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Interaction of European Chalcidoid Parasitoids with the Invasive Chestnut Gall Wasp, Dryocosmus kuriphilus Julja Ernst Submitted for the degree of Master of Philosophy The University of Edinburgh College of Science and Engineering School of Biological Science 2017 ii Declaration The work contained within this thesis has been composed by myself and is my own work unless otherwise stated. Aspects of this work were made possible by collaboration and data sharing with individuals and institutions presented here. Italy Data for D. kuriphilus and its parasitoid associates were made available by: The department of exploitation and protection of agricultural and forestry resources (DIVAPRA) in Turin (Italy). Prof. Alberto Alma, Dr. Ambra Quacchia and Dr. Chiara Ferrancini provided data from the North and Centre of Italy and suggested field sites for gall collections. They also provided space and staff hours to rear D. kuriphilus parasitoids collected by Marloes de Boer and myself in Sicily. The department of Agri-Food and Environmental Systems Management at the University of Catania (Italy), in particular, Prof. Santi Longo, Dr. Lucia Zappala and Dr. Antonio Biondi provided information on the distribution of D. kuriphilus in Sicily, suggested field sites for collections and shared preliminary research carried out after arrival of D. kuriphilus. Switzerland Dr. Alexandre Aebi at the research institute Agroscope Reckenholz-Taenikon of the University of Zurich suggested field sites for gall collections and provided data of D. kuriphilus parasitoids in Switzerland. Slovenia Dr. Katarina Kos at the Biotechnical Faculty of the Department of Agronomy at the University of Ljubljana (Slovenia) provided data and reared samples of D. kuriphilus parasitoids in Slovenia, gave suggestions for field sites to carry out gall collection and provided many person hours to aid with collections. Croatia Dr. Dinka Matosevic at the Croatian Forest Research Institute (Croatia) provided D. kuriphilus data and samples, field sites for gall collections and information on the spread of D. kuriphilus in Croatia. iii I have received samples of D. kuriphilus parasitoids, for molecular analyses, with permission, from the above stated institutions and it was agreed to return samples to their rightful owners after completion of this project. All data involving oak gall parasitoids have been collected and reared by myself and my field assistants under my supervision. Field assistants included Laura Riggi for spring collections 2011, Gethin Evans for autumn collections 2011 and Marloes de Boer for spring collections 2012. A subset of oak gall parasitoid identifications carried out by myself were verified by Dr. George Melika from the Plant Health and Molecular Biology Laboratory, National Food Chain Safety Office in Budapest, Hungary, an expert in cynipid taxonomy and their associates. Signed: _____________________________ Date:___________________ iv Acknowledgements There are too many people to thank and I hope I am covering everyone who has made this thesis possible. Each and every person has gone past what was necessary to make this massive undertaking as pleasurable as it was (at times ;) ) First, I would like to thank all the lovely collaborators stated in my declaration, above. They have given their time, data and so much valuable information to this thesis and I will forever remain in their debt. I would like to thank my family, who have not only brought me all the way here, but have been so patient (and sometimes pushing me) all the way through this undertaking which seemed so much bigger than myself at times. I also have so many friends to thank, those who have helped me with my fieldwork (Laura, Gethin and Marloes), those who have helped me proof-read (Frazer Sinclair, Kasia Mikolajczak, Esther Cabanillas) and those who have helped with intellectual and moral support and keeping me sane, well somewhat!!! (MaFe Gimenez, Doris Reineke, James Nicholls, Jack Hearn, Tom Godfrey, and so many more) First and foremost, I could never put into words the amount of gratitude I feel towards my supervisors Dr. Karsten Schönrogge and Prof. Graham Stone. They have taken me on as a lowly undergraduate student and have since shared with me the joys, excitement and gratifying work that is associated with studying gall wasp ecology. They have, since day one, been an incredible inspiration to what scientific work can entail including international collaboration on a grandiose scale and tending a closed knit community of warm-hearted and supportive scientists all sharing an absolutely beautiful passion. They have supported me throughout this, sometimes dooming, experience and made sure that I always saw the light at the end of the tunnel. Doktorväter through and through. I would have never managed without your unrelenting support. v Table of Contents Declaration ......................................................................................................... ii Acknowledgements ....................................................................................... iv Abstract ................................................................................................................ 1 Chapter 1 – Introduction ............................................................................... 2 1.1 Community Interactions and Biological Invasions .............................................. 2 1.2 Value of the Gall Wasp System in Studies of Community Interactions ........ 3 1.3 The Chestnut Gall Wasp Dryocosmus kuriphilus.................................................. 9 1.4 Classical Biological Control of D. kuriphilus........................................................ 15 1.5 Questions and Hypotheses ........................................................................................ 16 1.6 Thesis Outline............................................................................................................... 18 Chapter 2 – Methods .................................................................................... 19 2.1 Fieldwork ....................................................................................................................... 19 2.1.1 Sampling Strategy and Gall Collection ............................................................................... 19 2.1.2 Rearing of Gall Inhabitants ...................................................................................................... 21 2.2 Molecular Methods ..................................................................................................... 21 2.2.1 Sample Selection for DNA Barcoding ................................................................................. 21 2.2.2 DNA Extraction and PCR Amplification ............................................................................. 24 2.2.3 Allocation of Specimens to Molecular Operational Taxonomic Units ............... 26 2.3 Statistical Analyses ...................................................................................................... 27 2.3.1 Summary Statistics Used ........................................................................................................... 27 2.3.2 Subsets of Data Used to Define Parasitoid Assemblages .......................................... 31 Chapter 3 – Results ....................................................................................... 32 3.1 Qualitative Analysis of the Parasitoid Community Using Morpho-taxa ..... 32 3.1.1 Comparison of Parasitoid Assemblages Between Host Plants .............................. 36 3.1.2 Comparison of Parasitoid Assemblages Between Regions ..................................... 36 3.2 Barcode-informed Identification of Four Focal Species .................................... 37 3.2.1 Eupelmus urozonus ....................................................................................................................... 40 3.2.2 Eurytoma brunniventris ............................................................................................................. 40 3.2.3 Megastigmus dorsalis .................................................................................................................. 41 3.2.4 Torymus flavipes ............................................................................................................................ 42 3.3 The Impact of DNA Barcode Information on Community Composition ..... 43 3.3.1 Impact of DNA Barcode Identification on Differences in Parasitoid Assemblages between plant taxa ..................................................................................................... 43 3.3.2 Impact of DNA Barcode Identification