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When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk _________________________________________________________________________ UNIVERSITY OF SOUTHAMPTON FACULTY OF ENGINEERING, SCIENCE AND MATHEMATICS SCHOOL OF OCEAN AND EARTH SCIENCE MOLECULAR TECHNIQUES FOR INVESTIGATING TOXIC DINOFLAGELLATE SPECIES IN THE WESTERN ENGLISH CHANNEL, UK AND IN BAHRAIN COASTAL WATERS OF THE ARABIAN GULF. By Layla J. Hazeem Thesis for the degree of Doctor of Philosophy May 2009 a _________________________________________________________________________ UNIVERSITY OF SOUTHAMPTON FACULTY OF ENGINEERING, SCIENCE AND MATHEMATICS SCHOOL OF OCEAN AND EARTH SCIENCE Doctor of Philosophy MOLECULAR TECHNIQUES FOR INVESTIGATING TOXIC DINOFLAGELLATE SPECIES IN THE WESTERN ENGLISH CHANNEL, UK AND IN BAHRAIN COASTAL WATERS OF THE ARABIAN GULF. ABSTRACT Some species of marine microalgae produce toxins that have major impacts on aquaculture and fisheries and can cause human illnesses through their accumulation in shellfish. Dinoflagellates account for 75% of all toxic microalgal species and are considered primarily responsible for the current expansion and regional spreading of harmful algal bloom outbreaks in the sea. The aim of this investigation was to develop a protocol using a combination of molecular methods for specifically detecting, identifying and enumerating harmful algal species in natural water samples from the English Channel and Bahrain coastal waters. Initially, a fluorescent in situ hybridization (FISH) protocol with a monolabelled probe was assessed for detecting Alexandrium tamarense cultured cells, but was shown to produce a weak hybridization signal and only a small fraction of the target species were detected. Following this, two new species specific molecular primers were developed for use with polymerase chain reaction (PCR) to specifically differentiate between two closely related species ( Karenia mikimotoi and Karenia brevis ) of the harmful dinoflagellate genus Karenia. K. brevis produces brevetoxins and is responsible for neurotoxic shellfish poisoning in coastal regions of the Gulf of Mexico. Karenia mikimotoi is less toxic but has been linked with fish kills and is usually considered the species causing blooms in some Atlantic coastal regions of Europe. The 28S large subunit ribosomal RNA (28S LSU rDNA) of Karenia mikimotoi and its morphologically and genetically similar relative Karenia brevis were sequenced and the two sets of primers shown to be specific for detecting the two individual target species. Nested PCR allowed an unbiased identification of the bloom forming Karenia species occurring in Lugol’s preserved water samples previously collected from the western English Channel, identifying only Karenia mikimotoi to be present. In some preserved water samples collected from Bahrain coastal waters both K. brevis and K. mikimotoi were shown to be present using these primers. To allow specific enumeration of Karenia mikimotoi cells in preserved water samples, a protocol based on catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) was developed. The technique was optimized for quantification of the target species by epifluorescence microscopy and flow cytometry. Epifluorescence microscopy was shown to be superior for the quantification of the target species, although flow cytometry was demonstrated to be a promising technique for developing an automated detection system. Absolute numbers of K. mikimotoi cells estimated using the CARD-FISH probe did not correlate with inverted light microscope counts of Lugol’s preserved water samples from the western English Channel. However, positive hybridized relative cell counts compared to total DAPI stained phytoplankton cells did show a strong correlation indicating some loss of cells during processing of samples. These molecular methods were then sequentially applied to the potentially harmful species Bysmatrum granulosum isolated from Bahrain coastal waters using a newly designed 18S SSU rRNA probe. The CARD-FISH protocol was used to monitor changes in absolute cell concentrations of Bysmatrum granulosum in preserved water samples collected from Arad Bay, Bahrain from June to November 2006. CARD-FISH derived absolute cell counts were strongly correlated to those derived from inverted light microscope counts of Lugol’s preserved water samples, proving the useful application of this method. This investigation was one of the first to successfully use the CARD-FISH molecular technique for the detection and enumeration of harmful algal species in natural water samples. It could therefore be applied for monitoring a range of harmful dinoflagellate species providing a sensitive early warning method for their detection in coastal waters. b _________________________________________________________________________ Acknowledgments I would like to express my great thanks to my supervisors Duncan Purdie and Mike Zubkov for their guidance, encouragement and support during the course of my study. I am extremely grateful to my husband, my mother and my sisters to look after my daughters during my stay in the United Kingdom. I have to acknowledge that this work would not have been achieved without their support. Special thanks to the University of Bahrain for their full sponsoring and support of my study in the United Kingdom. I would like to acknowledge the support of the molecular Biology group: John Gittins, who helped a lot in the PCR Laboratory and to look after my cultures when I am away, Ross Holland for his help with flow cytometry analyses, Andrea Baker to construct a phylogenetic tree of Karenia mikimotoi using Phylip software, Manuela Hartmann to construct phylogenetic trees of Bysmatrum granulosum using ARB software and Ludwig to help with probe design of Bysmatrum granulosum and PCR amplification using single cells. My great thanks to my friends Suad Rashdan, Hanan Al-Buflasah, Turki Al-Said, Mohammed Al-Qurban and Humood Naser for their support and encouragements. I would like to thanks Fadheela Abdulla, Naeema Fakhroo and Ahmed Mohammed to collect water samples from Bahrain coastal water during summer 2006. Finally, I would like to thank everyone who has helped me, given me advice during the course of this research and provided me with information, which has enhanced this research. To all of those I mentioned I would like to express my appreciation and sincere thanks. c _________________________________________________________________________ Declaration of authorship I, Layla J. Hazeem, declare that the thesis entitled “ Molecular techniques for investigating toxic dinoflagellate species in the western English Channel, UK and in Bahrain coastal waters of the Arabian Gulf” and the work presented in the thesis are both my own, and have been generated by me as the result of my own original research. I confirm that: • This work was done wholly or mainly in candidature for a research degree at this University; • Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; • Where I have consulted the published work of others, this is always clearly attributed; • Where I have quoted from the work of others, the source is always given. With the exception of such quotation, the thesis is entirely my own work; • I have acknowledged all main sources of help; • Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; • None of this work has been published before submission. Signed: ……………………………………………………………… Date: ………………………………………………………………… d _________________________________________________________________________ List of Contents LIST OF CONTENTS ...................................................................................................................................... I LIST OF FIGURES ....................................................................................................................................... IV LIST OF TABLES ......................................................................................................................................... VI CHAPTER 1 ......................................................................................................................................................1 INTRODUCTION ................................................................................................................................................1 1.1 General introduction ...........................................................................................................................1 1.2 Harmful