UNIVERSITY OF CALIFORNIA, SAN DIEGO Bacterial Influence on the Bloom Dynamics of the Dinoflagellate Lingulodinium polyedrum A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Oceanography by Xavier Mayali Committee in charge: Professor Farooq Azam, Co-chair Professor Peter J. S. Franks, Co-chair Professor Douglas H. Bartlett Professor William Fenical Professor Milton H. Saier 2007 Copyright Xavier Mayali, 2007 All rights reserved The Dissertation of Xavier Mayali is approved, and it is acceptable in quality and form for publication on microfilm Co-chair Co-chair University of California, San Diego 2007 iii DEDICATION To my family iv EPIGRAPH The process of finding the pathogen responsible for an outbreak was “…you put up a candidate and then try to tear it down. And, if you can’t tear it down, it’s probably bona fide. That’s how we do science” Dr. Ian Lipkin, quoted by Elizabeth Kolbert, The New Yorker Magazine, August 6, 2007 I think it is really important to think of all of what we do as hypothesis-driven, it’s just that the hypothesis arrives at a different time. Dr. Jo Handelsman v TABLE OF CONTENTS Signature page ………………………………………………………………… iii Dedication …. …………………………………………………………………. iv Epigraph ……..…………………………………………………………………… v Table of contents ……………………………………………………………..…... vi List of Figures ……………………………………………………………………. viii List of Tables ……………………………………………………..………..…...… x Acknowledgements ………………………………………………………….....… xi Vita ……………………………………………………………………………...... xiii Abstract …………………………………………………………………...……… xiv I. Algicidal bacteria in the sea and their impact on algal blooms ..……..………. 1 Abstract …………………………………..…………………………… 2 Text ……………………………………..…………………………..… 3 Literature cited ………………………...………………………..……. 18 II. Bacterial induction of temporary cyst formation by the dinoflagellate Lingulodinium polyedrum ………………………………………………………. 24 Abstract ………………………………………………………………. 25 Introduction ………………………………………………………...… 26 Materials and methods ……………………………………….....……. 29 Results ………………………………………………………...........… 34 Discussion ……………………………………………………………. 39 Literature cited …… ………………………………………...………. 55 III. Attachment and population dynamics of alga-associated bacteria during blooms of Lingulodinium polyedrum (Dinophyceae) …………………………………… 59 Abstract …………………………..………………………….………. 60 Introduction …………………………...……………………………... 61 Materials and methods ………………………………………..…...… 63 Results ……………………………………………………………..… 67 Discussion ……………………………………………………..….…. 71 Literature cited ………………………..………………………..……. 85 vi IV. Bacteria-induced motility reduction in a marine dinoflagellate ………....… 89 Abstract …………………………………………………………..…. 90 Introduction ………………………………………………...…….…. 91 Materials and methods …………………………………………...…. 94 Results …………………………………………………………..……98 Discussion ………………………………………………………....... 102 Literature cited ……………….…………………………………....... 117 V. First cultivation of algicidal RCA cluster bacterua and their role in algal bloom dynamics ……………………………………………………………….……..... 120 Abstract ……………………………………………………………... 121 Introduction …………………………………………………………. 122 Materials and methods …………………………………………...…. 124 Results and discussion …………………………………………….... 128 Literature cited ……………………….……………………………... 145 VI. Conclusion …………………………………………………………………. 151 vii LIST OF FIGURES Figure 2.1 16S phylogenetic analysis of algicidal bacteria isolates …………….. 45 Figure 2.2 Growth of L. polyedrum with algicidal bacteria …………………….. 47 Figure 2.3 Growth of L. polyedrum with strain ALC1 ………………………….. 48 Figure 2.4 Dynamics of L. polyedrum cyst induction …………………….…….. 49 Figure 2.5 Effect of anoxia on cyst formation ……………………………….….. 51 Figure 2.6 Effect of bacteria and darkness of algal numbers ………..…….…….. 52 Figure 2.7 Excystment of temporary cysts …………………………….…….…... 53 Figure 2.8 Micrographs of L. polyedrum cysts ………………………………..… 54 Figure 3.1 Growth of xenic L. polyedrum cultures ……………………..……….. 78 Figure 3.2 Bacterial attachment in xenic L. polyedrum cultures ………..……..… 79 Figure 3.3 Dynamic of L. polyedrum and total bacteria in nature ……...……..…. 80 Figure 3.4 Dynamic of L. polyedrum and attachment intensity in nature ….....…. 81 Figure 3.5 Dynamic of L. polyedrum and attachment frequency in nature …..…. 82 Figure 3.6 Dynamic of L. polyedrum and chlorophyll a in nature …………....… 83 Figure 3.7 Bacterial colonization with depth ………………………………....… 84 Figure 4.1 L. polyedrum motility tracks ……………………………………...… 110 Figure 4.2 Effect o algicidal filtrates on L. polyedrum motility ………...…....… 111 Figure 4.3 Frequency distribution of L. polyedrum cell tracks …………….……. 112 Figure 4.4 Effect of pronase E on L. polyedrum motility ………………….…… 113 Figure 4.5 L. polyedrum motility in nature …………………………….....…..… 114 viii Figure 4.6 Regression of bacterial colonization and algal motility ………….…. 115 Figure 4.7 L. polyedrum motility in nature as a function of depth ……….…..…. 116 Figure 5.1 Phylogenetic analysis of algicidal strain LE17 ……………….….….. 138 Figure 5.2 Growth of strain LE17 in the laboratory ………………….….….…... 139 Figure 5.3 RCA cluster abundances in nature ……………………….….….…... 140 Figure 5.4 Algicidal activity and attachment of strain LE17…………….….…… 141 Figure 5.5 Temporal dynamics of RCA cluster in nature …...….……………...... 142 Figure 5.6 Temporal dynamics of RCA cluster colonization frequency…………. 143 Figure 5.7 Temporal dynamics of RCA cluster colonization intensity ………….. 144 ix LIST OF TABLES Table 1.1 Algicidal bacteria active against eukaryotic marine algae …………… 17 Table 2.1 Susceptibility of phytoplankton to algicidal bacteria ………………….44 Table 3.1 List of L. polyedrum strains used ………………………………….….. 77 Table 4.1 Summary of protease inhibitor experiments ………...……………….. 108 Table 4.2 Protease hydrolysis rates from axenic and xenic L. polyedrum ……… 109 Table 5.1 RCA cluster sequences from phytoplankton blooms ………………… 135 Table 5.2 RCA cluster sequences from non-bloom marine samples …………… 136 Table 5.3 16S bacterial diversity studies of phytoplankton cultures …………… 137 x ACKNOWLEDGEMENTS I would like to profoundly thank my dissertation co-chairs, Peter J. S. Franks and Farooq Azam, for their invaluable assistance, for allowing me the freedom to follow my interests, and for teaching me how to be an independent scientist. Peter brought me to SIO and gave me “carte blanche” during my first two years, a tremendously generous act. Farooq allowed me to join his laboratory and provided guidance during my early years as a PhD student, fostering creativity and inspiring me to continue my work on algicidal bacteria. I learned more than he will ever know from our interactions, but I am also proud to have produced an independent body of work that I can truly call my own. The rest of my committee, Douglass Bartlett, William Fenical, and Milton Saier, have been supportive throughout this long process and I appreciate their input. I have been fortunate to interact with many faculty members at SIO who have shaped my growth as a scientist through their exceptional teaching. Those that have made a lasting impression on me (other than my co-chairs who have each won teaching awards at SIO) include Doug Bartlett, Bill Fenical, Brian Palenik, Mark Ohman, and Bill Newman. Thanks to the Azam lab (Steve, Francesca, Johnny, Ty, Christine, Melissa, Jessica), the Franks lab (Erdem, Yuji, Drew), and undergraduate assistants Ellie Wallner and Ann Sugiura for assistance over the years. I have also enjoyed interacting with many SIO graduate students who are already world experts in their fields and I am honored to be part of their group. Of particular note are my close friends Ryan Mueller and Federico Lauro, with whom I have spent many enjoyable hours; I look forward to many more years of their friendship. xi Finally, I thank my family for tremendous support. My parents Laurent and Chantal have always been fully encouraging in all my endeavors. My children Max and Emma have provided perspective during scientifically frustrating times. Their mother and my better half, Michelle, has sacrificed a great deal for me to produce this dissertation and I am eternally grateful. Chapter 1, in full, is a reprint of the material as it appears in: Mayali X, Azam F (2004) Algicidal bacteria in the sea and their impact on algal blooms. Journal of Eukaryotic Microbiology 51: 139-144. Chapter 2, in part, has been accepted for publication: Mayali X, Franks PJS, Azam F, Bacterial induction of ecdysis in the dinoflagellate Lingulodinium polyedrum, accepted to Aquatic Microbial Ecology. The dissertation author was the primary investigator and author of these papers. xii VITA 1996 B. A. (Environmental Sciences), University of California Berkeley 2001 M. S. (Marine Biology), University of Charleston 2007 Ph.D (Oceanography), University of California San Diego Publications: Mayali X, Azam F (2004) Algicidal bacteria in the sea and their impact on algal blooms. Journal of Eukaryotic Microbiology 51: 139-144 Shankle AM, Mayali X, Franks PJS (2004) Temporal pattern in population genetic diversity of Prorocentrum micans (Dinophyceae). J Phycol 40: 239-247 Mayali X, Doucette GJ (2002) Microbial community interactions and population dynamics of an algicidal bacterium active against Karenia brevis (Dinophyceae). Harmful Algae 1: 277-293 Mayali X, Franks PJS, Azam F, Bacterial induction of ecdysis in the dinoflagellate Lingulodinium polyedrum, accepted to Aquatic Microbial Ecology xiii ABSTRACT OF THE DISSERTATION Bacterial Influence on the Bloom Dynamics of the Dinoflagellate Lingulodinium