University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School January 2012 Evidence for Viral Infection in the Copepods Labidocera aestiva and Acartia tonsa in Tampa Bay, Florida Darren Stephenson Dunlap University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the American Studies Commons, Other Oceanography and Atmospheric Sciences and Meteorology Commons, and the Virology Commons Scholar Commons Citation Dunlap, Darren Stephenson, "Evidence for Viral Infection in the Copepods Labidocera aestiva and Acartia tonsa in Tampa Bay, Florida" (2012). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/4032 This Thesis is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Evidence of Viruses in the Copepods Labidocera aestiva and Acartia tonsa in Tampa Bay, Florida By Darren S. Dunlap A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major Professor: Mya Breitbart, Ph.D Kendra Daly, Ph.D Ian Hewson, Ph.D Date of Approval: March 19, 2012 Key Words: Copepods, Single-stranded DNA Viruses, Mesozooplankton, Transmission Electron Microscopy, Metagenomics Copyright © 2012, Darren Stephenson Dunlap DEDICATION None of this would have been possible without the generous love and support of my entire family over the years. My parents, Steve and Jill Dunlap, have always encouraged my pursuits with support and love, and their persistence of throwing me into lakes and rivers is largely responsible for my passion for Marine Science. Thank you to my brothers, Alex and Russ Dunlap, who are always there with guidance, friendship, and competitive ribbing, and to all of my grandparents for their love and dedication. I would be remiss to forget to thank Scott and Rebecca Shaw, who have made me feel like a part of the family, and have supported me along the way. And finally, I lovingly dedicate this thesis to my wife, who patiently listened to all of my scientific ramblings, and still manages to tolerate me after 11 years of bad jokes. ACKNOWLEDGEMENTS There are a multitude of people that made this thesis possible to whom I am extremely grateful. I would like to thank my major professor, Dr. Mya Breitbart, for her guidance, support, and friendship over the course of my Master’s program. Her dedication to her students, scientific expertise, and optimism has made my experience here one of the most memorable times of my life. The support and guidance of my committee members, Dr. Kendra Daly and Dr. Ian Hewson, has also been a vital factor throughout the entire process and helped to shape this study into what it is today. The USF College of Marine Science is fortunate to have a variety of resources and helpful associates at its disposal. Thank you to Tony Greco for his transmission electron microscopy expertise and to Sennai Habtes, Christine Cass, Scott Burghart, and Ralph Kitzmiller for the help with zooplankton identification and collection. The Florida Fish and Wildlife Conservation Commission has also been extremely helpful, with Noretta Perry and the Histology Laboratory assisting with some of the sample processing and Theresa Cody allowing use of her laboratory equipment. Lastly, thank you to all of my labmates – Karyna Rosario, Camille Daniels, Kim Pause Tucker, Terry Ng, Bhakti Dwivedi, Erin Symonds, Dawn Goldsmith, Max Hopkins, Min Tan, Elizabeth Fahsbender, and Marco Padilla-Rodriguez - for their help and friendship over these few years. They have all helped to make me a better scientist and made this experience unforgettable. TABLE OF CONTENTS LIST OF TABLES .................................................................................................. ii LIST OF FIGURES ............................................................................................... iii ABSTRACT ........................................................................................................ iv INTRODUCTION ...................................................................................................1 Overall Research Objectives ........................................................................7 METHODS ..........................................................................................................8 Sample Collection ........................................................................................8 Sequencing of Viral Metagenomes ..............................................................9 Genome Completion and Annotation ........................................................10 Viral Load Estimation of LaCopCV ..........................................................11 Transcription of LaCopCV ........................................................................12 Prevalence of AcCopCV ............................................................................13 Transmission Electron Microscopy of L. aestiva and A. tonsa..................14 In-situ Hybridization of LaCopCV and AcCopCV in Copepod Tissue ....15 RESULTS ........................................................................................................19 Genome Discovery and Annotation ...........................................................19 AcCopCV and LaCopCV Viral Load and Prevalence ...............................20 Virus-like particles revealed by Transmission Electron Microscopy ........21 Linking AcCopCV and LaCopCV Genomes to Copepod Tissue with In-situ Hybridization ..................................................................22 DISCUSSION ........................................................................................................23 Genome Characterization and Phylogeny ..................................................23 Viral Load and Prevalence .........................................................................25 LaCopCV Transcription in L. aestiva ........................................................26 Virus-like Particles Observed by Transmission Electron Microscopy ......27 In-Situ Hybridization of AcCopCV ...........................................................28 CONCLUSIONS....................................................................................................30 TABLES AND FIGURES .....................................................................................32 REFERENCES ......................................................................................................42 i LIST OF TABLES Table 1: Primers, Probes, and qPCR Standards List .............................................32 Table 2: Genome Characteristics of the Labidocera aestiva Circovirus (LaCopCV) and the Acartia tonsa Circovirus (AcCopCV) ...........................33 Table 3: Detection of AcCopCV in A. tonsa in Tampa Bay, Florida ..................39 ii LIST OF FIGURES Figure 1: Sampling Locations in Tampa Bay, Florida ..........................................34 Figure 2: AcCopCV and LaCopCV Genome Organization .................................35 Figure 3: Condensed Neighbor-Joining Phylogenetic Tree of Rep Amino Acid Sequences from AcCopCV, LaCopCV, and the Viral Rep Family (PF02407) .......................................................................................................36 Figure 4: Viral Load and Prevalence of LaCopCV ..............................................38 Figure 5: Virus-like Particles Discovered in A. tonsa and L. aestiva Using Transmission Electron Microscopy ......................................................................................40 Figure 6: Detection of AcCopCV Using In-Situ Hybridization in A. tonsa .........41 iii ABSTRACT Mesozooplankton are of critical importance to marine food webs by transferring energy from the microbial food web to higher trophic levels and depositing energy to the deeper ocean layers through fecal deposition. While decades of research have shown that viruses have significant impacts in the oceans, and infect a wide range of organisms from bacteria to whales, there is still little known about the impacts of viruses on the mesozooplankton community. As copepods are the most abundant mesozooplankton group, this study sought to characterize the viruses present in natural populations of the calanoid copepods Acartia tonsa and Labidocera aestiva in Tampa Bay, Florida. Viral metagenomics revealed two virus genomes, named Acartia tonsa copepod circovirus (AcCopCV) and Labidocera aestiva copepod circovirus (LaCopCV), which were discovered in their respective copepod species. Both viruses show amino-acid similarities to known circoviruses, and phylogenetic and genomic analyses suggest they may be divergent members of the Circoviridae family. LaCopCV was found to be extremely prevalent in the L. aestiva population, with up to 100% of individuals infected. High viral loads for LaCopCV were observed by quantitative PCR, with an average viral load of 1.3x105 copies per individual. In addition, transcription of the LaCopCV replication gene was detected in L. aestiva, demonstrating active viral replication. AcCopCV could be detected sporadically in A. tonsa populations throughout the year. The circoviruses were specific to their respective hosts, and were not detected in the other copepod species or surrounding seawater.