The expression and regulation of lipid transport proteins in the desert locust, Schistocerca gregaria by Ahmed Sayed Ahmed M.Sc., Zagazig University, Egypt, 2006 B.Sc., Zagazig University, Egypt, 2001 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Biological Sciences Faculty of Science © Ahmed Sayed Ahmed 2018 SIMON FRASER UNIVERSITY Fall 2018 Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Approval Name: Ahmed Sayed Ahmed Degree: Doctor of Philosophy Title: The expression and regulation of lipid transport proteins in the desert locust, Schistocerca gregaria Examining Committee: Chair: Michael Silverman Professor Norbert Haunerland Senior Supervisor Professor Carl Lowenberger Supervisor Professor Inigo Novales Flamarique Supervisor Professor Jim Mattsson Internal Examiner Associate Professor Erwin Huebner External Examiner Professor Department of Biological Sciences University of Manitoba Date Defended/Approved: November 19, 2018 ii Abstract Lipids play a central role in insects, both for storage of nutrients and as an energy source during development and dispersal. Due to their low water solubility, special transport mechanisms are required for their efficient mobilization and utilization. In this thesis, I studied intra- and extracellular proteins involved in lipid transport in the desert locust, Schistocerca gregaria. Vitellogenins are very high-density lipoproteins produced by adult females and deposited into the developing eggs. Two different vitellogenins, named VG-A and VG-B, are expressed in locust fat body. Their complete cDNA transcripts of ~5.6 kb each have been sequenced, coding for two proteins of ~200 kDa each. VG-A and VG-B are co-expressed in similar amounts by mature females, commencing 11 days after adult eclosion, and continuing at high levels throughout the entire adult life. The expression of both proteins is dependent on the nuclear transcription factors Met or RXR, and knockdown of each of these proteins almost completely eliminates VG expression. A similar expression profile was observed in adult muscle for the cytosolic fatty acid binding protein FABP, albeit in both sexes. The direct knockdown of the strongly expressed FABP by RNA inference reduced its levels to less than 2% of what is normally found 3 weeks after adult eclosion. In a series of flight experiments, it was demonstrated that in the absence of FABP, insects are incapable of engaging in flight longer than 30 min; at this time, most carbohydrate resources have been depleted, and locusts normally switch to lipids as the sole fuel for muscle energy production. Short-term flight performance of FABP knockdown locusts was identical to control insects, suggesting that the lack of FABP does not interfere with carbohydrate metabolism. Moreover, the mobilization of lipids in the fat body and their transport by the major hemolymph lipoprotein lipophorin was indistinguishable from control animals. In contrast, knockdown of apolipophorin III, which is essential for lipid transport during flight, completely eliminated flight capability, even for short duration flights. Taken together, this thesis highlights the essential role of lipid transport proteins for locust reproduction and dispersal and identifies potential targets for insect control strategies. Keywords: vitellogenin; FABP; apoLp-III; Met; RXR; Schistocerca gregaria iii Dedication To my parents, my wife, and my dear sons Abdel Rahman and Omar. iv Acknowledgments I would like to express my gratitude and sincere thanks to everyone who has helped me over the past few years to finish this work. I may start by thanking Allah who has chosen me for this path and empowered me to complete this thesis. To my senior supervisor, Dr. Norbert Haunerland, thank you for every moment you have been there for me with your support, knowledge, patience, and encouragement; not only in the lab to overcome the scientific problems and challenges but with a significant impact in my personal life. Without you being there for me, I would not be able to come to this moment and finish this work. A Special thank goes to Dr. Jutta Haunerland for her daily support that extends beyond the vast knowledge of molecular techniques that I have learned from her to her soothing words which have enlightened my way during the hard times. I would like to thank my supervisory committee members Drs. Carl Lowenberger, Inigo Flamarique, and the former committee member; Allison Kermode. Dr. Carl has shown a great interest in our work. His suggestions and contributions have added a lot to this work. Dr. Carl has offered me the opportunity to attend his lab meetings as he treated me as one of his students. During these meetings, I have learned a lot about other molecular techniques and his amazing projects attempting to prevent the transmittance of different viruses by mosquitoes. His editing on the first manuscript of this thesis has solidified and enriched this work. Dr. Inigo, thank you for accepting the role to serve as a committee member on short notice. I have worked with Dr. Inigo on different projects where I have enjoyed learning about salmon and their vision mechanisms. During these projects, he has been following up with my thesis progress with valuable suggestions and support. Dr. Allison Kermode had served on the supervisory committee for a few years before she decided to resign, and then to step down from the supervision. We have sadly had to say thank you and goodbye and wishing her the best in the future out of the academia. Her valuable discussion and support during the annual committee meetings have helped us to finish the work. Many thanks to my former colleagues in Haunerland lab; Cecily Bernales, Katrina Salvante and Xin Jiang for their assistance in learning all the molecular techniques that I have used in my research and for creating that welcoming, friendly environment that I v have enjoyed since my first day in the lab. I would like to thank David Qu and Sanjeeva Rajapakse for their valuable contribution in chapter four of this thesis by designing the FABP-dsRNA and knockdown the expression of the FABP in the flight muscles. My sincere acknowledgment to the Egyptian government, Zagazig University in Egypt, and the Bureau of Cultural and Educational Affairs of Egypt in Canada for their support during the first two years of my research and choosing me for the joint-supervision scholarship which was entirely sponsored and financially supported by the Egyptian government. Last but not least, I would like to thank and acknowledge the great support of my family; my mother, brothers, and my wife “Nora” and my kids for being there with their unimaginable understanding, support, and love. Thank you, everyone. vi Table of Contents Approval .......................................................................................................................... ii Abstract .......................................................................................................................... iii Dedication ...................................................................................................................... iv Acknowledgments ........................................................................................................... v Table of Contents .......................................................................................................... vii List of Tables ................................................................................................................... x List of Figures................................................................................................................. xi List of Acronyms ............................................................................................................ xii Introduction .............................................................................................. 1 1.1. Lipid storage and functions .................................................................................... 2 1.2. Lipid Transport ...................................................................................................... 5 1.3. Cytosolic lipid binding proteins............................................................................... 6 1.4. Vertebrate Lipoproteins ......................................................................................... 6 1.5. Lipid Transport in Insects ....................................................................................... 8 1.5.1. Hemolymph lipophorins ................................................................................. 9 1.5.2. Storage lipoproteins ..................................................................................... 10 1.5.3. Vitellogenin (VG) ......................................................................................... 11 1.6. Endocrine regulation in insects ............................................................................ 12 1.6.1. Ecdysone hormone ...................................................................................... 12 1.6.2. Juvenile hormone (JH) ................................................................................. 13 1.7. Nuclear receptors as transcription factors ............................................................ 14 1.8. The desert locust, Schistocerca gregaria ............................................................