ABSTRACT CORRADINO, GABRIELLE. The Characterization and Ecology of a Heterotrophic Nanoflagellate from the Coastal Water of North Carolina. (Under the direction of Dr. Astrid Schnetzer). Nanoflagellates (2-20 µm) are key components of planktonic food webs and include single- celled eukaryotic autotrophs, heterotrophs and mixotrophs. The heterotrophic nanoflagellates (HNANs) are the primary top-down consumers of bacteria and other picoplankton through grazing. HNANs facilitate nutrient remineralization of organic matter and act as an intermediate for energy and carbon flow to higher trophic levels. While these organisms play pivotal ecological roles and are ubiquitous in marine and freshwater systems, limited information is available on species composition change over spatiotemporal scales or on trophic interactions between individual prey and predator taxa. A major obstacle to resolving these important dynamics, and a key reason why HNAN assemblages are commonly treated as a single predatory group (‘black box’), is the paucity of easily distinguishable morphological characteristics. Employing molecular tools has proven powerful in gaining insight into microbial community structure and functional diversity for HNANs and has, in combination with culturing and experimentation, become an essential tool. This thesis aimed to improve our general understanding of HNANs by characterizing a novel HNAN species and by describing its physiology and ecology. Chapter 1 of this dissertation focused on the isolation, culturing and characterization of a marine HNAN collected off the North Carolina coast. The HNAN was characterized by scanning and transmission electron microscopy, light microscopy and molecular methods. The small subunit ribosomal RNA gene (18S) was fully sequenced and supported the taxonomic placement of the a ii HNAN within the order Bicosoecida as a novel genus and species. On the basis of behavioral observations, morphology and molecular identification, the genus Coniuncta and species canzanellaia was proposed. Chapter 2 explored the feeding ecology of C. canzanellaia offering various prey types at increasing prey densities. My main research objectives were to i) determine whether C. canzanellaia selects for autotrophic or heterotrophic prey, ii) examine differences in growth (µ) and grazing responses feeding on varying prey, iii) determine how HNAN stoichiometry may be impacted, and iv) estimate gross growth efficiency (GGE) in relation to diet. To answer these research questions, prey was offered in form of a mixed heterotrophic bacterial assemblage and two common picophototrophs, the cyanobacterium Synechoccocus, and the picoeukaryote Ostreococcus. In an additional experiment, all three prey were made available as a mixed assemblage and changes in µ, ingestion rates (IR), flagellate biovolume (BV), cellular stoichiometry and GGE were examined for the HNAN. Our findings are discussed in context with previous flagellate feeding studies and the role that C. canzanellaia’s trophic interactions may play for carbon and nutrient transfer discussed. In chapter 3, predator-prey dynamics for C. canzanellaia feeding on heterotrophic bacteria and Ostreococcus were further explored over an ecologically pertinent temperature range (at 10°C, 15°C and 25°C). My main research objectives were to i) examine whether µ and IR rates would be prey- and/or temperature-dependent and ii) investigate if the flagellate could maintain GGEs across a temperature range characteristic for year-round ambient conditions in North Carolina coastal waters. Differences in the flagellate’s µ and grazing response and GGEs were compared to existing HNAN studies. In addition, the impact that HNANs, displayed in their feeding ecology, may have standing stocks of autotrophic and heterotrophic prey populations is discussed. This study is one of a limited number that describe the successful isolation and characterization a novel HNAN species and genus that further led to the interrogation of trophic interactions on a species-specific level and allowed for estimates of carbon and nutrient transfer from picoplankton to a nanograzer. It is my hope that the findings of this dissertation regarding C. canzanellaia can contribute to a growing overall understanding of HNAN ecology. © Copyright 2020 by Gabrielle Corradino All Rights Reserved The Characterization and Ecology of a Heterotrophic Nanoflagellate from the Coastal Water of North Carolina by Gabrielle L. Corradino A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Marine, Earth, and Atmospheric Sciences Raleigh, North Carolina 2020 APPROVED BY: _______________________________ _______________________________ Dr. Astrid Schnetzer Dr. Ryan Paerl Committee Chair _______________________________ _______________________________ Dr. Chris Osburn Dr. Adrian Marchetti DEDICATION This dissertation is dedicated to my parents and George who have been my support throughout my PhD. ii BIOGRAPHY Gabrielle Corradino was born in New Haven, Connecticut. She earned her Bachelor of Arts degree in Biology with a minor in Psychology from Salve Regina University. She went on to teach middle school science and work for several years before going back as a graduate student. In 2015, Gabrielle completed her Masters of Science degree in Biology and conducted her PhD at North Carolina State University with Dr. Astrid Schnetzer. After her PhD Gabrielle will be starting a Knauss marine policy fellowship in Washington, DC with NOAA. iii ACKNOWLEDGMENTS Pursuing my PhD at NC State was one of the best decisions I have made and I owe everything to my advisor Dr. Astrid Schnetzer. The support provided to me by Dr. Schnetzer is something that I simply cannot put into words and I am very appreciative of everything that she has done for me personally and academically. A big thank you to my committee members Dr. Chris Osburn, Dr. Ryan Paerl and Dr. Adrian Marchetti for their support and guidance throughout all stages of my research. This dissertation has been made better with their input and help, and for that, I am grateful. Additionally, I would like to thank Dr. Carmelo Tomas from UNC-W for his kindness and guidance through each step of characterizing this plankton. I would also like to thank the Associate Dean Dr. Mike Carter, and the whole group from the Dissertation Completion Grant for editing my thesis and making it stronger. I would like to acknowledge the Schnetzer lab members (past and current) whose laughter and support were always welcome. Also thank you to the crew of R/V Brown, R/V Pelican, RAPID and GOMECC collaborators, and their team members. This study was partially supported through funds from the National Science Foundation grant (OCE-1459406), Department of the Interior Southeast Climate Adaptation Science Center, Sigma Xi GIAR and by the National Geographic Society. Over the past decade, I also owe quite a bit to Dr. Jameson Chase of Salve Regina University for his continuous support of my work, Dr. David Vasseur of Yale University and Dr. John DeLong of the University of Nebraska. Dr. Vasseur and Dr. DeLong introduced me to freshwater iv plankton ecology in the EEB department at Yale University and I will forever be appreciative of their valuable teachings, kindness and support. Lastly, a special thanks goes to my wonderful family, parents, friends and my significant other. You have earned this degree with me, and I am so very thankful for each one of you. v TABLE OF CONTENTS LIST OF TABLES .................................................................................................................... .ix LIST OF FIGURES ................................................................................................................. xii CHAPTER 1 – Coniuncta canzanellaia gen. et sp. nov. (Bicosoecida), a Previously Uncultured Heterotrophic Nanoflagellate from North Carolina Coastal Waters ABSTRACT ............................................................................................................................... 1 INTRODUCTION ...................................................................................................................... 2 MATERIALS AND METHODS ................................................................................................ 3 Collection, Isolation and Growth .................................................................................... 3 Microscopy ..................................................................................................................... 4 Molecular Analyses ........................................................................................................ 6 Phylogenetic Analyses .................................................................................................... 7 RESULTS ................................................................................................................................... 8 Light- and Epifluorescence Microscopy ......................................................................... 8 Electron Microscopy ....................................................................................................... 9 Phylogenetic Analyses .................................................................................................. 10 DISCUSSION ........................................................................................................................... 11 Molecular