the i n t e r t i d a l habitat. 1. Invertebrate Grazing Grazing by various i n t e r t i d a l isopods and amphipods produces surface les i o n s on the host plant through which Havveyella spores penetrate and subse-quently germinate. The d i s t r i b u t i o n of Havveyella pustules on the base and i n the a x i l s of the host may be correlated with the greater frequency of grazing wounds i n these areas. 2. Water Movement Although'the hosts, Odonthalia and Rhodomela, are approximately continuous i n d i s t r i b u t i o n along the northwest coast of North America, Havveyella-ixifected host populations are discontinuous, confined to r e l a t i v e l y wave-sheltered 77 coa s t a l habitats. An explanation of t h i s phenomenon may involve water move-ment as i t r e l a t e s to spore d i s p e r s a l and s e t t l i n g . Water movement i s an important f a c t o r i n the transfer of reproductive stages (spores, gametes) of marine plants (Schwenke, 1971). Neushul (1972) speculated that p a r a s i t e germ c e l l s may be d i s t r i b u t e d with host germ c e l l s i n a slime produced by the l a t t e r . ' The slime would carry both host and p a r a s i t e germ c e l l s to uninfected host plants and thus insure successful propagation. The dependency of t h i s transfer process on water movement i s obvious. No evidence of such a concur-rent tra n s f e r was noted i n the present study of Harveyella. Rather, gametes and spores of Harveyella are transported by water without accompanying host c e l l s . I t i s probable that the spores and gametes are transferred only small distances, i n f e c t i n g another region of the same host or another c l o s e l y situated host. The v e l o c i t y of the water would therefore a f f e c t the a b i l i t y of Harveyella spores to s e t t l e within tissues of a s u i t a b l e host. In turbulent waters, the chance of a spore s e t t l i n g i n a wound of an appropriate host would be greatly reduced compared to a more sheltered s i t u a t i o n . 3. S a l i n i t y and Desiccation As previously discussed, s a l i n i t y f l u c t u a t i o n s , although p a r a l l e l i n g sporulation patterns at Parsons Point, cannot account for s i m i l a r sporulation patterns noted i n other northeast P a c i f i c populations (Appendix I ) . Although desiccation i n exposed i n t e r t i d a l areas has been noted to regulate spore s u r v i v a l , germination and adult development (Chapman, 1966; Reid, 1969), i t has l i t t l e or no e f f e c t on the reproduction of Harveyella at Parsons Point. During the winter when tetraspores would be .susceptible to desiccation, the low tides occur a f t e r sunset. In the spring and summer, extensive overgrowth by laminarian algae act to protect the underlying host plants and released 78 gametes and carpospores of Harveyella. The Importance of such "microclimates" to spore and gamete s u r v i v a l has been discussed by Gessner and Schram (1971). L i t t l e change i n developmental and reproductive patterns was.^ ; noted i n Harveyella growing i n areas stripped of the overgrowth community. 4. Temperature and Daylength The reproductive p e r i o d i c i t y of Harveyella may be correlated to annual v a r i a t i o n s i n temperature and daylength, although as previously mentioned, i t i s d i f f i c u l t to separate the two e f f e c t s i n f i e l d studies. The induction of tetraspore formation i n both the Plymouth and Parsons Point i n t e r t i d a l popula-tions (Figure 91) has been correlated to a l a t e winter increase i n seawater temperature, although t h i s r e l a t i o n s h i p has not been tested experimentally. A l a t e winter increase i n daylength (photoperiod) (Figure 91 ) may also be interpreted as inducing sporogenesis i n both the Parsons Point and Plymouth Harveyella mirabilis populations. [Both areas are situated at approximately the same l a t i t u d e (Parsons Point = 48°20'N, Plymouth = 50°25'N) and would therefore have s i m i l a r photoperiods.] Newton (1957) and Matsuura (1958) have noted r e l a t i o n s h i p s between seasonal changes i n water temperature and t e t r a -spore formation, whereas most other reports have correlated tetraspore forma-t i o n with photoperiod (Provasoli, 1963; West, 1968, 1969, 1972; Dixon and Richardson, 1970; Edwards, 1971). According to West (1972), tetrasporangia are formed p r i m a r i l y during short daylengths ( f a l l , winter) i n north temperate and subboreal l a t i t u d e s . It i s possible that temperature and daylength factors i n t e r a c t i n t e t r a -sporangial induction.! In h i s study of Pseudogloiophloea, Ramus (1969a) showed a complex i n t e r a c t i o n of l i g h t duration, i n t e n s i t y , and temperature i n t e t r a -sporangial i n i t i a t i o n . West (1968) has also demonstrated the i n t e r a c t i o n of 79 l i g h t and temperature i n tetrasporangial production i n • Ac roahaetium vectin-atvm K y l i n . Gametogenesis has been shown to be Independent of photoperiod i n a number of red algae (West, 1968, 1969, 1972; Ramus, 1969a;Richardson and Dixon, 1970). Preliminary studies have indicated that temperature may be a c o n t r o l -l i n g f a c t o r i n t h i s process (West, 1972). At Parsons Point, gametogenesis i n Earveyella occurred i n the spring, as the temperature increased from 9 to 11 C, and again i n the l a t e summer and f a l l as i t f e l l from 11 to 8.5 C. Although photoperiods were also s i m i l a r during both periods of gametogenesis, Earveyella was heavily shaded during the f a l l period and thus received con-siderably l e s s l i g h t than i n the spring. At Plymouth (as elsewhere i n the North A t l a n t i c with the exception of Greenland and Nova Scotia, gametes were produced over a s i m i l a r temperature range (9.5-12 C), although t h i s occurred during the winter instead of the spring and summer. 80 V. METABOLITE KXCHANGE BETWEEN EARVEYELLA AND ITS HOST Introduction The u n i l a t e r a l exchange of metabolites from host to p a r a s i t e i s another process c h a r a c t e r i s t i c of p a r a s i t i c associations (Scott, 1969). Recent re -views (Smith, Muscatine and Lewis, 1969; Bushell, 1972; Lewis, 1973; Taylor, 1973; Meyer, 1974) have summarized the information a v a i l a b l e on nutrient transfer i n a l g a l - f u n g a l , a l g a l - i n v e r t e b r a t e , fungal-higher plant, autotrophic higher p l a n t - p a r a s i t i c higher plant, and mycorrhizal symbiosis. Although much information i s a v a i l a b l e concerning the chemical nature of translocated organic compounds, there i s l i t t l e r e l a t e d to the method of metabolite transfer. Nutrient t r a n s l o c a t i o n has been only recently demonstrated i n marine algae. Carbon-14 and Phosphorus-32 labeled compounds have been shown to move through t r a n s l o c a t i n g "sieve elements" i n the laminarian algae Maorooystis pyrifeva (L.) C. A. Agardh (Sargent and L a n t r i p , 1952; Parker, 1965), M. integvifolia Bory (Schmitz and Srivastava, 1974), Nereooystis luetkeana (Mertensj) Postels and Ruprecht (Nicholson and Briggs, 1972), Laminaria digitata (L.) Lamouroux, L. hypevbovea (Gunn.) F o s l . , and L. saoohvina (L.) Lamouroux (Hellebust and Haug, 1972; Schmitz, LUning and Willenbrink, 1972; LUning, Schmitz and Willenbrink, 1973; Steinbiss and Schmitz, 1973). Translocation of metabolites within a red alga has only been observed i n " p h y l l o i d veins" of Delessevia sanguinea CL.) Lamourox and i n Cystoolonium purpureum (Hudson) Batt. (Hartmann and Eschrich, 1969). R e l a t i v e l y l i t t l e i s known of metabolic exchange .processes between symbiotic algae. Studies by Ende and Linskins (1962) and Linskens (1963) 81 provided the f i r s t evidence by traci n g the movement of P-32 labeled' compounds between Himanthalia elongata (L.) S e t c h e l l and i t s epiphytic algae and between Cutler-la multifida (Sm.) Grev., Codium diohotomum (Hudson) S e t c h e l l and Caulerpa prolifera Lamouroux and t h e i r epiphytes, Nitophyllum punotatum (Stackl.) Grev., Polysiphonia variegata (Ag.)Tam., Enteromorpha sp. and Viva laatuoa (L.). According to Linskins (1963), d i r e c t contact i s not necessary for metabolite exchange between the symbiont. This has been confirmed i n tran s l o c a t i o n studies (using 3 2 P and -^C) of the epiphytic red algae Snrithora naiddum (Anderson) Hollenberg and Punctaria orbiaulata Jao on the angiosperm / hosts Zostera marina L. and Phyllospadix soouleri Hooker, Mieroeladia coulteri Harvey on Grateloupia doryphorei (Montague) Howe and of the p a r a s i t i c red alga Gonimophyllum skottsbergii S e t c h e l l on Botryoglossum rupreehtiana ( J . G. Agardh) De Toni (Harlin, 1971, 1973a). H a r l i n experimentally demonstrated that r h i z o i d penetration i s not necessary for t r a n s l o c a t i o n of the isotopes tested. She concluded that the apparent proximity of two plants could enable nutrients to be transferred between the symbionts before being d i l u t e d by the sea. . In a d d i t i o n a l i n v e s t i g a t i o n s of a l g a l epiphytes on Zostera marina, McRoy and Goering (1974) demonstrated that labeled carbon (H - ^COa - ) and nitrogen (15N03~, -^NH^+j (15NH2)2C0) compounds are translocated from host leaves to attached a l g a l epiphytes. The r e s u l t s of p h y s i o l o g i c a l studies on the nature of the symbiosis between Ascophyllum nodosum (L.) Le J o l i s and Polysiphonia lanosa (L.) Tandy have recently been reported. C i t h a r e l (1972a, b) has described the transfer of glutamic acid from Ascophyllum toiPolysiphonia and Penot (1974) has demon-strated that mineral isotopes (H3 3 2 P O i t , 8 6 R b C l , Na2 "MOOI,., 2 4NaCl) are 82 transferred from Ascophyllum to Polysiphonia as w e l l as to the epiphytes Fuaus serratus L. and Cladophora serioea (Huds.) Kiitz. P r i o r to these p h y s i o l o g i c a l studies, Polysiphoni-a had been considered a "simple epiphyte" of Ascophyllum ( F r i t s c h , 1945) since i t possesses a l l the morphological c h a r a c t e r i s t i c s of an autotroph. The precise nature of t h i s symbiotic a s s o c i -ation has not yet been c l e a r l y established, but t h i s s i t u a t i o n emphasizes the importance of p h y s i o l o g i c a l as well as morphological studies i n a s c e r t a i n i n g the nature of symbiotic i n t e r a c t i o n . Kugrens (1971) has investigated the p o s s i b i l i t y of metabolite transloca-t i o n between several red a l g a l host-parasite associations. The r e s u l t s of h i s experiments are inconsistent but there are i n d i c a t i o n s that Gvaoilaviophila ovyzoides S e t c h e l l and Wilson, Levv-ingiella gavdnevi (Setchell) K y l i n , Plooamiooolax pulvinata S e t c h e l l , and Gardneviella tubevifera K y l i n obtain nutrients from t h e i r hosts. His findings on t r a n s l o c a t i o n by Gonimophyllum skottsbergii are not consistent with those of H a r l i n (1971, 1973a). Both Kugrens and H a r l i n have assumed that t r a n s l o c a t i o n may occur over r e l a t i v e l y great distances i n red a l g a l hosts and consequently t h e i r experimental designs were such that the parasite was situated at considerable distance on the host from the s i t e of i n i t i a l l a b e l incorporation. Indeed, long-distance i n t e r -c e l l u l a r t r a n s l o c a t i o n has been demonstrated i n the "veins" of Delessevia (Hartmanna and Eschrich, 1969), but t h i s may not be c h a r a c t e r i s t i c of a l l red algae, p a r t i c u l a r l y those lacking p h y l l o i d veins. The p o s s i b i l i t y that mass tra n s l o c a t i o n does not occur over great distances i n red a l g a l hosts could explain inconsistencies obtained i n Kugrens' study. In a recent i n v e s t i g a t i o n of the red a l g a l p a r a s i t e , Eolmsella paohiderma 83 (Reinsch) Sturch on Gracilaria verrucosa (Hudson) Papenfuss (Callow, Evans and Callow, 1972; Evans, Callow and Callow, 1973), t r a n s l o c a t i o n of Re-labeled compounds was demonstrated by isotope pulse-chase l a b e l i n g techniques and autoradiography. Attempts were made to i d e n t i f y the translocated com-pounds but no study was undertaken on the method of nutrient t r a n s l o c a t i o n . The only i n v e s t i g a t i o n of the mechanics of nutrient exchange i n a symbiotic red alga has been reported by H a r l i n (1971) i n her study of the epiphyte Smithora naiadum. She described crenulated epidermal "t r a n s f e r c e l l s " i n the angiosperm host, Phyllospadix scouleri, which she proposed may be involved with nutr i e n t transfer to the epiphytic alga. In the current study, vegetative c e l l s of Harveyella mirabili-s have been examined at both the l i g h t and electron microscopic l e v e l to determine i f there are any u l t r a s t r u c t u r a l modifications which may be r e l a t e d to the uptake of nutrients from adjacent host c e l l s . In addition, t r a n s l o c a t i o n of 1 R e -labeled metabolites has been investigated by l i q u i d s c i n t i l l a t i o n spectro-metry, radioautography and radiochromatography to as c e r t a i n i f translocation occurs and, i f so, what compounds may be involved i n the tr a n s f e r . 84 Results and Observations A. S t r u c t u r a l Investigations L i g h t - and electron-microscopic examinations of c o r t i c a l , medullary and r h i z o i d a l c e l l s of Harveyella mirabilis revealed each to be u l t r a s t r u c t u r a l l y d i s t i n c t . A comparison of c y t o l o g i c a l features i s presented i n Table VI. These features are discussed i n d e t a i l i n the following examination of each c e l l u l a r type. 1. C o r t i c a l C e l l s C o r t i c a l c e l l s i n the p e r i p h e r a l c e l l layer i n tangential section appear to be surrounded by a three-layered w a l l matrix (Figures 92, 93), s i m i l a r to that of Porphyra ( F r e i and Preston, 1964; Bourne, 1971), Smithora (McBride and Cole, 1969) and Erytkroeystis (Kugrens, 1971). This w a l l organization i s often obscured i n the w a l l region adjacent to the p i t connection. Character-i s t i c a l l y , an inner w a l l layer of m i c r o f i b r i l s occurs immediately external to the plasmalemma. The m i c r o f i b r i l s i n t h i s layer appear to be loosely aggregated and occasionally cross-connected (Figure 94, arrow 1). The micro-f i b r i l s assume a p a r a l l e l o r i e n t a t i o n i n the outer portion of t h i s layer. Surrounding the innermost layer i s an electron transparent zone composed of m i c r o f i b r i l s which are not as compactly arranged as i n the adjacent layers (Figures 93, 94, arrow 2). A t h i n dense band of compacted m i c r o f i b r i l s occupies the middle or t h i r d lamellar layer (Figures 92, 93, 94, arrow 3 ) . These three layers are recognized i n l o n g i t u d i n a l section i n a d d i t i o n to two layers at the external pustule boundary (Figure 95). The innermost of the two l a t t e r layers i s composed of l o o s e l y aggregated f i b r i l s and granules 85 TABLE VI Characteristic Ultrastructural features of Harveyella mirabilis vegetative c e l l s i n zones I, II, I I I . Co r t i c a l (zone I) Medullary (zone II) Rhizoidal (zone III) Wall P i t connections Plasmalemma Vacuolation Nucleus Plastids Mitochondria Dictyosomes Endoplasmic reticulum (ER) S-bodies Microbodies Microtubules Lipids Phospholipids Protein bodies (amorphous) Crystalline bodies 3-5-layer complex ty p i c a l smooth or with ex-tensions i n rapidly elongating c e l l s small, central, i f present t y p i c a l , uninucleate "proplastid", no thylakoids ty p i c a l , saccate smooth, frequent rough, infrequent rough and smooth, nuclear; c e l l periphery; associ-ated with p i t plug abundant • absent absent present, not abundant absent absent absent few m i c r o f i b r i l s *convoluted membrane both sides of plug *plasmalemmavilli large, central t y p i c a l , uninucleate *variable, occasion-a l l y with thylakoids t y p i c a l , saccate smooth, frequent rough, infrequent as i n c o r t i c a l c e l l s , abundant abundant absent absent present, not abundant absent absent absent amorphous *2°pit connection to host c e l l s *vacuole associated system *PAS positive 1° and 2° vacuoles t y p i c a l , uninucleate "proplastid", no thylakoids t y p i c a l , saccate smooth, frequent rough, infrequent *smooth, tubular associated with plasma-lemma, cuts off ve s i c l e s abundant *abundant, associated with plasmalemma vacuolar system *extra-nuclear and peripheral plasmalemma *abundant i n cytoplasm or i n vacuoles, f a t t y and neutral *abundant *abundant *abundant Unique characteristics 86 « Figures 92 - 94 C o r t i c a l c e l l s of Harveyella mirabilis. Figure 92. Tangential section through cortex of pustule. Dense c o r t i -c a l c e l l s with c e n t r a l l y - l o c a t e d nucleus (N) surrounded by three-layered w a l l matrix. Figure 93. Three w a l l layers surrounding c o r t i c a l c e l l . Inner layer of l o o s e l y aggregated m i c r o f i b r i l s (arrow 1) with p a r a l l e l l y orientated m i c r o f i b r i l s i n the outer portion of the layer; middle layer composed of les s densely compacted m i c r o f i b r i l s (arrow 2); t h i n dense band of compacted m i c r o f i b r i l s com-p r i s e s t h i r d (outermost) layer (arrow 3). Figure 94. Primary p i t connection between two c o r t i c a l c e l l s of Harveyella; adjacent w a l l layers indicated by numbers. T r i p a r t i t e membrane surrounding p i t plug (arrow a ) . A s t e r i s k 1 i n d i c a t e s where membrane j o i n s plasmalemma at aperture rim; a s t e r i s k 2 , possible continuation of plasma-lemma between the two c e l l s . Inner electron-dense, p i t plug-bounding layer i n d i c a t e d by arrow b and outer e l e c t r o n -transparent layer by arrow c. Cytoplasm adjacent to the p i t connection i s r i c h i n ribosomes and tubular ER. A l l measurements i n micrometers (ym) 8 6 87* Figures 95 - 98 C o r t i c a l c e l l s of H. mivabi'lis. Figure 95. Longitudinal s e c t i o n of outer c o r t i c a l c e l l and adjacent w a l l l a y e r s . The outer two w a l l layers (4, 5) and the inner three layers (1, 2, 3) are evident. Figure 96. Outer w a l l layers of pustule. Layer 4 i s composed of l o o s e l y aggregated f i b r i l s ; l a yer 5 i s a multilayered structure., Figure 97. Diagram of 5 c e l l w a l l layers surrounding outer c o r t i c a l c e l l (CC). Figure 98. Formation of primary p i t connection. Deposition of e l e c t r o n -dense matrix on ER membranes traversing septa. A l l measurements i n micrometers (ym) 8 7 88 whereas the outer boundary i s a dark amorphous or layered band (Figure 96) which may be analogous to the " c u t i c l e " described i n Porphyra ( F r e i and Preston, 1964; Hanic and C r a i g i e , 1969), Smithova (McBride and Cole, 1969), and'GonimophyHum and Janozewshia (Kugrens, 1971). The f i v e c e l l w a l l layers of the external.pustule boundary are shown diagrammatically i n Figure 97. Primary p i t connections are formed between c o r t i c a l c e l l s (Figure 94). The p i t connections are f i l l e d with an electron-dense matrix or plug (Myers, Preston and Ripley, 1959) which i s deposited on a series of f l a t t e n e d membranes which traverse the p i t septa (Figure 98) as described i n Batrachospevmum (Brown and Weier, 1970) and Pseudogloiophloea (Ramus, 1969b). P i t connections i n EaweyeVla are u l t r a s t r u c t u r a l l y s i m i l a r to those described i n other rhodophycean algae (Bouck, 1962; Peyriere, 1963; B i s a l p u t r a , Rusanowski and Walker, 1967; Ramus, 1969b, c; Brown and Weier, 1970; Lee, 1971, T r i p o d i , 1971a; Kugrens, 1971; Kugrens and West, 1973b; Hawkins, 1972; Duckett, Buchanan, Peel and Martin, 1974). The p i t connections i n the c o r t i c a l c e l l s are l e n t i c e l l u l a r with a deep groove circumscribing the short axis (Figure 94). The p i t plug consists of a homogeneous matrix of granules (4-8 nm i n diameter) i n which are dispersed l a r g e r , densely s t a i n i n g granules. The granules are i n close proximity to one another i n the plug c o n s t r i c t i o n thus increasing the apparent density of t h i s region. Surrounding t h i s matrix are two layers separated from the cytoplasm of adjacent c e l l s by an 8 nm t r i p a r t i t e membrane (Figure 94, arrow a). The density of the inner layer approximates the density of the condensed areas dispersed i n the plug (Figure 94, arrow b). Although the thickness of t h i s layer may vary considerably between d i f f e r e n t plugs (20-60 nm), i t i s of a uniform thickness i n any one plug. External to the inner layer i s a d i f f u s e zone, v a r i a b l e i n thickness and bounded exte r n a l l y by 89 a unit membrane (Figure 94, arrow c) . This membrane, which appears to j o i n the plasmalemma at the aperture rim (asterisk- 1), i s continuous from c e l l to c e l l (Figure 94, a s t e r i s k 2 ) . I t may either conform c l o s e l y to the convex plug surface or extend into the adjacent cytoplasm. The cytoplasm bordering the p i t plug i s r i c h i n ribosomes and tubular endoplasmic reticulum (Figure 94) but lacks cytoplasmic organelles. A s i m i l a r d i s t r i b u t i o n of endoplasmic reticulum and ribosomes was reported by Lee (1971), Brown and Weier (1970), and Duckett, Buchanan, Peel and Martin., (1974) i n t h e i r studies of red a l g a l p i t connections. However, Ramus (1969b, c) has reported that the area adjacent to the p i t plug i n the red alga Pseudogloioph-loea i s devoid of a l l structures. A s i n g l e , c e n t r a l l y - l o c a t e d nucleus with a mean diameter of 3.5 ym i s the most prominent organelle i n the c o r t i c a l c e l l s (Figure.99). A granular nucleolus i s found within some n u c l e i and complex nuclear pores are abundant i n the nuclear envelope. A t a n g e n t i a l section (Figure 100) shows that the pores are arranged i n a hexagonal pattern with a mean centre to centre distance of 100 nm. Rough tubular endoplasmic reticulum i s associated with the nucleus (Figures 92, arrow, 99) and concentrated around the c e l l periphery (Figure 101). Few connections occur between the endoplasmic reticulum and various c e l l organelles. Numerous mitochondria with saccate c r i s t a e are randomly d i s t r i b u t e d throughout the ribosome-rich cytoplasm or associated with the nucleus (Figures 99, 101, 102). Scattered throughout the c e l l are double membrane-bound• organelles of v a r i a b l e s i z e which lack i n t e r n a l membranes (Figure 103). Numer-ous electron transparent areas often containing condensed f i b r i l s are dispersed throughout the ground matrix of these organelles. The f i b r i l s are digested by 9 0 ft Figures 99 - 102 C o r t i c a l c e l l s of H. mirabilis. Figure 99. C o r t i c a l . c e l l nucleus (N) with.prominent nucleolus (Nu) , adjacent mitochondria. (M) , and rough ER (rER). Figure 100. Tangential section of nuclear envelope with hexagonally arranged nuclear, pores. A c e n t r a l i n c l u s i o n i s evident within each pore. Figure 101. X.S. of c o r t i c a l c e l l (non-medial) showing p e r i p h e r a l l y situated ER. C e l l wall (CW), mitochondria (M) and p l a s -t i d s (P) are indicated. Figure 102. Saccate mitochondrion associated with dictyosome (D) re l e a s i n g rough v e s i c l e s (RV). A l l measurements i n micrometers (ym) 90 9 1 * Figures 103 - 108 C o r t i c a l c e l l s of H. mirabilis. Figure 103. Double membrane-bound.organelle ( p l a s t i d , P).with electron transparent areas. Figure 104. Dictyosome r e l e a s i n g hypertrophied smooth v e s i c l e s (SV) containing f i b r i l l a r m a terial. Figures 105-106. Coalescence of dictyosome-derived smooth v e s i c l e s . The f i b r i l l a r material contained within these v e s i c l e s i s s i m i l a r to that of the c e l l w all (CW). Concentric body (cb) present i n large coalescent v e s i c l e . Figure 107. Tubular m u l t i v e s i c u l a r bodies (tmvb) i n cytoplasm, a s s o c i -ated with concentric body (cb) . Figure 108. Fusion of m u l t i v e s i c u l a r body (mvb) with the plasmalemma. A l l measurements i n micrometers (ym) 91 92 deoxyribonuclease and are thus presumably f i b r i l s of DNA s i m i l a r to those reported i n other red algae, including Laurenoia (Bisalputra and Bisa l p u t r a , 1967) and Pseudogloiophloea (Ramus, 1969). These double membrane-bound organelles may represent v e s t i g i a l chloroplasts as proposed by Peyriere (1972) and Kugrens and West (1973b) i n t h e i r studies of p a r a s i t i c red algae. Dictyosomes are common i n c o r t i c a l c e l l s and are found associated with mitochondria as described i n Covdltina (Bailey and Bisa l p u t r a , 1970), Griffithsia (Peyriere, 1969), Bonnemaisonia (Simon-Bichard-Breaud, 1972b), Polysiphonia (Tripodi, 1971a) and Ptilota (Scott and Dixon, 1973a). Two d i s t i n c t i v e dictyosome types may be recognized. The maturing face of the f i r s t type i s oriented toward the associated mitochondrion, whereas, i n the second type, i t i s oriented away from the mitochondrion. In the f i r s t dictyosome type, small s p h e r i c a l , granular v e s i c l e s ("rough v e s i c l e s " , Wolfe, 1972) are cut o f f from the maturing face and released into the cytoplasm (Figure 102). The second and most common type of dictyosome releases hyper-trophied smooth v e s i c l e s , f i l l e d with electron transparent, f i b r i l l a r material (Figure 104). V e s i c l e s derived from the second type of dictyosome fuse with one another to form small or large f i b r i l l a r v e s i c l e s which fuse d i r e c t l y with the. plasmalemma (Figures 105, 106). The f i b r i l l a r substance of these v e s i c l e s i s of the same electron density as that of the c e l l w a l l . Tubular m u l t i v e s i c u l a r bodies, s i m i l a r to those described i n Smithora (McBride, 1972) are common i n the c o r t i c a l c e l l s (Figure 107). These appear to fuse with the plasmalemma (Figure 108) and may be involved i n w a l l synthe-s i s or modification as hypothesized by Marchant and Robards (1968). Concen-t r i c bodies of various sizes occur frequently i n c o r t i c a l c e l l s . Although frequently associated with f i b r i l l a r v e s i c l e s (Figure 106), they may also 93 occur free i n the cytoplasm (Figure 107)-. Concentric bodies are more common i n older c o r t i c a l c e l l s than i n the a c t i v e l y growing a p i c a l and subapical c e l l s . A more frequent occurrence of concentric bodies i n older red a l g a l c e l l s has also been reported i n Batraehospermum (Brown and Weier, 1970), Porphyvidium (Gantt and Conti, 1965) and Smithova (McBride and Cole, 1969). 2. Medullary C e l l s A gradual increase i n s i z e , extent of vacuolation and quantity of stored f l o r i d e a n starch occurs as the outer, medullary c e l l s merge into the inner medullary c e l l s (Figure 109). Outer medullary c e l l s are embedded i n a very t h i c k c e l l w all matrix c o n s i s t i n g of randomly arranged m i c r o f i b r i l s which appear to lack the cross-connections evident between c o r t i c a l c e l l w a l l micro-f i b r i l s (Figure 110). Medullary c e l l s i n the inner medullary region are embedded i n a r e l a t i v e l y amorphous wall matrix with few, i f any, evident m i c r o f i b r i l s (Figure 111) . The medullary c e l l nucleus i s t y p i c a l l y eukaryotic and may be either s p h e r i c a l or i r r e g u l a r i n o u t l i n e (Figure 112). The granular nucleoplasm and a c e n t r i c nucleolus are bounded by a pore-fenestrated nuclear envelope (Figure 112, arrow). Floridean starch i s abundant i n the inner medullary c e l l s , commonly appressed t i g h t l y to the nucleus (Figure 111) as described i n Ptevocladia (Tripodi, 1971a). In medullary c e l l s containing large amounts of starch such as those i n mature or reproductive pustules, the starch i s dispersed throughout the cytoplasm and occasionally protrudes into the ce n t r a l vacuole. P l a s t i d - l i k e structures s i m i l a r to those described i n c o r t i c a l c e l l s , occur i n the dense, p a r i e t a l medullary c e l l cytoplasm (Figure 113). In her Figures 109 - 112 Medullary c e l l s of H. mirabilis. Figure 109. Plastic-embedded thick section, stained with PAS. Inner medulla (IM) i s composed of large, vacuolate c e l l s with large q u a n t i t i e s of f l o r i d e a n starch (FS), while c e l l s of the outer medulla (OM) are smaller and contain l e s s f l o r i -dean starch. Figure 110. Wall matrix (CW) surrounding outer medullary c e l l ; only a few, randomly-arranged m i c r o f i b r i l s (mf) are evident. The large c e n t r a l vacuole (V) i s shown. Figure 111. Inner medullary c e l l embedded i n amorphous w a l l matrix. Nucleus (N) with evident nucleolus (Nu) i s associated with f l o r i d e a n starch. Central vacuole (V) contains membranous i n c l u s i o n s . Figure 112. Nucleus of medullary c e l l bounded by pore-fenestrated nu-c l e a r envelope (NE). Arrows i n d i c a t e nuclear pores. A granular, a c e n t r i c a l l y positioned nucleolus (Nu)- i s present and dictyosomes (D) are ac t i v e i n producing hypertrophied smooth v e s i c l e s . Smooth ER (sER) i s common i n v i c i n i t y of nucleus. A l l measurements i n micrometers (ym) 94 95 <L Figures 113 - 117 Medullary c e l l s of H. mirabilis. Figure 113. Double membrane-bound p l a s t i d - l i k e structures (P) i n medullary c e l l s . Figure 114. Elongated dumbbell-shaped p l a s t i d s (P) associated with mitochondrion (M). Plasmalemmal extensions (plv) project into c e l l w a l l matrix (CW). Figure 115. Tangential section through c e l l periphery. Lobed p l a s t i d s (P) are abundant, as are mitochondria (M) and ER. Smooth v e s i c l e s (SV) produced by dictyosomes fuse with plasmalemma (asterisks) r e s u l t i n g i n convoluted c e l l membrane. Figure 116. P l a s t i d with a c e n t r i c a l l y positioned e n c i r c l i n g thylakoid (T) and electron-transparent areas from which DNA f i b r i l s have been digested with DNase ( a s t e r i s k ) . . Figure 117. P l a s t i d with e n c i r c l i n g thylakoid and one traversing t h y l a -koid (arrows). DNA f i b r i l s occur i n electron-transparent region. A l l measurements i n micrometers (ym) 96 examination of young and old vegetative c e l l s of Earveyella mirabilis on Rhodomela eonvervoides, Peyriere (1972) noted a structure which she described as "un a p p a r e i l p l a s t i d i a l forme de leucoplaste". She emphasized that no s t r u c t u r a l modifications, i . e . , i n t e r n a l thylakoids, were seen i n any of the " p l a s t i d s " . This contrasts with the v a r i a b i l i t y of p l a s t i d u l t r a s t r u c t u r e noted i n the present study of medullary c e l l s . Greatly elongated, dumb-bell shaped p l a s t i d s are commonly observed i n cross-sections of medullary c e l l s (Figure 114). In tangential section, these organelles appear to be lobed (Figure 115) and possess electron transparent areas i n which f i b r i l s resembl-ing deoxyribonucleic a c i d (DNA) are dispersed. E n c i r c l i n g thylakoids positioned either a c e n t r i c a l l y (Figure 116) or p e r i p h e r a l l y (Figure 117) are present i n some medullary c e l l p l a s t i d s . One or two a d d i t i o n a l thylakoids may traverse the organelle (Figure 116, arrows). Both the traversing and e n c i r c l i n g thylakoids lack phycobilosomes. In medullary c e l l s , dictyosomes with hypertrophied, smooth v e s i c l e s (Figure 118) are more common than those with granular v e s i c l e s . As i n the c o r t i c a l c e l l s , the dictyosomes are associated with mitochondria. Smooth dictyosome v e s i c l e s are released away from the mitochondrion. These v e s i c l e s may form d i r e c t connections with the vacuole tonoplast, r e l e a s i n g t h e i r contents into the c e n t r a l vacuole (Figure 119). Many small dictyosome v e s i c l e s may coalesce and fuse d i r e c t l y with the plasmalemma, as i n c o r t i c a l c e l l s . This process r e s u l t s i n the highly convoluted appearance of some medullary c e l l s (Figure 115). The plasmalemma may form cytoplasmic extensions which protrude into the c e l l w a l l (Figure 120). These extensions do not occur along the tonoplast of the c e n t r a l vacuole. Similar plasmalemmal projections have been reported along d i f f e r e n t i a t i n g procambial c e l l s i n the r a d i c l e of Aroeuthobium (Tainter, Figures 118 - 120 Medullary c e l l s of H. mirabilis. Figure 118. Dictyosomes (D) associated with nucleus (N). Smooth hyper-trophied v e s i c l e s (SV) released away from juxtaposed mitochondrion (M). Figure 119. Smooth v e s i c l e s forming d i r e c t connection with tonoplast of c e n t r a l vacuole (V) ( a s t e r i s k ) . Figure 120. Long plasmalemmal projections (plasmalemmavilli, plv) ex-tending i n t o c e l l w a l l matrix surrounding outer medullary c e l l . A s t e r i s k i n d i c a t e s d i r e c t connection with plasma-lemma. Bulbous v e s i c l e at end of p r o j e c t i o n i n d i c a t e d by arrow. A l l measurements i n micrometers (ym) 9 7 98 1971), i n the l i c h e n i z e d fungus Physeia avpolia (Brown and Wilson, 1968), i n Ricinus seedlings where they were termed "plasmic p a p i l l a e " (Frey-Wyssling, 1962), and i n the red alga Chondrus erispus CCott ler, 1971) where they were termed "plasmalemmavilli". The term plasmalemmavilli w i l l be applied to the plasmalemmal projections noted i n Harveyella. Plasmalemmavilli occur as extensions of both inner and outer medullary c e l l membranes, although they are a more regular feature of the outer medullary c e l l s . Long plasmalemma-v i l l i (500-1500 nm) occur on outer medullary c e l l s (Figure 120), while shorter projections (50-400 nm) occur on the inner medullary c e l l s (Figure 121). Both the long and short extensions end b l u n t l y , forming a d e f i n i t e band i n the c e l l w all (Figure 120). Only a few projections extend beyond t h i s band. Occasionally, v e s i c l e s form at the end of the plasmalemmavilli at the band i n t e r f a c e (Figure 120). In cross-section, the extensions appear s p h e r i c a l (Figure 122) and hollow, ranging i n diameter from 15-35 nm. The greatest development of the long plasmalemmavilli occurs i n the infolded areas of highly convoluted outer medullary c e l l s (Figure 123). P i t connections occur between c e l l s i n the medullary regions. These are s i m i l a r i n most respects to those described i n the c o r t i c a l region except for the unique, convoluted appearance of the membrane associated with the convex plug surface (Figures 124, 125). Small v e s i c l e s are often attached to the convoluted membrane (Figure 125, arrows), appearing either to form from the membrane or to coalesce with i t . Large amounts of smooth, tubular endoplasmic reticulum (ER) occur i n medullary c e l l s (Figure 115), frequently forming an extensive p e r i p h e r a l net-work, often concentrated near the p i t connection (Figure 126). Occasionally, long s i n g l e tubules are observed extending throughout the cytoplasm (Figure 127). 99* Figures 121 - 126 Medullary c e l l s of H. mirabilis. Figure 121. Short plasmalemmavilli (plv) projecting from membrane of inner medullary c e l l . Figure 122. X.S. of plasmalemmavilli (arrows). Figure 123. Long plasmalemmavilli i n the infolded areas of crenulate outer medullary c e l l s . Figure 124. Primary p i t connection between adjacent medullary c e l l s . Convoluted membrane occurs on both sides of p i t plug (arrows). Smooth ER (sER) i s common i n area adjacent to plug. Figure 125. Small v e s i c l e s attached to convoluted membrane surrounding p i t connection ( a s t e r i s k ) . Smooth ER (sER) i s abundant. Figure 126. Smooth ER near p i t connection (PC), non-medial cross-section through p i t connection. A l l measurements i n micrometers (ym) 9 9 l o o t Figures 127 - 130 Medullary c e l l s of H. mirabilis. Figure 127. Single tubule of ER (sER) extending throughout cytoplasm of medullary c e l l . F loridean starch (FS) i s present. Figure 128. C e n t r a l vacuole (V) containing granular m a t e r i a l and m u l t i -v e s i c u l a r bodies (mvb). The membrane surrounding the separate v e s i c l e s i s continuous with the tonoplast (aster-isk) . Figure 129. M u l t i v e s i c u l a r (mvb) and concentric bodies (cb) i n c e n t r a l vacuole. Osmiophilic granules (Og) appear to pinch i n t o vacuole. Figure 130. Concentric body i n c e n t r a l vacuole. A l l measurements i n micrometers (ym) 100 101 M u l t i v e s i c u l a r (Figure 128) and concentric bodies (Figures 129, 130) are common i n large c e n t r a l vacuoles and, occasionally, osmiophilic granules appear to pinch into the vacuole (Figure 129). The ce n t r a l vacuole i s c h a r a c t e r i s t i c a l l y electron transparent, but may be f i l l e d with granular or f i b r i l l a r substances (Figures 128, 130). Tubular m u l t i v e s i c u l a r bodies, common i n c o r t i c a l c e l l s , are not frequently encountered i n the cytoplasm of medullary c e l l s . 3. R h i z o i d a l C e l l s a. General U l t r a s t r u c t u r e R h i z o i d a l c e l l s i n the i n t e r d i g i t a t i o n zone (zone III) d i f f e r u l t r a s t r u c -t u r a l l y from c o r t i c a l and medullary c e l l s of the pustule as summarized i n Table VI (page 85). An extensive vacuolar system associated with the plasma-lemma and numerous storage metabolites are found e x c l u s i v e l y i n these c e l l s . Of the three c e l l types, the r h i z o i d a l c e l l s are i n close s t proximity to adjacent host c e l l s . The p o s s i b i l i t y thus e x i s t s that u l t r a s t r u c t u r a l modifications i n these c e l l s may be re l a t e d to nutrie n t exchange with adjacent host c e l l s . R h i z o i d a l c e l l s r e a d i l y e s t a b l i s h secondary p i t connections with other r h i z o i d a l c e l l s or with adjacent host medullary c e l l s (Figure 131). The c e l l w a l l layer surrounding the r h i z o i d a l c e l l s i s composed of an amorphous granular substance with few m i c r o f i b r i l s . The presence of carbohydrates has been demonstrated i n the w a l l matrix surrounding r h i z o i d a l and host c e l l s with the carbohydrate s p e c i f i c s t a i n , p e r i o d i c acid S c h i f f ' s reagent CPAS). The most prominent organelle i n the r h i z o i d a l c e l l i s the nucleus which contains one or two n u c l e o l i . Nuclear pores are abundant i n the double mem-brane of the nuclear envelope (Figure 132). Occasionally, microtubules are 102 « Figure 131 Rhizo i d a l c e l l of Earveyella mirabilis. Figure 131. Rh i z o i d a l c e l l connected to medullary c e l l of 0. flocoosa by a secondary p i t connection (2°CP). Numerous vacuoles (V) are scattered throughout the cytoplasm or associated with the plasmalemma. Host medullary c e l l (hmc) with c e n t r a l vacuole, p l a s t i d s (P) , and mitochondria (M). 102 103a Figures 132 - 134 R h i z o i d a l c e l l s of H. mivabilis. Figure 132. R h i z o i d a l c e l l nucleus with abundant nuclear pores (Np). C e l l w all (CW) and vacuole (V) are i n d i c a t e d . Figure 133. Nucleus with s i n g l e microtubule (mt) terminating at nuclear envelope. P l a s t i d (P) and mitochondrion (M) are adjacent to nuclear envelope. Figure 134. P a r a l l e l microtublues (mt) outside of nucleus. S o l i t a r y bodies (S-bodies, SB) are common i n cytoplasm. A l l measurements i n micrometers (urn) 103 1Q4 found outside the nucleus. These may occur either s i n g l y , ending b l u n t l y at the nuclear envelope (Figure 133) or i n groups near the nuclear envelope (Figure 134). As i n medullary c e l l s , the p l a s t i d - l i k e structure may be lobed and elongate (Figure 135). D i v i s i o n of these structures by simple f i s s i o n i s common i n act i v e r h i z o i d a l c e l l s (Figure 136). In older r h i z o i d a l c e l l s , both p l a s t i d s and mitochondria contain concentric lamellar bodies (Figure 137) s i m i l a r to those observed i n the p l a s t i d s of Batraohospermum (Brown and Weier, 1970). b. Vacuolar System Associated with Plasmalemma Probably the most d i s t i n g u i s h i n g feature of the r h i z o i d a l c e l l i s the complex membrane system con s i s t i n g of v e s i c l e s , vacuoles, plasmalemma, endo-plasmic reticulum, dictyosomes and microbody-like organelles which i s associated with a v a r i e t y of storage i n c l u s i o n s . A schematic summary of the i n t e r r e l a t i o n s h i p s of the s t r u c t u r a l components comprising the r h i z o i d a l c e l l membrane system i s presented i n Figure 138. C e l l s a c t i v e l y engaged i n i n t r u s i v e growth through the host w a l l matrix possess great amounts of smooth tubular ER (Figures 139, 140). The tubules are highly branched and positioned p e r i p h e r a l l y i n the c e l l . V e s i c l e s are cut o f f terminally from the tubules and appear to fuse with the plasmalemma (Figures 141, 138#7). Vacuoles are an i n t e g r a l part of the r h i z o i d a l c e l l membrane system. Large vacuoles, often associated with mitochondria, p l a s t i d s and dictyosomes (Figure 142), occur i n older c e l l s more frequently than i n young c e l l s . Small vacuoles are common i n young c e l l s , either scattered throughout the cytoplasm, or associated with l o c a l i z e d plasmalemma s i t e s (JFigure 143). The term second-ary vacuole (Wilson, 1973; Mahlberg, Turner, Walkinshaw and Venketeswaran, 1974) w i l l be applied to the small vacuoles to d i s t i n g u i s h them from the 1 0 5 «t Figures 135 - 137 Rhiz o i d a l c e l l s of H. mivabiZis. Figure 135. Lobed and elongate p l a s t i d s (P) and numerous mitochondria (M) i n electron-dense cytoplasm. Small vacuoles (V) are abundant and.multivesicular bodies (mvb) are associated with plasmalemma. Figure 136. D i v i s i o n of p l a s t i d ; S-bodies are ind i c a t e d . Figure 137. Concentric bodies (cb) i n p l a s t i d (P). A l l measurements i n micrometers (ym) 105 106 * Figure 138. Schematic summary of the i n t e r r e l a t i o n s h i p s of the struc-t u r a l components comprising the r h i z o i d a l cell.membrane system. Numbered pathways are discussed i n the text. 106 107 Figures 139 - 141 Rhizoidal c e l l s of H. mirabilis. Figure 139. Tangential section through c e l l periphery showing abundant smooth ER, Mitochondria (M) and m u l t i v e s i c u l a r body (mvb) are indicated. Figure 140. Non-medial cross-section of r h i z o i d a l c e l l . P e r i p h e r a l l y -situated smooth ER (sER) i s evident. Dictyosome (D), p l a s t i d (P) and mitochondrion (M) are i n d i c a t e d . Figure 141. Small v e s i c l e s derived from p e r i p h e r a l smooth ER (ERv) appear to fuse with plasmalemma. A l l measurements i n micrometers (ym) 107 108*s Figures 142 - 143 R h i z o i d a l c e l l s of H. mi-rabilis. Figure 142. Large primary vacuole (1°V) i s associated with mitochondria (M) and p l a s t i d s (P). Membranous debris within primary vacuole. Smaller secondary vacuoles (2°V) are scattered throughout cytoplasm and associated with plasmalemma. Figure 143. Secondary vacuoles are associated with plasmalemma and coalescing to form larger vacuoles ( a s t e r i s k s ) . Mitochond-r i a and p l a s t i d s are dispersed throughout cytoplasm. A l l measurements i n micrometers (ym) 108 109 larger primary vacuoles. The presence of soluble carbohydrates i n both the primary and secondary vacuoles has been cytochemically demonstrated with the s t a i n , p e r i o d i c a c i d ' S c h i f f 1 s reagent (Colour plates.1,2). Secondary vacuoles i n Harveyella r h i z o i d a l c e l l s are s i m i l a r to pinocyto-t i c or endocytotic vescles described i n root cap c e l l s of oats, corn and barley (Wheeler and Hanchey, 1971; Wheeler, Baker and Hanchey, 1972; Wheeler and Baker, 1973), r a d i c l e meristem c e l l s of Soorzonera (Coulomb, 1973), i n orchid mycorrhiza (Nieuwdorp, 1972), and i n c e l l s of the p a r a s i t i c angio-sperm Orabanehe ramosa (Db"rr and Kollmann, 1974). The contents of secondary vacuoles attached to the plasmalemma are confluent with the c e l l w all matrix (Figures 144, 138#1). A d i s t i n c t t r i p a r t i t e unit membrane surrounds the secondary vacuole (Figures 145, 146). In vacuoles attached to the plasmalemma the vacuolar membrane i s continuous with the plasmalemma but i s frequently thicker (Figure 146, arrow). The vacuolar membrane ranges from 12 to 17 nm i n width; other unit membranes t y p i c a l l y range from 7-8 nm. Similar membrane d i l a t a t i o n s have been reported i n p i n o c y t o t i c v e s i c l e s i n amoebae exposed to pinocytosis induction substances (Brandt and Freeman, 1967) and i n plant roots treated with uranyl and calcium s a l t s (Wheeler and Baker, 1973; Wheeler, Baker and Hanchey, 1972). Secondary vacuoles d i s s o c i a t e from the plasmalemma and others may form i n the same area, r e s u l t i n g i n multiple secondary vacuoles (Figures 146, 147). Mahlberg (1972) postulates that the tendency of membranes to form secondary vacuoles i n l o c a l i z e d areas may in d i c a t e that selected membrane l o c i are involved. Occasionally, a d d i t i o n a l membranes surround secondary vacuoles (Figure 144). These membranes are associated with peripheral tubular ER and circumscribe the v e s i c l e on the cytoplasmic side of the invaginated plasmalemma. 110 a Colour p l a t e s 1-8 Histochemical i d e n t i f i c a t i o n of compounds i n Havveyella mivabilis and host, Odonthalia flocoosa. Colour p l a t e s 1-16 P l a s t i c embedded thick sections. Colour p l a t e 1. PAS stained section. Floridean starch (fs) i s indicated within Havveyella mivabilis c e l l s (Hm) adjacent to host medullary c e l l s (HMC). Carbohydrates are also indicated i n the c e l l w a l l matrix. x800. Colour p l a t e 2. Colour p l a t e s 3-4. Colour p l a t e s 5-6. Colour p l a t e 7. PAS stained section. Carbohydrates are present within primary (l°vac.) and secondary vacuoles (2°vac.) of Havveyella c e l l s i n the i n t e r d i g i t a t i o n zone. x900. Fatty acids are present i n the autophagic v e s i c l e s (av) of Havveyella r h i z o i d a l c e l l s . Host c e l l p l a s t i d s (p) and a r h i z o i d a l c e l l nucleus (n) of Havveyella are i n d i c a t e d . Sections stained with N i l e blue A. x800. (Plate 3, phase contrast.) Floridean starch (fs) and polyphosphate bodies (pb) occur i n Havveyella r h i z o i d a l c e l l s and i n host medullary c e l l s (HMC). Stained with t o l u i d i n e blue 0. Plate 5, x900; p l a t e 6, x800. Host medullary c e l l (HMC) surrounded by Havveyella r h i z o i d a l c e l l s i n the i n t e r d i g i t a t i o n zone. Note the f l o r i d e a n starch i n both the host and Havveyella. Central vacuole i n host c e l l i s present. Stained with Sudan black B. x750. Colour p l a t e 8. Havveyella c e l l s penetrating a host medullary c e l l (HMC) i n the i n t e r d i g i t a t i o n zone. Note lack of f l o r i d e a n starch i n both Havveyella and host, and l o s s of c e n t r a l vacuolation i n host c e l l . Stained with Sudan black B. x750. I l l Colour plates 9-16 Cytohistochemical i d e n t i f i c a t i o n of compounds i n Earveyella mirabilis and host, Odonthalia fZoooosa. Colour plate 9. Floridean starch (fs) i n uninfected host c e l l . Note r i n g of starch around nucleus (n) . Stained with PAS. x700. (Phase contrast.) Colour plate 10. Phospholipids (pi) i n i s o l a t e d host c e l l (HC) i n Harveyella pustule. Nuclei are indic a t e d (n). Stained with Luxol's f a s t blue G and counter stained with neutral red. x800. (Phase contrast.) Colour plate 11. Host c e l l (HC) dispersed i n t e t r a s p o r i c pustule of EarveyeZZa. Tetraspores (T) are indi c a t e d . Note the difference i n c e l l w a l l s t a i n i n g i n walls surrounding host c e l l and EarveyeZZa c e l l s . Stained with t o l u i d i n e blue 0. x200. Colour plate 12. Floridean starch (fs) i n Harveyella c e l l . Note layers i n c e l l w all (cw). Stained with t o l u i d i n e blue 0. x700. Colour plates 12-16 Stained with a l c i a n blue and a l c i a n yellow. Colour plate 13. Sulfated polysaccharide (blue) present surrounding outer host medullary c e l l (HMC) but l i t t l e or none i s present i n c e l l walls of host c o r t i c a l c e l l s (HCC). x650. Colour plate 14. Sulfated polysaccharides are present i n w a l l layer surrounding large, inner host medullary c e l l (asterisk) Adjacent EarveyeZZa c e l l (Hm) i s in d i c a t e d . x650. Colour plate 15. Sulfated polysaccharides are absent from w a l l layer surrounding the host medullary c e l l s (HMC) dispersed i n the pustule ( a s t e r i s k ) . P l a s t i d s (p) are present i n host c e l l . x650. Colour plate 16. Sulfated polysaccharides form a d i f f u s e band surround-ing Harveyella mirabiZis (Hm) c e l l s i n pustule. Floridean starch (fs) and a nucleus (n) are in d i c a t e d . x650. 112 «-Figures 144 - 147 R h i z o i d a l c e l l s of H. mirabilis. Figure 144. Secondary vacuo l e (2"V) i s assoc ia ted with plasmalemma. Vacuolar contents are conf luent wi th c e l l w a l l m a t r i x . P e r i p h e r a l smooth ER i s assoc ia ted wi th membrane of vacuole (arrows). F igure 145. Plasmalemma-associated secondary vacuole surrounded by d i l a t e d t r i p a r t i t e membrane ( a s t e r i s k ) . F igure 146. Plasmalemma i s continuous w i t h d i l a t e d membrane surrounding secondary vacuole ( a s t e r i s k ) . I n i t i a l formation of m u l t i -p l e secondary v e s i c l e (arrow). F igure 147. M u l t i p l e secondary vacuoles forming at l o c a l i z e d membrane l o c i . V e s i c l e s are produced from ER (ERv) assoc ia ted w i t h secondary vacuoles . A p o l y p h o s p h a t e - l i k e granule (pp) i s i n d i c a t e d , as w e l l as the nucleus (N). A l l measurements i n micrometers (ym) 112 113 Secondary vacuoles dispersed i n the cytoplasm vary i n s i z e , shape, and i h the electron density of the vacuolar contents. S e r i a l sections through vacuolate cytoplasmic regions reveal that s p h e r i c a l secondary vacuoles invaginate to form crescent-shaped membrane-bound sacs (Figures 148, 138//1) . In cross-section, these appear as complete membrane-bound rings of electron transparent material (Figures 151, 138#3). A substance of s i m i l a r e l e c t r o n density to that of the invaginated sac appears external to the inner invagin-ated surface (Figure 149). The substance i s apparently released from the v e s i c l e during the process of invagination and eit h e r disperses into the cytoplasm or becomes delimited by a discontinuous double membrane. Remnants of the invaginated secondary vacuoles coalesce around the electron transparent areas to form the surrounding discontinuous double membrane (Figures 150, 151, 152, 138#1). Several of these regions may become adjoined or contiguous and open d i r e c t l y into the c e l l wall (Figure 153). Laminate membranes from the invaginated secondary vacuole may form- concentric bodies (Figures 154, 155, 138//4) which appear to fuse with the plasmalemma (Figure 156). Concen-t r i c lamellar bodies also occur' within double or sin g l e membrane-bound vacuoles (Figure 157). Single membrane-bound primary vacuoles occupy a large volume of some r h i z o i d a l c e l l s . An extensive membrane system i s contained within the vacuoles and, commonly, secondary vacuoles appear to invaginate d i r e c t l y into the primary vacuole (Figures 158, 138//5). The contents of the primary vacuole are s i m i l a r i n electron density to those of the secondary vacuoles and the i surrounding c e l l w a l l matrix. Primary vacuoles may also contain various quantities of osmiophilic membranous debris (Figure 159, arrows) suggestive of degrading cytoplasm. Similar substances associated with v e s i c l e s have Figures 148 - 151 Rhizoidal c e l l s of H. mivabilis. Figure 148. Figure 149. Figure 150. Figure 151. A l l measurements i n micrometers (ym) Secondary vacuoles (2°V) dispersed i n electron-dense cyto-plasm. I n i t i a t i o n of vacuole invagination and concomitant release of electron transparent vacuolar contents i n t o cytoplasm ( a s t e r i s k ) . Completion of invagination. Remnants of secondary vacuolar membrane remains surrounding electron-transparent substance (asterisk) released from vacuole. M u l t i v e s i c u l a r body (mvb) i s present. Release of secondary vacuolar contents i n t o l o c a l i z e d cyto-plasmic area ( a s t e r i s k ) . Remnants of vacuolar membranes remain surrounding area to form discontinuous bounding double membrane. X.S. of invaginated secondary vacuole. Continuous double membrane-bound r i n g with i n t e r n a l cytoplasm. 114 115 A Figures 152 - 153 R h i z o i d a l c e l l s of H. mirabilis. Figure 152. Remnants of secondary vacuoles (arrows) surrounding e l e c t r o n -transparent cytoplasmic region ( a s t e r i s k ) . Secondary vacuoles near plasmalemma are associated with p l a s t i d s (P). Polyphosphate-like granule (pp) i s i n d i c a t e d . Figure 153. Contiguous electron-transparent regions with surrounding secondary vacuole remnants ( a s t e r i s k s ) . These are continu-ous with the c e l l w a l l (arrow). Microbody-like structure (mb) i s present. A l l measurements i n micrometers (ym) 115 1 1 6 * Figures 154 - 157 R h i z o i d a l c e l l s of H. mirabilis. Figures 154-155. Concentric bodies (cb) forming from remnants of invag-inated secondary vacuoles (2°V). Figure 156. Concentric body i s associated with plasmalemma. Smooth v e s i c l e producing dictyosome (D) associated with mitochond-r i o n (M) . Figure 157. Concentric bodies (cb) within f i b r o u s - f i l l e d vacuole. A l l measurements i n micrometers (ym) 116 117" Figures 158 - 159 Rhizoidal c e l l s of H. mivabilis. Figure 158. Non-medial X.S. Secondary vacuoles associated with plasma-lemma, fusing with large primary vacuole ( a s t e r i s k ) . Mitochondrion(M) i s associated with plasmalemma. P i t con-nection (PC) i s indicated. Figure 159. Single membrane-bound primary vacuole (1°V) (autophagic vacuole) containing osmiophilic, granular and lamellar debris (de). ER, p i t connection (PC), S-bodies (SB) and dictyosomes (D) are indicated. A l l measurements i n micrometers (ym) 117 1 1 8 been reported' in the fungi Gilbevtella (Braker and Williams, 1966) and Phyaomyees (Thornton, 1968). Thornton termed the debris-containing vesicles "autophagic vesicles". Enzymatic digestion of cytoplasmic debris contained within autophagic vacuoles (vesicles) has been recently discussed' by Matile (1974). Electron transparent dictyosome vesicles coalesce forming vesicles similar to secondary vacuoles (Figures 148, 156). These may fuse with either primary or secondary vacuoles as described in root tip cells (Matile and Moor, 1968; Berjak and V i l l i e r s , 1970). Dictyosomes producing electron-dense granular vesicles occur infrequently in rhizoidal cells (Figure 160). They are usually associated with plasmalemma areas engaged in the formation of secondary vacuoles. As previously described, smooth tubular ER usually follows the contour of the plasmalemma but may also extend throughout the cytoplasm of active, vacuolated cells (Figure 161). From interpretations of serial sections, electron-dense vesicles appear to pinch off terminally from ER and become associated with primary or secondary vacuoles (Figures 162, 138). Tubular ER may also become greatly dilated to form extensive ER vacuoles containing either granular or f i b r i l l a r substances (Figures 163, 164, 138#10). D'drr (1972) has noted a similar occurrence of peripherally situated ER in cells of Cusauta (parasitic angiosperm) and has proposed that this ER may be the source of enzymes necessary for the active uptake of food from the host. Spherical, single membrane-bound bodies measuring 200 to 300 nm in diameter are dispersed throughout the cytoplasm of rhizoidal cells commonly associated with ER profiles (Figures 153, arrow, 165). These organelles possess a granular matrix of variable electron-density and are structurally 119a Figures 160 - 162 R h i z o i d a l c e l l s of.H. mivabiZis. Figure 160. Dictyosome producing electron-dense granular v e s i c l e s (RV) which are released toward the juxtaposed mitochondrion (M). Figure 161. Non-medial l o n g i t u d i n a l section. Large q u a n t i t i e s of smooth ER extending throughout cytoplasm. Numerous secondary vacuoles (2°V) are associated with plasmalemma and are d i s -persed throughout the cytoplasm. Several are invaginating and coalescing ( a s t e r i s k s ) . Concentric bodies (cb) are associated with multiple secondary vacuole and polyphos-phate-like granules (pp) occur i n the cytoplasm. Figure 162. ER-derived v e s i c l e s (ERv) appear to fuse with secondary vacuoles associated with the plasmalemma. P l a s t i d s (P), mitochondria (M) and dictyosomes (D) are present. A l l measurements i n micrometers (ym) 119 1 2 0 * Figures 163 - 165 Rh i z o i d a l c e l l s of H. mirabili-s. Figure 163. D i l a t a t i o n of ER to form extensive ER vacuole (V) containing f i b r i l l a r m a t e r i a l . A s t e r i s k i n d i c a t e s ER d i l a t a t i o n . Figure 164. D i l a t a t i o n of ER (a s t e r i s k ) to form complex lamellar vacuole (V) with associated osmiophilic granule (Og). S-bodies (SB) are abundant i n cytoplasm. Figure 165. Single membrane-bound, electron-dense microbody-like organ-e l l e s (mb) associated with ER. A l l measurements i n micrometers (ym) 120 121 s i m i l a r to microbodies described i n Porphyridium (Oakley and Dodge, 1974) and Nitella (Silverberg and Suwa, 1973), and i n Vaouolaria and Gonyostomum (Heywood, 1974). M u l t i v e s i c u l a r bodies are frequently observed i n r h i z o i d a l c e l l s as membrane-bound aggregations of v e s i c l e s within the cytoplasm (Figure 166) or associated with e i t h e r d i l a t e d ER vacuoles (Figure 167) or the plasmalemma (Figure 168). The v e s i c l e s vary i n s i z e and are usually surrounded by a s i n g l e unit membrane. Marchant and Robards (1968) have used the term lomasome for those m u l t i v e s i c u l a r bodies i n plants which a r i s e within the cytoplasm, and plasmalemmasomes for those a r i s i n g from the plasmalemma. M u l t i v e s i c u l a r bodies i n r h i z o i d a l c e l l s may be interpreted as plasmalemmasomes i f one assumes that they a r i s e from an invagination of the plasmalemma. They then may be transported through the cytoplasm and become associated with ER-derived vacuoles (Figures 167, 138#12-13), or v e s i c l e s derived from secondary vacuoles (Figures 169, 170). If the process i s reversed, the m u l t i v e s i c u l a r bodies could be termed lomasomes and thus be involved i n exocytosis instead of endo-c y t o s i s . Since i t has not been possible to a s c e r t a i n the d i r e c t i o n of move-ment of these organelles, they w i l l simply be r e f e r r e d to as m u l t i v e s i c u l a r bodies. I t should be noted that these bodies d i f f e r from the tubular m u l t i -v e s i c u l a r bodies which occur commonly i n c o r t i c a l c e l l s of the pustule (page 92). c. Cytoplasmic Storage Inclusions Various unique cytoplasmic storage i n c l u s i o n s have been observed i n Harveyella r h i z o i d a l c e l l s and the chemical nature of these structures has been ascertained using l i g h t microscopic s t a i n i n g techniques. Although not unique to only r h i z o i d a l c e l l s , small quantities of f l o r i d e a n starch occur 122 * Figures 166 - 170 Rhiz o i d a l c e l l s of H. mirabilis. Figure 166. Multivesicular, body (mvb) i n cytoplasm. Figure 167. M u l t i v e s i c u l a r body (mvb)' associated with d i l a t e d ER vacuole. Figure 168. Association of m u l t i v e s i c u l a r body.(mvb) with plasmalemma. Figure 169. M u l t i v e s i c u l a r body i n v i c i n i t y of invaginating secondary vacuole r e l e a s i n g vacuolar contents.(2°V). Figure 170. M u l t i v e s i c u l a r body associated with electron-transparent cytoplasmic region which i s surrounded by remnants of invaginated secondary vacuoles ( a s t e r i s k s ) . A l l measurements i n micrometers (ym) 122 123 within vacuoles and are occasionally dispersed i n the cytoplasm (Figure 171, colour p l a t e 12). Floridean starch has been cytochemically i d e n t i f i e d using p e r i o d i c a c i d S c h i f f ' s reagent (PAS) with the blocking agents DNPH (2,4-dinitrophenylhydrazine) or Dimedone (5,5-dimethylcyclohexane-l,3-dione) (Feder and O'Brien, 1968). Starch stained bright magenta (Figure 172, colour p l a t e 1), a p o s i t i v e reaction for carbohydrates. Spherical osmiophilic. granules are dispersed throughout the cytoplasm of r h i z o i d a l c e l l s (Figure 173). The granules lack a surrounding unit mem-brane (Figure 174) and often appear hollow (Figure 175). The osmiophilic granules are composed of l i p i d s as determined by s t a i n i n g with the l i p i d -s p e c i f i c l i g h t microscopic s t a i n , Sudan black B. Light microscopic examina-t i o n of stained g l y c o l methacrylate and Spurr's embedded sections revealed a p o s i t i v e r e a c t i o n for l i p i d s . Much more l i p i d was preserved using the g l y c o l methacrylate'embedding medium. It was dispersed throughout the cyto-plasm and occasionally occurred within vacuoles (Figure 176). Examination of acetone-extracted material embedded i n both media revealed the absence of Sudan black B p o s i t i v e granules (Figure 177). Presumably, the l i p i d substances were extracted by the acetone treatment. Much l i p i d i n r h i z o i d a l c e l l s i s l o s t during dehydrating/embedding procedures employed for electron microscopy. Hollow osmiophilic granules may be the r e s u l t of p a r t i a l chemical leaching of the l i p i d s . Most of the l i p i d s contained within vacuoles are l o s t during electron microscopic preparations, but remnants are seen oc c a s i o n a l l y . Fatty acids and neutral l i p i d s were distinguished with the l i p i d s t a i n N i l e blue A. N i l e blue A consists of two s t a i n i n g components, oxazine and oxazone. Oxazine stains f a t t y acids bright blue, and oxazone stains neutral 124 «• Figures 171 - 173 Storage i n c l u s i o n s i n r h i z o i d a l c e l l s of H. mivab-ilis. Figure 171. Floridean starch granule (FS) i n primary vacuole. Numerous v e s i c l e s (ve) surround the granule surface. Figure 172. Plastic-embedded t h i c k section, stained with PAS. Floridean starch (FS) (arrows) present i n cytoplasm and primary vacuoles. Figure 173. Spherical osmiophilic granules ( L i p i d s , L)' dispersed i n cytoplasm. Floridean starch (FS) also present i n cytoplasm. A l l measurements i n micrometers (ym) 124 125 K Figures 174 - 177 Storage i n c l u s i o n s i n r h i z o i d a l c e l l s of H. mirabilis. Figure 174. Osmiophilic granule (Og) l a c k i n g bounding membrane. Figure 175. Hollow osmiophilic granule, probably the r e s u l t of p a r t i a l chemical leaching of osmiophilic substance. Figure 176. G l y c o l methacrylate-embedded thick section, stained with Sudan black B. L i p i d s (L) are indic a t e d i n r h i z o i d a l c e l l s penetrating i n t o adjacent host medullary c e l l (hmc). Inset: L i p i d s i n vacuole and cytoplasm of r h i z o i d a l c e l l attached to host medullary c e l l by secondary p i t connection. Figure 177. Control treatment f o r l i p i d l o c a l i z a t i o n . L i p i d s absent i n acetone-extracted materials stained with Sudan black B. A connection i s shown between two host medullary c e l l s (hmc) separated by i n t e r d i g i t a t i n g Harveyella r h i z o i d a l c e l l s (arrow). A connection from an adjacent H. mirabilis c e l l j o i n s the connection between host c e l l s ( a s t e r i s k ) . A l l measurements i n micrometers (ym) 125 126 l i p i d s dark red. The oxazone reaction i s f a i r l y p o s i t i v e , but as pointed out by Tainter (1971), oxazine ( N i l e blue sulfate) which combines r e a d i l y with f a t t y acids, also binds at a number of other c e l l s i t e s . Thus, p o s i t i v e r e s u l t s for f a t t y acids must be interpreted with caution. Neutral l i p i d s appeared i n vacuoles of Harveyella r h i z o i d a l c e l l s and around the periphery of i n f e c t e d host c e l l s (Colour plate 3). A p o s i t i v e reaction for f a t t y acids occurred p r i m a r i l y i n large r h i z o i d a l c e l l vacuoles (Figure 178, colour p l a t e 3). C e l l u l a r debris i n the "autophagic vacuole" (page 118) reacted intensely with the oxazone component (Colour p l a t e 4). Dense osmiophilic i n c l u s i o n s with p e r i p h e r a l electron-transparent regions are found i n the cytoplasm and vacuoles of Harveyella r h i z o i d a l c e l l s and medullary c e l l s of 0. floeeosa and 0. washingtoniensis (Figures 179, 180, 181). The s p h e r i c a l granules vary l i t t l e i n diameter (0.3 to 0.7 ym) and are present only i n plants c o l l e c t e d i n the spring and early summer. They do not appear to be membrane-bound and consist of polyphosphate according to the t o l u i d i n e blue t e s t (Keck and S t i c h , 1957)* (Colour plate 5). Structur-a l l y s i m i l a r granules have been described i n Trebouxia erici Ahmadjian (Chlorophyceae) (Fisher, 1971), Gomphonema parvulvon Kiitz (Bacillariophyceae) (Dawson, 1973), Nostoc pruniforme Ag. (Cyanophyceae)(Jensen, 1968) and Anabaena sp. (Cyanophyceae) (Lang and Fisher, 1969). Dense granular areas frequently occur throughout the cytoplasm of r h i z o i d -a l c e l l s (Figures 182, 183). The electron dense material may lack bounding membranes or be delimited by d i l a t e d ER membranes (Figure 183). ER, micro-body-like organelles and "autophagic v e s i c l e s " are i n v a r i a b l y associated with the unbound granular substance (Figure 182). These granular regions s t a i n positive ' " (S lue)' f o r proteins with mercuric bromphenol blue (Figures 184a, b). 127 ^ Figures 178 - 181 Storage i n c l u s i o n s i n r h i z o i d a l c e l l s of H. mivabitis. Figure 178. Plastic-embedded thick section, stained with N i l e blue A. Fatty acids are indi c a t e d i n primary vacuole (1°V) corres-ponding to debris contained i n primary vacuole. C e l l w a l l (CW) i s i n d i c a t e d . Figure 179. Dense osmiophilic i n c l u s i o n (polyphosphate granule, pp) with p e r i p h e r a l electron-transparent regions. Figure 180. Polyphosphate granule (pp) i s associated with d i l a t e d ER near p i t connection with adjacent Harveyella. c e l l . Figure 181. Polyphosphate granule (pp) with p e r i p h e r a l e l e c t r o n -transparent regions and lacking a bounding membrane. A l l measurements i n micrometers (ym) 127 128 * Figures 182 - 183 Storage i n c l u s i o n s i n r h i z o i d a l c e l l s of H. mirabilis. Figure 182. Electron-dense granular material (GP, globular protein) i s common i n cytoplasm. ER, microbody-like organelles (mb) and primary vacuole with c e l l u l a r debris (de) i n surround-i n cytoplasm. Figure 183. Electron-dense granular material (GP) surrounded by d i l a t e d ER. A l l measurements i n micrometers (ym) 128 1 2 9 ^ Figures 184 - 185 Storage i n c l u s i o n s i n r h i z o i d a l c e l l s of H. mirabilis. Figure 184a,b. Plastic-embedded thick-sections, stained with mercuric chloride bromphenol blue. Granular electron-dense sub-stance seen i n corresponding EM sections s t a i n . p o s i t i v e l y f o r proteins (arrows, GP). Figure 185. Non-membrane-bound c r y s t a l l i n e body (CB) i n cytoplasm. C r y s t a l l i n e l a t t i c e structure evident. A l l measurements i n micrometers (ym) 129 130 A regular feature of r h i z o i d a l c e l l cytoplasm i s c r y s t a l l i n e bodies ranging i n s i z e from 0.2-0.8 ym and lacking a bounding membrane. A c r y s t a l l a t t i c e i s formed from linked subunits which are orientedoJ to form p a r a l l e l or cross-hatched l i n e s (Figure 185). The centre to centre distance between p a r a l l e l l i n e s measures 12.8-13.0 n/m and the angle of subunit i n t e r s e c t i o n i s 82-85°. The chemical nature of these structures has not been cytochemi-c a l l y determined. Similar c r y s t a l l i n e bodies have been reported i n the p l a s t i d , pyrenoid and cytoplasm of a number of algae (Holdsworth, 1968; Stein and Bi s a l p u t r a , 1969; Kowallik, 1969; B e r t a g n o l l i and Nadakavukaren, 1970; Retallack and Butler, 1970; Kugrens, 1971; ..Burr and West, 197If T r i p o d i , 1971; McBride and Cole, 1972; Kugrens and West, 1972a, 1973b; Markowitz and Hoffman, 1974). In the red algae membrane-bound c r y s t a l l i n e bodies occur i n mature carpospores and tetraspores where they are thought to serve as reserve protein ( T r i p o d i , 1971b; Kugrens, 1971). The only cytoplas-mic c r y s t a l l i n e bodies that are not membrane bound have been reported i n Gonimophyllum skottsbergii ((Kugrens, 1971) and i n the present study of Harveyella mirab-ilis. Cytoplasmic microtubules are found i n the peripheral cytoplasmic areas of r h i z o i d a l c e l l s of Harveyella but never i n great number. Tangential sections near the plasmalemma reveal the microtubules, arranged p a r a l l e l to the long axis of the c e l l and measuring approximately 14-15 nm i n diameter (Figure 186). These microtubules are most often encountered i n r a p i d l y elongating c e l l s of the r h i z o i d a l filament i n c e l l s engaged i n secondary vacuole formation. 131* Figures 186 - 189 Microtubules and s o l i t a r y - b o d i e s i n r h i z o i d a l c e l l s of H. mirabilis. Figure 186. Tangential section near plasmalemma. Cytoplasmic micro-tubules are arranged p a r a l l e l to the long axis of the c e l l ; microtubules i n cross-section are indicated by a s t e r i s k . Figure 187. Spherical cytoplasmic i n c l u s i o n s ( s o l i t a r y - b o d i e s or S-bodies, SB) associated with ER. C r y s t a l l i n e body (CB) i s present. Figure 188. S-body (SB) i n mitochondrion. Figure 189. S-body (SB) associated with mitochondrial membrane. A s t e r i s k i n d i c a t e s S-body connected d i r e c t l y to the outer mitochondrial membrane. A l l measurements i n micrometers (ym) 131 132 d. Spherical (S)-Bodies Unusual s p h e r i c a l structures, a l l s i m i l a r i n s i z e and u l t r a s t r u c t u r e , are abundant i n a l l H. mivabilis r h i z o i d a l c e l l s (Figures 187-199) and i n c o r t i c a l and medullary c e l l s of the pustule. They have been found i n carpo-spores, tetraspores and spermatia of H. mirabilis, and i n small numbers i n host c e l l s connected to Harveyella c e l l s by secondary p i t connections. I d e n t i c a l cytoplasmic i n c l u s i o n s have been reported i n the angiosperms, Epilobium (Anton-Lamprecht, 1965, 1966, 1967) and Tropaeolum (Ie, 1964, 1965, 1972), i n the p a r a s i t i c angiosperm Orabanohe ramosa (DSrr and Kollmann, 1974) and i n the lycopod Selaginella (Sigee,*1974). Because of the general tendency of these organelles to remain unassociated with one another, they have been termed " s o l i t a r y or S-bpdies" (Ie, 1965). In Harveyella r h i z o i d a l c e l l s , S-bodies are usually dispersed throughout the cytoplasm but may also be associated with tubular ER (Figure 187). They occur infrequently within the nucleus, p l a s t i d s , and mitochondria (Figures 188, 189) and primary and secondary vacuoles (Figures 190, 191). S-bodies i n Harveyella (and i n infec t e d host c e l l s ) vary i n diameter from 60-80 nm, s l i g h t l y larger than those described i n other, reports. In s e r i a l section they appear to be sp h e r i c a l or s l i g h t l y elongate (Figure 192) and consist of three zones (Figure 193): (a) a l i m i t i n g double membrane composed of two electron dense layers each having a mean thickness of 2 nm and separated by a 4 nm zone of low electron density; (b) a granular mantle zone with a mean radius of 20 nm; (c) an approximate 10 nm c e n t r a l core which may appear tubular with either an electron-transparent or—dense lumen. Several f i n e f i b r i l s , l e s s than 1 nm i n diameter, r r a d i a t e from the c e n t r a l core, traverse the mantle and appear to j o i n the inner membrane. 133^ Figures 190 - 193 S o l i t a r y - b o d i e s i n r h i z o i d a l c e l l s of H. mirabilis. Figures 190-191. S-bodies a s s o c i a t e d wi th secondary .vacuole . Tubular c e n t r a l core with r a d i a t i n g f i b r i l s i s e v i d e n t . F igures 192-193. U l t r a s t r u c t u r e of S-body: surrounding double membrane N (zone a ) , granular mantle zone (zone b) and c e n t r a l core wi th r a d i a t i n g f i b r i l s (zone c ) . A l l measurements i n micrometers (ym) 133 134 The three zones of the S-body can be distinguished i n negatively stained preparations as we l l as i n t h i n section. No differe n c e was noted i n preparations stained with uranyl acetate or potassium phosphotungstate. A " t a i l " s i m i l a r to that described by Anton-Lamprecht (1965), Ie (1972) and Dorr and Kollmann (1974) adjoined the l i m i t i n g membrane of S-bodies i n nega-t i v e l y stained preparations (Figure 194). This structure could not be found i n thin-sectioned material. The " t a i l " measures 10-12 nm i n diameter at the point of attachment and widens at i t s d i s t a l end. Two dense, oppositely convex bands appear at the attachment point of the " t a i l " (Figure 194, i n s e t ) . In negatively stained preparations, the dense core (zone 3) i s occasionally apparent and the S-body often assumes a hexagonal configuration (Figure 195). The c e n t r a l core and r a d i a t i n g f i b r i l s of S-bodies were s e n s i t i v e to deoxyribonuclease, but not ribonuclease. There appeared to be l i t t l e u l t r a -s t r u c t u r a l change a f t e r 14 hours d i g e s t i o n at 25 or 40 C i n 100 mg/ml RNase (Figures 196, 197). The c e n t r a l core remained i n t a c t and f i b r i l s were s t i l l v i s i b l e . A f t e r 14 hours digestion at 25 C i n 0.4 or 0.2 mg/ml DNase, few or no f i b r i l s remained (Figure 198), and the c e n t r a l core disappeared a f t e r 14 hours dig e s t i o n at 40 C (Figure 199). The a c t i v i t y of both enzymes could be monitored by observing the digestion of the nucleolus i n RNase-treated c e l l s and noting the absence of mitochondrial and p l a s t i d DNA f i b r i l s i n DNase-extracted c e l l s . It. thus appears that DNA may comprise part of a l l of the c e n t r a l core and r a d i a t i n g f i b r i l s of Harveyella S-bodies. These findings contradict those of Anton—Lamprecht i n her study of Epilobium S-bodies (1967) i n which she concluded that S-rbodies were s e n s i t i v e to RNase and a l k a l i n e phosphatase. 135* Figures 194 - 199 Negative staining and enzyme digestion of solitary-bodies in H.. mivabilis. Figure 194. Negative-stained preparation; stained with potassium phos-photungstate. S-body with " t a i l " (arrow) adjoining double membrane (zone a). Inset: two dense, oppositely-convex bands appear at point of attachment of t a i l to outer mem-brane (arrow). Tail widens at di s t a l end. Figure 195. S-body in negative-stained preparation (potassium phospho-tungstate). S-body hexagonal shape and central core (zone c) are evident. Figure 196. Thin-section of S-body (asterisk) after 14 hr digestion in RNase at 25 C. No ultrastructural change; central core s t i l l present. Figure 197. S-body (asterisk) after 14 hr RNase digestion at 40 C. Central tubule and f i b r i l s remain. Figure 198. S-body (asterisk) after 14 hr DNase digestion at 25 C. Central tubule remains but no radiating f i b r i l s are evident. Figure 199. S-body (asterisk) after 14 hr DNase digestion at 40 C. Absence of entire central core apparatus. A l l measurements in micrometers (ym) 135 136 B. Physiological Investigations 1. Analysis of Mass Translocation Plastids of Havveyella are reduced in size and structural complexity indicating that these cells may not be photosynthetically active. However, large quantities of storage metabolites including l i p i d s , proteins and carbo-hydrates have been cytochemically identified in these cells. Fluorescent microscopy failed to reveal the presence of photosynthetic pigments in H. mivabilis although chlorophyll was clearly evident in the cells of the host, Odonthalia. Spectrophotometry analysis of pigmentation was not attempted since i t was not possible to separate Havveyella and Odonthalia to the degree necessary for this analysis. Kugrens (1971) spectrophotometrically analyzed the pigments of the closely related parasitic red alga, Choveoaolax polysi-phoniae Reinsch. A small spectral peak corresponding to chlorophyll a was noted, although i t could not be determined i f this was due to pigments of Choveoaolax or to the inseparable contaminant host pigments. Autoradiography of Havveyella labeled with NaH11+C03 has demonstrated that i f photosynthesis occurs, i t does so at a greatly reduced level. Only background label could be detected in the pustule cells of Havveyella labeled under light conditions for 90 minutes (Figures 200a, b). The ^0 was readily incorporated into host cells dispersed amongst Havveyella cells. Ultrastructural, fluorescent microscopic and autoradiographic studies indicated that photosynthesis contributes l i t t l e to the fixation of carbon into storage metabolites within Havveyella c e l l s . An alternate possibility i s that Havveyella obtains organic carbon from i t s host. A series of trans-location experiments involving Havveyella and i t s host 0. floocosa were 137<L Figures 200, 207-209 Light microscopic autoradiography; uptake of 1 I +C and t r a n s l o c a t i o n i n H. mirabilis and 0. flocoosa. A l l autoradiographs photographed at two f o c a l planes: (a) above the specimen plane, focused on s i l v e r grains, (b) at the specimen plane. A l l incubated i n NaH11+C03 f o r 90 min i n l i g h t . Figure 200a,b. Q, hr t r a n s l o c a t i o n . S i l v e r grains ( i n d i c a t i n g B-emission from l l tC) associated with host c e l l s i n pustule cortex (asterisk) but are absent from adjacent Harveyella c e l l s . Figures 201-206 See pages 139, 140, 143. Figure 207a,b. 0 hr t r a n s l o c a t i o n . Greatest concentration of s i l v e r grains associated with illuminated cortex of Odonthalia. Figure 208a,b. 0 hr t r a n s l o c a t i o n . Few s i l v e r grains associated with 0. flocoosa medulla. Figure 209a,b. 4 hr t r a n s l o c a t i o n . Decrease i n s i l v e r grain density associated with i l l u m i n a t e d host c o r t i c a l c e l l s . A l l measurements i n micrometers (um) 137 138 conducted to ascertain (1) i f materials are translocated from the host to Harveyella, and, (2) what,substances are Involved i n the tr a n s l o c a t i o n process. L i q u i d s c i n t i l l a t i o n counting data of the t o t a l r a d i o a c t i v i t y i n both Odonthalia and Havveyella over various t r a n s l o c a t i o n periods are presented i n Figures 201 and 202. Dark-pretreated as w e l l as l i g h t - p r e t r e a t e d material was used i n t h i s procedure since, as discussed by Neilson and Lewin (1974), dark pretreatment lowers the endogenous l e v e l of storage products thus increas-ing the r e l a t i v e response to exogenous l a b e l , i n t h i s case NaH1 1 +C03. In general, the amount of ^^C i n Odonthalia decreased over 36 hours while i t increased i n Havveyella (Figures 201, 2020 • This o v e r a l l pattern occurred i n both l i g h t - p r e t r e a t e d and dark-pretreated materials. I n i t i a l uptake of i^ jG.4. was greater i n dark-pretreated than i n l i g h t - p r e t r e a t e d Odonthalia. Differences between l i g h t - and dark-pretreated materials also occurred i n the rate of i n i t i a l decrease of r a d i o a c t i v i t y i n Odonthalia.over the f i r s t s i x hours of t r a n s l o c a t i o n . No s i g n i f i c a n t d i f f e r e n c e was noted between dark-and l i g h t - p r e t r e a t e d Havveyella i n the i n i t i a l uptake of l a b e l , although a greater f l u c t u a t i o n i n the t o t a l r a d i o a c t i v i t y occurred a f t e r 6 hours i n l i g h t -pretreated Havveyella than i n dark-pretreated material. The seawater i n which Havveyella and Odonthalia were incubated during tr a n s l o c a t i o n was sampled to determine the amount of l a b e l l o s t to the medium from both dark- and l i g h t - p r e t r e a t e d material (Figure 203). The r a d i o a c t i v i t y of the seawater gradually increased over 36 hours. At the end of 36 hours tr a n s l o c a t i o n , more l a b e l was found i n the culture media from dark-pretreated mat e r i a l s than from l i g h t - p r e t r e a t e d plants. The amount of t o t a l r a d i o a c t i v i -ty i n the seawater a f t e r 36 hours t r a n s l o c a t i o n was approximately 100 times 139* Figures 201 - 202 Translocation of lhC between Odonthalia floooosa and Harveyella mirabilis over 36 h r 1 . Figure 201. Dark pretreatment p r i o r to l a b e l i n g . Figure 202. Ligh t pretreatment p r i o r to l a b e l i n g . Although t h i s experiment was repeated 3 times, the r e s u l t s presented i n Figures 201-205 represent the r e s u l t s of one experiment. P h y s i o l o g i c a l d i f f e r e n c e s i n plants c o l l e c t e d at d i f f e r e n t times of "the year caused diff e r e n c e s i n the amount of i n i t i a l uptake of 1 1 +C. When a comparison i s made of the percentage increase and decrease i n r a d i o a c t i v i t y i n H. mirabilis and 0. floooosa i n each experiment, the same trend of l l fC increase i n H. mirabilis and a concomitant decrease i n 0. floooosa i s evident. Each point of graphs 201-205 represents a mean value of r a d i o a c t i v i t y i n r e p l i c a t e samples. Since these values (dpm/gm) d i f f e r -ed only s l i g h t l y , no s t a t i s t i c a l t e s t s were conducted. 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Translocation time (hrs) 8H1 OH Light Pretreatment 90 min. label in NaH14COj F i g . 202 I • I I I I I I I I I I I I I I I I 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Translocation time (hrs) 140* Figures 203 - 205 Experimental c o n t r o l s . Figure 203. R a d i o a c t i v i t y i n seawater during 36 hr t r a n s l o c a t i o n . Figure 204. Light incubated c o n t r o l s . R a d i o a c t i v i t y i n i n f e c t e d and uninfected 0. flocoosa and i n H. mirabilis with and without subtending, host ti s s u e during 24 hr t r a n s l o c a t i o n . Figure 205. Dark incubated c o n t r o l s . R a d i o a c t i v i t y i n . i n f e c t e d and uninfected 0. floooosa and i n H. mirabilis with and without subtending host t i s s u e during 24 hr t r a n s l o c a t i o n . X E a T J >t •4-" « O •5 cc 140 Radioactivity in Seawater During Translocation - • Dark Pretreatment -O Light Pretreatment 12 16 20 24 28 Translocation time (hrs) I i I 32 36 F i g . 203 Light Incubated Controls DARK PRETREATMENT 0 + 0 12 16 F i g . 204 a -a 6-1 Harveyella with host Odonthalia. infected Dark Incubated Controls DARK PRETREATMENT 20 24 Translocation time (hrs) 12 16 20 24 F i g . 205 141 less than i n the tissues of Odonthalia and therefore suggests that, the medium was not d i r e c t l y involved i n the tra n s l o c a t i o n process. Controls co n s i s t i n g of infected and uninfected Odonthalia and Havveyella with and without subtending host material were incubated i n NaH-^GOs under l i g h t and dark conditions and were placed i n unlabeled medium for various tra n s l o c a t i o n periods. In light-incubated controls (Figure 204), l i t t l e l a b e l was incorporated into Havveyella which lacked subtending host t i s s u e s . During the f i r s t 24 hours a f t e r feeding i i +C, the t o t a l r a d i o a c t i v i t y gradually decreased, being l o s t to the culture medium. In contrast, the r a d i o a c t i v i t y i n Havveyella with subtending host t i s s u e s gradually increased over 24 hours. Uninfected light-incubated Odonthalia showed a small loss i n r a d i o a c t i v i t y over 24 hours t r a n s l o c a t i o n but s u b s t a n t i a l l y l e s s than infe c t e d Odonthalia. The i n i t i a l uptake of l a b e l was greater i n infected than i n uninfected Odonthalia (Figure 204, 0 h r s ) . As i n the light-incubated c o n t r o l , the r a d i o a c t i v i t y i n dark-incubated control Havveyella decreased gradually over 24 hours a f t e r feeding 1 4 C (Figure 205). In contrast, the r a d i o a c t i v i t y i n Havveyella with subtending host „ i n i t i a l l y decreased during the f i r s t 8 hours of tra n s l o c a t i o n and gradually increased between 8-24 hours. In both uninfected and infected dark-incubated Odonthalia, the i n i t i a l uptake of l a b e l was s u b s t a n t i a l l y l e s s than i n l i g h t -incubated Odonthalia. Uninfected Odonthalia showed a pattern of gradual los s of r a d i o a c t i v i t y s i m i l a r to light-incubated uninfected Odonthalia. A s i m i l a r pattern of r a d i o a c t i v i t y loss occurred i n both l i g h t - and dark-incubated infected' Odonthalia. Autoradiographic data of the tr a n s l o c a t i o n of -^C labeled compounds over 24 hours support the data obtained from l i q u i d s c i n t i l l a t i o n studies of trans-142 l o c a t i o n . To conserve as much soluble labeled compounds as possibl e , the material used f o r autoradiographic analysis was f i x e d according to the osmium v a p o u r - c r i t i c a l point drying method presented on page 13. The data obtained from counting s i l v e r grains deposited over sections of f i x e d and embedded Han)eyelid and subtending host t i s s u e are presented i n Figure 206a. In t h i s study, counts were made from ti s s u e zones indicated i n Figure 206b. Zones 1, 2 and 3 corresponding r e s p e c t i v e l y to shaded c o r t i c a l c e l l s , illuminated c o r t i c a l c e l l s and medullary c e l l s of Odonthalia, and zones 4-7 include v a r i -ous c e l l u l a r regions i n Havveyella. A f t e r 90 minutes l a b e l i n g i n NaH-^COs (0 hrs t r a n s l o c a t i o n ) , the greatest density of s i l v e r grains (corresponding to incorporated l t + C ) was associated with illuminated c o r t i c a l c e l l s of Odonthalia (zone 3) (Figures 206a, 207a, b). Few s i l v e r grains were associated with the medulla of Odonthalia (Figures 208a, b) or with c e l l s of Havveyella (Figures 200a, b). A f t e r 4 hours trans-l o c a t i o n , the density of s i l v e r grains i n the cortex decreased (Figures 206a, 209a, b), while i t increased i n the medulla of Odonthalia (zone 2) (Figures 206a, 210a, b), i n the medullary c e l l s adjacent to r h i z o i d a l c e l l s of Havveyella (zone 5) (Figures 206a, 211a, b) and i n Havveyella adjacent to illuminated c o r t i c a l c e l l s of Havveyella (zone 4) (Figures 206a). The density of s i l v e r grains continued to decrease i n the illuminated cortex of Odonthalia throughout the e n t i r e 24 hour t r a n s l o c a t i o n period (Figures 212a, b) while i t increased i n a l l other zones. Af t e r 8 hours, s i l v e r grains were associated with the wa l l i n t e r f a c e separating Odonthalia and Havveyella and were occasionally associated with the plasmalemma and adjacent secondary vacuole of Havveyella r h i z o i d a l c e l l s (Figures 213a, b). Aft e r 24 hours t r a n s l o c a t i o n , s i l v e r grains were associated with starch grains i n the 143 *~ Figure 206 a. Autoradiography of translocated 1 4 C . Density of s i l v e r grains associated with t i s s u e regions of H.. mirabilis and 0. floooosa a f t e r various t r a n s l o c a t i o n periods. Figure 206 b. Tissue regions from which s i l v e r grains were counted. 143 b a Translocation time (hrs) Fig. 206 1 4 4 ^ Figures 210 - 213 Light microscopic autoradiography; uptake of 1 L fC and t r a n s l o c a t i o n i n H. mirabilis and 0. floooosa Figure 210a,b. 4 hr t r a n s l o c a t i o n . Increase i n s i l v e r grain density associated with host medullary c e l l s . Figure 211a,b. 4 hr t r a n s l o c a t i o n . Increase i n s i l v e r grain concentra-t i o n over host medullary c e l l s i n i n t e r d i g i t a t i o n zone. Host medullary c e l l (hmc), Harveyella r h i z o i d a l c e l l (Hrc) and p i t connection (PC) in d i c a t e d . Figure 212a,b. 24 hr t r a n s l o c a t i o n . Continued decrease i n s i l v e r grain density associated with i l l u m i n a t e d host c o r t i c a l c e l l s . Figure 213a,b. 8 hr t r a n s l o c a t i o n . S i l v e r grains associated with w a l l i n t e r f a c e between Harveyella and Odonthalia c e l l s and with plasmalemma and plasmalemma-associated secondary vacuoles i n Harveyella (arrow). A l l measurements i n micrometers (ym) 144 1 4 5 r h i z o i d a l c e l l s of Earveyella (Figures 214a, b). A dense deposition of s i l v e r grains occurred a f t e r 24 hours over the medullary c e l l s of Earveyella (zone 6) (Figures 206a, 215a, b), decreasing i n density i n the cortex of Earveyella (zone 7) (Figures 206a). From these data, a hypothesized flow scheme for ^^C-labeled compounds may be proposed (Figure 216). A secondary f l u x of metabolites may occur between i s o l a t e d Odonthalia c e l l s i n the pustule of Earveyella and the adjacent c e l l s of Earveyella. A dense concentration of s i l v e r grains i s i n i t i a l l y associated with the i s o l a t e d host c e l l s i n the pustule (Figures 217a, b). S i l v e r grains are found i n Earveyella c e l l s adjacent to the i s o l a t e d host c e l l s as early as 4 hours a f t e r the s t a r t of translocation (Figures 218a, b). Thus t h i s secondary f l u x of metabolites may provide nutrients for Earveyella c e l l s i n l o c a l i z e d areas of the pustule, while the primary flow patterns (Figure 216) would account for the more massive movement of substances from the subtending host c e l l s to the c e l l s of the pustule. In both cases, the flow of nutrients from Odonthalia to Earveyella i s r e l a t i v e l y l o c a l i z e d and i s dependent upon Earveyella being i n close proximity to-O d o n t h a l i a . 2. Analysis of Translocated Compounds Compounds from Earveyella pustules and subtending Odonthalia were separ-ated by ion exchange chromatography, solvent extraction and enzyme digestion into component f r a c t i o n s to ascertain what labeled substances may be trans-located from Odonthalia to Earveyella. These data are presented i n Figures 219-222. The d i s t r i b u t i o n of l a b e l i n ether-soluble compounds and i n the residue that remained a f t e r ether and alcohol extractions followed by amylase digestion (see pages 22-23) are presented i n Figure 220; r a d i o a c t i v i t y i n 1 4 6 * Figures 214-215, 217-218 Light microscopic autoradiography; uptake of 1 1 +C and t r a n s l o c a t i o n i n H. mivabilis and 0. floocosa Figure 214a,b. 24 hr t r a n s l o c a t i o n . S i l v e r grains associated with starch grains i n r h i z o i d a l c e l l s of Havveyella (arrow). F l o r i -dean starch (FS), nucleus (N) and host medullary c e l l (hmc) i n d i c a t e d . Figure 215a,b. 24 hr t r a n s l o c a t i o n . Dense concentration of s i l v e r grains i n medulla of Havveyella pustule". Figure 216. See page 147. Figure 217a,b. Secondary f l u x of 1 I +C; 0 hr t r a n s l o c a t i o n . Dense con-centration of s i l v e r grains associated with host medu-l l a r y c e l l s i n outer pustule of Havveyella. Few s i l v e r grains associated with Havveyella c e l l s . Figure 218a,b. Secondary f l u x of l l tC; 4 hr t r a n s l o c a t i o n . S i l v e r grains associated with Havveyella c e l l s of pustule adjacent to i s o l a t e d host c e l l s . A l l measurements i n micrometers (ym) 146 147* Figure 216. Hypothesized flow scheme for ;.luC-labeled metabolites from 0. floocosa. to H. mivabilis . - • 147 Primary Nutrient Flow Pattern Secondary Nutrient Flow Pattern 148*-Figures 219-222. D i s t r i b u t i o n of i n separated f r a c t i o n s from Odonthalia floooosa. and Earveyella m i r a b i l i s a f t e r various periods of t r a n s l o c a t i o n . Figure 219. Change i n t o t a l r a d i o a c t i v i t y over 24 hr t r a n s l o c a t i o n (sum of a l l component f r a c t i o n s ) . Figure 220. Change i n r a d i o a c t i v i t y associated with the ether extract f r a c t i o n and the i n s o l u b l e residue. Figure 221. Change i n r a d i o a c t i v i t y associated with the c a t i o n i c and anionic f r a c t i o n s . Figure 222. Change i n r a d i o a c t i v i t y associated with the ne u t r a l sugars and starch f r a c t i o n s . TRANSLOCATION TIME (hours) 149 the cation and anion f r a c t i o n i s shown i n Figure 221; and neutral sugars and starch l a b e l i n Figure 222. The t o t a l r a d i o a c t i v i t y (sum of a l l component f r a c t i o n s over 24 hours translocation) i s presented i n Figure 219. Floridean starch may be excluded as a possible translocated substance because of i t s i n s o l u b l e nature, although i t s i n t e r a c t i o n i n metabolic exchange pools i n both H a r v e y e l l a and O d o n t h a l i a cannot be ignored. Radio-a c t i v i t y i n O d o n t h a l i a starch (Figure 222) markedly decreases over 24 hours. This may in d i c a t e that reserve starch i n O d o n t h a l i a i s being converted into soluble compounds or at l e a s t i t i s turning over. The s l i g h t increase i n the r a d i o a c t i v i t y i n f l o r i d e a n starch of H a r v e y e l l a may s i m i l a r l y i n d i c a t e that labeled soluble compounds are being converted into the storage compound, f l o r i d e a n starch. This p o s s i b i l i t y i s further substantiated by autoradio-graphic data (page 146 Figures 214a, b). The increase i n r a d i o a c t i v i t y of H a r v e y e l l a residue a f t e r 8 hours of tra n s l o c a t i o n may likewise denote a conversion of labeled soluble compounds into i n s o l u b l e residue. The sharp r i s e i n r a d i o a c t i v i t y of the neutral sugars i n H a r v e y e l l a and the sharp decrease i n r a d i o a c t i v i t y i n f l o r i d e a n starch of O d o n t h a l i a may in d i c a t e that labeled starch i n O d o n t h a l i a i s being converted to soluble n e u t r a l sugars and translocated to H a r v e y e l l a . A l t e r n a t e l y , the sharp increase i n r a d i o a c t i v i t y i n H a r v e y e l l a neutral sugars may simply r e f l e c t an intercon-version of substances within H a r v e y e l l a . S i m i l a r l y , the increase i n l a b e l i n the c a t i o n i c f r a c t i o n (Figure 221) of H a r v e y e l l a may in d i c a t e that i t i s involved i n tr a n s l o c a t i o n or i t may again represent an interconversion of labeled substances within H a r v e y e l l a . Of a l l f r a c t i o n s analyzed, the Increase i n r a d i o a c t i v i t y i n the neutral sugar f r a c t i o n of H a r v e y e l l a most c l o s e l y p a r a l l e l s the t o t a l increase i n 150 r a d i o a c t i v i t y f o r Earveyella over 24 hours tr a n s l o c a t i o n as represented i n Figure 219. These data i n d i c a t e that the neutral sugar f r a c t i o n may be involved.in the t r a n s l o c a t i o n process. In addition, cytochemical evidence (page 109) supports the hypothesis that the asoluble n e u t r a l f r a c t i o n may be involved i n t r a n s l o c a t i o n . An an a l y s i s of the neutral sugar f r a c t i o n was conducted to determine what s p e c i f i c substances might be involved i n t r a n s l o c a t i o n . The components of unlabeled n e u t r a l f r a c t i o n s of Earveyella and Odonthalia were i d e n t i f i e d using paper chromatography as previously described (page 23) . The components i d e n t i f i e d from each neutral f r a c t i o n are l i s t e d i n Tables VII and VIII. Radiochromatograms of ra d i o a c t i v e n e utral f r a c t i o n s from each t r a n s l o c a t i o n period were prepared and analyzed as described on page 23. In addition, the n e u t r a l f r a c t i o n from c o n t r o l Earveyella and Odonthalia samples were analyzed. Labeled compounds from Earveyella and Odonthalia n e u t r a l f r a c t i o n s were i d e n t i f i e d by c a l c u l a t i n g R g i u c o s e values f o r each radiochromatogram spot and comparing these values against standard R g j u c o s e values (Appendix IV) . A quantitative value representing the percentage of the t o t a l recovered radio-a c t i v i t y (percent recovery of t o t a l dpm) was calculated f o r each compound; The percent recovery of t o t a l dpm for each labeled compound from both Odonthalia and Harveyella n e u t r a l f r a c t i o n s a f t e r 0, 4, 8 and 24 hours tr a n s l o c a t i o n i s presented i n Figures 223-226. Some general trends i n the d i s t r i b u t i o n of r a d i o a c t i v i t y i n each n e u t r a l f r a c t i o n are obvious although i t must be noted that these changes may r e f l e c t transfer of carbon into other metabolic pools within an i n d i v i d u a l plant, as 151 TABLE VII Earveyella mirabilis Neutral Fraction Solvent system Compound . Basic Acidic Galacturonic acid +? Glucoronic acid + +? Trehalose + + Maltose + — Isofloridoside + + Floridoside/glucose + -Sucrose + + Glucose + — Mannitol + + Fructose/Arabinose + — Xylose - + + present +? presence questionable (overlapping) - absent TABLE VIII Odonthalia floocosa Neutral Fraction Solvent system Compound Basic Acidic Galacturonic acid +? + Glucuronic acid + — Trehalose + + Lactose + — m-inositol + +? Sucrose - + Isofloridoside - + Glucose + + Floridoside + + Mannitol + + Mannose + +? Xylose - + Fucose — + Ribose — + + present +? presence questionable (overlapping) - absent 1 5 2 « . Figures 223-224. Percentage recovery of t o t a l r a d i o a c t i v i t y from chromatographically separated f r a c t i o n . o f Earveyella mirabilis. Figure 223.. ,E. mirabilis neutral f r a c t i o n , a c i d i c solvent system. Figure 224. E. mirabilis neutral fraction., basic solvent system. 80 70 60 50 40 30 20 10 0 80 70 60 50 40 30 20 10 0 152 Harveyella mirabilis neutral fraction Cacidic solvent system ) Translocation Time (hrs) 0 V\\\\\\\\\\\ 4 H f l U E f e - l n Bs-ffll F i g . 223 TREHALOSE SUCROSE GALACTURONIC FL0R ID0S IDE MA NN IT0L UNKNOWN ACID & GLUCOSE & 8 GLUCOSE GLUCURONIC A. Harveyella mirabilis neutral fraction Cbasic solvent system ) F i g . 224 UNKNOWN GALACTURONIC UNKNOWN TREHALOSE SUCROSE GALACTOSE FLORIDOSIDE C & D * MANNITOL & y1* GLUCURONIC FLORIDOSIDE GLUCOSE 7T AC'° 153 w e l l as i n d i c a t i n g translocation of labeled compounds between Odonthalia and Harveyella. In Harveyella, an increase In r a d i o a c t i v i t y i n the f l o r i d o s i d e / glucose/glucuronic a c i d region occurs i n the a c i d i c solvent system during the f i r s t 8 hours of tr a n s l o c a t i o n (Figure 223), whereas i n the basic solvent system, the l a b e l increases markedly during the same tr a n s l o c a t i o n period i n an unknown compound (Figure 224). This unknown has a smaller R .. value r ° glucose (slower moving, R =6-8) than galacturonic and glucuronic acids. Evans, Callow and Callow (1973) i n t h e i r study of the red a l g a l p a r a s i t e Holmsella paohyderma also noted r a d i o a c t i v i t y associated with a low R - unknown glucose compound when run i n a basic solvent system s i m i l a r to that used i n the present study. The uronic acids, galacturonic and glucuronic, have small R , values b glucose i n the basic solvent system but have R , values f a l l i n g i n the f l o r i d o -glucose side/glucose region i n the a c i d i c system. The greatest percentage of t o t a l r a d i o a c t i v i t y appears i n the low R ., unknown i n the basic solvent system J glucose J but i s found i n the uronic a c i d region i n the a c i d i c solvent system. The unknown thus reacts chromatographically as a uronic a c i d although p o s i t i v e i d e n t i f i c a t i o n cannot be made on only t h i s c h a r a c t e r i s t i c . Although f l o r i d o -side and mannitol appeared to be involved i n tra n s l o c a t i o n i n Hoilmsella paohy-derma (Evans, Callow and Callow, 1973), no evidence of t h e i r involvement was found i n Harveyella. A d d i t i o n a l evidence i n support of the involvement of the low R , glucose unknown compound i n translocation i s provided by the con t r o l data. A f t e r 8 hours t r a n s l o c a t i o n , d i f f e r e n c e s are noted between Harveyella subtended by Odonthalia and control Harveyella w i t h Odonthalia. In the a c i d i c solvent system (Figure 223), s u b s t a n t i a l l y l e s s r a d i o a c t i v i t y i s found i n the f l o r i d o -154 side/glucose/glucuronic a c i d region i n Havveyella without host (control) than i n Havveyella with host. In addition, a greater percentage of l a b e l i s found i n mannitol i n con t r o l Havveyella than In Havveyella with subtending host. In the basic solvent system (Figure 224), differences are noted i n t o t a l r a d i o -a c t i v i t y between control Havveyella and Havveyella with subtending host i n -the low R .. unknown and i n the galacturonic/glucuronic acid region, glucose 6 ° • During 24 hours t r a n s l o c a t i o n i n Odonthalia, a large decrease i n the percent recovery of t o t a l dpm occurs i n the regions of a high ^ g ^ u c o s e (R =110-114) unknown and mannitol i n the a c i d i c solvent system (Figure 225). A general increase i n the percent r a d i o a c t i v i t y occurs i n the region of galacturonic acid/glucuronic a c i d / g l u c o s e / f l o r i d o s i d e . In the basic solvent system (Figure 226), an increase i n l a b e l occurs i n the g a l a c t o s e / f l o r i d o s i d e region, and i n the mannitol/glucose region, and a marked decrease i n l a b e l occurs i n an unknown with a high R .. value (R =128-135). ° glucose g The r a d i o a c t i v i t y of separated compounds i n con t r o l Odonthalia (uninfect-ed Odonthalia incubated f o r 8 hours) d i f f e r s from inf e c t e d Odonthalia. In the a c i d i c solvent system (Figure 225), a lower percentage of recovered radio-a c t i v i t y i s found i n the high R g i u c o s e unknown/mannitol region and i n the glucuronic acid/galacturonic a c i d / g l u c o s e / f l o r i d o s i d e region of the control Odonthalia than i n infected Odonthalia. In the basic solvent system (Figure 226), a great d i f f e r e n c e i s found i n the high R , (R =128-135) unknown. ' & _ glucose g A s u b s t a n t i a l l y greater percentage of r a d i o a c t i v i t y i s found i n t h i s region i n uninfected Odonthalia than i n infected Odonthalia. In addition, a much lower percentage of r a d i o a c t i v i t y i s found i n the galacturonic/glucuronic a c i d region of uninfected Odonthalia than i n infected .Odonthalia. 155 ^ Figures, 225-226. Percentage recovery of. t o t a l r a d i o a c t i v i t y from the chromatographically separated neutral f r a c t i o n of Odonthalia floooosa. Figure 225. 0. floooosa neutral f r a c t i o n , a c i d i c solvent system. Figure 226. 0. flocoosa neutral fraction,, basic solvent system. E a •o 80 70 60 H I 50 » 4 0 H > o o 30 o ON 20 H 1 0 H Translocation Tims (hrs) minimi o 4 1 8 |*8 155 Odonthalia floccosa neutral fraction Cacidic solvent system) E 3 24 >H8 Odonthalia-uninfected ORIGIN OF SPOT UNKNOWN TREHALOSE GALACTURONIC & GLUCURONIC ACID GLUCOSE FLORIDOSIDE F i g . 225 UNKNOWN F MANNITOL 80-F i g . 226 GALACTURONIC & TREHALOSE GALACTOSE MANNITOL UNKNOWN GLUCURONIC ACID M - I N 0 S I T 0 L FLORIDOSIDE GLUCOSE G l ORIGIN 156 Discussion S t r u c t u r a l and p h y s i o l o g i c a l studies i n d i c a t e that Harveyella mirabilis i s unable to f i x s u f f i c i e n t q uantities of carbon to sustain i t s growth requirements and must therefore r e l y on an external n u t r i e n t source. Photo-synthesis i s greatly reduced i n a l l c e l l s as evidenced by the absence of detectable c h l o r o p h y l l , the presence of "rudimentary p l a s t i d s " s i m i l a r to those described i n h o l o p a r a s i t i c angiosperms (Dodge and Lawes, 1974; Baccarini arid Melandri, 1967), and the reduced rate of carbon-14 f i x a t i o n . A. -1 ^ " F i x a t i o n and Translocation Some carbon f i x a t i o n does occur i n Harveyella pustules (Figures 204, 205). However, i t i s presently unclear i f t h i s f i x a t i o n i s the r e s u l t of photosynthetic a c t i v i t y i n the Harveyella c e l l s since i t i s possible that some host-fixed carbon-14 i s translocated to the pustule from the subtending host during the i n i t i a l 90-minute l a b e l i n g period. A l t e r n a t e l y or i n addition to the r a d i o a c t i v i t y associated with photosynthetic host, c e l l s i n the Harveyella pustule may account for much of the pustule's t o t a l l a b e l . F i x a t i o n of carbon-14 i n the dark occurs i n Harveyella at only a s l i g h t l y lower rate than i n Odonthalia (Figures 204, 205). Approximately 7% of the t o t a l carbon-14 f i x e d by Harveyella (without subtending host) (calculated from Figures IV and V: 1.41 x 10 3/2 x 10 6 dpm/gm) occurs i n the dark. The dark incorporation of -^C-bicarbonate into malate, c i t r a t e , aspartate and glutamate has been described i n a number of marine algae (Joshi, Dolan, Gee and Saltman, 1962; C r a i g i e , 1963; Yamaguchi, Ikawa and Nisizawa, 1966; Akagawa, Ikawa and Nisizawa, 1972a, 1972b). 157 Translocation of carbon-14 compounds i s the primary mechanism by which Harveyella receives organic carbon. Metabolite transfer between 0. floooosa and H. mirabilis has been c l e a r l y demonstrated (Figures 201, 202) although i t has not been determined i f t h i s i s s t r i c t l y a "one-way t r a n s f e r " or i f Harveyella r e c i p r o c a l l y transfers substances to Odonthalia. Translocation of metabolites between Harveyella and Odonthalia occurs d i r e c t l y across the i n t e r d i g i t a t i o n zone (zone III) and subsequently moves p e r i p h e r a l l y i n t o the host pustule (Figure 216). No evidence could be obtained (from monitoring the r a d i o a c t i v i t y of the culture medium throughout- translocation) to support the contention that carbon-14 i s simply released from the host into the medium where i t i s secondarily recovered by the parasite, as suggested by H a r l i n (1973a) i n her study of Gonimophyllum on Botryoglossum. According to Smith, Muscatine and Lewis (1969), the bulk of f i x e d carbon which moves from autotroph to heterotroph i n most symbiotic associations i s i n a s i n g l e compound, a carbohydrate. Evans, Callow and Callow (1973) deter-mined that the majority of translocated C-14 from Graoilaria to Holmsella was i n f l o r i d o s i d e but that t h i s compound was subsequently converted 'into mannitol i n Holmsella. No evidence of a s i m i l a r f l o r i d o s i d e exchange, mannitol conversion system, was found i n the present study. Rather, an increase i n l a b e l over 24 hours tr a n s l o c a t i o n occurred i n a low R .. unknown glucose (unknown C, basic system, Figure 224). This increase was also i d e n t i f i e d i n the a c i d i c system (Figure 223) i n the glucuronic a c i d / f l o r i d o s i d e / g l u c o s e zone. The unknown compound may not be translocated from the host but may merely represent a conversion substance formed by Harveyella a f t e r t r a n s l o c a t i o n . I t has been postulated that the conversions of translocated compounds into a l t e r n -ate forms, maintains concentration? gradients, bringing about a constant flow 158 of metabolites from host to parasite (Smith, Muscatine and Lewis, 1969; Tainter, 1971; Evans, Callow and Callow, 1973). A rapid decrease i n the r a d i o a c t i v i t y of a high R g ^ u c o s e compound (Figures 225, 226, unknowns F and G) i n Odonthalia may i n d i c a t e that t h i s compound i s involved i n tra n s l o c a t i o n or again, may simply represent a compound which i s converted to another before t r a n s l o c a t i o n . Gas chromatographic analysis of labeled compounds i n both Havveyella and Odonthalia must be undertaken to as c e r t a i n the nature of the various unknowns before any conclusions can be reached concerning the nature of the translocated compound. B. Proposed Nutrient Uptake System As previously mentioned, organic carbon i s translocated from host to para-s i t e c e l l s i n the i n t e r d i g i t a t i o n zone (zone I I I ) . An u l t r a s t r u c t u r a l study of Havveyella r h i z o i d a l c e l l s has revealed a high degree of u l t r a s t r u c t u r a l s p e c i a l i z a t i o n which may be r e l a t e d to the processes of nutrient procurement. P i t connections between adjacent c e l l s have been proposed to act as channels through which food i s transported ( F r i t s c h , 1945). Fan (1961) contends that t r a n s l o c a t i o n of metabolites from host to par a s i t e occurs across the p i t connection. U l t r a s t r u c t u r a l evidence does not support t h i s a s s e r t i o n . The p i t connections between c e l l s of Havveyella and Odonthalia are plugged with an electron-dense matrix s i m i l a r to that described i n Choveoaolax attached to Polysivhonia (Kugrens and West, 1973). A t r i p a r t i t e membrane i s present on the "Havveyella-slde" of the plug, separating i t from the cytoplasm. The presence of connections between host and par a s i t e c e l l s i s not axiomatic to metabolite t r a n s l o c a t i o n since parasites which lack host-parasite connections are s t i l l capable of obtaining translocated nutrients (Evans, 159 Callow and Callow, 1973). An a l t e r n a t i v e to d i r e c t cytoplasmic exchange would be that metabolites can move out of the donor c e l l and into the adjacent w a l l matrix. They then would move across the wa l l i n t e r f a c e and enter through the plasmalemma of the r e c i p i e n t p a r a s i t e c e l l . This mechanism has been proposed f o r other symbiotic systems (Smith, 1969) but presently there i s no d i r e c t evidence i n i t s support. In studies of pathogenic fungal a s s o c i -ations, Wheeler and Hanchey (1968) reported the induction of host c e l l permeability i n p a r a s i t e - i n f e c t e d t i s s u e . Such a system could provide f o r the exudation of carbohydrates from i n f e c t e d host c e l l s into the wa l l matrix, and into the adjacent p a r a s i t e c e l l s . This system would require a " f l u i d - t y p e " of c e l l w a l l . Although c e l l walls have been c l a s s i c a l l y conceived as highly structured " n o n - f l u i d " i n e r t layers of s t r u c t u r a l carbohydrates (Wheeler and Hanchey, 1968), current research has demonstrated that they may be composed of l i p i d s , proteins, and a v a r i e t y of carbohydrates, water-bound into " f l u i d " c o l l o i d a l arrays (Siegel and Si e g e l , 1973). The c e l l walls surrounding Harveyella r h i z o i d a l and medullary c e l l s as w e l l as Odonthalia medullary c e l l s i n infected regions are s t r u c t u r a l l y amorphous. They lack the complex array of interconnecting m i c r o f i b r i l s which occur i n the walls of Harveyella c o r t i -c a l c e l l s . It i s possible that nutrients are released from the host, Odonthalia, into and across t h i s " f l u i d " w a l l matrix. U l t r a s t r u c t u r a l studies of Harveyella c e l l s located at various distances from the nutrient source (host) have revealed a high degree of s t r u c t u r a l s p e c i a l i z a t i o n i n the r h i z o i d a l c e l l s of the i n t e r d i g i t a t i o n zone. The presence of a unique, membrane-associated vacuolar system and numerous storage i n c l u s i o n s not commonly found i n c o r t i c a l or medullary c e l l s may r e f l e c t the fun c t i o n a l s p e c i a l i z a t i o n of these c e l l s . A hypothetical system for n u t r i e n t 160 exchange between Odonthalia and Havveyella c e l l s i n the zone of i n t e r d i g i t a -t i o n (zone III) may be proposed. In t h i s system, host carbohydrates are released i n t o the surrounding amorphous wall matrix. This release may be a t t r i b u t e d to parasite-induced changes i n the permeability of the host plasma-lemma or to modifications of w a l l polysaccharide synthesis so that carbohyd-rates normally deposited as wall compounds are released as soluble carbohyd-rates. The carbohydrates would be dispersed across the amorphous wa l l , following a decreasing concentration gradient toward an adjacent parasite c e l l . Entry of the metabolites into Havveyella may occur d i r e c t l y across the plasmalemma by the processes of d i f f u s i o n , active transport, or both. A l t e r -nately, or i n addition, the complex membrane-associated vacuolar system may be involved i n the p i n o c y t o t i c uptake of the translocated carbohydrate. Secondary vacuoles, confluent with the c e l l w a l l matrix, would pinch into the cytoplasm, disperse and invaginate, r e l e a s i n g t h e i r contents of carbohydrates obtained from the w a l l matrix i n l o c a l i z e d cytoplasmic regions. The released organic carbon could then be used i n c e l l u l a r metabolism and i n the synthesis of such storage metabolites as l i p i d s , proteins and f l o r i d e a n starch. The presence of large primary vacuoles, s i m i l a r i n f i n e structure to autophagic v e s i c l e s described by Thornton (1968) may be involved i n autophag-i c a c t i v i t i e s i n r a p i d l y a s s i m i l a t i n g r h i z o i d a l c e l l s . Secondary (pinocytot-i c ) vacuoles may fuse with t h i s v e s i c l e (primary vacuole) as may ER- and dictyosome-derived v e s i c l e s . Lysosomal a c t i v i t y has been determined i n u l t r a s t r u c t u r a l l y s i m i l a r v e s i c l e s and vacuoles i n other plants (Poux, 1970; H a l l and Davie, 1971; Berjak, 1972; Marty, 1972; Wilson, 1973; Matile, 1974). Any conclusions concerning the lysosomic nature of these c e l l u l a r components i n Havveyella r h i z o i d a l c e l l s must await enzyme,cytochemical and c e l l f r a c t i o n a t i o n studies. 161 U l t r a s t r u c t u r a l evidence supports the p o s s i b i l i t y that such a hypotheti-c a l system may operate i n Harveyella r h i z o i d a l c e l l s i n procuring, processing and d i s t r i b u t i n g required organic carbon, although i t should be noted that the f u n c t i o n a l i n t e r p r e t a t i o n of t h i s system has been made from s t r u c t u r a l observations. I t i s also possible that the membrane-associated vacuolar system of the r h i z o i d a l c e l l s may be involved i n the production and release of host c e l l wall-degrading enzymes. Electron microscopic autoradiographic studies of r h i z o i d a l c e l l s i n Harveyella and other p a r a s i t i c red algae must be undertaken to resolve the question.' are materials (metabolites or enzymes) moving i n t o and/or out of r h i z o i d a l c e l l s ? C o r t i c a l and medullary c e l l s of Harveyella (zones I and II of the pustule) lack the membrane associated vacuolar system and storage i n c l u s i o n s which characterize r h i z o i d a l c e l l s (zone I I I ) . The only major s t r u c t u r a l modification which may r e f l e c t f u n c t i o n a l s p e c i a l i z a t i o n i n these c e l l s i s the presence of plasmalemma1 extensions (plasmalemmavilli), occurring on the inner and outer medullary c e l l s . Plasmalemmavilli are not found along the plasmalemma of c o r t i c a l c e l l s . Although s i m i l a r extensions have been report-ed i n other plants, t h e i r function i s not known. Their possible involvement i n c e l l w all formation has been discussed by Frey-Wyssling (1962), C o t t i e r (1971) and Tainter (1971). Brown and Wilson (1968) suggested that plasma-lemmal extensions may be involved i n nu t r i e n t uptake by the mycobiont i n the l i c h e n , Physeia aipolia. A s i m i l a r function ( i . e . , "short-distance transfer of metabolites") has been proposed for " t r a n s f e r - c e l l s " (Gunning and Pate, 1969),reported i n the p a r a s i t i c angiosperm Castilleja (Dobbins and K u i j t , 1973), the l i c h e n (mycobiontic portion) Lichina pygmaea (Peveling, 1973), the marine angiosperm Phyllospadix (Harlin, 1971), a v a r i e t y of fresh water 162 and t e r r e s t r i a l angiosperms (Pate and Gunning, 1969, review) and i n Phaseolus vulgaris inoculated with bean pod mottle v i r u s (BPMV) (Kim and Fulton, 1973). In a l l cases, the absorptive surface area of the protoplast i s increased considerably and thus could favour the transport of substances between adjacent c e l l s . The plasmalemmavilli of Harveyella may be interpreted as being involved i n e i t h e r c e l l w all synthesis or nut r i e n t uptake. The c e l l s of the outer medullary layer i n which plasmalemmavilli are most common, undergo transforma-t i o n from c o r t i c a l c e l l s to medullary c e l l s . Concomitant with t h i s change, the c e l l s increase markedly i n s i z e , vacuolation and w a l l synthesis. The numerous plasmalemmal extensions may be a c t i v e i n secreting new c e l l w all material. A l t e r n a t e l y , plasmalemmavilli may be involved i n n u t r i e n t uptake. I t i s i n t e r e s t i n g that the length of the plasmalemmavilli increases toward the periphery of the pustule. Assuming that nutrients p r i m a r i l y flow from the i n t e r d i g i t a t i o n zone p e r i p h e r a l l y i n t o the pustule, i s i t l i k e l y that a gradient e x i s t s throughout the pustule, i . e . , there i s a greater concentra-t i o n of n u t r i e n t s near the host c e l l s (zone III) than i n the pustule p e r i -phery. Thus, a greater increase i n absorptive surface area of the outer medullary c e l l s may be correlated to lower concentrations of nutrients i n the pustule periphery. In support of t h i s idea, i t has been noted that outer medullary c e l l s , c l o s e l y situated to photosynthetically a c t i v e host c e l l s , lack plasmalemmavilli. 1 6 3 VI. EFFECTS OF HARVEYELLA MIRABILIS ON ITS HOST ODONTHALIA FLOOCOSA Introduction A third characteristic used in defining a parasite i s that i t s presence affects the welfare of the host (Scott, 1969). Much information i s available concerning the effects of various fungal parasites on their hosts. Reference should be made to the recent reviews of Yarwood (1967), wheeler and Hanchey (1968) and Barnett and Binder (1973), and to selected papers of Williams and Yukawa (1967), Hess (1969), Calonge, Fielding, Byrde and Akinrefon (1969), Armentrout and Wilson (1969), Hanchey and Wheeler (1969) and Wheeler (1971, 1974). Comparatively l i t t l e i s known of the effects of parasitic higher-plant parasites on their hosts except for the economically important patho-gens (see review of Arceuthobium, Hawksworth and Wiens, 1972). In the study of host-parasite interactions involving algae, there i s l i t t l e information available concerning structural alterations induced in the host by the parasite. Richards (1891) noted the deleterious effects of Choreooolax polysiphoniae on Polysiphonia fastigiata stating that infected plants were less vigorous, had fewer growing tips and were often shriveled and rotted away, presumably from insufficient nourishment. Sturch (1924, 1926) reported that few,,if any, cells in the hosts of Choreooolax and Holmsella were actually penetrated by the parasite rhizoidal-cells but observed cells of Harveyella mirabilis entering through the wall of the host Rhodomela and growing inside the host c e l l . Cells of the parasite Ceratooolax hartzii have also been observed to penetrate and k i l l medullary cells of the host Phyllophora (Fritsch, 1945). 164 Only three papers treat the u l t r a s t r u c t u r a l aspects of parasite i n f e c t i o n on a l g a l host c e l l s . Kazama and F u l l e r (1970) described the pathological changes which occur i n the red alga Porphyra perforata i n f e c t e d by the marine fungus Pytkium marinum. The u l t r a s t r u c t u r a l e f f e c t s of Polysiphonia r h i z o i d a l penetration i n t o the fucalean host, Ascophyllum nodosum, i s reported by Rawlence (1972). In the most recent paper, Kugrens (1973a) b r i e f l y describes the e f f e c t s of parasitism by Choreooolax on i t s host Polysiphonia. The i n f e c t i v e response of the host Odonthalia to the presence of Earvey-ella was investigated at both the organismal and c e l l u l a r l e v e l . F i e l d observations were made of infected populations and the c e l l u l a r response studied mic r o s c o p i c a l l y . Penetration of Odonthalia by Earveyella has been characterized, i n addition to the process of s t r u c t u r a l connection formation between c e l l s of Earveyella and Odonthalia. 165 Results and Observations P h y s i o l o g i c a l i n t e r a c t i o n s between Harveyella and Odonthalia r e s u l t i n host responses d i s c e r n i b l e at both organismal and c e l l u l a r l e v e l s . Consider-ing f i r s t l y the e f f e c t of Harveyella on Odonthalia at an organismal l e v e l , there i s no evidence that i n f e c t i o n of Odonthalia by Harveyella reduces the a b i l i t y of infected Odonthalia to compete with uninfected Odonthalia. This d i f f e r s from the s i t u a t i o n described by Richards (1891) i n Polysiphonia i n f e c t e d by Choreooolax; i n f e c t e d Polysiphonia was always paler and l e s s vigorous than uninfected Polysiphonia. A. Response at Organismal Level Although i n f e c t i o n of Odonthalia by Harveyella does not r e s u l t i n the death of the host organism, i t may cause the death of medullary ti s s u e i n l o c a l i z e d regions. Localized death and decay of medullary c e l l s i n the basal branches of Odonthalia r e s u l t s i n the overwintering hollow basal branch described on page 64. Within these hollow branches, vegetative filaments of Harveyella p e r s i s t throughout the winter months, occasionally forming vege-t a t i v e pustules which project i n t o the lumen of the hollow branch. B. Response at C e l l u l a r Level At a c e l l u l a r l e v e l , a wide range of responses occurs i n Odonthalia c e l l s which are contiguous or c l o s e l y situated to Harveyella c e l l s . The type of response i n Odonthalia c e l l s d i f f e r s considerably depending on whether the c e l l s are situated i n the i n t e r d i g i t a t i o n zone (zone I I I , Figure 227), or dispersed throughout the pa r a s i t e pustule (Figure 228). The responses are summarized i n Table IX. 166<t Figures 227 - 230 C e l l u l a r i n t e r a c t i o n between Earveyella and host, 0. flocoosa. Figure 227. Plastic-embedded thick section, stained with Sudan black B. Earveyella r h i z o i d a l c e l l s and Odonthalia medullary c e l l s i n i n t e r d i g i t a t i o n zone. P i t connection (PC) between adjacent host c e l l s i n d i c a t e d . Figure 228. X.S. Earveyella pustule. Several host c e l l s containing numerous chloro p l a s t s are dispersed i n the" pustule (aster-isks) surrounded and attached to Earveyella c e l l s by second-ary p i t connections (PC) . Outer w a l l of pustule (OW) ind i c a t e d . Figure 229. Secondary p i t connection between host medullary c e l l (hmc) and Earveyella r h i z o i d a l c e l l (Hrc) i n i n t e r d i g i t a t i o n zone. Figure 230. T r i p a r t i t e membrane i s evident adjacent to p i t plug i n the r h i z o i d a l c e l l s of Harveyella (asterisk) but i s lacking from surface of p i t connection adjacent to host c e l l (hmc). A l l measurements i n micrometers (ym) 166 167 TABLE IX Structure C e l l u l a r responses In Odonthalia flocoosa infected by Earveyella uv'-rabilis Interdigitation zone (medullary c e l l s ) Light infection Heavy i n f e c t i o n Pustule-dispersed c e l l s P i t connection Plasmalemma Tonoplast ER Nuclei Nucleolus Plastids few 2° to Harveyella many 2° to Harveyella many 2° to Harveyella S-bodies C e l l wall C e l l d i v i s i o n Floridean starch Phospholipids Lipids l i t t l e membrane vesiculation, remains intact remains intact s l i g h t increase i n smooth ER few abberations no change reduction i n number of traversing thylakoids, loss p a r a l l e l arrange-ment of thylakoids, reduction i n number of phycobilisomes, increase in p l a s t i d osmiophilic granules few i n cytoplasm sulfated poly-saccharides present reduced present granules near 2° p i t connection (PC) to Harveyella much membrane v e s i -culation, ruptures ruptures, d i f f u s i o n of cytoplasm increase i n smooth ER, often dilated increase greatly i n diameter, number, formation of bleb nuclei increase i n diameter and stain a f f i n i t y loss of traversing thylakoids, hyper-trophy of thylakoids, vesiculation of pl a s t i d membrane many i n cytoplasm, associated with mitochondria and pl a s t i d membrane sulfated poly-saccharides present greatly reduced or absent . abundant granules adjacent to 2°PC to Harveyella associated with con-voluted membrane of 1°PC no increase i n vesicu-l a t i o n , remains in t a c t remains intact marked pr o l i f e r a t i o r . of rough ER no change no change few structural changes, interconnected travers-ing thylakoids, frequent d i v i s i o n , active C-14 assimilation many i n cytoplasm, associated with mito-chondria and p l a s t i d membrane lacking sulfated polysaccharides random c o r t i c a l c e l l d i v i s i o n forming c e l l packets reduced or absent absent granules near 1° and 2° PC 168 1. Formation of Secondary P i t Connections In both the i n t e r d i g i t a t i o n zone and the pustule regions, c e l l s of Odon-thalia and Havveyella are s t r u c t u r a l l y connected by secondary p i t connections (Figure 229) as previously described (page 101). The p i t connections between Odonthalia and Havveyella are u l t r a s t r u c t u r a l l y s i m i l a r i n most respects to primary p i t connections described from c o r t i c a l Havveyella t i s s u e (page 88). However, the t r i p a r t i t e membrane, situated between the p i t plug and cytoplasm, i s absent from the "Odonthalia-si&e" of the plug while i t i s u s u a l l y evident on the "Havveyella-slde" of the p i t plug (Figure 230). I t i s not known whether t h i s d i f f e r e n c e merely r e f l e c t s a d i f f e r e n c e i n membrane preservation between the two c e l l s . The complete developmental sequence of the formation of s t r u c t u r a l connections between Odonthalia and Havveyella remains to be elucidated. Preliminary u l t r a s t r u c t u r a l evidence indicates that the formation of the con-nections may be i n i t i a t e d by Odonthalia i n response to the presence of Havveyella. A s i m i l a r s i t u a t i o n has been described i n the p a r a s i t i c alga Choveooolax polysiphoniae (Kugrens and West, 1973). Cytoplasmic protuberances from Odonthalia c e l l s appear to grow toward the intruding Havveyella c e l l cytoplasm (Figures 231, 232). The c e l l w a l l separating Havveyella and Odonthalia becomes more electron transparent as the protuberance extends toward Havveyella (Figure 232, arrow), thus i n d i c a t i n g that w a l l d i s s o l u t i o n i n advance of the penetrating protuberance may be a chemical rather than mechanical process. In addition to forming s t r u c t u r a l connections with adjacent c e l l s of Havveyella, Odonthalia produces protuberances which form connections between adjacent Odonthalia c e l l s (Figure 233). The procedure of p i t connection f o r -169* Figures 231 - 235 C e l l u l a r i n t e r a c t i o n between Earveyella and host, 0. floooosa. Figure 231. Cytoplasmic protuberance from host c e l l toward adjacent i n -t r u s i v e l y growing Earveyella r h i z o i d a l c e l l . Figure 232. Wall adjacent to advancing host c e l l protuberance i s e l e c -tron-transparent (arrow). The adjacent r h i z o i d a l c e l l , plasmalemma forms c h a r a c t e r i s t i c concavity i n response to approaching protuberance. P i t connection (PC) and f l o r i -dean starch (FS) in d i c a t e d . Figure 233. Secondary p i t connection between two c l o s e l y situated host medullary c e l l s i n i n f e c t e d t i s s u e region of host. Figure 234. S-bodies (arrows) i n cytoplasm of host medullary c e l l attached to r h i z o i d a l c e l l of Earveyella by secondary p i t connection. Figure 235. S-bodies (arrows) associated with p l a s t i d (P) of host c e l l connected to Earveyella by secondary p i t connections. A l l measurements i n micrometers (ym) 169 170 mation between two Odonthalia c e l l s d i f f e r s from that occurring between Harveyella and Odonthalia. Adjacent Odonthalia c e l l s produce protuberances of approximately equal length which e s t a b l i s h contact midway across the wall i n t e r f a c e zone (Figure 227, arrow). In contrast, only the Odonthalia c e l l produces a protuberance when e s t a b l i s h i n g a connection with an adjacent Harveyella c e l l . U l t r a s t r u c t u r a l evidence in d i c a t e s that Harveyella may respond negatively to the advancing protuberance. The protuberance from Odonthalia often appears to contact a retracted or convex surface area of the Harveyella c e l l (Figures 229, 231). A continuous cytoplasmic connection between Harveyella and Odonthalia has hot been observed but t h i s may merely r e f l e c t the fac t that t h i s condition i s " s h o r t - l i v e d " , with a p i t plug deposited i n the cytoplasmic channel sh o r t l y a f t e r c e l l u l a r contact i s made. The d i s t r i b u t i o n of S-bodies (page 132) i n Odonthalia c e l l s connected to Harveyella provides i n d i r e c t evidence that, at some point i n development, the cytoplasm i s continuous between Odonthalia and Harveyella. S-bodies are a un i v e r s a l cytoplasmic component of Harveyella c e l l s but are only found i n Odonthalia c e l l s connected to c e l l s of Harveyella by secondary p i t connections. Although never i n abundance, S-bodies are r e a d i l y recognized i n the cytoplasm (Figure 234), nucleus (Figure 252) and associated with the chloroplast membrane (Figure 232). 2. C e l l u l a r Response i n Host C e l l s of I n t e r d i g i t a t i o n Zone Changes i n the c e n t r a l vacuole tonoplast, plasmalemma and various c e l l organelles are discernible i n Odonthalia c e l l s of the i n t e r d i g i t a t i o n zone (zone I I I ) . Uninfected host medullary and c o r t i c a l c e l l s possess a c e n t r a l vacuole and a l l the cytoplasmic organelles are confined i n the pe r i p h e r a l c e l l cytoplasm (Figure 236). As described i n Asoophyllim i n f e c t e d by 171 * Figures 236 - 238 C e l l u l a r i n t e r a c t i o n between Earveyella and host, 0. floooosa. Figure 236. Medullary c e l l of 0. floooosa from uninfected ti s s u e region. Central vacuole (V) i s i n t a c t and t y p i c a l f l o r i d e a n p l a s t i d s (P) are p e r i p h e r a l l y situated i n the electron-dense cytoplasm. Figure 237. Host medullary c e l l i n Earveyella-lnfected region. Ruptur-ing of c e n t r a l vacuole tonoplast r e s u l t s i n the formation of many small vacuoles (V) and d i f f u s i o n of cytoplasm. Figure 238. Increase i n plasmalemma v e s i c u l a t i o n i n i n f e c t e d host c e l l . A l l measurements i n micrometers (um) 171 172 Polysiphonia (Rawlence, 1972) and i n Polysiphonia i n f e c t e d by Choveooolax (Kugrens and West, 1973a), l y s i s of the c e n t r a l vacuole tonoplast i s one of the f i r s t responses to i n f e c t i o n . In medullary c e l l s of in f e c t e d Odonthalia, rupturing of the tonoplast r e s u l t s i n a d i f f u s i o n of cytoplasm into the c e n t r a l lumen (Figure 237). Many small vacuoles are scattered throughout the d i f f u s e cytoplasm (Figures 237, 239). An increase i n plasmalemma v e s i c u l a t i o n occurs (Figure 238). These v e s i c l e s appear to fuse with the i n t a c t plasma-lemma, re l e a s i n g substances into the adjacent c e l l w a l l matrix to form an electron-dense c e l l w a l l layer contiguous with the vesiculated plasmalemma (Figure 239) . The massive quantities^of f l o r i d e a n starch found i n uninfected medullary host c e l l s (Colour plate 9) are reduced i n host medullary c e l l s surrounded by Earveyella (Colour p l a t e 7). No f l o r i d e a n starch could be detected (Colour p l a t e 8) i n host c e l l s i n heavily in f e c t e d c e l l u l a r regions. L i p i d s (Sudan black B p o s i t i v e ) increase i n s i z e and number i n host c e l l s i n i n f e c t e d areas (Figures 240, 241, 242). These substances have been i d e n t i -f i e d as p r i m a r i l y phospholipids by the use of the phospholipid s p e c i f i c s t a i n , Luxol's f a s t blue G (Colour p l a t e 10, Figure 243). Phospholipids are not found i n uninfected host medullary c e l l s (Figure 244), or i n c o r t i c a l c e l l s of i n f e c t e d Odonthalia (Figure 245). Small q u a n t i t i e s of phospholipids are oc c a s i o n a l l y found i n the pustule c e l l s of Earveyella. A d d i t i o n a l osmiophi -l i e (Sudan black B p o s i t i v e ) substances appear i n the v i c i n i t y of secondary p i t connections between Earveyella and Odonthalia (Figure 246). The l i p i d granules are only found on the "Odonthalia-slde" of the p i t plug. They appear to unite, forming a large si n g l e granule which may become t i g h t l y appressed to the p i t plug (Figures 245, 246). In infected Odonthalia medul-1 7 3 ^ Figures 239 - 242 C e l l u l a r i n t e r a c t i o n between Earveyella and host, 0. floooosa. Figure 239. Infected host medullary c e l l . V e s i c l e s appear to fuse with plasmalemma, r e l e a s i n g contents into c e l l w all matrix. Electron-dense inner c e l l w a l l layer i s evident.(asterisks) Nucleus (N), mitochondria (M) , p l a s t i d s (P.) and small vacuoles (V) are scattered throughout c e l l . Figure 240. G l y c o l methacrylate-embedded thick section, stained with Sudan black B. L i p i d s (L) abundant i n host medullary c e l l i n i n f e c t e d t i s s u e region (hmc). Earveyella r h i z o i d a l c e l l (Hrc) i n d i c a t e d . Figure 241. Large osmiophilic ( l i p i d ) granule (Og) i n cytoplasm of i n f e c t e d host medullary c a l l . Mitochondrion (M) and dictyosome (D) are i n d i c a t e d . Figure 242. Increase i n number of osmiophilic ( l i p i d ) granules i n i n f e c t e d host c e l l . F loridean starch (FS) i s present. A l l measurements i n micrometers (ym) 173 T V 174«-Figures 243 - 249 C e l l u l a r i n t e r a c t i o n between Earveyella and host, 0. floooosa. Figures 243-245. Plastic-embedded thick sections, stained with Luxol's f a s t blue G. Figure 243. L o c a l i z a t i o n of phospholipids i n in f e c t e d host medullary c e l l s ( a s t e r i s k s ) . No phospholipids are indicated i n Earveyella r h i z o i d a l c e l l s (Hrc). Figure 244. No phospholipids i n uninfected host medullary c e l l . P i t connection (PC) i s evident. Figure 245. Phospholipids absent from c o r t i c a l c e l l s i n Earveyella-i n f e c t e d host. Figure 246. Osmiophilic granules (Og) ( l i p i d s ) i n v i c i n i t y of host secondary p i t connection (PC) to r h i z o i d a l c e l l of Earveyella. Figure 247. Aggregation of osmiophilic granules i n host medullary c e l l s (hmc) attached to Earveyella r h i z o i d a l c e l l (Hrc) by secondary p i t connection. Figure 248. Osmiophilic granule appressed to secondary p i t connection i n host c e l l . Figure 249. Convoluted membrane surrounding primary p i t connection (1°PC) between adjacent host c e l l s i n in f e c t e d t i s s u e region. Small osmiophilic granules (Og) are associated with membrane. A l l measurements i n micrometers (ym) 174 l a r y c e l l s , osmiophili'c• granules p r o l i f e r a t e i n the region of the primary p i t connection ( i . e . , a connection between two adjacent Odonthalia daughter c e l l s ) . The membrane bordering the p i t plug i s highly convoluted and small osmiophili'c granules appear associated with the membrane (Figure 249). This response i s unique to infected host c e l l s . The n u c l e i of an uninfected medullary c e l l are t y p i c a l l y eukaryotic, surrounded by a pore-fenestrated double membrane situated p e r i p h e r a l l y i n the c e l l cytoplasm (Figure 250). In inf e c t e d host c e l l s , the nuclear envelope l o c a l l y evaginates to form nuclear blebs (Figure 251) concomitant with the rupturing of the tonoplast of the c e n t r a l vacuole. Numerous bleb-nuclei may form and disperse throughout the cytoplasm upon l y s i s of the tonoplast. The bleb n u c l e i vary greatly i n diameter and are surrounded by a double membrane (Figure 252) . Large amounts of condensed chromatin c h a r a c t e r i s t i c a l l y occupy a c e n t r a l l o c a t i o n , surrounded by a l e s s electron-dense nucleoplasm. In addition to forming "blebs", the nucleus may become highly i r r e g u l a r i n ou t l i n e (Figure 253). In heavily infected host c e l l s , the nucleus may increase greatly i n diameter (Figure 254). The nucleolus becomes d i s t i n c t , increasing i n diameter proportionately to the increase i n nuclear diameter. Nuclear pores are evident i n the nuclear envelope (Figure 254). P l a s t i d s of uninfected Odonthalia are t y p i c a l of those u l t r a s t r u c t u r a l l y described from other higher red algae (Dodge, 1973). Thylakoids l i e s i n g l y i n the p l a s t i d stroma and are arranged p a r a l l e l to one another. One e n c i r c l -ing thylakoid i s present extending p a r a l l e l to the bounding double membrane (Figure 255). Phycobilosomes are abundant on both the p a r a l l e l traversing thylakoids and on the e n c i r c l i n g thylakoid (Figures 255, 256). P l a s t i d s i n s l i g h t l y infected Odonthalia are scattered throughout the d i f f u s e cytoplasm upon l y s i s of the tonoplast (Figure 257). The number of traversing t h y l a -176 t Figures 250 - 254 C e l l u l a r i n t e r a c t i o n between Havveyella and host.0. floeeosa; nuclear response. Figure 250. Uninfected host medullary c e l l nucleus (N). Chromatin (Ch) dispersed i n nucleus situated at c e l l periphery. Figure 251. L o c a l i z e d evagination i n nuclear envelope (asterisk) into vacuolar region (V) i n i n f e c t e d host medullary c e l l . Figure 252. Small bleb-nucleus with c e n t r a l l y located.condensed chro-matin (Ch). S-body present within nucleus (arrow). Figure 253. I r r e g u l a r l y shaped nucleus i n i n f e c t e d host c e l l . P l a s t i d s and mitochondria remain associated with nuclear envelope. Figure 254. Enlarged nucleus of i n f e c t e d host medullary c e l l . D i s t i n c t nucleolus (Nu) also increases i n diameter. Nuclear pore i s i n d i c a t e d . A l l measurements i n micrometers (ym) 176 177 * Figures 255 - 258 C e l l u l a r i n t e r a c t i o n between Havveyella and host 0. floocosa; p l a s t i d response. Figures 255-256. P l a s t i d showing no s t r u c t u r a l changes i n medullary c e l l of 0. floocosa.. Phycobilosomes (Pb). abundant on thylakoids. Figure 257. P l a s t i d i n i n f e c t e d host c e l l . Decreased number of tr a v e r s -ing thylakoids, loss of p a r a l l e l thylakoid arrangement, and increase i n p l a s t i d osmiophilic granules (Og). Figure 258. Degenerating p l a s t i d ; decrease i n t r a v e r s i n g thylakoids although e n c i r c l i n g thylakoid remains (arrow). A l l measurements i n micrometers (ym) 177 178 koids i s s u b s t a n t i a l l y reduced i n these p l a s t i d s and they become randomly arranged. Phycobilosomes decrease i n number and, as described i n the degener-at i n g p l a s t i d s of Rhodoohorton purpureum (Lightfoot) Rosenvinge ( L i c h t l e , 1973), the number of osmiophilic granules i n the p l a s t i d increases. The p l a s t i d s appear v a r i o u s l y contorted i n heavily infected c e l l s (Figure 258). The e n c i r c l i n g outer thylakoid i s s t i l l recognizable, but only remnants of the traversing thylakoids remain. The p e r s i s t i n g thylakoids are generally hypertrophied (Figure 259) . V e s i c l e s are formed from rupturing of the outer p l a s t i d membrane and inner thylakoids (Figure 260). These electron-transpar-ent, s i n g l e membrane-bound v e s i c l e s are released i n t o the d i f f u s e cytoplasm of the degenerating host c e l l . At the time the plasmalemma ruptures, the p l a s t i d s merely consist of the double outer membrane and remnants of the inner e n c i r c l i n g thylakoid (Figure 261). As the plasmalemma breaks down, degenerating cytoplasm becomes continu-ous with the c e l l w a l l matrix (Figure 262). B a c t e r i a l i n f e c t i o n i n these c e l l u l a r regions accelerates the f i n a l degeneration process r e s u l t i n g i n the hollowed medullary regions i n heavily i n f e c t e d Odonthalia. I t should be noted that r e l a t i v e l y few of the t o t a l i n f e c t e d c e l l s reach t h i s f i n a l state of degeneration. A wide range of path o l o g i c a l abnormalities may occur i n adjacent host c e l l s . Although Harveyella i s c h a r a c t e r i s t i c a l l y i n t e r c e l l u l a r i n i t s occurrence i n Odonthalia, i t w i l l o c casionally penetrate i n t o c e l l s of Odonthalia (Figure 263, Colour p l a t e 8) and e s t a b l i s h an i n t r a c e l l u l a r existence (Figure 264). The penetration of host c e l l s u s ually occurs i n the i n t e r d i g i t a t i o n zone (zone III) during periods of massive p r o l i f e r a t i o n of Harveyella. Changes i n the s t a i n i n g c h a r a c t e r i s t i c s of the host c e l l w all (Colour plate 8) 179 « Figures 259 - 262 C e l l u l a r i n t e r a c t i o n between,Havveyella and host 0. floeeosa', p l a s t i d response. Figure 259. Hypertrophied thylakoids i n infe c t e d host c e l l . p l a s t i d . Figure 260. Rupturing of outer membrane of in f e c t e d c e l l p l a s t i d and the formation.of p l a s t i d - d e r i v e d v e s i c l e s . (ve). Floridean starch (FS). Figure 261. Rupturing of plasmalemma., 'Remaining p l a s t i d s consist of e n c i r c l i n g thylakoid and osmiophilic granules. Figure 262. Presence of ba c t e r i a (B) i n degenerating host w a l l . Remnants of p l a s t i d (P) indicated. A l l measurements i n micrometers (ym) 179 180 t Figures 263.- 266 Penetration, of host c e l l by EaweyeZZa mivabiZis. Figure 263. Penetration of host medullary c e l l (hmc) by EaweyeZZa r h i z o i d a l c e l l s (Hrc). Note d i f f e r e n c e i n wall/matrix surrounding penetrating c e l l . P l a s t i d s . ( P ) i n host c e l l i n d i c a t e d . Figure 264. Penetrating EaweyeZZa c e l l . A t hick 2-layered c e l l w a l l c o n s i s t i n g of host c e l l w a l l (2) and r h i z o i d a l c e l l w a l l (1) surrounds the r h i z o i d a l c e l l . Cytoplasm (Cy) and f l o r i d e a n starch (FS) i n host c e l l indicated;.nucleus (N) and vacuole (V) are apparent i n EaweyeZZa. Figure 265. Fine w a l l s t r i a t i o n s (arrows) i n host w a l l near penetrating EaweyeZZa filaments. Figure 266. Plasmalemma of host c e l l s appears to be "pushed" i n by penetrating EaweyeZZa r h i z o i d a l c e l l . A l l measurements i n micrometers (ym) 180 181 and the development of fine wall striations near the penetrating Harveyella cells (Figure 265) suggest that the dissolution process may be chemical. Ultrastructural observations indicate a mechanical process may be additionally involved. As Harveyella penetrates into the cells of Odonthalia, the plasma-lemma appears to be "pushed" into the c e l l (Figures 266-269). A similar "pushing" inward of the c e l l wall interface has been reported in the penetra-tion of maize by the fungus Colletvotvichwn graminioola (Politis and Wheeler, 1973). It has also been reported that the haustoria of lichen fungi appear to penetrate algal cells by mechanical rather than chemical means (Ahmadjian, 1966). Elongation of the Havveyella c e l l protuberance results i n the f i n a l penetration of the wall and membrane of Odonthalia. When this occurs, the Odonthalia c e l l s are devoid of a l l recognizable organelles except nuclei. An identical situation has been described in onion c e l l s penetrated by the fungus Pyrenochaeta tevvestvis (Hess, 1969). A dense membranous material occurs in the wall interface zone between penetrating Havveyella cells and Odonthalia (Figures 267, 268). Hess (1969) has described similarly situated electron-dense products i n onion c e l l s , presumably derived from infection by the fungus Pyvenoohaeta. Rawlence (1972) has also described electron-dense areas in the c e l l walls of Ascophyllum digested by Polysiphonia. 3. Cellular Responses in Host Cells in Havveyella pustule Odonthalia cells in the pustule of Havveyella readily establish numerous pi t connections with adjacent Harveyella cells yet degeneration does not occur, as described in infected Odonthalia cells in the interdigitation zone. These isolated Odonthalia cells are surrounded by a thick c e l l wall and contain numerous discoid, parietal plastids (Figure 228). 182 a Figures 267 - 269 Penetration of host c e l l by Harvey ella mi-rabilis. Figures 267-268. El e c t r o n micrographs of penetration. Figure 267. Penetration of host medullary c e l l (hmc) by r h i z o i d a l c e l l of H. mirabil-i-s (Hrc) ( a s t e r i s k ) . Only remnant membranes and a nucleus (N) remain i n the host c e l l . Figure 268. Dense membranous material (arrows) i n the c e l l w a l l i n t e r -face between host and penetrating r h i z o i d a l c e l l s . Figure 269. Light micrograph. Penetration of host medullary c e l l s (hmc) i n i n t e r d i g i t a t i o n zone by Harveyella r h i z o i d a l c e l l s (Hrc). Several r h i z o i d a l c e l l s occupy the lumen of the penetrated host c e l l ( a s t e r i s k ) . A l l measurements i n micrometers (ym) 183 The host c e l l s were r e a d i l y i d e n t i f i e d i n sections stained with t o l u i d i n e blue 0 since the thick c e l l w a l l surrounding the dispersed Odonthalia c e l l s stained pink-purple (y-metachromasia) while the Earveyella pustule c e l l walls stained blue-purple (g-metachromasia) (Colour plate 11). Isolated host c e l l s d i f f e r chemically from the pustule c e l l s of Earveyella i n lacking sulfated polysaccharides i n t h e i r walls (Colour plate 15) as indicated with the cyto-chemical s t a i n s , a l c i a n blue and a l c i a n yellow. Large q u a n t i t i e s of sul f a t e d polysaccharides occur i n the walls surrounding medullary c e l l s of Earveyella (Colour p l a t e 16) and i n host c e l l s i n heavily in f e c t e d t i s s u e regions of the i n t e r d i g i t a t i o n zone. I t i s i n t e r e s t i n g that while d e f i n i t e w a l l bands of sulf a t e d polysaccharides occur i n the inner medullary c e l l s of uninfected Odonthalia, there are no sulfated polysaccharides i n the walls of c o r t i c a l c e l l s (Colour plate 13). P l a s t i d s i n Odonthalia medullary c e l l s dispersed i n the pustule of Earveyella, are frequently normal i n u l t r a s t r u c t u r e , e x h i b i t i n g few of the abnormalities described i n p l a s t i d s of host c e l l s i n the i n t e r d i g i t a t i o n zone. The p l a s t i d s i n these i s o l a t e d host c e l l s are apparently f u n c t i o n a l as shown by t h e i r a c t i v e a s s i m i l a t i o n of NaHlt+CC>3 (Figure 270). Occasionally, a l t e r a -tions of the thylakoid arrangement r e s u l t i n p l a s t i d s with interconnected traversing thylakoids (Figure 271). P l a s t i d s frequently divide i n i s o l a t e d medullary c e l l s of Odonthalia (Figure 272). P r o l i f e r a t i o n of rough ER may occur concomitantly with the increase i n p l a s t i d d i v i s i o n i n medullary c e l l s (Figure 273). A si m i l a r p r o l i f e r a t i o n of ER was noted i n c o r t i c a l c e l l s of the red alga Polyneuropsis stolonifera Wynne, McBride and West i n response to b a c t e r i a l i n f e c t i o n (McBride, Kugrens and West, 1974). U l t r a s t r u c t u r a l l y i s o l a t e d Odonthalia c o r t i c a l c e l l s appear s i m i l a r to 184^ Figures 270 - 273 C e l l u l a r i n t e r a c t i o n between Havveyella and pustule-dispersed host medullary c e l l s . Figure 270. Autoradiograph. Isolated host medullary c e l l i n pustule of Havveyella ( a s t e r i s k ) . P a r i e t a l p l a s t i d s a c t i v e i n assimi-l a t i n g l t fC phot o s y n t h e t i c a l l y . Figure 271. Interconnected tr a v e r s i n g thylakoids i n p l a s t i d of host medullary c e l l i n pustule of Havveyella. S-bodies (SB) occur i n the host c e l l cytoplasm. Figure 272. D i v i s i o n of p l a s t i d i n host medullary c e l l i n pustule ( a s t e r i s k s ) . Phycobilosomes occur on the thylakoids. Figure 273. P r o l i f e r a t i o n of rough ER i n host medullary c e l l i n pustule. A l l measurements i n micrometers (ym) 184 185 c o r t i c a l c e l l s observed i n uninfected Odonthalia. Much of the cytoplasmic volume i s occupied by the large p l a s t i d s and nucleus (Figure 274) and ribosomes are abundant (Figure 275). Floridean starch may occasionally occur i n associa-t i o n with the nucleus. Autoradiography reveals that these c e l l s are also very a c t i v e i n a s s i m i l a t i n g 1 4 C (Figure 276). Isolated Odonthalia c o r t i c a l c e l l s are capable of d i v i d i n g i n any plane r e s u l t i n g i n "packets" of 6-8 c o r t i c a l c e l l s randomly dispersed throughout the pustule of Harveyella (Figure 276). The c e l l s i n each "packet" are intercon-nected by p i t connections (Figure 277). Large quantities of osmiophilic -granules may be associated with these connections, s i m i l a r to the condition described i n inf e c t e d medullary c e l l s i n the i n t e r d i g i t a t i o n zone (page ,172). A s i m i l a r random c e l l d i v i s i o n response has been noted i n Odonthalia c o r t i c a l c e l l s surrounding host medullary ti s s u e i n f e c t e d by Harveyella. This r e s u l t s i n the addition of an asymetrically arranged palisade layer of c o r t i c a l c e l l s (Figure 278). A s i m i l a r wounding response has been reported to occur i n Polysiphonia i n f e c t e d by Choreooolax (Kugrens and West, 1973a). I t has not been ascertained whether t h i s response i n Odonthalia c o r t i c a l c e l l s i s d i r e c t l y i n i t i a t e d by Harveyella or i f i t i s a response to the death of the subtending medullary c e l l s and thus i n d i r e c t l y a t t r i b u t e d to the presence of Harveyella. I t should be noted i n t h i s regard that the response i s not l i m i t e d to host c e l l s d i r e c t l y adjacent to penetrating Harveyella filaments. 186^ Figures 274 - 276 C e l l u l a r i n t e r a c t i o n between Earveyella and pustule-dispersed host c o r t i c a l c e l l s . Figure 274. Host c o r t i c a l c e l l i n pustule appears s i m i l a r to uninfected host c o r t i c a l c e l l . Large p l a s t i d and c e n t r a l l y located nucleus occupy much of the c e l l volume. Floridean starch (FS) xassociated with nucleus (N). Figure 275. S-body i n cytoplasm of host c o r t i c a l c e l l connected to Earveyella by secondary p i t connection (2°PC). Figure 276a,b. Autoradiographs, 1 1 +C-labeled; two f o c a l planes, a - b. C o r t i c a l c e l l s i n pustule d i v i d e randomly to form "packets" of 6-8 c e l l s (b). These are a c t i v e i n a s s i m i l a t i n g l l +C photos y n t h e t i c a l l y (b). A l l measurements i n micrometers (vim) 186 187* Figures 277 - 278 C e l l u l a r i n t e r a c t i o n between..Havveyella and pustule-dispersed host c o r t i c a l c e l l s . . Figure 277. P i t connections between host, c o r t i c a l c e l l s dispersed i n Havveyella pustule. Osmiophilic granules '(Og) are associ-ated with .pit connection (PC). Figure 278. External palisade layer of c o r t i c a l c e l l s r e s u l t i n g from random d i v i s i o n of host c o r t i c a l c e l l s i n infected Odonthalia. A s t e r i s k s i n d i c a t e Havveyella r h i z o i d a l filaments. A l l measurements i n micrometers (ym) 187 188 Discussion Odonthalia cells may be described as lightly infected or heavily infected depending on the degree of cellular response. What factors cause the cellular response in infected host cells i s presently unclear. Secondary pit connec-tions are frequent between infected host cells and Havveyella rhizoidal cells although there i s no indication that their presence i s related to host c e l l degeneration. Numerous pit connections' occur between Havveyella and Odonthalia cells dispersed in the pustule (zone I, II) although there are few structural abnormalities noted i n these c e l l s . Host cells i n the parasite pustule are adjacent to Havveyella medullary and cortical cells which differ considerably in ultrastructure from the rhizoidal cells of the interdigitation zone. One important feature i s the absence of the membrane-associated ER-vacuolar system which characterizes rhizoidal cells. If this system i s involved in the production of l y t i c digestive substances (in addition to the process of nutrient uptake), i t s absence in Havveyella cortical and medullary cells could explain the reduction- in cellular response in adjacent host ce l l s . The possibility that S-bodies (solitary-bodies) may induce infective responses i n the recipient host c e l l i s an attractive hypothesis, but i t should be noted that S-bodies occur as commonly in host cells showing extreme structural abnormalities as they do in host cells exhibiting l i t t l e or no structural changes in the parasite pustule. The function of S-bodies in infected host cells and in Havveyella i s presently unknown. Circumstantial evidence indicates that Havveyella S-bodies or their precursors may be transmitted from parasite to host via a temporari-ly opened structural connection. These inclusions have not been reported i n any other red algal parasite although structures similar in size and struc-189 ture are seen i n electron micrographs of Choveoaolax polysiphoniae (Kugrens and West, 1973a-; Figures 11, 12). Anton-Lamprecht (1965, 1966, 1967) and Ie (1972) have concluded from studies of S-bodies i n two flowering plants, Epilobium and Tvopaeolum, that, although S-bodies s u p e r f i c i a l l y resemble s p h e r i c a l viruses described i n patho-l o g i c a l forms of plant and animal viruses as well as tumour inducing v i r u s e s , there i s no evidence to i n d i c a t e that they are v i r u s e s . They d i f f e r from viruses i n the structure of t h e i r c e n t r a l core apparatus, i n the f a c t that they seldom aggregate or associate with membranes, and that they are not transmitted by mechanical i n o c u l a t i o n and g r a f t i n g . From studies of r e c i p r o -c a l crosses between Tvopaeolum plants containing and lacking S-bodies, Ie (1972) concluded that S-bodies may be involved i n cytoplasmic inheritance. The present report of DNA i n the c e n t r a l core of Havveyella S-bodies would support t h i s . Whether S-bodies are v i r a l p a r t i c l e s i n Havveyella and Odonthalia cannot be concluded but i t should be noted that v i r u s - l i k e p a r t i c l e s have been frequently found i n cancerous tissues and tumours as well as i n normal c e l l s . These structures are often morphologically i n d i s t i n g u i s h a b l e from viruses and i n a l l cases there are ho i n d i c a t i o n s of i n f e c t i v i t y or transmission (Friedmann and B i r d , 1961; Moericke and Wohlfahrt-Bottermann, 1962; Siegel and Wellings, 1962; Zwellenberg, 1962). In a comprehensive paper on p a r a s i t i c red algae, Feldmann and Feldmann (1958) proposed that red a l g a l parasites c l o s e l y r e l a t e d taxonomically to t h e i r hosts (adelphoparasites) a f f e c t t h e i r hosts d i f f e r e n t l y than do para-s i t e s which are taxonomically unrelated ( a l l o p a r a s i t e s ) . They suggested that the p h y s i o l o g i c a l nature of the host-parasite i n t e r a c t i o n may be predicted 190 i f the taxonomic a f f i l i a t i o n of each i n d i v i d u a l i s known. This idea was based on the premise that taxonomic r e l a t i o n s h i p s i n the red algae imply b i o -chemical r e l a t i o n s h i p s . Red a l g a l c l a s s i f i c a t i o n has been described to be at the "pioneer phase" (Dixon, 1973), i n which taxonomy i s concerned with l i t t l e more than i d e n t i f i c a t i o n of i n d i v i d u a l s (Davis and Heywood, 1963, p. 3-4). Although a "workable" system at a "pioneer" l e v e l , the red a l g a l o r d i n a l c l a s s i f i c a t i o n scheme i s an a r t i f i c i a l system since i t i s based on only the morphological features of carposporophyte development and therefore may not r e f l e c t evolutionary r e l a t i o n s h i p s between plants. I t would be more l o g i c a l to study both the p h y s i o l o g i c a l nature of the host-parasite r e l a t i o n s h i p and the morphology of both organisms to determine how c l o s e l y these i n d i v i d u a l s may be r e l a t e d phylogenetically. In the current study, the possible taxonomic r e l a t i o n s h i p s of Earveyella to i t s rhodomelacean host Odonthalia have been examined by i n v e s t i g a t i n g the structure and development of the carposporophyte of Earveyella as well as the nature of the host-parasite i n t e r a c t i o n . C h a r a c t e r i s t i c s of carposporophyte development observed i n Earveyella support the contention that HavveyeZZa i s ' not c l o s e l y r e l a t e d to Odonthalia. Odonthalia i s included i n the r e l a t i v e l y uniform order Ceraniales, characterized by the generative a u x i l i a r y c e l l being cut o f f from the supporting c e l l a f t e r f e r t i l i z a t i o n (Papenfuss, 1951). In Earveyella, the supporting c e l l acts as the a u x i l i a r y , s i m i l a r to the Callymeniaceae (Cryptonemiales) and cannot be interpreted as forming a f t e r f e r t i l i z a t i o n . The precise o r d i n a l a s s o c i a t i o n of Earveyella s t i l l remains unclear, p r i m a r i l y because of problems of overlapping c h a r a c t e r i s t i c s of the G i g a r t i n a l e s and Cryptonemiales (Papenfuss, 1951; Dixon, 1973) although i t i s obviously not r e l a t e d to the Ceramiales. Since a l l developmental character-191 i s t i c s described i n the present study are i n general agreement with those previously observed i n Harveyella mirabilis on Rhodomela aonvervoides, no change i n i t s present o r d i n a l c l a s s i f i c a t i o n w i l l be proposed. Considering the foregoing data, Harveyella may be considered an a l i o -p a rasite which, according to Feldmann and Feldmann (1958), could be expected to form few i f any p i t connections to. adjacent host c e l l s . As has been noted, secondary p i t connections have been observed between Harveyella and Odonthalia, although only Odonthalia produces the cytoplasmic protuberance which traverses the w a l l matrix and establishes contact with Harveyella.. Harveyella may respond negatively to the approach of the protuberance, but does not i n i t i a t e protuberances to adjacent host c e l l s . This observation may be highly s i g n i f i -cant i n that i t indicates that, although p i t connections may be formed between an a l l o p a r a s i t e and i t s host, the process i s not n e c e s s a r i l y r e c i p r o c a l . Therefore, the presence of p i t connections between host and parasite c e l l s does not n e c e s s a r i l y imply the two c e l l s are biochemically s i m i l a r as surmised by Feldmann and Feldmann. Other a l l o p a r a s i t i c as well as adelophoparasitic associations should be examined to determine i f there i s any c o r r e l a t i o n between the degree of mutual response i n the formation of secondary p i t connections and the taxonomic a f f i l i a t i o n of the host and p a r a s i t e . Secondary p i t connections also form between adjacent host c e l l s i n i n f e c t e d and uninfected t i s s u e s . In t h i s process, both c e l l s i n i t i a t e pro-tuberances which "seek out each other" across the c e l l w all matrix. This process i s s i m i l a r to that recently described by Waaland and Cleland (1974) i n t h e i r i n v e s t i g a t i o n s of c e l l u l a r r e pair i n the wounded red alga, Griffithsia.paoifica K y l i n . In t h i s study, c e l l u l a r extensions were mutually induced from c e l l s at opposite ends of a dead, i n t e r c a l a r y c e l l , apparently 1 9 2 i n response to an u n i d e n t i f i e d e x t r a c e l l u l a r " a t t r a c t a n t " . I t i s possible that a s i m i l a r e x t r a c e l l u l a r substance may mediate mutual protuberance res-ponse i n the formation of secondary p i t connections between adjacent Odonthalia c e l l s . I t might also be speculated that c e l l s of the a l l o p a r a s i t e , Harveyella, are either " i n s e n s i t i v e " or react negatively ( r e t r a c t i o n of the plasmalemma) to the presence of such an " a t t r a c t a n t " . Feldmann and Feldmann (1958) predicted that an a l l o p a r a s i t e such as Harveyella would i n t e r a c t a n t a g o n i s t i c a l l y with i t s host. Host c e l l s i n the i n t e r d i g i t a t i o n zone have been noted to be d e l e t e r i o u s l y a f f e c t e d by the presence of i n t r u s i v e Harveyella c e l l s although l i t t l e or no deleterious e f f e c t s were noted i n host c e l l s i s o l a t e d i n the pustule of Harveyella. In f a c t , the pustule-dispersed host c e l l s may a c t u a l l y be stimulated to increase i n number (by random c e l l d i v i s i o n ) and metabolic a c t i v i t y . I t i s i n t e r e s t i n g that a greater incorporation of l l fC occurred i n i n f e c t e d rather than uninfe c t -ed Odonthalia flocoosa (Figure 204). This may be r e l a t e d to an increase i n p l a s t i d number or a c t i v i t y i n i n f e c t e d t i s s u e or i t may be the r e s u l t of induced host c o r t i c a l c e l l d i v i s i o n r e s u l t i n g i n the addition of palisade c o r t i c a l layers adjacent to heavily infected medullary t i s s u e . Autoradio-graphic studies have shown that these palisade ; c e l l s are extremely active i n 1 I +C-bicarbonate uptake. There are several reports of s i m i l a r increases i n photosynthesis as a r e s u l t of pathogen i n f e c t i o n (Allen, 1942; Livne, 1964; Yarwood, 1967). In a l l cases, increased photosynthesis was l i m i t e d to early i n f e c t i o n stages or to " l i g h t i n f e c t i o n s " . If only morphological c h a r a c t e r i s t i c s ( i . e . , development of the carpo-sporophyte) are considered, Harveyella may be c l a s s i f i e d as an a l l o p a r a s i t e , taxonomically unrelated to i t s host. It s a c t u a l phylogenetic a s s o c i a t i o n 193 with Odonthalia may also be r e f l e c t e d by such biochemical c h a r a c t e r i s t i c s as the tendency to form secondary p i t connections and the c e l l u l a r i n t e r a c t i o n which occurs between adjacent c e l l s . A possible biochemical d i s s i m i l a r i t y between Havveyella and Odonthalia may be r e f l e c t e d i n the fa c t that only 0. floocosa c e l l s i n i t i a t e the protuber-ance which subsequently forms the p i t connection to Havveyella. This may r e f l e c t a greater tendency for Odonthalia to form s t r u c t u r a l connections than Havveyella. Dixon (1973) has noted that secondary p i t connections are r e a d i l y formed i n many rhodomelacean genera, whereas they appear to be absent from vegetatively simple Crytonemiales. The fa c t that the host i s not always aff e c t e d d e l e t e r i o u s l y by Havveyella does not n e c e s s a r i l y i n d i c a t e that the i n d i v i d u a l s are biochemically s i m i l a r as suggested by Feldmann. and Feldmann (1958). Over a long period of evolution, a b i o t r o p i c a s s o c i a t i o n (Lewis, 1973) could have evolved i n which Havveyella derives nutrients from Odonthalia with only minimal tissue damage. The presence of i n t a c t , metabolically a c t i v e host c e l l s i n the pustule of Havveyella may be important i n the nourishment of adjacent Havveyella c e l l s , whereas host c e l l s i n the i n t e r d i g i t a t i o n zone may provide a greater release of metabolites upon degeneration. VII. FINAL DISCUSSION AND EVOLUTIONARY CONSIDERATION In the present study, the nature of the r e l a t i o n s h i p that e x i s t s between Harveyella mirabilis and i t s rhodomelacean hosts Odonthalia and Rhodomela has been examined using f i e l d , c u l t u r a l , cytohistochemical, electron microscopic, autoradiographic and p h y s i o l o g i c a l techniques. Harveyella had been defined previously as a parasite of Odonthalia and Rhodomela based only on morphologi-c a l c r i t e r i a . To determine i f i t i s a p h y s i o l o g i c a l parasite as well as a s t r u c t u r a l p a r a s i t e , the three questions o r i g i n a l l y proposed i n the introduc-t i o n : (1) Is the reproduction and development of Harveyella i n any way dependent on the presence of a s p e c i f i c host? (2) Is there an exchange of material between the host and Harveyellal (3) Does the presence of Harveyella a f f e c t the welfare of the host? have been investigated. From f i e l d , c u l t u r a l and chromosomal studies i t has been determined that the l i f e h i s t o r y of Harveyella i s of the "Polysiphonia-type", i n which t y p i c a l f l o r i d e a n spermatia, carpogonia, carpospores and tetraspores are produced annually. The l i f e h i s t o r y and phenology of Harveyella i n the east-ern P a c i f i c have been found to be dependent upon the presence of the host 0. floocosa, and to be affe c t e d by such environmental parameters as i n v e r t e -brate grazing of the host and seasonal changes i n water temperature and photoperiod. Although r e a d i l y attaching to the external host c o r t i c a l surface, Harveyella spores germinated only within ruptured host c e l l s . F i e l d and laboratory studies have revealed that host grazing by isopods and amphipods i s p r i m a r i l y responsible for the c h a r a c t e r i s t i c host c o r t i c a l wounds through which Harveyella spores gain entrance i n t o the host. In cult u r e , Harveyella 195 spores germinated only i n the presence of the hosts, Odonthalia or Rhodomela. Subsequent i n t e r n a l vegetative development and the production of reproductive pustules occurred within a 3-month period. In addition to the phenomenon of spore germination dependency, subsequent vegetative development of Harveyella occurred only within s p e c i f i c host t i s s u e s . S t r u c t u r a l evidence indicates that penetrating r h i z o i d a l c e l l s may produce enzymes (perhaps v i a a membrane-associated lysosome system) which digest the host wall as suggested by Hess (1969), Webber and Webber (1970), G r i f f i t h s (1970), and Dorr and Kollman (1974) i n t h e i r studies of host wall penetration by a v a r i e t y of plant symbionts. No apparent p h y s i c a l tearing of the host c e l l w a l l i s observed i n areas of i n t r u s i v e r h i z o i d a l c e l l growth. Rather, there i s a symmetrical i n t e r r u p t i o n of the host wall with a r e s u l t i n g smooth apposition of host and parasite walls, s i m i l a r to that described i n the l i c h e n Parmelia sulcata (Webber and.Webber, 1970). Assuming that c e l l w a l l penetration by Harveyella i s a chemical rather than mechanical process, the chemical s p e c i f i c i t y of wall degrading enzymes produced by Harveyella could be a determinant of host s p e c i f i c i t y . In support of t h i s hypothesis, Harveyella has been noted to penetrate and develop between host medullary c e l l s but r a r e l y i n the host c o r t i c a l c e l l w all region. This s p e c i f i c i t y may be due to the f a c t that the walls surrounding the medullary c e l l s chemically d i f f e r from the c o r t i c a l c e l l s i n possessing abundant quantities of s u l f a t e d polysaccharides. The p r o l i f e r a t i o n of Harveyella within the host medullary c e l l walls and r a r e l y i n the cortex indicates that Harveyella may produce the enzymes necessary for the digestion of the medullary but not the c o r t i c a l c e l l w a lls. Perhaps h o s t - s p e c i f i c i t y differences i n various populations of Harvey-1 9 6 ella may also be r e l a t e d to enzyme s p e c i f i c i t y . 0. floeeosa i s the most common host of Havveyella i n B r i t i s h Columbia and northern Washington waters, whereas 0. washingtoniensis i s most commonly infected i n southern Oregon (Cape Arago). Both hosts are abundant i n the two geographic areas. This diffe r e n c e i n host s p e c i f i c i t y may r e f l e c t populational differences i n enzyme s p e c i f i c i t y . Conclusions concerning the nature of host s p e c i f i c i t y i n Havveyella must await biochemical studies of the host c e l l w a l l and possible w a l l degrading enzymes produced by Havveyella. In a study of the e f f e c t s of environmental parameters on the reproductive p e r i o d i c i t y of Havveyella i n the i n t e r t i d a l environment, tetraspores have been observed i n the late-winter, early-spring , apparently produced i n r e s -ponse to increased daylength and water temperature. Gametogenesis appears to be temperature s e n s i t i v e , occurring between a narrow temperature range (8.5-12 C). Carpospores are produced i n the l a t e summer when both water temperature and daylength reach a peak. Havveyella overwinters as i n t e r n a l vegetative filaments i n the basal remnant stubs of the host 0. floocosa. I t would be i n t e r e s t i n g to compare the phenology of s u b t i d a l Havveyella with the i n t e r t i d a l populations to determine i f each population i s reproductively separate. I t i s possible that the s u b t i d a l population may be another overwintering stage of the i n t e r t i d a l population which may produce spores i n January and February, i n i t i a t i n g the new tetraspore i n f e c t i o n s i n the i n t e r t i d a l host plants. In the present study, Havveyella has been found on host plants situated i n r e l a t i v e l y protected i n t e r t i d a l areas, although . Odonthalia axidi':Rhodo-mela are abundant i n both protected and exposed areas. I t has been hypothe-sized that the s p e c i a l i z e d i n f e c t i v e process, i . e . , through injured c o r t i c a l 197 areas of the host,would be a limiting factor in the distribution of Earveyella. It i s unlikely that successful spore transfer and settling could be accomplish-ed i n an active surf zone since i n turbulent waters, the probability that a spore would settle in a wound of a suitable host would be reduced. Electron microscopic examination of Earveyella cells has revealed the lack of typical floridean plastids, and fluorescent light microscopic studies have demonstrated that chlorophyll i s either absent or greatly reduced in quantity in Earveyella c e l l s . Exchange of l l tC-labeled materials between Odonthalia and Earveyella has been demonstrated using liquid s c i n t i l l a t i o n , radiochromatographies and autoradiographic techniques. Although a s t r i c t one-way transfer of metabolites from Odonthalia to Earveyella has not been demonstrated, a substantial increase in 1 1 +C-labeled substances in Earveyella and a concomitant decrease in radioactivity in Odonthalia occurred over a 36 hour translocation period. Organic carbon, supplied by the host, i s the major source of metabolites required by Earveyella for metabolic assimilation and conversion into storage compounds. Autoradiographic studies have revealed that most of the l l fC-labeled compounds are supplied from the subtending host medullary cells and transfer-red to rhizoidal cells of Earveyella in the zone of interdigitation (zone I I I ) . From this area, the metabolites are transferred to Earveyella medullary and cortical cells of the pustule. Host c e l l s , dispersed throughout the pustule of Earveyella are also active in fixing carbon-14 which may be subsequently transferred to adjacent Earveyella cells and thus serve as a secondary nutrient source. In both cases, the incorporation of label into host tissues and transfer to Earveyella cells i s a localized process, dependent on the intimate association of Earveyella and host c e l l s : ., 198 Scintillation analysis of i 4C-labeled neutral, anionic and cationic fractions (separated after various translocation periods by alcohol extraction and ion exchange chromatography), floridean starch (separated from the insol-uble residue by amylase digestion), ether extracted compounds, and the insoluble residue, revealed that changes in the amount of radioactivity in the neutral sugar and starch fractions of Havveyella and Odonthalia most closely paralleled the overall change in label noted previously (increase in Havveyella and decrease in Odonthalia). Radiochromatography of the neutral sugar fraction has failed to demonstrate conclusively which sugar(s) may be translocated, although there are some indications that an unknown compound(s), perhaps a uronic acid, may be involved in the exchange. A light and electron microscopic study of the rhizoidal cells of Havveyella located between host medullary cells in the interdigitation zone (zone III) was undertaken to determine the structural mechanisms involved in nutrient transfer between the two symbionts. It has been noted that structur-a l connections are formed between the host cells and Havveyella, but the cytoplasm of the adjoining cells i s not continuous. Rather, a dense, membrane-bound plug f i l l s the connections, similar to that reported in p i t connections of other red algae. A more lik e l y route for material exchange has been proposed to exist along the plasmalemma of Havveyella. A membrane system con-sisting of pinocytotic vesicles, multivesicular and concentric lamellar bodies, peripheral endoplasmic reticulum, dictyosomes, microbodies and an extensive vacuolar system may be involved in the uptake, processing and distribution of nutrients throughout the c e l l . Histochemical identification has been made of proteins, lipi d s and carbohydrates associated with the vacuolar/vesicle system. Light autoradiographic studies support this proposed 199 membrane uptake mechanism. It i s also.possible;that the plasmalemma-associated vacuolar system may act as a lysosomal' system, producing l y t i c enzymes involved in'rthe digestion of metabolites brought into the c e l l by pinocytosis and digestive enzymes involved in the degradation of the host c e l l wall. Few structural modifications which might be related to nutrient uptake were noted i n Harveyella cells of the pustule except for the plasmalemmavilli (plasmalemmal projections) of both inner: and outer medullary ce l l s . These projections may act to increase the c e l l surface area thereby increasing the efficiency of nutrient uptake in areas of decreasing nutrient concentration. In the last part of this study, an investigation was made of the effect of Harveyella on 0. floocosa. Infection by•Harveye l l a did not result i n the death of the host, although degeneration of host medullary cells did occur in localized, heavily infected regions of Odonthalia. Cortical cells adjacent to the degenerating medullary cells divide randomly, forming an external palisade cortical c e l l layer. At, a cellular level, numerous responses to infection were noted in host cells'of the interdigitation zone, whereas very few changes were observed i n host cells dispersed in the parasite pustule (Table IX). Possible factors causing the cellular response in Odonthalia were investigated. The possibility.that cellular response might be related to the presence of pit connections to infecting -Harveyella cells and/or to the presence of S-bodies in the host c e l l cytoplasm was excluded since both were found associated with host cells in the pustule which showed few cellu-lar responses to infection. It i s more l i k e l y that the infectious response may be caused by the presence of wall degrading l y t i c enzymes, produced by Harveyella rhizoidal c e l l s . 200 In conclusion, the present study has demonstrated that Earveyella mira-bilis i s a " p h y s i o l o g i c a l " as w e l l as a " s t r u c t u r a l " p a r a s i t e on i t s host, Odonthalia. I t s development and reproduction i s i n e x t r i c a b l y dependent upon the presence of a s p e c i f i c host, and i t b e n e f i t s p h y s i o l o g i c a l l y from the ass o c i a t i o n ; but, i n the process, i t i n f l i c t s some deleterious e f f e c t s on the host. Several theories have been proposed to explain the evolution of red a l g a l p a rasites. Sturch (1926) proposed that a l l o p a r a s i t e s may have ar i s e n as epiphytes which, through the course of evolution, became progressively more dependent on the host as a source of n u t r i e n t s . According to a hypothesis proposed by S e t c h e l l (1918), adelphoparasites may have originated from mutated spores of the host which became separate plants yet remained p h y s i o l o g i c a l l y dependent on the host. Grubb (1925) and Chemin (1937) considered some red a l g a l parasites as merely g a l l s , or p r o l i f i c outgrowths of the host i n response to an i n f e c t i o u s agent. It has been demonstrated i n t h i s i n v e s t i g a t i o n that H. mirabilis i s taxonomically d i s t i n c t from i t s rhodomelacean hosts and i s thus an a l l o p a r a -s i t e . Therefore, according to Sturch's hypothesis, Harveyella may have originated as an epiphyte which became progressively more dependent on i t s host and reduced vegetatively. I t i s also possible that Harveyella may have originated as a tumour-like p r o l i f e r a t i o n of the host. A l g a l g a l l s have been reported on numerous algae, presumably caused by a range of inducing agents which include nematodes, copepods, b a c t e r i a and v i r u s e s (Dixon, 1973). I f S-bodies are v i r a l p a r t i c l e s , they may be remnants of a v i r u s that at one time i n i t i a t e d the p r o l i f e r a t i o n of host t i s s u e i n t o the c h a r a c t e r i s t i c H. mirabilis pustule. Once d i f f e r e n t i a t e d , the separate pustule could have assumed a 201 reproduction separate and d i s t i n c t from i t s host. A problem i n t h i s theory concerns the difference i n reproduction between EaweyeZZa and i t s hosts and the f a c t that EaweyeZZa and 0. fZoaoosa have been shown to be c y t o l o g i c a l l y d i s t i n c t , d i f f e r i n g i n chromosome number and morphology (Goff and Cole, 1973). I t i s d i f f i c u l t to hypothesize how such a c y t o l o g i c a l and reproductive conver-sion could have occurred unless EaweyeZZa originated on a d i f f e r e n t host and secondarily "adopted" 0. fZoeeosa as a more sui t a b l e or alternate host. Although one can only speculate on how EaweyeZZa became associated with a s p e c i f i c group of rhodomelacean hosts, i t can be concluded from t h i s study that EaweyeZZa i s dependent developmentally, reproductively and n u t r i t i o n a l l y on i t s host 0. fZoaoosa. During t h i s study, numerous secondary questions have ar i s e n which have not been adequately answered. Many of these questions are applicable to parasite systems i n general.. They include: 1. What i s the nature of h o s t - s p e c i f i c i t y i n EaweyeZZal a. Is i t r e l a t e d to the presence of a s p e c i f i c chemical substance i n the injured host which i n i t i a t e s spore germination? b. Are h o s t - s p e c i f i c wall degrading enzymes produced by i n t e r d i g i t a t i n g r h i z o i d a l c e l l s ? 2. What i s the function of the membrane-associated vacuolar system? 3. Is a lysosomal system present and f u n c t i o n a l i n r h i z o i d a l c e l l s ? 4. Does photosynthesis occur i n EaweyeZZal 5. How are secondary p i t connections formed between adjacent host and parasite c e l l s and what are t h e i r functions? 6. What are S-bodies? 202 7. 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Uber virusalmliche Teilchen i n den Schweigger-Seidelschen Hiilsen einer Hundemilz. Expevientia 18: 171-172. Appendix I, Part 1. New Distributional Records for Harveyella m i r a b i l i s i n the Northeast P a c i f i c . Map Ref. Location Host . Date Collected Collector Water Temp. C S a l i n i t y Notes 0.5 m Depth /oo Stages a" ? 0 12.5 - - a s a Goff 12.8 - - s^  a a Not abundant 12.9 a a at t h i s area Goff 13.3 -- — a s s _ Abundant Goff - - _ _. _ a 8.0 - a s s 11.5 - - s s a 9.3 33.42 s a a -8.5 30.59 . a a a _  • Goff 9.5 33.10 - a a - Abundant at 8.3 29.49 a s s - S. edge Cove Goff 12.0 30.52 - • s _ a 1974-Heavy 9.0 32.92 •- - a; a sea urchin Goff 8.5 29.94 - - . - •  - grazing 12.0 - - s - - Scarce . — - s - - -Goff - - s - . - Not abundant — - — s — a 9.5 - s a a _ 11.5 - - s s a 13.0 . - - - - a - - _ _ _ Goff 9.0 - s - - _ Not abundant 9.0 - s s — _ 10.0 - - >s s s 12.0 - - a a 13.0 - - - - a Goff 13.0 - • - a Not abundant 9.5 - - a - - (only i n tide 12.0 - . - - - a pools or i n 8.5 r a - - - sheltered 8.8 (old) areas, lower - than 0.5 m 12.5 - a - - . . - CHS) (old) 12.8 — - - - a On large bench Ore. 37 36 34 35 33 32 31 30 29 Harris Beach St. Pk. 0w(a);0f(s) P i s t o l River (near Crook Pt) North Cove, Cape Arago South Cove, Cape Arago Sunset Beach, Cape Arago Hecata Head Strawberry H i l l (S. Beach) Neptune Beach St. Pk. Cape Perpetua Rl(s);0f(s) Ow(a) Ow(a) Ow(a) Ow(a) Ow(a);Rl(s) Ow(a);0f(s) Ow (I T ( s ) Ow(a) Ow(a);Rl(a) Ow(a);Rl(s) absent Of(s) Of(a) Of(a);Rl(s) Of (a) Of(a) Of(a);Rl(a) Of (a) Of(a);Rl(a) Of(s) Of(a);Rl(a) Of(a);Rl(s) Of(s). Of(s);Rl(a) 8. VII.1972 5. VIII.1972 9. VII.1972 6. VIII.1972 23. V.1971 7. VIII.1971 13. V.1972 8. VII.1972 1. VI.1973 24. V.1974 3.VI.1973 25. V.1974 8.VII.1972 3.VI.1973 25.V.1974 7.VII.1972 27.V.1970 21.V.1971 - V.1971 2. VI.1972 14. VII.1972 6. VIII.1972 14.VI.1971 1.VI.1972 12.VI.1972 12.VII.1972 29.VII.1972 7. VIII.1972 Of(a);Rl(s) 14.VI.1971 Of(s) 1.VI.1972 Of(s) 12.VI.1972 Of(a);Rl(a) 13.VII.1972 Of(s) 28.VII.1972 6.VIII.1972 0f(a);0w(s) 10.VII.1972 Rl(a) 28 Seal Rock Of (a) 8.VIII.1972 Goff Of (a) 12.IV.1970 27 Yaquina Head Of(a);Ow(s) 23.IV.1970 Goff Of (a) 10.VIII.1972 26 Devils Punch Bowl Of (s) 7.VII.1972 Goff Of(a);Rl(s) 23.IV.1970 25 Boiler Bay Of (a) 1.IV.1971 Goff Of(a) 28.VI.1972 Wash. 2A ' Rosario Beach State Park 22 21 19 B.C. 17 16 15 14 13 12 10 7 Cattle Pt. (San Juan Is.) American Camp (SJI) Deadman Bay (SJI) Parson's Point (Sooke, Van. Is.) Otter Point Glacier Point Botany Beach (S. end) (near Pt. Renfrew) Botany Beach (N. end) Point Atkinson (Lighthouse Park) Cape Lazo Brandon Point (Robinson Is.) Of (a) Of(a) 0f(a) Of (s) Of (a) 0f(s) 0f(s) 0f(s) Of(s);Ow(s) Of(a);0w(s) Of(a) Of(s);Rl(a) Of(s);Rl(a) 18.III.1972 28.VI.1972 25.IX.1972 . 10.III.1973 14.VII.1973 10. XI.1973 2.III.1974 11. V.1974 21.X.1972 27.X.1973 21. X.1972 22. X.1972 27.X.1972 Goff Of(a) 1.V.1972 0f(a) 17.1.1972* *start 20-month study 0f(s) 29.III.1974 0f(a) 29.VI.1973 0f(a);0w(s) 31.III.1974 RKs) 0f(a);Rl(s) 19.VII.1974 0f(a) 30.VI.1973 0f(a) 6.II.1974 Of(a) 8.IV.1974 0f(a) 18.VII.1974 Of(a);Rl(s) 30.VI.1973 0f(s);Rl(s) 6.II.1974 0f(s) 8.VII.1974 0f(a);Rl(s) 18.VII.1974 . 0f(a) 4.VIII.1974 Of (a) Of(a) Goff Mumford Goff Goff Mumford Goff 14.IX.1972 28.11.1974 Goff Goff Goff Goff Goff Goff Goff Goff 14.5 - . - - a Behind rocks 7.0 - - - • a(old) Behind shelt-7.5 - - a s ering, rocks 10.5 - - - - only • 10.0 - - a - Scarce - - a a _ _ Abundant on .. - - a - s rock bench N. (old) side cove 0-1.5 M 7.0 - a 9.5 - high a a d r i f t Abundant 9.0 - high - - a 6.0 25 .39 a - - -10.0 31 .50 wall s s a 8.0 28 .06 s - - s 5.5 27 .01 a - - '• - Very scarce 9.0 30 .20 a a s - Very scarce 9.0 - - - s ( o l d ) D r i f t - - s - • - s 9.0 - -' - - a ( o l d ) D r i f t 9.3 - s - - a High tidepools • — s — s J a a s 0-1 M tide : l e v e l (CHS) 7.0 29 .25 s Scarce 12.0 - - s s a 8.0 29 .95 a - - -13.0 32 .10 _ a a a 12.5 - ' - s - a 7.5 29 .56 a - - - On rock bench 8.0 27 .16 a s - - 0.5 M tide 12.5 33, .25 a a a s l e v e l 11.5 - - - - a In coves 0-5.5 34. .41 a s - - 0.5 M tide 18.5 27. .16 a a s - l e v e l 13.0 32, .31 s a a a 13.8 - s a a a Subtidal depth 2-6 M - - - s s s Scarce 6.0 25, .32 a - - - Extremely abundant Burgess Island Of(a) 28.11.1974 Goff 5 Fox Island Channel Of(a) 27.11.1974 Goff 4 Dalkeith Point Of(a) 27.11.1974 ; Goff 3 Mignot Point Of(s);Rl(a) 26.11.1974 Goff (Belize Inlet) 2 Westerman Bay Rl(a) 26.11.1974 Goff 1 Chatham Sound Of(a);Rl(a) 21 .VII.1974 Nordin Key to Abbreviations: Of = Odonthalia floocosa 0w = 0. washingtoniensis Rl = Rhodomela larix CHS = Canadian Hydrographic Standard a = abundant s = scarce 0 = cystocarpic ® = tetrasporic o* = mature male gametophyte ? = mature female gametophyte 7.5 30.51 a s s - Very exposed rock - heavily grazed host Hm extremely abundant - s - - - Scarce 7.0 29.25 s - - - Scarce 5.5' 20.54 s - - -2.3 - s s - - Scarce - - - a s s Westside Ridley Is. mid-intertidal ho Location Appendix II. Distribution Records for Harveyella mirabilis'. Herbarium Annotations Host Reproductive Stage 0 o* ? Q Date Collected Subtidal (ST) • or I n t e r t i d a l (IT) Collector Specimen Location and Number B r i t i s h Columbia Inside Passage Of 50°05 3'N 125°15 2'W Discovery Passage Of (N. Duncan Bay) Quadra Is. (S) Of Whiffen Spit, Van.Is. Of Washington Goose Island Of 48°27'N Of 122°57'W Of Mt. Dallas Beach (San Juan Is.) Waadah Island 48°27'N 122°59'W (San Juan Is. ) American Camp Beach Planta Exsicatae A . B . ° ^ N E ^ Universitate Briton- Of(NE) niao-Columbiana Editae Series - Algae Fasciculus II. Of Of Of Of(NE) Of(NE) Of(NE) Of (NE) Of Of N. W. At l a n t i c Fortune Bay, Nfld. Rc Rc Channel, Newfoundland Rc x x X X X X X X X X X X X X X X X X 10.IX.1970 10.IX.1970 31.X.1973 25.X.1968 28. VI.1957 29. VI.1957 30. VII.1958 1.VII.1958 15.VIII.1958 21.VII. 14.VII. 14.VII. 14.VII. 14.VII. 14.VII. 14.VII. 14.VII. 14.VII. 1959 1958 1958 1958 1958 1958 1958 1958 1958 Rc 1.VII.1972 1.VII.1972 29.VII.1929 3.VIII.1901 ST ST IT(low) IT IT IT IT IT IT IT IT ST(upper) ST(upper) Pace Foreman Pace, Harrison Scagel Seagel Scagel Scagel Scagel Scagel Scagel Scagel Scagel Scagel Scagel Scagel Scagel Scagel Hooper, Roberge Hooper, Roberge Howe Howe HR UBC UBC UBC UBC UBC UBC UBC UBC UBC UBC L MIN LD PC GL US UC DS i n UC UBC UC NY wY Wet Stack(w) 43933 999(w) 38928 . 351(w) 1282(w) 31(w) 33(w) 60 (w) 970(w) m 107217 502509 49345 452161 1123 762(s) 743(s) K5 ON Kittery Pt., Maine Rc X IV.1893 Rc(NE) 6.IV.1893 Rc X X x. 6.IV.1893 Cutler, Maine Rc X 6.VII.1902 Harpswell, Maine Rl X X 10.VII.1906 Co l l i n s . Holden and _ ' VII.1906 Setchell, 1847 Kc X X Phyootheca Boreali-" R c X VII.1906 Americana Rc X X VII.1906 Rc(NE) VII.1906 Rci X VII.1906 Rc(NE) VII.1906 X VII.1906 L. Boar's Head, N.H. Rc- X 4.V.1902 Revere Beach, Mass. Rc X X 19.III.1882 Cape Ann, Mass. Rc X X III.1894 Newport, R.I. Rc X X X IV.1904 Rc V.1896 , E. Atlantic Lyme Regis, England Rc X IX.1889 Charmouth, England Rc IX.1889 Rc X IX.1889 Rc X X IX.1889 Berwick, England Rc X VIII.1884 Dover, England Rc X 22.IX.1970 Fi l e y , Yorkshire, Eng Rc X 1830-1860? Brook Bay, Isle of Wight, England Rc 19.IX.1896 Polysi-phonia elongulata Studland, England 12.IV.1889 Arbroath, Angus, Scotland Rc X 15.V.1893 St. Andrews, Scot. Rc X 15.XI.1973 Roscoff, France Rc X X 27.III.1929 Rc X 10.IX.1946 Rc X 23.111.1929 Rc X X 11.1974 Herdla, Norway Rc X 31.VII.1935 Bohuslan, Kristineberg Sweden Rc X 17.IV.1906 Thaxter Davis Davis Colli n s Collins C o l l i n s Collins Collins C o l l i n s Collins C o l l i n s C o l l i n s C o l l i n s C o l l i n s Thaxter Simmons Collins UC MICH C NY NY NY C MIN US MICH PC NY NY NY NY NY 94225 No 2-1 5515 4263 SD 30* Holmes Holmes Holmes Holmes Batters Fletcher, Gatty Farnham BM BM UC TCD BM BM STA N3 ho 020394 George BM Holmes BM Holmes STA Blackler STA Dangeard B0RD Dangeard B0RD Dangeard B0RD Cabioch UBC Levring LD Kylin LD Blekinge, Ronneby Sweden Rc X X Rc X Halland, FjondskSr Sweden Rc X X Kullen Penn.,Sweden Rc X Mecresalgen, Helgoland Rc X Rc X Katthammarsvlk, Helgoland Rc X Mb" en Rc X Rc X X Femerbelt: 0jct Rc Remmen 0f Bog6. Rc X Scoresby Sound, Greenland Rl Rl X X Rl X X 9.VI.1939 9.VI.1939 25.VII.1905 10.VI.1901 III.1900 V.1900 20. VI.1899 28.V.1895 28.V.1895 21. V.1895 2.V.1892 x 26.VII.1892 x 1.VII.1892 5.IV.1892 ST 15 M ST 15 M Levring Levrlng UC AHFH 737483 6269 - Kylin LD - Kylin c 5-- Kuckuck PC Kuckuck L Svedelius UPS - Rosenvinge L - Rosenvinge UC 478646 — Rosenvinge UPS 5787 Rosenvinge BM 240 - Hartz c 4-3 - Hartz c 3-4 - Hartz c 3-4 CO 2.29 Index to Standard Abbreviations (Lanjouw and Sta f l e u , 1956) Letter i n i t i a l s are standard abbreviations of herbaria (UBC - Univ e r s i t y of B r i t i s h Columbia, Vancouver; L - Rijksherberium, Leiden, Netherlands; Minn -Uni v e r s i t y of Minnesota, Minneapolis; LD - Lund, Sweden; PC - Museum National d'Histoire N a t u r e l l e , P a r i s ; GL - Univ e r s i t y of Glasgow, Scotland; US -Smithsonian. I n s t i t u t i o n , Washington, D. C.; UC - Un i v e r s i t y of C a l i f o r n i a , Berkeley; DS i n UC - Dudley Herbarium of Stanford i n Un i v e r s i t y of C a l i f o r n i a , Berkeley; NY - New York Botanical Gardens; MICH - Un i v e r s i t y of Michigan, Ann Arbor; C - Botanical Museum and Herbarium, Copenhagen; BM - B r i t i s h Museum, London; TCD - T r i n i t y College, Dublin; STA - Univ e r s i t y at St. . Andrews, Scotland; BORD - Jar d i n Botanique de Bordeaux, France; AHFH -Herbarium of A l l a n Hancock Foundation, Los Angeles; UPS - I n s t i t u t e of Systematic Botany, Uppsala, Sweden. 230 Appendix I I . D i s t r i b u t i o n a l Records of H. mirabilis 2. L i t e r a t u r e References Reference Location Kuckuck, 1894 Sturch, 1899 C o l l i n s , 1900 Sturch, 1924 Rbsenvinge, 1931 Sinova, 1940 K y l i n , 1944 Sinova, 1954 Wilce, 1959 Norton, 1970 Ed e l s t e i n , Chen and McLachlan, 1970 Helgoland {[Choreooolax albus) Stokes Bay, Gosport Maine, N. Mass., S. Mass., Rhode Island Cork Harbour, Ireland Denmark Svin^er, Norway (Lyngbye) Helsing^r, Sweden (Liebman) Holstein (West Coast), Germany (Reinke) FaerBe Island (B^rgensen) K r i s t i a n i a Fjord, Norway (Gran) BohuslMn, Sweden (Kylin) Halland, Sweden (Kylin) Gotland (Svedelius) Scoresby Sound, East Greenland (Rosenvinge) Sea of Japan: Nakhodka Bay Sweden (West Coast) Sea of Okhotsk: Shantar-Yakshina Bay Point Ola Anaur (TNPC) Labrador Peninsula Port Burwell, False River Bay, Koksoak River Ireland, Kilmore Quay Digby Neck Peninsula, Bay of Fundy, Nova Scotia Chemin, 1972 Couquet ( F i n i s t e r e , France) Roscoff Appendix III. Collection Data, Sooke, B. C , 1973-1974. Tide Date Level (Feet, CHS) Time P.S.T. A i r Temp. C Water Temp. C Sa l i n i t y loo Cloud Cover 0. floocosa Abundance (Relative) E.. mirabilis Abundance (Relative) H. mirabilis Reproductive Ratio: (%) 200 Plants « <? 5 0 V 16.1.1973 3.8 1605 5.0 6.0 _ R s s 30 6 0 4 60 13.11.1973 2.2 1745 2.0 6.0 - OC S-new growth s 51 18 5 4 22 13.III.1973 2.9 1625 4.5 8.2 - OC a+ a+ 65 23 9 3 0 7.IV.1973 1.9 1130 10.5 8.7 27.61 C a-H- a 45 32 10 3 10 6.V.1973 1.1 1110 12.5 9.0 29.56 R a-4-f a 24 33 26 8 9 18.V.1973 1.5 0915 12.0 9.3 29.46 0C a-H- a 19 35 30 10 6 16.VI.1973 1.4 0855 11.0 9.5 29.21 R a++ a+ 12 30 28 16 14 1.VII.1973 0.3 0900 15.5 10.0 30.29 ' C a++ a++ 12 27 25 19 17 15.VII.1973 1.8 0835 19.5 10.5 30.61 C 1 a++ a-H- 11 20 14 44 11 28.VII.1973 0.8 0715 18.0 11.5 31.53 C a-H- a 10 14 4 65 7 24.VII.1973 1.9 0520 13.0 11.5 32.80 C a-H- a 15 8 3 62 . 12 20.IX.1973 2.6 0235 10.0 9.0 31.97 R a+ s 22 16 15 47 0 15.X.1973 1.8 2300 3.0 8.6 32.06 R a+ s 22 28 26 6 18 11.XI.1973 1.1 2100 5.5 8.2 30.99 OC s s 19 22 16 10 33 9.XII.1973 1.0 2010 0.5 7.9 27.90 OC s s 19 6 2 12 61 8.1.1974 1.1 2040 - 3.0 4.7 30.56 C s s+ 26 5 0 7 62 6.II.1974 2.0 2015 4.0 6.5 20.23 OC s 6 67 a 0 2 23 29.III.1974 3.0 1245 18.0 8.5 27.95 C a a+ 62 20 7 4 7 25.IV.1974 1.9 1125 6.0 9.2 30.07 R a++ a++ 40 31 16 7 6 22.V.1974 1.1 0850 12.5 9.3 31.04 OC a++ a+++ 19 42 28 10 1 4.VI.1974 1.0 0810 10.0 9.5 30.89 R a-H- a+ 16 40 29 11 4 3.VI.1974 1.3 0750 14.5 10.0 30.983 R a-H- a ' 8 36 34 12 10 19.VII.1974 0.8 0815 15.5 10.4 30.99 C a++ - • - - • - - -. 16.VIII.74 1.6 0705 16.0 10.5 29.71 R ~ a s 6 10 15 60 9 Key to Abbreviations: 9 = tetrasporic a = abundant <f = male gametophyte a+ •= very abundant ? = female gametophyte a++ = extremely abundant © «• carposporic R: «* rain v •« vegetative 0C • overcast s = scarce C •* clear g+ r. very scarce 232 Appendix IV Temperature, S a l i n i t y and Hours Bright Sunshine at Selected Stations Sea Temperature C, 0.5 m Depth Parsons Point, B.C. Sooke, B.C. Neah Bay, Wash. (present study) 1965 - 1967 (U.S. Coast and M o n t h 1 9 7 3 1974 ( E l l i o t t , 1969a,b) Geodetic Survey : Publ., 1962) I 6.0 4.7 - 7.2 II 6.0 6.5 7.6 7.8 II I 8.2 8.5 7.9 9.0 IV 8.7 9.2 8.9 10.5 V 9.3 9.3 - 11.5 VI 9.5 9.5 9.3 12.0 VII 10.5 10.4 9.4 11.7 VIII 11.5 10.5 9.8 10.8 IX 9.0 - 10.8 11.0 X 8.6 - - 9.9 XI 8.2 - 10.1 -XII 7.9 - 9.6 8.4 233 S a l i n i t y /oo Month Parsons Point, B.C. (present study) 1973 1974 Sooke, B.C. 1965 - 1967 ( E l l i o t t , 1969a,b) Neah Bay, Wash. 1934 - 1960 . (U.sl' Coast and Geodetic Survey Publ., 1962) I - 30.56 - 31.7 II - 20.23 28.2 29.5 II I - 27.95 31.1 31.2 IV 27.61 30.07 30.1 31.0 V 29.56 31.04 - 31.5 VI 29.21 30.89 31.5 31.2 VII 30.61 30.98 31.7 29.1 VIII 32.80 29.71 31.8 29.5 IX 31.97 - 30.9 31.3 X 32.06 - - 31.8 XI 30.99 - 21.9 32.0 XII 27.90 - 31.0 * 29.5 234 Hours Bright Sunshine Month V i c t o r i a , B.C. (Gonzales Hts.) ( V i c t o r i a Weather Bureau, pers. comm., 1974) 1973 1974 V i c t o r i a , B.C. 1931 - 1960 (Environment Canada, 1973) Gla c i e r Point, Van. Is., B.C. (Meteorol. Branch Canada, 1962) I II I I I IV V VI VII VIII IX X XI XII 87.7 123.8. 154.3 244.3 314.9 262.9 324.1 313.6 207.6 117.1 77.8 98.6 81.7 144.2 143.8 258.3 305.5 284.6 320.2 258.1 206.3 70 97 156 210 275 277 337 298 208 141 81 66 65 90 140 200 260 278 340 290 195 130 60 40 235 Appendix V Modifications of Standard Techniques Modified Karpechenko's f i x a t i v e (based on Papenfuss's modification; Abbott, 1971). Solution A: Chromic acid, 1.g; g l a c i a l a c e t i c a c i d i 5 ml; seawater, 70 ml. Solution B: Commercial formaldehyde, 50 ml; seawater, 35 ml; DMSO, 2 ml. Mix equal portions of s o l u t i o n A and B immediately p r i o r to use. Material i s f i x e d 12-24 hr, washed i n running water 4-8 hr and dehydrated i n a graded ethanol seri e s to 70% ethanol. Tissue i s stored i n 70% ethanol to which small amounts of A n i l i n e blue may be added. A n i l i n e blue-HCl s t a i n i n g of reproductive structures ( a f t e r Johansen i n Smith, 1951). 1. Stain whole tissue pieces or sections i n a 1% aqueous solu t i o n of A n i l i n e blue for 3-5 minutes. 2. Wash i n water u n t i l s t a i n i n t e n s i t y appears to be c o r r e c t . 3. Add drop 1% HCl (aq) to water to f i x s t a i n . 4. Treat i n 1% HCl 15-30 minutes followed by washing i n water u n t i l a c i d i s removed. Aceto-iron-Haematoxylin-chloral hydrate s t a i n i n g of chromosomes (after Wittmann, 1965). 1. Stock Haematoxylin: Haematoxylin, 4 g; i r o n alum (FeNH^ (S0i+)2 • 12H 20), l g ; 45% a c e t i c a c i d , 100 ml. Allow to ripen at l e a s t 3 days. 2. Stain: Stock Haematoxylin, 5 ml; c h l o r a l hydrate, 2 g. Keep so l u t i o n r e f r i g e r a t e d and use within 2-3 weeks. 236 M-. Modified Erdschreiber Culture Medium Stock Amount added T o t a l to 1000 ml concentra-. seawater tion/1 S o i l water supernatant - 50 ml NaN03 . 2 g/100 ml SW1 . 1 ml 200 mg Na2HP0^ 2 g/200 ml DW2 1 ml 10 mg KN03 10 g/200 ml SW 1 ml 50 mg Fe(EDTA) 200 mg/200 ml SW 1 ml 1 mg T r i s - 500 mg 500 mg Vitamin B 12 10 mg/100 ml DW 1 ml 0.1 mg K2Te03 1 g/1000 ml SW 1 ml 1 mg Ge0 2 50 mg/100 ml SW 2 ml 1 mg *Tetracycline 500 mg/100 ml DW 20 ml 100 mg 1 Seawater . ( f i l t e r e d ) 2 D i s t i l l e d water Day 1: F i l t e r seawater through Whatman No. 1 paper; heat to 75 C for 1 hour; steam s o i l plus seawater 3 hours, decant, f i l t e r , saving supernatant. Day 2: Heat seawater to 75 C. M i l l i p o r e f i l t e r (0.22 ym) each stock s o l u t i o n . Day 3: Add above s p e c i f i e d volumes of each stock to 50 ml cold soil-water supernatant. Add soil-water (plus stocks) to cold seawater. Te t r a c y c l i n e may be added to the media with other stocks and used to help! c o n t r o T contaminants. 237 5. Hard Cure Modi f i c a t i o n of Spurr's Low V i s c o s i t y Epoxy Resin (Spurr, 1969) ERL 4206 (Vinyl cyclohexase dioxide) 10.0 gm DER 736 ( D i g l y c i d y l ether of poly-propylene glycol) 5.0 gm* NSA (Nonenylsuccinic anhydride) 26.0 gm S - l (Dimethylaminoethanol) 0.4 gm ( A l l components of r e s i n a v a i l a b l e through Polysciences, Inc., Warrington, Pennsylvania) * V a r i a t i o n i n the amount, of DER,736 determines the degree of hardness of the cured block Standard- medium (firm) = 6 gm Hard cure medium = 4-5 Soft cure medium = 7 Castings are polymerized for 8 hours at 70 C. 238 6. S c i n t i l l a t i o n Fluor (UNESCO, 1973) 1 1 2-ethoxyethanol 2 1 Toluene ( " S c i n t i l l a r " , Mallinckrodt Chemicals, St. Louis, Mo.) 5.5 g PPO (2,5-diphenyloxazole, Kent Laboratories Ltd., Vancouver, B. C.) 50 mg P0P0P (l,4-Bis-[2-(5-phenyloxazolyl)]-benzene, Kent Laboratories.Ltd.) A f t e r mixing,nitrogen gas i s passed through the. s o l u t i o n as a means of removing oxygen and thus reducing "oxygenquenching" (Wang and W i l l i s , 1965). 239 Appendix VI lglucose V a l u e s i n Two Solvent Systems Compound R g Basic Solvent g A c i d i c Solvent R Mean g Standard Deviation* Mean Standard Deviation Ribose 205.70 ± 26.61 Fucose 141.68 ± 2 . 0 9 195.28 5.30 Xylose 152.22 6.67 162.96 14.30 Rhamnose 233.06 26.46 Arabinose 119.36 8.37 147.19 5.12 Mannose 117.83 3.60 122.06 5.09 Fructose 121.17 140.39 5.53 Glucose 100.00 Standard 100.00 Standard Sucrose 75.22 3.35 L 55.14 5.56 Fl o r i d o s i d e 98.92 3.03 106.23 5.13 I s o f l o r i d o s i d e 78.76 3.67 93.28 3.01 Maltose 58.28 0.68 31.11 _ Trehalose 52.98 4.10 34.29 3.26 Lactose 46.63 5.21 25.29 2.0 Galactose 86.52 2.67 90.44 1.56 Glucuronic A 17.74 4.27 102.54 1.78 Galacturonic A 13.88 2.83 91.49 0.84 E r y t h r i t o l 239.99 20.97 Mannitol 94.19 2.50 122.98 2.58 S o r b i t o l 92.34 - 124.89 3.18 M - i n o s i t o l 34.78 3.72 38.15 4.19 Each mean R i s based on a minimum of 20 values. Where no standard deviation given, i n d i c a t e s l e s s than 20 values. 

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