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Examining the Physiology of Harmful Microalgae During EXAMINING THE PHYSIOLOGY OF HARMFUL MICROALGAE DURING ALGICIDAL CONTROL AND DIEL VERTICAL MIGRATION by Charles L. Tilney A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Studies Summer 2014 © 2014 Charles L. Tilney All Rights Reserved UMI Number: 3642365 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 complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI 3642365 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 EXAMINING THE PHYSIOLOGY OF HARMFUL MICROALGAE DURING ALGICIDAL CONTROL AND DIEL VERTICAL MIGRATION by Charles L. Tilney Approved: __________________________________________________________ Mark A. Moline, Ph.D. Director of the School of Marine Science and Policy Approved: __________________________________________________________ Nancy M. Targett, Ph.D. Dean of the College of Earth, Ocean, and Environment Approved: __________________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Mark E. Warner, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Kathryn J. Coyne, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Jonathan H. Cohen, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Christopher J. Gobler, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS Many people contributed to the successful completion of this dissertation, and I am much obliged to every one of them. First and foremost, I must thank Dr. Mark Warner for his support, sage guidance, and patience over the years, and for imparting his knowledge of biology, ecology, and science in general. I too must pay a special thanks to Dr. Kathryn Coyne, for her guidance throughout much of this dissertation, and for patiently training me in the realm of molecular biology. Certainly, everyone in the Warner and Coyne laboratories deserve my thanks for their help and friendship over the years, including: Dr. Sebastian Hennige, Dr. Michael McGinley, Dr. Matthew Aschaffenburg, Kenneth Hoadley, Dr. Tye Pettay, Kaytee Pokrzywinski, Chris Main, Colleen Bianco, Katherine Lee, Michelle Stuart and Dr. Jennifer Stewart. I also wish to thank my committee members, Dr. Jonathan Cohen and Dr. Christopher Gobler, for providing advice and direction. I must thank everyone on the Lewes campus, and should thank in particular Dr. Jonathan Sharp, and Dr. Edward Whereat. The participants of the University of Delaware’s Citizen Monitoring Program also deserve an acknowledgement. I would like to thank my family in Washington DC for their myriad offerings of support especially upon moving to Delaware. My sisters, parents, and grandparents in Great Britain deserve my eternal thanks for their love and support. Finally, without question, my wife Josée deserves my final words here for her patience and love. Thank you. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ ix LIST OF FIGURES ........................................................................................................ x ABSTRACT .............................................................................................................. xviii Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 What Are Harmful Algal Blooms? ............................................................ 1 1.2 A Brief Overview of HAB Dynamics ....................................................... 3 1.3 Circadian Rhythms and Diel Vertical Migration ...................................... 7 1.4 Control of HABs ....................................................................................... 9 1.5 Dissertation Chapters .............................................................................. 11 2 GROWTH, DEATH AND PHOTOBIOLOGY OF DINOFLAGELLATES UNDER BACTERIAL-ALGICIDE CONTROL ............................................. 13 2.1 Abstract ................................................................................................... 13 2.2 Introduction ............................................................................................. 15 2.3 Methods ................................................................................................... 19 2.3.1 Bacterial Filtrate Preparation ....................................................... 19 2.3.2 Algal Species ............................................................................... 19 2.3.3 Species Testing ............................................................................ 20 2.3.4 Algal Photochemistry .................................................................. 20 2.3.5 Dose Responses ........................................................................... 21 2.3.6 Membrane Permeability .............................................................. 22 2.3.7 Dark Incubation ........................................................................... 23 2.3.8 Statistical Analyses ...................................................................... 24 2.4 Results ..................................................................................................... 25 2.4.1 Species Comparison .................................................................... 25 2.4.2 Dose Response ............................................................................. 26 2.4.3 Membrane Permeability .............................................................. 27 2.4.4 Darkness ..................................................................................... 28 v 2.5 Discussion ............................................................................................... 29 2.5.1 General Dynamics of Photobiology & Cell Number .................. 29 2.5.2 Species Specificity & Membrane Exposure ................................ 30 2.5.3 Dose Responses ........................................................................... 32 2.5.4 Membrane Permeability and Fv/Fm .............................................. 33 2.5.5 Darkness ..................................................................................... 34 2.5.6 Conclusion ................................................................................... 35 3 EFFECTS OF A BACTERIAL ALGICIDE, IRI-160AA, ON DINOFLAGELLATES AND THE MICROBIAL COMMUNITY IN MICROCOSM EXPERIMENTS ..................................................................... 43 3.1 Abstract ................................................................................................... 43 3.2 Introduction ............................................................................................. 45 3.3 Methods ................................................................................................... 51 3.3.1 Bacterial Filtrate Preparation ....................................................... 51 3.3.2 Natural Waters, Incubation & Sampling ..................................... 51 3.3.3 Chlorophyll-a .............................................................................. 53 3.3.4 Active Chlorophyll-a Fluorescence ............................................. 54 3.3.5 Cell Enumeration ......................................................................... 54 3.3.6 DNA Extraction, PCR-DGGE, and Sequencing ......................... 55 3.3.7 Quantitative-PCR ........................................................................ 57 3.3.8 Statistical Analyses ...................................................................... 59 3.4 Results ..................................................................................................... 60 3.4.1 Bloom Experiments ..................................................................... 60 3.4.1.1 Photochemistry ............................................................. 60 3.4.1.2 Autotrophic Biomass & Target Cell Number ............... 61 3.4.1.3 Eukaryotic Community Structure ................................
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