The Microbial Associates and Putative Venoms of Seed Chalcid Wasps (Hymenoptera: Torymidae: Megastigmus) by Amber Rose Paulson BSc, Vancouver Island University, 2007 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of Biology Amber Rose Paulson, 2013 University of Victoria All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee The Microbial Associates and Putative Venoms of Seed Chalcid Wasps (Hymenoptera: Torymidae: Megastigmus) by Amber Rose Paulson BSc, Vancouver Island University, 2007 Supervisory Committee Dr. Steve Perlman, Co-supervisor (Department of Biology) Dr. Patrick von Aderkas, Co-supervisor (Department of Biology) Dr. Juergen Ehlting, Departmental Member (Department of Biology) iii Supervisory Committee Dr. Steve Perlman, Co-supervisor (Department of Biology) Dr. Patrick von Aderkas, Co-supervisor (Department of Biology) Dr. Juergen Ehlting, Departmental Member (Department of Biology) Abstract Conifer seed-infesting chalcids of the genus Megastigmus (Hymenoptera: Torymidae) are important forest pests. At least one species, M. spermotrophus Wachtl, has been shown to be able to manipulate the seed development of its host, Douglas-fir (Pseudotsuga menziesii) in remarkable ways, such as redirecting unfertilized ovules that would normally abort. The mechanism of host manipulation is currently unknown. Microbial associates and venoms are two potential mechanisms of host manipulation. Microbial associates are emerging as an important player in insect-plant interactions. There is also evidence that venoms may be important in gall- induction by phytophagous wasps. PCR and 16S rRNA pyrosequencing was used to characterize the microbial associates of Megastigmus and transcriptomic sequencing was used to identify putative venoms that were highly expressed in female M. spermotrophus. The common inherited bacterial symbionts Wolbachia and Rickettsia were found to be prevalent among several populations of Megastigmus spp. screened using a targeted PCR approach. A member of the Betaproteobacteria, Ralstonia, was identified as the dominant microbial associate of M. spermotrophus using 16S rRNA pyrosequencing. The transcriptome of M. spermotrophus was assembled de novo and three putative venoms transcripts were identified as highly expressed in females. One of these putative venoms transcripts, Aspartylglucosaminidase, (AGA) appears to have originated through gene duplication within the Hymenoptera and has been identified as a major venom component of two divergent parasitoid wasps. AGA was identified as a promising candidate for further investigation as a potential mechanism of early host manipulation by M. spermotrophus. iv Table of Contents Supervisory Committee .................................................................................................................. ii Abstract .......................................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Acknowledgments.......................................................................................................................... ix THE EVOLUTION OF PHYTOPHAGOUS HYMENOPTERA .................................................. 1 1.1 General evolutionary themes within the Hymenoptera ............................................. 1 1.2 Phytophagous Hymenoptera ...................................................................................... 3 1.2.1 Basal phytophagous Hymenoptera: Symphyta .......................................................... 3 1.2.2 The Secondary evolution of phytophagy: Apocrita ................................................... 4 1.2.3 Gall-induction in the Parasitica ................................................................................. 5 1.2.4 Cynipidae ................................................................................................................... 5 1.2.5 Chalcidoidea .............................................................................................................. 6 1.2.6 Agaonidae .................................................................................................................. 8 1.2.7 Seed-feeding chalcids ................................................................................................ 8 1.2.8 Braconidae ................................................................................................................. 9 1.2.9 Phytophagy within the Aculeata .............................................................................. 10 1.3 Nutritional considerations of phytophagous hymenopterans .................................. 10 1.4 Challenges associated with endophytophagy .......................................................... 12 1.5 Study-system: Megastigmus .................................................................................... 14 1.6 Thesis objectives ...................................................................................................... 16 Chapter 2. CULTURE-INDEPENDENT SURVEY OF THE MICROBIAL ASSOCIATES OF THE SEED-CHALCID WASPS (GENUS: MEGASTIGMUS) USING DIRECTED PCR SCREENING AND 454 PYROSEQUENCING .......................................................................... 17 2.1 Introduction ............................................................................................................. 17 2.1.1 Objectives ................................................................................................................ 20 2.2 Materials and methods ............................................................................................. 20 2.2.1 Insect samples .......................................................................................................... 20 2.2.2 DNA extraction ........................................................................................................ 22 2.2.3 Directed PCR ........................................................................................................... 22 2.2.4 Bacterial tag-encoded FLX amplicon pyrosequencing ........................................... 25 2.2.5 Qiime pipeline ......................................................................................................... 26 2.2.6 Phylogenetic analysis .............................................................................................. 27 2.3 Results ..................................................................................................................... 28 2.3.1 Inherited symbiont screening ................................................................................... 28 2.3.2 Phylogenetic analysis .............................................................................................. 28 2.3.3 Microbial associates of M. spermotrophus .............................................................. 33 2.4 Discussion ................................................................................................................ 43 2.4.1 Common heritable symbionts .................................................................................. 43 2.4.2 Microbial associates of M. spermotrophus .............................................................. 48 2.5 Conclusions ............................................................................................................. 52 v Chapter 3. DE NOVO TRANSCRIPTOME ASSEMBLY AND PUTATIVE VENOM DISCOVERY IN THE DOUGLAS-FIR SEED CHACLID, MEGASTIGMUS SPERMOTROPHUS (HYMENOPTERA: TORYMIDAE). ........................................................ 54 3.1 Introduction ............................................................................................................. 54 3.1.1 Objectives ................................................................................................................ 57 3.2 Materials and Methods: ........................................................................................... 58 3.2.1 RNA extraction ........................................................................................................ 58 3.2.2 Short read filtering and de novo assembly ............................................................... 59 3.2.3 Annotation ............................................................................................................... 59 3.2.4 Microsporidia investigation ..................................................................................... 60 3.2.5 Differential expression ............................................................................................ 61 3.2.6 Aspartylglucosaminidase protein phylogeny ........................................................... 61 3.3 Results ..................................................................................................................... 62 3.3.1 Short read filtering and de novo assembly ............................................................... 62 3.3.2 Annotation ..............................................................................................................