Protective Streptomyces in Beewolves

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Protective Streptomyces in Beewolves Protective Streptomyces in beewolves - Ecology, evolutionary history and specificity of symbiont-mediated defense in Philanthini wasps (Hymenoptera, Crabronidae) - Dissertation zur Erlangung des akademischen Grades „doctor rerum naturalium“ (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Dipl.-Biol. Sabrina Koehler geboren am 07.11.1982 in Zwickau Protective Streptomyces in beewolves - Ecology, evolutionary history and specificity of symbiont-mediated defense in Philanthini wasps (Hymenoptera, Crabronidae) - Seit 1558 Dissertation zur Erlangung des akademischen Grades „doctor rerum naturalium“ (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Dipl.-Biol. Sabrina Koehler geboren am 07.11.1982 in Zwickau Das Promotionsgesuch wurde eingereicht und bewilligt am: 14. Oktober 2013 Gutachter: 1) Dr. Martin Kaltenpoth, Max-Planck-Institut für Chemische Ökologie, Jena 2) Prof. Dr. Erika Kothe, Friedrich-Schiller-Universität, Jena 3) Prof. Dr. Cameron Currie, University of Wisconsin-Madison, USA Das Promotionskolloquium wurde abgelegt am: 03.März 2014 “There is nothing like looking, if you want to find something. You certainly usually find something, if you look, but it is not always quite the something you were after.” The Hobbit, J.R.R. Tolkien “We are symbionts on a symbiotic planet, and if we care to, we can find symbiosis everywhere.” Symbiotic Planet, Lynn Margulis CONTENTS LIST OF PUBLICATIONS ......................................................................................... 11 CHAPTER 1: GENERAL INTRODUCTION ........................................................... 13 1.1 The biology of Streptomyces ...................................................................................................... 13 1.1.1 Streptomyces life cycle ......................................................................................................... 13 1.1.2 Streptomyces secondary metabolism ................................................................................. 15 1.1.3 Streptomyces as symbionts .................................................................................................. 16 1.2 Evolution and maintenance of bacterial mutualisms ......................................................... 18 1.3 Defensive insect-Actionbacteria symbioses ......................................................................... 19 1.3.1 Protection of the host´s nourishment ........................................................................... 20 1.3.2 Protection of the insect host and its offspring ............................................................ 22 1.4 The biology of the European beewolf Philanthus triangulum (Hymenoptera, Crabronidae) ................................................................................................. 24 1.4.1 Geographic distribution of Philanthus triangulum (Hymenoptera, Crabronidae) ...... 24 1.4.2 Biology of female European beewolves ....................................................................... 24 1.4.3 The beewolf-Streptomyces symbiosis ................................................................................ 27 1.5 Thesis outline ........................................................................................................................... 28 1.5.1 Ecology of Streptomyces-mediated antimicrobial defense ............................................. 28 1.5.2 Evolutionary history of the beewolf-Streptomyces symbiosis ....................................... 28 1.6 References ................................................................................................................................. 30 CHAPTER 2: DYNAMICS OF SYMBIONT-MEDIATED ANTIBIOTIC PRODUCTION REVEAL EFFICIENT LONG-TERM PROTECTION FOR BEEWOLF OFFSPRING ............................................................................................ 43 2.1 Abstract ..................................................................................................................................... 43 2.2 Background ............................................................................................................................... 44 2.3 Methods .................................................................................................................................... 46 2.3.1 Beewolf rearing conditions ............................................................................................. 46 2.3.2 Sample preparation .......................................................................................................... 46 2.3.3 Methanol extraction ......................................................................................................... 46 2.3.4 GC-MS analysis of antibiotics ........................................................................................ 47 2.3.5 DNA/RNA extraction .................................................................................................... 47 2.3.6 Quantification of Streptomyces population size and gene expression ......................... 47 2.3.7 Statistical analysis ............................................................................................................. 49 2.4 Results ....................................................................................................................................... 49 2.4.1 Streptomyces population dynamics on beewolf cocoons ................................................ 49 2.4.2 Dynamics of the antibiotic cocktail and piericidin gene expression.......................... 50 2.4.3 Expression of housekeeping and sporulation genes .................................................... 52 2.5 Discussion ................................................................................................................................. 53 2.5.1 Growth and morphological differnentiation of beewolf symbionts on the cocoon ......................................................................................................................... 53 2.5.2 Dynamics of antibiotic production and PKS gene expression .................................. 54 2.5.3 Stability of antibiotics mediates long-term antimicrobial protection for wasp offspring ............................................................................................................................ 54 2.5.4 The importance of antibiotics for the wasp´s offspring ............................................. 55 2.6 Conclusions .............................................................................................................................. 56 2.7 Supplement ............................................................................................................................... 56 2.7.1 Supplementary methods ................................................................................................... 56 2.7.2 Supplementary figures ...................................................................................................... 60 2.8 References ................................................................................................................................. 61 CHAPTER 3: MATERNAL AND ENVIRONMENTAL EFFECTS ON SYMBIONT-MEDIATED ANTIMICROBIAL DEFENSE ..................................... 67 3.1 Abstract ...................................................................................................................................... 67 3.2 Introduction .............................................................................................................................. 68 3.3 Methods and Materials............................................................................................................. 69 3.3.1 Beewolf rearing conditions .............................................................................................. 69 3.3.2 Cocoon collection and experimental conditions .......................................................... 69 3.3.3 Quantification of antibiotics on beewolf cocoons by GC-MS ................................... 70 3.3.4 Quantification of Streptomyces bacterial cells on the cocoon using quantitative real-time PCR (qPCR) ..................................................................................................... 71 3.3.5 Collection of AGS ............................................................................................................ 71 3.3.6 Chemical analysis of the AGS by GC-MS ..................................................................... 72 3.3.7 Analysis of symbiont cell number and length in the AGS .......................................... 72 3.3.8 Statistical analysis .............................................................................................................. 73 3.4 Results ........................................................................................................................................ 74 3.4.1 Environmental effects on symbiont growth and antibiotic production ................... 74 3.4.2 Maternal investment: AGS .............................................................................................. 75 3.5 Discussion ................................................................................................................................. 76 3.5.1 Effect of temperature and humidity on antibiotic production................................... 76 3.5.2 Effect of pathogen
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