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Che Julius Ngwa from Bamenda, Cameroon

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Che Julius Ngwa from Bamenda, Cameroon The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions Die Moskitomitteldarmstadien des Malariaparasiten als Ziele für übertragungsblockierende Eingriffe Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Infection and Immunity Submitted by Che Julius Ngwa From Bamenda, Cameroon Würzburg, 2013 Members of the thesis committee: Chairperson: Prof. Dr. Thomas Dandekar Primary supervisor: PD. Dr. Gabriele Pradel Supervisor (Second): Prof. Dr. Ute Hentschel Humeida Supervisor (Third): Prof. Dr. Heribert Warzecha ii Affidavit I hereby confirm that my thesis entitled “The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions” is the result of my own work. I did not receive any or support from commercial consultants. All sources and/ or materials applied are listed and specified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical or similar form. Place, Date Signature ……………………………….. …………………………. Eidesstattliche Erklärung Hiermit erkläre ich an Eides statt, die Dissertation „Die Moskitomitteldarmstadien des Malariaparasiten als Ziele für übertragungsblockierende Eingriffe“ eigens- tändig, d.h. insbesondere selbständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen als die von mir angegebenen Quel- len und Hilfsmittel verwendet zu haben. Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat. Ort, Datum Unterschrift …………………………… .………………………… iii Acknowledgements I owe a lot to my primary supervisor PD. Dr. Gabriele Pradel for accepting to super- vise this piece of work and giving me the opportunity to work in a wonderful scientific environment. Her constructive advice, encouragements, and critisms helped in the re- alisation of this work. I also thank her for translating my summary from English to German. My heartfelt gratitude also goes to my second supervisor Prof. Dr. Ute Hentschel Hu- meida for giving me the opportunity to learn new techniques in her laboratory and also for her supervision and fruitful suggestions. Immense gratitude goes to Prof. Dr. Heribert Warzecha for his supervision and his stu- dent, Simone Reichwein for providing the recombinant peptides tested in this study. I am thankful to Prof. Dr. Jörg Vogel and Prof. Dr Rainer Fischer for allowing me to work in their Institutes, Prof Dr. Andreas Vilcinskas and PD. Dr. Jochen Wiesner for their laboratory support and for providing me with substances tested in this study. Special thanks to Dr. Matthias Scheuermayer for his enormous contribution in labora- tory work and in correcting of my thesis. I am grateful to Dr. Usama Ramadan Abdelmohsen and Mr. Volker Glöckner from the laboratory of Prof. Dr. Ute Hentschel Humeida for their training during my stay in their laboratory and also for their help in data analysis. I sincerely thank Prof. G. Krohne and his team for the assistance with the electron mi- croscopy and Dr. Gunnar Rudolf Mair for his support in transcriptome data analysis. I equally owe a debt of gratitude to all my colleagues especially Dr. Makoah Nigel, Andreas von Bohl, Thomas Brügl, Selina Kern, Christine Wirth, and Tim Weißbach for either providing me with anti-sera used in this study and/or correcting my thesis and also for providing a wonderful atmosphere in and out of the laboratory. Special thanks also go to Olivier Levy with whom I worked with during his Diploma thesis and to Ludmilla Sologub and Meike Lietzow for their technical assistance. I am sincerely thankful to the Graduate School of Life Sciences (GSLS) of the Unive- sity of Würzburg for the financial support through the Excellent Initiative fellowship. Finally special thanks go to my family and friends especially Abia Luther King for correcting my thesis and to God almighty for the strength given to me during the course of this work. iv Table of contents Table of contents Affidavit .............................................................................................................................. iii Acknowledgements .............................................................................................................. iv Table of contents ................................................................................................................... v Summary .............................................................................................................................. ix Zusammenfassung ................................................................................................................ xi 1. Introduction ...................................................................................................................... 1 1.1 Malaria ..................................................................................................................... 1 1.2 The P. falciparum life cycle ..................................................................................... 3 1.3 The malaria vector-Anopheles .................................................................................. 5 1.4 The malaria life cycle in the mosquito midgut ......................................................... 6 1.5 Anopheles mosquito midgut bacteria ....................................................................... 8 1.6 Effect of midgut bacteria on malaria infection in the mosquito ............................. 11 1.7 Gene expression and regulation in Plasmodium gametocytes ............................... 12 1.8 Transmission blocking strategies in the mosquito .................................................. 15 1.8.1 Malaria transmission blocking vaccines (TBVs) ............................................ 15 1.8.2 Transgenesis ................................................................................................... 18 1.8.3 Paratransgenesis ............................................................................................. 18 1.8.4 Transmission blocking drugs (TBDs) ............................................................. 21 1.9 Overview of antimicrobial molecules tested in study ............................................ 22 1.10 Objective of this study ........................................................................................... 24 2. Materials and Methods ................................................................................................... 26 2.1 Material .................................................................................................................. 26 2.1.1 Labwares ........................................................................................................ 26 2.1.2 Instruments ..................................................................................................... 27 2.1.3 Chemicals ....................................................................................................... 28 2.1.4 Antimicrobial molecules tested in this study .................................................. 28 2.1.5 Commercially available systems and enzymes............................................... 28 2.1.6 Antibodies and antisera .................................................................................. 29 2.1.7 Microorganisms and parasites ........................................................................ 30 2.1.8 Buffers, solutions and media .......................................................................... 31 2.1.9 Plasmids .......................................................................................................... 34 2.1.10 Genes investigated and oligonucleotides ........................................................ 35 2.1.11 Miscellaneous materials ................................................................................. 39 2.2 Methods .................................................................................................................. 39 v Table of contents 2.2.1 Entomology methods ...................................................................................... 39 2.2.1.1 Mosquito rearing and maintenance ......................................................... 39 2.2.1.2 Mosquito preparation and feed for bacteria diversity determination ...... 40 2.2.1.3 Transmission blocking assays using P. falciparum ................................. 41 2.2.1.4 Transmission blocking assays with P. berghei ........................................ 43 2.2.1.5 Mosquito midgut dissection for oocyst determination ............................ 43 2.2.1.6 Mosquito salivary gland dissection for sporozoites determination ......... 44 2.2.2 Cell biology methods ...................................................................................... 45 2.2.2.1 Thawing of P. falciparum parasites ........................................................ 45 2.2.2.2 Freezing of P. falciparum parasites ......................................................... 45 2.2.2.3 Preparation of blood smears ................................................................... 45 2.2.2.4 Estimation of parasitaemia ...................................................................... 46 2.2.2.5 Cultivation of P. falciparum .................................................................... 46 2.2.2.6 Synchronization of parasite cultures ......................................................
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