Evolution of mutualistic microbiome in firebugs and cotton stainers (Hemiptera; Pyrrhocoridae) -Characterizing the role of bacterial symbionts in diversification and niche expansion of Pyrrhocoridae Dissertation To Fulfill the Requirements for the Degree of „Doctor of Philosophy“ (PhD) Submitted to the Council of the Faculty of Biology and Pharmacy of the Friedrich Schiller University Jena by Sailendharan Sudakaran (M.Sc.) st born on Febrauray 21 , 1987 in Madurai (India) Reviewers 1. Prof. Dr. Rolf Beutel 2. Prof. Dr. Andreas Brune 3. Dr. Martin Kaltenpoth Date of defense: 18/05/2015 …from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved. The Origin of Species, Charles Darwin Table of Contents LIST OF PUBLICATIONS ........................................................................................................... 1 CHAPTER 1: GENERAL INTRODUCTION ............................................................................ 5 1.1 Symbioses: key drivers of niche expansion and evolutionary diversification .................... 5 1.2 Symbiotic interactions between microbes and insects ...................................................... 7 1.3 Evolutionary history and ecological factors shape symbiont composition ..................... 12 1.4 Role of host immunity in maintaining symbiosis equilibrium ........................................ 13 1.5 Symbiont-mediated resource acquisition ........................................................................ 15 1.6 Darwin’s blind spot: Symbiosis facilitates speciation and diversification in insects ...... 19 1.7 The Pyrrhocoridae – bacterial symbiosis ........................................................................ 22 1.7.1 Stinkbugs and their microbial symbionts ................................................................ 22 1.7.2 The family Pyrrhocoridae and its gut symbionts ..................................................... 23 1.8 Thesis outline .................................................................................................................. 26 1.9 References ....................................................................................................................... 28 CHAPTER 2: Geographic and ecological stability of the symbiotic mid-gut microbiota in European firebugs, Pyrrhocoris apterus (Hemiptera, Pyrrhocoridae) ................................ 41 2.1 Abstract ........................................................................................................................... 41 2.2 Introduction ..................................................................................................................... 43 2.3 Materials and Methods .................................................................................................... 47 2.3.1 Rearing conditions and sample collection ............................................................... 47 2.3.2 DNA extraction and amplification .......................................................................... 49 2.3.3 Bacterial tag-encoded FLX amplicon pyroseqencing (bTEFAP) and data analysis ............................................................................................................... 49 2.3.4 PCR amplification, cloning, and sequencing ........................................................... 50 2.3.5 Molecular phylogenetic analysis ............................................................................. 51 2.3.6 Quantitative PCR ..................................................................................................... 52 2.3.7 Fluorescence in-situ hybridization (FISH) ............................................................... 53 2.3.8 Microelectrode measurements ................................................................................. 54 2.4 Results ............................................................................................................................. 56 2.4.1 Bacterial communities in different gut regions of P. apterus .................................. 56 2.4.2 Phylogenetic placement of P. apterus midgut symbionts ....................................... 58 2.4.3 Ontogenetic changes of the P. apterus microbiota .................................................. 61 2.4.4 Inter-population differences in the mid-gut microbiota of P. apterus ..................... 63 2.4.5 Effect of different diets on the mid-gut microbiota of P. apterus ........................... 65 2.4.6 Transient microbes from ingested food material ..................................................... 66 2.4.7 Localisation of microbial symbionts ....................................................................... 67 2.4.8 Microelectrode measurements ................................................................................. 68 2.5 Discussion ....................................................................................................................... 69 2.5.1 Composition of the P. apterus gut microbiota ........................................................ 69 2.5.2 Transmission and establishment of the microbial mid-gut community in P. apterus ......................................................................................... 71 2.5.3 Ecological stability of the P. apterus gut microbiota .............................................. 72 2.5.4 Comparison of methods for the characterization of microbial communities .......... 73 i 2.5.5 Putative function of the P. apterus mid-gut microbiota ........................................... 74 2.6 Acknowledgements .......................................................................................................... 77 2.7 References ........................................................................................................................ 78 2.8 Supplement ...................................................................................................................... 87 CHAPTER 3: Actinobacteria as essential symbionts in firebugs and cotton stainers (Hemiptera,Pyrrhocoridae) ................................................................................ 93 3.1 Abstract ............................................................................................................................ 93 3.2 Introduction ...................................................................................................................... 95 3.3 Results .............................................................................................................................. 98 3.3.1 Midgut microbiota of D. fasciatus ........................................................................... 98 3.3.2 Success of symbiont manipulation procedure .......................................................... 99 3.3.3 Fitness of P. apterus and D. fasciatus following symbiont manipulation ............. 101 3.3.4 Reproductive success of adult D. fasciatus females following symbiont manipulation ................................................................................................... 103 3.3.5 Correlations linking individual strain abundances to fitness ................................. 105 3.3.6 Fitness of D. fasciatus fed on sunflower seeds ...................................................... 107 3.4 Discussion ...................................................................................................................... 108 3.5 Experimental procedures ............................................................................................... 113 3.5.1 Insect sampling and rearing ................................................................................... 113 3.5.2 Bacterial tag-encoded FLX amplicon pyroseqencing (bTEFAP) and data analysis ............................................................................................................. 113 3.5.3 Experimental manipulation of the microbial gut community ................................ 115 3.5.4 Fitness measurements ............................................................................................ 116 3.5.5 DNA extraction and PCR screening for C. glomerans .......................................... 116 3.5.6 Quantitative PCR ................................................................................................... 118 3.5.7 Statistical analysis .................................................................................................. 120 3.6 Acknowledgements ........................................................................................................ 120 3.7 References ...................................................................................................................... 121 3.8 Supplement .................................................................................................................... 128 CHAPTER 4: Evolutionary transition in symbiotic syndromes enabled adaptive radiation of phytophagous insects on an imbalanced diet ...................................... 135 4.1 Abstract .......................................................................................................................... 135 4.2 Introduction .................................................................................................................... 137 4.3 Materials and Methods ................................................................................................... 141 4.3.1 Insect sample collection and DNA extraction .......................................................