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Divergent Rnai Biology in Mites and Development of Pest Control Strategies

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Divergent Rnai Biology in Mites and Development of Pest Control Strategies The University of Southern Mississippi The Aquila Digital Community Dissertations Summer 8-1-2018 Divergent RNAi Biology in Mites and Development of Pest Control Strategies Md Mosharrof Hossain Mondal University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/dissertations Part of the Life Sciences Commons Recommended Citation Mondal, Md Mosharrof Hossain, "Divergent RNAi Biology in Mites and Development of Pest Control Strategies" (2018). Dissertations. 1539. https://aquila.usm.edu/dissertations/1539 This Dissertation is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Dissertations by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. Divergent RNAi Biology in Mites and Development of Pest Control Strategies by Md Mosharrof Hossain Mondal A Dissertation Submitted to the Graduate School, the College of Science and Technology and the Department of Biological Sciences at The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved by: Dr Alex Sutton Flynt, Committee Chair Dr. Shahid Karim Dr. Dmitri Mavrodi Dr. Faqing Huang Dr. Chaoyang Zhang ____________________ ____________________ ____________________ Dr. Alex Sutton Flynt Dr. Janet Donaldson Dr. Karen S. Coats Committee Chair Department Chair Dean of the Graduate School August 2018 COPYRIGHT BY Md Mosharrof Hossain Mondal 2018 Published by the Graduate School ABSTRACT RNA interference (RNAi) has transformed genetics research by revolutionizing reverse genetics in the nearly three decades that have passed since its discovery. ~19-31 nt small non-coding RNAs play a central role in RNAi biology, and are found in all multicellular eukaryotes. In these animals, three major classes of small RNAs have been described: microRNA (miRNA), small interfering RNA (siRNA), and Piwi interacting RNA (piRNA), which are produced in distinct yet occasionally overlapping pathways. While miRNAs are involved in tuning endogenous gene expression, piRNAs and siRNAs are essential for defense against viruses and transposons. Argonaute proteins are the main effectors in RNAi biology; they associate with small RNAs forming RNA induced silencing complex (RISC), which finds target transcripts by complementary base-pairing between small RNA and target leading to destruction or inhibition of expression. In the present study, we sought to investigate the RNAi pathways in two basal arthropods; a major allergy causing agent—dust mites, and the most polyphagous and pesticide resistant plant pest—spider mites. We have discovered that the piRNA pathway is absent in dust mite, and has been integrated into a derived siRNA pathway in spider mites. The spider mite siRNA pathway, which appears to work upstream of piRNA biogenesis, is gonad specific, and is a complete reversal of worm’s piRNA biology. Besides a laboratory tool, RNAi is being developed into an efficient pest-control technique to knock down gene expression in a single, targeted species. In such strategy, RNAi is triggered by long double-stranded RNAs, which get incorporated into the endogenous RNAi machinery producing siRNA, and trigger cleavage of complementary target transcripts. So far, RNAi technology is largely unsuccessful against spider mites, ii and the present study will help to design effective RNAi technology in future. Moreover, many species have been found insensitive to RNAi such as lepidopterans and hemipterans. Barriers in gut biology inhibit successful RNAi in these animals, which can be prevented if dsRNAs being delivered to epithelial cells effectively. To address this, we have developed a cationic polymeric delivery vehicle in this study that was successful in fall armyworm, a traditionally RNAi recalcitrant insect pest. iii ACKNOWLEDGMENTS I would like to thank my supervisor Dr. Alex Sutton Flynt for providing me the opportunity to work in some excellent projects, and mentoring my entire time at the Southern Miss. As an outstanding advisor, he offered full freedom to design, and carry out experiments, which helped me extensively to think independently, and will have enormous effect in my future career. In challenging projects, he offered his help and made himself always available making a graduate student’s life extraordinarily easy! I would also like to thank my dissertation committee members, Dr. Shahid Karim, Dr. Dmitri Mavrodi, Dr. Faqing Huang, Dr. Chaoyang Zhang, and Dr. Zheng Wang. They were always available for any discussion about my research and future career. I greatly appreciate their valuable insights, and expertise, which significantly helped me to understand my research from a different point of view. I also want to thank all my lab members for creating a friendly workplace environment, and engaging in scientific discussions, which was a key to success to everyone in the lab. I want to specially thank Matt DeCruz and Dr. Partha Sengupta for setting up the initial lab environment. I would also like to thank Kody Mansfield, Britton Strickland, and Obrie Scarbrough who worked as undergrad with me and helped me significantly to finish the projects. Finally, I want to thank Dr. Pavel Klimov at the University of Michigan, and Dr. Charles McCormick at the Department of Polymer Science and Engineering, The University of Southern Mississippi for offering their resources and expertise and allowing me to work collaboratively with their labs. I would also like to thank MS INBRE program for their continuous support in promoting research not only in the Southern Miss iv but also across the state of Mississippi. I thank John Lindner from MS INBRE Imaging Core for his support in my experiments. Special thanks to the Department of Biological Sciences at USM, and the entire USM community for providing necessary resources, and extending their love and affection to the students who come from other parts of the globe to Southern Miss to enrich themselves and the scientific community! v DEDICATION My parents, parents-in-law, siblings, and my wife Tumpa vi TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGMENTS ................................................................................................. iv DEDICATION ................................................................................................................... vi LIST OF TABLES ............................................................................................................ xii LIST OF ILLUSTRATIONS ........................................................................................... xiii LIST OF ABBREVIATIONS ........................................................................................... xv CHAPTER I – INTRODUCTION ...................................................................................... 1 1.1 RNA Interference (RNAi)......................................................................................... 1 1.2 A Short History of RNAi Discovery ......................................................................... 2 1.3 Major Classes of Small RNA .................................................................................... 6 1.3.1 MicroRNA (miRNA) Pathway .......................................................................... 7 1.3.2 Short Interfering RNA (siRNA) Pathway .......................................................... 8 1.3.3 Piwi Interfering RNA (piRNA) Pathway ......................................................... 10 1.4 Major RNAi Factors ............................................................................................... 14 1.4.1 Argonaute (Ago and Piwi) Proteins ................................................................. 14 1.4.2 Drosha .............................................................................................................. 15 1.4.3 Dicer ................................................................................................................. 15 1.4.4 RNA Dependent RNA Polymerase (Rdrp) ...................................................... 16 1.4.5 Systemic RNA Interference Deficient-1 (Sid-1).............................................. 17 vii 1.5 RNAi in Chelicerates and Aim of the Study ........................................................... 17 CHAPTER II – SMALL RNA BIOLOGY IN THE HOUSE DUST MITES .................. 21 2.1 Introduction ............................................................................................................. 21 2.2 Materials and Methods ............................................................................................ 22 2.2.1 Bulk Collection of Mixed Stages HDM........................................................... 22 2.2.2 High Molecular Weight (HMW) Genomic DNA Extraction .......................... 22 2.2.3 In vitro Transcription of dsRNA ...................................................................... 23 2.2.4 dsRNA Soaking of Mites ................................................................................. 24 2.2.5 Northern Blot ................................................................................................... 24 2.2.6 β-elimination .................................................................................................... 25 2.2.7 Terminal Exonuclease and CIP Treatment of Small RNA .............................
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