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Observations of higher fungi and protists associated with the marine red algae Rhodoglossum affine and… Phillips, Roger Edward 1982

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OBSERVATIONS OF HIGHER FUNGI AND PROTISTS ASSOCIATED WITH THE MARINE RED ALGAE RHODOGLOSSUM AFFINE AND GELIDIUM COULTRI by ROGER EDWARD PHILLIPS B.Sc. , C a l i f o r n i a Polytechnic State University, San Luis Obispo, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1982 © Roger Edward P h i l l i p s , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Abstract This d i s s e r t a t i o n reports a study of the fungi and 'protists* (Labyrinthulids, Thraustochytrids, Hyalochlorella  marina) associated with the i n t e r t i d a l red algae Rhodoglossum  a f f i n e and Gelidium c o u l t e r i . Research focused on laboratory i s o l a t i o n s from a l g a l t h a l l i c o l l e c t e d from i n s i t u populations. D i f f e r e n t i s o l a t i o n techniques and i s o l a t i o n media were employed to evaluate the abundance and d i v e r s i t y of fungi and p r o t i s t s associated with these red algae. A l g a l tissue surface s t e r i l i z a t i o n and rigorous r i n s i n g procedures were used to remove and/or enumerate surface-associated microbes. The results obtained from the d i f f e r e n t i s o l a t i o n techniques and a l g a l tissue pretreatment procedures are compared and discussed i n terms of th e i r usefulness for each member of the algal-associated microbiota. Natural populations of a f f i n e and c o u l t e r i support a r i c h fauna of marine p r o t i s t s . The most prevalent members of t h i s p r o t i s t fauna were Labyrinthula spp. resembling the "Vishniac Strains" and Thraustochytrium motivum. Schizochytrium  aggregatum, a new species of Labyrinthulid designated Labyrinthuloides sp. 1, and Hyalochlorella marina were also common depending upon the i s o l a t i o n method u t i l i z e d . These p r o t i s t s appear to be associated with the surfaces of the a l g a l t h a l l i , and exis t as saprobes and/or perthophytes rather than biotrophic parasites of the al g a l tissues. Isolations from f i e l d - c o l l e c t e d a l g a l tissues also yielded actinomycetes, yeasts, and a high d i v e r s i t y of imperfect fungi. Overall i s o l a t i o n frequencies for i n d i v i d u a l fungal taxa were low. Most of the mycelial fungi i s o l a t e d are considered to be of t e r r e s t r i a l o r i g i n and of questionable 'significance' i n the i n t e r t i d a l habitat. Only four, possibly five, are presently considered marine. The mycelial fungi most commonly is o l a t e d include: Acremonium sp. 019-78, Cladosporium cladosporioides, Dendryphiella salina, P e n i c i l l i u m spp., Phoma sp. (Group 1), Sigmoidea l i t t o r a l i s sp. nov., and Unidentified hyphomycete 044-78. Certain of these fungi may grow saprobically (as pertho-phytes) on reproductive and/or senescing a l g a l tissues i n the i n t e r t i d a l habitat, but t h e i r a c t i v i t i e s appear to be limited. F i e l d - c o l l e c t e d t h a l l i of Rhodoglossum a f f i n e and Gelidium  c o u l t e r i were allowed to decompose i n mesh bags placed i n the i n t e r t i d a l . The succession of higher fungi associated with the decomposing algae was followed by p l a t i n g representative bimonthly subsamples of the a l g a l tissues onto a Base Mineral Medium. Rhodoglossum a f f i n e deteriorated completely a f t e r 52 days of exposure, while a small amount of Gelidium c o u l t e r i remained a f t e r 71 days. Qualitative aspects of the mycobiota associated with the two a l g a l species were similar, however fungi were i s o l a t e d more frequently from c o u l t e r i . A dominant mycobiota was apparent aft e r 36 days of exposure on the beach. Acremonium sp. 019-78, Dendryphiella salina and Sigmoidea l i t t o r a l i s sp. nov. were active colonizers of the decomposing a l g a l tissues, and t h e i r i s o l a t i o n frequencies increased as decomposition proceeded. Several species of bacteria capable of u t i l i z i n g the c e l l i v w a l l polysaccharides of red algae (agar, carrageenan) were also present on the decomposing algae. It i s possible that the a c t i v i t i e s of these bacteria enhanced fungal development. Thraustochytrium motivum, Schizochytrium aggregatum and Ulkenia sp. RC02-80 were placed into s t e r i l e seawater cultures with s u r f a c e - s t e r i l i z e d tissues of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . After 72 hours of incubation, p o s i t i v e growth associations were examined using scanning electron microscopy. The three Thraustochytrids displayed luxuriant growth on a l l a l g a l tissue types, and produced extensive ectoplasmic networks on the a l g a l surfaces which functioned i n attachment and, presumably, i n the absorption of dissolved nutrients. Ectoplasmic net elements were resolved down to 0.02 pm i n diameter, but no obvious 'penetration' of the a l g a l tissues could be discerned. A l l of the p r o t i s t s (Labyrinthulids, Thraustochytrids, Hyalochlore11a marina) is o l a t e d from these red algae are described and i l l u s t r a t e d . Certain commonly encountered and/or poorly known mycelial fungi are also described, including a new species of marine hyphomycete, Sigmoidea l i t t o r a l i s sp. nov. V TABLE OF CONTENTS L i s t of Tables v i i i L i s t of Figures x i i Acknowledgement xiv I. INTRODUCTION AND REVIEW 1 I I . GENERAL RESEARCH INFORMATION AND METHODOLOGY A. THE ALGAE 8 B. MEDIA AND CULTURE CONDITIONS FOR THE ISOLATION, MAINTENANCE AND CHARACTERIZATION OF MICROORGANISMS 13 C. MICROSCOPIC EXAMINATION AND PHOTOMICROGRAPHY. . . . 15 D. THE MICROORGANISMS AND THEIR IDENTIFICATION . . . . 17 E. STATISTICS 19 III. OBSERVATIONS OF AND ISOLATIONS FROM FRESH ALGAE COLLECTED FROM i n s i t u POPULATIONS INTRODUCTION 21 MATERIALS AND METHODS A. FIELD COLLECTIONS 25 B. GENERAL LABORATORY PROCESSING AND PLATING OF FIELD SAMPLES 1. Surface s t e r i l i z a t i o n , r i n s i n g and pl a t i n g . . . 31 2. Media and Culture Conditions 34 3. Moist Chamber Incubation 35 C. SPECIFIC TECHNIQUES USED FOR EACH FIELD SAMPLE. . . 36 1. Sample 1 36 2. Sample 2 37 3. Sample 3 37 v i RESULTS A. OBSERVATIONS OF FIELD-COLLECTED ALGAE 42 B. ISOLATIONS FROM FIELD-COLLECTED ALGAE 1. Sample 1 (December, 1978) 44 2. Sample 2 (March, 1979) 49 3. Sample 3 (June, 1980) a. Alg a l Tissues Plated on Agar Media 63 b. Alg a l Tissue Rinse Water Plates 81 c. S t e r i l e Seawater/Pine Pollen Cultures . . . 87 4. Moist Chambers 92 DISCUSSION A. LABORATORY METHODS 1. Media, A n t i b i o t i c s and Culture Conditions . . . 95 2. Surface S t e r i l i z a t i o n and Rigorous Rinsing. . . 98 3. Iso l a t i o n Methods a. P r o t i s t s 101 b. Higher Fungi 104 B. ISOLATES 1. Labyrinthulids 108 2. Thraustochytrids 110 3. Hyalochlorella marina 112 4. Actinomycetes 114 5. Yeasts and Yeast-like Fungi 115 6. Mycelial Fungi 116 C. DIFFERENCES IN THE ISOLATION OF MICROORGANISMS BETWEEN THE ALGAE AND THEIR LIFE HISTORY STAGES 1. P r o t i s t s 120 2. Higher Fungi 122 CONCLUSIONS 123 IV. ISOLATION FROM DECOMPOSING, 'ARTIFICIALLY DRIFTED', Rhodoglossum a f f i n e and Gelidium c o u l t e r i INTRODUCTION 128 MATERIALS AND METHODS 131 RESULTS 133 DISCUSSION 140 CONCLUSIONS 143 v i i V . SCANNING ELECTRON MICROSCOPE OBSERVATIONS OF THRAUSTOCHYTRIDS ON THE SURFACES OF Rhodoglossum  a f f i n e and Gelidium c o u l t e r i INTRODUCTION 145 MATERIALS AND METHODS 146 RESULTS A. SURFACE STERILIZATION IN 0.3% CHLOROX 148 B. GROWTH OF HIGHER FUNGI 148 C. THRAUSTOCHYTRID DEVELOPMENT AND SCANNING ELECTRON MICROSCOPY 149 DISCUSSION 155 CONCLUSIONS 158 V I . FINAL DISCUSSION AND CONCLUSIONS 160 V I I . DESCRIPTIONS TABLE OF CONTENTS FOR DESCRIPTIONS 175 V I I I . BIBLIOGRAPHY 304 I X . APPENDICES A. APPENDIX A. DESCRIPTION OF ISOLATION MEDIA. . . . 323 B. APPENDIX B. EXAMPLES OF STATISTICAL CALCULATIONS 325 v i i i L i s t of Tables T a b l e 1. C o l l e c t i o n dates of a l g a l samples, ambient physical conditions, and estimated times that the algae were exposed to a i r p r i o r to c o l l e c t i o n 30 T a b l e 2. Laboratory processing of a l g a l tissues. Surface s t e r i l i z a t i o n and r i n s i n g treatments 32 T a b l e 3. Percent occurrence of Labyrinthula spp. isolated from the d i f f e r e n t l i f e h i s t o r y stages of Rhodoglossum  aff i n e . Sample 1, Dec. 1978. Isolation frequencies for s u r f a c e - s t e r i l i z e d and seawater-rinsed a l g a l tissues are l i s t e d by incubation temperature 45 T a b l e 4. Percent occurrence of actinomycetes, yeasts and mycelial fungi i s o l a t e d from Rhodoglossum affine. Sample 1, Dec. 1978. Isolation frequencies are l i s t e d for each organism by alga/tissue pretreatment regime and a l l regimes combined 47 T a b l e 5. Total i s o l a t i o n frequency (%) of actinomycetes, yeasts and mycelial fungi for each incubation temperature/tissue pretreatment regime l i s t e d by: 1) l i f e h i s t o r y stage of Rhodoglossum affine , and 2) i s o l a t i o n medium! Sample T~, Dec. T/978 48 T a b l e 6. Percent occurrence of Labyrinthula spp. isola t e d from Rhodoglossum af f i n e and Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d by i s o l a t i o n medium for s t e r i l i z e d and rinsed tissues of each alga 51 T a b l e 7. Percent occurrence of Labyrinthula spp. isolated from Rhodoglossum af f i n e and Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d by al g a l l i f e h i s t o r y stage for s t e r i l i z e d and rinsed tissues of each alga 52 T a b l e 8 . Percent occurrence of Thraustochytrid species i s o l a t e d from Rhodoglossum affine. Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d and rinsed a l g a l tissues by i s o l a t i o n medium and a l l media combined 54 T a b l e 9 . Percent occurrence of Thraustochytrid species i s o l a t e d from Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d and rinsed a l g a l tissues by i s o l a t i o n medium and a l l media combined 55 ix Table 10. Percent occurrence of Thraustochytrid species i s o l a t e d from Rhodoglossum affine. Sample 2, March 1979. Is o l a t i o n frequencies are l i s t e d for s t e r i l i z e d and rinsed tissues by a l g a l l i f e h i s t o r y stage and a l l stages combined 57 Table 11. Percent occurrence of Thraustochytrid species i s o l a t e d from Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d and rinsed tissues by a l g a l l i f e h i s t o r y stage and a l l stages combined 58 Table 12. Percent occurrence of actinomycetes, yeasts and mycelial fungi iso l a t e d from Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d for each organism by alga/tissue pretreatment regime and a l l regimes combined 60 Table 13. Total i s o l a t i o n frequency (%) of actinomycetes, yeasts and mycelial fungi for each alga/tissue pretreatment regime l i s t e d by: 1) l i f e h i s t o r y stage of the algae, and 2) i s o l a t i o n medium. Sample 2, March 1979 61 Table 14. Percent occurrence of Labyrinthulid species i s o l a t e d from Rhodoglossum af f i n e . Sample 3, June 1980. Isolation frequencies are l i s t e d for rigorous and l i g h t rinsed a l g a l tissues by i s o l a t i o n medium-a n t i b i o t i c combination and a l l media combined 64 Table 15. Percent occurrence of Labyrinthulid species i s o l a t e d from Gelidium c o u l t e r i . Sample 3, June 1980. Is o l a t i o n frequencies are l i s t e d for rigorous and l i g h t rinsed a l g a l tissues by i s o l a t i o n medium-a n t i b i o t i c combination and a l l media combined 6 5 Table 16. Percent occurrence of Labyrinthulid species is o l a t e d from Rhodoglossum affi n e . Sample 3, June 1980. Is o l a t i o n frequencies are l i s t e d for rigorous and l i g h t rinsed a l g a l tissues by a l g a l l i f e h i s t o r y stage and a l l stages combined 68 Table 17. Percent occurrence of Labyrinthulid species i s o l a t e d from Gelidium c o u l t e r i . Sample 3, June 1980. Is o l a t i o n frequencies are l i s t e d for rigorous and l i g h t rinsed a l g a l tissues by a l g a l l i f e h istory stage and a l l stages combined 69 Table 18. Percent occurrence of Thraustochytrid species and Hyalochlorella marina is o l a t e d from Rhodoglossum  af f i n e . Sample 3, June 1980. Iso l a t i o n frequencies are l i s t e d for rigorous and l i g h t rinsed a l g a l tissues by i s o l a t i o n medium-antibiotic combination and a l l media combined 71 X Table 19. Percent occurrence of Thraustochytrid species and Hyalochlorella marina is o l a t e d from Gelidium  c o u l t e r i . Sample 3, June 1980. Isolation frequencies are l i s t e d for rigorous and l i g h t rinsed a l g a l tissues by i s o l a t i o n medium-antibiotic combination and a l l media combined 72 Table 20. Percent occurrence of Thraustochytrid species and Hyalochorella marina is o l a t e d from Rhodoglossum  af f i n e . Sample 3, June 1980. I s o l a t i o n frequencies are l i s t e d for rigorous and l i g h t rinsed tissues by a l g a l l i f e h i s t o r y stage and a l l stages combined. . . . 75 Table 21. Percent occurrence of Thraustochytrid species and Hyalochlorella marina is o l a t e d from Gelidium  c o u l t e r i . Sample 3, June 1980. I s o l a t i o n frequencies are l i s t e d for rigorous and l i g h t rinsed tissues by a l g a l l i f e h i s t o r y stage and a l l stages combined. . . . 76 Table 22. Percent occurrence of yeasts and mycelial fungi i s o l a t e d from Rhodoglossum a f f i n e and Gelidium  c o u l t e r i . Sample 3, June 1980. I s o l a t i o n frequencies are l i s t e d for each organism by alga/tissue pretreatment regime and a l l regimes combined 78 Table 23. Total i s o l a t i o n frequency (%) of yeasts and mycelial fungi for each alga/tissue pretreatment regime l i s t e d by: 1) l i f e h i s t o r y stage of the algae, and 2) i s o l a t i o n medium-antibiotic combination. Sample 3, June 1980 79 Table 24. Organisms isol a t e d from the 3rd and 15th rinse waters of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Values given are the mean and standard error of colonies per culture plate. Sample 3, June 1980. . . . 83 Table 2 5. Percent occurrence of yeasts and mycelial fungi i s o l a t e d from the 3rd and 15th rinse waters of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 3, June 1980 86 Table 26. Percent occurrence of Labyrinthulids, Thraustochytrids and Hyalochlorella marina i s o l a t e d from s t e r i l e seawater/pine pollen cultures of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 3, June 1980. Isolation frequencies are l i s t e d for each organism by alga/tissue pretreatment regime 88 Table 27. Percent occurrence of yeasts and mycelial fungi i s o l a t e d from s t e r i l e seawater/pine pollen cultures of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 3, June 1980. I s o l a t i o n frequencies are l i s t e d for each organism by alga/tissue pretreatment regime 91 x i Table 28. Actinomycetes, yeasts and mycelial fungi developing on s u r f a c e - s t e r i l i z e d and/or rigorously-rinsed tissues of Rhodoglossum a f f i n e and Gelidium  c o u l t e r i incubated i n moist chambers. Data l i s t e d are the actual number of i s o l a t e s . Samples 1, 2, and 3 93 Table 29. Percent occurrence of the most common higher fungi i s o l a t e d from f i e l d - c o l l e c t e d a l g a l tissues. I s o l a t i o n frequencies are l i s t e d by i s o l a t i o n method. The actual number of is o l a t e s are given i n parentheses 118 Table 30. Percent occurrence of fungi and Labyrinthula spp. iso l a t e d from ' a r t i f i c i a l l y d r i f t e d ' Rhodoglossum  a f f i n e a f t e r 35 and 52 days of exposure. Also l i s t e d i s the r e l a t i v e abundance of nematodes. Experiment s t a r t i n g date - 27 Dec. 1978 136 Table 31. Percent occurrence of actinomycetes, fungi and Labyrinthula spp. isola t e d from ' a r t i f i c i a l l y d r i f t e d ' Gelidium c o u l t e r i a f t e r 35, 52 and 71 days of exposure. Also l i s t e d i s the r e l a t i v e abundance of nematodes. Experiment s t a r t i n g date - 27 Dec. 1978 137 Table 32. Colony diameter (cm) of Sigmoidea l i t t o r a l i s , S. marina and S^ p r o l i f e r a on Potato Dextrose Agar made up with tapwater and aged seawater adjusted to 15°/oo and 28°/oo. Values are expressed as colony diameter (cm) after 10 days of incubation at room temperature (22-25°C) 277 Table 33. Some distinguishing c h a r a c t e r i s t i c s of the four species described i n the genus Sigmoidea 299 x i i L i s t of Figures Figure 1. I l l u s t r a t i o n s of Gelidium c o u l t e r i and Rhodoglossum a f f i n e 11 Figure 2. P a c i f i c Coast of the United States and Monterey Bay, C a l i f o r n i a 27 Figure 3. C o l l e c t i o n s i t e s . Hopkins Marine Station (Stanford University), P a c i f i c Grove, C a l i f o r n i a . . . . 29 Figure 4. F i e l d and laboratory flow diagram for the pr o c e s s i n g of a l g a l samples. Sample 1 38 Figure 5. F i e l d and laboratory flow diagram for the pr o c e s s i n g of a l g a l samples. Sample 2 39 Figure 6. F i e l d and laboratory flow diagram for the pr o c e s s i n g of a l g a l samples. Sample 3 43 Figure 7. F i e l d and laboratory flow diagram for the processing of ' a r t i f i c i a l l y d r i f t e d ' decomposing a l g a l samples 134 Figures 8-11. Scanning electron micrographs of Thraustochytrium motivum and Schizochytrium  aggregatum on the surfaces of Rhodoglossum a f f i n e and Gelidium c o u l t e r i 152 Figures 12-15. Scanning electron micrographs of Thraustochytrium motivum and Ulkenia sp. RC02-79 on the surface of Rhodoglossum a f f i n e 154 Figures 16-22. Labyrinthula sp. Type 1 182 Figures 23-28. Labyrinthula sp. Type 2 189 Figures 29-35. Labyrinthuloides yorkensis Perkins 194 Figures 36-41. Labyrinthuloides sp. 1 199 Figures 42-45. Labyrinthuloides sp. 1 202 Figures 46-51. Labyrinthuloides sp. RV02-80 206 Figures 52-58. Labyrinthulid Unidentified. I s o l a t e SW/P GC03 213 Figures 59-65. Labyrinthulid Unidentified. I s o l a t e RC01 15-1 217 Figures 66-72. Labyrinthulid Unidentified. I s o l a t e GT04 3-1 220 x i i i Figures 73-79. Schizochytrium aggregatum Goldstein and Belsky 225 Figures 80-85. Schizochytrium aggregatum Goldstein and BelsTcy (pigmented s t r a i n ) . '. ". 230 Figures 86-92. Thraustochytrium aggregatum Ulken 235 Figures 93-100. Thraustochytrium motivum Goldstein 241 Figures 101-110. Ulkenia sp. RC02-80 246 Figures 111-117. Hyalochlorella marina Poyton 252 Figure 118. Acremonium sp. 019-78 258 Figure 119. T r a l i a ascophylli Sutherland. Ascospores. . . 262 Figure 120. T r a l i a ascophylli Sutherland. Ascocarps. . . . 264 Figures 121-122. Unidentified hyphomycete 044-78 264 Figures 123-128. Sigmoidea marina Haythorn and Jones. . . . 270 Figure 129. Sigmoidea marina Haythorn and Jones 272 Figure 130. Sigmoidea marina Haythorn and Jones 274 Figure 131. Colony diameter (cm) of Sigmoidea l i t t o r a l i s sp. nov., marina and S. p r o l i f e r a a f t e r 10 days of incubation on Potato Dextrose Agar made up with tap water and aged seawater adjusted to 15°/oo and 28^/oo 279 Figures 132-137. Sigmoidea l i t t o r a l i s sp. nov 285 Figures 138-141. Sigmoidea l i t t o r a l i s sp. nov 287 Figure 142. Sigmoidea l i t t o r a l i s sp. nov 289 Figure 143. Sigmoidea l i t t o r a l i s sp. nov 291 Figure 144. Sigmoidea l i t t o r a l i s sp. nov 293 Figures 145-148. Sigmoidea l i t t o r a l i s sp. nov. Pycnidia 296 xiv Acknowledgement I am very grateful to Dr. G i l b e r t C. Hughes for h i s persistent guidance and encouragement throughout the course of my graduate research, as well as for h i s understanding and friendship. My sincere appreciation goes to committee members Drs. Robert J. Bandoni, Robert F. Scagel and Thana Bisalputra for t h e i r i n s p i r a t i o n , assistance and constructive suggestions. My research at Hopkins Marine Station (Stanford University) would not have been possible without the support of Dr. Isabella A. Abbott. I would l i k e to thank Dr. Abbott for her patient guidance and friendship. I am indebted to Dr. Judith E. Hansen for her constant support, incentive and comradeship. Thanks also go to the C a l i f o r n i a Sea Grant Program for part-time support during my stay at HMS. Special thanks go to my dear and f a i t h f u l f r i e n d F a y l l a Chapman, who a s s i s t e d me i n many aspects of t h i s study. In par t i c u l a r , F a y l l a i s acknowledged for allowing me to take part i n her study of marine a l g a l decomposition, for her assistance i n the laboratory, and for her very thorough review of t h i s thesis. I am obliged to Chris Patton for i n s t r u c t i o n and assistance i n photography and electron microscopy. Fellow students who provided physical and/or i n t e l l e c t u a l assistance were Michael Dunn, B i l l Magruder, Karla McDermid, Jay Schlumpberger, Tim Thompson, James Watanabe and Lani West. Alan Baldridge and Susan Harris, of the HMS li b r a r y , are recognized for t h e i r unyielding assistance i n the acqui s i t i o n of l i t e r a t u r e . XV Dr. David Porter provided invaluable insight into the i s o l a t i o n and characterization of Labyrinthulid and Thraustochytrid organisms. I am g r a t e f u l to Dr. E. B. Gareth Jones for openly communicating information and advice concerning the hyphomycete genus Sigmoidea. John Hansen, director of Marine Bioassay Laboratories, and my fellow employees, are acknowledged for t h e i r support during the preparation of t h i s thesis. I would l i k e to give very s p e c i a l thanks to Sharon Nugent for her assistance i n preparing t h i s thesis, as well as for her emotional support, love, and understanding. I am deeply indebted to my father, mother, and f a m i l y f o r t h e i r consistent support, encouragement, and love throughout the many phases of my pursuit of an advanced degree. 1 I. INTRODUCTION AND REVIEW Fungi are ubiquitous organisms and i t should come as no surprise that they are common inhabitants of marine ecosystems. A considerable l i t e r a t u r e has accumulated concerning 'Fungi i n Oceans and Estuaries' (Hughes, 1975; Johnson and Sparrow, 1961; Jones, 1976a; Kohlmeyer and Kohlmeyer, 1979). However, compared to our knowledge of these microorganisms i n t e r r e s t r i a l ecosystems, we have only a l i m i t e d understanding of fungi i n the oceans. Much of the past research i n marine mycology has focused on wood and other c e l l u l o s i c substrata (including estuarine flowering plants) (Hughes, 1975; Johnson and Sparrow, 1961). Other areas which have attracted mycological research include: 1) fungi which cause disease i n commercially valuable f i s h and invertebrates (Alderman, 1976); 2) fungi found i n marine sediments (Lee and Baker, 1972; Sparrow, 1937), beach sands (Kohlmeyer, 1966; Steele, 1967; Wagner-Merner, 1973), and estuarine or oceanic waters (Muntanola-Cvetkovic and Ristanovic, 1980; Roth, et al., 1964; Steele, 1967); and 3) fungi associated with cert a i n substrata which man has introduced into marine habitats (e.g. polyurethane, o i l s p i l l s ) (Ahearn and Meyers, 1976; Jones, 1976b). These developments i n marine mycology have included many studies concerning the structure, development, phylogeny and taxonomy of marine fungi (see Kohlmeyer and Kohlmeyer, 1979). 2 There i s also a substantial amount of information on fungi associated with marine algae. The 'lower fungi' (Phycomycetes, Labyrinthulids and Thraustochytrids) include a large number of species known to be associated with, or saprobes and parasites of, marine algae (Fuller, et al., 1964; Huth 1974; Jones, 1976b; Kobayashi and Ookubo, 1953, 1954; Sparrow, 1936, 1969; Vishniac, 1956; Volz and Jerger, 1972). Most of the true phycomycetous fungi have been described from samples of microscopic phytoplankton or small filamentous algae, although a few larger macrophytic algae (especially Chlorophyta) have also been shown to harbor them. While Labyrinthulids and Thraustochytrids are often referred to as 'lower fungi', t h e i r true phylogenetic i d e n t i t y i s not fungal, and i t i s probably most appropriate that they be referred to as 'p r o t i s t s 1 * at t h i s time (Alderman, et al., 1974; Olive, 1975; Perkins, 1974b; Porter, 1974). These organisms are very conspicuous members of the marine microbiota which have been shown to occur on/in a wide variety of substrata; they are frequently iso l a t e d from marine algae (Bremer, 1976; Clokie, 1970; Pokorny, 1967; Vishniac, 1956). Hyalochlorella  marina Poyton i s another 'lower fungus'* of unknown taxonomic a f f i n i t i e s which i s known to be associated with marine a l g a l macrophytes (Alderman, et al., 1974; Perkins, 1974b; Poyton, 1970a, 1970b). *01ive (1975) places Labyrinthulids and Thraustochytrids i n the Kingdom Protista, Phylum Gymnomyxa, Subphylum Labyrinthulina. The best placement of Hyalochlorella marina may also be within t h i s Kingdom (Phylum Euprotista). Throughout t h i s thesis I s h a l l use the term 'protists' to refer to these organisms only. When 'other' members of t h i s Kingdom are discussed, they w i l l be referred to i n more s p e c i f i c terms (e.g. C i l i a t e s ) . 3 Among the 'higher fungi' (Ascomycetes, Basidiomycetes and Deuteromycetes), studies have shown that certain yeasts are very commonly associated (as saprobes?) with the surfaces of l i t t o r a l marine algae (Patel, 1975; Seshadri and Seiburth, 1971, 1975). Of the "known higher filamentous marine fungi (some 209 species), approximately one-third are associates of algae (Kohlmeyer and Kohlmeyer, 1979). The great majority of these algicolous fungi are Ascomycetes. No Basidiomycetes have been described from marine algae, and Kohlmeyer and Kohlmeyer (1979) included only 11 algicolous Deuteromycetes [although c e r t a i n l y more Deuteromycetes have been i s o l a t e d from marine algae (see M i l l e r and Whitney, 1981a)]. Much of the previous work on algicolous higher fungi has been taxonomic i n nature and consists of descriptions of fungi (mostly Ascomycetes) found f r u i t i n g on or i n various marine algae (see Kohlmeyer and Kohlmeyer, 1979). Very few thorough studies have concerned the nature of s p e c i f i c algal/fungal interactions (mutualistic, p a r a s i t i c , saprobic) or the ecological aspects of these associations (Schatz, 1980; Schatz and Kursar, 1980; Walker, et al., 1979; Webber, 1967; Wilson and Knoyle, 1961). There i s , therefore, l i t t l e information on the role(s) of fungi as pathogens (see Andrews, 1976) or saprobes of marine algae. Mycological investigations of wood and vascular plants ( l i g n o c e l l u l o s i c and c e l l u l o s i c substrata) i n marine and estuarine environments have demonstrated that fungi are involved i n the decomposition of these substrata (e.g. Anastasiou and Churchland, 1969; F e l l and Master, 1973; Gessner, 1980; Gessner 4 and Goos, 1973; Leightley, 1980; Meyers, et al., 1965; Newell, 1976). The l i t e r a t u r e available concerning the decomposition of marine algae implicates bacteria as the primary reducers of these macrophytes (Chan and McManus, 1967, 1969; Chesters, et al., 1956; Humm, 1946; Mann, 1976). However, so l i t t l e i s "known about the decomposition of marine and estuarine algae, that i t i s premature to exclude fungi (or 'protists') as potential contributors. Certain investigations have demonstrated the presence of fungi i n association with deteriorating algae (Chesters, et al., 1956; Haythorn, et al., 1980; M i l l e r and Whitney, 1981a; Nonomura, 1978; Schatz, 1980; Sutherland, 1916), but considerable work l i e s ahead before we w i l l understand the a c t i v i t i e s of these fungi i n or on a l g a l substrata. In comparison to wood and vascular plants, the biochemical substrates available i n marine algae which could support fungal growth are less w ell known and understood. Marine algae contain a wide range and d i v e r s i t y of c e l l w a l l polysaccharides, many of which are not encountered elsewhere. Some of the major c e l l w a l l polysaccharides which would be available to decomposers include: cel l u l o s e , mannans, xylans, a l g i n i c acid, and sulfated galactans (agar, agarose, carrageenan, etc.) (Mackie and Preston, 1974; McCandless, 1978). In addition, a l g a l storage products (including starches, laminarin, fl o r i d o s i d e s , and simple sugars) could be u t i l i z e d as well as other c e l l u l a r constituents such as proteins, f a t t y acids, l i p i d s , s t e r o l s and vitamins (see Stewart, 1974). Many of these constituents d i f f e r between members of the major groups of marine a l g a l macrophytes (Chlorophyta, Phaeophyta 5 and Rhodophyta), and even between algae within these groups. Therefore, one would anticipate that the saprobic microbiota associated with or involved i n the decomposition of the d i f f e r e n t types of algae might also vary. Studies have shown that c e r t a i n marine bacteria can u t i l i z e many of the polysaccharide compounds found i n marine algae (Breed, et al., 1957; Humm, 1946; Lehmann, 1919; Quatrano and Caldwell, 1978). Few studies have been undertaken to demonstrate the a b i l i t y of higher fungi to u t i l i z e a l g a l polysaccharides (Chesters, et al . , 1956; Fries, 1979; Lehmann, 1919; Payton, et al., 1976; Payton and Roberts, 1979; Tubaki, 1969; Wainwright, 1980; Wainwright and Sherbrock-Cox, 1981), and the results of cer t a i n of these studies are not conclusive. Detailed physiological studies (including substrate u t i l i z a t i o n ) of higher fungi found associated with marine algae are rare (Fries, 1979, 1980; M i l l e r and Whitney, 1981a). Further studies along these li n e s are necessary to interprete the nature of s p e c i f i c algal/fungal i n t e r r e l a t i o n s h i p s and the role(s) fungi play i n a l g a l decomposition processes. This thesis reports a study of the fungi and 'protists' (Labyrinthulids, Thraustochytrids, Hyalochlorella marina) found associated with two species of i n t e r t i d a l red algae. My research had several related goals which were as follows: 1) To determine the d i v e r s i t y and abundance of fungi and p r o t i s t s associated with these red algae i n t h e i r natural populations. 2) To evaluate the possible role(s) that fungi and p r o t i s t s 6 may have as parasites, perthophytes* or saprobes of these algae i n t h e i r natural populations. 3) To determine i f higher fungi have an active role i n the decomposition of marine (red) algae which have been cast upon the beach. 4) Ultimately, to gain some insight into the a c t i v i t i e s of fungi and p r o t i s t s i n marine a l g a l macrophyte communities and, thereby, i n temperate coastal ecosystems. Various f i e l d and laboratory i s o l a t i o n techniques were employed to address certain aspects of these goals, and each technique was evaluated as to i t s usefulness. The scope of t h i s project has been l i m i t e d to is o l a t i o n s , observations and descriptions of fungi and p r o t i s t s found associated with the a l g a l tissues. Much further f i e l d and laboratory research i s required to gain a more complete understanding of the results presented here. The body of t h i s thesis i s subdivided into seven sections. Section II, 'General Research Information and Methodology', i n c l u d e s a d e s c r i p t i o n of the two red algae studied, as w e l l as information concerning culture methodology, microscopy, i d e n t i f i c a t i o n of the microorganisms, and s t a t i s t i c s - a l l of which are of general application to subsequent sections. Research emphasis was placed on observations of, and i s o l a t i o n s from, a l g a l t h a l l i c o l l e c t e d from natural 'in s i t u ' populations. Various i s o l a t i o n techniques and a l g a l tissue *A 'perthophyte' i s an organism l i v i n g on dead tissues ( = Necrophyte) of a l i v i n g host (Ainsworth, 1971). 7 surface s t e r i l i z a t i o n or ri n s i n g procedures were incorporated into t h i s portion of the study which i s described i n Section III. A single experiment was performed i n which fungi were is o l a t e d from ' a r t i f i c i a l l y d r i f t e d ' decomposing a l g a l t h a l l i (Section IV). A 'mesh bag' technique was employed to 'strand' c o l l e c t i o n s of these red algae on the beach, and t h e i r decomposition as well as t h e i r associated mycobiotas were monitored over time. Section V describes a laboratory experiment i n which scanning electron microscopy was employed to examine the growth of several Thraustochytrids on the a l g a l tissues. In th i s experiment a surface s t e r i l i z a t i o n procedure was also evaluated for i t s effectiveness i n eliminating Thraustochytrids from f i e l d -c o l l e c t e d a l g a l tissues. The o v e r a l l r esults and conclusions from a l l f i e l d and laboratory experiments are drawn together and discussed i n Section VI. F i n a l l y , several undescribed species of Labyrinthulids, Thraustochytrids and higher fungi are characterized i n d e t a i l and i l l u s t r a t e d i n Section VII. Also included are descriptions of certai n frequently encountered organisms (both i d e n t i f i e d and unidentified), and several r a r e l y encountered organisms which are poorly known. 8 I I . GENERAL RESEARCH INFORMATION AND METHODOLOGY A. THE ALGAE The two red a l g a l species studied were Gelidium c o u l t e r i Harv. (Nemaliales) and Rhodoglossum a f f i n e (Harv.) Kyi. (Gigartinales). G^ c o u l t e r i and R^_ a f f i n e were chosen because concurrent f i e l d and laboratory studies of these two algae were being conducted at Hopkins Marine Station ( C a l i f o r n i a Sea Grant R/A-34; Abbott, 1980; Hansen, 1980). As a research assistant for t h i s Sea Grant program, I was able to c o l l e c t f i r s t hand information concerning seasonal aspects of the phenological structure of the a l g a l populations, standing crop biomass, regrowth from harvested portions of the populations, and phycocolloid yields. Both algae are of economic importance since G^ c o u l t e r i has been shown to contain a commercially valuable, high qu a l i t y agar [Marine C o l l o i d s (FMC), pers. comm.], and R^_ a f f i n e i s known to produce one of the best carrageenans found i n P a c i f i c Coast algae (Abbott and Chapman, 1981; McCandless, 1978). G. c o u l t e r i and R^_ af fine are conspicuous members of the i n t e r t i d a l f l o r a on rocky coasts of the P a c i f i c Northwest, occupying overlapping v e r t i c a l d i s t r i b u t i o n s from about 0.0 to +3.0 feet (0.0 to +1.0 meter) above MLLW. Both of these species have l i f e h i s t o r i e s involving an alternation of isomorphic generations, a l l the stages of which are more or less available i n the populations at any given time (Abbott and Hollenberg, 9 1976). While both algae grow i n s i m i l a r locations, there are differences i n habitats, morphologies, and seasonal aspects of population growth and structure (see Abbott, 1980). The following descriptions of these two algae pertain s p e c i f i c a l l y to the populations chosen for c o l l e c t i o n s i n the present study. These populations are immediately adjacent to the Hopkins Marine Station (HMS) study s i t e s of Abbott (1980) and Hansen (1980) (see Figs. 2 and 3). In t h i s study population, t h a l l i of Rhodoglossum a f f i n e occur i n small, bushy, erect or decumbent t u f t s or bands, attached to the tops and sides of rocks between +1.0 and +3.0 feet (+0.3 and +1.0 meter) above MLLW. Individual t h a l l i of R. a f f i n e consist of smooth blades with few to many orders of repeated dichotomous branching (Fig. lb). Blades of th i s alga were observed to at t a i n a height of up to 8.0 cm, the individual dichotomies ranging from 2.0-8.0 mm (or more) i n width. Studies of the Hopkins Marine Station R^ aff i n e population showed that: 1) a s i g n i f i c a n t standing crop was present year round; 2) tetrasporangial plants were r e l a t i v e l y rare (most of the f e r t i l e t h a l l i were cystocarpic); and 3) plants could mature, sporulate and begin senescence i n a r e l a t i v e l y short time (i.e. three or four months) (Abbott, 1980; Hansen, 1980). Gelidium c o u l t e r i occurs as small, dense, is o l a t e d clumps or large patches of erect and decumbent t h a l l i attached to rocks or to the surfaces of large colonies of the polychaete worms Dodecaceria fewkesi Berkeley and Berkeley and Phragmatopoma  c a l i f o r n i c a Fewkes. G^ c o u l t e r i i s found between the 0.0 and + 2.5 f e e t (0.0 and +0.7 meter) MLLW t i d a l l e v e l s , 1 foot (0.3 Figure 1 I l l u s t r a t i o n s of Gelidium c o u l t e r i and Rhodoglossum affine, approximately l i f e size. A. Gj_ c o u l t e r i (tetrasporangial) B. R. a f f i n e (vegetative/male) Figure ! 12 meter) or so below Rhodoglossum affi n e . This alga i s often associated with sand-scoured areas (sides of tide pools, sand channels, on rocks or polychaete worm colonies surrounded by sandy beach), and very commonly entraps sand around i t s base, obscuring the r h i z o i d a l holdfast. Individual t h a l l i of Gelidium  c o u l t e r i consist of thin erect axes with few major branches but with numerous distichous, once or twice pinnate, short l a t e r a l branchlets (Fig. la). T h a l l i were observed to atta i n a height of up to 10.0 cm, the main axes reaching a s i z e of up to 1.0 (-1.5) mm broad by 0.5 (-0.8) mm wide, the l a t e r a l branchlets generally being smaller. Population studies of Gelidium c o u l t e r i at Hopkins Marine Station showed that: 1) standing crop biomass fluctuated f a i r l y s i g n i f i c a n t l y over the year (1978-1979), being highest i n the f a l l and lowest i n the spring*; 2) cystocarpic plants were very rare i n t h i s population; 3) tetrasporangial plants dominated the population (especially i n the f a l l and winter); and 4) regrowth from harvested portions of the population was generally very slow (Abbott, 1980; Hansen, 1980). From a microbial standpoint, the differences i n the general morphology of these two algae are also important. G. c o u l t e r i o f f e r s much more surface area per unit volume than does Rj_ a f f i n e (Fig. 1). Although no attempts were made to estimate the difference i n the surface area/volume r a t i o s between *The spring decline i n standing crop was probably due to antecedent environmental conditions (i.e. death due to desiccation; extreme low tides on sunny days) and/or to the senescence of tetrasporangial plants. 13 these two algae, i t i s a f a c t o r t h a t must be considered when evaluating the frequency of i s o l a t i o n of microorganisms which may be surface associated. Observations of the conspicuous surface-associated f l o r a and fauna v i s i b l e at the l i g h t microscope l e v e l indicate that while both algae harbor s i m i l a r epiphytes, they are more common on Gelidium c o u l t e r i . Some of the epiphytes commonly encountered on these algae include: Dermocarpa sp. (= Entophysalis conferta Drouet and Daily) (Coccogonales, Chroococcaceae); Leucothrix sp. (Beggiatoales, Leucotrichaceae); various s e s s i l e and chain-forming (colonial) diatoms; filamentous blue-green algae; filamentous red and brown algae (e.g. Polysiphonia sp., Ectocarpales); small blade-like algae (e.g. juvenile red algae, Ulva sp., Enteromorpha sp.); stalked peritrichous c i l i a t e s (e.g. V o r t i c e l l a sp.); and others. B. MEDIA AND CULTURE CONDITIONS FOR THE ISOLATION, MAINTENANCE AND CHARACTERIZATION OF MICROORGANISMS Media used for i n i t i a l i s o l a t i o n s from f i e l d c o l l e c t i o n s included: Glucose-Yeast Extract Agar (GYSA) (Johnson and Sparrow, 1961); Modified Serum-Seawater Agar (SSA) (Porter, 1967; Watson and Ordal, 1957); Modified Vishniac's Medium (FUL) (Fuller, et al., 1964; Vishniac, 1956); Kazama's Modified Vishniac's Medium (KMV) (D. Porter, pers. comm.); Seawater/pine pollen cultures (SW/P) (Gaertner, 1972a); and Base Mineral Medium (Gunkel and Rheinheimer, 1972). A n t i b i o t i c s were incorporated into 'isolation' media to i n h i b i t the growth of bacteria. Either te t r a c y c l i n e HCL (Sigma) 14 or a combination of p e n i c i l l i n G (sodium salt) and streptomycin sulfate (both Sigma) were employed. A n t i b i o t i c s were added as dry powder to the media afte r they had been autoclaved and allowed to cool. Further d e t a i l s concerning the use of these media and antibotics are described i n the appropriate Materials and Methods sections. Numerous other culture media were u t i l i z e d throughout the course of t h i s study to maintain stock cultures of iso l a t e d organisms, to enhance developmental stages, and to allow i d e n t i f i c a t i o n of certain groups. Additional media used to culture higher fungi and yeasts included: Corn Meal Agar + ( F e l l and Master, 1975); Czapek's Agar (Thorn and Raper, 1945); Kirk's M-l (with or without birch applicator sticks) (Kirk, 1966); Malt Extract Agar (Stevens, 1974); Oatmeal Agar (Stevens, 1974); Potato Dextrose Agar (Difco); V-8 Juice Agar (Fuller, 1978); and Yeast Extract-Malt Extract Agar (Lodder, 1970). The source of the water incorporated into the media used for higher fungi varied Caged seawater (15 or 28°/oo), deionized, or tap] and i s e x p l i c i t l y stated i n descriptions where appropriate. Unless stated otherwise, descriptions are based on p e t r i plate cultures (15 x 100 mm) incubated at room temperature (22-25°C) i n d i f f u s e sunlight. For long term storage, i s o l a t e s of higher f u n g i and yeasts were maintained on t e s t tube (25 x 100 mm) slants of the above media i n a r e f r i g e r a t o r or cold room (4-6°C). Labyrinthulids and Thraustochytrids were maintained on and characterized from: Modified Serum-Seawater Agar containing 1% or 2% v/v horse serum (SSA-1 and SSA-2%) (Porter, 1967; Watson and Ordal, 1957); Kazama's Modified Vishniac's Agar and Slush (KMV-agar and KMV-slush) (D. Porter, pers. comm.); and Glucose-Serum Seawater Agar (GSSA) (unpubl. obs.). The formulae for the above three media are given i n Appendix A. Seawater/pine pollen (SW/P) cultures were also used i n the characterization of Thraustochytrids (Gaertner, 1972a). Media prepared for the c u l t i v a t i o n of Labyrinthulids and Thraustochytrids always contained seawater at 28°/oo. Labyrinthulids were maintained i n p e t r i plates (SSA-1 or 2%; GSSA) which were incubated i n a refrigerated room (14-16°C) or at room temperature (22-25°C). Thraustochyrids could be maintained at 4°C i n p e t r i p l a t e s of KMV-agar or on t e s t tube s l a n t s of KMV-agar with a KMV-slush overlay (5-10 ml). Hyalochlorella marina was maintained at room temperature on t e s t tube slants of KMV-agar with a KMV-slush overlay. The culture media u t i l i z e d for the characterization of s p e c i f i c Labyrinthulids and Thraustochytrids are stated i n t h e i r respective descriptions (Section VII). A l l descriptions are based on cultures incubated at room temperature i n diffuse sunlight. C. MICROSCOPIC EXAMINATION AND PHOTOMICROGRAPHY I n i t i a l microscopic observations of higher fungi were normally made on 'squash' s l i d e preparations. Squash mounts were prepared by removing a small block of agar containing the desired structures from an agar culture and placing i t surface down into 16 several drops of water [seawater (15°/oo or 28°/oo) or deionized, with or without 0.01% v/v Edwal "Quick Wet"] on a microscope s l i d e . A cover s l i p (No. 1.5, 22 x 22 mm) was then placed on top of the agar block and the p r e p a r a t i o n was squashed using a s o f t tipped, r i g i d instrument. Semi-permanent s l i d e s were prepared by replacing the water with glycerine-alcohol permanent s l i d e solution (30% glycerine, 50% water, 20% alcohol) (G. C. Hughes, pers. comm.). Mycological stains, such as phloxine or cotton blue, were added to the glycerine-alcohol s l i d e solution when staining was desired. Alternatively, squash mounts were prepared d i r e c t l y i n lactophenol mounting medium (Stevens, 1974). Several days a f t e r mounting, the area on the s l i d e surrounding the cover s l i p was cleaned w i t h 40% a l c o h o l and t i s s u e paper, and the cover s l i p sealed with clear f i n g e r n a i l polish. I f observations of in t a c t f r u i t i n g structures, conidiophore development or conidium ontogeny were required for complete characterization, s l i d e cultures were prepared using techniques very s i m i l a r to those described by Booth (1971). In certain cases, i f the f u n g i were c u l t u r e d on r e l a t i v e l y c l e a r media or on th i n agar plates, a cover s l i p could be placed d i r e c t l y onto the agar surface, and the culture observed under the compound microscope. Agar blocks containing f r u i t i n g bodies of coelomycetous fungi were sectioned at 4-8 um using an International Cryostat (IEC Model CTl) with 20% gum arabic i n seawater or deionized water as a c a r r i e r . Sections were mounted on s l i d e s i n glycerine-alcohol solution or lactophenol mounting medium. Observations of Thraustochytrids, Hyalochlorella marina, and 17 e s p e c i a l l y Labyrinthulids were made on thin agar cultures placed d i r e c t l y under the microscope. Developmental cycles of Thraustochytrids and Hyalochlorella marina were observed i n 'wet mounts' from l i q u i d cultures with or without the use of depression slides. An American Optical Microstar (One-Ten) b r i g h t f i e l d compound microscope was used to examine a l l i n i t i a l i s o l a t i o n plates and many axenic cultures and sli d e s . The 10X objective on thi s microscope has a working distance large enough to allow d i r e c t observation of organisms on the agar surface i n p l a s t i c p e t r i plates without removing the plate cover. An Olympus BHA compound microscope equipped with a halogen l i g h t source and phase optics was also used to examine sl i d e s and cultures. A l l photomicrographs were taken on t h i s microscope using an Olympus 0M-2n automatic exposure camera back and Kodak Panotomic X f i l m . D. THE MICROORGANISMS AND THEIR IDENTIFICATION A n t i b i o t i c s were incorporated into nearly a l l media u t i l i z e d i n t h i s study to i n h i b i t the growth of b a c t e r i a and actinomycetes. Occasionally, however, actinomycetes developed on the a l g a l tissues placed on agar media or i n moist chambers, and when encountered they were isolated. Several of these actinomycetes have been thoroughly examined, and i t appears that most, i f not a l l , of these i s o l a t e s belong i n the genus Streptomyces. Several d i s t i n c t species of t h i s large genus are included i n t h i s group of isol a t e s . Yeasts were isola t e d throughout t h i s study and were f a i r l y 18 common on cert a i n a l g a l samples. Only a few of these yeasts have been i d e n t i f i e d , or even distinguished from one another, i n the results. Most of the "Unidentified Yeasts" are believed to belong to the genera Candida and Rhodotorula, although several other genera are represented. With few exceptions, a sincere attempt was made to i d e n t i f y a l l i s o l a t e s of higher filamentous fungi to the species leve l . This was a cumbersome task, p a r t i c u l a r l y with certain groups. In most cases i s o l a t e s were separated to what I f e l t was the species l e v e l , whether or not they were a c t u a l l y 'identified' to species (or even genus). Unidentifiable fungi are distinguished i n the re s u l t s by t h e i r i s o l a t i o n numbers (e.g. Acremonium sp. 019-78; Unidentified hyphomycete 044-78). I f the fungus was i s o l a t e d more than once, i t i s consistently i d e n t i f i e d throughout the results by the same i s o l a t i o n number (i.e. the number applied to the f i r s t i s o l a t e obtained). This w i l l allow the reader to di s t i n g u i s h the same 'unidentifiable' fungus, i f present, i n d i f f e r e n t sections of the results. The genus P e n i c i l l i u m i s a notable exception to my i d e n t i f i c a t i o n e f f o r t s . While a few i s o l a t e s of t h i s genus have been i d e n t i f i e d , the majority of them have not. Some of these P e n i c i l l i u m species belong to Pj_ chrysogenum Thom [= P^ notatum (Samson, et a l . , 1977)3, which was the most common member of t h i s genus encountered. However, other species may also be well represented. Considerable time and e f f o r t was spent dwelling on coelomycetous fungi. Numerous d i s t i n c t coelomycetous fungi were 19 obtained from a l g a l tissues throughout the course of t h i s study; the m a j o r i t y of these were represented by one or only a few i s o l a t i o n s . Nearly a l l of these fungi have been thoroughly examined i n c u l t u r e , but I have yet to master the group w i t h s u f f i c i e n t confidence to apply names to many of these isolates. I d e n t i f i c a t i o n of Coelomycetes i n culture i s d i f f i c u l t since even the most comprehensive monographs (Sutton, 1980) are based on c h a r a c t e r i s t i c s of the fungi on natural substrata. In addition, very l i t t l e i s known about coelomycetous fungi i n marine habitats (B. C. Sutton, pers. comm.). E. STATISTICS S t a t i s t i c a l methods were employed to aid i n the inte r p r e t a t i o n of the results. The majority of the information gathered during t h i s study consists of 'presence - absence' data (i.e. either a p a r t i c u l a r fungus or type of organism was i s o l a t e d from a culture or i t was not). Tests of independence were performed between data sets (i.e. algae, tissue pretreatments, i s o l a t i o n media, etc.) and a G - s t a t i s t i c was calculated (Sokal and Rholf, 1969). Standard 95% confidence l i m i t s were used. S i g n i f i c a n t differences are expressed i n terms of the pr o b a b i l i t y of such a difference occurring purely as a matter of chance. For example, p < 0.025 indicates that less than 2.5% of the time would you expect to f i n d such a large difference between samples (p < 0.01, l e s s than 1.0%; p < 0.005, l e s s than 0.5%). Several representative examples of these s t a t i s t i c a l calculations are given i n Appendix B. These examples are referenced i n the text 20 when the corresponding results are described. 'Colony count' data were obtained i n Sample 3 of the f i e l d studies (Section III)/ and the s t a t i s t i c a l analyses of these data d i f f e r from those described above. These s t a t i s t i c a l analyses are explained further i n the results, adjacent to the data i t s e l f . 21 I I I . OBSERVATIONS OF AND ISOLATIONS FROM FRESH ALGAE COLLECTED FROM i n s i t u POPULATIONS INTRODUCTION The surfaces and/or tissues of marine a l g a l t h a l l i provide habitats for a wide variety of organisms, ranging from invertebrates and other algae to an abundance of heterotrophic microorganisms. Among the diverse community of heterotrophic microorganisms there are microbes which have been considered saprobes on, or parasites of, the a l g a l tissues themselves (including bacteria, 'protists' and fungi). While certain b i o l o g i c a l and physiological aspects of these microbes have been studied, few surveys of t h e i r abundance and d i s t r i b u t i o n on natural populations of marine algae have been conducted. We have a l i m i t e d knowledge of the interactions between these microbes and the algae, or of the ecological aspects of these associations. B a c t e r i a l f l o r a s have been described from the surfaces of l i t t o r a l seaweeds (Chan and McManus, 1967, 1969; Kong and Chan, 1979). Kong and Chan (1979) reported that a . . . " r e l a t i v e l y s p e c i f i c b a c t e r i a l f l o r a can be found to associate with d i f f e r e n t groups (Phylum level) of marine algae growing i n the same habitat." These investigators suggested that the excretion or elaboration of organic compounds by the algae provide favorable conditions for the growth of epiphytic, heterotrophic bacteria (Kong and Chan, 1979). 22 The ubiquitous marine Labyrinthulids and Thraustochytrids have been reported from f i e l d c o l l e c t i o n s of marine algae on numerous occasions (Bremer, 1976; F u l l e r , et al., 1964; Haythorn, et a l . , 1980; Sparrow, 1969). However, these reports are generally based on spotty c o l l e c t i o n s of a wide variety of algae, and the significance of these observations i s limited. No studies have been conducted concerning the d i v e r s i t y and abundance of these 'protists' associated with select species of marine algae. Seshadri and Sieburth (1971, 1975) have shown that the surfaces of l i t t o r a l seaweeds support somewhat s p e c i f i c yeast populations, the abundance of which may vary between a l g a l phyla and seasons. These investigators also considered the p o s s i b i l i t y that materials released from the a l g a l tissues support the yeast populations (Seshadri and Sieburth, 1971, 1975). Several studies have also demonstrated that a variety of marine and t e r r e s t r i a l Fungi Imperfecti can be found associated with natural populations of marine algae ( M i l l e r and Whitney, 1981a; Schatz, 1980). Certain of the fungi i s o l a t e d from attached a l g a l t h a l l i are also known from 'cast' or ' d r i f t ' seaweeds (Haythorn, et al., 1980; Sutherland, 1916). Recent studies have shown that several of these same fungi have at least l i m i t e d a b i l i t i e s to u t i l i z e a l g a l polysaccharides as carbon sources ( M i l l e r and Whitney, 1981a; Wainwright and Sherbrock-Cox, 1981). There i s , therefore, some accumulating evidence which suggests that certain imperfect fungi may be active saprobes of marine a l g a l tissues. The role(s) that these fungi, p a r t i c u l a r l y the ' t e r r e s t r i a l ' species, have i n association with natural 23 populations of marine algae and/or i n coastal ecosystems i s far from clear. Further surveys directed at determining the abundance and d i v e r s i t y of fungi and 'protists' associated with select species of marine algae are r e q u i r e d as an i n i t i a l step to evaluate and c l a r i f y possible roles for these microorganisms. From the r e s u l t s of these surveys, conspicuous members of the microbiota may be selected for further studies of t h e i r physiological a t t r i b u t e s and a b i l i t i e s to u t i l i z e a l g a l 'substrates', as well as a variety of other controlled experiments. In t h i s study a survey of the fungi and 'protists' associated with natural populations of the i n t e r t i d a l red algae Rhodoglossum a f f i n e and Gelidium c o u l t e r i has been conducted. The major polysaccharides found i n the c e l l walls of these two red algae d i f f e r (carrageenan, Rj_ a f f i n e ; agar, G^_ coulteri) and, i n Rj_ a f f i n e , these polysaccharides also vary somewhat between l i f e h i s t o r y stages (McCandless, 1978). Since the major constituents of a substratum may well influence the associated microbiota, the f i e l d - i d e n t i f i a b l e l i f e h i s t o r y stages of these two algae were studied separately. I t i s also possible that the d i f f e r e n t l i f e h i s t o r y stages of the algae could be i n d i f f e r e n t stages of maturation, reproduction and/or senescence at any given time. These factors may also influence the presence and/or abundance of associated microbes. The fungi and p r o t i s t s associated with f i e l d c o l l e c t i o n s of these red algae were p r i m a r i l y evaluated by means of d i r e c t observation, i s o l a t i o n from tissues plated on nutrient media, and 24 i s o l a t i o n from tissues incubated i n moist chambers. Prior to processing for i s o l a t i o n , some of the a l g a l t h a l l i were subjected to various surface s t e r i l i z i n g and/or rigorous r i n s i n g procedures i n attempts to eliminate or remove surface-associated microorganisms and/or spores. The results obtained from the surface s t e r i l i z a t i o n and/or rigorous r i n s i n g procedures are compared to one another and to a standard pretreatment of the al g a l tissues i n two rinses of s t e r i l e seawater. In F i e l d Sample 3, attempts were made to evaluate and enumerate the fungi and p r o t i s t s dislodged from the a l g a l surfaces by rigorous rinsing. S t e r i l e seawater/pine pollen (SW/P) cultures were also incorporated into F i e l d Sample 3 as a comparative technique for characterizing the p r o t i s t 'fauna'. Experimental results are expressed i n terms of frequency of occurrence or i s o l a t i o n of each fungus or p r o t i s t , and allow comparisons between i s o l a t i o n techniques (including media), tissue pretreatments ( s t e r i l i z e d , rinsed), f i e l d samples, a l g a l species, and the l i f e h i s t o r y stages of both a l g a l species. The nature and d i v e r s i t y of the fungi and p r o t i s t s found associated with these algae are evaluated. Organisms considered to be of regular and/or common occurrence are i d e n t i f i e d and discussed i n terms of t h e i r o r i g i n ( i f questionable) and t h e i r possible a c t i v i t i e s i n t h i s habitat. 25 MATERIALS AND METHODS A. FIELD COLLECTIONS Algae were collected from a rocky i n t e r t i d a l area within the Hopkins Marine Station (Stanford University) preserve i n P a c i f i c Grove, C a l i f o r n i a (36° 37.35'N; 121° 54.25'W) (Figs. 2 and 3). T h a l l i of Rhodoglossum a f f i n e and Gelidium c o u l t e r i were co l l e c t e d during low tides from rock configurations adjacent to si t e s established for population studies of these two species by Abbott (1980). When possible, equal numbers of individuals of each f i e l d - i d e n t i f i a b l e l i f e h i s t o r y stage (tetrasporangial, cystocarpic, vegetative/male) were coll e c t e d randomly and placed i n separate s t e r i l e p l a s t i c bags (Nasco Whirl-pac). The algae were returned to the l a b o r a t o r y i n an i c e chest and were stored i n a refrige r a t e d room (14-16°C) u n t i l processed for plat i n g (within 24 hours). The following physical conditions were also recorded i n the f i e l d : water temperature, a i r temperature, and water s a l i n i t y (American Optical T/C Refractometer). The length of time that the algae had been exposed to the a i r p r i o r to c o l l e c t i o n was estimated from: 1) the time of c o l l e c t i o n ; 2) record of the th e o r e t i c a l t i d a l cycle (local tide tables); 3) t i d a l height that the c o l l e c t i o n s were made; and 4) weather conditions. Physical conditions and estimated exposure times for each alga are l i s t e d by sampling date i n Table 1. Voucher specimens of the two a l g a l species under study were prepared and deposited i n the G. M. Smith Herbarium at Hopkins Figure 2 P a c i f i c Coast of the United States. Monterey Bay, C a l i f o r n i a . Hopkins Marine Station (Stanford University) on Point C a b r i l l o . Figure 3 Hopkins Marine Station (Stanford University), P a c i f i c Grove, C a l i f o r n i a . F i e l d c o l l e c t i o n s i t e s for Rhodoglossum af f i n e and Gelidium c o u l t e r i arrowed; D r i f t Study s i t e arrowed. Table 1 Sampl e 1 Collection dates of algal samples, ambient physical conditions, and estimated times that the algae were exposed to a i r prior to c o l l e c t i o n , Date 1 Dec. 1978 Ai r Temp (°Q 11 .5 H 20 Temp (°C) 13.0 H20 S a l i n i t y ( ° / o o ) 33.5 Time of Coll e c t i o n 1520-1610 Tide: Level/Time -1 .4 f t @ 1725 Estimated Maximum Time of Exposure (Hours) G. c o u l t e r i R. a f f i n e ND 1.3 2 17 March 1979 13.0 11.0 32.0 0720-0810 2 20 March 1 979 ND 12.5 ND 0900-0940 +0.7 f t @ 0623 ND +0.4 f t @ 0912 0.5 3.0 ND 3 3 4 June 1980 15.0 14.0 33.0 0830-0915 5 June 1980 14.0 13.0 34.0 0810-0840 -0.5 f t @ 0919 1 .8 -0.2 f t @ 1014 ND ND 1 .3 ND = no data U ) o 31 Marine Station. B. GENERAL LABORATORY PROCESSING AND PLATING OF FIELD SAMPLES 1. Surface S t e r i l i z a t i o n , Rinsing and Plating In the laboratory, i n d i v i d u a l l y c o l l e c t e d a l g a l t h a l l i were macroscopically examined for fungal f r u i t i n g structures and/or 'infected' areas. I f the a l g a l tissues were thought to harbor fungi, they were examined microscopically. A portion (2.5 to 3.5 cm length) of each specimen col l e c t e d was then selected for further processing and p l a t i n g on i s o l a t i o n media. The portion of a l g a l t h a l l u s selected for processing had to include s u f f i c i e n t material of s i m i l a r type (axes, branches, tips) and condition ( f e r t i l e , senescent, 'infected') to supply the required number of 'replicate' inocula (see S p e c i f i c Techniques used for each F i e l d Sample). The inocula removed from each a l g a l specimen were either taken from immediately adjacent areas of the thallus, or consisted of s i m i l a r structures (axes, branches, f r u i t i n g structures) taken i n close proximity to one another. The area of the a l g a l t h a l l u s and/or 'type' of tissue chosen varied between a l g a l specimens. An attempt was made to use a l l 'types' of tiss u e from each f i e l d c o l l e c t i o n of the algae. Notes were taken including area of t h a l l u s selected, general appearance, size, and reproductive status. P r i o r to dissection and plating, a l g a l t h a l l i were subjected to various washing and/or surface s t e r i l i z a t i o n procedures. The s p e c i f i c techniques used for each f i e l d sample are l i s t e d i n Table 2. A l g a l tissue was added to culture tubes Table 2 Laboratory processing of algal t i s s u e s . Surface s t e r i l i z a t i o n and r i n s i n g treatments.* Sample/ Procedure No. S t e r i l i z i n g Solution Exposure Time Rinse Exposure Time Reference 1 ;10,000 HgCl 2 in b% ETOH/deionized H 20 1 min. 2 X s t e r i l e SW 1 min. @ Fell & Master, 1973 Newell, 1976 1 :10,000 HgCl? in 5% 30 sec. ETOH/deionized H20 2 X s t e r i l e SW 1 min. @ Fel l & Master, 1973 Newell, 1976 (Modified) None 20 X s t e r i l e SW 1 min. @ Harley & Waid, 1955 (Modified) *See text for further d e t a i l s . co K> 33 (screw cap, 25 x 150 mm) c o n t a i n i n g 15 ml of the appropriate solution at room temperature (22-25°C). The culture tubes were mixed during s t e r i l i z a t i o n and r i n s i n g using a Vortex Mixer [ S c i e n t i f i c Products model S8223 or S c i e n t i f i c Industries model K-500(-4), at maximum setting(s)], with intermittant periods of rest. In Procedures 1 and 2 (Table 2), the s u r f a c e - s t e r i l i z i n g solution was poured o f f and replaced with 15 ml of s t e r i l e seawater (28°/oo). The a l g a l tissue was rinsed twice i n s t e r i l e seawater for one minute periods. In Procedure 3, the al g a l tissue was rinsed 20 times i n s t e r i l e seawater. During t h i s r i n s i n g sequence the tissue was transferred to new tubes of seawater between the 3rd and 4th, and the 15th and 16th rinses. Test tubes containing the 3rd and 15th rinse waters were set aside for further processing and p l a t i n g (see S p e c i f i c Techniques, Sample 3). S u r f a c e - s t e r i l i z e d and/or rinsed tissue was then removed from the culture tube, blotted, and dissected on s t e r i l e bibulous paper using s t e r i l e forceps and micro-scissors. Tissue sections 2 approximately 0.5-0.7 cm were taken from adjacent areas of each i n d i v i d u a l and placed on or i n appropriate media. For each f i e l d sample, portions of several a l g a l specimens (from each l i f e h i s t o r y stage, i f available) were selected and subjected to d i f f e r e n t treatment. The procedures were the same throughout except that these a l g a l samples were not exposed to a surface s t e r i l i z i n g solution or to rigorous rinsing. Instead, the a l g a l tissue was rinsed twice i n s t e r i l e seawater p r i o r to plating. A l g a l tissue subjected to only several seawater rinses 34 was for comparison, to evaluate the eff e c t s of the surface s t e r i l i z a t i o n or rigorous r i n s i n g techniques. Twice rinsed a l g a l tissues also acted as 'controls' for comparisons between f i e l d samples. 2. Media and Culture Conditions Three agar media were selected for general use i n the i s o l a t i o n of Fungi, Labyrinthulids and Thraustochytrids: 1) Glucose-Yeast Extract Seawater Agar (GYSA) (Johnson and Sparrow, 1961); 2) Modified Serum-Seawater Agar (SSA) (Porter, 1967; Watson and Ordal, 1957); and 3) Modified Vishniac's Medium (FUL) (Fuller, et al., 1964; Vishniac, 1956). The ingredients for each of these media are described i n Appendix A. Laboratory methods introduced i n Sample 3 also employed Kazama's Modified Vishniac's Medium (KMV) (D. Porter, pers. comm.) (see Appendix A), and s t e r i l e seawater/pine pollen (SW/P) cultures (Gaertner, 1972a). A l l i s o l a t i o n media were made up with l o c a l aged (2 months or more) seawater adjusted to a s a l i n i t y of 28°/oo with deionized water (Bremer, 1976; Hughes, 1975; Kirk, 1966; unpubl. obs.). A n t i b i o t i c s were added to media prepared for the i s o l a t i o n of fungi and p r o t i s t s to i n h i b i t b a c t e r i a l growth. Tetracycline HCl (Sigma) (200 mg/l) was used i n agar media prepared for i n i t i a l i s o l a t i o n s from f i e l d material (Hughes, 1975). A combination of p e n i c i l l i n G (sodium salt ) (500 mg/l) and streptomycin sulphate (500 mg/l) (both Sigma) was also u t i l i z e d for one h a l f of the i s o l a t i o n plates i n Sample 3 (see S p e c i f i c Techniques, Sample 3), and when persistent b a c t e r i a l contamination was encountered during i s o l a t i o n (Fuller, et a l . , 1964). A n t i b i o t i c s were added as dry powder to the media after they had been autoclaved and allowed to cool. P l a s t i c p e t r i p l a t e s (15 x 100 mm) of the i s o l a t i o n media inoculated with a l g a l tissue were placed i n p l a s t i c bags and incubated at 25°C i n the dark (unless s p e c i f i c a l l y stated otherwise). Isolation plates were examined for fungi approximately every 7-10 days for a period of 6-9 weeks. As fungi appeared they were either i d e n t i f i e d d i r e c t l y , or were transferred to s i m i l a r media for i s o l a t i o n u n t i l axenic cultures were obtained. 3. Moist Chamber Incubation Portions of several individuals from each available l i f e h i s t o r y stage of both a l g a l species were also placed i n moist chambers for incubation. A 90 mm c i r c u l a r f i l t e r pad (Whatman No. 1) was placed i n the bottom of a glass p e t r i plate (20 x 100 mm), soaked with 5 ml of seawater (28°/oo), and the closed plate autoclaved. A l g a l tissue (approximately 1-2 g wet weight) was surface s t e r i l i z e d , rinsed, and blotted as previously described (Procedures 1, 2, and 3, Table 2), p r i o r to being placed i n the chamber. Moist chambers were incubated at room temperature (22-25°C) i n di f f u s e sunlight and examined for the development of fungi over a period of two to three months. S t e r i l e deionized water was occasionally added to the moist chambers to prevent them from drying out. Fungi developing (producing a e r i a l hyphae and/or spores) on the a l g a l tissues were either i d e n t i f i e d 36 d i r e c t l y or transferred to i s o l a t i o n media for further characterization. C. SPECIFIC TECHNIQUES USED FOR EACH FIELD SAMPLE The general methods and processing procedures just described were adhered to throughout the study. The major differences beween samples were surface s t e r i l i z a t i o n and/or r i n s i n g techniques. However, certain of these procedures were considered unworthy of continued e f f o r t and/or stimulated new ideas, such that treatment of each f i e l d sample d i f f e r e d i n some aspect(s). It i s therefore necessary to further describe and c l a r i f y the procedures used for each f i e l d sample. 1. Sample 1 F i e l d c o l l e c t i o n s for Sample 1 included individuals of Rhodoglossum a f f i n e only. Laboratory methods were designed to evaluate the effects of surface s t e r i l i z a t i o n and incubation temperature on the recovery of fungi and p r o t i s t s from t h i s alga. D i s t i n c t aspects of the laboratory processes used for Sample 1 included the following: 1) Surface s t e r i l i z a t i o n (Procedure 1, Table 2). 2) Only two culture media were employed: Glucose-Yeast Extract Seawater Agar (GYSA) and Modified Serum-Seawater Agar (SSA). Tetracycline HCL was incorporated as a bacteriostat. 3) Four pieces of tissue were removed from each al g a l thallus. Each piece was placed on a separate p e t r i plate (2 GYSA; 2 SSA). One plate of each medium was incubated i n the dark 37 at 14-16°C, and one of each type was incubated i n the dark at 2 5°C. A flow diagram of t h i s sampling procedure i s given i n Figure 4. 2. Sample 2 F i e l d c o l l e c t i o n s for Sample 2 included individuals of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Laboratory procedures d i f f e r e d from Sample 1 i n that: 1) A new surface s t e r i l i z i n g regime was employed (Procedure 2, Table 2). 2) Three i s o l a t i o n media were employed - GYSA, SSA and FUL; t e t r a c y c l i n e HCL was incorporated as a bacteriostat. One tissue section of each a l g a l specimen was plated on each of these media. 3) A l l i s o l a t i o n plates were incubated i n the dark at 25°C. A flow diagram of the laboratory processes used i n Sample 2 i s presented i n Figure 5. 3. Sample 3 F i e l d c o l l e c t i o n s for t h i s sample included individuals of both R^ a f f i n e and G^ c o u l t e r i . The general laboratory procedures d i f f e r e d from Samples 1 and 2 i n the following respects: 1) The use of a surface s t e r i l i z i n g agent was replaced with a rigorous r i n s i n g technique designed to remove surface-associated microorganisms (Procedure 3, Table 2). 2) The p o s s i b i l i t y that t e t r a c y c l i n e HCL and pe n i c i l l i n / s t r e p t o m y c i n have d i f f e r e n t i a l e f f e cts on the ALGAL THALLI Figure 4. F i e l d and laboratory flow diagram for the processing of algal samples. Sample 1. Rhodoglossum a f f i n e . CO ALGAL THALLI Figure 5. F i e l d and laboratory flow diagram for the processing of algal samples. Sample 2. Rhodoglossum a f f i n e and Gelidium c o u l t e r i . LO 40 i s o l a t i o n of microorganisms was evaluated by p l a t i n g tissue sections from each alga on s i m i l a r media prepared with these d i f f e r e n t a n t i b i o t i c s (see Media and Culture Conditions). 3) Two culture media were used - GYSA and SSA; both were prepared with the a n t i b i o t i c s t e t r a c y c l i n e HCL (-T) or penicillin/streptomycin (-PS). 4) Alg a l tissue plates were incubated i n the dark at 25°C. Al g a l tissues were placed i n culture tubes and were rinsed using a Vortex mixer as previously described. The tissues were transferred to new test tubes of s t e r i l e seawater between the 3rd and 4th and the 15th and 16th rinses. Aliquots of the 3rd and 15th rinse water were subsequently spread on the surface of GYSA-PS culture plates i n an attempt to evaluate the effectiveness of rigorous r i n s i n g i n removing surface-associated microorganisms. Test tubes containing the rinse water were gently agitated by hand p r i o r to dispensing. Dispensing was performed using s t e r i l e , cotton-plugged pipettes, 1.0 ml of the rinse water being pipetted onto the surface of each agar plate. Spreading of the water sample was accomplished with a bent glass rod [3 mm diameter; 7.5 cm of surface contact (length)l while spinning the agar plate on a rotating c i r c u l a r pedestal. Glass rods were dipped i n alcohol and flamed between samples. T r i p l i c a t e agar cultures were prepared from water of the 3rd rinse; a single culture was inoculated with water from the 15th rinse. A l l rinse water plates were prepared within 30 minutes of the actual washing process. Rinse water plates were incubated at room temperature (22-25°C) i n dif f u s e l i g h t . These plates were 41 incubated i n an inverted position i n a laminar flow hood (Dexon Model HT74E-830) for three days, aft e r which time they were turned right-side-up and placed i n p l a s t i c bags. Individual colonies of organisms developing on rinse water plates were counted a f t e r 8, 16, and 24 days of incubation. Colony counts were made using a dissecting microscope (7-40X); a compound microscope (100X) was used to check i d e n t i f i c a t i o n s . Representative colonies of each organism were transferred to appropriate media for further characterization. S t e r i l e seawater/pine pollen (SW/P) cultures were also incorporated into Sample 3 as a comparative technique for the i s o l a t i o n of Labyrinthulid and Thraustochytrid organisms. Pine pollen (Pinus sp.) was sprinkled on the surface of 30 ml of s t e r i l e seawater (28°/oo; with pen i c i l l i n / s t r e p t o m y c i n - 500 mg/l each) contained i n p l a s t i c p e t r i plates (15 x 100 mm). These cultures were inoculated with portions of a l g a l tissue s i m i l a r to those plated on agar media, including tissues exposed to rigorous (20X) and l i g h t (2X) rinsing. Seawater/pollen cultures were incubated at room temperature (22-25°C) i n diffuse sunlight and examined for the growth of microorganisms every 3-5 days for 3 weeks. Fresh microscopic mounts of SW/P cultures were prepared by touching one surface of a glass cover s l i p (No. 1.5; 22 x 22 mm) to the seawater i n t e r f a c e and then p l a c i n g the cover s l i p on a microscope s l i d e . I s olation of microorganisms was accomplished by streaking an inoculation loop of the culture onto the surface of a KMV-PS agar plate. Streaking of these cultures on KMV-agar often f a c i l i t a t e d the i s o l a t i o n of organisms not observed i n sl i d e s prepared from the 42 l i q u i d cultures. The technique was, therefore, incorporated as a standard procedure: after 2 to 2.5 weeks of incubation, two inoculation loops of each SW/P culture were streaked on opposite sides of a KMV-PS agar plate. A l l i s o l a t i o n plates prepared from SW/P cultures were incubated at room temperature (22-25°C) i n d i f f u s e sunlight, and were examined for the development of organisms over a period of approximately 14 days. A flow diagram of the laboratory processes used i n Sample 3 i s presented i n Figure 6. RESULTS A. OBSERVATIONS OF FIELD-COLLECTED ALGAE Among the numerous t h a l l i of Rhodoglossum a f f i n e and Gelidium c o u l t e r i collected, examined and dissected i n t h i s study, none were observed to contain obvious fungal f r u i t i n g structures within t h e i r tissues. What appeared to be 'infected' areas were observed i n t h a l l i of R^_ affine. On one occasion, hand-cut cross sections of fresh tissue revealed a l i m i t e d fungal mycelium. More commonly, the tissues appeared discolored due to the presence of an endophytic or epiphytic alga. Areas of the R.  a f f i n e t h a l l u s which looked 'infected' were not p r e f e r e n t i a l l y selected as inocula for i s o l a t i o n s . ALGAL THALLI Figure 6. F i e l d and laboratory flow diagram f o r the processing of alga l samples. Sample 3. Rhodoglossum a f f i n e and Gelidium c o u l t e r i . (* -T = t e t r a c y c l i n e HCl; -PS = p e n i c i l l i n / s t r e p t o m y c i n ) LO 44 B. ISOLATIONS FROM FIELD-COLLECTED ALGAE 1. Sample 1 (December, 1978) a. A l g a l Tissues Plated on Agar Media i . Labyrinthulids Table 3 l i s t s the i s o l a t i o n frequency of Labyrinthula spp. from the d i f f e r e n t l i f e h i s t o r y stages of Rhodoglossum affine; data are given by incubation temperature (14°C or 25°C) and tissue pretreatment ( s t e r i l i z e d or rinsed). These organisms were not observed on Glucose-Yeast Extract Agar. Thus, the data presented i n Table 3 are based on i s o l a t i o n s from Serum-Seawater Agar (SSA) only. The i s o l a t i o n frequencies c l e a r l y indicate that the surface s t e r i l i z a t i o n procedure was e f f e c t i v e i n eliminating Labyrinthula spp. from the a l g a l tissues; no i s o l a t e s were obtained from s t e r i l i z e d tissues. Labyrinthula spp. were commonly is o l a t e d from tissues rinsed twice i n s t e r i l e seawater, having an o v e r a l l i s o l a t i o n frequency of 37.5%. The results from seawater-rinsed a l g a l tissues suggest that higher incubation temperatures (25°C) may enhance the recovery of Labyrinthula spp., but the difference between temperatures was not s t a t i s t i c a l l y s i g n i f i c a n t . Labyrinthula spp. were isola t e d with s i m i l a r frequency from the d i f f e r e n t l i f e h i story stages of Rj_ a f f i n e (Table 3) (see Appendix B, Example 1). i i . Thraustochytrids Thraustochytrids were not observed on a l g a l tissue-agar plates i n Sample 1. Table 3 Percent occurrence of Labyrinthula spp. isolated from the d i f f e r e n t l i f e history stages of Rhodoglossum a f f i n e . Sample 1, Dec. 1978.f Isolation frequencies for s u r f a c e - s t e r i l i z e d * and seawater-rinsed** algal tissues are l i s t e d by incubation temperature. 14°C 25°C L i f e History Stage S t e r i l i z e d * Rinsed** S t e r i l i z e d Rinsed of R. a f f i n e (n=12*r) (n=4) (n=12) (n=4) Vegetative/Male 0 25.0 0 50.0 Cystocarpic 0 0 0 50.0 Tetrasporangial 0 50.0 0 50.0 A l l Stages 0 25.0 0 50.0 fData for Modified Serum-Seawater Agar (SSA) only. • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 1 minute; followed by two one-minute rinses i n s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. •tm = the number of algal tissue pieces plated (inocula). 46 i i i . Higher Fungi and Actinomycetes Data pertaining to the i s o l a t i o n of actinomycetes, yeasts and mycelial fungi are presented i n Tables 4 and 5. Table 4 l i s t s the percent occurrence of the organisms is o l a t e d by incubation temperature/tissue pretreatment regime and a l l regimes combined. These data include a l l l i f e h i s t o r y stages of Rhodoglossum a f f i n e and both i s o l a t i o n media (SSA and GYSA). Table 5 shows possible differences i n the i s o l a t i o n of TOTAL actinomycetes, yeasts and mycelial fungi between the d i f f e r e n t l i f e h i s t o r y stages of R^  a f f i n e and between i s o l a t i o n media. As would be expected for a l g a l tissues plated on media containing a n t i b i o t i c s , the i s o l a t i o n frequencies for actinomycetes were low (1.6% overall). The data suggest that actinomycetes were eliminated from the a l g a l tissues by the surface s t e r i l i z a t i o n procedure (Table 4). The recovery of actinomycetes from rinsed a l g a l tissues was s i m i l a r for both incubation temperatures (Table 4). Yeasts also displayed f a i r l y low i s o l a t i o n frequencies (5.2% overall). The recovery of yeasts was not affected by incubation temperature, and was s t a t i s t i c a l l y s i m i l a r for s u r f a c e - s t e r i l i z e d and rinsed tissues of R^_ a f f i n e (Table 4). Yeasts did show some •preference' for the GYSA medium at the lower incubation temperature (14°C), but not at 25°C (Table 5). No obvious trends were seen between the i s o l a t i o n frequency of yeasts and the d i f f e r e n t l i f e h i s t o r y stages of R^_ a f f i n e (Table 5). Eighteen d i f f e r e n t mycelial fungi were isola t e d from Rhodoglossum a f f i n e i n Sample 1, including 12 (66.7%) Table 4 Percent occurrence of actinomycetes, yeasts and mycelial fungi i s o l a t e d from Rhodoglossum a f f i n e . Sample 1, Dec. 1978.f Isolation frequencies are l i s t e d f o r each organism by incubation temperature/tissue pretreatment regime and a l l regimes combined. Isolates 14°C S t e r i l i z e d * (n=72*) Rinsed** (n=24) 25°C S t e r i l i z e d (n=72) Rinsed (n=24) Combined (n=192) Actinomycetes 4.2 8.3 1.6 Yeasts and y e a s t - l i k e fungi Candida sp. Unidentified Yeasts 0 4.2 4.2 4.2 0 4.2 0 8.3 0.5 4.7 TOTAL YEASTS 4.2 8.3 4.2 8.3 5.2 Mycelial Fungi Acremonium sp. 012-78 Acremonium sp. 019-78 Acremonium sp. 029-78 Beauveria bassiana (Bals.) V u i l l . Cladosporium cladosporioides (Pers.) Link ex S.F. Gray Cladosporium sphaerospermum Penz. Dendryphiella s a l i n a (Suth.) Pugh et Nicot Fusarium concolor Reining Geotrichum sp. Pe n i c i l l i u m spp. Phialophora sp. 020-78 Phoma sp. (Group 1) Phoma sp. 033-78 V e r t i c i l ! i u m psal1iotae Treschow Unidentified hyphomycete 044-78 Unidentified coelomycete 005-78 Unidentified coelomycete 013-78 Unidentified coelomycete 035-78 Unidentified coelomycete 042-78 0 0 0 1.4 1.4 0 0 0 1.4 0 0 1.4 0 0 0 1.4 0 0 0 4.2 4.2 0 0 0 4.2 4.2 0 0 4.2 4.2 0 0 0 12.5 0 4.2 0 0 0 0 1.4 0 1.4 0 1.4 0 0 1.4 0 0 0 0 2.8 0 0 0 0 0 8.3 0 0 12.5 4.2 0 4.2 0 0 0 4.2 4.2 4.2 4.2 0 0 4.2 4.2 0.5 1.6 0.5 0.5 2.6 1.0 1.0 0.5 0.5 1.0 0.5 1.0 0.5 0.5 3.1 0.5 0.5 0.5 0.5 TOTAL MYCELIAL FUNGI 6.9 41.7 8.3 54.2 17.7 fData f o r a l l l i f e h istory stages of the alga and both i s o l a t i o n media combined. • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 1 minute; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). Table 5 Total i s o l a t i o n frequency {%) of actinomycetes, yeasts and mycelial fungi f o r each incubation temperature/tissue pretreatment regime l i s t e d by: 1) l i f e history stage of Rhodoglossum a f f i n e * and 2) i s o l a t i o n medium.** Sample 1, Dec. 1978. Incubation Tissue L i f e History Stage* Isolation Medium** Temperature Treatments Isolates Veg./d 9 GYSA SSA n=24& n=36& 14°C S t e r i 1 i z e d Actinomycetes 0 0 0 0 0 Yeasts 4.2 4.2 4.2 8.3 0 Mycelial Fungi 4.2 12.5 4.2 5.6 8.3 n=8 n=12 14°C Rinsed Actinomycetes 0 12.5 0 0 8.3 Yeasts 25.0 0 0 16.7 0 Mycelial Fungi 25.0 50.0 50.0 41.7 41.7 n=24 n=36 25°C S t e r i 1 i z e d Actinomycetes 0 0 0 0 0 Yeasts 0 4.2 8.3 5.6 2.8 Mycelial Fungi 8.3 8.3 8.3 2.8 13.9 n=8 n=12 25°C Rinsed Actinomycetes 12.5 12.5 0 0 16.7 Yeasts 0 0 25.0 8.3 8.3 Mycelial Fungi 62.5 25.0 75.0 66.7 41.7 *Both i s o l a t i o n media combined. **A11 three alg a l l i f e h i s t o r y stages combined. •tm = the number of algal t i s s u e pieces plated (inocula). 49 Hyphomycetes and 6 (33.3%) Coelomycetes (Table 4). Examination of the i n d i v i d u a l species and/or i s o l a t e s of mycelial fungi reveals that a l l individuals were r e l a t i v e l y rare; the highest o v e r a l l frequency of occurrence was 3.1% (Unidentified hyphomycete 044-78). Cladosporium cladosporioides (Pers.) Link ex S. F. Gray (2.6% o v e r a l l ) and Acremonium sp. 019-78 (1.6%) were the next two most common fungi. A l l other mycelial fungi were i s o l a t e d from 1.0% or less of the t o t a l a l g a l tissue inocula. Total i s o l a t i o n frequencies of mycelial fungi for each temperature/tissue pretreatment regime (Table 4, bottom) show that the surface s t e r i l i z a t i o n procedure was e f f e c t i v e i n diminishing the numbers of mycelial fungi i s o l a t e d from s t e r i l i z e d tissues. Isolation frequencies were s i g n i f i c a n t l y d i f f e r e n t between s t e r i l i z e d and rinsed tissues at both incubation temperatures (p < 0.005 for both) (see Appendix B, Example 2). Within each tissue pretreatment ( s t e r i l i z e d or rinsed), there were no differences i n t o t a l i s o l a t i o n frequencies between incubation temperatures. There were no s i g n i f i c a n t differences i n the occurrence of mycelial fungi between a l g a l l i f e h i s t o r y stages or i s o l a t i o n media (Table 5). 2. Sample 2 (March, 1979) a. A l g a l Tissues Plated on Agar Media i . Labyrinthulids Data pertaining to the i s o l a t i o n of Labyrinthula spp. from Rhodoglossum a f f i n e and Gelidium c o u l t e r i i n Sample 2 are given i n Tables 6 and 7. The results presented i n Table 6 show that, 50 of the three i s o l a t i o n media u t i l i z e d , Serum-Seawater Agar was the most e f f e c t i v e i n i s o l a t i n g Labyrinthula from these algae. Of the t o t a l Labyrinthula iso l a t e s , 89.5% were obtained from SSA, while only 7.0% and 3.5% were i s o l a t e d from GYSA and FUL respectively; these differences are highly s i g n i f i c a n t (p < 0.005) (but, see Discussion; Media, A n t i b i o t i c s and Culture Conditions). The data given i n Table 7 are based on i s o l a t i o n s from Serum-Seawater Agar only. Labyrinthula spp. were is o l a t e d with s i m i l a r frequency from s t e r i l i z e d (65.6%) and rinsed (66.7%) tissues of Gelidium c o u l t e r i (Table 7). However, s i g n i f i c a n t l y fewer Labyrinthula were obtained from s u r f a c e - s t e r i l i z e d tissues of Rj_ a f f i n e (36.1%) than from rinsed tissues of that alga (75.0%) (p < 0.025). Isolation frequencies from rinsed tissues of the two algae (Table 7) were s i m i l a r . The low o v e r a l l frequency of i s o l a t i o n for s t e r i l i z e d t i s s u e s of Rhodoglossum a f f i n e was due, i n large part, to the low number of Labyrinthula spp. i s o l a t e d from the tetrasporangial l i f e h i s t o r y stage of t h i s alga (Table 7). Isolation frequencies by a l g a l l i f e h i s t o r y stage were not only s i g n i f i c a n t l y d i f f e r e n t for s t e r i l i z e d tissues of R^ a f f i n e (p < 0.025, tetrasporangial low), but also for s t e r i l i z e d Gelidium c o u l t e r i (p < 0.025, cystocarpic low). i i . Thraustochytrids The i s o l a t i o n frequencies for each Thraustochytrid species encountered i n Sample 2 are l i s t e d by i s o l a t i o n medium and tissue pretreatment i n Tables 8 (R^ affine) and 9 (G^ c o u l t e r i ) . Table 6 Percent occurrence of Labyrinthula spp. isolated from Rhodoglossum affine and Gelidium coulteri Sample 2, March 1979. Isolation frequencies are l isted by isolation medium for s ter i l i zed* and rinsed** tissues of each alga. Medium R. affine Steri1ized* (n=36fc) Rinsed** (n=12) G. coulteri Steri1ized (n=32) Rinsed (n=12) SSA GYSA FUL 36.1 0 0 75.0 0 0 65.6 12.5 3.1 66.7 0 8.3 All Media 12.0 25.0 27.1 25.0 •Steri l ized = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s ter i le SW. **Rinsed = Two one-minute rinses in s ter i le SW. &n = the number of algal tissue pieces plated (inocula). Table 7 Percent occurrence of Labyrinthula spp. isolated from Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 2, March 1979.+ Isolation frequencies are l i s t e d by algal l i f e history stage for s t e r i l i z e d * and rinsed** tissues of each alga. R. a f f i n e G. c o u l t e r i L i f e History Stage S t e r i l i z e d * Rinsed** S t e r i l i z e d Rinsed of the Alga (n=12*) (n=4) (n=12) (n=4) Vegetative/Male 58.3 50.0 83.3 50.0 Cystocarpic 42.7 75.0 25.0* 100.0 Tetrasporangial 8.3 100.0 75.0 50.0 A l l Stages 36.1 75.0 65.6 66.7 tData for Modified Serum-Seawater Agar (SSA) only. • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. •im = the number of algal t i s s u e pieces plated (inocula). in = 8 for s u r f a c e - s t e r i l i z e d cystocarpic G. coul t e r i . 53 Schizochytrium aggregatum Goldstein and Belsky was of l i m i t e d and sporadic occurrence, amounting to 8.3% of the t o t a l Thraustochytrid isolates. S_^  aggregatum displayed s t a t i s t i c a l l y s i m i l a r (low) i s o l a t i o n frequencies from each of the three i s o l a t i o n media. Total i s o l a t i o n frequencies for each alga/tissue pretreatment regime (last two columns, Tables 8 and 9) show that the percent occurrence of aggregatum was s i g n i f i c a n t l y lower from s t e r i l i z e d than from rinsed tissues of both algae (p < 0.01, a f f i n e ; p < 0.025, G^ c o u l t e r i ) . Comparisons of rinsed and s t e r i l i z e d tissues between algae, show that Sj_ aggregatum; 1) was i s o l a t e d with equal frequency from both algae; and 2) was a f f e c t e d i n a s i m i l a r manner by t i s s u e pretreatments i n both algae. Thraustochytrium motivum Goldstein was the most common Thraustochytrid i s o l a t e d i n Sample 2, representing 90.5% of the t o t a l i s o l a t e s . Thraustochytrium motivum displayed disporportional i s o l a t i o n frequencies with respect to medium for s t e r i l i z e d tissues of Rhodoglossum a f f i n e (p < 0.005, Table 8) and rinsed tissues of Gelidium c o u l t e r i (p < 0.05, Table 9). In both of these cases T\_ motivum was recovered more frequently from Serum-Seawater Agar (SSA) than from GYSA or FUL. Thraustochytrium motivum was i s o l a t e d less frequently from s t e r i l i z e d tissues of both algae (p < 0.005, R^ af f i n e ; p < 0.025, G. c o u l t e r i ) ( l a s t two columns, Tables 8 and 9). I s o l a t i o n frequencies were s i g n i f i c a n t l y d i f f e r e n t for s t e r i l i z e d t i s s u e s of the two algae (p < 0.005; R. a f f i n e low); but f o r rinsed tissues they were not (0.1 > p < 0.05). These re s u l t s Table 8 Percent occurrence of Thraustochytrid species i s o l a t e d from Rhodoglossum a f f i n e . Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d * and r i n s e d * * algal tissues by i s o l a t i o n medium and a l l media combined. SSA GYSA FUL ALL MEDIA Isolates ST* R** ST R ST R ST R (n=36^r) (n=12) (n=36) (n=12) (n=36) (n=12) (n=108) (n=36) Schizochytrium  aggregatum Goldstein & Belsky 2.8 Thraustochytrium motivum Goldstein 22.2 Ulkenia sp. RC02-79 2.8 16.7 8.3 0 8.3 0.9 11.1 41 .7 0 41.7 0 25.0 7.4 36.1 0.9 0 • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). Table 9 Percent occurrence of Thraustochytrid species isolated from Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d * and r i n s e d * * algal tissues by i s o l a t i o n medium and a l l media combined. SSA GYSA FUL ALL MEDIA Isolates ST* R** ST R ST R ST R (n=32*)'(n=12) (n=32) (n=12) (n=32) (n=12) (n=96) (n=36) Schizochytrium  aggregatum Goldstein & Belsky 0 8.3 0 0 0 8.3 0 5.6 Thraustochytrium motivum Goldstein 34.4 83.3 37.5 58.3 34.4 33.3 35.4 58.3 • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. &n = the number of algal t issues pieces plated (inocula). suggest that surface s t e r i l i z a t i o n reduced the i s o l a t i o n of Thraustochytrium motivum i n both algae, but that the ef f e c t was more pronounced i n Rhodoglossum affi n e . A single i s o l a t e (1.2% of the t o t a l ) of an uni d e n t i f i e d species of Ulkenia (RC02-79) was obtained from s u r f a c e - s t e r i l i z e d t i s s u e s of R. a f f i n e (Table 8). Thraustochytrid i s o l a t i o n frequencies are l i s t e d for each l i f e h i s t o r y stage of the algae i n Tables 10 (Rj_ affine) and 11 (G. c o u l t e r i ) . Analyses of these data by tissue pretreatment for each alga show that aggregatum and T\_ motivum were isola t e d with s i m i l a r frequency from the d i f f e r e n t l i f e h i s t o r y stages of both algae. i i i . Higher Fungi and Actinomycetes Iso l a t i o n information for the actinomycetes, yeasts and mycelial fungi recovered from Sample 2 algae i s given i n Tables 12 and 13. Table 12 l i s t s the percent occurrence of each organism by alga/tissue pretreatment regime and for a l l regimes combined. Table 13 l i s t s TOTAL actinomycetes, yeasts and mycelial fungi for each alga/tissue pretreatment regime by: 1) a l g a l l i f e h i s t o r y stage; and 2) i s o l a t i o n medium. Actinomycetes continued to be i s o l a t e d infrequently i n Sample 2 (1.8% overall). These organisms were not i s o l a t e d from s u r f a c e - s t e r i l i z e d a l g a l tissues (Table 12). Seawater-rinsed t i s s u e s of the two algae gave r i s e to a s i m i l a r number of actinomycete i s o l a t e s (Table 12). Yeasts and yeast-like fungi were rather common i n Sample 2 (14.9% overall). These organisms also appear to have been Table 10 Percent occurrence of Thraustochytrid species isolated from Rhodoglossum a f f i n e . Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d * and r i n s e d * * tissues by algal l i f e history stage and a l l stages combined. Vegetative/Male Cystocarpic Tetrasporangial A l l Stages Isolates ST* R** ST R ST R ST R (n=36*) (n=12) (n=36) (n=12) (n=36) (n=12) (n=108) (n=36) Schizochytrium  aggregatum Goldstein & Bel sky 0 0 2.8 25.0 0 8.3 0.9 11.1 Thraustochytrium motivum Goldstein 5.6 50.0 11.1 33.3 5.6 25.0 7.4 36.1 Ulkenia sp. RC02-79 0 0 2.8 0 0 0 0.9 0 • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. •bn = the number of algal t i s s u e pieces plated (inocula). Table 11 Percent occurrence of Thraustochytrid species i s o l a t e d from Gelidium c o u l t e r i . Sample 2, March 1979. Isolation frequencies are l i s t e d for s t e r i l i z e d * and rinsed** tissues by algal l i f e history stage and a l l stages combined. Vegetative/Male Cystocarpic Tetrasporangial A l l Stages Isolates ST* R** ST R ST R ST R (n=36fc) (n=12) (n=24) (n=12) (n=36) (n=12) (n=96) (n=36) Schizochytrium  aggregatum Goldstein & Belsky 0 16.7 0 0 0 0 0 5.6 Thraustochytrium motivum Goldstein 38.9 58.3 37.5 58.3 30.6 58.3 35.4 58.3 • S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). reduced by the surface s t e r i l i z a t i o n procedure (Table 12). For Gelidium c o u l t e r i the difference between s u r f a c e - s t e r i l i z e d and seawater-rinsed a l g a l tissues was s i g n i f i c a n t (p < 0.005); for Rhodoglossum a f f i n e i t was not (0.1 < p > 0.05). Yeasts displayed s i m i l a r frequencies of i s o l a t i o n from seawater-rinsed tissues of both algae, but were recovered more frequently from s t e r i l i z e d tissues of Rj_ af f i n e than from tissues of G^ c o u l t e r i treated s i m i l a r l y (p < 0.05) (Table 12). Based on the data obtained for each alga/tissue pretreatment regime, yeasts were iso l a t e d with s i m i l a r frequency from the three d i f f e r e n t culture media (Table 13) (see Appendix B, Example 3A). Yeast i s o l a t i o n frequencies were generaly s i m i l a r for the d i f f e r e n t l i f e h i s t o r y stages of both algae, except for s t e r i l i z e d tissues of R^_ a f f i n e (p < 0.01, cystocarpic low or ?-vegetative/male high) (Table 13) (see Appendix B, Example 3B). Forty-three d i f f e r e n t mycelial fungi were i s o l a t e d from a l g a l tissues i n Sample 2. This t o t a l was composed of 18 (41.9%) Hyphomycetes, 20 (46.5%) Coelomycetes, 4 (9.3%) s t e r i l e mycelia, and a single (2.3%) Ascomycete (Table 12). Isolation frequencies for a l l mycelial fungi were low, a great many being represented by single i s o l a t i o n s . This i s es p e c i a l l y true for many of the as yet un i d e n t i f i e d coelomycetous fungi. Only 10 of the fungi were is o l a t e d at o v e r a l l frequencies of occurrence above 1.0%. The most common fungi encountered were: Cladosporium cladosporioides (6.5% o v e r a l l ) , Phoma sp. (Group 1) (4.7%), P e n i c i l l i u m spp. (2.5%), and Unidentified coelomycete 042-78 (2.2%). A l l other mycelial fungi were isola t e d from less than 2.0% of the t o t a l number of inocula. Table 12 Percent occurrence of actinomycetes, yeasts and mycelial fungi i s o l a t e d from Rhodoglossum a f f i n e and Gel idium c o u l t e r i . Sample 2, March 1979.f Isolation frequencies are l i s t e d f o r each organism by alga/tissue pretreatment regime and a l l regimes combined. R. a f f i n e G. c o u l t e r i Isolates S t e r i l i z e d * (n=108*) Rinsed** (n=36) S t e r i l i z e d (n=96) Rinsed (n=36) Combined (n=276) Actinomycetes 0 8.3 0 5.6 1.8 Yeasts and y e a s t - l i k e fungi Hormonema sp. 157-79 0 2.8 0 0 0.4 Trichosporon sp. 127-79 0.9 0 0 0 0.4 Unidentified Yeasts 13.9 25.0 6.3 25.0 14.1 TOTAL YEASTS 14.8 27.8 6.3 25.0 14.9 Mycelial Fungi Acremonium sp. 019-78 0 0 0 5.6 0.7 Acremonium sp. 010-79 0 0 1.0 0 0.4 Acremonium sp. 071-79 1.8 0 0 0 0.7 Acremonium sp. 102-79 0.9 0 0 0 0.4 Acremonium sp. 162-79 0 2.8 0 0 0.4 Acremonium sp. (not isolated) 1.8 0 0 0 0.7 Chalara sp. 148-79 0 2.8 0 0 0.4 Cladosporium cladosporioides (Pers.) Link ex S.F. Gray 8.3 2.8 5.2 8.3 6.5 Cladosporium herbarum Link ex Fries 0.9 0 0 2.8 0.7 Dendryphiella s a l i n a (Suth.) Pugh et Nicot 0 0 1.0 0 0.4 Gliomastix murorum (Corda) Hughes 0 0 0 2.8 0.4 Leptosphaeria sp. 089-79 0.9 0 0 0 0.4 Pe n i c i l l i u m spp. 4.6 0 2.1 0 2.5 Phialophora f a s t i g a t a (Lagerberg & Melin) Conant 0.9 0 0 0 0.4 Phialophora verrucosa Medlar 1.8 0 1.0 2.8 1.4 Phoma sp. (Group 1) 8.3 5.6 1.0 2.8 4.7 Phoma sp. 033-78 0.9 0 2.1 2.8 1.4 Phoma sp. 013-79 0.9 0 1.0 0 0.7 Phomopsis sp. 140-79 0 2.8 0 0 0.4 Sclerophoma sp. 003-79 0 0 1.0 0 0.4 Seiridium j u n i p e r i (Allesch.) Sutton 0 2.8 0 0 0.4 Sigmoidea l i t t o r a l i s sp. nov. 0 0 2.1 2.8 1.1 Unidentified hyphomycete 044-78 0.9 2.8 0 2.8 1.1 Unidentified hyphomycete 120-79 0.9 0 0 0 0.4 Unidentified coelomycete 035-78 0 0 1.0 0 0.4 Unidentified coelomycete 042-78 0 2.8 4.2 2.8 2.2 Unidentified coelomycete 029-79 0 0 1.0 0 0.4 Unidentified coelomycete 032-79 0 0 1.0 0 0.4 Unidentified coelomycete 039-79 0 0 0 2.8 0.4 Unidentified coelomycete 040-79 0 0 0 2.8 0.4 Unidentified coelomycete 051-79 0 0 0 8.3 1.1 Unidentified coelomycete 056-79 0 0 0 2.8 0.4 Unidentified coelomycete 063-79 0.9 0 0 2.8 0.7 Unidentified coelomycete 086-79 2.8 0 0 0 1.1 Unidentified coelomycete 095-79 0.9 0 0 0 0.4 Unidentified coelomycete 112-79 0.9 2.8 0 0 0.7 Unidentified coelomycete 119-79 1.8 0 0 0 0.7 Unidentified coelomycete 143-79 0 2.8 0 0 0.4 Unidentified mycelium ( S c l e r o t i a ) 144-79 0 2.8 0 0 0.4 S t e r i l e mycelium 048-79 0.9 0 0 2.8 0.7 S t e r i l e mycelium 060-79 0 0 0 2.8 0.4 S t e r i l e mycelium 116-79 0.9 0 0 0 0.4 TOTAL MYCELIAL FUNGI 43.5 33.3 25.0 63.9 38.4 fData f o r a l l l i f e h istory stages of the algae and a l l i s o l a t i o n media combined. * S t e r i l i z e d = 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses in s t e r i l e SW. **Rinsed = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). 60 Table 13 Total i s o l a t i o n frequency {%) of actinomycetes, yeasts and mycelial fungi for each alga/tissue pretreatment regime l i s t e d by: 1) l i f e history stage of the algae* and 2) i s o l a t i o n medium.** Sample 2, March 1979. Alga Tissue Treatments Isolates Alga L i f e History Stage* Veg./rj 9 $ Isolation Medium** GYSA SSA FUL n=36^ n=36-& Rhodoglossum S t e r i l i z e d Actinomycetes 0 0 0 0 0 0 a f f i n e Yeasts 27.8 2.8 13.9 16.7 13.9 13.9 Mycelial Fungi 58.3 41.7 30.6 52.8 52.8 25.0 n=12 n=12 Rhodoglossum Rinsed Actinomycetes 8.3 0 16.7 0 16.7 8.3 a f f i n e Yeasts 16.7 50.0 16.7 41.7 16.7 25.0 Mycelial Fungi 25.0 58.3 16.7 25.0 33.3 41.7 n=36 n=24 n=36 n=32 Gelidium S t e r i l i z e d Actinomycetes 0 0 0 0 0 0 c o u l t e r i Yeasts 8.3 0 8.3 3.1 3.1 12.5 Mycelial Fungi 38.9 20.8 13.9 28.1 28.1 18.8 n=12 n=12 Gelidium Rinsed Actinomycetes 0 8.3 8.3 8.3 8.3 0 c o u l t e r i Yeasts 8.3 25.0 41.7 33.3 8.3 33.3 Mycelial Fungi 91.7 50.0 50.0 58.3 50.0 83.3 *A11 three i s o l a t i o n media combined. **A11 three algal l i f e h i s t o r y stages combined. -im = the number of algal t i s s u e pieces plated (inocula). 62 Examination of the o v e r a l l i s o l a t i o n frequencies for mycelial fungi (Table 12, bottom) shows that s i g n i f i c a n t l y fewer mycelial fungi were obtained from s u r f a c e - s t e r i l i z e d than from seawater-rinsed tissues of Gelidium c o u l t e r i (p < 0.005). For Rhodoglossum affine, however, the numbers of fungi i s o l a t e d from s t e r i l i z e d and seawater-rinsed tissues were not s i g n i f i c a n t l y d i f f e r e n t . Fewer mycelial fungi were i s o l a t e d from seawater-rinsed tissues of Rj_ a f f i n e (33.3%) than from tissues of G.  c o u l t e r i treated s i m i l a r l y (63.9%) (p < 0.01). On the contrary, s t e r i l i z e d tissues of R^ a f f i n e gave r i s e to more i s o l a t e s (43.5%) than s t e r i l i z e d t i s s u e s of G. c o u l t e r i (25.0%) (p < 0.01) (Table 12, Bottom). Fewer fungi should have been i s o l a t e d from s t e r i l i z e d than from seawater-rinsed tissues of R^ affine. On the other hand, the differences between s t e r i l i z e d and rinsed tissues of G^ c o u l t e r i are rather large considering that Labyrinthula spp. were not adversely affected by surface s t e r i l i z a t i o n i n t h i s alga (see Table 7). These results r e f l e c t the extent to which careful evaluations are required when one attempts to couple numerical analyses with i n d i r e c t microbiological methods. Total i s o l a t i o n frequencies for mycelial fungi do show that exposing the a l g a l tissues to 1:10,000 w/v HgClj i n 5% Ethanol for one minute (Sample 1) was more e f f e c t i v e than exposure for 30 seconds (Sample 2) i n decreasing the number of mycelial fungi i s o l a t e d from s t e r i l i z e d tissues. Generally, mycelial fungi were i s o l a t e d with s i m i l a r frequency from a l l three i s o l a t i o n media tested. However, the 63 data obtained from s t e r i l i z e d tissues of Rhodoglossum af f i n e did vary s i g n i f i c a n t l y between media (p < 0.02 5, FUL low) (Table 13). Disproportional i s o l a t i o n frequencies were displayed between the d i f f e r e n t l i f e h i s t o r y stages of Gelidium c o u l t e r i (p < 0.05 for s t e r i l i z e d and rinsed; vegetative/male high) (Table 13). Based on the data obtained for each tissue pretreatment regime, i s o l a t i o n frequencies were s t a t i s t i c a l l y s i m i l a r from the d i f f e r e n t l i f e h i s t o r y stages of Rj_ affine. 3. Sample 3 (June, 1980) a. A l g a l Tissues Plated on Agar Media i . Labyrinthulids The Labyrinthulids i s o l a t e d from a l g a l tissue-agar plates i n Sample 3 are l i s t e d i n Tables 14 (Rhodoglossum affine) and 15 (Gelidium c o u l t e r i ) . Isolation frequencies are given by tissue pretreatment (20X or 2X rinsed) for each i s o l a t i o n medium-a n t i b i o t i c combination and a l l media combined. There was c l e a r l y an e f f e c t of i s o l a t i o n medium-antibiotic combination on the recovery of a l l organisms l i s t e d ; no isolates were obtained from Glucose-Yeast Extract Agar containing t e t r a c y c l i n e HCl (Tables 14 and 15). This presumed growth-i n h i b i t o r y e f f e c t was r e s t r i c t e d to the GYSA-T medium (not SSA-T), and was apparently a r e s u l t of the 'breakdown' of the t e t r a c y c l i n e HCl molecule upon exposure to l i g h t . Further description of t h i s i n h i b i t o r y phenomenon and the reasoning behind t h i s interpretation are included i n the discussion. No i s o l a t e s were obtained from GYSA-T, therefore the data for t h i s medium have been excluded from calculations of o v e r a l l i s o l a t i o n Table 14 Percent occurrence of Labyrinthulid species isolated from Rhodoglossum a f f i n e . Sample 3, June 1980. Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t (2X)** rinsed algal tissues by i s o l a t i o n medium-antibiotic combination and a l l media combined. Isolates SSA-T 20X* 2X** (n=24*) (n=6) SSA-PS 20X 2X (n=24) (n=6) GYSA-T 2 OX 2X (n=24) (n=6) GYSA-PS 20X 2X (n=24) (n=6) ALL MEDIAf 20X 2X (n=72) (n=18) Labyrinthula spp. Labyrinthula sp. Type LX Watson Labyrinthuloides  yorkensis Perkins Labyrinthuloides sp. 1 Labyrinthulid Unidentified 66.7 66.7 12.5 16.7 100.0 100.0 4.2 4.2 16.7 16.7 0 0 0 0 0 0 79.2 66.7 0 0 4.2 81.9 77.8 1 .4 4.2 2.8 9.7 11.1 1.4 *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. tValues for ' a l l media' do not include data for GYSA-T. -frn = the number of algal t i s s u e pieces plated (inocula). Table 15 Percent occurrence of Labyrinthulid species isolated from Gelidium c o u l t e r i . Sample 3, June 1980. Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t (2X)** rinsed algal tissues by i s o l a t i o n medium-antibiotic combination and a l l media combined. SSA-T SSA-PS GYSA-T GYSA-PS ALL MEDIAf Isolates 20X* 2X** 20X 2X 20X 2X 20X 2X 20X 2X (n=24*r) (n=6) (n=24) (n=6) (n=24) (n=6) (n=24) (n=6) (n=72) (n=18) Labyrinthula spp. 79.2 50.0 83.3 100.0 0 0 62.5 66.7 75.0 72.2 Labyrinthula sp. Type LX Watson 0 0 4.2 0 0 0 0 0 1.4 0 Labyrinthuloides sp. 1 0 0 4.2 0 0 0 0 0 1 .4 0 *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. fValues for ' a l l media 1 do not include data for GYSA-T. #n = the number of algal t i s s u e pieces plated (inocula). CTl U l 66 frequencies and comparisons between algae, a l g a l l i f e h i story stages, tissue pretreatments, etc. Labyrinthula spp. were the most common organisms isolated from a l g a l tissue-agar plates i n Sample 3; displaying an o v e r a l l i s o l a t i o n frequency of 77.8% (Tables 14 and 15). Labyrinthula spp. exhibited a s l i g h t 'preference' for the SSA-PS medium. However, the only regime i n which frequencies were s i g n i f i c a n t l y higher on t h i s medium was 20X rinsed tissues of R^_ a f f i n e (p < 0.005, SSA-PS vs. SSA-T; p < 0.01, SSA-PS vs. GYSA-PS) (Table 14). Labyrinthula spp. were recovered with equal frequency from both algae (last two columns, Tables 14 and 15). Rigorous r i n s i n g of the a l g a l tissues did not reduce the i s o l a t i o n frequency of these organisms. Several Labyrinthulids were i s o l a t e d i n Sample 3 which had not been encountered on a l g a l tissue-agar plates i n previous samples. Labyrinthula sp. Type LX Watson and Labyrinthuloides  yorkensis Perkins were i s o l a t e d rarely (1.1% o v e r a l l each) (Tables 14 and 15). A single i s o l a t e of Labyrinthula sp. Type LX was obtained from a SSA-PS plate of each alga. Labyrinthuloides  yorkensis was i s o l a t e d twice from tissues of Rj_ a f f i n e ; i t was r e s t r i c t e d to media containing p e n i c i l l i n / s t r e p t o m y c i n (Table 14). An undescribed species of the genus Labyrinthuloides (sp. 1) was i s o l a t e d from 8.3% of the Serum-Seawater Agar plates. This organism i s so small, and i t s colonies so t h i n and transparent, that i t may have been overlooked i n Samples 1 and 2. Isolation frequencies for Labyrinthuloides sp. 1 were low, but the data 67 obtained suggest that: 1) i t 'preferred' Serum-Seawater Agar ( s i g n i f i c a n t differences between media for 20X rinsed Rj_ a f f i n e ; p < 0.05) (Table 14); 2) i t was i s o l a t e d more frequently from tissues of Rhodoglossum af f i n e than Gelidium c o u l t e r i ( s i g n i f i c a n t between algae for 20X rinsed tissues; p < 0.025); 3) f o r each alga i t was i s o l a t e d with equal frequency from 20X and 2X rinsed tissues (Tables 14 and 15). Other Labyrinthulids were encountered (especially from rinse water and SW/P cultures, see below) which could not be iso l a t e d or would not grow well i n axenic culture using the methods described. Most of these organisms are believed to have been undescribed species of the genus Labyrinthuloides. Observations of several unidentified Labyrinthulids which were brought into pure culture are included i n the Descriptions (Section VII). A l l such i s o l a t e s are combined here under the category 'Labyrinthulid Unidentified' (Table 14). Is o l a t i o n frequencies for Labyrinthulid organisms are l i s t e d by l i f e h i s t o r y stage of each alga i n Tables 16 (Rhodoglossum  affine) and 17 (Gelidium c o u l t e r i ) . Generally, Labyrinthula spp. were i s o l a t e d with equal frequency from the d i f f e r e n t l i f e h i s t o r y stages of both algae. However, values obtained from 20X rinsed tissues of G^_ c o u l t e r i do vary s i g n i f i c a n t l y between l i f e h i s t o r y stage (p < 0.05) (Table 17). Labyrinthuloides sp. 1 was is o l a t e d with equal frequency from the d i f f e r e n t l i f e h i s t o r y stages of R^ a f f i n e (Table 16). A l l other Labyrinthulids were iso l a t e d so infrequently that analyses by alga l i f e h i s t o r y stage would be superfluous. Table 16 Percent occurrence of Labyrinthulid species isolated from Rhodoglossum a f f i n e . Sample 3, June 1980.+ Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t (2x)** rinsed t i s s u e s by alga l l i f e h istory stage and a l l stages combined. Vegetative/Male Cystocarpic Tetrasporangial ALL STAGES Isolates 20X* 2X** 20X 2X 20X 2X 20X 2X (n=24£) (n=6) (n=24) (n=6) (n=24) (n=6) (n=72) (n=18) Labyrinthula spp. 79.2 66.7 87.5 83.3 79.2 83.3 81.9 77.8 Labyrinthula sp. Type LX Watson 4.2 0 0 0 0 0 1.4 0 Labyrinthuloides yorkensis Perkins 8 . 3 0 0 0 0 0 2 . 8 0 Labyrinthuloides sp. 1 4.2 0 16.7 0 8.3 33.3 9.7 11 .1 Labyrinthulid Unidentified 0 0 4.2 0 0 0 1.4 0 fValues do not include data for the GYSA-T medium. *20X = Twenty one-minute rinses i n s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. &n = the number of alga l t i s s u e pieces plated (inocula). co Table 17 Percent occurrence of Labyrinthulid species isolated from Gelidium c o u l t e r i . Sample 3, June 1980.f Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t (2X)** rinsed tissues by algal l i f e history stage and a l l stages combined. Isolates Vegetative/Maie 20X* 2X** (n=6a) (n=6) Cystocarpic 20X 2X (n=18) (n=6) Tetrasporangial 20X 2X (n=48) (n=6) ALL STAGES 20X 2X (n=72) (n=18) Labyrinthula spp. Labyrinthula sp. Type LX Watson Labyrinthuloides sp. 1 100.0 66.7 55.6 50.0 5.6 79.2 100.0 2.1 75.0 72.2 1 .4 0 1.4 0 fValues do not include data f o r the GYSA-T medium. *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). 70 i i . Thraustochytrids and Hyalochlorella marina Isolates of Thraustochytrids and Hyalochlorella marina are l i s t e d by tissue pretreatment (20X or 2X rinsed) and i s o l a t i o n medium-antibiotic combination i n Tables 18 (Rhodoglossum affine) and 19 (Gelidium c o u l t e r i) . Growth of these organisms was also i n h i b i t e d on the GYSA-T medium. Consequently, data for t h i s medium-antibiotic combination have been excluded from further calculations. A d i s t i n c t 'strain' of Schizochytrium aggregatum was encountered i n Sample 3. Colonies of t h i s s t r a i n displayed a prominent pink/orange pigmentation on agar media, a t r a i t which prompted t h e i r further characterization. The subtle differences between i s o l a t e s of the two strains of S^ aggregatum are outlined i n t h e i r respective descriptions (Section VII). Since pigmentation i s known to be a variable character i n Thraustochytrids (Booth and M i l l e r , 1968), I cannot be certa i n that I was able to consistently separate these strains i n Sample 3; nor can I be sure that both strains were not present i n previous samples. I have l i s t e d the two strains separately to indicate t h e i r r e l a t i v e abundance. Combined, the two strains of S^ aggregatum were isol a t e d from 12.2% of the a l g a l tissues plated on agar media? the pigmented s t r a i n was isola t e d from 2.8% of the t o t a l inocula, whereas the 'normal' s t r a i n occurred 9.4% of the time. I s o l a t i o n frequencies of the S^ aggregatum strains showed s i g n i f i c a n t v a r i a b i l i t y between media-antibiotic combinations for 20X rinsed tissues of both algae (p < 0.025 for both); media containing p e n i c i l l i n / s t r e p t o m y c i n were 'preferred' (Tables 18 and 19). Table 18 Percent occurrence of Thraustochytrid species and Hyalochlorella marina 1 solated from Rhodoglossumaffine. Sample 3, June 1980. Is o l a t i o n frequencies are l i s t e d for rigorous (20X)* and l i g h t ^ X ) rinsed algal tissues by i s o l a t i o n medium-antibiotic combination and a l l media combined. Isolates SSA-T 20X* 2X** (n=24*) (n=6) SSA-PS 20X 2X (n=24) (n=6) GYSA-T 20X 2X (n=24) (n=6) GYSA-PS 20X 2X (n=24) (n=6) ALL MEDIAt 20X 2X (n=72) (n=18) Schizochytrium  aggregatum Goldstein & Bel sky S_. aggregatum Goldstein & Bel sky (pigmented) Thraustochytrium  motivum Goldstein Hyalochlorella  marina Poyton 0 0 29.2 0 0 16.7 0 8.3 16.7 0 16.7 29.2 50.0 75.0 66.7 16.7 4.2 16.7 16.7 41.7 83.3 11.1 1.4 5.6 25.0 22.2 41.7 55.6 *20X = Twenty one-minute rinses i n s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. tValues for ' a l l media' do not include data for GYSA-T. &n = the number of algal t i s s u e pieces plated (inocula). Table 19 Percent occurrence of Thraustochytrid species and Hyalochlorella marina isolated from Gelidium c o u l t e r i Sample 3, June 1980. Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t ( 2 X J * * rinsed algal tissues by i s o l a t i o n medium-antibiotic combination and a l l media combined. Isolates SSA-T 20X* 2X** (n=24fc) (n=6) SSA-PS 20X 2X (n=24) (n=6) GYSA-T 20X 2X (n=24) (n=6) GYSA-PS 20X 2X (n=24) (n=6) ALL MEDIAf 20X 2X (n=72) (n=18) Schizochytrium  aggregatum Goldstein & Bel sky S_. aggregatum Goldstein & Bel sky (pigmented) Thraustochytrium  motivum Goldstein Hyalochlorella  marina Poyton 8.3 0 0 0 54.2 50.0 29.2 0 16.7 16.7 4.2 0 58.3 33.3 37.5 66.7 0 0 0 0 8.3 8.3 33.3 16.7 12.5 33.3 11.1 5.6 4.2 48.6 33.3 26.4 33.3 *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. fValues f o r ' a l l media' do not include data for GYSA-T. •frn = the number of algal t i s s u e pieces plated (inocula). 73 These strains were is o l a t e d with equal frequency from both algae, and t h e i r numbers were not s i g n i f i c a n t l y reduced by rigorous r i n s i n g of the a l g a l tissues (last two columns, Tables 18 and 19). Thraustochytrium motivum was the most common Thraustochytrid isolated, having an o v e r a l l i s o l a t i o n frequency of 35.0%. There were no s i g n i f i c a n t differences between the i s o l a t i o n frequencies of T\_ motivum from the three i s o l a t i o n media-antibiotic combinations, although frequencies for GYSA-PS were comparatively low (Tables 18 and 19). For each alga, i s o l a t i o n frequencies were s i m i l a r for 20X and 2X rinsed tissues (last two columns, Tables 18 and 19). T\_ motivum was i s o l a t e d more often from 20X rinsed tissues of Gelidium c o u l t e r i (48.6%) than from s i m i l a r l y rinsed tissues of Rhodoglossum a f f i n e (25.0%) (p < 0.005). Total i s o l a t i o n frequencies from 2X rinsed tissues were s i m i l a r between algae. Hyalochlorella marina was the most common 'protist' i s o l a t e d from a l g a l tissue-agar plates i n Sample 3 (36.1% overall). I s o l a t i o n frequencies of H^ marina were s i g n i f i c a n t l y affected by medium-antibiotic combination for a l l alga/tissue pretreatment regimes except 20X rinsed G^ c o u l t e r i (p < 0.025 for 2X rinsed G. c o u l t e r i ; p < 0.05 f o r 2X and p < 0.005 f o r 20X r i n s e d R. aff i n e ) . Generally, the organism grew best on media containing penicillin/streptomycin, e s p e c i a l l y SSA-PS (Tables 18 and 19). The recovery of H^ marina was not s i g n i f i c a n t l y reduced by rigorous r i n s i n g of the tissues of either alga. I s o l a t i o n frequencies for H^ marina tended to be higher from tissues of 74 Rhodoglossum a f f i n e than Gelidium c o u l t e r i , but the values were not s i g n i f i c a n t l y d i f f e r e n t (at 95% confidence l i m i t s ) (last two columns, Tables 18 and 19). The percent occurrence of Thraustochytrids and Hyalochlorella marina i s l i s t e d by l i f e h i s t o r y stage of each alga i n Tables 20 (Rhodoglossum affine) and 21 (Gelidium  c o u l t e r i ) . The i s o l a t i o n frequency of the Schizochytrium  aggregatum strains was high for vegetative/male tissues of 20X r i n s e d R. a f f i n e (p < 0.005, Table 20). S. aggregatum was i s o l a t e d with equal frequency from the d i f f e r e n t l i f e h istory stages i n a l l other alga/tissue pretreatment regimes. Thraustochytrium motivum showed disproportional i s o l a t i o n from 20X rinsed tissues of Rj_ a f f i n e (p < 0.05; tetrasporangial low) and 2X rinsed tissues of G^ c o u l t e r i (p < 0.05; cystocarpic low) (Tables 20 and 21). Hyalochlorella marina was i s o l a t e d with equal frequency from the d i f f e r e n t l i f e h i s t o r y stages of each alga, regardless of tissue pretreatment. i i i . Higher Fungi The yeasts and mycelial fungi i s o l a t e d from the a l g a l tissue-agar plates of Sample 3 are l i s t e d i n Table 22. I s o l a t i o n frequencies are given for each rinse treatment of both algae and a l l a l g a l / r i n s e regimes combined. Table 23 shows differences i n the TOTAL i s o l a t i o n frequencies of yeasts and mycelial fungi from the d i f f e r e n t l i f e h i s t o r y stages of each alga and the d i f f e r e n t i s o l a t i o n media-antibiotic combinations. Actinomycetes were not i s o l a t e d from any a l g a l tissue-agar plates i n Sample 3. Table 20 Percent occurrence of Thraustochytrid species and Hyalochlorella marina isolated from Rhodoglossum a f f i n e . Sample 3, June 1980.t Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t (2X)** rinsed t i s s u e s by algal l i f e history stage and a l l stages combined. Vegetative/Male Cystocarpic Tetrasporangial ALL STAGES Isolates 20X* 2X** 20X 2X 20X 2X 20X 2X (n=24«) (n=6) (n=24) (n=6) (n=24) (n=6) (n=72) (n=18) Schizochytrium  aggregatum Goldstein & Bel sky 29.2 0 0 0 4.2 0 11.1 0 S_. aggregatum Goldstein & Bel sky (pigmented) 4.2 16.7 0 0 0 0 1.4 5.6 Thraustochytrium motivum Goldstein 37.5 50.0 29.2 0 8.3 16.7 25.0 22.2 Hyalochlorella marina Poyton 41.7 50.0 37.5 66.7 45.8 50.0 41.7 55.6 fValues do not include data f o r the GYSA-T medium. *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). Table 21 Percent occurrence of Thraustochytrid species and Hyalochlorella marina iso l a t e d from Gelidium c o u l t e r i . Sample 3, June 1980.f Isolation frequencies are l i s t e d for rigorous (20X)* and l i g h t (2X7** rinsed tissues by algal l i f e history stage and a l l stages combined. Vegetative/Male Cystocarpic Tetrasporangial ALL STAGES Isolates 20X* 2X** 20X 2X 20X 2X 20X 2X (n=6*) (n=6) (n=18) (n=6) (n=48) (n=6) (n=72) (n=18) Schizochytrium  aggregatum Goldstein & Belsky 33.3 0 11.1 16.7 8.3 0 11.1 5.6 S_. aggregatum Goldstein & Belsky (pigmented) 0 0 0 0 6.3 0 4.2 0 Thraustochytrium motivum Goldstein 50.0 50.0 22.2 16.7 58.3 33.3 48.0 33.3 Hyalochlorella marina Poyton 33.3 33.3 33.3 50.0 22.9 16.7 26.4 33.3 fValues do not include data for the GYSA-T medium. *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. #n = the number of algal t i s s u e pieces plated (inocula). 77 Yeasts were isol a t e d infrequently, exhibiting an o v e r a l l i s o l a t i o n frequency of 1.7% (Table 22). No differences were seen i n the recovery of yeasts between 20X and 2X r i n s e d t i s s u e s or algae. The i s o l a t i o n of yeasts was r e s t r i c t e d to the GYSA medium, but was not d i f f e r e n t i a l l y affected by the a n t i b i o t i c s t e t r a c y c l i n e HCl or penicillin/streptomycin (Table 23). Thirteen d i f f e r e n t mycelial fungi were is o l a t e d from al g a l tissue-agar plates of Sample 3 algae, including: 10 (76.9%) Hyphomycetes, 1 (7.7%) Coelomycete and 2 (15.4%) s t e r i l e mycelia (Table 22). As for the previous samples, nearly a l l of these fungi displayed low o v e r a l l frequencies of i s o l a t i o n . The one notable exception was Acremonium sp. 019-78 which was i s o l a t e d from 12.9% of the t o t a l number of inocula. Dendryphiella s a l i n a (Suth.) Pugh et Nicot (2.9% overall) and Sigmoidea l i t t o r a l i s sp. nov. (2.5%) were the next two most common fungi. A l l other mycelial fungi were isol a t e d from less than 1.0% of the t o t a l number of inocula (Table 22). The t o t a l i s o l a t i o n frequencies for mycelial fungi (Table 22, bottom) suggest that fewer fungi were is o l a t e d from tissues of Rhodoglossum a f f i n e than from tissues of Gelidium c o u l t e r i . Comparisons between algae for each rinse pretreatment show that for 20X rinsed tissues t o t a l i s o l a t i o n frequencies were s i g n i f i c a n t l y d i f f e r e n t between algae (p < 0.005), but for 2X rinsed tissues they were not (0.1 < p > 0.05). Acremonium sp. 019-78 contributed markedly to the higher o v e r a l l i s o l a t i o n frequencies obtained from tissues of G^ c o u l t e r i . Analyzed separately, Acremonium sp. 019-78 was i s o l a t e d more frequently from tissues of Gj_ c o u l t e r i regardless of rinse treatment Table 22 Percent occurrence of yeasts and mycelial fungi i s o l a t e d from Rhodoglossum a f f ^ g , and Gelidium c o u l t e r i . Sample 3, June 1980.t Is o l a t i o n frequencies are l i s t e d f o r ^ ^ o r l ^ f F - b y a l g a / t i s ue pretreatment regime and a l l regimes combined. R. a f f i n e G. c o u l t e r i Isolates Yeasts and y e a s t - l i k e fungi Mycelial Fungi Acremonium sp. 019-78 Acremonium sp. 020-80 Acremonium sp. 035-80 As p e r g i l l u s v e r s i c o l o r ( V u i l l . ) Tiraboschi Beauveria bassiana (Bals.) V u i l l . Dendryphiella s a l i n a (Suth.) Pugh et Nicot Phialophora verrucosa Medlar Phoma sp. 005-80 Sigmoidea marina Haythorn & Jones Sigmoidea l i t t o r a l i s sp. nov. Unidentified hyphomycete 044-78 S t e r i l e mycelium 055-80 S t e r i l e mycelium 066-80 20X* (n=96*) 2 x * * (n=24) 20X (n=96) 2X (n=24) Combined (n=240) TOTAL MYCELIAL FUNGI 0 4.2 3.1 0 1.7 3.1 0 24.0 20.8 12.9 0 0 1.0 0 0.4 0 0 1.0 0 0.4 0 0 1.0 0 0.4 1.0 0 0 0 0.4 1.0 0 6.3 0 2.9 0 4.2 1.0 0 0.8 0 0 1.0 0 0.4 0 0 1.0 0 0.4 1.0 0 4.2 4.2 2.5 1.0 0 0 4.2 0.8 2.1 0 0 0 0.8 0 4.2 0 0 0.4 9.4 8.3 40.6 29.2 23.8 fData f o r a l l l i f e h i s t o r y stages of the algae and a l l i s o l a t i o n media combined. *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses i n s t e r i l e SW. •frn = the number of alga l t i s s u e pieces plated (inocula). 00 Table 23 Total i s o l a t i o n frequency {%) of yeasts and mycelial fungi f o r each alga/tissue pretreatment regime l i s t e d by: 1) l i f e history stage of the algae* and 2) i s o l a t i o n medium-antibiotic combination.** Sample 3, June 1980. Alga Rinse Treatments Isolates Alga L i f e History Stage* Veg./o" 9 ® Isolation Med i urn-Ant i b i ot i c** SSA-T SSA-PS GYSA-T GYSA-PS Rhodoglossum a f f i n e Rhodoglossum a f f i n e Gelidium c o u l t e r i Gelidium c o u l t e r i Rigorous (20X) Light (2X) Rigorous (20X) Light (2X) n=32fc n=24& Yeasts 0 0 0 0 0 0 0 Myeelial Fungi 3.1 15.6 9.4 4.2 12.5 8.3 12.5 n=8 n=6 Yeasts 0 12.5 0 0 0 16.7 0 Mycelial Fungi 12.5 0 12.5 0 0 16.7 16.7 n=8 n=24 n=64 n=24 Yeasts 0 4.2 3.1 0 0 8.3 4.2 Mycelial Fungi 0 79.2 31.3 20.8 41.7 45.8 54.2 n=8 n=6 Yeasts 0 0 0 0 0 0 0 Mycelial Fungi 37.5 50.0 0 50.0 0 16.7 50.0 *A11 four i s o l a t i o n media-antibiotic mixtures combined. **A11 three algal l i f e history stages combined. <m = the number of algal t i s s u e pieces plated (inocula). 80 (p < 0.005, 20X ri n s e d ; p < 0.01, 2X ri n s e d ) . Analyses of ' a l l other mycelial fungi' combined, except Acremonium sp. 019-78, gave results s i m i l a r to those obtained for 'total mycelial fungi' [i.e. more common from 20X rinsed tissues of G^ c o u l t e r i than from 20X rinsed tissues of a f f i n e (p < 0.025); i s o l a t i o n frequencies from 2X rinsed tissues were s i m i l a r between algae]. Thus, Acremonium sp. 019-78 was not so l e l y responsible for the enhanced i s o l a t i o n frequency of mycelial fungi from 20X rinsed tissues of G^_ c o u l t e r i . For each alga, t o t a l i s o l a t i o n frequencies of mycelial fungi were s i m i l a r for 20X and 2X rinsed tissues (Table 22). These results show that 20 s t e r i l e seawater rinses did not e f f e c t i v e l y reduce the number of fungi i s o l a t e d from the a l g a l tissues. Total i s o l a t i o n frequencies of mycelial fungi were s i m i l a r for a l l four of the i s o l a t i o n media-antibiotic combinations u t i l i z e d (Table 23). When analyzed separately, the i s o l a t i o n frequencies of Acremonium sp. 019-78 and ' a l l other mycelial fungi' from Gelidium c o u l t e r i were also s i m i l a r for a l l four media-antibiotic combinations. Mycelial fungi were i s o l a t e d with s i m i l a r frequency from the di f f e r e n t l i f e h i s t o r y stages of Rhodoglossum a f f i n e (Table 23). However, disproportional i s o l a t i o n frequencies were obtained from the d i f f e r e n t l i f e h i story stages of Gelidium c o u l t e r i (p < 0.005, 20X ri n s e d ; p < 0.005, 2X ri n s e d ) . A higher frequency of i s o l a t i o n was obtained from the cystocarpic l i f e h i s t o r y stage of t h i s alga. Separate analyses of Acremonium sp. 019-78 and the t o t a l s for ' a l l other mycelial fungi' reveal that 81 the i s o l a t i o n frequencies for Acremonium sp. 019-78 were largely responsible for these differences (p < 0.005, 20X rinsed; p < 0.025, 2X rinsed). ' A l l other mycelial fungi' were isolated with s i m i l a r frequency from the d i f f e r e n t l i f e h i s t o r y stages of Gelidium c o u l t e r i (0.1 < p > 0.05). b. Al g a l Tissue Rinse Water Plates i . Propagules per M i l l i l i t e r Rinse Water The organisms i s o l a t e d from a l g a l tissue rinse water which was spread onto GYSA-PS plates are l i s t e d i n Table 24. The values given are the mean (X; + standard error) number of colonies per culture plate (which estimates propagules per ml rinse water). Values are l i s t e d for each organism i s o l a t e d by alga and seawater rinse (3rd and 15th). The s t a t i s t i c a l analyses of these data d i f f e r from those previously described. Methods were employed with the aim of comparing the means for i n d i v i d u a l i s o l a t e s between rinses (3rd and 15th) and algae. No s t a t i s t i c a l comparisons were made between the d i f f e r e n t organisms isolated. None of the raw data sets f o r each organism had equal variances ( F m a x Test; Solcal and Rohlf, 1969). Two commonly used data transformations Clog (Y + 1); /Y + 0.5 ] lessened, but did not a l l e v i a t e t h i s problem. As a result, the following tests were employed for each i s o l a t e using the raw data: 1) means for the 3rd rinse were compared between algae; 2) means for the 15th rinse were compared between algae; and 3-4) means for the 3rd and 15th rinses were compared within each alga. Prio r to these analyses, an F-test was performed on each pair of sample 82 variances. I f the variances were equal, a T-test was carried out; i f the variances were unequal, a "Test of equality of the means of two samples whose variances are assumed to be unequal." was performed (Sokal and Rohlf, 1969). Since i t i s claimed that T-tests are not additive, and should not be used when multiple tests are required, the results of these analyses could be c r i t i c i z e d (Sokal and Rohlf, 1969). Under these circumstances (i.e. these results), however, I do not believe that there w i l l be any question i n interpretation. The l i s t of p r o t i s t s i s o l a t e d (i.e. Labyrinthulids, Thraustochytrids and Hyalochlorella marina) from rinse water plates (Table 24) i s i d e n t i c a l to that obtained from a l g a l tissue-agar plates (see Tables 14-15 and 18-19). Comparisons of the values obtained for each rinse (3rd or 15th) between algae showed that most of the organisms were iso l a t e d with s i m i l a r frequency from the rinse waters of both algae. The Thraustochytrids were an exception. Schizochytrium  aggregatum ('normal' and pigmented combined) and Thraustochytrium  motivum produced larger numbers of colonies on plates prepared from the 3rd rinse water of Rhodoglossum a f f i n e than on s i m i l a r plates for Gelidium c o u l t e r i (p < 0.01, T\_ motivum; p < 0.001, S. aggregatum). The number of colonies produced on plates prepared from the 15th rinse water were s t a t i s t i c a l l y s i m i l a r between algae for both of these organisms (note high v a r i a b i l i t y for Tj_ motivum). Myc e l i a l fungi were more common on plates prepared from the 3rd rinse water of G^ c o u l t e r i than on s i m i l a r plates for R.  a f f i n e (p < 0.05). The r e l a t i v e l y high number of mycelial fungi Table 24 Organisms i s o l a t e d from the 3rd and 15th rinse waters of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Values given are the mean and standard error of colonies per culture plate. Sample 3, June 1980. R. af f i n e G. c o u l t e r i Isolates 3rd Rinse (n=36*r) X S.E. 15th X Rinse '12) S.E. 3rd Rinse (n=36) X S.E. 15th [n= X Rinse =12) S.E. Labyrinthula spp. 13.08 1.88 4.00 1.07 8.81 1.91 2.17 0.69 Labyrinthuloides yorkensis Perkins 0.30 0.12 0 0 0.14 0.06 0 0 Labyrinthuloides sp. 1 0.50 0.16 0.17 0.11 0.64 0.13 0.25 0.13 Labyrinthulid Unidentified 1.19 0.26 0.58 0.26 0.97 0.25 0.50 0.19 Schizochytrium aggregatum Goldstein & Belsky 0.28 0.08 0.17 0.11 0.11 0.07 0.08 0.08 S. aggregatum Goldstein & Belsky (pigmented) 0.25 0.08 0 0 0 0 0 0 Thraustochytrium motivum Goldstein 246.61 54.05 13.58 7.56 29.64 8.68 1.17 0.67 Hyalochlorella marina Poyton 6.61 1.06 1.33 0.73 4.67 0.94 0.83 0.47 Yeasts 0.03 0.03 0 0 0.06 0.04 0 0 Mycelial Fungi 0.08 0.05 0.50* 0.50 0.28 0.08 0.08 0.08 Diatoms 9.86 2.88 0.50 0.19 7.72 1.48 1.42 0.47 •frn = the number of ' r e p l i c a t e ' culture plates inoculated and counted. *?-suspect a i r contaminants. 84 recovered from the 15th rinse water of Rhodoglossum a f f i n e was due to the presence of six colonies on a single plate (five of which were Cladosporium sphaerospermum Penz.)- I t i s possible that these were a i r contaminants. In any case, the v a r i a b i l i t y for t h i s sample was so high that i t was not s i g n i f i c a n t l y d i f f e r e n t from the 1 5 th r i n s e water of c o u l t e r i or from the 3rd rinse water of Rj_ affine. Comparisons between 3rd and 15th rinses for each alga allowed evaluation of the effectiveness of the r i n s i n g technique i n removing each organism from the a l g a l surfaces. A l l isol a t e s displayed some reduction i n the mean number of colonies produced between waters of the 3rd and 15th rinses. However, only Labyrinthuloides yorkensis and yeasts showed signs of being eliminated from the a l g a l tissues by 15 seawater rinses. These organisms were also is o l a t e d very r a r e l y from waters of the 3rd rinse. Only those organisms which were abundant i n waters of the 3rd rinse (X > 4.00 colonies/plate) showed a s t a t i s t i c a l l y s i g n i f i c a n t reduction i n numbers by the 15th rinse. These included: Labyrinthula spp., Thraustochytrium motivum, Hyalochlorella marina and diatoms. A l l of these organisms were s i g n i f i c a n t l y reduced i n number between the 3rd and 15th rinse waters of both algae (p < 0.01). Excluding diatoms, these were also the most common p r o t i s t s i s o l a t e d from a l g a l tissue-agar plates i n Sample 3. S t a t i s t i c a l comparisons cannot be made between the data compiled from rinse water plates and that obtained from a l g a l 85 tissue-agar plates. An 'absolute percent occurrence' could be calculated for each organism observed on rinse water plates (based on the absolute presence or absence of an organism from each rinse water plate). However, the end re s u l t would not be comparable to a l g a l tissue-agar plates since considerably more a l g a l tissue was present i n the seawater rinse t e s t tubes than was ac t u a l l y plated out on agar media (see Materials and Methods). i i . Higher Fungi The yeasts and mycelial fungi i s o l a t e d from rinse water plates are l i s t e d by alga and rinse i n Table 25. The i s o l a t i o n data given are percentages calculated as follows: the number of in d i v i d u a l colonies of a fungus was divided by the number of spread plates for that alga/rinse regime (n); the r e s u l t was mul t i p l i e d by 100. The majority of the fungi l i s t e d i n Table 25 were isolated only once. The high i s o l a t i o n frequency shown for Cladosporium  sphaerospermum from the 15th rinse water of Rhodoglossum a f f i n e was due to the presence of f i v e c o l o n i e s of t h i s fungus on a single plate. The source of these spores i s suspect, and I f e e l that the t o t a l i s o l a t i o n frequency of mycelial fungi for t h i s alga/rinse regime i s high due to t h e i r presence. The data obtained for Gelidium c o u l t e r i suggest a decrease i n the number of fungi i s o l a t e d between the 3rd and 15th rinse. S t a t i s t i c a l l y , however, these i s o l a t i o n frequencies are s i m i l a r (Table 25, bottom). More mycelial fungi were i s o l a t e d from the 3rd rinse Table 25 Percent occurrence of yeasts and mycelial fungi i s o l a t e d from the 3rd and 15th rinse waters of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 3, June 1980. R. a f f i n e G. c o u l t e r i Isolates 3rd Rinse 15th Rinse 3rd Rinse 15th Rinse (n=36*r) (n=12) (n=36) (n=12) Yeasts and y e a s t - l i k e fungi Hormonema sp. 2.8 0 0 0 Unidentified Yeasts 0 0 5.6 0 TOTAL YEASTS 2.8 0 5.6 0 Mycelial Fungi Acremonium sp. 019-78 0 0 5.6 0 Cladosporium herbarum Link ex Fries 2.8 0 0 0 Cladosporium sphaerospermum Penz. 0 41.7* 0 0 Cladosporium sp. 088-80 0 8.3 0 0 Mariannaea elegans var. elegans (Corda) Samson 0 0 2.8 0 P e n i c i l l i u m chrysogenum Thorn 0 0 2.8 0 P e n i c i l l i u m vinaceum Gilman & Abbott 2.8 0 0 0 Sigmoidea l i t t o r a l i s sp. nov. 0 0 0 8.3 Trichoderma v i r i d e Pers. 0 0 2.8 0 Unidentified hyphomycete 044-78 0 0 5.6 0 Unidentified coelomycete 080-80 0 0 2.8 0 Unidentified coelomycete 089-80 2.8 0 0 0 Unidentified mycelium (not isolated) 0 0 5.6 0 TOTAL MYCELIAL FUNGI 8.3 50.0 27.8 8.3 -&n = the number of culture plates inoculated. *5 colonies on 1 plate. 87 water of Gelidium c o u l t e r i than from the 3rd rinse water of Rhodoglossum a f f i n e (p < 0.05). Comparison of these results with those from a l g a l t i s s u e -agar plates shows that most of these fungi were not is o l a t e d again when the a l g a l tissues were plated on agar media. The fungi present here which were also present on a l g a l tissue-agar plates include: yeasts, Acremonium sp. 019-78, Sigmoidea  l i t t o r a l i s sp. nov. and Unidentified hyphomycete 044-78. Acremonium sp. 019-78 was isola t e d from 24.0% of the a l g a l tissue-agar plates inoculated with 20X rinsed tissues of G.  c o u l t e r i (see Table 22). I f Acremonium sp. 019-78 was present only as spores on the a l g a l surface, one would anticipate recovering more than two colonies out of the 36 plates (5.6%) prepared from the 3rd rinse water of t h i s alga. Dendryphiella  s a l i n a was not is o l a t e d from rinse water culture plates. c S t e r i l e Seawater/Pine Pollen Cultures The results of observations and i s o l a t i o n s from seawater/pollen (SW/P) cultures are presented i n Tables 26 (Protists) and 27 (Fungi). i . Labyrinthulids, Thraustochytrids and Hyalochlorella marina The l i s t of p r o t i s t s i s o l a t e d from SW/P cultures i s very s i m i l a r that obtained from the a l g a l tissue-agar plate and seawater rinse plate techniques (Table 26). A second unide n t i f i e d species of the genus Labyrinthuloides (RV02-80), which was brought into axenic culture and characterized i n some d e t a i l , i s l i s t e d separately. This organism may be s i m i l a r to other i s o l a t e s which have been placed i n the 'Labyrinthulid Table 26 Percent occurrence of Labyrinthulids, Thraustochytrids and Hyalochlorella marina isolated from s t e r i l e seawater/pine pollen cultures of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Sample 3, June 1980. Isolation frequencies are l i s t e d f o r each organism by alga/tissue pretreatment regime.f JR. a f f i n e G. co u l t e r i Isolates 20X* (n=&v) 2X** (n=6) 20X (n=6) 2X (n=6) Labyrinthula spp. 83.3 66.7 50.0 50.0 Labyrinthuloides yorkensis Perkins 16.7 0 33.3 0 Labyrinthuloides sp. 1 83.3 33.3 66.7 66.7 Labyrinthuloides sp. RV02-80 16.7 0 0 0 Labyrinthulid Unidentified 0 0 16.7 0 Schizochytrium aggregatum Goldstein & Belsky 66.7 66.7 33.3 83.3 S. aggregatum Goldstein & Belsky (pigmented) 33.3 16.7 66.7 16.7 Thraustochytrium aggregatum Ulken 0 16.7 0 0 Thraustochytrium motivum Goldstein 66.7 50.0 100.0 66.7 Thraustochytrid Unidentified 16.7 16.7 0 16.7 Hyalochlorella marina Poyton 16.7 33.3 0 0 tFrequencies based on the presence of absence *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. #n = the number of SW/P cultures inoculated. of each i s o l a t e in each culture (n). CO CO 89 Unidentified' category [see Descriptions (Section VII)]. A single i s o l a t e of Thraustochytrium aggregatum Ulken, a Thraustochytrid which had not been encountered previously, was obtained from 2X rinsed tissues of Rhodoglossum affi n e . The i s o l a t i o n frequencies l i s t e d i n Table 26 are not considered quantitative since: 1) the sample sizes were r e l a t i v e l y small (n = 6 per alga/rinse regime); and 2) techniques were incorporated which could not be e a s i l y standardized (i.e. streaking of loops of the cultures onto KMV-agar). Very often the presence of an organism could not be confirmed u n t i l i t ' s colony development was observed on a KMV-agar plate. This was e s p e c i a l l y true for i s o l a t e s of the genus Labyrinthuloides. S i m i l a r d i f f i c u l t i e s were encountered with Thraustochytrids (probably Thraustochytrium motivum); i s o l a t e s were observed but could not be i d e n t i f i e d i n s l i d e s prepared from the SW/P cultures, and they did not develop colonies on KMV-agar plates. The 'Thraustochytrid Unidentified' category was established to accommodate these observations. Taking these shortcomings into consideration, the i s o l a t i o n frequencies of each organism appear to be s i m i l a r for both algae and both tissue pretreatments (20X and 2X rinsed) (Table 26). These results correspond to those of the p l a t i n g techniques previously described, indicating that 20 seawater rinses were i n s u f f i c i e n t to 'clean' the a l g a l tissues of these organisms. Compared to a l g a l tissue-agar plates and seawater rinse plates, a s l i g h t l y larger number of d i f f e r e n t p r o t i s t species were i s o l a t e d from SW/P cultures (higher d i v e r s i t y ) . Most 90 organisms, except Labyrinthula spp. and Hyalochlorella marina, displayed a higher frequency of i s o l a t i o n from SW/P cultures than from a l g a l tissue-agar plates (compare Table 26 to Tables 14-15 and 18-19). The recovery of Labyrinthuloides sp. 1 and Schizochytrium aggregatum (normal or pigmented) was higher from SW/P cultures than from both of the previously described i s o l a t i o n methods. i i . Higher Fungi Mycelial fungi were quite common i n SW/P cultures, displaying an o v e r a l l frequency of occurrence (58.3%) which was higher than that obtained from a l g a l tissue-agar plates (23.8%) (Table 27). Yeasts continued to be i s o l a t e d i n l i m i t e d numbers (8.3% o v e r a l l ) . For each alga, there were no differences i n the t o t a l i s o l a t i o n frequencies of mycelial fungi between 2X and 20X rinsed tissues (Table 27, bottom). Generally, more fungal i s o l a t e s were obtained from tissues of Gelidium c o u l t e r i than from tissues of Rhodoglossum a f f i n e [ s i g n i f i c a n t for 2X rinsed tissues (p < 0.005)]. Acremonium sp. 019-78 was the most common fungus isolated from SW/P cultures (29.2% overall). With the exception of Dendryphiella salina, a l l other fungi were is o l a t e d once only. A l l of the fungi l i s t e d , except Geotrichum sp., were obtained by at l e a s t one of the other i s o l a t i o n methods employed i n Sample 3. Acremonium sp. 019-78, Sigmoidea l i t t o r a l i s and Unidentified hyphomycete 044-78 were also i s o l a t e d from a l g a l tissue-agar plates and seawater rinse plates. Table 27 Percent occurrence of yeasts and mycelial fungi isolated from s t e r i l e seawater/pine pollen cultures of Rhodologssum a f f i n e and Gelidium c o u l t e r i . Sample 3, June 1980. Isol a t i o n frequencies are l i s t e d f o r each organism by alga/tissue pretreatment regime.f R_. a f f i n e G_. c o u l t e r i Isolates 20X* 2X** 20X 2X (n=6<r) (n=6) (n=6) (n=6) Yeasts & y e a s t - l i k e fungi 0 0 33.3 0 Mycelial Fungi Acremonium sp. 019-78 16.7 16.7 33.3 50.0 Cladosporium sphaerospermum Penz. 16.7 0 0 0 Dendryphiella s a l i n a (Suth.) Pugh et Nicot 0 0 0 33.3 Geotrichum sp. 0 0 0. 16.7 Phialophora verrucosa Medlar 0 0 16.7 0 Sigmoidea l i t t o r a l i s sp. nov. 16.7 0 0 0 Unidentified hyphomycete 044-78 0 0 16.7 0 TOTAL MYCELIAL FUNGI 50.0 16.7 66.7 100.0 fFrequencies based on the number of fungal isolates and the number of cultures (n). *20X = Twenty one-minute rinses in s t e r i l e SW. **2X = Two one-minute rinses in s t e r i l e SW. &n = the number of SW/P cultures inoculated. 92 4. Moist Chambers The fungi which developed on a l g a l tissues incubated i n moist chambers are l i s t e d for a l l f i e l d samples (1, 2, and 3) i n Table 28. The t o t a l number of moist chambers observed (n) for each sample was normally divided equally among the three l i f e h i s t o r y stages of each alga (vegetative/male, cystocarpic, tetrasporangial). These data have been combined since the sample sizes were very small, and there were no obvious differences between a l g a l l i f e h istory stages. Recall that the a l g a l tissues were surface s t e r i l i z e d (Samples 1 and 2) or rigorously (20X) rinsed (Sample 3) before being placed i n the moist chambers (see Materials and Methods). The most common mycelial fungi recovered from a l g a l tissues incubated i n moist chambers were: Acremonium sp. 019-78, Cladosporium cladosporioides and P e n i c i l l i u m spp. (Table 28). Six of the mycelial fungi l i s t e d were not encountered using other i s o l a t i o n methods; perhaps the most notable was Phialophorophoma  l i t t o r a l i s Linder. A l l six of these fungi were i s o l a t e d only once. Actinomycetes and yeasts also developed on a l g a l tissues incubated i n moist chambers, but they were of infrequent occurrence. The t o t a l number of i s o l a t e s obtained per moist chamber (Table 28, bottom) may seem high compared to the i s o l a t i o n frequencies obtained for a l g a l tissue-agar plates, but each moist chamber contained considerably more a l g a l tissue than was plated on each agar plate. For Rhodoglossum affine, the number of i s o l a t e s obtained per moist chamber for each sample follow a trend s i m i l a r to that Table 28 Actinomycetes, yeasts and mycelial fungi developing on s u r f a c e - s t e r i l i z e d and/or r i g o r o u s l y - r i n s e d * tissues of Rhodoglossum a f f i n e and Gelidium c o u l t e r i incubated in moist chambers. Data l i s t e d are the actual number of i s o l a t e s . Results f o r Samples 1, 2, and 3. Sample 1 Sample 2 Sample 3 Isolates R. a f f i n e R. a f f i n e G. c o u l t e r i R. a f f i n e G. c o u l t e r i (n=3*r) (H=6] T P 4 T (n=3) WT) Actinomycetes 0 0 0 0 1 Yeasts and y e a s t - l i k e fungi 0 1 0 0 0 Mycelial Fungi Acremonium sp. 019-78 0 3 1 0 2 Acremonium sp. 029-78 1 0 0 0 0 Acremonium sp. 071-79 0 0 1 0 0 Aspergillus v e r s i c o l o r ( V u i l l . ) Tiraboschi 0 0 0 1 0 Cladosporium cladosporioides (Pers.) Link ex S.F. Gray 0 4 0 0 0 Cladosporium sphaerospermum Penz. 2 0 0 0 0 Dendryphiella s a l i n a (Suth.) Pugh et Nicot 0 2 0 0 0 Geomyces pannorum (Link) S i g l e r & Carmichael var. pannorum 0 1 0 0 0 P e n i c i l l i u m chrysogenum Thorn 0 0 0 1 0 P e n i c i l l i u m spp. 1 8 0 0 0 Phialophora verrucosa Medlar 0 1 0 0 0 Phialophorophoma l i t t o r a l i s Linder 0 0 0 1 0 Sigmoidea l i t t o r a l i s sp. nov. 0 0 0 0 1 V e r t i c i l 1ium l a t e r i t i u m Berkeley 0 1 0 0 0 Unidentified hyphomycete 044-78 0 0 0 1 0 Unidentified coelomycete 063-79 0 2 0 0 0 Unidentified coelomycete 179-79 0 1 0 0 0 S t e r i l e mycelium 130-80 0 0 0 1 0 Unidentified mycelium (not isolated) 0 1 1 0 0 TOTAL ISOLATES 4 25 3 5 4 Isolates/Moisture Chamber 1.33 4.17 0.75 1.67 1.33 *Sample 1 - 1:10,000 HgCl 2 in 5% Ethanol, 1 minute; followed by two one-minute rinses in s t e r i l e SW. Sample 2 - 1:10,000 HgCl 2 in 5% Ethanol, 30 seconds; followed by two one-minute rinses i n s t e r i l e SW. Sample 3 - Twenty one-minute rinses in s t e r i l e SW. •&n = the number of moist chambers observed; each of which contained 2 ( r a r e l y 3) pieces of alga l t h a l l u s (approximately 1 to 2 grams wet weight). 94 shown by a l g a l tissue-agar plates. The fungi were: 1) uncommon on s t e r i l i z e d tissues i n Sample 1; 2) numerous on s t e r i l i z e d tissues i n Sample 2; and 3) r e l a t i v e l y uncommon from 20X rinsed tissues i n Sample 3. For Gelidium c o u l t e r i , however, the r e l a t i v e number of isol a t e s obtained per moist chamber was consistently low for both Samples 2 and 3. Incubation i n moist chambers i s generally not a sat i s f a c t o r y method for i s o l a t i n g fungi from tissues of Gelidium c o u l t e r i . A small piece (0.5-1.0 g wet weight) of G^ c o u l t e r i which has been s t e r i l i z e d and/or rinsed and blotted resembles a soaked, oversized a r t i s t ' s paint brush. The fine branches of the thallus coalesce and adhere together, entrapping excess moisture i n the i n t e r s t i t i a l spaces. As a result, a very small amount of the thallus surface area i s exposed to a i r c i r c u l a t i o n within the chamber. No a n t i b i o t i c s were used to i n h i b i t b a c t e r i a l growth in the moist chambers, and the development of bacteria was evident i n many of the Gelidium c o u l t e r i cultures. In certain cultures the tissues a c t u a l l y appeared to 'melt' and became coated with a thi n b a c t e r i a l slime. Under these circumstances i t i s possible that the i n t e r s t i t i a l spaces became anoxic. Thus, I attribute at least some of the inconsistency i n re s u l t s for these two algae to differences i n the micro-environment surrounding the tissues i n the moist chambers. 95 DISCUSSION A. LABORATORY METHODS 1. Media, Antibotics and Culture Conditions The r e s u l t s from Sample 3 demonstrate that, under c e r t a i n conditions, the a n t i b i o t i c t e t r a c y c l i n e HCL may become in h i b i t o r y to the growth of Labyrinthulids, Thraustochytrids and Hyalochlorella marina. My observations of t h i s phenomenon included the following. Agar media (e.g. GYSA, SSA) containing t e t r a c y c l i n e HCL (200 mg/l) slowly turned reddish/purple upon exposure to l i g h t (natural or a r t i f i c i a l ) . This color change was apparently i n d i c a t i v e of breakdown or a l t e r a t i o n of the t e t r a c y c l i n e HCL molecule. 'Red' agar plates l o s t t h e i r capacity to i n h i b i t the growth of bacteria associated with a l g a l tissue inocula. Concurrent with t h i s color change, the GYSA-T medium also became i n h i b i t o r y to the growth of Labyrinthulids, Thraustochytrids and Hyalochlorella marina. Serum-Seawater Agar (SSA) containing t e t r a c y c l i n e HCL also became red upon exposure to l i g h t and l o s t i t s b a c t e r i a l i n h i b i t o r y properties. Under these circumstances, however, SSA-T continued to support at least l i m i t e d growth of Labyrinthula spp., Labyrinthuloides sp. 1, Schizochytrium aggregatum, Thraustochytrium motivum and Hyalochlorella marina. B r i e f experiments were performed using Kazama's Modified Vishniac's-'slush' containing p e n i c i l l i n / s t r e p t o m y c i n (500 mg/l each) or t e t r a c y c l i n e HCL (200 mg/l). Two series of t e t r a c y c l i n e HCL-containing media were prepared, one which was kept i n the 96 dark and one which was exposed to l i g h t p r i o r to inoculation (exposed plates turned red within 36 hours). A l l media/antibiotic combinations were inoculated with axenic cultures of Schizochytrium aggregatum, Thraustochytrium motivum and Hyalochlorella marina. The cultures were incubated in the dark at room temperature. Growth of a l l three test organisms was i n h i b i t e d i n the 'red' t e t r a c y c l i n e HCL-containing medium which had been exposed to l i g h t . A l l the p r o t i s t s grew 'normally' i n the penicillin/streptomycin-containing medium, and S. aggregatum and T. motivum also grew well in the unexposed t e t r a c y c l i n e HCL medium. However, growth of H^ marina was apparently i n h i b i t e d i n the unexposed t e t r a c y c l i n e HCL-containing medium. Combined, these results indicate that the t e t r a c y c l i n e HCL molecule i s altered when exposed to l i g h t , and that t h i s reaction can produce toxic or growth-inhibitory e f f e c t s on Labyrinthulids and Thraustochytrids. Hyalochlorella marina displayed more d i r e c t i n h i b i t i o n which was not as dependent upon exposure conditions (note, however, that |L_ marina was isolated from SSA-T plates i n Sample 3). Poyton (1970b) noted that the a n t i b i o t i c streptomycin caused a s l i g h t decrease i n the growth of H. marina. While more convincing evidence i s needed, my observations suggest that the light-mediated t o x i c i t y of t e t r a c y c l i n e HCL was at least p a r t i a l l y buffered by c e r t a i n constituents i n the SSA medium. Further, more extensive studies concerning the e f f e c t s of a n t i b i o t i c s on these p r o t i s t s are desirable. I have not observed any obvious e f f e c t s of 97 t e t r a c y c l i n e HCL (even when agar plates have turned red) on the i s o l a t i o n of higher fungi or yeasts. These findings cast some doubt upon the r e l i a b i l i t y of the i s o l a t i o n r esults for p r o t i s t s i n Samples 1 and 2, when te t r a c y c l i n e HCL was the only a n t i b i o t i c u t i l i z e d . The absence of Thraustochytrids i n Sample 1 cannot be explained s o l e l y on the basis of t h i s medium-antibiotic e f f e c t (at least with the information at hand), since Thraustochytrids were absent from SSA-T plates as well as GYSA-T plates. Thraustochytrids were is o l a t e d from SSA-T, GYSA-T and FUL-T plates i n Sample 2, a fact t hat could be explained simply on the b a s i s of the p l a t e s being exposed to l i g h t for a shorter time period p r i o r to inoculation and incubation and/or during microscopic observation. Labyrinthula spp. were absent or very rarely i s o l a t e d from GYSA-T or FUL-T plates i n Samples 1 and 2. The premise that the GYSA medium w i l l not support the growth of Labyrinthula spp. was, however, contradicted by the results of Sample 3, when species of Labyrinthula were commonly is o l a t e d from GYSA containing penicillin/streptomycin. Indeed, i n Sample 3 several p r o t i s t s which had not been encountered i n previous samples (especially Labyrinthulids) were isol a t e d from media containing penicillin/streptomycin. These inconsistencies must be kept i n mind when the r e s u l t s are interpreted. The results of Sample 3 show that most Labyrinthulids were i s o l a t e d more frequently from the SSA-PS medium than from GYSA-PS. While several Labyrinthulids (especially Labyrinthula spp.) were commonly i s o l a t e d from GYSA-PS, they could not be maintained on t h i s medium for extended periods. These results may r e f l e c t 98 the observation that at least certain Labyrinthulas have a n u t r i t i o n a l requirement for steroids (Vishniac and Watson, 1953). Thraustochytrids and Hyalochlorella marina also exhibited a s l i g h t l y enhanced (nonsignificant) i s o l a t i o n frequency on the SSA-PS medium compared to GYSA-PS i n Sample 3. The usefulness of Fuller's Modified Vishniac's Medium (FUL) as an i s o l a t i o n medium for p r o t i s t s cannot be evaluated from the results of t h i s study, since, when tested (Sample 2), the FUL medium contained t e t r a c y c l i n e HCL. Overall, yeasts and mycelial fungi were i s o l a t e d with s i m i l a r frequency from a l l of the media u t i l i z e d . In several cases there was an indica t i o n (though not s i g n i f i c a n t ) that yeasts were i s o l a t e d more frequently from GYSA than SSA. Media r i c h i n simple sugars (e.g. glucose) are known to support optimal growth of many yeast species (Lodder, 1970). Mycelial fungi were iso l a t e d at comparable frequencies from a l l of the media tested. I s o l a t i o n frequencies for i n d i v i d u a l species of mycelial fungi were generally low, and no d i s t i n c t 'preferences' for p a r t i c u l a r media could be discerned. The use of two incubation temperatures (14° and 25°C) for a l g a l tissue-agar plates was discontinued af t e r Sample 1. The re s u l t s from Sample 1 indicated that there were no obvious differences between incubation temperatures i n either the types of organisms is o l a t e d or t h e i r percent occurrence. 2. Surface S t e r i l i z a t i o n and/or Rigorous Rinsing Of the surface s t e r i l i z a t i o n arid rigorous r i n s i n g techniques employed, the surface s t e r i l i z i n g procedure used i n Sample 1 99 [1:10,000 (w/v) HgCl 2 (mercuric chloride) i n 5% ethanol; 1 minute exposure] was the most e f f e c t i v e i n terms of reducing the number of microorganisms isol a t e d from the a l g a l tissues. Labyrinthula spp. were e f f e c t i v e l y eliminated from the a l g a l tissues by a one minute exposure to t h i s solution, and the number of mycelial fungi i s o l a t e d was d r a s t i c a l l y reduced (2X seawater-rinsed tissues served as comparative controls). However, observations also revealed that the a l g a l tissue i t s e l f was damaged by th i s surface s t e r i l i z a t i o n procedure. Conspicuous discol o r a t i o n of the a l g a l tissues within 24 hours was considered i n d i c a t i v e of damage caused by s t e r i l i z a t i o n . Damage to the reproductive structures of f e r t i l e a l g a l tissues and to tissues displaying grazing and/or abrasion scars was p a r t i c u l a r l y conspicuous. Spore release i n these red algae i s normally accompanied by the deterioration of certain tissues i n the immediate v i c i n i t y of the f r u i t i n g structures (unpubl. obs.). F e r t i l e areas of the a l g a l thallus, as well as grazed and/or abraded areas, would seem to be opportune l o c a l i t i e s for saprobic fungi to gain entrance int o the a l g a l tissues. I t i s not desirable for the surface s t e r i l i z i n g agent to penetrate and destroy tissues i n these areas, since p o t e n t i a l l y active fungi may be destroyed simultaneously. With t h i s opinion i n mind, the time that a l g a l tissues were exposed to 1:10,000 (w/v) HgCl 2 i n 5% ethanol was reduced from 1 minute to 30 seconds i n Sample 2. A 30 second exposure was not nearly as e f f e c t i v e i n reducing the microbial community isol a t e d from the a l g a l tissues. However, a certa i n amount of damage to 100 the a l g a l tissues was s t i l l apparent. New approaches to the problem of surface s t e r i l i z a t i o n were considered appropriate. S i m i l a r results (i.e. lack of convincing surface s t e r i l i z a t i o n , and obvious damage to a l g a l tissues) were obtained using a d i l u t e solution of Chlorox bleach (3% v/v) i n deionized water (see Section V). In Sample 3 of the f i e l d studies, surface s t e r i l i z a t i o n was abandoned and replaced by a rigorous washing technique (20 rinses i n s t e r i l e seawater). Plating of the actual rinse water demonstrated that s i g n i f i c a n t numbers of p r o t i s t s were being dislodged from the a l g a l surfaces by rinsing, and that the number of p r o t i s t propagules present i n the rinse water decreased as r i n s i n g progressed. Very few yeasts or mycelial fungi were is o l a t e d from a l g a l tissue rinse waters, but, generally, a s i m i l a r (decreasing with continued rinsing) pattern was displayed. When 20X and 2X (control) rinsed a l g a l tissues were plated on agar media the i s o l a t i o n frequencies obtained for p r o t i s t s , yeasts and mycelial fungi were s i m i l a r for both tiss u e pretreatments. These results imply that continued r i n s i n g may have been more e f f e c t i v e i n reducing the number of microbes iso l a t e d from a l g a l tissues plated on agar media. However, when a large number of tissue samples are being processed, more than 20 (or 30) rinses would not be p r a c t i c a l . Reducing the size of the o r i g i n a l tissue sample to be rinsed may be an effec i v e compromise. Modifications of t h i s procedure are c e r t a i n l y worthy of further evaluation. 101 3. I s o l a t i o n Methods a. P r o t i s t s i . Agar Plates Direct p l a t i n g of the a l g a l tissues onto agar media (especially SSA-PS) proved to be the best method for i s o l a t i n g Labyrinthula spp. and Labyrinthula sp. Type LX. Colonies of these r e l a t i v e l y large Labyrinthulids grew away from the a l g a l tissues f a i r l y rapidly and were e a s i l y recognized. Other Labyrinthulids were rar e l y encountered on a l g a l tissue-agar plates, probably due to t h e i r small size and slow growth c h a r a c t e r i s t i c s . Thraustochytrids were commonly i s o l a t e d from a l g a l t i s s u e -agar plates (disregarding a n t i b i o t i c effects). However, the i s o l a t i o n frequencies obtained for Thraustochytrids by this method undoubtedly underestimate t h e i r occurrence. The following d i f f i c u l t i e s contributed to t h e i r underestimation: 1) competition with other microbes; 2) v a r i a t i o n i n the micro-habitat surrounding the a l g a l tissue (e.g. + water fi l m , etc.); and 3) obscured observations (i.e. growth of other isolates, l a y e r i n g , etc.). The r e s u l t s from Sample 3 show that a l g a l tissue-agar plates are also an e f f e c t i v e means of detecting the presence of Hyalochlorella marina, provided the appropriate i s o l a t i o n medium/antibiotic combination i s u t i l i z e d . i i . Rinse Water P l a t i n g of a l g a l t i s s u e r i n s e water i n Sample 3 proved to be a very e f f e c t i v e means of i s o l a t i n g and characterizing the 102 Labyrinthulids, Thraustochytrids and Hyalochlorella marina associated with the a l g a l tissues. However, the quantitative r e s u l t s (propagules/ml) obtained for these organisms using this technique should be interpreted cautiously. Vishniac (1956) used s i m i l a r spread plate techniques to i s o l a t e Labyrinthulids and Thraustochytrids from seawater samples and water 'expressed' from algae. With regard to the quantitative nature of her results, Vishniac (1956) noted several defects i n t h i s type of procedure: 1) Spreading of the water sample e n t a i l s losing that portion of the sample which adhears to the bent glass rod. Loss must a l s o occur on the w a l l s of the s t e r i l e p i p e t t e s used to measure and transfer the water samples. 2) A colony may be formed by either a t h a l l u s or a spore, the ecological implications of which are quite d i f f e r e n t . 3) During the time between spreading and drying of the water sample, a t h a l l u s may produce zoospores. Each zoospore could then give r i s e to a separate colony. 4) Depending upon the select culture conditions, not every viable propagule i n the water sample may give r i s e to a colony (Vishniac, 1956). Of the p r o t i s t s i s o l a t e d from a l g a l tissue rinse water(s) i n t h i s study (see Table 24), i t i s easy to conceive that the r i n s i n g process stimulated zoosporulation i n Schizochytrium  aggregatum and Thraustochytrium motivum. It i s also possible, though less l i k e l y , that the colony counts for Hyalochlorella  marina, Labyrinthuloides yorkensis and the Unidentified 103 Labyrinthulids were s i m i l a r l y affected. The data for Thraustochytrium motivum occasionally displayed counts from t r i p l i c a t e platings which varied by over an order of magnitude (e.g. 28,243,191; 181,1,0). Counts such as these were responsible for the r e l a t i v e l y large v a r i a b i l i t y shown by the data for T;_ motivum, and are almost c e r t a i n l y attributable to zoosporulation a f t e r the rinse water was plated. Counts for the other organisms i s o l a t e d from rinse waters do not indicate that (zoo)sporulation occurred 'on' the agar plates. Despite the interpretive drawbacks, p l a t i n g of a l g a l tissue rinse water proved to be a r e l a t i v e l y simple and d i r e c t method for evaluating the d i v e r s i t y and r e l a t i v e abundance of p r o t i s t s associated with the a l g a l tissues. In the present study, GYSA-PS was shown to be an e f f e c t i v e 'all-around' medium for i s o l a t i n g microbes from a l g a l tissue rinse water. Other i s o l a t i o n media may have proven even more e f f e c t i v e for c e r t a i n p r o t i s t s [e.g. SSA-PS (especially Labyrinthuloides spp.), KMV-PS]. i i i . Seawater/Pine Pollen Cultures Incubation of the a l g a l tissues i n s t e r i l e seawater baited with pine pollen (Sample 3) was i n accordance with the c l a s s i c technique for i s o l a t i n g Thraustochytrids (Gaertner, 1972a). While many Thraustochytrids and Labyrinthulids could be i d e n t i f i e d i n s l i d e s prepared d i r e c t l y from the SW/P cultures, at least an equivalent number had to be brought into pure culture for further characterization. This involved streaking portions (loops) of the SW/P culture onto an agar plate, which, i n turn, commonly led to the i s o l a t i o n of yet another p r o t i s t which had 104 not been previously noticed. Given the technique as described, a conscientious researcher could obtain reasonably thorough and somewhat quantitative (i.e. presence or absence) results. Nearly a l l p r o t i s t s , except Labyrinthula spp. and Hyalochlorella marina, displayed higher i s o l a t i o n frequencies from SW/P cultures than from a l g a l tissue-agar plates. In addition, a greater d i v e r s i t y of p r o t i s t s was obtained from SW/P cultures than from any other i s o l a t i o n method. I f Thraustochytrid d i v e r s i t y alone i s of interest, SW/P culture methods coupled with i s o l a t i o n s onto agar media were superior to a l l other techniques u t i l i z e d . b. Higher Fungi i . Agar Plates A main goal of t h i s study was to i d e n t i f y p o t e n t i a l saprobes, perthophytes or parasites of the red algae Rhodoglossum  a f f i n e and Gelidium c o u l t e r i . With t h i s i n mind, the al g a l tissue-agar plate technique had certa i n shortcomings when used for i s o l a t i n g mycelial fungi. I t i s probable that many of the higher fungi i s o l a t e d by t h i s method were of t e r r e s t r i a l o r i g i n and were not a c t i v e l y growing i n the sea, but simply surviving (as spores) under saline conditions (Pugh, 1974; M i l l e r and Whitney, 1981a). Any 'inactive' fungal spores that were associated with the a l g a l tissues may have r e a d i l y germinated when the tissues were placed on nutrient agar media (cf. Kirk, 1980). I approach t h i s argument cautiously, since I f e e l that certain of the fungi isolated, which have previously been 105 considered ' t e r r e s t r i a l ' , may be active saprobes of marine algae (cf. Schatz, 1980). In addition, some of these fungi, which even I consider ' t e r r e s t r i a l ' , may grow saprobically on marine a l g a l tissues under certain conditions (i.e. when the a l g a l tissues are not regularly submerged i n seawater). The question at hand, however, i s . . . Were these fungi a c t i v e l y growing in/on the a l g a l tissues when they were c o l l e c t e d from the 'iri s i t u ' p o p ulations? My f e e l i n g i s that a great may of them were not. The d i v e r s i t y of mycelial fungi i s o l a t e d from a l g a l tissue-agar plates was very high, and a l l taxa were i s o l a t e d with low frequency. Based on these results alone, fungi which were p o t e n t i a l l y active i n t h i s habitat would be d i f f i c u l t to i d e n t i f y . During the 'Drift Study' (Section IV) p l a t i n g of a l g a l tissues on agar media gave results for mycelial fungi which were not so ambiguous. This exemplifies the e f f e c t s that inoculum and possibly i s o l a t i o n medium may have on the usefulness of t h i s technique. The r e s u l t s from previous studies of algicolous yeasts show that d i r e c t p l a t i n g on agar media i s not the most e f f e c t i v e technique for i s o l a t i n g and attempting to quantify the number of yeasts associated with a l g a l tissues. Methods considered superior include: 1) incubation of the tissues i n a f l u i d medium followed by inoculating the f l u i d medium onto an agar medium (Patel, 1975; Roth, et al., 1962); 2) t r i t u r a t i o n of the a l g a l tissues i n a blender at 4°C followed by f i l t e r i n g the suspension through a s t e r i l e membrane f i l t e r and placing the f i l t e r onto an agar medium (Seshadri and Sieburth, 1971). Using 106 the l a t e r technique, Seshadri and Sieburth (1971) reported yeast 'counts' 20 to 40 times higher than those obtained using other methods, including p l a t i n g of a l g a l t i s s u e rinse water. There are also reports that some a n t i b i o t i c s (streptomycin and chloramphenicol) i n h i b i t the growth of certain yeasts (see Seshadri and Sieburth, 1971). Therefore, I believe that the i s o l a t i o n frequencies obtained for yeasts i n t h i s study greatly underestimate t h e i r actual abundance. i i . Rinse Water Plates By p l a t i n g a l g a l tissue rinse water i n Sample 3 i t was my intent to evaluate the number of higher fungal propagules (mycelial fungi and yeasts) which could be dislodged from the a l g a l substratum during rigorous washing. I had hoped that these re s u l t s would aid i n my interpreting the 'origin' (i.e. i n tissues; on surface) of the fungi i s o l a t e d from a l g a l tissue-agar plates. The medium used i n t h i s experiment - GYSA-PS - was chosen with higher fungi i n mind. In the end, yeasts and mycelial fungi were among the most infrequent organisms isol a t e d from rinse water plates. S t r i c t l y speaking of mycelial fungi, however, rigorous r i n s i n g did remove a moderate number of fungal spores from the a l g a l surfaces. For example, i f the results obtained for Gelidium c o u l t e r i are e x t r a p o l a t e d over a l l 20 r i n s e s , from 20 to 30 or more fungal spores were washed from the surfaces of each G^ c o u l t e r i tissue sample. The removal of these fungal spores was not, however, re f l e c t e d by a reduction i n the i s o l a t i o n frequency of mycelial fungi from rigorous (20X) rinsed tissues plated on agar media. 107 I f e e l t h a t t h i s discrepancy was due to the f a c t t h a t r i n s e water test tubes contained considerably more a l g a l tissue than was plated on agar media (see Materials and Methods, Sample 3). i i i . Seawater/Pine Pollen Cultures The results of Sample 3 demonstrated that higher fungi w i l l grow and sporulate on a l g a l tissues incubated submerged i n SW/P cultures. In fact, mycelial fungi were i s o l a t e d more frequently from SW/P cultures than from a l g a l tissue-agar plates i n Sample 3. M i l l e r and Whitney (1981a) have recently reported s i m i l a r promising results using seawater incubation methods to i s o l a t e fungi from marine algae. Many of the fungi i s o l a t e d v i a seawater incubation methods were among those most commonly i s o l a t e d from a l g a l tissue-agar plates (Samples 1, 2, and 3 combined) and from the D r i f t Study (Section IV). Fungi iso l a t e d by t h i s method most ce r t a i n l y display the a b i l i t y to grow and reproduce on the a l g a l tissues under saline conditions. I regard seawater incubation as a valuable technique to be used i n studies of the fungi associated with marine algae or marine substrata i n general. However, not a l l marine algae decompose i n submerged conditions, and al t e r n a t i v e techniques which do not involve submergence should a l s o be used. i v . Moist Chambers The higher fungi i s o l a t e d from a l g a l tissues incubated i n moist chambers also exhibit the a b i l i t y to grow and reproduce under saline conditions, but not necessarily immersed i n 108 seawater. In t h i s study a n t i b i o t i c s were not incorporated into the seawater placed i n moist chambers. The a c t i v i t i e s of bacteria i n the moist chambers, together with the presence of a c e l l u l o s e f i l t e r disc, may have allowed the growth of fungi which otherwise would not have developed on the a l g a l tissues alone. For an equal amount of a l g a l tissue, fewer fungi were obtained from moist chambers than from a l g a l tissue-agar plates or SW/P cultures (cf. Haythorn, et al., 1980; M i l l e r and Whitney, 1981a). This was e s p e c i a l l y true for tissues of Gelidium c o u l t e r i (see Results). Moist chambers o f f e r a unique set of conditions which may vary between algae simply on the basis of t h e i r structure and morphology. Moist chamber incubation i s a useful technique for i s o l a t i n g higher fungi from marine a l g a l tissues, but i t should be evaluated against results obtained by other i s o l a t i o n methods (M i l l e r and Whitney, 1981a). I would recommend incorporating a n t i b i o t i c s into any seawater introduced into the moist chambers. For s t u d i e s of marine algae, I would a l s o suggest that the c e l l u l o s e f i l t e r (blotter) be replaced with some other rather in e r t substance (e.g. glass f i b e r f i l t e r s ) . B. ISOLATES 1. Labyrinthulids Labyrinthula spp. were by far the most common organisms iso l a t e d during the course of t h i s study. Algal tissues rinsed twice i n s t e r i l e seawater (standard controls), plated on SSA (-T or -PS) and incubated at 25°C gave r i s e to colonies of 109 Labyrinthula spp. approximately 73% of the time. These data also show that the i s o l a t i o n frequency of Labyrinthula spp. did not fluctuate s i g n i f i c a n t l y between f i e l d samples. The Labyrinthula spp. 'category' consists of two species which display developmental patterns very s i m i l a r to the Labyrinthula sp. Vishniac Strains described by Watson (1957) [see Descriptions (Section VII)]. The "Vishniac Strains" studied by Watson (1957) were i s o l a t e d from marine algae or water expressed from algae collected i n the v i c i n i t y of Woods Hole, Massachusetts. In my experience, these strains d i f f e r d i s t i n c t l y from the Labyrinthula spp. normally found associated with Zostera  marina L. ("eelgrass") (i.e. Labyrinthula sp. Type LX or Labyrinthula v i t e l l i n a - l i k e i s o l a t e s ) . Very l i t t l e information has accumulated concerning possible substratum 'preferences' of Labyrinthula species. It i s i n t e r e s t i n g that i s o l a t e s resembling the Vishniac Strains were so common on these red algae whereas other species are abundant on seagrasses. Labyrinthula sp. Type LX was also described by Watson (1957) who found t h i s Labyrinthulid to be very commonly associated with Zostera marina, Spartina sp. and a variety of marine algae c o l l e c t e d from Washington State and Massachusetts. In the present study Labyrinthula sp. Type LX was represented by only two i s o l a t e s obtained from SSA-PS plates i n Sample 3. The use of 'new' i s o l a t i o n methods (rinse water plates, SW/P cultures) and the a n t i b i o t i c s penicillin/streptomycin, enhanced the recovery of members of the genus Labyrinthuloides i n Sample 3. Labyrinthuloides yorkensis was i s o l a t e d from the a l g a l tissues infrequently. Perkins (1973a) described L^ _ yorkensis and 1 1 0 reported that t h i s species could be obtained from estuarine water samples, sediments and detritus. To my knowledge, no further accounts of L^ yorkensis have been published. Labyrinthuloides sp. 1 i s a very i n t e r e s t i n g and undoubtedly new species of t h i s genus. This organism was regularly encountered on rinse water plates and displayed an o v e r a l l i s o l a t i o n frequency of 62% from SW/P cultures i n Sample 3. Further comparative work i s required to investigate the presence and/or predominance of Labyrinthuloies sp. 1 on other substrata and i n other habitats before i t s occurrence on these algae can be evaluated. Labyrinthulids from Sample 3 which could not be i d e n t i f i e d [see Descriptions (Section VII)] augmented the already r i c h 'fauna' of these p r o t i s t s i s o l a t e d from the a l g a l tissues. Nearly a l l of the unidentified Labyrinthulids were encountered on a l g a l tissue rinse water plates, where t h e i r growth was often r e s t r i c t e d (presumably due to the i s o l a t i o n medium used - GYSA-PS). 2. Thraustochytrids The problems encountered with the use of the a n t i b i o t i c t e t r a c y c l i n e HCL and i t s e f f e c t on the growth of Thraustochytrids as well as the general drawbacks of the a l g a l tissue-agar plate technique for i s o l a t i n g these organisms have already been discussed. With t h i s i n mind, a discussion of differences i n the i s o l a t i o n frequencies of Thraustochytrids between samples i s unwarranted. The results from Sample 1, which indicate an absence of Thraustochytrids, are p a r t i c u l a r l y suspect. I l l Thraustochytrium motivum was c l e a r l y the most numerous Thraustochytrid associated with the two red algae studied. The o v e r a l l mean i s o l a t i o n frequency of T\_ movitum from control (2X rinsed) agar plates i n Samples 2 and 3 was 41%. In Sample 3 alone, T\_ motivum was isolated from 28% of the control a l g a l tissues plated on agar media, whereas the mean i s o l a t i o n frequency for s i m i l a r tissues incubated i n SW/P cultures was 71%. Again, these results point out the inadequacy of the a l g a l tissue-agar plate technique i n quantifying the presence of Thraustochytrids. Plating of the a l g a l tissue rinse water(s) i n Sample 3 also demonstrated that T\_ motivum was extremely abundant on the a l g a l tissues. Thraustochytrium motivum has been previously reported from l i t t o r a l marine algae (Booth and M i l l e r , 1968), as well as from various other habitats including marine and estuarine water samples and sediments (Bremer, 1976; Goldstein, 1963a; Sparrow, 1969; Volz, et al., 1976). In t h e i r studies of fungi and p r o t i s t s associated with marine algae, Haythorn and co-workers (1980) found that proliferous Thraustochytrids (i.e. with a basal p r o l i f e r a t i o n ) were the most common type isolated. While the signi f i c a n c e of t h i s observation i s not yet clear, my results also show that proliferous forms (i.e. T^ motivum) were most common. Schizochytrium aggregatum was also i s o l a t e d from a l g a l tissue-agar plates with regularity, but comparative i s o l a t i o n frequencies were low. The o v e r a l l mean i s o l a t i o n frequency of S.  aggregatum ('normal* and pigmented strains combined) from control 112 (2X rinsed) agar plates i n Samples 2 and 3 was 7%. The SW/P cultures of Sample 3 suggested that S^ aggregatum was actually much more common (X = 96% i s o l a t i o n ) . The results from a l g a l tissue rinse water plates i n Sample 3 did not r e f l e c t a s i m i l a r magnitude of enhanced i s o l a t i o n . Incubating the a l g a l tissues i n SW/P cultures was c l e a r l y the most e f f e c t i v e technique for detecting the presence of S^ aggregatum. Schizochytrium aggregatum i s an extremely common Thraustochytrid which has been i s o l a t e d from a wide variety of substrata; often being reported from marine algae (Booth and M i l l e r , 1969; Bremer, 1976; Clokie, 1970; Goldstein and Belsky, 1964; Sparrow, 1969; Volz and Jerger, 1972; Volz, et al., 1976). Thraustochytrium aggregatum was represented i n the present study by only a single i s o l a t e . This species has been previously reported from marine sediments, water samples and algae (Sparrow, 1969; Ulken, 1965; Volz and Jerger, 1972; Volz, et a l . , 1976). Previous reports of Thraustochytrids associated with marine algae include several studies i n which a wide variety of algae were examined, r e s u l t i n g i n the i s o l a t i o n of a high d i v e r s i t y of Thraustochytrids (Haythorn, et al . , 1980; Volz and Jerger, 1972). While the two red algae examined i n t h i s study supported a large number of Thraustochytrids, the d i v e r s i t y of these p r o t i s t s was very low. 3. Hyalochlorella marina Hyalochlorella marina was is o l a t e d from a l g a l tissues i n Sample 3 only. The results from a l g a l tissue-agar plates (as wel l as other observations) suggest that the a n t i b i o t i c 113 t e t r a c y c l i n e HCL may i n h i b i t the growth of H^ marina. However, several i s o l a t e s were obtained from agar plates containing t e t r a c y c l i n e HCL; a fact which confuses my hypothesis that i t was the use of t e t r a c y c l i n e which caused t h i s very d i s t i n c t organism to go unnoticed i n Samples 1 and 2. Poyton (1970b) presents data which indicate that H^ marina can be i s o l a t e d from t h i s locale and habitat during a l l seasons. Poyton (1970b) studied the occurrence and d i s t r i b u t i o n of Hyalochlorella marina, and also investigated various techniques which could be used to i s o l a t e t h i s organism. He concluded that H. marina . . . " i s p r e f e r e n t i a l l y attached to a l g a l filaments but exhibits no s p e c i f i c i t y with regard to p a r t i c u l a r algae." (Poyton, 1970b). The organism was absent from sediments and only ra r e l y i s o l a t e d from seawater samples. Poyton (1970b) also performed an experiment to assess the degree to which IL_ marina was bound to various a l g a l t h a l l i . He concluded that, for nearly a l l of the algae studied, between 77% and 100% of the Hj_ marina c e l l s were removed from the a l g a l surfaces by a one minute agita t i o n i n seawater (Vortex mixer). A d i s t i n c t exception to t h i s trend was Gelidium sp., where only 15-25% of the individuals were removed by a single rinse (Poyton, 1970b). In Sample 3 of the present study, Hyalochlorella marina was is o l a t e d from 62% of the a l g a l tissue-agar plates (SSA-PS and GYSA-PS) which were inoculated with tissues rinsed twice i n s t e r i l e seawater. Similar plates which were inoculated with tissues rinsed 20 times i n s t e r i l e seawater gave r i s e to H. marina 42% of the time. Based on the results for each alga, 114 the differences i n i s o l a t i o n frequency between 2X and 20X rinsed tissues were not s i g n i f i c a n t . The res u l t s from p l a t i n g a l g a l tissue rinse water(s) ce r t a i n l y show that numerous c e l l s of H.  marina were dislodged from the a l g a l tissues by rinsing, and that the number of c e l l s present i n the r i n s e water decreased as the ri n s i n g proceeded. However, a s i g n i f i c a n t number of c e l l s remained associated with the tissues even a f t e r 20 seawater rinses. Contrary to the studies of Poyton (1970b), my res u l t s indicate that H^ marina i s not e a s i l y removed from the (?-) surface of the a l g a l tissues. Hyalochlorella marina did not grow on pine pollen i n s t e r i l e seawater cultures and was i s o l a t e d i n r e l a t i v e l y low numbers using t h i s technique. Presumably t h i s was due to the lack of motile stages and/or to i t s a f f i n i t y for surfaces. Hyalochlorella marina has been reported from marine algae coll e c t e d from a variety of locations i n the United States (Poyton, 1970b) and England (Alderman, 1974; Haythorn, et al., 1980). 4. Actinomycetes I t has been stressed that the i s o l a t i o n methods u t i l i z e d i n t h i s study were not designed for actinomycetes (i.e. the various media contained a n t i b i o t i c s ) . Those actinomycetes that were iso l a t e d developed predominantly on the a l g a l tissues themselves rather than the antibiotic-containing media. Overall, actinomycetes were isol a t e d from 5% of the a l g a l tissue-agar plates inoculated with 2X rinsed a l g a l tissues ('controls'). They were not recovered from s t e r i l i z e d a l g a l tissues i n Samples 115 1 and 2. A single actinomycete was also obtained from a l g a l tissues incubated i n moist chambers (Sample 3). Concurrent studies of the bacteria associated with Rhodoglossum a f f i n e and Gelidium c o u l t e r i have yielded no actinomycetes (Faylla Chapman, pers. comm.). I t would appear that actinomycetes are not commonly associated with these red algae i n f i e l d populations. Further studies directed s p e c i f i c a l l y towards actinomycetes would c e r t a i n l y be desirable. 5. Yeasts and Yeast-like Fungi Yeasts and yeast-like fungi are commonly encountered i n marine and estuarine environments and are known to be associated with marine algae (Meyers and Ahearn, 1974; Patel, 1975; Seshadri and Sieburth, 1971, 1975; van Uden and Castelo Branco, 1963). Previous studies of yeasts associated with seaweeds have shown that those species i s o l a t e d from the algae are also present i n the surrounding seawater (Patel, 1975; Seshadri and Sieburth, 1971, 1975). However, the r e l a t i v e number of yeasts i s o l a t e d from seaweeds as compared to the surrounding seawater may vary considerably (cf. Roth, et al., 1962; Patel, 1975; Seshadri and Sieburth, 1975). Seshadri and Sieburth (1975) reported yeasts to be extremely common on natural populations of the red algae which they examined, and also demonstrated a s i g n i f i c a n t increase i n yeast populations with r i s i n g seawater temperatures (i.e. 15° up to 25°C). During the course of t h i s study, yeasts were i s o l a t e d from 14% of the a l g a l tissue-agar plates inoculated with control a l g a l tissues. Isolation frequencies for yeasts varied s i g n i f i c a n t l y 116 between f i e l d samples, being high i n Sample 2. This increase i n the i s o l a t i o n of yeasts was not correlated with warmer seawater temperatures (cf. Seshadri and Sieburth, 1975). Only i n Sample 2, when yeasts were abundant, did surface s t e r i l i z a t i o n procedures have any s i g n i f i c a n t e f f e c t i n reducing t h e i r i s o l a t i o n frequency. Few yeasts were i s o l a t e d from a l g a l t i s s u e rinse water plates or SW/P cultures i n Sample 3 (few yeasts were iso l a t e d from t h i s sample by any method). Since the majority of yeasts or y e a s t - l i k e fungi isolated during t h i s study have not been i d e n t i f i e d , t h e i r possible o r i g i n s and/or a c t i v i t i e s cannot be evaluated. Many of these yeasts are believed to be species of Candida and Rhodotorula, genera which are known to occur on marine algae and i n seawater (Kohlmeyer and Kohlmeyer, 1979; Patel, 1975; Roth, et al., 1962; Seshadri and Sieburth, 1975). Other i d e n t i f i e d genera include Trichosporon and Hormonema (? Aureobasidium), which are also known from marine habitats (Meyers and Ahearn, 1974; Roth, et a l . , 1962). 6. Mycelial Fungi Approximately 70 d i s t i n c t taxa of mycelial fungi were iso l a t e d from tissues of Rhodoglossum a f f i n e and Gelidium  c o u l t e r i c o l l e c t e d from natural populations. Among these 70 taxa there were 35 (50%) Hyphomycetes, 28 (40%) Coelomycetes, 1 (1%) Ascomycete, and 6 (9%) s t e r i l e mycelia. Only four, perhaps five, are presently considered marine, including: Dendryphiella salina, Phialophorophoma l i t t o r a l i s , Sigmoidea marina Haythorn and Jones, Sigmoidea l i t t o r a l i s sp. nov., and ?-Leptosphaeria sp. 089-79. 117 Over 60% of the fungi i s o l a t e d were represented by single i s o l a t i o n s . Those which were i s o l a t e d "abundantly* or with r e g u l a r i t y from a l g a l tissue-agar plates are l i s t e d i n Table 29. While cert a i n fungi were notably more common, there was no obvious cut-off point between fungi of 'scattered* and 'regular' occurrence. An o v e r a l l i s o l a t i o n frequency above 1% (over 7 i s o l a t i o n s ) has been selected as t h i s cut-off point. The i s o l a t i o n frequencies obtained for these fungi by other i s o l a t i o n methods are also l i s t e d i n Table 29. The only fungi whose ov e r a l l presence was underestimated by the results from a l g a l tissue-agar plates (and therefore are not l i s t e d i n Table 29) were other members of the genus Cladosporium. This i s p a r t i c u l a r l y true of C^ sphaerospermum which was i s o l a t e d by a l l methods, but displayed low o v e r a l l frequencies from a l g a l t i s s u e -agar plates. I have found that C^ sphaerospermum i s often d i f f i c u l t to d istinguish from Cj_ cladosporioides. Note that Penicilium spp. are included i n Table 29 only on a generic basis at t h i s time. These results are comparable to those of Haythorn and co-workers (Haythorn, et al., 1980) and M i l l e r and Whitney (1981a) who reported that the majority of fungi i s o l a t e d from l i v i n g and cast seaweeds were Fungi Imperfecti considered to be of t e r r e s t r i a l o r i g i n . M i l l e r and Whitney (1981a) i s o l a t e d over 30 d i f f e r e n t fungi from l i v i n g and cast seaweeds of which only f i v e were considered marine. A l l but f i v e fungal species were of low and variable occurrence. Those species considered to be of common occurrence included Cladosporium algarum Cooke et Masse, Table 29 Percent occurrence of the most common higher fungi isolated from f i e l d - c o l l e c t e d algal t i s s u e s . Isolation frequencies are l i s t e d by i s o l a t i o n method. The actual number of is o l a t e s are given in parentheses. Isolation Method Algal Tissue- Moist Algal Tissue Seawater/ Isolates Agar Plates Chambers Rinse Water Pine Pollen (n=708*r) (n=19a) (n=96) (n=24) Acremonium sp. 019-78 Cladosporium cladosporioides (Pers.) Link ex S.F. Gray Dendryphiella s a l i n a (Suth.) Pugh et Nicot P e n i c i l l i u m spp. Phoma sp. (Group 1) Sigmoidea l i t t o r a l i s sp. nov. Unidentified hyphomycete 044-78 5.1 (36) 3.2 (23) 1.4 (10) 1.3 (9) 2.1 (15) 1.3 (9) 1.6 (11) 31.6 (6) 21.0 (4) 10.5 (2) 47.4 (9) 0 5.3 (1) 5.3 (1) 2.1 (2) 0 0 2.1 (2) 0 0 0 29.2 (7) 0 8.3 (2) 0 0 4.2 (1) 4.2 (1) #n = the number of algal tissue inocula. For Moist Chambers, n = the total number of chambers inoculated. 119 Papuiaspora halima Anastasiou, P e n i c i l l i u m decumbens Thorn, Perconia cambrensis Mason et M. B. E l l i s , and Dendryphiella  arenaria Nicot. Dendryphiella salina, Cladosporium  cladosporioides, Phoma spp. and other P e n i c i l l i u m spp. were also w e l l represented ( M i l l e r and Whitney, 1981a). Using moist chamber incubation techniques Haythorn and co-workers (Haythorn, et al., 1980) i s o l a t e d 19 genera/species of fungi from cast marine algae, including the new species Sigmoidea  marina. Nine of the fungi i s o l a t e d were considered marine and one of these, Dendryphiella salina, was the only fungus found on a l l algae sampled. These investigators concluded that . . . "the number of higher marine fungi found growing on cast seaweed was low." (Haythorn, et a l . , 1980). In the present study Acremonium sp. 019-78 was the most common fungus i s o l a t e d from natural populations of the red algae examined. When a l l f i e l d samples and a l l i s o l a t i o n techniques are considered, Acremonium sp. 019-78 was is o l a t e d from 6% of the t o t a l inocula. Cladosporium cladosporioides was the second most common fungus (3% overall) followed by P e n i c i l l i u m spp. (2%). These fungi are a l l Fungi Imperfecti which are generally considered to be t e r r e s t r i a l . The remaining fungi l i s t e d i n Table 29 - Dendryphiella salina, Phoma sp. (Group 1), Sigmoidea  l i t t o r a l i s and Unidentified hyphomycete 044-78 - displayed o v e r a l l i s o l a t i o n frequencies between 1 and 2%. Taxonomic studies of Acremonium sp. 019-78, P e n i c i l l i u m spp., Phoma sp. (Group 1) and Unidentified hyphomycete 044-78 are s t i l l i n progress. 120 Total i s o l a t i o n frequencies for mycelial fungi exhibited s i g n i f i c a n t v a r i a b i l i t y between f i e l d samples for both algae. Comparisons were made between data obtained from twice rinsed (control) a l g a l tissues plated on agar media. For Rhodoglossum  affine , t o t a l i s o l a t i o n frequencies for mycelial fungi were s t a s t i c a l l y s i m i l a r i n Samples 1 and 2 (54% and 33% respectively), but declined s i g n i f i c a n t l y i n Sample 3 (8%). The results for Gelidium c o u l t e r i showed a s i m i l a r decline between Sample 2 (64%) and Sample 3 (29%). The 'relative d i v e r s i t y ' of mycelial fungi (i.e. the number of d i f f e r e n t fungi i s o l a t e d divided by the t o t a l number of a l g a l tissue inocula) also declined i n Sample 3. The higher d i v e r s i t y and i s o l a t i o n frequency of mycelial fungi i n Samples 1 and 2 may r e f l e c t the enhanced presence of 'extraneous' fungal spores of t e r r e s t r i a l o r i g i n (especially Sample 2, see Table 12). Even temporal changes i n c l i m a t i c conditions (i.e. wind, rain, runoff, etc.) could have a profound a f f e c t on the presence of ' t e r r e s t r i a l ' fungi i n t h i s habitat. C. DIFFERENCES IN THE ISOLATION OF MICROORGANISMS BETWEEN THE ALGAE AND/OR THEIR LIFE HISTORY STAGES 1. P r o t i s t s Comparative results from 'control' a l g a l tissues (Samples 1, 2, and 3) show that the i s o l a t i o n frequencies for Labyrinthula spp., Schizochytrium aggregatum and Thraustochytrium motivum were s i m i l a r from both algae as well as from the d i f f e r e n t l i f e h i s t o r y stages of each alga. In several instances surface s t e r i l i z a t i o n and/or rigorous r i n s i n g appeared to be more 121 e f f e c t i v e i n eliminating these organisms from cert a i n l i f e h i s t o r y stages of the algae. It i s possible that these procedures had d i f f e r e n t i a l e f f e c ts on f e r t i l e a l g a l tissues i n d i f f e r e n t stages of spore development and release. With the available data, I cannot extrapolate on t h i s p o s s i b i l i t y . The results from a l g a l tissue rinse water plates and SW/P cultures (Sample 3) also indicate that Labyrinthula spp. were iso l a t e d i n equal abundance from both algae. However, Schizochytrium aggregatum and Thraustochytrium motivum displayed higher colony counts on plates prepared from the 3rd rinse water(s) of Rhodoglossum a f f i n e than on s i m i l a r plates for Gelidium c o u l t e r i . These results suggest that the rigorous r i n s i n g technique was more e f f e c t i v e i n 'washing' Thraustochytrids from tissues of a f f i n e than from tissues of G. c o u l t e r i . The data for a l g a l tissue-agar plates i n Sample 3 support t h i s hypothesis, i n that T^ motivum was i s o l a t e d more frequently from rigorous rinsed tissues of G^_ c o u l t e r i than from s i m i l a r tissues of Rj_ affine. In addition, the results from Sample 2 show that the surface s t e r i l i z a t i o n technique employed i n t h i s sample was more e f f e c t i v e i n eliminating Labyrinthula spp. and T\_ motivum from tissues of R^_ a f f i n e than from tissues of G^ c o u l t e r i . These trends may r e f l e c t the less complex morphology of a f f i n e (i.e. blade-like), the fact that this alga generally harbors fewer surface-associated epiphytes (see Section II, 'The Algae'), or, perhaps, some less conspicuous in t e r a c t i o n . Comparisons of the data obtained from a l l i s o l a t i o n techniques u t i l i z e d i n Sample 3 (i.e. a l g a l tissue-agar plates, 122 rinse water plates, SW/P cultures) show that Labyrinthula sp. Type LX, Labyrinthuloides yorkensis and Labyrinthuloides sp. 1 were i s o l a t e d with s i m i l a r frequency from both algae. In Sample 3, Hyalochlorella marina displayed some tendency to be i s o l a t e d more frequently from tissues of Rhodoglossum  a f f i n e than from tissues of Gelidium c o u l t e r i . However, the differences between algae were never large enough to be s t a t i s t i c a l l y s i g n i f i c a n t . FL_ marina was i s o l a t e d with s i m i l a r frequency from the d i f f e r e n t l i f e h i s t o r y stages of each alga. 2. Higher Fungi For each f i e l d sample, yeasts were i s o l a t e d with s i m i l a r frequency from both Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Yeasts also showed s i m i l a r i s o l a t i o n frequencies from the d i f f e r e n t l i f e h i s t o r y stages of each alga (with only one exception; s t e r i l i z e d Rj_ affine, Sample 2). The r e s u l t s from a l g a l tissue-agar plates i n Samples 2 and 3 indicate that mycelial fungi were often i s o l a t e d more frequently from tissues of G^ c o u l t e r i than from tissues of Rj_ affine. However, i n only two of the four data comparisons were the i s o l a t i o n frequencies from G^ c o u l t e r i s t a t i s t i c a l l y higher. S i m i l a r results, suggesting that c o u l t e r i harbored more fungi, were also obtained from SW/P cultures i n Sample 3, from SW/P cultures i n the SEM Study (Section V), and from agar plates in the D r i f t Study (Section IV). In view of the p o s s i b i l i t y that many of the fungi i s o l a t e d from f i e l d c o l l e c t i o n s of these algae arose from spores or hyphae associated with the a l g a l surfaces (see M i l l e r and Whitney, 1981a), t h i s difference i n the i s o l a t i o n 123 of mycelial fungi between algae i s not surprising. Compared to Rhodoglossum af f i n e , Gelidium c o u l t e r i has a much fi n e r and more complex morphology, a larger surface area/volume ra t i o , and a more conspicuous e p i f l o r a and epifauna. I t seems that a l l of these c h a r a c t e r i s t i c s would enhance the p r o b a b i l i t y of 'wandering' fungal spores becoming entrapped by the G^ c o u l t e r i t h a l l u s . There were no differences i n the i s o l a t i o n frequencies of mycelial fungi between the d i f f e r e n t l i f e h i s t o r y stages of R.  a f f i n e (Samples 1, 2, and 3). However, the results for G.  c o u l t e r i displayed s i g n i f i c a n t v a r i a b i l i t y between l i f e h i s t o r y stage i n both Samples 2 and 3. In Sample 2 i s o l a t i o n frequencies of mycelial fungi were consistently high for vegetative/male tissues of t h i s alga. In Sample 3 i s o l a t i o n frequencies from cystocarpic tissues of G^ c o u l t e r i were obviously high; p r i m a r i l y due to a g r e a t e r recovery of Acremonium sp. 019-78 from t h i s l i f e h i s t o r y stage. Examination of the information available on the population biology of Gelidium c o u l t e r i at Hopkins Marine Station (Abbott, 1980) gives l i t t l e insight into a possible source for t h i s v a r i a b i l i t y . [Note that cystocarpic t h a l l i were extremely rare i n t h i s population of G^ c o u l t e r i (see Abbott, 1980)3 CONCLUSIONS The a n t i b i o t i c t e t r a c y c l i n e HCL was found to be unstable i n nutrient agar media which were exposed to l i g h t for r e l a t i v e l y short periods (< 36 hours). In certain media (GYSA, KMV) 'breakdown' of t e t r a c y c l i n e HCL was manifested not only by the 124 loss of i t s b a c t e r i a l i n h i b i t o r y properties and a medium color change (red), but also by the medium becoming i n h i b i t o r y to the growth of Labyrinthulids, Thraustochytrids and Hyalochlorella  marina. Mycelial fungi and yeasts were not noticeably affected. Tetracycline HCL i s not recommended for use i n studies of marine p r o t i s t s , and alternative a n t i b i o t i c s (penicillin/streptomycin, chloramphenicol) should be used at the lowest concentrations possible (i.e. 100-200 mg/l). The a l g a l tissue surface s t e r i l i z i n g and rigorous r i n s i n g techniques employed i n t h i s study are not considered e f f e c t i v e at accomplishing the following two goals simultaneously: 1) eliminating surface-associated microbes? 2) without causing damage to the a l g a l tissues and, therefore, p o t e n t i a l l y active microbes. Exposing the a l g a l tissues to a 1:10,000 (w/v) s o l u t i o n of HgCl2 i n 5% ethanol f o r one minute gave the best surface s t e r i l i z a t i o n results, but damage to the a l g a l tissues was conspicuous. Further studies are required to evaluate whether certa i n fungal spores are r e s i s t a n t to t h i s s t e r i l i z a t i o n procedure, and whether fungi growing i n the a l g a l tissues ( i f present) survive the exposure. Rigorous r i n s i n g (20X) of the a l g a l tissues i n s t e r i l e seawater released a large number of microbes (especially p r o t i s t s ) from the a l g a l surfaces, but did not reduce the absolute presence or absence of these organisms when the tissues were plated on nutrient agar media. Continued r i n s i n g (i.e. more than 20 rinses) i s not considered p r a c t i c a l when numerous samples require processing. Reducing the size of the i n i t i a l tissue sample to be rinsed (i.e. as small as 125 possible) may show t h i s technique to be more eff e c t i v e . A conspicuous 'protist fauna' was found associated with natural populations of the i n t e r t i d a l red algae Rhodoglossum  a f f i n e and Gelidium c o u l t e r i . The most prevalent and consistently i s o l a t e d members of t h i s p r o t i s t fauna were Labyrinthula spp. [resembling the "Vishniac Strains" (Watson, 1957)] and Thraustochytrium motivum. Schizochytrium aggregatum, Hyalochlorella marina and an undescribed species of the genus Labyrinthuloides (sp. 1) were also common, provided the appropriate i s o l a t i o n methods were u t i l i z e d . Other p r o t i s t s of infrequent or rare occurrence included: Labyrinthuloides  yorkensis; Labyrinthula sp. Type LX (Watson, 1957); Thraustochytrium aggregatum; Ulkenia sp. RC02-79; and 'Unidentified L a b y r i n t h u l i d s ' . Members of the genus Labyrinthula were most e f f e c t i v e l y i s o l a t e d by d i r e c t p l a t i n g of the a l g a l tissues onto Modified Serum-Seawater Agar (SSA) containing penicillin/streptomycin (Porter, 1967; Watson and Ordal, 1957). Direct p l a t i n g of a l g a l tissues on agar media was found to underestimate the occurrence of Thraustochytrids and members of the genus Labyrinthuloides. Seawater/pine pollen cultures (Gaertner, 1972a), coupled with i s o l a t i o n s onto Kazama's Modified Vishniac's Agar (KMV) containing penicillin/streptomycin (D. Porter, pers. comm.), yielded the highest i s o l a t i o n frequency and d i v e r s i t y of these organisms. P l a t i n g of a l g a l tissue rinse water(s) onto Glucose-Yeast Extract Agar (GYSA) containing p e n i c i l l i n / s t r e p t o m y c i n also proved to be a simple and d i r e c t method f o r i s o l a t i n g and 126 characterizing the 'protist fauna' associated with the a l g a l tissues. This technique may have proven even more e f f e c t i v e had other i s o l a t i o n media ( i . e . SSA, KMV) been u t i l i z e d . P l a t i n g of a l g a l tissue rinse water(s), or d i r e c t plating of the a l g a l tissues, onto nutrient agar media containing pen i c i l l i n / s t r e p t o m y c i n were the most e f f e c t i v e of the techniques employed for i s o l a t i n g Hyalochlorella marina. The results from surface s t e r i l i z a t i o n and rigorous r i n s i n g of the a l g a l tissues indicate that a l l of these 'protists' are predominantly associated with the a l g a l surface(s). Their o v e r a l l abundance, and the fact that they are not e a s i l y eliminated from the a l g a l tissues by rigorous rinsing, suggest that they are rather 'securely' associated with the a l g a l surface(s ). The data obtained from twice-rinsed 'control' a l g a l tissues show that each p r o t i s t was i s o l a t e d with s i m i l a r frequency from Rhodoglossum a f f i n e and Gelidium c o u l t e r i , as w ell as from the d i f f e r e n t l i f e h i s t o r y stages of these algae. Yeasts and yeast-like fungi were i s o l a t e d from f i e l d -c o llected a l g a l tissues with low to moderate frequency. Those genera which have been i d e n t i f i e d include: Candida, Rhodotorula, Trichosporon and Hormonema (? Aureobasidium). The i s o l a t i o n techniques u t i l i z e d i n t h i s study may have greatly underestimated the actual abundance of yeasts associated with the a l g a l tissues (cf. Seshadri and Sieburth, 1971). No obvious fungal 'associates' or parasites were observed i n t h a l l i of Rhodoglossum a f f i n e or Gelidium c o u l t e r i . However, 127 i s o l a t i o n s from f i e l d - c o l l e c t e d tissues of these algae yielded an abundance and high d i v e r s i t y of imperfect fungi. The majority of the fungal taxa were i s o l a t e d only once or at very low frequencies. Many of the species i s o l a t e d are considered to be of t e r r e s t r i a l o r i g i n and of questionable significance i n the i n t e r t i d a l habitat. Only four (?-five) of the mycelial fungi are presently considered marine. The mycelial fungi most frequently i s o l a t e d include: Acremonium sp. 019-78, Cladosporium  cladosporioides, Dendryphiella salina, P e n i c i l l i u m spp., Phoma sp. (Group 1), Sigmoidea l i t t o r a l i s sp. nov. and Unidentified hyphomycete 044-78. Certain of these species may be saprobes and/or perthophytes on attached a l g a l t h a l l i i n the i n t e r t i d a l habitat, but t h e i r a c t i v i t i e s would appear to be limited. Qualitative aspects of the fungal biota found associated with these two algae were simi l a r . Gelidium c o u l t e r i often harbored more fungi, a r e s u l t attributable to aspects of i t s morphology and 'habit' which may enhance the entrapment of 'wandering' fungal spores. Occasionally, i s o l a t i o n data showed s i g n i f i c a n t v a r i a b i l i t y between the d i f f e r e n t l i f e h i s t o r y stages of the algae. These observations may r e f l e c t an enhanced occurrence of 'saprobic' fungi on reproductive and/or senescing a l g a l tissues (e.g. Sample 3, cystocarpic G^ c o u l t e r i ) . 128 IV. ISOLATION FROM DECOMPOSING, 'ARTIFICIALLY DRIFTED", Rhodoglossum a f f i n e AND Gelidium c o u l t e r i INTRODUCTION Vascular plant materials, such as red mangrove (Rhizophora  mangle L.), cord grass (Spartina a l t e r n i f l o r a L o i s e l ) , t u r t l e grass (Thalassia testudinum Konig) and eelgrass (Zostera marina L.) contribute a major portion of the organic material that drives certain estuarine food chains. In many of these estuarine systems organic detritus i s the main l i n k between primary and secondary productivity, because only a small portion of the net production of the vascular plant i s grazed while i t i s a l i v e (Odum and De l a Cruz, 1967). The microbes which decompose t h i s vascular plant material could be considered the r e a l primary consumers i n these communities (Darnell, 1967). During decomposition and detritus formation the nitrogen or protein content of the organic matter increases due to the presence and a c t i v i t i e s of microbial communities. Thus, detritus r i c h i n microbes i s n u t r i t i o n a l l y a better food source for animals than the vascular plant tissue that forms the o r i g i n a l base f o r most of the p a r t i c u l a t e matter (Odum and De l a Cruz, 1967; Odum and Heald, 1972). In addition to bacteria, fungi have been shown to be active i n the decomposition of vascular plant substrata i n marine and estuarine environments (Anastasiou and Churchland, 1969; F e l l and Master, 1973, 1975; Meyers, 1974; Meyers, et al., 1965). Fungi are able to manufacture proteins 129 from dissolved nitrogen sources (urea, ammonia, nitrate, n i t r i t e ) (Jones and Irvine, 1972) and fungal protein may be an important food source for d e t r i t a l consumers. In the temperate coastal areas of North America, seaweeds predominate i n the available rocky habitats. The primary productivity of temperate coastal marine communities i s very high, and marine a l g a l macrophytes contribute a s i g n i f i c a n t biomass to these ecosystems (Josselyn and Mathieson, 1978; Mann, 1972). As for the estuarine systems, evidence suggests that only a small portion of t h i s a l g a l biomass i s u t i l i z e d by d i r e c t grazing, and that most of i t i s decomposed by microorganisms to be liberated as part i c u l a t e and dissolved organic matter (Khailov and Burlakova, 1969; Mann, 1976). The resultant detritus i s a prominent part of the diet of numerous marine invertebrates and f i s h which forage nourishment not only from the plant material i t s e l f , but also from the associated microorganisms (Mann, 1972, 1976). Available information indicates that the n u t r i t i v e value (nitrogen) of a l g a l detritus i s also enhanced by the presence of microbial communities (Hunter, 1976). The po t e n t i a l role(s) of fungi i n these a l g a l decomposition and detritus formation processes are e s s e n t i a l l y unknown. There are certain problems associated with eco l o g i c a l studies of marine a l g a l decomposition. Perhaps the most fundamental of these problems i s the fact that a l g a l biomass may be subjected to a wide variety of environmental conditions during decomposition. As an example, detached a l g a l t h a l l i may sink to the bottom and be abraded by wave a c t i o n or b u r i e d i n sand or sediments. Alternatively, algae are often washed up high-and-dry 130 on the beach, and may eventually become buried i n the sand, or perhaps, even be washed back out to sea again. Therefore, i t i s nearly impossible for a mycologist to follow the t o t a l 'natural' successional patterns during decomposition, and one i s forced to look at certain aspects of the process under somewhat less than i d e a l conditions. Mycological studies have been performed i n which 'cast' seaweeds were collected, transported to the laboratory, and subjected to various i s o l a t i o n and/or incubation techniques (Chesters, et al., 1956; Haythorn, et al., 1980; M i l l e r and Whitney, 1981a; Nonomura, 1978; present study). However, no studies have been reported i n which successional observations were made of the fungi associated with decomposing seaweeds, i.e. observations over time. This i s presumably due to the very unstable environment i n which seaweeds normally decompose. In t h i s experiment a 'mesh bag' technique was u t i l i z e d i n which t h a l l i of Rhodoglossum a f f i n e and Gelidium c o u l t e r i were a r t i f i c a i l l y stranded on a beach and t h e i r decomposition was monitored over time. This technique i s not without drawbacks, but i t has been used successfully i n studies of the fungi associated with decomposing vascular plant substrata i n marine and estuarine environments (Anastasiou and Churchland, 1969; F e l l and Master, 1973; Gessner and Goos, 1973; Newell, 1976). This collaborative experiment was performed i n conjunction with other studies supported by a C a l i f o r n i a Sea Grant (R/A-34; 1978-80 - Abbott, Stanford University). This Sea Grant research was aimed at monitoring the decomposition of the phycocolloids 131 contained i n Rj_ a f f i n e (carrageenan) and c o u l t e r i (agar) when these algae were stranded on the beach as d r i f t . MATERIALS AND METHODS Collections of cystocarpic Rhodoglossum a f f i n e and a l l l i f e h i s t o r y stages of Gelidium c o u l t e r i were made on the 24th, 26th and 27th of December 1978 from Point Pinos, Monterey Bay, C a l i f o r n i a (36° 38.50'N; 121° 55.90'W) (see F i g . IB). The algae were stored i n separate SCUBA "goodie bags" i n an outdoor flowing seawater tank u n t i l processed for use i n the experiment. On December 27th the algae were allowed to drain and were sorted, cleaned of major epiphytes, and weighed to obtain 4.5 kg wet weight each. Each species was then divided into three 1.5 kg aliquots and each of these was sewn into a fiberglass screen bag (approximately 30 x 46 cm; 1.2 mm mesh). The bags were placed i n a flowing seawater tank overnight. On December 28th, one 1.5 kg aliquot (screen bag) of each species was sewn into each of three SCUBA "goodie bags" (approximately 38 x 61 cm; 5 mm mesh). The bags containing the algae were then placed 6-12 meters apart on a sandy, northwest facing beach at Hopkins Marine Station between approximately +1.5 and +4.5 feet (+0.5 and +1.3 meters) MLLW (see Fig. 2); and were anchored to high i n t e r t i d a l rocks by 8 meter polypropylene lines. The ' d r i f t bags' were checked every two or three days f o r the d u r a t i o n of the experiment, and were repositioned i f they had been thrown high up on the beach. Representative subsamples (minimum 200 g wet weight) were taken from each aliquot of each species at approximately biweekly 132 i n t e r v a l s over a period of from 52 (Rj_ affine) to 71 days (G. c o u l t e r i ) . A l g a l subsamples were subjected to various analyses, including: percent composition of agar (G^ coulteri) or carrageenan (Rj_ af f i n e ) ; microbiological analysis for phycocolloid-digesting bacteria; and microbiological analysis for associated fungi. Only the l a t t e r of these studies (fungi) w i l l be reported on here. Mycological analyses of the a l g a l subsamples were not performed u n t i l the experiment had been i n progress for 36 days. Small portions of each a l g a l subsample were placed i n a test tube and washed once i n s t e r i l e seawater (33°/oo) using a Vortex Mixer for one minute periods. Two to four small pieces (approximately 1.0-1.5 cm i n length) of the decomposing algae from each subsample were plated on each of three s l i g h t l y d i f f e r e n t Base Mineral Media (Gunkel and Rheinheimer, 1972; see Appendix A). The Base Mineral Media d i f f e r e d only i n the phycocolloid u t i l i z e d to gel them: Difco Bacto agar; kappa carrageenan (K-7; Marine Colloids, Rockland, Maine); kappa carrageenan (K-13; Stauffer Chemical Co., Dobbs Ferry, New Jersey) (see Abbott and Chapman, 1981). A l l media contained t e t r a c y c l i n e HCl (250 mg/l) to i n h i b i t b a c t e r i a l growth. Culture plates were incubated i n diffuse sunlight at room temperature (22-25°C) and examined for the presence of fungi over a period of approximately 60 days. A post-experimental control consisted of fresh algae c o l l e c t e d from the same l o c a l i t y at Point Pinos on 1 A p r i l 1979 (24 days aft e r the termination of the decomposition experiment). Portions of representative t h a l l i from each a l g a l species were 133 processed and plated as described above. A flow chart of the ' d r i f t experiment' laboratory procedures i s presented i n Figure 7. RESULTS Weather conditions during the experimental period (28 December 1978 - 8 March 1979) remained f a i r l y cool (< 55°F) and generally overcast (approximately 60% of the time). Intermittant storms brought about 15 days of r a i n and produced heavy seas, causing considerable sand movement on the beach and depositing seaweed d r i f t i n the immediate v i c i n i t y of the ' d r i f t bags'. It i s estimated that the d r i f t bags were at least p a r t i a l l y buried i n the sand for 70% of the duration of the experiment. Rhodoglossum a f f i n e deteriorated more rapidly than Gelidium  c o u l t e r i , and by day 52 i t was almost completely decomposed. By day 71 the G^ c o u l t e r i was i n an advanced state of decomposition, but could have remained on the beach f o r another two week period. The experiment was terminated at t h i s time because there was i n s u f f i c i e n t biomass of G^ c o u l t e r i to allow for another sample. Observations of the microfauna associated with these decomposing algae revealed that nematodes (unidentified; probably two species) became established i n the bags afte r approximately 30 to 40 days on the beach. Relative numbers of these nematodes increased through the remainder of the experiment. A collembolid, tenatively i d e n t i f i e d as Anurida maritima Guerin, was a l s o present i n s e v e r a l of the d r i f t bags between day 16 and day 52, but was not found during subsequent observations. SAMPLES FROM DECOMPOSING ALGAE 3 f o r each alga per sampling date / / / / PHYCOCOLLOID ANALYSES INCUBATION 22-25°C -SUBSAMPLES^ ^PLATING -^ FOR FUNGI -•SW RINSE-1 minute -•TISSUE PORTIONS 2, 3 or 4/plate \ \ \ \ PLATING FOR BACTERIA POST-EXPERIMENTAL CONTROL SAMPLES BASE MEDIUM WITH DIFCO-BACTO AGAR -•K-7 KAPPA CARRAGEENAN 'K-13 KAPPA CARRAGEENAN Figure 7. F i e l d and laboratory flow diagram f o r the processing of ' a r t i f i c i a l l y d r i f t e d ' Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Ul 135 Results from microbiological analyses of the associated b a c t e r i a l f l o r a showed a general increase i n the number of bacteria as a l g a l decomposition proceeded. Several bacteria that were able to decompose or l i q u i f y the phycocolloids incorporated i n the Base Mineral Medium were consistently i s o l a t e d (Faylla Chapman, pers. comm.). The actinomycetes, fungi and Labyrinthula spp. i s o l a t e d from the decomposing algae are l i s t e d i n Tables 30 (Rhodoglossum  affine) and 31 (Gelidium c o u l t e r i ) . The values l i s t e d are an average frequency of i s o l a t i o n for a l l three d r i f t bags, and are based on the t o t a l number of i n d i v i d u a l a l g a l pieces plated (inocula) of each a l g a l species on each sampling date. Calculation of o v e r a l l averages was deemed appropriate since: 1) the i s o l a t i o n data from each of the three d r i f t bags was s i m i l a r ; and 2) the type of phycocolloid u t i l i z e d to s o l i d i f y the Base Mineral Medium had no apparent e f f e c t on the types or numbers of fungi isolated. Since nematodes developed i n the fungal cultures, t h e i r r e l a t i v e abundance i s also l i s t e d i n these tables. Not a l l i s o l a t e s of certain genera were f u l l y characterized i n t h i s study; however, some q u a l i t a t i v e observations were made. Cladosporium spp. included C^ herbarum, C. cladosporioides (most common) and C^ sphaerospermum. Acremonium spp. consisted mainly of two species: Acremonium sp. 215-79 and Acremonium sp. 019-78, of which the l a t e r was far more common. With the exception of Acremonium spp., Dendryphiella sal i n a and Sigmoidea l i t t o r a l i s sp. nov., the fungi i s o l a t e d displayed a discontinuous and rather low frequency of i s o l a t i o n (Tables 30 Table 30 Percent occurrence of fungi and Labyrinthula spp. isolated from ' a r t i f i c i a l l y d r i f t e d 1 Rhodoglossum a f f i n e a f t e r 35 and 52 days of exposure.* Also l i s t e d i s the r e l a t i v e abundance of nematodes.t Experiment s t a r t i n g date - 27 December 1978. Post-experimental 'Control' = s i m i l a r microorganisms i s o l a t e d from R. a f f i n e c o l l e c t e d from an in s i t u population. 36 days 52 days Control Isolates (n=36) (n=34) (n=27) Yeasts and y e a s t - l i k e fungi 0 14.7 0 Acremonium spp. 5.6 20.6 0 Cladosporium spp. 0 2.9 0 Dendryphiella s a l i n a (Suth.) Pugh et Nicot 5.6 23.5 0 Fusarium sp. 0 2.9 0 P e n i c i l l i u m spp. 2.8 0 22.2 Sigmoidea l i t t o r a l i s sp. nov. 11.1 58.8 0 Unidentified coelomycete 063-79 0 0 3.7 Labyrinthula spp. 0 2.9 0 Nematodes + +++ _ *Values are based on the number of algal t i s s u e pieces plated (inocula) f o r each sampling date. Except fo r the Control, values are the average of i s o l a t i o n s from 3 separate d r i f t bags. Isolation medium -Base Mineral Medium made up with natural aged seawater. fFor nematodes: - = not observed; + = few; +++ = thousands. i—• u> CTl Table 31 Percent occurrence of actinomycetes, fungi and Labyrinthula spp. i s o l a t e d from ' a r t i f i c i a l l y d r i f t e d 1  Gelidium c o u l t e r i a f t e r 35, 52 and 71 days of exposure.* Also l i s t e d i s the r e l a t i v e abundance of nematodes.f Experiment s t a r t i n g date - 27 December 1978. Post-experimental 'Control' = s i m i l a r microorganisms i s o l a t e d from G. c o u l t e r i c o l l e c t e d from an in s i t u population. 36 days 52 days 71 days Contro Isolates SW SW SW 10* SW (n=24) (n=24) (n=36) (n=36) (n=27 Actinomycetes 4.2 12.5 0 0 3.7 Yeasts and y e a s t - l i k e fungi 0 8.3 0 8.3 0 Acremonium spp. 12.5 70.8 83.3 83.3 22.2 Aspergillus sp. 0 0 0 0 3.7 Beauveria bassiana (Bals.) V u i l l . 0 0 0 0 3.7 Cladosporium spp. 0 16.7 0 0 22.2 Dendryphiella s a l i n a (Suth.) Pugh et Nicot 12.5 33.3 69.4 33.3 0 Fusarium sp. 0 4.2 0 0 0 Paecilomyces i n f l a t u s (Burnside) Carmichael 0 4.2 0 0 0 Paecilomyces spp. 0 0 0 0 11.1 P e n i c i l l i u m spp. 4.2 4.2 0 0 7.4 Phoma sp. 033-78 0 0 0 0 3.7 Sigmoidea l i t t o r a l i s sp. nov. 41.7 75.0 100.0 94.4 0 V e r t i c i l l i u m albo-atrum Reinke & Berth. 0 0 0 0 3.7 T r a l i a ascophylli Sutherland 0 0 0 2.8 0 Corollospora maritima Werdermann 0 0 0 2.8 0 Unidentified hyphomycete 207-79 0 0 0 0 3.7 Labyrinthula spp. 0 0 0 2.8 0 Nematodes _ ++ ++ ++ *Values are based on the number of algal t i s s u e pieces plated (inocula) f o r each sampling date. Except f o r the Control, values are the average of i s o l a t i o n s from 3 separate d r i f t bags. I s o l a t i o n medium - Base Mineral Medium made up with natural aged seawater (SW). At 71 days i s o l a t i o n s were re p l i c a t e d using the same medium made up with Instant Ocean (10) (Aquarium Systems, Eastlake, Ohio). fFor nematodes: - = not observed; ++ = hundreds. 138 and 31). On each sampling date (i.e. 36 and 52 days) i n o c u l a of Rhodoglossum a f f i n e gave r i s e to fewer fungi than did comparable tissues of Gelidium c o u l t e r i (day 36 - 0.3 i s o l a t e s per inoculum fo r Rj_ a f f i n e vs. 0.7 f o r G^ c o u l t e r i ; day 52 - 1.2 vs. 2.2). In both algae the i s o l a t i o n frequency of Acremonium spp., Dendryphiella sal i n a and Sigmoidea l i t t o r a l i s increased as the algae decomposed, u n t i l by the end of the experiment these fungi dominated the mycobiota observed using these techniques. This was e s p e c i a l l y true of S^ l i t t o r a l i s which was the most abundant fungus on both algae from day 36 u n t i l G. c o u l t e r i was terminated at day 71. The low i s o l a t i o n frequency of yeasts may have been due to the lack of appropriate nutrients (e.g. simple sugars) i n the i s o l a t i o n medium and/or to the i s o l a t i o n technique i t s e l f (cf. Patel, 1975; Seshadri and Sieburth, 1971, 1975). Similar nutrient and i s o l a t i o n technique l i m i t a t i o n s , including the use of t e t r a c y c l i n e HCL, could also explain the low recovery of Labyrinthula spp. (cf. Sykes and Porter, 1973; Vishniac and Watson, 1953) and the absence of Thraustochytrids (cf. Clokie, 1970; Gaertner, 1972a). The presence of T r a l i a ascophylli Sutherland and Corollospora maritima Werdermann may have been underestimated due to an unforseen oversight i n techniques. These two Ascomycetes were found among several i s o l a t i o n plates that were set aside at 4°C for approximately 45 days. I t i s e n t i r e l y possible that they were also present on other i s o l a t i o n plates that were not incubated for an extended period at low temperatures. I t i s also 139 possible that other ascomycetous fungi may have developed i f a l l i s o l a t i o n plates had been treated i n t h i s manner. The i s o l a t i o n data indicate that there were no obvious s h i f t s i n the mycobiota associated with the decomposing algae. Rather, the i s o l a t i o n frequency of Acremonium spp., Dendryphiella  s a l i n a and Sigmoidea l i t t o r a l i s gradually increased u n t i l these fungi became conspicuously dominant. This trend i s p a r t i c u l a r l y evident i n the results for Gelidium c o u l t e r i (Table 31). Data on the fungi present e a r l i e r i n the experiment (i.e. p r i o r to day 36) would be desirable to further i d e n t i f y and interpret possible patterns of succession. Indeed, the data suggest that fungal development may have been rather slow i n the early stages of decomposition, but that i t increased rapidly a f t e r approximately 36 days of exposure. Generally, the fungi i s o l a t e d from the post-experimental control samples did not resemble the dominant mycobiota found associated with the decomposing algae (Tables 30 and 31). The fungal species i s o l a t e d from the control samples were either: 1) not i s o l a t e d from the d r i f t algae; or 2) they were s i m i l a r to fungi which were of low and discontinuous occurrence (with the exception of Acremonium sp. 019-78 i s o l a t e d from the G^ c o u l t e r i control). Note also that control samples of G^ c o u l t e r i gave r i s e to mycelial fungi more frequently (81.5% overall) than s i m i l a r samples of R. a f f i n e (25.9%)(p < 0.005). 140 DISCUSSION The use of a mesh ' d r i f t bag' technique i n t h i s experiment deserves some cautionary comments. I t must be assumed that the microbial populations which developed on the decomposing a l g a l tissues i n the ' d r i f t bags' were s i m i l a r to those on algae outside the bags. However, the mesh bags undoubtedly offered a somewhat unique set of experimental conditions. During the experimental period naturally cast seaweeds did not remain on the beach for extended periods, i.e. not more than approximately 10 days. Certainly, however, d r i f t seaweeds can become stranded for considerable periods. F e l l and Master (1980) have noted that the mesh bag technique eliminates possible predation and/or grazing by macroinvertebrates; therefore the balance or succession of the community may be altered. Other considerations of the mesh bag method are discussed by Park (1974). The reader i s a l s o reminded t h a t screen bags of both Rhodoglossum a f f i n e and Gelidium c o u l t e r i were placed together i n each of the three larger mesh bags. The fungal populations observed i n each of the three mesh bags were extremely similar, suggesting no l o c a l i z e d s p a t i a l differences. However, i t must be assumed that the two algae being adjacent to one another did not influence the observation that s i m i l a r mycobiotas developed on the decomposing tissues. Bacteria which could digest the phycocolloids incorporated into the Base Mineral Medium were consistently i s o l a t e d from the a l g a l tissues throughout the experiment. Is o l a t i o n data show that b a c t e r i a l numbers increased with time. More bacteria 141 (including phycocolloid digesters) were is o l a t e d from the a l g a l tissues than from the a l g a l tissue rinse waters or from samples of beach sand. Si m i l a r bacteria were also obtained from the post-experimental control a l g a l samples (Faylla Chapman, pers. comm.). I t i s possible that the a c t i v i t i e s of these bacteria either prepared the a l g a l tissues for fungal assimilation, or provided metabolites which the fungi assimilated or possibly required. Considerable research i s needed before we w i l l understand the interactions between bacteria and fungi i n marine biodeterioration processes. In the mycology section of t h i s collaborative experiment, actinomycetes (Streptomyces spp.) were occasionally observed to develop on decomposing tissues of Gelidium c o u l t e r i (fungal i s o l a t i o n plates contained t e t r a c y c l i n e HCl). I t i s i n t e r e s t i n g that s i m i l a r actinomycete colonies were not observed on b a c t e r i a l i s o l a t i o n plates (similar media which did not contain a n t i b i o t i c s ) (Faylla Chapman, pers. comm.). I t i s possible, although unlikely, that actinomycetes were unable to grow on the Base Mineral Medium alone. Actinomycetes (Streptomyces spp.) have been shown to be active decomposers of brown a l g a l polysaccharides (Chesters, et al., 1956). I have also found various members of the genus Streptomyces to be very common on a i r 'dried', p a r t i a l l y decomposed, colloid-bearing red algae of foreign o r i g i n . Further studies of actinomycetes as decomposers of red algae are needed. There i s very l i t t l e doubt that the three dominant higher fungi, Acremonium spp., Dendryphiella s a l i n a and Sigmoidea 142 l i t t o r a l i s , were a c t i v e l y growing i n and obtaining nutriment from the decomposing a l g a l tissues. Few colonies of these fungi developed on plates inoculated with a l g a l tissue rinse waters (Faylla Chapman, pers. comm.), in d i c a t i n g that few spores were present and that vegetative mycelium was the source of the colonies produced on a l g a l tissue plates. When a l g a l tissues were incubated on the i s o l a t i o n medium, these fungi often produced a e r i a l mycelium and numerous conidia before s i g n i f i c a n t growth was apparent in/on the agar medium i t s e l f . Conidia of Sigmoidea l i t t o r a l i s were produced i n tangled masses which appeared to 'burst' through the surface of the a l g a l tissues [cf. Sigmoidea marina (Haythorn, et a l . , 1980)1. The abundance of nematodes found associated with these fungal-infested decomposing a l g a l tissues i s also of interest. Meyers and co-workers (Meyers and Hooper, 1966, 1967, 1973; Meyers, et a l . , 1963) have repeatedly demonstrated fungal and nematode biotas i n estuarine and marine habitats characterized by active degradation of c e l l u l o s i c material. Benthic and f o l i c o l o u s nematodes with d i f f e r e n t feeding habits were found during decomposition studies of Spartina a l t e r n i f l o r a and Thalassia testudinum. The marine nematode Metoncholaimus sp. exhibited a strong a t t r a c t i v e response to fungal-infested c e l l u l o s e f i l t e r discs 'planted' i n the f i e l d (Meyers and Hooper, 1966). A marine species of the nematode genus Aphelenchoides was able to develop and reproduce e f f e c t i v e l y when feeding on viable mycelia of various filamentous marine fungi, including Dendryphiella arenaria Nicot (Meyers, et al., 1963). Schatz (1980) has also noted the presence of nematodes i n fungal-143 infected stipe tissues of the brown alga Laminaria saccharina (L.) Lamour. These nematode-fungal i n t e r r e l a t i o n s h i p s are in d i c a t i v e of the complex interactions and trophic conversions associated with marine a l g a l decomposition and d e t r i t a l turnover. 'Wrack' i s a c h a r a c t e r i s t i c habitat of several Collembola species which feed d i r e c t l y on plant debris (Cheng, 1976). However, the collembolid encountered i n t h i s study, Anurida  maritima, i s carnivorous and feeds on a variety of small marine animals. A^_ maritima avoids t o t a l submersion and, therefore, moves up and down the i n t e r t i d a l zone i n accordance with the tides; i t i s often found clumped together i n hundreds. In thi s climate Anurida maritima i s active only during the day (Cheng, 1976). This information coincides with the timing of our d r i f t bag observations, i.e. during day time low tides. I t i s not known whether the A^_ maritima were a c t i v e l y feeding on the microfauna associated with the d r i f t algae, but t h e i r d a i l y wanderings are normally i n search of food (Cheng, 1976). CONCLUSIONS Contrary to previous reports suggesting that fungi are not active i n the decomposition of marine algae (Chesters, et al., 1956; Haythorn, et al., 1980), the results of the present study demonstrate that Acremonium spp. (especially # 019-78), Dendryphiella s a l i n a and Sigmoidea l i t t o r a l i s sp. nov. are active colonizers of decomposing red a l g a l tissues. I s o l a t i o n data indicate that these fungi were not abundant u n t i l a f t e r the algae had been exposed for 36 days, and i t i s possible that the 144 a c t i v i t i e s of phycocolloid-digesting bacteria were required for th e i r development. Observations of nematodes and a collembolid associated with the decomposing algae s i g n i f y the importance of al g a l biomass turnover i n coastal food webs. The 'mesh bag' technique has proven useful for studies concerning the successional development of microbial communities associated with decomposing algae. This technique i s to be recommended for future studies of marine a l g a l decomposition. 145 V. SCANNING ELECTRON MICROSCOPE OBSERVATIONS OF THRAUSTOCHYTRIDS ON THE SURFACES OF Rhodoglossum a f f i n e AND Gelidium c o u l t e r i INTRODUCTION Thraustochytrids are known to be associated with a variety of marine algae (Clokie, 1970; F u l l e r , et al., 1964; Haythorn, et al., 1980; Sparrow, 1969; Vishniac, 1956; Volz and Jerger, 1972). However, the nature of Thraustochytrid/algal associations (i.e. casual, saprobes, perthophytes, biotrophic parasites) has not been f u l l y elucidated. Presumably the growth of a Thraustochytrid on the surface of an alga includes the development of an e p i b i o t i c sporangium. A question which remains unresolved i s whether the ectoplasmic networks produced by the Thraustochytrid are able to a c t i v e l y penetrate and 'parasitize' the c e l l s of healthy a l g a l tissues. Alternatively, i f the Thraustochytrid i s unable to a c t i v e l y p a r a s i t i z e the al g a l tissues, then where does i t obtain the nutriment that i s required for i t s development? This experiment was designed to evaluate whether Thraustochytrids found associated with f i e l d - c o l l e c t e d t h a l l i of Rhodoglossum a f f i n e and Gelidium c o u l t e r i could be induced to grow on the surfaces of these algae i n s t e r i l e seawater cultures. The nature of posit i v e growth associations was examined using scanning electron microscopy (SEM). Laboratory processing of the a l g a l tissues included surface s t e r i l i z a t i o n using a d i l u t e solution of Chlorox bleach (sodium hypochlorite). This surface 146 s t e r i l i z a t i o n procedure was evaluated for i t s a b i l i t y to eliminate Thraustochytrids and/or higher fungi from f i e l d -c o l l e c t e d a l g a l tissues. MATERIALS AND METHODS Algae were co l l e c t e d from the i n t e r t i d a l populations at Hopkins Marine Station (see Fig. 3) on 22 October 1979. Individual plants representative of select l i f e h i s t o r y stages of Rhodoglossum a f f i n e and Gelidium c o u l t e r i were coll e c t e d and placed i n separate s t e r i l e p l a s t i c bags (Nasco Whirl-pac). The algae were returned to the l a b o r a t o r y i n an i c e chest, and were processed for use within f i v e hours of the time of co l l e c t i o n . C ollections were made at low tide (0.0 feet at 1705 hours). The estimated times that the algae were exposed to the a i r p r i o r to c o l l e c t i o n were 2.5 hours for R^ a f f i n e and 1.5 hours for G.  c o u l t e r i . Seawater temperature and s a l i n i t y at the time of c o l l e c t i o n were 16.2°C and 33°/oo respectively. In the laboratory, sections of the a l g a l t h a l l i approximately 3 cm long were surface s t e r i l i z e d for one minute i n a 0.3% solution of Chlorox i n deionized water (= 0.016% w/v NaOCl), followed by two rinses i n s t e r i l e seawater [Booth, 1971 (modified); unpubl. obs.]. Al g a l tissues were surface s t e r i l i z e d by p l a c i n g them i n c u l t u r e tubes (screw cap, 25 x 150 mm) containing 15 ml of 0.3% Chlorox solution at room temperature (22-25°C). The tubes were mixed on a Vortex mixer ( S c i e n t i f i c Products model S8223, at maximum setting) for one minute with intermittant periods of rest. The surface s t e r i l i z i n g solution 147 was then poured o f f and replaced with 15 ml of s t e r i l e seawater (28°/oo). The a l g a l tissues were rinsed twice i n s t e r i l e seawater for one minute periods. S u r f a c e - s t e r i l i z e d a l g a l tissues were then removed from the culture tubes using s t e r i l e forceps, blotted, and placed i n p l a s t i c p e t r i dishes (15 x 100 mm) containing 25 ml of s t e r i l e seawater (28°/oo, with pe n i c i l l i n / s t r e p t o m y c i n - 300 mg/l each). Experimental p e t r i dishes were inoculated with 1 ml of suspension from 6 day old KMV-slush (see Appendix A) cultures of Schizochytrium aggregatum, Thraustochytrium motivum and Ulkenia sp. RC02-79. Control plates with s u r f a c e - s t e r i l i z e d a l g a l tissue i n s t e r i l e seawater, but not inoculated with Thraustochytrids, were prepared. S t e r i l e seawater/pine pollen (SW/P) plates were inoculated with the Thraustochytrids to assure the v i a b i l i t y of these organisms. A l l plates were incubated at room temperature (22-25°C) i n diff u s e sunlight. After 72 hours of incubation, the following observations were made (to 100X magnification): growth of Thraustochytrids (in SW, on a l g a l tissues, on pollen); growth of higher fungi; and general condition of the a l g a l tissue i t s e l f . Select portions of the a l g a l tissue (0.5-1.0 cm long) were then removed and processed for scanning electron microscopy (SEM). Fixation was car r i e d out for 12 hours at 4°C [1% acrolein, 1% gluteraldehyde; 0.01 M sodium cacodylate buffer; i n 85% (approximately 28°/oo) seawater; pH 7.3 (Chris Patton, pers. comm.)]. The fixed tissues were rinsed i n deionized water and dehydrated i n a graded alcohol series (ethanol/deionized water: 10, 20, 30, 50, 70, 95, 100%) followed by a l / l solution of absolute ethanol/acetone. Al g a l 148 tissues were dehydrated at room temperature for 30 minutes each step. After c r i t i c a l - p o i n t - d r y i n g i n C0 2 (Dupont Sorvall), the tissue specimens were mounted on stubs using s i l v e r paint. Mounted tissue specimens were sputter-coated with gold [30 seconds at 25 mA; Denton (DV-502) vacume evaporator equipped with a DSM-5 sputter module] p r i o r to examination i n a Hitachi S450 scanning electron microscope operating at 20 kV. RESULTS A. SURFACE STERILIZATION IN 0.3% CHLOROX Thraustochytrids developed i n two out of four a l g a l tissue control cultures, i n d i c a t i n g that the surface s t e r i l i z a t i o n treatment was not very e f f e c t i v e i n eliminating these organisms from the a l g a l tissues. The a l g a l tissues themselves were i n many cases obviously damaged by the surface s t e r i l i z i n g solution. Within 24 hours of treatment, damage to the a l g a l tissues was noted as a bleaching of normal th a l l u s pigmentation (becoming green), followed by deterioration of the tissues. In a l l cases the detrimental e f f e c t s were r e s t r i c t e d to f e r t i l e a l g a l t h a l l i , and were l o c a l i z e d i n the immediate v i c i n i t y of carposporangia or tetrasporangia. The only vegetative/male a l g a l tissue used (Rhodoglossum affine) displayed no deterioration within the time of the experiment (72 hours). B. GROWTH OF HIGHER FUNGI Filamentous fungi often developed on the a l g a l tissues 149 incubated i n s t e r i l e seawater. In t h i s experiment, however, the fungi were not i s o l a t e d and further characterized. A l l cultures of Gelidium c o u l t e r i gave r i s e to at least one d i s t i n c t fungal 'colony'. Only a single culture of Rhodoglossum a f f i n e supported fungal development. These results show that: 1) while the s t e r i l i z a t i o n procedure damaged some of the a l g a l tissues i t did not eliminate mycelial fungi from within and/or on these tissues; and 2) when treated i n the same manner, tissues of G^ c o u l t e r i gave r i s e to more fungi than t i s s u e s of R. a f f i n e (p < 0.005). C. THRAUSTOCHYTRID DEVELOPMENT AND SCANNING ELECTRON MICROSCOPY After 72 hours of incubation, the v i a b i l i t y of the three 'introduced' Thraustochytrid species was confirmed by examination of t h e i r respective SW/P cultures. Development of Ulkenia sp. RC02-79 on pine pollen was noticeably slower than Schizochytrium  aggregatum and Thraustochytrium motivum. In a l l experimental cultures, growth of the Thraustochytrids could be observed (via l i g h t microscopy) on the a l g a l surfaces aft e r 72 hours of incubation. Thraustochytrid development was often most abundant on a l g a l tissue nearest the seawater/air interface. SEM observations confirmed the growth of the three Thraustochytrid species on the surfaces of a l l a l g a l tissue types (Figs. 8-15). Although mature sporangia of a l l Thraustochytrids were observed, they were rare i n comparison to the numerous young developing sporangia. This was presumably due to the r e l a t i v e l y short incubation period (72 hours). In some cases i t appeared 150 that a s l i g h t shrinkage of the a l g a l tissue (especially Rhodoglossum affine; buckled surface), and perhaps also the Thraustochytrid t h a l l i , occurred during c r i t i c a l - p o i n t - d r y i n g (Figs. 12, 13, and 15). Young developing sporangia of Thraustochytrium motivum and Ulkenia sp. RC02-79 had extensive branching and anastomosing ectoplasmic networks a r i s i n g from a single location on the thallus near the substratum (Figs. 12-15). Schizochytrium  aggregatum produced very extensive ectoplasmic networks which obscured the surface of the alga completely i n some areas (Figs. 9-11). The ectoplasmic networks produced by S^ aggregatum did not always aris e from a single d i s t i n c t location on the t h a l l u s surface, but instead often appeared to completely envelop the thallus, giving r i s e to small branches at various locations (most often near the substratum) (Fig. 11). In a l l cases examined, the ectoplasmic networks were observed to spread out upon the surface of the a l g a l tissue, often reaching considerable distances (up to 50 urn i n T\_ motivum; Fig. 12). Repeated branching resulted i n very fine ectoplasmic net elements, resolved down to 0.02 um and l e s s i n diameter (Figs. 11 and 13). Careful SEM examination of the three Thraustochytrids on the a l g a l surfaces revealed no obvious penetration of the a l g a l tissues by ectoplasmic networks. However, i t was often very d i f f i c u l t to resolve the exact termination of very fine ectoplasmic net elements. Also the nature of the substratum association beneath the ectoplasmic net elements and the Thraustochytrid t h a l l i could not be observed. Figures 8-11 Scanning electron micrographs of Thrasutochytrium motivum and Schizochytrium aggregatum on the surfaces of Rhodoglossum a f f i n e and Gelidium c o u l t e r i . Figure 8. Abundant growth of T. motivum on a small branch of G. c o u l t e r i (cystocarpicTT Bar = 5 um. Figure 9. Young sporangia (monads and diads) of S^ aggregatum on R^ a f f i n e (cystocarpic). Note the very extensive ectoplasmic network development on the surface of the R.  a f f i n e thallus, and extending onto the epiphytic diatom. Bar = 5 um. Figure 10. Clusters of S^ aggregatum on the surface of G.  c o u l t e r i (tetrasporangial). Bar = 5 um. Figure 11. Higher magnification of a portion of Figure 10. Note the extremely fine ectoplasmic net elements on the surface of G^ c o u l t e r i and also + surrounding the 'sporangia' of S^_ aggregatum. Bar = 5 pm. 152 Figures 12-15 Scanning electron micrographs of Thraustochytrium motivum and Ulkenia sp. RC02-79 on the surface of Rhodoglossum affine. Figure 12. Young sporangia of T\_ motivum on the surface of R.  a f f i n e (vegetative/male). Note buckled surface of the a l g a l thallus. Arrow indicates sporangium shown i n Figure 13. Bar = 5 pm. Figure 13. Higher magnification of a portion of Figure 12. Note highly branched ectoplasmic net systems. Bar = 5 pm. Figure 14. Sporangia of Ulkenia sp. RC02-79 intermixed with epiphytes (diatoms and ?-Leucothrix sp.) on the surface of R. a f f i n e ( c y s t o c a r p i c ) . Bar = 50 pm. Figure 15. Higher magnification of the central portion of Figure 14. Group of young sporangia displaying extensive ectoplasmic net development. Bar = 5 pm. 154 155 DISCUSSION It should be stated at the outset that the results of the present study were obscured by an oversight i n experimental methods. Inoculation of the s t e r i l e seawater-algal tissue cultures with Thraustochytrids should not have been performed using a KMV-slush culture of these organisms. I t i s becoming increasingly apparent that the n u t r i t i o n a l requirements of Thraustochytrids are extremely low (Vishniac, 1956). A 1 to 25 d i l u t i o n of KMV-slush i n s t e r i l e seawater may have supplied enough nutrients to support active growth of the Thraustochytrids throughout the time of the experiment (72 hours). Thus, the observations made i n t h i s experiment depict luxuriant growth of the Thraustochytrids on the a l g a l surfaces, but growth was probably enhanced by nutrients i n the seawater medium. Thraustochytrid development was much more conspicuous on the a l g a l t i s s u e s than i n the medium or on the w a l l s of the p e t r i plates. This 'preferred' d i s t r i b u t i o n may have been due to the release of dissolved organics from the a l g a l tissues, p a r t i c u l a r l y since they were somewhat damaged by the surface s t e r i l i z a t i o n procedure. It i s also possible that the observed d i s t r i b u t i o n was i n response to increased oxygen tension (cf. Vishniac, 1955). However, the a l g a l tissues did not f l o a t , and one might assume that the side wall of the p e t r i plate would o f f e r an equally suitable substrate. The a l g a l tissues themselves may have been l i b e r a t i n g a l i m i t e d amount of oxygen via photosynthesis. 156 SEM observations of these Thraustochytrid/algal associations revealed extensive development of ectoplasmic net systems which functioned i n attaching the Thraustochytrids to the a l g a l surfaces, but they did not obviously penetrate the tissues. I f any penetration occurred, i t was obscured from surface view, and transmission electron microscopy would have been necessary to detect i t . The very extensive development of surface-associated ectoplasmic networks i s also i n d i c a t i v e of the function of these structures i n the as s i m i l a t i o n of nutrients (Perkins, 1973b; cf. K l i e and Mack, 1968). The production of a large ectoplasmic membrane surface area undoubtedly enhances the absorption of available nutrients. S i m i l a r studies have been performed i n which various marine algal, marine invertebrate and marine or t e r r e s t r i a l vascular plant tissues were offered as substrata to Thraustochytrids and Labyrinthulids i n s t e r i l e seawater cultures. Goldstein and co-workers (Goldstein, 1963a, 1963b, 1963c; Goldstein and Belsky, 1964) examined f i v e Thraustochytrid species, including Thraustochytrium motivum and Schizochytrium aggregatum, and found v i r t u a l l y no development of mature sporangia on the marine a l g a l tissues tested. Several of these Thraustochytrids displayed l i m i t e d development on h e a t - k i l l e d portions of certain marine algae. In contrast, boiled vascular plant tissues (Zea mays, Poa  pratensis and Phragmites sp.) supported luxuriant sporangial development of those Thraustochytrids tested (Goldstein, 1963b, 1963c). Perkins (1973b) studied the growth of Thraustochytrids and Labyrinthulids on natural substrata i n estuarine water, and 157 further examined the nature of conclusive growth associations with transmission electron microscopy (TEM). His results agree with those of Goldstein (1963a, 1963b, 1963c) and Goldstein and Belsky (1964) i n that good growth of Thraustochytrium motivum and Schizochytrium aggregatum was observed on vascular plant substrata, whereas no or very l i m i t e d development occurred on the marine algae tested. S^ aggregatum did, however, exhibit "marginal" growth on the marine algae Bryopsis hypnoides Lamour. and G r i n n e l l i a americana Harvey. Both Thraustochytrids grew well on the three invertebrate tissue explants tested (Perkins, 1973b). Perkins' (1973b) TEM observations of these organisms demonstrate that the actual penetration of vascular plant and invertebrate tissues i s often accomplished by very f i n e (0.3 pm) branches of the ectoplasmic networks. He did not examine any of the marine algae tested v i a TEM. Studies concerning the physiological c a p a b i l i t i e s of Thraustochytrids to u t i l i z e s p e c i f i c carbon compounds should lend insight i n t o t h e i r colonization and u t i l i z a t i o n of d i f f e r e n t substrata i n natural habitats. Carbon compounds found to support good growth of most Thraustochytrids include: D-glucose, cellobiose, maltose, dextran, starch and glycerol. While starch i s generally hydrolized, c e l l u l o s e i s not (Bahnweg, 1979a, 1979b; Goldstein, 1963a, 1963b, 1963c; Goldstein and Belsky, 1964). The brown a l g a l storage product laminarin supports good growth of many species, including Thraustochytrium aggregatum, T. motivum and Schizochytrium aggregatum (Bahnweg, 1979b). Other sugars and 158 polysaccharides found i n the c e l l walls of marine algae (D-xylose, D-galactose, D-galacturonate, D-mannose, mannan, poly-galacturonate, xylan; see Mackie and Preston, 1974) support 'active' growth of r e l a t i v e l y few Thraustochytrids. The strains of Schizochytrium aggregatum studied by Bahnweg (1979b) were the most v e r s a t i l e i n t h i s respect, displaying good growth on D-galactose, D-galacturonate, D-mannose and mannan. Growth of Thraustochytrium motivum was not supported by any of these carbon sources (Bahnweg, 1979b; Goldstein, 1963a). There i s no information concerning the a b i l i t i e s of Thraustochytrids to hydrolize the complex a l g a l polysaccharides alginate, agar (agarose) or carrageenan. The results described above suggest that such c a p a b i l i t i e s are l i k e l y to be rare or absent. These physiological studies also indicate that c e l l u l o s e and c h i t i n are not hydrolized. However, other studies have demonstrated that Thraustochytrid ectoplasmic networks can a c t i v e l y penetrate pine pollen, vascular plant c e l l walls, and the c u t i c l e of brine shrimp (Bremer, 1976; Perkins, 1973b). Certainly further studies along these lines are desirable. CONCLUSIONS The accumulating evidence suggests that Thraustochytrids are ubiquitous marine (or h a l o p h i l i c ) microorganisms which can be i s o l a t e d from marine and estuarine water, s o i l s , sediments, invertebrates, vascular plants, algae, and other exotic substrata which fi n d t h e i r way into marine (or marine influenced) habitats (Amon, 1978; Bremer, 1976; Clokie, 1970; Polglase, 1980; Sparrow, 159 1969; Wagner-Merner, et al., 1980). Investigations of marine vascular plants and algae have most c e r t a i n l y demonstrated an abundance of Thraustochytrids (Booth and M i l l e r , 1968; Clokie, 1970; F u l l e r , et al., 1964; Haythorn, et al., 1980; Sparrow, 1969; Vishniac, 1956; Volz and Jerger, 1972; Volz, et al., 1976; present study). I t has yet to be demonstrated that the r e l a t i o n s h i p between Thraustochytrids and marine algae i s an 'intimate' one or that Thraustochytrids have the c a p a b i l i t y of a c t i v e l y extracting nutriment from healthy a l g a l tissues (Clokie, 1970; Sparrow, 1969). In such an association, the l i m i t e d n u t r i t i o n a l requirements of a Thraustochytrid could also be supported by substances obtained from seawater, dissolved organics leaching or d i f f u s i n g out of the a l g a l tissues, substances made available by the a c t i v i t i e s of other members of the microbial community. Most investigators of Thraustochytrid ecology would undoubtedly agree with a portion of the hypothesis presented by C l o k i e (1970) that . . . "the l i f e r o l e of these organisms i s that they are nondemanding saprobes with d i s t i n c t a f f i n i t i e s f o r s o l i d surfaces . . ." (Clokie, 1970; F u l l e r , et al., 1964; Goldstein, 1963a; Sparrow, 1969; Vishniac, 1956). The results of t h i s study are considered inconclusive, but they lend support to the premise that, at least for the Thraustochytrids and algae tested, Thraustochytrids are not active parasites of marine red algae. 160 VI. FINAL DISCUSSION AND CONCLUSIONS The results of t h i s study demonstrate that both attached and cast (decomposing) tissues of the marine red algae Rhodoglossum  a f f i n e and Gelidium c o u l t e r i support r e l a t i v e l y r i c h and diverse biotas of heterotrophic fungi and 'protists'. As a preface to a discussion of the significance of these observations, and the possible a c t i v i t i e s of these organisms, I would l i k e to summarize the sources from which these microbes could obtain nutriment. Those sources which are apparent to me include: 1) L i v i n g a l g a l tissues (biotroph). 2) Damaged and/or dead tissues on an otherwise 'healthy' a l g a l t h a l l u s (necrotroph, perthophyte). 3) Dead organic matter; p a r t i c u l a t e and/or dissolved organic matter (saprobe). In natural populations of these i n t e r t i d a l algae possible sources of POM and/or DOM include: A) Substances present i n the + surrounding seawater. B) Substances released from the a l g a l tissues themselves. C) Substances made available by the a c t i v i t i e s of other microorganisms present i n the community. 4) Other l i v i n g microorganisms. Perhaps a special type of biotrophy i n which other microbes are 'preyed-upon' and/or otherwise assimilated. Certainly none of these nutrient sources are mutually exclusive. I t i s very possible that a heterotrophic organism could obtain sustenance from any combination of these sources. 161 The release of dissolved organic matter by 'healthy' a l g a l tissues (point 3B) has been reported by numerous investigators (Hellebust, 1974; Khailov and Burlakova, 1969; Sieburth, 1969), and i t appears that marine algae can exude and/or r e l e a s e a substantial amount of t h e i r gross productivity to the surrounding environment. Khailov and Burlakova (1969) calculated that the amount of t o t a l dissolved organic matter released per year by the red algae which they studied (mostly filamentous forms) was approximately 38% of the gross production. Sieburth (1969) reported s i m i l a r carbon release rates (mg exudate/100 g dry wt./hour), and also demonstrated that the amount of DOM released not only varies between algae, but also with the physiological state of the alga (seasons of rapid growth, photosynthesis) as well as with 'physical' stress (water s a l i n i t y , water temperature, desiccation, reimmersion, rain). These 'extracellular products' not only include carbon compounds, but also nitrogen and 'growth factors' (Hellebust, 1974; Sieburth, 1969). This evidence suggests that the surfaces of marine algae are + constantly releasing organic 'nutrients' which could support a substantial population of surface-associated saprobic microbes (Kong and Chan, 1979; Seshadri and Sieburth, 1971, 1975; Sieburth, 1969). Antagonistic, syntrophic, and competitive interactions undoubtedly occur between marine heterotrophic microorganisms (saprobes), however there i s l i t t l e information available concerning t h i s subject. Of i n t e r e s t to t h i s study i s the p o s s i b i l i t y that bacteria either prepare the a l g a l substratum for u t i l i z a t i o n by, or release substances which are required by, other members of the microbial community (fungi, p r o t i s t s , other bacteria). Certainly bacteria are active decomposers i n thi s habitat (e.g. Quatrano and Caldwell, 1978), and such interactions may well exist. Since the r e l a t i v e l y recent description of the f i r s t Thraustochytrid (Sparrow, 1936) and the development of pure culture i s o l a t i o n techniques for Thraustochytrids and Labyrinthulids (Watson and Ordal, 1957), these organisms have become known to mycologists as not only a problematic group, but also as some of the most ubiquitous microbes i n coastal marine habitats (see Bremer, 1976; Pokorny, 1967). As more researchers employ techniques to i s o l a t e these organisms, more evidence accumulates to substantiate Vishniac's (1956) statement that . . . "the lower marine fungi [Labyrinthulids, Thraustochytrids and Phycomycetes] occupy e s s e n t i a l l y the same ecological niche as marine saprophytic bacteria." [my i n s e r t ] . Like bacteria, Thraustochytrids and Labyrinthulids have an a f f i n i t y for surfaces, but unlike some bacteria, they are s t r i c t l y aerobic (Porter, 1967; Vishniac, 1956; unpubl. obs.). While members of the genus Labyrinthula may not yet be considered quite as 'versatile' as Thraustochytrids, they are known from a wide variety of habitats (Amon, 1978; Anastasiou and Churchland, 1969; Meyers, et al., 1965; Newell, 1976; Perkins, 1973a; Pokorny, 1967). There i s l i t t l e or nothing known about substratum 'preferences' i n Thraustochytrids and Labyrinthulids, i f , i n fact, they occur. I t i s of intere s t that Labyrinthula spp., most 163 c l o s e l y resembling the "Vishniac Strains" described from marine algae by Watson (1957), were the most common organisms is o l a t e d from the i n t e r t i d a l red algae examined i n t h i s study. I t i s also noteworthy, i n fact surprising, that the d i v e r s i t y of Thraustochytrids i s o l a t e d from natural populations of these red algae was so low. Thraustochytrium motivum (especially) and Schizochytrium aggregatum were by far the predominant Thraustochytrids i s o l a t e d from two samples of these marine algae c o l l e c t e d 15 months apart. Previous reports of Thraustochytrids associated with marine algae have shown a wide variety of Thraustochytrid species i s o l a t e d from c o l l e c t i o n s of a wide variety of algae (Haythorn, et al., 1980; Sparrow, 1969; Volz and Jerger, 1972). Haythorn and co-workers (Haythorn, et al., 1980) reported that p r o l i f e r o u s Thraustochytrids (of which T\_ motivum i s one) were the most common type i s o l a t e d from 'cast' seaweeds. There may be some significance to the predominance of t h i s Thraustochytrid developmental pattern on what, at least to a Thraustochytrid, could be considered a stable substratum. Further studies concerning the 'persistence' (including abundance and species composition) of the Thraustochytrid/algal associations reported i n the present study would c e r t a i n l y be of inte r e s t . Previous inoculation studies i n which Thraustochytrid species were placed into culture with various natural substrata, including marine algae, have been discussed elsewhere (see Discussion, Section V). Generally, those Thraustochytrids tested, including Thraustochytrium motivum and Schizochytrium 164 aggregatum, grew well on vascular plant tissues and marine invertebrate tissue explants, but displayed "marginal" or no growth on the marine a l g a l t i s s u e s t e s t e d . In view of the observation of Vishniac (1956), that the amount of soluble organic material required to produce a single t h a l l u s of a Thraustochytrid i s on the order of 1 ng, the res u l t s of the present inoculation study are indeed questionable. Observations made during t h i s experiment did suggest that Thraustochytrids exhibit a chemotactic response to 'organic' substrata. The Thraustochytrids produced very extensive ectoplasmic networks on the a l g a l surfaces. Presumably these ectoplasmic networks functioned not only i n attaching the Thraustochytrids to the al g a l surface(s), but also i n the a s s i m i l a t i o n of dissolved nutrients. The n u t r i t i o n a l studies of Bahnweg (1979a, 1979b), Goldstein (1963a, 1963b, 1963c) and Goldstein and Belsky (1964) indicate that those Thraustochytrids tested would be unable to ac t i v e l y penetrate the polysaccharide c e l l walls of red algae. However, certai n Thraustochytrids, e s p e c i a l l y Schizochytrium aggregatum, were shown to u t i l i z e simple sugars and less complex molecules which are present i n the c e l l w a l l s of red algae (Bahnweg, 1979b). Different i s o l a t e s of the same species may also vary as to certain aspects of t h e i r n u t r i t i o n . There i s , as yet, no di r e c t evidence that Thraustochytrids are biotrophic parasites of marine a l g a l macrophytes. I suggest that the Thraustochytrids observed i n t h i s study occupy a saprobic mode of habit on the a l g a l surfaces, and that they obtain organic nutriment from one or a combination of the 165 following sources: 1) released from the a l g a l tissues; 2) present i n the + surrounding seawater; or 3) perhaps, made available by the a c t i v i t i e s of other microbes. Damaged and/or senescing a l g a l tissues i n early stages of decay may of f e r stronger chemotactic s t i m u l i for zoospore attraction, and Thraustochytrids are undoubtedly also associated with these t i s s u e s (necrophytes, ?-perthophytes). Many investigators have suggested that species of Labyrinthula may a c t i v e l y p a r a s i t i z e marine a l g a l macrophytes and estuarine vascular plants. This i s e s p e c i a l l y true of Labyrinthula's association with the 'wasting disease' of eelgrass (Zostera marina) (see Johnson and Sparrow, 1961). Perkins (1973b) demonstrated that the ectoplasmic nets produced by several Labyrinthulids can a c t i v e l y penetrate apparently l i v i n g c e l l s of Zostera marina and Spartina. The presence of Labyrinthula growing within c e l l s of Zj_ marina has also been demonstrated by other investigators (Porter, 1967). Evidence for 'parasitism' of marine a l g a l macrophytes i s less convincing, and consists mainly of observations of Labyrinthula spp. within c e l l s of brown algae (Laminarians) held i n aquaria (Jepps, 1931). Under these conditions the a l g a l tissues may have been predisposed to i n f e c t i o n (Andrews, 1976; Jepps, 1931). From the resu l t s of h i s inoculation studies, Perkins (1973b) reported that Labyrinthuloides minutum (as Labyrinthula minuta) displayed a "conclusive" growth response i n association with the marine red alga G r i n n e l l i a americana. Perkins (1973b) did not examine t h i s association using transmission electron microscopy. Certainly 166 more work i n t h i s area i s desirable. There i s considerable evidence that members of the genus Labyrinthula can 'graze' upon a variety of other microorganisms. The l i s t of microorganisms which can be u t i l i z e d as food sources includes: various bacteria, yeasts, diatoms, u n i c e l l u l a r green algae, and i n one instance, a member of the genus Thraustochytrium (Jepps, 1931; K l i e and Mach, 1968; Porter, 1967; Watson, 1957; Schnieder, 1969; unpubl. obs.). The s p e c i f i c microorganisms which can be u t i l i z e d may vary between di f f e r e n t species of the genus Labyrinthula. Very scanty evidence exists to suggest that members of the genus Labyrinthuloides have this c a b a b i l i t y (Perkins, 1974b), and i t has not been demonstrated f o r any Thraustochytrids. Unfortunately, data concerning the 'predatory' a c t i v i t i e s of the Vishniac Strains of Labyrinthula are not available. This evidence does suggest that species of Labyrinthula may exi s t as grazers or 'micro-predators' on a wide variety of microorganisms. The surfaces of marine algae c e r t a i n l y support a d i v e r s i t y of microorganisms which could serve as a source of nutriment for the species of Labyrinthula found i n t h i s habitat. There i s only l i m i t e d evidence to support the contention that Labyrinthulids are parasites of marine red algae (Perkins, 1974b). Studies have also shown that species of Labyrinthula can assimilate c e r t a i n u n i c e l l u l a r green algae i n laboratory culture (on agar plates) (unpubl. obs.). Further studies along these l i n e s with other algae under more 'natural' conditions are desirable. In the present study, Labyrinthula spp. were the most conspicuous members of the microbiota i s o l a t e d from Rhodoglossum 167 a f f i n e and Gelidium c o u l t e r i . My res u l t s also indicate that these organisms are p r i m a r i l y associated with the a l g a l surface(s). I f Labyrinthula spp. do p a r a s i t i z e healthy c e l l s of these red algae, t h e i r a c t i v i t i e s do not appear to have a detrimental e f f e c t on the o v e r a l l ' v i t a l i t y ' of the host. It seems more l i k e l y that Labyrinthulids occupy a saprobic habit on the surface(s) of marine red algae. In addition to the sources of dissolved organic matter l i s t e d above for Thraustochytrids, species of Labyrinthula may also exploit the r i c h microbiota of surface-associated microorganisms. Available information concerning the ecology of Hyalochlorella marina i s confused and limited. Poyton (1970b) demonstrated that t h i s organism apparently 'prefers' marine a l g a l surfaces; i t was not is o l a t e d from sediments and was rarely i s o l a t e d from the surrounding seawater. The results of this study show that at l e a s t some of the Hj_ marina c e l l s are more f i r m l y bound to the al g a l 'surfaces' than has been previously reported (Poyton 1970b). Studies of the morphology and ultras t r u c t u r e (Alderman, 1974) of t h i s organism give no ind i c a t i o n of how such a 'spherical' c e l l could become f i r m l y bound to the surface of an alga. My studies of IL_ marina i n culture include observations of amoeboid and plasmodial stages which may be of significance to i t s ecology and n u t r i t i o n a l habits. At present, Hyalochlorella marina appears to be a saprobe associated with the a l g a l surfaces. There has been considerable debate over what constitutes a 'marine fungus' and over the p o s s i b i l i t y that ' t e r r e s t r i a l ' fungi 168 can be 'active' i n marine habitats (Hughes, 1975; Kohlmeyer and Kohlmeyer, 1979). 'Terrestrial' species have been reported from a very diverse range of marine and estuarine environments (e.g. Meyers, et al., 1965; M i l l e r and Whitney, 1981a, 1981b; Muntanola-Cvetkovic and Ristanovic, 1980; Newell, 1976; Sparrow, 1936; Steele, 1967). However, evidence to support active roles for ' t e r r e s t r i a l ' species i n these habitats i s l i m i t e d (Meyers, et al., 1965; Newell, 1976). Studies suggesting possible roles for ' t e r r e s t r i a l ' species i n marine or open coastal environments were lacking u n t i l very recently ( M i l l e r and Whitney, 1981a, 1981b). Very few thorough surveys of the symbiotic or saprobic fungi associated with 'cast' and/or natural populations of marine a l g a l macrophytes have been conducted (Hyathorn, et al., 1980; M i l l e r and Whitney, 1981a; Sutherland, 1916). I t appears that the general consensus among marine microbiologists has been that, compared to other microorganisms (bacteria), higher marine fungi are not active as saprobes of marine a l g a l macrophytes and they play l i t t l e role i n the decomposition of these substrata (Chesters, et al., 1956; Haythorn, et al., 1980; Kohlmeyer and Kohlmeyer, 1979). T e r r e s t r i a l species i s o l a t e d from marine algae have generally been considered 'inactive* on these substrata. However, there i s accumulating evidence which suggests that both marine and ' t e r r e s t r i a l ' fungi may have at least l i m i t e d saprobic a b i l i t i e s i n association with marine algae ( M i l l e r and Whitney, 1981a; Schatz, 1980; Tubaki, 1969; Wainwright and Sherbrock-Cox, 1981). Bacteria found associated with marine algae are known to 169 produce enzymes capable of breaking down complex a l g a l polysaccharides (Humm, 1946; Kong and Chan, 1979). Actinomycetes have also been shown to u t i l i z e the brown a l g a l polysaccharides laminarin, Na-alginate and Ca-alginate (Chesters, et al., 1956). Many of the 'active' bacteria and actinomycetes reported i n these studies were ' t e r r e s t r i a l ' species, i s o l a t e d from the a l g a l fronds or from t e r r e s t r i a l s o i l s (Chesters, et al., 1956; Kong and Chan, 1979). D e f i n i t i v e research concerning the a b i l i t i e s of higher fungi to u t i l i z e a l g a l polysaccharides i s i n i t s early stages. Available reports show that c e r t a i n marine and t e r r e s t r i a l higher fungi have these c a p a b i l i t i e s , although they may be l i m i t e d ( M i l l e r and Whitney, 1981a; Payton, et a l . , 1976; Payton and Roberts, 1979; Wainwright, 1980; Wainwright and Sherbrock-Cox, 1981). A point which should be emphasized i s that microorganisms of both marine and t e r r e s t r i a l o r i g i n are capable of u t i l i z i n g the complex polysaccharides found i n marine algae. In the present study, i s o l a t i o n s from f i e l d - c o l l e c t e d a l g a l tissues yielded a very high d i v e r s i t y (70 + species) of higher fungi, a l l of which were isola t e d at low frequencies. The vast majority were Fungi Imperfecti considered to be of t e r r e s t r i a l o r i g i n . Only four (?-five) of the fungi i s o l a t e d are presently considered marine, and only two of these (Dendryphiella s a l i n a and Sigmoidea l i t t o r a l i s sp. nov.) were i s o l a t e d more than once. Other investigators who have surveyed fungi associated with natural populations of marine algae have reported s i m i l a r results ( M i l l e r and Whitney, 1981a). Most of the fungi which were i s o l a t e d r a r e l y (over 60% were 170 single i s o l a t i o n s ) are considered to be t e r r e s t r i a l s o i l or decay fungi, or parasites of higher plants, which were encountered because 'wandering' spores were present on the surfaces of the al g a l t h a l l i . Any 'activity' of these fungi i n t h i s habitat i s questionable. However, M i l l e r and Whitney (1981a) have shown that conidia of higher fungi may germinate and produce l i m i t e d (saprobic) growth on the surfaces of marine algae. This observation was attributed to the presence of l i m i t e d organics on the a l g a l surface(s), either exuded by the alga i t s e l f ( M i l l e r and Whitney, 1981a) or, perhaps, made a v a i l b l e by the a c t i v i t i e s of other microbes. M i l l e r and Whitney (1981a) suggest that 'released organics' might o b l i t e r a t e the mycostatic e f f e c t that seawater i s known to have on the spores of many t e r r e s t r i a l fungi (Kirk, 1980). Certain of the less frequent ' t e r r e s t r i a l ' fungi i s o l a t e d i n th i s study have been reported from marine or marine-influenced habitats by other investigators. Some may be active on other substrata (i.e. not algal) i n these habitats [e.g. Phialophora spp. and Leptosphaeria sp. on wood (Leightley, 1980)]. Other genera which are l i s t e d , but not i d e n t i f i e d to species, are known to have marine representatives [e.g. Phoma spp. (Kohlmeyer and Kohlmeyer, 1979)]. Based on i s o l a t i o n s from f i e l d - c o l l e c t e d algae, the following fungi were most commonly encountered: yeasts and yea s t - l i k e fungi, Acremonium sp. 019-78, Cladosporium  cladosporioides, Dendryphiella salina, P e n i c i l l i u m spp., Phoma sp. (Group 1), Sigmoidea l i t t o r a l i s sp. nov., and Unidentified hyphomycete 044-78. With the exception of Sigmoidea l i t t o r a l i s 171 and Unidentified hyphomycete 044-78, a l l of these genera/species have been reported from marine algae by other investigators (Haythorn, et al., 1980; M i l l e r and Whitney, 1981a; Sutherland, 1916). Sigmoidea l i t t o r a l i s i s a new species of marine fungus which has been is o l a t e d thus far only from marine algae. Dendryphiella s a l i n a i s a very common saprobic marine fungus known from vascular plants and algae (Haythorn, et al., 1980; Sutherland, 1916; Kolhmeyer and Kohlmeyer, 1979). The remaining taxa seem ubiquitous, and have been reported from a wide range of marine and estuarine habitats (Sparrow, 1936; Schatz, 1980; Newell, 1976; Meyers, et al., 1965; Muntanola-Cvetkovic and Ristanovic, 1980). Certain P e n i c i l l i u m and Cladosporium species have been implicated as potential saprobes of marine algae ( M i l l e r and Whitney, 1981a). Surveys of the 'saprobic' higher mycelial fungi associated with natural populations of marine algae have thus far demonstrated the presence of a very high d i v e r s i t y of imperfect fungi, most of which occur at low frequencies ( M i l l e r and Whitney, 1981a; present study). Certain of the most commonly is o l a t e d fungi display l i m i t e d a b i l i t i e s to u t i l i z e a l g a l polysaccharides as carbon sources, and, therefore, have some potent i a l as active saprobes of marine a l g a l tissues. However, even the most common fungi are of r e l a t i v e l y infrequent occurrence, and i t appears that t h e i r saprobic ' a c t i v i t i e s ' i n natural populations of marine algae are f a i r l y limited. There i s some evidence that these fungi may be growing as perthophytes i n damaged and/or senescent tissues of the a l g a l t h a l l i . 172 The results of the d r i f t study reported here o f f e r more conclusive evidence that certain of the fungi most commonly is o l a t e d from natural populations of marine algae, can be active saprobes involved i n a l g a l decomposition. In the present study, Acremonium sp. 019-78, Dendryphiella s a l i n a and Sigmoidea  l i t t o r a l i s were shown to be active colonizers of a r t i f i c i a l l y d r i f t e d decomposing tissues of Rhodoglossum a f f i n e and Gelidium  c o u l t e r i . However, these fungi were not conspicuous members of the microbial community associated with the decomposing algae u n t i l a f t e r the algae had been exposed on the beach for f i v e weeks. Again, i t i s possible that the growth of other microbes, p a r t i c u l a r l y bacteria, was required to 'condition' the a l g a l substrata for fungal assimilation. The use of the ' d r i f t bag' technique to study decomposition processes i s subject to c r i t i c i s m (Park, 1974). Perhaps very l i t t l e of the t o t a l 'seaweed' production which decomposes annually remains s t a b i l i z e d i n one place for such an extended period. This technique c e r t a i n l y requires further evaluation. Nonetheless, the present study demonstrates that higher fungi can be active saprobes of decomposing marine algae. Higher fungi (and p r o t i s t s ) as well as bacteria may provide an important source of nutriment for invertebrates which feed on decomposing algae and also for the numerous detritus consumers which exist i n coastal marine communities. The implication of an Acremonium species as an active saprobe i n a marine or marine-influenced habitat w i l l require further substantiation. This species requires i d e n t i f i c a t i o n and should be investigated as to i t s physiological responses 173 ( s a l i n i t y , etc.) and a b i l i t i e s to u t i l i z e a l g a l polysaccharides. Acremonium species (as Cephalosporium) have been previously reported from marine algae (Andrews, 1977; Schatz, 1980; M i l l e r and Whitney, 1981a; Haythorn, et a l . , 1980), and are also known from a variety of other marine or estuarine habitats (Meyers, et al., 1965; Newell, 1976; Sparrow, 1936). Studies have shown that Dendryphiella s a l i n a can u t i l i z e c ertain c e l l wall polysaccharides and storage products of brown algae (Chesters and B u l l , 1963; Wainwright and Sherbrock-Cox, 1981). Limited a b i l i t i e s to u t i l i z e red a l g a l polysaccharides have also been demonstrated for s a l i n a ( M i l l e r and Whitney, 1981a), but further studies are desirable. L i t t l e i s known about the ph y s i o l o g i c a l attributes of Sigmoidea l i t t o r a l i s , except that t h i s species displays enhanced growth on media prepared with seawater [28°/oo vs. 15°/oo or TAP (see Section VII)]. The ubiquitous genera P e n i c i l l i u m and Cladosporium are included i n nearly every l i s t of higher fungi i s o l a t e d from marine and estuarine habitats or substrata. These genera are also consistently found associated with marine algae, but l i t t l e s i g n i ficance has been placed on t h e i r presence. Recently, M i l l e r and Whitney (1981a) reported these genera as among the most common fungi i s o l a t e d from natural populations of marine algae. These investigators further demonstrated that P e n i c i l l i u m ochro- chloron, P e n i c i l l i u m simplicissimum, Cladosporium algarum, and C. cladosporioides have at least l i m i t e d a b i l i t i e s to u t i l i z e the c e l l w all polysaccharides of red and brown algae. In further studies, a 'marine* i s o l a t e of Cladosporium cladosporioides was shown to display a 'Phoma pattern' with respect to seawater s a l i n i t y and incubation temperature, a 'pattern' c h a r a c t e r i s t i c of marine fungi ( M i l l e r and Whitney, 1981b). In the present study, species of P e n i c i l l i u m and Cladosporium were among the most common fungi i s o l a t e d from natural populations of the algae, but were not among the dominant mycobiota which developed on the decomposing algae. Isolations from f i e l d - c o l l e c t e d algae show that these genera were most frequently encountered on tissues incubated i n moist chambers. Not a l l a l g a l biomass decomposes under conditions of constant or regular submergence, and I suggest that these genera may be active saprobes of a l g a l tissues cast high i n the i n t e r t i d a l which are not commonly submerged. A s i m i l a r suggestion was made by Sutherland (1916) who, based h i s surveys of fungi on decaying seaweeds, stated that . . . "Cercospora [Dendryphiella salina] plays very much the same par t along the t i d a l zone that Cladosporium [?-algarum] does above i t . " [my i n s e r t s ] . There are many possible conditions under which seaweeds can decay (e.g. Nonomura, 1978), and there i s c e r t a i n l y much more research which can be done i n t h i s area. 175 VII. DESCRIPTIONS TABLE OF CONTENTS INTRODUCTION 176 A. LABYRINTHULIDS 1. Labyrinthula 176 a. Group 1 i . Labyrinthula sp. Type LX 177 b. Group 2 177 i . Labyrinthula sp. (Type 1) 178 i i . Labyrinthula sp. (Type 2) 186 2. Labyrinthuloides a. Labyrinthuloides yorkensis Perkins 192 b. Labyrinthuloides sp. 1 196 c. Labyrinthuloides sp. RV02-80 204 3. Labyrinthulid Unidentified 210 a. Isolate SW/P GC03 210 b. Isolate RC01 15-1 214 c. Isolate GT04 3-1 218 B. THRAUSTOCHYTRIDS 1. Schizochytrium a. Schizochytrium aggregatum Goldstein and Belsky . T 223 b. Schizochytrium aggregatum Goldstein and Belsky (pigmented s t r a i n ) 22 7 2. Thraustochytrium a. Thraustochytrium aggregatum Ulken 232 b. Thraustochytrium motivum Goldstein 238 3. Ulkenia a. Ulkenia sp. RC02-80 243 C. Hyalochlorella marina Poyton 249 D. HIGHER FUNGI 1. Acremonium sp. 019-78 254 2. T r a l i a a scophylli Sutherland 259 3. Unidentified hyphomycete 044-78 260 4. Sigmoidea a. Sigmoidea marina Haythorn and Jones 266 b. Sigmoidea l i t t o r a l i s sp. nov 280 176 INTRODUCTION The number of fungi and 'protists' i s o l a t e d i n t h i s study which deserved description or taxonomic discussion far exceeded my microscope endurance and time r e s t r i c t i o n s . Indeed, certain of the descriptions included are rather cursory due to l i m i t e d observations. I have attempted to include those organisms which are: 1) poorly known and, therefore, of i n t e r e s t to marine mycologists; 2) obviously new species; 3) species of questionable taxonomic status and of i n t e r e s t to t h i s 'f i e l d * study as a whole. This l a s t group, I'm afraid, i s not f u l l y represented. As indicated i n the Table of Contents, there has been some pr e d i l e c t i o n towards the study of Labyrinthulid and Thraustochytrid organisms. I must confess that I am intrigued by these ' p r o t i s t s * . The descriptions of higher fungi a l l follow a s i m i l a r format. The c u l t u r a l c h a r a c t e r i s t i c s of the organism are described f i r s t , followed by morphology, and ending i n a discussion. In the descriptions of Labyrinthulids, Thraustochytrids and Hyalochlorella marina the c u l t u r a l c h a r a c t e r i s t i c s and morphology are often intermingled. A. LABYRINTHULIDS 1. Labyrinthula Members of the genus Labyrinthula were by far the most common organisms encountered during t h i s study. I t was not possible to bring a l l i s o l a t e s into pure culture for observation 177 and characterization, but examination of over 70 representative i s o l a t e s allowed t h e i r description and separation into two d i s t i n c t groups, including 3 types, of Labyrinthulas. a. Group 1 i . Labyrinthula sp. Type LX (Watson, 1957) Isolates of t h i s group were encountered very rarely i n Sample 3 only. In cultures grown on SSA (1% and 2%) c e l l s of these i s o l a t e s are always hyaline, fusiform or spindle-shaped, and measure 10.0-18.0 x (2.4-) 3.6-4.8 pm. Propagation i s by means of simple c e l l d i v i s i o n ( d i v i s i o n plane oblique), and no rounded c e l l s , c e l l u l a r aggregations (sori or pseudosori), nor zoospores were ever observed. Labyrinthula sp. Type LX i s o l a t e s were able to u t i l i z e the yeast Rhodotorula aurantiaca (Saito) Lodder as a food source i n monoxenic culture. Labyrinthula sp. Type LX w i l l not be described further. b. Group 2 Labyrinthula i s o l a t e s included i n t h i s group were easily distinguished from Type LX i s o l a t e s even i n 'rough' cultures by t h e i r c h a r a c t e r i s t i c formation of aggregations or mounds of rounded c e l l s [?-"pseudosori" (Watson, 1957)]. Two s i m i l a r yet d i s t i n c t strains were found associated with the algae studied, and are described further below. Of these, Type 1 i s very s i m i l a r to the group of i s o l a t e s thoroughly studied and described by Watson (1957) as Labyrinthula sp. Vishniac Strains. The i s o l a t e s described here as Type 2 have developmental c h a r a c t e r i s t i c s (colony development, c e l l d i v i s i o n , "pseudosorus" 178 formation) which are also very s i m i l a r to the Vishniac Strains, but t h e i r vegetative c e l l s are s i g n i f i c a n t l y smaller than those reported by Watson (1957). Unfortunately, Type 2 i s o l a t e s grew poorly i n culture and could not be maintained for extended periods of time. I t i s not possible to give an accurate frequency of occurrence for i s o l a t e s of Type 1 and Type 2. Type 1 i s o l a t e s were, however, noticeably more common and, judging from representative i s o l a t i o n s , amounted to 60-75% of the t o t a l i s o l a t e s referred to here as Labyrinthula spp. i . Labyrinthula sp. (Type 1) The following description i s based on cultures grown on SSA (1% and 2%) and GSSA media. Colonies a t t a i n a diameter of from 3.0 to 5.0 cm after 7 days of incubation at room temperature (22-25°C). Via transmitted l i g h t , colonies are diffuse; display a very f a i n t white or cream coloration i n older (central) portions; and are transparent/translucent i n the immediate area of the advancing margin (0.5-1.0 cm wide). The f a i n t coloration i n older portions of the culture results from growth beneath the agar surface and from the formation of "pseudosori". With the unaided eye the margin of the colony i s d i s t i n c t and gently undulate. Closer examination (40-200X) reveals the branching and anastomosing nature of the c o l o n i a l network (best observed just behind the advancing margin). The colony margin now appears serrate, with large clumps of vegetative 'spindle c e l l s ' often accumulating at the t i p s of major branches i n the c o l o n i a l 179 network (Fig. 16). Ectoplasmic networks are highly branched, anastomosing, and often appear as laminar sheaths or pathways (? tubes) (Figs. 17 and 18). At high magnification (400-1000X), these laminar structures were often observed to be composed of many smaller, filamentous ectoplasmic net elements aligned i n s i m i l a r orientation (Figs. 19 and 20). Sheath-like elements were also observed to break up into branching and anastomosing filaments within t h e i r outer boundaries, creating 'holes' i n the sheath. Motile spindle-shaped vegetative c e l l s are most abundant just behind the colony margin where they were observed advancing towards the outer p o r t i o n s of the colony at speeds up to 8-12 um/minute. C e l l s which were obviously motile appeared to be completely enveloped by elements of the ectoplasmic networks (within tubes?) (Fig. 17). When i n groups, motile c e l l s moved i n single f i l e along the networks or with two, rarely three, c e l l s abreast. S l i g h t l y larger, stationary c e l l s are also present just behind the colony margin. These c e l l s disassociate from the major pathways of the ectoplasmic network, cease m o t i l i t y , and enlarge, becoming subglobose, e l l i p s o i d or ovoid (Figs. 17, 19, and 20). In many instances these stationary c e l l s were observed to be i n various stages of c e l l u l a r d i v i s i o n (see below) (Fig. 20). The r e l a t i v e number of enlarged stationary c e l l s increases i n older portions of the colony u n t i l , i n central areas, i r r e g u l a r l y d i s t r i b u t e d clumps or aggregations of these c e l l s predominate and motile spindle-shaped c e l l s are absent (Figs. 21 and 22). The d i s t i n c t 'labyrinth' of major ectoplasmic pathways also 180 becomes thinner and less conspicuous i n older portions of the colony, corresponding with the decrease i n the r e l a t i v e number of motile c e l l s . In central areas of the colony the major pathways of the network dissolve and ectoplasmic networks are present only as branched filamentous elements radiating from i s o l a t e d clumps of c e l l s (Figs. 21 and 22). Motile c e l l s are fusiform, spindle-shaped, rarely + cuneiform; 7.2-12.0 x 3.2-4.8 urn; and contain numerous (2-15 +) small, opaque or r e f r a c t i l e inclusions up to 0.6 pm i n diameter. The nucleus i s prominent, 1.7-2.0 pm i n diameter, with a single c e n t r a l l y located nucleolus. 'Stationary' c e l l s are globose, subglobose, ovoid, broadly e l l i p s o i d ; 8.4-13.2 x 6.0-10.2 pm; and contain numerous (7-20 +) r e f r a c t i l e inclusions up to 1.8 pm i n diameter. Nuclei of stationary c e l l s were d i f f i c u l t to observe v i a b r i g h t f i e l d or phase microscopy of l i v e specimens because of the numerous large, highly r e f r a c t i l e , c e l l u l a r inclusions. Nuclei are generally s l i g h t l y larger (2.0-2.5 pm diameter i n uninucleate c e l l s ) than those of motile c e l l s . The size and prominence of r e f r a c t i l e c e l l u l a r inclusions was often well correlated with the presence or absence of m o t i l i t y . Motile spindle-shaped c e l l s r a r e l y contained r e f r a c t i l e inclusions over 0.6 pm i n diameter. Within several minutes afte r such a c e l l ceased m o t i l i t y , several (2-5 or more) of i t s r e f r a c t i l e inclusions were observed to have increased i n s i z e (to 1.0 pm diameter) (Figs. 17 and 20). C e l l u l a r d i v i s i o n (propagation) occurs by two d i s t i n c t Figures 16-22. Labyrinthula sp. Type 1. Figure 16. Colony margin of i s o l a t e RV03 3-1. Branching and anastomosing nature of colony structure, and clusters of c e l l s accumulating at the t i p s of major pathways. GSSA, day 12. Phase-contrast, 70X. Figure 17. Contrast between motile (in tubes?) and nonmotile c e l l s (some dividing, arrows) just behind the colony margin. Isolate RV03 3-1. GSSA, day 12. Phase-contrast, 350X. Figure 18. As Figure 17. Branching and anastomosing ectoplasmic networks. 120X. Figure 19. 'Stationary' c e l l s i n various stages of c e l l d i v i s i o n . Fine filamentous structure of ectoplasmic networks. Isolate RV03 3-1. GSSA, day 12. Phase-contrast, 370X. Figure 20. Four-celled tetrad r e s u l t i n g from successive b i p a r t i t i o n . Isolate RV03 3-1. GSSA, day 12. Phase-contrast, 1000X. Figure 21. Older (central) portion of a colony of i s o l a t e RV03 3-1. Stationary c e l l s enlarging and div i d i n g i n t e r n a l l y (young 'pseudosori'). GSSA, day 12. Phase-contrast, 380X. Figure 22. Similar to Figure 21, but i s o l a t e SW/P SW RV03. Note reduced radiating ectoplasmic networks. SSA, day 7. Phase-contrast, 320X. 182 183 processes i n these i s o l a t e s of Labyrinthula. Without the use of h i s t o l o g i c a l methods, however, i t was often d i f f i c u l t to follow nuclear behaviour and d i v i s i o n plane sequences. The following i s an interpretation from observations of l i v e specimens i n culture. Motile vegetative c e l l s were very r a r e l y observed to be i n the process of div i d i n g or to contain more than one nucleus (one occasion; oblique d i v i s i o n plane). C e l l s which had become stationary and enlarged were often observed i n various stages of a d i v i s i o n sequence which most commonly resulted i n the formation of four uninucleate c e l l s contained within a common membrane (Fig. 20). The f i r s t d i v i s i o n plane i s transverse across the median sector of the c e l l . This d i v i s i o n i s often followed immediately by the production of diagonal or transverse d i v i s i o n planes i n each of the daughter c e l l s [producing a 4-celled stage s i m i l a r to that described by Watson (1957, Fig. 71)]. Not uncommonly, however, the f i r s t (transverse) d i v i s i o n plane was observed to migrate into an oblique or nearly longitudinal orientation before further transverse or oblique d i v i s i o n planes became apparent (Fig. 17, arrows). What appeared to be 3-celled d i v i s i o n stages were also often observed. Whether 3-celled stages resulted from delayed d i v i s i o n in one of the primary daughter c e l l s or some other process i s unknown. In c e l l s apparently destined to remain stationary, the daughter c e l l s were observed to continue d i v i d i n g u n t i l clusters of f i v e to e i g h t or even more c e l l s were formed. The continuous membrane surrounding c e l l s d i viding i n t h i s manner became i n d i s t i n c t a f t e r the 4- or 6-celled stages. This d i v i s i o n process may be described as successive 184 b i p a r t i t i o n , although i t may not be synchronous or, perhaps, in some instances a second d i v i s i o n may not occur. Under normal circumstances [(3-) 4-celled stages], the resultant c e l l s remain encased within a common membrane u n t i l the d i v i s i o n sequence i s complete. They are then released as uninucleate (nucleus 1.5 um i n diameter), p o t e n t i a l l y motile vegetative c e l l s . Occasionally, structures which may have been the remains of outer membranes were f a i n t l y discernable on the agar surface. The d i v i s i o n process leading to 4-celled stages occurred predominantly i n the younger portions of the colony, within about 1.0 cm of the advancing margin. Stationary c e l l s which continued dividing (five or more c e l l s ) were more commonly observed i n older portions of the colony. This d i s t r i b u t i o n was presumably due to nutrient depletion or waste product accumulation i n the older (central) portions of the colony. The second type of d i v i s i o n process was observed i n c e l l s found i n older (central) portions of the colony. Subglobose, ovoid or e l l i p s o i d nonmotile c e l l s continue to enlarge, becoming multinucleate, and often i r r e g u l a r i n outline. Eventually these multinucleate individuals divide (presumably by progressive cleavage) into 5-12 uninucleate globose or ovoid c e l l s . The resultant c e l l s often remain together, the individuals enlarging repeatedly and d i v i d i n g i n the manner just described. Thus, i r r e g u l a r clusters or mounds of rounded c e l l s [the "pseudosori" of Watson (1957)] are formed (Figs. 21 and 22). Occasionally a few c e l l s produced from "pseudosori" reverted to motile spindle c e l l s . Although t h e i r subsequent development 185 was not observed, i t i s believed that these motile c e l l s moved only a short distance, became stationary, and enlarged to divide as described above. Under the given culture conditions, I believe that only two, or perhaps three, generations of c e l l s were produced by t h i s d i v i s i o n process before the culture stagnated. Because of numerous r e f r a c t i l e c e l l u l a r inclusions, nuclei were ra r e l y observed i n c e l l s involved i n t h i s second d i v i s i o n process. Observations were made of enlarged single c e l l s with nuclei up to 2.7 pm diameter (Watson reports up to 4.0 pm) and multinucleate stages with up to 5 nuclei at 1.5-2.0 pm diameter (Watson reports up to 16 at 1.5-2.0 pm). Enlarged multinucleate stages were found from 7.2-11.3 x 15.0-20.4 pm. Type 1 i s o l a t e s generally grew well i n culture when maintained between 15-25°C and transferred at least once a month. Growth was enhanced on GSSA (vs. SSA-1 and 2%). From my observations, the c h a r a c t e r i s t i c s displayed by Type 1 i s o l a t e s agree very well with the description of Labyrinthula sp. Vishniac Strains given by Watson (1957). Watson's (1957) description d i f f e r s from my observations i n the following respects: 1) nucleolus located adjacent to the nuclear membrane rather than i n a central location. 2) c e l l u l a r inclusions generally 1.0 pm or less i n diameter. 3) growth r e s t r i c t e d to the agar surface. 4) c e l l u l a r d i v i s i o n (successive b i p a r t i t i o n ) i n uninucleate c e l l s resulted i n four daughter c e l l s encased within 186 a single membrane (observations based on stained s l i d e preparations only). He did not describe any d i v i s i o n sequences (via successive b i p a r t i t i o n i n g ) which lead to three or more than four daughter c e l l s . i i . Labyrinthula sp. (Type 2) The following description i s based on cultures maintained on GSSA media. Growth i s restricted? agar block transfers form colonies which a t t a i n a diameter of 1.5 to 2.0 cm afte r 30 days. When the organism i s streaked onto the surface of the agar medium, smaller (to 2.0 mm af t e r 15 days), s t e l l a t e colonies are produced (Fig. 28). Via transmitted l i g h t , colonies are diffuse; display a fa i n t white to cream coloration i n older (central) portions; and are transparent/translucent at the outer margin. Faint coloration i n older areas of the colony results from growth beneath the agar surface and the formation of large, ir r e g u l a r clumps of c e l l s C?-"pseudosori" (Watson, 1957)]. The colony margin i s serrate (Figs. 23 and 28). Ectoplasmic networks are branched, anastomosing, often forming enlarged pathways (? tubes) along which spindle-shaped c e l l s move i n single f i l e (Figs. 23 and 24). Occasionally laminar sheaths or blebs of ectoplasmic material (to 7.5 um i n longest dimension) were observed adjacent to i s o l a t e d clumps of c e l l s . Motile vegetative c e l l s are e l l i p s o i d , fusiform or spindle-shaped; 3.4-6.0 (-8.0) x 2.3-5.5 um; and con t a i n n u c l e i 1.0-1.5 pm i n diameter (Figs. 23 and 26). The cytoplasm of young motile 187 c e l l s i s f a i r l y uniform, except for the presence of several (1-3) r e f r a c t i l e inclusions measuring 0.4-0.8 pm diameter. Motile c e l l s disassociate from the major pathways of the ectoplasmic network and become (?-) stationary. Stationary c e l l s enlarge to 6.0-9.5 x 5.3-7.5 um; become globose, subglobose or ovoid; and eventually divide v i a successive b i p a r t i t i o n into 4, (6) or 8 p o t e n t i a l l y motile vegetative c e l l s (Figs. 24, 25, and 27). The cytoplasm of stationary and/or di v i d i n g c e l l s contains several (1-3) large (1.3-1.8 pm diameter) and many (to 10+) smaller (0.5-0.8 pm diameter) r e f r a c t i l e inclusions. Owing to the r e f r a c t i l e nature of these c e l l s t h e i r nuclei were not e a s i l y discerned v i a l i g h t microscopy. I have interpreted the sequence of events i n c e l l u l a r d i v i s i o n as follows. Enlarged 'stationary' c e l l s divide i n h a l f by the formation of a transverse d i v i s i o n plane (Fig. 26, arrow). This primary d i v i s i o n plane was often observed to migrate into a longitudinal or oblique position p r i o r to further division(s). D i v i s i o n of the daughter c e l l s occurs by s i m i l a r , but often asynchronous, b i p a r t i t i o n ; the secondary d i v i s i o n planes also commonly migrating into longitudinal or oblique positions. The resultant 4-celled stages contain e l l i p s o i d or spindle-shaped c e l l s l y i n g adjacent to, or stacked upon, one another i n s i m i l a r orientation (Fig. 27). Occasionally c r u c i a t e l y divided 4-celled stages were observed; the daughter c e l l s were cuneiform i n outline (Fig. 24, arrow). Contiued asynchronous b i p a r t i t i o n often gives r i s e to (6-) 8-celled stages surrounded by a common membrane (Figs. 25-27). Divided 'sporangia' measure 4.5-7.5 x Figures 23-28. Labyrinthula sp. Type 2. Figure 23. Colony margin of i s o l a t e SW RT02 displaying predominantly motile c e l l s and the nature of the ectoplasmic networks. GSSA, day 27. Phase-contrast, 220X. Figure 24. Area behind the colony margin displaying predominantly stationary c e l l s , some of which are i n various stages of c e l l d i v i s i o n (arrow). Isolate SW RT02. GSSA, day 28. Phase-contrast, 490X. Figure 25. Colony margin of i s o l a t e GC03 15-1. Motile 'spindle-shaped' c e l l s and rounded c e l l s i n various stages of d i v i s i o n . GSSA, day 13. Phase-contrast, 630X. Figure 26. Colony margin of i s o l a t e GC03 15-1. C e l l s i n various stages of d i v i s i o n (2-celled stage, arrow). GSSA, day 11. Phase-contrast, 490X. Figure 27. As Figure 26. Enlarged d i v i d i n g c e l l s . One containing (?-) eight daughter c e l l s . Phase-contrast, 850X. Figure 28. Small, s t e l l a t e colony formed when i s o l a t e GC03 15-1 was streaked onto the agar medium. GSSA, day 13. Phase-contrast, 85X. 189 190 5.5-9.0 (-10.3) um. This d i v i s i o n process results i n motile vegetative c e l l s which, i f produced i n the outer portions of the colony, normally migrate out beyond the advancing margin. In older portions of the colony, such c e l l s move only a short distance before rounding up, enlarging, and repeating the process. Thus, large aggregations or mounds of c e l l s , s i m i l a r to the "pseudosori" of Watson (1957), are formed i n older, central portions of the colony. Simple c e l l u l a r d i v i s i o n ( b i p a r t i t i o n ) , r e s u l t i n g i n just two motile daughter c e l l s , was not observed i n motile or enlarged c e l l s of these isolates. Further d e t a i l s of c e l l u l a r development i n Type 2 isolates were not observed. Large aggregations or mounds of rounded c e l l s occurred i n older portions of the colony. These c e l l aggregations were extremely s i m i l a r to the "pseudosori" found i n the Type 1 i s o l a t e s . However, multinucleate c e l l s (i.e. more than two n u c l e i ) were not observed i n l i v e c u l t u r e s of the Type 2 i s o l a t e s v i a l i g h t mircoscopy. I t i s therefore presently assumed that these c e l l u l a r aggregations were produced by successive b i p a r t i t i o n only. C e l l u l a r m o t i l i t y i n Type 2 i s o l a t e s was observed to be slow and continuous (versus intermittant, rapid pulses). Commonly, spindle-shaped c e l l s were seen moving through what appeared to be tubular ectoplasmic net elements. Not uncommonly, however, is o l a t e d c e l l s displaying m o t i l i t y were not associated with major pathways of the ectoplasmic network, nor obviously enveloped by ectoplasmic elements. Perkins (1974a) has suggested that c e l l u l a r m o t i l i t y v i a enrobing (tubular) versus nonenrobing 191 ectoplasmic nets i s a useful c r i t e r i o n for separating i s o l a t e s of Labyrinthula from Labyrinthuloides. However, he also states . . ."that Labyrinthula sp. (Vishniac Strains) may form both enrobing and nonenrobing ectoplasmic nets i n d i f f e r e n t stages of t h e i r l i f e c y c l e s . . ." (Perkins, 1974a). E l e c t r o n microscopy i s required to c a r e f u l l y evaluate the nature of the ectoplasmic networks i n these Type 2 iso l a t e s , as well as i n the Vishniac Strains. Type 2 i s o l a t e s d i f f e r d i s t i n c t l y from those of Type 1 i n colony growth rates (as well as v i a b i l i t y ) and c e l l size. However, the general c h a r a c t e r i s t i c s of colony form and development as well as certain aspects of c e l l u l a r d i v i s i o n appear very s i m i l a r . Type 2 i s o l a t e s were best maintained by placing axenic cultures on GSSA and incubating between 15° and 25°C, t r a n s f e r r i n g them once a month. Even under these conditions growth of the Type 2 i s o l a t e s was generally 'sluggish', and culture transferes were often unsuccessful. These i s o l a t e s can be distinguished from any previously described species within the genus Labyrinthula on the basis of t h e i r diminutive c e l l size [except, perhaps, L^ valkanovii (Valkanov) Karling (Karling, 1944; Valkanov, 1929)]. Watson (1957) also noted s i m i l a r i t i e s between L^ valkanovii (as L.  zo p e f i i ) and the Vishniac strains. Type 2 i s o l a t e s resemble Labyrinthuloides minutum (Watson and Raper) Perkins (Watson and Raper, 1957) i n many respects, but appear d i s t i n c t on the basis of c u l t u r a l c h a r a c t e r i s t i c s (e.g. lack of orange pigment), c e l l size, and the lack of conspicuous enlarged multinucleate bodies. 192 Complete characterization of t h i s organism should provide further links between the genus Labyrinthula and the genus Labyrinthuloides. 2. Labyrinthuloides a. Labyrinthuloides yorkensis Perkins This organism was only encountered i n F i e l d Sample 3. I t occurred most commonly on rinse water plates and i n seawater/pine pollen (SW/P) cultures, although i t was rarely observed on a l g a l tissue-agar plates as well. The following description of this Labyrinthulid was compiled from observations of three i s o l a t e s brought into pure culture and grown on SSA (1% and 2%) and GSSA. Uninucleate motile vegetative c e l l s are globose, ovoid, cuneiform, or amoeboid; 3.0-13.0 um i n longest dimension; and glide very slowly by means of branching ectoplasmic networks. Uninucleate c e l l s become s e s s i l e and enlarge into globose or subglobose c e l l s up to 25 um i n diameter (Figs. 30 and 34). Nuclei are from 2.0-7.5 pm i n diameter depending on c e l l size; each nucleus contains a single c e n t r a l l y located nucleolus. Stationary c e l l s sometimes divide v i a successive b i p a r t i t i o n to form 2 to 32 uninucleate motile c e l l s which escape through a break i n the sporangial wall (Figs. 29-31, 33). Alternatively, repeated karyokinesis may occur to form a multinucleate cytoplasm which then divides by progressive cleavage [Fig. 31 (arrow) and 34]. Divided c e l l s measure from 5.0 x 7.5 pm (2-celled) to 25.0 pm i n diameter. The persistent sporangial wall was often conspicuous, e s p e c i a l l y when newly formed uninucleate c e l l s were released (Fig. 33). Uninucleate motile c e l l s are 3.0-4.3 pm i n Figures 29-35. Labyrinthuloides yorkensis Perkins. Figure 29. General nature of the colony margin, motile c e l l s and sporangia. Isolate GT07 3-1. GSSA, day 4. Phase-contrast, 105X. Figure 30. Enlarged uninucleate c e l l s (presporangia) and sporangia i n various stages of d i v i s i o n . Four-celled tetrad formed by successive b i p a r t i t i o n (arrow). Isolate GC03 3-3. GSSA, day 3. Phase-contrast, 350X. Figure 31. Enlarged presporangia and divided sporangia of is o l a t e GT07 3-1. Multinucleate c e l l undergoing i n t e r n a l cleavage (arrow). GSSA, day 4. Phase-contrast, 400X. Figure 32. Young colony of i s o l a t e GT07 3-1 formed after streaking the organism onto the agar surface. GSSA, day 4. B r i g h t f i e l d , 25X. Figure 33. Mature sporangium of i s o l a t e GT07 3-1 releasing six motile c e l l s . SSA, day 6. Phase-contrast, 880X. Figure 34. Enlarged uninucleate and multinucleate presporangia of i s o l a t e GC03 3-3. Also motile vegetative c e l l s escaping from mature divided sporangia. GSSA, day 3. Phase-contrast, 420X. Figure 35. Amoeboid and plasmodial stages intermixed with mature sporangia and vegetative c e l l s . Isolate GT07 3-1. GSSA, day 11. Phase-contrast, 340X. 194 195 diameter when released, and contain a nucleus 2.0-2.5 um i n diameter. C e l l s of a l l stages contain a various assortment of opaque or r e f r a c t i l e inclusions 0.2-0.8 pm i n diameter. Amoeboid (uninucleate) and plasmodial (multinucleate) c e l l s (6.5-13.0 x 7.5-20.8 pm) were occasionally observed i n agar cultures but t h e i r further development was not followed (Fig. 35). Zoospores were not observed. Colonies on nutrient agar are c i r c u l a r or s t e l l a t e with a granular surface and diffuse margins (Fig. 32); + translucent; and become cream to l i g h t tan colored, opaque and convex (especially i n the center). They a t t a i n a size of 1.5-2.5 mm i n diameter a f t e r 11 days (GSSA). Isolates of Labyrinthuloides yorkensis grew f a i r l y well i n KMV-slush. However, the maximum size attained by dividing c e l l s (13.0 pm diameter) was reduced, as was the number of uninucleate c e l l s produced by d i v i s i o n (2, 4, 6, and 8). Two- and 4-celled stages were observed i n which the uninucleate propagules were contracted away from the outer wall; tetrahedrally divided 4-c e l l e d stages were also common. From my observations, these i s o l a t e s agree extremely well with the description of Labyrinthuloides yorkensis given by Perkins (1973a). I do not regard the lack of zoosporulation i n my i s o l a t e s as a c h a r a c t e r i s t i c s i g n i f i c a n t enough to disallow my i d e n t i f y i n g them as L^ yorkensis. Zoosporulating i s o l a t e s of Labyrinthulid organisms are rarely encountered, and when found, they often lose t h e i r zoosporulating capacity a f t e r r e l a t i v e l y 196 short periods of time i n culture (D. Porter, pers. comm.; Perkins, 1973a; unpubl. obs.). Perkins (1973a) describes successive b i p a r t i t i o n leading to the formation of sporangia with 4, 8, 16, 32, or rarely 64 uninucleate c e l l s . "Twelve to 24-cell sporangia were occasionally found, possibly r e s u l t i n g from death of one c e l l at the four-celled stage." (Perkins, 1973a). I have also observed 6-celled sporangia, presumably formed as Perkins (1973a) suggests (Fig. 33). Perkins (1973a) isol a t e d Labyrinthuloides yorkensis from water samples, sediments, sand, detritus, and the mantle cavity of an oyster (Crassostrea v i r g i n i c a ) . To t h i s f a i r l y long l i s t of substrata I now add l i t t o r a l red algae (Gelidium c o u l t e r i and Rhodoglossum affi n e ) . To my knowledge, no observations of L.  yorkensis have been published since Perkins (1973a) described the organism. This i s also the f i r s t documented account of L.  yorkensis from the P a c i f i c Ocean. b. Labyrinthuloides sp. 1 This Labyrinthulid organism was commonly encountered on rinse water plates and i n SW/P cultures from F i e l d Sample 3. I t was very rarely i s o l a t e d from a l g a l tissue-agar plates and was not observed i n Samples 1 and 2. Four i s o l a t e s of t h i s organism brought into pure culture (SSA-1 and 2%) provided the basis for the following description. Uninucleate vegetative c e l l s are subglobose, oblate, fusiform, ovoid, crescent-shaped, cuneiform, or i r r e g u l a r i n o u t l i n e ; (3.0-) 4.0-8.0 (-9.5) x (1.8-) 2.9-4.3 (-5.5) um. 197 Nuclei are 1.0-2.0 um i n diameter, and contain a single c e n t r a l l y located nucleolus. Vegetative c e l l s also contain a very d i s t i n c t fusiform, crescent- or irregularly-shaped r e f r a c t i l e vacuolar inclusion. The vacuolar inclusions are quite large; 1.0-5.0 pm i n longest dimension; occupy a considerable portion of the c e l l volume; and are free to change configuration, but are normally elongate and oriented lengthwise i n the c e l l along one side (Figs. 36-38). C e l l s exhibit a g l i d i n g m o t i l i t y by means of ectoplasmic networks. Movement i s by means of short, f a i r l y r a p i d (4 pm i n approximately 5 seconds), intermittant advances. C e l l s are able to reverse d i r e c t i o n and also to pivot on one end. Ectoplasmic networks a r i s e from 1 to 3 (or more?) l o c a t i o n s on the c e l l surface, and often appear to influence c e l l shape. Normally two ectoplasmic networks were observed a r i s i n g from opposite ends of oblate, fusiform, or crescent-shaped c e l l s . Often, however, additi o n a l ectoplasmic networks arose from the attenuate t i p s of l a t e r a l protrusions i n c e l l u l a r outline (Fig. 38). Ectoplasmic networks are branched, anastomosing, often with small (0.5 pm diameter) subglobose or ovoid swellings along t h e i r length; they were occasionally observed to fuse with the networks of other c e l l s . The only d i s t i n c t i v e means of propagation observed i n t h i s organism was simple c e l l u l a r d i v i s i o n . Motile binucleate c e l l s become stationary p r i o r to dividing. In c e l l s of 'normal' configuration (oblate, fusiform, or crescent-shaped), the d i v i s i o n plane i s transverse or s l i g h t l y oblique; the vacuolar incl u s i o n often becomes aligned along the d i v i s i o n plane (Figs. Figures 36-41. Labyrinthuloides sp. 1. Figure 36. Young colony of i s o l a t e SW/P RV02. Vacuolate c e l l s i n center. SSA, day 6. Phase-contrast, 430X. Figure 37. Colony margin of i s o l a t e SW/P RV02 showing motile 'spindle c e l l s ' with vacuolar inclusions and ectoplasmic networks with small swellings. SSA, day 6. Phase-contrast, 1200X. Figure 38. Colony margin of i s o l a t e SW/P GC01 showing the range i n size of motile vegetative c e l l s , and larger c e l l s i n various stages of simple d i v i s i o n . SSA, day 9. Phase-contrast, 1200X. Figure 39. Margin of a large colony. Isolate SW/P RV02. SSA, day 8. Phase-contrast, approximately 100X. Figure 40. Trinucleate c e l l containing large vacuolar inclusions. Isolate SW/P GC01. SSA, day 9. Phase-contrast, 1500X. Figure 41. Binucleate and recently divided c e l l s . Isolate SW/P RV02. SSA, day 12. Phase-contrast, 1170X. 199 200 38 and 41). D i v i d i n g c e l l s are 3.4-6.0 x 6.2-8.7 um. On one occasion a trinucleate c e l l was observed (5.7 x 8.0 um) (Fig. 40). The fate of t h i s c e l l was not followed continuously, but apparently af t e r 36 hours i t divided into uninucleate c e l l s . Very rarely amoeboid or plasmodium-like c e l l s were observed (5.8-8.6 x 7.0-14.0 pm) (Fig. 42, arrow). Continued monitoring of such c e l l s for periods up to 96 hours yielded no movement or d i v i s i o n . Quite commonly, c e l l s were observed i n which the vacuolar i n c l u s i o n had enlarged considerably and become globose or subglobose. Such c e l l s measured 3.0-7.0 x 3.8-10.8 pm and contained vacuolar inclusions up to 7.0 pm i n longest dimension; often the vacuolar inclusion occupied 50% or more of the c e l l volume (Figs. 36, 42, and 43). In young cultures (4-5 days) these enlarged c e l l s were most commonly found i n the center of the colonies, and very often occurred i n discrete clumps (3-20 c e l l s ) . The number of such c e l l s was r e l a t i v e l y small, amounting to less than 5% of the t o t a l c e l l s . In older cultures (14 days) enlarged vacuolate c e l l s increased i n number and became more uniformly distributed, even i n regions of the colony margin (Fig. 43). It i s my opinion that these are scenescing c e l l s , since i n areas of t h e i r formation, c e l l 'ghosts' and what appeared to be c e l l wall debris were often observed (Fig. 44). Further examination i s required to be sure. Rounded c e l l s (2.1-4.0 pm diameter) without conspicuous vacuolar inclusions were also seen i n o l d e r c u l t u r e s (12 days) (Fig. 44). Subglobose, thick walled, c y s t - l i k e c e l l s (4.0-6.0 pm i n Figures 42-45. Labyrinthuloides sp. 1. Figure 42. Plasmodium-like c e l l (arrow) and a cluster of rounded c e l l s containing enlarged vacuolar inclusions surrounded by motile vegetative c e l l s . Isolate SW/P RV02. SSA, day 6. Phase-contrast, 600X. Figure 43. Cluster of six rounded c e l l s containing enlarged vacuolar inclusions. Isolate SW/P RV02. SSA, day 6. Phase-contrast, 1160X. Figure 44. Central portion of a 12 day old colony of i s o l a t e SW/P RV02. Many rounded c e l l s with small or inconspicuous vacuolar inclusions? several rounded vacuolate c e l l s apparently senescing? and (?-) c e l l 'ghosts' or c e l l w a l l debris (arrows). SSA, day 12. Phase-contrast, 1110X. Figure 45. An aggregation of rounded c y s t - l i k e c e l l s i n the central portion of a colony. Isolate SW/P GC01. SSA, day 9. Phase-contrast, 600X. 202 203 longest dimension) were observed on two occasions (9 and 16 day cultures). Under transmitted i l l u m i n a t i o n these 'cysts' displayed a rust to brown pigmentation. A mound or clump of such c e l l s (33 pm diameter) was observed on one occasion (Fig. 45). Periodic examination of t h i s structure for 72 hours revealed no increase i n size or c e l l number. On the second occasion single 'cysts' were observed di s t r i b u t e d randomly throughout an otherwise active colony. These c e l l s occurred rarely, and t h e i r actual function as cysts or resting c e l l s has not yet been determined. No zoospores were observed i n these i s o l a t e s . Colonies on SSA (1% and 2%) are c i r c u l a r or oval, thin, translucent (hyaline), marginate (smooth, scalloped, or occasionally i r r e g u l a r ) ; a t t a i n a diameter of 2-3 mm after 14 days, and 7-8 mm aft e r 32 days (Figs. 36 and 39). Growth beneath the agar surface does occur, but i s very limited. Labyrinthuloides sp. 1 would grow on KMV-agar, and i n KMV-slush, but i t i s not yet known i f i t can be maintained on/in these media. These i s o l a t e s were transferred to GSSA plates on several occasions, but never displayed any growth on t h i s medium. The presence and nature of the ectoplasmic networks, as well as other c h a r a c t e r i s t i c s , c l e a r l y demarcate t h i s organism as a Labyrinthulid most closely resembling members of the genus Labyrinthuloides (Perkins, 1973a). However, i t s c u l t u r a l c h a r a c t e r i s t i c s , the shape of the c e l l s , the 'vacuolar inclusion', and the apparent lack of sporangia distinguish t h i s organism from any of the described species of Labyrinthuloides. Labyrinthuloides sp. 1 i s to be described as a new organism, but 204 further i s o l a t i o n and examination i s required before t h i s can be done. This Labyrinthulid has been previously i s o l a t e d (? substrate) i n the v i c i n i t y of Mukilteo, Washington (D. Porter, pers. comm.). c. Labyrinthuloides sp. RV02-80 This i s o l a t e was obtained from a SW/P culture of Rhodoglossum a f f i n e (Sample 3). The organism was not observed i n sli d e s made from the SW/P culture i t s e l f , but was encountered when several inoculation loops of t h i s culture were streaked onto KMV-agar. The following description i s based on observations i n pure culture (SSA-2% and GSSA). Uninucleate motile vegetative c e l l s are globose, subglobose, or ovoid, becoming cuneiform or cuboidal i n crowded clusters (Figs. 47-49); 3.0-7.5 um i n longest dimension; and glide very slowly by means of branching ectoplasmic networks which often a r i s e from a single d i s t i n c t p i t on the c e l l surface (Fig. 47, arrows). Uninucleate c e l l s become stationary and enlarge into globose or subglobose c e l l s up to 10.0 (-14.0) um i n diameter. These c e l l s then divide by successive b i p a r t i t i o n (nuclear d i v i s i o n followed by cytokinesis) to form d i s t i n c t clusters of from 2 t o 8 (?-16) c e l l s (Figs. 47 and 49). Often such successive d i v i s i o n s are asynchronous, r e s u l t i n g i n asymetrical clusters containing c e l l s of various sizes (Fig. 49, arrows). Al t e r n a t i v e l y , repeated karyokinesis occurs to form a multinucleate cytoplasm (4, 6, and 8 nucleate c e l l s were observed) which then divides to form uninucleate c e l l s . Division Figures 46-51. Labyrinthuloides sp. RV02-80. Figure 46. A small colony of i s o l a t e RV02-80 showing the d i s t r i b u t i o n of c e l l s and the dif f u s e colony margin. GSSA, day 4. Phase-contrast, 280X. Figure 47. Advancing 'ray' at the colony margin showing motile vegetative c e l l s and a tetrad formed v i a successive b i p a r t i t i o n . Arrows indicate ectoplasmic networks a r i s i n g from what appear to be 'pits' i n the c e l l surface. GSSA, day 5. Phase-contrast, 670X. Figure 48. Enlarged, multinucleate 'plasmodium-like' c e l l s i n an older (central) portion of a colony. GSSA, day 13. Phase-contrast, 1100X. Figure 49. Colony margin displaying motile vegetative c e l l s and c e l l s i n various stages of d i v i s i o n . Note clusters of c e l l s which show asynchronous successive b i p a r t i t i o n i n g (arrow). GSSA, day 5. Phase-contrast, 640X. Figure 50. Colony displaying l o c a l i z e d subsurface growth i n an old culture. GSSA, day 37. B r i g h t f i e l d , 30X. Figure 51. Growth of i s o l a t e RV02-80 on pine pollen. SW/P culture, day 6. Phase-contrast, 550X. 206 207 of multinucleate c e l l s was observed to be nearly synchronous (progressive cleavage) or d i s t i n c t l y asynchronous (i.e. one c e l l being 'cut-off at a time). The walls presumably surrounding these d i v i d i n g c e l l s were inconspicuous. Multinucleate c e l l s a t t a i n a s i z e of up to 11.0 x 10.0 um (8 nucleate) (Fig. 48). Nuclei i n c e l l s of a l l stages (single c e l l e d or multinucleate) are from 1.2 to 1.6 um i n diameter; each contains a c e n t r a l l y located nucleolus. C e l l s of a l l stages contain various opaque or r e f r a c t i l e inclusions from 0.2 to 1.0 pm i n diameter. L a t e r a l l y b i f l a g e l l a t e Labyrinthulid- or Thraustochytrid-l i k e zoospores were observed (SSA-2%, water fi l m , day 3), but i t i s not known how they were produced. Presumably zoospores se t t l e d out to become uninucleate vegetative c e l l s . Colonies on nutrient agar are i r r e g u l a r or s t e l l a t e i n outline, with a f a i n t l y granular surface and sharp or dif f u s e margins; they are translucent, becoming white or cream colored and opaque i n the center; and at t a i n a diameter of up to 2.0 mm af t e r 14 days (SSA-2% and GSSA) (Fig. 46). Growth beneath the agar surface was observed i n old cultures (GSSA - 30 + days), but was very l o c a l i z e d and l i m i t e d (Fig. 50). Isolate RV02-80 displayed l i m i t e d growth on pine pollen i n s t e r i l e seawater cultures (Fig. 51). Presumably zoospores provided the is o l a t e s ' means of dispersal to the pollen grains. Growth on pine pollen was generally slow and inconspicuous, and consisted of i n d i v i d u a l c e l l s or small clumps. Four-celled stages (tetrads) and 'sporangia' containing six (?-eight) 208 daughter c e l l s were observed up to 8.5 um i n diameter (Fig. 51). I t i s not "known i f t h i s i s o l a t e can be maintained through successive transfers i n SW/P culture. Labyrinthuloides sp. RV02-80 displays c e r t a i n c h a r a c t e r i s t i c s which are s i m i l a r to several Thraustochytrids and Labyrinthulids. The 4-celled (tetrad) stages formed by successive b i p a r t i t i o n i n t h i s i s o l a t e are s i m i l a r to those found i n Schizochytrium aggregatum and Thraustochytrium aggregatum. I s o l a t e RV02-80 i s most s i m i l a r to T. aggregatum i n t h a t both of these organisms also exhibit i n t e r n a l d i v i s i o n of vegetative c e l l s (via progressive cleavage) which results i n four or more 'propagules' and/or vegetative c e l l s . However, the lack of conspicuous repeated zoosporulation and the presence of motile vegetative c e l l s , as well as other c h a r a c t e r i s t i c s , tend to remove i s o l a t e RV02-80 from these Thraustochytrid genera. Isolate RV02-80 d i f f e r s from Labyrinthuloides minutum i n vegetative c e l l size and shape as well as growth ch a r a c t e r i s t i c s (Watson and Raper, 1957; Quick, 1974b). A prominent s i m i l a r i t y between Labyrinthuloides saliens (Quick, 1974a) and the present i s o l a t e i s the ectoplasmic networks that often ari s e from d i s t i n c t p i t s on the surface of motile vegetative c e l l s . However, these two organisms d i f f e r considerably i n other aspects. Isolate RV02-80 also shares many s i m i l a r i t i e s with Labyrinthuloides yorkensis (Perkins, 1973a), but during my observations of these two Labyrinthulids I noted the following differences: 1) Enlarging 'presporangial' c e l l s of i s o l a t e RV02-80 (to 209 10.0 pm diameter) did not at t a i n the large size observed i n my isol a t e s of Lj_ yorkensis (to 25.0 pm diameter). In L^ yorkensis, the c e l l nuclei also enlarged (to 7.5 pm diameter). Such a d i s t i n c t increase i n nuclear size was not observed i n i s o l a t e RV02-80; the nuclei i n a l l stages of t h i s i s o l a t e remained between 1.2 to 1.6 pm i n diameter. The smallest nuclei observed i n c e l l s of my is o l a t e s of yorkensis were 2.0 pm i n diameter. 2) Successive b i p a r t i t i o n of sporangia was often d i s t i n c t l y asynchronous i n i s o l a t e RV02-80, whereas i t was predominantly synchronous i n L^ yorkensis. 3) In motile c e l l s of i s o l a t e RV02-80 the ectoplasmic networks commonly arise from a d i s t i n c t p i t i n the c e l l surface. This s i t u a t i o n was not observed i n yorkensis by Perkins (1973a), nor was i t apparent i n my i s o l a t e s of t h i s Labyrinthulid. In many respects, i s o l a t e RV02-80 also resembles the 'spindle-cell r i c h strains' of Labyrinthuloides schizochytrops (Quick, 1974b). Certain i s o l a t e s of Lj_ schizochytrops produce enlarged multinucleate Plasmodia and/or enlarged compartmentalized sporangia, but these stages were present i n only 20% of the is o l a t e s examined (Quick, 1974b). Determination of possible relationships between i s o l a t e RV02-80 and L.  schizochytrops or Lj_ yorkensis w i l l require further i s o l a t i o n and study of t h i s organism. 210 3. Labyrinthulid Unidentified During the processing of Sample 3 a r e l a t i v e l y large number of Labyrinthulid organisms were encountered which could not be isolated, or displayed very limited, slow growth under the culture conditions u t i l i z e d . Approximately 93 is o l a t e s or colonies of these unidentified Labyrinthulids were observed i n cultures prepared from Sample 3 algae. Of these, 91 colonies (98%) were obtained from a l g a l tissue rinse water which was plated on culture media. Attempts were made to bring eight representative i s o l a t e s of these organisms into pure culture. With some d i f f i c u l t y , three i s o l a t e s were maintained i n culture for up to nine months, and were c h a r a c t e r i z e d i n some d e t a i l . I do not know i f these three i s o l a t e s are representative of the remainding 90 colonies which were not isolated. A l l three of the following descriptions are based on observations of cultures grown on GSSA and SSA-2% media at room temperature (22-25°C). Attempts to grow these i s o l a t e s on KMV-agar, i n KMV-slush, and on pine pollen i n s t e r i l e seawater cultures were unsuccessful. a. Isolate SW/P GC03 This i s o l a t e was obtained from a SW/P culture of Gelidium  c o u l t e r i . The organism was not observed i n s l i d e s prepared from the SW/P culture i t s e l f , but was i s o l a t e d when several inoculation loops of t h i s culture were streaked onto KMV-agar. Uninucleate vegetative c e l l s are globose, subglobose, ovoid, 211 pyriform, cuneiform (in clusters) or i r r e g u l a r i n outline? 2.5-5.0 x 2.5-5.7 (-8.2) pm (Figs. 52-57). N u c l e i are 1.2 to 1.7 pm i n diameter, and contain a single c e n t r a l l y located nucleolus. C e l l s contain several to many (15+) opaque and/or r e f r a c t i l e inclusions which are 0.4 to 0.8 (-1.5) pm i n diameter. Ectoplasmic networks are branched, anastomosing? often form discrete bundles (to 1.8 pm wide) which radiate away from the colony (Figs. 53, 57, and 58)? and occasionally have globose, ovoid or oblate swellings along t h e i r length (to 5.0 pm i n longest dimension). Globose or subglobose i s o l a t e d 'blebs' or 'droplets' of what appeared to be ectoplasmic network material were also observed adjacent to some colonies of t h i s organism (to 3.8 pm i n longest dimension). Vegetative c e l l s display an active m o t i l i t y by means of ectoplasmic networks, but such m o t i l i t y was not a conspicuous c h a r a c t e r i s t i c of colony growth on the nutrient agar media u t i l i z e d . Vegetative c e l l s divide by synchronous successive b i p a r t i t i o n , and form 4-celled tetrads (some tetrahedrally divided) 6.3-11.0 x 5.9-9.3 pm (Figs. 52, 53, and 57). The daughter c e l l s r e s u l t i n g from tetrad formation often repeat the process, forming uniform clusters of 16 c e l l s (Figs. 52 and 57). Normally, the c e l l s i n a colony were so crowded and clumped that t h i s repeated process of successive b i p a r t i t i o n i n g was not discernable. Observations indicated that nuclei enlarged s l i g h t l y p r i o r to d i v i s i o n : nuclei i n large vegetative c e l l s were 1.6 to 1.7 pm i n diameter ( c e l l s 5.0-8.2 x 4.0-5.7 pm)? those i n newly divided c e l l s were 1.2 um i n diameter ( c e l l s 2.5 x 3.0-3.8 pm). C e l l d i v i s i o n v i a successive b i p a r t i t i o n i n g was the Figures 52-58. Labyrinthulid Unidentified. Isolate SW/P GC03. Figure 52. Enlarged single c e l l s and c e l l s i n various stages of successive b i p a r t i t i o n . GSSA, day 13. Phase-contrast, 430X. Figure 53. Tetrahedrally divided 4-celled stage at the colony margin. Note apparent lack of vegetative c e l l m o t i l i t y . GSSA, day 12. Phase-contrast, 850X. Figure 54. Irre g u l a r l y shaped, + amoeboid c e l l s at the colony margin. GSSA, day 12. Phase-contrast, 850X. Figure 55. Central (older) portion of a colony. Many c e l l s have an amoeboid appearance (arrows). GSSA, day 15. Phase-contrast, 430X. Figure 56. Small colony with uninucleate, apparently motile, vegetative c e l l s . GSSA, day 9. Phase-contrast, 340X. Figure 57. As Figure 56; same culture, (?-) a d i f f e r e n t colony approximately 24 hours later. The single c e l l s have divided into tetrads and the resultant daughter c e l l s are repeating the process. GSSA, day 10. Phase-contrast, 340X. Figure 58. A rather large colony consisting of discontinuous clumps of c e l l s . GSSA, day 15. Phase-contrast, 92X. 213 214 only means of propagation observed i n t h i s i s o l a t e . C e l l s at colony margins were often of i r r e g u l a r configuration (? amoeboid), and with a less r e f r a c t i l e cytoplasm (r e l a t i v e to other c e l l s ) (Fig. 54). Most probably these c e l l s soon divided v i a successive b i p a r t i t i o n . On one occasion a s i m i l a r c e l l was observed to be multinucleate [3 (?-4) n u c l e i ] , but i t s further development could not be followed. In old cultures (39 days; SSA-2%) + amoeboid c e l l s were formed (6.3-16.3 x 3.3-8.0 pm; nuclei 1.8-2.5 pm i n diameter) (Fig. 55, arrow). These c e l l s were not obviously motile, but were occasionally observed with ectoplasmic networks extending from i r r e g u l a r lobes i n t h e i r outline. This p a r t i c u l a r culture was i n an obvious stage of senescence (containing numerous dead c e l l 'ghosts'). Thus, these amoeboid c e l l s may have been the r e s u l t of ageing stress i n culture. No zoospores were observed i n t h i s i s o l a t e . Colonies on GSSA are c i r c u l a r or oval; often lobbed or s t e l l a t e ; translucent to f a i n t l y opaque white; and a t t a i n a diameter of 0.2 to 0.3 mm after 10 days, and 0.6 mm after 25 days (Fig. 58). Growth of Isolate SW/P GC03 on SSA-2% was even more r e s t r i c t e d and v i a b i l i t y was of r e l a t i v e l y short duration (approximately 30 days maximum). Occasionally colonies with d i f f u s e margins (? enhanced m o t i l i t y ) were produced on SSA-2%. b. Isolate RC01 15-1 Isolate RC01 15-1 was obtained from an agar culture which had been inoculated with the 15th rinse water from a Rhodoglossum  a f f i n e tissue sample. 215 Uninucleate vegetative c e l l s are globose, subglobose, ovoid or p y r i f o r m ; 2.8-4.5 x 3.0-5.0 pm. N u c l e i are 1.2 to 1.4 (-1.6) pm i n diameter, and contain a single central nucleolus. Motile c e l l s enlarge into globose or subglobose c e l l s 5.0-6.0 (-7.5) pm i n longest dimension, and become stationary. Enlarged c e l l s c o n t a i n n u c l e i 1.8 to 2.0 (-2.5) pm i n diameter. C e l l u l a r d i v i s i o n occurs by simple b i p a r t i t i o n of vegetative c e l l s r e s u l t i n g i n 2-celled stages (4.0-8.1 x 3.1-7.0 pm; Figs. 59 and 60), or by successive b i p a r t i t i o n of larger c e l l s r e s u l t i n g i n tetrads (5.0-8.0 pm i n diameter; Figs. 60 and 61). Continued asynchronous successive b i p a r t i t i o n i n g of daughter c e l l s which did not move apart often resulted i n ir r e g u l a r c l u s t e r s of c e l l s (Fig. 63). Rarely, i n t e r n a l d i v i s i o n of enlarged c e l l s (6.0-9.5 x 5.7-7.5 pm) was observed to resu l t i n (?-) 6 or 8 motile vegetative c e l l s (? zoospores) (Fig. 61, arrow). Presumably inte r n a l d i v i s i o n occurred by progressive cleavage. On rare occasions very large c e l l s were observed (15.0 x 14.3 pm; -27.5 pm i n diameter) which contained nuclei up to 5.0 (-8.8) pm i n diameter (Fig. 62). These c e l l s often appeared senescent, but there was also some i n d i c a t i o n that they could divide i n t e r n a l l y into motile vegetative c e l l s . Ectoplasmic networks are generally very thin, branching and anastomosing, occasionally with fusiform swellings (to 2.8 pm i n longest dimension) along t h e i r length. The ectoplasmic networks radiating out beyond the colony margins often displayed i r r e g u l a r swellings towards t h e i r d i s t a l ends (to 7.5 pm i n longest Figures 59-65. Labyrinthulid Unidentified. Isolate RC01 15-1. Figure 59. Globose or subglobose vegetative c e l l s at the colony margin. Many c e l l s undergoing d i v i s i o n processes. GSSA, day 32. Phase-contrast, 900X. Figure 60. As Figure 59. Four-celled tetrad formed via successive b i p a r t i t i o n (arrow). GSSA, day 32. Phase-contrast, 900X. Figure 61. Colony margin with c e l l s i n various stages of enlargement and d i v i s i o n , including one c e l l showing in t e r n a l d i v i s i o n (arrow). GSSA, day 13. Phase-contrast, 950X. Figure 62. A greatly enlarged single c e l l containing an enlarged nucleus. GSSA, day 13. Phase-contrast, 630X. Figure 63. Colony margin displaying motile vegetative c e l l s and c e l l s i n various stages of d i v i s i o n . GSSA, day 13. Phase-contrast, 660X. Figure 64. A large, dense colony with + dif f u s e margins. GSSA, day 20. Phase-contrast, 85X. Figure 65. A small colony with a s t e l l a t e appearance. GSSA, day 32. Phase-contrast, 160X. 217 218 dimension). Colonies on GSSA are c i r c u l a r or s t e l l a t e ; translucent to opaque white i n the center; diffuse or d i s t i n c t l y marginate; and a t t a i n a diameter of up to 0.6 mm afte r 20 days (Figs. 64 and 65). c. Isolate GT04 3-1 This i s o l a t e was obtained from an agar culture which had been inoculated with the 3rd rinse water from a tissue sample of Gelidium c o u l t e r i . Uninucleate motile vegetative c e l l s are globose, subglobose, ovoid, occasionally + e l l i p s o i d or i r r e g u l a r i n outline; 2.8-3.5 x 2.8-4.5 (-5.3) pm (Figs. 66 and 67). N u c l e i are 1.3 to 1.8 pm in diameter, and contain a single, c e n t r a l l y located nucleolus. Motile c e l l s enlarge (4.5-7.8 x 5.3-8.3 pm) and become stationary p r i o r to dividing. Nuclei i n enlarged c e l l s a t t a i n a size of up to 2.0-2.5 pm i n diameter. Further development of these enlarged c e l l s i s marked by simple (2-celled) or successive (tetrad) b i p a r t i t i o n , or, more commonly, continued enlargement and karyokinesis leading to multinucleate 'sporangia'. Multinucleate sporangia are 10.3-16.8 x 9.8-17.3 pm, and co n t a i n (6) 8 (12) nuclei (Figs. 66 and 68). 'Sporangia' divide v i a progressive cleavage into (6) 8 (12) motile vegetative c e l l s or zoospores (Figs. 66, 67, 69-72). The r e l a t i v e frequency of occurrence of zoospore production versus motile vegetative c e l l s i s not known. On those occasions when zoospores were produced, f l a g e l l a r a c t i v i t y was of l i m i t e d duration (approximately 25 minutes), and the zoospores did not escape the confines of the zoosporangium Figures 66-72. Labyrinthulid Unidentified. Isolate GT04 3-1. Figure 66. Colony margin with c e l l s i n various stages of enlargement and d i v i s i o n . Included are an enlarged multinucleate c e l l (arrow) and several divided sporangia. GSSA, day 15. Phase-contrast, 800X. Figure 67. As Figure 66. Several (zoo-?) sporangia containing more than eight (?-12) 'spores'. GSSA, day 15. Phase-contrast, 800X. Figure 68. Large i r r e g u l a r multinucleate c e l l s at the margin of a colony. Also visable are several 6- to 8-celled sporangia. GSSA, day 10. Phase-contrast, 700X. Figures 69-72. Photomicrograph sequence of active zoospores within a zoosporangium. Note the dark r e f r a c t i l e i n c l u s i o n i n each zoospore (Figs. 71 and 72); also f l a g e l l a (Figs. 70 and 72; arrows). Elapsed time between Figures 69 and 72 approximately 30 minutes. GSSA, day 15. Phase-contrast, 1800X. 220 (Figs. 69-72). After f l a g e l l a r a c t i v i t y ceased, the f l a g e l l a were apparently absorbed, and the zoospores converted into motile vegetative c e l l s producing ectoplasmic networks. Free swimming zoospores were not observed, and the following description i s based on zoospores clumped within the immediate v i c i n i t y of the zoosporangium. Zoospores are l a t e r a l l y b i f l a g e l l a t e ; ovoid, e l l i p s o i d , or frequently changing shape; 3.0-4.4 um long by 2.1-3.1 um wide; and contain nuclei 1.3-1.5 pm i n diameter. Anterior f l a g e l l a were observed up to 9.0 pm long (Figs. 70 and 72, arrows). The posterior f l a g e l l a were obviously shorter, but were inconspicuous and could not be measured. Each zoospore contains a very prominent black r e f r a c t i l e inclusion which measures 0.8-1.1 pm i n longest dimension (? eyespot) (Figs. 69-72). Enlarged, somewhat senescent looking c e l l s (10.3-14.0 x 10.0-12.5 pm), s i m i l a r to those described for i s o l a t e RC01 15-1, were often observed i n cultures of GT04 3-1. These c e l l s either contained a very enlarged single nucleus, or were multinucleate, containing up to 12 smaller nuclei (Fig. 68). Actual d i v i s i o n of such c e l l s was not observed. Ectoplasmic networks are generally very thin, branching and anastomosing, often aggregated into f a s i c l e s when radiating out from colony margins. Colonies on GSSA are thin, hyaline and translucent to opaque and white i n the center; with + d i f f u s e margins; and a t t a i n a diameter of 0.3-0.5 mm after 20 days. 222 From the preceeding descriptions i t should be apparent that the young vegetative c e l l s of a l l three 'Unidentified Labyrinthulid' i s o l a t e s and Labyrinthuloides sp. RV02-80 are very s i m i l a r i n size, shape and early development. The most prominent differences between the three 'unidentified' i s o l a t e s are displayed by t h e i r processes of c e l l d i v i s i o n and/or c e l l propagation. Isolate SW/P GC03 i s , perhaps, the most d i s t i n c t i n t h i s respect, exhibiting prominent repeated successive b i p a r t i t i o n r e s u l t i n g i n 4-celled tetrads. At the opposite extreme i s Isolate GT04 3-1. In t h i s organism the most common developmental pattern includes progressive cleavage of multinucleate presporangia into zoospores and/or motile vegetative c e l l s . Isolate RC01 15-1 appears intermediate between these two, displaying simple and successive b i p a r t i t i o n into vegetative c e l l s , as well as occasional progressive cleavage of multinucleate presporangia-like c e l l s into vegetative c e l l s (? zoospores). The c e l l c h a r a c t e r i s t i c s and developmental patterns of the three 'unidentified' i s o l a t e s include stages which, when combined, are very s i m i l a r to Labyrinthuloides sp. RV02-80. I have separated these i s o l a t e s from Labyrinthuloides sp. RV02-80 because of t h e i r developmental and morphological 'inconsistencies', r e s t r i c t e d colony growth rates, and generally less viable nature. The enlarged uninucleate c e l l s and/or multinucleate Plasmodia observed i n iso l a t e s RC01 15-1 and GT04 3-1 appear somewhat s i m i l a r to the enlarged presporangia produced by Labyrinthuloides schizochytrops (cf. stationary Plasmodia) and 223 Labyrinthuloides yorkensis (Perkins, 1973a; Quick, 1974b). Further studies of these 'aberrant' i s o l a t e s are required to decipher t h e i r relationships to other species. B. THRAUSTOCHYTRIDS 1. Schizochytrium a. Schizochytrium aggregatum Goldstein and Belsky Schizochytrium aggregatum was of regular occurrence on the two algae examined. The following description i s based on observations of ten i s o l a t e s which were brought into pure culture (SW/P, KMV-slush and KMV-agar). Zoospores are l a t e r a l l y b i f l a g e l l a t e ; subglobose, ovoid, or reniform, varying i n shape while swimming; 3.5-5.5 pm long x 1.8-3.5 pm wide. 'Encysted' zoospores and/or young s i n g l e - c e l l e d sporangia (monads) are globose or subglobose, 2.5-4.5 x 2.6-6.0 pm, and divide by successive b i p a r t i t i o n . Two-celled stages are 4.7-6.3 x 5.2-8.3 pm; t e t r a d s are 6.5-10.0 x 7.5-12.0 pm (Fig. 74). Individual c e l l s of sporangial clusters a t t a i n a size of up to 5.0 x 7.0 pm. Continued successive b i p a r t i t i o n i n g of the c e l l s r esults i n amorphous masses which a t t a i n a size of up to 130 pm i n diameter (Figs. 75 and 79). Sporangia eventually divide i n t e r n a l l y into several zoospores which escape through a crack i n the persistent sporangial wall. In SW/P cultures, Schizochytrium aggregatum often develops f r e e - f l o a t i n g i n the sea water, not i n contact with pine pollen. Branching ectoplasmic networks are well developed i n both SW/P Figures 73-79. Schizochytrium aggregatum Goldstein and Belsky. Figure 73. Recently s e t t l e d zoospores of i s o l a t e RW GT04. KMV-agar, day 2. Phase-contrast, 400X. Figure 74. Two-, 3-, and 4-celled stages r e s u l t i n g from successive b i p a r t i t i o n (often unequal and/or asynchronous). Isolate RW GT04. KMV-agar, day 4. Phase-contrast, 400X. Figure 75. Irregular clusters of c e l l s r e s u l t i n g from continued successive b i p a r t i t i o n i n g . Isolate RW GT04. KMV-agar, day 6. Phase-contrast, 400X. Figure 76. Small amorphous colony of i s o l a t e SW/P SW RT03 formed by continued successive divis i o n s . Radiating ectoplasmic networks with swellings. KMV-agar, day 2. Phase-contrast, 400X. Figure 77. Three day old colony of i s o l a t e SW/P SW RT03 displaying growth v i a zoospore release and encystment immediately surrounding the 'parent' colony. KMV-agar, day 3. Phase-contrast, 125X. Figure 78. Coalescing colonies of i s o l a t e RW GT04 displaying swellings and/or 'sheaths' of ectoplasmic material. KMV-agar, day 6. Phase-contrast, 210X. Figure 79. Free-floating sporangial cl u s t e r with radiating ectoplasmic networks. Contact with pine pollen due to s l i d e preparation (i.e. not endobiotic). Isolate SW GV01. SW/P culture, day 4. Phase-contrast, 430X. 225 226 and KMV-slush cultures. In SW/P cultures ectoplasmic networks are endobiotic, i n t e r b i o t i c (between pollen grains) or extend from f r e e - f l o a t i n g sporangia (Fig. 79). On KMV-agar, colonies of Schizochytrium aggregatum grow i n a ce n t r i f u g a l fashion due to zoospore release and settlement i n the immediate v i c i n i t y of the developing colony (Fig. 77). The various stages of development leading from monads to diads, tetrads and amorphous colonies are e a s i l y followed (Figs. 73-76). Branching and anastomosing ectoplasmic networks are conspicuous on KMV-agar. Very commonly the ectoplasmic networks display globose-, pyriform-, or spindle-shaped swellings along t h e i r length (Fig. 76). Often variously shaped sheaths or "blebs' of ectoplasmic network material are produced around rapidly growing colonies (Fig. 78). Amoeboid or ?-plasmodial c e l l s were occasionally found at colony margins. After three days on KMV-agar, colonies of these isol a t e s are opaque, white, g l i s t e n i n g and from 0.5 to 1.5 mm i n diameter. The i s o l a t e s described above resemble very cl o s e l y the S.  aggregatum strains described by Booth and M i l l e r (1969). The monad, diad and tetrad stages of these i s o l a t e s are only about one-half the siz e of the organism as o r i g i n a l l y described by Goldstein and Belsky (1964). Booth and M i l l e r (1969) emended the genus description of Schizochytrium to accommodate t h e i r i s o l a t e s . S i m i l a r l y , I presently regard my i s o l a t e s as within the concepts of Schizochytrium aggregatum Goldstein and Belsky emend Booth and M i l l e r . 227 b. Schizochytrium aggregatum Goldstein and Belsky (pigmented strain) In F i e l d Sample 3, a s t r a i n of Schizochytrium aggregatum was encountered which was d i s t i n c t from the previous i s o l a t e s i n colony morphology, pigmentation and c e l l dimensions. Studies of four i s o l a t e s of t h i s organism brought into pure culture (SW/P, KMV-slush and KMV-agar) provided the basis for the following description. Zoospores are l a t e r a l l y b i f l a g e l l a t e , subglobose, ovoid or reniform, varying i n shape while swimming; 4.5-6.0 pm long x 2.5-3.5 pm wide. Single- c e l l e d developing sporangia (monads) atta i n a s i z e of up to 12.5 x 17.0 pm (22.5 um diameter, KMV-agar) (Fig. 80); diads are 8.0-11.3 x 9.5-15.0 pm; t e t r a d s are 10.0-16.5 x 10.0-17.5 pm (Fig. 81). Individual c e l l s of large sporangial clus t e r s (cleaved into zoospores) reach a size of up to 18.8 x 22.3 pm. Unequal cleavage (bipartitioning) often results i n young sporangial clusters of very i r r e g u l a r configuration. Continued 'bipartitioning' of the c e l l s r esults i n large amorphous masses, up to 330 x 450 pm (Figs. 82-84). Clusters of sporangia display a l i g h t peach to orange pigmentation. Sporangia divide i n t e r n a l l y to form zoospores. Zoospores become motile within the sporangium just p r i o r to release; they escape through a crack i n the persistant sporangial wall. Branching and anastomosing ectoplasmic networks are normally prominent, except i n i s o l a t e RW RC01 where they were occasionally reduced (short, thin). In SW/P cultures 'sporangia' often develop f r e e - f l o a t i n g and not i n obvious contact with pine 228 pollen. When growth i s associated with pollen the ectoplasmic networks are endobiotic and/or i n t e r b i o t i c (Fig. 85). On KMV-agar, colonies of these pigmented i s o l a t e s grow i n a centr i f u g a l fashion s i m i l a r to that described for the nonpigmented str a i n . The developmental stages leading from single c e l l s through diad and tetrad stages to amorphous colonies are e a s i l y followed (Figs. 80-83). C e l l s displaying unequal and/or i r r e g u l a r d i v i s i o n planes are common. Branching and anastomosing ectoplasmic networks are generally well developed (Figs. 80, 82-84). As for the previously described i s o l a t e s of Schizochytrium aggregatum, globose or pyriform swellings often occur along the length of the ectoplasmic nets, and i r r e g u l a r laminar 'sheaths' of ectoplasmic material are commonly produced at the outer margins of sporangial clusters (Figs. 81, 83, and 84). Irregular + amoeboid c e l l s were also occasionally observed at colony margins. After three days on KMV-agar, colonies of these iso l a t e s are pigmented l i g h t peach to orange, noticeably elevated above the agar surface, and from 0.4 to 1.2 mm i n diameter. When these cultures were transferred to KMV-agar and placed i n a dark r e f r i g e r a t o r (4-5°C) for two weeks, several of the i s o l a t e s l o s t t h e i r d i s t i n c t pigmentation, and were cream colored when removed. Under s i m i l a r culture conditions, t h i s pigmented s t r a i n of Schizochytrium aggregatum produced noticeably fewer zoospores than the smaller celled, nonpigmented s t r a i n previously described. In one i s o l a t e of t h i s group (RW RC01), zoospore production was not observed. As a consequence, t h i s i s o l a t e grew much more Figures 80-85. Schizochytrium aggregatum (pigmented s t r a i n ) . Figure 80. Young s i n g l e - c e l l e d 'sporangia' (monads) and ectoplasmic 'rhizoids' of i s o l a t e SW/P GT02. KMV-agar, day 3. Phase-contrast, 400X. Figure 81. One to four (+) c e l l e d stages showing successive b i p a r t i t i o n sequences (often asynchronous and/or unequal) i n i s o l a t e SW/P GT02. Laminar 'sheaths' of ectoplasmic network material surrounding most c e l l clusters. KMV-agar, day 5. Phase-contrast, 400X. Figure 82. Young colony of clustered, d i v i d i n g 'sporangia'. Isolate RW RC01. KMV-agar, day 5. Phase-contrast, 400X. Figure 83. As Figure 82. S l i g h t l y larger colony with partitioned or lobed appearance. Note swelling(s) on radiating ectoplasmic networks. KMV-agar, day 5. Phase-contrast, 400X. Figure 84. Isolate SW/P GT02. Scattered colonies surrounded by young developmental stages. Colonies with radiating ectoplasmic networks displaying swellings and/or laminar 'sheaths'. KMV-agar, day 5. Phase-contrast, 95X. Figure 85. Sporangial cluster on pine pollen. Isolate SW/P GC03. SW/P culture, day 8. Phase-contrast, 770X. 230 231 slowly on KMV-agar, apparently by c e l l d i v i s i o n only. RW RC01 also grew very slowly i n KMV-slush and SW/P cultures, apparently propagating new t h a l l i by c e l l d i v i s i o n and colony fragmentation. In a l l other respects, except occasionally reduced ectoplasmic networks, i s o l a t e RW RC01 was indistinguishable from the others. In most respects these i s o l a t e s are s i m i l a r to the s t r a i n of Schizochytrium aggregatum f i r s t described by Goldstein and Belsky (1964). These authors do not describe pigment production i n t h e i r i s o l a t e s , while under 'normal' conditions colonies of my i s o l a t e s a l l displayed a l i g h t peach to orange pigmentation. Booth and M i l l e r (1968) have described v a r i a b i l i t y i n pigment production within s i m i l a r i s o l a t e s of Thraustochytrium spp. Therefore, I am not i n c l i n e d to regard pigment production as a sound taxonomic c r i t e r i o n . S l i g h t differences also exist i n the size of the various developmental stages, p a r t i c u l a r l y the larger size of s i n g l e -c e l l e d sporangia (to 22.5 pm diameter) i n my isolates. I presently regard such differences i n size, p a r t i c u l a r l y of a single stage, as within the v a r i a b i l i t y induced by culture conditions and/or the observer. It appears that the monotypic genus Schizochytrium contains a heterogenous group of organisms. Considerable further research i s required before we have the grounds to assign new species, v a r i e t a l or subspecies ranks to the d i f f e r e n t s t r a i n s . 232 2. Thraustochytrium a. Thraustochytrium aggregatum Ulken A single i s o l a t e of Thraustochytrium aggregatum was obtained from a SW/P culture of Rhodoglossum a f f i n e i n Sample 3. The organism was not discerned i n s l i d e s prepared from the SW/P culture i t s e l f , but was noted as a d i s t i n c t colony when several inoculation loops of the culture were streaked onto KMV-agar. The developmental cycle of t h i s Thraustochytrid i s somewhat complex and has not been elucidated to my s a t i s f a c t i o n . My observations of Tj_ aggregatum i n pure culture (GSSA, KMV-agar, KMV-slush, and SW/P) are given below. Zoospores are l a t e r a l l y b i f l a g e l l a t e ; e l l i p s o i d , ovoid or + reniform, varying i n shape; and often contain a d i s t i n c t r e f r a c t i l e inclusion. Zoospores display a rather large range i n size, measuring 2.0-3.8 x 2.8-6.0 um. Some zoospores also exhibited an odd swimming motion i n which the zoospore body undulated or v i b r a t e d more than normal, and the t i p of the anterior flagellum appeared to be bent back and attached to the spore body. The sequence of events leading from 'germinating' zoospores to mature divided zoosporangia appeared to be quite variable i n t h i s organism, and no single developmental pathway could be discerned. Successive b i p a r t i t i o n (often asynchronous and/or unequal) occurs r e s u l t i n g i n 2-, 3- and 4-celled stages (Fig. 86). The daughter c e l l s formed by t h i s process were not observed to be transformed into zoospores d i r e c t l y , but continued enlarging to become sporangia (Fig. 88). Alternatively, 233 developing 'monads' enlarge, become multinucleate, and divide via progressive cleavage into from four to approximately 30 zoospores (or daughter c e l l s ) . Four-celled sporangia formed by progressive cleavage are tetrahedrally divided (Fig. 87); larger sporangia display d i s t i n c t r a d i a l cleavage planes (Figs. 89 and 90) (not t r u l y r a d i a l since one to several zoospores are formed i n the center of the sporangium; the i n d i v i d u a l c e l l s being polyhedral in outline). From my observations, sporangia which divide by progressive cleavage normally release motile zoospores three or more hours aft e r cleavage. This was not always the case, however, and i n some instances the zoospores did not swim away but remained together and began development into sporangia. There was also evidence that the time between cleavage and zoospore release varied considerably, such that zoospores could begin enlarging i n the sporangium p r i o r to becoming motile and swimming away. This might explain the f a i r l y large discrepancy previously noted i n zoospore size. Due to the nature of i t s developmental cycle, t h a l l i of Thraustochytrium aggregatum are gregarious i n KMV-slush or on pine pollen i n seawater (Figs. 88 and 89). Zoospore release i s often semi-synchronous within clusters of 'daughter' sporangia (Fig. 91). The outer wall of mature sporangia appeared to dissolve completely p r i o r to zoospore release. In larger, 'radially divided' sporangia, the zoospores seemed to be attached to one another towards the center of the sporangium. Zoospores i n portions of the sporangium began beating t h e i r f l a g e l l a and vi b r a t i n g (either singly, i n pairs, or i n small groups) p r i o r to Figures 86-92. Thraustochytrium aggregatum Ulken. A l l photomicrographs of a single i s o l a t e - SW/P RV03. Figure 86. Enlarged single c e l l and 2-celled stage res u l t i n g from b i p a r t i t i o n . Free-floating i n SW/P culture, day 3. Phase-contrast, 2220X. Figure 87. Tetrahedrally divided 4-celled stage. KMV-slush, day 3. Phase-contrast, 2360X. Figure 88. Cluster of four variously divided sporangia which apparently had remained together since the 4-celled stage (as i n Figure 87, above). Free-floating i n SW/P culture, day 2. Phase-contrast, 970X. Figure 89. Young, developing colonies on KMV-agar. Note uneven sized c e l l s within large divided sporangia. KMV-agar, day 3. Phase-contrast, 400X. Figure 90. Gregarious nature of sporangium development on pine pollen. Several sporangia are r a d i a l l y divided. SW/P culture, day 3. Phase-contrast, 710X. Figure 91. Large mass of nonmotile zoospores and/or propagules apparently r e s u l t i n g from semi-synchronous sporangial release. SW/P culture, day 3. Phase-contrast, 640X. Figure 92. Small colony on agar media. GSSA, day 5. Phase-contrast, 3 50X. 235 236 swimming away (often 'tangled' together). In large sporangia (approximately 30 zoospores) 30 minutes to one hour or more passed before the majority of zoospores had departed. During th i s time the vibrating mass of zoospores remaining i n the immediate area of the sporangium spread out and became loosely organized. Observations also indicated that, i n some cases, not a l l zoospores a c t u a l l y swam away, but that some remained i n the immediate area of the sporangium to begin further development. Encysted zoospores and intermediate developmental stages leading to enlarged s i n g l e - c e l l e d presporangia range from 2.5 to 12.5 pm i n diameter. Diads r e s u l t i n g from b i p a r t i t i o n processes measure 3.0-5.0 x 4.5-6.5 pm (-8.0 x 9.0 pm; KMV-slush). Tetrads r e s u l t i n g from successive b i p a r t i t i o n and progressive cleavage are d i f f i c u l t to distinguish; they measure 4.8-6.3 x 5.0-6.8 pm (often remaining together and enlarging to 9.5-14.5 x 10.0-16.5 pm; KMV-slush). Mature sporangia which had divided v i a progressive cleavage into more than four zoospores ranged from 7.0 to 15.5 pm i n diameter. Branching and anastomosing ectoplasmic networks are generally well developed. Ectoplasmic nets occasionally have small (to 1.2 pm longest dimension) subglobose to fusiform swellings along t h e i r length. In SW/P cultures ectoplasmic networks are endobiotic, i n t e r b i o t i c (between pollen grains), or, quite often, extend from f r e e - f l o a t i n g single sporangia or clu s t e r s . On KMV-agar the development of Thraustochytrium aggregatum i s s i m i l a r to, but slower than, that described for the other Thraustochytrids presented herein. After 21 days on KMV-agar 237 colonies of t h i s i s o l a t e are c i r c u l a r , s l i g h t l y convex, opaque, creamy white with a g l i s t e n i n g surface, marginate, and from 1.0 to 1.5 mm i n diameter (maximum) (Fig. 92). It was the gregarious nature of t h i s organism and the fact that i t displayed successive b i p a r t i t i o n i n g which lead me to evaluate the developmental cycle of Thraustochytrium aggregatum. The information contained i n Ulken's (1964, 1965) o r i g i n a l descriptions of Thraustochytrium aggregatum i s , i n my opinion, i n s u f f i c i e n t to allow the i d e n t i f i c a t i o n of t h i s organism. Nowhere i n her descriptions do I f i n d mention of successive b i p a r t i t i o n or any other d i v i s i o n process occurring p r i o r to sporangium formation. There are, i n fact, no processes mentioned which would explain the gregarious nature of the sporangia i n t h i s organism. I t was only a f t e r a l i t e r a t u r e survey for other reports of the i s o l a t i o n of T\_ aggregatum that I became convinced that my i s o l a t e could be assigned to t h i s species. Thraustochytrium aggregatum i s described and/or i l l u s t r a t e d by Bahnweg (1973); Gaertner (1968, 1972a, 1972b, 1977), Sparrow (1969) and Volz, et a l . (1976). A l l of the available i l l u s t r a t i o n s [except Bahnweg (1973)] include clusters of r e l a t i v e l y small sporangia with d i s t i n c t i v e r a d i a l d i v i s i o n planes. In Gaertner's (1968, 1972b) drawings and photographs 2-celled and tetrad stages can also be seen. Among these reports only Bahnweg (1973) and Gaertner (1972a, 1972b, 1977) describe T^ aggregatum any further. These descriptions are very b r i e f and e s s e n t i a l l y state that i n T\_ aggregatum a zoospore may either form a sporangium d i r e c t l y , or divide v i a successive b i p a r t i t i o n 238 to form several sporangia. Thraustochytrium aggregatum displays very d i s t i n c t i v e c h a r a c t e r i s t i c s and I f e e l sure that my i s o l a t e i s s i m i l a r to those previously described as t h i s species. This Thraustochytrid i s c e r t a i n l y i n need of further characterization, and should, i n fact, be redescribed more completely (Sparrow, 1969). Successive b i p a r t i t i o n i s not generally considered to be a c h a r a c t e r i s t i c of the genus Thraustochytrium, although the amoeboid protoplasts of two Ulkenia species are capable of a s i m i l a r d i v i s i o n process (Gaertner, 1977; Raghu Kumar, 1979). Gaertner (1972a) describes successive b i p a r t i t i o n p r i o r to sporangium formation as an "obligate" process i n Schizochytrium. However, Booth and M i l l e r (1969) have described release of zoospores from the 'monad' stage i n Schizochytrium Goldstein and Belsky emend Booth and M i l l e r . In my i s o l a t e of T\_ aggregatum successive b i p a r t i t i o n i s of common occurrence, but i s not obligate; encysted zoospores may develop d i r e c t l y into mature zoosporangia. Further, the c e l l s r e s u l t i n g from progressive cleavage do not always become motile zoospores, but may enlarge to become sporangia. Thraustochytrium aggregatum displays c h a r a c t e r i s t i c s of both Schizochytrium and Thraustochytrium, and ce r t a i n l y appears intermediate between these two genera. Further evaluation i s required to determine i f the best placement of t h i s organism i s within the genus Thraustochytrium. b. Thraustochytrium motivum Goldstein Thraustochytrium motivum was by far the most common Thraustochytrid found associated with the two red algae studied. 239 Over 50 i s o l a t e s of t h i s organism were brought into pure culture for developmental observations. The following description was compiled from observations of these i s o l a t e s i n SW/P and KMV-slush cultures, and on KMV-agar. Zoospores are l a t e r a l l y b i f l a g e l l a t e ; reniform, e l l i p s o i d a l , subglobose, ovoid, or varying i n shape; 3.6-5.5 um long x 2.5-3.6 pm wide (Fig. 97). Immature sporangia p r i o r to d e l i m i t i n g basal p r o l i f e r a t i o n s a t t a i n a size of up to 25.0 pm wide x 24.0 pm t a l l . Mature sporangia p r i o r to zoospore release are 14.4-31.2 pm wide x 16.8-30.0 pm t a l l (Figs. 93 and 98). Basal p r o l i f e r a t i o n s measure 4.7-15.6 pm wide x 3.1-11.4 pm t a l l . In SW/P cultures the ectoplasmic networks are normally endobiotic i n pine pollen. Not uncommonly, however, sporangia develop f r e e - f l o a t i n g , and i n such instances the ectoplasmic networks are apparent as fine-branching elements. In KMV-slush cultures the ectoplasmic networks are often more prominent and occasionally display small globose-, ovoid-, or spindle-shaped swellings along t h e i r length. At zoospore release the upper portion of the divided sporangium swells and expands approximately 80% to 100% of i t s o r i g i n a l volume (Figs. 94 and 95). Once expanded, or occasionally during the process, the zoospores begin an active churning motion within the sporangial wall. Normally within about 30 seconds the sporangial wall s p l i t s open and the zoospores quickly swim away. Occasionally a few zoospores remain in the sporangium for several minutes. I cannot substantiate the observations of Goldstein (1963a) - that an active churning motion of the zoospores i s apparent within the sporangium for Figures 93-100. Thraustochytrium motivum Goldstein. Figures 93-96. Sequence of zoospore release from a mature sporangium. Isolate SW/P GT07. KMV-slush, day 3. Phase-contrast, 800X. 93. Mature divided sporangium with basal p r o l i f e r a t i o n . Time - zero. 94. Swelling of upper portion of the sporangium during the i n i t i a l stages of zoospore release. Zoospores a c t i v e l y churning and/or vibrating. Time - plus 2 minutes. 95. Continued swelling and zoospore a c t i v i t y . Zoospores just about to depart. Time - plus 2 minutes, 15 seconds. 96. Persistent sporangial wall and basal p r o l i f e r a t i o n remaining after zoospore release. Time - plus 3 minutes, 30 seconds. Figure 97. L a t e r a l l y b i f l a g e l l a t e zoospores of i s o l a t e SW RT05. SW/P culture. Phase-contrast, 1500X. Figures 98-99. Sporangium of i s o l a t e GT07 on pine pollen p r i o r to (Fig. 98) and af t e r (Fig. 99) zoospore release. SW/P culture, day 4. Phase-contrast, 690X. Figure 100. Young colonies of i s o l a t e SW/P RV02 on KMV-agar displaying enlarged mature sporangia surrounded by young developing sporangia. Radiating ectoplasmic networks. KMV-agar, day 3. Phase-contrast, 380X. 242 5-30 minutes p r i o r to discharge. The empited sporangium reveals the persistent sporangial wall and basal rudiment which w i l l develop into a new sporangium (Figs. 96 and 99). On one occasion two 4 - f l a g e l l a t e propagaules (9.6 um diameter) were observed within a sporangium, apparently the r e s u l t of incomplete cleavage. On KMV-agar the development of Thraustochytrium motivum i s s i m i l a r to that described above. Often, however, the d e t a i l s of zoospore release, the basal pr o l i f e r o u s rudiment, and the persistent sporangial wall are not e a s i l y discerned. Within 36 hours zoospores are released from mature sporangia. I f a f i l m of water i s present on the agar surface, the zoospores w i l l swim considerable distances. Normally, however, t h e i r movement i s r e s t r i c t e d to a f i l m of water immediately surrounding the parent sporangium or cluster. Thus, afte r a period of swimming the zoospores round up, produce ectoplasmic networks and develop i n t o sporangia, forming a ring around the parent sporangium or established colony (Fig. 100). Growth of the colony continues i n t h i s c e n t r i f u g a l fashion, forming opaque white or cream colored colonies 0.2-1.0 mm i n diameter a f t e r three days. Branching ectoplasmic networks with occasional swellings along t h e i r length can be seen extending from i n d i v i d u a l sporangia or colonies (Fig. 100). Very rarely i r r e g u l a r amoeboid or plasmodial c e l l s were observed on the agar surface at the outer margins of colonies. 243 3. Ulkenia a. Ulkenia sp. RC02-80 A single i s o l a t e of t h i s Thraustochytrid was obtained during Sample 2 from an a l g a l tissue-agar plate of Rhodoglossum affi n e . Continued observations of t h i s organism i n pure cultures (SW/P, KMV-slush, KMV-agar) have not yet allowed i t to be f u l l y characterized. The d e t a i l s of i t s developmental cycle which have been discerned are described below. Zoospores are l a t e r a l l y b i f l a g e l l a t e ; subglobose, ovoid, or e l l i p s o i d ; 2.8-4.3 (-5.5) um long by 2.0-2.8 um wide; o f t e n containing a r e f r a c t i l e inclusion (commonly i n the posterior end). Undivided sporangia a t t a i n a size of up to 12.5 um i n diameter (KMV-slush) (Figs. 104 and 110). Mature divided sporangia range i n size from 5.3 x 5.8 um (four zoospores) to 13.0 pm i n diameter (approximately 30 zoospores maximum) (Figs. 101, 105, and 110). The outer wall of mature sporangia i s very i n d i s t i n c t and i n most cases apparently dissolves sometime p r i o r to or during cleavage (or, perhaps, during the i n i t i a l stages of zoospore a c t i v i t y ) . During cleavage the sporangial protoplast contorts, exhi b i t i n g slow and l i m i t e d 'amoeboid' changes i n configuration (Fig. 110, arrow). From my observations of t h i s organism i n l i q u i d culture, the sporangial protoplast did not leave the area of the sporangium p r i o r to or during cleavage. Often, however, the cleaved mass of zoospores (or propagules) would swim or f l o a t a considerable distance away from the sporangium p r i o r to i t s breaking up into individuals. 244 In SW/P cultures, a mature divided sporangium could remain 'dormant' for a considerable length of time p r i o r to zoospore a c t i v i t y . In extreme cases, the propagules r e s u l t i n g from sporangial d i v i s i o n did not develop f l a g e l l a , but, i n time, passively separated themselves and floated away (Figs. 101-103). In such circumstances, at least some of the 'propagules' maintained the configuration of zoospores (i.e. ovoid), but i t i s not yet known i f they could develop f l a g e l l a at some l a t e r time. Zoospore release was observed on numerous occasions and i n no cases did a l l of the zoospores a c t i v e l y swim away from the sporangium (Fig. 110). In an 'active' mature sporangium, zoospores at the periphery began beating t h e i r f l a g e l l a and vibrating 20-45 minutes or more pr i o r to the departure of the f i r s t zoospore(s). After the f i r s t zoospore(s) had departed, the entangled mass of propagules continued vi b r a t i n g and wiggling i t s e l f apart for a further 20-45 minutes (Figs. 101-103). During t h i s time: 1) some zoospores became motile and swam away one by one; 2) certain zoospores with apparent f l a g e l l a managed to vibrate themselves free and move a short distance away (not swimming); 3) some propagules toward the center of the sporangium apparently developed f l a g e l l a and began v i b r a t i n g movements. At the end of the 20 to 45 minutes f l a g e l l a r action ceased. The remaining zoospores became motionless, and began rounding up. My observations suggested that even i n 'active' sporangia there was always at least one (to 5 or more) globose or sublogobse propagule(s) (2.5-3.0 um i n diameter) that never developed f l a g e l l a ( ? - p r o l i f e r a t i n g bodies). Figures 101-110. Ulkenia sp. RC02-80. Figures 101-103. Sequence of zoospore release on pine pollen. SW/P culture, day 4. Phase-contrast, 1400X. 101. Mature divided sporangium. Many zoospores motile. Time - zero. 102. Some zoospores have departed, the remainder are f l o a t i n g apart. Time - plus 35 minutes. 103. Zoospores s t i l l passively f l o a t i n g away; some begining to ?- 'round up'. Time - plus 60 minutes. Figures 104-105. A mature sporangium undergoing d i v i s i o n on KMV-agar. KMV-agar, day 8. Phase-contrast, 2100X. 104. Mature multinucleate sporangium just begining cleavage. Note central ?- vacuole. Time - zero. 105. Sporangium divided, f l a g e l l a not apparent. Time - plus 55 minutes. Figure 106. Irregular multinucleate 'plasmodium' apparently 'cutting o f f u n i c e l l s instead of div i d i n g i n t e r n a l l y . KMV-agar, day 9. Phase-contrast, 2100X. Figures 107-109. Sequence of div i d i n g 'plasmodia'. KMV-agar, day 8. Phase-contrast, 460X. 107. Very i r r e g u l a r multinucleate plasmodia at the colony margin 'pinching and pu l l i n g ' themselves apart. Time -zero. 108. A few motile fragments of the plasmodia remaining. Most have 'divided' into zoospores and/or ?- u n i c e l l u l a r propagules. The margin of an adjacent colony i s coming into view (bottom). Time - plus 5 1/2 hours. 109. Most of the o r i g i n a l plasmodia have divided completely, only a few amoeboid c e l l s remaining. Time - plus 18 hours. Figure 110. Sporangia i n various stages of development. Some of the divided sporangia 'contain' motile zoospores; most contain nonmotile spores. One sporangial protoplast i s undergoing amoeboid movements (arrow). SW/P culture, day 9. Phase-contrast, 560X. 246 Thus, at the end of zoospore release, 2 to 15 or more encysted zoospores and/or propagules remained i n the immediate area of the sporangium; these would eventually become separated and f l o a t away. In between the two extremes described above (i.e. d i v i s i o n but no m o t i l i t y ; d i v i s i o n , m o t i l i t y and departure of some zoospores) were cases i n which zoospores at the periphery of the divided sporangium became motile and somewhat spearated, but did not swim away. In SW/P cultures f r e e - f l o a t i n g sporangia were observed as frequently as sporangia on pine pollen. 'Encysted zoospores' and developing sporangia often occurred i n f a i r l y dense clusters (both f r e e - f l o a t i n g and on pollen), probably due to the nature of zoospore (propagule) release. Endobiotic ectoplasmic networks are produced by t h a l l i growing on pollen. Ectoplasmic networks were inconspicuous on f r e e - f l o a t i n g developmental stages (SW/P and KMV-slush), and when observed, existed as very fine branching and anastomosing filaments. 'Rhizoidal systems' were also comparatively reduced and inconspicuous on KMV-agar. Certain aspects of the development of t h i s organism on KMV-agar are quite d i s t i n c t i v e . Colonies develop i n the 'normal' ce n t r i f u g a l fashion due to 'zoospore' release and subsequent encystment i n the immediate area of the sporangium or colony. P r i o r to dividing, mature multinucleate sporangia very commonly became Plasmodia which displayed amoeboid movements. Plasmodia occurred most commonly at the margins of colonies. These multinucleate stages were normally of very i r r e g u l a r outline, and were capable of enlargement and 'fragmentation' p r i o r to dividing into uninucleate propagules (Figs. 106-109). When examined over 248 time, plasmodia were observed to contort and pinch and p u l l themselves apart forming: 1) small propagules which eventually became motile zoospores; 2) small propagules which apparently remained nonmotile (i.e. no f l a g e l l a ) ; 3) smaller multinucleate sectors which continued d i v i d i n g ( i t i s not known whether these always divided p r i o r to further development) (Figs. 107-109). Mature sporangia were also observed to divide i n a more 'normal' manner, without going through a conspicuous plasmodial t r a n s i t i o n (Figs. 104 and 105). Continued observations on KMV-agar indicated that nuclear d i v i s i o n was concomitant with size increase i n developing sporangia. Colonies on KMV-agar are c i r c u l a r ; convex; marginate; opaque; l i g h t cream colored with a f a i n t pink t i n t ; and a t t a i n a diameter of up to 0.7 mm after 8 days. The v a r i a b i l i t y described i n the zoospore release mechanisms of t h i s Thraustochytrid leads one to suspect that there may be something amiss with the conditions of observation. Although the other Thraustochytrids described i n t h i s thesis performed well under s i m i l a r conditions, i t i s possible that t h i s i s o l a t e has s p e c i f i c requirements or that i t s developmental cycle takes longer than the conditions of observation permitted. These p o s s i b i l i t i e s w i l l have to be examined before a 'normal' developmental cycle can be described for t h i s organism. The combination of c h a r a c t e r i s t i c s displayed by t h i s i s o l a t e indicate that i t belongs within the genus Ulkenia (Gaertner, 1977). I have not actually observed 'migration* of the naked amoeboid protoplast p r i o r to or during cleavage i n l i q u i d 249 cultures. However, the amoeboid movements of the protoplast during cleavage i n a l l culture conditions, and the motile naked Plasmodia commonly produced i n agar cultures, are evidence that a s i m i l a r process exists i n the developmental cycle of t h i s Thraustochyrtid. The i s o l a t e shows certa i n c h a r a c t e r i s t i c s which are s i m i l a r to several species within the genus Ulkenia (Gaertner, 1977), but further studies are required to determine i f i t can be assigned to one of these species. C. Hyalochlorella marina Poyton Hyalochlorella marina was only encountered i n F i e l d Sample 3, when i t was frequently i s o l a t e d from rinse water plates and a l g a l tissue-agar plates. Six i s o l a t e s of t h i s organism were brought into pure culture and maintained using Poyton's (1970a) GDY medium (agar and broth) and KMV (agar and slush). Young asexual spores [ c a l l e d autospores by Poyton (1970a) and aplanospores by Alderman (1974)] are hyaline; uninucleate; globose or r a r e l y subglobose; 3.5-6.5 um i n diameter; and frequently contain a large (2.0-4.0 um diameter) globose, centric or eccentric vacuolar incl u s i o n (Fig. 114). Autospores enlarge and become multinucleate by repeated nuclear d i v i s i o n , often with a concomitant increase i n the size of the vacuolar inclusion (Figs. I l l , 114, and 115). Continued development of presporangia i s marked by further nuclear d i v i s i o n and enlargement (dependent on the number of autospores to be produced). Multinucleate presporangia are normally globose, but occasionally subglobose or ovoid. 250 Sporangia divide by progressive cleavage into uninucleate protoplasts [preautospores (Poyton, 1970a)] (Figs. 112 and 114). Preautospores become spherical and form a wall p r i o r to t h e i r release from the sporangium by a l a t e r a l rupture of the sporangial wall. Autospores are liberated as individuals to repeat the developmental process, or often remain together i n a spherical clump and begin development into sporangia. Developing presporangia begin maturation at a wide range of sizes r e s u l t i n g i n mature sporangia which contain from 4 to 128 autospores; the size of the mature sporangium corresponds to the number of autospores produced. Immature multinucleate presporangia range from 6.5 (binucleate) to 35.0 um i n diameter (on rare occasions even larger multinucleate c e l l s were observed to 63 um i n diameter). Mature divided sporangia range from 13 um (4-celled) to 43 um (? 128-celled) i n diameter (occasionally up to 63 um or more i n diameter). Ecdysis, as described for t h i s organism by Goldstein and Moriber (1966) (as Dermocystidium sp.), was not observed to be a phenomenon required for the enlargement of developing sporangia (see Alderman, 1974). C e l l s with l a t e r a l protrusions s i m i l a r to those described by these investigators were occasionally encountered, but were not correlated with the subsequent release of the protoplast (Fig. 113). Colonies on KMV-agar are cream colored, convex, marginate, and a t t a i n a maximum diameter of about 2 mm a f t e r 14 days. A f t e r f i v e to eight days some iso l a t e s developed "watery edges" as described by Poyton (1970a). "Watery edges" r e s u l t from the deterioration of peripheral organisms, and appear as a th i n layer Figures 111-117. Hyalochorella marina Poyton. Figure 111. Multinucleate undivided sporangia with vacuolar inclusions and peripheral cytoplasm. Isolate GT05 3-2. KMV-slush. Phase-contrast, 490X. Figure 112. Multinucleate divided sporangium and an immature sporangium with peripheral cytoplasm (vacuole). Isolate GT05 3-2. KMV-slush, day 15. Phase-contrast, 490X. Figure 113. Multinucleate and divided sporangia of i s o l a t e GT05 3-2. One sporangium with a l a t e r a l 'protrusion'. KMV-slush, day 15. Phase-contrast, 490X. Figure 114. Numerous r e l a t i v e l y new 'autospores', mature (divided) and immature sporangia of i s o l a t e GT05 3-2. KMV-agar, day 3. Phase-contrast, 260X. Figure 115. Cluster of immature sporangia i n various stages of enlargement. Some containing vacuolar inclusions. Isolate GT05 3-2. KMV-agar, day 3. Phase-contrast, 175X. Figure 116. Small colony of i s o l a t e GT05 3-2 with "watery edges". KMV-agar, day 8. Phase-contrast, 200X. Figure 117. A 4-nucleate Plasmodium of i s o l a t e GT06 3-3. Also shown are + globose and i r r e g u l a r l y shaped sporangia at various stages of maturation. KMV-agar, day 10. Phase-contrast, 345X. 2 5 2 253 of multinucleate protoplasm i r r e g u l a r l y divided by r a d i a l and transverse walls (Fig. 116). When o r i g i n a l l y isolated, a l l six of these i s o l a t e s appeared i d e n t i c a l , except, perhaps, for the presence or absence of "watery edges". Two of the is o l a t e s d i f f e r e d from the developmental cycle described above by the consistent presence of a c t i v e l y motile amoebae and Plasmodia (Fig. 117). Orig i n a l l y , these stages were considered as belonging to a d i s t i n c t organism, but continued observation led me to believe that they were, in fact, stages of Hyalochlorella marina. On KMV-agar, colonies of these two is o l a t e s were cream colored, but diff u s e and thi n (rather than marginate and convex) owing to the active m o t i l i t y of amoeboid stages. In young cultures (five days) the colonies contained both active amoeboid stages and globose, subglobose, or ovoid sporangia (as previously described) i n various stages of development. Observations of these two cultures for the following 10 days revealed the following changes: 1) uninucleate amoebae enlarged and became multinucleate Plasmodia; 2) both amoebae and Plasmodia (at various stages) became stationary and rounded up, becoming globose, subglobose, ovoid, broadly e l l i p s o i d , or occasionally i r r e g u l a r i n outline. These stationary stages continued nuclear d i v i s i o n and developed into sporangia, eventually dividing up into autospores (Fig. 117). By the end of two weeks, a l l c e l l s were stationary and i n various stages of sporangium formation. When transferred back into l i q u i d culture (KMV-slush) these i s o l a t e s reverted to a developmental cycle which resembled more 254 c l o s e l y that described for the other four is o l a t e s . Even i n KMV-slush, however, the growth of these i s o l a t e s was much more dif f u s e (vs. small clumps); rare amoeboid stages continued to be observed; sporangia occasionally showed incomplete cleavage; and vacuolar inclusions were not as conspicuous i n autospores. In h i s studies of Hyalochlorella marina, Poyton (1970a) described uninucleate daughter protoplasts (preautospores) as . . ."capable of m o t i l i t y s i m i l a r to that of limax amoebae." Such amoeboid protoplasts [produced i n agar (GDY) cultures] moved a short d i s t a n c e from the colony and rounded up to continue development into sporangia (Poyton, 1970a). Continued study of these amoeboid and plasmodial stages to determine t h e i r r e l a t i o n s h i p to the developmental cycle of Hyalochlorella marina was beyond the scope of t h i s investigation. D. HIGHER FUNGI 1. Acremonium sp. 019-78 Numerous species referable to the hyphomycete genus Acremonium were encountered during the course of t h i s study. I have t r i e d to i d e n t i f y many of these species, but have met with considerable d i f f i c u l t y . I have, at least, succeeded i n separating these i s o l a t e s to what I f e l t was the species level, even though they are not named. This exercise has proven rewarding since i t has allowed me to di s t i n g u i s h i s o l a t e s encountered frequently from those which were of sporadic or infrequent occurrence (which, i n the end, most of the species of Acremonium i s o l a t e d during t h i s study were). However, a single 255 species of Acremonium (019-78) was encountered regularly, often displaying r e l a t i v e l y high frequencies of i s o l a t i o n (especially i n F i e l d Sample 3 and the D r i f t Study). The following description i s based on eighteen i s o l a t e s of t h i s species which were brought into axenic culture. Cultures were grown on Oatmeal Agar (tap water) at room temperature (22-25°C). For the f i r s t 12 days cultures were incubated i n the dark; they were then transferred into diffuse sunlight for two days p r i o r to observation (Gams, 1971). Further observations were made as the cultures aged. Colonies a t t a i n a diameter of 4.5-6.5 cm af t e r 14 days. Culture pigmentation i s creamy-flesh colored through shades of l i g h t pink to bright orange i n areas of heavy sporulation. A few iso l a t e s produced a f a i n t yellow (?-diffusable) pigment which cast a yellowish overtone to the medium. The reverse i s generally s i m i l a r , but orange pigments are subdued. The culture surface i s moist, glistening; smooth and matted or i r r e g u l a r l y roughened by dense, s l i g h t l y raised, concentric rings and/or clumps of spores. A e r i a l mycelium i s absent or very limited. I f present, i t consists of thin, short (1 mm t a l l ) , upright hyphae with a few associated phialides. Sporulation i s abundant to heavy; at the agar surface or immersed; often i n dense (+ orange) 'clumps'. Crystals are absent. Vegetative hyphae are hyaline; smooth and thi n walled; septate; branched; 1.0-3.5 (-5.0) um wide. Conidiophores a r i s e l a t e r a l l y on vegetative hyphae; they are mostly simple, but may have 1 or 2 orders of branching, i.e. conidiophores supporting 2-3 (-4) phialides (Fig. 118). Conidiophores are either few and widely spaced along a vegetative hypha, or, more commonly, aggregated and extending i n various directions from the hypha (not d i s t i n c t l y whorled). Phialides are hyaline; smooth and t h i n walled; elongate; slender; straight, curved or bent; 0-1 (-2) septate; 10.0-35.0 x 1.6-2.1 (-2.5) um, tapering to 0.9-1.4 um; with c o l l a r e t t e s absent or inconspicuous. Phialides are widest a short distance above the basal septum (Fig. 118). Conidia are produced i n large slimy clusters. Conidia are short c y l i n d r i c , e l l i p s o i d or obovoid; often apiculate at the basal end; hyaline; smooth; varying considerably i n size, (2.9-) 3.5-10.0 (-12.0) x 1.6-3.0 (-4.0) um; length/breadth 1.8-3.6 (Fig. 118). Chamydospores are absent. On the basis of condiophore and conidium morphology, Acremonium sp. 019-78 appears s i m i l a r to Acremonium c h a r t i c o l a (Lindu) W. Gams (Gams, 1971). However, these two fungi d i f f e r with respect to conidium size and culture c h a r a c t e r i s t i c s . After extended incubation, two i s o l a t e s of t h i s fungus developed c l e i s t o t h e c i a containing immature asci s i m i l a r to the genus Emericellopsis. Since mature a s c i and ascospores have not been observed, I have not been able to compare i t to the known species of Emericellopsis. I t i s noteworthy that a marine species of t h i s genus has been described - Ej_ maritima Belyakova (see Gams, 1971; page 30). Figure 118. Acremonium sp. 019-78. Camera lucida drawings of conidiophore and conidium morphology. Isolate 019-78 was designated as the 'type' or reference culture. Figure 118 includes drawings of several i s o l a t e s . Conidia. A l l i s o l a t e s produced a wide range of spore sizes. Upper rig h t . Branched conidiophore. Isolate 062-79. Bottom l e f t . Branched and simple conidiophores. Isolate 019-78. Bottom center and right. Simple and septate conidiophores. Isolate 039-78. CD CD crz> o a? o CP 259 2. T r a l i a ascophylli Sutherland This i n t e r e s t i n g Ascomycete was found associated with subsamples of a r t i f i c i a l l y d r i f t e d and decomposing Gelidium  c o u l t e r i which had been exposed on the beach f o r 71 days (8 March 1979). T r a l i a ascophylli was only observed on two of the i s o l a t i o n plates prepared for t h i s sampling period (one of which i s not l i s t e d among the data presented herein; GYSA-SW). In both culture plates ascocarps developed immersed i n the agar medium surrounding the decomposing a l g a l tissue; but only a f t e r prolonged incubation of these plates i n the cold (4°C; approximately 45 days). The growth and sporulation of t h i s fungus on a r t i f i c i a l media prompted attempts to bring i t into pure culture. Transfers of ascocarps and associated mycelium to several d i f f e r e n t media (Base Mineral Medium, GYSA, Kirk's M-l) brought negative results. However, some mycelial development occurred on GYSA and Kirk's M-l, and i t i s probable that a more sophisticated medium (cf. Fr i e s , 1979, 1980) w i l l allow one to grow t h i s fungus i n axenic culture. Ascocarps are immersed; spherical or subglobose; 120-205 pm high, 105-220 pm wide; os t i o l a t e ; with a very long neck; hyaline to l i g h t brown; thi n walled. The ascocarp neck i s 370-1085 pm long, 10.5-2 5.0 pm wide; c y l i n d r i c a l ; straight or curved; tapering; hyaline to cream colored (Fig. 120). Intact as c i were not observed. Ascospores are hyaline; f i l i f o r m ; bent double, curved and/or coiled; 75.0-120.0 pm long; 2.5-3.0 pm wide at the large end, tapering to 0.5-0.8 pm i n diameter at the opposite 260 end; one to three septate at the broad end (Fig. 119). Certain c h a r a c t e r i s t i c s of t h i s Ascomycete i s o l a t e d from a r t i f i c i a l media d i f f e r s l i g h t l y from previous descriptions (Kohlmeyer and Kohlmeyer, 1979). In p a r t i c u l a r , the ascocarp size range i s s l i g h t l y larger, and the ascocarp neck i s considerably longer. This Ascomycete has only been reported as associated with Ascophyllum nodosum and Fucus sp. i n B r i t a i n (Kohlmeyer, 1968; Sutherland, 1915; Wilson, 1951) and ascospores have been observed in seafoam col l e c t e d i n Maine, U.S.A. (Kohlmeyer, 1968). This i s apparently the f i r s t report of T r a l i a ascophylli from the P a c i f i c Ocean. 3. Unidentified hyphomycete 044-78 Unidentified hyphomycete 044-78 was regularly i s o l a t e d from a l g a l tissues throughout t h i s study. F i f t e e n i s o l a t e s were brought into pure culture for observation and characterization. Unfortunately, the only spores produced were asexual conidia which could not be assigned to any hyphomycete genus with which I am f a m i l i a r . Tight hyphal c o i l s were often produced i n cultures of t h i s fungus, perhaps an in d i c a t i o n that i t was tryi n g to produce f r u i t i n g bodies. A b r i e f description of these i s o l a t e s i s given below. Growth of Unidentified hyphomycete 044-78 was stimulated on media containing seawater; colonies a t t a i n a diameter of 2.0-3.0 cm a f t e r 14 days (OAT - 15°/oo). Colonies lack a e r i a l mycelium. The surface of the culture i s s l i g h t l y raised, densely matted. gure 119. T r a l i a ascophylli Sutherland. Camera lucida drawings of ascospores. 2 6 2 Figure 120. T r a l i a ascophylli Sutherland. Figure 120. 'Squash mount' of ascocarps from o r i g i n a l i s o l a t i o n culture. Note the extremely long ascocarp neck. GYSA, approximately day 45. Phase-contrast, 160X. Figures 121-122. Unidentified hyphomycete 044-78. Isolate 044-78 was designated as the 'type' or reference culture. These photomicrographs are of a d i f f e r e n t i s o l a t e of the same fungus, i s o l a t e 076-80. Figure 121. 'Squash mount' displaying clusters of conidia and conidia formed at the t i p s of long, th i n hyphal branches. Oatmeal Agar (15°/oo SW), day 20. Phase-contrast, 400X. Figure 122. As Figure 121. Nature of short conidiophores and associated conidia. Note the short, geniculate conidiogenous branch (arrow). Oatmeal Agar (15°/oo SW), day 20. Phase-contrast, 1150X. 264 265 moist, and glistening. Coloration i s hyaline to creamy fle s h or pink. Densely sporulating areas appear as scatterd, s l i g h t l y raised, small (approximately 2 mm diameter), pink to orange 'pustules' on the agar surface. The vegetative mycelium i s septate, sparsely to moderately branched, and t h i n [2.0-3.0 (-6.0) um diameter]. Occasionally single terminal or i n t e r c a l a r y hyaline swollen c e l l s were formed (to 14 pm longest dimension) which apparently lacked contents or contained peripheral cytoplasm. Conidia are holoblastic, with a wide basal scar; subglobose; short to long c y l i n d r i c or reniform (often s l i g h t l y constricted i n the middle); 6.5-18.5 x 2.0-3.8 pm. Conidia are produced at the t i p s of elongate and thin or short hyphal branches (Figs. 121 and 122). Attachment of the conidium i s normally basal, but not uncommonly o f f to one side or almost l a t e r a l . Dense clusters of conidia were formed by l a t e r a l 'proliferation' at the t i p of the conidiophore or (?-) from the conidium i t s e l f (Fig. 122). L a t e r a l p r o l i f e r a t i o n s were most commonly turned upwards and oriented i n a plane s i m i l a r to the primary conidiophore and/or conidium (geniculate). These conidiogenous p r o l i f e r a t i o n s remained f a i r l y short (5.0-12.5 x 2.0-2.5 pm). Occasionally secondary branches were formed, a h o l o b l a s t i c conidium being produced at each t i p . My observations suggested that conidia could also be formed successively from a conidiogenous c e l l . 266 4. Sigmoidea a. Sigmoidea marina Haythorn and Jones This marine hyphomycete was i s o l a t e d only once, from an a l g a l tissue-agar plate of Gelidium c o u l t e r i i n Sample 3 (isolate 007-80). The growth and sporulation of t h i s i s o l a t e i n pure culture has been examined and i s described below. A subculture of Sigmoidea marina, obtained from Dr. E. B. Gareth Jones (Portsmouth Polytechnic), has also been examined and certain c h a r a c t r i s t i c s of these two i s o l a t e s are compared. The following c u l t u r a l c h a r a c t e r i s t i c s describe cultures incubated at room temperature (22-25°C) i n diffuse sunlight. KMV-agar (28°/oo). Colonies a t t a i n a diameter of up to 6.0 cm aft e r 21 days. Pigmentation i s generally f a i n t i n these cultures, hyaline to l i g h t creamy yellow (5Y 9/2-9/4*); with very l i m i t e d dark olive-brown (5Y 4/2; 7.5Y 5/2, 4/2) splotches around the inoculum block; the colony reverse i s s i m i l a r . The colony surface i s mostly moist, glistening; the mycelium forming a tough mat at the agar surface. A few l o c a l i z e d areas were velvety, with very l i m i t e d a e r i a l mycelium. Sporulation i s very heavy on KMV-agar, tapering toward the colony margin. Spores accumulate i n dense r a i s e d clumps on the agar surface; the clumps of spores are moist, and glistening. PDA (28°/oo). Colonies a t t a i n a diameter of up to 3.1 cm a f t e r 22 days. Pigmentation i s f a i n t ; hyaline to creamy yellow (5Y 9/1-9/4); often with an immersed rin g of l i g h t olive-brown *Color reference codes are those of The Munsel Color Co. (1969). 267 (5Y 7/4-7/6) pigment around the inoculum. The reverse i s more prominently yellow due to the medium (approaching 5Y 9/8); i t becomes brownish toward the center (2.5Y 6/8). The surface i s s l i g h t l y raised around the inoculum; and generally moist, g l i s t e n i n g or f a i n t l y roughened. Often, a e r i a l , hyaline (to 5Y 9/2), funiculose hyphal ropes extend o f f the inoculum block. These hyphal ropes were a l s o observed out on the surface of the agar medium (procumbent); occasionally they stood erect (with or without associated spores). Sporulation i s abundant, as for KMV-agar. Growth i s r e s t r i c t e d on PDA prepared with 15°/oo seawater and tap water (see Fig. 131 and Table 32). Under these conditions the colony surface i s raised, undulate and/or i r r e g u l a r l y furrowed. On PDA-tap the surface became velvety with l o c a l i z e d wooly a e r i a l mycelium. Darker o l i v e pigments (7.5Y 5/4, 4/2-4/4) are also produced on less saline media. In older (4+ months) PDA cultures of a l l s a l i n i t i e s , dark pigments became more pronounced, ranging from dark olive-browns and chocolate browns to n e a r l y black (5Y 4/4, 3/2, 2.5/1; 2.5Y 3/2). OAT (28°/oo). Colonies a t t a i n a diameter of up to 4.0 cm a f t e r 22 days. Pigmentation and growth c h a r a c t e r i s t i c s are s i m i l a r to KMV-agar. Hyphae are hyaline or rarely f a i n t l y pigmented (brown) i n l o c a l i z e d areas; 2.3-4.5 (-5.3) um wide; smooth and thin walled; septate and branched. Localized swellings (6.3-7.5 pm wide) were observed i n c e l l s of vegetative hyphae; normally these were produced just behind a septum (Fig. 128). In old cultures (4 + 268 months; e s p e c i a l l y with 15°/oo SW and tap water) the c e l l s of vegetative hyphae often swell and take on a l i g h t to medium brown coloration. Normally these c e l l s are 4.5-7.3 um wide, but occasional 'blown-out' c e l l s may a t t a i n a size of up to 18.0 pm i n width. Conidiophores are colorless; smooth and t h i n walled; 5.0-87.5 (-120.0) pm long by 2.3-5.0 pm wide; produced l a t e r a l l y from c e l l s of vegetative hyphae; mononematous; simple or branched; erect or procumbent; straight or curved; and 0-9 septate (Figs. 126, 127, 129-A, 130). Conidiogenous c e l l s are terminal, and produce conidia singly at the apex. Conidia are separated from the conidiogenous c e l l s by a cross wall. Conidiogenous c e l l s are determinant or p r o l i f e r a t i n g v i a successive growing points produced to one side of the terminal conidium. This new conidiogenous growth may remain short, giving r i s e to a sympodial conidiophore with l a t e r a l denticles (conidial detachment scars) (Fig. 129-A); or i t may elongate and be cut o f f by a septum, giving r i s e to an elaborate condiophore with two or three orders of branching (Figs. 126, 127, 130). Conidia are acrogenous; f i l i f o r m (scolecosporous); falcate, sigmoid, U-shaped or looped; hyaline; smooth and t h i n walled; 5-9 (-14) septate; 72.5-137.5 (-165.0) pm long by 3.3-4.8 (-5.3) pm wide i n the middle, tapering to 1.5-3.3 pm wide at the base, 1.3-2.3 pm wide at the t i p (Figs. 123, 124, 129-B, 129-C). Conidia are generally attenuate at the t i p and rounded or truncate at the base. The terminal and basal c e l l s of each conidium are normally devoid of contents. Often the terminal c e l l at one end remained a l i v e (? normally the basal c e l l ) (Figs. 124 and 129-C), and Figures 123-128. Sigmoidea marina Haythorn and Jones. Isolate 007-80. Figure 123. Wet mount of conidia. KMV-agar, day 21. Phase-contrast, 180X. Figure 124. As Figure 123. Most conidia have 'dead' c e l l ( s ) at both t i p s . However, several conidia i n t h i s photomicrograph have l i v e (?-) basal c e l l s (arrow). KMV-agar, day 22. Phase-contrast, 340X. Figure 125. Germinating conidium. Note that the l i v e c e l l s are swollen and the conidium i s constricted at the septa. KMV-agar s l i d e culture, day 3. Phase-contrast, 850X. Figure 126. Elongate branching conidiophore. Origin of conidiophore at arrow. Note the conidia being produced on l a t e r a l denticles and the younger conidia at lower levels. KMV-agar s l i d e culture, day 13. Phase-contrast, 425X. Figure 127. Branched, complex conidiophore supporting numerous spores. KMV-agar s l i d e culture, day 15. Phase-contrast, 360X. Figure 128. Vegetative hyphae with swollen, vacuolate portions adjacent to septa. KMV-agar s l i d e culture, day 11. Phase-contrast, 350X. Figure 129 (A-C). Sigmoidea marina Haythorn and Jones. Isolate 007-80. Camera lucida drawings of conidiophore and conidium morphology. A. Various types of + simple conidiophores with attached conidia. The conidiophores i l l u s t r a t e d range from simple l a t e r a l protrusions of vegetative hyphae, through elongate septate l a t e r a l s , to those displaying + sympodial proliferous denticles. B. Conidia of various shapes and sizes with dead terminal c e l l s . C. Several conidia with l i v e terminal c e l l s at one end (?-base). Figure 130. Sigmoidea marina Haythorn and Jones. Isolate 007-80. Camera lucida drawing of a complex branched conidiophore with + attached conidia. 275 occasionally two (-3) c e l l s at the terminus were devoid of contents. End c e l l s devoid of contents are normally 10.0-35.0 um long, 1.8-2.8 pm wide at t h e i r attachment, and 1.3-2.3 pm wide at the t i p (Figs. 124, 129-B, 129-C). When f i r s t produced, the conidia of i s o l a t e 007-80 were not, or were only s l i g h t l y , constricted at the septa. Prior to germination (KMV-agar s l i d e culture) however, the i n d i v i d u a l c e l l s swelled considerably [becoming 5.0-9.2 (-10.5) pm wide] and the c o n i d i a l outline became d i s t i n c t l y constricted at the septa (Fig. 125). Any of the viable c e l l s i n a conidium could produce germ tubes (Fig. 125). The growth rates of the Sigmoidea marina i s o l a t e obtained from Dr. Gareth Jones were generally very s i m i l a r to those of i s o l a t e 007-80 and s i m i l a r trends were displayed with respect to s a l i n i t y (Table 32). Pigmentation and colony growth c h a r a c t e r i s t i c s were also nearly i d e n t i c a l to i s o l a t e 007-80. On less saline media (15°/oo, tap), the darkly pigmented areas produced by Dr. Jones' subculture took on a brownish hue [2.5Y 6/8, 5/6; 10YR 6/4-6/6 (PDA); 2.5Y 7/6-6/6 (OAT)] rather than olive-brown. The morphology and other c h a r a c t e r i s t i c s of the vegetative mycelium were also s i m i l a r to i s o l a t e 007-80; including l o c a l i z e d swellings (to 7.5 pm wide) of the hyphae. Occasionally large (to 30.0 pm longest dimension) globose or subglobose, terminal or intercalary swellings were produced by Dr. Jones' i s o l a t e (containing vacuoles or ? lacking contents). None of the culture conditions tested, including agar block submersion i n s t e r i l e seawater, induced sporulation i n t h i s 276 Sigmoidea marina subculture. Thus, the morphological c h a r a c t e r i s t i c s of the conidiogenous c e l l s and conidia were not observed. I t i s unfortunate that I have not been able to examine sporulation i n the culture of Sigmoidea marina obtained from England. Dr. Gareth Jones possesses photographs and a subculture of i s o l a t e 007-80. He agrees that the developmental stages of t h i s i s o l a t e are s i m i l a r to Sigmoidea marina. From my observations of the growth and colony c h a r a c t e r i s t i c s of these two i s o l a t e s , I f e e l assured that they are conspecific. However, i s o l a t e 007-80 displays several c h a r a c t e r i s t i c s which d i f f e r from S. marina as described by Haythorn and Jones (Haythorn, et al., 1980). These differences are: 1) When f i r s t produced, conidia of i s o l a t e 007-80 are thin and not noticeably constricted at the septa. Only p r i o r to germination (under certain conditions: KMV-agar s l i d e culture) do they swell to a width s i m i l a r to that described by Haythorn and Jones (Haythorn, et al., 1980) and become d i s t i n c t l y constricted at the septa. 2) In i s o l a t e 007-80, sympodial pro l i f e r o u s conidiophore development occurs via: a) short l a t e r a l protrusions of the conidiogenous c e l l (Figs. 126 and 129-A); b) short l a t e r a l (non-septate) branches of the conidiogenous c e l l (Fig. 129-A); and/or c) elongate l a t e r a l branches which originate from and are cut o f f from (septum) the i n i t i a l conidiogenous c e l l (Figs. 126, 127, 130). Haythorn and Jones (Haythorn, et al., 1980) describe only the f i r s t of these developmental patterns i n t h e i r i s o l a t e s 277 Table 32 Colony diameter (cm) of Sigmoidea l i t t o r a l i s , S_. marina and S_. prol i f era on Potato Dextrose Agar* made up with tap water and aged seawater adjusted to 15°/oo and 28°/oo. Values are expressed as colony diameter (cm) af t e r 10 days of incubation at room temperature (22-25°C). Water S a l i n i t y (°/oo) Species/Isolate Tap 15 28 Sigmoidea l i t t o r a l i s sp. nov. FHL-79 0.70 0.87 0.87 012-79 2.67 2.77 2.90 039-80 2.97 4.03 4.60 060-80 1.63 2.60 4.60 X 1.99 2.57 3.24 S.E. 0.45 0.56 0.77 Sigmoidea marina S. marina Haythorn & Jones 0.97 1.50 2.03 007-80 0.83 1.03 1.77 X 0.90 1.26 1.90 S.E. 0.05 0.17 0.09 Sigmoidea p r o l i f e r a S. p r o l i f e r a (ATCC #16660) 1.30 0.77 0.63 *DIFC0 prepared dehydrated medium. Figure 131. Colony diameter (cm) of Sigmoidea l i t t o r a l i s , S. marina and S.  p r o l i f e r a a f t e r 10 days of incubation on Potato Dextrose Agar* made up with tap water and aged seawater adjusted to 15°/oo and 28°/oo. Cultures were incubated at room temperature (22-25°C) in d i f f u s e sunlight. Values plotted are the mean (X) of three measurements? the second was taken at 90° to the f i r s t ? the t h i r d was taken at 45° between these two. Bars indicate standard error (S.E.) of the means where more than one i s o l a t e was observed (see Table 32). *DIFCO prepared dehydrated medium. o o Sigmoidea littoralis sp. nov. » • Sigmoidea marina A >A Sigmoidea prolifera SALINITY (%o) FIGURE 131 280 of Sigmoidea marina, and, therefore, do not describe branched conidiophores. In i s o l a t e 007-80 branched conidiophore development was observed i n KMV-agar s l i d e cultures, and may, therefore, r e f l e c t unusual conditions. However, short l a t e r a l branches were observed on many conidiogenous c e l l s i n 'wet mount' sl i d e s prepared from numerous agar cultures. Dr. Gareth Jones has observed the differences displayed by i s o l a t e 007-80, and also by another i s o l a t e of S^ marina from North Carolina. He believes that these d i s t i n c t i o n s do not vary s i g n i f i c a n t l y from the species concept of S^ marina, but that they should be documented (Dr. E. B. Gareth Jones, pers. comm.). b. Sigmoidea l i t t o r a l i s sp. nov. A new species of the genus Sigmoidea was discovered during the course of t h i s study. Many is o l a t e s of t h i s aquatic hyphomycete were obtained from tissues of Rhodoglossum a f f i n e and Gelidium c o u l t e r i v i a a l l of the i s o l a t i o n methods employed (agar plates, s t e r i l e seawater cultures, moist chambers). The fungus was most commonly isola t e d from a r t i f i c i a l l y d r i f t e d decomposing a l g a l tissues (27 Dec, 1978 - 8 March, 1979) and from the f i e l d -c o l l e c t e d a l g a l tissues of Sample 3 (4 June, 1980). A t o t a l of 14 i s o l a t e s brought into axenic culture have provided the basis for the following description. The following c u l t u r a l c h a r a c t e r i s t i c s describe cultures incubated at room temperature (22-25°C) i n diffuse sunlight. KMV-agar (28°/oo). Colonies a t t a i n a diameter of (4.4-) 6.0-7.5 cm a f t e r 16 days. Culture pigmentation i s hyaline to 281 various shades of yellow (7.5Y 9/2-9/10*); commonly an immersed ring and/or splotches of dark o l i v e green to black (7.5Y 5/4, 4/4, 3/2, 2.5/0; 5GY 3/2) coloration are produced around the inoculum. Transition pigments of intermediate yellow to o l i v e shades (7.5Y 8/8-8/10, 7/6-7/8). The colony reverse i s similar. The colony surface i s moist, s l i g h t l y roughened, or velvety. A e r i a l mycelium i s l i m i t e d or common; hyaline, wooly, funiculose; commonly associated with the inoculum and/or darkly pigmented areas; and occasionally throughout. Concentric 'growth rings' were displayed by some isolates. Sporulation i s sparse to heavy, occurring on the agar surface i n slimy a e r i a l 'stands', or raised clumps; i t was rarely immersed. With age (8+ months) dark brown (2.5Y 5/6, 4/4, 3/2) pigments were produced by some isolates. PDA (28°/oo). Colonies generally a t t a i n a diameter of 3.0-4.6 cm a f t e r 10 days; growth of two i s o l a t e s was r e s t r i c t e d t o 1.0 cm diameter a f t e r 10 days. Pigmentation i s hyaline (5Y 9/6-9/8) to creamy yellow (5Y 9/2-9/4); the colony reverse i s s i m i l a r or darker yellow due to the medium. The colony surface ranged from: 1) moist, glistening, and often s l i g h t l y raised and/or undulate around inoculum; 2) s i m i l a r to 1 with i r r e g u l a r l y d i s t r i b u t e d i s o l a t e d clumps (to 4 mm diameter, 2 mm high) of hyaline, wooly, funiculose a e r i a l mycelium; 3) to producing dense hyaline, + funiculose, a e r i a l (1-2 mm high) mycelium throughout, except at the margin (approximately 3 mm immersed). Sporulation i s common to abundant, on the agar surface, amongst a e r i a l mycelium, or i n dense raised clumps. *Color reference codes are those of The Munsel Color Co. (1969). 282 Growth rates were slower on PDA prepared with less saline water (15°/oo, tap) (Table 32, F i g . 131). Under these conditions i s o l a t e s often produced medium to dark brown (10YR 7/4, 6/4-6/6, 4/4, 3/2) and/or dark o l i v e green (5Y 4/4; 7.5Y 3/2) pigments i n older (central) portions of the culture. OAT (28°/oo). Colonies a t t a i n a diameter of 3.2-5.6 cm a f t e r 10 days. Pigmentation i s hyaline to yellow [5Y 9/2-9/6 (-9/8)], occasionally with limited, immersed, olive/brown (5Y 7/6, 6/6) coloration i n central areas. The surface i s moist, glistening, sometimes f i n e l y and i r r e g u l a r l y roughened and/or s l i g h t l y raised around the inoculum. Sporulation i s common to abundant, as for KMV-agar. Occasionally dense conidium production was associated with small (to 5 mm diameter), s l i g h t l y raised (convex), creamy tan (2.5Y 8/4-8/6) 'pustules' on the agar surface. Growth rates were slower on less saline Oatmeal Agar, and medium to dark brown, o l i v e green or black pigments were often produced i n older portions of the culture (7.5YR 4/4, 3/2, 2/0; 10YR 6/8, 5/6, 4/4, 3/2, 2.5/1; 2.5Y 6/8, 5/4-5/6, 4/4, 3/2; 5Y 5/4, 4/4; 7.5Y 6/4). Hyphae are hyaline; smooth and t h i n walled; 2.0-5.0 pm wide; septate; and branched. With age and under certain c u l t u r a l conditions, c e l l s of vegetative hyphae showed s l i g h t swelling, became c o n s t r i c t e d at the septa, and took on a l i g h t to medium brown coloration. Normally such c e l l s were 9.0 pm or less in width, but occasional i n t e r c a l a r y and/or terminal 'blown-out' c e l l s could a t t a i n a diameter of 18.0 pm. 283 Conidiophores originate as l a t e r a l branches of vegetative hyphae; they are mononematous; often somewhat aggregated; and 6.0-135.0 (-225.0) pm long by 2.3-5.0 (-7.5) pm wide. Conidiophores are simple or rarely branched; c y l i n d r i c a l ; straight or i r r e g u l a r l y curved; 0-14 septate; colorless; with smooth and th i n walls; and with acroauxic development (Figs. 136-143). In s l i d e cultures (KMV-agar), I occasionally observed very elongate (to 545 pm; 27 septate), simple (unbranched) l a t e r a l s which produced a spore at t h e i r t i p . I hesitate to include these i n the conidiophore length dimensions. Conidiogenous c e l l s are simple; apical; discrete; often with a rounded and s l i g h t l y swollen apex; determinate or p r o l i f e r a t i n g by growth beyond the point of attachment of the previous spore. Growth of p r o l i f e r a t i n g conidiogenous c e l l s i s + sympodial, with up to 10 or more spores being produced; detachment scars are i n d i s t i n c t or appear as s l i g h t l a t e r a l swellings (Figs. 138-140, 142, 143). Septa may be l a i d down as a p r o l i f e r a t i n g conidiogenous c e l l elongates. On rare occasions p r o l i f e r a t i n g conidiogenous c e l l s were observed to continue 'vegetative' growth. Conidia are acrogenous; terminal; s o l i t a r y ; f i l i f o r m (scolecosporous); sigmoid or U-shaped; (72.5-) 120.0-280.0 (-380.0) pm long by 2.3-5.0 pm wide (Figs. 132-134, 144). They are 4-16 septate (often i n d i s t i n c t ) ; colorless; with smooth and t h i n walls; and with i n d i s t i n c t detachment scars. The conidia are generally widest i n the middle, tapering very s l i g h t l y towards both ends. However, each t i p i s s l i g h t l y swollen and rounded, measuring 3.3-5.0 pm wide. Conidia germinated r e a d i l y Figures 132-137. Sigmoidea l i t t o r a l i s sp. nov. Figure 132. Conidia of i s o l a t e 046-80. KMV-agar, day 22. Phase-contrast, 200X. Figure 133. A conidium of i s o l a t e FHL-79. Note the s l i g h t l y swollen, rounded t i p s . Phase-contrast, 410X. Figure 134. A germinating conidium of i s o l a t e 012-79. Note the s l i g h t l y swollen c e l l s and constrictions at the septa. KMV-agar s l i d e culture, day 3. Phase-contrast, 680X. Figure 135. Two conidia of i s o l a t e 012-79 l y i n g adjacent to one another. They have apparently fused v i a short l a t e r a l protrusions. KMV-agar, day 23. Phase-contrast, 550X. Figure 136. Conidium produced on a short simple l a t e r a l ?-branch of a vegetative hypha. Isolate 060-80. KMV-agar s l i d e culture, day 7. Phase-contrast, 550X. Figure 137. Conidium produced on a simple, elongate, septate l a t e r a l branch. Isolate 012-79. KMV-agar s l i d e culture, day 13. Phase-contrast, 390X. 285 Figures 138-141. Sigmoidea l i t t o r a l i s sp. nov. Figure 138. Septate p r o l i f e r a t i n g conidiophore of i s o l a t e 060-80. KMV-agar s l i d e culture, day 13. Phase-contrast, 390X. Figure 139. An i r r e g u l a r l y branched conidiophore of i s o l a t e FHL-79. The T-shaped t i p of the conidiophore i s p r o l i f e r a t i n g conidia i n both directions. KMV-agar sl i d e culture, day 20. Phase-contrast, 390X. Figure 140. P r o l i f e r a t i n g conidiophore l y i n g adjacent to the vegetative hypha from which i t arose. Seven conidia have been produced. Isolate 060-80. KMV-agar s l i d e culture, day 9. Phase-contrast, 390X. Figure 141. What appears to be a highly branched, complex 'conidiophore-like* structure of i s o l a t e FHL-79. This branching structure originates from a vegetative hypha just below the agar surface (arrow). Seven branches are present, six of which have spores at t h e i r t i p . KMV-agar s l i d e culture, day 14. Phase-contrast, 190X. 287 Figure 142. Sigmoidea l i t t o r a l i s sp. nov. Camera lucida drawings of conidiophores and associated conidia. From KMV-agar s l i d e cultures. Upper l e f t . Elongate, septate, simple conidiophore. Isolate 060-80. Center. Two conidia produced on short, simple l a t e r a l branches. Isolate 039-80. Right. Conidiophore with a short l a t e r a l branch. Isolate 060-80. Bottom l e f t . Proliferous conidiophore with three conidia. Isolate 060-80. 289 Figure 143. Sigmoidea l i t t o r a l i s sp. nov. Camera lucida drawing of a septate p r o l i f e r a t i n g conidiophore with associated conidia. Isolate 060-80. From KMV-agar sl i d e culture. 291 Figure 144 (A&B). Sigmoidea l i t t o r a l i s sp. nov. Camera lucida drawings of conidia. Wet mounts from various cultures, including the following i s o l a t e s : FHL-79, 012-79, 025-79, 039-80 and 060-80. A. Morphology and size v a r i a t i o n i n conidia. B. Conidia containing 'dead' c e l l s i n various locations. 'Dead' c e l l s indicated by thinner walls. 294 on i s o l a t i o n media? they became s l i g h t l y swollen (to 5.8 pm diameter; 6.3 pm tips) and constricted at the septa p r i o r to germination (KMV-agar s l i d e culture). A l l c e l l s of a conidium, including the terminal c e l l s , could produce germ tubes (Fig. 134). I have observed conidia which contained c e l l s devoid of contents, but normally such conidia were produced under poor culture conditions and/or i n senescing cultures. C e l l s lacking contents were observed i n any position along the conidium; they were found i n central portions as frequently as toward or at the t i p s (Fig. 144-B). I have also observed conidia l y i n g p a r a l l e l to one another which had fused v i a short and t h i n l a t e r a l p r o t r u s i o n s (Fig. 135) [ c f . S. p r o l i f e r a (Crane, 1968; F i g . ID]. Chlamydospores are absent. Many i s o l a t e s of t h i s fungus also produced a coelomycetous f r u i t i n g structure on a r t i f i c i a l media. The c h a r a c t e r i s t i c s of t h i s coelmycetous stage, especially the conidiogenous c e l l s , have not been s a t i s f a c t o r i l y elucidated. Therefore, the taxonomic a f f i n i t i e s of t h i s stage remain, at present, undetermined. Those observations which have been made on t h i s coelomycete are described below. Conidiomata are pycnidial; t o t a l l y to p a r t i a l l y immersed or s u p e r f i c i a l (agar); separate or loosely aggregated; dark brown to black; globose to subglobose; 310-450 pm high, 290-400 pm wide (Figs. 147 and 148). Walls are thick (35-80 pm), composed of an outer layer of loose interwoven hyphae (textura i n t r i c a t a ) , pale to medium brown, and an inner l a y e r (8-30 pm t h i c k ) of compact Figures 145-148. Sigmoidea l i t t o r a l i s sp. nov. Figure 145. 'Pycnospore' of i s o l a t e 068-79. Phase-contrast, 370X. Figure 146. Pycnidium cross section (approximately 8 um thick) showing wall structure. Isolate 012-79. Phase-contrast, 370X. Figure 147. Cross section of a pycnidium displaying general morphology/structure. This pycnidium was formed on the g l a s s w a l l of a t e s t tube and was attached by a d i s c o i d base (subiculum). Isolate 068-70. KMV-agar. Phase-contrast, 100X. Figure 148. Culture of i s o l a t e 060-80 displaying pycnidium development. Most of the pycnidia i n t h i s culture are immersed or just penetrate the agar surface (erumpent). PDA (15°/oo SW), 9 months. About two-thirds actual size. 296 polyhedral c e l l s (textura angularis), pale to dark brown (Fig. 146). The (?-) single o s t i o l e i s c i r c u l a r and sometimes s l i g h t l y p a p i l l a t e . The nature and d i s t r i b u t i o n of the conidiophores and/or conidiogenous c e l l s are as yet undetermined. Conidia are hyaline; f i l i f o r m ; mostly curved or i r r e g u l a r l y bent (i.e. entangled and folded back and forth inside the pycnidium); smooth and th i n walled; multiseptate; 120.0-235.0 um long by 2.0-3.5 um wide (Fig. 145). Most conidia have s l i g h t l y swollen, rounded t i p s at both ends; occasionally swollen c e l l s (tips and/or central) attained a diameter of 6.3 (-10.0) pm. On a r t i f i c i a l agar media, pyc n i d i a l formation normally required two (+) months of incubation. Pycnidia were formed i n any location i n a agar culture plate or te s t tube slant. However, i n test tubes they were most often formed against the g l a s s w a l l of the tube, i n which case they were attached by a di s c o i d base (subiculum) of loose to t i g h t l y interwoven hyphae (textura i n t r i c a t a ) (Fig. 147). I have examined the following material: Sigmoidea p r o l i f e r a (Petersen) Crane: Subculture Ex-holotype (ATCC 16660). Sporulation was not observed. Sigmoidea marina Haythorn and Jones: Subculture (?-) Ex-holotype, Dr. E. B. Gareth Jones, Portsmouth Polytechnic, England. Sporulation was not observed. Isolate 007-80 obtained from the i n t e r t i d a l red alga Gelidium c o u l t e r i Harv.; P a c i f i c Grove, C a l i f o r n i a ; June 1980. 298 Sigmoidea sp. nov. (proposed Sigmoidea l i t t o r a l i s sp. nov.): Isolate FHL-79 obtained from the i n t e r t i d a l green alga Prasiola meridionalis S. & G.; Cattle Point, San Juan Island, Washington; July 1979. Numerous is o l a t e s obtained from the i n t e r t i d a l red algae Rhodoglossum a f f i n e (Harv.) Kyi. and Gelidium  c o u l t e r i Harv.; P a c i f i c Grove, C a l i f o r n i a ; January-March, 1979, June, 1980. A t o t a l of 14 is o l a t e s have been brought into axenic culture. Of these, i s o l a t e 012-79 w i l l be designated as the holotype. Isolate 012-79 was obtained from Gelidium  c o u l t e r i collected at Hopkins Marine Station (Stanford University), P a c i f i c Grove, Calif., 20 March, 1979. Cultures of Sigmoidea aurantia Descals were not available (Descals, pers. comm.). Only an unpublished description and i l l u s t r a t i o n s of t h i s species have been examined. This fungus i s placed i n the genus Sigmoidea because i t has simple or branched conidiophores; the conidiogenous c e l l s are teriminal, and determinate or with + sympodial p r o l i f e r a t i o n ; and the conidia are multiseptate, f i l i f o r m and/or sigmoid. Some of the major distinguishing c h a r a c t e r i s t i c s of the four species now placed i n the genus Sigmoidea are l i s t e d i n Table 33. Sigmoidea  l i t t o r a l i s can be distinguished from S^ p r o l i f e r a (Petersen) Crane and Sj_ aurantia Descals by i t s longer spores. The spores of S^ marina Haythorn and Jones are generally shorter than, but within the range of, S. l i t t o r a l i s . However, i n S. marina the Table 33 Some di s t i n g u i s h i n g c h a r a c t e r i s t i c s of the four species described in the genus Sigmoidea. Species Characteristic Sigmoidea p r o ! i f e r a Sigmoidea aurantia Sigmoidea marina Sigmoidea 1 i t t o r a l i s (Petersen) Crane Descals* Haythorn & Jones sp. nov.f Conidiophores and/or Conidiogenous c e l l s (ym) Conidia (um); Septation Growth Rate in Culture; Medium Pigmentation in Culture Additional D i s t i n c t i o n s 3.7-12.4 X 1.6-2.7 48.7-111.0 X 1.3-2.7 Multiseptate 1.0 cm/10 days OAT or PDA - Tapt Pale Cream, Gray, (Brownf) Chlamydospores 24.0-60.0 X 2.5-5.5 60.0-90.0 X 3.5-4.5 (0-)5(-9) septate 6.0 cm/18 weeks MEA (2%) - ? Orange Pycnidia; spores ?-shape 5.0-45.0 X 1.5-3.5 [5.0-87.5(-120.0) X 2.3-5.0]f (80.0-)110.0-180.0 (-231.0) X 2.0-10.0 ( 2 - ) 7 - l l septate 2.4 cm/10 days OAT - 28°/oo SWf Hyaline, Yellow or Orange (Olive Green, Brownf) Terminal c e l l s of conidia devoid of contents . Marine algae 6.0-135.0(-225.0) X 2.3-5.0(-7.3) (72.5-)120.0-280.0 (-380.0) X 2.3-5.0 4-16 septate 4.4 cm/10 days OAT - 280/oo SW Hyaline, Yellow, Rusty Brown, Brown, Olive Green, Black Pycnidia; spores f i l i f o r m Marine algae Habitat Freshwater; debris Freshwater; twig *Unpublished. fPersonal observations. 300 end c e l l s of mature conidia are normally devoid of contents, whereas those of Sigmoidea l i t t o r a l i s are viable and s l i g h t l y swollen. A description of t h i s new fungus w i l l be v a l i d l y published i n the near future. The holotype (isolate 012-79) w i l l be deposited with the American Type Culture Collection. Ex-holotypes w i l l also be deposited with the American Type Culture C o l l e c t i o n and the Commonwealth Mycological Institute. Petersen (1963) described the aquatic hyphomycete Flagellospora p r o l i f e r a as producing elongate, somewhat curved, u n i c e l l u l a r spores on short simple phialophores. In h i s o r i g i n a l description Petersen also noted several features of E\_ p r o l i f e r a which d i f f e r e d from other members of the genus Flagellospora. Most s i g n i f i c a n t l y , while phialospores were produced at f i r s t , the t i p of the p h i a l i d e could l a t e r grow v e g e t a t i v e l y and act as an aleuriophore. The aleuriospores were s o l i t a r y terminal spores . . ."with subsequent spores being produced by branching of the aleuriophore just below the point of attachment of the l a s t spore." (Petersen, 1963). Crane (1968) obtained several i s o l a t e s of a s i m i l a r aquatic fungus and, a f t e r examining the type material of F^ p r o l i f e r a , transferred t h i s species to a newly erected genus - Sigmoidea. Crane (1968) described the genus Sigmoidea as possessing the following special c h a r a c t e r i s t i c s : "The sporogenous c e l l may be a l a t e r a l phialide, and aleuriophore, or a p r o l i f e r a t i n g c e l l bearing successive growing points; the mature sigmoid spores are multiseptate." Petersen (1963) had f a i l e d to observe the 301 multiseptate nature of mature spores. From these descriptions of Sigmoidea p r o l i f e r a , and those of the other three species now placed i n the genus Sigmoidea, the major distinguishing c h a r a c t e r i s t i c s of t h i s genus are: 1) conidiogenous c e l l s able to p r o l i f e r a t e i n a + sympodial fashion by producing successive growing points to one side of the previous conidium; 2) multiseptate f i l i f o r m and/or sigmoid conidia. The spores produced by Sigmoidea species are s i m i l a r to those formed by members of the genus Anguillospora. However, i n Anguillospora there i s apparently only one 'aleuriospore' (see conidium development discussion, below) formed at the t i p of each sporophore (i.e. determinate) (Ingold, 1942; Ranzoni, 1953). In Flagellospora conidia are sigmoid or lunate, u n i c e l l u l a r or 1-septate, and are produced singly or successively from phialides (Ingold, 1942; Petersen, 1963; Ranzoni, 1953). The hyphomycete Condylospora spumigena Nawawi i s very s i m i l a r to members of the genus Sigmoidea i n that f i l i f o r m multiseptate spores are produced on determinate or indeterminate sympodially p r o l i f e r a t i n g conidiogenous c e l l s (Nawawi, 1976). Condylospora spumigena i s distinguished from Sigmoidea species based on spore shape; the conidia are bent back on themselves just past the half-way mark and bent out again a short distance away (Nawawi, 1976). Within the genus Sigmoidea, phialides have been described i n S. p r o l i f e r a [Crane, 1968; Petersen, 1963 (as Flagellospora  p r o l i f e r a ) ] • Examination of the l i t e r a t u r e available on the species within t h i s genus, as well as personal observations of sporulation i n two species, has given no other indications of 302 p h i a l i d i c spore development. For t h i s reason (and others) i t i s important that conidium ontogeny be examined again i n S.  p r o l i f e r a , and I w i l l continue my attempts to induce sporulation i n t h i s culture. Conidial development i n Sigmoidea aurantia and Sj_ marina has been described as h o l o b l a s t i c (Descals, unpubl.; Haythorn et al., 1980). E a r l i e r investigators of scoleoscosporous aquatic Hyphomycetes have described conidia produced i n . t h i s manner as aleuriospores (Crane, 1968; Ingold, 1942; Petersen, 1962, 1963; Ranzoni, 1953). In more recent years the term aleuriospore (as applied to these fungi) has been deemed unacceptable, and emphasis has been placed on t h a l l i c and b l a s t i c spore types (Kendrick, 1971; Ingold, 1979). In t h a l l i c development, . . . "any enlargement of the recognizable conidium i n i t i a l . . . occurs only a f t e r the i n i t i a l has been d e l i m i t e d by a septum or septa. The conidium d i f f e r e n t i a t e s from a whole c e l l . " (Kendrick, 1971). In b l a s t i c development, . . . "there i s marked enlargement of a recognizable i n i t i a l before the i n i t i a l i s delimited by a septum. The conidium d i f f e r e n t i a t e s from part of the c e l l . " (Kendrick, 1971). If we accept these d e f i n i t i o n s , then the spore development which I have observed i n my is o l a t e s of Sigmoidea marina and S. l i t t o r a l i s , as well as that shown i n certain i l l u s t r a t i o n s of S. p r o l i f e r a [Petersen, 1963; Figs. 2B-D (as F^ p r o l i f e r a ) ] , would be considered t h a l l i c . However, i t would appear that Descals' i l l u s t r a t i o n s of S^ aurantia depict b l a s t i c spore development (Descals, pers. comm.). Ingold (1979, 1981) has 303 questioned the v a l i d i t y of categorizing the spore development i n fungi producing thin-walled hyaline conidia, e s p e c i a l l y the conidia of aquatic species, as either b l a s t i c or t h a l l i c . I believe Ingold would agree with the interpretation that the spore development described herein for Sigmoidea marina and S. l i t t o r a l i s i s of the t h a l l i c type. However, spore development i n c e r t a i n other fungi i s much more d i f f i c u l t to categorize (see Ingold, 1981). I t i s Ingold's o p i n i o n that . . . " i f a fundamental d i s t i n c t i o n can be made, i t i s between the p h i a l i d i c and non-phialidic types." (Ingold, 1979). This provides further impetus to examine S^_ p r o l i f e r a for p h i a l i d i c spore development. Based on the major distinguishing c h a r a c t e r i s t i c s expressed above, the species placed within the genus Sigmoidea form a natural grouping. However, since spore development i s a major c r i t e r i o n i n hyphomycete taxonomy, we can expect further evaluation and discussion of the species placed within t h i s genus. 304 VIII. BIBLIOGRAPHY Abbott, I.A. 1980. Seasonal Population Biology of some Carrageenophytes and Agarophytes. In I.A. Abbott, M.S. Foster and L.E. Eklund, eds. " P a c i f i c Seaweed Aquaculture". C a l i f . 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The n u t r i t i o n a l requirements of i s o l a t e s of Labyrinthula spp. J. Gen. Microbiol. 12: 455-463. Vishniac, H.S. 1956. On the ecology of the lower marine fungi. B i o l . B u l l . I l l : 410-414. Vishniac, H.S. and Watson, S.W. 1953. The steroid requirements of Labyrinthula v i t e l l i n a var. p a c i f i c a . J. Gen. Microbiol. 8: 248-255. 321 Volz, P.A. and D.E. Jerger. 1972. A preliminary study of marine fungi from Abaco Island, the Bahamas. Mycopath. Mycol. Appl. 48(4): 271-274. Volz, P.A., Y-C. Hsu and C-H. L i u . 1976. The Thr a u s t o c h y t r i c e a e and other i n t e r t i d a l fungi of Taiwan. Taiwania 21(1): 1-5. Wagner-Merner, D.T. 1973. Arenicolous fungi from the south and central coast of Florida. Nova Hedwigia 23(4): 915-922. Wagner-Merner, D.T., W.R. Duncan and J.M. Lawrence. 1980. Preliminary comparison of Thraustochytriaceae i n the guts of a regular and i r r e g u l a r echinoid. Bot. Mar. 23: 95-97. Wainwright, M. 1980. Alginate degradation by the marine fungus Dendryphiella salina. Mar. B i o l . Letters 1: 351-354. Wainwright, M. and V. Sherbrock-Cox. 1981. Factors influencing alginate degradation by the marine fungi: Dendryphiella  s a l i n a and D. a r e n a r i a . Bot. Mar. 24: 489-491. Walker, D.C., G.C. Hughes and T. B i s a l p u t r a . 1979. A new interpretation of the i n t e r f a c i a l zone between Spathulospora (Ascomycetes) and B a l l i a (Florideophyceae). Trans. Br. Mycol. Soc. 73(2): 193-206. Watson, S.W. 1957. Cultural and c y t o l o g i c a l studies on species of Labyrinthula. Ph.D. Thesis. Univ. of Wisconsin, Madison. 165 p. Watson, S.W. and E.J. Ordal. 1957. Techniques for the i s o l a t i o n of Labyrinthula and Thraustochytrium i n pure culture. J. Bacteriol. 73: 589-590. Watson, S.W. and K.B. Raper. 1957. L a b y r i n t h u l a minuta sp. nov. J. Gen. M i c r o b i o l . 17: 368-377. 322 Webber, F.C. 1967. Observations on the structure, l i f e h istory and biology of Mycosphaerella ascophylli. Trans. Br. Mycol. Soc. 50(4): 583-601. Wilson, I.M. 1951. Notes on some marine fungi. Trans. Br. Mycol. Soc. 34: 540-543. Wilson, I.M. and J.M. Knoyle. 1961. Three species of Didymosphaeria on marine algae: danica (Berlese) comb. nov., D. p e l v e t i a n a Suth., and D. f u c i c o l a Suth. Trans- Br. Mycol. Soc. 44(1): 55-71. 323 APPENDIX A DESCRIPTION OF ISOLATION MEDIA (1) Glucose-Yeast Extract Seawater Agar (GYSA) (Johnson and Sparrow, 1961). Glucose (Dextrose-anhydrous) 1 g Yeast Extract (Difco-'Bacto') 0.01 g Bacto Agar (Difco) 15 g Sea Water (28°/oo) aged, f i l t e r e d 1 l i t e r (2) Modified Vishniac's Medium (FUL) (Fuller, et al., 1964; Vishniac, 1956). Glucose (Dextrose-anhydrous) 1 g Gelatin Hydrolysate-enzymatic (ICN) 1 g Yeast Extract (Difco-'Bacto') 0.1 g Liver Extract (1:20) (ICN) 0.01 g Bacto Agar (Difco) 12 g Sea Water (28°/oo) aged, f i l t e r e d 1 l i t e r (3) Modified Serum-Seawater Agar (SSA) (Porter, 1967; Watson and Ordal, 1957). Modified. Horse Serum (GIBCO) + 10 ml* Bacto Agar (Difco) 9 g Sea Water (28°/oo) aged, f i l t e r e d 1 l i t e r +Horse serum added to the cooled medium just p r i o r to pouring plates. *More can be added [e.g., 20 ml (SSA - 2%)] for maintaining cultures. (4) Kazama's Modified Vishniac's Medium (KMV) (D. Porter, pers. comm.). Glucose (Dextrose-anhydrous) 1 g Gelatin Hydrolysate-Enzymatic (ICN) 1 g Yeast Extract (Difco-'Bacto') 0.1 g Peptone (Difco-'Bacto') 0.1 g Bacto Agar (Difco) 12 g* Sea Water (28°/oo) aged, f i l t e r e d 1 l i t e r *KMV-slush i s prepared with 1 g of agar per l i t e r . 324 (5) Glucose-Serum Seawater Agar (GSSA) (unpubl. obs.). Horse Serum (GIBCO) + Glucose (Dextrose-Anhydrous) Gelatin Hydrolysate-Enzymatic (ICN) Yeast Extract (Difco-'Bacto') Liver Extract (1:20) (ICN) Bacto Agar (Difco) Sea Water (28°/oo) aged, f i l t e r e d +Horse serum added to the cooled medium just p r i o r to pouring plates. (6) Base Mineral Medium (Gunkel and Rheinheimer, 1972). K 2HP0 4 ' 3H20 NaN03 MgSO. ' 7H20 FeS0 4 * 7H20 Phycocolloid Sea Water (33°/oo) aged, f i l t e r e d ^The phycocolloids used included: Bacto Agar (Difco); Kappa Carrageenan (K-7; Marine Colloids, Rockland, Maine); Kappa Carrageenan (K-13; Stauffer Chemical Co., Dobbs Ferry, New Jersey) (see Abbott and Chapman, 1981). 20 ml 1 g 1 g 0. 02 g 0. 01 g 9 g 1 l i t e r i g 0.5 g 0.5 g Trace (0.01 g) 20 g 1 l i t e r APPENDIX B EXAMPLES OF STATISTICAL CALCULATIONS EXAMPLE 1 Sample 1, Labyrinthula spp. i s o l a t e d from rinsed tissues of Rhodoglossum affine. The comparison i s between the i s o l a t i o n frequencies obtained from the d i f f e r e n t l i f e h i s t o r y stages of Rj_ a f f i n e incubated at 14°C. Data (Table 3): L i f e History Stage n % Recovery # Isolations(t) # Negative(-) vegetative/ 4 male 25.0 1 3 cystocarpic 4 0 0 4 t e t r a -sporangial 4 50.0 2 2 Row X column (3 s t a t i s t i c (Sokal x 2) test of and Rohlf, independence using 1969; page 599). the G Positive (a) Negative Total vegetative/male 1 3 4 (b) cystocarpic 0 4 4 tetrasporangial 2 2 4 Totals 3 9 12 326 Compute the following: 1) sum of the transforms of the frequencies f o r the body of the contingency table b a = E Z f i j in f ± j = 1 In 1 + 3 In 3 + 0 In 0 + 4 In 4 + 2 In 2 + 2 In 2 = 0 + 3.296 + 0 + 5.545 + 1.386 + 1.386 = 11.614 2) sum of the transforms of the row t o t a l s b a a E ( E f ± j ) i n ( E f ) = 4 In 4 + 4 In 4 + 4 In 4 = 5.545 + 5.545 + 5.545 = 16.636 3) sum of the transforms of the column t o t a l s a b b E ( E f ) In ( E f ) = 3 In 3 + 9 In 9 = 3.296 + 19.775 = 23.071 4) transform the grand t o t a l = n In n = 12 In 12 = 29.819 5) G = 2[quantity 1 - quantity 2 - quantity 3 + quantity 4] = 2[11.614 - 16.636 - 23.071 + 29.819] = 2[1.726] = 3.452 327 Degrees of freedom = ( a - l ) ( b - l ) = (2-1)(3-1) = 2 Compare G with the c r i t i c a l value of a chi-sauare d i s t r i b u t i o n for two degrees of freedom. (X Q 05["2l = 5.991) (Rohlf and Sokal, 1969). G i s less than the c r i t i c a l value and the n u l l hypothesis can be accepted. The i s o l a t i o n of Labyrinthula spp. was independent of l i f e h i s t o r y stage of affine. However, had s i m i l a r results been obtained over a larger sample size (n), the outcome of t h i s test may well have been di f f e r e n t . EXAMPLE 2 Sample 1, t o t a l mycelial fungi i s o l a t e d from Rhodoglossum  af f i n e at 25 C. The comparison i s between s t e r i l i z e d and rinsed tissues. Data (Table 4, Bottom): (b) Tissue % # # Treatment n Recovery Isolations(+) Negative(-) S t e r i l i z e d 72 8.3 6 66 Rinsed 24 54.2 13 11 2 x 2 test of independence using the G s t a t i s t i c (Sokal and Rohlf, 1969; page 591). (a) Pos i t i v e Negative Total S t e r i l i z e d 6 66 72 Rinsed 13 11 24 Totals 19 77 96 Compute the following: 1) E f In f for the c e l l frequencies = 6 In 6 + 66 In 66 + 13 In 13 + 11 In 11 = 10.750 + 276.517 + 33.344 + 26.377 = 346.989 328 2) E f In f for the row and column t o t a l s = 72 In 72 + 24 In 24 + 19 In 19 + 77 In 77 = 307.920 + 76.273 + 55.944 + 344.473 = 774.611 3) n In n (n = t o t a l sample size) = 96 In 96 = 438.177 4) G = 2[quantity 1 - quantity 2 + quantity 3] = 2C346.989 - 774.611 + 438.177] = 2[10.556] = 21.112 Degrees of freedom = ( a - l ) ( b - l ) = (2-1)(2-1) = 1 Compare G with the c r i t i c a l value of X (chi-square) for one degree_of freedom. The observed G i s considerably higher than X 05[i] = 3.841. Therefore, we reject the n u l l hypothesis that the i s o l a t i o n of mycelial fungi at 2 5° was independent of a l g a l tissue pretreatment ( s t e r i l i e d or rinsed). S i g n i f i c a n t l y fewer mycelial fungi were isola t e d from s u r f a c e - s t e r i l i z e d a l g a l tissues (p < 0.005). 329 EXAMPLE 3 Sample 2 i s o l a t i o n frequency of yeasts. A) Iso l a t i o n media. Since the data obtained for rinsed tissues of Gelidium  c o u l t e r i display the largest v a r i a b i l i t y , t h i s regime w i l l be used as an example. Data (Table 13): % # # Medium n Recovery Isolations(+) Negative(-) GYSA 12 58.3 7 5 SSA 12 50.0 6 6 FUL 12 83.3 10 2 Row X column (3 x 2) test of independence using the G s t a t i s t i c (Sokal and Rohlf, 1969; page 599). Table and calculations similar to Example 1. (a) Pos i t i v e Negative Total GYSA 7 5 12 (b) SSA 6 6 12 FUL 10 2 12 Totals 23 13 36 Compute the following: 1) £ f In f for the c e l l frequencies = 7 In 7 + 5 In 5 + 6 In 6 + 6 In 6 + 10 In 10 + 2 In 2 = 13.621 + 8.047 + 10.750 + 10.750 + 23.026 + 1.386 = 67.582 330 2) E f In f for the row t o t a l s = 12 In 12 + 12 In 12 + 12 In 12 = 29.819 + 29.819 '+ 29.819 = 89.457 3) Z f In f for the column t o t a l s = 23 In 23 + 13 In 13 = 72.116 + 33.344 = 105.460 4) n In n = 36 In 36 = 129.007 5) G = 2[quantity 1 - quantity 2 - quantity 3 + quantity 4] = 2C67.582 - 89.457 - 100.460 + 129.007] = 2C1.672] = 3.344 Degrees of freedom = ( a - l ) ( b - l ) = (2-1)(3-1) = 2 Compare G with the c r i t i c a l value of a chi-^quare d i s t r i b u t i o n for two degrees of freedom (X 0,05[2] = 5.991). G i s nonsignificant. The n u l l hypothesis can"be accepted -the i s o l a t i o n of yeasts was independent of type of i s o l a t i o n medium (in t h i s alga/tissue pretreatment regime). 331 SAMPLE 3 Isolation frequencies of yeasts B) Algal l i f e h i s t o r y stage. The data obtained for s t e r i l i z e d tissues of Rhodoglossum  a f f i n e are used i n t h i s example. Data (Table 13): L i f e History % # # Stage n Recovery Isolations(+) Negative(-) vegetative/ 36 27.8 10 26 male cystocarpic 36 2.8 1 35 t e t r a -sporangial 36 13.9 5 31 Table format and computations as for Examples 1 and 3A. (a) Positive Negative Totals vegetative/male 10 26 36 (b) cystocarpic 1 35 36 tetrasporngial 5 31 36 Totals 16 92 108 Compute the following: 1) z f In f for the c e l l frequencies = 10 In 10 + 26 In 26 + 1 In 1 + 35 In 35 + 5 In 5 + 31 In 31 = 23.026 + 84.710 + 0 + 124.437 + 8.047 + 106.454 = 346.674 332 2) I f In f for the row t o t a l s = 36 In 36 + 36 In 36 + 36 In 36 = 129.007 + 129.007 + 129.007 = 387.020 3) £ f In f for the column t o t a l s = 16 In 16 + 92 In 92 = 44.361 + 416.005 = 460.366 4) n In n = 108 In 108 = 505.670 5) G = 2[quantity 1 - quantity 2 - quantity 3 + quantity 4] = 2[346.674 - 387.020 - 460.366 + 505.670] = 2[4.958] = 9.916 Degrees of freedom = ( a - l ) ( b - l ) = (2-1)(3-1) = 2 x 20.05[2] = 5 ' 9 9 1 G i s s i g n i f i c a n t at the 99% l e v e l (p < 0.01). Based on t h i s result, the n u l l hypothesis must be rejected. Isolation of yeasts was not independent of a l g a l tissue l i f e h i s t o r y stage. 

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