.• . • - • ...L-- • • --- _1 .. Sexual reproduction and ecophysiology of the marine dinoflagellate Alexandrium minutum Halim lan P. Probert Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy University of Westminster, London Ifremer, Brest June 1999 Jury Professor Chris Bucke Examiner Mr John Leftley Examiner Mrs Jane Lewis Director of Studies Mrs Evelyne Erard-Le Denn Second Supervisor Professor Jenny George Second Supervisor Sexual reproduction and ecophysiology of the marine dinoflagellate Alexandrium minutum Halim lan P. Probert Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy University of Westminster, London lfremer, Brest June 1999 Jury Professor Chris Bucke Examiner Mr John Leftley Examiner Mrs Jane Lewis Director of Studies Mrs Evelyne Erard-Le Denn Second Supervisor Professor Jenny George Second Supervisor Contents Acknowledgements Chapter 1 Introduction 1 A. Objectives of this study 1 B. Dinoflagellates 1 1. Evolutionary history 1 2. General characteristics 1 3. 'Red tides' 2 4. Dinoflagellate toxins 2 5. Economie impact 2 C. Life Cycles 3 1. Asexuallife history 3 2. Sexuallife history 3 3. Life cycle control 4 4. Environmental triggers for sexual reproduction 4 D. Nutrients and Sexuality 6 1. Microalgal nutrient physiology 6 2. Nutrient stress and the cell's responses 9 3. The sexual induction mechanism 10 4. Nutrient physiology through the time-course of transition from asexual to sexual reproduction 10 E. The Experimental Species 12 Chapter 2 The life history of Alexandrium minutum Halim 12 A. Introduction 12 1. The genusAlexandrium 12 2. Alexandrium minutum Halim 13 3. Dinoflagellate reproductive morphology and ultrastructure 13 B. Methods 14 1. Strain initiation and identification 14 2. Culturing 14 3. Direct observation 14 4. Video recording 1 image analysis 14 5. Photography 14 6. Protargol staining 14 7. Scanning electron microscopy 15 8. Crossing experiments 16 C. Results 16 1. Vegetative cell 16 2. Asexual division 16 3. The sexual cycle 17 D. Discussion 18 1. Taxonomie designation 18 2. Asexual division 18 3. The sexual cycle 18 Chapter 3 Laboratory Methods 27 A. Introduction 27 B. Culture techniques 28 1. Routine equipment preparation 28 2. Culture medium 28 3. Physical parameters 30 4. Isolation technique 30 5. The experimental strain 30 C. Cell enumeration and sizing 31 1. Cell counting 31 2. Measurement of cell dimensions 31 D. Dissolved nutrient analyses 32 1. Sample preparation 32 2. The Technicon Auto-Analyser system 32 3. Nitrite and nitrate analysis 32 4. Phosphate analysis 33 5. Total ammonia analysis 33 E. Intracellular nutrient analyses 34 1. CHN analysis 34 2. Particulate P analysis 34 3. InAA analysis 34 Chapter 4 Culture Experiments 37 A. Batch culture 37 1. Protocol 37 2. Zygote yield experiments 37 3. Time-course experiments 40 B. Semi-continuous culture 51 1. Automated system 51 2. Manual system 54 Chapter 5 Surveys of natural Alexandrium minutum populations in the estuaries of Northern Brittany 62 A. Introduction 62 B. Sampling 63 1. The sampling sites 63 2. Sampling procedure 63 3. Sample processing 63 C. Results 66 1. Aber Wrac'h 1995 66 2. Penzé 1997 69 D. Discussion 72 1. Bloom initiation 72 2. Population development and induction of sexuality 74 3. Bloom decline 81 E. Culture experiments with natural populations 86 1. Introduction 86 2. Methods 86 3. Results 86 4. Discussion 87 F. Conclusions 90 Chapter 6 Summary 91 A. Introduction 91 B. Alexandrium minutum 91 C. Culture experiments 91 D. Field Surveys 92 E. Perspectives for future research 92 References 93 Appendices 1. Batch culture experiment data 2. Automated semi-continuous culture experiment data 3. Manual semi-continuous culture experiment data 4. Aber Wrac'h 19951 Penzé 1997 survey data 5. Penzé 1997 natural population batch culture experiment data Acknowledgements 1 am in debt to a number of people for their support and encouragement throughout this research project. First and foremost 1 would like to thank both Dr Jane Lewis and Evelyne Erard-Le Denn for their organisational support, and for their advice, good humour and patience. 1 would like to acknowledge ali of the scientific and technical staff in the Laboratoire DEL/EC at IFREMER in Brest for welcoming me and making my time in France very enjoyable. Particular thanks go to Agnes Youenou, Jacqueline Le Grand, Marie-Pierre Crassous, Michel Lunven, Genevieve Arzul, Guy Bodennec and Patrick Gentien. 1 would also like to thank Marie­ Joseph Chrétiennot-Dinet for assistance with descriptive work and taxonomy, and Peter Hanaway from the University of Westminster for invaluable technical assistance with the HPLC system. Formally, 1 would like to acknowledge the Natural Environment Research Council in the UK for funding this research, and IFREMER Centre de Brest for hosting my visits to France and providing financial assistance. Chapter 1 Introduction A. Objectives of this study B. Dinoflagellates As part of their sexual life cycle sorne dinoflagellate species 1. Evolutionary history form resistant resting stages (hypnozygote cysts) which are Dinoflagellates are a geologically ancient algal group, with a important in their ecology. The factors inducing sexual fossil record dating back to the Upper Silurian period (Walker, reproduction (and hence encystment) have not been clearly 1984). They are classified alternatively as 'algae' (class defined and conflicting information exists from laboratory and Dinophyceae) by botanists an<;l 'protozoa' ( class field observations. Nutrient deficiency in growth medium is Phytomastigophorea) by zoologists, each emphasising either commonly used in laboratory dinoflagellate cultures to induce the plant or animal features of the group (Theobald, 1993). The sexual reproduction, but field observations of encystment have fact that dinoflagellates are an extremely successful class of been made in conditions of apparent nutrient sufficiency. The microalgae is evident not only from their widespread aim of this project is to investigate the factors which trigger distribution and occasional dominance in marine waters, but sexual reproduction in coastal dinoflagellates. also from the fact that they are one of the more primitive algal The experimental species, Alexandrium minutum Halim, is a classes and thus have survived millions of years of natural toxic dinoflagellate which is conducive to laboratory culture, selection (Anderson, 1983). and which is known to thrive in the estuaries of northern 2. General characteristics Brittany, France. The life cycle of this species has only been The dinoflagellates are a large group of eukaryotic, partially described, and the first part of the project therefore biflagellate organisms exhibiting great diversity in involves a detailed morphological and morphogenetic morphology, cellular organisation and behaviour. At present investigation of its life history, with characterisation of this diverse group is represented by over 2000 species in different life stages and the processes of asexual division and marine, estuarine and freshwater habitats world-wide (Taylor, sexual fusion. 1987a). The majority are free-living autotrophic planktonic and Determination of the mediating effects on sexual reproduction benthic species, found predominately in the euphotic zone of environmental variables such as temperature and light (Walker, 1984), but the group also includes heterotrophs, provide a framework within which to design the main phase of symbionts and parasitic forms. the laboratory part of this study, culture experiments which are Despite the diversity of habitats and modes of existence, ali concentrated on elucidating the role of nutrient stress in dinoflagellates share a number of unique characteristics. induction of sexuality. Only few studies of this kind have been Dinoflagellates are unicellular, although sorne may form chains conducted on other dinoflagellate species (Anderson, 1983; or pseudo-colonies. At sorne stage, ali dinoflagellates ( except Anderson & Lindquist, 1985; Anderson et al., 1985), ali using Noctiluca) also possess two dissimilar flagella (figure 1.1 ); the batch culture techniques, and only the latter study investigating longitudinal flagellum and the transverse flagellum which lies the effects of separate limitation of both nitrogen and in the groove or 'cingulum' surrounding the cell work together phosphorus. In this project, batch and semi-continuous culture to propel and stabilise the cell. experiments investigate the complex interactions between (a) (b) (cl external nutrients (both nitrogen and phosphorus), internai nutrient physiology, and gametogenesis. A more comprehensive approach to the measurement of internai nutrient status than previously employed in studies of dinoflagellate sexual reproduction involves the quantification of intracellular pools of amino acids, and in particular determination of the ratio of glutamine:glutamate, a biomarker which gives an independent measure of C:N status with which to compare cultures grown under different conditions of photon flux density and substrate type and concentration (Flynn, 1990). Figure 1.1 Basic dinoflagellate flagellar arrangements: (a) desmokont (e.g. Prorocentrum); (b) dinokont (e.g. An integral part of the proj ect is the in situ monitoring, using Alexandrium); (c) opisthokont (e.g. Oxyrrhis) (Taylor 1987a). protocols developed in culture experiments, of the bloom dynamics of natural populations of Alexandrium minutum in The size of individual