INVESTIGATION INTO THE MIGRATION OF LEISHMANIA WITHIN PHLEBOTOMINE SAND FLIES Yasmine Precious Kumordzi Lancaster University Division of Biology and Life Sciences Faculty of Health and Medicine A thesis submitted in partial fulfilment of the requirements for the degree of MSc (by Research) in Biomedical Sciences January 2019 Yasmine Precious Kumordzi DECLARATION I declare that the submitted work presented in this thesis is my own work and has not previously been submitted for a degree at this university or any other institution. 1 Yasmine Precious Kumordzi ACKNOWLEDGMENT First and foremost, I would like to thank Dr Rod Dillon and Dr Alexandre Benedetto, my Masters of Research supervisors for giving me this opportunity to work with their group and for their guidance and support during this work. I would like to thank Dr Rod Dillon, for his patience and useful discussions throughout this project. I would also like to thank Dr Alexandre Benedetto for teaching me about microfluidics. I am grateful to all the members of Dr Rod Dillon’s group especially Dr Raquel Vionette and Ms Monica Staniek, for teaching me everything I know about sandfly rearing and dissection. 2 Yasmine Precious Kumordzi TABLE OF CONTENTS DECLARATION ......................................................................................................... 1 ACKNOWLEDGMENT ............................................................................................... 2 TABLE OF CONTENTS ............................................................................................... 3 List of Figures ......................................................................................................... 6 List of TABles ........................................................................................................... 8 List of ImAges ......................................................................................................... 8 List of AbBreviAtions ............................................................................................... 9 AbstrAct ................................................................................................................ 11 CHAPTER ONE: GENERAL INTRODUCTION ............................................................. 13 1.1 Leishmaniasis ................................................................................................... 13 1.1.1 Epidemiology .................................................................................................................. 13 1.1.2 Clinical Presentation ....................................................................................................... 14 1.1.3 Causative Agent and Vector ........................................................................................... 18 1.1.4 Interventions .................................................................................................................. 18 1.2 Sandfly – The vector ......................................................................................... 21 1.2.1 Life cycle ......................................................................................................................... 22 1.2.2 Structure ......................................................................................................................... 23 1.2.3 Feeding ........................................................................................................................... 26 1.2.4 Microbiota ...................................................................................................................... 30 1.3 Leishmania protozoa - The parasite ................................................................. 35 1.3.1 Flagellum ........................................................................................................................ 36 1.3.2 Kinetoplast ..................................................................................................................... 38 1.3.3 Plasma membrane .......................................................................................................... 43 CHAPTER TWO: LITERATURE REVIEW .................................................................... 49 2.1 Leishmania Sand fly Interactions ...................................................................... 49 2.1.1 Metacyclogenesis of suprapylaria Leishmania ............................................................... 49 2.1.2 Developmental cycles in the sand fly ............................................................................. 53 2.1.3 Leishmania manipulation of the Phlebotomine host interaction ................................... 55 2.1.4 Important features of Leishmania for transmission ...................................................... 56 2.1.5 Transmission ................................................................................................................... 58 2.2 Chemotaxis ...................................................................................................... 59 2.2.1 Leishmania chemotaxic assay methods ......................................................................... 62 2.2.2 Microfluidics for studying Leishmania migration ........................................................... 68 2.3 Aims of the project ........................................................................................... 70 CHAPTER THREE: MATERIALS AND METHODS ....................................................... 72 3.1 General Methods ............................................................................................. 72 3.1.1 Insect rearing .................................................................................................................. 72 3.1.2 Parasites ......................................................................................................................... 73 3.1.3 Preparation of culture medium ...................................................................................... 73 3.1.4 Growth of parasites ........................................................................................................ 74 3 Yasmine Precious Kumordzi 3.2 Part A: Leishmania morphological studies of Leishmania tarentolae and Leishmania mexicana in axenic culture ......................................................................... 74 3.2.1 Slide preparation ............................................................................................................ 74 3.2.2 Morphological configuration and classification of promastigotes ................................. 75 3.3 Part B: Capillary assays of Leishmania challenged with an array of chemical compounds ................................................................................................................... 75 3.3.1 Preparation of Washing and Incubation Solution (WIS) buffer and promastigote suspension .................................................................................................................................... 75 3.3.2 Preparation of capillary tube solution ............................................................................ 76 3.3.3 Collecting results ........................................................................................................... 77 3.3.4 Experimental conditions ................................................................................................ 78 3.4 Part C: Development of a microfluidic method to analysing Leishmania chemotaxis ................................................................................................................... 79 3.4.1 Microfabrication ............................................................................................................ 79 3.4.2 Cell migration in microfluidic device .............................................................................. 80 3.5 Part D: Experimental infections of the Lutzomyia longipalpis with Leishmania tarentolae and Crithidia fasiculatA, and Aedes aegypti with Crithidia fasiculatA ........... 81 3.5.1 Heat treatment of sheep blood ..................................................................................... 81 3.5.2 Infection protocol .......................................................................................................... 81 3.5.3 Dissection ...................................................................................................................... 82 CHAPTER FOUR: RESULTS ...................................................................................... 83 4.1 Leishmania mexicana morphometric analysis in vitro ...................................... 83 4.2 Leishmania tarentolae morphometric analysis in vitro ..................................... 88 4.2.1 Bulgtomonad promastigotes .......................................................................................... 94 4.2.2 Kinetoplast Nucleus swapping promastigotes ............................................................... 96 4.3 Analysing chemotaxic migration ....................................................................... 98 4.3.1 Capillary assay ................................................................................................................ 98 4.3.2 Capillary assay morphometrics .................................................................................... 100 4.4 Microfluidic Chip design ................................................................................ 113 4.4.1 Design and operations of the microdevice