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Molecular and Morphological Characterization of Alexandrium Species (Dinophyceae) from the East Coast, Usa

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Molecular and Morphological Characterization of Alexandrium Species (Dinophyceae) from the East Coast, Usa MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF ALEXANDRIUM SPECIES (DINOPHYCEAE) FROM THE EAST COAST, USA Erika N. Schwarz A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the degree of Master of Science Department of Biology and Marine Biology University of North Carolina Wilmington 2011 Approved by Advisory Committee D. Wilson Freshwater R. Wayne Litaker Alison R. Taylor Carmelo R. Tomas Chair Accepted by Dean, Graduate School This thesis has been formatted in accordance to the author guidelines for the Journal of Phycology. ii TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iv ACKNOWLEDGMENTS ...............................................................................................................v DEDICATION ............................................................................................................................... vi LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii INTRODUCTION ...........................................................................................................................1 MATERIALS & METHODS ..........................................................................................................8 RESULTS ......................................................................................................................................14 DISCUSSION ................................................................................................................................16 LITERATURE CITED ..................................................................................................................23 TABLES ........................................................................................................................................28 FIGURES .......................................................................................................................................37 APPENDIX 1 .................................................................................................................................40 APPENDIX 2 .................................................................................................................................57 APPENDIX 3 .................................................................................................................................65 iii ABSTRACT The dinoflagellates (Dinophyceae) are a group of highly diverse unicellular organisms that inhabit fresh, estuarine and saltwater environments. Some dinoflagellates produce toxins that have negative impacts resulting as both economic and public health risks. This study focuses on Alexandrium peruvianum, a thecate, paralytic shellfish poisoning (PSP) toxin producing dinoflagellate. Alexandrium peruvianum was thought to be restricted to waters north of Cape Cod, MA but this species was recently isolated from the New River, NC. The aim of this study was to thoroughly characterize A. peruvianum using both morphological and molecular techniques in order to distinguish it from the closely related A. ostenfeldii. Calcofluor White staining was used to visualize thecal plates to determine morphological differences between A. peruvianum and A. ostenfeldii. Significant differences were found in the first apical (1’), the sixth precingular (6’’) and the anterior sulcal (s.a.) plates between the two species. The differences observed were consistent with original descriptions of A. peruvianum and A. ostenfeldii. Utilizing 5.8S, 18S, 28S and internal transcribed spacer (ITS) sequences generated in this study in addition to those submitted to GenBank, phylogenetic analysis resolved four clades between the two species; Clades 1 and 2 were comprised of A. peruvianum and Clades 3 and 4 were comprised of A. ostenfeldii. Isolates from Clades 1 and 3, including A. peruvianum from North Carolina, USA, Finland and A. ostenfeldii from Canada/Denmark/Scotland, respectively were used for comparison. Isolates from Clades 2 and 4, A. peruvianum from Spain and A. ostenfeldii from New Zealand, were not available for the morphological study. DNA sequence information for the Alexandrium isolates examined was used to develop a PCR-based method for detection and identification of A. peruvianum and A. ostenfeldii. iv ACKNOWLEDGMENTS First and foremost I would like to thank my advisor, Dr. Carmelo R. Tomas, who has believed in my abilities from the beginning. He has been a wonderful advisor and influence. He has shown me what it means to be a good advisor, professor and mentor and for that I am grateful. The only limiting factor is your mind and where it can take you when in his lab. In addition, the wonderful vegetables from his garden are always a welcome summertime surprise and lunches at Thai Spice will be missed. Thank you for everything! I would like to send many thanks to my wonderful committee members. To Dr. R. Wayne Litaker (NOAA) for always answering his phone to answer questions and offer advice or lend a helping hand when needed. Welcoming us into his lab and making the trek to Wilmington for our meetings are also things that are greatly appreciated. To Dr. D. Wilson Freshwater for his willingness to stop whatever he may be doing in order to answer questions without notice and to answer those questions in a way that is easy to understand. To Dr. Alison R. Taylor, not only for agreeing to serve as a committee member regardless of her busy schedule but for her wealth of knowledge and enthusiasm for science. Both traits make her someone to aspire to. I would like to thank Mark W. Vandersea (NOAA) for sharing his knowledge and testing the A. ostenfeldii primers. Also, Dr. Anke Kremp for sharing cultures and associated sequences. I would also like to thank my lab mates (past and present), Kristi Sommer, Lindsay Haus, Cory Dashiell, Brooke Stuercke, Bob York, Michelle Stuart, Harris Muhlstein and Tara Haney who have always been a wealth of information and very supportive. I would like to thank my best friend, Bart R. Frans, and family whose support throughout my educational endeavors has meant a great deal. Lastly, I would also like to thank MARBIONC for funding this thesis project. v DEDICATION I would like to dedicate this thesis to my daughter, Athena N. Schwarz, whose presence has been the driving force for my motivation. My desire to go to college first started when she was born in hopes of building a better life for us. However, it quickly turned from a stepping- stone to get a decent job into a true passion. Because of Athena I was able to find my true interests when I took my first biology class as a requirement for my Associate’s degree. If it weren’t for her, I would have never given school another chance and for that I am eternally grateful. vi LIST OF TABLES Table Page 1. Recognized Alexandrium species and present knowledge of toxicity ..............................28 2. Isolates of A. peruvianum and A. ostenfeldii used in this study including isolate code, location of sample, isolation date, isolator, growth temperature and salinity ................................................................................................................................29 3. Primers used in this study to generate partial SSU, ITS1, 5.8S, ITS2 and partial LSU sequences from A. peruvianum (AP0411-1) and A. ostenfeldii (CCMP1773) ..........30 4. Alexandrium D1-D2 rDNA sequences obtained from GenBank and used in combination with those of this study for the D1-D2 alignment and phylogenetic analysis. A. insuetum and A. tamutum were the most closely related sister taxa and used as outgroup taxa .......31 5. Primers designed to distinguish between A. peruvianum (AP) and A. ostenfeldii (AO). Grey regions indicate the variable regions between the two species .................................32 6. Genomic DNA used in cross-reactivity tests of A. peruvianum and A. ostenfeldii primer sets......................................................................................................................................33 7. Mean and standard deviation (sd) of plate measurements for A. peruvianum and A. ostenfeldii, number of samples (n) and significance (5% level) using a two-tailed student’s t-test. Measurements from AP0411-1, AOTVA4, AOF0933 were used to calculate A. peruvianum mean and sd and measurements from CCMP1773 and AONOR4 were used to calculate A. ostenfeldii mean and sd .............................................................34 8. Results of cross-reactivity tests with A. peruvianum primer set APF3/APR3 ...................35 9. Results of qPCR assays with A. ostenfeldii primer set AOF4/AOR3 ................................36 vii LIST OF FIGURES Figure Page 1. Calcofluor White stained cells representing A. peruvianum showing the anterior sulcal (s.a.) plates, the ventral pore (v.p.), first apical plate (1’) and the 6th precingular plate (6’’); a) AP0411-1, b) B2-NR, c) C10-NR, d) D4-NR, e) AOTVA4 and f) AOF0933 ....37 2. Calcofluor White stained cells representing A. ostenfeldii isolates showing the s.a. plates, the ventral pore (v.p.), first apical plate (1’) and the 6th precingular plate (6’’); a) CCMP1773 and b) AONOR4 ........................................................................................38
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