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Download (2MB) https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] EXPERIMENTAL STUDIES ON THE GENUS CHRYSOTILA (ORDER ISOCHRYSIDALES, CLASS HAPTOPHYCEAE) Thesis presented by JOHN R. STANIER, B.Sc. for the degree of Master of Science in the Faculty of Science in the UNIVERSITY of GLASGOW DECEMBER 1979 ProQuest Number: 10647160 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uesL ProQuest 10647160 Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 1 K ^sis b i t|-& Copy â f GI,A.5G0W I uN.VERsrriGi CONTENTS Page Acknowledgements SUMMARY n Chapter 1. INTRODUCTION 1.1, The Taxonomy of Chrysotila lamellosa Anand 1.2. The Structure of Ruttnera phase of Chrysotila lamellosa Chapter 2. MATERIALS and METHODS 2.1. Supply and Maintenance of Cultures 6 2.2. ASP6 Culture Medium 6 2.3. Culture Methods 9 2.4. Determination of Growth in Culture by Packed Cell Volume 10 2.5. Determination of Growth by Spectrophotometric Methods 12 2.6. Determination of Pigments 14 2.7. Determination of DNA 15 2.8. Production of Axenic Cultures 16 2.9. Production of Zoospores 20 Chapter 3. RESULTS 22 3.1, Growth Forms of Chrysotila lamellosa 22 3.2. The Effect of Salinity on Growth 24 Page 3.3. The Effect of Salinity on Pigment Concentration 25 3.4. The Effect of Salinity on DNA Content 25 3.5. Results of Long Term Growth at Different Salinities 26 3.6. Results of Observations on Growth at Different Temperatures , 27 3.7. Results of Exposures to Drying 28 3.8. Observations on Cultures in Different Media 29 3.9. Experiments on Zoospores Release 30 Chapter 4. DISCUSSION 32 REFERENCES 46 ACKNOWLEDGEMENTS I am indebted to Professor A. D. Boney for his advice, help, guidance and encouragement throughout this work. I wish to thank Professor M. B. Wilkins for providing the facilities necessary for carrying out this work in the Department of Botany. I am grateful to my mother Mrs E. Stanier for the time and e ffo rt which she spent in the typing of this thesis. The work in this thesis was partly financed by a grant from Lanarkshire Division Education Authority, and I am grateful for this support, and for the support from Calderhead High School during the final year of study. I wish, fin a lly , to express my thanks to my wife Marjory, and to both our families for their constant help, encouragement, and considerable patience during this work. SUMMARY Chrysotila lamellosa Anand. has been isolated from both fresh and marine habitats, and shows the a b ility to grow in salinities from 2?/oo to 32°/oo. The growth of the alga was measured within this range of s a lin ities , and i t was found that the growth rate at low salinities was greater than at high salinities. This was related to the production of zoospores in the cultures. The content of chlorophylls and carotenoids was also measured within this range of salinities and i t was shown that the pigment concentration tended to rise with increasing salinity. The release of zoospores was also investigated and was shown to be moderated by s a lin ity , temperature and aeration. These results were compared and indicate that the alga is under environmental stress at low salinities, and better adapted to growth at salinities above 16^/00 . n FIGURES Followinç 1.1 Chrysotila lamellosa 3* 1.2 Chrysotila lamellosa 4* 1.3 Chrysotila lamellosa 5* 2.1 Roux Culture Flask 9 2.2 Aeration System 9 2.3 Effect of Centrifuge Speed on Packed Cell Volume 11* 2.4 Turbidity at 700nm v Packed Cell Volume 12* 2.5 Calibration of Nephelometer 13* 2.6 Zoospore Pipette 19 3.1 Growth Over 21 Days v Salinity 23 3.2 Growth at 2°/oo 24 3.3 Growth at 4°/oo 24 3.4 Growth at 8°/oo 24 3.5 Growth at 16°/oo 24 3.6 Growth at 32°/oo 25* 3.7 Absorption Spectrum of Chlorophyll Extract 25 3.8 Chlorophyll a v Salinity 25 3.9 Chlorophyll c v Salinity 25 3.10 Carotenoids v Salinity 25 3.11 Total Chlorophylls/Carotenoids v Salinity 25 3.12 DMA V Salinity 26* 3.13 Zoospore Release at 2^/oo 31 3.14 Zoospore Release at 4°/oo 31 3.15 Zoospore Release at 8°/oo 31 3.16 Zoospore Release at 16°/oo 31 3.17 Zoospore Release at 32°/oo 31 * indicates facing page TABLES Following Page 2.1 Boney's Growth Medium 6* 2.2 ASP6 Culture Medium 6 2.3 Cell Packing by Centrifugation 9 2.4 Antibiotic Mix Number 1 T 2.5 Survival of Algae and Bacteria 1 2.6 Survival at 72 Hours Exposure 1 2.7 Survival of Algae and Bacteria with Penicillin 1 2.8 Survival of Algae and Bacteria with Streptomycin 1 2.9 Survival of Algae and Bacteria with Chloramphenicol 18 2.10 Survival of Algae and Bacteria with Neomycin 18 2.11 Antibiotic mix prepared 18 2.12 Results with Antibiotic Mix, 24 Hours Exposure 18 2.13 Subcultures from Antibiotic Mix after 7 Days 18 3.1 Salinity and growth over 21 Days 23 3.2 Statistical Results of Growth over 21 Days 24 3.3 Salinity and Growth Rate - Significance 24 3.4 Increase in Packed Cell Volume at Different Salinities 25 3.5 Variance Ratios - Chlorophyll a v Salinity 25 3.6 Variance Ratios - Chlorophyll c v Salinity 25 3.7 Variance Ratios - Carotenoids v Salinity 25 3.8 Variance Ratios - Total Chlorophylls/Carotenoids V Salinity 25 3.9 Zoospore Release 31 4.1 Growth Constants for Cultures 36* 4.2 Summary of Pigment Analysis 40* * indicates facing page CHAPTER ONE INTRODUCTION 1.1 The Taxonomy of Chryso tila lamellosa Anand Chrysotila lamellosa Anand was discovered during a study of the algal flora of British chalk cliffs(Anand, 1937). It was placed in the class Chrysophyceae family Chrysocapsaceae. The organism was subsequently placed in the Haptophyceae by Parke and Dixon (1964) and was fir s t placed in the order Prymnesiales (class Chrysotilaceae). The same organism was also discovered by G eitler, but from a terrestrial habitat in 1942 (G eitler, 1942). This form, which was discovered as golden yelTow mucilage covered masses of cells growing on damp rocks and paths in Vienna, was named Ruttnera spectabilis, and i t is under this name that most of the taxonomic studies have been made. Geitler noted that the vegetative phase of the organism was morphologically similar to that of members of the genus Chrysocapsa and placed R. spectabilis in the family Chrysocapsaceae, class Chrysophyceae, Later, a new family, Ruttneraceae, was erected for the genus (G eitler 1943), This was based on the observation that the zoids possess two equal to sub-equal fla g e lla , in contrast to the uniflagellate structure of the swarmers in Chrysocapsa. However Geitler was unable to state with certainty whether the flagella of the zoids were heterokont or isokont, and this le ft some doubt as to the systematic position of the species. In fact having observed them to be heterodynamic, he believed them to be heterokont. Bourelly (1968) included Ruttnera in the Ochromonadales (class Chrysophyceae) on the basis of Geitler*s belief that the flagella of the zoids are truly heterokont. The type material of Ruttnera was thus isolated from a freshwater habitat, whereas that of Chrysotila came from a marine habitat, and there is little doubt that this fact has been responsible for keeping the information on these two taxa separated. More recently, however, isolates of Ruttnera have been grown in seawater, and although the type species has since been lost (B illard and Gayral, 1972) the genus has been described from marine habitats. Green and Parke (1974) investigated the fine structure of a strain of R. spectabilis obtained from the locus classicus. This work has shown that the flagella of the zoids are equal to sub-equal, and that there is a much reduced haptonema of a very unusual type. This haptonema is so small that i t is undetectable by ligh t microscopy, and is only visible under the electron microscope when the cell is sectioned in certain ways. Local accumulations of material within the endoplasmic reticulum cisternae were also discovered in the cells.
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