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(Homarus Americanus) Experimentally Infected with White Spot Syndrome Virus

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(Homarus Americanus) Experimentally Infected with White Spot Syndrome Virus EFFECT OF WATER TEMPERATURE ON THE IMMUNE RESPONSE OF AMERICAN LOBSTER (HOMARUS AMERICANUS) EXPERIMENTALLY INFECTED WITH WHITE SPOT SYNDROME VIRUS A Thesis Submitted to the Graduate Faculty In Partial Fulfillment of the Requirements For the Degree of Master of Science In the Department of Biomedical Sciences Faculty of Veterinary Medicine University of Prince Edward Island Louise-Marie Roux Charlottetown, P.E.I. April 2017 © 2017, L.-M.D. Roux THESIS/DISSERTATION NON-EXCLUSIVE LICENSE Family Name: Roux Given Name, Middle Name (if applicable): Louise-Marie, Danielle Full Name of University: University of Prince Edward Island Faculty, Department, School: Faculty of Veterinary Medicine, Department of Biomedical Sciences, UPEI Degree for which thesis/dissertation was Date Degree Awarded: May 2017 presented: Master of Science Thesis/dissertation Title: Effect of water temperature on the immune response of American lobster (Homarus americanus) experimentally infected with white spot syndrome virus In consideration of my University making my thesis/dissertation available to interested persons, I, Louise-Marie Roux, hereby grant a non-exclusive, for the full term of copyright protection, license to my University, University of Prince Edward Island: (a) to archive, preserve, produce, reproduce, publish, communicate, convert into any format, and to make available in print or online by telecommunication to the public for non-commercial purposes; (b) to sub-license to Library and Archives Canada any of the acts mentioned in paragraph (a). I undertake to submit my thesis/dissertation, through my University, to Library and Archives Canada. Any abstract submitted with the thesis/dissertation will be considered to form part of the thesis/dissertation. I represent that my thesis/dissertation is my original work, does not infringe any rights of others, including privacy rights, and that I have the right to make the grant conferred by this non-exclusive license. If third party copyrighted material was included in my thesis/dissertation for which, under the terms of the Copyright Act, written permission from the copyright owners is required I have obtained such permission from the copyright owners to do the acts mentioned in paragraph (a) above for the full term of copyright protection I retain copyright ownership and moral rights in my thesis/dissertation, and may deal with the copyright in my thesis/dissertation, in any way consistent with rights granted by me to my University in this non-exclusive license. I further promise to inform any person to whom I may hereafter assign or license my copyright in my thesis/dissertation of the rights granted by me to my University in this non-exclusive license. Signature: Date: April 11th 2017 ii University of Prince Edward Island Faculty of Veterinary Medicine Charlottetown CERTIFICATION OF THESIS WORK We, the undersigned, certify that Louise-Marie Roux, candidate for the degree of Master of Science has presented his/her thesis with the following title: EFFECT OF WATER TEMPERATURE ON THE IMMUNE RESPONSE OF AMERICAN LOBSTER (Homarus americanus) EXPERIMENTALLY INFECTED WITH WHITE SPOT SYNDROME VIRUS that the thesis is acceptable in form and content, and that a satisfactory knowledge of the field covered by the thesis was demonstrated by the candidate through an oral examination held on: April 11th 2017. Examiners Dr. Luis Bate (Chair) ______________________________ Dr. Spencer Greenwood ______________________________ Dr. Glenda Wright ______________________________ Dr. Mark Fast ______________________________ Dr. Andy Tasker ______________________________ Date: iii Abstract Water temperature influences basic life history traits of the American lobster (Homarus americanus) such as survival, growth and reproduction. Yet, relatively little is known about the effects of water temperature on the immune response of H. americanus. White spot syndrome virus (WSSV) is currently one of the largest impediments to the shrimp aquaculture industry worldwide. The World Organization for Animal Health (Office International de Epizootic; OIE) lists WSSV as a notifiable disease with the potential to infect all crustacean decapods. This project investigates the constraints imposed by a range of temperatures (10 oC, 15 oC, 17.5 oC, 20 oC) on the clinical, tissue and molecular immune responses of H. americanus experimentally infected with WSSV. Haemocyte concentration and qPCR testing of haemolymph were used to monitor the host’s clinical response over time and to determine presence of viral infection, respectively. A decrease in clinical condition of H. americanus was observed at 17.5 oC and 20 oC but not at 10 oC and 15 oC. WSSV-qPCR revealed viral amplification was associated with higher ambient temperature. A combination of light and electron microscopy, as well as qPCR testing of various tissues was used to elucidate changes within infected H. americanus at the various temperatures. Of the 11 host tissues examined, hypertrophied nuclei (indicative of WSSV infection) were most easily identifiable within the antennal gland and intestine of infected animals at warmer temperatures (17.5 oC and 20 oC). At colder temperatures (10 oC) no WSSV associated histopathological changes were identified in any of the examined tissues. Electron microscopy was used to explore the general ultrastructure of the antennal gland and the confirmed presence of WSSV rod-shaped and enveloped virions. In infected animals the two main regions of the antennal gland, the coelomosac and labyrinth, contained randomly distributed infected nuclei with marginated host chromatin and WSSV virions in various stages of replication. In marine decapods, the antennal gland is involved in osmoregulation and excretion suggesting that the antennal gland may be a preferred site for WSSV viral replication and inclusion within the excretory product. Lastly, a lobster specific microarray was used to monitor transcriptomic changes across 14,592 genes during viral infection at the different temperatures. Using one-way ANOVA analyses, a total of 717 genes were identified as significantly differentially expressed between infected and control H. americanus at the various temperatures. Differentially expressed genes included, ribosomal proteins (L27a, L13, L11, and L39), mitogen-activated protein kinase organizer 1, prolyl-4-hydroxylase-alpha, laminin subunit gamma-3, short-chain dehydrogenase and acute phase serum amyloid A. Microarray results were verified using RT-qPCR. Clinical, tissue and molecular immune results from this study provide a thorough assessment of the impact of temperature and WSSV in H. americanus. The use of a range of approaches and experimental temperatures is critical for broadening our understanding of how temperature influences host pathogen interactions in the economically important American lobster H. americanus. iv Acknowledgements This project could not have been completed without the help of many individuals. First and foremost, I thank my co-supervisors Spencer Greenwood and Phil Byrne. Thank you Phil for starting things off, for introducing me to the world of government research and seeing the potential within me as a new researcher. You carry a wealth of information and I am honoured to have learned a small piece of it. Spencer, thank you for always being invested in my personal and professional success, for providing me with ample opportunities to discuss my research and for the light heartedness that accompanied rock star Fridays. I thank you both immensely for being incredibly patient, understanding and supportive as I worked towards completing my degree. It was not an easy journey. As co-supervisors and mentors, you both inspire me to be the most articulate, meticulous and professional researcher that I can be. I also thank my committee members Glenda Wright, Fraser Clark and Mark Fast for their patience, guidance and technical assistance. You have been wonderful professors, mentors and colleagues and I am grateful to have worked alongside, and learned from all of you. I would also like to thank the AVC Lobster Science Centre and the Gulf Biocontainment Unit for supporting this collaboration and allowing me to use both facilities. Thank you to the wonderful staff in each facility (Haili Wang, Phyllis Dixon and Greg McCallum). Thank you also the AVC Electron Microscopy laboratory and Dorota Wadowska for all your technical training and assistance. Lastly, I would like to thank my friends. Those who I have met in PEI (Ari, Laura, Ellen, Ryan, Marc, Alden, Dan), those who supported me from afar (Meghan, Becky, Maddie, Jess, Katie), my fellow graduate students (Denise, Bob, Laura, Logan) and my biggest supporters (members of the LMR fan club), Kevin Hodgson, Katie O’Hanley, and Matt MacFarlane. I am truly grateful for all of you. v Dedication My greatest achievements have been the result of Mama and Papa Roux instilling in me the belief that I can do anything. For that, I dedicate this thesis to them. vi Table of Contents Thesis/dissertation non-exclusive license ................................................................. ii Certification of thesis work ..................................................................................... iii Abstract ..................................................................................................................... iv Acknowledgements .................................................................................................... v Dedication ................................................................................................................
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