Host-microbiome-pathogen interactions in cockroaches Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry, Pharmacy of Freie Universität Berlin by Thorben Sieksmeyer 2019 The doctorate studies were conducted under supervision of Prof. Dr. Dino P. McMahon from December 2014 to September 2019 in the Institute of Biology. 1st Reviewer: Prof. Dr. Dino P. McMahon 2nd Reviewer: Prof. Dr. Jens Rolff date of defense: 19.08.2020 Hereby I confirm that I have prepared my doctoral thesis independently and without impermissible help. Acknowledgment First and foremost, I would like to express my deepest thanks to my supervisor Prof. Dr. Dino P. McMahon. Without his patient guidance, enthusiastic encouragement and useful critiques I would not have been able to finish this work. Furthermore, I would like to thank Dr. Paul Johnston for his contributions to this thesis, especially his help with analysing the 16S and transcriptome sequencing data. Without him I would have never been able to accomplish this on my own. At this point I also want to thank Susan and Sarah from BeGenDiv for their guidance and help with conducting both sequencing experiments. In addition, I want to thank the current and former members of the McMahon lab, namely Shulin, Alejandra, Annett and Ronald for their help in the lab, their helpful advices and last but not least their company which made work very enjoyable resulting in some priceless memories. I especially want to thank James and PhD Sophie Armitage for their help to improve this thesis. Further, I want to thank Prof. Dr. Jens Rolff for his guidance, his valuable professional comments and the permission to work in his lab, all of which extensively improved my work and expanded my knowledge. I also thank the other members of his group for their professional and personal advice and help during the work on my thesis. I especially thank Alex for his help with the proteomic experiment and the fun time spent in the lab while performing those experiments. This also includes Benno and Christoph for their help and expertise in proteomics. My appreciation also goes to the Bundesanstalt für Materialforschung und -prüfung for funding and the opportunity to perform most of my work there. My gratitude includes all the members of the department 4.0 and 4.1 for their personal and professional help. It was an honor to get to know them and work with them. The time and experiences shared with them will always be remembered. I especially want to mention Prof. Dr. Rüdiger Plarre here for his valuable advices and his support. I also want to thank all my friends for the time spent together and helping me to think different and refocus when feeling lost. Last but not least, I want to thank my beloved family, my mum Marianne, my dad Volker and my brother Nils. Without their love, their patience and their support I would not be the person I am today. Table of content List of abbreviations ........................................................................................................... 1 General introduction ........................................................................................................... 3 A new concept of the term ‘self’ – the host, the microbiome and the pathogens ....... 4 The role of the microbiome ............................................................................................. 5 The microbiome of insects .............................................................................................. 7 Nutritional role of the insect microbiota ........................................................................ 9 Microbial impacts on insect detoxification ...................................................................11 Microbial impacts on insect parasite and pathogen protection ..................................11 Nutritional impacts on insect pathogen protection ......................................................13 Cockroaches as a great model to study the tripartite of hosts, their microbiomes and pathogens .......................................................................................................................14 Description of the project ...............................................................................................15 Eating in a losing cause: anorexia and macronutrient manipulation by a cockroach fails to boost immunity ......................................................................................................17 Abstract ...........................................................................................................................18 Introduction .....................................................................................................................19 Materials and Methods ...................................................................................................21 Insects and bacteria ....................................................................................................21 Artificial diets ..............................................................................................................21 Bacterial inoculation ...................................................................................................22 Diet choice following sublethal infection ..................................................................22 Survival on enforced diet ............................................................................................22 Survival on enforced diet following lethal infection .................................................23 Hemolymph collection ................................................................................................23 Bacteria growth inhibition assay................................................................................23 Proteomic analysis by mass spectrometry ...............................................................24 Statistical analysis ......................................................................................................27 Results .............................................................................................................................28 Survival on enforced diet without infection ..............................................................28 Diet choice following sublethal infection ..................................................................28 Survival on enforced diet following infection............................................................31 Bacteria growth inhibition assay................................................................................33 Proteomic analysis by mass spectrometry ...............................................................35 Discussion ......................................................................................................................36 Chapter II .............................................................................................................................41 Cleaning to a higher standard: evaluating sterilization techniques for the creation of a germ-free Blatella germanica breeding line and the impact of the microbiome on host development .......................................................................................................................41 Abstract ...........................................................................................................................42 Introduction .....................................................................................................................44 Material and methods .....................................................................................................46 Insects ..........................................................................................................................46 Ootheca collection and establishment of the germ-free cockroach breeding ........47 Sterility testing by plating and 16S sequencing ........................................................48 Sequence quality control and taxonomic analyses ..................................................50 Comparison of the developmental time of conventional and germ-free cockroaches ................................................................................................................51 Results .............................................................................................................................51 Sterility testing by plating and 16S sequencing ........................................................51 Comparison of the developmental time of conventional and germ-free cockroaches ................................................................................................................57 Discussion ......................................................................................................................58 Chapter III ............................................................................................................................63 Be clean, be healthy? Impact of the microbiome on immune gene regulation and the outcome of a bacterial infection in the cockroach Blatella germanica ...........................63 Abstract ...........................................................................................................................64 Introduction .....................................................................................................................66 Material and