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Post-Feeding Physiology in Rhodnius Prolixus: the Possible Roles of Calcitonin-Like Diuretic Hormone (Or DH31) and Fglamide-Related Allatostatins

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Post-Feeding Physiology in Rhodnius Prolixus: the Possible Roles of Calcitonin-Like Diuretic Hormone (Or DH31) and Fglamide-Related Allatostatins Post-feeding physiology in Rhodnius prolixus: The possible roles of calcitonin-like diuretic hormone (or DH31) and FGLamide-related allatostatins by Meet Zandawala A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Cell and Systems Biology University of Toronto © Copyright by Meet Zandawala 2014 Post-feeding physiology in Rhodnius prolixus: The possible roles of calcitonin-like diuretic hormone (or DH31) and FGLamide-related allatostatins Meet Zandawala Doctor of Philosophy Department of Cell and Systems Biology University of Toronto 2014 Abstract Unfed Rhodnius prolixus (Order: Hemiptera) are in a state of arrested development. Blood- gorging results in short-term physiological/endocrinological changes that are essential to the survival of the insect, and also triggers several crucial developmental processes such as molting, growth and reproduction. Two neuropeptides that potentially play a role in this post-feeding physiology are FGLamide-related allatostatins (FGLa/ASTs) and calcitonin-like diuretic hormone (CT/DH). In order to determine the roles of these neuropeptides, I cloned and characterized their cDNA sequences and those of their cognate G protein-coupled receptors. I showed that both these neuropeptides are predominantly expressed in the central nervous system and are capable of being released into the haemolymph as hormones following feeding. I have also characterized two functionally different receptors for Rhopr-CT/DH, for the first time in any insect, and one receptor for Rhopr-FGLa/ASTs. Spatial expression analyses of these receptor transcripts revealed a wide distribution of the receptors in tissues associated with post-feeding physiology. Examination of their physiological effects using various biological assays revealed that these two neuropeptides may play indirect roles, but not direct roles during the rapid post- ii feeding diuresis and also may be involved with long term physiological changes such as metamorphosis and egg production. iii Acknowledgements I would like to begin by thanking my supervisor, Dr. Ian Orchard, for allowing me to carry out research in his laboratory, starting as an inexperienced second-year undergraduate. I am grateful and fortunate to have had such a wonderful and supportive mentor like you for the past 8 years. Thank you for always having faith in me and allowing me to pursue all the projects that I desired. I gladly move on to the next phase in my career knowing that I have learnt from the very best. I am also thankful to my immediate mentor and role-model, Dr. Jean-Paul Paluzzi, for training and guidance. Your selfless and continuing support throughout the course of my research has been invaluable. I would also like to thank my committee members, Dr. Angela Lange and Dr. Tim Westwood, for their advice and support. A special thanks to Dr. Lange for giving me the encouragement to always go a step further. I am very appreciative of all your input. Sincere thanks to all my past and present lab mates, colleagues and friend at UTM who have helped me achieve my goals and made my time memorable. I couldn’t have asked for a better work environment and colleagues. You all know who you are! Words cannot express how thankful I am to have such a supportive family. To my parents, thank you for all the sacrifices you have had to make to support my education. I wouldn’t be here if it wasn’t for your bravery to immigrate to Canada for the sake of my education. Dad, thank you for trying to learn about my research. Mom, even though it is hard for you to understand what it is that I do, I appreciate all your support and the wonderful meals you prepare when I come home from work. iv Finally, I would like to thank my best friend and confidant, my girlfriend, Aruna. This thesis would not have been possible without your undying love and support. Thank you for always being there for me. You always knew how to lift me up when I had been down countless times due to failed experiments. Thank you for respecting my dedication to work and believing in my goals, more than myself. I am lucky to have you in my life! v Table of Contents Abstract .........................................................................................................................................ii Acknowledgments ........................................................................................................................iv Table of Contents .........................................................................................................................vi Organization of the Thesis ...........................................................................................................x List of Figure and Tables ...........................................................................................................xii List of Appendices ......................................................................................................................xvi List of Abbreviations ................................................................................................................xvii Chapter 1: General Introduction ................................................................................................1 Rhodnius prolixus ..............................................................................................................2 Post-feeding physiology ....................................................................................................2 Neuropeptides ....................................................................................................................4 Diuretic and anti-diuretic hormones ...............................................................................6 Calcitonin-like diuretic hormone .....................................................................................7 FGLamide-related allatostatins (FGLa/ASTs) ...............................................................9 G protein-coupled receptors (GPCRs) ..........................................................................10 Objectives and organization of thesis ………………....................................................11 References ........................................................................................................................14 Chapter 2: Isolation and characterization of the cDNA encoding DH31 in the kissing bug, Rhodnius prolixus ........................................................................................................................18 Abstract ............................................................................................................................19 Introduction .....................................................................................................................20 Material and methods .....................................................................................................24 Results ..............................................................................................................................31 Discussion ........................................................................................................................53 References ........................................................................................................................58 Acknowledgements .........................................................................................................63 Appendices .......................................................................................................................64 vi Chapter 3: Isolation and functional characterization of calcitonin-like diuretic hormone receptors in Rhodnius prolixus ...................................................................................................68 Abstract ............................................................................................................................69 Introduction .....................................................................................................................70 Material and methods .....................................................................................................72 Results ..............................................................................................................................77 Discussion ........................................................................................................................97 References ......................................................................................................................101 Acknowledgements .......................................................................................................105 Appendices .....................................................................................................................106 Chapter 4: Structure-activity relationships of two Rhodnius prolixus calcitonin-like diuretic hormone analogs .......................................................................................................................118 Abstract ..........................................................................................................................119 Introduction ...................................................................................................................120 Material and methods ...................................................................................................121 Results ............................................................................................................................123
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