RNA VIRAL DIVERSITY AND DYNAMICS ALONG THE ANTARCTIC PENINSULA A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAIʻI AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN OCEANOGRAPHY MAY 2015 By Jaclyn A. Mueller Dissertation Committee: Grieg Steward, Chairperson Alexander Culley Matthew Church Craig Smith Guylaine Poisson, Outside member Key words: marine RNA viruses, viral ecology, metagenomes, viromes, nucleic acid extraction, reverse transcription quantitative PCR (RT-qPCR), Antarctica © Copyright 2015 – Jaclyn A Mueller All rights reserved. ii ACKNOWLEDGEMENTS This research would not have been possible without the generosity and support of a number of people and organizations, for which I am very thankful. I would first like to acknowledge the Department of Oceanography, the Center for Microbial Oceanography: Research and Education (C-MORE), and the National Science Foundation (NSF) for supporting my research and providing such a fulfilling and enriching graduate career. I am forever indebted to the C-MORE ‘Ohana for their unwavering support and enthusiasm, while providing me the opportunity to experience outreach and education, enhance my leadership and professional development skills, and conduct my doctoral research. In particular, I thank Dr. David Karl for his insights, enthusiasm for oceanography, and continued support of my research. It has been a pleasure to work with so many bright, motivated, and inspiring scientists. I am forever grateful for the patience, guidance, encouragement, and support of my advisor, Dr. Grieg Steward. His enthusiasm and creativity are contagious, and have made this experience quite an enjoyable one. Working with him has not only taught me to be more a perceptive and critical thinker, but also a better writer and scientist. I thank Dr. Alexander Culley for his mentorship over the years and for the inspiration to focus on marine RNA viruses. I am grateful for his patience, support, and countless insightful discussions. I thank Dr. Matthew Church for his continuous enthusiasm, perceptive discussions, and expertise in biogeochemical cycling. I thank Dr. Craig Smith for providing me the opportunity to join his lab group on a research cruise in the Larsen A Ice Shelf, Antarctica, and the ability to collect samples for an entire chapter of this dissertation. I thank Guylaine Poisson for her expertise in bioinformatics and collaboration with our earlier work on marine RNA viruses. It has been an honor to work with such a supportive and collaborative thesis committee. As a member of the Steward Laboratory, I have had the opportunity to interact and work with a number of excellent and talented colleagues. I offer my gratitude to past and present members for their friendship, support, and assistance in the lab and field sampling, in particular Dr. Elisha Wood-Charlson, Dr. Olivia Nigro, Chris Schvarcz, Gordon Walker, and Kirena Clah. I would also like to thank the scientists, technicians, crew, and staff associated with the Palmer LTER program and the NBP1203 cruise on board the R/V Nathaniel B. Palmer. Their support, expert iii advice, and assistance both at sea and onshore were instrumental to the success of instrument deployment and sample collection. Most importantly, I would like to thank my family and friends for their continued encouragement, support, and love that greatly contributed to my sanity and happiness. My parents have always supported whichever direction or path I chose, believing I could accomplish whatever I put my mind to. Much love and thanks to my siblings, grandparents, aunts, uncles, and cousins for always being a source of inspiration. I thank the Culleys, Grabowskis, and Foremans for being such wonderful families and sharing such a special part of their lives with me. Finally, I am most grateful for the love and sacrifice of my partner and biggest supporter, Javier Miranda. I thank him for the laughter and happiness, positive energy, always putting food on the table, and embarking with me on our greatest adventure. iv ABSTRACT Marine viruses are capable of driving species succession during plankton blooms and greatly influencing biogeochemical cycling and food web dynamics through cell lysis. Most of our knowledge has come from studies in temperate and sub-tropical waters, but whether they play a major role in eukaryotic phytoplankton blooms in polar waters has not been investigated. RNA viruses in particular have been shown to contribute up to half of the total virioplankton and are known to predominantly infect eukaryotes. The goals of this dissertation were to provide the first description of RNA viruses in the Antarctic, determine their relative and absolute abundances, and investigate the diversity and dynamics of these viruses to better understand how they contribute to the ecology of eukaryotic plankton. A temporal analysis of RNA viral metagenomes collected throughout the summer bloom in the Western Antarctic Peninsula (WAP) revealed an abundant and diverse community of positive-sense, single-stranded (+ss) RNA viruses within the order Picornavirales. RNA viral metagenomes from the Eastern Antarctic Peninsula were analyzed to investigate spatial diversity, and revealed a similar dominance of +ssRNA viruses in the order Picornavirales, but a much lower diversity than the WAP metagenomes. Novel RNA virus genomes were assembled and primers were designed to target the RNA-dependent RNA polymerase gene to track individual phylotypes throughout the season in the WAP. In order to determine the absolute abundance of these RNA viruses, two methods were evaluated and optimized. First, a method for extraction of viral nucleic acids from plankton harvested on nanoporous anodic aluminum oxide filters was improved and validated. Second, a reverse transcription quantitative PCR (RT-qPCR) assay was developed to accurately quantify specific RNA viruses in nucleic acid extracts. Both methods were applied to provide the first estimates of abundance of specific RNA virus phylotypes in the ocean, revealing three v distinct patterns of abundance from four unique virus phylotypes. The results of this dissertation not only suggest the pervasiveness and ecological significance of RNA viruses in polar waters, but also present molecular techniques that should be of interest to virologists, oceanographers, and microbial ecologists. vi TABLE OF CONTENTS Acknowledgements ...................................................................................................................... iii Abstract ...........................................................................................................................................v List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii Chapter 1: Introduction ...............................................................................................................1 1.1 Introduction to marine viruses ............................................................................................1 1.2 Viruses in the Antarctic .......................................................................................................3 1.3 Research objectives and structure ......................................................................................4 1.4 References ............................................................................................................................7 Chapter 2: RNA viruses as major contributors of Antarctic virioplankton ..........................11 2.1 Abstract ...............................................................................................................................12 2.2 Introduction ........................................................................................................................13 2.3 Methods ...............................................................................................................................14 2.3.1 Field sample collection and processing .........................................................................14 2.3.2 Buoyant density fraction analysis ..................................................................................15 2.3.3 Calculation of RNA and DNA virus abundance ...........................................................15 2.3.4 Metagenome construction .............................................................................................16 2.3.5 Metagenomic and genomic analyses .............................................................................17 2.3.6 Identification and analysis of RdRp phylotypes ............................................................18 2.3.7 Accession numbers ........................................................................................................19 2.4 Results .................................................................................................................................19 2.4.1 Buoyant density and relative abundance of RNA viruses .............................................19 2.4.2 Metagenomic assembly analysis ...................................................................................20 2.4.3 Genome reconstruction ..................................................................................................21