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Transcriptomic Exploration of the Vanessa Cardui Immune System

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University of Nevada, Reno Transcriptomic Exploration of the Vanessa cardui Immune System A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Ecology, Evolution, and Conservation Biology by Alexander Drake Selvey Dr. Angela Smilanich/Thesis Advisor August, 2020 THE GRADUATE SCHOOL We recommend that the thesis prepared under our supervision by entitled be accepted in partial fulfillment of the requirements for the degree of Advisor Committee Member Graduate School Representative David W. Zeh, Ph.D., Dean Graduate School i ABSTRACT Vanessa cardui, a polyphagous Nymphalid butterfly known as the painted lady, is an ideal species for understanding ecological interactions influencing immunity, but to date has few molecular resources available to examine the underlying physiological processes driving the regulatory mechanisms of these dynamics. Rapid technological advancements in sequencing technology have decreased sequencing costs to the point of feasibility for studying the molecular basis of ecological models such as V. cardui. To examine gene expression after infection with two unique pathogens, we acquired commercially available V. cardui, infected larvae in the lab with either Escherichia coli or Junonia coenia Densovirus (JcDNV), then sequenced the transcriptome to establish an immune profile for infected larvae and controls. Gene ontology (GO) and differential expression (DE) analyses pointed to an over-representation of genes associated with developmental and energy utilization pathways with no under-representation noted for any pathways after bacterial infection. For viral infection, there was also an over- representation of genes associated with metabolic and energy usage pathways and an under-representation of genes associated with pathways involved in regulatory processes and gene expression. When comparing viral and bacterial infections, there was an over- representation of genes associated with responses to biotic stimulus, defense responses, biosynthetic responses, and movement of cellular components, with an under- representation of genes associated with cellular processes and metabolic processes. Closer investigation revealed preexisting viral populations circulating in the commercially acquires specimens, potentially confounding results based on the proposed research design. However, the tools created from this investigation still hold value for ii further investigation into the immune response of V. cardui by providing another valuable transcriptomic resource that can be combined with the few existing, non-immune related, resources; making future investigations into any and all molecular functions more powerful and informative. In summary, this transcriptomic investigation pioneers efforts to bring powerful molecular biology tools to address ecological questions of caterpillar immunity. iii DEDICATION To my wife for being patient and understanding of my academic obligations while raising our children, making the best of our limited income, and always encouraging me to follow my intellectual instinct, even when it may have complicated my research and affected my overall results. Without your support, I would not have come close to being as successful as I am today, I love you with all my heart. To my children for being the main motivation for me to seek a higher education and show them that their possibilities are limitless if they attack their cause with dedication, enthusiasm, and integrity. Never give up on your dreams, even when faced with the most daunting of adversaries, for our Father did not give us a spirit of fear, but of power, and of love, and of a sound mind (2 Timothy 1:7). iv ACKNOWLEDGMENTS First and foremost, I would like to extend my utmost gratitude to Dr. Angela Smilanich, who willingly accepted a stray graduate student struggling to find their proper fit in higher education, into her lab group and gave him purpose. Without your compassion, this work would have never come to fruition, so I owe you much more than a thank you; I owe my future as an intellectual to your kindness. No matter how difficult this project became, throughout all the setbacks and unexpected discoveries, you maintained your positivity and encouraged me to continue making unexpected discoveries in search of the truth. For these things I am not only a better person, but a more well-rounded intellectual and I thank you dearly. Thank you to Dr. Andrew Nuss for your help in funding this project and for all your support throughout. Your insight and suggestions have been greatly appreciated. Thank you to Dr. Nuss’ lab, most notably Gurlaz Kaur and Rana Pooraiiouby, for all of your help with and suggestions on analyses throughout the project. A big thank you to Dr. Richard Tillett and the Nevada Center for Bioinformatics for supporting this project with an INBRE grant from the National Institute of General Medical Sciences (GM103440). The lessons learned have been invaluable to my growth as a graduate student and amateur bioinformatician. The assistance with best practices for RNA-seq analysis has been a learning experience that I will not soon forget I would also like to thank my committee (Dr. Pedro Miura and Dr. Andrew Nuss), for embracing the challenges that I have had and never losing hope that I would finally come to a coherent conclusion based on fact. Also, for assembling rapidly and allowing me to graduate after a major degree path switch, allowing me to pursue a career to v support my growing family. I thank you for your hard work and unrivaled dedication to making this happen on such a short turnaround. A special thanks to the wonderful graduate students and professors that I grew to know and love in the various classes and laboratories that I was fortunate enough to take and work in. As well as, those professors that allowed me to rotate through their laboratories to earn invaluable skills across a myriad of disciplines and technological specialties, to you I owe a large debt of gratitude and I thank you for the sacrifices you made on my behalf. vi TABLE OF CONTENTS INTRODUCTION ...............................................................................................................1 METHODS ..........................................................................................................................5 Focal Organism ...............................................................................................................5 Pathogens ........................................................................................................................5 Treatment Groups ............................................................................................................7 Caterpillar Rearing ..........................................................................................................7 RNA Extraction, Library Preparation, & Sequencing .....................................................8 Transcriptome Assembly .................................................................................................8 Transcriptome Annotation & Quality Assessment ..........................................................9 Differential Expression Analysis ...................................................................................10 Gene Ontology Enrichment Analysis ............................................................................10 Centrifuge Sequence Classification ..............................................................................10 RESULTS ...........................................................................................................................12 Transcriptome Assembly Quality & Annotation ...........................................................12 Immune System Components .......................................................................................14 Differential Expression & GO Analyses .......................................................................14 Profile of Viral Transcripts in V. cardui ........................................................................17 DISCUSSION ....................................................................................................................52 CONCLUSION ..................................................................................................................56 REFERENCES ..................................................................................................................57 APPENDIX ........................................................................................................................63 List of Supplementary Tables ........................................................................................63 List of Supplementary Figures ....................................................................................148 vii LIST OF TABLES Table 1. Number of reads before and after trimming .........................................................18 Table 2. BUSCO statistics and general transcriptome statistics obtained from EnTAP ....19 Table 3. Predicted set of immunity genes from the Nuss-Smilanich assembled transcriptome based on prior lepidopteran immunity gene annotations ......................20 Table 4. Kallisto alignment statistics: trimmed reads against Nuss-Smilanich transcriptome (final filtered). (Samples n=12, Transcripts: n=22,342) .......................38 Table 5. Top 5 up-regulated and top 5 down-regulated transcripts with log2 fold changes, false detection rate
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