HETEROSIGMA AKASHIWO AS A POTENTIAL SOURCE OF PREY FOR THE TOXIC DINOFLAGELLATE, DINOPHYSIS ACUMINATA by Amanda K. Williams A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Marine Studies Summer 2020 © 2020 Amanda K. Williams All Rights Reserved HETEROSIGMA AKASHIWO AS A POTENTIAL SOURCE OF PREY FOR THE TOXIC DINOFLAGELLATE, DINOPHYSIS ACUMINATA by Amanda K. Williams Approved: __________________________________________________________ Kathryn J. Coyne, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Mark A. Moline, Ph.D. Director of the School of Marine Science and Policy Approved: __________________________________________________________ Estella Atekwana, Ph.D. Dean of the College of Earth, Ocean, and Environment Approved: __________________________________________________________ Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education and Dean of the Graduate College ACKNOWLEDGMENTS Thank you to all members of my committee- Dr. Kathryn Coye, Dr. Jon Cohen, and Dr. Tye Pettay. Special thanks to Amanda Pappas who supported me both inside and outside of the lab. Thank you also to Yanfei Wang, Emmie Healey, Dr. Mark Warner, Dr. Sylvain Le Marchand, Dr. Matthew Johnson and Dr. Juliette Smith for helping me in various ways. Thanks to Dr. Ed Whereat and the University of Delaware Citizens Monitoring Program for insightful conversations and sharing data. Thank you to Kathy for taking me on as her student and welcoming me with support and guidance. I also want to acknowledge funding for this project, which was provided by the Carolyn Thompson Thoroughgood Graduate Student Research Award and Delaware Sea Grant R/HCE-32 to DTP and KJC. In addition, thank you to Delaware Sea Grant for sponsoring me to attend the U.S. Harmful Algae ID Course which took place at Bigelow Laboratories in Maine, it was a valuable experience that I will never forget. I would also like to acknowledge those who played a significant role in me getting to this point in my life. Thank you to my parents for their love and many sacrifices to ensure that I get the best education. Thank you to my late Nana Mary and Popop for supporting me and teaching me the honor and value of living a life of love towards others and all of life. Thank you to Nana Alice for the many conversations, notes, funny stories, and bouquets of flowers. Thank you to my brother, Dustin for always offering a smile and a shoulder to lean on. Thank you to Minnie, who has always been there for me to talk to and comfort me when I need it the most. And last but certainly not least, thank you to my dear love Amanda who has been here for me in every way and brought a smile to my face every day since I met her 2 years ago. In addition, thank you to the Indian River, which fostered in me a love of marine biology and nature. Thank you to the algae who have guided me the past 2 years, what glorious and immaculate beings! Thank you to the birds that sing every morning and the crickets that follow at night. Thanks to the many trees and rocks that share my love and the tiny tardigrades that inspire my dreams of outer space. Thanks to the stars, the sun and moon for being a constant- a rock to stand on in the midst of so much change. And finally, thank you to the plants, which have been my life-long teacher, it is to them that I commit my next steps, in hopes to unlock the healing and freedom I experience every day in the hearts of others. iii DEDICATION I dedicate the sum of this work and my efforts: To the waters of Delaware, where the algae dance. And to the algae who worship the sun, in eternal bliss. iv TABLE OF CONTENTS LIST OF TABLES ................................................................................................... vii LIST OF FIGURES ................................................................................................ viii ABSTRACT ............................................................................................................... x Chapter 1 LITERATURE REVIEW .............................................................................. 1 1.1 Dinophysis acuminata and its presence in the Delaware Inland Bays .. 1 1.2 Dinophysis acuminata ecology ............................................................. 2 1.3 Analysis of pigments in plastids of Dinophysis acuminata .................. 3 1.4 Research Objectives .............................................................................. 4 2 HETEROSIGMA AKASHIWO AS A POTENTIAL SOURCE OF PREY FOR THE TOXIC DINOFLAGELLATE DINOPHYSIS ACUMINATA ... 6 2.1 Abstract ................................................................................................. 6 2.2 Introduction ........................................................................................... 7 2.3 Methods................................................................................................. 9 2.3.1 Algal and ciliate cultures .......................................................... 9 2.3.2 Growth Curves ........................................................................ 10 2.3.3 Predation Experiments ............................................................ 10 2.3.4 Microscopic analysis ............................................................... 11 2.3.5 Analysis of accessory pigments .............................................. 11 2.3.6 Occurrence of Heterosigma akashiwo and Dinophysis acuminata at Torquay Canal ................................................... 12 2.3.7 Molecular analysis of bloom samples- looking for Heterosigma akashiwo ............................................................ 13 2.3.8 Statistical Analysis .................................................................. 14 2.4 Results ................................................................................................. 14 2.4.1 Growth Rates .......................................................................... 14 2.4.2 Ingestion Rates ........................................................................ 15 2.4.3 Analysis of nuclear inclusions ................................................ 15 2.4.4 A ratiometric approach using chlorophyll a emission to evaluate plastid origin ............................................................. 15 2.4.5 Relationships between D. acuminata and H. akashiwo abundances in Torquay Canal ................................................. 16 2.5 Discussion ........................................................................................... 17 v 2.6 Conclusions ......................................................................................... 23 REFERENCES ........................................................................................................ 39 Appendix DATA……………………………………………………………………………..... 43 vi LIST OF TABLES Table 2.1 Environmental metadata, cell density of D. acuminata (Pappas in prep), and presence of H. akashiwo during the winter 2019-2020 bloom of D. acuminata at site TQB in Torquay Canal, Rehoboth Beach, DE. .. 25 Table A1 Cell counts from the growth rate experiment where Dinophysis acuminata was offered either Heterosigma akashiwo, Heterosigma akashiwo and Mesodinium rubrum, or Mesodinium rubrum as prey. All values are representative of the mean of three biological replicates. ............................................................................................ 43 Table A2 Growth rates of Heterosigma akashiwo and Mesodinium rubrum in control treatment, used to offset ingestion rate of Dinophysis acuminata when offered these as prey. ............................................... 44 Table A3 Ingestion rates of Dinophysis acuminata when offered Heterosigma akashiwo or Mesodinium rubrum as prey. .......................................... 45 Table A4 Growth rates of Heterosigma akashiwo and Teleaulax amphioxeia in control treatment, used to offset ingestion rate of Dinophysis acuminata when offered these as prey. ............................................... 48 Table A5 Ingestion rates of Mesodinium rubrum when offered Heterosigma akashiwo or Teleaulax amphioxeia as prey. ....................................... 49 Table A6 Emission intensity of chlorophyll a in Dinophysis acuminata offered Heterosigma akashiwo, both Heterosigma akashiwo and Mesodinium rubrum, or Mesodinium rubrum as prey. ............................................ 50 Table A7 Ratio of intensity of emission of chlorophyll a when excited at 488 nm and 633 nm in plastids of Heterosigma akashiwo, Teleaulax amphioxeia, and Dinophysis acuminata that was given Mesodinium rubrum as prey. ................................................................................... 51 Table A8 Ratio of intensity of emission of chlorophyll a when excited at 488 nm and 561 nm in plastids of Dinophysis acuminata when offered Heterosigma akashiwo, Heterosigma akashiwo and Mesodinium rubrum, or Mesodinium rubrum as prey. ............................................ 52 vii LIST OF FIGURES Figure 2.1 Log Heterosigma akashiwo cell abundance vs. Log Dinophysis acuminata cell abundances in Torquay Canal between 2014 and 2017 for samples in which both species were present. A Pearson’s correlation revealed that Heterosigma akashiwo and Dinophysis acuminata cell abundance are not significantly correlated (p = 0.16). ..................................................................................................