Burkholderia as bacterial symbionts of Lagriinae beetles Symbiont transmission, prevalence and ecological significance in Lagria villosa and Lagria hirta (Coleoptera: Tenebrionidae) Dissertation To Fulfill the Requirements for the Degree of „doctor rerum naturalium“ (Dr. rer. nat.) Submitted to the Council of the Faculty of Biology and Pharmacy of the Friedrich Schiller University Jena by B.Sc. Laura Victoria Flórez born on 19.08.1986 in Bogotá, Colombia Gutachter: 1) Prof. Dr. Martin Kaltenpoth – Johannes-Gutenberg-Universität, Mainz 2) Prof. Dr. Martha S. Hunter – University of Arizona, U.S.A. 3) Prof. Dr. Christian Hertweck – Friedrich-Schiller-Universität, Jena Das Promotionskolloquium wurde abgelegt am: 11.11.2016 “It's life that matters, nothing but life—the process of discovering, the everlasting and perpetual process, not the discovery itself, at all.” Fyodor Dostoyevsky, The Idiot CONTENT List of publications ................................................................................................................ 1 CHAPTER 1: General Introduction ....................................................................................... 2 1.1. The significance of microorganisms in eukaryote biology ....................................................... 2 1.2. The versatile lifestyles of Burkholderia bacteria .................................................................... 4 1.3. Lagriinae beetles and their unexplored symbiosis with bacteria ................................................ 6 1.4. Thesis outline ........................................................................................................... 9 1.5. References ............................................................................................................. 10 CHAPTER 2: Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms ................................................................................................................... 16 1. Introduction.................................................................................................................. 16 2. Defensive animal-microbe symbioses .................................................................................... 17 3. Ecological and evolutionary implications................................................................................ 45 4. Outlook: current status, challenges and opportunities of defensive symbiosis research ......................... 49 5. References ................................................................................................................... 50 CHAPTER 3: An out-of-body experience: The extracellular dimension for the transmission of mutualistic bacteria in insects ........................................................................................ 61 3.1. Abstract .................................................................................................................... 61 3.2. Introduction ............................................................................................................... 61 3.3. Overview of extracellular transmission routes of insect symbionts .............................................. 62 3.4. Transition from a free-living state to symbiosis ..................................................................... 67 3.5. Evolutionary transitions among transmission routes ................................................................ 68 3.6. Implications of transmission ecology for symbiont genome evolution ........................................... 69 3.7. Host-symbiont co-evolutionary dynamics ............................................................................ 71 3.8. Conclusions and future perspectives .................................................................................. 72 3.10. Supplement .............................................................................................................. 77 CHAPTER 4: Antibiotic-producing beetle mutualists evolved from plant-pathogenic bacteria ................................................................................................................................ 88 4.1. Summary ................................................................................................................... 88 4.2. Main text................................................................................................................... 89 4.3. Methods .................................................................................................................... 94 4.4. References ................................................................................................................. 99 4.5. Extended Data ........................................................................................................... 102 4.6. Supplementary Information ............................................................................................ 110 CHAPTER 5: Symbiont dynamics and strain diversity in the defensive mutualism between Lagria beetles and Burkholderia ........................................................................................ 130 5.1. Summary .................................................................................................................. 130 5.2. Introduction .............................................................................................................. 130 5.3. Results and discussion .................................................................................................. 131 5.4. Experimental Procedures .............................................................................................. 140 5.5. References ................................................................................................................ 143 5.6. Supplementary Information ............................................................................................ 147 CHAPTER 6: Impact of the defensive symbiont Burkholderia gladioli on the performance of Lagria beetles in the absence of natural enemies. ............................................................ 155 6.1. Introduction .............................................................................................................. 155 6.2. Materials and methods .................................................................................................. 156 6.3. Results and discussion .................................................................................................. 159 6.4. Concluding remarks..................................................................................................... 166 6.5. References ................................................................................................................ 166 CHAPTER 7: Genome sequencing of two insect-associated Burkholderia gladioli strains isolated from Lagria hirta and Lagria villosa beetles. ....................................................... 167 7.1. Introduction .............................................................................................................. 167 7.2. Materials and methods .................................................................................................. 168 7.3. Results and discussion .................................................................................................. 169 7.4. Concluding remarks and perspectives ................................................................................ 172 7.5. References ................................................................................................................ 172 CHAPTER 8: General Discussion ....................................................................................... 174 8.1. Establishment of the Lagriinae - Burkholderia symbiosis ........................................................... 174 8.2. Persistence at an evolutionary timescale ............................................................................. 177 8.3. Research perspectives on the Lagriinae-Burkholderia symbiosis .................................................. 183 8.4. References ................................................................................................................ 184 9. Summary ........................................................................................................................ 188 10. Zusammenfassung........................................................................................................ 190 Appendix .......................................................................................................................... 192 Acknowledgements .......................................................................................................... 210 Originality statement / Eigenständigkeitserlärung ......................................................... 213 List of publications Flórez LV, Biedermann P, Engl T, Kaltenpoth M. (2015) Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms. Natural Product Reports, 32, 904-36 Salem H, Flórez LV, Gerardo N, Kaltenpoth M. (2015) An out-of-body experience: the extracellular dimension for the transmission of mutualistic bacteria in insects. Proceedings of the Royal Society B: Biological Sciences, 282, 20142957 Flórez LV, Gaube P, Scherlach K, Sitte E, Rodrigues A, Hertweck C, Kaltenpoth M. (In preparation) Antibiotic-producing beetle mutualists evolved from plant-pathogenic bacteria Flórez LV, Kaltenpoth